Some of the effects of insulin might also be mediated through its binding to insulin-like growth factor (IGF) receptors. IGF1 is a growth factor exerting trophic effects on neuronal regeneration. It stimulates protein synthesis in neurons, glia, oligodendrocytes, and Schwann cells, and favor neuronal survival while inhibiting apoptosis (31). Finally, insulin can promote neuronal survival by protecting the brain against neuroinflammation (32).

Inflammation plays a critical role in the pathogenesis of AD and T2D. AD pathology could be influenced by tissues involved in T2D (Liver, adipose tissue, pancreas), through the excessive trafficking of neurotoxic molecules such as proinflammatory mediators, generated by the diabetic condition, across the blood-brain barrier. Thus, periphery-derived proinflammatory molecules could aggravate AD pathogenesis in the central nervous system (**Figure 1**) (15, 16, 33).

However, despite intense research efforts, our knowledge of the cellular and molecular pathways linking AD and metabolic disorders remains incomplete. The understanding of the common molecular mechanisms associated with both AD and diabetes, are crucial because it could ultimately lead to the identification of common therapeutic targets for these two interconnected conditions.

### SHARED MOLECULAR PATHWAYS LINKING T2D TO AD

Abnormalities in insulin/IGF signaling pathways have been shown in brains with AD (34, 35). These abnormalities were associated with reduced levels of insulin receptor substrate (IRS) mRNA, tau mRNA, IRS-associated phosphotidylinositol 3-kinase, and phospho-Akt. They also increased glycogen synthase kinase-3β (GSK3β) activity, an enzyme involved in Tau phosphorylation, and APP mRNA expression (36). Hence, the disruption of insulin functions in diabetic condition interrupts insulin signaling involved in the clearance of Aβ plaques and in neurofibrillary tangles (NFTs) pathology. This participates

to the accelerated formation of neurotoxic Aβ and NFTs via various mechanisms including GSK3β and the dual-specificity tyrosine-(Y)-phosphorylation regulated kinase DYRK1A (37). Thus, insulin resistance and T2D can interact with key pathways involved in AD pathology.

Hyperphosphorylated Tau protein is the main constituent of NFTs, which alongside amyloid β plaques, have long been considered as key histopathological hallmarks of AD. Interestingly, amyloid deposition (amylin) and abnormal Tau processing may provide yet another link between diabetes and AD. Indeed, studies report a potential role of Aβ and Tau in peripheral tissues. In humans, pancreatic amyloid deposition, similar to its damaging effect in brain during AD, is associated with β cell loss and global dysmetabolism (38). High levels of phosphorylated Tau were found in pancreas from T2D patients (39). GSK3β is involved in the formation of both Aβ deposits and NFTs. GSK3β induces Tau hyperphoshorylation to form NFTs through PI3K/Akt/GSK3β signaling pathway (40). Importantly, GSK3β is also involved in many aspects of T2D pathogenesis. As a negative effector of the insulin signaling pathway, GSK3β is involved in insulin resistance (41). GSK3β expression and activity were shown to be increased in muscle of diabetic patients and was implicated in muscle insulin resistance (42). Furthermore, we and others have reported that beyond its implication in insulin resistance in target tissues, GSK3β acts as a negative regulator of β-cell growth and function, thus further implicating this enzyme in the relative insulin deficiency associated with T2D (43–46).

Peripheral insulin resistance, a hallmark of T2D, can cause brain insulin resistance via a liver/pancreas/brain axis. Elevated plasma homocysteine (Hcy) level is a risk factor for AD pathology and is also closely associated with metabolic syndrome (47–49). Elevated Hcy levels have been linked with gray and white matter volume reduction among individuals with mild cognitive impairment and AD (50). Plasma Hcy is highly dependent on intracellular Hcy metabolism in the liver and kidney, but it may also reflect one-carbon metabolism in a number of other cell types, notably in pancreas and brain. Hepatic insulin resistance during T2D leads to inflammation which could in turn results in excessive Hcy production. Cytotoxic Hcy generated in liver, traffics through the circulation following injury or cell death, and can cross the bloodbrain barrier and exert neurotoxic effects by impairing central insulin signaling and activating pro-inflammatory cytokines. These abnormalities establish or help propagate a cascade of neurodegeneration associated with oxidative stress, which exacerbate brain insulin resistance, cell death, and neuroinflammation (51) (**Figure 1**).

Another potentially important player in AD pathogenesis is DYRK1A (52). DYRK1A interacts with APP and APP processing by direct phosphorylation of APP at Thr-668 and indirect phosphorylation of the presenilin 1 (PS1) at Thr-354, promoting the pathological Aβ pathway (53, 54). Increased expression of DYRK1A seems to promote brain β-amyloidosis by enhancing the phosphorylation and the amyloidogenic cleavage of APP, increasing the amyloidogenic levels of Aβ40 and Aβ42 (54). It also promotes neurofibrillary degeneration directly through hyperphosphorylation of tau and indirectly through phosphorylation of alternative splicing factor, therefore participating to neurodegeneration and neuronal loss appearing in AD (54–56). Moreover, we have shown that AD patients exhibited a positive correlation between plasma DYRK1A levels and CSF tau and phosphorylated-tau proteins (57).

In recent years, increasing interest has been drawn to the role of DYRK1A in β cell biology, making it another possible molecular link between AD and T2D. Several studies show that inhibition of DYRK1A alone (58, 59) or associated with the inhibition of GSK3β (60), or with SMAD and Trithorax pathways (61) induces human β cell proliferation.

However, other data in mice model of DYRK1A overexpression showed expansion of β cell mass through increased proliferation and cell size (62), suggesting a positive effect of DYRK1A on β cell growth in this model, which contrasts with the data on human β cells cited above.

DYRK1A has been demonstrated to be involved in the cycle of Hcy (63, 64), and its overexpression was linked with BDNF reduction (65). BDNF, the most widely distributed neurotrophin in the central nervous system, has a pivotal role in maturation, synaptic connection, neuronal repair, and plasticity of the central nervous system (66). Loss of BDNF in neurodegenerative disorders is a key mediator of synaptic dysfunction, neurodegeneration and subsequent cognitive decline (67). AD subjects show reduced BDNF levels in the serum and brain as compared with healthy elderly controls (68–70). Interestingly, there was a notable increase in plasma Hcy level and significant decrease in serum BDNF level in amnestic mild cognitive impairment patients that converts to AD, especially in those with the APOE ε4 allele (71).

Higher expression of BDNF slows down cognitive decline in the elderly, especially in the setting of advancing AD neuropathology, indicating that the brain BDNF level could be used as a novel marker for evaluating AD progression (68, 72). Combined assessment of DYRK1A and the related markers BDNF and Hcy has been validated by our team, by logistic regression analysis as diagnostic marker for AD in two unrelated AD patient cohorts with age-matched controls (73).

FIGURE 2 | Age-dependent changes in plasma Dyrk1A levels. Blood was collected at the tail vein of Wistar and GK rats of 3 and 6 months of age, at 9:00 a.m. Analyses were performed in plasma. (A) Schematic representation of Dyrk1A with distinct epitopes recognized by different antibodies. Plasma levels of full-length Dyrk1A form (B) and full-length and truncated forms of Dyrk1A (C). The DYRK1A levels were assessed by a solid phase immobilized epitope- immunoassay set up for antibody 7D10 (Abnova; immunogen: 674 aa ∼763 aa) and antibody D1694 (Sigma; immunogen: 32 aa ∼51 aa) (73). After removal of unbound conjugates, bound enzyme activity was assessed by use of a chromogenic substrate for measurement at 450 nm by a microplate reader (Flex Station 3, Molecular Device, San Diego, CA, USA). All the assays were performed in duplicate. For multiple pairwise comparisons between genotypes and ages, statistical analysis was done with two-way ANOVA followed by Fisher's post-hoc test using Statview software. The results are expressed as means ± SEM (standard error of the mean). n = number of rats. Data were considered significant when p < 0.05.

Interestingly, several reports also documented an association between plasma BDNF and systemic or peripheral inflammatory conditions, notably T2D (74). Plasma BDNF levels were found to be decreased in T2D patients (75–78). Interestingly, the relationship between T2D, BDNF and dementia was reported in one study which demonstrated lower plasma BDNF levels in patient group with both T2D and dementia than in non-diabetic patients with dementia (79).

Treatments to alleviate brain insulin resistance, such as intranasal insulin administration, have been evaluated in mice models and AD patients (80–84). Insulinotropic hormones such as glucagon-like peptide-1 (GLP-1), have also been proposed as a treatment for neurodegenerative disorders. Indeed, exenatide, a glucagon-like peptide-1 (GLP-1) agonist used for the treatment of T2D led to improvements in motor assessments in patients with Parkinson's disease (85). The potential relevance of this drug for other neurodegenerative disorders (e.g., AD) has being assessed in pre-clinical studies. Exenatide was tested in different mice models of AD, in 3xTg-AD mice on a high-fat diet, in APP/PS1 mice, and in adult wild-type mice as a model of mid-life brain aging. Results demonstrate a beneficial effect of drug treatment not only on cognition but also on BDNF neurotrophic axis (86–88).

FIGURE 3 | Age-dependent changes in plasma BDNF. Blood was collected at the tail vein of Wistar and GK rats of 3 and 6 months of age, at 9:00 a.m. Analyses were performed in plasma. BDNF was assessed using sandwich ELISA (ELISA E-Max, Promega, Madison, WI, USA). After removal of unbound conjugates, bound enzyme activity was assessed by use of a chromogenic substrate for measurement at 450 nm by a microplate reader (Flex Station 3, Molecular Device, San Diego, CA, USA). All the assays were performed in duplicate. For multiple pairwise comparisons between genotypes and ages, statistical analysis was done with two-way ANOVA followed by Fisher's post-hoc test using Statview software. The results are expressed as means ± SEM (standard error of the mean). n = number of rats. Data were considered significant when p < 0.05.

### ANIMAL MODELS OF COMBINED AD AND T2D

Several animal models, mostly in rodents, have been designed to study the interconnection between AD and T2D. These models include high fat died-induced insulin resistance, streptozotocin-induced diabetes or monosodium glutamate (MSG)-treated rodents (89, 90). A variety of cognitive/behavioral impairments and/or histopathological defects have been reported in these studies, thus providing the experimental basis for the epidemiological data that link T2D to AD. However, most of available models have back draws since they do not replicate the progressive characteristics of T2D with a silent phase followed by the development of insulin resistance and relative insulin deficiency.

The Goto–Kakizaki (GK) rats is a spontaneous model of T2D with close similarities with human T2D (91). The chronology of the infra-clinical and clinical phases in the GK rat, ranging from primary defects in the endocrine pancreas, as early as the fetal stage, followed by a neonatal phase of pre-diabetes, and finally the occurrence of overt hyperglycemia in adult individuals has been extensively described by our team (91, 92). The relevance of the GK rat as a T2D model lies in the fact that it is a spontaneous model without genetic manipulation, in which diabetes develops through a gradual process following a well-characterized phase of pre-diabetes (93), similar to the human T2D pathology. We have previously demonstrated in GK rats, a strong association between Hcy metabolism and insulin via cystathionine beta synthase (CBS) activity, the enzyme implicated in the first step of the trans-sulfuration pathway (94). In addition to several metabolic defects, GK rats also display impairment in their learning abilities and memory capabilities. Interestingly, cognitive dysfunction in GK rats was correlated with their insulin resistance index (95). Another study have reported significant decrease in phosphorylation of Akt, as well as reduced expression of CREB, an important regulator in the expression of functional proteins associated with learning and memory in this model of T2D (96). Studies using transgenic models of AD have generated mounting evidence supporting alteration in neurogenesis (97). Previous studies by our group and other's showed that chronic hyperglycemia impairs hippocampus neurogenesis in adult diabetic GK rats (98, 99), showing similarity with defects reported transgenic mice models of AD.

TABLE 1 | Correlations between plasma levels of Dyrk1A, BDNF, and Tau determined by Spearman's rank correlation.

Based on the literature described above, the spontaneous GK model of T2D appears as a valuable tool to investigate the relationship between T2D and AD.

### STATEMENT OF HYPOTHESIS: PLASMA LEVELS OF DYRK1A, BDNF, AND TAU ARE MODIFIED IN GOTO-KAKIZAKI RATS

Taking advantage of the characteristics of this model, we sought to analyze the circulating levels of some of the biomarkers of AD, which could potentially be related to T2D, namely DYRK1A, BDNF and Tau, in 3 and 6 months old diabetic GK rats.

Jin et al. have reported that DYRK1A was truncated in the brains of AD patients resulting in formation of truncated forms due to increased calpain activity (100), associated with a decrease of the full-length form (**Figure 2A**). DYRK1A contains a PEST sequence, a signal peptide for protein degradation via calpain (101, 102). Recently, Souchet et al. found that this DYRK1A cleavage is a consequence of the amyloid pathology (103). Resulting truncated forms accumulate in astrocytes and exhibit increased affinity toward a regulator of inflammatory process (103). Here, we analyzed these different forms by the use of two different antibodies, one recognizing the full-length form, and the other the full-length and truncated forms of Dyrk1A (**Figure 2A**) in plasma of control Wistar (WT) and GK rats. No difference was found between WT and GK rats at 3 months (**Figures 2B,C**), while an increase of full-length and the truncated forms was found in GK rats at 6 months (**Figure 2C**). The full-length form was also increased at 6 months in WT rats compared to 3 months old WT rats (**Figure 2B**), suggesting an age-related effect.

BDNF levels were decreased in plasma of GK rats at 3 and 6 months, compared to age-matched WT rats (**Figure 3**). This was in keeping with studies showing decreased plasma levels of BDNF in diabetic patients (75–78). There is also solid evidence demonstrating a reduction in BDNF mRNA and protein levels in AD cortex and hippocampus (104, 105), and decreased BDNF levels contribute to cognitive dysfunction in AD (66). A significant decrease in BDNF serum concentration has been found in AD patients compared with healthy controls (106). Correlations were determined by using Spearman's rank correlation, as data were not normally distributed according to Shapiro-Wilk test. A negative correlation was found between plasma BDNF and full-length and truncated forms of Dyrk1A levels (**Table 1**). As Dyrk1A is involved in controlling numerous pathways, this result emphasizes the role of this kinase on BDNF signaling pathways, as previously suggested by our team (65, 73).

Tau protein truncated at amino acid D421 has been detected in AD (**Figure 4A**). This C-terminal truncation introduces a conformational change contributing to aggregation (107, 108). We therefore measured the levels of centrally-situated Tau epitope (**Figure 4B**) and levels of Tau 46 (**Figure 4C**), to evaluate the index of truncation. The index of C-terminal truncation was provided by the ratio of Tau46/Tau5 (**Figure 4D**). Tau levels (Tau5 immunoreactivity) increased in plasma of GK rats at 3 and 6 months, compared to age-matched WT rats. There was no difference of Tau levels between WT rats at 3 and 6 months (**Figure 4A**). Tau levels are correlated positively with full-length and truncated forms of Dyrk1A levels (**Table 1**) and negatively with BDNF levels (**Table 1**). Interestingly, we previously found a positive correlation between plasma Dyrk1A levels and CSF Tau proteins in AD patients (57).

An increased Tau46 immunoreactivity and Tau46/Tau 5 was found in plasma of GK rats as early as 3 months and persisted at 6 months of age compared to age-matched WT rats (**Figures 4C,D**). Interestingly, Tau46 immunoreactivity and Tau46/Tau5 ratio increased in an age-dependent manner within the WT group (**Figures 4C,D**). The Tau46 immunoreactivity also correlated positively with full-length form of Dyrk1A levels (**Table 1**), full-length and truncated forms of Dyrk1A levels (**Table 1**), and negatively with BDNF levels (**Table 1**). These results indicate that Tau undergoes increased C-terminal cleavage as early as 3 months in the GK rats, while this effect appears in non-diabetic Wistar rats only at 6 months.

### CONCLUSION

In this paper we used the type 2 diabetic GK rat as a tool to assess circulating biomarkers for AD. We show that plasma

### REFERENCES

BDNF and the index of C-terminal truncation of Tau could be considered as early biomarkers, while plasma Dyrk1A could represent a late biomarker. As a spontaneous model of T2D with gradual progression, the GK rat is acknowledged as a valuable tool to study the pathogenesis of diabetes. Here we propose that the GK rat could be a new model to investigate the link between T2D and AD. It could therefore be a useful tool for pre-clinical studies to assess drug efficacy in the onset of the disease. Currently, we are addressing the question of possible abnormalities in the expression/activity of the above markers in the brain and the pancreas of the GK rat, to validate the relevance of this model as a model of T2D-associated AD. These results need to be compared with those described in validated rodent models of AD with different grade pathology. Further longitudinal studies of metabolic and cognitive parameters with pharmacological intervention are warranted to comprehend the causal relationship underlining the progression of AD and T2D.

### DATA AVAILABILITY

The raw data supporting the conclusions of this manuscript will be made available by the authors, without undue reservation, to any qualified researcher.

### ETHICS STATEMENT

All procedures were carried out in accordance with the ethical standards of French and European regulations (European Communities Council Directive, 86/609/EEC). Official authorization from the French Ministry of Agriculture was granted to perform research and experiments on animals (authorization number B-75-13-17) and the experimental protocol was approved by the institutional animal care and use committee of the Paris Diderot University (CEEA40).

### AUTHOR CONTRIBUTIONS

JM and NJ designed the study, made the review of the literature, and wrote the manuscript. ED, JL, and YG performed experiments. All authors read and approved the manuscript.

### FUNDING

This work was supported by Lagafe Association and Fondation Jérome Lejeune (grant number 2017a-1634).

disease biomarkers: a pilot clinical trial. J Alzheimers Dis. (2017) 57:1325–34. doi: 10.3233/JAD-161256

**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Copyright © 2019 Movassat, Delangre, Liu, Gu and Janel. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

# Deciphering the Link Between Hyperhomocysteinemia and Ceramide Metabolism in Alzheimer-Type Neurodegeneration

Hervé Le Stunff 1,2, Julien Véret <sup>1</sup> , Nadim Kassis <sup>1</sup> , Jessica Denom<sup>1</sup> , Kelly Meneyrol <sup>1</sup> , Jean-Louis Paul <sup>3</sup> , Céline Cruciani-Guglielmacci <sup>1</sup> , Christophe Magnan<sup>1</sup> and Nathalie Janel <sup>1</sup> \*

<sup>1</sup> Université de Paris, BFA, UMR 8251, CNRS, Paris, France, <sup>2</sup> Institut des Neurosciences Paris-Saclay (Neuro-PSI), Université Paris-Sud, CNRS UMR 9197, Orsay, France, <sup>3</sup> AP-HP, Hôpital Européen Georges Pompidou, Service de Biochimie, Paris, France

#### Edited by:

Eugenio Barone, Sapienza University of Rome, Italy

#### Reviewed by:

Ilaria Piano, University of Pisa, Italy Bindu Diana Paul, Johns Hopkins University, United States

\*Correspondence: Nathalie Janel<EMAIL_ADDRESS>
#### Specialty section:

This article was submitted to Neurodegeneration, a section of the journal Frontiers in Neurology

Received: 05 March 2019 Accepted: 15 July 2019 Published: 31 July 2019

#### Citation:

Le Stunff H, Véret J, Kassis N, Denom J, Meneyrol K, Paul J-L, Cruciani-Guglielmacci C, Magnan C and Janel N (2019) Deciphering the Link Between Hyperhomocysteinemia and Ceramide Metabolism in Alzheimer-Type Neurodegeneration. Front. Neurol. 10:807. doi: 10.3389/fneur.2019.00807 Aging is one of the strongest risk factor for Alzheimer's disease (AD). However, several data suggest that dyslipidemia can either contribute or serve as co-factors in AD appearance. AD could be examined as a metabolic disorder mediated by peripheral insulin resistance. Insulin resistance is associated with dyslipidemia, which results in increased hepatic ceramide generation. Hepatic steatosis induces pro-inflammatory cytokine activation which is mediated by the increased ceramides production. Ceramides levels increased in cells due to perturbation in sphingolipid metabolism and upregulated expression of enzymes involved in ceramide synthesis. Cytotoxic ceramides and related molecules generated in liver promote insulin resistance, traffic through the circulation due to injury or cell death caused by local liver inflammation, and because of their hydrophobic nature, they can cross the blood-brain barrier and thereby exert neurotoxic responses as reducing insulin signaling and increasing pro-inflammatory cytokines. These abnormalities propagate a cascade of neurodegeneration associated with oxidative stress and ceramide generation, which potentiate brain insulin resistance, apoptosis, myelin degeneration, and neuro-inflammation. Therefore, excess of toxic lipids generated in liver can cause neurodegeneration. Elevated homocysteine level is also a risk factor for AD pathology and is narrowly associated with metabolic diseases and non-alcoholic fatty liver disease. The existence of a homocysteine/ceramides signaling pathway suggests that homocysteine toxicity could be partly mediated by intracellular ceramide accumulation due to stimulation of ceramide synthase. In this article, we briefly examined the role of homocysteine and ceramide metabolism linking metabolic diseases and non-alcoholic fatty liver disease to AD. We therefore analyzed the expression of mainly enzymes implicated in ceramide and sphingolipid metabolism and demonstrated deregulation of de novo ceramide biosynthesis and S1P metabolism in liver and brain of hyperhomocysteinemic mice.

Keywords: NAFLD, Alzheimer's disease, hyperhomocysteinemia, sphingolipid metabolism, ceramides

## INTRODUCTION

Insulin resistance is a major public health outcome by its association with the non-alcoholic fatty liver disease (NAFLD), metabolic syndrome, type 2 diabetes mellitus (T2DM), obesity, and Alzheimer's disease (AD)-type neurodegeneration epidemics. Aging is also a powerful AD risk factor. However, many data imply dyslipidemic conditions as contributor or cofactors in pathogenesis of AD. The classical "amyloid cascade" hypothesis in AD, actually deeply debated, demonstrates that cognitive defects and memory loss implicate the development of wide and insoluble beta amyloid plaques in various brain areas, resulting in apoptosis of neurons (1).

Apart from amyloid-β peptide (Aβ), many evidences suggest that impairing of insulin signaling and brain glucose metabolism act an important role in AD development. Human postmortem studies support this notion, indicating that insulin resistance in AD brain is systematically showed and increased with disease advancement (2–4). Another connection between T2DM and AD could be supplied by Tau processing failures. Neurofibrillary tangles (NFTs) are principally constituted by hyperphosporylated tau proteins. NFTs, such as amyloid beta plaques, account as important histopathological characteristics of AD. Some authors have called AD as "type 3 diabetes" in relation with the alterations, at the very early disease stages, of insulin signaling more specifically in the brain (2). Insulin resistance is linked with inflammation and dyslipidemia which results in increased ceramides generation notably in hepatic function (5, 6). For this reason, AD could be considered like metabolic diseases mediated by disorders due to peripheral insulin resistance. Progressive hepatic steatosis induces inflammation with activation of pro-inflammatory cytokines, leading not only to increased ceramide production, but also alteration in one carbon metabolism. Ceramides build up in cells because of disruptions in sphingolipid metabolism and activation of pro-ceramides genes (7). Cytotoxic ceramides and related molecules generated in liver promote insulin resistance, traffic through the circulation due to injury or apoptosis caused by local liver inflammation, and because of their hydrophobic nature, can pass through the blood-brain barrier (BBB), thereby exerting toxic responses as reducing insulin signaling and increasing pro-inflammatory cytokines. These defects initiate or support propagation of neurodegeneration with an oxidative stress and ceramide production, exacerbating brain insulin resistance, neuronal apoptosis, and neuro-inflammation. Therefore, neurodegeneration can be caused by toxic liver lipid production (5). In mice, increased production of toxic lipid/ceramide can be induced by liver/peripheral tissue-brain axis of neurodegeneration and be moved through the BBB resulting cognitive impairments (8).

Disturbances in one carbon metabolism, determined by enzyme failures integral to this process, comply with abnormally increased plasma homocysteine (Hcy) levels, namely hyperhomocysteinemia (HHcy). HHcy has been frequently linked with T2DM, cardiovascular diseases (CVD), atherosclerosis and present in NAFLD (9–11). Given that the majority of dietary methionine metabolism is made in liver, this organ represents the major place for Hcy metabolism (12). During liver failure, metabolism of Hcy was modified in association with lipid metabolism disturbance (13–15). Increased Hcy level is also associated with AD pathology (16–19). Taken as a whole, results suggest that Hcy/ceramides signaling pathway exists which suggests a link between Hcy toxicity and intracellular ceramide accumulation via the activation of ceramide synthase (20).

### HOMOCYSTEINE, A LINK BETWEEN NAFLD, T2DM, AND AD

In the world, NAFLD is the most shared hepatic disorder, its incidence reaching 70–90%. It is also linked with obesity, T2DM and related metabolic diseases (21). NAFLD is linked to hepatic insulin resistance and occurs frequently with obesity/T2DM.  Progression of NAFLD is more likely to take place in metabolic diseases patients (22). Oxidative stress associated with insulin resistance have an essential part in NAFLD (23). One-carbon metabolism is involved in methylation of notably proteins, DNA, RNA, and protects cells against oxidation (24). S-adenosylmethionine (SAM) is produced by methionine adenosylation and is the most important methyl-group donor in cellular metabolism (25) (**Figure 1**). DNA methylation capacity of cells can be modified by a reduction of SAM concentration associated with a reduction in SAM: S-adenosylhomocysteine (SAH) ratio (26). Onecarbon metabolism perturbation consequently participates to pathogenesis and NAFLD promotion.

In the liver, Hcy, a thiol-containing amino acid, is involved in metabolism of dietary methionine. SAH metabolism produces Hcy, SAH being produced through methylation reactions implicating SAM and methyltransferases. Hcy can be metabolized by conversion to cysteine via the transsulfuration pathway, the first step involving cystathionine beta synthase (CBS) (27) (**Figure 1**). In alcoholic fatty liver disease (28) but also in non-alcoholic steatohepatitis (NASH) (29), serum Hcy levels are elevated and good predictor of disease progression. Hepatic steatosis in human patients and in mice with CBS deficiency is associated with HHcy (29–32). In rats, HHcy due to decreased hepatic CBS activity is elicited by NAFLD induced by high fat diet (15). Therefore, NAFLD is early characterized by HHcy. The involvement of dual-specificity tyrosine-(Y) phosphorylation regulated kinase (DYRK1A) in one carbon metabolism is strengthened by the positive correlation between hepatic protein expression and CBS activity (33, 34). DYRK1A, a protein involved in development, growth and apoptosis (35), is also implicated in Hcy cycle (36, 37) (**Figure 1**). DYRK1A is also implicated in β-cell mass adjustment and involved in carbohydrate metabolism (38, 39). In mice, DYRK1A is also implicated in liver damage induced by alcohol consumption (34).

Individuals with NAFLD can exhibit neuropsychiatric dysfunction, including anxiety and depression, which frequently precede cognitive impairment and dementia. Factors affecting one-carbon metabolism and consequently elevated Hcy levels

have been linked to AD. Divers risk factors of sporadic AD have been characterized by long-term and prospective population and cross-sectional retrospective studies, as hypercholesterolemia, T2DM, and HHcy (40). Elevated proinflammatory cytokines have metabolic connotation (40). Older patients suffering from mild hypertension showed association between hippocampal atrophy, and white matter atrophy, with HHcy. Elderly patients with HHcy showed increased rate of hippocampal atrophy and cognitive decline (41, 42). In the aged population, elevated Hcy level can be established as a risk factor for cognitive decline (43). Progression of white matter hyperintensities and faster rates of total brain volume loss have been associated with increased plasma Hcy levels in patients with hypertension (19). Many studies have demonstrated that moderately elevated Hcy levels increased late-onset Alzheimer's disease (LOAD) risk, even if the value is close to the critical threshold (16–19, 44). According to recent international consensus statement (45), moderately elevated Hcy level can increase the relative risk of dementia in the elderly 1.15- to 2.5-fold, with the Population Attributable risk from 4.3 to 31%.

Many mechanisms have been suggested to connect elevated Hcy level with AD. Many experimental studies have demonstrated that elevated Hcy level can produce many neurotoxic effects implying excitotoxicity, oxidative stress, mitochondrial dysfunction, DNA damage and apoptosis. Therefore, HHcy can participate to AD neurodegeneration (46). Preclinical studies show that HHcy generates Aβ accumulation in brain (47–51) and increased hyperphosphorylation of tau (52). The link of Hcy to Aβ can lead to the development of interconnections and consequently to the development of aggregates (53). Elevation of Hcy contributes to the decrease of SAM levels. Demethylation of DNA can be produced by decreased SAM level, leading to overexpression of presenilin (PSEN1) and beta-secretase (BACE1), the β-site amyloid precursor protein (APP)-cleaving enzyme (54). Another study demonstrates that dimerization of apoE3 can be block by Hcy, reducing apoE3-mediated high-density lipoprotein (HDL) generation and consequently reducing microglial degradation of soluble Aβ (55). Patients with HHcy, compared to control subjects, have lower ratio of apoE3 dimers in their cerebrospinal fluid (CSF).

Many studies demonstrate the involvement of DYRK1A in AD (56). DYRK1A interacts with APP, and plays a role in APP processing by direct phosphorylation of APP at Thr-668 and indirect phosphorylation of PSEN1 at Thr-354, promoting the pathological Aβ pathway and the production of Aβ. Increased DYRK1A expression enhances APP phosphorylation and its cleavage, resulting in increased Aβ40 and Aβ42 levels and thus promoting brain β-amyloidosis (57). Its direct tau hyperphophorylation and indirect phosphorylation of alternative splicing factor promotes neurofibrillary degeneration, showing its involvement in neurodegenerative processes and neuronal depletion appearing in AD (57–60). We have previously shown, on the one hand, that plasma DYRK1A levels correlate positively with CSF tau and phosphorylated-tau proteins in AD (61), and on the other hand that combined assessment of plasma levels of DYRK1A and Hcy can be validate as diagnostic marker for AD (44). Lipidomics analysis showed that not only triglycerides (TG) content but also ceramide content were increased in HHcy mice which have decreased liver DYRK1A protein expression (36, 62). We previously demonstrated altered lipoprotein metabolism in mice overexpressing DYRK1A (63). Note that mice overexpressing DYRK1A have, on the contrary, not only a decreased plasma Hcy level (37) but also a decreased serum TG level (0.369 ± 0.04 vs. 0.519 ± 0.057 mmol/L; p < 0.05 by Student's t-test; n = 8 for each). It will be important to demonstrate if the effects of Hcy not only in liver but also in brain are mediated by ceramide signaling.

### SPHINGOLIPID METABOLISM AND AD

Considered for a long time as structural compounds, several evidences demonstrated that bioactive sphingolipids are involved as signaling molecules in the various tissues including the brain. In these tissues, sphingolipids play important role in several pathologies including neurodegenerative diseases, such as AD. Sphingolipids could be produced by several pathways. De novo sphingolipid synthesis is initiated in the cytoplasmic face of the endoplasmic reticulum (ER) which is started with the condensation of palmitoyl-CoA L-serine with to form 3 ketosphinganine (**Figure 1**). Serine palmitoyl-transferase (SPT) catalyzes this reaction (64). Two subunits, SPTLC1 and SPTLC2, showing a similarity at amino acid sequence of around 20%, compose the heterodimer SPT. SPTLC1 and SPTLC2 seem to be both required for enzyme activity. However, the SPTLC2 subunit contains a pyridoxal phosphate binding motif (65) and produces the common and major C18-sphingoid bases. A third SPT subunit, SPTLC3, has been identified, with 68% homology to the SPTLC2 subunit. However, SPTLC3 is involved in the production of C14- and C16-sphingoid bases. SPTLC2 and SPTLC3 are therefore distinct from a specificity point of view (66). The SPT substrate preference toward the requirement of longer acyl-CoA could be modulated by the differential expression of SPTLC2 and SPTLC3. Moreover, C16-sphingoid bases could be transformed into more complex sphingolipids, such as C16-ceramide and glycosphingolipid. Sphingolipids can be implicated in AβPP/Aβ metabolism and therefore AD development due to their structural roles in cellular membranes including lipid rafts (67). Conversely, ceramide formation can be promoted by accumulation of oligomerized Aβ in AD brain. Indeed, Aβ peptides can activate SPT, resulting in neurotoxic ceramide increase by the de novo synthesis pathway (68, 69). Interestingly, SPTLC2 was found to be up-regulated in AD (70, 71). However, a regulation of SPTLC3 subunit has not been explored in neurodegenerative diseases development, such as AD.

3-ketosphinganine is formed and rapidly reduced by 3 ketosphinganine reductase into dihydrosphingosine (DH-Sph) (**Figure 1**). The reaction results in DH-Sph, implicated in the production of various species of dihydro-ceramides by ceramide synthases (CerS) (**Figure 1**). This species will differ by the nature of acyl-CoA chain length used for the N-acylation of DH-Sph (72). The dihydroceramide desaturases (DEGS1) metabolizes dihydro-ceramides into ceramides (**Figure 1**). Interestingly, CerS1 and CerS2 are up-regulated in AD brains whereas CerS6 is reduced (70, 71), suggesting a remodeling of ceramide species during the development of AD. The ceramide produced are converted in the Golgi apparatus into sphingomyelin or glucosylceramides by sphingomyelin synthase and glucosyl-ceramide synthase, respectively (73) (**Figure 1**).

Ceramidases could deacylated ceramides to produce sphingosine (**Figure 1**). In cells, sphingosine but also other sphingoid bases can be phosphorylated by two sphingosine kinases (SphK 1 and SphK2) to form sphingosine-1-phopshate (S1P). S1P can be dephosphorylated to sphingosine by specific S1P phosphohydrolases, the reaction being reversible, or can be cleaved by a pyridoxal-dependent S1P lyase (SGPL) into ethanolamine phosphate and hexadecenal, the reaction being irreversible (74) (**Figure 1**). S1P, in contrary to ceramide, is known to be a pro-survival lipid for various cells including neurons (74). Interestingly, S1P metabolism has also been related to AD. Indeed, FTY720, a substrate of SphK2 that can bind S1P receptors has been shown to reduce neuronal Aβ generation (75). Loss of neuroprotective S1P and SPHK activity was found early in AD development prior to AD diagnosis with a decrease of Sphk2 activity in hippocampus (76). However, the role of SphK2 is still controversial in AD since S1P production by SphK2 and Aβ processing seemed to be positively correlated (77). This discrepancy could come from subcellular distribution of SphK2 between cytosol and nucleus which is altered in AD brains (78). These data suggest that the pro-survival cytosolic S1P may be less efficient by a shift in the subcellular localization of the S1P generating by SphK2 which will lead to the production of nuclear S1P associated with deleterious effects in AD pathogenesis. Up to date, SphK2 and SGPL (70) have been shown to be overexpressed whereas SphK1 was found to be down-regulated in AD brain suggesting a deregulation of S1P signaling in this pathology which remained to be clarified.

### CERAMIDES, A LINK BETWEEN NAFLD, T2DM, AND AD

Ceramides are also important mediators of insulin resistance in various peripheral tissues but also in pancreatic β cell deregulation induced by obesity (79, 80). Obesity is wellestablished as a predisposing factor for the appearance of hepatic steatosis, NAFLD being strongly associated with both hepatic and peripheral insulin resistance with a defect in the ability of insulin to suppress endogenous glucose production (81). In addition, NAFLD is linked to important hepatic changes in lipid metabolites, such as an increase in hepatic cholesterol. When excess of saturated fatty acids are poorly incorporated into hepatocyte triglycerides, they induce lipotoxicity, resulting in liver injuries (82). Excess of saturated fatty acids constitutes a preferential substrate for the de novo ceramide biosynthesis (83). It is known that ceramide levels contribute to the development of NAFLD by mediating obesity, inflammation, insulin resistance, and oxidative stress (84).

In addition to intracellular-based actions, circulating extracellular ceramides have roles in insulin resistance. In vitro studies showed that hepatocytes treated with palmitate could efficiently secrete newly synthesized ceramide in response to hyperlipidemia, demonstrated by increased extracellular ceramide concentration (85). Ceramide could be either transported by lipoproteins but also by cell-derived membrane shed both basally and under stress conditions called extracellular vesicles (86, 87). Interestingly, advanced pathological signs of AD and also induced neuronal apoptosis could be elicited by chronic NAFLD in mice (88). Therefore, chronic inflammation induced by obesity-associated NAFLD outside from the brain is sufficient to induce neurodegeneration in the absence of genetic predisposition. Since NAFLD is associated with an increase of circulating sphingolipid, such as ceramide, it is tempting to suggest that circulating ceramide originating from liver could also target brain tissues and favor the development of AD.

Some of sphingolipids could constitute biomarkers to identify individuals who are at risk to develop T2DM. Therefore, it will be more important to quantify circulating sphingolipid concentrations. Plasma dihydro-ceramides levels were showed effectively to be significantly elevated up to 9 years before the detection of the disease of individuals from two human cohorts who will progress to diabetes (89). Interestingly, deregulation of ceramide metabolism reflected by an increase of plasma ceramide level could arise in different stages of AD progression (90, 91). Moreover, it would be important to define whether AD is associated with a differential distribution of ceramides in lipoproteins, but also in exosomes, which could serve as biomarkers of disease progression.

S1P has been also shown as a potent regulator of NAFLD, treatment with S1P enhancing hepatic lipid storage (92). A 2 fold increase of SphK1 was determined in livers from humans with NAFLD but also in mice feeded with a high saturated fat diet (92, 93). These mice showed activation of NFκB, elevated cytokine production, and immune cell infiltration. Importantly, a total SphK1-null mice were protected from these outcomes. To date, the role of SphK2 has not been explored in the context of NALFD. In contrast, it has been shown that a total SPLnull mice resulted in a widespread change in lipid metabolism genes expression pattern, with a significant increase in the expression of PPARgamma, a key transcriptional regulator of lipid metabolism (94), suggesting that regulation of SGPL could be a potent regulator of NAFLD. The liver is known to be engaged in regulating the plasma level of S1P, as liver produces a chaperone for S1P transport, the apolipoprotein M (apoM) (95). Interestingly, polarized endothelial cells, composing the lining of the BBB, also express and secrete apoM toward the brain as well as to the circulation suggesting that circulating S1P could target the brain by this way (96). Therefore, as ceramide, secretion of S1P by hepatocytes could constitute a potent regulator of AD by targeting the brain.

### STATEMENT OF HYPOTHESIS: CERAMIDE AND SPHINGOLIPID METABOLISM IS MODIFIED IN LIVER AND HYPOTHALAMUS OF HYPERHOMOCYSTEINEMIC MICE ON A HIGH FAT DIET

Based on the results described above, we used mice heterozygous for targeted disruption of the Cbs gene (Cbs+/−) (30) and wild type (Cbs+/+) mice on the same background, fed on a standard diet supplemented with 0.5% L-methionine (Sigma-Aldrich, France) in drinking water to induce intermediate HHcy in Cbs+/<sup>−</sup> mice (97), and with a high-fat diabetogenic diet (HFD) (98). As expected, Cbs+/<sup>−</sup> mice showed a significant increase of plasma Hcy level (37.1 ± 3.2µM vs. 6.4 ± 0.4µM; p < 0.0001 by Student's t-test n = 8 for each). We previously evaluated plasma Hcy levels in transgenic mouse models of AD. No significant difference was observed in blood of transgenic mouse models of AD compared to control mice, indicating that Hcy is not a primary cause of AD (99). Animal studies using AD-like transgenic mouse models, on a methionine enriched diet, were used to provide potential mechanisms by which HHcy might influence AD development. Tg2576 transgenic female mice expressing hAPP with the Swedish mutation (K670N/M671L) on a methionine enriched diet exhibited an increase from 6 to 35 µM (51, 100).

### Effect of HHcy and HFD on Enzymes Expression Involved in Ceramide and Sphingolipid Metabolism in Liver

Gene expression of mainly enzymes implicated in ceramide and sphingolipid metabolism has first been analyzed by Q-PCR in mice liver. As expected, Cbs+/<sup>−</sup> mice showed a significant decrease of liver CBS activity (45.2 ± 11.1 vs. 102 ± 21.6; p < 0.05 by Student's t-test n = 4 for each), commensurate with a decrease in mRNA expression (13.5 ± 7.3 vs. 100 ± 26; p < 0.03 by Student's t-test n = 4 for each). Cbs+/<sup>−</sup> mice also showed a significant decrease of liver Dyrk1A level (40.6 ± 6.3 vs. 100 ± 16; p < 0.02 by Student's t-test n = 4 for each) (36), associated with a decrease in mRNA expression (18.9 ± 14 vs. 100 ± 21.3; p < 0.023 by Student's t-test n = 4 for each). SGPL1 and SPTlc2 expression were decreased in liver of Cbs+/<sup>−</sup> on methionine and HFD (**Table 1**) (**Figures 2A,B**). In agreement with the decreased gene expression of SPTlc2, we also found a decrease in protein level (**Figure 2C**). Hepatic expression of CBS and SGPL1 (r = 0.96, p < 0.02), Sphk2 (r = 0.83, p < 0.05), and SPTlc2 (r = 0.93, p < 0.03) were positively correlated, Dyrk1A and Cers5 expression being correlated negatively (r = −0.93, p < 0.02).

Ceramide is a potent regulator of cell proliferation, activation, and apoptosis. Ceramide plays an key role in different cellular functions, such as plasma lipoprotein metabolism and cell

TABLE 1 | Relative liver and hypothalamus mRNA expression based upon Q-PCR data obtained from wild-type (Cbs+/<sup>+</sup> mice) and mice heterozygous for targeted disruption of the Cbs (Cbs+/<sup>−</sup> mice) on a methionine enriched diet (Met) and high fat diet (HFD).

Total RNA was isolated from the hypothalamus and liver, reverse transcribed and real time quantitative PCR amplification reactions were carried as described using the LightCycler FastStart DNA Master plus SYBR Green I kit (Roche) (97). The mRNA transcript level was normalized against the mean of two genes: H1a and TBP (Table S1). Data were normalized to the mean of Cbs+/<sup>+</sup> mice on Met/HFD. Data are presented as mean ± SEM, and analyzed with the Student's t-test by using Statview software. n, number of mice. \*p < 0.05; \*\*p < 0.01. Data were considered significant when p < 0.05.

FIGURE 2 | Effect of hyperhomocysteinemia and high fat diet on liver (A) SGPL1 mRNA, (B) SPTlc2 mRNA, (C) SPTlc2 protein, (D) Cers2 hypothalamus mRNA, (E) Sphk2 hypothalamus mRNA, and (F) SPTlc1 hypothalamus mRNA level in wild type (Cbs+/<sup>+</sup> mice) and mice heterozygous for targeted disruption of the Cbs (Cbs+/<sup>−</sup> mice) on a methionine enriched diet (Met) and high fat diet (HFD). Relative liver and hypothalamus mRNA expression was described in Table 1. Liver SPTlc2 was determined by western blot and quantified by slot blotting [1/1,000 (101)]. β-actin (1/10,000) (Sigma-Aldrich, France) was used as an internal control for Western blot analysis or ponceau-S coloration for slot blot analysis. Data are presented as mean ± SEM, and analyzed with the Student's t-test by using Statview software. n, number of mice. Data were considered significant when p < 0.05.

membrane formation, known to contribute to the development of atherosclerosis and other sclerotic diseases, such as insulin resistance, obesity, and AD (102, 103). Previous results have demonstrated that de novo ceramide biosynthesis is implicated in induction of kidney NAD(P)H oxidase activity in HHcy rats fed a folate-free diet and report the important role of redox signaling catalyzed by ceramides in glomerular injury induced by HHcy in rats (104). Acid sphingomyelinase is also involved in the development of glomerular oxidative stress and injury induced by HHcy (105, 106).

In HHcy induced by supplementation of Hcy in drinking water for 6 weeks in mice, hepatic steatosis was found to be associated with a notable increase in ceramide-related metabolites and subsequent upregulation of ceramide synthesis genes including Sptlc3, Degs2, Cer4, and Smpd4 (62). Moreover, ceramide synthases were suggested to be involved in Hcyinduced ceramide production by the fact that abolishing the expression of Sptlc3 and Degs2 by omega-3 significantly ameliorated HHcy-mediated increases of hepatic ceramide (62). In our study, the increase of Cers5 (correlating negatively with Dyrk1A expression), the non-significant increase of Sptlc3 support an increased ceramide levels in liver of Cbs+/<sup>−</sup> mice on methionine and HFD mediated through specific ceramide synthases. The SPTLC3 subunit has been identified as generating short chain sphingoid bases (66) compared to SPTLC2. Commensurate with the increased Sptlc3 level, Sptlc2 was found to be decreased in liver of Cbs+/<sup>−</sup> mice on methionine and HFD, correlating with CBS expression. Palmitate-CoA is the predominant substrate for SPTLC2, whereas mysristoyl and lauryl-CoA is the preferential substrates for SPTLC3, which results in the production of different chain length of the sphingoid base. It is possible that these two subunits can be switched in a SPT enzyme complex to replenish the ceramide pool, knocking down all SPTLC subunits being necessary to decrease total ceramides significantly (107). The hepatic decrease of SPTLC2 in Cbs+/<sup>−</sup> mice on methionine and HFD could therefore lead to a decrease of ceramides with a C18-sphingoid bases backbone and promotes the synthesis of ceramide species through the action of SPLTC3 with a C16-sphingoid base backbone. Interestingly, SPTLC3 expression has been associated with NAFLD (108) and therefore could participate to its development under the context of HHcy. We also found a strong hepatic decrease of SGPL in Cbs+/<sup>−</sup> on methionine and HFD suggesting an altered catabolism of S1P. Knowing the novel role of S1P in hepatic injury, such as NAFLD (109), it will tempting to propose that S1P metabolism also could contribute to NAFLD in Cbs+/<sup>−</sup> mice on methionine and HFD (109). More importantly, down-regulation of SGPL could also contribute to increased circulating S1P levels since it has been shown that saturated fatty acids serve to the synthesis of S1P in hepatocytes which is released in the extracellular environment (110). Increasing S1P levels in HHcy mice could have repercussion on glucose homeostasis by targeting peripheral tissues but could also target the brain where S1P is a potent regulator of AD development (111). Hypothalamic insulin resistance and lipotoxicity have been previously reported to be induce by de novo ceramide biosynthesis (101). Therefore, we also analyzed the main enzymes implicated in ceramide and sphingolipid metabolism in hypothalamus of mice.

### Effect of HHcy and HFD on Enzymes Expression Involved in Ceramide and Sphingolipid Metabolism in Hypothalamus

As expected, Cbs+/<sup>−</sup> mice showed a significant decrease of CBS mRNA expression in hypothalamus (15 ± 10 vs. 100 ± 29; p < 0.03 by Student's t-test n = 4 for each). A significant increase of hypothalamus Dyrk1A level (259 ± 22.3 vs. 101.4 ± 25.2; p < 0.003 by Student's t-test n = 4 for each) was found as expected, without difference in mRNA level (58.9 ± 21.1 vs. 100 ± 30.7; n = 4 for each) (112). Expression of Cers2 was increased, with a decrease of Sphk2 and SPTlc1 in hypothalamus of Cbs+/<sup>−</sup> mice on methionine and HFD (**Table 1**) (**Figures 2D–F**). A positive correlation was found between hypothalamic expression of CBS and SGPL1 (r = 0.76, p < 0.04), Sphk2 (r = 0.81, p < 0.03), and SPTlc1 (r = 0.94, p < 0.04). We also found a positive correlation between liver expression of CBS and hypothalamic expression of Sphk2 (r = 0.86, p < 0.02), and SPTlc1 (r = 0.79, p < 0.04).

It has been demonstrated that Hcy-treatment of cerebral endothelial cells induces acid sphingomyelinase ceramide pathway (113). The increase of Cers2, the decrease of Sphk2 with the non-significant increase of Sptlc3 and the non-significant decrease of SGPL (Sphk2 and SGPL correlating positively with CBS expression) support increased hypothalamic ceramide levels in HHcy mice on HFD mediated through the regulation of specific ceramide synthases. Previous results found in human post-mortem brain and mouse transgenic AD model an increased mRNA level of Cers1, Cers2, and a decrease in Sphk1 and Sphk2 (68, 76, 114). Loss of neuroprotective S1P and SPHK activity was found early in AD pathogenesis prior to AD diagnosis (76). Altogether, our results suggest that local alteration of S1P metabolism also could contribute to AD development associated with HHcy.

## CONCLUSION

In this study, we used HHcy mice due to CBS deficiency to analyze expression of the main enzymes implicated in ceramide and sphingolipid metabolism. Our results support an increased ceramide levels in liver of HHcy mice, particularly implicated in NAFLD, and altered hepatic catabolism of S1P, which could target the brain where S1P is a potent regulator of AD development. Our results also support an increased hypothalamic ceramide levels in HHcy mice, with a local alteration of S1P metabolism. Altogether, our study emphasizes the role of Hcy/ceramides pathway in AD pathology.

## ETHICS STATEMENT

All procedures were carried out in accordance with the ethical standards of French and European regulations (European Communities Council Directive, 86/609/EEC). Official authorization from the French Ministry of Agriculture was granted to perform research and experiments on animals (authorization number 75-369) and the experimental protocol was approved by the Institutional Animal Care and Use Committee of the Paris Diderot University (CEEA40), and the agreement # 8728 was given to the project.

### AUTHOR CONTRIBUTIONS

HL and NJ made the review of the literature and wrote the manuscript. HL, CC-G, CM, and NJ designed the study. JV, NK, JD, KM, J-LP, and CC-G performed the experiments. All authors read and approved the manuscript.

### ACKNOWLEDGMENTS

This work was supported by Lagafe Association, Foundation Jerome Lejeune (grant number 2017a-1634), grant from National Research Agency (ANR-16-CE14-0026 Fat4Brain), and the

### REFERENCES

Innovative Medicines Initiative 2 Joint Undertaking under grant agreement No. 115881 (RHAPSODY).

### SUPPLEMENTARY MATERIAL

The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fneur. 2019.00807/full#supplementary-material

other neurodegenerative disorders. Mol Neurobiol. (2019) 56:5436–55. doi: 10.1007/s12035-018-1448-3

**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Copyright © 2019 Le Stunff, Véret, Kassis, Denom, Meneyrol, Paul, Cruciani-Guglielmacci, Magnan and Janel. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

# Brain Insulin Resistance and Hippocampal Plasticity: Mechanisms and Biomarkers of Cognitive Decline

#### Matteo Spinelli<sup>1</sup> , Salvatore Fusco<sup>2</sup> \* † and Claudio Grassi1,2 \* †

1 Institute of Human Physiology, Università Cattolica del Sacro Cuore, Rome, Italy, <sup>2</sup> Fondazione Policlinico Universitario Agostino Gemelli IRCCS, Rome, Italy

In the last decade, much attention has been devoted to the effects of nutrient-related signals on brain development and cognitive functions. A turning point was the discovery that brain areas other than the hypothalamus expressed receptors for hormones related to metabolism. In particular, insulin signaling has been demonstrated to impact on molecular cascades underlying hippocampal plasticity, learning and memory. Here, we summarize the molecular evidence linking alteration of hippocampal insulin sensitivity with changes of both adult neurogenesis and synaptic plasticity. We also review the epidemiological studies and experimental models emphasizing the critical role of brain insulin resistance at the crossroad between metabolic and neurodegenerative disease. Finally, we brief novel findings suggesting how biomarkers of brain insulin resistance, involving the study of brain-derived extracellular vesicles and brain glucose metabolism, may predict the onset and/or the progression of cognitive decline.

#### Keywords: brain insulin resistance, hippocampus, Alzheimer's disease, synaptic plasticity, adult neurogenesis

### INTRODUCTION

Since the discovery of insulin almost a century ago, much efforts have been conducted to study the effects of this hormone on all organs. Insulin was originally shown to act on the brain by stimulating the hypothalamic satiety center and inhibiting the feeding behavior (Debons et al., 1970). For long time the impact of insulin on other brain areas remained unknown because the central nervous system was considered a non-insulin dependent tissue. In the last decades, the discovery of insulin receptor (IR) expression in brain areas involved in functions different from the feeding control, such as learning and memory, has revolutionized this idea and paved the way for the understanding of how the brain is a highly insulin-sensitive organ (Hill et al., 1986; Zhao and Alkon, 2001). Brain plasticity, the capability of this organ to undergo structural and functional changes in response to environmental stimuli, is finely modulated by diet and nutrient-dependent hormones including insulin (Mainardi et al., 2015). Accordingly, alteration of insulin signaling into the central nervous system may accelerate brain aging, affect brain plasticity and promote neurodegeneration (Kullmann et al., 2016).

Here, we review the effects of insulin on hippocampus, a brain area playing a pivotal role in learning and memory and primarily affected in Alzheimer's disease (AD) (Bartsch and Wulff, 2015). First, we will describe the effects of insulin on both hippocampal synaptic plasticity and hippocampal adult neurogenesis. In addition, we will illustrate the crosstalk between insulin and neurotrophin signaling, the impact of insulin on cognitive function and the role of its signaling on brain aging. Moreover, we will describe how alteration of brain insulin signaling develops (i.e., brain insulin resistance, hereinafter named BIR) and we will summarize the effects of BIR on hippocampal plasticity, learning and memory along with the link between BIR and AD.

#### Edited by:

Eugenio Barone, Sapienza University of Rome, Italy

#### Reviewed by:

Andrea Giaccari, Fondazione Policlinico Universitario A. Gemelli, Università Cattolica del Sacro Cuore, Italy Viviana Triaca, Italian National Research Council (CNR), Italy João M. N. Duarte, Lund University, Sweden

#### \*Correspondence:

Salvatore Fusco<EMAIL_ADDRESS>Claudio Grassi<EMAIL_ADDRESS>†Shared last authorship

#### Specialty section:

This article was submitted to Neurodegeneration, a section of the journal Frontiers in Neuroscience

Received: 30 April 2019 Accepted: 15 July 2019 Published: 31 July 2019

#### Citation:

Spinelli M, Fusco S and Grassi C (2019) Brain Insulin Resistance and Hippocampal Plasticity: Mechanisms and Biomarkers of Cognitive Decline. Front. Neurosci. 13:788. doi: 10.3389/fnins.2019.00788

**94**

Finally, we will discuss novel evidence suggesting that both brain-derived extracellular vesicles and brain glucose metabolism may represent novel biomarkers of BIR able to predict and follow up cognitive decline.

### INSULIN AND BRAIN PLASTICITY

Neurons are high energy-consuming cells. Most energy is spent to generate action and postsynaptic potentials (Howarth et al., 2012), and for the biosynthesis of neurotransmitters (Dienel, 2012). Glucose is the main energy source used by brain cells and its transport across the plasma membrane is mediated by a specific family of membrane proteins known as glucose transporters (GLUTs) (Shepherd and Kahn, 1999). Though numerous GLUT isoforms (1–14) have been identified and characterized, only some of these transporters are expressed in the brain and can be involved in neuronal homeostasis and brain function (Duelli and Kuschinsky, 2001). Specifically, the insulinindependent transporters GLUT1 and GLUT3 mediate glucose uptake into glial and neuronal cells, respectively (Simpson et al., 2007), suggesting that the impact of insulin on synaptic plasticity should be independent of glucose uptake.

Moreover, GLUT2 and GLUT4 expression has been characterized in specific brain areas: GLUT2 is predominantly localized in the hypothalamus that regulates food intake (Eny et al., 2008), whereas GLUT4 has been identified in cerebellum, neocortex, and hippocampus, suggesting a role of GLUT-driven glucose uptake in neuronal activity (Vannucci et al., 1998; Sankar et al., 2002). GLUT4 is also expressed in astrocytes and insulin stimulation promotes both glucose uptake and glycogen accumulation in astrocyte cultures (Heni et al., 2011). However, not much data are available in the literature on the role of insulin on astrocytic functions.  GLUT6 and GLUT13, which have very low affinity to glucose, are also expressed in the brain, but their role in central nervous system has yet to be clarified (Joost and Thorens, 2001). Conversely, GLUT8 has been shown to drive hippocampal neuron proliferation during embryogenesis (Membrez et al., 2006). However, tissue/cell type specific expression of GLUTs in the brain still remains matter of debate. Further, several growth factors have been reported to modulate the insulin pathway, GLUT plasma membrane translocation and glucose uptake by transactivation of the IR downstream effectors (Assefa et al., 2017).

In this section, we brief the insulin cascade effectors and the effects of this hormone on both synaptic plasticity and adult neurogenesis in the hippocampus along with their impact on cognitive functions.

### Insulin Signaling in the Brain

Insulin and the insulin-like growth factor 1 (IGF-1) exert their biological effects through two tyrosine kinase receptors, the IR and the IGF-1 receptors (IGF-1R), which are closely related and highly distributed throughout the brain (Belfiore et al., 2009). In the mouse, IR is predominantly expressed in the olfactory bulb, hippocampus, neocortex, hypothalamus, and cerebellum, whereas IGF-1R is highly expressed in the hippocampus, neocortex, and thalamus, with lower expression in the hypothalamus, cerebellum, olfactory bulb, midbrain, and brainstem (Bruning et al., 2000; Fernandez and Torres-Aleman, 2012). As reported in other tissues, IRs and IGF-1Rs can heterodimerize in the brain and partially transactivate their signaling (Bailyes et al., 1997). Moreover, IR and IGF-1R share intracellular signaling machinery, and all major components of brain signaling cascades are similar to those present in peripheral tissues, including IR substrate 1 and 2 (IRS1 and IRS2, respectively), the major downstream phosphoinositide-3-kinase–protein kinase B/Akt (PI3K/Akt) pathway, the downstream effectors target of rapamycin (mTOR) and glycogen synthase kinase 3 beta (GSK3β), and the transcription factors cAMP response element-binding protein (CREB) and forkhead box O (FOXO) family (Fernandez and Torres-Aleman, 2012; **Figure 1**).

Many molecules involved in these signaling cascades have been demonstrated to have key roles in brain functions. Activation of PI3-kinase is required for glutamate receptor insertion at plasma membranes during synaptic plasticity (Man et al., 2003). GSK3β regulates neural progenitor cell proliferation and neuroplasticity, and its activation induces hyper-phosphorylation of tau protein that is considered a major determinant of AD pathogenesis (Salcedo-Tello et al., 2011). Insulin induces phosphorylation of GSK3β on inhibitory serine 9 residue, thus reducing its enzymatic activity. Moreover, mTOR complex 1 (mTORC1) is fundamental for both protein synthesis and autophagy, which are molecular processes involved in the regulation of long-term synaptic plasticity and degradation of misfolded proteins in neurons, respectively (Stoica et al., 2011; Son et al., 2012). Insulin/IGF-1 signaling also stimulates the growth factor receptor-bound protein 2- Son of Sevenless-Rat sarcoma-mitogen activated protein kinase (Grb2-SOS-Ras-MAPK) cascade, which plays a pivotal role in cytoskeletal modifications underlying the dendritic spine reorganization and memory formation (Adams and Sweatt, 2002).

The origin of insulin in the central nervous system is controversial. Insulin crosses the blood-brain barrier (BBB) via an IR-dependent transport operated by vascular endothelium and its concentration increases after meals (Woods et al., 2003). However, insulin can be also synthesized and secreted by neurons and adult neural progenitor cells of the hippocampus (Devaskar et al., 1994; Kuwabara et al., 2011), although no evidence clearly demonstrated that insulin synthesis in the brain is physiologically relevant.

However, as described in the next paragraphs, physiological levels of insulin play a neurotrophic action on both differentiated neurons and neural stem cells (NSCs).

### Insulin, Synaptogenesis and Hippocampal Synaptic Plasticity

Modifications of both activity and number of synapses are the functional and structural substrates, respectively, of brain plasticity underlying learning and memory

(Nakahata and Yasuda, 2018). Changes of the synaptic strength, either potentiation or depression, and generation of new dendritic spines are causally related to the acquisition and consolidation of behavioral modifications.

Insulin stimulation of hippocampal neurons induces both presynaptic and postsynaptic effects. Insulin increases basal neurotransmitter release from presynaptic terminals, as revealed by enhanced frequency of miniature excitatory postsynaptic currents (mEPSCs) (Lee et al., 2011). This effect is paralleled by a Rac1-mediated cytoskeleton rearrangement leading to increased density of dendritic spines (Lee et al., 2011). Moreover, insulin promotes synaptic plasticity by modulating long-term potentiation (LTP) or long-term depression (LTD) at hippocampal synapses through a metaplastic mechanism. Indeed, insulin administration reduces the stimulation frequency threshold required for inducing both LTP and LTD (Van Der Heide et al., 2005). Postsynaptic effects are mediated by PI3K activation (Van Der Heide et al., 2005) and increased membrane recruitment of N-methyl-D-aspartate receptors (NMDARs) (Skeberdis et al., 2001). Insulin impacts on glutamate receptor activity by multiple mechanisms. It increases NMDAR-mediated currents by enhancing phosphorylation of both NR2A and NR2B subunits (Christie et al., 1999; Liu et al., 1995). Insulin treatment of hippocampal cultures also increases phosphorylation and clathrin-dependent endocytosis of the GluA1 subunit of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors (AMPARs) (Adzovic and Domenici, 2014; Chen et al., 2014). Downregulation of AMPAR activity in excitatory synapse of hippocampal CA1 neurons is fundamental for insulin-induced LTD, which is a key step for memory consolidation and flexibility (Ge et al., 2010). IRs have also been demonstrated to modulate type A γ-aminobutyric acid (GABA) receptor activity by regulating both its membrane localization and expression in inhibitory synapses (Wan et al., 1997).

Furthermore, insulin may impinge on structural features of synapses. For instance, in thalamocortical organotypic slices this hormone stimulates maturation of silent synapses (Plitzko et al., 2001). Moreover, IR substrate p53 (IRSp53) interacts with the postsynaptic protein PSD-95 and enhances dendritic spine formation (Choi et al., 2005). Interestingly, IRS2 knockout mice show lower activation of NR2B subunits (Martin et al., 2012) and decreased LTP at the CA3–CA1 synapses in parallel with higher density of CA1 dendritic spines (Irvine et al., 2011). It is important to underline that these studies did not evaluate dendritic spine morphology, therefore our knowledge of the effects of IRSs manipulation on structural and functional plasticity still remains incomplete. Considering the physical and functional interaction between IR and IGF-1R, it is not surprising that IGF-1 stimulation can promote plasticity in the hippocampus by increasing spine density of CA1 basal dendrites in response to physical exercise (Glasper et al., 2010). Accordingly, IGF-I knockout mice show reduced density of glutamatergic synapses (Trejo et al., 2007). Importantly, IR expression and insulin activity in the brain are not restricted to neurons. Insulin has been demonstrated to influence proliferation and metabolism in insulin sensitive glial cells (Heni et al., 2011). Collectively, all the above mentioned evidence supports the positive effects of insulin on hippocampal synaptic and structural plasticity.

### Insulin and Hippocampal Adult Neurogenesis

Hippocampus is one of the brain areas where newborn neurons are generated throughout adulthood (Braun and Jessberger, 2014). Specifically, adult neurogenesis occurs in the subgranular zone of the hippocampus of all mammals including humans (Eriksson et al., 1998). NSCs populating this neurogenic niche proliferate and differentiate to generate new neurons (Kempermann et al., 2003). A proper balance between NSC proliferation and their differentiation/maturation underlies the maintenance of both the hippocampal stem cell niche and the supply of newborn neurons that integrate into existing circuits thus supporting cognitive functions under physiological conditions and brain repair after injury (Castilla-Ortega et al., 2011). Indeed, a growing number of studies indicates that hippocampal neurogenesis plays a critical role in learning, memory, and its impairment has been associated with cognitive dysfunction in neurodegenerative disorders including AD (van Praag et al., 2002; Taylor et al., 2013).

Insulin is a key trophic factor for brain development and control of neurogenic niches. Indeed, neuroblast exit from quiescence is regulated by insulin/IGF-I pathway activation (Chell and Brand, 2010; Sousa-Nunes et al., 2011). Evidence from in vitro and in vivo experiments indicate that insulin and IGF-I promote neurogenesis by modulating NSC proliferation, differentiation, and survival (Brooker et al., 2000; Åberg et al., 2003). However, a chronic hyper-activation of insulin/IGF-I signaling cascades can cause premature depletion of the NSC reservoir (Sun, 2006). Thus, insulin may produce either trophic or detrimental effects on neural stem niche based on the timing and the duration of stimulation.

Furthermore, calorie restriction has been clearly demonstrated to reduce plasma levels of both glucose and insulin, in parallel with increasing neurogenesis in the dentate gyrus (Lee et al., 2002) and counteracting the age-related decline of stem cell niche (Park et al., 2013). Nutrient deprivation may impact on NSC compartment by inducing the expression of the brainderived neurotrophic factor (Bdnf) gene (Maswood et al., 2004). In addition, calorie restriction may preserve the NSC capacity to self-renew and differentiate by cell-autonomous mechanisms involving metabolic sensors such as CREB and the NAD-dependent histone deacetylase Sirtuin 1 (SIRT1). In this regard, CREB is a nutrient-dependent transcription factor regulating genes promoting neuronal differentiation and survival (Lonze et al., 2002; Fusco et al., 2012b). Moreover, SIRT1 is as an epigenetic repressor that modulates adult neurogenesis in the subventricular zone and hippocampus (Saharan et al., 2013). Calorie restriction also induces the expression of SIRT1, which has been shown to functionally impinge on CREB-dependent gene expression, thus highlighting a novel molecular link between nutrient-dependent signaling and brain health (Fusco et al., 2012a). Under metabolic and oxidative stress, SIRT1 inhibits NSC self-renewal and induces their differentiation (Prozorovski et al., 2008; Ma et al., 2014). In summary, SIRT1 and CREB work as metabolic sensors regulating proliferation and self-renewal of NSCs and controlling their reservoir in the hippocampus (Fusco et al., 2016). Conversely, abolishing the expression of genes encoding the insulin-regulated FOXO transcription factors induces hyperproliferation of neural progenitors and rapid exhaustion of stem cell niche (Renault et al., 2009). Similarly, aberrant stimulation of the nutrient-dependent mTOR pathway causes reduced self-renewal and accelerates NSC differentiation (Magri et al., 2011). Together, this evidence confirms that nutrient related signals control NSC fate under both physiological and pathological conditions.

In addition, it is worth mentioning the close similarity between the intracellular signaling pathways activated by insulin and neurotrophins (Reichardt, 2006). In particular, CREB has been shown to play a critical role in the neurotrophintriggered effects on neuronal differentiation, survival, and plasticity, and it has been also characterized as metabolic sensor modulated by fasting-related stimuli (Finkbeiner, 2000; Altarejos and Montminy, 2011). Moreover, neurotrophic factors as BDNF, ciliary neurotrophic factor (CNTF), and glial cellderived neurotrophic factor (GDNF) regulate adult neurogenesis in multiple stages of NSC maturation and their expression is affected by overnutrition and metabolic stress (Lindsay et al., 1994). These evidence emphasizes the role of insulin as growth factor for neural niche especially during the early stages of life.

### Insulin Signaling and Hippocampus-Dependent Cognitive Task

The evidence summarized above suggest that all aspects of hippocampal plasticity (i.e., functional and structural synaptic

plasticity, and adult neurogenesis) are strongly sensitive to the modulation of insulin signaling into the brain. According to the key role of hippocampal plasticity in learning and memory, changes of insulin cascade in the hippocampus markedly affects cognitive functions.

Heterozygous knockout mice for IR display lower preference index in the novel object recognition (NOR) test (Nisticò et al., 2012). Accordingly, Zucker rats show lower performance in the Morris Water Maze (MWM) in parallel with impaired insulin sensitivity (Kamal et al., 2013). Moreover, a recent study performed on Goto-Kakizaki (GK) rats, a model of non-obese type 2 diabetes (T2D), display spatial memory impairment in Y-maze task and hippocampal synaptic dysfunction evaluated by LTP (Duarte et al., 2019). Further, GK rats show a reduction of SNAP25 and synaptophysin levels suggesting synapse degeneration (Duarte et al., 2019). In addition, IRSp53 knockout mice show impaired learning and memory when evaluated in both MWM and NOR tests (Kim et al., 2009). However, forebrain-specific IRS2 deficiency improved memory retention in the MWM task (Irvine et al., 2011), suggesting that molecular hubs of insulin signaling may differentially interfere on cognitive behavior.

In agreement with this finding, chronic brain stimulation by 8-week intranasal insulin administration improved memory in humans (Benedict et al., 2004). Moreover, lower values of both glycaemia and glycosylated hemoglobin (HbA1c) are associated with better performance in memory tasks in humans (Kerti et al., 2013). It has been shown that peripheral changes of insulin signaling and sensitivity may affect brain health and function although the results of these studies are controversial. Learning and memory deficits in MWM have been demonstrated in liver-specific IGF1 knockout mice (Trejo et al., 2007). In addition, IGF-I antiserum administration to young rats impairs learning in passive avoidance task (Lupien et al., 2003). Conversely, intraperitoneal injection of insulin impairs retention and spatial working memory in a dose-dependent manner (Kopf and Baratti, 1999; Akanmu et al., 2009). It seems that the negative impact of peripheral insulin administration on cognitive functions may be due to the lowering of blood glucose levels, as indicated by the positive effect of the simultaneous glucose infusion (Kopf et al., 1998). In line with this hypothesis, peripheral administration of insulin increases verbal memory and attention in healthy subjects under euglycemic conditions (Kern et al., 2001).

To avoid the side effects of systemic insulin administration, Park et al. (2000) studied the impact of intracerebroventricular injection of insulin in rats and found that insulin improved cognitive performance in passive avoidance test. Accordingly, intrahippocampal injection of insulin into the CA1 region has been reported to enhance memory of rats in passive avoidance test (Babri et al., 2007). More importantly, the effects of intrahippocampal injection of insulin on cognitive functions seem to be related to its dose. High doses of insulin significantly ameliorate spatial learning and memory in the MWM test, whereas low doses reduce cognitive performance (Moosavi et al., 2006). It has been hypothesized that the negative effects of insulin on spatial memory may be dependent on either upregulation of GABA<sup>A</sup> receptors or downregulation of AMPA receptors upon insulin treatment (Moosavi et al., 2006). Finally, McNay et al. (2010) demonstrated that endogenous intrahippocampal insulin signaling was required for memory processing. These authors showed that acute injection of insulin into the hippocampus at physiological doses enhanced spatial memory via a PI3K-dependent mechanism (McNay et al., 2010). Collectively, the results obtained in humans and rodents suggest that insulin is fundamental for both memory formation and retention.

### EFFECTS OF BRAIN INSULIN RESISTANCE ON HIPPOCAMPUS-DEPENDENT FUNCTIONS

Data reviewed in the previous paragraphs support the view that changes of either insulin signaling or insulin sensitivity in the hippocampus may alter molecular pathways involved in synaptic plasticity and adult neurogenesis, thereby leading to reduced "mindspan" (the maintenance of mental abilities throughout life) and increased risk of neurodegeneration (Kodl and Seaquist, 2008). Accordingly, while calorie restriction furthers neuronal survival and improves cognitive function (Fusco and Pani, 2013), the excess of nutrients harms the brain health and accelerates cognitive decline (Elias et al., 2012; Sellbom and Gunstad, 2012). Nutrient excess causes hyperactivation of insulin signaling in all tissues expressing IR, leading to the desensitization of IR-dependent molecular cascades. BIR decreases the ability of brain cells to respond to insulin and abolishes both metabolic and cognitive effects of this hormone (Kullmann et al., 2016). Specifically, this deficiency could be caused by lower expression of IR or poor activation of insulin signaling. IR downstream effectors may become insensitive to the insulin stimulation, resulting in inability of brain cells to respond to the hormone and leading to impairment of brain plasticity. In the Western world, the incidence of metabolic disorders, including insulin resistance, obesity and T2D, is increasing at alarming rates in parallel with the prevalence of cognitive decline (Cukierman-Yaffee, 2009). Obesity and inflammation affect the insulin transport to the brain (Ketterer et al., 2011) and low expression of IR has been reported in patients with T2D (Kullmann et al., 2016). However, patients with T2DM and/or obesity showed decreased insulin levels in the cerebrospinal fluid despite higher levels of this hormone in their plasma (Heni et al., 2014). In the following paragraphs, we will summarize the mechanisms underlying the detrimental effects of BIR on hippocampal plasticity and cognition, and the epidemiological and experimental evidence supporting a link between BIR and AD.

### Alterations of Hippocampal Plasticity in BIR Models

High-fat diet (HFD) is a well-established animal model of metabolic disorders (Wong et al., 2016). HFD induces obesity by compromising β-cell functions, promoting hyper-glycaemia,

whole-body insulin resistance, and dyslipidemia, and increasing free fatty acids in the blood. Many studies have investigated the structural and functional changes of neuroplasticity in experimental models of insulin resistance (Fadel and Reagan, 2016).

More specifically, HFD produces detrimental effects on brain functions including decreased neurogenesis in the dentate gyrus (Lindqvist et al., 2006), alteration of BBB integrity (Freeman and Granholm, 2012) and changes in both spine density and synapse formation (Stranahan et al., 2008a). HFD also impairs insulin signaling in the hippocampus and reduces the expression of synaptic proteins PSD-95 and synaptopodin (Arnold et al., 2014). However, the most significant effects occur on activity-dependent synaptic plasticity. Indeed, Zucker rats show impairment in LTP at CA3–CA1 synapses in parallel with loss of insulin sensitivity (Kamal et al., 2013). Moreover, IR heterozygous knockout mice display normal levels of both basal synaptic transmission and LTP that, however, fails to be consolidated due to reduced Akt activation (Nisticò et al., 2012).

Obesity and T2D have been demonstrated to induce hippocampal insulin resistance through different metabolic changes including alteration of hypothalamic-pituitary-adrenal (HPA) axis leading to elevated levels of glucocorticoids (Plotsky et al., 1992). Accordingly, glucocorticoids stimulation inhibits translocation of GLUT4 to the plasma membrane in the rat hippocampus (Piroli et al., 2007). Moreover, Stranahan et al. showed that restoring physiological levels of glucocorticoids in insulin resistant db/db mice rescued the impairment of hippocampal synaptic plasticity (Stranahan et al., 2008b). A different model of BIR is obtained by intracerebral injection of streptozotocin, which impairs cognitive function by reducing the activity of the neuroprotective protein SIRT1 (Du et al., 2014). As mentioned before, SIRT1 cooperates with the transcription factor CREB promoting the CREB-dependent expression of the neuroplasticity-related gene Bdnf (Jeong et al., 2012). To better clarify the functional role of hippocampal insulin resistance, Grillo et al. (2015) silenced the expression of IR in the hippocampus by injecting lentiviral particles harboring IR antisense sequence. This experimental model showed deficits in hippocampal synaptic transmission and spatial learning, in parallel with downregulation of NMDA subunit GluN2B expression and lower phosphorylation of AMPA subunit GluA1, without altering peripheral metabolic parameters (i.e., body weight, adiposity, and glucose homeostasis) (Grillo et al., 2015).

Recently, we described a novel link between BIR and altered glutamate receptor function underlying the HFDdependent impairment of hippocampal synaptic plasticity (Spinelli et al., 2017). In particular, we found that HFD induced accumulation of palmitic acid and increased FOXO3a-dependent expression of palmitoyl-transferase zDHHC3 leading to GluA1 hyper-palmitoylation in the hippocampus. Accordingly, in vitro stimulation of hippocampal neurons with a cocktail of insulin and palmitic acid replicated the in vivo molecular changes, inhibiting the GluA1 localization at the synaptic membrane and AMPA currents at glutamatergic synapses. Finally, either silencing of zDHHC3 or overexpression of the palmitoylation-deficient GluA1 mutant in the hippocampus abolished the insulin resistance-dependent impairment of synaptic plasticity (Spinelli et al., 2017). Of course, aberrant palmitoylation of other zDHHC3 targets (e.g., GABA<sup>A</sup> RG2) may contribute to the detrimental effects of HFD on hippocampusdependent learning and memory. However, our study adds a new layer to the hippocampal synaptic plasticity regulation by insulin signaling deterioration and proposes a novel molecular mechanism potentially linking BIR and cognitive decline. Other mechanisms underlying fatty acid-driven learning deficits involve cholesterol dysmetabolism, oxidative stress, endothelial dysfunctions, and neurotrophin depletion. Mice fed with HFD show higher levels of reactive oxygen species (ROS), superoxide, and peroxynitrite into the brain, leading to lower level of brain-derived neurotrophic factor (BDNF) and impaired cognition performance evaluated by spatial task (Wu et al., 2004). Moreover, epidemiological studies showed that diets enriched in cholesterol (HCD) were associated with poor cognitive performance in humans (Requejo et al., 2003). HCD diet also induced impairment of spatial and working memory due to microglial activation and alteration of the BBB integrity in rats (Chen et al., 2018). Interestingly, feeding obese rodents with HFD inhibited the transport through the BBB of neuroendocrine molecules, such as ghrelin and leptin, which promote synaptic plasticity and cognitive functions (Banks et al., 2008; Kanoski et al., 2013; Mainardi et al., 2017). Finally, HFD has been shown to induce activation of microglia and astrocytes, and increase of pro-inflammatory cytokines/mediators such as cyclooxygenase 2, TNF-α, IL-1-β, and IL-6 in the hippocampus of mice (Thirumangalakudi et al., 2008; Duffy et al., 2019).

In summary, metabolic diseases affecting insulin signaling may impair the synaptic function through a plethora of molecular mechanisms targeting neurons, astrocytes, endothelial or inflammatory cells.

### Cognitive Impairment in BIR Models

Epidemiological evidence indicate that metabolic alterations occurring in T2D, such as hyper-glycaemia and hyperinsulinaemia, positively correlate with cognitive impairment and diabetic patients exhibit higher susceptibility to develop dementia (Cukierman-Yaffee, 2009). Dysregulation of glucose homeostasis increases the risk of dementia in both diabetic and non-diabetic patients (Crane et al., 2013) and is associated with reduced hippocampal volume and cognitive decline (Kerti et al., 2013). Furthermore, longitudinal studies demonstrated that also Type 1 diabetes (T1D) patients were affected by mild-severe cognitive impairment related to the age of onset of the disease and the microvascular complications (Moheet et al., 2015; Nunley et al., 2015). Insulin administration is crucial to promote glucose homeostasis in these patients and to reduce the vascular complications but it increases the risk for hypoglycemic episodes, which negatively impact on cognitive functions (Desrocher and Rovet, 2004). However, the role of hypo- or hyper-insulinemia in T1D-related cognitive alterations has still to be clarified.

Numerous clinical studies revealed worse cognitive performance and earlier age incidence of all-cause dementia in subjects with T2D (Davis et al., 2017; Callisaya et al., 2019). Accordingly, meta-analysis studies showed that in diabetic

patients the risk for all types of dementia is increased by 60–73% (Gudala et al., 2013; Chatterjee et al., 2016).

However, alteration of brain insulin signaling may negatively impact on brain function also in the absence of T2D and before the onset of obesity. Several studies have demonstrated deficits in hippocampal-dependent learning and spatial memory associated with Western diet intake (Molteni et al., 2002; Kanoski and Davidson, 2010). Interestingly, when Kanoski and Davidson (2010) investigated both hippocampus-dependent and hippocampus-independent memory retention ability after different Western diet treatments, they found that only spatial memory impairments occurred after short-term consumption. This suggests that hippocampus is a brain area very sensitive to metabolic stress, and memory impairment may arise before the development of diet-induced metabolic alterations in peripheral tissues. Accordingly, few days of HFD regimen were sufficient to cause cognitive impairment in rats evaluated with MWM test (Murray et al., 2009). High caloric intake also affected hippocampus-dependent non-spatial learning and memory tasks and these results were related to changes of the BBB integrity. Specifically, high energy diet consumption reduced the expression of tight junction proteins selectively causing increased blood-to-brain permeability in the hippocampus (Kanoski and Davidson, 2010). The observed learning and memory deficits are strikingly similar to the poor task performance and cognitive impairment observed in patients with mild or severe metabolic derangements, which strengthens the hypothesis that hippocampal insulin resistance is a key mediator of dietdependent cognitive alterations. Therefore, cognitive dysfunction related to HFD or obesity in otherwise healthy individuals may be due to decreased insulin signaling and development of BIR in the hippocampus (McNay et al., 2010).

Nevertheless, peripheral insulin resistance and diet-induced obesity are correlated with some other changes that may cause neurocognitive dysfunction. Indeed, they induce systemic and central inflammation with high levels of circulating proinflammatory interleukins that have been linked to impaired executive function (Trollor et al., 2012). Obesity also alters HPA axis causing enhanced secretion of glucocorticoids, which have been associated with reduced hippocampal volume, memory impairment and mood alterations (MacQueen and Frodl, 2011). Moreover, mice specifically lacking the IR into the brain (NIRKO mice) display changes in dopamine turnover associated with anxiety and depressive-like behaviors (Kleinridders et al., 2015). In addition, diet-induced microbiota dysbiosis can impact on the gut-brain axis, thus promoting insulin resistance and cognitive impairment (Daulatzai, 2014). Finally, HFD exposure during early stages of life is associated with impaired learning and spatial memory (Boitard et al., 2012), suggesting that alteration of insulin signaling may negatively influence cognitive function at each stage of life.

### Brain Insulin Resistance, Brain Aging and Neurodegenerative Diseases

While diabetes is known to increase the risk for dementia, the underlying mechanisms linking insulin resistance, T2D and AD are poorly understood. Undoubtedly, micro- and macro-vascular complications of T2D may increase the risk of cerebrovascular disease, cognitive impairment and vascular dementia (Gorelick et al., 2011). Moreover, white matter disease, alteration of the BBB and neuro-inflammation may play a pathophysiologic role (Hsu and Kanoski, 2014). However, hyper-insulinaemia promotes the formation of advanced glucose end products and ROS causing neurotoxicity and brain damage (Brownlee, 2001). Despite insulin exerts a neurotrophic role at moderate concentrations, higher levels of the hormone may be associated with increased deposition of amyloid-β (Aβ) in the brain due to competition for their common and main clearance mechanism, the insulin-degrading enzyme (Farris et al., 2003). In this regard, AD has been defined a form of type 3 diabetes, based on the evidence of BIR development in the AD brain (Steen et al., 2005; Bedse et al., 2015; Sposato et al., 2019). The insulin synthesis decreases during aging and AD progression in brain areas such as frontal cortex, hippocampus, and hypothalamus (Frolich et al., 1998). In addition, Aβ inhibits insulin expression in astrocytes (Pitt et al., 2017). Together, these studies indicate a crosstalk between brain insulin signaling alteration and Aβ accumulation in neurodegenerative diseases. Accordingly, experimental data obtained from neuroimaging and biomarker studies revealed that T2D patients showed alterations of both brain glucose metabolism and cerebrospinal fluid including phosphorylated tau, which are reminiscent of changes observed in AD (Baker et al., 2011; Moran et al., 2015). In addition, analysis of AD postmortem brains revealed insulin signaling alterations in hippocampal tissues resembling the biochemical features of insulin resistance in parallel with histopathological hallmarks of neurodegeneration (Talbot et al., 2012; Tramutola et al., 2015). Moreover, tau is hyper-phosphorylated in the brain of NIRKO mice (Schubert et al., 2004) and BIR has been associated with tau pathology in AD human brains (Yarchoan and Arnold, 2014).

Interestingly, T2D and AD also share several metabolic derangements promoting brain aging. AD patients show hyperinsulinaemia and decreased peripheral insulin sensitivity (Craft et al., 1996), whereas insulin levels in cerebrospinal fluid are reduced (Craft et al., 1998). Accordingly, sustained peripheral hyper-insulinaemia can reduce the transport of insulin into the brain due to the lower expression of IR at the BBB (Schwartz et al., 1990). Brain insulin uptake is also impaired in both aging and AD independently by T2D (Frolich et al., 1998). Recent evidence suggests that insulin may influence Aβ deposition and AD-dependent impairment of both synaptic plasticity and memory formation (Cholerton et al., 2013). Intranasal insulin administration has been demonstrated to improve cognitive function in humans (Hallschmid et al., 2007; Reger et al., 2008). However, recent data about a clinical trial with mild cognitive impairment (MCI) or moderate AD patients revealed no significant effects of long-term intranasal insulin delivery on cognitive performance in memory task (Craft et al., 2017).

Finally, genetic and experimental data about insulin degrading enzyme and, more recently, the Aβ metabolism regulation by sortilin related VPS10 domain containing receptor 1 (SorCS1) gene suggest novel mechanistic links between BIR

and AD (Lane et al., 2010; Wang et al., 2015). Thus, BIR seems to play a pivotal role at the crossroad between metabolic and neurodegenerative diseases, independently from the cerebrovascular mechanisms.

### BIOMARKERS OF BRAIN INSULIN RESISTANCE

In view of the close relationship among metabolic diseases, BIR and cognitive decline, it is emerging the need to identify biomarkers able to detect BIR before, or possibly even in the absence of, peripheral insulin resistance, that may be predictive of age- and dementia-related cognitive impairment. Ideal biomarkers should be reliable, simple to measure, noninvasive and inexpensive (Noel-Storr et al., 2013). In this regard, the dosage of both Aβ and tau proteins in the cerebrospinal fluid is invasive and most likely indicative of a pathology already under development. For these reasons, in the last years several studies focused on evaluation of brain glucose metabolism and analysis of brain-derived extracellular vesicles extracted from the blood as biomarkers of BIR and early-phase cognitive decline.

Cerebral glucose metabolism is tightly correlated with neuronal activity (Simpson et al., 2007). Therefore, imaging of local brain hypo-metabolism can be used to visualize areas of reduced synaptic activity. The most frequently used method of brain metabolic imaging is positron emission tomography (PET) with (18F)fluorodeoxyglucose (FDG) (Cohen and Klunk, 2014). Reduced cerebral glucose metabolism represents one of the earliest signs of AD, and studies in both humans and experimental models suggest that altered brain glucose metabolism is associated with AD progression (Kapogiannis and Mattson, 2011; Ishibashi et al., 2015).

Recent work have identified in GK rats reduced glutamine synthesis and impairment of the glutamate-glutamine cycle between astrocytes and neurons, driving to diabetes-induced neurodegeneration and cognitive dysfunction (Girault et al., 2017). In a mouse model of AD, impaired glucose transport through the BBB and decreased cerebral lactate release during neuronal activity occur at early stages of the phenotype (Merlini et al., 2011). Dysregulated brain glucose metabolism resembling changes observed in AD patients has been observed in metabolic disorders such as obesity or T2D (Tschritter et al., 2006, 2007). However, whether neuroimaging changes of brain glucose metabolism anticipate the onset of neurodegeneration or are related to the development of BIR in the same brain areas remain still poorly understood.

More recently, molecular strategies have been developed to selectively isolate brain-derived exosomes (BDE) from biological fluids (Tschritter et al., 2006, 2007; Fiandaca et al., 2015; Goetzl et al., 2016). Exosomes are extracellular vesicles carrying information (e.g., proteins, lipids, and nucleic acids) to distant cells, which are emerging as novel potential biomarkers for human diseases (Tkach and Thery, 2016). Several pathogenic proteins that are involved in neurodegenerative diseases, including AD, are loaded into vesicles and then extracellularly secreted via exosomes (Rajendran et al., 2006; Sharples et al., 2008). More importantly, changes of insulin resistance molecular markers (i.e., higher serine phosphorylation and lower tyrosine phosphorylation of IRS-1) have been found in neural-derived exosomes extracted from blood of AD patients compared to age- and gender-matched patients with frontotemporal dementia or T2D (Kapogiannis et al., 2015). These differences were detectable up to 10 years before the onset of AD symptoms. Finally, exosomal biomarkers of BIR were associated with higher brain atrophy in AD patients (Mullins et al., 2017) emphasizing the potential role of brain derived microvesicles as detectors of brain insulin signaling and biomarkers of brain damage due to metabolic and neurodegenerative disorders.

### CONCLUSION

Molecules involved in metabolic homeostasis are now recognized to exert a great influence on hippocampal plasticity, and alteration of their equilibrium has a strong impact at the functional and behavioral levels. Insulin exerts a trophic role into the brain and it may also act as a signal of positive metabolic homeostasis promoting neuroplasticity, which is a high energy demanding process. Insulin plays a pivotal role in the regulation of central nervous system homeostasis and higher functions such as learning and memory, by controlling both NSC fate and the activity of neuronal network. In this regard, identifying the molecular targets that underlie the effects of insulin on brain plasticity may contribute to understand the mechanisms regulating neural plasticity in health and metabolic diseases and reveal novel targets in pathologies characterized by impaired neural plasticity, especially AD.

Actually, we do not yet have an exhaustive understanding of how systemic and brain insulin resistance are related to brain aging and AD, but clinical and experimental evidence indicates that insulin supplementation can be a therapeutic tool for patients with cognitive impairment and an added value in the treatment of dementia (Chapman et al., 2018; Santiago and Hallschmid, 2019). The availability of BDE, such as other biomarkers of brain metabolism detectable in the plasma, will foster clinical studies to identify novel therapeutic approaches for personalized medicine in neurodegenerative diseases.

### AUTHOR CONTRIBUTIONS

All authors conceived the work, took part to the scientific discussion, and wrote the manuscript.

### FUNDING

This research was supported by the Italian Ministry of University and Research (SIR 2014 RBSI14ZV59 to SF) and Universitá Cattolica del Sacro Cuore intramural grants (Linea D.3.2 2015 and Linea D.3.2 2017 to CG).

### REFERENCES

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in rats with short-term high-fat feeding. FASEB J. 23, 4353–4360. doi: 10.1096/ fj.09-139691

amyloid precursor protein processing correlate with loss of working memory in mice. J. Neurochem. 106, 475–485. doi: 10.1111/j.1471-4159.2008.05415.x

**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

The reviewer VT, declared a past co-authorship with one of the authors CG, and the reviewer AG, declared a shared affiliation, with no collaboration, with several of the authors, SF and CG, to the handling Editor at the time of review.

Copyright © 2019 Spinelli, Fusco and Grassi. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

# Insulin-Mediated Changes in Tau Hyperphosphorylation and Autophagy in a Drosophila Model of Tauopathy and Neuroblastoma Cells

Shreyasi Chatterjee1,2 \*, Suren. S. Ambegaokar1,3, George R. Jackson1,4 \* and Amritpal Mudher<sup>2</sup>

<sup>1</sup> Department of Neurology, Mitchell Center for Neurodegenerative Diseases, The University of Texas Medical Branch at Galveston, Galveston, TX, United States, <sup>2</sup> Department of Biological Sciences, University of Southampton, Southampton, United Kingdom, <sup>3</sup> Department of Botany and Microbiology, Ohio Wesleyan University, Delaware, OH, United States, <sup>4</sup> Department of Neurology, Michael E. DeBakey VA Medical Center, Parkinson's Disease Research Education and Clinical Center, Baylor College of Medicine, Houston, TX, United States

### Edited by:

Eugenio Barone, Sapienza University of Rome, Italy

#### Reviewed by:

Ines Moreno-Gonzalez, The University of Texas Health Science Center at Houston, United States Antonella Tramutola, Sapienza University of Rome, Italy

#### \*Correspondence:

Shreyasi Chatterjee<EMAIL_ADDRESS>George R. Jackson<EMAIL_ADDRESS>
#### Specialty section:

This article was submitted to Neurodegeneration, a section of the journal Frontiers in Neuroscience

Received: 15 April 2019 Accepted: 17 July 2019 Published: 02 August 2019

#### Citation:

Chatterjee S, Ambegaokar SS, Jackson GR and Mudher A (2019) Insulin-Mediated Changes in Tau Hyperphosphorylation and Autophagy in a Drosophila Model of Tauopathy and Neuroblastoma Cells. Front. Neurosci. 13:801. doi: 10.3389/fnins.2019.00801 Almost 50 million people in the world are affected by dementia; the most prevalent form of which is Alzheimer's disease (AD). Although aging is considered to be the main risk factor for AD, growing evidence from epidemiological studies suggests that type 2 diabetes mellitus (T2DM) increases the risk of dementia including AD. Defective brain insulin signaling has been suggested as an early event in AD and other tauopathies but the mechanisms that link these diseases are largely unknown. Tau hyperphosphorylation is a hallmark of neurofibrillary pathology and insulin resistance increases the number of neuritic plaques particularly in AD. Utilizing a combination of our Drosophila models of tauopathy (expressing the 2N4R-Tau) and neuroblastoma cells, we have attempted to decipher the pathways downstream of the insulin signaling cascade that lead to tau hyperphosphorylation, aggregation and autophagic defects. Using cell-based, genetic, and biochemical approaches we have demonstrated that tau phosphorylation at AT8 and PHF1 residues is enhanced in an insulin-resistant environment. We also show that insulin-induced changes in total and phospho-tau are mediated by the crosstalk of AKT, glycogen synthase kinase-3β, and extracellular regulating kinase located downstream of the insulin receptor pathway. Finally, we demonstrate a significant change in the levels of the key proteins in the mammalian target of rapamycin/autophagy pathway, implying an increased impairment of aggregated protein clearance in our transgenic Drosophila models and cultured neuroblastoma cells.

Keywords: Alzheimer's disease, type 2 diabetes, tau aggregation, autophagy, tau hyper-phosphorylation

**Abbreviations:** 4EIBP, eukaryotic initiation factor 4B; AD, Alzheimer's disease; ERK, extracellular regulating kinase; GMR, glass multimer reporter; GOF, gain of function; GSK, glycogen synthase kinase; HD, Huntington's disease; IRS, insulin receptor substrate; LOF, loss of function; mTOR, mammalian target of rapamycin; NFT, neurofibrillary tangle; p70S6K, p70S6kinase; PD, Parkinson's disease; T2DM, type 2 diabetes mellitus; UAS, GAL4-responsive upstream activating sequence; wt, wild type.

## INTRODUCTION

fnins-13-00801 August 1, 2019 Time: 18:37 # 2

Alzheimer's disease is a widely prevalent form of dementia that is characterized by loss of memory and other cognitive functions required to perform complex daily activities (Citron, 2004). Aging remains the most important risk factor for AD, but population-based studies have identified T2DM, an ageassociated chronic metabolic disorder, as a major risk factor for developing cognitive impairment and dementia (Barbagallo and Dominguez, 2014; Sridhar et al., 2015). Insulin resistance in peripheral tissues is a key feature of T2DM, and accumulating evidence suggests that insulin resistance also develops in AD and other tauopathies in early stages of the disease (Goncalves et al., 2019). Recent studies have also shown that brain insulin resistance can develop independently from systemic insulin resistance. However, the underlying mechanisms that lead to persistent brain insulin resistance and needs to be investigated (Triani et al., 2018). Several studies have demonstrated that both insulin and insulin receptors (IRs) are found in the brain, and IR are highly expressed in neurons of the central nervous system (CNS) (Talbot et al., 2012; O'Neill, 2013). FDG-PET studies in AD patients have shown a progressive impairment of cerebral glucose uptake and metabolism particularly in the parieto-temporal lobes and posterior cingulate cortical regions of the brain that correlates strongly with disease progression (Moloney et al., 2010). Moreover, insulin and insulin-sensitizing drugs improve cognitive performance in people at early stages of AD (Freiherr et al., 2013; Roberts et al., 2014). The physiological and pathological role of IR signaling in the CNS is still unknown, but a strong correlation between AD and dysfunction of the insulin-signaling pathway with regard to glucose metabolism in the brain has prompted some investigators to refer to AD as type 3 diabetes or an "insulin resistant" condition of the brain (de la Monte, 2012; Barone et al., 2019).

 GSK-3β is a key serine/threonine kinase that phosphorylates tau at pathological epitopes at proline directed serine and threonine residues (Jackson et al., 2002; Johnson and Bailey, 2002). Additionally, GSK-3β inactivates glycogen synthesis and forms an important component of the insulin signaling pathway (Doble and Woodgett, 2003; Jolivalt et al., 2008). Another non-proline directed kinase that acts downstream of the insulin signaling pathway and generates pathological phospho-tau epitopes under conditions of oxidative stress is ERK (Colucci-D'Amato et al., 2003; Diehl et al., 2017). Although evidence of tau hyperphosphorylation by GSK-3β and ERK strongly indicates an impairment of the IR pathway as a risk factor in AD, the underlying mechanisms that link insulin resistance to tau hyperphosphorylation remain unclear.

The disease and severity of AD correlates strongly with the spatial and temporal progression of the insoluble aggregated tau fragments (NFTs) in the vulnerable brain regions (Didonna and Legname, 2010). Recent studies have shown that the

clearance of these misfolded tau aggregates from the neurons is accomplished by autophagy (Smith, 2002; Lee, 2012). Thus, a prominent feature of AD is the massive accumulation of lysosomal vesicular structures in degenerating neurons, and pathological studies have suggested an impairment of macroautophagy in neurodegenerative disorders including AD and other tauopathies (Codogno and Meijer, 2010; Barnett and Brewer, 2011; Nassif and Hetz, 2012). Macroautophagy involves the formation of double membrane bound structures around cytosolic protein aggregates and forming autophagic vacuoles, which will subsequently fuse with the lysosomes for the degradation of protein aggregates and cellular organelles. The mTOR plays an important role in cellular homeostasis and is an inhibitor of autophagy (Jung et al., 2010). There are reports from a large body of literature, that insulin signaling leads to the activation of AKT and mTOR via a relay of phosphorylation events (Partridge et al., 2011). Phosphorylation of mTOR via the PI3K-AKT signaling pathway induces protein synthesis and downregulates autophagy, whereas dephosphorylation of mTOR has the opposite effect (Avet-Rochex et al., 2012). Several groups have shown that the PI3K/AKT/mTOR pathways are activated in the early stages of AD, while other groups have shown an upregulation of autophagy in the AD brains (Cai et al., 2012; O'Neill, 2013). However, the exact mechanism of autophagic induction and misfolded protein clearance in neurodegeneration especially with regard to insulin resistance, still remains uncertain.

Drosophila models misexpressing the full-length human tau have been used successfully by us and many others to recapitulate prominent features of human tauopathies that include progressive neurodegeneration and tau aggregation/phosphorylation at the disease-associated phospho-epitopes mediated by the key kinases (Jackson et al., 2002; Chatterjee et al., 2009).

We have previously demonstrated that the misexpression of full-length (2N4R) human tau under the control of a glass promoter in the fly retina causes a marked "rough eye" phenotype with reduced eye size and missing bristles (Chatterjee et al., 2009). This "rough eye" phenotype has been instrumental in performing genetic screens to identify the modifiers of tau toxicity. Interestingly, an unbiased genetic screen conducted with the gl-Tau flies identified "Chico" – the single fly homolog of the mammalian IRS as one of the suppressors of the tau-induced "rough eye" phenotype (Ambegaokar and Jackson, 2011).

We therefore investigated the effect of insulin on tau phosphorylation in vivo by co-expressing Chico with Tau in the Drosophila retina. Our findings demonstrate that Chico has a strong genetic interaction with Tau, causing a suppression of tau-induced toxicity in our model. We also observe that Chico decreases the level of total tau (T46) as well as phosphorylated tau at AT8 (phospho-Ser202/Thr205) and PHF1 (phospho-Thr231/Ser235) sites.

This effect correlates strongly with an elevation of inactive p-GSK-3βS9, while decreasing the level of active p-ERK (p-p44/42). We further demonstrate that these traits are reversed by Chico loss-of-function (Chico-LOF) indicating a Chico-specific effect on tau pathology. Finally, we show an increased proportion of soluble and insoluble tau aggregates in Tau transgenics that is accompanied by an induction of autophagy though not necessarily autophagic clearance. Interestingly, these effects are rescued by Chico but aggravated by Chico-LOF. We further validated our findings in vitro in human neuroblastoma cells under insulin resistant conditions. Collectively, these results show that the mechanisms by which insulin resistance impacts tau pathology are conserved in Drosophila and mammalian cell lines.

### MATERIALS AND METHODS

### Stocks and Genetics

Flies were grown on Jazz mix medium (Applied Scientific Jazz Mix, Fisher Scientific, Pittsburgh, PA, United States) at 25◦C. The GAL4 driver used in this study is GMR-GAL4 on the X chromosome. GMR-GAL4 (X) was placed in trans to gl-tau (A direct fusion construct of the human full-length tau cDNA to the eye specific glass promoter as described by (Jackson et al., 2002) to generate GMR-GAL4-gltau on the X chromosome. Transgenic fly lines carrying UASChico were established by amplifying target sequences by PCR using chico specific primers (chico forward: 5<sup>0</sup> -ATAATTCCGCACTGGCAAAG-3<sup>0</sup> ; chico reverse: 5<sup>0</sup> -CCATGCCATTAAGATGCTCA-3<sup>0</sup> ) and the resulting constructs were subcloned using Exelixis (San Francisco, CA, United States) modification of Drosophila upstream activation sequence (UAS) expression vector (pEx-UAS) and microinjected into fly embryos (BestGene, Chino Hills, CA, United States). UASChico-RNAi and Chico-LOF flies were a gift from Dr. Minoru Saitoe (Tokyo Metropolitan Institute of Medical Science). UASEGFP was ordered from Bloomington Fly Stock, Indiana, Stock no. 5431). The EGFP gene is located on the second chromosome.

### Light Microscopy

Light microscopy retinal images were acquired using a Nikon AZ100M light microscope (Melville, NY, United States). Extended depth of focus and volumetric images were taken using a Nikon DSFi1 camera and Nikon NIS-Elements AR 3.0 software as described previously (Chatterjee et al., 2009; Ambegaokar and Jackson, 2011).

### Histology and Immunohistochemistry

For SEM, flies were dehydrated in ethanol, incubated overnight in hexamethyldisilazane, dried under vacuum, attached to stubs with black nail polish and analyzed using a JSM-6510LV SEM (JEOL USA, Peabody, MA, United States) (Chatterjee et al., 2009). TRITC phalloidin (Sigma, St. Louis, MO, United States) whole-mount staining of adult retina was carried out as described previously (Sang and Ready, 2002). Briefly, adult retinas were dissected in cold PBS 1X pH 7.4, fixed in 4% formaldehyde/PBS for 45 min, then washed three times in PBT (0.5% Triton in PBS) and incubated for 40 min with TRITC-Phalloidin. After three washes in PBT, samples were mounted in vectashield for analysis with SP8 AOBS Laser Scanning Confocal Microscope (Leica Microsystems, Germany). 63× objective was used for scanning the retinas using a 561 nm laser spectral detector of detection bandwidth 550–650 nm.

### Immunoblotting and Reagents in Drosophila

Immunoblotting experiments were performed according to protocols previously described (Chatterjee et al., 2009). Briefly, about thirty freshly eclosed flies were collected and heads were decapitated and homogenized using an Argos battery-operated pestle mortar mixer for 1 min on ice in lysis buffer supplemented with complete protease inhibitors and PhosphoSTOP phosphatase inhibitor (Roche Diagnostics, Indianapolis, IN, United States). Extracts were then centrifuged at 4◦C for 10 min at 11,000 × g and the supernatants were collected while the pellets were discarded. Samples were then mixed with an equal volume of Laemmle sample buffer with β-mercaptomethanol (Bio-Rad Laboratories, Hercules, CA, United States) and resolved by appropriate SDS-PAGE gels before transfer to nitrocellulose membranes for antibody labeling. The membranes were then incubated with appropriate secondary antibodies and developed using enchanced chemiluminiscence (ECL). Following western blot detection the membranes were re-probed with antiβ-tubulin or anti-β-actin that was used as a loading control for each experiment. The following antibodies were used, anti-tau monoclonal antibody (1:1000; T46, Invitrogen), AT8 (1:500; Thermo Scientific), anti-P-AKT505, anti-AKT, anti-mTOR, anti-P-p70S6K, and anti-P-4E-BP (1:500; Cell Signaling), anti-P-GSK-3βS9 (1:500; Genetex), GSK-3β (1:1000; United States Biologicals), anti-P-p44mapk, and p44mapK (1:500; Promega), anti-Re f(2P) (1:500; Abcam), anti-ATG8 (1:400; EMD Millipore), anti-β-tubulin, and anti-β-actin (1:2000; Sigma). The blots were quantified by ImageJ (NIH). Western blots were repeated at least three times with different sets of animals.

### Cell Culture and Treatments

SY5Y human neuroblastoma cells were ordered from ATCC and grown in DMEM supplemented with 10% heat-inactivated fetal bovine serum, 2 mM L-glutamine, 100 U/ml penicillin, and 100 µg/ml streptomycin in 5% CO<sup>2</sup> atmosphere at 37◦C. Cells were plated at a density of 5 × 10<sup>3</sup> cells/cm<sup>2</sup> in 60 mm-diameter culture dishes with 10% FBS. From day 1 after plating, cells were differentiated in the presence of 10 µm all-trans retinoic acid for a week in the cell medium containing 1% FBS. For experiments, 4 × 10<sup>6</sup> cells were seeded on 60 mm dishes and cultured for 48 h (Jamsa et al., 2004). Briefly, the cells were serum-starved for 24 h and then incubated in the absence and presence of

insulin (100 nm) for time periods of 10 min, 30 min, 1, 2, and 4 h, respectively.

In parallel, the cells were serum starved for 24 h and then incubated with 100 nm insulin for a period of 48 h. Post-incubation the medium was removed and supplemented progressively with 20 nm insulin for a period of 4 h, respectively. For the study of autophagic flux the cells were incubated with 100 nm bafilomycin for 4 h prior to processing. DMEM, fetal bovine serum, L-glutamine, penicillin, streptomycin, and other cell culture reagents were obtained from Life Technologies, Inc. (Invitrogen), Recombinant human insulin and trans–retinoic acid was from Sigma.

### Immunoblotting in SY5Y Cells and Reagents

Following insulin treatment, cells were lysed in buffer containing 50 mM Tris–HCl, pH 8.0, 150 mM NaCl, 1% Triton-X-100, 1 mM EDTA, 10 mM NaF, 1 mM Na3VO4, and one complete protease inhibitor cocktail tablet (Roche Diagnostics)/10 ml buffer. Cell lysates were centrifuged at 15,000 × g (Eppendorf 5417R) for 10 min. The protein content of the supernatants was measured using BCA Protein Assay kit (Pierce). An equal amount of total protein was resolved on 4–20% SDSpolyacrylamide gel electrophoresis for immunoblotting analysis using standard protocols. Primary antibodies were incubated overnight at 4◦C at the following dilutions, T46 (1:1000; Invitrogen), AT8 (1:500; Thermo Scientific), pGSK-3βS9, pAKT473, mTOR, P-p70S6K, P-4E-BP1, GSK-3β, AKT, anti-IRS1-pSer616 (1:500; Cell Signaling), pERK, ERK (1:500; Promega), anti-LC3 (1:400; Novus Biologicals), and anti-GAPDH (1:500, Abcam), anti-β-actin (1:5000; Sigma). Horseradish peroxidase (HRP) conjugated secondary antibodies (Amersham Biosciences) were incubated for 1 h at room temperature in 5% milk at the dilution of 1:2000 for anti-mouse and anti-rabbit antibodies. The blots were developed by ECL.

### Immunocytochemistry and Reagents

Serum-starved SY5Y cells were stimulated at different time points with or without insulin (100 nm). After a brief PBS wash, cells were fixed for 30 min in 4% paraformaldehyde-PBS, washed three times in PBS, and blocked in 3% BSA for 1 h. Cells were then incubated with total tau antibody (A0024, DAKO) and p62 (NBL) at 1:200 dilution overnight at 4 ◦C. After extensive washing with PBS, cells were incubated with Alexa Fluor 568 nm conjugated goat anti-mouse IgG (Invitrogen) and Alexa Fluor 488 nm conjugated goat antirabbit IgG (Invitrogen) and counterstained with 4, 6-diamino-2-phenylindole (1:3000 dilution) to count the number of nuclei per field of view. Cells were mounted in coverslips with vectashield (Vector laboratories). Cell analysis was carried out using a Zeiss Axioplan 2 MOT upright fluorescence microscope equipped with filter sets for DAPI, FITC, and TRITC. The microscope settings were consistent for experimental and control sets.

### Statistical Analyses

Comparison between multiple groups in flies was done by one way ANOVA followed by Tukey-Kramer HSD test. Two experimental groups were analyzed using Student's t test. Comparison between multiple groups in SY5Y cells were carried out by one way ANOVA followed by Fisher's LSD post hoc test. Comparison between two experimental groups was done by Student's t test. Statistical analyses were performed using Graphpad Prism or Sigma Plot software.

### RESULTS

Several groups have shown that Chico plays a prominent role in the insulin-signaling pathway in Drosophila with Chico-LOF generating pronounced insulin resistance (Murillo-Maldonado et al., 2011; Naganos et al., 2012). Our previous studies from an unbiased genetic screen in Drosophila tauopathy models, have identified Chico as a strong modifier of the Tau phenotype. Hence, in the next sections we have investigated further the mechanistic pathways by which this interaction occurs.

### Misexpression of Chico in the Fly Eye Ameliorates Human Tau Induced Rough-Eye Phenotype

To investigate how insulin signaling impacts tau pathology, we utilized our well-established model of Tauopathy in which we expressed full-length wild type human Tau (2N4R) in Drosophila eyes using the pan-retinal GMR-GAL4 driver (Jackson et al., 2002; Chatterjee et al., 2009). As reported previously, Tau misexpression in the eye resulted in smaller eye size with a rough anterior and missing bristles (**Figure 1C**). Interestingly, co-expression of Chico with Tau ameliorated the "roughness" of the eye phenotype, resulting in larger eyes with fewer missing bristles (**Figure 1D**) while ChicoRNAi or Chico-LOF (null allele chico[1]) with Tau, resulted in a more severe worsening of the "rough-eye" phenotype (**Figures 1F,G**). As compared to the double transgenics, the Control eyes of (GMR/+), GMR/UASChico and GMR/UASChicoRNAi appeared perfectly normal (**Figures 1A,B,E**). Magnification of the SEM insets (400X) clearly demonstrates fused ommatidia and missing bristles in Tau flies (**Figure 1C**, lower panel) that are partially rescued by Chico (**Figure 1D**, lower panel) and exacerbated by Chico-LOF (**Figure 1F**, lower panel). Quantification of the percentage of rough area per eye in each genotype revealed an 80% rough-eye area in Tau and Tau+Chico-LOF dual transgenics as compared to 45% rougheye area in Tau+Chico transgenics compared to Controls (**Figure 1H**, lower panel). In contrast, UASEGFP co-expressed with Tau, failed to suppress the "rough-eye" phenotype confirming that the effect of Chico on Tau is specific (**Supplementary Figure S1A**).

In order to compare the internal retinal morphology, confocal imaging of adult retinas stained with TRITC-phalloidin was performed. Tangential optical sections display a normal

trapezoidal array of rhabdomeres in the driver-alone controls (**Supplementary Figure S2A**). In contrast, Tau flies produced disorganized ommatidia with massive loss of photoreceptor neurons (**Supplementary Figure S2B**). The retinas of transgenics co-expressing Chico (**Supplementary Figure S2C**) showed a relatively normal array of rhabdomeres other than some abnormal polarity.

Taken together our results show that the co-expression of Chico with Tau atleast partially rescues tau-induced neuronal loss and protects against tau-induced neurotoxicity.

### Misexpression of Chico With Tau Reduces the Levels of Total and Hyperphosphorylated Tau

We and others have previously shown that the "roughness" of the eye phenotype in Tau transgenics is associated with elevated tau expression as well as hyperphosphorylation at the disease epitopes (Jackson et al., 2002; Chatterjee et al., 2009). Therefore, in order to analyze the rescue of the "rough-eye" phenotype in Tau+Chico flies, we examined if there were any alterations in the levels of total and hyperphosphorylated tau in these transgenics.

Our results show that coexpression of Chico and Tau significantly decreased the total tau (T46) and phospho-tau at AT8 and PHF1 residues leading to >50% reduction of AT8/T46 and PHF1/T46 ratios as compared to Tau-alone flies. We further observed that these effects were reversed when Tau was coexpressed with Chico-LOF or ChicoRNAi (**Figure 2A** and **Supplementary Figure S3A**). Our data also showed that the coexpression of EGFP produced no alterations in the tau levels confirming that this is a Chico-specific effect (**Supplementary Figure S1B**). In summary, our results suggests that the ability of Chico to ameliorate the tau-induced "rough-eye" phenotype occurs by decreasing the level of total and phospho-tau at pathological epitopes.

### Misexpression of Chico With Tau Decreases the Formation of Soluble and Insoluble Tau Aggregates Compared to Tau and Tau+ChicoRNAi

Aggregation of hyperphosphorylated tau is one of the key features of AD and related tauopathies (Simic et al., 2016).

AT8 levels are reversed when Chico-RNAi is coexpressed with Tau. (A) Representative blots are probed with T46 (total tau), AT8, PHF1, (phospho-tau), and β-tubulin antibodies, respectively. All flies were 3 days after eclosion. Genotypes are as follows: GMR-GAL4/+; GMR-GAL4/UASChico; GMR-GAL4-gl-Tau/+; GMR-GAL4-gl-Tau/UASChico; GMR-GAL4/UASChico-RNAi; GMR-GAL4-gl-Tau/UASChico-RNAi. (B) Tau and Chico coexpression decreases the level of sarcosyl soluble and insoluble tau aggregates in the supernatant and pellet as compared to Tau alone and Tau+Chico-LOF dual transgenics. Western blot analysis from flies probed with T46 antibody against total tau. Genotypes: GMR-GAL4-gl-Tau/+; GMR-GAL4-gl-Tau/UASChico; GMR-GAL4-gl-Tau/UASChico-LOF. Densitometric analyses of AT8/T46 and PHF1/T46 levels and total tau distribution in soluble and pellet fractions are normalized to β-tubulin. (One way ANOVA with Tukey-Kramer HSD was used for multiple groups comparison or post hoc Dunnett's test for comparison against Tau, n = 3 sets of biological replicates for each genotype.) Data represents the mean ± SEM, ∗∗P < 0.01 and <sup>∗</sup>P < 0.05 relative to Tau.

Our previous studies in tauopathy models demonstrated the accumulation of sarcosyl soluble and –insoluble tau aggregates that contributed toward tau-induced neurotoxicity (Chatterjee et al., 2009). In line with these reports, we investigated whether loss or GOF of Chico has any effect on sarcosyl solubility of tau. Co-expression of Chico with Tau reduced the accumulation of sarcosyl-soluble and insoluble tau species in the supernatant and pellet fractions, respectively. However, co-expression of ChicoRNAi significantly altered the pattern of tau solubility and increased accumulation of sarcosylinsoluble tau (**Figure 2B**). Although recent studies have demonstrated that soluble tau aggregates are toxic for neuronal cells, sarcosyl-insoluble tau aggregates eventually form the NFTs detected in AD brains (Ren and Sahara, 2013). Taken together, our data shows that in addition to decreasing tau expression and hyperphosphorylation, Chico reduces the formation of sarcosyl-soluble and insoluble tau aggregates in Tau transgenics.

### Effect of Chico on Tau Hyperphosphorylation Is Mediated by GSK-3β

Given that the insulin signaling pathway in Drosophila is highly conserved with that of higher vertebrates, and the kinases GSK-3β and ERK lie downstream of Chico in the insulin signaling cascade; we investigated whether there is a direct effect of Chico on the regulation of these kinases in our model.

Our data showed a >50% reduction in the level of inactive p-GSK-3βS9 in Tau genotypes compared to controls (GMR/+