We then examined the tauopathy associated with chronic traumatic encephalopathy (CTE) and CTE with concurrent ALS (CTE-ALS) (Moszczynski et al., 2018). In both disease states, the neuropathological hallmark is a disseminated tauopathy including not only cortical neuronal and glial cytoplasmic inclusions, but also spinal cord tau protein pathology (McKee et al., 2009, 2010; Mez et al., 2017). Consistent with a role for pathological Thr175 tau phosphorylation, we observed pThr175 , pThr231, and T22 immunoreactivity in both hippocampal and spinal motor neurons. Fractionation of the hippocampal tau protein into sarkosyl-soluble and insoluble fractions yielded all six tau protein isoforms partitioning into both fractions, similar to that observed in ALSci. In support of the hypothesis that this pathway is directly related to the induction of tau protein cytoplasmic inclusions, we were able to replicate this pathology following a single cortical impact (moderate brain trauma model) in young adult Sprague Dawley rats (Moszczynski et al., 2018).

While we have suggested that pThr175 tau is critical to the induction of a tauopathy, this evidence does not prove it to be sufficient for the development of tauopathy in vivo. To test this, we utilized the somatic gene transfer of a pseudophosphorylated human tau protein construct mimicking

pThr175 tau. Young adult Sprague Dawley rats received bilateral stereotactic inoculums of a rAAV9 adenoviral construct containing one of either GFP tagged Thr175Asp-tau (pThr175 tau mimic), GFP-tagged WT-tau, GFP-tagged construct alone, or GFP-tagged Thr175Ala-tau (Moszcyznski et al., 2017). At 1 year following inoculation, expression of the viral vector remained prominent as assessed using anti eGFP immunohistochemistry. However, the brains of the Thr175Asp-tau inoculated rats demonstrated prominent neuronal tau protein pathology, including corkscrew neurites, and tau immunoreactive neuronal cytoplasmic inclusions.

### POSTULATED PATHOGENIC PATHWAY

In support of the pathogenicity of pThr175 tau, phosphorylation at this site is not observed in fetal human tissue (which is normally hyperphosphorylated) (Kenessey and Yen, 1993) or tissue from healthy-aged individuals (Moszczynski et al., 2017). We have shown in vitro that a pseudophosphorylated human tau construct (Thr175Asp tau) induces pathological tau protein inclusions accompanied by the activation of GSK3β along with phosphorylation of tau at Thr231 (Moszczynski et al., 2015). The in vivo observation of coexpression of pThr175 tau, pTyr216GSK3β, pThr231 tau, and immunoreactivity to a tau protein antibody recognizing oligomeric tau (T22) strongly suggests that this pathway is critical to the induction of pathological tauopathies, including ALSci, CTE and CTE-ALS (Moszczynski et al., 2017, 2018).

The mechanism of Thr175 phosphorylation is not known. Candidate kinases include mitogen activated protein kinases (MAPK), GSK3β, p38, and leucine-rich repeat kinase 2 (LRRK2) (Hanger et al., 1998; Reynolds et al., 2000; Atzori et al., 2001) (see also https://docs.google.com/spreadsheets/d/ 1hGYs1ZcupmTnbB7n6qs1r\_WVTXHt1O7NBLyKBN7EOUQ/ edit#gid=0). Amongst these, a member of the MAPK family of kinases, c-Jun N-terminal kinase [JNK; also known as stress activated protein kinase (SAPK)], is of specific interest given its increased activity in response to various cellular stresses in vitro (Namgung and Xia, 2000) and in the acute phases following traumatic brain injury (TBI) in vivo (Tran et al., 2012). Additionally, the inhibition of JNK can reduce pathological tau protein phosphorylation following TBI in a rodent model (Tran et al., 2012).

Although the mechanism(s) by which the phosphorylation of tau at Thr175 leads to the activation of GSK3β are not yet known, it is likely that such mechanism(s) will involve altering tau's global hairpin structure given that Thr175 lies within the Pro-rich domain/hinge region of the hairpin as it has been shown that the phosphorylation of other residues within this domain can open the hairpin structure (Jeganathan et al., 2006, 2008). There are at least two potential mechanisms of GSK3β activation which may result from tau hairpin structure opening (**Figure 2**). Firstly, opening of the hairpin may expose the N-terminus

Thr175 exposes SH3 homology domains within the proline-rich region (A). This interacts with and activates FYN kinase, which in turn activates GSK3β by phosphorylating at Tyr216. Alternatively, opening of the tau hairpin can expose the N-terminal phosphatase activating domain (PAD) (B), which then activates protein phosphatase 1 (PP1). Activated PP1 dephosphorylates GSK3β at Ser9 increasing its activity. The activation of GSK3β may exert toxic effects in the cell by the inhibition of fast axonal transport, and by the phosphorylation of tau protein at Thr231, which promotes the formation of neurofibrillary tangles (paired helical filaments; PHF). Additionally, increased activity of GSK3β can increase the formation of pThr231 tau which also contains an exposed PAD, thereby initiating a dysregulated positive feedback loop. Activated enzymes are indicated by an asterisk (\*).

proline-rich domain, which has been shown to interact with the SH3 domain of Src-family tyrosine kinase Fyn. In doing so, activated Fyn kinase may then phosphorylate GSK3β at the Tyr216 residue thereby enhancing its activity (Lee et al., 1998; Lesort et al., 1999; Klein et al., 2002). Secondly, opening of the hairpin conformation has been shown to expose the N-terminal phosphatase activating domain (PAD), consisting of amino acids 2–18. Exposure of this domain leads to activation of protein phosphatase-1 (PP1) which then dephosphorylates GSK3β at the Ser9 residue, enhancing GSK3β activity (Kanaan et al., 2011). In keeping with this mechanism, exposure of the PAD has been suggested to be important in tau protein mediated neuronal toxicity through interference with fast axonal transport, and may be an early event in the development of a number of tauopathies (Kanaan et al., 2011, 2016; Ward et al., 2012; Combs et al., 2016).

In the studies cited above, tau phosphorylation at Thr231 is a critical step in the induction of pathological fibrils. Several mechanisms have been proposed by which this may occur, with alterations in tau degradation being key in a process that is dependent on tau conformation. Of note, cis-pThr231 tau demonstrates specificity in the development of pathology over trans-pThr231 tau, in which the cis conformer is pathological and cannot be degraded, whereas the trans conformer is physiological and can be degraded (Nakamura et al., 2012). The isomerase, peptidyl-prolyl cis-trans isomerase NIMA-interacting 1 (PIN1), converts the pathologic cis conformer to the physiologic trans conformer, allowing for dephosphorylation at this site by protein phosphatase 1 (Lu et al., 1996, 1999). It is of note, therefore, that cis-pThr231 tau is closely associated with a number of tauopathies including, AD, CTE and experimental CTE (Lu et al., 1996, 1999; Liou et al., 2003; Pastorino et al., 2006; Sultana et al., 2006; Lu and Zhou, 2007; Lim et al., 2008; Lee et al., 2011; Chen et al., 2015; Kondo et al., 2015; Albayram et al., 2017). Given that both cis-pThr231 tau and pThr175 tau are observed exclusively in disease or stressed states, it is attractive to postulate that the phosphorylation of tau protein at Thr175 increases the presence of cis-pThr231. This may occur through either enhancing the probability of cis phosphorylation by GSK3β or by inhibiting the ability of PIN1 to isomerize the epitope from cis to trans. This is the subject of current studies.

### THE POTENTIAL ROLE OF COMORBID PATHOLOGIES

The fact that other cell stressors may induce Thr175 tau protein phosphorylation is of great significance to the mechanisms of neurodegenerative disease. Pathologies are rarely, if ever, observed in isolation, and it is common to identify comorbid pathologies in the same brain (Amador-Ortiz et al., 2007; Josephs et al., 2014a,b; Smith, 2017). While this high incidence of comorbidity has traditionally been attributed to the aging process, it is increasingly evident that multiple pathologies are present even in young cohorts of patients afflicted with neurodegenerative disease. Therefore, there is likely an interplay between pathological processes that drives these co-morbid pathologies (Spires-Jones et al., 2017; Tan et al., 2017). As such, the possibility of synergistic toxicity must be considered when trying to understand the mechanisms that drive neurodegenerative disease, and when attempting to treat such diseases.

As discussed earlier, the FTLD of ALSci is classified as a FTLD-TDP based on the prominence of pathological TDP-43 deposition. The association of TDP-43 pathology with ALS and ALS-FTSD is well described (Arai et al., 2006; Neumann et al., 2006). However, there is also an increasing literature describing comorbid TDP-43 and tau pathologies, such as that observed in AD and CTE (McKee et al., 2010; Josephs et al., 2014a,b). The relationship between tau protein and TDP-43 pathology is strengthened in the observation that the effective elimination of aberrantly phosphorylated tau protein in a rodent model of CTE

### REFERENCES

can also lead to the prevention of TDP-43 pathology (Albayram et al., 2017). It is possible that one pathological process primes the other. In this case, the toxicity of pseudophosphorylated Thr175Asp tau protein expression may be enhanced by the coexpression of TDP-43, serving as a second hit to the CNS. The potential of this synergistic toxicity is the focus of ongoing studies in our lab.

## SUMMARY

The presence of pathological tau processing is clearly evident across a broad range of ALS patients—including those found within the hyperendemic foci such as that seen in the western Pacific, a smattering of case reports, and ALSci as well as FTD-MND. This suggests that this is not a simple matter of incidental co-occurrence. Moreover, there is solid evidence to support the pathogenic role of pThr175 in the induction of a tauopathy, both in vitro and in vivo, and the pathological cascade induced by pThr175 which culminates in the generation of oligomeric tau has been observed in a broad range of tauopathies. The issue is therefore not whether a tauopathy exists in ALS-FTSD, but rather the extent to which it is the driving pathology—either alone, or in combination with the hallmark pathology of TDP-43—of the clinical and neuropathological phenotype.

Such a proposal however leaves many unanswered questions, including: the mechanism by which the Thr175 residue is initially phosphorylated; how pThr175 tau leads to the activation of GSK3β; whether pThr231 tau is necessary and sufficient to induce neuronal dysfunction and death, or whether additional tau protein pathological phosphorylation is needed; and what (if any) role for the co-existent TDP-43 pathology of ALS-FTSD is also necessary to drive the phenomenological aspects of pThr175 tau-mediated tauopathy.

# AUTHOR CONTRIBUTIONS

MS conceived and wrote the article. AM conceived and wrote the article. MH wrote the article.

# ACKNOWLEDGMENTS

Research funded by the Ontario Neurodegenerative Disorders Research Initiative (ONDRI) and the National Hockey League Players Association (NHLPA) Challenge Fund.

Initiative (GENFI) study: a cross-sectional analysis. Lancet Neurol. 14, 253–262. doi: 10.1016/S1474-4422(14)70324-2

**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 © 2018 Moszczynski, Hintermayer and Strong. 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 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.

# Association Between Autophagy and Neurodegenerative Diseases

### Nobuhiro Fujikake, Minkyoung Shin and Shigeomi Shimizu\*

Department of Pathological Cell Biology, Medical Research Institute, Tokyo Medical and Dental University, Tokyo, Japan

Autophagy is a phylogenetically conserved mechanism that controls the degradation of subcellular constituents, including misfolded proteins, and damaged organelles. The progression of many neurodegenerative diseases is thought to be driven by the aggregation of misfolded proteins; therefore, autophagic activity is thought to affect disease severity to some extent. In some neurodegenerative diseases, the suppression of autophagic activity accelerates disease progression. Given that the induction of autophagy can potentially mitigate disease severity, various autophagy-inducing compounds have been developed and their efficacy has been evaluated in several rodent models of neurodegenerative diseases.

Keywords: autophagy, neurodegenerative disease, Alzheimer disease, tauopathy, Parkinson disease, amyotrophic lateral sclerosis, polyglutamine disease

### Edited by:

Naruhiko Sahara, National Institute of Radiological Sciences (NIRS), Japan

### Reviewed by:

Gen Matsumoto, Nagasaki University, Japan Patrícia Maciel, Escola de Medicina da Universidade do Minho, Portugal Sabine Gilch, University of Calgary, Canada

### \*Correspondence:

Shigeomi Shimizu<EMAIL_ADDRESS>
### Specialty section:

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

Received: 04 December 2017 Accepted: 03 April 2018 Published: 22 May 2018

### Citation:

Fujikake N, Shin M and Shimizu S (2018) Association Between Autophagy and Neurodegenerative Diseases. Front. Neurosci. 12:255. doi: 10.3389/fnins.2018.00255 INTRODUCTION

A common pathology shared by several neurodegenerative diseases, including Alzheimer disease (AD), Parkinson disease (PD), amyotrophic lateral sclerosis (ALS), and the polyglutamine (polyQ) diseases is the accumulation of misfolded proteins. Because autophagy is a cellular function that degrades abnormal proteins, including those that are misfolded, the onset and progression of these diseases are affected by autophagic activity within neurons. In this review, we first summarize the molecular mechanism and physiological relevance of autophagy. Next, we review how autophagy is involved in the pathology of several neurodegenerative diseases. We also summarize the therapeutic effects of autophagy-modulating compounds on these neurodegenerative diseases.

# MOLECULAR MECHANISMS OF AUTOPHAGY

Autophagy is a catabolic process in which cellular contents, including proteins, lipids, and entire organelles, are degraded by lysosomal lytic enzymes. Autophagy constitutively functions at low levels but is highly induced by a variety of cellular stressors, such as nutrient starvation, growth factor withdrawal, DNA damage, the accumulation of abnormal proteins, and organelle damage. In many physiological and pathological contexts, autophagy protects cells by facilitating the degradation of superfluous or damaged cellular constituents, for the subsequent recycling of amino acids, lipids, nutrients, and metabolites.

There are at least three distinct autophagic mechanisms, namely, macroautophagy, microautophagy, and chaperone-mediated autophagy. Macroautophagy is believed to be the core pathway for degrading cytoplasmic proteins and organelles (Nakatogawa et al., 2009). In this process, cytoplasmic components and organelles are incorporated into double-membrane structures called autophagosomes. Subsequently, the outer membrane of the cellular constituents are broken down by acid hydrolases (Mizushima et al., 2002). The term "autophagy" is usually used synonymously with macroautophagy (hereafter referred to simply as "autophagy" unless otherwise indicated). The second type of autophagy is microautophagy, which occurs via direct invagination of the lysosomal membrane to engulf cellular constituents, followed by closure of the membrane pocket and degradation of the constituents within the lysososomal lumen (Li et al., 2012). Microautophagy can deliver entire organelles, such as peroxisomes, directly into lysosomes. The third type of autophagy is chaperone-mediated autophagy. During chaperone-mediated autophagy, cytoplasmic proteins with a targeting motif interact with the cytosolic heat shock cognate 70 chaperone, which delivers the proteins to the lysosomal membranes (Kaushik and Cuervo, 2012). Substrate proteins are subsequently recognized by the lysosomal receptor and are unfolded to penetrate the lysosomal membrane through a multimeric complex. After substrate translocation, rapid degradation of substrate proteins occurs in the lysosomal lumen. Thus, chaperone-mediated autophagy enables targeted protein degradation as opposed to the nonspecific or "bulk" protein degradation that occurs during macroautophagy.

Analysis of the molecular mechanisms involved in autophagy began with a genetic approach using autophagy-defective mutant yeast strains (Nakatogawa et al., 2009). Subsequently, many mammalian homologs of yeast autophagy proteins and their biochemical functions have been identified (**Figure 1**). It is now accepted that autophagy is driven by more than 30 autophagy-related proteins (Atgs) (Mizushima and Komatsu, 2011). Atg1 was the first such protein identified and shown to have intrinsic serine/threonine kinase activity, which is essential for the initiation of autophagy (Kabeya et al., 2005). Autophagy is regulated by phosphatidylinositol 3-kinase (PI3K) type I and type III. PI3K type I is activated by growth factors, and suppresses autophagy through the regulation of mammalian target of rapamycin (mTOR). Conversely, PI3K type III, which exists in a multiprotein complex including Atg6 (also called Beclin 1), facilitates the generation of the omegasomes, which is the initial membrane component of the isolation membrane (Axe et al., 2008). Isolation membranes subsequently expand, curve and close by two conjugation pathways, namely, the Atg5–Atg12 pathway and the microtubule-associated protein 1 light chain 3 (LC3) pathway (Mizushima and Komatsu, 2011). The ubiquitinlike conjugation of phosphatidylethanolamine (PE) to LC3 couples with the translocation of LC3 from the soluble fraction to autophagic membranes. Therefore, the localization of LC3- PE in autophagic membranes is a reliable marker of autophagy. The following lines of evidence indicate that the Atg5–Atg12 pathway is essential for the initiation of autophagy: (1) yeast Atg5 is crucial for autophagy, (2) autophagy is suppressed in some cell types from Atg5-deficient mice, and (3) LC3-PE formation has never been detected in Atg5-deficient cells. However, Atg5 deficient mouse embryos develop normally until the perinatal period (Kuma et al., 2004; Komatsu et al., 2005), suggesting that an alternative autophagic pathway may exist that compensates for the lack of Atg5-dependent autophagy in embryonic mutant mice.

In fact, we found that treatment with etoposide, a topoisomerase inhibitor that induces DNA strand breaks in mitotic cells, causes the formation of autophagic structures even in Atg5-deficient mouse embryonic fibroblasts (MEFs). The numbers and sizes of these autophagic vacuoles were equivalent in wild-type and Atg5-deficient MEFs, and the morphology of these etoposide-induced autophagic structures was indistinguishable from the autophagic vacuoles observed during starvation-induced autophagy (Nishida et al., 2009). Therefore, MEFs appear to perform two distinct types of autophagy, an Atg5-dependent conventional type and an Atg5 independent alternative type, which show similar morphological characteristics (**Figure 1**). Unlike Atg5-dependent autophagy, the molecules required for and the physiological roles of alternative autophagy have not yet been elucidated.

# THE ULK1 COMPLEX

Molecular mechanisms of conventional autophagy have been extensively studied using nutrient-starved cells, by referring to the information obtained from starved yeast cells, in which functional complexes containing Atg proteins drive for the formation of autophagic vacuoles. Starvation-induced autophagy begins from modifications in mTor and AMK-activated protein kinase (AMPK), which are well-established cellular nutritional sensors. mTor is a member of the serine/threonine protein kinase family and serves as a core component of two distinct protein complexes, mTor complex 1 (mTorc1) and mTorc2, which regulate different cellular processes. In healthy cells, mTorc1 suppresses autophagy by phosphorylating and inactivating Unc-51-like kinase 1 (Ulk1), a homolog of yeast Atg1 and an initiator of mammalian autophagy. AMPK is another kinase that plays a role in cellular energy homeostasis, and also suppresses autophagy by phosphorylating different amino acid residues of Ulk1. Upon nutrient starvation, Ulk1 is dephosphorylated by protein phosphatase 2A (PP2A) and translocates to limited membranes together with Fip200, Atg13, and Atg101. The membrane-associated ULK1 complex then phosphorylates multiple substrates, leading to the induction of conventional autophagy.

The Ulk1 protein is also crucial for alternative autophagy. Addition of etoposide induced the accumulation and autophosphorylation of Ulk1 in Atg5-deficient MEFs. Furthermore, no autophagic membranes were observed within Ulk1-silenced Atg5-deficient MEFs in response to etoposide. Similar results were also observed when Fip200, another component of the Ulk1 complex, was silenced. Thus, Ulk1 functions in the initiation of both conventional and alternative autophagy (**Figure 1**) (Nishida et al., 2009). The mechanism involved in the specific activation of each pathway by different stressors remains unclear. These two forms of autophagy may be selectively activated by the phosphorylation and activation of different Ulk1 substrates.

# THE PI3K COMPLEX

The class III PI3 kinase Vps34 phosphorylates PI at the 3′ hydroxyl position to produce PI3P, an abundant component of

autophagosomal membranes that is essential for autophagosome formation (Kihara et al., 2001). An autophagosome formationspecific PI3 kinase complex composed of Atg14L, Beclin 1, Vps15, and Vps34 was identified (Itakura et al., 2008). Two additional Vps34 complexes, Vps34-Vps15-Beclin 1-UVRAG and Vps34-Vps15-Beclin 1-UVRAG-Rubicon, function in the endocytic pathway and in the regulation of autophagosome/lysosome fusion, respectively (Matsunaga et al., 2009) (Zhong et al., 2009). Beclin 1 is also regulated by B-cell leukemia/lymphoma-2 (Bcl-2) family proteins, such as Bcl-2, Bcl-xL, and Bim (Levine et al., 2008). The protein double FYVE domain-containing protein 1 has been identified as a downstream effector of PI3P and it is concentrated within membrane domains of the endoplasmic reticulum (ER) that are sites of isolation membrane creation (Axe et al., 2008). The ULK1 complex was also reported to associate with the autophagosome formation-specific PI3K complex to generate isolation membranes. This PI3K complex is also crucial for alternative autophagy, as silencing Vps34 and Beclin 1 inhibited the alternative pathway (Nishida et al., 2009).

# ATG5-ATG12 AND LC3 PATHWAYS

Two unique ubiquitin-like conjugation systems, the Atg5–Atg12 pathway and the LC3 pathway, are believed to be essential for the induction of conventional autophagy (**Figure 1**). The Atg5–Atg12 pathway is initiated when Atg12 is conjugated to Atg5 by Atg7 (E1 enzyme) and Atg10 (E2 enzyme) (Mizushima and Komatsu, 2011), and the Atg5–Atg12 conjugate further binds with Atg16 to generate an Atg5–Atg12/Atg16 complex. The second protein conjugation system is controlled by the Atg8 homolog LC3, gamma-aminobutyric acid receptorassociated protein (GABARAP), and Golgi-associated ATPase enhancer of 16 kD (GATE-16). During autophagy, these proteins are cleaved by the cysteine protease Atg4, resulting in the exposure of a glycine residue, which is then catalyzed by Atg7 (E1 enzyme), Atg3 (E2 enzyme), and the Atg5– Atg12/Atg16 complex (E3 enzyme). PE then binds to the exposed glycine residue and acts to localize these proteins to isolation membranes and autophagosome membranes. The PE-conjugated forms of LC3, GABARAP, and GATE-16 (termed LC3-II, type II GABARAP, and type II GATE16) are reliable markers of conventional autophagy (Kabeya et al., 2000).

Although Ulk1 and PI3K complexes participate in both conventional and alternative autophagy, neither the Atg5– Atg12 nor the LC3 conjugation pathway is required for alternative autophagy. Furthermore, the conversion of LC3- I to PE-conjugated LC3-II does not occur in alternative autophagy (Nishida et al., 2009). Thus, it is unknown as to how extension and closure of autophagic membranes are accomplished during alternative autophagy without these two ubiquitin-like systems. Detailed morphological analysis has, however, provided some clues. We demonstrated that elongation and closure of isolation membranes, presumably derived from trans-Golgi membranes are generated by fusion with endosomal vesicular membranes (Nishida et al., 2009). The involvement Fujikake et al. Autophagy and Neurodegenerative Diseases

of trans-Golgi/endosomal fusion in the extension and closure of isolation membranes was confirmed by the colocalization of mannose-6-phosphate receptors (a trans-Golgi/late endosomal marker) and syntaxin 7 (a late endosomal marker) with Lamp2 positive vacuoles in etoposide-treated Atg5-deficient MEFs (Nishida et al., 2009). The formation of isolation membranes by trans-Golgi/endosomal fusion is also supported by studies showing a requirement for Rab9, a GTPase required for the trafficking of proteins from late endosomes to trans-Golgi membranes (Kabeya et al., 2000). First, GFP-Rab9 was colocalized with autolysosomes in etoposide-treated Atg5-deficient MEFs, and this colocalization was increased by the expression of GFP-Rab9Q66L, a constitutively active Rab9 mutant, and reduced by the expression of GFP-Rab9S21N, a guanosine diphosphate (GDP)-preferring dominant-negative Rab9 mutant (Nishida et al., 2009). Moreover, Rab9 silencing by a targeted siRNA reduced the number of autophagic vacuoles but induced the accumulation of isolation membranes. Numerous isolation membranes are normally generated by etoposide exposure, so siRab9 did not merely slow down the progression of autophagy but rather inhibited autophagosome maturation. The role of Rab9 in the extension and closure of isolation membranes in the alternative autophagy pathway is thought to replace the role of Atg5/Atg7/LC3 in conventional autophagy (**Figure 1**).

## PHYSIOLOGICAL ROLE OF AUTOPHAGY IN NEURODEGENERATION

Autophagy can be classified into constitutive and inducible types according to the mechanism of induction (**Table 1**). Whereas constitutive autophagy continuously degrades abnormal proteins to maintain cellular homeostasis, inducible autophagy occurs on a larger scale to suppress cellular injury against various stressors. Autophagy can also be classified into bulk and selective types based on the cargo that is degraded. Autophagy was originally considered to degrade subcellular constituents in a nonselective manner, i.e., bulk autophagy. However, specific molecules and organelles are also digested by selective autophagy during which cargo molecules are recognized by cargo receptors and subsequently enclosed by autophagic structures. According to these classification criteria, the well-studied starvation-induced type of autophagy is considered as inducible and bulk autophagy. Many phenotypes observed in systemic and tissue-specific Atg knockout mice, such as defects in preimplantation fetal development (Kuma et al., 2004), are due to the failure of constitutive and selective autophagy. As Atg7-deficient mice lack conventional autophagy, they demonstrate neurological defects, including the abnormal limb-clasping reflexes, because of the severe damage to cortical and cerebellar neurons (Komatsu et al., 2006). Furthermore, there was reactive gliosis was observed in association with neuronal loss. Importantly, ubiquitinpositive aggregates were found in neurons. These findings illustrate that conventional autophagy prevents neuronal damage by constitutively eliminating ubiquitin-positive aggregates.

TABLE 1 | Classification of autophagy from the view of inducers and substrates.

# NEURODEGENERATIVE DISEASES AND AUTOPHAGY IN HUMANS

Consistent with the findings observed in autophagydeficient mice showing neurodegenerative defects, an impairment of autophagy is often associated with human neurodegenerative diseases. In some cases, autophagy failure is considered as the primary underlying disease pathology. In most neurodegenerative diseases, including ALS, PD, AD, and the polyQ diseases, the accumulation of misfolded proteins is a common pathological hallmark. These misfolded proteins damage neurons, eventually causing their death. Because the reduction of autophagic activity directly affects the accumulation of misfolded proteins, autophagy is a key target pathway for the treatment of neurodegenerative diseases (**Figure 2**). In this section, we briefly describe the relationship between conventional autophagy and several major neurodegenerative diseases.

# ALS/ TDP-43 PROTEINOPATHIES

ALS, a fatal neurodegenerative disorder, is primarily a sporadic disease; however, approximately 5–10% of the cases are familial. In particular, mutations in several genes including superoxide dismutase 1 (SOD1), TAR DNA-binding protein-43 (TDP-43), fused in sarcoma/translocated in liposarcoma (FUS/TLC), and/or C9orf72, are thought to contribute to the progressive degeneration of motor neurons. A mutation in SOD1 was the first genetic mutation that was shown to be associated with ALS, and more than 170 SOD1 mutations have been identified to date. TDP-43, a nuclear factor that controls the fate of cellular RNAs, is found in an aggregated form in neurons of ALS patients, and genetic mutations in TDP-43 were recently identified in some ALS patients. These mutants commonly fail to fold properly, leading to the accumulation of misfolded proteins in neurons of ALS patients. Numerous studies suggest that autophagy might be a significant regulator of pathological aggregate formation in ALS. For example, the overexpression of transcription factor EB (TFEB), a master regulator of the transcription of autophagyrelated genes, led to increases in the expression of Beclin 1 and LC3 and suppressed the cellular toxicity induced by the expression of these mutant proteins (Chen Y. et al., 2015).

Recently, receptors for selective autophagy, such as sequestosome-1 (SQSTM1)/p62 and optineurin (OPTN), as well as TANK-binding kinase 1 (TBK1) were shown to be involved in the degeneration of motor neurons (Maruyama et al., 2010; Teyssou et al., 2013; Freischmidt et al., 2015). Insoluble protein aggregates of these proteins were found to accumulate in motor neurons and surrounding glial cells. Intriguingly, these inclusions were found in the brainstem, spinal cord, cerebellum, hippocampus, and frontal and temporal lobes of ALS patients. SQSTM1 interacts with and sequesters autophagy substrates to isolation membranes via its interaction with LC3, and ALS-causing SQSTM1 mutants increase the expression levels of TDP-43 (Teyssou et al., 2013). Consistently, mutations in the SQSTM1 were shown to suppress the recruitment of mutant SOD1 into autophagosomes (Gal et al., 2017). OPTN also plays a role in sequestering cargo to nascent autophagic vacuoles via its interaction with LC3 (Wild et al., 2011). In addition, OPTN interacts with myosin VI for the proper trafficking of autophagosomes (Sahlender et al., 2005). Interestingly, most of the ALS-causing mutations in OPTN are located in the myosin VI-binding domain (Sundaramoorthy et al., 2015). These mutants do not interact with myosin VI and are unable to eliminate mutant SOD1 and TDP-43. Finally, TBK1 promotes autophagy by phosphorylating both SQSTM1 and OPTN, and ALS-associated mutations in TBK1 reduce its binding affinity with OPTN and decrease the clearance of dysfunctional mitochondria (Richter et al., 2016).

Mutations in C9orf72, the most common cause of ALS, have been proposed to downregulate autophagy (Ji et al., 2017). C9orf72 is a core component of GDP/ GTP exchange factor (GEF) for Rab8 and Rab39, which is crucial for autophagosome maturation. This molecule also interacts with Ulk1 and advances the autophagy machinery; as such, ALSassociated mutations of C9orf72 do not interact with Rab proteins, and autophagy fails to proceed (Tang, 2016; Corbier and Sellier, 2017). These findings indicate that selective autophagy, which degrades SOD1 and TDP-43 mutants and requires SQSTM1, OPTN, and TBK1, is crucial for protection from ALS.

Autophagy facilitates the reduction of unfolded proteins. For example, progesterone and lithium were shown to activate autophagic flux, thereby decreasing SOD1 aggregates and improving motor dysfunction in SOD1-mutant mice (Motoi et al., 2014). Consistently, trehalose, a disaccharide that induces mTor-independent autophagy, was demonstrated to inhibit TDP-43 aggregate formation (Wang et al., 2010), whereas the activation of mTor-dependent autophagy by rapamycin also rescued motor dysfunction in TDP-43 transgenic mice (Wang et al., 2012). Not only misfolded protein aggregation but also mitophagy failure contributes to ALS pathology. Diseaseassociated mutants of OPTN, a key molecule in mitophagy, results in the failure to remove damaged mitochondria, as well as misfolded protein aggregates, and contribute to the neurodegeneration in ALS (Wong and Holzbaur, 2014). Despite the widespread evidence regarding the therapeutic outcomes of autophagy induction in diseased neurons, the role of autophagy is more complex, because n-butylidenephthalide (n-BP), an extract from Angelica sinensis, inhibits neuronal autophagy by activating the Akt/mTOR signaling but attenuates neuronal loss and improves motor function (Hsueh et al., 2016). Therefore, autophagy activators do not always show positive effects on ALS progression. The manner in which autophagy is induced is key to its protective or destructive effects in ALS. Studies to further our understanding of the autophagy pathway and its utility as a promising therapeutic strategy for ALS are important.

## POLYGLUTAMINE DISEASES

PolyQ diseases, including Huntington disease (HD), spinal and bulbar muscular atrophy, dentatorubral-pallidoluysian atrophy, spinocerebellar ataxia (SCA) types 1, 2, 3, 6, 7, and 17, and HD-like 2 are inherited progressive neurodegenerative diseases elicited by expansions of CAG triplet repeats within specific genes. PolyQ tracts in mutant proteins, i.e., mutant Huntingtin (Htt) in HD, encoded by the CAG repeat, are responsible for the formation of toxic oligomers and aggregates. The length of the polyQ tract is crucial, because longer tracts are more prone to form aggregates. These mutant proteins accumulate and form intracellular inclusions, and previous studies showed that autophagy degrades not only these inclusions but also soluble mutant proteins. Importantly, the degradation of mutant proteins was shown to decrease neuronal damage and improve neurodegenerative deficits in mouse models of the polyQ diseases.

Recent studies have increasingly focused on the perturbation of autophagy in the polyQ diseases, such as that observed in striatal neurons in HD mouse models and lymphocytes in HD patients. In HD cells, cargo recognition was shown to be significantly impaired due to the failure of p62 interaction with mutant Htt, which leads to the inefficient selective degradation of mutant Htt (Fu et al., 2017). Moreover, because the accumulated mutant Htt can potentially trap and inactivate Beclin 1 (Shibata et al., 2006), autophagy is significantly suppressed at the autophagosome-lysosome fusion stage. Mutant Htt also interacts with and inactivates Ras homolog enriched in striatum (Rhes), an autophagy regulator (Mealer et al., 2014). Rhes accelerates autophagy by inhibition of the interaction of Beclin 1 with Bcl-2. The inhibition of autophagy leads to the further accumulation of mutant Htt and neuronal injury. Consistently, in SCA7 mutant patients and mice, the accumulation of p62 and ubiquitin were observed, indicating the impairement of autophagic activity (Alves et al., 2014). Furthermore, the androgen receptor with an expanded polyQ repeat was shown to interact and attenuate the function of TFEB (Song et al., 2013; Cortes et al., 2014). Although studies suggesting that autophagic activity is altered in mouse models of polyQ disease are controversial, an impairment of autophagy is expected to occur in many of the neurons with polyQ accumulation.

PolyQ disease pathogenesis is strongly affected by neuronal autophagic activity. Therefore, the activation of autophagy can potentially prevent the accumulation of aggregateprone proteins, thus inhibiting or slowing the progression of neurodegeneration. Upregulating autophagy with rapamycin reduced the formation of aggregates and cytotoxicity in cells in vitro and in a fly model of HD in vivo (Ravikumar et al., 2002, 2004). CCI-779, a rapamycin analog, also improved behavioral and motor performance in mouse models of HD and SCA3 (Ravikumar et al., 2004). Other autophagy inducers, such as berberine, metformin, rilmenidine, and trehalose also activate autophagy, decrease polyQ aggregation, and improve behavioral performance in polyQ disease mouse models (Tanaka et al., 2004; Ma et al., 2007; Rose et al., 2010; Chen Z. Z. et al., 2015; Jiang et al., 2015). Metformin is an antidiabetic drug that activates AMPK-dependent autophagy (Poels et al., 2009), and rilmenidine is used for hypertension and activates mTor-independent autophagy (Rose et al., 2010). Both drugs are already used clinically in humans. In contrast, in a mouse model of SCA3, the combination of CCI-779 with LiCl showed no beneficial effects, but rather toxic effects (Duarte-Silva et al., 2016). Clinical trials have been performed to assess their utility in neurodegenerative diseases.

### ALZHEIMER DISEASE

AD is a chronic neurodegenerative disease that causes memory and cognitive deficits. AD is characterized by the accumulation of neurotoxic extracellular beta-amyloid (Aβ) plaques and hyperphosphorylated tau in intracellular neurofibrillary tangles (NFTs) in the brain. Aβ is generated from amyloid precursor protein (APP) by two cleavage events. Aβ homeostasis in the brain is believed to play a key role in the progression of neurodegeneration, and several studies demonstrated that various clearance mechanisms play pivotal roles in the development of AD. However, the role of autophagy in AD pathogenesis remains controversial. Numerous studies have reported the degradation of Aβ and APP by autophagy and the reduction of Aβ expression by the upregulation of autophagy, similar to that observed with other proteins associated with neurodegenerative diseases. However, some studies have reported the requirement of autophagy in AD pathogenesis. These contradictory findings stem from studies showing that Aβ generation was suppressed following the inhibition of autophagy by 3-methyl adenine (Zheng et al., 2011). Furthermore, this idea is supported by the presence of APP and Aβ in purified autophagic vacuoles from brains of AD patients. In these vacuoles, presenilin-1 (PS-1), an enzyme that cleaves APP, is highly enriched. Autophagy was also shown to contribute to Aβ secretion, a crucial event in AD pathogenesis. Delivery of Aβ to the extracellular space and plaque formation were significantly reduced due to the lack of Atg7 in APP transgenic mice, and restoration of autophagy restored Aβ secretion to normal levels (Nilsson et al., 2013).

The downregulation of autophagy was morphologically and genetically demonstrated in AD brains. Morphologically, numerous immature autophagic vacuoles were observed in dystrophic neurites of AD patients, suggesting the impairment of trafficking of autophagic vacuoles or autophagosome-lysosome fusion (Nixon et al., 2005). Genetically, Beclin 1, a major player in autophagy, was identified as a causative molecule in AD pathology. AD brains were reported to have decreased expression levels of Beclin 1 early in the disease process (Pickford et al., 2008). A reduction in Beclin 1 levels was shown to attenuate autophagic activity, which likely facilitates the proteolytic cleavage of APP. Another factor that was shown to be associated with AD is phosphatidylinositol-binding clathrin assembly protein (PICALM), which plays a role in autophagy (Tian et al., 2013) by the trafficking of soluble NSF attachment protein receptors. PICALM was also reported to play a role as an autophagy receptor that sequesters APP to LC3-positive autophagosomes (Tian et al., 2014). Genetic studies showing a reduction in PICALM indicate that not only bulk autophagy but also selective autophagy for APP might be suppressed in AD brains. Mutations in PS-1 cause familial AD via the alteration of APP processing. However, recently, another pathological role for mutant PS-1 was proposed, which includes inhibition of the maturation of the lysosomal v-ATPase that leads to an increase in lysosomal pH and a consequent reduction in autophagic cargo elimination (Lee et al., 2010). This process might be mediated by the failure of the ER chaperone function of PS-1, which facilitates the proper assembly of the V0a1 subunit of the v-ATPase.

Whereas the role of autophagy in Aβ generation remains controversial, various autophagy-related compounds were tested for potential therapeutic utility. Lithium and berberine, which are both autophagy inducers targeting the Ulk1-pathway, were demonstrated to induce autophagy and exert neuroprotective effects through the regulation of APP processing and a reduction in Aβ levels in the CRND8 transgenic mouse model of AD (Fiorentini et al., 2010; Durairajan et al., 2012). Both trehalose and carbamazepine were shown to exert protective effects on AD mice via mTor-independent autophagy (Du et al., 2013; Li et al., 2013). Carbamazepine was shown to significantly enhance autophagic flux in the APP(swe)/PS-1(deltaE9) AD mouse model and reduce the cerebral amyloid plaque burden and Aβ 1–42 levels (Li et al., 2013). Latrepirdine stimulates Atg5-dependent autophagy and reduces the accumulation of toxic Aß aggregates in AD mice (Steele et al., 2013). Rapamycin and SMER28, a small-molecule rapamycin enhancer, both activate autophagy and decrease the expression levels of Aß 1–42 (Spilman et al., 2010; Tian et al., 2011), which is the most amyloidogenic form of Aß, in PD-APP transgenic mice, one of the earliest mouse models of AD. Taken together, these findings suggest that, although the exact pathological role of autophagy in AD remains to be elucidated, autophagy inducers might provide a new effective therapeutic strategy by degrading aggregates in the early stages of AD. In contrast, the activation of autophagy might enhance disease severity during the late stages of AD, by accelerating Aβ production.

# TAUOPATHIES

Tau is a microtubule-binding protein that attaches to and stabilizes microtubules. Tauopathies are a constellation of neurodegeneration diseases, including AD, frontotemporal lobar degeneration (FTLD), progressive supranuclear palsy (PSP), and corticobasal degeneration (CBD), in which hyperphosphorylated tau accumulates in intracellular tangles and extracellular Aβ deposits. The phosphorylation of tau by glycogen synthase kinase 3β facilitates its accumulation within the cytosol (Sperber et al., 1995). Similar to that observed with other proteins implicated in neurodegenerative diseases, the inhibition of autophagy, such as that activated by chloroquine, causes a delay in tau clearance and aggregation (Hamano et al., 2008). Furthermore, phosphorylated tau accumulation is higher in brain-specific Atg7 knockout mice than control mice (Inoue et al., 2012), indicating that phosphorylated tau is a substrate of conventional autophagy. Consistently, phosphorylated tau colocalizes with LC3 and p62 proteins in postmortem brain specimens from patients with AD, PSP, and CBD, likely because these proteins are engulfed into autophagosomes but are not degraded by autolysosomes (Piras et al., 2016). These studies suggest that an impairment of autophagy plays a crucial role in the progression of tauopathies, and that autophagy facilitates the degradation of phosphorylated tau to maintain it at low levels.

Based on these findings, the induction of autolysosome formation appears to be a good therapeutic strategy for the tauopathies. Indeed, the activation of autophagy using rapamycin or temsirolimus, an analog of rapamycin, was shown to alleviate tau phosphorylation in neurons and to improve the progression of tau pathology in P301S mutant human tau transgenic mice (Ozcelik et al., 2013; Jiang et al., 2014). Furthermore, trehalose, and methylthioninium chloride were shown to reduce tau aggregates and improve neuronal survival in several mouse models of the tauopathies (Congdon et al., 2012; Schaeffer et al., 2012). Finally, metformin, which inhibits tau hyperphosphorylation via the inhibition of TORC1, is currently being tested for its utility against AD in several clinical trials (Kickstein et al., 2010).

## PARKINSON DISEASE

PD is the second most common neurodegenerative disease following AD. The main underlying pathology of PD is functional failure and subsequent death of dopaminergic neurons in the substantia nigra, which leads to muscle rigidity, bradykinesia, resting tremor, and even neurocognitive impairment. Many of the dopaminergic neurons in PD patients contain round, cytoplasmic inclusions called Lewy bodies that are comprised of α-synuclein and ubiquitin-positive proteins. Most PD cases are sporadic, and 5–10% are familial, due to mutations in various PDassociated genes that encode for PD-associated proteins (PARK).

One subtype of familial PD arises from the A53T mutation in α-synuclein, which is encoded by PARK1. The level of α-synuclein in neurons is a key to neurotoxicity in PD; therefore, efficient α-synuclein degradation is a crucial determinant of PD severity. Findings from recent studies indicate that deubiquitinated α-synuclein is the main target of autophagy (Rott et al., 2011), and that degradation of stress-increased, rather than basal α-synuclein was crucial in a transgenic mouse model (Wu et al., 2016). In contrast, inclusions containing α-synuclein were shown to reduce autophagic activity during the maturation of autophagosomes and their fusion with lysosomes (Button et al., 2017). Furthermore, α-synuclein itself was also reported to suppress autophagy by the induction of Atg9 mislocalization, which was observed in α-synuclein transgenic mice (Winslow et al., 2010). Interestingly, similar Atg9 mislocalization was observed in patients with the D620N mutation of vacuolar protein sorting-associated protein 35 (Vps35; PARK17) (Zavodszky et al., 2014). Atg9 is a Golgi-localizing protein, whereas Vps35 regulates various Golgi proteins by affecting the retromer complex. Therefore, the Vps35 mutant fails to appropriately localize Atg9 and leads to impaired autophagy.

Leucine-rich repeat serine-threonine protein kinase-2 (LRRK2; PARK8) is another PD-causing gene. LRRK2 colocalizes with Lewy bodies in some PD patient brains.  Loss of LRRK2 causes the accumulation of α-synuclein phosphorylated on Ser129 and the impairment of protein degradation pathways, leading to apoptotic cell death in aged mice (Tong et al., 2010). The G2019S LRRK2 mutation is found not only in familial PD patients but also in 1–2% of patients with sporadic PD (Goldwurm et al., 2005). LRRK2 was suggested to suppress autophagy, because silencing of LRRK2 or inhibition of its kinase activity was shown to increase autophagic flux (Alegre-Abarrategui et al., 2009).

Loss-of-function mutations in the P-type ATPase ATP13A2 (PARK9) causes early-onset PD (Ramirez et al., 2006). Because lysosomal ATPase is essential for the maintenance of lysosomal pH and autophagosome-lysosome fusion, mutations in ATP13A2 impair these processes, resulting in the accumulation of autophagosomes. A defect in autophagy accelerates the accumulation of α-synuclein, thereby worsening the disease.

As described above, autophagy contributes to the suppression of various neurodegenerative processes by degrading unfolded proteins. In addition, autophagy functions to inhibit certain PD types by degrading damaged mitochondria. Among the genes associated with familial PD, Parkin (PARK2) and Pink1 (PARK6) eliminate damaged mitochondria by autophagy (Clark et al., 2006; Park et al., 2006). This mechanism, termed mitophagy, starts with the accumulation of Pink1 on the outer mitochondrial membrane from the cytosol following mitochondrial depolarization. Next, Parkin, a cytosolic E3 like ligase, associates with Pink1 on the outer mitochondrial membrane, which leads to ubiquitination of depolarized mitochondria by the ubiquitin ligase activity of Parkin and recruitment of several autophagy components, including p62 (Geisler et al., 2010). Finally, ubiquitinated mitochondria are recognized by autophagic molecules and are digested by autophagy. Thus, genetic mutations in Parkin and Pink1, which cause early-onset familial and sporadic PD, fail to eliminate damaged mitochondria, which culminates in the degeneration of dopaminergic neurons. Importantly, Parkinmediated mitophagy is significantly suppressed by the lack of Atg5 (Huang et al., 2011).

Based on these data, reduction of α-synuclein aggregation and the induction of mitophagy by autophagy might be useful therapeutic approaches against PD. In cell-based assays, several polyphenols, including resveratrol demonstrated autophagyinducing activity and might be useful as therapeutic agents for PD. Kaempferol and celastrol also prevent rotenone-induced SH-SY5Y cell loss via autophagy activation (Filomeni et al., 2012; Deng et al., 2013). Furthermore, microencapsulated rapamycin improved motor function in mice overexpressing α-synuclein (Bai et al., 2015). Oral administration of trehalose also activated autophagy and suppressed insoluble α-synuclein accumulation (Tanji et al., 2015), whereas nilotinib, a receptor tyrosine kinase inhibitor approved for chronic myelogenous leukemia, was also shown to inhibit the protein phosphatase PP2A and induce AMPK phosphorylation and autophagy, resulting in modulation of the neuroimmune response to α-synuclein expression in mice (Yu et al., 2013).

# CLOSING REMARK

In this review, we described two distinct autophagic pathways, namely, conventional and alternative autophagy, and compared their characteristics. We also described the involvement of conventional autophagy in the progression of many neurodegenerative diseases. However, the association between alternative autophagy and neurodegenerative diseases has not been fully clarified to date. In this review, we also summarized the potential uses of conventional autophagy for the treatment of various neurodegenerative diseases. Given that the induction of autophagy can potentially mitigate disease severity, various autophagy-inducing compounds have been developed and evaluated in various rodent models of the neurodegenerative diseases. However, most compounds have multiple functions, and hence it is difficult to confirm that their therapeutic effects are a result of autophagy. Detailed analysis using rodent models are expected to clarify the effect of autophagy-inducing therapies on neurodegenerative diseases.

# AUTHOR CONTRIBUTIONS

NF, MS contributed to the design of this review. SS described the manuscript.

# FUNDING

This study was supported in part by a Grant-in-Aid for Grant-in-Aid for Scientific Research (A) (17H01533 and 17H06414), Grant-in-Aid for challenging Exploratory Research (16K15230), Grant-in-Aid for Scientific Research on Innovative Areas (17H05691), Grant-in-Aid for Scientific Research (C) (16K09668) from the MEXT of Japan, by the Project for Cancer Research and Therapeutic Evolution (P-CREATE) and by the Project for Psychiatric and Neurological Disorders from the Japan Agency for Medical Research and Development, AMED. This study was also supported by the Joint Usage/Research Program of Medical Research Institute, Tokyo Medical and Dental University.

### REFERENCES

**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 © 2018 Fujikake, Shin and Shimizu. 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 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.

# DnaJ/Hsp40 Family and Parkinson's Disease

Takafumi Hasegawa\*, Shun Yoshida, Naoto Sugeno, Junpei Kobayashi and Masashi Aoki

*Division of Neurology, Department of Neuroscience and Sensory Organs, Tohoku University Graduate School of Medicine, Sendai, Japan*

Parkinson's disease (PD) is the second most common devastating neurodegenerative disorder after Alzheimer's disease. The precise molecular and cellular basis underlying PD still remains uncertain; however, accumulating evidence suggests that neuronal cell death is caused by a combination of environmental and genetic factors. Over the previous two decades, more than 20 genes have been identified as the cause of and/or risk for PD. Because sporadic and familial forms of PD have many similarities in clinical and neuropathological features, common molecular pathways, such as aberrant mitochondrial and protein homeostasis, are likely to exist in both conditions. Of the various genes and proteins involved in PD, the versatile DnaJ/Hsp40 co-chaperones have attracted particular attention since several genes encoding this protein family have been successively identified as the cause of the familial forms of PD/Parkinsonism. In this review, we will introduce the current knowledge regarding the integratory and modulatory effect of DnaJ/Hsp40 in various cellular functions and argue how the failure of these proteins may initiate and/or facilitate of the disease.

### Edited by:

*Naruhiko Sahara, National Institute of Radiological Sciences (NIRS), Japan*

### Reviewed by:

*Takashi Nonaka, Tokyo Metropolitan Institute of Medical Science, Japan Chiara Zurzolo, Institut Pasteur, France*

\*Correspondence:

*Takafumi Hasegawa<EMAIL_ADDRESS>
### Specialty section:

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

Received: *21 October 2017* Accepted: *20 December 2017* Published: *10 January 2018*

### Citation:

*Hasegawa T, Yoshida S, Sugeno N, Kobayashi J and Aoki M (2018) DnaJ/Hsp40 Family and Parkinson's Disease. Front. Neurosci. 11:743. doi: 10.3389/fnins.2017.00743* Keywords: DnaJ protein, Hsp40, co-chaperones, Parkinson's disease, neurodegeneration

# INTRODUCTION

Newly synthesized proteins are transported to the destination site where they exert distinct functions; however, some proteins fail to properly fold, and misfolded proteins tend to form aggregates that may be potentially harmful to cells. To combat these perpetual threats, cells have evolved dexterous quality control mechanisms that facilitate degradation by the ubiquitinproteasome system and autophagy-lysosome pathway or refolding of misfolded proteins to normal tertiary structures by molecular chaperones (Oshima et al., 2016; Ciechanover and Kwon, 2017). The most well-known molecular chaperones are heat shock proteins (HSPs), which were named after the phenomenon of puffing in larval salivary gland chromosomes of Drosophila following heat exposure (Pauli et al., 1992). Heat shock and numerous noxious stimuli, including UV, oxidative stress, hypoxia, osmotic stress, and heavy metals, induce the expression of HSPs, which is considered as a fundamental biological defense mechanism (Piano et al., 2004). The family of HSPs are evolutionarily conserved proteins across species and are classified based on their molecular weights; different HSPs have distinct yet partially overlapping functions. For example, Hsp90, which is known to be one of the most abundantly expressed proteins (1–2% of total soluble proteins in an unstressed condition) in the cytosol, forms a complex with its client proteins, such as cell surface receptors, transcription factors, and protein kinases, to modulate their functions (Schopf et al., 2017). Thus, Hsp90 plays pivotal roles in various cellular processes, such as proliferation, differentiation, carcinogenesis, and neurodegeneration. Several Hsp90 inhibitors, including the geldanamycin and its analogs demonstrate anti-tumor effects in various cellular and animal models (Soga et al., 2013). The Hsp70 family is the eukaryotic homolog of the bacterial molecular chaperone DnaK and is widely expressed in various tissues and organs. The members of the Hsp70 family are characterized by their expression levels, activities, and subcellular distribution (Zuiderweg et al., 2017). Under physiological conditions, Hsp70 mainly resides in the cytoplasmic space, whereas in response to stress, it is upregulated and relocates from the cytosol to the nucleus and nucleoli. The heat shock cognate 70 (Hsc70) is, on the other hand, defined by its constitutive expression and cytoplasmic localization. Another Hsp70 member, BiP (also referred to as a 78-kDa of glucose-regulated protein: GRP78), expresses in the endoplasmic reticulum (ER) and acts as a chaperone for secreted or membrane proteins (Dudek et al., 2009). Hsp70 binds and shields the hydrophobic peptides of client proteins in an ATP-dependent manner, thus preventing protein aggregation and proper folding (Zuiderweg et al., 2017). Hsc70 chaperone mediates the lysosomal targeting of substrates with a KFERQlike sequence (i.e., chaperone-mediated autophagy; Cai et al., 2015). Furthermore, Hsp70 is involved in the intracellular traffic of membranes, including the involvement in regulation of endocytosis and exocytosis mechanisms (Goldfarb et al., 2006).

The complex and multifaceted functions of Hsp70 members are achieved by the assistance of their Hsp40/DnaJ co-chaperones (Qiu et al., 2006; Gorenberg and Chandra, 2017). Hsp40 family proteins were originally identified as mammalian homologs of bacterial DnaJ proteins and are subdivided into three distinct groups, i.e., DnaJA, DnaJB, and DnaJC. Structurally, DnaJ proteins consist of 4 functional domains, namely, an evolutionarily conserved J domain with a stretch of 70 amino acid residues, a glycine/phenylalanine-rich domain (G/F-rich domain), a zinc finger domain that contains CXXCXGXG motifs, and a cysteine-rich region (Qiu et al., 2006). In DnaJA and DnaJB proteins, the J domain is always located at the Nterminus which is followed by the G/F-rich domain. The DnaJC family usually lacks the G/F domain and the cysteine-rich region, and the J domain may be located at any location in the entire sequence. Through the intermediary of the J domain, Hsp40 binds to Hsp70 and promotes its ATPase activity, thus generating the ADP-bound Hsp70, which stably interacts with client proteins (**Figure 1**; Greene et al., 1998). Both the G/F-rich domain and c-terminal region of Hsp40/DnaJ proteins are likely to be important in their substrate recognition (Perales-Calvo et al., 2010). Although the Hsp40/DnaJ families are ubiquitously expressed, widespread distribution is also present in the central and peripheral nervous systems (Gorenberg and Chandra, 2017). Furthermore, recent genetic and biological studies suggest that Hsp40/DnaJ family genes/proteins directly or cooperatively influence on the initiation of familial Parkinson's disease (PD) and other inherited forms of parkinsonism (**Figure 2** and **Table 1**; Gorenberg and Chandra, 2017; Hasegawa et al., 2017; Puschmann, 2017). It remains obscure how the different mutated genes could result in the progressive loss of striatal dopaminergic innervation as well as the death of nigral neurons. Nevertheless, some relationships, involving the perturbation of cellular systems, now appear to be apparent. The affected systems include synaptic transmission, endosomal trafficking,

FIGURE 1 | Model of chaperone-assisted protein folding by Hsp70-DnaJ/Hsp40 complex. DnaJ/Hsp40 transiently associates with unfolded protein (substrate) for delivery of the substrate to Hsp70. Through the intermediary of the J domain, Hsp40 binds to Hsp70 and promotes its ATPase activity, thus generating the ADP-bound Hsp70, which stably interacts with client protein. After nucleotide exchange, the substrate is released from Hsp70 and leaves the cycle as a correctly folded protein.

protein-quality control, and/or mitochondrial systems. In this review, we will summarize the current understandings regarding the functional roles of Hsp40/DnaJ co-chaperones in the familial forms of PD/parkinsonism and discuss how these molecules may influence on the pathological consequence of this disease.

*RME-8, receptor-mediated endocytosis-8; JDP1, J domain-containing protein; CSP*α*, cysteine string protein* α*; AD, autosomal dominant; AR, autosomal recessive; PSP, progressive supranuclear palsy; PET, positron emission tomography; MRI, magnetic resonance imaging; LB, Lewy body; n.a., not available.*

### DNAJC13/RME-8

DNAJC13 is a human homolog of receptor-mediated endocytosis 8 (RME-8), which was originally identified in a genetic screening for mutants defective in the endocytic uptake of yolk protein in nematodes (Zhang et al., 2001). Recently, a p.N855S missense mutation of DNAJC13 was reported in a large Canadian Mennonite PD pedigree with Dutch-German-Russian ancestry (Appel-Cresswell et al., 2014; Vilariño-Güell et al., 2014). The pathogenicity of the N855S mutant is supported by segregation with disease, lack of healthy controls with mutation, and the high level of sequence conservation across species; however, genetic screening in a Caucasian series consisting of 1,938 patients with clinically diagnosed PD and 838 pathologically proven Lewy Body Disease failed to detect any coding variant in exon 24 containing Asn855 in DNAJC13 gene, indicating that mutations in this exon are not a common cause of PD or LBD among Caucasian populations (Lorenzo-Betancor et al., 2015). Subsequent genotyping in a Canadian cohort indicated that missense mutations, such as p.P336A, p.V722L, p.R1266Q, and P.T1895M, are only identified in affected members, and p.E1740Q, and p.L2170W may be associated with the risk variants for PD (Gustavsson et al., 2015). Clinically, DNAJC-linked PD (subsequently designated as PARK21) manifests as slowly progressive, late-onset, doparesponsive typical parkinsonism with an autosomal dominant inheritance (Vilariño-Güell et al., 2014). Of note, the N855S mutation in DNAJC13 gene has also been observed in patients with essential tremor, a monosymptomatic disorder characterized exclusively by postural- or action-type tremor, suggesting heterogeneity in the clinical manifestation with this mutation (Rajput et al., 2015). Neuropathological examination in three N855S mutation carriers indicated a brainstem or transitional type of Lewy body pathology (Vilariño-Güell et al., 2014). DNAJC13/RME-8 is a large protein with a high molecular mass of 254 kDa; it is widely expressed and is abundant in

nervous tissue. Structurally, it consists of four conserved IWN repeats, which are characterized by seven invariant residues, including isoleucine, tryptophan and asparagine, and a J domain in the center region (Fujibayashi et al., 2008). An in vitro pulldown assay demonstrated that the J domain of RME-8 binds to Hsc70 in the presence of ADP (Chang et al., 2004). Cell biological studies have demonstrated that DNAJC13 resides in the endosomal membrane and interacts with a family with sequence similarity 21 (FAM21) tail in the Wiskott–Aldrich syndrome protein and Scar homolog (WASH) complex and sorting nexin 1 (SNX1), thereby controlling the formation of the tubular structure on the endosome surface where retromermediated cargo transport occurs (Freeman et al., 2014). From a functional point of view, DNAJC13 plays important roles in the cell surface recycling and lysosomal degradation of transferrin and EGFR, as well as the retrograde transport of cation-independent mannose phosphate receptor (CI-MPR; Girard et al., 2005; Popoff et al., 2009; Shi et al., 2009; **Figure 3**). The over-expression of p.N855S mutant, but not wild-type, in cultured cells leads to aberrant retention of transferrin in endosomes (Vilariño-Güell et al., 2014), which indicates that a PD-linked DNAJC13 mutation may confer a toxic gain-of-function and hampers endosomal cargo trafficking. Intriguingly, immunofluorescence and co-immunoprecipitation studies demonstrate that vacuolar protein sorting 35 (VPS35), a vital element of retromer that is established as a causal gene for PARK17 PD, is co-localized with DNAJC13 in a primary cortical neuron culture obtained from mice (Vilariño-Güell et al., 2014). Further studies using appropriate animal models are required to better understand the precise molecular mechanisms by which DNAJC13 mutation leads to the dopaminergic degeneration with Lewy body pathology. In an independent study, Deng et al. identified loss-of-function mutations in TMEM230 gene in the same Canadian Mennonite group that Vilariño-Güell and his colleagues previously identified mutations in DNAJC13 (Deng et al., 2016). The TMEM230 transcript localizes to the secretory and recycling vesicle in the neuron and may be involved in synaptic vesicle trafficking and recycling. Another study has shown that TMEM230 is required for Rab8a-mediated transport of secretory vesicles and retromer-mediated cargo trafficking (Kim et al., 2017). Further discussion and reassessment are needed to understand this complex situation.

### DNAJC6/AUXILIN-1

In 2012, deleterious (c.801-2 A>G) and truncating (p.Q734X) mutations in the DNAJC6 gene were discovered in two consanguineous families with Palestinian and Turkish origins, respectively (Edvardson et al., 2012; Koroglu et al., 2013). The clinical phenotypes of DNAJC6-linked familial parkinsonism (termed PARK19) are characterized by a juvenile-onset, progressive parkinsonism, which includes bradykinesia, muscle

FIGURE 3 | DNAJC13/RME-8. At the surface of early endosome, DNAJC13 interacts with FAM21 tail in the WASH complex and SNX1, thereby controlling the formation of tubular structure on endosome surface where a retromer-mediated cargo transport (e.g., CI-MPR) occurs. DNAJC13 also play important roles in the recycling and the degradation of transferrin and EGFR, respectively. RME-8, receptor-mediated endocytosis-8; FAM21, family with sequence similarity 21; WASH, Wiskott–Aldrich syndrome protein and Scar homolog; SNX1, sorting nexin 1; EGF, epidermal growth factor; CI-MPR, cation-independent mannose 6-phosphate receptor.

rigidity, resting tremor, hypomimia, and postural instability. In addition to extrapyramidal signs, four members of the Turkish family exhibited hypomentia, pyramidal tract signs and/or seizures (Koroglu et al., 2013). Patients in the Palestinian family had dopa-refractory parkinsonism, whereas affected members in the Turkish family showed a favorable response to levodopa replenishment therapy. Subsequent genetic studies identified 2 different homozygous mutations in the DNAJC6 gene, i.e., a missense mutation (p.R927G) and a putative splice site mutation (c.2223A-T; Olgiati et al., 2016). These families accounted for 2 (2.2%) of 92 probands (mean age at onset: 54.65; range 19–84) with autosomal recessive PD who underwent DNAJC6 genetic screening. These patients showed milder phenotype compared to the patients with truncating mutations probably due to some residual activity of DNAJC6. Another novel non-sense mutation (exon 16, p.Q789∗ ) has also been reported in a consanguineous family with juvenile-onset PD (Elsayed et al., 2016). Of interest, a patient in this family manifested rigid-akinetic form of parkinsonism with visual hallucinations, which are frequently observed non-motor symptom in sporadic PD and dementia with Lewy bodies (Shoji et al., 2014). Because there is variation in the responsiveness of dopamine-replacement therapy, the term "parkinsonism" rather than PD would be suitable in DNAJC6-linked movement disorder. The DNAJC6 encodes the neuron-specific isoform of the co-chaperone auxilin-1, which has a crucial role in the detachment of the clathrin-coat after clathrin-mediated endocytosis (CME, **Figure 4**). Auxilin-1 is a 100 kDa protein and structurally consists of 3 distinct domains: the N-terminal phosphoinositide phosphatase (PTEN) like domain (residue 40–421), which is required for the recruitment to a clathrin-coated pit, the central clathrin-binding domain, and a C-terminal J domain, which enables its interaction with Hsc70 (Lee et al., 2006). The c.801-2 A>G homozygous splice-site mutation in DNAJC6 yields two misspliced abnormal transcripts that lack either a significant part of the J domain or the PTENlike domain, which indicates that this pathological mutation results in the defect of functionally active auxilin-1 (Edvardson et al., 2012). The homozygous mutation p.Q734X deletes the 180 amino acid residues of Auxilin-1 at the C-terminus, which are considered important for supporting Hsc70 function in clathrin dissociation from coated vesicles (Koroglu et al., 2013). Moreover, an in silico structural analysis predicted that that the p.R927G missense mutation in the J domain reduces the positive charge on the protein surface, which would be involved in the molecular interaction of auxilin-1 with Hsc70 or other partners (Olgiati et al., 2016). Interestingly, common variants in cyclin-Gassociated kinase (GAK), which encodes auxilin-2/DNAJC26, a ubiquitously expressed paralog of auxilin, are also considered a risk factor for idiopathic PD (Pankratz et al., 2009). Auxilin-null mice exhibited a high mortality rate in early life; however, they did not exhibit no obvious neuropathological alterations in the midbrain (Yim et al., 2010). These mice showed higher GAK levels in the brain; however, this compensation appeared to be insufficient for the lack of auxilin in clathrin uncoating since they exhibited an aberrant retention of clathrin-coated vesicles and empty clathrin cages at the synaptic terminals, which may be attributed to the parkinsonian phenotypes. Further investigation using genetically modified animal models is warranted to better understand the role of DNAJC6 in synaptic dopamine function.

### DNAJC12/JDP1

DNAJC12, a member of the Hsp70/Hsc70 co-chaperone proteins, has the J domain and a highly conserved C terminus; however, it lacks other canonical domains present in DnaJ family proteins. This protein is a mammalian ortholog of the J domain-containing protein (JDP) originally identified in Drosophila and is thus termed JDP1 (Hahn et al., 1999). In mammals, two transcript variants of DNAJC12/JDP1 have been identified: the primary isoform is 1.2 kb and encodes 198 amino acids, and the other isoform is 0.7 kb and is composed of 107 amino acids. Northern blot analysis showed that the major transcript of mouse JDP1 is ubiquitously expressed in various organs, including the brain (Hahn et al., 1999). Recently, biallelic mutations [del. 6943 (c.298-968\_503-2603del) and p.R72P] of the DNAJC12 gene have been identified as causes for phenylketonuria (PKU), the most frequent inherited metabolic disorder (Anikster et al., 2017). Segregation analysis confirmed autosomal-recessive inheritance in all families. Furthermore, a subsequent study of the genotypephenotype correlation in DNAJC12 mutations demonstrated that several homozygous null variants (c.187A>T and c.79- 2A>G) in DNAJC12 present young-onset, dopa-responsive non-progressive parkinsonism as the cardinal symptom and mild cognitive decline without apparent dystonia (Straniero et al., 2017). The autopsy studies of the proband's brain showed depigmentation in the substantia nigra, and no αsynuclein-positive Lewy pathology was observed. Thus, the PKU with the DNAJC12 mutation should be considered as a differential diagnosis of early-onset parkinsonism, since a strict diet therapy with low phenylalanine may prevent the progression of the disease. Most patients with PKU have mutations in a gene that encodes phenylalanine hydroxylase (PAH), a rate-limiting enzyme of the metabolic pathway that degrades excess phenylalanine to tyrosine (Blau, 2016). Patients with PKU do not show any abnormalities at birth; however, if left untreated, the accumulation of phenylalanine and the insufficient level of tyrosine insidiously lead to psychomotor retardation, microcephaly, epilepsy, generalized dystonia, and hypopigmentation in the skin and hair (Al Hafid and Christodoulou, 2015). A limited number (up to 2%) of PKU cases are caused by a deficiency of tetrahydrobiopterin (BH4), a co-factor of PAH and other members of aromatic amino acid hydroxylases (AAAHs), such as tyrosine hydroxylase (TH) and tryptophan hydroxylase (TPH), which are required for the biosynthesis of dopamine and serotonin, respectively (Blau, 2016). Affinity capture-mass spectrometry (AC-MS) and co-immunoprecipitation demonstrated that DNAJC12 interacts with AAAHs including PAH, TH, and peripheral and neuronal isoforms of TPH (Huttlin et al., 2015; Anikster et al., 2017). A molecular interaction between PAH and JDP, the ortholog of DNAJC12 in fruit fly, was also detected in the two-hybridbased protein-interaction datasets of Drosophila (the Biological General Repository for Interaction Datasets, BioGRID; Giot et al.,

2003). Furthermore, analysis of cerebrospinal fluid from affected individuals showed deficiencies of several catecholamines and their metabolites, including dopamine, serotonin, HVA, and 5-HIAA (van Spronsen et al., 2017). These findings suggest the importance of DNAJC12 for proper function of enzymes involved in catecholamine biosynthesis. The c.158-2A>T splice variant and entire deletion in exon 4 (del. 6943) are considered to be null mutants as the DNAJC12 protein was below the detection level in the fibroblasts obtained from individuals carrying these mutations (Anikster et al., 2017). Moreover, the p.R72P missense variant located in the J domain appears to affect the stability of DNAJC12 as the Arg72 residue is well-conserved across species and is indispensable for maintaining the 3D structure of the J domain through the interactions with the Ser25 residue (Anikster et al., 2017).

### DNAJC5/CSPα

DNAJC5, also referred to as cysteine string protein α (CSPα), is a 34 kDa protein and synaptic co-chaperone of the DnaJ/Hsp40 family (Burgoyne and Morgan, 2015). The name originates from the existence of a cysteine string domain that consists of 13–15 heavily palmitoylated cysteine residues within 25 amino acids. Structurally, DNAJC5/CSPα is divided into the following domains: a phosphorylation site for protein kinase A at an N-terminus, a conserved J domain, an adjacent linker region, the cysteine string domain, and a less conserved C-terminal domain (Burgoyne and Morgan, 2015). Mutations (p.L116del and p.L115R) in the cysteine string domain of DNAJC5 have previously been identified as causes for an autosomaldominant, adult-onset neuronal ceroid lipofuscinosis (ANCL, also referred to as Kufs disease), a neurodegenerative disorder characterized by abnormal accumulation of fluorogenic lipids, granular substances not only in the neuronal cells in the brain but also in some other tissues (Noskova et al., 2011; Velinov et al., 2012; Cadieux-Dion et al., 2013). Sanger sequencing revealed that p.L116del mutation was the only indel completely co-segregating with the phenotype in the pedigree. The clinical picture of individuals who carry pathogenic DNAJC5 mutations consists of tonic-clonic and myoclonic epileptic seizure, ataxia, and dementia with premature death. In some cases, patients subsequently began to manifest parkinsonism (Burneo et al., 2003; Cadieux-Dion et al., 2013). The over-expression of EGFPtagged DNAJC5 in the subclone of murine Cath.a differentiated neuronal cell line (CAD5) demonstrated cell surface localization of wild-type DNAJC5, whereas the p.L116del and p.L115R mutant proteins showed diffuse cytoplasmic localization in addition to aberrant accumulation in the ER and Golgi apparatus (Noskova et al., 2011). As expected, these mutants were less palmitoylated than wild-type DNAJC5, which may lead to impaired membrane tethering of DNAJC5/CSPα and a reduced protein level of DNAJC5/CSPα in the brains of affected individuals. Furthermore, p.L116del and p.L115R mutations in the cysteine-string domain cause DNAJC5/CSPα to form high molecular weight SDS-resistant aggregates, which are also present in post-mortem brain tissue from patients, suggesting a cluster of palmitoylated cysteines are essential for aggregation of CSPα (Diez-Ardanuy et al., 2017). In agreement with this, transgenic zebrafish model expressing the human mutant DNAJC5 gene under the control of a zebrafish neuron-specific promoter demonstrated mutant DNAJC5 protein aggregates in the affected neurons (Yao et al., 2017). However, the precise molecular mechanisms by which the mutant DNAJC5 leads to neurodegeneration accompanied by abnormal accumulation of autofluorescence materials in neuronal tissues still remain obscure. Accumulating evidence suggests that the DNAJC5/CSPα behaves as a critical regulator of synaptic proteostasis. Together with a small glutamine-rich tetratricopeptide repeat domain protein (SGT), DNAJC5/CSPα forms an enzymatically active chaperone complex and binds misfolded client proteins on synaptic vesicles, thereby preventing the buildup of misfolded proteins in the nerve terminal (Donnelier and Braun, 2014). For example, the DNAJC5-Hsc70-SGT chaperone complex stabilizes synaptosomal-associated protein 25 (SNAP-25) and facilitates its ability to assemble into the soluble NSF attachment protein receptor (SNARE) complex (Sharma et al., 2011). Intriguingly, the over-expression of α-synuclein, a culprit protein in PD, protects DNAJC5/CSPα-deficient mice from neurodegeneration (Chandra et al., 2005), which suggests that α-synuclein may compensate for the loss of DNAJC5/CSPα in the nervous system. This finding has been corroborated by a recent study showing that DNAJC5/CSPα removes neurodegeneration-related toxic proteins (e.g., abnormally expanded huntingtin and mutant superoxide dismutase 1) from neurons via extracellular vesicles (Deng et al., 2017).

# DNAJC10/ERDJ5

A recent comprehensive analysis of genetic variants in a well-characterized Han-Chinese cohort with sporadic PD demonstrated that the DNAJC10 gene variant rs13414223 decreased the risk of PD (Yuan et al., 2016). The statistical differences in genotypic and allelic frequencies between the PD and the controls were p = 0.004 and 0.002 (odds ratio = 0.652), respectively. The gene of DNAJC10 encodes the 90 kDa ER-resident co-chaperone ERdj5, which is a component of ERassociated degradation (ERAD), a quality-control machinery by which unfolded/misfolded proteins are degraded in eukaryotic cells (Ushioda et al., 2008). The ERdj family comprises five members, and each member contains a conserved N-terminal J domain; ERdj5 is the only member that has thioredoxin (Trx)-like domains with catalytic active CXXC motifs (Cunnea et al., 2003). A KDEL tetrapeptide sequence is present at the C terminus of DNAJC10/ERdj5, possibly mediating the targeting to the ER (Stornaiuolo et al., 2003). Via its reductase activity, DNAJC10/ERdj5 cleaves the disulfide bonds of misfolded proteins and thereby accelerates ERAD through the associations with EDEM (ER degradation-enhancing alpha-mannosidase-like protein) and the ER chaperone BiP/GRP78 (Ushioda et al., 2008). DNAJC10/ERdj5 is ubiquitously expressed across brain areas, including the cerebral cortex, striatum, hippocampus, hypothalamus, cerebellar cortex, and brainstem (Cunnea et al., 2003), which are the anatomical regions of neuronal cell loss and Lewy body formation in PD (Braak and Braak, 2000). Intriguingly, C. elegans DJ-27, an ortholog of mammalian ERdj5, showed a protective effect against aggregate formation, behavior abnormalities, and mitochondrial fragmentation in worm models of human Alzheimer, Parkinson, and Huntington diseases (Munoz-Lobato et al., 2014); these findings suggest that DNAJC5 may counteract protein misfolding/aggregation toxicity in neuronal cells. Studies in higher model organisms, such as vertebrates, would be useful and advantageous to figure out how the DNAJC10/ERdj5 mutation can be involved in the pathogenesis of PD.

# CONCLUDING REMARKS

Although more than 90% of PD cases occur sporadically, both familial and sporadic PD share common pathological features, such as cytoplasmic inclusions and dopaminergic cell loss (Hasegawa et al., 2004; Takeda et al., 2006). Evidence from recent genetic studies in rare familial forms of PD indicates that DnaJ/Hsp40 molecular chaperones are profoundly involved in the pathogenesis of PD. The diverse functions of the DnaJ/Hsp40 family in protein folding/unfolding, membrane trafficking, synaptic modulation and mitochondrial function are considered to not only affect the dopaminergic neurotransmission but also concomitantly influence on the PD-related neuropathological changes, such as nigral cell loss and Lewy body formation. HSPs are known to be localized with noxious proteinaceous aggregates in various neurodegenerative diseases including PD, Alzheimer's disease and prion disease (Matsuzaki et al., 2004; San Gil et al., 2017). Interestingly, several studies have shown that selective upregulation of HSPs such as DnaJ/Hsp40 and Hsp70 prevents clinicopathological progression in a variety of cellular and animal models (Popiel et al., 2012; Gao et al., 2015; Takeuchi et al., 2015). Thus, genetic as well as pharmacological manipulation of specific DnaJ/Hsp40 function may provide beneficial effects on protein and cellular homeostasis in neurodegenerative conditions. Deciphering the precise modes of DnaJ/Hsp40 functions in the pathological cascades in PD will shed light on the pathogenic mechanisms involved and may provide important clues regarding the diseasemodifying strategies for this devastating neurodegenerative disease.

### AUTHOR CONTRIBUTIONS

All authors (TH, SY, NS, JK, and MA) participated in the discussion of the paper. TH and SY mainly wrote the manuscript.

## FUNDING

This work was supported in part by a Grant-in-Aid for Scientific Research on Innovative Areas (Brain Protein Aging and Dementia Control) [grant number 17H05683] and a Grantin-Aid for Scientific Research (C) [grant number 17K09744] from the Ministry of Education, Culture, Sports, Science and Technology, MEXT; the Strategic Research Program for Brain Sciences from the Japan Agency for Medical Research and Development, AMED; Novartis Pharma Research Grants.

### REFERENCES

reticulum, intermediate compartment, and Golgi complex. Mol. Biol. Cell 14, 889–902. doi: 10.1091/mbc.E02-08-0468

**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 © 2018 Hasegawa, Yoshida, Sugeno, Kobayashi and Aoki. 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) or licensor 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.

# ER Dynamics and Derangement in Neurological Diseases

### Tomoyuki Yamanaka\* and Nobuyuki Nukina

*Laboratory of Structural Neuropathology, Graduate School of Brain Science, Doshisha University, Kyoto, Japan*

The endoplasmic reticulum (ER) is a morphologically dynamic organelle containing different membrane subdomains with distinct cellular functions. Numerous observations have revealed that ER stress response induced by disturbed ER homeostasis is linked to various neurological/neurodegenerative disorders. In contrast, recent findings unveil that ER structural derangements are linked to the progression of several neurological diseases. The derangements involve two distinct, and likely opposing pathways. One is dysfunction of ER dynamics machinery, leading to disruption of ER network organization. Another one is facilitation of pre-existing machinery, leading to generation of markedlyordered *de novo* membranous structure. Restoring the ER network can be the effective way toward the cure of ER-deranged neurological disorders.

Keywords: endoplasmic reticulum, ER architecture, ER dynamics, neurological disease, neurodegeneration, NF-Y

### Edited by:

*Gen Sobue, Nagoya University, Japan*

### Reviewed by:

*Corinne Lasmezas, Scripps Florida, United States Smitha Karunakaran, Indian Institute of Science, India*

### \*Correspondence:

*Tomoyuki Yamanaka<EMAIL_ADDRESS>
### Specialty section:

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

Received: *26 September 2017* Accepted: *05 February 2018* Published: *20 February 2018*

### Citation:

*Yamanaka T and Nukina N (2018) ER Dynamics and Derangement in Neurological Diseases. Front. Neurosci. 12:91. doi: 10.3389/fnins.2018.00091* INTRODUCTION

Endoplasmic reticulum (ER) is a continuous membrane organelle dispersing throughout the cells. It consist of two differentially shaped membranous domains; the nuclear envelope, a highly regulated membrane barrier that separates the nucleus from the cytoplasm, and the peripheral ERs including ribosome-studded rough ER (RER) and ribosome-free smooth ER (SER) (**Figure 1**). The RER plays a key role in synthesis and transport of secretary/membrane proteins. The SER is critical for synthesis of lipids / sterols, storage and regulated release of calcium, and metabolism and detoxification. These peripheral ERs are highly dynamic and change their shapes and volumes on the demand of cellular needs (Federovitch et al., 2005; Park and Blackstone, 2010; Westrate et al., 2015).

ER dysfunction causes deleterious effects on the cells and is associated with many diseases including various neurological/neurodegenerative disorders. The major pathway involved in this is ER stress responses induced by disturbance of ER homeostasis due to protein misfolding and aggregation (Matus et al., 2011; Remondelli and Renna, 2017). In contrast, recent findings highlight another ER abnormalities associated with ER morphological alteration in neurological diseases. These are mainly caused by dysregulations of ER-resident membrane proteins. In this review, we discussed the ER dynamics and its derangement in neuropathogenesis.

### Peripheral ER Organization and Dynamics

The peripheral ERs are morphologically subdivided into two domains, ER sheets and tubules (**Figure 1**). The sheets mainly located in perinuclear region and tend to be studded with ribosomes (RER), whereas tubules form cytoplasmic network and are largely devoid of ribosomes (SER). Different sets of proteins are involved in the organization of these distinct ER membrane domains (**Figure 2**; Park and Blackstone, 2010; Chen et al., 2013; Westrate et al., 2015). In the sheets, one of the key regulators is stubbed ribosome, which is elegantly shown by the experiment using two translation inhibitors, puromycin or cycloheximide; the ER sheets are disrupted by puromycin that quickly dissociates polysomes from ER membranes, but not by cycloheximide that stabilizes ribosomal association with translocons, in cultured cells (Puhka et al., 2007). Two transmembrane protein Climp63 and TMEM170A are also shown to be important regulators for RER sheet formation; knockdown of each one decreased ER sheets whereas its overexpression increased them (Shibata et al., 2010; Christodoulou et al., 2016). As for TMEM170A, its localization to nuclear envelopes and regulation of nuclear morphology are also reported (Christodoulou et al., 2016). Thus, the ER sheets are organized by different types of proteins (**Figure 2**).

On the contrary, the ER tubules are generated by a set of specific membrane proteins that contain one or two "hairpin" transmembrane domains. This domain is inserted

ERs are composed of two different subdomains; a ribosome-studded rough ER (RER) and a ribosome-free smooth ER (SER). The RERs form sheets in perinuclear region and play a role in protein synthesis and transport. The SERs form tubule networks in cytoplasm and are involved in lipids synthesis, calcium storage and detoxification.

into outer leaflet of the membrane bilayer and suggested to generate curvature to bend the membrane (**Figure 2**; Park and Blackstone, 2010; Chen et al., 2013; Westrate et al., 2015). The membrane proteins include Reticulons (Rtn1-4), DP1 (deleted in polyposis locus 1; also known as REEP5), other REEPs (receptor expression-enhancing proteins; REEP1-4 and 6), Atlastin-1, M1- Spastin and Lunapark. Among them, Reticulons, DP1 and Atlastin-1 are extensively analyzed and shown to be required for formation of ER tubular network in vitro (Hu et al., 2008; Wang et al., 2016; Powers et al., 2017) and in cultured cells (Voeltz et al., 2006; Hu et al., 2009). These three proteins interact with each other (Hu et al., 2009), and form immobile oligomer complex in the tubules (Shibata et al., 2008; Orso et al., 2009). Complex formation of REEP1 with Atlastin-1 and M1-Spastin and its contribution of ER tubule formation are also reported (Evans et al., 2006; Park et al., 2010). Thus, interconnected interactions of these membrane-inserting proteins mediate the ER tubule formation. Atlastin-1 and Lunapark also induce homotypic ER fusion to generate branched tubules (Orso et al., 2009; Chen et al., 2012, 2015). Binding to microtubule cytoskeleton through REEP1-4 and M1-spastin is further involved in establishment and maintenance of complicated tubular network (Park and Blackstone, 2010; Westrate et al., 2015).

### Disruption of ER Dynamics in Neurological Diseases

Notably, some of the ER tubule proteins have been shown to be mutated in hereditary spastic paraplegia (HSP), a group of genetic disorders caused by a length-dependent, distal axonopathy of corticospinal motor neurons, leading to lower limb spasticity and weakness. Currently, over 40 different genetic loci (SPG1–45) are reported (Blackstone et al., 2011; Lo Giudice et al., 2014). Despite the large number of loci, about 60% of HSP patients harbor pathogenic mutations in one of three proteins: Spastin (SPG4), Atlastin-1 (SPG3A), or REEP1 (SPG31), all of which are the regulators of ER tubule formation as described

above. In addition, another ER tubule protein Rtn2 is also mutated in SPG12 patients. Thus, these SPG proteins are critical for motor neuron integrity in vivo. Indeed, knockdown of Spastin or Atlastin-1 in zebrafish induces axonal degeneration in motor neurons and locomotion defects (Butler et al., 2010; Fassier et al., 2010; Patten et al., 2014). In mice, mutation in Spastin or REEP1 causes progressive axonal degeneration of corticospinal motor neurons and motor defects (Tarrade et al., 2006; Beetz et al., 2013; Renvoise et al., 2016). Furthermore, peripheral ER complexity decreases in primary motor neurons of REEP1 mutant mice (Beetz et al., 2013). These observations suggest that these SPG proteins are necessary for axonal development and/or maintenance probably by modulating ER architecture in motor neurons. Because Spastin, Atlastin-1, and REEP1 are coenriched in axonal growth cones in cultured neurons (Park et al., 2010), they may regulate axonal growth by modulating ER tubule network dynamics.

The other factors involved in the ER tubule formation are Rab GTPases, central regulators of vesicle budding, motility, and fusion (**Figure 2**). Two Rab GTPases, Rab10, and Rab18, are shown to localize to ER tubules and its dysfunction results in ER tubule disorganization in cultured cells (English and Voeltz, 2013; Gerondopoulos et al., 2014). Notably, loss-of-function mutations of Rab18 or its regulator RabGAP cause Warburg Micro Syndrome, a rare autosomal recessive genetic disorder characterized by severe eye and brain abnormalities (Bem et al., 2011). Knockdown of Rab18 in zebrafish causes developmental abnormalities including microphthalmia and microcephaly (Bem et al., 2011). In addition, knockdown of Rab10 or Rab18 induces defects in neuronal differentiation in mouse brain cortex (Wang et al., 2011; Wu et al., 2016). These observations reveal critical roles of these Rab GTPases in neuronal development in vivo.

An ER membrane protein FAM134B is also linked to a neurological disease named hereditary sensory and autonomic neuropathy (HSAN) (Kurth et al., 2009). Interestingly, this protein is shown to be an autophagy receptor to mediate degradation of ER through autophagic system. This phenomenon is called ER-phagy, which is considered to be involved in ER homeostasis (Khaminets et al., 2015). Being its localization mainly to ER sheets, FAM134B is thought to be an ER sheetspecific autophagy receptor. Recently, an ER tubule protein Rtn3 is identified as another ER-phagy receptor, which induce fragmentation and autophagic degradation of ER tubules independently of FAM134B (Grumati et al., 2017). Thus, FAM134B and Rtn3 may regulate different ER membranes to regulate overall ER homeostasis.

## The ER Tubule Network and Neurodegenerative Diseases

The ER tubules are interacting with other membranous organelles such as mitochondria, plasma membrane, peroxisomes and lysosomes. The mitochondria-associated ER membrane (MAM) mediates several fundamental cellular processes including calcium exchange, phospholipid exchange, intracellular trafficking and autophagy. The ER–mitochondria associations through MAM are shown to be disrupted in several neurodegenerative diseases such as Alzheimer's disease (AD), Parkinson's disease and amyotrophic lateral sclerosis with associated frontotemporal dementia (ALS/FTD), resulting in alteration in cellular functions regulated by MAM (Paillusson et al., 2016).

Direct implications of ER tubule proteins in disease pathogenesis have been reported. In superoxide dismutase 1 (SOD1) G93A transgenic mouse, a model for ALS, depletion of Rtn4 accelerates disease onset and progression possibly by disrupting normal distribution of protein disulfide isomerase (PDI), suggesting protective role of Reticulon in motor neuron degeneration (Yang et al., 2009). As for AD, Rtn3 binds to and colocalizes with BACE1, a beta-secretase involved in amyloid precursor protein (APP) cleavage and amyloid beta production. Interestingly, overexpression of Rtn3 reduces the production of amyloid beta while its knockdown enhances it in cultured cells. Furthermore, Rtn3 blocks BACE1 interactions with APP, suggesting that the Reticulon negatively modulates BACE1 activity and amyloid beta production (He et al., 2004). In addition, enhanced expression of Rtn3 suppresses amyloid plaque formation in transgenic mice expressing mutants for APP and presenilin-1 (Shi et al., 2009). In contrast to these beneficial effects, Rtn3 is found to be aggregated and accumulated in dystrophic neurites, named as Rtn3 immunoreactive dystrophic neurites (RIDNs) in brains of AD cases and mice brains expressing mutant APP. Furthermore, Rtn3 transgenic expression impairs spatial learning and memory as well as synaptic plasticity in mice, implying that RIDNs potentially contribute to AD cognitive dysfunction (Hu et al., 2007). Thus, Rtn3 may bidirectionally regulate AD pathogenesis in vivo.

# Biogenesis of Highly-Ordered ERs, the Stacked SERs

In addition to the tubular architecture as described above, the SER is known to form highly-organized membranous structures where cisternae are stacked with ordered arrays (Federovitch et al., 2005; Borgese et al., 2006). This "stacked SER" is observed in the cells highly demanding SER-related functions such as lipid biosynthesis and drug metabolism. These include adrenal cells that produce large amount of sterol lipids, and liver cells of animal treated with phenobarbital (Feldman et al., 1981; Federovitch et al., 2005). Treatment of statins (cholesterol synthesis inhibitors) also leads to stacked SER formation by induced expression of an ER-resident enzyme, hydroxy-methylglutaryl (HMG)-CoA reductase (Singer et al., 1988; Borgese et al., 2006). Interestingly, overexpression of ER resident membrane proteins such as cytochrome b(5), P450, aldehyde dehydrogenase, Sec61 and Calnexin in cultured cells is shown to be sufficient for stacked SER formation (Yamamoto et al., 1996; Snapp et al., 2003; Korkhov and Zuber, 2009). Thus, load of ER-resident membrane proteins is one of the factors for stacked SER biogenesis.

As for the molecular mechanism, selective activation of ATF6 and following lipid synthesis are suggested to be involved in stacked SER generation upon cytochrome b(5)

expression (Maiuolo et al., 2011). Because of no inductions of ER chaperone expression, XBP1 splicing or eIF2-alpha phosphorylation, the usual unfolded protein response (UPR) pathway may not be involved in ATF6-dependent pathway. Knockdown of Syntaxin 18, a SNARE component involved in ER-Golgi transport and ER-network organization, also induces stacked SER formation (Iinuma et al., 2009). Furthermore, knockdown of a membrane protein Yip1A, which cycles between ER and early Golgi, induces formation of stacked SER (Dykstra et al., 2010). A compound phenyl-2-decanoyl-amino-3-morpholino-1-propanol-hydrocholride (PDMP) that blocks membrane transport from ER to Golgi also induces generation of this type of ER (Sprocati et al., 2006). Experiments using several compounds further suggested that altering ionic homeostasis in ER is also an inducer of stacked SER formation independently of known UPR pathways (Varadarajan et al., 2012, 2013). Notably, the stacked SER formation often accompanies Golgi fragmentation and delay of ER export (Iinuma et al., 2009; Dykstra et al., 2010; Varadarajan et al., 2012). These observations suggest close relationship of ER-Golgi transport machineries, rather than canonical UPR pathways, to stacked SER biogenesis.

Despite the drastic alterations of ER and Golgi architectures, there is no report describing distinct reduction of cell viability. Importantly, the ER structure is reversible because depletion of the compounds such as PDMP leads to disappearance of stacked SER (Sprocati et al., 2006; Varadarajan et al., 2012). Thus, the stacked SER formation may not be the toxic inducer for cells but is suggested to be a novel stress response to cope with overload of ER membrane proteins independently of usual UPR pathways.

### Stacked SER Pathologies in Neurological Diseases

Recent observations, however, indicate pathogenic significance of stacked SER in neurological diseases. In familial ALS (ALS8), an ER-resident membrane protein, vesicle-associated membrane protein-associated protein B (VAPB) is mutated (**Figure 3**; Nishimura et al., 2004). Overexpressed mutant VAPB protein in cultured cells is insolubilized and aggregated in ER, leading to formation of ubiquitin-positive inclusions containing stacked SERs (**Figure 3**; Teuling et al., 2007; Fasana et al., 2010). In cultured neurons, the mutant VAPB overexpression induces Golgi fragmentation and cell death (Teuling et al., 2007). Furthermore, in VAPB mutant-transgenic mice ubiquitinpositive inclusions associated with stacked SER are developed in motor neurons (Tudor et al., 2010; Aliaga et al., 2013; Kuijpers et al., 2013; Qiu et al., 2013), and furthermore progressive loss of corticospinal motor neurons is observed in some of mice lines (Aliaga et al., 2013). Although the neurotoxic effect of mutant VAPB is still controversial because other transgenic mice lines do not show neuronal loss or motor phenotypes (Tudor et al., 2010; Kuijpers et al., 2013; Qiu et al., 2013), VAPB mutant knock-in mice are shown to display slow progression of motor behavior defects (Larroquette et al., 2015), suggesting a certain involvement of VAPB mutation on motor neuron dysfunction.

Torsion dystonia-1 (DYT1) is the most common inherited dystonia characterized by involuntary muscle contractions and abnormal postures, which is caused by mutation in TorsinA, an ER glycoprotein belonging to AAA family of proteins (Ozelius et al., 1997). The mutant TorsinA forms cytoplasmic inclusions containing stacked SER in cultured neuronal cells (Hewett et al., 2000; Gonzalez-Alegre and Paulson, 2004). In peripheral nervous system, mutation in peripheral myelin protein 22 (PMP22) is linked to Charcot-Marie-Tooth disease, a sensorineural peripheral polyneuropathy. PMP22 is expressed in Schwann cells and its mutation induces formation of cytoplasmic inclusion containing mutant protein in association with stacked SER formation (Dickson et al., 2002). These observations further provide the pathological significance of cytoplasmic inclusions containing stacked SER in neurological diseases.

A novel mouse model for stacked SER pathology in brain neurons was recently established (Yamanaka et al., 2014, 2016). Importantly, it does not involve disease-associated gene mutation but just caused by inactivation of CCAAT-binding factor NF-Y, a ubiquitous transcription factor shown to be affected in polyglutamine diseases (Yamanaka et al., 2008; Katsuno et al., 2010; Huang et al., 2011). The neuron-specific knockdown of NF-Y induces insolubilization of various membrane proteins including Calnexin, Reticulon, Atlastin-1, APP and Carboxypeptidase E together with ubiquitin and p62/Sqstm1, all of which are accumulated on ER (Yamanaka et al., 2014, 2016). It also accompanies perinuclear accumulation of ribosome-free SERs and Golgi disassembly (**Figure 4**). Chromatin immunoprecipitation identifies several genes involved in protein folding in ER and ER-associated degradation

### REFERENCES

(ERAD) as targets of NF-Y, suggesting a critical role of NF-Y in ER protein homeostasis to maintain normal ER architecture in brain neurons.

### CONCLUSIONS

Among the peripheral ERs, SERs appear to be deranged in several neurological diseases through at least two pathways. One is disturbance of peripheral ER network in the diseases such as HSPs, which is caused by mutations of genes required for SER tubule organization. The SER network disruption may also be involved in Alzheimer's disease and ALS pathogenesis. Another one is development of highly-ordered stacked SER in the diseases such as ALS8, which is caused by overload of misfolded and insolubilized proteins on ER membranes. NF-Ymediated gene regulatory pathway can be involved in this atypical ER pathogenesis. Finding the way to restore SER networks is a future challenging issue toward the cure of these ER-related neurological disorders.

## AUTHOR CONTRIBUTIONS

All authors listed have made a substantial, direct and intellectual contribution to the work, and approved it for publication.

### ACKNOWLEDGMENTS

This work was supported by Grant-in-Aid for Scientific Research on Innovative Areas (Brain Protein Aging and Dementia Control) (15H01567) from Ministry of Education, Culture, Sports, Science and Technology (MEXT), and Grant-in-Aids from MEXT/The Japan Society for the Promotion of Science (JSPS). (17H01564, 16H01345, 17KT0131, 15K06762), Takeda Science Foundation, and Center for Baby Science in Doshisha University (Joint Usage/Research Center accredited by MEXT). This work is partially supported by the Strategic Research Program for Brain Sciences from Japan Agency for Medical Research and Development, AMED.

**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 © 2018 Yamanaka and Nukina. 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 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.

# Imbalanced Expression of Tau and Tubulin Induces Neuronal Dysfunction in C. elegans Models of Tauopathy

Tomohiro Miyasaka1 \*, Yuki Shinzaki1 , Satomi Yoshimura1 , Sawako Yoshina2 , Eriko Kage-Nakadai2,3, Shohei Mitani2 and Yasuo Ihara1

1 Department of Neuropathology, Faculty of Life and Medical Sciences, Doshisha University, Kyoto, Japan, 2 Department of Physiology, School of Medicine, Tokyo Women's Medical University, Tokyo, Japan, 3 Graduate School of Human Life Science, Osaka City University, Osaka, Japan

Tauopathy is a type of dementia defined by the accumulation of filamentous tau inclusions in neural cells. Most types of dementia in the elderly, including Alzheimer's disease, are tauopathies. Although it is believed that tau protein abnormalities and/or the loss of its functions results in neurodegeneration and dementia, the mechanism of tauopathy remains obscure. Loss of microtubules and/or tubulin is a known consequence of tau accumulating in neurons in Alzheimer's disease. In other words, there is an excess level of tau relative to tubulin in tauopathy neurons. To test whether this imbalance of tau and tubulin expression results in the neurotoxicity of tau, we developed several transgenic C. elegans lines that express human tau at various levels in pan-neurons. These worms showed behavioral abnormalities in a tau expressiondependent manner. The knockdown of a tubulin-specific chaperon, or a subset of tubulin, led to enhanced tau toxicity even in low-expressing tau-transgenic worms that showed no abnormal behaviors. In addition, the suppression of tau expression in tubulin knockdown worms rescued neuronal dysfunction. Thus, not only the overexpression of tau but also a reduction in tubulin can trigger the neurotoxicity of tau. Tau expressed in worms was also highly phosphorylated and largely bound to tubulin dimers rather than microtubules. Relative amount of tubulin-unbound tau was increased in highexpressing tau-transgenic worms showing tau toxicity. We further demonstrated that tau aggregation was inhibited by co-incubation of purified tubulin in vitro, meaning sufficient amounts of tubulin can protect against the formation of tau inclusions. These results suggest that the expression ratio of tau to tubulin may be a determinant of the tauopathy cascade.

Keywords: tau, tauopathy, microtubule, tubulin, neurodegeneration, C. elegans, Alzheimer's disease

# INTRODUCTION

In certain types of neurodegenerative diseases, affected neurons or glial cells have filamentous tau inclusions called neurofibrillary tangles, neuropil threads, and glial fibrillary tangles (Serrano-Pozo et al., 2011; Spillantini and Goedert, 2013). These disorders, which constitute the majority of agedependent dementia, are called tauopathies and include Alzheimer's disease (AD), frontotemporal

### Edited by:

Naruhiko Sahara, National Institute of Radiological Sciences (NIRS), Japan

### Reviewed by:

Emmanuel Planel, Laval University, Canada Kentaro Tanemura, Tohoku University, Japan

\*Correspondence: Tomohiro Miyasaka<EMAIL_ADDRESS>
### Specialty section:

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

Received: 27 February 2018 Accepted: 31 May 2018 Published: 20 June 2018

### Citation:

Miyasaka T, Shinzaki Y, Yoshimura S, Yoshina S, Kage-Nakadai E, Mitani S and Ihara Y (2018) Imbalanced Expression of Tau and Tubulin Induces Neuronal Dysfunction in C. elegans Models of Tauopathy Front. Neurosci. 12:415. doi: 10.3389/fnins.2018.00415

dementia, progressive supranuclear palsy, and corticobasal degeneration. The severity of dementia in tauopathies has been shown to correlate with the abundance of tau inclusions and the extent of neuronal loss (Gomez-Isla et al., 1997; Delacourte et al., 1999). Furthermore, the pathogenic mutations in the tau gene have been identified from genetic studies of familial tauopathy, FTDP-17 (Iqbal et al., 2016). These findings indicate that changes in tau and its function lead to neurodegeneration and dementia.

Numerous studies revealed that pathological tau inclusions are composed of insoluble filaments, paired helical filaments (PHF) and straight filaments (Grundke-Iqbal et al., 1986; Greenberg and Davies, 1990; Lee et al., 1991; Fitzpatrick et al., 2017). This pathologically deposited tau is abnormally phosphorylated to a greater extent than physiological tau (Khatoon et al., 1992; Hanger et al., 2009). Since this abnormal phosphorylation disrupts the ability of tau to promote microtubule (MT) assembly (Yoshida and Ihara, 1993), it has been considered a key element of the pathology that underlies tauopathies. However, the pathological significance of the phosphorylation on individual sites of tau remains unclear. Other abnormal modifications, such as ubiquitination, acetylation, glycation, isomerization, and truncation, do not occur as often in physiological tau but were also identified (Miyasaka et al., 2005b; Iqbal et al., 2016). In contrast to the huge filamentous inclusions, it is currently assumed that the small soluble aggregates called oligomers are an essential part of the tau toxicity or substance that propagates tau pathology (Goedert et al., 2017; Shafiei et al., 2017). However, it is also suggested that unaggregated tau can affect neuronal function (Miyasaka et al., 2016; Xie and Miyasaka, 2016). Thus, the definitive trigger of tau, as well as which type of tau is toxic, is currently unknown.

Loss of MTs and/or tubulin is an invariable feature of tauaccumulating neurons in AD (Terry et al., 1964; Cash et al., 2003; Zhang et al., 2015). This inverse relationship is reproduced in the affected neurons of tauopathy animal models (Tatebayashi et al., 2002; Miyasaka et al., 2005a, 2016). Because the physiological function of tau is known to promote the assembly of tubulin or to stabilize MTs by binding to them, it is believed that the hyper-phosphorylation of tau may lead to MT loss (Bodea et al., 2016; Wang and Mandelkow, 2016). However, the finding that tau-knockout mice did not show obvious developmental abnormalities or brain function abnormalities, suggests that MT loss cannot simply be explained by a lack of tau (Harada et al., 1994; Kimura et al., 2014). Recently, we showed that the hyperphosphorylation of tau in hypothermic brains did not directly detach tau from MTs and that forced MT destruction induced simultaneous tau liberation and phosphorylation in cultured cells (Planel et al., 2008; Miyasaka et al., 2010). Furthermore, a reduction of MTs was found in the neurons of AD brains regardless of neurofibrillary tangle formation (Cash et al., 2003). These findings suggest that MT loss (or tubulin reduction) is not a passive event and is involved in the early pathogenesis of tauopathy.

We speculated that the amount of the tubulin in neural cells determines tau toxicity and that excess tau relative to tubulin triggers the tauopathy cascade. To confirm that the imbalanced expression of tau and tubulin can induce tauopathy, we developed several lines of transgenic C. elegans that express various levels of human tau pan-neuronally, and then, we assessed the effects of tubulin knockdown.

# MATERIALS AND METHODS

## Development and Maintenance of Worm Strain

Development and maintenance of integrant worm lines were performed as previously described (Xie et al., 2014; Miyasaka et al., 2016). Briefly, human tau or DsRed cDNA was subcloned into the site downstream of "pan-neuronal" unc-119 promoter (punc-119) of pFXneo-punc119 vector (Maduro and Pilgrim, 1995). The transgenes were injected into N2 together with a marker, pFXneo-Pges-1::EGFP. Germline transformation and generation of extrachromosomal arrays in C. elegans strains were performed according to the standard protocol (Mitani, 1995). Stable tau-transgenic (Tg)-lines, generated by UV irradiation, were backcrossed to N2 five times before analysis. Tg-strains used here were as follows: Mock-Tg (tmIs388), WT4R(L)-Tg (tmIs389), WT4R(H)-Tg (tmIs390), WT4R(ExH1)-Tg (tmIs763), and WT4R(ExH2)-Tg (tmIs765). For RNAi, tau-Tg worms were crossbred with rrf-3 mutant lines (Mock-Tg/rrf-3 and WT4R(L)/rrf-3). For mec-7 or mec-12 knockout, mec-7 (e1506) or mec-12 (tm5083) mutant lines were used. All C. elegans strains were maintained on a nematode growth medium (NGM) plate spread with E. coli OP50 under standard conditions (Miyasaka et al., 2016).

### Feeding RNAi

The RNA interference of C. elegans by feeding with dsRNAexpressing E. coli. was performed as previously described, with minor modifications (Kage-Nakadai et al., 2016). Complementary DNA fragments encoding human tau (748–1326 bp; corresponding to 2N4R isoform tau cDNA), K07H8.1 (tbce-1; 1066–1298 bp), and mec-12 (619–1058 bp) were subcloned into the multiple cloning site of the L4440 vector and transformed to the HT115 (DE3) E. coli. strain. The desired double-stranded RNA (dsRNA) was produced by bi-directional expression induced by 1 mM IPTG. The bacteria expressing dsRNA were spread on NGM media supplemented with ampicillin and IPTG. Synchronously cultured worms were grown on these RNAi plates for 4 days and subjected to behavioral and biochemical analyses. To ensure efficient knockdown in neuronal cells, the worm lines crossbred with rrf-3 were used for all RNAi experiments (Simmer et al., 2002).

## Behavioral Analyses

A touch assay was performed as previously described (Miyasaka et al., 2005a). Briefly, on the forth day after hatching the worms were isolated onto new 3.5-cm plates, and their escape reactions were assessed in response to gentle touch with an eyelash under a stereoscopic microscope. The number of responses to 10 touch trials, five for the anterior plus five for the posterior, were counted. Uncoordinated movements (Unc) were also assessed

under a microscope. The severity of Unc was scored as follows: normal, wide bending with fast movement (2 point); Unc, slow movement (1 point); severe Unc, negligible movement (0 point). Twenty worms were used in each assay for each experiment, and the experiment was performed three to four times. Thus, 60–80 worms were assessed for each line. For the abnormality in tbce-1 knockdown, the severities of Unc of all worms grown on knockdown plates were scored as follows: normal, wide bending with fast movement (2 point); Unc, slow movement (1 point); severe Unc, negligible movement and burst (0 point).

### Biochemical Analyses

For biochemical analysis, worms were grown on NGM plates at large rectangular plates (No. 2 square schale, Eiken chemical co ltd., Tokyo, Japan). Synchronized worms were harvested in M9 buffer and pelleted by brief centrifugation. After washing twice with M9 buffer (22 mM KH2PO4, 42 mM Na2HPO4, 85 mM NaCl, and 1 mM MgSO4), the worm pellets were weighed and stored at −80◦C. Total worm lysates were prepared as previously described (Miyasaka et al., 2016). The worm pellets were sonicated in sodium dodecyl sulfate (SDS) sample buffer (80-mM Tris-HCl, 2% SDS, 10% glycerol, 1% 2-mercaptoethanol, pH 6.8) and cleared by ultracentrifugation at 150,000 × g for 10 min at 20◦C. The protein concentration for each sample was verified by Coomassie Brilliant Blue (Wako Pure Chemical Industries, Ltd., Osaka, Japan) staining on SDS-poly acrylamide gel electrophoresis (PAGE). Soluble fractions were subjected to SDS-PAGE followed by Western blotting. The MT-binding of expressed tau was analyzed as previously described (Miyasaka et al., 2016). Freshly harvested worms were homogenized in MS buffer (0.1 M MES, 1 mM EGTA, 1 mM MgSO4, 2 mM DTT, and 0.5% Triton X-100, pH 6.8) containing 20 µM Taxol, 2 mM guanosine triphosphate (GTP), and protease inhibitors (cOmplete R , Sigma-Aldrich, St. Louis, MO, United States) and phosphatase inhibitors (1 mM Na3VO4, 1 mM NaF, 1 mM okadaic acid, and 1 mM b-glycerophosphate) at room temperature and then immediately chilled. After brief centrifugation, soluble fractions were centrifuged at 100,000 × g for 15 min at 2◦C to obtain the soluble free tubulin and insoluble MT fractions. MT-unbound tau in free tubulin fraction was immunoprecipitated with H-150 anti-tau antibody bound on ProteinG-conjugated Dynabeads (Thermo Fisher Scientific Inc., Waltham, MA, United States) at 4◦C for 3 h. After washing with MS buffer, bound proteins on the beads were solubilized in SDS-sample buffer. The amount of tau and tubulin were analyzed by Western blotting.

# Purification and Dephosphorylation of Tau Expressed in Worms and MT-Binding Assay

Purification and dephosphorylation of tau and an MT-binding assay were performed as previously described with minor modifications (Xie et al., 2014). Four-day-old WT4R-Tg worm pellets were homogenized in ×10 volume of Tris-saline buffer (TS; 50 mM Tris, 150 mM NaCl, pH 7.6) containing protease and phosphoatase inhibitors described above. After ultra-centrifugation at 120,000 × g for 15 min at 2◦C, the soluble fractions were adjusted in 0.5 M NaCl and 2% 2-mercaptoethanol and heated at 100◦C for 5 min. After the precipitates were removed by brief centrifugation, the soluble (heat-stable) fractions were precipitated by 50% ammonium sulfate. After

FIGURE 1 | Tau level-dependent neuronal dysfunction in WT4R-tau-Tg worms. Mock-Tg (a), WT4R(L)-Tg (b), WT4R(H)-Tg (c), WT4R(ExH1)-Tg (d), and WT4R(ExH2)-Tg (e) worms were grown on NGM plates and subjected to (A) Western blotting of tau (pool-2) and tubulin (DM1A) in the total lysate. Expression levels were quantified and shown against WT4R(L)-Tg (Lower panel). Average of four independent experiments are shown (means ± SEM, n = 4). Statistical significance was analyzed by Tukey's post hoc test ( ∗∗p < 0.01, ∗∗∗p < 0.001, vs. WT4R(L)-Tg). (B) The behavioral analysis of uncoordinated movement in each worm line was analyzed. Upper panel shows the population of healthy (open), weak Unc (hatched), and severe Unc (closed) worms (n = 60) in each line. Lower panel shows that the severity of Unc as described in Materials and Methods section. Average scores of three independent experiments are shown (means ± SEM, n = 3). Statistical significance was analyzed by Tukey's post hoc test (∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, vs. Mock-Tg).

centrifugation at 20,000 × g for 15 min at 2◦C, the resultant pellets were re-solubilized and incubated with or without Lambda protein phosphatase (New England BioLabs, Inc., Ipswich, MA, United States) for 30 min at 30◦C. Dephosphorylated and non-dephosphorylated tau were purified by using both heat and ammonium sulfate precipitation as described above. For the preparation of worm MT, fresh Mock-Tg worms were homogenized in ×5 volume of MS buffer containing protease and phosphatase inhibitors and centrifuged at 120,000 × g for 15 min at 2◦C. Then, the soluble fraction was incubated with 20 µM Taxol and 2 mM GTP for 30 min at 25◦C. After the addition of 0.5 M NaCl to remove the endogenous MAPs, polymerized MTs were collected by ultra-centrifugation and re-suspended into MS buffer containing Taxol and GTP. After the re-binding of purified tau on MTs at 25◦C for 20 min, MT-unbound and MT-bound fractions were separated by ultra-centrifugation.

### Morphological Analyses

Immunocytochemical analyses were performed by using the freeze-cracking method (Miyasaka et al., 2005a). Cracked worms put on PDL (poly-d-lysine)-coated glass slides were immersed in pre-cooled MeOH followed by hydration and permeabilization with 0.1% Triton X-100 in TS. After the worms were soaked with 10% goat serum in TS, the specimens were incubated with 1% BSA in TS containing primary antibodies. Bound antibodies were visualized using Alexa-conjugated secondary antibodies (Molecular Probes, Inc., Eugene, OR), and observed under an LSM700 microscope (Carl Zeiss Inc., Jena, Germany).

### Drug Treatment

Trimethylamine-N-oxide (TMAO) was dissolved in H2O and stored in −20◦C. For treatment, the compound was diluted with M9 buffer and directly added onto NGM plate. Worms were synchronized and grown on drug-containing plates for 3 days and subjected to biochemical analyses.

## In Vitro Tau Aggregation Assay

Purification of recombinant tau corresponding to the 0N4R isoform and in vitro tau aggregation assays, were performed as previously described (Xie et al., 2014, 2015). Briefly, the recombinant tau 0N4R isoform expressed in E. coli [BL21(DE3)] was solubilized in homogenization buffer (50 mM PIPES, 1 mM EGTA, 1 mM DTT, and pH 6.8) with protease inhibitors and charged onto a phosphocellulose column (P11, Whatman). Tau protein fraction eluted in 0.1–0.3 M NaCl was precipitated by ammonium sulfate precipitation. The resultant pellet was re-solubilized by homogenization buffer containing 0.5 M NaCl and 1% 2-mercaptoethanol and fractionated by heat stability. The heat-stable fraction was further purified using reverse-phase HPLC (Cosmosyl protein-R, Nacalai tesque, Kyoto, Japan).

plates showed Unc (b) or a frequent burst phenotype (c). (B) The severity of Unc was assessed as described in Materials and Methods section. Average scores of three independent experiments are shown (means ± SEM, n = 3). WT4R(L)-Tg/rrf-3 worms showed a significantly higher sensitivity to tubulin chaperon knockdown. (C) The levels of tbce-1 mRNA were quantified by quantitative real time RT-PCR (means ± SEM, n = 3). tbce-1 mRNA is suppressed to less than 25% compared to controls, regardless of tau expression. (D) The levels of tau and α-tubulin were analyzed by Western blotting. The percent expression of control worms is shown (means ± SEM, n = 3). Statistical significance was analyzed by Tukey's post hoc test (∗p < 0.05, ∗∗p < 0.01, and ∗∗∗p < 0.001) or Student's t-test (∗p < 0.05).

Purified porcine tubulin was purchased from Cytoskeleton Inc. (Denver, CO, United States). Purity and protein amount were analyzed by SDS-PAGE followed by Coomassie brilliant blue staining. For in vitro aggregation, 60 mg/ml of heparin was mixed with 10 mM purified tau, 100 mM NaCl, 10 mM thioflavine-T (Th-T), 10 mM HEPES (pH 7.4), and with/without tubulin. The time-dependent changes in Th-T fluorescence were measured at 465–635 nm (excitation) and 535–625 nm (emission) for 3 days. After incubation, the mixtures were centrifuged at 100,000 × g for 15 min before or after 1% Sarkosyl, treatment. The fractions were analyzed by SDS-PAGE followed by CBB staining and Western blotting.

### Antibodies and Compounds

The antibodies used here were as follows: pool-2 (anti-pan-tau, a generous gift of Dr. H. Mori), HT7 (anti-pan-tau, Innogenetics), H-150 (anti-human-tau, Santa Cruz Biotechnology, Dallas, TX, United States), AT8 (anti-phospho-tau, Thermo Fisher Scientific Inc.), tau-1 (anti-dephospho-tau, Merck Millipore, Billerica, MA, United States), PHF-1 (anti-phospho-tau, a generous gift of Dr. P Davies), pS262 (anti-phospho-tau, Sigma-Aldrich, St. Louis, MO, United States), DM1A (anti-α-tubulin, Sigma-Aldrich), KMX-1 (anti-β-tubulin, Merck Millipore), anti-mec-12 (6-11B1, Sigma-Aldrich), 1A4 (anti-α-actin, Sigma), and 6C5 (anti-GAPDH, Abcam). Taxol and trimethylamine-N-oxide were purchased from Sigma-Aldrich. Other materials were purchased from Nachalai tesque unless otherwise specified (Kyoto, Japan).

## Statistical Analysis

All statistical analyses were performed using IBM SPSS statistics 25 (IBM, Armonk, NY, United States). Analysis of variance (ANOVA) was used for comparisons made between three or more groups, followed by Tukey's post hoc test. All data were presented as the means ± SEM. In all cases, P values lower than 0.05 were considered as significant.

# RESULTS

To date, a number of Tg mouse lines have been developed (Noble et al., 2010), with varying neurological phenotypes. Although some of the unique phenotypes may be caused by

∗∗∗p < 0.001).

hoc test (∗p < 0.05,

the promoter used for exogenous tau expression in particular lines, it is generally understood that the expression level of tau correlates with the severity of the pathology in Tg mice. To probe whether the amount of tau expression affects its neuronal phenotype, we developed several lines of tau-expressing worms using the pan-neuronal punc-119 promoter (Xie et al., 2014; Miyasaka et al., 2016). Integration of extrachromosomal arrays in the worm genome allows for the introduction of various numbers of transgene copies (Mitani, 1995). We isolated four independent integrant human wild-type 0N4R-tau-Tg lines with different expression levels (**Figure 1A**). WT4R(H)-Tg, WT4R(ExH1)-Tg, and WT4R(ExH2)-Tg lines express tau at 2.17 ± 0.17, 2.98 ± 0.24, and 3.16 ± 0.31 fold than WT4R(L)- Tg respectively. We also found the amount of α-tubulin expression was slightly but significantly higher in WT4R(Ex2)- Tg line. As shown in **Figure 1**, WT4R(L)-Tg worms showed minimal Unc but the WT4R(H)-Tg worms expressing three times more tau had a more severe Unc phenotype as previously described (Miyasaka et al., 2016). Furthermore, most of the lines with extremely high tau expression levels, WT4R(ExH1)- Tg and WT4R(ExH2)-Tg, suffered from severe Unc and were immobile (**Figure 1B**). Therefore, in these lines, the severity of Unc seemed to be correlated with the expression levels of exogenous tau.