Around 200μg of mice tissues were lysed in 300μl of Lysis Buffer solution (LB) made up of 1% Triton X-100 (Serva, Germany), complete protease inhibitor cocktail solution (Serva, Germany), phosphatase inhibitor cocktail solution (Serva, Germany), 20 mM of NEM (Sigma–Aldrich) and the following components (mM): TRIS acetate, 20; sucrose, 0.27; EDTA, 1; EGTA, 1; Na Orthovanadate, 1; NaF, 50; Na Pyrophosphate, 5; Na β-glycerophosphate, 10; DTT, 1.

Samples were then kept for 30 min on ice to allow protein solubilization. Later a centrifugation step of 10 min at 12000 rpm was applied to the samples and the supernatant was collected and stored at −20◦C until needed.

## **WESTERN BLOT**

Protein concentrations for each sample were determined by Bradford assay and the samples were directly analyzed by immunoblotting following resuspension in Laemmli buffer.

Equal amount of proteins (∼15μg for each condition) were resolved by 10% SDS-polyacrylamide gels and blotted onto PVDF membrane (Serva, Germany). The proteins blotted on the membrane were then blocked for 1 h at room temperature using Tris-buffered saline-Tween (t-TBS) (M) Tris, 0, 02; NaCl, 0, 15; Tween 20, 0, 1%) containing 5% skimmed milk.

Later the membranes were treated with specific antibodies and the incubation last for 12 h at 4◦C with mild agitation. The primary antibody used for western blot analysis are: rabbit amyloid precursor protein (AβPP) 1:2000 (Sigma-Aldrich, USA), rabbit SUMO-1 1:1000 (Cell Signaling, USA), rabbit SUMO-2/3 (18H8) 1:1000 (Cell Signaling, USA), rabbit SENP1 1:500 (Thermo scientific, USA), mouse UBC9 (C12) 1:1000 (Santa Cruz Biotechnology, USA), mouse β-actin 1:30000 (Sigma-Aldrich, Italy).

Tris-buffer saline solution (TBS) with 0, 1% of Tween 20 was used to wash the membranes for 50 min of wash in t-TBS. Then the blots were incubated for 1 h at room temperature with peroxidase-conjugated goat anti-rabbit or anti-mouse IgG secondary antibodies (UCS Diagnostic), as needed.

After 50 min of washes in t-TBS bands immunoreactivity was detected by enhanced chemiluminescence (ECL; WESTAR, Cyanagen, Italy). For all experiments stripping procedure was applied when the control of loading, performed blotting for β-actin, was required (stripping buffer from SignaGen, USA).

For each time point, a western blot has been performed in which proteins from six samples for WT and Tg2576 have been separated and analyzed. Where possible, membranes have been stripped and re-blotted for different antibodies with the purpose of using same loading conditions.

## **RNA EXTRACTION AND REAL-TIME PCR**

Total RNA was extracted from brain cortex and hippocampus using the Trizol reagent (Invitrogen, Carlsbad, CA) according to the manufacturer's instruction. RNA yield and purity were determined by spectrophotometry absorption at 260 and 280 nm. To obtain cDNA, an equal amount of mRNA (1μg) underwent to Reverse Transcription (Promega, Madison, WI). The resulting cDNA was stored at −20◦C until used for the further analysis. Messenger RNA expression was quantitatively measured with quantitative (q) real time PCR using iCycler Bio-Rad. The reaction was performed in a 25μl volume using SensiMix SYBR Green and Fluorescein kit (Bioline, London, UK). All the measures were performed in triplicate. The reaction conditions were as follows: 95◦C for 10 min (Polymerase activation), followed by 40 cycles at 95◦C for 15, 55–50◦C (Temp. depends on the Tm of primers) for 15 s and 72◦C for 15 s. The reaction mixture without the cDNA was used as control.

The primer sequences used in this study were as following for SUMO-1: forward 5 -GCCTGGGACATGGGTTT-3 and reverse 5 -TTAATGAAGCTGGTACAGACGATG-3 ; SUMO-2: forward 5 -GGCAGGGTTTGTCAATGAGGC-3 and reverse 5 -CTGGAGTAAAGTA GTAGCAGGCTC-3 ; SUMO-3: forward 5 -GAGGCAGGGCTTGTCAATGAG-3 and reverse 5 -GGTC AGGACAACGGTTGGGTG-3 ; SENP1: forward 5 -AATGGCTG ATGATGATGTG-3 and reverse 5 -TTGGACAAGGATTAGA CTGAAT-3 ; UBC9: forward 5 -CATCCAGCCTTCGT AAA CC-3 and reverse 5 -GCTAACAGGCAGGGAGAT-3 ; glyceraldehydes-3-phosphate dehydrogenase (GAPDH): forward 5 -GCCA AGGCTGTGGGCAAGGT-3 and reverse 5 -TCTCCAGGCGGC ACGTCAGA-3 .

The *Ct* values of the specific gene of interest were normalized to the *Ct* value of the endogenous control, GAPDH, and the comparative *Ct* method (2−--*Ct*) was then applied using WT mice group as calibrator.

## **STATISTICAL ANALYSIS**

In the western blots and real time PCR experiments, *T*-test analysis was performed and *p* < 0.05 was considered statistically significant. Statistical analysis for biochemical experiments was performed using GraphPad PRISM 5. A number (as indicated in figure legends) of animal tissues has been used at the same time point for the experiments. Values shown represent the mean ± s.e.m. Final western blots have been run using representative sampling

## **RESULTS**

#### **AβPP INCREASES IN Tg2576 MICE**

The Tg2576 transgenic mouse carries a transgene coding for the 695-amino acid isoform of human AβPP derived from a large Swedish family with early-onset AD (Hsiao et al., 1996). This mouse model of AD expresses high concentrations of mutant Aβ, develops a significant number of amyloid plaques, and displays functional deficits like decreased dendritic spine density, impaired long-term potentiation (LTP), and behavioral deficits (Jacobsen et al., 2006; Balducci et al., 2011).

In order to validate the mice model used in this work we measured AβPP immunoreactivity in the cortical and hippocampal tissues from the brains of Tg2576 and WT mice. Tg2576 are expected to have an augmented expression of AβPP because, beside the murine endogenous AβPP, they genetically over-express human mutated AβPP. Indeed, both cortex and hippocampus prepared from Tg2576 mice showed a more intense immunoreactivity of the AβPP band corresponding to 95–100 kDa compared to WT (**Figure 1D**).

## **PROTEIN SUMOylation CHANGES IN THE CORTEX OF Tg2576 MICE DURING ONTOGENESIS**

The cerebral cortex is among the most vulnerable brain regions affected by the disease showing progressive plaque burden and tissue atrophy (Tosun et al., 2011). Although in a less severe manner, some biochemical hallmarks have also been reported in different AD mice models (Sclip et al., 2011; Izco et al., 2014).

In order to evaluate protein SUMOylation changes during disease progression, we dissected and lysated cortical tissues from 1.5, 3, 6, 17 months old Tg2576 and their age-matched WT littermates. Protein SUMOylation has been revealed by western blotting using an anti-SUMO-1 and an anti-SUMO-2/3 antibody. SUMO-1 and SUMO2/3 for each time point has been normalized against the actin. Interestingly, the ratio in protein SUMO-1 ylation between Tg2576 and WT mice is increased at early stages, and it later decreases following a bell-shaped curve. In fact, a peak of SUMO-1-ylation is observed at 3 and 6 months old animals (**Figure 1A**, 3 months: 1.65 ± 0.16 *p* < 0.05; 6 months: 1.78 ± 0.08 *p* < 0.01).

Noteworthy a protein SUMOylated band, absent in the other ages analyzed, appears at 6 months of age. The immunoreactivity of this band results more intense in the Tg2576 mice compared to controls (**Figure 1A**, black arrow). Further studies are required to identify which protein becomes target of SUMO-1 at this stage, and whether this protein modification has a role in AD pathology.

SUMO-2/3-ylation did not show any differences in the ratio between Tg2576 and WT mice at different ages except for 17 months old mice, where it resulted drastically decreased in transgenic mice (**Figure 1B**, 17 months: 0.35 ± 0.05 *p* < 0.01). Further experiments are required to understand what implication this reduction may have. As for the SUMO-1 western blot, also for SUMO-2/3 a more intense band is detectable (**Figure 1B**, black arrow).

## **PROTEIN SUMOylation CHANGES IN THE HIPPOCAMPUS OF Tg2576 MICE DURING ONTOGENESIS**

The hippocampus is the part of the brain that is involved in memory formation, being one of the first regions to suffer damage in AD. Since Tg2576 show cognitive impairment caused by hippocampal dysfunction even before the onset of the pathology (D'Amelio et al., 2011), we analyzed protein SUMOylation changes between Tg2576 and WT mice also in this brain area.

Similarly to the cortex, SUMO-1-ylation is increased in the hippocampus of 3 and 6 months old Tg2576 compared to WT (**Figure 2A**, 3 months: 1.71 ± 0.07 *p* < 0.01; 6 months: 2.07 ± 0.39 *p* < 0.01).

SUMO-2/3-ylation did not show any differences in the ratio between Tg2576 and WT mice at different ages except for 17 months old, where surprisingly it resulted decreased in transgenic mice (**Figure 2B**, 17 months: 0.65 ± 0.01 *p* < 0.01). This result needs more investigation in order to understand its potential implication in the pathophysiology of AD.

## **SUMO PATHWAY RELATED PROTEINS IN Tg2576 TRANSGENIC MICE ONTOGENESIS**

The protein conjugation by SUMO occurs through the activity of four key enzymes which form the SUMOylation pathway. Among them Ubc9 and SENP1 are the most important since they are dedicated exclusively to protein SUMOylation. On the one hand Ubc9 binds and transports SUMO to the target protein, on the other SENP1 is the isopeptidase that regulates the deSUMOylation (Droescher et al., 2013; Feligioni and Nisticò, 2013). Ubc9 and SENP1 are critical for the maintenance of SUMO/deSUMOylation balance, therefore their expression levels have been assessed by western blotting in cortical and hippocampal tissue from Tg2576 and WT mice. The expression level of the two proteins has been normalized on actin and the ratio between Tg2576 and WT has been analyzed.

Interestingly, we found that the expression level of Ubc9 is modulated both in cortical and hippocampal tissue. In fact, the western blots show for both WT and Tg2576 an increase of Ubc9 from 3 to 17 months with a peak at 6 months of age. Moreover Ubc9 seems to be highly expressed in Tg2576 compared to WT at different ages (**Figure 1C**, 6 months: 1.57 ± 0.16 *p* < 0.05) (**Figure 2C**, 3 months: 1.42 ± 0.24 *p* < 0.05; 6 months: 2.22 ± 0.37 *p* < 0.01). Conversely, immunoreactivity of SENP1 seems to be augmented in the cortex of transgenic mice at 3 and 6 months and only at 6 months in hippocampus of Tg2576 compared to age-matched WT animals (**Figure 1C**, 3 months: 1.75 ± 0.21 *p* < 0.05; 6 months: 2.51 ± 0.21 *p* < 0.01) (**Figure 2C**, 6 months: 2.15 ± 0.42 *p* < 0.01).

## **SUMO-1-, SUMO-2-, SUMO-3-, SENP1- AND Ubc9-mRNA IN BRAIN CORTEX AND HIPPOCAMPUS**

SUMO-1 mRNA levels in the cortex (**Figure 3A**) and hippocampus (**Figure 4A**) of Tg2576 mice at 1.5 months of age were similar to the levels measured in WT mice, whereas at 6 months of age their levels were significantly higher than that of WT mice.

On the contrary, we did not find any significant changes in SUMO-2, SUMO-3, SENP1, and Ubc9 mRNA levels in the cortex (**Figures 3B–E**) and hippocampus (**Figures 4B–E**) of Tg2576 and WT mice at the age of 1.5 and 6 months. The SUMO-1 mRNA increase is in accordance with the elevated level of SUMO-1-ylation that has been found in cortex and hippocampus at 6 months of age. It seems that SUMO-1 synthesis is augmented in order to be disposable for conjugation, whereas the Ubc9 and SENP1 protein levels are sufficient in both tissues to maintain SUMOylation pathway functional. The SUMO-2 and -3 mRNA level is in line with protein SUMO-2/3-ylation expression that resulted unchanged up to 6 months of age.

## **DISCUSSION**

Tg2576 mice model were developed to test the amyloid hypothesis of AD. These mice model answered partially the question whether AD pathology comes from the accumulation of Aβ species. Aβ species, although poorly detectable, can be found in the soluble form (monomers, dimers, trimmers or oligomers) already at a

of age) was compared with their age-matched Tg2576 mice.

young age (<5 months old) (Kawarabayashi et al., 2001; Klingner et al., 2003; Lesné et al., 2006), but they reach a highly detectable level around 6–8 months (Hsiao et al., 1996). Insoluble amyloid plaques start to form at around 9–10 months in hippocampus and cortex (Hsiao et al., 1996). This model also shows the typical AD cognitive impairment with a mild onset at 3 months, which

β-Actin was used as loading control.

is exacerbated starting from 6 months of age. However, Tg2576 do not resemble all the features of AD since they do not display any formation of neurofibrillary tangles, marked neuronal loss or gross brain atrophy, (Irizarry et al., 1997), but are anyway a very useful model to study AD amyloid-related pathology (Deacon et al., 2008).

Several AD-associated proteins undergo SUMO protein modification, leading to hypothesize that SUMO plays a fundamental role in AD pathogenesis. Despite that, we still lack information about how SUMO/deSUMOylation process is altered during AD onset and development. Therefore a better understanding of SUMO changes during AD pathogenesis could help to disclose the role of protein modification in the onset of AD. Notably, McMillan and co-authors (McMillan et al., 2011) reported that no clear differences in global protein SUMOylation can be measured in adult Tg2576 (9 months old) vs. their respective WT.

In this manuscript, we decided to investigate SUMOylation changes during mice lifespan analyzing four time points. To our knowledge this is the first ontogenetic study of SUMO/deSUMOylation profile in an AD model.

Yun and co-authors have shown in *in-vitro* experiments that SUMO-1 can be considered a modulator of the synthesis of Aβ oligomers (Yun et al., 2013). In fact SUMO-1, but not SUMO-2 or -3, when overexpressed in neuronal or cell culture, increases BACE1 level which mediates the amiloydogenic cleavage of AβPP. Moreover, as previously reported in 18 months old AβPP transgenic mice (Yun et al., 2013) SUMO-1 free protein is increased in their cortical tissue. So, it has been postulated that the increase of Aβ accumulation together with an accrual of cell oxidative stress can act synergistically to enhance protein SUMO-1-ylation. By conjugating BACE1, SUMO-1 intensifies Aβ oligomers production (Yun et al., 2013). Our data show that already at the age of 3 months, protein SUMO-1-ylation is augmented both in cortical and hippocampal tissue of Tg2576 model compared to their respective WT.

Therefore it can be hypothesized that at early stages (3 months) the "cell stress" might contribute more to the increase

in SUMO-1-ylation compared to Aβ oligomers which are present at low levels at this age. As a result, SUMO-1-ylation activates multiple signaling pathways, like BACE1, GSK3β, JNK activation, that can contribute to boost the production of Aβ oligomers. The feedback loop established between SUMO-1, Aβ oligomers and cell stress cooperate to the development of AD neuropathology. Interestingly, SUMO-2-ylation does not change between Tg2576 and WT except for 17 months old transgenic mice where it is drastically diminished both in cortex and hippocampus. This aspect also deserves a deeper investigation since very little is known about SUMO-2/3 contribution to AD.

Ubc9 and SENP1 are critical enzymes for protein SUMOylation (Droescher et al., 2013; Feligioni and Nisticò, 2013). An increase of SUMOylation is associated with the onset of several pathologies including cancer. For example, Ubc9 expression, the E2 enzyme which facilitates SUMOylation, has been reported increased in primary colon and prostate cancer compared with normal tissue (Moschos et al., 2010). We here demonstrate that the increase of SUMOylation in Tg2576 brain tissues corresponds to a high expression level of Ubc9 at the same time points. Therefore Ubc9 plays a fundamental role for SUMOylation event also in AD. On the other hand, the over-functionality of SUMOylation in Tg2576 is also supported by an elevated presence of the SUMO protease SENP1. Our results are in line with previous data where SENP-1 was found increased during oxygen-glucose deprivation (OGD) experiments, suggesting that the neuronal response could involve a complex interplay between SUMOylation and deSUMOylation (Cimarosti et al., 2012).

The analysis of mRNA expression has shown an increase of SUMO-1 RNA only at the age of 6 months both in cortex and hippocampus. This is in line with our western blot data where the major increase of SUMOylation was exactly found at 6 months of age in Tg2576 mice, where probably there is a need for more protein synthesis. In contrast, although we reported an increase in protein expression level of both Ubc9 and SENP1, the mRNA expression of both proteins was unchanged. We can therefore speculate that the degradation system of Ubc9 and SENP1 could be decreased during lifespan of mice. Further studies should be carried out in order to better clarify this aspect. In line with a previous work (McMillan et al., 2011), our experiments did not show significant changes in protein SUMO-2/3-ylation in AD mice up to 6 months of age. SUMO-2/3 role in AD remains still elusive. However, in *in-vitro* experiments it has been shown that SUMO-2 increases N-terminal ADAM-cleaved AβPP fragment (α-NTF) while a mutant form of SUMO-2 lacking the SUMOylation activity secreted significantly more N-terminal BACE1-cleaved AβPP fragment (β-NTF) and Aβ (Li et al., 2003). In line with this report, we can speculate that the reduction in protein SUMO-2/3-ylation that was observed in 17 months old Tg2576 mice is concomitant with an elevated amyloid deposition which occurs at this stage in AD mice model (Jacobsen et al., 2006). In conclusion, here we report for the first time that protein SUMO/deSUMOylation equilibrium is unbalanced in a mouse model of AD at a very early stage of the pathology. We believe that this event can contribute to the onset of AD pathogenesis. Future investigation on the target(s) of protein SUMOylation at an early stage could possibly lead to the identification of novel pharmacological targets.

## **ACKNOWLEDGMENTS**

We thank Miss Marisa Hipwood for excellent language revision and Mr. Guglielmo Barberini for technical support. This work was partly supported by grants from: "H. Lundbeck Foundation, Denmark" and from the Italian Ministry of Health to IRCCS Neurological Institute "C. Mondino" (Ricerca Corrente 2013– 2015).

## **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.

*Received: 28 February 2014; accepted: 19 March 2014; published online: 07 April 2014. Citation: Nisticò R, Ferraina C, Marconi V, Blandini F, Negri L, Egebjerg J and Feligioni M (2014) Age-related changes of protein SUMOylation balance in the A*β*PP Tg2576 mouse model of Alzheimer's disease. Front. Pharmacol. 5:63. doi: 10.3389/ fphar.2014.00063*

*This article was submitted to Experimental Pharmacology and Drug Discovery, a section of the journal Frontiers in Pharmacology.*

*Copyright © 2014 Nisticò, Ferraina, Marconi, Blandini, Negri, Egebjerg and Feligioni. 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.*

## HSV-1 and Alzheimer's disease: more than a hypothesis

## *Roberto Piacentini 1, Giovanna De Chiara2 , Domenica D. Li Puma1, Cristian Ripoli 1, Maria E. Marcocci 3 , Enrico Garaci 4 , Anna T. Palamara5,6 and Claudio Grassi 1\**

*1 Institute of Human Physiology, Medical School, Università Cattolica del Sacro Cuore, Rome, Italy*

*2 Institute of Translational Pharmacology, National Research Council, Rome, Italy*

*3 Department of Public Health and Infectious Diseases, Sapienza University of Rome, Rome, Italy*

*4 San Raffaele Pisana Scientific Institute for Research, Hospitalization and Health Care, Telematic University, Rome, Italy*

*5 Department of Public Health and Infectious Diseases, Institute Pasteur Cenci Bolognetti Foundation, Sapienza University of Rome, Rome, Italy*

*6 San Raffaele Pisana Scientific Institute for Research, Hospitalization and Health Care, Rome, Italy*

#### *Edited by:*

*Cesare Mancuso, Catholic University School of Medicine, Italy*

#### *Reviewed by:*

*Ottavio Arancio, Columbia University, USA*

*Roberto Manservigi, University of Ferrara, Italy*

#### *\*Correspondence:*

*Claudio Grassi, Institute of Human Physiology, Medical School, Università Cattolica del Sacro Cuore, Largo Francesco Vito 1, 00168 Rome, Italy e-mail<EMAIL_ADDRESS>
Among the multiple factors concurring to Alzheimer's disease (AD) pathogenesis, greater attention should be devoted to the role played by infectious agents. Growing epidemiological and experimental evidence suggests that recurrent herpes simplex virus type-1 (HSV-1) infection is a risk factor for AD although the underlying molecular and functional mechanisms have not been fully elucidated yet. Here, we review literature suggesting the involvement of HSV-1 infection in AD also briefly mentioning possible pharmacological implications of these findings.

**Keywords: HSV-1, Alzheimer's disease, recurrent infection, γ secretase, amyloid-β protein**

## **ALZHEIMER'S DISEASE, APP PROCESSING AND AMYLOID-β PRODUCTION/ACCUMULATION**

Alzheimer's disease (AD) is a neurodegenerative disorder characterized by progressive decline in cognitive functions leading to memory loss and dementia. It involves degeneration of limbic and cortical brain structures, especially in the temporal lobe. In 2010, it was estimated that 35.5 million individuals were affected by dementia in the world with a prediction that this number will increase to 65.7 million by 2030, and 115.4 million by 2050. The World Alzheimer Report 2010 (AD International) estimated that the total costs of dementia in 2010 were US\$ 604 billions. Most (>90%) AD cases are sporadic. Only a minority of them have a genetic inheritance and are referred to as familial AD. The main risk factor for AD is aging. The disease afflicts 10% of the population over the age of 65 and 50% of the population over the age of 85. Several factors may increase the chances to develop AD (Imtiaz et al., 2014). Among the "genetic" risk factors, the carriage of a type 4 allele of the apolipoprotein E gene (APOE-ε4) has been widely recognized (see references in Kim et al., 2009). Among the "environmental" risk factors, persistent brain infections, particularly those induced by herpes simplex virus type-1 (HSV-1), seem to play a key role in AD pathogenesis. In a multifactorial disease like AD, to identify the contribution made by each factor and the underlying mechanisms is critical to the development of new therapeutic strategies to prevent the disease and/or slow down its progression.

From the neuropathological point of view, the brains of AD sufferers are characterized by the presence of two major hallmarks mainly located in the hippocampus and cortex: extracellular amyloid plaques composed of insoluble aggregates of the amyloid-β protein (Aβ) and intraneuronal neurofibrillary

tangles, composed of hyperphosphorylated Tau protein (Kosik et al., 1986). Numerous other conditions, among which oxidative stress (Mattson et al., 1993) and inflammation, due to microglia activation and astrocytosis, may concur to produce the structural and functional alterations typically found in AD brain (Kitazawa et al., 2004).

Amyloid-β protein is a small protein (generally composed of 40 to 43 amino acids) generated by the proteolytic cleavage of the amyloid precursor protein (APP), a ubiquitous transmembrane protein whose functional role in cell biology has been only partially discovered. APP seems to be involved in neurite outgrowth and synaptogenesis, neuronal protein trafficking along the axon, transmembrane signal transduction, cell adhesion and Ca2+ signaling (Zheng and Koo, 2006; Octave et al., 2013). Among the various isoforms of APP, due to alternative splicing of *APP* gene located on chromosome 21 in humans, those of 695-amino acid length is prevalent in neurons whereas the other two forms, APP751 and APP770, are expressed in other tissues (Zhang et al., 2011).

Amyloid precursor protein is normally subjected to processing by specific enzymes called "secretases" (Nunan and Small, 2000). It may be processed along two different pathways. The first one, that is most frequent and"non amyloidogenic", involves sequential proteolytic cleavages by the α- and γ-secretases. The former cuts APP at a position 83 amino acids from the C-terminus, thus producing a large N-terminal domain (sAPPα), normally secreted into the extracellular medium, whose function is not well defined. On the opposite site, the other 83-amino-acid C-terminal fragment (C83) is retained in the membrane and it is subsequently cleaved by the γ-secretase complex (that consists of presenilins, nicastrin, anterior pharynx-defective 1 and presenilin enhancer 2), producing a short fragment that is generally considered non-toxic and known as "p3". The amyloidogenic pathway begins when APP undergoes cleavage by the β-secretase, also known as β-site APP cleaving enzyme 1 or BACE-1 (Vassar et al., 1999). β-secretase cuts APP 16 amino acids before α-secretase and yields two species, the large N-terminal ectodomain of the precursor and the 99-amino acid Cterminus stub (C99). Subsequent cleavage of the latter fragment by γ-secretase results in the formation of Aβ species containing 40 to 42 amino acids. This also means that APP cleavage by α-secretase prevents the Aβ formation. On the other C-terminal side of APP, γ-secretase (in both amyloidogenic and non-amyloidogenic pathways) also generate the "APP intracellular domain" (AICD), that has been reported to modulate the transcription of several genes (including APP itself, BACE-1 and the Aβ-degrading enzyme neprilysin), to regulate apoptosis and contribute to AD pathogenesis (Octave et al., 2013). In APP, the amino acid threonine in position 668 (Thr668) is an important site for its processing. Indeed, APP phosphorylation at Thr668 resulted in increased Aβ formation (Lee et al., 2003; Pierrot et al., 2006). This amino acid is on the C-terminal region of APP and its phosphorylation also regulates the activity of AICD. In fact, when AICD is phosphorylated at Thr668 it interacts with the Fe65 adapter protein (Bórquez and González-Billault, 2012) and enters the nucleus, where it may regulate gene transcription and induce neurodegeneration (Chang et al., 2006).

Amyloidogenic cleavage of APP is not confined to cell membrane; it also takes place in several cellular compartments (Vetrivel and Thinakaran, 2006), including the intermediate compartment of the endoplasmic reticulum (Cook et al., 1997), the trans-Golgi network (Choy et al., 2012), and the endosomal/lysosomal system, where APP processing is regulated by cytoplasmic phosphorylation at Thr668 (Lee et al., 2003). For these reasons, Aβ species may be secreted directly into the extracellular space, where their subsequent aggregation promotes senile plaque formation. Alternatively, it may remain within the cell or may be re-internalized in neurons, where it accumulates (Mohamed and Posse de Chaves, 2011). Some recent findings suggest that Aβ accumulating in neurons, particularly in the form of small oligomers (especially dimers and trimers) is the major determinant of the synaptic damage that highly correlates with the cognitive deficits characterizing the early phases of the disease preceding neuronal death (Tampellini et al., 2007; Mucke and Selkoe, 2012; Ripoli et al., 2013). However, Aβ is not a mere toxic peptide. It is constitutively produced and secreted by cells and, especially when present at very low concentration (in the range of pM), it even supports synapses by increasing synaptic strength in the hippocampus (Puzzo et al., 2008, 2011).

In addition to secretases, APP may be processed by caspases (in particular caspase-3), especially in cells undergoing apoptosis (Gervais et al., 1999; Pellegrini et al., 1999; Fiorelli et al., 2013). In neurons, caspase-induced processing of APP generates a C-terminal fragment (C31) with neurotoxic potential (Lu et al., 2003; Nguyen et al., 2008). Moreover, Fiorelli et al. (2013) showed that caspases cleavage generates two APP fragments (APP-Fs) of 25–35 kDa that are recognized by anti-Aβ antibodies.

Among the genetic risk factors for AD, the carriage of *APOE*ε*4* allele plays a major role. Apolipoproteins carry lipids in the circulation and regulate lipid metabolism. ApoE, expressed predominantly in astrocytes, is suggested to be involved in redistribution of cholesterol and phospholipids during membrane remodeling (Holtzman et al., 2012). Links between AD and*APOE*ε*4* are multiple: ApoE protein exists in 4 isoforms (E1 to E4), with ApoE3 being the most common allele, and it seems to play a role in Aβ fibrillogenesis and oligomerization as well as in Aβ clearance. Differently from the other three variants, ApoE4 exhibits scarce ability to bind Aβ, thus its expression contributes to Aβ accumulation and aggregation inside neurons and influences the formation of the parenchymal amyloid plaques (Holtzman et al., 2012; Verghese et al., 2013). When human ApoE isoforms were expressed in APP transgenic mice, differences in extracellular Aβ accumulation were observed in an isoform-dependent manner (E2 < E3 < E4; Holtzman et al., 2000). Similar results were also recently obtained by Hashimoto et al. (2012) who reported that the levels of Aβ oligomers in APOE ε4/ε4 AD brains were 2.7 times higher than those found in APOE ε3/ε3 AD brains, and that ApoE increased Aβ oligomer levels in an isoform-dependent manner (E2 < E3 < E4).

Finally, alterations of intracellular Ca2+ homeostasis and signaling have also been implicated in AD pathogenesis. It is well known that intracellular Ca2+, besides playing a pivotal role in a large number neuronal functions, is a critical determinant of neuronal survival and death (Piacentini et al., 2008a,b; Maiti et al., 2011; Bading, 2013). Several reports demonstrated correlation between Ca2+ homeostasis dysregulation leading to increased intracellular Ca2+ levels and AD (Green, 2009). Interestingly, ApoE expression in neuronal and glial cells correlates with increased intracellular Ca2+ concentrations in an isoform-dependent manner (E2 < E3 < E4; Müller et al., 1998).

## **HOW CAN HSV-1 BE INVOLVED IN AD?**

Herpes simplex type 1 virus is a neurotropic double-stranded DNA virus that primarily infects epithelial cells of oral and nasal mucosa. Here virus undergoes lytic replication; the newly produced viral particles may enter sensory neurons and, by axonal transport, reach the trigeminal ganglion where usually establishes a latent infection. The virus undergoes periodic reactivation cycles in which the newly formed viral particles are transported back to the site of primary infection through the sensory neurons, causing the well-known clinical lesions (i.e., cold sores and blisters). However, the bipolar trigeminal ganglion neurons also project to the trigeminal nuclei located in the brainstem. From here, neurons project to the thalamus to finally reach the sensory cortex. This is the path through which the reactivated virus may reach the central nervous system (CNS), where it may cause acute neurological disorders like encephalitis [herpes simplex encephalitis (HSE)] or a mild, clinically asymptomatic, infection, or establish life-long latent infection (Kastrukoff et al., 1982; Lewandowski et al., 2002; references in Dobson et al., 2003). The weakening of immune system occurring during aging may favor this process. In addition to the neuronal route, HSV-1 may enter the CNS through the blood stream, as demonstrated by Burgos et al. (2002, 2003, 2005). Many experimental evidence, described below, suggest that accumulation of intracellular damage caused by repeated cycles of viral reactivation may concur to neurodegeneration.

Some reports suggest that during infection herpes virus interacts with several human proteins that it uses to enter the cell and to move from plasma membrane to the nucleus and back (reviewed in Carter, 2008). HSV-1 also uses the host's transcriptional machinery to replicate and binds to proteins that control immune surveillance or apoptosis. Noteworthy, in the attempt to eliminate the virus, host may even cause cell damage via immune and inflammatory responses targeting the viruscontaining cells. If this happens in the CNS, HSV-1-induced inflammatory response may result in HSE or, in milder cases, in cell death and neurodegeneration.

Several epidemiological, immunological and molecular evidence link HSV-1 infections to AD pathogenesis. HSV-1 is a ubiquitous virus that affects more than 80% of people over 65 worldwide. The first evidence suggesting the involvement of HSV-1 in AD dates back to 1982 and is based on the observation that people surviving to HSE showed clinical signs reminiscent of AD (i.e., memory loss and cognitive impairment), and that brain regions primarily affected in HSE (limbic system, frontal and temporal cortices) were the same regions compromised in AD (Ball, 1982). During the last 30 years several research groups have conducted many studies providing solid support to the involvement of HSV-1 infection in AD pathogenesis. Here we will briefly summarize the main results of these researches.

#### **EPIDEMIOLOGICAL DATA**

Several studies have been performed to assess the presence of HSV-1 in the brain of AD patients. After the first observations made by Ball (1982), other studies have demonstrated that HSE affected the same brain areas involved in AD in humans as well as in rodent experimental models (Damasio and Van Hoesen, 1985; Caparros-Lefebvre et al., 1996; Beffert et al., 1998; Wu et al., 2003; Taylor et al., 2007; Ando et al., 2008). Jamieson et al. (1992) and Itzhaki et al. (1997) reported that a high proportion (about 60%) of brains of elderly people contained latent HSV-1 DNA, especially in the CNS regions critically involved in AD. When present in AD brains, HSV-1 DNA was primarily located within amyloid plaques (Wozniak et al., 2009a). Whether or not the virus contributes to activation of pro-neurodegenerative pathways may largely depend on several host factors, including genetic predisposition, as described in greater detail below. Many infectious agents whose pathogenetic role has been established in other CNS diseases (e.g., varicella zoster virus, causing meningitis or encephalitis; Epstein–Barr virus, associated to both multiple sclerosis and CNS lymphoma; human herpes virus 6, associated to seizure in children) may infect subjects without producing evident clinical signs. On the other hand, even in the presence of "clinically silent" infection, virus may periodically reactivate and replicate in the CNS (Peter and Sevall, 2001; Bearer, 2012). There is no clear evidence on whether HSV-1 reaching the brain and infecting neurons resides there in latent form. However, the detection of HSV-1 DNA in the cerebro-spinal fluid (Deatly et al., 1988; Peter and Sevall, 2001; Plentz et al., 2008) suggests that HSV-1 replicates in the CNS (Bearer, 2012). The presence of HSV-1 proteins in hippocampal neurons of mice infected intraperitoneally with HSV-1 was demonstrated by Burgos et al. (2006a) who also

showed that virus is reactivated by hyperthermia. After every reactivation, the newly produced viral particles generated by this "silent" replication and potentially reaching the brain might act, in a drop by drop fashion, to produce local brain damages that may largely differ from those of acute diseases like HSE. It has been recently reported that in brain of infected but asymptomatic mice, HSV-1 reactivation was associated to neuroinflammation and to the appearance of several markers of neurodegeneration including Tau hyperphosphorylation (Martin et al., 2013).

#### **IMMUNOLOGICAL DATA**

A number of studies have been conducted to demonstrate the association between AD and HSV-1 infection by searching for antibodies against HSV-1 in the blood of AD patients. These studies revealed a strong correlation between AD occurrence and HSV infection or reactivation, as addressed in a longitudinal study including 512 elderly persons looking for correlation between anti-HSV-1 IgM positivity (a marker of virus primary infection or its reactivation) and development of AD-like cognitive dysfunctions (Letenneur et al., 2008). In the same study, no correlation was found between anti-HSV-1 IgG positivity and early dementia, thus suggesting that recurrent infection, rather the primary one, is dangerous for the CNS. Anti-HSV IgM levels, and not those of IgG, have also been found to inversely correlate with lower plasma Aβ levels (Féart et al., 2011). It is believed that increased amyloid deposition in the brain is paralleled by a lowering of Aβ levels in plasma and that low plasma Aβ levels might be considered possible short-term risk marker of dementia (Schupf et al., 2008; Lambert et al., 2009; Blennow et al., 2010). Another study recently demonstrated that HSV-1 reactivation, assessed as anti-HSV IgG avidity index (that is a more accurate way to demonstrate viral reactivation) occurs in prodromal AD and highly correlates with symptoms of mild cognitive impairment (Kobayashi et al., 2013). Finally, Mancuso et al. (2014) recently found that elevated HSV-1 antibody titers were significantly more frequent in AD patients than in control healthy patients and that they positively correlated with cortical bilateral temporal and orbitofrontal gray matter volume, that may be considered an index of AD pathology (Whitwell et al., 2012).

#### **GENETIC DETERMINANTS**

Some studies have suggested that in people carrying the *APOE-*ε*4* allele and, therefore, predisposed to develop AD, HSV-1 infection markedly increases the risk of AD (Itzhaki et al., 1997; Itzhaki and Lin, 1998; references in Bearer, 2012). However, this correlation was not always confirmed (Beffert et al., 1998). APOE seems to affects the outcome of several different infections (Itzhaki et al., 1998; Dobson et al., 2003) and, interestingly, APOE-ε4 is a risk factor for cold sores (Itzhaki and Wozniak, 2008). Some studies also demonstrated that ApoE4 presence influences the viral load in the brain. Indeed, viral spreading into the brain of ApoE KO mice was lower than that occurring in wild-type mice and correlation was reported between ApoE expression and HSV-1 DNA concentration detected in the CNS (Burgos et al., 2002). In a subsequent study, the same authors showed that during acute infection

with HSV-1, ApoE4 was more efficient than ApoE3 in promoting viral colonization of the brain (Burgos et al., 2006b). However, many genes and proteins implicated in AD, other than ApoE, have been found to interact with herpes simplex viral genome or regulate its life cycle, further supporting the hypothesized synergy between host and pathogens in causing AD-like brain damage (Carter, 2008). From this point of view, genome wide association investigations (GWAs) carried out in a large cohort of AD and non-AD subjects also suggested an infective etiology for sporadic AD. In particular, the polymorphism association of genes located on the chromosome 19 (i.e., Nectin-2 [NC-2]; APOE, glycoprotein carcinoembryonic antigen related cell adhesion molecule-16 [CEACAM-16]; B-cell lymphoma-3 [Bcl-3]; translocase of outer mitochondrial membrane 40 homolog [TOMM-40]) and on the chromosome 8 (complement receptor-1 [CR-l]; APOJ, C-type lectin domain A family-16 member [CLEC-16A]), results in a genetic fingerprint that might modulate brain susceptibility to herpes virus infection and lead to neuronal loss, inflammation and Aβ deposition (Porcellini et al., 2010; Licastro et al., 2011).

#### **MOLECULAR DATA**

Several studies demonstrated that HSV-1 affects APP processing. HSV-1 infection may have profound effects on the host intracellular pathways leading to activation/inactivation of several signaling molecules and kinases involved in APP metabolism. Ruth Itzhaki's group found that in human SH-SY5Y neuroblastoma cells HSV-1 induces APP cleavage with the production of a 55-kDa fragment (recognized in Western blot analysis by an anti-C-terminal APP antibody) starting from 6 h post infection (hpi), and the concomitant reduction of band intensity relative to full-length APP (Shipley et al., 2005). These authors also reported that HSV-1 infection caused Aβ40 and Aβ42 accumulation in human neuroblastoma and glioblastoma cells *in vitro*, whereas in brains of mice infected with HSV-1 they only found an increase in Aβ42 (Wozniak et al., 2007). However, further studies on *in vivo* models of recurrent HSV-1 brain infections are needed to determine the structural and functional alterations induced by viral reactivation.

We have demonstrated that in cultured neuronal cells HSV-1 induces amyloidogenic APP cleavage, with production of several APP-Fs including Aβ (De Chiara et al., 2010; Piacentini et al., 2011). In particular, we found that infection of SH-SY5Y human neuroblastoma cells and rat cortical neurons with HSV-1 induces multiple cleavages of APP, which result in the intra- and/or extracellular accumulation of several APP-Fs with neurotoxic potential. Among them we found: (i) APP-Fs of 35 and 45 kDa (APP-F35 and APP-F45) that comprise portions of Aβ; (ii) N-terminal APP-Fs that are secreted extracellularly; (iii) intracellular C-terminal APP-Fs, including AICD; and (iv) Aβ40 and Aβ42 in the form of monomers and small oligomers (dimers and trimers). Notably, the fragment APP-F35 seems to be a large Aβ oligomer (probably a nonamer) that is typical of viral-induced APP processing given that it was only found in cells infected with HSV-1. Indeed, it was revealed with antibodies recognizing amino-acidic sequences in Aβ (e.g., 4G8 and M2◦, targeting amino acids 17–24 and 1–10 of Aβ, respectively), only 8–18 hpi (**Figures 1** and **2A**). Moreover, its formation was inhibited in the presence of β- and

γ-secretase inhibitors. APP-F45 was revealed by M2◦ only. On the contrary, neither of these APP-Fs were recognized by antibodies raised against the N- and C-terminals of APP (**Figure 1**), thus indicating that they are not APP cleavage end-products. We also demonstrated that APP-Fs were present in the extracellular space and they had the ability to induce apoptosis in the neighboring cells (De Chiara et al., 2010). The multiple cleavages of APP occurring in infected cells are produced in part by known components of the amyloidogenic APP processing pathway, i.e., host-cell β-secretase, γ-secretase, and caspase-3-like enzymes, and in part by other cellular or viral enzymes not yet identified. Incidentally, the increased production of Aβ also reflected in an increased production and nuclear accumulation of AICD fragment that it is known to play a role in AD by modulating gene transcription.

It has been hypothesized that viral glycoprotein B is responsible for Aβ aggregation because this glycoprotein shares a significant portion of amino acid sequence similarity with Aβ, and it might serve as"core" to trigger Aβ aggregation (Cribbs et al., 2000). However, we and others (Wozniak et al., 2007; Piacentini et al., 2011) found that HSV-1-induced increased Aβ immunoreactivity was not due to cross-reactivity of the anti-Aβ antibodies with this glycoprotein.

From a functional point of view, we reported that HSV-1 binding to neuronal membrane induced membrane depolarization leading to increased neuronal excitability and triggering action potentials. This depolarization was due to activation of persistent Na+ currents and inhibition of leak K+ currents. Neuronal hyperexcitability persisted over time and was observed in neurons also at 12 hpi. Downstream to this effect we observed increased intracellular Ca2+ signaling, mainly due to activation of L-type Ca2+ channels and opening of inositol trisphosphate receptors (InsP3Rs) that caused marked intracellular Ca2+ entry from extracellular medium and Ca2+ release from intracellular stores (Piacentini et al., 2011). As above described, this Ca2+ dysregulation may trigger neurodegeneration (Mattson and Chan, 2003; Piacentini et al., 2008a,b; Chakroborty et al., 2012). We also found that HSV-1 induced Ca2+-mediated APP phosphorylation at Thr668. This is a key event critically involved in APP processing and Aβ formation (Pierrot et al., 2006). We demonstrated that HSV-1 infection induces a significant accumulation of Aβ42 inside neurons (**Figure 2**), and this effect depended on Ca2+ signaling activation. Indeed, in the presence of nifedipine and/or 2-aminoethoxydiphenyl borate (2-APB), that are specific blockers of L-type Ca2+ channels and InsP3 receptors, respectively, intraneuronal Aβ accumulation was significantly reduced (Piacentini et al., 2011). Besides accumulating inside neurons, Aβ monomers and small oligomers (dimers and trimers) were also released in the culture medium of infected neurons and revealed by Western blot analysis.

Several other studies have investigated the link between HSV-1 and AD at molecular level, independently on APP processing. It has been demonstrated that HSV-1 infection causes several functional and molecular alterations including: neurodegeneration and AD-like phosphorylation of tau protein (Zambrano et al., 2008; Wozniak et al., 2009b), the latter involving the activation of the glicogen sinthase kinase 3; caspase-3-mediated cleavage of Tau

conditions. Double asterisk in the Western blot for 4G8 indicates the APP fragment F35, characteristic of HSV-1 infected cells. **(C)** Cartoon indicating the amino acid sequences targeted by the different antibodies we used for Western blot and immunofluorescence experiments: 4G8 recognizes all APP fragments containing the amino acid sequence 17–24 of Aβ, independently on its cleavage.  Western blots in the panel **B** refer to previously published data (De Chiara et al., 2010).

**FIGURE 2 | HSV-1 infection induces intracellular accumulation of several APP fragments.** Representative examples of mock and HSV-1-infected (18 hpi) rat cortical neurons immunostained for Aβ42 C-terminus (green) and HSV-1 (red; **A)**; pAPP*Thr*668/AICD*pThr*668 (green) and MAP-2 (red; **B)**. Nuclei are stained in blue with DAPI. White arrowheads in the panel **A** (right) indicate intracellular accumulation of Aβ42 in infected (HSV-1-positive) neurons. In the panel **B** (right), yellow arrowheads indicate nuclear accumulation of AICD*pThr*668 in infected neurons (MAP-2-positive) whereas white arrowheads indicate staining for putative APP full-length phosphorylated at Thr668. Images in the panels **A** and **B** refer to previously published data (Piacentini et al., 2011).

(Lerchundi et al., 2011); activation of the arachidonic acid cascade, which is involved in AD-type neuropathological changes (Hill et al., 2009). With regard to the interaction between herpes simplex viruses and APP, it has been recently shown that HSV-1 alters the distribution of cellular APP, and APP and HSV-1 capsid proteins physically interact to allow the migration of new viral particles inside infected cells by fast anterograde transport mechanisms (Cheng et al., 2011). This also suggests that HSV-1 affects APP dynamic and its intracellular distribution thus possibly causing alterations in its metabolism and processing.

### **POSSIBLE HSV-1 RELATED THERAPEUTIC INTERVENTIONS**

Experimental evidence reviewed above allows us to hypothesize that, in the near future, treatments aimed at preventing and/or delaying AD might include antiviral agents and/or target HSV-1 activated intracellular pathways. At the present, the main antiviral agent used for HSV-1 is Acyclovir. Acyclovir, that is the common name of acycloguanosine, targets infected cells and inhibits viral replication (Park et al., 1979). Wozniak et al. (2011) demonstrated that acyclovir reduced Aβ formation and Tau phosphorylation *in vitro*. This finding suggests that the appearance of molecular AD hallmarks depends on HSV-1 replication. Similarly, we observed that in rat cortical neurons, UV-inactivation of HSV-1 particles prevented Aβ42 formation and its intracellular accumulation, although the virus retained the ability to bind the neuronal membrane and trigger intracellular signaling leading to short-term APP phosphorylation (Piacentini et al., 2011). Itzhaki suggested that treatment with a variant of acyclovir, the valacyclovir, could be of great interest in treatment of HSV-1-induced neurodegeneration (Itzhaki and Wozniak, 2012). Indeed, valacyclovir displays greater bioavailability than acyclovir (5-fold to 10-fold), it crosses the blood brain barrier and has no toxicity when used in patients with multiple sclerosis (Friedman et al., 2005). Interestingly, lysine supplementation has been suggested to have a role in preventing the development of AD by reducing HSV-1 replication (Rubey, 2010).

Among the proposed pharmacological treatments for AD, the use of statins seems to be promising (Barone et al., 2013). It has been suggested that their beneficial effects may be related to their ability to modulate the entry of pathogens given that cholesterol synthesis inhibition blocks the entry, and limits neuronal spread, of HSV-1 (Hill et al., 2005).

Finally, some studies also suggested the use of GSK-3β inhibitors as possible candidates for AD treatment (King et al., 2014). Among them, lithium has been shown to have some beneficial effects on AD symptoms (see references in Forlenza et al., 2012; Nunes et al., 2013). Lithium has also been reported to inhibit HSV-1 replication both *in vitro* and *in vivo* (Ziaie and Kefalides, 1989; Ziaie et al., 1994; Amsterdam et al., 1996), thus allowing to speculate that its beneficial effects might also depend on its antiviral activity.

### **CONCLUSION**

Data reviewed here support the hypothesis that recurrent HSV-1 infection in the brain may have a critical role in AD pathogenesis by directly activating intracellular pathways leading to typical AD molecular hallmarks. Studies ongoing in our laboratory on *in vivo* models of recurrent mild brain HSV-1 infection will expectedly further support the causal relationship between HSV-1 reactivation in the CNS and AD-like cognitive decline. Collectively, data discussed in this manuscript indicate that greater attention should be paid to infectious and, especially, viral agents among the environmental factors contributing to AD pathogenesis.

#### **ACKNOWLEDGMENTS**

This work was supported by grant from the Italian Ministry of Research (PRIN-2009PM9B33) to Claudio Grassi and Anna T. Palamara.

## **REFERENCES**

seizure susceptibility in mice. *Eur. J. Neurosci.* 18, 3294–3304. doi: 10.1111/j.1460- 9568.2003.03075.x

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

*Received: 28 February 2014; accepted: 16 April 2014; published online: 07 May 2014. Citation: Piacentini R, De Chiara G, Li Puma DD, Ripoli C, Marcocci ME, Garaci E, Palamara AT and Grassi C (2014) HSV-1 and Alzheimer's disease: more than a hypothesis. Front. Pharmacol. 5:97. doi: 10.3389/fphar.2014.00097*

*This article was submitted to Experimental Pharmacology and Drug Discovery, a section of the journal Frontiers in Pharmacology.*

*Copyright © 2014 Piacentini, De Chiara, Li Puma, Ripoli, Marcocci, Garaci, Palamara and Grassi. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) 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.*

## The battle of Alzheimer's Disease – the beginning of the future Unleashing the potential of academic discoveries

*Johan Lundkvist 1, Magnus M. Halldin1, Johan Sandin1, Gunnar Nordvall 1, Pontus Forsell 1, Samuel Svensson1, Liselotte Jansson2 , Gunilla Johansson3 , BengtWinblad3 \* and Jonas Ekstrand1\**

*1 AlzeCure Foundation, Karolinska Institutet Science Park Novum, Huddinge, Sweden*

*2 Alzheimerfonden, Frösundavik, Solna, Sweden*

*3Center for Alzheimer Research at Karolinska Institutet and Swedish Brain Power, Novum, Huddinge, Sweden*

#### *Edited by:*

*Cesare Mancuso, Catholic University School of Medicine, Italy*

#### *Reviewed by:*

*Cedric Annweiler, University Hospital of Angers, France Domenico Valle, Eli Lilly Italia, Italy*

#### *\*Correspondence:*

*Bengt Winblad, Center for Alzheimer Research at Karolinska Institutet and Swedish Brain Power, Novum, Huddinge, Sweden e-mail<EMAIL_ADDRESS>Jonas Ekstrand, AlzeCure Foundation, Karolinska Institutet Science Park Novum, Huddinge, Sweden e-mail<EMAIL_ADDRESS>
Alzheimer's Disease (AD) is the most common form of dementia, affecting approximately 36 million people worldwide. To date there is no preventive or curative treatment available for AD, and in absence of major progress in therapeutic development, AD manifests a concrete socioeconomic threat.The awareness of the growing problem of AD is increasing, exemplified by the recent G8 Dementia Summit, a meeting held in order to set the stage and steer the compass for the future. Simultaneously, and paradoxically, we have seen key players in the pharmaceutical industry that have recently closed or significantly decreased their R&D spending on AD and other CNS disorders. Given the pressing need for new treatments in this area, other actors need to step-in and enter this drug discovery arena complementing the industrial efforts, in order to turn biological and technological progress into novel therapeutics. In this article, we present an example of a novel drug discovery initiative that in a non-profit setting, aims to integrate with both preclinical and clinical academic groups and pharmaceutical industry to explore the therapeutic potential of new concepts in patients, using novel biology, state of the art technologies and rapid concept testing.

**Keywords: Alzheimer's disease, drug discovery and development, technology, pharmaceutical, non-profit, collaborative research**

## **INTRODUCTION**

During the last 25 years the population of the world has increased from 5.3 to almost 7.2 billion people (World Population Review, 2013). While an increased standard of living has resulted in decreased mortality, a global increased awareness of healthy living in combination with major progress in several areas of medicine has also made significant contribution to the increase in world population. Indeed, the death caused by heart disease, stroke, and cancer have decreased in the USA from 68% (1980) to 53% (2010) (75 Years of Mortality in the United States, 1935–2010, 2012). In parallel with these encouraging numbers, another major medical threat is emerging which could result in a major socioeconomic chaos in the absence of medical progress. The threat is named Alzheimer's Disease (AD). Worldwide, approximately 36 million people are diagnosed having dementia and 7.7 million new cases are discovered every year. AD is the most common cause of dementia among the elderly and may contribute up to 60–70% of all cases (World Health Organization, 2012). Currently there is no curative or preventive treatment for the patients and only a few drugs, which all provide limited symptomatic relief during a relatively short time frame, are available. Age is by far the largest risk factor for developing AD and the incidence increases exponentially from about 1% of 65-year old people to 30% of all 85-year-old people. With the current global increase in average lifespan, approximately 115 million people are estimated to be suffering from AD in 2050. Besides the tremendous suffering for the affected individuals and their close relatives, the cost

for the society is estimated to be more than 200 billion USD in 2013 and 1200 billion USD by 2050 in the USA alone, a number that will put the healthcare system under enormous strain (Alzheimer's Association, 2013; World Alzheimer Report 2013, 2014).

Awareness, at the highest political level, of the emerging threat of dementia was highlighted with the recent G8 summit on dementia held in London in December 2013. The aim of the meeting was to develop a coordinated global action on dementia, and to shape an effective international response to dementia, resulting in the communication of a declaration (G8 Dementia Summit Agreements, 2013). This declaration included; "an ambition to identify a cure, or a disease-modifying therapy, for dementia by 2025." This will be achieved by, e.g., significantly increasing the amount spent on dementia research, increasing the number of people involved in clinical trials and studies on dementia as well as develop an international action plan for research. These efforts clearly state the sense of urgency in this field to move forward and for the research community to find a solution.

Dementia or senility as a concept or condition is a very old phenomenon, referred to in medical texts since antiquity. Today, we are aware of a large number of diseases that cause dementia but AD represents the most common one (Ballenger, 2006; Lane et al., 2012; Savonenko et al., 2012; Agis-Torres et al., 2014). A major milestone in the history of AD took place approximately 100 years ago (1907), when the German neuropsychiatrist Alois

Alzheimer first described the key neuropathological hallmarks of the disease (Hardy, 2006). During the examination of brain sections derived from his patient Auguste D, a middle-aged woman who suffered and died from dementia, Dr. Alzheimer discovered so-called senile plaques and neurofibrillary tangles in the brain parenchyma. These were seminal findings, linking for the first time the form of dementia now called "Alzheimer's disease," to specific pathological changes within the brain. However, the recognition that dementia was a result of abnormal pathological changes distinct from normal aging, did not really gain momentum until the 1970s, with the birth of the cholinergic hypothesis. A number of studies showed that, in AD brains, there was a particular loss of markers for cholinergic function as well as basal forebrain cholinergic neurons early on in the disease pathology (Francisa et al., 1999). These discoveries fueled an intense research effort to find drugs that could target cholinergic dysfunction and today three out of the four commonly prescribed drugs for AD (donepezil, rivastigmine and galantamine) are socalled cholinesterase inhibitors (Greenberg et al., 2013). These drugs inhibit the degradation of acetylcholine in the synaptic cleft, thereby sustaining the action of acetylcholine with a subsequent improvement in cognitive function in the patient. However, the underlying neurodegenerative cascade is not affected by these treatments, and the effect is thereby limited in time due to the continuous loss of cholinergic neurons and thus acetylcholine production in the disease. Moreover, gastrointestinal side effects, relating to muscarinic receptor engagement in the gut, as well as headache and effects on heart rate are also dose-limiting factors for the patients. The fourth described drug for AD is memantine, a weak NMDA receptor antagonist, prescribed for moderate to severe AD. Similar to the acetylcholinesterase inhibitors, memantine provides symptomatic relief for a limited time period, but is not believed to alter the neurodegenerative cascade of the disease.

## **ALZHEIMER'S DISEASE – NAVIGATION THROUGH COMPLEXITY**

Although the progress in the drug development for AD has been limited over the last 30–40 years, significant progress has been made in understanding the mechanisms behind the development of AD, e.g., the pathology, neurobiology, and genetics (Corbette et al., 2012; Dunkel et al., 2012; Kepp, 2012; Greenberg et al., 2013). Among these finding were the discoveries of the amyloid beta peptide (Aβ) and the tau protein, the principal components of senile plaques and neurofibrillary tangles, respectively. Furthermore, researchers were also able to identify more than 200 disease causing mutations localized to three genes directly involved in Aβ generation as well as unravel some of the molecular machinery and underlying mechanisms of Aβ production. Combined, these discoveries provided a strong rationale for a better understanding of the neurotoxic role of Aβ in AD and for the development of Aβ amyloid-directed therapies. Accordingly, during the last 15 years, both active and passive vaccines targeting Aβ as well as several inhibitors and modulators targeting the aggregation or synthesis of Aβ peptide has been developed and tested in AD clinical trials. In addition, a large number of other drug candidates exhibiting a range of different non-Aβ targeting therapeutic mechanism of

#### **BOX 1 | Some key challenges to be addressed in the development of novel therapeutics for Alzheimer's disease.**

Identify and link novel pathways and biomarkers to AD progression.

Improved preclinical modeling of disease relevant pathology. Engaging multiple targets might be required (polypharmacology) and thereby using a multi-drug regimen. Establish and conduct conclusive proof-of-concept (PoC) studies. Use appropriate patient inclusion criteria in clinical trials.

Achieve sufficient CNS exposure/target engagement of the drug. Establish appropriate biomarkers and/or surrogate markers for target engagement.

Establish surrogate markers for clinical effect to shortened the length of clinical trial.

Feedback to discovery from clinical PoC to refine hypothesis and improve research models.

Handle potential drug–drug interactions (patient population often on other medications).

action have been tested clinically. These include those that are primarily aiming for a symptomatic effect by increasing synaptic activity, e.g., compounds that interact with nicotinic, histaminergic, and serotonergic receptors (Misra and Medhi, 2013). Despite that many of these drug candidates have shown promising effect in preclinical models, the results from clinical trials have been disappointing, and no new drugs after the acetylcholine esterase inhibitors and the NMDA blocker memantine have made it all the way to the market. It is well recognized that drug discovery in the AD field has been hampered by the failure of preclinical models to recapitulate some of the key features of the AD pathogenesis (Savonenko et al., 2012). The use of these models to unravel the complex features of the neurodegenerative cascade in AD, as well as translational tools in, e.g., dose prediction studies, have therefore been limited.

The reasons for the clinical failures are likely many, and differ among the drug candidates tested (Mangialasche et al., 2010; Schneider et al., 2014). Major contributors to the general lack of success include insufficient target exposure to achieve a clinically meaningful effect, that the drug was not tested long enough, safety issues due to on- and off-target pharmacology and/or that the disease context was not appropriate for the therapeutic mechanism explored (see **Box 1**). The latter stems from recent progress in biomarker research, which has shown that the AD pathological cascade start early and takes place over decades prior to symptoms onset, and where Aβ amyloidosis dominates at early stages followed by more overt and outbread neuronal dysfunction and neuronal degeneration, implicating other pathogenic drivers of disease, once the disease becomes symptomatic and progresses into severe stages of dementia (Sperling et al., 2011). Therefore, a therapy tailored for a specific component of the AD pathogenic cascade may have to be given to patients at a specific stage of the disease in order to be efficacious. The recent development of specific radiopharmaceutical diagnostic tools for PET imaging

Aβ-amyloid plaque density (e.g., florbetapir and florbetaben) has been extremely useful in assessing amyloid load in patients at various stages of the disease (Herholz and Ebmeier, 2011). Thanks to these new methods with improved resolution combined with improvements in biomarker monitoring, the characterization and diagnosis of patients suspected to be suffering from AD pathogenesis have improved. Interestingly, such analyses have revealed that as many as 25–30% of the patients included in recent trials with therapeutic antibodies targeting Aβ amyloidosis were in fact negative for Aβ-amyloid in their brains (Doody et al., 2014; Salloway et al., 2014). These observations highlight the need to design trials where a lot of emphasis is put on the patient inclusion criteria in order to increase the odds to get meaningful clinical response of the therapeutic compound and/or mechanism tested.

## **FUTURE IDEAS TOWARDS NOVEL AD THERAPEUTICS THE POTENTIAL OF ACADEMIC DISCOVERIES IN AD**

The pharmaceutical industry (pharma) is currently under pressure from a range of different challenges in its environment, e.g., increased R&D costs, patent cliffs causing a major loss of income, high attrition of projects in the portfolio, increasing cost-constraints in the healthcare system mandating generics and parallel import, and not the least, more demanding regulatory requirements (Kola and Landis,2004;Cuatrecasas,2006; Schachter and Ramoni, 2007; Munos, 2009; Paul et al., 2010; Scannell et al., 2012; Slusher et al., 2013). In our view, an attractive alternative to the current way of working, as well as a way to offer a potential solution to the gaps in drug discovery and development pipelines, is to increase the research efforts in non-profit biopharmaceutical research institutions, such as universities and private research institutes, where creative and innovative science is not severely restricted by commercial objectives, but where novel hypothesis can be tried with industrial stringency in a dedicated way. These types of smaller research units could make significant contributions to the field since it is well recognized that small- and medium-sized biotechnological companies have been more successful than big pharmaceutical companies in moving candidate substances through the pipeline, and in particular, more successful in producing biological products, such as monoclonal antibodies, vaccines, and peptides (Munos, 2009). Non-profit organizations are well suited to address areas of large unmet need in rare CNSdisorders classified as orphan diseases. These diseases are, on the whole, not commercially blockbusters and, thus, research funded by philanthropists and public sources will be key to encourage work in these areas. It is also likely that certain therapeutics developed for an orphan indication may turn out to be efficacious in related disorders that share certain common features or principles of pathogenesis. The availability of clinically validated drugs is extremely valuable, and repositioning of existing drugs for novel indications opens up novel exciting possibilities to accelerate therapeutic development in complex CNS disorders such as AD.

Today when many major pharmaceutical companies are downsizing their R&D efforts in the CNS area (Alzheimer's Drug Failure: implication for Future R&D in Neuroscience, 2012), they have a strong need to get access to outside expertise in specific scientific areas such as for the identification of novel targets, increasing the knowledge in certain target families, advancing new platform

technologies for preclinical and clinical research, improving the diagnosis of disease, pharmacology and safety biomarker development, access to human cells and tissues for target validation, as well as how to perform earlier proof-of-mechanism (PoM) and proof-of-concept (PoC) studies, including clinical safety studies. Some of this will of course be possible through established collaborations between the pharmaceutical industry and third parties such as contract research organization (CRO) and academia, such an example is the Dominantly Inherited Alzheimer Network (DIAN, established in 2008), which test diagnostics and novel treatments in a specific patient population. Although many CROs now provide customers with various non-regulatory *in vitro* and *in vivo* experiments during early discovery, their main activities are regulatory-driven (in accordance with good laboratory practice (GLP) in later phases of drug development). However, we see an attractive alternative in non-profit biomedical research institutions, where scientific collaborations can be based on a close interaction rather than contracting. This collaborative network/partnership will facilitate a closer preclinical-to-clinical translation at the target, model, assay, substance, patient and clinical testing levels as well as in the development of new technologies. Innovation and progress driven in these areas by such constellations will certainly accelerate therapeutic development in AD, and thereby attract increased interest from the big pharmaceutical companies, given that the potential return of investment would be substantial.

## **AlzeCure FOUNDATION – AN EXAMPLE**

AlzeCure is a Swedish-based non-profit drug discovery foundation, whose aim is to develop novel therapeutics for AD and related disorders (see **Box 2** and **Figure 1**). This new organization was founded in 2013 by former AstraZeneca scientists with more than 30 years of experience from the CNS drug discovery and development area. The team exhibits complementary skills and expertise,from medicinal chemistry, screening, *in vivo* translational/biomarker competence to ADME and clinical development. The strategy of AlzeCure Foundation ("AlzeCure") is to use its wide experience and international network in the Alzheimer field to run collaborative projects with external expertise that has the potential to deliver real value for the patients. The team focuses on areas of research and development, which are of urgent need of innovative solutions, such as mechanistic diversity, preclinical models, drug delivery, clinical testing, and biomarker monitoring. We are convinced that rapid feedback to the drug discovery process from positive or failed PoC studies are essential in order to improve our understanding and to progress the development of efficacious therapeutics. Such backtranslation is also crucial in order to accelerate the development of follow-on compounds that address the same mechanism, but have improved drug-like characteristics, or to identify related mechanistic targets that will lead to new clinical studies. History has proven that there is a synergy between technology and biology innovation and in that interface, exciting discoveries take place that can result in novel products with a remarkable potential. Similarly, we strongly believe that technology breakthroughs will contribute significantly to speed up and improve the development of novel therapies in major CNS disorders.

Design early PoM and PoC clinical studies to test new hypothesis.

Optimize patient selection for earlier clinical safety and efficacy studies, based on qualified clinical biomarkers.

The laboratories of AlzeCure are located in direct vicinity to biotech companies and Karolinska Institute Alzheimer Research Center with academic groups of leading experts in neurobiology, preclinical and clinical laboratories with a long track record in AD translational research and in clinical trials. The strategic localization of AlzeCure has resulted in face-to-face interactions with these institutions and disciplines on a regular basis, which is a strong enabling factor for effective project progression and success. The non-profit nature of AlzeCure has turned-out to facilitate the establishment of joint efforts with both academia and industry and attract alternative funding. Today, AlzeCure manages several collaborations with international academic experts in both preclinical and clinical research. AlzeCure collaborates with several biotech companies, which provide unique chemistry and technology that in combination with neuronal screening assays based on induced pluripotent stem cells (iPS) and phenotypic endpoints, provide the basis for novel drug discovery. The most advanced collaboration aims at a novel way of administering therapeutic tau-directed antibodies to the brain, in order to increase target engagement and thereby therapeutic activity1. Tau is emerging as a new important molecular target in AD, and several anti-tau antibodies are rapidly approaching clinical trials. However, a major challenge with peripheral administration of antibodies is to reach the target in the brain to a sufficient extent. AlzeCure is together with NsGene, a Denmark and US-based biotechnology company, and Peter Davies (Feinstein Institute) evaluating a new concept to deliver therapeutic antibodies to the brain, using a novel proprietary Brain-Repair technology platform developed by NsGene (Lindvall and Wahlberg, 2008). The Brain-Repair platform is an implant comprised a semi-permeable hollow fiber containing a human cell line, which is engineered to locally produce therapeutic proteins of choice that diffuse into the target tissue. The implant has been clinically validated in patients diagnosed with AD through collaboration between NsGene and the Karolinska

Institute (Eriksdotter-Jönhagen et al., 2012). An opportunity with this device would be to more rapidly test the hypothesis in a PoC study in a well-defined tauopathy patient population for an orphan indication, before entering large expensive phase III trials. In this manner, we foresee getting both the technology, therapeutic mode of action, clinical effect, and safety evaluated, in an effective and conclusive manner within a shorter time frame compared to common standards of clinical testing in AD. Obviously the Brain-Repair implant also offers a very exciting concept to rapidly test other therapeutic hypothesis beyond tau-directed antibodies, and illustrates a concrete example where technology innovation together with new biology form a novel avenue for therapy development applicable for multiple CNS disorders.

A co-initiator and also major financial contributor to AlzeCure is the Swedish Alzheimer foundation, a charity organization. This type of direct financial commitment and interest in drug discovery and development in complex disorders such as CNS diseases from charity organizations is novel and may point to a new path for future organizations. In addition, the Swedish Brain Power program is sponsoring AlzeCure with two Ph.D. positions, which are highly integrated into the academic labs thereby providing the basis for a strong two-way communication between academic science and industrial development, which is of high mutual interest. Given the declining interest from many big pharmaceutical companies and venture capitalists alike in CNS disorders and the pressing need for new medications, initiatives like these are new ways of trying to directly stimulate drug discovery efforts in an area of great medical need. Interestingly, in line with the intention of AlzeCure, Alzheimer Research UK announced at the end of 2013 that they were to initiate a Drug Discovery Institute consisting of leading academic groups in the UK that will have close access to both preclinical and clinical research units and hospitals specialized in neurodegenerative diseases that cause dementia.

#### **CONCLUSION**

The problem we as a society are facing is of unseen proportions, highlighting the need of concerted actions at multiple levels of society. A firm political leadership paired with a well-designed regulatory framework and strong incentives for academia, the industry and the health care providers is needed in order to consolidate the resources and optimize the process required to make rapid and significant progress in therapeutic development for AD. In this light it may seem paradoxical that several major industrial actors, such as AstraZeneca, Bristol-Myers Squibb, GlaxoSmithKline, and Novartis have either abandoned the neuroscience therapeutic indication as such, or heavily decreased their head count within this area (Abbot, 2011). However, other companies, e.g., Eli Lilly, Roche, Pfizer have continued a strong interest in the area and are closely collaborating the regulatory agencies, which recently resulted in a Guidance for Industry from FDA on Alzheimer's disease: Developing drugs for the treatment of early stage disease (U.S. Food and Drug Administration, 2013). The medical need is however still unmet and rapidly growing, thus something radical needs to be done. For a start, we believe that new initiatives such as the AlzeCure Foundation and Drug Discovery Alzheimer Research UK group, both being non-profit and

1See www.alzecure.org

taking full advantage of the open-minded academic style of working with the stringent and goal-oriented industrial R&D, have the promise of being more innovative and effective unleashing the potential of academic discoveries and providing an exciting new framework for the long-term objective to deliver effective therapies to the benefit of the patients.

#### **REFERENCES**

Abbot, A. (2011).  *Nature* 480, 161–162. Agis-Torres, A., Sölhuber, M., Fernandez, M., and Sanchez-Montero, J. M. (2014). Multi-target-directed ligands and other therapeutic strategies in the search of

**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.

*Received: 04 March 2014; accepted: 18 April 2014; published online: 09 May 2014. Citation: Lundkvist J, Halldin MM, Sandin J, Nordvall G, Forsell P, Svensson S, Jansson L, Johansson G, Winblad B and Ekstrand J (2014) The battle of Alzheimer's Disease – the beginning of the future Unleashing the potential of academic discoveries. Front. Pharmacol. 5:102. doi: 10.3389/fphar.2014.00102*

*This article was submitted to Experimental Pharmacology and Drug Discovery, a section of the journal Frontiers in Pharmacology.*

*Copyright © 2014 Lundkvist, Halldin, Sandin, Nordvall, Forsell, Svensson, Jansson, Johansson, Winblad and Ekstrand. 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.*

## Sirtuin modulators control reactive gliosis in an in vitro model of Alzheimer's disease

## *Caterina Scuderi 1 †, Claudia Stecca1 †, Maria R. Bronzuoli 1, Dante Rotili 2 , Sergio Valente2 , Antonello Mai 2,3 and Luca Steardo1\**

*1 Vittorio Erspamer School of Physiology and Pharmacology, SAPIENZA University of Rome, Rome, Italy*

*2 Department of Drug Chemistry and Technologies, SAPIENZA University of Rome, Rome, Italy*

*3 Institute Pasteur – Cenci Bolognetti Foundation, SAPIENZA University of Rome, Rome, Italy*

#### *Edited by:*

*Cesare Mancuso, Catholic University School of Medicine, Italy*

#### *Reviewed by:*

*Alexej Verkhratsky, University of Manchester, UK Marcus Rattray, University of Bradford, UK*

#### *\*Correspondence:*

*Luca Steardo, Vittorio Erspamer School of Physiology and Pharmacology, SAPIENZA University of Rome, Piazzale Aldo Moro 5, 00185 Rome, Italy e-mail<EMAIL_ADDRESS>
†*Caterina Scuderi and Claudia Stecca have contributed equally to this work.* Among neurodegenerative disorders, Alzheimer's disease (AD) represents the most common cause of dementia in the elderly. Several genetic and environmental factors have been identified; however, aging represents the most important risk factor in the development of AD. To date, no effective treatments to prevent or slow this dementia are available. Sirtuins (SIRTs) are a family of NAD+-dependent enzymes, implicated in the control of a variety of biological processes that have the potential to modulate neurodegeneration. Here we tested the hypothesis that activation of SIRT1 or inhibition of SIRT2 would prevent reactive gliosis which is considered one of the most important hallmark of AD. Primary rat astrocytes were activated with beta amyloid 1-42 (Aβ 1-42) and treated with resveratrol (RSV) or AGK-2, a SIRT1 activator and a SIRT2-selective inhibitor, respectively. Results showed that both RSV and AGK-2 were able to reduce astrocyte activation as well as the production of pro-inflammatory mediators. These data disclose novel findings about the therapeutic potential of SIRT modulators, and suggest novel strategies for AD treatment.

**Keywords: resveratrol, AGK-2, sirtuins, beta-amyloid, astrocyte, reactive gliosis, Alzheimer's disease**

## **INTRODUCTION**

Alzheimer's disease (AD) represents one of the major health concern and it is a research priority since there is a pressing need to develop new agents to prevent or treat it. A part of the progressive deposition of beta amyloid peptide (Aβ) and accumulation of phosphorylated tau, several other alterations occur in AD brain, all concurring to neuronal loss. Among these, growing interest has been attracted by the role of inflammation in the onset and progression of this disorder. In fact, senile plaques and neurofibrillary tangles (which are considered the more characteristic hallmarks of AD) co-localize with activated astrocytes, suggesting for these cells a key role in the pathogenesis of AD (Meda et al., 2001; Craft et al., 2006). Along this line, in several experimental models it has been demonstrated that Aβ peptide fragments markedly alter astrocytes functions. This process is accompanied with a noticeable neuroinflammatory response, accounting for the synthesis of different cytokines and pro-inflammatory mediators which amplify neuropathological damage (Mrak and Griffin, 2001; Caricasole et al., 2003; Tuppo and Arias, 2005; Griffin, 2006; Scuderi et al., 2011, 2012, 2013). It is established that neuroinflammation is directly linked to neural dysfunction and cell death, representing a primary cause of neurodegeneration (Block and Hong, 2005). In fact, over-release of pro-inflammatory cytokines by glia cells causes neuronal dysfunction and loss of synapses, which correlates with memory decline. These phenomena are believed to precede neuronal death. Thus, research focused on developing therapeutic strategies directed at controlling the prolonged and uncontrolled glia activation should be encouraged.

An uncommon opportunity to improve inflammation and neurodegeneration simultaneously is provided by compounds able to modulate histone acetylation/deacetylation, since they participate in brain immune control and neuroprotection, in addition to their well-known effects on the molecular mechanisms associated to senescence and metabolic syndromes. Mounting evidence indicates sirtuins (SIRTs) exert neuroprotective effects in several models of neurodegeneration (Outeiro et al.,2008; Tang and Chua, 2008; de Oliveira et al., 2010). SIRTs, a family of NAD+-dependent enzymes with seven isoforms identified (SIRT1-7), are implicated in the control of a variety of biological processes including transcriptional silencing, chromosomal stability, cell cycle progression, apoptosis, autophagy, metabolism, growth suppression, inflammation, and stress response (Gan and Mucke, 2008; Haigis and Sinclair, 2010).

Recent observations indicate both SIRT1 and SIRT2 regulate neuronal survival, but with divergent functional outcomes. Indeed, activation of SIRT1 mainly exerts neuroprotective actions, while SIRT2 fosters neurodegeneration. The reason for such opposite effect may be due to their different sub-cellular localization, which gives SIRT1 and SIRT2 distinct molecular targets (Harting and Knöll, 2010). It has been demonstrated that the overexpression of SIRT1 prevents neuronal death in tissue culture models of AD, amyotrophic lateral sclerosis, and polyglutamine toxicity, and it reduces hippocampal degeneration in a mouse model of AD (Kim et al., 2007; Li et al., 2007). Moreover, treatment with resveratrol (RSV), a polyphenolic compound acting as a pharmacological activator of SIRT1, is protective in a number

of experimental neurodegeneration paradigms (Anekonda, 2006; Sun et al., 2010). Resveratrol, like other polyphenol compounds including curcumin, displays a plethora of actions, behaving as a potent antioxidant agent, increasing SUMOylation, and activating protein kinase C, all mechanisms able to counteract astrocyte reactivity and protect neurons (Jefremov et al., 2007; Hoppe et al., 2013; Menard et al., 2013). Finally, it has been observed that both SIRT1 overexpression and RSV treatment are able to significantly decrease the Aβ-induced activation of NF-κB, thus operating a simultaneous control on both neurodegeneration and neuroinflammation processes (Chen et al., 2005). Indeed, NF-κB is a transcription factor which controls the expression of gene products involved in key cellular signaling, including those associated to inflammatory and degenerative events. Post-mortem studies on cerebral cortices from AD patients have established a correlation between loss of SIRT1 and the accumulation of Aβ and hyperphosphorylated tau proteins (Julien et al., 2009). Growing evidence indicates that also SIRT2 is involved in regulating several brain processes including oligodendrocyte mitosis and differentiation, cytoskeletal dynamics necessary for trafficking, neurite outgrowth and synaptic remodeling. Unlike SIRT1, SIRT2 appears to promote neuronal death. In fact, blocking SIRT2 counteracted alpha synuclein toxicity in Parkinson's disease models (Outeiro et al., 2007). However, less is known about the role of SIRT2 in AD.

On the basis of these considerations, we explored the effects of modulators of SIRTs on astrocyte activation and the subsequent inflammatory process. In particular our experiments focalized the ability of RSV, a SIRT1 activator, and AGK-2, a SIRT2-selective inhibitor, to control astrocyte activation and to suppress the production of pro-inflammatory mediators in primary rat astrocytes exposed to Aβ peptide. These findings suggest that either RSV or AGK-2 may be an effective agent for neurodegenerative diseases initiated or maintained by inflammatory processes.

## **MATERIALS AND METHODS**

## **CELL CULTURES AND TREATMENTS**

Newborn Sprague-Dawley rats (1 or 2 days old) were used to obtain primary astroglial cultures (Vairano et al., 2002; Scuderi et al., 2011). Briefly, brain cortices were homogenized and processed to obtain single cells. Astrocytes were cultured at a density of 3 × 106 cells/75-cm2 flask and incubated at 37◦C in a humidified atmosphere containing 5% CO2. The culture medium used was DMEM supplemented with 5% inactivated fetal bovine serum, 100 IU/ml penicillin and 100 μg/ml streptomycin (all from Sigma–Aldrich, Milan, Italy), replaced 24 h after isolation and again one a week until astrocytes were grown to form a monolayer. Approximately 14–15 days after dissection, astrocytes were mechanically separated from microglia and oligodendrocytes. Obtained astrocytes were seeded onto 10-cm-diameter Petri dishes (1 × 106 cells/dish) or onto 24 well plates (1 x 105 cells/well). The monoclonal anti-glial fibrillary acidic protein (GFAP) was used to verify cell culture purity. Only cultures with more than 95% GFAP-positive cells were utilized. The 5% of non-astrocyte cells were microglia and oligodendrocytes.

All experiments were performed in accordance with the Italian Ministry of Health (DL 116/92), the Declaration of Helsinki, and the Guide for the Care and Use of Mammals in Neuroscience and Behavioral Research, and they were approved by the Institutional Animal Care and Use Committee at our institution.

Mature astrocytes were challenged with 0.23 μM Aβ 1-42 (Tocris Bioscience, Bristol, UK) in the presence or absence of the following substances: RSV (2 – 10 – 50 μM), a well-known SIRT1 activator, or AGK-2 (0.35 – 3.5 – 35 μM), a potent SIRT2-selective inhibitor (both from Sigma–Aldrich). After 24 (for viability and protein expression analyses) or 72 h (for proliferation assay) of treatment, astrocytes were collected for experiments. The concentration of the substances was chosen according to literature (Howitz et al., 2003; Outeiro et al., 2007; Scuderi et al., 2011, 2012).

## **ANALYSIS OF ASTROCYTE VIABILITY BY NEUTRAL RED UPTAKE ASSAY**

Astrocyte viability was evaluated 24 h after treatments by the neutral red uptake assay according to Repetto et al. (2008), with some modifications (Scuderi and Steardo, 2013). Cells were seeded in 24-well plates and treated as previously described. 24 h after treatments, the plates were incubated for 3 h at 37◦C with a neutral red working solution (50 μg ml−1 in PBS 1X without calcium and magnesium, Sigma-Aldrich). The cells were washed and the dye removed from each well through a destain solution (ethanol:deionized water: glacial acetic acid, 50:49:1, v/v). The absorbance was read at 540 nm using a microplate spectrophotometer (Epoch, Bio Teck, Winooski, VT, USA). The values of treated cells were referred to control non-exposed cultures, and expressed as percentage variation.

## **ANALYSIS OF ASTROCYTE PROLIFERATION BY TRYPAN BLUE ASSAY**

Trypan blue exclusion assay was performed to monitor astrocyte proliferation 72 h after treatments. This method is based on the principle that living cells do not take up the dye, whereas dead cells do. To determine the number of cells and their viability using trypan blue, 20 μl of trypsinized and re-suspended cells were mixed with 20 μl of 0.4% solution of trypan blue dye (Sigma–Aldrich) for 1 min. Cells were immediately counted using a Bürker chamber with a light microscope. All counts were done using four technical duplicates of each sample.

## **ANALYSIS OF PROTEIN EXPRESSION BY WESTERN BLOTTING**

Western blot analyzes were performed on extracts of cell cultures challenged as previously described. 24 h after treatment, cells were detached from petri dishes and each pellet was suspended in ice-cold hypotonic lysis buffer containing NaCl 150 mM; Tris/HCl pH 7.5 50 mM; Triton X-100 1%; ethylenediaminetetraacetic acid [EDTA] 1 mM, supplemented with PMSF 1 mM, Aprotinin 10 μg/ml, Leupeptin 0,1 mM (Roche, Mannheim, Germany). After incubation for 40 min at +4◦C, homogenates were centrifuged at 14000 rpm for 15 min and the supernatant removed and stored in aliquots at −80◦C until use. Equivalent amounts (70 μg) of each sample calculated by Bradford assay were resolved on 12% acrylamide SDS-PAGE precast gels (Bio-Rad Laboratories). Proteins were transferred onto nitro-cellulose. Membranes were blocked with 5% wt/vol no-fat dry milk powder in Tris-buffered saline-Tween 0,1% (TBS-T) for 1 h before

overnight incubation at 4◦C with one of the following primary antibodies: rabbit anti-GFAP (1:50000, Abcam plc, Cambridge, UK), rabbit anti-S100B (1:1000, Epitomics, Burlingame, CA, USA), rabbit anti-inducible nitric oxide synthase (iNOS; 1:9000, Sigma–Aldrich), rabbit anti-cyclooxygenase-2 (COX-2; 1:1000, Cell Signaling Technology, MA, USA), rabbit anti-β-actin (1:1500, Santa Cruz Biotechnology, Santa Cruz, CA, USA). After being extensively washed in TBS-T, membranes were incubated for 1 h at 25 ◦C with the secondary horseradish peroxidase-conjugated antibody (HRP conjugated goat anti-rabbit IgG, 1:30000, Jackson Immunoresearch Europe, Suffolk, UK). The immunocomplexes were visualized using an ECL kit (Amersham, Bucks, UK). Protein expression was quantified by densitometric scanning of the X-ray films with a GS 700 Imaging Densitometer (Bio-Rad laboratories) and a computer program (ImageJ software v1.44p, NIH, USA).

## **STATISTICAL ANALYSIS**

Analysis was performed using GraphPad Prism (GraphPad Software, San Diego, CA,USA). Data were analyzed by one way analysis of variance (ANOVA) to determine statistical differences between experimental groups. Multiple comparisons were performed with Bonferroni's test for *post hoc* analyzes. Differences between mean values were considered statistically significant when *p* < 0.05.

## **RESULTS**

## **EFFECT OF RSV AND AGK-2 ON ASTROCYTE VIABILITY AND PROLIFERATION.**

First of all, we decided to perform experiments to assess the effect of the SIRT modulators on astrocyte viability and proliferation after Aβ 1-42 challenge. In fact, it has been already demonstrated that Aβ peptides are able to affect cell viability and to induce astrocyte proliferation (Allaman et al., 2010; Scuderi et al., 2012). Our results highlighted a significant increase in cell viability after 24 h treatment with Aβ 1-42 (**Figures 1A,C**, *p* < 0.01). RSV and AGK-2 were able to reduce this effect at the two higher concentrations used (**Figures 1A,C**). In addition, we found a reduction in cell viability after treatment with AGK-2 at the concentration of 35 μM on un-stimulated cells, indicating a cytotoxic effect (**Figure 1C**). Trypan blue experiments revealed a significant astrocyte proliferation after 72 h treatment with Aβ 1-42 (**Figures 1B,D**, *p* < 0.01). Once again, both RSV and AGK-2 significantly controlled such increase at the two higher concentrations used. Surprisingly, RSV 50 μM and AGK-2 35 μM caused a reduction in proliferation rate also in un-challenged astrocytes (**Figures 1B,D**).

## **EFFECT OF RSV AND AGK-2 ON ASTROCYTE ACTIVATION**

In order to test the effect of RSV and AGK-2 on Aβ-induced astrogliosis, the expression of GFAP and S100B, specific markers of astrocyte activity, was explored. Reactive astrocytes display hypertrophied cell bodies and thickened processes exhibiting GFAP-immunoreactivity (O'Callaghan and Sriram, 2005; Olabarria et al., 2010). Using Western blot analysis, we observed a marked increase in the expression of GFAP after Aβ 1-42 challenge (*p* < 0.01; **Figure 2**). RSV was able to significantly attenuate such increase in a concentration dependent manner (**Figures 2A,B**). Likewise, the Aβ-induced GFAP overexpression was counteracted by AGK-2 at the three concentrations used (**Figures 2C,D**).

Similarly, the expression of S100B was investigated by Western blot. S100B is an astroglia-derived protein which acts as a neurotrophic factor and neuronal survival protein, even though the overproduction of S100B by activated astrocytes lead to further neurodegeneration. Elevated S100B levels are generally associated with a sustained reactive gliosis (Griffin, 2006; Donato and Heizmann, 2010). Results from cultured astrocytes showed a significant increase in S100B protein expression after Aβ 1-42 exposure (*p* < 0.01; **Figure 2**). Both RSV and AGK-2 controlled such increase. Also in this case, RSV exerted its effect in a concentration dependent manner (**Figures 2A,B**). Instead, all the AGK-2 concentrations completely abolished the Aβ-induced S100B increase (**Figures 2C,D**).

## **EFFECT OF RSV AND AGK-2 ON INFLAMMATION**

Another set of experiments was aimed at assessing the effect of RSV and AGK-2 on the production of inflammatory factors induced by Aβ 1-42 challenge. In fact, astrocyte activation is linked to the production of pro-inflammatory mediators which, in turn, stimulate gliosis and can kill neighboring neurons (Mrak and Griffin, 2001; Ferreira et al., 2014). Treatment with Aβ 1-42 resulted in an increase in iNOS expression, as determined by Western blot analysis (**Figure 3**; *p* < 0.05). Interestingly, this observed effect was reduced by both RSV and AGK-2 at the two higher concentrations used (**Figures 3A–D**). Parallel results were obtained with immunoblot experiments aimed at studying COX-2 expression. In fact, Aβ 1-42 significantly increased COX-2 protein expression (**Figure 3**; *p* < 0.05). Also in this case, both RSV and AGK-2 significantly decreased such effect at the two higher concentrations used (**Figures 3A–D**).

## **DISCUSSION**

The purpose of this study was to assess the efficacy of RSV, a well-known SIRT1 activator, and AGK-2, a potent SIRT2-selective inhibitor, in counteracting reactive gliosis, now considered one of the characteristic phenomena occurring in AD. AD leads to disability and death in a significant proportion of the world's aged population (Alzheimer's Association Report, 2013). However, the available treatments are limited and exert only symptomatic effects. Several promising drugs have recently failed to provide benefit, so there is urgent need to develop new, and hopefully more efficacious, drugs to affect AD course. To this attempt, in the last years researchers focused their attention on the role of reactive gliosis in the onset and progression of many neurodegenerative disorders, including AD. Produced results gave evidence that neuroinflammation and neurodegeneration mutually have a critical impact on AD course (Wyss-Coray and Rogers, 2012). For this reason, it is possible to assume that early combination of neuroprotective and anti-inflammatory treatments represents a particularly appropriate approach to AD (Di Filippo et al., 2008). Although neurodegenerative disorders have distinct clinical manifestations, many of the underlying pathogenic processes are similar (intraor extracellular accumulation of misfolded proteins, cytoskeletal abnormalities, disruption of calcium homeostasis, mitochondrial dysfunction, and inflammation), and most of them are strongly

influenced by and increased during aging. In particular, in both early- and late-onset sporadic AD, aging represents a major contributing factor for the disease development and progression, although the precise role remains still unclear. Transcriptional profiling studies revealed that expression of genes that play central roles in synaptic plasticity, vesicular transport and mitochondria function is reduced, whereas expression of genes encoding for stress, inflammatory or immune factors is increased in aged human frontal cortex (Lu et al., 2004). These findings implicate ongoing DNA damage, oxidative stress and inflammation as contributors to the functional decline occurring in age-related neurodegenerative diseases, including AD.

In this context, the discovery of SIRTs, indicated as class III histone deacetylases (HDACs), offers a close relationship between aging, metabolism and neurodegeneration, thereby representing an innovative target to develop therapeutic strategies (Outeiro et al., 2008). SIRTs play pleiotropic biological functions that range from repression of gene expression (through histone deacetylation) to regulation of cellular differentiation and/or apoptotic processes, from control of energetic cell metabolism to that of aging events. These enzymes have been extensively studied because of their involvement in mediating the effect of caloric restriction

evaluated by trypan blu assay **(B,D)**. Results are expressed as cell viability-fold increase versus unchallenged (open bars) or Aβ-challenged cells (black bars). Results are the mean ± SEM of four experiments in triplicate. Statistical analysis was performed by one-way ANOVA followed by Bonferroni multiple comparison test. *p* < 0.01 Aβ-challenged versus unchallenged cells; \**p* < 0.05; \*\**p* < 0.01; \*\*\**p* < 0.001 for multiple comparison among groups.

(CR) in fostering longevity and healthy aging. In addition, many data indicate that SIRTs are potentially able to delay neurodegenerative diseases related to senescence, including AD. (Michan and Sinclair, 2007). It has been demonstrated that CR reduces the content of Aβ in the temporal cortex of squirrel monkeys, and such effect is inversely linked to SIRT1 expression in the same brain region (Qin et al., 2006a). Moreover, in a transgenic mouse model of AD, the same authors previously demonstrated that CR antagonizes Aβ neuropathology by increasing the SIRT1 and NAD+/nicotinamide ratio (Qin et al., 2006b). Recently, SIRT2 inhibition has been proposed as a promising therapeutic strategy to achieve neuroprotection in *in vitro* and *in vivo* models of Parkinson's and Huntington's diseases (Outeiro et al., 2007; Luthi-Carter et al., 2010). Moreover, Spires-Jones et al. (2012) demonstrated that inhibition of SIRT2 is a safe and promising neuroprotective agent in both tau-associated frontotemporal dementia and AD.

It is recognized that Aβ affects cell viability and proliferation (Allaman et al., 2010; Scuderi et al., 2012). It is possible to speculate that these Aβ actions are due to its ability to enhance astrocyte metabolism turning on morpho-functional changes in such cells (Verkhratsky and Butt, 2007). Interestingly, our experiments highlighted alterations in astrocyte viability and proliferation after Aβ

1-42 challenge, and both RSV and AGK-2 markedly controlled these effects. SIRTs are considered as sensors of cell metabolic state because they finely modulate physiological processes. For this reason it is important to establish the appropriate concentrations to avoid dangerous unwanted consequences. In fact, in our conditions, we found that the highest concentrations used of both RSV and AGK-2 caused cytotoxic effects.

evaluated. Representative Western blots for GFAP and S100B proteins in lysates from astrocytes challenged with Aβ 1-42 (0.23 μM) in the presence of RSV (2 – 10 – 50 μM; **A**) or AGK-2 (0.35 – 3.5 – 35 μM; **C**). Densitometric

As a consequence of exogenous insults, glial cells lost their physiological functions and acquire a reactive phenotype, characterized by profound morphological and functional alterations, such GFAP and S100B overexpression (O'Callaghan and Sriram, 2005; Donato et al., 2013). In our model, we detected marked alteration of both these proteins. In fact, Western blot analysis showed that astrocytes express higher GFAP and S100B protein levels after

Aβ challenge. Interestingly, RSV and AGK-2 negatively modulated the expression of both GFAP and S100B.

\**p* < 0.05; \*\**p* < 0.01; for multiple comparison among groups.

As mentioned before, the direct correlation between the Aβ-induced toxicity and the production of pro-inflammatory mediators prompted us to investigate the expression of the two main inducible enzymes related to inflammation, iNOS and COX-2. In our experimental condition, we highlighted the existence of an inflammatory state induced by Aβ 1-42 treatment, as detected by the increased expression of both iNOS and COX-2. The alteration of these two proteins was significantly blunted by RSV and AGK-2, indicating a key role in regulating astrogliosis and important astrocyte changes, which contribute to disease progression. In the current study it was observed that SIRT1 and SIRT2 can represent promising targets, whose manipulation could prevent

over-activation of neuroglia upon pro-inflammatory stimulation. These data suggest a SIRT-dependent mechanism to restrain detrimental effects of excessive astrocyte activation. Moreover, the findings bear major implications in the context of several inflammatory conditions of the central nervous system where astroglia are known to mediate deleterious consequences. In conclusion, the results of the present study provide evidence that SIRT modulation can represent a strategy to counteract reactive gliosis, and suggest new avenues to walk for the discovery of novel and promising therapy for AD.

COX-2 proteins (**A,C** for RSV and AGK-2, respectively). Densitometric analyzes

## **AUTHOR CONTRIBUTIONS**

Caterina Scuderi, Claudia Stecca, Bronzuoli M. Rosanna, Dante Rotili, Sergio Valente, Antonello Mai, Luca Steardo contributed to the work design, the acquisition and interpretation of data. Caterina Scuderi, Claudia Stecca, Bronzuoli M. Rosanna, Dante Rotili, Sergio Valente drafted the manuscript and revised it. Antonello Mai and Luca Steardo approved the final version of the manuscript. Caterina Scuderi, Claudia Stecca, Bronzuoli M. Rosanna, Dante Rotili, Sergio Valente, Antonello Mai, Luca Steardo ensure that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved.

cells; \**p* < 0.05; \*\**p* < 0.01; for multiple comparison among groups.

#### **ACKNOWLEDGMENTS**

This work was supported by the Italian Ministry of Instruction, University and Research grants (MIUR; PON01-02512 and PRIN2009) to Luca Steardo, and by FIRB RBFR10ZJQT, Sapienza Ateneo Project 2012, IIT-Sapienza Project, and the FP7 Projects BLUEPRINT/282510 and COST/TD0905 grants to Antonello Mai.

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**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.

*Received: 21 February 2014; accepted: 11 April 2014; published online: 13 May 2014. Citation: Scuderi C, Stecca C, Bronzuoli MR, Rotili D, Valente S, Mai A and Steardo L (2014) Sirtuin modulators control reactive gliosis in an in vitro model of Alzheimer's disease. Front. Pharmacol. 5:89. doi: 10.3389/fphar.2014.00089*

*This article was submitted to Experimental Pharmacology and Drug Discovery, a section of the journal Frontiers in Pharmacology.*

*Copyright © 2014 Scuderi, Stecca, Bronzuoli, Rotili, Valente, Mai and Steardo. 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.*

## Cellular stress response, redox status, and vitagenes in glaucoma: a systemic oxidant disorder linked to Alzheimer's disease

*Angela Trovato Salinaro1 †, Carolin Cornelius 2 †, Guido Koverech1, Angela Koverech1, Maria Scuto1, Francesca Lodato1, Vincenzo Fronte1, Vera Muccilli 1, Michele Reibaldi 3 , Antonio Longo3 , Maurizio G. Uva3 and Vittorio Calabrese1\**

*1 Department of Biomedical Sciences, School of Medicine, University of Catania, Catania, Italy*

*2 Department of Chemistry, School of Medicine, University of Catania, Catania, Italy*

*3 Department of Ophthalmology, School of Medicine, University of Catania, Catania, Italy*

#### *Edited by:*

*Cesare Mancuso, Catholic University, Italy*

#### *Reviewed by:*

*Inga Kadish, University of Alabama at Birmingham, USA Benedetto Falsini, Catholic University, Italy*

#### *\*Correspondence:*

*Vittorio Calabrese, Department of Biomedical Sciences, School of Medicine, University of Catania, Viale Andrea Doria 6, 95100 Catania, Italy e-mail<EMAIL_ADDRESS>
†*Angela Trovato Salinaro and Carolin Cornelius have contributed equally to this work.*

Amyloid deposits, constituted of amyloid beta (Aβ) aggregates, are a characteristic feature of several neurodegenerative diseases, such as Alzheimer's, mild cognitive impairment and Parkinson's disease. They also have been recently implicated in the pathogenesis of retinal damage, as well as age-related macular degeneration and glaucoma. Glaucoma is a progressive optic neuropathy characterized by gradual degeneration of neuronal tissue due to retinal ganglion cell loss, associated to visual field loss over time resulting in irreversible blindness. Accumulation of Aβ characterizes glaucoma as a protein misfolding disease, suggesting a pathogenic role for oxidative stress in the pathogenesis of retinal degenerative damage associated to glaucoma. There is a growing body of evidence demonstrating a link between Alzheimer's disease and glaucoma. Further, several heat shock proteins (HSPs) members have been implicated both in neurodegenerative diseases and glaucomatous apoptosis. To maintain redox homeostasis vitagenes, as integrated mechanisms, operate actively to preserve cell survival under condition of stress. Vitagenes encode for sirtuin, thioredoxin and HSPs. The present study was designed to investigate cellular stress response mechanisms in the blood of patients with glaucoma, compared to control subjects. Levels of vitagenes HSP-72, heme oxygenase-1, as well as F2 isoprostanes were significantly higher in the blood of patients with glaucoma than in controls. Furthermore, in the same experimental group increased expression of Trx and sirtuin 1 were measured. Our results sustain the importance of redox homeostasis disruption in the pathogenesis of glaucoma and highlights the opportunity that new therapies that prevents neurodegeneration through non-immunomodulatory mechanisms might be synergistically associated with current glaucoma therapies, thus unraveling important targets for novel cytoprotective strategies.

**Keywords: free radicals, stress response, vitagenes, hormesis, antioxidants**

## **INTRODUCTION**

Glaucoma is a progressive optic neuropathy characterized by degeneration of neuronal tissue due to loss of retinal ganglion cells (RGCs), with accompanying compromission of visual field over time (Gupta et al., 2006; Quigley and Broman, 2006). It is a leading cause of irreversible blindness estimated to affect 79.6 million people worldwide by 2020 (Hinton et al., 1986; Yoneda et al., 2005). Research studies have demonstrated that RGC damage in glaucoma is not limited to the primary insulted neurons, but also involves neighboring neurons. The increase in the prevalence of glaucoma with age is not accounted for only by the increase in ocular hypertension alone, being accompanied by an increase in the vulnerability of the optic nerve to the effects of glaucoma risk factors which increase as function of age. In particular, factors such as tissue hypoxia, disturbed protein metabolism and oxidative stress have been identified to interact in a vicious cycle

underlying the pathogenesis of glaucoma (Calandrella et al., 2007; Gupta et al., 2008), ultimately leading to apoptotic retina ganglion cell death (Tatton et al., 2001; Soti et al., 2005; De la Monte and Wands, 2006). In view of these considerations glaucoma can be viewed as a neurodegenerative disease which, similarly to other neurodegenerative pathologies, i.e., Alzheimer's and Parkinson's disease, where irreversible functional deficit ensues as consequence of neuronal dysfunction and death. There is now a growing body of evidence demonstrating a link between AD and glaucoma.

Amyloid deposits, consisting of Aβ, which are a characteristic feature of several neurodegenerative diseases such as Alzheimer's (AD), mild cognitive impairment, and Parkinson's disease (Hinton et al., 1986) have been recently implicated in the pathogenesis of retinal damage, macular degeneration, and glaucoma (Yoneda et al., 2005). Accordingly, drugs designated to target β-amyloid (Aβ) has been found to reduce apoptotic degeneration of RGCs, as observed *in vitro* and *in vivo*. Furthermore, the presence of increased levels of Aβ characterizes glaucoma as a protein misfolding disease, also suggesting a role for oxidative stress in the pathogenesis of retinal degenerative damage associated to glaucome. Although oxidative stress has been recognized to play a critical role in the development and progression of glaucoma, yet, the exact mechanisms remain elusive. Oxidative stress can cause oxidative attack to DNA, proteins, and lipids, leading to DNA and protein modification, thus sustaining the pathophysiology of degenerative damage of RGCs (Gupta et al., 2006). Relevant to protein misfolding, of emerging interest are heat shock proteins (HSPs), specialized molecular chaperones which mediate various cellular functions. HSPs are up regulated in response to conditions of stress in order to restore normal cell integrity (Soti et al., 2005). The heat shock response, an important component of vitagene family, contributes to establishing a cytoprotective state in a wide variety of human diseases. Vitagenes include, besides HSPs 70 and 32, the latter also called heme oxygenase-1 (HO-1), thioredoxin and sirtuins (Butterfield et al., 2010, 2011). Several families of HSP have been implicated in neurodegenerative diseases and glaucomatous RGC apoptosis with increased levels of circulating autoantibodies to alpha-crystallins and HSP-27 and increased immunostaining of HSP-60, HSP-27 in RGCs and the retinal blood vessels in glaucoma patients (Shieds et al., 1996; Izzotti et al., 2006). In a rat glaucoma model, treatment with geranylgeranylacetone increases HSP-72 synthesis while reducing markedly RGC loss, possibly through interactions with different protein kinases, such as Akt kinase, and the inhibition of NF-kB. In this study we tested the hypothesis that there may be a causal relationship between AD and glaucoma that may be explained by systemic oxidative stress and dysregulation of cellular stress response. Present work elucidated cellular stress response in peripheral cells in patients with glaucoma as compared to healthy volunteers, as control, in order to gain insight into the pathogenic mechanisms operating in the neurodegenerative damage associated with this disease and exploit the possible role of vitagenes in opening up new therapeutic targets for limiting the oxidative damage associated to degeneration occurring in glaucoma.

## **MATERIALS AND METHODS**

#### **PATIENTS**

Eighteen patients (12 males and six females, mean age 60 ± 15 years) with diagnosis of hypertensive primary openangle glaucoma (POAG), with typical optic nerve head and visual field damage, were included in the study. Mean MD and PSD were respectively −7.5 ± 8.6 dB, and 4.2 ± 3.8 dB. Twenty age-matched healthy volunteers were recruited as controls. Patients and control subjects underwent IOP measurement by Goldmann applanation tonometer, optic nerve head examination by 78 D lens at the slit lamp, and visual field test 24-2 SITA standard, by a 750 Humprey perimeter (HFA II). Clinical characteristics of patients and control subjects are shown in **Table 1**. Patients with normal tension glaucoma, previous uveitis, diabetes, arterial hypertension were excluded. The study was conducted according to guidelines of local

Ethics Committee, and informed consent was obtained from all patients.

## **SAMPLING AND LYMPHOCYTE PURIFICATION**

Blood (5 ml) collected from controls and patients into tubes containing EDTA, was divided into two aliquots, 1 and 4 ml respectively. One aliquots (1 ml) was centrifuged at 3000 × *g* for 10 min at 4◦C to separate serum from red blood cells, while 4 ml aliquots, were utilized for lymphocytes purification, which was accomplished by using the Ficoll Paque System following the procedure provided by the manufacturer (GE Healthcare, Piscataway, NJ, USA).

## **WESTERN BLOT ANALYSIS**

HSP-70, HO-1, Trx, and Sirt-1 protein levels were estimated by Western blot analysis which was accomplished as previously described in Calabrese et al. (2012). Plasma samples were processed as such, while the isolated lymphocyte pellet was homogenized and centrifuged at 10,000 × *g* for 10 min. The supernatant was then used for analysis after determination of protein content. Proteins extracted for each sample, at equal concentration (40μg), were boiled for 3 min in sample buffer (containing 40 mM Tris-HCl pH 7.4, 2.5% SDS, 5% 2-mercaptoethanol, 5% glycerol, 0.025 mg/ml of bromophenol blue) and then separated on a polyacrylamide mini gels precasting 4–20% (cod NB10420 NuSept Ltd Australia). Separated proteins were transferred onto nitrocellulose membrane (BIO-RAD, Hercules, CA, USA) in transfer buffer containing (0.05% di SDS, 25 mM di Tris, 192 mM glycine and 20% v/v methanol). The transfer of the proteins on the nitrocellulose membrane was confirmed by staining with Ponceau Red which was then removed by three washes in PBS (phosphate buffered saline) for 5 min each. Membranes were then incubated for 1 h at room temperature in 20 mM Tris pH 7.4, 150 mM NaCl and Tween 20 (TBS-T) containing 2% milk powder and incubated with appropriate primary antibodies, namely anti-HSP-70, anti-HO-1, anti-Trx and anti Sirt-1 polyclonal antibody (Santa Cruz Biotech. Inc.), overnight at 4◦C in TBS-T. The same membrane was incubated with a goat polyclonal antibody anti-beta-actin (SC 1615 Santa Cruz Biotech. Inc., Santa Cruz, CA, USA, dilution 1:1000) to verify that the concentration of protein loaded in the gel was the same in each sample. Excess unbound antibodies were removed by three washes are with TBS-T for 5 min. After incubation with primary antibody, the membranes were washed three times for 5 min. in TBS-T and then incubated for 1 h at room temperature with the secondary polyclonal antibody conjugated with horseradish peroxidase (dilution 1:500). The membranes were then washed three times with TBS-T for 5 min. Finally, the membranes were incubated for 3 min with SuperSignal chemiluminiscence detection system kit (Cod 34080 Pierce Chemical Co, Rockford, IL, USA) to display the specific protein bands for each antibody. The immunoreactive bands were quantified by capturing the luminescence signal emitted from the membranes with the Gel Logic 2200 PRO (Bioscience) and analyzed with Molecular Imaging software for the complete analysis of regions of interest for measuring expression ratios. The molecular weight of proteins analyzed was determined using a standard curve prepared with protein molecular weight.

#### **Table 1 | Clinical data of glaucoma patients and control subjects.**

*Md, mean defect; psd, pattern standard deviation.*

#### **MEASUREMENT OF F2-ISOPROSTANES**

F2-isoprostanes were determined by HPLC according to the procedure of Ritov et al. (2002). F2-isoprostane content in plasma was expressed in nM.

#### **DETERMINATION OF PROTEIN**

Protein concentration in all experimental samples was determined by the bicinchoninic acid protein assay (Pierce, Rockford, IL, USA) according to Smith et al. (1985) and using serum bovine albumin as standard.

#### **STATISTICAL ANALYSIS**

Results were expressed as means ± S.E.M. Each experiment was performed, unless otherwise specified, in triplicate. Data were analyzed by one-way ANOVA,followed by inspection of all differences by Duncan's new multiple-range test. Differences were considered significant at *P* < 0.05.

## **RESULTS**

In our study we evaluated the expression of stress proteins in plasma and lymphocytes of glaucomatous patients compared to controls. Among the 70 kDa family of HSPs we evaluated the inducible HSP-70 (HSP-72) isoform and its expression. As shown in **Figure 1A**, a significant (*P* < 0.05) increase in HSP-70 was found in lymphocytes of patients with glaucoma with respect to healthy control subjects. A representative immunoblot is reported in **Figure 1B**. Western blot analysis of plasma probed for HSP-70 are reported in **Figure 2A**, which shows that HSP-70 expression increased significantly (*P* < 0.05) in patients with glaucoma, compared to controls. A representative immunoblot is shown in **Figure 2B**.

Heme oxygenase-1, another HSP-32 endowed with cytoprotective properties (Soti et al., 2005), was found expressed at significantly higher levels in lymphocytes of patients with glaucoma than in controls (**Figure 3A**). A representative immunoblot is illustrated in **Figure 3B**. Similarly to lymphocyte finding, patients with glaucoma exhibited higher level plasma HO-1 protein then healthy controls (**Figures 4A,B**).

Analysis of lymphocytes in patients with glaucoma, compared to control group, revealed a significant (*P* < 0.05) increase of Trx expression (**Figure 5A**), while in the plasma there was no statistically significant difference between the two experimental groups (**Figure 6A**). Representative immunoblots are reported in **Figures 5B** and **6B**, respectively.

Interestingly, we found significantly (*P* < 0.05) higher levels of sirtuin-1 in lymphocytes of patients with glaucoma than in the control group (**Figure 7A**). Consistent with the changes found in lymphocytes, analysis of plasma confirmed increased protein

levels of sirtuin-1, higher in patients with glaucoma as to compare with the healthy control group (**Figure 8A**). Representative immunoblots are shown in **Figures 7B** and **8B**, respectively. As to our knowledge this is the first evidence of changes in sirtuin-1 expression in glucomatous pathology, although this finding may not be a marker specific for this progressive chronic inflammatory systemic disease.

Further, we evaluated systemic pro-oxidant conditions, by measuring lipid-derived circulating F2 isoprostanes. We found a significant increase (*P* < 0.05) of total F2-isoprostanes in the plasma of patients with glaucoma (*P* < 0.05) with respect to controls (**Figure 9**).

## **DISCUSSION**

Glaucoma is one of the leading causes of vision loss worldwide. Open-angle glaucoma, the most common form of glaucoma, is characterized by a progressive loss of RGCs and atrophy of the optic nerve, resulting in loss of visual field (Hinton et al., 1986; Yoneda et al., 2005). Several theories have been proposed, including mechanical and vascular pathogenesis for the

**FIGURE 2 | (A)** HSP-70 protein levels in lymphocytes of glaucoma and control subjects. Samples from controls and glaucoma patients were assayed for HSP-70 expression by Western blot. A representative immunoblot is shown. β-actin has been used as loading control. **(B)** Densitometric evaluation: the bar graph shows the values are expressed as mean standard error of mean of 3 independent analyses. \**P* < 0.05 vs. control. D.U., densitometric units; CTRL, control.

**FIGURE 3 | (A)** HO-1 protein levels in plasma of glaucoma and control subjects. Samples from control and patients with glaucoma were assayed for HO-1 expression by Western blot. A representative immunoblot is shown. β-actin has been used as loading control. **(B)** Densitometric evaluation: the bar graph shows the values are expressed as mean standard error of mean of 3 independent analyses. \**P* < 0.05 vs. control. D.U., densitometric units; CTRL, control.

**FIGURE 4 | (A)** HO-1 protein levels in lymphocytes of glaucoma and control subjects. Samples from control and patients with glaucoma were assayed for HO-1 expression by Western blot. A representative immunoblot is shown. β-actin has been used as loading control. **(B)** Densitometric evaluation: the bar graph shows the values are expressed as mean standard error of mean of 3 independent analyses. \**P* < 0.05 vs. control. D.U., densitometric units; CTRL, control.

**FIGURE 5 | (A)** Sirtuin-1 protein levels in plasma of glaucoma and control subjects. Samples from control and patients with glaucoma were assayed for Sirt-1 expression by Western blot. A representative immunoblot is shown. β-actin has been used as loading control. **(B)** Densitometric evaluation: the bar graph shows the values are expressed as mean standard error of mean of three independent analyses. \**P* < 0.05 vs. control. D.U., densitometric units; CTRL, control.

glaucomatous optic neuropathy. Elevated intraocular pressure is a strong risk factor, but a subset of glaucoma patients has normal intraocular pressure designating a normal tension glaucoma. Clearly, other factors different from intraocular pressure, such as genetic factors, are thought to be involved in RGC apoptotic cell death in glaucoma. The e4 allele of the APOE gene has been

control subjects. Samples from control and patients with glaucoma were assayed for Sirt-1 expression by Western blot. A representative immunoblot is shown. β-actin has been used as loading control. **(B)** Densitometric evaluation: the bar graph shows the values are expressed as mean standard error of mean of 3 independent analyses. \**P* < 0.05 vs. control. D.U., densitometric units; CTRL, control.

**FIGURE 7 | (A)** Thioredoxin protein levels in plasma of glaucoma and control subjects. Samples from control and patients with glaucoma were assayed for Trx expression by Western blot. A representative immunoblot is shown. β-actin has been used as loading control. **(B)** Densitometric evaluation: the bar graph shows the values are expressed as mean standard error of mean of 3 independent analyses. *P* < 0.05 vs. control. D.U., densitometric units; CTRL, control.

**FIGURE 8 | (A)** Thioredoxin protein levels in lymphocytes of glaucoma and control subjects. Samples from control and patients with glaucoma were assayed for Trx expression by Western blot. A representative immunoblot is shown. β-actin has been used as loading control. **(B)** Densitometric evaluation: the bar graph shows the values are expressed as mean standard error of mean of 3 independent analyses. \**P* < 0.05 vs. control. D.U., densitometric units; CTRL, control.

also considered in the pathophysiology of open-angle glaucoma, although the question still remains elusive (Quigley and Broman, 2006).

Oxidative stress is considered an important risk factor for the development of primary angle-closure glaucoma and increased levels of oxidative stress products have been documented in primary angle-closure glaucoma (Shieds et al., 1996; Izzotti et al., 2006; Butterfield et al., 2011). Visual loss which often starts in the periphery and advances involving central vision, has devastating consequences to patient's quality of life (Gupta et al., 2006; Quigley and Broman, 2006).