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Autophagy in axonal degeneration in glaucomatous optic neuropathy
Yasunari Munemasa a' *■1, Yasushi Kitaoka a'b'1
a Department of Ophthalmology, St. Marianna University School of Medicine, 2-16-1 Sugao, Miyamae-ku, Kawasaki, Kanagawa 216-8511, Japan b Department of Molecular Neuroscience, St. Marianna University Graduate School of Medicine, 2-16-1 Sugao, Miyamae-ku, Kawasaki, Kanagawa 216-8511, Japan
The role of autophagy in retinal ganglion cell (RGC) death is still controversial. Several studies focused on RGC body death, although the axonal degeneration pathway in the optic nerve has not been well documented in spite of evidence that the mechanisms of degeneration of neuronal cell bodies and their axons differ. Axonal degeneration of RGCs is a hallmark of glaucoma, and a pattern of localized retinal nerve fiber layer defects in glaucoma patients indicates that axonal degeneration may precede RGC body death in this condition. As models of preceding axonal degeneration, both the tumor necrosis factor (TNF) injection model and hypertensive glaucoma model may be useful in understanding the mechanism of axonal degeneration of RGCs, and the concept of axonal protection can be an attractive approach to the prevention of neurodegenerative optic nerve disease. Since mitochondria play crucial roles in glau-comatous optic neuropathy and can themselves serve as a part of the autophagosome, it seems that mitochondrial function may alter autophagy machinery. Like other neurodegenerative diseases, optic nerve degeneration may exhibit autophagic flux impairment resulting from elevated intraocular pressure, TNF, traumatic injury, ischemia, oxidative stress, and aging. As a model of aging, we used senescence-accelerated mice to provide new insights. In this review, we attempt to describe the relationship between autophagy and recently reported noteworthy factors including Nmnat, ROCK, and SIRT1 in the degeneration of RGCs and their axons and propose possible mechanisms of axonal protection via modulation of autophagy machinery.
© 2015 The Authors. Published by Elsevier Ltd. This is an open access article under the CC BY-NC-ND
license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
Contents lists available at ScienceDirect
Progress in Retinal and Eye Research
journal homepage: www.elsevier.com/locate/prer
Article history: Received 17 December 2014 Received in revised form 14 March 2015 Accepted 19 March 2015 Available online xxx
Keywords: Autophagy Optic nerve Glaucoma Senescence
1. Introduction.......................................................................................................................00
2. Autophagy in retinal ganglion cell bodies.............................................................................................00
2.1. Optic nerve transection...................................................................................................... 00
3. Autophagy in retinal ganglion cell bodies.............................................................................................00
3.1. Optic nerve transection...................................................................................................... 00
3.2. Ocular hypertension.......................................................................................................... 00
3.3. Ischemia................................................................................................................... 00
3.4. RGC-5 cells.................................................................................................................. 00
4. Autophagy in optic nerve axons.....................................................................................................00
4.1. Optic nerve crush ............................................................................................................ 00
4.2. Ocular hypertension.......................................................................................................... 00
4.3. TNF-induced optic nerve damage.............................................................................................. 00
TNF-induced axonal degeneration and TNF has been implicated in Alzheimer's disease .............................................. 00
Autophagy in AD informs autophagic events in axons............................................................................ 00
Nmnat and axonal protection .................................................................................................... 00
* Corresponding author. Tel.: +81 44 977 8111; fax: +81 44 976 7435.
E-mail address: firstname.lastname@example.com (Y. Munemasa). 1 Percentage of work contributed by each author in the production of the manuscript is as follows: Yasunari Munemasa: 50%; Yasushi Kitaoka: 50%.
1350-9462/© 2015 The Authors. Published by Elsevier Ltd. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
ROCK and axonal regeneration................................................................................................... 00
5. Aging.............................................................................................................................00
5.1. Aging and glaucoma......................................................................................................... 00
5.2. Age-related changes in the optic nerve ......................................................................................... 00
5.3. Age-related change in RGC bodies............................................................................................. 00
6. Sirtl in aging......................................................................................................................00
6.1. Biological role of Sirtl ........................................................................................................ 00
6.2. Sirtl in axons ............................................................................................................... 00
6.3. Sirtl in the retina ........................................................................................................... 00
7. Autophagy and aging ..............................................................................................................00
Autophagy in cellular senescence................................................................................................. 00
Autophagy and SAM............................................................................................................ 00
Autophagy and Sirtl............................................................................................................. 00
Autophagy and other age-related neurodegenerative diseases........................................................................ 00
Conclusions and future directions ...................................................................................................00
Uncited references................................................................................................................. 00
Acknowledgments ................................................................................................................. 00
References........................................................................................................................ 00
1. Introduction
Autophagy, or cellular self-digestion, is a cellular pathway involved in protein and organelle degradation and has been associated with a cell-protective process and conversely linked to a role in cell death (Mizushima et al., 2008). Among the three types of autophagy (macroautophagy, microautophagy, and chaperone-mediated autophagy), macroautophagy (referred to as autophagy hereafter) is mediated by a unique organelle called the autopha-gosome, which is characterized by the formation of a double-membraned structure (Mizushima, 2007). The most typical inducer of autophagy is nutrient starvation. Under starvation or after rapamycin treatment, the numbers of all autophagic structures including isolation membranes, autophagosomes, and auto-lysosomes are increased and autophagic flux is also increased compared with the basal levels (Mizushima et al., 20l0). Importantly, the number of autophagosomes increases when steps in the autophagy pathway downstream of autophagosome formation are blockaded (Mizushima et al., 20l0), indicating that the increase in autophagosomes does not always result in autophagic activation.
Several autophagy-related (Atg) genes have been identified in yeast and many of these have been confirmed to be mammalian orthologues. Beclin l/Atg6 was shown to play a central role in autophagy, and sufficient levels of Beclin l are necessary for its autophagic function (Wirawan et al., 20l2). It regulates autopha-gosome formation as a Beclin l complex (Wirawan et al., 20l2). Among several Atg proteins, microtubule-associated protein light chain 3 (LC3)/Atg8 is known to occur on autophagosomes and is used as their marker (Kabeya et al., 2000). LC3 is specifically localized to the phagofore, the autophagosome, and the autolyso-some. LC3-1 is the cytosolic form, and LC3-11 is associated with autophagosome membranes. Mammalian Atg4 homologues are cysteine proteases required for the autophagy process, which cleave the C-terminal peptide of LC3 (Marino et al., 2003; Kabeya et al., 2004) to produce LC3-1, which resides in the cytosol (Kabeya et al., 2000). LC3-1 is conjugated to phosphatidylethanol-amine in a reaction involving Atg7 and Atg3 to form LC3-11 (Kabeya et al., 2000). The amount of LC3-11 correlates closely with autophagosome number (Mizushima et al., 20l0). However, the above finding that the increase in autophagosomes does not always result in autophagic activation means that the focus should be on interpreting the meaning of the amounts of LC3-11 present in the auto-phagosomes. That is, an increase in the LC3-11 level as shown by
immunoblotting indicates either autophagy induction or the suppression of a downstream step in autophagy.
To distinguish between autophagy induction and the suppression of a downstream step in autophagy, the measurement of autophagic flux, which refers to the entire process of autophagy, including autophagosome formation, maturation, and fusion with lysosomes, is useful; it is generally higher in the former condition and lower in the latter. One method for detecting autophagic flux is estimating LC3 immunoblotting with or without lysosomal inhibitors (Mizushima and Yoshimori, 2007). According to a review article, lysosomal protease inhibitors can partially inhibit the degradation of LC3-11, whereas they do not affect the degradation of LC3-1, thereby indicating that the amount of LC3-11 at a certain time point does not indicate total autophagic flux and that autophagic flux is more accurately represented by differences in the amount of LC3-11 between samples in the presence and absence of lysosomal protease inhibitors (Mizushima and Yoshimori, 2007). 1t is important to note that some antibodies against LC3 react preferentially with the LC3-11 form rather than the LC3-1 form, and therefore the signal ratio between LC3-1 and LC3-11 provides little information (Kimura et al., 2009). Another method for detecting autophagic flux is measuring levels of p62, which is also called sequestosome l (SQSTMl) (Bj0rk0y et al., 2005). 1t was demonstrated that LC3 colocalized with p62, and that these two proteins participated in the same complexes (Bj0rk0y et al., 2005). p62 is normally degraded by the lysosomal proteases through the interaction with LC3-11 (1chimura and Komatsu, 20l0). Since p62 accumulates when autophagy is inhibited, and decreased levels can be observed when autophagy is induced, p62 may be used as a marker of autophagic flux (Bj0rk0y et al., 2009). For example, Atg4-deficient mice reach adulthood with no excess mortality, are fertile, and do not present any obvious histopathological or biochemical alteration (Marino et al., 20l0). These mice are useful for autophagy research because they display a substantial systemic reduction in autophagic activity and reduced basal autophagic flux (Marino et al., 20l0). 1n that study, p62 protein levels were decreased after starvation in control mice and also decreased after starvation in Atg4-deficient mice skeletal muscle and liver. However, the p62 protein levels were always higher in the mutant mice tissue than in that from WT mice subjected to the same starvation conditions (Marino et al., 20l0), indicating an impairment of autophagic activity in these mutant mice. Therefore, p62 protein levels can reflect relatively accurately the degree of autophagic conditions in several tissue
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types. Thus, the examination of both LC3-II and p62 protein levels is important for understanding the status of autophagic activity.
2. Autophagy in retinal ganglion cell bodies
2.Í. Optic nerve transection
LC3 immunohistochemistry is predominantly expressed in the retinal ganglion cell (RGC) layer and in photoreceptors (Kim et al., 2008). The LC3-II protein level as shown by immunoblotting in isolated RGCs was significantly upregulated 1 day to 1 week, with the peak at 3 days, after optic nerve transection (Kim et al., 2008), implying a possible activation of autophagy in RGCs in optic nerve damage. This finding has been supported by a recent study demonstrating that autophagosomes were significantly increased in RGCs 3—10 days after optic nerve transection (Rodríguez-Muela et al., 2012). Rapamycin, an inducer of autophagy, significantly increased the number of Brn-3a-immunopositive RGCs compared with those in vehicle-treated mice 10 days after optic nerve transection (Rodríguez-Muela et al., 2012). Interestingly, p62 protein levels in the retina were higher in Atg4-deficient mice, which show reduced basal autophagic flux compared with WT mice (Rodríguez-Muela et al., 2012). Moreover, LC3-II levels in the retina were lower in Atg4-deficient mice compared with WT mice, indicating the reduction of autophagic activity in the mutant retina (Rodríguez-Muela et al., 2012). Furthermore, that study demonstrated a reduced number of surviving RGCs in Atg4-deficient mice after optic nerve transection compared with those in WT mice. These findings suggest that the activation of autophagy exerts a protective role on RGCs after traumatic optic nerve injury.
2.2. Ocular hypertension
Acute intraocular pressure (IOP) elevation (approximately 110 mmHg) is used for the retinal ischemia—reperfusion model in rats, and the ocular hypertension model usually means chronic mild IOP elevation (approximately 30—40 mmHg). Three general models have been described: 1) hypertonic saline injection to the aqueous humor outflow pathway; 2) limbal laser treatment; and 3) episcleral vein cautery (Morrison et al., 2005). Among them, the vein cautery model may produce less injury than the other two models, because the congestions of the choroidal vasculature, of veins surrounding the optic nerve head, or of the vasculature of the adjacent sclera may affect the nerve fibers (Morrison et al., 2005). Thus, these models may not all be equal, and caution should be used in interpreting the autophagic findings in these models. In the chronic hypertensive rat model with episcleral vein cautery, it was shown that autophagosomes were increased in the RGC layer 1—4 weeks after IOP elevation (Park et al., 2012). Similarly, LC3-II levels in the retina were increased 1 —4 weeks after IOP elevation. It is reasonable to speculate that those LC3-II levels in the retina reflect mainly autophagosomes in RGC bodies rather than autophago-somes in dendrites in the inner plexiform layer (IPL) because increased autophagosomes in the IPL were observed 1 and 2 weeks after IOP elevation but not at 4 weeks (Park et al., 2012). In primates, long periods (e.g., 40 weeks) of IOP elevation leads to optic disc cupping. A recent study has demonstrated increases in LC3-II levels in the retina in similar long-term periods of IOP elevation in a rhesus monkey glaucoma model (Deng et al., 2013). Even after such long periods, increased immunoreactivity of LC3B was observed in the RGC layer and IPL compared with that in normal controls (Deng et al., 2013). Therefore, it is possible that increased autophagosomes in RGCs may occur in glaucoma patients. However, it is puzzling to interpret the role of autophagy in RGC death because of the controversial results showing that 3-methyladenine
(3-MA), an autophagy inhibitor, is protective whereas rapamycin, an autophagic inducer, is also protective in the same hypertensive experimental glaucoma rat model (Park et al., 2012; Su et al., 2014). Another aspect is that tumor necrosis factor (TNF) was found to be increased in the mouse retina after IOP elevation (Nakazawa et al., 2006), and that upregulated TNF was significantly inhibited by rapamycin with its protective effect on RGC bodies in hypertensive glaucoma rats (Su et al., 2014).
2.3. Ischemia
In the retinal ischemia—reperfusion model, a previous study showed that LC3-11 levels were significantly decreased at the end of ischemia (reperfusion 0 h) and maintained at below basal levels in the retina during the first hour of reperfusion (Russo et al., 2011), although the possibility that under these circumstance all protein syntheses and cellular activity that require energy are severely compromised cannot be excluded. That study suggested the impairment of autophagy in the ischemic retina and a neuro-protective role of autophagy in RGCs (Russo et al., 2011). In the study by Russo et al. (2011), there was no difference in LC3-11 levels at 24 h between the nonischemic control and ischemic retina groups. However, another study found an increase in the LC3-11 level in the retina 24 h after ischemia—reperfusion compared with controls (Piras et al., 2011). Although it is difficult to explain this difference, one hypothesis is that LC3-11 levels may decrease in the early phase and return to the basal level or increase more at around 24 h after ischemic injury. In view of the role of autophagy, that study showed that the decrease in cell number in the RGC layer induced by ischemia was partially attenuated by 3-MA, an auto-phagy inhibitor, and that the positivity for apoptotic markers was suppressed by 3-MA, suggesting that autophagy and apoptosis can coexist in the same damaged neurons after ischemia—reperfusion (Piras et al., 2011). In accordance with this observation, post-ischemic induction of autophagy by intravitreal rapamycin administration did not provide protection against lesions induced by ischemic stress in the retina (Produit-Zengaffinen et al., 2014).
2.4. RGC-5 cells
It has been documented that RGC-5 cells are not derived from immortalized rat RGCs but represent a lineage of mouse neuronal precursor cells (Van Bergen et al., 2009). Recently, it has been shown that RGC-5 cells are not of RGC origin, but are the cell line 661W, a mouse SV-40 T antigen-transformed photoreceptor cell line (Krishnamoorthy et al., 2013). Therefore, one must be careful in interpreting the findings of several studies in RGC-5 cells, i.e., one can interpret the findings in RGC-5 cells as an in vitro model for retinal neuronal cells. Using this in vitro system, several studies demonstrated the autophagic machinery under starvation or some forms of stress. For example, serum deprivation resulted in increased LC3 distribution in autophagosomes in RGC-5 cells (Kim et al., 2008). Three different autophagy inhibitors, bafilomycin A1 (BafA1), 3-MA, and wortmannin, reduced cell viability under starvation conditions in RGC-5 cells (Kim et al., 2008). Other reports showed that serum deprivation resulted in increased LC3-11 levels in RGC-5 cells along with reduced RGC-5 cell survival (Russo et al., 2011). That study also showed that BafA1 and 3-MA reduced cell viability in starved RGC-5 cells (Russo et al., 2011). Since BafA1 prevents the maturation of autophagic vacuoles by inhibiting fusion between autophagosomes and lysosomes (Yamamoto et al., 1998), it is reasonable to find the accumulation of LC3-1 and -II in RGC-5 cells treated with BafA1 in the presence or absence of serum (Russo et al., 2011). The cumulative results suggest a protective role of autophagy in RGC-5 cells under starvation conditions. It is also
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interesting to note that rapamycin decreased intracellular reactive oxygen species (ROS) production and increased cell viability and that 3-MA increased ROS production and decreased cell viability in RGC-5 cells (Rodríguez-Muela et al., 2012). That study also showed that treatment with the ROS-inducing agent paraquat induced an increase in p62 levels in RGC-5 cells (Rodríguez-Muela et al., 2012), which is consistent with a previous finding that the upregulation of p62 protein was observed under oxidative stress (Jain et al., 2010). Another study demonstrated that a portion of p62 directly localizes within the mitochondria and supports the integrity of mitochon-drial functions in other tissues (Lee and Shin, 2011). Consistent with those findings, some p62-immunopositive dots were colocalized with MitoTracker Red in nonstarved RGC-5 cells, and this colocal-ization was more prominent in starved RGC-5 cells, indicating that p62 was present in mitochondria (Fig. 1). Since it was shown that rapamycin inhibited the increase in p62 levels induced by ROS in RGC-5 cells (Rodríguez-Muela et al., 2012), it is plausible that the activation of autophagy may exert protective effects with the inhibition of p62. The detailed relationship between mitochondrial function and p62 in the protective effect of rapamycin needs to be examined at the subcellular fraction level in immunoblotting and double immunofluorescence studies.
3. Autophagy in optic nerve axons
3.1. Optic nerve crush
In contrast to the findings of RGC body death after optic nerve transection, the intravitreal preinjection of 3-MA, an inhibitor of autophagy, resulted in a significant delay in axonal degeneration starting at 90 min, which continued until 360 min, after optic nerve crush compared with controls as evaluated using the axonal integrity ratio (Knoferle et al., 2010). That study also demonstrated that the application of calcium channel inhibitors significantly delayed the acute phase of axonal degeneration (up to 360 min) after optic nerve crush and resulted in a significant reduction in LC3-positive autophagosomes at 360 min (Knoferle et al., 2010). Therefore, it is likely that intracellular calcium levels are important in axonal degeneration in the acute phase after optic nerve crush with the involvement of autophagic machinery. Although it is unclear why rapamycin is protective for RGCs (Rodríguez-Muela et al., 2012) whereas 3-MA is protective for axons (Knoferle et al., 2010),
the main difference between those two studies was that the estimated time after injury was 10 days in the former and a few hours in the latter. Other differences were the injury type, i.e., transection or crush, but these injuries are very similar. In addition, the methodology for estimating RGC death was immunohistochem-istry and that for axonal degeneration was in vivo imaging. These differences may be associated with the opposing findings.
3.2. Ocular hypertension
Consistent with the findings of increased autophagosomes in RGCs in hypertensive glaucoma models (Park et al., 2012; Deng et al., 2013), our transmission electron microscopic study also showed noticeable autophagic vacuoles in axons in the hypertensive glaucoma rat model (Fig. 2B). Numerous normal mitochondria were observed inside axons of the laminar portion in the control group (Fig. 2A). On the other hand, abnormal mitochondria and autophagic vacuoles were observed in unmyelinated axons of the laminar portion 3 weeks after 1OP elevation (Fig. 2B). At higher magnification, degenerative changes such as neurofilament accumulation were observed in the experimental glaucoma groups in the myelinated portion (Fig. 2C). Autophagic vacuoles were observed in the glaucoma (Fig. 2D) and glaucoma + rapamycin groups (Fig. 2E, F). In spite of the appearance of autophagic vacuoles in these groups, degenerative changes were only noted in the glaucoma group (Fig. 2C, D). In the glaucoma + rapamycin group (Fig. 2E, F), myelin and microtubule structures were well preserved, and no apparent degenerative changes were seen. We also found significant increases in LC3-11 and p62 levels in optic nerves after 1OP elevation (Kitaoka et al., 2013). As mentioned above, an increase in the amount of LC3-11 shown by immunoblotting indicates either autophagy induction or the suppression of a downstream step in autophagy. Since p62 accumulates when autophagy is inhibited, and decreased levels can be observed when autophagy is induced (Bjorkoy et al., 2009), the increases in LC3-11 and p62 levels both suggest that autophagic flux impairment may occur in optic nerve degeneration after 1OP elevation.
1t was reported that rapamycin, an autophagy inducer that can upregulate flux, significantly ameliorated axonal degeneration after 1OP elevation as evidenced by light microscopic findings assisted by computer software morphometric analysis as well as electron microscopic findings (Kitaoka et al., 2013). This is not consistent
Fig. 1. Subcellular localization of p62 and mitochondria in nonstarved and starved RGC-5 cells. p62 was colocalized with Mito-Tracker Red, a marker of mitochondria in nonstarved (A) and starved (B) RGC-5 cells. Scale bar = 25 mm. Figure modified from Kitaoka et al. (2013).
Fig. 2. Electron microscopic findings 3 weeks after 1OP elevation. The laminar portions of the control (A) and experimental glaucoma (B) groups are shown. Abnormal mitochondria (black arrowheads) and autophagic vacuoles (black arrows) were noted in unmyelinated axons in experimental glaucoma (B). The myelinated portions in the experimental glaucoma (C, D) and glaucoma + rapamycin (E, F) groups are shown. Degenerative changes such as neurofilament accumulation (white asterisk) were noted in experimental glaucoma (C). The formation of multilamellar bodies is characteristic of autophagic vacuoles (white arrows, C, D). Preserved myelin and microtubule structures were noted in the glaucoma + rapamycin (E, F) groups. Autophagic vacuoles (white arrowheads) were observed in the glaucoma (D) and glaucoma + rapamycin (E, F) groups. Scale bar = 1 mm (A, B) and 200 nm (C—F). Figure modified from Kitaoka et al. (2013).
with the findings that DAP1-positive cells in the RGC layer were significantly increased by the application of the autophagy inhibitor 3-MA after 1OP elevation (Park et al., 2012). One possible explanation is the different roles of autophagy in axons and cell bodies. 1t was suggested that the protective effect of autophagy on axons may be prolonged (Lin and Kuang, 2014). On the other hand, with the progressive increase in 1OP, autophagy is predominantly activated in the cytoplasm in RGC bodies where it induces cell death (Lin and Kuang, 2014). However, a pattern of localized retinal nerve fiber layer defects in glaucoma patients indicates that axonal degeneration may precede RGC body death in this condition, suggesting that successful axonal protection can prevent further sequential RGC body death. Since we also found that rapamycin increased LC3-11 levels more than 1OP elevation alone, and that increased p62 levels induced by 1OP elevation were significantly inhibited by rapamycin in the optic nerve (Kitaoka et al., 2013), it is likely that autophagic flux impairment in the optic nerve after 1OP elevation may be improved by rapamycin, thereby leading to axonal protection. 1n other areas of the central nervous system, it is also
interesting to note a recent study demonstrating that there are two mechanisms of the neuroprotective effect of rapamycin: the first is stimulation of autophagy leading to impaired mitochondria removal; and the second is enhancement of mitochondrial fission to allow their elimination by mitophagy in axotomized precer-ebellar neurons (Cavallucci et al., 2014). Moreover, it has recently been proposed that enhanced mitochondrial function can attenuate the progression of glaucoma and other neurodegenerative diseases (Osborne et al., 2014). Taken together, the results indicate that the modulation of autophagic flux could be a target for the treatment of neurodegenerative diseases including glaucoma.
3.3. TNF-induced optic nerve damage
3.3.1. TNF-induced axonal degeneration and TNF has been implicated in Alzheimer's disease
A crucial role of TNF has been suggested in glaucomatous optic neuropathy (Yan et al., 2000; Yuan and Neufeld, 2000; Tezel et al., 2001). It was demonstrated that the glial production of TNF is
increased and its death receptor is upregulated on RGCs and optic nerve axons in glaucomatous eyes (Tezel, 2008). Not only the direct neurotoxicity of TNF to RGCs and their axons but also its indirect secondary neurodegeneration along with other cellular events may contribute to glaucomatous optic neuropathy (Tezel, 2008). A previous study of proteomic data from human glaucoma showed a prominent upregulation of TNF/TNF receptor 1 signaling in the glaucomatous retina (Yang et al., 2011). Intravitreal injection of TNF was shown to cause primary optic nerve axonal degeneration with subsequent slow RGC body death in rats and mice (Kitaoka et al., 2006; Nakazawa et al., 2006). In the degenerating optic nerve, CREB phosphorylation was found to be associated with axonal protection (Fujino et al., 2009). In the neonatal rat ischemic brain, rapamycin provided neuroprotection with CREB phosphorylation (Carloni et al., 2010). On the other hand, previous studies suggested that glaucoma may be correlated with Alzheimer's disease (AD) (Tamura et al., 2006; Wostyn et al., 2010). TNF is synthesized and released from astrocytes and microglia in the central nervous system and has been implicated in the pathogenesis and progression of AD (Paganelli et al., 2002; Alvarez et al., 2007). It was shown that presenilin is one of the proteases of g-secretase, consisting of presenilins (PS1 and PS2), nicastrin, Aph-1, and Pen-2, and is responsible for g-secretase activity, and that the inhibition of PS1 activity is a potential target for antiamyloidogenic therapy in AD (De Strooper et al., 1998). In addition, it was reported that increased expression of PS1 is sufficient to increase g-secretase activity (Li et al., 2011). Our previous study demonstrated that the increase in p-PS1 and activation of g-secretase in the optic nerve may be associated with TNF-induced axonal degeneration (Kojima et al., 2012).
3.3.2. Autophagy in AD informs autophagic events in axons
Autophagic impairment was found to stimulate PS1 expression and g-secretase activation in human embryonic kidney cells (Ohta et al., 2010). Rapamycin efficiently suppressed amyloid-ß peptide-induced neurite degeneration in PC12 cells (Yang et al., 2014a,b,c). Moreover, rapamycin prevented the synaptic failure induced by Aß oligomers in rat hippocampal neurons (Ramírez et al., 2014). It was proposed that impaired autophagy can induce the accumulation of dysfunctional mitochondria and cause disturbances in the processing of amyloid precursor protein (APP) and the clearance of tau proteins (Salminen et al., 2013). Thus, the improvement of autophagy seems to be beneficial in treating these neurodegenerative processes. Our recent study has demonstrated that rapamycin substantially protects axons in TNF-induced optic nerve degeneration (Kojima et al., 2014). In APP/PS1 transgenic mice, there was a significant increase in p62 protein levels compared with wild-type mice without any change in mRNA, implying that autophagic clearance of p62 is impaired in AD model neurons (Joshi et al., 2014). The level of p62 is significantly increased in the brain of AD patients relative to controls (Odagiri et al., 2012). However, it does not appear that upregulated p62 is specific to AD pathological processes, since autophagic deficiency caused accumulation of p62 aggregates, but the aggregates did not contain Aß in the AD model brain (Nilsson et al., 2014). In the optic nerve, p62 was upregulated after TNF injection, and the inhibition of p62 by siRNA exerted axonal protection in TNF-induced optic degeneration (Kojima et al., 2014). Therefore, we propose two possibilities: first, p62 itself may act as a neurodegenerative candidate since the overexpression of p62 promotes apoptosis with the activation of caspase-8, while knockdown of p62 reduces human glioma cell death (Zhang et al., 2013). The forced expression of p62 consistently decreased the number of neuronal cells under hypoxic stress (Tanabe et al., 2011). Second, p62 siRNA may affect autophagic activity, because a recent study has demonstrated that
p62 siRNA activated autophagic machinery as confirmed by increases in lysosome-associated membrane protein 1, LC3-11, and beclin 1 and by the formation of autophagosomes (Islam et al., 2014). Thus, p62 appears to have distinct roles depending on the physiological or pathological condition or the status of proteasome activity. Nonetheless, modulation of p62 protein levels including modulation of autophagy conditions may be an attractive approach for the treatment of neurodegenerative diseases.
3.4. Nmnat and axonal protection
Several studies suggested that nicotinamide mononucleotide adenylyltransferase 1 (Nmnat1), an enzyme predominantly located in the nucleus in the nicotinamide adenine dinucleotide (NAD) biosynthetic pathway, plays crucial roles in axonal protection against axotomy in a dorsal root ganglia (DRG) explant culture (Araki et al., 2004; Wang et al., 2005). Decreased NAD levels were observed in degenerating axons of cultured neurons (Wang et al., 2005) and in the spinal cord of experimental autoimmune encephalomyelitis model mice (Kaneko et al., 2006). Subcellular localization of Nmnat1, which was shown with cytNmnat1, an engineered mutant of Nmnat1 localized only to the cytoplasm and axon, was found to be critical for axonal protection in vivo (Sasaki et al., 2009). Our previous study found that Nmnat1 is located in the axoplasm of the optic nerve and that the depletion of Nmnat1 and decreased NAD levels are involved in optic nerve axonal degeneration (Kitaoka et al., 2009). Other mammalian Nmnat iso-forms such as Nmnat2 (located in the Golgi apparatus and cytosol) and Nmnat3 (located in mitochondria) were also reported to exert axonal protection. For example, the knockdown of endogenous Nmnat2 caused a significant reduction in the percentage of healthy neurites, and exogenous Nmnat2 protected transected neurites in superior cervical ganglia (SCG) culture (Gilley and Coleman, 2010). The regulation of overall cytosolic NAD metabolism by Nmnat2 was shown to be critical for axon survival (Milde et al., 2013). On the other hand, overexpression of Nmnat3 provided strong axonal protection after transection in DRG neurons (Sasaki et al., 2006). Moreover, overexpression of Nmnat3 was reported to protect against rotenone-mediated (mitochondrial dysfunction) axonal degeneration and to delay axonal degeneration induced by treatment with the oxidant H2O2 in DRG neurons (Press and Milbrandt, 2008). Furthermore, transgenic mice overexpressing NMNAT3 had a significant number of preserved axons in injured sciatic nerves, whereas wild-type mice had mostly degraded axons in injured nerves (Yahata et al., 2009). Those findings are consistent with those of our recent study demonstrating that overexpression of Nmnat3 exerted a significant protective effect against TNF-induced axonal loss in the optic nerve (Kitaoka et al., 2013). In addition, the downregulation of Nmnat3 accelerated axonal degeneration (Fig. 3), implying a pivotal role of endogenous Nmnat3 in certain conditions in optic nerve degeneration.
Some recent studies have proposed molecular mechanisms of Nmnat-regulated axonal protection. For example, it was demonstrated that Highwire ubiquitin ligase, an important regulator of axonal and synaptic degeneration, is a critical regulator of Nmnat and may play a central role in regulating the ability of a neuron to regenerate its connection (Xiong et al., 2012). SkpA functions with Highwire to regulate axonal stability, and SkpA mutants partially inhibit axon degeneration, demonstrating a role for SkpA in promoting axonal degeneration after injury (Brace et al., 2014). The axonal protection seen for SkpA mutants is abrogated upon knockdown of Nmnat, suggesting that this axonal protection requires Nmnat (Brace et al., 2014). On the other hand, it was demonstrated that molecular chaperones are key in Nmnat-regulated axonal protective functions (Rallis et al., 2013).
Nmnat3 siRNA
Fig. 3. Inhibition of Nmnat3 accelerated TNF-induced axon loss. Light microscopic findings 2 weeks after PBS injection (A), 10-ng TNF injection (B), or 10-ng TNF injection + simultaneous injection of 500 pmol of Nmnat3 siRNA (C) (sc-62696, Santa Cruz Biotechnology, Santa Cruz, CA). Scale bar = 10 mm (A—C). (D) Effect of Nmnat3 inhibition on axon numbers in the optic nerve. Each column represents mean ± SEM; n = 3—5 per group. *p < 0.05. Morphometric analysis of each optic nerve was performed as described previously with samples from 9 rats (Kitaoka et al., 2011).
Interestingly, a recent study has proposed a link between Nmnat-mediated protection and autophagy in dendrite degeneration (Wen et al., 2013). Our recent study has demonstrated that axonal protection by Nmnat3 transfection was suppressed by 3-MA in hypertensive glaucoma model rats (Kitaoka et al., 2013). Taken together with our observation of substantial autophagic vacuoles in the electron microscopy findings in Nmnat3 transfection plus experimental glaucoma, these findings suggest that the protective effect of Nmnat3 may be involved in the autophagy machinery in optic nerve axons after IOP elevation. Nmnat3 transfection decreased p62 protein levels and increased LC3-II protein levels in the optic nerve not only in the IOP elevation group, but also in the group without IOP elevation (Kitaoka et al., 2013). In vitro, substantial colocalization of Nmnat3 and p62 was observed in non-starved and starved RGC-5 cells (Fig. 4). It is particularly important to note that Nmnat3 transfection increased autophagic flux as shown in the LC3 turnover assay and decreased p62 protein levels in RGC-5 cells (Kitaoka et al., 2013). In addition, Nmnat3 trans-fection did not affect p62 mRNA levels in this in vitro system. Therefore, combining the in vivo and in vitro findings, it is likely that the protective effect of Nmnat3 is associated with the auto-phagy machinery as well as enhancement of outgoing p62 flux and induction of LC3-II, which means the enhancement of autophagic flux.
In the NAD biosynthetic pathway, nicotinamide phosphor-ibosyltransferase (Nampt, an enzyme) converts nicotinamide into nicotinamide mononucleotide (NMN). Nmnat subsequently converts NMN into NAD. Surprisingly, a very recent study has demonstrated that blocking Nampt with FK866 added 1 day before neurite transection potently promoted axonal survival despite lowering NAD in the SCG culture (Di Stefano et al., 2014). In that study, treatment with the Nampt inhibitor FK866 was found to
delay axonal degeneration markedly in an in vivo vertebrate (zebrafish) model organism. Considering the finding that FK866 induces autophagy in SH-SY5Y neuroblastoma cells (Billington et al., 2008), it would be particularly interesting to elucidate in future whether the axonal protection of FK866 is associated with the autophagy machinery.
3.5. ROCK and axonal regeneration
Two Rho-activated serine/threonine kinases have been identified: ROCK1, which is also known as ROKb and p160ROCK; and ROCK2, which is also known as ROKa and Rho kinase (Riento and Ridley, 2003). Many studies reported that the inhibition of ROCK leads to axonal regeneration of RGCs (Planchamp et al., 2008; Ahmed et al., 2009). For example, a significant increase in the number of regenerating axons was observed after the addition of ciliary neurotrophic factor (CNTF) or Y-27632, a ROCK1 inhibitor, to culture compared with PBS controls in a rat optic nerve crush model (Lingor et al., 2008). The effect on regeneration was more pronounced in the optic nerve crush model where animals administered a combination of Y-27632 and CNTF showed stronger regeneration compared with CNTF- or Y-27632-alone administration (Lingor et al., 2008). 1t was also demonstrated that the neurite number at the maximum effect of Y-39983, a ROCK1 inhibitor, was greater than that of Y-27632 in retinal piece culture, and that Y-39983 exerted substantial regenerative effect after optic nerve crush (Sagawa et al., 2007).
There is a discrepancy for the role of rapamycin on the axon of RGC between axonal protection and axonal regeneration. For example, the mammalian target of rapamycin (mTOR) activity was suppressed and new protein synthesis was impaired in axotomized RGCs, which may contribute to the failure to regenerate (Park et al.,
Fig. 4. Subcellular localization of p62 and Nmnat3 in nonstarved and starved RGC-5 cells. p62 was colocalized with Nmnat3 in nonstarved (A) and starved (B) RGC-5 cells. Scale bar = 25 mm. Figure modified from Kitaoka et al. (2013).
2008). Because rapamycin is an inhibitor of mTOR, it does not appear beneficial for axonal regeneration. However, we found that it exerted substantial axonal protection as mentioned above. This discrepancy suggests that axonal regeneration is not an inevitable outcome of survival but reflects a balance between direct mTOR-mediated effects on protein synthesis and links to other signaling pathways that may have a more profound influence on axonal protection than axonal regeneration (Morgan-Warren et al., 2013). In this regard, further study pointed out that the inhibition of autophagy by atg7 siRNA caused the elongation of axons, while activation of autophagy by rapamycin suppressed axonal growth in cortical neurons and that autophagy negatively regulated axonal extension via the RhoA-ROCK pathway (Ban et al., 2013). Unlike rapamycin, downregulation of ROCK2 promotes axonal regeneration and attenuates axonal degeneration after optic nerve crush and increases RGC survival after optic nerve axotomy (Koch et al., 2014), suggesting substantial beneficial roles of the inhibition of ROCK2 in both neuroprotection and neuroregeneration. Interestingly, it was shown that the inhibition of ROCK2 resulted in increased LC3-II levels and decreased p62 levels in RGCs (Koch et al., 2014). That study using BafA1 also found that ROCK2 downregulation increased autophagic flux in RGCs. This scenario is consistent with our previous findings suggesting that increased autophagic flux leads to axonal protection (Kitaoka et al., 2013).
4. Aging
4.1. Aging and glaucoma
Several risk factors have been reported for glaucoma development, but age is among the strongest and most consistent. Previous epidemiological studies, including the Baltimore Eye study (Tielsch et al., 1991), Beaver Dam Eye study (Klein et al., 1992), Blue Mountain Eye study (Mitchell et al., 1996), Melbourne Visual Impairment Project (Wensor et al., 1998), Rotterdam Study (Wolfs et al., 2000), Los Angeles Latino Eye Study (Jiang et al., 2012), and Tajimi study (Kawase et al., 2008), showed that age is an important risk factor for glaucoma development. In Caucasian patients, the prevalence begins to increase sharply after the age of 60, while in African-Americans, Asians, and Hispanics, the prevalence begins to increase at an earlier age, starting at around 40 years.
The impact of cellular senescence on tissue pathophysiology is still under debate. The accumulation of senescent cells has been
proposed to contribute to the loss of tissue function in aging and several age-related diseases including glaucoma by different mechanisms (Fossel, 2002; Campisi, 2005). Senescent cells in the optic nerve and trabecular meshwork (TM) can disrupt the local tissue microenvironment via the overexpression of several proin-flammatory cytokines and production of ROS (Li et al., 2010; Kernt et al., 2013). The increased production of ROS by senescent cells could potentially lead to an increase in the dysfunction of adjacent nonsenescent cells and therefore contribute to the development of glaucoma (Tanito et al., 2012; Feilchenfeld et al., 2008).
In the TM, senescent cells can induce important changes in the extracellular matrix composition, including the increased expression and degradation of fibronectin, which leads to the accumulation of degradation products that are believed to have noxious effects on tissue physiology (Robert and Labat-Robert, 2000; Labat-Robert and Robert, 2000). The accumulation of such degradation products in the TM could potentially result in increased outflow resistance and contribute to the development or progression of glaucoma. Coexistent with age-related increases in the prevalence of glaucoma is a decrease in the anterior segment outflow facility. That is, the resistance to fluid flow across the TM increases with age in a linear fashion and begins at a fairly young age (Gabelt and Kaufman, 2005). This decreased outflow facility is largely responsible for the elevated IOP encountered with increasing age. It is interesting to note that diminished autophagic flux in TM cells has been suggested to contribute to the pathogenesis of glaucoma (Porter et al., 2013). However, recent data from human TM cells have demonstrated that p62 levels are lower in individuals older than 60 years compared with younger ones (Pulliero et al., 2014). That study suggested that under physiological conditions, auto-phagy increases with age in human TM cells due to increased oxidative stress. Therefore, it is possible that the upregulation of autophagic flux may be a manifestation of endogenous defensive mechanisms.
Biochemical factors within the optic nerve head have been reported to play crucial roles in RGC physiology and contribute to the optic neuropathy of aging and glaucoma (Almasieh et al., 2012). The posterior sclera of aged monkeys is significantly stiffer than that of younger individuals and leads to axonal damage with the higher stresses related to IOP elevation (Yang et al., 2014a,b,c). This age-associated stiffening of the sclera significantly influences the biomechanical properties of the optic nerve head and may contribute to age-related susceptibility to glaucomatous optic
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Fig. 5. Axons of 3-month-old SAMR(A), SAMP8 (B), and SAMP10 (C). SAM was purchased from Japan SLC, Inc (Shizuoka Japan). Scale bar = 20 mm. Axon number of 1- and 3-month-old SAMR, SAMP8, and SAMP10. Morphometric analysis of each optic nerve was performed as described previously (Kitaoka et al., 2011). Axonal degeneration in 3 month old SAMP8 and SAMP10, compared with that in SAMR (D). Each column represents mean ± SEM; n = 4—6 per group. *p < 0.05.
damage. Furthermore, damaged phospholipids in mitochondrial membranes by free radicals due to 1OP elevation and aging factor in the axon can affect membrane integrity, fluidity and transmembrane potentials, resulting in RGC degeneration through decreased Trx2 and subsequent translocation of cytochrome C (cyt C) and apoptosis inducing factor (AIF) from mitochondria to cytosol and nuclei (Ju et al., 2009; Munemasa et al., 2010). These changes also occur in humans and may be an important underpinning of the relationship between aging and glaucoma.
4.2. Age-related changes in the optic nerve
The optic nerves of elderly rats display fewer nerve fiber profiles with reduced packing density as compared with younger rats. Age-associated changes are observed especially in the supporting neuroglial cells, such as oligodendrocytes, astrocytes, and microglia. They are responsible for potassium homeostasis, axon—glial signaling, and integrity of the nodes of Ranvier and blood—brain barrier (Rasband and Shrager, 2000). Oligodendrocytes develop dense inclusions, some of which occupy swellings of their processes. Astrocytes display hypertrophy and contain dense inclusions, which may contain phagocytosed myelin material. Microglia have heterochromatic nuclei and a granular cytoplasm with inclusions (Yassa, 2014). These changes may contribute to dysfunctional changes, such as a decline in axonal transport and axonal loss.
RGC axon number in the optic nerve declines consistently with age. While methodologic and strain differences or large intra-sample variability was reported, some studies simply found little axon loss with age (Repka and Quigley, 1989; Cepurna et al., 2005). The rate of axon loss per month depends on the total axon number and is 2.25% of total axons per month in mice, 1.5% of total axons per
month in rats, 0.2% of total axons per month in monkeys, and 0.1% of total axons per month in humans (Calkins, 2013). These findings correspond roughly to life span, so that mammalian species experience an approximately 40% loss of axons over their lifetime (Neufeld and Gachie, 2003).
Our laboratory focuses on age-related axonal degeneration and RGC body death in mice. Because the life spans of rats and mice are not longer than 24 months, it is difficult to detect significant molecular changes due to cellular senescence in aging mice and rats. The senescence accelerated mice (SAM) strain was developed from the AKR/J strain by Kyoto University researchers. The R-series (SAMR) shows normal aging. In contrast, the P-series (SAMP) exhibits accelerated senescence (Takeda et al., 1997). The early onset and irreversible advance of senescence manifested by several signs such as deficits in learning and memory and emotional disorders are characteristic of SAMP8 and -P10 (Takeda, 2009). Furthermore, our research showed that greater RGC axon loss occurred in 3-month-old SAMP8 and -P10 compared with SAMR (Fig. 5). Age-related pathological changes in RGC axons were observed in mice at least 12 months old and were more evident in 24-month-old C57/BL6 mice (Samuel et al., 2011). Since pathological changes in RGC axons in the SAM strain were more evident and occurred earlier than those in C57/BL6 and other mice strains, SAM are useful to analyze biological changes in RGCs due to senescence.
4.3. Age-related change in RGC bodies
Since RGC axons decline proportionally with age as described above, it could be assumed that a decline occurs in RGC bodies in the retina. In the monkey retina, however, over a nearly 30-year lifespan the number of RGC bodies does not change regardless of eccentricity from the fovea or retinal quadrant (Kim et al., 1996).
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Fig. 6. Fluorescence micrograph from whole mounted retinas of 3-month-old SAMR(A), SAMP8 (B), and SAMP10 (C). Retrograde labeling of RGCs with Fluoro-Gold (Fluorochrome, Denver, CO) was performed as described previously (Kitaoka et al., 2009). Scale bar = 20 mm. RGCs density of 1- and 3-month-old SAMR, SAMP8, and SAMP10. RGCs loss in 3-month-old SAMP8 and SAMP10, compared with that in SAMR (D). Each column represents mean ± SEM; n = 4—6 per group. *p < 0.05.
Furthermore, over a 2-year life span the number of RGC bodies does not change in the C57BL/6 mouse retina (Samuel et al., 2011).
However, the dendritic arbors of at least some types of mouse RGC shrink by approximately 20% with a concomitant decrease in the density of IPL synapses (Samuel et al., 2011). Another report (Danias et al., 2003) found age-related RGC body loss in C57 mouse retinas based on morphological analysis with retrograde Fluoro-Gold labeling from the superior colliculus. It is not surprising that the RGC body loss was similar to axon loss of approximately 40% with decreased axonal transport of Fluoro-Gold from the superior colliculus (Danias et al., 2003). A significant loss of RGC bodies in SAMP8 and -P10 was observed at 3 months (Fig. 6). Since RGCs were labeled with Fluoro-Gold 2 months before the mice were sacrificed, i.e., 1 month after birth, presumably there was no axonal damage at that time. Our current data indicate that RGC death was not due to decreased axonal flow. Cresyl violet, which is not specific for RGC, stains not only neuronal but also glial cells. Our morphological study from counting cells in the RGC layer distinguished neuronal cells from glial cells based on their shape under microscopy. Our cresyl violet staining results confirmed decreased neuronal cells (RGCs and displaced amacrine cells) in SAMP8 and -P10 at 3 months (Fig. 7). Although cell body death relies on predominantly axonal degeneration and decreased axonal flow in senescence as described above, the RGC body is also susceptible to various forms of age-related stress.
5. Sirt1 in aging
5.1. Biological role of Sirtl
The sirtuins are a highly conserved family of (NAD+)-depen-dent histone deacetylases that help regulate the life span of diverse organisms (Koltai et al., 2010). The human genome encodes seven different sirtuins (Sirtl—7) that share a common catalytic core domain but process distinct N- and C-terminal extensions (de Oliveira et al., 2010). Of the seven mammalian sirtuin proteins, Sirtl has been the most extensively characterized. Sirtl plays a pivotal role in the antiaging effects of caloric restriction (CR) (Bonda et al., 2011), the protective effect of CR against neurodegenerative disease (Chen et al., 2008), and enhancement of the proliferative state of neuronal stem cells in the rat hippocampus (Torres et al., 2011 ), as well as being involved in protection against cellular oxidative stress and DNA damage (Gorenne et al., 2013).
Several important clues about Sirt1 function have emerged from studies of knockout and transgenic mice. In general, Sirt1 knockdown or its inhibition results in decreased physical activity due to reduced secretion of growth hormone (Cohen et al., 2009) and reduced thyroid-stimulating hormone (Akieda-Asai et al., 2010) and results in many of molecular changes including induction of vascular leakage and inflammation (Wu et al., 2012).
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Fig. 7. Whole-mounted retinas of 3-month-old SAMR (A), SAMP8 (B), and SAMP10 (C), showing neuronal cells (RGCs and displaced amacrine cells) stained with 1% cresyl violet as described previously (Kitaoka et al., 2006). Scale bar = 20 mm. Neuronal cells numbers of 1- and 3-month-old SAMR, SAMP8, and SAMP10. Neuronal cells loss in 3-month-old SAMP8 and SAMP10, compared with that in SAMR (D). Each column represents mean ± SEM; n = 4—6 per group. *p < 0.05.
5.2. Sirt1 in axons
5.3. Sirt1 in the retina
The neuroprotective effect of Sirt1 against axonal degeneration was observed in a study of slow Wallerian degeneration (Wlds) mutant mice (Perry et al., 1990). Wlds mutant mice exhibit a significant delay in the onset of axonal degeneration after physical or chemical injury (Coleman and Perry, 2002; Coleman, 2005). Axonal protection by Wlds is mediated through overexpression of Nmnat1 via activation of a Sirt1-dependent process, while neuroprotection is blocked by the Sirt1 inhibitor sirtinol and by Sirt1 silencing with siRNA (Araki et al., 2004). It was demonstrated that NAD+ treatment protected certain types of axon by suppressing oxidative stress (Ding et al., 2013). Similarly, it was shown that NAD+ treatment protected axons from oxidative stress-induced degeneration of the spinal cord (Bros et al., 2014). In the eyes, a previous study indicated that accumulation of superoxide in the optic nerve was increased in crushed nerves compared with controls. This change was suppressed by Sirt1 overexpression and resveratrol treatment (Zuo et al., 2013). More recently, the administration of a sirt1-activating compound (SRTAW04) significantly reduced ROS levels and preserved axons in the optic nerve in an optic neuritis model (Khan et al., 2014). Our recent study showed that Sirt1 levels in the optic nerve of 1-month-old SAMP8 and -P10 mice were decreased compared with those in controls (Fig. 8). We therefore suspect the involvement of a Sirt1-dependent process in axonal degeneration in senescence-accelerated conditions. Our results and those of previous studies suggest that activation of the Sirt1 pathway may be a useful strategy for axonal protection via the suppression of oxidative stress.
Sirt1 is distributed in most retinal layers. 1t is mainly localized in the nucleus, although it can be translocated to the cytoplasm and stimulates downstream molecules under various forms of stress. Sirt1 maintains energy homeostasis and antiapoptotic mechanisms essential for ameliorating the effects of oxidative stress in the retina. A previous study reported that Sirt1 maintains survival pathways, balances energy homeostasis, and is involved in a physiological DNA repair mechanism in photoreceptor cells to treat inherited retinal degenerative disease (Jaliffa et al., 2009). The intravitreal injection of SRT647 and SRT501, which are Sirt1 activators, prevented RGC loss in a dose-dependent manner by stimulating Sirt1 enzymatic activity in a mouse optic neuritis model (Shindler et al., 2007). Furthermore, the upregulation of Sirt1 by resveratrol protects cultured retinal cells from antibody-induced apoptotic death (Anekonda and Adamus, 2008).
1ntravitreal injection of resveratrol exerts neuroprotective effect through sirt1 overexpression following optic nerve crush (Kim et al., 2013). This effect was diminished by co-injection of sirtinol, a Sirt1 inhibitor, indicating therapeutic potential of overexpression of Sirt1 (Kim et al., 2013). The accumulation of superoxide was reduced in mice overexpressing Sirt1 or treated with resveratrol, indicating the protective effect of Sirt1 via modulation of oxidative stress (Zuo et al., 2013). Sirtinol reduced RGC viability in hypoxia and increased apoptotic markers such as caspase 3 activity and phosphorylation of c-Jun N-terminal kinase (JNK) (Balaiya et al., 2012). Our study showed that Sirt1 in isolated RGCs was decreased in SAMP8 and -P10 compared with SAMR (Fig. 9). These
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Fig. 8. Immunoblotting was performed as described previously (Munemasa et al., 2008) using antibodies for Sirtl (Santa Cruz Biotechnology), LC3 (Medical & Biological Laboratories Co., Nagoya, Japan), p62 (Medical & Biological Laboratories Co), and) ß actin (Sigma—Aldrich) with optic nerve lysate of 1-and 3-month-old SAMR, SAMP8, and SAMP10 (A and C). Densitometry of the bands was analyzed with NIH Image software (National Institutes of Health, Bethesda, MD) (B and D). Significant decreases in Sirtl in 1- and 3-month-old SAMP8 and SAMP10, compared with that in SAMR. Significant increases in LC3-II in 1- and 3-month-old SAMP8 and SAMP10, compared with that in SAMR (B and D). Each column represents mean ± SEM.; n = 4 per group. *p < 0.05.
results are consistent with the data on Sirt1 changes in the optic nerve of SAM (Fig. 8). A decrease in Sirt1 may contribute to RGC death through increased susceptibility to oxidative stress.
6. Autophagy and aging
6.1. Autophagy in cellular senescence
The inhibition of autophagy induces neurodegenerative changes in mammalian neuronal tissues which resemble those associated with aging, and pathological aging is often associated with a reduction in the number of autophagic-related molecules (Fields
et al., 2013; He et al., 2013). Several reports indicated that Atg proteins or other proteins required for autophagy induction have reduced expression in aged tissues and that autophagy diminishes with aging (Yang et al., 2014a,b,c). This finding, for example, was observed in normal human brain aging with downregulated Atg5, Atg7, and Beclin1 (Bec-1). Likewise, inositol trisphosphate receptor signaling is increased in age-related neurodegenerative diseases such as AD, suggesting a possible decrease in autophagy in these conditions (Lipinski et al., 2010; Decuypere et al., 2011). Although merely phenomenological and correlative, the cumulative findings suggest that the exhaustion of the autophagic response may contribute to the aging phenotype.
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Fig. 9. Immunoblotting for Sirt1 with isolated RGCs of 1-month-old SAMR SAMP8, and SAMP10 (A). RGC isolation was carried out with magnetic beads as described previously (Munemasa et al., 2008). (B) Significant decrease in Sirt1 of SAMP8 and SAMP10, compared with that in SAMR. Each column represents mean ± SEM; n = 3 per group. *p < 0.05.
Loss-of-function mutations in Atg1, Atg7, Atg18, and Bec-1 results in damaged cytoplasmic constituents accumulating in all aging cells and the levels of those constituents subsequently showed decreased during the life span of the nematode Caenorhabditis elegans (Toth et al., 2008). Deficient expression of Atg1, Atg8, and Sestrin, another autophagy-related molecule, reduces the life span of the fruit fly Drosophila melanogaster (Lee et al., 2010; Simonsen et al., 2008). This is related to age-associated pathologies, including triglyceride accumulation, mitochondrial dysfunction, muscle degeneration, and cardiac malfunction (Lee et al., 2010).
Fig. 10. Schematic diagram of the relationship between Sirt1 and autophagy in aging. A decrease in Sirt1 influences autophagy activation and autophagic flux impairment.
Although autophagy is indispensable for mobilizing intracel-lular energy reserves during the transition from intrauterine metabolism to weaning, the knockout of Atg proteins is lethal during the early postnatal period (Levine and Kroemer, 2008). Conditional knockout of Atg genes has age-related phenotypes and precipitates the manifestation of multiple age-associated stigmata, including the accumulation of intracellular inclusion bodies containing ubiquitinylated proteins and lysosomes containing the aging pigment lipofuscin, dysfunctional mitochondria, and the oxidation of proteins related to their carbonylation (Komatsu et al., 2006; Hara et al., 2006). Thus normal aging may be associated with insufficient autophagy, which can explain at least part of the aging phenotype.
CR, i.e., reduced food intake without malnutrition, is the pivotal antiaging intervention that extends the life span of most animals tested so far, including rhesus monkeys, in which it reduces the incidence of diabetes, cardiovascular disease, cancer, and brain atrophy (Colman et al., 2009). CR is the most physiological inducer of autophagy (Levine and Kroemer, 2008), and inhibition of auto-phagy prevents the antiaging effect of CR in all species investigated in this respect.
6.2. Autophagy and SAM
A previous study showed increased LC3-II in the hippocampal neurons of 7-month-old SAMP8, similar to pathological changes seen in late-onset AD (Ma et al., 2011). In our study, increased LC3-II levels were also noted in the optic nerve of 1-month-old SAMP8 and -P10 (Fig. 8) compared with SAMR. On the other hand, there were no significant changes in p62 levels among 1-month-old SAMR, -P8, and -P10 (Fig. 8). Since axonal degeneration was not seen in 1-month old SAMP8 and -P10 in our morphological examination (Fig. 5), one hypothesis is that the increased LC3-II level was due to an endogenous protective reaction rather than auto-phagy flux impairment. Another possibility is that the unchanged p62 levels are a manifestation of the transition status from flux activation to flux impairment. At 3 months, p62 was accumulated in SAMP8 and -P10, implying that lysosomal dysfunction may occur with subsequent autophagic flux impairment, thereby leading to axonal degeneration during the aging process in the optic nerve (Fig. 8). The accumulation of p62 was seen in the hippocampus, and the dysregulation of autophagy may affect the turnover of aggregate-prone proteins in aged mice (Soontornniyomkij et al., 2012).
6.3. Autophagy and Sirtl
Autophagy and senescence share a number of characteristics, which suggests that both responses could serve to protect cells collaterally from the toxicity of external stress such as oxidative ROS and mitochondrial apoptotic factor (Ou et al., 2014). Recent studies have shown that autophagy regulates Sirt1, a key molecule in aging, in cellular senescence. Sirt1 could influence autophagy directly via its deacetylation of key components of the autophagy induction pathway, such as the products of autophagy genes Atg 5, 7, and 8 (Suzuki and Bartlett, 2014).
The link between Sirt1 and autophagy is supported by multiple findings. Resveratrol, a recognized Sirt1 activator, induces the activation of autophagy, and it has been shown that this occurs in a sirt1-dependent manner (Morselli et al., 2011). It was noted that resveratrol could suppress autophagy via the inhibition of S6 ki-nase (Hwang et al., 2010). Sirtinol, a classic inhibitor of Sirt1, also increases autophagy based on the elevation of LC3-II levels and autophagic cell death in MCF-7 cells (Wang et al., 2013). These observations indicate that Sirt1 modulates Atg proteins and
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Fig. 11. Schematic diagram of autophagic flux status and mitochondrial movement inside optic nerve axons.
subsequently activates autophagy machinery. Another possible scenario is represented by weak correlations within an experimental system in which autophagy and Sirt1 are simultaneously activated. The diterpenoid oridonin inhibits the proliferation of both Hela (Cui et al., 2006) and the multiple myeloma (MM) cell line RPMI 8266 (Zeng et al., 2012a). In both cell lines, autophagy and Sirt1 induction occurred, although subsequently Sirt1 levels decreased in the MM cell line. Furthermore, the Sirt1 level increased in the latter cell line by another type of autophagy induction, nutrient depletion (Zeng et al., 2012b). The inhibition of autophagy by 3-MA suppressed Sirt1 expression in all these cases. In contrast, autophagy induced by Sirt1 activation plays a pivotal role in protecting against prion-induced neuronal cell death (Jeong et al., 2013). That study also suggested that regulating autophagy including that by Sirt1 activation may be a therapeutic target for neurodegenerative disorders including prion disease.
We found that although LC3-II was increased in the optic nerve of 1-month-old SAMP8 and -P10, Sirt1 was decreased. This appears to contradict the results of previous studies showing that Sirt1 activity could conceivably influence autophagy activation. However, considering the p62 findings, decreased Sirt1 in the optic nerve precedes autophagic flux impairment, because thereafter increases in p62 levels were observed in 3-month-old SAMP8 and -P10, which may result in cellular senescence and age-related pathological changes (Fig. 10). Therefore, enhancement of the clearance of short-lived ubiquitin-proteasome system-specific substrates by Sirt1-modulated autophagy may have beneficial effects on age-related neurodegeneration.
6.4. Autophagy and other age-related neurodegenerative diseases
Significant accumulation of damaged protein, lipids, and DNA has been found in Parkinson's disease, indicating an insufficient clearance of these modified molecules. Genetic ablation of auto-phagy genes Atg5 and Atg7 has been shown to cause abnormal accumulation of cytoplasmic inclusion bodies (Hara et al., 2006; Komatsu et al., 2006). Mouse brains or primary neurons with deficiencies in Parkin, PINK1, and DJ-1, which can be regulated by the mitochondrial autophagy machinery, exhibited mitochondrial abnormalities that may be due to insufficient mitophagy (Mortiboys et al., 2008; Wang et al., 2011), increased protein oxidation, and lipid peroxidation (Palacino et al., 2004), increased mitochondrial or cytosolic ROS and mito-roGFP oxidation (Gandhi et al., 2009), and increased sensitivity to neurotoxic or inflammatory insults (Manning-Bog et al., 2007).
Animal studies in neurodegenerative diseases with modulation of autophagy have shown positive results. Rapamaycin and
trehalose decreased protein aggregation in animal models of amyotrophic lateral sclerosis, Huntington's disease, AD, and Parkinson's disease (Rodríguez-Navarro et al., 2010; Castillo et al., 2013; Ozcelik et al., 2013). Although it is hypothesized that autophagy activation decreases the widespread propagation of oxida-tive damage in neurons, many studies focused on demonstrating decreased accumulation of harmful proteins and cell death without directly examining the cellular redox status or oxidative damage. Future mechanistic investigations of the effects of autophagy upregulation on the oxidative damage of proteins, lipids, DNA, or organelles in animal models of neurodegenerative disease are therefore necessary.
7. Conclusions and future directions
The molecular mechanism of RGC death and axonal degeneration in glaucoma has been investigated in several laboratories. A recent study that used induced pluripotent stem cells (iPSCs) generated from dermal fibroblasts obtained from a patients with TANK binding kinase 1 (TBK1)-associated normal tension glaucoma showed that there was an increase in LC3-11 expression in iPSC-derived RGCs from an NTG patient with a TBK1 gene duplication compared with those from unrelated control subjects that do not have a diagnosis of glaucoma (Tucker et al., 2014), implicating an important role for this cellular process in the human glaucoma. Although the involvement of autophagy has recently been demonstrated in RGC body death, its role in axonal degeneration has not been elucidated thoroughly. Autophagy is a dynamic process, and it is important to comprehend the autophagic flux status in each pathological condition. As shown Fig. 11, an increase in autophagosome number can be seen in both the activation of autophagy and blockade of autophagosomal degradation. 1n addition to these dynamic states, in optic nerve axons, the condition of axonal flow may also affect the number of autophagosomes. Mitochondria are also moving, and their function and behavior may affect autophagic flux. 1f one could determine the increase in autophagosomes (i.e., LC3-11) as "autophagy" at a specific point, then "autophagy" means impaired or induced, and therefore both opposing findings could be observed. However, if one could determine flux, then more accurate molecular mechanisms could be investigated. Because enhanced autophagy flux leads to increasing clearance of unnecessary proteins and damaged mitochondria, it may be part of the neuronal survival pathway. Thus, the modulation of autophagy flux may be a potential target for neu-roprotective interventions in glaucomatous optic neuropathy.
This work was supported by Grants-in-Aid No. 24592683 (YK) and No. 23792016 (YM) from the Ministry of Education, Culture, Sports, Science, and Technology of Japan and by the Suzuken Memorial Foundation (YK) and by the Charitable Trust Fund for Ophthalmic Research in Commemoration of Santen Pharmaceutical's Founder (YK).
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