Axonal protection by a small molecule SIRT1 activator, SRT2104, with alteration of autophagy in TNF‑induced optic nerve degeneration

Yasushi Kitaoka · Kana Sase · Chihiro Tsukahara · Naoki Fujita · Naoto Tokuda · Jiro Kogo · Hitoshi Takagi
1 Department of Molecular Neuroscience, St. Marianna University Graduate School of Medicine, 2-16-1 Sugao, Miyamae-ku, Kawasaki, Kanagawa 216-8511, Japan
2 Department of Ophthalmology, St. Marianna University School of Medicine, Kawasaki, Japan

To examine the effects of SRT2104, an SIRT1 activator, in optic nerve degeneration induced by TNF and to inves- tigate whether it affects the autophagic status after induction of axonal degeneration.
Study design
Adult male Wistar rats received intravitreal injection of TNF alone, concomitant injection of SRT2104 and TNF, or injection of SRT2104 alone. The autophagic status in the optic nerve was evaluated to examine p62 and LC3-II expression by immunoblot analysis. The effect of SRT2104 on TNF-induced axon loss was determined by counting the number of axons.
Intravitreal injection of SRT2104 showed a modest protective tendency in the 2-pmol-treated groups against TNF- induced axon loss, although the tendency was not significant on quantitative analysis. However, significant protective effects were found in the 20- or 200-pmol-treated groups. Injection of SRT2104 alone significantly decreased the p62 levels and increased the LC3-II levels as compared with the basal levels. Similarly, concomitant injection of SRT2104 and TNF sig- nificantly decreased the p62 levels and increased the LC3-II levels as compared with the TNF-treated group. Upregulation of SIRT1 expression was observed in the optic nerve after SRT2104 treatment.
The SIRT1 activator SRT2104 exerts axonal protection in TNF-induced optic nerve degeneration. This effect may be associated with upregulated autophagic status in the optic nerve.

Sirtuin 1 (SIRT1) is a member of the sirtuin family of proteins, which has been implicated in several molecular cellular mechanisms such as aging, transcription, calo- rie restriction, stress tolerance, metabolism, exercise, and mitochondrial biogenesis [1]. Activation of SIRT1 report- edly exerted neuroprotective effects against retinal ganglion cell (RGC) death and optic nerve injury in several types of disease models. For example, it was shown that SRT647and SRT501, two structurally and mechanistically distinct activators of SIRT1, attenuated RGC loss in a mouse model of experimental autoimmune encephalomyelitis-optic neu- ritis [2]. Moreover, overexpression of SIRT1 was shown to prevent RGC loss after optic nerve crush in mice [3]. That study demonstrated that overexpression of SIRT1 prevented accumulation of reactive oxygen species (ROS) in crushed optic nerves [3]. In another optic nerve injury model, a multiple sclerosis-like model, the SIRT1-activating com- pound STRAW04 prevented RGC loss and reduced ROS in the optic nerve [4]. Thus, although reduced ROS may beone possible mechanism of the SIRT1-mediated protectiveeffect, other functions in axonal degeneration remain to be ascertained.
Several studies have suggested that autophagy is an important pathway in some distinct optic nerve damage conditions such as those in a hypertensive glaucoma model, an optic nerve transection model, and an optic nerve crush model [5–7]. Microtube-associated protein light chain 3 (LC3) is known to occur on the autophagosome, an quantification of changes in LC3-II is one of the most widely used methods in autophagy research. However, increases in the levels of LC3-II are not measures of autophagic flux per se but can reflect the induction of the autophagosome or inhibition of autophagosome clearance [8]. p62/SQSTM1 is also used as a protein marker, and decreased p62 levels are associated with autophagy activation [8]. We recently demonstrated that enhanced autophagy plays protective roles for axons in a tumor necrosis factor (TNF)-mediated optic nerve damage model [9–11]. Since a close relation- ship between SIRT1 and autophagy has been implicated [12–15], we hypothesized that a SIRT1 activator may affect the autophagy machinery of the optic nerve. In the present study, we investigated whether SRT2104, a SIRT1 activator, alters optic nerve axon loss induced by TNF and examined whether it affects the autophagic status in this process.

Materials and methods
Experiments were performed on 8-week-old male Wistar rats. All studies were conducted according to the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research and approved by the Ethics Committee of the Insti- tute of Experimental Animals of St. Marianna University School of Medicine. The rats were maintained in controlled rooms (23 ± 1 °C, humidity at 55 ± 5%, light from 6 AMto 6 PM).

Intravitreal injection
Intravitreal administration of 10 ng TNF (Sigma-Aldrich) in phosphate-buffered saline (PBS) was performed under anesthetization with intramuscular injection of a ketamine- xylazine mixture. SRT2104 was purchased from Selleck- chem, dissolved in dimethyl sulfoxide (DMSO), and diluted with PBS. The same amount of DMSO in PBS was admin- istered into the contralateral eye as a control. Simultaneous injection of 2, 20, or 200 pmol of SRT2104 and 10 ng TNF or injection of 200 pmol of SRT2104 alone was performed intravitreally. The rats were euthanized with an overdose of sodium pentobarbital 1 and 2 weeks after the intravitreal injection, and the optic nerves were collected.

Immunoblot analysis
One week after the intravitreal injection, optic nerve speci- mens (4-mm lengths) were collected and homogenized in protein extraction buffer. After centrifugation at 15,000g for 15 min at 4°C, the supernatants were collected and used for determination of the protein concentration. Equal-amount (3μg) samples were applied and loaded into a mini gel (Cat. #456-9036; Bio-Rad Laboratories). After being transferred to an enhanced chemiluminescent membrane (EMD Milli- pore Corporation), each membrane was blocked with Tris- buffered saline (TBS) containing 5% skim milk and reacted with anti-p62 antibody (MBL Life Science), anti-LC3 anti- body (MBL Life Science), anti-SIRT1 antibody (Santa Cruz Biotechnology), or anti-β-actin antibody (Sigma-Aldrich). After 3 washes, the membranes were reacted with antirabbit or antimouse peroxidase-labeled secondary antibody (MP Biochemicals). A chemiluminescence detection system (ECL Plus Western Blotting Detection Reagents) was used for signal visualization.

Morphometric analysis of optic nerve axons
Two weeks after the intravitreal injection, optic nerve speci- mens (4-mm lengths from 1 mm behind the globe) were col- lected and soaked in Karnovsky’s solution. After processing and embedding in acrylic resin, cross sections were made and stained with 1% paraphenylenediamine (Sigma-Aldrich) in absolute methanol. As described previously [16], 5 sep- arate areas were used for quantification using the image- processing software. The averaged axon numbers in each group were expressed as the number per square millimeters.

Statistical analysis
Data were expressed as means ± SEMs. Differences among groups were analyzed by 1-way ANOVA, followed by the Dunnett post hoc test. Probability values below < .05 were considered significant. Results Effects of SRT2104 on TNF‑induced axon loss in the optic nerve Light microscopy findings from cross-sectioned optic nerves showed that compared with the PBS-treated group (Fig. 1a), apparent degenerative changes and axon losses were observed in the TNF-treated group (Fig. 1b). Treatment with 2 pmol SRT2104 plus TNF showed a protective tendency (Fig. 1c); however, the morphometric analysis showed that this was not significant (P = .0834 vs TNF alone; Fig. 1f). Substantial protective effects were seen in the 20- and 200 pmol-treated groups (Fig. 1d and e, respectively) against TNF-induced axon loss. The morphometric analysis revealed that these protections were significant (20 pmol: P = .0265 vs TNF alone; 200 pmol: P = .0024 vs TNF alone; Fig. 1f). Effects of SRT2104 on p62 protein levels in the optic nerve Consistent with our previous findings [11], a significant increase in p62 protein levels was observed in the optic nerve 1 week after TNF injection (Fig. 2a). This increase was totally abolished by SRT2104 (Fig. 2a). Moreover, SRT2104-alone treatment significantly reduced p62 protein levels as compared with the basal levels (Fig. 2b). Effects of SRT2104 on LC3‑II protein levels in the optic nerve As previously reported [9], no significant change was found in the LC3-II levels in the TNF-treated group as comparedwith those in the PBS-treated group at 1 week (Fig. 3a). Treatment with SRT2104 plus TNF significantly upregulated the LC3-II levels as compared with TNF alone (Fig. 3a). Furthermore, SRT2104-alone treatment significantly increased the LC3-II levels as compared with the basal lev- els (Fig. 3b). Effects of SRT2104 on SIRT1 protein levels in the optic nerve We examined whether SRT2104 affects SIRT1 protein lev- els in the optic nerve. Although no significant change was observed in the TNF-treated group as compared with the PBS- treated group, treatment with SRT2104 plus TNF significantly upregulated the SIRT1 levels as compared with TNF alone Discussion A recent study demonstrated that optic nerve crush causes a significant reduction in SIRT1 mRNA levels from optic nerve samples 7 days after injury [17]. A previous studyalso reported time-dependent reduction in SIRT1 mRNA and protein levels in the optic nerve from 7 days to 21 days after optic nerve crush [18]. In another optic nerve injury, significant decreases in SIRT1 protein levels were observed in optic nerves in 1- and 3-month-old senescence-acceler- ated mice that exhibited significant optic nerve axon loss at the age of 3 months [5]. However, the present study did not find a significant change in SIRT1 expression in the optic nerve after TNF injection. Thus, SIRT1 expression may differ depending on the degree or type of optic nerve injury. Nonetheless, our present study found a significant increase in SIRT1 protein levels in the optic nerve after SRT2104 treatment. This finding is consistent with that of a recent study demonstrating that SRT2104 treatment induced a significant increase in SIRT1 protein levels in the hippocampus of chronic unpredictable mild stress-exposed mice [19]. Therefore, it is possible that intravitreal injection of SRT2104 exerted significant axonal protection against TNF-induced optic nerve degeneration with upregulation of SIRT1 expression in the optic nerve. In the present study, SRT2104 significantly increasedLC3-II protein levels and reduced p62 protein levels, impli- cating that upregulated SIRT1 expression leads to autophagy activation (Fig. 5). A previous review article suggested that SIRT1 activators stimulate autophagy, because SIRT1 reg- ulates several cellular processes, such as maintenance of energy homeostasis and cellular survival, which are also major autophagy functions [13]. Another study demonstrated that both transfection-enforced overexpression of SIRT1 and pharmacologic activation of SIRT1 by resveratrol stimulate the autophagic flux in a human colon cancer cell line as well as in nematode cells [14]. It has recently been reportedthat a SIRT1 activator, SRT1720, enhances autophagy and inhibits apoptosis in the myocardium of hypoxic mice [20]. In neurons, it was demonstrated that resveratrol attenuated β-amyloid-induced neurotoxicity by enhancing autophagy in PC12 cells and that this enhanced autophagy was SIRT1- dependent [21]. Moreover, it was demonstrated that resver- atrol upregulated autophagy and protected motor neurons after spinal cord injury in rats [22]. 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