Characterizing the protective effects of SHLP2, a mitochondrial-derived peptide, in macular degeneration

Mitochondrial-derived peptides (MDPs) are rapidly emerging therapeutic targets to combat development of neurodegenerative diseases. SHLP2 (small humanin-like peptide 2) is a newly discovered MDP that is coded from the MT-RNR2 (Mitochondrially encoded 16S rRNA) gene in mitochondrial DNA (mtDNA). In the current study, we examined the biological consequences of treatment with exogenously-added SHLP2 in an in vitro human transmitochondrial age-related macular degeneration (AMD) ARPE-19 cell model. In AMD cells, we observed significant down-regulation of the MDP-coding MT-RNR2 gene, and remarkably reduced levels of all five oxidative phosphorylation (OXPHOS) complex I-V protein subunits that are involved in the electron transport chain; these results suggested mitochondrial toxicity and abnormal OXPHOS complex protein subunits’ levels in AMD cells. However, treatment of AMD cells with SHLP2: (1) restored the normal levels of OXPHOS complex protein subunits, (2) prevented loss of viable cells and mitochondria, (3) increased the number of mtDNA copies, (4) induced anti-apoptotic effects, and (5) attenuated amyloid-β-induced cellular and mitochondrial toxicity. Cumulatively, our findings established the protective role of SHLP2 in AMD cells in vitro. In conclusion, this novel study supports the merit of SHLP2 in the treatment of AMD, a primary retinal disease that is a leading cause of blindness among the elderly population in the United States as well as worldwide.

been very well-characterized as a cytoprotective peptide in a plethora of disease models including neurodegenerative and retinal diseases 5 . Furthermore, our recent work highlighted the role of Humanin-G, a single amino acid Humanin variant, in protecting AMD transmitochondrial RPE cybrids against cellular and mitochondrial damage 4 . In addition to Humanin, the 16S region of the mtDNA also codes for six additional MDPs referred to as small humanin-like peptides (SHLP1, SHLP2, SHLP3, SHLP4, SHLP5, and SHLP6) that are 24-38 amino acids in length. Of these, SHLP2 is a 26 amino acid peptide (Molecular Weight-3017.54 D) that has been demonstrated to modulate cellular and mitochondrial functions in a recent study 8 . In that study, SHLP2 increased the number of viable cells and improved mitochondrial bioenergetics in pancreatic cell lines. Another study demonstrated that SHLP2 mediates chaperone-like effects 9 .
In the current study, we hypothesized that SHLP2 will preserve AMD cells and mitochondria from toxicity. Herein, we characterized the ability of SHLP2 to protect retinal cells in AMD, using the transmitochondrial ARPE-19 cybrid cells that contain identical nuclei (derived from mtDNA-deficient (Rho0) ARPE-19 cells) but differ in mtDNA, which is derived either from AMD patients (AMD cybrids) or normal subjects (normal cybrids). Therefore, each cybrid cell line represented one individual patient. Our study revealed that administration of SHLP2 confers cellular and mitochondrial protection in AMD, indicating the therapeutic potential of SHLP2 for treatment of AMD.

MT-RNR2 gene is down-regulated in AMD cybrids. To examine the expression of MT-RNR2 gene that
contains small open-reading frames (ORFs) for MDPs such as Humanin and SHLPs, we performed quantitative real-time polymerase chain reaction (qRT-PCR) using MT-RNR2 targeting TaqMan gene expression assays. We observed a 56% decrease in MT-RNR2 gene expression in untreated AMD cybrids (0.44 ± 0.17 a.u. (arbitrary unit)) compared to untreated normal cybrids (1 ± 0.13 a.u.) (P = 0.03, n = 5) (Fig. 1A). This suggests that down-regulation of MT-RNR2 gene might contribute to reduced production of MDPs which in turn leads to reduced cytoprotective effects and eventually unhealthy AMD cybrid cells. Furthermore, treatment with SHLP2 caused no significant change in the expression of MT-RNR2 gene in normal cybrids (NL UN: 1 ± 0.13 a.u., NL SHLP2: 0.84 ± 0.14 a.u.; P = 0.43, n = 5) (Fig. 1B). In contrast, AMD cybrids showed 42.3% decline in MT-RNR2 expression after treatment with SHLP2 (AMD UN: 1 ± 0.128 a.u., AMD SHLP2: 0.577 ± 0.10 a.u.; P = 0.04, n = 4) (Fig. 1C). This response to SHLP2 may indicate the presence of a feedback loop that counterbalances the production of MDPs within the cellular system. This counterbalancing effect was specific to AMD cybrids and was not observed in normal cybrids.

SHLP2 prevents loss of mitochondria.
To examine the effects of SHLP2 on mitochondrial abundance in AMD, cells were stained with a fluorescent probe that specifically targets mitochondria, followed by confocal imaging (Fig. 3A). SHLP2 led to a 153.02% increase in mtGFP fluorescence intensity in AMD cybrids (AMD UN: 0.232 ± 0.026 a.u.; AMD SHLP2: 0.587 ± 0.038 a.u.; P < 0.05, n = 4-5) (  In summary, these results indicate that SHLP2 increases mtDNA copy number and up-regulates PGC-1α gene in AMD cybrid cells.

SHLP2 prevents loss of viable cells and reduces apoptosis. Differences in cell viability between
untreated and SHLP2-treated AMD and normal groups were examined using MTT assay. Treatment with SHLP2 prevented loss of viable cells and led to a 21.79% increase in viable cell numbers in AMD cybrids (AMD UN: 0.78 ± 0.019 a.u.; AMD SHLP2: 0.95 ± 0.014 a.u.; P < 0.05, n = 4) ( To further investigate the effects of SHLP2 on Caspase-3 protein activation, cleaved Caspase-3 protein levels were detected using immunoblotting ( SHLP2 protects against Amyloid-β-induced cell death. The role of SHLP2 against the deleterious effects mediated by amyloid-β insult was characterized by exposing cybrid cells to amyloid-β 1-42 (active form) and amyloid-β 42-1 (inactive scrambled control (SC)) peptides and then measuring numbers of viable cells. As expected and as previously established, addition of amyloid-β 1-42 dramatically decreased cell viability in both normal (NL

SHLP2 protects against Amyloid-β-induced mitochondrial damage. To investigate if in addition
to preventing amyloid-β-induced cell death, SHLP2 also blocks amyloid-β-induced mitochondrial damage, the following experiments were conducted. The SHLP2-and amyloid-β-treated normal and AMD cybrids' mitochondria were labeled with a GFP stain followed by cell imaging (Fig. 7A)

Discussion
Identification of novel curative molecules that can mitigate retinal atrophy is critical to the development of a treatment regime for macular degeneration. Herein, we demonstrate that SHLP2, a naturally occurring MDP, plays a central role in protecting AMD retinal cells and mitochondria in an in vitro transmitochondrial ARPE-19 cell model.
The cytoprotective action of MDPs has been very well-characterized in various disease models including neurological diseases e.g., Alzheimer's disease 10 , and retinal diseases e.g., macular degeneration 11 ; and this has added new dimensions to the field of degenerative disease therapy. Humanin, the first characterized MDP, and its analogs have been shown to have therapeutic potential in in vitro and in vivo models of numerous diseases such as Alzheimer's disease, amyotrophic lateral sclerosis, Parkinson's disease, stroke, diabetes, cancer, cardiovascular diseases, and atherosclerosis 5,8,[12][13][14][15][16][17] . In the current study, we found that the mtDNA-encoded MT-RNR2 gene, which harbors ORFs for cytoprotective MDPs including Humanin and SHLPs, was down-regulated by 56% in AMD cybrids compared to normal cybrids. Other studies have reported that RPE cells from AMD patients have fragmented and damaged mtDNA 18,19 , which one can speculate lead to lower expression of MT-RNR2 gene. Consistent with our findings, previous studies have shown that mitochondrial dysfunction due to hepatotoxicity can also lead to reduced MT-RNR2 gene expression 20 . Furthermore, plasma levels of MDPs such as Humanin, SHLP2, and MOTS-c decline significantly with age, suggesting a correlation between loss of MDPs and the deteriorating biological processes associated with aging and diseases 8 . The mechanisms by which MDPs are regulated are unclear. Interestingly, in comparison to untreated cybrids, exogenous addition of SHLP2 decreased MT-RNR2 gene expression by 42.3% (P = 0.04) in AMD cybrids. This could support the idea that production of SHLP2 MDP may be regulated via a negative feedback loop, wherein the cellular system detects the exogenously added SHLP2 peptide and prevents the MT-RNR2 gene from being expressed to maintain a healthy balance of MDPs within the cell. Although further studies are required to verify this feedback loop, to our knowledge, this is the first report to demonstrate the involvement of such a counterbalancing effect in MDP regulation.
Recently, Hinton et al. demonstrated that Humanin enhanced mitochondrial respiration via an increase in ATP levels, reserve capacity, oxygen consumption rate, and proton leak in RPE cells 21 . SHLP2 also improves mitochondrial metabolism by significantly increasing cellular ATP production and mitochondrial oxygen consumption rate 8 . In the present study, we compared the protein levels of OXPHOS Complex I-V subunits between untreated and SHLP2-treated AMD and normal ARPE-19 cybrid cells. The AMD cybrids showed drastically diminished protein levels of complex I-V subunits, suggesting compromised mitochondrial bioenergetics in the untreated AMD group compared to untreated normal. However, AMD cybrids treated with SHLP2 showed substantially augmented OXPHOS complex protein subunit levels as represented by an increase in the levels of NADH-coenzyme Q oxidoreductase (Complex I subunit) by 24%, Succinate-coenzyme Q oxidoreductase (Complex II subunit) by 59%, Coenzyme Q-cytochrome c oxidoreductase (Complex III subunit) by 37%, Cytochrome c oxidase (Complex IV subunit) by 46%, and ATP synthase (Complex V subunit) by 38%. Since these respiratory chain complexes play a vital role in ATP generation and maintenance of mitochondrial membrane potential, these findings indicate that SHLP2 preserves OXPHOS complex I-V protein subunits, thereby stabilizing AMD mitochondria. Interestingly, addition of SHLP2 did not show any considerable effect on electron transport chain complex I-V protein subunits' levels in normal cybrids.
Prior MDP studies have shown that Humanin G increases mitochondria-targeted fluorescence staining intensity in AMD 4 . To test if SHLP2 has similar effects, cells were labeled with a baculovirus fusion construct stain containing GFP + mitochondrial leader peptide, which specifically targets mitochondria and stains independently of function (i.e., membrane potential). Our results revealed that SHLP2 treatment led to a 153.02% increase in mtGFP fluorescence intensity in AMD cybrids compared to their untreated counterparts. Furthermore, SHLP2-treated AMD cybrids showed 40.3% higher relative mtDNA copy numbers and 307.87% higher PGC-1α gene expression compared to untreated AMD cybrids. To our knowledge, this is the first study that reports the ability of SHLP2 peptide to increase mtDNA copies and enhance the gene expression of PGC-1α, a master regulator of mitochondrial biogenesis. These findings suggest that SHLP2 plays a crucial role in protecting mitochondrial function and number in AMD cybrids. This is important and supports therapeutic approaches because protecting mitochondrial structure and function is the key to maintaining cellular health in AMD.
One mechanism by which SHLP2 blocks cell death is through its inhibitory effects on apoptosis. AMD cybrids treated with SHLP2 showed a 21.79% increase in viable cell numbers compared to untreated AMD cybrids. The apoptosis pathway in SHLP2-treated cybrids was evaluated and showed reduced apoptosis, represented by down-regulation of effector caspases i.e., (1) decreased gene expression of Caspase-3 by 81.8% and of Caspase-7 by 72.48%, and (2) reduction in Cleaved Caspase-3 protein levels by 56.45% in AMD cybrids. These findings are consistent with a study by Cohen et al. demonstrating that SHLP2 enhanced cell survival and improved cell function in a pancreatic carcinoma cell line 8 . Moreover, other MDPs such as Humanin and its variants reportedly reduce apoptosis and improve cell survival in RPE cells 4,21 .
We found that another property of SHLP2 is to act as a rescue factor against amyloid-β induced cytotoxicity. Deposition of amyloid-β, a constituent of drusen, has been associated with AMD progression in the retina 22 and with Alzheimer's disease in the brain 23,24 . Sub-RPE deposition of amyloid-β increases with age and is associated with early and advanced stages of AMD 25 . Amyloid-β deposits lead to local RPE toxicity and inflammation, thereby contributing to drusen biogenesis and AMD pathogenesis. It is known that ocular amyloidosis also accumulates amyloid fibrils in the vitreous and the cornea 26 . In the present study, cybrids were treated with a form of active amyloid-β 1-42 peptide that is a key contributor to Alzheimer's disease pathology 27 . SHLP2 pretreatment in AMD cybrids led to 40.98% higher cell viability compared to the AMD cybrids treated with amyloid-β 1-42 peptide alone. As a control, the amyloid-β 42-1 scrambled peptide did not show any appreciable effect on the viable cell count of cybrids. In addition to improving the health of cells exposed to amyloid-β 1-42 , SHLP2 also protected against loss of AMD mitochondria induced by amyloid-β insult. Increased mitochondria-targeting mtGFP staining intensity was observed in SHLP2 + Amy-β 1-42 -treated cybrids. Our observations are consistent with a study by Ding et al. wherein anti-amyloid therapy served as a viable option for protection against retinal and visual function damage, and reduced ocular amyloid-β deposits in a macular degeneration model 28 .
In summary, our study presents novel findings that delineate the role of SHLP2 as a mitochondria-targeting protective molecule that confers both morphological and functional protection against cellular and mitochondrial toxicity. These observations make SHLP2 a promising prospect as a therapeutic for macular degeneration. Further studies are required for a smooth transition of the SHLP2 peptide as a therapeutic option from lab bench to clinic.

Methods
Human subjects. Our research that involved human subjects was approved by the Institutional Review Board of the University of California Irvine (Approval #2003-3131). Clinical investigations were performed according to the tenets of Declaration of Helsinki and informed written consent was obtained from all participants.
Cell culture. Normal and AMD ARPE-19 transmitochondrial cells used in this study were prepared by polyethylene glycol fusion of mitochondrial DNA-deficient APRE-19 (Rho0) cell line with platelets isolated from either AMD patients or age-matched normal subjects 29 , as described previously 4 . All ARPE-19 transmitochondrial cybrids belonged to the 'H' mitochondrial DNA haplogroup. Passage 5 cybrids were used for all experiments. Age-matched normal cybrids served as controls.
Treatment with SHLP2. Stock solution of SHLP2 was prepared by reconstitution of lyophilized SHLP2 (Anaspec, Fremont, CA) in water. SHLP2 stock solution was further dissolved in culture media to obtain a working concentration of 3.2 µM. In this study, all cybrids were treated with 3.2 µM SHLP2.
MTT assay. The colorimetric MTT assay that is based on reduction of tetrazolium salts was used to measure cell viability. This assay is based on the principle that actively metabolizing healthy cells contain NAD(P) H-dependent cellular oxidoreductases, which reduce the yellow tetrazolium MTT [3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide] dye to its insoluble purple-colored formazan. Dimethyl sulfoxide (DMSO) is then added to solubilize the formazan crystals and the colorimetric signal, which is proportional to the number of viable cells, is determined by measurement of optical density at 570 nm. For this assay, cells were plated in 96-well plates and were treated with MTT solution (Cat. # 30006, Biotium, CA, USA) at 37 °C for 1 hr. Signal absorbance was measured on a spectrophotometer at 570 nm and background absorbance was measured at 630 nm. Background absorbance was subtracted from signal absorbance to obtain normalized absorbance values. Absorbance at 630 nm is background absorbance of the non-reduced MTT itself, before it is reduced into the formazan that absorbs at 570 nm. Therefore the 630 nm reading is a measurement of the amount of MTT in each well. The colorimetric signal obtained was proportional to the cell number.
CellLight mtGFP staining and Confocal microscopy. The CellLight Mitochondrial GFP reagent (Cat. # C10600, Thermo Fisher Scientific, MA, USA) was used to estimate the mitochondrial number in normal and AMD cybrid cells. CellLight mtGFP is a baculovirus fusion construct having a mammalian promoter and a leader sequence of E1 alpha pyruvate dehydrogenase fused to GFP. It specifically targets mitochondria and mtGFP fluorescence can be quantified. Cells were plated in 4-well chamber slides, transduced with CellLight mtGFP reagent, and incubated overnight at 37 °C. Cells were then washed with 1X TBS (Tris buffered saline), fixed in paraformaldehyde, and mounted in DAPI. Confocal z-stack images were captured using the LSM-700 Confocal microscope (Zeiss, Thornwood, NY, USA). Images were quantified using ZEN 2 lite software (Zeiss). Each image was a maximum intensity projection. Each mtGFP image was normalized to DAPI (internal control).
Mitochondrial DNA (mtDNA) copy number. Total DNA was isolated from normal and AMD cells and qRT-PCR was performed using 18S (nuclear) and MT-ND2 (mitochondrial) TaqMan gene expression assays (Cat. # 4331182, Thermo Fisher Scientific) and TaqMan gene expression master mix (Cat.# 4369016, Thermo Fisher Scientific). Relative mtDNA copy numbers were determined using delta Cts. All samples were run in triplicates.  (Table S1) was used as a housekeeper gene. Data analysis was performed using ∆∆Ct method which was calculated by subtracting ∆Ct of the AMD group from ∆Ct of the normal group. ∆Ct was the difference between the Cts (threshold cycles) of the target gene and Cts of the housekeeper gene (reference gene). Fold change was calculated using the following formula: Fold change = 2 ΔΔCt .

Quantitative Real-Time PCR (qRT-PCR). RNA was extracted from cells using the RNeasy Mini Kit
Western blotting analyses. Cells were lysed using RIPA buffer (Cat. # 89900, Life Technologies), supernatant was transferred to a new microfuge tube, and Bio-Rad Dc protein assay kit (Bio-Rad Laboratories, Richmond, CA) was used to measure protein concentration. Total protein samples were loaded in equal concentrations into the wells of 4-12% Bolt mini gels (Life Technologies) followed by SDS-PAGE electrophoresis. Gels were then transferred onto PVDF membranes, blocked in 5% milk for 1 hr, then incubated overnight at 4°C in the following primary antibodies: Total OXPHOS Human WB Antibody Cocktail (Cat. # ab110411, Abcam, Cambridge, MA, USA) and Cleaved Caspase-3 Ab (Cat. # 9661 T, Cell Signaling Technology (CST), Danvers, MA, USA). Washing the blots with 1X TBST (Tris Buffered Saline-Tween20) was followed by incubation with the respective secondary antibodies: Anti-mouse IgG (HRP) Ab (Cat. # 7076, CST) for OXPHOS, and anti-rabbit IgG (HRP) Ab (Cat. # GTX 213110-01, Genetex, Irvine, CA, USA) for Cleaved Caspase-3. Primary and secondary antibodies were diluted in 1X TBST. Following secondary antibody incubation, the blots were washed with 1X TBST. Clarity ™ Western ECL Blotting Substrate (Cat. # 1705060, Bio-Rad) was used for development of blots and Versadoc imager (Bio-Rad) was used to detect protein bands. β-actin antibody (Cat. # GTX 110564, Genetex) was used as a loading control for all Western blotting experiments, and all data were normalized to β-actin before being normalized to NL untreated samples. Densitometry measurements were performed using Image J software (NIH Image).
Statistical analysis. Unpaired Student's t-test (2 groups) or one-way ANOVA (3 or more groups) followed by post-hoc Tukey-Kramer test (GraphPad Prism 5.0; GraphPad Software, CA, USA) were used to analyze data between groups. P values < 0.05 were considered statistically significant.