Articles, Neurobiology of Disease

Proteomic Survey Reveals Altered Energetic Patterns and Metabolic Failure Prior to Retinal Degeneration

Ana Griciuc, Michel J. Roux, Juliane Merl, Angela Giangrande, Stefanie M. Hauck, Liviu Aron and Marius Ueffing
Journal of Neuroscience 19 February 2014, 34 (8) 2797-2812; https://doi.org/10.1523/JNEUROSCI.2982-13.2014

Abstract

Inherited mutations that lead to misfolding of the visual pigment rhodopsin (Rho) are a prominent cause of photoreceptor neuron (PN) degeneration and blindness. How Rho proteotoxic stress progressively impairs PN viability remains unknown. To identify the pathways that mediate Rho toxicity in PNs, we performed a comprehensive proteomic profiling of retinas from Drosophila transgenics expressing Rh1P37H, the equivalent of mammalian RhoP23H, the most common Rho mutation linked to blindness in humans. Profiling of young Rh1P37H retinas revealed a coordinated upregulation of energy-producing pathways and attenuation of energy-consuming pathways involving target of rapamycin (TOR) signaling, which was reversed in older retinas at the onset of PN degeneration. We probed the relevance of these metabolic changes to PN survival by using a combination of pharmacological and genetic approaches. Chronic suppression of TOR signaling, using the inhibitor rapamycin, strongly mitigated PN degeneration, indicating that TOR signaling activation by chronic Rh1P37H proteotoxic stress is deleterious for PNs. Genetic inactivation of the endoplasmic reticulum stress-induced JNK/TRAF1 axis as well as the APAF-1/caspase-9 axis, activated by damaged mitochondria, dramatically suppressed Rh1P37H-induced PN degeneration, identifying the mitochondria as novel mediators of Rh1P37H toxicity. We thus propose that chronic Rh1P37H proteotoxic stress distorts the energetic profile of PNs leading to metabolic imbalance, mitochondrial failure, and PN degeneration and therapies normalizing metabolic function might be used to alleviate Rh1P37H toxicity in the retina. Our study offers a glimpse into the intricate higher order interactions that underlie PN dysfunction and provides a useful resource for identifying other molecular networks that mediate Rho toxicity in PNs.

Introduction

Accumulation of misfolded proteins and the ensuing formation of protein aggregates are pervasive features of neurodegenerative disease. In most instances, the misfolded proteins start accumulating decades before the clinical onset of symptoms, begging the question of how neurons can tolerate chronic proteotoxic stress. Accumulation of misfolded rhodopsin (Rho) in photoreceptor neurons (PNs) of the retina leads to retinitis pigmentosa (RP), a subtype of retinal dystrophy characterized by progressive loss of visual abilities leading to blindness. Mutations in >190 genes cause retinal degeneration (RD), making it the most complex genetic disease in man (Daiger et al., 2007). The most common mutations associated with RP are those that lead to Rho misfolding (Daiger et al., 2007; Bramall et al., 2010; Wright et al., 2010). The major, unresolved, question is how these abnormal Rho species lead to progressive dysfunction and degeneration of PNs.

Substitution of proline 23 by histidine in the visual pigment Rho (RhoP23H) generates a folding-deficient Rho variant that exhibits increased retention in the endoplasmic reticulum (ER) and self-aggregates (Illing et al., 2002; Saliba et al., 2002; Mendes et al., 2005). Studies in Drosophila melanogaster also revealed that misfolded Rh1 is retained in the ER, leading to ER expansion (Colley et al., 1995) and ER stress (Galy et al., 2005; Ryoo et al., 2007; Griciuc et al., 2012). We have recently found that misfolded Rho is cleared, both in mammalian cells (Griciuc et al., 2010a) and fly PNs (Griciuc et al., 2010b), by a process called ER-associated degradation (ERAD). Misfolded Rho interacts with the chaperone VCP/ter94, a major effector of the ERAD pathway, which promotes its extraction from the ER and proteasomal degradation (Griciuc et al., 2010a). Inhibition of VCP function, or attenuation of proteasome activity, strongly mitigates Rh1P37H-induced PN degeneration, suggesting that excessive ERAD is pathogenic for PNs (Griciuc et al., 2010b). In mice expressing RhoP23H and undergoing RD, Rho localized to both ER and plasma membrane; however, 90% of the mutant protein was cleared (Sakami et al., 2011), probably by ERAD.

How chronic ER stress and ERAD impact the cellular networks in PNs and engage the cell death machineries remains unknown (Griciuc et al., 2011). We hypothesized that trapping of misfolded Rho in the ER and chronic activation of ER stress/ERAD pathways exerts a distorting effect on multiple cellular pathways causing severe imbalances in critical homeostatic processes. To unravel such critical networks, we performed the first large-scale proteomic profiling of Drosophila melanogaster retinas expressing the RhoP23H equivalent mutation Rh1P37H. We uncovered an early upregulation of energy-producing pathways and attenuation of target of rapamycin (TOR) signaling, which were reversed at the onset of RD. Complementary pharmacological experiments suggest that chronic suppression of TOR signaling confers long-term protection against RD. Genetic analyses further suggest that chronic Rh1 proteotoxicity causes mitochondrial failure and activation of the APAF-1/caspase-9 pro-apoptotic axis, thus identifying the mitochondria as critical mediators of Rh1 proteotoxicity. We suggest that energetic and metabolic dysfunction represent a critical link between Rho misfolding/proteotoxicity and PN degeneration in RP.

Materials and Methods

Fly stocks, crosses, and rearing.

Drosophila lines p(w+;Rh1-Rh1WT) and p(w+;Rh1-Rh1P37H) referred to as Rh1WT and Rh1P37H, respectively, were previously described (Galy et al., 2005). Control flies were either wild-type (WT) or Rh1-Gal4 flies. DTRAF1ex1 referred to as Traf1LOF was a kind gift from J. Chung (Cha et al., 2003). IceΔ1 referred to as IceLOF was kindly provided by B.A. Hay (Muro et al., 2006). HidA206 referred to as HidLOF was a kind gift from H. Steller (Sandu et al., 2010). Bsk1 (stock no. 3088) referred to as bsk1LOF, Dark82 (stock no. 23285) referred to as DarkLOF, Dronc51 (stock no. 23284) referred to as DroncLOF and w1118 (stock no. 5905) flies were from the Bloomington stock center. Flies were raised on standard cornmeal agar medium, under moderate continuous illumination at 25°C. Moderate illumination was obtained by using photosynthetic fluorescent tubes (in total 170 cd/m2). All experimental groups included male and female flies (ratio 1:1). Fly progeny having same eye pigmentation was used throughout the study.

1D prefractionation of lysates.

After 2 d or 14 d of light exposure, 200 retinas were collected separately for each genotype and lysed in detergent-containing radioimmunoprecipitation assay (RIPA)-modified buffer (20 mm Tris-HCl, pH 8.0, 150 mm NaCl, 1 mm EDTA, 1% Triton X-100, 0.1% SDS, and 0.5% sodium deoxycholate) supplemented with protease inhibitors (Roche) and phosphatase inhibitors (Sigma-Aldrich). Lysates were centrifuged at 16,000 × g for 15 min at 4°C and the supernatants were kept for further analysis. The protein concentration of the lysates was measured using the DC protein kit based on the Lowry assay (Bio-Rad). For each postnatal day 2 and 14 (P2 and P14), three replicates for both Rh1WT and Rh1P37H (80 μg per lane) were loaded on a 4–15% precast gradient gel (TGX; Bio-Rad). After separation, the gel was fixed and stained using Coomassie dye (0.1% Coomassie Brilliant Blue R-250 in 50% methanol, 10% acetic acid). Each lane was cut into six bands corresponding to different molecular weights to allow sample prefractionation. Every band was subjected to in-solution tryptic digestion and subsequent nano-LC-MS/MS analysis.

In-gel digest with trypsin.

Excised bands of the prefractionation gel were cut in cubes of 1 mm3 and transferred into tubes (Eppendorf). After destaining the gel cubes for 10 min with 200 μl of 60% acetonitrile (ACN) and a 10 min wash using 200 μl H2O, the pieces were dehydrated using 200 μl of 100% ACN for 10 min. For protein reduction, 100 μl of 5 mm dithiothreitol (DTT) was added and incubated for 15 min at 60°C. After removal of DTT and dehydration using 100% ACN, 100 μl of freshly prepared 25 mm iodoacetamide solution was added for 15 min at room temperature in the dark. The gel pieces were washed for 5 min with 100 μl H2O and again dehydrated in 100% ACN for 10 min. After three wash steps of 10 min with 50 mm ammonium bicarbonate (ABC), 60% ACN, and 100% ACN, the gel cubes were air dried for 15 min at 37°C. One hundred microliters of a 0.01 μg/μl trypsin solution (Promega) in 50 mm ABC was added to the gel cubes and incubated for 10 min, and 25 mm ABC was added to cover the gel pieces completely during the digest at 37°C overnight (o/n). For elution, 100 μl of 60% ACN/0.1% TFA were added to the gel cubes and incubated for 15 min. The supernatant was transferred to a new tube and 100 μl of 99.9% ACN/0.1% TFA was added to the gel pieces. After 30 min of incubation, the supernatants containing the eluted peptides were pooled, dried in a speedvac (UniEquip), and stored at −20°C.

MS.

Dried digested samples were thawed and dissolved in 2% ACN/0.5% TFA. The samples were centrifuged for 5 min at 4°C. LC-MS/MS analysis was performed as described previously (Hauck et al., 2010). Every sample was automatically injected and loaded onto the trap column at a flow rate of 30 μl/min in 5% buffer B (98% ACN/0.1% formic acid (FA) in HPLC-grade water) and 95% buffer A (2% ACN/0.1% FA in HPLC-grade water). After 5 min, the peptides were eluted from the trap column and separated on the analytical column by a 170 min gradient from 5 to 31% of buffer B at 300 nl/min flow rate followed by a short gradient from 31 to 95% buffer B in 5 min. Between each sample, the gradient was set back to 5% buffer B and left to equilibrate for 20 min. From the MS prescan, the 10 most abundant peptide ions were fragmented in the linear ion trap if they showed an intensity of at least 200 counts and if they were at least +2 charged. During fragmentation a high-resolution (60,000 full-width half maximum) MS spectrum was acquired in the LTQ Orbitrap XL (Thermo Scientificwith a mass range from 200 to 1500 Da.

Label-free analysis.

Protein expression levels in Rh1P37H flies were compared relative to Rh1WT flies using a label-free LC-MS/MS-based strategy. Quantitative analysis was performed using the Progenesis LC-MS and Max-Quant software. Prefractionation improves the limits of quantification in complex samples, but is thought to potentially influence the accuracy of label-free quantifications (Bantscheff et al., 2007). Therefore, the technical variability of sample preparation was determined, including the MS measurements, expressed as coefficient of variation (CV). CV was <14% for the Progenesis and <24% for the MaxQuant analyses, confirming robust sample preparation, prefractionation, and MS analysis.

Label-free analysis using Progenesis LC-MS.

The acquired spectra were loaded to the Progenesis LC-MS software (version 2.5, Nonlinear) for label-free quantification and analyzed as described previously (Hauck et al., 2010; Merl et al., 2012). Features with only one charge or more than eight charges were excluded. Raw abundances of the remaining features were normalized to allow correction for factors resulting from experimental variation. Rank 1–3 MS/MS spectra were exported as Mascot generic file and used for peptide identification with Mascot (version 2.2) in the Ensembl Drosophila protein database (13136213 residues, 21886 sequences). Search parameters were as follows: 10 ppm peptide mass and 0.6 Da MS/MS tolerance, one missed cleavage allowed, carbamidomethylation as fixed modification, and methionine oxidation and asparagine/glutamine deamidation as variable modifications. A Mascot-integrated decoy database search calculated a false discovery rate of <1.4%, using an ion score cutoff of 30 and a significance threshold of p < 0.01 for all searches. Peptide assignments were re-imported into the Progenesis software. After summing up the abundances of all peptides allocated to each protein, the results of all fractions were combined to a total analysis set and grouped (WT vs P37H). Total normalized protein abundance values in all fractions were used for statistical calculations.

After alignment and data normalization all samples were allocated to their respective groups (Rh1WT or Rh1P37H) for both the P2 and the P14 analysis. The Progenesis LC-MS analyses provided a total of 2710 or 2046 protein identifications at P2 and P14, respectively. After filtering of proteins identified and quantified with <2 peptides, the list was narrowed down to 1916 and 1485 proteins, respectively. In the Student's t test analysis comparing Rh1WT and Rh1P37H mutant samples, 225 (P2) and 186 (P14) proteins reached a p value of 0.05 or below.

Label-free analysis using MaxQuant.

The raw files were loaded into the 1.2.0.13 version of the MaxQuant software (Max Planck Institute of Biochemistry, Martinsried; Cox and Mann, 2008; Cox et al., 2009) with its internal search engine Andromeda (Cox et al., 2011). Except for the precursor tolerance of 10 ppm, all settings were set as default and “multiplicity” was set to one. The Andromeda search engine was configured for the Ensembl Drosophila protein database. The software further includes a decoy database to determine the false discovery rate, as well as a common contaminants database to exclude false positive hits due to contamination. Feature-matching between raw files was enabled, using a retention time window of 2 min. “Discard unmodified counterpart peptides” was unchecked. Averaged label-free quantification intensity values were used for statistical analysis.

Through MaxQuant analysis, a total of 2931 protein groups were identified in the P2 analysis and 2327 in P14. After filtering, the list was reduced to 2518 or 2053 proteins, respectively. Of these, 266 (at P2) and 249 (at P14) protein groups showed significantly different abundance comparing Rh1WT and Rh1P37H mutant samples (p < 0.05).

Pathway enrichment analysis.

With the lists of significantly altered proteins, pathway enrichment analyses were performed, separately for the upregulated and downregulated proteins in the Rh1P37H mutant (relative to Rh1WT) at P2 or P14. We used the Ingenuity Pathway Analysis (IPA) tool (http://ingenuity.com/products/pathways_analysis.html) to obtain the overrepresented pathways of all four protein sets.

Histology, toluidine blue staining, and analysis.

Fly heads were dissected and postfixed in 2.5% glutaraldehyde in PBS overnight at 4°C. After washing with PBS, heads were incubated in 1% osmium tetraoxide solution (Science Services) and then dehydrated in ethanol solutions of increasing concentrations (25–100%), followed by 10 min incubation in propylene oxide (Sigma-Aldrich). Heads were then incubated overnight in a solution containing 50% propylene oxide and 50% Durcupan epoxy resin mixture. The epoxy resin mixture consisted of 48% Component A/M, 40% Hardener B, 2.25% Accelerator C, and 9% Plasticizer D (Sigma-Aldrich). Then, heads were incubated o/n in 100% Durcupan epoxy resin mixture. The next day, heads and fresh Durcupan epoxy resin mixture were transferred to molds; heads were oriented tangentially and cooked overnight at 60°C. The polymerized resin containing the specimens was cut using an ultramicrotome. Semithin sections of 2 μm were collected, mounted, and then stained with toluidine blue solution (0.1% toluidine blue, 2.5% sodium carbonate). Pictures at different retinal depths were acquired for each head at 40× magnification. To determine the number of photoreceptor neurons/ommatidium (P/O), at least 150 ommatidia were scored per animal from at least six animals per genotype.

Electron microscopy.

For electron microscopy, the specimens were embedded the same way as described above for toluidine blue staining experiments. Subsequently, 70 nm ultrathin sections (instead of 2 μm) were collected on nickel grids and contrasted with 5% uranyl acetate and lead citrate, and analyzed with a Zeiss electron microscope EM 10.

Western blotting.

Fly heads were homogenized in RIPA-modified buffer (20 mm Tris-HCl, pH 8.0, 150 mm NaCl, 1 mm EDTA, 1% Triton X-100, 0.1% SDS, and 0.5% sodium deoxycholate) supplemented with protease inhibitors (Roche) and phosphatase inhibitors (Sigma-Aldrich). Lysates were centrifuged at 16,000 × g for 15 min at 4°C and the supernatants containing detergent-soluble fractions were kept for further analysis. Samples were normalized for total protein using the DC protein kit (Bio-Rad) based on Lowry assay. An equal volume of 2× SDS sample buffer was added to samples that were separated by 1 mm thick gels for 10–12% SDS-PAGE and electroblotted onto PVDF membranes (GE Healthcare). Immunodetection was performed according to standard techniques using the following primary antibodies: anti-Rh1 (4C5, mouse monoclonal, 1/5000; Developmental Studies Hybridoma Bank), anti-Hsc3 (guinea pig, 1/2000; gift from H.D. Ryoo; Ryoo et al., 2007), anti-TRP (rabbit polyclonal, 1/10,000; gift from A. Huber; Voolstra et al., 2010), anti-INAD (rabbit polyclonal, 1/10,000; gift from C. Montell; Lee and Montell, 2004), anti-Calnexin (rabbit polyclonal, 1/5000; gift from N.J. Colley; Rosenbaum et al., 2006), anti-Gp93 (rabbit polyclonal, 1/10,000; gift from C.V. Nicchitta; Maynard et al., 2010), anti-mt-ND1 (rabbit polyclonal, 1/200; Abcam), anti-phospho-4E-BP1 (Thr37/46, rabbit monoclonal, 1/1000; Cell Signaling Technology), anti-4E-BP1 (rabbit monoclonal, 1/1000; Cell Signaling Technology), anti-phospho-Drosophila p70S6K kinase (Thr398, rabbit polyclonal, 1/1000; Cell Signaling Technology), anti-p70S6K (rabbit polyclonal, 1/500; generated by D.R. Alessi, University of Dundee, UK; Lizcano et al., 2003), and anti-β-Tubulin (mouse monoclonal, 1/4000; Millipore Bioscience Research Reagents). Secondary antibodies were horseradish peroxidase-coupled (1/8000; Jackson ImmunoResearch). Quantification of band intensity after ECL detection was performed using Image Quant TL software.

Pharmacological treatments.

Flies were treated with rapamycin (Sigma-Aldrich) or the JNK inhibitor-SP600125 (Calbiochem-Millipore) dissolved in fly food. Two doses of rapamycin (50 and 200 μm, final concentration in fly food) and two doses of SP600125 (200 μm and 1 mm, final concentration in fly food) were used. These compounds were first dissolved in dimethylsulfoxide (DMSO) and the resulting solution was then added to fly food cooled down to 30°C. Flies were transferred to vials containing modified food right after eclosion, were reared as described above, and transferred to fresh vials every day. The control food contained all the ingredients (including DMSO) except the active compound. After 30 d of light exposure, flies were collected and their retinal integrity was assessed histologically.

Electroretinogram analysis.

Cold-anesthetized flies were immobilized in clay. A tungsten electrode (0.5–1 MΩ; Intracell) was inserted into the posterior part of the head and a glass electrode filled with 3 m KCl (2–6 MΩ) was poked through the cornea. Flies were dark adapted for 2 min before recordings. An orange LED (591 nm, LY 5436-VBW-1; Osram) was placed at 1 cm from the head. The flash intensity reaching the eye was 650 μW/cm2, as measured with a PM100D power meter and S121C photodiode (Thorlabs). Six light pulses were applied and the recorded responses were averaged. Flash intensity and duration were controlled through pClamp and the Digidata analog output. Signals were filtered at 2 kHz and digitized at 10 kHz, using a MultiClamp 700A amplifier, a Digidata 1322A interface, and pClamp-8 software (Molecular Devices).

Statistical analysis.

Data were evaluated and statistics were performed using Excel software (Microsoft Office 2003 SP3). MaxQuant data were filtered for reverse identifications, contaminants, and “only identified by site.” All identifications in both analyses based on <2 unique peptides were eliminated. In both quantification approaches we performed a two-sided Student's t test using the normalized abundances of the identified proteins.

Results

Proteomic profiling of Rh1 transgenic flies

To explore the pathways linking Rho proteotoxic stress to PN degeneration in RP, we undertook a label-free LC-MS/MS-based comparative proteomic analysis of a Drosophila model expressing Rh1P37H (the equivalent of the most common RP-linked mutation, RhoP23H). We chose Rh1WT flies as the most appropriate control for Rh1P37H flies, to eliminate any confounding effects due to Rh1 overexpression. In both of these lines, transgenic Rh1 expression is driven by a promoter that mimics the endogenous Rh1 promoter (Galy et al., 2005; Griciuc et al., 2010b). As a result, both Rh1P37H and Rh1WT flies express moderate and equivalent levels of transgenic Rh1 (an additional 50% relative to the endogenous Rh1, totaling 150% Rh1 in their retinas; Griciuc et al., 2010b). Furthermore, we chose to perform this proteomic analysis in flies exposed to light for 2 d or 14 d after eclosion (corresponding to P2 and P14, respectively). Rh1P37H flies have a largely preserved retinal structure at P14, and a normal complement of PNs, but do display the first signs of RD, such as vacuoles and disturbed rhabdomere morphology (Galy et al., 2005 and see below).

Protein identification and quantification

The analysis of Rh1P37H and Rh1WT proteomes at P2 and P14 by label-free LC-MS/MS was performed using the MaxQuant quantitative proteomics software (Cox and Mann, 2008) and the Progenesis LC-MS platform (Hauck et al., 2010; Merl et al., 2012). Compilation of the Progenesis and MaxQuant data identified a total of 409 proteins whose levels were significantly altered in the Rh1P37H mutant at P2. Of these, 204 proteins were found to be more abundant in the Rh1P37H mutant and 205 proteins showed decreased levels in the Rh1P37H mutant. Of the 350 significantly different proteins at P14, 132 were less abundant and 218 proteins were more abundant in the Rh1P37H mutant (data not shown).

Early upregulation of energy-producing pathways in the Rh1P37H retina

To identify proteomic changes that show coordinated coregulation (i.e., networks), we performed a network analysis on the proteomic data using the IPA software (Thiele et al., 2012). The IPA analysis includes only those Drosophila genes with functional homology to their mammalian counterparts. When applying the IPA to identify cellular networks that were induced in 2-d-old Rh1P37H flies (relative to Rh1WT flies) we found a striking upregulation of energy-producing pathways, which included the glycolysis, the citric acid (Krebs) cycle, and oxidative phosphorylation (Table 1, Fig. 1A). Numerous proteins functioning in the respiratory chain complex I (NADH-coenzyme Q reductase; such as NDUFS1, NDUFV2, NDUFS8, NDUFS2, NDUFB8, NDUFB10, and NDUFS4) and complex IV (COX6A1) as well as those linked to ATPase function and ATP production (ATP6V1E1, ATP5H, and ATP5O) were upregulated in Rh1P37H retinas. The Parkinson's associated gene PARK7/DJ-1, encoding a major mitochondrially acting oxidative stress suppressor (Abou-Sleiman et al., 2006), was also upregulated.

View this table:
Table 1.

Summary of proteomic changes in Rh1P37H and Rh1WT-expressing retinas, at P2 and P14

Figure 1.
Figure 1.

Protein networks upregulated (A) and downregulated (B) in the Rh1P37H retina, at P2.

Early downregulation of protein synthesis and ubiquitination pathways in the Rh1P37H retina

When analyzing the genes that were downregulated in 2-d-old Rh1P37H retinas, we found a strong enrichment for gene products involved in EIF2 and eIF4/p70S6K signaling. This included genes encoding eukaryotic translation initiation factors (EIF1, EIF2S3, EIF3A, EIF3H, EIF4A2, and EIF4G1) and ribosomal proteins (RPS3A, RPLO, RPS23, RPL38, and PSMB7). With the exception of EIF1, these proteins represent components or targets of TOR signaling, a master regulator of cellular metabolism, which coordinates the biogenesis of ribosomes with the initiation of translation and the regulation of protein degradation machineries (Kapahi et al., 2010; Thoreen et al., 2012; Table 1, Fig. 1B). Two-day-old Rh1P37H flies also featured a prominent downregulation of proteasome components (PSMD11, PSMD7, PSMC6, PSMD7, PSMC6, PSMD14, PSMD4, and PSMC3), suggesting an attenuation of cellular degradative processes.

Collectively, these results are consistent with an early increase in energy production, featuring a coordinated upregulation of both aerobic and anaerobic processes and oxidative phosphorylation, coupled to attenuation of energy-consuming processes, such as proteasome biogenesis and mRNA translation in retinas expressing Rh1P37H.

Upregulation of TOR signaling and oxidative stress response pathways at the onset of RD

When categorizing the proteomic changes in 14-d-old flies, we found an unexpected upregulation of several TOR-signaling components (Table 1, Fig. 2A). This upregulation involved the following: (1) the eukaryotic translation initiation factor EIF4G1, which interacts with EIF4E and is critical for TOR/4E-BP1-mediated translational initiation (Thoreen et al., 2012) and might be phosphorylated downstream of TOR activation (Raught et al., 2000); (2) several ribosome components (RPS16, RPS18, RPLP2, RPS20, RPS25, RPL10A, and RPSA) whose translation was recently shown to depend on mTOR signaling activation (Thoreen et al., 2012); and (3) and other targets of TOR signaling (RHOC and PRKCA). We further detected an upregulation of caveolar-mediated endocytosis and NRF2-mediated signaling, a major controller of oxidative stress responses in cells (Kensler et al., 2007; Table 1, Fig. 2A).

Figure 2.
Figure 2.

Protein networks upregulated (A) and downregulated (B) in the Rh1P37H retina, at P14.

Downregulation of amino acid metabolism at the onset of RD

Fourteen-day-old Rh1P37H displayed a coordinated downregulation of several metabolic pathways, including glutamate metabolism, amino acid biosynthesis, glycerophospholipid metabolism, and oxidative phosphorylation (Table 1, Fig. 2B). Notably, several components of the ATP production network (SDHB, ATP5A1, UQCRC2, and UQCRFS1) were downregulated in 14-d-old Rh1P37H retinas.

These results suggest that, in stark contrast to the situation seen in P2 retinas, P14 Rh1P37H-expressing retinas activate TOR signaling and its associated proteasome biogenesis and translation initiation, while exhibiting enhanced endocytosis and an increased oxidative stress response. This is accompanied by a suppression of amino acid and glycerophospholipid metabolism and oxidative phosphorylation, suggesting that dysfunctional neurons exhibit a deregulated (inversed) metabolic and energetic pattern at the onset of retinal degeneration. It is interesting to note that enhanced endocytosis has been identified as a critical pathogenic event in several models of RD (Griciuc et al., 2012). Collectively, these proteomics observations raise the possibility that chronic Rh1P37H proteotoxic stress alters the pattern of energy consumption in PNs, leading to energy exhaustion and cellular and oxidative stress, and ultimately to cell death.

Validation of the proteomics results

We sought to validate our MS-based proteomic analysis by using a second independent method for protein quantification. We selected proteins for which antibodies were readily available and have been previously used for antigen detection by Western blotting in Drosophila. We tested the Rh1 protein; the rhabdomeric markers INAD and TRP; the mitochondrially encoded NADH dehydrogenase 1 (mt-ND1); and the ER chaperones Hsc3, Gp93, and Calnexin. The levels of these proteins in P2 and P14 Rh1P37H versus Rh1WT retinas–as assessed by Western blotting– showed a good correlation with those assessed by MS (Fig. 3). These results suggest that MS-based quantitation allows proper detection of proteomic alterations in Drosophila.

Figure 3.
Figure 3.

Validation of the proteomic results. A, Immunoblots showing the levels of the Rh1 protein; the rhabdomeric markers INAD and TRP; the mitochondrially encoded NADH dehydrogenase 1 (mt-ND1); and the ER chaperones Hsc3, Gp93, and Calnexin in flies of indicated genotypes at P2 and P14. β-Tubulin served as loading control. B, Quantification of protein levels by Western blotting (WB; normalized to β-tubulin levels) in Rh1P37H and Rh1WT retinas was averaged from 50 flies per genotype and represented as percentage increase/decrease in Rh1P37H relative to Rh1WT; the exact values are indicated for each protein. The percentage changes determined by MS for each protein are indicated at the bottom for comparison.

Altered mitochondrial structure and dynamics in Rh1P37H PNs

To analyze the cellular changes in the Rh1P37H retina with subcellular resolution, we performed electron microscopy studies on P14 flies (Fig. 4). This analysis revealed that retinal ultrastructure was grossly maintained in both Rh1WT and Rh1P37H flies, as evidenced by a normal complement of PNs (Fig. 4A,B). However, numerous rhabdomeres (light-transducing organelles, containing Rh1) exhibited structural abnormalities, suggesting that mutant Rh1P37H causes rhabdomere structural defects. In agreement with previous observations (Colley et al., 1995), we also found a marked expansion of the ER network in Rh1P37H PNs (Fig. 4C,D), indicating that misfolded Rh1P37H causes ER dysfunction in vivo.

Figure 4.
Figure 4.

Misfolded Rh1P37H leads to altered mitochondrial structure and dynamics in vivo. A, B, Electron microscopy analysis of 14-d-old Rh1WT and Rh1P37H retinas reveals an intact ommatidial network in Rh1WT retinas (A) and a largely preserved network in Rh1P37H retinas (B). a′, b′, Although Rh1P37H ommatidia display a normal set rhabdomeres, these organelles exhibit mild structural alterations (b′). a″, b″, Rh1P37H PNs display an increased number of mitochondria (60% relative to Rh1WT; arrows). C, D Electron microscopic analysis also reveals an expanded ER network in Rh1P37H PNs (D, arrows), consistent with a deleterious effect of Rh1P37H misfolding on ER homeostasis. E, F, higher resolution views of individual mitochondria reveals structural defects (altered or absent cristae; black arrows) in Rh1P37H PNs. F–H, Rh1P37H PNs also exhibit numerous autophagosomes (white arrows in G), autolysosomes (black arrows in G), and multivesicular bodies (white arrows in F and H), suggesting a defective autophagic degradation network.

We also detected a significant increase in the number of mitochondria in Rh1P37H flies relative to Rh1WT flies (60% Rh1P37H vs Rh1WT, n = 3, p < 0.05; Fig. 4a′,b″). Moreover, high-resolution images revealed that numerous mitochondria in Rh1P37H flies displayed structural abnormalities (such as absent or poorly patterned cristae; Fig. 4E,F. These observations support proteomic data suggesting that Rh1P37H misfolding leads to mitochondrial failure, before the onset of retinal degeneration in Rh1P37H flies, and raise the possibility that these structural defects in mitochondrial architecture induced by Rh1P37H toxicity contribute to PN demise.

Finally, we also detected a strong increase in the number of autophagic vacuoles, multivesicular bodies, and lysosomes in Rh1P37H PNs (Fig. 4G,H), pinpointing to an altered autophagic degradation network in Rh1P37H PNs. Collectively, these structural analyses suggest a tight correlation between Rh1 misfolding and ER retention and dysregulation of the mitochondrial/energetic networks and the autophagic network.

TOR inhibition confers robust long-term protection against Rh1P37H toxicity

The differential regulation of multiple TOR targets in P2 versus P14 Rh1P37H flies prompted us to investigate the activation status of 4E-BP1 and p70S6K, two critical downstream targets of TOR signaling. A master regulator of cellular metabolism, TOR is an atypical serine/threonine kinase that is part of the phosphoinositide-3-kinase-related kinase family. In mammals, mTOR recruits several adaptors to form two complexes, mTORC1 and mTORC2 (Laplante and Sabatini, 2012). mTORC1 activation promotes protein synthesis by two major mechanisms: (1) by phosphorylating and activating the p70S6 kinase (S6K kinase or p70S6K), which controls ribosome and mRNA biogenesis, translation initiation, and elongation via activation of the EIF2, EIF4B/EIF4A, and TIF1A/Pol1 axes and (2) by phosphorylating the EIF4E-binding protein 1 (4E-BP1), which prevents its association with the cap-binding protein EIF4E, thereby allowing it to initiate cap-dependent translation (Laplante and Sabatini, 2012). Activation of the p70S6K and inhibition of 4E-BP1/EIF4E activity are the most well established molecular events that occur upon mTOR activation. In Drosophila, TOR has also been shown to regulate ribosome biogenesis, translation initiation, autophagy, and cellular survival (Stanfel et al., 2009; Katewa and Kapahi, 2011).

We thus investigated the phosphorylation status of 4E-BP1 and p70S6K using specific antibodies to immunolabel retinal extracts. The levels of phosphorylated 4E-BP1 as well as p70S6K are decreased in P2 Rh1P37H retinas relative to Rh1WT retinas; in contrast, 4E-BP1 and p70S6K phosphorylation is increased in older (P14-P30) Rh1P37H retinas relative to Rh1WT retinas (Fig. 5A,B). The levels of phosphorylated 4E-BP1 were increased by 180% in P30 Rh1P37H retinas relative to Rh1WT retinas, while phospho-p70S6K levels increased by 82% (Fig. 5A,B). The levels of total as well as phosphorylated 4E-BP1 and p70S6K showed an age-dependent decline in Rh1WT retinas, hinting to a possible adaptive response to light exposure and/or aging. The initial decrease in 4E-BP1 and p70S6K signaling in young (P2) Rh1P37H retinas relative to Rh1WT retinas, which was reversed in older flies leading to a marked and sustained upregulation of 4E-BP1 and p70S6K signaling (in P14-P30 Rh1P37H retinas) is in agreement with the inversed pattern of TOR target expression identified by MS (Table 1).

Figure 5.
Figure 5.

TOR activation mediates Rh1P37H-induced retinal degeneration. A, Immunoblots showing the levels of phosphorylated 4E-BP and phosphorylated p70S6K, as well as total 4E-BP1 and total p70S6K in retinas from P2, P14, and P30 Rh1P37H and Rh1WT flies. β-Tubulin served as loading control. B, Quantification of phospho-4E-BP1 and phospho-p70S6K levels (normalized to the levels of total 4E-BP1 and total p70S6K, respectively, as well as β-tubulin levels) in Rh1P37H and Rh1WT retinas; results from three independent experiments and 50 flies/genotype/experiment were averaged. Values are represented as percentage increase/decrease in Rh1P37H relative to Rh1WT. **p < 0.01 and ***p < 0.001, Student's t test. C, Immunoblots showing the levels of phosphorylated 4E-BP1 and phosphorylated p70S6K, as well as total 4E-BP1 and total p70S6K in retinas from Rh1P37H flies that have been treated with 200 μm rapamycin for either 14 (P14) or 30 (P30) d. β-Tubulin served as loading control. D, Quantification of phospho-4E-BP1 and phospho-p70S6K levels (normalized to the levels of total 4E-BP1 and total p70S6K, respectively, as well as β-tubulin levels); results from three independent experiments and 50 flies/genotype/experiment were averaged. Values are represented as percentage increase/decrease in rapamycin-treated Rh1P37H relative to sham-treated Rh1P37H. ***p < 0.001, Student's t test. E–G, Photomicrographs of toluidine blue-stained eye sections of Rh1WT flies (E) and Rh1P37H flies (F) fed on control food, and of Rh1P37H flies reared on food containing 200 μm rapamycin (G) after 30 d of light exposure (P30). Scale bar, 50 μm. H, Quantification of average number of P/O (n > 7 animals/group, ***p < 0.001 t test). Treatment with both 50 and 200 μm doses of rapamycin leads to rescue of retinal degeneration in the Rh1P37H flies.

The antagonistic pattern of TOR activation in the Rh1P37H retinas of young and older flies prompted us to investigate the relevance of this signaling pathway to the process of Rh1P37H-mediated toxicity and cellular demise. To test the effects of a chronic suppression of TOR signaling on Rh1P37H-indued PN degeneration, we decide to employ a pharmacologic approach. We treated Rh1WT and Rh1P37H flies with the inhibitor rapamycin, which selectively inhibits TOR activity (Rubinsztein et al., 2007). We administered two doses of rapamycin, 50 and 200 μm, which were previously found to efficiently inhibit TOR signaling in Drosophila (Tain et al., 2009; Bjedov et al., 2010). As expected, rapamycin-treated Rh1P37H retinas exhibited a marked and sustained reduction in the levels of phosphorylated 4E-BP1 and p70S6K relative to retinas from sham-treated Rh1P37H flies (Fig. 5C,D) indicating that rapamycin treatment effectively inhibits TOR signaling in the Rh1P37H retina. To assess the effect of TOR signaling inhibition on Rh1P37H-mediated RD, we analyzed the retinal integrity on toluidine blue-stained eye sections. We found, remarkably, that both doses of rapamycin dramatically suppressed RD in the Rh1P37H retina (Fig. 5E–H), suggesting that TOR signaling mediates Rh1P37H-induced PN cell death.

The mitochondria-induced APAF-1/caspase-9 axis mediates Rh1P37H toxicity

To address a potential role of mitochondria in Rh1P37H-induced cell death we used genetic analysis using several mutations that target components of the mitochondria-induced apoptosis.

We first confirmed that Rh1P37H-induced cell death in the Drosophila compound eye proceeds via apoptosis by using mutants in which caspase-3, a central effector of apoptotic pathways, which integrates both the extracellular- and mitochondria-induced cell death signals (D'Amelio et al., 2010), is inactivated. We used the caspase-3 loss-of-function (LOF) allele IceΔ1 referred to as IceLOF (Muro et al., 2006) and found that Rh1P37H; IceLOF/+ flies exhibit a dramatic rescue of retinal degeneration (Fig. 6C).

Figure 6.
Figure 6.

Suppression of Rh1P37H-induced retinal degeneration by genetic inactivation of the APAF-1/caspase-9 and Traf1/JNK axis. A–G, Photomicrographs of toluidine blue-stained eye sections of Rh1WT (A), Rh1P37H (B), Rh1P37H;IceLOF (C), Rh1P37H;DarkLOF (D), Rh1P37H;DroncLOF (E), Rh1P37H;Traf1LOF (F), and Rh1P37H;Bsk1LOF (G) after 30 d of light exposure, i.e., P30. Scale bar, 50 μm. H, Quantification of average number of P/O (n > 7 animals/group, **p < 0.01 and ***p < 0.001 t test). Genetic inactivation of Ice/caspase-3 and of the APAF-1/caspase-9 axis (Dark and Dronc) potently suppresses retinal degeneration caused by Rh1P37H. Inactivation of the TRAF1/JNK axis (Traf1, Bsk1) also leads to rescue of retinal degeneration in the Rh1P37H retina.

To assess whether mitochondria mediates cell death in Rh1P37H flies we inactivated caspase-9, which is activated specifically downstream of failing mitochondria (Diaz et al., 2006), as well as APAF-1, which interacts with procaspase-9 to form the apoptosome, a critical activator of caspase-3 (Loudet et al., 2007; Riedl and Salvesen, 2007). We used the caspase-9 LOF allele Dronc51 referred to as DroncLOF (Chew et al., 2004; Waldhuber et al., 2005) and the APAF-1 LOF allele Dark82 referred to as DarkLOF (Sang et al., 2005). We found that Rh1P37H;DroncLOF/+ and Rh1P37H;DarkLOF/+ flies exhibited a strong suppression of RD (Fig. 6D,E), indicating that mitochondria represent a critical link between Rh1P37H toxicity and PN cell death.

Class II Rho mutations fail to fold properly and exhibit increased retention in the ER (Mendes et al., 2005; Griciuc et al., 2011). Repeated cycles of folding are thought to impair the overall folding capacity of the ER and lead to ER stress, in a variety of cellular systems and organisms (Griciuc et al., 2011). Recent evidence suggests that ER stress is tightly linked to mitochondrial function (Bravo et al., 2011, 2012). We explored the possibility that chronic ER stress in Rh1P37H-expressing retina causes mitochondrial dysfunction and contributes to mitochondria-induced apoptosis.

We inactivated TRAF1 and JNK, a major pathway that operates downstream of ER stress (Viornery et al., 2000) and has been found to contribute to mitochondria-induced apoptosis (Causse et al., 2002). Using the Traf1 LOF allele (Cha et al., 2003) and the JNK LOF allele bsk1 (Sluss et al., 1996), we found that Rh1P37H flies carrying each of these alleles displayed a strong rescue of eye degeneration (Fig. 6F,G). To further substantiate the critical role of JNK signaling, we inhibited the JNK protein using the SP600125 inhibitor, which inhibits JNK activity in a variety of cellular systems and organisms, and has been successfully used at 200 μm and 1 mm doses to inhibit JNK activation in Drosophila (Jimenez-Del-Rio et al., 2008; Chen et al., 2010). Using two doses of SP600125 (200 μm and 1 mm), we found that preventing JNK activation strongly suppressed Rh1P37H-induced cell death (data not shown), suggesting that the kinase activity of JNK is required for Rh1P37H-induced cell death. These results raise the possibility that the JNK/TRAF1 axis links Rh1P37H-induced ER stress to mitochondrial dysfunction and cell death.

Recovery of visual responses after suppression of mitochondria-induced cell death

We sought to independently confirm the rescue of retinal integrity in Rh1P37H mutants carrying the abovementioned alleles, by measuring the electric response of the fly eye to light stimulation (electroretinogram, ERG). In this experiment, individual flies were first allowed to adapt in the dark and then subjected to a brief light stimulation. The variations of electrical potential evoked by light stimulation were recorded. Fly ERG displays photoreceptor depolarization (Plateau), which corresponds to phototransduction cascade activation, and transient spikes following initiation and cessation of the light stimulus (ON and OFF), which results from synaptic activity.

As previously reported (Galy et al., 2005; Griciuc et al., 2010b), we found that 30-d-old Rh1P37H flies, unlike control and Rh1WT flies, exhibited a blunted electrical response to light stimulation (Fig. 7) consistent with their severe degenerative phenotype. Inactivation of caspase-3/Ice strongly restored visual responses (Fig. 7) in agreement with the rescue of retinal integrity. We also used Rh1P37H flies carrying the HidA206 LOF allele (referred to as HidLOF) of the head involution defective gene, a major pro-apoptotic gene in Drosophila (Sandu et al., 2010); Rh1P37H flies in a HidA206 mutant background exhibited a strong recovery of visual activity (Fig. 7).

Figure 7.
Figure 7.

Genetic suppression of APAF-1/caspase-9 and TRAF1/JNK prevents vision loss in Rh1P37H flies. ERG recordings were performed on each of the indicated genotypes and the results show the “OFF,” “PLATEAU,” and “ON” amplitudes for each group. Rh1P37H flies display a severe loss of ERG signal, which is restored after inactivation of the APAF-1/caspase-9 and TRAF1/JNK axes (n > 6 animals/group, *p < 0.05, **p < 0.01, and ***p < 0.001 t test).

Analysis of electrical responses in Rh1P37H flies carrying caspase-9/Dronc or APAF-1/Dark LOF alleles also revealed a dramatic rescue of visual functioning in agreement with our previous histological findings. Finally, inactivation of the TRAF1/JNK axis also led to a recovery of visual activity in 30-d-old Rh1P37H flies (Fig. 7). Collectively, these functional experiments establish the mitochondria as critical regulators of Rh1P37H-induced cell death and raise the possibility that the TRAF1/JNK axis provides a link between Rh1P37H misfolding and mitochondrial failure in PNs.

Discussion

A substantial body of literature deals with the downstream effects of chronic protein misfolding and the identity of the pathways that mediate chronic proteotoxicity and cellular demise in neurodegenerative disease. Comparatively little is known, however, about the earliest stages of the cellular response to the accumulation of misfolded proteins in neurons. Here, we used a comprehensive proteomic approach to explore the integrated cellular response to Rho misfolding and accumulation in the ER. We found an unexpected and intimate connection between Rh1 pathology and cellular metabolism involving mitochondrial energetics and TOR signaling. Remarkably, the interplay between Rh1 proteotoxicity and these aspects of metabolism is subject to change as the organism ages, such that the relationships seen after chronic exposure to Rh1 misfolding (P14 retina) are the opposite of those seen after a more acute exposure to Rh1P37H (P2 retina). Complementary mechanistic studies, using pharmacological and genetic approaches, further substantiated the critical link between mitochondrial- and TOR-mediated metabolic processes and Rh1P37H toxicity in PNs. We thus propose that chronic metabolic stress and metabolic failure are novel effectors of Rh1P37H pathology in PNs and might represent important cellular targets in RP.

Chronic activation of TOR signaling promotes Rh1P37H-induced cell death

Mounting evidence suggests a critical role for the interaction between ER stress and mTOR signaling in physiology and disease (Appenzeller-Herzog and Hall, 2012). In many physiological situations, the ER stress and mTOR signaling elicit opposite effects in cells: while the former promotes energy consumption, inhibits ribosome biogenesis and translation initiation, and promotes autophagy and cell death; the latter has the opposite effect on these processes (Hoyer-Hansen and Jäättelä, 2007; Rutkowski and Hegde, 2010; Zoncu et al., 2011; Appenzeller-Herzog and Hall, 2012).

Whether mTOR signaling promotes or prevents neurodegeneration might depend on the metabolic status of PNs. We detected a sustained upregulation of TOR signaling in the Rh1P37H retina, which was detected at the onset of RD (P14) and was maintained thereafter (Fig. 5A,B). P14 Rh1P37H retinas also exhibited mitochondrial structural defects and altered energetic patterns. The dramatic rescue of RD after rapamycin treatment (Fig. 5E–H) suggests that TOR signaling mediates ER stress-induced cell death in the Rh1P37H retina. Mounting evidence suggests that mTOR mediates apoptosis downstream of ER stress, in certain pathological situations (Appenzeller-Herzog and Hall, 2012). mTOR activation by chronic ER stress suppresses the phosphorylation of the prosurvival Akt kinase, an upstream negative regulator of the Ire1-ASK1-JNK pathway (Kato et al., 2012). Moreover, rescue of misfolded RhoT17M-induced RD in mice by caspase-7 ablation was associated with reprogramming of ER stress-associated gene expression, decreased mTOR expression, and attenuation of JNK signaling (Choudhury et al., 2013).

In contrast to these pro-apoptotic functions, mTOR was suggested to promote survival of nutrient-deprived cone PNs (Punzo et al., 2009). Cone photoreceptor starvation was shown to be a common feature of several models of RP, which also displayed reduced levels of activated (phosphorylated) mTOR during PN degeneration. Systemic insulin treatment mitigated cone degeneration in one of these models (the Pde6b−/− mouse); this effect was attributed to the activation of mTOR signaling by insulin and the subsequent mTOR-mediated autophagy inhibition, which would prevent an energetic crisis in cones (Punzo et al., 2009). However, insulin is also a key activator of prosurvival signaling pathways, including the Akt kinase–a major anti-apoptotic mediator. It remains to be determined whether mTOR signaling activation by insulin is neuroprotective in this model, or whether other mTOR-independent, insulin-mediated signaling pathways mediate this neuroprotective effect. It also remains to be determined whether the observed downregulation of mTOR signaling during PN degeneration was pathogenic or represented a prosurvival compensatory response. This study raises the interesting possibility that the effects of mTOR signaling on PN survival might be dependent on the metabolic status of PNs and the overall cellular energetic profile, a possibility that could be addressed by future studies.

Our present findings suggest that the ER stress/mTOR/JNK axis represents a critical link between chronic Rho proteotoxicity and PN demise in RP. A better understanding of the connection between the nutritional/metabolic status of PNs and the activation of the ER stress/mTOR/JNK axis might uncover important cellular targets and provide novel therapeutic approaches for RP.

Failing mitochondria mediate Rh1P37H toxicity

The present study identifies mitochondria as central regulators of chronic Rh1P37H proteotoxicity in PNs. We found an early acceleration of anaerobic and aerobic catabolism and mitochondrial energy production; this pattern was reversed before the onset of RD. Mitochondrial respiration is a major source of reactive oxygen species (ROS) and increased mitochondrial function leads to excess ROS production, should antioxidant systems fail to neutralize newly generated ROS (Sena and Chandel, 2012). Consistent with this, signaling by NRF2, the master regulator of oxidative stress responses in the cell, was increased in Rh1P37H retinas at the onset of RD; moreover, electron microscopic analysis of PNs revealed numerous mitochondria with defective cristae in P14 Rh1P37H flies (Fig. 4), suggesting the existence of oxidative stress and oxidative damage in the Rh1P37H retina. These observations are consistent with a model whereby misfolded Rh1P37H alters mitochondrial function, leading to the generation of oxidative stress and to mitochondrial damage.

Severely damaged mitochondria are potent activators of the apoptotic program; the highly conserved APAF-1/caspase-9 pathway represents a central link between failing mitochondria and executioner caspase activation (Riedl and Salvesen, 2007). Remarkably, inactivation of the APAF-1/caspase-9 axis dramatically suppressed Rh1P37H-induced RD, identifying the damaged mitochondria as novel mediators of Rh1P37H toxicity.

Defective communication between the ER and mitochondria is increasingly recognized as a pathogenic event in disease (Raturi and Simmen, 2013). To begin addressing whether the dysfunctional ER leads to mitochondrial pathology via activation of ER stress signaling in our Rh1P37H model of RP, we genetically inactivated the TRAF1/JNK axis, a well established mediator of ER stress (Ron and Walter, 2007). JNK signaling is enhanced in the Rh1P37H retina (Galy et al., 2005), but whether it directly contributes to Rh1P37H-induced apoptosis remained unknown. Genetic inactivation of JNK or TRAF1 strongly mitigated Rh1P37H toxicity (Figs. 6, 7) as did the pharmacological inhibition of JNK kinase activity using the specific inhibitor SP600125 (data not shown). Therefore, the kinase activity of JNK is required for Rh1P37H-induced apoptosis, and TRAF1 might represent the link between ER stress and JNK activation in the Rh1P37H retina.

Drugs that limit the deleterious effects of mitochondrial failure and caspase activation emerge as important drug targets in RP. The caspase-3 inhibitor Ac-DEVD-CHO transiently delays RD in the rd mouse (Yoshizawa et al., 2002); adeno-associated virus-mediated delivery of the X-linked inhibitor of apoptosis–which inhibits caspases-3, -7, and -9–protected PNs in the RhoP23H and RhoS334ter rat models of RP (Leonard et al., 2007); caspase-7 inhibition also mitigated RD in the RhoT17M mouse (Choudhury et al., 2013). Inhibitors of the APAF-1/caspase-9 axis identified in the present study could be tested in future studies.

PNs are among the most energy-consuming cell types in the organism. Based on our findings that mitochondrial and metabolic abnormalities are a central component of Rh1P37H toxicity, we surveyed the RD-linked genes and established that 13 genes that are functionally characterized play a role in cellular metabolism; moreover, chromosomal deletions that inactivate several respiratory chain components also cause RD (Table 2). It is interesting to note that inactivation of several mitochondrially encoded transfer RNAs cause defective mitochondrial respiration and RD in humans. We also detected a downregulation of glutaminyl-tRNA, phenylalanyl-tRNA, and phenylalanyl-tRNA synthetase subunits in 14-d-old Rh1P37H flies (Table 1). Thus, impaired amino acid synthesis might amplify Rh1P37H toxicity in fly PNs, a possibility that could be addressed by future studies.

View this table:
Table 2.

Genes linked to retinal degeneration in humans, which function in cellular metabolism

A model of metabolic failure in Rh1P37H-linked RD

Neurodegenerative diseases, such as Alzheimer's disease, Parkinson's disease, or RP, are chronic proteinopathies, whereby the presence of misfolded proteins induces long-term cellular alterations culminating with the demise of specific neuronal populations. It is currently unknown why such diseases only manifest after decades, although folding-deficient proteins are expressed early in life. We suggest that the breakdown of cellular homeostasis in RP is the result of the long-term modification of cellular metabolic and energetics output by misfolded Rho. Based on the present data, we propose that PN loss in the Rh1P37H fly model of RP is a two-step process in which an early, compensatory phase is followed by a late pro-apoptotic phase of metabolic dysregulation (Fig. 8). We believe the chronic presence of misfolded Rh1P37H in the ER and the highly energetic processes of Rh1 folding and ERAD ultimately lead to loss of energetic capacity and metabolic stress that culminate with PN cell death. This model underscores the importance of early compensatory changes in PNs and suggests that preventing high-anabolic states in PNs (by, for example, decreasing mTOR activity) might prevent energy crises and afford long-term protection against Rh1P37H toxicity. More generally, therapies effective at restoring metabolic function in PNs might be able to prevent or delay RP.

Figure 8.
Figure 8.

Model of Rh1P37H-induced metabolic failure in PNs (see text for details).

Footnotes

  • This study was supported by the European Community's Sixth Framework Program FP6 under grant agreement NEUROTRAIN (MEST-CT-2005-020235), the European Community's Seventh Framework Program FP7/2009 SYSCILIA under grant agreement number HEALTH-F5-2010-241955, and funds from the German Federal Ministry of Science and Education (BMBF) SysTec DYNAMO under grant agreement 0315513A. We thank Rüdiger Klein (Max-Planck Institute of Neurobiology, Munich-Germany) for providing training and for stimulating discussions. We are grateful to our fly (B.A. Hay, H. Steller, and J. Chung) and antibody (H.D. Ryoo, A. Huber, N.J. Colley, C. Montell, C.V. Nicchitta, and D.R. Alessi) donators. We thank Sandra Helm and Silke Becker for excellent technical assistance and Marcel Blindert for scripting. We thank Luise Jennen (Institute of Pathology, Helmholtz Zentrum Muenchen) for assistance with electron microscopy and the Ueffing lab for discussions.

  • The authors declare no financial interests or conflict of interest.

  • Correspondence should be addressed to Liviu Aron, Department of Genetics, Harvard Medical School, Boston, MA 02115, liviu{at}hms.harvard.edu; or Marius Ueffing, Center for Ophthalmology, Institute for Ophthalmic Research, University of Tübingen, 72076 Tübingen, Germany, marius.ueffing{at}uni-tuebingen.de

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Proteomic Survey Reveals Altered Energetic Patterns and Metabolic Failure Prior to Retinal Degeneration
Ana Griciuc, Michel J. Roux, Juliane Merl, Angela Giangrande, Stefanie M. Hauck, Liviu Aron, Marius Ueffing
Journal of Neuroscience 19 February 2014, 34 (8) 2797-2812; DOI: 10.1523/JNEUROSCI.2982-13.2014
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