Yaoyan Qiu1,2, Jingyu Yao1, Lin Jia1, Debra A Thompson1,3, David N Zacks4. 1. Department of Ophthalmology and Visual Sciences, University of Michigan Medical School, Kellogg Eye Center, Ann Arbor, MI, USA. 2. Department of Ophthalmology, Xiangya School of medicine, The Second Xiangya Hospital, Central South University, Changsha, Hunan, China. 3. Department of Biological Chemistry, University of Michigan Medical School, Ann Arbor, MI, USA. 4. Department of Ophthalmology and Visual Sciences, University of Michigan Medical School, Kellogg Eye Center, Ann Arbor, MI, USA. davzacks@med.umich.edu.
Abstract
The P23H variant of rhodopsin results in misfolding of the protein, and is a common cause of the blinding disease autosomal dominant retinitis pigmentosa (adRP). We have recently demonstrated that degeneration of photoreceptor cells in retinas of P23H mice is due to the endoplasmic reticulum stress (ERS)-induced activation of autophagy that leads to a secondary proteasome insufficiency and activation of cell death pathways. We propose that this increased level of autophagy flux relative to proteasome activity, which we term the A:P ratio, represents a marker of altered photoreceptor cell homeostasis, and that therapies aimed at normalizing this ratio will result in increased photoreceptor cell survival. To test this postulate, we treated P23H mice with a chemical chaperone (4-phenylbutyric acid) to improve rhodopsin folding, or with a selective phosphodiesterase-4 inhibitor (rolipram) to increase proteasome activity. P23H mice treated with either of these agents exhibited reduced ERS, decreased autophagy flux, increased proteasome activity, and decreased activation of cell death pathways. In addition, rates of retinal degeneration were decreased, and photoreceptor morphology and visual function were preserved. These findings support the conclusion that normalizing the A:P ratio, either by reducing the ERS-induced activation of autophagy, or by increasing proteasome activity, improves photoreceptor survival, and suggest a potential new therapeutic strategy for the treatment of adRP caused by protein folding defects.
The P23H variant of rhodopsin results in misfolding of the protein, and is a common cause of the blinding disease autosomal dominant retinitis pigmentosa (adRP). We have recently demonstrated that degeneration of photoreceptor cells in retinas of P23Hmice is due to the endoplasmic reticulum stress (ERS)-induced activation of autophagy that leads to a secondary proteasome insufficiency and activation of cell death pathways. We propose that this increased level of autophagy flux relative to proteasome activity, which we term the A:P ratio, represents a marker of altered photoreceptor cell homeostasis, and that therapies aimed at normalizing this ratio will result in increased photoreceptor cell survival. To test this postulate, we treated P23Hmice with a chemical chaperone (4-phenylbutyric acid) to improve rhodopsin folding, or with a selective phosphodiesterase-4 inhibitor (rolipram) to increase proteasome activity. P23Hmice treated with either of these agents exhibited reduced ERS, decreased autophagy flux, increased proteasome activity, and decreased activation of cell death pathways. In addition, rates of retinal degeneration were decreased, and photoreceptor morphology and visual function were preserved. These findings support the conclusion that normalizing the A:P ratio, either by reducing the ERS-induced activation of autophagy, or by increasing proteasome activity, improves photoreceptor survival, and suggest a potential new therapeutic strategy for the treatment of adRP caused by protein folding defects.
Inherited retinal degeneration (IRD) occurs in ~1 in 3000 people in the population and results from mutations in nearly 300 different genes[1,2]. This extreme genetic heterogeneity has complicated the development of therapies for individuals with IRD. Thus, there is an unmet need for therapies that target broadly shared pathophysiological mechanisms and that achieve lasting rescue in a mutation-independent manner. A significant subset of IRD-causing mutations result in misfolding, mistrafficking, or abnormal accumulation of proteins within photoreceptor cells, resulting in proteotoxic cell death, thus making the targeting of proteotoxicity an attractive therapeutic point of intervention for improving cell survival.Rhodopsin, the visual pigment of rod photoreceptor cells, is synthesized and folded in the endoplasmic reticulum (ER) of the rod cell inner segment before its subsequent processing in the Golgi and trafficking to the outer segment[3]. Mutations in the rhodopsin gene RHO, a common cause of IRD, often lead to misfolding or mistrafficking of the rhodopsin protein, resulting in proteotoxicity[4]. A mutation that results in the substitution of histidine for proline at amino acid residue 23 of rhodopsin (RHOP23H; hereafter referred to as P23H) is the most frequent RHO mutation identified in the United States[1]. The P23H mutation has been extensively studied, and represents a prototypical model for studying proteotoxicity[5,6].In both the human disease and corresponding transgenicmouse models, misfolding of the rhodopsinP23H variant results in intracellular accumulation of mutant protein, increased endoplasmic reticulum stress (ERS), and aberrations in rod outer segment formation[7-11]. To deal with this increased ERS, cells can employ one of two clearance mechanisms, the ubiquitin–proteasome system or the autophagy–lysosome pathway[12-14]. Under normal homeostatic conditions, channeling the misfolded protein to either one of these two clearance pathways is protective. However, when protein stress is chronic, as in IRD, this intrinsic response is insufficient to prevent cell death.Our previous studies examined the activity of autophagy in the P23Hmouse retina and its role in photoreceptor cell degeneration[15]. We found that misfolded rhodopsin results in the persistent activation of autophagy that contributes to secondary proteasome insufficiency. We also showed that increasing autophagy in P23Hmice further accelerates retinal degeneration, whereas inhibiting autophagy, either genetically or pharmacologically, improves proteasome levels and activity and reduces retinal degeneration. These findings highlight the reciprocal nature of autophagy and the proteasome, and suggest that the balance between their activity levels, which we term the A:P ratio, can serve as a marker of altered photoreceptor homeostasis.In the present study we sought to further study the interdependency of ERS, autophagy and proteasome to determine whether shifting protein degradation from autophagy to proteasome can relieve proteotoxic ERS and improve cell survival. P23Hmice were treated with either 4-phenylbutyric acid (4-PBA), a chemical chaperone that has been shown to reduce ERS by improving protein folding and shuttling to the proteasome degradation pathway[16-18], or with rolipram, a selective phosphodiesterase-4 inhibitor that can act to directly increase proteasome activity levels[19-21]. Assays of ERS activation, proteasome activity and autophagy flux showed that both treatments resulted in normalization of the A:P ratio and increased photoreceptor cell survival and retinal function. These observations suggest that modulating the flux of misfolded protein from autophagy to the proteasome represents a potential therapeutic option for reducing proteotoxicity and improving cell survival.
Methods
Animals and experimental treatments
All experiments were performed following the Association for Research in Vision and Ophthalmology statement for ethical care and use of animals and the guidelines approved by the University Committee on use and Care of Animals at University of Michigan. The RhoP23H/P23Hmice were bought from Jackson lab, and crossed with C57BL/6J mice to produce RhoP23H/+ mice (P23H). The RhoP23H/P23Hmice were also crossed with green fluorescent protein (GFP)-LC3 (Riken laboratories, Tsukuba, Japan) mice to produce RhoP23H/+-GFP-LC3mice. In all experiments on P23Hmice, heterozygous mice were used, as this represents the most common clinical presentation. Mice were housed under standard conditions with 12 h of light and 12 h of dark in the vivarium of the University of Michigan Kellogg Eye Center.4-phenylbutyric acid (PBA) (Santa Cruz, 200652) was dissolved in 0.9% saline at a concentration of 40 mg/ml and was given through daily intraperitoneal (IP) injections at a dose of 200 mg/kg from P14 up to 4 months. Control mice were given injections of vehicle alone (equivalent solution without 4-PBA).Rolipram (Enzo Life Science, BML-PD175-0050) was dissolved in DMSO at a concentration of 250 mg/mL as a stock solution. A working solution at 0.5 mg/mL was diluted in 0.9% saline freshly prior injection. Mice were given rolipram at 5 mg/kg or equivalent vehicle alone intraperitoneally daily from P14 up to 4 months. Mice were monitored daily.
Tissue collection
To control for the effect of autophagy flux, mice were given an IP injection of leupeptin (Sigma-Aldrich, L2884) (40 mg/kg body weight) 4 h before harvest[22,23]. All retinal samples were collected at the same time of the day, 1 p.m., to control for the time-dependent fluctuations in autophagy[24]. For better visualization of ubiquitinated proteins by western blot, mice were given an IP injection of MG132 (5 mg/kg) right after the last treatment of rolipram before collecting retinal samples.Eyes were enucleated immediately after euthanizing mice. Retinas were harvested under a dissection microscope as previously described[24]. Briefly, after removing the cornea and lens to form the eyecup, the retina was dissected free of the retinal pigment epithelium.
Chymotrypsin-like proteasome activity assay
An optimized assay was used for measuring chymotrypsin-like proteasome activity in the mouse retinas at 1 month of age using substrate Suc-LLVY-AMC (Enzo Life sciences, BML-P802-0005) as previously described[15]. Two retinas from each mouse were pooled as one sample. After adding 120 µl assay buffer (50 mM Hepes, pH 7.5, 20 mM KCl, 5 mM MgCl2, and 1 mM DTT), samples were sonicated and centrifuged at 4 °C at 10,000 g for 10 min to obtain cytosolic protein. Sixty micrograms of freshly prepared cytosolic protein and substrate Suc-LLVY-AMC were assessed in assay buffer containing 7 µM ATP (Dot Scientific, DSA30030-5) with or without the presence of the proteasome inhibitor lactacystin (Enzo Life sciences, BML-PI104-1000, Enzo Life Sciences) in a black-walled nine-well plate with a total volume of 250 µl in each well. An excitation wavelength of 380 nm and emission wavelength of 440 nm were used to scan the plate once per minute for 45 min in a Flexstation-II plate reader (Molecular Diagnostic). The readings at 40 min were used for analysis. All assays were repeated three times.
Western-blot analysis
Retinas were sonicated in RIPA buffer (Thermo Scientific,89900) containing a protease inhibitor (Roche, 11697498001). After centrifugation, the supernates were collected and the protein concentrations were measured using a Bio-Rad RC DC Protein Assay Kit (Cat. No. 500-0119,0120,0121,0122). Equal amount of total protein was added to 4–15% SDS-PAGE (Tris-HCl Ready Gels; Bio-Rad Laboratories, 4561086) and transferred to polyvinylidene fluoride membranes (Bio-Rad Laboratories, 162-0177). After blocking with 5% milk in Tris-buffered saline, Bio-rad, 170-6435 contains 0.1% Tween 20 (Sigma, P7949) at room temperature for 1 h, the membranes were incubated with primary antibodies overnight at 4 °C. Primary antibodies used in this study include: LC3 (Cell Signaling technology, 4108S;1:1000), P62/AQATM1 (Novus biologicals, NBP1-48320S;1:1500), Beclin1 (Cell Signaling technology, 3495; 1:1000), p-SQSTM1 (Gene Tex, GTX128171; 1:400), Rho 4D2 (Novus biologicals, NBP1-48334; 1:2000), Ubiquitin (Cell Signaling, 3933; 1:1000), proteasome 20S (Enzo, BML-PW8155; 1:1000), GAPDH (Ambion Applied Biosystems, AM4300; 1:80000), RIPK3 (ABGENT, AP7819B, 1:1000), pRIPK3 (Abcam, ab195117, 1:1000), MLKL (ABGENT, AP14272, 1:2000), CHOP (Cell Signaling technology, 2895, 1:1000), BIP (Cell Signaling technology, 3177, 1:1000) and pMLKL (Abcam, ab196436, 1:1000). After washing, the membranes were incubated with secondary antibodies (Dako, P0447, P0448; 1:2000) at room temperature for 1 h. Blots were developed using SuperSignal West Dura Sunstrate (Thermo Scientific, 34075). Quantitative densitometry of the bands was measured by ImageJ. All experiments were repeated at least three times.
Soluble and insoluble rhodopsin fractionation assay
Two retinas were pooled and lysed in 250 µl of ice-cold lysis buffer (PBS, pH 7.5, 5 mM EDTA, 1% TX-100) with protease inhibitor (Roche, 11697498001) for 30 min on ice with occasionally mixing. After centrifugation at 13,000 × g for 15 min, the supernate was collected as the soluble fraction. To prepare the insoluble fraction of the protein, the pellet was solubilized in 50 µl of 1% SDS in PBS for 10 min at room temperature. After additional 75 µl lysis buffer was added to the pellet, the sample was sonicated with a micro-tip sonicator[8].
Real-time polymerase chain reaction (RT-PCR)
A purification kit (Qiagen, 74104) was used for the isolation of RNA from mouse retinas. Five hundred nanograms of the total RNA was converted into cDNA with the SuperScript III Reverse Transcriptionase Kit (ThermoFisher Scientific, 18080093). The transcript levels were assayed in triplicate using a thermal cycler (Bio-Rad CFX96 Real Time System). The target gene was normalized to the expression level of rpl19 using a comparative Ct method. Specific primers were as follows: rho (forward 5′-GCCACACTTGGAGGTGAAATC-3′, reverse 5′- AAGCGGAAGTTGCTCATCG-3′); fas (forward 5′-ATG AGA TCG AGC ACA ACA GC-3′, reverse 5′-TTA AAG CTT GAC ACG CAC CA-3′); caspase 8 (forward 5′ –ATGGCGGAACTGTGTGACTCG-3′, reverse 5′-GTCACCGTGGGATAGGATACAGCA-3′); Xbp1s (forward 5′-GAGTCCGCAGCAGG TG-3′, reverse 5′-GTGTCAGAGTCCATGGGA-3′); rpl19 (forward 5′-ATGCCAACTCCCGTCAGCAG-3′; reverse 5′-TCATCCTTCTCATCCAGGTCACC-3′).
Immunohistochemistry
The superior cornea of the eyes was marked for orientation. To prepare samples for cryosectioning, the eyes were fixed in freshly prepared 4% paraformaldehyde solution overnight at 4 °C. After removal of the cornea and lens, the eyecup was washed in phosphate-buffered saline (PBS) three times, and transitioned through 5%, 10%, and 20% sucrose in PBS for 2 h each. Eyes were then embedded in an orientation-specific manner in the embedding mixture containing 1:1 ratio of Tissue-Tek embedding medium (Sakura Finetek, 4583) and 20% sucrose and then sectioned at 10-µm thickness using a cryostat. Retinal sections were blocked with 5% goat serum in PBS with 0.1% Triton-X100 (Sigma-Aldrich) and incubated with primary antibodies overnight at 4 °C. After washes, sections were incubated with secondary antibodies at room temperature for 1 h and then counterstained with ProLong Gold with DAPI (Invitrogen, P36941). Images were taken at comparable areas on sections with a fixed gain using Leica 6000 microscope (Leica Corp., Wetzlar, Germany) or a confocal microscope (Leica SP5, Leica Corp., Germany).
Photoreceptor cell counts
The orientation marked eyes were fixed in 4% paraformaldehyde overnight followed by paraffin embedding. Six-micrometer paraffin sections were obtained using a microtome (Shandon AS325, Thermo Scientific, Cheshire, England). After deparaffinization, sections were stained with hematoxylin (Fisher Scientific, Hercules, CA) and eosin (Fisher Scientific, Hercules, CA). Only sections crossing the optic nerve were used and images were captured with Leica DM6000 microscope. Four nonoverlapping sections crossing the optic nerve from each eye were used for photoreceptor counts. The total number of photoreceptors in the whole retinal section was counted in a masked fashion.
Quantification of GFP-LC3 puncta in photoreceptor cell inner segments
Three nonoverlapping cryosections of each GFP-LC3mouse were used for counting of GFP-positive puncta in photoreceptor inner segments as described previously[15]. Images were taken using confocal microscope with fixed ×60 magnification. For each section, the number of GFP-positive puncta were counted in three areas of 30-µm length of retina from both superior and inferior portions. The counting area consists of the inner segment up to the first row of the photoreceptor nuclei above the outer limiting membrane. At least four animals were used for each group.
TUNEL staining
TUNEL staining was performed on the cryosections using DeadEnd Colorimetric TUNEL System (Promega Corporation Madison, WI, USA) according to the manufacturer’s instructions. The total number of TUNEL-positive cells in the outer nuclear layer (ONL) of the whole retina section was counted and three nonoverlapping sections of each samples were used.
Optokinetic tracking responses
The optokinetic tracking response was used to measure the visual function of the mouse as previously described. Briefly, an awake mouse was placed in a drum projected with vertical black and white stripes at various spatial frequencies. The projected drum can be rotated clockwise and counter-clockwise. Mice were monitored using a camera to track head movements (i.e, the optokinetic reflex response) in response to the rotating drum. A genotype-masked observer recorded the maximum spatial frequency (in cycles/degree (c/d)) in 100% background contrast that stimulated a tracking movement of the mouse.
Spectral domain optical coherence tomography
Mice were anesthetized using a mixture of ketamine (80 mg/kg, Hopira, Lake Forest, IL) and xylazine (10 mg/kg, NAND, Lake Forest, IL). Pupils were dilated with topical 2.5% phenylephrine (Paragon BioTek, inc., Portland, OR) and 0.5% tropicamide (AKORN, Lake Forest, IL). After applying Systane Lubricant eye drops (Alcon. 9004494-0109) to the cornea of the mouse, optical coherence tomography (OCT) was performed using the spectral domain OCT system (Bioptigen, Inc., Durham, NC). The thickness of the ONL was measured in both superior and inferior of the retina with distances of 250 and 500 µm from the optic nerve.
Statistical analysis
ONL thickness measured by OCT at different age points was analyzed using two-way ANOVA with replicates for multiple comparisons. For western blot, rt-PCR and other statistical comparisons across more than two experimental groups, differences were analyzed using one-way ANOVA tests followed by Tukey multiple comparison test. Comparisons between two groups were analyzed using unpaired t-test. Statistical analysis was performed using Prism (GraphPad, Inc., La Jolla, CA) and Microsoft Office Excel (Richmond, WA). Results were expressed as mean ± SD. Differences were considered significant at P < 0.05.
Results
Chaperone treatment improves P23H folding and decreases ERS
To test the hypothesis that normalizing the A:P ratio results in decreased activation of cell death pathways and increased photoreceptor function, our studies focused on the P23Hmouse model of proteotoxic cell death. We began by evaluating whether improved folding and trafficking of P23H to the proteasome would result in decreased ERS and autophagy activation. P23Hmice were treated with the chemical chaperone 4-PBA, which has been shown to reduce ERS both in vivo and in vitro by correcting the folding of misfolded and unfolded proteins in the ER[16-18]. The effect of 4-PBA treatment on P23H folding was evaluated by assaying its detergent-solubility, as previous studies have shown that detergent-soluble rhodopsin correlates with correctly folded protein, while detergent-insoluble rhodopsin correlates with protein aggregates[8,25]. A significant reduction in insoluble rhodopsin was observed in 4-PBA-treated P23Hmice, consistent with improved rhodopsin folding and decreased aggregation (Fig. 1a, b). Analysis of the expression of proteins associated with ERS activation showed decreased levels of CHOP, BIP, and xbp1s in 4-PBA-treated mice relative to vehicle-treated controls (Fig. 1c, d, e), consistent with the conclusion that decreased ERS results from improved P23H folding.
Fig. 1
Treatment with the chaperone 4-PBA improves P23H-rhodopsin folding and reduces ERS.
a Representative western blots probed for rhodopsin (RHO) in the soluble and insoluble fraction of total retinal protein from P23H mice after 2 weeks of treatment with 4-PBA or vehicle-only as control (CTL). b Quantification of western-blot band intensities of soluble and insoluble RHO normalized to total RHO, and ratio of soluble RHO to insoluble RHO. Measured areas are indicated by the red rectangles in a. Statistical analyses with unpaired t-test. c Representative western blots probed for CHOP, BIP, and loading control GAPDH in retinas of C57 (wild-type) and P23H mice after 2 weeks of treatment with 4-PBA or vehicle-only (CTL). d Quantification of the western blots represented by c. e Transcript levels of xbp1s in retinas of P23H mice treated with 4-PBA or vehicle-only (CTL) for 2 weeks, normalized to aged-matched C57 mice. Statistical analyses with one-way ANOVA. Results are shown as individual symbols representing data values along with mean and standard deviation (SD). *p ≤ 0.05. **p ≤ 0.01. ***p ≤ 0.001. ****p ≤ 0.0001
Treatment with the chaperone 4-PBA improves P23H-rhodopsin folding and reduces ERS.
a Representative western blots probed for rhodopsin (RHO) in the soluble and insoluble fraction of total retinal protein from P23Hmice after 2 weeks of treatment with 4-PBA or vehicle-only as control (CTL). b Quantification of western-blot band intensities of soluble and insoluble RHO normalized to total RHO, and ratio of soluble RHO to insoluble RHO. Measured areas are indicated by the red rectangles in a. Statistical analyses with unpaired t-test. c Representative western blots probed for CHOP, BIP, and loading control GAPDH in retinas of C57 (wild-type) and P23Hmice after 2 weeks of treatment with 4-PBA or vehicle-only (CTL). d Quantification of the western blots represented by c. e Transcript levels of xbp1s in retinas of P23Hmice treated with 4-PBA or vehicle-only (CTL) for 2 weeks, normalized to aged-matched C57 mice. Statistical analyses with one-way ANOVA. Results are shown as individual symbols representing data values along with mean and standard deviation (SD). *p ≤ 0.05. **p ≤ 0.01. ***p ≤ 0.001. ****p ≤ 0.0001
Improved P23H folding decreases autophagy flux
Activation of ERS contributes to the elimination of misfolded proteins from cells, either through the ubiquitin–proteasome or the autophagy–lysosome clearance pathways. We have previously shown that misfolded rhodopsin in the P23H retina results in chronic activation of autophagy, leading to engulfment of proteasome components by autophagosomes, and relative proteasome insufficiency[15]. To determine whether decreasing ERS in P23Hmice results in decreased autophagy, the following standard measures of autophagy activation were evaluated: SQSTM1/p62, an autophagy receptor protein whose levels can serve as a metric of autophagy flux; Beclin1, an autophagy activator; and LC3-II, the lipidated form of LC3 protein[26]. As in our previous work, we observed increased SQSTM1/p62, Beclin1, and LC3-II to LC3-I ratios in the vehicle-treated P23H group compared with C57 controls (Fig. 2a, b), consistent with increased autophagy activation and autophagy flux. In contrast, retinas from P23Hmice treated with 4-PBA exhibited decreased SQSTM1/p62, Beclin1, and LC3-II/LC3-I ratios, consistent with decreased autophagy flux.
Fig. 2
Improved P23H folding reduces autophagy flux and increases proteasome activity.
a Representative western blots, with b quantification of bands probed with SQSTM1/p62, Beclin1, LC3, and loading control GAPDH in retinas of C57 (wild-type) and P23H mice after 2 weeks of treatment with 4-PBA or vehicle-only as control (CTL). Statistical analyses with one-way ANOVA. For determination of autophagy flux, animals received an intraperitoneal injection of leupeptin (40 mg/kg body weight) 4 h before tissue harvest. c Representative fluorescence micrographs of retinas from 1-month-old P23H GFP-LC3 mice treated with 4-PBA or vehicle-only (CTL) for 2 weeks. The GFP-LC3 puncta localize primarily to the photoreceptor inner segment (IS), with none localizing to the outer segments (OS). Nuclei of the photoreceptors in the outer nuclear layer (ONL) were stained with DAPI (blue). Scale bar: 25 µm. d Quantification of the number of GFP-LC3 puncta per counting unit indicated by the red square in c. Statistical analyses with unpaired t-test. e Chymotrypsin-like activities measured in the presence of ATP (7 µM) in retinal lysates from P23H mice treated with 4-PBA or vehicle-only (CTL) for 2 weeks, normalized to age-matched C57 (wild-type) mice. f Representative western blots and g quantification of 20S proteasome subunits and loading control GAPDH, in retinas of P23H mice treated with 4-PBA or control vehicle and untreated C57 control mice. Statistical analyses with one-way ANOVA. Results are shown as individual symbols representing data values along with mean and standard deviation (SD). *p ≤ 0.05. **p ≤ 0.01. ***p ≤ 0.001. ****p ≤ 0.0001
Improved P23H folding reduces autophagy flux and increases proteasome activity.
a Representative western blots, with b quantification of bands probed with SQSTM1/p62, Beclin1, LC3, and loading control GAPDH in retinas of C57 (wild-type) and P23Hmice after 2 weeks of treatment with 4-PBA or vehicle-only as control (CTL). Statistical analyses with one-way ANOVA. For determination of autophagy flux, animals received an intraperitoneal injection of leupeptin (40 mg/kg body weight) 4 h before tissue harvest. c Representative fluorescence micrographs of retinas from 1-month-old P23H GFP-LC3mice treated with 4-PBA or vehicle-only (CTL) for 2 weeks. The GFP-LC3 puncta localize primarily to the photoreceptor inner segment (IS), with none localizing to the outer segments (OS). Nuclei of the photoreceptors in the outer nuclear layer (ONL) were stained with DAPI (blue). Scale bar: 25 µm. d Quantification of the number of GFP-LC3 puncta per counting unit indicated by the red square in c. Statistical analyses with unpaired t-test. e Chymotrypsin-like activities measured in the presence of ATP (7 µM) in retinal lysates from P23Hmice treated with 4-PBA or vehicle-only (CTL) for 2 weeks, normalized to age-matched C57 (wild-type) mice. f Representative western blots and g quantification of 20S proteasome subunits and loading control GAPDH, in retinas of P23Hmice treated with 4-PBA or control vehicle and untreated C57 control mice. Statistical analyses with one-way ANOVA. Results are shown as individual symbols representing data values along with mean and standard deviation (SD). *p ≤ 0.05. **p ≤ 0.01. ***p ≤ 0.001. ****p ≤ 0.0001Autophagy activation was also evaluated by histological assessment of LC3-positive puncta formation, as LC3-II localizes to the autophagosome membrane. To facilitate the visualization of LC3-II-coated autophagosomes, P23H GFP-LC3mice were generated by crossing the P23Hmouse with the GFP-LC3mouse, in which LC3 is tagged with GFP[27]. Mice were then treated with 4-PBA or vehicle-only daily starting at P14. After 2 weeks of treatment, eyes were harvested for histological analysis of GFP-LC3 puncta formation. Significantly fewer GFP-LC3-positive puncta were present in the photoreceptor cells of 4-PBA-treated P23H GFP-LC3mice, as compared with vehicle-treated P23H GFP-LC3mice (Fig. 2c, d). These findings support the conclusion that 4-PBA treatment decreases autophagy activation and flux in the retina of the P23Hmouse.
Proteasome insufficiency has been shown to contribute to photoreceptor cell death in the P23H retina[28]. Our previous work demonstrated that this insufficiency results, at least in part, from increased proteasome degradation due to the increased levels of autophagy induced by misfolded rhodopsin[15]. When we suppressed autophagy activation and flux, degradation of the proteasome and loss of its activity were decreased, resulting in improved photoreceptor cell survival. Given that 4-PBA decreased ERS and autophagy activation, we wanted to determine if this also rescued proteasome levels and activity. Treatment of P23Hmice with 4-PBA resulted in increased chymotrypsin-like activity (Fig. 2e) and levels of 20S proteasome (Fig. 2f, g), consistent with increased proteasome activity and levels, respectively. These increases were not detected in vehicle-treated P23Hmouse retinas. These findings support the conclusion that reducing autophagy activity by decreasing ERS, can relieve proteasome insufficiency and improve proteasome activity.
Improved P23H folding decreases activation of cell death pathways
Both apoptosis[29,30] and necroptosis[31] have been reported to contribute to photoreceptor death resulting from rhodopsin misfolding in a ratP23H model. The peak of terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) of apoptotic photoreceptor cells in the P23Hmouse retina occurs at age P15. Thus, we initiated treatment with 4-PBA or vehicle starting at P14, and assessed whether the reduction in ERS correlated with a reduction in markers of apoptotic and necroptotic cell death. Treatment with 4-PBA resulted in a decreased number of TUNEL-positive photoreceptor cells as compared with vehicle-treated controls (Fig. 3a, b). There was also a significant decrease in transcript levels of the pro-apoptotic genes Fas and caspase 8 (Fig. 3c), consistent with decreased apoptotic cell death in 4-PBA-treated mice. Decreased phosphorylation of MLKL and RIPK3, which are markers of activation of necroptosis (Fig. 3d–f), was also observed in 4-PBA-treated mice, consistent with decreased necroptotic cell death as well.
Fig. 3
Improved P23H folding decreases activation of cell death pathways.
a Representative TUNEL staining images and b quantification of number of TUNEL-positive cells from retinal sections of P23H mice treated with 4-PBA or vehicle-only as control (CTL) for 2 weeks and aged-matched C57 (wild-type) mice. Scale bar: 50 µm. Transcript levels of c caspase 8 and Fas-receptor in retinas of P23H mice treated with 4-PBA or vehicle-only (CTL) for 2 weeks, normalized to aged-matched C57 mice. d Representative western blots probed for pMLKL, MLKL, pRIPK3, RIPK3, and loading control GAPDH in retinas of C57 (wild-type) or P23H mice treated with 4-PBA or vehicle-only (CTL) for 2 weeks. Quantification of phosphorylated pMLKL (e) and pRIPK3 (f) normalized to total MLKL and RIPK3, respectively. Results are shown as individual symbols representing data values along with mean and standard deviation (SD). Statistical analyses with one-way ANOVA. *p ≤ 0.05. **p ≤ 0.01. ***p ≤ 0.001. ****p ≤ 0.0001
Improved P23H folding decreases activation of cell death pathways.
a Representative TUNEL staining images and b quantification of number of TUNEL-positive cells from retinal sections of P23Hmice treated with 4-PBA or vehicle-only as control (CTL) for 2 weeks and aged-matched C57 (wild-type) mice. Scale bar: 50 µm. Transcript levels of c caspase 8 and Fas-receptor in retinas of P23Hmice treated with 4-PBA or vehicle-only (CTL) for 2 weeks, normalized to aged-matched C57 mice. d Representative western blots probed for pMLKL, MLKL, pRIPK3, RIPK3, and loading control GAPDH in retinas of C57 (wild-type) or P23Hmice treated with 4-PBA or vehicle-only (CTL) for 2 weeks. Quantification of phosphorylated pMLKL (e) and pRIPK3 (f) normalized to total MLKL and RIPK3, respectively. Results are shown as individual symbols representing data values along with mean and standard deviation (SD). Statistical analyses with one-way ANOVA. *p ≤ 0.05. **p ≤ 0.01. ***p ≤ 0.001. ****p ≤ 0.0001
Increased proteasome activity in the P23H retina decreases autophagy flux and cell death
Our finding that decreasing the activation of autophagy by ERS leads to improved proteasome function led us to test if the opposite was true; whether improving proteasome function results in decreased activation of autophagy. Recently, Lobanova et al. demonstrated that overexpression of proteasome subunits can decrease photoreceptor cell degeneration in the P23H retina[32]. However, in that report, no assessment of autophagy levels was made. We sought to increase proteasome function directly, through pharmacologic means, and determine the effect on autophagy activation and flux. P23Hmice were treated with rolipram, a selective phosphodiesterase inhibitor that increases intracellular cAMP levels and has been shown to increase proteasome activity[19-21]. Rolipram treatment significantly increased retinal chymotrypsin-like activity in the retinas of treated P23Hmice, compared with those treated with vehicle alone, with activity levels exceeding those found in the retinas of nontreated C57 mice (Fig. 4a). This increased chymotrypsin-like activity was associated with decreased levels of ubiquitinated proteins accumulating in the retina (Fig. 4b), and increased levels of the P20S subunit of the proteasome (Fig. 4c). To evaluate autophagy activation, we measured the levels of SQSTM1/p62, Beclin1, and LC3. Consistent with our hypothesis, we found significantly decreased Beclin1 and LC3-II/LC3-I in the retinas of rolipram-treated P23Hmice as compared with vehicle-only-treated mice (Fig. 4d, e) Interestingly, the level of total SQSTM1/p62 did not change, but there was a decrease in the level of phosphorylated SQSTM1/p62 (p-SQSTM1/p62) (Fig. 4f, g), consistent with decreased shuttling of proteins to the autophagy pathway[33]. A corresponding decrease in the level of autophagosome formation was also seen (Fig. 4h, i). These findings support the conclusion that increasing the proteasome level and activity results in decreased autophagy activation. Consistent with this notion, we found that treatment with rolipram also decreased the number of TUNEL-positive cells in the ONL, as well as decreased the activation of necroptotic cell death (Fig. 5a–d). We interpret these findings as evidence that the normalization of the A:P ratio results in improved retinal structure and function by decreasing the activation of cell death pathways.
Fig. 4
Increased proteasome activity in the P23H retina decreases autophagy flux.
a Chymotrypsin-like activities measured in the presence of ATP (7 µM) in retinal lysates from P23H mice treated with rolipram or vehicle-only as control (CTL) for 2 weeks, normalized to age-matched C57 (wild-type) mice. Representative western blots from retinas of untreated C57 and P23H mice treated with rolipram and vehicle-only (CTL) for 2 weeks, probed and quantified for b ubiquitin, c 20S proteasome and autophagy markers d, e Beclin1, LC3, f, g phosphorylated SQSTM1/p62 and total SQSTM1/p62. To allow for determination of autophagy flux, animals received an intraperitoneal injection of leupeptin (40 mg/kg body weight) 4 h before tissue harvest. Statistical analyses with one-way ANOVA. h Representative fluorescence micrographs of retinas from of 1-month-old P23H GFP-LC3 mice treated with rolipram or vehicle-only (CTL) for 2 weeks. GFP-LC3 puncta localizes primarily to the photoreceptor inner segment (IS), with none localizing to the outer segments (OS). Nuclei of the photoreceptors were stained with DAPI (blue). Scale bar: 25 µm. i Quantification of the number of GFP-LC3 puncta per counting unit indicated in (H) as a red rectangle. Results are shown as individual symbols representing data values along with mean and standard deviation (SD). Statistical analyses with unpaired t-test. *p ≤ 0.05. **p ≤ 0.01. ***p ≤ 0.001. ****p ≤ 0.0001
Fig. 5
Increased proteasome activity in the P23H retina decreases photoreceptor cell death.
a Representative TUNEL staining images and b quantification of TUNEL-positive photoreceptors from retinas of C57 (wild-type) mice or P23H mice treated with rolipram or vehicle-only as control (CTL) for 2 weeks. Scale bar: 50 µm. Representative western blots from retinas of C57 mice or P23H mice treated with rolipram or vehicle-only probed in c for pMLKL, total MLKL or GAPDH (loading control), with quantification of protein levels, or in d for pRIPK3, RIPK3 and GAPDH, with levels of pMLKL and pRIPK3 quantified by normalizing to levels of total MLKL and RIPK3 respectively. Results are shown as individual symbols representing data values along with mean and standard deviation (SD). Statistical analyses with one-way ANOVA. *p ≤ 0.05. **p ≤ 0.01. ***p ≤ 0.001. ****p ≤ 0.0001
Increased proteasome activity in the P23H retina decreases autophagy flux.
a Chymotrypsin-like activities measured in the presence of ATP (7 µM) in retinal lysates from P23Hmice treated with rolipram or vehicle-only as control (CTL) for 2 weeks, normalized to age-matched C57 (wild-type) mice. Representative western blots from retinas of untreated C57 and P23Hmice treated with rolipram and vehicle-only (CTL) for 2 weeks, probed and quantified for b ubiquitin, c 20S proteasome and autophagy markers d, e Beclin1, LC3, f, g phosphorylated SQSTM1/p62 and total SQSTM1/p62. To allow for determination of autophagy flux, animals received an intraperitoneal injection of leupeptin (40 mg/kg body weight) 4 h before tissue harvest. Statistical analyses with one-way ANOVA. h Representative fluorescence micrographs of retinas from of 1-month-old P23H GFP-LC3mice treated with rolipram or vehicle-only (CTL) for 2 weeks. GFP-LC3 puncta localizes primarily to the photoreceptor inner segment (IS), with none localizing to the outer segments (OS). Nuclei of the photoreceptors were stained with DAPI (blue). Scale bar: 25 µm. i Quantification of the number of GFP-LC3 puncta per counting unit indicated in (H) as a red rectangle. Results are shown as individual symbols representing data values along with mean and standard deviation (SD). Statistical analyses with unpaired t-test. *p ≤ 0.05. **p ≤ 0.01. ***p ≤ 0.001. ****p ≤ 0.0001
Increased proteasome activity in the P23H retina decreases photoreceptor cell death.
a Representative TUNEL staining images and b quantification of TUNEL-positive photoreceptors from retinas of C57 (wild-type) mice or P23Hmice treated with rolipram or vehicle-only as control (CTL) for 2 weeks. Scale bar: 50 µm. Representative western blots from retinas of C57 mice or P23Hmice treated with rolipram or vehicle-only probed in c for pMLKL, total MLKL or GAPDH (loading control), with quantification of protein levels, or in d for pRIPK3, RIPK3 and GAPDH, with levels of pMLKL and pRIPK3 quantified by normalizing to levels of total MLKL and RIPK3 respectively. Results are shown as individual symbols representing data values along with mean and standard deviation (SD). Statistical analyses with one-way ANOVA. *p ≤ 0.05. **p ≤ 0.01. ***p ≤ 0.001. ****p ≤ 0.0001
Calculating the A:P ratio in treated and untreated P23H retinas
The findings described above highlight the reciprocal nature of autophagy and the proteasome, and how modulating the activity of one affects the other. In previous studies, when we measured the levels of LC3-II/LC3-I in the presence of autophagy flux blockade, we detected ~1.25-times more LC3-II accumulating in the P23Hmouse retina compared with the C57 mouse retina[15]. Conversely, the P23Hmouse retina showed ~0.8-times proteasome activity as compared with the C57 mouse retina (as measured by chymotrypsin-like activity). This corresponds to an A:P ratio of ~1.25/0.8 = 1.56. This calculation indicates that even small shifts in autophagy and/or proteasome activity can result in significant disruption in overall cellular homeostasis that leads to photoreceptor cell degeneration.In this study, we replicated these results, finding an LC3-II/LC3-I ratio in the P23H retina that is ~1.34 times that found in the C57 control retina, and a proteasome activity in P23H retina that is 0.79 times that present in the C57 retina, thus resulting in an A:P ratio of ~1.69. Treatment of P23Hmice with 4-PBA resulted in an A:P ratio of 0.86/1.13 = 0.76, thus dramatically decreasing the A:P ratio and bringing it closer to one. Similarly, for the retinas of P23Hmice used in the rolipram treatment experiments, the vehicle-only treated group had an A:P ratio of 1.64/0.82 = 2.00, whereas the rolipram-treated animals had a much lower A:P ratio of 1.29/1.18 = 1.09. Thus, whether reducing ERS by improving P23H folding, or increasing proteasome activity directly, each modality worked to decrease autophagy activation and normalize the A:P ratio.
Normalizing the A:P ratio in the P23H retina increases photoreceptor cell survival and function
Ultimately, decreased activation of cell death pathways is meaningful only if it results in improved cell survival and function. In P23Hmice, there is a linear rate of photoreceptor degeneration over time, with more rapid loss of photoreceptors in the inferior retina as compared with the superior retina[10]. We assessed the rate of retinal degeneration in both superior and inferior portions of the retina by measuring the ONL thickness where the photoreceptor cell nuclei reside. Treatment of P23Hmice with either 4-PBA or rolipram resulted in marked preservation of ONL thickness in both superior and inferior retina as measured by OCT and compared with vehicle-treated control animals (Fig. 6a–d). This preservation of ONL was further documented by photoreceptor cell counts of HE-stained retinal sections (Fig. 6e–h). Consistent with increased photoreceptor survival, increased levels of rhodopsin and cone opsin in 4-PBA or rolipram-treated P23Hmice were also detected by immunostaining (Fig. 7a, b). The visual function of 4-PBA or rolipram-treated mice versus vehicle-treated mice was analyzed using an optokinetic tracking response system. After 4 months of treatment, the optokinetic tracking responses of 4-PBA or rolipram-treated P23Hmice were markedly higher than vehicle-treated controls, although lower than C57 mice (Fig. 8). These data suggest that increased photoreceptor cell preservation by 4-PBA or rolipram supports greater visual function. Taken together, our findings support the view that agents acting on different arms of the ERS–proteasome–autophagy pathways not only improve photoreceptor cell survival but also function.
Fig. 6
Normalizing the A:P ratio increases photoreceptor survival in P23H mice.
a Representative optical coherence tomography (OCT) images crossing the optic nerve and b quantification of the outer nuclear layer (ONL) thickness of both superior and inferior retinal regions measured by (OCT) in P23H mice treated for 4 months with 4-PBA (a, b) or rolipram (c, d) or their respective vehicle-only littermates as control (CTL). Red bars in the OCT images indicate the ONL layer that was measured for each mouse. Representative H&E staining images and the number of the photoreceptors in the retinal paraffin sections from 4-PBA (e, f) or rolipram (g, h) treated P23H groups. Results are shown as individual symbols representing data values along with mean and standard deviation (SD). Statistical analyses with unpaired t-test. **p ≤ 0.01. ***p ≤ 0.001. ****p ≤ 0.0001
Fig. 7
Normalizing the A:P ratio increases the expression of the opsin proteins in the photoreceptors of the P23H mice.
Representative fluorescence microscopy images of retinas from of P23H mice treated with 4-PBA (a) or rolipram (b) for 4 months and their control groups, stained for rhodopsin (RHO in red), cone opsin (m-Opsin in green) and cell nuclei (DAPI in blue). Scale bar: 50 µm
Fig. 8
Normalizing the A:P ratio increases visual function in the P23H mice.
Optokinetic tracking responses were recorded from P23H mice treated with 4-PBA (a) or rolipram (b) for 4 months, and their control groups. Responses were normalized to those of age-matched C57 (wild-type) mice. Spatial frequencies (expressed as cycles/degree) were recorded at 100% contrast. Results are shown as individual symbols representing data values along with mean and standard deviation (SD). Statistical analyses with unpaired t-test. **p ≤ 0.01. ***p ≤ 0.001
Normalizing the A:P ratio increases photoreceptor survival in P23H mice.
a Representative optical coherence tomography (OCT) images crossing the optic nerve and b quantification of the outer nuclear layer (ONL) thickness of both superior and inferior retinal regions measured by (OCT) in P23Hmice treated for 4 months with 4-PBA (a, b) or rolipram (c, d) or their respective vehicle-only littermates as control (CTL). Red bars in the OCT images indicate the ONL layer that was measured for each mouse. Representative H&E staining images and the number of the photoreceptors in the retinal paraffin sections from 4-PBA (e, f) or rolipram (g, h) treated P23H groups. Results are shown as individual symbols representing data values along with mean and standard deviation (SD). Statistical analyses with unpaired t-test. **p ≤ 0.01. ***p ≤ 0.001. ****p ≤ 0.0001
Normalizing the A:P ratio increases the expression of the opsin proteins in the photoreceptors of the P23H mice.
Representative fluorescence microscopy images of retinas from of P23Hmice treated with 4-PBA (a) or rolipram (b) for 4 months and their control groups, stained for rhodopsin (RHO in red), cone opsin (m-Opsin in green) and cell nuclei (DAPI in blue). Scale bar: 50 µm
Normalizing the A:P ratio increases visual function in the P23H mice.
Optokinetic tracking responses were recorded from P23Hmice treated with 4-PBA (a) or rolipram (b) for 4 months, and their control groups. Responses were normalized to those of age-matched C57 (wild-type) mice. Spatial frequencies (expressed as cycles/degree) were recorded at 100% contrast. Results are shown as individual symbols representing data values along with mean and standard deviation (SD). Statistical analyses with unpaired t-test. **p ≤ 0.01. ***p ≤ 0.001
Discussion
Activation of ERS typically protects cells from protein stress[12-14]. This response is often sufficient in the context of acute perturbations, such as when transient stressors affecting translational or post-translational events result in protein misfolding or unfolding. However, in the setting of chronic protein stress, such as in the case of P23H, where a genetic mutation results in the constant production of misfolded protein, the ERS system becomes dysregulated and can no longer provide adequate protection. In previous studies we have shown that activation of ERS by misfolded rhodopsin results in dysregulated activation of autophagy[15]. This increase in autophagy activity, in turn, results in catabolism of proteasome subunits, effectively blunting the protection afforded by this complementary mechanism of protein degradation. In the present studies, we find that the ratio of autophagy to proteasome activation (the A:P ratio) in the P23Hmouse retina is nearly 50% higher than the A:P ratio in the retinas of normal controls. Interventions that normalize the A:P ratio by decreasing autophagy flux relieve the proteasome insufficiency and increase photoreceptor cell survival. In addition, we demonstrate the therapeutic benefit of normalizing the A:P ratio either through interventions that decrease ERS and autophagy activation, or that directly increase proteasome capacity and activity. These outcomes establish that shifting protein degradation from autophagy to the proteasome is protective, and that acting appropriately on any of the three arms of the ERS–autophagy–proteasome relationship can serve to normalize the A:P ratio—findings that highlight the reciprocal nature of autophagy and the proteasome in regulating photoreceptor cell homeostasis.To assess autophagy activation in the degenerating retina relative to controls, we evaluated the levels of LC3-II in the retinas of P23H and C57 mice, as LC3-II represents a physiologic marker of autophagosome abundance. It is important to note that assays of LC3-II levels during periods of high-autophagy flux result in values that are less than those obtained during periods of low- autophagy flux[26]. This is because during periods of high-autophagy flux, LC3-II is degraded by the autophagy mechanism. Thus, LC3-II levels need to be assayed under conditions that block its degradation, such as with the use of leupeptin, chloroquine or hydroxychloroquine[26]. It would also be valid to use other measures of autophagy, such as SQSTM1/p62 or the number of autophagosome puncta. The measures of autophagy activation we report are not associated with any units. They are simply a relative measure of autophagy activation in the degenerating retina compared with the wild-type control, which is a genetically similar background strain but without the IRD-causing mutation. Similarly, the assay of proteasome activity used was the chymotrypsin-like activity of the proteasome, which reflects the proteasome content of the degenerating retina relative to the control.For visual function assessment we used the optokinetic tracking response, which serves as a quantitative measure of vision[34]. In this test, the optokinetic reflex response to vertical black-white stripes of varying spatial frequencies is measured. As the striped image is passed before the mouse, a reflex response is elicited. However, as the ability to distinguish the stripes is reduced, the reflex response becomes diminished. Thus, in degenerating retinas, the ability to elicit the reflex response to tightly spaced (high spatial frequency) stripes is reduced. Reflex responses at higher spatial frequency stripes reflect improved visual capacity. We chose this assay of vision, as it corresponds more directly to visual function than measures of electroretinogram recordings of the electrical activity of the retina. In addition, the variability of electrical activity measurements can be relatively high, even within the same animal, potentially masking differences in visual function between treatment cohorts.Our current findings are limited to studies of retinal degeneration secondary to rhodopsin misfolding, a form of proteotoxicity in which the abnormal processing of rhodopsin results in chronic activation of the ERS and disrupted autophagy–proteasome balance. We hypothesize that the A:P ratio will also serve as a marker of altered photoreceptor cell homeostasis in retinal degenerations secondary to other forms of proteotoxicity. This may be proteotoxicity secondary to primary defects in the protein itself, or defects that result in the accumulation or aggregation of otherwise normal proteins. Examples of the former would be mutations in RHO that lead to rhodopsin mistrafficking, rather than misfolding. Potential examples of the latter could be mutations that disrupt transport of proteins across the photoreceptor cell connecting cilium, resulting in accumulation of normal proteins in the inner segment of the cell, leading to increased ERS.Previous work has demonstrated the importance of apoptosis and necroptosis in photoreceptor cell death[35,36]. Effecting photoreceptor cell preservation by directly blocking these cell death pathways has remained an elusive goal, and has not yet resulted in useful neuroprotective therapies for IRD[37]. A major reason for this is that blocking one pathway of cell death often results in the shunting of the death cascade through other pathways. Our findings show that normalization of the A:P ratio reduces the activation of both the apoptotic and necroptotic cell death pathways, suggesting that altered homeostasis represented by the A:P ratio acts as an upstream activator of downstream executors of cell death. Thus, therapeutics that normalize the A:P ratio may represent a more effective strategy for the development of mutation-independent neuroprotective therapies for patients with IRD, as shunting between death pathways would be prevented. The molecular mechanism underlying the cross talk between the ERS–autophagy–proteasome axis and death-pathway activation in photoreceptor cells requires additional study, as this may represent another potential point of therapeutic intervention for decreasing retinal degeneration.Taken together, our data demonstrate that defects in protein folding cause chronic activation of the ERS that results in imbalanced activation of autophagy relative to the proteasome, which in turn promotes photoreceptor cell death. It follows that measures of the relative activity of autophagy versus the proteasome, which we have designated as the A:P ratio, represent an important readout, or biomarker, will be important in informing the development of mutation-independent therapies aimed at reducing proteotoxicity in IRD. We propose that the A:P ratio represents a measure of homeostasis within photoreceptor cells, and that treatments designed to normalize this important gatekeeper of cellular homeostasis have the potential to decrease retinal cell death in IRD. We predict that this concept can be extended beyond photoreceptor cells, and represents a novel perspective for assessing proteotoxicity across a spectrum of disease states.
Authors: Jingyu Yao; Lin Jia; Shameka J Shelby; Anna M Ganios; Kecia Feathers; Debra A Thompson; David N Zacks Journal: Invest Ophthalmol Vis Sci Date: 2014-04-29 Impact factor: 4.799
Authors: Wei-Chieh Chiang; Heike Kroeger; Sanae Sakami; Carissa Messah; Douglas Yasumura; Michael T Matthes; Judith A Coppinger; Krzysztof Palczewski; Matthew M LaVail; Jonathan H Lin Journal: Mol Neurobiol Date: 2014-10-02 Impact factor: 5.590
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Vojo Deretic; Benoît Derrien; Eric Deutsch; Timothy P Devarenne; Rodney J Devenish; Sabrina Di Bartolomeo; Nicola Di Daniele; Fabio Di Domenico; Alessia Di Nardo; Simone Di Paola; Antonio Di Pietro; Livia Di Renzo; Aaron DiAntonio; Guillermo Díaz-Araya; Ines Díaz-Laviada; Maria T Diaz-Meco; Javier Diaz-Nido; Chad A Dickey; Robert C Dickson; Marc Diederich; Paul Digard; Ivan Dikic; Savithrama P Dinesh-Kumar; Chan Ding; Wen-Xing Ding; Zufeng Ding; Luciana Dini; Jörg Hw Distler; Abhinav Diwan; Mojgan Djavaheri-Mergny; Kostyantyn Dmytruk; Renwick Cj Dobson; Volker Doetsch; Karol Dokladny; Svetlana Dokudovskaya; Massimo Donadelli; X Charlie Dong; Xiaonan Dong; Zheng Dong; Terrence M Donohue; Kelly S Doran; Gabriella D'Orazi; Gerald W Dorn; Victor Dosenko; Sami Dridi; Liat Drucker; Jie Du; Li-Lin Du; Lihuan Du; André du Toit; Priyamvada Dua; Lei Duan; Pu Duann; Vikash Kumar Dubey; Michael R Duchen; Michel A Duchosal; Helene Duez; Isabelle Dugail; Verónica I Dumit; Mara C Duncan; Elaine A Dunlop; William A Dunn; Nicolas Dupont; Luc Dupuis; Raúl V Durán; Thomas M Durcan; Stéphane Duvezin-Caubet; Umamaheswar Duvvuri; Vinay Eapen; Darius Ebrahimi-Fakhari; Arnaud Echard; Leopold Eckhart; Charles L Edelstein; Aimee L Edinger; Ludwig Eichinger; Tobias Eisenberg; Avital Eisenberg-Lerner; N Tony Eissa; Wafik S El-Deiry; Victoria El-Khoury; Zvulun Elazar; Hagit Eldar-Finkelman; Chris Jh Elliott; Enzo Emanuele; Urban Emmenegger; Nikolai Engedal; Anna-Mart Engelbrecht; Simone Engelender; Jorrit M Enserink; Ralf Erdmann; Jekaterina Erenpreisa; Rajaraman Eri; Jason L Eriksen; Andreja Erman; Ricardo Escalante; Eeva-Liisa Eskelinen; Lucile Espert; Lorena Esteban-Martínez; Thomas J Evans; Mario Fabri; Gemma Fabrias; Cinzia Fabrizi; Antonio Facchiano; Nils J Færgeman; Alberto Faggioni; W Douglas Fairlie; Chunhai Fan; Daping Fan; Jie Fan; Shengyun Fang; Manolis Fanto; Alessandro Fanzani; Thomas Farkas; Mathias Faure; Francois B Favier; Howard Fearnhead; Massimo Federici; Erkang Fei; Tania C Felizardo; Hua Feng; Yibin Feng; Yuchen Feng; Thomas A Ferguson; Álvaro F Fernández; Maite G Fernandez-Barrena; Jose C Fernandez-Checa; Arsenio Fernández-López; Martin E Fernandez-Zapico; Olivier Feron; Elisabetta Ferraro; Carmen Veríssima Ferreira-Halder; Laszlo Fesus; Ralph Feuer; Fabienne C Fiesel; Eduardo C Filippi-Chiela; Giuseppe Filomeni; Gian Maria Fimia; John H Fingert; Steven Finkbeiner; Toren Finkel; Filomena Fiorito; Paul B Fisher; Marc Flajolet; Flavio Flamigni; Oliver Florey; Salvatore Florio; R Andres Floto; Marco Folini; Carlo Follo; Edward A Fon; Francesco Fornai; Franco Fortunato; Alessandro Fraldi; Rodrigo Franco; Arnaud Francois; Aurélie François; Lisa B Frankel; Iain Dc Fraser; Norbert Frey; Damien G Freyssenet; Christian Frezza; Scott L Friedman; Daniel E Frigo; Dongxu Fu; José M Fuentes; Juan Fueyo; Yoshio Fujitani; Yuuki Fujiwara; Mikihiro Fujiya; Mitsunori Fukuda; Simone Fulda; Carmela Fusco; Bozena Gabryel; Matthias Gaestel; Philippe Gailly; Malgorzata Gajewska; Sehamuddin Galadari; Gad Galili; Inmaculada Galindo; Maria F Galindo; Giovanna Galliciotti; Lorenzo Galluzzi; Luca Galluzzi; Vincent Galy; Noor Gammoh; Sam Gandy; Anand K Ganesan; Swamynathan Ganesan; Ian G Ganley; Monique Gannagé; Fen-Biao Gao; Feng Gao; Jian-Xin Gao; Lorena García Nannig; Eleonora García Véscovi; Marina Garcia-Macía; Carmen Garcia-Ruiz; Abhishek D Garg; Pramod Kumar Garg; Ricardo Gargini; Nils Christian Gassen; Damián Gatica; Evelina Gatti; Julie Gavard; Evripidis Gavathiotis; Liang Ge; Pengfei Ge; Shengfang Ge; Po-Wu Gean; Vania Gelmetti; Armando A Genazzani; Jiefei Geng; Pascal Genschik; Lisa Gerner; Jason E Gestwicki; David A Gewirtz; Saeid Ghavami; Eric Ghigo; Debabrata Ghosh; Anna Maria Giammarioli; Francesca Giampieri; Claudia Giampietri; Alexandra Giatromanolaki; Derrick J Gibbings; Lara Gibellini; Spencer B Gibson; Vanessa Ginet; Antonio Giordano; Flaviano Giorgini; Elisa Giovannetti; Stephen E Girardin; Suzana Gispert; Sandy Giuliano; Candece L Gladson; Alvaro Glavic; Martin Gleave; Nelly Godefroy; Robert M Gogal; Kuppan Gokulan; Gustavo H Goldman; Delia Goletti; Michael S Goligorsky; Aldrin V Gomes; Ligia C Gomes; Hernando Gomez; Candelaria Gomez-Manzano; Rubén Gómez-Sánchez; Dawit Ap Gonçalves; Ebru Goncu; Qingqiu Gong; Céline Gongora; Carlos B Gonzalez; Pedro Gonzalez-Alegre; Pilar Gonzalez-Cabo; Rosa Ana González-Polo; Ing Swie Goping; Carlos Gorbea; Nikolai V Gorbunov; Daphne R Goring; Adrienne M Gorman; Sharon M Gorski; Sandro Goruppi; Shino Goto-Yamada; Cecilia Gotor; Roberta A Gottlieb; Illana Gozes; Devrim Gozuacik; Yacine Graba; Martin Graef; Giovanna E Granato; Gary Dean Grant; Steven Grant; Giovanni Luca Gravina; Douglas R Green; Alexander Greenhough; Michael T Greenwood; Benedetto Grimaldi; Frédéric Gros; Charles Grose; Jean-Francois Groulx; Florian Gruber; Paolo Grumati; Tilman Grune; Jun-Lin Guan; Kun-Liang Guan; Barbara Guerra; Carlos Guillen; Kailash Gulshan; Jan Gunst; Chuanyong Guo; Lei Guo; Ming Guo; Wenjie Guo; Xu-Guang Guo; Andrea A Gust; Åsa B Gustafsson; Elaine Gutierrez; Maximiliano G Gutierrez; Ho-Shin Gwak; Albert Haas; James E Haber; Shinji Hadano; Monica Hagedorn; David R Hahn; Andrew J Halayko; Anne Hamacher-Brady; Kozo Hamada; Ahmed Hamai; Andrea Hamann; Maho Hamasaki; Isabelle Hamer; Qutayba Hamid; Ester M Hammond; Feng Han; Weidong Han; James T Handa; John A Hanover; Malene Hansen; Masaru Harada; Ljubica Harhaji-Trajkovic; J Wade Harper; Abdel Halim Harrath; Adrian L Harris; James Harris; Udo Hasler; Peter Hasselblatt; Kazuhisa Hasui; Robert G Hawley; Teresa S Hawley; Congcong He; Cynthia Y He; Fengtian He; Gu He; Rong-Rong He; Xian-Hui He; You-Wen He; Yu-Ying He; Joan K Heath; Marie-Josée Hébert; Robert A Heinzen; Gudmundur Vignir Helgason; Michael Hensel; Elizabeth P Henske; Chengtao Her; Paul K Herman; Agustín Hernández; Carlos Hernandez; Sonia Hernández-Tiedra; Claudio Hetz; P Robin Hiesinger; Katsumi Higaki; Sabine Hilfiker; Bradford G Hill; Joseph A Hill; William D Hill; Keisuke Hino; Daniel Hofius; Paul Hofman; Günter U Höglinger; Jörg Höhfeld; Marina K Holz; Yonggeun Hong; David A Hood; Jeroen Jm Hoozemans; Thorsten Hoppe; Chin Hsu; Chin-Yuan Hsu; Li-Chung Hsu; Dong Hu; Guochang Hu; Hong-Ming Hu; Hongbo Hu; Ming Chang Hu; Yu-Chen Hu; Zhuo-Wei Hu; Fang Hua; Ya Hua; Canhua Huang; Huey-Lan Huang; Kuo-How Huang; Kuo-Yang Huang; Shile Huang; Shiqian Huang; Wei-Pang Huang; Yi-Ran Huang; Yong Huang; Yunfei Huang; Tobias B Huber; Patricia Huebbe; Won-Ki Huh; Juha J Hulmi; Gang Min Hur; James H Hurley; Zvenyslava Husak; Sabah Na Hussain; Salik Hussain; Jung Jin Hwang; Seungmin Hwang; Thomas Is Hwang; Atsuhiro Ichihara; Yuzuru Imai; Carol Imbriano; Megumi Inomata; Takeshi Into; Valentina Iovane; Juan L Iovanna; Renato V Iozzo; Nancy Y Ip; Javier E Irazoqui; Pablo Iribarren; Yoshitaka Isaka; Aleksandra J Isakovic; Harry Ischiropoulos; Jeffrey S Isenberg; Mohammad Ishaq; Hiroyuki Ishida; Isao Ishii; Jane E Ishmael; Ciro Isidoro; Ken-Ichi Isobe; Erika Isono; Shohreh Issazadeh-Navikas; Koji Itahana; Eisuke Itakura; Andrei I Ivanov; Anand Krishnan V Iyer; José M Izquierdo; Yotaro Izumi; Valentina Izzo; Marja Jäättelä; Nadia Jaber; Daniel John Jackson; William T Jackson; Tony George Jacob; Thomas S Jacques; Chinnaswamy Jagannath; Ashish Jain; Nihar Ranjan Jana; Byoung Kuk Jang; Alkesh Jani; Bassam Janji; Paulo Roberto Jannig; Patric J Jansson; Steve Jean; Marina Jendrach; Ju-Hong Jeon; Niels Jessen; Eui-Bae Jeung; Kailiang Jia; Lijun Jia; Hong Jiang; Hongchi Jiang; Liwen Jiang; Teng Jiang; Xiaoyan Jiang; Xuejun Jiang; Xuejun Jiang; Ying Jiang; Yongjun Jiang; Alberto Jiménez; Cheng Jin; Hongchuan Jin; Lei Jin; Meiyan Jin; Shengkan Jin; Umesh Kumar Jinwal; Eun-Kyeong Jo; Terje Johansen; Daniel E Johnson; Gail Vw Johnson; James D Johnson; Eric Jonasch; Chris Jones; Leo Ab Joosten; Joaquin Jordan; Anna-Maria Joseph; Bertrand Joseph; Annie M Joubert; Dianwen Ju; Jingfang Ju; Hsueh-Fen Juan; Katrin Juenemann; Gábor Juhász; Hye Seung Jung; Jae U Jung; Yong-Keun Jung; Heinz Jungbluth; Matthew J Justice; Barry Jutten; Nadeem O Kaakoush; Kai Kaarniranta; Allen Kaasik; Tomohiro Kabuta; Bertrand Kaeffer; Katarina Kågedal; Alon Kahana; Shingo Kajimura; Or Kakhlon; Manjula Kalia; Dhan V Kalvakolanu; Yoshiaki Kamada; Konstantinos Kambas; Vitaliy O Kaminskyy; Harm H Kampinga; Mustapha Kandouz; Chanhee Kang; Rui Kang; Tae-Cheon Kang; Tomotake Kanki; Thirumala-Devi Kanneganti; Haruo Kanno; Anumantha G Kanthasamy; Marc Kantorow; Maria Kaparakis-Liaskos; Orsolya Kapuy; Vassiliki Karantza; Md Razaul Karim; Parimal Karmakar; Arthur Kaser; Susmita Kaushik; Thomas Kawula; A Murat Kaynar; Po-Yuan Ke; Zun-Ji Ke; John H Kehrl; Kate E Keller; Jongsook Kim Kemper; Anne K Kenworthy; Oliver Kepp; Andreas Kern; Santosh Kesari; David Kessel; Robin Ketteler; Isis do Carmo Kettelhut; Bilon Khambu; Muzamil Majid Khan; Vinoth Km Khandelwal; Sangeeta Khare; Juliann G Kiang; Amy A Kiger; Akio Kihara; Arianna L Kim; Cheol Hyeon Kim; Deok Ryong Kim; Do-Hyung Kim; Eung Kweon Kim; Hye Young Kim; Hyung-Ryong Kim; Jae-Sung Kim; Jeong Hun Kim; Jin Cheon Kim; Jin Hyoung Kim; Kwang Woon Kim; Michael D Kim; Moon-Moo Kim; Peter K Kim; Seong Who Kim; Soo-Youl Kim; Yong-Sun Kim; Yonghyun Kim; Adi Kimchi; Alec C Kimmelman; Tomonori Kimura; Jason S King; Karla Kirkegaard; Vladimir Kirkin; Lorrie A Kirshenbaum; Shuji Kishi; Yasuo Kitajima; Katsuhiko Kitamoto; Yasushi Kitaoka; Kaio Kitazato; Rudolf A Kley; Walter T Klimecki; Michael Klinkenberg; Jochen Klucken; Helene Knævelsrud; Erwin Knecht; Laura Knuppertz; Jiunn-Liang Ko; Satoru Kobayashi; Jan C Koch; Christelle Koechlin-Ramonatxo; Ulrich Koenig; Young Ho Koh; Katja Köhler; Sepp D Kohlwein; Masato Koike; Masaaki Komatsu; Eiki Kominami; Dexin Kong; Hee Jeong Kong; Eumorphia G Konstantakou; Benjamin T Kopp; Tamas Korcsmaros; Laura Korhonen; Viktor I Korolchuk; Nadya V Koshkina; Yanjun Kou; Michael I Koukourakis; Constantinos Koumenis; Attila L Kovács; Tibor Kovács; Werner J Kovacs; Daisuke Koya; Claudine Kraft; Dimitri Krainc; Helmut Kramer; Tamara Kravic-Stevovic; Wilhelm Krek; Carole Kretz-Remy; Roswitha Krick; Malathi Krishnamurthy; Janos Kriston-Vizi; Guido Kroemer; Michael C Kruer; Rejko Kruger; Nicholas T Ktistakis; Kazuyuki Kuchitsu; Christian Kuhn; Addanki Pratap Kumar; Anuj Kumar; Ashok Kumar; Deepak Kumar; Dhiraj Kumar; Rakesh Kumar; Sharad Kumar; Mondira Kundu; Hsing-Jien Kung; Atsushi Kuno; Sheng-Han Kuo; Jeff Kuret; Tino Kurz; Terry Kwok; Taeg Kyu Kwon; Yong Tae Kwon; Irene Kyrmizi; Albert R La Spada; Frank Lafont; Tim Lahm; Aparna Lakkaraju; Truong Lam; Trond Lamark; Steve Lancel; Terry H Landowski; Darius J R Lane; Jon D Lane; Cinzia Lanzi; Pierre Lapaquette; Louis R Lapierre; Jocelyn Laporte; Johanna Laukkarinen; Gordon W Laurie; Sergio Lavandero; Lena Lavie; Matthew J LaVoie; Betty Yuen Kwan Law; Helen Ka-Wai Law; Kelsey B Law; Robert Layfield; Pedro A Lazo; Laurent Le Cam; Karine G Le Roch; Hervé Le Stunff; Vijittra Leardkamolkarn; Marc Lecuit; Byung-Hoon Lee; Che-Hsin Lee; Erinna F Lee; Gyun Min Lee; He-Jin Lee; Hsinyu Lee; Jae Keun Lee; Jongdae Lee; Ju-Hyun Lee; Jun Hee Lee; Michael Lee; Myung-Shik Lee; Patty J Lee; Sam W Lee; Seung-Jae Lee; Shiow-Ju Lee; Stella Y Lee; Sug Hyung Lee; Sung Sik Lee; Sung-Joon Lee; Sunhee Lee; Ying-Ray Lee; Yong J Lee; Young H Lee; Christiaan Leeuwenburgh; Sylvain Lefort; Renaud Legouis; Jinzhi Lei; Qun-Ying Lei; David A Leib; Gil Leibowitz; Istvan Lekli; Stéphane D Lemaire; John J Lemasters; Marius K Lemberg; Antoinette Lemoine; Shuilong Leng; Guido Lenz; Paola Lenzi; Lilach O Lerman; Daniele Lettieri Barbato; Julia I-Ju Leu; Hing Y Leung; Beth Levine; Patrick A Lewis; Frank Lezoualc'h; Chi Li; Faqiang Li; Feng-Jun Li; Jun Li; Ke Li; Lian Li; Min Li; Min Li; Qiang Li; Rui Li; Sheng Li; Wei Li; Wei Li; Xiaotao Li; Yumin Li; Jiqin Lian; Chengyu Liang; Qiangrong Liang; Yulin Liao; Joana Liberal; Pawel P Liberski; Pearl Lie; Andrew P Lieberman; Hyunjung Jade Lim; Kah-Leong Lim; Kyu Lim; Raquel T Lima; Chang-Shen Lin; Chiou-Feng Lin; Fang Lin; Fangming Lin; Fu-Cheng Lin; Kui Lin; Kwang-Huei Lin; Pei-Hui Lin; Tianwei Lin; Wan-Wan Lin; Yee-Shin Lin; Yong Lin; Rafael Linden; Dan Lindholm; Lisa M Lindqvist; Paul Lingor; Andreas Linkermann; Lance A Liotta; Marta M Lipinski; Vitor A Lira; Michael P Lisanti; Paloma B Liton; Bo Liu; Chong Liu; Chun-Feng Liu; Fei Liu; Hung-Jen Liu; Jianxun Liu; Jing-Jing Liu; Jing-Lan Liu; Ke Liu; Leyuan Liu; Liang Liu; Quentin Liu; Rong-Yu Liu; Shiming Liu; Shuwen Liu; Wei Liu; Xian-De Liu; Xiangguo Liu; Xiao-Hong Liu; Xinfeng Liu; Xu Liu; Xueqin Liu; Yang Liu; Yule Liu; Zexian Liu; Zhe Liu; Juan P Liuzzi; Gérard Lizard; Mila Ljujic; Irfan J Lodhi; Susan E Logue; Bal L Lokeshwar; Yun Chau Long; Sagar Lonial; Benjamin Loos; Carlos López-Otín; Cristina López-Vicario; Mar Lorente; Philip L Lorenzi; Péter Lõrincz; Marek Los; Michael T Lotze; Penny E Lovat; Binfeng Lu; Bo Lu; Jiahong Lu; Qing Lu; She-Min Lu; Shuyan Lu; Yingying Lu; Frédéric Luciano; Shirley Luckhart; John Milton Lucocq; Paula Ludovico; Aurelia Lugea; Nicholas W Lukacs; Julian J Lum; Anders H Lund; Honglin Luo; Jia Luo; Shouqing Luo; Claudio Luparello; Timothy Lyons; Jianjie Ma; Yi Ma; Yong Ma; Zhenyi Ma; Juliano Machado; Glaucia M Machado-Santelli; Fernando Macian; Gustavo C MacIntosh; Jeffrey P MacKeigan; Kay F Macleod; John D MacMicking; Lee Ann MacMillan-Crow; Frank Madeo; Muniswamy Madesh; Julio Madrigal-Matute; Akiko Maeda; Tatsuya Maeda; Gustavo Maegawa; Emilia Maellaro; Hannelore Maes; Marta Magariños; Kenneth Maiese; Tapas K Maiti; Luigi Maiuri; Maria Chiara Maiuri; Carl G Maki; Roland Malli; Walter Malorni; Alina Maloyan; Fathia Mami-Chouaib; Na Man; Joseph D Mancias; Eva-Maria Mandelkow; Michael A Mandell; Angelo A Manfredi; Serge N Manié; Claudia Manzoni; Kai Mao; Zixu Mao; Zong-Wan Mao; Philippe Marambaud; Anna Maria Marconi; Zvonimir Marelja; Gabriella Marfe; Marta Margeta; Eva Margittai; Muriel Mari; Francesca V Mariani; Concepcio Marin; Sara Marinelli; Guillermo Mariño; Ivanka Markovic; Rebecca Marquez; Alberto M Martelli; Sascha Martens; Katie R Martin; Seamus J Martin; Shaun Martin; Miguel A Martin-Acebes; Paloma Martín-Sanz; Camille Martinand-Mari; Wim Martinet; Jennifer Martinez; Nuria Martinez-Lopez; Ubaldo Martinez-Outschoorn; Moisés Martínez-Velázquez; Marta Martinez-Vicente; Waleska Kerllen Martins; Hirosato Mashima; James A Mastrianni; Giuseppe Matarese; Paola Matarrese; Roberto Mateo; Satoaki Matoba; Naomichi Matsumoto; Takehiko Matsushita; Akira Matsuura; Takeshi Matsuzawa; Mark P Mattson; Soledad Matus; Norma Maugeri; Caroline Mauvezin; Andreas Mayer; Dusica Maysinger; Guillermo D Mazzolini; Mary Kate McBrayer; Kimberly McCall; Craig McCormick; Gerald M McInerney; Skye C McIver; Sharon McKenna; John J McMahon; Iain A McNeish; Fatima Mechta-Grigoriou; Jan Paul Medema; Diego L Medina; Klara Megyeri; Maryam Mehrpour; Jawahar L Mehta; Yide Mei; Ute-Christiane Meier; Alfred J Meijer; Alicia Meléndez; Gerry Melino; Sonia Melino; Edesio Jose Tenorio de Melo; Maria A Mena; Marc D Meneghini; Javier A Menendez; Regina Menezes; Liesu Meng; Ling-Hua Meng; Songshu Meng; Rossella Menghini; A Sue Menko; Rubem Fs Menna-Barreto; Manoj B Menon; Marco A Meraz-Ríos; Giuseppe Merla; Luciano Merlini; Angelica M Merlot; Andreas Meryk; Stefania Meschini; Joel N Meyer; Man-Tian Mi; Chao-Yu Miao; Lucia Micale; Simon Michaeli; Carine Michiels; Anna Rita Migliaccio; Anastasia Susie Mihailidou; Dalibor Mijaljica; Katsuhiko Mikoshiba; Enrico Milan; Leonor Miller-Fleming; Gordon B Mills; Ian G Mills; Georgia Minakaki; Berge A Minassian; Xiu-Fen Ming; Farida Minibayeva; Elena A Minina; Justine D Mintern; Saverio Minucci; Antonio Miranda-Vizuete; Claire H Mitchell; Shigeki Miyamoto; Keisuke Miyazawa; Noboru Mizushima; Katarzyna Mnich; Baharia Mograbi; Simin Mohseni; Luis Ferreira Moita; Marco Molinari; Maurizio Molinari; Andreas Buch Møller; Bertrand Mollereau; Faustino Mollinedo; Marco Mongillo; Martha M Monick; Serena Montagnaro; Craig Montell; Darren J Moore; Michael N Moore; Rodrigo Mora-Rodriguez; Paula I Moreira; Etienne Morel; Maria Beatrice Morelli; Sandra Moreno; Michael J Morgan; Arnaud Moris; Yuji Moriyasu; Janna L Morrison; Lynda A Morrison; Eugenia Morselli; Jorge Moscat; Pope L Moseley; Serge Mostowy; Elisa Motori; Denis Mottet; Jeremy C Mottram; Charbel E-H Moussa; Vassiliki E Mpakou; Hasan Mukhtar; Jean M Mulcahy Levy; Sylviane Muller; Raquel Muñoz-Moreno; Cristina Muñoz-Pinedo; Christian Münz; Maureen E Murphy; James T Murray; Aditya Murthy; Indira U Mysorekar; Ivan R Nabi; Massimo Nabissi; Gustavo A Nader; Yukitoshi Nagahara; Yoshitaka Nagai; Kazuhiro Nagata; Anika Nagelkerke; Péter Nagy; Samisubbu R Naidu; Sreejayan Nair; Hiroyasu Nakano; Hitoshi Nakatogawa; Meera Nanjundan; Gennaro Napolitano; Naweed I Naqvi; Roberta Nardacci; Derek P Narendra; Masashi Narita; Anna Chiara Nascimbeni; Ramesh Natarajan; Luiz C Navegantes; Steffan T Nawrocki; Taras Y Nazarko; Volodymyr Y Nazarko; Thomas Neill; Luca M Neri; Mihai G Netea; Romana T Netea-Maier; Bruno M Neves; Paul A Ney; Ioannis P Nezis; Hang Tt Nguyen; Huu Phuc Nguyen; Anne-Sophie Nicot; Hilde Nilsen; Per Nilsson; Mikio Nishimura; Ichizo Nishino; Mireia Niso-Santano; Hua Niu; Ralph A Nixon; Vincent Co Njar; Takeshi Noda; Angelika A Noegel; Elsie Magdalena Nolte; Erik Norberg; Koenraad K Norga; Sakineh Kazemi Noureini; Shoji Notomi; Lucia Notterpek; Karin Nowikovsky; Nobuyuki Nukina; Thorsten Nürnberger; Valerie B O'Donnell; Tracey O'Donovan; Peter J O'Dwyer; Ina Oehme; Clara L Oeste; Michinaga Ogawa; Besim Ogretmen; Yuji Ogura; Young J Oh; Masaki Ohmuraya; Takayuki Ohshima; Rani Ojha; Koji Okamoto; Toshiro Okazaki; F Javier Oliver; Karin Ollinger; Stefan Olsson; Daniel P Orban; Paulina Ordonez; Idil Orhon; Laszlo Orosz; Eyleen J O'Rourke; Helena Orozco; Angel L Ortega; Elena Ortona; Laura D Osellame; Junko Oshima; Shigeru Oshima; Heinz D Osiewacz; Takanobu Otomo; Kinya Otsu; Jing-Hsiung James Ou; Tiago F Outeiro; Dong-Yun Ouyang; Hongjiao Ouyang; Michael Overholtzer; Michelle A Ozbun; P Hande Ozdinler; Bulent Ozpolat; Consiglia Pacelli; Paolo Paganetti; Guylène Page; Gilles Pages; Ugo Pagnini; Beata Pajak; Stephen C Pak; Karolina Pakos-Zebrucka; Nazzy Pakpour; Zdena Palková; Francesca Palladino; Kathrin Pallauf; Nicolas Pallet; Marta Palmieri; Søren R Paludan; Camilla Palumbo; Silvia Palumbo; Olatz Pampliega; Hongming Pan; Wei Pan; Theocharis Panaretakis; Aseem Pandey; Areti Pantazopoulou; Zuzana Papackova; Daniela L Papademetrio; Issidora Papassideri; Alessio Papini; Nirmala Parajuli; Julian Pardo; Vrajesh V Parekh; Giancarlo Parenti; Jong-In Park; Junsoo Park; Ohkmae K Park; Roy Parker; Rosanna Parlato; Jan B Parys; Katherine R Parzych; Jean-Max Pasquet; Benoit Pasquier; Kishore Bs Pasumarthi; Daniel Patschan; Cam Patterson; Sophie Pattingre; Scott Pattison; Arnim Pause; Hermann Pavenstädt; Flaminia Pavone; Zully Pedrozo; Fernando J Peña; Miguel A Peñalva; Mario Pende; Jianxin Peng; Fabio Penna; Josef M Penninger; Anna Pensalfini; Salvatore Pepe; Gustavo Js Pereira; Paulo C Pereira; Verónica Pérez-de la Cruz; María Esther Pérez-Pérez; Diego Pérez-Rodríguez; Dolores Pérez-Sala; Celine Perier; Andras Perl; David H Perlmutter; Ida Perrotta; Shazib Pervaiz; Maija Pesonen; Jeffrey E Pessin; Godefridus J Peters; Morten Petersen; Irina Petrache; Basil J Petrof; Goran Petrovski; James M Phang; Mauro Piacentini; Marina Pierdominici; Philippe Pierre; Valérie Pierrefite-Carle; Federico Pietrocola; Felipe X Pimentel-Muiños; Mario Pinar; Benjamin Pineda; Ronit Pinkas-Kramarski; Marcello Pinti; Paolo Pinton; Bilal Piperdi; James M Piret; Leonidas C Platanias; Harald W Platta; Edward D Plowey; Stefanie Pöggeler; Marc Poirot; Peter Polčic; Angelo Poletti; Audrey H Poon; Hana Popelka; Blagovesta Popova; Izabela Poprawa; Shibu M Poulose; Joanna Poulton; Scott K Powers; Ted Powers; Mercedes Pozuelo-Rubio; Krisna Prak; Reinhild Prange; Mark Prescott; Muriel Priault; Sharon Prince; Richard L Proia; Tassula Proikas-Cezanne; Holger Prokisch; Vasilis J Promponas; Karin Przyklenk; Rosa Puertollano; Subbiah Pugazhenthi; Luigi Puglielli; Aurora Pujol; Julien Puyal; Dohun Pyeon; Xin Qi; Wen-Bin Qian; Zheng-Hong Qin; Yu Qiu; Ziwei Qu; Joe Quadrilatero; Frederick Quinn; Nina Raben; Hannah Rabinowich; Flavia Radogna; Michael J Ragusa; Mohamed Rahmani; Komal Raina; Sasanka Ramanadham; Rajagopal Ramesh; Abdelhaq Rami; Sarron Randall-Demllo; Felix Randow; Hai Rao; V Ashutosh Rao; Blake B Rasmussen; Tobias M Rasse; Edward A Ratovitski; Pierre-Emmanuel Rautou; Swapan K Ray; Babak Razani; Bruce H Reed; Fulvio Reggiori; Markus Rehm; Andreas S Reichert; Theo Rein; David J Reiner; Eric Reits; Jun Ren; Xingcong Ren; Maurizio Renna; Jane Eb Reusch; Jose L Revuelta; Leticia Reyes; Alireza R Rezaie; Robert I Richards; Des R Richardson; Clémence Richetta; Michael A Riehle; Bertrand H Rihn; Yasuko Rikihisa; Brigit E Riley; Gerald Rimbach; Maria Rita Rippo; Konstantinos Ritis; Federica Rizzi; Elizete Rizzo; Peter J Roach; Jeffrey Robbins; Michel Roberge; Gabriela Roca; Maria Carmela Roccheri; Sonia Rocha; Cecilia Mp Rodrigues; Clara I Rodríguez; Santiago Rodriguez de Cordoba; Natalia Rodriguez-Muela; Jeroen Roelofs; Vladimir V Rogov; Troy T Rohn; Bärbel Rohrer; Davide Romanelli; Luigina Romani; Patricia Silvia Romano; M Isabel G Roncero; Jose Luis Rosa; Alicia Rosello; Kirill V Rosen; Philip Rosenstiel; Magdalena Rost-Roszkowska; Kevin A Roth; Gael Roué; Mustapha Rouis; Kasper M Rouschop; Daniel T Ruan; Diego Ruano; David C Rubinsztein; Edmund B Rucker; Assaf Rudich; Emil Rudolf; Ruediger Rudolf; Markus A Ruegg; Carmen Ruiz-Roldan; Avnika Ashok Ruparelia; Paola Rusmini; David W Russ; Gian Luigi Russo; Giuseppe Russo; Rossella Russo; Tor Erik Rusten; Victoria Ryabovol; Kevin M Ryan; Stefan W Ryter; David M Sabatini; Michael Sacher; Carsten Sachse; Michael N Sack; Junichi Sadoshima; Paul Saftig; Ronit Sagi-Eisenberg; Sumit Sahni; Pothana Saikumar; Tsunenori Saito; Tatsuya Saitoh; Koichi Sakakura; Machiko Sakoh-Nakatogawa; Yasuhito Sakuraba; María Salazar-Roa; Paolo Salomoni; Ashok K Saluja; Paul M Salvaterra; Rosa Salvioli; Afshin Samali; Anthony Mj Sanchez; José A Sánchez-Alcázar; Ricardo Sanchez-Prieto; 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