Rab32 connects ER stress to mitochondrial defects in multiple sclerosis
- Yohannes Haile†1, 6,
- Xiaodan Deng†2,
- Carolina Ortiz-Sandoval1,
- Nasser Tahbaz1,
- Aleksandra Janowicz1,
- Jian-Qiang Lu3,
- Bradley J. Kerr4,
- Nicholas J. Gutowski5,
- Janet E. Holley5,
- Paul Eggleton5,
- Fabrizio Giuliani2Email author and
- Thomas Simmen1Email authorView ORCID ID profile
© The Author(s). 2017
Received: 5 July 2016
Accepted: 29 December 2016
Published: 23 January 2017
Endoplasmic reticulum (ER) stress is a hallmark of neurodegenerative diseases such as multiple sclerosis (MS). However, this physiological mechanism has multiple manifestations that range from impaired clearance of unfolded proteins to altered mitochondrial dynamics and apoptosis. While connections between the triggering of the unfolded protein response (UPR) and downstream mitochondrial dysfunction are poorly understood, the membranous contacts between the ER and mitochondria, called the mitochondria-associated membrane (MAM), could provide a functional link between these two mechanisms. Therefore, we investigated whether the guanosine triphosphatase (GTPase) Rab32, a known regulator of the MAM, mitochondrial dynamics, and apoptosis, could be associated with ER stress as well as mitochondrial dysfunction.
We assessed Rab32 expression in MS patient and experimental autoimmune encephalomyelitis (EAE) tissue, via observation of mitochondria in primary neurons and via monitoring of survival of neuronal cells upon increased Rab32 expression.
We found that the induction of Rab32 and other MAM proteins correlates with ER stress proteins in MS brain, as well as in EAE, and occurs in multiple central nervous system (CNS) cell types. We identify Rab32, known to increase in response to acute brain inflammation, as a novel unfolded protein response (UPR) target. High Rab32 expression shortens neurite length, alters mitochondria morphology, and accelerates apoptosis/necroptosis of human primary neurons and cell lines.
ER stress is strongly associated with Rab32 upregulation in the progression of MS, leading to mitochondrial dysfunction and neuronal death.
KeywordsMultiple sclerosis Endoplasmic reticulum Mitochondria Unfolded protein response (UPR)
At an advanced stage of MS, immunomodulating therapies are no longer effective, highlighting the need to understand the molecular basis of this disease. Like other neurodegenerative diseases that are associated with mitochondrial impairment , MS mitochondria can be dysfunctional, especially during disease progression  and its neurodegenerative phase [2, 3]. For instance, mitochondria no longer respire normally in progressive MS patients . Dysfunctional mitochondria produce reactive oxygen species (ROS). As a consequence, mitochondrial ROS promote inflammation and shift mitochondrial dynamics towards fission . This latter process requires dynamin-related protein 1 (Drp1), a ubiquitous guanosine triphosphatase (GTPase) . While Drp1 is essential for post-mitotic neurons , its excessive activity can result in apoptosis . Indeed, mitochondria increase in number in MS neurons undergoing demyelination , thus accelerating axonal degeneration . Upstream causes of these mitochondrial defects are largely unknown. One potential mechanism involves the uncontrolled release of Ca2+ ions from the main cellular Ca2+ store, the endoplasmic reticulum (ER), within the diseased neurons. This intracellular signaling mechanism ultimately promotes cell death via a mitochondrial mechanism [11, 12]. Lethal ER-mitochondria Ca2+ transfer is observed for instance following axotomy  or spinal cord injury .
In addition to disrupted mitochondrial dynamics, the induction of ER stress is another hallmark of MS [15, 16]. This process not only promotes ER-mitochondria crosstalk in general by increasing the apposition of the ER and mitochondria at the so-called mitochondria-associated membrane (MAM) [17–19], but could also promote neuronal death, thus contributing to the MS pathology .
A prominent MAM regulatory protein is Rab32. This GTPase localizes to the ER and mitochondria [21, 22], where it regulates ER-mitochondria interactions and mitochondrial dynamics . Rab32 is induced upon brain inflammation in a mouse model . Consistent with an important role in neuroinflammation, our data indicate that ER stress induces Rab32 and occurs in the MS brain. These findings increase our understanding of Rab32 role in impairment of neuronal mitochondrial dynamics and cell survival.
Antibodies used in this study were purchased as follows: anti-actin, anti-phospho-Drp1 Ser637 (Cell Signaling, Danvers, MA), anti-amyloid precursor protein, anti-glucose-regulated protein of 94 kDA (GRP94), anti-α tubulin, anti-receptor-interacting protein kinase (RIPK) (EMD-Millipore, Billerica, MA), anti-immunoglobulin-binding protein/glucose-regulated protein of 78 kDA (BiP/GRP78) (BD Biosciences, Franklin Lakes, NJ), anti-BiP/GRP78, anti-Drp1 (abcam, Cambridge, UK), anti-CD68 (Dako/Agilent, Markham, ON), anti-CCAAT/enhancer-binding protein (C/EBP) homologous protein (CHOP), anti-GRP75 (Pierce/Thermo, Waltham, MA), anti-CHOP (Enzo, Farmingdale, NY), anti-phosphofurin acidic cluster sorting protein 2 (PACS-2) (Protein Tech, Chicago, IL), anti-Rab32 (Sigma/Aldrich, St. Louis, MO), and anti-FLAG (Rockland, Limerick, PA). The antibody against calnexin has been described previously .
Isolation and maintenance of primary neuronal cultures
Cultures of human fetal neurons (HFN) were generated from 15–19-week fetal brains (obtained with consent from the University of Alberta Ethics Committee) as described .
Human frozen brain tissues and EAE mice tissue
For immunohistochemistry, snap-frozen blocks of post-mortem normal control (NC) or MS cerebral sub-ventricular deep white matter samples were obtained from the NeuroResource Tissue Bank, UCL Institute of Neurology, London, and UK MS Biobank, with next-of-kin informed consent for tissue donation and ethical approval from Central London REC1 (I.D.08/H0718/62) and approval for the study from the Local Research Ethics Committee (I.D.04/Q2102/111), UK MS brain bank charity number 1139257. A total of 12 MS patients (9 females, 3 males) who had been affected from secondary progressive (10), primary progressive (1) or relapsing progressive MS were used for this part of the study. Control tissue from individuals who had not been affected by disease (7) and 2 individuals who had been affected by Parkinson’s disease was also examined. Control patients died of non-inflammatory diseases (cardiac failure, lung cancer, bladder cancer, prostate cancer, tongue cancer, myelodysplastic syndrome; for two control cases, the cause of death was not known). Further information is contained in Additional file 1.
For Western blot and immunofluorescence analysis, tissues of two frozen MS brains (patient 1: secondary progressive MS, aged 54, male; patient 2: relapsing-remitting MS, aged 45, male) were obtained from the MS Tissue Bank at the University of Alberta. Post-mortem brain tissues were collected and processed as described . Frozen brain and spinal cord tissues of triplicate experimental autoimmune encephalomyelitis (EAE) mice, an animal model of MS, were generated with proper approvals as described . Control samples showed no signs of nervous disease. Disease peak samples were from clinical grade 1, whereas post-peak samples were from clinical grade 4 (hind limb paralysis at time of dissection).
Lysate preparation and analysis from tissues and cell lines
Tissue lysates were prepared from the human frozen brain as well as from the spinal cords of EAE mice in 1× sodium dodecyl sulphate (SDS) extraction buffer (0.125 M Tris-HCK pH 6.8, 2% SDS, 10% glycerol, 5% β-ME), followed by sonication on a 550 Sonic Dismembrator (Fisher Scientific, Ottawa, ON). Supernatants were collected, and protein concentrations were measured by NanoDrop Spectrophotometer ND1000 (Thermo/Life Technologies) at an absorbance of 280 nm. Cellular lysates from SH-SY5Y cells were prepared as described .
SH-SY5Y cells were cultured in mild hypoxia (4% O2, grown in the presence of 4% O2, 5% CO2 balanced N2, as is typical for brain tissue) in the presence of thapsigargin. After 24 h in culture, total RNA was extracted. The primers used for RT-PCR were as follows: Rab32 forward AGCAGGACTCTGGTGCGCCTG (position 211-231); Rab32 reverse CGGGCAGCTTCCTCTATGTTTATGTTATC (position 557-529). The result was normalized against the ribosomal 18S.
Sample sections were stained with hematoxylin and eosin (H&E) and luxol fast blue (LFB) as described . Lesions were classified into acute (referring to tissue phenotype, see below), sub-acute, and chronic on the basis of the number and distribution of oil red-O-positive macrophages, the extent of demyelination, cellularity in the borders and parenchyma of lesions, and perivascular cuffing as described in our previous work . Briefly, acute lesions were identified via demyelination, invading macrophages, hypercellularity at the lesion border, and cuffing around the blood vessels. Sub-acute lesions showed a demyelinated plaque with fewer macrophages, mostly at the lesion border, and less perivascular cuffing. A chronic lesion consisted of a hypocellular demyelinated plaque completely lacking of oil red-O-stained macrophages. Examination of MS brain tissue for Rab32 expression in specific cell types was performed employing enzyme immunohistochemistry using a Vectastain ABC system® (Vector Laboratories, Peterborough, UK), as described .
Transfection of constructs and shRNA, immunofluorescence, and quantification of apoptosis
mCherry-labeled Rab32 shRNA psi-mH1 plasmids (HSH001118) as well as scrambled control (CSHCTR001) were purchased from Genecopoeia (Rockland MD). FLAG-tagged Rab32 constructs were expressed from pcDNA3 as published  (wt, wild type; Q85L, dominant-active; T39N, dominant-negative) or transferred into the bi-cistronic pIRES2-EGFP plasmid (Clontech-Takara, Mountain View, CA) that allows for the expression of any protein, in parallel with nuclear EGFP. To do so, the described constructs contained in pcDNA3 were PCR-amplified using the SP6 and TS484 (ATATGCTAGCACCATGGACTACAAGGACGACGATGACAAG) oligos following cuts with the 5’ Nhe1 and 3’ Xho1 sites. Primary neurons or SH-SY5Y neuronal cell lines were transfected by nucleofection (Lonza, Mississauga, ON). Immunofluorescence was performed as described . To assay neurotoxicity, nuclear EGFP was used to identify transfected HFNs and SH-SY5Y. Apoptosis was then detected by Cy5-annexin V binding (BD Biosciences). Assays were repeated in the presence of bafilomycin (100 nM, Sigma-Aldrich), necrostatin-1 (Nec-1, 50 μM, Cayman Chemical), carbobenzoxy-valyl-alanyl-aspartyl-[O-methyl]- fluoromethylketone (zVAD-fmk) (10 μM, Enzo Life Sciences, Farmingdale, NY), or with a combination of nec-1 and zVAD-fmk.
Cells were rinsed in PBS and fixed in 3% paraformaldehyde and 0.05% glutaraldehyde (GA) containing 2% sucrose. Next, free aldehyde groups were quenched with ammonium chloride (50 mM), and samples were permeabilized with saponin (0.1%). The samples were blocked (PBS + 1% BSA + 0.05% FSG + Saponin 0.05%) for an hour and then were incubated with mouse anti-FLAG in the blocking buffer overnight in a wet chamber. Following washes (0.2%BSA + 0.05%FSG + 0.05% saponin), the samples were incubated with the secondary antibody (Fluoronanogold Anti-mouse Fab’Alexa Fluor 488, cat. 7202; Nanoprobes, NY) for 3 h at RT and washed with PBS three times. The samples then were fixed (2% GA in PBS + 2% sucrose) for an hour, followed by three rinses in water. Following a 1-min incubation with GoldEnhance EM Plus (Cat. 2114; Nanoprobes, NY), the samples were rinsed in water, scraped in 100 mM sodium cacodylate and pelleted. The pellet was incubated for 1 h with osmium tetroxide (1%), followed by overnight staining with uranyl acetate. After dehydration in increasing concentrations of ethanol and then propylene oxide treatments, the pellets were transferred to resin (Embed 812 kit, cat. 14120; Electron Microscopy Sciences, Hatfield, PA) and incubated at 60 °C for 48 h. Blocks were sectioned (70 nm) using Ultracut E (Reichert Jung) and imaged with a Philips 310 electron microscope, equipped with a digital camera (Mega View III Soft Imaging System, Emsis Gmbh, Muenster, Germany).
Rab32 parallels ER stress within MS patient and EAE brains
Consistent with the previous identification of ER stress as a hallmark of the MS CNS [16, 36], we detected increased expression of calnexin, BiP/GRP78, GRP94, and the CCAAT/enhancer-binding protein (C/EBP) homologous protein (CHOP) in active, but not chronic lesions (Fig. 1d). Next, we tested whether other MAM regulatory proteins were upregulated as well. We probed for the tether proteins GRP75 and Mfn2 the MAM-associated mitochondria fission GTPase Drp1 and the MAM enrichment factor phosphofurin acidic cluster sorting protein 2 (PACS-2). Western blotting showed that all of the above proteins showed increased expression in active, but not chronic MS brain lesions (Fig. 1e). We extended our investigation into the animal model of MS, experimental autoimmune encephalomyelitis (EAE). Western blotting showed that high levels of brain-localized Rab32 occurred in the peak and post-peak period of EAE (Fig. 1f), reflecting the induction of Rab32 in both active and chronic lesions of MS brain.
Cell-type specific localization of Rab32 expression
Rab32 expression is under the control of the unfolded protein response (UPR)
Rab32 interferes with neuronal mitochondrial dynamics and growth
Long-term effect of Rab32 and its mutants on neuronal survival
Caspase inhibition and necrostatin can block Rab32-directed neuronal death
To determine the relative contribution of apoptosis and necroptosis to neuronal cell death upon Rab32 over-expression, we re-examined the survival of SH-SY5Y cells transfected with EGFP, shRab32, Rab32WT, and Rab32Q85L. We then repeated our survival assay in the presence of necrostatin-1 and zVAD-fmk and, as an additional control, bafilomycin that inhibits autophagy. Quantification of the surviving cells showed that the role or Rab32 in autophagy was not responsible for our observations. In contrast, necrostatin-1 and zVAD-fmk significantly inhibited the Rab32-induced neuronal damage and death, further increased upon combination of both inhibitors (Fig. 7c). Our results therefore indicate that Rab32 induces neuronal damage and death from a combination of apoptosis and necroptosis.
In our study, we report that Rab32 serves as a novel marker of neurodegeneration in MS lesions, consistent with its previously detected induction in response to pro-inflammatory lipopolysaccharide (LPS) . Interestingly, we found that Rab32 correlated with the inflammatory status of the tissues. In contrast to healthy tissue, which showed low levels of Rab32 as reported previously [34, 35], Rab32 was highly expressed in active lesions of both human MS patients and EAE mice; while not as high, expression of Rab32 was still elevated in chronic lesions. In terms of cell types, we have detected high amounts of Rab32 in neurons and microglial/macrophage cells.
Our investigation into a transcriptional regulation of Rab32 expression showed that this gene responds to ER stress. Since ER stress is well known to trigger inflammation and mitochondrial dysfunction, our observation that ER stress leads to Rab32 induction and subsequently alters mitochondrial dynamic as well as neuronal apoptosis induction identifies Rab32 as a protein of critical interest to MS research. Results presented in this study demonstrate that an increase of Rab32 in the inflamed brain directly promotes neuronal cell death from a combination of apoptosis and necroptosis. Interestingly, the putative role of Rab32 as an autophagy promoter  is not tied to this pro-death function of Rab32. While wild-type and active Rab32Q85L showed effects on mitochondrial morphology and neurite outgrowth, inactive Rab32T39N also compromised the survival of primary neurons as well as SH-SY5Y cells (Figs. 5 and 6), suggesting the mere upregulation of Rab32 is detrimental to neuronal function, potentially due to shared functions of active and inactive Rab32. Moreover, our results reinforce the role of ER stress as an upstream trigger of inflammation, which is one of the main pathology drivers in the MS context. Interestingly, the inhibition of the UPR can improve myelination of some disease models  and also plays a critical role in the most promising approaches to treat neurodegeneration .
Rab32 is induced in parallel with known mediators or regulators of the MAM, namely, Grp75, PACS-2, Mitofusin 2, and Drp1 (Fig. 1). In contrast to Rab32, however, these MAM modulatory proteins were only induced in active, but not in chronic lesions. Nevertheless, this suggests that MAM functions, including the exchange of Ca2+ between the two organelles and mitochondrial dynamics, actively control neuronal decay within active MS lesions but reach a new equilibrium in chronic lesions.
Taken together, our results identify Rab32 as a new marker for active and chronic inflammatory lesions of the CNS in MS whose ER stress-triggered transcription leads to altered mitochondrial dynamics and neuronal cell death. Future research will have to investigate whether the inhibition of Rab32 production in the inflamed CNS could inhibit neurodegeneration in vivo.
Amyotrophic lateral sclerosis
Immunoglobulin-binding protein/glucose-regulated protein of 78 kDA
CCAAT/enhancer-binding protein (C/EBP) homologous protein
Central nervous system
Dynamin-related protein 1
Experimental autoimmune encephalomyelitis
Enhanced green fluorescent protein
Glucose-regulated protein of 94 kDA
Hematoxylin and eosin
Human fetal neurons
Normal-appearing white matter
Phosphofurin acidic cluster sorting protein 2
Receptor-interacting protein kinase
Interfering ribonucleic acid
Reactive oxygen species
Reverse transcription polymerase chain reaction
Sodium dodecyl sulphate
Unfolded protein response
We thank Trefa M. Abdullah for the expert technical assistance and Sol Herrera for the useful discussions. We are indebted to Chris Power for providing some patient samples.
The study was supported by CIHR (MOP 133541 to TS and FG) and MS Society of Canada (FG) and a Royal Devon and Exeter Foundation Trust Hospital research grant (PE, JEH & NJG).
Availability of data and materials
All data generated or analyzed during this study are included in this published article [and its supplementary information files].
The experiments were performed by YH, XD, COS, NT, AJ, NJG, and JEH. The tissue samples from patients and animals were collected under the supervision of JQL, BJK, PE, and FG. FG and TS designed the study. TS wrote the manuscript, with editing assistance provided by PE and FG. All authors read and approved the final manuscript.
The authors declare that they have no competing interests.
Consent for publication
All authors have read the manuscript and consent of its publication.
Ethics approval and consent to participate
Cultures of human fetal neurons (HFN) were obtained with consent from the University of Alberta Ethics Committee as described . MS cerebral sub-ventricular deep white matter samples were obtained from the NeuroResource Tissue Bank, UCL Institute of Neurology, London, and UK MS Biobank, with next-of-kin informed consent for tissue donation and ethical approval from Central London REC1 (I.D.08/H0718/62) and approval for the study from the Local Research Ethics Committee (I.D.04/Q2102/111).
Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.
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