Manal A. Farg

Endoplasmic reticulum stress and induction of the unfolded protein response in human sporadic amyotrophic lateral sclerosis

The unfolded protein response (UPR) is induced at symptom onset and disease end stage in rodent models of familial amyotrophic lateral sclerosis (ALS) that express superoxide dismutase (SOD1) mutations. However, ninety percent of human ALS is sporadic and mutations in SOD1 account for only 2% of total ALS. Here we show that a full UPR, including induction of stress sensor kinases, chaperones and apoptotic mediators, is also present in spinal cords of human patients with sporadic disease. Furthermore, the UPR chaperone protein disulphide isomerase (PDI) was present in CSF and was aggregated and widely distributed throughout the motor neurons of these patients. We also show up-regulation of UPR prior to the onset of symptoms in SOD1 rodents, implying an active role in disease. This study offers new insights into pathogenesis, placing ER stress onto a generic pathophysiology for ALS.

Induction of the Unfolded Protein Response in Familial Amyotrophic Lateral Sclerosis and Association of Protein-disulfide Isomerase with Superoxide Dismutase 1

Mutations in Cu/Zn superoxide dismutase (SOD1) are linked to motor neuron death in familial amyotrophic lateral sclerosis (ALS) by an unclear mechanism, although misfolded SOD1 aggregates are commonly associated with disease. Proteomic analysis of the transgenic SOD1(G93A) ALS rat model revealed significant up-regulation of endoplasmic reticulum (ER)-resident protein-disulfide isomerase (PDI) family members in lumbar spinal cords. Expression of SOD1 mutants (mSOD1) led to an up-regulation of PDI in motor neuron-like NSC-34 cells but not other cell lines. Inhibition of PDI using bacitracin increased aggregate production, even in wild type SOD1 transfectants that do not readily form inclusions, suggesting PDI may protect SOD1 from aggregation. Moreover, PDI co-localized with intracellular aggregates of mSOD1 and bound to both wild type and mSOD1. SOD1 was also found in the microsomal fraction of cells despite being a predominantly cytosolic enzyme, confirming ER-Golgi-dependent secretion. In SOD1(G93A) mice, a significant up-regulation of unfolded protein response entities was also observed during disease, including caspase-12, -9, and -3 cleavage. Our findings therefore implicate unfolded protein response and ER stress-induced apoptosis in the patho-physiology of familial ALS. The possibility that PDI may be a therapeutic target to prevent SOD1 aggregation is also raised by this study.

C9ORF72, implicated in amytrophic lateral sclerosis and frontotemporal dementia, regulates endosomal trafficking

Intronic expansion of a hexanucleotide GGGGCC repeat in the chromosome 9 open reading frame 72 (C9ORF72) gene is the major cause of familial amyotrophic lateral sclerosis (ALS) and frontotemporal dementia. However, the cellular function of the C9ORF72 protein remains unknown. Here, we demonstrate that C9ORF72 regulates endosomal trafficking. C9ORF72 colocalized with Rab proteins implicated in autophagy and endocytic transport: Rab1, Rab5, Rab7 and Rab11 in neuronal cell lines, primary cortical neurons and human spinal cord motor neurons, consistent with previous predictions that C9ORF72 bears Rab guanine exchange factor activity. Consistent with this notion, C9ORF72 was present in the extracellular space and as cytoplasmic vesicles. Depletion of C9ORF72 using siRNA inhibited transport of Shiga toxin from the plasma membrane to Golgi apparatus, internalization of TrkB receptor and altered the ratio of autophagosome marker light chain 3 (LC3) II:LC3I, indicating that C9ORF72 regulates endocytosis and autophagy. C9ORF72 also colocalized with ubiquilin-2 and LC3-positive vesicles, and co-migrated with lysosome-stained vesicles in neuronal cell lines, providing further evidence that C9ORF72 regulates autophagy. Investigation of proteins interacting with C9ORF72 using mass spectrometry identified other proteins implicated in ALS; ubiquilin-2 and heterogeneous nuclear ribonucleoproteins, hnRNPA2/B1 and hnRNPA1, and actin. Treatment of cells overexpressing C9ORF72 with proteasome inhibitors induced the formation of stress granules positive for hnRNPA1 and hnRNPA2/B1. Immunohistochemistry of C9ORF72 ALS patient motor neurons revealed increased colocalization between C9ORF72 and Rab7 and Rab11 compared with controls, suggesting possible dysregulation of trafficking in patients bearing the C9ORF72 repeat expansion. Hence, this study identifies a role for C9ORF72 in Rab-mediated cellular trafficking.

Impaired Extracellular Secretion of Mutant Superoxide Dismutase 1 Associates with Neurotoxicity in Familial Amyotrophic Lateral Sclerosis

Mutations in the intracellular metalloenzyme superoxide dismutase 1 (SOD1) are linked to neurotoxicity in familial amyotrophic lateral sclerosis (ALS) by an unclear mechanism. Golgi fragmentation and endoplasmic reticulum stress are early hallmarks of spinal motor neuron pathology in transgenic mice overexpressing mutant SOD1, suggesting that dysfunction of the neuronal secretory pathway may contribute to ALS pathogenesis. We therefore proposed that mutant SOD1 directly engages and modulates the secretory pathway based on recent evidence of SOD1 secretion in diverse human cell lines. Here, we demonstrate that a fraction of active endogenous SOD1 is secreted by NSC-34 motor neuron-like cells via a brefeldin-A (BFA)-sensitive pathway. Expression of enhanced green fluorescent protein-tagged mutant human SOD1 (hSOD1-EGFP) in NSC-34 cells induced frequent cytoplasmic inclusions and protein insolubility that correlated with toxicity. In contrast, transfection of non-neuronal COS-7 cells resulted in mutant hSOD1-EGFP cytoplasmic inclusions, oligomerization, and fragmentation without detectable toxicity. Importantly, impaired secretion of hSOD1-EGFP was common to all 10 SOD1 mutants tested relative to wild-type protein in NSC-34 cells. Treatment with BFA inhibited hSOD1-EGFP secretion with pronounced BFA-induced toxicity in mutant cells. Extracellular targeting of mutant hSOD1-EGFP via SOD3 signal peptide fusion attenuated cytoplasmic inclusion formation and toxicity. The effect of elevated extracellular SOD1 was then evaluated in a transgenic rat model of ALS. Chronic intraspinal infusion of exogenous wild-type hSOD1 significantly delayed disease progression and endpoint in transgenic SOD1G93A rats. Collectively, these results suggest novel extracellular roles for SOD1 in ALS and support a causal relationship between mutant SOD1 secretion and intraneuronal toxicity.

Protein disulphide isomerase protects against protein aggregation and is S-nitrosylated in amyotrophic lateral sclerosis

Amyotrophic lateral sclerosis is a rapidly progressing fatal neurodegenerative disease characterized by the presence of protein inclusions within affected motor neurons. Endoplasmic reticulum stress leading to apoptosis was recently recognized to be an important process in the pathogenesis of sporadic human amyotrophic lateral sclerosis as well as in transgenic models of mutant superoxide dismutase 1-linked familial amyotrophic lateral sclerosis. Endoplasmic reticulum stress occurs early in disease, indicating a critical role in pathogenesis, and involves upregulation of an important endoplasmic reticulum chaperone, protein disulphide isomerase. We aimed to investigate the involvement of protein disulphide isomerase in endoplasmic reticulum stress induction, protein aggregation, inclusion formation and toxicity in amyotrophic lateral sclerosis. Motor neuron-like NSC-34 cell lines were transfected with superoxide dismutase 1 and protein disulphide isomerase encoding vectors and small interfering RNA, and examined by immunocytochemistry and immunoblotting. Expression of mutant superoxide dismutase 1 induced endoplasmic reticulum stress, predominantly in cells bearing mutant superoxide dismutase 1 inclusions but also in a proportion of cells expressing mutant superoxide dismutase 1 without visible inclusions. Over-expression of protein disulphide isomerase decreased mutant superoxide dismutase 1 aggregation, inclusion formation, endoplasmic reticulum stress induction and toxicity, whereas small interfering RNA targeting protein disulphide isomerase increased mutant superoxide dismutase 1 inclusion formation, indicating a protective role for protein disulphide isomerase against superoxide dismutase 1 misfolding. Aberrant modification of protein disulphide isomerase by S-nitrosylation of active site cysteine residues has previously been shown as an important process in neurodegeneration in Parkinsons and Alzheimers disease brain tissue, but has not been described in amyotrophic lateral sclerosis. Using a biotin switch assay, we detected increased levels of S-nitrosylated protein disulphide isomerase in transgenic mutant superoxide dismutase 1 mouse and human sporadic amyotrophic lateral sclerosis spinal cord tissues. Hence, despite upregulation, protein disulphide isomerase is also functionally inactivated in amyotrophic lateral sclerosis, which may prevent its normal protective function and contribute to disease. We also found that a small molecule mimic of the protein disulphide isomerase active site, (+/-)-trans-1,2-bis(mercaptoacetamido)cyclohexane, protected against mutant superoxide dismutase 1 inclusion formation. These studies reveal that endoplasmic reticulum stress is important in the formation of mutant superoxide dismutase 1 inclusions, and protein disulphide isomerase has an important function in ameliorating mutant superoxide dismutase 1 aggregation and toxicity. Functional inhibition of protein disulphide isomerase by S-nitrosylation may contribute to pathophysiology in both mutant superoxide dismutase 1-linked disease and sporadic amyotrophic lateral sclerosis. Protein disulphide isomerase is therefore a novel potential therapeutic target in amyotrophic lateral sclerosis and (+/-)-trans-1,2-bis(mercaptoacetamido)cyclohexane and other molecular mimics of protein disulphide isomerase could be of benefit in amyotrophic lateral sclerosis and other neurodegenerative diseases related to protein misfolding.

Ataxin-2 interacts with FUS and intermediate-length polyglutamine expansions enhance FUS-related pathology in amyotrophic lateral sclerosis

Fused in sarcoma (FUS) is mutated in both sporadic amyotrophic lateral sclerosis (ALS) and familial ALS patients. The mechanisms underlying neurodegeneration are not fully understood, but FUS redistributes from the nucleus to the cytoplasm in affected motor neurons, where it triggers endoplasmic reticulum (ER) stress. Ataxin-2 is a polyglutamine protein which normally contains 22 repeats, but expanded repeats (>34) are found in Spinocerebellar Ataxia type 2. Recently ataxin-2 with intermediate length repeats (27-33) was found to increase the risk of ALS. Here we show that ataxin-2 with an ALS-linked intermediate length repeat (Q31) is a potent modifier of FUS pathology in cellular disease models. Translocation of FUS to the cytoplasm and ER stress were significantly enhanced by co-expression of mutant FUS with ataxin-2 Q31. Ataxin-2 also co-localized with FUS in sporadic and FUS-linked familial ALS patient motor neurons, co-precipitated with FUS in ALS spinal cord lysates, and co-localized with FUS in the ER-Golgi compartments in neuronal cell lines. Fragmentation of the Golgi apparatus is linked to neurodegeneration in ALS and here we show that Golgi fragmentation is induced in cells expressing mutant FUS. Moreover, Golgi fragmentation was enhanced, and the early stages of apoptosis were triggered, when ataxin-2 Q31 was co-expressed with mutant FUS. These findings describe new cellular mechanisms linking ALS with ataxin-2 intermediate length polyQ expansions and provide further evidence linking disruption to ER-Golgi compartments and FUS pathology in ALS.

ALS-Associated TDP-43 Induces Endoplasmic Reticulum Stress, Which Drives Cytoplasmic TDP-43 Accumulation and Stress Granule Formation

In amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration, TAR DNA binding protein 43 (TDP-43) accumulates in the cytoplasm of affected neurons and glia, where it associates with stress granules (SGs) and forms large inclusions. SGs form in response to cellular stress, including endoplasmic reticulum (ER) stress, which is induced in both familial and sporadic forms of ALS. Here we demonstrate that pharmacological induction of ER stress causes TDP-43 to accumulate in the cytoplasm, where TDP-43 also associates with SGs. Furthermore, treatment with salubrinal, an inhibitor of dephosphorylation of eukaryotic initiation factor 2-α, a key modulator of ER stress, potentiates ER stress-mediated SG formation. Inclusions of C-terminal fragment TDP-43, reminiscent of disease-pathology, form in close association with ER and Golgi compartments, further indicating the involvement of ER dysfunction in TDP-43-associated disease. Consistent with this notion, over-expression of ALS-linked mutant TDP-43, and to a lesser extent wildtype TDP-43, triggers several ER stress pathways in neuroblastoma cells. Similarly, we found an interaction between the ER chaperone protein disulphide isomerase and TDP-43 in transfected cell lysates and in the spinal cords of mutant A315T TDP-43 transgenic mice. This study provides evidence for ER stress as a pathogenic pathway in TDP-43-mediated disease.

Mutant FUS induces endoplasmic reticulum stress in amyotrophic lateral sclerosis and interacts with protein disulfide-isomerase.

Mutations in the gene encoding fused in sarcoma (FUS) are linked to amyotrophic lateral sclerosis (ALS), but the mechanisms by which these mutants trigger neurodegeneration remain unknown. Endoplasmic reticulum (ER) stress is increasingly recognized as an important and early pathway to motor neuron death in ALS. FUS is normally located in the nucleus but in ALS, FUS redistributes to the cytoplasm and forms inclusions. In this study, we investigated whether FUS induces ER stress in a motor neuron like cell line (NSC-34). We demonstrate that ER stress is triggered in cells expressing mutant FUS, and this is closely associated with redistribution of mutant FUS to the cytoplasm. Mutant FUS also colocalized with protein disulfide-isomerase (PDI), an important ER chaperone, in NSC-34 cells and PDI was colocalized with FUS inclusions in human ALS lumbar spinal cords, in both sporadic ALS and mutant FUS-linked familial ALS tissues. These findings implicate ER stress in the pathophysiology of FUS, and provide evidence for common pathogenic pathways in ALS linked to the ER.

Mutant SOD1 inhibits ER-Golgi transport in amyotrophic lateral sclerosis

Cu/Zn‐superoxide dismutase is misfolded in familial and sporadic amyotrophic lateral sclerosis, but it is not clear how this triggers endoplasmic reticulum (ER) stress or other pathogenic processes. Here, we demonstrate that mutant SOD1 (mSOD1) is predominantly found in the cytoplasm in neuronal cells. Furthermore, we show that mSOD1 inhibits secretory protein transport from the ER to Golgi apparatus. ER‐Golgi transport is linked to ER stress, Golgi fragmentation and axonal transport and we also show that inhibition of ER‐Golgi trafficking preceded ER stress, Golgi fragmentation, protein aggregation and apoptosis in cells expressing mSOD1. Restoration of ER‐Golgi transport by over‐expression of coatomer coat protein II subunit Sar1 protected against inclusion formation and apoptosis, thus linking dysfunction in ER‐Golgi transport to cellular pathology. These findings thus link several cellular events in amyotrophic lateral sclerosis into a single mechanism occurring early in mSOD1 expressing cells.

Bim Links ER Stress and Apoptosis in Cells Expressing Mutant SOD1 Associated with Amyotrophic Lateral Sclerosis

Endoplasmic reticulum (ER) stress is an important pathway to cell death in amyotrophic lateral sclerosis (ALS). We previously demonstrated that ER stress is linked to neurotoxicity associated with formation of inclusions of mutant Cu,Zn-superoxide dismutase 1 (SOD1). Cells bearing mutant inclusions undergo mitochondrial apoptotic signalling. Here, we demonstrate that the BH3-only protein, Bim, is a direct link between ER stress and mitochondrial apoptosis. In the murine neuroblastoma cell line, Neuro2a, bearing mutant SOD1 inclusions, indicators of both ER stress and apoptosis are expressed. Bim knockdown by siRNA significantly reduced nuclear apoptotic features in these inclusion-bearing cells (but did not affect the proportion of cells overall that bear inclusions). Further, both Bax recruitment to mitochondria and cytochrome c redistribution were also decreased under Bim-depletion conditions. However, upregulation of CHOP, a marker of ER stress, was not reduced by Bim knockdown. Significantly, knockdown of CHOP by siRNA reduced the extent of apoptosis in cells bearing mutant SOD1 inclusions. These sequential links between ER stress, CHOP upregulation, and Bim activation of mitochondrial apoptotic signalling indicate a clear pathway to cell death mediated by mutant SOD1.