Bradley J. Turner

Transgenics, toxicity and therapeutics in rodent models of mutant SOD1-mediated familial ALS.

Gain-of-function mutations in the Cu,Zn-superoxide dismutase (SOD1) gene are implicated in progressive motor neuron death and paralysis in one form of inherited amyotrophic lateral sclerosis (ALS). At present, transgenic expression of 12 human SOD1 mutations driven by the endogenous promoter is disease-causative and uniformly lethal in mice and rats, despite tremendous biochemical and biophysical variation between the mutants tested. This contrasts with the subclinical motor neuron disease phenotypes of wild-type SOD1 transgenic and knockout mice. Molecular mechanisms such as glutamate-induced excitotoxicity, axonal transport blockade, mitochondrial dysfunction, neuroinflammation and apoptosis triggered by mutant SOD1 catalysed oxidative reactions and/or protein misfolding are proposed to drive ALS pathogenesis. Around 100 genetic cross-breeding experiments with transgenic mutant SOD1 mice have been performed to verify these mechanisms in vivo. Furthermore, mounting evidence from mice with cell restrictive, repressible or chimeric expression of mutant SOD1 transgenes and bone marrow transplants supports non-neuronal origins of neuroprotection in ALS. Transgenic mutant SOD1 rodents have also provided the benchmark preclinical tool for evaluation of over 150 potential therapeutic anti-oxidant, anti-aggregation, anti-glutamatergic, anti-inflammatory, anti-apoptotic and neurotrophic pharmacological agents. Recent promising findings from gene and antisense therapies, cell replacement and combinatorial drug approaches in transgenic mutant SOD1 rodents are also emerging, but await successful translation in patients. This review summarises the wealth of known genetic and therapeutic modifiers in rodent models with SOD1 mutations and discusses these in the wider context of ALS pathoetiology and treatment.

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.

Intercellular propagated misfolding of wild-type Cu/Zn superoxide dismutase occurs via exosome-dependent and -independent mechanisms.

Significance Amyotrophic lateral sclerosis (ALS), an incurable motor neuron disease, is associated with mutation and misfolding of the Cu/Zn superoxide dismutase (SOD1) protein. Prior studies found that mutant misfolded SOD1 can convert wild-type (WT) SOD1 to a misfolded form inside living cells in a prion-like fashion. We now report that misfolded WT SOD1 can be transmitted from cell to cell, and that propagated protein misfolding can be perpetuated. Misfolded SOD1 transmission between cells can be mediated through release and uptake of protein aggregates or via small membrane-bounded transport vesicles called exosomes. These mechanisms may help explain why sporadic ALS, without a known genetic cause, can spread systematically from region to region in a progressive manner. Amyotrophic lateral sclerosis (ALS) is predominantly sporadic, but associated with heritable genetic mutations in 5–10% of cases, including those in Cu/Zn superoxide dismutase (SOD1). We previously showed that misfolding of SOD1 can be transmitted to endogenous human wild-type SOD1 (HuWtSOD1) in an intracellular compartment. Using NSC-34 motor neuron-like cells, we now demonstrate that misfolded mutant and HuWtSOD1 can traverse between cells via two nonexclusive mechanisms: protein aggregates released from dying cells and taken up by macropinocytosis, and exosomes secreted from living cells. Furthermore, once HuWtSOD1 propagation has been established, misfolding of HuWtSOD1 can be efficiently and repeatedly propagated between HEK293 cell cultures via conditioned media over multiple passages, and to cultured mouse primary spinal cord cells transgenically expressing HuWtSOD1, but not to cells derived from nontransgenic littermates. Conditioned media transmission of HuWtSOD1 misfolding in HEK293 cells is blocked by HuWtSOD1 siRNA knockdown, consistent with human SOD1 being a substrate for conversion, and attenuated by ultracentrifugation or incubation with SOD1 misfolding-specific antibodies, indicating a relatively massive transmission particle which possesses antibody-accessible SOD1. Finally, misfolded and protease-sensitive HuWtSOD1 comprises up to 4% of total SOD1 in spinal cords of patients with sporadic ALS (SALS). Propagation of HuWtSOD1 misfolding, and its subsequent cell-to-cell transmission, is thus a candidate process for the molecular pathogenesis of SALS, which may provide novel treatment and biomarker targets for this devastating disease.

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.

Antisense peptide nucleic acid-mediated knockdown of the p75 neurotrophin receptor delays motor neuron disease in mutant SOD1 transgenic mice.

Re‐expression of the death‐signalling p75 neurotrophin receptor (p75NTR) is associated with injury and neurodegeneration in the adult nervous system. The induction of p75NTR expression in mature degenerating spinal motor neurons of humans and transgenic mice with amyotrophic lateral sclerosis (ALS) suggests a role of p75NTR in the progression of motor neuron disease (MND). In this study, we designed, synthesized and evaluated novel antisense peptide nucleic acid (PNA) constructs targeting p75NTR as a potential gene knockdown therapeutic strategy for ALS. An 11‐mer antisense PNA directed at the initiation codon, but not downstream gene sequences, dose‐dependently inhibited p75NTR expression and death‐signalling by nerve growth factor (NGF) in Schwann cell cultures. Antisense phosphorothioate oligonucleotide (PS‐ODN) sequences used for comparison failed to confer such inhibitory activity. Systemic intraperitoneal administration of this antisense PNA to mutant superoxide dismutase 1 (SOD1G93A) transgenic mice significantly delayed locomotor impairment and mortality compared with mice injected with nonsense or scrambled PNA sequences. Reductions in p75NTR expression and subsequent caspase‐3 activation in spinal cords were consistent with increased survival in antisense PNA‐treated mice. The uptake of fluorescent‐labelled antisense PNA in the nervous system of transgenic mice was also confirmed. This study suggests that p75NTR may be a promising antisense target in the treatment of ALS.

The bone morphogenetic protein axis is a positive regulator of skeletal muscle mass

The BMP signaling pathway promotes muscle growth and inhibits muscle wasting via SMAD1/5-dependent signaling.

Survival motor neuron deficiency enhances progression in an amyotrophic lateral sclerosis mouse model.

Mutations in the ubiquitously expressed survival motor neuron 1 (SMN1) and superoxide dismutase 1 (SOD1) genes are selectively lethal to motor neurons in spinal muscular atrophy (SMA) and familial amyotrophic lateral sclerosis (ALS), respectively. Genetic association studies provide compelling evidence that SMN1 and SMN2 genotypes encoding lower SMN protein levels are implicated in sporadic ALS, suggesting that SMN expression is a potential determinant of ALS severity. We therefore sought genetic evidence of SMN involvement in ALS by generating transgenic mutant SOD1 mice on an Smn deficient background. Partial genetic disruption of Smn significantly worsened motor performance and survival in transgenic SOD1(G93A) mice. Furthermore, ALS-linked mutant SOD1 expression severely reduced SMN protein levels, but not transcript, in neuronal culture and mouse models from early presymptomatic disease. SMN protein depletion was linked to the nuclear compartment and a physical interaction between SMN and mutant SOD1 was confirmed in mouse spinal cord. Treatment with the environmental toxin paraquat also depleted SMN protein, implicating oxidative stress in the mechanism underlying SMN deficiency in familial ALS and potentially sporadic disease. In contrast, transgenic SOD1(WT) overexpression in SMA type I mice was incapable of modulating SMN protein levels or disease progression. These data establish that SMN deficiency accelerates phenotypic severity in transgenic familial ALS mice, consistent with an enhancing genetic modifier role. We therefore propose that SMN replacement and upregulation strategies considered for SMA therapy may have protective potential for ALS.

Diacetylbis(N(4)-methylthiosemicarbazonato) Copper(II) (CuII(atsm)) Protects against Peroxynitrite-induced Nitrosative Damage and Prolongs Survival in Amyotrophic Lateral Sclerosis Mouse Model

Background: CuII(atsm) [(diacetylbis(N(4)-methylthiosemicarbazonato) copper(II)] was orally administrated to transgenic SOD1G93A mice. Results: Treatment significantly prolonged lifespan with preservation of motor neurons. Reduced protein oxidation, attenuated astrocyte, and microglial activation also resulted from treatment. Conclusion: CuII(atsm) is neuroprotective in this model even when treatment begins after the onset of disease symptoms. Significance: The drug has therapeutic potential for amyotrophic lateral sclerosis. Amyotrophic lateral sclerosis (ALS) is a progressive paralyzing disease characterized by tissue oxidative damage and motor neuron degeneration. This study investigated the in vivo effect of diacetylbis(N(4)-methylthiosemicarbazonato) copper(II) (CuII(atsm)), which is an orally bioavailable, blood-brain barrier-permeable complex. In vitro the compound inhibits the action of peroxynitrite on Cu,Zn-superoxide dismutase (SOD1) and subsequent nitration of cellular proteins. Oral treatment of transgenic SOD1G93A mice with CuII(atsm) at presymptomatic and symptomatic ages was performed. The mice were examined for improvement in lifespan and motor function, as well as histological and biochemical changes to key disease markers. Systemic treatment of SOD1G93A mice significantly delayed onset of paralysis and prolonged lifespan, even when administered to symptomatic animals. Consistent with the properties of this compound, treated mice had reduced protein nitration and carbonylation, as well as increased antioxidant activity in spinal cord. Treatment also significantly preserved motor neurons and attenuated astrocyte and microglial activation in mice. Furthermore, CuII(atsm) prevented the accumulation of abnormally phosphorylated and fragmented TAR DNA-binding protein-43 (TDP-43) in spinal cord, a protein pivotal to the development of ALS. CuII(atsm) therefore represents a potential new class of neuroprotective agents targeting multiple major disease pathways of motor neurons with therapeutic potential for 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.

ER stress and UPR in familial amyotrophic lateral sclerosis.

The primary mechanism by which mutations in Cu, Zn-superoxide dismutase (SOD1) contribute to progressive motor neuron loss in familial amyotrophic lateral sclerosis (FALS) remains unknown. Misfolded protein aggregates, ubiquitin-proteasome system impairment and neuronal apoptosis mediated by death receptor or mitochondrial-dependent pathways are implicated in mutant SOD1-induced toxicity. Recent evidence from cellular and transgenic rodent models of FALS proposes activation of a third apoptotic pathway linked to sustained endoplasmic reticulum (ER) stress. Here, we review the emerging role of ER stress and the unfolded protein response (UPR) in the pathogenesis of mutant SOD1-linked FALS. The UPR observed in FALS rodents is described which encompasses induction of key ER-resident chaperones during presymptomatic disease, leading to activation of stress transducers and pro-apoptotic molecules by late stage disease. Importantly, mutant SOD1 co-aggregates with UPR components and recruits to the ER, suggesting a direct adverse effect on ER function. By contrast, the opposing neuroprotective effects of wild-type SOD1 overexpression on UPR signalling are also highlighted. In addition, the potential impact of neuronal Golgi apparatus (GA) fragmentation and subsequent disturbances in intracellular protein trafficking on motor neuron survival in FALS is also discussed. We propose that ER stress and UPR may be coupled to GA dysfunction in mutant SOD1-mediated toxicity, promoting ER-initiated cell death signalling in FALS.