Advances in molecular pathology, diagnosis, and treatment of amyotrophic lateral sclerosis.

Although the past two decades have produced exciting discoveries in the genetics and pathology of amyotrophic lateral sclerosis (ALS), progress in developing an effective therapy remains slow. This review summarizes the critical discoveries and outlines the advances in disease characterization, diagnosis, imaging, and biomarkers, along with the current status of approaches to ALS care and treatment. Additional knowledge of the factors driving disease progression and heterogeneity will hopefully soon transform the care for patients with ALS into an individualized, multi-prong approach able to prevent disease progression sufficiently to allow for a dignified life with limited disability.

Targeting TNFα produced by astrocytes expressing amyotrophic lateral sclerosis-linked mutant fused in sarcoma prevents neurodegeneration and motor dysfunction in mice.

Genetic mutations that cause amyotrophic lateral sclerosis (ALS), a progressively lethal motor neuron disease, are commonly found in ubiquitously expressed genes. In addition to direct defects within motor neurons, growing evidence suggests that dysfunction of non-neuronal cells is also an important driver of disease. Previously, we demonstrated that mutations in DNA/RNA binding protein fused in sarcoma (FUS) induce neurotoxic phenotypes in astrocytes in vitro, via activation of the NF-κB pathway and release of pro-inflammatory cytokine TNFα. Here, we developed an intraspinal cord injection model to test whether astrocyte-specific expression of ALS-causative FUSR521G variant (mtFUS) causes neuronal damage in vivo. We show that restricted expression of mtFUS in astrocytes is sufficient to induce death of spinal motor neurons leading to motor deficits through upregulation of TNFα. We further demonstrate that TNFα is a key toxic molecule as expression of mtFUS in TNFα knockout animals does not induce pathogenic changes. Accordingly, in mtFUS-transduced animals, administration of TNFα neutralizing antibodies prevents neurodegeneration and motor dysfunction. Together, these studies strengthen evidence that astrocytes contribute to disease in ALS and establish, for the first time, that FUS-ALS astrocytes induce pathogenic changes to motor neurons in vivo. Our work identifies TNFα as the critical driver of mtFUS-astrocytic toxicity and demonstrates therapeutic success of targeting TNFα to attenuate motor neuron dysfunction and death. Ultimately, through defining and subsequently targeting this toxic mechanism, we provide a viable FUS-ALS specific therapeutic strategy, which may also be applicable to sporadic ALS where FUS activity and cellular localization are frequently perturbed.

Increased synthesis of pro-inflammatory cytokines in C9ORF72 patients.

C9ORF72 hexanucleotide expansion is the most common genetic cause of familial amyotrophic lateral sclerosis (ALS)/fronto-temporal dementia (FTD) disease spectrum. Even though three major mechanisms of disease pathogenesis have been proposed, we lack detailed understanding of the factors that influence disease onset and progression. We sought to characterize cerebrospinal fluid and sera of C9ORF72 patients via a multiplex assay of 41 chemokines and cytokines in comparison to neurological controls and sporadic ALS patients. We found an increase in synthesis of pro-inflammatory chemokines and cytokines in disease samples and particularly in C9ORF72 patients in comparison to controls. We provide evidence that a CSF pro-inflammatory signature is a feature of C9ORF72-mediated disease.

Overriding FUS autoregulation in mice triggers gain-of-toxic dysfunctions in RNA metabolism and autophagy-lysosome axis.

Mutations in coding and non-coding regions of FUS cause amyotrophic lateral sclerosis (ALS). The latter mutations may exert toxicity by increasing FUS accumulation. We show here that broad expression within the nervous system of wild-type or either of two ALS-linked mutants of human FUS in mice produces progressive motor phenotypes accompanied by characteristic ALS-like pathology. FUS levels are autoregulated by a mechanism in which human FUS downregulates endogenous FUS at mRNA and protein levels. Increasing wild-type human FUS expression achieved by saturating this autoregulatory mechanism produces a rapidly progressive phenotype and dose-dependent lethality. Transcriptome analysis reveals mis-regulation of genes that are largely not observed upon FUS reduction. Likely mechanisms for FUS neurotoxicity include autophagy inhibition and defective RNA metabolism. Thus, our results reveal that overriding FUS autoregulation will trigger gain-of-function toxicity via altered autophagy-lysosome pathway and RNA metabolism function, highlighting a role for protein and RNA dyshomeostasis in FUS-mediated toxicity.

Motoneuron Disease: Basic Science.

ALS is a relentless neurodegenerative disease in which motor neurons are the susceptible neuronal population. Their death results in progressive paresis of voluntary and respiratory muscles. The unprecedented rate of discoveries over the last two decades have broadened our knowledge of genetic causes and helped delineate molecular pathways. Here we critically review ALS epidemiology, genetics, pathogenic mechanisms, available animal models, and iPS cell technologies with a focus on their translational therapeutic potential. Despite limited clinical success in treatments to date, the new discoveries detailed here offer new models for uncovering disease mechanisms as well as novel strategies for intervention.

Motoneuron Disease: Clinical.

ALS is a neurodegenerative disease in which the primary symptoms result in progressive neuromuscular weakness. Recent studies have highlighted that there is significant heterogeneity with regard to anatomical and temporal disease progression. Importantly, more recent advances in genetics have revealed new causative genes to the disease. New efforts have focused on the development of biomarkers that could aid in diagnosis, prognosis, and serve as pharmacodynamics markers. Although traditional pharmaceuticals continue to undergo trials for ALS, new therapeutic strategies including stem cell transplantation studies, gene therapies, and antisense therapies targeting some of the familial forms of ALS are gaining momentum.

Mitochondria-associated membrane collapse is a common pathomechanism in SIGMAR1- and SOD1-linked ALS.

A homozygous mutation in the gene for sigma 1 receptor (Sig1R) is a cause of inherited juvenile amyotrophic lateral sclerosis (ALS16). Sig1R localizes to the mitochondria-associated membrane (MAM), which is an interface of mitochondria and endoplasmic reticulum. However, the role of the MAM in ALS is not fully elucidated. Here, we identified a homozygous p.L95fs mutation of Sig1R as a novel cause of ALS16. ALS-linked Sig1R variants were unstable and incapable of binding to inositol 1,4,5-triphosphate receptor type 3 (IP3R3). The onset of mutant Cu/Zn superoxide dismutase (SOD1)-mediated ALS disease in mice was accelerated when Sig1R was deficient. Moreover, either deficiency of Sig1R or accumulation of mutant SOD1 induced MAM disruption, resulting in mislocalization of IP3R3 from the MAM, calpain activation, and mitochondrial dysfunction. Our findings indicate that a loss of Sig1R function is causative for ALS16, and collapse of the MAM is a common pathomechanism in both Sig1R- and SOD1-linked ALS Furthermore, our discovery of the selective enrichment of IP3R3 in motor neurons suggests that integrity of the MAM is crucial for the selective vulnerability in ALS.

Therapeutic AAV9-mediated suppression of mutant SOD1 slows disease progression and extends survival in models of inherited ALS.

Mutations in superoxide dismutase 1 (SOD1) are linked to familial amyotrophic lateral sclerosis (ALS) resulting in progressive motor neuron death through one or more acquired toxicities. Involvement of wild-type SOD1 has been linked to sporadic ALS, as misfolded SOD1 has been reported in affected tissues of sporadic patients and toxicity of astrocytes derived from sporadic ALS patients to motor neurons has been reported to be reduced by lowering the synthesis of SOD1. We now report slowed disease onset and progression in two mouse models following therapeutic delivery using a single peripheral injection of an adeno-associated virus serotype 9 (AAV9) encoding an shRNA to reduce the synthesis of ALS-causing human SOD1 mutants. Delivery to young mice that develop aggressive, fatal paralysis extended survival by delaying both disease onset and slowing progression. In a later-onset model, AAV9 delivery after onset markedly slowed disease progression and significantly extended survival. Moreover, AAV9 delivered intrathecally to nonhuman primates is demonstrated to yield robust SOD1 suppression in motor neurons and glia throughout the spinal cord and therefore, setting the stage for AAV9-mediated therapy in human clinical trials.

Misfolded mutant SOD1 directly inhibits VDAC1 conductance in a mouse model of inherited ALS.

Mutations in superoxide dismutase (SOD1) cause amyotrophic lateral sclerosis (ALS), a neurodegenerative disease characterized by loss of motor neurons. With conformation-specific antibodies, we now demonstrate that misfolded mutant SOD1 binds directly to the voltage-dependent anion channel (VDAC1), an integral membrane protein imbedded in the outer mitochondrial membrane. This interaction is found on isolated spinal cord mitochondria and can be reconstituted with purified components in vitro. ADP passage through the outer membrane is diminished in spinal mitochondria from mutant SOD1-expressing ALS rats. Direct binding of mutant SOD1 to VDAC1 inhibits conductance of individual channels when reconstituted in a lipid bilayer. Reduction of VDAC1 activity with targeted gene disruption is shown to diminish survival by accelerating onset of fatal paralysis in mice expressing the ALS-causing mutation SOD1(G37R). Taken together, our results establish a direct link between misfolded mutant SOD1 and mitochondrial dysfunction in this form of inherited ALS.

Non-cell autonomous toxicity in neurodegenerative disorders: ALS and beyond.

Selective degeneration and death of one or more classes of neurons is the defining feature of human neurodegenerative disease. Although traditionally viewed as diseases mainly affecting the most vulnerable neurons, in most instances of inherited disease the causative genes are widely-usually ubiquitously-expressed. Focusing on amyotrophic lateral sclerosis (ALS), especially disease caused by dominant mutations in Cu/Zn superoxide dismutase (SOD1), we review here the evidence that it is the convergence of damage developed within multiple cell types, including within neighboring nonneuronal supporting cells, which is crucial to neuronal dysfunction. Damage to a specific set of key partner cells as well as to vulnerable neurons may account for the selective susceptibility of neuronal subtypes in many human neurodegenerative diseases, including Huntington's disease (HD), Parkinson's disease (PD), prion disease, the spinal cerebellar ataxias (SCAs), and Alzheimer's disease (AD).

Activated protein C therapy slows ALS-like disease in mice by transcriptionally inhibiting SOD1 in motor neurons and microglia cells.

Activated protein C (APC) is a signaling protease with anticoagulant activity. Here, we have used mice expressing a mutation in superoxide dismutase-1 (SOD1) that is linked to amyotrophic lateral sclerosis (ALS) to show that administration of APC or APC analogs with reduced anticoagulant activity after disease onset slows disease progression and extends survival. A proteolytically inactive form of APC with reduced anticoagulant activity provided no benefit. APC crossed the blood-spinal cord barrier in mice via endothelial protein C receptor. When administered after disease onset, APC eliminated leakage of hemoglobin-derived products across the blood-spinal cord barrier and delayed microglial activation. In microvessels, motor neurons, and microglial cells from SOD1-mutant mice and in cultured neuronal cells, APC transcriptionally downregulated SOD1. Inhibition of SOD1 synthesis in neuronal cells by APC required protease-activated receptor-1 (PAR1) and PAR3, which inhibited nuclear transport of the Sp1 transcription factor. Diminished mutant SOD1 synthesis by selective gene excision within endothelial cells did not alter disease progression, which suggests that diminished mutant SOD1 synthesis in other cells, including motor neurons and microglia, caused the APC-mediated slowing of disease. The delayed disease progression in mice after APC administration suggests that this approach may be of benefit to patients with familial, and possibly sporadic, ALS.

Messenger RNA oxidation occurs early in disease pathogenesis and promotes motor neuron degeneration in ALS.

BACKGROUND: Accumulating evidence indicates that RNA oxidation is involved in a wide variety of neurological diseases and may be associated with neuronal deterioration during the process of neurodegeneration. However, previous studies were done in postmortem tissues or cultured neurons. Here, we used transgenic mice to demonstrate the role of RNA oxidation in the process of neurodegeneration. METHODOLOGY/PRINCIPAL FINDINGS: We demonstrated that messenger RNA (mRNA) oxidation is a common feature in amyotrophic lateral sclerosis (ALS) patients as well as in many different transgenic mice expressing familial ALS-linked mutant copper-zinc superoxide dismutase (SOD1). In mutant SOD1 mice, increased mRNA oxidation primarily occurs in the motor neurons and oligodendrocytes of the spinal cord at an early, pre-symptomatic stage. Identification of oxidized mRNA species revealed that some species are more vulnerable to oxidative damage, and importantly, many oxidized mRNA species have been implicated in the pathogenesis of ALS. Oxidative modification of mRNA causes reduced protein expression. Reduced mRNA oxidation by vitamin E restores protein expression and partially protects motor neurons. CONCLUSION/SIGNIFICANCE: These findings suggest that mRNA oxidation is an early event associated with motor neuron deterioration in ALS, and may be also a common early event preceding neuron degeneration in other neurological diseases.

Mutant dynein (Loa) triggers proprioceptive axon loss that extends survival only in the SOD1 ALS model with highest motor neuron death.

Dominant mutations in cytoplasmic dynein (Loa or Cra) have been reported to provoke selective, age-dependent killing of motor neurons, while paradoxically slowing degeneration and death of motor neurons in one mouse model of an inherited form of ALS. Examination of Loa animals reveals no degeneration of large caliber alpha-motor neurons beyond an age-dependent loss (initiating only after 18 months) that was comparable in Loa and wild-type littermates. Absence of Loa-mediated alpha-motor neuron loss contrasted with dramatic, sustained, mutant dynein-mediated postnatal loss of lumbar proprioceptive sensory axons, accompanied by decreased excitatory glutamatergic inputs to motor neurons. In mouse models of inherited ALS caused by mutations in superoxide dismutase (SOD1), mutant dynein modestly prolonged survival in the one mouse model with the most extensive motor neuron loss (SOD(G93A)) while showing marginal (SOD(G85R)) or no (SOD(G37R)) benefit in models with higher numbers of surviving motor neurons at end stage. These findings support a noncell autonomous, excitotoxic contribution from proprioceptive sensory neurons that modestly accelerates disease onset in inherited ALS.

Increased ER stress during motor neuron degeneration in a transgenic mouse model of amyotrophic lateral sclerosis.

The endoplasmic reticulum (ER), which plays important roles in apoptosis, is susceptible to oxidative stress. ER stress is also thought to be involved in the pathogenesis of neurodegenerative diseases. In this study, we investigated whether ER stress is involved in the pathogenesis of amyotrophic lateral sclerosis (ALS) using the anterior part of the lumbar spinal cord of transgenic mice carrying a mutation (G93A) in the superoxide dismutase 1 (SOD1) gene. Western blot and immunohistochemical analyses demonstrated that the expressions of p-PERK and p-eIF2alpha were increased in the microsome fraction (P3) of the lumbar spinal cord at the pre-symptomatic age of 12 weeks (12W), while the expression of activated caspase-12 was increased in the cytoplasmic fraction (S3) of the lumbar spinal cord at both the pre-symptomatic age of 12W and the late symptomatic age of 20W. In contrast, GRP78 did not show any increases in the microsome fraction (P3) of the lumbar spinal cord at either the pre-symptomatic or symptomatic ages. Thus, the present results strongly suggest that the balance between anti- and pro-apoptotic proteins related to ER stress is impaired from the pre-symptomatic stage in this ALS mouse model, and that this imbalance may be related to the pathogenesis of motor neuron degeneration in ALS.

Therapeutic benefit of intrathecal injection of insulin-like growth factor-1 in a mouse model of Amyotrophic Lateral Sclerosis.

Insulin-like growth factor (IGF)-1 has been shown to have a protective effect on motor neurons both in vitro and in vivo, but has limited efficacy in patients with amyotrophic lateral sclerosis (ALS) when given subcutaneously. To examine the possible effectiveness of IGF-1 in a mouse model of familial ALS, transgenic mice expressing human Cu/Zn superoxide dismutase (SOD1) with a G93A mutation were treated by continuous IGF-1 delivery into the intrathecal space of the lumbar spinal cord. We found that the intrathecal administration of IGF-1 improved motor performance, delayed the onset of clinical disease, and extended survival in the G93A transgenic mice. Furthermore, it increased the expression of phosphorylated Akt and ERK in spinal motor neurons, and partially prevented motor neuron loss in these mice. Taken together, the results suggest that direct administration of IGF-1 into the intrathecal space may have a therapeutic benefit for ALS.

Hypoxic induction of vascular endothelial growth factor is selectively impaired in mice carrying the mutant SOD1 gene.

Localization and hypoxic induction of vascular endothelial growth factor (VEGF) was examined in the spinal cord of transgenic mice carrying a mutation in the superoxide dismutase 1 gene. Immunohistochemical and immunofluorescent study demonstrated that VEGF is mainly expressed in motor neurons before and after hypoxia. Baseline expression of VEGF was higher in transgenic (Tg) mice than in wild-type (Wt) littermates. However, VEGF was hardly induced after hypoxia in Tg mice, whereas Wt mice showed an approximate nine-fold increase. Impaired VEGF induction was evident in Tg mice at 12 weeks of age, when they were still presymptomatic. In contrast, baseline and hypoxic expression of brain-derived neurotrophic factor and glial cell line-derived neurotrophic factor did not differ between Tg and Wt mice. Thus, the present study demonstrates that hypoxic induction of VEGF in Tg mice is selectively impaired from a very early stage, suggesting profound involvement in the pathogenesis of motor neuron degeneration in this animal model of amyotrophic lateral sclerosis.

Sustained induction of survival p-AKT and p-ERK signals after transient hypoxia in mice spinal cord with G93A mutant human SOD1 protein.

Expression of survival p-AKT and p-ERK signals was examined by immunohistochemistry and Western blotting in the lumbar spinal cord of 12-week-old presymptomatic mice with human mutant G93A SOD1 gene (transgenic, Tg) and their wild-type (Wt) littermates during normoxia, and 0 and 6 h after 2 h of 9% hypoxia. During normoxia, a stronger p-AKT signal was detected in the nucleus of the motor neurons of Tg animals. At 0 h of recovery from 2 h of hypoxia, both p-AKT and p-ERK signals were induced at a slightly lower level in Tg (1.1-1.2-fold) compared to those of Wt (1.2-1.5-fold) animals. At 6 h of recovery, both p-AKT and p-ERK signals were sustained in the lumbar spinal motor neurons of Tg animals, while those in Wt animals quickly returned to baseline level. As a control, at 6 h of recovery, the hippocampus of Tg animals showed significantly sustained p-AKT levels, but not p-ERK levels, compared to Wt. The current results suggest that the presence of mutant SOD1 alters survival p-AKT and p-ERK signals, possibly to compensate for the acquired gain-of-function of the mutant protein.

Age-related changes in peroxisomal membrane protein 70 and superoxide dismutase 1 in transgenic G93A mice.

Peroxisomal membrane protein 70 (PMP70) and Cu/Zn superoxide dismutase (SOD1) were examined in the spinal cords of transgenic (Tg) mice expressing a human mutant SOD1 protein (G93A) and their age-matched controls at 8, 20 and 32 weeks by immunohistochemistry. At pre-symptomatic 20 weeks and symptomatic 32 weeks, PMP70 was reduced in the cytoplasm of motor neurons in Tg animals and increased in glial cells in anterior horn at late age. SOD1 showed a progressive increase of dot-like deposits in the neuropil of anterior horn of Tg mice, and a late decrease of signal intensity in the white matter and motor neurons at 32 weeks. It is conceivable that reduction of PMP70 might underlie decrease in peroxisomal functions and increase in oxidative stress that is well documented in this animal model.

Change in superoxide dismutase 1 protein localization towards mitochondria: an immunohistochemical study in transgenic G93A mice.

Localization of superoxide dismutase1 (SOD1) and mitochondrial glucose-regulated protein 75 (Grp75), were examined in the spinal cords of transgenic (Tg) mice expressing human mutant SOD1 protein (G93A) and wild-type (Wt) controls at 8, 20 and 32 weeks. SOD1 showed a progressive increase of dot-like deposits in the neuropil of anterior horn of Tg mice, and a late decrease of signal intensity in the white matter and motor neurons. Colocalization of Grp75 and SOD1 signals was demonstrated in Wt and presymptomatic Tg animals, while it was lost in Tg mice at a symptomatic age. The present results suggest that loss of SOD1 protein from mitochondria could contribute to motor neuron damage.