Systematically perturbed folding patterns of amyotrophic lateral sclerosis (ALS)-associated SOD1 mutants

Amyotrophic lateral sclerosis is a neurodegenerative syndrome associated with 114 mutations in the gene encoding the cytosolic homodimeric enzyme Cu/Zn superoxide dismutase (SOD). In this article, we report that amyotrophic lateral sclerosis-associated SOD mutations with distinctly different disease progression can be rationalized in terms of their folding patterns. The mutations are found to perturb the protein in multiple ways; they destabilize the precursor monomers (class 1), weaken the dimer interface (class 2), or both at the same time (class 1 + 2). A shared feature of the mutational perturbations is a shift of the folding equilibrium toward poorly structured SOD monomers. We observed a link, coupled to the altered folding patterns, between protein stability, net charge, and survival time for the patients carrying the mutations.     

Nonnative SOD1 trimer is toxic to motor neurons in a model of amyotrophic lateral sclerosis

Since the linking of mutations in the Cu,Zn superoxide dismutase gene (sod1) to amyotrophic lateral sclerosis (ALS) in 1993, researchers have sought the connection between SOD1 and motor neuron death. Disease-linked mutations tend to destabilize the native dimeric structure of SOD1, and plaques containing misfolded and aggregated SOD1 have been found in the motor neurons of patients with ALS. Despite advances in understanding of ALS disease progression and SOD1 folding and stability, cytotoxic species and mechanisms remain unknown, greatly impeding the search for and design of therapeutic interventions. Here, we definitively link cytotoxicity associated with SOD1 aggregation in ALS to a nonnative trimeric SOD1 species. We develop methodology for the incorporation of low-resolution experimental data into simulations toward the structural modeling of metastable, multidomain aggregation intermediates. We apply this methodology to derive the structure of a SOD1 trimer, which we validate in vitro and in hybridized motor neurons. We show that SOD1 mutants designed to promote trimerization increase cell death. Further, we demonstrate that the cytotoxicity of the designed mutants correlates with trimer stability, providing a direct link between the presence of misfolded oligomers and neuron death. Identification of cytotoxic species is the first and critical step in elucidating the molecular etiology of ALS, and the ability to manipulate formation of these species will provide an avenue for the development of future therapeutic strategies.Protein misfolding and aggregation are linked to cell death and disease progression in neurodegenerative diseases, such as Alzheimer’s disease, Parkinson’s disease, and amyotrophic lateral sclerosis (ALS). In these diseases and others, the formation of amyloid plaques, often observed post mortem, has long been thought to play a role in neurodegeneration, but toxicity has never been confirmed (1–3). Recent research has shown that small, soluble oligomers, rather than insoluble amyloids, are likely to be the cytotoxic species causing neurodegeneration (4–14). These small, soluble oligomers undergo aberrant interactions with cell machinery and activate cell death pathways, but their exact stoichiometry is not known and their properties have yet to be characterized. Recently, metastable soluble Cu,Zn superoxide dismutase (SOD1) oligomers have been identified that contain an epitope associated with disease-linked species of SOD1, mutants of which are implicated in a subset of ALS (15–18). Size exclusion chromatography (SEC) of these oligomers revealed a size range of two to four monomers, consistent with previous findings of potentially cytotoxic SOD1 oligomers (19–21).Knowledge of the structures of these species would not only allow for definitive testing of their toxicity but could potentially lead to an understanding of disease mechanism and therapeutic strategies against diseases for which no cure or effective treatment exists (22). By their nature, however, these oligomers are only metastable, and therefore difficult to isolate. More importantly, this instability, and consequent transient nature, combined with the proposed size range of the implicated oligomers, means that a high-resolution structure cannot be achieved using traditional methods. Although atomic structures have been explored for folding intermediates of small protein domains (23) or stable oligomeric structures of disease-relevant peptide sequences (24), structural characterization of metastable aggregation products of full-length disease-linked proteins has remained elusive.Here, we combined experimental and computational approaches to produce a molecular model of a toxic metastable protein oligomer. Using state-of-the-art high-speed atomic force microscopy (HS-AFM), we established the trimeric stoichiometry and highly dynamic nature of the isolated oligomers. We applied limited proteolysis to the isolated SOD1 trimers, allowing us to determine the solvent accessibility and rigidity of the structure. Using this information as constraints in molecular simulations, we evolved a structural model of the SOD1 trimer that is consistent with our experimental data. Using our model, we determined residues critical to trimer formation, and we used rational mutagenesis to verify our model and manipulate trimer formation in vitro and in living cells. We find that mutations that stabilize the trimeric form of SOD1 result in increased cell death over WT SOD1 or SOD1 mutants that inhibit trimer formation, demonstrating the neurotoxicity of the SOD1 trimer and its potential relevance to ALS etiology.     

SOD1基因与运动神经元病相关性的研究进展

肌萎缩侧索硬化(ALS)是病因不清的变性病,SOD1突变基因与其发生有相关性.SOD1基因突变如何造成运动神经元死亡尚不完全清楚,目前的研究认为SOD1基因造成运动神经元死亡不是由于基因产物功能丧失,而是通过基因产物直接毒性作用或形成聚集体影响细胞功能从而造成运动神经元死亡.虽然ALS中运动神经元死亡具有选择性,然而有多种细胞参与了疾病的发生,本文对SOD1在ALS发病中的作用机制进行了回顾.     

Common molecular signature in SOD1 for both sporadic and familial amyotrophic lateral sclerosis

Amyotrophic lateral sclerosis (ALS) is a devastating motor neuron degenerative disease whose etiology and pathogenesis remain poorly understood. Most cases of ALS ( approximately 90%) are sporadic (SALS), occurring in the absence of genetic associations. Approximately 20% of familial ALS (FALS) cases are due to known mutations in the copper, zinc superoxide dismutase (SOD1) gene. Molecular evidence for a common pathogenesis of SALS and FALS has remained elusive. Here we use covalent chemical modification to reveal an attribute of spinal cord SOD1 common to both SOD1-linked FALS and SALS, but not present in normal or disease-affected tissues from other neurodegenerative diseases, including Alzheimer's, Parkinson's, and Huntington's diseases and spinal muscular atrophy, a non-ALS motor neuron disease. Biotinylation reveals a 32-kDa, covalently cross-linked SOD1-containing protein species produced not only in FALS caused by SOD1 mutation, but also in SALS. These studies use chemical modification as a novel tool for the detection of a disease-associated biomarker. Our results identify a shared molecular event involving a known target gene and suggest a common step in the pathogenesis between SALS and FALS.     

Genome-wide association analysis reveals a SOD1 mutation in canine degenerative myelopathy that resembles amyotrophic lateral sclerosis

Canine degenerative myelopathy (DM) is a fatal neurodegenerative disease prevalent in several dog breeds. Typically, the initial progressive upper motor neuron spastic and general proprioceptive ataxia in the pelvic limbs occurs at 8 years of age or older. If euthanasia is delayed, the clinical signs will ascend, causing flaccid tetraparesis and other lower motor neuron signs. DNA samples from 38 DM-affected Pembroke Welsh corgi cases and 17 related clinically normal controls were used for genome-wide association mapping, which produced the strongest associations with markers on CFA31 in a region containing the canine SOD1 gene. SOD1 was considered a regional candidate gene because mutations in human SOD1 can cause amyotrophic lateral sclerosis (ALS), an adult-onset fatal paralytic neurodegenerative disease with both upper and lower motor neuron involvement. The resequencing of SOD1 in normal and affected dogs revealed a G to A transition, resulting in an E40K missense mutation. Homozygosity for the A allele was associated with DM in 5 dog breeds: Pembroke Welsh corgi, Boxer, Rhodesian ridgeback, German Shepherd dog, and Chesapeake Bay retriever. Microscopic examination of spinal cords from affected dogs revealed myelin and axon loss affecting the lateral white matter and neuronal cytoplasmic inclusions that bind anti-superoxide dismutase 1 antibodies. These inclusions are similar to those seen in spinal cord sections from ALS patients with SOD1 mutations. Our findings identify canine DM to be the first recognized spontaneously occurring animal model for ALS.Amyotrophic lateral sclerosis (ALS) refers to a heterogeneous group of adult onset human diseases, in which progressive neurodegeneration affecting both the upper and lower motor neuron systems causes advancing weakness and muscle atrophy, and culminates in paralysis and death. Approximately 5 to 10% of ALS cases are familial; the rest appear to be sporadic (1–3). Mutations in SOD1 account for ≈20% of the familial ALS cases and 1 to 5% of the cases of sporadic ALS (1–4); >120 different SOD1 mutations have been identified in ALS patients (http://alsod.iop.kcl.ac.uk/Als/index.aspx). Elucidation of mechanisms underlying ALS has been hampered by a paucity of biological material from affected individuals in early stages of the disease (5). To our knowledge, there are no previous reports of spontaneously occurring animal models of ALS. Thus, ALS research has relied heavily on transgenic rodents expressing mutant human SOD1 (hSOD1^m) to produce a motor neuron disease, which recapitulates many features of ALS (5–7). In contrast, nullizygous SOD1 knockout mice develop normally (8), suggesting that the neurodegeneration in hSOD1^m mice and in ALS patients results from a toxic gain of function (1, 5–8). Although the nature of the toxin is unclear, several experiments suggest that the neurodegeneration occurs because conformational changes in the mutant superoxide dismutase 1 protein (SOD1) alter the biological activity and/or promote the formation of intracellular SOD1 aggregates (1, 4, 9, 10).Canine degenerative myelopathy (DM) has been recognized for >35 years as a spontaneously occurring, adult-onset spinal cord disorder of dogs (11). When pelvic limb hyporeflexia and nerve root involvement were observed, the disease was termed chronic degenerative radiculomyelopathy (12). Initially thought to be specific to German Shepherds, it has also been called German Shepherd dog myelopathy (13). Since these early reports, DM has been diagnosed in several other breeds. The disease is common in certain breeds including the Pembroke Welsh corgi, Boxer, Rhodesian ridgeback, and Chesapeake Bay retriever (14).With DM, there is no sex predilection. Most dogs are at least 8 years old before the onset of clinical signs (11–18). The initial clinical sign is a spastic and general proprioceptive ataxia in the pelvic limbs. At this stage of the disease, the presence of spinal reflexes indicates an upper motor neuron paresis (11). The asymmetric weakness frequently reported at disease onset progresses to paraplegia (11, 12, 14, 16, 18). Hyporeflexia of the myotatic and withdrawal reflexes occur in the latter disease stage (11, 12, 14, 16, 18). The disease duration can exceed 3 years; however, dog owners usually elect euthanasia within a year of diagnosis when their dogs become paraplegic. If the disease is allowed to progress, clinical signs will ascend to affect the thoracic limbs (11, 14, 16). Because various common acquired compressive spinal cord diseases can mimic DM by compromising the upper motor neuron and general proprioceptive pathways, a definitive diagnosis of DM can only be accomplished postmortem by the histopathologic observation of axonal and myelin degeneration, which can occur at all levels of the spinal cord (16–18) and in all spinal cord funiculi, but are consistently most severe in the dorsal portion of the lateral funiculus within the middle to caudal thoracic region (11, 13–18).     

Dyspnea as a main symptom for visiting of amyotrophic lateral sclerosis (ALS) :a case report and literature review

目的 了解肌萎缩侧索硬化症患者呼吸系统障碍的临床特点和治疗方法.方法 通过对1例以呼吸困难为首诊症状肌萎缩侧索硬化症患者的诊治经过分析,结合相关文献进行讨论.结果 患者为57岁男性,因“进行性呼吸困难2年”来院就诊.以劳力性呼吸困难和端坐呼吸为主要表现.动脉血气分析提示Ⅱ型呼吸衰竭.肺功能检查表现为限制性为主的通气功能障碍.体检和X线检查提示双侧膈肌活动减弱.多导睡眠图检查示患者重度睡眠呼吸暂停综合征.肌电图和神经专科体检示多区域的运动神经元变性表现.经神经科会诊,患者诊断为肌萎缩侧索硬化症.结论 对以呼吸困难为首诊症状的肌萎缩侧索硬化症患者快速正确诊断存在一定难度.对于不能解释的呼吸困难和呼吸衰竭患者,要考虑神经源性疾病.有呼吸困难的肌萎缩侧索硬化症患者应该进行睡眠障碍相关检查.正确使用机械通气治疗,对患者有益.     

Chronic activation in presymptomatic amyotrophic lateral sclerosis (ALS) mice of a feedback loop involving Fas, Daxx, and FasL

The reasons for the cellular specificity and slow progression of motoneuron diseases such as ALS are still poorly understood. We previously described a motoneuron-specific cell death pathway downstream of the Fas death receptor, in which synthesis of nitric oxide (NO) is an obligate step. Motoneurons from ALS model mice expressing mutant SOD1 showed increased susceptibility to exogenous NO as compared with controls. Here, we report a signaling mechanism whereby NO leads to death of mutant, but not control, motoneurons. Unexpectedly, exogenous NO triggers expression of Fas ligand (FasL) in cultured motoneurons. In mutant SOD1(G93A) and SOD1(G85R), but not in control motoneurons, this up-regulation results in activation of Fas, leading through Daxx to phosphorylation of p38 and further NO synthesis. This Fas/NO feedback amplification loop is required for motoneuron death in vitro. In vivo, mutant SOD1(G93A) and SOD1(G85R) mice show increased numbers of positive motoneurons and Daxx nuclear bodies weeks before disease onset. Moreover, FasL up-regulation is reduced in the presence of transgenic dominant-negative Daxx. We propose that chronic low-level activation of the Fas/NO feedback loop may underlie the motoneuron loss that characterizes familial ALS and may help to explain its slowly progressive nature.     

Evidence for defective energy homeostasis in amyotrophic lateral sclerosis: benefit of a high-energy diet in a transgenic mouse model

Amyotrophic lateral sclerosis (ALS) is an adult-onset neurodegenerative disease characterized by selective loss of motor neurons and progressive muscle wasting. Growing evidence indicates that mitochondrial dysfunction, not only occurring in motor neurons but also in skeletal muscle, may play a crucial role in the pathogenesis. In this regard, the life expectancy of the ALS G93A mouse line is extended by creatine, an intracellular energy shuttle that ameliorates muscle function. Moreover, a population of patients with sporadic ALS exhibits a generalized hypermetabolic state of as yet unknown origin. Altogether, these findings led us to explore whether alterations in energy homeostasis may contribute to the disease process. Here, we show important variations in a number of metabolic indicators in transgenic ALS mice, which in all shows a metabolic deficit. These alterations were accompanied early in the asymptomatic phase of the disease by reduced adipose tissue accumulation, increased energy expenditure, and concomitant skeletal muscle hypermetabolism. Compensating this energetic imbalance with a highly energetic diet extended mean survival by 20%. In conclusion, we suggest that hypermetabolism, mainly of muscular origin, may represent by itself an additional driven force involved in increasing motor neuron vulnerability.     

Strategies for stabilizing superoxide dismutase (SOD1), the protein destabilized in the most common form of familial amyotrophic lateral sclerosis

Amyotrophic lateral sclerosis (ALS) is a disorder characterized by the death of both upper and lower motor neurons and by 3- to 5-yr median survival postdiagnosis. The only US Food and Drug Administration-approved drug for the treatment of ALS, Riluzole, has at best, moderate effect on patient survival and quality of life; therefore innovative approaches are needed to combat neurodegenerative disease. Some familial forms of ALS (fALS) have been linked to mutations in the Cu/Zn superoxide dismutase (SOD1). The dominant inheritance of mutant SOD1 and lack of symptoms in knockout mice suggest a "gain of toxic function" as opposed to a loss of function. A prevailing hypothesis for the mechanism of the toxicity of fALS-SOD1 variants, or the gain of toxic function, involves dimer destabilization and dissociation as an early step in SOD1 aggregation. Therefore, stabilizing the SOD1 dimer, thus preventing aggregation, is a potential therapeutic strategy. Here, we report a strategy in which we chemically cross-link the SOD1 dimer using two adjacent cysteine residues on each respective monomer (Cys111). Stabilization, measured as an increase in melting temperature, of ～20 °C and ～45 °C was observed for two mutants, G93A and G85R, respectively. This stabilization is the largest for SOD1, and to the best of our knowledge, for any disease-related protein. In addition, chemical cross-linking conferred activity upon G85R, an otherwise inactive mutant. These results demonstrate that targeting these cysteine residues is an important new strategy for development of ALS therapies.     

Conversion to the amyotrophic lateral sclerosis phenotype is associated with intermolecular linked insoluble aggregates of SOD1 in mitochondria

Twenty percent of the familial form of amyotrophic lateral sclerosis (ALS) is caused by mutations in the Cu, Zn-superoxide dismutase gene (SOD1) through the gain of a toxic function. The nature of this toxic function of mutant SOD1 has remained largely unknown. Here we show that WT SOD1 not only hastens onset of the ALS phenotype but can also convert an unaffected phenotype to an ALS phenotype in mutant SOD1 transgenic mouse models. Further analyses of the single- and double-transgenic mice revealed that conversion of mutant SOD1 from a soluble form to an aggregated and detergent-insoluble form was associated with development of the ALS phenotype in transgenic mice. Conversion of WT SOD1 from a soluble form to an aggregated and insoluble form also correlates with exacerbation of the disease or conversion to a disease phenotype in double-transgenic mice. This conversion, observed in the mitochondrial fraction of the spinal cord, involved formation of insoluble SOD1 dimers and multimers that are crosslinked through intermolecular disulfide bonds via oxidation of cysteine residues in SOD1. Our data thus show a molecular mechanism by which SOD1, an important protein in cellular defense against free radicals, is converted to aggregated and apparently ALS-associated toxic dimers and multimers by redox processes. These findings provide evidence of direct links among oxidation, protein aggregation, mitochondrial damage, and SOD1-mediated ALS, with possible applications to the aging process and other late-onset neurodegenerative disorders. Importantly, rational therapy based on these observations can now be developed and tested.     

Phenotypic effects of familial amyotrophic lateral sclerosis mutant Sod alleles in transgenic Drosophila

A subset of patients suffering from familial amyotrophic lateral sclerosis (FALS) exhibit point mutations in the gene encoding Cu-Zn superoxide dismutase [superoxide:superoxide oxidoreductase, EC (SOD)]. The human wild-type and five FALS Sod mutant transgenes were introduced into the fruit fly, Drosophila melanogaster, in a Cu-Zn Sod null background. Sod null flies had dramatically decreased life span, glutathione and methionine content, fertility, locomotor activity, and resistance to hyperoxic stress, compared with wild-type controls. All of these phenotypic manifestations were rescued fully by a single human wild-type allele, expressing 5-10% of wild-type SOD activity. Full recovery of wild-type life span was also observed when human mutant and wild-type alleles were placed together in the fly Sod null background. The FALS Sod mutations alone caused a recessive phenotype, usually involving low or undetectable levels of SOD activity, in which: (i) full restoration of the wild-type phenotype was observed among young adults, and (ii) older adults exhibited a sudden increase in oxidative stress, accompanied by physiological impairment of abrupt onset, and followed by premature death. Thus, the minimal SOD activity associated with the FALS Sod mutations appears to determine longevity, not by chronically increasing oxidative stress, but by limiting the time in which a viable redox environment can be maintained. However, the dominant gain of function by mutant SOD, which occurs in human patients and in the transgenic mouse model of FALS, is not observed in Drosophila.     

Neuroprotective efficacy of aminopropyl carbazoles in a mouse model of amyotrophic lateral sclerosis

We previously reported the discovery of P7C3, an aminopropyl carbazole having proneurogenic and neuroprotective properties in newborn neural precursor cells of the hippocampal dentate gyrus. We have further found that chemicals having efficacy in this in vivo screening assay also protect dopaminergic neurons of the substantia nigra following exposure to the neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine, a mouse model of Parkinson disease. Here, we provide evidence that an active analog of P7C3, known as P7C3A20, protects ventral horn spinal cord motor neurons from cell death in the G93A-SOD1 mutant mouse model of amyotrophic lateral sclerosis (ALS). P7C3A20 is efficacious in this model when administered at disease onset, and protection from cell death correlates with preservation of motor function in assays of walking gait and in the accelerating rotarod test. The prototypical member of this series, P7C3, delays disease progression in G93A-SOD1 mice when administration is initiated substantially earlier than the expected time of symptom onset. Dimebon, an antihistaminergic drug with significantly weaker proneurogenic and neuroprotective efficacy than P7C3, confers no protection in this ALS model. We propose that the chemical scaffold represented by P7C3 and P7C3A20 may provide a basis for the discovery and optimization of pharmacologic agents for the treatment of ALS.Amyotrophic lateral sclerosis (ALS), also known as Lou Gehrig’s disease, is a relatively rare, adult-onset, rapidly progressive and fatal disease that involves degeneration of spinal cord motor neurons (1). This disorder causes muscle weakness and atrophy throughout the body, and patients with ALS ultimately lose all voluntary movement. The earliest parts of the body affected in ALS reflect those motor neurons that are damaged first. Regardless of the region of onset, however, muscle weakness and atrophy invariably spread to other parts of the body as the disease progresses. Although disease progression varies between individuals, most patients are eventually unable to stand or walk, get in or out of bed on their own, or use their hands and arms. Difficulty with chewing, swallowing, and breathing leads to progressive weight loss and increased risk of choking and aspiration pneumonia. Toward the end stages of disease, as the diaphragm and intercostal muscles weaken, most patients require ventilator support. Individuals with ALS most commonly die of respiratory failure or pneumonia within 2–5 y of diagnosis. There are no current treatments for ALS.Approximately 20% of inherited cases of ALS, and 3% of sporadic cases, are associated with autosomal dominant mutations in the SOD1 gene on chromosome 21 (2–4), and about 150 different mutations dispersed throughout the gene have been identified thus far (5). SOD1 encodes cytosolic Cu/Zn superoxide dismutase, an antioxidant enzyme that protects cells by converting superoxide (a toxic free radical generated through normal metabolic activity of mitochondria) to hydrogen peroxide. Unchecked, free radicals damage both mitochondrial and nuclear DNA, as well as proteins within cells. In ALS linked to mutations in SOD1, cytotoxicity of motor neurons appears to result from a gain of toxic SOD1 function, rather than from loss of dismutase activity. Although the exact molecular mechanisms underlying toxicity are unclear, mutation-induced conformational changes in SOD1 lead to misfolding and subsequent aggregation of mutant SOD1 in cell bodies and axons (6–10). Aggregate accumulation of mutant SOD1 is thought to disrupt cellular functions and precipitate neuron death by damaging mitochondria, proteasomes, protein-folding chaperones, or other proteins (10).Transgenic animal models of mutant SOD1, such as G93A-SOD1 mutant mice, are currently used for research into the pathogenic mechanisms thought to broadly underlie ALS. Mice hemizygous for the G93A-SOD1 transgene express 18 ± 2.6 copies of a form of SOD1 found in some patients with inherited ALS (a substitution of glycine to alanine at codon 93). This was the first mutant form of SOD1 to be expressed in mice and is the most widely used and well-characterized mouse model of ALS. Superoxide dismutase activity in these mice is intact, and the pathogenic effect of the mutant transgene appears to be gain of function, as is thought to occur in human patients (11). Death of motor neurons in these mice occurs in the ventral horn of the spinal cord and is associated with paralysis and muscle atrophy (12). Around 100 d of age, G93A-SOD1 mice characteristically experience the onset of paralysis in one or more limbs, due to loss of spinal cord motor neurons. Paralysis spreads rapidly throughout the body, culminating in death of 50% of the mice within 7 wk of disease onset.We have previously reported the identification of a proneurogenic, neuroprotective aminopropyl carbazole (P7C3) discovered through a target-agnostic in vivo screen of postnatal hippocampal neurogenesis (13). Prolonged administration of P7C3 to mice suffering from pathologically high levels of neuronal apoptosis in the dentate gyrus (14) safely restored hippocampal structure and function with no observable physiologic side effects (13). Furthermore, extended administration of P7C3 to aged rats impeded hippocampal cell death and preserved cognitive ability as a function of terminal aging (13).We have synthesized and characterized a variant of P7C3, known as P7C3A20, which has greater potency and proneurogenic efficacy than the parent compound. P7C3A20 differs structurally by replacement of the hydroxyl group at the chiral center of the linker with a fluorine and the addition of a methoxy group to the aniline ring. P7C3A20 also displays a more favorable toxicity profile than P7C3, with no hERG channel binding, histamine receptor binding, or toxicity to HeLa cells (13, 15, 16). We have also found that Dimebon, an antihistaminergic drug that is reported to have anti-apoptotic and mitochondrial protective properties (17, 18), displays modest efficacy in the same biologic assays used to discover and characterize P7C3 and P7C3A20. However, it does so with substantially less potency and ceiling of efficacy (CoE) (13).Armed with three related chemicals, one having very high proneurogenic activity (P7C3A20), one having intermediate activity (P7C3), and one having only modest activity (Dimebon), we initiated efficacy studies in two animal models of neurodegenerative disease. In our companion article (19), we report evidence of significant neuroprotective activity of P7C3A20 in a rodent model of Parkinson disease (PD). P7C3 exhibited intermediate activity in the PD animal model, and Dimebon showed no evidence of efficacy. The correlative activities of chemicals tested in the neurogenesis and PD assays were extended to eight additional analogs of P7C3. In every case, derivatives of P7C3 that were active in the neurogenesis assay were also active in the animal model of PD, and inactive variants were inactive in both assays (19).Here, we have used the same approach to score the activities of P7C3A20, P7C3, and Dimebon in a model of neuron death outside of the brain. To address this question, we used G93A-SOD1 mutant mice, a model of ALS characterized by spinal motor neuron death associated with decreased motor functioning. As was observed for the rodent model of PD, we hereby report robust activity of P7C3A20 in the G93A-SOD1 mouse model of ALS, intermediate activity for P7C3, and no activity for Dimebon.     

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 α-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 α-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.     

An over-oxidized form of superoxide dismutase found in sporadic amyotrophic lateral sclerosis with bulbar onset shares a toxic mechanism with mutant SOD1

Recent studies suggest that Cu/Zn superoxide dismutase (SOD1) could be pathogenic in both familial and sporadic amyotrophic lateral sclerosis (ALS) through either inheritable or nonheritable modifications. The presence of a misfolded WT SOD1 in patients with sporadic ALS, along with the recently reported evidence that reducing SOD1 levels in astrocytes derived from sporadic patients inhibits astrocyte-mediated toxicity on motor neurons, suggest that WT SOD1 may acquire toxic properties similar to familial ALS-linked mutant SOD1, perhaps through posttranslational modifications. Using patients' lymphoblasts, we show here that indeed WT SOD1 is modified posttranslationally in sporadic ALS and is iper-oxidized (i.e., above baseline oxidation levels) in a subset of patients with bulbar onset. Derivatization analysis of oxidized carbonyl compounds performed on immunoprecipitated SOD1 identified an iper-oxidized SOD1 that recapitulates mutant SOD1-like properties and damages mitochondria by forming a toxic complex with mitochondrial Bcl-2. This study conclusively demonstrates the existence of an iper-oxidized SOD1 with toxic properties in patient-derived cells and identifies a common SOD1-dependent toxicity between mutant SOD1-linked familial ALS and a subset of sporadic ALS, providing an opportunity to develop biomarkers to subclassify ALS and devise SOD1-based therapies that go beyond the small group of patients with mutant SOD1.     

Lung function in the course of amyotrophic lateral sclerosis

We report a case of a 26 year old man who was diagnosticated of ALS. This case is a graphic example of the pulmonary function evolution through the flow-volume loop. In the first study the espirometric values were in the normal range. The Static pulmonary pressures were weackle decreased. This was the only sign of respiratory muscle impairement. Its important to study close enough the pulmonary function through the flow/volume loop, maximal respiratory pressures and maximal voluntary Ventilation in order to know the empairment rate, and also to detect intercurrent procedures as bulbar involvement, that can affect the prognosis of the disease.     

Progressive aggregation despite chaperone associations of a mutant SOD1-YFP in transgenic mice that develop ALS

Recent studies suggest that superoxide dismutase 1 (SOD1)-linked amyotrophic lateral sclerosis results from destabilization and misfolding of mutant forms of this abundant cytosolic enzyme. Here, we have tracked the expression and fate of a misfolding-prone human SOD1, G85R, fused to YFP, in a line of transgenic G85R SOD1-YFP mice. These mice, but not wild-type human SOD1-YFP transgenics, developed lethal paralyzing motor symptoms at 9 months. In situ RNA hybridization of spinal cords revealed predominant expression in motor neurons in spinal cord gray matter in all transgenic animals. Concordantly, G85R SOD-YFP was diffusely fluorescent in motor neurons of animals at 1 and 6 months of age, but at the time of symptoms, punctate aggregates were observed in cell bodies and processes. Biochemical analyses of spinal cord soluble extracts indicated that G85R SOD-YFP behaved as a misfolded monomer at all ages. It became progressively insoluble at 6 and 9 months of age, associated with presence of soluble oligomers observable by gel filtration. Immunoaffinity capture and mass spectrometry revealed association of G85R SOD-YFP, but not WT SOD-YFP, with the cytosolic chaperone Hsc70 at all ages. In addition, 3 Hsp110's, nucleotide exchange factors for Hsp70s, were captured at 6 and 9 months. Despite such chaperone interactions, G85R SOD-YFP formed insoluble inclusions at late times, containing predominantly intermediate filament proteins. We conclude that motor neurons, initially “compensated” to maintain the misfolded protein in a soluble state, become progressively unable to do so.