Deletions of the heavy neurofilament subunit tail in amyotrophic lateral sclerosis

Amyotrophic lateral sclerosis (ALS) is a progressive motor neuron degeneration resulting in paralysis and death, usually within 3 years of onset. Pathological and animal studies implicate neurofilament involvement in ALS, but whether this is primary or secondary is not clear. The heavy neurofilament subunit (NFH) tail is composed of a repeating amino acid motif, usually X-lysine-serine-proline-Y-lysine (XKSPYK), where X is a single amino acid and Y is one to three amino acids. There are two common polymorphic variants of 44 or 45 repeats. The tail probably regulates axonal calibre, with interfilament spacing determined by phosphorylation of the KSP motifs. A previous study suggested an association between sporadic cases of ALS and NFH tail deletions, but two subsequent studies have found none. We have analysed samples from two different populations (UK 207, Scandinavia 323) with age-matched controls for each group (UK 219, Scandinavia 228) and have found four novel NFH tail deletions, each involving a whole motif. These were found in three patients with sporadic ALS and a family with autosomal dominant ALS, although another was also found in two young controls. In all cases motif deletions were only associated with disease when paired with the long NFH allele. The deletions all occurred within a small region of the NFH tail. This has allowed us to propose a structural organization of the tail as well as allowing observed deletions both from this study and previous reports to be organized into logical groups. These results strongly suggest that NFH motif deletions can be a primary event in ALS but that they are not common.      

Bee venom attenuates neuroinflammatory events and extends survival in amyotrophic lateral sclerosis models

Background  

Amyotrophic lateral sclerosis (ALS) is a disease affecting the central nervous system that is either sporadic or familial origin and causing the death of motor neurons. One of the genetic factors contributing to the etiology of ALS is mutant SOD1 (mtSOD1), which induces vulnerability of motor neurons through protein misfolding, mitochondrial dysfunction, oxidative damage, cytoskeletal abnormalities, defective axonal transport, glutamate excitotoxicity, inadequate growth factor signaling, and neuroinflammation. Bee venom has been used in the practice of Oriental medicine and evidence from the literature indicates that BV plays an anti-inflammatory or anti-nociceptive role against inflammatory reactions associated with arthritis and other inflammatory diseases. The purpose of the present study was to determine whether bee venom suppresses motor neuron loss and microglial cell activation in hSOD1^G93A mutant mice.     

Motoneuron survival is promoted by specific exercise in a mouse model of amyotrophic lateral sclerosis

Several studies using transgenic mouse models of familial amyotrophic lateral sclerosis (ALS) have reported a life span increase in exercised animals, as long as animals are submitted to a moderate-intensity training protocol. However, the neuroprotective potential of exercise is still questionable. To gain further insight into the cellular basis of the exercise-induced effects in neuroprotection, we compared the efficiency of a swimming-based training, a high-frequency and -amplitude exercise that preferentially recruits the fast motor units, and of a moderate running-based training, that preferentially triggers the slow motor units, in an ALS mouse model. Surprisingly, we found that the swimming-induced benefits sustained the motor function and increased the ALS mouse life span by about 25 days. The magnitude of this beneficial effect is one of the highest among those induced by any therapeutic strategy in this disease. We have shown that, unlike running, swimming significantly delays spinal motoneuron death and, more specifically, the motoneurons of large soma area. Analysis of the muscular phenotype revealed a swimming-induced relative maintenance of the fast phenotype in fast-twitch muscles. Furthermore, the swimming programme preserved astrocyte and oligodendrocyte populations in ALS spinal cord. As a whole, these data are highly suggestive of a causal relationship not only linking motoneuron activation and protection, but also motoneuron protection and the maintenance of the motoneuron surrounding environment. Basically, exercise-induced neuroprotective mechanisms provide an example of the molecular adaptation of activated motoneurons.     

Variants of the heavy neurofilament subunit are associated with the development of amyotrophic lateral sclerosis

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disorderprimarily affecting motor neurons. The etiology of the majorityof cases remains unknown. Recent findings from several laboratoriessuggest a role for neurofilaments in the development of motorneuron disorders. The C-terminal region of the human neurofilamentheavy subunit (NEFH) contains a unique functional domain consistingof 43 repeat motifs of the amino acids Lys-Ser-Pro (KSP). ThisC-terminal region of NEFH forms the sidearm projections whichcross-link the neurofilaments. Previously, we have demonstratedpolymorphism in the C-terminal region of the human NEFH: anallelic variant of a slightly larger molecular size, containingan additional KSP phosphorylation motif. Novel mutations inthis region were found in five ALS patients. We propose thatchanges in the KSP-repeat domain may affect the cross-linkingproperties of the heavy neurofilament subunit and perhaps contributeto the development of neurofilamentous swellings in motor neurons,a hallmark of ALS.     

家族性肌萎缩侧索硬化小鼠保种过程中出现种系退化

背景：SOD1-G93A转基因小鼠是研究肌萎缩侧索硬化的经典代表性疾病模型动物,然而对于这种转基因小鼠在保种过程中所存在的种系退化现象却少有报道。 目的：研究家族性肌萎缩侧索硬化小鼠在保种过程中出现的种系退化现象。 方法：通过比较家族性肌萎缩侧索硬化疾病模型SOD1-G93A转基因小鼠在保种过程中不同代间的怀孕胎数、产仔数以及子代鼠中雄性和阳性小鼠所占得比例分析该转基因小鼠保种过程中生育能力的变化,并比较不同代间小鼠的起病时间、生存期以分析该转基因小鼠在保种过程中发病情况的变化。 结果与结论：第3,6代小鼠与第0代比较,其怀孕胎数、每胎产仔数、每只产仔数等均显著下降(P < 0.05),且第6代小鼠中有一半以上雌鼠无法受孕。其子代鼠中雄性小鼠以及阳性小鼠所占的比例有下降趋势,但是其差异无显著性意义。第6代小鼠与第1代比较其发病时间与死亡时间分别推迟了6.37 d (P=0.004)和9.67 d(P=0.022),差异有显著性意义。以上结果显示家族性肌萎缩侧索硬化小鼠在保种过程中存在生育能力下降、发病及死亡时间推迟等种系退化现象。     

Androgens affect muscle,motor neuron,and survival in a mouse model of SOD1-related amyotrophic lateral sclerosis

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease characterized by selective loss of upper and lower motor neurons and skeletal muscle atrophy. Epidemiologic and experimental evidence suggest the involvement of androgens in ALS pathogenesis, but the mechanism through which androgens modify the ALS phenotype is unknown. Here, we show that androgen ablation by surgical castration extends survival and disease duration of a transgenic mouse model of ALS expressing mutant human SOD1 (hSOD1-G93A). Furthermore, long-term treatment of orchiectomized hSOD1-G93A mice with nandrolone decanoate (ND), an anabolic androgenic steroid, worsened disease manifestations. ND treatment induced muscle fiber hypertrophy but caused motor neuron death. ND negatively affected survival, thereby dissociating skeletal muscle pathology from life span in this ALS mouse model. Interestingly, orchiectomy decreased androgen receptor levels in the spinal cord and muscle, whereas ND treatment had the opposite effect. Notably, stimulation with ND promoted the recruitment of endogenous androgen receptor into biochemical complexes that were insoluble in sodium dodecyl sulfate, a finding consistent with protein aggregation. Overall, our results shed light on the role of androgens as modifiers of ALS pathogenesis via dysregulation of androgen receptor homeostasis.     

UNC13A is a modifier of survival in amyotrophic lateral sclerosis

A large genome-wide screen in patients with sporadic amyotrophic lateral sclerosis (ALS) showed that the common variant rs12608932 in gene UNC13A was associated with disease susceptibility. UNC13A regulates the release of neurotransmitters, including glutamate. Genetic risk factors that, in addition, modify survival, provide promising therapeutic targets in ALS, a disease whose etiology remains largely elusive. We examined whether UNC13A was associated with survival of ALS patients in a cohort of 450 sporadic ALS patients and 524 unaffected controls from a population-based study of ALS in The Netherlands. Additionally, survival data were collected from individuals of Dutch, Belgian, or Swedish descent (1767 cases, 1817 controls) who had participated in a previously published genome-wide association study of ALS. We related survival to rs12608932 genotype. In both cohorts, the minor allele of rs12608932 in UNC13A was not only associated with susceptibility but also with shorter survival of ALS patients. Our results further corroborate the role of UNC13A in ALS pathogenesis.     

肌萎缩侧索硬化小鼠内源性神经干细胞膜突触蛋白表达

背景：成纤维细胞生长因子2是神经系统主要的神经营养因子之一,目前已经被证明有促进内源性神经干细胞分化的作用。 目的：观察成纤维细胞生长因子2干预下,肌萎缩侧索硬化SOD1G93A G1H转基因小鼠运动功能的改变,内源性神经干细胞的增殖情况,突触蛋白的水平改变及内源性神经干细胞增殖数目与突触蛋白水平改变的相关性。 方法：取新出生SOD1G93A G1H转基因小鼠(肌萎缩侧索硬化模型小鼠)60只分为肌萎缩侧索硬化组及成纤维细胞生长因子2组各30只,取新出生野生B6SJL小鼠30只作为正常对照组,成纤维细胞生长因子2组腹腔注入成纤维细胞生长因子2;肌萎缩侧索硬化组和正常对照组腹腔注入安慰剂生理盐水。分别于出生后60,90,120 d采用Rotarod方法评估小鼠运动功能的改变,采用免疫组织化学方法标记内源性神经干细胞和突触蛋白并计数,用spearman方法评估内源性神经干细胞增殖数目与突触蛋白水平的相关性。 结果与结论：与肌萎缩侧索硬化组相比,成纤维细胞生长因子2组小鼠运动功能明显改善,内源性神经干细胞增殖和突触蛋白水平显著增高。小鼠内源性神经干细胞的增加与突触蛋白水平的增呈正相关。提示成纤维细胞生长因子2神经保护机制可能与其促进内源性神经干细胞增殖和提升突触蛋白水平相关。     

Increased phospho-adducin immunoreactivity in a murine model of amyotrophic lateral sclerosis

Adducins alpha, beta and gamma are proteins that link spectrin and actin in the regulation of cytoskeletal architecture and are substrates for protein kinase C and other signaling molecules. Previous studies have shown that expressions of phosphorylated adducin (phospho-adducin) and protein kinase C are increased in spinal cord tissue from patients who died with amyotrophic lateral sclerosis, a neurodegenerative disorder of motoneurons and other cells. However, the distribution of phospho-adducin immunoreactivity has not been described in the mammalian spinal cord. We have evaluated the distribution of immunoreactivity to serine/threonine-dependent phospho-adducin at a region corresponding to the myristoylated alanine-rich C kinase substrate-related domain of adducin in spinal cords of mice over-expressing mutant human superoxide dismutase, an animal model of amyotrophic lateral sclerosis, and in control littermates. We find phospho-adducin immunoreactivity in control spinal cord in ependymal cells surrounding the central canal, neurons and astrocytes. Phospho-adducin immunoreactivity is localized to the cell bodies, dendrites and axons of some motoneurons, as well as to astrocytes in the gray and white matter. Spinal cords of mutant human superoxide dismutase mice having motoneuron loss exhibit significantly increased phospho-adducin immunoreactivity in ventral and dorsal horn spinal cord regions, but not in ependyma surrounding the central canal, compared with control animals. Increased phospho-adducin immunoreactivity localizes predominantly to astrocytes and likely increases as a consequence of the astrogliosis that occurs in the mutant human superoxide dismutase mouse with disease progression. These findings demonstrate increased immunoreactivity against phosphorylated adducin at the myristoylated alanine-rich C kinase substrate domain in a murine model of amyotrophic lateral sclerosis. As adducin is a substrate for protein kinase C at the myristoylated alanine-rich C kinase substrate domain, the increased phospho-adducin immunoreactivity is likely a consequence of protein kinase C activation in neurons and astrocytes of the spinal cord and evidence for aberrant phosphorylation events in mutant human superoxide dismutase mice that may affect neuron survival.     

Combined lithium and valproate treatment delays disease onset, reduces neurological deficits and prolongs survival in an amyotrophic lateral sclerosis mouse model

Lithium and valproic acid (VPA) are two primary drugs used to treat bipolar disorder, and have been shown to have neuroprotective properties in vivo and in vitro. A recent study demonstrated that combined treatment with lithium and VPA elicits synergistic neuroprotective effects against glutamate excitotoxicity in cultured brain neurons, and the synergy involves potentiated inhibition of glycogen synthase kinase-3 (GSK-3) activity through enhanced GSK-3 serine phosphorylation [Leng Y, Liang MH, Ren M, Marinova Z, Leeds P, Chuang DM (2008) Synergistic neuroprotective effects of lithium and valproic acid or other histone deacetylase inhibitors in neurons: roles of glycogen synthase kinase-3 inhibition. J Neurosci 28:2576-2588]. We therefore investigated the effects of lithium and VPA cotreatment on the disease symptom onset, survival time and neurological deficits in cooper zinc superoxide dismutase (SOD1) G93A mutant mice, a commonly used mouse model of amyotrophic lateral sclerosis (ALS). The G93A ALS mice received twice daily i.p. injections with LiCl (60 mg/kg), VPA (300 mg/kg) or lithium plus VPA, starting from the 30(th) day after birth and continuing until death. We found that combined treatment with lithium and VPA produced a greater and more consistent effect in delaying the onset of disease symptoms, prolonging the lifespan and decreasing the neurological deficit scores, compared with the results of monotreatment with lithium or VPA. Moreover, lithium in conjunction with VPA was more effective than lithium or VPA alone in enhancing the immunostaining of phospho-GSK-3beta(Ser9) in brain and lumbar spinal cord sections. To our knowledge, this is the first demonstration of enhanced neuroprotection by a combinatorial approach using mood stabilizers in a mouse ALS model. Our results suggest that clinical trials using lithium and VPA in combination for ALS patients are a rational strategy.     

Immunology of amyotrophic lateral sclerosis

Conclusion The antibody detected in the ALS sera with suppressive activity for terminal sprouting is likely directed against an antigen that is normally sequestered. Tolerance to the 56 kd antigen described here is not exhibited in rats, as syngenic immunizations induce an immune response (M. E. Gurney, unpublished observation). Freshly denervated muscle secretes very low amounts of the 56 kd antigen, and the level of secretion rises 2–3 days after denervation in vivo. Of the few antecedent events associated with ALS by case studies, mechanical injuries, participation in physical activities, or bone fractures might all produce nerve damage or periods of limb immobilization [48]. Similar physical insults are stimuli for sprouting in animal models [13, 39], and might result in elevated secretion of sprouting factor, resulting in induction of an immune response in susceptible individuals.The antibodies detected in this study could arise as a secondary response to neuromuscular destruction, as occurs following tissue injury in many organs. The absence of inhibitory activity in sera of patients with neuropathy suggests that denervation per se need not give rise to development of such antibodies, although the extent of denervation and muscle atrophy in the ALS patients was, in general, markedly greater than in patients with neuropathy. Whether sprouting inhibitory antibodies prove to be of primary pathogenic significance, or are only a secondary phenomenon, they provide potentially significant reagents with which to elucidate the existence and nature of human motor neuron growth factors.     

Noninvasive detection of brainstem and spinal cord axonal degeneration in an amyotrophic lateral sclerosis mouse model

Degeneration of motor neurons and their associated axons is a hallmark of amyotrophic lateral sclerosis, but reliable noninvasive lesion detection is lacking. In vivo diffusion tensor imaging was performed to evaluate neurodegeneration in the brainstem and cervical spinal cord of wild-type and G93A-SOD1 transgenic mice, an animal model of amyotrophic lateral sclerosis. A statistically significant reduction in the apparent diffusion coefficient was observed in the motor nuclei VII and XII of G93A-SOD1 transgenic mice relative to wild-type mice. No significant difference in diffusion anisotropy was observed in dorsal white or gray matter in cervical and lumbar segments of the spinal cord. In contrast, statistically significant decreases in axial diffusivity (diffusivity parallel to the axis of the spinal cord) and apparent diffusion coefficient were found in the ventrolateral white matter of G93A-SOD1 mice in both the cervical and lumbar spinal cord. The reduction in axial diffusivity, suggestive of axonal injury, in the white matter of the spinal cord of G93A-SOD1 mice was verified by immunostaining with nonphosphorylated neurofilament. The present study demonstrates that in vivo diffusion tensor imaging-derived axial diffusivity may be used to accurately evaluate axonal degeneration in an animal model of amyotrophic lateral sclerosis.     

Therapeutic effects of clenbuterol in a murine model of amyotrophic lateral sclerosis

We investigated the effects of clenbuterol, a beta2-adrenoceptor agonist with known anabolic and neuroprotective properties, on G93A-SOD1 mice, a transgenic murine model of familial amyotrophic lateral sclerosis (ALS). Relative to saline-treated vehicle controls (0.2 ml/kg/day; i.p.), early pathologic G93A-SOD1 mice treated with clenbuterol (1.5 mg/kg/day; i.p.) demonstrated a delayed onset of hindlimb signs as measured by rotarod performance, slowed disease progression, as well as trends toward mitigated losses of lumbar motoneurons and body weight. Responses in female G93A-SOD1 mice were favorable to those of males, suggesting synergistic effects between clenbuterol and sex-specific factors. Overall, our data suggest that clenbuterol offers therapeutic effects on ALS-related neuromuscular degeneration.     

Immunomodulation in the treatment of multiple sclerosis and amyotrophic lateral sclerosis: a model for autoimmune disorders.

Seventeen multiple sclerosis (MS) patients progressing under conventional therapy (average treatment duration: 3 years) with performance status 3-4 (mean Disability Status Scale [DSS]: 82) who demonstrated circulating lymphokine inhibitor factors were selected for a monthly immunomodulatory protocol using plasmapheresis, followed by 3 days of human intravenous immunoglobulin, and low-dose methylprednisolone, cyclophosphamide, interferon-a, and interferon-g, as well as octreide. Twelve of the 17 patients presented with visual problems, 12 had lower extremity weakness or paraperesis/paralysis, and 6 had bladder/bowel dysfunction. Following 4 months of therapy, 4 recovered completely, 7 showed loss of paralysis/paraparesis, and 5 had improvement in lower extremity weakness. One patient progressed (mean DSS: 51). Lymphokine inhibitor factors declined in 14 patients with concomitant normalization of circulating immune complexes. Eight patients experienced rises in CD4 levels with stabilization of CD8 levels. Hypotension and hypocalcemia were observed during plasmapheresis. Twelve patients with amyotrophic lateral sclerosis with poor performance status also were studied. Four of the 12 improved with the regimen, whereas six stabilized disease. Similar alterations in laboratory parameters were described. The rationale for this approach is discussed.     

Neuromuscular accumulation of mutant superoxide dismutase 1 aggregates in a transgenic mouse model of familial amyotrophic lateral sclerosis

Superoxide dismutase 1 (SOD1) aggregates are a histological and biochemical correlate of disease progression in neural tissues from mutant SOD1-linked forms of familial amyotrophic lateral sclerosis (FALS). In the present study, we assayed the monomeric and high molecular weight mutant SOD1 content of nervous, muscle and visceral tissues from transgenic SOD1(G93A) mice using immunoblotting and zymograms. A progressive age-dependent increase in mutant SOD1 level, aggregation and stabilisation by cross-species heterodimers was determined in lumbar spinal cord, sciatic nerve and gastrocnemius muscle. Such biochemical abnormalities were not present in cervical spinal cord, brainstem and diaphragm muscle, nor common to endogenous mouse SOD1. Mutant dismutase activity in general did not increase correspondingly with accumulating protein at later ages. These results suggest that peripheral targets such as hindlimb skeletal muscle and nerve accumulate mutant SOD1 aggregates and may therefore be susceptible to mutant SOD1-mediated toxicity, in addition to lower and upper motor neurons of the central nervous system in transgenic FALS mice.     

A mutation database for amyotrophic lateral sclerosis

An amyotrophic lateral sclerosis (ALS) mutation database has been constructed as a publicly accessible online resource for recording the nucleotide and amino acid variants identified in genes associated with ALS, along with corresponding clinical conditions. The database currently consists of more than 600 entries, including about 180 unique variants found in 25 disease‐causative or disease‐related genes. In addition to published data collected from literature, novel variants identified by microarray resequencing in our laboratory are incorporated into the database. Every reported gene has a respective page that provides information on its variation positions with various statistics, clinical characteristics, and primary references, as well as gene‐sequence and protein‐structure information that will assist in assessing variation significance. Users can access a homology search function to find variations in arbitrary sequences of interest and to check if they have already been described in the database. This database is expected to fulfill an essential need in terms of integrating comprehensive information on genetic and clinical data related to ALS, which will subsequently deepen our understanding of the possible mechanisms of the disease, as well as help with the clinical practice and treatment of ALS. The database is accessible at: https://reseq.lifesciencedb.jp/resequence/SearchDisease.do?targetId=1 . Data submission is open to all researchers and is highly encouraged. Hum Mutat 31:1003–1010, 2010. © 2010 Wiley‐Liss, Inc.     

In vivo diffusion MRI detects early spinal cord axonal pathology in a mouse model of amyotrophic lateral sclerosis

Diffusion magnetic resonance imaging (MRI) exhibits contrast that identifies macro‐ and microstructural changes in neurodegenerative diseases. Previous studies have shown that MR diffusion tensor imaging (DTI) can observe changes in spinal cord white matter in animals and humans affected with symptomatic amyotrophic lateral sclerosis (ALS). The goal of this preclinical work was to investigate the sensitivity of DTI for the detection of signs of tissue damage before symptoms appear. High‐field MRI data were acquired using a 9.4‐T animal scanner to examine the spinal cord of an ALS mouse model at pre‐ and post‐symptomatic stages (days 80 and 120, respectively). The MRI results were validated using yellow fluorescent protein (YFP) via optical microscopy of spinal cord tissue slices collected from the YFP,G93A‐SOD1 mouse strain. DTI maps of diffusion‐weighted imaging (DWI) signal intensity, mean diffusivity (MD), fractional anisotropy (FA), axial diffusivity (AD) and radial diffusivity (RD) were computed for axial slices of the lumbar region of the spinal cord. Significant changes were observed in FA (6.7% decrease, p < 0.01), AD (19.5% decrease, p < 0.01) and RD (16.1% increase, p < 0.001) at postnatal day 80 (P80). These differences were correlated with changes in axonal fluorescence intensity and membrane cellular markers. This study demonstrates the value of DTI as a potential tool to detect the underlying pathological progression associated with ALS, and may accelerate the discovery of therapeutic strategies for patients with this disease.