肌萎缩性侧索硬化症动物模型的应用进展

肌萎缩性侧索硬化症（ALS）是运动神经元选择性死亡而导致运动功能障碍的神经性疾病,是成年人运动神经元病中最常见的疾病。已有很多学说讨论其发病机制,并且建立了ALS动物模型。随着现代生物学的发展和不同学科间的相互渗入,各种治疗策略在ALS模型实验中得到实践并有望用于临床。简要综述了ALS治疗方法在转基因动物模型中的研究进展。     

Mitochondrial Degeneration in Amyotrophic Lateral Sclerosis

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disorder that causes motor neuron degeneration, progressive skeletal muscle atrophy, paralysis, and death. To understand the mechanism of motor neuron degeneration, we have analyzed the clinical disease progression and the pathological changes in a transgenic mouse model for ALS. We found massive mitochondrial vacuolation at the onset of disease. By detailed morphological observations, we have determined that this mitochondrial vacuolation is developed from expansion of mitochondrial intermembrane space and extension of the outer membrane and involves peroxisomes. Lysosomes do not actively participate at all stages of this vacuolation. We conclude that this mitochondrial vacuolation is neither classical mitochondrial permeability transition nor autophagic vacuolation. Thus, this appears to be a new form of mitochondrial vacuolation and we term this as mitochondrial vacuolation by intermembrane space expansion or MVISE.     

Defective Neurofilament Transport in Mouse Models of Amyotrophic Lateral Sclerosis: A Review

Neurofilament proteins synthesized in the cell body of neurons are assembled and transported into axons, where they influence axon radial growth, axonal transport, and nerve conduction velocities. In diseased states, neurofilaments accumulate in cell bodies and proximal axons of affected neurons, and these lesions are characteristic of many neurodegenerative diseases, including amyotrophic lateral sclerosis (ALS), Alzheimer's disease (AD), spinal muscular atrophy (SMA), Charcot-Marie-Tooth disease type 2 (CMT2), and hereditary sensory motor neuropathy. Although the molecular mechanisms that contribute to these accumulations are not yet identified, transgenic mouse models are beginning to provide insight into the role of neurofilament transport in disease-related dysfunction of neurons. This review addresses axonal transport in mouse models of ALS and the special significance of neurofilament transport in this disease.     

Anti-Ganglioside Antibodies in Amyotrophic Lateral Sclerosis Revisited

Background

Amyotrophic Lateral Sclerosis (ALS) is a devastating neurodegenerative disorder with typical onset in the 5^th- 6^th decade of life. The hypothesis of an autoimmune origin of ALS receives less attention today, but immunological phenomena still seem to be involved and mechanisms such as protective autoimmunity may be important. Detection of antibodies against a variety of gangliosides has been repeatedly described in ALS-patients by several authors, but widely differing frequencies and titres have been reported. Therefore, we investigated the presence of six common antibodies with a commercially available test panel for GA1, GM1, GM2, GD1a, GD1b and GQ1b in a large group of clinically well-characterized ALS patients and compared them to a collective of 200 healthy blood donors.

Methods

IgG and IgM antibodies to the six gangliosides asialoGM1 (GA1), GM1, GM2, GD1a, GD1b, GQ1b were determined by GanglioCombi ELISA in sera of 84 ALS patients. Results were expressed as a %-ratio of a highly positive control and categorized as negative (<30%), borderline (30–50%), moderately (50–100%) and strongly positive (>100%). The values obtained from 200 Swiss blood donors served as a reference group.

Results

In twenty-two (26.2%) ALS-patients elevated anti-ganglioside antibodies could be detected: Taking all subspecific antibodies together, IgG antibodies were found in 9/84 (10.7%) and IgM in 15/84 (17.9%) patients. There was no correlation between age, gender, site of onset or survival and anti-ganglioside-positive/-negative titres in ALS-patients. No statistically significant difference in the frequency of anti-ganglioside antibodies compared to the group of healthy blood donors was found.

Conclusion

Even with this more comprehensive approach, anti-ganglioside antibody frequencies and patterns in our ALS cohort closely resembled the values measured in healthy controls. In accordance with other studies, we did not observe any association of a distinct ALS phenotype with elevated anti-ganglioside antibodies or an impact on survival.     

DREAM-Dependent Activation of Astrocytes in Amyotrophic Lateral Sclerosis

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease of unknown origin and characterized by a relentless loss of motor neurons that causes a progressive muscle weakness until death. Among the several pathogenic mechanisms that have been related to ALS, a dysregulation of calcium-buffering proteins in motor neurons of the brain and spinal cord can make these neurons more vulnerable to disease progression. Downstream regulatory element antagonist modulator (DREAM) is a neuronal calcium-binding protein that plays multiple roles in the nucleus and cytosol. The main aim of this study was focused on the characterization of DREAM and glial fibrillary acid protein (GFAP) in the brain and spinal cord tissues from transgenic SOD1^G93A mice and ALS patients to unravel its potential role under neurodegenerative conditions. The DREAM and GFAP levels in the spinal cord and different brain areas from transgenic SOD1^G93A mice and ALS patients were analyzed by Western blot and immunohistochemistry. Our findings suggest that the calcium-dependent excitotoxicity progressively enhanced in the CNS in ALS could modulate the multifunctional nature of DREAM, strengthening its apoptotic way of action in both motor neurons and astrocytes, which could act as an additional factor to increase neuronal damage. The direct crosstalk between astrocytes and motor neurons can become vulnerable under neurodegenerative conditions, and DREAM could act as an additional switch to enhance motor neuron loss. Together, these findings could pave the way to further study the molecular targets of DREAM to find novel therapeutic strategies to fight ALS.     

Alsin and the Molecular Pathways of Amyotrophic Lateral Sclerosis

Autosomal recessive mutations in the ALS2 gene lead to a clinical spectrum of motor dysfunction including juvenile onset amyotrophic lateral sclerosis (ALS2), primary lateral sclerosis, and hereditary spastic paraplegia. The 184-kDa alsin protein, encoded by the full-length ALS2 gene, contains three different guanine-nucleotide-exchange factor-like domains, which may play a role in the etiology of the disease. Multiple in vitro biochemical and cell biology assays suggest that alsin dysfunction affects endosome trafficking through a Rab5 small GTPase family-mediated mechanism. Four ALS2-deficient mouse models have been generated by different groups and used to study the behavioral and pathological impact of alsin deficiency. These mouse models largely fail to recapitulate hallmarks of motor neuron disease, but the subtle deficits that are observed in behavior and pathology have aided in our understanding of the relationship between alsin and motor dysfunction. In this review, we summarize recent clinical and molecular reports regarding alsin and attempt to place these results within the larger context of motor neuron disease.     

Molecular Imaging of Microglial Activation in Amyotrophic Lateral Sclerosis

There is growing evidence of activated microglia and inflammatory processes in the cerebral cortex in amyotrophic lateral sclerosis (ALS). Activated microglia is characterized by increased expression of the 18 kDa translocator protein (TSPO) in the brain and may be a useful biomarker of inflammation. In this study, we evaluated neuroinflammation in ALS patients using a radioligand of TSPO, ¹⁸F-DPA-714. Ten patients with probable or definite ALS (all right-handed, without dementia, and untreated by riluzole or other medication that might bias the binding on the TSPO), were enrolled prospectively and eight healthy controls matched for age underwent a PET study. Comparison of the distribution volume ratios between both groups were performed using a Mann-Whitney’s test. Significant increase of distribution of volume ratios values corresponding to microglial activation was found in the ALS sample in primary motor, supplementary motor and temporal cortex (p = 0.009, p = 0.001 and p = 0.004, respectively). These results suggested that the cortical uptake of ¹⁸F-DPA-714 was increased in ALS patients during the “time of diagnosis” phase of the disease. This finding might improve our understanding of the pathophysiology of ALS and might be a surrogate marker of efficacy of treatment on microglial activation.     

Cortical Thinning and Clinical Heterogeneity in Amyotrophic Lateral Sclerosis

Amyotrophic lateral sclerosis (ALS) has heterogeneous clinical features that could be translated into specific patterns of brain atrophy. In the current study we have evaluated the relationship between different clinical expressions of classical ALS and measurements of brain cortical thickness. Cortical thickness analysis was conducted from 3D-MRI using FreeSurfer software in 29 ALS patients and 20 healthy controls. We explored three clinical traits of the disease, subdividing the patients into two groups for each of them: the bulbar or spinal onset, the higher or lower upper motor neuron burden, the faster or slower disease progression. We used both a whole brain vertex-wise analysis and a ROI analysis on primary motor areas. ALS patients showed cortical thinning in bilateral precentral gyrus, bilateral middle frontal gyrus, right superior temporal gyrus and right occipital cortex. ALS patients with higher upper motor neuron burden showed a significant cortical thinning in the right precentral gyrus and in other frontal extra-motor areas, compared to healthy controls. ALS patients with spinal onset showed a significant cortical thinning in the right precentral gyrus and paracentral lobule, compared to healthy controls. ALS patients with faster progressive disease showed a significant cortical thinning in widespread bilateral frontal and temporal areas, including the bilateral precentral gyrus, compared to healthy controls. Focusing on the primary motor areas, the ROI analysis revealed that the mean cortical thickness values were significantly reduced in ALS patients with higher upper motor neuron burden, spinal onset and faster disease progression related to healthy controls. In conclusion, the thickness of primary motor cortex could be a useful surrogate marker of upper motor neuron involvement in ALS; also our results suggest that cortical thinning in motor and non motor areas seem to reflect the clinical heterogeneity of the disease.     

Myostatin as a therapeutic target in Amyotrophic Lateral Sclerosis

Amyotrophic Lateral Sclerosis is a devastating neurological disease that is inevitably fatal after 3–5 years duration. Treatment options are minimal and as such new therapeutic modalities are required. In this review, we discuss the role of the myostatin pathway as a modulator of skeletal muscle mass and therapeutic approaches using biological based therapies. Both monoclonal antibodies to myostatin and a soluble receptor decoy to its high affinity receptor have been used in clinical trials of neuromuscular diseases and while there have been efficacy signals with the latter approach there have also been safety issues. Our approach is to target the high affinity receptor-binding site on myostatin and to develop a next generation set of therapeutic reagents built on a novel protein scaffold. This is the natural single domain VNAR found in sharks which is extremely versatile and has the ability to develop products with superior properties compared to existing therapeutics.     

Adipose Tissue Distribution Predicts Survival in Amyotrophic Lateral Sclerosis

Background

amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease that leads to death within a few years after diagnosis. Malnutrition and weight loss are frequent and are indexes of poor prognosis. Total body fat and fat distribution have not been studied in ALS patients.

Objectives

Our aim was to describe adipose tissue content and distribution in ALS patients.

Design

We performed a cross-sectional study in a group of ALS patients (n = 62, mean disease duration 22 months) along with age and gender matched healthy controls (n = 62) using a MRI-based method to study quantitatively the fat distribution.

Results

Total body fat of ALS patients was not changed as compared with controls. However, ALS patients displayed increased visceral fat and an increased ratio of visceral to subcutaneous fat. Visceral fat was not correlated with clinical severity as judged using the ALS functional rating scale (ALS-FRS-R), while subcutaneous fat in ALS patients correlated positively with ALS-FRS-R and disease progression. Multiple regression analysis showed that gender and ALS-FRS-R, but not site of onset, were significant predictors of total and subcutaneous fat. Increased subcutaneous fat predicted survival in male patients but not in female patients (p<0.05).

Conclusions

Fat distribution is altered in ALS patients, with increased visceral fat as compared with healthy controls. Subcutaneous fat content is a predictor of survival of ALS patients.     

Implications of Microglia in Amyotrophic Lateral Sclerosis and Frontotemporal Dementia

Amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) are neurodegenerative disorders with clear similarities regarding their clinical, genetic and pathological features. Both are progressive, lethal disorders, with no current curative treatment available. Several genes that correlated with ALS and FTD are implicated in the same molecular pathways. Strikingly, many of these genes are not exclusively expressed in neurons, but also in glial cells, suggesting a multicellular pathogenesis. Moreover, chronic inflammation is a common feature observed in ALS and FTD, indicating an essential role of microglia, the resident immune cells of the central nervous system, in disease development and progression. In this review, we will provide a comprehensive overview of the implications of microglia in ALS and FTD. Specifically, we will focus on the role of impaired phagocytosis and increased inflammatory responses and their impact on microglial function. Several genes associated with the disorders can directly be linked to microglial activation, phagocytosis and neuroinflammation. Other genes associated with the disorders are implicated in biological pathways involved in protein degradation and autophagy. In general such mutations have been shown to cause abnormal protein accumulation and impaired autophagy. These impairments have previously been linked to affect the innate immune system in the central nervous system through inappropriate activation of microglia and neuroinflammation, highlighted in this review. Although it has been well established that microglia play essential roles in neurodegenerative disorders, the precise underlying mechanisms remain to be elucidated.     

Cerebellar Integrity in the Amyotrophic Lateral Sclerosis - Frontotemporal Dementia Continuum

Amyotrophic lateral sclerosis (ALS) and behavioural variant frontotemporal dementia (bvFTD) are multisystem neurodegenerative disorders that manifest overlapping cognitive, neuropsychiatric and motor features. The cerebellum has long been known to be crucial for intact motor function although emerging evidence over the past decade has attributed cognitive and neuropsychiatric processes to this structure. The current study set out i) to establish the integrity of cerebellar subregions in the amyotrophic lateral sclerosis-behavioural variant frontotemporal dementia spectrum (ALS-bvFTD) and ii) determine whether specific cerebellar atrophy regions are associated with cognitive, neuropsychiatric and motor symptoms in the patients. Seventy-eight patients diagnosed with ALS, ALS-bvFTD, behavioural variant frontotemporal dementia (bvFTD), most without C9ORF72 gene abnormalities, and healthy controls were investigated. Participants underwent cognitive, neuropsychiatric and functional evaluation as well as structural imaging using voxel-based morphometry (VBM) to examine the grey matter subregions of the cerebellar lobules, vermis and crus. VBM analyses revealed: i) significant grey matter atrophy in the cerebellum across the whole ALS-bvFTD continuum; ii) atrophy predominantly of the superior cerebellum and crus in bvFTD patients, atrophy of the inferior cerebellum and vermis in ALS patients, while ALS-bvFTD patients had both patterns of atrophy. Post-hoc covariance analyses revealed that cognitive and neuropsychiatric symptoms were particularly associated with atrophy of the crus and superior lobule, while motor symptoms were more associated with atrophy of the inferior lobules. Taken together, these findings indicate an important role of the cerebellum in the ALS-bvFTD disease spectrum, with all three clinical phenotypes demonstrating specific patterns of subregional atrophy that associated with different symptomology.     

Cortical Dysfunction Underlies the Development of the Split-Hand in Amyotrophic Lateral Sclerosis

The split-hand phenomenon, a specific feature of amyotrophic lateral sclerosis (ALS), refers to preferential wasting of abductor pollicis brevis (APB) and first dorsal interosseous (FDI) with relative preservation of abductor digiti minimi (ADM). The pathophysiological mechanisms underlying the split-hand phenomenon remain elusive and resolution of this issue would provide unique insights into ALS pathophysiology. Consequently, the present study dissected out the relative contribution of cortical and peripheral processes in development of the split-hand phenomenon in ALS. Cortical and axonal excitability studies were undertaken on 26 ALS patients, with motor responses recorded over the APB, FDI and ADM muscles. Results were compared to 21 controls. Short interval intracortical inhibition (SICI), a biomarker of cortical excitability, was significantly reduced across the range of intrinsic hand muscles (APB_{SICI ALS} 0.3±2.0%, APB_{SICI controls} 16.0±1.9%, P<0.0001; FDI_{SICI ALS} 2.7±1.7%, FDI _{SICI controls} 14.8±1.9%, P<0.0001; ADM_{SICI ALS} 2.6±1.5%, ADM _{SICI controls} 9.7±2.2%, P<0.001), although the reduction was most prominent when recorded over APB/FDI. Changes in SICI were accompanied by a significant increase in motor evoked potential amplitude and reduction of cortical silent period duration, all indicative of cortical hyperexcitability, and these were most prominent from the APB/FDI. At a peripheral level, a significant increase in strength-duration time constant and reduction in depolarising threshold electrotonus were evident in ALS, although these changes did not follow a split-hand distribution. Cortical dysfunction contributed to development of the split-hand in ALS, thereby implying an importance of cortical hyperexcitability in ALS pathogenesis.     

Amyotrophic Lateral Sclerosis and Skeletal Muscle: An Update

Amyotrophic lateral sclerosis (ALS) is the most frequent adult-onset motor neuron disease characterized by degeneration of upper and lower motor neurons (MNs), generalized weakness and muscle atrophy. The “neurocentric” view of ALS assumes that the disease primarily affects motor neurons, while muscle alterations only represent a consequence, in the periphery, of motor neuron loss. However, this outlook was recently challenged by evidence suggesting that non-neural cells such as microglia, astrocytes, peripheral blood mononuclear cells (PBMCs) and skeletal muscle fibres participate in triggering motor neuron degeneration, and this stressed the concept that alterations in different cell types may act together to exacerbate the disease. In this review, we will summarize the most recent findings on the alterations of skeletal muscle fibres found in ALS, with particular attention to the relationship between mutant SOD1 and skeletal muscle. We will analyze changes in muscle function, in the expression of myogenic regulatory factors, and also mitochondrial dysfunction, SOD1 aggregation and proteasome activity.     

Superoxide Dismutase Concentration and Activity in Familial Amyotrophic Lateral Sclerosis

Abstract: Some cases of autosomal-dominant familial amyotrophic lateral sclerosis (FALS) have been associated with mutations in SOD1 , the gene that encodes Cu/Zn superoxide dismutase (Cu/Zn SOD). We determined the concentrations (µg of Cu/Zn SOD/mg of total protein), specific activities (U/µg of total protein), and apparent turnover numbers (U/µmol of Cu/Zn SOD) of Cu/Zn SOD in erythrocyte lysates from patients with known SOD1 mutations. We also measured the concentrations and activities of Cu/Zn SOD in FALS patients with no identifiable SOD1 mutations, sporadic ALS (SALS) patients, and patients with other neurologic disorders. The concentration and specific activity of Cu/Zn SOD were decreased in all patients with SOD1 mutations, with mean reductions of 51 and 46%, respectively, relative to controls. In contrast, the apparent turnover number of the enzyme was not altered in these patients. For the six mutations studied, there was no correlation between enzyme concentration or specific activity and disease severity, expressed as either duration of disease or age of onset. No significant alterations in the concentration, specific activity, or apparent turnover number of Cu/Zn SOD were detected in the FALS patients with no identifiable SOD1 mutations, SALS patients, or patients with other neurologic disorders. That Cu/Zn SOD concentration and specific activity are equivalently reduced in erythrocytes from patients with SOD1 mutations suggests that mutant Cu/Zn SOD is unstable in these cells. That concentration and specific activity do not correlate with disease severity suggests that an altered, novel function of the enzyme, rather than reduction of its dismutase activity, may be responsible for the pathogenesis of FALS.     

AMPK Signalling and Defective Energy Metabolism in Amyotrophic Lateral Sclerosis

Amyotrophic lateral sclerosis (ALS) is caused by selective loss of upper and lower motor neurons by complex mechanisms that are incompletely understood. Motor neurons are large, highly polarised and excitable cells with unusually high energetic demands to maintain resting membrane potential and propagate action potentials. This leads to higher ATP consumption and mitochondrial metabolism in motor neurons relative to other cells. Here, we review increasing evidence that defective energy metabolism and homeostasis contributes to selective vulnerability and degeneration of motor neurons in ALS. Firstly, we provide a brief overview of major energetic pathways in the CNS, including glycolysis, oxidative phosphorylation and the AMP-activated protein kinase (AMPK) signalling pathway, while highlighting critical metabolic interactions between neurons and astrocytes. Next, we review evidence from ALS patients and transgenic mutant SOD1 mice for weight loss, hypermetabolism, hyperlipidemia and mitochondrial dysfunction in disease onset and progression. Genetic and therapeutic modifiers of energy metabolism in mutant SOD1 mice will also be summarised. We also present evidence that additional ALS-linked proteins, TDP-43 and FUS, lead to energy disruption and mitochondrial defects in motor neurons. Lastly, we review emerging evidence including our own that dysregulation of the AMPK signalling cascade in motor neurons is an early and common event in ALS pathogenesis. We suggest that an imbalance in energy metabolism should be considered an important factor in both progression and potential treatment of ALS.