Protective effects of cardiotrophin-1 adenoviral gene transfer on neuromuscular degeneration in transgenic ALS mice

Amyotrophic lateral sclerosis (ALS) is mainly a sporadic neurodegenerative disorder characterized by loss of cortical and spinal motoneurons. Some familial ALS cases (FALS) have been linked to dominant mutations in the gene encoding Cu/Zn superoxide dismutase (SOD1). Transgenic mice overexpressing a mutated form of human SOD1 with a Gly93Ala substitution develop progressive muscle wasting and paralysis as a result of spinal motoneuron loss and die at 5 to 6 months. We investigated the effects of neurotrophic factor gene delivery in this FALS model. Intramuscular injection of an adenoviral vector encoding cardiotrophin-1 (CT-1) in SOD1G93A newborn mice resulted in systemic delivery of CT-1, supplying motoneurons with a continuous source of trophic factor. CT-1 delayed the onset of motor impairment as assessed in the rotarod test. Axonal degeneration was slowed and skeletal muscle atrophy was largely reduced by CT-1 treatment. By monitoring the amplitude of the evoked motor response, we showed that the time-course of motor impairment was significantly decreased by CT-1 treatment. Thus, adenovirus-mediated gene transfer of neurotrophic factors might delay neurogenic muscular atrophy and progressive neuromuscular deficiency in ALS patients.     

Downregulation of VAPB expression in motor neurons derived from induced pluripotent stem cells of ALS8 patients

Amyotrophic lateral sclerosis (ALS) is an incurable neuromuscular disease that leads to a profound loss of life quality and premature death. Around 10% of the cases are inherited and ALS8 is an autosomal dominant form of familial ALS caused by mutations in the vamp-associated protein B/C (VAPB) gene. The VAPB protein is involved in many cellular processes and it likely contributes to the pathogenesis of other forms of ALS besides ALS8. A number of successful drug tests in ALS animal models could not be translated to humans underscoring the need for novel approaches. The induced pluripotent stem cells (iPSC) technology brings new hope, since it can be used to model and investigate diseases in vitro. Here we present an additional tool to study ALS based on ALS8-iPSC. Fibroblasts from ALS8 patients and their non-carrier siblings were successfully reprogrammed to a pluripotent state and differentiated into motor neurons. We show for the first time that VAPB protein levels are reduced in ALS8-derived motor neurons but, in contrast to over-expression systems, cytoplasmic aggregates could not be identified. Our results suggest that optimal levels of VAPB may play a central role in the pathogenesis of ALS8, in agreement with the observed reduction of VAPB in sporadic ALS.     

VAPB interacts with the mitochondrial protein PTPIP51 to regulate calcium homeostasis

A proline to serine substitution at position 56 in the gene encoding vesicle-associated membrane protein-associated protein B (VAPB) causes some dominantly inherited familial forms of motor neuron disease including amyotrophic lateral sclerosis (ALS) type-8. VAPB is an integral endoplasmic reticulum (ER) protein whose amino-terminus projects into the cytosol. Overexpression of ALS mutant VAPBP56S disrupts ER structure but the mechanisms by which it induces disease are not properly understood. Here we show that VAPB interacts with the outer mitochondrial membrane protein, protein tyrosine phosphatase-interacting protein 51 (PTPIP51). ER and mitochondria are both stores for intracellular calcium (Ca(2+)) and Ca(2+) exchange between these organelles occurs at regions of ER that are closely apposed to mitochondria. These are termed mitochondria-associated membranes (MAM). We demonstrate that VAPB is a MAM protein and that loss of either VAPB or PTPIP51 perturbs uptake of Ca(2+) by mitochondria following release from ER stores. Finally, we demonstrate that VAPBP56S has altered binding to PTPIP51 and increases Ca(2+) uptake by mitochondria following release from ER stores. Damage to ER, mitochondria and Ca(2+) homeostasis are all seen in ALS and we discuss the implications of our findings in this context.     

P2X7 activation enhances skeletal muscle metabolism and regeneration in SOD1G93A mouse model of amyotrophic lateral sclerosis

Muscle weakness plays an important role in neuromuscular disorders comprising amyotrophic lateral sclerosis (ALS). However, it is not established whether muscle denervation originates from the motor neurons, the muscles or more likely both. Previous studies have shown that the expression of the SOD1G93A mutation in skeletal muscles causes denervation of the neuromuscular junctions, inability to regenerate and consequent atrophy, all clear symptoms of ALS. In this work, we used SOD1G93A mice, a model that best mimics some pathological features of both familial and sporadic ALS, and we investigated some biological effects induced by the activation of the P2X7 receptor in the skeletal muscles. The P2X7, belonging to the ionotropic family of purinergic receptors for extracellular ATP, is abundantly expressed in the healthy skeletal muscles, where it controls cell duplication, differentiation, regeneration or death. In particular, we evaluated whether an in vivo treatment in SOD1G93A mice with the P2X7 specific agonist 2′(3′)‐O‐(4‐Benzoylbenzoyl) adenosine5′‐triphosphate (BzATP) just before the onset of a pathological neuromuscular phenotype could exert beneficial effects in the skeletal muscles. Our findings indicate that stimulation of P2X7 improves the innervation and metabolism of myofibers, moreover elicits the proliferation/differentiation of satellite cells, thus preventing the denervation atrophy of skeletal muscles in SOD1G93A mice. Overall, this study suggests that a P2X7‐targeted and site‐specific modulation might be a strategy to interfere with the complex multifactorial and multisystem nature of ALS.     

Neurodegeneration and NLRP3 inflammasome expression in the anterior thalamus of SOD1(G93A) ALS mice

Nowadays, amyotrophic lateral sclerosis (ALS) is considered as a multisystem disorder, characterized by a primary degeneration of motor neurons as well as neuropathological changes in non‐motor regions. Neurodegeneration in subcortical areas, such as the thalamus, are believed to contribute to cognitive and behavioral abnormalities in ALS patients. In the present study, we investigated neurodegenerative changes including neuronal loss and glia pathology in the anterodorsal thalamic nucleus (AD) of SOD1(G93A) mice, a widely used animal model for ALS. We detected massive dendrite swelling and neuronal loss in SOD1(G93A) animals, which was accompanied by a mild gliosis. Furthermore, misfolded SOD1 protein and autophagy markers were accumulating in the AD. Since innate immunity and activation inflammasomes seem to play a crucial role in ALS, we examined protein expression of Nod‐like receptor protein 3 (NLRP3), apoptosis‐associated speck‐like protein containing a caspase‐1 recruitment domain (ASC) and the cytokine interleukin 1 beta (IL1β) in AD glial cells and neurons. NLRP3 and ASC were significantly up‐regulated in the AD of SOD1(G93A) mice. Finally, co‐localization studies revealed expression of NLRP3, ASC and IL1β in neurons. Our study yielded two main findings: (i) neurodegenerative changes already occur at an early symptomatic stage in the AD and (ii) increased inflammasome expression may contribute to neuronal cell death. In conclusion, neurodegeneration in the anterior thalamus may critically account for cognitive changes in ALS pathology.     

hVAPB, the causative gene of a heterogeneous group of motor neuron diseases in humans, is functionally interchangeable with its Drosophila homologue DVAP-33A at the neuromuscular junction

Motor neuron diseases (MNDs) are progressive neurodegenerative disorders characterized by selective death of motor neurons leading to spasticity, muscle wasting and paralysis. Human VAMP-associated protein B (hVAPB) is the causative gene of a clinically diverse group of MNDs including amyotrophic lateral sclerosis (ALS), atypical ALS and late-onset spinal muscular atrophy. The pathogenic mutation is inherited in a dominant manner. Drosophila VAMP-associated protein of 33 kDa A (DVAP-33A) is the structural homologue of hVAPB and regulates synaptic remodeling by affecting the size and number of boutons at neuromuscular junctions. Associated with these structural alterations are compensatory changes in the physiology and ultrastructure of synapses, which maintain evoked responses within normal boundaries. DVAP-33A and hVAPB are functionally interchangeable and transgenic expression of mutant DVAP-33A in neurons recapitulates major hallmarks of the human diseases including locomotion defects, neuronal death and aggregate formation. Aggregate accumulation is accompanied by a depletion of the endogenous protein from its normal localization. These findings pinpoint to a possible role of hVAPB in synaptic homeostasis and emphasize the relevance of our fly model in elucidating the patho-physiology underlying motor neuron degeneration in humans.     

Disease-dependent reciprocal phosphorylation of serine and tyrosine residues of c-Met/HGF receptor contributes disease retardation of a transgenic mouse model of ALS

Amyotrophic lateral sclerosis (ALS) is a fatal disease characterized by progressive degeneration of motoneurons. We have demonstrated that hepatocyte growth factor (HGF) attenuates loss of both spinal and brainstem motoneurons of ALS model mice expressing mutated human SOD1^G93A (G93A). This study was designed to assess disease-dependent regulatory mechanisms of c-Met/HGF receptor (c-Met) activation in the facial motoneurons of G93A mice. Using double transgenic mice expressing HGF and mutated SOD1^G93A (G93A/HGF), we showed that phosphorylation of c-Met tyrosine residues at positions 1230, 1234 and 1235 (phospho-Tyr), and thereby its activation, was slightly evident in G93A and highly obvious in G93A/HGF mice (but absent in WT and HGF-Tg mice). Phosphorylation of the c-Met serine residue at position 985 (phospho-Ser), a residue involved in the negative regulation of its activation, was evident in WT and HGF-Tg mice. Protein phosphatase 2A (PP2A), which is capable of dephosphorylating c-Met phospho-serine, is upregulated in the facial motoneurons of G93A and G93A/HGF mice compared with WT and HGF-Tg mice. Thus, c-Met activation is reciprocally regulated by phosphorylation between c-Met serine and tyrosine residues through PP2A induction in the presence or absence of mutant SOD1 expression, and HGF functions more efficiently in ALS and ALS-related diseases.     

Alteration of protein folding and degradation in motor neuron diseases: Implications and protective functions of small heat shock proteins

Motor neuron diseases (MNDs) are neurodegenerative disorders that specifically affect the survival and function of upper and/or lower motor neurons. Since motor neurons are responsible for the control of voluntary muscular movement, MNDs are characterized by muscle spasticity, weakness and atrophy. Different susceptibility genes associated with an increased risk to develop MNDs have been reported and several mutated genes have been linked to hereditary forms of MNDs. However, most cases of MNDs occur in sporadic forms and very little is known on their causes. Interestingly, several molecular mechanisms seem to participate in the progression of both the inherited and sporadic forms of MNDs. These include cytoskeleton organization, mitochondrial functions, DNA repair and RNA synthesis/processing, vesicle trafficking, endolysosomal trafficking and fusion, as well as protein folding and protein degradation. In particular, accumulation of aggregate-prone proteins is a hallmark of MNDs, suggesting that the protein quality control system (molecular chaperones and the degradative systems: ubiquitin-proteasome-system and autophagy) are saturated or not sufficient to allow the clearance of these altered proteins. In this review we mainly focus on the MNDs associated with disturbances in protein folding and protein degradation and on the potential implication of a specific class of molecular chaperones, the small heat shock proteins (sHSPs/HSPBs), in motor neuron function and survival. How boosting of specific HSPBs may be a potential useful therapeutic approach in MNDs and how mutations in specific HSPBs can directly cause motor neuron degeneration is discussed.     

Na channel density in extrajunctional sarcolemma of fast and slow twitch mouse skeletal muscle fibres: functional implications and plasticity after fast motoneuron transplantation on to a slow muscle

Summary Na channel densities were measured in fast and slow twitch mouse skeletal muscle fibres using the loose patch voltage clamp technique. It was found that Na channel density was approximately four times greater in fast twitch fibres than in slow. Computer simulations of action potential propagation in these fibres strongly suggest that the higher channel densities in fast twitch fibres are necessary to maintain action potential amplitude and fidelity of transmission across the neuromuscular junction, especially during the periods of rapid stimulation that these fibres are subjected to by their motoneurons. Transplantation of a foreign nerve containing axons which had previously innervated fast twitch fibres on to a slow twitch muscle resulted in an approximate doubling of the Na channel density in fibres innervated by the foreign nerve. These results suggest that motoneurons may exert considerable control over Na channel density in the muscle fibres they innervate.     

Low Ca2+ buffering in hypoglossal motoneurons of mutant SOD1 (G93A) mice

Mutations in the Cu/Zn superoxide dismutase (SOD1) gene are associated with amyotrophic lateral sclerosis (ALS), a fatal neurodegenerative disorder characterized by a selective degeneration of brainstem and spinal motoneurons. The pathomechanism of degeneration is still incompletely understood, but includes a disruption in cellular Ca2+ homeostasis. Here we report a quantitative microfluorometric analysis of the Ca2+ homeostasis in vulnerable hypoglossal motoneurons of neonatal mutant (G93A) SOD1 transgenic mice, a mouse model of human ALS. Ca2+ transient decay times (tau = 0.3 s), extrusion rates (gamma = 92 s(-1)) and exceptionally low intrinsic Ca2+ binding ratios (kappaS = 30) were found to be in the same range as compared to non-transgenic animals. Together with the previous observation of high Ca2+ binding ratios in ALS-resistant neurons (e.g. oculomotor), this supports the assumption that low Ca2+ buffering in vulnerable motoneurons represents a significant risk factor for degeneration. On the other hand, alterations in buffering properties by expression of mutant SOD1 are unlikely to be involved in disease initiation.     

Vesicle associated membrane protein B (VAPB) is decreased in ALS spinal cord

The aim of this study was to quantify spinal cord expression of genes known to cause familial amyotrophic lateral sclerosis (FALS) or influence survival in a large cohort of sporadic cases of ALS (SALS), in order to determine their relevance to pathogenic mechanisms occurring in SALS. The expression of superoxide dismutase 1 (SOD1), vesicle associated membrane protein (VAPB), senataxin (SETX), dynactin (DCTN1), vascular endothelial growth factor (VEGF), insulin-like growth factor-1 (IGF1), the small heat shock proteins, HSPB1 and HSPB8, and three genes activated during disease progression, caspases-1 and -3 and glial fibrillary acidic protein (GFAP), were quantified. Robust changes in the expression of four genes were found, VAPB mRNA levels were decreased in the spinal cord of ALS patients compared to controls (p<0.006), whilst HSPB1, HSPB8 and caspase-1 showed significant increases (1.5-2.3-fold). Expression of VAPB mRNA and protein was predominantly localised to large motor neurones further supporting the relevance of this finding to disease progression occurring in SALS.     

Mislocalization of TDP-43 in the G93A mutant SOD1 transgenic mouse model of ALS

Previous evidence demonstrates that TAR DNA binding protein (TDP-43) mislocalization is a key pathological feature of amyotrophic lateral sclerosis (ALS). TDP-43 normally shows nuclear localization, but in CNS tissue from patients who died with ALS this protein mislocalizes to the cytoplasm. Disease specific TDP-43 species have also been reported to include hyperphosphorylated TDP-43, as well as a C-terminal fragment. Whether these abnormal TDP-43 features are present in patients with SOD1-related familial ALS (fALS), or in mutant SOD1 over-expressing transgenic mouse models of ALS remains controversial. Here we investigate TDP-43 pathology in transgenic mice expressing the G93A mutant form of SOD1. In contrast to previous reports we observe redistribution of TDP-43 to the cytoplasm of motor neurons in mutant SOD1 transgenic mice, but this is seen only in mice having advanced disease. Furthermore, we also observe rounded TDP-43 immunoreactive inclusions associated with intense ubiquitin immunoreactivity in lumbar spinal cord at end stage disease in mSOD mice. These data indicate that TDP-43 mislocalization and ubiquitination are present in end stage mSOD mice. However, we do not observe C-terminal TDP-43 fragments nor TDP-43 hyperphosphorylated species in these end stage mSOD mice. Our findings indicate that G93A mutant SOD1 transgenic mice recapitulate some key pathological, but not all biochemical hallmarks, of TDP-43 pathology previously observed in human ALS. These studies suggest motor neuron degeneration in the mutant SOD1 transgenic mice is associated with TDP-43 histopathology.     

Amyloid precursor protein (APP) contributes to pathology in the SOD1G93A mouse model of amyotrophic lateral sclerosis

In amyotrophic lateral sclerosis (ALS), the progressive loss of motor neurons is accompanied by extensive muscle denervation, resulting in paralysis and ultimately death. Upregulation of amyloid beta (A4) precursor protein (APP) in muscle fibres coincides with symptom onset in both sporadic ALS patients and the SOD1(G93A) mouse model of familial ALS. We have further characterized this response in SOD1(G93A) mice and also revealed elevated levels of β-amyloid (Aβ) peptides in the SOD1(G93A) spinal cord, which were predominantly localized within motor neurons and their surrounding glial cells. We therefore examined the effect of genetic ablation of APP on disease progression in SOD1(G93A) mice, which significantly improved multiple disease parameters, including innervation, motor function, muscle contractile characteristics, motor unit and motor neuron survival. These results therefore strongly suggest that APP actively contributes to SOD1(G93A)-mediated pathology. Together with observations from ALS cases, this study indicates that APP may contribute to human ALS pathology.     

Autophagy dysregulation in amyotrophic lateral sclerosis

Autophagy is an intracellular lysosomal degradation process, which plays an important role in cell growth and development, and keeping cellular homeostasis in all eukaryotes. Autophagy has multiple physiological functions, including protein degradation, organelle turnover and response to stress. Emerging evidences support the notion that dysregulation of autophagy might be critical for pathogenesis of amyotrophic lateral sclerosis (ALS). The autophagy dysregulation in motor neurons of ALS may occur in different steps of the autophagic process. Recent studies have shown that two ALS associated proteins, TDP-43 and superoxide dismutase 1 (SOD1), are involved in the abnormal autophagy regulation. Furthermore, it is reported that several genetic mutations in ALS disturb the autophagic process in the motor neurons. This review will provide new evidence of autophagy dysregulation as a critical pathogenic process leading to ALS, and will discuss the prospect of future therapeutic targets using autophagic regulation to treat this disease.     

Origin and significance of small muscle fibres in neuromuscular disease

Summary Small muscle fibres, defined as those of less than 40 µm diameter in the male and 30 m in the female were encountered in muscle biopsies of patients with spinal muscular atrophy (SMA), amyotrophic lateral sclerosis (ALS), polymyositis (PM) and myopathy/dystrophy. Excessive reactivity with NADH-TR in small fibres did not discriminate between neurogenic and myopathic disorders. Quantification of perifascicular atrophic fibres, the number of nuclei in atrophic fibres, or the presence of isolated or grouped small fibres without histochemical kinship to their surrounding fibres did not aid recognition of the disease process in the groups studied. Small fibres which reacted strongly both with NADH-TR and ATPase at pH 9.4 (Type 3 fibres) constituted 38% of small fibres in the biopsies of SMA; 25% in ALS; but only 1% and 2.7% in PM and myopathy/dystrophy respectively. Thus, the presence of small Type 3 fibres in muscle biopsies may be a useful marker for neurogenic disorders in adults.     

Treatment of motoneuron degeneration by intracerebroventricular delivery of VEGF in a rat model of ALS

Neurotrophin treatment has so far failed to prolong the survival of individuals affected with amyotrophic lateral sclerosis (ALS), an incurable motoneuron degenerative disorder. Here we show that intracerebroventricular (i.c.v.) delivery of recombinant vascular endothelial growth factor (Vegf) in a SOD1(G93A) rat model of ALS delays onset of paralysis by 17 d, improves motor performance and prolongs survival by 22 d, representing the largest effects in animal models of ALS achieved by protein delivery. By protecting cervical motoneurons, i.c.v. delivery of Vegf is particularly effective in rats with the most severe form of ALS with forelimb onset. Vegf has direct neuroprotective effects on motoneurons in vivo, because neuronal expression of a transgene expressing the Vegf receptor prolongs the survival of SOD1(G93A) mice. On i.c.v. delivery, Vegf is anterogradely transported and preserves neuromuscular junctions in SOD1(G93A) rats. Our findings in preclinical rodent models of ALS may have implications for treatment of neurodegenerative disease in general.     

Amyotrophic lateral sclerosis-associated Vap33 is required for maintaining neuronal dendrite morphology and organelle distribution in Drosophila

VAMP-associated protein (VAP) is an endoplasmic reticulum (ER) membrane protein that functions as a tethering protein at the membrane contact sites between the ER and various intracellular organelles. Mutations such as P56S in human VAPB cause neurodegenerative diseases such as amyotrophic lateral sclerosis (ALS). However, VAP functions in neurons are poorly understood. Here, we utilized Drosophila olfactory projection neurons with a mosaic analysis with a repressible cell marker (MARCM) to analyze the neuronal function of Vap33, a Drosophila ortholog of human VAPB. In vap33 null mutant clones, the dendrites of projection neurons exhibited defects in the maintenance of their morphology. The subcellular localization of the Golgi apparatus and mitochondria were also abnormal. These results indicate that Vap33 is required for neuronal morphology and organelle distribution. Additionally, to examine the impact of ALS-associated mutations in neurons, we overexpressed human VAPB-P56S in vap33 null mutant clones (mosaic rescue experiments) and found that, in aged flies, human VAPB-P56S expression caused mislocalization of Bruchpilot, a presynaptic protein. These results implied that synaptic protein localization and ER quality control may be affected by disease mutations. We provide insights into the physiological and pathological functions of VAP in neurons.     

Preferential motor unit loss in the SOD1 G93A transgenic mouse model of amyotrophic lateral sclerosis

The present study investigated motor unit (MU) loss in a murine model of familial amyotrophic lateral sclerosis (ALS). The fast-twitch tibialis anterior (TA) and medial gastrocnemius (MG) muscles of transgenic SOD1(G93A) and SOD1(WT) mice were studied during the presymptomatic phase of disease progression at 60 days of age. Whole muscle maximum isometric twitch and tetanic forces were 80% lower (P < 0.01) in the TA muscles of SOD1(G93A) compared to SOD1(WT) mice. Enumeration of total MU numbers within TA muscles showed a 60% reduction (P < 0.01) within SOD1(G93A) mice (38 +/- 7) compared with SOD1(WT) controls (95 +/- 12); this was attributed to a lower proportion of the most forceful fast-fatigable (FF) MU in SOD1(G93A) mice, as seen by a significant (P < 0.01) leftward shift in the cumulative frequency histogram of single MU forces. Similar patterns of MU loss and corresponding decreases in isometric twitch force were observed in the MG. Immunocytochemical analyses of the entire cross-sectional area (CSA) of serial sections of TA muscles stained with anti-neural cell adhesion molecule (NCAM) and various monoclonal antibodies for myosin heavy chain (MHC) isoforms showed respective 65% (P < 0.01) and 28% (P < 0.05) decreases in the number of innervated IIB and IID/X muscle fibres in SOD1(G93A), which paralleled the 60% decrease (P < 0.01) in the force generating capacity of individual fibres. The loss of fast MUs was partially compensated by activity-dependent fast-to-slower fibre type transitions, as determined by increases (P < 0.04) in the CSA and proportion of IIA fibres (from 4% to 14%) and IID/X fibres (from 31% to 39%), and decreases (P < 0.001) in the CSA and proportion of type IIB fibres (from 65% to 44%). We conclude that preferential loss of IIB fibres is incomplete at 60 days of age, and is consistent with a selective albeit gradual loss of FF MUs that is not fully compensated by sprouting of the remaining motoneurons that innervate type IIA or IID/X muscle fibres. Our findings indicate that disease progression in fast-twitch muscles of SOD1(G93A) mice involves parallel processes: (1) gradual selective motor axon die-back of the FF motor units that contain large type IIB muscle fibres, and of fatigue-intermediate motor units that innervate type IID/X muscle fibres, and (2) activity-dependent conversion of motor units to those innervated by smaller motor axons innervating type IIA fatigue-resistant muscle fibres.