dnc-1/dynactin 1 Knockdown Disrupts Transport of Autophagosomes and Induces Motor Neuron Degeneration

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease characterized by the progressive loss of motor neurons. We previously showed that the expression of dynactin 1, an axon motor protein regulating retrograde transport, is markedly reduced in spinal motor neurons of sporadic ALS patients, although the mechanisms by which decreased dynactin 1 levels cause neurodegeneration have yet to be elucidated. The accumulation of autophagosomes in degenerated motor neurons is another key pathological feature of sporadic ALS. Since autophagosomes are cargo of dynein/dynactin complexes and play a crucial role in the turnover of several organelles and proteins, we hypothesized that the quantitative loss of dynactin 1 disrupts the transport of autophagosomes and induces the degeneration of motor neuron. In the present study, we generated a Caenorhabditis elegans model in which the expression of DNC-1, the homolog of dynactin 1, is specifically knocked down in motor neurons. This model exhibited severe motor defects together with axonal and neuronal degeneration. We also observed impaired movement and increased number of autophagosomes in the degenerated neurons. Furthermore, the combination of rapamycin, an activator of autophagy, and trichostatin which facilitates axonal transport dramatically ameliorated the motor phenotype and axonal degeneration of this model. Thus, our results suggest that decreased expression of dynactin 1 induces motor neuron degeneration and that the transport of autophagosomes is a novel and substantial therapeutic target for motor neuron degeneration.     

Neuromuscular effects of G93A-SOD1 expression in zebrafish

ABSTRACT: BACKGROUND: Amyotrophic lateral sclerosis (ALS) is a fatal disorder involving the degeneration and loss of motor neurons. The mechanisms of motor neuron loss in ALS are unknown and there are no effective treatments. Defects in the distal axon and at the neuromuscular junction are early events in the disease course, and zebrafish provide a promising in vivo system to examine cellular mechanisms and treatments for these events in ALS pathogenesis. RESULTS: We demonstrate that transient genetic manipulation of zebrafish to express G93A-SOD1, a mutation associated with familial ALS, results in early defects in motor neuron outgrowth and axonal branching. This is consistent with previous reports on motor neuron axonal defects associated with familial ALS genes following knockdown or mutant protein overexpression. We also demonstrate that upregulation of growth factor signaling is capable of rescuing these early defects, validating the potential of the model for therapeutic discovery. We generated stable transgenic zebrafish lines expressing G93A-SOD1 to further characterize the consequences of G93A-SOD1 expression on neuromuscular pathology and disease progression. Behavioral monitoring reveals evidence of motor dysfunction and decreased activity in transgenic ALS zebrafish. Examination of neuromuscular and neuronal pathology throughout the disease course reveals a loss of neuromuscular junctions and alterations in motor neuron innervations patterns with disease progression. Finally, motor neuron cell loss is evident later in the disease. CONCLUSIONS: This sequence of events reflects the stepwise mechanisms of degeneration in ALS, and provides a novel model for mechanistic discovery and therapeutic development for neuromuscular degeneration in ALS.     

EGFR Inhibitor Erlotinib Delays Disease Progression but Does Not Extend Survival in the SOD1 Mouse Model of ALS

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease that causes progressive paralysis due to motor neuron death. Several lines of published evidence suggested that inhibition of epidermal growth factor receptor (EGFR) signaling might protect neurons from degeneration. To test this hypothesis in vivo, we treated the SOD1 transgenic mouse model of ALS with erlotinib, an EGFR inhibitor clinically approved for oncology indications. Although erlotinib failed to extend ALS mouse survival it did provide a modest but significant delay in the onset of multiple behavioral measures of disease progression. However, given the lack of protection of motor neuron synapses and the lack of survival extension, the small benefits observed after erlotinib treatment appear purely symptomatic, with no modification of disease course.     

Increased Axonal Ribosome Numbers Is an Early Event in the Pathogenesis of Amyotrophic Lateral Sclerosis

Myelinating glia cells support axon survival and functions through mechanisms independent of myelination, and their dysfunction leads to axonal degeneration in several diseases. In amyotrophic lateral sclerosis (ALS), spinal motor neurons undergo retrograde degeneration, and slowing of axonal transport is an early event that in ALS mutant mice occurs well before motor neuron degeneration. Interestingly, in familial forms of ALS, Schwann cells have been proposed to slow disease progression. We demonstrated previously that Schwann cells transfer polyribosomes to diseased and regenerating axons, a possible rescue mechanism for disease-induced reductions in axonal proteins. Here, we investigated whether elevated levels of axonal ribosomes are also found in ALS, by analysis of a superoxide dismutase 1 (SOD1)^G93A mouse model for human familial ALS and a patient suffering from sporadic ALS. In both cases, we found that the disorder was associated with an increase in the population of axonal ribosomes in myelinated axons. Importantly, in SOD1^G93A mice, the appearance of axonal ribosomes preceded the manifestation of behavioral symptoms, indicating that upregulation of axonal ribosomes occurs early in the pathogenesis of ALS. In line with our previous studies, electron microscopy analysis showed that Schwann cells might serve as a source of axonal ribosomes in the disease-compromised axons. The early appearance of axonal ribosomes indicates an involvement of Schwann cells early in ALS neuropathology, and may serve as an early marker for disease-affected axons, not only in ALS, but also for other central and peripheral neurodegenerative disorders.     

Focal degeneration of astrocytes in amyotrophic lateral sclerosis

Astrocytes emerge as key players in motor neuron degeneration in Amyotrophic Lateral Sclerosis (ALS). Whether astrocytes cause direct damage by releasing toxic factors or contribute indirectly through the loss of physiological functions is unclear. Here we identify in the hSOD1(G93A) transgenic mouse model of ALS a degenerative process of the astrocytes, restricted to those directly surrounding spinal motor neurons. This phenomenon manifests with an early onset and becomes significant concomitant with the loss of motor cells and the appearance of clinical symptoms. Contrary to wild-type astrocytes, mutant hSOD1-expressing astrocytes are highly vulnerable to glutamate and undergo cell death mediated by the metabotropic type-5 receptor (mGluR5). Blocking mGluR5 in vivo slows down astrocytic degeneration, delays the onset of the disease and slightly extends survival in hSOD1(G93A) transgenic mice. We propose that excitotoxicity in ALS affects both motor neurons and astrocytes, favouring their local interactive degeneration. This new mechanistic hypothesis has implications for therapeutic interventions.     

Neurofilaments and motor neuron disease

Amyotrophic lateral sclerosis (ALS) is an adult-onset and heterogeneous neurological disorder that affects primarily motor neurons in the brain and spinal cord. Although multiple genetic and environmental factors might be implicated in ALS, the striking similarities in the clinical and pathological features of sporadic ALS and familial ALS suggest that similar mechanisms of disease may occur. A common and perhaps universal pathological finding in ALS is the presence of abnormal accumulations of neurofilaments (often called spheroids or Lewy body-like deposits) in the cell body and proximal axon of surviving motor neurons. Such neurofilament deposits have been widely viewed as a consequence of neuronal dysfunction, perhaps reflecting axonal transport defects. This review discusses the emerging evidence, based primarily on transgenic mouse studies and on the discovery of deletion mutations in a neurofilament gene associated with ALS, that neurofilament proteins can play a causative role in motor neuron disease.     

A mutation in dynein rescues axonal transport defects and extends the life span of ALS mice

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative condition characterized by motoneuron degeneration and muscle paralysis. Although the precise pathogenesis of ALS remains unclear, mutations in Cu/Zn superoxide dismutase (SOD1) account for approximately 20-25% of familial ALS cases, and transgenic mice overexpressing human mutant SOD1 develop an ALS-like phenotype. Evidence suggests that defects in axonal transport play an important role in neurodegeneration. In Legs at odd angles (Loa) mice, mutations in the motor protein dynein are associated with axonal transport defects and motoneuron degeneration. Here, we show that retrograde axonal transport defects are already present in motoneurons of SOD1(G93A) mice during embryonic development. Surprisingly, crossing SOD1(G93A) mice with Loa/+ mice delays disease progression and significantly increases life span in Loa/SOD1(G93A) mice. Moreover, there is a complete recovery in axonal transport deficits in motoneurons of these mice, which may be responsible for the amelioration of disease. We propose that impaired axonal transport is a prime cause of neuronal death in neurodegenerative disorders such as ALS.     

Mitochondrial dysfunction in amyotrophic lateral sclerosis

The etiology of motor neuron degeneration in amyotrophic lateral sclerosis (ALS) remains to be better understood. Based on the studies from ALS patients and transgenic animal models, it is believed that ALS is likely to be a multifactorial and multisystem disease. Many mechanisms have been postulated to be involved in the pathology of ALS, such as oxidative stress, glutamate excitotoxicity, mitochondrial damage, defective axonal transport, glia cell pathology and aberrant RNA metabolism. Mitochondria, which play crucial roles in excitotoxicity, apoptosis and cell survival, have shown to be an early target in ALS pathogenesis and contribute to the disease progression. Morphological and functional defects in mitochondria were found in both human patients and ALS mice overexpressing mutant SOD1. Mutant SOD1 was found to be preferentially associated with mitochondria and subsequently impair mitochondrial function. Recent studies suggest that axonal transport of mitochondria along microtubules and mitochondrial dynamics may also be disrupted in ALS. These results also illustrate the critical importance of maintaining proper mitochondrial function in axons and neuromuscular junctions, supporting the emerging “dying-back” axonopathy model of ALS. In this review, we will discuss how mitochondrial dysfunction has been linked to the ALS variants of SOD1 and the mechanisms by which mitochondrial damage contributes to the disease etiology.     

The wobbler mouse, an ALS animal model

This review article is focused on the research progress made utilizing the wobbler mouse as animal model for human motor neuron diseases, especially the amyotrophic lateral sclerosis (ALS). The wobbler mouse develops progressive degeneration of upper and lower motor neurons and shows striking similarities to ALS. The cellular effects of the wobbler mutation, cellular transport defects, neurofilament aggregation, neuronal hyperexcitability and neuroinflammation closely resemble human ALS. Now, 57 years after the first report on the wobbler mouse we summarize the progress made in understanding the disease mechanism and testing various therapeutic approaches and discuss the relevance of these advances for human ALS. The identification of the causative mutation linking the wobbler mutation to a vesicle transport factor and the research focussed on the cellular basis and the therapeutic treatment of the wobbler motor neuron degeneration has shed new light on the molecular pathology of the disease and might contribute to the understanding the complexity of ALS.     

VEGF reduces astrogliosis and preserves neuromuscular junctions in ALS transgenic mice

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease resulting from motor neuron loss in the spinal cord and brain stem. In the present study, we found that systemic administration of recombinant vascular endothelial growth factor (VEGF) significantly diminished astrogliosis and increased the number of neuromuscular junctions in a Cu/Zn superoxide dismutase (SOD1) transgenic mouse model of ALS. Our results thus demonstrate a novel regulatory role of VEGF on astrocytes and are suggestive of protective effects of VEGF both in the peripheral and central nervous system in the SOD1 transgenic mouse model. These findings warrant further evaluation of the mechanism(s) of regulatory effects of VEGF on neuronal and non-neuronal cells, and the relation of these events to motor neuron degeneration and the onset and progression of ALS.     

Peroxisome proliferator activator receptor gamma coactivator-1alpha (PGC-1α) improves motor performance and survival in a mouse model of amyotrophic lateral sclerosis

Background

Amyotrophic lateral sclerosis (ALS) is a devastating neurodegenerative disease that affects spinal cord and cortical motor neurons. An increasing amount of evidence suggests that mitochondrial dysfunction contributes to motor neuron death in ALS. Peroxisome proliferator-activated receptor gamma co-activator-1α (PGC-1α) is a principal regulator of mitochondrial biogenesis and oxidative metabolism.

Results

In this study, we examined whether PGC-1α plays a protective role in ALS by using a double transgenic mouse model where PGC-1α is over-expressed in an SOD1 transgenic mouse (TgSOD1-G93A/PGC-1α). Our results indicate that PGC-1α significantly improves motor function and survival of SOD1-G93A mice. The behavioral improvements were accompanied by reduced blood glucose level and by protection of motor neuron loss, restoration of mitochondrial electron transport chain activities and inhibition of stress signaling in the spinal cord.

Conclusion

Our results demonstrate that PGC-1α plays a beneficial role in a mouse model of ALS, suggesting that PGC-1α may be a potential therapeutic target for ALS therapy.     

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.     

Increased mitochondrial antioxidative activity or decreased oxygen free radical propagation prevent mutant SOD1-mediated motor neuron cell death and increase amyotrophic lateral sclerosis-like transgenic mouse survival

The molecular mechanisms of selective motor neuron degeneration in human amyotrophic lateral sclerosis (ALS) disease remain largely unknown and effective therapies are not currently available. Mitochondrial dysfunction is an early event of motor neuron degeneration in transgenic mice overexpressing mutant superoxide dismutase (SOD)1 gene and mitochondrial abnormality is observed in human ALS patients. In an in vitro cell culture system, we demonstrated that infection of mouse NSC-34 motor neuron-like cells with adenovirus containing mutant G93A-SOD1 gene increased cellular oxidative stress, mitochondrial dysfunction, cytochrome c release and motor neuron cell death. Cells pretreated with highly oxidizable polyunsaturated fatty acid elevated lipid peroxidation and synergistically exacerbated motor neuron-like cell death with mutant G93A-SOD1 but not with wild-type SOD1. Similarly, overexpression of mitochondrial antioxidative genes, MnSOD and GPX4 by stable transfection significantly increased NSC-34 motor neuron-like cell resistance to mutant SOD1. Pre-incubation of cells with spin trapping molecule, 5',5'-dimethylpryrroline-N-oxide (DMPO), prevented mutant SOD1-mediated mitochondrial dysfunction and cell death. Furthermore, treatment of mutant G93A-SOD1 transgenic mice with DMPO significantly delayed paralysis and increased survival. These findings suggest a causal relationship between enhanced oxidative stress and mutant SOD1-mediated motor neuron degeneration, considering that enhanced oxygen free radical production results from the SOD1 structural alterations. Molecular approaches aimed at increasing mitochondrial antioxidative activity or effectively blocking oxidative stress propagation can be potentially useful in the clinical management of human ALS disease.     

Proteomic analysis of 4-hydroxy-2-nonenal-modified proteins in G93A-SOD1 transgenic mice--a model of familial amyotrophic lateral sclerosis

Amyotrophic lateral sclerosis (ALS) is an age-related, fatal motor neuron degenerative disease occurring both sporadically (sALS) and heritably (fALS), with inherited cases accounting for approximately 10% of diagnoses. Although multiple mechanisms likely contribute to the pathogenesis of motor neuron injury in ALS, recent advances suggest that oxidative stress may play a significant role in the amplification, and possibly the initiation, of the disease. Lipid peroxidation is one of the several outcomes of oxidative stress. Since the central nervous system (CNS) is enriched with polyunsaturated fatty acids, it is particularly vulnerable to membrane-associated oxidative stress. Peroxidation of cellular membrane lipids or circulating lipoprotein molecules generates highly reactive aldehydes, among which is 4-hydroxy-2-nonenal (HNE). HNE levels are increased in spinal cord motor neurons of ALS patients, indicating that lipid peroxidation is associated with the motor neuron degeneration in ALS. In the present study, we used a parallel proteomic approach to identify HNE-modified proteins in the spinal cord tissue of a model of fALS, G93A-SOD1 transgenic mice, in comparison to the nontransgenic mice. We found three significantly HNE-modified proteins in the spinal cord of G93A-SOD1 transgenic mice: dihydropyrimidinase-related protein 2 (DRP-2), heat-shock protein 70 (Hsp70), and possibly alpha-enolase. These results support the role of oxidative stress as a major mechanism in the pathogenesis of ALS. Structural alteration and activity decline of functional proteins may consistently contribute to the neurodegeneration process in ALS.     

Inhibition of chaperone activity is a shared property of several Cu,Zn-superoxide dismutase mutants that cause amyotrophic lateral sclerosis

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease characterized by progressive motor neuron degeneration, paralysis, and death. Mutant Cu,Zn-superoxide dismutase (SOD1) causes a subset of ALS by an unidentified toxic property. Increasing evidence suggests that chaperone dysfunction plays a role in motor neuron degeneration in ALS. To investigate the relationship between mutant SOD1 expression and chaperone dysfunction, we measured chaperone function in central nervous system tissue lysates from normal mice and transgenic mice expressing human SOD1 variants. We observed a significant decrease in chaperone activity in tissues from mice expressing ALS-linked mutant SOD1 but not control mice expressing human wild type SOD1. This decrease was detected only in the spinal cord, became apparent by 60 days of age (before the onset of muscle weakness and significant motor neuron loss), and persisted throughout the late stages. In addition, this impairment of chaperone activity occurred only in cytosolic but not in mitochondrial and nuclear fractions. Furthermore, multiple recombinant human SOD1 mutants with differing biochemical and biophysical properties inhibited chaperone function in a cell-free extract of normal mouse spinal cords. Thus, mutant SOD1 proteins may impair chaperone function independent of gene expression in vivo, and this inhibition may be a shared property of ALS-linked mutant SOD1 proteins.     

Transgenic mouse models of amyotrophic lateral sclerosis

The discovery of missense mutations in the gene coding for the Cu/Zn superoxide dismutase 1 (SOD1) in subsets of familial cases was rapidly followed by the generation of transgenic mice expressing various forms of SOD1 mutants. The mice overexpressing high levels of mutant SOD1 mRNAs do develop motor neuron disease but unraveling the mechanisms of pathogenesis has been very challenging. Studies with mouse lines suggest that the toxicity of mutant SOD1 is unrelated to copper-mediated catalysis but rather to propensity of a subfraction of mutant SOD1 proteins to form misfolded protein species and aggregates. However, the mechanism of toxicity of SOD1 mutants remains to be elucidated. Involvement of cytoskeletal components in ALS pathogenesis is supported by several mouse models of motor neuron disease with neurofilament abnormalities and with genetic defects in microtubule-based transport. Here, we describe how transgenic mouse models have been used for understanding pathogenic pathways of motor neuron disease and for pre-clinical drug testing.     

Species-dependent neuropathology in transgenic SOD1 pigs

Mutations in the human copper/zinc superoxide dismutase 1 (hSOD1) gene cause familial amyotrophic lateral sclerosis (ALS). It remains unknown whether large animal models of ALS mimic more pathological events seen in ALS patients via novel mechanisms. Here, we report the generation of transgenic pigs expressing mutant G93A hSOD1 and showing hind limb motor defects, which are germline transmissible, and motor neuron degeneration in dose- and age-dependent manners. Importantly, in the early disease stage, mutant hSOD1 did not form cytoplasmic inclusions, but showed nuclear accumulation and ubiquitinated nuclear aggregates, as seen in some ALS patient brains, but not in transgenic ALS mouse models. Our findings revealed that SOD1 binds PCBP1, a nuclear poly(rC) binding protein, in pig brain, but not in mouse brain, suggesting that the SOD1-PCBP1 interaction accounts for nuclear SOD1 accumulation and that species-specific targets are key to ALS pathology in large mammals and in humans.     

Defective axonal transport of neurofilament proteins in neurons overexpressing peripherin

Peripherin is a type III neuronal intermediate filament detected in motor neuron inclusions of amyotrophic lateral sclerosis (ALS) patients. We previously reported that overexpression of peripherin provokes late-onset motor neuron dysfunction in transgenic mice. Here, we show that peripherin overexpression slows down axonal transport of neurofilament (NF) proteins, and that the transport defect precedes by several months the appearance of axonal spheroids in adult mice. Defective NF transport by peripherin up-regulation was further confirmed with dorsal root ganglia (DRG) neurons cultured from peripherin transgenic embryos. Immunofluorescence microscopy and western blotting revealed that excess peripherin provokes reduction in levels of hyperphosphorylated NF-H species in DRG neurites. Similarly the transport of a green fluorescent protein (GFP)-tagged NF-M, delivered by means of a lentiviral construct, was impaired in DRG neurites overexpressing peripherin. These results demonstrate that peripherin overexpression can cause defective transport of type IV NF proteins, a phenomenon that may account for the progressive formation of ALS-like spheroids in axons.     

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.     

Amyotrophic lateral sclerosis: all roads lead to Rome

Amyotrophic lateral sclerosis (ALS) is the most frequent adult-onset motor neuron disease characterized by degeneration of upper and lower motor neurons, generalized weakness and muscle atrophy. Most cases of ALS appear sporadically but some forms of the disease result from mutations in the gene encoding the antioxidant enzyme Cu/Zn superoxide dismutase (SOD1). Several other mutated genes have also been found to predispose to ALS including, among others, one that encodes the regulator of axonal retrograde transport dynactin. As all roads lead to the proverbial Rome, we discuss here how distinct molecular pathways may converge to the same final result that is motor neuron death. We critically review the basic research on SOD1-linked ALS to propose a pioneering model of a 'systemic' form of the disease, causally involving multiple cell types, either neuronal or non-neuronal. Contrasting this, we also postulate that other neuron-specific defects, as those triggered by dynactin dysfunction, may account for a primary motor neuron disease that would represent 'pure' neuronal forms of ALS. Identifying different disease subtypes is an unavoidable step toward the understanding of the physiopathology of ALS and will hopefully help to design specific treatments for each subset of patients.