Pathways to motor neuron degeneration in transgenic mouse models

Amyotrophic lateral sclerosis (ALS) is an adult-onset neurological disorder characterized by the selective loss of motor neurons. A pathological hallmark of both sporadic and familial ALS is the presence of abnormal accumulations of neurofilament and peripherin proteins in motor neurons. In the past decade, transgenic mouse approaches have been used to address the role of such cytoskeletal abnormalities in motor neuron disease and also to unravel the pathogenesis caused by mutations in the gene coding for superoxide dismutase 1 (SOD1) that account for ~20% of familial ALS cases. In mouse models, disparate effects could result from different types of intermediate filament (IF) aggregates. Perikaryal IF accumulations induced by the overexpression of any of the three wild-type neurofilament proteins were quite well tolerated by motor neurons. Indeed, perikaryal swellings provoked by NF-H overexpression can even confer protection against toxicity of mutant SOD1. Other types of IF aggregates seem neurotoxic, such as those found in transgenic mice overexpressing either peripherin or an assembly-disrupting NF-L mutant. Moreover, understanding the toxicity of SOD1 mutations has been surprisingly difficult. The analysis of transgenic mice expressing mutant SOD1 has yielded complex results, suggesting that multiple pathways may contribute to disease that include the involvement of non-neuronal cells.     

Role of Wnt1 and Fzd1 in the spinal cord pathogenesis of amyotrophic lateral sclerosis-transgenic mice

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease characterized by chronic progressive degeneration of motor neurons resulting in muscular atrophy, paralysis, and ultimately death. We have investigated the expression of Wnt1 and Fzd1 in the spinal cords of SOD1G93A ALS transgenic mice, SOD1G93A-transfected N2a cells, and primary cultured astrocytes from SOD1G93A transgenic mice. In addition, we provided further insight into the role of Wnt1 and Fzd1 in the pathogenesis of ALS transgenic mice and discuss the mechanisms underlying the Wnt signal pathway which may be useful in the treatment of ALS. The results indicate the involvement of Wnt1 and Fzd1 in the pathogenesis and development of ALS.     

Late onset of motor neurons in mice overexpressing wild-type peripherin

Peripherin, a type III intermediate filament (IF) protein, upregulated by injury and inflammatory cytokines, is a component of IF inclusion bodies associated with degenerating motor neurons in sporadic amyotrophic lateral sclerosis (ALS). We report here that sustained overexpression of wild-type peripherin in mice provokes massive and selective degeneration of motor axons during aging. Remarkably, the onset of peripherin-mediated disease was precipitated by a deficiency of neurofilament light (NF-L) protein, a phenomenon associated with sporadic ALS. In NF-L null mice, the overexpression of peripherin led to early- onset formation of IF inclusions and to the selective death of spinal motor neurons at 6 mo of age. We also report the formation of similar peripherin inclusions in presymptomatic transgenic mice expressing a mutant form of superoxide dismutase linked to ALS. Taken together, these results suggest that IF inclusions containing peripherin may play a contributory role in motor neuron disease.     

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.     

Deregulation of Cdk5 in a mouse model of ALS: toxicity alleviated by perikaryal neurofilament inclusions

Recent studies suggest that increased activity of cyclin-dependent kinase 5 (Cdk5) may contribute to neuronal death and cytoskeletal abnormalities in Alzheimer's disease. We report here such deregulation of Cdk5 activity associated with the hyperphosphorylation of tau and neurofilament (NF) proteins in mice expressing a mutant superoxide dismutase (SOD1(G37R)) linked to amyotrophic lateral sclerosis (ALS). A Cdk5 involvement in motor neuron degeneration is supported by our analysis of three SOD1(G37R) mouse lines exhibiting perikaryal inclusions of NF proteins. Our results suggest that perikaryal accumulations of NF proteins in motor neurons may alleviate ALS pathogenesis by acting as a phosphorylation sink for Cdk5 activity, thereby reducing the detrimental hyperphosphorylation of tau and other neuronal substrates.     

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.     

Apoptotic death of neurons exhibiting peripherin aggregates is mediated by the proinflammatory cytokine tumor necrosis factor-alpha

Peripherin, a neuronal intermediate filament protein associated with axonal spheroids in amyotrophic lateral sclerosis (ALS), induces the selective degeneration of motor neurons when overexpressed in transgenic mice. To further clarify the selectivity and mechanism of peripherin-induced neuronal death, we analyzed the effects of peripherin overexpression in primary neuronal cultures. Peripherin overexpression led to the formation of cytoplasmic protein aggregates and caused the death not only of motor neurons, but also of dorsal root ganglion (DRG) neurons that were cultured from dissociated spinal cords of peripherin transgenic embryos. Apoptosis of DRG neurons containing peripherin aggregates was dependent on the proinflammatory central nervous system environment of spinal cultures, rich in activated microglia, and required TNF-alpha. This synergistic proapoptotic effect may contribute to neuronal selectivity in ALS.     

Enhanced Cortical Metabolic Activity in Females and Males of a Slow Progressing Mouse Model of Amyotrophic Lateral Sclerosis

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disorder with selective degeneration of motor neurons in the central nervous system. The pathophysiology of ALS is not well understood. We have used ¹H-[¹³C]-NMR spectroscopy together with an administration of [1,6-¹³C₂]glucose and [2-¹³C]acetate in female and male SOD1^G37R mice to assess neuronal and astroglial metabolic activity, respectively, in the central nervous system in ALS condition. The female (p?=?0.0008) and male (p?<?0.0001) SOD1^G37R mice exhibited decreased forelimb strength when compared with wild-type mice. There was a reduction in N-acetylaspartylglutamate level, and elevation in myo-inositol in the spinal cord of female and male SOD1^G37R mice. The transgenic male mice exhibited increased acetate oxidation in the spinal cord (p?=?0.05) and cerebral cortex (p?=?0.03), while females showed an increase in the spinal cord (p?=?0.02) only. As acetate is transported and preferentially metabolized in the astrocytes, the finding of increased rate of acetate oxidation in the transgenic mice is suggestive of astrocytic involvement in the pathogenesis of ALS. The rates of glucose oxidation in glutamatergic (p?=?0.0004) and GABAergic neurons (p?=?0.0052) were increased in the cerebral cortex of male SOD1^G37R mice when compared with the controls. The female mice showed an increase in glutamatergic (p?=?0.039) neurometabolic activity only. The neurometabolic activity was unperturbed in the spinal cord of either sex. These data suggest differential changes in neurometabolic activity across the central nervous system in SOD1^G37R mice.

     

Overexpression of Abeta is associated with acceleration of onset of motor impairment and superoxide dismutase 1 aggregation in an amyotrophic lateral sclerosis mouse model

Transgenic mice carrying mutant Cu/Zn superoxide dismutase (SOD1) recapitulate the motor impairment of human amyotrophic lateral sclerosis (ALS). The amyloid-beta (Abeta) peptide associated with Alzheimer's disease is neurotoxic. To investigate the potential role of Abeta in ALS development, we generated a double transgenic mouse line that overexpresses SOD1(G93A) and amyloid precursor protein (APP)-C100. The transgenic mouse C100.SOD1(G93A) overexpresses Abeta and shows earlier onset of motor impairment but has the same lifespan as the single transgenic SOD1(G93A) mouse. To determine the mechanism associated with this early-onset phenotype, we measured copper and zinc levels in brain and spinal cord and found both significantly elevated in the single and double transgenic mice compared with their littermate control mice. Increased glial fibrillary acidic protein and decreased APP levels in the spinal cord of C100.SOD1(G93A) mice compared with the SOD1(G93A) mice agree with the neuronal damage observed by immunohistochemical analysis. In the spinal cords of C100.SOD1(G93A) double transgenic mice, soluble Abeta was elevated in mice at end-stage disease compared with the pre-symptomatic stage. Buffer-insoluble SOD1 aggregates were significantly elevated in the pre-symptomatic mice of C100.SOD1(G93A) compared with the age-matched SOD1(G93A) mice, correlating with the earlier onset of motor impairment in the C100.SOD1(G93A) mice. This study supports abnormal SOD1 protein aggregation as the pathogenic mechanism in ALS, and implicates a potential role for Abeta in the development of ALS by exacerbating SOD1(G93A) aggregation.     

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.     

Reduced expression of BTBD10, an Akt activator, leads to motor neuron death

BTBD10, an Akt interactor, activates Akt by decreasing the protein phosphatase 2A-mediated dephosphorylation and inactivation of Akt. Overexpression of BTBD10 suppresses motor neuron death that is induced by a familial amyotrophic lateral sclerosis (ALS)-linked superoxide dismutase 1 (SOD1) mutant, G93A-SOD1 in vitro. In this study, we further investigated the BTBD10-mediated suppression of motor neuron death. We found that the small interfering RNA-mediated inhibition of BTBD10 expression led to the death of cultured motor neurons. In Caenorhabditis elegans (C. elegans), disruption of the btbd-10 gene caused not only loss of neurons, including both motor and touch-receptor neurons, but also a locomotion defect. In addition, we found that the expression of BTBD10 was generally decreased in the motor neurons from patients of sporadic ALS and transgenic mice overexpressing G93A-SOD1 (G93A-SOD1-transgenic mice). Collectively, these results suggest that the reduced expression of BTBD10 leads to motor neuron death both in vitro and in vivo.     

An immunological epitope selective for pathological monomer-misfolded SOD1 in ALS

Misfolding of Cu/Zn-superoxide dismutase (SOD1) is emerging as a mechanism underlying motor neuron degeneration in individuals with amyotrophic lateral sclerosis (ALS) who carry a mutant SOD1 gene (SOD1 ALS). Here we describe a structure-guided approach to developing an antibody that specifically recognizes monomer-misfolded forms of SOD1. We raised this antibody to an epitope that is normally buried in the SOD1 native homodimer interface. The SOD1 exposed dimer interface (SEDI) antibody recognizes only those SOD1 conformations in which the native dimer is disrupted or misfolded and thereby exposes the hydrophobic dimer interface. Using the SEDI antibody, we established the presence of monomer-misfolded SOD1 in three ALS mouse models, with G37R, G85R and G93A SOD1 mutations, and in a human individual with an A4V SOD1 mutation. Despite ubiquitous expression, misfolded SOD1 was found primarily within degenerating motor neurons. Misfolded SOD1 appeared before the onset of symptoms and decreased at the end stage of the disease, concomitant with motor neuron loss.     

Peripherin-mediated death of motor neurons rescued by overexpression of neurofilament NF-H proteins

In previous studies, we showed that overexpression of peripherin, a neuronal intermediate filament (IF) protein, in mice deficient for neurofilament light (NF-L) subunits induced a progressive adult-onset degeneration of spinal motor neurons characterized by the presence of IF inclusion bodies reminiscent of axonal spheroids found in amyotrophic lateral sclerosis (ALS). In contrast, the overexpression of human neurofilament heavy (NF-H) proteins provoked the formation of massive perikaryal IF protein accumulations with no loss of motor neurons. To further investigate the toxic properties of IF protein inclusions, we generated NF-L null mice that co-express both peripherin and NF-H transgenes. The axonal count in L5 ventral roots from 6 and 8-month-old transgenic mice showed that NF-H overexpression rescued the peripherin-mediated degeneration of motor neurons. Our analysis suggests that the protective effect of extra NF-H proteins is related to the sequestration of peripherin into the perikaryon of motor neurons, thereby abolishing the development of axonal IF inclusions that might block transport. These findings illustrate the importance of IF protein stoichiometry in formation, localization and toxicity of neuronal inclusion bodies.     

Mutant SOD1-induced neuronal toxicity is mediated by increased mitochondrial superoxide levels

Amyotrophic lateral sclerosis (ALS), the most common motor neuron disease in adults, is characterized by the selective degeneration and death of motor neurons leading to progressive paralysis and eventually death. Approximately 20% of familial ALS cases are associated with mutations in SOD1, the gene encoding Cu/Zn-superoxide dismutase (CuZnSOD). Previously, we reported that overexpression of the mitochondrial antioxidant manganese superoxide dismutase (MnSOD or SOD2) attenuates cytotoxicity induced by expression of the G37R-SOD1 mutant in a human neuroblastoma cell culture model of ALS. In the present study, we extended these earlier findings using several different SOD1 mutants (G93C, G85R, and I113T). Additionally, we tested the hypothesis that mutant SOD1 increases mitochondrial-produced superoxide (O(2) (*)) levels and that SOD2 overexpression protects neurons from mutant SOD1-induced toxicity by reducing O(2) (*) levels in mitochondria. In the present study, we demonstrate that SOD2 overexpression markedly attenuates the neuronal toxicity induced by adenovirus-mediated expression of all four SOD1 mutants (G37R, G93C, G85R, or I113T) tested. Utilizing the mitochondrial-targeted O(2) (*)-sensitive fluorogenic probe MitoSOX Red, we observed a significant increase in mitochondrial O(2) (*) levels in neural cells expressing mutant SOD1. These elevated O(2) (*) levels in mitochondria were significantly diminished by the overexpression of SOD2. These data suggest that mitochondrial-produced O(2) (*) radicals play a critical role in mutant SOD1-mediated neuronal toxicity and implicate mitochondrial-produced free radicals as potential therapeutic targets in ALS.     

MTOR-independent,autophagic enhancer trehalose prolongs motor neuron survival and ameliorates the autophagic flux defect in a mouse model of amyotrophic lateral sclerosis

Amyotrophic lateral sclerosis (ALS) is a devastating neurodegenerative disorder caused by selective motor neuron degeneration. Abnormal protein aggregation and impaired protein degradation pathways may contribute to the disease pathogenesis. Although it has been reported that autophagy is altered in patients and animal model of ALS, little is known about the role of autophagy in motor neuron degeneration in this disease. Our previous study shows that rapamycin, an MTOR-dependent autophagic activator, accelerates disease progression in the SOD1^G93A mouse model of ALS. In the present report, we have assessed the role of the MTOR-independent autophagic pathway in ALS by determining the effect of the MTOR-independent autophagic inducer trehalose on disease onset and progression, and on motor neuron degeneration in SOD1^G93A mice. We have found that trehalose significantly delays disease onset prolongs life span, and reduces motor neuron loss in the spinal cord of SOD1^G93A mice. Most importantly, we have documented that trehalose decreases SOD1 and SQSTM1/p62 aggregation, reduces ubiquitinated protein accumulation, and improves autophagic flux in the motor neurons of SOD1^G93A mice. Moreover, we have demonstrated that trehalose can reduce skeletal muscle denervation, protect mitochondria, and inhibit the proapoptotic pathway in SOD1^G93A mice. Collectively, our study indicated that the MTOR-independent autophagic inducer trehalose is neuroprotective in the ALS model and autophagosome-lysosome fusion is a possible therapeutic target for the treatment of ALS.     

Death receptor 6 (DR6) antagonist antibody is neuroprotective in the mouse SOD1G93A model of amyotrophic lateral sclerosis

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease characterized by the death of motor neurons, axon degeneration, and denervation of neuromuscular junctions (NMJ). Here we show that death receptor 6 (DR6) levels are elevated in spinal cords from post-mortem samples of human ALS and from SOD1^G93A transgenic mice, and DR6 promotes motor neuron death through activation of the caspase 3 signaling pathway. Blocking DR6 with antagonist antibody 5D10 promotes motor neuron survival in vitro via activation of Akt phosphorylation and inhibition of the caspase 3 signaling pathway, after growth factor withdrawal, sodium arsenite treatment or co-culture with SOD1^G93A astrocytes. Treatment of SOD1^G93A mice at an asymptomatic stage starting on the age of 42 days with 5D10 protects NMJ from denervation, decreases gliosis, increases survival of motor neurons and CC1⁺ oligodendrocytes in spinal cord, decreases phosphorylated neurofilament heavy chain (pNfH) levels in serum, and promotes motor functional improvement assessed by increased grip strength. The combined data provide clear evidence for neuroprotective effects of 5D10. Blocking DR6 function represents a new approach for the treatment of neurodegenerative disorders involving motor neuron death and axon degeneration, such as ALS.     

MTOR-independent,autophagic enhancer trehalose prolongs motor neuron survival and ameliorates the autophagic flux defect in a mouse model of amyotrophic lateral sclerosis

Amyotrophic lateral sclerosis (ALS) is a devastating neurodegenerative disorder caused by selective motor neuron degeneration. Abnormal protein aggregation and impaired protein degradation pathways may contribute to the disease pathogenesis. Although it has been reported that autophagy is altered in patients and animal model of ALS, little is known about the role of autophagy in motor neuron degeneration in this disease. Our previous study shows that rapamycin, an MTOR-dependent autophagic activator, accelerates disease progression in the SOD1^G93A mouse model of ALS. In the present report, we have assessed the role of the MTOR-independent autophagic pathway in ALS by determining the effect of the MTOR-independent autophagic inducer trehalose on disease onset and progression, and on motor neuron degeneration in SOD1^G93A mice. We have found that trehalose significantly delays disease onset prolongs life span, and reduces motor neuron loss in the spinal cord of SOD1^G93A mice. Most importantly, we have documented that trehalose decreases SOD1 and SQSTM1/p62 aggregation, reduces ubiquitinated protein accumulation, and improves autophagic flux in the motor neurons of SOD1^G93A mice. Moreover, we have demonstrated that trehalose can reduce skeletal muscle denervation, protect mitochondria, and inhibit the proapoptotic pathway in SOD1^G93A mice. Collectively, our study indicated that the MTOR-independent autophagic inducer trehalose is neuroprotective in the ALS model and autophagosome-lysosome fusion is a possible therapeutic target for the treatment of ALS.     

Characterizing the multiple roles of FGF-2 in SOD1G93A ALS mice in vivo and in vitro

We have previously shown that knockout of fibroblast growth factor-2 (FGF-2) and potential compensatory effects of other growth factors result in amelioration of disease symptoms in a transgenic mouse model of amyotrophic lateral sclerosis (ALS). ALS is a rapidly progressive neurological disorder leading to degeneration of cortical, brain stem, and spinal motor neurons followed by subsequent denervation and muscle wasting. Mutations in the superoxide dismutase 1 (SOD1) gene are responsible for approximately 20% of familial ALS cases and SOD1 mutant mice still are among the models best mimicking clinical and neuropathological characteristics of ALS. The aim of the present study was a thorough characterization of FGF-2 and other growth factors and signaling effectors in vivo in the SOD1^G93A mouse model. We observed tissue-specific opposing gene regulation of FGF-2 and overall dysregulation of other growth factors, which in the gastrocnemius muscle was associated with reduced downstream extracellular-signal-regulated kinases (ERK) and protein kinase B (AKT) activation. To further investigate whether the effects of FGF-2 on motor neuron death are mediated by glial cells, astrocytes lacking FGF-2 were cocultured together with mutant SOD1 ^G93A motor neurons. FGF-2 had an impact on motor neuron maturation indicating that astrocytic FGF-2 affects motor neurons at a developmental stage. Moreover, neuronal gene expression patterns showed FGF-2- and SOD1 ^G93A-dependent changes in ciliary neurotrophic factor, glial-cell-line-derived neurotrophic factor, and ERK2, implying a potential involvement in ALS pathogenesis before the onset of clinical symptoms.