RNAi and neurological disease]

In many of autosomal dominant diseases such as familial amyotrophic lateral sclerosis (ALS) with Cu/Zn superoxide dismutase (SOD1) mutation, a missense point mutation may induce the disease by its gain of adverse property. Similar 'gain of toxic function' of mutant protein is predicted to cause cell death in other autosomal dominant neurodegenerative diseases such as familial Alzheimer disease, prion disease, polyglutamine diseases and Parkinson disease. In all these familial diseases, one rational approach to therapy is to develop a method to specifically eliminate the aberrant protein. Duplex of 21-nt RNA, known as siRNA, has recently emerged as a powerful tool to silence gene. Mutant-allele specific gene silencing with siRNA was showed in familial ALS and Machado-Joseph diseases. We made the transgenic (Tg) mouse of modified small interfering RNA (siRNA). By crossing this anti-SOD1 siRNA Tg mouse with a SOD1(G93A) Tg mouse as a model for ALS, siRNA halted the development of disease by inhibiting mutant G93A SOD1. Our results support the feasibility of utilizing siRNA-based gene therapy of neurodegenerative diseases of autosomal dominant inheritance.     

In vivo pathogenic role of mutant SOD1 localized in the mitochondrial intermembrane space

Mutations in Cu,Zn superoxide dismutase (SOD1) are associated with familial amyotrophic lateral sclerosis (ALS). Mutant SOD1 causes a complex array of pathological events, through toxic gain of function mechanisms, leading to selective motor neuron degeneration. Mitochondrial dysfunction is among the well established toxic effects of mutant SOD1, but its mechanisms are just starting to be elucidated. A portion of mutant SOD1 is localized in mitochondria, where it accumulates mostly on the outer membrane and inside the intermembrane space (IMS). Evidence in cultured cells suggests that mutant SOD1 in the IMS causes mitochondrial dysfunction and compromises cell viability. Therefore, to test its pathogenic role in vivo we generated transgenic mice expressing G93A mutant or wild-type (WT) human SOD1 targeted selectively to the mitochondrial IMS (mito-SOD1). We show that mito-SOD1 is correctly localized in the IMS, where it oligomerizes and acquires enzymatic activity. Mito-G93ASOD1 mice, but not mito-WTSOD1 mice, develop a progressive disease characterized by body weight loss, muscle weakness, brain atrophy, and motor impairment, which is more severe in females. These symptoms are associated with reduced spinal motor neuron counts and impaired mitochondrial bioenergetics, characterized by decreased cytochrome oxidase activity and defective calcium handling. However, there is no evidence of muscle denervation, a cardinal pathological feature of ALS. Together, our findings indicate that mutant SOD1 in the mitochondrial IMS causes mitochondrial dysfunction and neurodegeneration, but per se it is not sufficient to cause a full-fledged ALS phenotype, which requires the participation of mutant SOD1 localized in other cellular compartments.     

Enhanced expression of erythropoietin in the central nervous system of SOD1(G93A) transgenic mice

In the present study, we investigated the changes of erythropoietin (Epo) expression in the central nervous system (CNS) of SOD1(G93A) transgenic mice as an in vivo model of amyotrophic lateral sclerosis (ALS). In wild-type SOD1 (wtSOD1) transgenic mice, little immunoreactivity was found in all cortical regions. In the cerebral cortex of symptomatic SOD1(G93A) transgenic mice, there was a significant increase in Epo immunoreactivity. In the hippocampal formation, layer-specific alterations in the staining intensity were observed in the CA1-3 areas and dentate gyrus. Epo immunoreactivity was significantly increased in the midbrain, cerebellar cortex and brainstem of SOD1(G93A) transgenic mice. On the contrary, Epo immunoreactivity was moderately stained in the spinal cord and was not different between wtSOD1 and SOD1(G93A) transgenic mice at the age of 8 weeks, 13 weeks and 18 weeks. In the staining of Epo receptor (EpoR), the changing pattern was similar with that of Epo in the spinal cord and hippocampal formation in wtSOD1 and SOD1(G93A) transgenic mice. Although further studies of functional features of Epo in ALS are needed, the first demonstration of increased immunoreactivity for Epo in the CNS of SOD1(G93A) transgenic mice may provide initial insights into the development of interventional strategies to alleviate motor neuron degeneration in human ALS.     

Survival in transgenic ALS mice does not vary with CNS glutathione peroxidase activity

OBJECTIVE: Transgenic mice that overexpress a human gene encoding mutant cytosolic superoxide dismutase (SOD1) develop a progressive motor neuron loss that resembles human ALS. Why mutant SOD1 initiates motor neuron death is unknown. One hypothesis proposes that the mutant molecule has enhanced peroxidase activity, reducing hydrogen peroxide (H2O2) to form toxic hydroxyl adducts on critical targets. To test this hypothesis, the authors generated transgenic ALS mice with altered levels of glutathione peroxidase (GSHPx), the major soluble enzyme that detoxifies H2O2. METHODS: SOD1(G93A) ALS mice were bred with mice bearing a murine GSHPx transgene that have a four-fold elevation in brain GSHPx levels and with mice having targeted inactivation of the GSHPx gene and reduced brain GSHPx activity. RESULTS: Survival was not prolonged in ALS mice with elevated brain GSHPx activity (p = 0.09). ALS mice with decreased GSHPx brain activity (20% of normal) showed no acceleration of the disease course (p = 0.89). The age at disease onset in the ALS mice was unaffected by brain GSHPx activity. CONCLUSION: The level of GSHPx activity in the CNS of transgenic ALS mice does not play a critical role in the development of motor neuron disease.     

Immunohistochemical study on the distribution of insulin-like growth factor I (IGF-I) receptor in the central nervous system of SOD1(G93A) mutant transgenic mice

In the present study, we used the SOD1(G93A) mutant transgenic mice as an in vivo model of ALS and performed immunohistochemical studies to investigate the changes of insulin-like growth factor I (IGF-I) receptor in the central nervous system. IGF-I receptor-immunoreactive astrocytes were detected in the spinal cord, brainstem, central gray and cerebellar nuclei of SOD1(G93A) transgenic mice. In contrast to transgenic mice, no IGF-I receptor-immunoreactive astrocytes were observed in any brain region of wtSOD1 transgenic mice although a few moderately stained neurons were observed. In the hippocampal formation of SOD1(G93A) transgenic mice, IGF-I receptor immunoreactivity was increased in the pyramidal cells of the CA1-3 regions and granule cells of the dentate gyrus. The present study provides the first evidence that IGF-I receptor immunoreactivity was increased in reactive astrocytes in the central nervous system of SOD(G93A) transgenic mice, suggesting that reactive astrocytes may play an important role in the pathogenesis and progress of ALS. The mechanisms underlying the increased immunoreactivity for IGF-I receptor, and the functional implications of these increases, require elucidation.     

Human Cu/Zn Superoxide Dismutase (SOD1) Overexpression in Mice Causes Mitochondrial Vacuolization, Axonal Degeneration, and Premature Motoneuron Death and Accelerates Motoneuron Disease in Mice Expressing a Familial Amyotrophic Lateral Sclerosis Mutant SOD1

Cytosolic Cu/Zn superoxide dismutase (SOD1) is a ubiquitous small cytosolic metalloenzyme that catalyzes the conversion of superoxide anion to hydrogen peroxide (H₂O₂). Mutations in the SOD1 gene cause a familial form of amyotrophic lateral sclerosis (fALS). The mechanism by which mutant SOD1s causes ALS is not understood. Transgenic mice expressing multiple copies of fALS-mutant SOD1s develop an ALS-like motoneuron disease resembling ALS. Here we report that transgenic mice expressing a high concentration of wild-type human SOD1 (hSOD1_WT) develop an array of neurodegenerative changes consisting of (1) swelling and vacuolization of mitochondria, predominantly in axons in the spinal cord, brain stem, and subiculum; (2) axonal degeneration in a number of long fiber tracts, predominantly the spinocerebellar tracts; and (3) at 2 years of age, a moderate loss of spinal motoneurons. Parallel to the development of neurodegenerative changes, hSOD1_WT mice also develop mild motor abnormalities. Interestingly, mitochondrial vacuolization was associated with accumulation of hSOD1 immunoreactivity, suggesting that the development of mitochondrial pathology is associated with disturbed SOD1 turnover. In this study we also crossed hSOD1_WT mice with a line of fALS-mutant SOD1 mice (hSOD1_G93A) to generate “double” transgenic mice that express high levels of both wild-type and G93A mutant hSOD1. The “double” transgenic mice show accelerated motoneuron death, earlier onset of paresis, and earlier death as compared with hSOD1_G93A littermates. Thus in vivo expression of high levels of wild-type hSOD1 is not only harmful to neurons in itself, but also increases or facilitates the deleterious action of a fALS-mutant SOD1. Our data indicate that it is important for motoneurons to control the SOD1 concentration throughout their processes, and that events that lead to improper synthesis, transport, or breakdown of SOD1 causing its accumulation are potentially dangerous.     

Decreased expression of calretinin in the cerebral cortex and hippocampus of SOD1G93A transgenic mice

In the present study, we investigated the changes of calretinin (CR) expression in the central nervous system of SOD1G93A transgenic mice as an in vivo model of amyotrophic lateral sclerosis (ALS). In wild-type SOD1 (wtSOD1) transgenic mice, many CR-immunoreactive neurons were found in all cortical regions. In the cerebral cortex of SOD1G93A transgenic mice, the number and staining intensity of CR-positive neurons were decreased. In the hippocampal formation, layer-specific alterations in the staining intensity of CR-immunoreactive neurons were observed in the CA1-3 areas and dentate gyrus. In wtSOD1 transgenic mice, CR-immunoreactive neurons with long processes were found in the stratum oriens and stratum radiatum of CA1-3 areas, and heavily stained band-like molecular layer was prominent in the dentate gyrus. CR immunoreactivity was decreased in each layer of CA1-3 areas and dentate gyrus of SOD1G93A transgenic mice. The first demonstration of decreased immunoreactivity for CR in the cerebral cortex and hippocampus of SOD1G93A transgenic mice may provide insights into the pathogenesis of motor neuron degeneration in human ALS although further quantitative studies are needed.     

Aggregation of ubiquitin and a mutant ALS-linked SOD1 protein correlate with disease progression and fragmentation of the Golgi apparatus

Transgenic mice that express the G93A mutation of human Cu,Zn superoxide dismutase (SOD1(G93A)), found in familial amyotrophic lateral sclerosis (FALS), showed clinical symptoms and histopathological changes of sporadic ALS, including fragmentation of the neuronal Golgi apparatus (GA). The finding of fragmented neuronal GA in asymptomatic mice, months before the onset of paralysis, suggests that the GA is an early target of the pathological processes causing neuronal degeneration. Transgenic mice expressing human SOD1(G93A) have aggregates of mutant protein and ubiquitin in neuronal and glial cytoplasm; they appeared first in the neuropil and later in the perikarya of motor neurons, where they were adjacent to fragmented GA. The aggregates of SOD1(G93A) appeared in neuronal perikarya of asymptomatic mice containing fragmented GA. The numbers of neurons with deposits of SOD1(G93A) and fragmented GA progressively increased with age. Immuno-electron microscopy using colloidal gold showed labeling of ubiquitin and SOD1 over 13 nm thick cytoplasmic filaments. Spinal cord extracts showed a 20-fold increase of SOD1(G93A) in transgenic mice compared to the wild-type protein in controls. The results suggest a causal relationship between the aggregation of mutant SOD1 and ubiquitin, fragmentation of the Golgi apparatus of motor neurons and neurodegeneration.     

DJ-1 Changes in G93A-SOD1 Transgenic Mice: Implications for Oxidative Stress in ALS

Amyotrophic lateral sclerosis (ALS) is a progressive, lethal, neurodegenerative disorder. The causes of ALS are still obscure. Accumulating evidence supports the hypothesis that oxidative stress and mitochondrial dysfunction can be implicated in ALS pathogenesis. DJ-1 plays an important role in the oxidative stress response. The aim of this study was to discover whether there are changes in DJ-1 expression or in DJ-1-oxidized isoforms in an animal model of ALS. We used mutant SOD1^G93A transgenic mice, a commonly used animal model for ALS. Upregulation of DJ-1 mRNA and protein levels were identified in the brains and spinal cords of SOD1^G93A transgenic mice as compared to wild-type controls, evident from an early disease stage. Furthermore, an increase in DJ-1 acidic isoforms was detected, implying that there are more oxidized forms of DJ-1 in the CNS of SOD1^G93A mice. This is the first report of possible involvement of DJ-1 in ALS. Since DJ-1 has a protective role against oxidative stress, it may suggest a possible therapeutic target in ALS.     

The neuronal Golgi apparatus is fragmented in transgenic mice expressing a mutant human SOD1, but not in mice expressing the human NF-H gene

Fragmentation of the Golgi apparatus (GA) of motor neurons was first described in sporadic amyotrophic lateral sclerosis (ALS) and later confirmed in transgenic mice expressing the G93A mutation of the gene encoding the enzyme Cu,Zn superoxide dismutase (SOD1(G93A)) found in some cases of familial ALS. In these transgenic mice, however, the fragmentation of the neuronal GA was associated with cytoplasmic and mitochondrial vacuoles not seen in ALS. The present new series of transgenic mice expressing 14-17 trans gene copies of SOD1(G93A), compared to 25 copies in the mice we studied previously, showed consistent fragmentation of the GA of spinal cord motor neurons, axonal swellings, Lewy-like body inclusions in neurons and glia, but none of the cytoplasmic or mitochondrial vacuoles originally reported. Thus, this animal model recapitulates the clinical and most neuropathological findings of sporadic ALS. Neurofilaments (NF) accumulate in axons and, less often, in neuronal perikarya in most cases of sporadic ALS and they have been implicated in its pathogenesis. In order to investigate whether fragmentation of the neuronal GA also occurs in association with accumulation of perikaryal NFs, we studied the organelle in transgenic mice expressing the heavy subunit of human neurofilaments (NF-H) which developed a motor neuronopathy resembling ALS. The neuronal GA of mice expressing NF-H, however, was intact despite massive accumulation of NFs in both perikarya and axons of motor neurons. In contrast, in transgenic mice expressing SOD1(G93A), the GA was fragmented despite the absence of accumulation of perikaryal NFs. These findings suggest that, in transgenic mice with neuronopathies caused by the expression of mutant SOD1(G93A) or the human NF-H, the GA and the perikaryal NFs are independently involved in the pathogenesis. The evidence suggests that the GA plays a central role in the pathogenesis of the vast majority of sporadic ALS and in FALS with SOD1 mutations.     

Targeting of monomer/misfolded SOD1 as a therapeutic strategy for amyotrophic lateral sclerosis

There is increasing evidence that toxicity of mutant superoxide dismutase-1 (SOD1) in amyotrophic lateral sclerosis (ALS) is linked to its propensity to misfold and to aggregate. Immunotargeting of differently folded states of SOD1 has provided therapeutic benefit in mutant SOD1 transgenic mice. The specific region(s) of the SOD1 protein to which these immunization approaches target are, however, unknown. In contrast, we have previously shown, using a specific antibody [SOD1 exposed dimer interface (SEDI) antibody], that the dimer interface of SOD1 is abnormally exposed both in mutant SOD1 transgenic mice and in familial ALS cases associated with mutations in the SOD1 gene (fALS1). Here, we show the beneficial effects of an active immunization strategy using the SEDI antigenic peptide displayed on a branched peptide dendrimer to target monomer/misfolded in SOD1(G37R) and SOD1(G93A) mutant SOD1 transgenic mice. Immunization delayed disease onset and extended disease duration, with survival times increased by an average of 40 d in SOD1(G37R) mice. Importantly, this immunization strategy favored a Th2 immune response, thereby precluding deleterious neuroinflammatory effects. Furthermore, the beneficial effects of immunization correlated with a reduction in accumulation of both monomer/misfolded and oligomeric SOD1 species in the spinal cord, the intended targets of the immunization strategy. Our results support that SOD1 misfolding/aggregation plays a central role in SOD1-linked ALS pathogenesis and identifies monomeric/misfolded SOD1 as a therapeutic target for SOD1-related ALS.     

Absence of p53: no effect in a transgenic mouse model of familial amyotrophic lateral sclerosis

Familial amyotrophic lateral sclerosis (ALS) has been linked in some families to dominantly inherited mutations in the gene encoding copper-zinc superoxide dismutase 1 (Cu-Zn SOD1). Transgenic mice expressing a mutant human Cu-Zn SOD1 (G93A) develop a dominantly inherited adult-onset paralytic disorder that replicates many of the clinical and pathological features of familial ALS. Increased p53 immunoreactivity has been reported in the motor cortex and spinal ventral horns of postmortem tissue from ALS patients. The nuclear phosphoprotein p53 is an important regulator of cellular proliferation, and increasing evidence supports the role of p53 in regulating cellular apoptosis. To assess the role of p53-mediated apoptosis in amyotrophic lateral sclerosis, mice deficient in both p53 alleles (p53-/-) were crossed with transgenic mice expressing the G93A mutant (G93A+), creating novel transgenic knockout mice. The animals (p53 +/+G93A+, p53+/-G93A+, p53-/-G93A+) were examined at regular intervals for cage activity, upper and lower extremity strength, and mortality. At 120 days from birth mice from each genotype were sacrificed, and L2-L3 anterior horn motor neurons were counted. There was no significant difference in time to onset of behavioral decline, mortality, or motor neuron degeneration between the different genotypes. Despite evidence that p53 plays an important role after acute neuronal injury, the current study suggests that p53 is not significantly involved in cell death in the G93A+ transgenic mouse model of familial ALS.     

The microglial NLRP3 inflammasome is activated by amyotrophic lateral sclerosis proteins

Microglial NLRP3 inflammasome activation is emerging as a key contributor to neuroinflammation during neurodegeneration. Pathogenic protein aggregates such as β-amyloid and α-synuclein trigger microglial NLRP3 activation, leading to caspase-1 activation and IL-1β secretion. Both caspase-1 and IL-1β contribute to disease progression in the mouse SOD1^G93A model of amyotrophic lateral sclerosis (ALS), suggesting a role for microglial NLRP3. Prior studies, however, suggested SOD1^G93A mice microglia do not express NLRP3, and SOD1^G93A protein generated IL-1β in microglia independent to NLRP3. Here, we demonstrate using Nlrp3-GFP gene knock-in mice that microglia express NLRP3 in SOD1^G93A mice. We show that both aggregated and soluble SOD1^G93A activates inflammasome in primary mouse microglia leading caspase-1 and IL-1β cleavage, ASC speck formation, and the secretion of IL-1β in a dose- and time-dependent manner. Importantly, SOD1^G93A was unable to induce IL-1β secretion from microglia deficient for Nlrp3, or pretreated with the specific NLRP3 inhibitor MCC950, confirming NLRP3 as the key inflammasome complex mediating SOD1-induced microglial IL-1β secretion. Microglial NLRP3 upregulation was also observed in the TDP-43^Q331K ALS mouse model, and TDP-43 wild-type and mutant proteins could also activate microglial inflammasomes in a NLRP3-dependent manner. Mechanistically, we identified the generation of reactive oxygen species and ATP as key events required for SOD1^G93A-mediated NLRP3 activation. Taken together, our data demonstrate that ALS microglia express NLRP3, and that pathological ALS proteins activate the microglial NLRP3 inflammasome. NLRP3 inhibition may therefore be a potential therapeutic approach to arrest microglial neuroinflammation and ALS disease progression.     

Immunohistochemical study on the distribution of homocysteine in the central nervous system of transgenic mice expressing a human Cu/Zn SOD mutation

In the present study, we used the transgenic mice expressing a human Cu/Zn SOD mutation (SOD1(G93A)) as an in vivo model of ALS and performed immunohistochemical studies to investigate the changes of homocysteine in the central nervous system of symptomatic transgenic mice. In control and presymptomatic transgenic mice, homocysteine-immunoreactive astrocytes were not detected in any region. In symptomatic transgenic mice, homocysteine-immunoreactive astrocytes were distributed in the spinal cord, brainstem and cerebellar nuclei of transgenic mice. In the hippocampal formation of transgenic mice, pyramidal cells in the CA1-3 regions and granule cells in the dentate gyrus showed homocysteine immunoreactivity. The present study provides the first in vivo evidence that homocysteine immunoreactive astrocytes were found in the central nervous system of symptomatic SOD(G93A) transgenic mice, suggesting that reactive astrocytes may play an important role in the pathogenesis and progress of ALS. This study also suggests that increased expression of homocysteine in the hippocampal neurons might reflect a role of homocysteine in an abnormality of hippocampal function of ALS.     

Selective impairment of fast anterograde axonal transport in the peripheral nerves of asymptomatic transgenic mice with a G93A mutant SOD1 gene

Transgenic mice that express a mutant Cu/Zn superoxide dismutase (SOD1) gene have been provided a valuable model for human amyotrophic lateral sclerosis (ALS). We studied a possible impairment of fast axonal transport in transgenic mice carrying a Gly93-->Ala (G93A) mutant SOD1 gene found in human familial ALS (FALS). Left sciatic nerve was ligated for 6 h in transgenic (Tg) and age-matched wild-type (WT) mice. Immunohistochemical analyses were performed for accumulations of kinesin and cytoplasmic dynein on both sides of the ligation site. Clinical function and histology in the spinal cords, sciatic nerves and gastrocnemius muscles were also assessed. The mice were examined at an early asymptomatic stage (aged 19 weeks) and a late stage (30 weeks) just before the development of the symptoms. WT mice showed an apparent increase in immunoreactivities for kinesin and cytoplasmic dynein at proximal and distal of the ligation, respectively. In contrast, the young Tg mice showed a selective decrease of kinesin accumulation in the proximal of the ligation. The mice were asymptomatic with a mild histological change only in muscles. The old Tg mice showed a marked reduction of the immunoreactivity for kinesin and cytoplasmic dynein on both sides of the ligation. They had a significant loss of spinal motor neurons, relatively small myelinated fiber densities of sciatic nerves, and severe muscular changes. These results provide direct evidence that the SOD1 mutation leads to impaired fast axonal transport, particularly in the anterograde direction at an early, asymptomatic stage preceding loss of spinal motor neurons and peripheral axons. This impairment may contribute to subsequent selective motor neuron death in the present model implicated for human FALS.     

Immune reactivity in a mouse model of familial ALS correlates with disease progression

OBJECTIVE: The cause of motor neuron death in ALS is incompletely understood. This study aims to define the potential involvement of nonneuronal immune-inflammatory factors in the destruction of motor neurons in mutant superoxide dismutase-1 (SOD1) transgenic mice as a model of ALS. BACKGROUND: The presence of activated microglia, IgG and its receptor for Fc portion (FcgammaRI), and T lymphocytes in the spinal cord of both patients with ALS and experimental animal models of motor neuron disease strongly suggests that immune-inflammatory factors may be actively involved in the disease process. METHODS: The expression of immune-inflammatory factors was followed in both human mutant (G93A) SOD1 transgenic mice and human wild-type SOD1 transgenic mice, at different ages (40, 80, and 120 days). Fixed, frozen, free-floating sections of the lumbar spinal cord were stained with antibodies against CD11b, IgG, FcgammaRI, intercellular adhesion molecule-1 (ICAM-1), CD3, and glial fibrillary acidic protein. RESULTS: The earliest change observed was the upregulation of ICAM-1 in the ventral lumbar spinal cord of 40-day-old mutant SOD1 mice. IgG and FcgammaRI reactivities were detected on motor neurons as early as 40 days and on microglial cells at later stages. Microglial activation was first evident in the ventral horn at 80 days, whereas reactive astrocytes and T cells became most prominent in 120-day-old mutant SOD1 mice. CONCLUSION: The upregulation of proinflammatory factors during early presymptomatic stages as well as the expansion of immune activation as disease progresses in mutant SOD1 transgenic mice suggest that immune-inflammatory mechanisms could contribute to disease progression.