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.     

Progressive spinal axonal degeneration and slowness in ALS2-deficient mice

OBJECTIVE: Homozygous mutation in the ALS2 gene and the resulting loss of the guanine exchange factor activity of the ALS2 protein is causative for autosomal recessive early-onset motor neuron disease that is thought to predominantly affect upper motor neurons. The goal of this study was to elucidate how the motor system is affected by the deletion of ALS2. METHODS: ALS2-deficient mice were generated by gene targeting. Motor function and upper and lower motor neuron pathology were examined in ALS2-deficient mice and in mutant superoxide dismutase 1 (SOD1) mice that develop ALS-like disease from expression of an ALS-linked mutation in SOD1. RESULTS: ALS2-deficient mice demonstrated progressive axonal degeneration in the lateral spinal cord that is also prominent in mutant SOD1 mice. Despite the vulnerability of these spinal axons, lower motor neurons in ALS2-deficient mice were preserved. Behavioral studies demonstrated slowed movement without muscle weakness in ALS2(-/-) mice, consistent with upper motor neuron defects that lead to spasticity in humans. INTERPRETATION: The combined evidence from mice and humans shows that deficiency in ALS2 causes an upper motor neuron disease that in humans closely resembles a severe form of hereditary spastic paralysis, and that is quite distinct from amyotrophic lateral sclerosis.     

Expression of the ALS-causing variant hSOD1G93A leads to an impaired integrity and altered regulation of claudin-5 expression in an in vitro blood–spinal cord barrier model

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disorder characterized by progressive paralysis due to the loss of primary and secondary motor neurons. Mutations in the Cu/Zn-superoxide dismutase (SOD1) gene are associated with familial ALS and to date numerous hypotheses for ALS pathology exist including impairment of the blood–spinal cord barrier. In transgenic mice carrying mutated SOD1 genes, a disrupted blood–spinal cord barrier as well as decreased levels of tight junction (TJ) proteins ZO-1, occludin, and claudin-5 were detected. Here, we examined TJ protein levels and barrier function of primary blood–spinal cord barrier endothelial cells of presymptomatic hSOD1^G93A mice and bEnd.3 cells stably expressing hSOD1^G93A. In both cellular systems, we observed reduced claudin-5 levels and a decreased transendothelial resistance (TER) as well as an increased apparent permeability. Analysis of the β-catenin/AKT/forkhead box protein O1 (FoxO1) pathway and the FoxO1-regulated activity of the claudin-5 promoter revealed a repression of the claudin-5 gene expression in hSOD1^G93A cells, which was depended on the phosphorylation status of FoxO1. These results strongly indicate that mutated SOD1 affects the expression and localization of TJ proteins leading to impaired integrity and breakdown of the blood–spinal cord barrier.     

Nuclear localization of human SOD1 and mutant SOD1-specific disruption of survival motor neuron protein complex in transgenic amyotrophic lateral sclerosis mice

Amyotrophic lateral sclerosis (ALS) is a fatal adult-onset neurodegenerative disease that causes degeneration of motor neurons and paralysis. Approximately 20% of familial ALS cases have been linked to mutations in the copper/zinc superoxide dismutase (SOD1) gene, but it is unclear how mutations in the protein result in motor neuron degeneration. Transgenic (tg) mice expressing mutated forms of human SOD1 (hSOD1) develop clinical and pathological features similar to those of ALS. We used tg mice expressing hSOD1-G93A, hSOD1-G37R, and hSOD1-wild-type to investigate a new subcellular pathology involving mutant hSOD1 protein prominently localizing to the nuclear compartment and disruption of the architecture of nuclear gems. We developed methods for extracting relatively pure cell nucleus fractions from mouse CNS tissues and demonstrate a low nuclear presence of endogenous SOD1 in mouse brain and spinal cord, but prominent nuclear accumulation of hSOD1-G93A, -G37R, and -wild-type in tg mice. The hSOD1 concentrated in the nuclei of spinal cord cells, particularly motor neurons, at a young age. The survival motor neuron protein (SMN) complex is disrupted in motor neuron nuclei before disease onset in hSOD1-G93A and -G37R mice; age-matched hSOD1-wild-type mice did not show SMN disruption despite a nuclear presence. Our data suggest new mechanisms involving hSOD1 accumulation in the cell nucleus and mutant hSOD1-specific perturbations in SMN localization with disruption of the nuclear SMN complex in ALS mice and suggest an overlap of pathogenic mechanisms with spinal muscular atrophy.     

Isoform‐selective as opposed to complete depletion of fibroblast growth factor 2 (FGF‐2) has no major impact on survival and gene expression in SOD1G93A amyotrophic lateral sclerosis mice

We have previously shown that total knockout of fibroblast growth factor‐2 (FGF‐2) results in prolonged survival and improved motor performance in superoxide dismutase 1 (SOD1^G93A) mutant mice, the most widely used animal model of the fatal adult onset motor neuron disease amyotrophic lateral sclerosis (ALS). Moreover, we found differential expression of growth factors in SOD1^G93A mice, with distinct regulation patterns of FGF‐2 in spinal cord and muscle tissue. Within the present study we aimed to characterize FGF‐2‐isoform specific effects on survival, motor performance as well as gene expression patterns predominantly in muscle tissue by generating double mutant SOD1^G93AFGF‐2 high molecular weight‐ and SOD1^G93AFGF‐2 low molecular weight‐knockout mice. While isoform specific depletion was not beneficial regarding survival or motor performance of double mutant mice, we found isoform‐dependent differential gene expression of epidermal growth factor (EGF) in the muscle of SOD1^G93AFGF‐2 low molecular weight knockout mice compared to single mutant SOD1^G93A mice. This significant downregulation of EGF in the muscle tissue of double mutant SOD1^G93AFGF‐2 low molecular weight knockout mice implies that FGF‐2 low molecular weight knockout (or the presence of the FGF‐2 high molecular weight isoform) selectively impacts EGF gene expression in ALS muscle tissue.     

Prolonged survival and milder impairment of motor function in the SOD1 ALS mouse model devoid of fibroblast growth factor 2

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease characterized by selective motoneuron loss in brain and spinal cord. Mutations in the superoxide dismutase (SOD) 1 gene account for 10-20% of familial ALS patients. The ALS-mouse model over-expressing a mutant human SOD1 (G93A) gene closely mimics human ALS disease. The cause for the selective death of motoneurons is still unclear, but among several pathomechanisms discussed, loss of neurotrophic factors is one possibility. Basic fibroblast growth factor 2 (FGF-2) plays a prominent role in the motor system. In order to evaluate a role of FGF-2 in ALS pathogenesis, double mouse mutants transgenic for the human SOD1 mutation and lacking the endogenous FGF-2 gene were generated. Both heterozygous and homozygous FGF-2 deficient mutant SOD1 mice showed a significant delay in disease onset and less impaired motor performance in comparison to mutant SOD1 mice with normal FGF-2 levels. Survival of the double mouse mutants was significantly prolonged for two weeks. Motoneuron numbers were significantly higher in the double mutants and astrocytosis was diminished at disease endstage. While one would initially have expected that FGF-2 deficiency deteriorates the phenotype of mutant SOD1 animals, our results revealed a protective effect of FGF-2 reduction. In search of the underlying mechanisms, we could show up-regulation of other neurotrophic factors with proven protective effects in the ALS mouse model, ciliary neurotrophic factor (CNTF) and glial derived neurotrophic factor (GDNF) in muscle and spinal cord tissue of double mutant animals.     

The cellular mRNA expression of GABA and glutamate receptors in spinal motor neurons of SOD1 mice

ALS is a fatal neurodegenerative disorder characterized by a selective loss of upper motor neurons in the motor cortex and lower motor neurons in the brain stem and spinal cord. About 10% of ALS cases are familial, in 10-20% of these, mutations in the gene coding for superoxide dismutase 1 (SOD1) can be detected. Overexpression of mutated SOD1 in mice created animal models which clinically resemble ALS. Abnormalities in glutamatergic and GABAergic neurotransmission presumably contribute to the selective motor neuron damage in ALS. By in situ hybridization histochemistry (ISH), we investigated the spinal mRNA expression of the GABAA and AMPA type glutamate receptor subunits at different disease stages on spinal cord sections of mutant SOD1 mice and control animals overexpressing wild-type SOD1 aged 40, 80, 120 days and at disease end-stage, i.e. around 140 days) (n=5, respectively). We detected a slight but statistically significant decrease of the AMPA receptor subunits GluR3 and GluR4 only in end stage disease animals.     

Dorfin ameliorates phenotypes in a transgenic mouse model of amyotrophic lateral sclerosis

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease that is characterized by progressive motor neuron degeneration and leads to death within a few years of diagnosis. One of the pathogenic mechanisms of ALS is proposed to be a dysfunction in the protein quality‐control machinery. Dorfin has been identified as a ubiquitin ligase (E3) that recognizes and ubiquitinates mutant SOD1 proteins, thereby accelerating their degradation and reducing their cellular toxicity. We examined the effects of human Dorfin overexpression in G93A mutant SOD1 transgenic mice, a mouse model of familial ALS. In addition to causing a decrease in the amount of mutant SOD1 protein in the spinal cord, Dorfin overexpression ameliorated neurological phenotypes and motor neuron degeneration. Our results indicate that Dorfin overexpression or the activation or induction of E3 may be a therapeutic avenue for mutant SOD1‐associated ALS. © 2009 Wiley‐Liss, Inc.     

Early decrease of redox factor-1 in spinal motor neurons of presymptomatic transgenic mice with a mutant SOD1 gene

Oxidative stress has been proposed to play a pivotal role in pathogenesis of both sporadic and familial amyotrophic lateral sclerosis (ALS). Expression of DNA repair enzyme redox factor-1 (Ref-1) protein was examined in the spinal cord of transgenic mice with an ALS-linked mutant Cu/Zn superoxide dismutase (SOD1) gene. Immunoblotting and immunocytochemical analyses showed that the most spinal motor neurons lost the immunoreactivity for Ref-1 in the early presymptomatic stage that preceded significant loss of the neurons. The present result suggests that an early impairment of DNA repair in the spinal motor neurons may account for the mutant SOD1-mediated motor neuronal death in this model.     

Expression of the axon‐guidance protein receptor Neuropilin 1 is increased in the spinal cord and decreased in muscle of a mouse model of amyotrophic lateral sclerosis

Amyotrophic Lateral Sclerosis (ALS) is a degenerative motor neuron disorder. It is supposed that ALS is at least in part an axonopathy. Neuropilin 1 is an important receptor of the axon repellent Semaphorin 3A and a co‐receptor of vascular endothelial growth factor. It is probably involved in neuronal and axonal de‐/regeneration and might be of high relevance for ALS pathogenesis and/or disease progression. To elucidate whether the expression of either Neuropilin1 or Semaphorin3A is altered in ALS we investigated these proteins in human brain, spinal cord and muscle tissue of ALS‐patients and controls as well as transgenic SOD1^G93A and control mice. Neuropilin1 and Semaphorin3A gene and protein expression were assessed by quantitative real‐time PCR (qRT‐PCR), western blot and immunohistochemistry. Groups were compared using either Student t‐test or Mann–Whitney U test. We observed a consistent increase of Neuropilin1 expression in the spinal cord and decrease of Neuropilin1 and Semaphorin3A in muscle tissue of transgenic SOD1^G93A mice at the mRNA and protein level. Previous studies have shown that damage of neurons physiologically causes Neuropilin1 and Semaphorin3A increase in the central nervous system and decrease in the peripheral nervous system. Our results indicate that this also occurs in ALS. Pharmacological modulation of expression and function of axon repellents could be a promising future therapeutic option in ALS.     

Compensatory mechanism of motor defect in SOD1 transgenic mice by overactivation of striatal cholinergic neurons.

Expression of a mutant superoxide dismutase 1 (SOD1) gene in transgenic mice induces a gradual degeneration of cholinergic motor neurons in the spinal cord, causing progressive muscle weakness and hindlimb paralysis. Transgenic mice over-expressing the human SOD1 gene containing a Gly-->Ala substitution at position 93 (G93A) were employed to explore the effects of the SOD1 mutation on choline acetyltransferase (ChAT) expression in the striatum, and in the lumbar and cervical spinal cord. These mice showed a progressive loss of their spinal cord motor neurons, and at 130 days of age showed an up-regulation of ChAT mRNA expression in the striatum. On the other hand, ChAT mRNA decreased in cervical and lumbar motor neurons. These findings suggest that cholinergic interneurons in striatum in SOD1 transgenic mice are over-activated in an attempt to compensate for the death of spinal motor neurons.     

Neurochemical changes in the spinal cord in degenerative motor neuron diseases

Human amyotrophic lateral sclerosis (ALS), a typical motor neuron disease, is characterized pathologically by selective degenerative loss of motoneurons in the CNS. We have demonstrated significant reductions of neurotransmitter-related factors, such as acetylcholine-(ACh)-synthesizing enzyme activity and glutamate and aspartate contents in the ALS, compared to the non-ALS spinal cord obtained at autopsy. We have also shown considerable reductions in activities of cytochrome-c oxidase (CO), an enzyme contributing to aerobic energy production, and transglutaminase (TG), a Ca²⁺-dependent marker enzyme for tissue degeneration, in the ALS spinal cord. We found marked increases in fragmented glial fibrillary acidic protein (GFAP), a filamentous protein specifically associated with reactive astrocytes, in the ALS spinal cord relative to non-ALS tissue. These biochemical results corresponded well to pathomor-phological neuronal degenerative loss and reactive proliferation of astroglial components in the ALS spinal cord tissue. However, these results only indicate the final pathological and biochemical outcomes of ALS, and it is difficult to follow up cause and process in the ALS spinal cord during progression of the disease. Therefore, we used an animal model closely resembling human ALS, motor neuron degeneration (Mnd) mutant mice, a subline of C57BL/6 that shows late-onset progressive degeneration of lower motor neurons with paralytic gait beginning around 6.5 mo of age, to follow the biochemical and pathological alterations during postnatal development. We detected significant decreases in CO activity during early development and in activity of superoxide dismutase (SOD), an antioxidant enzyme, in later stages inMnd mutant spinal cord tissue. TG activity in theMnd spinal cord showed gradual increases during early development reaching a maximum at 5 mo, and then tending to decrease thereafter. Amounts of fragmented GFAPs increased continuously during postnatal development inMnd spinal cord. These biochemical changes were observed prior to the appearance of clinical motor dysfunctions in theMnd mutant mice. Such biochemical analyses using appropriate animal models will be useful for inferring the origin and progression of human ALS.     

Vascular endothelial growth factor counteracts the loss of phospho-Akt preceding motor neurone degeneration in amyotrophic lateral sclerosis

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disorder that results in the selective loss of motor neurones. In the present study, the involvement of the antiapoptotic protein, Akt (protein kinase B), was studied. We found that motor neurones of both sporadic and familial ALS patients lack phospho-Akt, and that motor neurones of mutant SOD1 mice lose activated Akt early in the disease, before the onset of clinical symptoms. In vitro, overexpression of constitutively active Akt protects against mutant SOD1-dependent cell death. In vivo, levels of phospho-Akt in the spinal cord increase after intracerebroventricular administration of vascular endothelial growth factor to mutant SOD1 rats, a treatment we previously described to significantly protect motor neurones. From these results, we conclude that the loss of phospho-Akt could be involved in motor neurone death in ALS, and that therapies upregulating phospho-Akt thus might be of clinical relevance.     

Peripherin is not a contributing factor to motor neuron disease in a mouse model of amyotrophic lateral sclerosis caused by mutant superoxide dismutase

Peripherin is a type III intermediate filament protein detected in axonal spheroids associated with amyotrophic lateral sclerosis (ALS). The overexpression of peripherin induces degeneration of spinal motor neurons during aging in transgenic mice and in cultured neuronal cells derived from peripherin transgenic embryos. Here, we investigated whether peripherin is a contributor of pathogenesis in mice overexpressing a mutant superoxide dismutase 1 (SOD1(G37R)) gene linked to familial ALS. This was done by the generation and analysis of SOD1(G37R) mice that either overexpress a peripherin transgene (G37R;TgPer mice) or lack the endogenous peripherin gene (G37R;Per-/- mice). Surprisingly, upregulation or suppression of peripherin expression had no effects on disease onset, mortality, and loss of motor neurons in SOD1(G37R) mice. These results provide compelling evidence that peripherin is not a key contributor of motor neuron degeneration associated with toxicity of mutant SOD1.     

Enhanced oxygen radical production in a transgenic mouse model of familial amyotrophic lateral sclerosis

Mutations of the SOD1 gene encoding copper/zinc superoxide dismutase (CuZnSOD) cause an inherited form of amyotrophic lateral sclerosis. When expressed in transgenic mice, the same SOD1 mutations cause progressive loss of spinal motor neurons with consequent paralysis and death. In vitro biochemical studies indicate that SOD1 mutations enhance free radical generation by the mutant enzyme. We investigated those findings in vivo by using a novel, brain-permeable spin trap, azulenyl nitrone. Reaction of azulenyl nitrone with a free radical forms a nitroxide adduct that then fragments to yield the corresponding azulenyl aldehyde. Transgenic mice expressing mutant SOD1-G93A show enhanced free radical content in spinal cord but not brain. This correlates with tissue-specific differences in the level of transgene expression. In spinal cord, the increase in free radical content is in direct proportion to the age-dependent increase in mutant human CuZnSOD expression. This increase precedes motor neuron degeneration. The higher level of human CuZnSOD expression seen in spinal cord compared with brain, and consequent difference in free radical generation, provides a basis for understanding the selective vulnerability of the spinal cord in this disease model.     

Flavin-Containing Monooxygenase mRNA Levels are Up-Regulated in ALS Brain Areas in SOD1-Mutant Mice

Flavin-containing monooxygenases (FMOs) are a family of microsomal enzymes involved in the oxygenation of a variety of nucleophilic heteroatom-containing xenobiotics. Recent results have pointed to a relation between Amyotrophic Lateral Sclerosis (ALS) and FMO genes. ALS is an adult-onset, progressive, and fatal neurodegenerative disease. We have compared FMO mRNA expression in the control mouse strain C57BL/6J and in a SOD1-mutated (G93A) ALS mouse model. Fmo expression was examined in total brain, and in subregions including cerebellum, cerebral hemisphere, brainstem, and spinal cord of control and SOD1-mutated mice. We have also considered expression in male and female mice because FMO regulation is gender-related. Real-Time TaqMan PCR was used for FMO expression analysis. Normalization was done using hypoxanthine-guanine phosphoribosyl transferase (Hprt) as a control housekeeping gene. Fmo genes, except Fmo3, were detectably expressed in the central nervous system of both control and ALS model mice. FMO expression was generally greater in the ALS mouse model than in control mice, with the highest increase in Fmo1 expression in spinal cord and brainstem. In addition, we showed greater Fmo expression in males than in female mice in the ALS model. The expression of Fmo1 mRNA correlated with Sod1 mRNA expression in pathologic brain areas. We hypothesize that alteration of FMO gene expression is a consequence of the pathological environment linked to oxidative stress related to mutated SOD1.     

The mitochondrial permeability transition pore in motor neurons: Involvement in the pathobiology of ALS mice

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease of motor neurons (MNs) that causes paralysis. Some forms of ALS are inherited, caused by mutations in the superoxide dismutase-1 (SOD1) gene. The mechanisms of human mutant SOD1 (mSOD1) toxicity to MNs are unresolved. Mitochondria in MNs might be key sites for ALS pathogenesis, but cause–effect relationships between mSOD1 and mitochondriopathy need further study. We used transgenic mSOD1 mice to test the hypothesis that the mitochondrial permeability transition pore (mPTP) is involved in the MN degeneration of ALS. Components of the multi-protein mPTP are expressed highly in mouse MNs, including the voltage-dependent anion channel, adenine nucleotide translocator (ANT), and cyclophilin D (CyPD), and are present in mitochondria marked by manganese SOD. MNs in pre-symptomatic mSOD1-G93A mice form swollen megamitochondria with CyPD immunoreactivity. Early disease is associated with mitochondrial cristae remodeling and matrix vesiculation in ventral horn neuron dendrites. MN cell bodies accumulate mitochondria derived from the distal axons projecting to skeletal muscle. Incipient disease in spinal cord is associated with increased oxidative and nitrative stress, indicated by protein carbonyls and nitration of CyPD and ANT. Reducing the levels of CyPD by genetic ablation significantly delays disease onset and extends the lifespan of G93A-mSOD1 mice expressing high and low levels of mutant protein in a gender-dependent pattern. These results demonstrate that mitochondria have causal roles in the disease mechanisms in MNs in ALS mice. This work defines a new mitochondrial mechanism for MN degeneration in ALS.