Male-Specific Differences in Proliferation,Neurogenesis, and Sensitivity to Oxidative Stress in Neural Progenitor Cells Derived from a Rat Model of ALS

Amyotrophic Lateral Sclerosis (ALS) is a fatal neurodegenerative disease characterized by progressive motor dysfunction and the loss of large motor neurons in the spinal cord and brain stem. A clear genetic link to point mutations in the superoxide dismutase 1 (SOD1) gene has been shown in a small group of familial ALS patients. The exact etiology of ALS is still uncertain, but males have consistently been shown to be at a higher risk for the disease than females. Here we present male-specific effects of the mutant SOD1 transgene on proliferation, neurogenesis, and sensitivity to oxidative stress in rat neural progenitor cells (rNPCs). E14 pups were bred using SOD1^G93A transgenic male rats and wild-type female rats. The spinal cord and cortex tissues were collected, genotyped by PCR using primers for the SOD1^G93A transgene or the male-specific Sry gene, and cultured as neurospheres. The number of dividing cells was higher in male rNPCs compared to female rNPCs. However, SOD1^G93A over-expression significantly reduced cell proliferation in male cells but not female cells. Similarly, male rNPCs produced more neurons compared to female rNPCs, but SOD1^G93A over-expression significantly reduced the number of neurons produced in male cells. Finally we asked whether sex and SOD1^G93A transgenes affected sensitivity to oxidative stress. There was no sex-based difference in cell viability after treatment with hydrogen peroxide or 3-morpholinosydnonimine, a free radical-generating agent. However, increased cytotoxicity by SOD1^G93A over-expression occurred, especially in male rNPCs. These results provide essential information on how the mutant SOD1 gene and sexual dimorphism are involved in ALS disease progression.     

Nerve Terminal Degeneration Is Independent of Muscle Fiber Genotype in SOD1G93A Mice

Background

Motor neuron degeneration in SOD1^G93A transgenic mice begins at the nerve terminal. Here we examine whether this degeneration depends on expression of mutant SOD1 in muscle fibers.

Methodology/Principal Findings

Hindlimb muscles were transplanted between wild-type and SOD1^G93A transgenic mice and the innervation status of neuromuscular junctions (NMJs) was examined after 2 months. The results showed that muscles from SOD1^G93A mice did not induce motor terminal degeneration in wildtype mice and that muscles from wildtype mice did not prevent degeneration in SOD1^G93A transgenic mice. Control studies demonstrated that muscles transplanted from SOD1^G93A mice continued to express mutant SOD1 protein. Experiments on wildtype mice established that the host supplied terminal Schwann cells (TSCs) at the NMJs of transplanted muscles.

Conclusions/Significance

These results indicate that expression of the mutant protein in muscle is not needed to cause motor terminal degeneration in SOD1^G93A transgenic mice and that a combination of motor terminals, motor axons and Schwann cells, all of which express mutant protein may be sufficient.     

The Legs at odd angles (Loa) Mutation in Cytoplasmic Dynein Ameliorates Mitochondrial Function in SOD1G93A Mouse Model for Motor Neuron Disease

Amyotrophic lateral sclerosis (ALS) is a debilitating and fatal late-onset neurodegenerative disease. Familial cases of ALS (FALS) constitute ∼10% of all ALS cases, and mutant superoxide dismutase 1 (SOD1) is found in 15–20% of FALS. SOD1 mutations confer a toxic gain of unknown function to the protein that specifically targets the motor neurons in the cortex and the spinal cord. We have previously shown that the autosomal dominant Legs at odd angles (Loa) mutation in cytoplasmic dynein heavy chain (Dync1h1) delays disease onset and extends the life span of transgenic mice harboring human mutant SOD1^G93A. In this study we provide evidence that despite the lack of direct interactions between mutant SOD1 and either mutant or wild-type cytoplasmic dynein, the Loa mutation confers significant reductions in the amount of mutant SOD1 protein in the mitochondrial matrix. Moreover, we show that the Loa mutation ameliorates defects in mitochondrial respiration and membrane potential observed in SOD1^G93A motor neuron mitochondria. These data suggest that the Loa mutation reduces the vulnerability of mitochondria to the toxic effects of mutant SOD1, leading to improved mitochondrial function in SOD1^G93A motor neurons.     

Expression of the protein chaperone, clusterin, in spinal cord cells constitutively and following cellular stress, and upregulation by treatment with Hsp90 inhibitor

Clusterin, a protein chaperone found at high levels in physiological fluids, is expressed in nervous tissue and upregulated in several neurological diseases. To assess relevance to amyotrophic lateral sclerosis (ALS) and other motor neuron disorders, clusterin expression was evaluated using long-term dissociated cultures of murine spinal cord and SOD1^G93A transgenic mice, a model of familial ALS. Motor neurons and astrocytes constitutively expressed nuclear and cytoplasmic forms of clusterin, and secreted clusterin accumulated in culture media. Although clusterin can be stress inducible, heat shock failed to increase levels in these neural cell compartments despite robust upregulation of stress-inducible Hsp70 (HspA1) in non-neuronal cells. In common with HSPs, clusterin was upregulated by treatment with the Hsp90 inhibitor, geldanamycin, and thus could contribute to the neuroprotection previously identified for such compounds in disease models. Clusterin expression was not altered in cultured motor neurons expressing SOD1^G93A by gene transfer or in presymptomatic SOD1^G93A transgenic mice; however, clusterin immunolabeling was weakly increased in lumbar spinal cord of overtly symptomatic mice. More striking, mutant SOD1 inclusions, a pathological hallmark, were strongly labeled by anti-clusterin. Since secreted, as well as intracellular, mutant SOD1 contributes to toxicity, the extracellular chaperoning property of clusterin could be important for folding and clearance of SOD1 and other misfolded proteins in the extracellular space. Evaluation of chaperone-based therapies should include evaluation of clusterin as well as HSPs, using experimental models that replicate the control mechanisms operant in the cells and tissue of interest.     

Intravenous mesenchymal stem cells improve survival and motor function in experimental amyotrophic lateral sclerosis

Despite some advances in the understanding of amyotrophic lateral sclerosis (ALS) pathogenesis, significant achievements in treating this disease are still lacking. Mesenchymal stromal (stem) cells (MSCs) have been shown to be effective in several models of neurological disease. To determine the effects of the intravenous injection of MSCs in an ALS mouse model during the symptomatic stage of disease, MSCs (1 × 10⁶) were intravenously injected in mice expressing human superoxide dismutase 1 (SOD1) carrying the G93A mutation (SOD1/G93A) presenting with experimental ALS. Survival, motor abilities, histology, oxidative stress markers and [³H]d-aspartate release in the spinal cord were investigated. MSC injection in SOD1/G93A mice improved survival and motor functions compared with saline-injected controls. Injected MSCs scantly home to the central nervous system and poorly engraft. We observed a reduced accumulation of ubiquitin agglomerates and of activated astrocytes and microglia in the spinal cord of MSC-treated SOD1/G93A mice, with no changes in the number of choline acetyltransferase– and glutamate transporter type 1–positive cells. MSC administration turned around the upregulation of metallothionein mRNA expression and of the activity of the antioxidant enzyme glutathione S-transferase, both associated with disease progression. Last, we observed that MSCs reverted both spontaneous and stimulus-evoked neuronal release of [³H]d-aspartate, a marker of endogenous glutamate, which is upregulated in SOD1/G93A mice. These findings suggest that intravenous administration of MSCs significantly improves the clinical outcome and pathological scores of mutant SOD1/G93A mice, thus providing the rationale for their exploitation for the treatment of ALS.     

Plasma neurofilament heavy chain levels correlate to markers of late stage disease progression and treatment response in SOD1(G93A) mice that model ALS

Background

Amyotrophic lateral sclerosis (ALS) is an incurable neurodegenerative disorder characterised by progressive degeneration of motor neurons leading to death, typically within 3–5 years of symptom onset. The diagnosis of ALS is largely reliant on clinical assessment and electrophysiological findings. Neither specific investigative tools nor reliable biomarkers are currently available to enable an early diagnosis or monitoring of disease progression, hindering the design of treatment trials.

Methodology/Principal Findings

In this study, using the well-established SOD1^G93A mouse model of ALS and a new in-house ELISA method, we have validated that plasma neurofilament heavy chain protein (NfH) levels correlate with both functional markers of late stage disease progression and treatment response. We detected a significant increase in plasma levels of phosphorylated NfH during disease progression in SOD1^G93A mice from 105 days onwards. Moreover, increased plasma NfH levels correlated with the decline in muscle force, motor unit survival and, more significantly, with the loss of spinal motor neurons in SOD1 mice during this critical period of decline. Importantly, mice treated with the disease modifying compound arimoclomol had lower plasma NfH levels, suggesting plasma NfH levels could be validated as an outcome measure for treatment trials.

Conclusions/Significance

These results show that plasma NfH levels closely reflect later stages of disease progression and therapeutic response in the SOD1^G93A mouse model of ALS and may potentially be a valuable biomarker of later disease progression in ALS.     

Multiple intravenous administrations of human umbilical cord blood cells benefit in a mouse model of ALS

Background

A promising therapeutic strategy for amyotrophic lateral sclerosis (ALS) is the use of cell-based therapies that can protect motor neurons and thereby retard disease progression. We recently showed that a single large dose (25×10⁶ cells) of mononuclear cells from human umbilical cord blood (MNC hUCB) administered intravenously to pre-symptomatic G93A SOD1 mice is optimal in delaying disease progression and increasing lifespan. However, this single high cell dose is impractical for clinical use. The aim of the present pre-clinical translation study was therefore to evaluate the effects of multiple low dose systemic injections of MNC hUCB cell into G93A SOD1 mice at different disease stages.

Methodology/Principal Findings

Mice received weekly intravenous injections of MNC hUCB or media. Symptomatic mice received 10⁶ or 2.5×10⁶ cells from 13 weeks of age. A third, pre-symptomatic, group received 10⁶ cells from 9 weeks of age. Control groups were media-injected G93A and mice carrying the normal hSOD1 gene. Motor function tests and various assays determined cell effects. Administered cell distribution, motor neuron counts, and glial cell densities were analyzed in mouse spinal cords. Results showed that mice receiving 10⁶ cells pre-symptomatically or 2.5×10⁶ cells symptomatically significantly delayed functional deterioration, increased lifespan and had higher motor neuron counts than media mice. Astrocytes and microglia were significantly reduced in all cell-treated groups.

Conclusions/Significance

These results demonstrate that multiple injections of MNC hUCB cells, even beginning at the symptomatic disease stage, could benefit disease outcomes by protecting motor neurons from inflammatory effectors. This multiple cell infusion approach may promote future clinical studies.     

Human Glial-Restricted Progenitor Transplantation into Cervical Spinal Cord of the SOD1G93A Mouse Model of ALS

Cellular abnormalities are not limited to motor neurons in amyotrophic lateral sclerosis (ALS). There are numerous observations of astrocyte dysfunction in both humans with ALS and in SOD1^G93A rodents, a widely studied ALS model. The present study therapeutically targeted astrocyte replacement in this model via transplantation of human Glial-Restricted Progenitors (hGRPs), lineage-restricted progenitors derived from human fetal neural tissue. Our previous findings demonstrated that transplantation of rodent-derived GRPs into cervical spinal cord ventral gray matter (in order to target therapy to diaphragmatic function) resulted in therapeutic efficacy in the SOD1^G93A rat. Those findings demonstrated the feasibility and efficacy of transplantation-based astrocyte replacement for ALS, and also show that targeted multi-segmental cell delivery to cervical spinal cord is a promising therapeutic strategy, particularly because of its relevance to addressing respiratory compromise associated with ALS. The present study investigated the safety and in vivo survival, distribution, differentiation, and potential efficacy of hGRPs in the SOD1^G93A mouse. hGRP transplants robustly survived and migrated in both gray and white matter and differentiated into astrocytes in SOD1^G93A mice spinal cord, despite ongoing disease progression. However, cervical spinal cord transplants did not result in motor neuron protection or any therapeutic benefits on functional outcome measures. This study provides an in vivo characterization of this glial progenitor cell and provides a foundation for understanding their capacity for survival, integration within host tissues, differentiation into glial subtypes, migration, and lack of toxicity or tumor formation.     

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.     

Unlike Physical Exercise,Modified Environment Increases the Lifespan of SOD1G93A Mice However Both Conditions Induce Cellular Changes

Background

Amyotrophic lateral sclerosis (ALS) is characterized by a gradual muscular paralysis resulting from progressive motoneurons death. ALS etiology remains unknown although it has been demonstrated to be a multifactorial disease involving several cellular partners. There is currently no effective treatment. Even if the effect of exercise is under investigation for many years, whether physical exercise is beneficial or harmful is still under debate.

Methods and Findings

We investigated the effect of three different intensities of running exercises on the survival of SOD1^G93A mice. At the early-symptomatic stage (P60), males were isolated and randomly assigned to 5 conditions: 2 sedentary groups (“sedentary” and “sedentary treadmill” placed on the inert treadmill), and 3 different training intensity groups (5 cm/s, 10 cm/s and 21 cm/s; 15 min/day, 5days/week). We first demonstrated that an appropriate “control” of the environment is of the utmost importance since comparison of the two sedentary groups evidenced an 11.6% increase in survival in the “sedentary treadmill” group. Moreover, we showed by immunohistochemistry that this increased lifespan is accompanied with motoneurons survival and increased glial reactivity in the spinal cord. In a second step, we showed that when compared with the proper control, all three running-based training did not modify lifespan of the animals, but result in motoneurons preservation and changes in glial cells activation.

Conclusions/Significance

We demonstrate that increase in survival induced by a slight daily modification of the environment is associated with motoneurons preservation and strong glial modifications in the lumbar spinal cord of SOD1^G93A. Using the appropriate control, we then demonstrate that all running intensities have no effect on the survival of ALS mice but induce cellular modifications. Our results highlight the critical importance of the control of the environment in ALS studies and may explain discrepancy in the literature regarding the effect of exercise 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.     

Experimental approach to the gene therapy of motor neuron disease with the use of genes hypoxia-inducible factors

Motor neuron disease (MND), or amyotrophic lateral sclerosis, is a fatal neurodegenerative disorder characterized by a progressive loss of motor neurons in the spinal cord and the brain. Several angiogenic and neurogenic growth factors, such as the vascular endothelial growth factor (VEGF), angiogenin (ANG), insulin-like growth factor (IGF) and others, have been shown to promote survival of the spinal motor neurons during ischemia. We constructed recombinant vectors using human adenovirus 5 (Ad5) carrying the VEGF, ANG or IGF genes under the control of the cytomegalovirus promoter. As a model for MND, we employed a transgenic mice strain, B6SJL-Tg(SOD1*G93A)dl1 Gur/J that develops a progressive degeneration of the spinal motor neurons caused by the expression of a mutated Cu/Zn superoxide dismutase gene SOD1 ^G93A. Delivery of the therapeutic genes to the spinal motor neurons was done using the effect of the retrograde axonal transport after multiple injections of the Ad5-VEGF, Ad5-ANG and Ad5-IGF vectors and their combinations into the limbs and back muscles of the SOD1 ^G93A mice. Viral transgene expression in the spinal cord motor neurons was confirmed by immunocytochemistry and RT-RCR. We assessed the neurological status, motor activity and lifespan of experimental and control animal groups. We discovered that SOD1 ^G93A mice injected with the Ad5-VEGF + Ad5-ANG combination showed a 2–3 week delay in manifestation of the disease, higher motor activity at the advanced stages of the disease, and at least a 10% increase in the lifespan compared to the control and other experimental groups. These results support the safety and therapeutic efficacy of the tested recombinant treatment. We propose that the developed experimental MND treatment based on viral delivery of VEGF + AGF can be used as a basis for gene therapy drug development and testing in the preclinical and clinical trials of the MND.     

Metformin treatment has no beneficial effect in a dose-response survival study in the SOD1(G93A) mouse model of ALS and is harmful in female mice

Background

Amyotrophic Lateral Sclerosis (ALS) is a devastating neurological disorder characterized by selective degeneration of upper and lower motor neurons. The primary triggers for motor neuron degeneration are unknown but inflammation, oxidative stress and mitochondrial defects have been identified as potential contributing factors. Metformin is an anti-type II diabetes drug that has anti-inflammatory and anti-oxidant properties, can bring about mitochondrial biogenesis and has been shown to attenuate pathology in mouse models of Huntington''s disease and multiple sclerosis. We therefore hypothesized that it might increase survival in the SOD1^G93A murine model of ALS.

Methodology/Principal Findings

Treatment of male and female SOD1^G93A mice (n = ≥6 per sex) with 2 mg/ml metformin in the drinking water from 35 days, resulted in a significant increase in motor unit survival, as measured by in vivo electrophysiology at 100 days, in male EDL muscles (24+/−2 vs. 14+/−2 motor units, p<0.005) and female TA muscles (21+/−1 vs. 15+/−2 motor units, P = 0.0134). We therefore continued to test the effect of 0.5, 2 and 5 mg/ml metformin in the drinking water from 35 days on disease onset and progression (identified by twice weekly determination of weight and neurological score) as well as survival in male and female SOD1^G93A mice (n = ≥14 per sex). Results for all groups were compared using Kaplan-Meier time to event analyses. In this survival study, metformin was unable to reduce pathology at any dose and had an unexpected dose-dependent negative effect on the onset of neurological symptoms (P = 0.0236) and on disease progression (P = 0.0362) in female mice.

Conclusions/Significance

This study suggests that metformin is a poor candidate for clinical trial in ALS patients and that the possibility of harmful effects of metformin in female ALS patients with type II diabetes should be investigated.     

Region-specific changes in the immunoreactivity of TRPV4 expression in the central nervous system of SOD1G93A transgenic mice as an in vivo model of amyotrophic lateral sclerosis

Transient receptor potential vanilloid 4 (TRPV4) is a broadly expressed Ca²⁺-permeable cation channel in the vanilloid subfamily of transient receptor potential channels. It is activated by warm temperature, lipids downstream of arachidonic acid metabolism, hypoosmolarity, or mechanical stimulation. In the present study, we used SOD1^G93A mutant transgenic mice as the animal model of amyotrophic lateral sclerosis (ALS) and investigated the changes of TRPV4 immunoreactivity in the central nervous system of these mice by immunohistochemical studies. An increased expression of TRPV4 was pronounced in the cerebral cortex, hippocampal formation, thalamus, cerebellum and spinal cord of symptomatic SOD1^G93A transgenic mice. In the cerebral cortex, TRPV4 immunoreactivity was significantly increased in pyramidal cells of SOD1^G93A transgenic mice. In the hippocampal formation, pyramidal cells of the CA1-3 areas and in the granule cells of the dentate gyrus demonstrated increased TRPV4 immunoreactivity. In addition, TRPV4 immunoreactivity was increased in the spinal cord, thalamus and cerebellum of the symptomatic SOD1^G93A transgenic mice. This study, which showed increased TRPV4 in different brain and spinal cord regions of SOD1^G93A transgenic mice, may provide clues to the understanding of many basic neuronal functions in ALS. These findings suggest a role for TRPV4 in the neuronal functions in ALS but the mechanisms and functional implications of increased TRPV4 require elucidation.     

Intraspinal Cell Transplantation for Targeting Cervical Ventral Horn in Amyotrophic Lateral Sclerosis and Traumatic Spinal Cord Injury

Respiratory compromise due to phrenic motor neuron loss is a debilitating consequence of a large proportion of human traumatic spinal cord injury (SCI) cases ¹ and is the ultimate cause of death in patients with the motor neuron disorder, amyotrophic laterals sclerosis (ALS) ².ALS is a devastating neurological disorder that is characterized by relatively rapid degeneration of upper and lower motor neurons. Patients ultimately succumb to the disease on average 2-5 years following diagnosis because of respiratory paralysis due to loss of phrenic motor neuron innnervation of the diaphragm ³. The vast majority of cases are sporadic, while 10% are of the familial form. Approximately twenty percent of familial cases are linked to various point mutations in the Cu/Zn superoxide dismutase 1 (SOD1) gene on chromosome 21 ⁴. Transgenic mice ^4,5 and rats ⁶ carrying mutant human SOD1 genes ^{(G93A, G37R, G86R, G85R)} have been generated, and, despite the existence of other animal models of motor neuron loss, are currently the most highly used models of the disease.Spinal cord injury (SCI) is a heterogeneous set of conditions resulting from physical trauma to the spinal cord, with functional outcome varying according to the type, location and severity of the injury ⁷. Nevertheless, approximately half of human SCI cases affect cervical regions, resulting in debilitating respiratory dysfunction due to phrenic motor neuron loss and injury to descending bulbospinal respiratory axons ¹. A number of animal models of SCI have been developed, with the most commonly used and clinically-relevant being the contusion ⁸.Transplantation of various classes of neural precursor cells (NPCs) is a promising therapeutic strategy for treatment of traumatic CNS injuries and neurodegeneration, including ALS and SCI, because of the ability to replace lost or dysfunctional CNS cell types, provide neuroprotection, and deliver gene factors of interest ⁹.Animal models of both ALS and SCI can model many clinically-relevant aspects of these diseases, including phrenic motor neuron loss and consequent respiratory compromise ^10,11. In order to evaluate the efficacy of NPC-based strategies on respiratory function in these animal models of ALS and SCI, cellular interventions must be specifically directed to regions containing therapeutically relevant targets such as phrenic motor neurons. We provide a detailed protocol for multi-segmental, intraspinal transplantation of NPCs into the cervical spinal cord ventral gray matter of neurodegenerative models such as SOD1^G93A mice and rats, as well as spinal cord injured rats and mice ¹¹.Download video file.^{(63M, mov)}     

Molecular Chaperone Mediated Late-Stage Neuroprotection in the SOD1G93A Mouse Model of Amyotrophic Lateral Sclerosis

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disorder characterized by the selective loss of motor neurons in the spinal cord, brain stem, and motor cortex. Mutations in superoxide dismutase (SOD1) are associated with familial ALS and lead to SOD1 protein misfolding and aggregation. Here we show that the molecular chaperone, HSJ1 (DNAJB2), mutations in which cause distal hereditary motor neuropathy, can reduce mutant SOD1 aggregation and improve motor neuron survival in mutant SOD1 models of ALS. Overexpression of human HSJ1a (hHSJ1a) in vivo in motor neurons of SOD1^G93A transgenic mice ameliorated disease. In particular, there was a significant improvement in muscle force, increased motor unit number and enhanced motor neuron survival. hHSJ1a was present in a complex with SOD1^G93A and led to reduced SOD1 aggregation at late stages of disease progression. We also observed altered ubiquitin immunoreactivity in the double transgenic animals, suggesting that ubiquitin modification might be important for the observed improvements. In a cell model of SOD1^G93A aggregation, HSJ1a preferentially bound to mutant SOD1, enhanced SOD1 ubiquitylation and reduced SOD1 aggregation in a J-domain and ubiquitin interaction motif (UIM) dependent manner. Collectively, the data suggest that HSJ1a acts on mutant SOD1 through a combination of chaperone, co-chaperone and pro-ubiquitylation activity. These results show that targeting SOD1 protein misfolding and aggregation in vivo can be neuroprotective and suggest that manipulation of DnaJ molecular chaperones might be useful in the treatment of ALS.     

Levels of membrane fluidity in the spinal cord and the brain in an animal model of amyotrophic lateral sclerosis

A mutant form of the copper/zinc superoxide dismutase (SOD1) protein is found in some patients with amyotrophic lateral sclerosis (ALS). Alteration of the activity of this antioxidant enzyme leads to an oxidative stress imbalance, which damages the structure of lipids and proteins in the CNS. Using fluorescence spectroscopy, we monitored membrane fluidity in the spinal cord and the brain in a widely used animal model of ALS, the SOD^G93A mouse, which develops symptoms similar to ALS with an accelerated course. Our results show that the membrane fluidity of the spinal cord in this animal model significantly decreased in symptomatic animals compared with age-matched littermate controls. To the best of our knowledge, this is the first report showing that membrane fluidity is affected in the spinal cord of a SOD^G93A animal model of ALS. Changes in membrane fluidity likely contribute substantially to alterations in cell membrane functions in the nervous tissue from SOD^G93A mice.     

Evidence of compromised blood-spinal cord barrier in early and late symptomatic SOD1 mice modeling ALS

Background

The blood-brain barrier (BBB), blood-spinal cord barrier (BSCB), and blood-cerebrospinal fluid barrier (BCSFB) control cerebral/spinal cord homeostasis by selective transport of molecules and cells from the systemic compartment. In the spinal cord and brain of both ALS patients and animal models, infiltration of T-cell lymphocytes, monocyte-derived macrophages and dendritic cells, and IgG deposits have been observed that may have a critical role in motor neuron damage. Additionally, increased levels of albumin and IgG have been found in the cerebrospinal fluid in ALS patients. These findings suggest altered barrier permeability in ALS. Recently, we showed disruption of the BBB and BSCB in areas of motor neuron degeneration in the brain and spinal cord in G93A SOD1 mice modeling ALS at both early and late stages of disease using electron microscopy. Examination of capillary ultrastructure revealed endothelial cell degeneration, which, along with astrocyte alteration, compromised the BBB and BSCB. However, the effect of these alterations upon barrier function in ALS is still unclear. The aim of this study was to determine the functional competence of the BSCB in G93A mice at different stages of disease.

Methodology/Principal Findings

Evans Blue (EB) dye was intravenously injected into ALS mice at early or late stage disease. Vascular leakage and the condition of basement membranes, endothelial cells, and astrocytes were investigated in cervical and lumbar spinal cords using immunohistochemistry. Results showed EB leakage in spinal cord microvessels from all G93A mice, indicating dysfunction in endothelia and basement membranes and confirming our previous ultrastructural findings on BSCB disruption. Additionally, downregulation of Glut-1 and CD146 expressions in the endothelial cells of the BSCB were found which may relate to vascular leakage.

Conclusions/Significance

Results suggest that the BSCB is compromised in areas of motor neuron degeneration in ALS mice at both early and late stages of the disease.