SOD1G93A Transgenic Mice Research Articles

Light therapy and supplementary riboflavin in the SOD1 transgenic mouse model of familial amyotrophic lateral sclerosis (FALS)

Familial amyotrophic lateral sclerosis (FALS) is a neurodegenerative disease characterized by progressive loss of motor neurons and death. Mitochondrial dysfunction and oxidative stress play an important role in motor neuron loss in ALS. Light therapy (LT) has biomodulatory effects on mitochondria. Riboflavin improves energy efficiency in mitochondria and reduces oxidative injury. The purpose of this study was to examine the synergistic effect of LT and riboflavin on the survival of motor neurons in a mouse model of FALS. G93A SOD1 transgenic mice were divided into four groups: Control, Riboflavin, Light, and Riboflavin+Light (combination). Mice were treated from 51 days of age until death. A single set of LT parameters was used: 810 nm diode laser, 140-mW output power, 1.4 cm(2) spot area, 120 seconds treatment duration, and 12 J/cm(2) energy density. Behavioral tests and weight monitoring were done weekly. At end stage of the disease, mice were euthanized, survival data was collected and immunohistochemistry and motor neuron counts were performed. There was no difference in survival between groups. Motor function was not significantly improved with the exception of the rotarod test which showed significant improvement in the Light group in the early stage of the disease. Immunohistochemical expression of the astrocyte marker, glial fibrilary acidic protein, was significantly reduced in the cervical and lumbar enlargements of the spinal cord as a result of LT. There was no difference in the number of motor neurons in the anterior horn of the lumbar enlargement between groups. The lack of significant improvement in survival and motor performance indicates study interventions were ineffective in altering disease progression in the G93A SOD1 mice. Our findings have potential implications for the conceptual use of light to treat other neurodegenerative diseases that have been linked to mitochondrial dysfunction.

An Animal Model of Oral Dysphagia in Amyotrophic Lateral Sclerosis

Relatively little is known about the underlying neuropathology of dysphagia in amyotrophic lateral sclerosis (ALS); thus, effective treatments remain elusive. Tremendous progress toward understanding and treating dysphagia in ALS may be possible through the use of an animal model of dysphagia in ALS research; however, no such animal model currently exists. The most logical candidate to consider is the SOD1-G93A transgenic mouse, the most widely investigated animal model of ALS. To investigate whether this animal model develops dysphagia, oral behaviors (lick and mastication rates) of SOD1-G93A transgenic mice (n = 30) were evaluated at three time points based on hind limb motor function: asymptomatic (60 days), disease onset (approximately 110 days), and disease end-stage (approximately 140 days). Age-matched nontransgenic littermates (n = 30) served as controls. At each time point, lick and mastication rates were significantly lower (p < 0.05) for transgenic mice compared with controls. Histologic analysis of the brainstem showed marked neurodegeneration (vacuolation) of the trigeminal and hypoglossal nuclei, two key motor components involved in mastication and licking behaviors. These results demonstrate a clinicopathologic correlation of oral dysfunction in SOD1-G93A transgenic mice, thereby establishing the SOD1-G93A transgenic mouse as a bona fide animal model of oral dysphagia in ALS.

Mutant SOD1 impairs axonal transport of choline acetyltransferase and acetylcholine release by sequestering KAP3

Mutations in the superoxide dismutase 1 (sod1) gene cause familial amyotrophic lateral sclerosis (FALS), likely due to the toxic properties of misfolded mutant SOD1 protein. Here we demonstrated that, starting from the pre-onset stage of FALS, misfolded SOD1 species associates specifically with kinesin-associated protein 3 (KAP3) in the ventral white matter of SOD1G93A-transgenic mouse spinal cord. KAP3 is a kinesin-2 subunit responsible for binding to cargos including choline acetyltransferase (ChAT). Motor axons in SOD1G93A-Tg mice also showed a reduction in ChAT transport from the pre-onset stage. By employing a novel FALS modeling system using NG108-15 cells, we showed that microtubule-dependent release of acetylcholine was significantly impaired by misfolded SOD1 species. Furthermore, such impairment was able to be normalized by KAP3 overexpression. KAP3 was incorporated into SOD1 aggregates in human FALS cases as well. These results suggest that KAP3 sequestration by misfolded SOD1 species and the resultant inhibition of ChAT transport play a role in the dysfunction of ALS.

Immunohistochemical study on the distribution of glycogen synthase kinase 3α in the central nervous system of SOD1G93A transgenic mice

Objective: To identify glycogen synthase kinase (GSK) 3α expression in a mouse model of familial amyotrophic lateral sclerosis (ALS), we investigated the changes of GSK3α in the central nervous system of SOD1G93A transgenic mice by immunohistochemistry.Methods: We used 12 SOD1G93A transgenic and ten wild-type (wt) SOD1 transgenic mice bred by 'The Jackson Laboratory' under the strain designations B6SJL-TgN (SOD1G93A) 1 Gur/J and B6SJL-TgN (SOD1) 2 Gur/J, respectively. Immunohistochemistry was performed in accordance with the free-floating method described earlier.Results: In symptomatic transgenic mice, GSK3α-immunoreactive astrocytes were detected in the spinal cord, brainstem and cerebellum of symptomatic SOD1G93A transgenic mice. In contrast to symptomatic mice, no GSK3α-immunoreactive astrocytes were observed in any brain region of wtSOD1 and pre-symptomatic mice, and the number and intensity of stained cells were not different at the age of 8 and 13 weeks.Discussion: These results provide the first evidence that GSK3α-immunoreactive astrocytes were found in the CNS of SOD1G93A transgenic mice after clinical symptoms, suggesting a possible role in the pathologic process of ALS. However, the mechanisms underlying the increased immunoreactivity for GSK3α and the functional implications require elucidation.

TDP-43 expression in mouse models of amyotrophic lateral sclerosis and spinal muscular atrophy

BackgroundRedistribution of nuclear TAR DNA binding protein 43 (TDP-43) to the cytoplasm and ubiquitinated inclusions of spinal motor neurons and glial cells is characteristic of amyotrophic lateral sclerosis (ALS) pathology. Recent evidence suggests that TDP-43 pathology is common to sporadic ALS and familial ALS without SOD1 mutation, but not SOD1-related fALS cases. Furthermore, it remains unclear whether TDP-43 abnormalities occur in non-ALS forms of motor neuron disease. Here, we characterise TDP-43 localisation, expression levels and post-translational modifications in mouse models of ALS and spinal muscular atrophy (SMA).ResultsTDP-43 mislocalisation to ubiquitinated inclusions or cytoplasm was notably lacking in anterior horn cells from transgenic mutant SOD1G93A mice. In addition, abnormally phosphorylated or truncated TDP-43 species were not detected in fractionated ALS mouse spinal cord or brain. Despite partial colocalisation of TDP-43 with SMN, depletion of SMN- and coilin-positive Cajal bodies in motor neurons of affected SMA mice did not alter nuclear TDP-43 distribution, expression or biochemistry in spinal cords.ConclusionThese results emphasise that TDP-43 pathology characteristic of human sporadic ALS is not a core component of the neurodegenerative mechanisms caused by SOD1 mutation or SMN deficiency in mouse models of ALS and SMA, respectively.

Progressive decrease in the level of YAPdeltaCs, prosurvival isoforms of YAP, in the spinal cord of transgenic mouse carrying a mutant SOD1 gene

Amyotrophic lateral sclerosis (ALS) is a progressive and fatal disease caused by the selective death of motor neurons. Between 5% and 10% of ALS patients have a genetically inherited form of the disease known as familial ALS (FALS), and approximately 20% of FALS patients have mutations in the SOD1 gene. Although the mechanism underlying motor neuron death has not yet been fully clarified, it is supposed to be not completely consistent with apoptosis, necrosis, or autophagic cell death. Recently, it was found that general transcriptional repression induces slowly progressive atypical cell death associated with the shift of balance between YAPdeltaCs as prosurvival factors and activated p73 promoting apoptosis. This type of neuronal death was named transcriptional repression-induced atypical death (TRIAD). Therefore, to investigate possible relationships between the mechanism of motor neuron death in ALS and TRIAD, G93ASOD1 transgenic mice (Tg) were examined as an ALS model. The levels of YAPdeltaCs in the spinal cords of Tg mice decreased with disease progression, even during the presymptomatic stage, whereas FL-YAP, a p73 cofactor that promotes apoptosis, was preserved until the late symptomatic stage. Although the expression of total p73 also decreased with age in Tg mice, the ratio of phosphorylated p73 to total p73 increased during the late symptomatic stage in Tg mice. These results suggest that the progressive decrease in the levels of YAPdeltaCs and the relative increase in phosphorylation of p73 over the time course are correlated with disease progression in ALS model animals.

Immunohistochemical study on the distribution of insulin-like growth factor-binding protein 4 in the central nervous system of SOD1 G93A transgenic mice as an in vivo model of amyotrophic lateral sclerosis

In the present study, we used the SOD1(G93A) mutant transgenic mice as an in vivo model of amyotrophic lateral sclerosis (ALS) and performed immunohistochemical studies to investigate the changes of insulin-like growth factor-binding protein 4 (IGFBP4) in the central nervous system. Decreased expression of IGFBP4 was obvious in the cerebral cortex, hippocampus, cerebellar cortex and inferior olive of SOD1(G93A) transgenic mice. In the cerebral cortex, there was a significant decrease in IGFBP4 immunoreactivity in the pyramidal cells. In the hippocampal formation, IGFBP4 immunoreactivity was also decreased in the pyramidal cells of CA1-3 areas and the granule cells of dentate gyrus. In the cerebellar cortex, IGFBP4 immunoreactivity was prominent in the granular layer in wtSOD1 transgenic mice, compared to that in SOD1(G93A) transgenic mice. IGFBP4 immunoreactivity was decreased in the inferior olive of SOD1(G93A) transgenic mice. This study, showing decreased IGFBP4 in different brain regions of SOD1(G93A) transgenic mice, may provide clues to understanding the differential susceptibility of neural structures in ALS, suggesting a role of IGFBP4 in an abnormality of cognitive and/or motor function in ALS. The mechanisms and functional implications of these decreases require elucidation.

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.

Nordihydroguaiaretic acid increases glutamate uptake in vitro and in vivo: Therapeutic implications for amyotrophic lateral sclerosis

Synaptic accumulation of glutamate causes neuronal death in many neurodegenerative pathologies including amyotrophic lateral sclerosis. Drugs capable of increasing glutamate uptake could therefore be therapeutically effective. We screened in a cell-based assay a library of 1040 FDA-approved drugs and nutrients for compounds that could enhance glutamate uptake. Nordihydroguaiaretic acid (NDGA), an anti-inflammatory drug that inhibits lipoxygensases, potently enhanced glutamate uptake in MN-1 cells. Given subcutaneously at 1 mg/day for 30 days in mice, NDGA increased glutamate uptake in spinal cord synaptosomes persistently throughout the treatment. However, when administered following the same regimen to the SOD1-G93A transgenic mouse model of ALS at disease onset, NDGA did not extend survival of these mice. We found that NDGA failed to sustain increased glutamate uptake in the SOD1-G93A mice despite an initial upregulation measured during the first 10 days of treatment. SOD1-G93A mice displayed a progressive increase in spinal cord expression levels of the efflux transporter P-glycoprotein beginning at disease onset. This increase was not mediated by the NDGA treatment because it was measured in untreated SOD1-G93A mice. Since P-glycoproteins control the extrusion of a broad range of toxins and xenobiotics and are responsible for drug resistance in many diseases including cancer and brain diseases such as epilepsy, we propose that the failure of NDGA in maintaining glutamate uptake upregulated in SOD1-G93A mice and its therapeutic inefficacy are due to acquired pharmacoresistance mediated by the increased expression of P-glycoprotein.

Deregulation of PKN1 activity disrupts neurofilament organisation and axonal transport

Neurofilaments are synthesised in neuronal cell bodies and then transported through axons. Damage to neurofilament transport is seen in amyotrophic lateral sclerosis (ALS). Here, we show that PKN1, a neurofilament head–rod domain kinase is cleaved and activated in SOD1G93A transgenic mice that are a model of ALS. Moreover, we demonstrate that glutamate, a proposed toxic mechanism in ALS leads to caspase cleavage and disruption of PKN1 in neurons. Finally, we demonstrate that a cleaved form of PKN1 but not wild-type PKN1 disrupts neurofilament organisation and axonal transport. Thus, deregulation of PKN1 may contribute to the pathogenic process in ALS.

Magnetic resonance imaging of mouse skeletal muscle to measure denervation atrophy

We assessed the potential of different MRI measures to detect and quantify skeletal muscle changes with denervation in two mouse models of denervation/neurogenic atrophy. Acute complete denervation and chronic partial denervation were examined in calf muscles after sciatic nerve axotomy and in transgenic SOD1 G93A mice, respectively. Serial T 2, diffusion tensor, and high resolution anatomical images were acquired, and compared to behavioral, histological, and electrophysiological data. Increase in muscle T 2 signal was first detected after sciatic nerve axotomy. Progressive muscle atrophy could be monitored with MRI-based volume measurements, which correlated strongly with postmortem muscle mass measurements. Significant increase in muscle fractional anisotropy and decreases in secondary and tertiary eigenvalues obtained from diffusion tensor imaging (DTI) were observed after denervation. In SOD1 G93A animals, muscle denervation was detected by elevated muscle T 2 and atrophy in the medial gastrocnemius at 10 weeks. Changes in T 2 and muscle volume were first observed in medial gastrocnemius and later in other calf muscles. Alterations in secondary and tertiary eigenvalues obtained from DTI were first observed in tibialis anterior and medial gastrocnemius muscles at age 12 weeks. We propose that MRI of skeletal muscle is a sensitive surrogate outcome measure of denervation atrophy in animal models of neuromuscular disorders, with potential applicability in preclinical therapeutic screening studies in rodents.

Isolated Cytochrome c Oxidase Deficiency in G93A SOD1 Mice Overexpressing CCS Protein

G93A SOD1 transgenic mice overexpressing CCS protein develop an accelerated disease course that is associated with enhanced mitochondrial pathology and increased mitochondrial localization of mutant SOD1. Because these results suggest an effect of mutant SOD1 on mitochondrial function, we assessed the enzymatic activities of mitochondrial respiratory chain complexes in the spinal cords of CCS/G93A SOD1 and control mice. CCS/G93A SOD1 mouse spinal cord demonstrates a 55% loss of complex IV (cytochrome c oxidase) activity compared with spinal cord from age-matched non-transgenic or G93A SOD1 mice. In contrast, CCS/G93A SOD1 spinal cord shows no reduction in the activities of complex I, II, or III. Blue native gel analysis further demonstrates a marked reduction in the levels of complex IV but not of complex I, II, III, or V in spinal cords of CCS/G93A SOD1 mice compared with non-transgenic, G93A SOD1, or CCS/WT SOD1 controls. With SDS-PAGE analysis, spinal cords from CCS/G93A SOD1 mice showed significant decreases in the levels of two structural subunits of cytochrome c oxidase, COX1 and COX5b, relative to controls. In contrast, CCS/G93A SOD1 mouse spinal cord showed no reduction in levels of selected subunits from complexes I, II, III, or V. Heme A analyses of spinal cord further support the existence of cytochrome c oxidase deficiency in CCS/G93A SOD1 mice. Collectively, these results establish that CCS/G93A SOD1 mice manifest an isolated complex IV deficiency which may underlie a substantial part of mutant SOD1-induced mitochondrial cytopathy.

Folic acid protects motor neurons against the increased homocysteine, inflammation and apoptosis in SOD1 G93A transgenic mice

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease caused by selective degeneration of motor neurons. Mutations in copper/zinc superoxide dismutase (SOD1) account for 20% cases of familial ALS (fALS), but the underlying pathogenetic mechanisms are largely unknown. Using SOD1 G93A mice model of ALS, we demonstrated that mutation in SOD1 caused a significant increase in the level of plasma homocysteine (Hcy). To investigate whether Hcy-lowering therapy is beneficial to this disease, we applied folic acid (FA) and vitamin B12 which are important factors involved in the Hcy metabolism to assess the neuroprotective effect of FA and B12 in the SOD1 G93A mice. Our results showed FA or FA + B12 treatment significantly delayed the disease onset and prolonged the lifespan, accompanied by the significant reduction of motor neuron loss. Furthermore, we found that FA or FA + B12 treatment significantly attenuated the plasma Hcy level, suppressed the activation of microglia and astrocytes, and inhibited the expression of inducible nitric oxide synthase (iNOS) and tumor necrosis factor-alpha (TNF-α) in spinal cord. Moreover, FA or FA + B12 treatment decreased the levels of cleaved caspase-3 and poly(ADP-ribose)polymerase (PARP) but up-regulated the level of anti-apoptotic protein Bcl-2. However, B12 treatment alone did not show any significant benefit to this disease. These results provide evidence to demonstrate that elevated Hcy is involved in the pathogenesis of fALS and FA therapy may have therapeutic potential for the treatment of the disease.

Vgf is a novel biomarker associated with muscle weakness in amyotrophic lateral sclerosis (ALS), with a potential role in disease pathogenesis

Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disease that affects nerve cells in the brain and the spinal cord. Previous proteomic evidence revealed that the content of certain peptide fragments including Vgf-derived peptide aa 398-411 (Vgf(398-411)) of the precursor Vgf protein in the cerebral spinal fluid (CSF) correctly identified patients with ALS from normal and disease controls. Using quantitative ELISA immunoassay we found that the CSF levels of Vgf decreases with muscle weakness in patients with ALS. In SOD1 G93A transgenic mice, loss of full-length Vgf content in CSF, serum and in SMI-32 immunopositive spinal cord motor neurons is noted in asymptomatic animals (approximately 75 days old) and continues to show a progressive decline as animals weaken. In vitro studies show that viral-mediated exogenous Vgf expression in primary mixed spinal cord neuron cultures attenuates excitotoxic injury. Thus, while Vgf may be a reliable biomarker of progression of muscle weakness in patients with ALS, restoration of Vgf expression in spinal cord motor neurons may therapeutically rescue spinal cord motorneurons against excitotoxic injury.

Peroxisome Proliferator‐Activated Receptor‐γ in Amyotrophic Lateral Sclerosis and Huntington′s Disease

Amyotrophic lateral sclerosis (ALS) is a debilitating and one of the most common adult-onset neurodegenerative diseases with the prevalence of about 5 per 100 000 individuals. It results in the progressive loss of upper and lower motor neurons and leads to gradual muscle weakening ultimately causing paralysis and death. ALS has an obscure cause and currently no effective treatment exists. In this review, a potentially important pathway is described that can be activated by peroxisome proliferator-activated receptor-γ (PPAR-γ) agonists and has the ability to block the neuropathological damage caused by inflammation in ALS and possibly in other neudegenerative diseases like Huntington's disease (HD). Neuroinflammation is a common pathological feature in neurodegenerative diseases. Therefore, PPAR-γ agonists are thought to be neuroprotective in ALS and HD. We and others have tested the neuroprotective effect of pioglitazone (Actos), a PPAR-γ agonist, in G93A SOD1 transgenic mouse model of ALS and found significant increase in survival of G93A SOD1 mice. These findings suggest that PPAR-γ may be an important regulator of neuroinflammation and possibly a new target for the development of therapeutic strategies for ALS. The involvement of PPAR-γ in HD is currently under investigation, one study finds that the treatment with rosiglitazone had no protection in R6/2 transgenic mouse model of HD. PPAR-γ coactivator-1α (PGC-1α) is a transcriptional coactivator that works together with combination of other transcription factors like PPAR-γ in the regulation of mitochondrial biogenesis. Therefore, PPAR-γ is a possible target for ALS and HD as it functions as transcription factor that interacts with PGC-1α. In this review, the role of PPAR-γ in ALS and HD is discussed based on the current literature and hypotheses.

A Caspase-3-cleaved Fragment of the Glial Glutamate Transporter EAAT2 Is Sumoylated and Targeted to Promyelocytic Leukemia Nuclear Bodies in Mutant SOD1-linked Amyotrophic Lateral Sclerosis

EAAT2 (excitatory amino acid transporter 2) is a high affinity, Na+-dependent glutamate transporter of glial origin that is essential for the clearance of synaptically released glutamate and prevention of excitotoxicity. During the course of human amyotrophic lateral sclerosis (ALS) and in a transgenic mutant SOD1 mouse model of the disease, expression and activity of EAAT2 is remarkably reduced. We previously showed that some of the mutant SOD1 proteins exposed to oxidative stress inhibit EAAT2 by triggering caspase-3 cleavage of EAAT2 at a single defined locus. This gives rise to two fragments that we termed truncated EAAT2 and COOH terminus of EAAT2 (CTE). In this study, we report that analysis of spinal cord homogenates prepared from mutant G93A-SOD1 mice reveals CTE to be of a higher molecular weight than expected because it is conjugated with SUMO-1. The sumoylated CTE fragment (CTE-SUMO-1) accumulates in the spinal cord of these mice as early as presymptomatic stage (70 days of age) and not in other central nervous system areas unaffected by the disease. The presence and accumulation of CTE-SUMO-1 is specific to ALS mice, since it does not occur in the R6/2 mouse model for Huntington disease. Furthermore, using an astroglial cell line, primary culture of astrocytes, and tissue samples from G93A-SOD1 mice, we show that CTE-SUMO-1 is targeted to promyelocytic leukemia nuclear bodies. Since one of the proposed functions of promyelocytic leukemia nuclear bodies is regulation of gene transcription, we suggest a possible novel mechanism by which the glial glutamate transporter EAAT2 could contribute to the pathology of ALS.

Familial amyotrophic lateral sclerosis-linked SOD1 mutants perturb fast axonal transport to reduce axonal mitochondria content.

Amyotrophic lateral sclerosis (ALS) is a late-onset neurological disorder characterized by death of motoneurons. Mutations in Cu/Zn superoxide dismutase-1 (SOD1) cause familial ALS but the mechanisms whereby they induce disease are not fully understood. Here, we use time-lapse microscopy to monitor for the first time the effect of mutant SOD1 on fast axonal transport (FAT) of bona fide cargoes in living neurons. We analyzed FAT of mitochondria that are a known target for damage by mutant SOD1 and also of membrane-bound organelles (MBOs) using EGFP-tagged amyloid precursor protein as a marker. We studied FAT in motor neurons derived from SOD1G93A transgenic mice that are a model of ALS and also in cortical neurons transfected with SOD1G93A and three further ALS-associated SOD1 mutants. We find that mutant SOD1 damages transport of both mitochondria and MBOs, and that the precise details of this damage are cargo-specific. Thus, mutant SOD1 reduces transport of MBOs in both anterograde and retrograde directions, whereas mitochondrial transport is selectively reduced in the anterograde direction. Analyses of the characteristics of mitochondrial FAT revealed that reduced anterograde movement involved defects in anterograde motor function. The selective inhibition of anterograde mitochondrial FAT enhanced their net retrograde movement to deplete mitochondria in axons. Mitochondria in mutant SOD1 expressing cells also displayed features of damage. Together, such changes to mitochondrial function and distribution are likely to compromise axonal function. These alterations represent some of the earliest pathological features so far reported in neurons of mutant SOD1 transgenic mice.

Stabilization of Hyperdynamic Microtubules Is Neuroprotective in Amyotrophic Lateral Sclerosis

Mutations in copper/zinc superoxide dismutase 1 (SOD1), a genetic cause of human amyotrophic lateral sclerosis, trigger motoneuron death through unknown toxic mechanisms. We report that transgenic SOD1G93A mice exhibit striking and progressive changes in neuronal microtubule dynamics from an early age, associated with impaired axonal transport. Pharmacologic administration of a microtubule-modulating agent alone or in combination with a neuroprotective drug to symptomatic SOD1G93A mice reduced microtubule turnover, preserved spinal cord neurons, normalized axonal transport kinetics, and delayed the onset of symptoms, while prolonging life by up to 26%. The degree of reduction of microtubule turnover was highly predictive of clinical responses to different treatments. These data are consistent with the hypothesis that hyperdynamic microtubules impair axonal transport and accelerate motor neuron degeneration in amyotrophic lateral sclerosis. Measurement of microtubule dynamics in vivo provides a sensitive biomarker of disease activity and therapeutic response and represents a new pharmacologic target in neurodegenerative disorders.

A role for the urokinase-type plasminogen activator system in amyotrophic lateral sclerosis

There is substantial evidence, implicating extracellular matrix (ECM) regulating enzymes in the pathogenesis of motor neuron degeneration in amyotrophic lateral sclerosis (ALS). The most important ECM-degrading proteases are serine proteases (plasminogen activators, PA) and matrix metalloproteinases (MMPs). Since the role of MMPs in ALS has been addressed recently, we investigated the expression of the serine protease urokinase-type plasminogen activator (uPA) and its receptor in ALS. Employing rtPCR, zymography and immunohistochemistry we analyzed the expression of uPA and its receptor uPAR in spinal cord tissue of ALS cases and in the G93A SOD1 transgenic mouse. In the ventral horn of the spinal cord of ALS cases we found increased uPAR staining of motor neurons. In G93A mice, the expression profile of uPA and uPAR mRNA was significantly increased starting at the age of 90 days as compared to non-transgenic littermates. The uPA-dependent plasminogen activation in G93A mice at endstage increased markedly compared with controls and immunostaining of the spinal cord from G93A mice revealed increased uPAR immunostaining in neurons. To determine the functional role of uPA, we investigated the effect of intraperitoneal (i.p.) administration of the uPA inhibitor WX-340 (10 mg/kg), starting at the age of 30 days ( n = 18). Treatment with WX-340 prolonged ( p < 0.05) survival of the animals (135 ± 2 vs. 126 ± 3) as well as improving rotarod performance. Our experiments demonstrate that uPA and its receptor are expressed in ALS patients and in an animal model of ALS. Early inhibition with a synthetic uPA inhibitor prolonged the life of the transgenic animals. These findings indicate that the urokinase-type plasminogen activator system may play a role in the complex pathogenesis of ALS.

Time course of preferential motor unit loss in the SOD1G93A mouse model of amyotrophic lateral sclerosis

Electromyographical analyses of pre-symptomatic motor unit loss in the SOD1 G93A transgenic mouse model of amyotrophic lateral sclerosis (ALS) have yielded contradictory findings as to the onset and time course. We recorded hindlimb muscle and motor unit isometric forces to determine motor unit number and size throughout the life span of the mice. Motor unit numbers in fast-twitch tibialis anterior, extensor digitorum longus and medial gastrocnemius muscles declined from 40 days of age, 50 days before reported overt symptoms and motoneuron loss. Motor unit numbers fell after overt symptoms in the slow-twitch soleus muscle. Muscle forces declined in parallel with motor unit numbers, indicating little or no functional compensation by sprouting. Early muscle-specific decline was due to selective preferential vulnerability of large, fast motor units, innervated by large motoneurons. Large motoneurons are hence the most vulnerable in ALS with die-back occurring prior to overt symptoms. We conclude that size of motoneurons, their axons, and their motor unit size are important determinants of motoneuron susceptibility in ALS.