Increased ER stress during motor neuron degeneration in a transgenic mouse model of amyotrophic lateral sclerosis

Abstract

The endoplasmic reticulum (ER), which plays important roles in apoptosis, is susceptible to oxidative stress. ER stress is also thought to be involved in the pathogenesis of neurodegenerative diseases. In this study, we investigated whether ER stress is involved in the pathogenesis of amyotrophic lateral sclerosis (ALS) using the anterior part of the lumbar spinal cord of transgenic mice carrying a mutation (G93A) in the superoxide dismutase 1 (SOD1) gene. Western blot and immunohistochemical analyses demonstrated that the expressions of p-PERK and p-eIF2α were increased in the microsome fraction (P3) of the lumbar spinal cord at the pre-symptomatic age of 12 weeks (12W), while the expression of activated caspase-12 was increased in the cytoplasmic fraction (S3) of the lumbar spinal cord at both the pre-symptomatic age of 12W and the late symptomatic age of 20W. In contrast, GRP78 did not show any increases in the microsome fraction (P3) of the lumbar spinal cord at either the pre-symptomatic or symptomatic ages. Thus, the present results strongly suggest that the balance between anti- and pro-apoptotic proteins related to ER stress is impaired from the pre-symptomatic stage in this ALS mouse model, and that this imbalance may be related to the pathogenesis of motor neuron degeneration in ALS.     

Increased 3-nitrotyrosine in both sporadic and familial amyotrophic lateral sclerosis

The pathogenesis of neuronal degeneration in both sporadic and familial amyotrophic lateral sclerosis (ALS) associated with mutations in superoxide dismutase may involve oxidative stress. A leading candidate as a mediator of oxidative stress is peroxynitrite, which is formed by the reaction of superoxide with nitric oxide. 3-Nitrotyrosine is a relatively specific marker for oxidative damage mediated by peroxynitrite. In the present study, biochemical measurements showed increased concentrations of 3-nitrotyrosine and 3-nitro-4-hydroxyphenylacetic acid in the lumbar and thoracic spinal cord of ALS patients. Increased 3-nitrotyrosine immunoreactivity was observed in motor neurons of both sporadic and familial ALS patients. Neurologic control patients with cerebral ischemia also showed increased 3-nitrotyrosine immunoreactivity. These findings suggest that peroxynitrite-mediated oxidative damage may play a role in the pathogenesis of both sporadic and familial ALS.     

Beta-amyloid 42 accumulation in the lumbar spinal cord motor neurons of amyotrophic lateral sclerosis patients

Amyotrophic lateral sclerosis (ALS) is characterized by a progressive loss of large motor neurons in the brain and spinal cord. Amyloid precursor protein (APP), the transmembrane precursor of beta-amyloid (A beta), accumulates in the anterior horn motor neurons of ALS patients with mild lesions. APP undergoes an alternative proteolysis mediated by caspase-3, which is activated in motor neurons in a mouse model of ALS. The ALS spinal cord motor neurons also show evidence of increased oxidative damage, which is thought to alter APP processing. We sought to determine whether A beta42, the more pathogenic A beta species, accumulates in the postmortem lumbar spinal cord of ALS patients. While there was little or no A beta42 labeling in control spinal cord tissues, elevated A beta42 immunoreactivity occurred in ALS motor neuronal perikarya and axonal swellings in the anterior horn. A few A beta42-positive neurons exhibited thioflavine S staining. No extracellular A beta42 deposits were found. A beta42 coexisted with the oxidative damage markers malondialdehyde, 8-hydroxydeoxyguanosine, heme oxygenase-1, and nitrotyrosine in abnormal neurons. The neurons with intracellular A beta42 accumulation also displayed robust cleaved caspase-3 immunoreactivity. Very little A beta40 immunoreactivity occurred in motor neurons of both control and ALS. These results suggest that aberrant accumulation of A beta42 in ALS spinal cord motor neurons is associated with oxidative stress, and may play a role in the pathogenesis of neurodegeneration in ALS.     

Discrete mitochondrial aberrations in the spinal cord of sporadic ALS patients

Amyotrophic lateral sclerosis (ALS) is an adult onset neurodegenerative disease characterized by progressive motor neuron degeneration in the brain and spinal cord leading to muscle atrophy, paralysis, and death. Mitochondrial dysfunction is a major contributor to motor neuron degeneration associated with ALS progression. Mitochondrial abnormalities have been determined in spinal cords of animal disease models and ALS patients. However, molecular mechanisms leading to mitochondrial dysfunction in sporadic ALS (sALS) patients remain unclear. Also, segmental or regional variation in mitochondrial activity in the spinal cord has not been extensively examined in ALS. In our study, the activity of mitochondrial electron transport chain complex IV was examined in post‐mortem gray and white matter of the cervical and lumbar spinal cords from male and female sALS patients and controls. Mitochondrial distribution and density in spinal cord motor neurons, lateral funiculus, and capillaries in gray and white matter were analyzed by immunohistochemistry. Results showed that complex IV activity was significantly decreased only in gray matter in both cervical and lumbar spinal cords from ALS patients. In ALS cervical and lumbar spinal cords, significantly increased mitochondrial density and altered distribution were observed in motor neurons, lateral funiculus, and cervical white matter capillaries. Discrete decreased complex IV activity in addition to changes in mitochondria distribution and density determined in the spinal cord in sALS patients are novel findings. These explicit mitochondrial defects in the spinal cord may contribute to ALS pathogenesis and should be considered in development of therapeutic approaches for this disease.     

Celastrol blocks neuronal cell death and extends life in transgenic mouse model of amyotrophic lateral sclerosis

There is substantial evidence that both inflammation and oxidative damage contribute to the pathogenesis of motor neuron degeneration in the G93A SOD1 transgenic mouse model of amyotrophic lateral sclerosis (ALS). Celastrol is a natural product from Southern China, which exerts potent anti-inflammatory and antioxidative effects. It also acts potently to increase expression of heat shock proteins including HSP70. We administered it in the diet to G93A SOD1 mice starting at 30 days of age. Celastrol treatment significantly improved weight loss, motor performance and delayed the onset of ALS. Survival of celastrol-treated G93A mice increased by 9.4% and 13% for 2 mg/kg/day and 8 mg/kg/day doses, respectively. Cell counts of lumbar spinal cord neurons confirmed a protective effect, i.e. 30% increase in neuronal number in the lumbar spinal cords of celastrol-treated animals. Celastrol treatment reduced TNF-alpha, iNOS, CD40, and GFAP immunoreactivity in the lumbar spinal cord sections of celastrol-treated G93A mice compared to untreated G93A mice. TNF-alpha immunoreactivity co-localized with SMI-32 (neuronal marker) and GFAP (astrocyte marker). HSP70 immunoreactivity was increased in lumbar spinal cord neurons of celastrol-treated G93A mice. Celastrol has been widely used in treating inflammatory diseases in man, and is well tolerated; therefore, it may be a promising therapeutic candidate for the treatment of human ALS.     

Amyotrophic lateral sclerosis: changes of noradrenergic and serotonergic transmitter systems in the spinal cord.

Noradrenaline (NA), dopamine (DA), serotonin (5-HT) and 5-hydroxyindoleacetic acid (5-HIAA) were measured in discrete subdivisions of cervical, thoracic and lumbar spinal cord segments obtained at autopsy of 4 subjects with amyotrophic lateral sclerosis (ALS) and 7 control patients. NA concentrations in thoracic and lumbar spinal cord of ALS patients were 2- to 4-fold higher compared with values obtained in control patients. 5-HT levels were unchanged at the cervical and thoracic level and slightly above normal in lumbar spinal cord, while the concentration of 5-HIAA was lowered in cervical and thoracic, but within the control range, in lumbar spinal cord. As a result, the molar ratios of 5-HT/5-HIAA were increased at all spinal levels in ALS. No difference in spinal DA concentration was found between ALS and control patients. The changes in the noradrenergic and serotonergic transmitter systems reported here most probably reflect a decreased release of these transmitter substances in ALS spinal cord. Since lack of the facilitatory monoaminergic influence would necessitate an increase in the excitatory, potentially neurotoxic glutamatergic input onto the motoneurones, we hypothesize that this could contribute to the progressive loss of spinal motoneurones in amyotrophic lateral sclerosis.     

Evidence of endoplasmic reticular stress in the spinal motor neurons exposed to CSF from sporadic amyotrophic lateral sclerosis patients

We have earlier reported that intrathecal injection of cerebrospinal fluid (CSF) from sporadic Amyotrophic Lateral Sclerosis patients (ALS-CSF) into neonatal rats and supplementation of rat spinal cord cultures with ALS-CSF induces motor neuron degeneration via aberrant neurofilament phosphorylation and Golgi apparatus fragmentation. Intracellular aggregates immunoreactive to ubiquitin, phosphorylated neurofilaments and choline acetyl transferase (ChAT) were prominently seen in NSC-34 cells exposed to ALS-CSF. Protein aggregation could cause stress on endoplasmic reticulum (ER) and may precede Golgi fragmentation. Here we assessed the effect of ALS-CSF on the expression of GRP-78 and caspase-12 proteins, the markers of ER stress responses, in NSC-34 cells and rat spinal cords by immunochemistry and immunoblotting. Both in vitro and in vivo, increased expression of these proteins accompanied elevated active caspase-12 levels. Apoptotic nuclei and nuclear translocation of caspase-12 were noted in some cells. In vitro, the occurrence of ER stress was supported by electron microscopic observations of numerous free polyribosomes and fragmented ER cisternae. Aggregated mSOD1 protein causes ER stress in familial ALS. ER stress is also reported in the autopsy samples of sporadic ALS. Thus our observation of ER stress may be linked to the protein aggregation, viz. phosphorylated neurofilaments and ChAT, reported earlier.     

Imbalanced excitatory to inhibitory synaptic input precedes motor neuron degeneration in an animal model of amyotrophic lateral sclerosis

Loss of motor neurons is the key neuropathological feature of amyotrophic lateral sclerosis (ALS). Although these neurons are targeted by many synapses, little is known about the importance of the pre-synaptic excitatory and inhibitory input for motor neuron degeneration. The present study aimed in determining the composition and abundance of neurochemically defined input on lumbar spinal cord motor neurons in the SOD1 mouse model of ALS by employing immunoreactivity (IR) for vesicular neurotransmitter transporter proteins. The first signs of motor neuron degeneration, visualized by vesicular acetylcholine transporter IR, were already evident in the pre-symptomatic phase at day 80 of life. With the beginning of the symptomatic phase at around day 110 of life, surviving motor neurons showed reductions in the abundances of vesicular glutamate transporter 1 and 2 appositions. This loss of excitatory input was paralleled by an essentially unchanged inhibitory input, visualized by vesicular inhibitory amino acid transporter IR. In addition, loss of excitatory and inhibitory fibers and terminals was also evident in non-motor neuron areas of the spinal cord. These data are indicative of an imbalanced synaptic input on spinal cord motor neurons with over-inhibition rather than over-excitation being a neuropathological feature during disease progression that may contribute to motor neuron death.     

Live imaging of amyotrophic lateral sclerosis pathogenesis: Disease onset is characterized by marked induction of GFAP in Schwann cells

Amyotrophic lateral sclerosis (ALS) is a late‐onset neurological disease characterized by progressive loss of motor neurons. At present, the pathological events precipitating disease onset and the exact pattern of disease progression are not fully understood. Recent studies suggest that glial cells, in particular activated astrocytes, can release factors that can directly kill motor neurons. To further investigate the involvement of glial cells (astrocytes and Schwann cells) in the pathogenesis of ALS, we generated ALS‐(GFAP‐luciferase/SOD^G93A) reporter mouse in which upregulation of glial fibrillary acidic protein (GFAP) can be visualized from live animals throughout the different stages of disease. Our results suggest that the disease in mice is initiated simultaneously in the spinal cord and in the peripheral nerves and is characterized by several cycles of GFAP upregulation. Immunohistochemical analysis confirmed that the induction GFAP bioluminescence signals were associated with the significant increases in GFAP immunoreactivity. The first pathological GFAP signals occurring at 25–30 days were asymptomatic and detectable at the level of lumbar spinal cord projections and at the periphery. These early events were then followed by GFAP promoter inductions that were associated with the distinct clinical symptoms. As expected, the onset of paralysis (112 days) was associated with the gradual and marked GFAP upregulation in the spinal cord. Interestingly, however, the disease onset (90 days) was characterized by sharp and synchronized induction of GFAP in peripheral nerve Schwann cells suggesting that peripheral nerves pathology/denervation and associated Schwann cell stress may play an important role in the ALS pathogenesis. © 2008 Wiley‐Liss, Inc.     

Amyotrophic lateral sclerosis: glutamate dehydrogenase and transmitter amino acids in the spinal cord.

Measurements were taken of the activity of glutamate dehydrogenase (GDH) and the levels of transmitter amino acids in anatomically dissected regions of cervical and lumbar spinal cord in eight patients dying with amyotrophic lateral sclerosis (ALS) and in 11 neurologically normal controls. GDH activity was considerably increased in lateral and ventral white matter and in the dorsal horn of the ALS cervical spinal cord, but normal in the ventral horn and the dorsal columns. Similar, although less pronounced, GDH changes were found in the lumbar enlargement. The mean concentrations of aspartate and glutamate were reduced in all regions of ALS spinal cord investigated. Taurine concentrations were significantly increased in several subdivisions of cervical spinal cord, but normal in lumbar regions. Glycine levels were significantly reduced in lumbar ventral and dorsal horns. There was no striking change in spinal cord GABA levels in our ALS patients. It is suggested that the reduced levels of glutamate and aspartate as well as the elevated GDH activity in the spinal cord of ALS patients may reflect an overactivity of the neurons releasing these potentially excitotoxic amino acids and thus may be causally related to the spinal neuro-degenerative changes characteristic of ALS.     

PARP expression is increased in astrocytes but decreased in motor neurons in the spinal cord of sporadic ALS patients

The evidence for increased oxidative stress and DNA damage in amyotrophic lateral sclerosis (ALS) prompted studies to determine if the expression of poly(ADP-ribose) polymerase (PARP) is increased in ALS. Using Western analyses of postmortem tissue, we demonstrated that PARP-immunoreactivity (PARP-IR) was increased 3-fold in spinal cord tissues of sporadic ALS (sALS) patients compared with non-neurological disease controls. Despite the increased PARP-IR, PARP mRNA expression was not increased significantly. Immunohistochemical analyses revealed PARP-IR was increased in both white and gray matter of sALS spinal cord. While PARP-IR was predominantly seen in astrocytes, large motor neurons displayed reduced staining compared with controls. This result contrasts sharply to the staining of Alzheimer and MPTP-induced Parkinson diseased tissue, where poly(ADP-ribose) (PAR)-IR was seen mostly in neurons, with little astrocytic staining. PARP-IR was increased in the pellet fraction of sALS homogenates compared with control homogenates, representing potential PARP binding to chromatin or membranes and suggesting a possible mechanism of PARP stabilization. The present results demonstrate glial alterations in sALS spinal cord tissue and support the role of glial alterations in sALS pathogenesis. Additionally, these results demonstrate differences in sALS spinal motor neurons and astrocytes compared to brain neurons and astrocytes in Alzheimer disease and MPTP-induced Parkinson disease despite the presence of markers for oxidative stress in all 3 diseases.     

Involvement of CHOP, an ER-stress apoptotic mediator, in both human sporadic ALS and ALS model mice

Endoplasmic reticulum (ER) stress-induced neuronal death may play a critical role in the pathogenesis of amyotrophic lateral sclerosis (ALS). However, whether CCAAT/enhancer binding protein (C/EBP) homologous protein (CHOP), an ER-stress apoptotic mediator, is involved in the pathogenesis of ALS is controversial. Here we demonstrate the expression levels and localization of CHOP in spinal cords of both sporadic ALS patients and ALS transgenic mice by immunohistochemistry. In the spinal cords of sporadic ALS patients, CHOP was markedly up-regulated but typically expressed at low levels in those of the control. Likewise, CHOP expression increased at 14 (symptomatic stage) and 18 to 20  weeks (end stage) in ALS transgenic mice spinal cords. Furthermore, localizations of CHOP were merged in motor neurons and glial cells, such as oligodendrocytes, astrocytes, and microglia. These results indicate that the up-regulation of CHOP in motor neurons and glial cells may play pivotal roles in the pathogenesis of ALS.     

Binding of IDPN (β,β′-iminodipropionitrile) to rat spinal cord: Possible implication in the mechanism of spheroid formation in amyotrophic lateral sclerosis

β,β′-iminodipropionitrile (IDPN) produces ‘spheroids’ similar to those in certain cases of amyotrophic lateral sclerosis (ALS). Therefore, the target molecule of IDPN could be important to the understanding of the molecular mechanism of spheroid formation in ALS. Wistar rats were injected ip with ¹⁴C-labeled IDPN (¹⁴C-IDPN) and killed at 0.5, 1, 3, 6, 12 and 24 h thereafter. The radioactivity in each organ increased rapidly and reached the maximum at 0.5–1 h after ¹⁴C-IDPN injection. Thereafter, a rapid decrease occurred until 6 h, followed by a gradual decline until 24 h. The radioactivity in the cerebral cortex, diencephalon and cerebellum was higher than in the pons, medulla oblongata and spinal cord. Although high in the visceral organs and skeletal muscles, no or little radioactivity was detected in fat tissue. Autoradiography also confirmed these results. In three rats, ¹⁴C-IDPN was injected to the lumbar enlargement of the spinal cord. Six hours after injection, the segment was removed and homogenized with physiological saline (PS). After centrifugation, the supernatant was obtained (PS fraction). The pellet was resuspended with 4 mol/L urea and the supernatant was obtained (urea fraction). Each fraction was analysed by gel filtration. A peak of radioactivity was observed at the elution fraction Nos 19 and 20 (consistent with free ¹⁴C-IDPN) when PS fraction was applied. On application of urea fraction, another peak was obtained at the elution fractions Nos 8 and 9 (MW 60～80 kDa). The present study demonstrates that ¹⁴C-IDPN does not selectively accumulate to the spinal cord and suggests that an IDPN-binding molecule with an MW of 60–80 kDa is present in the spinal cord. The molecule may be related to the pathological process of spheroid formation in ALS.     

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.     

Alterations in receptors for thyrotropin-releasing hormone, serotonin, and acetylcholine in amyotrophic lateral sclerosis

We utilized quantitative autoradiography to examine thyrotropin-releasing hormone (TRH) receptors, serotonin type 1A (5-HT1A) receptors, muscarinic cholinergic receptors, choline uptake sites, beta-adrenergic receptors, and norepinephrine uptake sites in discrete laminae of spinal cord from patients with amyotrophic lateral sclerosis (ALS) and non-neurologic controls. We found decreases of over 50% in the concentration of TRH receptors in lamina IX of cervical, thoracic, and lumbar spinal cord from ALS patients. Similar reductions were noted in concentrations of muscarinic cholinergic receptors in lamina IX of spinal cords from ALS patients. Significant increases of up to 140% in 5-HT1A receptor densities were noted in lamina IX of spinal cords from ALS patients. No differences were noted between the concentrations of beta-adrenergic receptors or norepinephrine uptake sites in patients with ALS and controls. These findings suggest that TRH and 5-HT may be involved in the pathophysiology of ALS, and act in a comodulatory role in the normal spinal cord.     

Additive neuroprotective effects of a histone deacetylase inhibitor and a catalytic antioxidant in a transgenic mouse model of amyotrophic lateral sclerosis

ALS is a devastating neurodegenerative disorder for which no effective treatment exists. Multiple molecular mechanisms are involved in the pathogenesis. We tested the catalytic antioxidant AEOL 10150, the histone deacetylase inhibitor phenylbutyrate (PBA), and the combination of PBA and AEOL 10150 in the G93A transgenic mouse model, administered from disease onset. AEOL 10150 alone improved motor function and extended survival by 11%, PBA alone significantly improved motor function and extended survival by 13%. PBA and AEOL 10150 together increased survival by 19%. Increased histone acetylation was confirmed by Western blot. Quantitative real-time RT-PCR analysis revealed upregulation of compounds capable of protecting cells against oxidative stress and apoptosis. Markers of oxidative damage were reduced in the lumbar spinal cord as compared to vehicle administration. These results suggest that agents inhibiting apoptosis and blocking oxidative stress show efficacy in treating mutant-SOD1-associated ALS and that a combination of agents targeting different disease mechanisms may exert additive therapeutic effects.     

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.     

Intrathecal application of neuroectodermally converted stem cells into a mouse model of ALS: limited intraparenchymal migration and survival narrows therapeutic effects

Summary Stem and progenitor cells provide a promising therapeutic strategy for amyotrophic lateral sclerosis (ALS). To comparatively evaluate the therapeutic potentials of human bone marrow-derived mesodermal stromal cells (hMSCs) and umbilical cord blood cells (hUBCs) in ALS, we transplanted hMSCs and hUBCs and their neuroectodermal derivatives (hMSC-NSCs and hUBC-NSCs) into the ALS mouse model over-expressing the G93A mutant of the human SOD1 gene. We used a standardized protocol similar to clinical studies by performing a power calculation to estimate sample size prior to transplantation, matching the treatment groups for gender and hSOD-G93A gene content, and applying a novel method for directly injecting 100,000 cells into the CSF (the cisterna magna). Ten days after transplantation we found many cells within the subarachnoidal space ranging from frontal basal cisterns back to the cisterna magna, but only a few cells around the spinal cord. hMSCs and hMSC-NSCs were also located within the Purkinje cell layer. Intrathecal cell application did not affect survival times of mice compared to controls. Consistently, time of disease onset and first pareses, death weight, and motor neuron count in lumbar spinal cord did not vary between treatment groups. Interestingly, transplantation of hMSCs led to an increase of pre-symptomatic motor performance compared to controls in female animals. The negative outcome of the present study is most likely due to insufficient cell numbers within the affected brain regions (mainly the spinal cord). Further experiments defining the optimal cell dose, time point and route of application and particularly strategies to improve the homing of transplanted cells towards the CNS region of interest are warranted to define the therapeutic potential of mesodermal stem cells for the treatment of ALS.     

Altered expression of bcl-2 and bax mRNA in amyotrophic lateral sclerosis spinal cord motor neurons

One of the primary neurodegenerative events occurring in amyotrophic lateral sclerosis (ALS) is the selective loss of spinal cord α motor neurons. To study the potential role of apoptosis in the degeneration of these motor neurons, in situ hybridization was used to measure the expression of two apoptotic cell death genes, bcl-2 and bax, in control and ALS lumbar spinal cord sections. The strongest hybridization signal for bcl-2 mRNA in neurological and nonneurological control spinal cords was found primarily in lamina IX α motor neurons, while a weaker hybridization signal was found in neurons of Clarke's nucleus and the proper sensory nucleus of the dorsal horn. Surviving lamina IX motor neurons in ALS spinal cord sections also expressed bcl-2 mRNA, but at levels that were significantly and selectively decreased (4.7-fold) compared with control. bax mRNA hybridization signal was detected in several cells throughout the gray matter in control and ALS lumbar spinal cord, but was significantly and selectively increased (2.8-fold) in ALS motor neurons. Given the proposed interactive roles of these genes in apoptosis, the present findings favor a scenario in which this mode of cell death would contribute to spinal cord motor neuron degeneration in ALS.