The prostate apoptosis response-4 protein participates in motor neuron degeneration in amyotrophic lateral sclerosis.

Prostate apoptosis response-4 (Par-4), a protein containing a leucine zipper domain within a death domain, is up-regulated in prostate cancer cells and hippocampal neurons induced to undergo apoptosis. Here, we report higher Par-4 levels in lumbar spinal cord samples from patients with amyotrophic lateral sclerosis (ALS) than in lumbar spinal cord samples from neurologically normal patients. We also compared the levels of Par-4 in lumbar spinal cord samples from wild-type and transgenic mice expressing the human Cu/Zn-superoxide dismutase gene with a familial ALS mutation. Relative to control samples, higher Par-4 levels were observed in lumbar spinal cord samples prepared from the transgenic mice at a time when they had hind-limb paralysis. Immunohistochemical analyses of human and mouse lumbar spinal cord sections revealed that Par-4 is localized to motor neurons in the ventral horn region. In culture studies, exposure of primary mouse spinal cord motor neurons or NSC-19 motor neuron cells to oxidative insults resulted in a rapid and large increase in Par-4 levels that preceded apoptosis. Pretreatment of the motor neuron cells with a Par-4 antisense oligonucleotide prevented oxidative stress-induced apoptosis and reversed oxidative stress-induced mitochondrial dysfunction that preceded apoptosis. Collectively, these data suggest a role for Par-4 in models of motor neuron injury relevant to ALS.     

Disease-related regressive alterations of forebrain cholinergic system in SOD1 mutant transgenic mice

Transgenic mice carrying the human mutated SOD1 gene with a glycine/alanine substitution at codon 93 (G93A) are a widely used model for the fatal human disease amyotrophic lateral sclerosis (ALS). In these transgenic mice, we carried out a neurochemical study not only restricted to the primarily affected regions, the cervical and lumbar segments of the spinal cord, but also to several other brain regions. At symptomatic (110 and 125 days of age), but not at pre-symptomatic (55 days of age) stages, we found significant decreases in catalytic activity of the cholinergic enzyme, choline acetyltransferase (ChAT) in the hippocampus, olfactory cortex and fronto-parietal cortex. In parallel, we observed a decreased number of basal forebrain cholinergic neurons projecting to these areas. No alterations of the cholinergic markers were noticed in the striatum and the cerebellum. A widespread marker for GABAergic neurons, glutamate decarboxylase (GAD), was unaffected in all the areas examined. Alteration of cholinergic markers in forebrain areas was paralleled by concomitant alterations in the spinal cord and brainstem, as a consequence of progressive apoptotic elimination of cholinergic motor neuron. Gestational supplementation of choline, while able to result in long-term enhancement of cholinergic activity, did not improve transgenic mice lifespan nor counteracted cholinergic impairment in brain regions and spinal cord.     

T-588 Protects Motor Neuron Death Following Axotomy

R(-)-1-(benzo [b] thiophen-5-yl)-2-[2-(N,N-diethylamino)ethoxy] ethanol hydrochloride) (T-588) enhances acetylcholine release. This compound slows the motor deterioration of wobbler mouse motor neuron disease and enhances neurite outgrowth and choline acetyltransferase activity in cultured rat spinal motor neurons. We examined the ability of T-588 on axotomized spinal motor neuron death in the rat spinal cord. After the postnatal unilateral section of sciatic nerve, there was approximately a 50% survival of motor neurons in the fourth lumbar segment. In comparison with vehicle, intraperitoneal injection of T-588 for 14 consecutive days rescued spinal motor neuron death. Our results showing in vivo neurotrophic activity of T-588 for motor neurons support the applicability of T-588 for the treatment of motor neuron diseases, such as amyotrophic lateral sclerosis and motor neuropathies.     

Regional and temporal alterations of ODC/polyamine system during ALS-like neurodegenerative motor syndrome in G93A transgenic mice

Natural polyamines (putrescine, spermidine and spermine) are ubiquitous molecules known to regulate a number of physiological processes and suspected to play a role also in various pathological conditions. Changes in polyamine levels and in their biosynthetic enzymes have been described for some neurodegenerative diseases but the available data are incomplete and somewhat contradictory. We report here alterations of the key enzyme of the polyamine pathway, ornithine decarboxylase (ODC) catalytic activity and polyamine levels in different CNS areas from SOD1 G39A transgenic mice, an animal model for amyotrophic lateral sclerosis (ALS). ODC catalytic activity, was found significantly increased both in the cervical and lumbar spinal cord and, to a lesser extent in the brain stem of transgenic mice at a symptomatic stage of the disease (125-day-old mice), while no differences were present at a pre-symptomatic stage (55-day-old mice). In parallel with the increase of ODC activity putrescine levels were several times increased in both cervical and lumbar spinal cord and in the brain stem of 125-day-old SOD1 G39A mice. Higher order polyamines were not increased except for a significant increase of spermidine in the cervical spinal cord. The present data demonstrate considerable alterations of the ODC/polyamine system in a reliable animal model of ASL, consistent with their role in neurodegeneration and in particular in motor neuron diseases.     

Neural mitochondrial Ca2+ capacity impairment precedes the onset of motor symptoms in G93A Cu/Zn-superoxide dismutase mutant mice

Mitochondrial respiratory chain dysfunction, impaired intracellular Ca2+ homeostasis and activation of the mitochondrial apoptotic pathway are pathological hallmarks in animal and cellular models of familial amyotrophic lateral sclerosis associated with Cu/Zn-superoxide dismutase mutations. Although intracellular Ca2+ homeostasis is thought to be intimately associated with mitochondrial functions, the temporal and causal correlation between mitochondrial Ca2+ uptake dysfunction and motor neuron death in familial amyotrophic lateral sclerosis remains to be established. We investigated mitochondrial Ca2+ handling in isolated brain, spinal cord and liver of mutant Cu/Zn-superoxide dismutase transgenic mice at different disease stages. In G93A mutant transgenic mice, we found a significant decrease in mitochondrial Ca2+ loading capacity in brain and spinal cord, as compared with age-matched controls, very early on in the course of the disease, long before the onset of motor weakness and massive neuronal death. Ca2+ loading capacity was not significantly changed in liver G93A mitochondria. We also confirmed Ca2+ capacity impairment in spinal cord mitochondria from a different line of mice expressing G85R mutant Cu/Zn-superoxide dismutase. In excitable cells, such as motor neurons, mitochondria play an important role in handling rapid cytosolic Ca2+ transients. Thus, mitochondrial dysfunction and Ca2+-mediated excitotoxicity are likely to be interconnected mechanisms that contribute to neuronal degeneration in familial amyotrophic lateral sclerosis.     

Fibroblast growth factor-1 induces heme oxygenase-1 via nuclear factor erythroid 2-related factor 2 (Nrf2) in spinal cord astrocytes: consequences for motor neuron survival

Fibroblast growth factor-1 (FGF-1) is highly expressed in motor neurons and can be released in response to sublethal cell injury. Because FGF-1 potently activates astroglia and exerts a direct neuroprotection after spinal cord injury or axotomy, we examined whether it regulated the expression of inducible and cytoprotective heme oxygenase-1 (HO-1) enzyme in astrocytes. FGF-1 induced the expression of HO-1 in cultured rat spinal cord astrocytes, which was dependent on FGF receptor activation and prevented by cycloheximide. FGF-1 also induced Nrf2 mRNA and protein levels and prompted its nuclear translocation. HO-1 induction was abolished by transfection of astrocytes with a dominant-negative mutant Nrf2, indicating that FGF-1 regulates HO-1 expression through Nrf2. FGF-1 also modified the expression of other antioxidant genes regulated by Nrf2. Both Nrf2 and HO-1 levels were increased and co-localized with reactive astrocytes in the degenerating lumbar spinal cord of rats expressing the amyotrophic lateral sclerosis-linked SOD1 G93A mutation. Overexpression of Nrf2 in astrocytes increased survival of co-cultured embryonic motor neurons and prevented motor neuron apoptosis mediated by nerve growth factor through p75 neurotrophin receptor. Taken together, these results emphasize the key role of astrocytes in determining motor neuron fate in amyotrophic lateral sclerosis.     

Focal dysfunction of the proteasome: a pathogenic factor in a mouse model of amyotrophic lateral sclerosis

Mutations in the Cu/Zn-superoxide dismutase (SOD-1) gene are responsible for a familial form of amyotrophic lateral sclerosis (fALS). The present study demonstrated impaired proteasomal function in the lumbar spinal cord of transgenic mice expressing human SOD-1 with the ALS-causing mutation G93A (SOD-1(G93A)) compared to non-transgenic littermates (LM) and SOD-1(WT) transgenic mice. Chymotrypsin-like activity was decreased as early as 45 days of age. By 75 days, chymotrypsin-, trypsin-, and caspase-like activities of the proteasome were impaired, at about 50% of control activity in lumbar spinal cord, but unchanged in thoracic spinal cord and liver. Both total and specific activities of the proteasome were reduced to a similar extent, indicating that a change in proteasome function, rather than a decrease in proteasome levels, had occurred. Similar decreases of total and specific activities of the proteasome were observed in NIH 3T3 cell lines expressing fALS mutants SOD-1(G93A) and SOD-1(G41S), but not in SOD-1(WT) controls. Although overall levels of proteasome were maintained in spinal cord of SOD-1(G93A) transgenic mice, the level of 20S proteasome was substantially reduced in lumbar spinal motor neurons relative to the surrounding neuropil. It is concluded that impairment of the proteasome is an early event and contributes to ALS pathogenesis.     

Messenger RNA oxidation occurs early in disease pathogenesis and promotes motor neuron degeneration in ALS

Background

Accumulating evidence indicates that RNA oxidation is involved in a wide variety of neurological diseases and may be associated with neuronal deterioration during the process of neurodegeneration. However, previous studies were done in postmortem tissues or cultured neurons. Here, we used transgenic mice to demonstrate the role of RNA oxidation in the process of neurodegeneration.

Methodology/Principal Findings

We demonstrated that messenger RNA (mRNA) oxidation is a common feature in amyotrophic lateral sclerosis (ALS) patients as well as in many different transgenic mice expressing familial ALS-linked mutant copper-zinc superoxide dismutase (SOD1). In mutant SOD1 mice, increased mRNA oxidation primarily occurs in the motor neurons and oligodendrocytes of the spinal cord at an early, pre-symptomatic stage. Identification of oxidized mRNA species revealed that some species are more vulnerable to oxidative damage, and importantly, many oxidized mRNA species have been implicated in the pathogenesis of ALS. Oxidative modification of mRNA causes reduced protein expression. Reduced mRNA oxidation by vitamin E restores protein expression and partially protects motor neurons.

Conclusion/Significance

These findings suggest that mRNA oxidation is an early event associated with motor neuron deterioration in ALS, and may be also a common early event preceding neuron degeneration in other neurological diseases.     

Neuroprotective Effects of (-)-Epigallocatechin-3-gallate in a Transgenic Mouse Model of Amyotrophic Lateral Sclerosis

The purpose of this study is to evaluate neuroprotective effects of (-)-Epigallocatechin-3-gallate (EGCG) in a transgenic mouse model of Amyotrophic lateral sclerosis (ALS). SOD1-G93A transgenic mice and wild-type mice were randomly divided into EGCG-treated groups (10 mg/kg, p.o) and vehicle-treated control groups. Rotarod measurement was performed to assess the motor function of mice starting at the age of 70 days. Nissl staining to examine the number of motor neurons and CD11b immunohistochemical staining to evaluate activation of microglia in the lumbar spinal cords were conducted at the age of 120 days. In addition, for further observation of regulation of cell signaling pathways by EGCG, we used immunohistochemical analysis for nuclear factor kappa B (NF-κB) and cleaved caspase-3 as well as western blot analysis to determine the expression of nitric oxide synthase (iNOS) and NF-κB in the spinal cord. This study demonstrated that oral administration of EGCG beginning from a pre-symptomatic stage significantly delayed the onset of disease, and extended life span. Furthermore, EGCG-treated transgenic mice showed increased number of motor neurons, diminished microglial activation, reduced immunohistochemical reaction of NF-κB and cleaved caspase-3 as well as reduced protein level of iNOS and NF-κB in the spinal cords. In conclusion, this study provides further evidences that EGCG has multifunctional therapeutic effects in the mouse model of ALS.     

Inducible Nitric Oxide Synthase Up-Regulation in a Transgenic Mouse Model of Familial Amyotrophic Lateral Sclerosis

Mutations in copper/zinc superoxide dismutase (SOD1) are associated with a familial form of amyotrophic lateral sclerosis (ALS), and their expression in transgenic mice produces an ALS-like syndrome. Here we show that, during the course of the disease, the spinal cord of transgenic mice expressing mutant SOD1 (mSOD1) is the site not only of a progressive loss of motor neurons, but also of a dramatic gliosis characterized by reactive astrocytes and activated microglial cells. These changes are absent from the spinal cord of age-matched transgenic mice expressing normal SOD1 and of wild-type mice. We also demonstrate that, during the course of the disease, the expression of inducible nitric oxide synthase (iNOS) increases. In both early symptomatic and end-stage transgenic mSOD1 mice, numerous cells with the appearance of glial cells are strongly iNOS-immunoreactive. In addition, iNOS mRNA level and catalytic activity are increased significantly in the spinal cord of these transgenic mSOD1 mice. None of these alterations are seen in the cerebellum of these animals, a region unaffected by mSOD1. Similarly, no up-regulation of iNOS is detected in the spinal cord of age-matched transgenic mice expressing normal SOD1 or of wild-type mice. The time course of the spinal cord gliosis and iNOS up-regulation parallels that of motor neuronal loss in transgenic mSOD1 mice. Neuronal nitric oxide synthase expression is only seen in neurons in the spinal cord of transgenic mSOD1 mice, regardless of the stage of the disease, and of age-matched transgenic mice expressing normal SOD1 and wild-type mice. Collectively, these data suggest that the observed alterations do not initiate the death of motor neurons, but may contribute to the propagation of the neurodegenerative process. Furthermore, the up-regulation of iNOS, which in turn may stimulate the production of nitric oxide, provides further support to the presumed deleterious role of nitric oxide in the pathogenesis of ALS. This observation also suggests that iNOS may represent a valuable target for the development of new therapeutic avenues for ALS.     

Overexpression of Abeta is associated with acceleration of onset of motor impairment and superoxide dismutase 1 aggregation in an amyotrophic lateral sclerosis mouse model

Transgenic mice carrying mutant Cu/Zn superoxide dismutase (SOD1) recapitulate the motor impairment of human amyotrophic lateral sclerosis (ALS). The amyloid-beta (Abeta) peptide associated with Alzheimer's disease is neurotoxic. To investigate the potential role of Abeta in ALS development, we generated a double transgenic mouse line that overexpresses SOD1(G93A) and amyloid precursor protein (APP)-C100. The transgenic mouse C100.SOD1(G93A) overexpresses Abeta and shows earlier onset of motor impairment but has the same lifespan as the single transgenic SOD1(G93A) mouse. To determine the mechanism associated with this early-onset phenotype, we measured copper and zinc levels in brain and spinal cord and found both significantly elevated in the single and double transgenic mice compared with their littermate control mice. Increased glial fibrillary acidic protein and decreased APP levels in the spinal cord of C100.SOD1(G93A) mice compared with the SOD1(G93A) mice agree with the neuronal damage observed by immunohistochemical analysis. In the spinal cords of C100.SOD1(G93A) double transgenic mice, soluble Abeta was elevated in mice at end-stage disease compared with the pre-symptomatic stage. Buffer-insoluble SOD1 aggregates were significantly elevated in the pre-symptomatic mice of C100.SOD1(G93A) compared with the age-matched SOD1(G93A) mice, correlating with the earlier onset of motor impairment in the C100.SOD1(G93A) mice. This study supports abnormal SOD1 protein aggregation as the pathogenic mechanism in ALS, and implicates a potential role for Abeta in the development of ALS by exacerbating SOD1(G93A) aggregation.     

Expression of nuclear hormone receptors, their coregulators and type I iodothyronine 5'-deiodinase gene in mammary tissue of nonlactating and postlactating rats

The aim of the study was to test the hypothesis that expression of retinoid receptors (RARalpha, RARbeta, RARgamma), rexinoid receptors (RXRalpha, RXRbeta), thyroid hormone receptors (TRalpha, TRbeta), estrogen receptors (ERalpha, ERbeta), nuclear receptor coregulators (N-CoR, SRC-1, SMRT), and in addition type I iodothyronine 5'-deiodinase (5'-DI), EGFR and erb-B2/neu would be different in mammary postlactating tissue in comparison with that of nonlactating mammary gland. Using RT-PCR, we have shown that expression of RARalpha, RXRalpha,TRalpha, ERalpha,ERbeta,N-CoR, SRC-1, SMRT and EGFR in rat was significantly increased in postlactating mammary gland when compared to that of nonlactating mammary tissue. Postlactating mammary glands were found to express all RAR and RXR subtypes studied when compared to nonlactating mammary tissues that express exclusively RARalpha and RXRalpha subtypes. Enhanced expression of a number of nuclear hormone receptors, their coregulators in mammary tissue of postlactating rats in comparison with nonlactating animals identify a potential role for retinoid, thyroid and estrogen signalling pathways also after lactation period.     

Induction of cyclooxygenase-2 in reactive glial cells by the CD40 pathway: relevance to amyotrophic lateral sclerosis

An inflammatory process in association with reactive gliosis has been suggested to play an important role in the pathogenesis of amyotrophic lateral sclerosis (ALS). One of the key findings is a marked increase in the level of cyclooxygenase-2 (COX-2), a therapeutic target of ALS. We investigated the expression of CD40 in the spinal cord of a transgenic mouse model of ALS (G93A mice), and its relevance to COX-2 upregulation. CD40 was predominantly expressed in neurons in normal spinal cord and upregulated in reactive glial cells in spinal cord injury. In the spinal cord of G93A mice, the expression of CD40 was increased in both reactive microglia and astrocytes, where COX-2 was especially increased. The level of COX-2 was upregulated in microglia and astrocytes by CD40 stimulation in vitro. CD40 stimulation in primary spinal cord cultures caused motor neuron loss that was protected by selective COX-2 inhibitor. These results suggest that CD40, which is upregulated in reactive glial cells in ALS, participates in motor neuron loss via induction of COX-2.