Modeling amyotrophic lateral sclerosis in hSOD1 transgenic swine.

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease that occurs in two clinically indistinguishable forms: sporadic (SALS) and familial (FALS), the latter linked to several gene mutations, mostly inheritable in a dominant manner. Nearly 20% of FALS forms are linked to mutations in the Cu/Zn superoxide dismutase (SOD1) gene. Research on ALS relies on transgenic models and particularly on mice carrying a glycine-to-alanine conversion at the 93rd codon (G93A) of the hSOD1 gene. Although G93A transgenic mice have been widely employed in clinical trials and basic research, doubts have been recently raised from numerous reliable sources about their suitability to faithfully reproduce human disease. Besides, the scientific community has already foreseen swine as an attractive and alternative model to nonhuman primates for modeling human diseases due to closer anatomical, physiological and biochemical features of swine rather than rodents to humans. On this basis, we have produced the first swine ALS model by in vitro transfection of cultured somatic cells combined with somatic cell nuclear transfer (SCNT). To achieve this goal we developed a SOD1(G93A) (superoxide dismutase 1 mutated in Gly93-Ala) vector, capable of promoting a high and stable transgene expression in primary porcine adult male fibroblasts (PAF). After transfection, clonal selection and transgene expression level assessment, selected SOD1(G93A) PAF colonies were used as nuclei donors in SCNT procedures. SOD1(G93A) embryos were transferred in recipient sows, and pregnancies developed to term. A total of 5 piglets survived artificial hand raising and weaning and developed normally, reaching adulthood. Preliminary analysis revealed transgene integration and hSOD1(G93A) expression in swine tissues and 360° phenotypical characterization is ongoing. We believe that our SOD1(G93A) swine would provide an essential bridge between the fundamental work done in rodent models and the reality of treating ALS.

Intracerebroventricular administration of human umbilical cord blood cells delays disease progression in two murine models of motor neuron degeneration.

The lack of effective drug therapies for motor neuron diseases (MND), and in general for all the neurodegenerative disorders, has increased the interest toward the potential use of stem cells. Among the cell therapy approaches so far tested in MND animal models, systemic injection of human cord blood mononuclear cells (HuCB-MNCs) has proven to reproducibly increase, although modestly, the life span of SOD1G93A mice, a model of familial amyotrophic lateral sclerosis (ALS), even if only few transplanted cells were found in the damaged areas. In attempt to improve the potential efficacy of these cells in the central nervous system, we examined the effect and distribution of Hoechst 33258-labeled HuCB-MNCs after a single bilateral intracerberoventricular injection in two models of motor neuron degeneration, the transgenic SOD1G93A and wobbler mice. HuCB-MNCs significantly ameliorated symptoms progression in both mouse models and prolonged survival in SOD1G93A mice. They were localized in the lateral ventricles, even 4 months after administration. However, HuCB-MNCs were not found in the spinal cord ventral horns. This evidence strengthens the hypothesis that the beneficial role of transplanted cells is not due to cell replacement but is rather associated with the production and release of circulating protective factors that may act both at the central and/or peripheral levels. In particular, we show that HuCB-MNCs release a series of cytokines and chemokines with antiinflammatory properties that could be responsible of the functional improvement of mouse models of motor neuron degenerative disorders.

Unraveling the complexity of amyotrophic lateral sclerosis: recent advances from the transgenic mutant SOD1 mice.

Amyotrophic Lateral Sclerosis (ALS), which accounts for the majority of motor neuron disorders, is a progressive and fatal neurodegenerative disease leading to complete paralysis of skeletal muscles and premature death usually from respiratory failure. About 10% of all ALS cases are inherited, with the responsible gene having been identified in approximately 25% of these individuals. Mutations in the copper-zinc superoxide dismutase (SOD1) gene were the first to be recognized nearly twenty years ago, and since then different animal models, in particular transgenic rodents, have been developed. They replicate many of the clinical, neuropathological and molecular features of ALS patients and have contributed significantly to our understanding of the pathogenic mechanisms of this disease. Although results obtained so far with mutant SOD1 mice have not translated into effective therapies in ALS patients, these models still represent the only experimentally accessible system to study multiple aspects of disease pathogenesis and to provide proof-of-principle for the development of new therapeutic strategies. This review will examine the most recent discoveries obtained from these animal models in an attempt to elucidate the complex mechanisms of the disease. In particular it will focus on the contribution of multiple cell types in governing the disease development and progression.

Lack of changes in the PI3K/AKT survival pathway in the spinal cord motor neurons of a mouse model of familial amyotrophic lateral sclerosis.

The vulnerability of motor neurons in transgenic SOD1G93A mice, a model of familial amyotrophic lateral sclerosis (ALS), may depend on the failure of these cells to activate survival mechanisms in response to the toxic mutant SOD1. To test this we investigated whether defects in the PI3K/Akt pathway, a survival signal, and of its neuron-specific activator, Rai, were important for motor neuron degeneration in these mice. No substantial changes were found in the levels of Rai, PI3K(p85) or phosphorylated Akt (P-Akt) in the ventral horn of spinal cord of SOD1G93A mice during disease progression. P-Akt immunoreactivity was the same in degenerating and healthy motor neurons. Rai ablation in SOD1G93A mice slightly accelerated the motor dysfunction without affecting their life span. Thus, motor neurons in SOD1G93A mice do not lose the pro-survival PI3K/Akt signal nor increase it in order to suppress the cell death mechanisms.

Activation of the p38MAPK cascade is associated with upregulation of TNF alpha receptors in the spinal motor neurons of mouse models of familial ALS.

Phosphorylated p38 mitogen-activated protein kinase (p38MAPK), but not activated c-jun-N-terminal kinase (JNK), increases in the motor neurons of transgenic mice overexpressing ALS-linked SOD1 mutants at different stages of the disease. This effect is associated with a selective increase of phosphorylated MKK3-6, MKK4 and ASK1 and a concomitant upregulation of the TNFalpha receptors (TNFR1 and TNFR2), but not IL1beta and Fas receptors. Activation of both p38 MAPK and JNK occurs in the activated microglial cells of SOD1 mutant mice at the advanced stage of the disease; however, this effect is not accompanied by the concomitant activation of the upstream kinases ASK1 and MKK3,4,6, while both the TNFRs are overexpressed in these cells. No changes of the upstream p38MAPK cascade kinases or TNFRs occur in reactive astrocytes. These findings highlight the activation of a selective intracellular signaling pathway in the motor neurons of SOD1 mutant mice, which is likely implicated in their death.

Rat brain serotonin neurones that express neuronal nitric oxide synthase have increased sensitivity to the substituted amphetamine serotonin toxins 3,4-methylenedioxymethamphetamine and p-chloroamphetamine.

Substituted amphetamines such as p-chloroamphetamine and the abused drug methylenedioxymethamphetamine cause selective destruction of serotonin axons in rats, by unknown mechanisms. Since some serotonin neurones also express neuronal nitric oxide synthase, which has been implicated in neurotoxicity, the present study was undertaken to determine whether nitric oxide synthase expressing serotonin neurones are selectively vulnerable to methylenedioxymethamphetamine or p-chloroamphetamine. Using double-labeling immunocytochemistry and double in situ hybridization for nitric oxide synthase and the serotonin transporter, it was confirmed that about two thirds of serotonergic cell bodies in the dorsal raphé nucleus expressed nitric oxide synthase, however few if any serotonin transporter immunoreactive axons in striatum expressed nitric oxide synthase at detectable levels. Methylenedioxymethamphetamine (30 mg/kg) or p-chloroamphetamine (2 x 10 mg/kg) was administered to Sprague-Dawley rats, and 7 days after drug administration there were modest decreases in the levels of serotonin transporter protein in frontal cortex, and striatum using Western blotting, even though axonal loss could be clearly seen by immunostaining. p-Chloroamphetamine or methylenedioxymethamphetamine administration did not alter the level of nitric oxide synthase in striatum or frontal cortex, determined by Western blotting. Analysis of serotonin neuronal cell bodies 7 days after p-chloroamphetamine treatment, revealed a net down-regulation of serotonin transporter mRNA levels, and a profound change in expression of nitric oxide synthase, with 33% of serotonin transporter mRNA positive cells containing nitric oxide synthase mRNA, compared with 65% in control animals. Altogether these results support the hypothesis that serotonin neurones which express nitric oxide synthase are most vulnerable to substituted amphetamine toxicity, supporting the concept that the selective vulnerability of serotonin neurones has a molecular basis.

Accumulation of human SOD1 and ubiquitinated deposits in the spinal cord of SOD1G93A mice during motor neuron disease progression correlates with a decrease of proteasome.

Mutations in SOD1 cause selective motor neuron degeneration in familial amyotrophic lateral sclerosis patients and transgenic mice overexpressing the mutant enzyme. Formation and accumulation of ubiquitinated aggregates in motor neurons are thought to be involved in the toxic gain of function of mutant SOD1. The present study shows that the accumulation of soluble and detergent-insoluble mutant SOD1 in spinal cord of symptomatic SOD1G93A transgenic mice is due to impaired degradation of mutant SOD1 rather than to increased transcript levels. This effect was accompanied by a decrease of constitutive proteasome levels and a concomitant increase of immunoproteasome in the spinal cord homogenate which resulted in overall unchanged proteasome activity. A decrease of constitutive proteasome occurred in the motor neurons of SOD1G93A mice at the presymptomatic stage and became remarkable with the progression of the disease. This provides further evidence for an involvement of proteasome impairment in the toxicity of mutant SOD1.

Inter- and intracellular signaling in amyotrophic lateral sclerosis: role of p38 mitogen-activated protein kinase.

The pathogenetic processes underlying the selective motor neuron degeneration in amyotrophic lateral sclerosis (ALS) are complex and still not completely understood even in the cases of inherited disease caused by mutations in the Cu/Zn superoxide dismutase-dependent (SOD1) gene. Recent evidence supports the view that ALS is not a cell-autonomous disease and that glial-neuron cross-talk, throughout cytokines and other toxic factors like the nitric oxide and superoxide, is a crucial determinant for the induction of motor neuron death. This cell-cell interaction may determine the progression of the disease through processes that are likely independent of the initial trigger and that may converge on the activation of intracellular death pathways in the motor neurons. In this review we provide support to the hypothesis that aberrant expression and activity of p38 mitogen protein-activated kinases cascade (p38MAPK) in motor neurons and glial cells may play a role in the development and progression of ALS. Increased activation of p38MAPK may phosphorylate neuron-specific substrates altering their physiological properties and it may turn on responsive genes leading to neurotoxicity.

Androgen 5-alpha-reductase type 2 is highly expressed and active in rat spinal cord motor neurones.

Spinal cord motoneurones express high levels of androgen receptor. However, in responsive tissue, the effects of testosterone is often mediated by the more potent androgenic derivative 5-alpha-dihydrotestosterone (DHT). This compound is formed in androgen target cells by the enzyme 5-alpha-reductase. Two isoforms of the 5-alpha-reductase, with limited degree of homology, have been cloned, type 1 and type 2. The low affinity-constitutive type 1 isoenzyme is widely distributed in the body; the high affinity-androgen regulated 5-alpha-reductase type 2 is confined to androgen-dependent structures and shows a peculiar pH optimum at acidic values. We have previously shown that high levels of 5-alpha-reductase activity are detectable in rat spinal cord. Here, using reverse transcriptase-polymerase chain reaction, we show that both isoforms are expressed in the whole spinal cord of the rat. The enzymatic pH optimum measured in immortalized spinal cord motoneurones (NSC34) is typical of the type 2 isoenzyme. Using in situ hybridization technique, we found that 5-alpha-reductase type 2 is confined to the motoneuronal cells of the anterior horns of the rat spinal cord, the cells that also are known to express high levels of androgen receptor. Because of the close association of androgen receptor and 5-alpha alpha-reductase type 2, motoneuronal cells should be considered as target cells for androgens.

Persistent activation of p38 mitogen-activated protein kinase in a mouse model of familial amyotrophic lateral sclerosis correlates with disease progression.

The p38 mitogen-activated protein kinase (p38MAPK) is activated via phosphorylation in neurones and glial cells by a variety of stimuli including oxidative stress, excitotoxicity, and inflammatory cytokines. Activated p38MAPK can in turn induce phosphorylation of cytoskeletal proteins and activation of cytokines and nitric oxide, thus contributing to neurodegeneration. We investigated the expression and distribution of p38MAPK in the spinal cord of transgenic mice expressing a superoxide dismutase 1 mutation (SOD1G93A), a model of familial amyotrophic lateral sclerosis (ALS). Accumulation of p38MAPK was found by immunoblotting in the spinal cord of G93A mice during the progression of disease, but no changes were detected in its mRNA levels. Immunostaining for phosphorylated p38MAPK in lumbar spinal cord sections of SOD1G93A mice at the presymptomatic and early stages of disease showed an increased labeling in motor neurones that colocalized with phosphorylated neurofilaments in vacuolized perikarya and neurites, as detected by confocal microscopy. As the disease progressed, activated p38MAPK also accumulated in hypertrophic astrocytes and reactive microglia, as demonstrated by colocalization with GFAP and CD11b immunostaining, respectively. These data suggest that activation of p38MAPK in motor neurons and then in reactive glial cells may contribute, respectively, to the development and progression of motor neuron pathology in SOD1G93A mice.

Overexpression of S100beta in transgenic mice does not protect from serotonergic denervation induced by 5,7-dihydroxytryptamine.

Transgenic mice overexpressing S100beta were used to examine whether the chronic elevation of this protein alters the response to selective partial serotonergic lesions produced by bilateral intracerebroventricular injections of 5,7-dihydroxytryptamine (5,7-DHT). Basal levels of S100beta mRNA examined by in situ hybridization were two- to threefold higher throughout the brain in transgenic than in control mice, whereas 5-HT levels in forebrain were similar in both. After the 5,7-DHT-induced lesions, no differences were found in the S100beta mRNA levels in either normal or transgenic mice. At 5 and 60 days after the lesion, forebrain 5-HT levels were reduced by 56% and 35%, respectively, in control mice and by 51% and 35%, respectively, in the transgenic mice. Analysis of the 5-HT immunostaining showed a marked decrease of the immunoreactivity in various brain regions, which was comparable at the two intervals postlesion. One exception was the medial hypothalamus, where an almost complete disappearance of 5-HT immunoreactivity was observed in the medial region at 5 days after lesion, followed by a marked reinnervation 60 days later. These hypothalamic changes were seen in both controls and S100beta-overexpressing transgenic mice. Quantitative analysis of the density of 5-HT transporter sites using [(3)H]citalopram binding, a marker of serotonergic terminals, showed a marked decrease in different brain regions at both 5 and 60 days after 5,7-DHT injections. No difference in basal and postlesion levels of [(3)H]citalopram binding was seen between transgenic and control mice. In conclusion, this study demonstrates that constitutive overexpression of S100beta in transgenic mice does not modify serotonin levels during development, nor does it protect the serotonergic neurons from selective neurotoxicity or modify the serotonergic sprouting induced by partial lesion.

Transgenic SOD1 G93A mice develop reduced GLT-1 in spinal cord without alterations in cerebrospinal fluid glutamate levels.

Glutamate-induced excitotoxicity is suggested to play a central role in the development of amyotrophic lateral sclerosis (ALS), although it is still unclear whether it represents a primary cause in the cascade leading to motor neurone death. We used western blotting, immunocytochemistry and in situ hybridization to examine the expression of GLT-1 in transgenic mice carrying a mutated (G93A) human copper-zinc superoxide dismutase (TgSOD1 G93A), which closely mimic the features of ALS. We observed a progressive decrease in the immunoreactivity of the glial glutamate transporter (GLT-1) in the ventral, but not in the dorsal, horn of lumbar spinal cord. This effect was specifically found in 14- and 18-week-old mice that had motor function impairment, motor neurone loss and reactive astrocytosis. No changes in GLT-1 were observed at 8 weeks of age, before the appearance of clinical symptoms. Decreases in GLT-1 were accompanied by increased glial fibrillary acidic protein (GFAP) levels and no change in the levels of GLAST, another glial glutamate transporter. The glutamate concentration in the cerebrospinal fluid (CSF) of TgSOD1 G93A mice was not modified at any of the time points examined, compared with age-matched controls. These findings indicate that the loss of GLT-1 protein in ALS mice selectively occurs in the areas affected by neurodegeneration and reactive astrocytosis and it is not associated with increases of glutamate levels in CSF. The lack of changes in GLT-1 at the presymptomatic stage suggests that glial glutamate transporter reduction is not a primary event leading to motor neurone loss.

Early vacuolization and mitochondrial damage in motor neurons of FALS mice are not associated with apoptosis or with changes in cytochrome oxidase histochemical reactivity.

Overexpression of mutated superoxide dismutase (SOD1) in transgenic mice causes a progressive motor neuron degeneration in the spinal cord similar to that in human amyotrophic lateral sclerosis (ALS). Ultrastructural analysis of motor neurons at different stages of the disease in transgenic C57BL/6 mice carrying the G93A mutation of SOD1 showed, at about 2 weeks of age, much earlier than the initial symptoms of the disease, microvacuoles in the cytoplasm, with marked swelling of the mitochondria. Nuclei with an apoptotic morphology were never observed in these motor neurons. Swollen mitochondria were also seen in the distal part of motor axons of phrenic nerves and in the large axons of sciatic nerves before the onset of the disease, but no mitochondrial alterations were seen in skeletal muscles or in the small sciatic nerve axons. Moreover, we found no apparent changes in the histochemical reactivity of cytochrome oxidase in motor neurons of transgenic mice even at the advanced stage of the disease, suggesting that partial neuronal activity in these cells may be maintained despite the altered mitochondria. Immunoreactivity for human SOD1 was high around vacuoles in the motor neurons of transgenic mice but no cytoplasmic intracellular SOD1 aggregates were observed. Our data indicate that mitochondrial swelling may be an important factor triggering the cascade leading to progressive motor neuron death. Activation of the mitochondrial permeability transition pore may be involved in this process, through excitotoxicity or other neurotoxic stimuli.

Differential expression of S100beta and glial fibrillary acidic protein in the hippocampus after kainic acid-induced lesions and mossy fiber sprouting in adult rat.

The expression of S100beta and glial fibrillary acidic protein (GFAP) was analyzed following bilateral injection of kainic acid (KA), a glutamate derivative, into the CA3 region of the adult rat hippocampus. This treatment produces a progressive degeneration of the pyramidal neurons of the hippocampus while sparing the granule cells of the dentate gyrus which undergo sprouting of their axons in the supragranular layer. Messenger RNA and protein levels were measured, by Northern blot and ELISA, in the hippocampus of lesioned and sham-operated rats 1, 7, and 30 days after KA injection. A significant increase of GFAP and its mRNA was demonstrated at each time point, whereas S100beta mRNA levels were significantly enhanced only 30 days after the KA injection and the levels of S100beta protein remained unchanged at all time points. However, when analyzed by immunohistochemistry the S100beta showed clear changes in its expression and distribution depending on the region considered. One month after KA injection, S100beta immunoreactivity was considerably reduced in the stratum radiatum of CA3 region, but there was increased S100beta immunoreactivity in the stratum moleculare. In particular, a notable band of S100beta positive, hypertrophic astrocytes appeared in the supragranular layer of the dentate gyrus where the sprouting of mossy fiber collaterals was detected by Timm's staining. These data show for the first time that an increase in S100beta expression in subpopulations of reactive astrocytes may be involved in the structural reorganization of the hippocampus following KA-induced neurodegeneration.

Intraregional variation in expression of serotonin transporter messenger RNA by 5-hydroxytryptamine neurons.

The distribution of the messenger RNA encoding the 5-hydroxytryptamine transporter was investigated in rat brain. 5-Hydroxytryptamine transporter messenger RNA was found exclusively in the B1-B9 cell groups containing the cell bodies of 5-hydroxytryptamine neurons. Combined in situ hybridization and 5-hydroxytryptamine immunocytochemistry demonstrated 5-hydroxytryptamine transporter gene expression in the majority of and exclusively in 5-hydroxytryptamine neurons. Cells differed in their levels of expression of 5-hydroxytryptamine transporter messenger RNA and 5-hydroxytryptamine immunofluorescence, but with a tight correlation between the two parameters. Image analysis of cells from B7, the dorsal raphe nucleus, and B8, the median raphe nucleus, revealed significant differences between groups in the mean cellular level of 5-hydroxytryptamine transporter gene expression. Cells in the ventromedial subdivision of B7 displayed higher levels of expression than cells in B8 or cells in the lateral wings of B7. There was also heterogeneity in the distribution of the cellular levels of expression for two other genes expressed by 5-hydroxytryptamine neurons: l-aromatic amino acid decarboxylase messenger RNA and tryptophan hydroxylase messenger RNA. However, the relative levels of expression of these two genes within the four regions studied differed from that of 5-hydroxytryptamine transporter messenger RNA. These results indicate intraregional differences between 5-hydroxytryptamine neurons with respect to 5-hydroxytryptamine transporter messenger RNA levels. Such differences may account for the differential sensitivity of 5-hydroxytryptamine neurons to cytotoxins.

S-100beta protein is upregulated in astrocytes and motor neurons in the spinal cord of patients with amyotrophic lateral sclerosis.

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease characterized by progressive motor neuron loss and astrogliosis. We studied the immunohistochemical expression of S-100beta, a calcium-binding protein with both neurotrophic and neurotoxic activities, in the spinal cord of patients with ALS. Adjacent sections were processed with an in situ end-labeling technique for the demonstration of apoptosis-related DNA fragmentation. In controls, low expression of S-100beta was found in astrocytes but not motor neurons. Compared to controls, S-100beta was overexpressed in ALS. Most stained cells were reactive astrocytes, but a minority of motor neurons was also labeled. Neuronal labeling was unrelated to the presence of signs of atrophy/degeneration. S-100beta expression was also unrelated to neuronal or glial apoptosis. S-100beta upregulation in ALS spinal cord suggests that the protein might be involved in cellular defense mechanisms against oxidative stress.