Superoxide Dismutase Transgenic Mice Research Articles

Activation of the stress-activated MAP kinase, p38, but not JNK in cortical motor neurons during early presymptomatic stages of amyotrophic lateral sclerosis in transgenic mice

Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disorder, characterized by the degeneration of upper and lower motor neurons (MNs). Central nervous system features include a loss of Betz cells and other pyramidal cells from sensorimotor cortex. The intrinsic mechanism underlying this selective motor neuron loss has not been identified. A recent in vitro study has provided evidence of a novel programmed cell death (PCD) pathway that is unique to spinal cord MNs and is exacerbated by superoxide dismutase (SOD) mutations. This PCD pathway is triggered through the Fas receptor and involves the apoptosis signal-regulating kinase 1 (ASK1), the p38 MAP kinase, and the neuronal form of nitric oxide synthase (nNOS). Previously, we found significant increases in the numbers of ventral horn MNs immunopositive for these enzymes in the spinal cords of mutant SOD transgenic (G93A) mice as early as 60 days of age, suggesting that this pathway may be active in vivo. Since the upper MNs of ALS patients and G93A mice are also known to degenerate, the purpose of the present study was to investigate the possible activation of this PCD pathway in the MNs of the sensorimotor cortex of G93A transgenic mice. Compared to non-transgenic littermates, the G93A mice showed significant increases in the numbers of MNs immunopositive for the active (phosphorylated) forms of ASK1, p38, MKK3/6 (the known activator of p38), and also active caspase-3, as early as 60 days of age. Another stress-activated protein kinase, c-Jun N-terminal kinase (JNK), commonly activated in other neurodegenerative disorders such as Alzheimer's disease, showed no increases in G93A mice at any age. These results suggest that, not only has a PCD pathway been activated in the cortical MNs, but one that may be unique to ALS. Moreover, these findings suggest that earlier diagnosis and therapeutic intervention may be possible for successful treatment of ALS. Consequently, these enzymes may provide the biochemical markers to enable earlier diagnosis of ALS and molecular targets for the development of new therapeutic compounds.

Integrative role of cPLA2 with COX‐2 and the effect of non‐steriodal anti‐inflammatory drugs in a transgenic mouse model of amyotrophic lateral sclerosis

Cyclooxygenase-2 (COX-2) is a key molecule in the inflammatory pathway in amyotrophic lateral sclerosis (ALS). Cytosolic phospholipase A (cPLA2) is an important enzyme providing substrate for cyclooxygenases. We therefore examined cPLA2 expression in human ALS and mutant Cu/Zn superoxide dismutase (SOD1) transgenic mice and its relation to COX-2. Immunohistochemistry and real-time RT-PCR revealed elevated cPLA2 protein and its mRNA levels in the lumbar spinal cord of mutant SOD1 mice. COX-2 immunoreactivity was increased in lumbar spinal cord sections from both familial ALS (FALS) and sporadic ALS (SALS) as compared to controls, and cPLA2 immunoreactivity was increased in a patient with FALS. Oral administration of the non-selective cyclooxygenase (COX) inhibitor, sulindac, extended the survival (by 10%) of G93A SOD1 mice as compared to littermate controls. Sulindac, as well as the selective COX-2 inhibitors, rofecoxib and celecoxib reduced cPLA2 immunoreactivity in the lumbar spinal cord of G93A transgenic mice. Sulindac treatment preserved motor neurons, and reduced microglial activation and astrocytosis, in the spinal cord of G93A SOD1 transgenic mice. These results suggest that cPLA2 plays an important role in supplying arachidonic acid to the COX-2 driven inflammatory pathway in ALS associated with SOD1 mutations.

Modulation of p53 Degradation via MDM2-Mediated Ubiquitylation and the Ubiquitin–Proteasome System during Reperfusion after Stroke: Role of Oxidative Stress

The tumor suppressor gene p53 plays an important role in the regulation of apoptosis through transcriptional activation of cell cycle control. Degradation of p53 hinders its role in apoptosis regulation. Recent studies have shown that MDM2-mediated ubiquitylation and the ubiquitin-proteasome system are critical regulating systems of p53 ubiquitylation. However, the mechanism regulating p53-mediated neuronal apoptosis after cerebral ischemia remains unknown. We examined the MDM2 pathway and the ubiquitin-proteasome system using a transient focal cerebral ischemia (tFCI) model and analyzed the interaction between p53 regulation and superoxide using copper/zinc superoxide dismutase (SOD1) transgenic mice after tFCI. p53 degradation and ubiquitylation were detected after tFCI. The accumulation of ubiquitylated p53 was inhibited and p53 degradation was facilitated by SOD1. Nuclear translocation and MDM2/Akt interaction were detected after tFCI and were inhibited by phosphatidylinositol 3-kinase inhibition and promoted by SOD1. Cytosolic translocation of the p53/MDM2 complex was detected after tFCI and was promoted by SOD1. Moreover, accumulation of multiubiquitin chains and direct oxidative injury to a proteasome were detected and inhibited by SOD1 after tFCI. These results suggest that SOD1 promotes the MDM2 pathway and the ubiquitin-proteasome system after tFCI and that production of reactive oxygen species after tFCI prevents p53 degradation by inhibiting both systems.

Protection by dietary zinc in ALS mutant G93A SOD transgenic mice

Mutations to the copper, zinc superoxide dismutase (SOD) gene are responsible for 2–3% of amyotrophic lateral sclerosis (ALS) cases. These mutations result in the protein having a reduced affinity for zinc. SOD becomes toxic to motor neurons when zinc is missing from its active site. Recently, high dosages of zinc (75 and 375 mg/kg/day) have been paradoxically reported to increase the death of G93A-mutant SOD transgenic mice [G.J. Groeneveld, J. de Leeuw van Weenen, F.L. van Muiswinkel, H. Veldman, J.H. Veldink, J.H. Wokke, P.R. Bar, L.H. van den Berg, Zinc amplifies mSOD1-mediated toxicity in a transgenic mouse model of amyotrophic lateral sclerosis, Neurosci. Lett. 352 (2003) 175–178]. In contrast, we have found that moderate supplementation of zinc (∼12 mg/kg/day) delayed death in G93A-mutant SOD mice by 11 days compared to mice on a zinc-deficient diet. Supplementing zinc with even 18 mg/kg/day resulted in a more rapid death of some mice, consistent with the results of Groenevelt et al. However, large amounts of zinc competitively inhibit copper absorption, which inhibits the copper-dependent ceruloplasmin, and can cause a lethal anemia. We found that supplementing the 18 mg/kg/day dosage of zinc with 0.3 mg/kg/day of copper prevented the early death from zinc treatment alone. These data support a role for moderate levels of dietary zinc potentially protecting against the toxicity of ALS-associated SOD and the protection does not result from depleting copper.

RNAi knockdown of Par‐4 inhibits neurosynaptic degeneration in ALS‐linked mice

Evidence from human amyotrophic lateral sclerosis (ALS) patients and ALS-linked Cu/Zn superoxide dismutase (Cu/Zn-SOD) transgenic mice bearing the mutation of glycine to alanine at position 93 (G93A) suggests that the pro-apoptotic protein prostate apoptosis response-4 (Par-4) might be a critical link in the chain of events leading to motor neuron degeneration. We now report that Par-4 is enriched in synaptosomes and post-synaptic density from the ventral horn of the spinal cord. Levels of Par-4 in synaptic compartments increased significantly during rapid and slow declining stages of muscle strength in hSOD1 G93A mutant mice. In the pre-muscle weakness stage, hSOD1 G93A mutation sensitized synaptosomes from the ventral horn of the spinal cord to increased levels of Par-4 expression following excitotoxic and apoptotic insults. In ventral spinal synaptosomes, Par-4-mediated production of pro-apoptotic cytosolic factor(s) was significantly enhanced by the hSOD1 G93A mutation. RNA interference (RNAi) knockdown of Par-4 inhibited mitochondrial dysfunction and caspase-3 activation induced by G93A mutation in synaptosomes from the ventral horn of the spinal cord, and protected spinal motor neurons from apoptosis. These results identify the synapse as a crucial cellular site for the cell death promoting actions of Par-4 in motor neurons, and suggest that targeted inhibition of Par-4 by RNAi may prove to be a neuroprotective strategy for motor neuron degeneration.

Activation of programmed cell death markers in ventral horn motor neurons during early presymptomatic stages of amyotrophic lateral sclerosis in a transgenic mouse model

The identification of the pathogenic mechanism of selective motor neuron (MN) death in amyotrophic lateral sclerosis (ALS) may lead to the development of new therapies to halt or slow the disease course. A novel, MN-specific, Fas-mediated programmed cell death (PCD) pathway has been reported in MNs which involves the activation of p38 MAP kinase (phospho-p38) and neuronal nitric oxide synthase (nNOS). PCD was found to be exacerbated in MNs expressing ALS-linked superoxide dismutase (SOD) mutations. Because this MN-specific pathway was investigated in vitro, we performed an in vivo study to evaluate its potential involvement in MN loss in the lumbar region of spinal cord of mutant SOD transgenic (G93A) mice. Compared to nontransgenic littermates, we found significant increases in the numbers of immunopositive ventral horn MNs of G93A mice as young as 60 days of age for several constituents of this putative PCD pathway, including phospho-p38, nNOS, phospho-ASK1 MAP kinase kinase, and active caspase-3. This study provides in vivo evidence of an MN-specific PCD pathway that may be a pathogenic mechanism of ALS and may be activated very early in the disease process, well before clinical symptoms are evident (200 days). These findings suggest that early diagnosis and therapeutic intervention may be critical for the successful treatment of the disease. These enzymes may provide new markers for earlier diagnosis of ALS and new molecular targets for therapeutic intervention.

Cervical spinal cord delivery of a rabies G protein pseudotyped lentiviral vector in the SOD-1 transgenic mouse. Invited submission from the Joint Section Meeting on Disorders of the Spine and Peripheral Nerves, March 2004.

Lentiviral vectors may constitute a vehicle for long-term therapeutic gene expression in the spinal cord. In amyotrophic lateral sclerosis, spinal cord sclerosis and altered axonal transport pose barriers to therapeutic gene distribution. In the present study the authors characterize gene expression distribution and the behavioral impact of the rabies G (RabG) protein pseudotyped lentiviral vector EIAV.LacZ through cervical spinal cord injection in control and Cu/Zn superoxide dismutase-1 (SOD-1) transgenic mice. Seven-week-old SOD-1 transgenic mice and their wild-type littermates underwent exposure of the cervicomedullary junction and microinjection of RabG.EIAV.LacZ or vehicle. The Basso-Beattie-Bresnahan locomotor score, grip strength meter, and Rotarod assays were used to assess the effects of disease progression, spinal cord microinjection, and lentiviral gene expression. Spinal cords were removed when the mice were in the terminal stage of the disease. The distribution of LacZ gene expression was histologically evaluated and quantified. Direct cervical spinal cord microinjection of RabG.EIAV.LacZ results in extensive central nervous system uptake in SOD-1 transgenic mice; these findings were statistically similar to those in wild-type mice (p > 0.05). Gene expression lasts for the duration of the animal's survival (132 days). The SOD-1 mutation does not prevent retrograde axonal transport of the vector. Three behavioral assays were used to demonstrate that long-term gene expression does not alter sensorimotor function. In comparison with normative data, vector injection and transgene expression do not accelerate disease progression. Direct spinal cord injection of RabG.EIAV vectors represents a feasible method for delivering therapeutic genes to upper cervical spinal cord and brainstem motor neurons. Distribution is not affected by the SOD-1 mutation or disease phenotype.

Endocannabinoids accumulate in spinal cord of SOD1 G93A transgenic mice.

Approximately 2% of amyotrophic lateral sclerosis (ALS) cases are caused by mutations in the super oxide dismutase 1 (SOD1) gene and transgenic mice for these mutations recapitulate many features of this devastating neurodegenerative disease. Here we show that the amount of anandamide (AEA) and 2-arachidonoylglycerol (2-AG), two endocannabinoids that have neuroprotective properties, increase in spinal cord of SOD1(G93A) transgenic mice. This increase occurs in the lumbar section of spinal cords, the first section to undergo neurodegeneration, and is significant before overt motor impairment. Our results show that chronic neurodegeneration induced by a genetic mutation increases endocannabinoid production possibly as part of an endogenous defense mechanism.

Local GDNF expression mediated by lentiviral vector protects facial nerve motoneurons but not spinal motoneurons in SOD1 G93A transgenic mice

Approximately 2% of amyotrophic lateral sclerosis (ALS) cases are associated with mutations in the cytosolic Cu/Zn superoxide dismutase 1 (SOD1) gene. Transgenic SOD1 mice constitute useful models of ALS to screen therapeutical approaches. Glial cell line-derived neurotrophic factor (GDNF) holds promises for the treatment of motoneuron disease. In the present study, GDNF expression in motoneurons of SOD1 G93A transgenic mice was assessed by facial nucleus or intraspinal injection of lentiviral vectors (LV) encoding GDNF. We show that lentiviral vectors allow the expression for at least 12 weeks of GDNF that was clearly detected in motoneurons. This robust intraspinal expression did, however, not prevent the loss of motoneurons and muscle denervation of transgenic mice. In contrast, LV-GDNF induced a significant rescue of motoneurons in the facial nucleus and prevented motoneuron atrophy. The differential effect of GDNF on facial nucleus versus spinal motoneurons suggests different vulnerability of motoneurons in ALS.

NEDL1, a Novel Ubiquitin-protein Isopeptide Ligase for Dishevelled-1, Targets Mutant Superoxide Dismutase-1

Approximately 20% of familial amyotrophic lateral sclerosis (FALS) arises from germ-line mutations in the superoxide dismutase-1 (SOD1) gene. However, the molecular mechanisms underlying the process have been elusive. Here, we show that a neuronal homologous to E6AP carboxyl terminus (HECT)-type ubiquitin-protein isopeptide ligase (NEDL1) physically binds translocon-associated protein-delta and also binds and ubiquitinates mutant (but not wild-type) SOD1 proportionately to the disease severity caused by that particular mutant. Immunohistochemically, NEDL1 is present in the central region of the Lewy body-like hyaline inclusions in the spinal cord ventral horn motor neurons of both FALS patients and mutant SOD1 transgenic mice. Two-hybrid screening for the physiological targets of NEDL1 has identified Dishevelled-1, one of the key transducers in the Wnt signaling pathway. Mutant SOD1 also interacted with Dishevelled-1 in the presence of NEDL1 and caused its dysfunction. Thus, our results suggest that an adverse interaction among misfolded SOD1, NEDL1, translocon-associated protein-delta, and Dishevelled-1 forms a ubiquitinated protein complex that is included in potentially cytotoxic protein aggregates and that mutually affects their functions, leading to motor neuron death in FALS.

Paradoxical Actions of Hydrogen Peroxide on Long-Term Potentiation in Transgenic Superoxide Dismutase-1 Mice

Hydrogen peroxide (H2O2), a reactive oxygen species, is assumed to have a detrimental effect on neuronal plasticity. Indeed, H2O2 suppresses long-term potentiation (LTP) in hippocampal slices of normal rats and wild-type (wt) mice. Transgenic mice overexpressing superoxide dismutase (SOD) 1 (tg-SOD), which maintain high ambient H2O2, have also been shown to be impaired in their ability to express hippocampal LTP. Paradoxically, H2O2, at a concentration (50 microm) that blocks LTP in wt mice, actually enhanced LTP in slices of 2-month-old tg-SOD mice. H2O2-dependent LTP in tg-SOD was blocked by the protein phosphatase calcineurin inhibitor FK506, but not by rapamycin, an FK-binding protein 12 (FKBP12) inhibitor or by 1-(5-isoquinolinesulfonyl)-2-methylpiperazine (H7), a serine-kinase inhibitor. Interestingly, wt and tg-SOD mice expressed similar levels of the antioxidant enzyme catalase and similar activity of glutathione peroxidase. An opposite situation was found in 2-year-old mice. Aged wt mice were impaired in LTP in a manner that could be reversed by the addition of H2O2. Surprisingly, aged tg-SOD mice exhibited larger LTP than that found in wt mice, but this was now reduced by 50 microm H2O2. Both young tg-SOD and aged control mice displayed altered protein phosphatase activity, compared with that of young controls; moreover, FK506 inhibited LTP in old tg-SOD as well as in old wt mice treated with H2O2. These data promoted a dual role for H2O2 in the regulation of LTP, and proposed that it is mediated by the protein phosphatase calcineurin.

Creatine supplementation and riluzole treatment provide similar beneficial effects in copper, zinc superoxide dismutase (G93A) transgenic mice

This study investigated the effects of riluzole (Ril), creatine (Cr) and a combination of these treatments on the onset and progression of clinical signs and neuropathology in an animal model of familial amyotrophic lateral sclerosis, the G93A transgenic mouse ( n=13–17 per group). The onset of clinical signs was delayed ( P<0.05) by about 12 days in all treatment groups compared with control; however, no differences occurred between treatments. All animals were killed at 199 days of age. At the end of the experimental period the severity of clinical signs was less ( P<0.05) with all treatments compared with control. Again no differences between treatments were observed. The treatments had no effect on the number of neurons in ventral horns of the lumbar region of the spinal cord. Transgenic mice ingesting Cr displayed elevated ( P<0.05) total Cr levels in cerebral hemispheres (5%) and spinal cord (8%), but not skeletal muscles. These data demonstrate that treatment with Ril and Cr were both effective in delaying disease onset and clinical disability. To the age of killing, no additional benefit was conferred by co-administration of Ril and Cr.

Copper-zinc superoxide dismutase affects Akt activation after transient focal cerebral ischemia in mice.

The serine-threonine kinase Akt is activated by phosphorylation at serine-473. After phosphorylation, activated Akt inactivates BAD or caspase-9 or other apoptogenic components, thereby inhibiting cell death. In this study we examined the relationship between Akt phosphorylation and oxidative stress after transient focal cerebral ischemia (FCI) using copper-zinc superoxide dismutase (SOD1) transgenic (Tg) mice. The mice were subjected to 60 minutes of middle cerebral artery occlusion by intraluminal suture blockade followed by 1, 4, and 24 hours of reperfusion. Phospho-Akt expression was examined by immunohistochemistry and Western blot analysis. Production of superoxide anion was assessed by the hydroethidine method in both wild-type mice and SOD1 Tg mice. DNA fragmentation was evaluated by terminal deoxynucleotidyl transferase-mediated uridine 5'-triphosphate-biotin nick end labeling (TUNEL). Immunohistochemistry demonstrated that phospho-Akt was constitutively expressed and was decreased in the ischemic core as early as 1 hour after reperfusion, whereas it was temporally increased in the cortex at 4 hours. Phospho-Akt expression was enhanced in the SOD1 Tg mice. Western blot analysis showed that phospho-Akt was maximized 4 hours after reperfusion in the wild-type mice, whereas phospho-Akt was increased as early as 1 hour after ischemia in the SOD1 Tg mice. There was a significant decrease in TUNEL-positive cells in the SOD1 Tg mice compared with the wild-type mice. The present study suggests that SOD1 may contribute to the early activation of the Akt cell survival signaling pathway and may attenuate subsequent DNA damage after transient FCI.

Expression of the low affinity neurotrophin receptor p75 in spinal motoneurons in a transgenic mouse model for amyotrophic lateral sclerosis

Amyotrophic lateral sclerosis is a lethal neurodegenerative disorder involving motoneuron loss in the cortex, brainstem and spinal cord, resulting in progressive paralysis. Aberrant neurotrophin signalling via the low affinity neurotrophin receptor p75 has been suggested to be involved in the motoneuron death by the activation of apoptotic pathways. In order to investigate the involvement of neurotrophin receptor p75 in the amyotrophic lateral sclerosis related motoneuron degeneration process, we have studied the expression of this receptor in the spinal cord of transgenic mice carrying a mutated human Cu, Zn superoxide dismutase gene. Mutations in the superoxide dismutase gene are one of the genetic causes for familiar amyotrophic lateras sclerosis and human superoxide dismutase-1 transgenic mice develop symptoms and pathology similar to those in human amyotrophic lateras sclerosis. Our study shows that in these mice, spinal motoneurons, which normally do not contain the neurotrophin receptor p75 receptor, express this receptor during the progress of the disease. Expression of the neurotrophin receptor p75 receptor coincides with the expression of activating transcription factor 3, a member of the activating transcription factor/cyclic AMP family of stress transcription factors. Only a minority of these spinal motoneurons actually showed co-expression of neurotrophin receptor p75 with caspase-3 activity, suggesting that expression of the neurotrophin receptor p75 receptor is not directly related to the execution phase of the apoptosis process.

Glutathione peroxidase-1 contributes to the neuroprotection seen in the superoxide dismutase-1 transgenic mouse in response to ischemia/reperfusion injury.

The authors hypothesized that glutathione peroxidase-1 (Gpx-1) contributes to the neuroprotection seen in the superoxide dismutase-1 transgenic (Sod-1 tg) mouse. To investigate this hypothesis, they crossed the Gpx-1 -/- mouse with the Sod-1 tg and subjected the cross to a mouse model of ischemia/reperfusion. Two hours of focal cerebral ischemia followed by 24 hours of reperfusion was induced via intraluminal suture. The Sod-1 tg/Gpx-1 -/- cross exhibited no neuroprotection when infarct volume was measured; indeed, infarct volume increased in the Sod-1 tg/Gpx-1 -/- cross compared with the wild-type mouse. Our results suggest that Gpx-1 plays an important regulatory role in the protection of neural cells in response to ischemia/reperfusion injury.

Regulation of intestinal nuclear factor-κB activity and E-selectin expression during sepsis: A role for peroxynitrite

Background & Aims: During sepsis, the production of both nitric oxide and superoxide are increased. Furthermore, NO and O2− may interact to produce peroxynitrite. The major aim of the present study was to assess the relative roles of NO, O2−, and ONOO− in the regulation of nuclear factor κB (NF-κB) activity and subsequent E-selectin expression during the early stages of sepsis. Methods: Mice were administered 5 μg lipopolysaccharide (LPS) intraperitoneally, and NF-κB activity and E-selectin expression in the small intestine were assessed 3 hours later. Results: In wild-type mice, increased levels of NF-κB in nuclear extracts were noted. By contrast, in both inducible nitric oxide synthase–deficient and transgenic Cu/Zn superoxide dismutase–overexpressing mice, NF-κB mobilization to the nucleus was diminished. Pretreatment with a ONOO− decomposition catalyst (5,10,15,20-tetrakis[4-sulfonatophenyl]porphyrinato iron [III] [FeTPPS]) resulted in a diminished impact of LPS on NF-κB activation. LPS increased vascular E-selectin expression in wild-type mice. E-selectin expression was diminished in inducible nitric oxide synthase–deficient mice after LPS challenge, but E-selectin expression remained elevated in both Cu/Zn superoxide dismutase transgenic mice and wild-type mice pretreated with FeTPPS. Conclusions: Our observations suggest that NO, O2−, and ONOO− production are all important mediators in the induction of NF-κB activity during endotoxemia and that, in vivo, E-selectin expression on endothelium may not always be associated with whole-organ NF-κB activation.GASTROENTEROLOGY 2003;124:118-128

Glutathione Peroxidase-1 Contributes to the Neuroprotection Seen in the Superoxide Dismutase-1 Transgenic Mouse in Response to Ischemia/Reperfusion Injury

Glutathione Peroxidase-1 Contributes to the Neuroprotection Seen in the Superoxide Dismutase-1 Transgenic Mouse in Response to Ischemia/Reperfusion Injury

Molecular biological approaches to neurological disorders including knockout and transgenic mouse models.

Advances of molecular biology have provided a great variety of new approaches to research on human disorders. This article gives an outline of molecular biological approaches to analysis of neurological disorders such as giant cell glioblastoma (GGBM) and amyotrophic lateral sclerosis (ALS), and their respective animal models: p53 knockout mice for GGBM and mutant superoxide dismutase-1 transgenic mice for ALS. Genomic DNA extracted from fresh-frozen tissue is examined by Southern blotting for screening mutations in a certain gene. Polymerase chain reaction (PCR) products of a gene in genomic DNA are examined by single-stranded conformation polymorphism, sequencing and agarose gel electrophoresis for identifying mutations, and for preparing and evaluating DNA probes used in Southern blotting and DNA in situ hybridization (ISH). Total RNA from tissue is examined by northern blotting for quantifying and verifying a certain mRNA. Reverse transcription-PCR products of a certain mRNA in total RNA are examined by sequencing and agarose gel electrophoresis for preparing and evaluating cDNA probes used in northern blotting and mRNA ISH. Tissue total protein is immunoblotted for quantifying and verifying a certain protein, and for evaluating the specificity of antibodies used in western blotting and immunohistochemistry. Immunoprecipitates are immunoblotted for evaluating a profile of protein or other substances. Enzyme-linked immunosorbent assay is used for measuring tissue concentration of protein or other substances, and for determining titers of specific antibodies. By these procedures, chronological analysis of animal models for human diseases contribute to elucidating pathogenic mechanisms and exploiting new therapies. Noticing both the similarity and difference between human and animal disorders will help understand the nature of disease.

Proteasomal inhibition by misfolded mutant superoxide dismutase 1 induces selective motor neuron death in familial amyotrophic lateral sclerosis.

Accumulating evidence indicates that abnormal conformation of mutant superoxide dismutase 1 (SOD1) is an essential feature underlying the pathogenesis of mutant SOD1-linked familial amyotrophic lateral sclerosis (ALS). Here we investigated the role of ubiquitin-proteasome pathway in the mutant SOD1-related cell death and the effect of oxidative stress on the misfolding of mutant SOD1. Transient overexpression of ubiquitin with human SOD1 (wild-type, ala4val, gly85arg, gly93ala) in Neuro2A cells decreased the amount of mutant SOD1, but not of wild-type, while only mutants were co-immunoprecipitated with poly-ubiquitin. Proteasome inhibition by lactacystin augmented accumulation of mutant SOD1 in the non-ionic detergent-insoluble fraction. The spinal cord lysates from mutant SOD1 transgenic mice showed multiple carbonylated proteins, including mutant SOD1 with SDS-resistant dimer formation. Furthermore, the treatment of hSOD1-expressing cells with hydrogen peroxide promoted the oligomerization, and detergent-insolubility of mutant SOD1 alone, and the oxidized mutant SOD1 proteins were more heavily poly-ubiquitinated. In Neuro2A cells stably expressing human SOD1 protein, the proteasome function measured by chymotrypsin-like activity, was decreased over time without a quantitative alteration of the 20S proteasomal component. Finally, primary motor neurons from the mouse embryonic spinal cord were more vulnerable to lactacystin than non-motor neurons. These results indicate that the sustained expression of mutant SOD1 leads to proteasomal inhibition and motor neuronal death, which in part explains the pathogenesis of mutant SOD1-linked ALS.

CDNA array analysis of gene expression profiles in the striata of wild-type and Cu/Zn superoxide dismutase transgenic mice treated with neurotoxic doses of amphetamine.

Amphetamine (AMPH) is a drug of abuse that causes the degeneration of striatal dopamine terminals in mammals. Superoxide radicals seem to participate in AMPH-induced damage because its toxicity is attenuated in Cu/Zn superoxide dismutase transgenic (SOD-tg) mice. To provide a detailed analysis of molecular changes associated with AMPH toxicity, we used cDNA arrays consisting of 1176 genes to detect differential changes in gene expression in the striata of wild-type and SOD-tg mice treated with neurotoxic doses of the drug. We found 42 genes that showed >1.8-fold changes in at least two consecutive time points during the course of the study and were differentially affected by AMPH in the two genotypes. Specifically, more transcription factors and genes involved in responses to injury/inflammation were affected in wild-type mice after AMPH administration. Some of these stimulant-induced superoxide-dependent alterations in gene expression might affect neuronal functions and promote neuronal damage. Other changes might help to provide some degree of protection against AMPH toxicity. These results support the view that the use of global array analysis of gene expression will help to identify novel molecular mediators of AMPH-induced neurodegeneration.