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Biomedical subjects

Michael J Strong

Publications and source records attributed to Michael J Strong.

28 records · Page 2Linked to original sources
PubMed2004

Amyotrophic lateral sclerosis: contemporary concepts in etiopathogenesis and pharmacotherapy.

Among the neurodegenerative diseases associated with ageing, amyotrophic lateral sclerosis (ALS) remains the most devastating. The disease inexorably progresses, the vast majority of pharmacotherapies have failed to modify the disease course, death ensues on average within 5 years of symptom onset and increasing numbers of individuals are afflicted with the disease. However, significant advances in our understanding of the natural history of ALS and of the fundamental nature of the biological defect underlying motor neuron degeneration have been gained, providing hope for the development of novel pharmacotherapies for ALS. Among these is the recognition that ALS is a biologically heterogeneous disorder in which genetics, environment and ageing all interrelate. The observation of clinical heterogeneity, with initial clinical manifestations serving as predictors of survivorship, is of considerable importance in designing therapeutic trials. The presence of frontotemporal dysfunction in a subset of patients has led to increased interest in the relationship between ALS and the degenerative tauopathies. Ultimately, the degenerating motor neurons do not die alone. The contribution of both microglia and astrocytes to the degenerative process are increasingly recognised. Understanding how these processes interrelate has become critical to understanding the pharmacotherapy of ALS and in the design of clinical trials. This review will highlight recent epidemiological and neurochemical advances in our understanding of ALS, and place them into the context of understanding the development of novel treatment avenues for this devastating disease.

Amyotrophic Lateral Sclerosis↗
PubMed2003

Microtubule-associated tau protein positive neuronal and glial inclusions in ALS.

BACKGROUND: The authors compared tau protein deposition in the frontal cortex of patients with cognitive impairment of amyotrophic lateral sclerosis (ALSci) (n = 6), cognitively intact patients with ALS (n = 6), and age-matched controls (n = 6) in order to determine the pathologic substrate of ALSci. METHODS: Archival paraffin-embedded tissue was examined using Gallyas staining and immunostaining for tau-1 (phosphorylation-dependent tau epitope), tau-2 (phosphorylation independent), Alzheimer-specific tau phosphoepitopes (AT 8; ser(396) phosphorylation), beta-amyloid, glial fibrillary acid protein, SMI 31 (recognizing phosphorylated NFH), alpha-synuclein, or ubiquitin. RESULTS: Tau immunoreactive astrocytic and dense neuronal inclusions were found in both ALS and ALSci, although to a greater extent in ALSci. Superficial linear spongiosis and Gallyas-positive intraneuronal aggregates, immunoreactive with tau-1 and AT 8 but rarely to ser(396) tau, were unique to ALSci. Dense extracellular aggregates were observed by both Gallyas staining and tau-1 immunostaining. Tufted degenerating astrocytes containing tau-1 and AT 8 immunoreactive aggregates and, rarely, dense Gallyas positive neuritic plaques immunoreactive with tau-1 and AT 8, but not with ser(396) tau or beta-amyloid, were observed in ALSci. Tau positive glial coiled bodies were observed in the deep cortical layers and adjacent subcortical white matter in ALSci. Although 3R and 4R tau mRNA isoforms were expressed to similar levels in the frontal cortex of all cases, the total amount of tau mRNA was increased in both ALS and ALSci. Both gray and white matter soluble tau protein expression was similar among control, ALS, and ALSci cases. CONCLUSIONS: Cognitive dysfunction in ALS may reflect abnormal tau protein metabolism.

Aged↗
PubMed2003

High threshold for induction of the stress response in motor neurons is associated with failure to activate HSF1.

Heat shock protein 70 (Hsp70) protects cultured motor neurons from the toxic effects of mutations in Cu/Zn-superoxide dismutase (SOD-1), which is responsible for a familial form of the disease, amyotrophic lateral sclerosis (ALS). Here, the endogenous heat shock response of motor neurons was investigated to determine whether a high threshold for activating this protective mechanism contributes to their vulnerability to stresses associated with ALS. When heat shocked, cultured motor neurons failed to express Hsp70 or transactivate a green fluorescent protein reporter gene driven by the Hsp70 promoter, although Hsp70 was induced in glial cells. No increase in Hsp70 occurred in motor neurons after exposure to excitotoxic glutamate or expression of mutant SOD-1 with a glycine--> alanine substitution at residue 93 (G93A), nor was Hsp70 increased in spinal cords of G93A SOD-1 transgenic mice or sporadic or familial ALS patients. In contrast, strong Hsp70 induction occurred in motor neurons with expression of a constitutively active form of heat shock transcription factor (HSF)-1 or when proteasome activity was sufficiently inhibited to induce accumulation of an alternative transcription factor HSF2. These results indicate that the high threshold for induction of the stress response in motor neurons stems from an impaired ability to activate the main heat shock-stress sensor, HSF1.

Amyotrophic Lateral Sclerosis↗
PubMed2003

NMDA induces NOS 1 translocation to the cell membrane in NGF-differentiated PC 12 cells.

Glutamatergic-mediated nitric oxide (NO) production occurs via the N-methyl-D-aspartic acid (NMDA) postsynaptic density protein 95 (PSD95)-neuronal nitric oxide synthase (NOS1) ternary complex. To determine whether NOS1 is targeted to the membrane subsequent to NMDA receptor activation, we examined the effect of NMDA on NOS1 subcellular localization in nerve growth factor (NGF) differentiated PC12 cells. No effect on cell viability was observed using a range of NMDA concentrations from 500 to 1000 microM. Within 3 min of stimulation with 750 microM NMDA, increased cytoplasmic NOS1 immunostaining was observed with rapid membrane staining thereafter. This was inhibited by NMDAR inhibition with MK801. This observation was confirmed using subcellular fractionation and immunoblotting. Using 4, 5-diaminofluorescein diacetate (DAF2-DA) staining and a diazotization assay, concurrent NO production was observed. When PC 12 cells were co-treated with either NMDA and N(6)-nitro-L-arginine methyl ester hydrochloride (L-NAME) or (5R, 10S)-(+)-5-methyl-10, 11-dihydro-5H-dibenzo [a, d] cyclohepten-5, 10-imine hydrogen maleate (MK-801), nitric oxide (NO) generation was inhibited. Stimulation in a calcium-free medium did not increase NO levels. Although no evidence of cytotoxicity was observed utilizing either the MTT assay or measures of apoptosis within the maximal interval of NOS1 translocation, cell viability was reduced following 10 h of continuous NMDA exposure. While it has been shown that NMDA triggers NOS1 activation, these results indicate that NMDAR activation also mediates NOS1 targeting to the membrane. Our data validate that NGF-differentiated PC12 cells may be employed as a useful in vitro model to further study the regulation of NOS1 subsequent to NMDAR activation.

Animals↗
PubMed2003

Selective loss of trans-acting instability determinants of neurofilament mRNA in amyotrophic lateral sclerosis spinal cord.

Neurofilament (NF) aggregates in motor neurons are a key neuropathological feature of amyotrophic lateral sclerosis (ALS). We have previously observed an alteration in the stoichiometry of NF subunit steady state mRNA levels in ALS spinal motor neurons using in situ hybridization and proposed that this led to aggregate formation. We have now examined the levels of NF mRNA in whole tissue homogenates of spinal cord using the RNase protection assay and real time reverse transcriptase-PCR and observed significant elevations of NF mRNA level in ALS. Compared with age-matched control, we observed a greater stability of heterogeneously expressed NFL mRNA in the presence of ALS spinal cord homogenates. Heat denaturing or protease K digestion of the control homogenates increased the stability of the NFL mRNA to levels observed in ALS homogenate. Increased NFL mRNA stability was also induced by increasing the percentage of ALS homogenate in an admixture of control and ALS homogenates. These observations suggest the presence of trans-acting NFL mRNA-destabilizing elements in control but not in ALS spinal cord homogenates. This was confirmed in gel retardation assays. We also observed that the destabilizing elements interact with the 3'-untranslated region of NFL mRNA. These findings suggest that the trans-acting NFL-destabilizing elements are selectively suppressed in ALS homogenates, resulting in an increased stability and level of NFL mRNA.

3' Untranslated Regions↗
PubMed2003

A neurotoxic peripherin splice variant in a mouse model of ALS.

Peripherin, a neuronal intermediate filament (nIF) protein found associated with pathological aggregates in motor neurons of patients with amyotrophic lateral sclerosis (ALS) and of transgenic mice overexpressing mutant superoxide dismutase-1 (SOD1G37R), induces the selective degeneration of motor neurons when overexpressed in transgenic mice. Mouse peripherin is unique compared with other nIF proteins in that three peripherin isoforms are generated by alternative splicing. Here, the properties of the peripherin splice variants Per 58, Per 56, and Per 61 have been investigated in transfected cell lines, in primary motor neurons, and in transgenic mice overexpressing peripherin or overexpressing SOD1G37R. Of the three isoforms, Per 61 proved to be distinctly neurotoxic, being assembly incompetent and inducing degeneration of motor neurons in culture. Using isoform-specific antibodies, Per 61 expression was detected in motor neurons of SOD1G37R transgenic mice but not of control or peripherin transgenic mice. The Per 61 antibody also selectively labeled motor neurons and axonal spheroids in two cases of familial ALS and immunoprecipitated a higher molecular mass peripherin species from disease tissue. This evidence suggests that expression of neurotoxic splice variants of peripherin may contribute to the neurodegenerative mechanism in ALS.

Alternative Splicing↗
PubMed2003

The basic aspects of therapeutics in amyotrophic lateral sclerosis.

Once thought to be a single pathological disease state, amyotrophic lateral sclerosis (ALS) is now recognized to be the limited phenotypic expression of a complex, heterogeneous group of biological processes, resulting in an unrelenting loss of motor neurons. On average, individuals affected with the disease live <5 years. In this article, the complex nature of the pathogenesis of ALS, including features of age dependency, environmental associations, and genetics, is reviewed. Once held to be uncommon, it is now clear that ALS is associated with a frontotemporal dementia and that this process may reflect disturbances in the microtubule-associated tau protein metabolism. The motor neuron ultimately succumbs in a state where significant disruptions in neurofilament metabolism, mitochondrial function, and management of oxidative stress exist. The microenvironment of the neuron becomes a complex milieu in which high levels of glutamate provide a source of chronic excitatory neurotoxicity, and the contributions of activated microglial cells lead to further cascades of motor neuron death, perhaps serving to propagate the disease once established. The final process of motor neuron death encompasses many features of apoptosis, but it is clear that this alone cannot account for all features of motor neuron loss and that aspects of a necrosis-apoptosis continuum are at play. Designing pharmacological strategies to mitigate against this process thus becomes an increasingly complex issue, which is reviewed in this article.

Amyotrophic Lateral Sclerosis↗
PubMed2002

Activated microglia (BV-2) facilitation of TNF-alpha-mediated motor neuron death in vitro.

We have studied the interactions between activated microglia and injured motor neurons using an immortalized murine microglial cell line (BV-2) stimulated with either lipopolysaccharide (LPS) (Escherichia coli) or supernatant from serum-deprived motor neurons (NSC-34 cell line). Both stimuli induced BV-2 activation. Although both BV-2 supernatants induced a subsequent increase in NO generation in otherwise healthy NSC-34 cells, only LPS-activated microglial supernatant induced NSC-34 cell death through a TNF-alpha-dependent pathway. However, we observed a 20-fold increase in the amount of TNF-alpha required to kill NSC-34 cells in the absence of LPS-activated BV-2 cell supernatant, indicating that microglia secrete factor(s) that facilitate TNF-alpha-mediated motor neuron death in vitro.

Amyotrophic Lateral Sclerosis↗