Review: The role of mitochondria in the pathogenesis of amyotrophic lateral sclerosis.

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease characterized by loss of upper and lower motor neurones leading to muscle weakness and paralysis. Despite recent advances in the genetics of ALS, the mechanisms underlying motor neurone degeneration are not fully understood. Mitochondria are known to be involved in the pathogenesis of ALS, principally through mitochondrial dysfunction, the generation of free radicals, and impaired calcium handling in ALS patients and models of disease. However, recent studies have highlighted the potential importance of altered mitochondrial morphology and defective axonal transport of mitochondria in ALS. Here, we review the evidence for mitochondrial involvement in ALS and discuss potential therapeutic strategies targeting mitochondria.

Characterization of the caspase cascade in a cell culture model of SOD1-related familial amyotrophic lateral sclerosis: expression, activation and therapeutic effects of inhibition.

There is increasing evidence that apoptosis or a similar programmed cell death pathway is the mechanism of cell death responsible for motor neurone degeneration in amyotrophic lateral sclerosis. Knowledge of the relative importance of different caspases in the cell death process is at present incomplete. In addition, there is little information on the critical point of the death pathway when the process of dying becomes irreversible. In this study, using the well-established NSC34 motor neurone-like cell line stably transfected with empty vector, normal or mutant human Cu-Zn superoxide dismutase (SOD1), we have characterized the activation of the caspase cascade in detail, revealing that the activation of caspases-9, -3 and -8 are important in motor neurone death and that the presence of mutant SOD1 causes increased activation of components of the apoptotic cascade under both basal culture conditions and following oxidative stress induced by serum withdrawal. Activation of the caspases identified in the cellular model has been confirmed in the G93A SOD1 transgenic mice. Furthermore, investigation of the effects of anti-apoptotic neuroprotective agents including specific caspase inhibitors, minocycline and nifedipine, have supported the importance of the mitochondrion-dependent apoptotic pathway in the death process and revealed that the upstream caspase cascade needs to be inhibited if useful neuro-protection is to be achieved.

Quantitative analysis of tau isoform transcripts in sporadic tauopathies.

A number of neurodegenerative diseases, including Alzheimer's disease (AD), are characterized by intraneuronal accumulation of the tau protein. Some forms of FTDP-17 are caused by mutations in the tau gene affecting exon 10 splicing. Therefore, dysregulation of tau pre-mRNA splicing may be a contributing factor to sporadic tauopathies. To address this question, we devised a real-time RT-PCR strategy based on the use of a single fluorogenic probe to evaluate the ratio between tau isoforms containing or lacking exon 10 (4R/3R ratio) in post-mortem brain samples. We found a two- to six-fold increase in the 4R/3R ratio in cases of FTDP-17 linked to a splice site mutation, hence confirming the validity of the strategy. The difference in the 4R/3R ratio in the superior temporal and superior frontal gyri between AD and control brains was not statistically significant. Similarly, there was no significant difference in the 4R/3R ratio between Pick's disease cases and controls, indicating that the predominance of tau3R protein in PiD reflects post-translational modifications of specific isoforms. This study indicates that post-translational events are likely to be the main factors controlling tau isoform composition in sporadic tauopathies and highlights the benefit of quantitative RT-PCR in the assessment of splicing abnormalities in tauopathies.

Axonal transport of neurofilaments in normal and disease states.

Neurofilaments are among the most abundant organelles in neurones. They are synthesised in cell bodies and then transported into and through axons by a process termed 'slow axonal transport' at a rate that is distinct from that driven by conventional fast motors. Several recent studies have now demonstrated that this slow rate of transport is actually the consequence of conventional fast rates of movement that are interrupted by extended pausing. At any one time, most neurofilaments are thus stationary. Accumulations of neurofilaments are a pathological feature of several human neurodegenerative diseases suggesting that neurofilament transport is disrupted in disease states. Here, we review recent advances in our understanding of neurofilament transport in both normal and disease states. Increasing evidence suggests that phosphorylation of neurofilaments is a mechanism for regulating their transport properties, possibly by promoting their detachment from the motor(s). In some neurodegenerative diseases, signal transduction mechanisms involving neurofilament kinases and phosphatases may be perturbed leading to disruption of transport.

Androgen induced cell death in SHSY5Y neuroblastoma cells expressing wild-type and spinal bulbar muscular atrophy mutant androgen receptors.

Spinal bulbar muscular atrophy (SBMA) is one of a family of inherited neurodegenerative diseases caused by expansion of CAG encoding polyglutamine repeats; in SBMA the affected gene is the androgen receptor. To understand further the mechanisms that lead to neuronal cell death in SBMA, we generated SHSY5Y neuroblastoma cell lines that stably express identical levels of wild-type (19 polyglutamine repeat) or SBMA (52 polyglutamine repeat) androgen receptor. Parental SHSY5Y cells do not express detectable levels of the androgen receptor. In the absence of androgen, the transfected cell lines have similar phenotypes and growth characteristics to parental SHSY5Y cells. However, upon treatment with androgen, both cell lines undergo a marked dose-dependent loss of viability; this loss was significantly greater in cells expressing the SBMA receptor. Morphological analyses of the androgen treated cells revealed that cell death bore hallmarks of apoptosis involving altered nuclear morphology and cleavage of poly(ADP-ribose) polymerase and of caspase 3 in both wild-type and SBMA cell lines. The caspase inhibitor VAD-fmk was able to decrease loss of viability of both cell lines on exposure to androgen.

Phosphorylation of thr(668) in the cytoplasmic domain of the Alzheimer's disease amyloid precursor protein by stress-activated protein kinase 1b (Jun N-terminal kinase-3).

Threonine(668) (thr(668)) within the carboxy-terminus of the Alzheimer's disease amyloid precursor protein (APP) is a known in vivo phosphorylation site. Phosphorylation of APPthr(668) is believed to regulate APP function and metabolism. Thr(668) precedes a proline, which suggests that it is targeted for phosphorylation by proline-directed kinase(s). We have investigated the ability of four major neuronally active proline-directed kinases, cyclin dependent protein kinase-5, glycogen synthase kinase-3 beta, p42 mitogen-activated protein kinase and stress-activated protein kinase-1b, to phosphorylate APPthr(668) and report here that SAPK1b induces robust phosphorylation of this site both in vitro and in vivo. This finding provides a molecular framework to link cellular stresses with APP metabolism in both normal and disease states.

Three different human tau isoforms and rat neurofilament light, middle and heavy chain proteins are cellular substrates for transglutaminase.

Abnormal aggregates of tau and neurofilaments are pathologies of Alzheimer's disease. Some of these aggregates are insoluble in chaotropic salts and ionic detergents but the mechanisms that lead to this are not clear. One suggestion is that it is due to crosslinking by tissue transglutaminase. Both tau and neurofilaments can be crosslinked by transglutaminase in vitro and one isoform of tau is now known to be a cellular transglutaminase substrate. However there is no evidence to demonstrate that neurofilaments are cellular substrates for transglutaminase and it is not clear whether other isoforms of tau are equally susceptible to transglutaminase crosslinking. Here, we demonstrate that three different tau isoforms and neurofilament light, middle and heavy chain proteins are all cellular substrates for transglutaminase.

Glutamate slows axonal transport of neurofilaments in transfected neurons.

Neurofilaments are transported through axons by slow axonal transport. Abnormal accumulations of neurofilaments are seen in several neurodegenerative diseases, and this suggests that neurofilament transport is defective. Excitotoxic mechanisms involving glutamate are believed to be part of the pathogenic process in some neurodegenerative diseases, but there is currently little evidence to link glutamate with neurofilament transport. We have used a novel technique involving transfection of the green fluorescent protein-tagged neurofilament middle chain to measure neurofilament transport in cultured neurons. Treatment of the cells with glutamate induces a slowing of neurofilament transport. Phosphorylation of the side-arm domains of neurofilaments has been associated with a slowing of neurofilament transport, and we show that glutamate causes increased phosphorylation of these domains in cell bodies. We also show that glutamate activates members of the mitogen-activated protein kinase family, and that these kinases will phosphorylate neurofilament side-arm domains. These results provide a molecular framework to link glutamate excitotoxicity with neurofilament accumulation seen in some neurodegenerative diseases.

An integrated map of chromosome 18 CAG trinucleotide repeat loci.

Expansions of trinucleotide CAG repeats have been demonstrated in at least eight neurodegenerative disorders, and suggested to occur in several others, including bipolar disorder and schizophrenia. Chromosome 18 loci have been implicated in bipolar disorder pedigrees by linkage analysis. To address this putative link between chromosome 18 CAG trinucleotide repeats and neuropsychiatric illness, we have screened a chromosome 18 cosmid library (LL18NCO2" AD") and identified 14 novel candidate loci. Characterisation of these loci involved repeat flank sequencing, estimation of polymorphism frequency and mapping using FISH as well as radiation hybrid panels. These mapped trinucleotide loci will be useful in the investigation of chromosome 18 in neurodegenerative or psychiatric conditions, and will serve to integrate physical and radiation hybrid maps of chromosome 18.

Polyglutamine repeat length influences human androgen receptor/c-Jun mediated transcription.

The androgen receptor and c-Jun are known to interact to modulate each others transcriptional activities. The androgen receptor contains a polymorphic polyglutamine repeat and expansion of this repeat to beyond approximately 40 causes spinobulbar muscular atrophy (SBMA; also known as Kennedy's disease), a genetic form of motor neurone disease. Here we show that the size of this polyglutamine tract influences both c-Jun regulation of androgen receptor-mediated transcription and androgen receptor regulation of c-Jun activity. c-Jun is a key mediator of neuronal survival and death by apoptosis. Inappropriate interactions between c-Jun and androgen receptors containing pathological length glutamine repeats may therefore be part of the pathogenic process in SBMA.

The gene for the human Src-like adaptor protein (hSLAP) is located within the 64-kb intron of the thyroglobulin gene.

In this study, we describe the molecular cloning and characterization of a Src-like adaptor protein gene embedded within the genomic organization of the human thyroglobulin (Tg) gene. This gene was identified by exon trapping on overlapping cosmids encompassing the largest Tg intron. A 2.6-kb transcript, with the highest levels of expression in fetal brain and lung, was detected on Northern blots. Two full-length cDNAs (one alternatively spliced) were isolated from a fetal brain library, both containing an open reading frame of 276 amino acids, but lacking a catalytic tyrosine kinase domain. The gene shows a high degree of cross-species similarity and appears to be transcribed in the direction opposite to Tg. This gene, designated hslap, appears to be the human ortholog of the recently described gene for the murine Src-like adaptor protein (mSLAP), a candidate intermediate in the signal-transduction pathway of the Eck receptor tyrosine kinase. Human slap is located in the candidate region for a recessive demyelinating neuropathy on chromosome 8q24, but sequence analysis failed to identify mutations, suggesting that it is not the gene for this disease.

Cyclin D2 interacts with cdk-5 and modulates cellular cdk-5/p35 activity.

Cyclin-dependent kinase-5 (cdk-5) is a serine/threonine kinase that displays neurone-specific activity. Experimental manipulation of cdk-5 expression in neurones has shown that cdk-5 is essential for proper development of the nervous system and, in particular, for outgrowth of neurites. Such observations suggest that cdk-5 activity must be tightly controlled during development of the nervous system. To identify possible regulators of cdk-5, we used the yeast two-hybrid system to search for proteins that interact with cdk-5. In two independent yeast transformation events, cyclin D2 interacted with cdk-5. Immunoprecipitation experiments confirmed that cyclin D2 and cdk-5 interact in mammalian cells. Cyclin D2 did not activate cdk-5 as assayed using three different substrates, which was in contrast to a known cdk-5 activator, p35. However, cyclin D2 expression led to a decrease in cdk-5/p35 activity in transfected cells. As cyclin D2 and cdk-5 are known to share overlapping patterns of expression during development of the CNS, the results presented here suggest a role for cyclin D2 in modulating cdk-5 activity in postmitotic developing neurones.

Investigation of the RB-1 tumour suppressor gene in a United Kingdom series of non-Hodgkin's lymphomas.

We have investigated the RB-1 tumour suppressor genes in a series of 20 non-Hodgkin's lymphomas (NHL). Polymerase chain reaction (PCR) amplification of polymorphic alleles indicated that there was evidence of allelic imbalance around 13q14, the site of the RB-1 gene, in at least 5 NHL. Immunohistochemical analysis of the RB-1 protein demonstrated wide variations in the percentage of cells exhibiting positive staining, but these usually correlated with differences in the proliferation index as indicated by staining of Ki67. Only 3/35 NHL exhibited significantly fewer cells expressing RB-1 protein than expressed Ki167. A comprehensive analysis of the mutation status of RB-1 in 20 NHL was carried out using PCR based strategies involving single strand conformational polymorphism (SSCP) gels. Most of the protein coding region was studied by analysing cDNA derived from its mRNA and the remaining 5'-end of the coding region investigated by analysing exon I of the gene. We also examined the promoter region of the gene. In none of the 20 NHL investigated were we able to identify a mutation: the only abnormal migrating fragment observed proved to be a polymorphism in exon I of the gene in 5 NHL. In one other case we detected instability at an intron repeat sequence, which had occurred during progression of the disease, but again no mutation of the protein coding region was found. The low levels of RB-1 protein expression that we had observed in a few of our NHL therefore did not appear to be due to mutation of the gene. These data suggest that mutation of RB-1 is not a common event in the evolution of NHL, but that there may be another, as yet unidentified, tumour suppressor gene near the RB-1 locus which is associated with NHL.

Gestational choriocarcinoma of the ovary diagnosed by analysis of tumour DNA.

Gestational choriocarcinoma of the ovary is a rare form of malignancy which can be difficult to distinguish from primary ovarian choriocarcinoma. The ability to make such a diagnosis could, however, have important implications for therapy. We report here a case of choriocarcinoma whose origins were difficult to determine and which behaved clinically more like a primary rather than a gestational choriocarcinoma. We have analysed DNA from this tumour by using polymerase chain reaction (PCR) amplification of a range of polymorphic alleles and have demonstrated that the tumour was in fact gestational. Furthermore, the lack of chromosome Y sequences and the presence of heterozygosity of the spouse's alleles, indicated that this tumour arose as a result of dispermic fertilisation of an empty ovum by sperm carrying the X chromosome.

Investigation of the activation state of the X chromosome in non-Hodgkin's lymphomas.

Cytogenetic analysis of non-Hodgkin's lymphomas (NHLs) has previously revealed a high incidence of numerical abnormalities involving the X chromosome. We have now used a combination of fluorescence in situ hybridization (FISH) and Southern blot analysis of methylation to examine the activation state of additional X chromosomes in NHL. Although FISH analysis of X chromosome centromeres in interphase nuclei was complicated by a number of factors, such as cell-cycle position, there was evidence that more than one X chromosome was present in the active state in 4/9 NHL. Methylation studies were carried out using the M27 beta probe, which also suggested that more than one activated X chromosome was present in at least 2/7 NHL cases. The two approaches therefore provided evidence that in some cases of NHL, unlike sex-chromosome-syndrome individuals, additional X chromosomes may be present in the active state. These data support the suggestion that NHL-associated oncogenes might be located on the X chromosome.