Lynnette J. Cook

Doxycycline attenuates and delays toxicity of the oculopharyngeal muscular dystrophy mutation in transgenic mice.

The muscular dystrophies are a heterogeneous group of disorders for which there are currently no cures. Oculopharyngeal muscular dystrophy (OPMD) is an autosomal dominant late-onset, progressive disease that generally presents in the fifth or sixth decade with dysphagia, ptosis and proximal limb weakness. OPMD is caused by the abnormal expansion of a (GCG)n trinucleotide repeat in the coding region of the poly-(A) binding protein nuclear 1 (PABPN1) gene. In unaffected individuals, (GCG)6 codes for the first six alanines in a homopolymeric stretch of ten alanines. In most individuals with OPMD this (GCG)6 repeat is expanded to (GCG)8–13, leading to a stretch of 12–17 alanines in mutant PABPN1. PABPN1 with an expanded polyalanine tract forms aggregates consisting of tubular filaments within the nuclei of skeletal muscle fibers. We have developed a transgenic mouse model of OPMD that manifests progressive muscle weakness accompanied by intranuclear aggregates and TUNEL-stained nuclei in skeletal muscle fibers. The onset and severity of these abnormalities were substantially delayed and attenuated by doxycycline treatment, which may exert its therapeutic effect by reducing aggregates and by distinct antiapoptotic properties. Doxycycline may represent a safe and feasible therapeutic for this disease.

Mammalian, yeast, bacterial, and chemical chaperones reduce aggregate formation and death in a cell model of oculopharyngeal muscular dystrophy.

Autosomal dominant oculopharyngeal muscular dystrophy (OPMD) is characterized pathologically by intranuclear inclusions in skeletal muscles and is caused by the expansion of a 10-alanine stretch to 12–17 alanines in the intranuclear poly(A)-binding protein 2 (PABP2). Whereas PABP2 is a major component of the inclusions in OPMD, the pathogenic mechanisms causing disease are unknown. Here we show that polyalanine expansions in PABP2 cause increased numbers of inclusions and enhance death in COS-7 cells. We observed similar increases of protein aggregation and cell death with nuclear-targeted green fluorescent protein linked to longerversus shorter polyalanine stretches. Intranuclear aggregates in our OPMD cell model were associated with heat shock protein (HSP) 40 (HDJ-1) and HSP70. Human HDJ-1, yeast hsp104, a bacterially derived GroEL minichaperone, and the chemical chaperone Me2SO reduced both aggregation and cell death in our OPMD model without affecting the levels of PABP2, and similar trends were seen with green fluorescent protein with long polyalanine stretches. Thus, polyalanine expansion mutations in different protein contexts cause proteins to misfold/aggregate and kill cells. The situation in OPMD appears to have many parallels with polyglutamine diseases, raising the possibility that misfolded, aggregate-prone proteins may perturb similar pathways, irrespective of the nature of the mutation or protein context.

The effect of polymorphism at the serotonin transporter gene on decision-making, memory and executive function in ecstasy users and controls

Rationale3, 4-Methylenedioxymethamphetamine (MDMA or “ecstasy”) is a popular drug of abuse known to result in depletions of the serotonin (5-HT) system. A number of studies have reported that ecstasy users differ from controls on a variety of measures of cognitive function. However, the literature is not consistent and many negative findings were also reported. One reason for such inconsistency might be interindividual variance in vulnerability to the deleterious effects of ecstasy due to a number of factors, both genetic and environmental.ObjectivesTo investigate the hypothesis that carriers of the s allele at the 5-HT transporter gene-linked polymorphic region (5-HTTLPR), which was associated with reduced serotonergic neurotransmission relative to the l allele, would be most vulnerable to the effects of ecstasy on cognitive function.MethodsWe assessed memory, decision-making, and executive function in ecstasy users and controls, stratifying by genotype at the 5-HTTLPR.ResultsWe observed that the 5-HTTLPR genotype groups differed on a number of measures in both the ecstasy users and the controls. While performing a risky decision-making task, ss and ls controls attended to differences in the probability of winning chosen gambles to a greater extent than the ll controls. However, this difference was dramatically attenuated in the ss ecstasy users. Furthermore, independent of ecstasy use, volunteers of the ss genotype outperformed the ll genotype on a visual planning task.ConclusionsThe results are consistent with the hypothesis that cognitive impairment in ecstasy users may depend on genetic variation at the 5-HTTLPR.

Candidate gene association studies of genes involved in neuronal cholinergic transmission in Alzheimer's disease suggests choline acetyltransferase as a candidate deserving further study

Consistent deficits in the cholinergic system are evident in the brains of Alzheimers Disease (AD) patients, including reductions in the activities of acetylcholine, acetylcholinesterase (AChE), and choline acetyltransferase (ChAT), increased butyrylcholinesterase (BChE) activity, and a selective loss of nicotinic acetylcholine receptors (nAChRs). Accordingly, we have analyzed polymorphisms in the genes encoding AChE, ChAT, BChE, and several of the subunit genes from neuronal nAChRs, for genetic associations with late‐onset AD. A significant association for disease was detected for a non‐coding polymorphism in ChAT (allele χ12 = 12.84, P = 0.0003; genotype χ22 = 11.89, P = 0.0026). Although replication analysis did not confirm the significance of this finding when the replication samples were considered alone (allele χ12 = 1.02, P=0.32; genotype χ22 = 1.101, P = 0.58) the trends were in the correct direction and a significant association remained when the two sample sets were pooled (allele χ12 = 12.37, P = 0.0004; genotype χ22 = 11.61, P = 0.003). Previous studies have reported significant disease associations for both the K‐variant of BChE and the coding ChAT rs3810950 polymorphism with AD. Replication analyses of these two loci failed to detect any significant association for disease in our case‐control samples.

Candidate gene association studies of the alpha 4 (CHRNA4) and beta 2 (CHRNB2) neuronal nicotinic acetylcholine receptor subunit genes in Alzheimer's disease

Consistent deficits in the cholinergic system are evident in Alzheimers disease (AD) patients, including selective loss of alpha4beta2 nicotinic acetylcholine receptors in the brains of AD patients. Knockout mice for the beta2 subunit have impaired neuronal survival in ageing. Accordingly, we have analysed polymorphisms in the genes that encode the alpha4 and beta2 subunits, CHRNA4 and CHRNB2 respectively, for genetic associations with late-onset AD. A significant association for disease was observed for a non-coding polymorphism in CHRNB2 (odds ratio=0.57, 95% confidence interval=0.35-0.95, P=0.024). Replication analysis was performed in two further sample sets. While these did not individually yield significant results, a significant association remained when all samples were pooled (odds ratio=0.70, 95% confidence interval=0.52-0.95, P=0.019). These data suggest that this variant warrants further examination in large case-control series.

No evidence for an association between Saitohin Q7R polymorphism and Alzheimer's disease.

The question that Nessler and colleagues raise on the potential stress effect of injecting minocycline intraperitoneally in rats with experimental autoimmune encephalomylelitis is important, yet we feel unproven as of yet. Any handling of laboratory animals can result in stress and in the case of testing potential therapies of experimental autoimmune encephalomylelitis, result in stress-induced immunosuppression. Indeed daily gavage might even be more stressful than intraperitoneal injections. Nonetheless, this point needs to be clarified. However, the methodologies involved in our report differ from that reported by Nessler and colleagues. They gave minocycline at 100mg/kg/day and we gave 45mg/kg/ day. They also did not measure plasma levels of minocycline, which would seem critical if a comparison between the two routes is being considered. The models used were different in both studies; in our study, rats were actively immunized whereas in their report, cell transfer was used. Note also that we used rats and they used mice, which also makes the comparison indirect. The growing literature on the effects of minocycline on a variety of neurological disorders, including in 1-methyl-4phenyl-1,2,3,6-tetrahydropyridine–induced Parkinson’s disease in mice, as reported by these authors, is extremely exciting. Minocycline has now been shown to have a wide variety of effects in vitro and in vivo on microglial activation and neuronal death. It has been shown to have anticaspase effects in a Huntington’s model as well as downregulating cytochrome oxidase in a motor neuron disease mouse model. In vitro, it has been shown in several models including excitotoxic disorders to inhibit p38 MAPK and finally to inhibit nitric oxide synthase induction. Although the exact mechanism of its effect on neuronal survival will be the subject of further study and discussion, it clearly has widespread and pleiotropic effects. In those situations in which minocycline was used in vivo, the drug was always given intraperitoneally (with the exception of Du and colleagues). Indeed, another group has shown similar data to us in mice with experimental autoimmune encephalomyleitis, implicating a major effect of minocycline on metalloproteinase induction. All of these studies therefore could be called into question if stress-induced immunosuppression is postulated to occur. We believe, however, that Nessler and colleagues raise an important point and one worthy of discussion. However, given its proven safety in humans, we do not believe the data reported by us preclude it from being tested in multiple sclerosis. In comparison with -interferon, which when first used in multiple sclerosis had an almost unknown mechanism of action, considerably more is known about minocycline clinically and experimentally that warrants its testing. Department of Medical Sciences, University of Wisconsin– Madison, Madison, WI

Decoding Darkness - The Search for the Genetic Causes of Alzheimer's Disease.

Decoding Darkness - The Search for the Genetic Causes of Alzheimers Disease. Rudolf E Tanzi, Ann B Parson. Cambridge, Massachusetts: Perseus Publishing, 2000. Alzheimers disease currently affects an estimated 14 million people world wide. As the world population and their longevity increase, it is predicted that this figure will continue to rise rapidly. Cognisant of this “time bomb”, scientists across the globe are endeavouring to elucidate the pathogenic mechanisms of the disorder with urgency. Within the past decade, major advances have been made in identifying the genetic causes that result in the abnormal accumulation of beta amyloid, the major component of senile plaques which form one of the neuropathological hallmarks of the disorder. Dr Tanzi is the Director of the Massachusetts General Hospitals (MGH) Genetics and Aging Unit. With the help of scientific journalist …

Fragmin60 encodes an actin-binding protein with a C2 domain and controls actin Thr-203 phosphorylation in Physarum plasmodia and sclerotia.

We report the isolation of a cDNA clone encoding a 60-kDa protein termed fragmin60 that cross-reacts with fragmin antibodies. Unlike other gelsolin-related proteins, fragmin60 contains a unique N-terminal domain that shows similarity with C2 domains of aczonin, protein kinase C, and synaptotagmins. The fragmin60 C2 domain binds three calcium ions, one with nanomolar affinity and two with micromolar affinity. Actin binding by fragmin60 requires higher calcium concentrations than does binding of actin by a fragmin60 mutant lacking the C2 domain, suggesting that the C2 domain secures the actin binding moiety in a conformation preventing actin binding at low calcium concentrations. The fragmin60 C2 domain does not bind phospholipids but interacts with the endogenous homologue ofSaccharomyces cerevisiae S-phase kinase-associated protein (Skp1), as shown by pull-down assays and co-expression in mammalian cells. Recombinant fragmin60 promotes in vitrophosphorylation of actin Thr-203 by the actin-fragmin kinase. We further show that in vivo phosphorylation of actin in the fragmin60-actin complex occurs in sclerotia, a dormant stage ofPhysarum development, as well as in plasmodia. Our findings indicate that we have cloned a novel type of gelsolin-related actin-binding protein that is involved in controlling regulation of actin phosphorylation in vivo.

Identification of three genes expressed primarily during development in Physarum polycephalum

Abstract During the life cycle of Physarum polycephalum, uninucleate amoebae develop into multinucleate syncytial plasmodia. These two cell types differ greatly in cellular organisation, behaviour and gene expression. Classical genetic analysis has identified the mating-type gene, matA, as the key gene controlling the initiation of plasmodium development, but nothing is known about the molecular events controlled by matA. In order to identify genes involved in regulating plasmodium formation, we constructed a subtracted cDNA library from cells undergoing development. Three genes that have their highest levels of expression during plasmodium development were identified: redA, redB (regulated in development) and mynD (myosin). Both redA and redB are single-copy genes and are not members of gene families. Although redA has no significant sequence similarities to known genes, redB has sequence similarity to invertebrate sarcoplasmic calcium-binding proteins. The mynD gene is closely related to type II myosin heavy-chain genes from many organisms and is one of a family of type II myosin genes in P. polycephalum. Our results indicate that many more red genes remain to be identified, some of which may play key roles in controlling plasmodium formation.