Reduction of plasma glutathione in psychosis associated with schizophrenia and bipolar disorder in translational psychiatry.

The establishment of mechanism-driven peripheral markers is important for translational psychiatry. Many groups, including ours, have addressed molecular alterations in peripheral tissues in association with symptomatic changes in major illnesses. Oxidative stress is implicated in the pathophysiology of schizophrenia (SZ) and bipolar disorder (BP) through studies of patient peripheral tissues and animal models. Although the relationship between peripheral changes and brain pathology remain elusive, oxidative stress may bridge such translational efforts. Nonetheless, the molecular substrates of oxidative stress are not well defined in mental conditions. Glutathione (GSH) is a non-enzymatic antioxidant that eliminates free radicals, and has been suggested to have a role in SZ. We performed a cross-sectional study of 48 healthy controls (CON), 52 SZ patients and 62 BP patients to compare the levels of peripheral GSH by a biochemical enzyme assay. We show a significant reduction of plasma GSH in both SZ and BP patients compared with CON. We evaluated possible influences of clinical characteristics on the level of GSH in SZ and BP. A decrease in GSH level correlated with Positive and Negative Syndrome Scale (PANSS) total and positive scores for SZ and correlated with the PANSS general for BP. Taken together, we provide evidence that SZ and BP display a common molecular signature in the reduction of peripheral GSH in the psychosis dimension.

Atrophin-1, the dentato-rubral and pallido-luysian atrophy gene product, interacts with ETO/MTG8 in the nuclear matrix and represses transcription.

Dentato-rubral and pallido-luysian atrophy (DRPLA) is one of the family of neurodegenerative diseases caused by expansion of a polyglutamine tract. The drpla gene product, atrophin-1, is widely expressed, has no known function or activity, and is found in both the nuclear and cytoplasmic compartments of neurons. Truncated fragments of atrophin-1 accumulate in neuronal nuclei in a transgenic mouse model of DRPLA, and may underlie the disease phenotype. Using the yeast two-hybrid system, we identified ETO/MTG8, a component of nuclear receptor corepressor complexes, as an atrophin-1-interacting protein. When cotransfected into Neuro-2a cells, atrophin-1 and ETO/MTG8 colocalize in discrete nuclear structures that contain endogenous mSin3A and histone deacetylases. These structures are sodium dodecyl sulfate-soluble and associated with the nuclear matrix. Cotransfection of ETO/MTG8 with atrophin-1 recruits atrophin-1 to the nuclear matrix, while atrophin-1 and ETO/MTG8 cofractionate in nuclear matrix preparations from brains of DRPLA transgenic mice. Furthermore, in a cell transfection-based assay, atrophin-1 represses transcription. Together, these results suggest that atrophin-1 associates with nuclear receptor corepressor complexes and is involved in transcriptional regulation. Emerging links between disease-associated polyglutamine proteins, nuclear receptors, translocation-leukemia proteins, and the nuclear matrix may have important repercussions for the pathobiology of this family of neurodegenerative disorders.

Nuclear targeting of mutant Huntingtin increases toxicity.

Huntington's disease is a neurodegenerative disorder resulting from expansion of the polyglutamine region in huntingtin. Although huntingtin is normally cytoplasmic, in affected brain regions proteolytic fragments of mutant huntingtin containing the polyglutamine repeat form intranuclear inclusions. Here, we examine the contribution of nuclear localization to toxicity by transiently transfecting neuro-2a cells with an N-terminal huntingtin fragment similar in size to that believed to be present in patients. The huntingtin fragment, HD-N63, was targeted either to the cytoplasm with a nuclear export signal (NES) or to the nucleus with a nuclear localization signal (NLS). The NES decreased the number of cells with aggregates in the nucleus while an NLS had the opposite effect. By cotransfecting HD-N63 with GFP as a marker, we observed direct cell loss with constructs containing expanded polyglutamine repeats. Compared to unmodified HD-N63-75Q, adding an NES reduced cell loss by 57% while an NLS increased cell loss by 111%. These results indicate that nuclear localization of mutant huntingtin fragments plays an important role in cell toxicity.

Polyglutamine pathogenesis.

An increasing number of neurodegenerative disorders have been found to be caused by expanding CAG triplet repeats that code for polyglutamine. Huntington's disease (HD) is the most common of these disorders and dentatorubral-pallidoluysian atrophy (DRPLA) is very similar to HD, but is caused by mutation in a different gene, making them good models to study. In this review, we will concentrate on the roles of protein aggregation, nuclear localization and proteolytic processing in disease pathogenesis. In cell model studies of HD, we have found that truncated N-terminal portions of huntingtin (the HD gene product) with expanded repeats form more aggregates than longer or full length huntingtin polypeptides. These shorter fragments are also more prone to aggregate in the nucleus and cause more cell toxicity. Further experiments with huntingtin constructs harbouring exogenous nuclear import and nuclear export signals have implicated the nucleus in direct cell toxicity. We have made mouse models of HD and DRPLA using an N-terminal truncation of huntingtin (N171) and full-length atrophin-1 (the DRPLA gene product), respectively. In both models, diffuse neuronal nuclear staining and nuclear inclusion bodies are observed in animals expressing the expanded glutamine repeat protein, further implicating the nucleus as a primary site of neuronal dysfunction. Neuritic pathology is also observed in the HD mice. In the DRPLA mouse model, we have found that truncated fragments of atrophin-1 containing the glutamine repeat accumulate in the nucleus, suggesting that proteolysis may be critical for disease progression. Taken together, these data lead towards a model whereby proteolytic processing, nuclear localization and protein aggregation all contribute to pathogenesis.

Differential cellular expression of isoforms of inositol 1,4,5-triphosphate receptors in neurons and glia in brain.

Inositol 1,4,5-trisphosphate receptors (IP3R) are mediators of second messenger-induced intracellular calcium release. Three isoforms are known to be expressed in brain, but their regional distributions and cellular localizations are little known. In order to better understand the roles of IP3 receptor isoforms in brain function, a first step is to define their distributions. We have used affinity-purified antibodies directed against peptides unique to each isoform to determine their sites of expression in rat brain. Type 1 IP3R (IP3R1) is dramatically enriched in Purkinje neurons in cerebellum and neurons in other regions, consistent with previous studies. By contrast, IP3R2 is only detected in glia, whereas IP3R3 is predominantly neuronal, with little detected in glia. IP3R3 is enriched in neuropil, especially in neuronal terminals (which often contain large dense core vesicles) in limbic and basal forebrain regions including olfactory tubercle, central nucleus of the amygdala, and bed nucleus of the stria terminalis. In addition, IP3R1 and IP3R3 have clearly distinct time courses of expression in developing brains. These data suggest separate roles for inositol 1,4,5-trisphosphate receptor isoforms in development, and for glial and neuronal function. The IP3R3 may be involved in regulation of neurotransmitter or neuropeptide release in terminals within specific nuclei of the basal forebrain and limbic system.

FKBP12 binds the inositol 1,4,5-trisphosphate receptor at leucine-proline (1400-1401) and anchors calcineurin to this FK506-like domain.

The immunophilin FKBP12 is one of the most abundant and conserved proteins in biology. It is the primary receptor for the immunosuppressant actions of the drug FK506 in whose presence FKBP12 binds to and inhibits calcineurin, disrupting interleukin formation in lymphocytes. The physiologic functions of FKBP12 are less clear, although the protein has been demonstrated to physiologically interact with the inositol 1,4,5-trisphosphate receptor (IP3R), the ryanodine receptor, and the type 1 transforming growth factor beta receptor. We now report that FKBP12 binds the IP3R at residues 1400-1401, a leucyl-prolyl dipeptide epitope that structurally resembles FK506. We further demonstrate that binding to IP3R at this site enables FKBP12 to interact with calcineurin, presumably to anchor the phosphatase to IP3R and modulate the receptor's phosphorylation status. We propose that FK506 promotes an FKBP12-calcineurin interaction by mimicking structurally similar dipeptide epitopes present within proteins that use FKBP12 to anchor calcineurin to the appropriate physiologic substrates.

Comparison of type 2 inositol 1,4,5-trisphosphate receptor distribution and subcellular Ca2+ release sites that support Ca2+ waves in cultured astrocytes.

We have examined the mechanisms that underlie Ca2+ wave propagation in cultured cortical astrocytes. Norepinephrine evoked Ca2+ waves in astrocytes that began at discrete initiation loci and propagated throughout the cell by regenerative amplification at a number of cellular sites, as shown by very high Ca2+ release rates at these regions. We have hypothesized previously that domains displaying elevated Ca2+ release kinetics in astrocytes may correspond to sites of high inositol 1,4,5-trisphosphate receptor (InsP3R) density. To examine this possibility, we compared the distribution pattern of endoplasmic reticulum (ER) and InsP3Rs with Ca2+ release kinetics in subcellular regions during propagation of norepinephrine-evoked waves. 3,3'-Dihexyloxacarbocyanine iodide staining revealed that the ER in astrocytes exists as a meshwork of membranes extending throughout the cells, including fine processes. A specific antibody directed against type 2 InsP3Rs (InsP3R2) detected a 260-kDa band in western blotting of astrocyte membranes. Immunocytochemistry using this antibody stained the entire ER system in a punctate, variegated manner. When Ca2+ responses and InsP3R2 immunofluorescence were compared in the same cell, domains of elevated Ca2+ response kinetics (high amplitude and rapid rate of rise) showed significant positive correlation with high local intensity of InsP3R2 staining. It appears, therefore, that specializations in the ER responsible for discrete local Ca2+ release sites that support regenerative wave propagation include increased levels of InsP3R2 expression.

Inositol 1,4,5-trisphosphate receptors in endocrine cells: localization and association in hetero- and homotetramers.

The inositol 1,4,5-trisphosphate receptor (IP3R) is an intracellular calcium channel involved in coupling cell membrane receptors to calcium signal transduction pathways within cells including endocrine cells. Several isoforms (I, II, and III) of IP3Rs have been identified, which are encoded by separate genes, and are expressed in many tissues with differing patterns of cellular expression. We have generated specific affinity-purified polyclonal anti-peptide antibodies to each of the three isoforms. Western blot analysis of RINm5F and ATt20 cells shows high levels of endogenously expressed type I and type III IP3R, but undetectable levels of type II. Immunofluorescence studies revealed an endoplasmic reticulum-like pattern similar to BiP, an ER marker. In contrast with previous claims, both type I and type III IP3Rs were absent from the secretory granules of ATt20 cells. Western blots of sucrose gradients and gel filtration probed with antibodies to either type I or type III showed a molecular weight of greater than 1,000 kDa consistent with a tetrameric structure. Co-immunoprecipitation experiments indicated that most of the receptors were present as heterotetramers. Homotetramers were identified for the type III IP3R; however, type I homotetramers were undetectable. These data suggest that molecular association of IP3Rs into heterotetrameric forms can contribute to the complexity of the regulation of Ca2+ release from ER by IP3Rs within cells.

A huntingtin-associated protein enriched in brain with implications for pathology.

Huntington's disease (HD) is an autosomal dominant neurodegenerative disorder caused by an expanding polyglutamine repeat in the IT15 or huntingtin gene. Although this gene is widely expressed and is required for normal development, the pathology of HD is restricted to the brain, for reasons that remain poorly understood. The huntingtin gene product is expressed at similar levels in patients and controls, and the genetics of the disorder suggest that the expansion of the polyglutamine repeat induces a toxic gain of function, perhaps through interactions with other cellular proteins. Here we report the identification of a protein (huntingtin-associated protein (HAP)-1) that binds to huntingtin. This binding is enhanced by an expanded polyglutamine repeat, the length of which is also known to correlate with the age of disease onset. The HAP-1 protein is enriched in the brain, suggesting a possible basis for the selective brain pathology of HD.

Molecular cloning of a cDNA for the human inositol 1,4,5-trisphosphate receptor type 1, and the identification of a third alternatively spliced variant.

The Inositol 1,4,5-trisphosphate receptor (IP3R) is an intracellular calcium channel involved in coupling cell membrane receptors to calcium signal transduction pathways within the cell. We have cloned a cDNA for the human type 1 inositol 1,4,5-trisphosphate receptor. The sequence contains the S2 splice site which appears to be the region most divergent between rat and human. We now report an additional alternatively spliced region in the coupling domain, that is 9 amino acids long, which we term S3. Alternatively spliced forms are found in both human and rat. PCR analysis of brain and peripheral tissues from human and rat shows both transcripts of the type 1 inositol 1,4,5-trisphosphate receptor in all tissues. The long form predominates in most brain regions (except the cerebellum) while the short form predominates in peripheral tissues. The sequence of the longer form in human appears to create an additional consensus protein kinase C phosphorylation site.