Synphilin-1 associates with alpha-synuclein and promotes the formation of cytosolic inclusions.

Parkinson disease (PD) is a neurodegenerative disease characterized by tremor, bradykinesia, rigidity and postural instability. Post-mortem examination shows loss of neurons and Lewy bodies, which are cytoplasmic eosinophilic inclusions, in the substantia nigra and other brain regions. A few families have PD caused by mutations (A53T or A30P) in the gene SNCA (encoding alpha-synuclein). Alpha-synuclein is present in Lewy bodies of patients with sporadic PD, suggesting that alpha-synuclein may be involved in the pathogenesis of PD. It is unknown how alpha-synuclein contributes to the cellular and biochemical mechanisms of PD, and its normal functions and biochemical properties are poorly understood. To determine the protein-interaction partners of alpha-synuclein, we performed a yeast two-hybrid screen. We identified a novel interacting protein, which we term synphilin-1 (encoded by the gene SNCAIP). We found that alpha-synuclein interacts in vivo with synphilin-1 in neurons. Co-transfection of both proteins (but not control proteins) in HEK 293 cells yields cytoplasmic eosinophilic inclusions.

Increased apoptosis of Huntington disease lymphoblasts associated with repeat length-dependent mitochondrial depolarization.

Huntington disease (HD) is a genetically dominant condition caused by expanded CAG repeats coding for glutamine in the HD gene product huntingtin. Although HD symptoms reflect preferential neuronal death in specific brain regions, huntingtin is expressed in almost all tissues, so abnormalities outside the brain might be expected. Although involvement of nuclei and mitochondria in HD pathophysiology has been suggested, specific intracellular defects that might elicit cell death have been unclear. Mitochondria dysfunction is reported in HD brains; mitochondria are organelles that regulates apoptotic cell death. We now report that lymphoblasts derived from HD patients showed increased stress-induced apoptotic cell death associated with caspase-3 activation. When subjected to stress, HD lymphoblasts also manifested a considerable increase in mitochondrial depolarization correlated with increased glutamine repeats.

Sequence and expression of mRNAs encoding the alpha 1 and alpha 2 subunits of a DHP-sensitive calcium channel.

Complementary DNAs were isolated and used to deduce the primary structures of the alpha 1 and alpha 2 subunits of the dihydropyridine-sensitive, voltage-dependent calcium channel from rabbit skeletal muscle. The alpha 1 subunit, which contains putative binding sites for calcium antagonists, is a hydrophobic protein with a sequence that is consistent with multiple transmembrane domains and shows structural and sequence homology with other voltage-dependent ion channels. In contrast, the alpha 2 subunit is a hydrophilic protein without homology to other known protein sequences. Nucleic acid hybridization studies suggest that the alpha 1 and alpha 2 subunit mRNAs are expressed differentially in a tissue-specific manner and that there is a family of genes encoding additional calcium channel subtypes.

Lymphocyte apoptosis: mediation by increased type 3 inositol 1,4,5-trisphosphate receptor.

B and T lymphocytes undergoing apoptosis in response to anti-immunoglobulin M antibodies and dexamethasone, respectively, were found to have increased amounts of messenger RNA for the inositol 1,4,5-trisphosphate receptor (IP3R) and increased amounts of IP3R protein. Immunohistochemical analysis revealed that the augmented receptor population was localized to the plasma membrane. Type 3 IP3R (IP3R3) was selectively increased during apoptosis, with no enhancement of type 1 IP3R (IP3R1). Expression of IP3R3 antisense constructs in S49 T cells blocked dexamethasone-induced apoptosis, whereas IP3R3 sense, IP3R1 sense, or IP3R1 antisense control constructs did not block cell death. Thus, the increases in IP3R3 may be causally related to apoptosis.

Localization of the inositol 1,4,5-trisphosphate receptor in synaptic terminals in the vertebrate retina.

Inositol 1,4,5-trisphosphate (InsP3) mobilizes internal Ca2+ in cells by binding to a receptor protein, which has recently been purified and molecularly cloned. To clarify those neuronal functions that are regulated by InsP3, we have localized this InsP3 receptor protein immunocytochemically in the retina, a neural tissue of well-defined structure and function. Positive staining in neurons is confined almost exclusively to the synaptic layers. Using dissociated retinal neurons, we have further localized the receptor to presynaptic terminals of photoreceptors and bipolar cells, as well as the synaptic processes of amacrine cells. The specific association of InsP3 receptors with synaptic terminals suggests a role for InsP3 in synaptic modulation, especially with respect to transmitter release.

Widespread expression of Huntington's disease gene (IT15) protein product.

Huntington's Disease (HD) is caused by expansion of a CAG repeat within a putative open reading frame of a recently identified gene, IT15. We have examined the expression of the gene's protein product using antibodies developed against the N-terminus and an internal epitope. Both antisera recognize a 350 kDa protein, the predicted size, indicating that the CAG repeat is translated into polyglutamine. The HD protein product is widely expressed, most highly in neurons in the brain. There is no enrichment in the striatum, the site of greatest pathology in HD. Within neurons, the protein is diminished in nuclei and mitochondria and is present in the soluble cytoplasmic compartment, as well as loosely associated with membranes or cytoskeleton, in cell bodies, dendrites, and axons. It is concentrated in nerve terminals, including terminals within the caudate and putamen. Thus, the normal HD gene product may be involved in common intracellular functions, and possibly in regulation of nerve terminal function. The product of the expanded allele is expressed, consistent with a gain of function mechanism for HD at the protein level.

Site-directed antibodies to low-voltage-activated calcium channel CaV3.3 (alpha1I) subunit also target neural cell adhesion molecule-180.

Synthetic peptides of defined amino acid sequence are commonly used as unique antigens for production of antibodies to more complex target proteins. We previously showed that an affinity-purified, site-directed polyclonal antibody (CW90) raised against a peptide antigen (CNGRMPNIAKDVFTKM) anticipated to be specific to a T-type voltage-dependent Ca(2+) channel subunit identified recombinant rat alpha1I/Ca(V)3.3 and two endogenous mouse proteins distinct in their developmental expression and apparent molecular mass (neonatal form 260 kDa, mature form 190 kDa) [Yunker AM, Sharp AH, Sundarraj S, Ranganathan V, Copeland TD, McEnery MW (2003) Immunological characterization of T-type voltage-dependent calcium channel Ca(V)3.1 (alpha 1G) and Ca(V)3.3 (alpha 1I) isoforms reveal differences in their localization, expression, and neural development. Neuroscience 117:321-335]. In the present study, we further characterize the biochemical properties of the CW90 antigens. We show for the first time that recombinant alpha1I/Ca(V)3.3 is modified by N-glycosylation. Using peptide:N-glycosidase F (PNGase F), an enzyme that removes polysaccharides attached at Asn residues, and endoneuraminidase-N (Endo-N), which specifically removes polysialic acid modifications, we reveal that differential glycosylation fully accounts for the large difference in apparent molecular mass between neonatal and adult CW90 antigens and that the neonatal form is polysialylated. As very few proteins are substrates for Endo-N, we carried out extensive analyses and herein present evidence that CW90 reacts with recombinant alpha1I/Ca(V)3.3 as well as endogenous neural cell adhesion molecule-180 (NCAM-180). We demonstrate the basis for CW90 cross-reactivity is a five amino acid epitope (AKDVF) present in both alpha1I/Ca(V)3.3 and NCAM-180. To extend these findings, we introduce a novel polyclonal anti-peptide antibody (CW678) that uniquely recognizes NCAM-180 and a new antibody (CW109) against alpha1I/Ca(V)3.3. Western blot analyses obtained with CW678, CW109 and CW90 on a variety of samples confirm that the endogenous CW90 signals are fully attributed to the two developmental forms of NCAM-180. Using CW678, we present novel data on differentiation-dependent NCAM-180 expression in human neuroblastoma IMR32 cells. These results strongly suggest the need for careful analyses to validate anti-peptide antibodies when targeting membrane proteins of low abundance.

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.

Huntington's disease and dentatorubral-pallidoluysian atrophy: proteins, pathogenesis and pathology.

Each of the glutamine repeat neurodegenerative diseases has a particular pattern of pathology largely restricted to the CNS. However, there is considerable overlap among the regions affected, suggesting that the diseases share pathogenic mechanisms, presumably involving the glutamine repeats. We focus on Huntington's disease (HD) and Dentatorubral-pallidoluysian atrophy (DRPLA) as models for this family of diseases, since they have striking similarities and also notable differences in their clinical features and pathology. We review the pattern of pathology in adult and juvenile onset cases. Despite selective pathology, the disease genes and their protein products (huntingtin and atrophin-1) are widely expressed. This presents a central problem for all the glutamine repeat diseases-how do widely expressed gene products give rise to restricted pathology? The pathogenic effects are believed to occur via a "gain of function" mechanism at the protein level. Mechanisms of cell death may include excitotoxicity, metabolic toxicity, apoptosis, and free radical stress. Emerging data indicate that huntingtin and atrophin-1 may have distinct protein interactions. The specific interaction partners may help explain the selective pathology of these diseases.

HAP1-huntingtin interactions do not contribute to the molecular pathology in Huntington's disease transgenic mice.

HAP1 (huntingtin associated protein) has previously been found to interact with huntingtin (htt) in a glutamine length dependent manner and has been proposed to play a role in the cell specific neurodegeneration observed in Huntington's disease (HD). We have isolated mouse HAP1 (hap1) and have shown that expression is not enriched in areas specifically affected in HD. We have used the yeast two hybrid system to demonstrate that htt amino acids 171-230 are necessary for the hap1-htt binding and that hapl does not interact with the transgene exon 1 protein in a transgenic model of HD.

Inositol 1,4,5-trisphosphate receptors: immunocytochemical localization in the dorsal cochlear nucleus.

In the cochlear nucleus of mammals, the relatively homogeneous responses of auditory nerve fibers are transformed into a variety of different response patterns by the different classes of resident neurons. The spectrum of these responses is hypothesized to depend on the types and distribution of receptors, ion channels, G proteins, and second messengers that form the signaling capabilities in each cell class. In the present study, we examined the immunocytochemical distribution of the inositol 1,4,5-triphosphate (IP3) receptor in the dorsal cochlear nucleus to better understand how this second messenger might be involved in shaping the neural signals evoked by sound. Affinity-purified polyclonal antibodies directed against the IP3 receptor labeled a homogeneous population of neurons in the dorsal cochlear nucleus of rats, guinea pigs, mustache bats, cats, New World owl monkeys, rhesus monkeys, and humans. These cells were all darkly immunostained except in the human where the labeling was less intense. Immunoblots of dorsal cochlear nucleus tissue from the rat revealed a single band of protein of molecular weight approximately 260 kD, which is the same size as the purified receptor, indicating that our antibodies reacted specifically with the IP3 receptor. These immunolabeled neurons were identified as cartwheel cells on the basis of shared characteristics across species, including cell body size and distribution, the presence of a highly invaginated nucleus, and a well-developed system of cisternae. Reaction product was localized along the membranes of rough and smooth endoplasmic reticulum, subsurface cisternae, and the nuclear envelope. This label was distributed throughout the cartwheel cell body and dendritic shafts but not within dendritic spines, axons, or axons terminals. The regular pattern of immunolabeling across mammals suggests that IP3 and cartwheel cells are conserved in evolution and that both play an important but as yet unknown role in hearing.

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.

Immunological characterization of T-type voltage-dependent calcium channel CaV3.1 (alpha 1G) and CaV3.3 (alpha 1I) isoforms reveal differences in their localization, expression, and neural development.

Low voltage-activated calcium channels (LVAs; "T-type") modulate normal neuronal electrophysiological properties such as neuronal pacemaker activity and rebound burst firing, and may be important anti-epileptic targets. Proteomic analyses of available alpha 1G/Ca(V)3.1 and alpha 1I/Ca(V)3.3 sequences suggest numerous potential isoforms, with specific alpha 1G/Ca(V)3.1 or alpha 1I/Ca(V)3.3 domains postulated to be conserved among isoforms of each T-type channel subtype. This information was used to generate affinity-purified anti-peptide antibodies against sequences unique to alpha 1G/Ca(V)3.1 or alpha 1I/Ca(V)3.3, and these antibodies were used to compare and contrast alpha 1G/Ca(V)3.1 and alpha 1I/Ca(V)3.3 protein expression by western blotting and immunohistochemistry. Each antibody reacted with appropriately sized recombinant protein in HEK-293 cells. Regional and developmental differences in alpha 1G/Ca(V)3.1 and alpha 1I/Ca(V)3.3 protein expression were observed when the antibodies were used to probe regional brain dissections prepared from perinatal mice and adult rodents and humans. Mouse forebrain alpha 1G/Ca(V)3.1 (approximately 240 kDa) was smaller than cerebellar (approximately 260 kDa) alpha 1G/Ca(V)3.1, and expression of both proteins increased during perinatal development. In contrast, mouse midbrain and diencephalic tissues evidenced an alpha 1I/Ca(V)3.3 immunoreactive doublet (approximately 230 kDa and approximately 190 kDa), whereas other brain regions only expressed the small alpha 1I/Ca(V)3.3 isoform. A unique large alpha 1I/Ca(V)3.3 isoform (approximately 260 kDa) was expressed at birth and eventually decreased, concomitant with the appearance and gradual increase of the small alpha 1I/Ca(V)3.3 isoform. Immunohistochemistry supported the conclusion that LVAs are expressed in a regional manner, as cerebellum strongly expressed alpha 1G/Ca(V)3.1, and olfactory bulb and midbrain contained robust alpha 1I/Ca(V)3.3 immunoreactivity. Finally, strong alpha 1I/Ca(V)3.3, but not alpha 1G/Ca(V)3.1, immunoreactivity was observed in brain and spinal cord by embryonic day 14 in situ. Taken together, these data provide an anatomical and biochemical basis for interpreting LVA heterogeneity and offer evidence of developmental regulation of LVA isoform expression.

Neuronal inositol 1,4,5-trisphosphate receptor localized to the plasma membrane of olfactory cilia.

Both the cyclic adenosine 3',5'-monophosphate and the phosphoinositide second messenger systems are involved in olfactory signal transduction. The inositol 1,4,5-trisphosphate receptor is one of the principal intracellular calcium channels responsible for mobilizing stored calcium. The precise location of the 1,4,5-trisphosphate receptor (endoplasmic reticulum vs surface) and its role in the events of olfactory signal transduction need to be defined. By light microscopic and confocal immunohistochemistry we show expression of the olfactory-enriched G-protein, G(olf), associated with cyclic AMP responses, and of the inositol 1,4,5-trisphosphate receptor in the dendritic projections and cilia of essentially all olfactory receptor neurons, indicating that individual neurons express components of both second messenger systems. By immunoelectron microscopy, we demonstrate that the inositol 1,4,5-trisphosphate receptor is localized to the surface membrane of rat olfactory cilia. This is the first morphological demonstration of the surface membrane localization of inositol 1,4,5-trisphosphate receptor in olfactory cilia. Our findings, taken in conjunction with electrophysiological data from other workers, are supportive of the inositol 1,4,5-trisphosphate receptor playing a novel role in regulating calcium flux at the ciliary surface membrane.

Inositol 1,4,5-trisphosphate receptors: immunohistochemical localization to discrete areas of rat central nervous system.

The second messenger inositol 1,4,5-trisphosphate triggers the release of intracellular Ca2+ stores upon binding to the inositol 1,4,5-trisphosphate receptor protein, a calcium channel that has been purified and molecularly cloned. To clarify the roles of inositol 1,4,5-trisphosphate receptor in the central nervous system, we have examined in detail the distribution of inositol 1,4,5-trisphosphate receptors in the rat brain and spinal cord using immunohistochemical methods. Inositol 1,4,5-trisphosphate receptors are present in neuronal cells, fibers and terminals in a wide distribution of areas throughout the central nervous system. These include a number of areas not previously reported, such as the olfactory bulb, thalamic nuclei and dorsal horn of the spinal cord. In addition, we have noted a strikingly high density of inositol 1,4,5-trisphosphate receptors in circumventricular organs and neuroendocrine structures such as the area postrema, choroid plexus, subcommisural organ, pineal gland and pituitary. The distribution of inositol 1,4,5-trisphosphate receptors in discrete structures throughout the central nervous system, including interconnected neuronal systems and neuroendocrine and circumventricular organ structures, presumably reflects the importance of Ca2+ release mediated by the phosphoinositide second messenger system in control of diverse physiological processes.

The endoplasmic reticulum of Purkinje neuron body and dendrites: molecular identity and specializations for Ca2+ transport.

Immunofluorescence and immunogold labeling, together with sucrose gradient separation and Western blot analysis of microsomal subfractions, were employed in parallel to probe the endoplasmic reticulum in the cell body and dendrites of rat cerebellar Purkinje neurons. Two markers, previously investigated in non-nerve cells, the membrane protein p91 (calnexin) and the lumenal protein BiP, were found to be highly expressed and widely distributed to the various endoplasmic reticulum sections of Purkinje neurons, from the cell body to dendrites and dendritic spines. An antibody (denominated anti-rough-surfaced endoplasmic reticulum), which recognized two membrane proteins, p14 and p40, revealed a similar immunogold labeling pattern. However, centrifugation results consistent with a widespread distribution were obtained for p14 only, while p40 was concentrated in the rough microsome-enriched subfractions. The areas enriched in the inositol 1,4,5-triphosphate receptor and thus presumably specialized in Ca2+ transport (stacks of multiple smooth-surfaced cisternae; the dendritic spine apparatus) also exhibited labeling for BiP and p91, and were positive for the anti-rough-surfaced endoplasmic reticulum antibody (presumably via the p14 antigen). Additional antibodies, that yielded inadequate immunocytochemical signals, were employed only by Western blotting of the microsomal subfractions, while the ryanodine receptor was studied by specific binding. The latter receptor and the Ca2+ ATPase, known in other species to be concentrated in Purkinje neurons, exhibited bimodal distributions with a peak in the light and another in the heavy subfractions. A similar distribution was also observed with another lumenal protein, protein disulfide isomerase. Taken as a whole, the results that we have obtained suggest the existence in the endoplasmic reticulum of Purkinje neurons of two levels of organization; the first identified by widespread, probably general markers (BiP, p91, possibly p14 and others), the second by specialization markers, such as the inositol 1,4,5-triphosphate receptor and, possibly, p40, which appear restricted to areas where specific functions appear to be localized.

Biochemical and anatomical evidence for specialized voltage-dependent calcium channel gamma isoform expression in the epileptic and ataxic mouse, stargazer.

Inherited forms of ataxia and absence seizures in mice have been linked to defects in voltage-dependent calcium channel subunits. However, a correlation between the sites of neuronal dysfunction and the impact of the primary lesion upon calcium channel subunit expression or function has not been clearly established. For example, the mutation in stargazer mice has pleiotropic consequences including synaptic alterations in cerebellar granule cells, hippocampal CA3/mossy fibers, and cortical neurons in layer V that, presumably, lead to ataxia and seizures. Genetic analysis of stargazer mice determined that the defective gene encodes a protein expressed in brain (gamma2) with limited homology to the skeletal muscle L-type calcium channel gamma1 subunit. Although additional gamma isoforms have been subsequently identified primarily in neural tissue, little was known about the proteins they encode. Therefore, this study explored the distribution and biochemical properties of gamma2 and other gamma isoforms in wild-type and stargazer brain. We cloned human gamma2, gamma3, and gamma4 isoforms, produced specific anti-peptide antibodies to gamma isoforms and characterized both heterologously expressed and endogenous gamma. We identified regional specificity in the expression of gamma isoforms by western analysis and immunohistochemistry. We report for the first time that the mutation in the stargazer gene resulted in the loss of gamma2 protein. Furthermore, no compensatory changes in the expression of gamma3 or gamma4 protein were evident in stargazer brain. In contrast to other voltage-dependent calcium channel subunits, gamma immunostaining was striking in that it was primarily detected in regions highly enriched in excitatory glutamatergic synapses and faintly detected in cell bodies, suggesting a role for gamma in synaptic functions. Sites of known synaptic dysfunction in stargazer (the hippocampal CA3 region, dentate gyrus, and cerebellar molecular layer) were revealed as relying primarily upon gamma2, as total gamma isoform expression was dramatically decreased in these regions. Electron microscopy localized anti-gamma antibody immunostaining to dendritic structures of hippocampal mossy fiber synapses, with enrichment at postsynaptic densities. To assess the association of native gamma with voltage-dependent calcium channel or alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) receptor subunits, gamma isoforms (gamma2, gamma3 and gamma4) were detergent solubilized from mouse forebrain. Antibodies against a highly conserved C-terminal epitope present in gamma2, gamma3 and gamma4 immunoprecipitated voltage-dependent calcium channel subunits (alpha1B), providing the first in vivo evidence that gamma and voltage-dependent calcium channels form stable complexes. Furthermore, both anti-gamma2 antibodies and anti-alpha1B antibodies independently immunoprecipitated the AMPA receptor subunit, GluR1, from mouse forebrain homogenates. In summary, loss of gamma2 immunoreactivity in stargazer is precisely localized so as to contribute to previously characterized synaptic defects. The data in this paper provide compelling evidence that gamma isoforms form complexes in vivo with voltage-dependent calcium channels as well as AMPA receptors, are selectively and differentially expressed in neuronal processes, and localize primarily to dendritic structures in the hippocampal mossy fiber region.

Pathogenesis of neurodegenerative diseases associated with expanded glutamine repeats: new answers, new questions.

Eight diseases are now known to be caused by an expansion mutation of the trinucleotide repeat CAG encoding glutamine. Each disease is caused by a CAG expansion in a different gene, and the genes bear no similarity to each other except for the presence of the repeat. Nonetheless, the essential feature of all of these disorders is neurodegeneration in a set of overlapping cortical and subcortical regions. Disease age of onset, and in some cases severity, is correlated with repeat length. These and other observations have led to the hypothesis that CAG expansion causes disease by a toxic gain-of-function of the encoded stretch of polyglutamine residues. Expansion-induced abnormalities of cytoskeletal function or neuronal signalling processes may contribute to the pathogenic process. In addition, theoretical and experimental analysis of the chemistry of uninterrupted stretches of glutamine residues suggest that polyglutamine-containing proteins or protein fragments may aggregate, via a "polar zipper", into beta pleated sheets. Recent findings have now established the presence of such aggregates in selected regions of brain from affected individuals, in transgenic mice expressing expanded repeats, and in isolated cells transfected with expanded repeats. The aggregates are most prominently manifest as neuronal intranuclear inclusion bodies. As the investigation of the link between these inclusions and cell dysfunction and death continues, it is possible that new avenues for therapeutic intervention will emerge.

32,000-Dalton subunit of the 1,4-dihydropyridine receptor.

Polyclonal antibodies to the 32,000-Da polypeptide of the 1,4-dihydropyridine receptor of the voltage-dependent Ca2+ channel have been produced and used to characterize the association of the 32,000-Da polypeptide (gamma subunit) with other subunits of the dihydropyridine receptor. The 32,000-Da polypeptide was found to copurify with alpha 1 and alpha 2 subunits at each step of the purification of the dihydropyridine receptor. Monoclonal antibodies against the alpha 1 and beta subunits immunoprecipitate the digitonin-solubilized dihydropyridine receptor as a multisubunit complex that includes the 32,000-Da polypeptide. Polyclonal antibodies generated against both the nonreduced and reduced forms of the alpha 2 subunit and the gamma subunit have been used to show that the 32,000-Da polypeptide is not a proteolytic fragment of a larger component of the dihydropyridine receptor and not disulfide linked to the alpha 2 subunit. Our results demonstrate that the 32,000-Da polypeptide (gamma subunit) is an integral and distinct component of the dihydropyridine receptor.