Alan H. Sharp

Formation of Neuronal Intranuclear Inclusions Underlies the Neurological Dysfunction in Mice Transgenic for the HD Mutation

Huntingtons disease (HD) is one of an increasing number of human neurodegenerative disorders caused by a CAG/polyglutamine-repeat expansion. The mutation occurs in a gene of unknown function that is expressed in a wide range of tissues. The molecular mechanism responsible for the delayed onset, selective pattern of neuropathology, and cell death observed in HD has not been described. We have observed that mice transgenic for exon 1 of the human HD gene carrying (CAG)115 to (CAG)156 repeat expansions develop pronounced neuronal intranuclear inclusions, containing the proteins huntingtin and ubiquitin, prior to developing a neurological phenotype. The appearance in transgenic mice of these inclusions, followed by characteristic morphological change within neuronal nuclei, is strikingly similar to nuclear abnormalities observed in biopsy material from HD patients.

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.

Synphilin-1 associates with α-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 α-synuclein; refs 3, 4, 5). α-synuclein is present in Lewy bodies of patients with sporadic PD (Refs 6,7), suggesting that α-synuclein may be involved in the pathogenesis of PD. It is unknown how α-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 α-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 α-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.

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

Huntingtons 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 genes 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.

The biochemistry and molecular biology of the dihydropyridine-sensitive calcium channel.

Abstract Calcium channels are known to exist in muscle, neuronal and secretory cells. The 1,4-dihydropyridines are potent blockers of L-type Ca 2+ channels, and have been used as specific probes in the study of dihydropyridine-sensitive Ca 2+ channels. The receptor for the 1,4-dihydropyridines has been purified from skeletal muscle in order to characterize the biochemistry and molecular biology of the dihydropyridine-sensitive Ca 2+ channel. This review summarizes recent findings on the subunit composition of the dihydropyridinesensitive Ca 2+ channel, and discusses the structure and possible function of the individual subunits.

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.

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. J. Comp. Neurol. 406:207–220, 1999.

Atrophin-1, the DRPLA gene product, interacts with two families of WW domain-containing proteins.

Atrophin-1 contains a polyglutamine repeat, expansion of which is responsible for dentatorubral and pallidoluysian atrophy (DRPLA). The normal function of atrophin-1 is unknown. We have identified five atrophin-1 interacting proteins (AIPs) which bind to atrophin-1 in the vicinity of the polyglutamine tract using the yeast two-hybrid system. Four of the interactions were confirmed using in vitro binding assays. All five interactors contained multiple WW domains. Two are novel. The AIPs can be divided into two distinct classes. AIP1 and AIP3/WWP3 are MAGUK-like multidomain proteins containing a number of protein-protein interaction modules, namely a guanylate kinase-like region, two WW domains, and multiple PDZ domains. AIP2/WWP2, AIP4, and AIP5/WWP1 are highly homologous, each having four WW domains and a HECT domain characteristic of ubiquitin ligases. These interactors are similar to recently isolated huntingtin-interacting proteins, suggesting possible commonality of function between two proteins responsible for very similar diseases.

Cleavage of Atrophin-1 at Caspase Site Aspartic Acid 109 Modulates Cytotoxicity

Dentatorubropallidoluysian atrophy (DRPLA) is one of eight autosomal dominant neurodegenerative disorders characterized by an abnormal CAG repeat expansion which results in the expression of a protein with a polyglutamine stretch of excessive length. We have reported recently that four of the gene products (huntingtin, atrophin-1 (DRPLA), ataxin-3, and androgen receptor) associated with these open reading frame triplet repeat expansions are substrates for the cysteine protease cell death executioners, the caspases. This led us to hypothesize that caspase cleavage of these proteins may represent a common step in the pathogenesis of each of these four neurodegenerative diseases. Here we present evidence that caspase cleavage of atrophin-1 modulates cytotoxicity and aggregate formation. Cleavage of atrophin-1 at Asp109 by caspases is critical for cytotoxicity because a mutant atrophin-1 that is resistant to caspase cleavage is associated with significantly decreased toxicity. Further, the altered cellular localization within the nucleus and aggregate formation associated with the expanded form of atrophin-1 are completely suppressed by mutation of the caspase cleavage site at Asp109. These results provide support for the toxic fragment hypothesis whereby cleavage of atrophin-1 by caspases may be an important step in the pathogenesis of DRPLA. Therefore, inhibiting caspase cleavage of the polyglutamine-containing proteins may be a feasible therapeutic strategy to prevent cell death.

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.