Vikramjit Chopra

HIP1 Functions in Clathrin-mediated Endocytosis through Binding to Clathrin and Adaptor Protein 2*

Polyglutamine expansion in huntingtin is the underlying mutation leading to neurodegeneration in Huntington disease. This mutation influences the interaction of huntingtin with different proteins, including huntingtin-interacting protein 1 (HIP1), in which affinity to bind to mutant huntingtin is profoundly reduced. Here we demonstrate that HIP1 colocalizes with markers of clathrin-mediated endocytosis in neuronal cells and is highly enriched on clathrin-coated vesicles (CCVs) purified from brain homogenates. HIP1 binds to the clathrin adaptor protein 2 (AP2) and the terminal domain of the clathrin heavy chain, predominantly through a small fragment encompassing amino acids 276–335. This region, which contains consensus clathrin- and AP2-binding sites, functions in conjunction with the coiled-coil domain to target HIP1 to CCVs. Expression of various HIP1 fragments leads to a potent block of clathrin-mediated endocytosis. Our findings demonstrate that HIP1 is a novel component of the endocytic machinery.

HIP12 is a non-proapoptotic member of a gene family including HIP1, an interacting protein with huntingtin

Abstract. Huntingtin-interacting protein 1 (HIP1) is a membrane-associated protein that interacts with huntingtin, the protein altered in Huntington disease. HIP1 shows homology to Sla2p, a protein essential for the assembly and function of the cytoskeleton and endocytosis in Saccharomyces cerevisiae. We have determined that the HIP1 gene comprises 32 exons spanning approximately 215 kb of genomic DNA and gives rise to two alternate splice forms termed HIP1-1 and HIP1-2. Additionally, we have identified a novel protein termed HIP12 with significant sequence and biochemical similarities to HIP1 and high sequence similarity to Sla2p. HIP12 differs from HIP1 in its pattern of expression both at the mRNA and protein level. However, HIP1 and HIP12 are both found within the brain and show a similar subcellular distribution pattern. In contrast to HIP1, which is toxic in cell culture, HIP12 does not confer toxicity in the same assay systems. Interestingly, HIP12 does not interact with huntingtin but can interact with HIP1, suggesting a potential interaction in vivo that may influence the function of each respective protein.

Identification of a set of genes showing regionally enriched expression in the mouse brain

BackgroundThe Pleiades Promoter Project aims to improve gene therapy by designing human mini-promoters (< 4 kb) that drive gene expression in specific brain regions or cell-types of therapeutic interest. Our goal was to first identify genes displaying regionally enriched expression in the mouse brain so that promoters designed from orthologous human genes can then be tested to drive reporter expression in a similar pattern in the mouse brain.ResultsWe have utilized LongSAGE to identify regionally enriched transcripts in the adult mouse brain. As supplemental strategies, we also performed a meta-analysis of published literature and inspected the Allen Brain Atlas in situ hybridization data. From a set of approximately 30,000 mouse genes, 237 were identified as showing specific or enriched expression in 30 target regions of the mouse brain. GO term over-representation among these genes revealed co-involvement in various aspects of central nervous system development and physiology.ConclusionUsing a multi-faceted expression validation approach, we have identified mouse genes whose human orthologs are good candidates for design of mini-promoters. These mouse genes represent molecular markers in several discrete brain regions/cell-types, which could potentially provide a mechanistic explanation of unique functions performed by each region. This set of markers may also serve as a resource for further studies of gene regulatory elements influencing brain expression.

Localization of the cell death genes CPP32 and Mch-2 to human chromosome 4q.

Programmed cell death, manifested as apoptosis, is a deliberate and systematic means of cell suicide characterized by several distinct biochemical and morphological changes including cell shrinkage, membrane blebbing, and chromatin condensation (Wyllie et al. 1980). While this mechanism for self-destruction can be triggered by different pathogenic stimuli including infectious agents, it can also occur physiologically to halt the spread of neighboring cells or to make room for new cell types. Thus, apoptosis plays an important role during normal development, helping to maintain the delicate balance between cell death and survival. However, this balance can go astray, resulting in disease. For instance, excessive apoptosis has been implicated in neurodegenerative diseases and ischemic damage, while insufficient apoptosis has been postulated to occur in cancers and autoimmune diseases (reviewed in Nicholson 1996). In the nematode Caenorhabditis elegans, 131 cells die during normal development by apoptosis (Hengartner and Horovitz 1994). This process is under the control of several genes including Ced-3, which encodes a key cell death protease that is absolutely necessary for apoptosis (Hengartner and Horovitz 1994). CPP32/apopain appears to be a key mammalian Ced-3 homolog acting early in the cell death pathway. Relative to other mammalian cysteine proteases, it shares a high level of homology with Ced-3 (Frenandes-Alnemri et al. 1994). Moreover, it is specifically responsible for cleavage and inactivation of key homeostatic proteins during apoptosis. These proteins include poly (ADP-ribose) polymerase (PARP), an enzyme involved in DNA repair particularly in response to environmental stress (Nicholson et al. 1995; Tewari et al. 1995). In addition, the catalytic subunit of DNAdependent protein kinase (DNA-PKcs), an enzyme essential for repair of DNA double-stranded breaks (Casciola-Rosen et al., 1995), and the U1-70 kDa small ribonucleoprotein (CasciolaRosen et al. 1994), which is necessary for RNA splicing, are also involved. Huntingtin, the gene product for the gene associated with Huntingtons Disease, is also cleared by apopain (Goldberg et al. 1996). Over-expression of CPP32 in vitro leads to apoptosis, which can be blocked by a specific peptide aldehyde inhibitor of CPP32 (Nicholson et al. 1995). However, no mutations in CPP32 have been shown to underlie any disease, perhaps owing to either functional redundancy in this enzyme family or to embryonic lethality. Using fluorescence in situ hybridization (FISH; Lichter et al. 1990) of a genomic clone isolated from a P1 library (Ioannou et al. 1994), we have mapped CPP32 to the tip of the long arm of human Chr 4 (Fig. 1) and have further refined its localization against a YAC contig from this region spanning at least 2 megabases (Mb).

rAAV-compatible MiniPromoters for restricted expression in the brain and eye

BackgroundSmall promoters that recapitulate endogenous gene expression patterns are important for basic, preclinical, and now clinical research. Recently, there has been a promising revival of gene therapy for diseases with unmet therapeutic needs. To date, most gene therapies have used viral-based ubiquitous promoters–however, promoters that restrict expression to target cells will minimize off-target side effects, broaden the palette of deliverable therapeutics, and thereby improve safety and efficacy. Here, we take steps towards filling the need for such promoters by developing a high-throughput pipeline that goes from genome-based bioinformatic design to rapid testing in vivo.MethodsFor much of this work, therapeutically interesting Pleiades MiniPromoters (MiniPs; ~4 kb human DNA regulatory elements), previously tested in knock-in mice, were “cut down” to ~2.5 kb and tested in recombinant adeno-associated virus (rAAV), the virus of choice for gene therapy of the central nervous system. To evaluate our methods, we generated 29 experimental rAAV2/9 viruses carrying 19 different MiniPs, which were injected intravenously into neonatal mice to allow broad unbiased distribution, and characterized in neural tissues by X-gal immunohistochemistry for icre, or immunofluorescent detection of GFP.ResultsThe data showed that 16 of the 19 (84 %) MiniPs recapitulated the expression pattern of their design source. This included expression of: Ple67 in brain raphe nuclei; Ple155 in Purkinje cells of the cerebellum, and retinal bipolar ON cells; Ple261 in endothelial cells of brain blood vessels; and Ple264 in retinal Müller glia.ConclusionsOverall, the methodology and MiniPs presented here represent important advances for basic and preclinical research, and may enable a paradigm shift in gene therapy.