Anindya Dehejia

The ubiquitin pathway in Parkinson's disease

Mutations of the α-synuclein gene, have been identified in some familial forms of Parkinsons disease, and α-synuclein protein has been shown to accumulate in the brains of patients with the disease. These findings suggest that Parkinsons disease may be caused by the abnormal aggregation of α-synuclein protein. Here we have identified in a German family with Parkinsons disease a missense mutation in the ubiquitin carboxy-terminal hydrolase L1 (UCH-L1) gene. We show that this mutation, Ile93Met, causes a partial loss of the catalytic activity of this thiol protease, which could lead to aberrations in the proteolytic pathway and aggregation of proteins.

Identification, localization and characterization of the human γ-synuclein gene

We have identified and characterized a new member of the human synuclein gene family, γ-synuclein (SNCG). This gene is composed of five exons, which encode a 127 amino acid protein that is highly homologous to α-synuclein, which is mutated in some Parkinson’s disease families, and to β-synuclein. The γ-synuclein gene is localized to chromosome 10q23 and is principally expressed in the brain, particularly in the substantia nigra. We have determined its genomic sequence, and established conditions for sequence analysis of each of the exons. The γ-synuclein gene, also known as BCSG1, was recently found to be overexpressed in advanced infiltrating carcinoma of the breast. Our survey of the EST database indicated that it might also be overexpressed in an ovarian tumor.

Alpha synuclein is present in Lewy bodies in sporadic Parkinson's disease

A missense mutation in the human alpha synuclein gene was recently identified in some cases of familial Parkinsons disease (FPD). We have developed an antibody that recognizes the C-terminal 12 amino acids of the human alpha synuclein protein and have demonstrated that alpha synuclein is an abundant component of the Lewy bodies found within the degenerating neurons of patients with Parkinsons disease (PD). The presence of alpha synuclein in Lewy bodies of sporadic PD patients suggests a central role for alpha synuclein in the pathogenesis of PD.

Serine Phosphorylation, Chromosomal Localization, and Transforming Growth Factor-β Signal Transduction by Human bsp-1

The transforming growth factor-β (TGF-β) superfamily regulates a multitude of cellular and developmental events. TGF-β family ligands signal through transmembrane serine/threonine kinase receptors whose downstream effectors are largely unknown. Using genetic data from the fruit fly, we have identified a downstream effector of TGF-β-induced signaling. TGF-β signaling protein-1 (BSP-1) is rapidly phosphorylated in response to TGF-β. Localization of bsp-1 to chromosome 4q28 suggests a role in carcinogenesis. These data suggest that BSP-1 is the prototype of a new class of signaling molecules.

Alpha-synuclein immunoreactivity of huntingtin polyglutamine aggregates in striatum and cortex of Huntington's disease patients and transgenic mouse models.

Polyglutamine expansions in proteins are implicated in at least eight inherited neurodegenerative disorders, including Huntingtons disease. These mutant proteins can form aggregates within the nucleus and processes of neurons possibly due to misfolding of the proteins. Polyglutamine aggregates are ubiquitinated and sequester molecular chaperone proteins and proteasome components. To investigate other protein components of polyglutamine aggregates, cerebral cortex and striata from patients with Huntingtons disease and full-length cDNA transgenic mouse models for this disease were examined immunohistochemically for alpha-synuclein reactivity. Our findings demonstrate that alpha-synuclein can be used as a marker for huntingtin polyglutamine aggregates in both human and mice. Moreover in the HD transgenic mice, the intensity of immunoreactivity increases with age. The significance of recruitment of alpha-synuclein into huntingtin aggregates and its translocation away from the synapses remains to be determined. We propose that aberrant interaction of mutant huntingtin with other proteins, including alpha-synuclein, may influence disease progression.

Characterization of the human extracellular matrix protein 1 gene on chromosome 1q21

Ecm1, the mouse gene encoding extracellular matrix protein 1, is highly expressed in bone and cartilage as well as in osteogenic, preosteoblastic and chondroblastic cell lines. Ecm1 was recently localized to a chromosomal region in mouse syntenic to human chromosome 1q21, establishing this gene as a prime candidate gene for pycnodysostosis, a rare, autosomal recessive sclerosing skeletal dysplasia. Shortly thereafter, it was determined that cathepsin K is the pycnodysostosis gene. We now report the radiation hybrid mapping of human ECM1 to 1q21, and the gene structure and coding sequence of human ECM1.

GONADOTROPIN‐INDUCED DESENSITIZATION OF LEYDIG CELLS IN VIVO AND IN VITRO: ESTROGEN ACTION IN THE TESTIS

It is now well recognized that an important aspect of peptide hormone action is the ability to regulate the concentration of specific receptor sites and metabolic functions of the target cell. Pulsatile secretion of luteinizing hormone at or near physiological levels exerts a trophic and supportive action on the induction and maintenance of LH receptors and steroidogenic enzymes in the Leydig cell. In contrast, studies performed on testis tissue and purified Leydig cells of animals treated with singlc supraphysiological doses of GnRH, LH, or hCG have indicated that important functional consequences occur as a result of end-organ desensitization by acute increases in exogenous or endogenous gonadotropin.l- These include loss of LH receptors and decrease in maximum steroidogcnic responses when Leydig cells are subsequently stimulated by LH or hCG in vitro. Gonadotropin receptor depletion was observed 18 hours after treatment and was preceded by an initial increase of receptors by 50% at six hours. This temporal sequence of upand down-regulation of receptors was observed with all treatment doses (0.1-10 p g ) of hCG.-! Although elevations of circulating gonadotropin initially stimulate testosterone production by testicular Leydig cells in both adult and immature rats, the early androgen response is soon followed (on or about 6 h) by a refractory period in which the testosterone responses to gonadotropin and exogenous or endogenous cyclic AMP are markedly impaired for several days after hormone treatment.6-g These findings indicated that a biosynthetic lesion occurs in such desensitized cells. beyond the receptor level and within the steroidogenic pathway leading to testosterone production., . - Thus, gonadotropin-induced down-regulation of LH receptors is preceded by a late steroidogenic lesion of the androgen biosynthetic pathway (after progesterone formation) following moderate elevations of gonadotropin, and by an additional early lesion (before pregnenolone formation) after marked elevations of gonadotropin. These lesions were most marked at two to three days after treatment, and began to recover at four days. In this paper we will describe the results of recent studies conducted to

Genomic Distribution and Gonadal mRNA Expression of Two Human Luteinizing Hormone Receptor Exon 1 Sequences in Random Populations

Exon 1 of the human luteinizing hormone receptor (LHR) gene coding region exhibits at least two forms of sequence heterogeneity between 37 and 60 bp, spanning the junction of the signal peptide and the amino terminus of the mature protein. The LHR 1 differs from the LHR 2 by the insertion of 6 bp in exon 1 but is of identical sequence in the 5′ flanking region. RFLP analysis of the two haplotypes within a random population of 63 individuals revealed allele frequencies of 0.37 and 0.63 for LHR 1 and LHR 2, respectively. 94% of the samples contained at least one LHR 2 allele, whereas only 68% contained the LHR 1 allele. No gender differences were observed, and both homozygotes and heterozygotes displayed apparently normal reproduction. Reverse-transcriptase polymerase chain-reaction analyses of LHR mRNA from testes and ovaries revealed that both haplotypes are transcribed in normal individuals, with no difference in tissue specific distribution. Thus, at least two functional polymorphic forms of exon 1 coding region of the same LHR gene are present in a random human population.

Purification of rat Leydig cells: functional and morphological evaluation.

The recent application of density gradient centrifugation to purification of testicular Leydig cells has clearly demonstrated the value of this technique for studies on hormonal regulation of gonadotropin receptors and the control of androgen biosynthesis at the cellular Ievel.l- In earlier studies, we employed a fraction of rat Leydig cells obtained by centrifugation of collagenase-dispersed interstitial cells on 0 to 80% Metrizamide gradients. This fraction consisted of nearly pure Leydig cells that were morphologically intact and retained normal steroidogenic capacity. The use of such pure Leydig cells is particularly important in studies on specific modes of hormonal control, such as nuclear translocation of estrogen receptors during human chorionic gonadotropin (hCG) induced desensitization and CAMP-mediated hormonal events, including occupancy of protein kinase regulatory subunits and phosphorylation of endogenous protein substrates.G It also permits valid comparative studies between specific hormonal stimulation of the target cell and the effects of choleragen ( a general stimulus of adenylate cyclase) , with the demonstration of functional compartmentalization of the hormonal stimulus. The use of a relatively homogeneous cell population has obvious advantages in the analysis of direct actions of hormones on their target cells, in particular, minimizing the complicating effects of metabolites produced by nonrelevant cell types during evaluation of the biochemical responses to hormonal stimulation. In more recent studies, we have employed 14-32% Metrizamide gradients to maximize the fractionation of interstitial cells into distinct Leydig cell bandss The bands obtained by this method were characterized by testosterone and CAMP responses, binding of [l?I]hCG, and cell morphology with autoradiography and electron microscopy. For these studies, we prepared interstitial cell suspensions by collagenase dispersion of decapsulated testes ( 12-min incubation with 0.25 mg/ml collagenase) obtained from adult male rats (55-65 days old) killed by cervical dislocation.9 After adjusting the total nucleated cell concentration of this preparation (methylene-blue staining cells) to 40 X 10 cells/5 ml, we applied 5 ml of the cell suspension to 14-32% Metrizamide gradients, followed by centrifugation at 6,300 x g for ten minutes. To obtain the hCG binding profile, the original cell preparation was incubated for one hour with [l?jI]hCG ( 3 2 X 108 cells/ 10) cpm labeled hCG) and washed free of unbound tracer hormone before separation. After centrifugation, the gradient was collected as fractions of 1 or 2 ml (FIGURE 1 ) . Binding of the preincubated hCG was determined in each fraction by counting in a gamma-spectrometer, then fresh medium was added to each tube and the cells were spun down,

Molecular cloning and mapping of a novel developmentally regulated human C2H2-type zinc finger

~Neuroimmunology Branch, Building 10, Room 5B16, National Institute of Neurological Diseases and Stroke, National Institutes of Health, Bethesda, Maryland 20892, USA 2Laboratory of Neurogenetics, National Institute of Neurological Diseases and Stroke, National Institutes of Health, Bethesda, Maryland 20892, USA 3Gene Mapping Unit, Laboratory of Genetic Disease Research, National Center for Human Genome Research, National Institute of Health, Bethesda, Maryland 20892, USA 4Laboratory of Neurogenetics, National Institute of Alcohol Abuse and Alcoholism, National Institutes of Health, Bethesda, Maryland 20892, USA