Joy G. Ghosh

Alzheimer's Disease Amyloid-β Links Lens and Brain Pathology in Down Syndrome

Down syndrome (DS, trisomy 21) is the most common chromosomal disorder and the leading genetic cause of intellectual disability in humans. In DS, triplication of chromosome 21 invariably includes the APP gene (21q21) encoding the Alzheimers disease (AD) amyloid precursor protein (APP). Triplication of the APP gene accelerates APP expression leading to cerebral accumulation of APP-derived amyloid-β peptides (Aβ), early-onset AD neuropathology, and age-dependent cognitive sequelae. The DS phenotype complex also includes distinctive early-onset cerulean cataracts of unknown etiology. Previously, we reported increased Aβ accumulation, co-localizing amyloid pathology, and disease-linked supranuclear cataracts in the ocular lenses of subjects with AD. Here, we investigate the hypothesis that related AD-linked Aβ pathology underlies the distinctive lens phenotype associated with DS. Ophthalmological examinations of DS subjects were correlated with phenotypic, histochemical, and biochemical analyses of lenses obtained from DS, AD, and normal control subjects. Evaluation of DS lenses revealed a characteristic pattern of supranuclear opacification accompanied by accelerated supranuclear Aβ accumulation, co-localizing amyloid pathology, and fiber cell cytoplasmic Aβ aggregates (∼5 to 50 nm) identical to the lens pathology identified in AD. Peptide sequencing, immunoblot analysis, and ELISA confirmed the identity and increased accumulation of Aβ in DS lenses. Incubation of synthetic Aβ with human lens protein promoted protein aggregation, amyloid formation, and light scattering that recapitulated the molecular pathology and clinical features observed in DS lenses. These results establish the genetic etiology of the distinctive lens phenotype in DS and identify the molecular origin and pathogenic mechanism by which lens pathology is expressed in this common chromosomal disorder. Moreover, these findings confirm increased Aβ accumulation as a key pathogenic determinant linking lens and brain pathology in both DS and AD.

Insights into the domains required for dimerization and assembly of human αB crystallin

Protein pin array technology was used to identify subunit–subunit interaction sites in the small heat shock protein (sHSP) αB crystallin. Subunit–subunit interaction sites were defined as consensus sequences that interacted with both human αA crystallin and αB crystallin. The human αB crystallin protein pin array consisted of contiguous and overlapping peptides, eight amino acids in length, immobilized on pins that were in a 96‐well ELISA plate format. The interaction of αB crystallin peptides with physiological partner proteins, αA crystallin and αB crystallin, was detected using antibodies and recorded using spectrophotometric absorbance. Five peptide sequences including 37LFPTSTSLSPFYLRPPSF54 in the N terminus, 75FSVNLDVK82 (β3), 131LTITSSLS138 (β8) and 141GVLTVNGP148 (β9) that form β strands in the conserved α crystallin core domain, and 155PERTIPITREEK166 in the C‐terminal extension were identified as subunit–subunit interaction sites in human αB crystallin using the novel protein pin array assay. The subunit–subunit interaction sites were mapped to a three‐dimensional (3D) homology model of wild‐type human αB crystallin that was based on the crystal structure of wheat sHSP16.9 and Methanococcus jannaschi sHSP16.5 (Mj sHSP16.5). The subunit–subunit interaction sites identified and mapped onto the homology model were solvent‐exposed and had variable secondary structures ranging from β strands to random coils and short α helices. The subunit–subunit interaction sites formed a pattern of hydrophobic patches on the 3D surface of human αB crystallin.

Interactive Domains in the Molecular Chaperone Human αB Crystallin Modulate Microtubule Assembly and Disassembly

Background Small heat shock proteins regulate microtubule assembly during cell proliferation and in response to stress through interactions that are poorly understood. Methodology Novel functions for five interactive sequences in the small heat shock protein and molecular chaperone, human αB crystallin, were investigated in the assembly/disassembly of microtubules and aggregation of tubulin using synthetic peptides and mutants of human αB crystallin. Principal Findings The interactive sequence 113FISREFHR120 exposed on the surface of αB crystallin decreased microtubule assembly by ∼45%. In contrast, the interactive sequences, 131LTITSSLSSDGV142 and 156ERTIPITRE164, corresponding to the β8 strand and the C-terminal extension respectively, which are involved in complex formation, increased microtubule assembly by ∼34–45%. The αB crystallin peptides, 113FISREFHR120 and 156ERTIPITRE164, inhibited microtubule disassembly by ∼26–36%, and the peptides 113FISREFHR120 and 131LTITSSLSSDGV142 decreased the thermal aggregation of tubulin by ∼42–44%. The 131LTITSSLSSDGV142 and 156ERTIPITRE164 peptides were more effective than the widely used anti-cancer drug, Paclitaxel, in modulating tubulin↔microtubule dynamics. Mutagenesis of these interactive sequences in wt human αB crystallin confirmed the effects of the αB crystallin peptides on microtubule assembly/disassembly and tubulin aggregation. The regulation of microtubule assembly by αB crystallin varied over a narrow range of concentrations. The assembly of microtubules was maximal at αB crystallin to tubulin molar ratios between 1∶4 and 2∶1, while molar ratios >2∶1 inhibited microtubule assembly. Conclusions and Significance Interactive sequences on the surface of human αB crystallin collectively modulate microtubule assembly through a dynamic subunit exchange mechanism that depends on the concentration and ratio of αB crystallin to tubulin. These are the first experimental results in support of the functional importance of the dynamic subunit model of small heat shock proteins.

Chaperone-like activity revealed in the matricellular protein SPARC

SPARC (Secreted Protein, Acidic and Rich in Cysteine) is a matricellular glycoprotein that modulates cell proliferation, adhesion, migration, and extracellular matrix (ECM) production. In this report chaperone‐like activity of SPARC was identified in a thermal aggregation assay in vitro. Ultraviolet circular dichroism (UVCD) spectroscopy determined that SPARC was stable at temperatures up to 50°C. Unfolding and aggregation of the chaperone target protein, alcohol dehydrogenase (ADH), were initiated at 50°C. SPARC inhibited the thermal aggregation of ADH in a concentration‐dependent manner, with maximal inhibition at a 1:4 molar ratio of SPARC:ADH. Synergy between the chaperone‐like activities of SPARC and αB‐crystallin, a small heat shock protein and molecular chaperone in the lens, was observed in SPARC‐αB‐crystallin double −/− mice. J. Cell. Biochem. 98: 701–705, 2006.

The function of the β3 interactive domain in the small heat shock protein and molecular chaperone, human αB crystallin

Abstract Knowledge of the interactive domains on the surface of small heat shock proteins (sHSPs) is necessary for understanding the assembly of complexes and the activity as molecular chaperones. The primary sequences of 26 sHSP molecular chaperones were aligned and compared. In the interactive β3 sequence, 73DRFSVNLDVKHFS85 of human αB crystallin, Ser-76, Asn-78, Lys-82, and His-83 were identified as nonconserved residues on the exposed surface of the α crystallin core domain. Site-directed mutagenesis produced the mutant αB crystallins: S76E, N78G, K82Q, and H83F. Domain swapping with homologous β3 sequences, 32EKFEVGLDVQFFT44 from Caenorhabditis elegans sHSP12.2 or 69DKFVIFLDVKHFS81 from αA crystallin, resulted in the mutant αB crystallins, CE1 and αA1, respectively. Decreased chaperone activity was observed with the point mutants N78G, K82Q, and H83F and with the mutant, CE1, in aggregation assays using βL crystallin, alcohol dehydrogenase (ADH), or citrate synthase (CS). The S76E mutant had minimal effect on chaperone activity, and domain swapping with αA crystallin had no effect on chaperone activity. The mutations that resulted in altered chaperone activity, produced minimal modification to the secondary, tertiary, and quaternary structure of human αB crystallin as determined by ultraviolet circular dichroism spectroscopy, chymotrypsin proteolysis, and size exclusion chromatography. Chaperone activity was influenced by the amount of unfolding of the target proteins and independent of complex size. The results characterized the importance of the exposed side chains of Glu-78, Lys-82, and His-83 in the interactive β3 sequence of the α crystallin core domain in αB crystallin for chaperone function.

P1-375: Alzheimer's disease beta-amyloid pathology in Down syndrome cataract

Lee E. Goldstein, Robert D. Moir, Roberto Pineda, Juliet A. Moncaster, Mark A. Burton, Joy Ghosh, Stephanie Soscia, Anca Mocofanescu, John I. Clark, Richard M. Robb, Rudolph E. Tanzi, David G. Hunter, Boston University School of Medicine, Boston, MA, USA; Massachusetts General Hospital/Harvard Medical School, Boston, MA, USA; Massachusetts Eye and Ear/ Harvard Medical School, Boston, MA, USA; University of Washington, Seattle, WA, USA; Children’s Hospital/Harvard Medical School, Boston, MA, USA. Contact e-mail: lgold@bu.edu