Faculty Bibliography

Widespread cerebral deposition of a 40-43-amino acid peptide called the amyloid beta-protein (Abeta) in the form of amyloid fibrils is one of the most prominent neuropathologic features of Alzheimer's disease. Numerous studies suggest that Abeta is toxic to neurons by free radical-mediated mechanisms. We have previously reported that melatonin prevents oxidative stress and death of neurons exposed to Abeta. In the process of screening indole compounds for neuroprotection against Abeta, potent neuroprotective properties were uncovered for an endogenous related species, indole-3-propionic acid (IPA). This compound has previously been identified in the plasma and cerebrospinal fluid of humans, but its functions are not known. IPA completely protected primary neurons and neuroblastoma cells against oxidative damage and death caused by exposure to Abeta, by inhibition of superoxide dismutase, or by treatment with hydrogen peroxide. In kinetic competition experiments using free radical-trapping agents, the capacity of IPA to scavenge hydroxyl radicals exceeded that of melatonin, an indoleamine considered to be the most potent naturally occurring scavenger of free radicals. In contrast with other antioxidants, IPA was not converted to reactive intermediates with pro-oxidant activity. These findings may have therapeutic applications in a broad range of clinical situations

Apolipoprotein J (apoJ, clusterin) is a multifunctional protein normally associated with lipids in plasma and cerebrospinal fluid, and secreted as lipoparticles by hepatocytes and astrocytes. To investigate whether the structural and functional properties of apoJ are modulated upon binding to lipids, we prepared apoJ high-density lipoprotein (HDL)-like particles employing either synthetic or plasma HDL-derived lipids. The majority of the resulting lipoparticles contained one molecule of apoJ per particle and exhibited the same alpha2 electrophoretic mobility characteristic of apoJ-containing plasma HDL. Particle size seemed to be dependent on the presence of cholesterol in the lipid mixture and ranged from diameters of 10 nm in the presence of cholesterol to 20 nm in the absence of cholesterol. CD analysis and Fourier-transform infrared spectroscopy revealed similar changes in the apoJ secondary structure induced by its incorporation into lipoparticles, namely a decrease in alpha-helix content and an increase in beta-turn structures. Two functional assays, the binding interaction with Alzheimer's amyloid beta peptides and the inhibitory activity of the complement membrane-attack complex, did not detect any changes in apoJ activity following its lipidation (P>0.05). On the contrary, the binding affinity to the cellular receptor megalin was enhanced significantly (P<0.01) after the association with lipids; the K(d) value decreased from 78.8+/-10.7 nM for the delipidated form to 37. 0+/-7.3 nM for apoJ-HDL. Although it is not known whether the structural changes observed are directly responsible for the higher receptor-binding affinity, the data suggest that the complement inhibition and amyloid beta-binding motifs are located in areas of the molecule different from those involved in the apoJ-megalin interaction

Light chain deposition disease (LCDD) and light chain amyloidosis (AL) are disorders of monoclonal immunoglobulin deposition in which normally soluble serum precursors form insoluble deposits in tissues. A common feature in both is the clonal proliferation of B-cells that produce pathogenic light chains. However, the deposits in LCDD differ from those in AL in that they are ultrastructurally granular rather than fibrillar and do not bind Congo red or colocalize with amyloid P component or apolipoprotein E. The reason(s) for their differences are unknown but are likely multifactorial and related to their protein conformation and their interaction with other molecules and tissue factors in the microenvironment. Knowledge of the primary structure of the light chains in LCDD is very limited. In the present study two new kappa(1) light chains from patients with LCDD are described and compared to seven other reported kappa-LCDD proteins. The N-terminal amino acid sequences of light chain GLA extracted from the renal biopsy and light chain CHO from myocardial tissue were each identical to the respective light chains isolated from the urines and to the V-region amino acid sequences translated from the cloned cDNAs obtained from bone marrow cells. The germline V-region sequences, determined from the genomic DNA in both and in MCM, a previously reported kappa(1) LCDD light chain, were identical and related to the L12a germline gene. The expressed light chains in all three exhibit amino acid substitutions that arise from somatic mutation and result in increased hydrophobicity with the potential for protein destabilization and disordered conformation

Disorders of immunoglobulin (Ig) synthesis that occur in malignant plasma-cell proliferation may result in either granular (LCDD) or fibrillar (AL) tissue deposition of light-chain monoclonal components. The structural features that govern the transition from soluble polypeptides to either fibrillar or granular conformational states remain undefined. Among the many factors presumed to play a role in these transitions the net charge of the molecule has been associated with folding conformation changes. The majority of the proteins involved in AL amyloidosis show acidic isoelectric points (pI 3.8-5.2), whereas most L chains with basic pIs deposit in granular patterns. In our studies a 12 kD VkappaIII fragment was purified as the main component of the fibrils isolated from myocardium and adipose tissue of the pericardium obtained post-mortem from an individual with systemic AL amyloidosis. An apparently identical 12 kD VL fragment with the same N-terminal sequence constituted the BJ protein present in the urine. This urinary protein exhibited strikingly cathodic electrophoretic mobility on agarose gels and lacked retention by anionic exchange chromatography matrices, indicative of a highly basic pI (>10). When it was subjected to in vitro fibril-formation experiments, the BJ protein adopted a fibrillar conformation only at acidic pHs, remaining aggregated but not fibrillar at physiological pH. The data indicate that a specific tissue deposition pattern involves not only structural properties of the protein but rather more complex mechanisms in which acidic micro-environments may contribute to the stabilization of amyloidogenic conformations

Familial British dementia (FBD), previously designated familial cerebral amyloid angiopathy-British type, is an autosomal dominant disorder of undetermined origin characterized by progressive dementia, spasticity, and cerebellar ataxia, with onset at around the fifth decade of life. Cerebral amyloid angiopathy, non-neuritic and perivascular plaques and neurofibrillary tangles are the predominant pathological lesions. Here we report the identification of a unique 4K protein subunit named ABri from isolated amyloid fibrils. This highly insoluble peptide is a fragment of a putative type-II single-spanning transmembrane precursor that is encoded by a novel gene, BRI, located on chromosome 13. A single base substitution at the stop codon of this gene generates a longer open reading frame, resulting in a larger, 277-residue precursor. Release of the 34 carboxy-terminal amino acids from the mutated precursor generates the ABri amyloid subunit. The mutation creates a cutting site for the restriction enzyme XbaI, which is useful for detecting asymptomatic carriers. Antibodies against the amyloid or homologous synthetic peptides recognize both parenchymal and vascular lesions in FBD patients. A point mutation at the stop codon of BRI therefore results in the generation of the ABri peptide, which is deposited as amyloid fibrils causing neuronal disfunction and dementia

About 90% of the soluble amyloid beta (sA beta) that circulates in normal human plasma is associated with lipoprotein particles. In sporadic Alzheimer's disease patients, free sA beta42 but not sA beta40 is increased approximately 2.3-fold compared with age-matched controls, although a more marked elevation (approximately 8-fold for free sA beta40 and about 20-fold for sA beta42) is found in Down's syndrome patients. The data suggest that lipoprotein-sA beta dissociation may contribute to the influx of sA beta into the brain as a result of decreased plasma clearance