alpha-chymotrypsin and Nerve-Degeneration

Alpha-synuclein (alpha-syn) is a major protein component of the neuropathological hallmarks of Parkinson's disease and related neurodegenerative disorders termed synucleinopathies. Neither the mechanism of alpha-syn fibrillization nor the degradative process for alpha-syn has been elucidated. Previously, we showed that wild-type, mutated, and fibrillar alpha-syn proteins are substrates of calpain I in vitro. In this study, we demonstrate that calpain-mediated cleavage near and within the middle region of soluble alpha-syn with/without tyrosine nitration and oxidation generates fragments that are unable to self-fibrillize. More importantly, these fragments prevent full-length alpha-syn from fibrillizing. Calpain-mediated cleavage of alpha-syn fibrils composed of wild-type or nitrated alpha-syn generate C-terminally truncated fragments that retain their fibrillar structure and induce soluble full-length alpha-syn to co-assemble. Therefore, calpain-cleaved soluble alpha-syn inhibits fibrillization, whereas calpain-cleaved fibrillar alpha-syn promotes further co-assembly. These results provide insight into possible disease mechanisms underlying synucleinopathies since the formation of alpha-syn fibrils could be causally linked to the onset/progression of these disorders.

Topics: alpha-Synuclein; Calpain; Chymotrypsin; Humans; Hydrolysis; Microscopy, Immunoelectron; Nerve Degeneration; Nerve Tissue Proteins; Nitrates; Parkinson Disease; Peptide Fragments; Peroxynitrous Acid; Recombinant Proteins; Solubility; Synucleins; Tyrosine

It is unclear how and when insoluble beta-amyloid in senile plaques exerts degenerative effects on distant hippocampal neurons in Alzheimer's disease. Racemization of Ser and Asp residues of insoluble beta-amyloid is a typical age-dependent process. In this study, we investigated the fibril formation activity and cytotoxic activity of beta-amyloid 1-40 racemized at the Asp or Ser residue. In contrast to beta-amyloid 1-40 and its derivative substituted with the D-Asp(1, 7 or 23) or D-Ser(8) residue, [D-Ser(26)]beta-amyloid 1-40 was non-toxic to PC12 cells, and did not exhibit significant fibril formation activity making it soluble. However, [D-Ser(26)]beta-amyloid 1-40, but not beta-amyloid 1-40, was converted in vitro to a potent neurotoxic and truncated peptide, [D-Ser(26)]beta-amyloid 25-35 or [D-Ser(26)]beta-amyloid 25-40, by chymotrypsin-like enzymes and aminopeptidase M. Soluble [D-Ser(26)]beta-amyloid 1-40 was injected into rat hippocampus with a non-toxic dose of ibotenic acid, an excitatory amino acid. Nissl staining and microtubule-associated protein-2 immunostaining revealed that [D-Ser(26)]beta-amyloid 1-40, as well as [D-Ser(26)]beta-amyloid 25-35, produced a drastic degeneration of the CA1 neurons with ibotenic acid although [D-Ser(26)]beta-amyloid 1-40 alone or ibotenic acid alone did not exert neuronal damage. This suggests the in vivo conversion of non-toxic [D-Ser(26)]beta-amyloid 1-40 to the toxic and truncated peptides which enhance the susceptibility of neurons to the excitatory amino acid.These results and the presence of [D-Ser(26)]beta-amyloid 25-35-like antigens in Alzheimer's disease brains suggest that soluble [D-Ser(26)]beta-amyloid 1-40, possibly formed during the aging process, is released from senile plaques, and converted by brain proteinases to truncated [D-Ser(26)]beta-amyloid 25-35(40)-like peptides, which degenerate hippocampal neurons by enhancing the susceptibility to excitatory amino acids in Alzheimer's disease brains. These findings may provide the basis for a new therapeutic approach to prevent the neurodegeneration in Alzheimer's disease.

Topics: Alzheimer Disease; Amino Acid Isomerases; Amino Acid Sequence; Aminopeptidases; Amyloid beta-Peptides; Animals; Aspartic Acid; Chymotrypsin; Coloring Agents; Endopeptidases; Excitatory Amino Acid Agonists; Hippocampus; Ibotenic Acid; Male; Nerve Degeneration; Neurofibrillary Tangles; Neurons; PC12 Cells; Peptide Fragments; Plaque, Amyloid; Rats; Rats, Sprague-Dawley; Serine

Injection of 150, 500, or 750 U of alpha chymotrypsin into the posterior chamber of clinically normal Beagles resulted in changes in intraocular pressure and iridocyclitis. In the 6 eyes treated with 500 or 750 U, lens subluxation occurred in 4 eyes, retinal detachment in 1 eye, retinal degeneration in 1 eye, and optic nerve degeneration in 6 eyes. Scanning electron microscopic changes included dose-related zonulolysis and zonulary remnants within the aqueous humor outflow pathways. Ultrastructural changes by transmission microscopy of the optic nerve degeneration revealed loss of axoplasm and organelles and disruption of the myelin sheaths. Retinal and optic nerve changes may represent the direct toxic effects of alpha chymotrypsin and/or the indirect effects of increased intraocular pressure.

Topics: Animals; Chymotrypsin; Dog Diseases; Dogs; Eye; Eye Diseases; Intraocular Pressure; Lens Subluxation; Microscopy, Electron; Nerve Degeneration; Ocular Physiological Phenomena; Optic Nerve; Optic Nerve Diseases; Retinal Degeneration; Retinal Detachment; Uveitis, Anterior

Topics: Animals; Carotid Arteries; Cats; Chymotrypsin; Collagen Diseases; Dura Mater; Endopeptidases; Inflammation; Injections, Spinal; Muscles; Necrosis; Nerve Degeneration; Subarachnoid Space