calpain and Lewy-Body-Disease

Topics: Aging; Alzheimer Disease; Amyloid beta-Peptides; Brain; Calpain; Endopeptidases; Humans; Lewy Body Disease; Membrane Proteins; Mutation; Nerve Tissue Proteins; Parkinson Disease; Phosphorylation; Presenilin-1; Presenilin-2; Protein Processing, Post-Translational; Synucleins; tau Proteins; Ubiquitin

There is evidence that accumulation of α-synuclein (α-syn) in Parkinson's disease (PD) and dementia with Lewy bodies (DLB) results from impaired removal of α-syn rather than its overproduction. Kallikrein-6 (KLK6), calpain-1 (CAPN1) and cathepsin-D (CTSD) are among a small number of proteases that cleave α-syn and are dysregulated in PD and DLB. Our aim in this study was to determine whether protease activity is altered in another α-synucleinopathy, multiple system atrophy (MSA), and might thereby modulate the regional distribution of α-syn accumulation.. mRNA and protein level and/or activity of KLK6, CAPN1 and CTSD were measured and assessed in relation to α-syn load in multiple brain regions (posterior frontal cortex, caudate nucleus, putamen, occipital cortex, pontine base and cerebellar white matter), in MSA (n = 20) and age-matched postmortem control tissue (n = 20).. CTSD activity was elevated in MSA in the pontine base and cerebellar white matter. KLK6 and CAPN1 levels were elevated in MSA in the putamen and cerebellar white matter. However, the activity or level of these proteolytic enzymes did not correlate with the regional distribution of α-syn.. Accumulation of α-syn in MSA is not due to reduced activity of the proteases we have studied. We suggest that their upregulation is likely to be a compensatory response to increased α-syn in MSA.

Topics: Aged; Aged, 80 and over; Brain; Calpain; Cathepsin D; Female; Humans; Kallikreins; Lewy Body Disease; Male; Middle Aged; Multiple System Atrophy; Parkinson Disease; Synucleinopathies

Parkinson's disease (PD) and dementia with Lewy bodies (DLB) are neurodegenerative disorders of the aging population characterized by the accumulation of α-synuclein (α-syn). The mechanisms triggering α-syn toxicity are not completely understood, however, c-terminus truncation of α-syn by proteases such as calpain may have a role. Therefore, inhibition of calpain may be of value. The main objective of this study was to evaluate the effects of systemically administered novel low molecular weight calpain inhibitors on α-syn pathology in a transgenic mouse model. For this purpose, non-tg and α-syn tg mice received the calpain inhibitors - Gabadur, Neurodur or a vehicle, twice a day for 30 days. Immunocytochemical analysis showed a 60% reduction in α-syn deposition using Gabadur and a 40% reduction using Neurodur with a concomitant reduction in c-terminus α-syn and improvements in neurodegeneration. Western blot analysis showed a 77% decrease in α-spectrin breakdown products (SBDPs) SBDPs with Gabadur and 63% reduction using Neurodur. There was a 65% reduction in the active calpain form with Gabadur and a 45% reduction with Neurodur. Moreover, treatment with calpain inhibitors improved activity performance of the α-syn tg mice. Taken together, this study suggests that calpain inhibition might be considered in the treatment of synucleinopathies.

Topics: alpha-Synuclein; Animals; Astrocytes; Calpain; Disease Models, Animal; Glycoproteins; Immunohistochemistry; Lewy Body Disease; Mice; Mice, Transgenic; Neuroglia; Neurons; Parkinson Disease

Kallikrein-6 and calpain-1 are amongst a small group of proteases that degrade α-synuclein. We have explored the possibility that reduction in the level or activity of these enzymes contributes to the accumulation of α-synuclein in Lewy body diseases. We measured calpain-1 activity by fluorogenic activity assay, kallikrein-6 level by sandwich ELISA, and levels of α-synuclein and α-synuclein phosphorylated at serine 129 (α-synuclein-P129), in post-mortem brain tissue in pure dementia with Lewy bodies (DLB, n=12), Alzheimer's disease (AD, n=20) and age-matched controls (n=19). Calpain-1 activity was significantly reduced in DLB within the cingulate and parahippocampal cortex, regions with highest α-synuclein and α-synuclein-P129 load, and correlated inversely with the levels of α-synuclein and α-synuclein-P129. Calpain-1 was unaltered in the thalamus and frontal cortex, regions with less α-synuclein pathology. Kallikrein-6 level was reduced in the cingulate cortex in the DLB cohort, and correlated inversely with α-synuclein and α-synuclein-P129. Kallikrein-6 was also reduced in DLB in the thalamus but not in relation to α-synuclein or α-synuclein-P129 load and was unaltered in the frontal and parahippocampal cortex. In SH-SY5Y cells overexpressing wild-type α-synuclein there was partial co-localisation of kallikrein-6 and calpain-1 with α-synuclein, and siRNA-mediated knock-down of kallikrein-6 and calpain-1 increased the amount of α-synuclein in cell lysates. Our results indicate that reductions in kallikrein-6 and calpain-1 may contribute to the accumulation of α-synuclein in DLB.

Topics: alpha-Synuclein; Alzheimer Disease; Brain; Calpain; Case-Control Studies; Cell Line, Tumor; Child; Child, Preschool; Enzyme-Linked Immunosorbent Assay; Female; Gene Expression Regulation; Humans; Kallikreins; Lewy Body Disease; Male; Neuroblastoma; Phosphorylation; RNA, Small Interfering; Serine

Parkinson's disease (PD) and dementia with Lewy bodies (DLB) are both characterized pathologically by the presence of neuronal inclusions termed Lewy bodies (LBs). A common feature found in LBs are aggregates of alpha-synuclein (alpha-Syn), and although it is now recognized that alpha-Syn is the major building block for these toxic filaments, the mechanism of how this occurs remains unknown. In the present study, we demonstrate that proteolytic processing of alpha-Syn by the protease calpain I leads to the formation of aggregated high-molecular weight species and adoption of a beta-sheet structure. To determine whether calpain-cleavage of alpha-Syn occurs in PD and DLB, we designed site-directed calpain-cleavage antibodies to alpha-Syn and tested their utility in several animal model systems. Detection of calpain-cleaved alpha-Syn was evident in mouse models of cerebral ischemia and PD and in a Drosophila model of PD. In the human PD and DLB brain, calpain-cleaved alpha-Syn antibodies immunolabeled LBs and neurites in the substantia nigra. Moreover, calpain-cleaved alpha-Syn fragments identified within LBs colocalized with activated calpain in neurons of the PD and DLB brains. These findings suggest that calpain I may participate in the disease-linked aggregation of alpha-Syn in various alpha-synucleinopathies.

Topics: Aged; alpha-Synuclein; Animals; Area Under Curve; Blotting, Western; Brain; Calpain; Cell Line, Tumor; Drosophila; Female; Fluorescent Antibody Technique; Humans; Immunohistochemistry; Immunoprecipitation; Lewy Body Disease; Male; Mice; Mice, Inbred C57BL; Mice, Transgenic; Middle Aged; Neurons; Parkinson Disease; Protein Structure, Secondary

The calcium-activated protease calpain cleaves a variety of biologically important proteins and serves, therefore, as a key regulator of many cellular functions. Activation of both main isoforms, calpain 1 and calpain 2, was demonstrated previously in Alzheimer's disease. In this report, antibodies specifically recognizing the active form of calpain 2 were used to investigate calpain 2 activation in a broad range of neurodegenerative diseases, utilizing multiple-label confocal immunofluorescence imaging. With rare exceptions, the active form of calpain 2 was found in colocalization with hyperphosphorylated tau protein. Aggregates of mutated huntingtin, alpha-synuclein, or unidentified protein in motor neuron disease type of frontotemporal dementia were always negative. These findings indicate that calpain 2 activation is not a general response to protein aggregation. In tauopathies, more pathological inclusions were labeled for hyperphosphorylated tau than for activated calpain 2. The extent of colocalization varied in both a disease-specific and cell-type specific manner. The active form of calpain 2 was detected in 50-75% of tau neurofibrillary pathology in Alzheimer's disease, Alzheimer neurofibrillary changes and Down's syndrome, as well as in the accompanying Alzheimer-type tau pathology in diffuse Lewy bodies disease, progressive supranuclear palsy, and corticobasal degeneration. For glial cells, only 10-25% of tuft-shaped astrocytes, glial plaques, or coiled bodies contained activated calpain 2. The majority of Pick bodies were negative. The association of calpain 2 activation with hyperphosphorylated tau might be the result of an attempt by the calpain proteolytic system to degrade the tau protein aggregates. Alternatively, calpain 2 could be directly involved in tau hyperphosphorylation by modulating protein kinase activities. Overall, these results provide evidence of the important role of the calpain proteolytic system in the pathogenesis of neurodegenerative diseases with tau neurofibrillary pathology.

Topics: Adolescent; Adult; Aged; Aged, 80 and over; Alzheimer Disease; Antibody Specificity; Calpain; Down Syndrome; Female; Fluorescent Antibody Technique; Humans; Huntington Disease; Lewy Body Disease; Male; Middle Aged; Neurodegenerative Diseases; Neurofibrillary Tangles; Pick Disease of the Brain; Supranuclear Palsy, Progressive