Upregulation of calpain activity precedes tau phosphorylation and loss of synaptic proteins in Alzheimer’s disease brain

Abstract

Alterations in calcium homeostasis are widely reported to contribute to synaptic degeneration and neuronal loss in Alzheimer’s disease. Elevated cytosolic calcium concentrations lead to activation of the calcium-sensitive cysteine protease, calpain, which has a number of substrates known to be abnormally regulated in disease. Analysis of human brain has shown that calpain activity is elevated in AD compared to controls, and that calpain-mediated proteolysis regulates the activity of important disease-associated proteins including the tau kinases cyclin-dependent kinase 5 and glycogen kinase synthase-3. Here, we sought to investigate the likely temporal association between these changes during the development of sporadic AD using Braak staged post-mortem brain. Quantification of protein amounts in these tissues showed increased activity of calpain-1 from Braak stage III onwards in comparison to controls, extending previous findings that calpain-1 is upregulated at end-stage disease, and suggesting that activation of calcium-sensitive signalling pathways are sustained from early stages of disease development. Increases in calpain-1 activity were associated with elevated activity of the endogenous calpain inhibitor, calpastatin, itself a known calpain substrate. Activation of the tau kinases, glycogen-kinase synthase-3 and cyclin-dependent kinase 5 were also found to occur in Braak stage II-III brain, and these preceded global elevations in tau phosphorylation and the loss of post-synaptic markers. In addition, we identified transient increases in total amyloid precursor protein and pre-synaptic markers in Braak stage II-III brain, that were lost by end stage Alzheimer's disease, that may be indicative of endogenous compensatory responses to the initial stages of neurodegeneration. These findings provide insight into the molecular events that underpin the progression of Alzheimer's disease, and further highlight the rationale for investigating novel treatment strategies that are based on preventing abnormal calcium homeostasis or blocking increases in the activity of calpain or important calpain substrates.Electronic supplementary materialThe online version of this article (doi:10.1186/s40478-016-0299-2) contains supplementary material, which is available to authorized users.

Membrane association and release of wild-type and pathological tau from organotypic brain slice cultures

“…29 Cortical extracts from 3xTg-AD mice showed overexpression of total tau, as expected on the basis of human tau overexpression (Figure 1a). At the ages examined here, no significant increases in tau phosphorylation at CP13 (Ser202) or PHF1 (Ser396/404) were observed, in line with previous reports.…”

“…29 White lines separating lanes in some immunoblots indicate removal of portions of the blots for clarity and/or splicing together of distinct blots. The following primary antibodies were used for western blotting.…”

Glucocorticoid receptors signaling impairment potentiates amyloid‐β oligomers‐induced pathology in an acute model of Alzheimer’s disease

“…Indeed, GR can be phosphorylated on several serine and threonine residues. 53 Calpain 1 is particularly involved in the activation of GSK-3β, 54 and in the maturation of p35 in p25 55 ( Figure 4A,F). 49,50 We first confirmed changed expression ratios of p(Ser9)GSK-3β/ GSK-3β and p(Tyr216)GSK-3β/GSK-3β 51 ( Figure 4A-C), reflecting an increase in GSK-3β activation.…”

“…We also measured increased levels of Calpain 1, a member of cysteine proteases family regulated by intracellular calcium and showing aberrant activity in AD. 53 Calpain 1 is particularly involved in the activation of GSK-3β, 54 and in the maturation of p35 in p25 55 (Figure 4A,F). Lastly, we observed increases in the levels of Fyn ( Figure 4A,G), a Src kinase associated with non-genomic effects of GR, 56 involved in the activation of GSK-3β and the phosphorylation of Tau.…”

The Deacetylase HDAC6 Mediates Endogenous Neuritic Tau Pathology

“…Therefore, we speculate that HDAC6 activity may become impaired within the dense beaded network along with other trapped vesicles and degenerated cytoskeletal components, eventually leading to increased tau acetylation, NR activation, and calcium deregulation. Increased intracellular calcium accumulation could further exacerbate neuronal toxicity via activation of downstream calcium-dependent enzymes including calpain and calcineurin, both of which have been implicated in Aβ-induced neurotoxicity (Kurbatskaya, et al 2016; Rozkalne, et al 2011). …”