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.

The spatiotemporal transmission of pathological tau in the brain is characteristic of Alzheimer's disease. Release of both soluble and abnormal tau species from healthy neurons is increased upon stimulation of neuronal activity. It is not yet understood whether the mechanisms controlling soluble tau release from healthy neurons is the same as those involved in the spread of pathological tau species. To begin to understand these events, we have studied tau distribution and release using organotypic brain slice cultures. The slices were cultured from postnatal wild-type and 3xTg-AD mice for up to 1 month. Tau distribution in subcellular compartments was examined by western blotting, and tau release into culture medium was determined using a sensitive sandwich ELISA. We show here that 3xTg-AD cultures have an accelerated development of pathological tau abnormalities including the redistribution of tau to synaptic and membrane compartments. The 3xTg-AD slice cultures show elevated basal tau release relative to total tau when compared with wild-type cultures. However, tau release from 3xTg-AD slices cannot be further stimulated when neuronal activity is increased with potassium chloride. Moreover, we report that there is an increased pool of dephosphorylated membrane-associated tau in conditions where tau release is increased. These data suggest that there may be differential patterns of tau release when using integrated slice culture models of wild-type and transgenic mouse brain, although it will be important to determine the effect of tau overexpression for these findings. These results further increase our knowledge of the molecular mechanisms underlying tau release and propagation in neurodegenerative tauopathies.

Increased production of amyloid β-peptide (Aβ) and altered processing of tau in Alzheimer's disease (AD) are associated with synaptic dysfunction, neuronal death and cognitive and behavioural deficits. Neuroinflammation is also a prominent feature of AD brain and considerable evidence indicates that inflammatory events play a significant role in modulating the progression of AD. The role of microglia in AD inflammation has long been acknowledged. Substantial evidence now demonstrates that astrocyte-mediated inflammatory responses also influence pathology development, synapse health and neurodegeneration in AD. Several anti-inflammatory therapies targeting astrocytes show significant benefit in models of disease, particularly with respect to tau-associated neurodegeneration. However, the effectiveness of these approaches is complex, since modulating inflammatory pathways often has opposing effects on the development of tau and amyloid pathology, and is dependent on the precise phenotype and activities of astrocytes in different cellular environments. An increased understanding of interactions between astrocytes and neurons under different conditions is required for the development of safe and effective astrocyte-based therapies for AD and related neurodegenerative diseases.

Alzheimer’s disease (AD) is a neurodegenerative disorder characterized by pathological deposits of β-amyloid (Aβ) in senile plaques, intracellular neurofibrillary tangles (NFTs) comprising hyperphosphorylated aggregated tau, synaptic dysfunction and neuronal death. Substantial evidence indicates that disrupted neuronal calcium homeostasis is an early event in AD that could mediate synaptic dysfunction and neuronal toxicity. Sodium calcium exchangers (NCXs) play important roles in regulating intracellular calcium, and accumulating data suggests that reduced NCX function, following aberrant proteolytic cleavage of these exchangers, may contribute to neurodegeneration. Here, we show that elevated calpain, but not caspase-3, activity is a prominent feature of AD brain. In addition, we observe increased calpain-mediated cleavage of NCX3, but not a related family member NCX1, in AD brain relative to unaffected tissue and that from other neurodegenerative conditions. Moreover, the extent of NCX3 proteolysis correlated significantly with amounts of Aβ1–42. We also show that exposure of primary cortical neurons to oligomeric Aβ1–42 results in calpain-dependent cleavage of NCX3, and we demonstrate that loss of NCX3 function is associated with Aβ toxicity. Our findings suggest that Aβ mediates calpain cleavage of NCX3 in AD brain and therefore that reduced NCX3 activity could contribute to the sustained increases in intraneuronal calcium concentrations that are associated with synaptic and neuronal dysfunction in AD.

Organotypic brain slice culture models provide an alternative to early stage in vivo studies as an integrated tissue system that can recapitulate key disease features, thereby providing an excellent platform for drug screening. We recently described a novel organotypic 3xTg-AD mouse brain slice culture model with key Alzheimer’s disease-like changes. We now highlight the potential of this model for testing disease-modifying agents and show that results obtained following in vivo treatment are replicated in brain slice cultures from 3xTg-AD mice. Moreover, we describe novel effects of the amyloid-binding tetra (ethylene glycol) derivative of benzothiazole aniline, BTA-EG4, on tau. BTA-EG4 significantly reduced tau phosphorylation in the absence of any changes in the amounts of amyloid precursor protein, amyloid-β or synaptic proteins. The reduction in tau phosphorylation was associated with inactivation of the Alzheimer’s disease-relevant major tau kinase, GSK-3. These findings highlight the utility of 3xTg-AD brain slice cultures as a rapid and reliable in vitro method for drug screening prior to in vivo testing. Furthermore, we demonstrate novel tau-directed effects of BTA-EG4 that are likely related to the ability of this agent to inactivate GSK-3. Our findings support the further exploration of BTA-EG4 as a candidate therapeutic for Alzheimer’s disease.