Cara L. Croft

Astrocytes and neuroinflammation in Alzheimer's disease

Increased production of amyloid β-peptide (Aβ) and altered processing of tau in Alzheimers 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.

Calpain cleavage and inactivation of the sodium calcium exchanger-3 occur downstream of Aβ in Alzheimer’s disease

Alzheimers 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.

Constant light enhances synchrony among circadian clock cells and promotes behavioral rhythms in VPAC2-signaling deficient mice

Individual neurons in the suprachiasmatic nuclei (SCN) contain an intracellular molecular clock and use intercellular signaling to synchronize their timekeeping activities so that the SCN can coordinate brain physiology and behavior. The neuropeptide vasoactive intestinal polypeptide (VIP) and its VPAC2 receptor form a key component of intercellular signaling systems in the SCN and critically control cellular coupling. Targeted mutations in either the intracellular clock or intercellular neuropeptide signaling mechanisms, such as VIP-VPAC2 signaling, can lead to desynchronization of SCN neuronal clocks and loss of behavioral rhythms. An important goal in chronobiology is to develop interventions to correct deficiencies in circadian timekeeping. Here we show that extended exposure to constant light promotes synchrony among SCN clock cells and the expression of ~24 h rhythms in behavior in mice in which intercellular signaling is disrupted through loss of VIP-VPAC2 signaling. This study highlights the importance of SCN synchrony for the expression of rhythms in behavior and reveals how non-invasive manipulations in the external environment can be used to overcome neurochemical communication deficits in this important brain system.

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

The spatiotemporal transmission of pathological tau in the brain is characteristic of Alzheimers 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.

Targeting psychologic stress signaling pathways in Alzheimer’s disease

Alzheimer’s Disease (AD) is the most prevalent progressive neurodegenerative disease; to date, no AD therapy has proven effective in delaying or preventing the disease course. In the search for novel therapeutic targets in AD, it has been shown that increased chronic psychologic stress is associated with AD risk. Subsequently, biologic pathways underlying psychologic stress have been identified and shown to be able to exacerbate AD relevant pathologies. In this review, we summarize the literature relevant to the association between psychologic stress and AD, focusing on studies investigating the effects of stress paradigms on transgenic mouse models of Amyloid-β (Aβ) and tau pathologies. In recent years, a substantial amount of research has been done investigating a key stress-response mediator, corticotropin-releasing hormone (CRH), and its interactions with AD relevant processes. We highlight attempts to target the CRH signaling pathway as a therapeutic intervention in these transgenic mouse models and discuss how targeting this pathway is a promising avenue for further investigation.

Inhibition of glycogen synthase kinase-3 by BTA-EG 4 reduces tau abnormalities in an organotypic brain slice culture model of Alzheimer’s disease

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.

Novel monoclonal antibodies targeting the microtubule-binding domain of human tau

Tauopathies including Alzheimer’s disease and Progressive Supranuclear Palsy are a diverse group of progressive neurodegenerative disorders pathologically defined by inclusions containing aberrantly aggregated, post-translationally modified tau. The tau pathology burden correlates with neurodegeneration and dementia observed in these diseases. The microtubule binding domain of tau is essential for its physiological functions in promoting neuronal cytoskeletal stability, however it is also required for tau to assemble into an amyloid structure that comprises pathological inclusions. A series of novel monoclonal antibodies were generated which recognize the second and fourth microtubule-binding repeat domain of tau, thus enabling the identification specifically of 4-repeat tau versus 3-/4-repeat tau, respectively. These antibodies are highly specific for tau and recognize pathological tau inclusions in human tauopathies including Alzheimer’s disease and Progressive Supranuclear Palsy and in transgenic mouse models of tauopathies. These new antibodies will be useful for identifying and characterizing different tauopathies and as tools to target tau pathology in these diseases.

TARGETING TAU AGGREGATION IN BRAIN SLICE CULTURE MODELS OF TAUOPATHIES

Background:Amyloid-b (Ab) toxicity in Alzheimer’s disease (AD) is considered to be mediated by phosphorylated tau protein. Methods:Our study used mouse genetics, adeno-associated virus-mediated gene delivery, primary neuronal culture, biochemical, imaging and histological techniques as well as telemetric electroencephalography and behavioural tests including standard memory testing and touch screen operant chambers. Results: In contrast to previous assumptions on tau phosphorylation, we found that, at least in early disease, site-specific phosphorylation of tau inhibited Ab toxicity. This specific tau phosphorylation was mediated by the neuronal p38 mitogen-activated protein kinase p38g and interfered with postsynaptic excitotoxic signaling complexes engaged by Ab. Accordingly, depletion of p38g exacerbated neuronal circuit aberrations, cognitive deficits, and premature lethality in a mouse model of AD, whereas increasing the activity of p38g abolished these deficits. Furthermore, mimicking site-specific tau phosphorylation alleviated Abinduced neuronal death and offered protection from excitotoxicity. Conclusions: Our work provides insights into postsynaptic processes in AD pathogenesis and challenges a purely pathogenic role of tau phosphorylation in neuronal toxicity.

The amyloid-binding agent bta-eg4 reduces pathological tau species in a novel organotypic 3xTg-AD brain slice culture model that recapitulates key in vivo degenerative phenotypes

Background:The progressive development of amyloid and tau deposits, synaptic dysfunction and cognitive decline are well characterised in 3xTg-AD mice. 3xTg-AD mice thus provide an excellent model for testing the potential benefit of new disease-modifying treatments. Organotypic brain slice culture models provide an alternative to early stage in vivo testing since they recapitulate key disease features, and provide a higher throughput platform for drug screening. Here, we describe a novel organotypic 3xTg-AD brain slice culture model, and highlight its potential for researching the effects of disease-modifying agents such as the amyloid-binding tetra (ethylene glycol) derivative of benzothiazole aniline, BTAEG4. Methods: Organotypic brain slice cultures were prepared from neonatal 3xTg-AD and WT mice and cultured for several weeks. Slices were characterised for changes in total and phosphorylated tau and APP processing, with respect to aged mice. Slice cultures were treated with disease-modifying agents such as BTA-EG4 and the effects on abnormal tau species and APP processing were determined. Results:Organotypic brain slice cultures from 3xTgAD mice show elevated tau phosphorylation and the presence of high molecular weight tau species soon after culture, in comparison to slice cultures from WT mice. The novel amyloid-binding agent BTA-EG4, previously reported to reduce levels of beta-amyloid (Ab), increase dendritic spine density and ameliorate cognitive decline in mouse models of AD, was found here to also reduce the presence of abnormal tau species and other neurodegenerative features in 3xTg-AD brain slice cultures. Conclusions:We have successfully established a method to produce and maintain brain slices from 3xTg-AD mice in culture long-term. Our observations of rapid development of tau pathology in 3xTg-AD brain slices highlights the potential of this model as a more rapid and reliable in vitro method for drug screening prior to in vivo testing. Indeed, we demonstrate here novel tau-directed effects of BTA-EG4. The precise mode of action of this drug is currently being explored. Our findings support the further exploration of BTA-EG4 as a candidate therapeutic for Alzheimer’s disease.