P2X7R influences tau aggregate burden in human tauopathies and shows distinct signalling in microglia and astrocytes.

The purinoceptor P2X7R is a promising therapeutic target for tauopathies, including Alzheimer's disease (AD). Pharmacological inhibition or genetic knockdown of P2X7R ameliorates cognitive deficits and reduces pathological tau burden in mice that model aspects of tauopathy, including mice expressing mutant human frontotemporal dementia (FTD)-causing forms of tau. However, disagreements remain over which glial cell types express P2X7R and therefore the mechanism of action is unresolved. Here, we show that P2X7R protein levels increase in human AD post-mortem brain, in agreement with an upregulation of P2RX7 mRNA observed in transcriptome profiles from the AMP-AD consortium. P2X7R protein increases mirror advancing Braak stage and coincide with synapse loss. Using RNAScope we detect P2RX7 mRNA in microglia and astrocytes in human AD brain, including in the vicinity of senile plaques. In cultured microglia, P2X7R activation modulates the NLRP3 inflammasome pathway by promoting the formation of active complexes and release of IL-1ß. In astrocytes, P2X7R activates NFκB signalling and increases production of the cytokines CCL2, CXCL1 and IL-6 together with the acute phase protein Lcn2. To further explore the role of P2X7R in a disease-relevant context, we expressed wild-type or FTD-causing mutant forms of tau in mouse organotypic brain slice cultures. Inhibition of P2X7R reduces insoluble tau levels without altering soluble tau phosphorylation or synaptic localisation, suggesting a non-cell autonomous role of glial P2X7R on pathological tau aggregation. These findings support further investigations into the cell-type specific effects of P2X7R-targeting therapies in tauopathies.

Astrocytic C-X-C motif chemokine ligand-1 mediates ß-amyloid-induced synaptotoxicity.

BACKGROUND: Pathological interactions between ß-amyloid (Aß) and tau drive synapse loss and cognitive decline in Alzheimer's disease (AD). Reactive astrocytes, displaying altered functions, are also a prominent feature of AD brain. This large and heterogeneous population of cells are increasingly recognised as contributing to early phases of disease. However, the contribution of astrocytes to Aß-induced synaptotoxicity in AD is not well understood. METHODS: We stimulated mouse and human astrocytes with conditioned medium containing concentrations and species of human Aß that mimic those in human AD brain. Medium from stimulated astrocytes was collected and immunodepleted of Aß before being added to naïve rodent or human neuron cultures. A cytokine, identified in unbiased screens of stimulated astrocyte media and in postmortem human AD brain lysates was also applied to neurons, including those pre-treated with a chemokine receptor antagonist. Tau mislocalisation, synaptic markers and dendritic spine numbers were measured in cultured neurons and organotypic brain slice cultures. RESULTS: We found that conditioned medium from stimulated astrocytes induces exaggerated synaptotoxicity that is recapitulated following spiking of neuron culture medium with recombinant C-X-C motif chemokine ligand-1 (CXCL1), a chemokine upregulated in AD brain. Antagonism of neuronal C-X-C motif chemokine receptor 2 (CXCR2) prevented synaptotoxicity in response to CXCL1 and Aß-stimulated astrocyte secretions. CONCLUSIONS: Our data indicate that astrocytes exacerbate the synaptotoxic effects of Aß via interactions of astrocytic CXCL1 and neuronal CXCR2 receptors, highlighting this chemokine-receptor pair as a novel target for therapeutic intervention in AD.

Stress-Related Immune Markers in Depression: Implications for Treatment.

Major depression is a serious psychiatric disorder; however, the precise biological basis of depression still remains elusive. A large body of evidence implicates a dysregulated endocrine and inflammatory response system in the pathogenesis of depression. Despite this, given the heterogeneity of depression, not all depressed patients exhibit dysregulation of the inflammatory and endocrine systems. Evidence suggests that inflammation is associated with depression in certain subgroups of patients and that those who have experienced stressful life events such as childhood trauma or bereavement may be at greater risk of developing depression. Consequently, prolonged exposure to stress is thought to be a key trigger for the onset of a depressive episode. This review assesses the relationship between stress and the immune system, with a particular interest in the mechanisms by which stress impacts immune function, and how altered immune functioning, in turn, may lead to a feed forward cascade of multiple systems dysregulation and the subsequent manifestation of depressive symptomology. The identification of stress-related immune markers and potential avenues for advances in therapeutic intervention is vital. Changes in specific biological markers may be used to characterize or differentiate depressive subtypes or specific symptoms and may predict treatment response, in turn facilitating a more effective, targeted, and fast-acting approach to treatment.

C-reactive protein predicts fatigue independently of depression in breast cancer patients prior to chemotherapy.

Heightened inflammatory activity has been proposed as a mechanism for the development of cancer-related fatigue (CRF), a common and distressing condition that can negatively affect quality of life. Inflammation is also implicated in the pathogenesis of depression, and depression is a strong predictor of CRF. Thus, the role of the pro-inflammatory cytokine network in CRF may be mediated by depression or both conditions may share similar underlying physiological processes. The current study investigated associations between fatigue, depression and inflammatory cytokine (IFN-γ, IL-6, TNF-α) and CRP concentrations, as well as kynurenine pathway (KP) activation, in 61 breast cancer patients prior to chemotherapy. Changes in inflammatory markers and KP activation over time were also explored, and associations with changes in fatigue and depression were examined. Higher levels of CRP were significantly correlated with fatigue and depression before chemotherapy; nevertheless, CRP predicted fatigue independently of depression. Although greater kynurenine concentrations were associated with increased immune activation, there was no evidence that the KP played a role in fatigue or depression. Furthermore, no relationships emerged between either fatigue or depression and IFN-γ, IL-6, or TNF-α before chemotherapy. Nevertheless, kynurenine levels pre- and post-treatment significantly predicted changes in depression, suggesting that heightened KP activation may contribute to depressive symptoms in patients treated for cancer. In addition, IL-6 significantly covaried with fatigue. These preliminary findings provide some support for the idea that low-grade inflammation contributes to the development of CRF, independently of depression; however, there was no evidence that this is mediated by KP activity.

Tryptophan depletion in depressed patients occurs independent of kynurenine pathway activation.

The kynurenine pathway (KP) and its rate-limiting tryptophan degrading enzyme indolamine 2,3-dioxygenase (IDO) have been implicated in the pathogenesis of depression. IDO expression is driven by inflammatory cytokines, and has been suggested as the link between inflammation and a serotonergic deficit in depression. Studies also indicate that inflammatory cytokines upregulate the serotonin transporter (SERT), representing another mechanism by which inflammation could influence serotonin availability. Here we examined circulating concentrations of inflammatory cytokines (IFN-γ, TNF-α, IL-1ß, IL-6), and the acute phase protein CRP alongside plasma tryptophan, kynurenine, kynurenic acid (KYNA) and 3-hydroxyanthranilic acid (3-HAA) concentrations, and whole blood mRNA expression of IDO, kynurenine aminotransferases (KAT I and II), kynurenine-3-monooxygenase (KMO), kynureninase and SERT in patients with major depressive disorder (MDD) compared with age and sex-matched controls. Whilst no changes in TNF-α or IL-1ß were observed, plasma concentrations of IL-6, IFN-γ and CRP were increased in the depressed cohort. Despite this inflammatory phenotype, IDO expression or plasma kynurenine were not significantly different between MDD patients and controls. In addition, there was no difference between controls and depressives in concentrations of KYNA and 3-HAA, or in expression of enzymes KAT, KMO or kynureninase that drive their production. Nonetheless, a depletion in tryptophan was evident in depressed patients and was correlated with HAM-D scores. In addition, we failed to observe any difference in SERT mRNA expression in the blood cells from patients with MDD relative to controls. These data support the idea that a mild inflammatory signature is evident in MDD and is accompanied by reduced circulating tryptophan concentrations. However, we found no indication of KP activation in the depressed cohort suggesting that an alternative mechanism mediates the depletion of tryptophan observed. Taken together these data question the ability of the mild inflammatory phenotype observed in depression to induce molecules such as IDO and SERT that could negatively impact upon serotonergic functioning.

Microglia in the degenerating brain are capable of phagocytosis of beads and of apoptotic cells, but do not efficiently remove PrPSc, even upon LPS stimulation.

Despite the phagocytic machinery available to microglia the aberrant amyloid proteins produced during Alzheimer's and prion disease, amyloid-ß and PrP(Sc), are inefficiently cleared. We have shown that microglia in the ME7 model of prion disease show morphological evidence of activation, synthesize low levels of pro-inflammatory cytokines and are primed to produce exaggerated responses to subsequent inflammatory challenges. Whether these microglia engage in significant phagocytic activity in the disease per se, or upon subsequent inflammatory challenge is not clear. In the present study we show transcriptional activation of a large number of scavenger receptors (SRs), matrix metalloproteinases (MMPs), oxidative enzymes, and cathepsins in ME7 animals. Hippocampally-injected inert latex beads (6 µm) are efficiently phagocytosed by microglia of ME7 prion-diseased animals, but not by microglia in normal animals. Stimulation of ME7 animals with systemic bacterial endotoxin (lipopolysaccharide, LPS) induced further increases in SR-A2, MMP3, and urokinase plasminogen activator receptor (uPAR) but decreased, or did not alter, transcription of most phagocytosis-related genes examined and did not enhance clearance of deposited PrP(Sc). Furthermore, intracerebral injection with LPS (0.5 µg) induced marked microglial production of IL-1ß, robust cellular infiltration and marked apoptosis but also did not induce further clearance of PrP(Sc). These data indicate that microglia in the prion-diseased brain are capable of phagocytosis per se, but show limited efficacy in removing PrP(Sc) even upon marked escalation of CNS inflammation. Furthermore, microglia/macrophages remain IL-1ß-negative during phagocytosis of apoptotic cells. The data demonstrate that phagocytic activity and pro-inflammatory microglial phenotype do not necessarily correlate.

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 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.

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

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.