Biology subjects

Whiting, P.

Publications and source records attributed to Whiting, P..

7 recordsLinked to original sources

bioRxiv2024

Mutations in PSEN1 predispose inflammation in an astrocyte model of familial Alzheimer's disease through disrupted regulated intramembrane proteolysis

Mutations in PSEN1 cause familial Alzheimers disease with almost complete penetrance. Age at onset is highly variable between different PSEN1 mutations and even within families with the same mutation. Current research into late onset Alzheimers disease implicates inflammation in both disease onset and progression. PSEN1 is the catalytic subunit of {gamma}-secretase, responsible for regulated intramembrane proteolysis of numerous substrates that include cytokine receptors. For this reason, we tested the hypothesis that mutations in PSEN1 impact inflammatory responses in astrocytes, thereby contributing to disease progression. Here, using iPSC-astrocytes, we show that PSEN1 is upregulated in response to inflammatory stimuli, and this upregulation is disrupted by pathological PSEN1 mutations. Using transcriptomic analyses, we demonstrate that PSEN1 mutant astrocytes have an augmented inflammatory profile in their basal state, concomitant with an upregulation of genes coding for regulated intramembrane proteolytic and robust activation of JAK-STAT signalling. Using JAK-STAT2 as an example signalling pathway, we show altered phosphorylation cascades in PSEN1 mutant astrocytes, reinforcing the notion of altered cytokine signalling cascades. Finally, we use small molecule modulators of {gamma}-secretase to confirm a role for PSEN1/{gamma}-secretase in regulating the astrocytic response to inflammatory stimuli. Together, these data suggest that mutations in PSEN1 enhance cytokine signalling via impaired regulated intramembrane proteolysis, thereby predisposing astrocytic inflammatory profiles. These findings support a two-hit contribution of PSEN1 mutations to fAD pathogenesis, not only impacting APP and A{beta} processing but also altering the cellular response to inflammation.

neuroscience↗

bioRxiv2024

Sodium channel inhibitors alter the progress of tangle development in a mouse model of dementia

Sodium channel inhibitors have been reported to protect against a range of neuroinflammatory and neurodegenerative diseases. Here the effect of chronic administration of two Na+ channel inhibitors with different mechanisms of action, phenytoin and GS967 are tested in mouse models of different stages of Alzheimers disease. Subtle changes in the distribution of plaque sizes were observed in AppNLGF/NLGF mouse at 3 months of age, after being fed control or drug-supplemented chow from weaning onwards, with phenytoin treatment resulting in a significant increase in the frequency of the smallest plaques and a decrease in large plaques. The later pathology of neurofibrillary tangles was studied, in old age, by supplementing the food of transgenic mice with a P301L mutation in Tau. Chronic administration of Na+ inhibitors from 15 months of age resulted in a decrease in the density of MC1-positive neurofibrillary tangles, possibly due to effects on microglial Na+ channels. The density of microglial cells was strongly correlated with the density of neurofibrillary tangles but only in mice treated with the Na+ inhibitors.

neuroscience↗

bioRxiv2024

Identification of novel small molecule chaperone activators for neurodegenerative disease treatment

A pathological hallmark of neurodegenerative disease is the accumulation of aberrant protein aggregates which contribute to the cytotoxicity and are therefore a target for new therapy development. One key machinery to manage cellular protein homeostasis is the chaperones proteins, the heat shock proteins (HSPs) which are known to target aberrant proteins including TDP-43, tau and amyloid and rescue neurodegeneration in various disease models. As an attempt to target HSP activation for neurodegeneration therapy, we develop a drug screening assay to search compounds that activate the master regulator of HSPs, the transcription factor heat shock factor 1 (HSF1). As HSF1 is bound by HSP90 which prevents its activation, we develop a NanoBRET assay, which allows us to monitor and quantify the HSF1-HSP90 interaction in living cells to screen for compounds disrupting this interaction and thereby releasing HSF1 for activation. After the assay was optimised and validated for its robustness and reliability, a two thousand compound library was screened which produced 10 hits including a couple of known HSP90 inhibitors. Follow-up functional study showed that one of the hit oxyphenbutazone (OPB) significantly reduces the accumulation of insoluble TDP-43 in a cell model, displaying no signs of stress or toxicity. Further works are needed to characterise the mode-of-action of OPB in HSF1 activation and aberrant protein reduction, and the physiological impact of OPB on TDP-43 needs to be examined in an in vivo model. However, this study demonstrates a viable strategy for new drug discovery in targeting aberrant proteins and identifies potential candidates for translation into neurodegenerative disease treatment.

molecular biology↗

bioRxiv2022

A primary rodent triculture model to investigate the role of glia-neuron crosstalk in regulation of neuronal activity.

Neuroinflammation and hyperexcitability have been implicated in the pathogenesis of neurodegenerative disease, and new models are required to investigate the cellular crosstalk involved in these processes. We developed an approach to generate a quantitative and reproducible triculture system that is suitable for pharmacological studies. While primary rodent cells were previously grown in a coculture medium formulated to support only neurons and astrocytes, we now optimised a protocol to generate tricultures containing neurons, astrocytes and microglia by culturing in a medium designed to support all three cell types and adding exogenous microglia to cocultures. Immunocytochemistry was used to confirm the intended cell types were present. The percentage of ramified microglia in the tricultures decreases as the number of microglia present increases. Multi-electrode array (MEA) recordings indicate that microglia in the triculture model suppress neuronal activity in a dose-dependent manner. Neurons in both cocultures and tricultures were responsive to the potassium channel blocker 4-aminopyridine (4-AP) suggesting that neurons remained viable and functional in the triculture model. Furthermore, suppressed neuronal activity in tricultures correlated with decreased densities of dendritic spines and of the postsynaptic protein Homer1 along dendrites, indicative of a direct or indirect effect of microglia on synapse function. We thus present a functional triculture model, which, due to its more complete cellular composition, is a more relevant model than standard cocultures. The model can be used to probe glia-neuron interactions and subsequently aid the development of assays for drug discovery, using neuronal excitability as a functional endpoint.

neuroscience↗

bioRxiv2021

Epigenetic repression of Wnt receptors in AD: a role for Sirtuin2-induced H4K16ac deacetylation of Frizzled1 and Frizzled7 promoters

Growing evidence supports a role for deficient Wnt signalling in Alzheimer's disease (AD). First, the Wnt antagonist DKK1 is elevated in AD brains and is required for amyloid-{beta}-induced synapse loss. Second, LRP6 Wnt co-receptor is required for synapse integrity and three variants of this receptor are linked to late-onset AD. However, the expression/role of other Wnt signalling components remain poorly explored in AD. Wnt receptors Frizzled1 (Fzd1), Fzd5, Fzd7 and Fzd9 are of interest due to their role in synapse formation/plasticity. Our analyses showed reduced FZD1 and FZD7 mRNA levels in the hippocampus of human early AD stages and in the hAPPNLGF/NLGF mouse model. This transcriptional downregulation was accompanied by reduced levels of the pro-transcriptional histone mark H4K16ac and a concomitant increase of its deacetylase Sirt2 at Fzd1 and Fzd7 promoters in AD. In vitro and in vivo inhibition of Sirt2 rescued Fzd1 and Fzd7 mRNA expression and H4K16ac levels at their promoters. In addition, we showed that Sirt2 recruitment to Fzd1 and Fzd7 promoters is dependent on FoxO1 activity in AD, thus acting as a co-repressor. Finally, we found reduced levels of Sirt2 inhibitory phosphorylation in nuclear samples from human early AD stages with a concomitant increased in the Sirt2 phosphatase PP2C. This results in hyperactive nuclear Sirt2 and favours Fzd1 and Fzd7 repression in AD. Collectively, our findings define a novel role for nuclear hyperactivated Sirt2 in repressing Fzd1 and Fzd7 expression via H4K16ac deacetylation in AD. We propose Sirt2 as an attractive target to ameliorate AD pathology.

neuroscience↗

bioRxiv2020

Regulation of mitophagy by the NSL complex underlies genetic risk for Parkinson's disease at Chr16q11.2 and on the MAPT H1 allele.

Parkinsons disease (PD) is a common incurable neurodegenerative disease. The identification of genetic variants via genome-wide association studies (GWAS) has considerably advanced our understanding of the PD genetic risk. Understanding the functional significance of the risk loci is now a critical step towards translating these genetic advances into an enhanced biological understanding of the disease. Impaired mitophagy is a key causative pathway in familial PD, but its relevance to idiopathic PD is unclear. We used a mitophagy screening assay to evaluate the functional significance of risk genes identified through GWAS. We identified two new regulators of PINK1-mitophagy, KAT8 and KANSL1, previously shown to modulate lysine acetylation. We show that KAT8 and KANSL1 modulate PINK1 gene expression and subsequent PINK1-mitophagy. These findings suggest PINK1-mitophagy is a contributing factor to idiopathic PD. KANSL1 is located on chromosome 17q21 where the risk associated gene has long been considered to be MAPT. While our data does not exclude a possible association between the MAPT gene and PD, it provides strong evidence that KANSL1 plays a crucial role in the disease. Finally, these results enrich our understanding of physiological events regulating mitophagy and establish a novel pathway for drug targeting in neurodegeneration.

neuroscience↗

bioRxiv2018

Alzheimer’s disease Phospholipase C-gamma-2 (PLCG2) protective variant is a functional hypermorph

Recent Genome Wide Association Studies (GWAS) have identified novel rare coding variants in immune genes associated with late onset AD (LOAD). Amongst these, a polymorphism in Phospholipase C-gamma 2 (PLCG2) P522R, has been reported to be protective against LOAD. PLC enzymes are key elements in signal transmission networks and are potentially druggable targets. PLCG2 is highly expressed in the hematopoietic system. Hypermorphic mutations in PLCG2 in humans have been reported to cause autoinflammation and immune disorders, suggesting a key role for this enzyme in the regulation of immune cell function.\n\nWe confirmed that PLCG2 expression is restricted primarily to microglia in both the healthy and AD brain. Functional analysis of the P522R variant in heterologous systems demonstrated a small hypermorphic effect of the mutation on enzyme function. PLC{gamma}2 is therefore a potential target for modulating microglia function in AD, and a small molecule drug that weakly activates PLC{gamma}2 may be one potential therapeutic approach.\n\nSUMMARYThe PLCG2 P522R variant is protective against Alzheimers disease (AD). We show that PLCG2 is expressed in CNS-resident myeloid cells, and the P522R polymorphism weakly activates enzyme function. These data suggest that activation of PLCG2 and not inhibition could be therapeutically beneficial in AD.

neuroscience↗