Biology subjects

King, A. E.

Publications and source records attributed to King, A. E..

5 recordsLinked to original sources

bioRxiv2023

HDAC6 inhibition as a mechanism to prevent axon degeneration in the mSOD1G93A mouse model of ALS.

The loss of upper and lower motor neurons, and their axons is central to the loss of motor function and death in amyotrophic lateral sclerosis (ALS). Due to the diverse range of genetic and environmental factors that contribute to the pathogenesis of ALS, there have been difficulties in developing effective therapies for ALS. One dichotomy emerging in the field is that protection of the neuronal cell soma itself does not prevent axonal vulnerability and degeneration, suggesting the need for targeted therapeutics to prevent axon degeneration. Post-translational modifications of protein acetylation can alter the function, stability and half-life of individual proteins, and can be enzymatically modified by histone acetyltransferases (HATs) and histone deacetyltransferases (HDACs), which add, or remove acetyl groups, respectively. Maintenance of post-translational microtubule acetylation has been suggested as a potential mechanism to stabilise axons and prevent axonal loss and neurodegeneration in ALS. This study has utilized an orally dosed HDAC6 specific inhibitor, ACY-738, prevent deacetylation and stabilize microtubules in the mSOD1G93A mouse model of ALS. Furthermore, co-treatment with riluzole was performed to determine any effects or drug interactions and potentially enhance preclinical research translation. This study shows ACY-738 treatment increased acetylation of microtubules in the spinal cord of mSOD1G93A mice, reduced lower motor neuron degeneration in the lumbar spinal cord of female mice, ameliorated reduction in peripheral nerve axon puncta size, but did not prevent overt motor function decline. The current study also shows peripheral nerve axon puncta size to be partially restored after treatment with riluzole and highlights the importance of co-treatment to measure the potential effects of therapeutics in ALS. HighlightsO_LIACY-738 inhibits HDAC6 and leads to increased microtubule acetylation in spinal cord of mSOD1G93A mice. C_LIO_LIACY-738 treatment reduces lower motor neuron degeneration in the lumbar spinal cord of mSOD1G93A mice. C_LIO_LIACY-738 treatment restores peripheral nerve axon puncta size of mSOD1G93A mice. C_LIO_LIACY-738 treatment does not prevent overt motor function decline mSOD1G93A mice. C_LIO_LIRiluzole treatment partially restores peripheral nerve axon puncta size in mSOD1G93A mice. C_LI

neuroscience↗

bioRxiv2022

Microglia associations with brain pericytes and the vasculature are reduced in Alzheimer's disease

Cerebral blood flow is important for the maintenance of brain function and its dysregulation has been implicated in Alzheimers disease (AD). Subpopulations of microglia have well-characterised associations with the vasculature in the central nervous system but the precise relationship between microglia and cells which exist on the vasculature is not yet clear. In this study we explored the relationship between microglia and pericytes, a vessel-resident cell type that has a major role in the regulation of cerebral blood flow and maintenance of the blood brain barrier. Using fixed tissue sections and in vivo live imaging, we discovered a subset of microglia that closely associated with pericytes, termed PEricyte-associated Microglia (PEM). PEM are present throughout all regions of the brain and spinal cord in NG2DsRed x CX3CR1+/GFP mice, and in the human frontal cortex. They reside adjacent to pericytes at all levels of the capillary tree and can maintain their position for at least 28 days. PEM associate with pericytes lacking astroglial endfeet coverage but are segregated from pericytes by capillary basement membranes and capillary vessel width is similarly increased beneath pericytes with or without an associated PEM. Deletion of the microglia fractalkine receptor (CX3CR1) did not disrupt the association between pericytes and PEM, suggesting the association is not reliant on fractalkine signalling. Finally, we found that the proportion of microglia that are capillary-associated and PEM declines in the superior frontal gyrus (SFG) in AD, which is exacerbated by the APOE {varepsilon}3/{varepsilon}4 genotype. In summary, we identify and characterise a subpopulation of microglia that specifically associate with pericytes and find this population is reduced in the SFG in AD. This reduction may be a novel mechanism contributing to vascular dysfunction in diseases such as AD.

neuroscience↗

bioRxiv2022

Lysosomal alterations and decreased electrophysiological activity in CLN3 disease (966 bp deletion, E295K) patient-derived cortical neurons

CLN3 disease is a lysosomal storage disorder associated with fatal neurodegeneration that is caused by mutations in CLN3. Most individuals with CLN3 disease carry at least one allele with a 966 bp deletion in CLN3 which results in the deletion of exons 7 and 8. There is a need for more physiologically relevant human cell-based CLN3 disease models to better understand the cellular changes during the disease process. Using CRISPR/Cas9, we corrected the 966 bp deletion mutation in human induced pluripotent stem cells (iPSCs) of a compound heterozygous patient (CLN3 {Delta} 966 bp and E295K). The isogenic deletion-corrected and unedited CLN3 patient iPSCs were used for disease modeling. iPSC-derived neurons carrying this particular CLN3 mutation (CLN3 neurons) had lower functional activity as recorded using microelectrode arrays for most of the culture period. Proteomics analysis showed downregulation of proteins related to axon guidance and endocytosis at day in vitro (DIV) 14 and 42 in CLN3 neurons. This was accompanied by an increase in lysosomal-related proteins in CLN3 neurons. Western blot analysis revealed hyperglycosylation of the lysosomal marker, Lysosome Associated Membrane Protein 1 (LAMP1) in CLN3 neurons at DIV 14, 28 and 42, which was not apparent in control neurons. Ultrastructural analysis of CLN3 neurons showed numerous membrane-bound vacuoles containing diverse types of storage material, ranging from curvilinear deposits, multilamellar structures to osmiophilic deposits. Our findings suggest alterations in lysosomal function and neurodevelopment involving axon guidance and synaptic transmission in CLN3-deficient neuronal derivatives, which could be potential targets for therapy.

neuroscience↗

bioRxiv2020

Enhanced anti-amyloid effect of combined leptin and pioglitazone in APP/PS1 transgenic mice

BackgroundAlzheimers disease (AD) has challenged single-target therapeutic strategies, raising the possibility that combined therapies may offer a more effective treatment strategy. ObjectiveThere is substantial evidence for the efficacy of leptin (L) (neuroprotective hormone) and pioglitazone (P) (anti-inflammatory agent) as monotherapies in AD. We have previouly shown that combination treatment of L+P in APP/PS1 mice at the onset of pathology significantly improved memory and reduced brain A{beta} levels relative to control mice. In this new study, we sought to replicate our previous findings in a new cohort of APP/PS1 mouse to further confirm whether the combined treatment of L+P is superior to each treatment individually. MethodsWe have re-evaluated the effects of L+P co-treatment in APP/PS1 mice using thioflavin-S staining, MOA{beta} immunolabeling and enzyme-linked immunosorbent assay (ELISA) to examine effects on A{beta} levels and pathology, relative to animals that received L or P individually. To explore mechanism of regulation, we used Western blotting to examine the expression of the peroxisome-proliferator activated receptor {gamma} (PPAR{gamma}), due to its potential role in the regulation of the inflammatory response. ResultsWe demonstrated that combining L and P significantly enhances the anti-A{beta} effect of L or P in the hippocampus of APP/PS1 mice. Western blot analysis indicated that A{beta} reduction was accompanied by up-regulation of the PPAR{gamma} levels. ConclusionOur findings suggest that combining L and P significantly enhances the anti-A{beta} effect of L or P in the hippocampus of APP/PS1 mice, and may be a potential new effective strategy for AD therapy.

neuroscience↗

bioRxiv2018

Efficacy and dynamics of self-targeting CRISPR/Cas constructs for gene editing in the retina.

Safe delivery of CRISPR/Cas endonucleases remains one of the major barriers to the widespread application of in vivo genome editing including the anticipatory treatment of monogenic retinal diseases. We previously reported the utility of adeno-associated virus (AAV)-mediated CRISPR/Cas genome editing in the retina; however, with this type of viral delivery system, active endonucleases will remain in the retina for an extended period, making genotoxicity a significant consideration in clinical applications. To address this issue, we have designed a self-destructing \"kamikaze\" CRISPR/Cas system that disrupts the Cas enzyme itself following expression. Four guide RNAs (sgRNAs) were designed to target Streptococcus pyogenes Cas9 (SpCas9), after in situ validation, the selected sgRNAs were cloned into a dual AAV vector. One construct was used to deliver SpCas9 and the other delivered sgRNAs directed against SpCas9 and the target locus (yellow fluorescent protein, YFP), in the presence of mCherry. Both constructs were packaged into AAV2 vector and intravitreally administered in C57BL/6 and Thy1-YFP transgenic mice. After 8 weeks the expression of SpCas9, the efficacy of YFP gene disruption was quantified. A reduction of SpCas9 mRNA was found in retinas treated with AAV2-mediated-YFP/SpCas9 targeting CRISPR/Cas compared to those treated with YFP targeting CRISPR/Cas alone. We also show that AAV2-mediated delivery of YFP/SpCas9 targeting CRISPR/Cas significantly reduced the number of YFP fluorescent cells among mCherry-expressing cells (~85.5% reduction compared to LacZ/SpCas9 targeting CRISPR/Cas) in transfected retina of Thy1-YFP transgenic mice. In conclusion, our data suggest that a self-destructive \"kamikaze\" CRISPR/Cas system can be used as a robust tool for refined genome editing in the retina, without compromising on-target efficiency.

genetics↗