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Mortiboys, H. J.

Publications and source records attributed to Mortiboys, H. J..

8 recordsLinked to original sources

Identification and small molecule rescue of mitochondrial dysfunction phenotype which converges across Leigh Syndrome and Huntington's Disease patient fibroblasts.

Mitochondrial dysfunction is implicated in a variety of complex neurological disorders. Primary mitochondrial diseases are caused directly by mutations in genes encoding mitochondrial proteins, leading to mitochondrial dysfunction and disease. Mitochondrial dysfunction is also a key contributor to pathogenesis in multiple neurodegenerative diseases. Rescue of mitochondrial function is therefore an attractive therapeutic target in both groups of diseases. In this study, we used primary fibroblasts derived from patients with the primary mitochondrial disease Leigh syndrome (LS) and the neurodegenerative disease Huntingtons disease (HD) to investigate mitochondrial phenotypes in these patients. We used these to identify modifiable measures of mitochondrial phenotype using a high content imaging screen. Despite having distinct underlying disease causes, different mitochondrial phenotypes in LS and HD patient derived cells converged on an imbalance between functional and dysfunctional mitochondria. Through multi-parameter screening of the mitochondrial phenotype we identified the AMPK activator A769662 as a small molecule able to rescue this imbalance in both LS and HD patient derived fibroblasts via different pathways. Our findings indicate that high throughput screening for mitochondrial phenotypes could identify novel therapeutic agents to rescue mitochondrial dysfunction in complex neurological disorders. Research in ContextO_ST_ABSEvidence before this studyC_ST_ABSMitochondrial dysfunction is the primary driver of mitochondrial disease and key contributing factor to neurodegenerative disease pathogenesis. Rescuing mitochondrial function is a promising therapeutic strategy, yet strategies to identify mitochondrial modulators in patient cells are limited. Added value of this studyOur in-depth mitochondrial characterisation of Leigh syndrome and Huntingtons disease patient fibroblasts shows the potential for using this methodology to identify mitochondrial therapeutics. We identify a mitochondrial phenotype common across diseases and an AMPK activator capable of rescuing this phenotype. Implications of all the available evidenceOur work extends our understanding of the mitochondrial dysfunction associated with Leigh syndrome and Huntingtons disease and expands the tool set available for identifying modulators of mitochondrial health as potential therapeutics for complex neurological disorders.

neuroscience↗

Secondary nucleation of α-Synuclein drives Mitochondria dysfunctions and Lewy body formation in Parkinson's Disease

The seeding of -Synuclein (Syn) is a key driver of Lewy pathology propagation in Parkinsons disease (PD) and forms the basis for recent diagnostic advances. However, it remains unclear how the structural and biochemical features of Syn seeds dictate their propagation efficiency, capacity to induce Lewy body formation, and resulting cellular toxicity. Using genetic and idiopathic PD cell models, we map the pathogenic cascade beginning with the seed-driven conversion of endogenous Syn, followed by impaired degradation, mitochondrial dysfunction, and ultimately Lewy body formation. By coupling kinetic modelling of aggregation with functional readouts, we identify secondary nucleation as the predominant mechanism generating toxic Syn aggregation intermediates, identifying the critical process that links seeding to pathology. Extending this framework to PD brain, we quantitatively correlate seeding capacity with the spatiotemporal spread and severity of Lewy pathology, revealing a mechanistic connection between Syn aggregation dynamics and disease progression at molecular, cellular, and anatomical levels. By unifying molecular mechanism with clinicopathological progression, our work identifies catalytic Syn fibrillar seeds as tractable targets for both disease-modifying therapy and biomarker development in PD. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=193 HEIGHT=200 SRC="FIGDIR/small/676873v1_ufig1.gif" ALT="Figure 1"> View larger version (79K): org.highwire.dtl.DTLVardef@1eca299org.highwire.dtl.DTLVardef@a714d4org.highwire.dtl.DTLVardef@1486db5org.highwire.dtl.DTLVardef@1aa895_HPS_FORMAT_FIGEXP M_FIG C_FIG HighlightsO_LISyn fibril-oligomer interplay drives mitochondrial abnormalities and Lewy pathology C_LIO_LIFibrillar Syn catalyse toxic aggregate formations via secondary nucleation C_LIO_LIPhosphorylated Syn evades lysosomal clearance and drives enhanced dysfunctions C_LIO_LISeeding capacity of Syn predicts Lewy pathology burden and disease progression C_LI

neuroscience↗

C9orf72-ALS mutation drives mitophagy impairments in iNeurons

IntroductionALS is a neurodegenerative disorder characterised by progressive upper and lower motor neuron loss. A GGGGCC hexanucleotide repeat expansion (HRE) in the C9orf72 gene is the most common mutation found in populations of European descent. Mitochondrial dysfunction has been observed in C9orf72-ALS patients and models of the disease, however reports on mitochondrial clearance via mitophagy in C9orf72-ALS are limited. ResultsiNeurons from C9orf72-ALS patients displayed reduced mitochondrial membrane potential and reduced mitophagy, due to reductions in autophagosome production and reduced ULK1 recruitment to mitochondria. No consistent changes to PINK1/Parkin or BNIP3 mitophagy pathways were observed. ConclusionsOur data show that mitochondrial function is impaired in C9orf72-ALS patient iNeurons. An in-depth characterisation of mitophagy suggests that a deficit in autophagosome production is responsible and provides further evidence that toxic gain-of-function mechanisms in C9orf72-ALS are responsible for autophagy deficits.

neuroscience↗

The Parkinson's Disease related mutant VPS35 (D620N) amplifies the LRRK2 response to endolysosomal stress

The identification of multiple genes linked to Parkinsons Disease invites the question as to how they may cooperate. We have generated isogenic cell lines that inducibly express either wild-type or a mutant form of the retromer component VPS35 (D620N), which has been linked to Parkinsons Disease. This has enabled us to test proposed effects of this mutation in a setting where the relative expression reflects the physiological occurrence. We confirm that this mutation compromises VPS35 association with the WASH complex, but find no defect in WASH recruitment to endosomes, nor in the distribution of lysosomal receptors, cation-independent mannose-6-phosphate receptor and Sortilin. We show VPS35 (D620N) enhances the activity of the Parkinsons associated kinase LRRK2 towards RAB12 under basal conditions. Furthermore, VPS35 (D620N) amplifies the LRRK2 response to endolysosomal stress resulting in enhanced phosphorylation of RABs 10 and 12. By comparing different types of endolysosomal stresses such as the ionophore nigericin and the membranolytic agent LLOMe, we are able to dissociate phospho-RAB accumulation from membrane rupture.

cell biology↗

Deficits in mitochondrial function and glucose metabolism seen in sporadic and familial Alzheimers disease derived Astrocytes are ameliorated by increasing hexokinase 1 expression.

BackgroundAstrocytes have multiple roles including providing neurons with metabolic substrates and maintaining neurotransmitter synaptic homeostasis. Astrocyte glucose metabolism plays a key role in learning and memory with astrocytic glycogen a key substrate supporting memory encoding. The neuronal support provided by astrocytes has a high metabolic demand. Deficits in astrocytic mitochondrial metabolic functioning and glycolysis could impair neuronal function. Changes to cellular metabolism are seen early in Alzheimers disease (AD). Understanding cellular metabolism changes in AD astrocytes could be exploited as a new biomarker or synergistic therapeutic agent when combined with anti-amyloid treatments in AD. MethodsIn this project, we characterised mitochondrial and glycolytic function in astrocytes derived from patients with sporadic (n=6) and familial (PSEN1, n=3) forms of AD. Astrocytes were derived using direct reprogramming methods. Astrocyte metabolic outputs: ATP, and extracellular lactate levels were measured using luminescent and fluorescent protocols. Mitochondrial respiration and glycolytic function were measured using a Seahorse XF Analyzer. Hexokinase deficits identified where corrected by transfecting astrocytes with an adenovirus viral vector containing the hexokinase 1 gene. ResultsThere was a reduction of total cellular ATP of 20% (p=0.05 in sAD astrocytes) and of 48% (p<0.01) in fAD. A 44% reduction (p<0.05), and 80% reduction in mitochondrial spare capacity was seen in sAD and fAD astrocytes respectively. Reactive oxygen species (ROS) were increased in both AD astrocyte types (p=0.05). Mitochondrial complex I and II was significantly increased in sAD (p<0.05) but not in fAD. Astrocyte glycolytic reserve and extracellular lactate was significantly reduced when compared to controls in both sAD and fAD (p<0.05). We identified a deficit in the glycolytic pathway enzyme hexokinase, and correcting this deficit restored most of the metabolic phenotype in sAD but not fAD astrocytes. ConclusionAD astrocytes have abnormalities in functional capacity of mitochondria and the process of glycolysis. These functional deficits can be improved by correcting hexokinase expression deficits with adenoviral vectors. This suggests that hexokinase 1 deficiency could potentially be exploited as a new therapeutic target for AD.

neuroscience↗

FBXL4 deficiency promotes mitophagy by elevating NIX.

The selective autophagy of mitochondria is linked to mitochondrial quality control and is critical to a healthy organism. We have conducted a CRISPR/Cas9 screen of human E3 ubiquitin ligases for influence on mitophagy under both basal cell culture conditions and following acute mitochondrial depolarisation. We identify two Cullin RING ligases, VHL and FBXL4 as the most profound negative regulators of basal mitophagy. We show that these converge through control of the mitophagy adaptors BNIP3 and BNIP3L/NIX through different mechanisms. FBXL4 suppression of BNIP3 and NIX levels is mediated via direct interaction and protein destabilisation rather than suppression of HIF1-mediated transcription. Depletion of NIX but not BNIP3 is sufficient to restore mitophagy levels. Our study enables a full understanding of the aetiology of early onset mitochondrial encephalomyopathy that is supported by analysis of a disease associated mutation. We further show that the compound MLN4924, which globally interferes with Cullin RING ligase activity, is a strong inducer of mitophagy providing a research tool in this context and a candidate therapeutic agent for conditions linked to mitochondrial dysfunction.

cell biology↗

A p21-GFP zebrafish model of senescence for rapid testing of senolytics in vivo.

Senescence drives the onset and severity of multiple ageing-associated diseases as well as frailty. As a result, there has been an increased interest in mechanistic studies and in the search for compounds targeting senescent cells, known as senolytics. Mammalian models are commonly used to test senolytics and generate functional and toxicity data at the level of organs and systems, yet this is expensive and time consuming. Zebrafish share high homology in genes associated with human ageing and disease. They can be genetically-modified relatively easily. In larvae, most organs develop within 5 days of fertilisation and are transparent, which allows tracking of fluorescent cells in vivo in real time, testing drug off-target toxicity and assessment of cellular and phenotypic changes. Here, we have generated a transgenic zebrafish line that expresses green fluorescent protein (GFP) under the promoter of a key senescence marker, p21. We show an increase in p21:GFP+ cells in larvae following exposure to ionising radiation and with natural ageing. p21:GFP+ cells display other markers of senescence, including senescence-associated {beta}-galactosidase and IL6. The observed increase in senescent cells following irradiation is associated with a reduction in the thickness of muscle fibres and mobility, two important ageing phenotypes. We also show that quercetin and dasatinib, two senolytics currently in clinical trials, reduce the number of p21:GFP+ cells, in a rapid 5-day assay. This model provides an important tool to study senescence in a living organism, allowing the rapid selection of senolytics before moving to more expensive and time-consuming mammalian systems.

pharmacology and toxicology↗

Co-aggregation with Apolipoprotein E modulates the function of Amyloid-β in Alzheimer's disease

Which isoforms of apolipoprotein E (apoE) we inherit determine our risk of developing late-onset Alzheimers Disease (AD), but the mechanism underlying this link is poorly understood. In particular, the relevance of direct interactions between apoE and amyloid-{beta} (A{beta}) remains controversial. Here, single-molecule imaging shows that all isoforms of apoE associate with A{beta} in the early stages of aggregation and then fall away as fibrillation happens. ApoE-A{beta} co-aggregates account for [~]50% of the mass of soluble A{beta} aggregates detected in the frontal cortices of homozygotes with the higher-risk APOE4 gene. Our results connect inherited APOE genotype with the risk of developing AD by demonstrating how, in an isoform- and lipidation-specific way, apoE modulates the aggregation, clearance and toxicity of A{beta}. Selectively removing non-lipidated apoE4-A{beta} co-aggregates enhances clearance of toxic A{beta} by glial cells, and reduces inflammation and membrane damage, demonstrating a clear path to AD therapeutics.

neuroscience↗