5 research outputs found
Oxidative switch drives mitophagy defects in dopaminergic parkin mutant patient neurons
Mutations in PRKN are the most common cause of early onset Parkinsonās disease. Parkin is an E3 ubiquitin ligase, functioning in mitophagy. Mitochondrial abnormalities are present in PRKN mutant models. Patient derived neurons are a promising model in which to study pathogenic mechanisms and therapeutic targets. Here we generate induced neuronal progenitor cells from PRKN mutant patient fibroblasts with a high dopaminergic neuron yield. We reveal changing mitochondrial phenotypes as neurons undergo a metabolic switch during differentiation. Fibroblasts from 4 controls and 4 PRKN mutant patients were transformed into induced neuronal progenitor cells and subsequently differentiated into dopaminergic neurons. Mitochondrial morphology, function and mitophagy were evaluated using live cell fluorescent imaging, cellular ATP and reactive oxygen species production quantification. Direct conversion of control and PRKN mutant patient fibroblasts results in induced neuronal progenitor and their differentiation yields high percentage of dopaminergic neurons. We were able to observe changing mitochondrial phenotypes as neurons undergo a metabolic switch during differentiation. Our results show that when pre-neurons are glycolytic early in differentiation mitophagy is unimpaired by PRKN deficiency. However as neurons become oxidative phosphorylation dependent, mitophagy is severely impaired in the PRKN mutant patient neurons. These changes correlate with changes in mitochondrial function and morphology; resulting in lower neuron yield and altered neuronal morphology. Induced neuronal progenitor cell conversion can produce a high yield of dopaminergic neurons. The mitochondrial phenotype, including mitophagy status, is highly dependent on the metabolic status of the cell. Only when neurons are oxidative phosphorylation reliant the extent of mitochondrial abnormalities are identified. These data provide insight into cell specific effects of PRKN mutations, in particular in relation to mitophagy dependent disease phenotypes and provide avenues for alternative therapeutic approaches
Applications of machine learning to diagnosis and treatment of neurodegenerative diseases
Globally, there is a huge unmet need for effective treatments for neurodegenerative diseases. The complexity of the molecular mechanisms underlying neuronal degeneration and the heterogeneity of the patient population present massive challenges to the development of early diagnostic tools and effective treatments for these diseases. Machine learning, a subfield of artificial intelligence, is enabling scientists, clinicians and patients to address some of these challenges. In this Review, we discuss how machine learning can aid early diagnosis and interpretation of medical images as well as the discovery and development of new therapies. A unifying theme of the different applications of machine learning is the integration of multiple high-dimensional sources of data, which all provide a different view on disease, and the automated derivation of actionable insights
Directly converted astrocytes retain the ageing features of the donor fibroblasts and elucidate the astrocytic contribution to human CNS health and disease
Astrocytes are highly specialised cells, responsible for CNS homeostasis and neuronal activity. Lack of human in vitro systems able to recapitulate the functional changes affecting astrocytes during ageing represents a major limitation to studying mechanisms and potential therapies aiming to preserve neuronal health. Here, we show that induced astrocytes from fibroblasts donors in their childhood or adulthood display ageārelated transcriptional differences and functionally diverge in a spectrum of ageāassociated features, such as altered nuclear compartmentalisation, nucleocytoplasmic shuttling properties, oxidative stress response and DNA damage response. Remarkably, we also show an ageārelated differential response of induced neural progenitor cells derived astrocytes (iNPCāAs) in their ability to support neurons in coāculture upon proāinflammatory stimuli. These results show that iNPCāAs are a renewable, readily available resource of human glia that retain the ageārelated features of the donor fibroblasts, making them a unique and valuable model to interrogate human astrocyte function over time in human CNS health and disease
Translating SOD1 gene silencing towards the clinic: A highly efficacious, off-target free and biomarker-supported strategy for familial ALS
Twenty per cent of familial amyotrophic lateral sclerosis (fALS) cases are caused by mutations in
the gene encoding human cytosolic Cu/Zn superoxide dismutase (hSOD1). Efficient translation of
the therapeutic potential of interfering RNA (RNAi) for the treatment of SOD1-ALS patients
requires the development of vectors that are free of significant off-target effects and with reliable
biomarkers to discern sufficient target engagement and correct dosing. Using adeno-associated virus
serotype 9 to deliver RNAi against hSOD1 in the SOD1G93A mouse model, we found that intrathecal
injection of the therapeutic vector via the cisterna magna delayed onset of disease, decreased motor
neuron death at end stage by up to 88% and prolonged the median survival of SOD1G93A mice by up
to 42%. To our knowledge this is the first report to demonstrate no significant off-target effects
linked to hSOD1 silencing, providing further confidence in the specificity of this approach. We also
report the measurement of cerebrospinal fluid (CSF) hSOD1 protein levels as a biomarker of
effective dosing and efficacy of hSOD1 knockdown. Together, these data provide further confidence
in the safety of the clinical therapeutic vector. The CSF biomarker will be a useful measure of
biological activity for translation into human clinical trials
Astrocytic CāXāC motif chemokine ligand-1 mediates Ī²-amyloid-induced synaptotoxicity
Background
Pathological interactions between Ī²-amyloid (AĪ²) and tau drive synapse loss and cognitive decline in Alzheimerās 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 AĪ²-induced synaptotoxicity in AD is not well understood.
Methods
We stimulated mouse and human astrocytes with conditioned medium containing concentrations and species of human AĪ² that mimic those in human AD brain. Medium from stimulated astrocytes was collected and immunodepleted of AĪ² before being added to naĆÆve 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 CāXāC motif chemokine ligand-1 (CXCL1), a chemokine upregulated in AD brain. Antagonism of neuronal CāXāC motif chemokine receptor 2 (CXCR2) prevented synaptotoxicity in response to CXCL1 and AĪ²-stimulated astrocyte secretions.
Conclusions
Our data indicate that astrocytes exacerbate the synaptotoxic effects of AĪ² via interactions of astrocytic CXCL1 and neuronal CXCR2 receptors, highlighting this chemokineāreceptor pair as a novel target for therapeutic intervention in AD