Ataxia Telangiectasia patient-derived neuronal and brain organoid models reveal mitochondrial dysfunction and oxidative stress.

Ataxia Telangiectasia (AT) is a rare disorder caused by mutations in the ATM gene and results in progressive neurodegeneration for reasons that remain poorly understood. In addition to its central role in nuclear DNA repair, ATM operates outside the nucleus to regulate metabolism, redox homeostasis and mitochondrial function. However, a systematic investigation into how and when loss of ATM affects these parameters in relevant human neuronal models of AT was lacking. We therefore used cortical neurons and brain organoids from AT-patient iPSC and gene corrected isogenic controls to reveal levels of mitochondrial dysfunction, oxidative stress, and senescence that vary with developmental maturity. Transcriptome analyses identified disruptions in regulatory networks related to mitochondrial function and maintenance, including alterations in the PARP/SIRT signalling axis and dysregulation of key mitophagy and mitochondrial fission-fusion processes. We further show that antioxidants reduce ROS and restore neurite branching in AT neuronal cultures, and ameliorate impaired neuronal activity in AT brain organoids. We conclude that progressive mitochondrial dysfunction and aberrant ROS production are important contributors to neurodegeneration in AT and are strongly linked to ATM's role in mitochondrial homeostasis regulation.

Reduced acetylated α-tubulin in SPAST hereditary spastic paraplegia patient PBMCs.

HSP-SPAST is the most common form of hereditary spastic paraplegia (HSP), a neurodegenerative disease causing lower limb spasticity. Previous studies using HSP-SPAST patient-derived induced pluripotent stem cell cortical neurons have shown that patient neurons have reduced levels of acetylated α-tubulin, a form of stabilized microtubules, leading to a chain of downstream effects eventuating in increased vulnerability to axonal degeneration. Noscapine treatment rescued these downstream effects by restoring the levels of acetylated α-tubulin in patient neurons. Here we show that HSP-SPAST patient non-neuronal cells, peripheral blood mononuclear cells (PBMCs), also have the disease-associated effect of reduced levels of acetylated α-tubulin. Evaluation of multiple PBMC subtypes showed that patient T cell lymphocytes had reduced levels of acetylated α-tubulin. T cells make up to 80% of all PBMCs and likely contributed to the effect of reduced acetylated α-tubulin levels seen in overall PBMCs. We further showed that mouse administered orally with increasing concentrations of noscapine exhibited a dose-dependent increase of noscapine levels and acetylated α-tubulin in the brain. A similar effect of noscapine treatment is anticipated in HSP-SPAST patients. To measure acetylated α-tubulin levels, we used a homogeneous time resolved fluorescence technology-based assay. This assay was sensitive to noscapine-induced changes in acetylated α-tubulin levels in multiple sample types. The assay is high throughput and uses nano-molar protein concentrations, making it an ideal assay for evaluation of noscapine-induced changes in acetylated α-tubulin levels. This study shows that HSP-SPAST patient PBMCs exhibit disease-associated effects. This finding can help expedite the drug discovery and testing process.

An Alzheimer's Disease Patient-Derived Olfactory Stem Cell Model Identifies Gene Expression Changes Associated with Cognition.

An early symptom of Alzheimer's disease (AD) is an impaired sense of smell, for which the molecular basis remains elusive. Here, we generated human olfactory neurosphere-derived (ONS) cells from people with AD and mild cognitive impairment (MCI), and performed global RNA sequencing to determine gene expression changes. ONS cells expressed markers of neuroglial differentiation, providing a unique cellular model to explore changes of early AD-associated pathways. Our transcriptomics data from ONS cells revealed differentially expressed genes (DEGs) associated with cognitive processes in AD cells compared to MCI, or matched healthy controls (HC). A-Kinase Anchoring Protein 6 (AKAP6) was the most significantly altered gene in AD compared to both MCI and HC, and has been linked to cognitive function. The greatest change in gene expression of all DEGs occurred between AD and MCI. Gene pathway analysis revealed defects in multiple cellular processes with aging, intellectual deficiency and alternative splicing being the most significantly dysregulated in AD ONS cells. Our results demonstrate that ONS cells can provide a cellular model for AD that recapitulates disease-associated differences. We have revealed potential novel genes, including AKAP6 that may have a role in AD, particularly MCI to AD transition, and should be further examined.

Single-Cell RNA-Seq Analysis of Olfactory Mucosal Cells of Alzheimer's Disease Patients.

Olfaction is orchestrated by olfactory mucosal cells located in the upper nasal cavity. Olfactory dysfunction manifests early in several neurodegenerative disorders including Alzheimer's disease, however, disease-related alterations to the olfactory mucosal cells remain poorly described. The aim of this study was to evaluate the olfactory mucosa differences between cognitively healthy individuals and Alzheimer's disease patients. We report increased amyloid-beta secretion in Alzheimer's disease olfactory mucosal cells and detail cell-type-specific gene expression patterns, unveiling 240 differentially expressed disease-associated genes compared to the cognitively healthy controls, and five distinct cell populations. Overall, alterations of RNA and protein metabolism, inflammatory processes, and signal transduction were observed in multiple cell populations, suggesting their role in Alzheimer's disease-related olfactory mucosa pathophysiology. Furthermore, the single-cell RNA-sequencing proposed alterations in gene expression of mitochondrially located genes in AD OM cells, which were verified by functional assays, demonstrating altered mitochondrial respiration and a reduction of ATP production. Our results reveal disease-related changes of olfactory mucosal cells in Alzheimer's disease and demonstrate the utility of single-cell RNA sequencing data for investigating molecular and cellular mechanisms associated with the disease.

Ataxia Telangiectasia iPSC line generated from a patient olfactory biopsy identifies novel disease-causing mutations.

Ataxia Telangiectasia is a rare autosomal recessive disorder caused by a mutated ATM gene. The most debilitating symptom of Ataxia Telangiectasia is the progressive neurodegeneration of the cerebellum, though the molecular mechanisms driving this degeneration remains unclear. Here we describe the generation and validation of an induced pluripotent stem cell (iPSC) line from an olfactory biopsy from a patient with Ataxia Telangiectasia. Sequencing identified two previously unreported disease-causing mutations in the ATM gene. This line can be used to generate 2D and 3D patient-specific neuronal models enabling investigations into the mechanisms underlying neurodegeneration.

Reprogramming of human olfactory neurosphere-derived cells from olfactory mucosal biopsies of a control cohort.

Human olfactory neurosphere-derived (ONS) cells are derived from the olfactory mucosa and display some progenitor- and neuronal cell-like properties, making them useful models of neurological disorders. However, they lack several important characteristics of true neurons, which can be overcome using induced pluripotent stem cell (iPSC) -derived neurons. Here we describe, for the first time, the generation and validation of an iPSC line from an olfactory biopsy from a control cohort member. This data lays the groundwork for future reprogramming of ONS cells, which can be used to generate neuronal models and compliment current ONS cell-based investigations into numerous neurological disorders.

Directional Persistence of Cell Migration in Schizophrenia Patient-Derived Olfactory Cells.

Cell migration is critical for brain development and linked to several neurodevelopmental disorders, including schizophrenia. We have shown previously that cell migration is dysregulated in olfactory neural stem cells from people with schizophrenia. Although they moved faster than control cells on plastic substrates, patient cells were insensitive to regulation by extracellular matrix proteins, which increase the speeds of control cells. As well as speed, cell migration is also described by directional persistence, the straightness of movement. The aim of this study was to determine whether directional persistence is dysregulated in schizophrenia patient cells and whether it is modified on extracellular matrix proteins. Directional persistence in patient-derived and control-derived olfactory cells was quantified from automated live-cell imaging of migrating cells. On plastic substrates, patient cells were more persistent than control cells, with straighter trajectories and smaller turn angles. On most extracellular matrix proteins, persistence increased in patient and control cells in a concentration-dependent manner, but patient cells remained more persistent. Patient cells therefore have a subtle but complex phenotype in migration speed and persistence on most extracellular matrix protein substrates compared to control cells. If present in the developing brain, this could lead to altered brain development in schizophrenia.

Single cell morphology distinguishes genotype and drug effect in Hereditary Spastic Paraplegia.

A central need for neurodegenerative diseases is to find curative drugs for the many clinical subtypes, the causative gene for most cases being unknown. This requires the classification of disease cases at the genetic and cellular level, an understanding of disease aetiology in the subtypes and the development of phenotypic assays for high throughput screening of large compound libraries. Herein we describe a method that facilitates these requirements based on cell morphology that is being increasingly used as a readout defining cell state. In patient-derived fibroblasts we quantified 124 morphological features in 100,000 cells from 15 people with two genotypes (SPAST and SPG7) of Hereditary Spastic Paraplegia (HSP) and matched controls. Using machine learning analysis, we distinguished between each genotype and separated them from controls. Cell morphologies changed with treatment with noscapine, a tubulin-binding drug, in a genotype-dependent manner, revealing a novel effect on one of the genotypes (SPG7). These findings demonstrate a method for morphological profiling in fibroblasts, an accessible non-neural cell, to classify and distinguish between clinical subtypes of neurodegenerative diseases, for drug discovery, and potentially for biomarkers of disease severity and progression.

Inhibition of the cGAS-STING pathway ameliorates the premature senescence hallmarks of Ataxia-Telangiectasia brain organoids.

Ataxia-telangiectasia (A-T) is a genetic disorder caused by the lack of functional ATM kinase. A-T is characterized by chronic inflammation, neurodegeneration and premature ageing features that are associated with increased genome instability, nuclear shape alterations, micronuclei accumulation, neuronal defects and premature entry into cellular senescence. The causal relationship between the detrimental inflammatory signature and the neurological deficiencies of A-T remains elusive. Here, we utilize human pluripotent stem cell-derived cortical brain organoids to study A-T neuropathology. Mechanistically, we show that the cGAS-STING pathway is required for the recognition of micronuclei and induction of a senescence-associated secretory phenotype (SASP) in A-T olfactory neurosphere-derived cells and brain organoids. We further demonstrate that cGAS and STING inhibition effectively suppresses self-DNA-triggered SASP expression in A-T brain organoids, inhibits astrocyte senescence and neurodegeneration, and ameliorates A-T brain organoid neuropathology. Our study thus reveals that increased cGAS and STING activity is an important contributor to chronic inflammation and premature senescence in the central nervous system of A-T and constitutes a novel therapeutic target for treating neuropathology in A-T patients.

Novel STMN2 Variant Linked to Amyotrophic Lateral Sclerosis Risk and Clinical Phenotype.

OBJECTIVE: There is a critical need to establish genetic markers that explain the complex phenotypes and pathogenicity of ALS. This study identified a polymorphism in the Stathmin-2 gene and investigated its association with sporadic ALS (sALS) disease risk, age-of onset and survival duration. METHODS: The candidate CA repeat was systematically analyzed using PCR, Sanger sequencing and high throughput capillary separation for genotyping. Stathmin-2 expression was investigated using RT-PCR in patient olfactory neurosphere-derived (ONS) cells and RNA sequencing in laser-captured spinal motor neurons. RESULTS: In a case-control analysis of a combined North American sALS cohort (n = 321) and population control group (n = 332), long/long CA genotypes were significantly associated with disease risk (p = 0.042), and most strongly when one allele was a 24 CA repeat (p = 0.0023). In addition, longer CA allele length was associated with earlier age-of-onset (p = 0.039), and shorter survival duration in bulbar-onset cases (p = 0.006). In an Australian longitudinal sALS cohort (n = 67), ALS functional rating scale scores were significantly lower in carriers of the long/long genotype (p = 0.034). Stathmin-2 mRNA expression was reduced in sporadic patient ONS cells. Additionally, sALS patients and controls exhibited variable expression of Stathmin-2 mRNA according to CA genotype in laser-captured spinal motor neurons. CONCLUSIONS: We report a novel non-coding CA repeat in Stathmin-2 which is associated with sALS disease risk and has disease modifying effects. The potential value of this variant as a disease marker and tool for cohort enrichment in clinical trials warrants further investigation.

Hereditary Spastic Paraplegia: From Genes, Cells and Networks to Novel Pathways for Drug Discovery.

Hereditary spastic paraplegia (HSP) is a diverse group of Mendelian genetic disorders affecting the upper motor neurons, specifically degeneration of their distal axons in the corticospinal tract. Currently, there are 80 genes or genomic loci (genomic regions for which the causative gene has not been identified) associated with HSP diagnosis. HSP is therefore genetically very heterogeneous. Finding treatments for the HSPs is a daunting task: a rare disease made rarer by so many causative genes and many potential mutations in those genes in individual patients. Personalized medicine through genetic correction may be possible, but impractical as a generalized treatment strategy. The ideal treatments would be small molecules that are effective for people with different causative mutations. This requires identification of disease-associated cell dysfunctions shared across genotypes despite the large number of HSP genes that suggest a wide diversity of molecular and cellular mechanisms. This review highlights the shared dysfunctional phenotypes in patient-derived cells from patients with different causative mutations and uses bioinformatic analyses of the HSP genes to identify novel cell functions as potential targets for future drug treatments for multiple genotypes.

Mitochondrial Function in Hereditary Spastic Paraplegia: Deficits in SPG7 but Not SPAST Patient-Derived Stem Cells.

Mutations in SPG7 and SPAST are common causes of hereditary spastic paraplegia (HSP). While some SPG7 mutations cause paraplegin deficiency, other SPG7 mutations cause increased paraplegin expression. Mitochondrial function has been studied in models that are paraplegin-deficient (human, mouse, and Drosophila models with large exonic deletions, null mutations, or knockout models) but not in models of mutations that express paraplegin. Here, we evaluated mitochondrial function in olfactory neurosphere-derived cells, derived from patients with a variety of SPG7 mutations that express paraplegin and compared them to cells derived from healthy controls and HSP patients with SPAST mutations, as a disease control. We quantified paraplegin expression and an extensive range of mitochondrial morphology measures (fragmentation, interconnectivity, and mass), mitochondrial function measures (membrane potential, oxidative phosphorylation, and oxidative stress), and cell proliferation. Compared to control cells, SPG7 patient cells had increased paraplegin expression, fragmented mitochondria with low interconnectivity, reduced mitochondrial mass, decreased mitochondrial membrane potential, reduced oxidative phosphorylation, reduced ATP content, increased mitochondrial oxidative stress, and reduced cellular proliferation. Mitochondrial dysfunction was specific to SPG7 patient cells and not present in SPAST patient cells, which displayed mitochondrial functions similar to control cells. The mitochondrial dysfunction observed here in SPG7 patient cells that express paraplegin was similar to the dysfunction reported in cell models without paraplegin expression. The p.A510V mutation was common to all patients and was the likely species associated with increased expression, albeit seemingly non-functional. The lack of a mitochondrial phenotype in SPAST patient cells indicates genotype-specific mechanisms of disease in these HSP patients.

Antibody-Free Targeted Proteomics Assay for Absolute Measurement of α-Tubulin Acetylation.

Acetylation of α-tubulin at conserved lysine 40 (K40) amino acid residue regulates microtubule dynamics and controls a wide range of cellular activities. Dysregulated microtubule dynamics characterized by differential α-tubulin acetylation is a hallmark of cancer, neurodegeneration, and other complex disorders. Hence, accurate quantitation of α-tubulin acetylation is required in human disease and animal model studies. We developed a novel antibody-free proteomics assay to measure α-tubulin acetylation targeting protease AspN-generated peptides harboring K40 site. Using the synthetic unmodified and acetylated stable isotope labeled peptides DKTIGGG and DKTIGGGD, we demonstrate assay linearity across 4 log magnitude and reproducibility of <10% coefficient of variation. The assay accuracy was validated by titration of 10-80% mixture of acetylated/nonacetylated α-tubulin peptides in the background of human olfactory neurosphere-derived stem (ONS) cell matrix. Furthermore, in agreement with antibody-based high content microscopy analysis, the targeted proteomics assay reported an induction of α-tubulin K40 acetylation upon Trichostatin A stimulation of ONS cells. Independently, we found 35.99% and 16.11% α-tubulin acetylation for mouse spinal cord and brain homogenate tissue, respectively, as measured by our assay. In conclusion, this simple, antibody-free proteomics assay enables quantitation of α-tubulin acetylation, and is applicable across various fields of biology and medicine.

Urban air particulate matter induces mitochondrial dysfunction in human olfactory mucosal cells.

BACKGROUND: The adverse effects of air pollutants including particulate matter (PM) on the central nervous system is increasingly reported by epidemiological, animal and post-mortem studies in the last decade. Oxidative stress and inflammation are key consequences of exposure to PM although little is known of the exact mechanism. The association of PM exposure with deteriorating brain health is speculated to be driven by PM entry via the olfactory system. How air pollutants affect this key entry site remains elusive. In this study, we investigated effects of urban size-segregated PM on a novel cellular model: primary human olfactory mucosal (hOM) cells. RESULTS: Metabolic activity was reduced following 24-h exposure to PM without evident signs of toxicity. Results from cytometric bead array suggested a mild inflammatory response to PM exposure. We observed increased oxidative stress and caspase-3/7 activity as well as perturbed mitochondrial membrane potential in PM-exposed cells. Mitochondrial dysfunction was further verified by a decrease in mitochondria-dependent respiration. Transient suppression of the mitochondria-targeted gene, neuronal pentraxin 1 (NPTX1), was carried out, after being identified to be up-regulated in PM2.5-1 treated cells via RNA sequencing. Suppression of NPTX1 in cells exposed to PM did not restore mitochondrial defects resulting from PM exposure. In contrast, PM-induced adverse effects were magnified in the absence of NPTX1, indicating a critical role of this protein in protection against PM effects in hOM cells. CONCLUSION: Key mitochondrial functions were perturbed by urban PM exposure in a physiologically relevant cellular model via a mechanism involving NPTX1. In addition, inflammatory response and early signs of apoptosis accompanied mitochondrial dysfunction during exposure to PM. Findings from this study contribute to increased understanding of harmful PM effects on human health and may provide information to support mitigation strategies targeted at air pollution.

Oxidative Stress-Induced Axon Fragmentation Is a Consequence of Reduced Axonal Transport in Hereditary Spastic Paraplegia SPAST Patient Neurons.

Hereditary spastic paraplegia (HSP) is a group of inherited disorders characterized by progressive spasticity and paralysis of the lower limbs. Autosomal dominant mutations in SPAST gene account for ∼40% of adult-onset patients. We have previously shown that SPAST patient cells have reduced organelle transport and are therefore more sensitive to oxidative stress. To test whether these effects are present in neuronal cells, we first generated 11 induced pluripotent stem (iPS) cell lines from fibroblasts of three healthy controls and three HSP patients with different SPAST mutations. These cells were differentiated into FOXG1-positive forebrain neurons and then evaluated for multiple aspects of axonal transport and fragmentation. Patient neurons exhibited reduced levels of SPAST encoded spastin, as well as a range of axonal deficits, including reduced levels of stabilized microtubules, lower peroxisome transport speed as a consequence of reduced microtubule-dependent transport, reduced number of peroxisomes, and higher density of axon swellings. Patient axons fragmented significantly more than controls following hydrogen peroxide exposure, suggesting for the first time that the SPAST patient axons are more sensitive than controls to the deleterious effects of oxidative stress. Treatment of patient neurons with tubulin-binding drugs epothilone D and noscapine rescued axon peroxisome transport and protected them against axon fragmentation induced by oxidative stress, showing that SPAST patient axons are vulnerable to oxidative stress-induced degeneration as a consequence of reduced axonal transport.

Patient-Derived Stem Cell Models in SPAST HSP: Disease Modelling and Drug Discovery.

Hereditary spastic paraplegia is an inherited, progressive paralysis of the lower limbs first described by Adolph Strümpell in 1883 with a further detailed description of the disease by Maurice Lorrain in 1888. Today, more than 100 years after the first case of HSP was described, we still do not know how mutations in HSP genes lead to degeneration of the corticospinal motor neurons. This review describes how patient-derived stem cells contribute to understanding the disease mechanism at the cellular level and use this for discovery of potential new therapeutics, focusing on SPAST mutations, the most common cause of HSP.

Expanding the spectrum of PEX16 mutations and novel insights into disease mechanisms.

Zellweger syndrome spectrum disorders are caused by mutations in any of at least 12 different PEX genes. This includes PEX16, an important regulator of peroxisome biogenesis. Using whole genome sequencing, we detected previously unreported, biallelic variants in PEX16 [NM_004813.2:c.658G>A, p.(Ala220Thr) and NM_004813.2:c.830G>A, p.(Arg277Gln)] in an individual with leukodystrophy, spastic paraplegia, cerebellar ataxia, and craniocervical dystonia with normal plasma very long chain fatty acids. Using olfactory-neurosphere derived cells, a population of neural stem cells, we showed patient cells had reduced peroxisome density and increased peroxisome size, replicating previously reported findings in PEX16 cell lines. Along with alterations in peroxisome morphology, patient cells also had impaired peroxisome function with reduced catalase activity. Furthermore, patient cells had reduced oxidative stress levels after exposure to hydrogen-peroxide (H2O2), which may be a result of compensation by H2O2 metabolising enzymes other than catalase to preserve peroxisome-related cell functions. Our findings of impaired catalase activity and altered oxidative stress response are novel. Our study expands the phenotype of PEX16 mutations by including dystonia and provides further insights into the pathological mechanisms underlying PEX16-associated disorders. Additional studies of the full spectrum of peroxisomal dysfunction could improve our understanding of the mechanism underlying PEX16-associated disorders.

DNA methylation in schizophrenia in different patient-derived cell types.

DNA methylation of gene promoter regions represses transcription and is a mechanism via which environmental risk factors could affect cells during development in individuals at risk for schizophrenia. We investigated DNA methylation in patient-derived cells that might shed light on early development in schizophrenia. Induced pluripotent stem cells may reflect a "ground state" upon which developmental and environmental influences would be minimal. Olfactory neurosphere-derived cells are an adult-derived neuro-ectodermal stem cell modified by developmental and environmental influences. Fibroblasts provide a non-neural control for life-long developmental and environmental influences. Genome-wide profiling of DNA methylation and gene expression was done in these three cell types from the same individuals. All cell types had distinct, statistically significant schizophrenia-associated differences in DNA methylation and linked gene expression, with Gene Ontology analysis showing that the differentially affected genes clustered in networks associated with cell growth, proliferation, and movement, functions known to be affected in schizophrenia patient-derived cells. Only five gene loci were differentially methylated in all three cell types. Understanding the role of epigenetics in cell function in the brain in schizophrenia is likely to be complicated by similar cell type differences in intrinsic and environmentally induced epigenetic regulation.

A Patient-Specific Stem Cell Model to Investigate the Neurological Phenotype Observed in Ataxia-Telangiectasia.

The molecular pathogenesis of ataxia-telangiectasia (A-T) is not yet fully understood, and a versatile cellular model is required for in vitro studies. The occurrence of continuous neurogenesis and easy access make the multipotent adult stem cells from the olfactory mucosa within the nasal cavity a potential cellular model. We describe an efficient method to establish neuron-like cells from olfactory mucosa biopsies derived from A-T patients for the purpose of studying the cellular and molecular aspects of this debilitating disease.

Cell migration in schizophrenia: Patient-derived cells do not regulate motility in response to extracellular matrix.

Schizophrenia is a highly heritable psychiatric disorder linked to a large number of risk genes. The function of these genes in disease etiology is not fully understood but pathway analyses of genomic data suggest developmental dysregulation of cellular processes such as neuronal migration and axon guidance. Previous studies of patient-derived olfactory cells show them to be more motile than control-derived cells when grown on a fibronectin substrate, motility that is dependent on focal adhesion kinase signaling. The aim of this study was to investigate whether schizophrenia patient-derived cells are responsive to other extracellular matrix (ECM) proteins that bind integrin receptors. Olfactory neurosphere-derived cells from nine patients and nine matched controls were grown on ECM protein substrates at increasing concentrations and their movement was tracked for 24h using automated high-throughput imaging. Control-derived cells increased their motility as the ECM substrate concentration increased, whereas patient-derived cell motility was little affected by ECM proteins. Patient and control cells had appropriate integrin receptors for these ECM substrates and detected them as shown by increases in focal adhesion number and size in response to ECM proteins, which also induced changes in cell morphology and cytoskeleton. These observations indicate that patient cells failed to translate the detection of ECM proteins into appropriate changes in cell motility. In a sense, patient cells act like a moving car whose accelerator is jammed, moving at the same speed without regard to the external environment. This focuses attention on cell motility regulation rather than speed as key to impairment of neuronal migration in the developing brain in schizophrenia.