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SPG7 is the most common form of autosomal recessive hereditary spastic paraplegia (HSP). There is a lack of HSP-SPG7 human neuronal models to understand the disease mechanism and identify new drug treatments. We generated a human neuronal model of HSP-SPG7 using induced pluripotent stem (iPS) cell technology. We first generated iPS cells from three HSP-SPG7 patients carrying different disease-causing variants and three healthy controls. The iPS cells were differentiated to form neural progenitor cells (NPCs) and then from NPCs to mature cortical neurons. Mitochondrial and neuronal defects were measured using a high throughout imaging and analysis-based assay in live cells. Our results show that compared to control NPCs, patient NPCs had aberrant mitochondrial morphology with increased mitochondrial size and reduced membrane potential. Patient NPCs develop to form mature cortical neurons with amplified mitochondrial morphology and functional defects along with defects in neuron morphology − reduced neurite complexity and length, reduced synaptic gene, protein expression and activity, reduced viability and increased axonal degeneration. Treatment of patient neurons with Bz-423, a mitochondria permeability pore regulator, restored the mitochondrial and neurite morphological defects and mitochondrial membrane potential back to control neuron levels and rescued the low viability and increased degeneration in patient neurons. This study establishes a direct link between mitochondrial and neuronal defects in HSP-SPG7 patient neurons. We present a strategy for testing mitochondrial targeting drugs to rescue neuronal defects in HSP-SPG7 patient neurons.

Summary: High-throughput imaging allows in vitro assessment of neuron morphology for screening populations under developmental, homeostatic, and/or disease conditions. Here, we present a protocol to differentiate cryopreserved human cortical neuronal progenitors into mature cortical neurons for high-throughput imaging analysis. We describe the use of a notch signaling inhibitor to generate homogeneous neuronal populations at densities amenable to individual neurite identification. We detail neurite morphology assessment via measuring multiple parameters including neurite length, branches, roots, segments and extremities, and neuron maturation. : Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.

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.

Abstract 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.

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.

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.

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. Keywords: Whole genome sequencing, PEX16, Peroxisomes, Leukodystrophy, Dystonia

Hereditary Spastic Paraplegia (HSP) is a genetically heterogeneous group of disorders, diagnosed by progressive gait disturbances with muscle weakness and spasticity, for which there are no treatments targeted at the underlying pathophysiology. Mutations in spastin are a common cause of HSP. Spastin is a microtubule-severing protein whose mutation in mouse causes defective axonal transport. In human patient-derived olfactory neurosphere-derived (ONS) cells, spastin mutations lead to lower levels of acetylated α-tubulin, a marker of stabilised microtubules, and to slower speed of peroxisome trafficking. Here we screened multiple concentrations of four tubulin-binding drugs for their ability to rescue levels of acetylated α-tubulin in patient-derived ONS cells. Drug doses that restored acetylated α-tubulin to levels in control-derived ONS cells were then selected for their ability to rescue peroxisome trafficking deficits. Automated microscopic screening identified very low doses of the four drugs (0.5 nM taxol, 0.5 nM vinblastine, 2 nM epothilone D, 10 µM noscapine) that rescued acetylated α-tubulin in patient-derived ONS cells. These same doses rescued peroxisome trafficking deficits, restoring peroxisome speeds to untreated control cell levels. These results demonstrate a novel approach for drug screening based on high throughput automated microscopy for acetylated α-tubulin followed by functional validation of microtubule-based peroxisome transport. From a clinical perspective, all the drugs tested are used clinically, but at much higher doses. Importantly, epothilone D and noscapine can enter the central nervous system, making them potential candidates for future clinical trials.

SUMMARY Hereditary spastic paraplegia (HSP) leads to progressive gait disturbances with lower limb muscle weakness and spasticity. Mutations in SPAST are a major cause of adult-onset, autosomal-dominant HSP. Spastin, the protein encoded by SPAST, is a microtubule-severing protein that is enriched in the distal axon of corticospinal motor neurons, which degenerate in HSP patients. Animal and cell models have identified functions of spastin and mutated spastin but these models lack the gene dosage, mutation variability and genetic background that characterize patients with the disease. In this study, this genetic variability is encompassed by comparing neural progenitor cells derived from biopsies of the olfactory mucosa from healthy controls with similar cells from HSP patients with SPAST mutations, in order to identify cell functions altered in HSP. Patient-derived cells were similar to control-derived cells in proliferation and multiple metabolic functions but had major dysregulation of gene expression, with 57% of all mRNA transcripts affected, including many associated with microtubule dynamics. Compared to control cells, patient-derived cells had 50% spastin, 50% acetylated α-tubulin and 150% stathmin, a microtubule-destabilizing enzyme. Patient-derived cells were smaller than control cells. They had altered intracellular distributions of peroxisomes and mitochondria and they had slower moving peroxisomes. These results suggest that patient-derived cells might compensate for reduced spastin, but their increased stathmin expression reduced stabilized microtubules and altered organelle trafficking. Sub-nanomolar concentrations of the microtubule-binding drugs, paclitaxel and vinblastine, increased acetylated α-tubulin levels in patient cells to control levels, indicating the utility of this cell model for screening other candidate compounds for drug therapies.

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.