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.

Pharmacological rescue of mitochondrial and neuronal defects in SPG7 hereditary spastic paraplegia patient neurons using high throughput assays.

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.

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.

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.