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

Abstract

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.

Keywords: SPAST; SPG7; hereditary spastic paraplegia; mitochondria; oxidative phosphorylation; paraplegin; spastin.

FIGURE 1

(A) Sagittal T1-weighted fast field echo and (B) transverse T2-weighted MRI images showing mild cerebellar atrophy of the patient (patient 3) carrying novel SPG7 variant. (C) Pedigree of patient 3 (arrow); both the proband and his affected sister carried two heterozygote variants in SPG7: a common mutation [NM_003119.3:c.1529C > T (p.Ala510Val)] and novel canonical splice variant (NM_003119.3:c.1449 + 1G > A). (D) Linear protein domain diagram of paraplegin. The AAA+ domain is shown as two subdomains (ATPase and Lid), separated by a linker region. Three of the four variants in the SPG7 patients in this study locate to the AAA+ ATPase domain and one to the M41 peptidase domain.

FIGURE 2

Paraplegin expression, mitochondrial morphology, and membrane potential in control, SPG7, and SPAST patient cells. (A) Western blot analysis of paraplegin and GAPDH expression. (B) Representative flow cytometry histogram of paraplegin fluorescence intensities. IC: Isotype control. (C) Western blot densitometry band quantification showing paraplegin expression normalized to GAPDH expression. (D) Paraplegin mean fluorescence intensity quantified by flow cytometry. (E) Representative images of MitoTracker-labeled mitochondria in cells from control, SPG7, and SPAST patient cells. Scale bar: 10 μm. (F,G) Mitochondria morphological analysis showing aspect ratio (mitochondria length) and form factor (degree of branching). (H) Mitochondrial mass was calculated as the fluorescence intensity of MitoTracker green (MTG) labeled mitochondria. (I) Mitochondrial membrane potential was measured using tetramethylrhodamine methyl ester (TMRM) fluorescence and normalized to mitochondrial mass (MTG fluorescence). Data presented as mean ± SEM. The bars present data from the three technical replicates. One-way analysis of variance with Tukey’s multiple comparisons test was performed to compare control, SPG7, and SPAST groups. n.s., not significant.

FIGURE 3

Oxidative respiration and oxidative stress in control, SPG7, and SPAST patient cells. (A) Representative graph of mitochondrial respiration determined by measuring OCR (mean ± SE) using the Seahorse-based assay. (B–E) Comparison of different aspects of mitochondrial respiration (F) ATP content in the cells was measured using the ATPlite assay kit and normalized to mitochondrial mass [MitoTracker green (MTG) fluorescence]. (G) Mitochondrial oxidative stress was measured using MitoSOX (mitochondrial superoxide indicator) fluorescence and normalized to mitochondrial mass (MTG fluorescence). (H) General oxidative stress was measured using the CM-H₂DCFDA (general oxidative stress indicator) assay kit and normalized to mitochondrial mass (MTG fluorescence). (I) Cellular proliferation was measured for on day 0 (4 h after cell seeding), day 1, and day 2 using CyQUANT assay. Data presented as mean ± SEM. The bars present data from three technical replicates. One-way analysis of variance with Tukey’s multiple comparisons test was performed to compare control, SPG7, and SPAST groups. n.s., not significant.