GBA mutation promotes early mitochondrial dysfunction in 3D neurosphere models.

Glucocerebrosidase (GBA) mutations are the most important genetic risk factor for the development of Parkinson disease (PD). GBA encodes the lysosomal enzyme glucocerebrosidase (GCase). Loss-of-GCase activity in cellular models has implicated lysosomal and mitochondrial dysfunction in PD disease pathogenesis, although the exact mechanisms remain unclear. We hypothesize that GBA mutations impair mitochondria quality control in a neurosphere model.We have characterized mitochondrial content, mitochondrial function and macroautophagy flux in 3D-neurosphere-model derived from neural crest stem cells containing heterozygous and homozygous N370SGBA mutations, under carbonyl cyanide-m-chlorophenyl-hydrazine (CCCP)- induced mitophagy.Our findings on mitochondrial markers and ATP levels indicate that mitochondrial accumulation occurs in mutant N370SGBA neurospheres under basal conditions, and clearance of depolarised mitochondria is impaired following CCCP-treatment. A significant increase in TFEB-mRNA levels, the master regulator of lysosomal and autophagy genes, may explain an unchanged macroautophagy flux in N370SGBA neurospheres. PGC1α-mRNA levels were also significantly increased following CCCP-treatment in heterozygote, but not homozygote neurospheres, and might contribute to the increased mitochondrial content seen in cells with this genotype, probably as a compensatory mechanism that is absent in homozygous lines.Mitochondrial impairment occurs early in the development of GCase-deficient neurons. Furthermore, impaired turnover of depolarised mitochondria is associated with early mitochondrial dysfunction.In summary, the presence of GBA mutation may be associated with higher levels of mitochondrial content in homozygous lines and lower clearance of damaged mitochondria in our neurosphere model

Bioenergetic Consequences of PINK1 Mutations in Parkinson Disease

BACKGROUND: Mutations of the gene for PTEN-induced kinase 1 (PINK1) are a cause of familial Parkinson's disease (PD). PINK1 protein has been localised to mitochondria and PINK1 gene knockout models exhibit abnormal mitochondrial function. The purpose of this study was to determine whether cells derived from PD patients with a range of PINK1 mutations demonstrate similar defects of mitochondrial function, whether the nature and severity of the abnormalities vary between mutations and correlate with clinical features. METHODOLOGY: We investigated mitochondrial bioenergetics in live fibroblasts from PINK1 mutation patients using single cell techniques. We found that fibroblasts from PINK1 mutation patients had significant defects of bioenergetics including reduced mitochondrial membrane potential, altered redox state, a respiratory deficiency that was determined by substrate availability, and enhanced sensitivity to calcium stimulation and associated mitochondrial permeability pore opening. There was an increase in the basal rate of free radical production in the mutant cells. The pattern and severity of abnormality varied between different mutations, and the less severe defects in these cells were associated with later age of onset of PD. CONCLUSIONS: The results provide insight into the molecular pathology of PINK1 mutations in PD and also confirm the critical role of substrate availability in determining the biochemical phenotype--thereby offering the potential for novel therapeutic strategies to circumvent these abnormalities

Increase in mitochondrial ROS production in fibroblasts with PINK1 mutations.

<p>Fibroblasts with PINK1 mutations displayed a higher basal rate of increase in Mitosox fluorescence, demonstrating a higher basal production of ROS compared to controls. Inhibition of complex I with 5 µM rotenone induced a significant increase in ROS production in control fibroblasts but only a small increase in ROS production in fibroblasts with PINK1 mutation. Histogram demonstrates %age values of rate of Mitosox fluorescence compared to 100% for control (C3) fibroblasts.</p

Characteristics of mitochondrial membrane potential (Δψ_m) in human fibroblasts with PINK1 mutations.

<p>A-L2122 fibroblasts exhibited a 20% increase (p<0.001) in TMRM fluorescence (i,e, an increased Δψ_m) compared to controls. Fibroblasts with PINK1 mutations (L2123, L2124, L2126 amd L1703) showed a significant decrease in Δψ_m compared to control cells. B–G In control, L2122 and L2126 fibroblasts (B–C, F), oligomycin did not affect Δψ_m; rotenone induced a partial depolarisation; FCCP induced complete depolarisation. In L2123, L2124 and L1703 fibroblasts (D–E, G), oligomycin caused a mitochondrial depolarisation.</p

Level of FAD autofluorescence in fibroblasts with PINK1 mutations and control cells.

<p>Quantification of the %age change in FAD⁺⁺ fluorescence: 100 is response to FCCP and 0% is response to cyanide, averaged traces for control L2132 and fibroblasts with PINK1 mutation L2122 presented in A–B. C-Fibroblasts with PINK1 mutations have increased FAD⁺⁺ fluorescence than controls. This can be reversed by application of 5 mM Me-succinate to PINK1 mutated cells. D- Values of mitochondrial FAD autofluorescence.</p

Redox state and NADH level in fibroblasts with PINK1 mutations and control cells.

<p>A–B Graphs demonstrate averaged trace of NADH autofluorescence in control L2132 fibroblasts (A) and L1703 PINK1 mutation (B). Estimation of the %age change in mitochondrial redox level in control cells – L2132 (A) and cells with mutations in PINK1 (B–C). C-Redox state was estimated as: 0 is response to FCCP (maximal rate of respiration and lowest level of mitochondrial NADH) and 100% is response to cyanide (inhibition of respiration with no consumption of NADH in mitochondria; Fibroblasts with PINK1 mutations have lower NADH redox state compared to control fibroblasts (except L2126) that can be normalized by application of 5 mM glutamate (C). D – Total pool of mitochondrial NADH was also significantly lower in fibroblasts with PINK1 mutations.</p

A rise in [Ca²⁺]_c induces mitochondrial depolarisation in fibroblasts with PINK1 mutations.

<p>Arrows mark UV-induced flash photolysis of cells loaded with Ca-NP-EGTA, fluo-4 and TMRM. In control C3 (A), L2132 (B) and in fibroblast with L2122 mutation (C) demonstrated an increase in [Ca²⁺]c in response to flash photolysis, with no change in Δψ_m. In fibroblasts with PINK1 mutations (L2123 –D; L2124 – E; L2126 – F; and L1703-G), flash photolysis induced an increase in [Ca²⁺]_c with profound depolarisation of the mitochondria.</p

Effect of mitochondrial substrates on mechanism of maintenance of Δψ_m in human fibroblast with PINK1 mutations.

<p>Application of pyruvate (5 mM) or/and methyl succinate (5 mM) to fibroblasts increased Δψ_m; Substrate provision prevented the oligomycin induced mitochondrial depolarisation in L2123, L2124 and L1703 fibroblasts.</p

Clinical and biochemical characteristics of samples studied.

<p>Clinical and biochemical characteristics of samples studied.</p