Himesha Vandebona

Mutant Parkin Impairs Mitochondrial Function and Morphology in Human Fibroblasts

Background Mutations in Parkin are the most common cause of autosomal recessive Parkinson disease (PD). The mitochondrially localized E3 ubiquitin-protein ligase Parkin has been reported to be involved in respiratory chain function and mitochondrial dynamics. More recent publications also described a link between Parkin and mitophagy. Methodology/Principal Findings In this study, we investigated the impact of Parkin mutations on mitochondrial function and morphology in a human cellular model. Fibroblasts were obtained from three members of an Italian PD family with two mutations in Parkin (homozygous c.1072delT, homozygous delEx7, compound-heterozygous c.1072delT/delEx7), as well as from two relatives without mutations. Furthermore, three unrelated compound-heterozygous patients (delEx3-4/duplEx7-12, delEx4/c.924C>T and delEx1/c.924C>T) and three unrelated age-matched controls were included. Fibroblasts were cultured under basal or paraquat-induced oxidative stress conditions. ATP synthesis rates and cellular levels were detected luminometrically. Activities of complexes I-IV and citrate synthase were measured spectrophotometrically in mitochondrial preparations or cell lysates. The mitochondrial membrane potential was measured with 5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethylbenzimidazolylcarbocyanine iodide. Oxidative stress levels were investigated with the OxyBlot technique. The mitochondrial network was investigated immunocytochemically and the degree of branching was determined with image processing methods. We observed a decrease in the production and overall concentration of ATP coinciding with increased mitochondrial mass in Parkin-mutant fibroblasts. After an oxidative insult, the membrane potential decreased in patient cells but not in controls. We further determined higher levels of oxidized proteins in the mutants both under basal and stress conditions. The degree of mitochondrial network branching was comparable in mutants and controls under basal conditions and decreased to a similar extent under paraquat-induced stress. Conclusions Our results indicate that Parkin mutations cause abnormal mitochondrial function and morphology in non-neuronal human cells.

Prevalence and Clinical Features of Common LRRK2 Mutations in Australians with Parkinson's Disease

We determined the prevalence of two common leucine‐rich repeat kinase 2 (LRRK2) gene mutations in Australian patients with Parkinsons disease (PD). Of 830 affected patients, eight were heterozygous for the G2019S mutation, and two were heterozygous for the R1441H (4,322 G > A) mutation. In addition, one familial patient had a novel A1442P (4,324 G > C) mutation. Haplotype analysis showed that all LRRK2 G2019S‐positive individuals carried the common founder haplotype 1 and a putative founder haplotype for the R1441H mutation carriers. Clinically, patients with LRRK2 mutations had typical levodopa responsive Parkinsonism with tremor being the commonest presenting feature. Patients with the G2019S mutation in our series had a similar age of onset of symptoms when compared with patients with other LRRK2 mutations or sporadic PD, although they were more likely to have a family history of PD (2.4% of Australian patients with familial PD and 0.3% of Australian patients with sporadic PD). Our results demonstrate that the G2019S mutation carriers share the same ancestors who migrated to Australia originally from Europe and that other LRRK2 mutations (R1441H and A1442P) can be found in this population.

Mitochondrial DNA haplogroups J and K are not protective for Parkinson's disease in the Australian community

MtDNA haplogroups J and K have been associated with a decreased risk of developing Parkinsons disease (PD). To confirm this finding, we compared the distribution of mtDNA haplogroups J and K in a large sample of Australian patients with PD (n = 890) to population‐based controls (n = 3,491). We assigned subjects to haplogroups J or K using standard PCR/RFLP techniques. Of the 890 subjects with PD, 10.6% were haplogroup J (95% CI 8.6–12.8, n = 94) and 7.1% were haplogroup K (95% CI 5.5–8.9, n = 63). In our controls, 10.2% belonged to haplogroup J (95% CI 9.2–11.2, n = 356), and 7.8% were in haplogroup K (95% CI 6.9–8.7, n = 272). There was no significant difference in the prevalence of mtDNA haplogroup J or K in PD patients compared to population‐based controls. Our findings indicate that mtDNA haplogroups J and K are not associated with a lower risk of PD.

POLG mutations in Australian patients with mitochondrial disease

The nuclear POLG gene encodes the catalytic subunit of DNA polymerase gamma (polγ), the only polymerase involved in the replication and proofreading of mitochondrial DNA. As a consequence, POLG mutations can cause disease through impaired replication of mitochondrial DNA. To date, over 150 different mutations have been identified, with a growing number of associated phenotypes described. The aim of this study was to determine the prevalence of POLG mutations in an adult population of Australian patients with mitochondrial disease, displaying symptoms commonly associated with POLG‐related diseases.

SPAST mutations in Australian patients with hereditary spastic paraplegia

Hereditary spastic paraplegia (HSP) is often caused by mutations in the SPAST gene. The frequency of SPAST mutations causing HSP in Australian patients is currently unknown.

A NOVEL CLCN1 MUTATION (G1652A) CAUSING A MILD PHENOTYPE OF THOMSEN DISEASE

We investigated a 62‐year‐old man who had mild clinical features of myotonia congenita. He was found to have a novel heterozygous G‐to‐A nucleotide substitution at position 1652 in exon 15 of the CLCN1 gene. Clinicogenetic studies performed on his family revealed that his asymptomatic son also shared the mutation. We conclude that a novel chloride channel mutation (G1652A) has caused a mild form of autosomal‐dominant myotonia congenita (Thomsen disease) in this family. Muscle Nerve, 2010