Justus C. Dachsel

VPS35 Mutations in Parkinson Disease

The identification of genetic causes for Mendelian disorders has been based on the collection of multi-incident families, linkage analysis, and sequencing of genes in candidate intervals. This study describes the application of next-generation sequencing technologies to a Swiss kindred presenting with autosomal-dominant, late-onset Parkinson disease (PD). The family has tremor-predominant dopa-responsive parkinsonism with a mean onset of 50.6 ± 7.3 years. Exome analysis suggests that an aspartic-acid-to-asparagine mutation within vacuolar protein sorting 35 (VPS35 c.1858G>A; p.Asp620Asn) is the genetic determinant of disease. VPS35 is a central component of the retromer cargo-recognition complex, is critical for endosome-trans-golgi trafficking and membrane-protein recycling, and is evolutionarily highly conserved. VPS35 c.1858G>A was found in all affected members of the Swiss kindred and in three more families and one patient with sporadic PD, but it was not observed in 3,309 controls. Further sequencing of familial affected probands revealed only one other missense variant, VPS35 c.946C>T; (p.Pro316Ser), in a pedigree with one unaffected and two affected carriers, and thus the pathogenicity of this mutation remains uncertain. Retromer-mediated sorting and transport is best characterized for acid hydrolase receptors. However, the complex has many types of cargo and is involved in a diverse array of biologic pathways from developmental Wnt signaling to lysosome biogenesis. Our study implicates disruption of VPS35 and retromer-mediated trans-membrane protein sorting, rescue, and recycling in the neurodegenerative process leading to PD.

DCTN1 mutations in Perry syndrome

Perry syndrome consists of early-onset parkinsonism, depression, severe weight loss and hypoventilation, with brain pathology characterized by TDP-43 immunostaining. We carried out genome-wide linkage analysis and identified five disease-segregating mutations affecting the CAP-Gly domain of dynactin (encoded by DCTN1) in eight families with Perry syndrome; these mutations diminish microtubule binding and lead to intracytoplasmic inclusions. Our findings show that DCTN1 mutations, previously associated with motor neuron disease, can underlie the selective vulnerability of other neuronal populations in distinct neurodegenerative disorders.

Translation Initiator EIF4G1 Mutations in Familial Parkinson Disease

Genome-wide analysis of a multi-incident family with autosomal-dominant parkinsonism has implicated a locus on chromosomal region 3q26-q28. Linkage and disease segregation is explained by a missense mutation c.3614G>A (p.Arg1205His) in eukaryotic translation initiation factor 4-gamma (EIF4G1). Subsequent sequence and genotype analysis identified EIF4G1 c.1505C>T (p.Ala502Val), c.2056G>T (p.Gly686Cys), c.3490A>C (p.Ser1164Arg), c.3589C>T (p.Arg1197Trp) and c.3614G>A (p.Arg1205His) substitutions in affected subjects with familial parkinsonism and idiopathic Lewy body disease but not in control subjects. Despite different countries of origin, persons with EIF4G1 c.1505C>T (p.Ala502Val) or c.3614G>A (p.Arg1205His) mutations appear to share haplotypes consistent with ancestral founders. eIF4G1 p.Ala502Val and p.Arg1205His disrupt eIF4E or eIF3e binding, although the wild-type protein does not, and render mutant cells more vulnerable to reactive oxidative species. EIF4G1 mutations implicate mRNA translation initiation in familial parkinsonism and highlight a convergent pathway for monogenic, toxin and perhaps virally-induced Parkinson disease.

Impaired dopaminergic neurotransmission and microtubule-associated protein tau alterations in human LRRK2 transgenic mice.

Mutations in the Leucine Rich Repeat Kinase 2 (LRRK2) gene, first described in 2004 have now emerged as the most important genetic finding in both autosomal dominant and sporadic Parkinsons disease (PD). While a formidable research effort has ensued since the initial gene discovery, little is known of either the normal or the pathological role of LRRK2. We have created lines of mice that express human wild-type (hWT) or G2019S Lrrk2 via bacterial artificial chromosome (BAC) transgenesis. In vivo analysis of the dopaminergic system revealed abnormal dopamine neurotransmission in both hWT and G2019S transgenic mice evidenced by a decrease in extra-cellular dopamine levels, which was detected without pharmacological manipulation. Immunopathological analysis revealed changes in localization and increased phosphorylation of microtubule binding protein tau in G2019S mice. Quantitative biochemical analysis confirmed the presence of differential phospho-tau species in G2019S mice but surprisingly, upon dephosphorylation the tau isoform banding pattern in G2019S mice remained altered. This suggests that other post-translational modifications of tau occur in G2019S mice. We hypothesize that Lrrk2 may impact on tau processing which subsequently leads to increased phosphorylation. Our models will be useful for further understanding of the mechanistic actions of LRRK2 and future therapeutic screening.

Parkinsonism, Lrrk2 G2019S, and tau neuropathology.

Lrrk2 G2019S is predominantly associated with α-synuclein–immunopositive Lewy body pathology. We have identified Family SK where Lrrk2 G2019S segregates with slowly progressive parkinsonism and the affected proband has tau-immunopositive neurofibrillary tangle pathology. Thus α-synucleinopathy and tauopathy, the predominant pathologies associated with parkinsonism, may be alternate outcomes of the same underlying genetic cause. Intriguingly, we observe no evidence of a direct interaction between either the tau or α-synuclein protein with Lrrk2.

LRRK2 and Parkinson Disease

OBJECTIVES To review the molecular genetics and functional biology of leucine-rich repeat kinase 2 (LRRK2) in parkinsonism and to summarize the opportunities and challenges to develop interventions for Parkinson disease (PD) based on this genetic insight. DATA SOURCES Publications cited are focused on LRRK2 biology between 2004 and March 2009. STUDY SELECTION Literature selected was based on original contributions, seminal observations, and thoughtful reviews. DATA EXTRACTION Unless stated otherwise, data was primarily abstracted from peer-reviewed literature appearing on PubMed. DATA SYNTHESIS Genetic mutations that predispose PD are diagnostically useful in early or atypical presentations. The molecular pathways identified suggest therapeutic interventions for Lrrk2 and idiopathic PD and the rationale and opportunity to develop physiologically relevant biomarkers and experimental models with which to test them. CONCLUSIONS Both affected and asymptomatic LRRK2 carriers now provide the opportunity to define the natural history of PD. This includes the frequency, penetrance, and rate of motor symptoms, nonmotor comorbidities, and their associated biomarkers.

LRRK2 knockout mice have an intact dopaminergic system but display alterations in exploratory and motor co-ordination behaviors

Mutations in the LRRK2 gene are the most common cause of genetic Parkinson’s disease. Although the mechanisms behind the pathogenic effects of LRRK2 mutations are still not clear, data emerging from in vitro and in vivo models suggests roles in regulating neuronal polarity, neurotransmission, membrane and cytoskeletal dynamics and protein degradation.We created mice lacking exon 41 that encodes the activation hinge of the kinase domain of LRRK2. We have performed a comprehensive analysis of these mice up to 20 months of age, including evaluation of dopamine storage, release, uptake and synthesis, behavioral testing, dendritic spine and proliferation/neurogenesis analysis.Our results show that the dopaminergic system was not functionally comprised in LRRK2 knockout mice. However, LRRK2 knockout mice displayed abnormal exploratory activity in the open-field test. Moreover, LRRK2 knockout mice stayed longer than their wild type littermates on the accelerated rod during rotarod testing. Finally, we confirm that loss of LRRK2 caused degeneration in the kidney, accompanied by a progressive enhancement of autophagic activity and accumulation of autofluorescent material, but without evidence of biphasic changes.

Novel Pathogenic LRRK2 p.Asn1437His Substitution in Familial Parkinson's Disease

Genealogical investigation of a large Norwegian family (F04) with autosomal dominant parkinsonism has identified 18 affected family members over four generations. Genetic studies have revealed a novel pathogenic LRRK2 mutation c.4309 A>C (p.Asn1437His) that co‐segregates with disease manifestation (LOD = 3.15, θ = 0). Affected carriers have an early age at onset (48 ± 7.7 SD years) and are clinically asymmetric and levodopa responsive. The variant was absent in 623 Norwegian control subjects. Further screening of patients from the same population identified one additional affected carrier (1 of 692) with familial parkinsonism who shares the same haplotype. The mutation is located within the Roc domain of the protein and enhances GTP‐binding and kinase activity, further implicating these activities as the mechanisms that underlie LRRK2‐linked parkinsonism.

Mutations in LRRK2 increase phosphorylation of peroxiredoxin 3 exacerbating oxidative stress‐induced neuronal death

Mutations in the leucine rich repeat kinase 2 (LRRK2) gene are responsible for autosomal dominant and sporadic Parkinson disease (PD), possibly exerting their effects via a toxic gain of function. A common p.G2019S mutation (rs34637584:A>G) is responsible for up to 30–40% of PD cases in some ethnic populations. Here, we show that LRRK2 interacts with human peroxiredoxin 3 (PRDX3), a mitochondrial member of the antioxidant family of thioredoxin (Trx) peroxidases. Importantly, mutations in the LRRK2 kinase domain significantly increased phosphorylation of PRDX3 compared to wild‐type. The increase in PRDX3 phosphorylation was associated with decreased peroxidase activity and increased death in LRRK2‐expressing but not in LRRK2‐depleted or vector‐transfected neuronal cells. LRRK2 mutants stimulated mitochondrial factors involved in apoptosis and induced production of reactive oxygen species (ROS) and oxidative modification of macromolecules. Furthermore, immunoblot and immunohistochemical analysis of postmortem human PD patients carrying the p.G2019S mutation showed a marked increase in phosphorylated PRDX3 (p‐PRDX3) relative to normal brain. We showed that LRRK2 mutations increase the inhibition of an endogenous peroxidase by phosphorylation promoting dysregulation of mitochondrial function and oxidative damage. Our findings provide a mechanistic link between the enhanced kinase activity of PD‐linked LRRK2 and neuronal cell death. 32:1390–1397, 2011. ©2011 Wiley Periodicals, Inc.

A comparative study of Lrrk2 function in primary neuronal cultures.

OBJECTIVE To assess the contribution of wild-type, mutant and loss of leucine-rich repeat kinase-2 (LRRK2; Lrrk2) on dendritic neuronal arborization. BACKGROUND LRRK2 mutations are recognized as the major genetic determinant of susceptibility to Parkinsons disease for which a cellular assay of Lrrk2 mutant function would facilitate the development of targeted molecular therapeutics. METHODS Dendritic neuronal arborization (neurite length, branching and the number of processes per cell) was quantified in primary hippocampal and midbrain cultures derived from five lines of recombinant LRRK2 mice, including human BAC wild-type and mutant overexpressors (Y1699C and G2019S), murine knock-out and G2019S knock-in animals. RESULTS Neuronal arborization in cultures from BAC Lrrk2 wild-type animals is comparable to non-transgenic littermate controls, despite high levels of human transgene expression. In contrast, primary neurons from both BAC mutant overexpressors presented with significantly reduced neuritic outgrowth and branching, although the total number of processes per cell remained comparable. The mutant-specific toxic gain-of-function observed in cultures from BAC mutant mice may be partially rescued by staurosporine treatment, a non-specific kinase inhibitor. In contrast, neuronal arborization is far more extensive in neuronal cultures derived from murine knock-out mice that lack endogenous Lrrk2 expression. In Lrrk2 G2019S knock-in mice, arguably the most physiologically relevant system, neuritic arborization is not impaired. CONCLUSIONS Impairment of neuritic arborization is an exaggerated, albeit mutant specific, consequence of Lrrk2 over-expression in primary cultures. The phenotype and assay described provides a means to develop therapeutic agents that modulate the toxic gain-of-function conferred by mutant Lrrk2.