Ruey-Meei Wu

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.

Lrrk2 pathogenic substitutions in Parkinson's disease

Leucine-rich repeat kinase 2 (LRRK2) mutations have been implicated in autosomal dominant parkinsonism, consistent with typical levodopa-responsive Parkinsons disease. The gene maps to chromosome 12q12 and encodes a large, multifunctional protein. To identify novel LRRK2 mutations, we have sequenced 100 affected probands with family history of parkinsonism. Semiquantitative analysis was also performed in all probands to identify LRRK2 genomic multiplication or deletion. In these kindreds, referred from movement disorder clinics in many parts of Europe, Asia, and North America, parkinsonism segregates as an autosomal dominant trait. All 51 exons of the LRRK2 gene were analyzed and the frequency of all novel sequence variants was assessed within controls. The segregation of mutations with disease has been examined in larger, multiplex families. Our study identified 26 coding variants, including 15 nonsynonymous amino acid substitutions of which three affect the same codon (R1441C, R1441G, and R1441H). Seven of these coding changes seem to be pathogenic, as they segregate with disease and were not identified within controls. No multiplications or deletions were identified.

Association of LRRK2 exonic variants with susceptibility to Parkinson's disease: A case-control study

BACKGROUND Background The leucine-rich repeat kinase 2 gene (LRRK2) harbours highly penetrant mutations that are linked to familial parkinsonism. However, the extent of its polymorphic variability in relation to risk of Parkinsons disease (PD) has not been assessed systematically. We therefore assessed the frequency of LRRK2 exonic variants in individuals with and without PD, to investigate the role of the variants in PD susceptibility. METHODS LRRK2 was genotyped in patients with PD and controls from three series (white, Asian, and Arab-Berber) from sites participating in the Genetic Epidemiology of Parkinsons Disease Consortium. Genotyping was done for exonic variants of LRRK2 that were identified through searches of literature and the personal communications of consortium members. Associations with PD were assessed by use of logistic regression models. For variants that had a minor allele frequency of 0·5% or greater, single variant associations were assessed, whereas for rarer variants information was collapsed across variants. FINDINGS 121 exonic LRRK2 variants were assessed in 15 540 individuals: 6995 white patients with PD and 5595 controls, 1376 Asian patients and 962 controls, and 240 Arab-Berber patients and 372 controls. After exclusion of carriers of known pathogenic mutations, new independent risk associations were identified for polymorphic variants in white individuals (M1646T, odds ratio 1·43, 95% CI 1·15-1·78; p=0·0012) and Asian individuals (A419V, 2·27, 1·35-3·83; p=0·0011). A protective haplotype (N551K-R1398H-K1423K) was noted at a frequency greater than 5% in the white and Asian series, with a similar finding in the Arab-Berber series (combined odds ratio 0·82, 0·72-0·94; p=0·0043). Of the two previously reported Asian risk variants, G2385R was associated with disease (1·73, 1·20-2·49; p=0·0026), but no association was noted for R1628P (0·62, 0·36-1·07; p=0·087). In the Arab-Berber series, Y2189C showed potential evidence of risk association with PD (4·48, 1·33-15·09; p=0·012). INTERPRETATION The results for LRRK2 show that several rare and common genetic variants in the same gene can have independent effects on disease risk. LRRK2, and the pathway in which it functions, is important in the cause and pathogenesis of PD in a greater proportion of patients with this disease than previously believed. These results will help discriminate those patients who will benefit most from therapies targeted at LRRK2 pathogenic activity. FUNDING Michael J Fox Foundation and National Institutes of Health.

Analysis of Lrrk2 R1628P as a risk factor for Parkinson's disease

Common genetic variants that increase the risk for Parkinsons disease may differentiate patient subgroups and influence future individualized therapeutic strategies. Herein we show evidence for leucine‐rich repeat kinase 2 (LRRK2) c.4883G>C (R1628P) as a risk factor in ethnic Chinese populations. A study of 1,986 individuals from 3 independent centers in Taiwan and Singapore demonstrates that Lrrk2 R1628P increases risk for Parkinsons disease (odds ratio, 1.84; 95% confidence interval, 1.20–2.83; p = 0.006). Haplotype analysis suggests an ancestral founder for carriers approximately 2,500 years ago. These findings support the importance of LRRK2 variants in sporadic Parkinsons disease. Ann Neurol 2008

DNAJC13 mutations in Parkinson disease

A Saskatchewan multi-incident family was clinically characterized with Parkinson disease (PD) and Lewy body pathology. PD segregates as an autosomal-dominant trait, which could not be ascribed to any known mutation. DNA from three affected members was subjected to exome sequencing. Genome alignment, variant annotation and comparative analyses were used to identify shared coding mutations. Sanger sequencing was performed within the extended family and ethnically matched controls. Subsequent genotyping was performed in a multi-ethnic case-control series consisting of 2928 patients and 2676 control subjects from Canada, Norway, Taiwan, Tunisia, and the USA. A novel mutation in receptor-mediated endocytosis 8/RME-8 (DNAJC13 p.Asn855Ser) was found to segregate with disease. Screening of cases and controls identified four additional patients with the mutation, of which two had familial parkinsonism. All carriers shared an ancestral DNAJC13 p.Asn855Ser haplotype and claimed Dutch-German-Russian Mennonite heritage. DNAJC13 regulates the dynamics of clathrin coats on early endosomes. Cellular analysis shows that the mutation confers a toxic gain-of-function and impairs endosomal transport. DNAJC13 immunoreactivity was also noted within Lewy body inclusions. In late-onset disease which is most reminiscent of idiopathic PD subtle deficits in endosomal receptor-sorting/recycling are highlighted by the discovery of pathogenic mutations VPS35, LRRK2 and now DNAJC13. With this latest discovery, and from a neuronal perspective, a temporal and functional ecology is emerging that connects synaptic exo- and endocytosis, vesicular trafficking, endosomal recycling and the endo-lysosomal degradative pathway. Molecular deficits in these processes are genetically linked to the phenotypic spectrum of parkinsonism associated with Lewy body pathology.

Apparent antioxidant effect of l-deprenyl on hydroxyl radical formation and nigral injury elicited by MPP+ in vivo

Using a modified microdialysis procedure, we confirmed that intrastriatal administration of 1-methyl-4-phenylpyridinium ion (MPP+) induced a sustained overflow of dopamine accompanied by increased formation of hydroxyl free radicals (.OH) as reflected by salicylate hydroxylation. Pretreatment with l-deprenyl (selegiline 60 pmol, intrastriatal perfusion) significantly decreased the .OH formation elicited by MPP+ (75 nmol). There was a small decrease of dopamine efflux and an insignificant change of the ratio of 3,4-dihydroxyphenylacetic acid (DOPAC)/dopamine following l-deprenyl pretreatment. These in vivo findings support prior in vitro data that an unique antioxidant property of l-deprenyl may be independent of its inhibition of type B monoamine oxidase. In addition, intranigral co-administration of l-deprenyl (4.2 nmol) with MPP+ (4.2 nmol) completely protected nigral neurons from probable oxidative injuries induced by MPP+ (4.2 nmol), as reflected by a near 50% loss of striatal dopamine ipsilateral to the side of infusion of drug into the substantia nigra. This apparent neuroprotective effect of l-deprenyl on midbrain nigral neurons was also confirmed by histological findings. The present in vivo data clearly demonstrate that l-deprenyl can protect nigral neurons against dopamine neurotoxicity produced by MPP+, as suggested by an earlier in vitro study. Thus, l-deprenyl can preserve the function of MPP(+)-damaged nigral neurons perhaps by its apparent antioxidant property in addition to its blockade of the bioactivation of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) to toxic pyridinium metabolites by type B monoamine oxidase.

Minimal Detectable Change of the Timed “Up & Go” Test and the Dynamic Gait Index in People With Parkinson Disease

Background The minimal detectable change (MDC) is the smallest amount of difference in individual scores that represents true change (beyond random measurement error). The MDCs of the Timed “Up & Go” Test (TUG) and the Dynamic Gait Index (DGI) in people with Parkinson disease (PD) are largely unknown, limiting the interpretability of the change scores of both measures. Objective The purpose of this study was to estimate the MDCs of the TUG and the DGI in people with PD. Design This investigation was a prospective cohort study. Methods Seventy-two participants were recruited from special clinics for movement disorders at a university hospital. Their mean age was 67.5 years, and 61% were men. All participants completed the TUG and the DGI assessments twice, about 14 days apart. The MDC was calculated from the standard error of measurement. The percentage MDC (MDC%) was calculated as the MDC divided by the mean of all scores for the sample. Furthermore, the intraclass correlation coefficient was used to examine the reproducibility between testing sessions (test-retest reliability). Results The respective MDC and MDC% of the TUG were 3.5 seconds and 29.8, and those of the DGI were 2.9 points and 13.3. The test-retest reliability values for the TUG and the DGI were high; the intraclass correlation coefficients were .80 and .84, respectively. Limitations The study sample was a convenience sample, and the participants had mild to moderately severe PD. Conclusions The results showed that the TUG and the DGI have generally acceptable random measurement error and test-retest reliability. These findings should help clinicians and researchers determine whether a change in an individual patient with PD is a true change.

In Vivo Generation of Hydroxyl Radicals and MPTP‐Induced Dopaminergic Toxicity in the Basal Ganglia

The in vivo generation of .OH free radicals in specific brain regions can be measured by intracerebral microdialysis perfusion of salicylate, avoiding many of the pitfalls inherent in systemic administration of salicylate. Direct infusion of salicylate into the brain can minimize the hepatic hydroxylation of salicylate and its contribution to brain levels of 2,5-DHBA. Levels of 2,5-DHBA detected in the brain dialysate may reflect the .OH adduct plus some enzymatic hydroxylation of salicylate in the brain. After minimizing the contribution of enzyme and/or blood-borne 2,5-DHBA, the present data demonstrate the validity of the use of 2,3-DHBA and apparently 2,5-DHBA as indices of .OH formation in the brain. Therefore, intracranial microdialysis of salicylic acid and measurement of 2,3-DHBA appears to be a useful .OH trapping procedure for monitoring the time course of .OH generation in the extracellular fluid of the brain. These results indicate that nonenzymatic and/or enzymatic oxidation of the dopamine released by MPTP analogues in the extracellular fluid may play a key role in the generation of .OH free radicals in the iron-rich basal ganglia. Moreover, a site-specific generation of cytotoxic .OH free radicals and quinone/semiquinone radicals in the striatum may cause the observed lipid peroxidation, calcium overload, and retrograde degeneration of nigrostriatal neurons. This free-radical-induced nigral injury can be suppressed by antioxidants (i.e., U-78517F, DMSO, and deprenyl) and possibly hypothermia as well. In the future, this in vivo detection of .OH generation may be useful in answering some of the fundamental questions concerning the relevance of oxidants and antioxidants in neurodegenerative disorders during aging. It could also pave the way for the research and development of novel neuroprotective antioxidants and strategies for the early or preventive treatment of neurodegenerative disorders, such as Parkinsons disease (Wu et al., this issue), amyotrophic lateral sclerosis, head trauma, and possibly Alzheimers cognitive dysfunction as well. In conclusion, this in vivo free-radical trapping procedure provides evidence to support a current working hypothesis that a site-specific formation of cytotoxic .OH free radicals in the basal ganglia may be one of the neurotoxic mechanisms underlying nigrostriatal degeneration and Parkinsonism caused by the dopaminergic neurotoxin MPTP. Addendum added in proof: The controversy concerning possible neurotoxic and/or neuroprotective roles of NO. in cell cultures was discussed and debated at the symposium (Wink et al., this issue; Dawson et al., this issue; Lipton et al., this issue).(ABSTRACT TRUNCATED AT 400 WORDS)

LRRK2 G2019S Mutation Induces Dendrite Degeneration through Mislocalization and Phosphorylation of Tau by Recruiting Autoactivated GSK3β

Intraneuronal tau aggregations are distinctive pathological features of Parkinsons disease (PD) with autosomal-dominant mutations in leucine-rich repeat kinase 2 (LRRK2). The most prevalent LRRK2 mutation, G2019S (glycine to serine substitution at amino acid 2019), causes neurite shrinkage through unclear pathogenetic mechanisms. We found that expression of G2019S mutant in Drosophila dendritic arborization neurons induces mislocalization of the axonal protein tau in dendrites and causes dendrite degeneration. G2019S-induced dendrite degeneration is suppressed by reducing the level of tau protein and aggravated by tau coexpression. Additional genetic analyses suggest that G2019S and tau function synergistically to cause microtubule fragmentation, inclusion formation, and dendrite degeneration. Mechanistically, hyperactivated G2019S promotes tau phosphorylation at the T212 site by the Drosophila glycogen synthase kinase 3β homolog Shaggy (Sgg). G2019S increases the recruitment of autoactivated Sgg, thus inducing hyperphosphorylation and mislocalization of tau with resultant dendrite degeneration.

Gene therapy for aromatic L-amino acid decarboxylase deficiency.

Gene therapy can restore some motor function in patients with aromatic l-amino acid decarboxylase deficiency. Gene Therapy for AADC Deficiency Patients with aromatic l-amino acid decarboxylase (AADC) deficiency cannot produce the neurotransmitter dopamine from its precursor l-DOPA in the brain. Dopamine is a crucial molecule required for normal motor function. There are few treatment options for AADC deficiency, and most patients afflicted with this rare disease die in childhood. In a phase 1 clinical trial, Hwu and colleagues use an adeno-associated virus (AAV) type 2 vector to deliver the AADC gene into a brain area called the putamen in four children with AADC deficiency. Although at first the patients exhibited dyskinesias (abnormal muscle movements), these resolved after a few months and the patients showed improved motor function. One patient after 16 months was able to stand, and the other three patients were able to sit upright with support. Several other symptoms improved as well including mood and oculogyric crises. There were a number of translational challenges for this gene therapy clinical trial. For example, the authors had to work out how to deliver the viral vector carrying the therapeutic gene directly into the putamen, and even then only a small part of the putamen became transduced with the AADC gene. Also, because the patients’ brains had not been able to make dopamine, it was not clear how the neurons would respond once dopamine started to be produced. Despite these challenges, this first-in-human gene therapy clinical trial suggests that targeting localized areas in the brain with a therapeutic gene delivered by an AAV vector could help ameliorate the symptoms of AADC deficiency and may also be useful for treating other diseases caused by lack of a crucial enzyme in brain tissue. Aromatic l-amino acid decarboxylase (AADC) is required for the synthesis of the neurotransmitters dopamine and serotonin. Children with defects in the AADC gene show compromised development, particularly in motor function. Drug therapy has only marginal effects on some of the symptoms and does not change early childhood mortality. Here, we performed adeno-associated viral vector–mediated gene transfer of the human AADC gene bilaterally into the putamen of four patients 4 to 6 years of age. All of the patients showed improvements in motor performance: One patient was able to stand 16 months after gene transfer, and the other three patients achieved supported sitting 6 to 15 months after gene transfer. Choreic dyskinesia was observed in all patients, but this resolved after several months. Positron emission tomography revealed increased uptake by the putamen of 6-[18F]fluorodopa, a tracer for AADC. Cerebrospinal fluid analysis showed increased dopamine and serotonin levels after gene transfer. Thus, gene therapy targeting primary AADC deficiency is well tolerated and leads to improved motor function.