Wencheng Liu

PINK1-dependent recruitment of Parkin to mitochondria in mitophagy

Phosphatase and tensin homolog (PTEN)-induced putative kinase 1 (PINK1) and PARK2/Parkin mutations cause autosomal recessive forms of Parkinsons disease. Upon a loss of mitochondrial membrane potential (ΔΨm) in human cells, cytosolic Parkin has been reported to be recruited to mitochondria, which is followed by a stimulation of mitochondrial autophagy. Here, we show that the relocation of Parkin to mitochondria induced by a collapse of ΔΨm relies on PINK1 expression and that overexpression of WT but not of mutated PINK1 causes Parkin translocation to mitochondria, even in cells with normal ΔΨm. We also show that once at the mitochondria, Parkin is in close proximity to PINK1, but we find no evidence that Parkin catalyzes PINK1 ubiquitination or that PINK1 phosphorylates Parkin. However, co-overexpression of Parkin and PINK1 collapses the normal tubular mitochondrial network into mitochondrial aggregates and/or large perinuclear clusters, many of which are surrounded by autophagic vacuoles. Our results suggest that Parkin, together with PINK1, modulates mitochondrial trafficking, especially to the perinuclear region, a subcellular area associated with autophagy. Thus by impairing this process, mutations in either Parkin or PINK1 may alter mitochondrial turnover which, in turn, may cause the accumulation of defective mitochondria and, ultimately, neurodegeneration in Parkinsons disease.

Mutant LRRK2R1441G BAC transgenic mice recapitulate cardinal features of Parkinson's disease

Mutations in leucine-rich repeat kinase 2 (LRRK2) are the most common genetic cause of Parkinsons disease. We created a LRRK2 transgenic mouse model that recapitulates cardinal features of the disease: an age-dependent and levodopa-responsive slowness of movement associated with diminished dopamine release and axonal pathology of nigrostriatal dopaminergic projection. These mice provide a valid model of Parkinsons disease and are a resource for the investigation of pathogenesis and therapeutics.

Pink1 regulates the oxidative phosphorylation machinery via mitochondrial fission

Mutations in PTEN-induced kinase 1 (PINK1), a mitochondrial Ser/Thr kinase, cause an autosomal recessive form of Parkinsons disease (PD), PARK6. To investigate the mechanism of PINK1 pathogenesis, we used the Drosophila Pink1 knockout (KO) model. In mitochondria isolated from Pink1-KO flies, mitochondrial respiration driven by the electron transport chain (ETC) is significantly reduced. This reduction is the result of a decrease in ETC complex I and IV enzymatic activity. As a consequence, Pink1-KO flies also display a reduced mitochondrial ATP synthesis. Because mitochondrial dynamics is important for mitochondrial function and Pink1-KO flies have defects in mitochondrial fission, we explored whether fission machinery deficits underlie the bioenergetic defect in Pink1-KO flies. We found that the bioenergetic defects in the Pink1-KO can be ameliorated by expression of Drp1, a key molecule in mitochondrial fission. Further investigation of the ETC complex integrity in wild type, Pink1-KO, PInk1-KO/Drp1 transgenic, or Drp1 transgenic flies indicates that the reduced ETC complex activity is likely derived from a defect in the ETC complex assembly, which can be partially rescued by increasing mitochondrial fission. Taken together, these results suggest a unique pathogenic mechanism of PINK1 PD: The loss of PINK1 impairs mitochondrial fission, which causes defective assembly of the ETC complexes, leading to abnormal bioenergetics.

PINK1 Defect Causes Mitochondrial Dysfunction, Proteasomal Deficit and α-Synuclein Aggregation in Cell Culture Models of Parkinson's Disease

Mutations in PTEN induced kinase 1 (PINK1), a mitochondrial Ser/Thr kinase, cause an autosomal recessive form of Parkinsons disease (PD), PARK6. Here, we report that PINK1 exists as a dimer in mitochondrial protein complexes that co-migrate with respiratory chain complexes in sucrose gradients. PARK6 related mutations do not affect this dimerization and its associated complexes. Using in vitro cell culture systems, we found that mutant PINK1 or PINK1 knock-down caused deficits in mitochondrial respiration and ATP synthesis. Furthermore, proteasome function is impaired with a loss of PINK1. Importantly, these deficits are accompanied by increased α-synclein aggregation. Our results indicate that it will be important to delineate the relationship between mitochondrial functional deficits, proteasome dysfunction and α-synclein aggregation.

Microglial internalization and degradation of pathological tau is enhanced by an anti-tau monoclonal antibody

Microglia have been shown to contribute to the clearance of brain amyloid β peptides (Aβ), the major component of amyloid plaques, in Alzheimer’s disease (AD). However, it is not known whether microglia play a similar role in the clearance of tau, the major component of neurofibrillary tangles (NFTs). We now report that murine microglia rapidly internalize and degrade hyperphosphorylated pathological tau isolated from AD brain tissue in a time-dependent manner in vitro. We further demonstrate that microglia readily degrade human tau species released from AD brain sections and eliminate NFTs from brain sections of P301S tauopathy mice. The anti-tau monoclonal antibody MC1 enhances microglia-mediated tau degradation in an Fc-dependent manner. Our data identify a potential role for microglia in the degradation and clearance of pathological tau species in brain and provide a mechanism explaining the potential therapeutic actions of passively administered anti-tau monoclonal antibodies.

Genomic View of Bipolar Disorder Revealed by Whole Genome Sequencing in a Genetic Isolate

Bipolar disorder is a common, heritable mental illness characterized by recurrent episodes of mania and depression. Despite considerable effort to elucidate the genetic underpinnings of bipolar disorder, causative genetic risk factors remain elusive. We conducted a comprehensive genomic analysis of bipolar disorder in a large Old Order Amish pedigree. Microsatellite genotypes and high-density SNP-array genotypes of 388 family members were combined with whole genome sequence data for 50 of these subjects, comprising 18 parent-child trios. This study design permitted evaluation of candidate variants within the context of haplotype structure by resolving the phase in sequenced parent-child trios and by imputation of variants into multiple unsequenced siblings. Non-parametric and parametric linkage analysis of the entire pedigree as well as on smaller clusters of families identified several nominally significant linkage peaks, each of which included dozens of predicted deleterious variants. Close inspection of exonic and regulatory variants in genes under the linkage peaks using family-based association tests revealed additional credible candidate genes for functional studies and further replication in population-based cohorts. However, despite the in-depth genomic characterization of this unique, large and multigenerational pedigree from a genetic isolate, there was no convergence of evidence implicating a particular set of risk loci or common pathways. The striking haplotype and locus heterogeneity we observed has profound implications for the design of studies of bipolar and other related disorders.

A population-based study of KCNH7 p.Arg394His and bipolar spectrum disorder

We conducted blinded psychiatric assessments of 26 Amish subjects (52 ± 11 years) from four families with prevalent bipolar spectrum disorder, identified 10 potentially pathogenic alleles by exome sequencing, tested association of these alleles with clinical diagnoses in the larger Amish Study of Major Affective Disorder (ASMAD) cohort, and studied mutant potassium channels in neurons. Fourteen of 26 Amish had bipolar spectrum disorder. The only candidate allele shared among them was rs78247304, a non-synonymous variant of KCNH7 (c.1181G>A, p.Arg394His). KCNH7 c.1181G>A and nine other potentially pathogenic variants were subsequently tested within the ASMAD cohort, which consisted of 340 subjects grouped into controls subjects and affected subjects from overlapping clinical categories (bipolar 1 disorder, bipolar spectrum disorder and any major affective disorder). KCNH7 c.1181G>A had the highest enrichment among individuals with bipolar spectrum disorder (χ2 = 7.3) and the strongest family-based association with bipolar 1 (P = 0.021), bipolar spectrum (P = 0.031) and any major affective disorder (P = 0.016). In vitro, the p.Arg394His substitution allowed normal expression, trafficking, assembly and localization of HERG3/Kv11.3 channels, but altered the steady-state voltage dependence and kinetics of activation in neuronal cells. Although our genome-wide statistical results do not alone prove association, cumulative evidence from multiple independent sources (parallel genome-wide study cohorts, pharmacological studies of HERG-type potassium channels, electrophysiological data) implicates neuronal HERG3/Kv11.3 potassium channels in the pathophysiology of bipolar spectrum disorder. Such a finding, if corroborated by future studies, has implications for mental health services among the Amish, as well as development of drugs that specifically target HERG3/Kv11.3.

Vectored Intracerebral Immunization with the Anti-Tau Monoclonal Antibody PHF1 Markedly Reduces Tau Pathology in Mutant Tau Transgenic Mice

Passive immunization with anti-tau monoclonal antibodies has been shown by several laboratories to reduce age-dependent tau pathology and neurodegeneration in mutant tau transgenic mice. These studies have used repeated high weekly doses of various tau antibodies administered systemically for several months and have reported reduced tau pathology of ∼40–50% in various brain regions. Here we show that direct intrahippocampal administration of the adeno-associated virus (AAV)-vectored anti-phospho-tau antibody PHF1 to P301S tau transgenic mice results in high and durable antibody expression, primarily in neurons. Hippocampal antibody levels achieved after AAV delivery were ∼50-fold more than those reported following repeated systemic administration. In contrast to systemic passive immunization, we observed markedly reduced (≥80–90%) hippocampal insoluble pathological tau species and neurofibrillary tangles following a single dose of AAV-vectored PHF1 compared with mice treated with an AAV-IgG control vector. Moreover, the hippocampal atrophy observed in untreated P301S mice was fully rescued by treatment with the AAV-vectored PHF1 antibody. Vectored passive immunotherapy with an anti-tau monoclonal antibody may represent a viable therapeutic strategy for treating or preventing such tauopathies as frontotemporal dementia, progressive supranuclear palsy, or Alzheimers disease. SIGNIFICANCE STATEMENT We have used an adeno-associated viral (AAV) vector to deliver the genes encoding an anti-phospho-tau monoclonal antibody, PHF1, directly to the brain of mice that develop neurodegeneration due to a tau mutation that causes frontotemporal dementia (FTD). When administered systemically, PHF1 has been shown to modestly reduce tau pathology and neurodegeneration. Since such antibodies do not readily cross the blood–brain barrier, we used an AAV vector to deliver antibody directly to the hippocampus and observed much higher antibody levels and a much greater reduction in tau pathology. Using AAV vectors to deliver antibodies like PHF1 directly to brain may constitute a novel approach to treating various neurodegenerative disorders, such as FTD and Alzheimers disease.

Kinase Signaling Dysfunction in Parkinson's Disease: A Reverse Genetic Approach in Drosophila

Abstract: Drosophila genetics is one of the most powerful tools in modern biology. For many years, the “forward genetic” approach using Drosophila has been extraordinarily successful in elucidating the molecular pathways of many physiological processes and behaviors. Recently, the “reverse genetic” approach in Drosophila is increasingly being developed as a major tool for research in biology, especially in the study of human diseases. Parkinsons disease (PD) is the second most common neurodegenerative disease. Kinase signaling has been directly implicated in PD pathogenesis. Mutations in PTEN-induced kinase 1 (PINK1) cause PARK6 type PD, in which mitochondrial deficits are at the center of pathogenesis. Mutations in leucine-rich repeat kinase 2 (LRRK2) are the most prevalent genetic cause of both familial (PARK8 type with autosomal dominant inheritance) and sporadic PD. To understand the mechanism of PINK1- and LRRK2- mediated pathogenesis, reverse-engineered Drosophila models have been critical tools. Here the authors will discuss the usage of Drosophila models in their and other laboratories, and share scientific insights that originate from these studies, and discuss their experimental results of the effect of PINK1 on proteasome function. The authors will also comment on the different approaches taken in these lines of investigation.

596. AAV Gene Delivery of the Anti-Tau Antibody PHF1 Reduces Brain Tau Pathology in P301L Mice

Anti-tau immunotherapy has been proposed as a promising therapy for various tauopathies including Alzheimers disease (AD), frontotemporal dementia (FTD) and progressive supranuclear palsy (PSP). However, there are limitations to passive immunization including the need for repeated administration and the very low levels of antibody that get into brain from the circulation. To circumvent the disadvantages of passive immunization, we have used serotype rh. 10 adeno-associated virus vectors to deliver an anti-tau monoclonal antibody PHF1, known to reduce tau pathology following passive immunization in several tauopathy mouse models directly to the CNS. To accomplish this, we stereotactically administered 1010 viral genomes (vg) of AAVrh. 10-PHF1 or AAVrh. 10-mCherry as a control, bilaterally into the hippocampus of young (3.5-month old) homozygous P301L mice, a model that develops robust tau pathology in an age- and brain region-dependent manner. Six months after injection, mice were sacrificed to measure the presence of anti-tau antibody as well as the effects of treatment on tau pathology in several brain regions using specific ELISAs and immunohistochemistry (IHC) for quantifying pathological and normal tau. In pilot experiments using wild-type C57BL/6 and P301L mice, we observed much higher levels of PHF1 antibody in the hippocampus when delivered via the AAVrh. 10 vector compared to passive immunization (3100-fold higher). We observed that treatment with AAVrh. 10-PHF1 resulted in a marked antibody-dependent reduction in tau pathology in P301L mice, including a highly significant reduction in tau pathology in the hippocampus (≥80-90% p=0.001) compared to the control vector. There was also no reduction in total tau measured by ELISA in any brain region examined. Based on these observations, AAVrh. 10 gene delivery of anti-tau monoclonal antibodies to the CNS may represent a novel therapeutic strategy for treating various tauopathies including FTD, PSP and AD.