Deletion of the WD40 Domain of LRRK2 in Zebrafish Causes Parkinsonism-Like Loss of Neurons and Locomotive Defect

LRRK2 plays an important role in Parkinson's disease (PD), but its biological functions are largely unknown. Here, we cloned the homolog of human LRRK2, characterized its expression, and investigated its biological functions in zebrafish. The blockage of zebrafish LRRK2 (zLRRK2) protein by morpholinos caused embryonic lethality and severe developmental defects such as growth retardation and loss of neurons. In contrast, the deletion of the WD40 domain of zLRRK2 by morpholinos targeting splicing did not induce severe embryonic developmental defects; rather it caused Parkinsonism-like phenotypes, including loss of dopaminergic neurons in diencephalon and locomotion defects. These neurodegenerative and locomotion defects could be rescued by over-expressing zLRRK2 or hLRRK2 mRNA. The administration of L-dopa could also rescue the locomotion defects, but not the neurodegeneration. Taken together, our results demonstrate that zLRRK2 is an ortholog of hLRRK2 and that the deletion of WD40 domain of zLRRK2 provides a disease model for PD

Mitochondrial Uncoupling Protein-2 (UCP2) Mediates Leptin Protection Against MPP+ Toxicity in Neuronal Cells

Mitochondrial dysfunction is involved in the pathogenesis of neurodegenerative diseases, including Parkinson’s disease (PD). Uncoupling proteins (UCPs) delink ATP production from biofuel oxidation in mitochondria to reduce oxidative stress. UCP2 is expressed in brain, and has neuroprotective effects under various toxic insults. We observed induction of UCP2 expression by leptin in neuronal cultures, and hypothesize that leptin may preserve neuronal survival via UCP2. We showed that leptin preserved cell survival in neuronal SH-SY5Y cells against MPP+ toxicity (widely used in experimental Parkinsonian models) by maintaining ATP levels and mitochondrial membrane potential (MMP); these effects were accompanied by increased UCP2 expression. Leptin had no effect in modulating reactive oxygen species levels. Stable knockdown of UCP2 expression reduced ATP levels, and abolished leptin protection against MPP+-induced mitochondrial depolarization, ATP deficiency, and cell death, indicating that UCP2 is critical in mediating these neuroprotective effects of leptin against MPP+ toxicity. Interestingly, UCP2 knockdown increased UCP4 expression, but not of UCP5. Our findings show that leptin preserves cell survival by maintaining MMP and ATP levels mediated through UCP2 in MPP+-induced toxicity

The role of UCP5 in mitochondrial dysfunction in Parkinsonian models

published_or_final_versionMedicineDoctoralDoctor of Philosoph

Naturally Occurring Anti-Escherichia coli Protein Antibodies in the Sera of Healthy Humans Cause Analytical Interference in a Recombinant Nucleocapsid Protein-Based Enzyme-Linked Immunosorbent Assay for Serodiagnosis of Severe Acute Respiratory Syndrome

We reported the analytical interference of anti-Escherichia coli protein (EP) antibodies in human sera and residual EP in a recombinant nucleocapsid protein-based enzyme-linked immunosorbent assay as a possible source of false positives in severe acute respiratory syndrome serodiagnosis. The rate of false positives was significantly reduced by adding mouse anti-EP antiserum in the blocking step

Genome-Wide Linkage, Exome Sequencing and Functional Analyses Identify <i>ABCB6</i> as the Pathogenic Gene of Dyschromatosis Universalis Hereditaria

<div><p>Background</p><p>As a genetic disorder of abnormal pigmentation, the molecular basis of dyschromatosis universalis hereditaria (DUH) had remained unclear until recently when ABCB6 was reported as a causative gene of DUH.</p><p>Methodology</p><p>We performed genome-wide linkage scan using Illumina Human 660W-Quad BeadChip and exome sequencing analyses using Agilent SureSelect Human All Exon Kits in a multiplex Chinese DUH family to identify the pathogenic mutations and verified the candidate mutations using Sanger sequencing. Quantitative RT-PCR and Immunohistochemistry was performed to verify the expression of the pathogenic gene, Zebrafish was also used to confirm the functional role of ABCB6 in melanocytes and pigmentation.</p><p>Results</p><p>Genome-wide linkage (assuming autosomal dominant inheritance mode) and exome sequencing analyses identified <i>ABCB6</i> as the disease candidate gene by discovering a coding mutation (c.1358C>T; p.Ala453Val) that co-segregates with the disease phenotype. Further mutation analysis of <i>ABCB6</i> in four other DUH families and two sporadic cases by Sanger sequencing confirmed the mutation (c.1358C>T; p.Ala453Val) and discovered a second, co-segregating coding mutation (c.964A>C; p.Ser322Lys) in one of the four families. Both mutations were heterozygous in DUH patients and not present in the 1000 Genome Project and dbSNP database as well as 1,516 unrelated Chinese healthy controls. Expression analysis in human skin and mutagenesis interrogation in zebrafish confirmed the functional role of <i>ABCB6</i> in melanocytes and pigmentation. Given the involvement of <i>ABCB6</i> mutations in coloboma, we performed ophthalmological examination of the DUH carriers of <i>ABCB6</i> mutations and found ocular abnormalities in them.</p><p>Conclusion</p><p>Our study has advanced our understanding of DUH pathogenesis and revealed the shared pathological mechanism between pigmentary DUH and ocular coloboma.</p></div

Phenotypes of the microinjected zebrafish.

<p>Morpholinos against exon 6 and exon 8 of <i>zABCB6</i> and wildtype <i>zABCB6</i> mRNA transcript (in case of rescue) were designed and injected into one- to two-cell stage embryos. No obvious phenotypic changes were observed in the morphants (C and D) and rescued (E and F) embryos after 5 days (5pdf) with reference to their uninjected counterparts (A and B).</p

Number of mature melanocytes in the head region in the morphants and rescued embryos (5 dpf).

<p>Immobilized embryos were dorsally oriented and the number of mature melanocytes was counted in the defined region of the head. Reduction in melanocyte number was observed in both exon 6- and exon 8-morphants (B and E). Co-injection of wildtype <i>hABCB6</i> mRNA transcript significantly rescued the loss of melanocyte phenotype by increasing the number of mature melanocytes (C and F).</p

Family trees and clinical manifestations.

<p><b>A</b> Shown are the pedigree of family 1 and 2 with autosomal dominant DUH. “#”represents the individuals used in exome sequencing analysis, “☆”represents the individuals used in the linkage analysis; “▴”represents the individuals subjected to Sanger sequencing analysis. <b>B</b>: Clinical manifestations of DUH patients, both hands and abdomen with hyper- and hypo-pigmented macules in variable distribution and patterns.</p