A Caenorhabditis elegans model to study dopamine transporter deficiency syndrome.

Dopamine transporter deficiency syndrome (DTDS) is a novel autosomal recessive disorder caused by mutations in the dopamine transporter (DAT), which leads to the partial or total loss of function of the protein. DTDS is a pharmacoresistant syndrome and very little is known about its neurobiology, in part due to the lack of relevant animal models. The objective of this study was to establish the first animal model for DTDS with strong construct validity, using Caenorhabditis elegans, and to investigate the in vivo role played by DTDS-related mutations found in human DAT (hDAT). We took advantage of a C. elegans knockout for the hDAT orthologue, cedat-1, to obtain genetically humanized animals bearing hDAT, in the wild type and in two mutated forms (399delG and 941C>T), in a null background. In C. elegans transgenic animals expressing the human wild-type form, we observed a rescue of the knockout phenotype, as assessed using two well-established paradigms, known to be regulated by the endogenous uptake of dopamine or 6-hydroxydopamine (6-OHDA) by DAT. The less severe mutation (941C>T) was able to partially rescue only one of the knockout phenotypes, whereas the 399delG mutation impaired DAT function in both phenotypic paradigms. Our in vivo phenotypic findings demonstrate a functional conservation between human and nematode DAT and validate previous in vitro indications of the loss of function of hDAT in carriers of DTDS-related mutations. Taken together, these observations establish C. elegans as a novel animal model for fast and inexpensive screening of hDAT mutations in functional and in vivo tests.

Co-occurring WARS2 and CHRNA6 mutations in a child with a severe form of infantile parkinsonism.

OBJECTIVE: To investigate the molecular cause(s) underlying a severe form of infantile-onset parkinsonism and characterize functionally the identified variants. METHODS: A trio-based whole exome sequencing (WES) approach was used to identify the candidate variants underlying the disorder. In silico modeling, and in vitro and in vivo studies were performed to explore the impact of these variants on protein function and relevant cellular processes. RESULTS: WES analysis identified biallelic variants in WARS2, encoding the mitochondrial tryptophanyl tRNA synthetase (mtTrpRS), a gene whose mutations have recently been associated with multiple neurological phenotypes, including childhood-onset, levodopa-responsive or unresponsive parkinsonism in a few patients. A substantial reduction of mtTrpRS levels in mitochondria and reduced OXPHOS function was demonstrated, supporting their pathogenicity. Based on the infantile-onset and severity of the phenotype, additional variants were considered as possible genetic modifiers. Functional assessment of a selected panel of candidates pointed to a de novo missense mutation in CHRNA6, encoding the α6 subunit of neuronal nicotinic receptors, which are involved in the cholinergic modulation of dopamine release in the striatum, as a second event likely contributing to the phenotype. In silico, in vitro (Xenopus oocytes and GH4C1 cells) and in vivo (C. elegans) analyses demonstrated the disruptive effects of the mutation on acetylcholine receptor structure and function. CONCLUSION: Our findings consolidate the association between biallelic WARS2 mutations and movement disorders, and suggest CHRNA6 as a genetic modifier of the phenotype.

Silencing of Syntaxin 1A in the Dopaminergic Neurons Decreases the Activity of the Dopamine Transporter and Prevents Amphetamine-Induced Behaviors in C. elegans.

The dopamine transporter (DAT) is a cell membrane protein whose main function is to reuptake the dopamine (DA) released in the synaptic cleft back into the dopaminergic neurons. Previous studies suggested that the activity of DAT is regulated by allosteric proteins such as Syntaxin-1A and is altered by drugs of abuse such as amphetamine (Amph). Because Caenorhabditis elegans expresses both DAT (DAT-1) and Syntaxin-1A (UNC-64), we used this model system to investigate the functional and behavioral effects caused by lack of expression of unc-64 in cultured dopaminergic neurons and in living animals. Using an inheritable RNA silencing technique, we were able to knockdown unc-64 specifically in the dopaminergic neurons. This cell-specific knockdown approach avoids the pleiotropic phenotypes caused by knockout mutations of unc-64 and ensures the transmission of dopaminergic specific unc-64 silencing to the progeny. We found that, similarly to dat-1 knockouts and dat-1 silenced lines, animals with reduced unc-64 expression in the dopaminergic neurons did not respond to Amph treatment when tested for locomotor behaviors. Our in vitro data demonstrated that in neuronal cultures derived from animals silenced for unc-64, the DA uptake was reduced by 30% when compared to controls, and this reduction was similar to that measured in neurons isolated from animals silenced for dat-1 (40%). Moreover, reduced expression of unc-64 in the dopaminergic neurons significantly reduced the DA release elicited by Amph. Because in C. elegans DAT-1 is the only protein capable to reuptake DA, these data show that reduced expression of unc-64 in the dopaminergic neurons decreases the capability of DAT in re-accumulating synaptic DA. Moreover, these results demonstrate that decreased expression of unc-64 in the dopaminergic neurons abrogates the locomotor behavior induced by Amph. Taken together these data suggest that Syntaxin-1A plays an important role in both functional and behavioral effects caused by Amph.

Variegated silencing through epigenetic modifications of a large Xq region in a case of balanced X;2 translocation with Incontinentia Pigmenti-like phenotype.

Molecular mechanisms underlying aberrant phenotypes in balanced X;autosome translocations are scarcely understood. We report the case of a de novo reciprocal balanced translocation X;2(q23;q33) presenting phenotypic alterations highly suggestive of Incontinentia Pigmenti (IP) syndrome, a genodermatosis with abnormal skin pigmentation and neurological failure, segregating as X-linked dominant disorder. Through molecular studies, we demonstrated that the altered phenotype could not be ascribed to chromosome microdeletions or to XIST-mediated inactivation of Xq24-qter. Interestingly, we found that the Xq24-qter region, which translocated downstream of the heterochromatic band 2q34, undergoes epigenetic silencing mediated by DNA methylation and histone alterations. Among the downregulated genes, we found the inhibitor of kappa light polypeptide gene enhancer in B cells, kinase gamma (IKBKG/NEMO), the causative gene of IP. We hypothesize that a mosaic functional nullisomy of the translocated genes, through a Position Effect Variegation-like heterochromatization, might be responsible for the proband's phenotypic anomalies. Partial silencing of IKBKG may be responsible for the skin anomalies observed, thereby mimicking the IP pathological condition. In addition to its clinical relevance, this paper addresses fundamental issues related to the chromatin status and nuclear localization of a human euchromatic region translocated proximally to heterochromatin. In conclusion, the study provides new insight into long-range gene silencing mechanisms and their direct impact in human disease.