Developmental exposure to the organochlorine pesticide dieldrin causes male-specific exacerbation of α-synuclein-preformed fibril-induced toxicity and motor deficits.

Human and animal studies have shown that exposure to the organochlorine pesticide dieldrin is associated with increased risk of Parkinson's disease (PD). Previous work showed that developmental dieldrin exposure increased neuronal susceptibility to MPTP toxicity in male C57BL/6 mice, possibly via changes in dopamine (DA) packaging and turnover. However, the relevance of the MPTP model to PD pathophysiology has been questioned. We therefore studied dieldrin-induced neurotoxicity in the α-synuclein (α-syn)-preformed fibril (PFF) model, which better reflects the α-syn pathology and toxicity observed in PD pathogenesis. Specifically, we used a "two-hit" model to determine whether developmental dieldrin exposure increases susceptibility to α-syn PFF-induced synucleinopathy. Dams were fed either dieldrin (0.3 mg/kg, every 3-4 days) or vehicle corn oil starting 1 month prior to breeding and continuing through weaning of pups at postnatal day 22. At 12 weeks of age, male and female offspring received intrastriatal α-syn PFF or control saline injections. Consistent with the male-specific increased susceptibility to MPTP, our results demonstrate that developmental dieldrin exposure exacerbates PFF-induced toxicity in male mice only. Specifically, in male offspring, dieldrin exacerbated PFF-induced motor deficits on the challenging beam and increased DA turnover in the striatum 6 months after PFF injection. However, male offspring showed neither exacerbation of phosphorylated α-syn aggregation (pSyn) in the substantia nigra (SN) at 1 or 2 months post-PFF injection, nor exacerbation of PFF-induced TH and NeuN loss in the SN 6 months post-PFF injection. Collectively, these data indicate that developmental dieldrin exposure produces a male-specific exacerbation of synucleinopathy-induced behavioral and biochemical deficits. This sex-specific result is consistent with both previous work in the MPTP model, our previously reported sex-specific effects of this exposure paradigm on the male and female epigenome, and the higher prevalence and more severe course of PD in males. The novel two-hit environmental toxicant/PFF exposure paradigm established in this project can be used to explore the mechanisms by which other PD-related exposures alter neuronal vulnerability to synucleinopathy in sporadic PD.

Basic Concepts in Viral Vector-Mediated Gene Therapy.

Today any researcher with the desire can easily purchase a viral vector. However, despite the availability of viral vectors themselves, the requisite knowledge that is absolutely essential to conducting a gene therapy experiment remains somewhat obscure and esoteric. To utilize viral vectors to their full potential, a large number of decisions must be made, in some instances prior to even obtaining the vector itself. For example, critical decisions include selection of the proper virus, selection of the proper expression cassette, whether to produce or purchase a viral vector, proper viral handling and storage, the most appropriate delivery method, selecting the proper controls, how to ensure your virus is expressing properly, and many other complex decisions that are essential to performing a successful gene therapy experiment. The need to make so many important decisions can be overwhelming and potentially prohibitive, especially to the novice gene therapist. In order to aid in this challenging process, here we provide an overview of basic gene therapy modalities and a decision tree that can be used to make oneself aware of the options available to the beginning gene therapist. This information can be used as a road map to help navigate the complex and perhaps confusing process of designing a successful gene therapy experiment.

Author Correction: Defective Gpsm2/Gαi3 signalling disrupts stereocilia development and growth cone actin dynamics in Chudley-McCullough syndrome.

This corrects the article DOI: 10.1038/ncomms14907.

Induction of alpha-synuclein pathology in the enteric nervous system of the rat and non-human primate results in gastrointestinal dysmotility and transient CNS pathology.

Alpha-Synuclein (α-syn) is by far the most highly vetted pathogenic and therapeutic target in Parkinson's disease. Aggregated α-syn is present in sporadic Parkinson's disease, both in the central nervous system (CNS) and peripheral nervous system (PNS). The enteric division of the PNS is of particular interest because 1) gastric dysfunction is a key clinical manifestation of Parkinson's disease, and 2) Lewy pathology in myenteric and submucosal neurons of the enteric nervous system (ENS) has been referred to as stage zero in the Braak pathological staging of Parkinson's disease. The presence of Lewy pathology in the ENS and the fact that patients often experience enteric dysfunction before the onset of motor symptoms has led to the hypothesis that α-syn pathology starts in the periphery, after which it spreads to the CNS via interconnected neural pathways. Here we sought to directly test this hypothesis in rodents and non-human primates (NHP) using two distinct models of α-syn pathology: the α-syn viral overexpression model and the preformed fibril (PFF) model. Subjects (rat and NHP) received targeted enteric injections of PFFs or adeno-associated virus overexpressing the Parkinson's disease associated A53T α-syn mutant. Rats were evaluated for colonic motility monthly and sacrificed at 1, 6, or 12 months, whereas NHPs were sacrificed 12 months following inoculation, after which the time course and spread of pathology was examined in all animals. Rats exhibited a transient GI phenotype that resolved after four months. Minor α-syn pathology was observed in the brainstem (dorsal motor nucleus of the vagus and locus coeruleus) 1 month after PFF injections; however, no pathology was observed at later time points (nor in saline or monomer treated animals). Similarly, a histopathological analysis of the NHP brains revealed no pathology despite the presence of robust α-syn pathology throughout the ENS which persisted for the entirety of the study (12 months). Our study shows that induction of α-syn pathology in the ENS is sufficient to induce GI dysfunction. Moreover, our data suggest that sustained spread of α-syn pathology from the periphery to the CNS and subsequent propagation is a rare event, and that the presence of enteric α-syn pathology and dysfunction may represent an epiphenomenon.

Movement disorder in GNAO1 encephalopathy associated with gain-of-function mutations.

OBJECTIVE: To define molecular mechanisms underlying the clinical spectrum of epilepsy and movement disorder in individuals with de novo mutations in the GNAO1 gene. METHODS: We identified all GNAO1 mutations reported in individuals with epilepsy (early infantile epileptiform encephalopathy 17) or movement disorders through April 2016; 15 de novo mutant alleles from 25 individuals were introduced into the Gαo subunit by site-directed mutagenesis in a mammalian expression plasmid. We assessed protein expression and function in vitro in HEK-293T cells by Western blot and determined functional Gαo-dependent cyclic adenosine monophosphate (cAMP) inhibition with a coexpressed α2A adrenergic receptor. RESULTS: Of the 15 clinical GNAO1 mutations studied, 9 show reduced expression and loss of function (LOF; <90% maximal inhibition). Six other mutations show variable levels of expression but exhibit normal or even gain-of-function (GOF) behavior, as demonstrated by significantly lower EC50 values for α2A adrenergic receptor-mediated inhibition of cAMP. The GNAO1 LOF mutations are associated with epileptic encephalopathy while GOF mutants (such as G42R, G203R, and E246K) or normally functioning mutants (R209) were found in patients with movement disorders with or without seizures. CONCLUSIONS: Both LOF and GOF mutations in Gαo (encoded by GNAO1) are associated with neurologic pathophysiology. There appears to be a strong predictive correlation between the in vitro biochemical phenotype and the clinical pattern of epilepsy vs movement disorder.

Primary cilium migration depends on G-protein signalling control of subapical cytoskeleton.

In ciliated mammalian cells, the precise migration of the primary cilium at the apical surface of the cells, also referred to as translational polarity, defines planar cell polarity (PCP) in very early stages. Recent research has revealed a co-dependence between planar polarization of some cell types and cilium positioning at the surface of cells. This important role of the primary cilium in mammalian cells is in contrast with its absence from Drosophila melanogaster PCP establishment. Here, we show that deletion of GTP-binding protein alpha-i subunit 3 (Gαi3) and mammalian Partner of inscuteable (mPins) disrupts the migration of the kinocilium at the surface of cochlear hair cells and affects hair bundle orientation and shape. Inhibition of G-protein function in vitro leads to kinocilium migration defects, PCP phenotype and abnormal hair bundle morphology. We show that Gαi3/mPins are expressed in an apical and distal asymmetrical domain, which is opposite and complementary to an aPKC/Par-3/Par-6b expression domain, and non-overlapping with the core PCP protein Vangl2. Thus G-protein-dependent signalling controls the migration of the cilium cell autonomously, whereas core PCP signalling controls long-range tissue PCP.