Congenital disorders of glycosylation (CDG) comprise a family of human multisystemic diseases caused by recessive mutations in genes required for protein N-glycosylation. More than 100 distinct forms of CDGs have been identified and most of them cause severe neurological impairment. The Conserved Oligomeric Golgi (COG) complex mediates tethering of vesicles carrying glycosylation enzymes across the Golgi cisternae. Mutations affecting human COG1, COG2 and COG4-COG8 cause monogenic forms of inherited, autosomal recessive CDGs. We have generated a COG7-CDG model that closely parallels the pathological characteristics of COG7-CDG patients, including pronounced neuromotor defects associated with altered N-glycome profiles. Consistent with these alterations, larval neuromuscular junctions of mutants exhibit a significant reduction in bouton numbers. We demonstrate that the COG complex cooperates with Rab1 and Golgi phosphoprotein 3 to regulate Golgi trafficking and that overexpression of Rab1 can rescue the cytokinesis and locomotor defects associated with loss of Cog7 Our results suggest that the COG7-CDG model can be used to test novel potential therapeutic strategies by modulating trafficking pathways.

Modulation of neurotransmission by the catecholamine dopamine (DA) is conserved across phylogeny. In the nematode C. elegans, excess DA signaling triggers Swimming-Induced Paralysis (Swip), a phenotype first described in animals with loss of function mutations in the presynaptic DA transporter (dat-1). Swip has proven to be a phenotype suitable for the identification of novel dat-1 mutations as well as the identification of novel genes that impact DA signaling. Pharmacological manipulations can also induce Swip, though the reagents employed to date lack specificity and potency, limiting their use in evaluation of dat-1 expression and function. Our lab previously established the mammalian norepinephrine transporter (NET) inhibitor nisoxetine to be a potent antagonist of DA uptake conferred by DAT-1 following heterologous expression. Here we demonstrate the ability of low (μM) concentrations of nisoxetine to trigger Swip within minutes of incubation, with paralysis dependent on DA release and signaling, and non-additive with Swip triggered by dat-1 deletion. Using nisoxetine in combination with genetic mutations that impact DA release, we further demonstrate the utility of the drug for demonstrating contributions of presynaptic DA receptors and ion channels to Swip. Together, these findings reveal nisoxetine as a powerful reagent for monitoring multiple dimensions of DA signaling in vivo, thus providing a new resource that can be used to evaluate contributions of DAT-1 and other genes linked to DA signaling without the potential for compensations that attend constitutive genetic mutations.

The monoamine neurotransmitter dopamine (DA) acts across phylogeny to modulate both simple and complex behaviors. The presynaptic DA transporter (DAT) is a major determinant of DA signaling capacity in ensuring efficient extracellular DA clearance. In humans, DAT is also a major target for prescribed and abused psychostimulants. Multiple structural determinants of DAT function and regulation have been defined, though largely these findings have arisen from heterologous expression or ex vivo cell culture studies. Loss of function mutations in the gene encoding the Caenhorhabditis elegans DAT (dat-1) produces rapid immobility when animals are placed in water, a phenotype termed Swimming-induced paralysis (Swip). The ability of a DA neuron-expressed, GFP-tagged DAT-1 fusion protein (GFP::DAT-1) to localize to synapses and rescue Swip in these animals provides a facile approach to define sequences supporting DAT somatic export and function in vivo. In prior studies, we found that truncation of the last 25 amino acids of the DAT-1 C-terminus (Δ25) precludes Swip rescue, supported by a deficit in GFP::DAT-1 synaptic localization. Here, we further defined the elements within Δ25 required for DAT-1 export and function in vivo. We identified two conserved motifs (584KW585 and 591PYRKR595) where mutation results in a failure of GFP::DAT-1 to be efficiently exported to synapses and restore DAT-1 function. The 584KW585 motif conforms to a sequence proposed to support SEC24 binding, ER export from the endoplasmic reticulum (ER), and surface expression of mammalian DAT proteins, whereas the 591PYRKR595 sequence conforms to a 3R motif identified as a SEC24 binding site in vertebrate G-protein coupled receptors. Consistent with a potential role of SEC24 orthologs in DAT-1 export, we demonstrated DA neuron-specific expression of a sec-24.2 transcriptional reporter. Mutations of the orthologous C-terminal sequences in human DAT (hDAT) significantly reduced transporter surface expression and DA uptake, despite normal hDAT protein expression. Although, hDAT mutants retained SEC24 interactions, as defined in co-immunoprecipitation studies. However, these mutations disrupted the ability of SEC24D to enhance hDAT surface expression. Our studies document an essential role of conserved DAT C-terminal sequences in transporter somatic export and synaptic localization in vivo, that add further support for important roles for SEC24 family members in efficient transporter trafficking.

Dopamine (DA) is a neurotransmitter with actions across phylogeny that modulate core behaviors such as motor activity, reward, attention, and cognition. Perturbed DA signaling in humans is associated with multiple disorders, including addiction, ADHD, schizophrenia, and Parkinson’s disease. The presynaptic DA transporter exerts powerful control on DA signaling by efficient clearance of the neurotransmitter following release. As in vertebrates, Caenorhabditis elegans DAT (DAT-1) constrains DA signaling and loss of function mutations in the dat-1 gene result in slowed crawling on solid media and swimming-induced paralysis (Swip) in water. Previously, we identified a mutant line, vt34 , that exhibits robust DA-dependent Swip. vt34 exhibits biochemical and behavioral phenotypes consistent with reduced DAT-1 function though vt34; dat-1 double mutants exhibit an enhanced Swip phenotype, suggesting contributions of the vt34 -associated mutation to additional mechanisms that lead to excess DA signaling. SNP mapping and whole genome sequencing of vt34 identified the site of the molecular lesion in the gene B0412.2 that encodes the Runx transcription factor ortholog RNT-1. Unlike dat-1 animals, but similar to other loss of function rnt-1 mutants, vt34 exhibits altered male tail morphology and reduced body size. Deletion mutations in both rnt-1 and the bro-1 gene, which encodes a RNT-1 binding partner also exhibit Swip. Both vt34 and rnt-1 mutations exhibit reduced levels of dat-1 mRNA as well as the tyrosine hydroxylase ortholog cat-2 . Although reporter studies indicate that rnt-1 is expressed in DA neurons, its re-expression in DA neurons of vt34 animals fails to fully rescue Swip. Moreover, as shown for vt34 , rnt-1 mutation exhibits additivity with dat-1 in generating Swip, as do rnt-1 and bro-1 mutations, and vt34 exhibits altered capacity for acetylcholine signaling at the neuromuscular junction. Together, these findings identify a novel role for rnt-1 in limiting DA neurotransmission and suggest that loss of RNT-1 may disrupt function of both DA neurons and body wall muscle to drive Swip.

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