Characterization of the zebrafish gabra1sa43718/sa43718 germline loss of function allele confirms a function for Gabra1 in motility and nervous system development.

Mutation of the GABRA1 gene is associated with neurodevelopmental defects and epilepsy. GABRA1 encodes for the α1 subunit of the γ-aminobutyric acid type A receptor (GABAAR), which regulates the fast inhibitory impulses of the nervous system. Multiple model systems have been developed to understand the function of GABRA1, but these models have produced complex and, at times, incongruent data. Thus, additional model systems are required to validate and substantiate previous results. We sought to provide initial phenotypic analysis of a novel germline mutant allele. Our analysis provides a solid foundation for the future use of this allele to characterize gabra1 functionally and pharmacologically using zebrafish. We investigated the behavioral swim patterns associated with a nonsense mutation of the zebrafish gabra1 (sa43718 allele) gene. The sa43718 allele causes a decrease in gabra1 mRNA expression, which is associated with light induced hypermotility, one phenotype previously associated with seizure like behavior in zebrafish. Mutation of gabra1 was accompanied by decreased mRNA expression of gabra2, gabra3, and gabra5, indicating a reduction in the expression of additional α sub-units of the GABAAR. Although multiple sub-units were decreased, larvae continued to respond to pentylenetetrazole (PTZ), indicating that a residual GABAAR exists in the sa43718 allele. Proteomics analysis demonstrated that mutation of gabra1 is associated with abnormal expression of proteins that regulate synaptic vesicle fusion, vesicle transport, synapse development, and mitochondrial protein complexes. These data support previous studies performed in a zebrafish nonsense allele created by CRISPR/Cas9 and validate that loss of function mutations in the gabra1 gene result in seizure-like phenotypes with abnormal development of the GABA synapse. Our results add to the existing body of knowledge as to the function of GABRA1 during development and validate that zebrafish can be used to provide complete functional characterization of the gene.

Abnormal chondrocyte development in a zebrafish model of cblC syndrome restored by an MMACHC cobalamin binding mutant.

Variants in the MMACHC gene cause combined methylmalonic acidemia and homocystinuria cblC type, the most common inborn error of intracellular cobalamin (vitamin B12) metabolism. cblC is associated with neurodevelopmental, hematological, ocular, and biochemical abnormalities. In a subset of patients, mild craniofacial dysmorphia has also been described. Mouse models of Mmachc deletion are embryonic lethal but cause severe craniofacial phenotypes such as facial clefts. MMACHC encodes an enzyme required for cobalamin processing and variants in this gene result in the accumulation of two metabolites: methylmalonic acid (MMA) and homocysteine (HC). Interestingly, other inborn errors of cobalamin metabolism, such as cblX syndrome, are associated with mild facial phenotypes. However, the presence and severity of MMA and HC accumulation in cblX syndrome is not consistent with the presence or absence of facial phenotypes. Thus, the mechanisms by which mutations in MMACHC cause craniofacial defects are yet to be completely elucidated. Here we have characterized the craniofacial phenotypes in a zebrafish model of cblC (hg13) and performed restoration experiments with either a wildtype or a cobalamin binding deficient MMACHC protein. Homozygous mutants did not display gross morphological defects in facial development but did have abnormal chondrocyte nuclear organization and an increase in the average number of neighboring cell contacts, both phenotypes were fully penetrant. Abnormal chondrocyte nuclear organization was not associated with defects in the localization of neural crest specific markers, sox10 (RFP transgene) or barx1. Both nuclear angles and the number of neighboring cell contacts were fully restored by wildtype MMACHC and a cobalamin binding deficient variant of the MMACHC protein. Collectively, these data suggest that mutation of MMACHC causes mild to moderate craniofacial phenotypes that are independent of cobalamin binding.

Characterization of the zebrafish gabra1sa43718/sa43718 germline loss of function allele confirms a function for Gabra1 in motility and nervous system development.

Mutation of the GABRA1 gene is associated with neurodevelopmental defects and epilepsy. GABRA1 encodes for the α1 subunit of the gamma-aminobutyric acid type A receptor (GABAAR), which regulates the fast inhibitory impulses of the nervous system. Multiple model systems have previously been developed to understand the function of GABRA1 during development, but these models have produced complex and at times incongruent data. Thus, additional model systems are required to validate and substantiate previously published results. We investigated the behavioral swim patterns associated with a nonsense mutation of the zebrafish gabra1 (sa43718 allele) gene. The sa43718 allele causes a decrease in gabra1 mRNA expression, which is associated with light induced hypermotility, one phenotype associated with seizure like behavior in zebrafish. Mutation of gabra1 was accompanied by decreased mRNA expression of gabra2, gabra3, and gabra5, indicating a reduction in the expression of additional alpha sub-units of the GABAAR. Although multiple sub-units were decreased in total expression, larvae continued to respond to pentylenetetrazole (PTZ) indicating that a residual GABAAR exists in the sa43718 allele. Proteomics analysis demonstrated that nonsense mutation of gabra1 is associated with abnormal expression of proteins that regulate proton transport, ion homeostasis, vesicle transport, and mitochondrial protein complexes. These data support previous studies performed in a zebrafish nonsense allele created by CRISPR/Cas9 and validate that loss of function mutations in the gabra1 gene result in seizure like phenotypes with abnormal function of inhibitory synapses.

Abnormal chondrocyte intercalation in a zebrafish model of cblC syndrome restored by an MMACHC cobalamin binding mutant.

Variants in the MMACHC gene cause combined methylmalonic acidemia and homocystinuria cblC type, the most common inborn error of intracellular cobalamin (vitamin B12) metabolism. cblC is associated with neurodevelopmental, hematological, ocular, and biochemical abnormalities. In a subset of patients, mild craniofacial dysmorphia has also been described. Mouse models of Mmachc deletion are embryonic lethal but cause severe craniofacial phenotypes such as facial clefts. MMACHC encodes an enzyme required for cobalamin processing and variants in this gene result in the accumulation of two metabolites: methylmalonic acid (MMA) and homocysteine (HC). Interestingly, other inborn errors of cobalamin metabolism, such as cblX syndrome, are associated with mild facial phenotypes. However, the presence and severity of MMA and HC accumulation in cblX syndrome is not consistent with the presence or absence of facial phenotypes. Thus, the mechanisms by which mutation of MMACHC cause craniofacial defects have not been completely elucidated. Here we have characterized the craniofacial phenotypes in a zebrafish model of cblC ( hg13 ) and performed restoration experiments with either wildtype or a cobalamin binding deficient MMACHC protein. Homozygous mutants did not display gross morphological defects in facial development, but did have abnormal chondrocyte intercalation, which was fully penetrant. Abnormal chondrocyte intercalation was not associated with defects in the expression/localization of neural crest specific markers, sox10 or barx1 . Most importantly, chondrocyte organization was fully restored by wildtype MMACHC and a cobalamin binding deficient variant of MMACHC protein. Collectively, these data suggest that mutation of MMACHC causes mild to moderate craniofacial phenotypes that are independent of cobalamin binding.

Overexpression of MMACHC Prevents Craniofacial Phenotypes Caused by Knockdown of znf143b.

ZNF143 is a sequence-specific DNA binding protein that regulates the expression of protein-coding genes and small RNA molecules. In humans, ZNF143 interacts with HCFC1, a transcriptional cofactor, to regulate the expression of downstream target genes, including MMACHC, which encodes an enzyme involved in cobalamin (cbl) metabolism. Mutations in HCFC1 or ZNF143 cause an inborn error of cobalamin metabolism characterized by abnormal cbl metabolism, intellectual disability, seizures, and mild to moderate craniofacial abnormalities. However, the mechanisms by which ZNF143 mutations cause individual phenotypes are not completely understood. Defects in metabolism and craniofacial development are hypothesized to occur because of decreased expression of MMACHC. But recent results have called into question this mechanism as the cause for craniofacial development. Therefore, in the present study, we implemented a loss of function analysis to begin to uncover the function of ZNF143 in craniofacial development using the developing zebrafish. The knockdown of znf143b, one zebrafish ortholog of ZNF143, caused craniofacial phenotypes of varied severity, which included a shortened and cleaved Meckel's cartilage, partial loss of ceratobranchial arches, and a distorted ceratohyal. These phenotypes did not result from a defect in the number of total chondrocytes but were associated with a mild to moderate decrease in mmachc expression. Interestingly, expression of human MMACHC via endogenous transgene prevented the onset of craniofacial phenotypes associated with znf143b knockdown. Collectively, our data establishes that knockdown of znf143b causes craniofacial phenotypes that can be alleviated by increased expression of MMACHC.

Overexpression of corticotropin-releasing factor in the nucleus accumbens enhances the reinforcing effects of nicotine in intact female versus male and ovariectomized female rats.

This study assessed the role of stress systems in the nucleus accumbens (NAc) in promoting sex differences in the reinforcing effects of nicotine. Intravenous self-administration (IVSA) of various doses of nicotine was compared following overexpression of corticotropin-releasing factor (CRF) in the NAc of female and male rats. Ovariectomized (OVX) females were also included to assess the role of ovarian hormones in promoting nicotine reinforcement. Rats received intra-NAc administration of an adeno-associated vector that overexpressed CRF (AAV2/5-CRF) or green fluorescent protein (AAV2/5-GFP). All rats were then given extended access (23 h/day) to an inactive and an active lever that delivered nicotine. Separate groups of rats received intra-NAc AAV2/5-CRF and saline IVSA. Rats were also allowed to nose-poke for food and water during IVSA testing. At the end of the study, the NAc was dissected and rt-qPCR methods were used to estimate CRF overexpression and changes in CRF receptors (CRFr1, CRFr2) and the CRF receptor internalizing protein, ß-arrestin2 (Arrb2). Overexpression of CRF in the NAc increased nicotine IVSA to a larger extent in intact female versus male and OVX females. Food intake was increased to a larger extent in intact and OVX females as compared to males. The increase in CRF gene expression was similar across all groups; however, in females, overexpression of CRF resulted in a larger increase in CRFr1 and CRFr2 relative to males. In males, overexpression of CRF produced a larger increase in Arrb2 than females, suggesting greater CRF receptor internalization. Our results suggest that stress systems in the NAc promote the reinforcing effectiveness of nicotine in female rats in an ovarian hormone-dependent manner.