Phox2b-expressing neurons contribute to breathing problems in Kcnq2 loss- and gain-of-function encephalopathy models.

Loss- and gain-of-function variants in the gene encoding KCNQ2 channels are a common cause of developmental and epileptic encephalopathy, a condition characterized by seizures, developmental delays, breathing problems, and early mortality. To understand how KCNQ2 dysfunction impacts behavior in a mouse model, we focus on the control of breathing by neurons expressing the transcription factor Phox2b which includes respiratory neurons in the ventral parafacial region. We find Phox2b-expressing ventral parafacial neurons express Kcnq2 in the absence of other Kcnq isoforms, thus clarifying why disruption of Kcnq2 but not other channel isoforms results in breathing problems. We also find that Kcnq2 deletion or expression of a recurrent gain-of-function variant R201C in Phox2b-expressing neurons increases baseline breathing or decreases the central chemoreflex, respectively, in mice during the light/inactive state. These results uncover mechanisms underlying breathing abnormalities in KCNQ2 encephalopathy and highlight an unappreciated vulnerability of Phox2b-expressing ventral parafacial neurons to KCNQ2 pathogenic variants.

Disordered breathing in a Pitt-Hopkins syndrome model involves Phox2b-expressing parafacial neurons and aberrant Nav1.8 expression.

Pitt-Hopkins syndrome (PTHS) is a rare autism spectrum-like disorder characterized by intellectual disability, developmental delays, and breathing problems involving episodes of hyperventilation followed by apnea. PTHS is caused by functional haploinsufficiency of the gene encoding transcription factor 4 (Tcf4). Despite the severity of this disease, mechanisms contributing to PTHS behavioral abnormalities are not well understood. Here, we show that a Tcf4 truncation (Tcf4tr/+) mouse model of PTHS exhibits breathing problems similar to PTHS patients. This behavioral deficit is associated with selective loss of putative expiratory parafacial neurons and compromised function of neurons in the retrotrapezoid nucleus that regulate breathing in response to tissue CO2/H+. We also show that central Nav1.8 channels can be targeted pharmacologically to improve respiratory function at the cellular and behavioral levels in Tcf4tr/+ mice, thus establishing Nav1.8 as a high priority target with therapeutic potential in PTHS.

Differential responsiveness of a splice variant of the human type I interferon receptor to interferons.

Chinese hamster ovary cells containing the yeast artificial chromosome F136C5 (alphaYAC) respond to all type I human interferons including IFN-alphaA, IFN-beta, and IFN-omega. The alphaYAC contains at least two genes encoding interferon-alpha receptor (IFN-alphaR) chains that are required for response to type I human interferons: Hu-IFN-alphaR1 and Hu-IFN-alphaR2. We previously isolated a splice variant of the Hu-IFN-alphaR1 chain designated Hu-IFN-alphaR1s. Chinese hamster ovary cells containing a disrupted alphaYAC, which contains a deletion in the human IFNAR1 gene, were transfected with expression vectors for the Hu-IFN-alphaR1 and Hu-IFN-alphaR1s chains. With these cells, two type I interferons have been identified which can interact with the splice variant (Hu-IFN-alphaR1s) and with the Hu-IFN-alphaR1 chains: Hu-IFN-alphaA and IFN-omega. Two other type I interferons, Hu-IFN-alphaB2 and Hu-IFN-alphaF, are capable of signaling through the Hu-IFN-alphaR1 chain only and cannot utilize the splice variant Hu-IFN-alphaR1s. Hu-IFN-alphaR1 and Hu-IFN-alphaR1s differ in that the latter is missing a single subdomain of the receptor extracellular domain encoded by exons 4 and 5 of the IFNAR1 gene. These results therefore indicate that different type I interferons require different subdomains of the Hu-IFN-alphaR1 receptor chain, and that the splice variant chain (Hu-IFN-alphaR1s) is functional.

Knockout and reconstitution of a functional human type I interferon receptor complex.

The functional subunits of the human Type I interferon (IFN) receptor complex have not been defined. Using site-specific recombination in a yeast artificial chromosome (YAC), we have produced a deletion within the human IFN-alpha receptor (Hu-IFN-alpha R1) gene which eliminates exon II of the gene. This deletion effectively eliminates the MHC Class I antigen induction and antiviral activity previously reported for this fully functional parental YAC clone (Soh, J., Mariano, T. M., Lim, J.-K., Izotova, L., Mirochnitchenko, O., Schwartz, B., Langer, J., and Pestka, S. (1994c) J. Biol. Chem. 269, 18102-18110). We have successfully reconstituted this activity by expression of the cDNA encoding the Hu-IFN-alpha R1 component (Uzé, G., Lutfalla, G., and Gresser, I. (1990) Cell 60, 225-234) in cells containing the YAC with this deletion. The Hu-IFN-alpha R1 subunit thus plays a critical role in the functional human Type I IFN receptor complex, whose components are encoded on this YAC. In addition, as binding of ligands is retained in the cells containing the YAC with the deletion, it is clear a second subunit encoded on the YAC is responsible for ligand binding activity. This system will now allow the identification of additional subunits involved in the response to the Type I IFNs and the functional significance of each.

Amplification of c-Ki-ras-2 oncogene sequences in human carcinoma of pancreas.

c-Ki-ras-2 sequences were visualized in paraffin embedded sections from normal adult human pancreases and 24 carcinomas of pancreas by an in situ hybridization technique. A biotinylated 1 kbp EcoRI fragment of pHiHi3 DNA was used as probe and the oncogene was visualized as one or two large grains of reaction products produced in more than 9% of normal pancreas nuclei by streptavidin-peroxidase complex and diaminobenzidine tetrachloride. Its amplification in pancreatic carcinomas was detected as one or more large grains in 54% of the nuclei. In addition, tumor cells showed small nuclear and cytoplasmic grains scarcely seen in normal pancreatic cells. The differential transcriptional activity of this oncogene in cancer cells and the adjacent normal pancreatic cells on the same section was evident in sections from 5 cases where normal pancreas was present.

Visualization of O6-methylguanine in target cell nuclei of dimethylnitrosamine-treated human pancreas by a murine monoclonal antibody.

A murine monoclonal IgG2b with high affinity and specificity for O6-methylguanine was developed. This allowed visualization of this adduct in paraffin-embedded sections by indirect fluorescence or immunoperoxidase methods. The antibody was used to study and compare the formation and persistence of this adduct detected by immunohistochemistry and the presence of 14C methyl by autoradiography in sections from adult human pancreas explants exposed to [14C methyl]dimethylnitrosamine (DMN) and organ-cultured for up to 7 days. O6-methylguanine was detectable randomly and with varied intensity in parenchymal cell nuclei and cytoplasms, 6 h after exposure to DMN. Following RNase treatment, however, only 7.5% of nuclei revealed measurable staining. The positive nuclei decreased to 2.5% within 24 h and remained about the same throughout the 7 days of culture. This correlated well with the change in percentage of positive nuclei for 14C methyl autoradiographic grains which were selectively condensed over the O6-methylguanine-stained nuclei. The data demonstrate a subpopulation of repair-deficient parenchymal cells of human pancreas exposed to DMN.

Visualization of c-Ki-ras-2 oncogene sequences in human pancreas, a chemically induced transplantable carcinoma, and carcinomas of pancreas by in situ hybridization.

c-Ki-ras-2 sequences were visualized in paraffin-embedded sections from normal fetal and adult human pancreases, a chemically induced transplantable human pancreas carcinoma (PT-1) and three carcinomas of pancreas by in situ hybridization technique. A biotinylated 1-kilobase-pair (kb) EcoRI fragment of pHiHi3 DNA was used as probe and the oncogene was visualized as one or two large grains of reaction products produced by streptavidin-peroxidase complex and diaminobenzidine tetrachloride in more than 9% of normal pancreas nuclei. Its amplification in the chemically induced cell line was detected as one or more large grains in 72% of the nuclei and numerous cytoplasmic grains. The detection of oncogene in normal pancreases and its amplification in PT-1 cells was validated by Southern analysis of EcoRI digests of genomic DNA extracted from normal pancreases and PT-1 cell line. The oncogene was also demonstrated to be equally amplified in two adenocarcinomas and one undifferentiated carcinoma of human pancreas by in situ hybridization.

Hamster pancreas acinar cell post-differentiation antigen detected by murine monoclonal antibody.

For identification of hamster acinar cells, murine monoclonal antibodies to dispersed adult hamster acinar cells were developed. One of these, Ham-Acl, with strong affinity for a membrane-associated adult acinar cell antigen, was purified by HPLC, isotyped as IgG1 and used for the characterization of a species-specific, immunoprecipitable post-differentiation antigen on adult acinar cells. This antigen of 98 kDa was identified on paraffin sections of adult hamster acinar cells by an indirect immunofluorescence technique. It was undetectable in fetal hamster pancreas, but was present on 2-week-old and older hamster acinar cells.