TAR DNA-binding protein-43 (TDP-43) is known to accumulate in ubiquitinated inclusions of amyotrophic lateral sclerosis affected motor neurons, resulting in motor neuron degeneration, loss of motor functions, and eventually death. Rapamycin, an mTOR inhibitor and a commonly used immunosuppressive drug, has been shown to increase the survivability of Amyotrophic Lateral Sclerosis (ALS) affected motor neurons. Here we present a transgenic, TDP-43-A315T, mouse model expressing an ALS phenotype and demonstrate the presence of ubiquitinated cytoplasmic TDP-43 aggregates with > 80% cell death by 28 days post differentiation in vitro. Embryonic stem cells from this mouse model were used to study the onset, progression, and therapeutic remediation of TDP-43 aggregates using a novel microfluidic rapamycin concentration gradient generator. Results using a microfluidic device show that ALS affected motor neuron survival can be increased by 40.44% in a rapamycin dosage range between 0.4-1.0 µM.

BACKGROUND:Orofacial clefts (OFCs) are common birth defects with complex etiology. Genome wide association studies for OFC have identified SNPs in and near MAFB. MAFB is a transcription factor critical for structural development of digits, kidneys, skin, and brain. MAFB is also expressed in the craniofacial region. Previous sequencing of MAFB in a Filipino population revealed a novel missense variant significantly associated with an increased risk for OFC. This MAFB variant, leading to the amino acid change H131Q, was knocked into the mouse Mafb, resulting in the MafbH131Q allele. The MafbH131Q construct was engineered to allow for deletion of Mafb ("Mafbdel ").RESULTS:Mafbdel/del animals died shortly after birth. Conversely, MafbH131Q/H131Q mice survived into adulthood at Mendelian ratios. Mafbdel/del and MafbH131Q/H131Q heads exhibited normal macroscopic and histological appearance at all embryonic time points evaluated. The periderm was intact based on expression of keratin 6, p63, and E-cadherin. Despite no effect on craniofacial morphogenesis, H131Q inhibited the Mafb-dependent promoter activation of Arhgap29 in palatal mesenchymal, but not ectodermal-derived epithelial cells in a luciferase assay.CONCLUSIONS:Mafb is dispensable for murine palatogenesis in vivo, and the cleft-associated variant H131Q, despite its lack of morphogenic effect, altered the expression of Arhgap29 in a cell-dependent context.

Non‐syndromic cleft lip with or without cleft palate (NSCL/P) is a common birth defect with complex etiology. Amongst the numerous genes involved, mutations in MAFB (MAF BZIP Transcription Factor B) were recently associated with increased risk for NSCL/P. MAFB, a single‐exon gene of 3.3kb, encodes the protein MAFB, which is a transcription factor expressed in the craniofacial region at time points critical for palatal development. Previous sequencing of MAFB in cases revealed a novel missense mutation which was significantly associated with an increased risk of NSCL/P. This patient‐derived MAFB mutation, leading to the amino acid change H131Q, was knocked‐into the mouse Mafb, resulting in the allele MafbH131Q. In contrast to Mafb nulls, which die shortly after birth, MafbH131Q mice survived into adulthood at Mendelian ratios. MafbH131Q embryos were harvested for serial sectioning and histological characterization at time points critical for craniofacial development. One MafbH131Q/H131Q animal at embryonic day (e) 18.5 (N = 12) exhibited highly abnormal craniofacial morphology that included a cleft palate and shortened snout. At e13.5, our preliminary data indicate the presence of mild oral adhesions in MafbH131Q/+ and MafbH131Q/H131Q mice that were absent in wild type animals. However, immunofluorescence for periderm markers did not indicate altered expression of keratin 6 and keratin 17. These data demonstrate functional significance of the patient‐derived H131Q mutation and its role in the etiology of NSCL/P.Support or Funding InformationMD ‐ NIH/NIAMS AR067739. Additional partial financial support was provided by a grant from the NIH R37DE08559 and by the FaceBase consortium (grants DE020052 and DE020057)CP ‐ Iowa Biosciences Academy, funded by NIH/NIGMS R25GM058939, the University of Iowa (UI) Office of the Vice President for Research, and the UI Chief Diversity Office.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Non‐syndromic cleft lip and/or palate (NSCLP) is among the most frequent major birth defects. The etiology is complex, drawing influence from environmental and genetic factors. Previous studies have identified mutations affecting ARHGAP29 on chromosome 1p22 as a factor for increased risk of NSCLP. To investigate the effects of altered ARHGAP29 in vivo, we generated a novel murine allele by replacing Arhgap29 with a mutated sequence from a human NSLCLP patient. This single‐nucleotide variation of ARHGAP29 led to a nonsense mutation (K326X) in the 11th exon of 31 total, presumably resulting in a nonfunctional protein product. Arhgap29K326X/+ mice were crossed and embryos were harvested for analysis at various time points. We hypothesize that this allele will lead to altered craniofacial development, supporting a critical role for ARHGAP29 in this process. Our results show the absence of homozygous ArhgapK326X animals in all the analyzed litters (N=36), assessed as early as embryonic day 9.5 (e9.5). At e14.5, 54.8% of Arhgap29K326X/+ mice (N=31) exhibited the presence of oral adhesions between the maxilla and mandible or maxilla and tongue, compared to 12.5% of wild type animals (N=8) (P=0.0489). Further quantification of the oral adhesions revealed that 15.94% of the length of the oral epithelium was in improper contact with another oral epithelium in Arhgap29K326X/+ compared with only 3.26% in wild type embryos (P=0.0029). Previous reports have implicated alterations in the periderm as the potential cause of oral adhesions. We performed immunofluorescent analyses at sites of adhesions and detected the presence of p63‐negative, Keratin 17‐positive cells at those sites. This suggests that periderm cells are still present, yet whether or not they are functioning properly remains unknown. Evaluation of E‐cadherin expression at adhesion sites shows proper location at cell‐cell boundaries. Oral adhesions did not appear to impair palatogenesis, as all analyzed embryos show confluent palatal mesenchyme and epithelium at e18.5 (N=22). Collectively, our data demonstrate that ARHGAP29 is required for embryonic survival, and that heterozygosity for Arhgap29 loss of function variants increases the incidence and length of oral adhesions at a critical time point during palatogenesis. In conclusion, we validate the damaging function of the human K326X mutation in vivo and reveal a previously unknown effect of altered Arhgap29 in murine embryonic and craniofacial development.Support or Funding InformationPartial financial support was provided by a grant from the National Institute of Health R37DE08559, R01AR067739, and by the FaceBase consortium (grants DE020052 and DE020057). Brian Paul was a recipient of a fellowship from the Iowa Center for Research by Undergraduates.

Spontaneously arising mouse mutations have served as the foundation for understanding gene function for more than 100 years. We have used exome sequencing in an effort to identify the causative mutations for 172 distinct, spontaneously arising mouse models of Mendelian disorders, including a broad range of clinically relevant phenotypes. To analyze the resulting data, we developed an analytics pipeline that is optimized for mouse exome data and a variation database that allows for reproducible, user-defined data mining as well as nomination of mutation candidates through knowledge-based integration of sample and variant data. Using these new tools, putative pathogenic mutations were identified for 91 (53%) of the strains in our study. Despite the increased power offered by potentially unlimited pedigrees and controlled breeding, about half of our exome cases remained unsolved. Using a combination of manual analyses of exome alignments and whole-genome sequencing, we provide evidence that a large fraction of unsolved exome cases have underlying structural mutations. This result directly informs efforts to investigate the similar proportion of apparently Mendelian human phenotypes that are recalcitrant to exome sequencing.