ABSTRACTBackgroundPathogenic variants in subunits of succinyl‐CoA synthetase (SCS) are associated with mitochondrial encephalomyopathy in humans. SCS catalyses the conversion of succinyl‐CoA to succinate coupled with substrate‐level phosphorylation of either ADP or GDP in the TCA cycle. This report presents a muscle‐specific conditional knock‐out (KO) mouse model of Sucla2, the ADP‐specific beta subunit of SCS, generating a novel in vivo model of mitochondrial myopathy.MethodsThe mouse model was generated using the Cre‐Lox system, with the human skeletal actin (HSA) promoter driving Cre‐recombination of a CRISPR‐Cas9–generated Sucla2 floxed allele within skeletal muscle. Inactivation of Sucla2 was validated using RT‐qPCR and western blot, and both enzyme activity and serum metabolites were quantified by mass spectrometry. To characterize the model in vivo, whole‐body phenotyping was conducted, with mice undergoing a panel of strength and locomotor behavioural assays. Additionally, ex vivo contractility experiments were performed on the soleus (SOL) and extensor digitorum longus (EDL) muscles. SOL and EDL cryosections were also subject to imaging analyses to assess muscle fibre‐specific phenotypes.ResultsMolecular validation confirmed 68% reduction of Sucla2 transcript within the mutant skeletal muscle (p < 0.001) and 95% functionally reduced SUCLA2 protein (p < 0.0001). By 3 weeks of age, Sucla2 KO mice were 44% the size of controls by body weight (p < 0.0001). Mutant mice also exhibited 34%–40% reduced grip strength (p < 0.01) and reduced spontaneous exercise, spending about 88% less cumulative time on a running wheel (p < 0.0001). Contractile function was also perturbed in a muscle‐specific manner; although no genotype‐specific deficiencies were seen in EDL function, SUCLA2‐deficient SOL muscles generated 40% less specific tetanic force (p < 0.0001), alongside slower contraction and relaxation rates (p < 0.001). Similarly, a SOL‐specific threefold increase in mitochondria (p < 0.0001) was observed, with qualitatively increased staining for both COX and SDH, and the proportion of Type 1 myosin heavy chain expressing fibres within the SOL was nearly doubled (95% increase, p < 0.0001) in the Sucla2 KO mice compared with that in controls.ConclusionsSUCLA2 loss within murine skeletal muscle yields a model of SCS‐deficient mitochondrial myopathy with reduced body weight, muscle weakness and exercise intolerance. Physiological and morphological analyses of hindlimb muscles showed remarkable differences in ex vivo function and cellular consequences between the EDL and SOL muscles, with SOL muscles significantly more impacted by Sucla2 inactivation. This novel model will provide an invaluable tool for investigations of muscle‐specific and fibre type–specific pathogenic mechanisms to better understand SCS‐deficient myopathy.

Lysine succinylation is a subtype of protein acylation associated with metabolic regulation of succinyl-CoA in the tricarboxylic acid cycle. Deficiency of succinyl-CoA synthetase (SCS), the tricarboxylic acid cycle enzyme catalyzing the interconversion of succinyl-CoA to succinate, results in mitochondrial encephalomyopathy in humans. This report presents a conditional forebrain-specific knockout (KO) mouse model of Sucla2, the gene encoding the ATP-specific beta isoform of SCS, resulting in postnatal deficiency of the entire SCS com-plex. Results demonstrate that accumulation of succinyl-CoA in the absence of SCS leads to hypersuccinylation within the murine cerebral cortex. Specifically, increased succinylation is associated with functionally significant reduced activity of respiratory chain complex I and widescale alterations in chromatin landscape and gene expression. Integrative analysis of the transcriptomic data also reveals perturbations in regulatory networks of neuronal transcription in the KO forebrain. Together, these findings provide evidence that protein succinylation plays a significant role in the pathogenesis of SCS deficiency.

Though biallelic variants in SLC13A5 are known to cause severe encephalopathy, the mechanism of this disease is poorly understood. SLC13A5 protein deficiency reduces citrate transport into the cell. Downstream abnormalities in fatty acid synthesis and energy generation have been described, though biochemical signs of these perturbations are inconsistent across SLC13A5 deficiency patients. To investigate SLC13A5-related disorders, we performed untargeted metabolic analyses on the liver, brain, and serum from a Slc13a5-deficient mouse model. Metabolomic data were analyzed using the connect-the-dots (CTD) methodology and were compared to plasma and CSF metabolomics from SLC13A5-deficient patients. Mice homozygous for the Slc13a5tm1b/tm1b null allele had perturbations in fatty acids, bile acids, and energy metabolites in all tissues examined. Further analyses demonstrated that for several of these molecules, the ratio of their relative tissue concentrations differed widely in the knockout mouse, suggesting that deficiency of Slc13a5 impacts the biosynthesis and flux of metabolites between tissues. Similar findings were observed in patient biofluids, indicating altered transport and/or flux of molecules involved in energy, fatty acid, nucleotide, and bile acid metabolism. Deficiency of SLC13A5 likely causes a broader state of metabolic dysregulation than previously recognized, particularly regarding lipid synthesis, storage, and metabolism, supporting SLC13A5 deficiency as a lipid disorder.

Biallelic pathogenic variants inDYNC2H1are the cause of short-rib thoracic dysplasia type III with or without polydactyly (OMIM #613091), a skeletal ciliopathy characterized by thoracic hypoplasia due to short ribs. In this report, we review the case of a patient who was admitted to the Neonatal Intensive Care Unit (NICU) of Indiana University Health (IUH) for respiratory support after experiencing respiratory distress secondary to a small, narrow chest causing restrictive lung disease. Additional phenotypic features include postaxial polydactyly, short proximal long bones, and ambiguous genitalia were noted. Exome sequencing (ES) revealed a maternally inherited likely pathogenic variant c.10322C > T p.(Leu3448Pro) in theDYNC2H1gene. However, there was no variant found on the paternal allele. Microarray analysis to detect deletion or duplication inDYNC2H1was normal. Therefore, there was insufficient evidence to establish a molecular diagnosis. To further explore the data and perform additional investigations, the patient was subsequently enrolled in the Undiagnosed Rare Disease Clinic (URDC) at Indiana University School of Medicine (IUSM). The investigators at the URDC performed a reanalysis of the ES raw data, which revealed a paternally inheritedDYNC2H1deep-intronic variant c.10606-14A > G predicted to create a strong cryptic acceptor splice site. Additionally, the RNA sequencing of fibroblasts demonstrated partial intron retention predicted to cause a premature stop codon and nonsense-mediated mRNA decay (NMD). Droplet digital RT-PCR (RT-ddPCR) showed a drastic reduction by 74% ofDYNCH2H1mRNA levels. As a result, the intronic variant was subsequently reclassified as likely pathogenic resulting in a definitive clinical and genetic diagnosis for this patient. Reanalysis of ES and fibroblast mRNA experiments confirmed the pathogenicity of the splicing variants to supplement critical information not revealed in original ES or CMA reports. The NICU and URDC collaboration ended the diagnostic odyssey for this family; furthermore, its importance is emphasized by the possibility of prenatally diagnosing the mother's current pregnancy.

Islet amyloid deposits contribute to beta cell dysfunction and death in most individuals with type 2 diabetes but non-invasive methods to determine the presence of these pathological protein aggregates are currently not available. Therefore, we examined whether florbetapir, a radiopharmaceutical agent used for detection of amyloid-β deposits in the brain, also allows identification of islet amyloid in the pancreas.

Significance The discovery of MLH1 -rheMac hereditary nonpolyposis colorectal cancer syndrome in rhesus macaques ( MLH1 -rheMac HNPCC), which is an orthologue of Lynch syndrome in humans, is highly significant in the field of oncology. The hereditary nature of this disease should allow for planned cross-breeding of rhesus macaques to assess the effects of homozygous versus heterozygous MLH1 gene mutations, as well as other comutations and environmental factors that may affect the development of colon cancers. Also, the MLH1 -rheMac HNPCC syndrome in rhesus macaques can serve as an important model for development of novel approaches to diagnosis and therapy of Lynch syndrome in human patients.

Many studies have been published regarding suicidal hanging deaths, and most forensic pathologists and coroners are very familiar with such causes of death. Forensic pathologists are challenged over their rulings regarding manner of death in part because the general public has a limited scope of knowledge. One such challenge centers on the question of whether a hanging can be a suicide if the individual is not fully suspended. The authors designed a retrospective study to review suspension in hangings and to analyze other criteria used to help in deciding manner of death. We examined 229 suicidal hanging deaths over an 11-year period (1997 through early 2009) using the data from two separate jurisdictions in Ohio. In conclusion, we found that the vast majority (83.4%) of people who hanged themselves were found partially suspended. Among other criteria analyzed, only the presence of petechial hemorrhages and acute neck injury was statistically significant.

Mutations in Prophet of PIT1 (Prop1), one of several homeodomain transcription factors that are required for the development of the anterior pituitary gland, are the predominant cause of MPHD (multiple pituitary hormone deficiency) in humans. We show that deletion of Prop1 in mice causes severe pituitary hypoplasia with failure of the entire Pit1 lineage and delayed gonadotrope development. The pituitary hormone deficiencies cause secondary endocrine problems and a high rate of perinatal mortality due to respiratory distress. Lung atelectasis in mutants correlates with reduced levels of NKX2.1 and surfactant. Lethality of mice homozygous for either the null allele or a spontaneous hypomorphic allele is strongly influenced by genetic background. Prop1-null mice are an excellent model for MPHD and may be useful for testing the efficacy of pharmaceutical intervention for neonatal respiratory distress.

Sodium channel β-subunits modulate channel gating, assembly, and cell surface expression in heterologous cell systems. We generated β2 −/− mice to investigate the role of β2 in control of sodium channel density, localization, and function in neurons in vivo . Measurements of [ 3 H]saxitoxin (STX) binding showed a significant reduction in the level of plasma membrane sodium channels in β2 −/− neurons. The loss of β2 resulted in negative shifts in the voltage dependence of inactivation as well as significant decreases in sodium current density in acutely dissociated hippocampal neurons. The integral of the compound action potential in optic nerve was significantly reduced, and the threshold for action potential generation was increased, indicating a reduction in the level of functional plasma membrane sodium channels. In contrast, the conduction velocity, the number and size of axons in the optic nerve, and the specific localization of Na v 1.6 channels in the nodes of Ranvier were unchanged. β2 −/− mice displayed increased susceptibility to seizures, as indicated by reduced latency and threshold for pilocarpine-induced seizures, but seemed normal in other neurological tests. Our observations show that β2-subunits play an important role in the regulation of sodium channel density and function in neurons in vivo and are required for normal action potential generation and control of excitability.

The accumulation of unfolded protein in the endoplasmic reticulum (ER) attenuates protein synthesis initiation through phosphorylation of the α subunit of eukaryotic translation initiation factor 2 (eIF2α) at Ser51. Subsequently, transcription of genes encoding adaptive functions including the glucose-regulated proteins is induced. We show that eIF2α phosphorylation is required for translation attenuation, transcriptional induction, and survival in response to ER stress. Mice with a homozygous mutation at the eIF2α phosphorylation site (Ser51Ala) died within 18 hr after birth due to hypoglycemia associated with defective gluconeogenesis. In addition, homozygous mutant embryos and neonates displayed a deficiency in pancreatic β cells. The results demonstrate that regulation of translation through eIF2α phosphorylation is essential for the ER stress response and in vivo glucose homeostasis.

The fucose alpha (1 -->2) galactose beta structure is expressed by uterine epithelial cells in the mouse and has been implicated in blastocyst adhesion events thought to be required for murine implantation. Fuc alpha (1 -->2)Gal beta moieties and cognate fucosyltransferases are also expressed by epithelial cells of the male reproductive tract and have been implicated in sperm maturation events that may contribute to fertilization. To determine directly if Fuca(1 -->2)Gal beta moieties are required for fertility, we have generated strains of mice that are deficient in genes encoding FUT1 and FUT2, a pair of GDP-L-fucose:beta (1 -->4)-D-galactosyl-R 2-alpha -L-fucosyl-transferase enzymes (EC 2.4.1.69) responsible for Fuc alpha (1 -->2)Galp synthesis and expression. FUT1 null mice and FUT2 null mice develop normally and exhibit no gross phenotypic abnormalities. The Fuc alpha (1 -->2)Gal beta epitope is absent from the uterine epithelia of FUT2 null mice and from the epithelia of the epididymis of FUT1 null mice. Fully normal fertility is observed in FUT1. null intercrosses and in FUT2 null intercrosses. These observations indicate that Fuc alpha (1 -->2)Galp moieties are not essential to blastocyst-uterine epithelial cell interactions required for implantation and are not required for sperm maturation events that permit fertilization and that neither the FUT loci nor their cognate fucosylated glycans are essential to normal development.

Glomerular epithelial protein 1 (GLEPP1) is a receptor tyrosine phosphatase present on the apical cell surface of the glomerular podocyte. The GLEPP1 gene (Ptpro) was disrupted at an exon coding for the NH2-terminal region by gene targeting in embryonic stem cells. Heterozygote mating produced the expected genotypic ratio of 1:2:1, indicating that the Ptpro(-/-) genotype does not lead to embryonic or neonatal lethality. Kidney and glomerular structure was normal at the gross and Light microscopic levels. Scanning and transmission electron microscopy showed that Ptpro(-/-) mice had an amoeboid rather than the typical octopoid structure seen in the wild-type mouse podocyte and that there were blunting and widening of the minor (foot) processes in association with altered distribution of the podocyte intermediate cytoskeletal protein vimentin. Reduced filtration surface area in association with these structural changes was confirmed by finding reduced glomerular nephrin content and reduced glomerular filtration rate in Ptpro(-/-) mice. There was no detectable increase in the urine albumin excretion of Ptpro(-/-) mice. After removal of one or more kidneys, Ptpro(-/-) mice had higher blood pressure than did their wild-type littermates. These data support the conclusion that the GLEPP1 (Ptpro) receptor plays a role in regulating the glomerular pressure/filtration rate relationship through an effect on podocyte structure and function.

Cholecystokinin (CCK) is a regulatory peptide that is primarily expressed in two adult cell types: endocrine cells of the intestine and neurons of the central nervous system. To determine the ontogeny of CCK expression during intestinal organogenesis, we created a mouse strain in which the CCK gene was replaced by a lacZ reporter cassette using homologous recombination in embryonic stem cells. Initially, CCK expression in the developing intestine was limited to the myenteric plexus of the enteric nervous system. This expression pattern was widespread, extending from the proximal stomach into the colon, yet transient, being detected soon after gut tube closure [embryonic day 10.5 (E10.5)] through E15.5. Since enteric neurons are derived from the neural crest, we examined earlier (E8.5-9.5) embryos and concluded that lacZ was expressed in subpopulations of neural tube and neural crest cells. Endocrine cell expression in the intestinal epithelium occurred later, beginning at E15.5 as enteric neuronal expression was dwindling. This expression persisted to yield the adult pattern of scattered single endocrine cells in the upper small intestine. The data show that CCK is a very early marker of both neuronal and endocrine cell lineages in the developing gastrointestinal tract. Furthermore, reverse transcriptase polymerase chain reaction (RT-PCR) analysis showed that CCK receptor transcripts were detected in embryos as early as E10.5, suggesting that CCK signaling is established early in mouse development. Dev Dyn 1999;216:190-200.

A CCK-deficient mouse mutant generated by gene targeting in embryonic stem cells was analyzed to determine the importance of CCK for growth and function of the exocrine pancreas and for pancreatic adaptation to dietary changes. RIAs confirmed the absence of CCK in mutant mice and demonstrated that tissue concentrations of the related peptide gastrin were normal. CCK-deficient mice are viable and fertile and exhibit normal body weight. Pancreas weight and cellular morphology appeared normal, although pancreatic amylase content was elevated in CCK-deficient mice. We found that a high-protein diet increased pancreatic weight, protein, DNA, and chymotrypsinogen content similarly in CCK-deficient and wild-type mice. This result demonstrates that CCK is not required for protein-induced pancreatic hypertrophy and increased proteolytic enzyme content. This is a novel finding, since CCK has been considered the primary mediator of dietary protein-induced changes in the pancreas. Altered somatostatin concentrations in brain and duodenum of CCK-deficient mice suggest that other regulatory pathways are modified to compensate for the CCK deficiency.

To further understand the role of the peptide hormone gastrin in the development and function of the stomach, we have generated gastrin-deficient mice by gene targeting in embryonic stem cells. Mutant mice were viable and fertile, without obvious visible abnormalities. However, gastric function was severely affected by the loss of gastrin. Basal gastric acid secretion was abolished and could not be induced by histamine, carbachol, or gastrin. Histological analysis revealed alterations in the two cell types primarily involved in acid secretion, parietal and enterochromaffin-like (ECL) cells. Parietal cells were reduced in number with an accumulation of immature cells lacking H(+)-K(+)-adenosinetriphosphatase (H(+)-K(+)-ATPase). ECL cells were positioned closer to the base of the gastric glands, with markedly lower expression of histidine decarboxylase. Gastrin administration for 6 days reversed the effects of the gastrin deficiency, leading to an increase in the number of mature, H(+)-K(+)-ATPase-positive parietal cells and a partial restoration of acid secretion. The results show that gastrin is critically important for the function of the acid secretory system.

Gene targeting in mouse embryonic stem (ES) cells generally includes the analysis of numerous colonies to identify a few with mutations resulting from homologous recombination with a targeting vector. Thus, simple and efficient screening methods are needed to identify targeted clones. Optimal screening approaches require probes from outside of the region included in the targeting vector to avoid detection of the more common random insertions. However, the use of large genomic fragments in targeting vectors can limit the availability of cloned DNA, thus necessitating a strategy to obtain unique flanking sequences. We describe a rapid method to identify sequences adjacent to cloned DNA using long-range polymerase chain reaction (PCR) amplification from a genomic DNA library, followed by direct nucleotide sequencing of the amplified fragment. We have used this technique in two independent gene targeting experiments to obtain genomic DNA sequences flanking the mouse cholecystokinin (CCK) and gastrin genes. The sequences were then used to design primers to characterize ES cell lines with CCK or gastrin targeted gene mutations, employing a second long-range PCR approach. Our results show that these two long-range PCR methods are generally useful to rapidly and accurately characterize allele structures in ES cells.