Standardized,systemic phenotypic analysis of Slc12a1I299F mutant mice

Background

Type I Bartter syndrome is a recessive human nephropathy caused by loss-of-function mutations in the SLC12A1 gene coding for the Na⁺-K⁺-2Cl⁻ cotransporter NKCC2. We recently established the mutant mouse line Slc12a1^I299F exhibiting kidney defects highly similar to the late-onset manifestation of this hereditary human disease. Besides the kidney defects, low blood pressure and osteopenia were revealed in the homozygous mutant mice which were also described in humans. Beside its strong expression in the kidney, NKCC2 has been also shown to be expressed in other tissues in rodents i.e. the gastrointestinal tract, pancreatic beta cells, and specific compartments of the ear, nasal tissue and eye.

Results

To examine if, besides kidney defects, further organ systems and/or metabolic pathways are affected by the Slc12a1^I299F mutation as primary or secondary effects, we describe a standardized, systemic phenotypic analysis of the mutant mouse line Slc12a1^I299F in the German Mouse Clinic. Slc12a1^I299F homozygous mutant mice and Slc12a1^I299F heterozygous mutant littermates as controls were tested at the age of 4–6 months. Beside the already published changes in blood pressure and bone metabolism, a significantly lower body weight and fat content were found as new phenotypes for Slc12a1^I299F homozygous mutant mice. Small additional effects included a mild erythropenic anemia in homozygous mutant males as well as a slight hyperalgesia in homozygous mutant females. For other functions, such as immunology, lung function and neurology, no distinct alterations were observed.

Conclusions

In this systemic analysis no clear primary effects of the Slc12a1^I299F mutation appeared for the organs other than the kidneys where Slc12a1 expression has been described. On the other hand, long-term effects additional and/or secondary to the kidney lesions might also appear in humans harboring SLC12A1 mutations.     

Pou4f1 and Pou4f2 Are Dispensable for the Long-Term Survival of Adult Retinal Ganglion Cells in Mice

Purpose

To investigate the role of Pou4f1 and Pou4f2 in the survival of adult retinal ganglion cells (RGCs).

Methods

Conditional alleles of Pou4f1 and Pou4f2 were generated (Pou4f1^loxP and Pou4f2^loxP respectively) for the removal of Pou4f1 and Pou4f2 in adult retinas. A tamoxifen-inducible Cre was used to delete Pou4f1 and Pou4f2 in adult mice and retinal sections and flat mounts were subjected to immunohistochemistry to confirm the deletion of both alleles and to quantify the changes in the number of RGCs and other retinal neurons. To determine the effect of loss of Pou4f1 and Pou4f2 on RGC survival after axonal injury, controlled optic nerve crush (CONC) was performed and RGC death was assessed.

Results

Pou4f1 and Pou4f2 were ablated two weeks after tamoxifen treatment. Retinal interneurons and Müller glial cells are not affected by the ablation of Pou4f1 or Pou4f2 or both. Although the deletion of both Pou4f1 and Pou4f2 slightly delays the death of RGCs at 3 days post-CONC in adult mice, it does not affect the cell death progress afterwards. Moreoever, deletion of Pou4f1 or Pou4f2 or both has no impact on the long-term viability of RGCs at up to 6 months post-tamoxifen treatment.

Conclusion

Pou4f1 and Pou4f2 are involved in the acute response to damage to RGCs but are dispensable for the long-term survival of adult RGC in mice.     

Microphthalmia, parkinsonism, and enhanced nociception in Pitx3 416insG mice

A new spontaneous mouse mutant was characterized by closed eyelids at weaning and without apparent eyes (provisional gene name, eyeless; provisional gene symbol, eyl). The mutation follows a recessive pattern of inheritance and was mapped to the region of chromosome 19 containing Pitx3. Genetic complementation tests using Pitx3 ^ak/+ mice confirmed eyl as a new allele of Pitx3 (Pitx3 ^eyl ). Sequencing of the Pitx3 gene in eyl mutants identified an inserted G after cDNA position 416 (416insG; exon 4). The shifted open reading frame is predicted to result in a hybrid protein still containing the Pitx3 homeobox, but followed by 121 new amino acids. The novel Pitx3 ^eyl/eyl mutants expressed ophthalmological and brain defects similar to Pitx3 ^ak/ak mice: microphthalmia or anophthalmia and loss of dopamine neurons of the substantia nigra. In addition, we observed in the homozygous eyeless mutants increased extramedullary hematopoiesis in the spleen, frequently liver steatosis, and reduced body weight. There were also several behavioral changes in the homozygous mutants, including reduced forelimb grip strength and increased nociception. In addition to these alterations in both sexes, we observed in female Pitx3 ^eyl/eyl mice increased anxiety-related behavior, reduced locomotor activity, reduced object exploration, and increased social contacts; however, we observed decreased anxiety-related behavior and increased arousal in males. Most of these defects identified in the new Pitx3 mutation are observed in Parkinson patients, making the Pitx3 ^eyl mutant a valuable new model. It is the first mouse mutant carrying a point mutation within the coding region of Pitx3.     

Distinct Acute Lymphoblastic Leukemia (ALL)-associated Janus Kinase 3 (JAK3) Mutants Exhibit Different Cytokine-Receptor Requirements and JAK Inhibitor Specificities

JAK1 and JAK3 are recurrently mutated in acute lymphoblastic leukemia. These tyrosine kinases associate with heterodimeric cytokine receptors such as IL-7 receptor or IL-9 receptor, in which JAK1 is appended to the specific chain, and JAK3 is appended to the common gamma chain. Here, we studied the role of these receptor complexes in mediating the oncogenic activity of JAK3 mutants. Although JAK3^V674A and the majority of other JAK3 mutants needed to bind to a functional cytokine receptor complex to constitutively activate STAT5, JAK3^L857P was unexpectedly found to not depend on such receptor complexes for its activity, which was induced without receptor or JAK1 co-expression. Introducing a mutation in the FERM domain that abolished JAK-receptor interaction did not affect JAK3^L857P activity, whereas it inhibited the other receptor-dependent mutants. The same cytokine receptor independence as for JAK3^L857P was observed for homologous Leu⁸⁵⁷ mutations of JAK1 and JAK2 and for JAK3^L875H. This different cytokine receptor requirement correlated with different functional properties in vivo and with distinct sensitivity to JAK inhibitors. Transduction of murine hematopoietic cells with JAK3^V674A led homogenously to lymphoblastic leukemias in BALB/c mice. In contrast, transduction with JAK3^L857P induced various types of lymphoid and myeloid leukemias. Moreover, ruxolitinib, which preferentially blocks JAK1 and JAK2, abolished the proliferation of cells transformed by the receptor-dependent JAK3^V674A, yet proved much less potent on cells expressing JAK3^L857P. These particular cells were, in contrast, more sensitive to JAK3-specific inhibitors. Altogether, our results showed that different JAK3 mutations induce constitutive activation through distinct mechanisms, pointing to specific therapeutic perspectives.     

Disc1 Variation Leads to Specific Alterations in Adult Neurogenesis

Disrupted in schizophrenia 1 (DISC1) is a risk factor for a spectrum of neuropsychiatric illnesses including schizophrenia, bipolar disorder, and major depressive disorder. Here we use two missense Disc1 mouse mutants, described previously with distinct behavioural phenotypes, to demonstrate that Disc1 variation exerts differing effects on the formation of newly generated neurons in the adult hippocampus. Disc1 mice carrying a homozygous Q31L mutation, and displaying depressive-like phenotypes, have fewer proliferating cells while Disc1 mice with a homozygous L100P mutation that induces schizophrenia-like phenotypes, show changes in the generation, placement and maturation of newly generated neurons in the hippocampal dentate gyrus. Our results demonstrate Disc1 allele specific effects in the adult hippocampus, and suggest that the divergence in behavioural phenotypes may in part stem from changes in specific cell populations in the brain.     

Exome sequencing identifies a nonsense mutation in <Emphasis Type="Italic">Fam46a</Emphasis> associated with bone abnormalities in a new mouse model for skeletal dysplasia

We performed exome sequencing for mutation discovery of an ENU (N-ethyl-N-nitrosourea)-derived mouse model characterized by significant elevated plasma alkaline phosphatase (ALP) activities in female and male mutant mice, originally named BAP014 (bone screen alkaline phosphatase #14). We identified a novel loss-of-function mutation within the Fam46a (family with sequence similarity 46, member A) gene (NM_001160378.1:c.469G>T, NP_001153850.1:p.Glu157*). Heterozygous mice of this mouse line (renamed Fam46a ^E157*Mhda) had significantly high ALP activities and apparently no other differences in morphology compared to wild-type mice. In contrast, homozygous Fam46a ^E157*Mhda mice showed severe morphological and skeletal abnormalities including short stature along with limb, rib, pelvis, and skull deformities with minimal trabecular bone and reduced cortical bone thickness in long bones. ALP activities of homozygous mutants were almost two-fold higher than in heterozygous mice. Fam46a is weakly expressed in most adult and embryonic tissues with a strong expression in mineralized tissues as calvaria and femur. The FAM46A protein is computationally predicted as a new member of the superfamily of nucleotidyltransferase fold proteins, but little is known about its function. Fam46a ^E157*Mhda mice are the first mouse model for a mutation within the Fam46a gene.     

Pou4f2‐GFP knock‐in mouse line: A model for studying retinal ganglion cell development

Pou4f2 acts as a key node in the comprehensive and step‐wise gene regulatory network (GRN) and regulates the development of retinal ganglion cells (RGCs). Accordingly, deletion of Pou4f2 results in RGC axon defects and apoptosis. To investigate the GRN involved in RGC regeneration, we generated a mouse line with a POU4F2‐green fluorescent protein (GFP) fusion protein expressed in RGCs. Co‐localization of POU4F2 and GFP in the retina and brain of Pou4f2‐GFP/+ heterozygote mice was confirmed using immunofluorescence analysis. Compared with those in wild‐type mice, the expression patterns of POU4F2 and POU4F1 and the co‐expression patterns of ISL1 and POU4F2 were unaffected in Pou4f2‐GFP/GFP homozygote mice. Moreover, the quantification of RGCs showed no significant difference between Pou4f2‐GFP/GFP homozygote and wild‐type mice. These results demonstrated that the development of RGCs in Pou4f2‐GFP/GFP homozygote mice was the same as in wild‐type mice. Thus, the present Pou4f2‐GFP knock‐in mouse line is a useful tool for further studies on the differentiation and regeneration of RGCs.     

Biochemical and genetic evidence for a macromolecular -glucuronidase complex in microsomal membranes

In the mouse β-glucuronidase is present in both microsomes and lysosomes and the enzyme at both sites is coded by the same structural gene. Electrophoresis on polyacrylamide gels showed that liver, kidney and lung from normal strains contained five enzyme forms designated L, M1, M2, M3 and M4 in order of decreasing mobility toward the anode. Band L is found primarily in lysosomes and is a tetramer of 260,000 molecular weight. Bands M1 to M4 are found exclusively in microsomes and range in molecular weight from 310,000 to 470,000. The increase in molecular weight is due to sequential addition of an accessory protein chain. When glucuronidase is highly induced in kidneys of female mice by injection of dihydrotestosterone, a sixth electrophoretic form of glucuronidase, designated X, appears. Form X appears early in induction, is localized in microsomes, and has a molecular weight (260,000) equal to that of the tetramer form L.Mice homozygous for the eg ° mutation, and thus deficient in microsomal glucuronidase, completely lack the microsomal forms M1 to M4. They do contain form X, and this increases after testosterone induction in kidney. The form X present in eg ° mice is indistinguishable from the form X seen in normal induced kidney.It appears that mice synthesize two different tetrameric forms of glucuronidase from the same structural gene. One, form L, is lysosomal; the other, form X, gives rise to microsomal enzyme forms M1 to M4 by the successive addition of up to four accessory protein chains. The eg ° mutant is blocked in the conversion of X to M1.     

Generation and Characterization of a Mouse Model Harboring the Exon-3 Deletion in the Cardiac Ryanodine Receptor

A large genomic deletion in human cardiac ryanodine receptor (RYR2) gene has been detected in a number of unrelated families with various clinical phenotypes, including catecholaminergic polymorphic ventricular tachycardia (CPVT). This genomic deletion results in an in-frame deletion of exon-3 (Ex3-del). To understand the underlying disease mechanism of the RyR2 Ex3-del mutation, we generated a mouse model in which the RyR2 exon-3 sequence plus 15-bp intron sequences flanking exon-3 were deleted. Heterozygous Ex3-del mice (Ex3-del^+/−) survived, but no homozygous Ex3-del mice were born. Unexpectedly, the Ex3-del^+/− mice are not susceptible to CPVT. Ex3-del^+/− cardiomyocytes exhibited similar amplitude but altered dynamics of depolarization-induced Ca²⁺ transients compared to wild type (WT) cells. Immunoblotting analysis revealed markedly reduced expression of RyR2 protein in the Ex3-del^+/− mutant heart, indicating that Ex3-del has a major impact on RyR2 protein expression in mice. Cardiac specific, conditional knockout of the WT RyR2 allele in Ex3-del^+/− mice led to bradycardia and death. Thus, the absence of CPVT and other phenotypes in Ex3-del^+/− mice may be attributable to the predominant expression of the WT RyR2 allele as a result of the markedly reduced expression of the Ex3-del mutant allele. The effect of Ex3-del on RyR2 protein expression is discussed in relation to the phenotypic variability in individuals with the RyR2 exon-3 deletion.     

Proteomic analysis of differentially expressed skin proteins in iRhom2Uncv mice

A mouse homozygous for the spontaneous mutation uncovered (Uncv) has a hairless phenotype. A 309-bp non-frameshift deletion mutation in the N-terminal cytoplasmic domain of iRhom2 was identified in Uncv mice (iRhom2^Uncv) using target region sequencing. The detailed molecular basis for how the iRhom2 mutation causes the hairless phenotype observed in the homozygous iRhom2^Uncv mouse remains unknown. To identify differentially expressed proteins in the skin of wild-type and homozygous iRhom2^Uncv littermates at postnatal day 5, proteomic approaches, including two-dimensional gel electrophoresis and mass spectrometry were used. Twelve proteins were differentially expressed in the skin in a comparison between wild-type and homozygous iRhom2^Uncv mice. A selection of the proteomic results were tested and verified using qRT-PCR, western blot and immunohistochemistry. These data indicate that differentially expressed proteins, especially KRT73, MEMO1 and Coro-1, might participate in the mechanism by which iRhom2 regulates the development of murine skin. [BMB Reports 2015; 48(1): 19-24]     

Sequential functioning of Sym-13 and Sym-31, two genes affecting symbiosome development in root nodules of pea (Pisum sativum L.)

Two Fix^? mutants of pea (Pisum sativum L.) which are unable to fix molecular nitrogen, E135f (sym-13) and Sprint-2Fix^? (sym-31), were crossed to create the doubly homozygous recessive line, named RBT (sym-13, sym-31). The ultrastructural organization of nodules of the RBT line was compared with that of each of the two parental mutant lines and with the original wild-type genotypes of the cultivars Sparkle and Sprint-2. It was shown that the RBT line is similar to the mutant line Sprint-2Fix^? in having abnormal symbiosome composition and bacteroids with relatively undifferentiated morphology. Because the phenotypic manifestation of the sym-31 mutant allele suppresses the phenotypic manifestation of the sym-13 mutant allele, it is concluded that the function of the gene Sym-31 (which is mutated in the Sprint-2Fix^? line) is necessary at an earlier stage of symbiosome development than the gene Sym-13 (which is mutant in the E135f line).     

Essential amino acid residues in the central transmembrane domains and loops for energy coupling of Streptomyces coelicolor A3(2) H-pyrophosphatase

The H⁺-translocating inorganic pyrophosphatase is a proton pump that hydrolyzes inorganic pyrophosphate. It consists of a single polypeptide with 14−17 transmembrane domains, and is found in a range of organisms. We focused on the second quarter region of Streptomyces coelicolor A3(2) H⁺-pyrophosphatase, which contains long conserved cytoplasmic loops. We prepared a library of 1536 mutants that were assayed for pyrophosphate hydrolysis and proton translocation. Mutant enzymes with low substrate hydrolysis and proton-pump activities were selected and their DNAs sequenced. Of these, 34 were single-residue substitution mutants. We generated 29 site-directed mutant enzymes and assayed their activity. The mutation of 10 residues in the fifth transmembrane domain resulted in low coupling efficiencies, and a mutation of Gly¹⁹⁸ showed neither hydrolysis nor pumping activity. Four residues in cytoplasmic loop e were essential for substrate hydrolysis and efficient H⁺ translocation. Pro¹⁸⁹, Asp²⁸¹, and Val³⁵¹ in the periplasmic loops were critical for enzyme function. Mutation of Ala³⁵⁷ in periplasmic loop h caused a selective reduction of proton-pump activity. These low-efficiency mutants reflect dysfunction of the energy-conversion and/or proton-translocation activities of H⁺-pyrophosphatase. Four critical residues were also found in transmembrane domain 6, three in transmembrane domain 7, and five in transmembrane domains 8 and 9. These results suggest that transmembrane domain 5 is involved in enzyme function, and that energy coupling is affected by several residues in the transmembrane domains, as well as in the cytoplasmic and periplasmic loops. H⁺-pyrophosphatase activity might involve dynamic linkage between the hydrophilic and transmembrane domains.