Neuregulin 3 Knockout Mice Exhibit Behaviors Consistent with Psychotic Disorders.

Neuregulin 3 (NRG3) is a paralog of NRG1. Genetic studies in schizophrenia demonstrate that risk variants in NRG3 are associated with cognitive and psychotic symptom severity, and several intronic single nucleotide polymorphisms in NRG3 are associated with delusions in patients with schizophrenia. In order to gain insights into the biological function of the gene, we generated a novel Nrg3 knockout (KO) mouse model and tested for neurobehavioral phenotypes relevant to psychotic disorders. KO mice displayed novelty-induced hyperactivity, impaired prepulse inhibition of the acoustic startle response, and deficient fear conditioning. No gross cytoarchitectonic or layer abnormalities were noted in the brain of KO mice. Our findings suggest that deletion of the Nrg3 gene leads to alterations consistent with aspects of schizophrenia. We propose that KO mice will provide a valuable animal model to determine the role of the NRG3 in the molecular pathogenesis of schizophrenia and other psychotic disorders.

Enhanced conversion of induced neuronal cells (iN cells) from human fibroblasts: Utility in uncovering cellular deficits in mental illness-associated chromosomal abnormalities.

The novel technology of induced neuronal cells (iN cells) is promising for translational neuroscience, as it allows the conversion of human fibroblasts into cells with postmitotic neuronal traits. However, a major technical barrier is the low conversion rate. To overcome this problem, we optimized the conversion media. Using our improved formulation, we studied how major mental illness-associated chromosomal abnormalities may impact the characteristics of iN cells. We demonstrated that our new iN cell culture protocol enabled us to obtain more precise measurement of neuronal cellular phenotypes than previous iN cell methods. Thus, this iN cell culture provides a platform to efficiently obtain possible cellular phenotypes caused by genetic differences, which can be more thoroughly studied in research using other human cell models such as induced pluripotent stem cells.

Mutation analysis of PEX7 in 60 probands with rhizomelic chondrodysplasia punctata and functional correlations of genotype with phenotype.

PEX7 encodes the cytosolic receptor for the set of peroxisomal matrix enzymes targeted to the organelle by the peroxisome targeting signal 2 (PTS2). Mutations in PEX7 cause rhizomelic chondrodysplasia punctata (RCDP), a distinct peroxisome biogenesis disorder. In previous work we described three novel PEX7 mutant alleles, including one, L292X, with a high frequency due to a founder effect. We have now extended our analysis to 60 RCDP probands and identified a total of 24 PEX7 alleles, accounting for 95% of the mutant PEX7 genes in our sample. Of these, 50% are L292X, 13% are IVS9+1G>C, and the remainder are mostly private. IVS9+1G>C occurs on at least three different haplotypes and thus appears to result from recurrent mutation. The phenotypic spectrum of RCDP is broader than commonly recognized and includes minimally affected individuals at the mild end of the spectrum. To relate PEX7 genotype and phenotype, we evaluated the consequence of the disease mutation on PEX7 RNA by Northern analysis and RT/PCR. We evaluated the function of the encoded Pex7 protein (Pex7p) by expressing selected alleles in fibroblasts from RCDP patients and assaying their ability to restore import of a PTS2 marker protein. We find that residual activity of mutant Pex7p and reduced amounts of normal Pex7p are associated with milder and variant phenotypes.

X chromosome cDNA microarray screening identifies a functional PLP2 promoter polymorphism enriched in patients with X-linked mental retardation.

X-linked Mental Retardation (XLMR) occurs in 1 in 600 males and is highly genetically heterogeneous. We used a novel human X chromosome cDNA microarray (XCA) to survey the expression profile of X-linked genes in lymphoblasts of XLMR males. Genes with altered expression verified by Northern blot and/or quantitative PCR were considered candidates. To validate this approach, we documented the expected changes of expression in samples from a patient with a known X chromosome microdeletion and from patients with multiple copies of the X chromosome. We used our XCA to survey lymphoblast RNA samples from 43 unrelated XLMR males and found 15 genes with significant (>or=1.5-fold) reduction in expression in at least one proband. Of these, subsequent analysis confirmed altered expression in 12. We followed up one, PLP2, at Xp11.23, which exhibits approximately fourfold decreased expression in two patients. Sequencing analysis in both patients revealed a promoter variant, -113C>A, that alters the core-binding site of the transcription factor ELK1. We showed that PLP2-(-113C>A) is sufficient to cause reduced expression using a luciferase reporter system and is enriched in a cohort of males with probable XLMR (14 of 239, 5.85%) as compared to normal males (9 of 577, 1.56%) (chi2=11.07, P<0.001). PLP2 is expressed abundantly in the pyramidal cells of hippocampus and granular cells of the cerebellum in the brain. We conclude that our XCA screening is an efficient strategy to identify genes that show significant changes in transcript abundance as candidate genes for XLMR.

Overexpression of proline oxidase induces proline-dependent and mitochondria-mediated apoptosis.

Proline oxidase (POX), a mitochondrial inner-membrane protein, catalyzes the rate-limiting oxidation of proline to pyrroline- 5-carboxylate (P5C). Previously we showed that overexpression of POX is associated with generation of reactive oxygen species (ROS) and apoptosis in POX-inducible colorectal cancer cells, DLD-1.POX. We also showed expression of mitochondrial MnSOD partially blunts POX-induced ROS generation and apoptosis. To further investigate the molecular basis of POX-induced apoptosis, we utilized the DLD-1.POX cells to show that cells overproducing POX exhibit an L-proline-dependent apoptotic response. The apoptotic effect is specific for L-proline, detectable at 0.2 mM, maximal at 1 mM, and occurs during 48-72 h following the addition of L-proline to cells with maximally induced POX. The apoptotic response is mitochondria-mediated with release of cytochrome c, activation of caspase-9, chromatin condensation/DNA fragmentation, and cell shrinkage. We conclude that in the presence of proline, high POX activity is sufficient to induce mitochondria-mediated apoptosis.

Alternative splicing suggests extended function of PEX26 in peroxisome biogenesis.

Matsumoto and colleagues recently identified PEX26 as the gene responsible for complementation group 8 of the peroxisome biogenesis disorders and showed that it encodes an integral peroxisomal membrane protein with a single C-terminal transmembrane domain and a cytosolic N-terminus that interacts with the PEX1/PEX6 heterodimer through direct binding to the latter. They proposed that PEX26 functions as the peroxisomal docking factor for the PEX1/PEX6 heterodimer. Here, we identify new PEX26 disease alleles, localize the PEX6-binding domain to the N-terminal half of the protein (aa 29-174), and show that, at the cellular level, PEX26 deficiency impairs peroxisomal import of both PTS1- and PTS2-targeted matrix proteins. Also, we find that PEX26 undergoes alternative splicing to produce several splice forms--including one, PEX26- delta ex5, that maintains frame and encodes an isoform lacking the transmembrane domain of full-length PEX26 (PEX26-FL). Despite its cytosolic location, PEX26- delta ex5 rescues peroxisome biogenesis in PEX26-deficient cells as efficiently as does PEX26-FL. To test our observation that a peroxisomal location is not required for PEX26 function, we made a chimeric protein (PEX26-Mito) with PEX26 as its N-terminus and the targeting segment of a mitochondrial outer membrane protein (OMP25) at its C-terminus. We found PEX26-Mito localized to the mitochondria and directed all detectable PEX6 and a fraction of PEX1 to this extraperoxisomal location; yet PEX26-Mito retains the full ability to rescue peroxisome biogenesis in PEX26-deficient cells. On the basis of these observations, we suggest that a peroxisomal localization of PEX26 and PEX6 is not required for their function and that the interaction of PEX6 with PEX1 is dynamic. This model predicts that, once activated in an extraperoxisomal location, PEX1 moves to the peroxisome and completes the function of the PEX1/6 heterodimer.