Paternally expressed gene 3 (Pw1/Peg3) promotes sexual dimorphism in metabolism and behavior.

The paternally expressed gene 3 (Pw1/Peg3) is a mammalian-specific parentally imprinted gene expressed in stem/progenitor cells of the brain and endocrine tissues. Here, we compared phenotypic characteristics in Pw1/Peg3 deficient male and female mice. Our findings indicate that Pw1/Peg3 is a key player for the determination of sexual dimorphism in metabolism and behavior. Mice carrying a paternally inherited Pw1/Peg3 mutant allele manifested postnatal deficits in GH/IGF dependent growth before weaning, sex steroid dependent masculinization during puberty, and insulin dependent fat accumulation in adulthood. As a result, Pw1/Peg3 deficient mice develop a sex-dependent global shift of body metabolism towards accelerated adiposity, diabetic-like insulin resistance, and fatty liver. Furthermore, Pw1/Peg3 deficient males displayed reduced social dominance and competitiveness concomitant with alterations in the vasopressinergic architecture in the brain. This study demonstrates that Pw1/Peg3 provides an epigenetic context that promotes male-specific characteristics through sex steroid pathways during postnatal development.

An allograft mouse model for the study of hearing loss secondary to vestibular schwannoma growth.

Vestibular schwannoma is a benign neoplasm arising from the Schwann cell sheath of the auditory-vestibular nerve. It most commonly affects both sides in the genetic condition Neurofibromatosis type 2, causing progressive high frequency sensorineural hearing loss. Here, we describe a microsurgical technique and stereotactic coordinates for schwannoma cell grafting in the vestibular nerve region that recapitulates local tumor growth in the cerebellopontine angle and inner auditory canal with resulting hearing loss. Tumor growth was monitored by bioluminescence and MRI in vivo imaging, and hearing assessed by auditory brainstem responses. These techniques, by potentially enabling orthotopic grafting of a variety of cell lines will allow studies on the pathogenesis of tumor-related hearing loss and preclinical drug evaluation, including hearing endpoints, for NF2-related and sporadic schwannomas.

The zinc finger transcription factor PW1/PEG3 restrains murine beta cell cycling.

AIMS/HYPOTHESIS: Pw1 or paternally-expressed gene 3 (Peg3) encodes a zinc finger transcription factor that is widely expressed during mouse embryonic development and later restricted to multiple somatic stem cell lineages in the adult. The aim of the present study was to define Pw1 expression in the embryonic and adult pancreas and investigate its role in the beta cell cycle in Pw1 wild-type and mutant mice. METHODS: We analysed PW1 expression by immunohistochemistry in pancreas of nonpregant and pregnant mice and following injury by partial duct ligation. Its role in the beta cell cycle was studied in vivo using a novel conditional knockout mouse and in vitro by lentivirus-mediated gene knockdown. RESULTS: We showed that PW1 is expressed in early pancreatic progenitors at E9.5 but becomes progressively restricted to fully differentiated beta cells as they become established after birth and withdraw from the cell cycle. Notably, PW1 expression declines when beta cells are induced to proliferate and loss of PW1 function activates the beta cell cycle. CONCLUSIONS/INTERPRETATION: These results indicate that PW1 is a co-regulator of the beta cell cycle and can thus be considered a novel therapeutic target in diabetes.

Tubular proteinuria in patients with HNF1α mutations: HNF1α drives endocytosis in the proximal tubule.

Hepatocyte nuclear factor 1α (HNF1α) is a transcription factor expressed in the liver, pancreas, and proximal tubule of the kidney. Mutations of HNF1α cause an autosomal dominant form of diabetes mellitus (MODY-HNF1A) and tubular dysfunction. To gain insights into the role of HNF1α in the proximal tubule, we analyzed Hnf1a-deficient mice. Compared with wild-type littermates, Hnf1a knockout mice showed low-molecular-weight proteinuria and a 70% decrease in the uptake of ß2-microglobulin, indicating a major endocytic defect due to decreased expression of megalin/cubilin receptors. We identified several binding sites for HNF1α in promoters of Lrp2 and Cubn genes encoding megalin and cubilin, respectively. The functional interaction of HNF1α with these promoters was shown in C33 epithelial cells lacking endogenous HNF1α. Defective receptor-mediated endocytosis was confirmed in proximal tubule cells from these knockout mice and could be rescued by transfection of wild-type but not mutant HNF1α. Transfection of human proximal tubule HK2 cells with HNF1α was able to upregulate megalin and cubilin expression and to increase endocytosis of albumin. Low-molecular-weight proteinuria was consistently detected in individuals with HNF1A mutations compared with healthy controls and patients with non-MODY-HNF1A diabetes mellitus. Thus, HNF1α plays a key role in the constitutive expression of megalin and cubilin, hence regulating endocytosis in the proximal tubule of the kidney. These findings provide new insight into the renal phenotype of individuals with mutations of HNF1A.

mTORC1 inhibition delays growth of neurofibromatosis type 2 schwannoma.

BACKGROUND: Neurofibromatosis type 2 (NF2) is a rare autosomal dominant genetic disorder, resulting in a variety of neural tumors, with bilateral vestibular schwannomas as the most frequent manifestation. Recently, merlin, the NF2 tumor suppressor, has been identified as a novel negative regulator of mammalian target of rapamycin complex 1 (mTORC1); functional loss of merlin was shown to result in elevated mTORC1 signaling in NF2-related tumors. Thus, mTORC1 pathway inhibition may be a useful targeted therapeutic approach. METHODS: We studied in vitro cell models, cohorts of mice allografted with Nf2(-/-) Schwann cells, and a genetically modified mouse model of NF2 schwannoma in order to evaluate the efficacy of the proposed targeted therapy for NF2. RESULTS: We found that treatment with the mTORC1 inhibitor rapamycin reduced the severity of NF2-related Schwann cell tumorigenesis without significant toxicity. Consistent with these results, in an NF2 patient with growing vestibular schwannomas, the rapalog sirolimus induced tumor growth arrest. CONCLUSIONS: Taken together, these results constitute definitive evidence that justifies proceeding with clinical trials using mTORC1-targeted agents in selected patients with NF2 and in patients with NF2-related sporadic tumors.

Therapeutic potential of HSP90 inhibition for neurofibromatosis type 2.

PURPOSE: The growth and survival of neurofibromatosis type 2 (NF2)-deficient cells are enhanced by the activation of multiple signaling pathways including ErbBs/IGF-1R/Met, PI3K/Akt, and Ras/Raf/Mek/Erk1/2. The chaperone protein HSP90 is essential for the stabilization of these signaling molecules. The aim of the study was to characterize the effect of HSP90 inhibition in various NF2-deficient models. EXPERIMENTAL DESIGN: We tested efficacy of the small-molecule NXD30001, which has been shown to be a potent HSP90 inhibitor. The antiproliferative activity of NXD30001 was tested in NF2-deficient cell lines and in human primary schwannoma and meningioma cultures in vitro. The antitumor efficacy of HSP90 inhibition in vivo was verified in two allograft models and in one NF2 transgenic model. The underlying molecular alteration was further characterized by a global transcriptome approach. RESULTS: NXD30001 induced degradation of client proteins in and suppressed proliferation of NF2-deficient cells. Differential expression analysis identified subsets of genes implicated in cell proliferation, cell survival, vascularization, and Schwann cell differentiation whose expression was altered by NXD30001 treatment. The results showed that NXD30001 in NF2-deficient schwannoma suppressed multiple pathways necessary for tumorigenesis. CONCLUSIONS: HSP90 inhibition showing significant antitumor activity against NF2-related tumor cells in vitro and in vivo represents a promising option for novel NF2 therapies.

Increased neuronal glutathione and neuroprotection in GTRAP3-18-deficient mice.

Glutathione (GSH) is an important neuroprotective molecule in the brain. The strategy to increase neuronal GSH level is a promising approach to the treatment of neurodegenerative diseases. However, the regulatory mechanism by which neuron-specific GSH synthesis is facilitated remains elusive. Glutamate transporter-associated protein 3-18 (GTRAP3-18) is an endoplasmic reticulum protein interacting with excitatory amino acid carrier 1 (EAAC1), which is a neuronal glutamate/cysteine transporter. To investigate the potential regulatory mechanism to increase neuronal GSH level in vivo, we generated GTRAP3-18-deficient (GTRAP3-18(-/-)) mice using a gene-targeting approach. Disruption of the GTRAP3-18 gene resulted in increased EAAC1 expression in the plasma membrane, increased neuronal GSH content and neuroprotection against oxidative stress. In addition, GTRAP3-18(-/-) mice performed better in motor/spatial learning and memory tests than wild-type mice. Therefore, the suppression of GTRAP3-18 increases neuronal resistance to oxidative stress by increasing GSH content and also facilitates cognitive function. The present results may provide a molecular basis for the development of treatments for neurodegenerative diseases.

The transcription factor HNF1α regulates expression of chloride-proton exchanger ClC-5 in the renal proximal tubule.

The Cl(-)/H(+) exchanger ClC-5 is essential for the endocytic activity of the proximal tubule cells and the tubular clearance of proteins filtered in the glomeruli. The mechanisms that regulate the expression of ClC-5 in general and its specific expression in the proximal tubule are unknown. In this study, we investigated the hypothesis that the hepatocyte nuclear transcription factor HNF1α, which is predominantly expressed in proximal tubule segments, may directly regulate the expression of ClC-5. In situ hybridization demonstrated that the expression of Clcn5 overlaps with that of Hnf1α in the developing kidney as well as in absorptive epithelia, including the digestive tract and yolk sac. Multiple binding sites for HNF1 were mapped in the 5'-regulatory sequences of the mouse and human Clcn5/CLCN5 genes. The transactivation of the Clcn5/CLCN5 promoter by HNF1α was verified in vitro, and the binding of HNF1α to the Clcn5 promoter in vivo was confirmed by chromatin immunoprecipitation in mouse kidney. The expression of Clcn5 was reduced in the proximal tubule segments of HNF1α-null kidneys, and it was rescued upon transfection of HNF1α-null cells with wild-type but not with mutant HNF1α. These data demonstrate that HNF1α directly regulates the expression of ClC-5 in the renal proximal tubule and yield insights into the mechanisms governing epithelial differentiation and specialized transport activities in the kidney.

Reduced renal ClC-5 Cl- channel expression in spontaneously hypertensive rats with microalbuminuria.

Mutations in a renal-specific Cl(-) channel, ClC-5, result in low-molecular-weight proteinuria. Herein we studied ClC-5 expression in the kidneys of spontaneously hypertensive rats (SHR) to identify possible causes of their increased urinary excretion of albumin. The amount of ClC-5 protein was significantly reduced in 3-month-old SHR as compared with normotensive Wistar/Kyoto (WKY) rats. The ClC-5 protein level was partially restored by short term administration of perindopril, an inhibitor of angiotensin-converting enzyme. Corresponding to the increase in ClC-5 expression, the albuminuria in SHR improved to the control level. These results implicate the ClC-5 Cl(-) channel reduction in the development of albuminuria in the early stage of essential hypertension.