NOTCH1 Gain of Function in Germ Cells Causes Failure of Spermatogenesis in Male Mice

NOTCH1 is a member of the NOTCH receptor family, a group of single-pass trans-membrane receptors. NOTCH signaling is highly conserved in evolution and mediates communication between adjacent cells. NOTCH receptors have been implicated in cell fate determination, as well as maintenance and differentiation of stem cells. In the mammalian testis expression of NOTCH1 in somatic and germ cells has been demonstrated, however its role in spermatogenesis was not clear. To study the significance of NOTCH1 in germ cells, we applied a cre/loxP approach in mice to induce NOTCH1 gain- or loss-of function specifically in male germ cells. Using a Stra8-icretransgene we produced mice with conditional activation of the NOTCH1 intracellular domain (NICD) in germ cells. Spermatogenesis in these mutants was progressively affected with age, resulting in decreased testis weight and sperm count. Analysis of downstream target genes of NOTCH1 signaling showed an increased expression of Hes5, with a reduction of the spermatogonial differentiation marker, Neurog3 expression in the mutant testis. Apoptosis was significantly increased in mouse germ cells with the corresponding elevation of pro-apoptotic Trp53 and Trp63genes\u27 expression. We also showed that the conditional germ cell-specific ablation of Notch1 had no effect on spermatogenesis or male fertility. Our data suggest the importance of NOTCH signaling regulation in male germ cells for their survival and differentiation

DJ1 expression downregulates in neuroblastoma cells (SK-N-MC) chronically exposed to HIV-1 and cocaine

Background: HIV-associated neurological disorder (HAND) has long been recognized as a consequence of human immunodeficiency virus (HIV) infection in the brain. The pathology of HAND gets more complicated with the recreational drug use such as cocaine. Recent studies have suggested multiple genetic influences involved in the pathology of addiction and HAND but only a fraction of the entire genetic risk has been investigated so far. In this regard, role of DJ1 protein (a gene linked to autosomal recessive early-onset Parkinson’s disease) in regulating dopamine (DA) transmission and reactive oxygen species (ROS) production in neuronal cells will be worth investigating in HIV-1 and cocaine exposed microenvironment. Being a very abundant protein in the brain, DJ1 could serve as a potential marker for early detection of HIV-1 and/or cocaine related neurological disorder. Methods: In vitro analysis was done to observe the effect of HIV-1 and/or cocaine on DJ1 protein expression in neuroblastoma cells (SK-N-MC). Gene and protein expression analysis of DJ1 was done on the HIV infected and/or cocaine treated SK-N-MC and compared to untreated cells using real time PCR, Western Blot and flow cytometry. Effect of DJ1 dysregulation on oxidative stress was analyzed by measuring ROS production in these cells. Results: Gene expression and protein analysis indicated that there was a significant decrease in DJ1 expression in SK-N-MC chronically exposed to HIV-1 and/or cocaine which is inversely proportional to ROS production. Conclusion: This is the first study to establish that DJ1 expression level in the neuronal cells significantly decreased in presence of HIV-1 and/or cocaine indicating oxidative stress level of DA neurons

Mechanisms of INSL3 signaling in male reproductive organs

Global ablation of INSL3 hormone or its receptor RXFP2 in male mice results in cryptorchidism and infertility. Using novel LacZ knock-in Rxfp2 allele we demonstrated a strong expression of this gene in postmeiotic germ cells. RXFP2 was expressed in embryonic and neonatal gubernaculum. No RXFP2 expression was detected in cremaster muscles in adult mice. We produced a floxed allele of Rxfp2 and then deleted this gene in male germ cells in testes located in normal scrotal position. No differences in fertility or spermatogenesis of such males were found, suggesting non-essential role of INSL3 signaling in germ cell differentiation in adult males. We have also produced shRNA transgenic mice with reduced RXFP2 expression Such males manifested various degree of uni- and bilateral cryptorchidism. Total gene expression analysis of the mutant cremasteric sacs indicated misexpression of a significant number of genes in Wnt/β-catenin and NOTCH pathways. Conditional deletion of β-catenin or Notch1 genes in male gubernacular ligament resulted in its abnormal development. Our data suggest that β-catenin and NOTCH1 pathways are potential targets of INSL3 signaling during gubernacular development

Rescue of auditory function by a single Administration of AAV-TMPRSS3 Gene Therapy in Aged Mice of Human Recessive Deafness DFNB8

Patients with mutations in the TMPRSS3 gene suffer from recessive deafness DFNB8/DFNB10. For these patients, cochlear implantation is the only treatment option. Poor cochlear implantation outcomes are seen in some patients. To develop biological treatment for TMPRSS3 patients, we generated a knockin mouse model with a frequent human DFNB8 TMPRSS3 mutation. The Tmprss3A306T/A306T homozygous mice display delayed onset progressive hearing loss similar to human DFNB8 patients. Using AAV2 as a vector to carry a human TMPRSS3 gene, AAV2-hTMPRSS3 injection in the adult knockin mouse inner ear results in TMPRSS3 expression in the hair cells and the spiral ganglion neurons. A single AAV2-hTMPRSS3 injection in Tmprss3A306T/A306T mice of an average age of 18.5 months leads to sustained rescue of the auditory function to a level similar to wild-type mice. AAV2-hTMPRSS3 delivery rescues the hair cells and the spiral ganglions neurons. This study demonstrates successful gene therapy in an aged mouse model of human genetic deafness. It lays the foundation to develop AAV2-hTMPRSS3 gene therapy to treat DFNB8 patients, as a standalone therapy or in combination with cochlear implantation

Germ cell-specific NOTCH1 gain-of-function in male mice.

<p>A. A transgene encoding an intracellular portion of the mouse <i>Notch1</i> gene (NICD) is inserted into the <i>ROSA</i> locus. The transcription of the transgene is disrupted by the presence of the floxed <i>Neo</i> cassette (PGK-Neo-pA:3pA). After cre-mediated removal of the <i>Neo</i> cassette, the transgene expresses the NICD. IRES, internal ribosome entry site, separates NICD-encoding sequence from nuclear enhanced green fluorescent protein (nEGFP) sequence. B. Genotyping of mice with <i>ROSA-Notch1^fl</i> allele (320 bp) and wild-type allele (235 bp) using ear DNA PCR. C. Detection of activated <i>ROSA-Notch1</i> allele in genomic DNA isolated from mutant (<i>ROSA-Notch1^fl/+, Stra8-icre/+</i>) adult testes and epidydimal sperm but not control (<i>ROSA-Notch1^fl/+, +</i>) DNA. The 650 bp fragment is specific for the activated <i>ROSA-Notch1</i> allele. Control PCR with primers specific for <i>Ctnnb1</i> locus was used to confirm the quality of isolated DNA. D. The <i>NICD</i> expression was increased in mutant testis. The QRT-PCR analysis of <i>NICD</i> expression in RNA isolated from control (n = 4, 3, and 3, respectively) and gain-of-function NOTCH1 mutants (n = 4, 4, and 3, respectively) at day 50, 140, and 400 after birth. The data are normalized to the expression of <i>Actb</i> gene and the level of gene expression in control samples was assumed to be equal 1 at each time point. *P<0.05. E. Expression of NICD protein in mutant testis. Western blot analysis was used to verify transgenic NICD expression in testicular lysates of adult mutant and normal males. The transgenic NICD (MW  = 58.69 kDa) is expressed only in mutant. f. Increased <i>Hes5</i> gene expression in mutant testis. The QRT-PCR analysis of <i>Hes5</i> expression in RNA isolated from control (n = 4, 3, and 3, respectively) and gain-of-function NOTCH1 mutants (n = 4, 4, and 3, respectively) at day 50, 140, and 400 after birth. The data are normalized to the expression of <i>Actb</i> gene and the level of gene expression in control samples was assumed to be equal 1 at each time point. *P<0.05.</p

Characterization of reproductive phenotype of male mice with conditional deletion of <i>Notch1</i> in germ cells.

<p>Characterization of reproductive phenotype of male mice with conditional deletion of <i>Notch1</i> in germ cells.</p

Expression of spermatogonial stem cell marker Neurog3 decreases in mutant testis.

<p>A. Expression of spermatogonial stem cell markers in control and NOTCH1 gain-of-function mutants. The expression of SSC markers in adult (140 day) control and mutant testes were compared by QRT-PCR. The <i>Neurog3</i> gene is significantly down-regulated in mutant testis (*P<0.05). QRT-PCR results were normalized to the <i>Plzf</i> gene expression. The expression of each gene in control samples was assumed to be equal to 1. Number of samples N≥3 in each group. B. The expression of <i>Neurog3</i> gene is reduced in mutant testes of different ages. *P<0.05. Number of samples N≥3 in each group. QRT-PCR results were normalized to <i>Plzf</i> gene expression.</p

The effect of transgenic activation of NOTCH1 signaling in spermatogonial germ cells.

<p>In male spermatogonial cells, the known target of NOTCH1, HES5 is up-regulated and causes a decrease of NEUROG3 expression. This leads to a failure of spermatogonial cell differentiation. Simultaneously, the activation of NOTCH1 signaling induces an increased apoptosis with overexpression of pro-apoptotic genes TRP53 and TRP63. Both pathways contribute to the progressive decrease in sperm number and testis weight.</p

NOTCH1 expression in testis.

<p>Immunohistochemistry images of wild-type testis sections stained with anti-NOTCH antibodies. The staining was detected in pre-spermatogonial cells in P1 testis (arrows) and at later stages of spermatogenesis in adult (140 days old) testes. A weak non-specific staining was detected in interstitial testicular cells, but not in germ cells without primary antibody control sections. All images were acquired under the same magnification. The scale bar in control is 20 μm.</p