Developmental neuronal abnormalities in mouse models of PWS

Rachel Wevrick¹, Syann Lee¹, Christine L. Walker¹, Barbara Karten², Alysa A. P. Tennese¹, Sharee L. Kuny¹, Silvia Pagliardini³ and John J. Greer³ 

¹Department of Medical Genetics, University of Alberta, Edmonton, Alberta, Canada and ²Centre for Neuroscience, Department of Physiology, University of Alberta, Edmonton, Alberta, Canada.

     Only a few genes are functionally inactivated in individuals with PWS, although the typical 15q11-q13 deletion extends over several Mb.  Most PWS candidate genes are conserved in mice, and all are located in an orthologous region on mouse chromosome 7C.  Simultaneous inactivation of PWS-equivalent genes in the mouse is usually lethal in the first postnatal week, because of failure to thrive.  Several mouse models in which one or more of the PWS candidate genes are silenced partially recapitulate various aspects of the PWS phenotype.    The analysis of expression patterns of murine orthologues of human disease genes is valuable for identifying sites of gene expression that correlate with disease phenotype. We examined the patterns of PWS gene expression throughout the development of the murine brain, including the hypothalamus, pituitary, forebrain and hindbrain.  Snrpn, Ipw and Ndn are widely expressed at high levels throughout the mouse brain, whereas Magel2, Mkrn3 and the snoRNA MBII-85 are preferentially expressed in specific brain regions. Magel2 is most specifically expressed in developing hypothalamus, the region of the brain implicated in PWS hyperphagia and obesity. Although Snrpn, Ipw and MBII-85 are transcribed from the same promoter, the transcripts are differentially detected in neural tissues.

We now concentrate our studies on two related PWS candidate genes, Magel2 and Ndn.  Necdin has been proposed to act in the survival of post-mitotic neurons, perhaps supporting the maintenance of the postmitotic state and/or in preventing apoptosis.  Newborn pups from several strains of mice with a paternally inherited necdin deletion succumb to respiratory insufficiency.  We previously showed that the defect is due to abnormal neuronal activity within the putative respiratory rhythm-generating center, the pre-Bötzinger complex. Specifically, the rhythm is unstable with prolonged periods of depression of respiratory rhythmogenesis. These observations suggest that the developing respiratory center is particularly sensitive to loss of necdin activity. 

To further define the nervous system defect in the necdin-null mice, we analyzed embryonic brain development in the necdin-null mice using a combination of microarray-based expression analysis, immunohistochemistry with neuronal markers, and RNA in situ hybridization.  To determine alterations in gene expression caused by loss of necdin function, we used an Affymetrix Gene Array to compare levels of RNA in the medulla of necdin-null versus wild-type littermates, at embryonic day 18.  A set of genes encoding structural, regulatory and developmentally important proteins was altered in expression level, as confirmed by quantitative RT-PCR and/or immunoblotting.  We noted changes in a set of genes encoding cytoskeletal proteins specific to neurons, glia and their precursor cells, and in extracellular matrix and secreted proteins.  We also detected changes consistent with a loss of or delay in maturation of oligodendrocytes and reduced myelination.  Together these changes point to a possible disruption of cytoskeletal elements in neurons and glia, which could together lead to a defect in axonogenesis. 

As necdin is expressed in neurons, we hypothesized that changes in gene expression may be due to changes in neuronal identity, development or differentiation.  We identified molecular interactions between necdin and proteins important in axonal outgrowth and integrity.  The axons of necdin-null sympathetic embryonic neurons have reduced elongation and grow aberrantly in compartmented culture. We identified morphological abnormalities in necdin-null mouse embryos that point to an essential role for necdin in the outgrowth and fasciculation of axons in multiple neuronal subtypes in the central nervous system.  These data point to a novel necdin-mediated intracellular process essential for neurite outgrowth in the central nervous system. We now propose a model whereby defects in axonal elongation and fasciculation may also contribute to the phenotype in Prader-Willi syndrome. 

June 2004