Research Notes: Genes - MAGEL2

Nat Genet. 2007 Sep 23.
The imprinted gene Magel2 regulates normal circadian output.
Kozlov SV, Bogenpohl JW, Howell MP, Wevrick R, Panda S, Hogenesch JB, Muglia LJ, Van Gelder RN, Herzog ED, Stewart CL.
Cancer and Developmental Biology Laboratory, National Cancer Institute, Frederick, Maryland, USA.

Mammalian circadian rhythms of activity are generated within the suprachiasmatic nucleus (SCN). Transcripts from the imprinted, paternally expressed Magel2 gene, which maps to the chromosomal region associated with Prader-Willi Syndrome (PWS), are highly enriched in the SCN. The Magel2 message is circadianly expressed and peaks during the subjective day. Mice deficient in Magel2 expression entrain to light cycles and express normal running-wheel rhythms, but with markedly reduced amplitude of activity and increased daytime activity. These changes are associated with reductions in food intake and male fertility. Orexin levels and orexin-positive neurons in the lateral hypothalamus are substantially reduced, suggesting that some of the consequences of Magel2 loss are mediated through changes in orexin signaling. The robust rhythmicity of Magel2 expression in the SCN and the altered behavioral rhythmicity of null mice reveal Magel2 to be a clock-controlled circadian output gene whose disruption results in some of the phenotypes characteristic of PWS.

Hum Mol Genet. 2007 Aug 29.
Inactivation of the mouse Magel2 gene results in growth abnormalities similar to Prader-Willi Syndrome.
Bischof JM, Stewart CL, Wevrick R.
Department of Medical Genetics, University of Alberta, Edmonton, AB Canada.

Prader-Willi syndrome (PWS) is an imprinted genetic obesity disorder characterized by abnormalities of growth and metabolism. Multiple mouse models with deficiency of one or more PWS candidate genes have partially correlated individual genes with aspects of the PWS phenotype, although the genetic origin of defects in growth and metabolism has not been elucidated. Gene-targeted mutation of the PWS candidate gene Magel2 in mice causes altered circadian rhythm output and reduced motor activity. We now report that Magel2-null mice exhibit neonatal growth retardation, excessive weight gain after weaning, and increased adiposity with altered metabolism in adulthood, recapitulating fundamental aspects of the PWS phenotype. Magel2-null mice provide an important opportunity to examine the physiological basis for PWS neonatal failure to thrive and post-weaning weight gain and for the relationships among circadian rhythm, feeding behavior, and metabolism.

Hum Mol Genet. 2005 Mar 1.
Essential role for the Prader-Willi syndrome protein necdin in axonal outgrowth.
Lee S, Walker CL, Karten B, Kuny SL, Tennese AA, O'Neill MA, Wevrick R.
Department of Medical Genetics, University of Alberta, Edmonton, Alberta, Canada.
[ Free full text ]

Necdin and Magel2 are related proteins inactivated in Prader-Willi syndrome (PWS), a sporadic chromosomal deletion disorder. We demonstrate that necdin and Magel2 bind to and prevent proteasomal degradation of Fez1, a fasciculation and elongation protein implicated in axonal outgrowth and kinesin-mediated transport, and also bind to the Bardet-Biedl syndrome (BBS) protein BBS4 in co-transfected cells. The interactions among necdin, Magel2, Fez1 and BBS4 occur at or near centrosomes. Centrosomal or pericentriolar dysfunction has previously been implicated in BBS and may also be important in the features of PWS that overlap with BBS, such as learning disabilities, hypogonadism and obesity. Morphological abnormalities in axonal outgrowth and fasciculation manifest in several regions of the nervous system in necdin null mouse embryos, including axons of sympathetic, retinal ganglion cell, serotonergic and catecholaminergic neurons. These data demonstrate that necdin mediates intracellular processes essential for neurite outgrowth and that loss of necdin impinges on axonal outgrowth. We further suggest that loss of necdin contributes to the neurological phenotype of PWS, and raise the possibility that co-deletion of necdin and the related protein Magel2 may explain the lack of single gene mutations in PWS.

Excerpts from the full text article:

Introduction

NDN (encoding necdin) and MAGEL2 are two of four protein-coding genes inactivated in individuals with Prader–Willi syndrome (PWS), a sporadic chromosome deletion disorder marked by profound neonatal hypotonia, global developmental delay, hypoventilation, childhood onset hyperphagia, obesity and hypogonadism (1). Hypothalamic dysfunction underlies many aspects of this disorder and, at least one of the inactivated PWS genes, is predicted to be critical for the normal development and function of the hypothalamus (2). Decreased fetal movements, decreased myelination, hypoplasia of cortical commissures and enlarged lateral ventricles are found in fetuses and young children with PWS (3,4). The partial resolution of both neonatal hypotonia and failure to thrive suggested to us that a delay in the maturation of neuronal circuits might underlie the early course of PWS. In adults, additional abnormalities include chronically elevated levels of the orexigenic hormone ghrelin, elevated levels of serotonin metabolites and, to a lesser extent, dopamine metabolites in the cerebrospinal fluid (2,5) and peripheral insensitivity to pain. Defects in parasympathetic innervation of the gut and/or failure of serotonergic neuronal transmission could contribute to the voracious appetite of individuals with PWS.

Simultaneous deletion of all PWS-equivalent genes in mice is usually lethal in the first postnatal week because of hypoventilation and failure to thrive (6,7). Necdin and MAGEL2 are part of a multiprotein family related by a MAGE homology domain. This family also includes MAGED1 (NRAGE), which interacts with the p75 neurotrophin receptor and facilitates nerve growth factor (NGF)-mediated apoptosis through a Jun kinase-dependent pathway (8,9). Expression of murine Magel2 is highest in neurons of the developing hypothalamus, particularly the suprachiasmatic nucleus and supraoptic tract. Magel2 null mice have not been described. Murine Ndn is expressed in many but not all postdifferentiation stage neurons (10,11), and also in developing muscle, skin and cartilage (11). A subset of necdin null mice exhibit a defect in respiratory rhythm generation in the medulla, causing hypoventilation with high neonatal mortality (12–15). A role for necdin in neuronal terminal differentiation is supported by experiments showing that necdin-transfected PC12 cells display increased differentiation and accelerated neurite extension in response to nerve growth factor (16), that ectopic necdin expression induces neurite outgrowth in neuroblastoma cells (17) and that repression of necdin in embryonic dorsal root ganglion cells suppresses their differentiation (18).

We now present a novel role for necdin and Magel2 in neuronal function. We identified an interaction between both necdin and Magel2 and fasciculation and elongation (Fez) proteins implicated in centrosome-mediated cytoskeletal rearrangement after neuronal differentiation and in axonal outgrowth. We identified a second interaction of necdin and Magel2 with BBS4, another protein implicated in centrosome function. BBS4 is one of several genes mutated in Bardet–Biedl syndrome (BBS), a complex disorder in which affected individuals display learning disabilities, retinopathy and obesity, together with hypogonadism, cardiac, limb and kidney malformations (19). We show that necdin null embryos have defects in cortical commissural formation and axonal extension, bundling and pathfinding. These results suggest that necdin is required to facilitate the intracellular processes that underlie neurite and axonal outgrowth in embryonic neurons, leading us to propose that loss of necdin impairs these processes in necdin null mice and in PWS. Furthermore, if the functions of necdin and Magel2 are partially redundant in key neurons, their combined loss in PWS may abrogate this shared function. We postulate that PWS is one of an emerging class of neurodevelopmental disorders that includes BBS, schizophrenia and lissencephaly, which are in part caused by defects in centrosome function in cytoskeletal rearrangement during neurite extension.

Results

Necdin and Magel2 interact with fasciculation and elongation proteins Fez1 and Fez2

We performed a screen for cytoplasmic proteins interacting with necdin using the yeast two-hybrid Ras rescue system (16). We identified a necdin-interacting protein corresponding to amino acids 46–282 of the 353 amino acid Fez2 protein. Fez2 (UniGene Cluster Hs.258563), which is widely expressed, and Fez1 (UniGene Cluster Hs.79226), which is primarily expressed in the brain, are mammalian homologues of Caenorhabditis elegans and Drosophila melanogaster unc-76 (20–26). UNC-76 protein is found in the cell body and axonal cytoplasm of C. elegans neurons during development, and is essential for axon bundling (C. elegans) and transport mediated by the microtubule motor protein kinesin (D. melanogaster). The unc-76 mutation can be partially rescued by ectopic expression of human Fez1, suggesting retention of function between species (21).

We confirmed the interaction of Fez1 and Fez2 (together called Fez1/2) with necdin by co-immunoprecipitation of transiently transfected epitope-tagged full-length proteins. [...]

Necdin and Magel2 may increase Fez1 levels by increasing Fez1 transcription or stability. We first determined by RT–PCR that equivalent amounts of Fez1 RNA are present in cells transfected with pHAFez1 with or without pXNdn (data not shown). Co-transfected cells were then treated with each of three different proteasome inhibitors. In the presence of proteasome inhibitors, HAFez1 is 3-fold more abundant than with DMSO alone (Fig. 1G), and is slightly more abundant than when co-expressed with XNdn (Fig. 1D). Similarly, co-transfection of pXNdn with pHAFez2 increases the amount of HAFez2, and proteasome inhibitors stabilize HAFez2 in the absence of necdin co-transfection (Fig. 1G). This suggests that necdin stabilizes Fez1/2 by preventing their degradation by the proteasomal pathway.

Co-localization of necdin and Fez1 near centrosomes

Endogenous Fez1 is present in SK-N-SH neuroblastoma cells in a punctate fashion in the cytosol, along organized filamentous structures (23). In cultured rat hippocampal neurons, Fez1 also colocalizes with F-actin in growth cones (23). Endogenous necdin is highly concentrated in the cytoplasm of differentiated neurons, and moves to the nucleus under specific conditions (27). [...]

Interaction of necdin with BBS4

We next investigated whether necdin or Magel2 interact with other proteins located in centrosomes. One of the proteins implicated in the genetic disorder BBS, BBS4, localizes to basal bodies of ciliated cells and to centrosomes. BBS4 is proposed to act in intracellular microtubule-associated transport but not in the formation of the cilia themselves (19,28). Given the overlapping phenotypes of PWS and BBS and the common centrosomal localization of BBS4 and the necdin/Magel2-interacting protein Fez1, we explored the interactions among necdin, Magel2, Fez1 and BBS4. We co-transfected HEK293 cells with pMycBBS4 and/or pHAFez1. As expected, staining for both epitopes revealed an overlap in centrosomes, with HAFez1 also present in the cytoplasm (Fig. 2C). On co-transfection of pHABBS4 and pXpressNdn, both proteins were present in centrosomes, although transfected necdin is also widely distributed in the cell (Fig. 2D). Transfected Magel2 is present in a punctate pattern in the cytoplasm, and is also detected in an overlapping juxtanuclear location with transfected HABBS4 (Fig. 2E).

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Axonal extension and bundling of primary sympathetic neurons are impaired in cell culture

Because Fez-related proteins are essential for axonal elongation in C. elegans (21) and kinesin-dependent axonal transport in D. melanogaster (25) and because of the role of the centrosome in organizing microtubules during axonal outgrowth in postmitotic neurons (29), we investigated whether the absence of necdin caused a defect in neuronal fasciculation and elongation in the developing murine embryonic nervous system. We assayed superior cervical ganglion (SCG) neurons, which extend axons in a reproducible manner when plated in compartmented chambers (30); these neurons normally express necdin (11). In this assay, dissociated neurons are plated in the central compartment of a tissue culture dish, and their axons extend along collagen tracks and cross under grease barriers into side compartments that contain different culture medium. To observe differences in neuronal bundling, we isolated SCGs from control or necdin null embryonic day 17.5 (E17.5) mouse embryos, then dissociated and plated the neurons in compartmented culture dishes supplemented with rat serum in the center compartment and NGF in the side compartments. The necdin null ganglia were typically more difficult to dissect, and the neurons grew poorly in culture, with thinner, less bundled and more branched axons in surviving neurons (Fig. 3A). At higher magnification, we observed necdin null axons with varicosities and atypically localized thickenings, changes in direction without branching and backtracking towards the barrier. These anomalies are rarely seen in control axons (Fig. 3B). The necdin null growth cones are swollen rather than flattened, with ruffles as is typical of control growth cones (Fig. 3C). Overall, the outgrowth, bundling and morphology of axons are significantly compromised in necdin null SCG neurons.

Axon projections and tracts are reduced or misrouted in necdin null mice

The compartmented culture data established the importance of necdin for axon outgrowth in vitro. We then compared the histology of axon tracts in necdin null embryonic brain sections with those of control littermates. Thionin labels neuronal Nissl substance and cell nuclei, and is excluded from axonal tracts. On sagittal sections at E18.5, the axonal bundle leading into anterior commissure is typically reduced or missing in the necdin null embryos (Fig. 4A'). In contrast, the corpus callosum axonal bundle and the hippocampal commissure axonal bundle are variably affected in the mutant embryos. The lateral and fourth ventricles were consistently moderately enlarged in necdin null embryos, from E13.5 to birth. Abnormalities in the corpus callosum and fornix were also apparent by anti-neurofilament immunohistochemistry of transverse brain sections at E18.5 (Fig. 4B). To visualize the optic chiasm, we traced the trajectory of retinal ganglion cell (RGC) axons in E16.5 mice by placing DiA powder in the optic cup of the right eye, then 10 days later examining coronal sections through the telencephalon. The optic chiasm is larger and less compact as visualized by thionin staining in the necdin null embryos (Figs 4C and 5E), and the DiA labeling shows that most of the axons reach the contralateral side, as in the control (Fig. 4D). We then examined the fasciculus retroflexus (Fr), a long fasciculated tract that carries axons within its core from the medial habenula to the interpeduncular nucleus, and carries axons within its sheath from the lateral habenula to specific midbrain targets. In thionin-stained sagittal (Fig. 4E) and coronal (Fig. 4F) E18.5 control sections, the Fr is visualized as a single, non-staining bundle of axons. In contrast, the Fr is less tightly bundled in the necdin null (Fig. 4E') and appears as a cluster of non-staining ectopic circular axonal bundles (arrowheads in Fig. 4F') that surround what is normally a tight axonal bundle. This confirmed a fasciculation defect in both cultured and in vivo neurons, and led us to perform a more detailed immunohistological investigation of the necdin null brain.

On thionin-stained sections of necdin null embryos from E14.5 onwards, an ectopic axonal bundle was detected in the anterior hypothalamic region dorsolateral to the optic chiasm and rostral to the zona incerta and lateral hypothalamus (Lh) (Fig. 5A' and B'). At E16.5, this bundle has not increased in its extent, the internal capsule is poorly defined and ectopic axonal whorls become evident in the striatum of the necdin null embryos (Fig. 5C'). We supposed that the ectopic bundle could be misrouted thalamocortical axons (Tca), which normally course ventrally from the thalamus, then rostrally through the internal capsule before extending dorsolaterally into the cortex (31), or alternatively could be misrouted RGC axons (32). At E16.5, the necdin null thalamic axons labeled with the L1 cell adhesion molecule do not form an internal capsule, and a reduced number of projections extend to the intermediate zone of the cortex (Fig. 5D', arrow). Both the ectopic bundle in the rostral lateral hypothalamus and the smaller ectopic whorls in the thalamus stain strongly with L1, which labels both thalamic efferent projections and RGC axons. RGC axons are normally fasciculated within the optic nerve, course through the optic chiasm at the base of the ventral hypothalamus and partially defasciculate as they grow over the diencephalon to reach the superior colliculus (32) (Fig. 5E). In the necdin null E16.5 embryos, DiA-labeled RGC axons reach the optic chiasm and extend partway along the lateral border of the thalamus, but we failed to detect any axonal labeling, with DiA extending into the superior colliculus on coronal sections (Fig. 5E') or on transverse sections. Some of the RGC axons deviate dorsomedially into the dorsal aspect of the ectopic patch (Fig. 5E'). In both the control and the necdin null brain, a minority of RGC axons does not cross at the optic chiasm but instead course along the ipsilateral aspect of the hypothalamus. In summary, the defects in the thalamus and hypothalamus of necdin null embryos include failures in both extension and routing of thalamocortical and retinal ganglion cell axons towards their respective targets.

Developmental timing of the axonal defect

We analyzed mid-gestation embryos to define the timing of the axonal defects, particularly the ectopic bundle in the developing hypothalamus. The hypothalamus is of interest because of its major role in endocrine function and appetite regulation, which are profoundly affected in PWS. Necdin and Fez1 are co-expressed in the developing hypothalamus by E10.5 (Fig. 6A–D). We then investigated whether the expression of early hypothalamic markers was perturbed in the necdin null mice. The obesity-related transcription factor Sim1 and Magel2 are expressed in regions overlapping with Ndn expression in the early hypothalamic neuroepithelium (11,33), and Sim1 is also expressed in the zona limitans. The expression of Sim1 and Magel2 in the necdin null embryo is comparable to control in serial sections through the hypothalamus at E12.5 (Fig. 6E–G). At E13.5, a misrouting of thalamocortical L1-positive axons is evident in the ventral part of the dorsal thalamus (Fig. 6H'), the region later to become part of the ectopic bundle shown in Figure 5C. Thus no evidence for defects in hypothalamic neuroepithelial differentiation was found at E12.5, but misrouting of hypothalamic axons was seen in necdin null embryos by E13.5.

Serotonergic and catecholaminergic projections are reduced in the necdin null embryo

The rostral and caudal projections of the serotonergic raphe nuclei participate in many functional systems, and are implicated in behavioral disorders including PWS (34). At E12.5, we detected a comparable number and placement of rostral serotonergic cells using RNA in situ hybridization with a probe for the serotonin transporter (SERT) in control and necdin null embryos (Fig. 7A). Immunohistochemistry with an anti-serotonin antibody reveals that while these cell bodies were indeed producing serotonin, the ascending fibers containing serotonin are not detectable in the mesencephalon of the necdin null embryo (Fig. 7B'). Furthermore, serotonin immunohistochemistry on sagittal sections at E15.5 revealed a continuing paucity of ascending serotonergic-positive fibers (Fig. 7C'). Immunohistochemistry with anti-tyrosine hydroxylase (TH) antibody at E15.5 normally detects the noradrenergic neurons of the brainstem and their rostral and caudal projections, and the mesencephalic dopaminergic neurons and their striatal, cortical and limbic projections. In a section adjacent to a thionin-stained section showing the ectopic thalamocortical bundle (Fig. 8A), we noted a severe disruption of the TH-positive projections in the nigrostriatal pathway and caudoputamen (Nsp) (Fig. 8B). In a section 600 µm rostral to that in Figure 8B, few TH-positive axons were detected in the necdin null section (Fig. 8C), but a section 600 µm caudal to the ectopic bundle had fairly normal TH staining of the cell bodies in the dorsal medial hypothalamus (Dmh) and the Nsp (Fig. 8D). Thus both the serotonergic and catecholaminergic projection systems are disrupted in the necdin null embryo.

Discussion

We propose that deficiency of necdin causes a delay or dysfunction in axonal extension, which accounts for the consistent finding of profound hypotonia, reduced myelination, enlarged ventricles and commissural defects described in fetuses and children with PWS. A defect in parasympathetic innervation of the stomach has been suggested in PWS (2), and parallels with the axonal defects we see in necdin null sympathetic neurons. Defects in serotonin pathways and altered responses to psychoactive drugs targeting dopaminergic pathways are consistently seen in PWS, mirroring the dysfunction in the late embryonic necdin null embryo. In this model, hypoventilation and central sleep apneas prevalent in individuals with PWS are also ascribed to necdin deficiency (15,35).

We propose that necdin and the related, co-deleted protein Magel2 act in Fez and BBS4 centrosome-related activities that lead to cytoskeletal rearrangements during neurite outgrowth. Centrosomes are critically dependent on the function of BBS proteins (including BBS4) (19), providing a tantalizing mechanistic link between these two rare disorders that each result in impaired mental development and obesity (19). BBS4 is thought to transport the scaffold protein PCM1 to centrosomal satellites through interactions with the dynein microtubule-based molecular motor, allowing for formation of the centrosomal microtubule organizing center (19). In D. melanogaster, the interaction of the Fez orthologue UNC-76 with the molecular motor kinesin is essential for axonal transport (25), and loss of UNC-76 causes disruption of fast axonal transport and 'axon clogs' similar in appearance to the varicosities we describe in necdin null cultured sympathetic neurons. Necdin enhances neurite outgrowth in NGF-stimulated PC12 cells, whereas Fez1, when phosphorylated by PKC zeta, causes neurite outgrowth in PC12 cells (22). We identified a necdin- and Magel2-mediated protection of Fez proteins from proteasomal degradation, and co-localization of necdin and Fez to a juxtanuclear compartment overlapping centrosomes. Moreover, Ndn, Magel2 and Fez1 mRNAs are co-expressed in the embryonic ventral and caudal hypothalamus. Unfortunately, Magel2 null mice are not available to test whether specific hypothalamic neurons are dependent on Magel2 function for appropriate axonal extension. Together, our data support a model whereby up-regulation of necdin/Magel2 in postmitotic neurons stabilizes Fez proteins to facilitate centrosome-mediated cytoskeletal rearrangements required for axonal outgrowth and kinesin-mediated transport.

The necdin null pathology also emulates a subset of neuronal patterning and specification mutant phenotypes that cause ectopic whorls and bundles (32,36). Although we have clearly shown an axonal extension defect in necdin null mice, it remains possible that some of the axonal misrouting and whorling defects are related to dysfunctional specification or patterning of the ventral telencephalon. However, the up-regulation of necdin in most postmitotic, differentiated neurons, the localization in proximal axons and cell bodies but not in distal axons in SCG cultures and the role of necdin in cytoskeletal rearrangements and the dysfunction of necdin null neurons in culture are more strongly suggestive of an intrinsic growth defect in cytoskeletal dynamics than of a direct role in patterning and/or specification of subsets of neurons during development.

Necdin and Fez1 interact in a juxta–centrosomal compartment also containing the schizophrenia candidate protein DISC1 in a complex with the cytoplasmic dynein-regulating proteins LIS1 and NUDEL (37,38). Although we have not fully characterized the domains of Fez1/2 interacting with necdin, in the initial yeast two-hybrid screen necdin interacted with amino acids 46–282 of the 353 amino acid Fez2 protein, while the more C-terminal domain of this protein family is implicated in binding to the KHC tail of kinesin (25) and to DISC1 (23). LIS1 mutations cause neuronal migration defects and lissencephaly in humans and mice, through a disruption of dynein and microtubular dynamics mediated in part through centrosomes (37). This complex set of interactions points to overlapping dysfunctions in centrosome–microtubule dynamics in PWS, BBS, lissencephaly and possibly other psychiatric disorders. The possibility that concurrent deletion of necdin and Magel2 in PWS may abrogate the functional redundancy of these two related proteins in specific neurons in the hypothalamus provides a plausible explanation for the lack of single gene mutations in PWS. The neurons involved in the hypothalamic circuits implicated in PWS may indeed have delayed or insufficient axonal connections. Therefore, strategies applied early in postnatal life that functionally restore some of these connections could provide a novel line of therapy for infants diagnosed with PWS.

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