Research Activities
Neurons withdraw from the cell cycle after differentiation from neural stem cells. Thereafter, neurons become permanently quiescent (postmitotic) and terminally differentiated to acquire their specific functions. On the other hand, massive amounts of neuronal death (apoptosis) occur under physiological and pathological conditions. Our goal is to gain a unified view about cell cycle regulation, differentiation, and apoptosis during neuronal development.
1. Expression of Necdin in Neurons and Other Cell Types
Murine embryonal carcinoma P19 cells differentiate into postmitotic neurons in response to retinoic acid treatment. In 1991, we isolated a novel cDNA sequence encoding a 325 amino acid residue protein, designated necdin (for neurally differentiated embryonal carcinoma-derived protein), from a subtraction cDNA library of retinoic acid-treated P19 cells (Maruyama, K. et al., Biochem Biophys Res Commun, 178, 291-296, 1991). Necdin mRNA is expressed in postmitotic neurons in most of the brain regions, especially in the hypothalamus and brain stem at the highest levels (Aizawa, T. et al., Brain Res Dev Brain Res, 68, 265-274, 1992; Uetsuki, T. et al., J Biol Chem, 271, 918-924, 1996). Necdin is also expressed in non-neuronal postmitotic cells such as skeletal muscle cells and adipocytes. Ectopic expression of necdin in proliferative cells suppresses cell growth without affecting cell viability (Hayashi, Y. et al., Biochem Biophys Res Commun, 213, 317-324, 1995), suggesting that necdin is involved in the molecular processes by which neurons become permanently quiescent. In collaboration with other laboratories, we have found that necdin is expressed in vessel-derived stem cells or mesoangioblasts (Brunelli, S. et al., Circ Res, 94, 1571-1578, 2004), brown preadipocytes (Tzeng, Y-H. et al., Nat Cell Biol, 7, 601-611, 2005) and hematopoietic stem cells (Kubota, Y. et al., Blood, 114, 4383-4392, 2009). These findings suggest that necdin regulates the proliferation and differentiation of these non-neuronal stem cells. We have recently found that mesenchymal stem cells residing in white adipose tissues express high levels of necdin, and that enforced reduction of endogenous necdin expression markedly increases fat mass in juvenile mice fed a high-fat diet until adulthood, suggesting that necdin prevents excessive preadipocyte proliferation induced by adipogenic stimulation to control white adipocyte number during adipose tissue development (Fujiwara, K. et al., PLoS One, 7, e30948, 2012).
The human necdin gene (NDN) is mapped to chromosome 15q11-12, a region deleted in Prader-Willi syndrome (PWS), a classic example of the genomic imprinting-associated neurodevelopmental disorder (Nakada, Y. et al., Gene, 213, 65-72, 1998). Earlier studies have demonstrated that NDN is maternally imprinted, expressed only from the paternal allele, and not expressed in PWS. The major symptoms of PWS are hyperphagia, hypogonadism, short stature, and abnormal behavior, which are indicative of impaired neuronal development in the hypothalamus. The fact that necdin is abundantly expressed in the hypothalamus suggests that the absence of necdin expression in PWS impairs neuronal differentiation in this brain area. Indeed, necdin gene knockout mice have been reported to show several symptoms reminiscent of PWS. Therefore, necdin may contribute, at least in part, to the pathogenesis of PWS.
We have established mutant mice defective in the paternal Ndn (Ndntm1Ky), which show no necdin expression in all somatic cells, and used them for the functional studies on necdin (Kuwako, K. et al., J Neurosci, 25, 7090-7099, 2005; Kuwajima, T. et al., ibid, 26, 5383-5392, 2006; Kurita, M. et al., ibid, 26, 12003-12013, 2006; Hasegawa, K. & Yoshikawa, K., ibid, 28, 8772-87841, 2008; Kuwajima, T. et al., ibid, 30, 3709-3714, 2010). These mutant mice show abnormal phenotypes such as increased apoptosis and abnormal neuronal differentiation. Using these mutant mice, we are also studying the epigenetic control of necdin gene expression such as DNA methylation and histone modifications. It is noteworthy that necdin is expressed in the telencephalic subventricular zone and hippocampal subependymal zone, where specific neurogenic systems have been described (Wu, H. et al., Science 329, 444-448, 2010; Rolando, C. et al., Eur J Neurosci, 31, 1340-1351, 2010; Gajera, CR. et al., J Cell Sci, 123, 1922-1930, 2010; Tepavcevic, V. et al., J Clin Invest, 121, 4722-4734, 2011; de Chevigny, A. et al., Front Cell Neurosci, 6, 6, 2012).
2. Necdin as a Hub Protein Required for Protein-protein Interaction Networks
Necdin binds to the tumor virus oncoproteins simian virus 40 (SV40) large T antigen and adenovirus E1A and the cellular transcription factor E2F-1, all of which are involved in cell cycle progression (Taniura, H. et al., J Biol Chem, 273, 720-728, 1998). Furthermore, necdin represses E2F1-dependent cdc2 gene transcription and attenuates apoptosis of cerebellar granule neurons (Kurita, M. et al., J Neurosci, 26, 12003-12013, 2006), suggesting that necdin attenuates apoptosis by suppressing the E2F1-Cdc2 system in differentiated postmitotic neurons. These characteristics resemble those of the retinoblastoma protein (Rb), a well-characterized tumor suppressor gene product. Necdin also interacts with p53, a transcription factor possessing tumor suppressor function (Taniura, H. et al., J Biol Chem, 274, 16242-16248, 1999). Thus, necdin may serve as a transcriptional co-repressor that interacts with multiple cell cycle regulatory factors. Necdin also binds to NEFA, nucleobindin (both cytoplasmic Ca2+-binding proteins) (Taniguchi, N. et al., J Biol Chem, 275, 31674-31681, 2000) and the nuclear matrix-binding protein hnRNP U (Taniura, H. & Yoshikawa, K., J Cell Biochem, 84, 545-555, 2002). Moreover, necdin also serves as a direct transcriptional suppressor that interacts with a specific DNA sequence termed GN boxes (Matsumoto, K. et al., Gene, 272, 173-179, 2001). We have defined the functional domains of necdin for protein-protein interaction, nuclear matrix binding, and cell growth suppression (Taniura, H. et al., J Cell Biochem, 94, 804-815, 2005).
We have demonstrated that necdin interacts with Dlx homeodomain proteins via MAGE-D1 to promote the differentiation of GABAergic neurons in mouse embryonic forebrain (Kuwajima, T. et al., J Neurosci, 26, 5383-5392, 2006). Necdin-deficient (paternal Ndn-deficient) mice lacking the paternal necdin allele showed a significant reduction in the differentiation of forebrain GABAergic neurons in vivo and in vitro. We also found that necdin-deficient mice show impaired tangential migration of forebrain Dlx2-expressing GABAergic interneurons originating from the medial ganglionic eminence at embryonal stages (Kuwajima, T. et al., J Neurosci, 30, 3709-3714, 2010). These data suggest that paternally expressed necdin facilitates the differentiation and specification of GABAergic neurons in cooperation with Dlx homeodomain proteins. We are currently investigating the physiological significance of necdin in Dlx2-dependent differentiation of GABAergic neurons born at adult stages.
There is a growing body of information about the involvement of necdin and necdin-like MAGE proteins in neuronal fate decisions. We suppose that necdin and other MAGE family members are involved in the modulations of neuronal apoptosis. Necdin interacts with p53, a typical apoptosis inducer whose activity is regulated through posttranslational modifications (Taniura, H. et al., J Biol Chem, 274, 16242-16248, 1999). We have demonstrated that necdin and necdin-like 2 (MAGE-G1) interact with the neuronal proapoptotic transcription factor E2F1 and the neuronal death receptor p75, and that these two MAGE proteins control neuronal apoptosis through interactions with these proapoptotic proteins (Kuwako, K. et al., J Biol Chem, 279, 1703-1712, 2004). Analyses of mutant mice lacking the paternal necdin allele revealed that paternally expressed necdin facilitates TrkA signaling to promote the survival of NGF-dependent nociceptive neurons (Kuwako, K. et al., J Neurosci, 25, 7090-7099, 2005). These mutant mice show a high tolerance to heat-induced pain, which is also seen in individuals with PWS. Necdin interacts with intracellular domains of transmembrane proteins such as the neurotrophin receptors TrkA and p75NTR (Kuwako, K. et al., J Neurosci, 25, 7090-7099, 2005) and Nogo-A (Liu, X. et al., Mol Cell Neurosci, 41, 51-61, 2009). We assume that necdin potentiates intracellular signal transduction from the surface membrane to the nucleus.
We have found that necdin regulates the acetylation status of p53 via Sirt1 to suppress p53-dependent apoptosis in postmitotic neurons (Hasegawa, K. & Yoshikawa, K., J Neurosci, 28, 8772-87841, 2008). Sirt1 is an NAD-dependent histone deacetylase involved in energy homeostasis, DNA repair, cell survival, and lifespan extension. Necdin forms a stable ternary complex with p53 and Sirt1 in postmitotic neurons. Because necdin interacts with many regulatory proteins, it is possible that necdin regulates their acetylation statuses that are associated with their biological activities. We are currently studying the effects of necdin on posttranslational modifications of regulatory proteins involved in cell differentiation and survival.
3. Necdin as a MAGE Superfamily Member
Necdin is the first identified member of MAGE (melanoma antigen) protein superfamily. MAGE family proteins are encoded by >30 genes in human and mouse genomes and commonly possess a large central region termed MAGE homology domain (MHD). MAGE proteins can be divided into two groups based on the homology of MHD. MAGE-A, B, C subfamilies, which encode precursors of tumor rejection antigens recognized by cytolytic T lymphocytes, are expressed in various cancers or undifferentiated cells, but their function remains largely unknown. In contrast, MAGE proteins such as necdin, MAGE-D1, E1, F1, G1, H1, and L2 are expressed in differentiated cells such as neurons and skeletal muscle cells. The necdin-homologous protein necdin-like 2 suppresses cell growth and interacts with both E2F1 and p75 (Kuwako, K. et al., J Biol Chem, 279, 1703-1712, 2004). Furthermore, we found that necdin associates with the Msx homeodomain proteins via MAGE-D1 to potentiate skeletal muscle differentiation by suppressing Msx2 (Kuwajima, T. et al., J Biol Chem, 279, 40484-40493, 2004).
Remarkably, only one MAGE gene exists in genomes from Drosophila and non-mammalian vertebrates such as chicken (Lopez-Sanchez, N. et al., Physiol Genomics, 30, 156-171, 2007). Furthermore, non-mammalian MAGE protein shows a closest similarity to mammalian necdin-like 2. Drosophila MAGE gene is highly expressed in neural stem cells (neuroblasts) and their progeny (ganglion mother cells and postmitotic neurons) at larval and pupal stages, and moderately in postmitotic neurons including mushroom body neurons and retinal photoreceptors in adulthood (Nishimura, I. et al., Gene Expr Patterns, 7, 244-251, 2007). To elucidate the physiological significance of MAGE expression in Drosophila neural stem cells, we knocked down MAGE in developing mushroom bodies in vivo by RNAi-mediated gene silencing using the OK107-GAL4 driver (Nishimura, I. et al., Neuroscience, 154, 572-581, 2008). MAGE RNAi increased the population of proliferative neural precursors in larval mushroom bodies. At the pupal stage, RNAi-mediated MAGE knockdown led to a significant enlargement of the mushroom bodies as a result of increased neuronal population. These findings suggest that evolutionally conserved necdin-like MAGE is involved in the control of proliferation and differentiation of neural stem cells during nervous system development.
Updated
4/21/12