TGFβ-Smad2 Signaling Regulates the Cdh1-APC/SnoN Pathway of Axonal Morphogenesis

Abstract

Axon growth is critical to the establishment of neuronal connectivity. The E3 ubiquitin ligase Cdh1-anaphase-promoting complex (Cdh1-APC) and its substrate the transcriptional modulator SnoN form a cell-intrinsic pathway that orchestrates axonal morphogenesis in the mammalian brain. How the Cdh1-APC/SnoN pathway is controlled in the nervous system remained unknown. Here, we report that the TGF␤-regulated signaling protein Smad2 plays a key role in regulating the Cdh1-APC/SnoN pathway in neurons. We find that Smad2 is expressed in primary granule neurons of the developing rat cerebellar cortex. The Smad signaling pathway is basally activated in neurons. Endogenous Smad2 is phosphorylated, localized in the nucleus, and forms a physical complex with endogenous SnoN in granule neurons. Inhibition of Smad signaling by several distinct approaches, including genetic knock-down of Smad2, stimulates axonal growth. Biochemical evidence and genetic epistasis analyses reveal that Smad2 acts upstream of SnoN in a shared pathway with Cdh1-APC in the control of axonal growth. Remarkably, Smad2 knock-down also overrides the ability of adult rat myelin to inhibit axonal growth. Collectively, our findings define a novel function for Smad2 in regulation of the Cdh1-APC/SnoN cell-intrinsic pathway of axonal morphogenesis, and suggest that inhibition of Smad signaling may hold therapeutic potential in stimulating axonal growth after injury in the CNS.

Smad2 Protein Disruption in the Central Nervous System Leads to Aberrant Cerebellar Development and Early Postnatal Ataxia in Mice

“…As shown in Fig. 3, Smad2 is preferentially expressed in neuronal cells such as PCs and GCs in the cerebellum, which is consistent with previous studies showing neuron-specific expression of Smad2 (8,9,10). Together, apoptotic cells induced in Smad2-CNS-KO mice are likely to be neuronal cells.…”

“…These findings suggest that Smad2-mediated TGF-␤ signaling plays critical roles in development, function, and/or maintenance of neuronal cells and the brain. Indeed, Smad2 has been shown to regulate proliferation and differentiation of cultured hippocampal granule neurons (11) along with axonal morphogenesis of cultured cerebellar granule neurons (10). SMAD2 has also been suggested to be involved in the pathogenesis of Tau-related neurodegenerative diseases in humans, such as Alzheimer disease (8,12,13) and Pick disease (14).…”

The ubiquitin ligase Smurf2 suppresses TGFβ-induced epithelial–mesenchymal transition in a sumoylation-regulated manner

“…70,73 The plasmids were confirmed by DNA sequence analyses (University of Calgary Core Sequencing Facility, Calgary, AB, Canada). The Smad2 and Smad3 RNAi vectors 74,75 and the FLAG/SENP1 and FLAG/SENP2 expression plasmids were kind gifts from Dr. Azad Bonni (Washington University School of Medicine St. Louis, MO, USA). To establish NMuMG stable cell lines, a pCaGip vector was employed to generate constructs containing cDNA encoding WT Smurf2 or a SUMO loss-of-function (KdR) Smurf2 protein.…”

Initiating and Growing an Axon

“…In postmitotic neurons, APC and its activator protein, Cdh1, stimulate degradation of the transcription factor SnoN (Konishi et al 2004;Stegmuller et al 2006). SnoN levels are also regulated by TGF-b/SMAD-2 signaling (Stegmuller et al 2008). Knockdown experiments in vivo show that reduction of Cdh1 enhances axon growth of cerebellar granule cells and, conversely, reduction of SnoN expression reduces granule cell axon growth.…”