Highlights d G9a expression and nuclear enrichment occur early in polarizing neurons d G9a is required for neuronal migration and axon formation d G9a inhibits RhoA/ROCK activity during neuronal polarization d G9a targets Lfc, a GEF for RhoA that impairs axon growth

Endocytic recycling is an intracellular process that returns internalized molecules back to the plasma membrane and plays crucial roles not only in the reuse of receptor molecules but also in the remodeling of the different components of this membrane. This process is required for a diversity of cellular events, including neuronal morphology acquisition and functional regulation, among others. The recycling endosome (RE) is a key vesicular component involved in endocytic recycling. Recycling back to the cell surface may occur with the participation of several different Rab proteins, which are master regulators of membrane/protein trafficking in nerve cells. The RE consists of a network of interconnected and functionally distinct tubular subdomains that originate from sorting endosomes and transport their cargoes along microtubule tracks, by fast or slow recycling pathways. Different populations of REs, particularly those formed by Rab11, Rab35, and Arf6, are associated with a myriad of signaling proteins. In this review, we discuss the cumulative evidence suggesting the existence of heterogeneous domains of REs, controlling different aspects of neurogenesis, with a particular focus on the commonalities and singularities of these REs and their contribution to nerve development and differentiation in several animal models.

Summary The establishment of polarity is crucial for the physiology and wiring of neurons. Therefore, monitoring the axo-dendritic specification allows the mechanisms and signals associated with development, growth, and disease to be explored. Here, we describe major and minor steps to study polarity acquisition, using primary cultures of hippocampal neurons isolated from embryonic rat hippocampi, for in vitro monitoring. Furthermore, we use in utero electroporated, GFP-expressing embryonic mouse brains for visualizing cortical neuron migration and polarization in situ . Some underreported after-protocol steps are also included. For complete details on the use and execution of this protocol, please refer to Wilson et al. (2020 ).

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The development and maintenance of multicellular organisms require specialized coordination between external cellular signals and the proteins receiving stimuli and regulating responses. A critical role in the proper functioning of these processes is played by endosomal trafficking, which enables the transport of proteins to targeted sites as well as their return to the plasma membrane through its essential components, the endosomes. During this trafficking, signaling pathways controlling functions related to the endosomal system are activated both directly and indirectly. Although there are a considerable number of molecules participating in these processes, some are more known than others for their specific functions. Toward the end of the 1990s, Smad anchor for receptor activation (SARA) protein was described to be controlling and to facilitate the localization of Smads to transforming growth factor β (TGF-β) receptors during TGF-β signaling activation, and, strikingly, SARA was also identified to be one of the proteins that bind to early endosomes (EEs) participating in membrane trafficking in several cell models. The purpose of this review is to analyze the state of the art of the contribution of SARA in different cell types and cellular contexts, focusing on the biological role of SARA in two main processes, trafficking and cellular signaling, both of which are necessary for intercellular coordination, communication, and development.

The acquisition of neuronal polarity is a complex molecular process that depends on changes in cytoskeletal dynamics and directed membrane traffic, regulated by the Rho and Rab families of small GTPases, respectively. However, during axon specification, a molecular link that couples these protein families has yet to be identified. In this paper, we describe a new positive feedback loop between Rab8a and Cdc42, coupled by Tuba, a Cdc42-specific guanine nucleotide-exchange factor (GEF), that ensures a single axon generation in rodent hippocampal neurons from embryos of either sex. Accordingly, Rab8a or Tuba gain-of-function generates neurons with supernumerary axons whereas Rab8a or Tuba loss-of-function abrogated axon specification, phenocopying the well-established effect of Cdc42 on neuronal polarity. Although Rab8 and Tuba do not interact physically, the activity of Rab8 is essential to generate a proximal to distal axonal gradient of Tuba in cultured neurons. Tuba-associated and Rab8a-associated polarity defects are also evidenced in vivo, since dominant negative (DN) Rab8a or Tuba knock-down impairs cortical neuronal migration in mice. Our results suggest that Tuba coordinates directed vesicular traffic and cytoskeleton dynamics during neuronal polarization.

Nerve growth factor (NGF) stimulates numerous cellular physiological processes, including growth, differentiation, and survival, and maintains the phenotype of several neuronal types. Most of these NGF-induced processes require adaptation of the secretory pathway since they involve extensive remodeling of membranes and protein redistribution along newly formed neuritic processes. CREB3 transcription factors have emerged as signaling hubs for the regulation of numerous genes involved in the secretory pathway and Golgi homeostasis, integrating stimuli from multiple sources to control secretion, posttranslational modifications and trafficking of proteins. Although recent studies have focused on their role in the central nervous system, little is known about their participation in cell differentiation. Therefore, we aimed to analyze the expression and signaling mechanism of CREB3 transcription factor family members, using the NGF-induced PC12 cell differentiation model. Results show that NGF treatment causes Golgi enlargement and a parallel increased expression of proteins and mRNAs encoding for proteins required for membrane transport (transport factors). Additionally, a significant increase in CREB3L2 protein and mRNA levels is detected in response to NGF. Both MAPK and cAMP signaling pathways are required for this response. Interestingly, CREB3L2 overexpression hampers the NGF-induced neurite outgrowth while its inhibition enhances the morphological changes driven by NGF. In agreement, CREB3L2 overexpressing cells display higher immunofluorescence intensity of Rab5 GTPase (a negative regulator of PC12 differentiation) than control cells. Also, Rab5 immunofluorescence levels decrease in CREB3L2-depleted cells. Taken together, our findings imply that CREB3L2 is an important downstream effector of NGF-activated pathways, leading to neuronal differentiation.

Neural development is a complex process that involves critical events, including cytoskeleton dynamics and selective trafficking of proteins to defined cellular destinations. In this regard, Smad Anchor for Receptor Activation (SARA) is an early endosome resident protein, where perform trafficking-associated functions. In addition, SARA is also involved in cell signaling, including the TGFβ-dependent pathway. Accordingly, SARA, and TGFβ signaling are required for proper axonal specification and migration of cortical neurons, unveiling a critical role for neuronal development. However, the cooperative action between the TGFβ pathway and SARA to this process has remained understudied. In this work, we show novel evidence suggesting a cross-talk between SARA and TGFβ pathway needed for proper polarization, axonal specification, growth and cortical migration of central neurons both in vitro and in vivo. Using microscopy tools and cultured hippocampal neurons, we show a local interaction between SARA and TβRI (TGFβ I receptor) at endosomes. In addition, SARA loss of function, induced by the expression of the dominant-negative SARA-F728A, over-activates the TGFβ pathway, most likely by preserving phosphorylated TβRI. Consequently, SARA-mediated activation of TGFβ pathway impacts on neuronal development, promoting axonal growth and cortical migration of neurons during brain development. Moreover, our data suggests that SARA basally prevents the activation of TβRI through the recruitment of the inhibitory complex PP1c/GADD34 in polarizing neurons. Together, these results propose that SARA is a negative regulator of the TGFβ pathway, being critical for a proper orchestration for neuronal development.