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. SIGNIFICANCE STATEMENT The morphologic, biochemical, and functional differences observed between axon and dendrites, require dramatic structural changes. The extension of an axon that is 1 µm in diameter and grows at rates of up to 500 µm/d, demands the confluence of two cellular processes: directed membrane traffic and fine-tuned cytoskeletal dynamics. In this study, we show that both processes are integrated in a positive feedback loop, mediated by the guanine nucleotide-exchange factor (GEF) Tuba. Tuba connects the activities of the Rab GTPase Rab8a and the Rho GTPase Cdc42, ensuring the generation of a single axon in cultured hippocampal neurons and controlling the migration of cortical neurons in the developing brain. Finally, we provide compelling evidence that Tuba is the GEF that mediates Cdc42 activation during the development of neuronal polarity.

Recessive gene mutations underlie many developmental disorders and often lead to disabling neurological problems. Here, we report identification of a homozygous c.170G>A (p.Cys57Tyr or C57Y) mutation in the gene coding for protein disulfide isomerase A3 (PDIA3, also known as ERp57), an enzyme that catalyzes formation of disulfide bonds in the endoplasmic reticulum, to be associated with syndromic intellectual disability. Experiments in zebrafish embryos show that PDIA3C57Y expression is pathogenic and causes developmental defects such as axonal disorganization as well as skeletal abnormalities. Expression of PDIA3C57Y in the mouse hippocampus results in impaired synaptic plasticity and memory consolidation. Proteomic and functional analyses reveal that PDIA3C57Y expression leads to dysregulation of cell adhesion and actin cytoskeleton dynamics, associated with altered integrin biogenesis and reduced neuritogenesis. Biochemical studies show that PDIA3C57Y has decreased catalytic activity and forms disulfide-crosslinked aggregates that abnormally interact with chaperones in the endoplasmic reticulum. Thus, rare disease gene variant can provide insight into how perturbations of neuronal proteostasis can affect the function of the nervous system.

Fragile X Syndrome (FXS) is the most common inherited form of intellectual disability. It is produced by mutation of the Fmr1 gene that encodes for the Fragile Mental Retardation Protein (FMRP), an important RNA-binding protein that regulates the expression of multiple proteins located in neuronal synapses. Individuals with FXS exhibit abnormal sensory information processing frequently leading to hypersensitivity across sensory modalities and consequently a wide array of behavioral symptoms. Insects and mammals engage primarily their sense of smell to create proper representations of the external world and guide adequate decision-making processes. This feature in combination with the exquisitely organized neuronal circuits found throughout the olfactory system (OS) and the wide expression of FMRP in brain regions that process olfactory information makes it an ideal model to study sensory alterations in FXS models. In the last decade several groups have taken advantage of these features and have used the OS of fruit fly and rodents to understand neuronal alteration giving rise to sensory perception issues. In this review article, we will discuss molecular, morphological and physiological aspects of the olfactory information processing in FXS models. We will highlight the decreased inhibitory/excitatory synaptic balance and the diminished synaptic plasticity found in this system resulting in behavioral alteration of individuals in the presence of odorant stimuli.

Rab35 is a key protein for cargo loading in the recycling endosome. In neuronal immortalized cells, Rab35 promotes neurite differentiation. Here we describe that Rab35 favors axon elongation in rat primary neurons in an activity-dependent manner. In addition, we show that the p53-related protein kinase (PRPK) negatively regulates axonal elongation by reducing Rab35 protein levels through the ubiquitin-proteasome degradation pathway. PRPK-induced Rab35 degradation is regulated by its interaction with microtubule-associated protein 1B (MAP1B), a microtubule stabilizing binding protein essential for axon elongation. Consistently, axon defects found in MAP1B knock-out neurons were reversed by Rab35 overexpression or PRPK inactivation suggesting an epistatic relationship among these proteins. These results define a novel mechanism to support axonal elongation, by which MAP1B prevents PRPK-induced Rab35 degradation. Such a mechanism allows Rab35-mediated axonal elongation and connects the regulation of actin dynamics with membrane trafficking. In addition, our study reveals for the first time that the ubiquitin-proteasome degradation pathway regulates a Rab GTPase.

The capacity of the nervous system to generate neuronal networks relies on the establishment and maintenance of synaptic contacts. Synapses are composed of functionally different presynaptic and postsynaptic compartments. An appropriate synaptic architecture is required to provide the structural basis that supports synaptic transmission, a process involving changes in cytoskeletal dynamics. Actin microfilaments are the main cytoskeletal components present at both presynaptic and postsynaptic terminals in glutamatergic synapses. However, in the last few years it has been demonstrated that microtubules (MTs) transiently invade dendritic spines, promoting their maturation. Nevertheless, the presence and functions of MTs at the presynaptic site are still a matter of debate. Early electron microscopy (EM) studies revealed that MTs are present in the presynaptic terminals of the central nervous system (CNS) where they interact with synaptic vesicles (SVs) and reach the active zone. These observations have been reproduced by several EM protocols; however, there is empirical heterogeneity in detecting presynaptic MTs, since they appear to be both labile and unstable. Moreover, increasing evidence derived from studies in the fruit fly neuromuscular junction proposes different roles for MTs in regulating presynaptic function in physiological and pathological conditions. In this review, we summarize the main findings that support the presence and roles of MTs at presynaptic terminals, integrating descriptive and biochemical analyses, and studies performed in invertebrate genetic models.

CDK5 is a serine/threonine kinase that is involved in the normal function of the adult brain and plays a role in neurotransmission and synaptic plasticity. However, its over-regulation has been associated with Tau hyperphosphorylation and cognitive deficits. Our previous studies have demonstrated that CDK5 targeting using shRNA-miR provides neuroprotection and prevents cognitive deficits. Dendritic spine morphogenesis and forms of long-term synaptic plasticity—such as long-term potentiation (LTP)—have been proposed as essential processes of neuroplasticity. However, whether CDK5 participates in these processes remains controversial and depends on the experimental model. Using wild-type mice that received injections of CDK5 shRNA-miR in CA1 showed an increased LTP and recovered the PPF in deficient LTP of APPswe/PS1Δ9 transgenic mice. On mature hippocampal neurons CDK5, shRNA-miR for 12 days induced increased dendritic protrusion morphogenesis, which was dependent on Rac activity. In addition, silencing of CDK5 increased BDNF expression, temporarily increased phosphorylation of CaMKII, ERK, and CREB; and facilitated calcium signaling in neurites. Together, our data suggest that CDK5 downregulation induces synaptic plasticity in mature neurons involving Ca2+ signaling and BDNF/CREB activation.

Microtubule-associated protein 1B (MAP1B) is expressed predominantly during the early stages of development of the nervous system, where it regulates processes such as axonal guidance and elongation. Nevertheless, MAP1B expression in the brain persists in adult stages, where it participates in the regulation of the structure and physiology of dendritic spines in glutamatergic synapses. Moreover, MAP1B expression is also found in presynaptic synaptosomal preparations. In this work, we describe a presynaptic phenotype in mature neurons derived from MAP1B knockout (MAP1B KO) mice. Mature neurons express MAP1B, and its deficiency does not alter the expression levels of a subgroup of other synaptic proteins. MAP1B KO neurons display a decrease in the density of presynaptic and postsynaptic terminals, which involves a reduction in the density of synaptic contacts, and an increased proportion of orphan presynaptic terminals. Accordingly, MAP1B KO neurons present altered synaptic vesicle fusion events, as shown by FM4-64 release assay, and a decrease in the density of both synaptic vesicles and dense core vesicles at presynaptic terminals. Finally, an increased proportion of excitatory immature symmetrical synaptic contacts in MAP1B KO neurons was detected. Altogether these results suggest a novel role for MAP1B in presynaptic structure and physiology regulation in vitro.

X Pablo Muñoz-Llancao,1,3* X Daniel R. Henríquez,1* Carlos Wilson,1 Felipe Bodaleo,1 X Erik W. Boddeke,2 Frank Lezoualc’h,4,5 Martina Schmidt,3 and Christian González-Billault1 1Laboratory of Cell and Neuronal Dynamics, Department of Biology, Faculty of Sciences, Universidad de Chile, 7800024 Santiago, Chile, 2Department of Medical Physiology, University Medical Center Groningen, University of Groningen, The Netherlands, 3Department of Molecular Pharmacology, University of Groningen, The Netherlands, 4Inserm UMR-1048, Institut des Maladies Métaboliques et Cardiovasculaires, 31432 Toulouse, France, and 5Université de Toulouse III, Paul Sabatier, 31062 Toulouse, France

Microtubule-associated protein 1B (MAP1B) is a neuronal protein involved in the stabilization of microtubules both in the axon and somatodendritic compartments. Acute, genetic inactivation of MAP1B leads to delayed axonal outgrowth, most likely due to changes in the post-translational modification of tubulin subunits, which enhances microtubule polymerization. Furthermore, MAP1B deficiency is accompanied by abnormal actin microfilament polymerization and dramatic changes in the activity of small GTPases controlling the actin cytoskeleton. In this work, we showed that MAP1B interacts with a guanine exchange factor, termed Tiam1, which specifically activates Rac1. These proteins co-segregated in neurons, and interact in both heterologous expression systems and primary neurons. We dissected the molecular domains involved in the MAP1B-Tiam1 interaction, and demonstrated that pleckstrin homology (PH) domains in Tiam1 are responsible for MAP1B binding. Interestingly, only the light chain 1 (LC1) of MAP1B was able to interact with Tiam1. Moreover, it was able to increase the activity of the small GTPase, Rac1. These results suggest that the interaction between Tiam1 and MAP1B, is produced by the binding of LC1 with PH domains in Tiam1. The formation of such a complex impacts on the activation levels of Rac1 confirming a novel function of MAP1B related with the control of small GTPases. These results also support the idea of cross-talk between cytoskeleton compartments inside neuronal cells.