RGMa and Neogenin control dendritic spine morphogenesis via WAVE Regulatory Complex-mediated actin remodeling.

Structural plasticity, the ability of dendritic spines to change their volume in response to synaptic stimulation, is an essential determinant of synaptic strength and long-term potentiation (LTP), the proposed cellular substrate for learning and memory. Branched actin polymerization is a major force driving spine enlargement and sustains structural plasticity. The WAVE Regulatory Complex (WRC), a pivotal branched actin regulator, controls spine morphology and therefore structural plasticity. However, the molecular mechanisms that govern WRC activation during spine enlargement are largely unknown. Here we identify a critical role for Neogenin and its ligand RGMa (Repulsive Guidance Molecule a) in promoting spine enlargement through the activation of WRC-mediated branched actin remodeling. We demonstrate that Neogenin regulates WRC activity by binding to the highly conserved Cyfip/Abi binding pocket within the WRC. We find that after Neogenin or RGMa depletion, the proportions of filopodia and immature thin spines are dramatically increased, and the number of mature mushroom spines concomitantly decreased. Wildtype Neogenin, but not Neogenin bearing mutations in the Cyfip/Abi binding motif, is able to rescue the spine enlargement defect. Furthermore, Neogenin depletion inhibits actin polymerization in the spine head, an effect that is not restored by the mutant. We conclude that RGMa and Neogenin are critical modulators of WRC-mediated branched actin polymerization promoting spine enlargement. This study also provides mechanistic insight into Neogenin's emerging role in LTP induction.

Presynaptic targeting of botulinum neurotoxin type A requires a tripartite PSG-Syt1-SV2 plasma membrane nanocluster for synaptic vesicle entry.

The unique nerve terminal targeting of botulinum neurotoxin type A (BoNT/A) is due to its capacity to bind two receptors on the neuronal plasma membrane: polysialoganglioside (PSG) and synaptic vesicle glycoprotein 2 (SV2). Whether and how PSGs and SV2 may coordinate other proteins for BoNT/A recruitment and internalization remains unknown. Here, we demonstrate that the targeted endocytosis of BoNT/A into synaptic vesicles (SVs) requires a tripartite surface nanocluster. Live-cell super-resolution imaging and electron microscopy of catalytically inactivated BoNT/A wildtype and receptor-binding-deficient mutants in cultured hippocampal neurons demonstrated that BoNT/A must bind coincidentally to a PSG and SV2 to target synaptic vesicles. We reveal that BoNT/A simultaneously interacts with a preassembled PSG-synaptotagmin-1 (Syt1) complex and SV2 on the neuronal plasma membrane, facilitating Syt1-SV2 nanoclustering that controls endocytic sorting of the toxin into synaptic vesicles. Syt1 CRISPRi knockdown suppressed BoNT/A- and BoNT/E-induced neurointoxication as quantified by SNAP-25 cleavage, suggesting that this tripartite nanocluster may be a unifying entry point for selected botulinum neurotoxins that hijack this for synaptic vesicle targeting.

Radial contractility of actomyosin rings facilitates axonal trafficking and structural stability.

Most mammalian neurons have a narrow axon, which constrains the passage of large cargoes such as autophagosomes that can be larger than the axon diameter. Radial axonal expansion must therefore occur to ensure efficient axonal trafficking. In this study, we reveal that the speed of various large cargoes undergoing axonal transport is significantly slower than that of small ones and that the transit of diverse-sized cargoes causes an acute, albeit transient, axonal radial expansion, which is immediately restored by constitutive axonal contractility. Using live super-resolution microscopy, we demonstrate that actomyosin-II controls axonal radial contractility and local expansion, and that NM-II filaments associate with periodic F-actin rings via their head domains. Pharmacological inhibition of NM-II activity significantly increases axon diameter by detaching the NM-II from F-actin and impacts the trafficking speed, directionality, and overall efficiency of long-range retrograde trafficking. Consequently, prolonged NM-II inactivation leads to disruption of periodic actin rings and formation of focal axonal swellings, a hallmark of axonal degeneration.

WNT5a Regulates Epithelial Morphogenesis in the Developing Choroid Plexus.

The choroid plexus (CP) is the predominant supplier of cerebral spinal fluid (CSF) and the site of the blood-CSF barrier and is thus essential for brain development and central nervous system homeostasis. Despite these crucial roles, our understanding of the molecular and cellular processes giving rise to the CPs within the ventricles of the mammalian brain is very rudimentary. Here, we identify WNT5a as an important regulator of CP development, where it acts as a pivotal factor driving CP epithelial morphogenesis in all ventricles. We show that WNT5a is essential for the establishment of a cohesive epithelium in the developing CP. We find that in its absence all CPs are substantially reduced in size and complexity and fail to expand into the ventricles. Severe defects were observed in the epithelial cytoarchitecture of all Wnt5a-/- CPs, exemplified by loss of apicobasally polarized morphology and detachment from the ventricular surface and/or basement membrane. We also present evidence that the WNT5a receptor, RYK, and the RHOA kinase, ROCK, are required for normal CP epithelial morphogenesis. Our study, therefore, reveals important insights into the molecular and cellular mechanisms governing CP development.

STXBP1 encephalopathy: Connecting neurodevelopmental disorders with α-synucleinopathies?

De novo pathogenic variants in STXBP1 encoding syntaxin1-binding protein (STXBP1, also known as Munc18-1) lead to a range of early-onset neurocognitive conditions, most commonly early infantile epileptic encephalopathy type 4 (EIEE4, also called STXBP1 encephalopathy), a severe form of epilepsy associated with developmental delay/intellectual disability. Other neurologic features include autism spectrum disorder and movement disorders. The progression of neurologic symptoms has been reported in a few older affected individuals, with the appearance of extrapyramidal features, reminiscent of early onset parkinsonism. Understanding the pathologic process is critical to improving therapies, as currently available antiepileptic drugs have shown limited success in controlling seizures in EIEE4 and there is no precision medication approach for the other neurologic features of the disorder. Basic research shows that genetic knockout of STXBP1 or other presynaptic proteins of the exocytic machinery leads to widespread perinatal neurodegeneration. The mechanism that regulates this effect is under scrutiny but shares intriguing hallmarks with classical neurodegenerative diseases, albeit appearing early during brain development. Most critically, recent evidence has revealed that STXBP1 controls the self-replicating aggregation of α-synuclein, a presynaptic protein involved in various neurodegenerative diseases that are collectively known as synucleinopathies, including Parkinson disease. In this review, we examine the tantalizing link among STXBP1 function, EIEE, and the neurodegenerative synucleinopathies, and suggest that neural development in EIEE could be further affected by concurrent synucleinopathic mechanisms.

Branching mechanisms shaping dendrite architecture.

A neuron's contribution to the information flow within a neural circuit is governed by the structure of its dendritic arbor. The geometry of the dendritic arbor directly determines synaptic density and the size of the receptive field, both of which influence the firing pattern of the neuron. Importantly, the position of individual dendritic branches determines the identity of the neuron's presynaptic partner and thus the nature of the incoming sensory information. To generate the unique stereotypic architecture of a given neuronal subtype, nascent branches must emerge from the dendritic shaft at preprogramed branch points. Subsequently, a complex array of extrinsic factors regulates the degree and orientation of branch expansion to ensure maximum coverage of the receptive field whilst constraining growth within predetermined territories. In this review we focus on studies that best illustrate how environmental cues such as the Wnts and Netrins and their receptors sculpt the dendritic arbor. We emphasize the pivotal role played by the actin cytoskeleton and its upstream regulators in branch initiation, outgrowth and navigation. Finally, we discuss how protocadherin and DSCAM contact-mediated repulsion prevents inappropriate synapse formation between sister dendrites or dendrites and the axon from the same neuron. Together these studies highlight the clever ways evolution has solved the problem of constructing complex branch geometries.

5-HT1A receptor-dependent modulation of emotional and neurogenic deficits elicited by prolonged consumption of alcohol.

Repeated episodes of binge-like alcohol consumption produce anxiety, depression and various deleterious effects including alterations in neurogenesis. While the involvement of the serotonin receptor 1 A (5-HT1A) in the regulation of anxiety-like behavior and neurogenesis is well documented, its contribution to alcohol withdrawal-induced anxiety and alcohol-induced deficits in neurogenesis is less documented. Using the Drinking-In-the-Dark (DID) paradigm to model chronic long-term (12 weeks) binge-like voluntary alcohol consumption in mice, we show that the selective partial activation of 5-HT1A receptors by tandospirone (3 mg/kg) prevents alcohol withdrawal-induced anxiety in a battery of behavioral tests (marble burying, elevated-plus-maze, open-field), which is accompanied by a robust decrease in binge-like ethanol intake (1 and 3 mg/kg). Furthermore, using triple immunolabelling of proliferation and neuronal differentiation markers, we show that long-term DID elicits profound deficits in neurogenesis and neuronal fate specification in the dorsal hippocampus that are entirely reversed by a 2-week chronic treatment with the 5-HT1A partial agonist tandospirone (3 mg/kg/day). Together, our results confirm previous observations that 5-HT1A receptors play a pivotal role in alcohol drinking behavior and the associated emotional and neurogenic impairments, and suggest that 5-HT1A partial agonists represent a promising treatment strategy for alcohol abuse.

Dendritic spine actin cytoskeleton in autism spectrum disorder.

Dendritic spines are small actin-rich protrusions from neuronal dendrites that form the postsynaptic part of most excitatory synapses. Changes in the shape and size of dendritic spines correlate with the functional changes in excitatory synapses and are heavily dependent on the remodeling of the underlying actin cytoskeleton. Recent evidence implicates synapses at dendritic spines as important substrates of pathogenesis in neuropsychiatric disorders, including autism spectrum disorder (ASD). Although synaptic perturbations are not the only alterations relevant for these diseases, understanding the molecular underpinnings of the spine and synapse pathology may provide insight into their etiologies and could reveal new drug targets. In this review, we will discuss recent findings of defective actin regulation in dendritic spines associated with ASD.

ENA/VASP proteins regulate exocytosis by mediating myosin VI-dependent recruitment of secretory granules to the cortical actin network.

In neurosecretory cells, myosin VI associated with secretory granules (SGs) mediates their activity-dependent recruitment to the cortical actin network and is necessary to sustain exocytosis. The mechanism by which myosin VI interacts with SGs is unknown. Using a myosin VI pull-down assay and mass spectrometry we identified Mena, a member of the ENA/VASP family, as a myosin VI binding partner in PC12 cells, and confirmed that Mena colocalized with myosin VI on SGs. Using a knock-sideways approach to inactivate the ENA/VASP family members by mitochondrial relocation, we revealed a concomitant redistribution of myosin VI. This was ensued by a reduction in the association of myosin VI with SGs, a decreased SG mobility and density in proximity to the plasma membrane as well as decreased evoked exocytosis. These data demonstrate that ENA/VASP proteins regulate SG exocytosis through modulating the activity of myosin VI.

The Wnt receptor Ryk is a negative regulator of mammalian dendrite morphogenesis.

The unique dendritic architecture of a given neuronal subtype determines its synaptic connectivity and ability to integrate into functional neuronal networks. It is now clear that abnormal dendritic structure is associated with neuropsychiatric and neurodegenerative disorders. Currently, however, the nature of the extrinsic factors that limit dendritic growth and branching within predetermined boundaries in the mammalian brain is poorly understood. Here we identify the Wnt receptor Ryk as a novel negative regulator of dendritic arborisation. We demonstrate that loss of Ryk in mouse hippocampal and cortical neurons promotes excessive dendrite growth and branching in vitro. Conversely, overexpression of wildtype Ryk restricts these processes, confirming that Ryk acts to restrain dendrite arborisation. Furthermore, we identify a hitherto uncharacterized membrane proximal subdomain crucial for Ryk-mediated suppression of dendrite morphogenesis, suggesting that it may act through a novel signalling pathway to constrain dendrite complexity. We also demonstrate that Ryk performs a similar function in vivo as Ryk haploinsufficient postnatal animals exhibit excessive dendrite growth and branching in layer 2/3 pyramidal neurons of the somatosensory cortex. These findings reveal an essential role for Ryk in regulating dendrite complexity and raise the intriguing possibility that it may influence neural plasticity by modifying dendritic structure.