Tubulinopathy mutations in TUBA1A that disrupt neuronal morphogenesis and migration override XMAP215/Stu2 regulation of microtubule dynamics

ABSTRACTHeterozygous, missense mutations in α- or β-tubulin genes are associated with a wide range of human brain malformations, known as tubulinopathies. We seek to understand whether a mutation’s impact at the molecular and cellular levels scale with the severity of brain malformation. Here we focus on two mutations at the valine 409 residue of TUBA1A, V409I and V409A, identified in patients with pachygyria or lissencephaly, respectively. We find that ectopic expression of TUBA1A-V409I/A mutants disrupt neuronal migration in mice and promote excessive neurite branching and delayed retraction in primary neuronal cultures, accompanied by increased microtubule acetylation. To determine the molecular mechanisms, we modeled the V409I/A mutants in budding yeast and found that they promote intrinsically faster microtubule polymerization rates in cells and in reconstitution experiments with purified tubulin. In addition, V409I/A mutants decrease the recruitment of XMAP215/Stu2 to plus ends and ablate tubulin binding to TOG domains. In each assay tested, the TUBA1A-V409I mutant exhibits an intermediate phenotype between wild type and the more severe TUBA1A-V409A, reflecting the severity observed in brain malformations. Together, our data support a model in which the V409I/A mutations may limit tubulin conformational states and thereby disrupt microtubule regulation during neuronal morphogenesis and migration.

MicroRNAs as Critical Biomarkers of Major Depressive Disorder: A Comprehensive Perspective

Major Depressive Disorder (MDD) represents a major global health concern, a body-mind malady of rising prevalence worldwide nowadays. The complex network of mechanisms involved in MDD pathophysiology is subjected to epigenetic changes modulated by microRNAs (miRNAs). Serum free or vesicles loaded miRNAs have starred numerous publications, denoting a key role in cell-cell communication, systematically and in brain structure and neuronal morphogenesis, activity and plasticity. Upregulated or downregulated expression of these signaling molecules may imply the impairment of genes implicated in pathways of MDD etiopathogenesis (neuroinflammation, brain-derived neurotrophic factor (BDNF), neurotransmitters, hypothalamic-pituitary-adrenal (HPA) axis, oxidative stress, circadian rhythms...). In addition, these miRNAs could serve as potential biomarkers with diagnostic, prognostic and predictive value, allowing to classify severity of the disease or to make decisions in clinical management. They have been considered as promising therapy targets as well and may interfere with available antidepressant treatments. As epigenetic malleable regulators, we also conclude emphasizing lifestyle interventions with physical activity, mindfulness and diet, opening the door to new clinical management considerations.

Computational simulations reveal that Abl activity controls cohesiveness of actin networks in growth cones

Extensive studies of growing axons have revealed many individual components and protein interactions that guide neuronal morphogenesis. Despite this, however, we lack any clear picture of the emergent mechanism by which this nanometer-scale biochemistry generates the multi-micron scale morphology and cell biology of axon growth and guidance in vivo. To address this, we studied the downstream effects of the Abl signaling pathway using a computer simulation software (MEDYAN) that accounts for mechanochemical dynamics of active polymers. Previous studies implicate two Abl effectors, Arp2/3 and Enabled, in Abl-dependent axon guidance decisions. We now find that Abl alters actin architecture primarily by activating Arp2/3, while Enabled plays a more limited role. Our simulations show that simulations mimicking modest levels of Abl activity bear striking similarity to actin profiles obtained experimentally from live-imaging of actin in wild type axons in vivo. Using a graph-theoretical filament-filament contact analysis, moreover, we find that networks mimicking hyperactivity of Abl (enhanced Arp2/3) are fragmented into smaller domains of actin that interact weakly with each other, consistent with the pattern of actin fragmentation observed upon Abl overexpression in vivo. Two perturbative simulations further confirm that high Arp2/3 actin networks are mechanically disconnected and fail to mount a cohesive response to perturbation. Taken together, these data provide a molecular-level picture of how the large-scale organization of the axonal cytoskeleton arises from the biophysics of actin networks.

KLC4 shapes axon arbors during development and mediates adult behavior

Development of elaborate and polarized neuronal morphology requires precisely regulated transport of cellular cargos by motor proteins such as kinesin-1. Kinesin-1 has numerous cellular cargos which must be delivered to unique neuronal compartments. The process by which this motor selectively transports and delivers cargo to regulate neuronal morphogenesis is poorly understood. Our work implicates one kinesin light chain subunit, KLC4, as an essential regulator of axon branching and arborization pattern of sensory neurons during development. Using several live imaging approaches in klc4 mutant zebrafish, we show that KLC4 is required for stabilization of nascent axon branches and for proper microtubule (MT) dynamics. Furthermore, KLC4 is required for the contact repulsion necessary for tiling of peripheral axon arbors: in klc4 mutants, peripheral axons showed abnormal fasciculation, a behavior characteristic of central axons, suggesting that KLC4 patterns axonal compartments and helps define axon identity. Finally, we find that klc4 mutant adults show anxiety-like behavior in a novel tank test, implicating klc4 as a novel gene involved in stress response circuits.

The Hidden Side of NCAM Family: NCAM2, a Key Cytoskeleton Organization Molecule Regulating Multiple Neural Functions

Although it has been over 20 years since Neural Cell Adhesion Molecule 2 (NCAM2) was identified as the second member of the NCAM family with a high expression in the nervous system, the knowledge of NCAM2 is still eclipsed by NCAM1. The first studies with NCAM2 focused on the olfactory bulb, where this protein has a key role in axonal projection and axonal/dendritic compartmentalization. In contrast to NCAM1, NCAM2’s functions and partners in the brain during development and adulthood have remained largely unknown until not long ago. Recent studies have revealed the importance of NCAM2 in nervous system development. NCAM2 governs neuronal morphogenesis and axodendritic architecture, and controls important neuron-specific processes such as neuronal differentiation, synaptogenesis and memory formation. In the adult brain, NCAM2 is highly expressed in dendritic spines, and it regulates synaptic plasticity and learning processes. NCAM2’s functions are related to its ability to adapt to the external inputs of the cell and to modify the cytoskeleton accordingly. Different studies show that NCAM2 interacts with proteins involved in cytoskeleton stability and proteins that regulate calcium influx, which could also modify the cytoskeleton. In this review, we examine the evidence that points to NCAM2 as a crucial cytoskeleton regulation protein during brain development and adulthood. This key function of NCAM2 may offer promising new therapeutic approaches for the treatment of neurodevelopmental diseases and neurodegenerative disorders.

Development of motor neurons and motor activity in zebrafish requires F-actin nucleation by Fmn2b

Background: Cytoskeletal remodelling plays a pivotal role in the establishment of neuronal connectivity during development and in plasticity in adults. Mutations in the cytoskeleton regulatory protein Formin-2 (Fmn2) are associated with neurodevelopmental disorders like intellectual disability, though its function in neuronal morphogenesis has not been characterised in vivo. Results: Here we develop a loss-of-function model for fmn2b, the zebrafish orthologue of Fmn2, using CRISPR/Cas9-mediated gene editing. fmn2b mutants display motor deficits starting from the earliest motor responses in the embryo. We find that fmn2b is expressed in motor neurons and its loss reduces motor neuron innervation of the axial muscles without affecting myotome integrity. The translocation of caudal primary (CaP) motor neuron outgrowth is compromised in fmn2b mutants, while rostral primary (RoP) motor neurons have missing soma or stall at the horizontal myoseptum. Strikingly, axon collateral branching of the motor neurons is severely compromised and results in reduced synaptic coverage of the myotome. Rescue experiments identify the requirement for Fmn2-mediated actin nucleation for motor neuron outgrowth and arborisation. Conclusions: The zebrafish loss-of-function model of Fmn2 reveals the specific requirement of F-actin polymerisation by Fmn2 in neuromuscular development. It also underscores the role of Fmn2 in motor neuropathies, especially as a proportion of individuals harbouring mutations in Fmn2 present with hypotonia.

Ankyrin2 is required for neuronal morphogenesis and long-term memory and interacts genetically with HDAC4

Dysregulation of HDAC4 expression and/or subcellular distribution results in impaired neuronal morphogenesis and long-term memory in Drosophila melanogaster. A recent genetic screen for genes that interact in the same molecular pathway as HDAC4 identified the cytoskeletal adapter Ankyrin2 (Ank2). Here we sought to investigate the role of Ank2 in neuronal morphogenesis, learning and memory, and to examine the nature of interaction with HDAC4. We found that Ank2 is expressed widely throughout the Drosophila brain where it localizes predominantly to axon tracts. Pan-neuronal knockdown of Ank2 in the mushroom body, a region critical for memory formation, resulted in defects in axon morphogenesis, and similarly reduction of Ank2 in lobular plate tangential neurons of the optic lobe disrupted dendritic branching and arborization. Conditional knockdown of Ank2 in the mushroom body of adult Drosophila significantly impaired long-term courtship memory, and this requirement for Ank2 was isolated to gamma (γ) neurons of the mushroom body. As overexpression of HDAC4 in γ neurons also impairs the formation of long-term courtship memory, this suggests that any functional relationship between these proteins during LTM likely occurs in γ neurons. We determined that the genetic interaction requires the presence of nuclear HDAC4 and is not dependent on a conserved putative ankyrin-binding motif present in HDAC4. In summary, we provide the first characterization of the expression pattern of Ank2 in the adult Drosophila brain and demonstrate that Ank2 is critical for morphogenesis of the mushroom body and for the molecular processes required in the adult brain for formation of long-term memories.

New Partners Identified by Mass Spectrometry Assay Reveal Functions of NCAM2 in Neural Cytoskeleton Organization

Neuronal cell adhesion molecule 2 (NCAM2) is a membrane protein with an important role in the morphological development of neurons. In the cortex and the hippocampus, NCAM2 is essential for proper neuronal differentiation, dendritic and axonal outgrowth and synapse formation. However, little is known about NCAM2 functional mechanisms and its interactive partners during brain development. Here we used mass spectrometry to study the molecular interactome of NCAM2 in the second postnatal week of the mouse cerebral cortex. We found that NCAM2 interacts with >100 proteins involved in numerous processes, including neuronal morphogenesis and synaptogenesis. We validated the most relevant interactors, including Neurofilaments (NEFs), Microtubule-associated protein 2 (MAP2), Calcium/calmodulin kinase II alpha (CaMKIIα), Actin and Nogo. An in silico analysis of the cytosolic tail of the NCAM2.1 isoform revealed specific phosphorylation site motifs with a putative affinity for some of these interactors. Our results expand the knowledge of NCAM2 interactome and confirm the key role of NCAM2 in cytoskeleton organization, neuronal morphogenesis and synaptogenesis. These findings are of interest in explaining the phenotypes observed in different pathologies with alterations in the NCAM2 gene.

Neuronal extracellular vesicles mediate BDNF-dependent dendritogenesis and synapse maturation via microRNAs

Extracellular vesicles (EVs) have emerged as novel regulators of several biological processes, in part via the transfer of EV content such as microRNA; small non-coding RNAs that regulate protein production, between cells. However, how neuronal EVs contribute to trans-neuronal signaling is largely elusive. We examined the role of neuron-derived EVs in neuronal morphogenesis downstream signaling induced by brain-derived neurotrophic factor (BDNF). We found that EVs perpetuated BDNF induction of dendrite complexity and synapse maturation in naive hippocampal neurons, which was dependent on the activity of three microRNAs, miR-132-5p, miR-218 and miR-690. These microRNAs were up-regulated in BDNF-stimulated EVs. Moreover, supplementation with BDNF-EVs rescued the block of BDNF-induced phenotypes upon inhibition of miRNA activity. Our data therefore suggest a major role for EVs in BDNF-dependent morphogenesis, and provide new evidence for the functional transfer of microRNAs between neurons. This is not only an important step towards understanding the function of EVs in inter-neuronal signaling, but is also relevant for many disorders characterized by decreased BDNF signaling, such as major depression or cognitive impairment.

Defective metabolic programming impairs early neuronal morphogenesis in neural cultures and an organoid model of Leigh syndrome

Leigh syndrome (LS) is a severe manifestation of mitochondrial disease in children and is currently incurable. The lack of effective models hampers our understanding of the mechanisms underlying the neuronal pathology of LS. Using patient-derived induced pluripotent stem cells and CRISPR/Cas9 engineering, we developed a human model of LS caused by mutations in the complex IV assembly gene SURF1. Single-cell RNA-sequencing and multi-omics analysis revealed compromised neuronal morphogenesis in mutant neural cultures and brain organoids. The defects emerged at the level of neural progenitor cells (NPCs), which retained a glycolytic proliferative state that failed to instruct neuronal morphogenesis. LS NPCs carrying mutations in the complex I gene NDUFS4 recapitulated morphogenesis defects. SURF1 gene augmentation and PGC1A induction via bezafibrate treatment supported the metabolic programming of LS NPCs, leading to restored neuronal morphogenesis. Our findings provide mechanistic insights and suggest potential interventional strategies for a rare mitochondrial disease.