Bruno Sterlini

The development and maintenance of the nervous system depend on a tightly regulated intracellular transport network in which kinesin superfamily (KIF) motor proteins drive microtubule-based delivery of synaptic vesicle precursors, organelles, mRNAs, and signaling components along axons and dendrites. Disruption of this machinery underlies a clinically heterogeneous spectrum of neurodevelopmental disorders (NDDs), including intellectual disability, epilepsy, autism spectrum disorder, microcephaly, malformations of cortical development, spasticity, and axonal neuropathy. Here, we synthesize current knowledge on how kinesin dysfunction shapes neurodevelopment. We outline the physiological roles of kinesins in neuronal polarity, organelle and mitochondrial positioning, synaptogenesis, and progenitor division, and survey principal disease-associated genes, including KIF1A, KIF5A, KIF7, KIF11, KIF2A, KIF5C, and emerging members such as KIF14, KIF15, and KIF16B. We detail how distinct pathogenic mechanisms, such as loss of motility, impaired cargo coupling, motor hyperactivity, mitotic spindle defects, and disrupted ciliary signaling, converge on shared cellular endpoints, and how tubulin isotypes and posttranslational modifications further modulate motor output. In this review, we discuss translational implications, including variant-resolved diagnosis and precision strategies to restore transport, dampen pathological hyperactivity, or stabilize the microtubule track. Collectively, these advances reframe kinesinopathies as mechanistically stratified disorders of neuronal transport.

The development and maintenance of the nervous system depend on a tightly regulated intracellular transport network in which kinesin superfamily (KIF) motor proteins drive microtubule-based delivery of synaptic vesicle precursors, organelles, mRNAs, and signaling components along axons and dendrites. Disruption of this machinery underlies a clinically heterogeneous spectrum of neurodevelopmental disorders (NDDs), including intellectual disability, epilepsy, autism spectrum disorder, microcephaly, malformations of cortical development, spasticity, and axonal neuropathy. Here, we synthesize current knowledge on how kinesin dysfunction shapes neurodevelopment. We outline the physiological roles of kinesins in neuronal polarity, organelle and mitochondrial positioning, synaptogenesis, and progenitor division, and survey principal disease-associated genes, including KIF1A, KIF5A, KIF7, KIF11, KIF2A, KIF5C, and emerging members such as KIF14, KIF15, and KIF16B. We detail how distinct pathogenic mechanisms, such as loss of motility, impaired cargo coupling, motor hyperactivity, mitotic spindle defects, and disrupted ciliary signaling, converge on shared cellular endpoints, and how tubulin isotypes and posttranslational modifications further modulate motor output. In this review, we discuss translational implications, including variant-resolved diagnosis and precision strategies to restore transport, dampen pathological hyperactivity, or stabilize the microtubule track. Collectively, these advances reframe kinesinopathies as mechanistically stratified disorders of neuronal transport.

Induced glutamatergic neurons (iGluNeurons) generated by Neurogenin-2 (NGN2) overexpression in human pluripotent stem cells are a powerful model for studying human neuronal maturation and function; however, NGN2-based protocols still lack standardized culture conditions that critically affect neuronal development and function. Three key factors have been identified by previous literature, namely the composition of extracellular matrix coating, the initial plating density, and the choice of culture medium, but the differential effects of their combination have not been thoroughly analyzed. Here, we investigated the combinatorial effects of these three variables, testing eight distinct culture conditions resulting from the combinations of two coatings (poly-L-ornithine and polyethyleneimine), two media (BrainPhys and Neurobasal), and two cell densities (4800 and 1200 cells/mm²). We assessed electrophysiological properties at the single-cell and network levels, characterized morphofunctional and proteomic features across multiple developmental stages. Electrophysiological data indicate that medium composition and plating density, rather than substrate coating, determine neuronal maturation dynamics, with BrainPhys and high density promoting rapid but transient maturation while Neurobasal and low density supporting gradual and sustained network development. Morphofunctional analyzes of synapses and the axon initial segment, together with neuronal maturation markers, support an early BrainPhys-driven acceleration of development that is later exceeded by Neurobasal. To enable accurate proteome profiling of the iGluNeuron system—comprising human neurons and rat astrocytes—we developed a robust taxonomic filtering algorithm that selectively identifies human-specific proteins. This approach confirmed the presence of a conserved core of NGN2-driven differentiation pathways across all settings, in addition to condition-specific signatures. Finally, in the optimal conditions identified through our experimental analyzes, robust spontaneous and evoked synaptic activity was observed. These results provide a framework for optimizing iGluNeuron cultures, balancing rapid maturation and long-term functional stability, and establishing a benchmark for human neuronal models in disease research and drug screening.

Pathogenic variants in the SCN2A gene, encoding the α-subunit type 2 of the voltage-gated sodium channel Na_V1.2, cause a phenotypic spectrum including 4 major disorders as benign familial infantile seizures, developmental and epileptic encephalopathy, intellectual disability, and autism. Gain-of-function variants resulting phenotypes may be treated with sodium channel blockers, while loss-of-function (LoF) conditions are non-respondent. We focused on the effects of the pathogenic SCN2A variant c.4976C>T (p.A1659V) found in heterozygosity in 3 patients affected by DEE non responsive to SCB. We functionally investigated this previously uncharacterized SCN2A variant. Three individuals with the SCN2A c.4976C>T (p.A1659V) variant were studied. This variant was detected by next-generation sequencing (NGS). The nucleotide substitution was inserted by site-directed mutagenesis in a stabilized SCN2A plasmid encoding Na_V1.2. Expression and functional characterization of the Na_V1.2 A1659V variant was performed in HEK293 cells by western blotting, confocal microscopy, and patch clamp electrophysiology. The same de novo pathogenic SCN2A variant was detected in 3 patients with DEE characterized by early onset, severe ID, and seizures unresponsive to SCB. In 2 patients, the variant is in a mosaic state. The Na_V1.2 A1659V variant did not affect channel protein expression while exhibiting significant effects on its function as shown by the reduced Na⁺ currents, a shift of the activation curve toward more negative potentials, a shift of the inactivation curve to more negative voltages, and slower kinetics of inactivation compared with native Na_V1.2 in HEK293 cells. Simulations suggested that the variant increases excitability in neurons. These results revealed the multifaceted functional effect of A1659V variant on channel activity and highlighted the complex genotype-phenotype correlation underlying significant clinical and pharmacological variability in SCN2A-related encephalopathies.

Progress of human brain in vitro models stands as a keystone in neurological and psychiatric research, addressing the limitations posed by species-specific differences in animal models. The generation of human neurons from induced pluripotent stem cells (iPSCs) using transcription factor reprogramming protocols has been shown to reduce heterogeneity and improve consistency across different stem cell lines. Despite notable advancements, the current protocols still exhibit several shortcomings. This study focuses on standardizing and optimizing the procedure for iPSC-derived glutamatergic neurons generation through the inducible overexpression of Neurogenin-2. Noteworthy refinements include stringent scrutiny of genomic rearrangements post-fibroblast reprogramming, selection of a homogeneously integrated NGN2-cassettes population, and the incorporation of an intermediate step during neuronal differentiation to store neuronal progenitors. The neural culture showed a high degree of neuronal maturation and consistency, as shown by single-cell and network electrophysiological recordings. These advancements aim to provide more reliable tools for disease modelling and drug screening in neurological disorders. Graphical Abstract

The opioidergic system and intrinsic brain activity, as organized in large-scale networks such as the salience network (SN), sensorimotor network (SMN), and default-mode network (DMN), play core roles in healthy behavior and psychiatric disorders. This work aimed to investigate how opioidergic signaling affects intrinsic brain activity in healthy individuals by reviewing relevant neuroanatomical, molecular, functional, and pharmacological magnetic resonance imaging studies in order to clarify their physiological links and changes in psychiatric disorders. The SN shows dense opioidergic innervations of subcortical structures and high expression levels of opioid receptors in subcortical-cortical areas, with enhanced or reduced activity with low or very high doses of opioids, respectively. The SMN shows high levels of opioid receptors in subcortical areas and functional disconnection caused by opioids. The DMN shows low levels of opioid receptors in cortical areas and inhibited or enhanced activity with low or high doses of opioids, respectively. Finally, we proposed a working model. Opioidergic signaling enhances SN and suppresses SMN (and DMN) activity, resulting in affective excitation with psychomotor inhibition; stronger increases in opioidergic signaling attenuate the SN and SMN while disinhibiting the DMN, dissociating affective and psychomotor functions from the internal states; the opposite occurs with a deficit of opioidergic signaling.

The study of the pathomechanisms by which gene mutations lead to neurological diseases has benefit from several cellular and animal models. Recently, induced Pluripotent Stem Cell (iPSC) technologies have made possible the access to human neurons to study nervous system disease-related mechanisms, and are at the forefront of the research into neurological diseases. In this review, we will focalize upon genetic epilepsy, and summarize the most recent studies in which iPSC-based technologies were used to gain insight on the molecular bases of epilepsies. Moreover, we discuss the latest advancements in epilepsy cell modeling. At the two dimensional (2D) level, single-cell models of iPSC-derived neurons lead to a mature neuronal phenotype, and now allow a reliable investigation of synaptic transmission and plasticity. In addition, functional characterization of cerebral organoids enlightens neuronal network dynamics in a three-dimensional (3D) structure. Finally, we discuss the use of iPSCs as the cutting-edge technology for cell therapy in epilepsy.