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Early-onset epileptic encephalopathies are severe disorders often associated with specific genetic mutations. In this context, the CDKL5 deficiency disorder (CDD) is a neurodevelopmental condition characterized by early-onset seizures, intellectual delay, and motor dysfunction. Although crucial for proper brain development, the precise targets of CDKL5 and its relation to patients' symptoms are still unknown. Here, induced pluripotent stem cells derived from individuals deficient in CDKL5 protein were used to generate neural cells. Proteomic and phosphoproteomic approaches revealed disruption of several pathways, including microtubule-based processes and cytoskeleton organization. While CDD-derived neural progenitor cells have proliferation defects, neurons showed morphological alterations and compromised glutamatergic synaptogenesis. Moreover, the electrical activity of CDD cortical neurons revealed hyperexcitability during development, leading to an overly synchronized network. Many parameters of this hyperactive network were rescued by lead compounds selected from a human high-throughput drug screening platform. Our results enlighten cellular, molecular, and neural network mechanisms of genetic epilepsy that could ultimately promote novel therapeutic opportunities for patients.

The evolutionarily conserved splicing regulator neuro-oncological ventral antigen 1 (NOVA1) plays a key role in neural development and function. NOVA1 also includes a protein-coding difference between the modern human genome and Neanderthal and Denisovan genomes. To investigate the functional importance of an amino acid change in humans, we reintroduced the archaic allele into human induced pluripotent cells using genome editing and then followed their neural development through cortical organoids. This modification promoted slower development and higher surface complexity in cortical organoids with the archaic version of NOVA1 Moreover, levels of synaptic markers and synaptic protein coassociations correlated with altered electrophysiological properties in organoids expressing the archaic variant. Our results suggest that the human-specific substitution in NOVA1, which is exclusive to modern humans since divergence from Neanderthals, may have had functional consequences for our species' evolution.

It is well known that oligodendrocyte-associated Nogo-A protein is an important regulator of axonal outgrowth and an important inhibitor of functional recovery and anatomical plasticity after central nervous system (CNS) injury. Abundant studies of oligodendrocyte-associated Nogo-A function in the uninjured rodent have suggested a role in neuronal development and synaptic function. On the other hand, the roles of neuron-associated (i.e., neuronal) Nogo-A have not been fully investigated. We have previously shown that neuronal Nogo-A influence dendritic spine density and morphology in pyramidal neurons of the intact neocortex. To further examine the role of neuronal Nogo-A in this synaptic population, we designed an RNAi directed against Nogo-A, delivered to the developing rat sensorimotor cortex using a neurotropic viral vector adeno-associated virus (AAV) 2/8. We examined the transduced neocortex for molecules important for synaptic plasticity, including N-Methyl-D-Aspartate (NMDA) receptor subunits GRIN2A; glutamate receptor subunit epsilon-1 (NR2A), and GRIN2B; glutamate receptor subunit epsilon-2 (NR2B), as well as postsynaptic density-95 (PSD-95). Furthermore, we also determined the density of excitatory synapses by examining the presynaptic protein vesicular glutamate transporter 1 (vGLut1) as a marker for potential excitatory synapses. Our results showed that neuronal Nogo-A knockdown in postnatal pyramidal neurons of the sensorimotor cortex led to a significant decrease in NMDA receptor subunits NR2A and NR2B messenger RNA when examined as adults. However, there was no difference in PSD-95 expression in comparison to controls. In addition, the decrease in the number of vGlut1(+) puncta on branches of apical dendrites of pyramidal neurons indicated the loss of synapses that have a strong influence on direct current entering the dendrite. Taken together, these results indicate that neuronal Nogo-A may regulate synaptic plasticity by modulating the components of excitatory synapses. This finding represents a novel role in excitatory synaptic formation for neuronal Nogo-A in developing neurons of the uninjured CNS. [ABSTRACT FROM AUTHOR]