Disrupted Excitatory Synaptic Contacts and Altered Neuronal Network Activity Underpins the Neurological Phenotype in PCDH19-Clustering Epilepsy (PCDH19-CE).

PCDH19-Clustering Epilepsy (PCDH19-CE) is an infantile onset disorder caused by mutation of the X-linked PCDH19 gene. Intriguingly, heterozygous females are affected while hemizygous males are not. While there is compelling evidence that this disorder stems from the coexistence of WT and PCDH19-null cells, the cellular mechanism underpinning the neurological phenotype remains unclear. Here, we investigate the impact of Pcdh19 WT and KO neuron mosaicism on synaptogenesis and network activity. Using our previously established knock-in and knock-out mouse models, together with CRISPR-Cas9 genome editing technology, we demonstrate a reduction in excitatory synaptic contacts between PCDH19-expressing and PCDH19-null neurons. Significantly altered neuronal morphology and neuronal network activities were also identified in the mixed populations. In addition, we show that in Pcdh19 heterozygous mice, where the coexistence of WT and KO neurons naturally occurs, aberrant contralateral axonal branching is present. Overall, our data show that mosaic expression of PCDH19 disrupts physiological neurite communication leading to abnormal neuronal activity, a hallmark of PCDH19-CE.

Functional screening of GATOR1 complex variants reveals a role for mTORC1 deregulation in FCD and focal epilepsy.

Mutations in the GAP activity toward RAGs 1 (GATOR1) complex genes (DEPDC5, NPRL2 and NPRL3) have been associated with focal epilepsy and focal cortical dysplasia (FCD). GATOR1 functions as an inhibitor of the mTORC1 signalling pathway, indicating that the downstream effects of mTORC1 deregulation underpin the disease. However, the vast majority of putative disease-causing variants have not been functionally assessed for mTORC1 repression activity. Here, we develop a novel in vitro functional assay that enables rapid assessment of GATOR1-gene variants. Surprisingly, of the 17 variants tested, we show that only six showed significantly impaired mTORC1 inhibition. To further investigate variant function in vivo, we generated a conditional Depdc5 mouse which modelled a 'second-hit' mechanism of disease. Generation of Depdc5 null 'clones' in the embryonic brain resulted in mTORC1 hyperactivity and modelled epilepsy and FCD symptoms including large dysmorphic neurons, defective migration and lower seizure thresholds. Using this model, we validated DEPDC5 variant F164del to be loss-of-function. We also show that Q542P is not functionally compromised in vivo, consistent with our in vitro findings. Overall, our data show that mTORC1 deregulation is the central pathological mechanism for GATOR1 variants and also indicates that a significant proportion of putative disease variants are pathologically inert, highlighting the importance of GATOR1 variant functional assessment.

IGF-1 receptor regulates dynamic changes in neuronal polarity during cerebral cortical migration.

During cortical development, neurons undergo polarization, oriented migration and layer-type differentiation. The biological and biochemical mechanisms underlying these processes are not completely understood. In neurons in culture we showed that IGF-1 receptor activation is important for growth cone assembly and axonal formation. However, the possible roles of the insulin like growth factor-1 receptor (IGF-1R) on neuronal differentiation and polarization in vivo in mammals have not yet been studied. Using in utero electroporation, we show here that the IGF-1R is essential for neocortical development. Neurons electroporated with a shRNA targeting IGF-1 receptor failed to migrate to the upper cortical layers and accumulated at the ventricular/subventricular zones. Co-electroporation with a constitutively active form of PI3K rescued migration. The change of the morphology from multipolar to bipolar cells was also attenuated. Cells lacking the IGF-1 receptor remain arrested as multipolar forming a highly disorganized tissue. The typical orientation of the migrating neurons with the Golgi complex oriented toward the cortical upper layers was also affected by electroporation with shRNA targeting IGF-1 receptor. Finally, cells electroporated with the shRNA targeting IGF-1 receptor were unable to form an axon and, therefore, neuron polarity was absent.

Protein interacting with NIMA (never in mitosis A)-1 regulates axonal growth cone adhesion and spreading through myristoylated alanine-rich C kinase substrate isomerization.

Axonal growth cone motility requires precise regulation of adhesion to navigate the complex environment of the nervous system and reach its target. Myristoylated alanine-rich C kinase substrate (MARCKS) protein is enriched in the developing brain and plays an important, phosphorylation-dependent role in the modulation of axonal growth cone adhesion. The ratio of phospho-MARCKS (MARCKS-P) to total MARCKS controls adhesion modulation and spreading of the axonal growth cone. Pin1, a peptidyl-prolyl cis/trans isomerase (PPIase) that recognizes and binds to phosphorylated serine/threonine residues preceded by a proline (pSer/Thr-Pro) is also expressed in the developing brain. Here, we show that Pin1 is present in the growth cone, interacts with MARCKS-P, and regulates its dephosphorylation. We also described morphological alterations in the corpus callosum and cerebral cortex fibers of the Pin1 knockout mouse brain that may be caused by the misregulation of MARCKS-P and alterations of neuronal adhesion. We have shown that MARCKS, a critical protein in the movement of neuronal growth cones, is in turn regulated by both phosphorylation and cis-trans peptidyl isomerization mediated by Pin1. In the absence of Pin1, MARCKS is hyperphosphorylated, leading to loss of adhesions, and collapse of the growth cone. The Pin1 KO mice exhibited disturbed neuronal projections from the cerebral cortex and reduced white matter tracks such as the corpus callosum. This study highlights a novel function of Pin1 in neurodevelopment.

The insulin-like growth factor 1 receptor is essential for axonal regeneration in adult central nervous system neurons.

Axonal regeneration is an essential condition to re-establish functional neuronal connections in the injured adult central nervous system (CNS), but efficient regrowth of severed axons has proven to be very difficult to achieve. Although significant progress has been made in identifying the intrinsic and extrinsic mechanisms involved, many aspects remain unresolved. Axonal development in embryonic CNS (hippocampus) requires the obligate activation of the insulin-like growth factor 1 receptor (IGF-1R). Based on known similarities between axonal growth in fetal compared to mature CNS, we decided to examine the expression of the IGF-1R, using an antibody to the ßgc subunit or a polyclonal anti-peptide antibody directed to the IGF-R (C20), in an in vitro model of adult CNS axonal regeneration, namely retinal ganglion cells (RGC) derived from adult rat retinas. Expression of both ßgc and the ß subunit recognized by C20 antibody were low in freshly isolated adult RGC, but increased significantly after 4 days in vitro. As in embryonic axons, ßgc was localised to distal regions and leading growth cones in RGC. IGF-1R-ßgc co-localised with activated p85 involved in the phosphatidylinositol-3 kinase (PI3K) signaling pathway, upon stimulation with IGF-1. Blocking experiments using either an antibody which neutralises IGF-1R activation, shRNA designed against the IGF-1R sequence, or the PI3K pathway inhibitor LY294002, all significantly reduced axon regeneration from adult RGC in vitro (∼40% RGC possessed axons in controls vs 2-8% in the different blocking studies). Finally, co-transfection of RGC with shRNA to silence IGF-1R together with a vector containing a constitutively active form of downstream PI3K (p110), fully restored axonal outgrowth in vitro. Hence these data demonstrate that axonal regeneration in adult CNS neurons requires re-expression and activation of IGF-1R, and targeting this system may offer new therapeutic approaches to enhancing axonal regeneration following trauma.