PCDH19-related epilepsy is a rare genetic disease caused by defective function of PCDH19, a calcium-dependent cell–cell adhesion protein of the cadherin superfamily. This disorder is characterized by a heterogeneous phenotypic spectrum, with partial and generalized febrile convulsions that are gradually increasing in frequency. Developmental regression may occur during disease progression. Patients may present with intellectual disability (ID), behavioral problems, motor and language delay, and a low motor tone. In most cases, seizures are resistant to treatment, but their frequency decreases with age, and some patients may even become seizure-free. ID generally persists after seizure remission, making neurological abnormalities the main clinical issue in affected individuals. An effective treatment is lacking. In vitro studies using patient-derived induced pluripotent stem cells (iPSCs) reported accelerated neural differentiation as a major endophenotype associated with PCDH19 mutations. By using this in vitro model system, we show that accelerated in vitro neurogenesis is associated with a defect in the cell division plane at the neural progenitors stage. We also provide evidence that altered PCDH19 function affects proper mitotic spindle orientation. Our findings identify an altered equilibrium between symmetric versus asymmetric cell division as a previously unrecognized mechanism contributing to the pathogenesis of this rare epileptic encephalopathy.
The development of the nervous system requires cytoskeleton-mediated processes coordinating self-renewal, migration, and differentiation of neurons. It is not surprising that many neurodevelopmental problems and neurodegenerative disorders are caused by deficiencies in cytoskeleton-related genes. For this reason, we focus on the cytoskeletal dynamics in proliferating iPSCs and in iPSC-derived neurons to better characterize the underpinnings of cytoskeletal organization looking at actin and tubulin repolymerization studies using the cell permeable probes SiR-Actin and SiR-Tubulin. During neurogenesis, each neuron extends an axon in a complex and changing environment to reach its final target. The dynamic behavior of the growth cone and its capacity to respond to multiple spatial information allows it to find its correct target. We decided to characterize various parameters of the actin filaments and microtubules. Our results suggest that a rapid re-organization of the cytoskeleton occurs 45 minutes after treatments with de-polymerizing agents in iPSCs and 60 minutes in iPSC-derived neurons in both actin filaments and microtubules. The quantitative data confirm that the actin filaments have a primary role in the re-organization of the cytoskeleton soon after de-polymerization, while microtubules have a major function following cytoskeletal stabilization. In conclusion, we investigate the possibility that de-polymerization of the actin filaments may have an impact on microtubules organization and that de-polymerization of the microtubules may affect the stability of the actin filaments. Our results suggest that a reciprocal influence of the actin filaments occurs over the microtubules and vice versa in both in iPSCs and iPSC-derived neurons.
Kinesins are motor proteins involved in microtubule-mediated intracellular transport. They contribute to key cellular processes, including intracellular trafficking, organelle dynamics and cell division. Pathogenic variants in kinesin-encoding genes underlie several human diseases characterized by an extremely variable clinical phenotype, ranging from isolated neurodevelopmental/neurodegenerative disorders to syndromic phenotypes belonging to a family of conditions collectively termed as ‘ciliopathies’. Among kinesins, kinesin-1 is the most abundant microtubule motor for transport of cargoes towards the plus end of microtubules. Three kinesin-1 heavy chain isoforms exist in mammals. Different from KIF5A and KIF5C, which are specifically expressed in neurons and established to cause neurological diseases when mutated, KIF5B is an ubiquitous protein. Three de novo missense KIF5B variants were recently described in four subjects with a syndromic skeletal disorder characterized by kyphomelic dysplasia, hypotonia and DD/ID. Here, we report three dominantly acting KIF5B variants (p.Asn255del, p.Leu498Pro and p.Leu537Pro) resulting in a clinically wide phenotypic spectrum, ranging from dilated cardiomyopathy with adult-onset ophthalmoplegia and progressive skeletal myopathy to a neurodevelopmental condition characterized by severe hypotonia with or without seizures. In vitro and in vivo analyses provide evidence that the identified disease-associated KIF5B variants disrupt lysosomal, autophagosome and mitochondrial organization, and impact cilium biogenesis. All variants, and one of the previously reported missense changes, were shown to affect multiple developmental processes in zebrafish. These findings document pleiotropic consequences of aberrant KIF5B function on development and cell homeostasis, and expand the phenotypic spectrum resulting from altered kinesin-mediated processes.
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