Altered synaptic connectivity in anin vitrohuman model of STXBP1 encephalopathy

Abstract

Early infantile developmental and epileptic encephalopathies are devastating conditions, generally of genetic origin, but the pathological mechanisms often remain obscure. A major obstacle in this field of research is the difficulty of studying cortical brain development in humans, at the relevant time period in utero. To address this, we established an in vitro assay to study the impact of gene variants on the developing human brain, using living organotypic cultures of the human subplate and neighbouring cortical regions, prepared from ethically sourced, 14-17 post conception week brain tissue (www.hdbr.org). We were able to maintain cultures for several months, during which time, the gross anatomical structures of the cortical plate, subplate and marginal zone persisted, while neurons continued to develop morphologically, and form new synaptic networks. This preparation thus permits the study of genetic manipulations, and their downstream effects upon an intact developing human cortical network. We focused upon STXBP1 haploinsufficiency, which is among the most common genetic causes of developmental and epileptic encephalopathy. This was induced using shRNA interference, leading to impaired synaptic function and a reduced density of glutamatergic synapses. We thereby provide a critical proof-of-principle for how to study the impact of any gene of interest on the development of the human cortex.

Opposing roles of physiological and pathological amyloid-β on synapses in live human brain slice cultures

“…We also recently showed that oligomeric tau derived from brains of individuals with Progressive Supranuclear Palsy (PSP) can be taken up into synapses, and induced gliosis in HBSCs 63 . Other studies have established HBSCs as an excellent tool to investigate: physiological properties of human neurons 12,60,61,[106][107][108][109][110][111][112] , tumour environments [113][114][115] , neurodegenerative disease 98,99,[116][117][118][119][120][121][122] , epileptic activity 123,124 and developmental disorders 125 . By correlating responses to treatments, as well as changes in endogenous dynamics, to patient age, sex, brain region, genetics and lifestyle risk factors, we propose that HBSCs represent an extremely powerful tool for both discovery and translational research.…”

“…Dissection and culture methods to generate human brain slice cultures (HBSCs) were adapted from published studies 60,106,[126][127][128] B9001), 1 ug/ml laminin (APExBIO: A1023), 1X penicillin/streptomycin (ThermoFisher: 15140122), 3 units/ ml nystatin (Merck: N1638) and 20 mM HEPES. For all samples, the time taken from surgical tissue removal to slices being plated in Wash Solution 2 was kept to a minimum (< 2 hours).…”

Cortico-thalamic development and disease: From cells, to circuits, to schizophrenia

“…For instance, traditional ex vivo methods such as organotypic cultures have been exploited as a valid system to investigate human neurodevelopmental alterations of specific networks. Indeed, organotypic brain slice cultures represent a physiological threedimensional model of the brain that can be interrogated to define the impact of genetic and environmental insults and pathophysiological responses (Onorati et al, 2016;Grønning Hansen et al, 2020;McLeod et al, 2022). This method, indeed, allows the culture of neural complex tissue preserving the architecture and cell interactions, even if the obtainment and longterm maintenance of the culture may be challenging.…”

Can in vitro studies aid in the development and use of antiseizure therapies? A report of the ILAE/AES Joint Translational Task Force

“…For longer term use, including the application of viral vectors, human brain slices can be maintained in organotypic culture 109,110 . This approach has been extended recently to the study of the developing human brain 111 . Resected human slices do suffer some disadvantages.…”

“…109,110 This approach has been extended recently to the study of the developing human brain. 111 Resected human slices do suffer some disadvantages. Patient donors and tissue samples are highly heterogeneous with key variables including patient age, sex, ASM history as well as the anatomic location of the seizure-onset zone and the viability of the resected tissue.…”