Publications

Abstract

Rare de novo heterozygous loss-of-function SETBP1 variants lead to a neurodevelopmental disorder characterized by speech deficits, indicating a potential involvement of SETBP1 in human speech. However, the expression pattern of SETBP1 in brain regions associated with vocal learning remains poorly understood, along with the underlying molecular mechanisms linking it to vocal production. In this study, we examined SETBP1 expression in the brain of male zebra finches, a well-established model for studying vocal production learning. We demonstrated that zebra finch SETBP1 exhibits a greater number of exons and isoforms compared to its human counterpart. We characterized a SETBP1 antibody and showed that SETBP1 colocalized with FoxP1, FoxP2, and Parvalbumin in key song nuclei. Moreover, SETBP1 expression in neurons in Area X is significantly higher in zebra finches singing alone, than those singing courtship song to a female, or non-singers. Importantly, we found a distinctive neuronal protein expression of SETBP1 and FoxP2 in Area X only in zebra finches singing alone, but not in the other conditions. We demonstrated SETBP1´s regulatory role on FoxP2 promoter activity in vitro. Taken together, these findings provide compelling evidence for SETBP1 expression in brain regions to be crucial for vocal learning and its modulation by singing behavior.

Abstract

The language network of the human brain has core components in the inferior frontal cortex and superior/middle temporal cortex, with left-hemisphere dominance in most people. Functional specialization and interconnectivity of these neocortical regions is likely to be reflected in their molecular and cellular profiles. Excitatory connections between cortical regions arise and innervate according to layer-specific patterns. Here we generated a new gene expression dataset from human postmortem cortical tissue samples from core language network regions, using spatial transcriptomics to discriminate gene expression across cortical layers. Integration of these data with existing single-cell expression data identified 56 genes that showed differences in laminar expression profiles between frontal and temporal language cortex together with upregulation in layer II/III and/or layer V/VI excitatory neurons. Based on data from large-scale genome-wide screening in the population, DNA variants within these 56 genes showed set-level associations with inter-individual variation in structural connectivity between left-hemisphere frontal and temporal language cortex, and with predisposition to dyslexia. The axon guidance genes SLIT1 and SLIT2 were consistently implicated. These findings identify region-specific patterns of laminar gene expression as a feature of the brain’s language network.

Abstract

The establishment of a reliable model for the study of Purkinje cells in vitro is of particular importance, given their central role in cerebellar function and pathology. Recent advances in induced pluripotent stem cell (iPSC) technology offer the opportunity to generate multiple neuronal subtypes for study in vitro. However, to date, only a handful of studies have generated Purkinje cells from human pluripotent stem cells, with most of these protocols proving challenging to reproduce. Here, we describe a simplified method for the reproducible generation of Purkinje cells from human iPSCs. After 21 days of treatment with factors selected to mimic the self-inductive properties of the isthmic organiser—insulin, fibroblast growth factor 2 (FGF2), and the transforming growth factor β (TGFβ)-receptor blocker SB431542—hiPSCs could be induced to form En1-positive cerebellar progenitors at efficiencies of up to 90%. By day 35 of differentiation, subpopulations of cells representative of the two cerebellar germinal zones, the rhombic lip (Atoh1-positive) and ventricular zone (Ptf1a-positive), could be identified, with the latter giving rise to cells positive for Purkinje cell progenitor-specific markers, including Lhx5, Kirrel2, Olig2 and Skor2. Further maturation was observed following dissociation and co-culture of these cerebellar progenitors with mouse cerebellar cells, with 10% of human cells staining positive for the Purkinje cell marker calbindin by day 70 of differentiation. This protocol, which incorporates modifications designed to enhance cell survival and maturation and improve the ease of handling, should serve to make existing models more accessible, in order to enable future advances in the field.

Abstract

Spinocerebellar ataxia type 14 (SCA14) is a subtype of the autosomal dominant cerebellar ataxias that is characterized by slowly progressive cerebellar dysfunction and neurodegeneration. SCA14 is caused by mutations in the PRKCG gene, encoding protein kinase C gamma (PKCγ). Despite the identification of 40 distinct disease-causing mutations in PRKCG, the pathological mechanisms underlying SCA14 remain poorly understood. Here we report the molecular neuropathology of SCA14 in post-mortem cerebellum and in human patient-derived induced pluripotent stem cells (iPSCs) carrying two distinct SCA14 mutations in the C1 domain of PKCγ, H36R and H101Q. We show that endogenous expression of these mutations results in the cytoplasmic mislocalization and aggregation of PKCγ in both patient iPSCs and cerebellum. PKCγ aggregates were not efficiently targeted for degradation. Moreover, mutant PKCγ was found to be hyper-activated, resulting in increased substrate phosphorylation. Together, our findings demonstrate that a combination of both, loss-of-function and gain-of-function mechanisms are likely to underlie the pathogenesis of SCA14, caused by mutations in the C1 domain of PKCγ. Importantly, SCA14 patient iPSCs were found to accurately recapitulate pathological features observed in post-mortem SCA14 cerebellum, underscoring their potential as relevant disease models and their promise as future drug discovery tools.