Cytomegalic parvalbumin neurons in fetal cases of hemimegalencephaly

Abstract

Objective: Mutations in genes of the mTOR pathway have been identified as a major cause of hemimegalencephaly (HMG), focal cortical dysplasia type II, and tuberous sclerosis, cortical malformations associated with epilepsy. These conditions are characterized at the cellular level by increased size of pyramidal neurons that grow with dysmorphic features and in some cases by the presence of giant balloon cells. Our previous research in tuberous sclerosis has shown that parvalbumin (Pvalb) and calbindin immunoreactive cells in cortical and subcortical tuberal lesions show cytomegalic features, suggesting the involvement of GABAergic cells in mTOR-related pathologies. In the present report, we propose to deepen our understanding of the role of interneurons in mTOR-related cortical malformations by analyzing the maturation of Pvalb neurons in fetal samples of HMG.

Methods: We performed immunohistochemical staining of cortical samples from individuals with HMG from 21 gestational weeks to 10 postnatal months. The study focused on Pvalb cells, and pS6 counterstaining was performed to assess the activation of the mTOR pathway. To investigate the pathomechanisms behind the cytomegalic features, we examined mTOR pathway gene expression in Pvalb interneurons and cortical projection neurons using a single-cell transcriptomic atlas of the human neocortex.

Results: Our results revealed cytomegalic features in Pvalb interneurons, indicating abnormal development in HMG patients compared to controls. This phenotype progressively worsened over time, suggesting ongoing developmental abnormalities associated with mTOR dysregulation, which may underlie the pathology of cortical malformations in HMG. Our transcriptomic data revealed similar expression patterns of mTOR and its upstream regulators in both Pvalb and glutamatergic neurons during development, suggesting that mTOR pathway disorders may induce similar phenotypes in both cell types.

Significance: The present data suggest that Pvalb interneurons are involved in the development of mTOR-related cortical dysplasia and that they may be a contributor to the clinical phenotype of these patients.

Keywords: cortical developmental disorders; interneurons; mTOR pathway; neocortex.

FIGURE 1

Parvalbumin (Pvalb)‐positive neurons in hemimegalencephaly (HMG). (A) Immunostaining of cortical sections illustrating the presence of cytomegalic interneurons in HMG hemisphere (bottom) as compared to controls (top). Scale bar = 20 μm. (B) Violin plots illustrating the soma size of control and HMG Pvalb‐positive neurons at different developmental ages (soma size in μm²). ****p < .00001 when compared with same developmental age controls. In controls, n of cells = 77 (31 gestational weeks [GW]) 235 (33 GW), 24 (36 GW), 14 (39 GW), 130 (41 GW). In HMG, n of cells = 182 (31 GW), 199 (34 GW), 120 (37 GW), 136 (40 GW), 204 (10 months [mo]). (C) Single regression lines (mean soma size in μm² vs. age) showing significant differences in slopes of HMG (21.33 ± 1.24) compared to controls (2.577 ± .33; F = 222.1, df = 1, df = 973, p < .0001).

FIGURE 2

Double staining of hemimegalencephaly sections with parvalbumin (Pvalb) and phosphorylated S6 (pS6) antibodies. All sections were 4,6‐diamidino‐2‐phenylindole (Dapi) counterstained. Cytomegalic Pvalb neurons were pS6 immunopositive (red arrows), whereas the smaller Pvalb neurons were pS6 negative (white arrows). Scale bars = 25 μm². GW, gestational weeks; mo, months.

FIGURE 3

Temporal expression of genes associated with cortical dysplasia, hemimegalencephaly/megalencephaly, and focal cortical dysplasia type II across neuronal subtypes. Dot plots illustrate the temporal gene expression profiles in three neuronal subtypes: parvalbumin (Pvalb; A), intratelencephalic (IT; B), and extratelencephalic (ET; C) neurons. The x‐axis represents developmental stages, whereas the y‐axis denotes the selected genes. For each of the 11 genes, the dots encode mean gene expression (color intensity) and the percentage of cells expressing the gene (dot size). GlutN, glutamatergic neuron; GW, gestational week.