Case Reports
doi: 10.1073/pnas.2023333118.
Early role for a Na+,K+-ATPase (ATP1A3) in brain development
Affiliations
- PMID: 34161264
- PMCID: PMC8237684
- DOI: 10.1073/pnas.2023333118
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Case Reports
Early role for a Na+,K+-ATPase (ATP1A3) in brain development
Proc Natl Acad Sci U S A.
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Abstract
Osmotic equilibrium and membrane potential in animal cells depend on concentration gradients of sodium (Na+) and potassium (K+) ions across the plasma membrane, a function catalyzed by the Na+,K+-ATPase α-subunit. Here, we describe ATP1A3 variants encoding dysfunctional α3-subunits in children affected by polymicrogyria, a developmental malformation of the cerebral cortex characterized by abnormal folding and laminar organization. To gain cell-biological insights into the spatiotemporal dynamics of prenatal ATP1A3 expression, we built an ATP1A3 transcriptional atlas of fetal cortical development using mRNA in situ hybridization and transcriptomic profiling of ∼125,000 individual cells with single-cell RNA sequencing (Drop-seq) from 11 areas of the midgestational human neocortex. We found that fetal expression of ATP1A3 is most abundant to a subset of excitatory neurons carrying transcriptional signatures of the developing subplate, yet also maintains expression in nonneuronal cell populations. Moving forward a year in human development, we profiled ∼52,000 nuclei from four areas of an infant neocortex and show that ATP1A3 expression persists throughout early postnatal development, most predominantly in inhibitory neurons, including parvalbumin interneurons in the frontal cortex. Finally, we discovered the heteromeric Na+,K+-ATPase pump complex may form nonredundant cell-type-specific α-β isoform combinations, including α3-β1 in excitatory neurons and α3-β2 in inhibitory neurons. Together, the developmental malformation phenotype of affected individuals and single-cell ATP1A3 expression patterns point to a key role for α3 in human cortex development, as well as a cell-type basis for pre- and postnatal ATP1A3-associated diseases.
Keywords: ATP1A3; cortex development; cortical malformation; developmental channelopathy; polymicrogyria.
Copyright © 2021 the Author(s). Published by PNAS.
Conflict of interest statement
The authors declare no competing interest.
Figures
ATP1A3 variants associated with cerebral cortex malformations. (A, Left) Schematic of the cortical malformation, PMG, and resulting macroscopic disorganization of cortical gyri and sulci. (Right) MRI images of Cases A, B, C, and D showing PMG. White arrows denote gross location of affected brain regions. “Flat Neocortex” image provides alternative view of PMG surrounding the perisylvian region (green arrows) if the neocortex were unwrapped from around the subcortical structures and laid flat. The control image comes from an unaffected 11-y-old. (Scale bar, 1 cm.) (B) Pedigrees with novel de novo pathogenic single-nucleotide variants in ATP1A3: Case A (missense, p.Arg901Met) with bilateral frontoparietal PMG, Case B (splice donor site, c.2921+1G > A) with extensive bilateral PMG, Case C (missense, p.Leu924Pro) with unilateral PMG, and Case D (missense, p.Gly851Arg) with extensive bilateral PMG, more pronounced in the right hemisphere. Individual features: Genotype (±, de novo heterozygous), PMG, infantile seizures, developmental delay, and postnatal microcephaly shown in pedigree summary. Square, male; circle, female; black and/or gray shading, affected individual. See SI Appendix for genetic validation and comprehensive clinical phenotyping. (C) Overview of the α-subunit (green) of the Na,K-ATPase with novel PMG alleles mapped (colored amino acids) and previous case report of PMG-associated allele (Leu888Pro) (17). Red, p.Leu888Pro (L888P); orange, p.Arg901Met (R901M); magenta, p.Leu924Pro (L924P); blue, p.Gln851Arg (Q851R). Image generated with PyMOL using Protein Databank 2ZXE (21). (Right) TM topology schematic of Na,K-ATPase for visualization of PMG causing variants, including α-, β-, and FXYD-subunits. We also denote a variant enriched region TM7/TM8, where β-subunits interact with the α3-subunit in the extracellular TM7/TM8 segment Glu899, Gln904, and Gln905 (21). See SI Appendix, Figs. S7 and S8 and Table S4 for complete allele-to-protein topology breakdown.
ATP1A3 is differentially enriched to the human fetal period across cortical layers. (A) Chromosome locations for CNS-enriched Na+,K+-ATPase α-isoforms, ATP1A1 and ATP1A2 (Chr1), and ATP1A3 (Chr19). RPKM, reads per kilobase and million mapped reads. (B) RNA transcriptome analysis of bulk brain regions revealed ATP1A3 transcripts are high during fetal gestation weeks (WKSG) and persist postnatally. CNS-expressed paralogous ATP1A1 and ATP1A2 paralogs show relative expression-pattern changes during development, while ATP1A4 is not expressed significantly in the brain. (C, Left) ATP1A3 mRNA chromogenic ISH performed on 19 wpc coronal brain sections demonstrate highest ATP1A3 expression in the human CP and SP regions. (Scale bar, Left, 500 µm.) (Right) Corresponding fluorescence imaging of 19 wpc fetal brain with cell-type–specific markers for intermediate progenitors (TBR2) and neural progenitors (vimentin, VIM) demonstrates ATP1A3 transcripts are not present within cells of the SVZ and VZ. Arrows indicate high expressing neurons in deep cortical layers and SP region. Scale bar, 50 µm. (D) Magnified image from C depicting high ATP1A3-expressing cells within the SP layer indicated with arrows. Scale bar, 50 µm. (E, Left) Schematic of developing sagittal fetal cerebellum tissue section at 19 wpc demonstrates enrichment of ATP1A3 in Purkinje cell layer. (i) ATP1A3 mRNA fluorescence in situ at 19 wpc demonstrates highest expression in the Purkinje cell layer, colocalizing with Purkinje cell marker calbindin (CALB1, green). (ii) Zoomed fluorescence image, including labeling of external granule layer. (Scale bar, Left, 50 µm.) DAPI stain for nuclei in blue. EGL, external granule layer; PCL, Purkinje cell layer. (F) Cerebellum RNA data revealed ATP1A3 transcripts are high during fetal gestational weeks (WKSG) and persist postnatally. Raw transcriptome data for B and F from Allen Brain Atlas, presented as log2 RPKM values and a polynomial fit to average across time points (23).
Single-cell ATP1A3 expression atlas in the developing human cerebral cortex. (A, Left) Schematic showing a 21-wpc forebrain and location of regions sampled (for anatomical detail, see SI Appendix, Fig. S2A ). (Upper) Dorsolateral view; (Lower) sagittal view. (Right) Uniform Manifold Approximation and Projection (UMAP) of 125,943 single cells profiled by Drop-seq. Each dot represents a cell, color-coded by sample of origin. Key: blue-to-red: frontal-to-caudal. CGE, caudal ganglionic eminence; Hp, hippocampus; Ins, insula; MGE, medial ganglionic eminence; Occ, occipital; Orb, orbital; Par, parietal; PFC, prefrontal cortex; Str, striatum; Temp, temporal; V1, primary visual cortex. (B) UMAP plot of all profiled cells color-coded by level of ATP1A3 expression. (Inset) UMAP region highlighting highest ATP1A3 expression, which also maps onto ENs within the SP. (C, Left) Histogram showing relative expression of ATP1A3 (x axis) across cell clusters (y axis). Notice enriched expression of ATP1A3 in cluster EN.4-SP, containing SP EN neurons (see H and SI Appendix, Fig. S2C for SP markers enriched in this cluster). Mean ATP1A3 expression was aggregated by cluster, then rescaled from 0 to 1. Cell clusters are color-coded by cell type (blue: EN; gold: glia; blue: IN; red: NPC). See SI Appendix, Fig. S2 D and E for cluster markers and assignments. (Right) Dot-plot showing ATP1A3 expression across clusters split by areas of origin (x axis; sample 1–11, ordered from caudal to rostral). Color scale codes for mean expression by group; size of the dots codes for percentage of cells expressing ATP1A3 in each group. (D, Upper) Violin-box plot showing differential expression of ATP1A3 (y axis) in the EN-4–SP cluster across three main cortical partitions (x axis; caudal: samples 1 to 3, occipital cortex); medial: samples 4 to 8 (including parietal and temporal cortex, and subcortical structures); frontal: samples 9 to 11 (frontal cortex). Dots represent individual cells. Violins show probability density distributions; boxes show interquartile ranges; notches show confidence intervals around the median (horizontal lines). Outliers were trimmed. Mean expression of ATP1A3 was aggregated by region, then log10-transformed. Notice the highest expression of ATP1A3 in cells sampled from the frontal cortex, and lowest expression in cells sampled from caudal cortex. (Lower) Nissl-stained frontal coronal section of a 21-wpc forebrain highlighting anatomical position of SP. Source: Atlas of the Developing Human Brain, BrainSpan (www.brainspan.org ) (64). ATP1A3 mRNA ISH performed on 20-wpc coronal brain sections within the SP region to depict ATP1A3 coexpression with EN.4_SP clade marker (CRYM). (Scale bar, 10 µm.) (E) ATP1A3 expression across cells in the EN clusters (color-coded in blue in C, Left) ordered by pseudotime. Dots represent individual cells. y axis: log-transformed, scaled expression of ATP1A3 by cell. x axis: pseudotime score (color-coded) for each cell, calculated using the Monocle3 algorithm. Trend-line shows the increase of ATP1A3 expression as function of pseudotime, calculated by fitting a quasipoisson model to the data. (F) UMAP of all profiled cells color-coded by cluster (see C, Left). The EN-4–SP cluster is highlighted. (G) UMAP of cells in the EN-4–SP cluster (highlighted in F) color-coded by rostral, medial, and caudal anatomical partition of origin (see also D, Upper). Notice that cells segregate by origin. (H) Relative expression of areal marker genes in the EN-4–SP cluster (see G). Relative gene expression is coded by color and size. Notice that ATP1A3 is enriched in cells expressing SP markers (CRYM, WNT7B, HS3ST4) and frontal cortex markers (GRP, SEMA3E), but not caudal cortex markers (NPY, LPL, NEFL).
Single-cell analysis of ATP1A3 expression in the infant human neocortex. (A, Left) Schematic showing an 8-mo-old neocortex and location of regions sampled. BA: Brodmann area; PFC: prefrontal cortex; V1: primary visual cortex. (Right) t-distributed stochastic neighbor embedding (tSNE) of 51,878 single nuclei profiled by Drop-seq. Each dot represents a nucleus, color-coded by cluster (Left), cell type (Center), and origin sample (Right). See C for cluster and cell-class assignments. (B, Left) Dendrogram summarizing hierarchical clustering of data. Clusters are color-coded as in A, Left. See SI Appendix, Fig. S4 for cluster markers and assignments. (Middle) Bar graph showing relative expression of ATP1A3 (y axis) across cell clusters (x axis). Mean expression was aggregated by cluster, then rescaled from 0 to 1 across all clusters. Data are color-coded by cell type (as in A, Center: blue, EN; gold, IN; brown, glia). (Top Right) Violin-box plot showing expression of ATP1A3 (y axis) within EN and IN clusters across four main cortical partitions. x axis: caudal sample from occipital cortex (V1); medial samples from parietal (Par) and temporal (Temp) cortex; frontal samples from prefrontal cortex (PFC). Dots represent individual cells. Violins show probability density distributions; boxes show interquartile ranges; notches show confidence intervals around the median (horizontal lines). Outliers were trimmed. Mean expression of ATP1A3 was aggregated by region, then log10-transformed. Notice the broad higher expression of ATP1A3 in IN across x axis cortical regions, and lower expression in ENs. (C) tSNE plots showing specific enrichment of cortical marker genes including genes associated with early activity in the developing cortex (MEFC2, SLC17A7, and SLC17A6) (VGLU1 and VGLU2), and MCD-associated gene TUBB2A. (D) Venn diagram showing intersection between the top 100 genes correlating with ATP1A3 in the EN cluster (blue) and IN cluster (gold); 47 of 100 genes are shared between the two groups. Top 50 genes correlating with ATP1A3 in the EN and IN clusters are shown. Dots indicate Pearson’s R for each gene in the EN cluster (Upper; blue) and IN cluster (Lower; gold). Genes found in both groups are indicated in red, and the respective correlation coefficients are connected by red lines. GO analysis for biological function of the 47 genes shared between EN and IN clusters indicates several components of the somatodendritic compartment, including synapses and dendrites (see SI Appendix, Tables S2 and S3 for entire GO table and P values).
