Unique molecular features and cellular responses differentiate two populations of motor cortical layer 5b neurons in a preclinical model of ALS

Abstract

Many neurodegenerative diseases, such as amyotrophic lateral sclerosis (ALS), lead to the selective degeneration of discrete cell types in the CNS despite the ubiquitous expression of many genes linked to disease. Therapeutic advancement depends on understanding the unique cellular adaptations that underlie pathology of vulnerable cells in the context of disease-causing mutations. Here, we employ bacTRAP molecular profiling to elucidate cell type-specific molecular responses of cortical upper motor neurons in a preclinical ALS model. Using two bacTRAP mouse lines that label distinct vulnerable or resilient projection neuron populations in motor cortex, we show that the regulation of oxidative phosphorylation (Oxphos) pathways is a common response in both cell types. However, differences in the baseline expression of genes involved in Stem and the handling of reactive oxygen species likely lead to the selective degeneration of the vulnerable cells. These results provide a framework to identify cell-type-specific processes in neurodegenerative disease.

Keywords: ALS; CP: Neuroscience; G93A; RNA-seq; SOD1; TRAP; corticopontine; corticospinal; motor cortex.

Figure 1.. Colgalt2 and Gprin3 bacTRAP lines label molecularly and anatomically distinct populations of projection neurons in layer 5b of M1

(A) Anti-GFP immunostaining (green) showing expression of eGFP-L10a in deep-layer cells in M1 of Colgalt2-bacTRAP DU9 (left) and Gprin3-bacTRAP ES152 (right) animals. Red staining indicates NeuN + cells. Scale bar, 200 μm. (B) Frequency distribution of laminar depth of labeled cells in Colgalt2-bacTRAP (purple, n = 209 cells), Gprin3-bacTRAP (green, n = 163 cells), S100a10-bacTRAP (blue, n = 248 cells), and Ntsr1-bacTRAP (black, n = 146 cells) mice in M1 and in neighboring S1 (L5b populations only, Colgalt2-bacTRAP, n = 125; Gprin3-bacTRAP, n = 95 cells). (C) Box and whisker plots of population distribution for soma sizes (area in μm²) of S100a10 (blue, mean ± SEM 144.2 ± 1.7 μm²), Colgalt2 (purple, 233.3 ± 2.6 μm²), Gprin3 (green, 244.0 ± 2.7 μm²), and Ntsr1 (black, 122.3 ± 1.3 μm²) cells. ****p < 0.0001 by one-way ANOVA and subsequent Tukey multiple comparison test. (D) Immunostaining for CTIP2 (red) and GFP (green) in Colgalt2-bacTRAP (left) and Gprin3-bacTRAP (right) animals. Scale bar, 50 μm. (E) Scatterplot showing Log₂ fold enrichment values from DE analyses of Colgalt2 TRAP vs. M1 input (x axis) and Gprin3 TRAP vs. M1 input (y axis). Genes enriched in Colgalt2 only (purple), Gprin3 only (green), or both (red) are indicated. L5b PT cell marker genes (black) are labeled. (F) Heatmap showing relative expression of glial genes (gray) and a subset of known L5b marker genes that showed enrichment in either Colgalt2 cells only (purple), both cell types (red), or Gprin3 cells only (green) across M1 input, Colgalt2 TRAP, and Gprin3 TRAP datasets. Values are reported as z-scores of normalized CPM, averaged across biological replicates, and scaled for each gene. (G) Images showing FISH for Vat1l in UL5b (left column), Lypd1 in LL5b (middle column), and Nefh in both sublayers (right column) co-labeled with anti-GFP immunofluorescence in M1 of bacTRAP mice. Arrows indicate cells that were double-positive for GFP and the probed marker gene. Scale bar, 100 μm. See also Figures S1 and S2 and Tables S1 and S2.

Figure 2.. Retrograde tracing reveals overlapping and distinct axonal projection targets of Colgalt2 and Gprin3 cells

(A) Schematic illustration of the strategy for retrograde tracing of corticopontine neurons with cholera toxin B (CTB; left), and a representative image of the injection site (right) showing anti-CTB (red) and anti-NeuN (cyan) immunofluorescence. (B) Immunofluorescent images showing anti-CTB-labeled cells (red) in both sublayers of L5b in M1 following an injection into the pons (right). Staining with anti-GFP (green) revealed overlap of labeled cells in UL5b of Colgalt2-bacTRAP animals (middle) and LL5b in Gprin3-bacTRAP (right) animals. Scale bar, 200 μm. (C) Schematic of the strategy for retrograde labeling of corticospinal neurons (left) and a representative image of the injection site in C6 spinal cord (right) showing anti-CTB (red) and anti-NeuN (cyan) immunofluorescence. (D) Immunofluorescent images, as in (B), showing CTB- and GFP-labeled cells in LL5b of M1 (left) in Gprin3-bacTRAP (right) and Colgalt2-bacTRAP (center) mice following C6 injections. Scale bar, 200 μm. (E) Histogram of the quantification of laminar depth of GFP+ Colgalt2 (purple, n = 209 cells) and Gprin3 (green, n = 163) cells alongside CTB+ corticopontine (red left, n = 651 cells) and corticospinal cells (red right, n = 192 cells). Frequency reported as percentage of total cells found at each depth. (F) Quantification (mean ± SEM) of the percentage of M1 CTB+ cells that were GFP+ in Colgalt2-bacTRAP (purple) or Gprin3-bacTRAP (green) animals across all of L5b (left), or within each individual sublayer (middle) after pons injections. Right: the percentage (mean ± SEM) of double-labeled cells in M1 of each bacTRAP line following injections into C6. See also Figure S3.

Figure 3.. Loss of Gprin3 cells, but not Colgalt2 cells, in symptomatic SOD1-G93A mice

(A) Schematic of breeding paradigm for SOD1-G93A and bacTRAP animals (top) and the timeline of disease progression used for histology and sequencing (bottom). (B) Immunofluorescent images of M1 from Colgalt2-bacTRAP and Gprin3-bacTRAP animals at the symptomatic time point, showing GFP (green) and the activated astrocyte marker, GFAP (red), across healthy (WT) or disease (SOD) conditions. Scale bar, 100 μm. (C) Frequency distribution of laminar depth of GFAP+-activated astrocytes across healthy (WT, gray, n = 83 cells) and disease (SOD, blue, n = 130 cells) conditions at age >P110. Frequency is reported as absolute number of GFAP+ cells found at each depth. (D) Representative images showing GFP+ cells in M1 of Colgalt2-bacTRAP (top row) or Gprin3-bacTRAP (bottom row) mice at pre-symptomatic (P70) and symptomatic (P110) time points in WT and symptomatic SOD animals. Scale bars, 100 μm. (E and F) Quantification (mean ± SEM) of the number of GFP+ cells along the rostrocaudal axis of M1 at pre-symptomatic (left) and symptomatic (right) time points in healthy (WT, grays) and disease (SOD, blues) conditions for (E) the Colgalt2 bacTRAP line (WT P70 n = 5 animals, SOD P70 n = 8 animals; Colgalt2 WT P110 n = 7 animals, SOD P110, n = 5), and (F) the Gprin3 bacTRAP line (Gprin3 WT P70 n = 8 animals, SOD P70 n = 5 animals; Gprin3 WT P110 n = 7 animals, SOD P110 n = 5 animals). *p < 0.05 by unpaired t test with Holm-Sidak correction for multiple comparisons. See also Figure S4.

Figure 4.. Gprin3 cells show a robust molecular response to SOD1-G93A expression

(A) Bar graphs showing relative expression (mean ± SD CPM) of glial and L5b marker genes across healthy (WT) and diseased (SOD) TRAP samples from Colgalt2 and Gprin3 cells compared with sample-matched M1 input. (B) Volcano plots identifying DE genes between WT and SOD littermates for M1 input (left), Colgalt2 TRAP (middle), and Gprin3 TRAP (right) samples. Genes significantly up-regulated (red) or down-regulated (cyan) in SOD are indicated. Dotted lines represent significance thresholds of 0.0 log₂ fold change (vertical line) and 1.3 −log₁₀ padj (horizontal line). (C) Scatterplot comparing SOD-mediated log₂ fold changes in Colgalt2 TRAP (x axis) and Gprin3 TRAP (y axis) samples for all genes that had mean CPM >100 in Gprin3 cells. Purple and green dots highlight genes that were significantly changed only in Colgalt2 cells or Gprin3 cells, respectively; genes that were changed in both cell types are highlighted in red (up-regulated) or cyan (down-regulated). Dotted lines represent log₂ fold change of ±0.5. Insets show Venn diagrams indicating the total number of genes that were uniquely or commonly upregulated (top right) or downregulated (bottom left) across both cell types. (D) Summary of GO enrichment analysis of DE genes in Colgalt2 TRAP data from (B). (E) Summary of GO enrichment analysis of DE genes in Gprin3 TRAP data from (B). Red bars in (D) and (E) indicate a category that was common to both cell types. See also Figure S5 and Tables S1 and S3.

Figure 5.. Gprin3 cells modulate ribosomal, axon morphogenesis, and synapse structure and function genes in disease

(A) Heatmaps showing relative expression of genes that encode synaptic (left) and axon morphogenesis (right) proteins in M1 input, Colgalt2 TRAP, and Gprin3 TRAP samples across WT and SOD replicates. Values are reported as z-scores for normalized CPM, scaled for each gene. (B) Violin plots of SOD-mediated log₂ fold change values for all synapse (left) and axonal (right) genes that showed a significant change in Gprin3 cells across M1 input, Colgalt2, and Gprin3 cells. (C) Scatterplot showing SOD-mediated log₂ fold changes in Colgalt2 (x axis) and Gprin3 (y axis) TRAP samples for cytosolic ribosomal subunit genes (top, blue and lilac dots) and mitochondrial ribosome genes (bottom, red dots) among all genes that had mean CPM >100 in Gprin3 cells. (D) Violin plots of SOD-mediated log₂ fold change values of cytoplasmic (left) and mitochondrial (right) ribosome genes from (C) across M1 input, Colgalt2 TRAP, and Gprin3 TRAP samples. For (B) and (D), *p < 0.05 and ****p < 0.00001 by one-way ANOVA and subsequent Tukey multiple comparison test. See also Figures S5 and S6.

Figure 6.. SOD1-G93A-mediated regulation of oxidative phosphorylation in layer 5b PT neurons

(A) Heatmap showing relative expression of genes encoding each subunit of the Oxphos complexes in each experimental condition. Values are reported as z-scores for normalized CPM, scaled for each gene. (B) Violin plots of SOD-mediated log₂ fold change values for all Oxphos core subunit and glycolysis genes (top), as well as all mitochondrial transmembrane transport and mitophagy genes (bottom) across M1 input, Colgalt2 TRAP, and Gprin3 TRAP samples. *p < 0.05, ****p < 1 × 10⁻⁷; n.s., not significant by one-way ANOVA and subsequent Tukey multiple comparison test. (C) Scatterplot comparing the enrichment of Oxphos genes in Colgalt2 (x axis) and Gprin3 (y axis) at baseline. Genes enriched in Gprin3 only (green), Colgalt2 only (purple), both L5b cell types (magenta), or depleted from each cell type relative to M1 input (orange) are indicated. See also Figures S6 and S7.

Figure 7.. Oxidative stress response genes are modulated in Gprin3 cells, and hypoxia- and oxidative stress-responsive transcription factors are depleted in healthy Gprin3 cells

(A) Box and whisker plot showing ratio of CPMs of genes that comprise the Oxphos core subunits relative to WT M1 input for WT and SOD datasets across M1 input, Colgalt2 TRAP, and Gprin3 TRAP samples. Gray lines indicate trajectory of change for each individual Oxphos gene. Dotted red line highlights a theoretical threshold above which Oxphos gene expression levels may trigger oxidative stress pathways. (B) Heatmap showing relative expression across all samples of genes involved in oxidative stress and antioxidant responses that were significantly changed in Gprin3 cell. Genes that code for transcription factors are in bolded text. Values are reported as z-scores for normalized CPM, scaled for each gene. (C) Scatterplot comparing enrichment of genes encoding transcription factors associated with activation of hypoxia and oxidative stress response pathways between Gprin3 and. Colgalt2 at baseline (x axis) and Gprin3 cells in disease (y axis). Genes significantly depleted in Gprin3 compared with Colgalt2 and significantly down-regulated in disease (blue), down-regulated in disease but not enriched in either cell type (cyan), depleted in Gprin3 cells but not changed in disease (purple), and up-regulated in disease but not enriched in either cell type (red) are indicated. See also Figure S8.