Extended Data Fig. 10: Layer 2/3 neurons are selectively lost in the grey matter sulcus and do not associate with tau pathology.

a. CUX2/LAMP5 proportion from snRNAseq against total years of football play colored by pathological group. Statistical analysis performed by linear regression, depicted as black line. Test was two-tailed. n = 28 individuals. b. CUX2/LAMP5 density from in situ hybridization compared to highest level of football played. Statistical analysis performed by linear regression. Dots represent single individuals; line shows linear regression. Test was two-tailed. n = 23 individuals. c. CUX2/LAMP5 cells identified by in situ experiment compared to proportion of CUX2/LAMP5 neurons from snRNAseq experiment. Statistical analysis performed by linear regression, depicted as black line with 95% confidence intervals in grey. Test was two-tailed. n = 7 individuals. d. Representative image showing the annotation strategy used to identify the sulcus (yellow line) and crest (red line) layer 2/3. Scale bar = 1 mm. e. (left) Layer 2/3 CUX2+/LAMP5+ neuronal density in the sulcus and crest. Statistical analysis performed by paired t-test. Tests were two tailed. n = 18 individuals. (right) Layer 2/3 CUX2+/LAMP5+ neuronal density in the crest compared to years of football play, colored by pathological group. Statistical analysis performed by linear regression. Black line represents general linear model regression, grey shows 95% confidence interval. Tests were two tailed. n = 18 individuals. f. Total CUX2 populations and subpopulations in snRNAseq (right panel) and in situ hybridization (left panel) experiments compared to years of football play. Statistical analysis performed by linear regression and represented by lines. Tests were two-tailed. left panel n = 22 individuals, right panel n = 28 individuals. g, h, i. Scatter plots depicting (g) Sulcus layers 4–6 Nissl+ density compared to binned years of football play, (h) Crest layer 2/3 Nissl+ density compared to binned years of football play, (i) L2/3 Nissl+ density compared to log tau+ density, Statistical analysis performed by linear regression, (g, h) corrected for age and staining batch. n = 86 individuals. j. CUX2+/LAMP5+ density in situ compared to log AT8+ tau+ density, Colored by pathological group. Statistical analysis performed by linear regression. Test were two-tailed. Black line represents general linear model regression, grey shows 95% confidence interval. n = 22 individuals. k, l. Representative images of Nissl staining across cortical layers depicting neuronal loss in superficial layers in RHI and CTE individuals. Scale bars, 100 μm.