Gene Profiling of Nucleus Basalis Tau Containing Neurons in Chronic Traumatic Encephalopathy: A Chronic Effects of Neurotrauma Consortium Study

Abstract

Military personnel and athletes exposed to traumatic brain injury may develop chronic traumatic encephalopathy (CTE). Brain pathology in CTE includes intracellular accumulation of abnormally phosphorylated tau proteins (p-tau), the main constituent of neurofibrillary tangles (NFTs). Recently, we found that cholinergic basal forebrain (CBF) neurons within the nucleus basalis of Meynert (nbM), which provide the major cholinergic innervation to the cortex, display an increased number of NFTs across the pathological stages of CTE. However, molecular mechanisms underlying nbM neurodegeneration in the context of CTE pathology remain unknown. Here, we assessed the genetic signature of nbM neurons containing the p-tau pretangle maker pS422 from CTE subjects who came to autopsy and received a neuropathological CTE staging assessment (Stages II, III, and IV) using laser capture microdissection and custom-designed microarray analysis. Quantitative analysis revealed dysregulation of key genes in several gene ontology groups between CTE stages. Specifically, downregulation of the nicotinic cholinergic receptor subunit β-2 gene (CHRNB2), monoaminergic enzymes catechol-O-methyltransferase (COMT) and dopa decarboxylase (DDC), chloride channels CLCN4 and CLCN5, scaffolding protein caveolin 1 (CAV1), cortical development/cytoskeleton element lissencephaly 1 (LIS1), and intracellular signaling cascade member adenylate cyclase 3 (ADCY3) was observed in pS422-immunreactive nbM neurons in CTE patients. By contrast, upregulation of calpain 2 (CAPN2) and microtubule-associated protein 2 (MAP2) transcript levels was found in Stage IV CTE patients. These single-population data in vulnerable neurons indicate alterations in gene expression associated with neurotransmission, signal transduction, the cytoskeleton, cell survival/death signaling, and microtubule dynamics, suggesting novel molecular pathways to target for drug discovery in CTE.

Keywords: CTE; TBI; basal forebrain; brain trauma; expression profiling; genes.

FIG. 1.

Box plot showing the percentage of pS422-positive relative to total p75^NTR -positive nucleus basalis of Meynert (nbM) neurons in chronic traumatic encephalopathy (CTE) Stages II, III, and IV. Percentage of pS422-positive neurons was significantly increased in Stage IV compared with Stage II but not Stage III (Kruskal–Wallis follows Dunn's post-hoc test; *p < 0.01).

FIG. 2.

Low power images showing pre (A, C, E) and post (B, D, F) laser capture microdissection (LCM) extraction of pS422-positive nucleus basalis of Meynert (nbM) neurons in a chronic traumatic encephalopathy (CTE) stage II, III, and IV subject. Black ovals outline the location of pS422-positive nbM after LCM retrieval (B, D, E). All sections were counterstained with Cresyl Violet. Scale bar = 50 μm.

FIG. 3.

Linear representation showing the mean values of the gene expression level changes for neurotransmission (DDC, CHRNB2, COMT) (A), cytoskeleton/cell metabolism (CAPN2, LIS1, MAP2) (B), and cell homeostasis/endocytosis and intracellular signaling (CLN5, CLN4, CAV1, ADCY3) (C) transcript across CTE stages. Note that MAP2 and CAPN2 mRNA levels were upregulated in CTE Stage IV compared with Stages II (p = 0.019) and III (p = 0.004), respectively, whereas the expression of the other genes was downregulated during the pathological progression of CTE (one way analysis of variance [ANOVA]). Holm–Sidak comparisons indicated by: *II vs. III; #II vs. IV, and $III vs. IV. p < 0.05.

FIG. 4.

Histogram illustrating gene expression for neurotransmission (DDC, CHRNB2, COMT) (A), cytoskeleton/cell metabolism (CAPN2, L1S1, MAP2) (B), and cell homeostasis/endocytosis and intracellular signaling (CLN5, CLN4, CAV1, ADCY3) (C) transcripts in non-tangle-bearing nucleus basalis of Meynert (nbM) p75^NTR-positive neurons across chronic traumatic encephalopathy (CTE) stages. Note that mRNA levels were unchanged in non-tangle-bearing neurons across the CTE stages (one way analysis of variance [ANOVA]). p < 0.05.

FIG. 5.

CLCN5 in-situ hybridization images showing differential labeling of nucleus basalis of Meynert (nbM) neurons from chronic traumatic encephalopathy (CTE) Stage II (A) and Stage IV (B) subjects. Note that there are many more CLCN5 mRNA copies (brown dots; arrows) in CTE Stage II than in CTE Stage IV. Sections were counterstained with hematoxylin. Scale bar = 10 μm.

FIG. 6.

DDC transcript levels correlated positively with COMT (A) and CHRNB2 (B) expression l within the pS422-positive nucleus basalis of Meynert (nbM) across chronic traumatic encephalopathy (CTE) stages. Likewise, CAV1 mRNA levels correlated positively with ADCY3 (C) and LIS1 (D) levels in CTE. MAP2 (E) transcript levels correlated positively with the age at which sport began, whereas CHRNB2 (F) correlated negatively with years of nplay in CTE. Stage II, red circles; Stage III, blue circles; Stage IV, green circles.