Locus coeruleus vulnerability to tau hyperphosphorylation in a rat model

Abstract

Post-mortem investigations indicate that the locus coeruleus (LC) is the initial site of hyperphosphorylated pretangle tau, a precursor to neurofibrillary tangles (NFTs) found in Alzheimer's disease (AD). The presence of pretangle tau and NFTs correlates with AD progression and symptomatology. LC neuron integrity and quantity are linked to cognitive performance, with degeneration strongly associated with AD. Despite their importance, the mechanisms of pretangle tau-induced LC degeneration are unclear. This study examined the transcriptomic and mitochondrial profiles of LC noradrenergic neurons after transduction with pseudophosphorylated human tau. Tau hyperphosphorylation increased the somatic expression of the L-type calcium channel (LTCC), impaired mitochondrial health, and led to deficits in spatial and olfactory learning. Sex-dependent alterations in gene expression were observed in rats transduced with pretangle tau. Chronic LTCC blockade prevented behavioral deficits and altered mitochondrial mRNA expression, suggesting a potential link between LTCC hyperactivity and mitochondrial dysfunction. Our research provides insights into the consequences of tau pathology in the originating structure of AD.

Keywords: L‐type calcium channel; electron microscopy; locus coeruleus; mitochondria; single nuclei RNA sequencing; tau.

FIGURE 1

Human pretangle tau mimic in the rat locus coeruleus results in impaired spatial and olfactory discrimination learning. (a) Schematic diagram demonstrating the experimental flow. (b) Construct of htauE14. Human tau protein contains four domains as shown. The presence or absence of exons 2, 3 at the N‐terminal and exon 10 (E10) at the microtubule‐binding domain, form six isoforms of tau. We used 0N4R isoform of human tau with 14 sites of pseudophosphorylation, as shown. The majority of the phosphorylation sites are within the proline‐rich domain. (c) Example of AAV uptake in the LC, indexed by GFP staining. The inserts show enlargement of the LC. Red arrowheads indicate cannula tracks. (d). Example of a brainstem section containing dopamine β‐hydroxylase (DBH⁺) cells (red) in the LC. Scale bar, 500 μm. (e) GFP (green) and DBH (red) double‐labeled noradrenergic neurons in the LC. Scale bar, 50 μm. (f) Tyrosine hydroxylase (TH) expression in one GFP and three htauE14 (briefly E14) LCs. (g) Human tau HT7 expression in the htauE14 LC. (h, i) Expression of phosphorylated tau at S262 (h) and S356 (i) in the htauE14 LC. In panels (f–i) the molecular weight label is overlaid on the image, separated from the gel bands by a black box. (J1‐J3) Distance traveled (J1), percentage of time spent freezing (J2) and percentage of time spent rearing (J3) in an open‐field test. (k) Percentage of time spent in the close arm of an elevated maze. (l) Number of marbles buried. (m) Percentage of sucrose water consumed. (n) Discrimination index in a spontaneous location recognition test. (o) Discrimination index in an odor discrimination test. N = 3F/5M (GFP), 7F/1M (htau) and 4F/5M (htauE14). *p < 0.05; **p < 0.01.

FIGURE 2

HtauE14 incubation in the LC leads to impaired mitochondrial morphology. (a). Example electron microscopy images of mitochondria showing intact (yellow arrows), mildly deranged (magenta arrow) and severely deranged (red arrow) mitochondria. Scale bars, 500 nm. (b) Index of cristae derangement. (c) Percentage of mitochondria with broken membranes. (d) Percentage of mitochondria of various shapes. (e) Numbers of mitochondria per cell. (f) Mean area of mitochondria per cell. (g) Distance to the nucleus. (h) Percentage of mitochondria showing pinching. (i) Correlation between cristae derangement and spontaneous location recognition (SLR) performance. (j) Examples images of L‐type calcium channel subunit Cav1.2 staining and GFP‐expressing cells. Scale bar, 50 μm. (k) Relative optical density (ROD) of Cav1.2 in GFP vs. htauE14 rats. N = 3F/2M (GFP), 7F/1M (htau) and 2F/3M (htauE14). *p < 0.05; **p < 0.01.

FIGURE 3

Single nuclei RNA sequencing demonstrates sex‐dependent differentially expressed genes in the LC induced by htauE14. (a) Schematic diagram demonstrating the experimental flow. (b) Cell‐type specific clustering of 76,178 nuclei from 12 samples (6 GFP and 6 htauE14, with 3 biological replicates for each sex). (c) Expression levels of canonical gene markers in each cell cluster. (d–f) Volcano plots displaying differentially expressed genes in all samples (d), female only (e) and male only (f) samples. Grey dots represent non‐significant (NS) data. Green dots indicate data with absolute log2 fold change (Log2FC) >0.50. Red dots represent data with adjusted p‐value (Adj‐p‐Val) <0.05 and absolute log2FC >0.50. F, female. M, Male. (g) Gene oncology (GO) analysis showing biological processes enriched in adult male htauE14 LC. FDR, false discovery rate.

FIGURE 4

Chronic LTCC blockade by nimodipine prevents learning deficits and alters LC mRNA expression. (a) Schematic diagram demonstrating the experimental flow. (b, c) Spatial (b) and odor (c) discrimination index in adult rats. Veh, vehicle. Nimo, nimodipine. N = 3F/3M (GFP + Veh), 2F/4M (htauE14 + Veh) and 4F/4M (htauE14 + nimodipine). (d, e) Spatial (d) and odor (e) discrimination index in the aged rats. N = 4F/2M (GFP + Veh), 3F/3M (htauE14 + Veh) and 3F/4M (htauE14 + nimodipine). (f) Heatmap showing fold changes of various gene markers among groups in adult rats by qPCR measurement. Genes related to mitochondrial function are highlighted in green. (g) Heatmap showing fold changes of various gene markers among groups in aged rats by qPCR measurement. Genes related to mitochondrial function are highlighted in green. N = 3–6 for each group per marker, sex mixed. *p < 0.05; **p < 0.01.