The Neurotoxin DSP-4 Dysregulates the Locus Coeruleus-Norepinephrine System and Recapitulates Molecular and Behavioral Aspects of Prodromal Neurodegenerative Disease

DSP-4 induces degeneration of noradrenergic terminals and oxidative stress but leaves LC cell bodies intact. Mice received saline or DSP-4 (2 × 50 mg/kg, i.p.) and assessed for locus coeruleus (LC) neuron damage one week later. a, DSP-4 results in substantial loss of axon terminals as measured by norepinephrine transporter (NET) immunoreactivity in the dentate gyrus (DG) and anterior cingulate cortex (ACC), with a similar decrease in NET also present in the LC itself. b, Representative images of silver-stained brain tissue indicates neurodegenerative processes in the LC, ACC, and DG following DSP-4. c, The oxidative stress marker 3-nitrotyrosine (3-NT) was increased in the ACC and DG but decreased in the LC by DSP-4. d, Despite NE fiber damage and the evidence of neurodegenerative and oxidative processes following DSP-4, LC cell body number was unaffected as measured by TH and NeuroTrace Nissl immunoreactivity. Images acquired at 20×. Data shown as mean ± SEM, N = 3–4 per group; **p < 0.01, ***p < 0.001, ****p < 0.0001.

DSP-4 alters astrocyte and microglia activation across the LC-NE system. Mice received saline or DSP-4 (2 × 50 mg/kg, i.p.) and assessed for neuroinflammation one week later. a, Astrocyte reactivity, as measured by GFAP immunostaining, was significantly increased in the locus coeruleus (LC) with a trend in the dentate gyrus (DG) following DSP-4 treatment, while the anterior cingulate cortex (ACC) showed a decreased astrocytic response. b, Microglial response, indicated by Iba-1 immunoreactivity, was increased across all regions assessed. Images acquired at 20×. Data shown as mean ± SEM, N = 3–4 per group; *p < 0.05, **p < 0.01.

DSP-4 triggers changes in the LC transcriptome. Mice received saline or DSP-4 (2 × 50 mg/kg, i.p.) and LC gene expression was assessed one week later. a, TRAP allows for purification of mRNA from LC neurons through immunoprecipitation (TRAP, gray), resulting in enrichment of noradrenergic genes compared with mRNA from the entire hindbrain sample (input, black). b, Differential gene expression (DGE) was assessed between DSP-4 (blue) and saline control (gray) TRAP samples, and revealed that noradrenergic-specific genes, including galanin (Gal), norepinephrine transporter (Slc6a2), dopamine β-hydroxylase (Dbh), and tyrosine hydroxylase (Th), were among the most significantly and robustly downregulated transcripts in the LC of DSP-4 treated mice. Data for a and b shown as mean ± SEM, N = 2–6 per group. c, List of top 15 downregulated and top 15 upregulated DGE, sorted by fold change (logFC) with p < 0.05. d, Volcano plot of all filtered, normalized genes (∼11,500) with genes from WGCNA-defined module in red. Labeled genes from this module are those of interest based on published connections to neurodegenerative disease. e, Volcano plots (as shown in d) highlighting genes from three significantly enriched KEGG pathways in our LC data, with GSEA-identified “core enrichment genes” colored magenta, and remaining pathway genes colored light pink.

DSP-4 treatment does not alter baseline or footshock-induced LC activity. Mice received saline or DSP-4 (2 × 50 mg/kg, i.p.) and assessed for locus coeruleus (LC) neuron firing under anesthesia one week later. No differences were found for (a) baseline tonic firing rates (in Hertz). b, There was no main effect of treatment on footshock-evoked firing rates of LC neurons 0–60, 60–100, or 200–400 ms following the stimulus. Data shown as mean ± SEM, N = 5 mice per group 42–53 neurons/group.