A noradrenergic lesion exacerbates neurodegeneration in a Down syndrome mouse model

Abstract

Individuals with Down syndrome (DS) acquire Alzheimer's-like dementia (AD) and associated neuropathology earlier and at significantly greater rates than age-matched normosomic individuals. However, biological mechanisms have not been discovered and there is currently limited therapy for either DS- or AD-related dementia. Segmental trisomy 16 (Ts65Dn) mice provide a useful model for many of the degenerative changes which occur with age in DS including cognitive deficits, neuroinflammation, and degeneration of basal forebrain cholinergic neurons. Loss of noradrenergic locus coeruleus (LC) neurons is an early event in AD and in DS, and may contribute to the neuropathology. We report that Ts65Dn mice exhibit progressive loss of norepinephrine (NE) phenotype in LC neurons. In order to determine whether LC degeneration contributes to memory loss and neurodegeneration in Ts65Dn mice, we administered the noradrenergic neurotoxin N-(2-chloroethyl)-N-ethyl-2-bromobenzylamine (DSP-4; 2 doses of 50 mg/kg, i.p.) to Ts65Dn mice at four months of age, prior to working memory loss. At eight months of age, Ts65Dn mice treated with DSP-4 exhibited an 80% reduction in hippocampal NE, coupled with a marked increase in hippocampal neuroinflammation. Noradrenergic depletion also resulted in accelerated cholinergic neuron degeneration and a further impairment of memory function in Ts65Dn mice. In contrast, DSP-4 had minimal effects on normosomic littermates, suggesting a disease-modulated vulnerability to NE loss in the DS mouse model. These data suggest that noradrenergic degeneration may play a role in the progressive memory loss, neuroinflammation, and cholinergic loss occurring in DS individuals, providing a possible therapeutic avenue for future clinical studies.

Fig. 1

Locus Coeruleus neurons degenerate in Ts65Dn mice with aging. A–H) TH immunostaining of the LC demonstrated that at 4 mo NS mice (A) showed only mildly stronger staining than in Ts65Dn mice (B), while in 12 mo animals, Ts65Dn mice (D, F, H) exhibited altered neuronal morphology, axonal swellings, and fewer axonal processes relative to NS mice (C, E, G). I–J) A reduction in TH-positive neurons was present at 12 mo (J) but not at 4 mo (I) of age in Ts65Dn mice as evidenced by stereological counting. K, L) An analysis of the rostrocaudal distribution of TH neurons in the LC indicated that neuron reductions in Ts65Dn mice occurred primarily in rostral LC (mean ± SEM **p < 0.01; n = 5 per group for 4 mo, n = 9–10 per group for 12 mo). L = ratio of Ts65Dn versus normosomic mice at each portion of the LC, rostral to caudal.

Fig. 2

Altered Calbindin D-28k morphology with age in Ts65Dn mice. A–F) Cal 28k immunostaining of the hippocampus in 4 mo (A, B), 12 mo (C, D), and 18 mo (E, F) mice demonstrates the effects of aging on hippocampal morphology in NS (A, C, E) and Ts65Dn (B, D, F) mice. G) Densitometric analysis revealed that overall Cal 28k-IR in CA1 pyramidal neurons of the hippocampus was reduced with age in Ts65Dn mice, while density in NS mice remained stable. H) Cal 28-IR in the dentate gyrus was not altered at 18 mo (mean ± SEM; ⁺p = 0.08; ***p < 0.001; n = 5 per group for 4 mo, n = 9–10 for 12 mo, n = 4 for 18 mo).

Fig. 3

NE depletion further disrupted spatial memory function and activity in Ts65Dn mice. A) Performance in the WRAM is indicated by errors per trial across days (mean ± SEM, *p < 0.05 relative to NS Sal, ^{^}p < 0.05 relative to Ts65Dn Sal). B) Analysis of errors across blocks (made up of three trials) demonstrate performance within each day of testing, and indicate that DSP-4 treatment alters “overnight remembering” on the first block of testing of days 2 and 3. C) Total activity was measured over a one-hour session (distance traveled, cm). D) Time spent in the center region, as a percentage of total time, showing that Ts65Dn mice spent less time in the center relative to NS mice (mean±SEM, *p < 0.05 relative to NS Sal; NS Sal n = 8; NS DSP-4 n = 8; Ts65Dn Sal n = 6; Ts65Dn DSP-4 n = 5).

Fig. 4

DSP-4 administration resulted in a long-term NE depletion. HPLC analysis of monamines showed reduced NE levels in DSP-4 treated mice regardless of genotype, with 50% reductions in the frontal cortex and cerebellum and a 75% loss in the hippocampus. DSP-4 did not alter levels of other monoamines, such as serotonin (5-HT), dopamine, or their metabolites (data not shown).

Fig. 5

DSP-4 administration induced LC degeneration. A–H) TH immunostaining of NS Sal (A, E) and Ts65Dn Sal (C, G) mice revealed that saline did not disrupt noradrenergic neurons of the LC, while NS DSP-4 (B, F) and Ts65Dn DSP-4 (D, H) mice showed significant reductions in TH-positive nerve fibers and LC neurons. E–H represents a higher magnification of images shown in A–D, and the inset in H shows axonal swellings in the Ts65Dn DSP-4 group, representing neurite degeneration frequently observed in this group. I, J) Densitometry confirmed that TH-immunoreactivity was significantly reduced following DSP-4 injection (I), while stereologic cell counts showed a 20% loss in LC neurons (J; mean ± SEM; *p < 0.05).

Fig. 6

Microglial activation was increased in Ts65Dn mice treated with DSP-4. A–D) Microglial immunostaining in the hippocampus with CD45, a pan-microglial marker, showed that NE depletion in NS mice did not induce enhanced microglial activity (NS Sal, A; NS DSP-4, B), while alterations in microglial morphology in Ts65Dn Sal mice (C) were aggravated by DSP-4 (Ts65Dn DSP-4, D). E) Densitometry revealed a significant increase in microglial immunoreactivity in the hippocampus of Ts65Dn DSP-4 mice compared to Ts65Dn Sal mice (mean ± SEM; *p < 0.05 and **p < 0.01 compared to NS Sal; ^p < 0.05 compared to Ts65Dn Sal). F) IL-1β mRNA analysis by quantitative RT-PCR in the hippocampus showed a step-wise increase in the levels of this pro-inflammatory cytokine in Ts65Dn treatment groups with or without DSP-4 lesions.

Fig. 7

Cal 28k was affected by DSP-4 administration in Ts65Dn mice. A–C) Cal 28k immunostaining of NS Sal (A), Ts65Dn Sal (B), and Ts65Dn DSP-4 (C) showed that the distribution of Cal 28k was altered in the CA1 region of Ts65Dn DSP-mice, with greater loss in pyramidal neurons (arrowhead) than in interneurons of stratum oriens (arrow). D) Densitometry confirmed decreased Cal 28k-IR in the Ts65Dn DSP-4 group (mean ± SEM, *p < 0.05 relative to NS Sal). E) Hippocampal gene expression of BDNF mRNA was reduced in the Ts65Dn saline and DSP-4 groups compared to NS groups (mean ± SEM; *p < 0.05).

Fig. 8

Reduced cell size and number of BFCNs in Ts65Dn mice after DSP-4 administration. A–F) TrkA-positive cholinergic neurons in the MSN are shown in NS Sal (A, D), Ts65Dn Sal (B, E), and Ts65Dn DSP-4 (C, F) at 20× and 40×, respectively. Cell body atrophy was observed in Trk-A I.R. neurons in Ts65Dn mice (E), which was further exacerbated by the DSP-4 lesion (F). Also note that TrkA-I.R. cells in both Ts65Dn gropus exhibit fewer processes and less robust labeling with the TrkA antibody overall. G) Cell size measurements confirmed that Ts65Dn DSP-4 average cell area was significantly lower than in all other groups, including Ts65Dn Sal mice. H) Stereology of TrkA-positive neurons in the MSN depicted a significant reduction in the number of neurons in Ts65Dn DSP-4 mice compared to NS mice (mean ± SEM, *p < 0.05 relative to NS Sal, ^{^}p < 0.05 relative to Ts65Dn Sal).