Long-lasting rescue of age-associated deficits in cognition and the CNS cholinergic phenotype by a partial agonist peptidomimetic ligand of TrkA - PubMed (original) (raw)

Comparative Study

Long-lasting rescue of age-associated deficits in cognition and the CNS cholinergic phenotype by a partial agonist peptidomimetic ligand of TrkA

Martin A Bruno et al. J Neurosci. 2004.

Abstract

Previously, we developed a proteolytically stable small molecule peptidomimetic termed D3 as a selective ligand of the extracellular domain of the TrkA receptor for the NGF. Ex vivo D3 was defined as a selective, partial TrkA agonist. Here, the in vivo efficacy of D3 as a potential therapeutic for cholinergic neurons was tested in cognitively impaired aged rats, and we compared the consequence of partial TrkA activation (D3) versus full TrkA/p75 activation (NGF). We show that in vivo D3 binds to TrkA receptors and affords a significant and long-lived phenotypic rescue of the cholinergic phenotype both in the cortex and in the nucleus basalis. The cholinergic rescue was selective and correlates with a significant improvement of memory/learning in cognitively impaired aged rats. The effects of the synthetic ligand D3 and the natural ligand NGF were comparable. Small, proteolytically stable ligands with selective agonistic activity at a growth factor receptor may have therapeutic potential for neurodegenerative disorders.

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Figures

Figure 1.

Peptidomimetic D3 efficiently penetrates the CNS parenchyma. Images of the lateral ventricle of fresh, unfixed tissue after D3-biotin injection at time points: 15 min (A); 2 hr (B); 4 hr (C); 8 hr (D); 12 hr (E); 24 hr (F). At 2 hr after injection, D3-biotin started to penetrate the CNS parenchyma and gave a maximal signal at the 8 and 12 hr. At 24 hr, no D3-biotin is apparent in the choroid plexus or ventricular surface. Scale bar, 200 μm.

Figure 2.

Neuronal binding by D3 and colocalization with TrkA receptors. Confocal microscopy of parietal cortex lamina V (A, B) or nucleus basalis (C) 24 hr after injection of D3-biotin. Brains were perfused, and sections were immunostained. A, Colocalization of D3-biotin and NeuN. B, Lack of colocalization of D3-biotin and GFAP. C, Colocalization of D3-biotin and TrkA. Scale bar, 20 μm.

Figure 3.

Preselection of cognitively impaired aged rats. A, TrkA partial agonist D3 peptidomimetic. B, Segregation of cohorts of aged impaired rats in the Morris water maze (see Materials and Methods for details). Latency scores were subjected to a two-way ANOVA with two factors: days (within-subject) and group (between-subject). This confirmed that the three groups did indeed differ. The main effects of day and group, and their interaction, were all highly significant (F = 99.0; df 2, 228; p < 0.0001; _F_ = 18.1; df 4, 228; _p_ < 0.0001; _F_ = 10.3; df 8, 228; _p_ < 0.0001, respectively). In the visual platform task (day 6), all three groups reached the platform rapidly. _C_, Graphic representation of the pool divided into three concentric zones (Zone A, 20 cm diameter; Zone B, 45 cm width; Zone C, 15 cm from the outer wall). The location of the escape platform is indicated in the northeast, with its center 45 cm from the outer wall. _D_, The young, aged unimpaired, and aged impaired groups spend similar relative amounts of time in the three zones averaged for days 2-5 of the acquisition phase. In particular, there was no group difference in the proportion of time spent in the outer Zone C, which is a measure of thigmotaxis (_F_ = 0.99; df 2.57; _p_ > 0.3).

Figure 4.

D3 and NGF improve performance in cognitively impaired aged rats. A, Cognitively impaired rats were treated as indicated and then were retested, as described in Materials and Methods, in the Morris water maze on week 4 after initiation of drug delivery. ANOVA of latencies to find the platform revealed significant main effects of group (F = 44.8; df = 2, 15; p < 0.0001) and trial (F = 18.6; df = 6, 90; p < 0.0001). The latencies of D3- and NGF-treated rats were similar in all trials, whereas both groups diverged significantly from the vehicle-treated control group (group × linear trend of trial: F = 4.62; df = 2, 15; p < 0.05). Drug effects were evident, for example, for data pooled across the last four trials (Tukey's test: D3 or NGF vs vehicle, p < 0.0001). B, Schematic graph of the pool showing the quadrants and the position of the submerged escape platform. Data for Figure 4 A were evaluated by quadrant analyses and presented as the percentage of time spent in the trained quadrant (where the platform is located) versus the other three quadrants. C, Analyses for the vehicle, NGF, and D3 groups for trial 1. D, Analyses for the vehicle, NGF, and D3 groups for trial 7. Statistical comparisons refer to Tukey's test.

Figure 5.

D3 and NGF improve memory in cognitively impaired aged rats. A, Typical swim path for individual rats recorded over a 1 min period in week 12. Target quadrant is where the escape platform had been located formerly (northeast quadrant). The summary of quadrant analysis for the vehicle and D3 groups and NGF group at week 5 (B) and week 12 (C) after drug treatment. Statistical comparisons refer to Tukey's test.

Figure 6.

Selective effect of D3 on the cholinergic phenotype of cortical neurons. A, Number (boutons/1000 μm2) ± SEM of cholinergic presynaptic boutons (VAChT immunoreactive). B, Total presynaptic boutons (synaptophysin immunoreactive) in lamina V of the parietal cortex of aged Fischer-344 rats. In A and B, a total area of 80,000 μm2 was analyzed per rat (n = 4 or 5). Aged impaired versus aged impaired plus vehicle were not different from each other but presented a significantly decreased number of cholinergic presynaptic boutons versus young. Restoration of the size in the cholinergic presynaptic boutons in aged impaired plus NGF and aged impaired plus D3 when compared with aged impaired and aged impaired plus vehicle is shown. For synaptophysin staining, there were no significant differences between any groups. C, Representative pictures of lamina V of the parietal cortex immunostained with anti-VAChT antibodies. Statistical comparisons refer to Tukey's test.

Figure 7.

Selective effect of D3 on the size and cholinergic phenotype of nucleus basalis neurons. A, Cell size of the soma (μm2) ± SEM of VAChT-IR cholinergic neurons in the nucleus basalis of Fischer-344 rats. For cell size analyses, a total of 10,000 VachT-positive cells per rat (n = 4 or 5) were studied. Aged impaired and aged impaired plus vehicle were not different from each other but displayed significantly lower cell size than young. Restoration of the cell size of cholinergic neurons after receiving either D3 or NGF when compared with aged impaired and aged impaired plus vehicle. B, Density of VAChT-IR neurons, representative of neuronal numbers in the nucleus basalis. Statistical comparisons refer to Tukey's test. For VAChT density, a total of 50,000 VachT-positive cells per rat (n = 4 or 5) were studied, and data are expressed per 1000 μm2.

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Long-lasting rescue of age-associated deficits in cognition and the CNS cholinergic phenotype by a partial agonist peptidomimetic ligand of TrkA - PubMed (original) (raw)