Targeting Alzheimer's Disease Neuro-Metabolic Dysfunction with a Small Molecule Nuclear Receptor Agonist (T3D-959) Reverses Disease Pathologies

Abstract

Background: Alzheimer's disease (AD) could be regarded as a brain form of diabetes since insulin resistance and deficiency develop early and progress with severity of neurodegeneration. Preserving insulin's actions in the brain restores function and reduces neurodegeneration. T3D-959 is a dual nuclear receptor agonist currently in a Phase 2a trial in mild-to-moderate AD patients (ClinicalTrials.gov identifier NCT02560753). Herein, we show that T3D-959 improves motor function and reverses neurodegeneration in a sporadic model of AD.

Methods: Long Evans rats were administered intracerebral (i.c.) streptozotocin (STZ) or normal saline (control) and dosed orally with T3D-959 (1.0 mg/kg/day) or saline for 21 or 28 days. Rotarod tests evaluated motor function. Histopathology with image analysis was used to assess neurodegeneration.

Results: T3D-959 significantly improved motor performance, and preserved both cortical and normalized white matter structure in i.c STZ-treated rats. T3D-959 treatments were effective when dosed therapeutically, whether initiated 1 day or 7 days after i.c. STZ.

Conclusion: T3D-959's targeting neuro-metabolic dysfunctions via agonism of PPAR delta and PPAR gamma nuclear receptors provides potential disease modification in AD.

Keywords: Alzheimer; Cerebellum; Motor function; Neurodegeneration; PPAR agonist; Streptozotocin; T3D-959; Type 3 diabetes.

Figures

Figure 1

Image analysis approach. (A) Histological sections of the cerebellar vermis were stained with LHE to highlight the cerebellar cortex and white matter. Image J/Fiji software was used to measure (highlighted in red) (B) Total cross-sectional areas of the vermis, (C) The molecular layer, (D) Granule cell layer, and (E) White matter.

Figure 2

T3D-959 rescue of i.c. STZ-mediated of motor impairment. Long Evans rats were administered i.c. STZ or normal saline (Control). The control rats and a subset of the i.c. STZ rats were gavage with saline. Two other sub-groups of i.c. STZ rats were gavage with a single daily dose of 1.0 mg/kg of T3D-959 beginning 1 day or 7 days after the i.c. STZ injections. Sixteen days later, the rats were subjected to Rotarod testing with 10 incremental speed trials. Latency to fall was measured optically. Data culled from Trials (A) 1–4, (B) 5–6, and (C) 8–10 are depicted in the graphs (mean ± S.E.M. per group/trial). Inter-group comparisons were made using the Mann-Whitney test. *P

Figure 3

Effects of T3D-959 on i.c. STZ-induced alterations in cerebellar structure. Long Evans male rats (4-weeks old) were used to generate a sporadic model of AD by i.c. STZ. The rats (n=6–8 per group) were treated with saline or 1 mg/kg of T3D-959 by daily oral gavage. Rats were sacrificed on Experimental day 28. (A) Fresh cerebellar weights in rats treated with i.c. saline (Con), ic-STZ (STZ), T3D-959 beginning 1 day after i.c. STZ (STZ T3D-1), or T3D-959 beginning 7 days after i.c.-STZ (STZ T3D-7). After formalin-fixation, cerebella were cut in the mid-sagittal plane and embedded in paraffin. Histological sections (4 μm thick) stained with hematoxylin and eosin (H&E) were used for image analysis (Image-J) to measure relative cross-sectional areas (percentages of overall area) of the (B) molecular layer, (C) granule cell layer, and (D) white matter. All graphs depict the mean ± S.E.M. of results. Inter-group comparisons were made by one-way ANOVA with the Tukey post-hoc significance test. F-ratios are indicated in the panels. Significant inter-group differences are specified.

Figure 4

T3D-959 Prevents STZ-induced neurodegeneration. H&E stained sections of the cerebellar vermis from rats treated with (A, E) i.c. saline or (B–D, F–H) i.c. STZ, and then treated with (A, B, E, F) vehicle (saline) or (C, D, G, H) 1 mg/kg/day T3D by gavage. T3D after delays of (C, G) 1 day or (D, H) 7 days. Rats were sacrificed 4 weeks after i.c. STZ or vehicle injections. Cerebella were fixed in formalin, embedded in paraffin and sectioned (4 μthick). (A, E) Control cerebella had relatively thick and uniform molecular (black bars) and granule cell (red bars, yellow ‘G’), (E) well populated Purkinje cell layers (black arrows), and uniform white matter cores (green bars). Relative to control, i.c. STZ treatment caused cortical atrophy with (B) thinning of the molecular and (B, F) granule cell layers, (F) loss (bracket) and on-going necrosis (red arrows) of Purkinje cells, and modest reductions in white matter thickness. (C, D, G, H) T3D-959 normalized or expanded the molecular layer and increased white matter core thickness, reduced cell loss and necrosis in the Purkinje layer, but did not reverse the irregular thinning and neuronal loss in the granule cell layer. Original magnification: (A–D) 100x; (E–H) 425x.

Figure 5

Semi-quantitative grading of cerebellar histopathology. H&E stained sections of cerebellar vermis were scored under code using semi-quantitative assessments of the uniformity and thickness of the molecular layer, cellularity within the Purkinje layer, and thickness and cellularity of the granule cell layer (Table 1). The mean ± S.D. scores corresponding were compared across experimental groups by two-way ANOVA (results tabulated below graph) with the Tukey post-hoc multiple comparisons test (*P