Targeting Alzheimer's disease with multimodal polypeptide-based nanoconjugates

Abstract

Alzheimer's disease (AD), the most prevalent form of dementia, remains incurable mainly due to our failings in the search for effective pharmacological strategies. Here, we describe the development of targeted multimodal polypeptide-based nanoconjugates as potential AD treatments. Treatment with polypeptide nanoconjugates bearing propargylamine moieties and bisdemethoxycurcumin or genistein afforded neuroprotection and displayed neurotrophic effects, as evidenced by an increase in dendritic density of pyramidal neurons in organotypic hippocampal culture. The additional conjugation of the Angiopep-2 targeting moiety enhanced nanoconjugate passage through the blood-brain barrier and modulated brain distribution with nanoconjugate accumulation in neurogenic areas, including the olfactory bulb. Nanoconjugate treatment effectively reduced neurotoxic β amyloid aggregate levels and rescued impairments to olfactory memory and object recognition in APP/PS1 transgenic AD model mice. Overall, this study provides a description of a targeted multimodal polyglutamate-based nanoconjugate with neuroprotective and neurotrophic potential for AD treatment.

Fig. 1. Schematic representation of the polymeric structures used within this study.

(i, vi, and ix) N,N′-diisopropylcarbodiimide/4-dimethylaminopyridine, N,N-dimethylformamide, 72 hours, room temperature. (ii, iv, vii, and x) 4-(4,6-Dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride, cysteamine pyridyl dithiol, H₂O, 24 hours, room temperature. (iii, v, viii, and xi) Hepes buffer (pH 7.4), 16 hours, room temperature.

Fig. 2. Neural cell uptake of St-Cl-Pr-Cy5.5-ANG after passage through the BBB.

(A) Schematic of an adult mouse brain representing the two antero-posterior coronal levels studied for the presence of St-Cl-Pr-Cy5.5-ANG. AON, anterior olfactory nucleus; Ctx, cerebral cortex; CB, cerebellum; CC, corpus callosum; HP, hippocampus; LV, lateral ventricle; OB, olfactory bulb. (B) The presence of St-Cl-Pr-Cy5.5-ANG in the cerebral cortex, showing abundant accumulation in the blood vessel (BV) walls and surrounding GFAP⁺ astrocytes (left), and in punctate regions (arrows) in the cytoplasm of NeuN⁺ neurons (middle) and Iba1⁺ microglia (right). (C and D) Distribution of St-Cl-Pr-Cy5.5-ANG (arrows) in GFAP⁺ astrocytes (left), NeuN⁺ neurons (middle), and Iba1⁺ microglia (right) in the granule cell layer (GCL) of the dentate gyrus of the hippocampus (C) and the olfactory bulb (D). (E) Quantification of Cy5.5 fluorescent intensity between different brain regions at short (30 min) and long (4 hours) time points after intravenous administration of St-Cl-Pr-Cy5.5-ANG. All data shown are relative fluorescence intensity values corrected for background autofluorescence of the same brain regions in vehicle-injected mice (means ± SEM; n = 4 mice per time point). Two-way ANOVA followed by Šidák’s post hoc test, *P < 0.05. (F) St-Cl-Pr-Cy5.5-ANG (arrows) localizes to lysosomes (Lamp1, green) in cells of the cerebral cortex (top) and olfactory bulb (bottom). Scale bars, 20 μm in panoramic images and 10 μm in insets.

Fig. 3. In vitro cell viability, drug release, and fibril formation inhibition capacity of St-Cl-Pr-BDMC.

(A) Cell viability of BDMC and St-Cl-Pr-BDMC in the SH-SY5Y cell line as measured by MTS cell viability assay. n > 3, means ± SEM. (B) Cell viability of St-Cl-Pr-BDMC and St-Cl-Pr-BDMC-ANG in primary neural culture as measured by PI-labeled nuclei. n > 3, means ± SEM. (C) Drug release profiles at different pH values (5.0, 6.5, and 7.4) for St-Cl-Pr-BDMC. Time course experiments were carried out in triplicate. n > 3, means ± SEM. (D) ThT fluorescence intensity changes over time in HEWL samples incubated with BDMC and St-Cl-Pr-BDMC at 10 μM BDMC-equivalents n > 3, means ± SEM. a.u., arbitrary units. (E) Quantification of fibril length obtained by analyzing TEM micrographs using ImageJ. Photo credit: Aroa Duro-Castaño, CIPF. (F) Image depicts an example of unimers and fibrils used in the experiments. (G) Images of HEWL unimers and HEWL fibrils upon heating at 60°C and vigorous stirring for 24 hours (pH 2.0) (20). TEM image visualization of the effect of free BDMC, St-Cl-Pr-ANG and St-Cl-Pr-BDMC-ANG on HEWL fibril formation or disruption. Scale bars, 500 nm, in all cases, apart from HEWL fibrils (second-down), 200 nm. Photo credit: I. Conejos-Sánchez (CIPF).

Fig. 4. Analysis of St-Cl-Pr-BDMC capability to inhibit Aβ-induced neurotoxicity and to induce a neurotrophic effect in hippocampal organotypic cultures.

(A) Changes in density (nuclei/1000 μm²) of PI-stained nuclei in the pyramidal layer of the CA1 region of hippocampal organotypic cultures comparing control cultures treated with vehicle and cultures treated with different concentrations of St-Cl-Pr-BDMC (0.005, 0.05, 0.2, and 0.5 μM drug-equivalents). Asterisks indicate statistically significant differences after ANOVA analyses followed Bonferroni’s post hoc tests. n > 3, means ± SEM (20). (B) Changes in the density (nuclei/1000 μm²) of PI-stained nuclei in the pyramidal layer of the CA1 region of hippocampal organotypic cultures when comparing control cultures treated with vehicle and cultures treated with the polymer St-Cl-Pr, St-Cl-Pr-BDMC, or free BDMC (0.05 μM). The distinct groups were subsequently treated with vehicle (no Aβ) or Aβ_1–42 peptide (Aβ). Asterisks indicate statistically significant differences after ANOVA analyses followed Bonferroni’s post hoc tests. n > 3, means ± SEM (20). (C) Graph representing the changes in dendrite density of CA1 pyramidal neurons measured in the stratum radiatum (optical density, arbitrary units) of hippocampal organotypic cultures when compared with control cultures treated with St-Cl-Pr, St-Cl-Pr-BDMC, or free BDMC (0.05 μM). The distinct groups were subsequently treated with vehicle (no Aβ) or Aβ_1–42 peptide (Aβ). Asterisks in bars indicate statistically significant differences between groups after ANOVA analyses followed by Tukey or Games-Howell post hoc tests. (D) Confocal microscopic analysis of dendrite density of pyramidal neurons in the stratum radiatum of CA1 of hippocampal organotypic cultures. All microphotograph images are derived from single confocal planes. Scale bar, 25 μm.

Fig. 5. In vivo activity of LRP1-targeted conjugates bearing BDMC or genistein in AβPP/PS1 Alzheimer’s murine model.

(A and B) Aβ40 or Aβ42 levels determined in whole brain homogenates by enzyme-linked immunosorbent assay (ELISA). (C) Plasma leptin levels determined by the Quantikine ELISA Leptin Immunoassay Kit. (D) Hippocampal learning evaluated using the Hebb-Williams maze. (E) Recognition memory studied using the novel object recognition test. (F) Olfactory deficits assessed by the odor habituation test. Data are expressed as means ± SD. Significance is shown as *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, and ****P < 0.0001 versus TG, n = 5 to 10 animals per group.