Sara Montoya

Background: Alzheimer’s Disease (AD) is a progressive neurodegenerative disorder responsible for up to 80% of dementia cases.¹ It impairs language, memory, critical thinking, and problem-solving.² About 55 million people worldwide have AD, a number expected to double every five years.³ Two key pathological features—amyloid-β (Aβ) plaque buildup and hyperphosphorylated tau tangles—damage neural tissue.^1,2 While AD remains incurable, current treatments aim to relieve symptoms.¹ Emerging research targets disease pathology, especially by reducing Aβ aggregation.^4-8 Peptide-based therapeutics show promise due to their high affinity and selectivity for Aβ, but poor blood-brain barrier (BBB) penetration limits their use.^5-7 Cell-penetrating peptides (CPPs), small fragments with diverse physicochemical properties, are widely used to improve intracellular delivery and BBB penetration. As such, CPPs are strong candidates for pairing with peptide-based therapies targeting Aβ in AD.^4-8

Objectives: In this narrative review, we explored the effects of varying protein therapeutic-CPP fusion treatments on Aβ aggregation and BBB penetration, with particular emphasis on the electrostatic mechanisms that govern CPP function.

Search Methods: An online search in the PubMed database was conducted from 2018-2024 using the following keywords: “Alzheimer’s Disease”, “amyloid-beta”, “cell-penetrating peptide”, and “blood-brain barrier penetration”.

Results: NCAM1-PrP inhibits Aβ plaque formation by “promoting off-pathway amorphous aggregation”, as shown by ELISA, and dot blot results, as well as TEM imaging. A high positive charge density adds stability to Aβ:NCAM1-PrP between the cationic residues of the CPP and the aspartic acid residues of Aβ monomers, preventing toxic Aβ oligomerization and plaque formation.⁴ When assessed by ThT and S assay, TTR-PrP showed minimal fluorescence, implying a strong reduction in Aβ aggregation.⁵ Similarly, results from AFM-IR show that TTR-PrP alters the morphology of Aβ aggregates by disbanding oligomers.⁵ When assessed by ThT assay, KTP, a recombinant molecular candidate including Pen, showed the least fluorescence, indicating the most potent inhibition of Aβ aggregation in comparison to other treatment candidates lacking a CPP component. This is similarly attributed to Pen’s cationic nature. ⁶ BBB penetration efficiency of KTP also showed 15.7% penetrance at 3 hours and 35.0% at 6 hours, compared to TTR (2.3% at 3 hours and 6.5% at 6 hours), when assessed by transwell experimentation using a monolayer of bEnd.3 mouse brain cells.⁶ Assessed similarly, TTR-Pen (TP) was shown to exhibit 10 times higher BBB permeability than TTR.⁷ Molecular docking experiments utilizing electrostatic affinity showed greater electrostatic interactions in the TP-Aβ complex (-812 kJ/mol) compared to TTR-Aβ (-584.8 kJ/mol).⁷ These extensive electrostatic interactions between the positively charged TP and negatively charged Aβ species confer greater stability to the Aβ-fusion therapeutic complex, preventing Aβ self-association and aggregation. Lastly, injection of AAV-aMTD-Parkin demonstrated cognitive function recovery rates of 62%, 57%, and 127% at 4,6, and 8 weeks, respectively in AD mice, as assessed by Y-maze behavioral tests.⁸ AAV-aMTD-Parkin also showed effective removal of Aβ plaques, as confirmed by dot blot analysis and immunohistochemistry of post-mortem animal model brain lysate, whereas AAV-Parkin did not show such effects. BBB penetrance is configured by aMTD’s cationic profile.⁸

Conclusions: Each assessed AD treatment candidate targets AD pathology by reducing neurotoxic Aβ aggregation and have BBB penetration abilities across multiple experimental models, a feature which is conferred via electrostatic and membrane-dependent hydrophobic interactions.^4-7 CPP and protein therapeutic variability is complex, yet conjugation of CPP:protein therapeutic yields promising results for future treatment of AD pathology.

Works Cited:

1. Weller J, Budson A. Current understanding of Alzheimer’s disease diagnosis and treatment. F1000Res. 2018;7: F1000 Faculty Rev-1161. Published 2018 Jul 31. doi:10.12688/f1000research.14506.1
2. Breijyeh Z, Karaman R. Comprehensive Review on Alzheimer’s Disease: Causes and Treatment. Molecules. 2020;25(24):5789. Published 2020 Dec 8. doi:10.3390/molecules25245789
3. Twarowski B, Herbet M. Inflammatory Processes in Alzheimer’s Disease-Pathomechanism, Diagnosis and Treatment: A Review. Int J Mol Sci. 2023;24(7):6518. Published 2023 Mar 30. doi:10.3390/ijms24076518
4. Henning-Knechtel A, Kumar S, Wallin C, et al. Designed cell-penetrating peptide inhibitors of amyloid-beta aggregation and cytotoxicity. Cell Reports Physical Science. 2020;1(2):100014. doi:10.1016/j.xcrp.2020.100014
5. Porosk L, Härk HH, Bicep RN, et al. Aggregation Limiting Cell-Penetrating Peptides Derived from Protein Signal Sequences. Int J Mol Sci. 2023;24(5):4277. Published 2023 Feb 21. doi:10.3390/ijms24054277
6. Lan H, Wang Y, Liu W, Dong X, Sun Y. Composite of KLVFF-Transthyretin-Penetratin and Manganese Dioxide Nanoclusters: A Multifunctional Agent against Alzheimer’s β-Amyloid Fibrillogenesis. Molecules. 2024;29(6):1405. Published 2024 Mar 21. doi:10.3390/molecules29061405
7. Wang Y, Liu W, Sun Y, Dong X. Transthyretin-Penetratin: A Potent Fusion Protein Inhibitor against Alzheimer’s Amyloid-β Fibrillogenesis with High Blood Brain Barrier Crossing Capability. Bioconjug Chem. 2024;35(3):419-431.
8. Lee S, Kang M, Lee S, et al. AAV-aMTD-Parkin, a therapeutic gene delivery cargo, enhances motor and cognitive functions in Parkinson’s and Alzheimer’s diseases. Pharmacological Research. 2024;208:107326. Published 2024 Aug 28. doi:10.1016/j.phrs.2024.107326