Inhibition of β-amyloid (1-40) aggregation and neurotoxicity by dendritic polymers

Palanivelu, Manoj Kumar (2012). Inhibition of β-amyloid (1-40) aggregation and neurotoxicity by dendritic polymers PhD Thesis, School of Pharmacy, The University of Queensland.

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Author Palanivelu, Manoj Kumar
Thesis Title Inhibition of β-amyloid (1-40) aggregation and neurotoxicity by dendritic polymers
School, Centre or Institute School of Pharmacy
Institution The University of Queensland
Publication date 2012
Thesis type PhD Thesis
Supervisor Ben Ross
Ross McGeary
Nick Shaw
Total pages 248
Total colour pages 73
Total black and white pages 175
Language eng
Subjects 0601 Biochemistry and Cell Biology
0304 Medicinal and Biomolecular Chemistry
Formatted abstract
Alzheimer’s disease (AD) is an irreversible, progressive neurodegenerative disorder, which gradually leads to cognitive disorder and loss of independence. Amyloid precursor protein (APP) is a type I transmembrane protein, whose proteolysis generates the beta amyloid (Aβ) peptide. According to the amyloid cascade hypothesis, Alzheimer’s disease pathogenesis can be attributed to the abnormal production and deposition of Aβ. Once formed, Aβ self-assembles to β–sheet rich oligomers, filaments/protofibrils, fibrils and amyloid plaques. Glycosaminoglycans (GAGs) such as heparan sulfate (HS), a high molecular weight linear anionic heteropolysaccharide, bind to Aβ and inhibit its proteolytic degradation, thus contributing to the persistence and accumulation of plaques. Current drugs used for AD are symptomatic treatments and there is an urgent need for disease-modifying drugs to halt the progression of AD. Recently, dendrimers, mono-disperse branched polymers, have been shown to inhibit the aggregation of Aβ28 and consequently they are potential disease-modifying drugs. Cationic PAMAM dendrimers consisting of tertiary amines in the core and primary amines on the surface alter the aggregation kinetics of Aβ28 and inhibit fibril formation in the presence of heparin at acidic pH 5.5. It was demonstrated that generation 3 (G3) of PAMAM dendrimer decreased the elongation rate, whereas higher generations of PAMAM dendrimers (G4 and G5) inhibit the fibril formation. Despite the wealth of data on the Aβ28-PAMAM systems, no study has examined the influence of cationic PAMAM dendrimers on Aβ40 aggregation at physiological pH, which is a better model of AD. Similarly, linear poly-L-lysine has been shown to inhibit the formation of amyloid fibrils and to dissolve preformed fibrils. However, the interaction of branched surface capped poly-L-lysine with Aβ40 has not been studied.

The first part of this thesis examines the effect of G1-4 PAMAM dendrimers on Aβ40 aggregation in the presence and absence of heparin at pH 7.4 and 6.5. Thioflavin T (ThT) assay showed that G4 PAMAM dendrimer at pH 6.5 decreased the total fibril formation whereas no such effect was observed at pH 7.4 in the presence and absence of heparin. This difference in activity can be attributed to the high degree of ionisation of G4 PAMAM dendrimer at acidic pH, which enhanced the electrostatic interaction of PAMAM with Aβ40 and interfered with amyloid aggregation resulting in decreased fibril formation. ThT assay results for PAMAM dendrimers reveal the importance of electrostatic interactions playing a major role in inhibition of Aβ aggregation. Therefore, quaternised PAMAM (QPAMAM) dendrimers were synthesised to introduce a permanent cationic charge and their interaction with Aβ40 was investigated. This study involved the synthesis, purification, characterisation and in vitro evaluation of QPAMAM by ThT assay, transmission electron microscopy (TEM) and neurotoxicity studies using mouse primary cortical neurons. G4 QPAMAM was synthesised by reacting PAMAM with methyl iodide in N,N-dimethylformamide. Pure compound was obtained after dialysis with 2 M NaCl and subsequently with water. Examination of the aggregation kinetics of Aβ40 via ThT fluorescence showed a large and significant delay in lag time with G4 QPAMAM whereas only a slight delay in lag time was observed for G4 PAMAM. TEM analysis revealed that fewer amyloid fibrils were formed in presence of G4 QPAMAM dendrimers than for G4 PAMAM dendrimers both with and without heparin. G4 QPAMAM substantially decreased Aβ40 induced neurotoxicity when compared to the parent G4 PAMAM in post treatment study. From the PAMAM and QPAMAM studies, it was observed that electrostatic interactions are likely to play major role in inhibition of Aβ aggregation.

The second part of this thesis involved the preparation of poly-L-lysine core dendrimers, which may be considerably less toxic than the PAMAM core but maintain the activity of PAMAM. It is also known that linear poly-L-lysine has the capacity to dissolve preformed Aβ fibrils. The surface of poly-L-lysine dendrimers were modified with anionic (succinate and sulfopropionate), cationic (alaninamide), and neutral (methoxyacetate and [(methoxyethoxy)ethoxy]acetate) molecules to yield diverse dendrimer surfaces. Fmoc solid phase peptide synthesis was used to synthesize the dendrimers, which were purified by RP-HPLC and characterised by NMR, LR/HR-MS and analytical RP-HPLC. Cationic alaninamide, anionic succinate and neutral methoxyacetate capped poly-L-lysine dendrimers were synthesised from G1 to G4 while sulfopropionate and [(methoxyethoxy)ethoxy]acetate) capped poly-L-lysine dendrimers were synthesised from G1 to G3. ThT assay, TEM and neurotoxicity studies were performed. ThT assay results revealed that succinate and alaninamide capped poly-L-lysine dendrimers are significant in delaying the lag time of Aβ40 aggregation in the presence and absence of heparin. TEM and neurotoxicity studies were performed for both succinate and alaninamide dendrimers; cationic alaninamide capped dendrimers produced less amyloid fibrils than anionic succinate capped dendrimers. Similarly, cationic alaninamide dendrimers decreased Aβ40 induced neurotoxicity in mouse primary neuronal cells when compared to anionic succinate dendrimers.

In conclusion, cationic surface engineered poly-L-lysine and G4 QPAMAM dendrimers inhibit the Aβ40 aggregation better than the PAMAM dendrimers. The electrostatic interaction of dendrimer with Aβ40 is important for activity.
Keyword Alzheimers disease

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Created: Sun, 26 Aug 2012, 21:24:55 EST by Manoj Kumar Palanivelu on behalf of Scholarly Communication and Digitisation Service