Strain-specific profiling of Amyloid-β in Alzheimer´s disease: functional and clinical signature
by Aneeqa Noor
Date of Examination:2020-05-25
Date of issue:2020-05-29
Advisor:Dr. Saima Zafar
Referee:Prof. Dr. Inga Zerr
Referee:Prof. Dr. Thomas Meyer
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Abstract
English
The molecular culprits driving the atypical clinical variants of Alzheimer’s disease (AD), including the recently discovered rapidly progressive AD (rpAD), are unknown to date. Of the several mechanisms being studied in this regard, the fibrillization of the amyloid-β (Aβ) peptide is most frequently targeted. The Aβ peptide can exist as multiple proteoforms that vary with respect to their sequences, post-translational modifications, capabilities to generate amyloids and mechanisms of toxicity. The current study was designed to target these variations in AD patients exhibiting classical and rapid progression, with the primary aim of establishing if these variants can constitute strains that underlie the phenotypic variability of AD. The differences in sequences of pathophysiological proteoforms among sporadic AD (sAD), rpAD and non-demented controls were established using hybrid-immunoprecipitation followed by 2D gel electrophoresis and top-down MALDI mass spectrometry. A total of 33 Aβ proteoforms were identified. Aβ40, Aβ42, Aβ4-42, Aβ11-42 and pyroglutamate Aβ11-42 were common in all AD cases however, several shorter N and C-terminally truncated proteoforms showed subtype-specific involvement. sAD showed a greater variety among monomeric species of proteoforms in comparison to rpAD. Although no significant differences were evident in the quantities of various Aβ-cleaving enzymes that were analyzed to explain the variations in the signature of proteoforms, the ratio of β-secretase/α-secretase was significantly higher in rpAD in comparison to sAD indicating higher cleavage of Aβ via the amyloidogenic pathway. The aggregation of common sAD and rpAD-derived proteoforms and variations in the generated fibrils were assessed through a combination of RT-QuIC, Infrared spectroscopy and Atomic force microscopy. Although spectroscopy showed that the secondary structure of Aβ fibrils from both subtypes of AD was highly similar, the conversion of monomeric species to β-sheet rich fibrils was faster in sAD cases in comparison to rpAD. The latter group presented significantly larger aggregates highlighting the presence of more hydrophobic, albeit decelerated, Aβ seeds. Applications of these fibrils to neuronal cells resulted in no significant differences in the survival, implicating that Aβ from sAD and rpAD were equally toxic. Co-IP experiments, on the other hand, validated differences in Aβ-modulated toxic pathways in sAD and rpAD. Aβ proteoforms from the former group mainly affected transcription and metabolism while Aβ proteoforms isolated from rpAD primarily modulated neurogenesis and neurotransmission. This study gives a comprehensive insight into the constituents of Aβ proteome, their relative quantities and their generation in sAD and rpAD brains and, for the first time, establishes differences in aggregation kinetics and 3D morphologies of fibrils associated with distinct clinical variants of AD. Further validation of reported targets and mechanisms will aid in establishing potential points of intervention in the diagnosis and therapy of AD.
Keywords: Proteoforms; Strains; Aβ; Rapidly progressive AD