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Age-modified forms of Amyloid-β as initiator of Alzheimer Disease pathogenesis and mediator of Aβ-tau interaction: a study in a Drosophila model and Aβ immunized human Alzheimer patients

Final Report Summary - ALZPROTAGEING (Age-modified forms of Amyloid-β as initiator of Alzheimer Disease pathogenesis and mediator of Aβ-tau interaction: a study in a Drosophila model and Aβ immunized human Alzheimer patients)

Alzheimer’s disease (AD) is the most common form of dementia. Aggregation of amyloid-beta (Ab), upstream of tau phosphorylation, is considered a key driver of AD pathogenesis and the major target of the clinical trials of amyloid immunotherapy.
Ageing is the main risk factor for AD, but the link between physiological ageing and protein misfolding of Ab remains unclear. Furthermore, a rapidly ageing population makes AD an economic and social burden for our society, thus investigating the pathological mechanisms of AD to identify pharmacological targets are research priorities.

AlzProtAgeing project has proposed to investigate, for the first time, the neurodegeneration in the context of molecular and physiological ageing, verifying the hypothesis that protein ageing of Ab, namely the accumulation of age-related amino acid modifications, is directly associated with AD. The project has used Drosophila to explore Ab molecular ageing and to study its association with misfolding and toxicity. In human brain tissues of AD cases and cognitive normal people, AlzProtAgeing has examined Ab and its precursor APP to identify changes specifically associated with either physiological ageing and/or AD.
In particular, two age-modified forms of Ab, pyroglutamate-Ab (pE-Ab) and isoAspartate Ab (isoAsp-Ab) have been investigated to assess whether they spontaneously increase with physiological ageing, or become prevalent in AD; and whether their accumulation correlates with hyperphosphorylated tau (p-Tau), the other hallmark of the disease.
Therapies targeting Ab have not shown improvement of the cognition in treated patients, possibly due to the absence of clearance of aged modified Ab from the brain. Taking advantage of the unique cohort of human AD brains actively immunized against Ab1-42 (iAD), available at the University of Southampton (UoS), AlzProtAgeing has also analysed the effect of active Ab immunotherapy on the brain accumulation of both pE-Ab and isoAsp-Ab.

Drosophila study: In the year 1, Dr Moro has acquired the skills to set-up genetic crosses of transgenic Ab fly stocks to drive the expression of human Ab in the nervous system of Drosophila. She has improved both the Ab level of expression and the sensitivity of the Ab detection protocol. She has introduced in Mudher’s laboratory at UoS, new Drosophila stocks with different genetic constructs containing human Ab. She has screened the progeny generated from all of the different crosses to identify the best transgenic flies with detectable monomers and aggregated forms of human Ab. Using a specifically developed Western Blot (WB) protocol, Dr Moro has screened for Ab, pE-Ab and IsoAsp-Ab the progeny of the established crosses. During year 2, she concluded the screening for monomeric and oligomeric Ab species using WB analysis of SDS soluble and insoluble protein fractions extracted from the fly heads. In parallel, she performed longevity assay to assess the toxicity due to the expression of Ab in these flies. She has identified in the double transgenic flies expressing two copies of human Ab1-42, a significant reduction of the lifespan compared to controls flies, associated with detectable levels of monomers and oligomers Ab . No signal of modified Ab was detectable in these flies. This part of the project has confirmed the relevance of the Drosophila model to explore characteristics of Ab misfolding, typical of AD, like the association between oligomers Ab and toxicity. At the same time limitations have been identified in the use of Drosophila to mimic age-related diseases. Indeed, part of AlzProtAgeing results showed that a complex, spontaneous and slow process, like Ab protein ageing, could not be tracked in the short lifespan of an invertebrate, like Drosophila.

Human study: formalin fixed paraffin embedded brain tissues of 13 iAD cases were sourced from Dr Boche’s brain collection at UoS, while tissues of 11 young controls (under 65 years old, with no neurological and cerebrovascular disorders), 32 old-controls (over 65 years old, with no neurological and cerebrovascular disorders) and 27 AD cases were obtained from BRAIN UK and Brains for Dementia Research (year 1). Immunohistochemistry protocols were optimized for the detection of amyloid deposits on human tissue and the immunostaining quantified.
To investigate the key markers of protein ageing described in the project, two antibodies, directed against pE-Ab and IsoAsp-Ab were identified, optimised and used to screen the 83 cases. In addition, a qualitative and quantitative assessment was performed using three antibodies directed against: pan-amyloid, Ab specific, and amyloid precursor protein (APP) specific. Since AlzProtAgeing has proposed to investigate the association between markers of protein ageing and the typical hallmarks of AD, p-Tau immunostaining was also performed and quantified in all cases.

This study has showed that APP is uniformly distributed in the cytoplasm of neurons, in young, cognitively normal people. This characteristic is lost with ageing while extracellular accumulation of Ab increases, becoming significantly abundant in AD cases. Deposits of pEAb and IsoAsp-Ab are present in the brain of old controls, but significantly increased only in AD. The only significant correlation identified in relation to p-Tau was with pE-Ab, both in old controls and AD cases . No significant difference in the level of pE-Ab and IsoAsp-Ab were identified between AD and iAD cases. This might indicate a lack of clearance of these forms despite the immunotherapy treatment. In conclusion, AlzProtAgeing has demonstrated that ageing affects cellular localization of APP and that age-related amino acid modifications of Ab are linked to AD pathology.

Since protein ageing takes place earlier than the first signs of physiological ageing appear, it may represent both an early pathogenic process as well as an obstacle to the effectiveness of therapies for AD. The findings of AlzProtAgeing have showed that is possible to identify markers of ageing in Ab and APP and that a link exists between protein ageing and AD. It has also provided additional helpful information to understand failure and success of Ab immunotherapies. Therefore, AlzProtAgeing outcomes can contribute to the further characterization of Ab as a pharmacological target in AD, and to the rational design of a second generation of “Ab immunotherapies”.

Maria Luisa Moro, PhD, Clinical and Experimental Sciences, Southampton General Hospital, University of Southampton, Southampton SO16 6YD, UK. Email: