Insertion of the protective APP A673T mutation by CRISPR/Cas9 base editing or PRIME editing

There is currently no treatment for Alzheimer disease (AD). However, the Icelandic mutation in the APP gene (A673T) has been shown to confer a protection against the onset and development of AD (Jonsson et al. Nature 2012). This single nucleotide mutation in APP exon 16 reduces the cleavage of the APP protein by the beta-secretase by 40% thus preventing the development of AD even in persons more than 95 years old. Our research group has initially shown that the presence of the A673T mutation in an APP gene reduced the secretion of beta-amyloid peptides even if there is also a FAD mutation in the gene. This is the case for 14 different FAD mutations. We have used CRISPR/Cas9 base editing and PRIME editing technologies to insert the A673T mutation in the APP gene. We have compared several different cytidine base editor complexes to achieve the most effective and accurate genome modification possible in HEK293T cells and in SH-SY5Y neuroblastomas. The insertion of the A673T mutation in cells containing the London mutation reduced the secretion of beta-amyloid peptides. We are currently using lentiviral vectors to infect neurons from a mouse model and human neurons induced from fibroblasts of a patient with the London mutation. The insertion of the protective Icelandic mutation in the APP gene using these editing technologies opens a new potential therapeutic avenue not only for Familial Alzheimer’s diseases but also for sporadic Alzheimer’s disease.

An isogenic panel of single App knock-in mouse models of Alzheimer's disease confers differential profiles of β-secretase inhibition and endosomal abnormalities

We previously developed single App knock-in mouse models of Alzheimer's disease (AD) that harbor the Swedish and Beyreuther/Iberian mutations with or without the Arctic mutation (AppNL-G-F and AppNL-F mice). These models showed the development of amyloid We previously developed single App knock-in mouse models of AD that harbor the Swedish and Beyreuther/Iberian mutations with or without the Arctic mutation (AppNL-G-F and AppNL-F mice). We have now generated App knock-in mice devoid of the Swedish mutations (AppG-F mice) and some additional mutants to address the following two questions: [1] Do the Swedish mutations influence the mode of beta-secretase inhibitor action in vivo? [2] Does the quantity of C-terminal fragment of amyloid precursor protein (APP) generated by beta-secretase (CTF-beta) affect endosomal properties as previously reported as well as other pathological events? Abeta pathology was exhibited by AppG-F mice from 6 to 8 months of age, and was accompanied by microglial and astrocyte activation. We found that a beta-secretase inhibitor, verubecestat, inhibited Abeta production in AppG-F mice, but not in AppNL-G-F mice, indicating that the AppG-F mice are more suitable for preclinical studies of beta-secretase inhibition given that most AD patients do not carry Swedish mutations. We also found that the quantity of CTF-beta generated by various App knock-in mutants failed to correlate with endosomal alterations or enlargement, implying that CTF-beta, endosomal abnormalities, or both are unlikely to play a major role in AD pathogenesis. This is the first AD mouse model ever described that recapitulates amyloid pathology in the brain without the presence of Swedish mutations and without relying on the overexpression paradigm. Thus, experimental comparisons between different App knock-in mouse lines will potentially provide new insights into our understanding of the etiology of AD.

Trehalose Reduces the Secreted Beta-Amyloid Levels in Primary Neurons Independently of Autophagy Induction

The disaccharide trehalose was described as possessing relevant neuroprotective properties as an mTORC1-independent inducer of autophagy, with the ability to protect cellular membranes and denaturation, resulting from desiccation, and preventing the cellular accumulation of protein aggregates. These properties make trehalose an interesting therapeutic candidate against proteinopathies such as Alzheimer’s disease (AD), which is characterized by deposits of aggregated amyloid-beta (Aβ) and hyperphosphorylated tau. In this study, we observed that trehalose was able to induce autophagy in neurons only in the short-term, whereas long-term treatment with trehalose provoked a relevant anti-amyloidogenic effect in neurons from an AD mouse model that was not mediated by autophagy. Trehalose treatment reduced secreted Aβ levels in a manner unrelated to its intracellular accumulation or its elimination through endocytosis or enzymatic degradation. Moreover, the levels of Aβ precursor protein (APP) and beta-secretase (BACE1) remained unaltered, as well as the proper acidic condition of the endo-lysosome system. Instead, our results support that the neuroprotective effect of trehalose was mediated by a reduced colocalization of APP and BACE1 in the cell, and, therefore, a lower amyloidogenic processing of APP. This observation illustrates that the determination of the mechanism, or mechanisms, that associate APP and BACE is a relevant therapeutic target to investigate.

IDENTIFICAÇÃO DE POTENCIAIS INIBIDORES TRIPLOS FRENTE A ACETILCOLINESTERASE, BUTIRILCOLINESTERASE E BETA- SECRETASE 1 POR MODELO FARMACOFÓRICO

Doença de Alzheimer (DA) é uma doença neurodegenerativa, responsável por60 a 70% dos casos de demência no mundo (OMS, 2017). O tratamento disponível paraDA é limitado (galantamina, tacrina, rivastigmina e donepezila), ineficaz esintomatológico, ou seja, não conseguem inibir o processo de neurodegeneração. Poresse motivo, a busca por novas alternativas terapêuticas é emergencial.

Potential Phyto Therapeutic Approaches for the Treatment of Alzheimer’s Disease: An Overview

: Alzheimer’s disease is one of the most prevailing age-dependent neurodegenerative disease and the most common cause of dementia. The pharmacological therapies available for the disease provide only symptomatic relief. Plants are being extensively investigated for Alzheimer’s as they are relatively safer and cheaper. This review summarizes recent findings suggesting anti-Alzheimer potential of the plants along with compounds or mechanisms responsible for their efficacy and their therapeutic targets. The findings of recent studies revealed that the plants or the compounds isolated from them exhibit mitigative potential in Alzheimer’s disease by targeting amyloid beta, tau protein, cholinergic pathways via various enzymes like beta secretase, gamma secretase, acetylcholinesterase or receptors involved in these pathways. A number of putative compounds revealed from these studies can be investigated further for the mitigation of Alzheimer’s disease.

Epigenetic profiling of Italian patients identified methylation sites associated with hereditary transthyretin amyloidosis

Hereditary transthyretin (TTR) amyloidosis (hATTR) is a rare life-threatening disorder caused by amyloidogenic coding mutations located in TTR gene. To understand the high phenotypic variability observed among carriers of TTR disease-causing mutations, we conducted an epigenome-wide association study (EWAS) assessing more than 700,000 methylation sites and testing epigenetic difference of TTR coding mutation carriers vs. non-carriers. We observed a significant methylation change at cg09097335 site located in Beta-secretase 2 (BACE2) gene (standardized regression coefficient = −0.60, p = 6.26 × 10–8). This gene is involved in a protein interaction network enriched for biological processes and molecular pathways related to amyloid-beta metabolism (Gene Ontology: 0050435, q = 0.007), amyloid fiber formation (Reactome HSA-977225, q = 0.008), and Alzheimer’s disease (KEGG hsa05010, q = 2.2 × 10–4). Additionally, TTR and BACE2 share APP (amyloid-beta precursor protein) as a validated protein interactor. Within TTR gene region, we observed that Val30Met disrupts a methylation site, cg13139646, causing a drastic hypomethylation in carriers of this amyloidogenic mutation (standardized regression coefficient = −2.18, p = 3.34 × 10–11). Cg13139646 showed co-methylation with cg19203115 (Pearson’s r2 = 0.32), which showed significant epigenetic differences between symptomatic and asymptomatic carriers of amyloidogenic mutations (standardized regression coefficient = −0.56, p = 8.6 × 10–4). In conclusion, we provide novel insights related to the molecular mechanisms involved in the complex heterogeneity of hATTR, highlighting the role of epigenetic regulation in this rare disorder.