Implication of Adult Hippocampal Neurogenesis in Alzheimer’s Disease and Potential Therapeutic Approaches

Alzheimer’s disease is the most common neurodegenerative disease, affecting more than 6 million US citizens and representing the most prevalent cause for dementia. Neurogenesis has been repeatedly reported to be impaired in AD mouse models, but the reason for this impairment remains unclear. Several key factors play a crucial role in AD including Aβ accumulation, intracellular neurofibrillary tangles accumulation, and neuronal loss (specifically in the dentate gyrus of the hippocampus). Neurofibrillary tangles have been long associated with the neuronal loss in the dentate gyrus. Of note, Aβ accumulation plays an important role in the impairment of neurogenesis, but recent studies started to shed a light on the role of APP gene expression on the neurogenesis process. In this review, we will discuss the recent approaches to neurogenesis in Alzheimer disease and update the development of therapeutic methods.

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.

Τhe Greek Variant in APP Gene: The Phenotypic Spectrum of APP Mutations

Mutations in the gene encoding amyloid precursor protein (APP) cause autosomal dominant inherited Alzheimer’s disease (AD). We present a case of a 68-year-old female who presented with epileptic seizures, neuropsychiatric symptoms and progressive memory decline and was found to carry a novel APP variant, c.2062T>G pLeu688Val. A comprehensive literature review of all reported cases of AD due to APP mutations was performed in PubMed and Web of Science databases. We reviewed 98 studies with a total of 385 cases. The mean age of disease onset was 51.3 ± 8.3 (31–80 years). Mutations were most often located in exons 17 (80.8%) and 16 (12.2%). The most common symptoms were dementia, visuospatial symptoms, aphasia, epilepsy and psychiatric symptoms. Mutations in the β-amyloid region, and specifically exon 17, were associated with high pathogenicity and a younger age of disease onset. We describe the second reported APP mutation in the Greek population. APP mutations may act variably on disease expression and their phenotype is heterogeneous.

Effect of Gene Editing on Alzheimer's Disease

Background Alzheimer’s disease is an irreversible, progressive brain disorder that slowly destroys memory and thinking skills, and, eventually, the ability to carry out the simplest tasks. In most people with Alzheimer’s, symptoms first appear in their mid-60s. Current estimates suggest that 44 million people live with dementia worldwide at present. This is predicted to more than triple by 2050 as the population ages. Alzheimer’s disease is currently ranked as the sixth leading cause of death in the United States, but recent estimates indicate that the disorder may rank third, just behind heart disease and cancer, as a cause of death for older people Treatment is currently targeted toward symptomatic therapy, although trials are underway that aim to reduce the production and overall burden of pathology within the brain. Recently, the Clustered Regular Interspaced Short Palindromic Repeats (CRISPR-cas9) has shown promise in certain neurological disorders, it provides a precise editing to human genome and reflect an efficient curative therapy. We aimed to investigate the efficacy of knock-out of the APP gene “Amyloid precursor protein gene(APBA2)that consequently modify the expression of Amyloid protein in leucocytes cell line using CRISPR-cas9 technology. Methods The gene expression profile of Alzheimer's disease was downloaded from biological bioinformatics databases,and based on bioinformatics analysis, we figured out that APP gene was overexpressed in Alzheimer's disease in both brain and peripheral tissues such as plasma, fibroblast and PMNLs. We used PMNLs as the source of gene for edition in our study .We knocked out the APP gene in leucocytes cell lines using CRISPR-cas9 technology. Finally, the gene editing efficacy was evaluated by cell viability assay, the gene expression was measured by qPCR and the Amyloid protein expression was proved by Immunofluorescence. Results knockout of APP gene int Leucocytes Cell line resulted in reduction in cell viability that was associated with marked reduction in the amyloid protein and gene expressions. Conclusion knockout of APP(APBA2) gene represents a promising therapeutic strategy in Alzheimer's disease.

Cell line construction and maintenance for Lyso-IP and Endo-IP analysis of amyloid precursor protein processing v1

Lyso-IP is a method that allows for the isolation of lysosomes for proteomics and metabolomics (dx.doi.org/10.17504/protocols.io.bybjpskn; dx.doi.org/10.17504/protocols.io.bx9hpr36). We have developed an analogous approach for purification of early/sorting endosomes (Endo-IP). In addition, we have found that endolysosomal purification via Lyso-IP and Endo-IP can be coupled with a quantitative proteomics workflow to obtain snapshots of Amyloid Precursor Protein (APP) processing to its Aβ products (Park et al. in submission). Here, we describe methods for cell line construction and maintenance of 293 cells with TMEM192-3xHA and 3xFLAG-EEA1, which are used for lysosome and endosome purification, respectively, with the addition of patient mutations to APP promotes processing. Cells with endogenously tagged TMEM192 and stably expressing FLAG-EEA1 are referred to as 293EL cells, for Endo-IP and Lyso-IP. These cells were also prepared in a form that has a deletion of the APP gene (293EL;APP-/-) and the same cells reconstituted with a lentivirus stably expressing APPSw;T700N to allow functional analysis of APP processing.

Investigating drug–target interactions in frontotemporal dementia using a network pharmacology approach

Background Frontotemporal dementia (FTD) is the second most common type of dementia in individuals aged below 65 years with no current cure. Current treatment plan is the administration of multiple medications. This has the issue of causing adverse effects due to unintentional drug–drug interactions. Therefore, there exists an urgent need to propose a novel targeted therapy that can maximize the benefits of FTD-specific drugs while minimizing its associated adverse side effects. In this study, we implemented the concept of network pharmacology to understand the mechanism underlying FTD and highlight specific drug–gene and drug–drug interactions that can provide an interesting perspective in proposing a targeted therapy against FTD. Results We constructed protein–protein, drug–gene and drug–drug interaction networks to identify highly connected nodes and analysed their importance in associated enriched pathways. We also performed a historeceptomics analysis to determine tissue-specific drug interactions. Through this study, we were able to shed light on the APP gene involved in FTD. The APP gene which was previously known to cause FTD cases in a small percentage is now being extensively studied owing to new reports claiming its participation in neurodegeneration. Our findings strengthen this hypothesis as the APP gene was found to have the highest node degree and betweenness centrality in our protein–protein interaction network and formed an essential hub node between disease susceptibility genes and neuroactive ligand–receptors. Our findings also support the study of FTD being presented as a case of substance abuse. Our protein–protein interaction network highlights the target genes common to substance abuse (nicotine, morphine and cocaine addiction) and neuroactive ligand–receptor interaction pathways, therefore validating the cognitive impairment caused by substance abuse as a symptom of FTD. Conclusions Our study abandons the one-target one-drug approach and uses networks to define the disease mechanism underlying FTD. We were able to highlight important genes and pathways involved in FTD and analyse their relation with existing drugs that can provide an insight into effective medication management.

Neuronal Dynamics and miRNA Signaling Differ between SH-SY5Y APPSwe and PSEN1 Mutant iPSC-Derived AD Models upon Modulation with miR-124 Mimic and Inhibitor

Neuronal miRNA dysregulation may have a role in the pathophysiology of Alzheimer’s disease (AD). miRNA(miR)-124 is largely abundant and a critical player in many neuronal functions. However, the lack of models reliably recapitulating AD pathophysiology hampers our understanding of miR-124’s role in the disease. Using the classical human SH-SY5Y-APP695 Swedish neuroblastoma cells (SH-SWE) and the PSEN1 mutant iPSC-derived neurons (iNEU-PSEN), we observed a sustained upregulation of miR-124/miR-125b/miR-21, but only miR-124 was consistently shuttled into their exosomes. The miR-124 mimic reduced APP gene expression in both AD models. While miR-124 mimic in SH-SWE neurons led to neurite outgrowth, mitochondria activation and small Aβ oligomer reduction, in iNEU-PSEN cells it diminished Tau phosphorylation, whereas miR-124 inhibitor decreased dendritic spine density. In exosomes, cellular transfection with the mimic predominantly downregulated miR-125b/miR-21/miR-146a/miR-155. The miR-124 inhibitor upregulated miR-146a in the two experimental cell models, while it led to distinct miRNA signatures in cells and exosomes. In sum, though miR-124 function may be dependent on the neuronal AD model, data indicate that keeping miR-124 level strictly controlled is crucial for proper neuronal function. Moreover, the iNEU-PSEN cellular model stands out as a useful tool for AD mechanistic studies and perhaps for the development of personalized therapeutic strategies.