Alzheimer's genetic risk factor FERMT2 (Kindlin-2) controls axonal growth and synaptic plasticity in an APP-dependent manner.

Although APP metabolism is being intensively investigated, a large fraction of its modulators is yet to be characterized. In this context, we combined two genome-wide high-content screenings to assess the functional impact of miRNAs and genes on APP metabolism and the signaling pathways involved. This approach highlighted the involvement of FERMT2 (or Kindlin-2), a genetic risk factor of Alzheimer's disease (AD), as a potential key modulator of axon guidance, a neuronal process that depends on the regulation of APP metabolism. We found that FERMT2 directly interacts with APP to modulate its metabolism, and that FERMT2 underexpression impacts axonal growth, synaptic connectivity, and long-term potentiation in an APP-dependent manner. Last, the rs7143400-T allele, which is associated with an increased AD risk and localized within the 3'UTR of FERMT2, induced a downregulation of FERMT2 expression through binding of miR-4504 among others. This miRNA is mainly expressed in neurons and significantly overexpressed in AD brains compared to controls. Altogether, our data provide strong evidence for a detrimental effect of FERMT2 underexpression in neurons and insight into how this may influence AD pathogenesis.

The new genetic landscape of Alzheimer's disease: from amyloid cascade to genetically driven synaptic failure hypothesis?

A strong genetic predisposition (60-80% of attributable risk) is present in Alzheimer's disease (AD). In view of this major genetic component, identification of the genetic risk factors has been a major objective in the AD field with the ultimate aim to better understand the pathological processes. In this review, we present how the genetic risk factors are involved in APP metabolism, ß-amyloid peptide production, degradation, aggregation and toxicity, innate immunity, and Tau toxicity. In addition, on the basis of the new genetic landscape, resulting from the recent high-throughput genomic approaches and emerging neurobiological information, we propose an over-arching model in which the focal adhesion pathway and the related cell signalling are key elements in AD pathogenesis. The core of the focal adhesion pathway links the physiological functions of amyloid precursor protein and Tau with the pathophysiological processes they are involved in. This model includes several entry points, fitting with the different origins for the disease, and supports the notion that dysregulation of synaptic plasticity is a central node in AD. Notably, our interpretation of the latest data from genome wide association studies complements other hypotheses already developed in the AD field, i.e., amyloid cascade, cellular phase or propagation hypotheses. Genetically driven synaptic failure hypothesis will need to be further tested experimentally within the general AD framework.

BIN1 recovers tauopathy-induced long-term memory deficits in mice and interacts with Tau through Thr348 phosphorylation.

The bridging integrator 1 gene (BIN1) is a major genetic risk factor for Alzheimer's disease (AD). In this report, we investigated how BIN1-dependent pathophysiological processes might be associated with Tau. We first generated a cohort of control and transgenic mice either overexpressing human MAPT (TgMAPT) or both human MAPT and BIN1 (TgMAPT;TgBIN1), which we followed-up from 3 to 15 months. In TgMAPT;TgBIN1 mice short-term memory deficits appeared earlier than in TgMAPT mice; however-unlike TgMAPT mice-TgMAPT;TgBIN1 mice did not exhibit any long-term or spatial memory deficits for at least 15 months. After killing the cohort at 18 months, immunohistochemistry revealed that BIN1 overexpression prevents both Tau mislocalization and somatic inclusion in the hippocampus, where an increase in BIN1-Tau interaction was also observed. We then sought mechanisms controlling the BIN1-Tau interaction. We developed a high-content screening approach to characterize modulators of the BIN1-Tau interaction in an agnostic way (1,126 compounds targeting multiple pathways), and we identified-among others-an inhibitor of calcineurin, a Ser/Thr phosphatase. We determined that calcineurin dephosphorylates BIN1 on a cyclin-dependent kinase phosphorylation site at T348, promoting the open conformation of the neuronal BIN1 isoform. Phosphorylation of this site increases the availability of the BIN1 SH3 domain for Tau interaction, as demonstrated by nuclear magnetic resonance experiments and in primary neurons. Finally, we observed that although the levels of the neuronal BIN1 isoform were unchanged in AD brains, phospho-BIN1(T348):BIN1 ratio was increased, suggesting a compensatory mechanism. In conclusion, our data support the idea that BIN1 modulates the AD risk through an intricate regulation of its interaction with Tau. Alteration in BIN1 expression or activity may disrupt this regulatory balance with Tau and have direct effects on learning and memory.

Genome-wide, high-content siRNA screening identifies the Alzheimer's genetic risk factor FERMT2 as a major modulator of APP metabolism.

Genome-wide association studies (GWASs) have identified 19 susceptibility loci for Alzheimer's disease (AD). However, understanding how these genes are involved in the pathophysiology of AD is one of the main challenges of the "post-GWAS" era. At least 123 genes are located within the 19 susceptibility loci; hence, a conventional approach (studying the genes one by one) would not be time- and cost-effective. We therefore developed a genome-wide, high-content siRNA screening approach and used it to assess the functional impact of gene under-expression on APP metabolism. We found that 832 genes modulated APP metabolism. Eight of these genes were located within AD susceptibility loci. Only FERMT2 (a ß3-integrin co-activator) was also significantly associated with a variation in cerebrospinal fluid Aß peptide levels in 2886 AD cases. Lastly, we showed that the under-expression of FERMT2 increases Aß peptide production by raising levels of mature APP at the cell surface and facilitating its recycling. Taken as a whole, our data suggest that FERMT2 modulates the AD risk by regulating APP metabolism and Aß peptide production.

Growth arrest-specific 1 binds to and controls the maturation and processing of the amyloid-beta precursor protein.

Alzheimer's disease (AD), the most common neurodegenerative disorder, is characterized by cerebral deposition of amyloid-ß (Aß), a series of peptides derived from the processing of the amyloid-ß precursor protein (APP). To identify new candidate genes for AD, we recently performed a transcriptome analysis to screen for genes preferentially expressed in the hippocampus and located in AD linkage regions. This strategy identified CALHM1 (calcium homeostasis modulator 1), a gene modulating AD age at onset and Aß metabolism. Here, we focused our attention on another candidate identified using this screen, growth arrest-specific 1 (Gas1), a gene involved in the central nervous system development. We found that Gas1 formed a complex with APP and controlled APP maturation and processing. Gas1 expression inhibited APP full glycosylation and routing to the cell surface by leading to a trafficking blockade of APP between the endoplasmic reticulum and the Golgi. Gas1 expression also resulted in a robust inhibition of APP transport into multivesicular bodies, further demonstrating that Gas1 negatively regulated APP intracellular trafficking. Consequently, Gas1 overexpression led to a reduction in Aß production, and conversely, Gas1 silencing in cells expressing endogenously Gas1 increased Aß levels. These results suggest that Gas1 is a novel APP-interacting protein involved in the control of APP maturation and processing.

High-Content Screening of Synaptic Density Modulators in Primary Neuronal Cultures.

The synapse, which represents the structural and functional basis of neuronal communication, is one of the first elements affected in several neurodegenerative diseases. To better understand the potential role of gene expression in synapse loss, we developed an original high-content screening (HCS) model capable of quantitatively assessing the impact of gene silencing on synaptic density. Our approach is based on a model of primary neuronal cultures (PNCs) from the neonatal rat hippocampus, whose mature synapses are visualized by the relative localization of the presynaptic protein Synaptophysin with the postsynaptic protein Homer1. The heterogeneity of PNCs and the small sizes of the synaptic structures pose technical challenges associated with the level of automation necessary for HCS studies. We overcame these technical challenges, automated the processes of image analysis and data analysis, and carried out tests under real-world conditions to demonstrate the robustness of the model developed. In this article, we describe the screening of a custom library of 198 shRNAs in PNCs in the 384-well plate format. © 2023 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Culture of primary hippocampal rat neurons in 384-well plates Basic Protocol 2: Lentiviral shRNA transduction of primary neuronal culture in 384-well plates Basic Protocol 3: Immunostaining of the neuronal network and synaptic markers in 384-well plates Basic Protocol 4: Image acquisition using a high-throughput reader Basic Protocol 5: Image segmentation and analysis Basic Protocol 6: Synaptic density analysis.

The Alzheimer susceptibility gene BIN1 induces isoform-dependent neurotoxicity through early endosome defects.

The Bridging Integrator 1 (BIN1) gene is a major susceptibility gene for Alzheimer's disease (AD). Deciphering its pathophysiological role is challenging due to its numerous isoforms. Here we observed in Drosophila that human BIN1 isoform1 (BIN1iso1) overexpression, contrary to human BIN1 isoform8 (BIN1iso8) and human BIN1 isoform9 (BIN1iso9), induced an accumulation of endosomal vesicles and neurodegeneration. Systematic search for endosome regulators able to prevent BIN1iso1-induced neurodegeneration indicated that a defect at the early endosome level is responsible for the neurodegeneration. In human induced neurons (hiNs) and cerebral organoids, BIN1 knock-out resulted in the narrowing of early endosomes. This phenotype was rescued by BIN1iso1 but not BIN1iso9 expression. Finally, BIN1iso1 overexpression also led to an increase in the size of early endosomes and neurodegeneration in hiNs. Altogether, our data demonstrate that the AD susceptibility gene BIN1, and especially BIN1iso1, contributes to early-endosome size deregulation, which is an early pathophysiological hallmark of AD pathology.

New insights into the genetic etiology of Alzheimer's disease and related dementias.

Characterization of the genetic landscape of Alzheimer's disease (AD) and related dementias (ADD) provides a unique opportunity for a better understanding of the associated pathophysiological processes. We performed a two-stage genome-wide association study totaling 111,326 clinically diagnosed/'proxy' AD cases and 677,663 controls. We found 75 risk loci, of which 42 were new at the time of analysis. Pathway enrichment analyses confirmed the involvement of amyloid/tau pathways and highlighted microglia implication. Gene prioritization in the new loci identified 31 genes that were suggestive of new genetically associated processes, including the tumor necrosis factor alpha pathway through the linear ubiquitin chain assembly complex. We also built a new genetic risk score associated with the risk of future AD/dementia or progression from mild cognitive impairment to AD/dementia. The improvement in prediction led to a 1.6- to 1.9-fold increase in AD risk from the lowest to the highest decile, in addition to effects of age and the APOE ε4 allele.

CALHM1 P86L polymorphism modulates CSF Aß levels in cognitively healthy individuals at risk for Alzheimer's disease.

The calcium homeostasis modulator 1 (CALHM1) gene codes for a novel cerebral calcium channel controlling intracellular calcium homeostasis and amyloid-ß (Aß) peptide metabolism, a key event in the etiology of Alzheimer's disease (AD). The P86L polymorphism in CALHM1 (rs2986017) initially was proposed to impair CALHM1 functionally and to lead to an increase in Aß accumulation in vitro in cell lines. Recently, it was reported that CALHM1 P86L also may influence Aß metabolism in vivo by increasing Aß levels in human cerebrospinal fluid (CSF). Although the role of CALHM1 in AD risk remains uncertain, concordant data have now emerged showing that CALHM1 P86L is associated with an earlier age at onset of AD. Here, we have analyzed the association of CALHM1 P86L with CSF Aß in samples from 203 AD cases and 46 young cognitively healthy individuals with a positive family history of AD. We failed to detect an association between the CALHM1 polymorphism and CSF Aß levels in AD patients. Our data, however, revealed a significant association of CALHM1 P86L with elevated CSF Aß42 and Aß40 in the normal cohort at risk for AD. This work shows that CALHM1 modulates CSF Aß levels in presymptomatic individuals, strengthening the notion that CALHM1 is involved in AD pathogenesis. These data further demonstrate the utility of endophenotype-based approaches focusing on CSF biomarkers for the identification or validation of risk factors for AD.

Alternative glycosylation controls endoplasmic reticulum dynamics and tubular extension in mammalian cells.

The endoplasmic reticulum (ER) is a central eukaryotic organelle with a tubular network made of hairpin proteins linked by hydrolysis of guanosine triphosphate nucleotides. Among posttranslational modifications initiated at the ER level, glycosylation is the most common reaction. However, our understanding of the impact of glycosylation on the ER structure remains unclear. Here, we show that exostosin-1 (EXT1) glycosyltransferase, an enzyme involved in N-glycosylation, is a key regulator of ER morphology and dynamics. We have integrated multiomics and superresolution imaging to characterize the broad effect of EXT1 inactivation, including the ER shape-dynamics-function relationships in mammalian cells. We have observed that inactivating EXT1 induces cell enlargement and enhances metabolic switches such as protein secretion. In particular, suppressing EXT1 in mouse thymocytes causes developmental dysfunctions associated with the ER network extension. Last, our data illuminate the physical and functional aspects of the ER proteome-glycome-lipidome structure axis, with implications in biotechnology and medicine.

Association study of the NEDD9 gene with the risk of developing Alzheimer's and Parkinson's disease.

Alzheimer's disease (AD) and Parkinson's disease (PD), the two most common neurodegenerative disorders in the elderly, have been hypothesized to share genetic determinants. Recently, Li et al. proposed that a variant in the NEDD9 gene may be one of these common genetic factors. We attempted to confirm this initial observation by conducting an equivalent analysis in terms of pathologies and sample size. We genotyped the NEDD9 rs760678 SNP in three independent AD case-control studies (n = 3176) and two independent PD case-control studies (n = 1855). However, we failed to detect an association of this SNP with the risk of developing AD or PD, in any of these populations. In conclusion, these data indicate that the rs760678 SNP of the NEDD9 gene is at best a weak genetic determinant of AD or PD.

Pyk2 overexpression in postsynaptic neurons blocks amyloid ß1-42-induced synaptotoxicity in microfluidic co-cultures.

Recent meta-analyses of genome-wide association studies identified a number of genetic risk factors of Alzheimer's disease; however, little is known about the mechanisms by which they contribute to the pathological process. As synapse loss is observed at the earliest stage of Alzheimer's disease, deciphering the impact of Alzheimer's risk genes on synapse formation and maintenance is of great interest. In this article, we report a microfluidic co-culture device that physically isolates synapses from pre- and postsynaptic neurons and chronically exposes them to toxic amyloid ß peptides secreted by model cell lines overexpressing wild-type or mutated (V717I) amyloid precursor protein. Co-culture with cells overexpressing mutated amyloid precursor protein exposed the synapses of primary hippocampal neurons to amyloid ß1-42 molecules at nanomolar concentrations and induced a significant decrease in synaptic connectivity, as evidenced by distance-based assignment of postsynaptic puncta to presynaptic puncta. Treating the cells with antibodies that target different forms of amyloid ß suggested that low molecular weight oligomers are the likely culprit. As proof of concept, we demonstrate that overexpression of protein tyrosine kinase 2 beta-an Alzheimer's disease genetic risk factor involved in synaptic plasticity and shown to decrease in Alzheimer's disease brains at gene expression and protein levels-selectively in postsynaptic neurons is protective against amyloid ß1-42-induced synaptotoxicity. In summary, our lab-on-a-chip device provides a physiologically relevant model of Alzheimer's disease-related synaptotoxicity, optimal for assessing the impact of risk genes in pre- and postsynaptic compartments.

Is the ornithine transcarbamylase gene a genetic determinant of Alzheimer's disease?

Expression of ornithine transcarbamylase (OTC) is strongly induced in the brain of individuals suffering from Alzheimer's Disease (AD). Association studies in a population from northern France have revealed that two SNPs -389 G/A (rs5963409) and -241 A/G (rs5963411) located in the promoter of the OTC gene are associated with the risk of developing AD. In the present work, these association studies were extended to a population of 2113 AD cases and 1580 controls from northern France, western France, the United Kingdom and Italy. The rs5963409 minor allele was weakly but significantly associated with an increased risk of developing AD (OR=1.19, p=0.004). This association was independent of age and ApoE status. Our results support that the OTC gene may be a minor genetic determinant of AD.

Association study of the GAB2 gene with the risk of developing Alzheimer's disease.

The first genome-wide association in Alzheimer's disease (AD) suggested that the GAB2 gene rs2373115 polymorphism may be a strong risk factor in APOE varepsilon4-carriers. We failed to detect an association of rs2373115 with the risk of developing AD in three populations (totalling 1406 controls and 1749 AD cases) whatever the APOE status, even if we observed a slight tendency for an increase of the GG genotype (OR (GG versus GT+TT)=1.3, 95% CI 1.0-1.6, p=0.09) and the G allele frequency (OR=1.3, 95%CI 1.0-1.6, p=0.05) in varepsilon4-carriers. In addition, the rs2373115 did not modulate the extent of tau phosphorylation in the brain of 89 AD cases. The GAB2 gene is at best a minor genetic determinant of AD.

Using High-Throughput Animal or Cell-Based Models to Functionally Characterize GWAS Signals.

PURPOSE OF REVIEW: The advent of genome-wide association studies (GWASs) constituted a breakthrough in our understanding of the genetic architecture of multifactorial diseases. For Alzheimer's disease (AD), more than 20 risk loci have been identified. However, we are now facing three new challenges: (i) identifying the functional SNP or SNPs in each locus, (ii) identifying the causal gene(s) in each locus, and (iii) understanding these genes' contribution to pathogenesis. RECENT FINDINGS: To address these issues and thus functionally characterize GWAS signals, a number of high-throughput strategies have been implemented in cell-based and whole-animal models. Here, we review high-throughput screening, high-content screening, and the use of the Drosophila model (primarily with reference to AD). SUMMARY: We describe how these strategies have been successfully used to functionally characterize the genes in GWAS-defined risk loci. In the future, these strategies should help to translate GWAS data into knowledge and treatments.

Proximity Ligation Assay: A Tool to Study Endogenous Interactions Between Tau and Its Neuronal Partners.

Tau is a microtubule associated protein (MAP) that is expressed in neurons of the central nervous system. Tau proteins are deregulated in a group of pathologies, including Alzheimer's disease, commonly called tauopathies. Therefore intensive research has been conducted to understand both the regulation of Tau and its involvement in neuronal cellular pathways. Since its originally described interactor tubulin, Tau has been described to interact with several other proteins, including tyrosine kinases (Src, Fyn, Lck) and Phospholipase C-γ. In this chapter, we describe the use of proximity ligation assay as a versatile method to study the endogenous interaction of Tau with these different neuronal partners and use the recently identified Tau interactor Bin1 as case study.

Association study of the PIN1 gene with Alzheimer's disease.

Genetic linkage studies indicate evidence for one or more Alzheimer's disease (AD) genes on chromosome 19 independently of the apolipoprotein E gene, a well-characterized AD-risk factor. Recently, the PIN1 gene on chromosome 19p13.2 has been proposed as a candidate gene for AD. Here, we have investigated the potential impact of two promoter polymorphisms (rs2233678 and rs2233679) within this gene on the risk of developing AD. No association of these polymorphisms or haplotypes with the disease was observed in a large French case-control population. Our data suggest that these genetic variants in PIN1 do not make a significant contribution to AD risk.

ADAM30 Downregulates APP-Linked Defects Through Cathepsin D Activation in Alzheimer's Disease.

Although several ADAMs (A disintegrin-like and metalloproteases) have been shown to contribute to the amyloid precursor protein (APP) metabolism, the full spectrum of metalloproteases involved in this metabolism remains to be established. Transcriptomic analyses centred on metalloprotease genes unraveled a 50% decrease in ADAM30 expression that inversely correlates with amyloid load in Alzheimer's disease brains. Accordingly, in vitro down- or up-regulation of ADAM30 expression triggered an increase/decrease in Aß peptides levels whereas expression of a biologically inactive ADAM30 (ADAM30(mut)) did not affect Aß secretion. Proteomics/cell-based experiments showed that ADAM30-dependent regulation of APP metabolism required both cathepsin D (CTSD) activation and APP sorting to lysosomes. Accordingly, in Alzheimer-like transgenic mice, neuronal ADAM30 over-expression lowered Aß42 secretion in neuron primary cultures, soluble Aß42 and amyloid plaque load levels in the brain and concomitantly enhanced CTSD activity and finally rescued long term potentiation alterations. Our data thus indicate that lowering ADAM30 expression may favor Aß production, thereby contributing to Alzheimer's disease development.

Functional complementation in Drosophila to predict the pathogenicity of TARDBP variants: evidence for a loss-of-function mechanism.

The human TAR DNA binding protein 43 (TDP-43), encoded by the gene TARDBP, plays a central role in amyotrophic lateral sclerosis (ALS) and frontotemporal dementia. TDP-43 inclusions are also found in up to approximately 60% of Alzheimer's disease (AD) brains. Although ALS-causing TARDBP mutations cluster in the C-terminal glycine-rich region of the protein, the pathogenic nature of the atypical missense variants p.A90V (located between the bipartite nuclear localization signal) and p.D169G (located in the first RNA-binding domain) is unclear. In addition, whether causal ALS mutations represent gain or loss-of-function alleles remains unknown. We recently reported that loss-of-function of the highly conserved TARDBP ortholog in Drosophila (called TBPH) leads to death of bursicon neurons resulting in adult maturation and wing expansion defects. Here, we compared wild-type TARDBP, 2 typical ALS-causing mutations (p.G287S and p.A315T) and 2 atypical variants (p.A90V and p.D169G), for their ability to complement neuronal TBPH loss-of-function. Although p.D169G rescued organismal pupal lethality and neuronal loss to a similar extent as wild-type TARDBP, p.A90V, p.G287S, and p.A315T were less efficient. Accordingly, p.A90V, p.G287S, and p.A315T but not p.D169G or wild-type protein promoted a shift of TDP-43 from the nucleus to the cytoplasm in approximately 12%-14% of bursicon neurons. Finally, we found that the carrier frequency of rare variant p.A90V was higher in French-Belgian AD cases (5/1714, 0.29%) than in controls of European descent (5/9436, 0.05%) (odds ratio = 5.5; 95% confidence interval, 1.6-19.0; p = 0.009). We propose that pathogenic TARDBP mutations have partial loss-of-function properties and that TARDBP p.A90V may increase AD risk by the same mechanism.