Search

The Amyloid precursor protein (APP) is a transmembrane glycoprotein from which amyloid-β (Aβ) peptides are generated after proteolytic cleavage. Aβ peptides are the main constituent of amyloid plaques in Alzheimer's Disease (AD). The physiological functions of APP in the human adult brain are very diverse including intracellular signaling, synaptic and neuronal plasticity, and cell adhesion, among others. There is growing evidence that APP becomes dysfunctional in AD and that this dyshomeostasis may impact several APP functions beyond Aβ generation. The vast majority of current anti-amyloid approaches in AD have focused on reducing the synthesis of Aβ or increasing the clearance of brain Aβ aggregates following a paradigm in which Aβ plays a solo in APP dyshomeostasis. A wider view places APP at the center stage in which Aβ is an important, but not the only, factor involved in APP dyshomeostasis. Under this paradigm, APP dysfunction is universal in AD, but with some differences across different subtypes. Little is known about how to approach APP dysfunction therapeutically beyond anti-Aβ strategies. In this review, we will describe the role of APP dyshomeostasis in AD beyond Aβ and the potential therapeutic strategies targeting APP. [ABSTRACT FROM AUTHOR]

Background: Extracellular vesicles (EVs) participate in the spread of pathological proteins between cells, contributing to the progression of neurodegenerative disorders, including Alzheimer's disease (AD). As cerebrospinal fluid (CSF) reflects the biochemical environment of the brain, EVs from CSF constitutes a promising source to investigate novel biomarkers and therapeutic targets. We aimed to explore the unbiased proteome profile of CSF‐derived EVs from individuals with AD by label‐free quantitative liquid chromatography mass spectrometry (LC‐MS/MS). Method: EVs were obtained from 500 µL of CSF from sex and age‐matched cognitively healthy donors (HC, n = 10, mean age = 73±1.4) and patients with AD dementia (AD, n = 10, mean age = 76.2±0.9) by a high‐throughput size exclusion chromatography method (SmartSEC). Resultant EV fractions were characterized by nanoparticle tracking analysis (NTA). EVs were lysed and proteins were trypsinized overnight via S‐Trap filters (Protifi). The peptides were separated on an Evosep nano LC system and analyzed by the TimsTOF Pro (Bruker) mass spectrometer. Peptides were identified using PEAKS Online software (1% FDR). DAVID Bioinformatics Resources 6.8 was used to determine the gene ontology (GO) enrichment. We normalized log2 peptide intensities (quantiles) and determined the differential protein expression across groups by a linear mixed effects (fixed effects = diagnosis, random effects = peptide sequence) model using the MsqRob package in R. P‐values were adjusted using Benjamini‐Hochberg. Result: NTA showed homogeneous EV size profiles (mean size = 113.4±1.41nm). We identified 2096 proteins with at least 1 proteotypic peptide. GO enrichment analyses showed "extracellular exosome" as the most statistically significant enriched term (p = 1.8×10−212), confirming the enrichment of EVs. Statistical inference analysis revealed 18 differentially expressed proteins between both diagnostic groups (adj.p<0.05), all of them increased in the AD group. (Fig 1). Some of the greatest changes include CLUS (log2FC = 0.61, adj.p = 8.38×10−4, Fig.2), SPB1 (log2FC = 1.15, adj.p = 1.23×10−2, Fig.3), LRP1 (log2FC = 3.43, adj.p = 1.23×10−2), KLK7 (log2FC = 2.59, adj.p = 1.23×10−2), SAP3 (log2FC = 2.09, adj.p = 3.1×10−2) and SHPS1 (log2FC = 1.03, adj.p = 4.25×10−3), which are proteins that have been previously associated with inflammation. Conclusion: Our results reveal novel protein changes in CSF‐derived EVs from individuals with AD, which could be explored further to their applicability in distinguishing the different clinical stages across the AD continuum. [ABSTRACT FROM AUTHOR]

Background: Synapse degeneration is one of the earliest changes in Alzheimer's disease (AD) and is the major neuropathologic correlate of cognitive impairment. The aim of this study was to characterize microRNA (miRNA) dysregulation at AD synapses post‐mortem brain tissue and explore the specificity for AD. Method: We prepared synaptosomes (SYN) by serial ultracentrifugation of 10 AD (mean age = 77.4±13.3) and 10 control (CN; mean age = 72.3±18.8) temporal cortex samples over a sucrose gradient and saved the homogenates (H). We extracted and quantified miRNA from SYN and H fractions by TaqMan Advanced miRNA Assay‐Openarray system. We calculated deltaCt and performed linear regression to identify differentially expressed (DE) miRNA across AD and CN SYN, adjusting for multiple testing using Benjamini‐Hochberg method (α<0.05). We identified gene targets of the DE miRNA using two predictive databases (TargetScan and miRDB) and performed pathway analysis using the Panther database. We selected miRNA candidates that are DE in AD synapses and performed a follow‐up study in 4 different brain regions of 10 CN (mean age = 80.7±12.9), 10 AD (mean age = 77.4±13.3) and 10 non‐AD tauopathy (mean age = 70.6±6.4) cases to determine the regional distribution and the AD specificity. Result: Synapse fractions were isolated demonstrating a 3.2‐fold enrichment in the synaptic protein VAMP‐2 (p<0.001) compared to homogenates. We detected 411 miRNA in H and 432 in SYN of the total in the panel with 89% overlap between AD and CN in SYN. Three miRNA from the same family (miR‐132‐3p, miR‐132‐5p and miR‐212‐3p) were under‐expressed and 2 miRNA (miR‐181a‐3p and miR‐1260a) were over‐expressed in AD compared to CN SYN. The gene targets of these miRNA were overrepresented in pathways regulating synaptic and mitochondrial function. In addition, targets of miR‐132 also were overrepresented in pathways related to tau pathology, Aβ production, interleukin production, apoptosis and oxidative stress. We will describe the regional distribution in AD brains post‐mortem and the specificity of the changes to AD by comparing to non‐pathological controls and non‐AD tauopathies. Conclusion: miRNA that are dysregulated in AD synapses target genes that are involved in pathways related to AD pathogenesis. Future studies will focus on the use of these miRNA as therapeutic targets. [ABSTRACT FROM AUTHOR]

Background: Synaptic degeneration is a primary neuropathological factor associated with cognitive decline in Alzheimer's disease (AD). In 2021, we generated a synaptic Polygenic Risk Score (PRS) that comprised only 8 variants within 6 synaptic genes (APOE, PICALM, BIN1, PTK2B, DLG2 and MINK1) that predicted AD with 72% accuracy in two neuropathological cohorts. This supports the hypothesis that genetic variants that regulate an individual's vulnerability to AD‐related synapse degeneration could be used to identify individuals at‐risk for AD prior to the appearance of clinical symptoms. The aim of this study was to determine whether the constituent variants of the linkage disequilibrium (LD) blocks represented by the PRS have regulatory activity in vitro. Method: We cloned an oligonucleotide library of 137 putative regulatory variants each represented by 5 barcodes per allele into pMPRA1 vector. Then we transfected the plasmids into HEK293 cells (n = 5). We extracted DNA and RNA from the cells and sequenced on an Illumina MiSeq. Using the mpra package in R, we normalized the tag counts to a common size of 10 million reads and computed paired log ratios of RNA/DNA counts for each barcode. We used weighted linear models to test for differential activity of the minor versus the major allele of each SNP using the mpralm function, adjusting for multiple testing using the Benjamini‐Hochberg method. Result: We acquired approximately 15 million reads of DNA and RNA from 5 independent experiments. We found that 35 out of the 137 SNPs tested had differential activity between alleles (adj. p <0.05, Figure 1A). Three of the SNPs that showed regulatory activity were included in the PRS (BIN1: rs17014923 and rs35114168; DLG2: rs286043) and the remaining 32 are captured on LD blocks within the synaptic PRS. Conclusion: All LD captured by the synaptic PRS contain SNPs that impact on regulatory activity, thus supporting a potential mechanism by which changes in the expression of these specific loci that encode synaptic proteins could lead to a modified cumulative risk for AD. Further studies to determine the precise mechanism involved in this regulatory activity at the synapse could guide future therapeutic strategies for AD. [ABSTRACT FROM AUTHOR]

Aims: Amyloid precursor protein (APP) 훽‐C‐terminal fragment (훽CTF) may have a neurotoxic role in Alzheimer's disease (AD). 훽CTF accumulates in the brains of patients with sporadic (SAD) and genetic forms of AD. Synapses degenerate early during the pathogenesis of AD. We studied whether the 훽CTF accumulates in synapses in SAD, autosomal dominant AD (ADAD) and Down syndrome (DS). Methods: We used array tomography to determine APP at synapses in human AD tissue. We measured 훽CTF, A훽40, A훽42 and phosphorylated tau181 (p‐tau181) concentrations in brain homogenates and synaptosomes of frontal and temporal cortex of SAD, ADAD, DS and controls. Results: APP colocalised with pre‐ and post‐synaptic markers in human AD brains. APP 훽CTF was enriched in AD synaptosomes. Conclusions: We demonstrate that 훽CTF accumulates in synapses in SAD, ADAD and DS. This finding might suggest a role for 훽CTF in synapse degeneration. Therapies aimed at mitigating 훽CTF accumulation could be potentially beneficial in AD.Using array tomography in post‐mortem brain tissue from the temporal and frontal cortex, we found that APP is located in synaptic compartments in different forms of AD. We also describe that APP βCTF accumulates in synaptosomal fractions in different forms of AD and that this accumulation correlates with Aβ and tau concentrations. [ABSTRACT FROM AUTHOR]

Background: A key pathological event in frontotemporal lobar degeneration (FTLD) is the alteration of the RNA metabolism. Despite this, no study has characterized the diversity of RNA species using high‐throughput sequencing approaches and correlated them with the main neuropathological hallmarks across FTLD subtypes. Method: Total and small RNA sequencing was performed in the frontal cortex of patients neuropathologically diagnosed with FTLD‐TDP (including non‐mutation carriers [sFTLD‐TDP;n = 9], and carriers of the C9orf72 repeat expansion [FTLD‐C9;n = 11]), FTLD‐tau (n = 13, six carrying the p.P301L mutation in MAPT) and controls without neuropathological alterations in the same brain region (n = 7). Gene and miRNA co‐expression modules were identified using WGCNA. Cell‐type proportions were estimated through cell‐type deconvolution using MuSiC. Gene ontology enrichment analyses were performed using Metascape. We assessed in the frontal cortex the presence of pTDP43 (in sFTLD‐TDP and FTLD‐C9), dipeptide repeats and RNA foci (in FTLD‐C9), and tau aggregates (in FTLD‐tau) through quantitative immunohistochemistry and correlated their density with transcriptome‐wide RNA alterations. Result: Our results indicate statistically significant correlations between gene and miRNA co‐expression modules, neuropathological changes and cell‐type proportions specific for each FTLD subtype. The most significant findings include: in sFTLD‐TDP, the density of pTDP43 positively correlated with a gene co‐expression module (R = 0.9,p = 0.04) enriched with splicing functions (p<1×10−8), which directly correlated with a miRNA co‐expression module (R = 0.77,p = 5×10−4) and the proportion of a microglial subpopulation (R = 0.69,p = 4×10−3). In FTLD‐C9, the density of poly(GP) repeats inversely correlated with the proportion of a neuronal subpopulation (R = ‐0.62,p = 0.041), and negatively correlated with a gene co‐expression module (R = ‐0.67,p = 0.024) enriched with protein phosphorylation functions (p<1×10−4). In FTLD‐tau, the density of tau aggregates negatively correlated with the proportion of a neuronal subpopulation (Fig.1A;R = ‐0.77,p = 0.003) and positively correlated with a miRNA (Fig.1B;R = 0.88,p = 8.8×10−5) and a gene (Fig.1C;R = 0.65,p = 0.016) co‐expression module enriched in genes with neuron ensheathing functions (Fig.1D;p<1×10−20). Conclusion: Our data demonstrate selective vulnerability of cell‐subtypes to neuropathological changes. In addition, we describe striking correlations between the main neuropathological hallmarks of each FTLD subtype and specific gene and miRNA co‐expression modules, including their hub genes and miRNAs which might be used as biomarkers to identify the FTLD neuropathological substrate in vivo, and reveal novel molecular mechanisms amenable for therapeutic intervention in FTLD. [ABSTRACT FROM AUTHOR]

Background: Synapse loss is the major neuropathologic correlate of cognitive impairment in Alzheimer's disease (AD). MicroRNA (miRNA) regulate synaptic plasticity and play a role in AD pathogenesis and therefore represent potential therapeutic targets. The aim of this study is to determine the role of miRNA‐mediated dysfunction at AD synapses. Method: We prepared homogenates (H) and synaptosomes (SYN) from frozen post‐mortem temporal cortex tissue from 10 AD (mean age = 77.4±4.2) and 10 non‐pathological controls (CN; mean age = 72.3±5.9). We extracted (mirVana) and quantified miRNA using the TaqManR Advanced miRNA Assay, QuantstudioTM and OpenarrayTM system. Samples were normalized (median Ct). DeltaCt values calculated relative to control hsa‐miR‐16‐5p. We performed linear regression to identify differentially expressed (DE) miRNA across groups, controlling the false discovery rate (FDR) using the Benjamini‐Hochberg method. We considered DE miRNA with fold changes <0.8 or >1.2 (adj.p<0.05) in AD vs CN. We searched two predictive databases (TargetScan and miRDB) for gene targets of the DE miRNA and identified overrepresented target genes using the Panther database (PantherDB.org) after controlling the FDR. Result: Of the 751 miRNA included on the Openarray, we detected 411 in H and 432 in SYN, with 89% overlap between AD and CN in SYN. To enrich our analyses for miRNA that are predominantly present at the synapse, we took forward only the 47 miRNA with >1.2‐foldchange (adj.p<0.2) in SYN vs H in either AD or CN. miR‐132‐3p and miR‐132‐5p were under‐expressed (0.17‐fold, p = 0.001 and 0.35‐fold, p = 0.006, respectively) and miR‐181a‐3p was over‐expressed (2.13‐fold, p = 0.04) in AD compared to CN SYN. The 594 gene targets of upregulated synaptic miRNA (miR‐181a‐3p) were overrepresented in pathways regulating synaptic function and mitochondrial bioenergetics and integrity. The 474 and 1230 targets of downregulated synaptic miRNA (miR‐132‐3p and miR‐132‐5p, respectively) were overrepresented in the same pathways as well as pathways related to interleukin production, tau pathology, Abeta production, apoptotic signalling and oxidative stress. Conclusion: Three synapse‐specific miRNA are differentially expressed at AD synapses and target genes involved in core AD‐related processes, thus making them promising therapeutic targets. Future studies will seek to validate these findings and determine the specificity for AD over other neurodegenerative diseases. [ABSTRACT FROM AUTHOR]

Background: Deposition of fibrillar forms of tau‐neurofibrillary tangles (NFT) and neuropil threads (NT) follow a progressive pattern across brain regions as reflected by Braak stages. However, oligomeric forms of tau have been proposed to precede the appearance of fibrillar forms. Synapses are the earliest structure linked to Azheimer's Disease cognitive loss, but whether tau pathology, fibrillar or oligomeric affects early this structure in each region remains unclear. This is mainly explained by the technical limitations to study oligomeric forms of tau and the visualization of synaptic terminals. We aim to investigate the presence of oligomeric tau at synapses of human AD cases and its relation to the presence of fibrillar forms of tau in different brain regions. Method: We used array tomography microscopy to visualize single synaptic terminals on postmortem human samples from BA20/21 and BA17 brain regions of 10 AD and 10 controls with diverse Braak stages. Pre‐ and post‐synaptic terminals (synaptophysin and PSD95) and oligomeric tau (T22 antibody) were labelled. To compare synaptic tau with total fibrillar tau burden, NFTs and NTs were quantified from immunohistochemical sections (AT8 antibody) of the same cases and areas. We also studied the presence of tau in synapses using Western Blot experiments in synaptoneurosomes enriched fractions from the same cases and areas (Tau5 antibody). Finally, the presence at synapses of oligomeric and fibrillar tau was confirmed with other superresolution techniques such as electron microscopy, STORM and DNA‐PAINT. Result: We found that 1) Oligomeric tau was found in both pre‐ and postsynaptic terminals, in a higher proportion of presynaptic terminals. 2) Oligomeric tau at synapses was found in areas without abundant fibrillar forms of tau. 3) A higher proportion of oligomeric tau when compared to phosphorylated tau was found at synaptic terminals in early affected cases. 4) Early synaptic oligomeric tau was found also by western blot of synaptoneurosomes, electron microscopy and STORM. Conclusion: Our results suggest that synaptic oligomeric tau may be an early event at tau‐affected brain regions, preceding the phosphorylation of tau and the formation of fibrillar forms. Also, the findings may support the transsynaptic spreading of tau at early stages. [ABSTRACT FROM AUTHOR]