Citations

Abstract Background Neurodegenerative tauopathies, characterized by the aggregation of misfolded tau protein, poses a significant clinical and scientific challenge. Tau interaction with other proteins affects the formation of ‘tau‐seed’ but less is known about its effects on tau‐seed properties. Our previous study revealed that Bassoon (BSN), a presynaptic protein, interacts with tau‐seed, exacerbating its toxicity. A parallel study also associated missense mutations in BSN with tau aggregation in patients, prompting an investigation into the influence of BSN and its mutations in neurodegeneration for potential therapeutic insights. Method We generated a knock‐in mouse model (BSNKI) harboring the disease‐associated p.Pro3866Ala mutation in endogenous Bsn. Cognitive and motor abilities were evaluated throughout the lifespan of heterozygous and homozygous BSNKI mice, along with analyses of BSN and tau patterns, gliosis, and gene expression changes in their brains. Additionally, we conducted single‐nucleic RNA sequencing to identify changes at the cellular level. Result BSNKI mice displayed progressive motor and memory impairments on the rotarod and fear conditioning assays, respectively. At 10 months, their brains displayed somatic BSN and pathological tau accumulation, with the gene expression changes indicating alterations of microglia, classical complement cascade, and synapse pruning pathways, as well as dysregulation of protein quality control pathways. Neuropathological data suggests that the BSN P3866A mutation instigates robust microglia activation, increased microglia‐mediated synapse engulfment, and a co‐deposition of proteasomal subunits with BSN. Furthermore, snRNA‐seq data suggest changes in neuronal populations and cell signaling. Conclusion Our BSNKI mouse model revealed the contributions of BSN P3866A mutation to tau pathogenesis and neurodegeneration. BSNKI animals displayed progressive motor and memory impairments, deposits of endogenous tau and BSN, as well as changes in gene expression and neuronal populations. Notably, BSN likely plays a dual role, promoting tau aggregation and sequestering protein degradation molecules, leading to tau and protein accumulation at the soma and synapse, which in turn could trigger neuroinflammation and other cellular changes. These findings propose BSN as a promising therapeutic target for tauopathies, underscoring the need for further exploration to elucidate underlying mechanisms and therapeutic implications.

Abstract Background We previously identified a novel mechanism describing how pathological tau transfers via extracellular vesicles (EVs) in Alzheimer's disease (AD). Targeting EV secretion to mitigate tau transfer is, therefore, a promising therapeutic approach for AD. P2X purinoreceptor 7 (P2RX7), an ATP‐gated cationic channel predominantly expressed in microglia, regulates the secretion and biogenesis of EVs. Here we investigated the effect of P2rx7 deficiency in PS19 tauopathy mouse model and by proteomic profiling of brain EVs. Method PS19: P2rx7 –/– mice at 9‐10 months of age were tested for fear conditioning and their brain sections were assessed for brain atrophy. Tau pathology was evaluated using ELISA and immunofluorescence against phosphorylated (AT8) and misfolded (Alz50) tau. Brain tissues were further assessed by bulk and single cell RNA‐seq (scRNA‐seq) and brain‐EVs were subjected to proteomic profiling by mass‐spectrometry. P2rx7 –/– mice were intracranially injected with viral vectors expressing P301L tau in neurons and mEmerald‐CD9 in microglia to visualize the secretion of microglial EVs in vivo . Mice conditional knockout (cKO) for P2rx7 in microglia, neurons and astrocytes were injected with a viral vector expressing P301L tau in entorhinal cortex for tau propagation assessment. Result PS19: P2rx7 –/– mice showed significant improvement in contextual and cued memory compared to age‐matched PS19 mice, which was accompanied by preserved cortical and hippocampal volume, and significant reduction of hippocampal synapse loss and tau pathology. By weighted gene co‐expression network analysis (WGCNA) we identified a microglial and EV‐related module strongly correlated with tau pathology which was remarkably downregulated in PS19: P2rx7 –/– mice hippocampus. PS19 mice showed increased secretion of EVs containing tau and mitochondrial proteins in the mouse brain parenchyma, which was significantly downregulated in PS19: P2rx7 –/– mice. scRNA‐seq analysis showed that P2rx7 is required for microglia conversion to inflammatory phenotype with increased expression of EV genes in PS19 mice. P2rx7 deficiency reduced microglial mEmerald‐CD9 + ‐EV secretion in mice expressing P301L tau. Moreover, microglia and neuron‐specific deletion of P2rx7 inhibit tau propagation from the entorhinal cortex to the mouse hippocampus. Conclusion Our study demonstrated a microglial P2RX7‐EV axis with potential implications on neuroinflammation and neurodegeneration associated with tau pathology, further indicating the therapeutic potential of targeting P2rx7 to ameliorate AD progression.

Abstract Background pTau217 is a well validated plasma biomarker for Alzheimer's disease (AD), sensitive to the accumulation of amyloid pathology in the preclinical stages of disease. We leveraged whole blood RNA sequencing and plasma pTau217 measures of amyloid burden to identify blood transcriptomic gene‐network changes that relate to plasma pTau217 levels in cognitively normal older adults. We then quantified the abundance of cell populations from the transcriptomic data to identify blood cell abundance alterations that correlate with plasma ptau217 levels. Method Whole blood RNA sequencing and plasma pTau217 data were obtained from 1739 participants in the Anti‐Amyloid Treatment in Asymptomatic Alzheimer's Disease (A4) Study. RNAseq data were aligned, sorted, counted, batch normalized, and iteratively adjusted for covariates. 20,718 genes were available for analysis following quality control. Plasma samples ( n = 1597) were analyzed for pTau217 using the Eli Lilly and Company Diagnostics Laboratory immunoassay. Samples from 724 participants (63% females, mean age 71, 61% amyloid+) with RNA sequencing and biomarker data were used for analysis. Cell fractions were quantified using CIBERSORTx (LM22 used as reference) and WGCNA was used to calculate gene co‐expression modules. Linear regression related gene module expression or cell fraction to mean pTau217 levels covarying for age, sex, education, APOE ε2 and ε4 status. Correction for multiple comparisons used the false discovery rate. Gene enrichment was performed using Gene Ontology. Result Five gene modules were associated with plasma pTau217. For the turquoise module (β=‐0.81, p.fdr=0.005), higher expression was associated with higher p ‐tau217 while higher expression in the blue (β=‐0.70, p.fdr=0.01), lightcyan (β=‐0.68, p.fdr=0.01), grey60 (β=‐0.64, p.fdr=0.02), and brown (β=‐0.55, p.fdr=0.04) modules were associated with lower p ‐tau217. These modules were enriched for phagocytosis, ribonucleoprotein complex biogenesis, immunoglobulin mediated immune response, nucleosome assembly, and cytoplasmic translation pathway genes. In cell fraction analysis, higher predicted abundance of neutrophils related to ptau217 levels (β=0.33, p.fdr=0.03). Conclusion Our results demonstrate an association between immune response and epigenetic pathway genes with plasma pTau217 levels in preclinical AD. Future work will seek to clarify whether these signatures also relate to brain amyloid PET to evaluate blood transcription networks as complementary biomarkers to improve preclinical prediction.

Abstract Background Early mechanisms of neuronal damage in Alzheimer's Disease (AD) include mitochondrial dysfunction and abnormal energy homeostasis. AD‐associated mitochondrial signaling was shown to occur upstream of amyloid precursor protein processing, Ab production, apolipoprotein E (APOE) expression, and tau pathology. However, whether alterations in mitochondrial function are sufficient to instigate AD remains unclear. Mitochondrial complex I (mtCI) is a rate‐limiting enzyme in the oxidative phosphorylation (OXPHOS) machinery. Changes in mtCI activity influence ATP and ROS production, cellular energy and redox homeostasis. We examined how reduced activity of mtCI due to a knockout of one of its accessory subunits Ndufs4 in mice affects transcriptional networks in the brain. We also determined whether reduced activity of mtCI abrogates neuroprotective properties of a small molecule CP2. Method RNA‐sequencing of the brain tissue was performed using Ndufs4 ‐/‐ and control mice ( n = 3–5 per group). The co‐expression modules of differentially expressed genes were compared to the AMP‐AD patient cohort data. Key mechanisms, including mitophagy signaling, were confirmed using ELISA and western blot analyses; the effect on mtCI activity was confirmed using functional assays. Results In Ndufs4 ‐/‐ mice, a residual activity of mtCI was ∼50% of the control. This loss of function led to a global disruption of mitochondrial homeostasis, energy metabolism, synaptic function, significant increase in mitophagy and a reduction in biogenesis. Transcriptomic signatures in brain tissue of male and female Ndufs4 ‐/‐ mice overlapped with changer reported for AD patients and AD mouse models with Ab and p ‐tau pathologies. These changes were partially rescued by a neuroprotective mitochondria‐targeting small molecule CP2. Consistent with studies in AD mice, CP2 treatment in Ndufs4 ‐/‐ mice augmented the expression of genes associated with mitochondrial biogenesis and turnover, synaptic activity, autophagy, redox balance, and reduced expression of genes related to inflammation. Sex‐specific differences included better adaptation to a reduced activity of mtCI and to CP2 treatment in female mice. Conclusion Reduced mtCI activity was sufficient to induce the AD‐like transcriptomic changes in brain. Residual mtCI function was sufficient to mediate neuroprotective effect of CP2, emphasizing a key role of mitochondria in AD development and treatment.

Abstract Background Some cases of Alzheimer's Disease (AD) and Frontotemporal Dementia (FTD) feature the mislocalization of the normally nuclear TAR DNA binding protein (TDP‐43) to the cytoplasm where it is incorporated into inclusions in affected brain regions. TDP‐43 plays a crucial role in the suppression of cryptic exons in the mature RNA of many targets in both AD and FTD. One such target, Stathmin‐2 (STMN2), is misspliced in the absence of TDP‐43 leading to a reduction of STMN2 RNA and protein while the levels of the cryptic splice variant is increased. Cryptic exons are not conserved across species, meaning previous Tdp‐43 models did not capture Stmn2 processing defects and Stmn2 loss of function (LOF). We hypothesize that STMN2 LOF contributes to cognitive phenotypes observed in AD and FTD. Method We have generated a novel mouse that more accurately models human disease mechanisms by expressing short‐hairpin (shRNA) via adeno associated virus in the central nervous system of a recently described humanized Stmn2 mouse model in which Stmn2 splicing is dependent on Tdp‐43. In this mouse we will simultaneously model Tdp‐43 loss of function and aberrant Stmn2 splicing. Result Our preliminary experiments demonstrate that humanized Stmn2 mice expressing shRNA against Tardbp have a reduction of Tardbp RNA and TDP‐43 protein compared to mice expressing a non‐targeting shRNA. Tdp‐43 knock‐down is accompanied by cryptic splicing of endogenous mouse targets, Sortilin 1 and Translin . As anticipated, we also detected cryptic Stmn2 splicing, suggesting that correct Stmn2 splicing is dependent on Tdp‐43 in this model. We will further characterize motor and cognitive functions in addition to neurodegeneration and gliosis in these animals. Conclusion We anticipate this novel model, and our subsequent studies will identify relevant disease mechanisms and provide an invaluable tool with which to investigate therapies for AD and FTD.

Abstract Background The interplay between vascular dysfunction and brain amyloidosis in driving Alzheimer's disease (AD) pathogenesis and cognitive decline is increasingly recognized. The retina, sharing an embryonic origin with the brain, offers an accessible CNS organ for high‐resolution, noninvasive imaging of AD‐related vascular pathology. However, the exact nature of retinal vascular damage in AD—especially in early disease stages—and its relationship with cerebral amyloid angiopathy (CAA) and cognitive impairment remain understudied. Method We examined vascular abnormalities in retinas of MCI and AD via histopathology and in‐vivo imaging studies. Postmortem retinas from 53–62 individuals (CN: 22; MCI/AD: 31–40) underwent immunohistochemistry for pericyte integrity, vascular Aβ42/40, and endothelial TJ proteins, with dysregulated proteins quantified by mass spectrometry in 12 additional AD/CN cases. Retinal curcumin‐amyloid imaging by scanning‐laser ophthalmoscope in 28–34 live participants (CN[MOCA>26]: 8–15; MCI/AD[MOCA≤26]: 13–19) assessed peri‐arteriolar/venular amyloid plaques (APs). Perivascular AP burden was correlated with MRI measures (hippocampal volume, WMH lesions) and cognitive/neuropsychiatric parameters. Result Retinal pericyte loss, reflected by PDGFRβ deficiency and apoptosis, was evident in MCI and AD patients and correlated with vascular Aβ42/40 accumulation and CAA severity (r=0.68–0.84; p <0.01–0.0001). Retinal endothelial ZO‐1 and claudin‐5 levels were significantly reduced, correlating with arteriolar Aβ40 deposition, suggesting impaired retinal Aβ clearance. Retinal capillary claudin‐5 and arteriolar Aβ40 strongly associated with CAA (r=0.75–0.77, p <0.0001), while ZO‐1 moderately correlated with brain amyloidosis/tauopathy, CAA, and cognitive decline (r=0.43–0.57, p <0.01–0.001). Both histological and in‐vivo imaging studies revealed greater Aβ deposition in retinal arterioles, particularly in secondary and tertiary branches, compared to venules or capillaries ( p <0.01–0.0001). Retinal perivascular APs in secondary/tertiary branches imaged in living patients correlated with cognitive impairment (global CDR, MOCA), hippocampal atrophy, and WMH lesions (r=0.51–0.66, p <0.05–0.001). Perivascular APs in the secondary (β=0.397, p = 0.033) and tertiary (β=0.756, p = 0.017) arteriolar branches showed significant interactions with hippocampal volumetry for visual memory. Conclusion Retinal arteriolar Aβ accumulation, pericyte loss, and TJ dysfunction contribute to early AD vascular pathology. If validated in larger cohorts, these findings support retinal vascular biomarkers as tools for predicting cognitive decline, monitoring AD progression, and assessing CAA severity.

Abstract Background Our group recently performed the largest‐to‐date X‐chromosome wide association study (XWAS) of Alzheimer's disease (AD). Here, we expand on this with additional samples, novel sex‐ and APOE *4‐stratified analyses, and evaluate escape from random X chromosome inactivation (eXCI) to better capture female‐biased risk genes. We then integrate these results with XWASs of various AD‐relevant traits to elucidate shared genetic signals and ultimately uncover novel AD risk genes and mechanisms. Method The AD XWAS design is shown in Figure 1A and includes random X chromosome inactivation (rXCI) and eXCI models. We further assessed suggestive AD signals ( p <1x10 ‐3 ) for pleiotropy using genetic colocalization with 2852 AD‐relevant traits (Figure 2A). Loci exhibiting X chromosome‐wide significance ( p <1x10 ‐5 ) or apparent trait colocalization underwent quantitative trait locus (QTL) colocalization analyses across 62 tissues and cell types to functionally prioritize AD risk genes. Result Full sample analyses reproduced Belloy et al. 2024 findings and identified 3 additional loci, including a male‐specific signal near GLUD2 that impacts glutamate/glutamine metabolism (Figure 1B). APOE *4‐stratified analyses revealed 9 additional loci, including 3 eXCI hits in APOE *4 carriers (Figure 1C‐D). Pleiotropy colocalization identified 38 loci, most being novel and many driven by eXCI and/or female‐specific AD XWAS, including MOSPD2, PNMA3 and GABRE loci that showed extensive colocalization with brain imaging traits (Figure 2B). The most significant XWAS locus, SLC9A7 , colocalized with total testosterone, HDL‐C, and various brain imaging traits. Only the NLGN4X locus colocalized with amyloid positivity, linking its proposed role in synaptic dysfunction to amyloid pathology. The locus at KDM6A , previously implicated as an AD resilience gene, colocalized with a functional MRI (fMRI) phenotype. QTL colocalization analyses confirmed gene prioritizations in Belloy et al. 2024, and additionally provided support for PNMA3 , an antigen highly expressed in the brain, and 3 APOE ‐stratified loci, including ACLS4 , which impacts lipid metabolism. Conclusion This study provides novel insights into biological mechanisms that contribute to X‐linked genetic risk for AD in an APOE *4 and sex‐specific manner, identifying various novel risk loci. Notably, 29 female‐biased risk loci were uncovered, which may shed light on sex disparities in AD. Overall, our findings will aid in elucidating AD pathways and drug targets relevant to personalized medicine.

Abstract Background Alzheimer's disease (AD) affects all brain cells and has complex genomic and immunological alterations. Previous research discovered missense AD risk or protective variants in microglial genes ABI3 and PLCG2, respectively. Expression levels of these genes are altered in AD and can influence microglial function. This study aims to uncover protective, and risk microglial molecular signatures associated with these variants to determine their role in microglial subtypes and states in AD by single cell expression and functional studies. Method We generated microglia‐enriched snRNAseq data from donors harboring either AD protective PLCG2 or AD risk ABI3 missense mutations, or neither mutation. After standard snRNAseq QC, we performed differential expressed gene (DEG) analysis of all microglial cells between variant carriers and non‐carriers using MAST. We defined protective signature as genes that are both down in ABI3 and up in PLCG2 mutation‐carriers. In contrast, risk signature genes are up in ABI3 and down in PLCG2 mutation‐carriers. We investigated the conservation of protective and risk signatures across multiple datasets, including scRNAseq data from iPSC‐derived microglia cells carrying PLCG2 protective variant, snRNAseq data from AD‐resilient donors, and data from AD and other diagnostic groups across multiple brain regions sourced from external datasets. Result We obtained snRNAseq profiles of 35,000 microglia from AD variant carriers. Our DEG analysis among all microglia cells provided 227 microglial protective and 293 risk signature genes defined by these variants. Using integrated analysis of multiple internal and external datasets, we further narrowed down these signatures. We determined that these high‐confidence protective signature genes are downregulated in early AD, upregulated in late AD brains and positively‐correlated with protective variant load in in vitro models. In contrast, risk signature genes are upregulated in early AD, downregulated in late AD and resilient donors. Risk signature expression is decreased with protective PLCG2 variant load, and altered with Aβ treatment in in vitro models. Conclusion Our study uncovers microglia specific protective and risk signatures associated with AD using sn/scRNAseq datasets from multiple sources and models. These findings nominate novel immune targets and pathways with implications for microglial function in health and disease, and ultimately therapeutic potential.

Abstract Background Disparities in structural and social determinants of health in Latino and Black communities may increase the burden of Alzheimer disease and related dementias (ADRD). Identifying dementia risk factors, which may account for up to 45% of ADRD risk, is crucial to implement effective risk‐reduction strategies. This study aimed to assess ADRD risk factors in community‐dwelling, cognitively normal, Latino and Black Americans by measuring the frequency of modifiable dementia risk factors through subjective and objective measures. Method Community‐dwelling, cognitively normal, Latino and Black adults (≥45‐years‐old) were enrolled in a longitudinal study of memory and aging at Mayo Clinic in Jacksonville, Florida. Baseline assessments conducted between Mach 2022 and November 2024 included demographics, self‐reported race/ethnicity, and history of dementia risk factors. Socioeconomic deprivation [Area Deprivation Index (ADI) State scores] and modifiable dementia risk factors were reported by participants and directly measured, including hypertension (BP >130/80 mmHg), obesity (BMI≥30), hyperlipidemia (LDL>100 mg/dL), hyperglycemia (HbA1C >5.6%), low vitamin B12 (B12<350 pg/mL), hearing loss (bedside assessment), vision loss (bedside assessment), and depression (Geriatric Depression Scale ≥5). Result 105 participants (47 Latino, 55 Black), 64.7 (±8.8) years‐old, female 66 (63%), with 16 (±2.3) years of education were assessed. Black participants experienced higher socioeconomic deprivation than Latino participants (median ADI 7 vs 3; p <0.001). Dementia risk factors were common in Latino and Black participants. These included, hypertension (55.3%, 70.3%; p = 0.291), hyperlipidemia (58.7%, 55.8%; p = 0.908), obesity (29.8%, 45.5%; p = 0.263), hyperglycemia (51.1%, 49.1%; p = 0.834), low vitamin B12 (23.9%, 28.0%; p = 0.270), hearing loss (19.1%, 7.3%; p = 0.111), vision loss (85.1%, 85.2%; p = 0.771), and depression (6.4%, 5.5%; p = 0.168). Objective measures revealed a higher frequency of risk factors compared with participant‐provided history, highlighting a substantial proportion of unrecognized or inadequately treated risk factors: hypertension (62.3%, 45.1%; p = 0.067), hyperlipidemia (57.6%, 47.5%; p = 0.004), hyperglycemia (49%, 20%; p = <0.001), vitamin B12 deficiency (27.8%, 9.3%; p = 0.170), and depression (6.9%, 11.9%; p = 0.009). Conclusion Modifiable dementia risk factors were detected in most cognitively normal Latino and Black adults engaged in a longitudinal study of memory and aging. Objective measures highlighted unrecognized and undertreated risk factors. Active surveillance programs are needed to detect and treat modifiable ADRD risk factors in underrepresented populations.