Journals
2025 EN
Huhmer Andreas · Zhang Zhengjian · Budamagunta Vivek
+6 more
Abstract Background The microtubule‐associated protein Tau (MAPT) is implicated in various progressive neurodegenerative diseases, including Alzheimer's Disease (AD). With six primary splicing isoforms and over 70 different post‐translational modifications (PTMs) identified on Tau, its proteoform diversity is extensive. However, a significant knowledge gap remains regarding the prevalence of specific proteoforms and their role in the progression of neurodegenerative diseases. Method We employed a novel single‐molecule assay utilizing the Nautilus proteome analysis platform to investigate the Tau proteoform landscape in human brain samples. This platform immobilizes individual protein molecules on a hyper‐dense flow cell, which are then probed iteratively with splice‐variant specific or PTM‐specific antibodies. Each Tau molecule's proteoform is estimated based on the pattern of antibody binding, with the overall abundance of each proteoform quantified by advanced data‐processing software that corrects for potential binding errors and off‐target interactions. Result We analyzed brain samples from seven individuals, five diagnosed with Alzheimer's and two controls, assessing the diversity and abundance of Tau proteoforms. Our findings reveal a complex proteoform landscape with distinct differences in the maturity and phosphorylation patterns of Tau isoforms between affected individuals and controls. Notably, hyperphosphorylation was prevalent in samples from Alzheimer's patients, with clear differentiation between proteoforms with minimal phosphorylation and those with extensive modifications. Conclusions The single molecule platform has proven effective in quantifying the molecular heterogeneity of Tau proteoforms in human brain tissue. This analysis begins to enhance our understanding of Tau's role in Alzheimer's disease, highlighting its potential for developing more sensitive diagnostic approaches based on proteoform heterogeneity. This study sets the stage for future research on the impact of specific Tau proteoforms on neurodegenerative disease progression and their utility as biomarkers for AD.
Journals
2025 EN
Sands Mark · Benitez Bruno A. · Wallace Clare E
+17 more
Abstract Background Although lysosome dysfunction has been implicated in Alzheimer's disease (AD), it is unclear what level or type of dysfunction is pathogenic. We hypothesize that haploinsufficiency of lysosomal enzymes is associated with AD and that CNS‐directed, AAV‐mediated gene transfer can be an effective treatment. Method Although lysosome dysfunction has been implicated in Alzheimer's disease (AD), it is unclear what level or type of dysfunction is pathogenic. We hypothesize that haploinsufficiency of lysosomal enzymes is associated with AD and that CNS‐directed, AAV‐mediated gene transfer can be an effective treatment. Result Heterozygous protein‐damaging mutations in several lysosomal enzyme genes are enriched in AD patients compared to matched controls. Proteomic analysis shows that the lysosomal storage disease pathway is activated in the brains of AD patients. There is a gene‐dosage effect on Ab 40 levels in brain ISF between WT, heterozygous, and homozygous PPT1 deficient mice. Although APP is not changed, the levels of a‐, b‐, and g‐secretases are altered in PPT1 heterozygous mice in a pattern that favors an amyloidogenic pathway. Heterozygosity of PPT1 increases Ab plaques, insoluble Ab40 and Ab42 levels, and decreases the life span of 5xFAD mice. Consistent with the human genetic and murine data, the AD pathway is activated in heterozygous PPT1 sheep. AAV‐mediated gene therapy in 5xFAD/PPT1+/‐ mice decreased the Ab burden, increased life span, and improved cognitive function. The effects of heterozygosity of the NAGLU, GALC, IDUA , and GUSB genes on Ab pathology were nearly identical to those observed for PPT1 heterozygosity. Conclusion These data strongly implicate heterozygosity of at least five, likely more, lysosomal enzyme genes in the development of AD and these genes might be effective therapeutic targets in certain genetically‐defined forms of AD.
Journals
2025 EN
Dixon Steven G · Cook Maxwell · Carroll Steven L
+1 more
Abstract Background Epidemiological studies have identified an association between the exposure to neurotoxic heavy metals such as cadmium (Cd) and an increased risk for Alzheimer's disease (AD). However, the cellular and molecular mechanisms by which Cd or lead (Pb) exposure affects AD pathogenesis and progression are largely unknown. The goal of this study was to determine how Cd and Pb exposure impact cognitive function and AD progression using the 5xFAD transgenic mouse model of AD. Methods Juvenile (5‐weeks old) 5xFAD mice were challenged with 0.2% Pb or 0.002% Cd in drinking water for 6 weeks to model chronic environmental exposures. Y‐maze tests were performed to assess spatial memory and learning ability. The Open Field Maze test was employed to examine anxiety‐like behaviors. Senescent cells were determined using senescence‐associated β‐galactosidase staining. Quantitative RT‐PCR assays were performed to measure expression levels of senescence markers and senescence‐associated secretory phenotype (SASP) related cytokines. Immunofluorescence and confocal microscopy were used to analyze senescent cells and neuroinflammation markers in brain tissues. Results Cd and Pb both can accelerate the pace of amyloid beta (Aβ) plaque formation and the decline of cognitive functions in 5xFAD mice. Even at a 100‐fold lower dose, chronic Cd exposure causes a greater level of Aβ deposition and cognitive deficits than Pb does, indicating that Cd is more potent than Pb in exacerbating AD progression. Mechanistically, we found that the increased expression of the p16 Ink4a senescence marker was more pronounced in the hippocampus of Cd‐exposed mice compared to those treated with Pb. Notably, Cd upregulates the expression of the SASP marker IL‐6 preferentially in the hippocampus over the cortex, suggesting a spatial difference in Cd‐induced increase in neuroinflammation. Furthermore, in vitro mechanistic studies reveal that Cd exposure induces premature senescence in human microglial cells, and senescent microglia release high levels of IL‐6, a major modulator of neuroinflammation. Conclusion Our studies demonstrate that chronic exposure to Cd or Pb can exacerbate cognitive deficits and AD progression in 5xFAD mice. Mechanistically, we discovered that Cd exposure‐induced acceleration of AD progression was associated with increased levels of senescent cell burden and neuroinflammation in the hippocampus.
Journals
2025 EN
Zhou Weichen · Mumm Camille · Gan Yanming
+11 more
Abstract Background Somatic mutations in individual cells lead to genomic mosaicism, contributing to the intricate regulatory landscape of genetic disorders and cancers. To evaluate and refine the detection of somatic mosaicism across different technologies with personalized donor‐specific assembly (DSA), we obtained tissue from the dorsolateral prefrontal cortex (DLPFC) of a post‐mortem neurotypical 31‐year‐old individual. Method We sequenced bulk DLPFC tissue using Oxford Nanopore Technologies (∼60X), NovaSeq (∼30X), and linked‐read sequencing (∼28X). Additionally, we applied Cas9 capture methodology coupled with long‐read sequencing (TEnCATS), targeting active transposable elements. We also isolated and amplified DNA from flow‐sorted single DLPFC neurons using MALBAC, sequencing 115 of these MALBAC libraries on Nanopore and 94 on NovaSeq. Result We constructed a haplotype‐resolved assembly with a total length of 5.77 Gb and a phase block length of 2.67 Mb (N50) to facilitate cross‐platform analysis of somatic genetic variations. We observed an increase in the phasing rate from 11.6% to 38.0% between short‐read and long‐read technologies. By generating a catalog of phased germline SNVs, CNVs, and TEs from the assembled genome, we applied standard approaches to recall these variants across sequencing technologies. We achieved aggregated recall rates from 97.3% to 99.4% based on long‐read bulk tissue data, setting an upper bound for detection limits. Moreover, utilizing haplotype‐based analysis from DSA, we achieved a remarkable reduction in false positive somatic calls in bulk tissue, ranging from 14.9% to 72.4%. We developed pipelines leveraging DSA information to enhance somatic large genetic variant calling in long‐read single cells. By examining somatic variation using long‐reads in 115 individual neurons, we identified 468 candidate somatic heterozygous large deletions (1.5Mb ‐ 20Mb), 137 of which intersected with short‐read single‐cell data. Additionally, we identified 61 putative somatic TEs (60 Alu s, one LINE‐1) in the single‐cell data. Conclusion Collectively, our analysis spans personalized assembly to single‐cell somatic variant calling, providing a comprehensive ab initio ad finem approach and resource in real human tissue.
Journals
2025 EN
Abeer Muhammad I · Boone Steven · Gitcho Michael A.
Abstract Background There are currently more than 6 million Americans age 65 or older diagnosed with Alzheimer's disease (AD) which is the seventh leading cause of death in the United States and the most common cause of dementia among older adults [1]. Phenotypically mimicking AD, limbic‐ predominant age‐related TDP‐43 encephalopathy (LATE) accounts for 30‐50% of dementia cases in those older than 80 years of age [2]. Apolipoprotein E epsilon 4 (APOE4) is a major genetic risk factor for AD. Genetic, clinical, and biochemical studies are incomplete in understanding the relationship between APOE4 and TDP‐43 pathology in AD. The overall goal of this study is to investigate the functional relationship between TDP‐43 and APOE. Method We utilized primary mouse astrocytes and human hippocampal tissue from AD and LATE cases to study APOE and TDP‐43 interactions. We employed Western blot, co‐immunoprecipitation, immunofluorescence and proteomics to study this interaction. The plasmid for pLX304‐V5 vector (Addgene plasmid #25890) was used as a control and pCMV4‐ APOEe4 was provided from Bradley Hyman (Addgene plasmid # 87087). Results We identified an interaction between TDP‐43 and APOE through immunoprecipitation followed by proteomics in mice, verified in human brain by co‐ immunoprecipitation. In addition to that we identified a difference in post translational modifications of APOE between AD patients without TDP‐43 pathology and LATE patients with TDP‐43 pathology. We further show in a cellular model of APOE4 overexpression that endogenous TDP‐43 interacts in complex with APOE4. In addition, primary mouse astrocytes challenged with oxidative stress show a significant increase in endogenous APOE/TDP‐43 colocalization. Conclusion Our results indicate a functional relationship between TDP‐43 and APOE which could offer more insights into the pathology of these diseases.
Journals
2025 EN
Spyropoulos Demetri · Jenkins Dorea P · Carroll Steven L
Abstract Background Frontotemporal dementia (FTD) involves progressive deterioration of behavior, executive function, personality/traits and is evident structurally as frontotemporal lobar degeneration (FTLD). FTLD with pathological TDP43 neural/glial inclusions (FTLD‐TDP), often coexists with TDP43‐positive amyotrophic lateral sclerosis (ALS). Several families have been identified that inherit autosomal dominant FTLD/ALS or ALS alone and mutations in the gene encoding the ERBB4 receptor tyrosine kinase (RTK). Although the neuropathology associated with ERBB4 mutations remains obscure, ERBB4 with p.R927Q or p.I712M mutations show reduced phosphorylation when stimulated with the ERBB4 ligand neuregulin‐1β, suggesting that reduced ERBB4 activity causes FTLD/ALS or ALS. Method We generated mice carrying p.I712M (familial FTLD/ALS) or p.R927Q (familial ALS only) Erbb4 mutations. We performed initial studies of behavior (Barnes Maze), gait‐mobility (Catwalk‐XT) and body composition (DXA scans) on Erbb4 heterozygous and homozygous mutants and wild‐type littermates. Result Barnes Maze Acquisition was performed on males ( N = 4‐5/genotype) after habituation and several days training. Heatmaps of grouped averages for acquisition day 5 show that ErbB4‐R927Q mutants (homozygotes > heterozygotes) take longer finding the goal box, suggesting a gene dosage‐dependent defect in spatial learning. CatWalk‐XT was performed on Erbb4‐R927Q mutant females ( N = 4‐6/genotype), with six complaint post‐training walks/animal. Significantly lower mean intensities of the most intense paw prints occurred in the hindlimbs of mutants (Tukey's 2‐way ANOVA). Multiple gene‐dosage‐dependent trends in altered gait metrics occurred, including footfall patterns, 2D/3D mean/max intensities, footfall sequence, print positions, standing and diagonal mean phase dispersions. DXA scans on Erbb4‐R927Q mutant and wild‐type males ( N = 5‐9 per genotype) showed decreased body weights and fat percentages in homozygotes. Whole body and limb regions of interest showed decreases in bone mineral density and content that was greater in homozygotes than in heterozygotes. Conclusion Initial findings suggest gene dosage‐dependent abnormalities in Erbb4‐R927Q mutants that have features of FTLD and ALS. A wider battery of tests on more animals, at different ages and including the Erbb4‐I712M mutants are underway. Neuropathology is being assessed following consensus recommendations for FTLD and ALS. Stereology/morphometry and other methods will assess loss of ErbB4 ‐expressing inhibitory interneurons and synaptic loss and how this evolves temporally.
Journals
2025 EN
Fisher Elizabeth S · Tubbesing Kate · Stevens Katherine
+7 more
Abstract Background Genome‐wide association studies (GWAS) have identified single‐nucleotide polymorphisms (SNPs) influencing the development and progression of Late‐Onset Alzheimer's Disease (LOAD), including low‐frequency variants. Phospholipase D3 (PLD3), an atypical phospholipase, harbors a rare variant, p.A442A, previously shown to double the risk of LOAD. PLD3 has been implicated in Amyloid Precursor Protein (APP) processing and Aβ development, potentially affecting amyloid deposition. Our research using bulk RNA sequencing (RNA‐seq) in mice suggested that PLD3 plays a role in vascular function; however, its specific role in endothelial cells (ECs) and supporting mural cells (pericytes, smooth muscle cells, and fibroblasts) remains unexplored. Methods We investigated the impact of PLD3 using APP/PS1xPLD3 KO mice and analyzing RNA‐seq from the cortex and comparing it to controls. Additionally, we utilized human‐induced pluripotent stem cells (iPSCs) to examine the effects of the PLD3 variant and PLD3 knockdown in ECs and mural cells. iPSCs were differentiated into ECs and mural cells and cultured separately in 2D or together in 3D vascular networks, which model EC‐mural cell interactions. We assessed PLD3 variant and knockdown effects in purified ECs and mural cells through RNA‐seq, immunohistochemistry, and cytokine release. EC barrier function was evaluated using electric cell‐substrate impedance sensing (ECIS). Results In APP/PS1xPLD3 KO mouse brains, pathways related to vascular function and inflammation were downregulated, while protein degradation pathways were upregulated. In iPSC models, PLD3 variant and knockdown ECs exhibited altered cell division and APP processing pathways, with specific changes in AD‐GWAS genes ADAM23, APOC1 , and NRCAM . PLD3 variant mural cells showed disruptions in extracellular matrix remodeling, including FN1 , and MMP25 . In 3D vascular models, the PLD3 variant led to altered IL‐8 release, which may have implications for vascular remodeling. Conclusion Our findings demonstrate that PLD3 influences vascular function and inflammation in mice, potentially contributing to disease pathogenesis. In iPSC‐derived vascular models, PLD3 regulates EC proliferation and APP processing as well as mural cell matrix organization, suggesting that this variant may lead to vascular remodeling or instability. Future studies will explore the underlying mechanisms of these alterations and assess whether they can be targeted to prevent vascular dysfunction.
Journals
2025 EN
Jenkins Dorea P · Davis Laura · Belk Jennifer
+4 more
Abstract Background South Carolina has an extraordinarily high prevalence of Alzheimer's disease (AD) and AD‐related dementias (ADRDs), with approximately 122,699 individuals living with dementia. Mapping of ICD‐10 codes shows that these diseases are non‐uniformly distributed, with multiple “hot spots” in the state. Due to the complexity of these diseases, a post‐mortem examination of the brain is required for an accurate diagnosis. The Carroll A. Campbell, Jr. Neuropathology Laboratory (CCNL) at the Medical University of South Carolina (MUSC) has thus performed detailed histologic examination of 343 cases to define the causes of dementia in South Carolina, determine the prevailing ADRD type across SC and whether these deviated from national averages and establish whether specific kinds of neurodegenerative diseases were concentrated in the “hot spots”. Method Donors were recruited via physicians, community outreach, and our website. Gross and microscopic examination of the brain was performed following consensus recommendations and using a panel of stains including hematoxylin and eosin (H&E), modified Bielschowsky stains, and immunostains for Abeta, hyperphosphorylated tau, alpha‐synuclein, p62, p ‐TDP43, 3‐repeat and 4‐repeat tau. All cases were reviewed by a board‐certified neuropathologist. Diagnoses were mapped at a zip‐code level. Result The 343 cases included 195 male and 148 female donors, with the average age of donors being 73.8 years. 148 cases (43.1%) had an AD diagnosis alone or in combination with another disease. 20.3% of the AD cases were early onset AD, as opposed to the usual 5‐10%. Frontotemporal lobar degeneration (FTLD)‐tau represented 63.6% of the FTLD cases (normally 45%) of cases, with FTLD‐TDP representing 26.4% of cases (normally 45%). Multiple system atrophy also occurred at a higher‐than‐expected rate. Pure AD was uncommon, with the majority of donors with AD having multiple neurodegenerative diseases. Mapping of these diagnoses indicated that certain types of neurodegenerative diseases clustered in specific regions of the state. Conclusion The results of these autopsies suggest that South Carolina has a higher‐than‐expected representation of early onset AD and FTLD‐tau and that certain types of neurodegenerative disease cluster at specific locations. We are currently performing whole genome sequencing to determine whether specific disease‐causing genetic variants contribute to these diagnostic skews.
Journals
2025 EN
Marx Hannah · Jacova Claudia · Robertson Frank
+3 more
Abstract Background In recent years, a significant effort has been made toward digitizing neuropsychological measures in an effort to increase accessibility. Results of this research demonstrate that digitized versions, when conducted in a supervised setting, are comparable to traditional paper‐and‐pencil tests. The validity of unsupervised online tests is less well known. In the present study we examined how an unsupervised, self‐administered online version of a list learning task compares to traditional versions among a cognitively healthy, older adult sample. Method A community‐based sample ( n = 279) of English‐speaking older adults, aged 60‐96 ( M = 73.8), with a mean education of 14.9 (range=10‐20), majority female (52%), without existing neurocognitive diagnoses, self‐administered a Qualtrics online survey from home, which included a digitized list learning task based on the Rey Auditory Verbal Learning Task (RAVLT). Participants were visually exposed to, and asked to recall, a novel list of 15 words over the course of three learning trials. Then they were presented with an interference list, and later asked to recall the initial list of words following a delay. Total correct words recalled from each trial were summed, and mean score differences were compared across gender, years of education, and age quartiles (Q2=68, Q3=74, Q4=80). Result Participants recalled an average of 5.4 (SD=2.7), 8.8 (SD=3.1), 10.0 (SD=3.2), and 7.0 (SD=3.9) words on Trials 1‐3 and Delayed Recall respectively. Females recalled more words across all trials (all t tests p < .05). Neither education or age were significantly correlated with performance; however, upon further inspection, those in the second quartile (ages 68‐73) recalled approximately one word more than the other age groups. Conclusion Our results generally align well with published AVLT norms. They are also consistent with existing research suggesting that females perform better on verbal learning tasks. However, the absence of a relationship between performance and age or education suggests that the utilization of an unsupervised, digital verbal learning task must be approached with caution.
Journals
2025 EN
Kang Moonil · Karjadi Cody · Ang Ting Fang Alvin
+9 more
Abstract Background Digital cognitive assessment may offer earlier Alzheimer's disease (AD) and related dementias (ADRD) detection compared with traditional neuropsychological (NP) tests. Subjective cognitive decline (SCD) is recognized as a preclinical AD/ADRD feature. We hypothesized that among cognitively unimpaired (CU) individuals by traditional NP tests, the digital clock drawing test (dCDT) would be associated with concurrent SCD and with future objective cognitive impairment. Method Participants from the Framingham Heart Study were followed longitudinally with assessment for SCD and NP testing, including dCDT, and were surveilled for mild cognitive impairment (MCI), AD, and all‐cause dementia. This study included CU participants at analytic baseline (first visit at/after age 60). Participants with SCD at baseline or in the future, defined by SCD‐plus criteria, were matched on age, sex, and education with participants without SCD. Using data from the latest CU visit, we conducted cross‐sectional analyses to test associations between dCDT (overall and four composite scores—drawing efficiency, information processing, simple and complex motor, spatial reasoning—in command and copy conditions) and SCD. Additionally, we examined associations between dCDT at baseline and time to MCI, AD, and all‐cause dementia. Models were adjusted for traditional NP performance ( i . e ., cognitive scores for executive function, language, memory), age, sex, education, APOE4 and APOE2 carrier status, and familial relatedness. Result Among 1,601 participants (mean age at the latest CU visit: 71.6, women: 54.7%, SCD: 32.5%), 9.5%, 2.0%, and 2.6% developed MCI, AD, and all‐cause dementia, respectively. Better performance on dCDT—but not traditional NP tests—was associated with reduced odds of SCD: overall (OR=0.86, p = .007); drawing efficiency, command (OR=0.83, p = .002); information processing, command (OR=0.75, p = 1.14×10 ‐5 ) and copy (OR=0.83, p = .005). Better dCDT performance, after traditional NP performance adjustment, was associated with reduced hazards of future MCI and all‐cause dementia: overall dCDT (MCI: HR=0.88, p = .03; all‐cause dementia: HR=0.78, p = .02), drawing efficiency, command (MCI: HR=0.88, p = .03), and information processing, command (MCI: HR=0.85, p = .01; all‐cause dementia: HR=0.79, p = .03). Conclusion In this community‐based study of CU individuals, dCDT was associated with SCD and time to MCI and all‐cause dementia, independent of traditional NP test performance. dCDT may capture subtle AD/ADRD cognitive changes not detected by traditional NP assessment.