Postmortem Alzheimer's Disease Research Articles

Probing Alzheimer's pathology: Exploring the next generation of FDDNP analogues for amyloid β detection

Fluorescent probes are a powerful tool for imaging amyloid β (Aβ) plaques, the hallmark of Alzheimer’s disease (AD). Herein, we report the synthesis and comprehensive characterization of 21 novel probes as well as their optical properties and binding affinities to Aβ fibrils. One of these dyes, 1Ae, exhibited several improvements over FDDNP, an established biomarker for Aβ- and Tau-aggregates. First, 1Ae had large Stokes shifts (138–213 nm) in various solvents, thereby reducing self-absorption. With a high quantum yield ratio (φ(dichloromethane/methanol) = 104), 1Ae also ensures minimal background emission in aqueous environments and high sensitivity. In addition, compound 1Ae exhibited low micromolar binding affinity to Aβ fibrils in vitro (Kd = 1.603 µM), while increasing fluorescence emission (106-fold) compared to emission in buffer alone. Importantly, the selective binding of 1Ae to Aβ1–42 fibrils was confirmed by an in cellulo assay, supported by ex vivo fluorescence microscopy of 1Ae on postmortem AD brain sections, allowing unequivocal identification of Aβ plaques. The intermolecular interactions of fluorophores with Aβ were elucidated by docking studies and molecular dynamics simulations. Density functional theory calculations revealed the unique photophysics of these rod-shaped fluorophores, with a twisted intramolecular charge transfer (TICT) excited state. These results provide valuable insights into the future application of such probes as potential diagnostic tools for AD in vitro and ex vivo such as determination of Aβ1–42 in cerebrospinal fluid or blood.

Role of Neurocellular Endoplasmic Reticulum Stress Response in Alzheimer's Disease and Related Dementias Risk.

Currently, more than 55 million people around the world suffer from dementia, and Alzheimer's Disease and Related Dementias (ADRD) accounts for nearly 60-70% of all those cases. The spread of Alzheimer's Disease (AD) pathology and progressive neurodegeneration in the hippocampus and cerebral cortex is strongly correlated with cognitive decline in AD patients; however, the molecular underpinning of ADRD's causality is still unclear. Studies of postmortem AD brains and animal models of AD suggest that elevated endoplasmic reticulum (ER) stress may have a role in ADRD pathology through altered neurocellular homeostasis in brain regions associated with learning and memory. To study the ER stress-associated neurocellular response and its effects on neurocellular homeostasis and neurogenesis, we modeled an ER stress challenge using thapsigargin (TG), a specific inhibitor of sarco/endoplasmic reticulum Ca2+ ATPase (SERCA), in the induced pluripotent stem cell (iPSC)-derived neural stem cells (NSCs) of two individuals from our Mexican American Family Study (MAFS). High-content screening and transcriptomic analysis of the control and ER stress-challenged NSCs showed that the NSCs' ER stress response resulted in a significant decline in NSC self-renewal and an increase in apoptosis and cellular oxidative stress. A total of 2300 genes were significantly (moderated t statistics FDR-corrected p-value ≤ 0.05 and fold change absolute ≥ 2.0) differentially expressed (DE). The pathway enrichment and gene network analysis of DE genes suggests that all three unfolded protein response (UPR) pathways, protein kinase RNA-like ER kinase (PERK), activating transcription factor-6 (ATF-6), and inositol-requiring enzyme-1 (IRE1), were significantly activated and cooperatively regulated the NSCs' transcriptional response to ER stress. Our results show that IRE1/X-box binding protein 1 (XBP1) mediated transcriptional regulation of the E2F transcription factor 1 (E2F1) gene, and its downstream targets have a dominant role in inducing G1/S-phase cell cycle arrest in ER stress-challenged NSCs. The ER stress-challenged NSCs also showed the activation of C/EBP homologous protein (CHOP)-mediated apoptosis and the dysregulation of synaptic plasticity and neurotransmitter homeostasis-associated genes. Overall, our results suggest that the ER stress-associated attenuation of NSC self-renewal, increased apoptosis, and dysregulated synaptic plasticity and neurotransmitter homeostasis plausibly play a role in the causation of ADRD.

Seizures exacerbate excitatory: inhibitory imbalance in Alzheimer's disease and 5XFAD mice.

Approximately 22% of Alzheimer's disease (AD) patients suffer from seizures, and the co-occurrence of seizures and epileptiform activity exacerbates AD pathology and related cognitive deficits, suggesting that seizures may be a targetable component of AD progression. Given that alterations in neuronal excitatory:inhibitory (E:I) balance occur in epilepsy, we hypothesized that decreased markers of inhibition relative to those of excitation would be present in AD patients. We similarly hypothesized that in five times familial AD (5XFAD) mice, the E:I imbalance would progress from an early stage (prodromal) to later symptomatic stages, and be further exacerbated by pentylenetetrazol (PTZ) kindling. Post-mortem AD temporal cortical tissue from patients with or without seizure history were examined for changes in several markers of E:I balance, including levels of the inhibitory GABAA receptor, the sodium potassium chloride cotransporter 1 (NKCC1) and potassium chloride cotransporter 2 (KCC2), and the excitatory NMDA and AMPA type glutamate receptors. We performed patch clamp electrophysiological recordings from CA1 neurons in hippocampal slices and examined the same markers of E:I balance in prodromal 5XFAD mice. We next examined 5XFAD mice at chronic stages, after PTZ or control protocols, and in response to chronic mTORC1 inhibitor rapamycin, administered following kindled seizures, for markers of E:I balance. We found that AD patients with comorbid seizures had worsened cognitive and functional scores and had decreased GABAA receptor subunit expression, and increased NKCC1/KCC2 ratios, indicative of depolarizing GABA responses. Patch clamp recordings of prodromal 5XFAD CA1 neurons showed increased intrinsic excitability, along with decreased GABAergic inhibitory transmission and altered glutamatergic neurotransmission, indicating that E:I imbalance may occur in early disease stages. Furthermore, seizure induction in prodromal 5XFAD mice led to later dysregulation of NKCC1/KCC2 and a reduction in GluA2 AMPA glutamate receptor subunit expression, indicative of depolarizing GABA receptors and calcium permeable AMPA receptors. Finally, we found that chronic treatment with the mTORC1 inhibitor, rapamycin, at doses we have previously shown to attenuate seizure-induced β-amyloid pathology and cognitive deficits, could also reverse elevations to NKCC1/KCC2 ratio in these mice. Our data demonstrate novel mechanisms of interaction between AD and epilepsy and indicate that targeting E:I balance, potentially with FDA-approved mTOR inhibitors, hold therapeutic promise for AD patients with a seizure history.

Ultrastructural localization of Porphyromonas gingivalis gingipains in the substantia nigra of Parkinson’s disease brains

Gingipains are protease virulence factors produced by Porphyromonas gingivalis, a Gram-negative bacterium best known for its role in chronic periodontitis. Gingipains were recently identified in the middle temporal gyrus of postmortem Alzheimer’s disease (AD) brains, where gingipain load correlated with AD diagnosis and tau and ubiquitin pathology. Since AD and Parkinson’s disease (PD) share some overlapping pathologic features, including nigral pathology and Lewy bodies, the current study explored whether gingipains are present in the substantia nigra pars compacta of PD brains. In immunohistochemical techniques and multi-channel fluorescence studies, gingipain antigens were abundant in dopaminergic neurons in the substantia nigra of both PD and neurologically normal control brains. 3-dimensional reconstructions of Lewy body containing neurons revealed that gingipains associated with the periphery of alpha-synuclein aggregates but were occasionally observed inside aggregates. In vitro proteomic analysis demonstrated that recombinant alpha-synuclein is cleaved by lysine-gingipain, generating multiple alpha-synuclein fragments including the non-amyloid component fragments. Immunogold electron microscopy with co-labeling of gingipains and alpha-synuclein confirmed the occasional colocalization of gingipains with phosphorylated (pSER129) alpha-synuclein. In dopaminergic neurons, gingipains localized to the perinuclear cytoplasm, neuromelanin, mitochondria, and nucleus. These data suggest that gingipains localize in dopaminergic neurons in the substantia nigra and interact with alpha-synuclein.

Unveiling the Role of Novel miRNA PC-5P-12969 in Alleviating Alzheimer's Disease.

The intricate and complex molecular mechanisms that underlie the progression of Alzheimer's disease (AD) have prompted a concerted and vigorous research endeavor aimed at uncovering potential avenues for therapeutic intervention. This study aims to elucidate the role of miRNA PC-5P-12969 in the pathogenesis of AD. We assessed the differential expression of miRNA PC-5P-12969 in postmortem AD brains, AD animal and cell models using real-time reverse-transcriptase RT-PCR, we also checked the gene and protein expression of GSK3α and APP. Our investigation revealed a notable upregulation of miRNA PC-5P-12969 in postmortem brains of AD patients, in transgenic mouse models of AD, and in mutant APP overexpressing-HT22 cells. Additionally, our findings indicate that overexpression of miRNA PC-5P-12969 exerts a protective effect on cell survival, while concurrently mitigating apoptotic cell death. Further-more, we established a robust and specific interaction between miRNA PC-5P-12969 and GSK3α. Our luciferase reporter assays provided confirmation of the binding between miRNA PC-5P-12969 and the 3'-UTR of the GSK3α gene. Manipulation of miRNA PC-5P-12969 levels in cellular models of AD yielded noteworthy alterations in the gene and protein expression levels of both GSK3α and APP. Remarkably, the manipulation of miRNA PC-5P-12969 levels yielded significant enhancements in mitochondrial respiration and ATP production, concurrently with a reduction in mitochondrial fragmentation, thus unveiling a potential regulatory role of miRNA PC-5P-12969 in these vital cellular processes. In summary, this study sheds light on the crucial role of miRNA PC-5P-12969 and its direct interaction with GSK3α in the context of AD.

125I]IPC-Lecanemab: Synthesis and Evaluation of Aβ-Plaque-Binding Antibody and Comparison with Small-Molecule [18F]Flotaza and [125I]IBETA in Postmortem Human Alzheimer's Disease.

Therapeutic antibodies for reducing Aβ plaque load in Alzheimer's disease (AD) is currently making rapid progress. The diagnostic imaging of Aβ plaque load in AD has been underway and is now used in clinical studies. Here, we report our preliminary findings on imaging a therapeutic antibody, Lecanemab, in a postmortem AD brain anterior cingulate. [125I]5-iodo-3-pyridinecarboxamido-Lecanemab ([125I]IPC-Lecanemab) was prepared by coupling N-succinimidyl-5-([125I]iodo)-3-pyridinecarboxylate with Lecanemab in modest yields. The distinct binding of [125I]IPC-Lecanemab to Aβ-rich regions in postmortem human AD brains was higher in grey matter (GM) containing Aβ plaques compared to white matter (WM) (GM/WM was 1.6). Anti-Aβ immunostaining was correlated with [125I]IPC-Lecanemab regional binding in the postmortem AD human brains. [125I]IPC-Lecanemab binding was consistent with the binding of Aβ small molecules, [18F]flotaza and [125I]IBETA, in the same subjects. [18F]Flotaza and [125I]IBETA, however, exhibited significantly higher GM/WM ratios (>20) compared to [125I]IPC-Lecanemab. Our results suggest that radiolabeled [125I]IPC-Lecanemab retains the ability to bind to Aβ in human AD and may therefore be useful as a PET imaging radiotracer when labeled as [124I]IPC-Lecanemab. The ability to directly visualize in vivo a promising therapeutic antibody for AD may be useful in treatment planning and dosing and could be complimentary to small-molecule diagnostic imaging to assess outcomes of therapeutic interventions.

TREM1 disrupts myeloid bioenergetics and cognitive function in aging and Alzheimer disease mouse models.

Human genetics implicate defective myeloid responses in the development of late-onset Alzheimer disease. A decline in peripheral and brain myeloid metabolism, triggering maladaptive immune responses, is a feature of aging. The role of TREM1, a pro-inflammatory factor, in neurodegenerative diseases is unclear. Here we show that Trem1 deficiency prevents age-dependent changes in myeloid metabolism, inflammation and hippocampal memory function in mice. Trem1 deficiency rescues age-associated declines in ribose 5-phosphate. In vitro, Trem1-deficient microglia are resistant to amyloid-β42 oligomer-induced bioenergetic changes, suggesting that amyloid-β42 oligomer stimulation disrupts homeostatic microglial metabolism and immune function via TREM1. In the 5XFAD mouse model, Trem1 haploinsufficiency prevents spatial memory loss, preserves homeostatic microglial morphology, and reduces neuritic dystrophy and changes in the disease-associated microglial transcriptomic signature. In aging APPSwe mice, Trem1 deficiency prevents hippocampal memory decline while restoring synaptic mitochondrial function and cerebral glucose uptake. In postmortem Alzheimer disease brain, TREM1 colocalizes with Iba1+ cells around amyloid plaques and its expression is associated with Alzheimer disease clinical and neuropathological severity. Our results suggest that TREM1 promotes cognitive decline in aging and in the context of amyloid pathology.

Micro-RNA profiles of pathology and resilience in posterior cingulate cortex of cognitively intact elders.

The posterior cingulate cortex (PCC) is a key hub of the default mode network underlying autobiographical memory retrieval, which falters early in the progression of Alzheimer's disease (AD). We recently performed RNA sequencing of post-mortem PCC tissue samples from 26 elderly Rush Religious Orders Study participants who came to autopsy with an ante-mortem diagnosis of no cognitive impairment but who collectively displayed a range of Braak I-IV neurofibrillary tangle stages. Notably, cognitively unimpaired subjects displaying high Braak stages may represent cognitive resilience to AD pathology. Transcriptomic data revealed elevated synaptic and ATP-related gene expression in Braak Stages III/IV compared with Stages I/II, suggesting these pathways may be related to PCC resilience. We also mined expression profiles for small non-coding micro-RNAs (miRNAs), which regulate mRNA stability and may represent an underexplored potential mechanism of resilience through the fine-tuning of gene expression within complex cellular networks. Twelve miRNAs were identified as differentially expressed between Braak Stages I/II and III/IV. However, the extent to which the levels of all identified miRNAs were associated with subject demographics, neuropsychological test performance and/or neuropathological diagnostic criteria within this cohort was not explored. Here, we report that a total of 667 miRNAs are significantly associated (rho > 0.38, P < 0.05) with subject variables. There were significant positive correlations between miRNA expression levels and age, perceptual orientation and perceptual speed. By contrast, higher miRNA levels correlated negatively with semantic and episodic memory. Higher expression of 15 miRNAs associated with lower Braak Stages I-II and 47 miRNAs were associated with higher Braak Stages III-IV, suggesting additional mechanistic influences of PCC miRNA expression with resilience. Pathway analysis showed enrichment for miRNAs operating in pathways related to lysine degradation and fatty acid synthesis and metabolism. Finally, we demonstrated that the 12 resilience-related miRNAs differentially expressed in Braak Stages I/II versus Braak Stages III/IV were predicted to regulate mRNAs related to amyloid processing, tau and inflammation. In summary, we demonstrate a dynamic state wherein differential PCC miRNA levels are associated with cognitive performance and post-mortem neuropathological AD diagnostic criteria in cognitively intact elders. We posit these relationships may inform miRNA transcriptional alterations within the PCC relevant to potential early protective (resilience) or pathogenic (pre-clinical or prodromal) responses to disease pathogenesis and thus may be therapeutic targets.

In vitro evaluation of [3H]PI-2620 and structural derivatives in non-Alzheimer's tauopathies

Alzheimer's disease (AD) and non-AD tauopathies such as chronic traumatic encephalopathy (CTE), progressive supranuclear palsy (PSP), and corticobasal degeneration (CBD) are characterized by the abnormal aggregation of three-repeat (3R) and/or four-repeat (4R) tau isoforms. Several tau-PET tracers have been applied for human imaging of AD and non-AD tauopathies including [18F]PI-2620. Our objective is to evaluate [3H]PI-2620 and two promising structural derivatives, [3H]PI-2014 and [3H]F-4, using in vitro saturation assays and competitive binding assays against new chemical entities based on this scaffold in human AD tissues for comparison with PSP, CBD and CTE tissues. Thin section autoradiography was employed to assess specific binding and distribution of [3H]PI-2620 and [3H]F-4 in fresh-frozen human post-mortem AD, PSP, CBD and CTE tissues. Immunohistochemistry was performed for phospho-tau (AT8) and 4R-tau (RD4). Homogenate filtration binding assays were performed for saturation analysis and competitive binding studies against [3H]PI-2620. All compounds bound with high affinity in AD tissue. In PSP tissue [3H]PI-2620 demonstrated the highest affinity (5.3 nM) and in CBD tissue [3H]F-4 bound with the highest affinity (9.4 nM). Over 40 fluorinated derivatives based on PI-2620 and F-4 were screened in AD and PSP tissue. Notably, compound 2 was the most potent derivative in PSP tissue (Ki = 7.3 nM). By autoradiography, [3H]PI-2620 and [3H]F-4 demonstrated positive signals similar in intensity in AD, PSP and CTE tissues that were displaced by homologous blockade. Binding of both radiotracers aligned with immunostaining for 4R-tau. This work demonstrates that [3H]PI-2620 and [3H]F-4 show promise for imaging 4R-tau aggregates in non-AD tauopathies. PI-2620 continues to serve as a structural scaffold for PET radiotracers with higher affinity for non-AD tau over AD tau.

Signal peptide peptidase-like 2b modulates the amyloidogenic pathway and exhibits an Aβ-dependent expression in Alzheimer's disease

Alzheimer's disease (AD) is a multifactorial disorder driven by abnormal amyloid β-peptide (Aβ) levels. In this study, we investigated the role of presenilin-like signal peptide peptidase-like 2b (SPPL2b) in AD pathophysiology and its potential as a druggable target within the Aβ cascade. Exogenous Aβ42 influenced SPPL2b expression in human cell lines and acute mouse brain slices. SPPL2b and its AD-related substrate BRI2 were evaluated in the brains of AppNL-G-F knock-in AD mice and human postmortem AD brains. An early high cortical expression of SPPL2b was observed, followed by a downregulation in late AD pathology in AppNL-G-F mice, correlating with synaptic loss. To understand the consequences of pathophysiological SPPL2b dysregulation, we found that SPPL2b overexpression significantly increased APP cleavage, while genetic deletion reduced APP cleavage and Aβ production. Notably, postmortem AD brains showed higher levels of SPPL2b's BRI2 substrate compared to healthy control samples. These results strongly support the involvement of SPPL2b in AD pathology. The early Aβ-induced upregulation of SPPL2b may enhance Aβ production in a vicious cycle, further aggravating Aβ pathology. Therefore, SPPL2b emerges as a potential anti-Aβ drug target.

Metabolic Bypass Rescues Aberrant S-nitrosylation-Induced TCA Cycle Inhibition and Synapse Loss in Alzheimer's Disease Human Neurons.

In Alzheimer's disease (AD), dysfunctional mitochondrial metabolism is associated with synaptic loss, the major pathological correlate of cognitive decline. Mechanistic insight for this relationship, however, is still lacking. Here, comparing isogenic wild-type and AD mutant human induced pluripotent stem cell (hiPSC)-derived cerebrocortical neurons (hiN), evidence is foundfor compromised mitochondrial energy in AD using the Seahorse platform to analyze glycolysis and oxidative phosphorylation (OXPHOS). Isotope-labeled metabolic flux experiments revealed a major block in activity in the tricarboxylic acid (TCA) cycle at the α-ketoglutarate dehydrogenase (αKGDH)/succinyl coenzyme-A synthetase step, metabolizing α-ketoglutarate to succinate. Associated with this block, aberrant protein S-nitrosylation of αKGDH subunits inhibited their enzyme function. This aberrant S-nitrosylation is documented not only in AD-hiN but also in postmortem human AD brains versus controls, as assessed by two separate unbiased mass spectrometry platforms using both SNOTRAP identification of S-nitrosothiols and chemoselective-enrichment of S-nitrosoproteins. Treatment with dimethyl succinate, a cell-permeable derivative of a TCA substrate downstream to the block, resulted in partial rescue of mitochondrial bioenergetic function as well as reversal of synapse loss in AD-hiN. These findings have therapeutic implications that rescue of mitochondrial energy metabolism can ameliorate synaptic loss in hiPSC-based models of AD.

Advancements in high-resolution 3D microscopy analysis of endosomal morphology in postmortem Alzheimer's disease brains.

Abnormal endo-lysosomal morphology is an early cytopathological feature of Alzheimer's disease (AD) and genome-wide association studies (GWAS) have implicated genes involved in the endo-lysosomal network (ELN) as conferring increased risk for developing sporadic, late-onset AD (LOAD). Characterization of ELN pathology and the underlying pathophysiology is a promising area of translational AD research and drug development. However, rigorous study of ELN vesicles in AD and aged control brains poses a unique constellation of methodological challenges due in part to the small size of these structures and subsequent requirements for high-resolution imaging. Here we provide a detailed protocol for high-resolution 3D morphological quantification of neuronal endosomes in postmortem AD brain tissue, using immunofluorescent staining, confocal imaging with image deconvolution, and Imaris software analysis pipelines. To demonstrate these methods, we present neuronal endosome morphology data from 23 sporadic LOAD donors and one aged non-AD control donor. The techniques described here were developed across a range of AD neuropathology to best optimize these methods for future studies with large cohorts. Application of these methods in research cohorts will help advance understanding of ELN dysfunction and cytopathology in sporadic AD.

Comprehensive Analysis of Metabolites in Postmortem Brains of Patients with Alzheimer's Disease.

Disturbed metabolism has been proposed as being involved in the pathogenesis of Alzheimer's disease (AD), and more evidence from human AD brains is required. In this study, we attempted to identify or confirm modulations in the levels of metabolites associated with AD in postmortem AD brains. We performed metabolomics analyses using a gas chromatography mass spectrometry system in postmortem brains of patients with confirmed AD, patients with CERAD score B, and control subjects. Impaired phosphorylation of glucose and elevation of several tricarboxylic acid (TCA) metabolites, except citrate, were observed and the degree of impaired phosphorylation and elevation in the levels of the TCA cycle metabolites were negatively and positively correlated, respectively, with the clinical phenotypes of AD. The levels of uronic acid pathway metabolites were modulated in AD and correlated positively with the amyloid-β content. The associations of nucleic acid synthesis and amino acid metabolites with AD depended on the kinds of metabolites; in particular, the contents of ribose 5-phosphate, serine and glycine were negatively correlated, while those of ureidosuccinic acid and indole-3-acetic acid were positively modulated in AD. Comprehensive statistical analyses suggested that alterations in the inositol pathway were most closely associated with AD. The present study revealed many novel associations between metabolites and AD, suggesting that some of these might serve as novel potential therapeutic targets for AD.

PCSK6 exacerbates Alzheimer's disease pathogenesis by promoting MT5-MMP maturation

Proprotein convertase subtilisin/kexin type 6 (PCSK6) is a calcium-dependent serine proteinase that regulates the proteolytic activity of various precursor proteins and facilitates protein maturation. Dysregulation of PCSK6 expression or function has been implicated in several pathological processes including nervous system diseases. However, whether and how PCSK6 is involved in the pathogenesis of Alzheimer's disease (AD) remains unclear. In this study, we reported that the expression of PCSK6 was significantly increased in the brain tissues of postmortem AD patients and APP23/PS45 transgenic AD model mice, as well as N2AAPP cells. Genetic knockdown of PCSK6 reduced amyloidogenic processing of APP in N2AAPP cells by suppressing the activation of membrane-type 5-matrix metalloproteinase (MT5-MMP), referred to as η-secretase. We further found that PCSK6 cleaved and activated MT5-MMP by recognizing the RRRNKR sequence in its N-terminal propeptide domain in N2A cells. The mutation or knockout of this cleavage motif prevented PCSK6 from interacting with MT5-MMP and performing cleavage. Importantly, genetic knockdown of PCSK6 with adeno-associated virus (AAV) reduced Aβ production and ameliorated hippocampal long-term potentiation (LTP) and long-term spatial learning and memory in APP23/PS45 transgenic mice. Taken together, these results demonstrate that genetic knockdown of PCSK6 effectively alleviate AD-related pathology and cognitive impairments by inactivating MT5-MMP, highlighting its potential as a novel therapeutic target for AD treatment.

Downregulation of peripheral luteinizing hormone rescues ovariectomy-associated cognitive deficits in APP/PS1 mice

Alzheimer's disease (AD) is more prevalent in women than men, supposing due to the decline of estrogens in menopause, accompanied by increased gonadotropins such as luteinizing hormone (LH). We and others found that the transcription factor early growth response-1 (EGR1) regulates cholinergic function including the expression of acetylcholinesterase (AChE) and plays a significant role in cognitive decline of AD. Here we investigated in APP/PS1 mice by ovariectomy (OVX) and estradiol (E2) supplementation or inhibition of LH the effect on hippocampus-related cognition and related molecular changes. We found that OVX-associated cognitive impairment was accompanied by increased dorsal hippocampal EGR1 expression, which was rescued by downregulating peripheral LH rather than by supplementing E2. We also found in postmortem AD brains a higher expression of pituitary LH-mRNA and higher EGR1 expression in the posterior hippocampus. Both, in human and mice, there was a significant positive correlation between respectively posterior/dorsal hippocampal EGR1 and peripheral LH expression. We conclude that peripheral increased LH and increased posterior hippocampal EGR1 plays a significant role in AD pathology.

Multitracer Approach to Understanding the Complexity of Reactive Astrogliosis in Alzheimer's Brains.

A monoamine oxidase B (MAO-B) selective positron emission tomography (PET) tracer [11C]-deuterium-l-deprenyl holds promise for imaging reactive astrogliosis in neurodegenerative diseases, such as Alzheimer's disease (AD). Two novel PET tracers ([11C]-BU99008 and [18F]-SMBT-1) have recently been developed to assess the complexity of reactive astrogliosis in the AD continuum. We have investigated the binding properties of SMBT-1, l-deprenyl, and BU99008 in AD and cognitively normal control (CN) brains. Competition binding assays with [3H]-l-deprenyl and [3H]-BU99008 versus unlabeled SMBT-1 in postmortem AD and CN temporal and frontal cortex brains demonstrated that SMBT-1 interacted with [3H]-deprenyl at a single binding site (nM range) and with [3H]-BU99008 at multiple binding sites (from nM to μM). Autoradiography studies on large frozen postmortem AD and CN hemisphere brain sections demonstrated that 1 μM SMBT-1 almost completely displaced the [3H]-l-deprenyl binding (>90%), while SMBT-1 only partly displaced the [3H]-BU99008 binding (50-60% displacement) in cortical regions. In conclusion, SMBT-1, l-deprenyl, and BU99008 interact at the same MAO-B binding site, while BU99008 shows an additional independent binding site in AD and CN brains. The high translational power of our studies in human AD and CN brains suggests that the multitracer approach with SMBT-1, l-deprenyl, and BU99008 could be useful for imaging reactive astrogliosis.

Glutamate's Effects on the N-Methyl-D-Aspartate (NMDA) Receptor Ion Channel in Alzheimer's Disease Brain: Challenges for PET Radiotracer Development for Imaging the NMDA Ion Channel.

In an effort to further understand the challenges facing in vivo imaging probe development for the N-methyl-D-aspartate (NMDA) receptor ion channel, we have evaluated the effect of glutamate on the Alzheimer's disease (AD) brain. Human post-mortem AD brain slices of the frontal cortex and anterior cingulate were incubated with [3H]MK-801 and adjacent sections were tested for Aβ and Tau. The binding of [3H]MK-801 was measured in the absence and presence of glutamate and glycine. Increased [3H]MK-801 binding in AD brains was observed at baseline and in the presence of glutamate, indicating a significant increase (>100%) in glutamate-induced NMDA ion channel activity in AD brains compared to cognitively normal brains. The glycine effect was lower, suggesting a decrease of the co-agonist effect of glutamate and glycine in the AD brain. Our preliminary findings suggest that the targeting of the NMDA ion channel as well as the glutamate site may be appropriate in the diagnosis and treatment of AD. However, the low baseline levels of [3H]MK-801 binding in the frontal cortex and anterior cingulate in the absence of glutamate and glycine indicate significant hurdles for in vivo imaging probe development and validation.

Herpes Simplex Virus‐1 Proteins Drive Alzheimer’s Disease Pathologies in Humans

AbstractBackgroundDNA and RNA of Herpes Simplex Virus 1 (HSV‐1) were found in the brains and serological samples of Alzheimer’s disease (AD) patients. Such molecular presence of HSV‐1 in AD patients is especially intriguing as HSV‐1 virions are rarely detected in AD brains.MethodsTo follow the molecular footsteps detected, we imaged viral proteins in postmortem human AD brains at superior resolution using expansion microscopy, a tissue manipulation method that physically expands the samples by a factor of 4.5x, allowing a 40 nm imaging resolution, and immunolabeled herpetic proteins, AD pathologies and cell markers.ResultsWe found an abundance of herpetic proteins, previously undetectable with standard methods, across large brain areas. Importantly, we found that HSV‐1 proteins strongly co‐localized with AD pathologies. Consequently, we hypothesized that expression of HSV‐1 proteins during latency may be linked to AD pathology. We are now in the process of characterizing the HSV‐1 proteome in AD brains by imaging key proteins in expanded AD brain slices and examining their colocalization with AD pathologies across brain areas and disease stages. As a complementary system to the fixed human brain slices, we introduced HSV‐1 in human derived brain organoids and imaged the relationships between viral proteins and the formation of AD pathologies via expansion microscopy. We found that HSV‐1 infection triggered these pathologies, pointing out that viruses may be triggers of immune responses driving AD.Conclusionsthis study sheds light on one common pathogen, HSV‐1, while serving as a framework to unveiling molecular causation between infectious agents and AD hallmarks.

A multi‐omics approach to investigate the synapse dysfunction in Alzheimer’s disease

AbstractBackgroundSynapse dysfunction is an early event for Alzheimer’s disease (AD) progression. Synapse dysfunctions are caused by multiple cellular and pathologic factors such as Amyloid beta, p‐tau, inflammation and aging. However, exact molecular mechanism of synapse dysfunction is largely unknown in AD. Therefore, to understand the molecular basis of synapse dysfunction in AD, we conducted a high throughput multi‐omics analysis of synaptosome fraction in AD and cognitively normal postmortem brain samples.MethodSynaptosomes were extracted from healthy control (HC) postmortem brain (n = 14) and AD postmortem brains (n = 27). Next, we performed miRNAs and mRNAs HiSeq analysis on synaptosomal RNAs. We also used HC (n = 10) and AD (n = 10) synaptosome samples for mass spectroscopy proteomic analysis. We conducted the synaptosomal miRNAs and mRNA sequencing and mass spec analysis on the same groups of samples. Further, we conducted a high throughput multi‐comics analysis to understand the molecular interaction of deregulated synapse miRNAs, mRNAs and proteins at AD synapse.ResultMiRNA HiSeq analysis showed the significant deregulation of several miRNAs in synaptosome fraction in AD vs HC. Our study also identified the several novel potential candidate miRNAs those were deregulated in AD vs HC synaptosomes. Similarly, several protein coding genes and novel transcript were also found to be deregulated in AD vs HC synaptosomes. Further, mass spec analysis showed significant deregulation of several synaptic proteins in AD vs HC synaptosomes. Further, to understand the molecular interaction of synapse miRNAs‐mRNA‐proteins and their deregulation in AD, we conducted high throughput multi‐Omics analysis. Study revealed the involvement of omics targets in several biological process and molecular function such as signal transduction, protein binding, GABAergic synapse and synaptic vesicle cycle etc.ConclusionOur study unveiled the synapse centered novel omics candidates those could be potential therapeutic targets for synapse dysfunction in AD.

Characteristics of AD‐brain tau aggregate internalisation by human astrocytes and the responses induced

AbstractBackgroundHighly phosphorylated tau aggregates are deposited in tauopathy brain as disease progresses, driven by a prion‐like spread of tau seeds. Evidence suggests that astrocytes may influence tau spread. However, the efficiency of uptake of disease‐associated tau species is not well defined, nor are the effects of tau uptake on astrocyte reactivity and function. We investigate human astrocyte ability to internalise and clear tau aggregates derived from multiple Alzheimer’s disease (AD) cases, and their impact on astrocyte function and reactivity. We further explore alterations in endogenous tau expression, APP processing and cytoskeletal proteins following tau uptake.MethodControl human iPSC‐derived astrocytes were cultured for 70 days. Aggregated (sarkosyl‐insoluble) tau was isolated from postmortem AD brain. Post‐translational modifications (PTM) of isolated AD‐brain tau aggregates were analysed by mass spectrometry. Tau (0.1 ng/µL) and extracts from control human brain were spiked into astrocyte cultures. Internalisation of aggregated tau and the localised response of S100B and GFAP were quantified by ICC. Changes to tau, APP, functional and reactivity related genes were measured by qPCR and/or proteins by SDS‐PAGE.ResultAggregated tau from AD brain is readily internalised by human astrocytes, and sequesters GFAP and S100B. Differences in the rate of uptake, clearance and seeding of tau correlates with cytoskeletal protein accumulation and specific changes in astrocyte mRNA and protein levels. Work is in progress to relate these changes to characteristics of tau aggregates.ConclusionOur data shows that the capacity of AD brain aggregates to be internalised, cleared and induce seeding of endogenous tau varies by case. Investigations are underway to pinpoint the tau characteristics that underlie these differences. We also show that seeding ability is associated with changes in cytoskeletal elements and reactivity markers at mRNA and protein level. This work agrees with growing evidence that tau variability in AD is linked with specific cell type vulnerabilities that contribute to tau spread and disease progression.