Background:We previously demonstrated that 1,10-phenanthroline-5-amine (PAA) significantly reduced the number and size of amyloid plaques in one-year-old APP/TAU mice. The primary objective of the present study was to validate these findings in the APP/PS1 mouse model using a larger cohort of animals. A second objective was to determine whether PAA binds directly to amyloid plaques in brain tissue sections. Methods:For the in vivo studies, APP/PS1 mice received daily oral PAA or vehicle treatment and were euthanized at one year of age. Brains were collected, fixed, cryosectioned, and stained with hydroxyquinoline oxalate (HQ-O) to visualize amyloid plaques. For the in vitro studies, brain tissue sections were incubated in a PAA solution. Double labeling with PAA and HQ-O was performed on the same tissue sections to compare plaque labeling patterns. Results:Daily oral administration of PAA produced a significant reduction in both the number and size of amyloid plaques compared with untreated control mice. In vitro incubation of tissue sections with PAA resulted in red fluorescent labeling of all amyloid plaques. Double-labeling studies showed that PAA labeled plaques are more extensive than HQ-O in frozen tissue sections, whereas no such difference was observed in paraffin-embedded sections. Conclusions:These findings extend our previous observation that chronic oral administration of PAA significantly reduces amyloid plaque burden in vivo. In addition, the in vitro studies demonstrate that PAA binds directly to amyloid plaques. The mechanism of PAA binding may involve interactions with transition metals incorporated within amyloid plaques and/or the sialic acid moieties of plaque-associated gangliosides.

Alzheimer's disease (AD) is characterized by amyloid-β (Aβ) peptide plaques and neurofibrillary tangles from hyperphosphorylated tau, though factors linking amyloid and tau pathology remain unclear. We investigated whether microRNA-181d-5p (miR-181d) associates with AD-related brain changes and regulates neprilysin and tau. Modeling miR-181d across individuals with no cognitive impairment, mild cognitive impairment, and AD revealed region- and sex-specific associations. Higher miR-181d levels associated with greater AD probability in the temporal lobe and cerebellum, and lower probability in the posterior cingulate cortex of males; miR-181c attenuated these probabilities. SNPs near MIR181 associated with altered entorhinal cortical thickness. In cellular models, miR-181 reduced neprilysin 3'-UTR activity, mRNA, protein, and enzymatic activity, while increasing tau mRNA and protein. Neprilysin diminution impairs Aβ clearance and elevates tau, contributing to AD. RNA sequencing identified miR-181d-responsive neurodegenerative pathways. These findings identify miR-181 as a regulator of AD-relevant amyloid and tau pathways, providing novel targets.

Non-coding RNA species, such as microRNA (miRNA), regulate multiple biological and pathological processes by binding to target mRNAs and facilitating alteration of translation levels via complexes such as RNA-induced silencing complex (RISC). Disrupting this process could contribute to AD pathogenesis by fostering aggregation of hyperphosphorylated microtubule-associated protein tau and amyloid-β (Aβ) peptides, and neuroinflammation. Understanding how these pathological changes are regulated remains our research focus. We report that miR298 plays a vital role in maintaining APP and tau homeostasis and that miR298 imbalances may impact AD progression. Levels of miR298 from non-cognitively impaired (NCI) and AD subject brain tissue samples from different recognized sources were measured by qRT-PCR and assessed for associations with AD risk and potential covariates such as age and APOE genotype. Other biomarkers were assessed in cortical samples from the same subjects, as we previously described. Further, APP, tau, and cytokines were profiled in miR298 mimic- or its antagomiR-expressing human neuronal and astrocyte cultures. Levels of miR298 varied in postmortem temporal lobe between AD patients and age-matched NCI controls. Higher brain miR298 levels were associated with a reduced risk of AD. Subject age and APOE genotype altered this association; specifically, greater age and dose of the APOEε4 allele were associated with an increased risk of AD. However, APOEε4 dose-associated risk reduced as age increased. We identified putative binding sites for miR298 on APP, BACE1, MAPT, IL1α, and IL6 mRNAs to form RISC. We showed that treatment by miR298 reduced tau, APP, and BACE1 proteins and mRNA levels in cell cultures. These studies suggest that miR298 regulates a coordinated network of AD-related proteins APP, BACE1, and tau. Hence, such network regulation may represent a rational therapeutic target for reducing AD risk and disease modification. In addition to late-onset cases, we will profile miR298 in brain tissue samples from early-onset AD cases. Future work involves testing miR298 in AD animal models, such as in human tau-overexpressing transgenic mice. We sincerely thank grant support from NIA/NIH.

Major contributors to AD pathogenesis include aggregates of amyloid-β (Aβ) peptides, hyperphosphorylated tau protein, and neuroinflammation. No currently approved treatment stops or significantly slows the progression of AD. Nevertheless, one class of agents that has shown promise is metal chelators. For the assessment of a novel effect of oral administration of 1,10-Phenanthroline-5-amine (PAA) on the severity of amyloid plaque load, we used a transgenic (Tg) mouse model with inserted human autosomally dominant (familial) AD genes: amyloid-beta (Aβ) protein precursor (APP) and tau protein. APP/Tau transgenic mice that model AD were allotted into one of two groups. The control group received no treatment while the experimental group received 1,10-phenanthroline-5-amine (PAA) in their drinking water, starting at 4 months. All animals were sacrificed at 1 year of age, and their brains were stained with 2 different markers of amyloid plaques, Amylo-Glo+ and HQ-O, as we have recently described (Schmued et al, 2023). PAA administered as a daily oral dose for 9 months resulted in no changes in weight or behavior and resulted in no observed pathologies. Control animals exhibited numerous dense core plaques throughout the neo- and allo-cortical brain regions. PAA administered as a daily dose for 9 months resulted in roughly 2/3 the amyloid plaque burden compared to untreated transgenic mice. Oral daily dosing with PAA significantly reduced the amyloid plaque burden in transgenic AD model mice. The mode of action of PAA may be attributed to its ability to chelate transition metals and to inhibit either metalloprotease enzymes or the metal-seeded auto aggregation of Aβ. The underlying mechanism for this protection is not fully known; one possible mechanism would be to inhibit the “metal-seeding” of Aβ. Dyshomeostasis of certain transition metals in brain microenvironment contributes to amyloidosis. Although this dysregulation may be attributable to impaired metal transporter function, there exists no known treatment that would modify such dysfunction. Further research is underway to confirm these results in another mouse model of AD (APP/PS1). Altogether, reducing regional metal levels via chelation with PAA may be a feasible and viable strategy for suppressing the formation of amyloid plaques in AD patients.

The most common neurodegenerative disorders include Alzheimer's disease (AD), Lewy body and related dementias (ADRDs). Triggers of pathobiochemical changes in ADRDs remains unknown and appear numerous. Short non-coding RNAs, microRNA (miRNA), play a vital role in regulating biological and pathological processes leading to neurodegenerative diseases. Amyloid plaques, major hallmarks of AD, comprise abnormal aggregation of extracellular amyloid-β peptides (Aβ) derived from Aβ precursor protein (APP). Neurofibrillary tangles consist of filamentous hyper-phosphorylated tau proteins. Alpha-synuclein (SNCA) plays a critical role in the pathogenesis of Parkinson's and other synucleinopathies. Repressor Element 1-Silencing Transcription (REST) factor is altered in ADRDs. We studied the role of miR-153-3p in AD risk and in regulating levels of critical proteins. miR-153-3p reduced APP, SNCA and We measured miR153 levels in non-cognitively impaired (NCI) and AD subject brain tissue samples from different recognized sources by qRT-PCR as described (Wang et al). We utilized autopsy brain tissues and ADNI participants' genotyping and performed association studies of miR-153-3p and its single nucleotide polymorphisms (SNPs) with AD risk, and nine endophenotypes. We used iPSC-derived neuronal cells, human cell lines and miRNA transfections to study the mechanism of miR-153-3p Elevation of miR-153-3p is associated with a reduced probability of AD, while elevated REST associated with a greater likelihood of AD. MiR-153 gene SNPs are associated with nine AD-related endophenotypes. MiR-153-3p reduced REST, APP and SNCA 3’-UTR activities and respective protein levels. MiR-153-3p treatment altered REST and neuronal differentiation in iPSC-derived neuronal stem cells. RNA sequencing proteomics and interactome analysis revealed the role of miR-153-3p in axonal guidance. With the increased emphasis on comorbidities of AD and other neurodegenerative diseases, we identified that miR-153-3p, as a master regulator, reduced a key group of neurodegeneration-related proteins. MiR-153-3p reduces APP, SNCA and REST expression, all pointing towards a therapeutic and biomarker potential in ADRDs. In addition to late-onset cases, we will profile miR153 in brain tissue samples from early-onset AD cases.

Amyloid-β precursor protein (APP) and apolipoprotein E (APOE) are both involved in AD. APP is proteolytically cleaved to produce neurotoxic amyloid fragments. Screening a variety of compounds for their ability to reduce the amyloid plaque burden in mice models of AD provides potential novel drug targets. One promising class of compounds are certain metal chelators including phenanthroline-amine (PAA), a metal chelator, an aromatic cation and a metalloprotease inhibitor shown to significantly reduce the size and number of plaques seen in year old APP/PS1 mice (Schmued et al-2024). Our objective was to expand the previous report of APP administration using a double transgenic model (N=16) that express considerably more plaques. The second objective of this study was to determine whether PAA will bind directly to amyloid plaques in tissue sections. At 1 year of age both PAA-dosed and control APP/PS1 mice were euthanized, and their fixed brains removed and freeze-sectioned. Some sections were stained with hydroxyquinoline oxalate to label all amyloid plaques. In parallel labeling studies, the tissue sections were immersed in PAA dissolved in a pH 4.5 buffer at 55 o C. Double labeling was also achieved by combining these two staining methods on the same tissue section. Daily oral dosing with PAA resulted in a significant reduction in the number and size of amyloid plaques vs. the vehicle treated control mice. The average plaque areas of the PAA treated group exhibited 67.6% of the plaque burden seen in the control animals. Also, immersing tissue sections in a PAA solution resulted in the red fluorescent labeling of all amyloid plaques. Double labeling revealed that PAA labeled the plaques more extensively than HQ-O. Our study confirmed that chronic oral dosing with PAA resulted in plaque reductions comparable to that seen in the original study that used a smaller number of a less common mouse model of AD. This study also showed that PAA is capable of binding directly to amyloid plaques. Possible endogenous target molecules, based on the chemical properties of PAA, include certain transition metals, certain glycosylated molecules of AD relevance (APOE, NCAM1, APP and gangliosides) and metalloproteases.

Background: Most preclinical studies on glioblastoma (GBM) fail to provide translational utility in the clinic. Fluorescence-guided surgery using 5-aminolevulinic acid (5-ALA) improves tumor resection, disease prognosis, and, thus, patient outcomes. Given the critical role of surgery in managing recurrent GBM, it is essential to incorporate surgical elements into preclinical models to accurately reflect clinical scenarios and enhance translational success. However, existing protocols for 5-ALA-guided resection in preclinical models are limited and often lack clinical relevance. Methods: To address this gap, we developed a novel protocol for the 5-ALA-guided resection in two mouse GBM models: TRP-mCherry-FLuc and GL261 Red-FLuc. Results: The resection of TRP-mCherry-FLuc tumors significantly extended survival and mitigated weight loss compared to controls. Similarly, GL261 Red-FLuc tumor resection increased survival, reduced body weight loss, and slowed tumor progression. Conclusions: This study presents a clinically relevant protocol for 5-ALA-guided resection in preclinical GBM models, providing a platform for future research to integrate adjuvant therapies and enhance their potential translation into clinical practice.

Blood–brain barrier dysfunction is one characteristic of Alzheimer’s disease (AD) and is recognized as both a cause and consequence of the pathological cascade leading to cognitive decline. The goal of this study was to assess markers for barrier dysfunction in postmortem tissue samples from research participants who were either cognitively normal individuals (CNI) or diagnosed with AD at the time of autopsy and determine to what extent these markers are associated with AD neuropathologic changes (ADNC) and cognitive impairment. We used postmortem brain tissue and plasma samples from 19 participants: 9 CNI and 10 AD dementia patients who had come to autopsy from the University of Kentucky AD Research Center (UK-ADRC) community-based cohort; all cases with dementia had confirmed severe ADNC. Plasma samples were obtained within 2 years of autopsy. Aβ40, Aβ42, and tau levels in brain tissue samples were quantified by ELISA. Cortical brain sections were cleared using the X-CLARITY™ system and immunostained for neurovascular unit-related proteins. Brain slices were then imaged using confocal microscopy and analyzed for microvascular diameters and immunoreactivity coverage using Fiji/ImageJ. Isolated human brain microvessels were assayed for tight-junction protein expression using the JESS™ automated Western blot system. S100 calcium-binding protein B (S100β), matrix metalloproteinase (MMP)-2, MMP-9, and neuron-specific enolase (NSE) levels in plasma were quantified by ELISA. All outcomes were assessed for linear associations with global cognitive function (MMSE, CDR) and cerebral atrophy scores by Pearson, polyserial, or polychoric correlation, as appropriate, along with generalized linear modeling or generalized linear mixed-level modeling. As expected, we detected elevated Aβ and tau pathology in brain tissue sections from AD patients compared to CNI. However, we found no differences in microvascular diameters in cleared AD and CNI brain tissue sections. We also observed no differences in claudin-5 protein levels in capillaries isolated from AD and CNI tissue samples. Plasma biomarker analysis showed that AD patients had 12.4-fold higher S100β plasma levels, twofold lower NSE plasma levels, 2.4-fold higher MMP-9 plasma levels, and 1.2-fold lower MMP-2 plasma levels than CNI. Data analysis revealed that elevated S100β plasma levels were predictive of AD pathology and cognitive impairment. Our data suggest that among different markers relevant to barrier dysfunction, plasma S100β is the most promising diagnostic biomarker for ADNC. Further investigation is necessary to assess how plasma S100β levels relate to these changes and whether they may predict clinical outcomes, particularly in the prodromal and early stages of AD.

INTRODUCTION:Small non-coding microRNAs (miRNAs) play essential roles in Alzheimer's disease (AD) pathogenesis. Repressor element 1-silencing transcription factor (REST) is involved in AD, though its regulation remains unclear. METHODS:We performed real-time quantitative polymerase chain reaction (qPCR) in autopsied brain tissues to determine miR-153-3p and AD associations. A reporter-based assay measured the activity of REST mRNA 3'-untranslated region (3'-UTR). Induced pluripotent stem cells (iPSC)-derived neurons and human cell lines were applied to determine miR-153-3p regulation of endogenous proteins. RESULTS:Elevation of miR-153-3p is associated with a reduced probability of AD, while elevated REST is associated with a greater probability of AD. The 3'-UTR functional assay pinpointed the miR-153-3p binding sites. miR-153-3p treatment reduced REST, amyloid precursor protein (APP), and α-synuclein (SNCA) 3'-UTR activities and protein levels. miR-153-3p treatment altered REST and neuronal differentiation in iPSC-derived neuronal stem cells. RNA-sequencing and proteomics revealed miR-153-3p-associated networks. DISCUSSION:miR-153-3p reduces REST, APP, and SNCA expression, pointing toward its therapeutic and biomarker potential in neurodegenerative diseases. HIGHLIGHTS:With the increased emphasis on comorbidities of Alzheimer's disease (AD) and other neurodegenerative diseases, we identified that miR-153-3p, as a master regulator, reduced a group of neurodegeneration related proteins: REST, amyloid precursor protein (APP) and α-synuclein (SNCA) levels. The elevation of miR-153-3p levels is associated with reduced probability of AD in posterior cingulate cortex (PCC), while REST, by contrast, is associated with a greater probability of AD. miR-153-3p treatment alters REST protein levels and neuronal differentiation in induced pluripotent stem cells (iPSC) derived neuronal cells. RNA sequencing proteomics and interactome analysis revealed the role of miR-153-3p in axonal guidance.

INTRODUCTION:MicroRNA (miRNA) activity is increasingly appreciated as a key regulator of pathophysiologic pathways in Alzheimer's disease (AD). However, the role of miRNAs during the progression of AD, including resilience and prodromal syndromes such as mild cognitive impairment (MCI), remains underexplored. METHODS:We performed miRNA-sequencing on samples of posterior cingulate cortex (PCC) obtained post mortem from Rush Religious Orders Study participants diagnosed ante mortem with no cognitive impairment (NCI), MCI, or AD. NCI subjects were subdivided as low pathology (Braak stage I/II) or high pathology (Braak stage III/IV), suggestive of resilience. Bioinformatics approaches included differential expression, messenger RNA (mRNA) target prediction, interactome modeling, functional enrichment, and AD risk modeling. RESULTS:We identified specific miRNA groups, mRNA targets, and signaling pathways distinguishing AD, MCI, resilience, ante mortem neuropsychological test performance, post mortem neuropathological burden, and AD risk. DISCUSSION:These findings highlight the potential of harnessing miRNA activity to manipulate disease-modifying pathways in AD, with implications for precision medicine. HIGHLIGHTS:MicroRNA (MiRNA) dysregulation is a well-established feature of Alzheimer's disease (AD). Novel miRNAs also distinguish subjects with mild cognitive impairment and putative resilience. MiRNAs correlate with cognitive performance and neuropathological burden. Select miRNAs are associated with AD risk with age as a significant covariate. MiRNA pathways include insulin, prolactin, kinases, and neurite plasticity.

Abstract Background Patients with Alzheimer's disease (AD) develop blood–brain barrier dysfunction to varying degrees. How aging impacts Aβ pathology, blood–brain barrier function, and cognitive decline in AD remains largely unknown. In this study, we used 5xFAD mice to investigate changes in Aβ levels, barrier function, and cognitive decline over time. Methods 5xFAD and wild-type (WT) mice were aged between 9.5 and 15.5 months and tested for spatial learning and reference memory with the Morris Water Maze (MWM). After behavior testing, mice were implanted with acute cranial windows and intravenously injected with fluorescent-labeled dextrans to assess their in vivo distribution in the brain by two-photon microscopy. Images were processed and segmented to obtain intravascular intensity, extravascular intensity, and vessel diameters as a measure of barrier integrity. Mice were sacrificed after in vivo imaging to isolate brain and plasma for measuring Aβ levels. The effect of age and genotype were evaluated for each assay using generalized or cumulative-linked logistic mixed-level modeling and model selection by Akaike Information Criterion (AICc). Pairwise comparisons were used to identify outcome differences between the two groups. Results 5xFAD mice displayed spatial memory deficits compared to age-matched WT mice in the MWM assay, which worsened with age. Memory impairment was evident in 5xFAD mice by 2–threefold higher escape latencies, twofold greater cumulative distances until they reach the platform, and twice as frequent use of repetitive search strategies in the pool when compared with age-matched WT mice. Presence of the rd1 allele worsened MWM performance in 5xFAD mice at all ages but did not alter the rate of learning or probe trial outcomes. 9.5-month-old 15.5-month-old 5xFAD mice had twofold higher brain Aβ40 and Aβ42 levels (p < 0.001) and 2.5-fold higher (p = 0.007) plasma Aβ40 levels compared to 9.5-month-old 5xFAD mice. Image analysis showed that vessel diameters and intra- and extravascular dextran intensities were not significantly different in 9.5- and 15.5-month-old 5xFAD mice compared to age-matched WT mice. Conclusion 5xFAD mice continue to develop spatial memory deficits and increased Aβ brain levels while aging. Given in vivo MP imaging limitations, further investigation with smaller molecular weight markers combined with advanced imaging techniques would be needed to reliably assess subtle differences in barrier integrity in aged mice.

BACKGROUND:Alzheimer's disease (AD) is the most prevalent age-related dementia, and, despite numerous attempts to halt or reverse its devastating progression, no effective therapeutics have yet been confirmed clinically. However, one class of agents that has shown promise is certain metal chelators. OBJECTIVE:For the novel assessment of the effect of oral administration of 1,10-phenanthroline-5-amine (PAA) on the severity of amyloid plaque load, we used a transgenic (Tg) mouse model with inserted human autosomally dominant (familial) AD genes: amyloid-β protein precursor (AβPP) and tau. METHODS:AβPP/Tau transgenic mice that model AD were allotted into one of two groups. The control group received no treatment while the experimental group received PAA in their drinking water starting at 4 months of age. All animals were sacrificed at 1 year of age and their brains were stained with two different markers of amyloid plaques, Amylo-Glo+ and HQ-O. RESULTS:The control animals exhibited numerous dense core plaques throughout the neo- and allo- cortical brain regions. The experimental group treated with PAA, however, showed 62% of the amyloid plaque burden seen in the control group. CONCLUSIONS:Oral daily dosing with PAA will significantly reduce the amyloid plaque burden in transgenic mice that model AD. The underlying mechanism for this protection is not fully known; however, one proposed mechanism involves inhibiting the "metal-seeding" of Aβ.

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.

Several proteins play critical roles in vulnerability or resistance to neurodegenerative disorders such as Alzheimer’s disease (AD), Parkinson’s disease (PD), and frontotemporal dementia (FTD). Regulation of these proteins is critical to maintaining healthy neurohomeostasis. In addition to transcription factors regulating gene transcription and microRNAs regulating mRNA translation, natural antisense transcripts (NATs) regulate mRNA levels, splicing, and translation. NATs’ roles are significant in regulating key protein-coding genes associated with neurodegenerative disorders. Elucidating the functions of these NATs could prove useful in treating or preventing diseases. NAT activity is not restricted to mRNA translation; it can also regulate DNA (de)methylation and other gene expression steps. NATs are noncoding RNAs (ncRNAs) encoded by DNA sequences overlapping the pertinent protein genes. These NATs have complex structures, including introns and exons, and therefore bind their target genes, precursor mRNAs (pre-mRNAs), and mature RNAs. They can occur at the 5’- or 3’-ends of a mRNA-coding sequence or internally to a parent gene. NATs can downregulate translation, e.g., microtubule-associated protein tau (MAPT) antisense-1 gene (MAPT-AS1), or upregulate translation, e.g., β-Amyloid site Cleaving Enzyme 1 (BACE1) antisense gene (BACE1-AS). Regulation of NATs can parallel pathogenesis, wherein a “pathogenic” NAT (e.g., BACE1-AS) is upregulated under pathogenic conditions, while a “protective” NAT (e.g., MAPT-AS1) is downregulated under pathogenic conditions. As a relatively underexplored endogenous control mechanism of protein expression, NATs may present novel mechanistic targets to prevent or ameliorate aging-related disorders.

Alzheimers disease (AD) is a progressive degenerative disease characterized by a significant loss of neurons and synapses in cognitive brain regions and is the leading cause of dementia worldwide. AD pathology comprises extracellular amyloid plaques and intracellular neurofibrillary tangles. However, the triggers of this pathology are still poorly understood. Repressor element 1-silencing transcription/neuron-restrictive silencer factor (REST/NRSF), a transcription repressor of neuronal genes, is dysregulated during AD pathogenesis. How REST is dysregulated is still poorly understood, especially at the post-transcriptional level. MicroRNAs (miRNAs), a group of short non-coding RNAs, typically regulate protein expression by interacting with target mRNA transcript 3-untranslated region (UTR) and play essential roles in AD pathogenesis. Herein, we demonstrate that miR-153-3p reduces REST 3-UTR activities, mRNA, and protein levels in human cell lines, along with downregulating amyloid β precursor protein (APP) and α-synuclein (SNCA). We determine by mutational analyses that miR-153-3p interacts with specific targets via the seed sequence present within the respective mRNA 3-UTR. We show that miR-153-3p treatment alters the expression of these specific proteins in human neuronally differentiated cells and human induced pluripotent stem cells and that miR-153-3p is itself dysregulated in AD. We further find that single nucleotide polymorphisms (SNPs) within 5kb of the MIR153-1 and MIR153-2 genes are associated with AD-related endophenotypes. Elevation of miR-153-3p is associated with reduced AD probability, while elevated REST may associate with a greater AD probability. Our work suggests that a supplement of miR-153-3p would reduce levels of toxic protein aggregates by reducing APP, SNCA, and REST expression, all pointing towards a therapeutic and biomarker potential of miR-153-3p in AD and related dementias.

Glioblastoma (GBM) is the most aggressive brain cancer. To model GBM in research, orthotopic brain tumor models, including syngeneic models like GL261 and genetically engineered mouse models like TRP, are used. In longitudinal studies, tumor growth and the treatment response are typically tracked with in vivo imaging, including bioluminescence imaging (BLI), which is quick, cost-effective, and easily quantifiable. However, BLI requires luciferase-tagged cells, and recent studies indicate that the luciferase gene can elicit an immune response, leading to tumor rejection and experimental variation. We sought to optimize the engraftment of two luciferase-expressing GBM models, GL261 Red-FLuc and TRP-mCherry-FLuc, showing differences in tumor take, with GL261 Red-FLuc cells requiring immunocompromised mice for 100% engraftment. Immunohistochemistry and MRI revealed distinct tumor characteristics: GL261 Red-FLuc tumors were well-demarcated with densely packed cells, high mitotic activity, and vascularization. In contrast, TRP-mCherry-FLuc tumors were large, invasive, and necrotic, with perivascular invasion. Quantifying the tumor volume using the HALO® AI analysis platform yielded results comparable to manual measurements, providing a standardized and efficient approach for the reliable, high-throughput analysis of luciferase-expressing tumors. Our study highlights the importance of considering tumor engraftment when using luciferase-expressing GBM models, providing insights for preclinical research design.

MicroRNA (miRNA) dysregulation is linked to Alzheimer’s disease (AD) pathophysiology. We recently reported a role for miR-298 and miR-20b in AD risk and amyloid and tau metabolism. To examine the extent to which miRNAs are dysregulated during the earliest stages of AD, we quantified miRNA transcript levels in postmortem samples of posterior cingulate cortex (PCC), a default mode network (DMN) hub that underlies autobiographical memory, in normal subjects and those who died within the AD continuum. PCC samples were obtained from Rush ROS/MAP participants who came to autopsy with a diagnosis of a) no cognitive impairment and low pathology (NCI-LP, Braak stage I/II, n = 12), b) NCI with high pathology (NCI-HP, Braak stage IV, representing cognitive resilience, n = 8), c) mild cognitive impairment (MCI, n = 10), or d) dementia due to AD (n = 9). Illumina-based sequencing of total RNA was performed and trimmed reads with a size of 15-31 bases were analyzed. Differential expression analysis (FDR ≤ 0.04) revealed 42 miRNAs significantly dysregulated among the NCI (independent of Braak stage), MCI, and AD groups. Two miRNAs, miR-99a (FDR-adjusted p = 0.01) and miR-664b (p = 0.005), were downregulated in AD vs NCI and MCI, while miR-30a (p = 0.006), miR-374a (p = 0.004), and miR-501 (p = 0.005) were significantly upregulated in MCI vs. NCI and AD. Three miRNAs were significantly downregulated in NCI-HP vs. NCI-LP: miR-103a (p = 0.04), miR-211 (p = 0.03), and miR-4443 (p = 0.03). These miRNAs may operate in resilience-related pathways. Correlation analysis revealed that decreasing PCC levels of miR-664b levels were associated with poorer performance on antemortem tests of episodic memory (r = 0.41, p = 0.009), semantic memory (r = 0.44, p = 0.004), and visuospatial ability (r = 0.45, p = 0.004). Taken together, these data may identify potential pathogenic or protective miRNA-related mechanisms contributing to PCC and DMN function during the progression of AD, which may inform biomarker and intervention strategies. Ongoing analysis will identify functional pathway enrichment and miRNA targets.

Small non-coding microRNA (miRNA) play a vital role in regulating various biological and pathological processes, including Alzheimer’s disease (AD), brain injury, and head trauma. AD hallmarks include brain amyloid plaques and neurofibrillary tangles (NFTs). Amyloid plaques are the abnormal aggregation of amyloid-β peptides (Aβ) derived from Aβ precursor protein (APP), and NFTs consist mostly of hyper-phosphorylated tau proteins. We have recently shown that miRNAs, such as miR-20b, miR-153, and miR-298, can independently regulate AD-related protein expression (Wang et al-Mol Psychiatry-2022). APOE ε4 increases the risk of AD; however, the role of brain miR101 levels in APOE’s influence is presently unknown. Here, we studied the role of miR-101-3p (miR-101) in AD risk and in regulating levels of critical proteins such as APP, GSK3β, and ECE1 implicated in AD. Temporal lobes (TL), cerebellum (CB), and posterior cingulate cortices (PCC) were obtained from non-cognitively impaired (NCI) and AD subjects, and quantitative RT-PCR measured miR-101 levels. The target specificity of miR-101 was tested using dual reporter clones expressing the 3'-UTRs of either APP, ECE1, or GSK3β mRNAs. Human microglia and differentiated neuroblastoma (SKNSH) cells were transfected with miR-101 for 72 hours, and cellular RNA, total proteins, and secreted proteins were analyzed by ELISA and Westerns to understand miR-101-mediated regulation. TL and CB regions showed that increased miR-101 levels were associated with reduced AD risk in the absence of the APOEε4 allele. Interestingly, miR-101 may increase AD risk in the presence of two APOEε4 alleles. Transfection of miR-101 altered the translation of several AD-related proteins, including aggregation-prone proteins, processing enzymes, and cytokines. Mechanistically, miR-101 treatment reduced ECE1 and GSK3β mRNA 3’-UTR activities in reporter clones and protein levels. We have shown that miR-101 levels modify AD risk in an APOE allele-dependent manner. Notably, miR-101 targets the 3’-UTRs of ECE1 and GSK3β and reduces their expression, making it a key regulator of AD-related proteins across species and cell types. Our combined results suggest that miR-101 is critical in regulating essential proteins relevant to AD pathogenesis and could potentially be a novel “multi-hit” drug target for AD and other neurodegenerative diseases. NIH grants to DKL and SEC.

Background Loss of P-glycoprotein (P-gp) at the blood–brain barrier contributes to amyloid-β (Aβ) brain accumulation in Alzheimer’s disease (AD). Using transgenic human amyloid precursor protein (hAPP)-overexpressing mice (Tg2576), we previously showed that Aβ triggers P-gp loss by activating the ubiquitin–proteasome pathway, which leads to P-gp degradation. Furthermore, we showed that inhibiting the ubiquitin-activating enzyme (E1) prevents P-gp loss and lowers Aβ accumulation in the brain of hAPP mice. Based on these data, we hypothesized that repurposing the FDA-approved proteasome inhibitor, bortezomib (Velcade ® ; BTZ), protects blood–brain barrier P-gp from degradation in hAPP mice in vivo. Methods We treated hAPP mice with the proteasome inhibitor BTZ or a combination of BTZ with the P-gp inhibitor cyclosporin A (CSA) for 2 weeks. Vehicle-treated wild-type (WT) mice were used as a reference for normal P-gp protein expression and transport activity. In addition, we used the opioid receptor agonist loperamide as a P-gp substrate in tail flick assays to indirectly assess P-gp transport activity at the blood–brain barrier in vivo. We also determined P-gp protein expression by Western blotting, measured P-gp transport activity levels in isolated brain capillaries with live cell confocal imaging and assessed Aβ plasma and brain levels with ELISA. Results We found that 2-week BTZ treatment of hAPP mice restored P-gp protein expression and transport activity in brain capillaries to levels found in WT mice. We also observed that hAPP mice displayed significant loperamide-induced central antinociception compared to WT mice indicating impaired P-gp transport activity at the blood–brain barrier of hAPP mice in vivo. Furthermore, BTZ treatment prevented loperamide-induced antinociception suggesting BTZ protected P-gp loss in hAPP mice. Further, BTZ-treated hAPP mice had lower Aβ40 and Aβ42 brain levels compared to vehicle-treated hAPP mice. Conclusions Our data indicate that BTZ protects P-gp from proteasomal degradation in hAPP mice, which helps to reduce Aβ brain levels. Our data suggest that the proteasome system could be exploited for a novel therapeutic strategy in AD, particularly since increasing Aβ transport across the blood–brain barrier may prove an effective treatment for patients.