Drivers of Memory Loss Underreport in Mild Cognitive Impairment Due to Alzheimer Versus Vascular Disease.

BACKGROUND: We examined drivers of self and study partner reports of memory loss in mild cognitive impairment (MCI) from Alzheimer (AD-MCI) and vascular disease (Va-MCI). METHODS: We performed retrospective cross-sectional analyses of participants with AD-MCI (n=2874) and Va-MCI (n=376) from the National Alzheimer Coordinating Center data set. Statistical analysis utilized 2-sided t test or the Fisher exact test. RESULTS: Compared with AD-MCI, Va-MCI subjects (24.5% vs. 19.7%, P =0.031) and study partners (31.4% vs. 21.6%, P <0.0001) were more likely to deny memory loss. Black/African Americans were disproportionately represented in the group denying memory loss in AD-MCI (20.0% vs. 13.2%, P <0.0001) and Va-MCI (33.7% vs. 18.0%, P =0.0022). Study partners of participants with these features also disproportionately denied memory loss: female (AD-MCI: 60.1% vs. 51.7%, P =0.0002; Va-MCI: 70.3% vs. 52.3%, P =0.0011), Black/African American (AD-MCI: 23.5% vs. 11.98%, P <0.0001; Va-MCI: 48.8% vs. 26.5%, P =0.0002), and <16 years of education (AD-MCI only: 33.9% vs. 16.3%, P =0.0262). In AD-MCI and Va-MCI, participants with anxiety were disproportionately represented in the group endorsing memory loss (AD: 28.2% vs. 17.4%, P <0.0001; Va: 31.5% vs. 16.1%, P =0.0071), with analogous results with depression. CONCLUSION: The findings would suggest extra vigilance in interview-based MCI detection of persons at-risk for self-based or informant-based misreport.

Impact of white matter hyperintensities on subjective cognitive decline phenotype in a diverse cohort of cognitively normal older adults.

OBJECTIVES: Subjective cognitive decline (SCD) is a preclinical stage of AD. White matter hyperintensities (WMH), an MRI marker of cerebral small vessel disease, associate with AD biomarkers and progression. The impact of WMH on SCD phenotype is unclear. METHODS/DESIGN: A retrospective, cross-sectional analysis was conducted on a diverse cohort with SCD evaluated at the NYU Alzheimer's Disease Research Center between January 2017 and November 2021 (n = 234). The cohort was dichotomized into none-to-mild (n = 202) and moderate-to-severe (n = 32) WMH. Differences in SCD and neurocognitive assessments were evaluated via Wilcoxon or Fisher exact tests, with p-values adjusted for demographics using multivariable logistic regression. RESULTS: Moderate-to-severe WMH participants reported more difficulty with decision making on the Cognitive Change Index (1.5 SD 0.7 vs. 1.2 SD 0.5, p = 0.0187) and worse short-term memory (2.2 SD 0.4 vs. 1.9 SD 0.3, p = 0.0049) and higher SCD burden (9.5 SD 1.6 vs. 8.7 SD 1.7, p = 0.0411) on the Brief Cognitive Rating Scale. Moderate-to-severe WMH participants scored lower on the Mini-Mental State Examination (28.0 SD 1.6 vs. 28.5 SD 1.9, p = 0.0491), and on delayed paragraph (7.2 SD 2.0 vs. 8.8 SD 2.9, p = 0.0222) and designs recall (4.5 SD 2.3 vs. 6.1 SD 2.5, p = 0.0373) of the Guild Memory Test. CONCLUSIONS: In SCD, WMH impact overall symptom severity, specifically in executive and memory domains, as well as objective performance on global and domain-specific tests in verbal memory and visual working/associative memory.

Sensitized 1-Acyl-7-nitroindolines with Enhanced Two-Photon Cross Sections for Release of Neurotransmitters.

Precise photochemical control, using two-photon excitation (2PE), of the timing and location of activation of glutamate is useful for studying the molecular and cellular physiology of the brain. Antenna-based light harvesting strategies represent a general method to increase the sensitivity to 2PE of otherwise insensitive photoremovable protecting groups (PPGs). This was applied to the most commonly used form of "caged" glutamate, MNI-Glu. Computational investigation showed that a four- or six-carbon linker attached between the 4-position of thioxanthone (THX) and the 4-position of the 5-methyl derivative of MNI-Glu (MMNI-Glu) would position the antenna and PPG close to one another to enable Dexter energy transfer. Nine THX-MMNI-Glu conjugates were prepared and their photochemical properties determined. Installation of the THX antenna resulted in a red shift of the absorption (λmax = 385-405 nm) along with increased quantum yield compared to the parent compound MNI-Glu (λmax = 347 nm). The THX-MMNI-Glu conjugate with a four-carbon linker and attachment to the 4-position of THX underwent photolysis via 1PE at 405 and 430 nm and via 2PE at 770 and 860 nm, yielding glutamate. The two-photon uncaging action cross section (δu) was 0.11 and 0.29 GM at 770 and 860, respectively, which was greater than for MNI-Glu (0.06 and 0.072 GM at 720 and 770 nm, respectively). The THX sensitizer harvested the light via 2PE and transferred its resulting triplet energy to MMNI-Glu. Release of glutamate through 2PE at 860 nm from the compound (100 µM) activated iGluSnFR, a genetically encoded, fluorescent glutamate sensor, on the surface of cells in culture, portending its usefulness in studies of neurophysiology in acute brain slice.

DICER-ARGONAUTE2 complex in continuous fluorogenic assays of RNA interference enzymes.

Mechanistic studies of RNA processing in the RNA-Induced Silencing Complex (RISC) have been hindered by lack of methods for continuous monitoring of enzymatic activity. "Quencherless" fluorogenic substrates of RNAi enzymes enable continuous monitoring of enzymatic reactions for detailed kinetics studies. Recombinant RISC enzymes cleave the fluorogenic substrates targeting human thymidylate synthase (TYMS) and hypoxia-inducible factor 1-α subunit (HIF1A). Using fluorogenic dsRNA DICER substrates and fluorogenic siRNA, DICER+ARGONAUTE2 mixtures exhibit synergistic enzymatic activity relative to either enzyme alone, and addition of TRBP does not enhance the apparent activity. Titration of AGO2 and DICER in enzyme assays suggests that AGO2 and DICER form a functional high-affinity complex in equimolar ratio. DICER and DICER+AGO2 exhibit Michaelis-Menten kinetics with DICER substrates. However, AGO2 cannot process the fluorogenic siRNA without DICER enzyme, suggesting that AGO2 cannot self-load siRNA into its active site. The DICER+AGO2 combination processes the fluorogenic siRNA substrate (Km=74 nM) with substrate inhibition kinetics (Ki=105 nM), demonstrating experimentally that siRNA binds two different sites that affect Dicing and AGO2-loading reactions in RISC. This result suggests that siRNA (product of DICER) bound in the active site of DICER may undergo direct transfer (as AGO2 substrate) to the active site of AGO2 in the DICER+AGO2 complex. Competitive substrate assays indicate that DICER+AGO2 cleavage of fluorogenic siRNA is specific, since unlabeled siRNA and DICER substrates serve as competing substrates that cause a concentration-dependent decrease in fluorescent rates. Competitive substrate assays of a series of DICER substrates in vitro were correlated with cell-based assays of HIF1A mRNA knockdown (log-log slope=0.29), suggesting that improved DICER substrate designs with 10-fold greater processing by the DICER+AGO2 complex can provide a strong (~2800-fold) improvement in potency for mRNA knockdown. This study lays the foundation of a systematic biochemical approach to optimize nucleic acid-based therapeutics for Dicing and ARGONAUTE2-loading for improving efficacy.

Novel HIV-1 miRNAs stimulate TNFα release in human macrophages via TLR8 signaling pathway.

PURPOSE: To determine whether HIV-1 produces microRNAs and elucidate whether these miRNAs can induce inflammatory response in macrophages (independent of the conventional miRNA function in RNA interference) leading to chronic immune activation. METHODS: Using sensitive quantitative Real Time RT-PCR and sequencing, we detected novel HIV-derived miRNAs in the sera of HIV+ persons, and associated with exosomes. Release of TNFα by macrophages challenged with HIV miRNAs was measured by ELISA. RESULTS: HIV infection of primary alveolar macrophages produced elevated levels of viral microRNAs vmiR88, vmiR99 and vmiR-TAR in cell extracts and in exosome preparations from conditioned medium. Furthermore, these miRNAs were also detected in exosome fraction of sera from HIV-infected persons. Importantly, vmiR88 and vmiR99 (but not vmiR-TAR) stimulated human macrophage TNFα release, which is dependent on macrophage TLR8 expression. These data support a potential role for HIV-derived vmiRNAs released from infected macrophages as contributing to chronic immune activation in HIV-infected persons, and may represent a novel therapeutic target to limit AIDS pathogenesis. CONCLUSION: Novel HIV vmiR88 and vmiR99 are present in the systemic circulation of HIV+ persons and could exhibit biological function (independent of gene silencing) as ligands for TLR8 signaling that promote macrophage TNFα release, and may contribute to chronic immune activation. Targeting novel HIV-derived miRNAs may represent a therapeutic strategy to limit chronic immune activation and AIDS progression.

HIV-derived ssRNA binds to TLR8 to induce inflammation-driven macrophage foam cell formation.

Even though combined anti-retroviral therapy (cART) dramatically improves patient survival, they remain at a higher risk of being afflicted with non-infectious complications such as cardiovascular disease (CVD). This increased risk is linked to persistent inflammation and chronic immune activation. In this study, we assessed whether this complication is related to HIV-derived ssRNAs inducing in macrophages increases in TNFα release through TLR8 activation leading to foam cell formation. HIV ssRNAs induced foam cell formation in monocyte-derived macrophages (MDMs) in a dose-dependent manner. This response was reduced when either endocytosis or endosomal acidification was inhibited by dynasore or chloroquine, respectively. Using a flow cytometry FRET assay, we demonstrated that ssRNAs bind to TLR8 in HEK cells. In MDMs, ssRNAs triggered a TLR8-mediated inflammatory response that ultimately lead to foam cell formation. Targeted silencing of the TLR8 and MYD88 genes reduced foam cell formation. Furthermore, foam cell formation induced by these ssRNAs was blocked by an anti-TNFα neutralizing antibody. Taken together in MDMs, HIV ssRNAs are internalized; bind TLR8 in the endosome followed by endosomal acidification. TLR8 signaling then triggers TNFα release and ultimately leads to foam cell formation. As this response was inhibited by a blocking anti-TNFα antibody, drug targeting HIV ssRNA-driven TLR8 activation may serve as a potential therapeutic target to reduce chronic immune activation and inflammation leading to CVD in HIV+ patients.

An in vivo approach to structure activity relationship analysis of peptide ligands.

PURPOSE: The goals in this study were several-fold. First, to optimize the in vivo phage display methodology by incorporating phage pharmacokinetic properties, to isolate peptides that target the brain microvasculature, and then to build focused libraries to obtain structure activity relationship information in vivo to identify the optimal targeting motif. MATERIALS AND METHODS: The blood pharmacokinetics of filamentous and T7 phage were evaluated to choose the optimal platform. A randomized peptide library with a motif CX(10)C was constructed in T7 phage and used for in vivo panning. Focused peptide libraries around each structural element of the brain-specific peptide were constructed to perform kinetic structure activity relationship (kSAR) analysis in vivo. To determine potential function, sepsis was induced in mice by LPS administration and four hours later the effect of GST-peptide on adhesion of rhodamine-labelled lymphocytes or CFDA-labelled platelets to pial microvasculature was observed by intravital microscopy. RESULTS: The blood phamacokinetics of T7 was rapid (half-life of 12 min) which aids the clearance of non-specific phage. In vivo panning in brain enriched for isolates expressing the motif CAGALCY. Kinetic analysis of focused libraries built around each structural element of the peptide provided for rapid pharmacophore mapping. The computer modeling data suggested the peptide showed similarities to peptide mimetics of adhesion molecule ligands. GST-CAGALCY but not GST control protein was able to inhibit the rolling and adhesion of labeled platelets to inflamed pial vasculature. GST-CAGALCY had no effect on lymphocyte adhesion. CONCLUSIONS: Incorporating normal blood phamacokinetics of T7 phage into in vivo phage display improves the ability to recover targeting peptide motifs and allows effective lead optimization by kSAR. This approach led to the isolation of a brain-specific peptide, CAGALCY, which appears to function as an effective antagonist of platelet adhesion to activated pial microvasculature.