Terminally differentiated effector memory T cells associate with cognitive and AD-related biomarkers in an aging-based community cohort.

Background and Purpose: The immune response changes during aging and the progression of Alzheimer's disease (AD) and related dementia (ADRD). Terminally differentiated effector memory T cells (called TEMRA) are important during aging and AD due to their cytotoxic phenotype and association with cognitive decline. However, it is not clear if the changes seen in TEMRAs are specific to AD-related cognitive decline specifically or are more generally correlated with cognitive decline. This study aimed to examine whether TEMRAs are associated with cognition and plasma biomarkers of AD, neurodegeneration, and neuroinflammation in a community-based cohort of older adults. Methods: Study participants from a University of Kentucky Alzheimer's Disease Research Center (UK-ADRC) community-based cohort of aging and dementia were used to test our hypothesis. There were 84 participants, 44 women and 40 men. Participants underwent physical examination, neurological examination, medical history, cognitive testing, and blood collection to determine plasma biomarker levels (Aß42/Aß40 ratio, total tau, Neurofilament Light chain (Nf-L), Glial Fibrillary Acidic Protein (GFAP)) and to isolate peripheral blood mononuclear cells (PBMCs). Flow cytometry was used to analyze PBMCs from study participants for effector and memory T cell populations, including CD4+ and CD8+ central memory T cells (TCM), Naïve T cells, effector memory T cells (TEM), and effector memory CD45RA+ T cells (TEMRA) immune cell markers. Results: CD8+ TEMRAs were positively correlated with Nf-L and GFAP. We found no significant difference in CD8+ TEMRAs based on cognitive scores and no associations between CD8+ TEMRAs and AD-related biomarkers. CD4+ TEMRAs were associated with cognitive impairment on the MMSE. Gender was not associated with TEMRAs, but it did show an association with other T cell populations. Conclusion: These findings suggest that the accumulation of CD8+ TEMRAs may be a response to neuronal injury (Nf-L) and neuroinflammation (GFAP) during aging or the progression of AD and ADRD. As our findings in a community-based cohort were not clinically-defined AD participants but included all ADRDs, this suggests that TEMRAs may be associated with changes in systemic immune T cell subsets associated with the onset of pathology.

Recruitment methods and yield rates for a multisite clinical trial exploring risk reduction for Alzheimer's disease (rrAD).

INTRODUCTION: The risk reduction for Alzheimer's disease (rrAD) trial was a multisite clinical trial to assess exercise and intensive vascular pharmacological treatment on cognitive function in community-dwelling older adults at increased risk for Alzheimer's disease. METHODS: Eligibility, consent, and randomization rates across different referral sources were compared. Informal interviews conducted with each site's project team were conducted upon study completion. RESULTS: Initially, 3290 individuals were screened, of whom 28% were eligible to consent, 805 consented to participate (87.2% of those eligible), and 513 (36.3% of those consented) were randomized. Emails sent from study site listservs/databases yielded the highest amount (20.9%) of screened individuals. Professional referrals from physicians yielded the greatest percentage of consented individuals (57.1%). Referrals from non-professional contacts (ie, friends, family; 75%) and mail/phone contact from a site (73.8%) had the highest yield of randomization. DISCUSSION: Professional referrals or email from listservs/registries were most effective for enrolling participants. The greatest yield of eligible/randomized participants came from non-professional and mail/phone contacts. Future trials should consider special efforts targeting these recruitment approaches. Highlights: Clinical trial recruitment is commonly cited as a significant barrier to advancing our understanding of cognitive health interventions.The most cited referral source was email, followed by interviews/editorials on the radio, television, local newspapers, newsletters, or magazine articles.The referral method that brought in the largest number of contacts was email but did not result in the greatest yield of consents or eligible participants.The sources that yielded the greatest likelihood of consent were professional referrals (ie, physician), social media, and mail/phone contact from study site.The greatest yield of eligible/randomized participants came from non-professional contacts and mail/phone contact from a site.Findings suggest that sites may need to focus on more selective referral sources, such as using contact mailing and phone lists, rather than more widely viewed recruitment sources, such as social media or TV/radio advertisements.

A blunted TH17 cytokine signature in women with mild cognitive impairment: insights from inflammatory profiling of a community-based cohort of older adults.

People with dementia have an increase in brain inflammation, caused in part by innate and adaptive immune cells. However, it remains unknown whether dementia-associated diseases alter neuro-immune reflex arcs to impact the systemic immune system. We examined peripheral immune cells from a community-based cohort of older adults to test if systemic inflammatory cytokine signatures associated with early stages of cognitive impairment. Human peripheral blood mononuclear cells were cultured with monocyte or T-cell-targeted stimuli, and multiplex assays quantitated cytokines in the conditioned media. Following T-cell-targeted stimulation, cells from women with cognitive impairment produced lower amounts of TH17 cytokines compared with cells from cognitively healthy women, while myeloid-targeted stimuli elicited similar amounts of cytokines from cells of both groups. This TH17 signature correlated with the proportion of circulating CD4+ and CD8+ T cells and plasma glial fibrillary acidic protein and neurofilament light concentrations. These results suggest that decreases in TH17 cytokines could be an early systemic change in women at risk for developing dementia. Amelioration of TH17s cytokines in early cognitive impairment could, in part, explain the compromised ability of older adults to respond to vaccines or defend against infection.

Neuronal binding by antibodies can be influenced by low pH stress during the isolation procedure.

Low pH stress and its influence on antibody binding is a common consideration among chemists, but is only recently emerging as a consideration in Immunological studies. Antibody characterizations in Multiple Sclerosis (MS), an autoimmune disease of the Central Nervous System (CNS) has revealed that antibodies in the cerebrospinal fluid (CSF) of patients with Multiple Sclerosis bind to myelin-related and non-myelin antigen targets. Many laboratories have used molecular biology techniques to generate recombinant human antibodies (rhAbs) expressed by individual B cells from healthy donors and patients with systemic autoimmune disease to identify antigen targets. This approach has been adapted within the Neuroimmunology research community to investigate antigen targets of individual B cells in the CSF of MS patients. Our laboratory determines which antibodies to clone based on their immunogenetics and this method enriches for cloning of rhAbs that bind to neurons. However, newer technologies to assist in purification of these rhAbs from culture supernatants use an acidic elution buffer which may enhance low pH stress on the antibody structure. Our laboratory routinely uses a basic elution buffer to purify rhAbs from culture supernatants to avoid low pH stress to the antibody structure. Our goal was to investigate whether acidic elution of our rhAbs using Next Generation Chromatography would impact the rhAbs' ability to bind neurons. The limited data presented here for two neuron-binding rhAbs tested indicated that acidic elution buffers used during rhAb purification impacted the ability of rhAbs with low CDR3 charge to maintain binding to neuronal targets. Reproducibility in a larger panel of rhAbs and factors underlying these observations remain untested.

Inflammatory-associated proteomic predictors of cognitive outcome in subjects with ELVO treated by mechanical thrombectomy.

BACKGROUND: Emergent Large Vessel Occlusion (ELVO) stroke causes devastating vascular events which can lead to significant cognitive decline and dementia. In the subset of ELVO subjects treated with mechanical thrombectomy (MT) at our institution, we aimed to identify systemic and intracranial proteins predictive of cognitive function at time of discharge and at 90-days. These proteomic biomarkers may serve as prognostic indicators of recovery, as well as potential targets for novel/existing therapeutics to be delivered during the subacute stage of stroke recovery. METHODS: At the University of Kentucky Center for Advanced Translational Stroke Sciences, the BACTRAC tissue registry (clinicaltrials.gov; NCT03153683) of human biospecimens acquired during ELVO stroke by MT is utilized for research. Clinical data are collected on each enrolled subject who meets inclusion criteria. Blood samples obtained during thrombectomy were sent to Olink Proteomics for proteomic expression values. Montreal Cognitive Assessments (MoCA) were evaluated with categorical variables using ANOVA and t-tests, and continuous variables using Pearson correlations. RESULTS: There were n = 52 subjects with discharge MoCA scores and n = 28 subjects with 90-day MoCA scores. Several systemic and intracranial proteins were identified as having significant correlations to discharge MoCA scores as well as 90-day MoCA scores. Highlighted proteins included s-DPP4, CCL11, IGFBP3, DNER, NRP1, MCP1, and COMP. CONCLUSION: We set out to identify proteomic predictors and potential therapeutic targets related to cognitive outcomes in ELVO subjects undergoing MT. Here, we identify several proteins which predicted MoCA after MT, which may serve as therapeutic targets to lessen post-stroke cognitive decline.

Sex-based differences in effector cells of the adaptive immune system during Alzheimer's disease and related dementias.

Neurological conditions such as Alzheimer's disease (AD) and related dementias (ADRD) present with many challenges due to the heterogeneity of the related disease(s), making it difficult to develop effective treatments. Additionally, the progression of ADRD-related pathologies presents differently between men and women. With two-thirds of the population affected with ADRD being women, ADRD has presented itself with a bias toward the female population. However, studies of ADRD generally do not incorporate sex-based differences in investigating the development and progression of the disease, which is detrimental to understanding and treating dementia. Additionally, recent implications for the adaptive immune system in the development of ADRD bring in new factors to be considered as part of the disease, including sex-based differences in immune response(s) during ADRD development. Here, we review the sex-based differences of pathological hallmarks of ADRD presentation and progression, sex-based differences in the adaptive immune system and how it changes with ADRD, and the importance of precision medicine in the development of a more targeted and personalized treatment for this devastating and prevalent neurodegenerative condition.

Spared Premotor Areas Undergo Rapid Nonlinear Changes in Functional Organization Following a Focal Ischemic Infarct in Primary Motor Cortex of Squirrel Monkeys.

Recovery of motor function after stroke is accompanied by reorganization of movement representations in spared cortical motor regions. It is widely assumed that map reorganization parallels recovery, suggesting a causal relationship. We examined this assumption by measuring changes in motor representations in eight male and six female squirrel monkeys in the first few weeks after injury, a time when motor recovery is most rapid. Maps of movement representations were derived using intracortical microstimulation techniques in primary motor cortex (M1), ventral premotor cortex (PMv), and dorsal premotor cortex (PMd) in 14 adult squirrel monkeys before and after a focal infarct in the M1 distal forelimb area. Maps were derived at baseline and at either 2 (n = 7) or 3 weeks (n = 7) postinfarct. In PMv the forelimb maps remained unchanged at 2 weeks but contracted significantly (-42.4%) at 3 weeks. In PMd the forelimb maps expanded significantly (+110.6%) at 2 weeks but contracted significantly (-57.4%) at 3 weeks. Motor deficits were equivalent at both time points. These results highlight two features of plasticity after M1 lesions. First, significant contraction of distal forelimb motor maps in both PMv and PMd is evident by 3 weeks. Second, an unpredictable nonlinear pattern of reorganization occurs in the distal forelimb representation in PMd, first expanding at 2 weeks, and then contracting at 3 weeks postinjury. Together with previous results demonstrating reliable map expansions in PMv several weeks to months after M1 injury, the subacute time period may represent a critical window for the timing of therapeutic interventions.SIGNIFICANCE STATEMENT The relationship between motor recovery and motor map reorganization after cortical injury has rarely been examined in acute/subacute periods. In nonhuman primates, premotor maps were examined at 2 and 3 weeks after injury to primary motor cortex. Although maps are known to expand late after injury, the present study demonstrates early map expansion at 2 weeks (dorsal premotor cortex) followed by contraction at 3 weeks (dorsal and ventral premotor cortex). This nonlinear map reorganization during a time of gradual behavioral recovery suggests that the relationship between map plasticity and motor recovery is much more complex than previously thought. It also suggests that rehabilitative motor training may have its most potent effects during this early dynamic phase of map reorganization.

Thermosensitive Biodegradable Hydrogels for Local and Controlled Cerebral Delivery of Proteins: MRI-Based Monitoring of In Vitro and In Vivo Protein Release.

Hydrogels have been suggested as novel drug delivery systems for sustained release of therapeutic proteins in various neurological disorders. The main advantage these systems offer is the controlled, prolonged exposure to a therapeutically effective dose of the released drug after a single intracerebral injection. Characterization of controlled release of therapeutics from a hydrogel is generally performed in vitro, as current methods do not allow for in vivo measurements of spatiotemporal distribution and release kinetics of a loaded protein. Importantly, the in vivo environment introduces many additional variables and factors that cannot be effectively simulated under in vitro conditions. To address this, in the present contribution, we developed a noninvasive in vivo magnetic resonance imaging (MRI) method to monitor local protein release from two injected hydrogels of the same chemical composition but different initial water contents. We designed a biodegradable hydrogel formulation composed of low and high concentration thermosensitive polymer and thiolated hyaluronic acid, which is liquid at room temperature and forms a gel due to a combination of physical and chemical cross-linking upon injection at 37 °C. The in vivo protein release kinetics from these gels were assessed by MRI analysis utilizing a model protein labeled with an MR contrast agent, i.e. gadolinium-labeled albumin (74 kDa). As proof of principle, the release kinetics of the hydrogels were first measured with MRI in vitro. Subsequently, the protein loaded hydrogels were administered in male Wistar rat brains and the release in vivo was monitored for 21 days. In vitro, the thermosensitive hydrogels with an initial water content of 81 and 66% released 64 ± 3% and 43 ± 3% of the protein loading, respectively, during the first 6 days at 37 °C. These differences were even more profound in vivo, where the thermosensitive hydrogels released 83 ± 16% and 57 ± 15% of the protein load, respectively, 1 week postinjection. Measurement of volume changes of the gels over time showed that the thermosensitive gel with the higher polymer concentration increased more than 4-fold in size in vivo after 3 weeks, which was substantially different from the in vitro behavior where a volume change of 35% was observed. Our study demonstrates the potential of MRI to noninvasively monitor in vivo intracerebral protein release from a locally administered in situ forming hydrogel, which could aid in the development and optimization of such drug delivery systems for brain disorders.

Aß efflux impairment and inflammation linked to cerebrovascular accumulation of amyloid-forming amylin secreted from pancreas.

Impairment of vascular pathways of cerebral ß-amyloid (Aß) elimination contributes to Alzheimer disease (AD). Vascular damage is commonly associated with diabetes. Here we show in human tissues and AD-model rats that bloodborne islet amyloid polypeptide (amylin) secreted from the pancreas perturbs cerebral Aß clearance. Blood amylin concentrations are higher in AD than in cognitively unaffected persons. Amyloid-forming amylin accumulates in circulating monocytes and co-deposits with Aß within the brain microvasculature, possibly involving inflammation. In rats, pancreatic expression of amyloid-forming human amylin indeed induces cerebrovascular inflammation and amylin-Aß co-deposits. LRP1-mediated Aß transport across the blood-brain barrier and Aß clearance through interstitial fluid drainage along vascular walls are impaired, as indicated by Aß deposition in perivascular spaces. At the molecular level, cerebrovascular amylin deposits alter immune and hypoxia-related brain gene expression. These converging data from humans and laboratory animals suggest that altering bloodborne amylin could potentially reduce cerebrovascular amylin deposits and Aß pathology.

Rodent Stroke Models to Study Functional Recovery and Neural Repair.

Rodent ischemic stroke models are essential research tools for studying this highly prevalent disease and represent a critical element in the translational pipeline for development of new therapies. The majority of ischemic stroke models have been developed to study the acute phase of the disease and neuroprotective strategies, but a subset of models is better suited for studying stroke recovery. Each model therefore has characteristics that lend itself to certain types of investigations and outcome measures, and it is important to consider both explicit and implicit details when designing experiments that utilize each model. The following chapter briefly summarizes the known aspects of the main rodent stroke models with emphasis on their clinical relevance and suitability for studying recovery and neural repair following stroke.

FACS to Identify Immune Subsets in Mouse Brain and Spleen.

Flow cytometry enables the multi-parametric quantification of cell types, especially in immunophenotyping of unique immune cell subsets that can either contribute to or ameliorate pathology. For tissues to be used in such analyses, single-cell suspensions must be created. Here we describe protocols for preparing single-cell suspensions of mouse spleen and brain tissue, as well as the steps for fluorescently activated cell staining/sorting (FACS). Specifically, this protocol enables the isolation of lymphocytes for the study of immune responses during various diseases, such as long-term neuroinflammation following ischemic stroke.

A Guide on Analyzing Flow Cytometry Data Using Clustering Methods and Nonlinear Dimensionality Reduction (tSNE or UMAP).

Flow cytometry has been used for the last two decades to identify which immune cell subsets diapedese from the periphery into the brain parenchyma following injuries, including ischemic and hemorrhagic stroke. Recent developments have moved the analysis of high-parameter flow cytometry data sets from the traditional analysis method of manual gating to using unbiased analyses to improve scientific rigor. This chapter gives a step-by-step guide on using modern computational approaches to analyze complex flow cytometry data sets in FlowJo™ Software v10. The section will describe pre-processing and outline the steps needed to perform unsupervised clustering of your data set in addition to using nonlinear dimensionality reduction for visualizing your analysis. While these methods can identify long-term neuroinflammatory responses after stroke, the methods could be applied to a variety of flow cytometry data sets.

Co-culturing Immune Cells and Mouse-Derived Mixed Cortical Cultures with Oxygen-Glucose Deprivation to In Vitro Simulate Neuroinflammatory Interactions After Stroke.

Studying interactions between neural cells and glial cells in vitro remains an essential tool for scientists worldwide, and with the addition of oxygen-glucose deprivation (OGD) can be particularly useful for identifying mechanisms related to ischemic stroke-related injury and repair. In developing these protocols in the lab, however, we discovered the limitation of co-culturing immune cells with pure neuronal cultures as the standard media for immune cells impair neuronal growth and vice versa. Thus, we optimized a mixed cortical cell culture system that does not require the use of glial-conditioned media to support the viability and growth of neurons but can nonetheless be used to quantify neuronal survival and dendritic arborization. The following methods provide a guide as to how to culture mixed cortical cells from mouse pups (postnatal day 0-2). Additionally, we demonstrate how to co-culture mixed cortical cells with immune cells (e.g., B cells) to study neuro-immune interactions in vitro.

Use of an Automated Mouse Touchscreen Platform for Quantification of Cognitive Deficits After Central Nervous System Injury.

Analyzing cognitive performance is an important aspect of assessing physiological deficits after stroke or other central nervous system (CNS) injuries in both humans and in basic science animal models. Cognitive testing on an automated touchscreen operant platform began in humans but is now increasingly popular in preclinical studies as it enables testing in many cognitive domains in a highly reproducible way while minimizing stress to the laboratory animal. Here, we describe the step-by-step setup and application of four operant touchscreen tests used on adult mice. In brief, mice are trained to touch a graphical image on a lit screen and initiate subsequent trials for a reward. Following initial training, mice can be tested on tasks that probe performance in many cognitive domains and thus infer the integrity of brain circuits and regions. There are already many outstanding published protocols on touchscreen cognitive testing. This chapter is designed to add to the literature in two specific ways. First, this chapter provides in a single location practical, behind-the-scenes tips for setup and testing of mice in four touchscreen tasks that are useful to assess in CNS injury models: Paired Associates Learning (PAL), a task of episodic, associative (object-location) memory; Location Discrimination Reversal (LDR), a test for mnemonic discrimination (also called behavioral pattern separation) and cognitive flexibility; Autoshaping (AUTO), a test of Pavlovian or classical conditioning; and Extinction (EXT), tasks of stimulus-response and response inhibition, respectively. Second, this chapter summarizes issues to consider when performing touchscreen tests in mouse models of CNS injury. Quantifying gross and fine aspects of cognitive function is essential to improved treatment for brain dysfunction after stroke or CNS injury as well as other brain diseases, and touchscreen testing provides a sensitive, reliable, and robust way to achieve this.

Using Operant Reach Chambers to Assess Mouse Skilled Forelimb Use After Stroke.

Skilled forelimb reaching and grasping are important components of rodent motor performance. The isometric pull task can serve as a tool for quantifying forelimb function following stroke or other CNS injury as well as in forelimb rehabilitation. This task has been extensively developed for use in rats. Here, we describe methods of setup and training of an operant reach chamber for mice. Using a reward of peanut oil, mice are adaptively trained to pull a handle positioned slightly outside of an operant chamber, with automated recording of the number of attempts, force generated, success rate, and latency to maximal force.

The immunopathology of B lymphocytes during stroke-induced injury and repair.

B cells, also known as B lymphocytes or lymphoid lineage cells, are a historically understudied cell population with regard to brain-related injuries and diseases. However, an increasing number of publications have begun to elucidate the different phenotypes and roles B cells can undertake during central nervous system (CNS) pathology, including following ischemic and hemorrhagic stroke. B cell phenotype is intrinsically linked to function following stroke, as they may be beneficial or detrimental depending on the subset, timing, and microenvironment. Factors such as age, sex, and presence of co-morbidity also influence the behavior of post-stroke B cells. The following review will briefly describe B cells from origination to senescence, explore B cell function by integrating decades of stroke research, differentiate between the known B cell subtypes and their respective activity, discuss some of the physiological influences on B cells as well as the influence of B cells on certain physiological functions, and highlight the differences between B cells in healthy and disease states with particular emphasis in the context of ischemic stroke.

Evaluation of noise regression techniques in resting-state fMRI studies using data of 434 older adults.

Subject motion is a well-known confound in resting-state functional MRI (rs-fMRI) and the analysis of functional connectivity. Consequently, several clean-up strategies have been established to minimize the impact of subject motion. Physiological signals in response to cardiac activity and respiration are also known to alter the apparent rs-fMRI connectivity. Comprehensive comparisons of common noise regression techniques showed that the "Independent Component Analysis based strategy for Automatic Removal of Motion Artifacts" (ICA-AROMA) was a preferred pre-processing technique for teenagers and adults. However, motion and physiological noise characteristics may differ substantially for older adults. Here, we present a comprehensive comparison of noise-regression techniques for older adults from a large multi-site clinical trial of exercise and intensive pharmacological vascular risk factor reduction. The Risk Reduction for Alzheimer's Disease (rrAD) trial included hypertensive older adults (60-84 years old) at elevated risk of developing Alzheimer's Disease (AD). We compared the performance of censoring, censoring combined with global signal regression, non-aggressive and aggressive ICA-AROMA, as well as the Spatially Organized Component Klassifikator (SOCK) on the rs-fMRI baseline scans from 434 rrAD subjects. All techniques were rated based on network reproducibility, network identifiability, edge activity, spatial smoothness, and loss of temporal degrees of freedom (tDOF). We found that non-aggressive ICA-AROMA did not perform as well as the other four techniques, which performed table with marginal differences, demonstrating the validity of these techniques. Considering reproducibility as the most important factor for longitudinal studies, given low false-positive rates and a better preserved, more cohesive temporal structure, currently aggressive ICA-AROMA is likely the most suitable noise regression technique for rs-fMRI studies of older adults.

Plasma protein alterations during human large vessel stroke: A controlled comparison study.

BACKGROUND: Stroke is a major cause of death and disability in the United States. Mechanical thrombectomy (MT) and tissue plasminogen activator are the current treatments for ischemic stroke, which have improved clinical outcomes. Despite these treatments, functional and cognitive deficits still occur demonstrating a need for predictive biomarkers for beneficial clinical outcomes which can be used as therapeutic targets for pharmacotherapy. The aim of this study compares the proteomic expression of systemic arterial blood collected at the time of MT to those from a matched cerebrovascular disease (CVD) control cohort. METHODS: The Blood And Clot Thrombectomy Registry And Collaboration (BACTRAC) (clinicaltrials.gov NCT03153683) collects and banks arterial blood, both distal and proximal to the thrombus, from ischemic stroke subjects undergoing MT. Arterial blood from patients undergoing a diagnostic angiogram was also collected and banked as CVD controls. Changes in cardiometabolic and inflammatory proteins between stroke and CVD controls were analyzed via Olink Proteomics. RESULTS: Proteins including ARTN, TWEAK, HGF, CCL28, FGF-5, CXCL9, TRANCE and GDNF were found to be decreased in stroke subjects when compared to CVD controls. CXCL1, CCL5, OSM, GP1BA, IL6, MMP-1, and CXCL5 were increased in stroke subjects when compared to CVD controls. These proteins were also significantly correlated to stroke outcome metrics such as NIHSS, infarct volume and MoCA scoring. CONCLUSION: Overall, acute stroke patients had an increase in inflammatory proteins with a decrease in trophic proteins systemically compared to matched CVD controls. Using our CVD controls, proteins of interest were directly compared to stroke patients with the same cerebrovascular risk factors instead of statistically controlling for comorbidities. The novel methodology of matching an arterial blood CVD control group to a stroke group, as well as controlling for age and comorbid status add to the literature on prognostic stroke biomarkers, which are specific targets for future therapeutics.