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Traumatic brain injury (TBI) is a risk factor for later-life dementia. Clinical and pre-clinical studies have elucidated multiple mechanisms through which TBI may influence or exacerbate multiple pathological processes underlying Alzheimer's disease and Alzheimer's disease-related dementias (AD/ADRD). The National Institutes of Health hosts triennial ADRD Summits to inform a national research agenda, and the 2019 ADRD Summit was the first to highlight "TBI and AD/ADRD Risk" as an emerging topic in the field. A multidisciplinary committee of TBI researchers with relevant expertise reviewed extant literature, identified research gaps and opportunities, and proposed draft research recommendations at the 2019 ADRD Summit. These research recommendations, further refined after broad stakeholder input at the Summit, cover four overall areas: 1) Encourage crosstalk and interdisciplinary collaboration between TBI and dementia researchers; 2) Establish infrastructure to study TBI as a risk factor for AD/ADRD; 3) Promote basic and clinical research examining the development and progression of TBI AD/ADRD neuropathologies and associated clinical symptoms; and 4) Characterize the clinical phenotype of progressive dementia associated with TBI and develop non-invasive diagnostic approaches. These recommendations recognize a need to strengthen communication and build frameworks to connect the complexity of TBI with rapidly evolving AD/ADRD research. Recommendations acknowledge TBI as a clinically and pathologically heterogeneous disease whose associations with AD/ADRDs remain incompletely understood. The recommendations highlight the scientific advantage of investigating AD/ADRD in the context of a known TBI exposure, the study of which can directly inform on disease mechanisms and treatment targets for AD/ADRDs with shared common pathways.

To describe the protocol and findings of the instrumental validation of three imaging-based biomarker kits selected by the MarkVCID consortium: free water (FW) and peak width of skeletonized mean diffusivity (PSMD), both derived from diffusion tensor imaging (DTI), and white matter hyperintensity (WMH) volume derived from fluid attenuation inversion recovery and T1-weighted imaging.The instrumental validation of imaging-based biomarker kits included inter-rater reliability among participating sites, test-retest repeatability, and inter-scanner reproducibility across three types of magnetic resonance imaging (MRI) scanners using intra-class correlation coefficients (ICC).The three biomarkers demonstrated excellent inter-rater reliability (ICC 0.94,The three biomarker kits demonstrated very high inter-rater reliability, test-retest repeatability, and inter-scanner reproducibility, offering robust biomarkers suitable for future multi-site observational studies and clinical trials in the context of vascular cognitive impairment and dementia (VCID).

The MarkVCID consortium was formed under cooperative agreements with the National Institute of Neurologic Diseases and Stroke (NINDS) and National Institute on Aging (NIA) in 2016 with the goals of developing and validating biomarkers for the cerebral small vessel diseases associated with the vascular contributions to cognitive impairment and dementia (VCID). Rigorously validated biomarkers have consistently been identified as crucial for multicenter studies to identify effective strategies to prevent and treat VCID, specifically to detect increased VCID risk, diagnose the presence of small vessel disease and its subtypes, assess prognosis for disease progression or response to treatment, demonstrate target engagement or mechanism of action for candidate interventions, and monitor disease progression during treatment. The seven project sites and central coordinating center comprising MarkVCID, working with NINDS and NIA, identified a panel of 11 candidate fluid- and neuroimaging-based biomarker kits and established harmonized multicenter study protocols (see companion paper "MarkVCID cerebral small vessel consortium: I. Enrollment, clinical, fluid protocols" for full details). Here we describe the MarkVCID neuroimaging protocols with specific focus on validating their application to future multicenter trials. MarkVCID procedures for participant enrollment; clinical and cognitive evaluation; and collection, handling, and instrumental validation of fluid samples are described in detail in a companion paper. Magnetic resonance imaging (MRI) has long served as the neuroimaging modality of choice for cerebral small vessel disease and VCID because of its sensitivity to a wide range of brain properties, including small structural lesions, connectivity, and cerebrovascular physiology. Despite MRI's widespread use in the VCID field, there have been relatively scant data validating the repeatability and reproducibility of MRI-based biomarkers across raters, scanner types, and time intervals (collectively defined as instrumental validity). The MRI protocols described here address the core MRI sequences for assessing cerebral small vessel disease in future research studies, specific sequence parameters for use across various research scanner types, and rigorous procedures for determining instrumental validity. Another candidate neuroimaging modality considered by MarkVCID is optical coherence tomography angiography (OCTA), a non-invasive technique for directly visualizing retinal capillaries as a marker of the cerebral capillaries. OCTA has theoretical promise as a unique opportunity to visualize small vessels derived from the cerebral circulation, but at a considerably earlier stage of development than MRI. The additional OCTA protocols described here address procedures for determining OCTA instrumental validity, evaluating sources of variability such as pupil dilation, and handling data to maintain participant privacy. MRI protocol and instrumental validation The core sequences selected for the MarkVCID MRI protocol are three-dimensional T1-weighted multi-echo magnetization-prepared rapid-acquisition-of-gradient-echo (ME-MPRAGE), three-dimensional T2-weighted fast spin echo fluid-attenuated-inversion-recovery (FLAIR), two-dimensional diffusion-weighted spin-echo echo-planar imaging (DWI), three-dimensional T2*-weighted multi-echo gradient echo (3D-GRE), three-dimensional T2 -weighted fast spin-echo imaging (T2w), and two-dimensional T2*-weighted gradient echo echo-planar blood-oxygenation-level-dependent imaging with brief periods of CO2 inhalation (BOLD-CVR). Harmonized parameters for each of these core sequences were developed for four 3 Tesla MRI scanner models in widespread use at academic medical centers. MarkVCID project sites are trained and certified for their instantiation of the consortium MRI protocols. Sites are required to perform image quality checks every 2 months using the Alzheimer's Disease Neuroimaging Initiative phantom. Instrumental validation for MarkVCID MRI-based biomarkers is operationally defined as inter-rater reliability, test-retest repeatability, and inter-scanner reproducibility. Assessments of these instrumental properties are performed on individuals representing a range of cerebral small vessel disease from mild to severe. Inter-rater reliability is determined by distribution of an independent dataset of MRI scans to each analysis site. Test-retest repeatability is determined by repeat MRI scans performed on individual participants on a single MRI scanner after a short (1- to 14-day) interval. Inter-scanner reproducibility is determined by repeat MRI scans performed on individuals performed across four MRI scanner models. OCTA protocol and instrumental validation The MarkVCID OCTA protocol uses a commercially available, Food and Drug Administration-approved OCTA apparatus. Imaging is performed on one dilated and one undilated eye to assess the need for dilation. Scans are performed in quadruplicate. MarkVCID project sites participating in OCTA validation are trained and certified by this biomarker's lead investigator. Inter-rater reliability for OCTA is assessed by distribution of OCTA datasets to each analysis site. Test-retest repeatability is assessed by repeat OCTA imaging on individuals on the same day as their baseline OCTA and a different-day repeat session after a short (1- to 14-day) interval. Methods were developed to allow the OCTA data to be de-identified by the sites before transmission to the central data management system. The MarkVCID neuroimaging protocols, like the other MarkVCID procedures, are designed to allow translation to multicenter trials and as a template for outside groups to generate directly comparable neuroimaging data. The MarkVCID neuroimaging protocols are available to the biomedical community and intended to be shared. In addition to the instrumental validation procedures described here, each of the neuroimaging MarkVCID kits will undergo biological validation to determine its ability to measure important aspects of VCID such as cognitive function. The analytic methods for the neuroimaging-based kits and the results of these validation studies will be published separately. The results will ultimately determine the neuroimaging kits' potential usefulness for multicenter interventional trials in small vessel disease-related VCID.

The concept of vascular contributions to cognitive impairment and dementia (VCID) derives from more than two decades of research indicating that (1) most older individuals with cognitive impairment have post mortem evidence of multiple contributing pathologies and (2) along with the preeminent role of Alzheimer's disease (AD) pathology, cerebrovascular disease accounts for a substantial proportion of this contribution. Contributing cerebrovascular processes include both overt strokes caused by etiologies such as large vessel occlusion, cardioembolism, and embolic infarcts of unknown source, and frequently asymptomatic brain injuries caused by diseases of the small cerebral vessels. Cerebral small vessel diseases such as arteriolosclerosis and cerebral amyloid angiopathy, when present at moderate or greater pathologic severity, are independently associated with worse cognitive performance and greater likelihood of dementia, particularly in combination with AD and other neurodegenerative pathologies. Based on this evidence, the US National Alzheimer's Project Act explicitly authorized accelerated research in vascular and mixed dementia along with frontotemporal and Lewy body dementia and AD itself. Biomarker development has been consistently identified as a key step toward translating scientific advances in VCID into effective prevention and treatment strategies. Validated biomarkers can serve a range of purposes in trials of candidate interventions, including (1) identifying individuals at increased VCID risk, (2) diagnosing the presence of cerebral small vessel disease or specific small vessel pathologies, (3) stratifying study participants according to their prognosis for VCID progression or treatment response, (4) demonstrating an intervention's target engagement or pharmacodynamic mechanism of action, and (5) monitoring disease progression during treatment. Effective biomarkers allow academic and industry investigators to advance promising interventions at early stages of development and discard interventions with low success likelihood. The MarkVCID consortium was formed in 2016 with the goal of developing and validating fluid- and imaging-based biomarkers for the cerebral small vessel diseases associated with VCID. MarkVCID consists of seven project sites and a central coordinating center, working with the National Institute of Neurologic Diseases and Stroke and National Institute on Aging under cooperative agreements. Through an internal selection process, MarkVCID has identified a panel of 11 candidate biomarker "kits" (consisting of the biomarker measure and the clinical and cognitive data used to validate it) and established a range of harmonized procedures and protocols for participant enrollment, clinical and cognitive evaluation, collection and handling of fluid samples, acquisition of neuroimaging studies, and biomarker validation. The overarching goal of these protocols is to generate rigorous validating data that could be used by investigators throughout the research community in selecting and applying biomarkers to multi-site VCID trials. Key features of MarkVCID participant enrollment, clinical/cognitive testing, and fluid biomarker procedures are summarized here, with full details in the following text, tables, and supplemental material, and a description of the MarkVCID imaging biomarker procedures in a companion paper, "MarkVCID Cerebral small vessel consortium: II. Neuroimaging protocols." The procedures described here address a range of challenges in MarkVCID's design, notably: (1) acquiring all data under informed consent and enrollment procedures that allow unlimited sharing and open-ended analyses without compromising participant privacy rights; (2) acquiring the data in a sufficiently wide range of study participants to allow assessment of candidate biomarkers across the various patient groups who might ultimately be targeted in VCID clinical trials; (3) defining a common dataset of clinical and cognitive elements that contains all the key outcome markers and covariates for VCID studies and is realistically obtainable during a practical study visit; (4) instituting best fluid-handling practices for minimizing avoidable sources of variability; and (5) establishing rigorous procedures for testing the reliability of candidate fluid-based biomarkers across replicates, assay runs, sites, and time intervals (collectively defined as the biomarker's instrumental validity). Participant Enrollment Project sites enroll diverse study cohorts using site-specific inclusion and exclusion criteria so as to provide generalizable validation data across a range of cognitive statuses, risk factor profiles, small vessel disease severities, and racial/ethnic characteristics representative of the diverse patient groups that might be enrolled in a future VCID trial. MarkVCID project sites include both prospectively enrolling centers and centers providing extant data and samples from preexisting community- and population-based studies. With approval of local institutional review boards, all sites incorporate MarkVCID consensus language into their study documents and informed consent agreements. The consensus language asks prospectively enrolled participants to consent to unrestricted access to their data and samples for research analysis within and outside MarkVCID. The data are transferred and stored as a de-identified dataset as defined by the Health Insurance Portability and Accountability Act Privacy Rule. Similar human subject protection and informed consent language serve as the basis for MarkVCID Research Agreements that act as contracts and data/biospecimen sharing agreements across the consortium. Clinical and Cognitive Data Clinical and cognitive data are collected across prospectively enrolling project sites using common MarkVCID instruments. The clinical data elements are modified from study protocols already in use such as the Alzheimer's Disease Center program Uniform Data Set Version 3 (UDS3), with additional focus on VCID-related items such as prior stroke and cardiovascular disease, vascular risk factors, focal neurologic findings, and blood testing for vascular risk markers and kidney function including hemoglobin A1c, cholesterol subtypes, triglycerides, and creatinine. Cognitive assessments and rating instruments include the Clinical Dementia Rating Scale, Geriatric Depression Scale, and most of the UDS3 neuropsychological battery. The cognitive testing requires ≈60 to 90 minutes. Study staff at the prospectively recruiting sites undergo formalized training in all measures and review of their first three UDS3 administrations by the coordinating center. Collection and Handling of Fluid Samples Fluid sample types collected for MarkVCID biomarker kits are serum, ethylenediaminetetraacetic acid-plasma, platelet-poor plasma, and cerebrospinal fluid (CSF) with additional collection of packed cells to allow future DNA extraction and analyses. MarkVCID fluid guidelines to minimize variability include fasting morning fluid collections, rapid processing, standardized handling and storage, and avoidance of CSF contact with polystyrene. Instrumental Validation for Fluid-Based Biomarkers Instrumental validation of MarkVCID fluid-based biomarkers is operationally defined as determination of intra-plate and inter-plate repeatability, inter-site reproducibility, and test-retest repeatability. MarkVCID study participants both with and without advanced small vessel disease are selected for these determinations to assess instrumental validity across the full biomarker assay range. Intra- and inter-plate repeatability is determined by repeat assays of single split fluid samples performed at individual sites. Inter-site reproducibility is determined by assays of split samples distributed to multiple sites. Test-retest repeatability is determined by assay of three samples acquired from the same individual, collected at least 5 days apart over a 30-day period and assayed on a single plate. The MarkVCID protocols are designed to allow direct translation of the biomarker validation results to multicenter trials. They also provide a template for outside groups to perform analyses using identical methods and therefore allow direct comparison of results across studies and centers. All MarkVCID protocols are available to the biomedical community and intended to be shared. In addition to the instrumental validation procedures described here, each of the MarkVCID kits will undergo biological validation to determine whether the candidate biomarker measures important aspects of VCID such as cognitive function. Analytic methods and results of these validation studies for the 11 MarkVCID biomarker kits will be published separately. The results of this rigorous validation process will ultimately determine each kit's potential usefulness for multicenter interventional trials aimed at preventing or treating small vessel disease related VCID.

See related article, p 2341 Stroke risk and poststroke disability have steadily decreased in the United States over the past 2 decades because of improved prevention and access to reperfusion therapies for acute ischemic stroke, such as tPA (tissue-type plasminogen activator; alteplase) and endovascular thrombectomy. Despite the efficacy and safety of thrombolysis and thrombectomy, not all patients who receive the treatment improve to full, independent recovery, and most patients are ineligible for treatment. Additionally, there are no efficacious treatments to improve long-term outcomes for patients after the acute phase of ischemic stroke or to reduce brain injury induced by acute intracerebral hemorrhage. Therefore, development of new therapies for both acute and chronic stroke is sorely needed. Stroke occurs because of a variety of vascular pathologies and injury mechanisms, some of which are difficult to model in animals. With the exception of reperfusion therapy, preclinical research end points do not generally reflect clinical outcomes. Pharmacodynamics, pharmacokinetics, and target engagement in the human brain need to be further developed and optimized for stroke interventions so that drug level in brain tissue, time to initiation, and duration of treatment can be accurately measured in clinical trials. Many variables, such as heterogeneity of vascular pathologies, patient demographics, and a host of comorbid conditions, as well as the lack of validated biomarkers to stratify patient populations, limit the ability of typical stroke clinical trials to detect a treatment effect. To address these gaps, the National Institute of Neurological Disorders and Stroke organized and sponsored the workshop Translational Stroke Research: Vision and Opportunities, which was held in Bethesda, Maryland, on November 1 to 2, 2016. The workshop gathered over 180 registered participants from academia, industry, the Food and Drug Administration, and other public and private funding agencies. …

White matter hyperintensities (WMHs) are frequently seen on brain magnetic resonance imaging scans of older people. Usually interpreted clinically as a surrogate for cerebral small vessel disease, WMHs are associated with increased likelihood of cognitive impairment and dementia (including Alzheimer's disease [AD]). WMHs are also seen in cognitively healthy people. In this collaboration of academic, clinical, and pharmaceutical industry perspectives, we identify outstanding questions about WMHs and their relation to cognition, dementia, and AD. What molecular and cellular changes underlie WMHs? What are the neuropathological correlates of WMHs? To what extent are demyelination and inflammation present? Is it helpful to subdivide into periventricular and subcortical WMHs? What do WMHs signify in people diagnosed with AD? What are the risk factors for developing WMHs? What preventive and therapeutic strategies target WMHs? Answering these questions will improve prevention and treatment of WMHs and dementia.

The research into vascular contributions to cognitive impairment and dementia (VCID) aims to understand the importance of cerebrovascular biology in cognitive decline. Prevention and treatment of VCID is poised to have major impact on dementia-related disease burden and is thus a critical emerging objective in dementia research. This article presents VCID consortia focused on multidisciplinary approaches to identify key pathologic targets and develop diagnostic tools with the goal of bridging the divide between basic research and clinical trials. Members of these multi-institute, multidisciplinary consortia provide a prospective on the history and emerging science of VCID and how VCID consortia can address some of the more complex questions in VCID and drive the field forward. These consortia, and others like them, are uniquely suited to tackle some of the most difficult obstacles in translating research to the clinic.

The World Health Organization reports that 47.5 million people are affected by dementia worldwide. With aging populations and 7.7 million new cases each year, the burden of illness due to dementia approaches crisis proportions. Despite significant advances in our understanding of the biology of Alzheimer’s disease, the leading dementia diagnosis, the actual causes of dementia in affected individuals are unknown except for rare fully penetrant genetic forms. Evidence from epidemiology and pathology studies indicates that damage to the vascular system is associated with an increased risk of many types of dementia. Both Alzheimer’s pathology and cerebrovascular disease increase with age. How Alzheimer’s disease affects small blood vessel function and how vascular dysfunction contributes to the molecular pathology of Alzheimer’s are areas of intense research. The science of vascular contributions to cognitive impairment and dementia (VCID) integrates diverse aspects of biology and incorporates the roles of multiple cell types that support the function of neural tissue. Because of the proven ability to prevent and treat cardiovascular disease and hypertension with population benefits for heart and stroke outcomes, it is proposed that understanding and targeting the biological mechanisms of VCID can have a similarly positive impact on public health.

Goal 1 of the National Plan to Address Alzheimer’s Disease is to prevent and effectively treat Alzheimer disease and Alzheimer disease–related dementias by 2025. To help inform the research agenda toward achieving this goal, the NIH hosts periodic summits that set and refine relevant research priorities for the subsequent 5 to 10 years. This proceedings article summarizes the 2016 Alzheimer's Disease–Related Dementias Summit, including discussion of scientific progress, challenges, and opportunities in major areas of dementia research, including mixed-etiology dementias, Lewy body dementia, frontotemporal degeneration, vascular contributions to cognitive impairment and dementia, dementia disparities, and dementia nomenclature.

The National Alzheimer's Project Act, signed into law in 2011, mandates a National Plan to Address Alzheimer's Disease that is updated annually. In the Plan, the term Alzheimer disease includes not only Alzheimer disease (AD) proper, but also several specified related dementias, namely, frontotemporal, Lewy body, vascular, and mixed dementia. In response to a specific action item in the 2012 National Plan, the National Institute of Neurological Disorders and Stroke, in collaboration with the National Institute on Aging, convened panels of experts and conducted a 2-day public conference to develop research priorities and timelines for addressing Alzheimer disease–related dementias (ADRD) in 5 topic areas: multiple etiology dementias, health disparities, Lewy body dementias including dementia with Lewy bodies and Parkinson disease dementia, frontotemporal dementia and related tauopathies, and vascular contributions to ADRD. By design, the product was up to 8 prioritized research recommendations in each topic area including estimated timelines from when work on a recommendation is started to completion or to full implementation of an ongoing activity, and recognition of shared research themes across recommendations. These included increased education and training of both researchers and health care professionals, addressing health disparities, fundamental neurobiology research, advanced diagnostics, collaborative biosample repositories, and a focus on developing effective interventions to prevent or treat ADRD by the year 2025 as targeted by the National Plan.

Naturally occurring cell death is a universal feature of developing nervous systems that plays an essential role in determining adult brain function. Yet little is known about the decisions that select a subset of CNS neurons for survival and cause others to die. We report that postnatal day 0 NMDA receptor subunit 1 (NMDAR1) knockout mice display an ≈2-fold increase in cell death in the brainstem trigeminal complex (BSTC), including all four nuclei that receive somatosensory inputs from the face (principalis, oralis, interpolaris, and caudalis). Treatment with the NMDA receptor antagonist dizocilpine maleate (MK-801) for 24 h before birth also caused an increase in cell death that reached statistical significance in two of the four nuclei (oralis and interpolaris). The neonatal sensitivity to NMDA receptor hypofunction in the BSTC, and in its main thalamic target, the ventrobasal nucleus (VB), coincides with the peak of naturally occurring cell death and trigeminothalamic synaptogenesis. At embryonic day 17.5, before the onset of these events, NMDAR1 knockout does not affect cell survival in either the BSTC or the VB. Immunostaining for active caspase-3 and the neuronal marker Hu specifically confirms the presence of dying neurons in the BSTC and the VB of NMDAR1 knockout neonates. Finally, genetic deletion of Bax rescues these structures from the requirement for NMDA receptors to limit naturally occurring cell death. Taken together, the results indicate that NMDA receptors play a survival role for somatosensory relay neurons during synaptogenesis by inhibiting Bax-dependent developmental cell death.

Signaling through gap junctions (electrical synapses) is important in the development of the mammalian central nervous system. Abundant between neurons during postnatal development, gap junction coupling subsequently decreases and remains low in the adult, confined to specific subsets of neurons. Here we report that developmental uncoupling of gap junctions in the rat hypothalamus in vivo and in vitro is associated with a decrease in connexin 36 (Cx36) protein expression. Both developmental gap junction uncoupling and Cx36 downregulation are prevented by the blockade of NMDA glutamate receptors, action potentials and the calcium-cyclic AMP response element binding protein (CREB), and are accelerated by CREB overexpression. Developmental gap junction uncoupling and Cx36 downregulation are not affected by blockade of non-NMDA glutamate receptors, and do not occur in hypothalamic neurons from NMDA receptor subunit 1 (NMDAR1) knockout mice. These results demonstrate that NMDA receptor activity contributes to the developmental uncoupling of gap junctions via CREB-dependent downregulation of Cx36.

Genetic deletion of NMDA glutamate receptors disrupts development of whisker-related neuronal patterns in the somatosensory system. Independent studies have shown that NMDA receptor antagonists increase cell death among developing neurons. Here, we report that a dramatic feature of the developing somatosensory system in newborn NMDA receptor 1 (NMDAR1) knock-out mice is increased cell death in the ventrobasal nucleus (VB) of the thalamus. Sections were subject to terminal deoxynucleotidyl transferase dUTP nick end labeling staining for apoptotic DNA fragmentation, thionine staining for pyknotic nuclei, silver staining for degenerating cells, and immunostaining for caspase-3. All four methods demonstrated that deletion of NMDAR1 causes a large (on the order of threefold to fivefold) increase in cell death in the VB. The NMDA receptor antagonists dizocilpine maleate (MK-801) and phencyclidine also increase cell death in this structure. The onset of increased cell death in the VB in the absence of NMDA receptor function is approximately the time of birth, overlaps with naturally occurring cell death and synaptogenesis, and displays some anatomical specificity. For example, there was no increase in cell death in the hippocampus or neocortex of NMDAR1 knock-out mice at any of the time points examined: embryonic day 15.5 (E15.5), E17.5, and postnatal day 0. We also report a significant reduction in the size of the VB that is evident starting at E17.5. The results indicate that NMDA receptors play a major role in cell survival during naturally occurring cell death in the VB and demonstrate that cell death is a consideration in NMDA receptor knock-out studies.

One of the most abundant nicotinic acetylcholine receptors expressed in the central and peripheral nervous systems is a species that contains the alpha 7 gene product, binds alpha-bungarotoxin with high affinity, and has a high relative permeability to calcium. The alpha 7 gene is also expressed at low levels in embryonic muscle tissue. We show here that the alpha 7 gene is expressed in tendon fibroblasts and periosteal cells during development. In situ hybridizations identify alpha 7 transcripts in tissue sections containing embryonic tendon and periosteum. RNase protection experiments demonstrate alpha 7 mRNA in primary tendon cells grown in culture. Immunofluorescence with subunit-specific monoclonal antibodies reveals alpha 7 protein in embryonic tendon. Immunoprecipitation assays with the antibodies indicate that the alpha 7-containing species in tendon is capable of binding alpha-bungarotoxin and that a similar species can be identified at low levels on the surface of fibroblasts in culture. The results show that the alpha 7 gene product is expressed in a range of tissues, including cells thought to be nonexcitable. The distribution of alpha 7 expression early in development and the ability of alpha 7-containing receptors to elevate intracellular calcium suggest that the gene may influence a variety of calcium-dependent events during embryogenesis.

Scientific evidence continues to demonstrate the linkage of vascular contributions to cognitive impairment and dementia such as Alzheimer's disease. In December, 2013, the Alzheimer's Association, with scientific input from the National Institute of Neurological Disorders and Stroke and the National Heart, Lung and Blood Institute from the National Institutes of Health, convened scientific experts to discuss the research gaps in our understanding of how vascular factors contribute to Alzheimer's disease and related dementia. This manuscript summarizes the meeting and the resultant discussion, including an outline of next steps needed to move this area of research forward.

Mammalian brain development requires the transmission of electrical signals between neurons via theN-methyl-d-aspartate (NMDA) class of glutamate receptors. However, little is known about how NMDA receptors carry out this role. Here we report the first genes shown to be regulated by physiological levels of NMDA receptor function in developing neuronsin vivo: NMDA receptor-regulated gene 1(NARG1), NARG2, and NARG3. These genes share several striking regulatory features. All three are expressed at high levels in the neonatal brain in regions of neuronal proliferation and migration, are dramatically down-regulated during early postnatal development, and are down-regulated by NMDA receptor function. NARG2 and NARG3 appear to be novel, while NARG1 is the mammalian homologue of a yeast N-terminal acetyltransferase that regulates entry into the Go phase of the cell cycle. The results suggest that highly specific NMDA receptor-dependent regulation of gene expression plays an important role in the transition from proliferation of neuronal precursors to differentiation of neurons.

Over the past several decades, anatomical and electrophysiological analyses have demonstrated that the electrical activity of neurons is required for development of the precise patterns of synaptic connectivity found in the adult central nervous system. However, knowledge of the molecular cascades that underlie activity-dependent synaptic development remains rudimentary. As a result, many fundamental issues remain unresolved. Recent advances in differential cloning have begun to provide the tools and insight necessary to bring a molecular level of understanding to principles of activity-dependent synaptic development established via classic systems approaches. © 1999 John Wiley & Sons, Inc. J Neurobiol 41: 148–157, 1999

During visual system development, neural activity regulates structural changes in connectivity including axonal branching and dendritic growth. Here we have examined a role for the candidate plasticity gene 15 (cpg15), which encodes an activity-regulated molecule that can promote dendritic growth, in this process. We report that cpg15 is expressed in the cat visual system at relatively high levels in the lateral geniculate nucleus (LGN) but at very low levels in its synaptic target, layer 4 of the visual cortex. Prenatally, when cpg15 mRNA in the LGN is most abundant, expression is insensitive to action potential blockade by tetrodotoxin. Postnatally, activity regulation of cpg15 emerges in the LGN coincident with development of ocular dominance columns in the visual cortex. cpg15 can be detected in layers 2/3 and 5/6 of visual cortex postnatally, and expression in layers 2/3 is activity-regulated during known periods of activity-dependent plasticity for these layers. Localization and regulation of cpg15 expression in the visual system are consistent with a presynaptic role for CPG15 in shaping dendritic arbors of target neurons during activity-dependent synaptic rearrangements, both in development and adulthood.

Background: Genetic ancestry is known to impact outcomes of genotype-phenotype studies that are designed to identify risk for common diseases in human populations. Failure to control for population stratification due to genetic ancestry can significantly confound results of disease association studies. Moreover, ancestry is a critical factor in assessing lifetime risk of disease, and can play an important role in optimizing treatment. As modern medicine moves towards using personal genetic information for clinical applications, it is important to determine genetic ancestry in an accurate, cost-effective and efficient manner. Self-identified race is a common method used to track and control for population stratification; however, social constructs of race are not necessarily informative for genetic applications. The use of ancestry informative markers (AIMs) is a more accurate method for determining genetic ancestry for the purposes of population stratification. Methodology/Principal Findings: Here we introduce a novel panel of 36 microsatellite (MSAT) AIMs that determines continental admixture proportions. This panel, which we have named Continental Ancestry Informative Markers or CoAIMs, consists of MSAT AIMs that were chosen based upon their measure of genetic variance (Fst), allele frequencies and their suitability for efficient genotyping. Genotype analysis using CoAIMs along with a Bayesian clustering method (STRUCTURE) is able to discern continental origins including Europe/Middle East (Caucasians), East Asia, Africa, Native America, and Oceania. In addition to determining continental ancestry for individuals without significant admixture, we applied CoAIMs to ascertain admixture proportions of individuals of self declared race. Conclusion/Significance: CoAIMs can be used to efficiently and effectively determine continental admixture proportions in a sample set. The CoAIMs panel is a valuable resource for genetic researchers performing case-control genetic association studies, as it can control for the confounding effects of population stratification. The MSAT-based approach used here has potential for broad applicability as a cost effective tool toward determining admixture proportions.

We recently used Western blots for connexin 36 and neuronal dye coupling with neurobiotin to measure developmental decrease in neuronal gap junction coupling in cell cultures. To ask whether Ca2+ imaging also can be used to measure changes in the amount of neuronal gap junction coupling, we defined a Ca2+ coupling coefficient as the percentage of neurons with bicuculline-induced increases in intracellular Ca2+ that are suppressed by blocking gap junctions. We demonstrate in rat and mouse hypothalamic neuronal cultures that the Ca2+ coupling coefficient decreases during culture development, this decrease is prevented by manipulations that also prevent developmental decrease in neuronal gap junction coupling, and the coefficient is low in cultures lacking connexin 36. The results indicate that Ca2+ imaging is a useful tool to quantify the amount of neuronal gap junction coupling in cultures.

Amyotrophic lateral sclerosis (ALS) is the most common form of motor neuron disease (MND). It is currently incurable and treatment is largely limited to supportive care. Family history is associated with an increased risk of ALS, and many Mendelian causes have been discovered. However, most forms of the disease are not obviously familial. Recent advances in human genetics have enabled genome-wide analyses of single nucleotide polymorphisms (SNPs) that make it possible to study complex genetic contributions to human disease. Genome-wide SNP analyses require a large sample size and thus depend upon collaborative efforts to collect and manage the biological samples and corresponding data. Public availability of biological samples (such as DNA), phenotypic and genotypic data further enhances research endeavors. Here we discuss a large collaboration among academic investigators, government, and non-government organizations which has created a public repository of human DNA, immortalized cell lines, and clinical data to further gene discovery in ALS. This resource currently maintains samples and associated phenotypic data from 2332 MND subjects and 4692 controls. This resource should facilitate genetic discoveries which we anticipate will ultimately provide a better understanding of the biological mechanisms of neurodegeneration in ALS.

A number of studies have indicated an important role for N-methyl-d-aspartate (NMDA) receptors in cell survival versus cell death decisions during neuronal development, trauma, and ischemia. Coupling of neurons by electrical synapses (gap junctions) is high or increases in neuronal networks during all three of these conditions. However, whether neuronal gap junctions contribute to NMDA receptor–regulated cell death is not known. Here we address the role of neuronal gap junction coupling in NMDA receptor–regulated cell death in developing neurons. We report that inactivation or hyperactivation of NMDA receptors induces neuronal cell death in primary hypothalamic cultures, specifically during the peak of developmental gap junction coupling. In contrast, increasing or decreasing NMDA receptor function when gap junction coupling is low has no or greatly reduced impact on cell survival. Pharmacological inactivation of gap junctions or knockout of neuronal connexin 36 prevents the cell death caused by NMDA receptor hypofunction or hyperfunction. The results indicate the critical role of neuronal gap junctions in cell death caused by increased or decreased NMDA receptor function in developing neurons. Based on these data, we propose the novel hypothesis that NMDA receptors and gap junctions work in concert to regulate neuronal survival.

We previously identified a partial expressed sequence tag clone corresponding to NARG2 in a screen for genes that are expressed in developing neurons and misexpressed in transgenic mice that lack functional N-methyl-d-aspartate receptors. Here we report the first characterization of the mouse and human NARG2 genes, cDNAs and the proteins that they encode. Mouse and human NARG2 consist of 988 and 982 amino acids, respectively, and share 74% identity. NARG2 does not display significant homology to other known genes, and lower organisms such as Saccharomyces cerevisiae, Drosophila melanogaster and Fugu rubripes appear to lack NARG2 orthologs. In vitro translation of the mouse cDNA yields a 150 kDa protein. NARG2 localizes to the nucleus in transfected cells, and deletion of a canonical basic nuclear localization signal suggests that this and other sequences in the protein cooperate for nuclear targeting. NARG2 consists of 16 exons in both mice and humans, 11 of which are identical in length, and alternative splicing is evident in both species. Exon 10 is the largest, and exhibits a much higher rate of nonsynonymous nucleotide substitution than the others. In addition, NARG2 contains (S/T)PXX motifs (11 in mouse NARG2, six in human NARG2). Northern blot analysis and RNase protection demonstrated that NARG2 is expressed at relatively high levels in dividing and immature cells, and that it is down-regulated upon terminal differentiation. The results indicate that NARG2 encodes a novel (S/T)PXX motif-containing nuclear protein, and suggest that NARG2 may play an important role in the early development of a number of different cell types.

We previously identified mNAT1 (murine N-terminal acetyltransferase 1) as an embryonic gene that is expressed in the developing brain and subsequently down-regulated, in part, by the onset of N-methyl-d-aspartate (NMDA) receptor function. By searching the data base we discovered a second closely related gene, mNAT2. mNAT1 and mNAT2 are highly homologous to yeast NAT1, a gene known to regulate entry into the G0 phase of the cell cycle. However, in the absence of further characterization, including evidence that mammalian homologues of NAT1 encode functional acetyltransferases, the significance of this relationship has been unclear. Here we focus on mNAT1. Biochemical analysis demonstrated that mNAT1 and its evolutionarily conserved co-subunit, mARD1, assemble to form a functional acetyltransferase. Transfection of mammalian cells with mNAT1 and mARD1 followed by immunofluorescent staining revealed that these proteins localize to the cytoplasm in both overlapping and separate compartments. In situ hybridization demonstrated that throughout brain development mNAT1 and mARD1 are highly expressed in areas of cell division and migration and are down-regulated as neurons differentiate. Finally, mNAT1 and mARD1 are expressed in proliferating mouse P19 embryonic carcinoma cells; treatment of these cells with retinoic acid initiates exit from the cell cycle, neuronal differentiation, and down-regulation of mNAT1 and mARD1 as the NMOA receptor 1 gene is induced. The results provide the first direct evidence that vertebrate homologues of NAT1 and ARD1 form an evolutionarily conserved N-terminal acetyltransferase and suggest that expression and down-regulation of this enzyme complex plays an important role in the generation and differentiation of neurons.

Native acetylcholine (ACh) receptors that contain the α7 gene product function as ligand-gated ion channels that are cation-selective, prefer nicotine over ACh, and rapidly desensitize. Activation of the receptors elevates intracellular calcium levels in neurons, triggering calcium-dependent events such as neurite retraction and the release of arachidonic acid. Arachidonic acid, in turn, reversibly inhibits the receptors, suggesting negative feedback regulation. The α7 gene product is expressed not only in neurons but also in developing muscle, raising the possibility that such receptors mediate cholinergic regulation of calcium-dependent events in a variety of cells.

Neurons can express a number of acetylcholine receptor genes simultaneously and assemble several receptor subtypes distinguished by subunit composition. One class of such receptors is largely extrasynaptic in location and contains the α7 gene product. It functions as a ligand-gated ion channel that is cation-selective, prefers nicotine over ACh, and rapidly desensitizes. Activation of the receptor elevates intracellular calcium levels in neurons, triggering calcium-dependent events such as neurite retraction and the release of arachidonic acid. Arachidonic acid, in turn, reversibly inhibits the receptors, suggesting negative feedback regulation. The α7 gene product is expressed not only in neurons but also in developing muscle, raising the possibility that such receptors mediate cholinergic regulation of calcium-dependent events in a variety of cells.

Of the 10 neuronal nicotinic acetylcholine receptor (AChR) genes identified in chick, five are expressed by ciliary ganglion neurons in vivo (α3, α5, α7, β2, and β4), and the mRNA levels produced increase during development approximately in parallel with the two major classes of AChRs present. Here we report that when chick ciliary ganglion neurons from 8-day embryos are transferred to dissociated cell culture, they express the same five genes but at much lower levels. The α3 and α7 transcripts, chosen for detailed analysis because they encode subunits segregated between the two AChR species, decrease rapidly in abundance on transfer to culture and, after 1 week, are at levels less than a 20th of those found in vivo for neurons of the same age. Co-culturing the neurons with skeletal myotubes did not increase the levels of AChR transcripts in the neurons. Despite low amounts of mRNA from all five genes, neither class of AChRs was much reduced in culture compared to in vivo. The numbers of AChRs on the cell surface actually increased with time in culture. Several culture conditions known to down-regulate the receptors in culture did not reduce the abundance of the α3 and α7 mRNAs. The results suggest that post-transcriptional controls can play an important role in determining AChR abundance on the neurons. © 1994 John Wiley & Sons, Inc. 1994 John Wiley & Sons, Inc.

To elucidate molecular mechanisms underlying activity-dependent synaptic remodeling in the developing mammalian visual system, we screened for genes whose expression in the lateral geniculate nucleus (LGN) is regulated by spontaneously generated action potentials present prior to vision. Activity blockade did not alter expression in the LGN of 32 known genes. Differential mRNA display, however, revealed a decrease in mRNAs encoding class I major histocompatibility complex antigens (class I MHC). Postnatally, visually driven activity can regulate class I MHC in the LGN during the final remodeling of retinal ganglion cell axon terminals. Moreover, in the mature hippocampus, class I MHC mRNA levels are increased by kainic acid–induced seizures. Normal expression of class I MHC mRNA is correlated with times and regions of synaptic plasticity, and immunohistochemistry confirms that class I MHC is present in specific subsets of CNS neurons. Finally, β2-microglobulin, a cosubunit of class I MHC, and CD3ζ, a component of a receptor complex for class I MHC, are also expressed by CNS neurons. These observations indicate that class I MHC molecules, classically thought to mediate cell–cell interactions exclusively in immune function, may play a novel role in neuronal signaling and activity-dependent changes in synaptic connectivity.

Objective: The objective of this study was to assess the level of agreement between stroke subtype classifications made using the Trial of Org 10172 Acute Stroke Treatment (TOAST) and Causative Classification of Stroke (CCS) systems. Methods: Study subjects included 13,596 adult men and women accrued from 20 US and European genetic research centers participating in the National Institute of Neurological Disorders and Stroke (NINDS) Stroke Genetics Network (SiGN). All cases had independently classified TOAST and CCS stroke subtypes. Kappa statistics were calculated for the 5 major ischemic stroke subtypes common to both systems. Results: The overall agreement between TOAST and CCS was moderate (agreement rate, 70%; kappa = 0.59, 95% confidence interval [CI] 0.58-0.60). Agreement varied widely across study sites, ranging from 28% to 90%. Agreement on specific subtypes was highest for large-artery atherosclerosis (kappa = 0.71, 95% CI 0.69-0.73) and lowest for small-artery occlusion (kappa = 0.56, 95% CI 0.54-0.58). Conclusion: Agreement between TOAST and CCS diagnoses was moderate. Caution is warranted when comparing or combining results based on the 2 systems. Replication of study results, for example, genome-wide association studies, should utilize phenotypes determined by the same classification system, ideally applied in the same manner.