Prediction of neuropathologic lesions from clinical data.

INTRODUCTION: Post-mortem analysis provides definitive diagnoses of neurodegenerative diseases; however, only a few can be diagnosed during life. METHODS: This study employed statistical tools and machine learning to predict 17 neuropathologic lesions from a cohort of 6518 individuals using 381 clinical features (Table S1). The multisite data allowed validation of the model's robustness by splitting train/test sets by clinical sites. A similar study was performed for predicting Alzheimer's disease (AD) neuropathologic change without specific comorbidities. RESULTS: Prediction results show high performance for certain lesions that match or exceed that of research annotation. Neurodegenerative comorbidities in addition to AD neuropathologic change resulted in compounded, but disproportionate, effects across cognitive domains as the comorbidity number increased. DISCUSSION: Certain clinical features could be strongly associated with multiple neurodegenerative diseases, others were lesion-specific, and some were divergent between lesions. Our approach could benefit clinical research, and genetic and biomarker research by enriching cohorts for desired lesions.

Enantiomers of 4-aminopentanoic acid act as false GABAergic neurotransmitters and impact mouse behavior.

Imbalance in the metabolic pathway linking excitatory and inhibitory neurotransmission has been implicated in multiple psychiatric and neurologic disorders. Recently, we described enantiomer-specific effects of 2-methylglutamate, which is not decarboxylated to the corresponding methyl analogue of gamma-aminobutyric acid (GABA): 4-aminopentanoic acid (4APA). Here, we tested the hypothesis that 4APA also has enantiomer-specific actions in brain. Mouse cerebral synaptosome uptake (nmol/mg protein over 30 min) of (R)-4APA or (S)-4APA was time and temperature dependent; however, the R enantiomer had greater uptake, reduction of endogenous GABA concentration, and release following membrane depolarization than did the S enantiomer. (S)-4APA exhibited some weak agonist (GABAA α4ß3δ, GABAA α5ß2γ2, and GABAB B1/B2) and antagonist (GABAA α6ß2γ2) activity while (R)-4APA showed weak agonist activity only with GABAA α5ß2γ2. Both 4APA enantiomers (100 mg/kg IP) were detected in mouse brain 10 min after injection, and by 1 hr had reached concentrations that were stable over 6 hr; both enantiomers were cleared rapidly from mouse serum over 6 hr. Two-month-old mice had no mortality following 100-900 mg/kg IP of each 4APA enantiomer but did have similar dose-dependent reduction in distance moved in a novel cage. Neither enantiomer at 30 or 100 mg/kg impacted outcomes in 23 measures of well-being, activity chamber, or withdrawal from hot plate. Our results suggest that enantiomers of 4APA are active in mouse brain, and that (R)-4APA may act as a novel false neurotransmitter of GABA. Future work will focus on disease models and on possible applications as neuroimaging agents.

Mass-tag barcoding for multiplexed analysis of human synaptosomes and other anuclear events.

Mass-tag cell barcoding has increased the throughput, multiplexing, and robustness of multiple cytometry approaches. Previously, we adapted mass cytometry for cells to analyze synaptosome preparations (mass synaptometry or SynTOF), extending mass cytometry to these smaller, anuclear particles. To improve throughput and individual event resolution, we report here the application of palladium-based barcoding in human synaptosomes. Up to 20 individual samples, each with a unique combinatorial barcode, were pooled for labeling with an antibody cocktail. Our synaptosome protocol used six palladium-based barcoding reagents, and in combination with sequential gating increased the identification of presynaptic events approximately fourfold. These same parameters also efficiently resolved two other anuclear particles: human red blood cells and platelets. The addition of palladium-based mass-tag barcoding to our approach improves mass cytometry of synaptic particles.

An improved ATAC-seq protocol reduces background and enables interrogation of frozen tissues.

We present Omni-ATAC, an improved ATAC-seq protocol for chromatin accessibility profiling that works across multiple applications with substantial improvement of signal-to-background ratio and information content. The Omni-ATAC protocol generates chromatin accessibility profiles from archival frozen tissue samples and 50-µm sections, revealing the activities of disease-associated DNA elements in distinct human brain structures. The Omni-ATAC protocol enables the interrogation of personal regulomes in tissue context and translational studies.

The basis of cellular and regional vulnerability in Alzheimer's disease.

Alzheimer's disease (AD) differentially and specifically affects brain regions and neuronal cell types in a predictable pattern. Damage to the brain appears to spread and worsens with time, taking over more regions and activating multiple stressors that can converge to promote vulnerability of certain cell types. At the same time, other cell types and brain regions remain intact in the face of this onslaught of neuropathology. Although neuropathologic descriptions of AD have been extensively expanded and mapped over the last several decades, our understanding of the mechanisms underlying how certain regions and cell populations are specifically vulnerable or resistant has lagged behind. In this review, we detail what is known about the selectivity of local initiation of AD pathology in the hippocampus, its proposed spread via synaptic connections, and the diversity of clinical phenotypes and brain atrophy patterns that may arise from different fibrillar strains of pathologic proteins or genetic predispositions. We summarize accumulated and emerging knowledge of the cellular and molecular basis for neuroanatomic selectivity, consider potential disease-relevant differences between vulnerable and resistant neuronal cell types and isolate molecular markers to identify them.

Human Striatal Dopaminergic and Regional Serotonergic Synaptic Degeneration with Lewy Body Disease and Inheritance of APOE ε4.

Cognitive impairment in older individuals is a complex trait that in population-based studies most commonly derives from an individually varying mixture of Alzheimer disease, Lewy body disease, and vascular brain injury. We investigated the molecular composition of synaptic particles from three sources: consecutive rapid autopsy brains from the Adult Changes in Thought Study, a population-based cohort; four aged nonhuman primate brains optimally processed for molecular investigation; and targeted replacement transgenic mice homozygous for APOE ε4. Our major goal was to characterize the molecular composition of human synaptic particles in regions of striatum and prefrontal cortex. We performed flow cytometry to measure six markers of synaptic subtypes, as well as amyloid ß 42 and paired helical filament tau. Our results showed selective degeneration of dopaminergic terminals throughout the striatum in individuals with Lewy body disease, and serotonergic degeneration in human ventromedial caudate nucleus from individuals with an APOE ε4 allele. Similar results were seen in mouse caudate nucleus homozygous for APOE ε4 via targeted replacement. Together, extension of these clinical, pathologic, and genetic associations from tissue to the synaptic compartment of cerebral cortex and striatum strongly supports our approach for accurately observing the molecular composition of human synapses by flow cytometry.

Clinical-pathologic correlations in vascular cognitive impairment and dementia.

The most common causes of cognitive impairment and dementia are Alzheimer's disease (AD) and vascular brain injury (VBI), either independently, in combination, or in conjunction with other neurodegenerative disorders. The contribution of VBI to cognitive impairment and dementia, particularly in the context of AD pathology, has been examined extensively yet remains difficult to characterize due to conflicting results. Describing the relative contribution and mechanisms of VBI in dementia is important because of the profound impact of dementia on individuals, caregivers, families, and society, particularly the stability of health care systems with the rapidly increasing age of our population. Here we discuss relationships between pathologic processes of VBI and clinical expression of dementia, specific subtypes of VBI including microvascular brain injury, and what is currently known regarding contributions of VBI to the development and pathogenesis of the dementia syndrome. This article is part of a Special Issue entitled: Vascular Contributions to Cognitive Impairment and Dementia edited by M. Paul Murphy, Roderick A. Corriveau and Donna M. Wilcock.

Precision Medicine: Clarity for the Complexity of Dementia.

Three key elements to precision medicine are stratification by risk, detection of pathophysiological processes as early as possible (even before clinical presentation), and alignment of mechanism of action of intervention(s) with an individual's molecular driver(s) of disease. Used for decades in the management of some rare diseases and now gaining broad currency in cancer care, a precision medicine approach is beginning to be adapted to cognitive impairment and dementia. This review focuses on the application of precision medicine to address the clinical and biological complexity of two common neurodegenerative causes of dementia: Alzheimer disease and Parkinson disease.

Prostaglandin E2 receptor subtype 2 regulation of scavenger receptor CD36 modulates microglial Aß42 phagocytosis.

Recent studies underline the potential relevance of microglial innate immune activation in Alzheimer disease. Primary mouse microglia that lack prostaglandin E2 receptor subtype 2 (EP2) show decreased innate immune-mediated neurotoxicity and increased amyloid ß (Aß) peptide phagocytosis, features that were replicated in vivo. Here, we tested the hypothesis that scavenger receptor CD36 is an effector of EP2-regulated Aß phagocytosis. CD36 expression was 143-fold greater in mouse primary microglia than in primary astrocytes. Three different means of suppressing EP2 signaling increased and an agonist of EP2 decreased CD36 expression in primary wild-type microglia. Activation of Toll-like receptor (TLR) 3, TLR4, and TLR7, but not TLR2 or TLR9, reduced primary microglial CD36 transcription and cell surface CD36 protein and reduced Aß42 phagocytosis as well. At each step, the effects of innate immune activation on CD36 were reversed by at least 50% by an EP2 antagonist, and this partial rescue of microglia Aß42 phagocytosis was largely mediated by CD36 activity. Finally, we showed in hippocampus of wild-type mice that innate immune activation suppressed CD36 expression by an EP2-dependent mechanism. Taken together with results of others that found brain clearance of Aß peptides and behavioral improvements mediated by CD36 in mice, regulation of CD36-mediated Aß phagocytosis by suppression of EP2 signaling may provide a new approach to suppressing some aspects of Alzheimer disease pathogenesis.

Different mechanisms of apolipoprotein E isoform-dependent modulation of prostaglandin E2 production and triggering receptor expressed on myeloid cells 2 (TREM2) expression after innate immune activation of microglia.

Several lines of evidence support immune response in brain as a mechanism of injury in Alzheimer disease (AD). Moreover, immune activation is heightened in apolipoprotein E (APOE) ε4 carriers; inhibitors of prostaglandin (PG) synthesis show a partially protective effect on AD risk from APOE ε4; and genetic variants in triggering receptor expressed on myeloid cells 2 (TREM2) are a rare but potent risk for AD. We tested the hypothesis that APOE ε4 inheritance modulates both the PGE2 pathway and TREM2 expression using primary murine microglia from targeted replacement (TR) APOE3/3 and APOE4/4 mice. Microglial cyclooxygenase-2, microsomal PGE synthase, and PGE2 expression were increased 2- to 25-fold in both genotypes by TLR activators; however, this induction was significantly (P < 0.01) greater in TR APOE4/4 microglia with TLR3 and TLR4 activators. Microglial TREM2 expression was reduced approximately 85% by all TLR activators; this reduction was approximately one-third greater in microglia from TR APOE4/4 mice. Importantly, both receptor-associated protein and a nuclear factor κ-light-chain-enhancer inhibitor blocked TR APOE4/4-dependent effects on the PGE2 pathway but not on TREM2 expression. These data demonstrate complementary, but mechanistically distinct, regulation of pro- and anti-inflammatory mediators in TR APOE4/4 murine microglia that yields a more proinflammatory state than with TR APOE3/3.

Partial depletion of striatal dopamine enhances penetrance of cognitive deficits in a transgenic mouse model of Alzheimer's disease.

Parkinson's disease and Alzheimer's disease (AD) are recognized to coexist on a spectrum of neurodegeneration, and it has been proposed that molecular interactions among pathogenic proteins are a basis for the overlap between these two diseases. We instead hypothesized that degeneration of the nigrostriatal dopaminergic system enhances the clinical penetrance of early-stage AD. To determine the effect of striatal dopamine (DA) on the pathological effects in an experimental model of AD, APPSWE /PS1ΔE9 mice received striatal injections of the neurotoxin 6-hydroxydopamine (6OHDA). Animals were tested in a Barnes maze protocol and in a water T-maze protocol at different ages to determine the onset of cognitive impairment. APPSWE /PS1ΔE9 mice that received 6OHDA injections showed significant impairment in Barnes maze performance at an earlier age than controls. Additionally, at 12 months of age, APPswe /PS1ΔE9 + 6OHDA mice demonstrated worse behavioral flexibility than other groups in a task-switch phase of the water T-maze. To determine the neuroprotective effects of dopaminergic neurotransmission against amyloid-ß42 (Aß42 ) toxicity, neuronal branch order and dendrite length were quantified in primary medium spiny neuron (MSN) cultures pretreated with increasing doses of the D1 and D2 receptor agonists before being exposed to oligomerized Aß42 . Although there were no differences in Aß peptide levels or plaque burden among the groups, in murine MSN culture dopaminergic agonists prevented a toxic response to Aß42. Depletion of DA in the striatum exacerbated the cognitive impairment seen in a mouse model of early-stage AD; this may be due to a protective effect of dopaminergic innervation against Aß striatal neurotoxicity.

Cognitive profile of LRRK2-related Parkinson's disease.

BACKGROUND: Increasing evidence suggests that genetic factors play a role in the variability associated with cognitive performance in Parkinson's disease (PD). Mutations in the LRRK2 gene are the most common cause of monogenic PD; however, the cognitive profile of LRRK2-related PD is not well-characterized. METHODS: A cohort of 1,447 PD patients enrolled in the PD Cognitive Genetics Consortium was screened for LRRK2 mutations and completed detailed cognitive testing. Associations between mutation carrier status and cognitive test scores were assessed using linear regression models. RESULTS: LRRK2 mutation carriers (n = 29) demonstrated better performance on the Mini Mental State Examination (P = 0.03) and the Letter-Number Sequencing Test (P = 0.005). A smaller proportion of LRRK2 carriers were demented (P = 0.03). CONCLUSIONS: Our cross-sectional study demonstrates better performance on certain cognitive tests, as well as lower rates of dementia in LRRK2-related PD. Future longitudinal studies are needed to determine whether LRRK2 mutation carriers exhibit slower cognitive decline. © 2015 International Parkinson and Movement Disorder Society.

APOE3, but not APOE4, bone marrow transplantation mitigates behavioral and pathological changes in a mouse model of Alzheimer disease.

Apolipoprotein E4 (APOE4) genotype is the strongest genetic risk factor for late-onset Alzheimer disease and confers a proinflammatory, neurotoxic phenotype to microglia. Here, we tested the hypothesis that bone marrow cell APOE genotype modulates pathological progression in experimental Alzheimer disease. We performed bone marrow transplants (BMT) from green fluorescent protein-expressing human APOE3/3 or APOE4/4 donor mice into lethally irradiated 5-month-old APPswe/PS1ΔE9 mice. Eight months later, APOE4/4 BMT-recipient APPswe/PS1ΔE9 mice had significantly impaired spatial working memory and increased detergent-soluble and plaque Aß compared with APOE3/3 BMT-recipient APPswe/PS1ΔE9 mice. BMT-derived microglia engraftment was significantly reduced in APOE4/4 recipients, who also had correspondingly less cerebral apoE. Gene expression analysis in cerebral cortex of APOE3/3 BMT recipients showed reduced expression of tumor necrosis factor-α and macrophage migration inhibitory factor (both neurotoxic cytokines) and elevated immunomodulatory IL-10 expression in APOE3/3 recipients compared with those that received APOE4/4 bone marrow. This was not due to detectable APOE-specific differences in expression of microglial major histocompatibility complex class II, C-C chemokine receptor (CCR) type 1, CCR2, CX3C chemokine receptor 1 (CX3CR1), or C5a anaphylatoxin chemotactic receptor (C5aR). Together, these findings suggest that BMT-derived APOE3-expressing cells are superior to those that express APOE4 in their ability to mitigate the behavioral and neuropathological changes in experimental Alzheimer disease.

A pathologist-AI collaboration framework for enhancing diagnostic accuracies and efficiencies.

In pathology, the deployment of artificial intelligence (AI) in clinical settings is constrained by limitations in data collection and in model transparency and interpretability. Here we describe a digital pathology framework, nuclei.io, that incorporates active learning and human-in-the-loop real-time feedback for the rapid creation of diverse datasets and models. We validate the effectiveness of the framework via two crossover user studies that leveraged collaboration between the AI and the pathologist, including the identification of plasma cells in endometrial biopsies and the detection of colorectal cancer metastasis in lymph nodes. In both studies, nuclei.io yielded considerable diagnostic performance improvements. Collaboration between clinicians and AI will aid digital pathology by enhancing accuracies and efficiencies.

Eicosanoid receptor subtype-mediated opposing regulation of TLR-stimulated expression of astrocyte glial-derived neurotrophic factor.

A major therapeutic target for Parkinson's disease (PD) is providing increased glial-derived neurotrophic factor (GDNF) to dopaminergic neurons. We tested the hypothesis that innate immune activation increases astrocyte GDNF production and that this is regulated by specific eicosanoid receptors. Innate immune-activated primary murine astrocytes were assayed for GDNF expression and secretion. Controls were agent vehicle exposure and wild-type mice. Rank order for up to 10-fold selectively increased GDNF expression was activators of TLR3 > TLR2 or TLR4 > TLR9. TLR3 activator-stimulated GDNF expression was selectively JNK-dependent, followed cyclooxygenase (COX)-2, was coincident with membranous PGE(2) synthase, and was not significantly altered by a nonspecific COX- or a COX-2-selective inhibitor. Specific eicosanoid receptors had opposing effects on TLR3 activator-induced GDNF expression: ∼60% enhancement by blocking or ablating of PGE(2) receptor subtype 1 (EP1), ∼30% enhancement by activating PGF(2α) receptor or thromboxane receptor, or ∼15% enhancement by activating EP4. These results demonstrate functionally antagonistic eicosanoid receptor subtype regulation of innate immunity-induced astrocyte GDNF expression and suggest that selective inhibition of EP1 signaling might be a means to augment astrocyte GDNF secretion in the context of innate immune activation in diseased regions of brain in PD.

Perivascular, but not parenchymal, cerebral engraftment of donor cells after non-myeloablative bone marrow transplantation.

Myeloablative (MyA) bone marrow transplantation (BMT) results in robust engraftment of BMT-derived cells in the central nervous system (CNS) and is neuroprotective in diverse experimental models of neurodegenerative diseases of the brain and retina. However, MyA irradiation is associated with significant morbidity and mortality and does not represent a viable therapeutic option for the elderly. Non-myeloablative (NMyA) BMT is less toxic, but it is not known if the therapeutic efficacy observed with MyA BMT is preserved. As a first step to address this important gap in knowledge, we evaluated and compared engraftment characteristics of BMT-derived monocytes/microglia using several clinically relevant NMyA pretransplant conditioning regimens in C57BL/6 mice. These included chemotherapy (fludarabine and cyclophosphamide) with or without 2 Gy irradiation, and 5.5 Gy irradiation alone. Each regimen was followed by transplantation of whole bone marrow from green fluorescent protein-expressing wild type (wt) mice. While stable hematopoietic engraftment occurred, to varying degrees, in all NMyA regimens, only 5.5 Gy irradiation resulted in significant engraftment of BMT-derived cells in the brain, where these cells were exclusively localized to perivascular, leptomeningeal, and related anatomic regions. Engraftment in retina under 5.5 Gy NMyA conditions was significantly reduced compared to MyA, but robust engraftment was identified in the optic nerve. Advancing the therapeutic applications of BMT to neurodegenerative diseases will require identification of the barrier mechanisms that MyA, but not NMyA, BMT is able to overcome.

Ablation of the microglial protein DOCK2 reduces amyloid burden in a mouse model of Alzheimer's disease.

Alzheimer's disease (AD) neuropathology is characterized by innate immune activation primarily through prostaglandin E2 (PGE2) signaling. Dedicator of cytokinesis 2 (DOCK2) is a guanyl nucleotide exchange factor expressed exclusively in microglia in the brain and is regulated by PGE2 receptor EP2. DOCK2 modulates microglia cytokine secretion, phagocytosis, and paracrine neurotoxicity. EP2 ablation in experimental AD results in reduced oxidative damage and amyloid beta (Aß) burden. This discovery led us to hypothesize that genetic ablation of DOCK2 would replicate the anti-Aß effects of loss of EP2 in experimental AD. To test this hypothesis, we crossed mice that lacked DOCK2 (DOCK2-/-), were hemizygous for DOCK2 (DOCK2+/-), or that expressed two DOCK2 genes (DOCK2+/+) with APPswe-PS1Δe9 mice (a model of AD). While we found no DOCK2-dependent differences in cortex or in hippocampal microglia density or morphology in APPswe-PS1Δe9 mice, cerebral cortical and hippocampal Aß plaque area and size were significantly reduced in 10-month-old APPswe-PS1Δe9/DOCK2-/- mice compared with APPswe-PS1Δe9/DOCK2+/+ controls. DOCK2 hemizygous APPswe-PS1Δe9 mice had intermediate Aß plaque levels. Interestingly, soluble Aß42 was not significantly different among the three genotypes, suggesting the effects were mediated specifically in fibrillar Aß. In combination with earlier cell culture results, our in vivo results presented here suggest DOCK2 contributes to Aß plaque burden via regulation of microglial innate immune function and may represent a novel therapeutic target for AD.

Paradigm Shift: Multiple Potential Pathways to Neurodegenerative Dementia.

Neurodegenerative dementia can result from multiple underlying abnormalities, including neurotransmitter imbalances, protein aggregation, and other neurotoxic events. A major complication in identifying effective treatment targets is the frequent co-occurrence of multiple neurodegenerative processes, occurring either in parallel or sequentially. The path towards developing effective treatments for Alzheimer's disease (AD) and other dementias has been relatively slow and until recently has focused on disease symptoms. Aducanumab and lecanemab, recently approved by the FDA, are meant to target disease structures but have only modest benefit on symptom progression and remain unproven in reversing or preventing dementia. A third, donanemab, appears more promising but awaits FDA approval. Ongoing trials include potential cognition enhancers, new combinations of known drugs for synergistic effects, prodrugs with less toxicity, and increasing interest in drugs targeting neuroinflammation or microbiome. Scientific and technological advances offer the opportunity to move in new therapy directions, such as modifying microglia to prevent or suppress underlying disease. A major challenge, however, is that underlying comorbidities likely influence the effectiveness of therapies. Indeed, the full range of comorbidity, today only definitively identified postmortem, likely contributes to failed clinical trials and overmedication of older adults, since it is difficult to exclude (during life) people unlikely to respond. Our current knowledge thus signals that a paradigm shift towards individualized and multimodal treatments is necessary to effectively advance the field of dementia therapeutics.

Understanding the molecular basis of resilience to Alzheimer's disease.

The cellular and molecular distinction between brain aging and neurodegenerative disease begins to blur in the oldest old. Approximately 15-25% of observations in humans do not fit predicted clinical manifestations, likely the result of suppressed damage despite usually adequate stressors and of resilience, the suppression of neurological dysfunction despite usually adequate degeneration. Factors during life may predict the clinico-pathologic state of resilience: cardiovascular health and mental health, more so than educational attainment, are predictive of a continuous measure of resilience to Alzheimer's disease (AD) and AD-related dementias (ADRDs). In resilience to AD alone (RAD), core features include synaptic and axonal processes, especially in the hippocampus. Future focus on larger and more diverse cohorts and additional regions offer emerging opportunities to understand this counterforce to neurodegeneration. The focus of this review is the molecular basis of resilience to AD.

Human cerebrospinal fluid single exosomes in Parkinson's and Alzheimer's diseases.

Exosomes are proposed to be important in the pathogenesis of prevalent neurodegenerative diseases. We report the first application of solid-state technology to perform multiplex analysis of single exosomes in human cerebrospinal fluid (CSF) obtained from the lumbar sac of people diagnosed with Alzheimer's disease dementia (ADD, n=30) or Parkinson's disease dementia (PDD, n=30), as well as age-matched health controls (HCN, n=30). Single events were captured with mouse monoclonal antibodies to one of three different tetraspanins (CD9, CD63, or CD81) or with mouse (M) IgG control, and then probed with fluorescently labeled antibodies to prion protein (PrP) or CD47 to mark neuronal or presynaptic origin, as well as ADD- and PDD-related proteins: amyloid beta (Aß), tau, α-synuclein, and Apolipoprotein (Apo) E. Data were collected only from captured events that were within the size range of 50 to 200 nm. Exosomes were present at approximately 100 billion per mL human CSF and were similarly abundant for CD9+ and CD81+ events, but CD63+ were only 22% to 25% of CD9+ (P<0.0001) or CD81+ (P<0.0001) events. Approximately 24% of CSF exosomes were PrP+, while only 2% were CD47+. The vast majority of exosomes were surface ApoE+, and the number of PrP-ApoE+ (P<0.001) and PrP+ApoE+ (P<0.01) exosomes were significantly reduced in ADD vs. HCN for CD9+ events only. Aß, tau, and α-synuclein were not detected on the exosome surface or in permeabilized cargo. These data provide new insights into single exosome molecular features and highlight reduction in the CSF concentration of ApoE+ exosomes in patients with ADD.