The vast majority of archived research and clinical pathological specimens are stored in the form of formalin fixed, paraffin-embedded (FFPE) tissues, but, unlike fresh frozen tissue samples, highly ...quantitative measures in FFPE tissues are limited to immunohistochemical and immunofluorescence thresholding image analysis studies, cell counting, and ordinal ranking systems. This poses a significant obstacle for clinical investigations that aim to correlate diagnostic markers of neurodegenerative diseases like Alzheimer's disease (AD) with parameters like age, gender, drug exposures, genotype, disease stage, co-morbidities, or environmental factors. To overcome this limitation, we have developed Luminex-based techniques and protocols for the quantification of amyloid β and hyperphosphorylated Tau in FFPE brain sections. We validated the Luminex assay in FFPE sections from prefrontal cortex, hippocampus, and neostriatum from 30 cases that underwent prior neuropathological diagnostic assessment of AD following the current NIA–AA recommendations for AD: 10 cases diagnosed as not or low, 10 cases as intermediate, and 10 cases as high AD neuropathologic change. Consistent with the neuropathologic assessment, Luminex assay detected high amounts of amyloid beta in the frontal cortex and striatum, and high amounts of hyperphosphorylated Tau in the frontal cortex and hippocampus, of cases with high AD neuropathologic change. This assay can be expanded to detect diverse antigenic targets of interest, as we show here with IBA1 and GFAP. This novel approach supports multiplexed highly quantitative, molecularly specific neuropathology measures to further explore mechanisms of neurodegeneration in AD.
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 β (Aβ) peptide phagocytosis, features that were replicated in vivo . Here, we tested the hypothesis that scavenger receptor CD36 is an effector of EP2-regulated Aβ 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 Aβ42 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 Aβ42 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 Aβ peptides and behavioral improvements mediated by CD36 in mice, regulation of CD36-mediated Aβ phagocytosis by suppression of EP2 signaling may provide a new approach to suppressing some aspects of Alzheimer disease pathogenesis.
•Mass synaptometry method for single synapse multiplexed molecular profiling.•Synapse by Time Of Flight (SynTOF) panel of 34 antibodies for wide application.•Optimized synaptosome preparation and ...CyTOF acquisition parameters.•Proof of principle validation in 4 Lewy body disease and 2 Alzheimer’s disease cases.
Synaptic alterations, especially presynaptic changes, are cardinal features of neurodegenerative diseases and strongly correlate with cognitive decline.
We report “Mass Synaptometry” for the high-dimensional analysis of individual human synaptosomes, enriched nerve terminals from brain. This method was adapted from cytometry by time-of-flight mass spectrometry (CyTOF), which is commonly used for single-cell analysis of immune and blood cells.
Here we overcome challenges for single synapse analysis by optimizing synaptosome preparations, generating a ‘SynTOF panel,’ recalibrating acquisition settings, and applying computational analyses. Through the analysis of 390,000 individual synaptosomes, we also provide proof-of principle validation by characterizing changes in synaptic diversity in Lewy Body Disease (LBD), Alzheimer’s disease and normal brain.
Current imaging methods to study synapses in humans are capable of analyzing a limited number of synapses, and conventional flow cytometric techniques are typically restricted to fewer than 6 parameters. Our method allows for the simultaneous detection of 34 parameters from tens of thousands of individual synapses.
We applied Mass Synaptometry to analyze 34 parameters simultaneously on more than 390,000 synaptosomes from 13 human brain samples. This new approach revealed regional and disease-specific changes in synaptic phenotypes, including validation of this method with the expected changes in the molecular composition of striatal dopaminergic synapses in Lewy body disease and Alzheimer’s disease. Mass synaptometry enables highly parallel molecular profiling of individual synaptic terminals.
Departments of 1 Rehabilitation Medicine, and
2 Physiology and Biophysics,
3 Graduate Program in Neurobiology and
Behavior, and 4 Regional Primate Research Center,
University of Washington, ...Seattle, Washington, 98195
Anderson, Marjorie E.,
Nadia Postupna, and
Mark Ruffo.
Effects of High-Frequency Stimulation in the Internal Globus
Pallidus on the Activity of Thalamic Neurons in the Awake Monkey. J. Neurophysiol. 89: 1150-1160, 2003. The
reduction in symptoms of Parkinson's disease produced by
high-frequency stimulation (HFS) in the internal globus pallidus (GPi)
has been proposed to be due to stimulus-induced inactivation of
pallidal neurons and resulting disinhibition of thalamic neurons. We
tested this in awake Macaca fascicularis by
stimulating between pairs of electrodes inserted into GPi under
electrophysiological control and recording the responses evoked in
thalamic neurons. HFS produced a reduction, not an increase, in
discharge frequency during the stimulus train in 77% of the responsive
thalamic neurons. Only 16% of the responsive cells showed an increase
in discharge during stimulation and, for some of these, stimulation at
a similar intensity produced contralateral muscle contraction, a
probable sign of current spread to the internal capsule. The few
thalamic neurons studied during bursting had a reduction in burst
frequency and duration during HFS. We conclude that high-frequency
stimulation within GPi does not necessarily facilitate thalamic
discharge, and it may act, instead, to interrupt abnormal patterns of
thalamic discharge associated with parkinsonian symptoms.
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.
Comparing brain structure across species and regions enables key functional insights. Leveraging publicly available data from a novel mass cytometry-based method, synaptometry by time of flight ...(SynTOF), we applied an unsupervised machine learning approach to conduct a comparative study of presynapse molecular abundance across three species and three brain regions. We used neural networks and their attractive properties to model complex relationships among high dimensional data to develop a unified, unsupervised framework for comparing the profile of more than 4.5 million single presynapses among normal human, macaque, and mouse samples. An extensive validation showed the feasibility of performing cross-species comparison using SynTOF profiling. Integrative analysis of the abundance of 20 presynaptic proteins revealed near-complete separation between primates and mice involving synaptic pruning, cellular energy, lipid metabolism, and neurotransmission. In addition, our analysis revealed a strong overlap between the presynaptic composition of human and macaque in the cerebral cortex and neostriatum. Our unique approach illuminates species- and region-specific variation in presynapse molecular composition.
Cerebral white matter injury (WMI) contributes to cognitive dysfunction associated with pathological aging. Because reactive astrocyte-related factors contribute to remyelination failure after WMI, ...we sought accurate, cost-effective, and reproducible histopathological approaches for quantification of morphometric features of reactive astrogliosis in aged human white matter in patients with vascular brain injury (VBI). We compared 7 distinct approaches to quantify the features of glial fibrillary acidic protein (GFAP)-labeled astrocytes in the prefrontal white matter of brains from patients with VBI (n = 17, mean age 88.8 years) and controls that did not exhibit VBI (n = 11, mean age 86.6 years). Only modern stereological techniques (ie, optical fractionator and spaceballs) and virtual process thickness measurements demonstrated significant changes in astrocyte number, process length, or proximal process thickness in cases with VBI relative to controls. The widely employed methods of neuropathological scoring, antibody capture assay (histelide), area fraction fractionator, and Cavalieri point counting failed to detect significant differences in GFAP expression between the groups. Unbiased stereological approaches and virtual thickness measurements provided the only sensitive and accurate means to quantify astrocyte reactivity as a surrogate marker of WMI in human brains with VBI.
The late neuropathological effects of traumatic brain injury have yet to be fully elucidated, particularly with respect to community-based cohorts. To contribute to this critical gap in knowledge, we ...designed a multimodal neuropathological study, integrating traditional and quantitative approaches to detect pathologic changes in 532 consecutive brain autopsies from participants in the Adult Changes in Thought (ACT) study. Diagnostic evaluation including assessment for chronic traumatic encephalopathy (CTE) and quantitative immunoassay-based methods were deployed to examine levels of pathological (hyperphosphorylated) tau (pTau) and amyloid (A) β in brains from ACT participants with (
= 107) and without (
= 425) history of remote TBI with loss of consciousness (w/LOC). Further neuropathological assessments included immunohistochemistry for α-synuclein and phospho-TDP-43 pathology and astro- (GFAP) and micro- (Iba1) gliosis, mass spectrometry analysis of free radical injury, and gene expression evaluation (RNA sequencing) in a smaller sub-cohort of matched samples (49 cases with TBI and 49 non-exposed matched controls). Out of 532 cases, only 3 (0.6%-none with TBI w/LOC history) showed evidence of the neuropathologic signature of chronic traumatic encephalopathy (CTE). Across the entire cohort, the levels of pTau and Aβ showed expected differences for brain region (higher levels in temporal cortex), neuropathological diagnosis (higher in participants with Alzheimer's disease), and
genotype (higher in participants with one or more
ε4 allele). However, no differences in PHF-tau or Aβ
were identified by Histelide with respect to the history of TBI w/LOC. In a subset of TBI cases with more carefully matched control samples and more extensive analysis, those with TBI w/LOC history had higher levels of hippocampal pTau but no significant differences in Aβ, α-synuclein, pTDP-43, GFAP, Iba1, or free radical injury. RNA-sequencing also did not reveal significant gene expression associated with any measure of TBI exposure. Combined, these findings suggest long term neuropathological changes associated with TBI w/LOC may be subtle, involve non-traditional pathways of neurotoxicity and neurodegeneration, and/or differ from those in autopsy cohorts specifically selected for neurotrauma exposure.
Synaptic dysfunction is thought to have an important role in the pathophysiology of neurodegenerative diseases, such as Alzheimer's disease (AD) and Lewy body disease (LBD). To improve our ...understanding of synaptic alterations in health and disease, we investigated synaptosomes prepared from post-mortem human cerebral cortex, putamen (PT), and two regions of the caudate nucleus, dorso-lateral (DL) and ventro-medial (VM), regions commonly affected in AD and LBD. We observed that the fraction of synaptosomal particles with reactivity for dopamine transporter (DAT) was significantly reduced in the PT and VM caudate of patients with neuropathological diagnosis of LBD. As expected, these differences also were reflected in direct measurements of dopamine (DA) and its metabolite, 3,4-dihydroxyphenylacetic acid (DOPAC), in caudate and PT of LBD patients. The fraction of synaptosomal particles positive for amyloid β (Aβ) was significantly increased in frontal cortical samples of patients with the neuropathological diagnosis of severe AD, and was positively correlated with disease progression. We also prepared synaptosomes from the striatum of mice with severe loss of DA neurons (Slc6a3-DTR mice) and wild-type littermate controls. We observed markedly reduced levels of DAT-positive synaptosomes in Slc6a3-DTR mice following exposure to diphtheria toxin (DT). Striatal levels of DA and DOPAC in Slc6a3-DTR mice also were reduced significantly following DT exposure. We conclude that flow cytometric analysis of synaptosomes prepared from human or mouse brain provides an opportunity to study expression of pathology-associated proteins and also the specific loss of dopaminergic nerve terminals. Hence, we believe it is a valid method to detect pathological changes at the level of the synapse in LBD as well as AD.
Parkinson's disease (PD) is characterized by the loss of dopaminergic neurons and produces a movement disorder and cognitive impairment that becomes more extensive with the duration of the disease. ...To what extent cognitive impairment in advanced PD can be attributed to severe loss of dopamine (DA) signaling is not well understood. Furthermore, it is unclear if the loss of DA neurons contributes to the cognitive impairment caused by the reduction in DA signaling. We generated genetic mouse models with equally severe chronic loss of DA achieved by either extensive ablation of DA neurons or inactivation of DA synthesis from preserved neurons and compared their motor and cognitive performance. Motor behaviors were equally blunted in both models, but we observed that DA neuron ablation caused more severe cognitive deficits than DA depletion. Both models had marked deficits in cue-discrimination learning. Yet, deficits in cue-discrimination learning were more severe in mice with DA neuron ablation and only mice with DA neuron ablation had drastically impaired performance in spatial learning, spatial memory and object memory tests. These results indicate that while a severe reduction in DA signaling results in motor and cognitive impairments, the loss of DA neurons promotes more extensive cognitive deficits and suggest that a loss of additional factors that depend on DA neurons may participate in the progressive cognitive decline found in patients with PD.
•Severe loss of striatal dopamine causes cognitive impairment resembling advanced PD.•Loss of dopamine neurons causes more cognitive deficits than loss of dopamine alone.•Severe loss of striatal dopamine is sufficient to cause extensive motor defects.
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