Simultaneous isolation of intact brain cells and cell-specific extracellular vesicles from cryopreserved Alzheimer's disease cortex.

BACKGROUND: The neuronal and gliaI populations within the brain are tightly interwoven, making isolation and study of large populations of a single cell type from brain tissue a major technical challenge. Concurrently, cell-type specific extracellular vesicles (EVs) hold enormous diagnostic and therapeutic potential in neurodegenerative disorders including Alzheimer's disease (AD). NEW METHOD: Postmortem AD cortical samples were thawed and gently dissociated. Following filtration, myelin and red blood cell removal, cell pellets were immunolabeled with fluorescent antibodies and analyzed by flow cytometry. The cell pellet supernatant was applied to a triple sucrose cushion for brain EV isolation. RESULTS: Neuronal, astrocyte and microglial cell populations were identified. Cell integrity was demonstrated using calcein AM, which is retained by cells with esterase activity and an intact membrane. For some experiments cell pellets were fixed, permeabilized, and immunolabeled for cell-specific markers. Characterization of brain small EV fractions showed the expected size, depletion of EV negative markers, and enrichment in positive and cell-type specific markers. COMPARISON WITH EXISTING METHODS AND CONCLUSIONS: We optimized and integrated established protocols, aiming to maximize information obtained from each human autopsy brain sample. The uniqueness of our method lies in its capability to isolate cells and EVs from a single cryopreserved brain sample. Our results not only demonstrate the feasibility of isolating specific brain cell subpopulations for RNA-seq but also validate these subpopulations at the protein level. The accelerated study of EVs from human samples is crucial for a better understanding of their contribution to neuron/glial crosstalk and disease progression.

Multi-Omics Analysis of Microglial Extracellular Vesicles From Human Alzheimer's Disease Brain Tissue Reveals Disease-Associated Signatures.

Alzheimer's disease (AD) is the most common cause of dementia, yet there is no cure or diagnostics available prior to the onset of clinical symptoms. Extracellular vesicles (EVs) are lipid bilayer-delimited particles that are released from almost all types of cell. Genome-wide association studies have linked multiple AD genetic risk factors to microglia-specific pathways. It is plausible that microglia-derived EVs may play a role in the progression of AD by contributing to the dissemination of insoluble pathogenic proteins, such as tau and Aß. Despite the potential utility of EVs as a diagnostic tool, our knowledge of human brain EV subpopulations is limited. Here we present a method for isolating microglial CD11b-positive small EVs from cryopreserved human brain tissue, as well as an integrated multiomics analysis of microglial EVs enriched from the parietal cortex of four late-stage AD (Braak V-VI) and three age-matched normal/low pathology (NL) cases. This integrated analysis revealed 1,000 proteins, 594 lipids, and 105 miRNAs using shotgun proteomics, targeted lipidomics, and NanoString nCounter technology, respectively. The results showed a significant reduction in the abundance of homeostatic microglia markers P2RY12 and TMEM119, and increased levels of disease-associated microglia markers FTH1 and TREM2, in CD11b-positive EVs from AD brain compared to NL cases. Tau abundance was significantly higher in AD brain-derived microglial EVs. These changes were accompanied by the upregulation of synaptic and neuron-specific proteins in the AD group. Levels of free cholesterol were elevated in microglial EVs from the AD brain. Lipidomic analysis also revealed a proinflammatory lipid profile, endolysosomal dysfunction, and a significant AD-associated decrease in levels of docosahexaenoic acid (DHA)-containing polyunsaturated lipids, suggesting a potential defect in acyl-chain remodeling. Additionally, four miRNAs associated with immune and cellular senescence signaling pathways were significantly upregulated in the AD group. Our data suggest that loss of the homeostatic microglia signature in late AD stages may be accompanied by endolysosomal impairment and the release of undigested neuronal and myelin debris, including tau, through extracellular vesicles. We suggest that the analysis of microglia-derived EVs has merit for identifying novel EV-associated biomarkers and providing a framework for future larger-scale multiomics studies on patient-derived cell-type-specific EVs.

Exosomal tau with seeding activity is released from Alzheimer's disease synapses, and seeding potential is associated with amyloid beta.

Synaptic transfer of tau has long been hypothesized from the human pathology pattern and has been demonstrated in vitro and in vivo, but the precise mechanisms remain unclear. Extracellular vesicles such as exosomes have been suggested as a mechanism, but not all tau is exosomal. The present experiments use a novel flow cytometry assay to quantify depolarization of synaptosomes by KCl after loading with FM2-10, which induces a fluorescence reduction associated with synaptic vesicle release; the degree of reduction in cryopreserved human samples equaled that seen in fresh mouse synaptosomes. Depolarization induced the release of vesicles in the size range of exosomes, along with tetraspanin markers of extracellular vesicles. A number of tau peptides were released, including tau oligomers; released tau was primarily unphosphorylated and C-terminal truncated, with Aß release just above background. When exosomes were immunopurified from release supernatants, a prominent tau band showed a dark smeared appearance of SDS-stable oligomers along with the exosomal marker syntenin-1, and these exosomes induced aggregation in the HEK tau biosensor assay. However, the flow-through did not seed aggregation. Size exclusion chromatography of purified released exosomes shows faint signals from tau in the same fractions that show a CD63 band, an exosomal size signal, and seeding activity. Crude synaptosomes from control, tauopathy, and AD cases demonstrated lower seeding in tauopathy compared to AD that is correlated with the measured Aß42 level. These results show that AD synapses release exosomal tau that is C-terminal-truncated, oligomeric, and with seeding activity that is enhanced by Aß. Taken together with previous findings, these results are consistent with a direct prion-like heterotypic seeding of tau by Aß within synaptic terminals, with subsequent loading of aggregated tau onto exosomes that are released and competent for tau seeding activity.

Pharmacological inhibition of nSMase2 reduces brain exosome release and α-synuclein pathology in a Parkinson's disease model.

AIM: We have previously reported that cambinol (DDL-112), a known inhibitor of neutral sphingomyelinase-2 (nSMase2), suppressed extracellular vesicle (EV)/exosome production in vitro in a cell model and reduced tau seed propagation. The enzyme nSMase2 is involved in the production of exosomes carrying proteopathic seeds and could contribute to cell-to-cell transmission of pathological protein aggregates implicated in neurodegenerative diseases such as Parkinson's disease (PD). Here, we performed in vivo studies to determine if DDL-112 can reduce brain EV/exosome production and proteopathic alpha synuclein (αSyn) spread in a PD mouse model. METHODS: The acute effects of single-dose treatment with DDL-112 on interleukin-1ß-induced extracellular vesicle (EV) release in brain tissue of Thy1-αSyn PD model mice and chronic effects of 5 week DDL-112 treatment on behavioral/motor function and proteinase K-resistant αSyn aggregates in the PD model were determined. RESULTS/DISCUSSION: In the acute study, pre-treatment with DDL-112 reduced EV/exosome biogenesis and in the chronic study, treatment with DDL-112 was associated with a reduction in αSyn aggregates in the substantia nigra and improvement in motor function. Inhibition of nSMase2 thus offers a new approach to therapeutic development for neurodegenerative diseases with the potential to reduce the spread of disease-specific proteopathic proteins.

Dual Neutral Sphingomyelinase-2/Acetylcholinesterase Inhibitors for the Treatment of Alzheimer's Disease.

We report the discovery of a novel class of compounds that function as dual inhibitors of the enzymes neutral sphingomyelinase-2 (nSMase2) and acetylcholinesterase (AChE). Inhibition of these enzymes provides a unique strategy to suppress the propagation of tau pathology in the treatment of Alzheimer's disease (AD). We describe the key SAR elements that affect relative nSMase2 and/or AChE inhibitor effects and potency, in addition to the identification of two analogs that suppress the release of tau-bearing exosomes in vitro and in vivo. Identification of these novel dual nSMase2/AChE inhibitors represents a new therapeutic approach to AD and has the potential to lead to the development of truly disease-modifying therapeutics.

Apolipoprotein E/Amyloid-ß Complex Accumulates in Alzheimer Disease Cortical Synapses via Apolipoprotein E Receptors and Is Enhanced by APOE4.

Apolipoprotein E (apoE) colocalizes with amyloid-ß (Aß) in Alzheimer disease (AD) plaques and in synapses, and evidence suggests that direct interactions between apoE and Aß are important for apoE's effects in AD. The present work examines the hypothesis that apoE receptors mediate uptake of apoE/Aß complex into synaptic terminals. Western blot analysis shows multiple SDS-stable assemblies in synaptosomes from human AD cortex; apoE/Aß complex was markedly increased in AD compared with aged control samples. Complex formation between apoE and Aß was confirmed by coimmunoprecipitation experiments. The apoE receptors low-density lipoprotein receptor (LDLR) and LDLR-related protein 1 (LRP1) were quantified in synaptosomes using flow cytometry, revealing up-regulation of LRP1 in early- and late-stage AD. Dual-labeling flow cytometry analysis of LRP1- and LDLR positives indicate most (approximately 65%) of LDLR and LRP1 is associated with postsynaptic density-95 (PSD-95)-positive synaptosomes, indicating that remaining LRP1 and LDLR receptors are exclusively presynaptic. Flow cytometry analysis of Nile red labeling revealed a reduction in cholesterol esters in AD synaptosomes. Dual-labeling experiments showed apoE and Aß concentration into LDLR and LRP1-positive synaptosomes, along with free and esterified cholesterol. Synaptic Aß was increased by apoE4 in control and AD samples. These results are consistent with uptake of apoE/Aß complex and associated lipids into synaptic terminals, with subsequent Aß clearance in control synapses and accumulation in AD synapses.

Kv2 Ion Channels Determine the Expression and Localization of the Associated AMIGO-1 Cell Adhesion Molecule in Adult Brain Neurons.

Voltage-gated K+ (Kv) channels play important roles in regulating neuronal excitability. Kv channels comprise four principal α subunits, and transmembrane and/or cytoplasmic auxiliary subunits that modify diverse aspects of channel function. AMIGO-1, which mediates homophilic cell adhesion underlying neurite outgrowth and fasciculation during development, has recently been shown to be an auxiliary subunit of adult brain Kv2.1-containing Kv channels. We show that AMIGO-1 is extensively colocalized with both Kv2.1 and its paralog Kv2.2 in brain neurons across diverse mammals, and that in adult brain, there is no apparent population of AMIGO-1 outside of that colocalized with these Kv2 α subunits. AMIGO-1 is coclustered with Kv2 α subunits at specific plasma membrane (PM) sites associated with hypolemmal subsurface cisternae at neuronal ER:PM junctions. This distinct PM clustering of AMIGO-1 is not observed in brain neurons of mice lacking Kv2 α subunit expression. Moreover, in heterologous cells, coexpression of either Kv2.1 or Kv2.2 is sufficient to drive clustering of the otherwise uniformly expressed AMIGO-1. Kv2 α subunit coexpression also increases biosynthetic intracellular trafficking and PM expression of AMIGO-1 in heterologous cells, and analyses of Kv2.1 and Kv2.2 knockout mice show selective loss of AMIGO-1 expression and localization in neurons lacking the respective Kv2 α subunit. Together, these data suggest that in mammalian brain neurons, AMIGO-1 is exclusively associated with Kv2 α subunits, and that Kv2 α subunits are obligatory in determining the correct pattern of AMIGO-1 expression, PM trafficking and clustering.

Stimulation of the medial septum improves performance in spatial learning following pilocarpine-induced status epilepticus.

Temporal lobe epilepsy often leads to hippocampal sclerosis and persistent cognitive deficits, including difficulty with learning and memory. Hippocampal theta oscillations are critical in optimizing hippocampal function and facilitating plasticity. We hypothesized that pilocarpine-induced status epilepticus would disrupt oscillations and behavioral performance and that electrical neuromodulation to entrain theta would improve cognition specifically in injured rats. Rats received a pilocarpine (n=30) or saline injection (n=27) and unilateral bi-polar electrodes were implanted into the medial septum and hippocampus the following day. Hippocampal and septal theta were recorded in a Plexiglas box over the first week following implantation. Control and pilocarpine-treated rats were split into stimulation (continuous 7.7Hz, 80µA, 1ms pulse width) and non-stimulation groups for behavioral analysis. Continuous stimulation was initiated one-minute prior to and throughout an object exploration task (post-injury day seven) and again for each of six trials on the Barnes maze (post-injury days 12-14). There was a significant reduction in hippocampal theta power (p<0.05) and percentage of time oscillating in theta (p<0.05). In addition there was a significant decrease in object exploration in rats post-pilocarpine (p<0.05) and an impairment in spatial learning. Specifically, pilocarpine-treated rats were more likely to use random search strategies (p<0.001) and had an increase in latency to find the hidden platform (p<0.05) on the Barnes maze. Stimulation of the medial septum at 7.7Hz in pilocarpine-treated rats resulted in performance similar to shams in both the object recognition and Barnes maze tasks. Stimulation of sham rats resulted in impaired object exploration (p<0.05) with no difference in Barnes maze latency or strategy. In conclusion, pilocarpine-induced seizures diminished hippocampal oscillations and impaired performance in both an object exploration and a spatial memory task in pilocarpine-treated rats. Theta stimulation at 7.7Hz improved behavioral outcome on the Barnes maze task; this improvement in function was not related to a general cognitive enhancement, as shams did not benefit from stimulation. Therefore, stimulation of the medial septum represents an exciting target to improve behavioral outcome in patients with epilepsy.

Septohippocampal Neuromodulation Improves Cognition after Traumatic Brain Injury.

Traumatic brain injury (TBI) often results in persistent attention and memory deficits that are associated with hippocampal dysfunction. Although deep brain stimulation (DBS) is used to treat neurological disorders related to motor dysfunction, the effectiveness of stimulation to treat cognition remains largely unknown. In this study, adult male Harlan Sprague-Dawley rats underwent a lateral fluid percussion or sham injury followed by implantation of bipolar electrodes in the medial septal nucleus (MSN) and ipsilateral hippocampus. In the first week after injury, there was a significant decrease in hippocampal theta oscillations that correlated with decreased object exploration and impaired performance in the Barnes maze spatial learning task. Continuous 7.7 Hz theta stimulation of the medial septum significantly increased hippocampal theta oscillations, restored normal object exploration, and improved spatial learning in injured animals. There were no benefits with 100 Hz gamma stimulation, and stimulation of sham animals at either frequency did not enhance performance. We conclude, therefore, that there was a theta frequency-specific benefit of DBS that restored cognitive function in brain-injured rats. These data suggest that septal theta stimulation may be an effective and novel neuromodulatory therapy for treatment of persistent cognitive deficits following TBI.