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.

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.

Genetic removal of synaptic Zn2+ impairs cognition, alters neurotrophic signaling and induces neuronal hyperactivity.

Vesicular Zn2+ (zinc) is released at synapses and has been demonstrated to modulate neuronal responses. However, mechanisms through which dysregulation of zinc homeostasis may potentiate neuronal dysfunction and neurodegeneration are not well-understood. We previously reported that accumulation of soluble amyloid beta oligomers (AßO) at synapses correlates with synaptic loss and that AßO localization at synapses is regulated by synaptic activity and enhanced by the release of vesicular Zn2+ in the hippocampus, a brain region that deteriorates early in Alzheimer's disease (AD). Significantly, drugs regulating zinc homeostasis inhibit AßO accumulation and improve cognition in mouse models of AD. We used both sexes of a transgenic mouse model lacking synaptic Zn2+ (ZnT3KO) that develops AD-like cognitive impairment and neurodegeneration to study the effects of disruption of Zn2+ modulation of neurotransmission in cognition, protein expression and activation, and neuronal excitability. Here we report that the genetic removal of synaptic Zn2+ results in progressive impairment of hippocampal-dependent memory, reduces activity-dependent increase in Erk phosphorylation and BDNF mRNA, alters regulation of Erk activation by NMDAR subunits, increases neuronal spiking, and induces biochemical and morphological alterations consistent with increasing epileptiform activity and neurodegeneration as ZnT3KO mice age. Our study shows that disruption of synaptic Zn2+ triggers neurodegenerative processes and is a potential pathway through which AßO trigger altered expression of neurotrophic proteins, along with reduced hippocampal synaptic density and degenerating neurons, neuronal spiking activity, and cognitive impairment and supports efforts to develop therapeutics to preserve synaptic zinc homeostasis in the brain as potential treatments for AD.

Genome sequencing analysis identifies new loci associated with Lewy body dementia and provides insights into its genetic architecture.

The genetic basis of Lewy body dementia (LBD) is not well understood. Here, we performed whole-genome sequencing in large cohorts of LBD cases and neurologically healthy controls to study the genetic architecture of this understudied form of dementia, and to generate a resource for the scientific community. Genome-wide association analysis identified five independent risk loci, whereas genome-wide gene-aggregation tests implicated mutations in the gene GBA. Genetic risk scores demonstrate that LBD shares risk profiles and pathways with Alzheimer's disease and Parkinson's disease, providing a deeper molecular understanding of the complex genetic architecture of this age-related neurodegenerative condition.

Low Cost Electrode Assembly for EEG Recordings in Mice.

Wireless electroencephalography (EEG) of small animal subjects typically utilizes miniaturized EEG devices which require a robust recording and electrode assembly that remains in place while also being well-tolerated by the animal so as not to impair the ability of the animal to perform normal living activities or experimental tasks. We developed simple and fast electrode assembly and method of electrode implantation using electrode wires and wire-wrap technology that provides both higher survival and success rates in obtaining recordings from the electrodes than methods using screws as electrodes. The new wire method results in a 51% improvement in the number of electrodes that successfully record EEG signal. Also, the electrode assembly remains affixed and provides EEG signal for at least a month after implantation. Screws often serve as recording electrodes, which require either drilling holes into the skull to insert screws or affixing screws to the surface of the skull with adhesive. Drilling holes large enough to insert screws can be invasive and damaging to brain tissue, using adhesives may interfere with conductance and result in a poor signal, and soldering screws to wire leads results in fragile connections. The methods presented in this article provide a robust implant that is minimally invasive and has a significantly higher success rate of electrode implantation. In addition, the implant remains affixed and produces good recordings for over a month, while using economical, easily obtained materials and skills readily available in most animal research laboratories.

Metabolic and Growth Rate Alterations in Lymphoblastic Cell Lines Discriminate Between Down Syndrome and Alzheimer's Disease.

BACKGROUND: Deficits in mitochondrial function and oxidative stress play pivotal roles in Down syndrome (DS) and Alzheimer's disease (AD) and these alterations in mitochondria occur systemically in both conditions. OBJECTIVE: We hypothesized that peripheral cells of elder subjects with DS exhibit disease-specific and dementia-specific metabolic features. To test this, we performed a comprehensive analysis of energy metabolism in lymphoblastic-cell-lines (LCLs) derived from subjects belonging to four groups: DS-with-dementia (DSAD), DS-without-dementia (DS), sporadic AD, and age-matched controls. METHODS: LCLs were studied under regular or minimal feeding regimes with galactose or glucose as primary carbohydrate sources. We assessed metabolism under glycolysis or oxidative phosphorylation by quantifying cell viability, oxidative stress, ATP levels, mitochondrial membrane potential (MMP), mitochondrial calcium uptake, and autophagy. RESULTS: DS and DSAD LCLs showed slower growth rates under minimal feeding. DS LCLs mainly dependent on mitochondrial respiration exhibited significantly slower growth and higher levels of oxidative stress compared to other groups. While ATP levels (under mitochondrial inhibitors) and mitochondrial calcium uptake were significantly reduced in DSAD and AD cells, MMP was decreased in DS, DSAD, and AD LCLs. Finally, DS LCLs showed markedly reduced levels of the autophagy marker LC3-II, underscoring the close association between metabolic dysfunction and impaired autophagy in DS. CONCLUSION: There are significant mitochondrial functional changes in LCLs derived from DS, DSAD, and AD patients. Several parameters analyzed were consistently different between DS, DSAD, and AD lines suggesting that metabolic indicators between LCL groups may be utilized as biomarkers of disease progression and/or treatment outcomes.