Unique Properties of Synaptosomes and Prospects for Their Use for the Treatment of Alzheimer's Disease.

Alzheimer's disease (AD) is a severe neurodegenerative condition affecting millions worldwide. Prevalence of AD correlates with increased life expectancy and aging population in the developed countries. Considering that AD is a multifactorial disease involving various pathological processes such as synaptic dysfunction, neuroinflammation, oxidative stress, and improper protein folding, a comprehensive approach targeting multiple pathways may prove effective in slowing the disease progression. Cellular therapy and its further development in the form of cell vesicle and particularly mitochondrial transplantation represent promising approaches for treating neurodegeneration. The use of synaptosomes, due to uniqueness of their contents, could mark a new stage in the development of comprehensive therapies for neurodegenerative diseases, particularly AD. Synaptosomes contain unique memory mitochondria, which differ not only in size but also in functionality compared to the mitochondria in the neuronal soma. These synaptosomal mitochondria actively participate in cellular communication and signal transmission within synapses. Synaptosomes also contain other elements such as their own protein synthesis machinery, synaptic vesicles with neurotransmitters, synaptic adhesion molecules, and microRNAs - all crucial for synaptic transmission and, consequently, cognitive processes. Complex molecular ensemble ensures maintenance of the synaptic autonomy of mitochondria. Additionally, synaptosomes, with their affinity for neurons, can serve as an optimal platform for targeted drug delivery to nerve cells. This review discusses unique composition of synaptosomes, their capabilities and advantages, as well as limitations of their suggested use as therapeutic agents for treating neurodegenerative pathologies, particularly AD.

Rescue of synaptosomal glutamate release defects in tau transgenic mice by the tau aggregation inhibitor hydromethylthionine.

Glutamatergic neurotransmission, important for learning and memory, is disrupted in different ways in patients with Alzheimer's disease (AD) and frontotemporal dementia (FTD) tauopathies. We have previously reported that two tau transgenic mouse models, L1 and L66, produce different phenotypes resembling AD and FTD, respectively. The AD-like L1 model expresses the truncated core aggregation domain of the AD paired helical filament (PHF) form of tau (tau296-390) whereas the FTD-like L66 model expresses full-length tau carrying two mutations at P301S/G335D. We have used synaptosomes isolated from these mice to investigate K+-evoked glutamate release and, if abnormal, to determine responsiveness to hydromethylthionine, a tau aggregation inhibitor previously shown to reduce tau pathology in these models. We report that the transgenes in these two mouse lines cause opposite abnormalities in glutamate release. Over-expression of the core tau unit in L1 produces a significant reduction in glutamate release and a loss of Ca2+-dependency compared with wild-type control mice. Full-length mutant tau produces an increase in glutamate release that retains normal Ca2+-dependency. Chronic pre-treatment with hydromethylthionine normalises both reduced (L1) and excessive glutamate (L66) and restores normal Ca2+-dependency in L1 mice. This implies that both patterns of impairment are the result of tau aggregation, but that the direction and Ca2+-dependency of the abnormality is determined by expression of the disease-specific transgene. Our results lead to the conclusion that the tauopathies need not be considered a single entity in terms of the downstream effects of pathological aggregation of tau protein. In this case, directionally opposite abnormalities in glutamate release resulting from different types of tau aggregation in the two mouse models can be corrected by hydromethylthionine. This may help to explain the activity of hydromethylthionine on cognitive decline and brain atrophy in both AD and behavioural-variant FTD.

Cystatin C loaded in brain-derived extracellular vesicles rescues synapses after ischemic insult in vitro and in vivo.

Synaptic loss is an early event in the penumbra area after an ischemic stroke. Promoting synaptic preservation in this area would likely improve functional neurological recovery. We aimed to detect proteins involved in endogenous protection mechanisms of synapses in the penumbra after stroke and to analyse potential beneficial effects of these candidates for a prospective stroke treatment. For this, we performed Liquid Chromatography coupled to Mass Spectrometry (LC-MS)-based proteomics of synaptosomes isolated from the ipsilateral hemispheres of mice subjected to experimental stroke at different time points (24 h, 4 and 7 days) and compared them to sham-operated mice. Proteomic analyses indicated that, among the differentially expressed proteins between the two groups, cystatin C (CysC) was significantly increased at 24 h and 4 days following stroke, before returning to steady-state levels at 7 days, thus indicating a potential transient and intrinsic rescue mechanism attempt of neurons. When CysC was applied to primary neuronal cultures subjected to an in vitro model of ischemic damage, this treatment significantly improved the preservation of synaptic structures. Notably, similar effects were observed when CysC was loaded into brain-derived extracellular vesicles (BDEVs). Finally, when CysC contained in BDEVs was administered intracerebroventricularly to stroked mice, it significantly increased the expression of synaptic markers such as SNAP25, Homer-1, and NCAM in the penumbra area compared to the group supplied with empty BDEVs. Thus, we show that CysC-loaded BDEVs promote synaptic protection after ischemic damage in vitro and in vivo, opening the possibility of a therapeutic use in stroke patients.

Phillygenin Suppresses Glutamate Exocytosis in Rat Cerebrocortical Nerve Terminals (Synaptosomes) through the Inhibition of Cav2.2 Calcium Channels.

Glutamate is a major excitatory neurotransmitter that mediates neuronal damage in acute and chronic brain disorders. The effect and mechanism of phillygenin, a natural compound with neuroprotective potential, on glutamate release in isolated nerve terminals (synaptosomes) prepared from the rat cerebral cortex were examined. In this study, 4-aminopyridine (4-AP), a potassium channel blocker, was utilized to induce the release of glutamate, which was subsequently quantified via a fluorometric assay. Our findings revealed that phillygenin reduced 4-AP-induced glutamate release, and this inhibitory effect was reversed by removing extracellular Ca2+ or inhibiting vesicular transport with bafilomycin A1. However, exposure to the glutamate transporter inhibitor dl-threo-beta-benzyl-oxyaspartate (dl-TOBA) did not influence the inhibitory effect. Moreover, phillygenin did not change the synaptosomal membrane potential but lowered the 4-AP-triggered increase in intrasynaptosomal Ca2+ concentration ([Ca2+]i). Antagonizing Cav2.2 (N-type) calcium channels blocked the inhibition of glutamate release by phillygenin, whereas pretreatment with the mitochondrial Na+/Ca2+ exchanger inhibitor, CGP37157 or the ryanodine receptor inhibitor, dantrolene, both of which block intracellular Ca2+ release, had no effect. The effect of phillygenin on glutamate release triggered by 4-AP was completely abolished when MAPK/ERK inhibitors were applied. Furthermore, phillygenin attenuated the phosphorylation of ERK1/2 and its major presynaptic target, synapsin I, a protein associated with synaptic vesicles. These data collectively suggest that phillygenin mediates the inhibition of evoked glutamate release from synaptosomes primarily by reducing the influx of Ca2+ through Cav2.2 calcium channels, thereby subsequently suppressing the MAPK/ERK/synapsin I signaling cascade.

Complement tunes glutamate release and supports synaptic impairments in an animal model of multiple sclerosis.

BACKGROUND AND PURPOSE: To deepen our knowledge of the role of complement in synaptic impairment in experimental autoimmune encephalomyelitis (EAE) mice, we investigated the distribution of C1q and C3 proteins and the role of complement as a promoter of glutamate release in purified nerve endings (synaptosomes) and astrocytic processes (gliosomes) isolated from the cortex of EAE mice at the acute stage of the disease (21 ± 1 day post-immunization). EXPERIMENTAL APPROACH: EAE cortical synaptosomes and gliosomes were analysed for glutamate release efficiency (measured as release of preloaded [3H]D-aspartate ([3H]D-ASP)), C1q and C3 protein density, and for viability and ongoing apoptosis. KEY RESULTS: In healthy mice, complement releases [3H]D-ASP from gliosomes more efficiently than from synaptosomes. The releasing activity occurs in a dilution-dependent manner and involves the reversal of the excitatory amino acid transporters (EAATs). In EAE mice, the complement-induced releasing activity is significantly reduced in cortical synaptosomes but amplified in cortical gliosomes. These adaptations are paralleled by decreased density of the EAAT2 protein in synaptosomes and increased EAAT1 staining in gliosomes. Concomitantly, PSD95, GFAP, and CD11b, but not SNAP25, proteins are overexpressed in the cortex of the EAE mice. Similarly, C1q and C3 protein immunostaining is increased in EAE cortical synaptosomes and gliosomes, although signs of ongoing apoptosis or altered viability are not detectable. CONCLUSION AND IMPLICATIONS: Our results unveil a new noncanonical role of complement in the CNS of EAE mice relevant to disease progression and central synaptopathy that suggests new therapeutic targets for the management of MS.

Akt activation ameliorates deficits in hippocampal-dependent memory and activity-dependent synaptic protein synthesis in an Alzheimer's disease mouse model.

Protein kinase-B (Akt) and the mechanistic target of rapamycin (mTOR) signaling pathways are implicated in Alzheimer's disease (AD) pathology. Akt/mTOR signaling pathways, activated by external inputs, enable new protein synthesis at the synapse and synaptic plasticity. The molecular mechanisms impeding new protein synthesis at the synapse in AD pathogenesis remain elusive. Here, we aimed to understand the molecular mechanisms prior to the manifestation of histopathological hallmarks by characterizing Akt1/mTOR signaling cascades and new protein synthesis in the hippocampus of WT and amyloid precursor protein/presenilin-1 (APP/PS1) male mice. Intriguingly, compared to those in WT mice, we found significant decreases in pAkt1, pGSK3ß, pmTOR, pS6 ribosomal protein, and p4E-BP1 levels in both post nuclear supernatant and synaptosomes isolated from the hippocampus of one-month-old (presymptomatic) APP/PS1 mice. In synaptoneurosomes prepared from the hippocampus of presymptomatic APP/PS1 mice, activity-dependent protein synthesis at the synapse was impaired and this deficit was sustained in young adults. In hippocampal neurons from C57BL/6 mice, downregulation of Akt1 precluded synaptic activity-dependent protein synthesis at the dendrites but not in the soma. In three-month-old APP/PS1 mice, Akt activator (SC79) administration restored deficits in memory recall and activity-dependent synaptic protein synthesis. C57BL/6 mice administered with an Akt inhibitor (MK2206) resulted in memory recall deficits compared to those treated with vehicle. We conclude that dysregulation of Akt1/mTOR and its downstream signaling molecules in the hippocampus contribute to memory recall deficits and loss of activity-dependent synaptic protein synthesis. In AD mice, however, Akt activation ameliorates deficits in memory recall and activity-dependent synaptic protein synthesis.

Utilities of Isolated Nerve Terminals in Ex Vivo Analyses of Protein Translation in (Patho)physiological Brain States: Focus on Alzheimer's Disease.

Synapses are the cellular substrates of higher-order brain functions, and their dysfunction is an early and primary pathogenic mechanism across several neurological disorders. In particular, Alzheimer's disease (AD) is categorized by prodromal structural and functional synaptic deficits, prior to the advent of classical behavioral and pathological features. Recent research has shown that the development, maintenance, and plasticity of synapses depend on localized protein translation. Synaptosomes and synaptoneurosomes are biochemically isolated synaptic terminal preparations which have long been used to examine a variety of synaptic processes ex vivo in both healthy and pathological conditions. These ex vivo preparations preserve the mRNA species and the protein translational machinery. Hence, they are excellent in organello tools for the study of alterations in mRNA levels and protein translation in neuropathologies. Evaluation of synapse-specific basal and activity-driven de novo protein translation activity can be conveniently performed in synaptosomal/synaptoneurosomal preparations from both rodent and human brain tissue samples. This review gives a quick overview of the methods for isolating synaptosomes and synaptoneurosomes before discussing the studies that have utilized these preparations to study localized synapse-specific protein translation in (patho)physiological situations, with an emphasis on AD. While the review is not an exhaustive accumulation of all the studies evaluating synaptic protein translation using the synaptosomal model, the aim is to assemble the most relevant studies that have done so. The hope is to provide a suitable research platform to aid neuroscientists to utilize the synaptosomal/synaptoneurosomal models to evaluate the molecular mechanisms of synaptic dysfunction within the specific confines of mRNA localization and protein translation research.

Early-life stress and amyloidosis in mice share pathogenic pathways involving synaptic mitochondria and lipid metabolism.

INTRODUCTION: Early-life stress (ES) increases the risk for Alzheimer's disease (AD). We and others have shown that ES aggravates amyloid-beta (Aß) pathology and promotes cognitive dysfunction in APP/PS1 mice, but underlying mechanisms remain unclear. METHODS: We studied how ES affects the hippocampal synaptic proteome in wild-type (WT) and APP/PS1 mice at early and late pathological stages, and validated hits using electron microscopy and immunofluorescence. RESULTS: The hippocampal synaptosomes of both ES-exposed WT and early-stage APP/PS1 mice showed a relative decrease in actin dynamics-related proteins and a relative increase in mitochondrial proteins. ES had minimal effects on older WT mice, while strongly affecting the synaptic proteome of advanced stage APP/PS1 mice, particularly the expression of astrocytic and mitochondrial proteins. DISCUSSION: Our data show that ES and amyloidosis share pathogenic pathways involving synaptic mitochondrial dysfunction and lipid metabolism, which may underlie the observed impact of ES on the trajectory of AD.

Density of Sphingosine-1-Phosphate Receptors Is Altered in Cortical Nerve-Terminals of Insulin-Resistant Goto-Kakizaki Rats and Diet-Induced Obese Mice.

Sphingosine-1-phosphate (S1P) is a phosphosphingolipid with pleiotropic biological functions. S1P acts as an intracellular second messenger, as well as extracellular ligand to five G-protein coupled receptors (S1PR1-5). In the brain, S1P regulates neuronal proliferation, apoptosis, synaptic activity and neuroglia activation. Moreover, S1P metabolism alterations have been reported in neurodegenerative disorders. We have previously reported that S1PRs are present in nerve terminals, exhibiting distinct sub-synaptic localization and neuromodulation actions. Since type 2 diabetes (T2D) causes synaptic dysfunction, we hypothesized that S1P signaling is modified in nerve terminals. In this study, we determined the density of S1PRs in cortical synaptosomes from insulin-resistant Goto-Kakizaki (GK) rats and Wistar controls, and from mice fed a high-fat diet (HFD) and low-fat-fed controls. Relative to their controls, GK rats showed similar cortical S1P concentration despite higher S1P levels in plasma, yet lower density of S1PR1, S1PR2 and S1PR4 in nerve-terminal-enriched membranes. HFD-fed mice exhibited increased plasma and cortical concentrations of S1P, and decreased density of S1PR1 and S1PR4. These findings point towards altered S1P signaling in synapses of insulin resistance and diet-induced obesity models, suggesting a role of S1P signaling in T2D-associated synaptic dysfunction.

Pathological Deficit of Cystatin B Impairs Synaptic Plasticity in EPM1 Human Cerebral Organoids.

Cystatin B (CSTB) is a small protease inhibitor protein being involved in cell proliferation and neuronal differentiation. Loss-of-function mutations in CSTB gene cause progressive myoclonic epilepsy 1 (EPM1). We previously demonstrated that CSTB is locally synthesized in synaptic nerve terminals from rat brain and secreted into the media, indicating its role in synaptic plasticity. In this work, we have further investigated the involvement of CSTB in synaptic plasticity, using synaptosomes from human cerebral organoids (hCOs) as well as from rodents' brain. Our data demonstrate that CSTB is released from synaptosomes in two ways: (i) as a soluble protein and (ii) in extracellular vesicles-mediated pathway. Synaptosomes isolated from hCOs are enriched in pre-synaptic proteins and contain CSTB at all developmental stages analyzed. CSTB presence in the synaptic territories was also confirmed by immunostaining on human neurons in vitro. To investigate if the depletion of CSTB affects synaptic plasticity, we characterized the synaptosomes from EPM1 hCOs. We found that the levels of presynaptic proteins and of an initiation factor linked to local protein synthesis were both reduced in EPM1 hCOs and that the extracellular vesicles trafficking pathway was impaired. Moreover, EPM1 neurons displayed anomalous morphology with longer and more branched neurites bearing higher number of intersections and nodes, suggesting connectivity alterations. In conclusion, our data strengthen the idea that CSTB plays a critical role in the synapse physiology and reveal that pathologically low levels of CSTB may affect synaptic plasticity, leading to synaptopathy and altered neuronal morphology.

Mitochondrial bioenergetics and cytometric characterization of a synaptosomal preparation from mouse brain cortex.

Mitochondrial function at synapses can be assessed in isolated nerve terminals. Synaptosomes are structures obtained in vitro by detaching the nerve endings from neuronal bodies under controlled homogenization conditions. Several protocols have been described for the preparation of intact synaptosomal fractions. Herein a fast and economical method to obtain synaptosomes with optimal intrasynaptic mitochondria functionality was described. Synaptosomal fractions were obtained from mouse brain cortex by differential centrifugation followed by centrifugation in a Ficoll gradient. The characteristics of the subcellular particles obtained were analyzed by flow cytometry employing specific tools. Integrity and specificity of the obtained organelles were evaluated by calcein and SNAP-25 probes. The proportion of positive events of the synaptosomal preparation was 75 ± 2 % and 48 ± 7% for calcein and Synaptosomal-Associated Protein of 25 kDa (SNAP-25), respectively. Mitochondrial integrity was evaluated by flow cytometric analysis of cardiolipin content, which indicated that 73 ± 1% of the total events were 10 N-nonylacridine orange (NAO)-positive. Oxygen consumption, ATP production and mitochondrial membrane potential determinations showed that mitochondria inside synaptosomes remained functional after the isolation procedure. Mitochondrial and synaptosomal enrichment were determined by measuring synaptosomes/ homogenate ratio of specific markers. Functionality of synaptosomes was verified by nitric oxide detection after glutamate addition. As compared with other methods, the present protocol can be performed briefly, does not imply high economic costs, and provides an useful tool for the isolation of a synaptosomal preparation with high mitochondrial respiratory capacity and an adequate integrity and function of intraterminal mitochondria.

STIM2 regulates NMDA receptor endocytosis that is induced by short-term NMDA receptor overactivation in cortical neurons.

Recent findings suggest an important role for the dysregulation of stromal interaction molecule (STIM) proteins, activators of store-operated Ca2+ channels, and the prolonged activation of N-methyl-D-aspartate receptors (NMDARs) in the development of neurodegenerative diseases. We previously demonstrated that STIM silencing increases Ca2+ influx through NMDAR and STIM-NMDAR2 complexes are present in neurons. However, the interplay between NMDAR subunits (GluN1, GluN2A, and GluN2B) and STIM1/STIM2 with regard to intracellular trafficking remains unknown. Here, we found that the activation of NMDAR endocytosis led to an increase in STIM2-GluN2A and STIM2-GluN2B interactions in primary cortical neurons. STIM1 appeared to migrate from synaptic to extrasynaptic sites. STIM2 silencing inhibited post-activation NMDAR translocation from the plasma membrane and synaptic spines and increased NMDAR currents. Our findings reveal a novel molecular mechanism by which STIM2 regulates NMDAR synaptic trafficking by promoting NMDAR endocytosis after receptor overactivation, which may suggest protection against excessive uncontrolled Ca2+ influx through NMDARs.

Presynaptic Release-Regulating Sphingosine 1-Phosphate 1/3 Receptors in Cortical Glutamatergic Terminals: Adaptations in EAE Mice and Impact of Therapeutic FTY720.

This study provides evidence of the existence of presynaptic inhibitory sphingosine-1-phosphate receptor 1 (S1P1R) and facilitatory S1P3R in cortical nerve endings (synaptosomes) of healthy mice. The conclusion relies on the findings that (i) the S1P1R agonist CS-2100 (0.1-30 nM) inhibits the 12 mM KCl-evoked glutamate exocytosis (quantified as the release of [3H]D-aspartate) while the S1P3R allosteric agonist CYM-5541 potentiates it and (ii) these effects are inhibited by the S1P1R antagonist Ex 26 (30-300 nM) and the S1P3R antagonist TY-52156 (100-1000 nM), respectively. Confocal microscopy and western blot analysis confirmed the presence of S1P1R and S1P3R proteins in cortical glutamatergic synaptosomes, which were scarcely accessible to biotin in a biotinylation study. Then, we demonstrated that S1P1R and S1P3R densities and their release activity are amplified in cortical synaptosomes of mice suffering from experimental autoimmune encephalomyelitis (EAE), despite receptors maintain their preferential internal distribution. Receptor changes recover following chronic oral therapeutic FTY720 (0.03 mg/Kg/day). These results improve our knowledge of the role of presynaptic release-regulating S1P1Rs and S1P3Rs controlling glutamate transmission in the CNS also unravelling functional adaptations during EAE that recover following chronic FTY720. In a whole, these findings provide new information on the central neuroprotectant activities of FTY720.

Unveiling the nongenomic actions of thyroid hormones in adult mammalian brain: The legacy of Mary B. Dratman.

A comprehensive review was conducted to compile the contributions of Mary B. Dratman and studies by other researchers in the field of nongenomic actions of thyroid hormones in adult mammalian brain. Dratman and her collaborators authored roughly half of the papers in this area. It has been almost fifty years since Dratman introduced the novel concept of thyroid hormones as neurotransmitters for the first time. The characterization of unique brain-region specific accumulation of thyroid hormones within the nerve terminals in adult mammals was a remarkable contribution by Dratman. It suggested a neurotransmitter- or neuromodulator-like role of thyroid hormone and/or its derivative, 3-iodothyronamine within adrenergic systems in adult mammalian brain. Several studies by other researchers using synaptosomes as a model system, have contributed to the concept of direct nongenomic actions of thyroid hormones at synaptic regions by establishing that thyroid hormones or their derivatives can bind to synaptosomal membranes, alter membrane functions including enzymatic activities and ion transport, elicit Ca2+/NO-dependent signaling pathways and induce substrate-protein phosphorylation. Such findings can help to explain the physiological and pathophysiological roles of thyroid hormone in psychobehavioral control in adult mammalian brain. However, the exact mode of nongenomic actions of thyroid hormones at nerve terminals in adult mammalian brain awaits further study.

Neuroanatomical zones of human traumatic brain injury reveal significant differences in protein profile and protein oxidation: Implications for secondary injury events.

Traumatic brain injury (TBI) causes significant neurological deficits and long-term degenerative changes. Primary injury in TBI entails distinct neuroanatomical zones, i.e., contusion (Ct) and pericontusion (PC). Their dynamic expansion could contribute to unpredictable neurological deterioration in patients. Molecular characterization of these zones compared with away from contusion (AC) zone is invaluable for TBI management. Using proteomics-based approach, we were able to distinguish Ct, PC and AC zones in human TBI brains. Ct was associated with structural changes (blood-brain barrier (BBB) disruption, neuroinflammation, axonal injury, demyelination and ferroptosis), while PC was associated with initial events of secondary injury (glutamate excitotoxicity, glial activation, accumulation of cytoskeleton proteins, oxidative stress, endocytosis) and AC displayed mitochondrial dysfunction that could contribute to secondary injury events and trigger long-term degenerative changes. Phosphoproteome analysis in these zones revealed that certain differentially phosphorylated proteins synergistically contribute to the injury events along with the differentially expressed proteins. Non-synaptic mitochondria (ns-mito) was associated with relatively more differentially expressed proteins (DEPs) compared to synaptosomes (Syn), while the latter displayed increased protein oxidation including tryptophan (Trp) oxidation. Proteomic analysis of immunocaptured complex I (CI) from Syn revealed increased Trp oxidation in Ct > PC > AC (vs. control). Oxidized W272 in the ND1 subunit of CI, revealed local conformational changes in ND1 and the neighboring subunits, as indicated by molecular dynamics simulation (MDS). Taken together, neuroanatomical zones in TBI show distinct protein profile and protein oxidation representing different primary and secondary injury events with potential implications for TBI pathology and neurological status of the patients.

Levels of Synaptic Proteins in Brain and Neurofilament Light Chain in Cerebrospinal Fluid and Plasma of OVT73 Huntington's Disease Sheep Support a Prodromal Disease State.

BACKGROUND: Synaptic changes occur early in patients with Huntington's disease (HD) and in mouse models of HD. An analysis of synaptic changes in HD transgenic sheep (OVT73) is fitting since they have been shown to have some phenotypes. They also have larger brains, longer lifespan, and greater motor and cognitive capacities more aligned with humans, and can provide abundant biofluids for in vivo monitoring of therapeutic interventions. OBJECTIVE: The objective of this study was to determine if there were differences between 5- and 10-year-old OVT73 and wild-type (WT) sheep in levels of synaptic proteins in brain and in neurofilament light chain (NfL) in cerebrospinal fluid (CSF) and plasma. METHODS: Mutant huntingtin (mHTT) and other proteins were measured by western blot assay in synaptosomes prepared from caudate, motor, and piriform cortex in 5-year-old and caudate, putamen, motor; and piriform cortex in 10-year-old WT and OVT73 sheep. Levels of NfL, a biomarker for neuronal damage increased in many neurological disorders including HD, were examined in CSF and plasma samples from 10-year-old WT and OVT73 sheep using the Simoa NfL Advantage kit. RESULTS: Western blot analysis showed mHTT protein expression in synaptosomes from OVT73 sheep was  23% of endogenous sheep HTT levels at both ages. Significant changes were detected in brain levels of PDE10A, SCN4B, DARPP32, calmodulin, SNAP25, PSD95, VGLUT 1, VAMP1, and Na+/K+-ATPase, which depended on age and brain region. There was no difference in NfL levels in CSF and plasma in OVT73 sheep compared to age-matched WT sheep. CONCLUSIONS: These results show that synaptic changes occur in brain of 5- and 10-year-old OVT73 sheep, but levels of NfL in biofluids are unaffected. Altogether, the data support a prodromal disease state in OVT73 sheep that involves the caudate, putamen and cortex.

Increased hippocampal cannabinoid 1 receptor expression is associated with protection from severe seizures in pregnant mice with reduced uterine perfusion pressure.

Eclampsia, new-onset seizures in pregnancy, can complicate preeclampsia, a hypertensive pregnancy disorder. The mechanisms contributing to increased risk of seizures in preeclampsia are not fully known. One mechanism could be abnormal endocannabinoid system (ECS) activity and impaired neuromodulation. Indeed, increased placental cannabinoid receptor 1 (CB1R) expression and reduced serum anandamide, a CB1R ligand, have been reported in preeclampsia patients. We hypothesized that reduced uterine perfusion pressure (RUPP), used to mimic preeclampsia, leads to changes in hippocampal CB1R expression, and that manipulating CB1R activity will change seizure severity in RUPP mice. Pregnant mice underwent sham or RUPP surgery on gestational day (GD)13.5. On GD18.5, mice received: no drug treatment, pentylenetetrazol (PTZ, 40 mg/kg), Rimonabant (10 mg/kg) + PTZ, or 2-AG (1 mg/kg) + PTZ. Behaviors were video recorded (15 min for Rimonabant and 2-AG, followed by 30 min for PTZ), and the hippocampus was harvested. The expression of CB1R and ECS proteins was measured in hippocampal homogenates, synaptosomes, and cytosol. Hippocampal CB1R increased in homogenates and cytosolic fraction, and was unchanged in synaptosomes of RUPP mice. Increased CB1R colocalization on glutamate-releasing neurons within hippocampal CA1 was observed in RUPP mice. Rimonabant modestly increased seizure scores over time in RUPP mice. PTZ after rimonabant pretreatment increased seizure scores and duration, while reducing latency in sham mice, with little to no change in RUPP mice. Furthermore, RUPP mice had lower seizure scores over time than sham following CB1R blockade and activation. These data suggest that RUPP modifies CB1R activity prior to seizure induction, which protects mice from worse seizure outcomes.

APP family member dimeric complexes are formed predominantly in synaptic compartments.

BACKGROUND: The amyloid precursor protein (APP), a key player in Alzheimer's disease (AD), is part of a larger gene family, including the APP like proteins APLP1 and APLP2. They share similar structures, form homo- and heterotypic dimers and exhibit overlapping functions. RESULTS: We investigated complex formation of the APP family members via two inducible dimerization systems, the FKBP-rapamycin based dimerization as well as cysteine induced dimerization, combined with co-immunoprecipitations and Blue Native (BN) gel analyses. Within the APP family, APLP1 shows the highest degree of dimerization and high molecular weight (HMW) complex formation. Interestingly, only about 20% of APP is dimerized in cultured cells whereas up to 50% of APP is dimerized in mouse brains, independent of age and splice forms. Furthermore, we could show that dimerized APP originates mostly from neurons and is enriched in synaptosomes. Finally, BN gel analysis of human cortex samples shows a significant decrease of APP dimers in AD patients compared to controls. CONCLUSIONS: Together, we suggest that loss of full-length APP dimers might correlate with loss of synapses in the process of AD.

Cycloartane Saponins from Astragalus glycyphyllos and Their In Vitro Neuroprotective, Antioxidant, and hMAO-B-Inhibiting Effects.

Astragalus glycyphyllos (Fabaceae) is used in the traditional medicine of many countries against hepatic and cardiac disorders. The plant contains mainly flavonoids and saponins. From a defatted methanol extract from its overground parts, a new triterpenoid saponin, 3-O-[α-L-rhamnopyranosyl-(1→2)]-ß-D-xylopyranosyl]-24-O-α-L-arabinopyranosyl-3ß,6α,16ß,24(R),25-pentahydroxy-20R-cycloartane, together with the rare saponin astrachrysoside A, were isolated using various chromatography methods. The compounds were identified via extensive high resolution electrospray ionisation mass spectrometry (HRESIMS) and NMR analyses. Both saponins were examined for their possible antioxidant and neuroprotective activity in three different in vitro models. Rat brain synaptosomes, mitochondria, and microsomes were isolated via centrifugation using Percoll gradient. They were treated with the compounds in three different concentrations alone, and in combination with 6-hydroxydopamine or tert-butyl hydroperoxide as toxic agents. It was found that the compounds had statistically significant dose-dependent in vitro protective activity on the sub-cellular fractions. The compounds exhibited a weak inhibitory effect on the enzyme activity of human recombinant monoamine oxidase type B (hMAO-B), compared to selegiline.

A method to study human synaptic protein-protein interactions by using flow cytometry coupled to proximity ligation assay (Syn-FlowPLA).

BACKGROUND: Synapses are highly specialized sites characterized by intricate networks of protein-protein interactions (PPIs) important to maintain healthy synapses. Therefore, mapping these networks could address unsolved questions about human cognition, synaptic plasticity, learning, and memory in physiological and pathological conditions. The limitation of analyzing synaptic interactions in living humans has led to the development of methods to isolate synaptic terminals (synaptosomes) from cryopreserved human brains. NEW METHOD: Here, we established a method to detect synaptic PPIs by applying flow cytometric proximity ligation assay (FlowPLA) to synaptosomes isolated from frozen human frontal cortex (FC) and hippocampus (HP) (Syn-FlowPLA). RESULTS: Applying this method in synaptosomes, we were able to detect the known post-synaptic interactions between distinct subtypes of N-methyl-D-aspartate glutamate receptors (NMDARs) and their anchoring postsynaptic density 95 protein (PSD95). Moreover, we detected the known pre-synaptic interactions between the SNARE complex proteins synaptosomal-associated protein of 25 kDa (SNAP25), synaptobrevin (VAMP2), and syntaxin 1a (STX1A). As a negative control, we analyzed the interaction between mitochondrial superoxide dismutase 2 (SOD2) and PSD95, which are not expected to be physically associated. COMPARISON WITH EXISTING METHODS: PPIs have been studied in vitro primarily by co-immunoprecipitation, affinity chromatography, protein-fragment complementation assays (PCAs), and flow cytometry. All these are valid approaches; however, they require more steps or combination with other techniques. PLA technology identifies PPIs with high specificity and sensitivity. CONCLUSIONS: The Syn-FlowPLA described here allows rapid analyses of PPIs, specifically within the synaptic compartment isolated from frozen autopsy specimens, achieving greater target sensitivity. Syn-FlowPLA, as presented here, is therefore a useful method to study human synaptic PPI in physiological and pathological conditions.