Mouse Hippocampal Neurons Research Articles

Reducing polypyrimidine tract‑binding protein 1 fails to promote neuronal transdifferentiation on HT22 and mouse astrocyte cells under physiological conditions.

In contrast to prior findings that have illustrated the conversion of non-neuronal cells into functional neurons through the specific targeting of polypyrimidine tract-binding protein 1 (PTBP1), accumulated evidence suggests the impracticality of inducing neuronal transdifferentiation through suppressing PTBP1 expression in pathological circumstances. Therefore, the present study explored the effect of knocking down PTBP1 under physiological conditions on the transdifferentiation of mouse hippocampal neuron HT22 cells and mouse astrocyte (MA) cells. A total of 20 µM negative control small interfering (si)RNA and siRNA targeting PTBP1 were transfected into HT22 and MA cells using Lipo8000™ for 3 and 5 days, respectively. The expression of early neuronal marker βIII-Tubulin and mature neuronal markers NeuN and microtubule-associated protein 2 (MAP2) were detected using western blotting. In addition, βIII-tubulin, NeuN and MAP2 were labeled with immunofluorescence staining to evaluate neuronal cell differentiation in response to PTBP1 downregulation. Under physiological conditions, no significant changes in the expression of βIII-Tubulin, NeuN and MAP2 were found after 3 and 5 days of knockdown of PTBP1 protein in both HT22 and MA cells. In addition, the immunofluorescence staining results showed no apparent transdifferentiation in maker levels and morphology. The results suggested that the knockdown of PTBP1 failed to induce neuronal differentiation under physiological conditions.

Canagliflozin ameliorates neuronal injury after cerebral ischemia reperfusion by targeting SGLT1 and AMPK-dependent apoptosis

Currently, the treatment of acute ischemic stroke is still limited. In clinical studies, the effect of sodium-glucose cotransporter-2 (SGLT2) inhibitors on stroke risk is controversial. This preclinical study was conducted to determine whether canagliflozin, a SGLT2 inhibitor, has neuroprotective effects on ischemic stroke and the underlying mechanisms. The middle cerebral artery occlusion (MCAO) model was established in Sprague Dawley rats, and canagliflozin was administered by gavage before reperfusion. Canagliflozin treatment significantly reduced infarct volume, improved neurological deficits, lessened brain edema, alleviated neuronal damage and reduced apoptosis. SGLT2 inhibitors which are commonly used in the clinic have different degrees of affinity for SGLT1 and SGLT2 receptors, the expression levels of SGLT1 and SGLT2 were detected using immunofluorescence in the cortex and hippocampus. SGLT2 was barely detectable in these regions regardless of MCAO or canagliflozin treatment or not. The SGLT1 expression levels were significantly elevated in the cortex and hippocampus after MCAO, and further significantly reduced by canagliflozin administration. In vitro experiments, the oxygen-glucose deprivation/reoxygenation (OGD/R) model of the HT-22 mouse hippocampal neurons was established. Canagliflozin treatment increased cell viability and attenuated lactate dehydrogenase (LDH) release. Additionally, canagliflozin activated AMP-activated protein kinase (AMPK) and subsequently inhibited apoptosis in a SGLT1 inhibition dependent manner. The present study shows that canagliflozin could attenuate cerebral ischemia-reperfusion injury in experimental ischemic stroke via SGLT1/AMPK signaling, leading to anti-apoptotic effects in neuron. These findings might serve as evidence for the further application of canagliflozin in the treatment of acute ischemic stroke.

The effect of hippocampal overexpression of dopamine neurotrophic factor (CDNF) on the behavior of mice with genetic predisposition to depressive-like behavior

Cerebral dopamine neurotrophic factor (CDNF) is the promising tool for treatment of Parkinson’s disease. However, its role in the regulation of non-motor behavior, including various psychopathologies, remains unclear. In this regard, the aim of present work was to study the effect of CDNF overexpression in the hippocampus on the behavior of ASC mice (Antidepressant Sensitive Cataleptics) with a genetic predisposition to depression-like behavior. CDNF overexpression in mouse hippocampal neurons was induced using an adeno-associated viral vector. Four weeks after stereotaxic injection of the AAV-CDNF construct into the dorsal hippocampus, home cage behavior, exploratory, anxiety and depressive-like behaviors, as well as spatial and associative learning were assessed. We found significant improvements in the dynamics of spatial learning in the Morris water maze in CDNF-overexpressing animals. At the same time, no effect of CDNF was found on other studied behaviors. The behavior of the experimental animals in the home cage conditions did not differ from that in the control group, except for a decrease in the total amount of food eaten and a slight increase in the number of sleep episodes in the light phase of the day. In the present study we also attempted to determine the molecular basis for the above mentioned changes by genes expression assessment. We did not find significant changes in the mRNA level of key kinases, genes involved in neuroplasticity and neuronal survival as well as genes encoding receptors for the main neurotransmitter systems. However, animals overexpressing CDNF show increased lever of spliced Xbp indicating activation of the Ire1α/Xbp1 pathway traditionally associated with ER stress. Immunohistochemical analysis showed that CDNF was co-localized with the ER marker calreticulin. Thus, the effects of endogenous CDNF on behavior that we have found may be mediated by a specific molecular cascade, which emphasizes its difference from classical neurotrophic factors.

Investigation of the functioning of a neurohybrid system based on the FitzHugh–Nagumo radio generator and mouse hippocampal neurons

Currently, in the treatment of neurodegenerative diseases that are difficult to respond to drug therapy, electrical stimulation of the brain is performed through invasive intervention in damaged structures of the nervous tissue. The development and development of invasive technologies of closed-loop neural interfaces have made it possible to achieve great success in restoring neural connections, since they have finer and more precise stimulation settings that respond to changes in the physiological state, which is important in the process of restoring the functions of the nervous tissue. Advances in these areas open up prospects for the treatment of a wide range of diseases of the motor system and neurodegenerative diseases of the brain. In this study, a neurohybrid closed system was used, consisting of a FitzHugh-Nagumo radio generator and live surviving slices of the mouse brain hippocampus. For the preparation of surviving slices of the hippocampus, sexually mature males aged 2-3 months of the C57BL/6 mouse line were used. For the preparation and incubation of slices of the hippocampus, a solution of artificial cerebrospinal fluid (ACSF) was used, composition in (mM): 126 NaCl; 3.5 KCl; 1.2 KH2PO4; 26 NaHCO3; 1.3 MgCl * 6H2O; 2 CaCl2 * 6H2O; 10 D-glucose at constant carbogen saturation (95% O2 and 5% CO2). Registration of the electrical activity of brain neurons was carried out using optical and electrophysiological methods. In experiments on pairing a neuron-like FitzHugh-Nagumo generator and biological nerve cells in a closed circuit, an effect was obtained when the activity of brain nerve cells switched the generator to a self-oscillating mode. The evoked oscillations in the neuron-like generator provided an effective stimulus for the activation of nerve fibers in the perforant pathway of the hippocampus. As a result, it was possible to fix a decrease in the frequency of the generator impulses, which was provoked by the responses of living neurons to the incoming stimulus from the neuron-like generator. These results show the ability of live neural networks to control an artificial signal by adjusting its parameters by changing their own activity and confirm the efficiency of using closed loop systems when combining live and artificial neurons. The present study requires further experiments to create more physiological conditions for the functioning of the proposed neurohybrid system. In addition, this neurohybrid system will be improved and have adaptive properties through the use of memristive devices. Advances in this direction will help solve the urgent problem of restoring lost brain functions at the cellular and network levels.

Dynamics of space coding by mouse hippocampal CA1 field neurons in a free navigation task in different environments

The development of persistent cognitive adaptations in neurons is a key area of focus in contemporary neuroscience. A significant illustration of such adaptations occurs through spatial specializations in place cells with one or more receptive fields that are sensitive to spatial cues (place fields) [1]. In prior research, we extensively examined the dynamics of place field formation in granular layer neurons of the CA1 field in the hippocampus of mice during arbitrary free navigation in a circular track [2]. As the primary factor for maintaining stability in the spatial specialization of place cells, we have introduced dynamic selectivity. This approach enables us to monitor the latency of each place field’s formation and the selectivity dynamics of place cells to their respective fields throughout one or multiple shooting sessions. However, in one-dimensional settings, like the circular track referenced previously, place fields can demonstrate direction-specificity with regards to animal movement [3]. This factor considerably complicates the analysis for arbitrary movement trajectories of animals. In this study, additional experiments were conducted to capture the neural activity of mice in two-dimensional environments. The experiments were conducted in both a rectangular field with objects and a circular arena with a varying number of obstacles. In this paper, we conduct a comparative analysis to examine the critical parameters governing the development of spatial specializations in a one-dimensional circle track and two-dimensional arenas. These parameters include specialization latency, average dynamic selectivity, initial increase in selectivity, and the proportion of “immediate” place fields that remain stable from the first animal visit. The study discovered that the mean selectivity of the place fields escalated in each session, with higher rates observed in subsequent sessions versus the initial session in a novel setting. Additionally, there was a significant occurrence of “immediate” place fields, representing 11% of all place fields in the rounded arena with obstacles, and 25% of all place fields in the circular track. Additionally, a population analysis of neural activity was performed on the first session in a circular track and a round arena with obstacles. Using Laplace eigenmaps for the dimension reduction of population vectors, the trajectory of animals was reconstructed, and the accuracy of this reconstruction agreed with the average dynamic selectivity for each animal included in the analysis. Thus, dynamic selectivity was verified as a measure of the spatial coding quality of the entire registered population of neurons.

Effects of microRNA-320 on learning and memory in mice with vascular cognitive impairment caused via cerebral ischemia.

We aimed to explore microRNA (miR)-320's impacts on learning and memory in mice with vascular cognitive impairment induced via cerebral ischemia. After establishment of a cerebral small vessel disease (CSVD) cognitive impairment model, application of corresponding treatment methods was in the model mice to inject miR-320 antagomir/agomir and their negative controls to the lateral ventricles: Test of the learning and memory abilities of mice was conducted; Detection of oxidative stress, inflammation, miR-320, Vascular endothelial growth factor (VEGF) and endostatin (ES) was implemented; Taking mouse hippocampal neuron cells was to detect the cell advancement. MiR-320 was elevated in the CSVD model; MiR-320 was negatively linked with the learning and memory abilities of mice; Repressing miR-320 was available to memorably elevate the learning and memory abilities of CSVD mice; Depressing miR-320 clearly drove CSVD mouse neovascular protein VEGF, but reduced inflammation, oxidative stress response and ES; Restraining miR-320 was available to contribute to mouse neuronal cell advancement. MiR-320 mitigates the learning and memory abilities of cerebral ischemia-induced vascular cognitive dysfunction mice to a certain extent.

Tetrabromobisphenol A induces neuronal cytotoxicity by inhibiting PINK1-Parkin-mediated mitophagy via upregulating ATF3 expression

Tetrabromobisphenol A (TBBPA), as a widely used brominated flame retardant, has been implicated as a potential neurotoxicant. However, the mechanism of TBBPA-induced neurotoxicity has not been fully elucidated yet. In this study, using mouse hippocampal neuron cell HT22 as the in vitro model, the neuronal cytotoxicity of TBBPA and the mechanism by focusing on mitophagy have been studied. We found that neuronal cytotoxic effects were indeed induced by TBBPA exposure at concentrations of >20 μM for 24 h, including decreased cell viability (to 92.38 % at 20 μM; 18.25 % at 80 μM), enhanced ROS (enhanced 53.26 % at IC50 of 60 μM, compared with that in the control group) and mitochondrial ROS (mtROS) levels (enhanced 24.12 % at 60 μM), reduced mitochondrial membrane potential (MMP) (decreased 33.60 % at 60 μM). As a protective mechanism in cells, autophagy was initiated; however, mitophagy was inhibited, where PINK1 (PINK1-Parkin activation is critical in the depolarized MMP-induced mitophagy) expression was found to be repressed and decreased, further leading to the failure of Parkin recruitment to the damaged mitochondria. Mitophagy activator, nicotinamide mononucleotide (β-NMN) that activates the PINK1-Parkin pathway, could alleviate TBBPA-induced mitophagy deficiency and further reduce the neuronal cytotoxicity, demonstrating that TBBPA-induced PINK1-Parkin-mediated mitophagy deficiency contributed to the neuronal cytotoxicity. Furthermore, we found TBBPA caused the upregulation of Atf3 (activating transcription factor 3) gene transcription and expression levels, alongside reduced Pink1 levels; whereas enhanced Pink1 transcript levels were observed after ATF3 depletion even under TBBPA treatment, demonstrating TBBPA-induced overexpression of ATF3 should be responsible for the reduced PINK1 expression. Therefore, for the first time, here we demonstrate that TBBPA can inhibit PINK1-Parkin-mediated mitophagy via upregulating ATF3 expression, which further contributes to its neuronal cytotoxicity. This study should be able to improve our understanding of the mechanism of TBBPA-induced neuronal cytotoxicity.

Palmatine Protects PC12 Cells and Mice from Aβ25-35-Induced Oxidative Stress and Neuroinflammation via the Nrf2/HO-1 Pathway.

Alzheimer's disease is a common degenerative disease which has a great impact on people's daily lives, but there is still a certain market gap in the drug research about it. Palmatine, one of the main components of Huangteng, the rattan stem of Fibraurea recisa Pierre (Menispermaceae), has potential in the treatment of Alzheimer's disease. The aim of this study was to evaluate the neuroprotective effect of palmatine on amyloid beta protein 25-35-induced rat pheochromocytoma cells and AD mice and to investigate its mechanism of action. CCK8 assays, ELISA, the Morris water maze assay, fluorescent probes, calcein/PI staining, immunofluorescent staining and Western blot analysis were used. The experimental results show that palmatine can increase the survival rate of Aβ25-35-induced PC12 cells and mouse hippocampal neurons, reduce apoptosis, reduce the content of TNF-α, IL-1β, IL-6, GSH, SOD, MDA and ROS, improve the learning and memory ability of AD mice, inhibit the expression of Keap-1 and Bax, and promote the expression of Nrf2, HO-1 and Bcl-2. We conclude that palmatine can ameliorate oxidative stress and neuroinflammation produced by Aβ25-35-induced PC12 cells and mice by modulating the Nrf2/HO-1 pathway. In conclusion, our results suggest that palmatine may have a potential therapeutic effect on AD and could be further investigated as a promising therapeutic agent for AD. It provides a theoretical basis for the development of related drugs.

Tangzhiqing decoction attenuates cognitive dysfunction of mice with type 2 diabetes by regulating AMPK/mTOR autophagy signaling pathway

Ethnopharmacological relevanceTangzhiqing decoction (TZQD) is an effective prescription developed by Jiangsu Province Hospital of Chinese Medicine for the treatment of diabetes mellitus (DM) and its complications, which has a clear cerebral protective effect on mice with diabetic cognitive dysfunction, but its specific mechanism has not been well elucidated. Aims of the studyThis study aims to verify the protection of TZQD on cognitive function in mice with type 2 diabetes mellitus (T2DM) and explore the possible underlying mechanisms. Materials and methodsSix active ingredients in TZQD were detected using high-performance liquid chromatography analysis. In vivo experiments, the protection of TZQD on cognitive function and hippocampal neurons in type 2 diabetes mice was verified to obtain the optimal intervention dose of TZQD. TZQD and 3-methyladenine (3 MA) respectively or jointly intervened in mice with T2DM for 12 weeks, followed by detecting the cognitive difference, hippocampus cornu ammonis 1 (CA1) region injury, and hippocampal neuronal apoptosis in each group. Simultaneously, the investigation of autophagosome formation and organelle impairment in hippocampal neurons, along with the examination of AMPK/mTOR pathway proteins and autophagy-related proteins, was conducted to elucidate the potential mechanisms, through which TZQD modulates autophagy and enhances cognitive function. In vitro experiments, TZQD-containing serum and AMPK inhibitor Compound C (CC) were used to intervene in mouse hippocampal neuron HT22 cells under high glucose environment, further clarifying the regulatory role of TZQD on the AMPK/mTOR pathway and its impact on HT22 cell apoptosis and autophagy. ResultsIn vivo experiment results showed that TZQD had an obvious hypoglycemic effect. Different doses of TZQD could improve cognitive function and hippocampus damage in diabetes mice, with the middle dose of TZQD showing the best effect. TZQD increased the swimming speed of diabetes mice, improved their spatial recognition and memory ability, and reduced hippocampal neuronal apoptosis, Nissl body injury, and p-tau217 protein deposition. In addition, through transmission electron microscopy (TEM), immunofluorescence, and Western blot (WB) detection, TZQD significantly improved the organelle damage of hippocampal neurons in diabetes mice, promoted the formation of autophagy lysosomes, increased the expression of autophagy-related proteins like Beclin 1, LC3II/LC3I, LAMP1, and LAMP2, reduced the level of P62 and promoted autophagy flow, which, however, were all significantly weakened by 3 MA. Meanwhile, TZQD regulated the expressions of AMPK/mTOR pathway proteins. In vitro experimental study results showed that TZQD can regulate the expression ratio of p-AMPK/AMPK alpha 1 and p-mTOR/mTOR in HT22 cells under high glucose conditions and improved the morphology and vitality of HT22 cells. By employing techniques such as monodansylcadaverine (MDC) staining, Lysosomal red fluorescent probe staining, and Annexin V-FITC/PI double staining, the investigation revealed that TZQD administration resulted in enhanced autophagosome formation, preservation of a lysosomal acidic milieu, and consequent mitigation of HT22 cell apoptosis under high glucose conditions. ConclusionsTZQD can regulate the AMPK/mTOR pathway to activate autophagy to attenuate hippocampal neuronal apoptosis, thereby protecting cognitive function in diabetic mice.

Investigating the synaptic mechanisms of the spread of wild type and P301S 1N4R human tau in in vitro and in vivo models

AbstractBackgroundTau aggregates propagate across functionally connected neuronal networks in tauopathies as Alzheimer’s disease. However, the mechanisms underlying this process are poorly understood. Different studies showed that tau release is dependent on neuronal activity and that pathological tau can be found in the extracellular space confined in extracellular vesicles or in a free form. We recently showed that metabotropic glutamate receptors and SNAP25 modulate the release of pathologic tau from human and mouse synaptosomes. Here, with the use of blocking agents such as botulinum neurotoxins (BoNTs), we plan to test the effect on the synaptic release of tau in vitro and in vivo. BoNTs enter synapses and cleave specific synaptic SNARE proteins, impairing synaptic vesicle fusion and release. We aim at demonstrating the potential of BoNTs as tools to study neurodegenerative diseases.MethodsPrimary mouse hippocampal neurons were cultured in microfluidic chambers, transduced with lentiviruses expressing human tau (hTau) isoforms, and treated with BoNTs and stimulators. ELISA was used to quantify hTau in the culture media. AAVs expressing hTau were injected in the vitreous of wild‐type mice, and BoNT type A (BoNT/A) was injected in the superior colliculus (SC). Retina, SC and lateral geniculate nucleus (LGN) were analysed through immunohistochemistry.ResultsOverexpression of hTau in primary neurons increases the expression of synaptic markers as compared to non‐transduced cells, while any difference is detected between wild‐type and P301S hTau. Neuronal stimulation significantly increases the release of P301S hTau, whereas BoNT/A blocks it. Preliminary in vivo data show no difference in the expression and transfer of wild type and P301S hTau in the SC and LGN. The ongoing analysis of BoNT/A injected mice will reveal any effect on tau spread in the brain.ConclusionsThe release of hTau is modulated by specific SNARE proteins and differs depending on the isoform in vitro. The use of BoNT/A in vivo will help us to clarify the mechanisms modulating tau release in a complex system. Moreover, with the use of other BoNTs, we will provide novel insights into the mechanisms of synaptic tau release and identify novel molecular targets for the development of therapeutic interventions to treat tauopathies.

Dissection of alpha‐synuclein specific synaptic functions by gene editing

AbstractBackgroundDespite almost two decades of research, the physiologic role of α‐synuclein remains unclear. Mice lacking α‐syn only show mild phenotypes, and most mechanistic studies have been done either in α‐syn over‐expressing systems, or in mice lacking all three synuclein genes (α/β/γ). However, over‐expression of proteins can induce unwanted phenotypes, and ‘triple‐knockout’ synuclein mice have structural and physiologic changes that are not specific to α‐syn – complicating interpretations.MethodsWe used CRISPR technology to systematically inactivate or activate each synuclein gene in cultured mouse hippocampal neurons, ensuring that our manipulations did not lead to compensatory changes in the non‐targeted synucleins. Gene‐editing was followed by unbiased evaluation of physiology and ultrastructure using optical synaptic vesicle (SV) recycling assays, electrophysiologic recordings, single‐molecule trafficking assays, super‐resolution imaging, and electron microscopy.ResultsNeurotransmission is maintained at a range of different activity patterns, motivating us to evaluate the role of α‐syn under basal states as well as conditions involving trains of action potentials. Elimination of α‐syn led to substantial increases in basal exocytosis. Single‐molecule trafficking of SVs in these neurons indicated an increase in mobility of reserve pool vesicles when α‐syn was absent. Increasing endogenous α‐syn levels by CRISPRa had largely reciprocal effects. Recent studies in our lab found that phosphorylation of α‐syn at the Ser129‐site is a trigger for α‐syn function at the synapse. Augmenting α‐syn functionality by constitutive phosphorylation of the α‐syn Ser129‐site also triggered clustering of distal SV‐pools, consistent with a role for α‐syn in physiologic clustering of reserve pool vesicles. However, when neuronal activity was augmented by trains of action potentials, loss of α‐syn selectively attenuated endocytosis, with no effect on exocytosis. Super‐resolution imaging revealed that α‐syn was translocated to peri‐active zones upon stimulation, suggesting that the altered localization of α‐syn under these conditions resulted in facilitation of endocytosis. Finally, mass‐spectrometry data also indicate that α‐syn binds to an endocytosis‐enriched proteome under conditions that favor neuronal activity.ConclusionOur data provide clarity of the repertoire of a‐syn specific functions under different physiologic conditions.

Dynamic imaging and targeted degradation of the RNA‐binding protein hnRNP A2B1 using nanobodies

AbstractBackgroundRNA‐binding proteins (RBP) are a primary component of stress granules, a key indicator of various cell stress responses. Recently, the heterogeneous nuclear ribonucleoprotein hnRNP A2B1 emerged as a primary protein that preferentially interacts with tau oligomers than tau monomers. Here we studied the dynamics of hnRNP A2B1 in cultured cells and primary neurons using a novel nanobody reagent that enables live‐cell imaging.MethodWe screened a recombinant nanobody library to identify nanobodies (Nbs) that bind to hnRNP A2B1. The Nbs were designed to bind to all isoforms of hnRNP A2B1. We applied protein engineering approaches to improve the binding affinity of the identified Nbs. Live cell imaging was conducted in cultured cell lines and primary mouse hippocampal neurons. We also generated Nb‐E3 ligase fusions for targeted degradation of hnRNP A2B1.ResultThe Nbs revealed an unexpected localization of hnRNP A2B1 previously not observed using GFP fusions. We observed bright puncta throughout the cytoplasm in addition to typical nucleoplasmic distribution of hnRNP A2B1. The hnRNP A2B1 puncta were co‐localized with exosomal markers. In cell lines, the nucleoplasmic hnRNP A2B1 was dramatically reduced during mitosis, while the cytoplasmic puncta did not change, suggesting cell cycle‐dependent nucleocytoplasmic translocation. The Nb‐E3 ligase fusion constructs caused a significant reduction in hnRNP A2B1, lowering the level by 60%.ConclusionThese novel tools will greatly aid the study of endogenous hnRNP A2B1 dynamics and function. Isoform‐dependent localization of hnRNP A2B1 and whether it is involved in protein loading to exosomes remains to be studied.

Muscone inhibits opening of mPTP to alleviate OGD/R-induced injury of HT22 cells

This study aims to investigate the mechanism of muscone in inhibiting the opening of mitochondrial permeability transition pore(mPTP) to alleviate the oxygen and glucose deprivation/reoxygenation(OGD/R)-induced injury of mouse hippocampal neurons(HT22). An in vitro model of HT22 cells injured by OGD/R was established. CCK-8 assay was employed to examine the viability of HT22 cells, fluorescence microscopy to measure the mitochondrial membrane potential, the content of reactive oxygen species(ROS), and the opening of mPTP in HT22 cells. Enzyme-linked immunosorbent assay was employed to determine the level of ATP and the content of cytochrome C(Cyt C) in mitochondria of HT22 cells. Flow cytometry was employed to determine the Ca~(2+) content and apoptosis of HT22 cells. The expression of Bcl-2(B-cell lymphoma-2) and Bcl-2-associated X protein(Bax) was measured by Western blot. Molecular docking and Western blot were employed to examine the binding between muscone and methyl ethyl ketone(MEK) after pronase hydrolysis of HT22 cell proteins. After the HT22 cells were treated with U0126, an inhibitor of MEK, the expression levels of MEK, p-ERK, and CypD were measured by Western blot. The results showed that compared with the OGD/R model group, muscone significantly increased the viability, mitochondrial ATP activity, and mitochondrial membrane potential, lowered the levels of ROS, Cyt C, and Ca~(2+), and reduced mPTP opening to inhibit the apoptosis of HT22 cells. In addition, muscone up-regulated the expression of MEK, p-ERK, and down-regulated that of CypD. Molecular docking showed strong binding activity between muscone and MEK. In conclusion, muscone inhibits the opening of mPTP to inhibit apoptosis, thus exerting a protective effect on OGD/R-injured HT22 cells, which is associated with the activation of MEK/ERK/CypD signaling pathway.

NTRK1 knockdown induces mouse cognitive impairment and hippocampal neuronal damage through mitophagy suppression via inactivating the AMPK/ULK1/FUNDC1 pathway

Hippocampal neuronal damage may induce cognitive impairment. Neurotrophic tyrosine kinase receptor 1 (NTRK1) reportedly regulates neuronal damage, although the underlying mechanism remains unclear. The present study aimed to investigate the role of NTRK1 in mouse hippocampal neuronal damage and the specific mechanism. A mouse NTRK1-knockdown model was established and subjected to pre-treatment with BAY-3827, followed by a behavioral test, Nissl staining, and NeuN immunofluorescence (IF) staining to evaluate the cognitive impairment and hippocampal neuronal damage. Next, an in vitro analysis was conducted using the CCK-8 assay, TUNEL assay, NeuN IF staining, DCFH-DA staining, JC-1 staining, ATP content test, mRFP-eGFP-LC3 assay, and LC3-II IF staining to elucidate the effect of NTRK1 on mouse hippocampal neuronal activity, apoptosis, damage, mitochondrial function, and autophagy. Subsequently, rescue experiments were performed by subjecting the NTRK1-knockdown neurons to pre-treatment with O304 and Rapamycin. The AMPK/ULK1/FUNDC1 pathway activity and mitophagy were detected using western blotting (WB) analysis. Resultantly, in vivo analysis revealed that NTRK1 knockdown induced mouse cognitive impairment and hippocampal tissue damage, in addition to inactivating the AMPK/ULK1/FUNDC1 pathway activity and mitophagy in the hippocampal tissues of mice. The treatment with BAY-3827 exacerbated the mouse depressive-like behavior induced by NTRK1 knockdown. The results of in vitro analysis indicated that NTRK1 knockdown attenuated viability, NeuN expression, ATP production, mitochondrial membrane potential, and mitophagy, while enhancing apoptosis and ROS production in mouse hippocampal neurons. Conversely, pre-treatment with O304 and rapamycin abrogated the suppression of mitophagy and the promotion of neuronal damage induced upon NTRK1 silencing. Conclusively, NTRK1 knockdown induces mouse hippocampal neuronal damage through the suppression of mitophagy via inactivating the AMPK/ULK1/FUNDC1 pathway. This finding would provide insight leading to the development of novel strategies for the treatment of cognitive impairment induced due to hippocampal neuronal damage.

Differential tyrosine and serine/threonine phosphorylation/dephosphorylation pathways regulate the expression of α7 versus α3β4 nicotinic receptor subtypes in mouse hippocampal neurons

We have recently reported that α7 and α3β4 nicotinic acetylcholine receptor (nAChR) subtypes are expressed in human chromaffin cells in the plasma membrane where they colocalize and physically interact. The present study was designed to evaluate whether those receptor subtypes also colocalize at the central nervous system to mutually interact, and whether their expression and colocalization are regulated by phosphorylation/dephosphorylation processes, as they are in human chromaffin cells. We have here found that in isolated and maintained in culture mouse hippocampal neurons, nAChR expression and colocalization of α7, but not α3β4, nAChR subtypes decreased by tyrosine (Tyr)- and serine/threonine (Ser/Thr)-phosphatase inhibition. However, Tyr-kinase inhibition or protein-phosphatase 2A (PP2A) activation increased α3β4 nAChR expression, diminishing receptor subtypes colocalization. Furthermore, colocalization is not recovered if the inhibitors of Tyr-phosphatase and kinases, or the inhibitor of Ser/Thr-phosphatases and the activator of PP2A are applied together. Therefore, regulation of α7 and α3β4 nAChR subtypes expression by Tyr- and Ser/Thr kinases and phosphatases exhibit differential mechanisms in mouse hippocampal neurons. Colocalization of nAChR subtypes, however, is altered by any maneuver that affects these kinases or phosphatases, which might have consequences in the functional activity of nAChR subtypes.

Secretagogin regulates asynchronous and spontaneous glutamate release in hippocampal neurons through interaction with Doc2α

Abstract Synaptic vesicle (SV) exocytosis is orchestrated by protein machineries consisting of the SNARE complex, Ca2+ sensors, and their partners. Secretagogin (SCGN) is a Ca2+-binding protein involved in multiple forms of vesicle secretion. Although SCGN is implicated in multiple neurological disorders, its role in SV exocytosis in neurons remains unknown. Here, using knockout and knockdown techniques, we report that SCGN could regulate the asynchronous and spontaneous forms of excitatory but not inhibitory SV exocytosis in mouse hippocampal neurons. Furthermore, SCGN functioned in glutamate release via directly interacting with Doc2α, a high-affinity Ca2+ sensor specific for asynchronous and spontaneous SV exocytosis. Conversely, the interaction with SCGN is also required for Doc2α to execute its Ca2+ sensor function in SV release. Together, our study revealed that SCGN plays an important role in asynchronous and spontaneous glutamate release through its interaction with Doc2α.

Theiler's Murine Encephalomyelitis Virus Replicates in Primary Neuron Cultures and Impairs Spine Density Formation.

In this study, we examined infection with the highly neurovirulent GDVII, the less neurovirulent DA strains, and with a mutant DA, which lacks the L* protein (L*-1) involved in viral persistence and demyelinating disease, to analyze the direct effects of Theiler's murine encephalomyelitis virus (TMEV) replication using primary cultures of mouse brain hippocampal neurons. All viruses replicate in cultured neurons, with GDVII having the highest titers and L*-1 the lowest. Accordingly, all were positive for viral antigen staining 3 days postinfection (dpi), and DA and L*-1 were also positive after 12 dpi. NeuN+immunostaining showed an early and almost complete absence of positive cells in cultures infected with GDVII, an approximately 50% reduction in cultures infected with DA, and fewer changes in L*-1 strains at 3 dpi. Accordingly, staining with chloromethyltetramethylrosamine orange (Mitotracker OrangeTM) as a parameter for cell viability showed similar results. Moreover, at 1 dpi, the strain DA induced higher transcript levels of neuroprotective genes such as IFN-Iβ, IRF7, and IRF8. At 3 dpi, strains GDVII and DA, but not the L*-1 mutant, showed lower PKR expression. In addition, confocal analysis showed that L*-1-infected neurons exhibited a decrease in spine density. Treatment with poly (I:C), which is structurally related to dsRNA and is known to trigger IFN type I synthesis, reduced spine density even more. These results confirmed the use of mouse hippocampal neuron cultures as a model to study neuronal responses after TMEV infection, particularly in the formation of spine density.

Aerobic exercise improves cognitive impairment in mice with type 2 diabetes by regulating the MALAT1/miR-382-3p/BDNF signaling pathway in serum-exosomes

BackgroundIt has been documented that aerobic exercise (AE) has a positive effect on improving cognitive function in type 2 diabetes (T2DM) patients. Here, we tried to explore how AE regulates the expression of long non-coding RNA in serum-exosomes (Exos), thereby affecting cognitive impairment in T2DM mice as well as its potential molecular mechanism.MethodsT2DM mouse models were constructed, and serum-Exos were isolated for whole transcriptome sequencing to screen differentially expressed lncRNA and mRNA, followed by prediction of downstream target genes. The binding ability of miR-382-3p with a long non-coding RNA MALAT1 and brain-derived neurotrophic factor (BDNF) was explored. Then, primary mouse hippocampal neurons were collected for in vitro mechanism verification, as evidenced by the detection of hippocampal neurons' vitality, proliferation, and apoptosis capabilities, and insulin resistance. Finally, in vivo mechanism verification was performed to assess the effect of AE on insulin resistance and cognitive disorder.ResultsTranscriptome sequencing analysis showed that MALAT1 was lowly expressed and miR-382-3p was highly expressed in serum-Exos samples of T2DM mice. There were targeted binding sites between MALAT1 and miR-382-3p and between miR-382-3p and BDNF. In vitro experiments showed that MALAT1 upregulated BDNF expression by inhibiting miR-382-3p. Silencing MALAT1 or overexpressing miR-382-3p could reduce the expression of INSR, IRS-1, IRS-2, PI3K/AKT, and Ras/MAPK, inhibit neuronal proliferation, and promote apoptosis. In vivo experiments further confirmed that AE could increase the expression of MALAT1 in serum-Exos to competitively inhibit miR-382-3p and upregulate BDNF expression, thereby improving cognitive impairment in T2DM mice.ConclusionAE may upregulate the expression of MALAT1 in serum-Exos to competitively inhibit miR-382-3p and upregulate BDNF expression, thus improving cognitive impairment in T2DM mice.

VEGF-A ameliorates ischemia hippocampal neural injury via regulating autophagy and Akt/CREB signaling in a rat model of chronic cerebral hypoperfusion

ObjectiveChronic cerebral hypoperfusion (CCH) can cause a series of pathophysiological processes, including neuronal autophagy and apoptosis. VEGF-A has been reported to affect angiogenesis and neurogenesis in many CNS diseases. However, its effects on neuronal autophagy and apoptosis, as well as the underlying mechanisms in CCH remain unclear. MethodsTo address these issues, the CCH model was established by permanent bilateral common carotid artery occlusion (2VO). Rats were sacrificed at different stages of CCH. Hippocampal morphological and ultrastructural changes were detected using HE staining and electron microscopy. The immunoreactivities of microtubule-associated protein 1 light chain 3 (LC3) and phospho-cAMP response element binding protein (p-CREB) were examined by immunofluorescence staining. The neuronal apoptosis was detected via TUNEL staining. The levels of LC3-II, Beclin-1, Akt, p-Akt, CREB, p-CREB, Caspase-3, and Bad were accessed by Western blotting. Furthermore, mouse hippocampal HT22 neurons received the oxygen and glucose deprivation (OGD) treatment, VEGF-A treatment, and GSK690693 (an Akt inhibitor) treatment, respectively. ResultsLC3-II protein started to increase at 3 days of CCH, peaked at 4 weeks of CCH, then decreased. CCH increased the levels of LC3-II, Caspase-3, and Bad, and decreased the levels of p-Akt, CREB, and p-CREB, which were reversed by VEGF-A treatment. VEGF-A also improved CCH-induced neuronal ultrastructural injuries and apoptosis in the hippocampus in vitro. In HT22, the anti-apoptosis and pro-phosphorylation of VEGF-A were reversed by GSK690693. ConclusionPresent results provide a novel neuroprotective effect of VEGF-A in CCH that is related to the inhibition of neuronal autophagy and activation of the Akt/CREB signaling, suggesting a potential therapeutic strategy for ischemic brain damage.

Adaptor protein AP-3 produces synaptic vesicles that release at high frequency by recruiting phospholipid flippase ATP8A1.

Neural systems encode information in the frequency of action potentials, which is then decoded by synaptic transmission. However, the rapid, synchronous release of neurotransmitters depletes synaptic vesicles (SVs), limiting release at high firing rates. How then do synapses convey information about frequency? Here, we show in mouse hippocampal neurons and slices that the adaptor protein AP-3 makes a subset of SVs that respond specifically to high-frequency stimulation. Neurotransmitter transporters slot onto these SVs in different proportions, contributing to the distinct properties of release observed at different excitatory synapses. Proteomics reveals that AP-3 targets the phospholipid flippase ATP8A1 to SVs; loss of ATP8A1 recapitulates the defect in SV mobilization at high frequency observed with loss of AP-3. The mechanism involves recruitment of synapsin by the cytoplasmically oriented phosphatidylserine translocated by ATP8A1. Thus, ATP8A1 enables the subset of SVs made by AP-3 to release at high frequency.