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Background: Alzheimer’s disease (AD) is a degenerative disorder, accompanied by progressive cognitive decline, for which there is no cure. Recently, the close correlation between AD and type 2 diabetes mellitus (T2DM) has been noted, and a promising anti-AD strategy is the use of anti-T2DM drugs. Objective: To investigate if the novel glucagon-like peptide-1 (GLP-1)/glucose-dependent insulinotropic polypeptide (GIP) receptor agonist DA4-JC shows protective effects in the triple APP/PS1/tau mouse model of AD. Methods: A battery of behavioral tests were followed by in vivo recording of long-term potentiation (LTP) in the hippocampus, quantified synapses using the Golgi method, and biochemical analysis of biomarkers. Results: DA4-JC improved cognitive impairment in a range of tests and relieved pathological features of APP/PS1/tau mice, enhanced LTP in the hippocampus, increased numbers of synapses and dendritic spines, upregulating levels of post-synaptic density protein 95 (PSD95) and synaptophysin (SYP), normalized volume and numbers of mitochondria and improving the phosphatase and tensin homologue induced putative kinase 1 (PINK1) - Parkin mitophagy signaling pathway, while downregulating amyloid, p-tau, and autophagy marker P62 levels. Conclusion: DA4-JC is a promising drug for the treatment of AD.

Alcohol hangover is defined as the combination of mental and physical symptoms experienced the day after a single episode of heavy drinking, starting when blood alcohol concentration approaches zero. We previously evidenced increments in free radical generation and an imbalance in antioxidant defences in non-synaptic mitochondria and synaptosomes during hangover. It is widely known that acute alcohol exposure induces changes in nitric oxide (NO) production and blocks the binding of glutamate to NMDAR in central nervous system. Our aim was to evaluate the residual effect of acute ethanol exposure (hangover) on NO metabolism and the role of NMDA receptor-PSD95-nNOS pathway in non-synaptic mitochondria and synaptosomes from mouse brain cortex. Results obtained for the synaptosomes fraction showed a 37% decrease in NO total content, a 36% decrease in NOS activity and a 19% decrease in nNOS protein expression. The in vitro addition of glutamate to synaptosomes produced a concentration-dependent enhancement of NO production which was significantly lower in samples from hangover mice than in controls for all the glutamate concentrations tested. A similar patter of response was observed for nNOS activity being decreased both in basal conditions and after glutamate addition. In addition, synaptosomes exhibited a 64% and 15% reduction in NMDA receptor subunit GluN2B and PSD-95 protein expression, respectively. Together with this, glutamate-induced calcium entry was significant decreased in synaptosomes from alcohol-treated mice. On the other hand, in non-synaptic mitochondria, no significant differences were observed in NO content, NOS activity or nNOS protein expression. The expression of iNOS remained unaltered in synaptosomes and non-synaptic mitochondria. Here we demonstrated that hangover effects on NO metabolism are strongly evidenced in synaptosomes probably due to a disruption in NMDAR/PSD-95/nNOS pathway.

Previous midlife estradiol treatment, like continuous treatment, improves memory and results in lasting increases in hippocampal levels of estrogen receptor (ER) α and ER-dependent transcription in ovariectomized rodents. We hypothesized that previous and continuous midlife estradiol act to specifically increase levels of nuclear ERα, resulting in transcriptional regulation of proteins that mediate estrogen effects on memory. Ovariectomized middle-aged rats received estradiol or vehicle capsule implants. After 40 days, rats initially receiving vehicle received another vehicle capsule (Vehicle). Rats initially receiving estradiol received either another estradiol (Continuous Estradiol) or a vehicle (Previous Estradiol) capsule. One month later, hippocampal genes and proteins were analyzed. Continuous and previous estradiol increased levels of nuclear, but not membrane or cytosolic ERα and had no effect on Esr1. Continuous and previous estradiol impacted gene expression and/or protein levels of mediators of estrogenic action on memory including ChAT, BDNF, and PSD-95. Findings demonstrate a long-lasting role for hippocampal ERα as a transcriptional regulator of memory following termination of previous estradiol treatment in a rat model of menopause.

Bright light has been reported to improve spatial memory of diurnal rodents, yet how it will influence the spatial memory of nocturnal rodents is unknown. Here, we found that dynamic changes in spatial memory and anxiety were induced at different time point after bright light treatment. Mice maintained in brighter light exhibited impaired memory in Y maze at one day after bright light exposure, but showed significantly improved spatial memory in the Y maze and Morris water maze at four weeks after bright light exposure. We also found increased anxiety one day after bright light exposure, which could be the reason of impaired memory. However, no change of anxiety was detected after four weeks. Thus, we further explore the underlying mechanism of the beneficial effects of long term bright light on spatial memory. Golgi staining indicated that the structure of dendritic spines changed, accompanied by increased expression of synaptophysin and postsynaptic density 95 in the hippocampus. Further research has found that bright light treatment leads to elevated CaMKII/CREB phosphorylation levels in the hippocampus, which are associated with synaptic function. Moreover, higher expression of brain-derived neurotrophic factor (BDNF) was followed by increased phosphorylated TrkB levels in the hippocampus, indicating that BDNF/TrkB signaling is also activated during this process. Taken together, these findings revealed that bright light exposure with different duration exert different effects on spatial memory in nocturnal rodents, and the potential molecular mechanism by which long term bright light regulates spatial memory was also demonstrated.

Pituitary adenylate cyclase-activating polypeptide (PACAP) has beneficial effects in learning and memory. However, the mechanism by which PACAP improves cognitive impairment of vascular dementia (VaD) is not clear. We established a VaD model by bilateral common carotid stenosis (BCAS) to investigate the molecular mechanism of cognitive impairment. Protein levels of PACAP, Sirtuin 3 (Sirt3), brain-derived neurotrophic factor (BDNF), and postsynaptic density 95 (PSD-95) were assessed by Western blot. In vitro, oxygen glucose deprivation (OGD) was used to simulate the ischemia/hypoxia state. HT22 cells were transfected with Sirt3 knockdown and overexpression to study the relationship between PACAP, Sirt3, and BDNF. In vivo, PACAP was administered intranasally to assess its protective effects on BCAS. The study showed that the levels of PACAP, Sirt3, BDNF, and PSD-95 were decreased in the BCAS model of VaD. PACAP increased the protein levels of Sirt3, BDNF, PSD-95, Bcl-2, and Bax under OGD condition in vitro. Sirt3 regulated BDNF and synaptic plasticity. Intranasal PACAP increased the protein levels of PAC1, Sirt3, BDNF, and PSD-95 in vivo. This study provides evidence that PACAP regulates synaptic plasticity and plays an antiapoptotic role through Sirt3.

Neuropathic pain is a very complex chronic pain state, the detailed molecular mechanisms of which remain unclear. In the present study, Shank3 was found to play an important role in neuropathic pain in rats following spared nerve injury (SNI). Shank3 was upregulated in the spinal dorsal horn of rats subjected to SNI, and mechanical hypersensitivity to noxious stimuli in these rats could be alleviated by knock down of Shank3. Shank3 also interacted with hyperpolarization-activated cyclic nucleotide-gated channel 2 (HCN2) and promoted the expression of HCN2 in central neurons of the spinal dorsal. Together with the SNI-dependent increase of HCN2, we also found that the postsynaptic protein of excitatory synapse (PSD95) was increased in rats following SNI. Taken together, our results showed that Shank3 modulated neuropathic pain by facilitating the SNI-dependent increase of HCN2 and the expression of PSD95 in spinal dorsal horn neurons. Our findings revealed new synaptic remodeling mechanisms linking Shank3 with neuropathic pain.

Anesthesia of neonates with propofol induces persistent behavioral abnormalities in adulthood. Although propofol-triggered apoptosis of neurons in the developing brain may contribute to the development of cognitive deficits, the mechanism of neurotoxicity induced by neonatal exposure to propofol remains unclear. In this study, the effects of neonatal propofol anesthesia on synaptic plasticity and neurocognitive function were investigated. Postnatal day 7 (PND-7) Sprague-Dawley rats were intraperitoneally injected with fat emulsion or 20, 40 or 60 mg/kg propofol for three consecutive days. The expression of brain-derived neurotrophic factor (BDNF), tropomyosin-related kinase B (TrkB) and postsynaptic density protein 95 (PSD-95) in the rat hippocampus at PND-10 and PND-12 was measured by Western blotting. The number of dendritic branches, total dendritic length and dendritic spine density were observed by Golgi-Cox staining 24 h and 72 h after the last propofol administration. Long-term potentiation (LTP) was measured electrophysiologically in hippocampus of PND-60 rats to evaluate the synaptic function. The learning and memory abilities of rats were evaluated by Morris water maze (MWM) experiments, Novel object recognition test (NORT) and Object location test (OLT) at PND-60. Our results showed that neonatal exposure to propofol significantly inhibited the expression of BDNF, TrkB and PSD-95 in the rat hippocampus. The number of dendritic branches, total dendritic length and dendritic spine density of neurons in the rat hippocampus were markedly reduced after neonatal propofol anesthesia. LTP was significantly diminished in hippocampus of PND-60 rats after repeated exposure to propofol in the neonatal period. Morris water maze experiments showed that repeated neonatal exposure to propofol significantly prolonged the escape latency and decreased the time spent in the target quadrant and the number of platform crossings. NORT and OLT showed that repeated neonatal exposure to propofol markedly reduced the Investigation Time for novel object or location. All of the results above indicate that repeated exposure to propofol in the neonatal period can impair hippocampal synaptic plasticity and the recognition function of rats in adulthood.

The health effects of prolonged exposure to embedded metal fragments, such as those found in shrapnel wounds sustained by an increasing number of military personnel, are not well known. As part of a large collaborative effort to expand this knowledge, we use an animal model of shrapnel wounds originally developed to investigate effects of embedded depleted uranium to investigate effects of military-relevant metals tungsten, nickel, cobalt, iron, copper, aluminum, lead, and depleted uranium compared to an inert control, tantalum. Rats are surgically implanted with pellets of one of the metals of interest in the gastrocnemius (leg) muscle and tracked until 1 month, 3 months, 6 months, or 12 months from the time of implant, at which point they are euthanized and multiple organs and tissue samples are collected for inspection. Here we focus on four regions of the brain: frontal cortex, hippocampus, amygdala, and cerebellum. We examined changes in accumulated metal concentration in each region as well as changes in expression of proteins related to blood brain barrier tight junction formation, occludin and ZO-1, and synapse function, PSD95, spinophilin, and synaptotagmin. We report few changes in metal accumulation or blood brain barrier protein expression, but a large number of synapse proteins have reduced expression levels, particularly within the first 6 months of exposure, but there are regional and metal-specific differences in effects.

We recently demonstrated that NMDA receptors (NMDARs) are capable of ion-flux independent signaling through conformational change in the NMDAR intracellular domain resulting in long-term depression of synaptic transmission (LTD). Here we show that PSD-95 overexpression blocks agonist induced conformational movement in the NMDAR intracellular domain as well as LTD that is NMDAR-dependent and ion-flux independent. Interestingly, previous studies indicate that overexpressed PSD-95 does not block NMDAR-dependent LTD. These data support a model where ion-flux independent LTD is predominant in young animals, which have synapses with low amounts of PSD-95, whereas only ion flux dependent LTD occurs at more mature synapses, which have more PSD-95 that would block ion-flux independent LTD. These results may reconcile different findings regarding ion-flux independent LTD.

S-oxiracetam (S-ORC), a nootropic drug, was used to protect against ischemic stroke by lessening the blood brain barrier dysfunction and inhibiting neuronal apoptosis. However, the potential effects of S-ORC in the recovery of cognitive functions after ischemic stroke and the underlying mechanisms remains unclear. In this study, middle cerebral artery occlusion/reperfusion (MCAO/R) in rats was used as the animal model. By using Y-maze test, Morris water maze, triphenyl tetrazolium chloride (TTC) staining, terminal deoxynucleotidyl transferase-mediated 2'-deoxyuridine 5'-triphosphate (dUTp) nick end labeling (TUNEL) assay, hematoxylin and eosin, immunohistochemical staining and western blot to evaluate the protective effect of S-ORC on cognitive recovery, we were able to confirm that S-ORC ameliorated spatial learning impairment, tissue loss, and hippocampal neuronal apoptosis and injury induced by MCAO/R in rats. These cognitive effects were achieved by restoring the normal function of synaptophysin and increasing PSD95 expression in the hippocampus. Furthermore, we found that methyllycaconitine, the antagonist of α7 nicotinic acetylcholine receptor (α7nAChR), and LY294002, the inhibitor of phosphoinositide 3-kinase (PI3K), were able to block the cognitive effects of S-ORC after MCAO/R in rats. In conclusion, α7nAChR and PI3K are key molecules that mediated the signaling pathway leading to S-ORC-induced cognitive restoration after MCAO/R.

Background: Local protein synthesis and mRNA metabolism mediated by mRNP granules in the dendrites and the postsynaptic compartment is essential for synaptic remodeling and plasticity in neuronal cells. Dysregulation of these processes caused by TDP-43 proteinopathy leads to neurodegenerative diseases, such as frontotemporal lobar degeneration and amyotrophic lateral sclerosis. Methods: Using biochemical analysis and imaging techniques, including super-resolution microscopy, we provide evidence, for the first time, for the postsynaptic localization of TDP-43 in mammalian synapses and we show that TDP-43 is a component of neuronal mRNP granules. Results: With activity stimulation and various molecular approaches, we further demonstrate activity-dependent mRNP granule dynamics involving disassembly of mRNP granules, release of mRNAs, activation of local protein translation, and the impairment of granule disassembly in cellular, animal and human models of TDP-43 proteinopathy. Conclusion: Our study elucidates the interplay between TDP-43 and neuronal mRNP granules in normal physiology and TDP-43 proteinopathy in the regulation of local protein translation and mRNA metabolism in the postsynaptic compartment.

Adolescent alcohol drinking is widely recognized as a significant public health problem, and evidence is accumulating that sufficient levels of consumption during this critical period of brain development have an enduring impact on neural and behavioral function. Recent studies have indicated that adolescent intermittent ethanol (AIE) exposure alters astrocyte function, astrocyte-neuronal interactions, and related synaptic regulation and activity. However, few of those studies have included female animals, and a broader assessment of AIE effects on the proteins mediating astrocyte-mediated glutamate dynamics and synaptic function is needed. We measured synaptic membrane expression of several such proteins in the dorsal and ventral regions of the hippocampal formation (DH, VH) from male and female rats exposed to AIE or adolescent intermittent water. In the DH, AIE caused elevated expression of glutamate transporter 1 (GLT-1) in both males and females, elevated postsynaptic density 95 expression in females only, and diminished NMDA receptor subunit 2A expression in males only. AIE and sex interactively altered ephrin receptor A4 (EphA4) expression in the DH. In the VH, AIE elevated expression of the cystine/glutamate antiporter and the glutamate aspartate transporter 1 (GLAST) in males only. Compared to males, female animals expressed lower levels of GLT-1 in the DH and greater levels of ephrin receptor B6 (EphB6) in the VH, in the absence of AIE effects. These results support the growing literature indicating that adolescent alcohol exposure produces long-lasting effects on astrocyte function and astrocyte-neuronal interactions. The sex and subregion specificity of these effects have mechanistic implications for our understanding of AIE effects generally.

The effects of X-irradiation on developing neurons and their functions are unclear. We used primary cultures of mouse hippocampal neurons to investigate the effects of X-irradiation on cell death in developing neurons by analyzing caspase-3, MAP2 and DAPI-labeled cells, and the phenotypes and function of surviving neurons, by examining GAD67-positive cells as a GABAergic marker, and the synaptic markers synapsin 1, drebrin and PSD-95 through its maturation. One-day in vitro (DIV 1) cells were exposed to 0.5 Gy or 1 Gy of X-rays. A significant increase in the percentage of activated caspase-3, a decrease in the number of MAP2/DAPI-positive cells and change in the percentage of GAD67 positive neurons, compared with sham controls, were found 6 days after 1 Gy and 13 days after 0.5 Gy of X-rays. The expression of PSD-95 and drebrin, as well as drebrin clusters, in the remaining neurons was decreased at DIV 21, in both 0.5 Gy and on 1 Gy-irradiation there was a reduced number of dendritic intersection as well. Together, our findings show that 0.5 Gy and 1 Gy of X-irradiation at DIV 1 not only causes neuronal cell death but elicits an increase in the percentage of inhibitory neurons, changes in the dendrites and decrease in expression of important synaptic proteins in the surviving neurons at maturity 3 weeks after exposure.

The abnormal function of the voltage-gated potassium channel Kv10.2 can induce epilepsy. However, the physiological function of Kv10.2 in the central nervous system remains unclear. In this study, we found that Kv10.2 knockout (KO) increased the complexity of neurons in the CA3 subarea of hippocampus. Kv10.2 KO led to enlarged somata, elongated dendritic length, and increased the number of dendritic tips in cultured rat hippocampus neurons. Kv10.2 KO also increased Synapsin I and PSD95 protein density in cultured rat hippocampal neurons. Whole cell patch-clamp recordings of brain slices in the CA3 subarea of hippocampus revealed that Kv10.2 KO increased the amplitude of spontaneous excitatory postsynaptic currents (sEPSC) and miniature excitatory postsynaptic currents (mEPSC), depolarized the resting membrane potential and increased the action potential firing, reduced the rheobase and increased the input resistance, which results in enhanced neuronal excitability. Furthermore, we made electroencephalogram (EEG) recordings of brain activity in freely moving rats before and after inducing seizures by pentylenetetrazole (PTZ) injection. Kv10.2 KO rats dramatically increased the EEG amplitude during epilepsy. Behavioral observation after seizure induction revealed that Kv10.2 KO rats demonstrated shortened onset latency, prolonged duration, and increased seizure severity when compared with wild type rats. Therefore, this study provides a new link between Kv10.2 and neuronal morphology and higher intrinsic excitability.

Cerebrovascular abnormalities have emerged as a preclinical manifestation of Alzheimer's disease and frontotemporal dementia, diseases characterized by the accumulation of hyperphosphorylated forms of the microtubule-associated protein tau. However, it is unclear whether tau contributes to these neurovascular alterations independent of neurodegeneration. We report that mice expressing mutated tau exhibit a selective suppression of neural activity-induced cerebral blood flow increases that precedes tau pathology and cognitive impairment. This dysfunction is attributable to a reduced vasodilatation of intracerebral arterioles and is reversible by reducing tau production. Mechanistically, the failure of neurovascular coupling involves a tau-induced dissociation of neuronal nitric oxide synthase (nNOS) from postsynaptic density 95 (PSD95) and a reduced production of the potent vasodilator nitric oxide during glutamatergic synaptic activity. These data identify glutamatergic signaling dysfunction and nitric oxide deficiency as yet-undescribed early manifestations of tau pathobiology, independent of neurodegeneration, and provide a mechanism for the neurovascular alterations observed in the preclinical stages of tauopathies.

α-Synuclein (α-syn)-induced neurotoxicity has been generally accepted as a key step in the pathogenesis of Parkinson’s disease (PD). Microtubule-associated protein tau, which is considered second only to α-syn, has been repeatedly linked with PD in association studies. However, the underlying interaction between these two PD-related proteins in vivo remains unclear. To investigate how the expression of tau affects α-syn-induced neurodegeneration in vivo, we generated triple transgenic mice that overexpressed α-syn A53T mutation in the midbrain dopaminergic neurons (mDANs) with different expression levels of tau. Here we found that tau had no significant effect on the A53T α-syn-mediated mDANs degeneration. However, tau knockout could modestly promote the formation of α-syn aggregates, accelerate severe and progressive degeneration of parvalbumin-positive (PV(+)) neurons in substantia nigra pars reticulata (SNR), accompanied with anxiety-like behavior in aged PD-related α-syn A53T mice. The mechanisms may be associated with A53T α-syn-mediated specifically successive impairment of N-methyl-D-aspartate receptor subunit 2B (NR2B), postsynaptic density-95 (PSD-95) and microtubule-associated protein 1A (MAP1A) in PV(+) neurons. Our study indicates that MAP1A may play a beneficial role in preserving the survival of PV(+) neurons, and that inhibition of the impairment of NR2B/PSD-95/MAP1A pathway, which may be a novel and preferential option to ameliorate α-syn-induced neurodegeneration.

Background Experimental evidence shows postnatal exposure to anesthesia negatively affects brain development. The PDZ2 domain, mediating protein–protein interactions of the postsynaptic density-95 protein, serves as a molecular target for several inhaled anesthetics. The authors hypothesized that early postnatal disruption of postsynaptic density-95 PDZ2 domain interactions has persistent effects on dendritic spines and cognitive function. Methods One-week-old mice were exposed to 1.5% isoflurane for 4 h or injected with 8 mg/kg active postsynaptic density-95 wild-type PDZ2 peptide along with their respective controls. A subset of these mice also received 4 mg/kg of the nitric oxide donor molsidomine. Hippocampal spine density, long-term potentiation, novel object recognition memory, and fear learning and memory were evaluated in mice. Results Exposure of 7-day-old mice to isoflurane or postsynaptic density-95 wild-type PDZ2 peptide relative to controls causes: (1) a long-term decrease in mushroom spines at 7 weeks (mean ± SD [spines per micrometer]): control (0.8 ± 0.2) versus isoflurane (0.4 ± 0.2), P < 0.0001, and PDZ2MUT (0.7 ± 0.2) versus PDZ2WT (0.4 ± 0.2), P < 0.001; (2) deficits in object recognition at 6 weeks (mean ± SD [recognition index]): naïve (70 ± 8) versus isoflurane (55 ± 14), P = 0.010, and control (65 ± 13) versus isoflurane (55 ± 14), P = 0.045, and PDZ2MUT (64 ±11) versus PDZ2WT (53 ± 18), P = 0.045; and (3) deficits in fear learning at 7 weeks and memory at 8 weeks (mean ± SD [% freezing duration]): Learning, control (69 ± 12) versus isoflurane (52 ± 13), P < 0.0001, and PDZ2MUT (65 ± 14) versus PDZ2WT (55 ± 14) P = 0.011, and Memory, control (80 ± 17) versus isoflurane (56 ± 23), P < 0.0001 and PDZ2MUT (73 ± 18) versus PDZ2WT (44 ± 19) P < 0.0001. Impairment in long-term potentiation has fully recovered here at 7 weeks (mean ± SD [% baseline]): control (140 ± 3) versus isoflurane (137 ± 8), P = 0.560, and PDZ2MUT (136 ± 17) versus PDZ2WT (128 ± 11), P = 0.512. The isoflurane induced decrease in mushroom spines was preventable by introduction of a nitric oxide donor. Conclusions Early disruption of PDZ2 domain-mediated protein–protein interactions mimics isoflurane in decreasing mushroom spine density and causing learning and memory deficits in mice. Prevention of the decrease in mushroom spine density with a nitric oxide donor supports a role for neuronal nitric oxide synthase pathway in mediating this cellular change associated with cognitive impairment. Editor’s Perspective What We Already Know about This Topic What This Article Tells Us That Is New

Mapping the molecular composition of individual excitatory synapses across the mouse brain reveals high synapse diversity with each brain region showing a distinct composition of synapse types. As a first step towards systematic mapping of synapse diversity across the human brain, we have labelled and imaged synapses expressing the excitatory synapse protein PSD95 in twenty human brain regions, including 13 neocortical, two subcortical, one hippocampal, one cerebellar and three brainstem regions, in four phenotypically normal individuals. We quantified the number, size and intensity of individual synaptic puncta and compared their regional distributions. We found that each region showed a distinct signature of synaptic puncta parameters. Comparison of brain regions showed that cortical and hippocampal structures are similar, and distinct from those of cerebellum and brainstem. Comparison of synapse parameters from human and mouse brain revealed conservation of parameters, hierarchical organization of brain regions and network architecture. This work illustrates the feasibility of generating a systematic single‐synapse resolution atlas of the human brain, a potentially significant resource in studies of brain health and disease.

BACKGROUND Anesthesia in pregnant rodents causes neurotoxicity in fetal and offspring rodents. However, the underlying mechanisms and targeted treatments remain largely to be determined. Isoflurane and propofol are among commonly used anesthetics. Thus, we set out to investigate whether propofol can mitigate the isoflurane-induced neurotoxicity in mice. METHODS Pregnant C57BL/6 mice at gestational day 15 (G15) were randomly assigned to 4 groups: control, isoflurane, propofol, and isoflurane plus propofol. Levels of interleukin (IL)-6 and poly-ADP ribose polymerase (PARP) fragment were measured in the brains of G15 embryos, and levels of postsynaptic density (PSD)-95 and synaptophysin were determined in the hippocampal tissues of postnatal day 31 (P31) offspring using Western blotting and immunohistochemical staining. Learning and memory functions in P31 offspring were determined using a Morris water maze test. RESULTS Isoflurane anesthesia in pregnant mice at G15 significantly increased brain IL-6 (222.6% ± 36.45% vs 100.5% ± 3.43%, P < .0001) and PARP fragment (384.2% ± 50.87% vs 99.59% ± 3.25%, P < .0001) levels in fetal mice and reduced brain PSD-95 (30.76% ± 2.03% vs 100.8% ± 2.25%, P < .0001) and synaptophysin levels in cornu ammonis (CA) 1 region (57.08% ± 4.90% vs 100.6% ± 2.20%, P < .0001) and dentate gyrus (DG; 56.47% ± 3.76% vs 99.76% ± 1.09%, P < .0001) in P31 offspring. Isoflurane anesthesia also impaired cognitive function in offspring at P31. Propofol significantly mitigated isoflurane-induced increases in brain IL-6 (117.5% ± 10.37% vs 222.6% ± 36.45%, P < .0001) and PARP fragment (205.1% ± 35.99% vs 384.2% ± 50.87%, P < .0001) levels in fetal mice, as well as reductions in PSD-95 (49.79% ± 3.43% vs 30.76% ± 2.03%, P < .0001) and synaptophysin levels in CA1 region (85.57% ± 2.97% vs 57.08% ± 4.90%, P < .0001) and DG (85.05% ± 1.87% vs 56.47% ± 3.76%, P < .0001) in hippocampus of P31 offspring. Finally, propofol attenuated isoflurane-induced cognitive impairment in offspring. CONCLUSIONS These findings suggest that gestational isoflurane exposure in mice induces neuroinflammation and apoptosis in embryos and causes cognitive impairment in offspring. Propofol can attenuate these isoflurane-induced detrimental effects.

Major depression disorder is one of the most common psychiatric disorders that greatly threaten the mental health of a large population worldwide. Previous studies have shown that endoplasmic reticulum (ER) stress plays an important role in the pathophysiology of depression, and current research suggests that brain-derived neurotrophic factor precursor (proBDNF) is involved in the development of depression. However, the relationship between ER and proBDNF in the pathophysiology of depression is not well elucidated. Here, we treated primary hippocampal neurons of mice with corticosterone (CORT) and evaluated the relationship between proBDNF and ERS. Our results showed that CORT induced ERS and upregulated the expression of proBDNF and its receptor, Follistatin-like protein 4 (FSTL4), which contributed to significantly decreased neuronal viability and expression of synaptic-related proteins including NR2A, PSD95, and SYN. Anti-proBDNF neutralization and ISRIB (an inhibitor of the ERS) treatment, respective ly, protected neuronal viabilities and increased the expression of synaptic-related proteins in corticosterone-exposed neurons. ISRIB treatment reduced the expression of proBDNF and FSTL4, whereas anti-proBDNF treatment did not affect ERS markers (Grp78, p-PERK, ATF4) expression. Our study presented evidence that CORT-induced ERS negatively regulated the neuronal viability and the level of synaptic-related protein of primary neurons via the proBDNF/FSTL4 pathway.

Accurate quantification of synaptic changes is essential for understanding the molecular mechanisms of synaptogenesis, synaptic plasticity, and synaptic toxicity. Here we demonstrate a robust high-content imaging method for the assessment of synaptic changes and apply the method to brain homogenates from an Alzheimer’s disease mouse model. Our method uses serial imaging of endogenous fluorescent labeled presynaptic VAMP2 and postsynaptic PSD95 in long-term cultured live primary neurons in 96 well microplates, and uses automatic image analysis to quantify the number of colocalized mature synaptic puncta for the assessment of synaptic changes in live neurons. As a control, we demonstrated that our synaptic puncta assay is at least 10-fold more sensitive to the toxic effects of glutamate than the MTT assay. Using our assay, we have compared synaptotoxic activities in size-exclusion chromatography fractioned protein samples from 3xTg-AD mouse model brain homogenates. Multiple synaptotoxic activities were found in high and low molecular weight fractions. Amyloid-beta immunodepletion alleviated some but not all of the synaptotoxic activities. Although the biochemical entities responsible for the synaptotoxic activities have yet to be determined, these proof-of-concept results demonstrate that this novel assay may have many potential mechanistic and therapeutic applications.

Excessive drinking in adolescence continues to be a problem, and almost a quarter of young Canadians have reported consuming five or more alcoholic drinks in one occasion in recent surveys. The consequences of such drinking may be more pronounced when commenced in adolescence, given the ongoing brain development during this period of life. Here, we investigated the consequences of 3 weeks’ intermittent access to ethanol in mid-adolescence to early adulthood in rats, and the extent to which a stress history moderated the negative consequences of ethanol access. In experiment 1, male rats that underwent adolescent social instability stress (SS; daily 1 h isolation + return to unfamiliar cage partner every day from postnatal day [PND] 30–45) did not differ from control (CTL) rats in intake of 10% ethanol sweetened with 0.1% saccharin (access period; PND 47–66). Ethanol drinking reduced proteins relevant for synaptic plasticity (αCaMKII, βCaMKII, and PSD-95) in the dorsal hippocampus, and in CTL rats only in the prefrontal cortex (αCaMKII and PSD 95), attenuating the difference between CTL and SS rats in the water-drinking group. In experiment 2, ethanol also attenuated the difference between SS and CTL rats in a social interaction test by reducing social interaction in SS rats; CTL rats, however, had a higher intake of ethanol than did SS rats during the access period. Ethanol drinking reduced baseline and fear recall recovery concentrations of corticosterone relative to those exposed only to water, although there was no effect of either ethanol or stress history on fear conditioning. Ethanol drinking did not influence intake after 9 days of withdrawal; however, ethanol-naive SS rats drank more than did CTL rats when given a 24-h access in adulthood. These results reveal a complex relationship between stress history and ethanol intake in adolescence on outcomes in adulthood.

Toll-like receptor 4 (TLR4) is a crucial receptor in neuroinflammation and apoptotic neuronal death, and increasing evidences indicated that β2-microglobulin (B2M) is thought to be a major contributor to age-related cognitive decline. In present study, we designed to investigate the effects of TLR4 on B2M-induced age-related cognitive decline. Wild-type (WT) C57BL/6, TLR4 knockout (TLR4 -KO) mice and hippocampal neurons from the two type mice were respectively divided into two groups: (1) Veh group; (2) B2M-treated group. The behavioral responses of mice were measured using Morris Water Maze. Hippocampal neurogenesis and neuronal damage, inflammatory response, apoptosis, synaptic proteins and neurotrophic factors, and TLR4/MyD88/NF-κB signaling pathway proteins were examined using molecular biological or histopathological methods. The results showed that WT mice received B2M in the DG exhibited age-related cognitive declines, increased TLR4 mRNA expression and high levels of interleukin-1β (IL-1β), tumor necrosis factor-alpha (TNF-α) and apoptotic neuronal death in the hippocampus, which were partially attenuated in TLR4-KO mice. Moreover, in absence of TLR4, B2M treatment improved hippocampus neurogenesis and increased synaptic related proteins. Our cell experiments further demonstrated that deletion of TLR4 could significantly increase synaptic related protein, decrease neuroinflammatory fators, inhibited apoptotic neuronal death, and regulated MyD88/NF-κB signal pathway after B2M treatment. In summary, our results support the TLR4 contributes to B2M-induced age-related cognitive decline due to neuroinflammation and apoptosis through TLR4/MyD88/NF-κB signaling pathway via a modulation of hippocampal neurogenesis and synaptic function. This may provide an important neuroprotective mechanism for improving age-related cognitive decline.

Epidemiological studies have found that diabetes and cognitive dysfunction are closely related. Quercetin has been certified with the effect on improving diabetes mellitus (DM) and cognitive impairment. However, the effect and related mechanism of quercetin on diabetic encephalopathy (DE) are still ambiguous. In this study, we used the db/db mice (diabetic model) to discover whether quercetin could improve DE through the Sirtuin1/NLRP3 (NOD‐, LRR‐ and pyrin domain‐containing 3) pathway. Behavioural results (Morris water maze and new object recognition tests) showed that quercetin (70 mg/kg) improved the learning and memory. Furthermore, quercetin alleviated insulin resistance and the level of fasting blood glucose. Besides, Western blot analysis also showed that quercetin increased the protein expressions of nerve‐ and synapse‐related protein, including postsynapticdensity 93 (PSD93), postsynapticdensity 95 (PSD95), brain‐derived neurotrophic factor (BDNF) and nerve growth factor (NGF) in the brain of db/db mice. Quercetin also increased the protein expression of SIRT1 and decreased the expression of NLRP3 inflammation‐related proteins, including NLRP3, the adaptor protein ASC and cleaved Caspase‐1, the pro‐inflammatory cytokines IL‐1β and IL‐18. In conclusion, the present results indicate that the SIRT1/NLRP3 pathway may be a crucial mechanism for the neuroprotective effect of quercetin against DE.

Mechanisms of tissue damage in Huntington’s disease involve excitotoxicity, mitochondrial damage, and inflammation, including microglia activation. Immunomodulatory and anti-protein aggregation properties of tetracyclines were demonstrated in several disease models. In the present study, the neuroprotective and anti-inflammatory effects of the tetracycline doxycycline were investigated in the mouse model of HD disease R6/2. Transgenic mice were daily treated with doxycycline 20 mg/kg, starting from 4 weeks of age. After sacrifice, histological and immunohistochemical studies were performed. We found that doxycycline-treated R6/2 mice survived longer and displayed less severe signs of neurological dysfunction than the saline-treated ones. Primary outcome measures such as striatal atrophy, neuronal intranuclear inclusions, and the negative modulation of microglial reaction revealed a neuroprotective effect of the compound. Doxycycline provided a significantly increase of activated CREB and BDNF in the striatal neurons, along with a down modulation of neuroinflammation, which, combined, might explain the beneficial effects observed in this model. Our findings show that doxycycline treatment could be considered as a valid therapeutic approach for HD. Electronic supplementary material The online version of this article (10.1007/s12035-019-01847-8) contains supplementary material, which is available to authorized users.

Anesthesia in pregnant women may cause adverse effects in the hippocampus of unborn babies and fetal brain development. The mechanisms underlying pathological changes resulting from anesthetics are unclear. This study tested the hypothesis that exposure to desflurane during pregnancy may impair cognition and memory functions of juvenile offspring. Pregnant mice (at gestational day 14) were administered 10% desflurane for 3 h and compared to sham control and sciatic nerve hemi-transection surgery. Hippocampal tissues of both fetal (G14) and offspring mice (postnatal day 31) were collected and analyzed by real-time qPCR and Western blot. Functional tests were performed to assess fear and memory functions in offspring mice. Primary hippocampal neuronal cultures from postnatal day 0 (without desflurane exposure) were examined for neuronal and synaptic development under desflurane treatment in vitro. In this acute experiment, we showed that neuronal cultures exposed to desflurane significantly increased interleukin (IL)-6 expression and apoptotic gene caspase-3 activation. Desflurane exposure significantly reduced PSD-95 expression in hippocampal neurons. Similar changes were observed in hippocampal tissues from juvenile offspring mice. Inhaled desflurane impaired memory functions in offspring mice compared to sham control. These mice displayed higher sensitivity to fear conditioning. Neurons isolated from the mice exposed to desflurane exhibited significantly lower levels of synaptophysin expression. These results suggest that anesthetic exposure together with surgery during pregnancy may induce detrimental effects in juvenile offspring mice via the induction of cell death and disruption of synaptic integrity.

PSD-95 (Dlg4) is an ionotropic glutamate receptor scaffolding protein essential in synapse stability and neurotransmission. PSD-95 levels are reduced during aging and in neurodegenerative diseases like Huntington’s disease (HD), and it is believed to contribute to synaptic dysfunction and behavioral deficits. However, the mechanism responsible for PSD-95 dysregulation under these conditions is unknown. The Heat Shock transcription Factor 1 (HSF1), canonically known for its role in protein homeostasis, is also depleted in both aging and HD. Synaptic protein levels, including PSD-95, are influenced by alterations in HSF1 levels and activity, but the direct regulatory relationship between PSD-95 and HSF1 has yet to be determined. Here, we showed that HSF1 chronic or acute depletion in cell lines and mice decreased PSD-95 expression. Furthermore, HSF1(+/-) mice had reduced PSD-95 synaptic puncta that paralleled a loss in thalamo-striatal excitatory synapses, an important circuit disrupted early in HD. We demonstrated that HSF1 binds to regulatory elements present in the PSD-95 gene and directly regulates PSD-95 expression. HSF1 DNA-binding on the PSD-95 gene was disrupted in an age-dependent manner in WT mice and worsened in HD cells and mice, leading to reduced PSD-95 levels. These results demonstrate a direct role of HSF1 in synaptic gene regulation that has important implications in synapse maintenance in basal and pathological conditions.

Hypobaric hypoxia (HH) is a typical characteristic of high altitude environment and causes a spectrum of pathophysiological effects, including headaches, gliovascular dysfunction and cognitive retardation. Here, we sought to understand the mechanisms underlying cognitive deficits under HH exposure. Our results showed that hypobaric hypoxia exposure impaired cognitive function and suppressed dendritic spine density accompanied with increased neck length in both basal and apical hippocampal CA1 region neurons in mice. The expression of PSD95, a vital synaptic scaffolding molecule, is down-regulated by hypobaric hypoxia exposure and post-transcriptionally regulated by cold-inducible RNA-binding protein (Cirbp) through 3′-UTR region binding. PSD95 expressing alleviates hypoxia-induced dendritic spine morphology changes of hippocampal neurons and memory deterioration. Moreover, overexpressed Cirbp in hippocampus rescues HH-induced abnormal expression of PSD95 and attenuates hypoxia-induced dendritic spine injury and cognitive retardation. Thus, our findings reveal a novel mechanism that Cirbp-PSD-95 axis appears to play an essential role in HH-induced cognitive dysfunction in mice. Supplementary Information The online version contains supplementary material available at 10.1186/s13041-021-00827-1.

Objective To investigate the effects of 7,8-dihydroxycoumarin on the myelin morphological changes and PSD-95 protein expression in mice with sciatic nerve injury, and to explore the relationship between PSD-95 protein and myelin regeneration after nerve myelin injury. Methods One hundred twenty-seven male adult Balb/c mice were selected and randomly divided into high, medium and low 7,8-dihydroxycoumarin dose groups and blank control group. Anastomosis was then carried out for the amputated right sciatic nerve, and intraperitoneal injection of 7,8-dihydroxycoumarin was applied postoperatively. At weeks 1, 2, 4 and 8 after surgery, nervous tissues from the injury side were taken for immunohistochemical Luxol Fast Blue staining, so as to observe the morphological changes of the locally injured nerve myelin. Meanwhile, PSD-95 mRNA and protein expression were determined using real-time PCR and western blotting. Results The nerve myelin recovery in injury side of mice at all time points showed a definite dose-effect relationship with the dose of 7,8-dihydroxycoumarin. Moreover, 7,8-dihydroxycoumarin could inhibit the PSD-95 mRNA level and protein expression. At the same time, there was a dose-effect of the inhibition. Conclusions 7,8-Dihydroxycoumarin can affect nerve recovery in mice with sciatic nerve injury, which shows a definite dose-effect relationship with its dose. Besides, PSD-95 protein expression can suppress the regeneration of the injured nerve myelin.

We investigated the alterations of hippocampal and reticulo-thalamic (RT) GABAergic parvalbumin (PV) interneurons and their synaptic re-organizations underlying the prodromal local sleep disorders in the distinct rat models of Parkinson’s disease (PD). We demonstrated for the first time that REM sleep is a predisposing state for the high-voltage sleep spindles (HVS) induction in all experimental models of PD, particularly during hippocampal REM sleep in the hemiparkinsonian models. There were the opposite underlying alterations of the hippocampal and RT GABAergic PV+ interneurons along with the distinct MAP2 and PSD-95 expressions. Whereas the PD cholinopathy enhanced the number of PV+ interneurons and suppressed the MAP2/PSD-95 expression, the hemiparkinsonism with PD cholinopathy reduced the number of PV+ interneurons and enhanced the MAP2/PSD-95 expression in the hippocampus. Whereas the PD cholinopathy did not alter PV+ interneurons but partially enhanced MAP2 and suppressed PSD-95 expression remotely in the RT, the hemiparkinsonism with PD cholinopathy reduced the PV+ interneurons, enhanced MAP2, and did not change PSD-95 expression remotely in the RT. Our study demonstrates for the first time an important regulatory role of the hippocampal and RT GABAergic PV+ interneurons and the synaptic protein dynamic alterations in the distinct rat models of PD neuropathology.

Rationale Deregulated attack behaviors have devastating social consequences; however, satisfactory clinical management for the behavior is still an unmet need so far. Social isolation (SI) has been common during the COVID-19 pandemic and may have detrimental effects on mental health, including eliciting heightened attack behavior. Objectives This study aims to explore whether injection of ZL006 can alleviate SI-induced escalation of attack behavior in mice. Methods Pharmacological tools, biochemical methods, and behavioral tests were used to explore the potential therapeutic effects of ZL006 targeting postsynaptic density 95 (PSD95)/neuronal nitric oxide synthase (nNOS) pathway on escalation of attack behavior induced by SI in mice. Results ZL006 mitigated SI-induced escalated attack behaviors and elevated nitric oxide (NO) level in the cortex of the SI mice. The beneficial effects of ZL006 lasted for at least 72 h after a single injection of ZL006. Potentiation of NO levels by L-arginine blocked the effects of ZL006. Moreover, a sub-effective dose of 7-NI in combination with a sub-effective dose of ZL006 decreased both SI-induced escalated attack behaviors and NO levels in mice subjected to SI. Conclusions Our study highlights the importance of the PSD95/nNOS pathway in mediating SI-induced escalation of attack behavior. ZL006 may be a promising therapeutic strategy for treating aggressive behaviors. Supplementary Information The online version contains supplementary material available at 10.1007/s00213-021-06000-9.

Fluorescence imaging at single-cell resolution is a crucial approach to analyzing the spatiotemporal regulation of proteins within individual cells of complex neural networks. Here we present a nonviral strategy that enables the tagging of endogenous loci by CRISPR/Cas9-mediated genome editing combined with a nucleofection technique. The method allowed expression of fluorescently tagged proteins at endogenous levels, and we successfully achieved tagging of a presynaptic protein, synaptophysin (Syp), and a postsynaptic protein, PSD-95, in cultured postmitotic neurons. Superresolution fluorescence microscopy of fixed neurons confirmed the identical localization patterns of the tagged proteins to those of endogenous ones verified by immunohistochemistry. The system is also applicable for multiplexed labeling and live-cell imaging. Live imaging with total internal reflection fluorescence microscopy of a single dendritic process of a neuron double-labeled with Syp-mCherry and PSD-95-EGFP revealed the previously undescribed dynamic localization of the proteins synchronously moving along dendritic shafts. Our convenient and versatile strategy is potent for analysis of proteins whose ectopic expressions perturb cellular functions.

Impulsivity is a multifaceted behavioral manifestation with implications in several neuropsychiatric disorders. Glutamate neurotransmission through the N-methyl-D-aspartate receptors (NMDARs) in the medial prefrontal cortex (mPFC), an important brain region in decision-making and goal-directed behaviors, plays a key role in motor impulsivity. We discovered that inherent motor impulsivity predicted responsiveness to D-cycloserine (DCS), a partial NMDAR agonist, which prompted the hypothesis that inherent motor impulsivity is associated with the pattern of expression of cortical NMDAR subunits (GluN1, GluN2A, GluN2B), specifically the protein levels and synaptosomal trafficking of the NMDAR subunits. Outbred male Sprague-Dawley rats were identified as high (HI) or low (LI) impulsive using the one-choice serial reaction time task. Following phenotypic identification, mPFC synaptosomal protein was extracted from HI and LI rats to assess the expression pattern of the NMDAR subunits. Synaptosomal trafficking and stabilization for the GluN2 subunits were investigated by co-immunoprecipitation for postsynaptic density 95 (PSD95) and synapse associated protein 102 (SAP102). HI rats had lower mPFC GluN1 and GluN2A, but higher GluN2B and pGluN2B synaptosomal protein expression versus LI rats. Further, higher GluN2B:PSD95 and GluN2B:SAP102 protein:protein interactions were detected in HI versus LI rats. Thus, the mPFC NMDAR subunit expression pattern and/or synaptosomal trafficking associates with high inherent motor impulsivity. Increased understanding of the complex regulation of NMDAR balance within the mPFC as it relates to inherent motor impulsivity may lead to a better understanding of risk factors for impulse-control disorders.

Cleavage of amyloid precursor protein (APP) by β-secretase BACE1 initiates the production and accumulation of neurotoxic amyloid-β peptides, which is widely considered an essential pathogenic mechanism in Alzheimer's disease (AD). Here, we report that BACE1 is essential for normal auditory function. Compared with wild-type littermates, BACE1−/−mice of either sex exhibit significant hearing deficits, as indicated by increased thresholds and reduced amplitudes in auditory brainstem responses (ABRs) and decreased distortion product otoacoustic emissions (DPOAEs). Immunohistochemistry revealed aberrant synaptic organization in the cochlea and hypomyelination of auditory nerve fibers as predominant neuropathological substrates of hearing loss in BACE1−/−mice. In particular, we found that fibers of spiral ganglion neurons (SGN) close to the organ of Corti are disorganized and abnormally swollen. BACE1 deficiency also engenders organization defects in the postsynaptic compartment of SGN fibers with ectopic overexpression of PSD95 far outside the synaptic region. During postnatal development, auditory fiber myelination in BACE1−/−mice lags behind dramatically and remains incomplete into adulthood. We relate the marked hypomyelination to the impaired processing of Neuregulin-1 when BACE1 is absent. To determine whether the cochlea of adult wild-type mice is susceptible to AD treatment-like suppression of BACE1, we administered the established BACE1 inhibitor NB-360 for 6 weeks. The drug suppressed BACE1 activity in the brain, but did not impair hearing performance and, upon neuropathological examination, did not produce the characteristic cochlear abnormalities of BACE1−/−mice. Together, these data strongly suggest that the hearing loss of BACE1 knock-out mice represents a developmental phenotype.SIGNIFICANCE STATEMENTGiven its crucial role in the pathogenesis of Alzheimer's disease (AD), BACE1 is a prime pharmacological target for AD prevention and therapy. However, the safe and long-term administration of BACE1-inhibitors as envisioned in AD requires a comprehensive understanding of the various physiological functions of BACE1. Here, we report that BACE1 is essential for the processing of auditory signals in the inner ear, as BACE1-deficient mice exhibit significant hearing loss. We relate this deficit to impaired myelination and aberrant synapse formation in the cochlea, which manifest during postnatal development. By contrast, prolonged pharmacological suppression of BACE1 activity in adult wild-type mice did not reproduce the hearing deficit or the cochlear abnormalities of BACE1 null mice.

Cognitive decline and dementia in neurodegenerative diseases are associated with synapse dysfunction and loss, which may precede neuron loss by several years. While misfolded and aggregated α-synuclein is recognized in the disease progression of synucleinopathies, the nature of glutamatergic synapse dysfunction and loss remains incompletely understood. Using fluorescence-activated synaptosome sorting (FASS), we enriched excitatory glutamatergic synaptosomes from mice overexpressing human alpha-synuclein (h-αS) and wild-type littermates to unprecedented purity. Subsequent label-free proteomic quantification revealed a set of proteins differentially expressed upon human alpha-synuclein overexpression. These include overrepresented proteins involved in the synaptic vesicle cycle, ER-Golgi trafficking, metabolism and cytoskeleton. Unexpectedly, we found and validated a steep reduction of eukaryotic translation elongation factor 1 alpha (eEF1A1) levels in excitatory synapses at early stages of h-αS mouse model pathology. While eEF1A1 reduction correlated with the loss of postsynapses, its immunoreactivity was found on both sides of excitatory synapses. Moreover, we observed a reduction in eEF1A1 immunoreactivity in the cingulate gyrus neuropil of patients with Lewy body disease along with a reduction in PSD95 levels. Altogether, our results suggest a link between structural impairments underlying cognitive decline in neurodegenerative disorders and local synaptic defects. eEF1A1 may therefore represent a limiting factor to synapse maintenance.

The aim of this study investigates whether β-asarone can improve cognition deficits in dizocilpine (MK-801) treated mice. Six-week-old male C57BL/6 mice were divided into four groups: control group (CON), MK-801-treated group (MK-801), MK-801 plus β-asarone group (MK-801+β-asa) and β-asarone group (β-asa). Behavioral tests, including sociability test, open field test (OPT) and Morris water-maze (MWM), were performed. Extracellular field excitatory postsynaptic potentials were recorded in the hippocampal dentate gyrus (DG) region. Western blot was employed to measure the expression of cognitive function-associated proteins and pro-inflammatory cytokines in the hippocampus. Immunofluorescence was performed to assess the microglial activation in the hippocampus DG region. The data show that social interactions and spatial learning and memory were impaired by MK-801. However, β-asarone significantly mitigated the impairments. Furthermore, it was found that MK-801 aggravated the hyperactivity and anxiety-like behavior, but β-asarone alleviated them. Moreover, β-asarone alleviated the impairments of hippocampal synaptic plasticity and enhanced the expression of hippocampal synaptophysin (SYP) and postsynaptic density protein 95 (PSD95) in MK-801-treated mice. In addition, it suppressed the expression of interleukin-6 (IL-6), interleukin-1β (IL-1β), inducible nitric oxide synthase (i-Nos) and cyclo-oxygenase-2 (COX-2) expression in MK-801-treated mice. The results suggest that β-asarone improved the impairment of cognition and synaptic plasticity possibly through modulating the excess release of pro-inflammatory cytokines and microglia activation in MK-801-treated mice.

Apolipoprotein E (apoE) colocalizes with amyloid-β (Aβ) in Alzheimer disease (AD) plaques and in synapses, and evidence suggests that direct interactions between apoE and Aβ are important for apoE's effects in AD. The present work examines the hypothesis that apoE receptors mediate uptake of apoE/Aβ complex into synaptic terminals. Western blot analysis shows multiple SDS-stable assemblies in synaptosomes from human AD cortex; apoE/Aβ complex was markedly increased in AD compared with aged control samples. Complex formation between apoE and Aβ 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 Aβ concentration into LDLR and LRP1-positive synaptosomes, along with free and esterified cholesterol. Synaptic Aβ was increased by apoE4 in control and AD samples. These results are consistent with uptake of apoE/Aβ complex and associated lipids into synaptic terminals, with subsequent Aβ clearance in control synapses and accumulation in AD synapses.

Over 130 brain diseases are caused by mutations that disrupt genes encoding the proteome of excitatory synapses. These include neurological and psychiatric disorders with early and late onset such as autism, schizophrenia and depression and many other rarer conditions. The proteome of synapses is highly complex with over 1000 conserved proteins which are differentially expressed generating a vast, potentially unlimited, number of synapse types. The diversity of synapses and their location in the brain are described by the synaptome. A recent study has mapped the synaptome across the mouse brain, revealing that synapse diversity is distributed into an anatomical architecture observed at scales from individual dendrites to the whole systems level. The synaptome architecture is built from the hierarchical expression and assembly of proteins into complexes and supercomplexes which are distributed into different synapses. Mutations in synapse proteins change the synaptome architecture leading to behavioral phenotypes. Mutations in the mechanisms regulating the hierarchical assembly of the synaptome, including transcription and proteostasis, may also change synapse diversity and synaptome architecture. The logic of synaptome hierarchical assembly provides a mechanistic framework that explains how diverse genetic disorders can converge on synapses in different brain circuits to produce behavioral phenotypes.

Background Apolipoprotein E4 (ApoE4) is the major genetic risk factor of Alzheimer's disease (AD). ApoE4 carriers have cerebral hypometabolism which is thought as a harbinger of AD. Our previous studies indicated ketones improved mitochondria energy metabolism via sirtuin 3 (Sirt3). However, it is unclear whether ketones upregulate Sirt3 and improve ApoE4-related learning and memory deficits. Results Ketones improved learning and memory abilities of ApoE4 mice but not ApoE3 mice. Sirt3, synaptic proteins, the NAD+/ NADH ratio, and ATP production were significantly increased in the hippocampus and the cortex from ketone treatment. Methods Human ApoE3 and ApoE4 transgenic mice (9-month-old) were treated with either ketones or normal saline by daily subcutaneous injections for 3 months (ketones, beta-hydroxybutyrate (BHB): 600 mg/kg/day; acetoacetate (ACA): 150 mg/kg/day). Learning and memory ability of these mice were assessed. Sirt3 protein, synaptic proteins (PSD95, Synaptophysin), the NAD+/ NADH ratio, and ATP levels were measured in the hippocampus and the cortex. Conclusion Our current studies suggest that ketones improve learning and memory abilities of ApoE4 transgenic mice. Sirt3 may mediate the neuroprotection of ketones by increasing neuronal energy metabolism in ApoE4 transgenic mice. This provides the foundation for Sirt3's potential role in the prevention and treatment of AD.

In the adult hippocampus new neurons are generated in the dentate gyrus from neural progenitor cells. Adult-born neurons integrate into the hippocampal circuitry and contribute to hippocampal function. PSD95 is a major postsynaptic scaffold protein that is crucial for morphological maturation and synaptic development of hippocampal neurons. Here we study the function of PSD95 in adult hippocampal neurogenesis by downregulating PSD95 expression in newborn cells using retroviral-mediated RNA interference. Retroviruses coding for a control shRNA or an shRNA targeting PSD95 (shPSD95) were stereotaxically injected into the dorsal dentate gyrus of 2-month-old C57BL/6 mice. PSD95 knockdown did not affect neuronal differentiation of newborn cells into neurons, or migration of newborn neurons into the granule cell layer. Morphological analysis revealed that newborn neurons expressing shPSD95 showed increased dendritic length and increased number of high-order dendrites. Concomitantly, dendrites from shPSD95-expressing newborn granule neurons showed a reduction in the density of dendritic spines. These results suggest that PSD95 is required for proper dendritic and spine maturation of adult-born neurons, but not for early stages of neurogenesis in the hippocampus.

Metabolic conditions during brain development may have long-term consequences on brain metabolism, thereby influencing the risk of neurodegenerative disease in later life. To ascertain the long-term consequences of omega-3 (ω3) fatty acid deficiency during brain development on oxidative fatty acid degradation in the brain and the development of Alzheimer-like pathology, wild-type (WT) female mice were fed diets that were either replete or deficient in ω3 fatty acids for 5 weeks. These females were then mated with hemizygous 5xFAD male transgenic (TG) mouse models of Alzheimer's disease, and the progeny were continued on diets that were either ω3-replete or ω3-deficient. When the progeny were 6 months of age, they received radiolabeled arachidonic acid (ARA) by intracerebroventricular injection. Five days after these injections, the brains were harvested and oxidative degradation of the radiolabeled ARA was characterized. Among the progeny of female mice on an ω3-replete diet, TG progeny had lower PSD-95 expression and higher oxidative ARA degradation than WT progeny. Progeny on an ω3-deficient diet, however, had no significant differences in PSD-95 expression between TG and WT mice, or in the extent of ARA degradation. In TG mice, an ω3-deficient diet reduced oxidative ARA degradation to a greater extent than in WT mice. The reductions in oxidative ARA degradation occurred even if the progeny of female mice on an ω3-deficient diet resumed an ω3-replete diet immediately on weaning. These results demonstrate that dietary ω3 fatty acid deficiency during development can cause long-term changes in the expression of a synaptic marker and long-term reductions in the rate of ARA degradation in the WT brain, which are not completely alleviated by an ω3-replete diet after weaning. The elimination of differences between TG and WT mice by an ω3-deficient diet suggests that mechanisms regulating PSD-95 expression and the oxidative degradation of ARA are related and that the timing of dietary ω3 intake during development may influence Alzheimer's disease-related pathological changes later in life.

Glutamate receptors play a crucial pathogenic role in brain damage induced by status epilepticus (SE). SE may initiate NMDAR-dependent excitotoxicity through the production of oxidative damage mediated by the activation of a ternary complex formed by the NMDA receptor, the post-synaptic density scaffolding protein 95 (PSD95) and the neuronal NO synthase (nNOS). The inhibition of the protein-protein-interaction (PPI) of the NMDAR-PSD95-nNOS complex is one of the most intriguing challenges recently developed to reduce neuronal death in both animal models and in patients with cerebral ischemia. We took advantage of this promising approach to verify whether early administration of a neuroprotective NMDAR-PSD95-nNOS PPI inhibitor preserves the brain from SE-induced damage in a model of acquired cortical dysplasia, the methylazoxymethanol (MAM)/pilocarpine rat. Pilocarpine-induced SE rapidly determined neurodegenerative changes mediated by a NMDAR-downstream neurotoxic pathway in MAM rats. We demonstrated that SE rapidly induces NMDAR activation, nNOS membrane translocation, PSD95-nNOS molecular interaction associated with neuronal and glial peroxynitrite accumulation in the neocortex of MAM-pilocarpine rats. These changes were paralleled by rapid c-fos overexpression and by progressive spectrin proteolysis, suggestive of calpain activity and irreversible cytoskeletal damage. Early administration of a cell-penetrating Tat-N-dimer peptide inhibitor of NMDAR-PSD95-nNOS PPI during SE significantly rescued the MAM-pilocarpine rats from SE-induced mortality, reduced the number of degenerating neurons, decreased neuronal c-fos activation, peroxynitrite formation and cytoskeletal degradation and prevented astrogliosis. Our findings suggest an overall neuroprotective effect of blocking PSD95-nNOS protein-protein-interaction against SE insult.

Atrazine (ATR), a widely used herbicide, has been previously shown to damage spatial memory capability and the hippocampus of male rats during the development. It has also been indicated that physical exercise can improve learning and memory in both humans and animals, as a neuroprotective method. Our aim here was to investigate the effect of maternal ATR exposure during gestation and lactation on spatial learning and memory function and hippocampal morphology in offspring and to further evaluate the neuroprotective effect of swimming training and identify possible related learning and memory signaling pathways. Using Sprague-Dawley rats, we examined behavioral and molecular biology effects associated with maternal ATR exposure, as well as the effects of 8 or 28 days swimming training. Maternal exposure to ATR was found to impair spatial learning and memory by behavioral test, damage the hippocampal morphology, and reduce related genes and proteins expression of learning and memory in the hippocampus. The extended, 28 days, period of swimming training produced a greater amelioration of the adverse effects of ATR exposure than the shorter, 8 days, training period. Our results suggest that maternal ATR exposure may damage the spatial learning and memory of offspring male rats via PSD95/NR2B signaling pathway. The negative effect of ATR could be at least partially reversed by swimming training, pointing to a potential neuroprotective role of physical exercise in nervous system diseases accompanying by learning and memory deficit.

Fragile X syndrome (FXS) is the leading monogenic cause of autism and intellectual disability. FXS is caused by loss of expression of fragile X mental retardation protein (FMRP), an RNA-binding protein that regulates translation of numerous mRNA targets, some of which are present at synapses. While protein synthesis deficits have long been postulated as an etiology of FXS, how FMRP loss affects distributions of newly synthesized proteins is unknown. Here we investigated the role of FMRP in regulating expression of new copies of the synaptic protein PSD95 in an in vitro model of synaptic plasticity. We find that local BDNF application promotes persistent accumulation of new PSD95 at stimulated synapses and dendrites of cultured neurons, and that this accumulation is absent in FMRP-deficient mouse neurons. New PSD95 accumulation at sites of BDNF stimulation does not require known mechanisms regulating FMRP-mRNA interactions but instead requires the PI3K-mTORC1-S6K1 pathway. Surprisingly, in FMRP-deficient neurons, BDNF induction of new PSD95 accumulation can be restored by mTORC1-S6K1 blockade, suggesting that constitutively high mTORC1-S6K1 activity occludes PSD95 regulation by BDNF and that alternative pathways exist to mediate induction when mTORC1-S6K1 is inhibited. This study provides direct evidence for deficits in local protein synthesis and accumulation of newly synthesized protein in response to local stimulation in FXS, and supports mTORC1-S6K1 pathway inhibition as a potential therapeutic approach for FXS.

The non-genomic actions of androgen-induced synaptic plasticity have been extensively studied. However, the underlying mechanisms remain controversial. We recently found that testosterone-fetal bovine serum albumin (T-BSA), a cell membrane-impermeable complex, led to a rapid increase in the postsynaptic density 95 (PSD95) protein level through a transcription-independent mechanism in mouse hippocampal HT22 cells. Using T-BSA conjugated FITC, we verified the presence of membrane androgen-binding sites. Here, we show that T-BSA-induced PSD95 expression is mediated by G-protein-coupled receptor (GPCR)-zinc transporter ZIP9 (SLC39A9), one of the androgen membrane binding sites, rather than the membrane-localized androgen receptor. Furthermore, we found that T-BSA induced an interaction between ZIP9 and Gnα11 that lead to the phosphorylation of Erk1/2 MAPK and eIF4E, which are critical in the mRNA translation process. The PSD95 and p-eIF4E expression decreased when knockdown of ZIP9 or Gnα11 expression or inhibition of Erk1/2 activation. Taken together, these findings suggest that ZIP9 mediates the non-genomic action of androgen on synaptic protein PSD95 synthesis through the Gnα11/Erk1/2/eIF4E pathway in HT22 cells. This novel mechanism provides a theoretical basis to understand the neuroprotective mechanism of androgen.

The serotoninergic system plays a key role in environmental sensitivity, potentially through down-stream effects on the GABAergic and glutamatergic systems. We previously demonstrated that juvenile serotonin transporter knockout (SERT−/−) rats, showing increased environmental sensitivity, exhibit a decreased GABA-mediated inhibitory tone in the cortex. Since the GABAergic and glutamatergic systems are tightly interconnected, we here analyzed glutamatergic markers in the prefrontal cortex of SERT−/− rats, from the early stages of life until adulthood. We found that SERT inactivation in pre-weaning, juvenile, and adult rats was associated with reduced expression of proteins essential for the glutamatergic synapses such as GluN1, PSD95, CDC42, and SEPT7. These lifelong molecular changes may destabilize glutamatergic signaling and possibly contribute to stress sensitivity and vulnerability to stress-related disorders associated with SERT alteration.

Background Piper nigrum L. and Piper longum L. consist a classic formula in traditional Chinese Hui medicine and are widely used in treatment of stroke. To examine the therapeutic effect of neuron injury after apoplexy, we used a permanent middle cerebral artery occlusion model in rats to investigate the effects of dichloromethane fraction (DF) of Piper nigrum L. and Piper longum L. Materials and Methods After subjecting the rats to permanent middle cerebral artery occlusion, DF (100 and 200 mg/kg) were administered for 14 days. Neurological deficits and the degree of cerebral tissue injury was detected by 2,3,5-Triphenyltetrazolium Chloride Staining Hematoxylin and eosin staining and Nissl staining. Postsynaptic density protein 95 (PSD-95), synapsin-I (syn-I), and α-synuclein (α-syn) were stained by immunohistochemistry. PSD-95, Ca2+/calmodulin (CaM)-dependent protein kinase II (CaMK II), phosphorylated CaMK II (p-CaMK II), CaM, N-methyl D-aspartate receptor subtype 2B (NR2B) expression were detected by Western blot. Meanwhile, phytochemical profile of DF was determined through ultrahigh-performance liquid chromatography-quadrupole time-of-flight mass spectrometry (UPLC-Q-TOF/MS). Results DF alleviated neurological deficits and markedly prevented ischemia-induced cellular damage. Immunohistochemical micrographs revealed that PSD-95 and syn-I proteins increased, and α-syn presented reduced expression in brain samples from the sham group. Western blot analyses revealed that the model group exhibited a noticeable reduction in PSD-95, p-CaMK II, CaM, and NR2B. The DF-treated model group exhibited increased PSD-95, p-CaMK II, CaM, and NR2B. UPLC-Q-TOF/MS analysis revealed eight main components of DF, of which piperine accounted for the largest proportion.

Background/aims Pterostilbene (Pt; trans-3,5-dimethoxy-4'-hydroxystilbene) is a natural phenol found in blueberries and grapevines. It shows remarkable biomedical activities similar to those of resveratrol. Its high bioavailability is a major advantage for possible biomedical applications. The goal of the study was to evaluate the effects of chronic pterostilbene administration on cognitive performance in aged rats with mild cognitive impairment. Methods 18-month-old animals were subjected to behavioral tests to establish the "baseline", then divided into treatment and control groups. The former were chronically fed Pt (22.5 mg/kg-day) for 20 consecutive days. At the end of this period all animals were tested again and sacrificed. The dentate gyrus, the hippocampus and the prefrontal and perirhinal cortices were then collected, and RT-qPCR and/or Western blot analyses were performed on a few transcripts/proteins involved in synaptic remodeling. Mitochondrial content was also assessed. Results Pt administration improved performance in behavioral tests and positively affected memory consolidation. We found increased levels of REST, PSD-95 and mitochondrial porin1 in the dentate gyrus and a positive correlation between T-maze test score and levels of cAMP responsive element binding protein (CREB) phosphorylation. Conclusion These results underscore the therapeutic potential of Pt supplementation for age-related cognitive decline.

Serotonin (5-HT) and the habenula (Hb) contribute to motivational and emotional states such as depression and drug abuse. The dorsal raphe nucleus, where 5-HT neurons originate, and the Hb are anatomically and reciprocally interconnected. Evidence exists that 5-HT influences Hb glutamatergic transmission. Using serotonin transporter knockout (SERT-/- ) rats, which show depression-like behavior and increased cocaine intake, we investigated the effect of SERT reduction on expression of genes involved in glutamate neurotransmission under both baseline conditions as well as after short-access or long-access cocaine (ShA and LgA, respectively) intake. In cocaine-naive animals, SERT removal led to reduced baseline Hb mRNA levels of critical determinants of glutamate transmission, such as SLC1A2, the main glutamate transporter and N-methyl-D-aspartate (Grin1, Grin2A and Grin2B) as well as α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (Gria1 and Gria2) receptor subunits, with no changes in the scaffolding protein Dlg4. In response to ShA and LgA cocaine intake, SLC1A2 and Grin1 mRNA levels decreased in SERT+/+ rats to levels equal of those of SERT-/- rats. Our data reveal that increased extracellular levels of 5-HT modulate glutamate neurotransmission in the Hb, serving as critical neurobiological substrate for vulnerability to cocaine addiction.