Osteocalcin ameliorates cognitive dysfunctions in a mouse model of Alzheimer's Disease by reducing amyloid ß burden and upregulating glycolysis in neuroglia.

Alzheimer's disease (AD) is the most common neurodegenerative disease characterized by the accumulation of amyloid ß peptides (Aß) and impaired glucose metabolism in the brain. Osteocalcin (OCN), an osteoblast-derived protein, has been shown to modulate brain functions but whether it has any effect on AD is undetermined. In this study, daily intraperitoneal injection of OCN for 4 weeks ameliorated the anxiety-like behaviors and cognitive dysfunctions in the APP/PS1 transgenic AD mice model, as shown in the increased entries into the central area in open field test, the increased time and entries into open arms in elevated plus maze test, the increased time spent in the light chamber in light-dark transition test, as well as the reduced escape latency and the increased preference for target quadrant in Morris water maze test. Aß burden in the hippocampus and cortex of AD mice was ameliorated by OCN. Besides, OCN improved the neural network function of the brain, mainly in the enhanced power of high gamma band in the medial prefrontal cortex of AD mice. The proliferation of astrocytes in the hippocampus in AD mice was also inhibited by OCN as demonstrated by immunofluorescence. Furthermore, OCN enhanced glycolysis in astrocytes and microglia, as evidenced by elevated glucose consumption, lactate production, and increased extracellular acidification rate. Such an effect was abolished when the receptor of OCN - Gpr158 was knockdown in astrocytes. Our study revealed OCN as a novel therapeutic factor for AD potentially through reducing Aß burden and upregulation of glycolysis in neuroglia.

Kininogen-Nitric Oxide Signaling at Nearby Nonexcited Acupoints after Long-Term Stimulation.

Acupuncture treatment is based on acupoint stimulation; however, the biological basis is not understood. We stimulated one acupoint with catgut embedding for 8 weeks and then used isobaric tags for relative and absolute quantitation to screen proteins with altered expression in adjacent acupoints of Sprague Dawley rats. We found that kininogen expression was significantly upregulated in the stimulated and the nonstimulated adjacent acupoints along the same meridian. The enhanced kininogen expression was meridian dependent and was most apparent among small vessels in the subcutaneous layer. Enhanced signals of nitric oxide synthases, cGMP-dependent protein kinase, and myosin light chain were also observed at the nonstimulated adjacent acupoints along the same meridian. These findings uncover biological changes at acupoints and suggest the critical role of the kininogen-nitric oxide signaling pathway in acupoint activation.

Gut microbiota-derived propionate mediates the neuroprotective effect of osteocalcin in a mouse model of Parkinson's disease.

BACKGROUND: Parkinson's disease (PD) is a neurodegenerative disorder with no absolute cure. The evidence of the involvement of gut microbiota in PD pathogenesis suggests the need to identify certain molecule(s) derived from the gut microbiota, which has the potential to manage PD. Osteocalcin (OCN), an osteoblast-secreted protein, has been shown to modulate brain function. Thus, it is of interest to investigate whether OCN could exert protective effect on PD and, if yes, whether the underlying mechanism lies in the subsequent changes in gut microbiota. RESULTS: The intraperitoneal injection of OCN can effectively ameliorate the motor deficits and dopaminergic neuronal loss in a 6-hydroxydopamine-induced PD mouse model. The further antibiotics treatment and fecal microbiota transplantation experiments confirmed that the gut microbiota was required for OCN-induced protection in PD mice. OCN elevated Bacteroidetes and depleted Firmicutes phyla in the gut microbiota of PD mice with elevated potential of microbial propionate production and was confirmed by fecal propionate levels. Two months of orally administered propionate successfully rescued motor deficits and dopaminergic neuronal loss in PD mice. Furthermore, AR420626, the agonist of FFAR3, which is the receptor of propionate, mimicked the neuroprotective effects of propionate and the ablation of enteric neurons blocked the prevention of dopaminergic neuronal loss by propionate in PD mice. CONCLUSIONS: Together, our results demonstrate that OCN ameliorates motor deficits and dopaminergic neuronal loss in PD mice, modulating gut microbiome and increasing propionate level might be an underlying mechanism responsible for the neuroprotective effects of OCN on PD, and the FFAR3, expressed in enteric nervous system, might be the main action site of propionate. Video abstract.

Protective effects of ß- nicotinamide adenine dinucleotide against motor deficits and dopaminergic neuronal damage in a mouse model of Parkinson's disease.

The level of nicotinamide adenine dinucleotide (NAD) decreases in Parkinson's disease (PD), and its reduction has been reported to be involved in many age-associated neurodegenerative pathologies. Thus, we investigated whether NAD replenishment is beneficial in a 6-hydroxydopamine (6-OHDA)-induced mouse model of PD. Preinjection with NAD in the striatum ameliorated motor deficits and dopaminergic neuronal damage in the substantia nigra and striatum of a mouse model of PD. Moreover, preincubation with NAD protected PC12 cells against the loss of cell viability, morphological damage, oxidative stress and mitochondrial dysfunction caused by 6-OHDA. These results add credence to the beneficial role of NAD against parkinsonian neurodegeneration in mouse models of PD, provide evidence for the potential of NAD for the prevention of PD, and suggest that NAD prevents pathological changes in PD via decreasing mitochondrial dysfunctions.

Gluconate suppresses seizure activity in developing brains by inhibiting CLC-3 chloride channels.

Neonatal seizures are different from adult seizures, and many antiepileptic drugs that are effective in adults often fail to treat neonates. Here, we report that gluconate inhibits neonatal seizure by inhibiting CLC-3 chloride channels. We detect a voltage-dependent outward rectifying Cl- current mediated by CLC-3 Cl- channels in early developing brains but not adult mouse brains. Blocking CLC-3 Cl- channels by gluconate inhibits seizure activity both in neonatal brain slices and in neonatal animals with in vivo EEG recordings. Consistently, neonatal neurons of CLC-3 knockout mice lack the outward rectifying Cl- current and show reduced epileptiform activity upon stimulation. Mechanistically, we demonstrate that activation of CLC-3 Cl- channels alters intracellular Cl- homeostasis and enhances GABA excitatory activity. Our studies suggest that gluconate can suppress neonatal seizure activities through inhibiting CLC-3 Cl- channels in developing brains.

Roles for osteocalcin in brain signalling: implications in cognition- and motor-related disorders.

It is now generally accepted that the extra-skeleton functionalities of bone are multifaceted. Its endocrine functions came first to light when it was realized that osteoblasts, the bone forming cells, maintain energy homeostasis by improving glucose metabolism, insulin sensitivity and energy expenditure through osteocalcin, a multipurpose osteokine secreted by osteoblasts. Recently, the emerging knowledge on the functional aspects of this osteokine expanded to properties including adult and maternal regulation of cognitive functions. Therapeutic potential of this osteokine has also been recently reported in experimental Parkinson's disease models. This review highlights such findings on the functions of osteocalcin in the brain and emphasizes on exploring and analyzing much more in-depth basic and clinical studies.

Osteocalcin Ameliorates Motor Dysfunction in a 6-Hydroxydopamine-Induced Parkinson's Disease Rat Model Through AKT/GSK3ß Signaling.

Osteoblasts derived osteocalcin (OCN) is recently reported to be involved in dopaminergic neuronal development. As dopaminergic neuronal injury in the substantia nigra (SN) is a pathological hallmark of Parkinson's disease (PD), we investigated whether OCN could exert protective effects on 6-hydroxydopamine (6-OHDA)-induced PD rat model. Our data showed that the OCN level in the cerebrospinal fluid (CSF) in PD rat models was significantly lower than that in controls. Intervention with OCN could improve the behavioral dysfunction in PD rat models and reduce the tyrosine hydroxylase (TH) loss in the nigrostriatal system. In addition, OCN could inhibit the astrocyte and microglia proliferation in the SN of PD rats. In vitro studies showed that OCN significantly ameliorated the neurotoxicity of 6-OHDA through the AKT/GSK3ß signaling pathway. In summary, OCN plays a protective role against parkinsonian neurodegeneration in the PD rat model, suggesting a potential therapeutic use of OCN in PD.

Extrasynaptic NMDA receptor dependent long-term potentiation of hippocampal CA1 pyramidal neurons.

In the adult mouse hippocampus, NMDA receptors (NMDARs) of CA1 neurons play an important role in the synaptic plasticity. The location of NMDARs can determine their roles in the induction of long-term potentiation (LTP). However, the extrasynaptic NMDARs (ES-NMDARs) dependent LTP haven't been reported. Here, through the use of a 5-Hz stimulation and MK-801 (an irreversible antagonist of NMDARs) in the CA1 neurons of adult mice hippocampal slices, synaptic NMDARs were selectively inhibited and NMDAR-mediated excitatory postsynaptic currents were not recovered. We found that a robust LTP was induced by 3-train 100-Hz stimulation when the synaptic NMDARs and extrasynaptic NR2B containing NMDARs were blocked, but not in the any of the following conditions: blocking of all NMDARs (synaptic and extrasynaptic), blocking of the synaptic NMDARs, and blocking of the synaptic NMDARs and extrasynaptic NR2A-containing NMDARs. The results indicate that this LTP is ES-NMDARs dependent, and NR2B-containing ES-NMDARs modulates the threshold of LTP induction.

The effects of early-life predator stress on anxiety- and depression-like behaviors of adult rats.

Childhood emotional trauma contributes significantly to certain psychopathologies, such as post-traumatic stress disorder. In experimental animals, however, whether or not early-life stress results in behavioral abnormalities in adult animals still remains controversial. Here, we investigated both short-term and long-term changes of anxiety- and depression-like behaviors of Wistar rats after being exposed to chronic feral cat stress in juvenile ages. The 2-week predator stress decreased spontaneous activities immediately following stress but did not increase depression- or anxiety-like behaviors 4 weeks after the stimulation in adulthood. Instead, juvenile predator stress had some protective effects, though not very obvious, in adulthood. We also exposed genetic depression model rats, Wistar Kyoto (WKY) rats, to the same predator stress. In WKY rats, the same early-life predator stress did not enhance anxiety- or depression-like behaviors in both the short-term and long-term. However, the stressed WKY rats showed slightly reduced depression-like behaviors in adulthood. These results indicate that in both normal Wistar rats and WKY rats, early-life predator stress led to protective, rather than negative, effects in adulthood.

Synaptic and extrasynaptic glutamate signaling in ischemic stroke.

Stroke is a leading cause of human mortality and disability where most cases of stroke are ischemic. The central nervous system (CNS) is extremely vulnerable to ischemic stroke particularly due to its unique ability: synaptic transmission. Not only does elaborate synaptic transmission consume extravagant energy that constrains neuronal viability under ischemic conditions, but glutamate, the most predominant neurotransmitter in the CNS, also triggers several catastrophic signaling cascades at both synaptic and extrasynaptic sites when excessively released. These signaling cascades accelerate neuronal death and exacerbate cerebral injuries during ischemic stroke. In this review, we discuss the complete picture of synaptic and extrasynaptic glutamate signaling in ischemic stroke. We hope to provide substantial insights into potential therapies by reviewing recent discoveries that have advanced our understanding of the complex glutamate signaling mechanisms in ischemic stroke.

Quantitative assessment of the association between rs2046210 at 6q25.1 and breast cancer risk.

Genome-wide association studies (GWAS) have identified several genetic susceptibility loci for breast cancer (BC). One of them, conducted among Chinese women, found an association of rs2046210 at 6q25.1 with the risk of BC recently. Since then, numerous association studies have been carried out to investigate the relationship between this polymorphism and BC risk in various populations. However, these have yielded contradictory results. We therefore performed a meta-analysis to clarify this inconsistency. Overall, a total of 235003 subjects based on 13 studies were included in our study. Significantly increased BC risk was detected in the pooled analysis [allele contrast: OR = 1.13, 95%CI = 1.10-1.17, P(Z) <10(-5), P(Q) <10(-4); dominant model: OR = 1.21, 95%CI = 1.14-1.27, P(Z) <10(-5), P(Q) <10(-4); recessive model: OR = 1.18, 95%CI = 1.12-1.24, P(Z) <10(-5), P(Q) = 0.04]. In addition, our data revealed that rs2046210 conferred greater risk in estrogen receptor (ER)-negative tumors [OR = 1.27, 95%CI = 1.15-1.40, P(Z) <10(-5), P(Q) <10(-4)] than in ER-positive ones [OR = 1.18, 95%CI = 1.09-1.28, P(Z) <10(-4), P(Q) = 0.0003]. When stratified by ethnicity, significant associations were found in Caucasian and Asian populations, but not detected among Africans. There was evidence of heterogeneity (P<0.05), however, the heterogeneity largely disappeared after stratification by ethnicity. The present meta-analysis demonstrated that the rs2046210 polymorphism may be associated with increased BC susceptibility, but this association varies in different ethnicities.

Activation of extrasynaptic NMDA receptors induces LTD in rat hippocampal CA1 neurons.

In the adult rat hippocampus, activation of N-methyl-d-aspartate receptors (NMDARs) is required for the induction of certain forms of synaptic plasticity, including long-term potentiation (LTP) and long-term depression (LTD). Several studies have indicated the opposing role of synaptic NMDARS (S-NMDARs) versus extrasynaptic NMDARs (ES-NMDARs) in CREB-dependent gene regulation and neuronal survival/death. The contribution of ES-NMDARs in synaptic plasticity, however, remains unclear. Here we investigated the contribution of ES-NMDARs on LTD induction in CA1 neurons of rat hippocampal slices. ES-NMDARs were selectively activated by theta burst stimulation (TBS) after selective blockade of S-NMDARs with pairing of 5 Hz stimulation and MK-801, an irreversible use-dependent antagonist of NMDARs. Application of TBS in naïve slices evoked a transient potentiation. In contrast, the activation of ES-NMDARs evoked a robust LTD. These results suggest the involvement of ES-NMDARs in LTD induction.

[Effect of acute stress stimulation on the seizure induction in epileptic model rats].

This study was undertaken to observe the effect of acute stress on seizure occurrence in chronic period of epileptic model rats. Lithium-pilocarpine (LiCl-PILO)-induced epileptic rat model was constructed. At the spontaneous recurrent seizure period, acute stress stimulations such as cat's urine and foot electrical shock were applied to observe the behavioral changes and seizure occurrence. The results showed that after the cat's urine stimulation, the self-directed behaviors of the epileptic model rats decreased significantly, while the risk assessment behaviors increased significantly. The seizure occurrence, however, was not observed during the 45 min after the stimulation. Applying electrical foot shocks also did not evoke seizures in epileptic model rats. On the contrast, intra-peritoneal injection of low dose of pentylenetetrazole (PTZ, 30 mg/kg) evoked seizure more efficiently, and the duration of seizure activity was extensively prolonged in epileptic model rats than that of control rats. Taken together, these results indicate that although applying stress stimulations such as cat's urine and electrical foot shock cause several behavioral changes, they are not severe enough to evoke seizure in epileptic model rats.

Functional roles of synaptic and extrasynaptic NMDA receptors in physiological and pathological neuronal activities.

The N-methyl-D-aspartate (NMDA) receptor is a major type of ionotropic glutamate receptor. Many studies have shown that NMDA receptors play a pivotal role in the central nervous system (CNS) under both physiological and pathological conditions. The functional diversity of NMDA receptors can be mainly attributed to their different subunit compositions that perform multiple functions in various situations. Furthermore, recent reports have indicated that synaptic and extrasynaptic NMDA receptors have distinct compositions and couple with different signaling pathways: while synaptic NMDA receptors tend to promote cell survival, extrasynaptic NMDA receptors promote cell death. Currently, intensive efforts are being made to study the pathological role of extrasynaptic NMDA receptors in order to find a more effective approach for the treatment of neurologic disorders. Here we reviewed some recent progress on the participation of synaptic and extrasynaptic NMDA receptors in neurologic diseases including epilepsy, ischemia, schizophrenia, depression and some neurodegenerative diseases.

Hippocampal endocannabinoids play an important role in induction of long-term potentiation and regulation of contextual fear memory formation.

Recent studies show contradictory results regarding the contribution of endocannabinoids in fear memory formation and long-term synaptic plasticity. In this study, we investigated the effects of both cannabinoid receptor type 1 (CB1 receptor) antagonist AM281 and anandamide reuptake inhibitor AM404 on the formation of contextual fear memory in adult mice. Both i.p. and intra-hippocampal injections of AM281 promoted contextual fear memory while a high dose of AM404 inhibited it. These findings demonstrate that CB1 receptor-mediated signaling negatively contributes to contextual fear memory formation. We further investigated the induction of long-term potentiation (LTP) in CA1 pyramidal neurons of hippocampal slices and found that AM281 impaired the induction of LTP. Additionally, the blockade of LTP by AM281 was completely prevented by bath application of picrotoxin, a selective antagonist of GABA(A) receptor. Taken together, these results indicate that activation of CB1 receptor contributes to induction of LTP via a GABA(A) receptor-mediated mechanism.

Forebrain-specific constitutively active CaMKKα transgenic mice show deficits in hippocampus-dependent long-term memory.

The Ca(2+)/calmodulin (CaM) kinase cascade is activated by Ca(2+) influx through the voltage-dependent Ca(2+) channels and the NMDA receptor. CaM kinase kinase (CaMKK), the most upstream kinase of the CaM kinase cascade, phosphorylates and activates both CaM kinase I (CaMKI) and CaMKIV, resulting in activation of cyclic AMP-responsive element binding protein (CREB)-dependent gene transcription. Using transgenic techniques, we created mutant mice in which a constitutively active form of CaMKK1, the autoinhibitory domain truncated protein, is over-expressed specifically in the forebrain. In these mice, although performance was normal in basal activity and short-term memory, specific impairments were shown in hippocampus-dependent long-term memory after training in spatial memory tasks and after contextual fear conditioning. In cultured neurons of these mice, phosphorylation of CaMKI was significantly increased in basal states, whereas the activity range of CaMKI phosphorylation by brain-derived neurotrophic factor (BDNF) and KCl stimulation was significantly diminished in mutant mice. Our results define a critical role for CaMKKα in synaptic plasticity and the retention of hippocampus-dependent long-term memory.

Hippocampal synaptic metaplasticity requires the activation of NR2B-containing NMDA receptors.

The potential to exhibit synaptic plasticity itself is modulated by previous synaptic activity, which has been termed as metaplasticity. In this paper, we demonstrated that the activation of N-methyl-d-aspartate (NMDA) receptor 2B (NR2B) subunit in NNDA receptors was required for hippocampal metaplasticity at Schaffer collateral-commissural fiber-CA1 synapses. Brief 5 Hz priming stimulation did not cause long-term synaptic plasticity; however, it could result in the inhibition of subsequently evoked long-term potentiation (LTP). Meanwhile, the application of selective antagonists for NR2B subunit of NMDA receptors after delivering priming stimulation could block the metaplasticity. In contrast, LTP induction was not affected by NR2B antagonists in slices without pre-treatment of priming stimulation. These results indicated that the activation of NR2B-containing NMDA receptors was required for metaplasticity.

Time-dependent changes in learning ability and induction of long-term potentiation in the lithium-pilocarpine-induced epileptic mouse model.

To explore the mechanism underlying the development of learning deficits in patients with epilepsy, we generated a mouse model for temporal lobe epilepsy by intraperitoneally injecting mice with pilocarpine with lithium chloride, and investigated time-dependent changes in both contextual fear memory of those model mice and long-term potentiation (LTP) in hippocampal CA1 neurons 1 day, 2 weeks, and 6 weeks after the onset of status epilepticus (SE). Fear memory formation did not change 1 day and 2 weeks after the onset of SE, but was significantly reduced after 6 weeks. Induction of LTP was enhanced 1 day after the onset of SE, but returned to the normal level 2 weeks later, and was almost completely attenuated after 6 weeks. The enhancement of LTP was accompanied by an increase in output responses of excitatory postsynaptic potentials, whereas suppression of LTP was accompanied by alteration of the ratio of paired pulse facilitation. These results indicate that time-dependent changes of LTP induction have a causal role in the development of learning deficits of epilepsy patients.