Hypoxia-sensing VGLL4 promotes LDHA-driven lactate production to ameliorate neuronal dysfunction in a cellular model relevant to Alzheimer's disease.

Alzheimer's disease (AD) is a neurodegenerative disease where abnormal amyloidogenic processing of amyloid-ß precursor protein (APP) occurs and has been linked to neuronal dysfunction. Hypometabolism of glucose in the brain can lead to synaptic loss and neuronal death, which in turn exacerbates energy deficiency and amyloid-ß peptide (Aß) accumulation. Lactate produced by anaerobic glycolysis serves as an energy substrate supporting neuronal function and facilitating neuronal repair. Vestigial-like family member 4 (VGLL4) has been recognized as a key regulator of the hypoxia-sensing pathway. However, the role of VGLL4 in AD remains unexplored. Here, we reported that the expression of VGLL4 protein was significantly decreased in the brain tissue of AD model mice and AD model cells. We further found that overexpression of VGLL4 reduced APP amyloidogenic processing and ameliorated neuronal synaptic damage. Notably, we identified a compromised hypoxia-sensitive capability of LDHA regulated by VGLL4 in the context of AD. Upregulation of VGLL4 increased the response of LDHA to hypoxia and enhanced the expression levels of LDHA and lactate by inhibiting the ubiquitination and degradation of LDHA. Furthermore, the inhibition of lactate production by using sodium oxamate, an inhibitor of LDHA, suppressed the neuroprotective function of VGLL4 by increasing APP amyloidogenic processing. Taken together, our findings demonstrate that VGLL4 exerts a neuroprotective effect by upregulating LDHA expression and consequently promoting lactate production. Thus, this study suggests that VGLL4 may be a novel player involved in molecular mechanisms relevant for ameliorating neurodegeneration.

Inhibiting NLRP3 inflammasome signaling pathway promotes neurological recovery following hypoxic-ischemic brain damage by increasing p97-mediated surface GluA1-containing AMPA receptors.

BACKGROUND: The nucleotide-binding oligomeric domain (NOD)-like receptor protein 3 (NLRP3) inflammasome is believed to be a key mediator of neuroinflammation and subsequent secondary brain injury induced by ischemic stroke. However, the role and underlying mechanism of the NLRP3 inflammasome in neonates with hypoxic-ischemic encephalopathy (HIE) are still unclear. METHODS: The protein expressions of the NLRP3 inflammasome including NLRP3, cysteinyl aspartate specific proteinase-1 (caspase-1) and interleukin-1ß (IL-1ß), the α-amino-3-hydroxy-5-methyl-4-isoxazole-propionicacid receptor (AMPAR) subunit, and the ATPase valosin-containing protein (VCP/p97), were determined by Western blotting. The interaction between p97 and AMPA glutamate receptor 1 (GluA1) was determined by co-immunoprecipitation. The histopathological level of hypoxic-ischemic brain damage (HIBD) was determined by triphenyltetrazolium chloride (TTC) staining. Polymerase chain reaction (PCR) and Western blotting were used to confirm the genotype of the knockout mice. Motor functions, including myodynamia and coordination, were evaluated by using grasping and rotarod tests. Hippocampus-dependent spatial cognitive function was measured by using the Morris-water maze (MWM). RESULTS: We reported that the NLRP3 inflammasome signaling pathway, such as NLRP3, caspase-1 and IL-1ß, was activated in rats with HIBD and oxygen-glucose deprivation (OGD)-treated cultured primary neurons. Further studies showed that the protein level of the AMPAR GluA1 subunit on the hippocampal postsynaptic membrane was significantly decreased in rats with HIBD, and it could be restored to control levels after treatment with the specific caspase-1 inhibitor AC-YVAD-CMK. Similarly, in vitro studies showed that OGD reduced GluA1 protein levels on the plasma membrane in cultured primary neurons, whereas AC-YVAD-CMK treatment restored this reduction. Importantly, we showed that OGD treatment obviously enhanced the interaction between p97 and GluA1, while AC-YVAD-CMK treatment promoted the dissociation of p97 from the GluA1 complex and consequently facilitated the localization of GluA1 on the plasma membrane of cultured primary neurons. Finally, we reported that the deficits in motor function, learning and memory in animals with HIBD, were ameliorated by pharmacological intervention or genetic ablation of caspase-1. CONCLUSION: Inhibiting the NLRP3 inflammasome signaling pathway promotes neurological recovery in animals with HIBD by increasing p97-mediated surface GluA1 expression, thereby providing new insight into HIE therapy.

Orexin-A increases the activity of globus pallidus neurons in both normal and parkinsonian rats.

Orexin is a member of neuropeptides which was first identified in the hypothalamus. The globus pallidus is a key structure in the basal ganglia, which is involved in both normal motor function and movement disorders. Morphological studies have shown the expression of both OX1 and OX2 receptors in the globus pallidus. Employing single unit extracellular recordings and behavioural tests, the direct in vivo electrophysiological and behavioural effects of orexin-A in the globus pallidus were studied. Micro-pressure administration of orexin-A significantly increased the spontaneous firing rate of pallidal neurons. Correlation analysis revealed a negative correlation between orexin-A induced excitation and the basal firing rate. Furthermore, application of the specific OX1 receptor antagonist, SB-334867, decreased the firing rate of pallidal neurons, suggesting that endogenous orexinergic systems modulate the firing activity of pallidal neurons. Orexin-A increased the excitability of pallidal neurons through both OX1 and OX2 receptors. In 6-hydroxydopamine parkinsonian rats, orexin-A-induced increase in firing rate of pallidal neurons was stronger than that in normal rats. Immunostaining revealed positive OX1 receptor expression in the globus pallidus of both normal and parkinsonian rats. Finally, postural test showed that unilateral microinjection of orexin-A led to contralateral deflection in the presence of systemic haloperidol administration. Further elevated body swing test revealed that pallidal orexin-A and SB-334867 induced contralateral-biased swing and ipsilateral-biased swing respectively. Based on the electrophysiological and behavioural findings of orexin-A in the globus pallidus, the present findings may provide a rationale for the pathogenesis and treatment of Parkinson's disease.

PCSK6 exacerbates Alzheimer's disease pathogenesis by promoting MT5-MMP maturation.

Proprotein convertase subtilisin/kexin type 6 (PCSK6) is a calcium-dependent serine proteinase that regulates the proteolytic activity of various precursor proteins and facilitates protein maturation. Dysregulation of PCSK6 expression or function has been implicated in several pathological processes including nervous system diseases. However, whether and how PCSK6 is involved in the pathogenesis of Alzheimer's disease (AD) remains unclear. In this study, we reported that the expression of PCSK6 was significantly increased in the brain tissues of postmortem AD patients and APP23/PS45 transgenic AD model mice, as well as N2AAPP cells. Genetic knockdown of PCSK6 reduced amyloidogenic processing of APP in N2AAPP cells by suppressing the activation of membrane-type 5-matrix metalloproteinase (MT5-MMP), referred to as η-secretase. We further found that PCSK6 cleaved and activated MT5-MMP by recognizing the RRRNKR sequence in its N-terminal propeptide domain in N2A cells. The mutation or knockout of this cleavage motif prevented PCSK6 from interacting with MT5-MMP and performing cleavage. Importantly, genetic knockdown of PCSK6 with adeno-associated virus (AAV) reduced Aß production and ameliorated hippocampal long-term potentiation (LTP) and long-term spatial learning and memory in APP23/PS45 transgenic mice. Taken together, these results demonstrate that genetic knockdown of PCSK6 effectively alleviate AD-related pathology and cognitive impairments by inactivating MT5-MMP, highlighting its potential as a novel therapeutic target for AD treatment.

miR-429-3p mediates memory decline by targeting MKP-1 to reduce surface GluA1-containing AMPA receptors in a mouse model of Alzheimer's disease.

Alzheimer's disease (AD) is a leading cause of dementia in the elderly. Mitogen-activated protein kinase phosphatase 1 (MKP-1) plays a neuroprotective role in AD. However, the molecular mechanisms underlying the effects of MKP-1 on AD have not been extensively studied. MicroRNAs (miRNAs) regulate gene expression at the post-transcriptional level, thereby repressing mRNA translation. Here, we reported that the microRNA-429-3p (miR-429-3p) was significantly increased in the brain of APP23/PS45 AD model mice and N2AAPP AD model cells. We further found that miR-429-3p could downregulate MKP-1 expression by directly binding to its 3'-untranslated region (3' UTR). Inhibition of miR-429-3p by its antagomir (A-miR-429) restored the expression of MKP-1 to a control level and consequently reduced the amyloidogenic processing of APP and Aß accumulation. More importantly, intranasal administration of A-miR-429 successfully ameliorated the deficits of hippocampal CA1 long-term potentiation and spatial learning and memory in AD model mice by suppressing extracellular signal-regulated kinase (ERK1/2)-mediated GluA1 hyperphosphorylation at Ser831 site, thereby increasing the surface expression of GluA1-containing α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors (AMPARs). Together, these results demonstrate that inhibiting miR-429-3p to upregulate MKP-1 effectively improves cognitive and synaptic functions in AD model mice, suggesting that miR-429/MKP-1 pathway may be a novel therapeutic target for AD treatment.

Lack of interferon regulatory factor 3 leads to anxiety/depression-like behaviors through disrupting the balance of neuronal excitation and inhibition in mice.

Disrupting the balance of neuronal excitation and inhibition (E/I) is an important pathogenic mechanism of anxiety and depression. Interferon regulatory factor 3 (IRF3) plays a key role in the innate immune response, and activation of IRF3 triggers the expression of type I interferons and downstream interferon-stimulated genes, which are associated with anxiety and depression. However, whether IRF3 participates in the pathogenesis of anxiety/depression by regulating E/I balance remains poorly understood. Here, we reported that global knockout (KO) of IRF3 (IRF3-/-) significantly increased anxiety/depression-like behaviors, but did not affect normal spatial learning and memory. Compared with wild type (WT) control mice, the E/I balance was disrupted, as reflected by enhanced glutamatergic transmission and decreased GABAergic transmission in the neurons of hippocampal CA1 and medial prefrontal cortex (mPFC) in IRF3-KO mice. Importantly, genetic rescue of IRF3 expression by adeno-associated virus (AAV) was sufficient to alleviate anxiety/depression-like behaviors and restore the neuronal E/I balance in IRF3-KO mice. Taken together, our results indicate that IRF3 is critical in maintaining neuronal E/I balance, thereby playing an essential role in ensuring emotional stability.

Octadecaneuropeptide Ameliorates Cognitive Impairments Through Inhibiting Oxidative Stress in Alzheimer's Disease Models.

BACKGROUND: Alzheimer's disease (AD) is a neurodegenerative disorder characterized by amyloid-ß peptide (Aß) deposition. Aß accumulation induces oxidative stress, leading to mitochondrial dysfunction, apoptosis, and so forth. Octadecaneuropeptide (ODN), a diazepam-binding inhibitor (DBI)-derived peptide, has been reported to have antioxidant properties. However, it is unclear whether ODN has neuroprotective effects in AD. OBJECTIVE: To profile the potential effects of ODN on AD. METHODS: We established a mouse model of AD via microinjection of Aß in the lateral ventricle. Utilizing a combination of western blotting assays, electrophysiological recordings, and behavioral tests, we investigated the neuroprotective effects of ODN on AD. RESULTS: DBI expression was decreased in AD model mice and cells. Meanwhile, ODN decreased Aß generation by downregulating amyloidogenic AßPP processing in HEK-293 cells stably expressing human Swedish mutant APP695 and BACE1 (2EB2). Moreover, ODN could inhibit Aß-induced oxidative stress in primary cultured cells and mice, as reflected by a dramatic increase in antioxidants and a decrease in pro-oxidants. We also found that ODN could reduce oxidative stress-induced apoptosis by restoring mitochondrial membrane potential, intracellular Ca2+ and cleaved caspase-3 levels in Aß-treated primary cultured cells and mice. More importantly, intracerebroventricular injection of ODN attenuated cognitive impairments as well as long-term potentiation in Aß-treated mice. CONCLUSION: These results suggest that ODN may exert a potent neuroprotective effect against Aß-induced neurotoxicity and memory decline via its antioxidant effects, indicating that ODN may be a potential therapeutic agent for AD.

Genetic inhibition of glutamate allosteric potentiation of GABAARs in mice results in hyperexcitability, leading to neurobehavioral abnormalities.

The imbalance between neuronal excitation and inhibition (E/I) in neural circuit has been considered to be at the root of numerous brain disorders. We recently reported a novel feedback crosstalk between the excitatory neurotransmitter glutamate and inhibitory γ-aminobutyric acid type A receptor (GABAAR)-glutamate allosteric potentiation of GABAAR functions through a direct binding of glutamate to the GABAAR itself. Here, we investigated the physiological significance and pathological implications of this cross-talk by generating the ß3E182G knock-in (KI) mice. We found that ß3E182G KI, while had little effect on basal GABAAR-mediated synaptic transmission, significantly reduced glutamate potentiation of GABAAR-mediated responses. These KI mice displayed lower thresholds for noxious stimuli, higher susceptibility to seizures and enhanced hippocampus-related learning and memory. Additionally, the KI mice exhibited impaired social interactions and decreased anxiety-like behaviors. Importantly, hippocampal overexpression of wild-type ß3-containing GABAARs was sufficient to rescue the deficits of glutamate potentiation of GABAAR-mediated responses, hippocampus-related behavioral abnormalities of increased epileptic susceptibility, and impaired social interactions. Our data indicate that the novel crosstalk among excitatory glutamate and inhibitory GABAAR functions as a homeostatic mechanism in fine-tuning neuronal E/I balance, thereby playing an essential role in ensuring normal brain functioning.

Scopolamine causes delirium-like brain network dysfunction and reversible cognitive impairment without neuronal loss.

Delirium is a severe acute neuropsychiatric syndrome that commonly occurs in the elderly and is considered an independent risk factor for later dementia. However, given its inherent complexity, few animal models of delirium have been established and the mechanism underlying the onset of delirium remains elusive. Here, we conducted a comparison of three mouse models of delirium induced by clinically relevant risk factors, including anesthesia with surgery (AS), systemic inflammation, and neurotransmission modulation. We found that both bacterial lipopolysaccharide (LPS) and cholinergic receptor antagonist scopolamine (Scop) induction reduced neuronal activities in the delirium-related brain network, with the latter presenting a similar pattern of reduction as found in delirium patients. Consistently, Scop injection resulted in reversible cognitive impairment with hyperactive behavior. No loss of cholinergic neurons was found with treatment, but hippocampal synaptic functions were affected. These findings provide further clues regarding the mechanism underlying delirium onset and demonstrate the successful application of the Scop injection model in mimicking delirium-like phenotypes in mice.

Dihydroartemisinin Induces O-GlcNAcylation and Improves Cognitive Function in a Mouse Model of Tauopathy.

BACKGROUND: Tauopathies are a group of neurodegenerative disorders, including Alzheimer's disease (AD) and frontotemporal lobar degeneration with tau pathology. Hyperphosphorylation modification promotes tau protein misfolding and aggregation into neurofibrillary tangles, leading to impairments of synaptic plasticity and learning and memory. However, very limited therapeutic strategies are available. OBJECTIVE: In the present study, we wanted to investigate the potential effects of Dihydroartemisinin (DHA) on tauopathies. METHODS: We constructed adeno-associated virus carrying hTau cDNA (AAVhTau) to establish a mouse model of tauopathy through intrahippocampal microinjection. Using a combination of behavioral test, electrophysiological recording, and western blotting assay, we examined the neuroprotective effects of DHA on learning and memory deficits in mice with tauopathy. RESULTS: DHA improved learning and memory and increased hippocampal CA1 long-term potentiation (LTP) in mice overexpressed human tau (hTau) in the hippocampus. More importantly, further study revealed that DHA could induce protein O-GlcNAcylation modification and reduce protein phosphorylation. O-GlcNAc transferase inhibitor alloxan could suppress DHA-induced protein O-GlcNAcylation, and subsequently prevent therapeutic effect of DHA on the deficits of learning and memory as well as synaptic plasticity in hTau mice. CONCLUSION: These results indicate that DHA may exert neuroprotective role in tauopathy through a crosstalk between O-GlcNAcylation and phosphorylation, suggesting a potential therapeutic for learning and memory deficits associated with tau pathology.

Aagab acts as a novel regulator of NEDD4-1-mediated Pten nuclear translocation to promote neurological recovery following hypoxic-ischemic brain damage.

Hypoxic-ischemic encephalopathy (HIE) is a main cause of mortality and severe neurologic impairment in the perinatal and neonatal period. However, few satisfactory therapeutic strategies are available. Here, we reported that a rapid nuclear translocation of phosphatase and tensin homolog deleted on chromosome TEN (PTEN) is an essential step in hypoxic-ischemic brain damage (HIBD)- and oxygen-glucose deprivation (OGD)-induced neuronal injures both in vivo and in vitro. In addition, we found that OGD-induced nuclear translocation of PTEN is dependent on PTEN mono-ubiquitination at the lysine 13 residue (K13) that is mediated by neural precursor cell expressed developmentally downregulated protein 4-1 (NEDD4-1). Importantly, we for the first time identified α- and γ-adaptin binding protein (Aagab) as a novel NEDD4-1 regulator to regulate the level of NEDD4-1, subsequently mediating Pten nuclear translocation. Finally, we demonstrated that genetic upregulation of Aagab or application of Tat-K13 peptide (a short interference peptide that flanks K13 residue of PTEN) not only reduced Pten nuclear translocation, but also significantly alleviated the deficits of myodynamia, motor and spatial learning and memory in HIBD model rats. These results suggest that Aagab may serve as a regulator of NEDD4-1-mediated Pten nuclear translocation to promote functional recovery following HIBD in neonatal rats, and provide a new potential therapeutic target to guide the clinical treatment for HIE.

Nicotine Promotes AßPP Nonamyloidogenic Processing via RACK1-Dependent Activation of PKC in SH-SY5Y-AßPP695 Cells.

BACKGROUND: Accumulation of amyloid-ß (Aß) peptides, generated from amyloid-ß precursor protein (AßPP) amyloidogenic processing, is one of the most salient disease hallmarks of Alzheimer's disease (AD). Nicotine is able to promote α-secretase-mediated AßPP nonamyloidogenic processing and increase the release of sAßPPα and C-terminal fragment of 83 amino acids (C83). However, the potential molecular mechanism remains elusive. OBJECTIVE: The aim of the present study was to investigate the effect of nicotine on AßPP processing in SH-SY5Y cells that stably express human Swedish mutant AßPP695 (SH-SY5Y-AßPP695). METHODS: The expression of AßPP and its C-terminal fragments including C99, C89, and C83, was measured in SH-SY5Y-AßPP695 cells treated with nicotine for 6 h. Protein kinase C (PKC) antagonist Ro30-8220 or agonist PMA was used to determine the role of PKC in AßPP processing. Lentivirus-mediated shRNA targeting receptor for activated C-kinase 1 (RACK1) gene was added into the media to knockdown RACK1 expression, and then AßPP processing was examined. RESULTS: The results showed that 6 h of nicotine exposure increased the expression of α-secretase (ADAM10) and C83 in a dose dependent manner. While the ß-secretase (BACE1), AßPP amyloidogenic processing products C89 and C99 as well as Aß peptides (including Aß40 and Aß42) remained unchanged. We also found that nicotine elevated the expression of phosphorylated PKC (P-PKC) and RACK1 on the cytomembrane. PKC antagonist Ro30-8220 treatment prevented the increase of ADAM10 and C83 by nicotine. Genetic knockdown RACK1 significantly inhibited P-PKC, and consequently abolished the increase of ADAM10 and C83 by nicotine. CONCLUSION: Taken together, these results indicate that nicotine effectively promotes AßPP nonamyloidogenic processing via RACK1-dependent activation of PKC in SH-SY5Y-AßPP695 cells and could be a potential molecule for AD treatment.

TRPV1 activation alleviates cognitive and synaptic plasticity impairments through inhibiting AMPAR endocytosis in APP23/PS45 mouse model of Alzheimer's disease.

Alzheimer's disease (AD) is one of the most common causes of neurodegenerative diseases in the elderly. The accumulation of amyloid-ß (Aß) peptides is one of the pathological hallmarks of AD and leads to the impairments of synaptic plasticity and cognitive function. The transient receptor potential vanilloid 1 (TRPV1), a nonselective cation channel, is involved in synaptic plasticity and memory. However, the role of TRPV1 in AD pathogenesis remains largely elusive. Here, we reported that the expression of TRPV1 was decreased in the brain of APP23/PS45 double transgenic AD model mice. Genetic upregulation of TRPV1 by adeno-associated virus (AAV) inhibited the APP processing and Aß deposition in AD model mice. Meanwhile, upregulation of TRPV1 ameliorated the deficits of hippocampal CA1 long-term potentiation (LTP) and spatial learning and memory through inhibiting GluA2-containing α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPAR) endocytosis. Furthermore, pharmacological activation of TRPV1 by capsaicin (1 mg/kg, i.p.), an agonist of TRPV1, dramatically reversed the impairments of hippocampal CA1 LTP and spatial learning and memory in AD model mice. Taken together, these results indicate that TRPV1 activation effectively ameliorates cognitive and synaptic functions through inhibiting AMPAR endocytosis in AD model mice and could be a novel molecule for AD treatment.

MKP-1 reduces Aß generation and alleviates cognitive impairments in Alzheimer's disease models.

Mitogen-activated protein kinase (MAPK) phosphatase 1 (MKP-1) is an essential negative regulator of MAPKs by dephosphorylating MAPKs at both tyrosine and threonine residues. Dysregulation of the MAPK signaling pathway has been associated with Alzheimer's disease (AD). However, the role of MKP-1 in AD pathogenesis remains elusive. Here, we report that MKP-1 levels were decreased in the brain tissues of patients with AD and an AD mouse model. The reduction in MKP-1 gene expression appeared to be a result of transcriptional inhibition via transcription factor specificity protein 1 (Sp1) cis-acting binding elements in the MKP-1 gene promoter. Amyloid-ß (Aß)-induced Sp1 activation decreased MKP-1 expression. However, upregulation of MKP-1 inhibited the expression of both Aß precursor protein (APP) and ß-site APP-cleaving enzyme 1 by inactivating the extracellular signal-regulated kinase 1/2 (ERK)/MAPK signaling pathway. Furthermore, upregulation of MKP-1 reduced Aß production and plaque formation and improved hippocampal long-term potentiation (LTP) and cognitive deficits in APP/PS1 transgenic mice. Our results demonstrate that MKP-1 impairment facilitates the pathogenesis of AD, whereas upregulation of MKP-1 plays a neuroprotective role to reduce Alzheimer-related phenotypes. Thus, this study suggests that MKP-1 is a novel molecule for AD treatment.

Effects of secretin on neuronal activity and feeding behavior in central amygdala of rats.

Previous studies have shown that secretin and secretin receptors are expressed in central amygdala neurons. By using both in vivo extracellular recording as well as behavioral test, we investigated the direct electrophysiological effects of secretin in the central amygdala and its involvement in feeding behavior. Micro-pressure ejection of secretin increased the spontaneous firing rate by 104.22±26.18% in 13 out of the 27 central amygdala neurons. In other 6 out of the 27 neurons, secretin decreased the firing rate by 68.80±12.10%. Firing patter analysis showed that secretin did not change the firing pattern significantly. Further electrophysiological recordings revealed that secretin decreased the firing rate of glucose-sensitive neurons. In behavioral test, microinjection of secretin into the central amygdala significantly reduced cumulative food intake through cAMP-activated protein kinase activation. Based on the present electrophysiological and behavioral findings, we hypothesized that secretin may suppress food intake by its modulation of spontaneous firing of central amygdala neurons.