Sodium butyrate activates the KATP channels to regulate the mechanism of Parkinson's disease microglia model inflammation.

BACKGROUND: Parkinson's disease (PD) is a common neurodegenerative disorder. Microglia-mediated neuroinflammation has emerged as an involving mechanism at the initiation and development of PD. Activation of adenosine triphosphate (ATP)-sensitive potassium (KATP ) channels can protect dopaminergic neurons from damage. Sodium butyrate (NaB) shows anti-inflammatory and neuroprotective effects in some animal models of brain injury and regulates the KATP channels in islet ß cells. In this study, we aimed to verify the anti-inflammatory effect of NaB on PD and further explored potential molecular mechanisms. METHODS: We established an in vitro PD model in BV2 cells using 1-methyl-4-phenylpyridinium (MPP+ ). The effects of MPP+ and NaB on BV2 cell viability were detected by cell counting kit-8 assays. The morphology of BV2 cells with or without MPP+ treatment was imaged via an optical microscope. The expression of Iba-1 was examined by the immunofluorescence staining. The intracellular ATP content was estimated through the colorimetric method, and Griess assay was conducted to measure the nitric oxide production. The expression levels of pro-inflammatory cytokines and KATP channel subunits were evaluated by reverse transcription-quantitative polymerase chain reaction and western blot analysis. RESULTS: NaB (5 mM) activated the KATP channels through elevating Kir6.1 and Kir6.1 expression in MPP+ -challenged BV2 cells. Both NaB and pinacidil (a KATP opener) suppressed the MPP+ -induced activation of BV2 cells and reduced the production of nitrite and pro-inflammatory cytokines in MPP+ -challenged BV2 cells. CONCLUSION: NaB treatment alleviates the MPP+ -induced inflammatory responses in microglia via activation of KATP channels.

Frameshift mutation of Timm8a1 gene in mouse leads to an abnormal mitochondrial structure in the brain, correlating with hearing and memory impairment.

BACKGROUND: Deafness-dystonia-optic neuronopathy (DDON) syndrome is a progressive X-linked recessive disorder characterised by deafness, dystonia, ataxia and reduced visual acuity. The causative gene deafness/dystonia protein 1 (DDP1)/translocase of the inner membrane 8A (TIMM8A) encodes a mitochondrial intermembrane space chaperon. The molecular mechanism of DDON remains unclear, and detailed information on animal models has not been reported yet. METHODS AND RESULTS: We characterized a family with DDON syndrome, in which the affected members carried a novel hemizygous variation in the DDP1 gene (NM_004085.3, c.82C>T, p.Q28X). We then generated a mouse line with the hemizygous mutation (p.I23fs49X) in the Timm8a1 gene using the clustered regularly interspaced short palindromic repeats /Cas9 technology. The deficient DDP1 protein was confirmed by western blot assay. Electron microscopic analysis of brain samples from the mutant mice indicated abnormal mitochondrial structure in several brain areas. However, Timm8a1I23fs49X/y mutation did not affect the import of mitochondria inner member protein Tim23 and outer member protein Tom40 as well as the biogenesis of the proteins in the mitochondrial oxidative phosphorylation system and the manganese superoxide dismutase (MnSOD / SOD-2). The male mice with Timm8a1I23fs49X/y mutant exhibited less weight gain, hearing impairment and cognitive deficit. CONCLUSION: Our study suggests that frameshift mutation of the Timm8a1 gene in mice leads to an abnormal mitochondrial structure in the brain, correlating with hearing and memory impairment. Taken together, we have successfully generated a mouse model bearing loss-of-function mutation in Timm8a1.

Untargeted metabolomics coupled with chemometrics analysis reveals potential non-volatile markers during oolong tea shaking.

Shaking is a critical process in the formation of oolong tea quality, although the metabolites and their changes in this sensitive process have not yet been determined. In this study, untargeted analysis based on ultrahigh-performance liquid chromatography/quadrupole-Orbitrap mass spectrometry was conducted to comprehensively profile metabolite changes in different cultivars. Theanine glucoside was identified for the first time in oolong tea. Hierarchical cluster analysis indicated that shaking caused major changes in metabolite levels in oolong tea. Seventy-one, 83 and 73 potential features showed significant differences between pre- and post-shaking samples for var. sinensiscv. "Zimudan", "Shuixian", and "Huangmeigui," respectively. Chemometrics analysis of the three cultivars led to the identification of 18 shared metabolites, including epigallocatechin gallate, phenylalanine, tryptophan, proline, and hydroxy-jasmonic acid, as potential markers. This study identified the metabolites that allow monitoring of tea quality formation during both processing and preservation, and it provides a novel strategy for data reduction in studies to discover key metabolites.

AAV9-Mediated Cdk5 Inhibitory Peptide Reduces Hyperphosphorylated Tau and Inflammation and Ameliorates Behavioral Changes Caused by Overexpression of p25 in the Brain.

BACKGROUND: Under stress stimulation, p25 is generated by cleavage of p35 and acts as an activator of cyclin-dependent kinase 5 (Cdk5) like p35. Unlike Cdk5/p35, which is important for brain development, aberrant activity of Cdk5/p25 plays a pathological role in neurodegenerative diseases, such as Alzheimer's disease, by inducing hyperphosphorylation of downstream substrates related to pathological progression. A truncated fragment of the c-terminus of p35, the Cdk5 inhibitory peptide (CIP), selectively inhibits Cdk5/ p25 activity in cultured neurons and in CIP/p25 tetra-transgenic mice. OBJECTIVE: First, we aimed to establish a p25 overexpression adult mouse model, then to evaluate whether CIP delivered by adeno-associated virus serotype 9 (AAV9) can ameliorate neuronal toxicity induced by p25. METHODS: The p25 overexpression mouse model was established by intracerebroventricular (i.c.v.) injection of AAV8-GFP-p25 in 8-week-old mice. One month later, these mice were i.c.v. injected with AAV9-CIP-T2A-mCherry or AAV9 vector as control. Pathological and behavioral changes were assessed 3-months post-injection in all mice. RESULTS: The p25 overexpression mice displayed hyperphosphorylation of tau at multiple sites, activation of astrocytes, and elevated inflammatory factors, including IL-1 and TNF-α, which were significantly decreased by the administration of CIP. However, Aß deposition and microgliosis were not obvious in p25 overexpression mice. In addition, a significant learning decline and anxiety-like behavior were induced by p25 toxicity, and CIP treatment improved learning ability in p25 mice. CONCLUSION: AAV-mediated p25 overexpression mouse model is easy to construct to study p25-induced neuronal toxicity. Application of CIP after p25 insult reverses the pathological changes and behavioral abnormalities.

Glucosylation of (Z)-3-hexenol informs intraspecies interactions in plants: A case study in Camellia sinensis.

Plants emit a variety of volatiles in response to herbivore attack, and (Z)-3-hexenol and its glycosides have been shown to function as defence compounds. Although the ability to incorporate and convert (Z)-3-hexenol to glycosides is widely conserved in plants, the enzymes responsible for the glycosylation of (Z)-3-hexenol remained unknown until today. In this study, uridine-diphosphate-dependent glycosyltransferase (UGT) candidate genes were selected by correlation analysis and their response to airborne (Z)-3-hexenol, which has been shown to be taken up by the tea plant. The allelic proteins UGT85A53-1 and UGT85A53-2 showed the highest activity towards (Z)-3-hexenol and are distinct from UGT85A53-3, which displayed a similar catalytic efficiency for (Z)-3-hexenol and nerol. A single amino acid exchange E59D enhanced the activity towards (Z)-3-hexenol, whereas a L445M mutation reduced the catalytic activity towards all substrates tested. Transient overexpression of CsUGT85A53-1 in tobacco significantly increased the level of (Z)-3-hexenyl glucoside. The functional characterization of CsUGT85A53 as a (Z)-3-hexenol UGT not only provides the foundation for the biotechnological production of (Z)-3-hexenyl glucoside but also delivers insights for the development of novel insect pest control strategies in tea plant and might be generally applicable to other plants.

Cdk5 Inhibitory Peptide Prevents Loss of Dopaminergic Neurons and Alleviates Behavioral Changes in an MPTP Induced Parkinson's Disease Mouse Model.

Parkinson's disease (PD) is one of the most affected neurodegenerative diseases in the world. Deregulation of cyclin-dependent kinase 5 (Cdk5) is believed to play an important role in neurodegenerative diseases including PD. p25 is a cleavage peptide of p35, a physiologic activator of Cdk5. p25 combines to Cdk5 and leads to the hyperactivity of Cdk5, which in turn hyperphosphorylates downstream substrates and leads to neuroinflammation and apoptosis of neurons. Previously, we have demonstrated that adeno-associated virus serotype-9 (AAV9) mediated Cdk5 inhibitory peptide (CIP) inhibits the activity of Cdk5/p25 complex and alleviates pathologic and behavioral changes in Alzheimer's disease mouse model. In this study, we evaluated whether AAV9-CIP protected dopaminergic (DA) neurons in 1-methyl-4-phe-nyl-1,2,3,6-tetrahydropyridine-probenecid (MPTP/p) induced PD mouse model. The data showed that administration of AAV9-CIP by intracerebroventricular injection 1 week before MPTP/p exposure protected loss of DA neurons in substantia nigra compact of the model mice. Importantly, AAV9-CIP also alleviated the motor and anxiety-like symptoms of the disease animals. In summary, AAV9 mediated CIP might be a potential intervention for PD.

Disruption of AT-hook 1 domain in MeCP2 protein caused behavioral abnormality in mice.

MECP2 is the causative gene for autism spectrum disorders, including Rett syndrome, a regressive neurodevelopmental rare disease mainly occurring in girls. Except for the distinct methyl-CpG binding domain and the transcriptional repression domain in MeCP2, three AT-hook-like domains have recently been identified. Several mutations in AT-hook 1 domain have been reported in autism cases or Rett database. However, the role of AT-hook 1 domain is still unclear. In this study, we generated a mouse line carrying deletion of eight conserved amino acids in AT-hook 1 domain by clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 technology. Mecp2ΔAT-hook1/y mutant male mice exhibited low locomotor activity, motor incoordination and cognitive deficit. In addition, these mutant mice exhibited increased anxiety. Moreover, pain insensitivity was noted in the mutant males. However, the social interactions were unaffected in AT-hook 1 mutant mice. Thinner CA1 region of the hippocampus was observed in the mutant mice. On the molecular basis, Western blot analysis showed increased expression of mutant MeCP2 protein in the cortex. Additionally, several genes expressed specifically in inhibitory neurons were markedly changed in the cerebrum. Taken together, these data demonstrate that disruption of AT-hook 1 domain in MeCP2 caused behavioral abnormality in mice, which suggests that AT-hook 1 is a critical region for the function of MeCP2 protein.

A novel mutation R190H in the AT-hook 1 domain of MeCP2 identified in an atypical Rett syndrome.

BACKGROUND: Mutations in Methyl-CpG binding protein 2 (MECP2) have been identified as the disease-causing mutations in Rett Syndrome (RTT). However, no mutation in the AT-hook 1 domain of MECP2 has been reported in RTT yet. The function of AT-hook 1 domain of MECP2 has not been described either. METHODS: The clinical and radiological features of a girl with progressive hyperactivity and loss of acquired linguistic and motor functions were presented. Next generation sequencing was used to screen the causative gene. Effect of the mutant protein on histone 3 methylation was assessed in vitro experiment. RESULTS: The patient was diagnosed with an atypical RTT at the age of nine. Magnetic resonance imaging revealed a loss of whole-brain volume and abnormal myelination. Genetic analysis identified a de novo novel missense mutation of MECP2 (NM_004992, c.570G->A, p.Arg190His). This mutation is located in the AT-hook 1 domain of MeCP2 protein. Overexpression of the mutant MeCP2 in cultured neuroblastoma cells SH-SY5Y revealed increased level of dimethylated histone 3 lysine 9, a transcriptional repressor marker. CONCLUSION: A novel missense mutation in AT-hook 1 domain of MeCP2 was identified in a patient with atypical RTT. Clinical data and in vitro experiment result imply that R190H mutation in AT-hook1 may cause dysfunction of MeCP2 and be a pathogenic variant.

Adeno-associated virus 9-mediated Cdk5 inhibitory peptide reverses pathologic changes and behavioral deficits in the Alzheimer's disease mouse model.

Cyclin-dependent kinase 5 (Cdk5), which binds to and is activated by p35, phosphorylates multiple substrates and plays an essential role in the development and function of the CNS; however, proteolytic production of p25 from p35 under stress conditions leads to the inappropriate activation of Cdk5 and contributes to hyperphosphorylation of τ and other substrates that are related to the pathogenesis of Alzheimer's disease. Selective inhibition of aberrant Cdk5 activity via genetic overexpression of Cdk5 inhibitory peptide (CIP) reduces pathologic changes and prevents brain atrophy and memory loss in p25-transgenic mice. In the present study, we delivered adeno-associated virus 9 carrying green fluorescent protein-CIP (AAV9-GFP-CIP) to brain cells via intracerebroventricular infusion in amyloid precursor protein/presenilin 1 (APP/PS1) double-transgenic 3-mo-old mice after the occurrence of ß-amyloid (Aß) aggregation and the hyperphosphorylation of τ. Three months of treatment of AAV9-GFP-CIP reduced pathologic changes, including τ hyperphosphorylation, (Aß) deposit, astrocytosis, and microgliosis, which were correlated with the reversal of memory loss and anxiety-like behavior observed in APP/PS1 mice. The neuroprotection effect of AAV9-GFP-CIP lasted an additional 7 mo-the end point of the study. These findings provide a novel strategy to selectively target Cdk5 for the treatment of Alzheimer's disease.-He, Y., Pan, S., Xu, M., He, R., Huang, W., Song, P., Huang, J., Zhang, H.-T., Hu, Y. Adeno-associated virus 9-mediated Cdk5 inhibitory peptide reverses pathologic changes and behavioral deficits in the Alzheimer's disease mouse model.

Cdk5/p25 specific inhibitory peptide TFP5 rescues the loss of dopaminergic neurons in a sub-acute MPTP induced PD mouse model.

Parkinson's disease (PD) is pathologically characterized by progressively loss of dopaminergic (DA) neurons in the substantia nigra pars compacta (SNpc) and the formation of Lewy bodies. In 1-methyl-4-phenyl-1, 2, 3, 6-tetrahydropyridine (MPTP) induced PD mice models, the calpain- cyclin-dependent kinase 5 (Cdk5)-myocyte enhancer factor 2 (MEF2) signaling has been proven in governing dopaminergic neuronal death. Under MPTP insult, p35 is cleaved by calpain into p25, which binds to Cdk5 and exhibits hyperactivity of Cdk5/p25. Cdk5/p25 inactivates MEF2, a survivor factor, which is critical for DA neuronal death. In this study, neuroprotective effect of the Cdk5/p25 specific peptide, TFP5, was evaluated in sub-acute MPTP induced PD mouse model by intraperitoneal (i.p.) injection of MPTP for five consecutive days. The results indicated that the levels of p35 and p25, and p25/p35 ratio increased in the sub-acute MPTP mice. TFP5 broadly reached cortex neuron, hippocampus and SNpc areas after i.p. injections. Pretreatment with 45mg/kg/day TFP5, as well as 10mgkg/day Cdk5 inhibitor roscovitine, for three days significantly rescued DA neuronal loss up to 9.8% or 9.7% respectively compared to the saline treated group. Treatment of TFP5 and roscovitine reduced the levels of inactive form of MEF2 and cleaved caspase 3, thus protected apoptosis of DA neurons against MPTP insult. Our results propose that TFP5 might be a potential therapeutic candidate for PD.

Neurodegeneration-Like Pathological and Behavioral Changes in an AAV9-Mediated p25 Overexpression Mouse Model.

BACKGROUND: The transgenic mice models overexpressing human p25 contribute greatly to the in vivo neurotoxic mechanism of p25 in neurodegenerative diseases. However, it is time-consuming to manipulate existing transgenic mice models. OBJECTIVE: Here we aim to establish a novel mouse model of neurodegeneration by overexpressing p25 mediated by recombinant adeno-associated virus serotype 9 (rAAV9). METHODS: AAV9-GFP-p25 encoding GFP-fused p25 driven by synapsin promoter, and the control, AAV9-GFP, were delivered in mice by tail-vein injection. Assessments of p25 expression, neurodegenerative pathology, and behavioral changes were performed. RESULTS: GFP expression was detected by in vivo imaging as early as one week after virus injection. Notably, widespread expression of p25 was obviously found in cortex, hippocampus, and cerebellum in AAV9-GFP-p25 mice. Moreover, decreased hippocampus volumes in AAV9-GFP-p25 mice were detected by 7T MRI examination about one month after injection. Further, these AAV9-GFP-p25 mice exhibited progressive memory impairment from three-month to six-month after virus injection. At last, hyperphosphorylated tau, neurofibrillary tangles, activated astrocytes and microglia cells were elevated in these p25 mice at about six months after virus delivery. However, amyloid-ß plaques, overt neuronal loss, and apoptosis in the hippocampus and cortex were not significantly induced by AAV9-mediated p25 overexpression. CONCLUSION: The AAV9-mediated p25 overexpression mouse model, which is a practical model exhibiting neurodegeneration-like pathological and behavioral changes, provides an easier and time-saving method to explore the functions of p25 in vivo, as well as an alternative tool for development of drugs against neurotoxic of p25.