Role of Different Doses of Ketamine in Postoperative Neurocognitive Function in Aged Mice Undergoing Partial Hepatectomy by Regulating the Bmal1/NMDA/NF-Κb Axis

<b><i>Background:</i></b> The current study set out to probe the function of different doses of ketamine in postoperative neurocognitive disorder (PND) in aged mice undergoing partial hepatectomy (PH) with the involvement of the brain and muscle aryl hydrocarbon receptor nuclear translocator-like protein 1 (Bmal1)/n-methyl-D-aspartate (NMDA)/nuclear factor-kappa B (NF-κB) axis. <b><i>Methods:</i></b> First, aged mice were intraperitoneally injected with different doses of ketamine prior to surgery, followed by hepatic lobectomy. Afterward, mice cognitive function was assessed. In addition, Bmal1 mRNA expression patterns were quantified, while NMDA 2B receptor, NF-κB p65, synapsin 1, and postsynaptic density 95 (PSD95) levels were determined; the release of inflammatory factors was detected, and ionized calcium-binding adapter molecule-1 expression was measured to quantify microglia activation. In addition, Bmal1-knockout (Bmal1-KO) mice were intraperitoneally injected with a subanesthetic dose of ketamine to verify the mechanism of Bmal1 in regulating the NMDA 2B subunit (NR2B)/NF-κB axis to affect PH in aged patients. <b><i>Results:</i></b> After PH, hippocampal-dependent memory was impaired, and synapsin 1 and PSD95 levels were downregulated. On the other hand, PH diminished Bmal1 expression but elevated NR2B and NF-κB p65 levels and anesthetic doses of ketamine further regulated the Bmal1/NMDA/NF-κB axis. In Bmal1-KO mice, the NMDA/NF-κB axis was activated, the release of inflammatory cytokines was promoted, and hippocampus-dependent memory was impaired, which were reversed by a subanesthetic dose of ketamine. <b><i>Conclusion:</i></b> Altogether, findings obtained in our study indicated that a subanesthetic dose of ketamine activated Bmal1, downregulated the NMDA/NF-κB axis, and reduced inflammation and microglia activation to alleviate PND in aged mice undergoing PH.

Probing the multimodal fungiform papilla: complex peripheral nerve endings of chorda tympani taste and mechanosensitive fibers before and after Hedgehog pathway inhibition

The fungiform papilla (FP) is a gustatory and somatosensory structure incorporating chorda tympani (CT) nerve fibers that innervate taste buds (TB) and also contain somatosensory endings for touch and temperature. Hedgehog (HH) pathway inhibition eliminates TB, but CT innervation remains in the FP. Importantly, after HH inhibition, CT neurophysiological responses to taste stimuli are eliminated, but tactile responses remain. To examine CT fibers that respond to tactile stimuli in the absence of TB, we used Phox2b-Cre; Rosa26LSL−TdTomato reporter mice to selectively label CT fibers with TdTomato. Normally CT fibers project in a compact bundle directly into TB, but after HH pathway inhibition, CT fibers reorganize and expand just under the FP epithelium where TB were. This widened expanse of CT fibers coexpresses Synapsin-1, β-tubulin, S100, and neurofilaments. Further, GAP43 expression in these fibers suggests they are actively remodeling. Interestingly, CT fibers have complex terminals within the apical FP epithelium and in perigemmal locations in the FP apex. These extragemmal fibers remain after HH pathway inhibition. To identify tactile end organs in FP, we used a K20 antibody to label Merkel cells. In control mice, K20 was expressed in TB cells and at the base of epithelial ridges outside of FP. After HH pathway inhibition, K20 + cells remained in epithelial ridges but were eliminated in the apical FP without TB. These data suggest that the complex, extragemmal nerve endings within and disbursed under the apical FP are the mechanosensitive nerve endings of the CT that remain after HH pathway inhibition.

Synuclein Regulates Synaptic Vesicle Clustering and Docking at a Vertebrate Synapse

Neurotransmission relies critically on the exocytotic release of neurotransmitters from small synaptic vesicles (SVs) at the active zone. Therefore, it is essential for neurons to maintain an adequate pool of SVs clustered at synapses in order to sustain efficient neurotransmission. It is well established that the phosphoprotein synapsin 1 regulates SV clustering at synapses. Here, we demonstrate that synuclein, another SV-associated protein and synapsin binding partner, also modulates SV clustering at a vertebrate synapse. When acutely introduced to unstimulated lamprey reticulospinal synapses, a pan-synuclein antibody raised against the N-terminal domain of α-synuclein induced a significant loss of SVs at the synapse. Both docked SVs and the distal reserve pool of SVs were depleted, resulting in a loss of total membrane at synapses. In contrast, antibodies against two other abundant SV-associated proteins, synaptic vesicle glycoprotein 2 (SV2) and vesicle-associated membrane protein (VAMP/synaptobrevin), had no effect on the size or distribution of SV clusters. Synuclein perturbation caused a dose-dependent reduction in the number of SVs at synapses. Interestingly, the large SV clusters appeared to disperse into smaller SV clusters, as well as individual SVs. Thus, synuclein regulates clustering of SVs at resting synapses, as well as docking of SVs at the active zone. These findings reveal new roles for synuclein at the synapse and provide critical insights into diseases associated with α-synuclein dysfunction, such as Parkinson’s disease.

Bassoon controls synaptic vesicle pools via regulation of presynaptic phosphorylation and cAMP homeostasis

Neuronal presynaptic terminals contain hundreds of neurotransmitter-filled synaptic vesicles (SVs). The morphologically uniform SVs differ in their release competence segregating into functional pools that differentially contribute to neurotransmission. The presynaptic scaffold bassoon is required for neurotransmission, but the underlying molecular mechanisms are unknown. We report that glutamatergic synapses lacking bassoon featured a decreased SV release competence and increased resting pool of SV as observed by imaging of SV release in cultured neurons. Further analyses in vitro and in vivo revealed a dysregulation of CDK5/calcineurin and cAMP/PKA presynaptic signalling resulting in an aberrant phosphorylation of their downstream effectors synapsin 1 and SNAP25, which are well-known regulators of SV release competence. An acute pharmacological restoration of physiological CDK5 and cAMP/PKA activity fully normalised the SV pools in neurons lacking bassoon. Finally, we demonstrated that CDK5-dependent regulation of PDE4 activity controls SV release competence by interaction with cAMP/PKA signalling. These data reveal that bassoon organises SV pools via regulation of presynaptic phosphorylation and indicate an involvement of PDE4 in the control of neurotransmitter release.

Sirtuin 1 Regulates Synapsin 1 in POMC-Producing N43-5 Neurons via FOXO1

The nutrient-sensor protein Sirtuin 1 (Sirt1; silent mating type information regulation 2 homolog 1) has been shown to have significant and opposing effects on insulin resistance, leptin resistance, and body weight in the periphery and the brain. In the hypothalamic arcuate nucleus (ARC) of the brain, Sirt1 increases in the obese state and acts to promote weight gain as well as insulin and leptin resistance by increasing the orexigenic neuropeptides Agouti-related protein (AgRP) and neuropeptide Y (NPY), and in a distinct set of ARC neurons, by decreasing POMC and thus its anorexigenic derivative alpha-melanocyte stimulating hormone (alpha-MSH) (1). Sirt1’s actions on these neuropeptides are mediated at least in part by the deacetylation of the transcription factor forkhead box O1 (FOXO1). Another mechanism by which Sirt1 regulates body weight appears to be through mediating changes in the synapses of these neuropeptide-producing ARC neurons. For example, a previous study demonstrated that Sirt1 inhibition with the specific Sirt1 inhibitor, Ex-527, decreased AgRP-NPY inhibitory synaptic input on POMC neurons, which suggests that the obesity-induced increase in ARC Sirt1 would increase AgRP-NPY inhibition of POMC neurons thus promoting weight gain (2). The present study investigated how Sirt1 regulates synapses specifically in POMC-producing N43-5 neurons. Results reveal that inhibition of Sirt1 with Ex-527 significantly increased the presynaptic marker Synapsin 1 (Syn1) in N43-5 neurons. Furthermore, we investigated whether the Sirt1 target, FOXO1, mediates these synaptic changes. FOXO1 overexpression significantly decreased Syn1 and transfection of mutant FOXO1 significantly increased Syn1. Overall, our results suggest that Sirt1 regulates synapses of POMC neurons and does so in a manner that differs from Sirt1’s regulation of AgRP-NPY neuronal synapses. Future work will elucidate the mechanisms and consequences of Sirt1 and FOXO1 regulation of POMC neuron synapses under different nutritional conditions in vitro and in vivo. (1) Cyr, N. E., Steger, J. S., Toorie, A. M., Yang, J. Z., Stuart, R., Nillni, E. A. (2014). Central Sirt1 Regulates Body Weight and Energy Expenditure Along With the POMC-Derived Peptide α-MSH and the Processing Enzyme CPE Production in Diet-Induced Obese Male Rats, Endocrinology, 155(7), 2423–2435. (2) Dietrich, M. O., Antunes, C., Geliang, G., Liu, Z., Borok, E., Nie, Y., . . . Horvath, T. L. (2010). Agrp neurons mediate Sirt1’s action on the melanocortin system and energy balance: Roles for Sirt1 in neuronal firing and synaptic plasticity. The Journal of Neuroscience, 30(35), 11815–11825.

A Novel Role of Nuclear and Membrane Receptor on Isoflavone-Induced Neuritogenesis and Synaptogenesis

Thyroid hormone (TH) receptor (TR) and estrogen receptor (ER) play crucial roles in brain development. TR and ER are involved in dendrite growth, spines, and synapse formation in neurons. Soybean isoflavones, such as genistein, daidzein, and daidzein metabolite, S-equol are known to exert their action through TR, ER, and GPER1, a G-protein-coupled ER. However, the mechanisms of isoflavones action on brain development, especially during neuritogenesis and synaptogenesis, have not yet been extensively studied. We evaluated the effects of isoflavones using mouse primary cerebellar culture, astrocyte-enriched culture, Neuro-2A clonal cells, and co-culture with neurons and astrocytes. Soybean isoflavone augmented TH- or estradiol (E2)-mediated dendrite arborization of Purkinje cells. Such augmentation was suppressed by G15, a selective GPER1 antagonist, and ICI 182.780, an antagonist for ERs in both cultures. The knockdown of nuclear TRs or ERs also significantly reduced the dendrite arborization of Purkinje cells. It also increased the mRNA levels of TH-responsive genes, including Mbp, Bdnf, Rc3, Ntf3, Camk2b, Hr, and also Syn1, Syp, and Psd95 that are involved in synaptic plasticity. Isoflavones also increased the protein levels of synapsin-1, synaptophysin, and PSD95 in dendrite and membrane fraction of the cerebellar culture. To study further the molecular mechanism, we used Neuro-2A clonal cells. Isoflavones also induced neurite growth of Neuro-2A. The knockdown of TRs, ERs, and GPR30 by RNAi reduced isoflavones-induced neurite growth. Moreover, the co-culture study of Neuro-2A and astrocytes also showed an increase in isoflavones-induces neurite growth. In addition, isoflavones increased the localization of synapsin-1 or synaptophysin and F-actin in filopodia tips during Neuro-2A differentiation. The knockdown of nuclear ERs or GPR30 significantly reduced the number of filopodia and synapsin-1 or synaptophysin expression levels in neurite and membrane fractions. However, there are no significant effects of filopodia formation after co-culture with astrocytes. These results indicate that nuclear ERs and TRs play an essential role in isoflavones-induces neuritogenesis. Non-genomics signaling through membrane receptor and F-actin are necessary for the isoflavones-induces synaptogenesis. Astrocytes-neurons communication also increased isoflavones-induced neuritogenesis, but not synaptogenesis.

Aβ1-16 controls synaptic vesicle pools at excitatory synapses via cholinergic modulation of synapsin phosphorylation

Amyloid beta (Aβ) is linked to the pathology of Alzheimer’s disease (AD). At physiological concentrations, Aβ was proposed to enhance neuroplasticity and memory formation by increasing the neurotransmitter release from presynapse. However, the exact mechanisms underlying this presynaptic effect as well as specific contribution of endogenously occurring Aβ isoforms remain unclear. Here, we demonstrate that Aβ1-42 and Aβ1-16, but not Aβ17-42, increased size of the recycling pool of synaptic vesicles (SV). This presynaptic effect was driven by enhancement of endogenous cholinergic signalling via α7 nicotinic acetylcholine receptors, which led to activation of calcineurin, dephosphorylation of synapsin 1 and consequently resulted in reorganization of functional pools of SV increasing their availability for sustained neurotransmission. Our results identify synapsin 1 as a molecular target of Aβ and reveal an effect of physiological concentrations of Aβ on cholinergic modulation of glutamatergic neurotransmission. These findings provide new mechanistic insights in cholinergic dysfunction observed in AD.

Human brain organoids assemble functionally integrated bilateral optic vesicles

During embryogenesis, optic vesicles develop from the diencephalon via a complex process of organogenesis. Using iPSC-derived human brain organoids, we attempted to simplify the complexities and demonstrate the formation of forebrain-associated bilateral optic vesicles, cellular diversity, and functionality. Around day thirty, brain organoids could assemble optic vesicles, which progressively develop as visible structures within sixty days. These optic vesicle-containing brain organoids (OVB-Organoids) constitute a developing optic vesicle's cellular components, including the primitive cornea and lens-like cells, developing photoreceptors, retinal pigment epithelia, axon-like projections, and electrically active neuronal networks. Besides, OVB-Organoids also display synapsin-1, CTIP-positive, myelinated cortical neurons, and microglia. Interestingly, various light intensities could trigger photoreceptor activity of OVB-Organoids, and light sensitivities could be reset after a transient photo bleach blinding. Thus, brain organoids have the intrinsic ability to self-organize forebrain-associated primitive sensory structures in a topographically restricted manner and can allow conducting interorgan interaction studies within a single organoid.

Suppressing Cdk5 Activity by Luteolin Inhibits MPP+-Induced Apoptotic of Neuroblastoma through Erk/Drp1 and Fak/Akt/GSK3β Pathways

Parkinson’s disease (PD) is characterized by the progressive degeneration of dopaminergic neurons. The cause of PD is still unclear. Oxidative stress and mitochondrial dysfunction have been linked to the development of PD. Luteolin, a non-toxic flavonoid, has become interested in an alternative medicine, according to its effects on anti-oxidative stress and anti-apoptosis, although the underlying mechanism of luteolin on PD has not been fully elucidated. This study aims to investigate whether luteolin prevents neurotoxicity induction by 1-methyl-4-phenylpyridinium iodide (MPP+), a neurotoxin in neuroblastoma SH-SY5Y cells. The results reveal that luteolin significantly improved cell viability and reduced apoptosis in MPP+-treated cells. Increasing lipid peroxidation and superoxide anion (O2ˉ), including mitochondrial membrane potential (Δψm) disruption, is ameliorated by luteolin treatment. In addition, luteolin attenuated MPP+-induced neurite damage via GAP43 and synapsin-1. Furthermore, Cdk5 is found to be overactivated and correlated with elevation of cleaved caspase-3 activity in MPP+-exposed cells, while phosphorylation of Erk1/2, Drp1, Fak, Akt and GSK3β are inhibited. In contrast, luteolin attenuated Cdk5 overactivation and supported phosphorylated level of Erk1/2, Drp1, Fak, Akt and GSK3β with reducing in cleaved caspase-3 activity. Results indicate that luteolin exerts neuroprotective effects via Cdk5-mediated Erk1/2/Drp1 and Fak/Akt/GSK3β pathways, possibly representing a potential preventive agent for neuronal disorder.