Neuroprotection of Kaji-Ichigoside F1 via the BDNF/Akt/mTOR Signaling Pathways against NMDA-Induced Neurotoxicity

Kaji-ichigoside F1 (KF1), a natural oleanane-type triterpenoid saponin, is the main active constituent from Rosa roxburghii. In the southwest regions of China, particularly in Guizhou Province, this plant was used as a Miao ethnic medicine to prevent and treat dyspepsia, dysentery, hypoimmunity, and neurasthenia. In the present study, the neuroprotective effect of KF1 was evaluated against N-methyl-D-aspartate (NMDA)-induced neurotoxicity in vivo and in vitro. An NMDA-induced PC12 cell neurotoxicity assay showed that KF1 effectively improved cellular viability, inhibited the release of lactate dehydrogenase (LDH), and reduced cell apoptosis. Furthermore, KF1-treated NMDA-induced excitotoxicity mice displayed a remarkable capacity for improving spatial learning memory in the Y-maze and Morris water maze tests. In addition, KF1 increased the levels of the neurotransmitters 5-hydroxytryptamine, dopamine, and monoamine oxidase and reduced the calcium ion concentration in the hippocampus of mice. Hematoxylin and eosin and Nissl staining indicated that KF1 effectively reduced the impairment of neurons. Furthermore, Western blot assays showed that KF1 decreased NMDAR1 expression. In contrast, the NMDAR2B (NR2B), glutamate receptor (AMPA), TrkB, protein kinase B (AKT), mammalian target of rapamycin (mTOR), PSD95, and synapsin 1 were upregulated in NMDA-induced PC12 cells and an animal model. These results suggest that KF1 has a remarkable protective effect against NMDA-induced neurotoxicity, which is directly related to the regulation of the NMDA receptor and the activation of the α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid receptor (AMPAR) and BDNF/AKT/mTOR signaling pathways.     

Reduced Glx and GABA Inductions in the Anterior Cingulate Cortex and Caudate Nucleus Are Related to Impaired Control of Attention in Attention-Deficit/Hyperactivity Disorder

Attention-deficit/hyperactivity disorder (ADHD) is a neurodevelopmental disorder that impairs the control of attention and behavioral inhibition in affected individuals. Recent genome-wide association findings have revealed an association between glutamate and GABA gene sets and ADHD symptoms. Consistently, people with ADHD show altered glutamate and GABA content in the brain circuitry that is important for attention control function. Yet, it remains unknown how glutamate and GABA content in the attention control circuitry change when people are controlling their attention, and whether these changes can predict impaired attention control in people with ADHD. To study these questions, we recruited 18 adults with ADHD (31–51 years) and 16 adults without ADHD (28–54 years). We studied glutamate + glutamine (Glx) and GABA content in the fronto-striatal circuitry while participants performed attention control tasks. We found that Glx and GABA concentrations at rest did not differ between participants with ADHD or without ADHD. However, while participants were performing the attention control tasks, participants with ADHD showed smaller Glx and GABA increases than participants without ADHD. Notably, smaller GABA increases in participants with ADHD significantly predicted their poor task performance. Together, these findings provide the first demonstration showing that attention control deficits in people with ADHD may be related to insufficient responses of the GABAergic system in the fronto-striatal circuitry.     

Perineuronal Nets in the Prefrontal Cortex of a Schizophrenia Mouse Model: Assessment of Neuroanatomical,Electrophysiological, and Behavioral Contributions

Schizophrenia is a neurodevelopmental disorder whose etiopathogenesis includes changes in cellular as well as extracellular structures. Perineuronal nets (PNNs) associated with parvalbumin-positive interneurons (PVs) in the prefrontal cortex (PFC) are dysregulated in schizophrenia. However, the postnatal development of these structures along with their associated neurons in the PFC is unexplored, as is their effects on behavior and neural activity. Therefore, in this study, we employed a DISC1 (Disruption in Schizophrenia) mutation mouse model of schizophrenia to assess these developmental changes and tested whether enzymatic digestion of PNNs in the PFC affected schizophrenia-like behaviors and neural activity. Developmentally, we found that the normal formation of PNNs, PVs, and colocalization of these two in the PFC, peaked around PND 22 (postnatal day 22). However, in DISC1, mutation animals from PND 0 to PND 60, both PNNs and PVs were significantly reduced. After enzymatic digestion of PNNs with chondroitinase in adult animals, the behavioral pattern of control animals mimicked that of DISC1 mutation animals, exhibiting reduced sociability, novelty and increased ultrasonic vocalizations, while there was very little change in other behaviors, such as working memory (Y-maze task involving medial temporal lobe) or depression-like behavior (tail-suspension test involving processing via the hypothalamic pituitary adrenal (HPA) axis). Moreover, following chondroitinase treatment, electrophysiological recordings from the PFC exhibited a reduced proportion of spontaneous, high-frequency firing neurons, and an increased proportion of irregularly firing neurons, with increased spike count and reduced inter-spike intervals in control animals. These results support the proposition that the aberrant development of PNNs and PVs affects normal neural operations in the PFC and contributes to the emergence of some of the behavioral phenotypes observed in the DISC1 mutation model of schizophrenia.     

Optical Activation of TrkB (E281A) in Excitatory and Inhibitory Neurons of the Mouse Visual Cortex

The activation of tropomyosin receptor kinase B (TrkB), the receptor of brain-derived neurotrophic factor (BDNF), plays a key role in induced juvenile-like plasticity (iPlasticity), which allows restructuring of neural networks in adulthood. Optically activatable TrkB (optoTrkB) can temporarily and spatially evoke iPlasticity, and recently, optoTrkB (E281A) was developed as a variant that is highly sensitive to light stimulation while having lower basal activity compared to the original optoTrkB. In this study, we validate optoTrkB (E281A) activated in alpha calcium/calmodulin-dependent protein kinase type II positive (CKII⁺) pyramidal neurons or parvalbumin-positive (PV⁺) interneurons in the mouse visual cortex by immunohistochemistry. OptoTrkB (E281A) was activated in PV⁺ interneurons and CKII⁺ pyramidal neurons with blue light (488 nm) through the intact skull and fur, and through a transparent skull, respectively. LED light stimulation significantly increased the intensity of phosphorylated ERK and CREB even through intact skull and fur. These findings indicate that the highly sensitive optoTrkB (E281A) can be used in iPlasticity studies of both inhibitory and excitatory neurons, with flexible stimulation protocols in behavioural studies.     

NGF Eye Administration Recovers the TrkB and Glutamate/GABA Marker Deficit in the Adult Visual Cortex Following Optic Nerve Crush

Eye-drop recombinant human nerve growth factor (ed-rhNGF) has proved to recover the retina and optic nerve damage in animal models, including the unilateral optic nerve crush (ONC), and to improve visual acuity in humans. These data, associated with evidence that ed-rhNGF stimulates the brain derived neurotrophic factor (BDNF) in retina and cortex, suggests that NGF might exert retino-fugal effects by affecting BDNF and its receptor TrkB. To address these questions, their expression and relationship with the GABAergic and glutamatergic transmission markers, GAD65 and GAD67, vesicular inhibitory amino acid transporter (VGAT), and vesicular glutamate transporters 1 and 2 (VGLUT-1 and VGLUT-2) were investigated in adult ONC rats contralateral and ipsilateral visual cortex (VCx). Ed-rhNGF recovers the ONC-induced alteration of GABAergic and glutamatergic markers in contralateral VCx, induces an upregulation of TrkB, which is positively correlated with BDNF precursor (proBDNF) decrease in both VCx sides, and strongly enhances TrkB+ cell soma and neuronal endings surrounded by GAD65 immuno-reactive afferents. These findings contribute to enlarging the knowledge on the mechanism of actions and cellular targets of exogenously administrated NGF, and suggest that ed-rhNGF might act by potentiating the activity-dependent TrkB expression in GAD+ cells in VCx following retina damage and/or ONC.     

Intranasal Administration of Rotenone Reduces GABAergic Inhibition in the Mouse Insular Cortex Leading to Impairment of LTD and Conditioned Taste Aversion Memory

The pesticide rotenone inhibits mitochondrial complex I and is thought to cause neurological disorders such as Parkinson’s disease and cognitive disorders. However, little is known about the effects of rotenone on conditioned taste aversion memory. In the present study, we investigated whether intranasal administration of rotenone affects conditioned taste aversion memory in mice. We also examined how the intranasal administration of rotenone modulates synaptic transmission and plasticity in layer V pyramidal neurons of the mouse insular cortex that is critical for conditioned taste aversion memory. We found that the intranasal administration of rotenone impaired conditioned taste aversion memory to bitter taste. Regarding its cellular mechanisms, long-term depression (LTD) but not long-term potentiation (LTP) was impaired in rotenone-treated mice. Furthermore, spontaneous inhibitory synaptic currents and tonic GABA currents were decreased in layer V pyramidal neurons of rotenone-treated mice compared to the control mice. The impaired LTD observed in pyramidal neurons of rotenone-treated mice was restored by a GABA_A receptor agonist muscimol. These results suggest that intranasal administration of rotenone decreases GABAergic synaptic transmission in layer V pyramidal neurons of the mouse insular cortex, the result of which leads to impairment of LTD and conditioned taste aversion memory.     

Imaging of C-fos Activity in Neurons of the Mouse Parietal Association Cortex during Acquisition and Retrieval of Associative Fear Memory

The parietal cortex of rodents participates in sensory and spatial processing, movement planning, and decision-making, but much less is known about its role in associative learning and memory formation. The present study aims to examine the involvement of the parietal association cortex (PtA) in associative fear memory acquisition and retrieval in mice. Using ex vivo c-Fos immunohistochemical mapping and in vivo Fos-EGFP two-photon imaging, we show that PtA neurons were specifically activated both during acquisition and retrieval of cued fear memory. Fos immunohistochemistry revealed specific activation of the PtA neurons during retrieval of the 1-day-old fear memory. In vivo two-photon Fos-EGFP imaging confirmed this result and in addition detected specific c-Fos responses of the PtA neurons during acquisition of cued fear memory. To allow a more detailed study of the long-term activity of such PtA engram neurons, we generated a Fos-Cre-GCaMP transgenic mouse line that employs the Targeted Recombination in Active Populations (TRAP) technique to detect calcium events specifically in cells that were Fos-active during conditioning. We show that gradual accumulation of GCaMP3 in the PtA neurons of Fos-Cre-GCaMP mice peaks at the 4th day after fear learning. We also describe calcium transients in the cell bodies and dendrites of the TRAPed neurons. This provides a proof-of-principle for TRAP-based calcium imaging of PtA functions during memory processes as well as in experimental models of fear- and anxiety-related psychiatric disorders and their specific therapies.     

Deficiency in the Treatment Description of mTOR Inhibitor Resistance in Medulloblastoma,a Systematic Review

Medulloblastoma is a common fatal pediatric brain tumor. More treatment options are required to prolong survival and decrease disability. mTOR proteins play an essential role in the disease pathogenesis, and are an essential target for therapy. Three generations of mTOR inhibitors have been developed and are clinically used for immunosuppression and chemotherapy for multiple cancers. Only a few mTOR inhibitors have been investigated for the treatment of medulloblastoma and other pediatric tumors. The first-generation mTOR, sirolimus, temsirolimus, and everolimus, went through phase I clinical trials. The second-generation mTOR, AZD8055 and sapanisertib, suppressed medulloblastoma cell growth; however, limited studies have investigated possible resistance pathways. No clinical trials have been found to treat medulloblastoma using third-generation mTOR inhibitors. This systematic review highlights the mechanisms of resistance of mTOR inhibitors in medulloblastoma and includes IDO1, T cells, Mnk2, and eIF4E, as they prolong malignant cell survival. The findings promote the importance of combination therapy in medulloblastoma due to its highly resistant nature.     

Current Understanding of the Involvement of the Insular Cortex in Neuropathic Pain: A Narrative Review

Neuropathic pain is difficult to cure and is often accompanied by emotional and psychological changes. Exploring the mechanisms underlying neuropathic pain will help to identify a better treatment for this condition. The insular cortex is an important information integration center. Numerous imaging studies have documented increased activity of the insular cortex in the presence of neuropathic pain; however, the specific role of this region remains controversial. Early studies suggested that the insular lobe is mainly involved in the processing of the emotional motivation dimension of pain. However, increasing evidence suggests that the role of the insular cortex is more complex and may even be related to the neural plasticity, cognitive evaluation, and psychosocial aspects of neuropathic pain. These effects contribute not only to the development of neuropathic pain, but also to its comorbidity with neuropsychiatric diseases. In this review, we summarize the changes that occur in the insular cortex in the presence of neuropathic pain and analgesia, as well as the molecular mechanisms that may underlie these conditions. We also discuss potential sex-based differences in these processes. Further exploration of the involvement of the insular lobe will contribute to the development of new pharmacotherapy and psychotherapy treatments for neuropathic pain.     

mTOR Signaling in the Inner Ear as Potential Target to Treat Hearing Loss

Hearing loss affects many people worldwide and occurs often as a result of age, ototoxic drugs and/or excessive noise exposure. With a growing number of elderly people, the number of people suffering from hearing loss will also increase in the future. Despite the high number of affected people, for most patients there is no curative therapy for hearing loss and hearing aids or cochlea implants remain the only option. Important treatment approaches for hearing loss include the development of regenerative therapies or the inhibition of cell death/promotion of cell survival pathways. The mammalian target of rapamycin (mTOR) pathway is a central regulator of cell growth, is involved in cell survival, and has been shown to be implicated in many age-related diseases. In the inner ear, mTOR signaling has also started to gain attention recently. In this review, we will emphasize recent discoveries of mTOR signaling in the inner ear and discuss implications for possible treatments for hearing restoration.     

MicroRNA Expression Profile in TSC Cell Lines and the Impact of mTOR Inhibitor

The aim of this study was to assess the potential implication of microRNA on tuberous sclerosis (TSC) pathogenesis by performing microRNA profiling on cell lines silencing TSC1 or TSC2 genes using qPCR panels, before and after incubation with rapamycin. Significant differences in expression were observed between samples before and after rapamycin treatment in nineteen miRNAs in TSC1, five miRNAs in TSC2 and seven miRNAs in controls. Of miRNAs dysregulated before rapamycin treatment, three normalized after treatment in the TSC1 group (miR-21-3p, miR-433-3p, let-7g-3p) and one normalized in the TSC2 group (miR-1224-3p). Of the miRNAs dysregulated before rapamycin treatment in the TSC1 and TSC2 groups, two did not normalize after treatment (miR-33a-3p, miR-29a-3p). The results of the possible targets indicated that there are four common genes with seed regions susceptible to regulation by those miRNAs: ZBTB20, PHACTR2, PLXNC1 and ATP1B4. Our data show no changes in mRNA expression of these targets after rapamycin treatment. In conclusion, results of our study indicate the involvement of miRNA dysregulation in the pathogenesis of TSC. Some of the miRNA might be used as markers of treatment efficacy and autonomic miRNA as a target for future therapy.     

Cell Proliferation in the Piriform Cortex of Rats with Motor Cortex Ablation Treated with Growth Hormone and Rehabilitation

Traumatic brain injury represents one of the main health problems in developed countries. Growth hormone (GH) and rehabilitation have been claimed to significantly contribute to the recovery of lost motor function after acquired brain injury, but the mechanisms by which this occurs are not well understood. In this work, we have investigated cell proliferation in the piriform cortex (PC) of adult rats with ablation of the frontal motor cortex treated with GH and rehabilitation, in order to evaluate if this region of the brain, related to the sense of smell, could be involved in benefits of GH treatment. Male rats were either ablated the frontal motor cortex in the dominant hemisphere or sham-operated and treated with GH or vehicle at 35 days post-injury (dpi) for five days. At 36 dpi, all rats received daily injections of bromodeoxyuridine (BrdU) for four days. We assessed motor function through the paw-reaching-for-food task. GH treatment and rehabilitation at 35 dpi significantly improved the motor deficit caused by the injury and promoted an increase of cell proliferation in the PC ipsilateral to the injury, which could be involved in the improvement observed. Cortical ablation promoted a greater number of BrdU+ cells in the piriform cortex that was maintained long-term, which could be involved in the compensatory mechanisms of the brain after injury.     

Psychedelic-Induced Serotonin 2A Receptor Downregulation Does Not Predict Swim Stress Coping in Mice

Serotoninergic psychedelics such as psilocybin have been reported to elicit a long-lasting reduction in depressive symptoms. Although the main target for serotoninergic psychedelics, serotonin type 2A receptor (5-HT_2A), has been established, the possible mechanism of the antidepressant action of psychedelics remains unknown. Using the mouse forced swim test model, we examined whether the administration of the synthetic serotoninergic psychedelic 2,5-dimethoxy-4-iodoamphetamine (DOI) would modulate 5-HT_2A receptor levels in the medial prefrontal cortex (mPFC) and revert stress-induced changes in behavior. Mice subjected to swim stress developed a passive stress-coping strategy when tested in the forced swim test 6 days later. This change in behavior was not associated with the hypothesized increase in 5-HT_2A receptor-dependent head twitch behaviors or consistent changes in 5-HT_2A receptor levels in the mPFC. When DOI was administered 1 day before the forced swim test, a low dose (0.2 mg/kg i.p.) unexpectedly increased immobility while a high dose (2 mg/kg i.p.) had no significant effect on immobility. Nevertheless, DOI evoked a dose-dependent decrease in 5-HT_2A levels in the mPFC of mice previously exposed to swim stress. Our findings do not support the hypothesis that the downregulation of 5-HT_2A receptors in the mPFC contributes to the antidepressant-like properties of serotoninergic psychedelics.     

Postnatal Changes of Neural Stem Cells in the Mammalian Auditory Cortex

Our previous study reported neural stem cells (NSCs) in the auditory cortex (AC) of postnatal day 3 (P3) mice in vitro. It is unclear whether AC-NSCs exist in vivo. This study aims to determine the presence and changes of AC-NSCs during postnatal development and maturation both in vitro and in vivo. P3, postnatal day 14 (P14), 2-month-old (2M), and 4-month-old (4M) mouse brain tissues were fixed and cryosectioned for NSC marker immunostaining. In vitro, P3, P14, and 2M AC tissues were dissected and cultured in suspension to study NSCs. NSC proliferation was examined by EdU incorporation and cell doubling time assays in vitro. The results show that Nestin and Sox2 double expressing NSCs were observed in the AC area from P3 to 4M in vivo, in which the number of NSCs remarkably reduced with age. In vitro, the neurosphere forming capability, cell proliferation, and percentage of Nestin and Sox2 double expressing NSCs significantly diminished with age. These results suggest that AC-NSCs exist in the mouse AC area both in vitro and in vivo, and the percentage of AC-NSCs decreases during postnatal development and maturation. The results may provide important cues for the future research of the central auditory system.     

PSA Depletion Induces the Differentiation of Immature Neurons in the Piriform Cortex of Adult Mice

Immature neurons are maintained in cortical regions of the adult mammalian brain. In rodents, many of these immature neurons can be identified in the piriform cortex based on their high expression of early neuronal markers, such as doublecortin (DCX) and the polysialylated form of the neural cell adhesion molecule (PSA-NCAM). This molecule plays critical roles in different neurodevelopmental events. Taking advantage of a DCX-CreERT2/Flox-EGFP reporter mice, we investigated the impact of targeted PSA enzymatic depletion in the piriform cortex on the fate of immature neurons. We report here that the removal of PSA accelerated the final development of immature neurons. This was revealed by a higher frequency of NeuN expression, an increase in the number of cells carrying an axon initial segment (AIS), and an increase in the number of dendrites and dendritic spines on the immature neurons. Taken together, our results demonstrated the crucial role of the PSA moiety in the protracted development of immature neurons residing outside of the neurogenic niches. More studies will be required to understand the intrinsic and extrinsic factors affecting PSA-NCAM expression to understand how the brain regulates the incorporation of these immature neurons to the established neuronal circuits of the adult brain.     

SIRT4 Expression Ameliorates the Detrimental Effect of Heat Stress via AMPK/mTOR Signaling Pathway in BMECs

Sirtuin 4 (SIRT4), a member of the SIRT family, has been reported to be a key factor involved in antioxidant defense in mitochondria. This study aimed to explore the potential molecular mechanism via which SIRT4 regulates heat stress-induced oxidative stress and lactoprotein synthesis in bovine mammary epithelial cells (BMECs). Our results showed that SIRT4 was significantly decreased in heat stressed mammary tissue. Depletion of SIRT4 in BMECs induced the generation of ROS, which, as exhibited by the decreased activity of antioxidant enzymes, changed mitochondrial morphology through mediating protein and mRNA levels related to mitochondrial fission and fusion. Moreover, we found that depletion of SIRT4 or stress conditions inhibited the expression of milk proteins, as well as lipid and glucose synthesis-related genes, and activated the AMPK/mTOR signaling pathway. Increased SIRT4 expression was found to have the opposite effect. However, blocking the AMPK/mTOR signaling pathway could inhibit the regulatory function of SIRT4 in milk synthesis-related gene expression. In summary, our results suggest that SIRT4 may play critical roles in maintaining mammary gland function by regulating the AMPK/mTOR signaling pathway in dairy cows, indicating that SIRT4 may be a potential molecular target for curing heat stress-induced BMEC injury and low milk production in dairy cows.