Small G-Protein Rheb Gates Mammalian Target of Rapamycin Signaling to Regulate Morphine Tolerance in Mice.

BACKGROUND: Analgesic tolerance due to long-term use of morphine remains a challenge for pain management. Morphine acts on µ-opioid receptors and downstream of the phosphatidylinositol 3-kinase signaling pathway to activate the mammalian target of rapamycin (mTOR) pathway. Rheb is an important regulator of growth and cell-cycle progression in the central nervous system owing to its critical role in the activation of mTOR. The hypothesis was that signaling via the GTP-binding protein Rheb in the dorsal horn of the spinal cord is involved in morphine-induced tolerance. METHODS: Male and female wild-type C57BL/6J mice or transgenic mice (6 to 8 weeks old) were injected intrathecally with saline or morphine twice daily at 12-h intervals for 5 consecutive days to establish a tolerance model. Analgesia was assessed 60 min later using the tail-flick assay. After 5 days, the spine was harvested for Western blot or immunofluorescence analysis. RESULTS: Chronic morphine administration resulted in the upregulation of spinal Rheb by 4.27 ± 0.195-fold (P = 0.0036, n = 6), in turn activating mTOR by targeting rapamycin complex 1 (mTORC1). Genetic overexpression of Rheb impaired morphine analgesia, resulting in a tail-flick latency of 4.65 ± 1.10 s (P < 0.0001, n = 7) in Rheb knock-in mice compared to 10 s in control mice (10 ± 0 s). Additionally, Rheb overexpression in spinal excitatory neurons led to mTORC1 signaling overactivation. Genetic knockout of Rheb or inhibition of mTORC1 signaling by rapamycin potentiated morphine-induced tolerance (maximum possible effect, 52.60 ± 9.56% in the morphine + rapamycin group vs. 16.60 ± 8.54% in the morphine group; P < 0.0001). Moreover, activation of endogenous adenosine 5'-monophosphate-activated protein kinase inhibited Rheb upregulation and retarded the development of morphine-dependent tolerance (maximum possible effect, 39.51 ± 7.40% in morphine + metformin group vs. 15.58 ± 5.79% in morphine group; P < 0.0001). CONCLUSIONS: This study suggests spinal Rheb as a key molecular factor for regulating mammalian target of rapamycin signaling.

Interregulation between fragile X mental retardation protein and methyl CpG binding protein 2 in the mouse posterior cerebral cortex.

Several X-linked neurodevelopmental disorders including Rett syndrome, induced by mutations in the MECP2 gene, and fragile X syndrome (FXS), caused by mutations in the FMR1 gene, share autism-related features. The mRNA coding for methyl CpG binding protein 2 (MeCP2) has previously been identified as a substrate for the mRNA-binding protein, fragile X mental retardation protein (FMRP), which is silenced in FXS. Here, we report a homeostatic relationship between these two key regulators of gene expression in mouse models of FXS (Fmr1 Knockout (KO)) and Rett syndrome (MeCP2 KO). We found that the level of MeCP2 protein in the cerebral cortex was elevated in Fmr1 KO mice, whereas MeCP2 KO mice displayed reduced levels of FMRP, implicating interplay between the activities of MeCP2 and FMRP. Indeed, knockdown of MeCP2 with short hairpin RNAs led to a reduction of FMRP in mouse Neuro2A and in human HEK-293 cells, suggesting a reciprocal coupling in the expression level of these two regulatory proteins. Intra-cerebroventricular injection of an adeno-associated viral vector coding for FMRP led to a concomitant reduction in MeCP2 expression in vivo and partially corrected locomotor hyperactivity. Additionally, the level of MeCP2 in the posterior cortex correlated with the severity of the hyperactive phenotype in Fmr1 KO mice. These results demonstrate that MeCP2 and FMRP operate within a previously undefined homeostatic relationship. Our findings also suggest that MeCP2 overexpression in Fmr1 KO mouse posterior cerebral cortex may contribute to the fragile X locomotor hyperactivity phenotype.

Dynamic tripartite construct of interregional engram circuits underlies forgetting of extinction memory.

Fear extinction allows for adaptive control of learned fear responses but often fails, resulting in a renewal or spontaneous recovery of the extinguished fear, i.e., forgetting of the extinction memory readily occurs. Using an activity-dependent neuronal labeling strategy, we demonstrate that engram neurons for fear extinction memory are dynamically positioned in the medial prefrontal cortex (mPFC), basolateral amygdala (BLA), and ventral hippocampus (vHPC), which constitute an engram construct in the term of directional engram synaptic connectivity from the BLA or vHPC to mPFC, but not that in the opposite direction, for retrieval of extinction memory. Fear renewal or spontaneous recovery switches the extinction engram construct from an accessible to inaccessible state, whereas additional extinction learning or optogenetic induction of long-term potentiation restores the directional engram connectivity and prevents the return of fear. Thus, the plasticity of engram construct underlies forgetting of extinction memory.

Phosphoregulated FMRP phase separation models activity-dependent translation through bidirectional control of mRNA granule formation.

Activity-dependent translation requires the transport of mRNAs within membraneless protein assemblies known as neuronal granules from the cell body toward synaptic regions. Translation of mRNA is inhibited in these granules during transport but quickly activated in response to neuronal stimuli at the synapse. This raises an important question: how does synaptic activity trigger translation of once-silenced mRNAs? Here, we demonstrate a strong connection between phase separation, the process underlying the formation of many different types of cellular granules, and in vitro inhibition of translation. By using the Fragile X Mental Retardation Protein (FMRP), an abundant neuronal granule component and translational repressor, we show that FMRP phase separates in vitro with RNA into liquid droplets mediated by its C-terminal low-complexity disordered region (i.e., FMRPLCR). FMRPLCR posttranslational modifications by phosphorylation and methylation have opposing effects on in vitro translational regulation, which corroborates well with their critical concentrations for phase separation. Our results, combined with bioinformatics evidence, are supportive of phase separation as a general mechanism controlling activity-dependent translation.

The γ-Protocadherins Regulate the Survival of GABAergic Interneurons during Developmental Cell Death.

Inhibitory interneurons integrate into developing circuits in specific ratios and distributions. In the neocortex, inhibitory network formation occurs concurrently with the apoptotic elimination of a third of GABAergic interneurons. The cell surface molecules that select interneurons to survive or die are unknown. Here, we report that members of the clustered Protocadherins (cPCDHs) control GABAergic interneuron survival during developmentally-regulated cell death. Conditional deletion of the gene cluster encoding the γ-Protocadherins (Pcdhgs) from developing GABAergic neurons in mice of either sex causes a severe loss of inhibitory populations in multiple brain regions and results in neurologic deficits such as seizures. By focusing on the neocortex and the cerebellar cortex, we demonstrate that reductions of inhibitory interneurons result from elevated apoptosis during the critical postnatal period of programmed cell death (PCD). By contrast, cortical interneuron (cIN) populations are not affected by removal of Pcdhgs from pyramidal neurons or glial cells. Interneuron loss correlates with reduced AKT signaling in Pcdhg mutant interneurons, and is rescued by genetic blockade of the pro-apoptotic factor BAX. Together, these findings identify the PCDHGs as pro-survival transmembrane proteins that select inhibitory interneurons for survival and modulate the extent of PCD. We propose that the PCDHGs contribute to the formation of balanced inhibitory networks by controlling the size of GABAergic interneuron populations in the developing brain.SIGNIFICANCE STATEMENT A pivotal step for establishing appropriate excitatory-inhibitory ratios is adjustment of neuronal populations by cell death. In the mouse neocortex, a third of GABAergic interneurons are eliminated by BAX-dependent apoptosis during the first postnatal week. Interneuron cell death is modulated by neural activity and pro-survival pathways but the cell-surface molecules that select interneurons for survival or death are unknown. We demonstrate that members of the cadherin superfamily, the clustered γ-Protocadherins (PCDHGs), regulate the survival of inhibitory interneurons and the balance of cell death. Deletion of the Pcdhgs in mice causes inhibitory interneuron loss in the cortex and cerebellum, and leads to motor deficits and seizures. Our findings provide a molecular basis for controlling inhibitory interneuron population size during circuit formation.

Identification of a molecular locus for normalizing dysregulated GABA release from interneurons in the Fragile X brain.

Principal neurons encode information by varying their firing rate and patterns precisely fine-tuned through GABAergic interneurons. Dysregulation of inhibition can lead to neuropsychiatric disorders, yet little is known about the molecular basis underlying inhibitory control. Here, we find that excessive GABA release from basket cells (BCs) attenuates the firing frequency of Purkinje neurons (PNs) in the cerebellum of Fragile X Mental Retardation 1 (Fmr1) knockout (KO) mice, a model of Fragile X Syndrome (FXS) with abrogated expression of the Fragile X Mental Retardation Protein (FMRP). This over-inhibition originates from increased excitability and Ca2+ transients in the presynaptic terminals, where Kv1.2 potassium channels are downregulated. By paired patch-clamp recordings, we further demonstrate that acutely introducing an N-terminal fragment of FMRP into BCs normalizes GABA release in the Fmr1-KO synapses. Conversely, direct injection of an inhibitory FMRP antibody into BCs, or membrane depolarization of BCs, enhances GABA release in the wild type synapses, leading to abnormal inhibitory transmission comparable to the Fmr1-KO neurons. We discover that the N-terminus of FMRP directly binds to a phosphorylated serine motif on the C-terminus of Kv1.2; and that loss of this interaction in BCs exaggerates GABA release, compromising the firing activity of PNs and thus the output from the cerebellar circuitry. An allosteric Kv1.2 agonist, docosahexaenoic acid, rectifies the dysregulated inhibition in vitro as well as acoustic startle reflex and social interaction in vivo of the Fmr1-KO mice. Our results unravel a novel molecular locus for targeted intervention of FXS and perhaps autism.

Rectification of radiotherapy-induced cognitive impairments in aged mice by reconstituted Sca-1+ stem cells from young donors.

BACKGROUND: Radiotherapy is widely used and effective for treating brain tumours, but inevitably impairs cognition as it arrests cellular processes important for learning and memory. This is particularly evident in the aged brain with limited regenerative capacity, where radiation produces irreparable neuronal damage and activation of neighbouring microglia. The latter is responsible for increased neuronal death and contributes to cognitive decline after treatment. To date, there are few effective means to prevent cognitive deficits after radiotherapy. METHODS: Here we implanted hematopoietic stem cells (HSCs) from young or old (2- or 18-month-old, respectively) donor mice expressing green fluorescent protein (GFP) into old recipients and assessed cognitive abilities 3 months post-reconstitution. RESULTS: Regardless of donor age, GFP+ cells homed to the brain of old recipients and expressed the macrophage/microglial marker, Iba1. However, only young cells attenuated deficits in novel object recognition and spatial memory and learning in old mice post-irradiation. Mechanistically, old recipients that received young HSCs, but not old, displayed significantly greater dendritic spine density and long-term potentiation (LTP) in CA1 neurons of the hippocampus. Lastly, we found that GFP+/Iba1+ cells from young and old donors were differentially polarized to an anti- and pro-inflammatory phenotype and produced neuroprotective factors and reactive nitrogen species in vivo, respectively. CONCLUSION: Our results suggest aged peripherally derived microglia-like cells may exacerbate cognitive impairments after radiotherapy, whereas young microglia-like cells are polarized to a reparative phenotype in the irradiated brain, particularly in neural circuits associated with rewards, learning, and memory. These findings present a proof-of-principle for effectively reinstating central cognitive function of irradiated brains with peripheral stem cells from young donor bone marrow.

The ED50 and ED95 of oxytocin infusion rate for maintaining uterine tone during elective caesarean delivery: a dose-finding study.

BACKGROUND: The 90% effective dose (ED90) of oxytocin infusion has been previously estimated to be 16.2 IU h- 1. However, bolus administration of oxytocin prior to the infusion may decrease the infusion dose required. The aim of this study was to estimate the ED95 for oxytocin infusion after a bolus at elective caesarean delivery (CD) in nonlaboring parturients. METHODS: We performed a randomized, triple blinded study in 150 healthy termparturients scheduled for elective CD under epidural anaesthesia. After delivery of the infant and i.v. administration of 1 IU oxytocin as a bolus, Participants were randomized to receive oxytocin infusion at a rate of 0, 1, 2, 3, 5, or 8 IU h- 1, to be given for a total of 1 h. Uterine tone assessed by the blinded obstetrician as either adequate or inadequate. Secondary outcomes included estimated blood loss (EBL), requirement for supplemental uterotonic agents, and development of side effects. RESULTS: The 95% effective dose (ED95) of oxytocin infusion was estimated to be 7.72 IU h- 1 (95% confidence interval 5.80-12.67 IU h- 1). With increasing oxytocin infusion rate, the proportion of parturients who needed rescue oxytocin bolus or secondary uterotonic agents decreased. No significant among-group differences in the EBL and oxytocin-related side effects were observed. CONCLUSIONS: In parturients who receive a 1 IU bolus of oxytocin during elective cesarean delivery, an infusion rate of oxytocin at 7.72 IU h- 1 will produce adequate uterine tone in 95% of parturients. These results suggest that the total dose of oxytocin administered in the postpartum period can be decreased when administered as an infusion after oxytocin bolus.

Structural basis for calcium and magnesium regulation of a large conductance calcium-activated potassium channel with ß1 subunits.

Large conductance Ca(2+)- and voltage-activated potassium (BK) channels, composed of pore-forming α subunits and auxiliary ß subunits, play important roles in diverse physiological activities. The ß1 is predominately expressed in smooth muscle cells, where it greatly enhances the Ca(2+) sensitivity of BK channels for proper regulation of smooth muscle tone. However, the structural basis underlying dynamic interaction between BK mSlo1 α and ß1 remains elusive. Using macroscopic ionic current recordings in various Ca(2+) and Mg(2+) concentrations, we identified two binding sites on the cytosolic N terminus of ß1, namely the electrostatic enhancing site (mSlo1(K392,R393)-ß1(E13,T14)), increasing the calcium sensitivity of BK channels, and the hydrophobic site (mSlo1(L906,L908)-ß1(L5,V6,M7)), passing the physical force from the Ca(2+) bowl onto the enhancing site and S6 C-linker. Dynamic binding of these sites affects the interaction between the cytosolic domain and voltage-sensing domain, leading to the reduction of Mg(2+) sensitivity. A comprehensive structural model of the BK(mSlo1 α-ß1) complex was reconstructed based on these functional studies, which provides structural and mechanistic insights for understanding BK gating.

Remodelling at the calyx of Held-MNTB synapse in mice developing with unilateral conductive hearing loss.

Structure and function of central synapses are profoundly influenced by experience during developmental sensitive periods. Sensory synapses, which are the indispensable interface for the developing brain to interact with its environment, are particularly plastic. In the auditory system, moderate forms of unilateral hearing loss during development are prevalent but the pre- and postsynaptic modifications that occur when hearing symmetry is perturbed are not well understood. We investigated this issue by performing experiments at the large calyx of Held synapse. Principal neurons of the medial nucleus of the trapezoid body (MNTB) are innervated by calyx of Held terminals that originate from the axons of globular bushy cells located in the contralateral ventral cochlear nucleus. We compared populations of synapses in the same animal that were either sound deprived (SD) or sound experienced (SE) after unilateral conductive hearing loss (CHL). Middle ear ossicles were removed 1 week prior to hearing onset (approx. postnatal day (P) 12) and morphological and electrophysiological approaches were applied to auditory brainstem slices taken from these mice at P17-19. Calyces in the SD and SE MNTB acquired their mature digitated morphology but these were structurally more complex than those in normal hearing mice. This was accompanied by bilateral decreases in initial EPSC amplitude and synaptic conductance despite the CHL being unilateral. During high-frequency stimulation, some SD synapses displayed short-term depression whereas others displayed short-term facilitation followed by slow depression similar to the heterogeneities observed in normal hearing mice. However SE synapses predominantly displayed short-term facilitation followed by slow depression which could be explained in part by the decrease in release probability. Furthermore, the excitability of principal cells in the SD MNTB had increased significantly. Despite these unilateral changes in short-term plasticity and excitability, heterogeneities in the spiking fidelity among the population of both SD and SE synapses showed similar continuums to those in normal hearing mice. Our study suggests that preservations in the heterogeneity in spiking fidelity via synaptic remodelling ensures symmetric functional stability which is probably important for retaining the capability to maximally code sound localization cues despite moderate asymmetries in hearing experience.

Synergistic hyperactivation of both mTORC1 and mTORC2 underlies the neural abnormalities of PTEN-deficient human neurons and cortical organoids.

Mutations in the phosphatase and tensin homolog (PTEN) gene are associated with severe neurodevelopmental disorders. Loss of PTEN leads to hyperactivation of the mechanistic target of rapamycin (mTOR), which functions in two distinct protein complexes, mTORC1 and mTORC2. The downstream signaling mechanisms that contribute to PTEN mutant phenotypes are not well delineated. Here, we show that pluripotent stem cell-derived PTEN mutant human neurons, neural precursors, and cortical organoids recapitulate disease-relevant phenotypes, including hypertrophy, electrical hyperactivity, enhanced proliferation, and structural overgrowth. PTEN loss leads to simultaneous hyperactivation of mTORC1 and mTORC2. We dissect the contribution of mTORC1 and mTORC2 by generating double mutants of PTEN and RPTOR or RICTOR, respectively. Our results reveal that the synergistic hyperactivation of both mTORC1 and mTORC2 is essential for the PTEN mutant human neural phenotypes. Together, our findings provide insights into the molecular mechanisms that underlie PTEN-related neural disorders and highlight novel therapeutic targets.

High-resolution electron cryomicroscopy of V-ATPase in native synaptic vesicles.

Intercellular communication in the nervous system occurs through the release of neurotransmitters into the synaptic cleft between neurons. In the presynaptic neuron, the proton pumping vesicular- or vacuolar-type ATPase (V-ATPase) powers neurotransmitter loading into synaptic vesicles (SVs), with the V1 complex dissociating from the membrane region of the enzyme before exocytosis. We isolated SVs from rat brain using SidK, a V-ATPase-binding bacterial effector protein. Single particle electron cryomicroscopy allowed high-resolution structure determination of V-ATPase within the native SV membrane. In the structure, regularly spaced cholesterol molecules decorate the enzyme's rotor and the abundant SV protein synaptophysin binds the complex stoichiometrically. ATP hydrolysis during vesicle loading results in loss of V1 from the SV membrane, suggesting that loading is sufficient to induce dissociation of the enzyme.

Measuring endogenous levels of unconjugated bilirubin released from isolated murine brain tissue during oxygen-glucose deprivation.

Quantitative assessment of endogenously synthesized and released bilirubin from brain tissue remains a challenge. Here, we present a sensitive and reproducible experimental paradigm to quantify, in real time, unconjugated bilirubin (UCB) from isolated murine brain tissue during oxygen-glucose deprivation (OGD). We describe steps for perfusion, brain dissection, brain slice preparation and incubation, glucose depletion, and OGD processing. We then detail procedures for standard calibration plotting and sample UCB measurement. For complete details on the use and execution of this protocol, please refer to Liu et al.1.

Redistribution of the astrocyte phenotypes in the medial vestibular nuclei after unilateral labyrinthectomy.

Astrocytes are highly heterogeneous and involved in different aspects of fundamental functions in the central nervous system (CNS). However, whether and how this heterogeneous population of cells reacts to the pathophysiological challenge is not well understood. To investigate the response status of astrocytes in the medial vestibular nucleus (MVN) after vestibular loss, we examined the subtypes of astrocytes in MVN using single-cell sequencing technology in a unilateral labyrinthectomy mouse model. We discovered four subtypes of astrocytes in the MVN with each displaying unique gene expression profiles. After unilateral labyrinthectomy, the proportion of the astrocytic subtypes and their transcriptional features on the ipsilateral side of the MVN differ significantly from those on the contralateral side. With new markers to detect and classify the subtypes of astrocytes in the MVN, our findings implicate potential roles of the adaptive changes of astrocyte subtypes in the early vestibular compensation following peripheral vestibular damage to reverse behavioral deficits.

Bilirubin gates the TRPM2 channel as a direct agonist to exacerbate ischemic brain damage.

Stroke prognosis is negatively associated with an elevation of serum bilirubin, but how bilirubin worsens outcomes remains mysterious. We report that post-, but not pre-, stroke bilirubin levels among inpatients scale with infarct volume. In mouse models, bilirubin increases neuronal excitability and ischemic infarct, whereas ischemic insults induce the release of endogenous bilirubin, all of which are attenuated by knockout of the TRPM2 channel or its antagonist A23. Independent of canonical TRPM2 intracellular agonists, bilirubin and its metabolic derivatives gate the channel opening, whereas A23 antagonizes it by binding to the same cavity. Knocking in a loss of binding point mutation for bilirubin, TRPM2-D1066A, effectively antagonizes ischemic neurotoxicity in mice. These findings suggest a vicious cycle of stroke injury in which initial ischemic insults trigger the release of endogenous bilirubin from injured cells, which potentially acts as a volume neurotransmitter to activate TRPM2 channels, aggravating Ca2+-dependent brain injury.

A designer peptide against the EAG2-Kvß2 potassium channel targets the interaction of cancer cells and neurons to treat glioblastoma.

Glioblastoma (GBM) is an incurable brain cancer that lacks effective therapies. Here we show that EAG2 and Kvß2, which are predominantly expressed by GBM cells at the tumor-brain interface, physically interact to form a potassium channel complex due to a GBM-enriched Kvß2 isoform. In GBM cells, EAG2 localizes at neuron-contacting regions in a Kvß2-dependent manner. Genetic knockdown of the EAG2-Kvß2 complex decreases calcium transients of GBM cells, suppresses tumor growth and invasion and extends the survival of tumor-bearing mice. We engineered a designer peptide to disrupt EAG2-Kvß2 interaction, thereby mitigating tumor growth in patient-derived xenograft and syngeneic mouse models across GBM subtypes without overt toxicity. Neurons upregulate chemoresistant genes in GBM cells in an EAG2-Kvß2-dependent manner. The designer peptide targets neuron-associated GBM cells and possesses robust efficacy in treating temozolomide-resistant GBM. Our findings may lead to the next-generation therapeutic agent to benefit patients with GBM.

Mechanosensitive brain tumor cells construct blood-tumor barrier to mask chemosensitivity.

Major obstacles in brain cancer treatment include the blood-tumor barrier (BTB), which limits the access of most therapeutic agents, and quiescent tumor cells, which resist conventional chemotherapy. Here, we show that Sox2+ tumor cells project cellular processes to ensheathe capillaries in mouse medulloblastoma (MB), a process that depends on the mechanosensitive ion channel Piezo2. MB develops a tissue stiffness gradient as a function of distance to capillaries. Sox2+ tumor cells perceive substrate stiffness to sustain local intracellular calcium, actomyosin tension, and adhesion to promote cellular process growth and cell surface sequestration of ß-catenin. Piezo2 knockout reverses WNT/ß-catenin signaling states between Sox2+ tumor cells and endothelial cells, compromises the BTB, reduces the quiescence of Sox2+ tumor cells, and markedly enhances the MB response to chemotherapy. Our study reveals that mechanosensitive tumor cells construct the BTB to mask tumor chemosensitivity. Targeting Piezo2 addresses the BTB and tumor quiescence properties that underlie treatment failures in brain cancer.