Natural Killer Cells Degenerate Intact Sensory Afferents following Nerve Injury.

Sensory axons degenerate following separation from their cell body, but partial injury to peripheral nerves may leave the integrity of damaged axons preserved. We show that an endogenous ligand for the natural killer (NK) cell receptor NKG2D, Retinoic Acid Early 1 (RAE1), is re-expressed in adult dorsal root ganglion neurons following peripheral nerve injury, triggering selective degeneration of injured axons. Infiltration of cytotoxic NK cells into the sciatic nerve by extravasation occurs within 3 days following crush injury. Using a combination of genetic cell ablation and cytokine-antibody complex stimulation, we show that NK cell function correlates with loss of sensation due to degeneration of injured afferents and reduced incidence of post-injury hypersensitivity. This neuro-immune mechanism of selective NK cell-mediated degeneration of damaged but intact sensory axons complements Wallerian degeneration and suggests the therapeutic potential of modulating NK cell function to resolve painful neuropathy through the clearance of partially damaged nerves.

The impact of isoflurane anesthesia on brain metabolism in mice: An MRI and electroencephalography study.

Isoflurane is one of the most widely used anesthetic agents in rodent imaging studies. However, the impact of isoflurane on brain metabolism has not been fully characterized to date, primarily due to a scarcity of noninvasive technologies to quantitatively measure the brain's metabolic rate in vivo. In this study, using noncontrast MRI techniques, we dynamically measured cerebral metabolic rate of oxygen (CMRO2) under varying doses of isoflurane anesthesia in mice. Concurrently, systemic parameters of heart and respiration rates were recorded alongside CMRO2. Additionally, electroencephalogram (EEG) recording was used to identify changes in neuronal activities under the same anesthetic regimen employed in the MRI experiments. We found suppression of the CMRO2 by isoflurane in a dose-dependent manner, concomitant with a diminished high-frequency EEG activity. The degree of metabolic suppression by isoflurane was strongly correlated with the respiration rate, which offers a potential approach to calibrate CMRO2 measurements. Furthermore, the metabolic level associated with neural responses of the somatosensory and motor cortices in mice was estimated as 308.2 µmol/100 g/min. These findings may facilitate the integration of metabolic parameters into future studies involving animal disease models and anesthesia usage.

All-or-none disconnection of pyramidal inputs onto parvalbumin-positive interneurons gates ocular dominance plasticity.

Disinhibition is an obligatory initial step in the remodeling of cortical circuits by sensory experience. Our investigation on disinhibitory mechanisms in the classical model of ocular dominance plasticity uncovered an unexpected form of experience-dependent circuit plasticity. In the layer 2/3 of mouse visual cortex, monocular deprivation triggers a complete, "all-or-none," elimination of connections from pyramidal cells onto nearby parvalbumin-positive interneurons (Pyr→PV). This binary form of circuit plasticity is unique, as it is transient, local, and discrete. It lasts only 1 d, and it does not manifest as widespread changes in synaptic strength; rather, only about half of local connections are lost, and the remaining ones are not affected in strength. Mechanistically, the deprivation-induced loss of Pyr→PV is contingent on a reduction of the protein neuropentraxin2. Functionally, the loss of Pyr→PV is absolutely necessary for ocular dominance plasticity, a canonical model of deprivation-induced model of cortical remodeling. We surmise, therefore, that this all-or-none loss of local Pyr→PV circuitry gates experience-dependent cortical plasticity.

The pathological effects of connexin 26 variants related to hearing loss by in silico and in vitro analysis.

Gap junctions (GJs) are intercellular channels associated with cell-cell communication. Connexin 26 (Cx26) encoded by the GJB2 gene forms GJs of the inner ear, and mutations of GJB2 cause congenital hearing loss that can be syndromic or non-syndromic. It is difficult to predict pathogenic effects using only genetic analysis. Using ionic and biochemical coupling tests, we evaluated the pathogenic effects of Cx26 variants using computational analyses to predict structural abnormalities. For seven out of ten variants, we predicted the variation would result in a loss of GJ function, whereas the others would completely fail to form GJs. Functional studies demonstrated that, although all variants were able to function normally as hetero-oligomeric GJ channels, six variants (p.E47K, p.E47Q, p.H100L, p.H100Y, p.R127L, and p.M195L) did not function normally as homo-oligomeric GJ channels. Interestingly, GJs composed of the Cx26 variant p.R127H were able to function normally, even as homo-oligomeric GJ channels. This study demonstrates the particular location and property of an amino acid are more important mainly than the domain where they belong in the formation and function of GJ, and will provide information that is useful for the accurate diagnosis of hearing loss.

Mitochondrial dysfunction and calcium deregulation by the RanBP9-cofilin pathway.

Mitochondrial dysfunction and synaptic damage are important features of Alzheimer's disease (AD) associated with amyloid ß (Aß) and tau. We reported previously that the scaffolding protein RanBP9, which is overall increased in brains of patients with AD and in mutant APP transgenic mice, simultaneously promotes Aß generation and focal adhesion disruption by accelerating the endocytosis of APP and ß1-integrin, respectively. Moreover, RanBP9 induces neurodegeneration in vitro and in vivo and mediates Aß-induced neurotoxicity. Here we show in primary hippocampal neurons that RanBP9 potentiates Aß-induced reactive oxygen species (ROS) overproduction, apoptosis, and calcium deregulation. Analyses of calcium-handling measures demonstrate that RanBP9 selectively delays the clearance of cytosolic Ca(2+) mediated by the mitochondrial calcium uniporter through a process involving the translocation of cofilin into mitochondria and oxidative mechanisms. Further, RanBP9 retards the anterograde axonal transport of mitochondria in primary neurons and decreases synaptic mitochondrial activity in brain. These data indicate that RanBP9, cofilin, and Aß mimic and potentiate each other to produce mitochondrial dysfunction, ROS overproduction, and calcium deregulation, which leads to neurodegenerative changes reminiscent of those seen in AD.

Pivotal role of RanBP9 in integrin-dependent focal adhesion signaling and assembly.

Accumulation of the amyloid ß (Aß) peptide derived from the amyloid precursor protein (APP) plays a central role in the pathogenesis of Alzheimer's disease (AD). We previously reported that the scaffolding protein RanBP9 is markedly increased in AD brains and promotes Aß generation by scaffolding APP/BACE1/LRP complexes together and accelerating APP endocytosis. Because APP, LRP, and RanBP9 all physically interact with ß-integrins, we investigated whether RanBP9 alters integrin-dependent cell adhesion and focal adhesion signaling. Here, we show that RanBP9 overexpression dramatically disrupts integrin-dependent cell attachment and spreading in NIH3T3 and hippocampus-derived HT22 cells, concomitant with strongly decreased Pyk2/paxillin signaling and talin/vinculin localization in focal adhesion complexes. Conversely, RanBP9 knockdown robustly promotes cell attachment, spreading, and focal adhesion signaling and assembly. Cell surface biotinylation and endocytosis assays reveal that RanBP9 overexpression and RanBP9 siRNA potently reduces and increases surface ß1-integrin and LRP by accelerating and inhibiting their endocytosis, respectively. Primary hippocampal neurons derived from RanBP9-transgenic mice also demonstrate severely reduced levels of surface ß1-integrin, LRP, and APP, as well as neurite arborization. Therefore, these data indicate that RanBP9 simultaneously inhibits cell-adhesive processes and enhances Aß generation by accelerating APP, LRP, and ß1-integrin endocytosis.

Sleep and circadian rhythm disruption by NPTX2 loss of function.

Sleep and circadian rhythm disruption (SCRD) is commonly observed in aging, especially in individuals who experience progressive cognitive decline to mild cognitive impairment (MCI) and Alzheimer's disease (AD). However, precise molecular mechanisms underlying the association between SCRD and aging are not fully understood. Orexin A is a well-characterized "sleep neuropeptide" that is expressed in hypothalamic neurons and evokes wake behavior. The importance of Orexin is exemplified in narcolepsy where it is profoundly down-regulated. Interestingly, the synaptic immediate early gene NPTX2 is co-expressed in Orexin neurons and is similarly reduced in narcolepsy. NPTX2 is also down-regulated in CSF of some cognitively normal older individuals and predicts the time of transition from normal cognition to MCI. The association between Orexin and NPTX2 is further evinced here where we observe that Orexin A and NPTX2 are highly correlated in CSF of cognitively normal aged individuals and raises the question of whether SCRD that are typically attributed to Orexin A loss of function may be modified by concomitant NPTX2 down-regulation. Is NPTX2 an effector of sleep or simply a reporter of orexin-dependent SCRD? To address this question, we examined NPTX2 KO mice and found they retain Orexin expression in the brain and so provide an opportunity to examine the specific contribution of NPTX2 to SCRD. Our results reveal that NPTX2 KO mice exhibit a disrupted circadian onset time, coupled with increased activity during the sleep phase, suggesting difficulties in maintaining states. Sleep EEG indicates distinct temporal allocation shifts across vigilance states, characterized by reduced wake and increased NREM time. Evident sleep fragmentation manifests through alterations of event occurrences during Wake and NREM, notably during light transition periods, in conjunction with an increased frequency of sleep transitions in NPTX2 KO mice, particularly between Wake and NREM. EEG spectral analysis indicated significant shifts in power across various frequency bands in the wake, NREM, and REM states, suggestive of disrupted neuronal synchronicity. An intriguing observation is the diminished occurrence of sleep spindles, one of the earliest measures of human sleep disruption, in NPTX2 KO mice. These findings highlight the effector role of NPTX2 loss of function as an instigator of SCRD and a potential mediator of sleep disruption in aging.

Tagging active neurons by soma-targeted Cal-Light.

Verifying causal effects of neural circuits is essential for proving a direct circuit-behavior relationship. However, techniques for tagging only active neurons with high spatiotemporal precision remain at the beginning stages. Here we develop the soma-targeted Cal-Light (ST-Cal-Light) which selectively converts somatic calcium rise triggered by action potentials into gene expression. Such modification simultaneously increases the signal-to-noise ratio of reporter gene expression and reduces the light requirement for successful labeling. Because of the enhanced efficacy, the ST-Cal-Light enables the tagging of functionally engaged neurons in various forms of behaviors, including context-dependent fear conditioning, lever-pressing choice behavior, and social interaction behaviors. We also target kainic acid-sensitive neuronal populations in the hippocampus which subsequently suppress seizure symptoms, suggesting ST-Cal-Light's applicability in controlling disease-related neurons. Furthermore, the generation of a conditional ST-Cal-Light knock-in mouse provides an opportunity to tag active neurons in a region- or cell-type specific manner via crossing with other Cre-driver lines. Thus, the versatile ST-Cal-Light system links somatic action potentials to behaviors with high temporal precision, and ultimately allows functional circuit dissection at a single cell resolution.

A biomarker-authenticated model of schizophrenia implicating NPTX2 loss of function.

Schizophrenia is a polygenetic disorder whose clinical onset is often associated with behavioral stress. Here, we present a model of disease pathogenesis that builds on our observation that the synaptic immediate early gene NPTX2 is reduced in cerebrospinal fluid of individuals with recent onset schizophrenia. NPTX2 plays an essential role in maintaining excitatory homeostasis by adaptively enhancing circuit inhibition. NPTX2 function requires activity-dependent exocytosis and dynamic shedding at synapses and is coupled to circadian behavior. Behavior-linked NPTX2 trafficking is abolished by mutations that disrupt select activity-dependent plasticity mechanisms of excitatory neurons. Modeling NPTX2 loss of function results in failure of parvalbumin interneurons in their adaptive contribution to behavioral stress, and animals exhibit multiple neuropsychiatric domains. Because the genetics of schizophrenia encompasses diverse proteins that contribute to excitatory synapse plasticity, the identified vulnerability of NPTX2 function can provide a framework for assessing the impact of genetics and the intersection with stress.

Direct translation of climbing fiber burst-mediated sensory coding into post-synaptic Purkinje cell dendritic calcium.

Climbing fibers (CFs) generate complex spikes (CS) and Ca2+ transients in cerebellar Purkinje cells (PCs), serving as instructive signals. The so-called 'all-or-none' character of CSs has been questioned since the CF burst was described. Although recent studies have indicated a sensory-driven enhancement of PC Ca2+ signals, how CF responds to sensory events and contributes to PC dendritic Ca2+ and CS remains unexplored. Here, single or simultaneous Ca2+ imaging of CFs and PCs in awake mice revealed the presynaptic CF Ca2+ amplitude encoded the sensory input's strength and directly influenced post-synaptic PC dendritic Ca2+ amplitude. The sensory-driven variability in CF Ca2+ amplitude depended on the number of spikes in the CF burst. Finally, the spike number of the CF burst determined the PC Ca2+ influx and CS properties. These results reveal the direct translation of sensory information-coding CF inputs into PC Ca2+, suggesting the sophisticated role of CFs as error signals.

Transendothelial migration (TEM) of in vitro generated dendritic cell vaccine in cancer immunotherapy.

Many efforts have been made to improve the efficacy of dendritic cell (DC) vaccines in DC-based cancer immunotherapy. One of these efforts is to deliver a DC vaccine more efficiently to the regional lymph nodes (rLNs) to induce stronger anti-tumor immunity. Together with chemotaxis, transendothelial migration (TEM) is believed to be a critical and indispensable step for DC vaccine migration to the rLNs after administration. However, the mechanism underlying the in vitro-generated DC TEM in DC-based cancer immunotherapy has been largely unknown. Currently, junctional adhesion molecules (JAMs) were found to play an important role in the TEM of in vitro generated DC vaccines. This paper reviews the TEM of DC vaccines and TEM-associated JAM molecules.

Junctional adhesion molecules mediate transendothelial migration of dendritic cell vaccine in cancer immunotherapy.

In vitro generated dendritic cells (DCs) have been studied in cancer immunotherapy for decades. However, the detailed molecular mechanism underlying transendothelial migration (TEM) of DC vaccine across the endothelial barrier to regional lymph nodes (LNs) remains largely unknown. Here, we found that junctional adhesion molecule (JAM)-Like (JAML) is involved in the TEM of mouse bone marrow-derived DCs (BMDCs). Treatment with an anti-JAML antibody or JAML knock-down significantly reduced the TEM activity of BMDCs, leading to impairment of DC-based cancer immunotherapy. We found that the interaction of JAML of BMDCs with the coxsackie and adenovirus receptor of endothelial cells plays a crucial role in the TEM of BMDCs. On the other hand, human monocyte-derived DCs (MoDCs) did not express the JAML protein but still showed normal TEM activity. We found that MoDCs express only JAM1 and that the homophilic interaction of JAM1 is essential for MoDC TEM across a HUVEC monolayer. Our findings suggest that specific JAM family members play an important role in the TEM of in vitro-generated mouse and human DCs from the inoculation site to regional LNs in DC-based cancer immunotherapy.

Cortical astrocytes rewire somatosensory cortical circuits for peripheral neuropathic pain.

Long-term treatments to ameliorate peripheral neuropathic pain that includes mechanical allodynia are limited. While glial activation and altered nociceptive transmission within the spinal cord are associated with the pathogenesis of mechanical allodynia, changes in cortical circuits also accompany peripheral nerve injury and may represent additional therapeutic targets. Dendritic spine plasticity in the S1 cortex appears within days following nerve injury; however, the underlying cellular mechanisms of this plasticity and whether it has a causal relationship to allodynia remain unsolved. Furthermore, it is not known whether glial activation occurs within the S1 cortex following injury or whether it contributes to this S1 synaptic plasticity. Using in vivo 2-photon imaging with genetic and pharmacological manipulations of murine models, we have shown that sciatic nerve ligation induces a re-emergence of immature metabotropic glutamate receptor 5 (mGluR5) signaling in S1 astroglia, which elicits spontaneous somatic Ca2+ transients, synaptogenic thrombospondin 1 (TSP-1) release, and synapse formation. This S1 astrocyte reactivation was evident only during the first week after injury and correlated with the temporal changes in S1 extracellular glutamate levels and dendritic spine turnover. Blocking the astrocytic mGluR5-signaling pathway suppressed mechanical allodynia, while activating this pathway in the absence of any peripheral injury induced long-lasting (>1 month) allodynia. We conclude that reawakened astrocytes are a key trigger for S1 circuit rewiring and that this contributes to neuropathic mechanical allodynia.

Lipid rafts serve as signaling platforms for mGlu1 receptor-mediated calcium signaling in association with caveolin.

BACKGROUND: Group I metabotropic glutamate receptors (mGlu1/5 receptors) have important roles in synaptic activity in the central nervous system. They modulate neuronal excitability by mobilizing intracellular Ca2+ following receptor activation. Also, accumulating evidence has indicated the association of Ca2+ signaling with lipid rafts. Caveolin, an adaptor protein found in a specialized subset of lipid rafts, has been reported to promote the localization of membrane proteins to lipid rafts. RESULTS: In the present study, we investigated the role of lipid rafts on the mGlu1α receptor-mediated Ca2+ signaling in association with caveolin in hippocampal primary neurons and HEK293 cells. We show that the disruption of lipid rafts using methyl-ß-cyclodextrin markedly decreased mGlu1α receptor-mediated Ca2+ transients and lipid rafts localization of the receptor. Furthermore, transfection of mGlu1α receptor with mutated caveolin-binding domain reduced localization of the receptor to lipid rafts. Also, application of a peptide blocker of mGlu1α receptor and caveolin binding reduced the Ca2+ signaling and the lipid rafts localization. CONCLUSIONS: Taken together, these results suggest that the binding of mGlu1α receptor to caveolin is crucial for its lipid rafts localization and mGlu1α receptor-mediated Ca2+ transients.

The ganglioside GQ1b regulates BDNF expression via the NMDA receptor signaling pathway.

Gangliosides are sialic acid-containing glycosphingolipids which play a role in neuronal functions. Among the gangliosides, tetrasialoganglioside GQ1b shows neurotrophic factor-like actions, such as increasing neurite outgrowth, cell proliferation, and long-term potentiation. In addition, we recently reported that GQ1b improves spatial learning and memory performance in naïve rats. However, it is still unknown how GQ1b exerts its diverse neuronal functions. Thus, we hypothesized that GQ1b might influence synaptic activity by regulating brain-derived neurotrophic factor (BDNF) expression, which is an important protein for synaptic plasticity and cognition. Interestingly, GQ1b treatment increased BDNF expression in GQ1b-null SH-SY5Y cell lines and rat primary cortical neurons. Additionally, we confirmed whether the observed effects were due to GQ1b or due to a ganglioside with fewer sialic acid molecules (GT1b and GD1b) created by the sialidases present on the plasma membranes, by directly applying GT1b and GD1b or GQ1b co-treated with a sialidase inhibitor. Treatment with GT1b or GD1b had no effect on BDNF expression, whereas co-treatment with a sialidase inhibitor and GQ1b significantly increased BDNF levels. Moreover, GQ1b restored the decreased BDNF expression induced by the ganglioside synthesis inhibitor, D-PDMP, in rat primary cortical neurons. GQ1b treatment significantly increased BDNF levels, whereas pretreatment with the N-methyl-d-aspartate (NMDA) receptor antagonist D-AP5 blocked the effects of GQ1b on BDNF expression, suggesting that GQ1b regulates BDNF expression via the NMDA receptor signaling. Finally, we performed an intracerebroventricular GQ1b injection, which resulted in increased prefrontal and hippocampal BDNF expression in vivo. These findings demonstrate, for the first time, that tetrasialoganglioside GQ1b regulates BDNF expression in vitro and in vivo.

Activation of TRPC4ß by Gαi subunit increases Ca2+ selectivity and controls neurite morphogenesis in cultured hippocampal neuron.

The ubiquitous transient receptor potential canonical (TRPC) channels function as non-selective, Ca(2+)-permeable channels. TRPC channels are activated by stimulation of Gαq-PLC-coupled receptors. Here, we report that TRPC4/TRPC5 can be activated by Gαi. We studied the essential role of Gαi subunits in TRPC4 activation and investigated changes in ion selectivity and pore dilation of the TRPC4 channel elicited by the Gαi2 subunit. Activation of TRPC4 by Gαi2 increased Ca2+ permeability and Ca2+ influx through TRPC4 channels. Co-expression of the muscarinic receptor (M2) and TRPC4 in HEK293 cells induced TRPC4-mediated Ca2+ influx. Moreover, both TRPC4ß and the TRPC4ß-Gαi2 signaling complex induced inhibition of neurite growth and arborization in cultured hippocampal neurons. Cells treated with KN-93, a CaMKII inhibitor, prevented TRPC4- and TRPC4-Gαi2(Q205L)-mediated inhibition of neurite branching and growth. These findings indicate an essential role of Gαi proteins in TRPC4 activation and extend our knowledge of the functional role of TRPC4 in hippocampal neurons.

The Interface between Cytoskeletal Aberrations and Mitochondrial Dysfunction in Alzheimer's Disease and Related Disorders.

The major defining pathological hallmarks of Alzheimer's disease (AD) are the accumulations of Aß in senile plaques and hyperphosphorylated tau in neurofibrillary tangles and neuropil threads. Recent studies indicate that rather than these insoluble lesions, the soluble Aß oligomers and hyperphosphorylated tau are the toxic agents of AD pathology. Such pathological protein species are accompanied by cytoskeletal changes, mitochondrial dysfunction, Ca(2+) dysregulation, and oxidative stress. In this review, we discuss how the binding of Aß to various integrins, defects in downstream focal adhesion signaling, and activation of cofilin can impact mitochondrial dysfunction, cytoskeletal changes, and tau pathology induced by Aß oligomers. Such pathological consequences can also feedback to further activate cofilin to promote cofilin pathology. We also suggest that the mechanism of Aß generation by the endocytosis of APP is mechanistically linked with perturbations in integrin-based focal adhesion signaling, as APP, LRP, and ß-integrins are physically associated with each other.