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Repeat-associated non-AUG (RAN) translation of mRNAs/transcripts responsible for polyglutamine (polyQ) diseases may generate peptides containing different mono amino acid tracts such as polyserine (polyS) and polyleucine (polyL). The propagation of aggregated polyQ from one cell to another is also an intriguing feature of polyQ proteins. However, whether the RAN translation-related polyS and polyL have the ability to propagate remains unclear, and if they do, whether the exogenous polyS and polyL exert toxicity on the recipient cells is also not known yet. In the present study, we found that aggregated polyS and polyL peptides spontaneously enter neuron-like cells and astrocytes in vitro. Aggregated polyS led to the degeneration of the differentiated neuron-like cultured cells. Likewise, the two types of aggregates taken up by astrocytes induced aberrant differentiation and cell death in vitro. Furthermore, injection of each of the two types of aggregates into the ventricles of adult mice resulted in their behavioral changes. The polyS-injected mice showed extensive vacuolar degeneration in the brain. Thus, the RAN translation-related proteins containing polyS and polyL have the potential to propagate and the proteins generated by all polyQ diseases might exert universal toxicity in the recipient cells.

The bidirectional controller of the thermoregulatory center in the preoptic area (POA) is unknown. Using rats, here, we identify prostaglandin EP3 receptor–expressing POA neurons (POA EP3R neurons) as a pivotal bidirectional controller in the central thermoregulatory mechanism. POA EP3R neurons are activated in response to elevated ambient temperature but inhibited by prostaglandin E 2 , a pyrogenic mediator. Chemogenetic stimulation of POA EP3R neurons at room temperature reduces body temperature by enhancing heat dissipation, whereas inhibition of them elicits hyperthermia involving brown fat thermogenesis, mimicking fever. POA EP3R neurons innervate sympathoexcitatory neurons in the dorsomedial hypothalamus (DMH) via tonic (ceaseless) inhibitory signaling. Although many POA EP3R neuronal cell bodies express a glutamatergic messenger RNA marker, their axons in the DMH predominantly release γ-aminobutyric acid (GABA), and their GABAergic terminals are increased by chronic heat exposure. These findings demonstrate that tonic GABAergic inhibitory signaling from POA EP3R neurons is a fundamental determinant of body temperature for thermal homeostasis and fever.

Exercise including locomotion requires appropriate autonomic cardiovascular adjustments to meet the metabolic demands of contracting muscles, yet the functional brain architecture underlying these adjustments remains unknown. Here, we demonstrate brainstem circuitry that plays an essential role in relaying volitional motor signals, i.e., central command, to drive locomotor activities and sympathetic cardiovascular responses. Mesencephalic locomotor neurons in rats transmit central command-driven excitatory signals onto the rostral ventrolateral medulla at least partially via glutamatergic processes, to activate both somatomotor and sympathetic nervous systems. Optogenetic excitation of this monosynaptic pathway elicits locomotor and cardiovascular responses as seen during running exercise, whereas pathway inhibition suppresses the locomotor activities and blood pressure elevation during voluntary running without affecting basal cardiovascular homeostasis. These results demonstrate an important subcortical pathway that transmits central command signals, providing a key insight into the central circuit mechanism required for the physiological conditioning essential to maximize exercise performance.

CRISPR-Cas9 are widely used for gene targeting in mice and rats. The non-homologous end-joining (NHEJ) repair pathway, which is dominant in zygotes, efficiently induces insertion or deletion (indel) mutations as gene knockouts at targeted sites, whereas gene knock-ins (KIs) via homology-directed repair (HDR) are difficult to generate. In this study, we used a double-stranded DNA (dsDNA) donor template with Cas9 and two single guide RNAs, one designed to cut the targeted genome sequences and the other to cut both the flanked genomic region and one homology arm of the dsDNA plasmid, which resulted in 20–33% KI efficiency among G0 pups. G0 KI mice carried NHEJ-dependent indel mutations at one targeting site that was designed at the intron region, and HDR-dependent precise KIs of the various donor cassettes spanning from 1 to 5 kbp, such as EGFP, mCherry, Cre, and genes of interest, at the other exon site. These findings indicate that this combinatorial method of NHEJ and HDR mediated by the CRISPR-Cas9 system facilitates the efficient and precise KIs of plasmid DNA cassettes in mice and rats. Electronic supplementary material The online version of this article (10.1007/s00439-020-02198-4) contains supplementary material, which is available to authorized users.

Repeat-associated non-AUG translation (RAN translation) is observed in transcripts that are causative for polyglutamine (polyQ) diseases and generates proteins with mono amino acid tracts such as polyalanine (polyA), polyleucine (polyL) and polyserine (polyS) in neurons, astrocytes and microglia. We have previously shown that microglia with aggregated polyQ led to defective differentiation and degeneration of neuron-like cells. However, it has not been determined whether only microglia containing a specific RAN product, but not other RAN products, is harmful in vitro and in vivo. Here we show that polyL-incorporating microglia specifically led to altered startle response in mice. Aggregated polyA, polyS and polyL induced aberrant differentiation of microglia-like BV2 cells. Differentiated PC12 cells treated with conditioned medium (CM) of polyS- and polyL- but not polyA-incorporating microglia-like BV2 cells showed retraction of neurites and loss of branch of neurites. Injection of the polyL-CM, but not polyA-CM and polyS-CM, into the lateral ventricle lowered startle response in mice. Consistently, polyL induced the highest expression of CD68 in BV2 cells. The lowered startle response was replicated in mice given the polyL-CM in the caudal pontine reticular nucleus (PnC), the key region of startle response. Thus, endogenous RAN proteins having polyL derived from polyQ diseases-causative genes in microglia might specifically impair startle response.

In addition to maintaining a constant core body temperature, the mammalian thermoregulatory system, including that in humans deviates body temperature from the normally defended range as necessary to protect the organism from various environmental stressors, such as ambient temperature (heat and cold), pathogens, natural enemies, and starvation. Recent studies have reported that neural circuits of the brain are involved in thermoregulatory responses to environmental stresses. This article summarizes the most recent findings on the central neural network associated with thermoregulatory physiological responses to environmental stresses.

In polyalanine (PA) diseases, the disease-causing transcription factors contain an expansion of alanine repeats. While aggregated proteins that are responsible for the pathogenesis of neurodegenerative disorders show cell-to-cell propagation and thereby exert toxic effects on the recipient cells, whether this is also the case with expanded PA has not been studied. It is also not known whether the internalized PA is toxic to recipient cells based on the degree of aggregation. In this study, we therefore prepared different degrees of aggregation of a peptide having 13 alanine repeats without flanking sequences of PA disease-causative proteins (13A). The aggregated 13A was spontaneously taken up by neuron-like cultured cells. Functionally, strong aggregates but not weak aggregates displayed a deficit in neuron-like differentiation in vitro. Moreover, the injection of strong but not weak 13A aggregates into the ventricle of mice during the neonatal stage led to enhanced spontaneous motor activity later in life. Thus, PA in the extracellular space has the potential to enter adjacent cells, and may exert toxicity depending on the degree of aggregation.

Various environmental stressors, such as extreme temperatures (hot and cold), pathogens, predators and insufficient food, can threaten life. Remarkable progress has recently been made in understanding the central circuit mechanisms of physiological responses to such stressors. A hypothalamomedullary neural pathway from the dorsomedial hypothalamus (DMH) to the rostral medullary raphe region (rMR) regulates sympathetic outflows to effector organs for homeostasis. Thermal and infection stress inputs to the preoptic area dynamically alter the DMH → rMR transmission to elicit thermoregulatory, febrile and cardiovascular responses. Psychological stress signalling from a ventromedial prefrontal cortical area to the DMH drives sympathetic and behavioural responses for stress coping, representing a psychosomatic connection from the corticolimbic emotion circuit to the autonomic and somatic motor systems. Under starvation stress, medullary reticular neurons activated by hunger signalling from the hypothalamus suppress thermogenic drive from the rMR for energy saving and prime mastication to promote food intake. This Perspective presents a combined neural network for environmental stress responses, providing insights into the central circuit mechanism for the integrative regulation of systemic organs. Environmental stressors, including extreme ambient temperature, the presence of pathogens or predators, and a lack of food, can profoundly influence animal behaviour. In this Perspective, Nakamura, Nakamura and Kataoka present a hypothalamomedullary network model for physiological responses to various environmental stressors.

Oxytocin alters autonomic functions besides social behaviors. However, the central neuronal links between hypothalamic oxytocinergic neurons and the autonomic nervous system remain unclear. Here we show that oxytocinergic neurons in the rat paraventricular hypothalamic nucleus (PVH), a pivotal site for energy homeostasis, innervate sympathetic premotor neurons in the rostral medullary raphe region (rMR) to stimulate brown adipose tissue (BAT) thermogenesis and cardiovascular functions. Oxytocin receptor stimulation in the rMR evokes BAT thermogenesis and tachycardia. In vivo optogenetic stimulation of the PVH→rMR long-range oxytocinergic pathway, using a virus-mediated system for amplified gene expression in oxytocinergic neurons, not only elicits BAT thermogenic and cardiac responses but also potentiates sympathetic responses evoked by glutamatergic transmission in the rMR. The PVH→rMR oxytocinergic pathway connects the hypothalamic circuit for energy homeostasis to thermogenic and cardiac sympathetic outflow, and, therefore, its defects may cause obesity and impaired thermoregulation, as seen in Prader-Willi syndrome.

In mammals, many types of psychological stressors elicit a variety of sympathoexcitatory responses paralleling the classic fight-or-flight response to a threat to survival, including increased body temperature via brown adipose tissue thermogenesis and cutaneous vasoconstriction, and increased skeletal muscle blood flow via tachycardia and visceral vasoconstriction. Although these responses are usually supportive for stress coping, aberrant sympathetic responses to stress can lead to clinical issues in psychosomatic medicine. Sympathetic stress responses are mediated mostly by sympathetic premotor drives from the rostral medullary raphe region (rMR) and partly by those from the rostral ventrolateral medulla (RVLM). Hypothalamomedullary descending pathways from the dorsomedial hypothalamus (DMH) to the rMR and RVLM mediate important, stress-driven sympathoexcitatory transmission to the premotor neurons to drive the thermal and cardiovascular responses. The DMH also likely sends an excitatory input to the paraventricular hypothalamic nucleus to stimulate stress hormone release. Neurons in the DMH receive a stress-related excitation from the dorsal peduncular cortex and dorsal tenia tecta (DP/DTT) in the ventromedial prefrontal cortex. By connecting the corticolimbic emotion circuit to the central sympathetic and somatic motor systems, the DP/DTT → DMH pathway plays as the primary mediator of the psychosomatic signaling that drives a variety of sympathetic and behavioral stress responses. These brain regions together with other stress-related regions constitute a central neural network for physiological stress responses. This network model is relevant to understanding the central mechanisms by which stress and emotions affect autonomic regulations of homeostasis and to developing new therapeutic strategies for various stress-related disorders.

Precise evaluation of motor functions using simple and reproducible tests for mouse models of spinal cord injury (SCI) are required. Overground walking of SCI mice has been tested by Basso Mouse Scale for locomotion (BMS). In contrast, only a few works quantify walking performances of SCI mice on narrow beams, a different task. Here, we established a novel scoring system using a single beam walking apparatus for SCI mice. The scoring system uses binary judgments of values such as retention, moving forward and reaching the goal on a beam for rating. In addition, high score was given to SCI mouse when the mouse efficiently used hindlimbs for locomotion on the beam. A high rate of concordance of the score derived from positions of hindlimbs between two observers was obtained. Mice displayed the lowest total score on the beam immediately after the SCI, then the score gradually increased like time course of BMS score. Furthermore, the total scores reflected gradation of severity of SCI in 2 strains of mice. The beam walking score proved to be strongly correlated with that of BMS score, indicating that performances between overground walking and beam walking are partly correlated in SCI mice. Collectively, the novel scoring system offers an opportunity to easily evaluate motor performances of mice with SCI.

Adaptive thermogenesis is essential for survival, and therefore is tightly regulated by a central neural circuit. Here, we show that microRNA (miR)-33 in the brain is indispensable for adaptive thermogenesis. Cold stress increases miR-33 levels in the hypothalamus and miR-33−/− mice are unable to maintain body temperature in cold environments due to reduced sympathetic nerve activity and impaired brown adipose tissue (BAT) thermogenesis. Analysis of miR-33f/f dopamine-β-hydroxylase (DBH)-Cre mice indicates the importance of miR-33 in Dbh-positive cells. Mechanistically, miR-33 deficiency upregulates gamma-aminobutyric acid (GABA)A receptor subunit genes such as Gabrb2 and Gabra4. Knock-down of these genes in Dbh-positive neurons rescues the impaired cold-induced thermogenesis in miR-33f/f DBH-Cre mice. Conversely, increased gene dosage of miR-33 in mice enhances thermogenesis. Thus, miR-33 in the brain contributes to maintenance of BAT thermogenesis and whole-body metabolism via enhanced sympathetic nerve tone through suppressing GABAergic inhibitory neurotransmission. This miR-33-mediated neural mechanism may serve as a physiological adaptive defense mechanism for several stresses including cold stress., 褐色脂肪細胞の燃焼を促す新たなメカニズムを解明 --体の熱産生にマイクロRNA-33が関与--. 京都大学プレスリリース. 2021-02-17.

Proteins containing an expanded polyglutamine tract tend to aggregate, leading to the neuronal damage observed in polyglutamine diseases. We recently reported that free electron laser (FEL) irradiation markedly dissociates naked polyglutamine aggregates as well as the aggregate in the 293 T cells. In the present study, we investigated whether FEL irradiation of neuron-like cells with polyglutamine aggregates would restore the cellular damage and dysfunction. The aggregated polyglutamine peptides induced neurite retraction of differentiated SH-SY5Y cells. Upon FEL irradiation, the polyglutamine aggregates in the SH-SY5Y cells were dissociated, and the shorter length of individual neurite, fewer number of neurites per cell and shorter total length of neurite by polyglutamine were inhibited. Same results were essentially obtained in PC12 cells. Moreover, when FEL irradiation was applied to undifferentiated SH-SY5Y cells, the deficits in neuron-like differentiation seen in expanded polyglutamine peptide-containing cells were also rescued. Thus, FEL irradiation restored both the damage and differentiation caused by polyglutamine in neuron-like cells.

Structure of amyloid β (Aβ) fibrils is rigidly stacked by β-sheet conformation, and the fibril state of Aβ is profoundly related to pathogenesis of Alzheimer’s disease (AD). Although mid-infrared light has been used for various biological researches, it has not yet been known whether the infrared light changes the fibril structure of Aβ. In this study, we tested the effect of irradiation of intense mid-infrared light from a free-electron laser (FEL) targeting the amide bond on the reduction of β-sheet content in Aβ fibrils. The FEL reduced entire contents of proteins exhibiting β-sheet structure in brain sections from AD model mice, as shown by synchrotron-radiation infrared microscopy analysis. Since Aβ1-42 fibril absorbed a considerable FEL energy at amide I band (6.17 μm), we irradiated the FEL at 6.17 μm and found that β-sheet content of naked Aβ1-42 fibril was decreased using infrared microscopic analysis. Consistent with the decrease in the β-sheet content, Congo-red signal is decreased after the irradiation to Aβ1-42 fibril. Furthermore, electron microscopy analysis revealed that morphologies of the fibril and proto-fibril were largely changed after the irradiation. Thus, mid-infrared light dissociates β-sheet structure of Aβ fibrils, which justifies exploration of possible laser-based therapy for AD.

Energy homeostasis of mammals is maintained by balancing energy expenditure within the body and energy intake through feeding. Several lines of evidence indicate that brown adipose tissue (BAT), a sympathetically activated thermogenic organ, turns excess energy into heat to maintain the energy balance in rodents and humans, in addition to its thermoregulatory role for the defense of body core temperature in cold environments. Elucidating the central circuit mechanism controlling BAT thermogenesis dependent on nutritional conditions and food availability in relation to energy homeostasis is essential to understand the etiology of symptoms caused by energy imbalance, such as obesity. The central thermogenic command outflow to BAT descends through an excitatory neural pathway mediated by hypothalamic, medullary and spinal sites. This sympathoexcitatory thermogenic drive is controlled by tonic GABAergic inhibitory signaling from the thermoregulatory center in the preoptic area, whose tone is altered by body core and cutaneous thermosensory inputs. This circuit controlling BAT thermogenesis for cold defense also functions for the development of fever and psychological stress-induced hyperthermia, indicating its important role in the defense from a variety of environmental stressors. When food is unavailable, hunger-driven neural signaling from the hypothalamus activates GABAergic neurons in the medullary reticular formation, which then block the sympathoexcitatory thermogenic outflow to BAT to reduce energy expenditure and simultaneously command the masticatory motor system to promote food intake-effectively commanding responses to survive starvation. This article reviews the central mechanism controlling BAT thermogenesis in relation to the regulation of energy and thermal homeostasis dependent on food availability.

Background and purpose Experimental subarachnoid hemorrhage (SAH) by endovascular filament perforation method is used widely in mice, but it sometimes present acute cerebral infarctions with varied magnitude and anatomical location. This study aimed to determine the prevalence and location of the acute ischemic injury in this experimental model. Methods Male C57BL/6 mice were subjected to SAH by endovascular perforation. Distribution of SAH was defined by T2*-weighted images within 1 h after SAH. Prevalence and location of acute infarction were assessed by diffusion-weighted MR images on day 1 after the induction. Results Among 72 mice successfully acquired post-SAH MR images, 29 (40%) developed acute infarction. Location of the infarcts was classified into either single infarct (ipsilateral cortex, n = 12; caudate putamen, n = 3; hippocampus, n = 1) or multiple lesions (cortex and caudate putamen, n = 6; cortex and hippocampus, n = 2; cortex, hippocampus and thalamus/hypothalamus, n = 3; bilateral cortex, n = 2). The mortality rate within 24 h was significantly higher in mice with multiple infarcts than those with single lesion (30% versus 0%; P = 0.03). Distribution of the ischemic lesion positively correlated with MRI-evidenced SAH grading (r2 = 0.31, P = 0.0002). Conclusion Experimental SAH immediately after the vessel perforation can induce acute cerebral infarction in varying vascular territories, resulting in increased mortality. The present model may in part, help researchers to interpret the mechanism of clinically-evidenced early multiple combined infarction.

A major psychosocial stress circuit Psychological stress induces various physiological responses by activating the sympathetic nervous system. The brain circuits involved in these functions are still not completely understood. In a rat model, Kataoka et al. combined anatomical tracing, immediate early gene expression analysis, pharmacology, optogenetics, electrophysiology, and genetic cell ablation to provide evidence for the prominent role of a ventral part of the medial prefrontal cortex in sympathetic responses to social defeat stress. This brain region sends excitatory projections to the dorsomedial hypothalamus as a central coordinator of the psychosocial stress responses. This pathway is crucial for understanding how psychosocial stress influences a variety of body functions. Science , this issue p. 1105

Masking is a direct behavioral response to environmental changes and plays an important role in the temporal distribution of activity. However, the mechanisms responsible for masking remain unclear. Here we identify thermosensors and a possible neural circuit regulating temperature-dependent masking behavior in mice. Analysis of mice lacking thermosensitive transient receptor potential (TRP) channels (Trpv1/3/4 and Trpm2/8) reveals that temperature-dependent masking is impaired in Trpm2- and Trpm8-null mice. Several brain regions are activated during temperature-dependent masking, including the preoptic area (POA), known as the thermoregulatory center, the suprachiasmatic nucleus (SCN), which is the primary circadian pacemaker, the paraventricular nucleus of the thalamus (PVT), and the nucleus accumbens (NAc). The POA, SCN, PVT are interconnected, and the PVT sends dense projections to the NAc, a key brain region involved in wheel-running activity. Partial chemical lesion of the PVT attenuates masking, suggesting the involvement of the PVT in temperature-dependent masking behavior.

SummaryBackground LL-37 is an antimicrobial peptide with pleiotropic effects on the immune system, angiogenesis and tissue remodelling. These are cardinal pathological events in systemic sclerosis (SSc). Objectives To elucidate the potential role of LL-37 in SSc. Methods The expression of target molecules was evaluated by immunostaining and quantitative reverse-transcription real-time polymerase chain reaction in human and murine skin. The mechanisms regulating LL-37 expression in endothelial cells were examined by gene silencing and chromatin immunoprecipitation. Serum LL-37 levels were determined by enzyme-linked immunosorbent assay. Results In SSc lesional skin, LL-37 expression was increased in dermal fibroblasts, perivascular inflammatory cells, keratinocytes and, particularly, dermal small vessels. Expression positively correlated with interferon-α expression, possibly reflecting LL-37-dependent induction of interferon-α. In SSc animal models, bleomycin-treated skin exhibited the expression pattern of CRAMP, a murine homologue of LL-37, similar to that of LL-37 in SSc lesional skin. Furthermore, Fli1+/− mice showed upregulated expression of CRAMP in dermal small vessels. Fli1 binding to the CAMP (LL-37 gene) promoter and Fli1 deficiency-dependent induction of LL-37 were also confirmed in human dermal microvascular endothelial cells. In the analysis of sera, patients with SSc had serum LL-37 levels significantly higher than in healthy controls. Furthermore, serum LL-37 levels positively correlated with skin score and the activity of alveolitis and were significantly elevated in patients with digital ulcers compared with those without. Conclusions LL-37 upregulation, induced by Fli1 deficiency at least in endothelial cells, potentially contributes to the development of skin sclerosis, interstitial lung disease and digital ulcers in SSc.

Summary Aims Spinocerebellar ataxia type 1 (SCA1) is caused by the ataxin-1 protein (ATXN1) with an abnormally expanded polyglutamine tract and is characterized by progressive neurodegeneration. We previously showed that intrathecal injection of mesenchymal stem cells (MSCs) during the nonsymptomatic stage mitigates the degeneration of the peripheral nervous system (PNS) neurons in SCA1-knock-in (SCA1-KI) mice. We tested in this study whether the therapeutic effects of MSCs in SCA1-KI mice could be reproduced with MSC-releasing factor(s). Methods To test the effects of MSC-releasing factor(s), we used MSC-conditioned medium (MSC-CM). MSC-CM was intrathecally and/or intravenously injected into young SCA1-KI mice, and the therapeutic effects were assessed in the PNS at later ages using immunostaining, electrophysiology, and behavioral tests. Results MSC-CM attenuated the degeneration of axons and myelin of spinal motor neurons. Consequently, the injected SCA1-KI mice exhibited smaller reductions in nerve conduction velocity in spinal motor neurons and reduced motor incoordination than the untreated mice. Conclusions These results suggest that factors released from MSC mitigate the morphological and functional abnormalities in the PNS that are observed in SCA1-KI mice in a paracrine manner.

Body core temperature of mammals is regulated by the central nervous system, in which the preoptic area (POA) of the hypothalamus plays a pivotal role. The POA receives peripheral and central thermosensory neural information and provides command signals to effector organs to elicit involuntary thermoregulatory responses, including shivering thermogenesis, nonshivering brown adipose tissue thermogenesis, and cutaneous vasoconstriction. Cool-sensory and warm-sensory signals from cutaneous thermoreceptors, monitoring environmental temperature, are separately transmitted through the spinal-parabrachial-POA neural pathways, distinct from the spinothalamocortical pathway for perception of skin temperature. These cutaneous thermosensory inputs to the POA likely impinge on warm-sensitive POA neurons, which monitor body core (brain) temperature, to alter thermoregulatory command outflows from the POA. The cutaneous thermosensory afferents elicit rapid thermoregulatory responses to environmental thermal challenges before they impact body core temperature. Peripheral humoral signals also act on neurons in the POA to transmit afferent information of systemic infection and energy storage to induce fever and to regulate energy balance, respectively. This chapter describes the thermoregulatory afferent mechanisms that convey cutaneous thermosensory signals to the POA and that integrate the neural and humoral afferent inputs to the POA to provide descending command signals to thermoregulatory effectors.

Maintenance of a homeostatic body core temperature is a critical brain function accomplished by a central neural network. This orchestrates a complex behavioral and autonomic repertoire in response to environmental temperature challenges or declining energy homeostasis and in support of immune responses and many behavioral states. This review summarizes the anatomical, neurotransmitter, and functional relationships within the central neural network that controls the principal thermoeffectors: cutaneous vasoconstriction regulating heat loss and shivering and brown adipose tissue for heat production. The core thermoregulatory network regulating these thermoeffectors consists of parallel but distinct central efferent pathways that share a common peripheral thermal sensory input. Delineating the neural circuit mechanism underlying central thermoregulation provides a useful platform for exploring its functional organization, elucidating the molecular underpinnings of its neuronal interactions, and discovering novel therapeutic approaches to modulating body temperature and energy homeostasis.

Causal relationships between central cardiovascular pathways and sympathetic vasomotor tone have not been evidenced. This study aimed to verify the sympathoexcitatory role of hypothalamic paraventricular nucleus neurons that project to the rostral ventrolateral medulla (PVN-RVLM neurons). By using optogenetic techniques, we demonstrated that stimulation of PVN-RVLM glutamatergic neurons increased renal sympathetic nerve activity and arterial pressure via, at least in part, stimulation of RVLM C1 neurons in rats. This monosynaptic pathway may function in acute sympathetic adjustments to stressors and/or be a component of chronic sympathetic hyperactivity in pathological conditions such as heart failure.The rostral ventrolateral medulla (RVLM), which is known to play an important role in regulating sympathetic vasomotor tone, receives axonal projections from the hypothalamic paraventricular nucleus (PVN). However, no studies have proved that excitation of the PVN neurons that send axonal projections to the RVLM (PVN-RVLM neurons) causes sympathoexcitation. This study aimed to directly examine the sympathoexcitatory role of PVN-RVLM neurons. Male rats received microinjections into the PVN with an adeno-associated virus (AAV) vector that encoded a hybrid of channelrhodopsin-2/1 with the reporter tdTomato (ChIEF-tdTomato), or into the RVLM with a retrograde AAV vector that encoded a channelrhodopsin with green fluorescent protein (ChR2-GFP

Spinocerebellar ataxia type 1 (SCA1) is a progressive neurodegenerative disease caused by extension of a CAG repeat in the Sca1gene. Although the mechanisms underlying the symptoms of SCA1 have not been determined, aberrant neuronal activation potentially contributes to the neuronal cell death characteristic of the disease. Here we examined the potential involvement of extrasynaptic N-methyl-d-aspartate receptor (NMDAR) activation in the pathogenesis of SCA1 by administering memantine, a low-affinity noncompetitive NMDAR antagonist, in SCA1 knock-in (KI) mice. In KI mice, the exon in the ataxin 1 gene is replaced with abnormally expanded 154CAG repeats. Memantine was administered orally to the SCA1 KI mice from 4 weeks of age until death. The treatment significantly attenuated body-weight loss and prolonged the life span of SCA1 KI mice. Furthermore, memantine significantly suppressed the loss of Purkinje cells in the cerebellum and motor neurons in the dorsal motor nucleus of the vagus, which are critical for motor function and parasympathetic function, respectively. These findings support the contribution of aberrant activation of extrasynaptic NMDARs to neuronal cell death in SCA1 KI mice and suggest that memantine may also have therapeutic benefits in human SCA1 patients.

The recent discovery of the medullary circuit driving "hunger responses" - reduced thermogenesis and promoted feeding - has greatly expanded our knowledge on the central neural networks for energy homeostasis. However, how hypothalamic hunger and satiety signals generated under fasted and fed conditions, respectively, control the medullary autonomic and somatic motor mechanisms remains unknown. Here, in reviewing this field, we propose two hypothalamomedullary neural pathways for hunger and satiety signaling. To trigger hunger signaling, neuropeptide Y activates a group of neurons in the paraventricular hypothalamic nucleus (PVH), which then stimulate an excitatory pathway to the medullary circuit to drive the hunger responses. In contrast, melanocortin-mediated satiety signaling activates a distinct group of PVH neurons, which then stimulate a putatively inhibitory pathway to the medullary circuit to counteract the hunger signaling. The medullary circuit likely contains inhibitory and excitatory premotor neurons whose alternate phasic activation generates the coordinated masticatory motor rhythms to promote feeding.

Thermoregulatory behavior in homeothermic animals is an instinctive behavior to reduce the cost of their homeothermy, such as warm-seeking behavior to reduce metabolic energy for heat production in cold environments. A recent study identified the central neural pathway that transmits cutaneous thermosensory information on environmental temperature to drive thermoregulatory behavior. This study proposed that the brain mechanism that drives thermoregulatory behavior does not require conscious"feeling"(i.e., perception)of skin temperature mediated by the spinothalamocortical pathway, whereas thermosensory information that ascends through the spinal cord and the lateral parabrachial nucleus of the pons generates thermal comfort and discomfort that drive thermoregulatory behavior.

Thermoregulatory behaviour, such as migration to a comfortable thermal environment, is a representative innate animal behaviour and facilitates effective autonomic regulation of body temperature with a reduced cost of resources. Here we determine the central thermosensory ascending pathway that transmits information on environmental temperature from cutaneous thermoreceptors to elicit thermoregulatory behaviour. To examine the contribution of the spinothalamocortical pathway, which is known to mediate thermosensory transmission for perception of skin temperature, we lesioned thalamic regions mediating this pathway in rats. Thalamic-lesioned rats showed compromised electroencephalographic responses in the primary somatosensory cortex to changes in skin temperature, indicating functional ablation of the spinothalamocortical pathway. However, these lesioned rats subjected to a two-floor innocuous thermal plate preference test displayed intact heat- and cold-avoidance thermoregulatory behaviours. We then examined the involvement of the lateral parabrachial nucleus (LPB), which mediates cutaneous thermosensory signaling to the thermoregulatory center for autonomic thermoregulation. Inactivation of neurons in the LPB eliminated both heat- and cold-avoidance thermoregulatory behaviours and ablated heat defense. These results demonstrate that the LPB, but not the thalamus, mediates the cutaneous thermosensory neural signaling required for behavioural thermoregulation, contributing to understanding of the central circuit that generates thermal comfort and discomfort underlying thermoregulatory behaviours.

Psychological stress-induced hyperthermia (PSH) is a fundamental autonomic stress response observed in many mammalian species. Here we show a hypothalamomedullary, glutamatergic neural pathway for psychological stress signaling that drives the sympathetic thermogenesis in brown adipose tissue (BAT) that contributes to PSH. Using invivo drug nanoinjections into rat brain and thermotelemetry, we demonstrate that the rostral medullary raphe region (rMR) and dorsomedial hypothalamus (DMH) mediate a psychosocial stress-induced thermogenesis in BAT and PSH. Functional neuroanatomy indicates that the DMH functions as a hub for stress signaling, with monosynaptic projections to the rMR for sympathetic outputs and to the paraventricular hypothalamic nucleus for neuroendocrine outputs. Optogenetic experiments showed that the DMH-rMR monosynaptic pathway drives BAT thermogenesis and cardiovascular responses. These findings make an important contribution to our understanding of the central autonomic circuitries linking stress coping with energy homeostasis-potentially underlying the etiology of psychogenic fever, a major psychosomatic symptom., 心理ストレスを受けたときに体温を上昇させる脳神経回路を解明 -ストレス疾患の発症機構の解明に期待- 京都大学プレスリリース. 2014-06-27.

The anxiolytic diazepam selectively inhibits psychological stress-induced autonomic and behavioral responses without causing noticeable suppression of other central performances. This pharmacological property of diazepam led us to the idea that neurons that exhibit diazepam-sensitive, psychological stress-induced activation are potentially those recruited for stress responses. To obtain neuroanatomical clues for the central stress circuitries, we examined the effects of diazepam on psychological stress-induced neuronal activation in broad brain regions. Rats were exposed to a social defeat stress, which caused an abrupt increase in body temperature by up to 2°C. Pretreatment with diazepam (4mg/kg, i.p.) attenuated the stress-induced hyperthermia, confirming an inhibitory physiological effect of diazepam on the autonomic stress response. Subsequently, the distribution of cells expressing Fos, a marker of neuronal activation, was examined in 113 forebrain and midbrain regions of these rats after the stress exposure and diazepam treatment. The stress following vehicle treatment markedly increased Fos-immunoreactive (IR) cells in most regions of the cerebral cortex, limbic system, thalamus, hypothalamus and midbrain, which included parts of the autonomic, neuroendocrine, emotional and arousal systems. The diazepam treatment significantly reduced the stress-induced Fos expression in many brain regions including the prefrontal, sensory and motor cortices, septum, medial amygdaloid nucleus, medial and lateral preoptic areas, parvicellular paraventricular hypothalamic nucleus, dorsomedial hypothalamus, perifornical nucleus, tuberomammillary nucleus, association, midline and intralaminar thalami, and median and dorsal raphe nuclei. In contrast, diazepam increased Fos-IR cells in the central amygdaloid nucleus, medial habenular nucleus, ventromedial hypothalamic nucleus and magnocellular lateral hypothalamus. These results provide important information for elucidating the neural circuitries that mediate the autonomic and behavioral responses to psychosocial stressors.

Postnatal hematopoietic progenitor cells do not contribute to microglial homeostasis in adult mice under normal conditions. However, previous studies using whole-body irradiation and bone marrow (BM) transplantation models have shown that adult BM cells migrate into the brain tissue and differentiate into microglia (BM-derived microglia; BMDM). Here, we investigated whether cranial irradiation alone was sufficient to induce the generation of BMDM in the adult mouse brain. Transgenic mice that express green fluorescent protein (GFP) under the control of a murine stem cell virus (MSCV) promoter (MSCV-GFP mice) were used. MSCV-GFP mice express GFP in BM cells but not in the resident microglia in the brain. Therefore, these mice allowed us to detect BM-derived cells in the brain without BM reconstitution. MSCV–GFP mice, aged 8–12 weeks, received 13.0 Gy irradiation only to the cranium, and BM-derived cells in the brain were quantified at 3 and 8 weeks after irradiation. No BM-derived cells were detected in control non-irradiated MSCV-GFP mouse brains, but numerous GFP-labeled BM-derived cells were present in the brain stem, basal ganglia and cerebral cortex of the irradiated MSCV-GFP mice. These BM-derived cells were positive for Iba1, a marker for microglia, indicating that GFP-positive BM-derived cells were microglial in nature. The population of BMDM was significantly greater at 8 weeks post-irradiation than at 3 weeks post-irradiation in all brain regions examined. Our results clearly show that cranial irradiation alone is sufficient to induce the generation of BMDM in the adult mouse.

Spinocerebellar ataxia type 1 (SCA1) is a progressive neurodegenerative disorder caused by the expansion of a polyglutamine tract in the ataxin-1 protein. To date, no fundamental treatments for SCA1 have been elucidated. However, some studies have shown that mesenchymal stem cells (MSCs) are partially effective in other genetic mouse models of cerebellar ataxia. In this study, we tested the efficacy of the intrathecal injection of MSCs in the treatment of SCA1 in transgenic (SCA1-Tg) mice. We found that intrathecal injection of only 3 × 10(3) MSCs greatly mitigated the cerebellar neuronal disorganization observed in SCA1 transgenic mice (SCA1-Tg mice). Although the Purkinje cells (PCs) of 24-week-old nontreated SCA1-Tg mice displayed a multilayer arrangement, SCA1-Tg mice at a similar age injected with MSCs displayed monolayer PCs. Furthermore, intrathecal injection of MSCs suppressed the atrophy of PC dendrites in SCA1-Tg mice. Finally, behavioral tests demonstrated that MSCs normalized deficits in motor coordination in SCA1-Tg mice. Future studies should be performed to develop optimal protocols for intrathecal transplantation of MSCs in SCA1 model primates with the aim of developing applications for SCA1 patients.

Spinocerebellar ataxia type 1 (SCA1) is an autosomal dominant neurodegenerative disorder caused by the expansion of polyglutamine repeats in the Ataxin-1 protein. An accumulating body of cerebellar, histological and behavioral analyses has proven that SCA1-knock-in mice (in which the endogenous Atxn1 gene is replaced with mutant Atxn1 that has abnormally expanded 154 CAG repeats) work as a good tool, which resembles the central nervous system pathology of SCA1 patients. However, the peripheral nervous system pathology of the model mice has not been studied despite the fact that the clinical manifestation is also characterized by peripheral involvement. We show here that spinal motor neurons are degenerated in SCA1-knock-in mice. Histologically, some spinal motor neurons of the SCA1-knock-in mice have polyglutamine aggregates in their nuclei and also thinner and demyelinated axons. Electrophysiological examinations of the mice showed slower nerve conduction velocities in spinal motor neurons and lower amplitudes of muscle action potential, compared to wild-type mice. Consistently, the mice displayed decrease in rearing number and total rearing time. These results suggest that the knock-in mice serve as a definite model that reproduces peripheral involvement and are therefore useful for research on the peripheral nervous system pathology in SCA1 patients.

Background Autotaxin (ATX), secreted mainly from adipose tissue, functions as a lysophospholipase D (lysoPLD) to hydrolyze lysophosphatidylcholine (LPC) into lysophosphatidic acid (LPA). ATX–LPA signaling is implicated in a wide range of physiological and pathophysiological processes including immune response. Methods The present study measured serum ATX antigen levels in patients with various autoimmune diseases using a recently developed automated enzyme immunoassay. In addition, serum lysoPLD activity was assessed by measuring choline liberation from the substrate LPC. Moreover, the effect of prednisolone (PSL) on mRNA expression of ATX was evaluated using cultured adipose tissue from mice. Results Decreased serum ATX antigen levels were observed after the initiation of treatment with PSL. The decreased levels recovered during tapering of PSL dose in a dose-dependent manner without exacerbation of disease activity. Moreover, decreased ATX mRNA expression in PSL-treated cultured murine adipose tissue suggested that the effect of PSL on serum ATX may have resulted from changes in adipose tissue ATX expression. Conclusions Our results suggest that measurement of serum ATX antigen level may be clinically useful for the assessment of steroid treatment effect and drug compliance with steroids. Furthermore, our findings provide many novel insights into the biosynthesis, physiological functions, pathological roles, and clinical significance of circulating ATX.

Acute cerebral hypoperfusion following subarachnoid hemorrhage (SAH) is highly related to the pathogenesis of delayed cerebral ischemia (DCI), but the therapeutic option is poorly available. This study aimed to clarify the effect of milrinone (MIL) on cerebral blood flow (CBF) and related outcomes after experimental SAH.Twenty-seven male C57BL/6 mice were assigned to either sham surgery (SAH-sham; n=6), SAH induced by endovascular perforation (control; n=10), or SAH followed by cardiac support with intravenous MIL (n=11) performed 1.5-h after SAH induction. CBF, neurobehavioral function, occurrence of DCI were assessed by MR-continuous arterial spin labeling, daily neurological score testing, and diffusion- and T2-weighted MR images on days 1 and 3, respectively.Initial global CBF depression was notable in mice of control and MIL groups as compared to the SAH-sham group (P0.05). MIL raised CBF in a dose-dependent manner (P0.001), resulted in lower incidence of DCI (P=0.008) and better recovery from neurobehavioral decline than control (P0.001). The CBF values on day 1 predicted DCI with a cut-off of 42.5ml/100g/min (82% specificity and 83% sensitivity), which was greater in mice treated with MIL than those of control (51.7 versus 37.6ml/100g/min; P0.001).MIL improves post-SAH acute hypoperfusion that can lead to the prevention of DCI and functional worsening, acting as a neurocardiac protective agent against EBI.

Early brain injury/ischaemia (EBI) is a serious complication early after subarachnoid haemorrhage (SAH) that contributes to development of delayed cerebral ischaemia (DCI). This study aimed to determine the role of inotropic cardiac support using milrinone (MIL) on restoring acute cerebral hypoperfusion attributable to EBI and improving outcomes after experimental SAH. Forty-three male C57BL/6 mice were assigned to either sham surgery (SAH-sham), SAH induced by endovascular perforation plus postconditioning with 2% isoflurane (Control), or SAH plus isoflurane combined with MIL with and without hypoxia-inducible factor inhibitor (HIF-I) pretreatment. Cardiac output (CO) during intravenous MIL infusion (0.25-0.75 μg/kg/min) between 1.5 and 2.5 hours after SAH induction was monitored with Doppler echocardiography. Magnetic resonance imaging (MRI)-continuous arterial spin labelling was used for quantitative cerebral blood flow (CBF) measurements. Neurobehavioral function was assessed daily by neurological score and open field test. DCI was analyzed 3 days later by determining infarction on MRI. Mild reduction of cardiac output (CO) and global cerebral blood flow (CBF) depression were notable early after SAH. MIL increased CO in a dose-dependent manner (P

Early brain injury/ischemia is a recent therapeutic target that contributes to triggering delayed cerebral ischemia (DCI) in the setting of subarachnoid hemorrhage (SAH). This study aimed to determine the role of dobutamine for inotropic cardiac support in improving cerebral blood flow (CBF) and outcomes after experimental SAH, mediated by hypoxia-inducible factor (HIF). Thirty-one mice were subjected to SAH by endovascular perforation, and assigned to either 2% isoflurane postconditioning performed between 1 and 2.5h after SAH induction or concomitant intravenous dobutamine infusion (15μg/kg/min) with or without HIF inhibitor 2-methoxyestradiol (2ME2) (10mg/kg) administered intraperitoneally. Neurobehavioral function was assessed daily by neurological scores and open field testing. DCI was defined 3days later by detecting a new infarction on MRI. Global CBF depression was notable early after SAH, but dobutamine showed significant improvement in CBF, lower incidence of DCI, and better recovery of neuroscores and open field test variables compared with isoflurane postconditioning (P

Hypothalamic neuropeptide Y (NPY) elicits hunger responses to increase the chances of surviving starvation: an inhibition of metabolism and an increase in feeding. Here we elucidate a key central circuit mechanism through which hypothalamic NPY signals drive these hunger responses. GABAergic neurons in the intermediate and parvicellular reticular nuclei (IRt/PCRt) of the medulla oblongata, which are activated by NPY-triggered neural signaling from the hypothalamus, potentially through the nucleus tractus solitarius, mediate the NPY-induced inhibition of metabolic thermogenesis in brown adipose tissue (BAT) via their innervation of BAT sympathetic premotor neurons. Intriguingly, the GABAergic IRt/PCRt neurons innervating the BAT sympathetic premotor region also innervate the masticatory motor region, and stimulation of the IRt/PCRt elicits mastication and increases feeding as well as inhibits BAT thermogenesis. These results indicate that GABAergic IRt/PCRt neurons mediate hypothalamus-derived hunger signaling by coordinating both autonomic and feeding motor systems to reduce energy expenditure and to promote feeding.

From fiscal year 2012 to 2014, we surveyed the concentration of radioactive cesium in 39 wild animal meats obtained from 20 wild boars and 19 deer caught in Chiba prefecture, using a germanium semiconductor detector. Four wild boar meats in the fiscal years 2012 and 2013 exceeded the radioactive cesium limit in general foods (100 Bq/kg), whereas none of the deer meats exceeded the limit. The left side of the wild boar that showed a radioactive cesium concentration above 100 Bq/kg was divided into 14 parts. We compared the radioactive cesium concentration in the ham used for the screening test with those in other parts. The concentration was highest in ham, among the edible parts.

The Wnt/β-catenin pathway promotes proliferation of neural progenitor cells (NPCs) at early stages and induces neuronal differentiation from NPCs at late stages, but the molecular mechanisms that control this stage-specific response are unclear. Pin1 is a prolyl isomerase that regulates cell signaling uniquely by controlling protein conformation after phosphorylation, but its role in neuronal differentiation is not known. Here we found that whereas Pin1 depletion suppresses neuronal differentiation, Pin1 overexpression enhances it, without any effects on gliogenesis from NPCs in vitro. Consequently, Pin1-null mice have significantly fewer upper layer neurons in the motor cortex and severely impaired motor activity during the neonatal stage. A proteomic approach identified β-catenin as a major substrate for Pin1 in NPCs, in which Pin1 stabilizes β-catenin. As a result, Pin1 knockout leads to reduced β-catenin during differentiation but not proliferation of NPCs in developing brains. Importantly, defective neuronal differentiation in Pin1 knockout NPCs is fully rescued in vitro by overexpression of β-catenin but not a β-catenin mutant that fails to act as a Pin1 substrate. These results show that Pin1 is a novel regulator of NPC differentiation by acting on β-catenin and provides a new postphosphorylation signaling mechanism to regulate developmental stage-specific functioning of β-catenin signaling in neuronal differentiation.

SummaryKinase inhibitors have limited success in cancer treatment because tumors circumvent their action. Using a quantitative proteomics approach, we assessed kinome activity in response to MEK inhibition in triple-negative breast cancer (TNBC) cells and genetically engineered mice (GEMMs). MEK inhibition caused acute ERK activity loss, resulting in rapid c-Myc degradation that induced expression and activation of several receptor tyrosine kinases (RTKs). RNAi knockdown of ERK or c-Myc mimicked RTK induction by MEK inhibitors, and prevention of proteasomal c-Myc degradation blocked kinome reprogramming. MEK inhibitor-induced RTK stimulation overcame MEK2 inhibition, but not MEK1 inhibition, reactivating ERK and producing drug resistance. The C3Tag GEMM for TNBC similarly induced RTKs in response to MEK inhibition. The inhibitor-induced RTK profile suggested a kinase inhibitor combination therapy that produced GEMM tumor apoptosis and regression where single agents were ineffective. This approach defines mechanisms of drug resistance, allowing rational design of combination therapies for cancer.

Previous studies have examined the therapeutic effect of endothelial progenitor cells (EPCs) during the chronic phase of cerebral infarction in rats; however, few studies have investigated the effects of EPCs during the acute phase of infarction. In this study, we evaluated the therapeutic effect of EPCs with low aldehyde dehydrogenase activity (Alde-Low EPCs) in rats with acute cerebral infarction, and our results provide insight that may help to identify a therapeutic mechanism of EPCs for acute cerebral infarction. The administration of Alde-Low EPCs into rats with acute cerebral infarction results in the accumulation and migration of the Alde-Low EPCs into the infarct area and the subsequent decrease of infarct volume. Moreover, we found that the stromal cell-derived factor-1 (SDF-1) and CXC chemokine receptor 4 (CXCR4) signaling pathway may regulate the accumulation of Alde-Low EPCs. The transplantation of Alde-Low EPCs may represent a potential treatment strategy for acute cerebral infarction.

An inflammatory reaction in vascular tissue is a potential factor linking restenosis after angioplasty. Although cilostazol, a selective phosphodiesterase type 3 inhibitor that is a unique antiplatelet drug and vasodilator, has been reported to be anti-inflammatory, its effect on the inflammatory action of mononuclear cells homing to endothelial cells is not clearly understood. In this study, whether cilostazol inhibits neointimal formation and improves inflammatory actions by inhibiting sialyl Lewis X (SLX) expression on mononuclear cells and E-selectin expression on endothelial cells was evaluated.The effect of cilostazol (1, 3, 10, 30 μM) on expression of E-selectin in human umbilical vein endothelial cells and SLX in rat mononuclear cells stimulated with lipopolysaccharide by immunofluorescence and real-time polymerase chain reaction (n = 3) was studied. Additionally, a double-balloon injury model was used on rat carotid arteries to evaluate vascular intimal hyperplasia. 0.1% cilostazol was administered 3 days before the first balloon injury, and the second balloon injury was performed 7 days after the first injury. Cilostazol administration was continued until rats were sacrificed 14 days after the second angioplasty. The expression of SLX on mononuclear cells and E-selectin on endothelial cells by immunofluorescence (n = 10) and real-time polymerase chain reaction (n = 5) were studied.Cilostazol effectively inhibited the expression of SLX on mononuclear cells and E-selectin on endothelial cells. Cilostazol inhibited the migration of mononuclear cells in neointimal regions and neointimal hyperplasia after balloon injury. The numbers of macrophages and T-lymphocytes and the hyperplasia area in neointimal regions decreased from 71.06 ± 20.04, 1121 ± 244.4 cells per section, 206,400 ± 96,150 mm(2) to 29.65 ± 16.73, 374.2 ± 124.5 cells per section, and 101,900 ± 16,150 mm(2) due to the administration of cilostazol.These results demonstrate that the protective effect of cilostazol against neointimal hyperplasia may be mediated by its anti-inflammatory actions of mononuclear cells homing to endothelial cells by decreasing SLX and E-selectin expression.It is reported that cilostazol inhibits neointimal hyperplasia by decreasing the expression of some cell-adhesion molecules. We evaluated the effects of cilostazol for the expression of sialyl Lewis X (SLX) on mononuclear cells and E-selectin on endothelial cells, which interaction is the first step of inflammation action. Cilostazol was thought to show the anti-inflammatory actions by decreasing SLX and E-selectin expression in addition to decreasing the expression of some cell-adhesion molecules.

Analysis of patient tumors suggests that multiple MAP3 kinases (MAP3Ks) are critical for growth and metastasis of cancer cells. MAP3Ks selectively control the activation of extracellular signal-regulated kinase 1/2 (ERK1/2), Jun N-terminal kinase (JNK), p38 and ERK5 in response to receptor tyrosine kinases and GTPases. We used MDA-MB-231 cells because of their ability to metastasize from the breast fat pad to distant lymph nodes for an orthotopic xenograft model to screen the function of seven MAP3Ks in controlling tumor growth and metastasis. Stable short hairpin RNA (shRNA) knockdown was used to inhibit the expression of each of the seven MAP3Ks, which were selected for their differential regulation of the MAPK network. The screen identified two MAP3Ks, MEKK2 and MLK3, whose shRNA knockdown caused significant inhibition of both tumor growth and metastasis. Neither MEKK2 nor MLK3 have been previously shown to regulate tumor growth and metastasis in vivo. These results demonstrated that MAP3Ks, which differentially activate JNK, p38 and ERK5, are necessary for xenograft tumor growth and metastasis of MDA-MB-231 tumors. The requirement for MAP3Ks signaling through multiple MAPK pathways explains why several members of the MAPK network are activated in cancer. MEKK2 was required for epidermal growth factor receptor and Her2/Neu activation of ERK5, with ERK5 being required for metastasis. Loss of MLK3 expression increased mitotic infidelity and apoptosis in vitro. Knockdown of MEKK2 and MLK3 resulted in increased apoptosis in orthotopic xenografts relative to control tumors in mice, inhibiting both tumor growth and metastasis; MEKK2 and MLK3 represent untargeted kinases in tumor biology for potential therapeutic development.

In the embryonic neocortex, neuronal precursors are generated in the ventricular zone (VZ) and accumulate in the cortical plate. Recently, the subventricular zone (SVZ) of the embryonic neocortex was recognized as an additional neurogenic site for both principal excitatory neurons and GABAergic inhibitory neurons. To gain insight into the neurogenesis of GABAergic neurons in the SVZ, we investigated the characteristics of intermediate progenitors of GABAergic neurons (IPGNs) in mouse neocortex by immunohistochemistry, immunocytochemistry, single-cell RT-PCR and single-cell array analysis. IPGNs were identified by their expression of some neuronal and cell cycle markers. Moreover, we investigated the origins of the neocortical IPGNs by Cre-loxP fate mapping in transgenic mice and the transduction of part of the telencephalic VZ by Cre-reporter plasmids, and found them in the medial and lateral ganglionic eminence. Therefore, they must migrate tangentially within the telencephalon to reach the neocortex. Cell-lineage analysis by simple-retrovirus transduction revealed that the neocortical IPGNs self-renew and give rise to a small number of neocortical GABAergic neurons and to a large number of granule and periglomerular cells in the olfactory bulb. IPGNs are maintained in the neocortex and may act as progenitors for adult neurogenesis.

The development of antagonists of the transient receptor potential vanilloid-1 (TRPV1) channel as pain therapeutics has revealed that these compounds cause hyperthermia in humans. This undesirable on-target side effect has triggered a surge of interest in the role of TRPV1 in thermoregulation and revived the hypothesis that TRPV1 channels serve as thermosensors. We review literature data on the distribution of TRPV1 channels in the body and on thermoregulatory responses to TRPV1 agonists and antagonists. We propose that two principal populations of TRPV1-expressing cells have connections with efferent thermoeffector pathways: 1) first-order sensory (polymodal), glutamatergic dorsal-root (and possibly nodose) ganglia neurons that innervate the abdominal viscera and 2) higher-order sensory, glutamatergic neurons presumably located in the median preoptic hypothalamic nucleus. We further hypothesize that all thermoregulatory responses to TRPV1 agonists and antagonists and thermoregulatory manifestations of TRPV1 desensitization stem from primary actions on these two neuronal populations. Agonists act primarily centrally on population 2; antagonists act primarily peripherally on population 1. We analyze what roles TRPV1 might play in thermoregulation and conclude that this channel does not serve as a thermosensor, at least not under physiological conditions. In the hypothalamus, TRPV1 channels are inactive at common brain temperatures. In the abdomen, TRPV1 channels are tonically activated, but not by temperature. However, tonic activation of visceral TRPV1 by nonthermal factors suppresses autonomic cold-defense effectors and, consequently, body temperature. Blockade of this activation by TRPV1 antagonists disinhibits thermoeffectors and causes hyperthermia. Strategies for creating hyperthermia-free TRPV1 antagonists are outlined. The potential physiological and pathological significance of TRPV1-mediated thermoregulatory effects is discussed.

The central mechanism of fever induction is triggered by an action of prostaglandin E(2) (PGE(2)) on neurons in the preoptic area (POA) through the EP3 subtype of prostaglandin E receptor. EP3 receptor (EP3R)-expressing POA neurons project directly to the dorsomedial hypothalamus (DMH) and to the rostral raphe pallidus nucleus (rRPa), key sites for the control of thermoregulatory effectors. Based on physiological findings, we hypothesize that the febrile responses in brown adipose tissue (BAT) and those in cutaneous vasoconstrictors are controlled independently by separate neuronal pathways: PGE(2) pyrogenic signaling is transmitted from EP3R-expressing POA neurons via a projection to the DMH to activate BAT thermogenesis and via another projection to the rRPa to increase cutaneous vasoconstriction. In this case, DMH-projecting and rRPa-projecting neurons would constitute segregated populations within the EP3R-expressing neuronal group in the POA. Here, we sought direct anatomical evidence to test this hypothesis with a double-tracing experiment in which two types of the retrograde tracer, cholera toxin b-subunit (CTb), conjugated with different fluorophores were injected into the DMH and the rRPa of rats and the resulting retrogradely labeled populations of EP3R-immunoreactive neurons in the POA were identified with confocal microscopy. We found substantial numbers of EP3R-immunoreactive neurons in both the DMH-projecting and the rRPa-projecting populations. However, very few EP3R-immunoreactive POA neurons were labeled with both the CTb from the DMH and that from the rRPa, although a substantial number of neurons that were not immunoreactive for EP3R were double-labeled with both CTbs. The paucity of the EP3R-expressing neurons that send collaterals to both the DMH and the rRPa suggests that pyrogenic signals are sent independently to these caudal brain regions from the POA and that such pyrogenic outputs from the POA reflect different control mechanisms for BAT thermogenesis and for cutaneous vasoconstriction by distinct sets of POA neurons.

Both suppressive and promoting roles of NKT cells have been reported in the pathogenesis of systemic lupus erythematosus (SLE). Herein, we found that although New Zealand mice have normal frequencies of NKT cells, their in vitro potential to produce IL-4 and IFN-γ in response to α-galactosylceramide was remarkably impaired in New Zealand Black (NZB) mice prone to mild SLE, while production was highly up-regulated in nonautoimmune New Zealand White (NZW) mice and at intermediate levels in (NZB × NZW)F1 mice, which are prone to severe SLE. Because this aberration is evident in young mice before disease onset, genetic mechanisms are thought to be involved. Genome-wide quantitative trait locus analysis and association studies revealed that a locus linked to D11Mit14 on chromosome 11 may be involved in the difference in cytokine-producing potential between NZB and NZW NKT cells. Additionally, (NZB × NZW)F1 × NZB backcross progeny with the NZW genotype for D11Mit14 showed significantly increased frequencies of age-associated SLE phenotypes, such as high serum levels of IgG, IgG anti-DNA Abs, and lupus nephritis. In coculture studies, α-galactosylceramide-stimulated NKT cells from NZW and (NZB × NZW)F1 mice, but not from NZB mice, showed significantly enhanced Ig synthesis by B cells. These findings suggest that the D11Mit14-linked NZW locus may contribute to the development of SLE in (NZB × NZW)F1 mice through a mechanism that up-regulates NKT cell function. Thus, this NZW allele may be a candidate of the NZW modifiers that act to promote (NZB × NZW)F1 disease.

Monoamines exert diverse functions in various cells in peripheral organs as well as in the central nervous system. 5-Hydroxy-l-tryptophan (5-HTP) has been simply regarded as a precursor of serotonin, and it is believed that the biological significance of 5-HTP is essentially ascribable to the production of serotonin. Systemic treatment with 5-HTP is often applied to patients with low serotonin levels in the brain. Here we show that endogenous and exogenous 5-HTP but not serotonin induced the development of microvilli in the gut villi epithelium. In contrast, serotonin but not 5-HTP regulated phagocytosis by macrophages. 5-HTP specifically induced actin remodeling and decreased phosphorylation of extracellular signal-regulated kinase (ERK) in the gut, whereas serotonin stimulated actin remodeling and increased ERK phosphorylation in macrophages. Functionally, inhibition of ERK activity promoted the development of microvilli in the gut and ameliorated phagocytosis by macrophages. Thus, 5-HTP and serotonin contribute to distinct cell-type-specific functions via common mediators. Our study might create an opportunity to explore the effects of exogenously applied 5-HTP in humans.

A series of experiments were performed to investigate why two peaks (D and E) of the five dissolved phase peaks in hyperpolarized 129Xe rat head spectra appeared inconsistently in previous work. Specifically, spectra were acquired under conditions of various shim states, anaesthetics, and arterial ligation. The shimming experiments showed that slice-shimming can be used to improve resolution of the dissolved phase peaks, but even so, subtle changes in the shim state that may dramatically alter the shape of peak E remain poorly understood. Also, the inability to shim gas spaces and tissue simultaneously may explain why inconsistent chemical shift values have been reported in the literature. A possible solution for this problem is suggested. The results of pre- and postligation spectra from the same animal indicated that two peaks (A and E) originate from brain. Changing the anaesthetic was found to have no effect on the number of dissolved peaks in xenon spectra. Magn Reson Med 57:791–797, 2007. © 2007 Wiley-Liss, Inc.

The serotonin 5-hydroxytryptamine (5-HT) neurotransmitter system contributes to various physiological and pathological conditions. 5-HT is the first neurotransmitter for which a developmental role was suspected. Tryptophan hydroxylase (TPH) catalyzes the rate-limiting reaction in the biosynthesis of 5-HT. Both TPH1 and TPH2 have tryptophan hydroxylating activity. TPH2 is abundant in the brain, whereas TPH1 is mainly expressed in the pineal gland and the periphery. However, TPH1 was found to be expressed predominantly during the late developmental stage in the brain. Recent advances have shed light on the kinetic properties of each TPH isoform. TPH1 showed greater affinity for tryptophan and stronger enzymic activity than TPH2 under conditions reflecting those in the developing brain stem. Transient alterations in 5-HT homeostasis during development modify the fine wiring of brain connections and cause permanent changes to adult behavior. An increasing body of evidence suggests the involvement of developmental brain disturbances in psychiatric disorders. These findings have revived a long-standing interest in the developmental role of 5-HT-related molecules. This article summarizes our understanding of the kinetics and possible neuronal functions of each TPH during development and in the adult.