Suppression of excitatory synaptic transmission in the centrolateral amygdala via presynaptic histamine H3 heteroreceptors.

The central histaminergic system has a pivotal role in emotional regulation and psychiatric disorders, including anxiety, depression and schizophrenia. However, the effect of histamine on neuronal activity of the centrolateral amygdala (CeL), an essential node for fear and anxiety processing, remains unknown. Here, using immunostaining and whole-cell patch clamp recording combined with optogenetic manipulation of histaminergic terminals in CeL slices prepared from histidine decarboxylase (HDC)-Cre rats, we show that histamine selectively suppresses excitatory synaptic transmissions, including glutamatergic transmission from the basolateral amygdala, on both PKC-δ- and SOM-positive CeL neurons. The histamine-induced effect is mediated by H3 receptors expressed on VGLUT1-/VGLUT2-positive presynaptic terminals in CeL. Furthermore, optoactivation of histaminergic afferent terminals from the hypothalamic tuberomammillary nucleus (TMN) also significantly suppresses glutamatergic transmissions in CeL via H3 receptors. Histamine neither modulates inhibitory synaptic transmission by presynaptic H3 receptors nor directly excites CeL neurons by postsynaptic H1, H2 or H4 receptors. These results suggest that histaminergic afferent inputs and presynaptic H3 heteroreceptors may hold a critical position in balancing excitatory and inhibitory synaptic transmissions in CeL by selective modulation of glutamatergic drive, which may not only account for the pathophysiology of psychiatric disorders but also provide potential psychotherapeutic targets. KEY POINTS: Histamine selectively suppresses the excitatory, rather than inhibitory, synaptic transmissions on both PKC-δ- and SOM-positive neurons in the centrolateral amygdala (CeL). H3 receptors expressed on VGLUT1- or VGLUT2-positive afferent terminals mediate the suppression of histamine on glutamatergic synaptic transmission in CeL. Optogenetic activation of hypothalamic tuberomammillary nucleus (TMN)-CeL histaminergic projections inhibits glutamatergic transmission in CeL via H3 receptors.

Oxytocin Receptor in Cerebellar Purkinje Cells Does Not Engage in Autism-Related Behaviors.

The classical motor center cerebellum is one of the most consistent structures of abnormality in autism spectrum disorders (ASD), and neuropeptide oxytocin is increasingly explored as a potential pharmacotherapy for ASD. However, whether oxytocin targets the cerebellum for therapeutic effects remains unclear. Here, we report a localization of oxytocin receptor (OXTR) in Purkinje cells (PCs) of cerebellar lobule Crus I, which is functionally connected with ASD-implicated circuits. OXTR activation neither affects firing activities, intrinsic excitability, and synaptic transmission of normal PCs nor improves abnormal intrinsic excitability and synaptic transmission of PCs in maternal immune activation (MIA) mouse model of autism. Furthermore, blockage of OXTR in Crus I in wild-type mice does not induce autistic-like social, stereotypic, cognitive, and anxiety-like behaviors. These results suggest that oxytocin signaling in Crus I PCs seems to be uninvolved in ASD pathophysiology, and contribute to understanding of targets and mechanisms of oxytocin in ASD treatment.

Proinflammatory activation of microglia in the cerebellum hyperexcites Purkinje cells to trigger ataxia.

Specific medications to combat cerebellar ataxias, a group of debilitating movement disorders characterized by difficulty with walking, balance and coordination, are still lacking. Notably, cerebellar microglial activation appears to be a common feature in different types of ataxic patients and rodent models. However, direct evidence that cerebellar microglial activation in vivo is sufficient to induce ataxia is still lacking. Here, by employing chemogenetic approaches to manipulate cerebellar microglia selectively and directly, we found that specific chemogenetic activation of microglia in the cerebellar vermis directly leads to ataxia symptoms in wild-type mice and aggravated ataxic motor deficits in 3-acetylpyridine (3-AP) mice, a classic mouse model of cerebellar ataxia. Mechanistically, cerebellar microglial proinflammatory activation induced by either chemogenetic M3D(Gq) stimulation or 3-AP modeling hyperexcites Purkinje cells (PCs), which consequently triggers ataxia. Blockade of microglia-derived TNF-α, one of the most important proinflammatory cytokines, attenuates the hyperactivity of PCs driven by microglia. Moreover, chemogenetic inhibition of cerebellar microglial activation or suppression of cerebellar microglial activation by PLX3397 and minocycline reduces the production of proinflammatory cytokines, including TNF-α, to effectively restore the overactivation of PCs and alleviate motor deficits in 3-AP mice. These results suggest that cerebellar microglial activation may aggravate the neuroinflammatory response and subsequently induce dysfunction of PCs, which in turn triggers ataxic motor deficits. Our findings thus reveal a causal relationship between proinflammatory activation of cerebellar microglia and ataxic motor symptoms, which may offer novel evidence for therapeutic intervention for cerebellar ataxias by targeting microglia and microglia-derived inflammatory mediators.

Targeting presynaptic H3 heteroreceptor in nucleus accumbens to improve anxiety and obsessive-compulsive-like behaviors.

Anxiety commonly co-occurs with obsessive-compulsive disorder (OCD). Both of them are closely related to stress. However, the shared neurobiological substrates and therapeutic targets remain unclear. Here we report an amelioration of both anxiety and OCD via the histamine presynaptic H3 heteroreceptor on glutamatergic afferent terminals from the prelimbic prefrontal cortex (PrL) to the nucleus accumbens (NAc) core, a vital node in the limbic loop. The NAc core receives direct hypothalamic histaminergic projections, and optogenetic activation of hypothalamic NAc core histaminergic afferents selectively suppresses glutamatergic rather than GABAergic synaptic transmission in the NAc core via the H3 receptor and thus produces an anxiolytic effect and improves anxiety- and obsessive-compulsive-like behaviors induced by restraint stress. Although the H3 receptor is expressed in glutamatergic afferent terminals from the PrL, basolateral amygdala (BLA), and ventral hippocampus (vHipp), rather than the thalamus, only the PrL- and not BLA- and vHipp-NAc core glutamatergic pathways among the glutamatergic afferent inputs to the NAc core is responsible for co-occurrence of anxiety- and obsessive-compulsive-like behaviors. Furthermore, activation of the H3 receptor ameliorates anxiety and obsessive-compulsive-like behaviors induced by optogenetic excitation of the PrL-NAc glutamatergic afferents. These results demonstrate a common mechanism regulating anxiety- and obsessive-compulsive-like behaviors and provide insight into the clinical treatment strategy for OCD with comorbid anxiety by targeting the histamine H3 receptor in the NAc core.

Suppression of microglial activation and monocyte infiltration ameliorates cerebellar hemorrhage induced-brain injury and ataxia.

Ataxia, characterized by uncoordinated movement, is often found in patients with cerebellar hemorrhage (CH), leading to long-term disability without effective management. Microglia are among the first responders to CNS insult. Yet the role and mechanism of microglia in cerebellar injury and ataxia after CH are still unknown. Using Ki20227, an inhibitor for colony-stimulating factor 1 receptor which mediates the signaling responsible for the survival of microglia, we determined the impact of microglial depletion on cerebellar injury and ataxia in a murine model of CH. Microglial depletion reduced cerebellar lesion volume and alleviated gait abnormality, motor incoordination, and locomotor dysfunction after CH. Suppression of CH-initiated microglial activation with minocycline ameliorated cerebellum infiltration of monocytes/macrophages, as well as production of proinflammatory cytokines and chemokine C-C motif ligand-2 (CCL-2) that recruits monocytes/macrophages. Furthermore, both minocycline and bindarit, a CCL-2 inhibitor, prevented apoptosis and electrophysiological dysfunction of Purkinje cells, the principal neurons and sole outputs of the cerebellar cortex, and consequently improved ataxia-like motor abnormalities. Our findings suggest a detrimental role of microglia in neuroinflammation and ataxic motor symptoms after CH, and pave a new path to understand the neuroimmune mechanism underlying CH-induced cerebellar ataxia.

Hyperphosphorylation determines both the spread and the morphology of tau pathology.

INTRODUCTION: Neurofibrillary pathology of abnormally hyperphosphorylated tau (P-tau) is a hallmark of Alzheimer's disease (AD) and other tauopathies. Tau pathology can be experimentally induced and propagated. However, what induces the prion-like transmission character to tau and produces morphologically distinct tau lesions remains elusive. METHODS: We investigated the role of hyperphosphorylation in the spread of tau pathology in hTau transgenic mice. RESULTS: We found that intrahippocampal injection with AD P-tau, but not nonphosphorylated tau, produced numerous P-tau tangles and neuropil threads locally and in neocortex lateral to injection and upstream to the hippocampus. Dephosphorylation of AD P-tau with protein phosphatase-2A dramatically reduced and switched tau pathology from neurofibrillary tangles to argyrophilic grain-like morphology. CONCLUSIONS: Our findings show that abnormal hyperphosphorylation of tau determines the spread and morphology of tau lesions and that the propagation of tau pathology takes place both locally and in axonally connected areas and highlight tau hyperphosphorylation as a potential drug target.

IL-17RA is essential for optimal localization of follicular Th cells in the germinal center light zone to promote autoantibody-producing B cells.

Germinal centers (GCs) provide a microenvironment that promotes and regulates the interactions of B cells with follicular Th (TFH) cells. In this study, we show that there are significantly higher frequencies of CXCR5(+)ICOS(+) TFH cells in autoimmune BXD2 mice, and these cells express both IL-21R and IL-17RA. Although IL-17 and IL-21 are both important for the formation of spontaneous GCs and development of pathogenic autoantibodies, IL-21, but not IL-17, is required for the proper development of TFH cells in BXD2 mice. The total numbers of TFH cells and their ability to induce B cell responses in vitro were not affected by a deficiency of IL-17RA in BXD2-Il17ra(-/-) mice, the majority of CXCR5(+) TFH cells from BXD2-Il17ra(-/-) mice were, however, not localized in the GC light zone (LZ). Interruption of IL-17 signaling, either acutely by AdIL-17R:Fc or chronically by Il17ra(-/-), disrupted TFH-B interactions and abrogated the generation of autoantibody-forming B cells in BXD2 mice. IL-17 upregulated the expression of regulator of G-protein signaling 16 (RGS16) to promote the ability of TFH to form conjugates with B cells, which was abolished in TFH cells from BXD2-Rgs16(-/-) mice. The results suggests that IL-17 is an extrinsic stop signal that it acts on postdifferentiated IL-17RA(+) TFH to enable its interaction with responder B cells in the LZ niche. These data suggest a novel concept that TFH differentiation and its stabilization in the LZ are two separate checkpoints and that IL-21 and IL-17 act at each checkpoint to enable pathogenic GC development.

Structural insight into the autoinhibition mechanism of AMP-activated protein kinase.

The AMP-activated protein kinase (AMPK) is characterized by its ability to bind to AMP, which enables it to adjust enzymatic activity by sensing the cellular energy status and maintain the balance between ATP production and consumption in eukaryotic cells. It also has important roles in the regulation of cell growth and proliferation, and in the establishment and maintenance of cell polarity. These important functions have rendered AMPK an important drug target for obesity, type 2 diabetes and cancer treatments. However, the regulatory mechanism of AMPK activity by AMP binding remains unsolved. Here we report the crystal structures of an unphosphorylated fragment of the AMPK alpha-subunit (KD-AID) from Schizosaccharomyces pombe that contains both the catalytic kinase domain and an autoinhibitory domain (AID), and of a phosphorylated kinase domain from Saccharomyces cerevisiae (Snf1-pKD). The AID binds, from the 'backside', to the hinge region of its kinase domain, forming contacts with both amino-terminal and carboxy-terminal lobes. Structural analyses indicate that AID binding might constrain the mobility of helix alphaC, hence resulting in an autoinhibited KD-AID with much lower kinase activity than that of the kinase domain alone. AMP activates AMPK both allosterically and by inhibiting dephosphorylation. Further in vitro kinetic studies demonstrate that disruption of the KD-AID interface reverses the autoinhibition and these AMPK heterotrimeric mutants no longer respond to the change in AMP concentration. The structural and biochemical data have shown the primary mechanism of AMPK autoinhibition and suggest a conformational switch model for AMPK activation by AMP.

Extension of the germinal center stage of B cell development promotes autoantibodies in BXD2 mice.

OBJECTIVE: Regulator of G protein signaling (RGS) proteins inhibit chemokine signaling by desensitizing G protein-coupled receptor signals. This study was undertaken to determine the mechanisms by which RGS13 promotes the generation of pathogenic autoantibodies in germinal centers (GCs), using BXD2-Rgs13-/- mice. METHODS: Confocal and light microscopy imaging techniques were used to determine the location of cells that express RGS13 and activation-induced cytidine deaminase (AID) in the mouse spleen, and the number of plasmablasts. The levels of GC and plasma cell program transcripts in GC B cells were determined by real-time quantitative polymerase chain reaction (qPCR). Differential interleukin-17 (IL-17)-mediated expression of RGS13 in GC versus non-GC B cells was analyzed using A20 and 70Z/3 B cells. RESULTS: In the spleens of BXD2 mice, RGS13 was mainly expressed by GC B cells and was stimulated by IL-17 but not IL-21. IL-17 up-regulated RGS13 in A20 GC cells but not 70Z/3 non-GC B cells. BXD2- Rgs13-/- mice exhibited smaller GCs and lower AID levels, suggesting lower somatic hypermutation and affinity maturation. However, GC B cells from BXD2- Rgs13-/- mice showed increased levels of IgMbright plasmablasts, up-regulation of the genes encoding plasma program, including interferon regulatory factor 4, B lymphocyte-induced maturation protein 1, and X-box binding protein 1 and the p-CREB target genes Fosb and Obf1, and down-regulation of the GC program genes Aid, Pax5, and Bach2 compared to BXD2 mice. BXD2-Rgs13-/- mice had lower titers of IgG autoantibodies and IgG deposits in the glomeruli, suggesting reduced autoantibody pathogenicity. CONCLUSION: RGS13 deficiency is associated with a reduction in GC program genes and the exit of fewer pathogenic IgM plasmablasts in BXD2 mice. Our findings indicate that prolonged GC program, mediated by up-regulation of RGS13, enhances AID expression and enables the generation of pathogenic autoantibodies in autoreactive GCs.

Expression, purification, and crystal structure of N-terminal domains of human ubiquitin-activating enzyme (E1).

Ubiquitin-activating enzyme (E1) is a key regulator in protein ubiquitination, which lies on the upstream of the ubiquitin-related pathways and determines the activation of the downstream enzyme cascade. Thus far, no structural information about the human ubiquitin-activating enzyme has been reported. We expressed and purified the N-terminal domains of human E1 and determined their crystal structures, which contain inactive adenylation domain (IAD) and the first catalytic cysteine half-domain (FCCH). This study presents the crystal structure of human E1 fragment for the first time. The main structure of both IAD and FCCH superimposed well with their corresponding domains in yeast Uba1, but their relative positions vary significantly. This work provides new structural insights in understanding the mechanisms of ubiquitin activation in humans.

A role for the cerebellum in motor-triggered alleviation of anxiety.

Physical exercise is known to reduce anxiety, but the underlying brain mechanisms remain unclear. Here, we explore a hypothalamo-cerebello-amygdalar circuit that may mediate motor-dependent alleviation of anxiety. This three-neuron loop, in which the cerebellar dentate nucleus takes center stage, bridges the motor system with the emotional system. Subjecting animals to a constant rotarod engages glutamatergic cerebellar dentate neurons that drive PKCδ+ amygdalar neurons to elicit an anxiolytic effect. Moreover, challenging animals on an accelerated rather than a constant rotarod engages hypothalamic neurons that provide a superimposed anxiolytic effect via an orexinergic projection to the dentate neurons that activate the amygdala. Our findings reveal a cerebello-limbic pathway that may contribute to motor-triggered alleviation of anxiety and that may be optimally exploited during challenging physical exercise.

Calpain, Atg5 and Bak play important roles in the crosstalk between apoptosis and autophagy induced by influx of extracellular calcium.

Calcium (Ca(2+)) signals are involved in important checkpoints in cell death pathways and promote both apoptosis and autophagy. However, the relationship between autophagy and apoptosis in response to Ca(2+) level elevation is poorly understood. Here, we provided evidence that the influx of extracellular Ca(2+) triggered by Trichokonin VI (TK VI), an antimicrobial peptide, induced calpain-dependent apoptosis and autophagy in hepatocellular carcinoma (HCC) cells. Remarkably, TK VI preferentially induced apoptosis that was associated with calpain-mediated Bax and Atg5 cleavage, which resulted in the collapse of the mitochondrial membrane potential and cytochrome c release. Interestingly, truncated, but not full-length Atg5, associated with Bcl-xL and promoted the intrinsic pathway. Moreover, TK VI treatment induced reactive oxygen species (ROS) accumulation, an effect in which Bak might play a major role. This accumulation of ROS resulted in the subsequent disposal of damaged mitochondria within autophagosomes via Atg5-mediated and mitochondria-selective autophagy. Both the inhibition of calpain activity and Bax deficiency activated a switch that promoted an enhancement of autophagy. The inhibition of both apoptosis and autophagy significantly attenuated the TK VI cytotoxicity, indicating that the two processes had stimulatory effects during TK VI-meditated cell death. These results suggested that calpain, Bak and Atg5 were molecular links between autophagy and apoptosis and revealed novel aspects of the crosstalk between these two processes. The potential of TK VI is proposed as a promising anticancer agent for its well-characterized activity of Ca(2+) agonist and as a possible novel therapeutic strategy that acts on cancer cell mitochondria.

Lipid-accumulated reactive astrocytes promote disease progression in epilepsy.

Reactive astrocytes play an important role in neurological diseases, but their molecular and functional phenotypes in epilepsy are unclear. Here, we show that in patients with temporal lobe epilepsy (TLE) and mouse models of epilepsy, excessive lipid accumulation in astrocytes leads to the formation of lipid-accumulated reactive astrocytes (LARAs), a new reactive astrocyte subtype characterized by elevated APOE expression. Genetic knockout of APOE inhibited LARA formation and seizure activities in epileptic mice. Single-nucleus RNA sequencing in TLE patients confirmed the existence of a LARA subpopulation with a distinct molecular signature. Functional studies in epilepsy mouse models and human brain slices showed that LARAs promote neuronal hyperactivity and disease progression. Targeting LARAs by intervention with lipid transport and metabolism could thus provide new therapeutic options for drug-resistant TLE.

IL-17 activates the canonical NF-kappaB signaling pathway in autoimmune B cells of BXD2 mice to upregulate the expression of regulators of G-protein signaling 16.

We previously identified that autoreactive B cells from BXD2 mice can be targeted by IL-17, leading to upregulation of the expression of regulators of G-protein signaling (Rgs) genes that facilitated the development of spontaneous germinal centers. Little is known about the signaling pathway used by IL-17 to upregulate RGS. In the current study, we found that IL-17 rapidly activates the canonical NF-kappaB signaling pathway and that BXD2 B cells exhibit higher basal and activated phosphorylated p65 levels than B6 or BXD2-Il17ra(-/-) B cells. Inhibition of p65 phosphorylation downregulated RGS16 expression and abrogated the IL-17-induced chemotactic arrest of B cells in response to CXCL12. Knockdown of TNFR-associated factor 6 or NF-kappaB activator 1 in 70Z/3 pre-B cells led to decreased Rgs16 expression, indicating that both of these two genes are involved in IL-17-mediated activation of NF-kappaB signaling in B cells. These findings identify the signaling pathway regulated by IL-17 to contribute to the development of spontaneous germinal centers in autoimmune BXD2 mice.

Marginal zone precursor B cells as cellular agents for type I IFN-promoted antigen transport in autoimmunity.

The pathogenic connection of type I IFN and its role in regulating the migration response of Ag delivery by B cells into lymphoid follicles in an autoimmune condition has not been well-identified. Here, we show that there was a significantly larger population of marginal zone precursor (MZ-P) B cells, defined as being IgM(hi)CD1d(hi)CD21(hi)CD23(hi) in the spleens of autoimmune BXD2 mice compared with B6 mice. MZ-P B cells were highly proliferative compared with marginal zone (MZ) and follicular (FO) B cells. The intrafollicular accumulation of MZ-P B cells in proximity to germinal centers (GCs) in BXD2 mice facilitated rapid Ag delivery to the GC area, whereas Ag-carrying MZ B cells, residing predominantly in the periphery, had a lower ability to carry Ag into the GCs. IFN-alpha, generated by plasmacytoid dendritic cells, induced the expression of CD69 and suppressed the sphingosine-1-phosphate-induced chemotactic response, promoting FO-oriented Ag transport by MZ-P B cells. Knockout of type I IFN receptor in BXD2 (BXD2-Ifnalphar(-/-)) mice substantially diffused the intrafollicular MZ-P B cell conglomeration and shifted their location to the FO-MZ border near the marginal sinus, making Ag delivery to the FO interior less efficient. The development of spontaneous GCs was decreased in BXD2-Ifnalphar(-/-) mice. Together, our results suggest that the MZ-P B cells are major Ag-delivery B cells and that the FO entry of these B cells is highly regulated by type I IFN-producing plasmacytoid dendritic cells in the marginal sinus in the spleens of autoimmune BXD2 mice.

A comparative study of vestibular improvement and gastrointestinal effect of betahistine and gastrodin in mice.

Betahistine and gastrodin are the first-line medications for vestibular disorders in clinical practice, nevertheless, their amelioration effects on vestibular dysfunctions still lack direct comparison and their unexpected extra-vestibular effects remain elusive. Recent clinical studies have indicated that both of them may have effects on the gastrointestinal (GI) tract. Therefore, we purposed to systematically compare both vestibular and GI effects induced by betahistine and gastrodin and tried to elucidate the mechanisms underlying their GI effects. Our results showed that betahistine and gastrodin indeed had similar therapeutic effects on vestibular-associated motor dysfunction induced by unilateral labyrinthectomy. However, betahistine reduced total GI motility with gastric hypomotility and colonic hypermotility, whereas gastrodin did not influence total GI motility with only slight colonic hypermotility. In addition, betahistine, at normal dosages, induced a slight injury of gastric mucosa. These GI effects may be due to the different effects of betahistine and gastrodin on substance P and vasoactive intestinal peptide secretion in stomach and/or colon, and agonistic/anatgonistic effects of betahistine on histamine H1 and H3 receptors expressed in GI mucosal cells and H3 receptors distributed on nerves within the myenteric and submucosal plexuses. Furthermore, treatment of betahistine and gastrodin had potential effects on gut microbiota composition, which could lead to changes in host-microbiota homeostasis in turn. These results demonstrate that gastrodin has a consistent improvement effect on vestibular functions compared with betahistine but less effect on GI functions and gut microbiota, suggesting that gastrodin may be more suitable for vestibular disease patients with GI dysfunction.

Antimicrobial peptaibols, novel suppressors of tumor cells, targeted calcium-mediated apoptosis and autophagy in human hepatocellular carcinoma cells.

BACKGROUND: Hepatocellular carcinoma (HCC) is one of the most common cancers in the world which is highly chemoresistant to currently available chemotherapeutic agents. Thus, novel therapeutic targets are needed to be sought for the successful treatment of HCC. Peptaibols, a family of peptides synthesized non-ribosomally by the Trichoderma species and other fungi, exhibit antibiotic activities against bacteria and fungi. Few studies recently showed that peptaibols exerted cytotoxicity toward human lung epithelial and breast carcinoma cells. However, the mechanism involved in peptaibol-induced cell death remains poorly understood. RESULTS: Here, we showed that Trichokonin VI (TK VI), a peptaibol from Trichoderma pseudokoningii SMF2, induced growth inhibition of HCC cells in a dose-dependent manner. It did not obviously impair the viability of normal liver cells at lower concentration. Moreover, the suppression of cell viability resulted from the programmed cell death (PCD) with characteristics of apoptosis and autophagy. An influx of Ca2+ triggered the activation of mu-calpain and proceeded to the translocation of Bax to mitochondria and subsequent promotion of apoptosis. On the other hand, typically morphological characteristics consistent with autophagy were also observed by punctate distribution of MDC staining and the induction of LC3-II, including extensive autophagic vacuolization and enclosure of cell organelles by these autophagosomes. More significantly, specific depletion of Bak expression by small RNA interfering (siRNA) could partly attenuate TK VI-induced autophagy. However, siRNA against Bax led to increased autophagy. CONCLUSION: Taken together, these findings showed for the first time that peptaibols were novel regulators involved in both apoptosis and autophagy, suggesting that the class of peptaibols might serve as potential suppressors of tumor cells.

Tip30-induced apoptosis requires translocation of Bax and involves mitochondrial release of cytochrome c and Smac/DIABLO in hepatocellular carcinoma cells.

TIP30 (Tat-interacting protein 30), a newly found proapoptotic factor, appears to be involved in multiple functions including metabolic suppression, apoptosis induction, and diminishing angiogenic properties. In the present study, we reported that mitochondrial events were required for apoptosis induced by TIP30 in hepatocellular carcinoma cells (HCC cells). Translocation of Bax was essential for TIP30-induced apoptosis, whereas overexpression of the anti-apoptotic protein Bcl-xL delayed both second mitochondria-derived activator of caspases (Smac/DIABLO) release and onset of apoptosis. Furthermore, TIP30-induced apoptosis was dependent on caspase activity because the pan-caspase inhibitor benzyloxycarbonyl-Val-Ala-Asp (OMe)-fluoromethyl ketone (z-VAD-fmk) blocked DNA fragmentation. Release of Smac/DIABLO from the mitochondria through the TIP30-P53-Bax cascade was required to remove the inhibitory effect of XIAP (X-linked Inhibitor of Apoptosis) and allowed apoptosis to proceed. Our results showed for the first time that Bax-dependent release of Smac/DIABLO, cytochrome c and AIF from the mitochondria mediated the contribution of the mitochondrial pathway to TIP30-mediated apoptosis. Our data suggested that adenovirus-mediated overexpression of TIP30 was capable of inducing therapeutic programmed cell death in vitro by activating the mitochondrial pathway of apoptosis. On the basis of these studies, elucidating the mechanism by which TIP30 induces cell death might establish it as an anticancer approach.

Solid-state fermentation for Trichokonins production from Trichoderma koningii SMF2 and preparative purification of Trichokonin VI by a simple protocol.

Trichokonins are peptaibols produced by Trichoderma koningii SMF2. The main isoforms are Trichokonin VI, Trichokonin VII and Trichokonin VIII. The solid-state fermentation (SSF) was applied for the production of Trichokonin VI. The fermentation factors, which included inoculum size, incubation temperature, initial moisture content and initial pH, were investigated and optimized by response surface methodology. The maximum Trichokonin VI production (4.07mg/g dry substrate) was achieved by employing inoculum size of 18%, incubation temperature at 24.3 degrees C, initial moisture content of 77.5% and initial pH at 5.0. Furthermore, gel filtration and preparative HPLC were used for separation of Trichokonin VI from a crude extract of the T. koningii SMF2 culture. With this preparative purification protocol under optimized fermentation conditions, 146.20mg Trichokonin VI was obtained from 1kg solid cultures. It has been shown that the obtained Trichokonin VI is more than 95% in purity. This is the first report on optimization of peptaibols production in SSF with high content. An efficient method for the preparative purification of Trichokonin VI is also proposed.

GSK-3ß is Dephosphorylated by PP2A in a Leu309 Methylation-Independent Manner.

Hyperphosphorylation of tau is pivotally involved in the pathogenesis of Alzheimer's disease (AD) and related tauopathies. Glycogen synthase kinase-3ß (GSK-3ß) and protein phosphate 2A (PP2A) are crucial enzymes to regulate tau phosphorylation. GSK-3ß activity is regulated by its inhibitory phosphorylation at Ser9. We previously reported the cross-talk between GSK-3ß and PP2A signaling and showed that PP2A could dephosphorylate GSK-3ß at Ser9. Here, we investigated the dephosphorylation of GSK-3ß in brain extracts in the presence of phosphatase inhibitors and found that a PP2A-like phosphatase activity was required for dephosphorylation of GSK-3ß at Ser9. PP2A interacted with GSK-3ß and suppressed its Ser9 phosphorylation in vitro and in HEK-293FT cells. Activity of PP2A negatively correlated to the level of phosphorylated GSK-3ß in kainic acid-induced excitotoxic mouse brain. Alteration of methylation of the catalytic subunit of PP2A (PP2Ac) at Leu309 did not affect GSK-3ß phosphorylation. These findings suggest that Leu309 methylation is not required for PP2A to dephosphorylate GSK-3ß at Ser9.