HFD-exacerbated Metabolic Side Effects of Olanzapine Are Suppressed by ER Stress Inhibitor.

OBJECTIVE: Numerous schizophrenic patients are suffering from obesity primarily attributed to antipsychotic medication and poor dietary habits. This study investigated the progressive deterioration of olanzapine-induced metabolic disorders in the presence of a high-fat diet (HFD) and explored the involvement of endoplasmic reticulum (ER) stress. METHODS: Female Sprague-Dawley rats fed on a standard chow diet or HFD were treated with olanzapine (3 mg/kg/day) and the ER stress inhibitor 4-phenylbutyric acid (4-PBA, 1 and 0.5 g/kg/day) for 8 days. Changes in body weight, food intake, and plasma lipids were assessed. Hepatic fat accumulation was evaluated using oil red O staining. Western blotting and immunofluorescence assays were employed to examine the expression of ER stress markers, NOD-like receptor pyrin domain-containing protein 3 (NLRP3), and proopiomelanocortin (POMC) in the hypothalamus or liver. RESULTS: Compared to olanzapine alone, olanzapine+HFD induced greater weight gain, increased hyperlipidemia, and enhanced hepatic fat accumulation (P<0.05). Co-treatment with 4-PBA exhibited a dose-dependent inhibition of these effects (P<0.05). Further mechanistic investigations revealed that olanzapine alone activated ER stress, upregulated NLRP3 expression in the hypothalamus and liver, and downregulated hypothalamic POMC expression. The HFD exacerbated these effects by 50%-100%. Moreover, co-administration of 4-PBA dose-dependently attenuated the olanzapine+HFD-induced alterations in ER stress, NLRP3, and POMC expression in the hypothalamus and liver (P<0.05). CONCLUSION: HFD worsened olanzapine-induced weight gain and lipid metabolic disorders, possibly through ER stress-POMC and ER stress-NLRP3 signaling. ER stress inhibitors could be effective in preventing olanzapine+HFD-induced metabolic disorders.

Substitutive proprioception feedback of a prosthetic wrist by electrotactile stimulation.

Objective: Sensory feedback of upper-limb prostheses is widely desired and studied. As important components of proprioception, position, and movement feedback help users to control prostheses better. Among various feedback methods, electrotactile stimulation is a potential method for coding proprioceptive information of a prosthesis. This study was motivated by the need for proprioception information for a prosthetic wrist. The flexion-extension (FE) position and movement information of the prosthetic wrist are transmitted back to the human body through multichannel electrotactile stimulation. Approach: We developed an electrotactile scheme to encode the FE position and movement of the prosthetic wrist and designed an integrated experimental platform. A preliminary experiment on the sensory threshold and discomfort threshold was performed. Then, two proprioceptive feedback experiments were performed: a position sense experiment (Exp 1) and a movement sense experiment (Exp 2). Each experiment included a learning session and a test session. The success rate (SR) and discrimination reaction time (DRT) were analyzed to evaluate the recognition effect. The acceptance of the electrotactile scheme was evaluated by a questionnaire. Main results: Our results showed that the average position SRs of five able-bodied subjects, amputee 1, and amputee 2 were 83.78, 97.78, and 84.44%, respectively. The average movement SR, and the direction and range SR of wrist movement in five able-bodied subjects were 76.25, 96.67%, respectively. Amputee 1 and amputee 2 had movement SRs of 87.78 and 90.00% and direction and range SRs of 64.58 and 77.08%, respectively. The average DRT of five able-bodied subjects was less than 1.5 s and that of amputees was less than 3.5 s. Conclusion: The results indicate that after a short period of learning, the subjects can sense the position and movement of wrist FE. The proposed substitutive scheme has the potential for amputees to sense a prosthetic wrist, thus enhancing the human-machine interaction.

Tone evaluation of Ling sound test in Mandarin tone version.

BACKGROUND: The Ling sound test cannot provide the test of Chinese tone for preschool children with hearing aid and cochlear implants. OBJECTIVE: The paper tries to design a new tone test method composed of the Ling sound test and four Chinese tones to evaluate the hearing level of Chinese hearing-impaired children. METHODS: The tone identification rates of 20 cochlear implant children were statistically analyzed to verify the validity of the Ling sound test in the Chinese tone version. In addition, this paper analyzed the pronunciation characteristics of the Ling sound test in the Chongqing-accented Mandarin version of 20 subjects. RESULTS: The identification rate of Ling six sounds was more than 97.0%, the identification rate of tone was more than 81.0%, and the identification rate of vowels was 83.1%, which was higher than that of consonants 79.0%. The Ling sound test n the Chongqing-accented Mandarin version has a narrower frequency range. CONCLUSION: The results verify the effectiveness and feasibility of the Ling sound test in the Chinese tone version in the assessment of frequency range and tone identification for cochlear implant users.

The role of hypothalamic endoplasmic reticulum stress in schizophrenia and antipsychotic-induced weight gain: A narrative review.

Schizophrenia (SCZ) is a serious mental illness that affects 1% of people worldwide. SCZ is associated with a higher risk of developing metabolic disorders such as obesity. Antipsychotics are the main treatment for SCZ, but their side effects include significant weight gain/obesity. Despite extensive research, the underlying mechanisms by which SCZ and antipsychotic treatment induce weight gain/obesity remain unclear. Hypothalamic endoplasmic reticulum (ER) stress is one of the most important pathways that modulates inflammation, neuronal function, and energy balance. This review aimed to investigate the role of hypothalamic ER stress in SCZ and antipsychotic-induced weight gain/obesity. Preliminary evidence indicates that SCZ is associated with reduced dopamine D2 receptor (DRD2) signaling, which significantly regulates the ER stress pathway, suggesting the importance of ER stress in SCZ and its related metabolic disorders. Antipsychotics such as olanzapine activate ER stress in hypothalamic neurons. These effects may induce decreased proopiomelanocortin (POMC) processing, increased neuropeptide Y (NPY) and agouti-related protein (AgRP) expression, autophagy, and leptin and insulin resistance, resulting in hyperphagia, decreased energy expenditure, and central inflammation, thereby causing weight gain. By activating ER stress, antipsychotics such as olanzapine activate hypothalamic astrocytes and Toll-like receptor 4 signaling, thereby causing inflammation and weight gain/obesity. Moreover, evidence suggests that antipsychotic-induced ER stress may be related to their antagonistic effects on neurotransmitter receptors such as DRD2 and the histamine H1 receptor. Taken together, ER stress inhibitors could be a potential effective intervention against SCZ and antipsychotic-induced weight gain and inflammation.

NLRP3/Caspase-1-Mediated Pyroptosis of Astrocytes Induced by Antipsychotics Is Inhibited by a Histamine H1 Receptor-Selective Agonist.

Emerging data indicate that antipsychotic treatment causes brain volume loss and astrocyte death, but the mechanisms remain elusive. Pyroptosis, inflammatory cell death characterized by the formation of inflammatory bodies, increased expression of nod-like receptor proteins (NLRPs) such as NLRP3, and activation of caspases and gasdermin D (GSDMD) are largely associated with innate immunity, inflammation, and cell injury/death. However, the main effect of antipsychotics on astrocyte pyroptotic signaling and the molecular mechanisms remain obscure. In the present study, 72-h treatment with olanzapine, quetiapine, risperidone, or haloperidol significantly decreased the viability of astrocytes. Twenty-four hour treatment with olanzapine, quetiapine, risperidone, or haloperidol dose-dependently increased the protein expression of astrocytic NLRP3, NLRP6, caspase-1, caspase-4, and GSDMD. Co-treatment with a histamine H1 receptor agonist, 2-(3-trifluoromethylphenyl) histamine (FMPH), dose-dependently reduced the increased expression of NLRP3, caspase-1 and GSDMD induced by olanzapine, quetiapine, risperidone, or haloperidol. Moreover, olanzapine, quetiapine, risperidone, or haloperidol treatment induced pore formation in the membranes of astrocytes, and these effects were inhibited by FMPH co-treatment. Taken together, antipsychotic treatment activated astrocyte pyroptotic signaling, and these effects may be related to antipsychotic-induced astrocyte death. H1 receptor activation is an effective treatment strategy to suppress antipsychotic-induced astrocyte pyroptosis and inflammation.

Modulation of ERK5 is a novel mechanism by which Cdc42 regulates migration of breast cancer cells.

Members of Rho family GTPases including Cdc42 are known to play pivotal roles in cell migration. Cell migration is also known to be regulated by many protein kinases. Kinetworks KPSS 11.0 phospho-site screening of Cdc42-silenced Hs578T breast cancer cells revealed most dramatic change in ERK5 MAP kinase. In the present study, we set out to determine the relationship between Cdc42 and ERK5 and its significance in breast cancer cell migration and invasion. Specific siRNAs were used for knocking down Cdc42 or ERK5 in breast cancer cells. Increased ERK5 phosphorylation in breast cancer cells was achieved by infection of constitutively active MEK5 adenovirus. The cells were then subjected to cell migration or invasion assay without the presence of serum or any growth factor. We found that Cdc42 negatively regulated phosphorylation of ERK5, which in turn exhibited an inverse relationship with migration and invasiveness of breast cancer cells. To find out some in vivo relevance of the results of our in vitro experiments we also examined the expression of ERK5 in the breast cancer tissues and their adjacent normal control tissues by real-time RT-PCR and immunocytochemistry. ERK5 expression was found to be reduced in breast cancer tissues as compared with their adjacent uninvolved mammary tissues. Therefore, Cdc42 may promote breast cancer cell migration and invasion by inhibiting ERK5 phosphorylation and ERK5 expression may be inversely correlated with the progression of some breast tumors.

Cdc42 negatively regulates intrinsic migration of highly aggressive breast cancer cells.

The small GTPase Cdc42 has been implicated as an important regulator of cell migration. However, whether Cdc42 plays similar role in all cancer cells irrespective of metastatic potential remains poorly defined. Here, we show by using three different breast cancer cell lines with different metastatic potential, the role of Cdc42 in cell migration/invasion and its relationship with a number of downstream signaling pathways controlling cell migration. Small interfering RNA (siRNA)-mediated knockdown of Cdc42 in two highly metastatic breast cancer cell lines (MDA-MB-231 and C3L5) resulted in enhancement, whereas the same in moderately metastatic (Hs578T) cell line resulted in inhibition of intrinsic cellular migration/invasion. Furthermore, Cdc42 silencing in MDA-MB-231 and C3L5 but not Hs578T cells was shown to be accompanied by increased RhoA activity and phosphorylation of protein kinase C (PKC)-δ, extracellular signal regulated kinase1/2 (Erk1/2), and protein kinase A (PKA). Pharmacological inhibition of PKCδ, MEK-Erk1/2, or PKA was shown to inhibit migration of both control and Cdc42-silenced MDA-MB-231 cells. Furthermore, introduction of constitutively active Cdc42 was shown to decrease migration/invasion of MDA-MB-231 and C3L5 but increase migration/invasion of Hs578T cells. This decreased migration/invasion of MDA-MB-231 and C3L5 cells was also shown to be accompanied by the decrease in the phosphorylations of PKCδ, Erk1/2, and PKA. These results suggested that endogenous Cdc42 could exert a negative regulatory influence on intrinsic migration/invasion and some potentially relevant changes in phosphorylation of PKCδ, Erk1/2, and PKA of some aggressive breast cancer cells.

Role of Rho GTPases in human trophoblast migration induced by IGFBP1.

Insulin-like growth factor binding protein 1 (IGFBP1), the main secretory product of the decidualized endometrium of a pregnant woman, has previously been shown to interact with the alpha(5)beta(1) integrin of extravillous trophoblast (EVT) cell surface to stimulate its migration in an IGF-independent manner. This migration stimulation has also been shown to require activation of extracellular signal regulated kinases 1 and 2 (ERK1/2; mitogen-activated protein kinase [MAPK] 3/1]) and focal adhesion kinase. The present study examined the roles of Rho GTPases RHOA, RHOC, RAC1, and CDC42 as well as RHO kinases ROCK1 and ROCK2 in IGFBP1-mediated migration of an immortalized EVT cell line HTR-8/SVneo. A nonselective RHO kinase inhibitor, Y27632, as well as siRNAs selective for ROCK1 and ROCK2 decreased the migration of these cells in a Transwell migration assay, and this inhibition could not be restored by IGFBP1. Clostridium difficile toxin B, which inhibits all the Rho GTPases, RAC inhibitor NSC23766, RAC1 siRNA, and CDC42 siRNA, decreased their basal migration, but none of these inhibitions except CDC42 siRNA-induced inhibition could be restored by IGFBP1. Clostridium botulinum C3 exoenzyme that inhibits RHOA, RHOB, and RHOC inhibited basal migration but not IGFBP1-induced migration. IGFBP1-induced activation of ERK1/2 (MAPK3/1), which did not require RHO proteins, might function as an alternate pathway for RHO action. However, selective siRNA-mediated downregulation of RHOA inhibited basal, but not IGFBP1-mediated, migration, whereas that of RHOC inhibited both basal and IGFBP1-mediated migration of these EVT cells. Therefore, RHO kinase, RHOC, and RAC1 are essential, but RHOA and CDC42 are not essential, for IGFBP1-induced EVT migration.

ERK5 Contributes to VEGF Alteration in Diabetic Retinopathy.

Diabetic retinopathy is one of the most common causes of blindness in North America. Several signaling mechanisms are activated secondary to hyperglycemia in diabetes, leading to activation of vasoactive factors. We investigated a novel pathway, namely extracellular signal regulated kinase 5 (ERK5) mediated signaling, in modulating glucose-induced vascular endothelial growth factor (VEGF) expression. Human microvascular endothelial cells (HMVEC) were exposed to glucose. In parallel, retinal tissues from streptozotocin-induced diabetic rats were examined after 4 months of follow-up. In HMVECs, glucose caused initial activation followed by deactivation of ERK5 and its downstream mediators myocyte enhancing factor 2C (MEF2C) and Kruppel-like factor 2 (KLF2) mRNA expression. ERK5 inactivation further led to augmented VEGF mRNA expression. Furthermore, siRNA mediated ERK5 gene knockdown suppressed MEF2C and KLF2 expression and increased VEGF expression and angiogenesis. On the other hand, constitutively active MEK5, an activator of ERK5, increased ERK5 activation and ERK5 and KLF2 mRNA expression and attenuated basal- and glucose-induced VEGF mRNA expression. In the retina of diabetic rats, depletion of ERK5, KLF2 and upregulation of VEGF mRNA were demonstrated. These results indicated that ERK5 depletion contributes to glucose induced increased VEGF production and angiogenesis. Hence, ERK5 may be a putative therapeutic target to modulate VEGF expression in diabetic retinopathy.

Glucose-induced endothelin-1 expression is regulated by ERK5 in the endothelial cells and retina of diabetic rats.

Upregulation of endothelin 1 (ET-1) causing blood flow alteration and increased extracellular matrix production are characteristic features of diabetic angiopathy. Several glucose-induced signaling mechanisms cause ET-1 upregulation in diabetic angiopathy. Extracellular signal-regulated kinase 5 (ERK5) is a member of the MAPK family, which plays a key role in cardiovascular development. ERK kinase (MEK) 5 is the specific MEK for ERK5 activation. In this study we examined the role of glucose-induced ERK5 signaling in mediating ET-1 expression in diabetic angiopathy. We investigated retinas from 1-month STZ-induced diabetic rats and human macro- and microvascular endothelial cells to study ERK5-dependent ET-1 alterations. Glucose (25 mmol/L) caused significant upregulation of ET-1 mRNA and downregulation of ERK5 and Kruppel-like factor 2 (KLF2) after 24 h treatment in the endothelial cells. Simultaneously, phospho-ERK5 proteins were reduced. Activation of ERK5 by constitutively active MEK5 (caMEK5) upregulated KLF2 and suppressed ET-1 expression in both cell lines, whereas ERK5 siRNA transfection resulted in decreased ERK5 and KLF2 and increased ET-1 mRNA expression. In addition, caMEK5 prevented glucose-induced upregulation of ET-1. Furthermore, 1 month of diabetes caused a significant increase in retinal ET-1 mRNA and decrease in ERK5 mRNA expression. These data indicate that ERK5 signaling regulates glucose-induced ET-1 expression in diabetes. The ERK5/ET-1 pathway may provide a potential novel target for the treatment of diabetic angiopathy.

[Construction, expression and functional characterization of single chain variable fragments (scFv) against human CD33 antigen].

AIM: To construct and express the single chain variable fragments (scFv) gene against human CD33 antigen, and characterize its bioactivity. METHODS: The genes encoding the light and heavy chain variable regions were cloned by RT-PCR from a murine hybridoma cell line, which could produce monoclonal antibody(mAb) against human CD33 antigen. Then the light and heavy chain variable regions were fused together by a short peptide linker containing 15 amino acid (Gly(4)Ser)(3) using splice-overlap extensive PCR. The recombinant anti-CD33 scFv was subcloned into the expression vector pET28a(+) and expressed in E.coli Rosetta after induction by IPTG. RESULTS: SDS-PAGE and Western blot analysis showed that the recombinant anti-CD33 scFv gene was expressed in the form of inclusion body in E.coli Rosetta, and the purified fusion protein was obtained after a series of purification steps including cell lysis, inclusion body solubilization, Ni(2+) metal affinity chromatography and protein refolding. Flow cytometry(FCM) analysis showed that the scFv could react with human CD33 antigen. CONCLUSION: Recombinant anti-CD33 scFv gene has been successfully constructed and expressed in E.coli Rosetta, which could provide foundation for the future target therapy to the myeloid leukemia.

Enhanced intrinsic migration of aggressive breast cancer cells by inhibition of Rac1 GTPase.

Rac GTPases are known to play a crucial role in regulating cytoskeletal changes necessary for cell migration. Migration has been shown to be positively regulated by Rac in most cell types. However, there is also a large body of conflicting evidence in some other cell types with respect to the role of Rac in migration, suggesting that Rac GTPases regulate cell migration in a cell type-dependent manner. In the present study, we have characterized the effects of Rac1 GTPase inhibition on the migratory abilities of a number of breast cancer cell lines with differential degrees of tumorigenic and metastatic potentials. We show that Rac1 inhibition in non-metastatic (MCF-7, T-47D) or moderately metastatic (Hs578T) cell lines results in inhibition of migration, whereas in highly metastatic cell lines (MDA-MB-435, MDA-MB-231, and C3L5) Rac1 inhibition results in stimulation of migration. This stimulation of migration following Rac1 inhibition is also accompanied by the enhanced RhoA activity, suggesting a possible existence of a dominating role of RhoA over Rac1 in regulating intrinsic migration of the highly metastatic breast cancer cells.