Induction of heat shock proteins in cerebral cortical cultures by celastrol

Alzheimer’s disease, Parkinson’s disease and amyotrophic lateral sclerosis (ALS) are ‘protein misfolding disorders’ of the mature nervous system that are characterized by the accumulation of protein aggregates and selective cell loss. Different brain regions are impacted, with Alzheimer’s affecting cells in the cerebral cortex, Parkinson’s targeting dopaminergic cells in the substantia nigra and ALS causing degeneration of cells in the spinal cord. These diseases differ widely in frequency in the human population. Alzheimer’s is more frequent than Parkinson’s and ALS. Heat shock proteins (Hsps) are ‘protein repair agents’ that provide a line of defense against misfolded, aggregation-prone proteins. We have suggested that differing levels of constitutively expressed Hsps (Hsc70 and Hsp27) in neural cell populations confer a variable buffering capacity against ‘protein misfolding disorders’ that correlates with the relative frequencies of these neurodegenerative diseases. The high relative frequency of Alzheimer’s may due to low levels of Hsc70 and Hsp27 in affected cell populations that results in a reduced defense capacity against protein misfolding. Here, we demonstrate that celastrol, but not classical heat shock treatment, is effective in inducing a set of neuroprotective Hsps in cultures derived from cerebral cortices, including Hsp70, Hsp27 and Hsp32. This set of Hsps is induced by celastrol at ‘days in vitro’ (DIV) 13 when cultured cortical cells reached maturity. The inducibility of a set of neuroprotective Hsps in mature cortical cultures at DIV13 suggests that celastrol is a potential agent to counter Alzheimer’s disease, a neurodegenerative ‘protein misfolding disorder’ of the adult brain that targets cells in the cerebral cortex.     

Induction of heat shock proteins in differentiated human and rodent neurons by celastrol

Neurodegenerative disorders such as Alzheimer's disease, Parkinson's disease, and amyotrophic lateral sclerosis have been termed protein misfolding disorders that are characterized by the neuronal accumulation of protein aggregates. Manipulation of the cellular stress-response involving induction of heat shock proteins (Hsps) in differentiated neurons offers a therapeutic strategy to counter conformational changes in neuronal proteins that trigger pathogenic cascades resulting in neurodegenerative diseases. Hsps are protein repair agents that provide a line of defense against misfolded, aggregation-prone proteins. These proteins are not induced in differentiated neurons by conventional heat shock. We have found that celastrol, a quinine methide triterpene, induced expression of a wider set of Hsps, including Hsp70B', in differentiated human neurons grown in tissue culture compared to cultured rodent neuronal cells. Hence the beneficial effect of celastrol against human neurodegenerative diseases may exceed its potential in rodent models of these diseases.     

Localization of heat shock proteins in cerebral cortical cultures following induction by celastrol

Hsp70, Hsp32, and Hsp27 were induced by celastrol in rat cerebral cortical cultures at dosages that did not affect cell viability. Pronounced differences were observed in the cellular localization of these heat shock proteins in cell types of cerebral cortical cultures. Celastrol-induced Hsp70 localized to the cell body and cellular processes of neurons that were identified by neuron-specific βIII-tubulin. Hsp70 was not detected in adjacent GFAP-positive glial cells that demonstrated a strong signal for Hsp27 and Hsp32 in both glial cell bodies and cellular processes. Cells in the cerebral cortex region of the brain are selectively impacted during the progression of Alzheimer’s disease which is a “protein misfolding disorder.” Heat shock proteins provide a line of defense against misfolded, aggregation-prone proteins. Celastrol is a potential agent to counter this neurodegenerative disorder as recent evidence indicates that in vivo administration of celastrol in a transgenic model of Alzheimer’s reduces an important neuropathological hallmark of this disease, namely, amyloid beta pathology that involves protein aggregation.     

Activation of hepatocytes by extracellular heat shock protein 72

Heat shock protein (HSP) 72 is released by cells during stress and injury. HSP-72 also stimulates the release of cytokines in macrophages by binding to Toll-like receptors (TLR) 2 and 4. Circulating levels of HSP-72 increase during hepatic ischemia-reperfusion injury. The role of extracellular HSP-72 (eHSP-72) in the injury response to ischemia-reperfusion is unknown. Therefore, the objective of the present study was to determine whether eHSP-72 has any direct effects on hepatocytes. Primary mouse hepatocytes were treated with purified human recombinant HSP-72. Conditioned media were evaluated by ELISA for the cytokines, TNF-alpha, IL-6, and macrophage inflammatory protein 2 (MIP-2). Stimulation of hepatocytes with eHSP-72 did not induce production of TNFalpha or IL-6 but resulted in dose-dependent increases in MIP-2 production. To evaluate the pathway responsible for this response, expression of TLR2 and TLR4 was confirmed on hepatocytes by immunohistochemistry. Hepatocyte production of MIP-2 was significantly decreased in hepatocytes obtained from TLR2 or TLR4 knockout mice. MIP-2 production was found to be partially dependent on NF-kappaB because inhibition of NF-kappaB with Bay 11-7085 significantly decreased eHSP-72-induced MIP-2 production. Inhibitors of p38 mitogen-activated protein kinase or c-Jun NH(2)-terminal kinase had no effect on production of MIP-2 induced by eHSP-72. The data suggest that eHSP-72 binds to TLR2 and TLR4 on hepatocytes and signals through NF-kappaB to increase MIP-2 production. The fact that eHSP-72 did not increase TNF-alpha or IL-6 production may be indicative of a highly regulated signaling pathway downstream from TLR.     

Novel transcriptional responses to heat revealed by turning up the heat at night

Plant Molecular Biology - Genome-wide association study of maize plant architecture using F1 populations can better dissect various genetic effects that can provide precise guidance for genetic...     

Activation of natural killer cells in vitro by a product of beta-hemolytic streptococci

Streptokinase-streptodornase (SK-SD), a product of Group A and Group C β-hemolytic streptococci, activates natural killer (NK) cells in vitro. This effect appears to occur without the induction of interferon. It is not present in every individual but is reproducible. There appears to be no correlation between NK activation and assays for cellular immunity such as in vivo delayed skin reactions and/or in vitro cell proliferation.     

Hippocampal and cortical neuronal growth mediated by the small molecule natural product clovanemagnolol

The use of small molecule surrogates of growth factors that directly or indirectly promote growth represents an attractive approach to regenerative medicine. With synthetic access to clovanemagnolol, a small molecule initially isolated from the bark of the Bigleaf Magnolia tree, we have examined the small molecule’s ability to promote growth of embryonic hippocampal and cortical neurons in serum-free medium. Comparisons with magnolol, a known promoter of growth, reveals that clovanmagnolol is a potent neurotrophic agent, promoting neuronal growth at concentrations of 10 nM. In addition, both clovanemagnolol and magnolol promote growth through a biphasic dose response.     

Role of cytochrome P-450 in schisandrin B-induced antioxidant and heat shock responses in mouse liver

In order to explore the role of cytochrome P-450 (P-450) in schisandrin B (Sch B)-induced antioxidant and heat shock responses, the effect of 1-aminobenzotriazole (ABT, a broad spectrum inhibitor of P-450) on hepatic mitochondrial glutathione antioxidant status (mtGAS) and heat shock protein (Hsp)25/70 expression was examined in Sch B-treated mice. The non-specific and partial inhibition of cytochrome P-450 (P-450) by ABT pretreatment significantly caused a protraction in the time-course of Sch B-induced enhancement in hepatic mitGAS and Hsp25/70 expression in mice. Using mouse liver microsomes as a source of P-450, Sch B, but not dimethyl diphenyl bicarboxylate (a non-hepatoprotective analog of Sch B), was found to serve as a co-substrate for the P-450-catalyzed NADPH oxidation reaction, with a concomitant production of oxidant species. Taken together, the results suggest that oxidant species generated from P-450-catalyzed reaction with Sch B may trigger the antioxidant and heat shock responses in mouse liver.     

Synthesis and antioxidant properties of diphenylmethane derivative bromophenols including a natural product

Bromination of bis(3,4-dimethoxyphenyl)methanone (5) gave four products (6–9) with mono, di, tri, and tetra Br under different conditions. Reduction and demethylation reactions of product 9 with tetra Br were performed, consecutively and a natural product, 5,5′-methylene bis(3,4-dibrombenzene-1,2-diol) (1), was obtained with a 53% yield. Five derivatives, (13–17) (bromophenols), of 1 were also synthesised. The antioxidant and radical scavenging activities of bromophenols 1 and 13–17 were determined by employing various in vitro assays such as 1,1-diphenyl-2-picryl-hydrazyl free radical (DPPH^?), 2,2′-azino-bis(3-ethylbenzthiazoline-6-sulphonic acid (ABTS^?+), N,N-dimethyl-p-phenylenediamine dihydrochloride radical cation (DMPD^?+), and superoxide anion radical (O₂^?-) scavenging, reducing ability determination by the Fe³⁺-Fe²⁺ and Cu²⁺-Cu⁺ cupric reducing antioxidant capacity (CUPRAC) transformation methods, hydrogen peroxide scavenging, and ferrous ion (Fe²⁺) chelating activities. Moreover, these activities were compared to those of synthetic standard antioxidant compounds such as butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), α-tocopherol, and trolox. The results showed that the synthesised bromophenols had effective antioxidant power.     

Dietary intake of antioxidant supplements modulate antioxidant status and heat shock protein 70 synthesis

The physiological effects and efficacy of dietary intake of antioxidant supplements in humans remains controversial. Experiments involving dietary, often high, intake of a single antioxidant or vitamin may be seriously flawed given the interactive nature of antioxidants in vivo. The present studies were conducted on individuals (35-60 years of age) taking a commercial antioxidant mixture in a double-blind, placebo-controlled, cross-over study. Intake was two capsules per day, for 4 weeks, with a 4-week washout period in between active dose or placebo. Intake of antioxidants was associated with little change in superoxide dismutase activity, but an increase in glutathione peroxidase was noted. Haemolysis of red blood cells (erythrocytes) induced by the free radical generator AAPH was significantly reduced in individuals on antioxidant supplements. In lymphocytes isolated from individuals taking supplements, there was a marked increase, as compared with individuals on placebo, in the synthesis of heat shock protein 70 (hsp70) following heat shock from 37 degrees C to 42.5 degrees C. We conclude that dietary intake of a mixed antioxidant supplement leads to modulation of cellular redox status resulting in decreased oxidative stress and increased ability of lymphocytes to mount a stress response.