mTORC2/AKT/HSF1/HuR constitute a feed-forward loop regulating Rictor expression and tumor growth in glioblastoma.

Overexpression of Rictor has been demonstrated to result in increased mechanistic target of rapamycin C2 (mTORC2) nucleation and activity leading to tumor growth and increased invasive characteristics in glioblastoma multiforme (GBM). However, the mechanisms regulating Rictor expression in these tumors is not clearly understood. In this report, we demonstrate that Rictor is regulated at the level of mRNA translation via heat-shock transcription factor 1 (HSF1)-induced HuR activity. HuR is shown to directly bind the 3' untranslated region of the Rictor transcript and enhance translational efficiency. Moreover, we demonstrate that mTORC2/AKT signaling activates HSF1 resulting in a feed-forward cascade in which continued mTORC2 activity is able to drive Rictor expression. RNAi-mediated blockade of AKT, HSF1 or HuR is sufficient to downregulate Rictor and inhibit GBM growth and invasive characteristics in vitro and suppress xenograft growth in mice. Modulation of AKT or HSF1 activity via the ectopic expression of mutant alleles support the ability of AKT to activate HSF1 and demonstrate continued HSF1/HuR/Rictor signaling in the context of AKT knockdown. We further show that constitutive overexpression of HuR is able to maintain Rictor expression under conditions of AKT or HSF1 loss. The expression of these components is also examined in patient GBM samples and correlative associations between the relative expression of these factors support the presence of these signaling relationships in GBM. These data support a role for a feed-forward loop mechanism by which mTORC2 activity stimulates Rictor translational efficiency via an AKT/HSF1/HuR signaling cascade resulting in enhanced mTORC2 activity in these tumors.

Heat shock proteins in hypoxic-ischemic brain injury: a perspective.

There is much to suggest that the induction of heat shock protein synthesis is an important response to injury and stress in the brain. The role of heat shock proteins in neurological disease has been approached from two points-of-view. First, the induction and synthesis of specific proteins after brain cell injury provide a window through which insight on the regulation of gene expression in pathological tissue can be obtained. These studies have broad implications for understanding pathophysiological mechanisms of disease. Second, putative cell protective effects of heat shock proteins in brain tissue provide insight into biochemical mechanisms of selective neuronal vulnerability. These studies have extremely important clinical implications since cell sensitivity to injury can seemingly be modified. The role of heat shock proteins in hypoxic-ischemic brain injury is discussed forthwith.

Neurologic complications of Gaucher's disease, type 3.

We discuss the clinical features of two patients with type 3 Gaucher's disease. Glucocerebroside levels in liver biopsy specimens were measured in both patients and the findings are discussed in regard to the pathogenesis of the disease.

Expression of antisense hsp70 is a major determining factor in heat-induced cell death of P-19 carcinoma cells.

Overexpressed heat shock protein 70 (Hsp70) is known to be associated with thermoprotection in a number of cell lines and transgenic animals. We hypothesized that because overexpression of Hsp70 protects cells from lethal heat stress, inhibition of expression should make cells susceptible to heat stress. The model used for this study was a stably transfected P-19 carcinoma cell line expressing antisense hsp70 under the control of the hsp70b promoter. The results showed marked inhibition of Hsp70 expression after heat shock correlated with heat-induced cell death. Hsp90 and Hsc70 protein expression were not affected by the antisense construct. Unexpectedly, heme oxygenase (HO-1), another highly inducible heat shock protein, was not induced after heat shock in the antisense hsp70 cell line. Heat shock transcription factor-1 (HSF-1) was in a highly phosphorylated state in the antisense cell line before and after heat shock. This was in contrast to the untransfected control P-19 cells where HSF-1 was primarily highly phosphorylated after heat shock. A control cell line expressing only the vector, pMAMneo, without the antisense construct also showed partial loss of Hsp70 induction but not increased cell death after heat shock. The findings support the role of Hsp70 in thermoresistance.

Evidence for different mechanisms of induction of HSP70i: a comparison of cultured rat cortical neurons with astrocytes.

This study is a follow-up of previous work which demonstrated that cultured cortical neurons did not synthesize HSP70i immediately after heat stress when compared with cultured cortical astrocytes. We have extended the period of observation for HSP70i induction of cultured cortical neurons and astrocytes up to 24 h after heat stress. Cultured rat cortical neurons derived from 16-day-old fetal rats respond differently to heat stress than cultured rat astrocytes derived from newborn rats. They showed a delayed HSP70i induction in the majority of cultured neurons and the response was heterogeneous and was absent in most smaller neurons. The delayed neuronal induction was accompanied by a prolonged activation of heat-shock transcription factor 1 (HSF-1) and prolonged transcription of HSP70i mRNA. In comparison astrocytes showed a marked early induction of HSP70i mRNA and protein. In addition the induction of HSP70i in astrocytes was followed by translocation of the protein into the nucleus, a finding which we failed to demonstrate in neurons. Immunostaining for HSP70i was more uniform in astrocytes than neurons. Many neurons did not stain for up to 24 h after heat shock in this study. Immunocytochemical staining of HSF-1 and 2 showed major differences between neurons and astrocytes. Astrocytes showed localization of HSF-1 to the nucleus before and after heat stress, while neurons showed HSF-1 localization to the cytoplasm and nucleus before and after heat stress. Finally HSF-2 was undetectable in neurons when compared with astrocytes by Western immunoblot analysis. However, astrocytes and neurons revealed weak immunostaining of HSF-2 in the cytoplasm and nucleus. The staining in the neurons was likely secondary to cross-reactivity to an unidentified protein. We conclude that HSP70i expression after heat shock is delayed in rat cortical neurons when compared with rat cortical astrocytes. In addition most small neurons did not synthesize HSP70i after heat shock. This difference in induction of HSP70i may be secondary to localization and activation of HSF-1 but not HSF-2. Neuronal susceptibility to injury may be related to the delayed induction of HSP70i and also the possible failure of newly synthesized HSP70i to translocate into the nucleus.

Differential expression of heme oxygenase-1 in cultured cortical neurons and astrocytes determined by the aid of a new heme oxygenase antibody. Response to oxidative stress.

Heme oxygenase exists as two isoenzymes designated heme oxygenase-1 (HO-1) and heme oxygenase-2 (HO-2). HO-2 is made constitutively in many cell types whereas HO-1 is a stress protein inducible by heat, heavy metals, ultraviolet irradiation, and oxidative stress. Recombinant rat HO-1 was expressed in bacteria and antiserum designated HO-1713 was raised against the purified protein. HO-1713 detected recombinant rat HO-1 and recombinant rat HO-2. In rat tissues it detected HO-1 and a second, unidentified band designated HO-L (heme oxygenase-like immunoreactivity) which was not HO-2. Cultured rat cortical neurons and forebrain astrocytes were exposed to hydrogen peroxide (0.14-0.7 micromolar for 30 or 60 min). Neurons which contained little detectable HO-1 and which were sensitive to hydrogen peroxide at the high end of the dose curve failed to induce HO-1 by Western blot analysis. In contrast, cultured rat forebrain astrocytes which contained HO-1 under normal culture conditions and which were resistant to injury by hydrogen peroxide, increased their content of immunoreactive HO-1 by 7-fold within 3 h after exposure. Our results support a protective role for HO-1 in oxidative injury and suggest that the relative inability of neurons to increase HO-1 after oxidative stress may contribute to their selective vulnerability vis-a-vis astrocytes. They also suggest that differential expression of heme oxygenase in studies utilizing CNS cultures may alter normal cell physiology and cell survival.

Transient induction of heme oxygenase after cortical stab wound injury.

Heme oxygenase (HO) exists as two isoenzymes designated heme oxygenase-1 (HO-1) and heme oxygenase-2 (HO-2). HO-1 has been identified as a heat shock or stress protein and is inducible whereas HO-2 is largely refractory to induction. HO-2 is the predominant isoenzyme in normal brain and appears to have a predominantly neuronal distribution in cerebral cortex. Cortical stab wound injury resulted in HO-1 induction as determined by Western blot analysis. Immunohistochemical analysis suggested that induced HO-1 was largely restricted to reactive astrocytes and macrophage-like cells. Enhanced HO-1 immunoreactivity was observed in hypertrophied, GFAP+ reactive astrocytes near the wound margin as early as 12 h after injury. Very rarely were HO-1+ neurons observed and then only up to 6 h after stabbing. Maximal numbers of HO-1+ astrocytes were found 3 days after stabbing. Their numbers declined thereafter. By 5 days after stab injury few HO-1+ reactive astrocytes were observed although GFAP+ reactive astrocytes were still prominent near the wound margin. HO-1+ macrophage-like cells were initially observed between 1 and 3 days after injury and they persisted in the margin of the wound for at least 14 days. The proximity of HO-1+ cells to the wound margin suggests that factors associated with injury contribute to the regulation of HO-1 in injured cortex.

Comparison of the heat shock response in cultured cortical neurons and astrocytes.

Cultured cortical neurons and astrocytes were compared for synthesis of the major inducible 68 kDa heat shock protein. By one- and two-dimensional electrophoresis the inducible 68 kDa protein appeared similar, but astrocytes produced greater amounts of the protein by 3 h than did neurons. Antibodies raised against HeLa cell inducible 72 and constitutive 73 kDa heat shock proteins were used to characterizes the inducible heat shock proteins in neurons and astrocytes. Unlike the gels, major differences were noted of the major inducible heat shock protein in astrocytes compared with neurons when analyzed by Western immunoblots. Heat shock protein 68 kDa mRNA induction in neurons was less than astrocytes suggesting an attenuated inducible 68 kDa heat shock protein response in neurons. The neuronal protein may be a different isoform of the 70 kDa family of heat shock proteins.

Characterization of the major 68 kDa heat shock protein in a rat transformed astroglial cell line.

The heat shock response in a transformed astrocyte line was compared with nontransformed astrocytes. The synthesis of HSP 68, the major inducible heat shock protein (HSP 68) was induced by a non-lethal 45 degrees C, 10 min heat shock. Although the incorporation of [35S]methionine into HSP 68 suggested that similar amounts of protein were being synthesized after heat shock, Western immunoblotting demonstrated striking differences in the HSP immunostaining between the two cell types. By one- and 'two-dimensional gel electrophoresis the major 68 kDa heat shock protein (HSP 68) was similar in both cell types. However, HSP 68 from heat shocked, transformed astrocytes did not immunostain with the monoclonal antibody, C-92, which is specific for the major inducible heat shock protein of HeLa cells. In contrast HSP 68 from heat shocked, nontransformed astrocytes immunostained quite well. A polyclonal antibody raised against the inducible 72 kDa heat shock protein of HeLa cells immunostained the HSP 68 from both astrocytes and transformed astrocytes. Analysis of the mRNA from the two cell types after heat shock revealed two bands of approximately 2.5 and 2.8 kb in astrocytes but only a single 2.5 kb band in the heat shocked transformed astroglia. These results suggest that structural differences in the HSP 68 may be present in the transformed astrocytes compared to the normal astrocytes.

Differential localization of heme oxygenase and NADPH-diaphorase in spinal cord neurons.

Western blot analysis using several antibodies showed that rat spinal cord contained abundant immunostainable heme oxygenase-2 (HO-2) and barely detectable levels of heme oxygenase-1 (HO-1). Anti-HO-2 antibody stained large anterior horn motoneurones and numerous smaller neurons throughout spinal cord gray matter including the dorsal root entry zone. HO-2+ astrocytes were not evident in gray matter although their presence cannot be ruled out. The distribution of HO-2+ neurons was compared with the distribution of cells containing NADPH-diaphorase (NADPH-d) activity, a marker for nitric oxide synthase. NADPH-d activity was restricted to far fewer neurons, many of which were close to the central canal and dorsal root entry zone.

Neuroprotection against CA1 injury with metalloporphyrins.

The hippocampal slice was used to examine neuroprotection with metalloporphyrins, a class of drug which inhibits heme oxygenase and which has been found to be effective in the treatment of neonatal hyperbilirubinemia. Tin-protoporphyrin given during hypoxia significantly improved recovery of CA1 antidromic PS to a mean of 82 +/- 2% of initial amplitude, while unmedicated slices regained only 6 +/- 3% of initial amplitude. Tin-protoporphyrin also protected against fluid percussion injury with an EC50 of 10 microM when given after trauma. This protection extended to induction of long-term potentiation. Tin-mesoporphyrin and zinc-protoporphyrin protected against trauma with EC50's of 4 and 32 microM. Treatment with Sn-PP also protected against exposure to hydrogen peroxide, but not NMDA, AMPA, glycine or nitric oxide. These findings indicate that metalloporphyrins protect against CA1 neuronal injury through direct neural effects.

Myelination of mouse cerebellar explants by rat cultured oligodendrocytes.

It has been shown that transplanted central nervous system tissue containing oligodendrocytes will myelinate neuronal processes in vitro and in situ. In this study we propose to show that cultured rat oligodendrocytes have the capacity to myelinate mouse cerebellar neuronal processes in vitro. Cultured rat oligodendrocytes were transplanted to cytosine arabinoside-treated mouse cerebellar explant cultures, then observed for myelination. Ultrastructural examination showed myelin and myelin-like figures in co-cultures. Control cytosine arabinoside-treated cultures and cultured oligodendroglia were without compact myelin.

Localization of heme oxygenase in rat retina: effect of light adaptation.

Heme oxygenase-2 isozyme is the predominant form of heme oxygenase in rat brain by western blot analysis. Heme oxygenase-1 isozyme is not induced by light adaptation in rat retina by western blot analysis. Immunocytochemistry localizes heme oxygenase-2 in three areas of the retina: the retinal pigment epithelium, inner segment and external nuclear layers of the rat retina. Ganglion cells and cell bodies of the internal nuclear layer of the retina and Müller cells were largely unstained for heme oxygenase-2. The localization of heme oxygenase-2 in the retina implies that its function is not associated with phototransduction. Also, light adaptation does not appear to induce heme oxygenase-1, a measure of oxidative injury.

Phenytoin neurotoxicity in developing mouse cerebellum in tissue culture.

Phenytoin applied to developing neonatal mouse cerebellar cultures at concentrations of 9-46 micrograms/ml of nutrient medium from the day of explantation to 16 days in vitro induced cerebellar cortical degeneration. The degree of neurotoxicity correlated with drug concentration. Purkinje cells were the most susceptible of the cerebellar elements, and intracerebellar nucleus neurons were the most resistant. In contrast, mature mouse cerebellar explants were resistant to chronic exposure to high concentrations of phenytoin.

Quantitation of myelination and demyelination by the measurement of myelin basic protein by ELISA.

Utilizing a competitive inhibition enzyme-linked immunosorbent assay (ELISA) we measured the amount of myelin basic protein (MBP) in brain, liver, and kidney of newborn mouse and compared these values with myelinated cerebellar explant cultures under various conditions. Explant cultures demyelinated with anti-myelin serum showed a 90% decrease in MBP when compared with controls. These results correlated with myelination and demyelination as observed by light microscopy. Newborn tissues including brain showed only negligible activity for MBP. This technique provides a sensitive and objective method for quantitating in-vitro demyelination.

Induction of cell death by L-alpha-aminoadipic acid exposure in cultured rat astrocytes: relationship to protein synthesis.

The excitotoxin, L-alpha-aminoadipic acid (L-AAA), kills primary astrocytes in the brain. The mechanism underlying the induction of cell death is not well understood although many possible mechanisms are theorized. Previous studies have reported that astrocytes die after prolonged exposure to L-AAA suggesting a delayed programmed cell death and apoptosis. In this study rat cortical astrocytes exposed to continuous 1 mM L-AAA exposure for 24-, 48-, or 72 hours demonstrated increased DNA laddering, a characteristic of apoptosis. Unexpectedly, this was not ameliorated by the presence of cycloheximide at 0.1 microg/ml medium. Because of our interest in cytoprotective heat shock proteins induced by excitoxic stress, we studied the effect of prolonged exposure of L-AAA on the synthesis of stress proteins and protein synthesis in rat cortical astrocytes. Protein synthesis as measured by [35S]-methionine labeling showed a marked and significant decrease in incorporation of radiolabel after 24 hours of exposure to L-AAA and prior to induction of significant cell death noted at 48- and 72 hours of L-AAA exposure. The inhibition of protein synthesis was partially reversible at 24 hours if cells were labeled in medium without L-AAA during the radiolabeling period. Heat shock or stress proteins, HSP70 and heme oxygenase-1 (HO-1), were analyzed after a 24 hour exposure to L-AAA and showed no significant induction of HSP70 or HO-1. The findings suggest that the prolonged inhibition of protein synthesis and associated lack of induction of HSP70 and HO-1 synthesis contributed to apoptotic cell death induced by the excitoxin L-AAA.

Pharmacological induction of heat shock protein 68 synthesis in cultured rat astrocytes.

The induction of the highly inducible 70-kDa heat shock protein (HSP 70) is associated with thermotolerance and survival from many other types of stress. This investigation studied the pharmacological induction of HSP 68 (HSP 68 is the rat homolog of human HSP 70) by 1,10-phenanthroline in cultured rat astrocytes under conditions that activated heat shock transcription factor-1 without inducing HSP 68 synthesis. Two conditions that activate heat shock transcription factor-1 and promote its binding to the heat shock element without subsequent transcription of HSP 68 mRNA, intracellular acidosis and exposure to salicylate, showed synthesis of HSP 68 when 1,10-phenanthroline was added to culture medium after the activation of heat shock transcription factor-1. 1,10-phenanthroline mimicked heat shock by inducing HSP 68 mRNA and protein under both conditions. 1,10-phenanthroline added alone to culture medium did not induce the synthesis of HSP 68 or activate heat shock transcription factor-1. These findings strongly suggest a multistep activation for HSP 68 synthesis and also demonstrate that the synthesis of HSP 68 can be pharmacologically regulated.

Cerebral hypoxia-ischemia in immature rats: methodological considerations.

We used a model of perinatal hypoxic/ischemic brain damage which combines unilateral common carotid artery ligation and hypoxia (8% O2). Protein synthesis inhibition and cell loss were found in the ipsilateral forebrain of 11-day-old rats when hypoxia was initiated 4 h but not 24 h after carotid ligation. [14C]Iodoantipyrine uptake studies suggest that compensating vascular changes which protect the ipsilateral forebrain occur within 24 h of carotid ligation.

Heme oxygenase in the experimental ALS mouse.

Heme oxygenase-1 (HO-1) is a stress protein inducible in some cells by oxidative stress. The status of heme oxygenase was investigated in a transgenic mouse model of amyotrophic lateral sclerosis (ALS) since oxidative mechanisms are postulated in neuronal injury. Three ALS mice [(SOD1-G93A)1Gur] and three controls [(SOD-1)2Gur] were obtained from The Jackson Laboratory. Behavioral differences suggestive of neurodegeneration in ALS mice developed at 4-5 months of age. All mice were killed at 7-8 months of age. Tissue vacuolation, cell loss, and the presence of GFAP+ cells were noted in the spinal cords of ALS mice. Spinal cord motor neurons in both control and ALS mice stained positive for heme oxygenase-2 (HO-2). While not precluding the presence of low levels of HO-1 neither immunohistochemical staining nor Western blot analysis provided evidence for significant HO-1 induction in degenerating spinal cord.