Dual Inhibition of PI3K/AKT and MEK/ERK Pathways Induces Synergistic Antitumor Effects in Diffuse Intrinsic Pontine Glioma Cells

Diffuse intrinsic pontine glioma (DIPG) is a devastating disease with an extremely poor prognosis. Recent studies have shown that platelet-derived growth factor receptor (PDGFR) and its downstream effector pathway, PI3K/AKT/mTOR, are frequently amplified in DIPG, and potential therapies targeting this pathway have emerged. However, the addition of targeted single agents has not been found to improve clinical outcomes in DIPG, and targeting this pathway alone has produced insufficient clinical responses in multiple malignancies investigated, including lung, endometrial, and bladder cancers. Acquired resistance also seems inevitable. Activation of the Ras/Raf/MEK/ERK pathway, which shares many nodes of cross talk with the PI3K/AKT pathway, has been implicated in the development of resistance. In the present study, perifosine, a PI3K/AKT pathway inhibitor, and trametinib, a MEK inhibitor, were combined, and their therapeutic efficacy on DIPG cells was assessed. Growth delay assays were performed with each drug individually or in combination. Here, we show that dual inhibition of PI3K/AKT and MEK/ERK pathways synergistically reduced cell viability. We also reveal that trametinib induced AKT phosphorylation in DIPG cells that could not be effectively attenuated by the addition of perifosine, likely due to the activation of other compensatory mechanisms. The synergistic reduction in cell viability was through the pronounced induction of apoptosis, with some effect from cell cycle arrest. We conclude that the concurrent inhibition of the PI3K/AKT and MEK/ERK pathways may be a potential therapeutic strategy for DIPG.     

Inhibition of aminoacyl-tRNA synthetases by p-chloroamphetamine and its role in protein synthesis inhibition

p-Chloroamphetamine inhibited to some degree all amino acid-dependent pyrophosphate-exchange activities which could be detected in a rabbit reticulocyte extract. A detailed kinetic analysis of the reaction catalyzed by reticulocyte leucyl-tRNA synthetase demonstrated that the inhibitor affected only amino acid binding. Less rigorous studies of other synthetases from both rabbit reticulocyte and Escherichia coli could be similarly interpreted, suggesting that this compound interacts in a common manner with these several enzymes. The contribution of such effects to the inhibition of protein synthesis by the drug was investigated using cell-free translation systems in which rates of amino acid incorporation were limited to varying degrees by the synthesis and availability of aminoacyl-tRNA. In a wheat germ system programmed with globin mRNA, in which levels of amino acids and aminoacyl-tRNAs were shown to limit the rate of protein synthesis, the inhibition produced by p-chloroamphetamine could be partially reversed by increasing the concentration of the limiting amino acid. In a reticulocyte lysate, in which amino acid concentrations were not limiting, inhibition failed to show an amino acid-reversible component. Thus, while the inhibition of aminoacyl-tRNA synthetases by amphetamines can be shown in some cases to play a role in the effects of these compounds on in vitro protein synthesis, other sites of interference with initiation and/ or elongation reactions may predominate, depending on the construction of the system under study.     

Physical growth and development, neurological maturation and behavioral functioning in two Ecuadorian Andean communities in which goiter is endemic. I. Outline of the problem of endemic goiter and cretinism. Physical growth and neurological maturation in the adult population of La Esperanza

Animal experiments indicate that chronic dietary iodine deficiency can have a marked effect on neurological maturation and the consequent behavioral capacities of the individuals so affected. Goiter, enlargement of the thyroid gland, is hyperendemic throughout the Andean region of Ecuador and is associated with an insufficient dietary intake of iodine. In these populations there is also a strikingly high prevalence of gross mental and physical developmental retardation that has been classified under the rubric of “endemic cretinism.” Anthropometric measurements and Bender Gestalt productions were collected from 44 deaf-mute “cretin” and 344 “normal” adult individuals in the parroquia of La Esperanza. The anthropometric data were generally comparable to that of other Ecuadorian Andean populations which have been studied. The deaf-mute “cretin” group showed retardation in physical growth and marked retardation in visual motor maturation compared to the “normal” population. Seventeen percent of the “normal” population showed clear signs of neurological deficit in visual motor perception. The data suggest that in addition to the large number of grossly retarded “cretins” in these populations, many of the “normal” individuals show some degree of neurological deficit, thus forming a continuum from frankly retarded, through moderately and slightly affected, to normal individuals. The socio-cultural adaptations to the presence of a large number of retarded individuals in the community are discussed.     

Beta-lactam antibiotics are multipotent agents to combat neurological diseases

Recently, Rothstein et al. reported that beta-lactam antibiotics, including penicillin and ceftriaxone, are potential therapeutic drugs to treat some neurological disorders, e.g., amyotrophic lateral sclerosis (ALS), by modulating the expression of glutamate transporter GLT1 via gene activation. However, considering the facts that: (i) many neurological diseases (including ALS) are associated with transition metal ions and redox stress, and ALS can be efficiently prevented by metal chelators, e.g., diethyl-dithiocarbamate (DDC); (ii) beta-lactam antibiotics have long been known as metal chelators, we argue that the beneficial effect of beta-lactam antibiotics on ALS likely involves Cu(II)-attenuating ability. This is partially supported by our theoretical calculations.     

Diazepam Increases γ-Aminobutyric Acid in Human Cerebrospinal Fluid

In 11 neurological patients, levels of the inhibitory neurotransmitter gamma-aminobutyric acid (GABA) were determined in cerebrospinal fluid (CSF) before and 1, 3, 5, and 8 min after intravenous injection of diazepam (2 or 5 mg). GABA levels increased progressively after intravenous injection of 5 but not 2 mg of the benzodiazepine, the differences from preinjection values being significant at 3, 5, and 8 min. Furthermore, when relative CSF GABA alterations determined after injection of diazepam were compared to those determined in sequential CSF aliquots of 10 patients without diazepam injection, mean GABA increases after diazepam were significantly different from controls in all CSF fractions. The data suggest that, in addition to its well-known effects on postsynaptic GABA function, diazepam may exert effects on endogenous GABA concentrations and/or on GABA release in the human CNS as reflected by elevation of GABA levels in human CSF.     

Expression of Myelin Proteolipid Protein and Basic Protein in Normal and Dysmyelinating Mutant Mice

Expression of myelin proteins was studied in the brains of 21-day-old normal mice and three dysmyelinating mutants-jimpy, quaking, and shiverer. Total brain polyribosomes and poly(A)+ mRNA were translated in two cell-free systems and the levels of synthesis of the myelin basic proteins (MBPs) and proteolipid protein (PLP) were determined. Synthesis of the MBPs in quaking homozygotes was at or above normal levels but PLP synthesis was significantly reduced to approximately 15% of control values, indicating independent effects on the expression of these proteins in this mutant. Immunoblot analysis of 21-day-old quaking brain homogenates showed a reduction in the steady-state levels of MBPs and PLP, suggesting a failure of newly synthesized MBPs to be incorporated into a stable membrane structure such as myelin. In the shiverer mutant very little synthesis of MBPs was observed, whereas greater synthesis of PLP occurred (approximately 50% of control). Almost no MBP, and low levels of PLP, were detected in the immunoblots, suggesting the possibility of a partial failure of PLP to be assembled into myelin in shiverer. In the jimpy mutant, low levels of MBP synthesis were observed in vitro (approximately 26% of controls) and very little synthesis of PLP was evident. The immunoblots of 21-day jimpy brain homogenates revealed no appreciable steady-state levels of PLP or MBP, again indicating that most newly synthesized MBPs were not incorporated into a stable membrane structure in this mutant. In sum, the data show that in the three cases examined, the mutation appears to affect the expression of the MBPs and PLP independently. Furthermore, regardless of their absolute levels of synthesis these proteins may or may not be assembled into myelin.     

A CHIPotle in physiology and disease

The carboxy-terminus of Hsc70 interacting protein (CHIP) is known to function as a chaperone associated E3 ligase for several proteins and regulates a variety of physiological processes. Being a connecting link between molecular chaperones and 26S proteasomes, it is widely regarded as the central player in the cellular protein quality control system. Recent analyses have provided new insights on the biochemical and functional dynamics of CHIP. In this review article, we give a comprehensive account of our current knowledge on the biology of CHIP, which apart from shedding light on fundamental biological questions promises to provide a potential target for therapeutic intervention.     

Copper mediated neurological disorder: Visions into amyotrophic lateral sclerosis,Alzheimer and Menkes disease

Copper (Cu) is a vital redox dynamic metal that is possibly poisonous in superfluous. Metals can traditionally or intricately cause propagation in reactive oxygen species (ROS) accretion in cells and this may effect in programmed cell death. Accumulation of Cu causes necrosis that looks to be facilitated by DNA damage, followed by activation of P53. Cu dyshomeostasis has also been concerned in neurodegenerative disorders such as Alzheimer, Amyotrophic lateral sclerosis (ALS) or Menkes disease and is directly related to neurodegenerative syndrome that usually produces senile dementia. These mortal syndromes are closely related with an immense damage of neurons and synaptic failure in the brain. This review focuses on copper mediated neurological disorders with insights into amyotrophic lateral sclerosis, Alzheimer and Menkes disease.     

Reevaluation of Phosphorylation Sites in the Parkinson Disease-associated Leucine-rich Repeat Kinase 2

Mutations in the leucine-rich repeat kinase 2 (LRRK2) gene have been identified as an important cause of late-onset, autosomal dominant familial Parkinson disease and contribute to sporadic Parkinson disease. LRRK2 is a large complex protein with multiple functional domains, including a Roc-GTPase, protein kinase, and multiple protein-protein interaction domains. Previous studies have suggested an important role for kinase activity in LRRK2-induced neuronal toxicity and inclusion body formation. Disease-associated mutations in LRRK2 also tend to increase kinase activity. Thus, enhanced kinase activity may therefore underlie LRRK2-linked disease. Similar to the closely related mixed-lineage kinases, LRRK2 can undergo autophosphorylation in vitro. Three putative autophosphorylation sites (Thr-2031, Ser-2032, and Thr-2035) have been identified within the activation segment of the LRRK2 kinase domain based on sequence homology to mixed-lineage kinases. Phosphorylation at one or more of these sites is critical for the kinase activity of LRRK2. Sensitive phopho-specific antibodies to each of these three sites have been developed and validated by ELISA, dot-blot, and Western blot analysis. Using these antibodies, we have found that all three putative sites are phosphorylated in LRRK2, and Ser-2032 and Thr-2035 are the two important sites that regulate LRRK2 kinase activity.     

Membrane-type Matrix Metalloproteinase-3 Regulates Neuronal Responsiveness to Myelin through Nogo-66 Receptor 1 Cleavage

Nogo-66 receptor 1 (NgR1) is a glycosylphosphatidylinositol-anchored receptor for myelin-associated inhibitors that restricts plasticity and axonal regrowth in the CNS. NgR1 is cleaved from the cell surface of SH-SY5Y neuroblastoma cells in a metalloproteinase-dependent manner; however, the mechanism and physiological consequence of NgR1 shedding have not been explored. We now demonstrate that NgR1 is shed from multiple populations of primary neurons. Through a loss-of-function approach, we found that membrane-type matrix metalloproteinase-3 (MT3-MMP) regulates endogenous NgR1 shedding in primary neurons. Neuronal knockdown of MT3-MMP resulted in the accumulation of NgR1 at the cell surface and reduced the accumulation of the NgR1 cleavage fragment in medium conditioned by cortical neurons. Recombinant MT1-, MT2-, MT3-, and MT5-MMPs promoted NgR1 shedding from the surface of primary neurons, and this treatment rendered neurons resistant to myelin-associated inhibitors. Introduction of a cleavage-resistant form of NgR1 reconstitutes the neuronal response to these inhibitors, demonstrating that specific metalloproteinases attenuate neuronal responses to myelin in an NgR1-dependent manner.