Shlomit Erlich

Rapamycin is a neuroprotective treatment for traumatic brain injury

The mammalian target of rapamycin, commonly known as mTOR, is a serine/threonine kinase that regulates translation and cell division. mTOR integrates input from multiple upstream signals, including growth factors and nutrients to regulate protein synthesis. Inhibition of mTOR leads to cell cycle arrest, inhibition of cell proliferation, immunosuppression and induction of autophagy. Autophagy, a bulk degradation of sub-cellular constituents, is a process that keeps the balance between protein synthesis and protein degradation and is induced upon amino acids deprivation. Rapamycin, mTOR signaling inhibitor, mimics amino acid and, to some extent, growth factor deprivation. In the present study we examined the effect of rapamycin, on the outcome of mice after brain injury. Our results demonstrate that rapamycin injection 4 h following closed head injury significantly improved functional recovery as manifested by changes in the Neurological Severity Score, a neurobehavioral testing. To verify the activity of the injected rapamycin, we demonstrated that it inhibits p70S6K phosphorylation, reduces microglia/macrophages activation and increases the number of surviving neurons at the site of injury. We therefore suggest that rapamycin is neuroprotective following traumatic brain injury and as a drug used in the clinic for other indications, we propose that further studies on rapamycin should be conducted in order to consider it as a novel therapy for traumatic brain injury.

Differential Interactions Between Beclin 1 and Bcl-2 Family Members

Autophagy, a cellular degradation system, promotes both cell death and survival. The interaction between Bcl-2 family proteins and Beclin 1, a Bcl-2 interacting protein that promotes autophagy, can mediate crosstalk between autophagy and apoptosis. We investigated the interaction between anti-and pro-apoptotic Bcl-2 proteins with Beclin 1. Our results show that Beclin 1 directly interacts with Bcl-2, Bcl-xL, Bcl-w and to a lesser extent with Mcl-1. Beclin 1 does not bind the pro-apoptotic Bcl-2 proteins. The interaction between Beclin 1 and the anti-apoptotic protein Bcl-xL was inhibited by BH3-only proteins, but not by multi-domain proteins. Sequence alignment and structural modeling suggest that Beclin 1 contains a putative BH3-like domain which may interact with the hydrophobic grove of Bcl-xL. Mutation of the Beclin 1 amino acids predicted to mediate this interaction inhibited the association of Beclin 1 with Bcl-xL. Our results suggest that BH3 only proapoptotic Bcl-2 proteins may modulate the interactions between Bcl-xL and Beclin 1.

ErbB-4 activation promotes neurite outgrowth in PC12 cells.

Abstract: Neu differentiation factor (NDF; also known as neuregulin) induces a pleiotropic cellular response that is cell type‐dependent. NDF and its receptor ErbB‐4 are highly expressed in neurons, implying important roles in neuronal cell functions. In the present study we demonstrate that ErbB‐4 receptors expressed in PC12 cells mediate NDF‐induced signals and neurite outgrowth that are indistinguishable from those mediated by the nerve growth factor‐activated Trk receptors. In PC12‐ErbB‐4 cells but not in PC12 cells, NDF induced an initial weak mitogenic signal and subsequently neurite outgrowth. The NDF‐induced differentiation in PC12‐ErbB‐4 cells was mimicked by the pan‐ErbB ligand betacellulin but not by other epidermal growth factor‐like ligands. Thus, NDF and betacellulin mediate similar activities through the ErbB‐4 receptor. Indeed, only these ligands induced strong phosphorylation of the ErbB‐4 receptors. Neurite outgrowth induced by NDF in PC12‐ErbB‐4 cells was accompanied by sustained activation of mitogen‐activated protein kinase (MAPK) and induction of the neural differentiation marker GAP‐43. Inhibition of the MAPK kinase MEK or of protein kinase C (PKC) blocked NDF‐induced differentiation, whereas elevation of cyclic AMP levels enhanced the response. Taken together, these results indicate that neurite outgrowth induced by ErbB‐4 in PC12 cells requires MAPK and PKC signaling networks.

Neurodegeneration Induces Upregulation of Beclin 1

Autophagy, a bulk degradation of subcellular constituents, is activated in normal cell growth and development, and represents the major pathway by which the cell maintains a balance between protein synthesis and protein degradation. Autophagy was documented in several neurodegenerative diseases, and under stress conditions the autophagic process can lead to cell death (type II programmed cell death). Beclin 1 is a Bcl-2 interacting protein that was previously found to promote autophagy. We have used Beclin 1 protein as a marker for autophagy following traumatic brain injury in mice. We demonstrated a dramatic elevation in Beclin 1 levels near the injury site. Interestingly Beclin 1 elevation starts at early stages post injury (4 h) in neurons and 3 days later in astrocytes. In both cell types it lasts for at least three weeks. Neuronal cells, but not astrocytes, that overexpress Beclin 1 may exhibit damaged DNA but without changes in nuclear morphology. These observations may indicate that not all the Beclin 1 overexpressing cells will die. The elevation of Beclin 1 at the site of injury may represent enhanced autophagy as a mechanism to discard injured cells and reduce damage to cells by disposing of injured components. Addenda to: Closed Head Injury Induces Upregulation of Beclin 1 at the Cortical Site of Injury T. Diskin, P. Tal-Or, S. Erlich, L. Mizrachy, A. Alexandrovich, E. Shohami and R. Pinkas-Kramarski J Neurotrauma 2005; 22:750-62

ErbB-4 activation inhibits apoptosis in PC12 cells.

Neuregulins, a large family of polypeptide growth factors, exert various distinctive effects in the nervous system. neuregulins and their receptors are widely expressed in neurons implying important roles in neuronal cell functions. Recently, we have shown that ErbB-4 receptors expressed in PC12 cells mediate neuregulin-induced differentiation. In the present study we demonstrate that in the PC12-ErbB-4 cells, neuregulin rescues cells from apoptosis induced by serum deprivation or tumor necrosis factor (TNF)alpha treatment. The neuregulin-induced survival is comparable to the effect mediated by the neurotrophic factor nerve growth factor (NGF). Both neuregulin and NGF protect cells from apoptosis induced by serum deprivation and TNF alpha treatment. Moreover, neuregulin like NGF induces the survival of neuronal differentiated PC12-ErbB-4 cells. The survival effect of neuregulin is probably mediated by the phosphoinositide 3-kinase (PI3K) and protein kinase B/Akt signaling pathways. Neuregulin induces the activation of PI3K and prolonged activation of protein kinase B/Akt. In addition, inhibition of the PI3K activity prevented the neuregulin-induced survival effect. Taken together, these results indicate that survival induced by neuregulin in PC12-ErbB-4 cells requires PI3K signaling networks.

Neuregulin induces sustained reactive oxygen species generation to mediate neuronal differentiation

Neuregulins (NRGs), which are highly expressed in the nervous system, bind and activate two receptor tyrosine kinases, ErbB-3 and ErbB-4. Recently, we have shown that ErbB-4 receptors expressed in PC12 cells mediate NRG-induced differentiation through the MAPK signaling pathway. Here we demonstrate that NRG induces an increase in the intracellular concentration of reactive oxygen species (ROS). N-acetylcysteine, a ROS scavenger, inhibited NRG-induced activation of Ras and Erk and PC12-ErbB-4 cell differentiation. These results suggest that ROS production is involved in NRG-mediated neuronal differentiation and that ROS can regulate activation of Ras and Erk. Constitutively active Ras enhanced ROS production and dominant negative Ras inhibited NRG-induced ROS production, suggesting, a positive regulatory loop between Ras and ROS. The mitogen, EGF, induced short-term ROS production whereas NRG and NGF, which induce cell differentiation, induced prolonged ROS production. These results strongly suggest that the kinetics of ROS production may determine whether the cells will differentiate or proliferate.

Closed Head Injury Induces Up-Regulation of ErbB-4 Receptor at the Site of Injury

ErbB-4 receptor tyrosine kinase and its ligand neu differentiation factor (NDF/neuregulin) are widely expressed in the brain. The closed head injury model was used to investigate the possible role of ErbB-4 receptor in neurodegeneration. It is demonstrated that levels of ErbB-4 are dramatically increased at the site of injury. Activated microglia/macrophages constitute the major population of cells with the highest receptor levels at the site of injury. In addition ErbB-4 expression after injury is elevated also in neurons but not in astrocytes. Confocal microscopy analysis suggests that the high level of ErbB-4 protein in activated microglia/macrophages is probably due to phagocytosis of neuronal cells. These findings show for the first time that ErbB-4 receptors play a role in brain responses to head trauma. Overexpression of ErbB-4 receptors may be important for directing activated microglia/macrophages to the lesion site.

Beclin 1 self‐association is independent of autophagy induction by amino acid deprivation and rapamycin treatment

Autophagy, a process of self‐digestion of cellular constituents, regulates the balance between protein synthesis and protein degradation. Beclin 1 represents an important component of the autophagic machinery. It interacts with proteins that positively regulate autophagy, such as Vps34, UVRAG, and Ambra1, as well as with anti‐apoptotic proteins such as Bcl‐2 via its BH3‐like domain to negatively regulate autophagy. Thus, Beclin 1 interactions with several proteins may regulate autophagy. To identify novel Beclin 1 interacting proteins, we utilized a GST‐Beclin 1 fusion protein. Using mass spectroscopic analysis, we identified Beclin 1 as a protein that interacts with GST‐Beclin 1. Further examination by cross linking and co‐immunoprecipitation experiments confirmed that Beclin 1 self‐interacts and that the coiled coil and the N‐terminal region of Beclin 1 contribute to its oligomerization. Importantly, overexpression of vps34, UVRAG, or Bcl‐xL, had no effect on Beclin 1 self‐interaction. Moreover, this self‐interaction was independent of autophagy induction by amino acid deprivation or rapamycin treatment. These results suggest that full‐length Beclin 1 is a stable oligomer under various conditions. Such an oligomer may provide a platform for further protein–protein interactions. J. Cell. Biochem. 110: 1262–1271, 2010. Published 2010 Wiley‐Liss, Inc.

Ligand-Independent Regulation of ErbB4 Receptor Phosphorylation by Activated Ras

The ErbB family of receptor tyrosine kinases regulates cell growth, differentiation and survival. Activation of the receptors is induced by specific growth factors in an autocrine, paracrine or juxtacrine manner. The activated ErbB receptors turn on a large variety of signaling cascades, including the prominent Ras‐dependent signaling pathways. The activated Ras can induce secretion of growth factors such as EGF and neuregulin, which activate their respective receptors. In the present study, we demonstrate for the first time that activated Ras can activate ErbB4 receptor in a ligand‐independent manner. Expression of constitutively active H‐Ras(12V), K‐Ras(12V) or N‐Ras(13V) in PC12‐ErbB4 cells induced ErbB4‐receptor phosphorylation, indicating that each of the most abundant Ras isoforms can induce receptor activation. NRG‐induced phosphorylation of ErbB4 receptor was blocked by the soluble ErbB4 receptor, which had no effect on the Ras‐induced receptor phosphorylation. Moreover, conditioned medium from H‐Ras(12V)‐transfected PC12‐ErbB4 cells had no effect on receptor phosphorylation. It thus indicates that Ras induces ErbB4 phosphorylation in a ligand‐independent manner. Each of the Ras effector domain mutants, H‐Ras(12V)S35, H‐Ras(12V)C40, and H‐Ras(12V)G37, which respectively activate Raf1, PI3K, and RalGEF, induced a small but significant receptor phosphorylation. The PI3K inhibitor LY294002 and the MEK inhibitor PD98059 caused a partial inhibition of the Ras‐induced ErbB4 receptor phosphorylation. Using a mutant ErbB4 receptor, which lacks kinase activity, we demonstrated that the Ras‐mediated ErbB4 phosphorylation depends on the kinase activity of the receptor and facilitates ligand‐independent neurite outgrowth in PC12‐ErbB4 cells. These experiments demonstrate a novel mechanism controlling ErbB receptor activation. Ras induces ErbB4 receptor phosphorylation in a non‐autocrine manner and this activation depends on multiple Ras effector pathways and on ErbB4 kinase activity.