Download Roux P, Blenis J. ERK and p38 MAPK

Extracellular HSP70
Brain ischemia, or stroke, claims the lives of 150,000 Americans each year, making it the
third leading cause of death in the United States (Rosamond et al., 2003). In a typical nonhemorrhagic stroke, an embolis or thrombus occludes a blood vessel causing hypoxia, glucose
deprivation, and buildup of toxins. The only FDA approved treatments revolve around removing
the occlusion in order to reperfuse ischemic tissue. This involves either mechanical
thrombecotmy or chemical thrombolysis via tissue plasminogen activator. In either case, the
therapeutic window to perform these procedures is quite small and less than 1% of eligible
patients receive these therapies. Even following aggressive rehabilitation, patients experience a
wide and often unpredictable range of outcomes. Enhancing our ability to predict outcomes and
provide neuroprotective agents to patients during and after ischemic injury requires a far more
comprehensive understanding of the neuronal stress response in order to develop new drugs to
protect the brain from ischemia and minimize induced cell death.
The McLaughlin lab investigates a process known as ischemic preconditioning, in which
cells exposed to a sublethal event can gain temporary resistance to subsequent hypoxia or
toxicity. It has been observed that those who suffer from transient ischemic attacks (TIAs)
shortly before a stroke display decreased morbidity and less cell death than individuals who have
no history of TIA. Thus, cells have an endogenous neuroprotective mechanism that can be
studied for eventual design of more effective stroke drug targets. The presence of molecules
associated with neuroprotection could also be useful markers of the level of ischemic stress for
and help predict the outcome of a stroke.
The McLaughlin lab uses several in vivo and in vitro models of ischemia and
preconditioning. In particular, the lab’s first in vitro model uses mixed cultures of rat cortical
neurons and glia given 90 minutes of oxygen and glucose deprivation along with KCN, an
electron transport chain complex IV inhibitor. When given sixty minutes of an excitotoxic level
of NMDA twenty-four hours later, preconditioned cells experienced much less cell death than
those not given preconditioning. In surviving cells, molecules normally associated with cell
death such as cleaved caspase 3 and ROS actually generate part of the protective cell signaling
pathways essential for survival (McLaughlin et al., 2003). Importantly, upregulation of the 70
kDa heat shock protein, HSP70, is necessary but not sufficient to induce protection. That is, all
cells induce HSP70 with preconditioning, but only half of these survive. Maximal expression of
HSP70 occurs twenty-four hours after preconditioning and plays a vital role in refolding
polypeptides and targeting proteins that are damaged beyond repair for subsequent proteasomal
degradation.
HSP70 functions with other molecules to determine the fate of client proteins. For
instance, binding of the Carbxy-terminus of HSC70 interacting protein (CHIP) to HSP70 blocks
the ATPase activity of HSP70, thereby attenuating HSP70’s substrate affinity and preventing
protein refolding. Given CHIP’s role as an E3-ubiquitin ligase, CHIP will also promote the
tagging of client proteins with ubiquitin, thereby leading to their proteasomal degredation
(Hohfeld et al., 2001). Balancing protein refolding and protein degradation is an essential part of
the cell’s protein quality control mechanism. The failure to curb the buildup of misfolded and
damaged proteins in the form of protein aggregates is a primary cause underlying the activation
of cell death pathways.
Unfortunately, the neuron-glia mixed cell culture is a difficult model with which to
elucidate the specific role of HSP70 in specifically neurons. While the mixed culture provides a
more accurate brain-like environment, the data can be misleading due to the vastly different
behavior of different cell types. Consequently, the McLaughlin lab has recently gathered new
data on HSP70 in a new, neuron enriched preconditioning model. Here, neuron cultures were
preconditioned with five minutes of oxygen glucose deprivation per day over three days. This is
intended to mimic repeated bouts of transient ischemia. Twenty-four hours later, the cells were
given ninety minutes of hypoxia, an ordinarily lethal event. In this model, preconditioning did
not cause the intracellular level of HSP70 to change appreciably. Instead, HSP70 is found in
large quantities in the extracellular media.
Release of HSP70 is a common but poorly understood phenomenon in other pathologies,
but it is only just being investigated as it pertains to hypoxic brain tissue (Asea et al., 2005). For
example, HSP70 has long been implicated as a signaling molecule for the inflammatory immune
response and as a marker of cell compromise (Anjea et al., 2006). As such, it is key evidence for
the immunology “danger hypothesis” which proposes the immune system is capable of
recognizing self-originating danger signals which target a local immune response. Here,
extracellular HSP70 has been found to act as an endogenous adjuvant, signaling the maturation
of antigen presenting dendritic cells (Vabulas et al., 2002).
Within the realm of neuroprotection, the role of HSP70 has been investigated in glioma
and neuroblastoma cells (Guzhova et al., 2001). It was shown that glioma cells export HSP70
into the extracellular media under both normal and heat shock conditions. Under heat shock
stress, not only is the release of HSP70 elevated, but neuroblastoma cells that take up HSP70 are
more resistant to cell death. The link between protection afforded to neuroblastoma cells from
HSP70 and the increased release of HSP70 by glioma cells under precisely the same conditions
is a powerful connection between extracellular HSP70 and neuroprotective pathways. The
mechanism for the release of this large protein and its interaction in the extracellular space still
has yet to be determined. Additionally, the connection between glial release of HSP70 and
neuronal uptake and subsequent protection has not yet been demonstrated in healthy neuron
cultures. It has, however, been hinted at by our preliminary data and thus is an exciting prospect
for furthering the study of neuronal preconditioning.
The primary goal of preconditioning studies is to establish ways to activate the benefits of
preconditioning without the obvious dangers of recurring, transient ischemia. Having established
that cells secrete protective molecules, identification of the essential components in the media
could uncover a mechanism for cells to survive hypoxic events. Ultimately, this could become a
route for mimicking preconditioning in a therapeutic setting. My thesis will follow in the
footsteps of such preconditioning studies in order to determine whether HSP70 signaling can be
used to provide mimicked preconditioning. The first experiments of my thesis project will
investigate the link between the buildup of HSP 70 extracellularly and improvements in neuron
survival during hypoxia. I will then evaluate how the release of HSP70 is different in neuronrich, neuron-glia mixed, and glia only cultures following preconditioning. It will then become
important to investigate the source of this specific preconditioning response. This will involve
investigating whether the typical blocks to preconditioning such as antioxidants or caspase
inhibitors also knock down extracellular HSP70. Also, the binding partners of HSP70 will need
to be analyzed as they correlate with the level of protection.
REFERENCES
Anjea R, Odoms K, Dunsmore K, Shanley T, Wong H. Extracellular Heat Shock Protein-70
Induces Endotoxin Tolerance in THP-1 Cells. J. Immunol. 177: 7184-7192, 2006.
Asea, A., and SK Calderwood. "Regulation of Signal Transduction by Intracellular and
Extracellular HSP70." Molecular Chaperones and Cell Signaling. New York:
Cambridge, 2005. 133-39.
Bijur, G. N., and Jope, R. S. Opposing actions of phosphatidylinositol 3-kinase and glycogen
synthase kinase-3{beta} in the regulation of HSF-1 activity J Neurochem 75: 2401-2408,
2000.
Guzhova I, Kislyakova K, Moskaliova O, Fridlanskaya I, Tytell M, Cheetham M, Margulis B. In
vitro studies show that Hsp70 can be released by glia and that exogenous Hsp70 can
enhance neuronal stress tolerance. Brain Res 914: 66-73, 2001.
Hohfeld J, Cyr D, Patterson C. From the cradle to the grave: molecular chaperones that may
choose between folding and degredation. EMBO Rep. 21: 885-890, 2001.
McLaughlin B, Hartnett KA, Erhardt JA, Legos JJ, White RF, Barone FC, Aizenman E. Caspase
3 activation is essential for neuroprotection in preconditioning. Proc Natl Acad Sci USA
100: 715–720, 2003.
Rosamond, et al. Heart Disease and Stroke Statistics_2008 Update: A Report From the American
Heart Association Statistics Committee and Stroke Statistics Subcommittee. Circulation.
117: 25-146, 2008.
Roux P, Blenis J. ERK and p38 MAPK-Activated Protein Kinases: a Family of Protein Kinases
with Diverse Biological Functions. Microbiol Mol Biol R. 68: 320-324, 2004.
Turman M, Kahn D, Rosenfeld S, Apple C, Bates C. Characterization of Human Proximal
Tubular Cells after Hypoxic Preconditioning: Constitutive and Hypoxia-Induced
Expression of Heat Shock Proteins HSP70 (A, B, and C), HSC70, and HSP90. Biochem
Mol Med. 60: 49-58, 1997.
Vabulas R, Ahman-Nejad P, Ghose S, Kirschning C, Issels R, Wagner H. HSP70 as exogenous
stimulus of the toll/interleukin-1 receptor signal pathway. J. Biol. Chem. 277: 1510715112, 2002.