Calpain proteases play an undisputed but incompletely characterized role in neuronal injury. Calpain inhibitors are protective to varying degrees in animal models of stroke (Bartus et al. 1994
;Markgraf et al. 1998
;Tsubokawa et al. 2006
), traumatic brain injury (Saatman et al. 1996
;Buki et al. 2003
;Ai et al. 2007
), Alzheimer's disease (Trinchese et al. 2008
), and Parkinson's disease (Crocker et al. 2003
). Additionally, calpain-generated α-spectrin breakdown products in cerebrospinal fluid are associated with traumatic brain injury severity in humans (Pineda et al. 2007
). However, despite the early promise of calpain inhibitors, data on long-term outcome in animal studies remains sparse and successful clinical studies in humans have yet to materialize. It is clear that multiple cell death mechanisms contribute to the spatiotemporal profile of neuronal loss in vivo
and a better understanding of the participation of calpain proteases in the pathogenesis of different injury paradigms is required for the development of targeted treatments. In this study we set out to: 1) identify whether the major activation of calpain proteases in the cytoplasm occurs up or downstream of glutamate-induced intracellular calcium deregulation and 2) determine whether calpain activity is required for excitotoxic calcium deregulation or cell death in cortical neurons.
Using a genetically encoded calpain sensor, we demonstrated that somal cytoplasmic calpain activity becomes detectable via fluorescent substrate within a very narrowly defined time window at approximately 40 min after the onset of DCD (). Once initiated, cleavage of the FRET sensor progressed at a constant rate until the loss of FRET was complete (), suggesting that this event represented `major' unchecked calpain activation.
The mechanisms of calpain activation in intact neurons are incompletely understood. In vitro
, both μ-calpain (3-50 μM) and m-calpain (0.4-0.8 mM) require supra-physiological calcium concentrations for activation (Goll et al. 2003
). Furthermore, the constitutive expression of an endogenous inhibitor protein, calpastatin, is predicted to limit calpain activity in response to transient pulses of high calcium while ultimately succumbing to proteolysis only if elevated calcium is sustained (Goll et al. 2003
). Thus, unchecked calpain activation in neurons is expected only after a period of prolonged calcium deregulation. Our data in are consistent with a model where the calpastatin-mediated check on calpain activity is relieved following 40 min of sustained DCD, allowing unimpeded calpain proteolysis of cytoplasmic substrates to progress. Because calpain is reportedly sequestered in subcellular membrane fractions including mitochondria (Hewitt et al. 1998
;Garcia et al. 2005
;Ozaki et al. 2007
;Kar et al. 2008
), an alternative possibility is that the delayed but distinct cytoplasmic calpain activation with respect to DCD represents a subcellular translocation event that brings active calpain in contact with the fluorescent sensor.
Spectrin breakdown products and a decrease in FRET were reported in pYSCS-transfected hippocampal neuron dendritic spines within 5 min of glutamate treatment (Vanderklish et al. 2000
). In our experiments, glutamate treatment of cortical neurons caused an initial calpain-independent, reversible decrease in FRET, suggesting that conformational changes of the probe affect FRET efficiency. While the spectral unmixing and three-wavelength FRET calculation avoided errors originating from spectral bleed-through and variations of FRET acceptor fluorescence yield, these calculations also decreased signal-to-noise ratio. Due to the high calpain-insensitive background we cannot confirm or reject the possibility of localized calpain activation (e.g. in sub-plasma membrane regions) prior to DCD. However, our data do not support a generalized early increase in calpain activity.
Here we found that calpeptin effectively inhibited calpain-mediated α-spectrin breakdown () without decreasing the incidence or onset of glutamate-induced DCD (, ). In fact, a small but significant increase in the incidence of DCD was detected when large numbers of neurons were considered, possibly due to inhibition of local calpain processing of NMDA NR2 subunits (Wu et al. 2005
). Calpeptin was also unable to improve the rate of sustained [Ca2+
recovery following glutamate-triggered DCD-like reversible calcium elevations ( and Supplemental Movie 3
), or protect against necrotic cell death (). Apoptotic death was not observed in the present study. Because calpain inhibition protects against neuronal apoptosis (Ray et al. 2006
;Cao et al. 2007
), we hypothesize that the percentage of neurons undergoing apoptosis vs
. necrosis in response to a given excitotoxic challenge ultimately determines the ability of calpain inhibitors to effectively delay glutamate receptor-related injury.
Our primary findings contrast with a recent report that overexpression of calpastatin inhibits DCD in cerebellar granule neurons by preventing calpain-mediated NCX3 cleavage (Bano et al. 2005
). The onset of DCD in cerebellar granule neurons is considerably delayed relative to cortical neurons (e.g. compare to (Bano et al. 2005
)) and it is possible that different mechanisms (apoptotic vs. necrotic) contribute to injury. The expression of NCX3 relative to NCX1 also differs between cortical and cerebellar granule neurons (Kiedrowski et al. 2004
). We failed to see the appearance of 58-60 kD NCX3 breakdown products or a degradation of full-length NCX3 in glutamate-treated cortical neurons even after 1.5 hr of continuous glutamate treatment (and ~1 hr after DCD, ). The NCX antagonist KB-R7943 delayed rather than shortened the time to DCD (), suggesting that in our model NCX activity following glutamate exposure was deleterious rather than protective. Calculation of the ΔG for Na+
exchange () revealed that NCX operates in reverse mode favoring Ca2+
entry during glutamate exposure, in agreement with previous reports (Kiedrowski et al. 1994
;Hoyt et al. 1998
;Czyz and Kiedrowski 2002
;Kiedrowski et al. 2004
;Araujo et al. 2007
;Storozhevykh et al. 2007
). Consistent with reverse mode NCX operation, KB-R7943 acutely decreased intracellular Ca2+
when administered after glutamate addition (). Thus, NCX failure cannot explain the sudden loss of calcium homeostasis in glutamate-exposed cortical neurons although failure of other calcium extrusion pathways (e.g. the PMCA (Pottorf et al. 2006
)) may contribute.
In summary, our studies for the first time imaged somal calpain activation relative to intracellular calcium changes in live glutamate-treated neurons, revealing the onset of major activity following ~40 min of sustained DCD. Modifications of this technique, particularly ones that reduce pH-sensitive and calpeptin-insensitive changes in fluorescence, should be useful for characterizing calpain activation in less acute models of neuronal injury where calpain inhibitors are protective.