JOurn.31 ofr in.nerr.3du.3a- R-ese-arch
..Oluin 6, issue 1 - Sept, n-irer 21i11:14
Functional Degradomics of Proteolytic Processing of Cytoskeletal Proteins
By Caspase-3 and Calpain Following Traumatic Brain Injury in the Immature
Jada Aikman, Jeremy Flint, Barbara O'Steen, Clair Ringger, Kevin Wang, Ron Hayes, and Jose Pineda
Traumatic brain injury (TBI) is a leading cause of death and disability among children and young adults
(MacKenzie, 2000; Prins, 2003; Hoyert, 2001). This is especially true for children under the age of four, a group
in which TBI results in poor outcome in greater than 50% of the cases (Levin, 1992). Our understanding of
the mechanisms of damage to the immature brain after TBI remains quite limited (Adelson, 1999; Lea, 2001). It
is likely that the mechanisms of cellular injury and death in the immature brain are different from those described
to date in the mature brain. That such differences exist seems intuitive given critical periods of vulnerability for
the developing nervous system of animals and humans (Rice, 2000).
After TBI, brain cells can deteriorate following more than one pathway. Proteolytic processing plays an important
role in regulating a wide range of important cellular functions, including processing of cytoskeletal proteins.
Integrity of the cytoskeleton is essential for cell survival and function, and possible functional consequences
of cytoskeletal loss include cell death. Cysteine proteases such as calpain are highly involved in the breakdown
of cytoskeletal proteins (Kampfl et al 1996, Posmantur, et al. 1998). Loss of cytoskeletal proteins such as
spectrin and tau is an important characteristic in a variety of acute central nervous system injuries
including ischemia, spinal cord injury, and traumatic brain injury (Wang, 2000). Virtually all of these
investigations have been done in mature brain animal models. The literature contains extensive information on
the proteolytic degradation of two important cytoskeletal proteins, aII-spectrin and tau, after TBI in the adult
brain. In the mature brain, calpain and caspase-3 generate signature 145 kDa and 120 kDa BDP of all-
spectrin (SBDPs) respectively (Pike et al, 2000; Pike et al 1998; Beer et al 2000; Pike et al, 2001). Notably,
The proteolytic processing of the microtubule-associated protein tau after TBI is much less characterized. By
contrast, little is known about the characteristics and relevance of these important processes in the immature
brain (Figure 1).
Figure 1: Simple Schematic of secondary pathology of brain injury.
For this study, we hypothesized that TBI leads to over-activation of two major cytosolic protease systems in
the immature brain: calpain and caspase-3. Distinct proteolytic products readily detectable in injured brain
tissue result from degradation of the cytoskeletal protein all-spectrin and microtubule-associated protein tau
by caplain and caspase-3. Our work examined TBI-induced proteolytic processing of these cytoskeletal proteins
after TBI in the immature rat brain.
Postnatal day (PND) 9 male Sprague-Dawley rats (19-25 g) were randomized into one of three groups:
controlled cortical impact (n=8), sham-craniotomy control (n=8), and nafve (n=6). This protocol was approved
by the Institutional Animal Care and Use Committee of the University of Florida in compliance with NIH guidelines.
Animals were anesthetized via nosecone with 4% isoflourane. After a right lateral craniotomy between lambda
and bregma was performed with the dura left intact, brain trauma was induced by impacting the right tempo-
parietal cortex (ipsilateral cortex) with a 3mm diameter aluminum impactor tip (speed of 3.23 m/s, dwell time of
150 ms). Compression depth was set at 1.3mm. Sham (craniotomy control) animals underwent a craniotomy
without controlled cortical impact. Core temperature was maintained at 37.5-38ooC. After the induction of
general anesthesia with Isoflourane, the animals were killed by decapitation at 24, 48 or 72 hours post TBI.
After collection, tissue samples were snap frozen with liquid N02 and stored at -80ooC. Brain tissue samples
were homogenized in Triton Buffer with the addition of a protease inhibitor to prevent natural activation of
proteases and subsequent artificial degradation of spectrin/tau during tissue processing. Protein concentrations
were determined by bicinchoninic acid protein assays. Protein balanced samples were prepared for western
blotting. Twenty micrograms of protein were added to each lane. The protein was resolved in either 4-12%
Tris-Glycine gels for SBDPs or 4-20% Tris-Glycine gels for tau breakdown products (TBDPs). After separation by
> Primar injur ..
TBDPl *TBDP2 SBDP1 SBDP2
SDS-PAGE, the proteins were transferred to PVDF membranes and probed with either Anti-_-II spectrin
breakdown product antibody 1:4000 for brain tissue or Anti-Tau 1 monoclonal antibody 1:2000 for brain tissue.
Semiquantitative evaluation of immunoreactivity as seen in Western Blotting was performed using computer-
assisted two-dimensional densitometric scanning on a computer using the public domain NIH program
Image. Relative band densities were expressed as arbitrary densitometric units and values are shown as
percent craniotomy controls and are given as mean ï¿½ SEM. Statistical analysis was performed using
Statviewï¿½. Differences were considered significant when p<0.05.
Cytoskeletal protein breakdown product (BDP) expression was detected at several timepoints. TBI produced
elevated levels of spectrin break down products (SBDPs). Both caspase and calpain produce _-II spectrin
breakdown products in the ipsilateral cortex following controlled cortical impact in PN9 rats. Significant increases
in the amount of all-SBDPs (p< 0.05) were noted at 24h post injury. At 48h post injury significant increases
were seen in the 145 kDa and 150 kDa. At 72h post injury only the levels of 145 kDa expression were
statistically significant (Figure 2). We also observed that TBI resulted in the robust generation of a 10 kDa TBDP
24 hours after TBI (Figure 2). Significant formation of additional TBDPs (17kDa, 18kDa and 26kDa) was noted at
48 to 72 hours post-injury.
, 150 k \4
Shls 24h 428 72h
Shm 4th h 74H
Nave Sham CCI
am 48 h
Figure 2. Time course for the production of a II-spectrin BDP's after controlled cortical impact in 9 day
old rats. Significant increases in the levels of the calpain specific proteolytic fragment (145 kDa)
were noted in the ipsilateral cortex (A) and hippocampus (B) at 24 (cotex and hippocampus), 48
hours (cortex) and 72 hours (cortex) post-controlled cortical impact. Significant increases in caspase-
3 specific BDP's were detected at 24 hours post injury in the ipsilateral cortex and hypoccampus (A and
B respectively). C: representative Western Blot gels for the ipsilateral at 24, 48 and 72 hours post
injury. Densitometry data are expressed as percentage of s ham values. N=8 per group (*=p<0.05).
Our experiments further emphasize the importance of exploring the effect of age and the relative contribution
of caspase-3 and calpain to the degradation of this important developmentally regulated microtubule-
associated protein after TBI.
As hypothesized, TBI results in significant formation of all-spectrin BDPs in brain tissue. Both caspase-3 and
calpain produce _-II spectrin breakdown products in the ipsilateral cortex and hippocampus following
controlled cortical impact in PN9 rats (Figure 2). Our experiments demonstrate the formation of both calpain
and caspase-3 specific BDPs of all-spectrin in the first 72 hours in the ipsilateral cortex. A similar pattern was
found in the hippocampus with significant increase in BDPs at 24 hours post-injury. No significant increases
were seen in the contralateral cortex or hippocampus therefore, for brevity, the data will not be addressed in
We conducted additional experiments to demonstrate that there is no significant formation of all-spectrin BDPs as
a result of our craniotomy procedure in our craniotomy control animals when compared with brain tissue
samples obtained from naive animals (data not shown). These experiments are the first to demonstrate the
formation of brain injury related _-II spectrin breakdown products after traumatic brain injury in the immature
brain. Our findings are consistent with the work of Bittigau et al using the weight-drop model of closed head
injury. The authors reported PND-7 rats showed increased caspase-3 (CPP 32)-like proteolytic activity and
apoptotic cell death as early as 6 hours, peaked at 24 hours, and subsided by 5 days. The severity of trauma
induced apoptosis in the brain of 3 to 30 day old rats was age dependent, demonstrating the increased
vulnerability of the younger brain to apoptotic cell death (Bittigau et al, 1999). It is important to note that
previous experiments in our laboratory provided evidence for a more prominent role of calpain over caspase-3
after TBI in the mature brain, as evidenced by a relatively higher ratio of calpain produced SBDP145 over caspase-
3-produced SBDP120 (SBDP145/SBDP120 = 12.5:1) in the cortex 24 hours after CCI (Pike et al, 1998). In
contrast, our preliminary results in the immature rat brain suggest a more balanced attack by capsase-3 and
calpain after TBI, as evidenced by the relatively low SBDP145/SBDP12 ratio (2.4:1) obtained 24 hours after 1.3
mm cortical impact in PND-9 rats (Table 1). Future studies will further confirm this observation at different
severities of injury in two developmental stages (PND 9 and PND 17).
Ratios for Relative Contributions of Caspase-3 and Calpain to SBDP Production.
145 120 Ratio
Hours After P9 Adul P9 Adul P9 Adul
24 4.5 +/- 25.0 +/- 1.9 +/- 2.0 4+- 2.4 12.5
48 3.4 +/- 30.0 +/- 0.8 +/- 2.0 +/- 4.6 15.0
72 0.9 +/- 18.5 +/- 0.7 +/- 3.2 +1- 1.2 5.8
Although there are no in vitro experiments in this experiment, it is important to note our in vitro tau
digestion experiments (data gained from a collaborating laboratory) demonstrating the formation of several
low molecular weight (LMW) tau BDPs (26 kDa, 18 kDa, 17 kDa and 10 kDa) after calpain proteolysis.
Importantly, these BDPs match up perfectly with the LMW Tau BDP's observed in the immature rat brain after
TBI. These data suggest the clear involvement of calpain in tau proteolyis in immature rat brain post-
TBI. Additionally, in vitro digestion of tau by caspase-3 yields a cluster of high molecular weight (HMW) fragments
of about 40-44 kDa, due to N-terminal truncation . Since the size of these HMW fragments overlapped with the
intact tau isoform bands (45-55 kDa), we suspected that these HMW tau BDPs, although present in immature
rat brain after TBI, could not be definitively identified (Figure 3). Our laboratory and a collaborating laboratory
are already in the process of raising antibodies specific to this caspase3-generated Tau BDP (40 kDa; Tau
BDP2), which can be used to unambiguously detect such caspase-3-mediated attack on tau. In parallel,
antibody specific to calpain generated Tau BDP1 (17 kDa) is also being raised for comparative immunoblotting
and immunohistochemical studies.
Sham 24h 48h 72h
3 - --- -CC I.qCsDA
0 . ---------------- . ------ . --------
Sham 24h 48h 72h
Figure 3. Time course for the production of tau BDP's after controlled cortical impact in 9 day old rats.
We observed that TBI resulted in the robust generation of a 10 kDa tau BDP 24 hours after TBI in
the ipsilateral cortex (A). Significant formation of additional BDPs (17kDa, 18kDa and 26kDa) was
noted at 48 and 72 hours post-injury (B). The formation of the se BDPs is characteristic of
calpain activation. C: representative Western Blot gel for the ipsilateral cortex at 24, 48 and 72
hours post injury. Densitometry data are expressed as percentage of sham values. N=8 per
There are challenges to modeling TBI in the immature brain. Our experiments do not intend to make direct
age comparisons between rodents and humans. Rather, the work will further define relevant components of
the pathophysiology of TBI that are influenced by brain development and will lay the ground for future
proposals aimed at studying such components of brain injury in children of different ages. To overcome the
problem posed by variations in biomechanics of brain injury at different developmental stages, future efforts
are already planned to assess two severities of injury in each age group. This approach will help us
differentiate between injury severity and age related variations in the response of the immature brain to TBI.
In summary, our findings emphasize the importance of further defining the role of calpain and caspase-3 in
the pathophysiology of TBI in the immature brain.
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