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. 2014 Dec 10:8:421.
doi: 10.3389/fncel.2014.00421. eCollection 2014.

Altering endoplasmic reticulum stress in a model of blast-induced traumatic brain injury controls cellular fate and ameliorates neuropsychiatric symptoms

Aric Flint Logsdon  1 , Ryan Coddington Turner  2 , Brandon Peter Lucke-Wold  2 , Matthew James Robson  3 , Zachary James Naser  4 , Kelly Elizabeth Smith  5 , Rae Reiko Matsumoto  6 , Jason Delwyn Huber  1 , Charles Lee Rosen  2
Affiliations

Altering endoplasmic reticulum stress in a model of blast-induced traumatic brain injury controls cellular fate and ameliorates neuropsychiatric symptoms

Aric Flint Logsdon et al. Front Cell Neurosci. .

Abstract

Neuronal injury following blast-induced traumatic brain injury (bTBI) increases the risk for neuropsychiatric disorders, yet the pathophysiology remains poorly understood. Blood-brain-barrier (BBB) disruption, endoplasmic reticulum (ER) stress, and apoptosis have all been implicated in bTBI. Microvessel compromise is a primary effect of bTBI and is postulated to cause subcellular secondary effects such as ER stress. What remains unclear is how these secondary effects progress to personality disorders in humans exposed to head trauma. To investigate this we exposed male rats to a clinically relevant bTBI model we have recently developed. The study examined initial BBB disruption using Evan's blue (EB), ER stress mechanisms, apoptosis and impulsive-like behavior measured with elevated plus maze (EPM). Large BBB openings were observed immediately following bTBI, and persisted for at least 6 h. Data showed increased mRNA abundance of stress response genes at 3 h, with subsequent increases in the ER stress markers C/EBP homologous protein (CHOP) and growth arrest and DNA damage-inducible protein 34 (GADD34) at 24 h. Caspase-12 and Caspase-3 were both cleaved at 24 h following bTBI. The ER stress inhibitor, salubrinal (SAL), was administered (1 mg/kg i.p.) to investigate its effects on neuronal injury and impulsive-like behavior associated with bTBI. SAL reduced CHOP protein expression, and diminished Caspase-3 cleavage, suggesting apoptosis attenuation. Interestingly, SAL also ameliorated impulsive-like behavior indicative of head trauma. These results suggest SAL plays a role in apoptosis regulation and the pathology of chronic disease. These observations provide evidence that bTBI involves ER stress and that the unfolded protein response (UPR) is a promising molecular target for the attenuation of neuronal injury.

Keywords: CHOP; apoptosis; blast-induced traumatic brain injury; blood-brain barrier; endoplasmic reticulum stress; prefrontal cortex; salubrinal.

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Figures

Figure 1
Figure 1
Schematic shows primary and secondary effects of blast injury. Blast-induced traumatic brain injury (bTBI) can burst brain microvessels and shear axons. Both primary effects lead to increased intracellular calcium levels which triggers a variety of secondary effects including endoplasmic reticulum (ER) stress. Endoplasmic reticulum stress activates the unfolded protein response (UPR) which consists of three separate adaptive arms that play a time-dependent role in maintaining cellular homeostasis. Following neural injury the protein kinase-like ER kinase (PERK)-mediated UPR is considered the acute phase adaptive arm. This mechanism, along with a link to ER-mediated apoptosis, is displayed with the proposed effects of salubrinal (SAL) included.
Figure 2
Figure 2
Blood-brain barrier (BBB) disruption observed in the contra coup brain after bTBI. (A) Blood-Brain Barrier disruption as evidenced by increased Evan’s Blue (EB) absorbance in the left prefrontal cortex (PFC) at 0.5 h post-bTBI (***p < 0.001 vs. Ctrl) and 6 h post-bTBI (*p < 0.01 vs. Ctrl) (values represent mean ± s.e.m.) (n = 4). (B) Raw images of EB bound albumin in the left brain parenchyma at acute time points post-bTBI.
Figure 3
Figure 3
Blast exposure upregulates stress response genes atf4, ddit3, ppp1R15A and gfap. Real-time quantitative PCR time course showed a significant increase in mRNA abundance in the left PFC post-bTBI of stress response genes (A) atf4 (activating transcription factor 4) (ATF4) at 3 h (***p < 0.001 vs. Ctrl at 3 h) and (B) ddit3 (CHOP) at 3 h (***p < 0.001 vs. Ctrl at 3 h). No real differences were observed in mRNA abundance in the right PFC for (C) atf4 (p > 0.05) and (D) ddit3 (p > 0.05). A significant increase in mRNA abundance was also observed in the left PFC for UPR gene (E) ppp1R15A (GADD34) at 6 h (***p < 0.001 vs. Ctrl at 6 h), and astrocyte activation marker (F) gfap (GFAP) at 24 h (**p < 0.01 vs. Ctrl at 24 h) (values represent mean ± s.e.m.) (n = 4).
Figure 4
Figure 4
Salubrinal attenuates ER stress markers in the contra coup PFC after bTBI. (A) Immunoblots show a significant increase in CHOP expression at 24 h post-bTBI in the left PFC (*p < 0.05 vs. Ctrl). (B) No significant differences were observed in the right PFC (p > 0.05 vs. Ctrl). (C) CHOP expression was significantly increased at 24 h post-bTBI compared to SAL administered alone (*p < 0.05 vs. SAL). Salubrinal administration prior to blast exposure attenuated CHOP expression (#p < 0.05 vs. bTBI). (D) A significant increase in Caspase-12 cleavage was observed at 24 h in bTBI rats (*p < 0.05 vs. SAL), as well as in SAL+bTBI rats at 24 h (*p < 0.05 vs. SAL). (E) GADD34 protein expression was significantly increased at 24 h post-bTBI (*p < 0.05 vs. SAL). Salubrinal administration prior to blast exposure attenuated GADD34 protein expression (#p < 0.05 vs. bTBI) (values represent mean ± s.e.m.; normalized to β-actin) (n = 4–5).
Figure 5
Figure 5
Salubrinal modulates fluorescence of apoptosis markers after bTBI. Column of panels display immunofluorescence of apoptosis markers: CHOP (green), Caspase-12 (red) and Caspase-3 (red) (top to bottom). Row of Panels are separated by experimental group: CTRL, bTBI and SAL+bTBI (left to right). All images are from the left PFC region and display the blue nuclear counterstain 4’,6-diamidino-2-phenylindole (DAPI). Images are displayed at 20x; arrows demarcate insets at 40x. (Scale bars = 50 μm). (A) Immunohistochemistry (IHC) shows bTBI augmented CHOP fluorescence in the left PFC at 24 h (***p < 0.001 vs. Ctrl). Salubrinal administration prior to bTBI attenuated CHOP fluorescence at 24 h (###p < 0.001 vs. bTBI). (B) Immunohistochemistry shows bTBI increased Caspase-12 fluorescence in the left PFC at 24 h (***p < 0.001 vs. Ctrl). Salubrinal administration prior to bTBI had no significant effect on Caspase-12 fluorescence (p > 0.05 vs. bTBI). (C) Immunohistochemistry also shows bTBI augmented Caspase-3 fluorescence in the left PFC at 24 h (**p < 0.01 vs. Ctrl). Salubrinal administration prior to bTBI mitigated Caspase-3 fluorescence at 24 h (##p < 0.01 vs. bTBI) (values represent mean ± s.e.m.) (n = 4; 12 randomly selected areas from left PFC).
Figure 6
Figure 6
Salubrinal reduces CHOP and MAP2 colocalization after blast exposure. Column of panels display immunofluorescence of UPR marker CHOP (green), neuron-specific marker MAP2 (red), nuclear counterstain DAPI (blue), and those three markers merged (yellow) (left to right). Row of panels are separated by experimental group: CTRL, bTBI and SAL+bTBI (top to bottom). All images are from the left PFC region with colocalization determined by levels of yellow in merged images. Images are displayed at 20x; arrows demarcate insets at 40x. (Scale bars = 50 μm).
Figure 7
Figure 7
CHOP and Caspase-12 colocalize regardless of SAL administration prior to bTBI. Column of panels display immunofluorescence of UPR marker CHOP (green), Caspase-12 (red), nuclear counterstain DAPI (blue), and those three markers merged (yellow) (left to right). Row of panels are separated by experimental group: CTRL, bTBI and SAL+bTBI (top to bottom). All images are from the left PFC region with colocalization determined by levels of yellow in merged images. Images are displayed at 20x; arrows demarcate insets at 40x. (Scale bars = 50 μm).
Figure 8
Figure 8
Salubrinal reduces apoptosis in the left PFC following blast exposure. (A) Caspase-3 cleavage increased in the left PFC of bTBI rats at 24 h (*p < 0.05 vs. SAL), but was not increased in SAL+bTBI rats at 24 h (p > 0.05 vs. SAL) (values represent mean ± s.e.m.; normalized to β-actin) (n = 5). (B,C) Evidence of increased cell membrane blebbing, an indicator of early apoptosis, was observed at 24 h in the left PFC of bTBI rats compared to control rats. (D) Membrane blebbing was reduced in the left PFC of SAL+bTBI rats. Images displayed at 20x; arrows demarcate insets at 63x. (Scale bars = 30 μm) (n = 4).
Figure 9
Figure 9
Salubrinal ameliorates impulsive-like behavior indicative of PFC damage after Blast. (A) A motion capture track plot of one animal from each experimental group during a single elevated plus maze (EPM) trial using AnyMaze Software™. (B) Percentage of time spent in the open arms of the EPM was significantly increased in bTBI rats compared to SAL only rats at 7 d (*p < 0.05 vs. SAL). Salubrinal administration prior to bTBI decreased the percent time spent in the open arms of the EPM (#p < 0.05 vs. bTBI) (values represent mean ± s.e.m.) (n = 12). No significant differences were observed between groups for (C) Speed, (D) Closed-Arm Entries, or (E) Movement within the EPM trials (5 min).
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