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Protein Citrullination: A Proposed Mechanism for Pathology in Traumatic Brain Injury.

Lazarus RC, Buonora JE, Flora MN, Freedy JG, Holstein GR, Martinelli GP, Jacobowitz DM, Mueller GP - Front Neurol (2015)

Bottom Line: The present investigation addressed this gap by examining the effects of TBI on the distribution of protein citrullination and on the specific cell types involved.This response was exclusively seen in astrocytes; no such effects were observed on the status of protein citrullination in neurons, oligodendrocytes or microglia.Further, proteomic analyses demonstrated that the effects of TBI on citrullination were confined to a relatively small subset of neural proteins.

View Article: PubMed Central - PubMed

Affiliation: Program in Neuroscience, Uniformed Services University of the Health Sciences , Bethesda, MD , USA.

ABSTRACT
Protein citrullination is a calcium-driven post-translational modification proposed to play a causative role in the neurodegenerative disorders of Alzheimer's disease, multiple sclerosis (MS), and prion disease. Citrullination can result in the formation of antigenic epitopes that underlie pathogenic autoimmune responses. This phenomenon, which is best understood in rheumatoid arthritis, may play a role in the chronic dysfunction following traumatic brain injury (TBI). Despite substantial evidence of aberrations in calcium signaling following TBI, there is little understanding of how TBI alters citrullination in the brain. The present investigation addressed this gap by examining the effects of TBI on the distribution of protein citrullination and on the specific cell types involved. Immunofluorescence revealed that controlled cortical impact in rats profoundly up--regulated protein citrullination in the cerebral cortex, external capsule, and hippocampus. This response was exclusively seen in astrocytes; no such effects were observed on the status of protein citrullination in neurons, oligodendrocytes or microglia. Further, proteomic analyses demonstrated that the effects of TBI on citrullination were confined to a relatively small subset of neural proteins. Proteins most notably affected were those also reported to be citrullinated in other disorders, including prion disease and MS. In vivo findings were extended in an in vitro model of simulated TBI employing normal human astrocytes. Pharmacologically induced calcium excitotoxicity was shown to activate the citrullination and breakdown of glial fibrillary acidic protein, producing a novel candidate TBI biomarker and potential target for autoimmune recognition. In summary, these findings demonstrate that the effects of TBI on protein citrullination are selective with respect to brain region, cell type, and proteins modified, and may contribute to a role for autoimmune dysfunction in chronic pathology following TBI.

No MeSH data available.


Related in: MedlinePlus

Simulated brain injury in normal human astrocytes reveals a spectrum of GFAP breakdown products and the hyper-citrullination of one GFAP species. Normal human astrocytes were treated with ionomycin (10 μM; 4 h) and analyzed for GFAP (left panel) and protein-bound citrulline immunoreactivity (right panel) by western blot. The results of three independent experiments (A–C) are presented, showing the immunoreactivity in extracts prepared from untreated control cells (Control) and cells treated with ionomycin (+Iono). The blue arrows indicate intact GFAP, while the orange arrows indicate the hyper-citrullinated GFAP breakdown product.
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Figure 9: Simulated brain injury in normal human astrocytes reveals a spectrum of GFAP breakdown products and the hyper-citrullination of one GFAP species. Normal human astrocytes were treated with ionomycin (10 μM; 4 h) and analyzed for GFAP (left panel) and protein-bound citrulline immunoreactivity (right panel) by western blot. The results of three independent experiments (A–C) are presented, showing the immunoreactivity in extracts prepared from untreated control cells (Control) and cells treated with ionomycin (+Iono). The blue arrows indicate intact GFAP, while the orange arrows indicate the hyper-citrullinated GFAP breakdown product.

Mentions: To further investigate the cellular mechanisms of injury-induced protein citrullination, a model of simulated TBI was established. In this model, normal human astrocytes (obtained commercially) were incubated in control medium or in medium containing the calcium ionophore, ionomycin, to induce calcium excitotoxicity. Figure 9 shows the results of three separate experiments investigating the effects of ionomycin treatment on the proteolytic processing of GFAP (left panel) and the generation of citrullinated proteins (right panel). The data show that treatment with ionomycin consistently activated the proteolytic processing of intact GFAP (left panel; blue arrows) to produce a distinctive pattern of breakdown products. Probing with mAb 6B3 indicated that one of the GFAP breakdown products is preferentially citrullinated (right panel; orange arrow) in response to simulated TBI. In addition, several other protein features appeared to be heavily citrullinated in response to ionomycin treatment. The identity of these signals remains to be determined.


Protein Citrullination: A Proposed Mechanism for Pathology in Traumatic Brain Injury.

Lazarus RC, Buonora JE, Flora MN, Freedy JG, Holstein GR, Martinelli GP, Jacobowitz DM, Mueller GP - Front Neurol (2015)

Simulated brain injury in normal human astrocytes reveals a spectrum of GFAP breakdown products and the hyper-citrullination of one GFAP species. Normal human astrocytes were treated with ionomycin (10 μM; 4 h) and analyzed for GFAP (left panel) and protein-bound citrulline immunoreactivity (right panel) by western blot. The results of three independent experiments (A–C) are presented, showing the immunoreactivity in extracts prepared from untreated control cells (Control) and cells treated with ionomycin (+Iono). The blue arrows indicate intact GFAP, while the orange arrows indicate the hyper-citrullinated GFAP breakdown product.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4585288&req=5

Figure 9: Simulated brain injury in normal human astrocytes reveals a spectrum of GFAP breakdown products and the hyper-citrullination of one GFAP species. Normal human astrocytes were treated with ionomycin (10 μM; 4 h) and analyzed for GFAP (left panel) and protein-bound citrulline immunoreactivity (right panel) by western blot. The results of three independent experiments (A–C) are presented, showing the immunoreactivity in extracts prepared from untreated control cells (Control) and cells treated with ionomycin (+Iono). The blue arrows indicate intact GFAP, while the orange arrows indicate the hyper-citrullinated GFAP breakdown product.
Mentions: To further investigate the cellular mechanisms of injury-induced protein citrullination, a model of simulated TBI was established. In this model, normal human astrocytes (obtained commercially) were incubated in control medium or in medium containing the calcium ionophore, ionomycin, to induce calcium excitotoxicity. Figure 9 shows the results of three separate experiments investigating the effects of ionomycin treatment on the proteolytic processing of GFAP (left panel) and the generation of citrullinated proteins (right panel). The data show that treatment with ionomycin consistently activated the proteolytic processing of intact GFAP (left panel; blue arrows) to produce a distinctive pattern of breakdown products. Probing with mAb 6B3 indicated that one of the GFAP breakdown products is preferentially citrullinated (right panel; orange arrow) in response to simulated TBI. In addition, several other protein features appeared to be heavily citrullinated in response to ionomycin treatment. The identity of these signals remains to be determined.

Bottom Line: The present investigation addressed this gap by examining the effects of TBI on the distribution of protein citrullination and on the specific cell types involved.This response was exclusively seen in astrocytes; no such effects were observed on the status of protein citrullination in neurons, oligodendrocytes or microglia.Further, proteomic analyses demonstrated that the effects of TBI on citrullination were confined to a relatively small subset of neural proteins.

View Article: PubMed Central - PubMed

Affiliation: Program in Neuroscience, Uniformed Services University of the Health Sciences , Bethesda, MD , USA.

ABSTRACT
Protein citrullination is a calcium-driven post-translational modification proposed to play a causative role in the neurodegenerative disorders of Alzheimer's disease, multiple sclerosis (MS), and prion disease. Citrullination can result in the formation of antigenic epitopes that underlie pathogenic autoimmune responses. This phenomenon, which is best understood in rheumatoid arthritis, may play a role in the chronic dysfunction following traumatic brain injury (TBI). Despite substantial evidence of aberrations in calcium signaling following TBI, there is little understanding of how TBI alters citrullination in the brain. The present investigation addressed this gap by examining the effects of TBI on the distribution of protein citrullination and on the specific cell types involved. Immunofluorescence revealed that controlled cortical impact in rats profoundly up--regulated protein citrullination in the cerebral cortex, external capsule, and hippocampus. This response was exclusively seen in astrocytes; no such effects were observed on the status of protein citrullination in neurons, oligodendrocytes or microglia. Further, proteomic analyses demonstrated that the effects of TBI on citrullination were confined to a relatively small subset of neural proteins. Proteins most notably affected were those also reported to be citrullinated in other disorders, including prion disease and MS. In vivo findings were extended in an in vitro model of simulated TBI employing normal human astrocytes. Pharmacologically induced calcium excitotoxicity was shown to activate the citrullination and breakdown of glial fibrillary acidic protein, producing a novel candidate TBI biomarker and potential target for autoimmune recognition. In summary, these findings demonstrate that the effects of TBI on protein citrullination are selective with respect to brain region, cell type, and proteins modified, and may contribute to a role for autoimmune dysfunction in chronic pathology following TBI.

No MeSH data available.


Related in: MedlinePlus