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. 2018 Nov 9;15(1):312.
doi: 10.1186/s12974-018-1350-y.

Age-related Deregulation of TDP-43 After Stroke Enhances NF-κB-mediated Inflammation and Neuronal Damage

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Free PMC article

Age-related Deregulation of TDP-43 After Stroke Enhances NF-κB-mediated Inflammation and Neuronal Damage

Sai Sampath Thammisetty et al. J Neuroinflammation. .
Free PMC article

Abstract

Background: TDP-43 has been identified as a disease-associated protein in several chronic neurodegenerative disorders and increasing evidence suggests its potentially pathogenic role following brain injuries. Normally expressed in nucleus, under pathological conditions TDP-43 forms cytoplasmic ubiquitinated inclusions in which it is abnormally phosphorylated and cleaved to generate a 35 and a 25 kDa C-terminal fragments. In the present study, we investigated age-related expression patterns of TDP-43 in neurons and glia and its role as modulator of inflammation following ischemic injury.

Methods: Wild-type and TDP-43 transgenic mice of different age groups were subjected to transient middle cerebral artery occlusion. The role of TDP-43 in modulation of inflammation was assessed using immunofluorescence, Western blot analysis, and in vivo bioluminescence imaging. Finally, post-mortem stroke human brain sections were analyzed for TDP-43 protein by immunohistochemistry.

Results: We report here an age-related increase and formation of ubiquitinated TDP-43 cytoplasmic inclusions after stroke. The observed deregulation in TDP-43 expression patterns was associated with an increase in microglial activation and innate immune signaling as revealed by in vivo bioluminescence imaging and immunofluorescence analysis. The presence of ubiquitinated TDP-43 aggregates and its cleaved TDP-35 and TDP-25 fragments was markedly increased in older, 12-month-old mice leading to larger infarctions and a significant increase in in neuronal death. Importantly, unlike the hallmark neuropathological features associated with chronic neurodegenerative disorders, the TDP-43-positive cytoplasmic inclusions detected after stroke were not phosphorylated. Next, we showed that an increase and/or overexpression of the cytoplasmic TDP-43 drives the pathogenic NF-κB response and further increases levels of pro-inflammatory markers and ischemic injury after stroke in age-dependent manner. Finally, analyses of the post-mortem stroke brain tissues revealed the presence of the cytoplasmic TDP-43 immunoreactive structures after human stroke.

Conclusion: Together, our findings suggest that the level of cytoplasmic TDP-43 increases with aging and may act as an age-related mediator of inflammation and neuronal injury after stroke. Thus, targeting cytoplasmic TDP-43 may have a therapeutic potential after stroke.

Keywords: Acute neurodegeneration; Aging; Innate immune response; Microglia; Neuronal injury; Post-stroke inflammation.

Conflict of interest statement

Ethics approval and consent to participate

All the experimental procedures were approved by the Laval University Animal care Ethics Committee (Number of the protocol 2017-063-02, approbation ID number: 114887), and are in accordance with the Guide to the Care and Use of Experimental Animals of the Canadian Council on Animal Care. We used brain tissue of ischemic stroke patients who died at the Stroke Unit of Hospital Clínic de Barcelona. We obtained written consent from the families for tissue removal after death for diagnostic and research purposes at the Neurological Tissue bank of the Biobank-Hospital Clínic-Institut d’Investigacions Biomèdiques August Pi i Sunyer (IDIBAPS). We also obtained tissue from control subjects stored at this Biobank. The study had the approval of the Ethics Committee of Hospital Clínic de Barcelona (CEIm). Features of the cases are shown in Additional file 1: Table S1.

Consent for publication

Not applicable at this time.

Competing interests

The authors declare that they have no competing interests.

Publisher’s Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Figures

Fig. 1
Fig. 1
Characterization of TDP-43 expression patterns in aging and stroke. a Immunofluorescence of the brain cortex of wild-type 3-month-old mice using TDP-43 antibody at different time points after MCAO reveals mislocalization of TDP-43 protein into cytoplasm starting 72 h after MCAO. b Double immunofluorescence of the brain cortex sections of 3- and c 12-month-old mice in control conditions CTL (upper panels) and 72 h after MCAO using TDP-43 antibody (green) and NeuN antibody (red) show nuclear localization of TDP-43 in control conditions and cytoplasmic mislocalization of TDP-43 in neuronal cells in both age groups. d, e Western blot of nuclear lysates from 3- and 12-month-old mice using TDP-43 antibody in control and 72 h post MCAO does not reveal any significant changes in the levels of whole length TDP-43. P84 is used as loading control. f Western blot of cytoplasmic lysates from 3- and 12-month-old mice using TDP-43 antibody in control and 72 h after MCAO show expression of whole length TDP-43, fragmented TDP-35, and TDP-25. g Western blot of cytoplasmic lysates from 3- and 12-month-old mice using phospho-TDP-43 antibody in control and 72 h after MCAO show no expression of whole length P-TDP-43, fragmented P-TDP-35, or P-TDP-25. h Normalized densitometry values of immunoblots from control and 72 h after MCAO reveal significant increase in the levels of whole length TDP-43, pathological TDP-35, and TDP-25 fragments in aging. Actin is used as loading control. Quantified data in the figure was presented as mean ± SEM and statistical significance between the groups was achieved using ANOVA followed by Tukey’s multiple comparison test and depicted as ***p < 0.001. Scale bar represents 10 and 20 μm
Fig. 2
Fig. 2
Cytoplasmic TDP-43 shows ubiquitin positivity and microglia/macrophages exhibit TDP-43 mislocalization. a Double immunofluorescence of the brain cortex sections of 3- and 12-month-old mice 72 h after MCAO using TDP-43 antibody (green) and ubiquitin (red) reveal the formation of cytoplasmic TDP-43-ubiquinated aggregates 72 h after MCAO in 3- and 12-month-old mice. b Co-immunoprecipitation of ubiquitin using rabbit polyclonal TDP-43 from the cytoplasmic lysates of 3- and 12-month-old control and MCAO operated mice reveal the ubiquitination of TDP-43 after 72 h post MCAO. Note that TDP-43 ubiquitination is increased in 12-month-old transgenic mice after MCAO. Western blot of TDP-43 using rabbit polyclonal TDP-43 is shown as 15% input to confirm the presence of TDP-43 protein in the cytoplasmic lysate we used for Co-immunoprecipitation and actin is shown as loading control. c Double immunofluorescence of the brain cortex sections of 3- and 12-month-old mice 72 h after MCAO using TDP-43 antibody (red) and CD11b antibody (green) show mislocalization of TDP-43 in microglial cells in both age groups. Scale bar represents 10 μm
Fig. 3
Fig. 3
Cytoplasmic mislocalization of TDP-43 in neuronal and glial cells causes upregulation of inflammatory response 72 h after MCAO in aging. a Representative photographs 48 h after MCAO after real-time imaging of TLR2 induction. The color calibrations at the right are photon counts. b Data plotted was obtained measuring the photon emission. Red (3 month old) and green (12 month old) lines indicate TLR2 induction at different time points post MCAO. c Western blot of cortical lysates from 3- and 12-month-old mice using Iba1 in control and 72 h after MCAO reveals an increase in the levels of Iba1 in 12-month-old mice. Actin is used as loading control. d Western blot of nuclear lysates from 3- and 12-month-old wild-type mice using phospho-P65 in control and 72 h after MCAO reveals an increase in the levels of phospho-P65 in 12-month-old mice. P84 is used as loading control. e Levels of inflammatory cytokines like IL-1β, IL-4, IL-6, IL-10, IL-17, TNF-α, CCL5, and GM-CSF were significantly increased in 12-month-old mice 72 h after MCAO. Quantified data in the figure was presented as mean ± SEM and statistical significance between the groups was achieved using one-way ANOVA followed by Tukey’s multiple comparison test and depicted as ***p < 0.001, *p < 0.05
Fig. 4
Fig. 4
Increased stroke area and neuronal apoptosis was observed 72 h after MCAO in 12-month-old mice. a Representative images of cresyl violet staining showing stroke area in 3- and 12-month-old mice 72 h after MCAO. b Quantification of stroke area reveals an increase in the ischemic region in 12-month-old mice 72 h after MCAO. c Western blots of cytoplasmic lysates from 3- and 12-month-old mice using cleaved caspase-3 72 h after MCAO. d Normalized densitometry values of immunoblots 72 h after MCAO reveal a significant increase in the levels of cleaved caspase-3 in 12-month-old mice suggesting more tissue damage 72 h after MCAO. e Double immunofluorescence of the brain cortex sections of 3 and 12-month-old mice 72 h after MCAO using caspase-3 antibody (green) and NeuN (red) antibody show neuronal apoptosis in both age groups. Quantified data in the figure was presented as mean ± SEM and statistical significance between the groups was achieved using unpaired t test and depicted as ***p < 0.001, **p < 0.01
Fig. 5
Fig. 5
Overexpression of TDP-43 increases brain damage and inflammatory response after stroke. a Representative image of cresyl violet staining showing stroke area in 3-month-old WT and TDP-43A315T 72 h after MCAO. b Quantification of stroke area reveals an increase in the ischemic lesion in TDP-43A315T compared to WT mice. c Western blots of cytoplasmic lysates coming from 3- and 12-month-old WT and TDP-43A315T mice using cleaved caspase-3. d Quantification of immunoblots 72 h after MCAO reveal a significant increase in the levels of cleaved caspase-3 in 12-month-old TDP-43A315T mice when compared to the 3-month TDP-43A315T or the 12-month-old WT control. e Double immunofluorescence of the brain cortex sections of 3- and 12-month-old TDP-43A315T mice 72 h after MCAO using caspase-3 antibody (green) and NeuN (red) antibody show neuronal apoptosis in both age mice. f Expression of P65 by revealed by Western blot in control mice (3- and 12-month-old WT and TDP-43A315T). g Expression of P65 by revealed by Western blot 72 h after MCAO (3- and 12-month-old WT and TDP-43A315T). Normalized densitometry values of Western blot reveal a general significant increase in the levels of P65 after MCAO in either age group or mice. 3- and 12-month-old TDP-43A315T show a higher P65 base level compared to the 3-month-old WT control. Quantified data in the figure was presented as mean ± SEM and statistical significance between the groups was achieved using unpaired t test and depicted as ***p < 0.001, **p < 0.01
Fig. 6
Fig. 6
TDP-43 immunoreactivity in human stroke. a Immunohistochemistry of a control contralateral and non-affected hemisphere of the human subjects using anti human TDP-43 antibody reveals the localization of TDP-43 in the nucleus. b TDP-43 immunohistochemistry on cortical sections of human subjects died 1 day (stroke 4) and 5 days (stroke 5, 6, 7, 8) after ischemic stroke reveals the localization of TDP-43 in the cytoplasm of neurons in the periphery and core regions of the ischemia, which is indicated with arrow marks in black. Scale bar represents 20 μm

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