BACKGROUND: Neuronal cytoplasmic inclusions containing TAR DNA-binding protein 43 (TDP-43) are a neuropathological feature of several neurodegenerative diseases, including amyotrophic lateral sclerosis (ALS), frontotemporal dementia (FTD), and Alzheimer's Disease (AD). Emerging evidence also indicates that systemic inflammation may be a contributor to the pathology progression of these neurodegenerative diseases.
METHODS: To investigate the role of systemic inflammation in the progression of neuronal TDP-43 pathology, AAV9 particles driven by the UCHL1 promoter were delivered to the frontal cortex of wild-type aged mice via intracranial injections to overexpress TDP-43 or green fluorescent protein (GFP) in corticospinal motor neurons. Animals were then subjected to a low-dose (500 μg/kg) intraperitoneal E. coli lipopolysaccharide (LPS) administration challenge for 2 weeks to mimic a chronically altered low-grade systemic inflammatory state. Mice were then subjected to neurobehavioral studies, followed by biochemical and immunohistochemical analyses of the brain tissue.
RESULTS: In the present study, we report that elevated neuronal TDP-43 levels induced microglial and astrocytic activation in the cortex of injected mice followed by increased RANTES signaling. Moreover, overexpression of TDP-43 exerted abundant mouse immunoglobulin G (IgG), CD3, and CD4+ T cell infiltration as well as endothelial and pericyte activation suggesting increased blood-brain barrier permeability. The BBB permeability in TDP-43 overexpressing brains yielded the frontal cortex vulnerable to the systemic inflammatory response following LPS treatment, leading to marked neutrophil infiltration, neuronal loss, reduced synaptosome-associated protein 25 (SNAP-25) levels, and behavioral impairments in the radial arm water maze (RAWM) task.
CONCLUSIONS: These results reveal a novel role for TDP-43 in BBB permeability and leukocyte recruitment, indicating complex intermolecular interactions between an altered systemic inflammatory state and pathologically prone TDP-43 protein to promote disease progression.
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This work was supported by seed grant from the College of Pharmacy and Byrd Alzheimer’s Institute at the University of South Florida.
All relevant data are available upon request directed to the corresponding author.
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Additional file 1: Figure S1. TDP-43 oligomers and c-terminal fragments are not altered by TDP-43 overexpression. (A) Western blots of RIPA and urea-soluble brain homogenates probed for total TDP-43 at a high exposure to visualize oligomers and C-terminal fragments. (B) Quantification of oligomers and C-terminal fragments in the RIPA and urea-soluble fractions (n = 4 mice/group). Statistical analysis was carried out using a one-way ANOVA with Bonferroni post-hoc test.
Additional file 2: Figure S2. TDP-43 overexpression increases astrocytic activation. (A) Representative images depicting GFAP levels (red channel) in the frontal cortex of mouse brain tissue. Scale bar = 20 μm. (B) Quantification of GFAP levels. Statistical analysis was carried out using a one-way ANOVA with Bonferroni post-hoc test (n = 4 mice/group; * p < 0.05). (C) Representative blots probed for GFAP, aquaporin 4, and actin. (D) Quantification of GFAP and (E) aquaporin 4 levels normalized to actin. Statistical analysis was carried out using a one-way ANOVA with Bonferroni post-hoc test (n = 4 mice/group; ** p < 0.01).
Additional file 3: Figure S3. Regional differences in LPS-driven neuroinflammation. (A) Representative blots probed for p-CamKIIβ, RGS14, and iNOS in hippocampal and frontal cortex tissue lysates. (B) Quantification of p-CamKIIβ, RGS14, and iNOS normalized to actin. Statistical analysis was carried out using one-way ANOVAs with Bonferroni post-hoc test (n = 4 mice/group).
Additional file 4: Figure S4. TDP-43 overexpression induces pericyte activation and does not alter tight junction protein levels. (A) Representative blots probed for CD13, PDGFRβ, and GAPDH. (B) Quantification of CD13 and PDGFRβ levels normalized to GAPDH. Statistical analysis was carried out using a one-way ANOVA with Bonferroni post-hoc test (n = 4 mice/group, * p < 0.05). (C) Representative blots probed for laminin B, ZO-1, occludin, claudin 5, claudin 3, and actin. (D) Quantification of laminin B, ZO-1, occludin, claudin 5, and claudin 3 levels normalized to actin. Statistical analysis was carried out using a one-way ANOVA with Bonferroni post-hoc test (n = 4 mice/group).
Zamudio, Frank; Loon, Anjanet R.; Smeltzer, Shayna; Benyamine, Khawla; Navalpur Shanmugam, Nanda K.; Stewart, Nicholas J. F.; Lee, Daniel C.; Nash, Kevin; and Selenica, Maj-Linda B., "TDP-43 Mediated Blood-Brain Barrier Permeability and Leukocyte Infiltration Promote Neurodegeneration in a Low-Grade Systemic Inflammation Mouse Model" (2020). Sanders-Brown Center on Aging Faculty Publications. 144.