Inflammation Induces TDP-43 Mislocalization and Aggregation

RESEARCH ARTICLE Inflammation Induces TDP-43 Mislocalization and Aggregation Ana Sofia Correia1, Priyanka Patel2, Kallol Dutta2, Jean-Pierre Julien2* 1 Centre de Recherche du Centre Hospitalier Universitaire de Québec, 2705 Boulevard Laurier, Québec, QC, G1V 4G2, Canada, 2 Research Centre of Institut universitaire en santé mentale de Québec, Department of Psychiatry and Neuroscience, Laval University, Québec, QC, Canada * Abstract OPEN ACCESS Citation: Correia AS, Patel P, Dutta K, Julien J-P (2015) Inflammation Induces TDP-43 Mislocalization and Aggregation. PLoS ONE 10(10): e0140248. doi:10.1371/journal.pone.0140248 Editor: Xu Gang Xia, Department of Pathology, Anatomy & Cell Biology, Thomas Jefferson University, UNITED STATES Received: April 24, 2015 Accepted: September 23, 2015 Published: October 7, 2015 Copyright: © 2015 Correia et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability Statement: All relevant data are within the paper and its Supporting Information file. Funding: This work was supported by the Canadian Institutes of Health Research (CIHR) and the Fondation André-Delambre. Competing Interests: The authors have declared that no competing interests exist. TAR DNA-binding protein 43 (TDP-43) is a major component in aggregates of ubiquitinated proteins in amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD). Here we report that lipopolysaccharide (LPS)-induced inflammation can promote TDP-43 mislocalization and aggregation. In culture, microglia and astrocytes exhibited TDP-43 mislocalization after exposure to LPS. Likewise, treatment of the motoneuron-like NSC-34 cells with TNF-alpha (TNF-α) increased the cytoplasmic levels of TDP-43. In addition, the chronic intraperitoneal injection of LPS at a dose of 1mg/kg in TDP-43A315T transgenic mice exacerbated the pathological TDP-43 accumulation in the cytoplasm of spinal motor neurons and it enhanced the levels of TDP-43 aggregation. These results suggest that inflammation may contribute to development or exacerbation of TDP-43 proteinopathies in neurodegenerative disorders. Introduction Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disorder characterized by the loss of motor neurons in the brain and spinal cord, causing progressive muscle weakness and typically leading to death by paralysis within a few years. Mutations in over twenty genes are known to be associated with familial forms of ALS [1–2] which account for 10% of all ALS cases. In both familial and sporadic ALS, degenerating neurons are known to present an abnormal accumulation of cytoplasmic inclusions containing ubiquitinated proteins [3]. TAR DNAbinding protein (TDP-43) has been identified as a major component of cytoplasmic inclusions in sporadic and most familial ALS cases, as well as in frontotemporal lobar dementia (FTLD) with ubiquitinated inclusions, coupling these two diseases as TDP-43 proteinopathies [4–9]. Various dominant mutations in TDP-43 have also been linked with familial cases of both ALS and FTLD, confirming the importance of TDP-43 in the pathology of these diseases [10–16]. Under normal conditions TDP-43 is mostly localized in the nucleus, where it is mainly involved in RNA processing [17–19]. In degenerating neurons of patients with ALS and FTLD, TDP-43 accumulates in the cytoplasm and forms insoluble aggregates in the nucleus, cytoplasm or processes [4, 7]. Aberrant cytoplasmic TDP-43 is known to be truncated into C-terminal fragments (CTFs), phosphorylated and/or ubiquitinated [9, 7, 20]. The cellular pathways PLOS ONE | DOI:10.1371/journal.pone.0140248 October 7, 2015 1 / 19 Inflammation Induces TDP-43 Proteinopathy causing TDP-43 proteinopathy are not fully elucidated albeit some factors are known to induce TDP-43 mislocalization in the cytoplasm including axotomy, cell stress, TDP-43 gene mutations and overexpression [17, 21, 22]. Previously, we reported that levels of messenger RNA (mRNA) and protein for TDP-43 and nuclear factor κ B (NF-κB) p65 were higher in the spinal cord of ALS patients than of control individuals [23]. Surprisingly, TDP-43 was found to interact with NF-κB p65 in glia and neurons of ALS patients and of transgenic mice overexpressing human wild-type or mutant TDP43 species. NF-κB is a key component of the innate immune response. This led us to investigate the potential effects of NF-κB activation by inflammatory stimuli on TDP-43 redistribution in various cultured cells including microglia, astrocytes and neurons. It is well established that dysfunction glial cells can contribute to motor neuron damage [24–26]. Moreover, it is noteworthy that ALS patients exhibit increased levels of lipopolysaccharides (LPS) in the blood as well as an up-regulation of LPS/TLR-4 signaling associated genes in peripheral blood monocytes [27–28]. Here, we report that LPS exposure induced cytoplasmic redistribution of TDP-43 in cultured microglia and astrocytes. Similarly, NF-κB activation in motor neuron-like cell line NSC-34 by TNF-α enhanced TDP-43 cytoplasmic level. We also tested the in vivo effect of chronic LPS administration in transgenic mice expressing genomic fragment of human TDP43 A315T gene (hTDP-43A315T) [11–12]. Interestingly, the chronic LPS treatment enhanced the cytoplasmic mislocalization and aggregation of TDP-43 in the spinal cord of TDP-43 A315T transgenic mice. These results suggest that chronic brain inflammation may contribute to TDP-43 proteinopathies. Materials and Methods Animals used The heterozygous transgenic mouse line expressing the human mutant TDP-43A315T (hTDP43A315T) has been generated and characterized by us [29, 23]. All experimental procedures were approved by the Laval University Animal Care Ethics Committee and are in accordance with the Guide to the Care and Use of Experimental Animals of the Canadian Council on Animal Care. Astroglia cultures Primary astroglial cultures from brain tissues of neonatal (P2-P3) mice were prepared as described previously [30]. In brief, the brain tissues were stripped of their meninges and minced with scissors under a dissecting microscope in Dulbecco’s modified Eagle medium (DMEM). After trypsinization (0.25% trypsin-EDTA (Life Technologies), 10 min, 37°C, 5% CO2), the tissue was triturated. The cell suspension was washed in glial culture medium (DME supplemented with 10% FBS, 1 mM l-glutamine, 1 mM Na pyruvate, 100 U/ml penicillin, and 100 mg/ml streptomycin, non-essential amino acids (all from Life Technologies) and cultured at 37°C, 5% CO2 in 25 cm2 Falcon tissue culture flasks (BD, one brain per flask) coated with 10 mg/ml poly-d-lysine (PDL; Sigma-Aldrich) for overnight and then rinsed thoroughly with sterile distilled water. Four to five days later medium was changed and supplemente (...truncated)