Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2017 Apr;14(2):447-462.
doi: 10.1007/s13311-016-0499-2.

Withania somnifera Reverses Transactive Response DNA Binding Protein 43 Proteinopathy in a Mouse Model of Amyotrophic Lateral Sclerosis/Frontotemporal Lobar Degeneration

Kallol Dutta  1 , Priyanka Patel  2 , Reza Rahimian  1 , Daniel Phaneuf  1 , Jean-Pierre Julien  3   4
Affiliations

Withania somnifera Reverses Transactive Response DNA Binding Protein 43 Proteinopathy in a Mouse Model of Amyotrophic Lateral Sclerosis/Frontotemporal Lobar Degeneration

Kallol Dutta et al. Neurotherapeutics. 2017 Apr.

Abstract

Abnormal cytoplasmic mislocalization of transactive response DNA binding protein 43 (TARDBP or TDP-43) in degenerating neurons is a hallmark of amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration with ubiquitin-positive inclusions (FTLD-U). Our previous work suggested that nuclear factor kappa B (NF-κB) may constitute a therapeutic target for TDP-43-mediated disease. Here, we investigated the effects of root extract of Withania somnifera (Ashwagandha), an herbal medicine with anti-inflammatory properties, in transgenic mice expressing a genomic fragment encoding human TDP-43A315T mutant. Ashwagandha extract was administered orally to hTDP-43A315T mice for a period of 8 weeks starting at 64 and 48 weeks of age for males and females, respectively. The treatment of hTDP-43A315T mice ameliorated their motor performance on rotarod test and cognitive function assessed by the passive avoidance test. Microscopy examination of tissue samples revealed that Ashwagandha treatment of hTDP-43A315T mice improved innervation at neuromuscular junctions, attenuated neuroinflammation, and reduced NF-κB activation. Remarkably, Ashwagandha treatment reversed the cytoplasmic mislocalization of hTDP-43 in spinal motor neurons and in brain cortical neurons of hTDP-43A315T mice and it reduced hTDP-43 aggregation. In vitro evidence is presented that the neuronal rescue of TDP-43 mislocalization may be due to the indirect effect of factors released from microglial cells exposed to Ashwagandha. These results suggest that Ashwagandha and its constituents might represent promising therapeutics for TDP-43 proteinopathies.

Keywords: Amyotrophic lateral sclerosis; NF-κB.; TDP-43; Withania somnifera.

PubMed Disclaimer

Figures

Fig. 1
Fig. 1
Effective inhibition of nuclear factor kappa B (NF-κB) activity by Ashwagandha (ASH) extract in mouse microglial BV2 cells and modulation of cytokine/chemokine activity in primary microglia. No significant alteration in BV2 cell survival was observed by addition of lipopolysaccharide (LPS) or ASH extract in dimethyl sulfoxide (DMSO) at varying concentrations (500, 250, 100, 10, and 1 μg/ml) (a). BV2 cells were stably transfected with pGL4.32[luc2P/NF-κB–RE/Hygro] plasmid carrying 5 copies of an NF-κB response element that drives transcription of the luciferase reporter gene luc2P. When treated with bacterial LPS (100 ng/ml) there was an approximately 3-fold increase in NF-κB luciferase activity compared with nontreated controls. When LPS-stimulated cells were further treated with varying concentrations of ASH–DMSO extract, a significant reduction was observed in luciferase activity at 500 and 250 μg/ml doses(b). Data were analyzed by 1-way analysis of variance with Bonferroni’s multiple comparison test as the post-test (***p < 0.001). Cytokines and chemokines secreted from primary microglia post-LPS challenge and the effect of ASH treatment was evaluated by commercially available array. The cytokines/chemokines whose levels were found to be significantly modulated were (c) FAS ligand, (d) interferon (IFN)-γ, (e) interleukin (IL)-1β (not statistically significant), (f) IL-4, (g) IL-6, (h) IL-17A, (i) monocyte chemoattractant protein-1 (MCP-1/CCL2), (j) macrophage inflammatory protein (MIP)-1α and (k) MIP-1γ, (l) monokine induced by IFN-γ (MIG), (m) the chemokine LIX, (n) regulated on activation, normal T cell-expressed and secreted (RANTES), (o) soluble TNF receptor 1 (R1), (p) soluble TNF receptor 2 (R2), and (q) chemokine (C motif) ligand 1 (XCL1). Data are represented as mean ± SEM of cytokine/chemokine levels normalized against positive controls. Values are expressed as arbitrary units (*p < 0.05; **p < 0.01; ***p < 0.001). IOD = integrated optical density; CTRL = control
Fig. 2
Fig. 2
Oral Ashwagandha (ASH) administration ameliorated performance of hTDP-43A315T mice on rotarod and passive avoidance tasks. ASH or vehicle (Veh)-treated male or female mice were trained to run on an accelerating rotarod (speed 3 rpm; acceleration 0.2 rpm/s) and the latency to fall was recorded. The maximum score of 3 trials for each mouse is represented. A significant increase was observed in the latency to fall in mice treated with ASH as compared with Veh-treated controls from 5 weeks post-treatment onwards. Discontinuation of ASH treatment resulted in gradual decrease in latency in both male and female mice (a, b). Data were compared using an unpaired t test with Welch’s correction (#p < 0.01; **p < 0.001; *p < 0.0001). After 8 weeks of treatments, male (c) and female (d) mice were subjected to the passive avoidance test to assess memory function based on the association formed with an nociceptive stimulus. The results clearly demonstrated a significant increase in memory retention in ASH-treated mice of either sex in comparison to Veh-treated controls. These data were analyzed by Kruskal–Wallis test with Dunn's multiple comparison post-test (##p < 0.01)
Fig. 3
Fig. 3
Increase of innervated neuromuscular junctions (NMJs) in Ashwagandha (ASH)-treated hTDP-43A315T mice. Serial sections of gastrocnemius muscle of ASH- or vehicle (Veh)-treated hTDP-43A315T mice were done with a cryostat. The sections were stained with antibodies against neurofilament-heavy chain (NF-H) and synaptic vesicle glycoprotein 2A (SV2) followed by Alexa-488 conjugated secondary antibody. Prior to mounting, the sections were briefly incubated with α-bungarotoxin conjugated with tetramethylrhodamine. The numbers of innervated, partially innervated, or denervated NMJs were counted based on the merged colored composite images of the sections (a). The data are represented as percent of total NMJs counted (b). ASH-treated mice possessed significantly more innervated NMJs than the Veh-treated controls in which roughly 50% of NMJs were denervated. Data are representative of total NMJs counted from 7 sections per mice, 3 mice per group (n = 1062 for ASH; n = 1501 for Veh) and were analyzed for statistics by 1-way analysis of variance with Bonferroni's multiple comparison post-test (*p < 0.001)
Fig. 4
Fig. 4
Attenuation of astrogliosis and microgliosis by Ashwagandha (ASH) treatment. Spinal cord sections from ASH- and vehicle (Veh)-treated hTDP-43A315T mice were immunostained for glial fibrillary acidic protein (GFAP; astrocytic marker) or Iba-1 (microglial marker). A marked difference in the phenotype of ASH-treated (a–c) and Veh-treated (d–f) astrocytes were observed. Similarly, microglia also showed decreased activation post-ASH treatment (i–k) as compared with Veh-treated (l–n). All images are of 20× magnification. Scale bar = 20 μm. The single-channel images are in grayscale so as to give better contrast. The merged colored images are 150% enlargements. (Inset) A single cell has been enlarged to 300%. Overall integrated density (IOD) analysis of GFAP (g) and Iba-1(h) staining intensities revealed significant reduction of 28% and 23%, respectively, in the spinal cord of ASH -treated mice. Data are mean ± SEM of multiple sections of at least 3 animals per group. Statistical significance was analyzed by 2-tailed t test with Welch’s correction (*p < 0.05; ***p < 0.001). Western blot analysis also clearly showed more GFAP expression in the spinal cords of Veh-treated mice vs that in ASH-treated mice (o). The expression levels of 2 M2a microglial phenotypic markers (Ym-1 and arginase-1) were found to be increased in the spinal cord of ASH-treated hTDP-43A315T mice when compared with Veh-treated controls. However, the tumor necrosis factor (TNF)-α levels remained the same in the 2 groups (o). DAPI = 4,6-diamidino-2-phenylindole
Fig. 5
Fig. 5
Reduction of p65 nuclear factor kappa B (NF-κB) activation in spinal cord of Ashwagandha (ASH)-treated hTDP-43A315T mice. Spinal cord sections from ASH- and vehicle (Veh)-treated hTDP-43A315T mice were immunostained for detection of phophorylated p65 NF-κB. A significantly weaker staining signal was observed in the nucleus of neurons (including motor neurons) from the ASH-treated hTDP-43A315T mice (a–d) compared with Veh-treated hTDP-43A315T mice (e–h). All images are of 20× magnification. Scale bar = 20 μm. The single-channel images are grayscale so as to give better contrast. The merged colored images are 150% enlargements. (Inset) A single cell with phosphoP65 staining has been enlarged to 300%. To further confirm this, the nuclear signal intensities from neurons of both groups were measured using ImageJ after demarcation of the nuclear area using 4,6-diamidino-2-phenylindole (DAPI) in Adobe Photoshop (outline drawn in blue). Upon comparison there was significantly lower intensity of nuclear signal in ASH-treated mice than in Veh-treated mice (i). Data are mean ± SEM of signal intensities per unit nuclear area from at least 6 sections per mouse, 3 mice per group. Data were analyzed by 2-tailed t test with Welch’s correction (***p < 0.001). Immunoblot performed from whole spinal cord lysate also revealed lower expression levels of P65 NF-κB protein in ASH-treated mice than in Veh-treated controls (j)
Fig. 6
Fig. 6
Alleviation of human transactive response DNA binding protein 43 (hTDP-43) mislocalization and aggregation in Ashwagandha (ASH)-treated hTDP-43A315T mice. In hTDP-43A315T mice, there is a clear nuclear depletion of hTDP-43 occurring in spinal motor neurons at about 11 months of age (a–d). Therefore, ASH treatment was initiated after this age. After 8 weeks of treatment, the spinal cord of hTDP-43A315T mice (~500 days old) exhibited intense immunodetection of hTDP-43 in the nucleus of motor neurons (e–h). In contrast, the Veh-treated mice exhibited diffused hTDP-43 in the cytoplasm of spinal cord neurons (i–l). All images are of 20× magnification. Scale bar = 20 μm. The single-channel images are grayscale so as to give better contrast. The merged colored images are 150% enlargements. (Inset) A single cell with hTDP-43 staining has been enlarged to 300%. Nuclear:cytoplasmic hTDP-43 stain intensity was calculated from motor neurons (neurons with diameter ≥4 μm) present in ventral horn of spinal cord sections using ImageJ after demarcation of the nuclear (outline drawn in red) and whole cell area (outline drawn in yellow) using 4,6-diamidino-2-phenylindole (DAPI) in Adobe Photoshop. Results showed a significant increase of about 3.5-fold in the nuclear to cytoplasmic ratio in ASH-treated spinal motor neurons compared with Veh-treated samples (m). Data were analyzed by 2-tailed t test with Welch’s correction (***p < 0.001). On subjecting detergent-soluble and insoluble fractions of spinal cord lysate to immunoblotting, no major difference was observed in the soluble hTDP-43 levels between the 2 groups. However, hTDP-43 was present at lower levels in the insoluble fraction of ASH-treated hTDP-43A315T mice when compared with Veh-treated hTDP-43A315T mice. ASH treatment caused also a reduction in levels of the toxic 35-kDa TDP-43 species (cleaved fragment/splice variant) (n)
Fig. 7
Fig. 7
Evidence that nuclear redistribution of human transactive response DNA binding protein 43 (hTDP-43) may result from soluble factors released from microglia in response to Ashwagandha (ASH) treatment. To study the role of microglial factors on TDP-43 redistribution in motor neurons in vitro, a model was devised wherein motor neuron-like immortalized cell line NSC34 was stably transfected with hTDP-43A315T-HA plasmid DNA (a). The stable cells showed about 30% increase in TDP-43 expression (b). Data were analyzed by t test with Welch’s correction (*p < 0.05). hTDP-43 was found to be localized predominantly inside the nucleus of these stably transfected cells (c). Various treatments were tested for their potency to induce hTDP-43 cytoplasmic mislocalization. The cells were treated with either glutamate (2 mM) or H2O2 (50 μM) or tumor necrosis factor (TNF)-α (40 ng/ml). Glutamate was the most effective inducer of hTDP-43 mislocalization (d). Mouse microglial cell line BV2 was treated with dimethyl sulfoxide (DMSO), lipopolysaccharide (LPS; 100 ng/ml), or ASH (250 μg/ml) in solution in DMSO. The conditioned media from these cells were used to treat glutamate-pretreated stably transfected NSC34 cells. The conditioned media from BV2 cells induced cytoplasmic mislocalization of hTDP-43 in NSC34 cells. However, hTDP-43 was found to be cleared off from the cytosolic fraction of NSC34 cells exposed to conditioned media from ASH-treated microglia (e). Heat inactivation of the conditioned media prior to exposure to NSC34 cells abolished the cytoplasmic clearance of hTDP-43 by media of ASH-treated BV2 cells (f). The stably transfected NSC34 cells were found to tolerate exposure to ASH solution poorly. On incubation with 250 and 100 μg/ml ASH, the viability of these NSC34 cells was significantly reduced to 50% within 6 h. However, dosages of 10 and 1 μg/ml were found to be tolerated (g). Data were analyzed by 1-way analysis of variance (ANOVA) with Bonferroni’s post-test (**p < 0.01). The efficacy of 10 and 1 μg/ml doses of ASH on reduction of P65 activity in stably transfected NSC34 cells were studied by luciferase assay. Results showed that the 10 μg/ml was sufficient to significantly reduce P65 activity post-TNF-α challenge (h). Data were analyzed by 1-way ANOVA with Bonferroni’s post-test (***p < 0.05). However, ASH treatment of NSC34 cells at 10 μg/ml failed to eliminate the cytosolic mislocalization of hTDP-43 induced by glutamate (i). HA = Hemagglutinin antigen ; GAPDH = glyceraldehyde 3-phosphate dehydrogenase
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Ou SH, Wu F, Harrich D, Garcia-Martinez LF, Gaynor RB. Cloning and characterization of a novel cellular protein, TDP-43, that binds to human immunodeficiency virus type 1 TAR DNA sequence motifs. J Virol. 1995;69:3584–3596. - PMC - PubMed
    1. Benajiba L, Le Ber I, Camuzat A, et al. TARDBP mutations in motoneuron disease with frontotemporal lobar degeneration. Ann Neurol. 2009;65:470–473. - PubMed
    1. Ayala YM, Zago P, D'Ambrogio A, et al. Structural determinants of the cellular localization and shuttling of TDP-43. J Cell Sci. 2008;121:3778–3785. - PubMed
    1. Lee EB, Lee VM, Trojanowski JQ. Gains or losses: molecular mechanisms of TDP43-mediated neurodegeneration. Nat Rev Neurosci. 2012;13:38–50. - PMC - PubMed
    1. Neumann M, Sampathu DM, Kwong LK, et al. Ubiquitinated TDP-43 in frontotemporal lobar degeneration and amyotrophic lateral sclerosis. Science. 2006;314:130–133. - PubMed

MeSH terms

Grants and funding