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. 2021 Jan;18(1):286-296.
doi: 10.1007/s13311-020-00952-0. Epub 2020 Oct 19.

Withaferin-A Treatment Alleviates TAR DNA-Binding Protein-43 Pathology and Improves Cognitive Function in a Mouse Model of FTLD

Sunny Kumar  1 , Daniel Phaneuf  1 , Jean-Pierre Julien  2   3
Affiliations

Withaferin-A Treatment Alleviates TAR DNA-Binding Protein-43 Pathology and Improves Cognitive Function in a Mouse Model of FTLD

Sunny Kumar et al. Neurotherapeutics. 2021 Jan.

Abstract

Withaferin-A, an active withanolide derived from the medicinal herbal plant Withania somnifera induces autophagy, reduces TDP-43 proteinopathy, and improves cognitive function in transgenic mice expressing mutant TDP-43 modelling FTLD. TDP-43 is a nuclear DNA/RNA-binding protein with cellular functions in RNA transcription and splicing. Abnormal cytoplasmic aggregates of TDP-43 occur in several neurodegenerative diseases including amyotrophic lateral sclerosis (ALS), frontotemporal lobar degeneration (FTLD), and limbic-predominant age-related TDP-43 encephalopathy (LATE). To date, no effective treatment is available for TDP-43 proteinopathies. Here, we tested the effects of withaferin-A (WFA), an active withanolide extracted from the medicinal herbal plant Withania somnifera, in a transgenic mouse model of FTLD expressing a genomic fragment encoding mutant TDP-43G348C. WFA treatment ameliorated the cognitive performance of the TDP-43G348C mice, and it reduced NF-κB activity and neuroinflammation in the brain. WFA alleviated TDP-43 pathology while it boosted the levels of the autophagic marker LC3BII in the brain. These data suggest that WFA and perhaps other autophagy inducers should be considered as potential therapy for neurodegenerative diseases with TDP-43 pathology.

Keywords: Amyotrophic lateral sclerosis; NF-κB; TDP-43; Withania somnifera; autophagy; frontotemporal dementia; withaferin-A.

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Figures

Fig. 1
Fig. 1
WFA treatment Inhibits NF-κB Activation in both microglial and neuronal cultured cells. (a) BV2/NF-κB luc cells was pretreated with LPS bacterial LPS (500 ng/ml) for 2 h. Posttreatment of LPS stimulated cells were treated with WFA in LPS containing media for 2 h (n = 4; one way ANOVA; Tukey multiple comparison test). (b) NSC-34/NF-κB luc cells were pretreated with 1 μM WFA for 20 min. After the treatment, cells were exposed to TNF-α (40 ng/ml) (n = 4, one-way ANOVA; Tukey’s multiple comparison test). (c) Cell death assay performed with BV2 cell by addition of lipopolysaccharide (LPS) and withaferin-A (WFA) in dimethyl sulfoxide (DMSO) (one-way ANOVA; Bonferroni’s multiple comparison test; n = 6). (d) Cell death difference observed in NSC-34 cells by addition of TNF-α and withaferin-A (WFA) dimethyl sulfoxide (DMSO) (n = 4; one-way ANOVA; Bonferroni’s multiple comparison test). P < 0.0001 ***)
Fig. 2
Fig. 2
WFA treatment reduces TDP-43 inclusions and improves cognitive function in TDP-43G348C mice. (a) Passive avoidance test performed after 8 weeks of saline and withaferin-A treatment in hTDP-43G348C mice. Graphs showing freezing time in second spent by mice (n = 7; Unpaired t-test * p < 0.05). (b) Representative pictures showing TDP-43 (red), NeuN (green), and DAPI (blue) in the brain sections of vehicle or WFA-treated mutant hTDP-43G348C mice. (c) Data showing stereology of % neurons showing TDP-43 cytoplasmic aggregation in vehicle or WFA-treated mutant hTDP-43 mice. Five to six cortical sections from each mouse were analyzed. Further, to make a comparison and perform statistical analysis, 3 mice were used from each group. (d) Representative immunoblots and quantification of RIPA-insoluble and RIPA-soluble fractions of brain from n = 3 to 4 independent mice from both hTDP-43G348C mice. RIPA-insoluble TDP-43 or RIPA-soluble TDP-43 was normalized with ponceau or actin, respectively. Statistical analysis used was unpaired t test. Data represents mean ± sem. ** p < 0.01
Fig. 3
Fig. 3
WFA reduced microglial and astrocyte activation in the brain of TDP-43G348C mice. (a) Representative immunoblots and quantification of NF-ĸB and phospho-NF-ĸB levels in the brain from n = 4 independent mice from both saline and WFA-treated hTDP-43G348C mice. (b) Representative immunostaining showing astrocytes in the hippocampal region of 1-year-old TDP-43G348C mouse brains treated with vehicle or WFA. The graph represents quantification of % area covered by the GFAP+ cells, using multiple sections from n = 3 mice, unpaired t test; * p < 0.05. Representative immunostaining of Iba1+ staining in the (b) hippocampus and in the (c) cortex of vehicle or WFA-treated mice (n = 3). The graph represents the % area fraction, unpaired t test; *** p < 0.001. (d) Representative immunoblots and quantification of YM1 and arginase1 levels in the brain from n = 4 independent mice from both saline and WFA-treated hTDP-43G348C mice
Fig. 4
Fig. 4
WFA activated autophagic pathways. (a) Representative immunoblots showing autophagic markers LC3BII and (b) quantitative data of LC3BII blot in the brain of hTDP-43G348C mice received vehicle or WFA treatment. Representative immunoblot of (c) autophagic markers and data showing normalized density of (d) Beclin-1, (e) ATG 5, and (f) p62 in TDP-43G348C mice that received either saline or WFA (n = 3 to 4). Protein levels were normalized using actin a Statistical analysis used was unpaired t test. Data represents mean ± sem. * p < 0.05
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