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.
Highlights
Transactive response DNA binding protein was originally described as a regulatory element involved in modulating HIV-1 gene expression [1]
A number of amyotrophic lateral sclerosis (ALS)-linked genes encode proteins that may interact with the NF-κB signaling pathway: 1) transactive response DNA binding protein 43 (TDP-43) and FUS can bind and activate p65 NF-κB [12, 30]; 2) ALSlinked mutations have been discovered in the optineurin gene, which encodes a protein activating the suppressor of NF-κB [31]; 3) mutations in valosin-containing protein activates NF-κB signalling [32, 33]; 4) overt inflammation was present in mice deficient for progranulin, a negative regulator of NF-κB activity [34]; 5) p62 (SQSTM1) can be associated with exacerbated inflammatory responses [35] and mutations in ALS/FTLD increase p62 levels [36]; 6) NF-kB signaling pathway is upregulated in ALS induced pluripotent stem cellderived motor neurons [37]; 7) ablation of NF-κB signaling in microglia extended survival of superoxide dismutase 1 mutant mice [38]
To attenuate activation of the NF-κB signaling pathway in a transgenic mouse model of ALS, we tested an extract of W. somnifera (ASH) that had the potency to reduce in a dose-dependent fashion the activity of NF-κB P65–luciferase reporter in the microglial cultured BV2 cells (Fig. 1)
Summary
Transactive response DNA binding protein was originally described as a regulatory element involved in modulating HIV-1 gene expression [1]. The 414 amino acid-containing protein has a molecular weight of 43 kDa ( it is commonly referred to as TDP-43), and structurally consists of 2 RNA recognition motifs, a nuclear localization sequence, a nuclear export domain, and a glycine-rich C-terminal domain [2]. TDP-43 is predominantly a nuclear protein, even though it is capable of shuttling between the nucleus and cytoplasm—a process partly regulated by nuclear localization signal and nuclear export signal motifs [3]. TDP-43 is a DNA/RNA binding protein and it is involved in regulating RNA transcription, splicing, trafficking, and microRNA biogenesis [4]. Aggregation, and neurotoxicity enhancing mutations in the TARDBP have been reported from both familial and sporadic ALS cases [8]
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