Tianma (Gastrodia elata Blume) is a traditional Chinese medicine (TCM) often used for the treatment of headache, convulsions, hypertension and neurodegenerative diseases. Tianma also modulates the cleavage of the amyloid precursor protein App and cognitive functions in mice. The neuronal actions of tianma thus led us to investigate its specific effects on neuronal signalling. Accordingly, this pilot study was designed to examine the effects of tianma on the proteome metabolism in differentiated mouse neuronal N2a cells using an iTRAQ (isobaric tags for relative and absolute quantitation)-based proteomics research approach. We identified 2178 proteins, out of which 74 were found to be altered upon tianma treatment in differentiated mouse neuronal N2a cells. Based on the observed data obtained, we hypothesize that tianma could promote neuro-regenerative processes by inhibiting stress-related proteins and mobilizing neuroprotective genes such as Nxn, Dbnl, Mobkl3, Clic4, Mki67 and Bax with various regenerative modalities and capacities related to neuro-synaptic plasticity.
Discussion
Orchids and their derivatives have been used for many years in clinical studies to treat various neuronal disorders and demonstrated a powerful effect [4,6]. In our previous study, we could demonstrate the effect of tianma on cognitive functions in mice [12]. Here, we provide an additional interesting insight into the molecular and cellular mechanisms of herbal medicine by disclosing the effect of tianma on the full neuronal proteome changes upon stimulation of differentiated mouse neuronal N2a cells. In the following sections we briefly discuss the identified proteins that were found to be altered upon neuronal tianma stimulation and we hypothesize potential applications of tianma that may emerge from our data obtained:
Increased neuro-protective protein levels in differentiated neuronal N2a cells upon tianma activation
Nxn: Nucleoredoxin is a novel thioredoxin family member that is involved in cell growth and differentiation where it sustains Wnt/β-catenin signalling by retaining a pool of inactive dishevelled protein [27-29]. Its activation by tianma allows the herb to influence pivotal neuronal differentiation pathways. In fact, we observed slightly enhanced neurite extension formation after adding tianma to the neuronal cells (Figure 2C).
Dbnl: Similarly, tianma partakes in cell differentiation processes by mobilizing Dbnl [30]. Dbnl deficiency leads to tissue and behavioral abnormalities and impaired vesicle transport [31]. It is a cytoskeletal protein that may serve as a signal-responsive link between the dynamic cortical actin cytoskeleton and regions of membrane dynamics such as neurite-outgrowth processes and synaptic plasticity [32].
Mobkl3: Mobkl3 is both a member and a putative substrate of striatin family-protein phosphatase 2A (PP2A) complexes [33], an enzyme that belongs to a highly regulated family of serine/threonine phosphatases implicated in cell growth and signalling [34] which has been shown to participate in various signalling events crucially involved in neurodegenerative processes [35,36]. This adds a further interesting aspect on tianma’s potential application for a possible treatment of neurological diseases [4,12].
Clic4: Clic4 (chloride intracellular channel 4) is a multifunctional protein that localizes to the mt and cytoplasm and also traffics between the cytoplasm and nucleus while it interacts with Schnurri-2, a transcription factor in the bone morphogenetic protein (BMP) signalling pathway. Transforming growth factor beta (TGF-beta) promotes the expression of Clic4 and Schnurri-2 as well as their association in the cytoplasm and their translocation to the nucleus. In the absence of Clic4 or Schnurri-2, TGF-beta signalling is abrogated. Direct nuclear targeting of Clic4 enhances TGF-beta signalling and removes the requirement for Schnurri-2. Nuclear Clic4 associates with phospho (p)-Smad2 and p-Smad3, protecting them from dephosphorylation by nuclear phosphatases. These result in newly identified Clic4 as modifier of TGF-beta signalling through its function as stabilizer of p-Smad2 and 3 in the nucleus which is essential for Clic4-mediated growth-arrest and differentiation [37]. In addition, Clic4 mediates TGF-beta1-induced fibroblast-to-myofibroblast transdifferentiation [38] and is required for Ca2+-induced keratinocyte differentiation [39]. Proteomic analysis of vascular endothelial growth factor-induced endothelial cell differentiation reveals a role for Clic4 in tubular morphogenesis also hinting at its involvement in neuronal differentiation processes [40]. Furthermore, Clic4 could be involved in mt-membrane potential generation in mtDNA-depleted cells, a feature required to prevent apoptosis and to drive continuous protein import into mt [41]. Besides, in response to cellular stress Clic4 translocates to the nucleus for the control of apoptotic processes [42] making it another pivotal protein of the tianma-activated signalling cascade.
Mki67: The up-regulation of Mki67 (though rather considered as a proliferative marker) has also been observed previously for ginkgo biloba during the stimulation of neurogenesis [43]. The significance of this finding, however, still needs further detailed investigations.
Bax: Bax is a nuclear-encoded protein present in higher eukaryotes that is able to pierce the mt-outer membrane to mediate cell death by apoptosis [44]. However, a recent report demonstrated a non-apoptotic function of Bax in long-term depression of synaptic transmission with caspase-3 activation and Bax modulation as pivotal elements during synaptic plasticity [45]. Thus, fine tuning of bax and caspase-3 may contribute to tianma-mediated synaptic plasticity as part of tianma’s effect on cognitive functions [12].
Decreased levels of GTPases and stress-related proteins in differentiated neuronal N2a cells upon tianma stimulation
Sept2: Septins are an evolutionarily conserved group of GTP-binding and filament-forming proteins that belong to the large superclass of Ploop GTPases. Their expression is tightly regulated to maintain proper filament assembly and normal cellular functions. Septins perform diverse cellular functions according to tissue expression and their interacting partners. Functions identified to date include cell apoptosis, DNA damage response and alterations of these septin scaffolds, by mutation or expression changes, have been associated with a variety of neurological diseases such as AD and Parkinson’s disease (PD) [46,47]. As other Rho GTPases [48,49], Sept2 is crucially involved in modeling neurite outgrowth during neuronal differentiation and a tight regulation of its expression is necessary [50].
Dnm2: Dynamin 2 (Dnm2) is a large GTPase mainly involved in membrane trafficking through its function in the formation and release of nascent vesicles from biological membranes. Additionally, it tightly interacts with and is involved in the regulation of actin and microtubule networks, independent from membrane trafficking processes. Functional data on Dnm2 reveals the possible pathophysiological mechanisms via which Dnm2 mutations can lead to two distinct neuromuscular disorders. Dnm2 mutations cause autosomal dominant centronuclear myopathy, a rare form of congenital myopathy, and intermediate and axonal forms of Charcot-Marie-Tooth disease, a peripheral neuropathy [51,52]. Furthermore, altered expression of Dnm2 has been observed in AD [53].
Wnk1: Wnk1 is a Ser/Thr protein kinase and mutations in the nervous system-specific HSN2 exon of Wnk1 cause hereditary sensory neuropathy type II [54]. Moreover, Wnk1 was identified to interact with Rho-GDI1 to regulate Lingo1-mediated inhibition of neurite extension [55].
Prdx2: Peroxiredoxins are antioxidant enzymes involved in protein and lipid protection against oxidative injury and in cellular signalling pathways regulating apoptosis. In the CNS, Prdx2 has been shown to be expressed in neurons and its de-regulation has been associated with several neurodegenerative diseases such as AD and PD [56-59].
Tianma modulates (ER-resident) molecular chaperone proteins in differentiated neuronal N2a cells
Skp1a: Decreased expressions of the ubiquitin-proteasome/E3 ligase component Skp1a and the chaperone Hsc-70 can lead to a wide impairment in the function of an entire repertoire of proteins in neurons [60] suggesting a new structural role of Skp1a in dopaminergic neuronal functions besides its E3 ligase activity [61]. The close relation between apoptotic and neuronal differentiation pathways raises the question about the significance of tianma-mediated inhibition of Skp1a protein expression in differentiated neuronal N2a cells [62,63].
Hsp90aa1, Hsp90ab1, Hspa4, Hspa5: The heat shock protein (HSP) family has long been associated with a generalized cellular stress response, particularly in terms of recognizing and chaperoning misfolded proteins. HSPs are induced in response to many injuries including stroke, neurodegenerative diseases, epilepsy, and trauma. Hsp70 has a multifaceted role in neurons. It serves a protective role in several different models of nervous system injury. For instance, Hsp70 functions as a chaperone and protects neurons from protein aggregation and toxicity (in PD, AD, polyglutamine diseases, and amyotrophic lateral sclerosis), protects cells from apoptosis (PD), is a stress marker (temporal lobe epilepsy), and also protects cells from cerebral ischemic injury. However, it has also been linked to a deleterious role in some diseases [64,65]. In particular, it has been shown very recently that Hsp70 can suppress AD phenotypes in mice [66]. The main function of Hsp90 complexes is to maintain protein quality control and to assist in protein degradation via proteasomal and autophagic-lysosomal pathways. As such it plays a major role in the pathology of AD where it is crucially involved (with co-chaperones such as the immunophilins FKBP51 and FKBP52) in the control of aberrant phosphorylated tau protein [67]. Thus, alongside Mobkl3 and PP2A, tianma can eventually influence aberrant tau phosphorylation by modulating Hsp90 action [35,36,68].
Canx: Calnexin is an ER-resident molecular chaperone that plays an essential role in the correct folding of membrane proteins and a component of the quality control of the secretory pathway. Canx gene-deficient mice showed that Canx deficiency leads to myelinopathy [69]. In addition, Canx (-/-) cells have an increased constitutively active unfolded protein response (UPR). Importantly, Canx (-/-) cells have significantly increased proteasomal activity, which may play a role in the adaptive mechanisms addressing the acute ER stress observed in the absence of Canx [70]. Besides, caspase-3 or caspase-7 cleaves Canx, whose cleaved product, very interestingly, leads to the attenuation of apoptosis [71].
Trim28: In neurons disruption of Trim28, a key component of transcriptional repressor complexes in the brain, results in increased anxiety-like behavior and sensitivity to stress [72].
Calr: Calreticulin is a soluble calcium-binding chaperone of the ER that is also detected on the cell surface and in the cytosol. The protein is involved in the regulation of intracellular Ca2+ homeostasis and ER Ca2+storage capacity. Calr is also an important molecular chaperone involved in quality control within secretory pathways. As such, it is involved in the folding of newly synthesized proteins and glycoproteins and, together with calnexin (an integral ER membrane chaperone similar to Calr) and Pdia3 (ERp57, an ER protein of 57 kDa; a PDI (protein disulfide-isomerase)-like ER-resident protein), it constitutes the ‘calreticulin/calnexin cycle’ that is responsible for folding and quality control of newly synthesized glycoproteins. In fact, during recent years, Calr has been implicated to play a pivotal role in many biological systems, including functions inside and outside the ER, indicating that the protein is a multi-process molecule [73-75] that might be involved as an ER-resident chaperone in AD and PD [76-78].
Pdia3: Pdia3 is an ER-resident thiol-disulfide oxidoreductase which is modulating Stat3 (signal transducer and activator of transcription) signalling from the lumen of the ER together with Calr [79,80] that might be affected by PD [81].
Gnb2l1: This guanine nucleotide binding protein (G protein), also known as Rack1 (receptor for activated protein kinase C 1), regulates intracellular Ca2+ levels, potentially contributing to processes such as learning, memory and synaptic plasticity by binding specifically to an ionotropic glutamate receptor and thereby dictating neuronal excitation and sensitivity [82].
Atp5a1: Mt-ATP synthase catalyzes ATP synthesis, utilizing an electrochemical gradient of protons across the inner membrane during oxidative phosphorylation. It seems obvious that even intermittent and minor impairment of this highly important enzyme could deprive the brain tissue of energy at crucial times, which may predispose or contribute to neurological diseases [83].
Concluding, our data has shed new insights on the possible involvement of the herb tianma on neuronal functions and its potential effect on signalling molecules critically involved in common neurorestorative processes related to neurodegenerative diseases such as AD, PD or Huntington’s disease (Figure 10). However, further systemic functional in/ex vivo biology studies are required to decipher the functional significance of the individual bioactive components of tianma, by phytochemistry, to unravel their direct effect on neuronal activities related to neuroprotective activities in order to open new potential avenues based on tianma for the possible treatment of neurodegenerative diseases such as AD [4,84,85].
There has been success in our office with PD patients to combine Glutathione or GlutaMax with tianma. Please contact us for more information.
-synuclein. These results indicate that chronic exposure to a common pesticide can reproduce the anatomical, neurochemical, behavioral and neuropathological features of PD.
