Alzheimer Disease: Clues from traditional and complementary medicine Journal of Traditional and Complementary Medicine

Despite modern medicine's incredible innovation and resulting accumulation of valuable knowledge, many of the world's most problematic diseases such as Alzheimer Disease (AD) still lack effective cures and treatments. Western medicine has revealed many genetic, cellular, and molecular processes that characterize AD such as protein aggregation and in ﬂ ammation. As the need for novel and effective treatments increases, researchers have turned towards traditional medicine as a resource. Modern, ev- idence based research examining traditional and complementary remedies for AD has generated promising results within the last decade. Animal based products inhibiting cellular toxicity, anti- in ﬂ ammatory nutraceuticals such as omega-3 fatty acids, and plant based compounds derived from herbal medicine demonstrate viability as neuroprotective treatments and possible application in developing pharmaceuticals. Analysis of antioxidant, anti-in ﬂ ammatory, and neuroprotective phytochemicals used in various traditional medicines around the world reveal potential to ameliorate and prevent the devastating neurodegeneration observed in AD.

Alzheimer Disease (AD): general views
Alzheimer, a progressive neurodegenerative disease an affliction of the elderly, is the most common form of dementia, affecting over 150 million patients. 1 Advances in research and technology have increased our quality of life and achieved increased longevity. Unfortunately, increased longevity is accompanied by elevated incidences of age-related diseases such as Alzheimer Disease (AD). AD is the sixth leading cause of death, and a leading cause of dementia amongst aged populations in the USA. It is a progressive neurodegenerative disorder, characterized by the prevalence of extracellular Ab plaques and intracellular neurofibrillary tangles, derived from the proteolysis of amyloid precursor protein and hyperphosphorylation of microtubule-associated protein tau respectively. Multiple biological processes such as depletion or insufficient synthesis of neurotransmitters, oxidative stress, and abnormal ubiquitination are linked to neurodegenerative diseases. 2 ( Fig. 1) AD has received substantial attention as researchers seek to understand associated debilitating cognitive decline and dementia in patients. Despite mounting research, using simpler animal models such as the fruit fly, Drosophila, and the tiny round worm, Caenorhabditis elegans, molecular mechanisms that underlie pathology of AD still remain unclear. 3 As one of the most complicated neurodegenerative diseases, AD is still an unsolved social and medical problem without effective treatment. 3e5 Understanding and modulating chronic activation of innate inflammatory responses could lead to new approaches for preventing and treating AD. Formation of neurofibrillary tangles and misfolding of amyloid-b peptides are reasonably clear as the devastating basis of neurodegeneration in AD. 2,3 However, the role of neuroinflammation has only been recently identified as an important component. Experimental, genetic, and epidemiological data have revealed the immune system's active role as a disease promoting factor. 1 Binding of misfolded and aggregated proteins to receptors on microglia and astroglia may trigger an innate immune response, setting off a cascade of inflammation in the brain which in effect contributes to disease progression. Several genes associated with misfolding of proteins and inflammatory responses have been identified. External factors such as diabetes and obesity that exacerbate inflammatory processes may also contribute to disease progression. Thus, evidence indicates that the pathology of AD extends beyond its effects on neurons, and is closely related to immunological mechanisms and neuroinflammation. 7 Immune reactions that may contribute to progression of AD however are not entirely clear. The immune system plays a complex role in progression of neurodegenerative disorders, exhibiting both neurodestructive and neuroprotective responses. The destructive or protective mechanisms are dependent on the relative numerical and functional dominance of effector or regulatory T cells. Immune responses may exhibit different migratory, regulatory, and effector functions after being triggered by misfolded and aggregated proteins and cell-specific stimuli. This in turn alters glial and neuronal behaviors that affect neuroinflammation and neuronal survival, altering the disease course. Consequently, the discovery of these destructive or protective immune mechanisms could lead to development of novel treatments by targeting therapeutic pathways that affect neuronal survival and slow disease progression. 8

Utilizing animal models
Our elderly population is rapidly growing, and as needs for novel therapeutics and methods of preventing AD escalate, analysis from an evolutionary perspective may prove valuable. In searching for answers and treatments, considering why AD exists and how it has evolved may expand our understanding of disease pathology. 9 Due to the complex nature of immune and genetic mechanisms involved in AD, using certain animal models has advantages. Turning to a known model, Lee et al (2014) acknowledge that the fruit fly Drosophila is one of the oldest and most powerful genetic models, and a novel source of insights into numerous biological processes. Drosophila has emerged as a model system to analyze human diseases, which now include several that affect the nervous system. Due to genomic similarity between Drosophila and humans, neurodegenerative disease models that employ Drosophila exhibit various human-disease-like phenotypes (Fig. 2). As a result there is rapid and cost-effective in vivo genetic modifier for screening and drug evaluation. These models reveal many disease-associated genetic factors that facilitate the identification of potentially useful drugs. 10 Despite numerous advantages of Drosophila, its usage in evaluating traditional medicines is relatively new. Thus Drosophila can now be introduced for analyzing neurodegenerative diseases modeled after examples that demonstrate successful application of Drosophila models in evaluating traditional medicines. 10 Development of new genomic technologies has potential to advance animal model research even further. 10,11 When viewing animal models of disease and treatment, the fruit fly is often the first species to be examined. However, an even simpler model is becoming equally popular. 3 Model organisms, such as the nematode, C. elegans, offer a complementary approach to numerous questions. C. elegans is advantageous for several reasons to analyze AD and other neurodegenerative diseases in depth. Like their mammalian counterparts, these nematode worms possess complex conserved biochemical pathways. Gene mutations that correlate with AD possess worm counterparts in C. elegans. These include an amyloid precursor protein related gene, apl-1, a tau homolog, ptl-1, and presenilin homologs, such as sel-12 and hop-1. With neuronal connectivity in C. elegans established, it is now advantageous as a model for learning and memory impairments that occur during AD. 3 Earthworms, an emerging model, have been widely used as sources of food and medicine in the practice of traditional medicine. Forging a new research direction recently has resulted from analyses of innate immunity. While interest in and recognition of earthworms' beneficial properties stems from ancient cultures, earthworms are now being examined in modern medicine for their biomedical potential, advancing how we understand innate The multifaceted molecular pathology of AD. AD has been linked to many possible causes on genetic, molecular, and cellular levels. Each node in this figure represents a possible cause of AD. These causal events may work in concert and form an intricate cross-talking network, eventually resulting in neuronal death among patients. 6 immunity. In fact, one of the first studies devoted to the histopathology of senescence revealed characteristics in earthworms with uniform amyloid deposits. 12 This may be an important path and prove useful in analyzing and understanding AD, revealing even more undiscovered insights into immunity and medicinal properties. 13 We recognize that AD is a complex and progressive neurodegenerative disorder. Available therapy is limited to treating symptoms, thus its efficacy remains somewhat unsatisfactory. Aware of the prevalence and predicted increase in incidence of AD, is it essential to develop effective therapy so crucial for public health? Following this lead and due to multifactorial etiology of AD, the multi-target-directed ligand approach is promising as we augment our search for new anti-AD drugs. Guzior et al (2015) review current development of multifunctional potential of anti-AD agents, most of which are acetylcholinesterase inhibitors that extend the pharmacological profile. Thus compounds that offer hope are symptomatic and suggest causal treatment of AD.
Advantageous properties include the amyloid-b antiaggregation activity, inhibition of b-secretase and monoamine oxidase, an antioxidant and metal chelating activity, NO releasing ability and interaction with cannabinoid, NMDA or histamine H3 receptors.
These unusual molecules possess heterodimeric structures that interact with multiple targets which in turn might combine different pharmacophores, original or derived from natural products or existing therapeutics (tacrine, donepezil, galantamine, memantine). There is minimal reaction to these findings since several described compounds may be promising drug candidates.
Others may be valuable inspirations as we continue to search for new effective AD therapies. 14

CAM and AD
Various natural animal and plant based products have enormous potential. We can therefore expect improved treatment and creation of drugs for certain diseases: cancer, diabetes, and heart disease, and expect that AD will be added. Natural products from invertebrates have already begun to be integrated into modern biomedicine, e.g. leeches are employed for plastic surgery or snail venom is used as an alternative to opioids for humans. 15 For example, alterations in uptake and release of glutamate, predominant excitatory neurotransmitter in the central nervous system, have been observed in various neurodegenerative diseases, including AD. Bee venom, which has been used in Traditional Korean Medicine, exerts anti-inflammatory effects, and has been assessed for its inhibition of glutamate as related to neurotoxicity. Protecting against cell death and inhibiting cellular toxicity, bee venom is a promising compound that may be helpful as a treatment against glutamatergic neurotoxicity for neurodegenerative diseases. 16 Moreover, compounds isolated from marine invertebrates have shown equally promising leads. Acetylcholinesterase (AChE) inhibition seems relevant for treating AD. Marine natural products may be effective as AChE inhibitors with curative potential. 17

Application of phytochemicals and nutraceuticals
Synthetic drugs are useful for managing AD and many other chronic illnesses; still there are side effects. Consequently, attention of researchers has inclined toward phytochemicals as promising therapeutic agents. Many are anti-inflammatory, antioxidative and possess anticholinesterase activities with minimal side effects. 2 Approaches to Traditional Chinese Medicine (TCM) remedies reveal first that traditional herbs and phytochemicals may delay AD onset and slow its progression but also allow recovery by targeting multiple pathological causes that possess antioxidative, antiinflammatory, and antiamyloidogenic properties. 4 Furthermore herbs regulate mitochondrial stress, apoptotic factors, free radical scavenging systems, and neurotrophic factors. Neurotrophins such as BDNF, NGF, NT3, and NT4/5 may also participate in neuronal and nonneuronal responses to AD.
Neurotrophin depletion accelerates progression of AD and therefore, replacing such neurotrophins could serve as a potential treatment for certain neurodegenerative diseases. Emphasis concerning mechanisms rests on phytochemicals that mediate signaling pathways involved in neuroprotection specifically neurotrophin-mediated activation of Trk receptors and members of the p75(NTR) superfamily. Research conducted by Venkatesan et al (2015) focused on representative phenolic derivatives, iridoid glycosides, terpenoids, alkaloids, and steroidal saponins as regulators of neurotrophin-mediated neuroprotection. There is evidence derived from encouraging advances, since these phytochemicals have attracted attention due to their in vitro neurotrophin potentiating activity, yet there is still a need for in vivo and clinical efficacy trials. Currently, proof of neuroprotective effects in certain preclinical models and in humans is unclear. 4 Here is where we are with respect to therapeutic approaches. According to Frautschy and Cole (2010), AD involves a complex pathological cascade perhaps triggered initially by accumulation of beta-amyloid peptide aggregates or aberrant amyloid precursor protein processing. Moreover, there is credible information concerning factors initiating AD process significantly before onset of cognitive deficits. However, there is an unclear understanding of any events that immediately precede and precipitate cognitive decline. Without these events defined or at least clarified more adequately we are left with a major limiting factor that hinders rapid development of adequate prevention and treatment strategies. If we agree with the hypothesis of inhibiting certain pathways, clearly efforts have focused on pleiotropic activities of omega-3 fatty acids and anti-inflammatory, antioxidant, and anti-amyloid activity of curcumin in multiple models that consider many steps in the AD pathogenic cascade. 18 AD reveals that inflammation contributes to neurodegenerative disease. Knowing this it is therefore suggested that early prevention and management of inflammation might conceivably delay onset or reduce symptoms of AD, but what is a likely target? With aging, normal physiological changes in the brain include depletion of long chain omega-3 fatty acids. Analyses have shown that brains of AD patients possess lower levels of docosahexaenoic acid (DHA). In agreement, Thomas et al (2015) report that DHA supplementation reduces markers of inflammation. Research devoted to epidemiological, dietary intervention, and supplementation support roles of long chain omega-3 fatty acids in preventing or delaying cognitive decline in AD during its early stages. These results support further investigation of long chain omega-3 supplementation in early stage AD, and maintains the importance of overall quality and composition of diet to protect against AD and dementia. 19 Complementary and alternative medicine reveals nutraceuticals whose properties are both anti-inflammatory and anti-cancer. For example, mangosteen, the fruit from a tropical evergreen tree native to Southeast Asia, has nutraceuticals which possess multiple beneficial properties, especially neuroprotective, anti-oxidative, and anti-inflammatory effects. Treatment with mangosteen based nutraceuticals decreased cell death and increased brain-derived levels of neurotrophic factor. Moreover, mice fed a mangosteen supplemented diet showed improved inflammation related cognitive function, demonstrating its promise in treating AD. 20 Another nutraceutical that has garnered interest as an antioxidant is quercetin. Oxidative stress plays a role in the progression of various diseases and conditions including AD. Thus quercetin has received attention as a food derived antioxidant for its promising biological effects and ability to prevent oxidative damage. 21

Traditional medicine and AD
Despite increasing prevalence of AD worldwide, in addition to extensive research efforts to find a cure, we still have no long term solution. Effective therapeutic and preventative treatments are urgently needed to combat the devastating cognitive decline observed in patients with AD. This is especially pertinent as many potential remedies and medications for neurodegenerative diseases have been derived from traditional medicine.
According to Liu et al (2014) Traditional Chinese Medicine (TCM) has been in use for more than 2,000 years. Recently herbal medicines employed to treat AD in China are based on TCM or modern pharmacological theories; this approach has resonated with respect to etiology and pathogenesis of AD, TCM therapy, and herbal extracts useful in treating AD. Evidence suggests that TCM therapy may offer certain complementary cognitive benefits for treating AD. Moreover, Chinese herbs may be advantageous if we consider multiple target regulation especially compared with single-target antagonist. 22 (Fig. 3).
One neuroprotective plant that is widely used in TCM as herbal medicine is Evodia rutaecarpa Bentham. Evodiamine (Evo), an extract of E. rutaecarpa Bentham, presents an extensive array of beneficial properties. First, Evo exhibits anti-AD and antiinflammatory functions. Second, a surfeit exists. There are anticancer, antiobesity, antinomic, antinociceptive, and antimetastatic functions. These results are enormously positive for treating neurodegenerative disorders. 24 Moreover, TCM has also given us compounds that require improvement or alternative substitutes for existing AD. Retinoid X Receptor (RXR) agonist, Targetin, is an effective treatment for AD in mouse models. The TCM compounds b -lipoic acid and sulfanilic acid are also strong candidates as RXR agonists. Forming viable bonds with the RXR protein receptors, these TCM compounds exhibit potential to be developed into antidementia drugs. 25 Research on the promising compound Nobiletin employed in Traditional Japanese Medicine (Kampo) has yielded promising results. Nobiletin, a citrus flavonoid, exhibited memory improving functions when tested in several animal models. Demonstrating beneficial effects against oxidative stress, cholinergic neurodegeneration, dysfunction of synaptic plasticity-related signaling, and formation of plaques, nobiletin shows potential as a natural anti-dementia compound. In addition, nobiletin exerts certain possible novel pharmaceutical and preventative applications. When tested in mice, Nobiletin enhanced learning and memory, reversing the impairment inflicted on short-term memory and recognition memory. 26,27 Further investigation of Kampo treatments has revealed another herbal medicine ninjin'yoeito (NYT). NYT has been analyzed in clinical trials for its long term effects on cognitive function and mood. Twenty-three patients at varying stages of disease progression who had all shown insufficient responses to treatment with donzepil alone were tested. Eleven patients were treated with donzepil alone, whereas twelve received a combined combination of donzepil supplemented with NYT for two years. After this period, patients receiving donzepil and NYT showed both cognitive improvement and a reduction of AD related depression. 28 Ayurveda, Traditional Indian Medicine, has also contributed to the growing list of valuable compounds. Results have validated ancient remedies for nervous system disorders, including memory related conditions such as dementia. Efforts have been made to analyze Ayurvedic medicine experimentally and to understand its effects on geriatric diseases such as Alzhiemer Disease. 29 Traditionally used as Ayurvedic brain tonic medicine, the plant Centella asiatica (L.) Urban, native to Southeast Asia, exerts various neuroprotective effects. Reducing oxidative stress, inhibiting enzymes, and preventing the formation of amyloid plaques in AD, C. asiatica exhibits comprehensive neuroprotection, a potential phytopharmaceutical. 30

Application of integrative approaches
Modern biomedicine mostly utilizes a Western approach towards solving current medical problems, and has revealed many of the genetic, cellular, and molecular processes that characterize AD such as protein aggregation and inflammation. The unmistakable influence and value of primitive animal models in advancing our understanding of neurodegenerative disease is evident in studies that continue to analyze Drosophila and C. elegans. Animal models serve as cost-effective genetic sources for evaluating treatments, enhancing our understanding of conserved biochemical pathways. However, in spite recent insights into mechanisms that underlie AD pathology, current treatments lack efficacy and adequacy, and a significant cure is yet to be founddstill, we persevere! As the need for novel and effective treatments increases, researchers have turned towards ancient knowledge and alternative practices. Using a rigorous, evidence based approach to analyze compounds attributed in CAM and various traditional medicines, researchers revealed viable alternate treatments that show promising anti-inflammatory, memory improving, and neuroprotective effects. They utilize similar animal models to evaluate their potency and value, a confirmation of their still largely untapped utility. Most products are animal or plant based, and their compounds have possible applications whose development into pharmaceuticals reveal promising cures and potential prevention. Acceptance and practice of theory based medicines in Western biomedicine may require significant time to first become integrated or even totally accepted. However, utilizing Western methodology and approaches to medicine to investigate Traditional Medicine and CAM is a more pragmatic approach to integration. Evidence based research and successful application of alternative and traditional compounds have shed light on potential methods and cures for preventing AD, yielding encouraging results that point towards progress and even beyond.

Conflict of interest
There are no conflicts of interest.


extfieldsofstudy: Biology Medicine

text:
3-Hydroxykynurenine and 3-Hydroxyanthranilic Acid Enhance the Toxicity Induced by Copper in Rat Astrocyte Culture

Copper is an integral component of various enzymes, necessary for mitochondrial respiration and other biological functions. Excess copper is related with neurodegenerative diseases as Alzheimer and is able to modify cellular redox environment, influencing its functions, signaling, and catabolic pathways. Tryptophan degradation through kynurenine pathway produces some metabolites with redox properties as 3-hydroxykynurenine (3-HK) and 3-hydroxyanthranilic acid (3-HANA). The imbalance in their production is related with some neuropathologies, where the common factors are oxidative stress, inflammation, and cell death. This study evaluated the effect of these kynurenines on the copper toxicity in astrocyte cultures. It assessed the CuSO4 effect, alone and in combination with 3-HK or 3-HANA on MTT reduction, ROS production, mitochondrial membrane potential (MMP), GHS levels, and cell viability in primary cultured astrocytes. Also, the chelating copper effect of 3-HK and 3-HANA was evaluated. The results showed that CuSO4 decreased MTT reduction, MMP, and GSH levels while ROS production and cell death are increasing. Coincubation with 3-HK and 3-HANA enhances the toxic effect of copper in all the markers tested except in ROS production, which was abolished by these kynurenines. Data suggest that 3-HK and 3-HANA increased copper toxicity in an independent manner to ROS production.

Introduction
Metals have a vital participation in some cellular processes as enzyme cofactors, as structural and antioxidant components, and also as part of metabolism. For this reason, their balance in cell environment is important and their unbalance causes damage in elemental cell structures as lipids and DNA; therefore, metals excess can be toxic mainly by oxidative stress production [1][2][3]. Copper is the third most abundant essential transition metal that is naturally found in human liver [4] and the most abundant in the brain [5]; this metal works as structural and functional part in various systems. In CNS, copper can act as cofactor of some antioxidant enzymes like + copper/zinc-dependent superoxide dismutase (SOD-1) and has big importance in respiration mitochondrial respiration as part of cytochrome c oxidase structure [6,7]. In addition, this metal participates in the neurotransmitter biosynthesis (noradrenaline) and can be stored in ceruloplasmin [8][9][10]. Copper can be transported into the brain through copper transporter 1 (Ctr1) and toward inner of brain cells by the ATPase copper transporter (ATP7A) and the divalent metal transporter (DMT1) [11][12][13][14][15]. It has been reported that astrocytes have a great influence in cerebral copper homeostasis and they can store big amounts of this metal due to specific characteristics as vast amounts of DMT1, ferritin, metallothionein, and antioxidants as glutathione (GSH) [13,16,17]. However, alterations in copper metabolism have been related with neurodegenerative diseases as Alzheimer's (AD), Parkinson's (PD), Menkes (MD), and Wilson's (WD) diseases, triggering an oxidative stress state in cell environment, resulting in disturbance of energy metabolism and reactive oxygen species (ROS) production [18][19][20][21]. ROS can also stimulate endogenous pathways that can be modulated by redox environment as the kynurenine pathway (KP).
KP is the main route of tryptophan (Trp) catabolism. Trp is an essential amino acid, which can be metabolized through different pathways to form important substances as serotonin and melatonin, but more than 95% is degraded through KP [22], whose main aim is NAD + production, an electron carrier and cofactor in some redox reactions [23]. KP is present in the liver, kidney, and brain of various mammals such as mice, rats, guinea pigs, rabbits, monkeys, and humans [22,24,25]. Along the pathway, different metabolites with neuroactive activity and/or redox properties are produced. KP is highly regulated by redox status of the cellular environment, but its metabolites can also modify this environment due to their redox properties [26]. The alteration in KP metabolite levels has been associated with aging and several neurodegenerative diseases as Huntington, Parkinson, and Alzheimer [27]. Specifically, 3-hydroxykynurenine (3-HK) and 3-hydroxyanthranilic acid (3-HANA) have been studied by various research groups, which describe controversial results. The metabolite 3-HK is found at nanomolar concentrations in CNS in normal conditions, but its levels are modified in neurodegenerative diseases. In fact, the amount increases as much as three times in Huntington's disease [28,29]. It has been reported that 3-HK is able to induce cell death through apoptosis in brain regions as well as in cell cultures and in vivo experiments (with DNA fragmentation and chromatin condensation) [30][31][32][33]. In addition, it has been shown that 3-HK generates oxidative stress besides that it triggers protein aggregates in human lens and finally cataract formation because of its interaction with metals [33][34][35]. On the other hand, there are reports where it was observed that 3-HK (0-100 μM) works as an antioxidant. In this context, 3-HK and 3-HANA were able to decrease lipid peroxidation and GSH oxidation in brain cortex homogenates [36]; in Aldrichina grahami homogenates, 3-HK was able to trap superoxide [37]. In other reports, it has been demonstrated that 3-HK can capture hydroxyl and peroxyl radicals [36,38]. Recently, it was shown that 3-HK can have chelating properties with metals as ferrous and also can scavenge OH• and ONOO − in chemical combinatory assays [39].
Moreover, 3-HANA has also ambiguous characteristics which cause toxicity in neuronal cultures and can produce protein damage due to its interaction with metals and with the ability to generate hydroxyl radicals through Fenton's reaction. Besides, it has been reported that 3-HANA can have uncoupling effect in oxidative phosphorylation and is able to decrease oxygen consumption-activating astrocytes and neuron death [33,34,[40][41][42]. Nevertheless, 3-HANA is described as scavenger of OH• and ONOO − in chemical combinatory assays and can act as a chelator of ferrous ion [36,39]. In addition, 3-HANA can be an inflammatory and neuroprotector molecule since it induces hemeoxygenase-1 and suppresses cytokine and chemokine production stimulated by IL-1/IFN-γ and toll-like receptor (TLR) ligands leading to neuroprotection [43].
Due to the fact that 3-HK and 3-HANA influence the redox environment and knowing that copper can be toxic to the cell, the aim of this work was to determinate the effect of the coincubation of copper with these two kynurenine metabolites, in the toxicity induced by this metal.

Copper Chelation Assays.
Chelation capacity of both KP metabolites was assessed according to previous report [44], where different concentrations of 3-HK and 3-HANA (0-1 mM) were tested. Briefly, a solution of chelator (50 μl of 3-HK or 3-HANA in different concentrations) was mixed with CuSO 4 (50 μl) in HEPES buffer (50 μl). After 2 minutes, 50 μl of hematoxylin or DMSO (blank) was added and mixed for 3 minutes. Then, the absorbance was measured during 4 min. The wavelength used was different for each pH tested. Three different pH (5.5, 6.8, and 7.5) and 2 different buffers were tested (sodium acetate buffer pH 5.5 and HEPES buffer pH 6.8 and 7.5), considering previous reports where it was demonstrated that copper accumulation, as in the pathologies, can change pH environment [45,46].

Primary Astrocyte Cultures.
Rat-cultured cortical astrocytes were obtained from the brains of 3 days postnatal Wistar rats (PND). Cells were seeded in Roux flasks at a 9 × 10 6 cells/ml density. The cells were maintained in DMEM supplemented with FBS at 10% under incubation at 37°C with CO 2 (5%), until the cells were again seeded in 24-well plates to be used. Over 95% of the cells were immunoreactive for glial fibrillary acidic protein, an astrocyte-specific marker [39].

MTT Reduction
Assay. According to previous reports [39,47,48], cellular function was evaluated by MTT reduction assay. This assay is employed as a functional status test through the formation of formazan salts by the action of dehydrogenases in viable cells [39]. Briefly, astrocytes (100,000 per well) were treated with different copper concentrations (0-500 μM), to stablish the toxic copper concentration. Then, CuSO 4 (350 μM) was coincubated with 3-HK    After treatment, the medium was removed and 500 μl MTT (1 mg/ml in DMEM medium) was added to each well. MTT was incubated for 3 h at 37°C, then medium was removed, and acid isopropanol was added to dissolve the blue formazan salts. Quantification of resulting blue formazan salts was done at a wavelength of 570 nm in a plate reader (EON, BioTek). The results were expressed as the percentage of MTT reduction versus control values.

ROS Production
Determination. ROS were evaluated through DCF-DA oxidation [49]. Astrocytes (100,000 per well) were treated with different copper concentrations (0-500 μM), and then copper (350 μM) was coincubated with 3-HK and 3-HANA (100 μM) in DMEM medium for 24 h at 37°C. After that, medium was removed, and cells were washed with saline solution and were added with 75 μl of trypsin. Cells were recollected, and 100 μl of DCF-DA (75 μM) was added to the tubes and reincubated for 20 min at 37°C in darkness. After incubation, ROS formation was quantified by flow cytometry at 488 nm excitation and 532 emission considering 10,000 total events in FlowJo programm. Data are presented as percentage of ROS production versus control.

Mitochondrial
Membrane Potential (MMP) Assay. Mitochondrial membrane potential is a marker of healthy cells, and JC-1, a lipophilic cation, is used to evaluate because it is   selective to changes in mitochondrial membrane potential and can form red fluorescence aggregates (FL-1 channel, emission length 525 nm) with high MMP, whereas when MMP is low, JC-1 is in its monomeric form (FL-2 channel, emission length at 590 nm) and displays a green fluorescence [50,51]. After treatments, medium was removed, cells were washed with saline solution, and then 75 μl of trypsin was added to each well. Cells were recollected and centrifuged at 2000 rpm for 10 min. Medium was discarded, and mitochondrial membrane potential was evaluated through the label of cells with 3 μM of 5,5 ′ ,6,6 ′ -tetrachloro-1,1 ′ ,3,3 ′ -tetraethylbenzimi-dazolylcarbocyanine iodide (JC-1) for 15 min at 37°C in darkness. Then, cells were washed with buffer assay two times. After washing, cells were resuspended and analyzed by flow cytometry. 10,000 events were assessed. Data are expressed as mean fluorescence intensity (MFI) in FL-2 channel and the percentage of cells that decreased MMP [39].
2.6. GSH Determination. GSH concentration was measured with a glutathione detection assay kit (Abcam 65322). Briefly, astrocytes were incubated with copper (350 μM), 3-HK and 3-HANA (100 μM), and with combinations of both in DMEM medium for 24 h at 37°C. After that, medium was removed, cells were washed with saline solution, and then 75 μl trypsin was added to each well. Cells were recollected (100,000 cells) and centrifuged at 2000 rpm for 10 min. Medium was discarded, cells were washed with cold PBS, resuspended in cell lysis buffer, homogenized, and centrifuged 10 min at 4°C, and supernatant was collected. The cells were deproteinized with perchloric acid and potassium hydroxide. Once deproteinized, samples were ready to use in the GSH determination assay according to the kit's instructions. Briefly, standard curve was prepared from 0.1 μg/μl of GSH and dilutions were done in lysis buffer. 50 μl of standard and 100 μl of each sample were added to each well, and then 2 μl of GST reagent and 2 μl of monochlorobimane (MCB) were added. The plate was mixed, and fluorescence in samples was immediately measured in a plate reader at 360 nm excitation and 460 nm emission in a kinetic mode, every 3 minutes for 1 hour at 37°C. The results were expressed as the percentage change in glutathione levels in treated versus untreated control samples.
2.7. Viability Test Assay. Cellular death was assessed with propidium iodide according to Magana-Maldonado et al. [52]. Astrocytes were incubated with the combinations of CuSO 4 (350 μM) with 3-HK or 3-HANA (100 μM) in DMEM medium for 24 h at 37°C. Then, medium was removed, cells were washed with saline solution, and trypsin (75 μl) was added to each well. Cells were recollected and centrifuged at 2000 rpm for 10 min. Medium was discarded, and propidium iodide (PI) was added (5 μg/ml) and incubated for 15 min in darkness. After incubation with PI, samples were analyzed by flow cytometry, and a total of 10,000 events were assessed. PI fluorescence was determinated with a FACSCalibur instrument, and data collection was performed using unstained cells and positive controls for single color. The results were expressed as cells death percentage.

Concentration-Response Effects of Copper Toxicity in
Astrocyte Cultures. To evaluate copper toxicity, astrocytes were incubated during 24 h with different copper concentrations (0-500 μM) in DMEM medium. Astrocytes showed decrease on MTT reduction in a concentration-dependent manner in all used concentrations, being the major effect (80%) with 500 μM of copper (Figure 1(a)). After it was proven that copper had effect on cellular function and knowing that the dehydrogenases are responsible of MTT reduction, we evaluated how mitochondrial function was affected and if ROS were implied in copper toxicity on astrocytes.
Copper reduced MMP in a concentration-dependent manner (Figure 1(c)) and increased ROS production around 50% versus control; however, this effect was not concentration dependent (Figure 1(b)). After the toxicity pattern was observed, we evaluated cell death through propidium iodide (PI), which is capable of binding and labeling DNA. After incubation during 24 h with different concentrations of copper, the cell death increased significantly since the lower concentration (10 uM) was tested and this effect was concentration dependent (Figure 1(d)).

Copper-Chelating Ability of 3-HK and 3-HANA.
With the purpose of testing if copper had an interaction with 3-HK or 3-HANA, we assessed the chelation capacity of 3-HK and 3-HANA for copper. The assay was carried out at three different pH (5.5, 6.8, and 7.5) since we knew that copper is able to modify the pH. Figure 2 shows the 3-HK ability to form a chelating complex with copper in all pH tested. This 3-HK ability to catch copper was more efficient in acid pH being that the IC50 is at pH 5.5 = 56.223 ± 10.322 μM, IC50 at pH 6.8 = 74.731 ± 9.3231 μM, and IC50 at pH 7.5 = 74.232 ± 16.769 μM.
On the other hand, 3-HANA had chelating capacity for copper too, three different conditions were tested, and in all of them, 3-HANA was able to catch copper (Figure 3). At pH 6.8, the most efficient ability to chelate copper with an IC50 = 112.491 ± 7.212 μM took place, following at pH 5.5 with an IC50 = 146.637 ± 4.922 μM and the pH where it was observed less effect was at pH 7.5 with at IC50 = 559.497 ± 31.422 μM.

Effect of 3-HK and 3-HANA in the Cellular Dysfunction
Induced by Copper in Astrocytes. After copper toxicity was evaluated, we determined the kynurenine effect in the presence of this metal (350 μM). Figure 4 shows that copper decreases cellular function (around 50% versus control), evaluated by MTT reduction assay, and the coincubation with the kynurenines enhances this effect (around 80% versus control). The kynurenines alone do not have effect on MTT reduction. alone do not induce effect on this parameter compared with the control group. However, copper (350 μM) is able to reduce around 40% the MMP, while the coincubation of copper with both kynurenines reduced around 60% the MMP versus control.

3-HK and 3-HANA Reduce ROS Production Induced by
Copper. Considering redox properties of 3-HK and 3-HANA, the next experiment was to know if the potentiation in the copper toxicity induced by the kynurenines was through ROS production. Copper (350 μM) induces around 60% ROS production, and coincubation with 3-HK and 3-HANA abolished this effect. Incubation of 3-HK and 3-HANA alone did not have effect in ROS production ( Figure 6). Representative pictures are shown in Figures 6(a), 6(b), 6(c), and 6(d).
3.6. GSH Depletion Is Involved in Toxicity Pattern Induced by the Coincubation of Copper and Kynurenines. As we know, both copper and kynurenines can interact with GSH, and then we evaluated the levels of this endogenous antioxidant that it is in high concentration in astrocytes. 3-HK decreased GSH levels around 20%, while copper decreased them around 55%; 3-HANA did not have effect in this parameter. However, the coincubation of copper with these kynurenines decreased GSH levels around 70% versus control, in both cases (Figure 7).

Effect of Copper Coincubation with Kynurenines on Cell
Viability. Figure 8 shows the effect of copper and kynurenines on cell viability. Representative pictures are shown in Figures 8(a), 8(b), 8(c), 8(d), 8(e), and 8(f). 3-HK and 3-HANA alone did not have effect on cell death, while copper was able to increase the percentage of dead cells around 45%. Coincubation of copper with kynurenines enhanced the number of dead cells (Figure 8(g)). Figure 9 shows representative bright field micrographs of the different treatments. 3-HK and 3-HANA did not show difference compared with control. However, in copper alone (Figure 9(d)) and the copper combination with 3-HK (Figure 9(e)) and 3-HANA (Figure 9(f)), a considerable number of dead cells compared with control can be seen.

Discussion
3-HK and 3-HANA are metabolites of tryptophan catabolism, which possess redox properties and have been associated with neurodegenerative diseases as HD and AD. These kynurenines are produced through KP, which is highly regulated by redox environment. In this context, copper is an integral part of many important enzymes involved in cellular metabolism; however, its dyshomeostasis can generate oxidative stress and it has been related with some neurodegenerative diseases in which also KP metabolites are involved [53,54]. In the present work, we evaluated the effect of 3-HK and 3-HANA in the copper toxicity on astrocytes. We performed the experiments in astrocytes since in the brain, these cells are thought to play a key role in copper homeostasis; in fact, it has been proposed that astrocytes can normally accumulate this metal which will be used by themselves or routed to neurons [53], and the second reason to use astrocytes is that 3-HK and 3-HANA cannot be enzymatically degraded in these cells. First, we demonstrate that copper had toxic effects on astrocytes as some previous reports showed [55][56][57]. Copper was able to decrease cell functionality and MMP and increase ROS production; these factors may be closely related and be dependent on each other. Despite the fact that copper has important functions as cofactor of antioxidant enzymes as SOD 1, copper is also cytotoxic considering that it can participate in ROS production through Fenton's reaction and to displace other elemental metals [58,59]. Besides, mitochondrial alterations and changes in redox environment induced by copper can lead to decrease cell viability as was observed in this work.
On the other hand, it has been shown that 3-HK and 3-HANA are able to scavenge hydroxyl radical and peroxynitrite in chemical combinatory assays and also are able to chelate some metals such as iron [39]. Keeping in mind this background, we explore if 3-HK and 3-HANA would have  copper-chelating capability. Copper-chelating probes were placed at three different pH (5.5, 6.8, and 7.5) by two reasons: (1) in the method that we are using to determinate copper chelation, the affinity of hematoxylin for cupric ions is decreased when the pH is also reduced, and mainly (2) because it has been described in previous reports that copper accumulation, as in some pathologies, can change environment pH and this changes can influence the chelating capacity of various molecules [44][45][46]. Our data show that both kynurenine metabolites were able to chelate copper under different conditions of pH; this results may be due to the nature of 3-HK and 3-HANA since it has been reported that these metabolites are good electron donors in electrochemical experiments [60].
According to redox and chelating properties of 3-HK and 3-HANA, we decided to evaluate their effect on the copper toxicity. Both kynurenines were able to abolish ROS production induced by copper; however, the toxic effect on mitochondrial and cellular function was enhanced by the coincubation of copper with both kynurenines. These effects may be due to the fact that both 3-HK and 3-HANA are able to affect respiratory control (oxygen consumption in states 2 and 3 of mitochondrial respiration) [61] in addition to copper toxicity in mitochondria. Both sceneries affect ATP production and can lead to cell death as can be observed in Figure 9. In this context, a previous report showed that 3-HK and 3-HANA produced cellular damage but in an independent way of ROS production, and actually, the oxidative stress parameters evaluated were even below of basal levels [39]. Moreover, the toxic copper effect enhanced by the kynurenines can be due to the fact that these kynurenines can be oxidized in the presence of copper as copper is reduced, promoting cross-linking in important proteins [34]. Besides, autoxidation of these hydroxykynurenines can form compounds as xanthommatin radical, p-quinone, and 4,6dihydroxyquinolinequinonecarboxylic acid (DHQCA) which are reactive [62] and can interfere with the mitochondrial function and subsequently provoke cell toxicity. Other parameter evaluated was the effect of copper and kynurenines on GSH, which is in high amounts in astrocytes [17]. GSH is responsible to form complexes with copper being copper a natural pool in astrocytes; this complex is required for the incorporation of copper into metallothionein and for SOD activation. In the case of copper was overmuch, GSH would be the first antioxidant to catch it and avoid a triggering of oxidative effects [63][64][65]. Our data show that copper (350 μM) decreased GSH levels and this effect was also enhanced by 3-HK and 3-HANA. This could be explained by the fact that in astrocytes, 3-HK can suffer a deamination and this could occur slowly at physiological pH or by the action of kynurenine aminotransferase (KAT), forming 3-hydroxykynurenine glucoside (3-OHKG) in a nonoxidative way, which in turn can form adducts with GSH [66][67][68]. With the knowledge that KAT is the most abundant KP enzyme in astrocytes, it is not hard to think that 3-HK deamination is taking place and 3-OHKG can form adducts with the large amounts of GSH in this cell type, decreasing GSH available to catch copper and allowing that free copper causes the greater damage observed. Although that process has not been described with 3-HANA, it has been shown that this metabolite is also able to decrease GSH levels in some kind of cells [69]. It is important to take into consideration that 3-HK and 3-HANA could be good ROS scavengers and chelating agents; however, their interaction with other cellular components could increase the cell vulnerability to damage toward other agents, as in this case, to copper.

Conclusion
This research provides important evidence about how two endogen KP metabolites can intensify the cellular damage induced by copper. It is relevant, because in some neurodegenerative diseases, they are found as common factors, the alteration in copper concentrations or in other metals, as well as KP metabolite alterations. The challenge for the future research would be to know the precise modulation of KP metabolites by metals and try to identify therapeutic targets in diseases where these components are present.


extfieldsofstudy: Psychology Medicine

s2fieldsofstudy: Biology Medicine Engineering

text:
Electromyographic Pattern during Gait Initiation Differentiates Yoga Practitioners among Physically Active Older Subjects

During gait initiation, postural adjustments are needed to deal with balance and movement. With aging, gait initiation changes and reflects functional degradation of frailty individuals. However, physical activities have demonstrated beneficial effects of daily motor tasks. The aim of our study was to compare center of pressure (COP) displacement and ankle muscle co-activation during gait initiation in two physically active groups: a group of walkers (n = 12; mean age ± SD 72.6 ± 3.2 years) and a yoga group (n = 11; 71.5 ± 3.8 years). COP trajectory and electromyography of leg muscles were recorded simultaneously during five successive trials of gait initiation. Our main finding was that yoga practitioners had slower COP displacements (p < 0.01) and lower leg muscles % of coactivation (p < 0.01) in comparison with walkers. These parameters which characterized gait initiation control were correlated (r = 0.76; p < 0.01). Our results emphasize that lengthy ankle muscle co-activation and COP path in gait initiation differentiate yoga practitioners among physically active subjects.

INTRODUCTION
In older adults, physiological changes and physical inactivity are associated with a decrease in personal independence in general and in walking ability in particular. Indeed, older subjects with impaired gait are scared of falls and thus limit their physical activity and activities of daily living (Alexander, 1994;Maki and McIlroy, 1996). For example, individuals adopt a more conservative, basic gait pattern (Menz et al., 2003). Because of socio-economics impact of falling, several studies were lead to determine predictive factors of falling in older population. Gait initiation parameters (as described below) may be sensitive indicators of balance dysfunction and the risk of falls in older adults (Chang and Krebs, 1999). Indeed, gait initiation is a transient phase during which postural control and balance maintenance systems are highly active. Specifically, gait initiation consists in creating forward momentum from a quiet stance. Muscle activity creates internal forces that dissociate the center of pressure (COP) from the center of gravity (COG), in order to produce the initiation step (Mann et al., 1979;Brunt et al., 1991). While momentum creation disturbs balance, anticipatory postural adjustments (APA) are needed to deal with balance and allow efficient step initiation.
Several studies have evidenced the degradation of gait initiation with age. The older people show a decrease in the peak moment arm between the body's COG and the COP (Brunt et al., 1991;Polcyn et al., 1998;Chang and Krebs, 1999). The decrease in this forward momentum is associated with lower COG velocity and lower first step amplitude (Patchay et al., 1997(Patchay et al., , 2002Halliday et al., 1998). It has been suggested that this change in kinetic gait initiation parameters occurs because older subjects do not tolerate a deviation of the body as a whole (COG) from the ground reaction forces (COP). In order to initiate gait and compensate for impairments in the moment arm, older subjects have to perform additional trunk movements (Martin et al., 2002).
The observed changes in kinetic parameters have also been explained in terms of impaired muscle activation patterns. The role of muscle activity is to create internal forces in order to dissociate the COP from the COG (Mann et al., 1979;Brunt et al., 1991). For that reason, the electromyographic (EMG) sequence recorded during gait initiation can be considered as ''a motor program that adjusts the configuration of external forces, which acts directly on the position of the COP and joint position'' (Crenna and Frigo, 1991). Several studies described a stereotypical pattern of muscle activation in healthy, younger adults: (i) starting from a postural quiet stance, postural muscles are activated in order to maintain postural alignment; and (ii) in order to initiate gait per se, postural muscle activity is inhibited and motor muscles are then activated (Mann et al., 1979;Brunt et al., 1991Brunt et al., , 1999. Difficulties in inhibiting antigravity muscles prior to movement are correlated with the age-related loss of Betz cells in the motor cortex (Scheibel et al., 1977;Scheibel, 1985). Higher levels of muscle coactivation have been reported in older subjects during activities of daily living-such as bipedal stance (Laughton et al., 2003;Nagai et al., 2011) and dynamic balance (Hortobágyi and DeVita, 2000;Larsen et al., 2008;Schmitz et al., 2009;Pereira and Goncalves, 2011). During gait initiation, the duration of postural and motor muscle coactivation increases with age. Indeed, it appears that the degradation of central mechanisms in gait initiation translates into a decrease in the ankle muscle antagonist coordination pattern (Polcyn et al., 1998;Henriksson and Hirschfeld, 2005), i.e., coactivation of the tibialis anterior (TA) and triceps surae muscles. The difference between young adults and older adults is more obvious for the swing limb (SWL), which shows greater degradation than the stance limb (STL) does (Polcyn et al., 1998).
It has often been suggested that physical activity improves functional ability in older people (Lord and Castell, 1994;Maki and McIlroy, 1996;Lelard and Ahmaidi, 2015). As an indicator of functional status, physically active olders show higher walking velocity compared to aged-matched sedentary adults. However, it seems that the characteristics of the physical activity, duration and frequency of training sessions induce differentiated effects on motor ability. Tai Chi training also showed an enhancement of gait initiation control (Hass et al., 2004;Vallabhajosula et al., 2014). These studies focused on CoP-CoG displacements during APA. These findings demonstrated that proprioceptive physical activity could help to improve gait initiation control via better COP displacement. Gait initiation is a pre-programmed task (Fiolkowski et al., 2002) which need APA controlled by an open-loop mechanism (Massion, 1992). As neuromuscular activity reflects motor program, previous results may explain this improvement by enhanced neuromuscular coordination after proprioceptive training (Chen et al., 2012a,b). Indeed, Nagai et al. (2012) have demonstrated significant changes in muscle coactivation after balance training (relative to an untrained group). Most of these studies reported beneficial effects of proprioceptive practice on postural control, however, differential effects depending on the type of practice were described on postural control (Gauchard et al., 2003). As described by these authors, proprioceptive activities such as yoga consist to produce a sequence of postures that need to deal with balance and movements improving body schema knowledge. To our knowledge, no study was performed to compare the effects of different types of physical activity on gait initiation.
Since differentiated effects on postural control were previously reported according the type of training program, we hypothesize that practicing different type of physical activity would show differences in APA during gait initiation. To initiate the first step, the central nervous system has to deal with the antigravity function of the posture and production of movements. Motor program has to switch from postural activity (Triceps Surae) to focal movements (TA). A time course analysis of co-activation and COP displacement might then be an effective indicator of efficiency of gait initiation. The aim of the present study was thus to investigate potentially differentiating patterns of leg muscle co-activation and COP displacements during gait initiation in yoga practitioners and physically active group (regular walkers).

Subjects
Twenty-three volunteers healthy subjects aged from 68 years to 78 years took part in the study. Regular yoga practitioners (n = 11; 71.5 ± 3.8 years old; 1.59 ± 0.08 m height; 66.8 ± 9.3 kg weight) and walkers subjects (n = 12; 72.6 ± 3.2 years old; 1.61 ± 0.03 m height; 64.3 ± 8.6 kg weight) were recruited from community dwelling. The group of yoga practitioners was standardized in terms of time (more than 1 year) weekly sessions (1) and sessions duration (1 h) of practice. The physically active group was selected on the basis of a questionnaire. Their physical activity level was equal or higher than 1 h of walking per week. All subjects signed prior the study an informed consent which was accepted by the local ethical committee (Comité de protection des Personnes Nord Ouest 2, Amiens, France) in accordance with Helsinki Declaration of 1975. Prior to taking part in the study, all participants reported their medical history. Clinical examination and questioning was conducted in order to exclude subjects showing any possible causes of balance alteration (medication or disease). Subjects were free from any disease which could influence postural maintaining and were able to walk without external help. Subjects did not show cognitive impairments with a score superior to 24 points in Mini-Mental State Evaluation (Folstein et al., 1975). The participants were independent as revealed by a maximal score in the Activities of Daily Living (Katz et al., 1963).

Experimental Devices
A Piezoelectic force plate (Kistler type 9281 B11, Kistler AG, Winterthur, Switzerland) associated with a calculator was used to assess the temporal time-course of the COP displacements in antero-posterior (AP) and medio-lateral (ML) from the ground reaction forces and their moments in the three planes. The analogic signal was digitized at a sampling frequency of 1000 Hz.
Electromyograms (EMG) of leg muscles were collected using bipolar Ag/AgCl surface electrodes (Beckmann, 8-mm diameter) on the first leg that leaves the floor (initial SW limb). Before the electrode positioning, the skin was slightly abraded and cleaned with an alcohol solution in order to reduce the interelectrode resistance to below 5 kΩ. An electrolytic gel was placed between the skin and the electrodes to insure electrical contact. The electrodes were fixed (2 cm apart center to center) over the muscle bellies for TA and lateral gastrocnemius (LG) muscles. The EMG signals were amplified and filtered using a bandwidth of 10-1000 Hz (Gould 6600), and then addressed to an analog-digital converter piloted by the Turbolab software (SM2I, France) for their digitization at a sampling rate of 1000 Hz.

Procedure
After two learning trials, the subjects performed five gait initiation trials. In order to standardize the starting conditions, the subjects placed their bare feet on foot marks drawn on the platform. They had to keep their arms by their sides, relax their jaw and fix a spot on a wall 10 m in front. The experiments took place in a quiet room with no visual perturbations.
In each test, the experimenter triggered the acquisition of the COP displacement and EMG signals. The order to initiate gait was given by the experimenter after at least 2 s of steadystate postural recording (judged by visual inspection of the EMG signal).

Data Analysis
Usually, based on COP displacement, three gait initiation phases were described (Halliday et al., 1998;Martin et al., 2002;Hass et al., 2004), the first phase represents the backward COP displacements that create the forward momentum thanks to COP-COG dissociation, the second the weight bearing transfer from SWL to STL, and the third the forward COP displacement.
These phases were identified in our study thanks to the gait initiation events using landmarks on the COP trajectory (Figure 1). Landmark 1 (L1) represents the mean position of COP during quiet stance. Landmark 2 (L2) represents the most posterior and lateral position toward the SWL of COP location. Landmark 3 (L3) represents the most posterior and lateral position toward the STL of COP location. The end of the recording of COP displacement represents the outgoing of FIGURE 1 | Kinetic and electromyographic (EMG) data used to describe gait initiation. Center of pressure (COP) displacement during gait initiation. L1 represents the position of the COP in normal quiet standing. L2 represents the backward of COP. L3 represents the COP displacement toward the stance limb (STL). Parameters used were the backward COP position during the phase 1 (L1-L2), the medio-lateral (ML) excursion, the velocity (path reported to phase duration). Insert represents tibialis anterior (TA) and lateral gastrocnemius (LG) vs. time during the weight bearing transfer from swing limb (SWL) to STL (From L2 to L3). The bold lines represent when muscle are activated (>25 ms consecutive above the threshold corresponding to the mean ± 3 standard deviation). Gray area represents the phase of coactivation of LG and TA muscles.
COP from the force plate. The identification of these landmarks was conducted with an application of Matlab Program (The Mathworks, Inc., Natick, MA, USA), under visual control. In order to characterize the COP displacement, we calculated the excursion separating two landmarks, the average velocity (sum of distance separating two points of acquisition related to time elapsed in the phase (Figure 1).
For EMG data, we have analyzed the period corresponding to weight bearing transfer from STL to SWL during which an inhibition of postural muscle and activation of motor muscles were reported. From an algorithm developed with Matlab program, we determined phasic activation of LG and TA of the SWL under visual control. The Matlab application was developed using previous methodological studies (Mickelborough et al., 2004). EMG data were smoothed with low-pass filter cut off frequency of 20 Hz. The threshold for EMG activation detection was defined as mean added with 3 standard deviations of reference phase. The muscle was considered as active after 25 ms of consecutive above the threshold. Then, we calculated relative time of coactivation of LG and TA muscles during weight bearing transfer from SWL to STL (ratio of time of coactivation with time elapse in the phase).

Statistics
Statistical analysis was carried out with Statview software (SAS Institute, Cary, NC, USA). Given that the data were normally distributed (according to a Kolmogorov-Smirnov test) and the equality of variance Levene median test) was confirmed, an unpaired t-test was used to compare the physically active group and yoga group in terms of kinetic and EMG values. The threshold for statistical significance was set to p < 0.05.
We also assessed the relationship between kinetic COP displacement parameters and muscle coactivation by applying Pearson's correlation test.

COP Movements
The yoga and physically active groups do not differ significantly in terms of displacements during the first phase of gait initiation (Figure 2A). The mean ± SD backward movement of COP position shows non-significant lower values in the yoga group than in the group of walkers (−27.8 ± 5.3 vs. −31.1 ± 13.6 mm respectively).
For the second phase of gait initiation, we characterized the excursion and velocity of COP displacement from the SWL to the STL. We did not find significant differences in COP excursion during this phase (110.8 ± 12.1 vs. 102.5 ± 19.7 mm for the yoga and physically active groups, respectively; Figure 2B). The COP velocity was significantly lower in yoga practitioners than in the walkers (555.2 ± 32.5 vs. 624.0 ± 50.3 mm.s −1 , respectively; p < 0.01; Figure 2C).

Leg Muscle Co-Activation
During gait initiation, we found significant differences in leg muscle co-activation between the two physically active groups ( Figure 2D). In fact, leg muscle co-activation is lower in yoga practitioners than in the walkers (3.9 ± 6.0% vs. 10.5 ± 5.1%, respectively; p < 0.01). The relationships between kinetic variables and leg muscle co-activation reveal that muscle co-activation is correlated with the velocity measured during the SWL-to-STL phase (r = 0.76; p < 0.01).

DISCUSSION
The objective of the present study was to assess the effect of regular yoga practice in gait initiation control in the elderly. This was characterized in terms of COP excursion, COP velocity and muscle co-activation. We hypothesized that Yoga practitioners present differences in APA during gait initiation compared to physically active older adults. Indeed, Gauchard et al. (2003) demonstrated that proprioceptive activities enhance sensory inputs and improve the subject's knowledge of the body scheme and gravity effects. Significant results were reported during the SWL to STL transfer for the COP velocity and % of leg muscle coactivation between the two groups. Lastly, the observed link between COP path and muscle co-activation duration confirmed the interaction between muscle co-activation and the COP trajectory during gait initiation.
During gait initiation, the central postural control system has two main functions: (i) maintenance of balance; and (ii) creation of the anteroposterior moment needed to produce the first step (via a stereotyped pattern of leg muscle activation). In the present study, we did not observe any significant intergroup differences in the rearward COP excursion during the first phase of gait initiation. Previous studies have found that older and disabled subjects exhibit a lower arm peak moment (i.e., a smaller COP-COG distance; Polcyn et al., 1998;Chang and Krebs, 1999;Martin et al., 2002). Based on COP displacements, the present study does not allow to demonstrate differentiated effect between type of physical activity practiced on COP rearward during the first phase of APA.
The second phase of gait initiation consisted in transferring body weight from the initial SWL to initial STL. In the present study, we reported higher average velocity of the COP in the physically active group as compared to the yoga group for a similar ML excursion. In previous studies, smoother COP displacement during gait initiation was seen after 48 weeks of tai chi practice (Hass et al., 2004) and was also described in healthy older subjects (relative to disabled subjects; Martin et al., 2002). The reason for this significant difference remains unclear: the lower COP velocity in the yoga group might be related to the specific, slow movements performed during this activity. Then, it might be the consequence of a behavioral change in yoga practitioners.
However, our results might also be related to adaptations of the central nervous system in the yoga group (relative to the physically active group). To our knowledge, the present study is the first investigation conducted to explore differentiated coactivation of the leg muscles during gait initiation in two types of physical activity practitioners. The time course analysis of EMG data assessed the time during which muscles were involved in postural maintaining (LG) and movement (TA) (Polcyn et al., 1998;Henriksson and Hirschfeld, 2005). The duration of coactivation (expressed as a % of APA duration) might be an indicator of CNS adaptation to deal with postural constraints induced by gravity and movement production during the preparation phase of gait. This muscle pattern (inhibition of postural muscle) was described in healthy young adults (Mann et al., 1979;Brunt et al., 1991Brunt et al., , 1999 and seem to be altered with aging and/or disability (Scheibel et al., 1977;Scheibel, 1985;Polcyn et al., 1998;Henriksson and Hirschfeld, 2005). In the present study, Yoga practitioners present lower TA/LG muscles coactivation than the physically active group. The result should be related to a previous study that demonstrated that muscle activation during gait initiation can reflect different behavior in counteracting and using gravity (Honeine et al., 2014).
One specific feature of the present study was the observed association between COP displacement and the EMG motor program. As previously described, the EMG sequence recorded during gait initiation can be considered as a motor program that acts on the COP's position (Crenna and Frigo, 1991). APA are pre-programmed by an open-loop mechanism, then their improvements need better body scheme knowledge. In our study, we reported a significant correlation (r = 0.76; p < 0.01) between TA/LG coactivation and COP displacement. The relationship obtained between muscle co-activation (time of coactivation/time) and velocity (path/time), demonstrates that the path length is greater when the duration of muscle co-activation is longer. Leg muscle coactivation previously reported in frailty adults might also reflect hesitation or difficulties in initiating gait (i.e., pain, motor impairment, fear of falls, etc.). Several studies have shown that changes in ankle control occur with age and may be associated with an increased risk of falling. A previous study has shown that ankle dorsiflexion was significantly delayed and its amplitude was lower for fallers during gait (Kemoun et al., 2002). Simoneau et al. (2007) explained the decrease in dorsiflexion maximal voluntary contraction torque with aging by neural factor (Simoneau et al., 2007). The question of the benefit of reducing muscle co-activation in frailty adults should then be explored.
Our results suggest that the neuro-adaptation related to yoga performance can modify leg muscle pattern during gait initiation.
This result should also be related to the reduction in muscle coactivation during other dynamic tasks after practice (Chu et al., 2009;Chen et al., 2012a,b;Nagai et al., 2012).
However, the present study may have some limitations inherent to cross-sectional studies. Even if our inclusion criteria allow us to select a healthy elderly population in this crosssectional study, the two groups might not present similar health conditions. Comparison with other groups should help to understand the effects of physical activity on gait initiation by comparing our results with groups of young adults, sedentary older adults or with other type of physical activity (exercises at a fast pace way requiring balance). We can also note that due to the absence of kinematic data, we could not determine the position of the COG. Therefore, the COP-COG dissociation could not be assessed in the present study.

CONCLUSION
The actual results obtained did not accurately demonstrated that gait initiation is more efficient (in terms of COP-COG dissociation and in term of amplitude of the first step) in yoga practitioners than in regular walkers. However, Yoga practitioners showed slower COP displacement associated with lower leg muscle coactivation. This last point result might be a marker of the use of a different strategy to prepare the first step. Further studies are needed to explore the clinical interests of proprioceptive activities on muscle coactivation during gait initiation and the relationship between gait initiation efficiency (COP-COG, step length) and leg muscle coactivation.

AUTHOR CONTRIBUTIONS
TL, P-LD and SA: project creation. TL: data collection. TL, P-LD and AT: data analysis. TL, P-LD, AT and SA: discussed the results; revised the manuscript. TL and AT: drafted the manuscript.

FUNDING
This work was funded by Université de Picardie Jules Verne and Centre hospitalier Universitaire d'Amiens.