International ISSN: 2471-0016 ICPJL

Clinical Pathology Journal
Mini Review
Volume 2 Issue 3 - 2016
SOD1 Pathology in ALS: TDP or not TDP that is the Question
Hortle E*, Don EK, Stoddart JJ, Radford R, Laird AS, Morsch M, Chung R and Cole NJ
Department of Biomedical Sciences, Macquarie University, Australia
Received: April 15, 2016 | Published: May 09, 2016

*Corresponding author: Elinor Hortle, Department of Biomedical Sciences, Faculty of Medicine and Health Sciences, Ground Floor, 2 Technology Place, F10A Building, Macquarie University, NSW 2109, Australia, T: +61298502723; F: +61298502701; Email:

Citation: Hortle E, Don EK, Stoddart JJ, Radford R, Laird AS, et al. (2016) SOD1 Pathology in ALS: TDP or not TDP that is the Question. Int Clin Pathol J 2(3): 00038. DOI: 10.15406/icpjl.2016.02.00038

Abstract

Amyotrophic Lateral Sclerosis (ALS) is a fatal motor neuron disease with no cure. Patients experience degeneration of both upper and lower motor neurons, which leads to paralysis and eventual death, usually within 2-5 years of onset. Although ALS was first described in 1824, there remains a lack of a detailed understanding of the mechanisms that culminate in the progressive spread of ALS pathology and subsequent motor neuron loss. Current understanding highlights Cu/Zn superoxide dismutase (SOD1) and transitive response DNA binding protein 43kDa (TDP-43) proteopathies as two of the main pathologies observed in ALS. However, despite the similarities between the two, they have historically been studied in isolation. Here we consider the emerging body of evidence that suggests that the disease mechanisms of the two proteopathies may be linked. We discuss the possibility that insights might be gained from studying the interactions between the two pathologies, instead of continuing to examine them in isolation in order to truly understand ALS pathology.

Keywords: ALS; MND; SOD1; TDP-43; Pathology; Proteopathy

Abbreviations

ALS: Amyotrophic Lateral Sclerosis; C9ORF72: Chromosome 9 Open Reading frame 72; FALS: Familiar ALS; FUS: Fused In Sarcoma; HEK293: Human Embryonic Kidney 293; SALS: Sporadic ALS; shRNA: Small Hairpin RNA; siRNA: Small Interfering RNA; SOD1: Cu/Zn Superoxide Dismutase; TDP-43: Transactive Response DNA Binding Protein 43 kDa; VHL: Von Hippel Lindau

Introduction

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease, with no cure, that affects both the upper and lower motor neurons. ALS patients experience muscle weakness that starts focally, but spreads, culminating in paralysis and in most cases death 2-5 years after diagnosis [1]. Currently we do not have a detailed understanding of what triggers ALS, or the mechanisms responsible for the progressive spreading of ALS pathology. The majority of cases are sporadic (SALS), occurring with no known genetic cause; around 10% of cases are familiarly inherited (FALS). Mutations in over 33 genes have been found to cause ALS [2-4], notably C9ORF72, FUS, TARDBP (which encodes TDP-43) and Cu/Zn superoxide dismutase (SOD1) [5-8].

A key pathological hallmark of ALS-including in those patients with no known genetic cause - is the aggregation of ubiquitinated proteins in brain and spinal cord neurons. In 2006 TDP-43 was identified as a major component of ubiquitinated inclusions in FTLD and ALS cases [9,10]. In the majority of cases (all SALS patients and most SOD-1-negative FALS patients, but not in SOD1 related ALS [11,12] these aggregates (also known as inclusions) consist largely of TDP-43. TDP-43 and SOD1 proteopathies have previously been considered to represent separate parts of the same spectrum of the disease, largely because TDP-43 inclusions have not been observed in SOD1 FALS cases [11,13,14], and early studies did not detect SOD1 in SALS protein inclusions [15]. Therefore, most research focuses on either TDP-43 or SOD1 pathology separately, and very little examines the effect that each may have on the other. Now, a new body of literature is emerging suggesting that TDP-43 dysregulation may induce SOD1 pathology, and that the two may be linked.

Discussion

SOD1 is a detoxification enzyme localized to the cytoplasm and outer mitochondrial membrane, where it converts superoxide to hydrogen peroxide. In SOD1 FALS and SOD1 SALS cases (about 5% of ALS cases), inclusions containing mutant protein are observed in both astrocytes and neurons [15,16]. Although the precise cause of SOD1 aggregation has not been elucidated, many studies have implicated molecular chaperones in this process [17-19]. SOD1 transgenic rodents-expressing human FALS associated SOD1 mutations-have been extensively studied, and recapitulate many features of ALS, including axonal and mitochondrial dysfunction, progressive neuromuscular dysfunction, gliosis, motor neuron loss, and the formation of neuronal protein inclusions containing mutant SOD1 [20].

TDP-43 is a primarily nuclear localised transcription repressor that functions in RNA metabolism, splicing and translational regulation [21]. Briefly 97% of ALS cases [9] - including those with and without mutations in TARDBP - display characteristic pathology in neurons and glial cells: mislocalisation and accumulation of dense, insoluble aggregates containing phosphorylated TDP-43 in the cytoplasm. Numerous animal models both of TDP-43 over- and under-expression have been described [20,21]. One recent model, in which mice express a form of cytoplasmic human TDP-43, recapitulates several of the features of ALS, including cytoplasmic TDP-43 aggregation, nuclear clearance of mouse TDP-43, brain atrophy, muscle denervation, motor neuron loss, and progressive motor impairment leading to death [22].

SOD1 and TDP-43; are they connected?

Despite the fact that SOD1 and TDP-43 proteins have very different roles within the cell, there are marked similarities between the two resulting proteopathies. For example, both mutant SOD1 and mutant TDP-43 are able to induce misfolding and aggregation of their respective wild-type (wt) proteins [23,24]. Both are able to spread misfolded and toxic protein from cell to cell [14,23,25-30]. Both have been shown to induce motor neuron death in a non-cell autonomous fashion; that is, when mutant protein is introduced to astrocytes in mixed cultures or in vivo models, these astrocytes can then induce selective motor neuron death [31,32] (although this finding for TDP-43 is contested [33]).

A small number of studies have suggested that SOD1 pathology may be relevant to all ALS cases; not just the 5% with known mutations in SOD1. Some have detected misfolded wild-type SOD1 in both non-SOD1 FALS, as well as SALS patients [34-37], suggesting that SOD1 mutations are not required for the formation of SOD1 pathology. Similarly, in mixed primary cell cultures derived from both FALS and SALS patients, shRNA knockdown of wild-type SOD1 resulted in protection of motor neurons, in the absence of any previously reported disease variants in SOD1, FUS or TDP-43 [38], supporting early hypotheses that treating SOD1 pathology may be beneficial in all ALS cases, regardless of mutation status [39].

A direct connection between SOD1 and TDP-43 proteopathies has also been suggested by several in vitro studies. Somalinga et al. [40] showed that in HeLa TetOn cells, the amount of soluble SOD1 was increase by siRNA knockdown of TDP-43, and decreased by TDP-43 over-expression [40], suggesting TDP-43 can regulate the expression of SOD1. Xia et al showed that in both human embryonic kidney 293 (HEK293) cells and mouse neuroblastoma cells, knockdown of TDP-43 impaired SOD1 aggresome formation, resulting in a higher number of smaller SOD1 aggregates within the cell [41]. Similarly, Uchida et al. found that in HEK293 cells loss of TDP-43 could lead to an increase in von Hippel Lindau (VHL) protein levels, and that VHL overexpression increased the number of inclusion harbouring mutant SOD1 cells [42]. Together these studies suggest that TDP-43 can induce SOD1 pathology (although it is unclear whether changes to the number or size of SOD1 inclusions results in increased toxicity).

Perhaps the most intriguing evidence of a connection between SOD1, TDP-43 and the spreading of ALS pathology, comes from two recent studies by Pokrishevsky et al. [43], who were able to show that in mouse primary spinal cord cultures, transfection of both wild-type TDP-43, as well as TDP-43 carrying disease linked mutations, induced misfolding of wild-type SOD1. Moreover, they demonstrated that this aberrant SOD1 protein could propagate from cell-to-cell via conditioned media, and seed cytotoxic misfolding of wild-type SOD1 in the recipient cells. Interestingly, in this system, no transmission of TDP-43 pathology was observed [35,43] as previously reported in other systems [14].

Some caveats

The studies listed above suggest a connection between SOD1 and TDP43 proteopathies in ALS, but further work is required before they can be deemed conclusive. It is not yet clear whether misfolded wild-type SOD1 really is present in non-SOD1 ALS cases. Some studies have found no mutant or misfolded SOD1 in samples from non-SOD1 FALS and SALS patients [44-46]. Some have suggested that this discrepancy may be due to the different affinities of the various antibodies used in these experiments; an ‘inferior’ antibody may fail to detect misfolded SOD1, even if it is present [45]. However, given that even when using the same antibody, some studies have detected misfolded SOD134, and others have not [46], the finding remains unclear.

Most of the in vitro studies listed here propose that TDP-43 pathology is upstream of, and induces, SOD1 pathology. However, there is some evidence that the reverse may also be true: the accumulation of ubiquitinated TDP-43 has been detected in both humans and mice carrying SOD1 mutations [47,48]. In these cases, SOD1 pathology is presumably inducing TDP-43 pathology. Given the small number of studies conducted to date, it is difficult to conclude which of these alternatives is most relevant to human disease.

The ability to draw conclusions about the role of TDP-43 in inducing SOD1 pathology is also hampered by our lack of understanding of the effect of TDP-43 in disease. Both over- and under-expression of wild-type and mutant TDP-43 can induce ALS-like phenotypes in animal models20; equally, over-expression of the truncated C- terminal fragment can induce an ALS phenotype, without changes to the abundance of full length TDP-43 protein [49]. This lack of clarity has, in some cases, led to confusion within the literature. For example, both Xia et al and Cheng et al tested the effect of rapamycin on their respective drosophila models of TDP-43 induced ALS. While Xia et al. [41] found that rapamycin worsened ALS symptoms, Cheng et al found that it alleviated ALS symptoms [41,50]. This apparent contradiction can perhaps be explained by the fact that the former were knocking down TDP-43 to induce an ALS-like phenotype, and the latter were over-expressing TDP-43 to achieve the same effect. Therefore, it is difficult to directly relate the above mentioned knock-down and over expression models to human biology, and each study has to be considered in its own context.

Conclusion

The hypothesis that TDP-43 and SOD1 pathology are linked through the propagation of misfolded protein is yet to be confirmed, but is a promising avenue for further research. More than twenty years of studying TDP-43 and SOD1 in isolation has yielded key insights into the cellular and molecular pathology of ALS, but an unfortunate lack of translational findings from the lab to the clinic [51,52]. It has recently become clear that the pathogenesis of ALS is converging on two linked pathways - RNA processing and protein homeostasis-suggesting that there are conserved pathological mechanisms in this genetically heterogeneous disease [9,12]. As more evidence is compiled towards this hypothesis, it is becoming increasingly important to study the pathogenesis of ALS in the context of linked disease mechanisms. In order to understand the true pathological mechanism of this disease, it is of great importance to study the interactions between all ALS pathologies, instead of continuing to examine these proteopathies in isolation.

We are in an exciting time for ALS and neurodegenerative disease research. As the pace of discovery accelerates, it is important to keep an open mind. In vivo models may prove to be key in resolving the interactions between currently known genes, proteins, and mechanisms. All information that can add to our knowledge of the biology of ALS is critical to ending forever the slings and arrows of this devastating disease. It is clear that SOD1 and TDP-43 are key players in ALS; recent and future discovery of new genes to the ALS stage can only help us understand whether there is a divergence or a convergence of TDP-43 and SOD1 mechanisms in ALS.

Acknowledgment

We would like to thank The Snow Foundation, The Rebecca Cooper Medical Research Foundation and BitFury.org for their funding. Dr Nicholas Cole is supported by the National Health and Medical Research Council (NHMRC), project grant [GNT1034816], and Macquarie University. Dr Angela Laird is supported by the National Health and Medical Research Council (NHMRC), project grant (GNT1069235) and the Machado Joseph Disease Foundation.

References

  1. Kiernan MC, Vucic S, Cheah BC, Turner MR, Eisen A, et al. (2011) Amyotrophic lateral sclerosis. Lancet 377(9769): 942-955.
  2. Finsterer J, Stöllberger C, Güler N (2014) Non-compaction delineates amyotrophic lateral sclerosis from metabolic myopathy. Int J Cardiol 176(1): 277-279.
  3. Renton AE, Chiò A, Traynor BJ (2014) State of play in amyotrophic lateral sclerosis genetics. Nat neurosci 17(1): 17-23.
  4. Boylan K (2015)Familial Amyotrophic Lateral Sclerosis. Neurol Clin 33(4): 807-830.
  5. Lattante S, Ciura S, Rouleau GA, Kabashi E (2015) Defining the genetic connection linking amyotrophic lateral sclerosis (ALS) with frontotemporal dementia (FTD). Trends Genet 31(5): 263-273.
  6. Mancuso R, Navarro X (2015) Amyotrophic lateral sclerosis: Current perspectives from basic research to the clinic. Prog neurobiol 133: 1-26.
  7. De Jesus-Hernandez M, Mackenzie IR, Boeve BF, Boxer AL, Baker M, et al. (2011) Expanded GGGGCC hexanucleotide repeat in noncoding region of C9ORF72 causes chromosome 9p-linked FTD and ALS. Neuron 72(2): 245-256.
  8. Renton AE, Majounie E, Waite A, Simón-Sánchez J, Rollinson S, et al. (2011) A hexanucleotide repeat expansion in C9ORF72 is the cause of chromosome 9p21-linked ALS-FTD. Neuron 72(2): 257-268.
  9. Arai T, Hasegawa M, Akiyama H, Ikeda K, Nonaka T, et al. (2006) TDP-43 is a component of ubiquitin-positive tau-negative inclusions in frontotemporal lobar degeneration and amyotrophic lateral sclerosis. Biochem Biophys Res Commun 351(3): 602-611.
  10. Neumann M, Sampathu DM, Kwong LK, Truax AC, Micsenyi MC, et al. (2006)Ubiquitinated TDP-43 in frontotemporal lobar degeneration and amyotrophic lateral sclerosis. Science 314(5796): 130-133.
  11. Mackenzie IR, Bigio EH, Ince PG, Geser F, Neumann M, et al. (2007) Pathological TDP-43 distinguishes sporadic amyotrophic lateral sclerosis from amyotrophic lateral sclerosis with SOD1 mutations. Ann Neurol 61(5): 427-434.
  12. Tan CF, Eguchi H, Tagawa A, Onodera O, Iwasaki T, et al. (2007) TDP-43 immunoreactivity in neuronal inclusions in familial amyotrophic lateral sclerosis with or without SOD1 gene mutation. Acta Neuropathol 113(5): 535-542.
  13. Farrawell NE, Lambert-Smith IA, Warraich ST, Blair IP, Saunders DN, et al. (2015) Distinct partitioning of ALS associated TDP-43, FUS and SOD1 mutants into cellular inclusions. Sci Rep 5: 13416.
  14. Radford RA, Morsch M, Rayner SL, Cole NJ, Pountney DL et al. (2015) The established and emerging roles of astrocytes and microglia in amyotrophic lateral sclerosis and frontotemporal dementia. Front Cell Neurosci 9: 414.
  15. Kato S, Takikawa M, Nakashima K, Hirano A, Cleveland DW, et al. (2000) New consensus research on neuropathological aspects of familial amyotrophic lateral sclerosis with superoxide dismutase 1 (SOD1) gene mutations: inclusions containing SOD1 in neurons and astrocytes. Amyotroph Lateral Scler Other Motor Neuron Disord 1(3): 163-184.
  16. Bruijn LI, Houseweart MK, Kato S, Anderson KL, Anderson SD, et al. (1998) Aggregation and motor neuron toxicity of an ALS-linked SOD1 mutant independent from wild-type SOD1. Science 281(5384): 1851-1854.
  17. Novoselov SS,Mustill WJ, Gray AL, Dick JR, Kanuga N, et al. (2013) Molecular chaperone mediated late-stage neuroprotection in the SOD1(G93A) mouse model of amyotrophic lateral sclerosis. PloS one 8(8): e73944.
  18. Wang J, Farr GW, Zeiss CJ, Rodriguez-Gil DJ, Wilson JH, et al. (2009) Progressive aggregation despite chaperone associations of a mutant SOD1-YFP in transgenic mice that develop ALS. Proc Natl Acad Sci U S A 106(5): 1392-1397.
  19. Nagy M, Fenton WA, Li D, Furtak K, Horwich AL (2016) Extended survival of misfolded G85R SOD1-linked ALS mice by transgenic expression of chaperone Hsp110. Proc Natl Acad Sci U S A 13.
  20. McGoldrick P, Joyce PI, Fisher EM, Greensmith L (2013) Rodent models of amyotrophic lateral sclerosis. Biochim Biophys Acta 1832(9): 1421-1436.
  21. van Eersel J, Ke YD, Gladbach A, Bi M, Götz J, et al. (2011) Cytoplasmic accumulation and aggregation of TDP-43 upon proteasome inhibition in cultured neurons. PloS One 6(7): e22850.
  22. Walker AK, Spiller KJ, Ge G, Zheng A, Xu Y, et al. (2015) Functional recovery in new mouse models of ALS/FTLD after clearance of pathological cytoplasmic TDP-43. Acta neuropathol 130(5): 643-660.
  23. Grad LI, Guest WC, Yanai A, Pokrishevsky E, O'Neill MA, et al. (2011) Intermolecular transmission of superoxide dismutase 1 misfolding in living cells. Proc Natl Acad Sci U S A 108(39): 16398-16403.
  24. Winton MJ, Igaz LM, Wong MM, Kwong LK, Trojanowski JQ, L et al. (2008) Disturbance of nuclear and cytoplasmic TAR DNA-binding protein (TDP-43) induces disease-like redistribution, sequestration, and aggregate formation. T J Biol Chem 283(19): 13302-13309.
  25. Maniecka Z, Polymenidou (2015) From nucleation to widespread propagation: A prion-like concept for ALS. Virus Res 207: 94-105.
  26. Smethurst P, Sidle KC, Hardy J (2015) Prion-like mechanisms of transactive response DNA binding protein of 43 kDa (TDP-43) in amyotrophic lateral sclerosis (ALS). Neuropathol Appl Neurobiol 41(5): 578-597.
  27. Lee S, Kim HJ (2015) Prion-like Mechanism in Amyotrophic Lateral Sclerosis: are Protein Aggregates the Key? Exp Neurobiol 24(1): 1-7.
  28. Münch C, O'Brien J, Bertolotti A (2011) Prion-like propagation of mutant superoxide dismutase-1 misfolding in neuronal cells. Proc Natl Acad Sci U S A 108(9): 3548-3553.
  29. Grad LI, Yerbury JJ, Turner BJ, Guest WC, Pokrishevsky E, et al. (2014) Intercellular propagated misfolding of wild-type Cu/Zn superoxide dismutase occurs via exosome-dependent and -independent mechanisms. Proc Natl Acad Sci U S A 111(9): 3620-3625.
  30. Ludolph AC, Brettschneider J (2015) TDP-43 in amyotrophic lateral sclerosis - is it a prion disease? Eur J Neurol 22(5): 753-761.
  31. Tong J, Huang C, Bi F, Wu Q, Huang B, et al. (2013) Expression of ALS-linked TDP-43 mutant in astrocytes causes non-cell-autonomous motor neuron death in rats. EMBO 32(13): 1917-1926.
  32. Marchetto MC, Muotri AR, Mu Y, Smith AM, Cezar GG, et al. (2008) Non-cell-autonomous effect of human SOD1 G37R astrocytes on motor neurons derived from human embryonic stem cells. Cell Stem Cell 3(6): 649-657.
  33. Serio A, Bilican B, Barmada SJ, Ando DM, Zhao C, et al. (2013) Astrocyte pathology and the absence of non-cell autonomy in an induced pluripotent stem cell model of TDP-43 proteinopathy. Proc Natl Acad Sci U S A 110(12): 4697-4702.
  34. Bosco DA, Morfini G, Karabacak NM, Song Y, Gros-Louis F, et al. (2010) Wild-type and mutant SOD1 share an aberrant conformation and a common pathogenic pathway in ALS. Nat Neurosci 13(11): 1396-1403.
  35. Pokrishevsky E, Grad LI, Yousefi M, Wang J, Mackenzie IR, et al. (2012) Aberrant localization of FUS and TDP43 is associated with misfolding of SOD1 in amyotrophic lateral sclerosis. PloS One 7(4): e35050.
  36. Forsberg K, Jonsson PA, Andersen PM, Bergemalm D, Graffmo KS, et al. (2010) Novel antibodies reveal inclusions containing non-native SOD1 in sporadic ALS patients. PloS One 5(7): 11552.
  37. Guareschi S, Cova E, Cereda C, Ceroni M, Donetti E, et al. (2012) An over-oxidized form of superoxide dismutase found in sporadic amyotrophic lateral sclerosis with bulbar onset shares a toxic mechanism with mutant SOD1. Proc Natl Acad Sci U S A 109(13): 5074-5079.
  38. Haidet-Phillips AM, Hester ME, Miranda CJ, Meyer K, Braun L, et al. (2011) Astrocytes from familial and sporadic ALS patients are toxic to motor neurons. Nat Biotechnol 29(9): 824-828.
  39. K Kabashi E, Valdmanis PN, Dion P, Rouleau GA (2007) Oxidized/misfolded superoxide dismutase-1: the cause of all amyotrophic lateral sclerosis? Ann Neurol 62(6): 553-559.
  40. Somalinga BR, Day CE, Wei S, Roth MG, Thomas PJ (2012) TDP-43 identified from a genome wide RNAi screen for SOD1 regulators. PloS One 7(4): e35818.
  41. Xia Q, Wang H, Zhang Y, Ying Z, Wang G (2015) Loss of TDP-43 Inhibits Amyotrophic Lateral Sclerosis-Linked Mutant SOD1 Aggresome Formation in an HDAC6-Dependent Manner. Journal of Alzheimer's disease : J Alzheimers Dis 45(2): 373-386.
  42. Uchida T, Tamaki Y, Ayaki T, Shodai A, Kaji S, et al. (2016) CUL2-mediated clearance of misfolded TDP-43 is paradoxically affected by VHL in oligodendrocytes in ALS. Sci Rep 6: 19118.
  43. Pokrishevsky E, Grad LI, Cashman NR (2016) TDP-43 or FUS-induced misfolded human wild-type SOD1 can propagate intercellularly in a prion-like fashion. Sci Reps 6: 22155.
  44. Liu HN, Sanelli T, Horne P, Pioro EP, Strong MJ, et al. (2009) Lack of evidence of monomer/misfolded superoxide dismutase-1 in sporadic amyotrophic lateral sclerosis. Ann Neurol 66(1): 75-80.
  45. Rotunno MS, Bosco DA (2013) An emerging role for misfolded wild-type SOD1 in sporadic ALS pathogenesis. Front Cell Neurosci 7: 253.
  46. Brotherton TE, Li Y, Cooper D, Gearing M, Julien JP, et al. (2012) Localization of a toxic form of superoxide dismutase 1 protein to pathologically affected tissues in familial ALS. Proc Natl Acad Sci U S A 109(14): 5505-5510.
  47. Cai M, Lee KW, Choi SM, Yang EJ (2015) TDP-43 modification in the hSOD1(G93A) amyotrophic lateral sclerosis mouse model. Neurol Res 37(3): 253-262.
  48. Maekawa S, Leigh PN, King A, Jones E, Steele JC, et al. (2009) TDP-43 is consistently co-localized with ubiquitinated inclusions in sporadic and Guam amyotrophic lateral sclerosis but not in familial amyotrophic lateral sclerosis with and without SOD1 mutations. Neuropathology 29(6): 672-683.
  49. Caccamo A, Shaw DM, Guarino F, Messina A, Walker AW, et al. (2015) Reduced protein turnover mediates functional deficits in transgenic mice expressing the 25 kDa C-terminal fragment of TDP-43. Hum Mol Genet 24(16): 4625-4635.
  50. Cheng CW, Lin MJ, Shen CK (2015) Rapamycin alleviates pathogenesis of a new Drosophila model of ALS-TDP. J Neurogenet 29(2-3): 59-68.
  51. Gordon PH, Meininger V (2011) How can we improve clinical trials in amyotrophic lateral sclerosis? Nat Rev Neurol 7(11): 650-654.
  52. Clerc P, Lipnick S, Willett C (2015) A look into the future of ALS research. Drug Discov Today.
© 2014-2016 MedCrave Group, All rights reserved. No part of this content may be reproduced or transmitted in any form or by any means as per the standard guidelines of fair use.
Creative Commons License Open Access by MedCrave Group is licensed under a Creative Commons Attribution 4.0 International License.
Based on a work at http://medcraveonline.com
Best viewed in Mozilla Firefox | Google Chrome | Above IE 7.0 version | Opera |Privacy Policy