Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disorder with no cure, and elucidation of the mechanisms mediating neuronal death in this neuropathology is crucial to develop effective treatments. It has recently been demonstrated in animal models that the Wnt family of proteins is involved in this neuropathology, although its potential involvement in case of humans is almost unknown. We analyzed the expression of Wnt signaling components in healthy and ALS human spinal cords by quantitative RT-PCR, and we found that most Wnt ligands, modulators, receptors, and co-receptors were expressed in healthy controls. Moreover, we observed clear alterations in the mRNA expression of different components of this family of proteins in human spinal cord tissue from ALS cases. Specifically, we detected a significant increase in the mRNA levels of Wnt3, Wnt4, Fz2, and Fz8, together with several non-significant increases in the mRNA expression of other genes such as Wnt2b, Wnt5a, Fz3, Lrp5, and sFRP3. Based on these observations and on previous reports of studies performed in animal models, we evaluated with immunohistochemistry the protein expression patterns of Fz2 and Fz5 receptors and their main ligand Wnt5a in control samples and ALS cases. No substantial changes were observed in Fz5 protein expression pattern in ALS samples. However, we detected an increase in the amount of Fz2+ astrocytes in the borderline between gray and white matter at the ventral horn in ALS samples. Finally, Wnt5a expression was observed in neurons and astrocytes in both control and ALS samples, although Wnt5a immunolabeling in astroglial cells was significantly increased in ALS spinal cords in the same region where changes in Fz2 were observed. Altogether, these observations strongly suggest that the Wnt family of proteins, and more specifically Fz2 and Wnt5a, might be involved in human ALS pathology.
Wnt Frizzled ALS Human Spinal cord
This is a preview of subscription content, log in to check access.
We would like to thank Sandra Vázquez and Virginia Pérez for their outstanding technical help, as well as Dr. Daniel García-Ovejero from the Group of Neuroinflammation for sharing with us his deep knowledge and methodology on the human histology used in the study. We are extremely grateful to all individuals who agreed to donate their tissues to research.
This work was funded by the Fondo de Investigaciones Sanitarias (FIS) (Grant PI12-02895, co-funded by Fondo Europeo de Desarrollo Regional (FEDER)) from the Instituto de Salud Carlos III (ISCIII).
Compliance with Ethical Standards
Statement on the Welfare of Animals
This article does not contain any studies with animals performed by any of the authors.
Conflict of Interest
The authors declare they have no conflict of interests.
Statement on Sample Extraction and Processing from ALS Patients
Postmortem samples from all individual participants were obtained with written informed consent prior to inclusion in the study, which has been conducted according to 1964 Declaration of Helsinki principles and its later amendments, following the ethical rule of the Hospital Universitari de Bellvitge (Spain) and according to the Directive 2004/23/EC of the European Parliament and of the Council. All samples were handled after approval by the Clinical Research Ethical Committee (CEIC) in Toledo (Spain) and in accordance with Spanish law and International Guidelines (LOPD 15/1999; RD 1720/2007; 1964 Helsinki declaration and its later amendments or comparable ethical standards).
Pre-incubation of Fz2, Fz5 and Wnt5a antibodies with their corresponding blocking peptides. The pre-incubation was performed with 10-fold weight/weight excess for Fz2 and Fz5, and 20-fold weight/weight excess for Wnt5a. Representative images showing the immunostaining blockade of Fz2 (b1- b3), Fz5 (a1- a3) and Wnt5a (c1- c3). Scale bars = 100μm. (PNG 1436 kb)
Lacomblez L, Bensimon G, Leigh PN, Guillet P, Meininger V (1996) Dose-ranging study of riluzole in amyotrophic lateral sclerosis. Amyotrophic Lateral Sclerosis/Riluzole Study Group II. Lancet 347(9013):1425–1431Google Scholar
Gonzalez-Fernandez C, Fernandez-Martos CM, Shields S, Arenas E, Rodriguez FJ (2013) Wnts are expressed in the spinal cord of adult mice and are differentially induced after injury. J Neurotrauma 31(6):565–581. https://doi.org/10.1089/neu.2013.3067Google Scholar
Tapia-Rojas C, Inestrosa NC (2017) Wnt signaling loss accelerates the appearance of neuropathological hallmarks of Alzheimer’s disease in J20-APP transgenic and wild-type mice. J Neurochem 144:443–465. https://doi.org/10.1111/jnc.14278Google Scholar
Li X, Guan Y, Chen Y, Zhang C, Shi C, Zhou F, Yu L, Juan J et al (2013) Expression of Wnt5a and its receptor Fzd2 is changed in the spinal cord of adult amyotrophic lateral sclerosis transgenic mice. Int J Clin Exp Pathol 6(7):1245–1260Google Scholar
Chen Y, Guan Y, Liu H, Wu X, Yu L, Wang S, Zhao C, Du H et al (2012) Activation of the Wnt/beta-catenin signaling pathway is associated with glial proliferation in the adult spinal cord of ALS transgenic mice. Biochem Biophys Res Commun 420(2):397–403. https://doi.org/10.1016/j.bbrc.2012.03.006Google Scholar
Gonzalez-Fernandez C, Mancuso R, Del Valle J, Navarro X, Rodriguez FJ (2016) Wnt signaling alteration in the spinal cord of amyotrophic lateral sclerosis transgenic mice: special focus on Frizzled-5 cellular expression pattern. PLoS One 11(5):e0155867. https://doi.org/10.1371/journal.pone.0155867Google Scholar
Minami Y, Oishi I, Endo M, Nishita M (2010) Ror-family receptor tyrosine kinases in noncanonical Wnt signaling: their implications in developmental morphogenesis and human diseases. Dev Dyn 239(1):1–15. https://doi.org/10.1002/dvdy.21991Google Scholar
Garcia-Ovejero D, Arevalo-Martin A, Paniagua-Torija B, Florensa-Vila J, Ferrer I, Grassner L, Molina-Holgado E (2015) The ependymal region of the adult human spinal cord differs from other species and shows ependymoma-like features. Brain 138(Pt 6):1583–1597. https://doi.org/10.1093/brain/awv089Google Scholar
Johann S, Heitzer M, Kanagaratnam M, Goswami A, Rizo T, Weis J, Troost D, Beyer C (2015) NLRP3 inflammasome is expressed by astrocytes in the SOD1 mouse model of ALS and in human sporadic ALS patients. Glia 63(12):2260–2273. https://doi.org/10.1002/glia.22891Google Scholar
Schindelin J, Arganda-Carreras I, Frise E, Kaynig V, Longair M, Pietzsch T, Preibisch S, Rueden C et al (2012) Fiji: an open-source platform for biological-image analysis. Nat Methods 9(7):676–682. https://doi.org/10.1038/nmeth.2019Google Scholar
Paniagua-Torija B, Arevalo-Martin A, Ferrer I, Molina-Holgado E, Garcia-Ovejero D (2015) CB1 cannabinoid receptor enrichment in the ependymal region of the adult human spinal cord. Sci Rep 5:17745. https://doi.org/10.1038/srep17745Google Scholar
Kawamata T, Akiyama H, Yamada T, McGeer PL (1992) Immunologic reactions in amyotrophic lateral sclerosis brain and spinal cord tissue. Am J Pathol 140(3):691–707Google Scholar
Schiffer D, Cordera S, Cavalla P, Migheli A (1996) Reactive astrogliosis of the spinal cord in amyotrophic lateral sclerosis. J Neurol Sci 139(Suppl):27–33Google Scholar
Vucic S, Kiernan MC (2009) Pathophysiology of neurodegeneration in familial amyotrophic lateral sclerosis. Curr Mol Med 9(3):255–272Google Scholar
Caricasole A, Copani A, Caraci F, Aronica E, Rozemuller AJ, Caruso A, Storto M, Gaviraghi G et al (2004) Induction of Dickkopf-1, a negative modulator of the Wnt pathway, is associated with neuronal degeneration in Alzheimer's brain. J Neurosci 24(26):6021–6027. https://doi.org/10.1523/JNEUROSCI.1381-04.2004Google Scholar
Wei H, Qin ZH, Senatorov VV, Wei W, Wang Y, Qian Y, Chuang DM (2001) Lithium suppresses excitotoxicity-induced striatal lesions in a rat model of Huntington’s disease. Neuroscience 106(3):603–612Google Scholar
L'Episcopo F, Tirolo C, Testa N, Caniglia S, Morale MC, Cossetti C, D'Adamo P, Zardini E et al (2011) Reactive astrocytes and Wnt/beta-catenin signaling link nigrostriatal injury to repair in 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine model of Parkinson’s disease. Neurobiol Dis 41(2):508–527. https://doi.org/10.1016/j.nbd.2010.10.023Google Scholar
Parish CL, Castelo-Branco G, Rawal N, Tonnesen J, Sorensen AT, Salto C, Kokaia M, Lindvall O et al (2008) Wnt5a-treated midbrain neural stem cells improve dopamine cell replacement therapy in parkinsonian mice. J Clin Invest 118(1):149–160. https://doi.org/10.1172/JCI32273Google Scholar
Miyashita T, Koda M, Kitajo K, Yamazaki M, Takahashi K, Kikuchi A, Yamashita T (2009) Wnt-Ryk signaling mediates axon growth inhibition and limits functional recovery after spinal cord injury. J Neurotrauma 26(7):955–964. https://doi.org/10.1089/neu.2008.0776Google Scholar
Chen Y, Wang Q, Wang Q, Liu H, Zhou F, Zhang Y, Yuan M, Zhao C et al (2017) DDX3 binding with CK1epsilon was closely related to motor neuron degeneration of ALS by affecting neurite outgrowth. Am J Transl Res 9(10):4627–4639Google Scholar
de Oliveira GP, Maximino JR, Maschietto M, Zanoteli E, Puga RD, Lima L, Carraro DM, Chadi G (2014) Early gene expression changes in skeletal muscle from SOD1(G93A) amyotrophic lateral sclerosis animal model. Cell Mol Neurobiol 34(3):451–462. https://doi.org/10.1007/s10571-014-0029-xGoogle Scholar
Bhinge A, Namboori SC, Zhang X, VanDongen AMJ, Stanton LW (2017) Genetic correction of SOD1 mutant iPSCs reveals ERK and JNK activated AP1 as a driver of neurodegeneration in amyotrophic lateral sclerosis. Stem Cell Rep 8(4):856–869. https://doi.org/10.1016/j.stemcr.2017.02.019Google Scholar
Varela-Nallar L, Alfaro IE, Serrano FG, Parodi J, Inestrosa NC (2010) Wingless-type family member 5A (Wnt-5a) stimulates synaptic differentiation and function of glutamatergic synapses. Proc Natl Acad Sci U S A 107(49):21164–21169. https://doi.org/10.1073/pnas.1010011107Google Scholar
Turner MR, Cagnin A, Turkheimer FE, Miller CC, Shaw CE, Brooks DJ, Leigh PN, Banati RB (2004) Evidence of widespread cerebral microglial activation in amyotrophic lateral sclerosis: an [11C](R)-PK11195 positron emission tomography study. Neurobiol Dis 15(3):601–609. https://doi.org/10.1016/j.nbd.2003.12.012Google Scholar
Alexianu ME, Kozovska M, Appel SH (2001) Immune reactivity in a mouse model of familial ALS correlates with disease progression. Neurology 57(7):1282–1289Google Scholar
Hall ED, Oostveen JA, Gurney ME (1998) Relationship of microglial and astrocytic activation to disease onset and progression in a transgenic model of familial ALS. Glia 23(3):249–256Google Scholar
Keller AF, Gravel M, Kriz J (2009) Live imaging of amyotrophic lateral sclerosis pathogenesis: disease onset is characterized by marked induction of GFAP in Schwann cells. Glia 57(10):1130–1142. https://doi.org/10.1002/glia.20836Google Scholar
Levine JB, Kong J, Nadler M, Xu Z (1999) Astrocytes interact intimately with degenerating motor neurons in mouse amyotrophic lateral sclerosis (ALS). Glia 28(3):215–224Google Scholar
Valencia J, Martinez VG, Hidalgo L, Hernandez-Lopez C, Canseco NM, Vicente A, Varas A, Sacedon R (2014) Wnt5a signaling increases IL-12 secretion by human dendritic cells and enhances IFN-gamma production by CD4+ T cells. Immunol Lett 162(1 Pt A):188–199. https://doi.org/10.1016/j.imlet.2014.08.015Google Scholar
Pereira C, Schaer DJ, Bachli EB, Kurrer MO, Schoedon G (2008) Wnt5A/CaMKII signaling contributes to the inflammatory response of macrophages and is a target for the antiinflammatory action of activated protein C and interleukin-10. Arterioscler Thromb Vasc Biol 28(3):504–510. https://doi.org/10.1161/ATVBAHA.107.157438Google Scholar
Blumenthal A, Ehlers S, Lauber J, Buer J, Lange C, Goldmann T, Heine H, Brandt E et al (2006) The Wingless homolog WNT5A and its receptor Frizzled-5 regulate inflammatory responses of human mononuclear cells induced by microbial stimulation. Blood 108(3):965–973. https://doi.org/10.1182/blood-2005-12-5046Google Scholar