Abstract
Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease of unknown cause. Absence of specific targets and biomarkers compromise the development of new therapeutic strategies and of innovative tools to stratify patients and assess their responses to treatment. Here, we investigate changes in neuroprotective-neuroinflammatory actions in the spinal cord of SOD1 G93A mice, at presymptomatic and symptomatic stages to identify stage-specific biomarkers and potential targets. Results showed that in the presymptomatic stage, there are alterations in both astrocytes and microglia, which comprise decreased expression of GFAP and S100B and upregulation of GLT-1, as well as reduced expression of CD11b, M2-phenotype markers, and a set of inflammatory mediators. Reduced levels of Connexin-43, Pannexin-1, CCL21, and CX3CL1 further indicate the existence of a compromised intercellular communication. In contrast, in the symptomatic stage, increased markers of inflammation became evident, such as NF-κB/Nlrp3-inflammasome, Iba1, pro-inflammatory cytokines, and M1-polarizion markers, together with a decreased expression of M2-phenotypic markers. We also observed upregulation of the CX3CL1-CX3CR1 axis, Connexin-43, Pannexin-1, and of microRNAs (miR)-124, miR-125b, miR-146a and miR-21. Reduced motor neuron number and presence of reactive astrocytes with decreased GFAP, GLT-1, and GLAST further characterized this inflammatory stage. Interestingly, upregulation of miR-155 and downregulation of MFG-E8 appear as consistent biomarkers of both presymptomatic and symptomatic stages. We hypothesize that downregulated cellular interplay at the early stages may represent neuroprotective mechanisms against inflammation, SOD1 aggregation, and ALS onset. The present study identified a set of inflamma-miRNAs, NLRP3-inflammasome, HMGB1, CX3CL1-CX3CR1, Connexin-43, and Pannexin-1 as emerging candidates and promising pharmacological targets that may represent potential neuroprotective strategies in ALS therapy.
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Abbreviations
- ALS:
-
Amyotrophic lateral sclerosis
- Arg1:
-
Arginase 1
- Cebpa:
-
CCAAT/enhancer-binding protein alpha
- CCL21:
-
Chemokine (C-C motif) ligand 21
- CCR7:
-
Chemokine (C-C motif) receptor type 7
- CSF:
-
Cerebrospinal fluid
- CX3CL1:
-
Chemokine (C-X3-C motif) ligand 1
- CX3CR1:
-
Chemokine (C-X3-C motif) receptor 1
- Cx43:
-
Connexin-43
- GFAP:
-
Glial fibrillary acidic protein
- GLT-1:
-
Glutamate transporter 1
- Fizz1:
-
Found in inflammatory zone 1
- HMGB1:
-
High mobility group box 1
- H&E:
-
hematoxylin–eosin
- Iba1:
-
Ionized calcium-binding adapter molecule 1
- IL:
-
Interleukin
- miR:
-
MicroRNA
- MFG-E8:
-
Milk fat globule-EGF factor 8
- NeuN:
-
Neuronal nuclei
- NF-κB:
-
Nuclear factor kappa B
- NLRP3:
-
NOD-like receptor family pyrin domain containing 3
- Panx1:
-
Pannexin-1
- PBS:
-
Phosphate buffer saline
- pNF-κB:
-
Phosphorylated NF-κB
- qRT-PCR:
-
Quantitative reverse transcription polymerase chain reaction
- RT:
-
Room temperature
- S100B:
-
S100 calcium-binding protein B
- SC:
-
Spinal cord
- SOCS1:
-
Suppressor of cytokine signaling 1
- SOD1:
-
Superoxide dismutase 1
- TLRs:
-
Toll-like receptors
- TGF-β:
-
Transforming growth factor beta
- TNF-α:
-
Tumor necrosis factor alfa
- WB:
-
Western blot
- WT:
-
Wild type
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Acknowledgements
This work was funded by the Research Grant of the Santa Casa Scientific Research Program on ALS, by Santa Casa da Misericórdia de Lisboa (SCML), Portugal, Project Ref. ELA-2015-002 (to DB), by the project PTDC/SAU-FAR/118787/2010 (to D.B.) and, in part, by iMed.ULisboa (UID/DTP/04138/2013) from Fundação para a Ciência e a Tecnologia (FCT). A.R.V. holds a postdoctoral research fellowship (SFRH/BPD/76590/2011), and C.C. and C.G. are recipients of PhD fellowships (SFRH/BD/91316/2012 and SFRH/BD/102718/2014, respectively), all from FCT. The funding organization had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. We thank to Dr. Rui Gomes (Instituto de Medicina Molecular, Faculdade de Medicina de Lisboa, Universidade de Lisboa, Portugal) for genotyping mice.
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D.B, conceived the study; D.B. and A.R.V. designed the experiments; A.R.V. gave technical support; C.C., C.G., A.R.V., A.F. and C.S. acquired data; A.S. and A.M.C. contributed to conceptual advice and data acquisition on mice related with its usage, management, and maintenance; A.R.V., C.C., and D.B. analyzed data; C.C. and A.R.V. drafted figures and participated in the manuscript writing; D.B. wrote and edited the final version. The manuscript has been read and approved by all named authors.
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Cunha, C., Santos, C., Gomes, C. et al. Downregulated Glia Interplay and Increased miRNA-155 as Promising Markers to Track ALS at an Early Stage. Mol Neurobiol 55, 4207–4224 (2018). https://doi.org/10.1007/s12035-017-0631-2
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DOI: https://doi.org/10.1007/s12035-017-0631-2