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Fractalkine Induces Hepcidin Expression of BV-2 Microglia and Causes Iron Accumulation in SH-SY5Y Cells

Pandur, Edina, Tamási, Kitti, Pap, Ramóna, Varga, Edit, Miseta, Attila, Sipos, Katalin·2019·Cellular and Molecular Neurobiology
DOI 10.1007/s10571-019-00694-4PMC PMC6711953
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Abstract

Fractalkine (CX3CL1) is a potent inflammatory mediator of the central nervous system, which is expressed by neurons and regulates microglial functions by binding to fractalkine receptor (CX3CR1). It has been demonstrated that neuroinflammation plays an important role in iron accumulation of the brain leading to neuronal cell death. The major regulator of iron homeostasis is the peptide hormone hepcidin. Hepcidin expression is triggered by inflammatory conditions, which may contribute to the neuronal iron accumulation. In the present study, we established a bilaminar co-culture system of differentiated SH-SY5Y cells and BV-2 microglia as a neuronal model to examine the effect of soluble fractalkine on iron homeostasis of microglia and SH-SY5Y cells. We determined the hepcidin expression of fractalkine-treated microglia which showed significant elevation. We examined the relation between increased hepcidin secretion, the known hepcidin regulators and the signalling pathways controlled by fractalkine receptor. Our data revealed that TMPRSS6 and alpha 1-antitrypsin levels decreased due to fractalkine treatment, as well as the activity of NFκB pathway and the tyrosine phosphorylation of STAT5 factor. Moreover, fractalkine-induced hepcidin production of microglia initiated ferroportin internalisation of SH-SY5Y cells, which contributed to iron accumulation of neurons. Our results demonstrate that soluble form of fractalkine regulates hepcidin expression of BV-2 cells through fractalkine-mediated CX3CR1 internalisation. Moreover, fractalkine indirectly contributes to the iron accumulation of SH-SY5Y cells by activating ferroportin internalisation and by triggering the expressions of divalent metal transporter-1, ferritin heavy chain and mitochondrial ferritin.

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Figures

Fig. 1 mRNA expression levels of IL-6, TNFα and IL-1β and concentrations of the secreted IL-6 and TNFα in fractalkine-treated co-cultured BV-2 cells. Real-time PCR was performed with SYBR green protocol using gene-specific primers. β-actin was used as housekeeping gene for the normalisation and relative expression of controls was regarded as 1. Secreted IL-6 and TNFα were measured with ELISA according to the manufacturer’s protocols. a Relative mRNA expression levels of IL-6, TNFα and IL-1β. b Concentrations of the secreted IL-6 and TNFα measured from the cell culture medium. The bars represent mean values and error bars represent standard errors of the mean (S.E.M.) for three independent experiments (n = 3). The asterisks mark p < 0.05 compared to the controls
Fig. 1 mRNA expression levels of IL-6, TNFα and IL-1β and concentrations of the secreted IL-6 and TNFα in fractalkine-treated co-cultured BV-2 cells. Real-time PCR was performed with SYBR green protocol using gene-specific primers. β-actin was used as housekeeping gene for the normalisation and relative expression of controls was regarded as 1. Secreted IL-6 and TNFα were measured with ELISA according to the manufacturer’s protocols. a Relative mRNA expression levels of IL-6, TNFα and IL-1β. b Concentrations of the secreted IL-6 and TNFα measured from the cell culture medium. The bars represent mean values and error bars represent standard errors of the mean (S.E.M.) for three independent experiments (n = 3). The asterisks mark p < 0.05 compared to the controlsCC BY · PubMed Central
Fig. 2 mRNA expression levels of HAMP and concentration of the secreted hepcidin in fractalkine-treated co-cultured BV-2 cells. Real-time PCR was performed with SYBR green protocol using gene specific primers. The β-actin was used as housekeeping gene for the normalisation and the relative expression of the controls was regarded as 1. Secreted hepcidin was quantified with ELISA according to the manufacturer’s instructions. a Relative mRNA levels of HAMP. b Concentration of the secreted hepcidin measured from the cell culture medium. The bars represent mean values and error bars represent standard errors of the mean (S.E.M.) for three independent experiments (n = 3). The asterisks indicate p < 0.05 compared to the controls
Fig. 2 mRNA expression levels of HAMP and concentration of the secreted hepcidin in fractalkine-treated co-cultured BV-2 cells. Real-time PCR was performed with SYBR green protocol using gene specific primers. The β-actin was used as housekeeping gene for the normalisation and the relative expression of the controls was regarded as 1. Secreted hepcidin was quantified with ELISA according to the manufacturer’s instructions. a Relative mRNA levels of HAMP. b Concentration of the secreted hepcidin measured from the cell culture medium. The bars represent mean values and error bars represent standard errors of the mean (S.E.M.) for three independent experiments (n = 3). The asterisks indicate p < 0.05 compared to the controlsCC BY · PubMed Central
Fig. 3 Analyses of the mRNA and protein levels of hepcidin regulators. Real-time PCR was performed with SYBR green protocol using gene specific primers. The β-actin was used as housekeeping gene for the normalisation and the relative expression of the controls was considered as 1. Pelleted BV-2 cells from the co-cultures were fractionated. Protein contents of the fractions were measured. The same amount of protein (15 µg) from each sample was loaded onto 12% SDS-PAGE and transferred by electroblotting to nitrocellulose membranes and was probed with TMPRSS6, A1AT and β-actin polyclonal rabbit antibodies according to the manufacturer’s protocols. a Relative mRNA expression levels of the negative hepcidin regulators TMPRSS6 and alpha-1 antitrypsin. b Protein levels of negative hepcidin regulators TMPRSS6 and A1AT. c Optical densities of Western blot analyses of TMPRSS6 and A1AT. The analyses were done using ImageJ software; the optical density of the examined proteins was expressed as percentage of target protein/β-actin abundance. The bars represent mean values and error bars represent standard errors of the mean (SEM) for three independent experiments (n = 3). The asterisks indicate p < 0.05 compared to the controls
Fig. 3 Analyses of the mRNA and protein levels of hepcidin regulators. Real-time PCR was performed with SYBR green protocol using gene specific primers. The β-actin was used as housekeeping gene for the normalisation and the relative expression of the controls was considered as 1. Pelleted BV-2 cells from the co-cultures were fractionated. Protein contents of the fractions were measured. The same amount of protein (15 µg) from each sample was loaded onto 12% SDS-PAGE and transferred by electroblotting to nitrocellulose membranes and was probed with TMPRSS6, A1AT and β-actin polyclonal rabbit antibodies according to the manufacturer’s protocols. a Relative mRNA expression levels of the negative hepcidin regulators TMPRSS6 and alpha-1 antitrypsin. b Protein levels of negative hepcidin regulators TMPRSS6 and A1AT. c Optical densities of Western blot analyses of TMPRSS6 and A1AT. The analyses were done using ImageJ software; the optical density of the examined proteins was expressed as percentage of target protein/β-actin abundance. The bars represent mean values and error bars represent standard errors of the mean (SEM) for three independent experiments (n = 3). The asterisks indicate p < 0.05 compared to the controlsCC BY · PubMed Central
Fig. 4 Western blot analysis of the proteins in the NFκB signalling pathways. Pelleted BV-2 cells from the co-cultures were fractionated and protein concentrations of the fractions were measured. The same amount of protein (15 µg) from each sample was loaded onto 12% SDS-PAGE and transferred by electro blotting to nitrocellulose membranes and were probed with CX3CR1, p50 (NFκB) and RELA (NFκB) polyclonal rabbit antibodies according to the manufacturer’s protocols. β-actin was used as loading control. a Protein levels of CX3CR1, p50 (NFκB) and RELA (NFκB) in the different protein fractions of BV-2 cells. b Optical densities of the Western blot analyses of CX3CR1, p50 (NFκB) and RELA (NFκB). The analyses were carried out using ImageJ software; the optical density of the examined proteins was expressed as percentage of target protein/β-actin abundance. The bars represent mean values and error bars represent standard errors of the mean (SEM) for three independent determinations (n = 3). The asterisks indicate p < 0.05 compared to the controls
Fig. 4 Western blot analysis of the proteins in the NFκB signalling pathways. Pelleted BV-2 cells from the co-cultures were fractionated and protein concentrations of the fractions were measured. The same amount of protein (15 µg) from each sample was loaded onto 12% SDS-PAGE and transferred by electro blotting to nitrocellulose membranes and were probed with CX3CR1, p50 (NFκB) and RELA (NFκB) polyclonal rabbit antibodies according to the manufacturer’s protocols. β-actin was used as loading control. a Protein levels of CX3CR1, p50 (NFκB) and RELA (NFκB) in the different protein fractions of BV-2 cells. b Optical densities of the Western blot analyses of CX3CR1, p50 (NFκB) and RELA (NFκB). The analyses were carried out using ImageJ software; the optical density of the examined proteins was expressed as percentage of target protein/β-actin abundance. The bars represent mean values and error bars represent standard errors of the mean (SEM) for three independent determinations (n = 3). The asterisks indicate p < 0.05 compared to the controlsCC BY · PubMed Central
Fig. 5 Western blot analysis of STAT5 and STAT3 tyrosine phosphorylation due to fractalkine treatment of co-cultured BV-2 cells. Pelleted BV-2 cells from the co-cultures were fractionated and protein concentrations of the fractions were measured. The same amount of protein (15 µg) from each sample was loaded onto 10% SDS-PAGE and transferred to nitrocellulose membranes and was probed with p-STAT5 or p-STAT3 polyclonal rabbit antibodies according to the manufacturer’s protocols. The β-actin was used as loading control. a Protein expression levels of p-STAT5 and p-STAT3 in the nuclear protein fractions of BV-2 cells. b Optical densities of the Western blot analyses of p-STAT5 and p-STAT3. The analyses were carried out using ImageJ software; the optical density of the examined proteins was expressed as percentage of target protein/β-actin abundance. The bars represent mean values and error bars represent standard errors of the mean (S.E.M.) for three independent experiments (n = 3). The asterisks indicate p < 0.05 compared to the controls
Fig. 5 Western blot analysis of STAT5 and STAT3 tyrosine phosphorylation due to fractalkine treatment of co-cultured BV-2 cells. Pelleted BV-2 cells from the co-cultures were fractionated and protein concentrations of the fractions were measured. The same amount of protein (15 µg) from each sample was loaded onto 10% SDS-PAGE and transferred to nitrocellulose membranes and was probed with p-STAT5 or p-STAT3 polyclonal rabbit antibodies according to the manufacturer’s protocols. The β-actin was used as loading control. a Protein expression levels of p-STAT5 and p-STAT3 in the nuclear protein fractions of BV-2 cells. b Optical densities of the Western blot analyses of p-STAT5 and p-STAT3. The analyses were carried out using ImageJ software; the optical density of the examined proteins was expressed as percentage of target protein/β-actin abundance. The bars represent mean values and error bars represent standard errors of the mean (S.E.M.) for three independent experiments (n = 3). The asterisks indicate p < 0.05 compared to the controlsCC BY · PubMed Central
Fig. 6 Western blot analysis of the iron exporter ferroportin (Fp) in BV-2 and differentiated SH-SY5Y cells after fractalkine treatment. SH-SY5Y cells were collected from Thermanox coverslips and BV-2 cells were collected from the cell culture dish with trypsinization. Cells were lysed and protein concentrations of the samples were measured. The same amount of protein (15 µg) from each sample was loaded onto 12% SDS-PAGE and transferred to nitrocellulose membranes and was probed with Fp polyclonal rabbit antibody according to the manufacturer’s protocol. The β-actin was used as loading control. a Protein expression levels of ferroportin in the BV-2 and SH-SY5Y cells. b Optical densities of the Western blot analyses of ferroportin. The analyses were carried out using ImageJ software; the optical density of the ferroportin was expressed as percentage of ferroportin protein/β-actin abundance. The bars represent mean values and error bars represent standard errors of the mean (S.E.M.) for three independent experiments (n = 3). The asterisks mark p < 0.05 compared to the controls
Fig. 6 Western blot analysis of the iron exporter ferroportin (Fp) in BV-2 and differentiated SH-SY5Y cells after fractalkine treatment. SH-SY5Y cells were collected from Thermanox coverslips and BV-2 cells were collected from the cell culture dish with trypsinization. Cells were lysed and protein concentrations of the samples were measured. The same amount of protein (15 µg) from each sample was loaded onto 12% SDS-PAGE and transferred to nitrocellulose membranes and was probed with Fp polyclonal rabbit antibody according to the manufacturer’s protocol. The β-actin was used as loading control. a Protein expression levels of ferroportin in the BV-2 and SH-SY5Y cells. b Optical densities of the Western blot analyses of ferroportin. The analyses were carried out using ImageJ software; the optical density of the ferroportin was expressed as percentage of ferroportin protein/β-actin abundance. The bars represent mean values and error bars represent standard errors of the mean (S.E.M.) for three independent experiments (n = 3). The asterisks mark p < 0.05 compared to the controlsCC BY · PubMed Central
Fig. 7 mRNA and Western blot analyses of genes responsible for iron uptake and storage in the co-cultured SH-SY5Y cells. Real-time PCR was performed with SYBR green protocol using gene-specific primers. The β-actin was used as housekeeping gene for the normalisation and the relative expression of the controls was considered as 1. Pelleted SH-SY5Y cells from the co-cultures were lysed and protein contents of the samples were measured. The same amount of protein (15 µg) from each sample was loaded onto 12% SDS-PAGE and transferred by electro blotting to nitrocellulose membranes and was probed with DMT-1, FTH and FTMT polyclonal rabbit antibodies according to the manufacturer’s protocols. The β-actin was used as loading control. a Relative mRNA expression levels of DMT-1, FTH and FTMT. b Protein levels of DMT-1, FTH and FTMT showing increased expressions. c Optical densities of the Western blot analyses of the DMT-1, FTH and FTMT. The analyses were carried out using ImageJ software; the optical densities of investigated proteins were expressed as percentage of target protein/β-actin abundance. The bars represent mean values and error bars represent standard errors of the mean for three independent experiments. The asterisks mark p < 0.05 compared to the controls
Fig. 7 mRNA and Western blot analyses of genes responsible for iron uptake and storage in the co-cultured SH-SY5Y cells. Real-time PCR was performed with SYBR green protocol using gene-specific primers. The β-actin was used as housekeeping gene for the normalisation and the relative expression of the controls was considered as 1. Pelleted SH-SY5Y cells from the co-cultures were lysed and protein contents of the samples were measured. The same amount of protein (15 µg) from each sample was loaded onto 12% SDS-PAGE and transferred by electro blotting to nitrocellulose membranes and was probed with DMT-1, FTH and FTMT polyclonal rabbit antibodies according to the manufacturer’s protocols. The β-actin was used as loading control. a Relative mRNA expression levels of DMT-1, FTH and FTMT. b Protein levels of DMT-1, FTH and FTMT showing increased expressions. c Optical densities of the Western blot analyses of the DMT-1, FTH and FTMT. The analyses were carried out using ImageJ software; the optical densities of investigated proteins were expressed as percentage of target protein/β-actin abundance. The bars represent mean values and error bars represent standard errors of the mean for three independent experiments. The asterisks mark p < 0.05 compared to the controlsCC BY · PubMed Central
Fig. 8 Iron measurements and cell viability determinations of the co-cultured SH-SY5Y and BV-2 cells. Intracellular iron contents of SH-SY5Y and BV-2 cells were determined using a colorimetric ferrozine-based assay and were expressed as % of the controls. Changes in the intracellular iron contents of the fractalkine-treated cells were expressed as µM iron/mg protein. The values are calculated from the differences between the treated and the control cells. Viability of the SH-SY5Y cells was measured using CCK-8 cell viability assay after the separation of the two cells types from the treated co-cultures. Viability is expressed as percentile of the total cell number of the untreated control cells. a Comparison of the total iron contents of fractalkine-treated, co-cultured SH-SY5Y and BV-2 cells. b Comparison of the changes of the total iron contents of fractalkine-treated, co-cultured SH-SY5Y and BV-2 cells. c Cell viability measurements of co-cultured SH-SY5Y and BV-2 cells after fractalkine treatment. d Protein levels of cleaved-caspase-3 in co-cultured SH-SY5Y and BV-2 cells. The bars represent mean values and error bars represent standard errors of the mean (S.E.M.) for four independent experiments (n = 4). The asterisks indicate p < 0.05 compared to the controls, while crosses mark difference p < 0.05 between the two cell types
Fig. 8 Iron measurements and cell viability determinations of the co-cultured SH-SY5Y and BV-2 cells. Intracellular iron contents of SH-SY5Y and BV-2 cells were determined using a colorimetric ferrozine-based assay and were expressed as % of the controls. Changes in the intracellular iron contents of the fractalkine-treated cells were expressed as µM iron/mg protein. The values are calculated from the differences between the treated and the control cells. Viability of the SH-SY5Y cells was measured using CCK-8 cell viability assay after the separation of the two cells types from the treated co-cultures. Viability is expressed as percentile of the total cell number of the untreated control cells. a Comparison of the total iron contents of fractalkine-treated, co-cultured SH-SY5Y and BV-2 cells. b Comparison of the changes of the total iron contents of fractalkine-treated, co-cultured SH-SY5Y and BV-2 cells. c Cell viability measurements of co-cultured SH-SY5Y and BV-2 cells after fractalkine treatment. d Protein levels of cleaved-caspase-3 in co-cultured SH-SY5Y and BV-2 cells. The bars represent mean values and error bars represent standard errors of the mean (S.E.M.) for four independent experiments (n = 4). The asterisks indicate p < 0.05 compared to the controls, while crosses mark difference p < 0.05 between the two cell typesCC BY · PubMed Central
Fig. 9 Effect of fractalkine on BV-2 monocultures. Real-time PCR was performed with SYBR green protocol using gene-specific primers. The β-actin was used as housekeeping gene for the normalisation and the relative expression of the controls was considered as 1. Pelleted BV-2 cells were fractionated and protein contents of the fractions were measured. The same amount of protein (15 µg) from each sample was loaded onto 12% SDS-PAGE and transferred to nitrocellulose membranes and was probed with CX3CR1, TMPRSS6 and A1AT polyclonal rabbit antibodies according to the manufacturer’s protocols. The β-actin was used as the loading control. Secreted hepcidin was quantified with ELISA kit according to the manufacturer’s instructions. a Relative mRNA expression levels of CX3CR1 and the negative hepcidin regulators TMPRSS6 and A1AT. b Protein levels of TMPRSS6, A1AT and CX3CR1. c Optical densities of the Western blot analyses of TMPRSS6, A1AT and CX3CR1. The analyses were done using ImageJ software; the optical density of the examined proteins was expressed as percentage of target protein/β-actin abundance. d Relative HAMP mRNA expression of the fractalkine-treated BV-2 cells. e Hepcidin secretion of the fractalkine-treated BV-2 cells. The bars represent mean values and error bars represent standard errors of the mean (SEM) for three independent experiments (n = 3 in each experiment). The asterisks indicate p < 0.05 compared to the controls
Fig. 9 Effect of fractalkine on BV-2 monocultures. Real-time PCR was performed with SYBR green protocol using gene-specific primers. The β-actin was used as housekeeping gene for the normalisation and the relative expression of the controls was considered as 1. Pelleted BV-2 cells were fractionated and protein contents of the fractions were measured. The same amount of protein (15 µg) from each sample was loaded onto 12% SDS-PAGE and transferred to nitrocellulose membranes and was probed with CX3CR1, TMPRSS6 and A1AT polyclonal rabbit antibodies according to the manufacturer’s protocols. The β-actin was used as the loading control. Secreted hepcidin was quantified with ELISA kit according to the manufacturer’s instructions. a Relative mRNA expression levels of CX3CR1 and the negative hepcidin regulators TMPRSS6 and A1AT. b Protein levels of TMPRSS6, A1AT and CX3CR1. c Optical densities of the Western blot analyses of TMPRSS6, A1AT and CX3CR1. The analyses were done using ImageJ software; the optical density of the examined proteins was expressed as percentage of target protein/β-actin abundance. d Relative HAMP mRNA expression of the fractalkine-treated BV-2 cells. e Hepcidin secretion of the fractalkine-treated BV-2 cells. The bars represent mean values and error bars represent standard errors of the mean (SEM) for three independent experiments (n = 3 in each experiment). The asterisks indicate p < 0.05 compared to the controlsCC BY · PubMed Central
Fig. 10 mRNA and Western blot analyses of fractalkine in the fractalkine-treated mono- and co-cultured SH-SY5Y cells. Real-time PCR was performed with SYBR green protocol using gene specific primers. The β-actin was used as housekeeping gene for the normalisation and the relative expression of the controls was considered as 1. Pelleted SH-SY5Y cells were lysed and protein contents of the samples were measured. The same amount of protein (15 µg) from each sample was loaded onto 10% SDS-PAGE and transferred by electro blotting to nitrocellulose membranes and was probed with fractalkine polyclonal rabbit antibodies according to the manufacturer’s protocol. The β-actin was used as loading control. a Relative mRNA expression levels of fractalkine in mono-and co-cultured SH-SY5Y cells. b Protein levels of fractalkine in fractalkine-treated SH-SY5Y cells. c Optical densities of the Western blot analysis of fractalkine. The analysis was carried out using ImageJ software, the optical density of the investigated protein was expressed as percentage of fractalkine protein/β-actin abundance. The bars represent mean values and error bars represent standard errors of the mean (SEM) for three independent experiments (n = 3). The asterisks mark p < 0.05 compared to the controls
Fig. 10 mRNA and Western blot analyses of fractalkine in the fractalkine-treated mono- and co-cultured SH-SY5Y cells. Real-time PCR was performed with SYBR green protocol using gene specific primers. The β-actin was used as housekeeping gene for the normalisation and the relative expression of the controls was considered as 1. Pelleted SH-SY5Y cells were lysed and protein contents of the samples were measured. The same amount of protein (15 µg) from each sample was loaded onto 10% SDS-PAGE and transferred by electro blotting to nitrocellulose membranes and was probed with fractalkine polyclonal rabbit antibodies according to the manufacturer’s protocol. The β-actin was used as loading control. a Relative mRNA expression levels of fractalkine in mono-and co-cultured SH-SY5Y cells. b Protein levels of fractalkine in fractalkine-treated SH-SY5Y cells. c Optical densities of the Western blot analysis of fractalkine. The analysis was carried out using ImageJ software, the optical density of the investigated protein was expressed as percentage of fractalkine protein/β-actin abundance. The bars represent mean values and error bars represent standard errors of the mean (SEM) for three independent experiments (n = 3). The asterisks mark p < 0.05 compared to the controlsCC BY · PubMed Central
Fig. 11 Regulation of HAMP mRNA expression and hepcidin maturation/secretion in microglia through activation/internalisation of CX3CR1 receptor at a normal and b inflammatory conditions. The key molecule of hepcidin regulation is CX3CR1 which controls cell signalling pathways that play a role in the transcriptional regulation of the two negative hepcidin regulators TMPRSS6 and A1AT. Internalisation of the receptor will cause the downregulation of TMPRSS6 and A1AT mRNA expressions and as a consequence HAMP transcriptional rate will decrease. Decreasing level of A1AT enhances prohepcidin–hepcidin conversion. For further explanation, see discussion section
Fig. 11 Regulation of HAMP mRNA expression and hepcidin maturation/secretion in microglia through activation/internalisation of CX3CR1 receptor at a normal and b inflammatory conditions. The key molecule of hepcidin regulation is CX3CR1 which controls cell signalling pathways that play a role in the transcriptional regulation of the two negative hepcidin regulators TMPRSS6 and A1AT. Internalisation of the receptor will cause the downregulation of TMPRSS6 and A1AT mRNA expressions and as a consequence HAMP transcriptional rate will decrease. Decreasing level of A1AT enhances prohepcidin–hepcidin conversion. For further explanation, see discussion sectionCC BY · PubMed Central
Fig. 12 Effect of microglial hepcidin on differentiated SH-SY5Y cells. Secreted microglial hepcidin binds to its cognate receptor ferroportin expressed by SH-SY5Y cells. Upon hepcidin binding ferroportin is internalised reducing iron release and increasing iron retention. FTH and FTMT expression levels increased, suggesting iron accumulation
Fig. 12 Effect of microglial hepcidin on differentiated SH-SY5Y cells. Secreted microglial hepcidin binds to its cognate receptor ferroportin expressed by SH-SY5Y cells. Upon hepcidin binding ferroportin is internalised reducing iron release and increasing iron retention. FTH and FTMT expression levels increased, suggesting iron accumulationCC BY · PubMed Central

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Metadata

Authors
Pandur, E., Tamási, K., Pap, R., Varga, E., Miseta, A., & Sipos, K.
ORCID
Pandur, Edina
Venue
Cellular and Molecular Neurobiology
Publisher
Springer Science and Business Media LLC
Published
2019-06-06
Volume
39
Issue
7
Pages
985-1001
ISSN
  • 0272-4340
  • 1573-6830
DOI
10.1007/s10571-019-00694-4
PMC
PMC6711953
Cited by
37
References
15
Language
en
Licence

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