The VGF gene encodes a protein precursor (617/615 AA rat/man, respectively), which is processed in vivo to various low MW peptides with different biologic activities. These include attenuation of excytotoxic injury on spinal motor neurons from G93A-SOD-1 mice, upon overexpression of the VGF precursor, or an anti-apoptotic action of the VGF peptide TLQP-21 on cerebellar granules. Recently, the C3a and gC1q have been identified as TLQP-21 receptors. Using the mouse motoneuronal cell line NSC-34 and the primary human fibroblast cell colture, we studied the expression and modulation of certain VGF peptides (VGFp) in ALS and oxidative stress and their possible neuroprotective effect. Oxidative stress was induced with Na Arsenite (SA: 0.5mM, 30/60 min) while the MTT test was used to assess cell viability. Antibodies used (for either ICC or ELISA) were specific for: the N/C-terminus of the VGF precursor, NERP-1, rat PGH, NAPPE-19 and TLQP-21, and for the C3a/gC1q TLQP receptors. Both antibody receptors were also used for ICC and western blot analysis. In NSC-34, almost all VGFp were selectively localized in the growth cones, perinuclear region, and/or in a specific paranuclear region. Upon oxidative stress, most cells showed a rounded morphology, with loss of growth cones, while almost all VGFp were found mainly in a paranuclear region compatible with the Golgi apparatus. In ELISA, untreated cellular extracts showed the presence of the majority VGFp with a significant reduction in TLQP and NERP-1 levels versus untreated cells (58% and 65% of reduction, respectively) after SA treatment, while the culture medium showed a marginal increase of the same peptides. Cell viability studies showed a significant protective action of TLQP-21 on stressed neurons (SA alone: 74.7% 0.08, SA and TLQP-21 together: 82.1% 0.08 of untreated controls, p0.018), with no detectable effect for NERP-1. Results from immunocytochemistry showed the presence in NSC-34 of gC1q receptor only, confirmed also by western blot analysis. In human fibroblasts, VGFp were selectively localized in citoplasmic and paranuclear region morfologically related to the membranous cellular system, without obvious differences between healthy and ALS subjects (with TARDBPA382T and without known mutation). In ELISA, N-terminus and NERP-1 showed a significant reduction in both groups of ALS patients (TARDBPA382T: 30% and 61% of reduction, respectively; ALS without known mutation: 37% and 62% of reduction, respectively) versus healty controls, while TLQP-21 and C-terminus showed a reduction only in certain ALS patients (TLQP-21: 37% of reduction, for ALS without known mutation; C-term: 62% of reduction, only for ALS TARDBPA382T) versus healty cells. In conclusion, the VGFp studied are present in NSC-34, but only TLQPp are modulated and secreted after oxidative stress, with a potential neuroprotective role. In fibroblasts, VGFp showed a significant reduction in ALS cell extracts. Hence, the research in the VGF field using NSC-34 could open new perspectives for potential therapeutic tools, while the results obtained through fibroblasts could be helpful for diagnostic purposes.
VGF e stress ossidativo: alterazioni cellulari e modulazione nella Sclerosi Laterale Amiotrofica
PILLERI, ROBERTA
2016-03-15
Abstract
The VGF gene encodes a protein precursor (617/615 AA rat/man, respectively), which is processed in vivo to various low MW peptides with different biologic activities. These include attenuation of excytotoxic injury on spinal motor neurons from G93A-SOD-1 mice, upon overexpression of the VGF precursor, or an anti-apoptotic action of the VGF peptide TLQP-21 on cerebellar granules. Recently, the C3a and gC1q have been identified as TLQP-21 receptors. Using the mouse motoneuronal cell line NSC-34 and the primary human fibroblast cell colture, we studied the expression and modulation of certain VGF peptides (VGFp) in ALS and oxidative stress and their possible neuroprotective effect. Oxidative stress was induced with Na Arsenite (SA: 0.5mM, 30/60 min) while the MTT test was used to assess cell viability. Antibodies used (for either ICC or ELISA) were specific for: the N/C-terminus of the VGF precursor, NERP-1, rat PGH, NAPPE-19 and TLQP-21, and for the C3a/gC1q TLQP receptors. Both antibody receptors were also used for ICC and western blot analysis. In NSC-34, almost all VGFp were selectively localized in the growth cones, perinuclear region, and/or in a specific paranuclear region. Upon oxidative stress, most cells showed a rounded morphology, with loss of growth cones, while almost all VGFp were found mainly in a paranuclear region compatible with the Golgi apparatus. In ELISA, untreated cellular extracts showed the presence of the majority VGFp with a significant reduction in TLQP and NERP-1 levels versus untreated cells (58% and 65% of reduction, respectively) after SA treatment, while the culture medium showed a marginal increase of the same peptides. Cell viability studies showed a significant protective action of TLQP-21 on stressed neurons (SA alone: 74.7% 0.08, SA and TLQP-21 together: 82.1% 0.08 of untreated controls, p0.018), with no detectable effect for NERP-1. Results from immunocytochemistry showed the presence in NSC-34 of gC1q receptor only, confirmed also by western blot analysis. In human fibroblasts, VGFp were selectively localized in citoplasmic and paranuclear region morfologically related to the membranous cellular system, without obvious differences between healthy and ALS subjects (with TARDBPA382T and without known mutation). In ELISA, N-terminus and NERP-1 showed a significant reduction in both groups of ALS patients (TARDBPA382T: 30% and 61% of reduction, respectively; ALS without known mutation: 37% and 62% of reduction, respectively) versus healty controls, while TLQP-21 and C-terminus showed a reduction only in certain ALS patients (TLQP-21: 37% of reduction, for ALS without known mutation; C-term: 62% of reduction, only for ALS TARDBPA382T) versus healty cells. In conclusion, the VGFp studied are present in NSC-34, but only TLQPp are modulated and secreted after oxidative stress, with a potential neuroprotective role. In fibroblasts, VGFp showed a significant reduction in ALS cell extracts. Hence, the research in the VGF field using NSC-34 could open new perspectives for potential therapeutic tools, while the results obtained through fibroblasts could be helpful for diagnostic purposes.File | Dimensione | Formato | |
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