Glycoursodeoxycholic Acid Reduces Matrix Metalloproteinase-9 and Caspase-9 Activation in a Cellular Model of Superoxide Dismutase-1 Neurodegeneration

Abstract

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease that affects mainly motor neurons (MNs). NSC-34 MN-like cells carrying the G93A mutation in human superoxide dismutase-1 (hSOD1(G93A)) are a common model to study the molecular mechanisms of neurodegeneration in ALS. Although the underlying pathways of MN failure still remain elusive, increased apoptosis and oxidative stress seem to be implicated. Riluzole, the only approved drug, only slightly delays ALS progression. Ursodeoxycholic acid (UDCA), as well as its glycine (glycoursodeoxycholic acid, GUDCA) and taurine (TUDCA) conjugated species, have shown therapeutic efficacy in neurodegenerative models and diseases. Pilot studies in ALS patients indicate safety and tolerability for UDCA oral administration. We explored the mechanisms associated with superoxide dismutase-1 (SOD1) accumulation and MN degeneration in NSC-34/hSOD1(G93A) cells differentiated for 4 days in vitro (DIV). We examined GUDCA efficacy in preventing such pathological events and in restoring MN functionality by incubating cells with 50 μM GUDCA at 0 DIV and at 2 DIV, respectively. Increased cytosolic SOD1 inclusions were observed in 4 DIV NSC-34/hSOD1(G93A) cells together with decreased mitochondria viability (1.2-fold, p < 0.01), caspase-9 activation (1.8-fold, p < 0.05), and apoptosis (2.1-fold, p < 0.01). GUDCA exerted preventive effects (p < 0.05) while also reduced caspase-9 levels when added at 2 DIV (p < 0.05). ATP depletion (2-fold, p < 0.05), increased nitrites (1.6-fold, p < 0.05) and metalloproteinase-9 (MMP-9) activation (1.8-fold, p < 0.05), but no changes in MMP-2, were observed in the extracellular media of 4 DIV NSC-34/hSOD1(G93A) cells. GUDCA inhibited nitrite production (p < 0.05) while simultaneously prevented and reverted MMP-9 activation (p < 0.05), but not ATP depletion. Data highlight caspase-9 and MMP-9 activation as key pathomechanisms in ALS and GUDCA as a promising therapeutic strategy for slowing disease onset and progression.

Exosomes from NSC-34 Cells Transfected with hSOD1-G93A Are Enriched in miR-124 and Drive Alterations in Microglia Phenotype

“…Mouse NSC-34 is a hybrid cell line produced by the fusion of MNs from the spinal cord embryos with N18TG2 neuroblastoma cells (Cashman et al, 1992) that exhibit properties of MNs after differentiation and maturation protocols. Thus, NSC-34 cells were grown in proliferation media [Dulbecco's modified Eagle's medium (DMEM) high glucose with glutamine, w/o pyruvate, supplemented with 10% of fetal bovine serum (FBS) and 1% of Penicillin/Streptomycin] and selection was made with Geneticin 418 sulfate (G418) at 0.5 mg/ml (Vaz et al, 2015). Medium was changed every 2–3 days.…”

“…After 48 h in proliferation medium, differentiation was induced by changing medium for DMEM-F12 plus 1% of FBS-exosome depleted, 1% of non-essential amino acids (NEAA), 1% of Penicillin/Streptomycin and 0.5 mg/ml of G418, as indicated by Cho et al (2011). Cells were maintained in culture with differentiation medium for 4 days in vitro (DIV) to induce SOD1 accumulation (Vaz et al, 2015). DMEM, DMEM-F12, FBS, Penicillin/Streptomycin, and NEAA were purchased from Biochrom AG (Berlin, Germany).…”

“…Transmission electron microscopy (TEM) technique used the Jeol JEM 1400 Transmission Electron Microscope (Peabody, MA, USA). Western blot analysis was performed as usual in our lab (Vaz et al, 2015) to evaluate the expression of alix, flotillin-1 and CD63 by using 20 μg of total protein and specific antibodies (mouse anti-Alix, Cell Signaling, #2171; mouse anti-flotillin-1, BD Biosciences, #6108; goat anti-CD63, Santa Cruz Biotechnology, #sc-31214). Normalization was made by using Amido Black staining as loading control.…”

Microglia centered pathogenesis in ALS: insights in cell interconnectivity

“…Interestingly, when working with the NSC-34/hSOD1G93A cells we found that one of the most sensitive indicators of SOD1 accumulation and neuronal dysfunction was the elevation of MMP-9, but not of MMP-2 that remained unchanged (Cunha, 2012; Vaz et al, 2014). Elevation of the MMP-9 levels was previously observed in the SC of ALS mice from pre-symptomatic phase, predominantly in MNs but also in glia (Soon et al, 2010).…”

Bile Acids in Neurodegenerative Disorders

“…NSC-34 cells modified to carry the human form of SOD1 with the G93A mutation (hSOD1 G93A ) have thus been used as a model for ALS. GUDCA was reported to reduce cell death in the NSC-34 hSOD1 G93A cell model and to block caspase-9 activation (Vaz et al, 2015). …”