Prevention of neuronal apoptosis by astrocytes through thiol-mediated stress response modulation and accelerated recovery from proteotoxic stress

The development of drugs directly interfering with neurodegeneration has proven to be astonishingly difficult. Alternative therapeutic approaches could result from a better understanding of the supportive function of glial cells for stressed neurons. Therefore, here, we investigated the mechanisms involved in the endogenous neuro-defensive activity of astrocytes. A well-established model of postmitotic human dopaminergic neurons (LUHMES cells) was used in the absence ('LUHMES' mono-culture) or presence ('co-culture') of astrocytes. Inhibition of the LUHMES proteasome led to proteotoxic (protein aggregates; ATF-4 induction) and oxidative (GSH-depletion; NRF-2 induction) stress, followed by neuronal apoptosis. The presence of astrocytes attenuated the neuronal stress response, and drastically reduced neurodegeneration. A similar difference between LUHMES mono- and co-cultures was observed, when proteotoxic and oxidative stress was triggered indirectly by inhibitors of mitochondrial function (rotenone, MPP+). Human and murine astrocytes continuously released glutathione (GSH) into the medium, and transfer of glia-conditioned medium was sufficient to rescue LUHMES, unless it was depleted for GSH. Also, direct addition of GSH to LUHMES rescued the neurons from inhibition of the proteasome. Both astrocytes and GSH blunted the neuronal ATF-4 response and similarly upregulated NRF-1/NFE2L1, a transcription factor counter-regulating neuronal proteotoxic stress. Astrocyte co-culture also helped to recover the neurons’ ability to degrade aggregated poly-ubiquitinated proteins. Overexpression of NRF-1 attenuated the toxicity of proteasome inhibition, while knockdown increased toxicity. Thus, astrocytic thiol supply increased neuronal resilience to various proteotoxic stressors by simultaneously attenuating cell death-related stress responses, and enhancing the recovery from proteotoxic stress through upregulation of NRF-1.


Fig. S1: Accumulation of ubiquitinated proteins (UBI) by different stressors A/B/C:
To test for proteasomal dysfunction, cells were treated with the indicated concentrations of rotenone for 24 h, or MPP + for 48 h or MG-132 for 3 h. The caspase inhibitor Q-VD-Oph [5 µM] was used as media supplement to prevent cells from dying. Then, cells were lysed and analysed by Western blot with anti-ubiquitin and anti-GAPDH (loading control) antibodies . D: To confirm proteasomal dysfunction, cells were treated with 100 nM MG-132 for the indicated time periods. After incubation cells were lysed and analysed by Western blot with anti-ubiquitin and anti-GAPDH antibodies.  Human astrocytes (hu-Astro) were differentiated from induced pluripotent stem cells following established protocols (Chandrasekaran 2016). The cells were obtained as Astro.4U from Ncardia (Cologne, Germany) and plated according to manufacturer's instructions. More than 85% of the cells were GFAP positive (immunostaining). LUHMES were plated on top of the cells as described earlier (Efremova 2016). A: Differential toxicity of MG-132 in co-and mono-culture was assessed by immunocytochemistry staining against β-III tubulin, GFAP and H-33342 after cultures were exposed for 24 h to MG-132 at the indicated concentrations. Toxicity of MG-132 on LUHMES and astrocyte-LUHMES co-culture was assessed by measuring the neurite integrity after cells were exposed for 24 h to MG-132 at the indicated concentrations. Data are means ± SD of three independent experiments, ***: p<0.001. B: Exemplary images of the different experimental conditions are displayed for the 24 h time point.

Fig. S4: Stress response and cell death signals triggered by MG-132 in neurons A:
To analyse the stress response following proteasome inhibition, cells were treated with 100 nM MG-132 for the indicated time periods. After incubation, cells were lysed and analysed by Western blot with anti-NRF-1, anti-ATF-4, anti-GADD34 and anti-GAPDH antibodies. The blots shown are representative for three experiments with similar results. B: LUHMES (d6) cells were exposed to MG-132 [100 nM] for 20 h in the presence or absence of z-Vad-fmk [100 µM]. The different cell death/viability endpoints were measured. LDH-release is indicated as the percentage of total enzymatic activity in the well that is found in the media supernatants. Resazurin data reflects viable cells (100% corresponds to all cells are viable). Apoptosis refers to counting of nuclei with apoptotic morphology relative to all nuclei. Neurites refers to the integrity of the neuronal network as determined by high content imaging. It is a viability measure (100% = healthy cells) like resazurin. Data were analysed for statistical differences between treatments by two-way ANOVA, followed by a Dunnett's post-hoc test, ***: p < 0.001.

Fig. S8: Induction of ATF-4 target genes by MG-132
Differentiated (d6) LUHMES cells were exposed to MG-132 [100 nM] for the indicated time periods in the presence or absence of L-cysteine [1 mM]. Changes in mRNA levels were monitored by qPCR for activating transcription factor 4 (ATF-4) and its target genes phosphoserine phosphatase (PSPH), DNA damage inducible transcript 4 (DDIT-4) and phorbol-12-myristate-13-acetate-induced protein 1 (NOXA). Data are means ± SEM of three independent experiments. Detected differences were tested for significance by two-way ANOVA comparing the time points of the different treatments, followed by a Bonferroni posthoc test to correct for multiple comparisons, *: p<0.05 **: p<0.01; ***: p<0.001.

Fig. S9: Elevation of neuronal GSH and protection against MG-132 by astrocyteconditioned medium
A: Differentiated (d6) LUHMES cells were exposed for 4 h to astrocyte-conditioned medium, diluted with fresh LUHMES culture medium. Neuronal intracellular total glutathione (GSH+GSSG) was measured after incubation. Data are means ± SD of three independent experiments. B: Differentiated (d6) LUHMES cells were exposed to astrocyte-conditioned medium from mAGES, diluted with fresh LUHMES culture medium in the presence or absence of MG-132 [100 nM]. Viability was assessed 24 h after start of MG-132 exposure. For measurement of viability, cells were stained with the vital dye calcein-AM and the DNA stain H-33342. Neurite area of the cells was assessed by automated microscopy. Data are means ± SD of three independent experiments. C: Differentiated (d6) LUHMES cells were exposed to astrocyte-conditioned medium from hu-Astro (see Fig. S3) in the presence or absence of MG-132 [100 nM]. Viability was assessed as in B. Data are means ± SD of three independent experiments. For A-C, the significance of individual data points was tested by ANOVA followed by Dunnett's post-hoc test (*: p < 0.05). In addition, all curves showed a significant overall rising trend (p < 0.05 for slope non-equal to zero). D: Experimental setup of thiol scavanging. Astrocyte conditioned medium from mAGES was harvested and treated either with 10 µM DTNB (5,5-dithio-bis-2-nitrobenzoic acid/ Ellman's reagent) or solvent control. Conditioned medium was transferred onto LUHMES cells.

Fig. S16: Stress response in astrocytes triggered by MG-132
We observed in Figure 6A that the NRF-2 response was stronger in the co-culture compared to neuronal mono-cultures. This is consistent with published literature that astrocytes are the main cells in the brain showing an NRF-2 response (Vargas 2009), and in co-cultures we measured mainly astrocytic NRF-2. This was supported by the stress response observed in astrocytic mono-culture (Fig. S15A), and by resistance of astrocytes against MG-132 exposure (Fig. S15B). A: To analyse the stress response following proteasome inhibition, astrocytic cells ( CRISPR/Cas technology was used to cut the Nrf-1 gene of HEK293 cells within exon 2 (at the position coding for tyrosine-50, in the PSSAY amino acid stretch). Single cell clone picking and testing yielded cells with a homozygous identical deletion of 22 base pairs, as confirmed by PCR strategies and by sequencing (cells here termed Nrf-1 knockout (ko)). The PSSAY sequence was modified to code for PSSTT, followed by a TGA stop codon (arising from the frame shift). For the experiment, the wild-type (wt) and Nrf-1 ko cells were cultured under identical standard conditions in 96-well dishes, and cell numbers were carefully controlled to be identical for testing. A: Wild type and Nrf-1 knock-out (ko) cells were treated for 48 h with the indicated concentrations of MG-132. Cell viability was assessed by a resazurin reduction assay (and an LDH-release assay, not shown). Knock-out cells were found to be about 3-fold more sensitive (IC50(wt)= 0.9 µM; IC50(ko)= 0.3 µM) to MG-132 treatment than wt cells (red lines indicate IC50). Data are means ± SEM of three experiments run with entirely different start cultures. B: Cells were treated with MG-132 (2 µM) for the indicated times. Then, the drug was washed out and cysteine (1 mM) was added. Viability was assessed for all cultures (irrespective of washout time point) by resazurin reduction at 48 h after the start of the MG-132 treatment. Data were normalized to corresponding controls in which MG-132 was washed out directly after its addition (i.e. after 0 h). In addition to the individual data points, the means (large horizontal line) and the SEM (small horizontal line above) are depicted. For A+B, statistical significances were evaluated by two-way ANOVA for connected measures to compare different cells and treatments, followed by a Bonferroni post-hoc test to correct for multiple comparisons; ** p< 0.01, *** p< 0.001. In brief, Nrf-1 ko cells were more susceptible to MG-132 treatment and a minimum exposure to MG-132 for 7 h was necessary to trigger cell damage, but under this condition, the separation between wt and ko was not significant. At later time points (9-12 h), wt cells were rescued to a significant extent (i.e. maintaining > 80% viability), while ko cells could not be rescued. At late time points (15 h and beyond), cell death had occurred in wt and ko cells and rescue was not possible anymore. ** *** ***

MG-132 [µM]
Fig. S18: Differential regulation of ATF-4 target genes in co-and monocultures following MPP + exposure To address differences in the stress responses following MPP + exposure of co-and mono-cultures, gene expression was monitored after 36 h exposure to MPP + [5 µM]. Relative regulation (to GAPDH) in co-and mono-culture is displayed in a x-y-graph. Data are means ± SEM of three independent experiments. To determine, whether cocultures differed from mono-cultures, two statistical approaches were used (both based on mean regulation levels for each gene): a) two-way ANOVA indicated a influence of co-culture (p < 0.01). Posthoc testing was not performed; b) regulation data were compared by a pairwise Student's t-test (pairs were formed for each gene of the two treatments), p = 0.013. Fig. S17: Protection by astrocyte-conditioned medium against MPP + LUHMES cells (d6) were cultured in fresh LUHMES culture medium only, or in astrocyte-conditioned medium from mAGES, diluted with fresh LUHMES culture medium (1:1). They were exposed to MPP + [5 µM], and viability (resazurin reduction) was assessed 72 h after start of MPP + exposure. Data are means ± SD from three experiments. The difference in viability was tested by Student's t-test and it was significant at the p < 0.001 level.