CDNF rescues motor neurons in models of amyotrophic lateral sclerosis by targeting endoplasmic reticulum stress.

Amyotrophic lateral sclerosis is a progressive neurodegenerative disease that affects motor neurons (MNs) in the spinal cord, brainstem, and motor cortex, leading to paralysis and eventually to death within 3 to 5 years of symptom onset. To date, no cure or effective therapy is available. The role of chronic endoplasmic reticulum (ER) stress in the pathophysiology of amyotrophic lateral sclerosis, as well as a potential drug target, has received increasing attention. Here, we investigated the mode of action and therapeutic effect of the ER-resident protein cerebral dopamine neurotrophic factor (CDNF) in three preclinical models of amyotrophic lateral sclerosis, exhibiting different disease development and etiology: (i) the conditional choline acetyltransferase (ChAT)-tTA/TRE-hTDP43-M337V rat model previously described, (ii) the widely used SOD1-G93A mouse model, and (iii) a novel slow-progressive TDP43-M337V mouse model. To specifically analyse the ER stress response in MNs, we used three main methods: (i) primary culture of MNs derived from E13 days embryos, (ii) immunohistochemical analyses of spinal cord sections with ChAT as spinal MNs marker, and (iii) qPCR analyses of lumbar MNs isolated via laser microdissection. We show that intracerebroventricular administration of CDNF significantly halts the progression of the disease and improves motor behavior in TDP43-M337V and SOD1-G93A rodent models of amyotrophic lateral sclerosis. CDNF rescues motor neurons in vitro and in vivo from ER stress-associated cell death and its beneficial effect is independent of genetic disease etiology. Notably, CDNF regulates the unfolded protein response (UPR) initiated by transducers IRE1α, PERK, and ATF6, thereby enhancing MN survival. Thus, CDNF holds great promise for the design of new rational treatments for amyotrophic lateral sclerosis

Altered Immunomodulatory Responses in the CX3CL1/CX3CR1 Axis Mediated by hMSCs in an Early In Vitro SOD1^G93A Model of ALS

Amyotrophic lateral sclerosis (ALS) is a fatal motor neuron (MN) disease characterized by progressive MN loss and muscular atrophy resulting in rapidly progressive paralysis and respiratory failure. Human mesenchymal stem/stromal cell (hMSC)-based therapy has been suggested to prolong MN survival via secretion of growth factors and modulation of cytokines/chemokines. We investigated the effects of hMSCs and a hMSC-conditioned medium (CM) on Cu/Zn superoxidase dismutase 1G93A (SOD1G93A) transgenic primary MNs. We found that co-culture of hMSCs and MNs resulted in slightly higher MN numbers, but did not protect against staurosporine (STS)-induced toxicity, implying marginal direct trophic effects of hMSCs. Aiming to elucidate the crosstalk between hMSCs and MNs in vitro, we found high levels of vascular endothelial growth factor (VEGF) and C-X3-C motif chemokine 1 (CX3CL1) in the hMSC secretome. Co-culture of hMSCs and MNs resulted in altered gene expression of growth factors and cytokines/chemokines in both MNs and hMSCs. hMSCs showed upregulation of CX3CL1 and its receptor CX3CR1 and downregulation of interleukin-1 β (IL1β) and interleukin-8 (IL8) when co-cultured with SOD1G93A MNs. MNs, on the other hand, showed upregulation of growth factors as well as CX3CR1 upon hMSC co-culture. Our results indicate that hMSCs only provide moderate trophic support to MNs by growth factor gene regulation and may mediate anti-inflammatory responses through the CX3CL1/CX3CR1 axis, but also increase expression of pro-inflammatory cytokines, which limits their therapeutic potential

Therapeutic potential of mesenchymal stromal cells and MSC conditioned medium in Amyotrophic Lateral Sclerosis (ALS)--in vitro evidence from primary motor neuron cultures, NSC-34 cells, astrocytes and microglia.

Administration of mesenchymal stromal cells (MSC) improves functional outcome in the SOD1G93A mouse model of the degenerative motor neuron disorder amyotrophic lateral sclerosis (ALS) as well as in models of other neurological disorders. We have now investigated the effect of the interaction between MSC and motor neurons (derived from both non-transgenic and mutant SOD1G93A transgenic mice), NSC-34 cells and glial cells (astrocytes, microglia) (derived again from both non-transgenic and mutant SOD1G93A ALS transgenic mice) in vitro. In primary motor neurons, NSC-34 cells and astrocytes, MSC conditioned medium (MSC CM) attenuated staurosporine (STS) - induced apoptosis in a concentration-dependent manner. Studying MSC CM-induced expression of neurotrophic factors in astrocytes and NSC-34 cells, we found that glial cell line-derived neurotrophic factor (GDNF) and ciliary neurotrophic factor (CNTF) gene expression in astrocytes were significantly enhanced by MSC CM, with differential responses of non-transgenic and mutant astrocytes. Expression of Vascular Endothelial Growth Factor (VEGF) in NSC-34 cells was significantly upregulated upon MSC CM-treatment. MSC CM significantly reduced the expression of the cytokines TNFα and IL-6 and iNOS both in transgenic and non-transgenic astrocytes. Gene expression of the neuroprotective chemokine Fractalkine (CX3CL1) was also upregulated in mutant SOD1G93A transgenic astrocytes by MSC CM treatment. Correspondingly, MSC CM increased the respective receptor, CX3CR1, in mutant SOD1G93A transgenic microglia. Our data demonstrate that MSC modulate motor neuronal and glial response to apoptosis and inflammation. MSC therefore represent an interesting candidate for further preclinical and clinical evaluation in ALS

Protective effects of MSC CM against STS toxicity in NSC-34 cells.

<p>NSC-34 cells were incubated with DMEM conditioned by MSC (CM) starting 4 h before exposure to STS (0.03 µM). A: STS-induced apoptosis was attenuated best by a 50% dilution of CM, as shown by MTT assay. B: Quantification of cell death by immunocytochemical analysis similarly revealed most neuroprotection at a 10–50% dilution of MSC CM. Values represent means ± SEM, ***<i>p</i><0.001, **<i>p</i><0.01, *<i>p</i><0.05, one-way ANOVA with Bonferroni post-test. C: Immunostainings showed a reduction of cell number due to induction of apoptosis by STS, as well as the protective effect of MSC CM. a: NSC-34 cell monoculture without any treatment. b: Reduction of ß-III tubulin positive NSC-34 cells following exposure to 0.03 µM STS. c: Increase in NSC-34 cell survival due to CM treatment. NSC-34 cells were stained by an antibody against ß-III tubulin (green). Stained nuclei of cultured cells appear in blue (DAPI). Scale bar 100 µm.</p

Influence of MSC CM on astrocytic and microglial gene expression.

<p>The relative mRNA expression of the pro-inflammatory cytokines TNFα and IL-6, the anti-inflammatory cytokine IL-10, the pro-inflammatory enzymes iNOS and COX2, and the neuroprotective chemokine CX3CL1 and its receptor CX3CR1 were examined in non-transgenic and SOD1G93A transgenic astrocyte cultures incubated for 28 h in 100% MSC CM or regular medium without CM and with or without LPS stimulation (500 ng/mL, added during the last 4 h) (A). The relative mRNA expression of the corresponding receptor CX3CR1 was analysed in non-transgenic and SOD1G93A transgenic microglia cultures co-cultured with or without MSC in a transwell system (B). LPS-induced upregulation of the pro-inflammatory cytokines IL-6 and TNFα and of the pro-inflammatory enzyme iNOS was significantly higher in transgenic than in non-transgenic astrocytes and was significantly attenuated by MSC CM (A). MSC CM significantly upregulated CX3CL1 expression in SOD1G93A transgenic astrocytes, while the MSC CM induced increase in CX3CL1 in non-transgenic astrocytes was not statistically significant. MSC significantly increased the expression of CX3CR1 in transgenic microglial cells, and slightly but not significantly upregulated it also in non-transgenic ones (B). Values represent means ± SEM, §§§/^###/***p<0.001, ^#/*p<0.05, two-way ANOVA with Bonferroni post-test.</p

Influence of MSC CM on astrocytes under STS treatment.

<p>Astrocyte cultures were examined at high magnification by fluorescence microscopy after GFAP staining. Cultures were incubated for 48 h in conditioned medium, 1 µM STS was added during the last 24 h. A: MTT assay showed significantly increased survival of non-transgenic astrocytes in presence of 20% and 30% CM. B: Immunocytochemical quantification of astrocyte survival showed most efficient attenuation of STS-induced apoptosis with a 30% dilution of CM, whereas concentrations higher than 50% CM did not mediate significant protective effects. STS-sensitivity of non-transgenic and SOD1G93A transgenic astrocytes did not significantly differ. Values represent means ± SEM, ***<i>p</i><0.001, **<i>p</i><0.01, *<i>p</i><0.05, two-way ANOVA with Bonferroni post-test (Fig. 4B); one-way ANOVA with Bonferroni post-test (Fig. 4A). C: Immunostainings revealed STS-induced apoptosis of astrocytes (a as compared to b) and a protective effect of MSC CM (c).</p

Assessment of staurosporine-induced toxicity in motor neurons, NSC-34 cells and astrocytes.

<p>Dose dependent neurotoxic effect of STS on non-transgenic motor neurons cultured in monoculture as quantified by immunocytochemistry at DIV 7 (n = 8, Fig. 1A). Significant toxic effect of different concentrations of STS on viability of NSC-34 cells (Fig. 1B) and non-transgenic astrocytes (Fig. 1C) as detected by MTT assay. Values represent means ± SEM, ***<i>p</i><0.001, *<i>p</i><0.05. One-way ANOVA with Bonferroni post-test.</p

Involvement of the MAPK/Erk1/2 and PI3-K/Akt pathways in MSC CM-mediated protection.

<p>10 µM MAPK/Erk1/2 inhibitor PD98059 and 10 µM PI3-K/Akt inhibitor LY294002 were applied to both NSC-34 cells (A) and astrocytes (B) 1 h before CM co-incubation and 5 h before induction of apoptosis by STS (0.03 µM for NSC-34 cells, 1 µM for astrocytes). This pre-incubation with PD98059 and LY294002 lowered the protective effect of CM against STS-induced apoptosis resulting in a no longer significant increase in cell survival as compared to STS-exposure. Values represent means ± SEM, ***<i>p</i><0.001, **<i>p</i><0.01, *<i>p</i><0.05, one-way ANOVA with Bonferroni post-test.</p

Protective effects of MSC-co-culture and of MSC conditioned medium (MSC CM) against STS toxicity in motor neurons.

<p>A: The co-cultivation of motor neurons on MSC showed a slight but not significant protective effect against STS toxicity, with greater STS-sensitivity of SOD1G93A motor neurons. Values represent means ± SEM, ***<i>p</i><0.001, *<i>p</i><0.05, two-way ANOVA with Bonferroni post-test. B: Photographs show representative motor neuron cultures at DIV 7 (B). a: SOD1G93A transgenic motor neuron monoculture without any treatment. b: Reduction of SMI 32 positive motor neurons following exposure to 0.1 µM STS. c: Increase in motor neuron survival due to co-cultivation on MSC. Motor neurons were stained by an antibody against SMI 32 (red). MSC were immunopositive for CD44 (green). Stained nuclei of cultured cells appear in blue (DAPI). Scale bar 100 µm. C: STS-induced apoptosis was attenuated best by 40% dilution of MSC CM, whereas at concentrations higher than 50% CM did not mediate neuroprotection. SOD1G93A motor neurons were significantly more sensitive to STS than non-transgenic motor neurons. Values represent means ± SEM, ***<i>p</i><0.001, **/^##<i>p</i><0.01, *<i>p</i><0.05, two-way ANOVA with Bonferroni post-test. D: Immunostainings revealed the apoptotic morphology of motor neurons after STS treatment (b) and the protective effect of MSC CM (c).</p