Introduction and objectives:Amyotrophic Lateral Sclerosis (ALS) is a devastating disease in which patients suffer increasing paralysis due to the loss of motor neurons (MNs). 97% of all ALS cases show TDP-43 pathogenic inclusions, and mutations in the gene TARDBP, encoding TDP-43, cause familial ALS, suggesting that TDP43 pathology plays a causal role in MN degeneration in most ALS patients.TDP-43 is an mRNA-binding protein that predominantly resides in the nucleus. Pathogenic modifications include its cytoplasmic mislocalization, cleavage, phosphorylation and aggregation. Here, we differentiated isogenic iPSC reporter lines expressing DENDRA2-tagged TDP-43 in its wild-type (WT) or A315T mutant form into MN precursors and MNs and evaluated TDP-43 behaviour under unstressed conditions and following proteasome blockage. Results: Differentiated MN precursors stained positive for Nestin, while MNs expressed Smi-32 and a small fraction for MN-specific marker HB9. DENDRA2-tagged TDP-43 WT and A315T showed no differences in localization nor signal intensity when analysed by fluorescence microscopy. Western blot (WB) and filter retardation assay showed that untagged and DENDRA2-tagged TDP-43 protein levels were similar among cell lines. Aging is a major risk factor for ALS pathogenesis and is associated with proteasome dysfunction. Treatment with the proteasome inhibitor MG-132 increased the amount of detected TDP-43 fragments as well as the TDP-43 insoluble species, in all cases. Additionally, proteasome inhibition allowed the detection of phosphorylated TDP-43 in all cell lines. Under unstressed conditions, untagged TDP-43 and DENDRA2-tagged TDP-43 in MNs showed nuclear localization. In contrast, after proteasome inhibition, untagged TDP-43 mislocalized to the cytoplasm, while DENDRA2-tagged TDP-43 was still nuclear. MG-132 treatment increased MN degeneration as marked by cleaved-caspase 3. Conclusions: Our results show that differentiated isogenic reporter cell lines MNs represent a valuable model to study and validate novel molecular mechanisms related to TDP-43 pathology in ALS as well as related diseases.

Differentiation of isogenic iPSC reporter lines to motoneurons as tools for familial and sporadic amyotrophic lateral sclerosis / B. Tedesco, M.E. Cicardi, V. Crippa, V. Tripathy, R. Cristofani, P. Rusmini, V. Ferrari, E. Casarotto, M. Chierichetti, M. Cozzi, L. Marrone, J. Sterneckert, A. Poletti. ((Intervento presentato al convegno New Perspectives in Neuroscience : Research Results of Young Italian Neuroscientist - National meeting of PhD students in Neuroscience tenutosi a online nel 2020.

Differentiation of isogenic iPSC reporter lines to motoneurons as tools for familial and sporadic amyotrophic lateral sclerosis

B. Tedesco;M. E. Cicardi;V. Crippa;R. Cristofani;P. Rusmini;V. Ferrari;E. Casarotto;M. Chierichetti;M. Cozzi;A. Poletti
2020-09-30

Abstract

Introduction and objectives:Amyotrophic Lateral Sclerosis (ALS) is a devastating disease in which patients suffer increasing paralysis due to the loss of motor neurons (MNs). 97% of all ALS cases show TDP-43 pathogenic inclusions, and mutations in the gene TARDBP, encoding TDP-43, cause familial ALS, suggesting that TDP43 pathology plays a causal role in MN degeneration in most ALS patients.TDP-43 is an mRNA-binding protein that predominantly resides in the nucleus. Pathogenic modifications include its cytoplasmic mislocalization, cleavage, phosphorylation and aggregation. Here, we differentiated isogenic iPSC reporter lines expressing DENDRA2-tagged TDP-43 in its wild-type (WT) or A315T mutant form into MN precursors and MNs and evaluated TDP-43 behaviour under unstressed conditions and following proteasome blockage. Results: Differentiated MN precursors stained positive for Nestin, while MNs expressed Smi-32 and a small fraction for MN-specific marker HB9. DENDRA2-tagged TDP-43 WT and A315T showed no differences in localization nor signal intensity when analysed by fluorescence microscopy. Western blot (WB) and filter retardation assay showed that untagged and DENDRA2-tagged TDP-43 protein levels were similar among cell lines. Aging is a major risk factor for ALS pathogenesis and is associated with proteasome dysfunction. Treatment with the proteasome inhibitor MG-132 increased the amount of detected TDP-43 fragments as well as the TDP-43 insoluble species, in all cases. Additionally, proteasome inhibition allowed the detection of phosphorylated TDP-43 in all cell lines. Under unstressed conditions, untagged TDP-43 and DENDRA2-tagged TDP-43 in MNs showed nuclear localization. In contrast, after proteasome inhibition, untagged TDP-43 mislocalized to the cytoplasm, while DENDRA2-tagged TDP-43 was still nuclear. MG-132 treatment increased MN degeneration as marked by cleaved-caspase 3. Conclusions: Our results show that differentiated isogenic reporter cell lines MNs represent a valuable model to study and validate novel molecular mechanisms related to TDP-43 pathology in ALS as well as related diseases.
Settore BIO/13 - Biologia Applicata
Settore BIO/09 - Fisiologia
Italian Society for Neuroscience
Differentiation of isogenic iPSC reporter lines to motoneurons as tools for familial and sporadic amyotrophic lateral sclerosis / B. Tedesco, M.E. Cicardi, V. Crippa, V. Tripathy, R. Cristofani, P. Rusmini, V. Ferrari, E. Casarotto, M. Chierichetti, M. Cozzi, L. Marrone, J. Sterneckert, A. Poletti. ((Intervento presentato al convegno New Perspectives in Neuroscience : Research Results of Young Italian Neuroscientist - National meeting of PhD students in Neuroscience tenutosi a online nel 2020.
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Utilizza questo identificativo per citare o creare un link a questo documento: http://hdl.handle.net/2434/785359
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