Question

How could you differentiate between a neurite and filopodia-like protusion in differentiating NSC34 cells?

Answer 1

Alpha-motoneurons appear to be exceedingly affected in neurodegenerative diseases such as amyotrophic lateral sclerosis (ALS). Morphological and physiological degeneration of this neuronal phenotype is typically characterized by a marked decrease of neuronal markers and by alterations of cholinergic metabolism such as reduced choline acetyltransferase (ChAT) expression. The motoneuron-like cell line NSC-34 is a hybrid cell line produced by fusion of neuroblastoma with mouse motoneuron-enriched primary spinal cord cells. In order to further establish this cell line as a valid model system to investigate cholinergic neurodegeneration, NSC-34 cells were differentiated by serum deprivation and additional treatment with all-trans retinoic acid (atRA). Cell maturation was characterized by neurite outgrowth and increased expression of neuronal and cholinergic markers, including MAP2, GAP-43 and ChAT. Subsequently, we used differentiated NSC-34 cells to study early degenerative responses following exposure to various neurotoxins (H2O2, TNF-α, and glutamate). Susceptibility to toxin-induced cell death was determined by means of morphological changes, expression of neuronal marker proteins, and the ratio of pro-(Bax) to anti-(Bcl-2) apoptotic proteins. NSC-34 cells respond to low doses of neurotoxins with increased cell death of remaining undifferentiated cells with no obvious adverse effects on differentiated cells. Thus, the different vulnerability of differentiated and undifferentiated NSC-34 cells to neurotoxins is a key characteristic of NSC-34 cells and has to be considered in neurotoxic studies. Nonetheless, application of atRA induced differentiation of NSC-34 cells and provides a suitable model to investigate molecular events linked to neurodegeneration of differentiated neurons.The nsulin receptor substrate protein of 53 kDa (IRSp53) is critically involved in the formation of filopodia and neurites through mechanisms that have only in part been clarified. Here, we investigated the role of the small scaffold protein LIN7, an interactor of IRSp53. We found that formation of actin-filled protrusions in neuronal NSC34 cells and neurites in neuroblastoma N2A depends on motifs mediating the LIN7:IRSp53 association, as both the coexpression of LIN7 with IRSp53 or the expression of the L27-IRSp53 chimera (a fusion protein between IRSp53 and the LIN7L27 domain for plasma membrane protein complexes association) prevented actin-deficient protrusions induced by overexpressed IRSp53, and enhanced the formation of actin-filled protrusions. The regulatory role of LIN7 in IRSp53-mediated extension of filopodia was demonstrated by live-cell imaging experiments in neuronal N2A cells. Moreover, LIN7 silencing prevented the extension of filopodia and neurites, induced by ectopic expression of IRSp53 or serum starvation, respectively in undifferentiated and differentiated N2A cells. The expression of full length IRSp53 or the LIN7ΔPDZ mutant lacking the domain for association with IRSp53 was unable to restore neuritogenesis in LIN7 silenced cells. Conversely, defective neuritogenesis could be rescued by the expression of RNAi-resistant full length LIN7 or chimeric L27-IRSp53. Finally, LIN7 silencing prevented the recruitment of IRSp53 in Triton X-100 insoluble complexes, otherwise occurring in differentiated cells.