Flupirtine derivatives as potential treatment for the neuronal ceroid lipofuscinoses

Abstract

Abstract Objective Neuronal Ceroid Lipofuscinoses ( NCL ) are fatal inherited neurodegenerative diseases with established neuronal cell death and increased ceramide levels in brain, hence, a need for disease‐modifying drug candidates, with potential to enhance growth, reduce apoptosis and lower ceramide in neuronal precursor PC 12 cells and human NCL cell lines using enhanced flupirtine aromatic carbamate derivatives in vitro. Methods Aromatic carbamate derivatives were tested by establishing growth curves under pro‐apoptotic conditions and activity evaluated by trypan blue and JC ‐1 staining, as well as a drop in pro‐apoptotic ceramide in neuronal precursor PC 12 cells following si RNA knockdown of the CLN 3 gene, and CLN 1‐/ CLN 2‐/ CLN 3‐/ CLN 6‐/ CLN 8 patient‐derived lymphoblasts. Ceramide levels were determined in CLN 1‐/ CLN 2‐/ CLN 3‐/ CLN 6‐/ CLN 8 patient‐derived lymphoblasts before and after treatment. Expression of BCL ‐2 , ceramide synthesis enzymes ( CERS 2/ CERS 6/ SMPD 1/ DEGS 2 ) and Caspases 3/8/9 levels were compared in treated versus untreated CLN 3‐deficient PC 12 cells by qRT ‐ PCR . Results Retigabine, the benzyl‐derivatized carbamate and an allyl carbamate derivative were neuroprotective in CLN 3‐defective PC 12 cells and rescued CLN 1‐/ CLN 2‐/ CLN 3‐/ CLN 6‐/ CLN 8 patient‐derived lymphoblasts from diminished growth and accelerated apoptosis. All drugs decreased ceramide in CLN 1‐/ CLN 2‐/ CLN 3‐/ CLN 6‐/ CLN 8 patient‐derived lymphoblasts. Increased BCL ‐2 and decreased ceramide synthesis enzyme expression were established in CLN 3‐derived PC 12 cells treated with the benzyl and allyl carbamate derivatives. They down‐regulated Caspase 3/Caspase 8 expression. Caspase 9 expression was reduced by the benzyl‐derivatized carbamate. Interpretation These findings establish that compounds analogous to flupirtine demonstrate anti‐apoptotic activity with potential for treatment of NCL disease and use of ceramide as a marker for these diseases.