Flupirtine derivatives as potential treatment for the neuronal ceroid lipofuscinoses

Abstract

ObjectiveNeuronal 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 PC12 cells and human NCL cell lines using enhanced flupirtine aromatic carbamate derivatives in vitro.MethodsAromatic 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 PC12 cells following siRNA knockdown of the CLN3 gene, and CLN1‐/CLN2‐/CLN3‐/CLN6‐/CLN8 patient‐derived lymphoblasts. Ceramide levels were determined in CLN1‐/CLN2‐/CLN3‐/CLN6‐/CLN8 patient‐derived lymphoblasts before and after treatment. Expression of BCL‐2, ceramide synthesis enzymes (CERS2/CERS6/SMPD1/DEGS2) and Caspases 3/8/9 levels were compared in treated versus untreated CLN3‐deficient PC12 cells by qRT‐PCR.ResultsRetigabine, the benzyl‐derivatized carbamate and an allyl carbamate derivative were neuroprotective in CLN3‐defective PC12 cells and rescued CLN1‐/CLN2‐/CLN3‐/CLN6‐/CLN8 patient‐derived lymphoblasts from diminished growth and accelerated apoptosis. All drugs decreased ceramide in CLN1‐/CLN2‐/CLN3‐/CLN6‐/CLN8 patient‐derived lymphoblasts. Increased BCL‐2 and decreased ceramide synthesis enzyme expression were established in CLN3‐derived PC12 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.InterpretationThese 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.

Small Molecules that Rescue Multiple Phenotypic Aberrations in an iPSC-Derived Neuron Model of CLN3 Disease

“…We have previously reported small molecule aromatic carbamates that possess multi-functional neuroprotective activity by inducing Bcl-2 levels and increasing autophagy (33). The neuroprotective effect of selected analogues was further 15 demonstrated in lymphoblasts derived from CLN1, CLN2, CLN3, CLN6 and CLN8 patients (32). Here, using neurons differentiated from CLN3 patient iPSCs, we demonstrate that selected compounds from our small molecule library translate their mechanistic effect to induce Bcl-2 and enhance autophagy in a highly phenotypic model of Batten disease.…”

“…25 Propidium iodide toxicity: Neurons derived from CLN3 patient iPSCs were incubated with the indicated compound at 3 µM for 48 hours. Cells were dissociated and incubated with 50 µg/mL PI solution (Life Technologies-# P3566) in NMM medium for 15 minutes at 37 o C. PI Stained cells were analyzed by flow cytometry as previously described (32).…”

“…Indeed, the lysosomal storage disorders (30), such as CLN3 disease, can be thought off as primarily autophagy disorders as lysosomes play a fundamental role in the autophagy pathway by fusing with autophagosomes and digesting their content (31). We have previously disclosed compounds designed in our laboratory that are protective in CLN3-patient derived lymphoblasts and healthy IMR90-c4-induced pluripotent stem cell (iPSC)-derived neurons (32)(33)(34). The compounds upregulate expression of the anti-apoptotic protein Bcl-2 and suppress ceramide levels, directly countering two of the phenotypes of CLN3 disease.…”

Small Molecules that Rescue Multiple Phenotypic Aberrations in an iPSC-Derived Neuron Model of CLN3 Disease

“…We have previously reported small molecule aromatic carbamates that possess multi-functional neuroprotective activity by inducing Bcl-2 levels and increasing autophagy (33). The neuroprotective effect of selected analogues was further demonstrated in lymphoblasts derived from CLN1, CLN2, CLN3, CLN6 and CLN8 patients (32). Here, using neurons differentiated from CLN3 patient iPSCs, we demonstrate that selected compounds from our small molecule library translate their mechanistic effect to induce Bcl-2 and enhance autophagy in a highly phenotypic model of Batten disease.…”

“…Neurons derived from CLN3 patient iPSCs were incubated with the indicated compound at 3 µM for 48 hours. Cells were dissociated and incubated with 50 µg/mL PI solution (Life Technologies-# P3566) in NMM medium for 15 minutes at 37 o C. PI Stained cells were analyzed by flow cytometry as previously described (32).…”