A novel glucosylceramide synthase inhibitor attenuates alpha synuclein pathology and lysosomal dysfunction in preclinical models of synucleinopathy

Mutations in the lysosomal enzyme glucocerebrosidase (GCase, GBA1 gene) are the most common genetic risk factor for developing Parkinson's disease (PD). GCase metabolizes the glycosphingolipids glucosylceramide (GlcCer) and glucosylsphingosine (GlcSph). Mutations in GBA1 reduce enzyme activity and the resulting accu-mulation of glycosphingolipids may contribute to the underlying pathology of PD, possibly via altering lysosomal function. While reduction of GCase activity exacerbates alpha-synuclein (alpha-syn) aggregation, it has not been deter-mined that this effect is the result of altered glycosphingolipid levels and lysosome function or some other effect of altering GCase. The glycosphingolipid GlcCer is synthesized by a single enzyme, glucosylceramide synthase (GCS), and small molecule inhibitors (GCSi) reduce cellular glycosphingolipid levels. In the present studies, we utilize a preformed fibril (PFF) rodent primary neuron in vitro model of alpha-syn pathology to investigate the relationship between glycosphingolipid levels, alpha-syn pathology, and lysosomal function. In primary cultures, pharmacological inhibition of GCase and D409V GBA1 mutation enhanced accumulation of glycosphingolipids and insoluble phosphorylated alpha-syn. Administration of a novel small molecule GCSi, benzoxazole 1 (BZ1), significantly decreased glycosphingolipid concentrations in rodent primary neurons and reduced alpha-syn pathol-ogy. BZ1 rescued lysosomal deficits associated with the D409V GBA1 mutation and alpha-syn PFF administration, and attenuated alpha-syn induced neurodegeneration of dopamine neurons. In vivo studies revealed BZ1 had pharmacological activity and reduced glycosphingolipids in the mouse brain to a similar extent observed in neuronal cultures. These data support the hypothesis that reduction of glycosphingolipids through GCS inhibition may impact progression of synucleinopathy and BZ1 is useful tool to further examine this important biology.

Automated summary

Mutations in the GBA1 gene that encodes the enzyme glucocerebrosidase are the most common genetic risk factor for Parkinson's disease. Reduction of GCase activity due to these mutations leads to accumulation of glycosphingolipids, which may contribute to the pathology of Parkinson's disease by altering lysosomal function. Pharmacological inhibition of GCase and a GBA1 mutation were shown to enhance glycosphingolipid accumulation and insoluble phosphorylated alpha-synuclein, a protein associated with Parkinson's disease.