Lack of Prenylated Proteins, Autophagy Impairment and Apoptosis in SH-SY5Y Neuronal Cell Model of Mevalonate Kinase Deficiency

Abstract

Background/Aims: Mevalonate Kinase Deficiency (MKD), is a hereditary disease due to mutations in mevalonate kinase gene (MVK). MKD has heterogeneous clinical phenotypes: the correlation between MVK mutations and MKD clinical phenotype is still to be fully elucidated. Deficiency of prenylated proteins has been hypothesized as possible MKD pathogenic mechanism. Based on this hypothesis and considering that neurologic impairment characterizes Mevalonic Aciduria (MA), the most severe form of MKD, we studied the effects of I268T and N301T MVK mutations on protein prenylation, autophagy and programmed cell death in SH-SY5Y neuroblastoma cell lines. Methods: SH-SY5Y cells were transiently transfected, with the pCMV-6 plasmid containing MVK wild type and the two mutated sequences. Protein prenylation levels were evaluated using GFP-RhoA-F to assess farnesylation, and GFP-RhoA to evaluate geranylgeranylation; autophagy was measured by evaluating LC3 and p62 protein levels, while Annexin V-FITC and Propidium Iodide staining allowed apoptosis detection. Results: MVK mutants’ over-expression causes decreased levels of farnesylation and geranylgeranylation, and also increased LC3 lipidation in SH-SY5Y, with concomitant p62 accumulation. Treatment with bafilomycin A1 (an inhibitor of vacuolar H+-ATPase, a late autophagy inhibitor) further increase LC3-II and p62 levels, suggesting that degradation of autophagolysosome could be impaired. SH-SY5Y, with both MVK mutants, showed apoptosis increase; the presence of N301T associated with augmented cell death. Conclusions: We hypothesize that mevalonate pathway impairment causes alteration of farnesylation and geranylgeranylation proteins and alteration of the autophagic flux; these changes can induce apoptosis, possibly more relevant in the presence of N301T mutation.

Compromised Protein Prenylation as Pathogenic Mechanism in Mevalonate Kinase Deficiency

“…Moreover, induction of autophagy decreased the LPS-induced IL-1b release in PBMCs from healthy controls, whereas induction of autophagy had no significant effect on LPS-induced IL-1b release in PBMCs from MKD patients (79). In line with this, transient overexpression of MVK variants I268T and N301T in neuronal SH-SY5Y cells was found to increase cytosolic RhoA levels, impair autophagy and increase apoptosis (80). In another study, however, the transient expression of CAAX-deficient RhoA in THP-1 cells did not affect mitochondrial membrane potential and autophagy, although it resulted in mitochondrial elongation (58).…”

Mechanisms and function of autophagy in intestinal disease

“…59 Impaired autolysosomal turnover and increased IL1B secretion have been observed in the context of MKD mutations, suggesting these pathways are critical in disease. 60,61 This observation suggests that lysosomal function directly affects intestinal homeostasis, potentially through an autophagy-dependent mechanism. Furthermore, recent studies have identified a role for GPR65, an H + -sensing G protein-coupled receptor, in maintaining lysosomal pH and thus lysosomal function.…”

Effects of mevalonate kinase interference on cell differentiation, apoptosis, prenylation and geranylgeranylation of human keratinocytes are attenuated by farnesyl pyrophosphate or geranylgeranyl pyrophosphate

“…In addition, the increase in apoptosis following MVK interference was significantly attenuated by GGPP, whereas the overexpression of MVK significantly decreased the apoptotic rate of human keratinocytes. Autophagy impairment, apoptosis, and lack of prenylated proteins were observed in SH-SY5Y neuronal cell model of MVK deficiency (17). In addition, MVK mutation and deficiency were shown to cause a rare autosomal recessive disease called mevalonic aciduria.…”