Impaired geranylgeranyltransferase-I regulation reduces membrane-associated Rho protein levels in aged mouse brain

Abstract

Synaptic impairment rather than neuronal loss may be the leading cause of cognitive dysfunction in brain aging. Certain small Rho-GTPases are involved in synaptic plasticity, and their dysfunction is associated with brain aging and neurodegeneration. Rho-GTPases undergo prenylation by attachment of geranylgeranylpyrophosphate (GGPP) catalyzed by GGTase-I. We examined age-related changes in the abundance of Rho and Rab proteins in membrane and cytosolic fractions as well as of GGTase-I in brain tissue of 3- and 23-month-old C57BL/6 mice. We report a shift in the cellular localization of Rho-GTPases toward reduced levels of membrane-associated and enhanced cytosolic levels of those proteins in aged mouse brain as compared with younger mice. The age-related reduction in membrane-associated Rho proteins was associated with a reduction in GGTase-Iβ levels that regulates binding of GGPP to Rho-GTPases. Proteins prenylated by GGTase-II were not reduced in aged brain indicating a specific targeting of GGTase-I in the aged brain. Inhibition of GGTase-I in vitro modeled the effects of aging we observed in vivo. We demonstrate for the first time a decrease in membrane-associated Rho proteins in aged brain in association with down-regulation of GGTase-Iβ. This down-regulation could be one of the mechanisms causing age-related weakening of synaptic plasticity.

Keywords: aging; brain; isoprenoid; rho protein; small GTPases; synaptic markers.

Figure 1. Abbreviated mevalonate/isoprenoid/cholesterol pathway

The mevalonate pathway is a crucial metabolic pathway in eukaryotic cells for which mevalonate is the precursor of several compounds including the isoprenoids farnesyl- (FPP), geranylgeranyl-pyrophosphate (GGPP) and cholesterol. The synthesis of GGPP and FPP is catalyzed by farnesylpyrophosphate synthase (FDPS) and geranylgeranylpyrophosphate synthase (GGPPS), respectively. GGPP is substrate for post-translational geranylgeranylation of small GTPases by geranylgeranyltransferases (GGTase). GGTase-I prenylates Rho-GTPases (Rac-1, RhoA, Cdc42) and GGTase-II prenylates Rab-GTPases (Rab3A, Rab11B). Geranylgeranylation of these proteins (-GG) is critical for membrane localization and optimal function. GGTI-2133 is a specific inhibitor of GGTase-I. Aging reduces the relative GGTas-I activity leading to reduced levels of prenylated Rho-proteins.

Figure 2. Brain Rho-GTPase membrane-associated and total protein levels

Western blot analysis of brain membrane preparations (a, c, e), and of total homogenates (b, d, f) isolated from brains of young (3 months old) and aged (23 months old) mice was used to characterize the prenylation of Rac1 (a, b), RhoA (c, d), and Cdc42 (e, f). Levels of membrane-associated, geranylgeranylated Rho-GTPases (membrane Rho-GTPases) were normalized to the membrane marker Flotillin1. Levels of total homogenates (total Rho-GTPases) were normalized to GAPDH. Each graph shows representive Western blots. Mean ±SEM, unpaired t-test (p*<0,05), n=6.

Figure 3. Brain Rho/Rab-GTPase cytosolic protein levels

Western blot analysis was used to characterize the the pool of of Rac1 (a), RhoA (b), Cdc42 (c), Rab11B (d) and Rab3A (e) in cytosolic fractions (isolated from brains of young (3 months old) and aged (23 months old) mice). Small GTPase levels were normalized to GAPDH. Each graph shows representive Western blots. Mean ±SEM, unpaired t-test (p*<0,05), n=6.

Figure 4. Brain RhoGDIα and RabGDIα protein levels

Western blot analysis of RhoGDIα (a) and RabGDIα (b) in brain cytosol preparations isolated from brains of 3 months old and 23 months old mice. Protein levels were normalized to GAPDH. Each graph shows representive Western blots. Mean ±SEM, unpaired t-test (ns=not significant), n=6.

Figure 5. Brain Rab-GTPase membrane-associated and total protein levels

Western blot analysis of brain membrane preparations (a, c), and of total homogenates (b, d) isolated from brains of young (3 months old) and aged (23 months old) mice was used to characterize the prenylation of Rab11B (a, b) and Rab3A (c, d). Levels of membrane-associated, geranylgeranylated Rab-GTPases (membrane Rab-GTPases) were normalized to the membrane marker Flotillin1. Levels of total homogenates (total Rho-GTPases) were normalized to GAPDH. Each graph shows representive Western blots. Mean ±SEM, unpaired t-test (p*<0,05), n=6.

Figure 6. Brain GGPP levels, relative GGTase activity, GGTase-I protein and gene expression

(a) GGPP levels (pmol/mg protein) of brains from 3 and 23 months old C57BL/6mice were analysed using HPLC-FD as described in Material & Methods. Western blot analysis (b) and qRT-PCR (c) of brain homogenates isolated from brains of 3 and 23 months old C57BL/6 mice were used to assess protein (b) and mRNA (c) levels of the GGTase-I subunit. Relative GGTase activity (d) was assayed as described in the Materials & Methods section. GGTase-I protein and mRNA levels were normalized to GAPDH. Mean ±SEM, unpaired t-test (p*<0.05; p***<0.001), n=8 (GGPP levels), n=4 (relative GGTase activity), n=6 (GGTase-Iβ protein and gene expression levels).

Figure 7. Effects of GGTase-I inhibition on membrane localization of Rac1 and GGPP levels in human SH-SY5Y neuroblastoma cells

Cells were treated with 10 and 25 μmol/L GGTI-2133 for 48 hrs. Western blot analysis of membrane preparations (a), cytosol preparations (b) and total homogenate (c) was used to characterize the cellular localization of Rac1 (a, b, c) in human SH-SY5Y neuroblastoma cells, treated with GGTI-2133. Levels of membrane-associated, geranylgeranylated Rac1 (membrane Rac1) were normalized to the membrane marker Flotillin1. Levels of cytosol and total Rac1 in cytosol preparations and cell homogenate were normalized to GAPDH. Western blot analysis of RhoGDIα(d) and RabGDIα(e) in cytosol preparations. Each graph shows representive Western blots. GGPP (f) levels were determined using HPLC-FD as described in Material & Methods. Mean ±SEM, p*<0.05; p**<0.01; p***<0.001; ns=not significant; ANOVA & Tukey's post test), n=6.

Figure 8. Effects of GGTase-I inhibition on synaptic markers in SH-SY5Y neuroblastoma cells

Cells were treated with 10 μmol/L GGTI-2133 for 48 hrs. Western blot analysis of total homogenates was used to assess protein levels of synaptophysin (a) and GAP43 (b). Effects of aging on brain synaptic markers. Western blot analysis of total homogenates isolated from brains of young (3 months old) and aged (23 months old) mice was used to assess protein levels of synaptophysin (c) and GAP43 (d). Protein levels were normalized to GAPDH. Each graph shows a representive Western blot. Mean ±SEM, unpaired t-test (p*<0.05; p**<0.01; p***<0.001), n=6. Cover image suggestion. Rho-GTPases are geranylgeranylated by transferase GGTase-I. Their prenylation is essential for their localization in membranes, the site of their activation and function. Even that we found GGPP levels elevated in brains of aged (23 months) mice compared to younger (3 months) mice as well as in GGTI-2133 treated SH-SY5Y cells, their amount of total (homogenate) Rho-GTPases (Rac1, RhoA and Cdc42) was unchanged. To our surprise the prenylated Rho-GTPases were decreased in membrane preparations of aged mice brains and SH-SY5Y, treated with the GGTaseI-inhibitor GGTI-2133. These findings directly correlate with the reduction of relative GGTase activity, GGTaseIβ protein and mRNA levels. Since Rac1, RhoA and Cdc42 are associated with synaptogenesis, we examined GAP43 and synaptophysin, two common synaptic marker proteins. GAP43 and synaptophysin declined in an age-related manner in the mouse brain and were also reduced in our in vitro model. Faulty regulation of Rho proteins in aged brain is associated with a specific deficit in GGTase-I , which could contribute to age-related deficits in neuronal outgrowth.