Mutations in glucose transporter 10 (GLUT10) alter angiogenesis and cause arterial tortuosity syndrome (ATS); however, the mechanisms by which these mutations cause disease remain unclear. It has been reported that in most cells, mitochondria are the major source of reactive oxygen species (ROS). Moreover, mitochondria are known to incorporate as well as recycle vitamin C, which plays a critical role in redox homeostasis, although the molecular mechanism(s) underlying mitochondrial vitamin C uptake are poorly understood. We report here that GLUT10 localizes predominantly to the mitochondria of smooth muscle cells and insulin-stimulated adipocytes, where GLUT10 is highly expressed. We further demonstrate that GLUT10 facilitates transport of l-dehydroascorbic acid (DHA), the oxidized form of vitamin C, into mitochondria, and also increases cellular uptake of DHA, which in turn protects cells against oxidative stress. This protection is compromised when GLUT10 expression in mitochondria is inhibited. In addition, we found that aortic smooth muscle cells from GLUT10-mutant mice have higher ROS levels than those from wild-type mice. Our results identify the physiological role of GLUT10 as the mitochondrial DHA transporter, and demonstrate that GLUT10 protects cells from oxidative injury. Furthermore, our findings provide a mechanism to explain the ascorbate in mitochondria and show how loss-of-function GLUT10 mutations may lead to arterial abnormalities in ATS. These results also reinforce the importance of vitamin C and ROS in degenerative diseases.

Neuronal ceroid lipofuscinoses represent the most common childhood neurodegenerative storage disorders. Infantile neuronal ceroid lipofuscinosis (INCL) is caused by palmitoyl protein thioesterase-1 (PPT1) deficiency. Although INCL patients show signs of abnormal neurotransmission, manifested by myoclonus and seizures, the molecular mechanisms by which PPT1 deficiency causes this abnormality remain obscure. Neurotransmission relies on repeated cycles of exo-and endocytosis of the synaptic vesicles (SVs), in which several palmitoylated proteins play critical roles. These proteins facilitate membrane fusion, which is required for neurotransmitter exocytosis, recycling of the fused SV membrane components, and regeneration of fresh vesicles. However, palmitoylated proteins require depalmitoylation for recycling. Using postmortem brain tissues from an INCL patient and tissue from the PPT1-knockout (PPT1-KO) mice that mimic INCL, we report here that PPT1 deficiency caused persistent membrane anchorage of the palmitoylated SV proteins, which hindered the recycling of the vesicle components that normally fuse with the presynaptic plasma membrane during SV exocytosis. Thus, the regeneration of fresh SVs, essential for maintaining the SV pool size at the synapses, was impaired, leading to a progressive loss of readily releasable SVs and abnormal neurotransmission. This abnormality may contribute to INCL neuropathology.

Infantile neuronal ceroid lipofuscinosis (INCL), a neurodegenerative storage disorder of childhood, is caused by mutations in the palmitoyl-protein thioesterase-1 (PPT1) gene. PPT1 cleaves thioester linkages in S-acylated (palmitoylated) proteins and its mutation causes abnormal intracellular accumulation of fatty-acylated proteins and peptides leading to INCL pathogenesis. Although apoptosis is the suggested cause of neurodegeneration in INCL, the molecular mechanism(s) of apoptosis remains unclear. Using the PPT1-knockout (PPT1-KO) mice that mimic INCL, we previously reported that one mechanism of apoptosis involves endoplasmic reticulum (ER) stress-induced caspase-12 activation. However, the human caspase-12 gene contains several mutations, which make it functionally inactive. Thus, it has been suggested that human caspase-4 is the counterpart of murine caspase-12. Here we report that in the human INCL brain ER stress-induced activation of unfolded protein response (UPR) mediates caspase-4 and caspase-3 activation and apoptosis. Moreover, we show that the INCL brain contains high level of growth-associated protein-43 (GAP-43), which is known to undergo palmitoylation. We also demonstrate that transfection of cultured INCL cells with a green fluorescent protein-GAP-43 cDNA construct shows abnormal localization of this protein in the ER. Further, INCL cells manifest evidence of ER stress and UPR (elevated levels of Grp-78/Bip and GADD153), caspase-4 as well as caspase-3 activation and cleavage of poly(ADP)-ribose polymerase, a compelling sign of apoptosis. Most importantly, we show that inhibition of caspase-4 activity protects INCL cells from undergoing apoptosis. Our results provide insight into at least one of the molecular mechanisms of apoptosis in INCL and may allow the identification of potential targets for therapeutic intervention.

Numerous proteins undergo modification by palmitic acid (S-acylation) for their biological functions including signal transduction, vesicular transport and maintenance of cellular architecture. Although palmitoylation is an essential modification, these proteins must also undergo depalmitoylation for their degradation by lysosomal proteases. Palmitoyl-protein thioesterase-1 (PPT1), a lysosomal enzyme, cleaves thioester linkages in S-acylated proteins and removes palmitate residues facilitating the degradation of these proteins. Thus, inactivating mutations in the PPT1 gene cause infantile neuronal ceroid lipofuscinosis (INCL), a devastating neurodegenerative storage disorder of childhood. Although rapidly progressing brain atrophy is the most dramatic pathological manifestation of INCL, the molecular mechanism(s) remains unclear. Using PPT1-knockout (PPT1-KO) mice that mimic human INCL, we report here that the endoplasmic reticulum (ER) in the brain cells of these mice is structurally abnormal. Further, we demonstrate that the level of growth-associated protein-43 (GAP-43), a palmitoylated neuronal protein, is elevated in the brains of PPT1-KO mice. Moreover, forced expression of GAP-43 in PPT1-deficient cells results in the abnormal accumulation of this protein in the ER. Consistent with these results, we found evidence for the activation of unfolded protein response (UPR) marked by elevated levels of phosphorylated translation initiation factor, eIF2alpha, increased expression of chaperone proteins such as glucose-regulated protein-78 and activation of caspase-12, a cysteine proteinase in the ER, mediating caspase-3 activation and apoptosis. Our results, for the first time, link PPT1 deficiency with the activation of UPR, apoptosis and neurodegeneration in INCL and identify potential targets for therapeutic intervention in this uniformly fatal disease.

The molecular mechanisms that initiate and control the metabolic activities of seed germination are largely unknown. Sugars may play important roles in regulating such metabolic activities in addition to providing an essential carbon source for the growth of young seedlings and maintaining turgor pressure for the expansion of tissues during germination. To test this hypothesis, we investigated the physiological role of sugars in the regulation of alpha-amylase gene expression and carbohydrate metabolism in embryo and endosperm of germinating rice seeds. RNA gel blot analysis revealed that in the embryo and aleurone cells, expression of four alpha-amylase genes was differentially regulated by sugars via mechanisms beyond the well-known hormonal control mechanism. In the aleurone cells, expression of these alpha-amylase genes was regulated by gibberellins produced in the embryo and by osmotically active sugars. In the embryo, expression of two alpha-amylase genes and production of gibberellins were transient, and were probably induced by depletion of sugars in the embryo upon imbibition, and suppressed by sugars influx from the endosperm as germination proceeded. The deferential expression of the four alpha-amylase genes in the embryo and aleurone cells was probably due to their markedly different sensitivities to changes in tissue sugar levels. Our study supports a model in which sugars regulate the expression of alpha-amylase genes in a tissue-specific manner: via a feedback control mechanism in the embryo and via an osmotic control mechanism in the aleurone cells. An interactive loop among sugars, gibberellins, and alpha-amylase genes in the germinating cereal grain is proposed.