Our accomplishments for the last year are: (1)We discovered that, in cultured INCL fibroblasts and in the brain tissues of Ppt1-KO mice, the NAD(+)/NADH ratio, the levels of phosphorylated-AMPK (p-AMPK), peroxisome proliferator-activated receptor- (PPAR) coactivator-1 (PGC-1) and Silent Information Regulator T1 (SIRT1) are markedly down-regulated. This suggested an abnormality in AMPK/SIRT1/PGC-1 signaling pathway of energy metabolism. Moreover, we found that, in INCL fibroblasts and in the Ppt1-KO mice, phosphorylated-S6K-1 (p-S6K1) levels, which inversely correlate with lifespan, are markedly elevated. Most importantly, resveratrol (RSV), an antioxidant polyphenol, elevated the NAD(+)/NADH ratio, levels of ATP, p-AMPK, PGC-1 and SIRT1 while decreasing the level of p-S6K1 in both INCL fibroblasts and in Ppt1-KO mice, which showed a modest increase in lifespan. Our results show that disruption of adaptive energy metabolism and increased levels of p-S6K1 are contributing factors in INCL pathogenesis and provide the proof of principle that small molecules such as RSV, which alleviate these abnormalities, may have therapeutic potential. (2)Nonsense mutations in a gene generate premature termination codons producing truncated, nonfunctional or deleterious proteins. PPT1 nonsense-mutations account for approximately 31% of INCL patients in the US. Currently, there is no effective treatment for this disease. While aminoglycosides such as gentamycin suppress nonsense mutations, inherent toxicity of aminoglycosides prohibits chronic use in patients. PTC124 is a non-toxic compound that induces ribosomal read-through of premature termination codons. We sought to determine whether PTC124-treatment of cultured cells from INCL patients carrying nonsense mutations in the PPT1 gene would correct PPT1 enzyme-deficiency with beneficial effects. Our results showed that PTC124-treatment of cultured cells from INCL patients carrying PPT1 nonsense-mutations induced PPT1 enzymatic activity in a dose- and time-dependent manner. This low level of PPT1 enzyme activity induced by PTC124 is virtually identical to that induced by gentamycin-treatment. Even though only a modest increase in PPT1 activity was achieved by PTC124-treatment of INCL cells, this treatment reduced the levels of thioester (constituent of ceroid) load. Our results suggest that PTC124-treatment induces PPT1 enzymatic activity in cultured cells from INCL patients carrying PPT1 nonsense-mutations, and this modest enzymatic activity has demonstrable beneficial effects on these cells. The clinical relevance of these effects may be tested in animal models of INCL carrying nonsense mutations in the PPT1 gene. (3)Omega-3 and omega-6 fatty acids suppress ER- and oxidative stress in cultured neurons and neuronal progenitor cells from mice lacking PPT1. Reactive oxygen species (ROS) damage brain lipids, carbohydrates, proteins, as well as DNA and may contribute to neurodegeneration. We previously reported that ER- and oxidative stress cause neuronal apoptosis in infantile neuronal ceroid lipofuscinosis (INCL), a lethal neurodegenerative storage disease, caused by palmitoyl-protein thioesterase-1 (PPT1) deficiency. Polyunsaturated fatty acids (PUFA) are essential components of cell membrane phospholipids in the brain and excessive ROS may cause oxidative damage of PUFA leading to neuronal death. Using cultured neurons and neuroprogenitor cells from mice lacking Ppt1, which mimic INCL, we demonstrate that Ppt1-deficient neurons and neuroprogenitor cells contain high levels of ROS, which may cause peroxidation of PUFA and render them incapable of providing protection against oxidative stress. We tested whether treatment of these cells with omega-3 or omega-6 PUFA protects the neurons and neuroprogenitor cells from oxidative stress and suppress apoptosis. We report here that both omega-3 and omega-6 fatty acids protect the Ppt1-deficient cells from ER- as well as oxidative stress and suppress apoptosis. Our results suggest that PUFA supplementation may have neuroprotective effects in INCL. (4) Evaluation of Neurodegeneration in a Mouse Model of Infantile Batten Disease by Magnetic Resonance Imaging and Magnetic Resonance Spectroscopy. The effectiveness of potentiall therapies in vivo could be evaluated using Ppt1-knockout (Ppt1-KO) mice, which recapitulate virtually all clinical and pathological features of INCL. However, such studies will require non-invasive methods that could be used to perform repeated evaluations on the same animal. Thus, the development of non-invasive method(s) of evaluation is urgently needed. Here we report that magnetic resonance imaging and magnetic resonance spectroscopy may be used to evaluate potential therapeutic agents on Ppt1-KO mice to determine their effectiveness in vivo. (5) We are continuing a bench-to bedside clinical trial to determine the beneficial effects of a combination therapy of INCL patients with cystagon and mucomyst. The study results are expected to be published within the coming year. We also conducted a study which we have been interested in for many years: (6) Lack of an endogenous anti-inflammatory protein in mice enhances colonization of B16F10 melanoma cells in the lungs. Emerging evidence indicates a link between inflammation and cancer metastasis, but the molecular mechanism(s) remains unclear. Uteroglobin (UG), a potent anti-inflammatory protein, is constitutively expressed in the lungs of virtually all mammals. UG-knock-out (UG-KO) mice, which are susceptible to pulmonary inflammation, and B16F10 melanoma cells, which preferentially metastasize to the lungs, provide the components of a model system to determine how inflammation and metastasis are linked. We report here that B16F10 cells, injected into the tail vein of UG-KO mice, form markedly elevated numbers of tumor colonies in the lungs compared with their wild type littermates. Remarkably, UG-KO mouse lungs overexpress two calcium-binding proteins, S100A8 and S100A9, whereas B16F10 cells express the receptor for advanced glycation end products (RAGE), which is a known receptor for these proteins. Moreover, S100A8 and S100A9 are potent chemoattractants for RAGE-expressing B16F10 cells, and pretreatment of these cells with a blocking antibody to RAGE suppressed migration and invasion. Interestingly, in UG-KO mice S100A8/S100A9 concentrations in blood are lowest in tail vein and highest in the lungs, which most likely guide B16F10 cells to migrate to the lungs. Further, B16F10 cells treated with S100A8 or S100A9 overexpress matrix metalloproteinases, which are known to promote tumor invasion. Most notably, the metastasized B16F10 cells in UG-KO mouse lungs express MMP-2, MMP-9, and MMP-14 as well as furin, a pro-protein convertase that activates MMPs. Taken together, our results suggest that a lack of an anti-inflammatory protein leads to increased pulmonary colonization of melanoma cells and identify RAGE as a potential anti-metastatic drug target. (7) We published a minireview in the Journal of Biological Chemistry describing the contributions of genetic and environmental factors in allergic asthma.
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