We have accomplished the following advances during the past year: (A) The availability of Ppt1-knockout (Ppt1-KO) mice, which recapitulate virtually all clinical and pathological features of INCL, provides an opportunity to test the effectiveness of novel therapeutic strategies in vivo. However, such studies will require noninvasive methods that can be used to perform serial evaluations of the same animal receiving an experimental therapy. Thus, the development of noninvasive method(s) of evaluation is urgently needed. We developed a non-invasive method for the evaluation of the progression of neurodegeneration in Ppt1-KO mice starting at 3 months of age by using MRI and MR spectroscopy (MRS) and repeating these tests using the same mice at 4, 5 and 6 months of age. Our findings provide a method to serially examine the brains of mouse models of neurodegenerative diseases (e.g. Ppt1-KO mice) using noninvasive and nonlethal procedures such as MRI and MRS. These methods may be useful in studies to understand the progression of neuropathology in animal models of neurodegenerative diseases as they allow repeated evaluations of the same animal in which experimental therapies are tested.(B) Disruption of the blood-brain barrier (BBB) is a serious complication frequently encountered in neurodegenerative disorders. As stated above, INCL is a devastating childhood neurodegenerative LSD caused by palmitoyl-protein thioesterase-1 (PPT1) deficiency. However, it remains unclear whether BBB is disrupted in INCL and if so, what might be the molecular mechanism(s) of this complication. Using Ppt1-KO mice we have delineated that T(H)17 lymphocytes producing IL-17A mediate disruption of the BBB by stimulating production of matrix metalloproteinases (MMPs), which degrade the tight junction proteins essential for maintaining BBB integrity. Importantly, dietary supplementation of resveratrol (RSV), a naturally occurring antioxidant/anti-inflammatory polyphenol, markedly reduced the levels of T(H)17 cells, IL-17A and MMPs, and elevated the levels of tight junction proteins, which improved the BBB integrity in Ppt1-KO mice. Intriguingly, we found that RSV suppressed the differentiation of CD4(+) T lymphocytes to IL-17A-positive T(H)17 cells. Our findings uncover a mechanism by which T(H)17 lymphocytes mediate BBB disruption and suggest that small molecules such as RSV that suppress T(H)17 differentiation are therapeutic targets for neurodegenerative disorders such as INCL. (C) We have previously reported that children with INCL have increased risk of hypothermia during anesthesia. Moreover, we have shown that PPT1-deficiency is associated with disruption of adaptive energy metabolism, downregulation of peroxisome roliferator-activated receptor γcoactivator 1α(PGC-1α), and mitochondrial dysfunction. In a collaborative study with Dr. Quezado and colleagues, we tested a hypothesis PPT1-deficiency in mice impair thermoregulation observed in children with INCL involving the upregulation of PGC-1αand uncoupling protein 1 (UCP-1) in brown adipose tissue. We found that the Ppt1-KO mice, a well-studied model of INCL, have lower basal body temperature as they age and develop hypothermia during cold exposure. This inability to maintain body temperature during cold exposure in Ppt1-KO mice was associated with upregulation of PGC-1αand UCP-1 but with lower levels of sympathetic neurotransmitters in brown adipose tissue. The results of our experiments uncover previously unknown phenotypes associated with PPT1-deficiency and suggest that in patients with this disease, impaired thermoregulation and hypothermia are potential risk factors.(D)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 treatmentfor this disease. While aminoglycosides such as gentamycin suppress nonsense mutations, inherent toxicity of aminoglycosides prohibits chronic use inpatients. 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. Most recently, we have identified a thioesterase mimetic small molecule that is non-toxic, ameliorates neuropathology and extends lifespan in Ppt1-KO mice.This compound promises to have clinical implications for the therapy of INCL patients. 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. Our ongoing studies are attempting to generate a mouse model of INCL in which the most common nonsense mutations in the Ppt1 gene is knocked-in (Ppt1-KI mice). We plan to test whether PTC124 can induce PPT1 activity in these mice and ameliorate INCL pathogenesis.(E) PPT1 is a lysosomal enzyme that catalyzes the cleavage of thioester linkage in palmitoylated (S-acylated) proteins and its deficiency impairs degradation of fatty-acylated proteins by lysosomal proteases. Consequently, accumulation of these lipid-modified proteins (constituents of ceroid) in lysosomes leads to INCL pathogenesis. Given that thioester linkages are susceptible to nucleophilic attack, drugs that can cleave such linkages may have therapeutic potential for INCL. The results of our laboratory studies showed that two drugs, phosphocysteamine and N-acetylcysteine, cleave thioester linkage in 14C palmitoyl-CoA, a model substrate of PPT1. Moreover, these small molecules also mediate depletion of ceroid from cultured lymphoblasts and fibroblasts from INCL patients. The drugs also inhibit apoptosis in these cells. These laboratory results prompted us to initiate a bench-to-bedside clinical trial to determine whether a combination of Cystagon (cysteamine bitartrate) and Mucomyst (N-acetylcysteine) is beneficial for patients with INCL. To date, we have treated 9 INCL patients and the study results are being analyzed to evaluate whether recruitment of more patients are needed to arrive at a meaningful conclusion of this study. Thus, at present our protocol is not recruiting any patients until our analysis of the data is complete and a determination is made as to whether we need to admit more patients to this protocol.
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