During the past year we have accomplished the following: (a) We have previously reported that phosphocysteamine and N-acetylcysteine mediate ceroid depletion in cultured cells from patients with this disease. We completed a bench-to-bedside clinical trial to assess whether a combination of oral cysteamine bitartrate and N-acetylcysteine is beneficial for INCL patients. Children between 6 months and 3 years of age with INCL carrying any combination two of two the seven most lethal CLN1/PPT1 mutations were included in this study. Patients received oral cysteamine bitartrate (60 mg/kg per day) and N-acetylcysteine (60 mg/kg per day) and were assessed every 6-12 months until they had an isoelectric electroencephalogram (EEG, attesting to a vegetative state, or were too ill to travel). Patients were also assessed by electroretinography, brain MRI and magnetic resonance spectroscopy (MRS), and electron microscopic analyses of leukocytes for granular osmiophilic deposits (GRODs). Children also underwent physical and neurodevelopmental assessments on the Denver scale. Outcomes were compared with the reported natural history of infantile neuronal ceroid lipofuscinosis and that of affected older siblings. We recruited ten children with infantile neuronal ceroid lipofuscinosis;one child was lost to follow-up after the first visit and nine patients (five girls and four boys) were followed up for 8 to 75 months. The results of this study suggest that a combination therapy with cysteamine bitartrate and N-acetylcysteine is associated with delayed onset of isoelectric EEG, depletion of ceroid (GRODs) accumulation, and subjective benefits as reported by parents and physicians. Our through systematic and quantitative report of the natural history of a cohort of INCL patients carrying the most lethal CLN1 mutations provide a guide for future evaluation(s) of experimental therapies;(b) Towards our goals to identify and characterize non-toxic small molecules as potential therateutic agents for INCL we sought to identify and characterize such molecules using Ppt1-/- mice, which are a reliable animal model of INCL. As described above, PPT1-deficiency impairs the cleavage of thioester linkage in palmitoylated proteins (constituents of ceroid), preventing degradation by lysosomal hydrolases. Consequently, accumulation of lysosomal ceroid leads to INCL. Thioester linkage is cleaved by nucleophilic attack. Hydroxylamine, a potent nucleophilic cellular metabolite, may have therapeutic potential for INCL, but its toxicity precludes clinical application. We found that a hydroxylamine derivative, N-(tert-Butyl) hydroxylamine (NtBuHA), was non-toxic, cleaved thioester linkage in palmitoylated proteins and mediated lysosomal ceroid depletion in cultured cells from INCL patients. In Ppt1-/- mice, NtBuHA crossed the blood-brain barrier, depleted lysosomal ceroid, suppressed neuronal apoptosis, slowed neurological deterioration and extended lifespan. Thus, our findings provide a proof of concept that thioesterase-mimetic and antioxidant small molecules such as NtBuHA are potential drug targets for thioesterase deficiency diseases such as INCL. A US patent application has been filed for this discovery;(c) 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) Dynamic palmitoylation (palmitoylation-depalmitoylation) has emerged as an important regulatory mechanism for the function of many proteins. It requires both palmitoyltransferases (PATs) that catalyze palmitoylation and thioesterases, which facilitate depalmitoylation. Although much is known about the PATs, our knowledge on thioesterases is limited. Acyl-protein thioesterase-1 (APT1) and APT2 are cytosolic enzymes that catalyze depalmitoylation of membrane-anchored, palmitoylated H-Ras and growth-associated protein-43 (GAP-43), respectively. However, the mechanism(s) of cytosol-membrane shuttling of APT1 and APT2, required for depalmitoylating their substrates H-Ras and GAP-43, respectively, remained largely unknown. Here, we report that both APT1 and APT2 undergo palmitoylation on Cys-2. Moreover, blocking palmitoylation adversely affects membrane localization of both APT1 and APT2 and that of their substrates. We also demonstrate that APT1 not only catalyzes its own depalmitoylation but also that of APT2 promoting dynamic palmitoylation (palmitoylation-depalmitoylation) of both thioesterases. Furthermore, shRNA suppression of APT1 expression or inhibition of its thioesterase activity by palmostatin B markedly increased membrane localization of APT2, and shRNA suppression of APT2 had virtually no effect on membrane localization of APT1. In addition, mutagenesis of the active site Ser residue to Ala (S119A), which renders catalytic inactivation of APT1, also increased its membrane localization. Taken together, our findings provide insight into a novel mechanism by which dynamic palmitoylation links cytosol-membrane trafficking of APT1 and APT2 with that of their substrates, facilitating steady-state membrane localization and function of both.

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Bagh, Maria B; Peng, Shiyong; Chandra, Goutam et al. (2017) Misrouting of v-ATPase subunit V0a1 dysregulates lysosomal acidification in a neurodegenerative lysosomal storage disease model. Nat Commun 8:14612
Baker, E H; Levin, S W; Zhang, Z et al. (2017) MRI Brain Volume Measurements in Infantile Neuronal Ceroid Lipofuscinosis. AJNR Am J Neuroradiol 38:376-382
Baker, Eva H; Levin, Sondra W; Zhang, Zhongjian et al. (2015) Evaluation of disease progression in INCL by MR spectroscopy. Ann Clin Transl Neurol 2:797-809
Peng, Shiyong; Xu, Jianhua; Pelkey, Kenneth A et al. (2015) Suppression of agrin-22 production and synaptic dysfunction in Cln1 (-/-) mice. Ann Clin Transl Neurol 2:1085-104
Chandra, Goutam; Bagh, Maria B; Peng, Shiyong et al. (2015) Cln1 gene disruption in mice reveals a common pathogenic link between two of the most lethal childhood neurodegenerative lysosomal storage disorders. Hum Mol Genet :
Levin, Sondra W; Baker, Eva H; Zein, Wadih M et al. (2014) Oral cysteamine bitartrate and N-acetylcysteine for patients with infantile neuronal ceroid lipofuscinosis: a pilot study. Lancet Neurol 13:777-87
Bouchelion, Ashleigh; Zhang, Zhongjian; Li, Yichao et al. (2014) Mice homozygous for c.451C>T mutation in Cln1 gene recapitulate INCL phenotype. Ann Clin Transl Neurol 1:1006-23
Sarkar, Chinmoy; Chandra, Goutam; Peng, Shiyong et al. (2013) Neuroprotection and lifespan extension in Ppt1(-/-) mice by NtBuHA: therapeutic implications for INCL. Nat Neurosci 16:1608-17
Kong, Eryan; Peng, Shiyong; Chandra, Goutam et al. (2013) Dynamic palmitoylation links cytosol-membrane shuttling of acyl-protein thioesterase-1 and acyl-protein thioesterase-2 with that of proto-oncogene H-ras product and growth-associated protein-43. J Biol Chem 288:9112-25
Saha, Arjun; Sarkar, Chinmoy; Singh, Satya P et al. (2012) The blood-brain barrier is disrupted in a mouse model of infantile neuronal ceroid lipofuscinosis: amelioration by resveratrol. Hum Mol Genet 21:2233-44

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