Disruption of lysosomal nutrient sensing scaffold contributes to pathogenesis of a fatal neurodegenerative lysosomal storage disease.

The ceroid lipofuscinosis neuronal 1 (CLN1) disease, formerly called infantile neuronal ceroid lipofuscinosis, is a fatal hereditary neurodegenerative lysosomal storage disorder. This disease is caused by loss-of-function mutations in the CLN1 gene, encoding palmitoyl-protein thioesterase-1 (PPT1). PPT1 catalyzes depalmitoylation of S-palmitoylated proteins for degradation and clearance by lysosomal hydrolases. Numerous proteins, especially in the brain, require dynamic S-palmitoylation (palmitoylation-depalmitoylation cycles) for endosomal trafficking to their destination. While 23 palmitoyl-acyl transferases in the mammalian genome catalyze S-palmitoylation, depalmitoylation is catalyzed by thioesterases such as PPT1. Despite these discoveries, the pathogenic mechanism of CLN1 disease has remained elusive. Here, we report that in the brain of Cln1-/- mice, which mimic CLN1 disease, the mechanistic target of rapamycin complex-1 (mTORC1) kinase is hyperactivated. The activation of mTORC1 by nutrients requires its anchorage to lysosomal limiting membrane by Rag GTPases and Ragulator complex. These proteins form the lysosomal nutrient sensing scaffold to which mTORC1 must attach to activate. We found that in Cln1-/- mice, two constituent proteins of the Ragulator complex (vacuolar (H+)-ATPase and Lamtor1) require dynamic S-palmitoylation for endosomal trafficking to the lysosomal limiting membrane. Intriguingly, Ppt1 deficiency in Cln1-/- mice misrouted these proteins to the plasma membrane disrupting the lysosomal nutrient sensing scaffold. Despite this defect, mTORC1 was hyperactivated via the IGF1/PI3K/Akt-signaling pathway, which suppressed autophagy contributing to neuropathology. Importantly, pharmacological inhibition of PI3K/Akt suppressed mTORC1 activation, restored autophagy, and ameliorated neurodegeneration in Cln1-/- mice. Our findings reveal a previously unrecognized role of Cln1/Ppt1 in regulating mTORC1 activation and suggest that IGF1/PI3K/Akt may be a targetable pathway for CLN1 disease.

Ppt1-deficiency dysregulates lysosomal Ca++ homeostasis contributing to pathogenesis in a mouse model of CLN1 disease.

Inactivating mutations in the PPT1 gene encoding palmitoyl-protein thioesterase-1 (PPT1) underlie the CLN1 disease, a devastating neurodegenerative lysosomal storage disorder. The mechanism of pathogenesis underlying CLN1 disease has remained elusive. PPT1 is a lysosomal enzyme, which catalyzes the removal of palmitate from S-palmitoylated proteins (constituents of ceroid lipofuscin) facilitating their degradation and clearance by lysosomal hydrolases. Thus, it has been proposed that Ppt1-deficiency leads to lysosomal accumulation of ceroid lipofuscin leading to CLN1 disease. While S-palmitoylation is catalyzed by palmitoyl acyltransferases (called ZDHHCs), palmitoyl-protein thioesterases (PPTs) depalmitoylate these proteins. We sought to determine the mechanism by which Ppt1-deficiency may impair lysosomal degradative function leading to infantile neuronal ceroid lipofuscinosis pathogenesis. Here, we report that in Ppt1-/- mice, which mimic CLN1 disease, low level of inositol 3-phosphate receptor-1 (IP3R1) that mediates Ca++ transport from the endoplasmic reticulum to the lysosome dysregulated lysosomal Ca++ homeostasis. Intriguingly, the transcription factor nuclear factor of activated T-cells, cytoplasmic 4 (NFATC4), which regulates IP3R1-expression, required S-palmitoylation for trafficking from the cytoplasm to the nucleus. We identified two palmitoyl acyltransferases, ZDHHC4 and ZDHHC8, which catalyzed S-palmitoylation of NFATC4. Notably, in Ppt1-/- mice, reduced ZDHHC4 and ZDHHC8 levels markedly lowered S-palmitoylated NFATC4 (active) in the nucleus, which inhibited IP3R1-expression, thereby dysregulating lysosomal Ca++ homeostasis. Consequently, Ca++ -dependent lysosomal enzyme activities were markedly suppressed. Impaired lysosomal degradative function impaired autophagy, which caused lysosomal storage of undigested cargo. Importantly, IP3R1-overexpression in Ppt1-/- mouse fibroblasts ameliorated this defect. Our results reveal a previously unrecognized role of Ppt1 in regulating lysosomal Ca++ homeostasis and suggest that this defect contributes to pathogenesis of CLN1 disease.

Ablation of microRNA-155 and neuroinflammation in a mouse model of CLN1-disease.

Infantile neuronal ceroid lipofuscinosis (INCL), also known as CLN1-disease, is a devastating neurodegenerative lysosomal storage disorder (LSD), caused by inactivating mutations in the CLN1 gene. The Cln1-/- mice, which mimic INCL, manifest progressive neuroinflammation contributing to neurodegeneration. However, the underlying mechanism of neuroinflammation in INCL and in Cln1-/- mice has remained elusive. Previously, it has been reported that microRNA-155 (miR-155) regulates inflammation and miR profiling in Cln1-/- mouse brain showed that the level of miR-155 was upregulated. Thus, we sought to determine whether ablation of miR-155 in Cln1-/- mice may suppress neuroinflammation in these mice. Towards this goal, we generated Cln1-/-/miR-155-/- double-knockout mice and evaluated the inflammatory signatures in the brain. We found that the brains of double-KO mice manifest progressive neuroinflammatory changes virtually identical to those found in Cln1-/- mice. We conclude that ablation of miR-155 in Cln1-/- mice does not alter the neuroinflammatory trajectory in INCL mouse model.

In a mouse model of INCL reduced S-palmitoylation of cytosolic thioesterase APT1 contributes to microglia proliferation and neuroinflammation.

S-palmitoylation is a reversible posttranslational modification in which a 16-carbon saturated fatty acid (generally palmitate) is attached to specific cysteine residues in polypeptides via thioester linkage. Dynamic S-palmitoylation (palmitoylation-depalmitoylation), like phosphorylation-dephosphorylation, regulates the function of numerous proteins, especially in the brain. While a family of 23 palmitoyl-acyl transferases (PATS), commonly known as ZDHHCs, catalyze S-palmitoylation of proteins, the thioesterases, localized either in the cytoplasm (eg, APT1) or in the lysosome (eg, PPT1) mediate depalmitoylation. Previously, we reported that APT1 requires dynamic S-palmitoylation for shuttling between the cytosol and the plasma membrane. APT1 depalmitoylated H-Ras to regulate its signaling pathway that stimulates cell proliferation. Although we demonstrated that APT1 catalyzed its own depalmitoylation, the ZDHHC(s) that S-palmitoylated APT1 had remained unidentified. We report here that ZDHHC5 and ZDHHC23 catalyze APT1 S-palmitoylation. Intriguingly, lysosomal Ppt1-deficiency in Cln1-/- mouse, a reliable animal model of INCL, markedly reduced ZDHHC5 and ZDHHC23 levels. Remarkably, in the brain of these mice decreased ZDHHC5 and ZDHHC23 levels suppressed membrane-bound APT1, thereby, increasing plasma membrane-localized H-Ras, which activated its signaling pathway stimulating microglia proliferation. Increased inflammatory cytokines produced by microglia together with increased complement C1q level contributed to the transformation of astrocytes to neurotoxic A1 phenotype. Importantly, neuroinflammation was ameliorated by treatment of Cln1-/- mice with a PPT1-mimetic small molecule, N-tert(Butyl)hydroxylamine (NtBuHA). Our results revealed a novel pathway to neuropathology in an INCL mouse model and uncovered a previously unrecognized mechanism of the neuroprotective actions of NtBuHA and its potential as a drug target.

Cln1-mutations suppress Rab7-RILP interaction and impair autophagy contributing to neuropathology in a mouse model of infantile neuronal ceroid lipofuscinosis.

Infantile neuronal ceroid lipofuscinosis (INCL) is a devastating neurodegenerative lysosomal storage disease (LSD) caused by inactivating mutations in the CLN1 gene. CLN1 encodes palmitoyl-protein thioesterase-1 (PPT1), a lysosomal enzyme that catalyzes the deacylation of S-palmitoylated proteins to facilitate their degradation and clearance by lysosomal hydrolases. Despite the discovery more than two decades ago that CLN1 mutations causing PPT1-deficiency underlies INCL, the precise molecular mechanism(s) of pathogenesis has remained elusive. Here, we report that autophagy is dysregulated in Cln1-/- mice, which mimic INCL and in postmortem brain tissues as well as cultured fibroblasts from INCL patients. Moreover, Rab7, a small GTPase, critical for autophagosome-lysosome fusion, requires S-palmitoylation for trafficking to the late endosomal/lysosomal membrane where it interacts with Rab-interacting lysosomal protein (RILP), essential for autophagosome-lysosome fusion. Notably, PPT1-deficiency in Cln1-/- mice, dysregulated Rab7-RILP interaction and preventing autophagosome-lysosome fusion, which impaired degradative functions of the autolysosome leading to INCL pathogenesis. Importantly, treatment of Cln1-/- mice with a brain-penetrant, PPT1-mimetic, small molecule, N-tert (butyl)hydroxylamine (NtBuHA), ameliorated this defect. Our findings reveal a previously unrecognized role of CLN1/PPT1 in autophagy and suggest that small molecules functionally mimicking PPT1 may have therapeutic implications.

Cln3-mutations underlying juvenile neuronal ceroid lipofuscinosis cause significantly reduced levels of Palmitoyl-protein thioesterases-1 (Ppt1)-protein and Ppt1-enzyme activity in the lysosome.

Mutations in at least 13 different genes (called CLNs) underlie various forms of neuronal ceroid lipofuscinoses (NCLs), a group of the most common neurodegenerative lysosomal storage diseases. While inactivating mutations in the CLN1 gene, encoding palmitoyl-protein thioesterases-1 (PPT1), cause infantile NCL (INCL), those in the CLN3 gene, encoding a protein of unknown function, underlie juvenile NCL (JNCL). PPT1 depalmitoylates S-palmitoylated proteins (constituents of ceroid) required for their degradation by lysosomal hydrolases and PPT1-deficiency causes lysosomal accumulation of autofluorescent ceroid leading to INCL. Because intracellular accumulation of ceroid is a characteristic of all NCLs, a common pathogenic link for these diseases has been suggested. It has been reported that CLN3-mutations suppress the exit of cation-independent mannose 6-phosphate receptor (CI-M6PR) from the trans Golgi network (TGN). Because CI-M6PR transports soluble proteins such as PPT1 from the TGN to the lysosome, we hypothesized that CLN3-mutations may cause lysosomal PPT1-insufficiency contributing to JNCL pathogenesis. Here, we report that the lysosomes in Cln3-mutant mice, which mimic JNCL, and those in cultured cells from JNCL patients, contain significantly reduced levels of Ppt1-protein and Ppt1-enzyme activity and progressively accumulate autofluorescent ceroid. Furthermore, in JNCL fibroblasts the V0a1 subunit of v-ATPase, which regulates lysosomal acidification, is mislocalized to the plasma membrane instead of its normal location on lysosomal membrane. This defect dysregulates lysosomal acidification, as we previously reported in Cln1 -/- mice, which mimic INCL. Our findings uncover a previously unrecognized role of CLN3 in lysosomal homeostasis and suggest that CLN3-mutations causing lysosomal Ppt1-insuffiiciency may at least in part contribute to JNCL pathogenesis.

Cln1 gene disruption in mice reveals a common pathogenic link between two of the most lethal childhood neurodegenerative lysosomal storage disorders.

Neurodegeneration is a devastating manifestation in the majority of >50 lysosomal storage disorders (LSDs). Neuronal ceroid lipofuscinoses (NCLs) are the most common childhood neurodegenerative LSDs. Mutations in 13 different genes (called CLNs) underlie various types of NCLs, of which the infantile NCL (INCL) and congenital NCL (CNCL) are the most lethal. Although inactivating mutations in the CLN1 gene encoding palmitoyl-protein thioesterase-1 (PPT1) cause INCL, those in the CLN10 gene encoding cathepsin D (CD) underlie CNCL. PPT1 is a lysosomal thioesterase that cleaves the thioester linkage in S-acylated proteins required for their degradation by lysosomal hydrolases like CD. Thus, PPT1 deficiency causes lysosomal accumulation of these lipidated proteins (major constituents of ceroid) leading to INCL. We sought to determine whether there is a common pathogenic link between INCL and CNCL. Using biochemical, histological and confocal microscopic analyses of brain tissues and cells from Cln1(-/-) mice that mimic INCL, we uncovered that Cln10/CD is overexpressed. Although synthesized in the endoplasmic reticulum, the CD-precursor protein (pro-CD) is transported through endosome to the lysosome where it is proteolytically processed to enzymatically active-CD. We found that despite Cln10 overexpression, the maturation of pro-CD to enzymatically active-CD in lysosome was disrupted. This defect impaired lysosomal degradative function causing accumulation of undegraded cargo in lysosome leading to INCL. Notably, treatment of intact Cln1(-/-) mice as well as cultured brain cells derived from these animals with a thioesterase-mimetic small molecule, N-tert-butyl-hydroxylamine, ameliorated the CD-processing defect. Our findings are significant in that they define a pathway in which Cln1 mutations disrupt the maturation of a major degradative enzyme in lysosome contributing to neuropathology in INCL and suggest that lysosomal CD deficiency is a common pathogenic link between INCL and CNCL.

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.

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.

The blood-brain barrier is disrupted in a mouse model of infantile neuronal ceroid lipofuscinosis: amelioration by resveratrol.

Disruption of the blood-brain barrier (BBB) is a serious complication frequently encountered in neurodegenerative disorders. Infantile neuronal ceroid lipofuscinosis (INCL) is a devastating childhood neurodegenerative lysosomal storage disorder caused by palmitoyl-protein thioesterase-1 (PPT1) deficiency. It remains unclear whether BBB is disrupted in INCL and if so, what might be the molecular mechanism(s) of this complication. We previously reported that the Ppt1-knockout (Ppt1-KO) mice that mimic INCL manifest high levels of oxidative stress and neuroinflammation. Recently, it has been reported that CD4(+) T-helper 17 (T(H)17) lymphocytes may mediate BBB disruption and neuroinflammation, although the precise molecular mechanism(s) remain unclear. We sought to determine: (i) whether the BBB is disrupted in Ppt1-KO mice, (ii) if so, do T(H)17-lymphocytes underlie this complication, and (iii) how might T(H)17 lymphocytes breach the BBB. Here, we report that the BBB is disrupted in Ppt1-KO mice and 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.

Disruption of adaptive energy metabolism and elevated ribosomal p-S6K1 levels contribute to INCL pathogenesis: partial rescue by resveratrol.

The infantile neuronal ceroid lipofuscinosis (INCL) is a devastating neurodegenerative lysosomal storage disease. Despite our knowledge that palmitoyl-protein thioesterase-1 (PPT1)-deficiency causes INCL, the molecular mechanism(s) of neurodegeneration and the drastically reduced lifespan of these patients remain poorly understood. Consequently, an effective treatment for this disease is currently unavailable. We previously reported that oxidative stress-mediated abnormality in mitochondria activates caspases-9 pathway of apoptosis in INCL fibroblasts and in neurons of Ppt1-knockout (Ppt1-KO) mice, which mimic INCL. Since mitochondria play critical roles in maintaining cellular energy homeostasis, we hypothesized that oxidative stress-mediated disruption of energy metabolism and homeostasis may contribute to INCL pathogenesis. We report here 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-γ (PPARγ) coactivator-1α (PGC-1α) and Silent Information Regulator T1 (SIRT1) are markedly down-regulated. This suggested an abnormality in AMPK/SIRT1/PGC-1α 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-1α 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.

Allergic asthma: influence of genetic and environmental factors.

Allergic asthma is a chronic airway inflammatory disease in which exposure to allergens causes intermittent attacks of breathlessness, airway hyper-reactivity, wheezing, and coughing. Allergic asthma has been called a "syndrome" resulting from a complex interplay between genetic and environmental factors. Worldwide, >300 million individuals are affected by this disease, and in the United States alone, it is estimated that >35 million people, mostly children, suffer from asthma. Although animal models, linkage analyses, and genome-wide association studies have identified numerous candidate genes, a solid definition of allergic asthma has not yet emerged; however, such studies have contributed to our understanding of the multiple pathways to this syndrome. In contrast with animal models, in which T-helper 2 (T(H)2) cell response is the dominant feature, in human asthma, an initial exposure to allergen results in T(H)2 cell-dependent stimulation of the immune response that mediates the production of IgE and cytokines. Re-exposure to allergen then activates mast cells, which release mediators such as histamines and leukotrienes that recruit other cells, including T(H)2 cells, which mediate the inflammatory response in the lungs. In this minireview, we discuss the current understanding of how associated genetic and environmental factors increase the complexity of allergic asthma and the challenges allergic asthma poses for the development of novel approaches to effective treatment and prevention.

Evaluation of neurodegeneration in a mouse model of infantile batten disease by magnetic resonance imaging and magnetic resonance spectroscopy.

Neuronal ceroid lipofuscinoses (NCLs) represent a group of common hereditary childhood neurodegenerative storage disorders that have no effective treatment. Mutations in eight different genes cause various forms of NCLs. Infantile NCL (INCL), the most lethal disease, is caused by inactivating mutations in the palmitoyl-protein thioesterase-1 (PPT1) gene. 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. Here, we report our evaluation of the progression of neurodegeneration in Ppt1-KO mice starting at 3 months of age by MRI and MR spectroscopy (MRS) and repeating these tests using the same mice at 4, 5 and 6 months of age. Our results showed progressive cerebral atrophy, which was associated with histological loss of neuronal content and increase in astroglia. Remarkably, while the brain volumes in Ppt1-KO mice progressively declined with advancing age, the MRS signals, which were significantly lower than those of their wild-type littermates, remained virtually unchanged from 3 to 6 months of age. In addition, our results also showed an abnormality in cerebral blood flow in these mice, which showed progression with age. Our findings provide methods 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.

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.

Uteroglobin: a steroid-inducible immunomodulatory protein that founded the Secretoglobin superfamily.

Blastokinin or uteroglobin (UG) is a steroid-inducible, evolutionarily conserved, secreted protein that has been extensively studied from the standpoint of its structure and molecular biology. However, the physiological function(s) of UG still remains elusive. Isolated from the uterus of rabbits during early pregnancy, UG is the founding member of a growing superfamily of proteins called Secretoglobin (Scgb). Numerous studies demonstrated that UG is a multifunctional protein with antiinflammatory/ immunomodulatory properties. It inhibits soluble phospholipase A(2) activity and binds and perhaps sequesters hydrophobic ligands such as progesterone, retinols, polychlorinated biphenyls, phospholipids, and prostaglandins. In addition to its antiinflammatory activities, UG manifests antichemotactic, antiallergic, antitumorigenic, and embryonic growth-stimulatory activities. The tissue-specific expression of the UG gene is regulated by several steroid hormones, although a nonsteroid hormone, prolactin, further augments its expression in the uterus. The mucosal epithelia of virtually all organs that communicate with the external environment express UG, and it is present in the blood, urine, and other body fluids. Although the physiological functions of this protein are still under investigation, a single nucleotide polymorphism in the UG gene appears to be associated with several inflammatory/autoimmune diseases. Investigations with UG-knockout mice revealed that the absence of this protein leads to phenotypes that suggest its critical homeostatic role(s) against oxidative damage, inflammation, autoimmunity, and cancer. Recent studies on UG-binding proteins (receptors) provide further insight into the multifunctional nature of this protein. Based on its antiinflammatory and antiallergic properties, UG is a potential drug target.

Palmitoyl protein thioesterase-1 deficiency impairs synaptic vesicle recycling at nerve terminals, contributing to neuropathology in humans and mice.

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.

Stop codon read-through with PTC124 induces palmitoyl-protein thioesterase-1 activity, reduces thioester load and suppresses apoptosis in cultured cells from INCL patients.

Infantile neuronal ceroid lipofuscinosis (INCL), a lethal hereditary neurodegenerative lysosomal storage disorder, affects mostly children. It is caused by inactivating mutations in the palmitoyl-protein thioesterase-1(PPT1) gene. 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 asgentamycin 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. 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.

Uteroglobin represses allergen-induced inflammatory response by blocking PGD2 receptor-mediated functions.

Uteroglobin (UG) is an antiinflammatory protein secreted by the epithelial lining of all organs communicating with the external environment. We reported previously that UG-knockout mice manifest exaggerated inflammatory response to allergen, characterized by increased eotaxin and Th2 cytokine gene expression, and eosinophil infiltration in the lungs. In this study, we uncovered that the airway epithelia of these mice also express high levels of cyclooxygenase (COX)-2, a key enzyme for the production of proinflammatory lipid mediators, and the bronchoalveolar lavage fluid (BALF) contain elevated levels of prostaglandin D2. These effects are abrogated by recombinant UG treatment. Although it has been reported that prostaglandin D2 mediates allergic inflammation via its receptor, DP, neither the molecular mechanism(s) of DP signaling nor the mechanism by which UG suppresses DP-mediated inflammatory response are clearly understood. Here we report that DP signaling is mediated via p38 mitogen-activated protein kinase, p44/42 mitogen-activated protein kinase, and protein kinase C pathways in a cell type-specific manner leading to nuclear factor-kappaB activation stimulating COX-2 gene expression. Further, we found that recombinant UG blocks DP-mediated nuclear factor-kappaB activation and suppresses COX-2 gene expression. We propose that UG is an essential component of a novel innate homeostatic mechanism in the mammalian airways to repress allergen-induced inflammatory responses.

ER and oxidative stresses are common mediators of apoptosis in both neurodegenerative and non-neurodegenerative lysosomal storage disorders and are alleviated by chemical chaperones.

It is estimated that more than 40 different lysosomal storage disorders (LSDs) cumulatively affect one in 5000 live births, and in the majority of the LSDs, neurodegeneration is a prominent feature. Neuronal ceroid lipofuscinoses (NCLs), as a group, represent one of the most common (one in 12,500 births) neurodegenerative LSDs. The infantile NCL (INCL) is the most devastating neurodegenerative LSD, which is caused by inactivating mutations in the palmitoyl-protein thioesterase-1 (PPT1) gene. We previously reported that neuronal death by apoptosis in INCL, and in the PPT1-knockout (PPT1-KO) mice that mimic INCL, is at least in part caused by endoplasmic reticulum (ER) and oxidative stresses. In the present study, we sought to determine whether ER and oxidative stresses are unique manifestations of INCL or they are common to both neurodegenerative and non-neurodegenerative LSDs. Unexpectedly, we found that ER and oxidative stresses are common manifestations in cells from both neurodegenerative and non-neurodegenerative LSDs. Moreover, all LSD cells studied show extraordinary sensitivity to brefeldin-A-induced apoptosis, which suggests pre-existing ER stress conditions. Further, we uncovered that chemical disruption of lysosomal homeostasis in normal cells causes ER stress, suggesting a cross-talk between the lysosomes and the ER. Most importantly, we found that chemical chaperones that alleviate ER and oxidative stresses are also cytoprotective in all forms of LSDs studied. We propose that ER and oxidative stresses are common mediators of apoptosis in both neurodegenerative and non-neurodegenerative LSDs and suggest that the beneficial effects of chemical/pharmacological chaperones are exerted, at least in part, by alleviating these stress conditions.

Children with infantile neuronal ceroid lipofuscinosis have an increased risk of hypothermia and bradycardia during anesthesia.

BACKGROUND: Neuronal ceroid lipofuscinoses (NCLs) are a group of autosomal recessive neurodegenerative diseases characterized by lysosomal accumulation of autofluorescent material in neurons and other cell types. The infantile NCL (INCL) subtype is rare (1 in >100,000 births), the most devastating of childhood subtypes, and is caused by mutations in the gene CLN1, which encodes palmitoyl-protein thioesterase-1. METHODS: To investigate the incidence of hypothermia and bradycardia during general anesthesia in patients with INCL, we conducted a case-control study to examine the perianesthetic course of patients with INCL and of controls receiving anesthesia for diagnostic studies. RESULTS: Eight children with INCL (mean age 25 mo [range, 10-32] at first anesthetic) and 25 controls (mean age 44 mo [range, 18-92]) underwent 62 anesthetics for nonsurgical procedures. Patients with INCL had neurologic deficits including developmental delay, myoclonus, and visual impairment. Patients with INCL had lower baseline temperature (36.4 +/- 0.1 vs 36.8 +/- 0.1, INCL versus controls, P < 0.007), and during anesthesia, despite active warming techniques, had significantly more hypothermia (18 vs 0 episodes, P < 0.001) and sinus bradycardia (10 vs 1, P < 0.001) compared with controls. INCL diagnosis was significantly associated with temperature decreases during anesthesia (P < 0.001), whereas age, sex, and duration of anesthesia were not (P = NS). CONCLUSIONS: We report that patients with INCL have lower baseline body temperature and during general anesthesia, despite rewarming interventions, are at increased risk for hypothermia and bradycardia. This suggests a previously unknown INCL phenotype, impaired thermoregulation. Therefore, when anesthetizing these children, careful monitoring and routine use of warming interventions are warranted.

Emerging new roles of the lysosome and neuronal ceroid lipofuscinoses.

Neuronal Ceroid Lipofuscinoses (NCLs), commonly known as Batten disease, constitute a group of the most prevalent neurodegenerative lysosomal storage disorders (LSDs). Mutations in at least 13 different genes (called CLNs) cause various forms of NCLs. Clinically, the NCLs manifest early impairment of vision, progressive decline in cognitive and motor functions, seizures and a shortened lifespan. At the cellular level, all NCLs show intracellular accumulation of autofluorescent material (called ceroid) and progressive neuron loss. Despite intense studies the normal physiological functions of each of the CLN genes remain poorly understood. Consequently, the development of mechanism-based therapeutic strategies remains challenging. Endolysosomal dysfunction contributes to pathogenesis of virtually all LSDs. Studies within the past decade have drastically changed the notion that the lysosomes are merely the terminal degradative organelles. The emerging new roles of the lysosome include its central role in nutrient-dependent signal transduction regulating metabolism and cellular proliferation or quiescence. In this review, we first provide a brief overview of the endolysosomal and autophagic pathways, lysosomal acidification and endosome-lysosome and autophagosome-lysosome fusions. We emphasize the importance of these processes as their dysregulation leads to pathogenesis of many LSDs including the NCLs. We also describe what is currently known about each of the 13 CLN genes and their products and how understanding the emerging new roles of the lysosome may clarify the underlying pathogenic mechanisms of the NCLs. Finally, we discuss the current and emerging therapeutic strategies for various NCLs.