HERITABLE NEURODEGENERATIVE AND AUTOIMMUNE DISODERS
, Head, Section on Developmental Genetics
Zhongjian Zhang, MD, PhD, Staff Scientist
Sung-Jo Kim, PhD, Visiting Fellow
Yi-ching Lee, PhD, Visiting Fellow
Rabindranath Ray, PhD, Visiting Fellow
Arjun Saha, PhD, Visiting Fellow
Chinmoy Sarkar, PhD, Visiting Fellow
Hui Wei, PhD, Visiting Fellow
Moonsuk S. Choi, PhD, Adjunct Scientist
Sondra W. Levin, MD, Adjunct Scientist
Aprile Pilon, PhD, Adjunct Scientist
Pei Chen Tsai, MS, Technical Training Fellow
With the aim of developing novel and rational therapeutic approaches, we conduct both laboratory and clinical investigations to understand the molecular mechanisms of complex genetic disorders of inflammation, autoimmunity, and neurodegeneration. To that end, our research focuses on understanding the regulation and physiological functions of primarily two proteins: uteroglobin (UG), which is also known as Clara cell 10 kDa protein (CC10); and palmitoyl-protein thioesterase-1 (PPT1), mutations of which are the genetic basis of infantile neuronal ceroid lipofuscinosis (INCL), a devastating neurodegenerative storage disease of childhood. Investigations in both areas have led to ongoing clinical trials. Recently, using PPT1-knockout mice, which recapitulate virtually all clinical and pathological features of INCL, we discovered that this enzyme deficiency leads to abnormal accumulation of S-acylated proteins in the endoplasmic reticulum (ER), causing ER stress. ER stress in turn mediates activation of caspase-12, a cysteine proteinase in the ER, that leads to caspase-3 activation and apoptosis. This discovery provides insight into a complex mechanism of neurodegeneration in INCL and identifies several potential targets for the development of rational therapeutic approaches for this uniformly fatal disease.
Uteroglobin suppresses allergen-induced TH2 differentiation
The molecular mechanism(s) of inflammatory diseases of the respiratory system, such as asthma, is complex. In the United States alone, 6,000 deaths annually are attributable to airway inflammatory diseases. One of the precipitating factors in these diseases is exaggerated response to allergens, which leads to airway inflammation and bronchoconstriction. Uteroglobin (UG), the founding member of the Secretoglobin superfamily, is a potent anti-inflammatory protein constitutively expressed at high levels in the airway epithelia of all mammals. We previously reported that the lungs of UG-knockout (UG-KO) mice express elevated levels of TH2 cytokines (e.g., interleukin (IL)-4 and IL-13), which are augmented by allergen sensitization and challenge leading to exaggerated airway inflammation. Notably, treatment with recombinant UG suppresses these responses. Exposure of the respiratory system to antigen (allergen) stimulates the expression of acute-phase proteins such as serum amyloid A (SAA). It has been reported that dendritic cells (DCs), which are the major antigen-presenting cells (APCs), express fMLP-receptor (FPR2), which promotes SAA-binding on DCs. Thus, interaction of SAA with FPR2 on DCs may stimulate chemotactic migration of APCs to the antigen site, facilitating the processing and presentation of antigens. Recently, it has been shown that the direction of TH2 differentiation, which requires input from the DCs, is determined by the cytokine environment at the site of initial antigenic (allergic) activation.
To determine the molecular mechanism(s) by which UG suppresses the allergen-induced TH2 cytokine production that causes airway inflammation, we studied, in UG-KO mice, the expression of genes critically important in the differentiation of TH2 cell lineage. Our results demonstrate that UG binds to FPR2, inhibits chemotaxis, and downregulates SOCS-3 gene expression and STAT-1 activation, which are all reported to play critical roles in the differentiation of TH2 cells.
Ray R, Zhang Z, Lee YC, Gao JL, Mukherjee AB. Uteroglobin suppresses allergen-induced TH2 differentiation by down-regulating the expression of serum amyloid A and SOCS-3 genes. FEBS Lett 2006;580:6022-6.
Mice lacking uteroglobin are highly susceptible to pulmonary fibrosis
We previously noted that UG-KO mice, generated by targeted disruption of the UGgene in embryonic stem (ES) cells, sporadically develop focal pulmonary fibrosis (PF) with age. PF is a complex disease with high mortality and morbidity. Its molecular mechanism(s) remains poorly understood, although the mucosal epithelial cells that line the airways of all mammals’ cells have emerged as the key site of initial injury that precedes development of PF. Thus, UG may play a protective role against development of PF.
Bleomycin, an anticancer drug, is limited in its clinical use in humans because it causes widespread alveolar epithelial cell injury and mediates development of PF. It is those properties that have led bleomycin to find broad application in generating animal models of PF. The pathologic features of PF include progressive alveolar inflammation, deposition of extracellular matrix proteins such as collagen, and proliferation of fibroblasts. It appears that a breakdown of homeostatic mechanisms that would otherwise prevent pulmonary inflammation plays critical pathogenic roles in PF. In fact, reports indicate that injury to the mucosal epithelial layer that lines the mammalian airways is one of the primary events leading to PF. Recombinant UG treatment of UG-KO mice, which manifest exaggerated pulmonary inflammation when challenged with allergens such as ovalbumin, abrogates allergen-induced inflammation in the lungs of the mice. Given that airway inflammation precedes PF and that UG is a potent anti-inflammatory protein present in the airways, we sought to determine whether UG plays protective roles against PF. We obtained evidence that UG plays a pivotal role in maintaining homeostasis in the airways by preventing TH2-mediated inflammation and consequent development of PF.
Lee YC, Zhang Z, Mukherjee AB. Mice lacking uteroglobin are highly susceptible to developing pulmonary fibrosis. FEBS Lett 2006;580:4515-20.
Lack of palmitoyl-protein thioesterase-1 activates unfolded protein response mediating neuronal apoptosis in INCL
In a process commonly known as palmitoylation or S-acylation, several proteins undergo post-translational modification by palmitic acid. This fatty acid modification of proteins facilitates anchorage to membranes, a process that plays important roles in diverse biological functions, including vesicular transport, signal transduction, and maintenance of cellular architecture. To date, at least 100 proteins have been reported to undergo palmitoylation, including the transferrin receptor, nitric oxide synthase, neuronal growth-associated protein-43 (GAP-43), acetylcholine esterase, postsynaptic density protein-95 (PSD-95), and the synaptosomal-associated protein-25. A critical function of palmitoylation in signaling proteins is to confine the proteins to cellular membranes. Research has suggested that cycles of palmitoylation and depalmitoylation control the proteins’ distribution between the membrane and cytoplasm and/or between subdomains of the plasma membrane, modulating the coupling of specific signaling proteins to cell-surface receptors or intracellular effectors.
Soon after the molecular cloning and characterization of both the cDNA and the gene for palmitoyl-protein thioesterase-1 (PPT1) and its chromosomal localization, it was reported that inactivating mutations in the PPT1 gene are the genetic basis of INCL. PPT1 catalyzes the cleavage of thioester linkages in S-acylated (palmitoylated) proteins, thereby facilitating their degradation and/or recycling. The lack of PPT1 activity leads to INCL pathogenesis, although the molecular mechanism(s) by which PPT1 deficiency mediates neurodegeneration remains unclear. While INCL is a rare disease (1 in 100,000 births), it belongs to the most common group (1 in 12,500 births) of heritable, neurodegenerative storage disorders of childhood, collectively known as neuronal ceroid lipofuscinoses (NCLs) (more commonly, Batten disease). Some of the characteristic pathological manifestations of all NCL types are rapidly progressive brain atrophy, retinal blindness, and accumulation of autofluorescent lipopigments in neurons as well as in other cell types. On the basis of age of onset and cellular ultrastructure and composition of the storage material, NCLs are classified into four major subtypes: infantile (INCL), late-infantile (LNCL), juvenile (JNCL), and adult onset (ANCL). Recent reports indicate that mutations in at least six genes underlie the various forms of NCLs known to date.
INCL is the most lethal and devastating of these diseases. Although normal at birth, children afflicted with INCL undergo complete retinal degeneration and blindness by two years of age and brain death by age four. The children remain in a vegetative state for several more years, with death occurring around 10 to 12 years of age. Autopsy reveals characteristic autofluorescent intracellular storage material, known as granular osmiophilic deposits, in the brain and other tissues. Increased levels of apoptosis in the brain biopsy tissues and in cultured cells from INCL patients and in the brains of PPT1-knockout (PPT1-KO) mice have been reported. Moreover, others have recently shown that increased apoptosis is associated with loss of neuronal cells in the brains of PPT1-KO mice. However, the molecular mechanism(s) of apoptosis in INCL remains poorly understood. In our current study, we have amassed compelling evidence linking PPT1 deficiency with abnormal accumulation of S-acylated proteins in the endoplasmic reticulum (ER). The accumulation activates the unfolded protein response (UPR) associated with elevated levels of the phosphorylated translation initiation factor eIF2a and glucose-regulated protein-78 (Grp-78/Bip) and causes activation of caspase-12 leading to caspase-3 activation, apoptosis, and neurodegeneration. Our results explain at least one of the major molecular mechanisms of apoptosis that leads to neurodegeneration in INCL and identifies potential targets for developing novel therapeutic strategies for this always fatal disease.
Zhang Z, Lee YC, Kim SJ, Choi MS, Tsai PC, Xu Y, Xiao YJ, Zhang P, Heffer A, Mukherjee AB. Palmitoyl-protein thioesterase-1 deficiency mediates the activation of the unfolded protein response and neuronal apoptosis in INCL Hum Mol Genet 2006;15:337-46.
In humans, PPTi deficiency mediates ER stress and caspase-4 and caspase-9 activation causing neuronal apoptosis
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 ER stress–induced caspase-12 activation. However, the human caspase-12 gene contains several mutations that make the gene functionally inactive. Thus, it has been suggested that human caspase-4 is the counterpart of murine caspase-12. We discovered that, in the human INCL brain, ER stress–induced activation of UPR mediates caspase-4 and caspase-3 activation and apoptosis. Moreover, we showed that the INCL brain contains a high level of growth-associated protein-43 (GAP-43), which is known to undergo palmitoylation. We also demonstrated that transfection of cultured INCL cells with a green fluorescent protein–GAP-43 cDNA construct showed abnormal localization of the protein in the ER. Furthermore, INCL cells manifested evidence of ER stress and UPR (elevated levels of Grp-78/Bip and GADD153), caspase-4 and caspase-3 activation, and cleavage of poly(ADP)-ribose polymerase, which is a compelling sign of apoptosis. Most important, we showed that inhibition of caspase-4 activity protects INCL cells from apoptosis. Our results provide insight into at least one of the molecular mechanisms of apoptosis in INCL and may permit the identification of potential targets for therapeutic intervention.
ER stress may result in disruption of calcium signaling and lead to activation of the caspase-9 pathway of apoptosis. In addition, it has been reported that the ER function is sensitive to oxidative stress. Interestingly, the ER is one of the organelles that generates reactive oxygen species (ROS), in addition to functioning as a major storage organelle for calcium. During ER stress, both ROS production and Ca2+ release are elevated, increasing the potential for activation of the mitochondrial pathway of apoptosis in neurodegenerative diseases such as Parkinson’s disease. We sought to determine whether ER stress activates the caspase-9–mediated apoptotic pathway in INCL. We found that that the brain tissues of both an INCL patient and a PPT1-KO mouse contained elevated levels of superoxide dismutase (SOD) protein, along with SOD enzymatic activity and increased cleavage (activation) of caspase-9. We further demonstrated that cultured neurospheres derived from PPT1-KO mouse fetuses showed elevated levels of reactive oxygen species (ROS), increased SOD-protein and SOD activity, elevated levels of cleaved (activated) caspase-9 and, indicative of apoptosis, elevated levels of both caspase-3 and cleaved poly(ADP-ribose) polymerase (PARP). We propose that ER stress in neurons leads to elevated levels of ROS, stimulates SOD-2 production and activation, and disrupts calcium homeostasis. Together, these abnormalities mediate the activation of caspase-9 and therefore apoptosis, as demonstrated by activation of caspase-3 and cleavage of PARP, thereby causing the rapid brain atrophy that is characteristic of INCL.
Kim SJ, Zhang Z, Hitomi E, Lee YC, Mukherjee AB. Endoplasmic reticulum stress-induced caspase-4 activation mediates apoptosis and neurodegeneration in INCL. Hum Mol Genet 2006;15:1826-34.
Kim SJ, Zhang Z, Lee YC, Mukherjee AB. Palmitoyl-protein thioesterase-1 deficiency leads to the activation of caspase-9 and contributes to rapid neurodegeneration in INCL. Hum Mol Genet 2006;15:1580-6.
Lysophosphatidylcholine mediates phagocyte recruitment in the brain of mice lacking PPT1
In multicellular organisms, phagocytes efficiently remove dead cells after apoptosis, thereby preventing inflammation, suppressing autoimmunity, and maintaining homeostasis. Accumulating evidence indicates that, in the majority of neurodegenerative lysosomal storage disorders, neuronal death by apoptosis is followed by infiltration of phagocytes (e.g., activated microglia, astroglia, and macrophages) that may cause further damage to adjoining viable cells. Microglia represent the largest phagocyte population in the central nervous system and account for about 10 percent of the brain’s non-neuronal cells. These phagocytes possess many characteristics of myeloid cells, and their activation is associated with upregulated expression of marker genes commonly found on macrophages and dendritic cells. In response to neuronal injury and/or death, microglia undergo rapid morphological and functional changes. For example, they acquire myeloid cell properties, including antigen presentation, matrix metalloproteinase expression, generation of ROS, and phagocytosis. In addition, reports suggest that excessive phagocyte infiltration may damage viable neurons, leading to rapid progression of neurodegeneration. However, the molecular signal(s) that mediates the recruitment of phagocytes in neurodegenerative storage disorders remains poorly understood.
The infiltration of phagocytes in the brain has been reported in more than 40 neurodegenerative lysosomal storage diseases. Recently, another laboratory discovered pronounced astrocytosis in autopsy brain tissues from patients with juvenile NCL, commonly known as Batten disease. However, the molecular signal(s) that mediates the recruitment of these phagocytic cells in the brain is undefined.
We showed in PPT1-KO mouse brain that age-related increased expression and activation of cytosolic phospholipase A2 (cPLA2) catalyzes the production of elevated levels of lysophosphatidylcholine (LPC). Previously, LPC was reported to be a lipid attraction signal for phagocyte migration in vitro. We also showed that increased production of LPC positively correlates with elevated levels of expression of phagocyte markers in the brain of PPT1-KO mice. Further, using an in vitro assay, we demonstrated (1) that conditioned media derived from cultured PPT1-KO brain slices promoted chemotactic migration of phagocytes and (2) more important, that the same conditioned media pretreated with a cPLA2 inhibitor failed to manifest the chemo-attractant property. Taken together, the results demonstrate that increased LPC production in the brain of PPT1-KO mice is at least one of the mediators of phagocyte recruitment in vivo, raising the possibility that reduction of LPC levels may have beneficial effects on INCL patients.
Zhang Z, Lee YC, Kim SJ, Choi MS, Tsai PC, Saha A, Wei H, Xu Y, Xiao YJ, Zhang P, Heffer A, Mukherjee AB. Production of lysophosphatidyl-choline by cPLA2 in the brain of mice lacking PPT1 is a signal for phagocyte infiltration. Hum Mol Genet 2006;16:837-47.
A combination therapy with Cystagon™ and N-acetylcysteine (Mucomyst®) for INCL patients
PPT catalyzes the hydrolysis of thioester linkages in S-acylated polypeptides, and its deficiency leads to abnormal accumulation of acylated proteins, called ceroids, in lysosomes, thereby leading to INCL. Given that thioester bonds are susceptible to nucleophilic attack, drugs with nucleophilic property have therapeutic potential for INCL. Accordingly, we tested several compounds with this property (i.e., cysteamine, phosphocysteamine, cysteamine bitartrate, and N-acetylcysteine, the latter sold as Mucomyst®) and found that they disrupt thioester linkages in the model PPT1-substrate [14C] palmitoyl~CoA, releasing [14C] palmitic acid. Among the drugs tested, we characterized cysteamine (Cystagon™) in further detail because (1) anecdotal evidence indicates that it crosses the blood brain barrier; (2) it functions at a low pH in cleaving thioester linkages; (3) it prevents apoptosis in INCL lymphoblasts; and (4) it is relatively nontoxic (Zhang et al., Nat Med 2001;7:478). Furthermore, our laboratory studies have shown that phosphocysteamine not only disrupts thioester linkages in S-acylated polypeptides in cultured cells from INCL patients but also mediates the depletion of intracellular ceroid deposits and prevents their re-accumulation. For the last five years, we have been conducting a clinical trial to determine whether Cystagon™ is beneficial for INCL patients. Given that preliminary results showed slower disease progression, we are currently testing a combination of two drugs on our patients. Mucomyst® has anti-apoptotic and neuroprotective effects and, like Cystagon™, a proven safety record. Our current combination therapy protocol is approved for 20 INCL patients who include those originally enrolled in the Cystagon™ monotherapy protocol.
1 Allison Heffer, former Summer Intern
2 Emiko Hitomi, MD, former Guest Researcher
COLLABORATOR
Eva Baker, MD, PhD, Diagnostic Radiology Department, NIH Clinical Center, Bethesda, MD
Rafael Caruso, MD, Ophthalmic and Visual Function Branch, NEI, Bethesda, MD
Eli Eisenstein, PhD, Hadassah Medical Center, Jerusalem, Israel
Jiliang Gao, PhD, Laboratory of Molecular Immunology, NIAID, Bethesda, MD
Andrea Gropman, MD, Georgetown University Medical Center, Washington, DC
Sandra L. Hofmann, MD, PhD, University of Texas Southwestern Medical Center, Dallas, TX
Shau-Ku Huang, PhD, The Johns Hopkins University Medical School, Baltimore, MD
Alan Koretsky, PhD, NMR Center, NINDS, Bethesda, MD
Ning Miao, MD, Department of Anesthesia and Surgical Services, NIH Clinical Center, Bethesda, MD
Jeeva Munasinghe, PhD, NMR Center, NINDS, Bethesda, MD
Nagarajan Pattabiraman, PhD, Georgetown University Medical Center, Washington, DC
Zenaide Quezado, MD, Department of Anesthesia and Surgical Services, NIH Clinical Center, Bethesda, MD
Yi-Jin Xiao, PhD, Cleveland Clinic, Cleveland, OH
Yan Xu, PhD, Indiana University Medical School, Indianapolis, IN
For further information, contact mukherja@mail.nih.gov.
