Heritable Neurodegenerative and Autoimmune Disorders

Anil B. Mukherjee, MD, PhD, Head, Section on Developmental Genetics
Zhongjian Zhang, MD, PhD, Staff Scientist
Sung-Jo Kim, PhD, Visiting Fellow
Arjun Saha, PhD, Visiting Fellow
Chinmoy Sarkar, PhD, Visiting Fellow
Hui Wei, PhD, Visiting Fellow
Sondra W. Levin, MD, Adjunct Scientist
Aprile Pilon, PhD, Adjunct Scientist
Matthew Moralle, BA, Postbaccalaureate Fellow

We conduct laboratory and clinical investigations into the molecular mechanisms underlying genetic disorders of neurodegeneration, inflammation, and autoimmunity for the purpose of developing therapeutic approaches to diseases for which no effective treatment exists. We focus on the regulation and physiological functions of two genes: (1) palmitoyl-protein thioesterase-1 (PPT1), mutations of which result in infantile neuronal ceroid lipofuscinosis, a devastating neurodegenerative storage disease of childhood; and (2) uteroglobin (UG), also known as Clara cell 10 kDa protein (CC10), which manifests potent anti-inflammatory and immunomodulatory properties. UG deficiency in mice causes IgA-nephropathy and focal pulmonary fibrosis and predisposes the animals to invasive cancers. Our investigations into both genes have led to ongoing clinical trials. Using PPT1-knockout mice, which recapitulate virtually all clinical and pathological features of INCL, we discovered that deficiency in this enzyme leads to endoplasmic reticulum stress, which in part mediates neuronal apoptosis, a discovery that provides insight into the complex mechanism of neurodegeneration in INCL and identifies several potential therapeutic targets. We also delineated the mechanism by which PPT1 deficiency leads to disrupted recycling of synaptic vesicle (SV) components and prevents the regeneration of SVs required for maintaining a steady SV pool size at neuronal synapses; SVs are essential for uninterrupted neurotransmission.

Synaptic vesicle recycling at the nerve terminals contributes to INCL neuropathology

Kim, Zhang, Sarkar, Tsai,¹ Lee,² Mukherjee; in collaboration with Dye

Neuronal ceroid lipofuscinoses (NCL) are the most common (1 in 12,500 births) neurodegenerative storage disorders of childhood. The infantile form of NCL, or INCL, is caused by palmitoyl protein thioesterase-1 (PPT1) deficiency. We previously reported that PPT1 deficiency causes endoplasmic reticulum (ER) stress, which at least in part mediates neuronal apoptosis and contributes to neurodegeneration in INCL. Although INCL patients show signs of abnormal neurotransmission as 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 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 PPT1-knockout (PPT1-KO) mice that mimic INCL, we found that PPT1 deficiency caused persistent membrane anchorage of the palmitoylated SV proteins, hindering 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.

Kim SJ, Zhang Z, Sarkar C, Tsai PC, Lee YC, Dye L, Mukherjee AB. Palmitoyl protein thioesterase-1 deficiency impairs synaptic vesicle recycling at nerve terminals, contributing to neuropathology in humans and mice. J Clin Invest 2008;118:3075-3086.
Zhang Z, Lee Y-C, Kim S-J, 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;15:337-346.

ER and oxidative stresses are common mediators of apoptosis in lysosomal storage disorders and are alleviated by chemical chaperones

Wei, Kim, Zhang, Tsai,¹ Mukherjee; in collaboration with Wisniewski

It is estimated that over 40 lysosomal storage disorders (LSD) cumulatively affect 1 in 5,000 live births; indeed, neurodegeneration is a prominent feature of the majority of such disorders. As a group, NCLs represent one of the most common (1 in 12,500 births) neurodegenerative LSDs. INCL is the most devastating neurodegenerative LSD. We previously reported that ER and oxidative stresses at least in part cause neuronal death by apoptosis in both INCL and PPT1-KO mice that mimic INCL. Over the past year, we unexpectedly found that ER and oxidative stresses are not unique manifestations of INCL but are common to both neurodegenerative and non-neurodegenerative LSDs. Moreover, all LSD cells studied in our laboratory show extraordinary sensitivity to brefeldin A–induced apoptosis, suggesting pre-existing ER stress conditions. Further, we found that chemical disruption of lysosomal homeostasis in normal cells causes ER stress, suggesting cross-talk between lysosomes and the ER. Most important, we found chemical chaperones that alleviate ER and oxidative stresses to be 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 posit that, at least in part, alleviation of stress conditions permits chemical/pharmacological chaperones to exert their beneficial effects.

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;15:1580-1586.
Wei H, Kim S-J, Zhang Z, Tsai PC, Wisniewski KE, Mukherjee AB. ER and oxidative stresses are common mediators of apoptosis in both neurodegenerative and non-neurodegenerative lysosomal storage disorders and are alleviated by chemical chaperones. Hum Mol Genet 2008;17:469-477.

Cytosolic PLA₂ activation in the brain of mice lacking PPT1 mediates phagocyte infiltration

Zhang, Lee,² Kim, Choi,³ Tsai,¹ Saha, Wei, Zhang, Heffer,4 Mukherjee; in collaboration with Xiao, Xu

In the majority of neurodegenerative storage disorders, neuronal death in the brain is followed by infiltration of phagocytic cells (e.g., activated microglia, astroglia, and macrophages) for the efficient removal of dead cells. However, it is increasingly evident that the phagocytes may also cause death of adjoining viable neurons, thus contributing to rapid progression of neurodegeneration. PPT1 catalyzes the cleavage of thioester linkages in S-acylated (palmitoylated) proteins while a deficiency in the enzyme leads to abnormal accumulation of thioesterified polypeptides (ceroid) in lysosomes, thereby causing INCL pathogenesis. As noted, PPT1-KO mice mimic the clinical and pathological features of human INCL, including rapid neuronal death by apoptosis and phagocyte infiltration. We previously reported that, in PPT1-KO mice, neurons undergo ER stress, activating the unfolded protein response, which mediates caspase-12 activation and apoptosis. However, the molecular mechanism(s) by which the phagocytic cells are recruited in the PPT1-KO mouse brain remains poorly understood. We found that increased production of lysophosphatidylcholine (LPC), catalyzed by the activation of cytosolic phospholipase A₂ (cPLA₂) in the PPT1-KO mouse brain, is a “lipid signal” for phagocyte recruitment. We also report that an age-dependent increase in LPC levels in the PPT1-KO mouse brain positively correlates with elevated expression of the genes characteristically associated with phagocytes. We propose that increased cPLA₂-catalyzed LPC production in the brain is at least one of the mechanisms that mediates phagocyte infiltration contributing to INCL neuropathology.

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 lysophosphatidylcholine by cPLA₂ in the brain of mice lacking PPT1 is a signal for phagocyte infiltration. Hum Mol Genet 2007;16:837-847.
Zhang Z, Lee Y-C, Kim S-J, 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;15:337-346.

Clinical trial of a combination of Cystagon™ and Mucomyst® for INCL patients

Levin, Zhang, Mukherjee; in collaboration with Baker, Caruso, Quezado, Miao, Gropman

Thioester-linkaged S-acylated polypeptides are susceptible to nucleophilic attack. Thus drugs with nucleophilic property have therapeutic potential for INCL. Several such compounds (e.g., cysteamine, phosphocysteamine, cysteamine bitartrate [Cystagon™], and N-acetylcysteine [Mucomyst®]) disrupt thioester linkages in the model PPT1-substrate [¹⁴C] palmitoyl~CoA, releasing [¹⁴C] palmitic acid. Among the drugs tested, we previously characterized phosphocysteamine, Cystagon™, and Mucomyst® in detail and found that they do indeed cleave thioester linkages and deplete ceroid in cultured cells from INCL patients (Zhang et al., Nat Med 2002;7:478). It has also been reported that cysteamine is lysosomotrophic, crosses the blood-brain barrier, and is relatively non-toxic. Thus, we have been conducting a clinical trial involving 20 INCL patients to determine whether a combination of Cystagon™ and Mucomyst® is beneficial. Given that INCL is a fatal disease, that Mucomyst® has anti-apoptotic effects, and that both drugs are neuroprotective and have a proven record of safety, we received approval for combination therapy of those patients who were originally enrolled in the Cystagon™ monotherapy protocol.

Uteroglobin is the founder of the Secretoglobin superfamily of proteins

Mukherjee, Zhang; in collaboration with Chilton

UG, also known as blastokinin, is a steroid-inducible, evolutionarily conserved, secreted protein whose structure and molecular biology have undergone extensive study. However, the physiological function(s) of UG remains elusive. Isolated from the uterus of rabbits during early pregnancy, UG is the founding member of a growing superfamily of proteins called Secretoglobins (Scgb). Numerous studies demonstrated that UG is a multifunctional protein with anti-inflammatory/immunomodulatory properties. It inhibits soluble phospholipase A₂ activity and binds to and perhaps sequesters hydrophobic ligands such as progesterone, retinols, polychlorinated biphenyls, phospholipids, and prostaglandins. In addition, UG manifests anti-chemotactic, anti-allergic, anti-tumorigenic, and embryonic growth–stimulatory activities. Several steroidal hormones regulate the tissue-specific expression of the UG gene, although a non-steroid hormone, prolactin, further augments the gene’s expression in the uterus. The mucosal epithelia of virtually all organs that communicate with the external environment express UG, which is present in blood, urine, and other body fluids. Although UG’s physiological functions 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 the protein leads to phenotypes suggestive of the protein’s critical homeostatic role(s) against oxidative damage, inflammation, autoimmunity, and cancer. Recent studies on UG-binding proteins (receptors) provide further insight into the protein’s multifunctional nature. Given its anti-inflammatory and anti-allergic properties, UG is a potential drug target.

Mukherjee AB, Zhang Z, Chilton BS. Uteroglobin: a steroid-inducible immuno-modulatory protein that founded the Secretoglobin superfamily. Endocr Rev 2007;28:707-725.

UG prevents fibronectin-IgA complex formation found in IgA-nephropathy

Zhang, Mukherjee; in collaboration with Chowdhury

Immunoglobulin A (IgA) nephropathy (IgAN) is the most common primary renal glomerular disease in the world without effective treatment. High levels of circulating IgA-fibronectin (Fn) complexes characteristic of IgAN patients are thought to cause abnormal deposition of IgA and Fn in the renal glomeruli of these patients, causing renal failure. We previously reported that binding of Fn to UG inhibits Fn-IgA heteromerization (Zheng et al., Nat Med 1999;5:1018). However, the specific site of Fn-UG interaction was unknown until now. We report that UG interacts with the heparin-binding site of Fn and propose that small molecules competing for interaction with this site may reduce the level of circulating Fn-IgA complexes in IgAN.

¹ Pei Chen Tsai, MS, former Technical Training Fellow
² Yi-ching Lee, PhD, former Visiting Fellow
³ Moonsuk S. Choi, PhD, former Adjunct Scientist
⁴ Allison Heffer, former Summer Intern

Collaborators

Eva Baker, MD, PhD, Clinical Center, NIH, Bethesda, MD
Rafael Caruso, MD, Ophthalmic and Visual Function Branch, NEI, Bethesda, MD
Beverly S. Chilton, PhD, Texas Tech University Health Sciences Center, Lubbock, TX
Bhabadeb Chowdhury, PhD, Laboratory of Molecular Immunology, NIAID, Bethesda, MD
Louis Dye, BS, Microscopy and Imaging Core Facility, NICHD, Bethesda, MD
Andrea Gropman, MD, Georgetown University, 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, Clinical Center, NIH, Bethesda, MD
Jeeva Munasinghe, PhD, NMR Center, NINDS, Bethesda, MD
Nagarajan Pattabiraman, PhD, Lombardi Cancer Center, Georgetown University Medical Center, Washington, DC
Zenaide Quezado, MD, Clinical Center, NIH, Bethesda, MD
Krystyna Wisniewski, MD, PhD, New York State Institute for Basic Research in Developmental Disabilities, Staten Island, NY
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.