SCD | SDG | SGDDM
| UMD | Main
Page
HERITABLENEURODEGENERATIVE
AND
AUTOIMMUNE DISODERS
|
Anil B. Mukherjee,
MD, PhD, Head, Section on
Developmental Genetics Moonsuk Choi, PhD, Postdoctoral
Fellow Rabindranath Ray,
PhD, Postdoctoral Fellow Bhabadeb Chowdhury, PhD, CRADA Fellowa Sung-Jo Kim, PhD, Visiting Fellow Yi-ching Lee,
PhD, Visiting Fellow Aperna
Mital, PhD, Visiting Fellowa Sondra
Levin, MD, Guest Researcherb |
|
|
We conduct basic and clinical investigations into
the molecular mechanisms of heritable neurodegenerative and
inflammatory/autoimmune disorders in order to develop novel therapeutic
approaches. We focus primarily on
uteroglobin (UG), a multifunctional secreted protein with potent
anti-inflammatory and anti-chemotactic properties, and on palmitoyl-protein thioesterase 1 (PPT-1). Targeted disruption of the murine
UG gene yields three phenotypes: immunoglobulin A nephropathy (IgAN), the most common primary glomerular disease;
exaggerated allergen-induced pulmonary inflammation, reminiscent of allergic
asthma in humans; and increased susceptibility to pulmonary adenomas when
exposed to NNK, a potent carcinogen in tobacco smoke. We are currently
delineating the molecular mechanisms of the UG-knockout phenotypes.
Inactivating mutations of PPT-1 lead to infantile neuronal ceroid
lipofuscinosis (INCL), also known as infantile Batten disease. We found that
drugs with nucleophilic properties may mimic PPT-1 function and benefit INCL
patients. We are currently determining the effectiveness of one such drug,
Cystagon™, to treat INCL. We also continued our efforts to understand
the natural history and molecular mechanisms of INCL pathogenesis by using a
PPT-knockout mouse model. Overexpression of SCCA2, a gene associated
with allergic asthma, in mice lacking uteroglobin Ray, Zhang, Choi, Mukherjee; in collaboration with Silverman Uteroglobin (UG), the first member of the secretoglobin superfamily to be discovered, is a
steroid-inducible, multifunctional protein with potent anti-inflammatory and antichemotactic properties. It is secreted by the mucosal
epithelia of all mammalian organs that communicate with the external
environment. It has also been reported that some of the biological functions
of the protein may be mediated via its cell surface binding sites (putative
receptors). We previously reported that mice lacking UG manifest exaggerated
inflammatory response in the airways when sensitized and challenged with
ovalbumin (OVA), a commonly used allergen in animal models of allergic
asthma. Most important, treatment of these mice with recombinant UG abrogates
the airway inflammatory response. Recent reports indicate that the expression
of squamous cell carcinoma antigen-2 (SCCA-2), a serine protease inhibitor of
the ovalbumin-serpin family, is upregulated in the
airway epithelia of patients with bronchial asthma. In the present study, we
used OVA-sensitized and -challenged UG-knockout mice to determine whether UG
plays a role in altering SCCA-2 gene expression. We found that, compared with
wild-type litter mates, UG-knockout mice express markedly higher levels of
the SCCA-2 gene in the lungs, which is further augmented by OVA
sensitization and challenge. We further demonstrate that the treatment of
these mice with recombinant UG suppresses SCCA-2 gene expression.
Taken together, our results suggest that UG is a potent inhibitor of SCCA-2
expression, which is associated with bronchial asthma, raising the
possibility that UG and its cell surface binding proteins play critical roles
in suppressing allergen-mediated airway inflammatory responses. Critical role of UG in allergic asthma Mandal,c Zhang, Chowdhury,
Ray, Mukherjee; in collaboration with Pattabiraman Asthma is a complex heritable inflammatory disease
of the respiratory system. In the Mandal AK, Ray R, Zhang Z, Chowdhury B, Ray R, Choi MS, Mukherjee AB. Uteroglobin represses allergen-induced
inflammation by blocking PGD2 receptor-mediated functions. J Exp Med 2004;99:1317-1330. Increased susceptibility of UG-knockout mice
to lung tumorigenesis by NNK Zhang, Mukherjee,
Yang; in collaboration with Linnoila, Yang We previously reported that one of the phenotypes
of mice lacking Clara cell 10 kDa (CC10) protein, also known as UG, is the
development of multiorgan tumors. It has been reported that CC10 expression
is rarely detectable in human nonsmall cell cancers
despite abundant production by progenitors of normal airway epithelial cells.
Moreover, CC10 expression is drastically reduced
following exposure of animals to methyl-nitrosamine (NNK), the potent
carcinogen in tobacco smoke. Furthermore, we previously reported that forced
expression of CC10 in cancer cells reverses the transformed phenotype. Given
that tobacco smoke and lung tumorigenesis are associated with
reduced levels of CC10 and that 90 percent of all human lung cancers are
related to cigarette smoking, we sought to determine whether the mice lacking
UG (CC10) are more susceptible to NNK-induced lung tumors. We found that,
compared with wild-type controls, UG-knockout mice exposed to NNK are highly
susceptible (30 percent knockout versus 5 percent wild type) to increased
proliferation of airway epithelial cells and the development of adenoma of
the lung within five to 12 months of NNK exposure. Interestingly, the
hyperproliferation and tumorigenesis in NNK-treated knockout mice were
associated with activation of Ras and the mitogen-activated protein
kinase while 50 percent of the adenomas had K-Ras mutation. Our
results indicate that UG-knockout mice are highly susceptible to NNK-induced
lung adenomas, raising the possibility that UG has tumor
suppressor–like properties. We have now generated transgenic mice that overexpress human UG in the lungs, providing an animal
model to test our hypothesis further that UG has tumor-suppressor activity.
We are currently characterizing these mice. Yang Y, Zhang Z, Characterization of a novel secretoglobin gene Choi, Ray, Zhang,
Mukherjee The human secretoglobin
cluster of genes map to chromosome 11q12-13, a region in which candidate
genes for inflammatory/autoimmune disorders and cancer are co-localized. It
has been previously reported that interferon gamma (IFN-gamma), a proinflammatory cytokine, stimulates the expression of
UG, a possible homeostatic mechanism to counteract the proinflammatory
effects of IFN-gamma. In the present study, we sought to determine whether
this cytokine might stimulate the expression of any other secretoglobin.
We report that IFN-gamma stimulates a novel secretoglobin
gene that bears sequence similarity to lipophilin-B,
another member of the same superfamily. A BLAST search revealed that the gene
is also similar to YGB, which encodes a lymphocyte-specific secretoglobin. However, unlike YGB, the gene we
characterized is not only expressed in lymphoblasts but also in other cell
types, including colon carcinoma cells. The deduced protein sequence of IIS
(interferon-inducible secretoglobin) reveals that
the protein lacks the C-terminal cysteine residue characteristic of YGB.
Given that IFN-gamma plays critical roles in microbial defense, IIS may have
important functions. Choi M, Ray R, Zhang Z, Clinical trial to determine whether
Cystagon™ is beneficial for children with infantile Batten disease Levin,
Zhang, Mukherjee; in
collaboration with Caruso, Gropman Neuronal ceroid lipofuscinoses (NCLs) are the most common (1 in 12,500) heritable
progressive encephalopathies of children. Infantile NCL (INCL), also known as
Batten disease, is caused by lysosomal palmitoyl-protein thioesterase
(PPT) deficiency. PPT catalyzes the hydrolysis of thioester linkages in
S-acylated polypeptides, and its deficiency causes abnormal accumulation of
these polypeptides, leading to INCL. Given that thioester bonds are
susceptible to nucleophilic attack, drugs with nucleophilic properties (e.g.,
Cystagon™) may have therapeutic potential for INCL. Last year, we
demonstrated that the drug 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 reaccumulation. Taken together, the results raised the
possibility that the drug is an effective treatment for INCL. Given that INCL
is a uniformly fatal disease for which there is currently no effective
treatment and that the active compound of phosphocysteamine has been in
clinical use for more than two decades with a proven record of safety, we
have implemented a pilot study to determine whether cysteamine bitartrate
(Cystagon™) is beneficial for INCL patients. We have received approval
to treat 20 INCL patients in our study. We have so far recruited two patients
to the protocol, with preliminary evaluations of the patients indicating
stabilization of the retinal functions and cortical degeneration. The most
dramatic results were obtained from the electron-microscopic analyses of the
patients’ white blood cells. Compared with the cells before treatment,
the mononuclear cells show virtually no ceroid deposits in their lysosomes
after the initiation of Cystagon™ treatment. So far, the treated patients
have not required antiepileptic medications and have not exhibited adverse
effects. The study will continue until we recruit 20 patients, as approved,
and we will complete final analyses when all data are available. Zhang Z, Butler JD, Levin SW, Wisniewski
KE, Brooks SS, Mukherjee AB. Lysosomal ceroid
depletion by drugs: therapeutic implications for a hereditary
neurodegenerative disease of childhood. Nat Med 2002;7:478-484. Development of novel therapeutic approaches
for INCL using a mouse model of INCL Zhang, Mukherjee;
in collaboration with Hofmann, Koretsky, Munasinghe PPT facilitates the degradation (recycling) of
post-translationally acylated polypeptides by cleaving thioester linkages
that connect the lipids with the polypeptides that are acylated (see above).
A deficiency of this enzyme leads to abnormal deposition of acylated proteins
or peptides (ceroids), causing INCL pathogenesis.
Given that the PPT gene is expressed at high levels in the retina and
brain, children afflicted with INCL become blind by two years and brain-dead
by age four. Recently, we used gene targeting to develop a mouse model of
INCL, allowing detailed pathophysiologic analyses and the development of
novel therapeutic approaches for INCL. Evaluation of potential therapeutic
approaches would require techniques to assess the outcome of
new treatment modalities. Last year, we reported that, in a preliminary
study, we investigated five age- and sex-matched PPT knockout mice and
five wild-type litter mates by MRI, using a 7 Tesla horizontal scanner
operating on a Bruker Avance
platform. We conducted the preliminary study to assess whether MRI could
measure degenerative changes in the mouse brain. Our preliminary results
indicate that results obtained from MRI combined with CT scanning and
spectroscopy are an excellent means to evaluate neurodegenerative changes in
the PPT knockout mice, thereby setting the stage for studies to screen
and evaluate potential drug candidates and other novel therapeutic approaches
(gene therapy and neuronal stem-cell implantation) by using this animal
model. The Animal Care and Use Committee recently approved our animal study
protocol, permitting us to initiate investigations to evaluate novel drugs
and other therapeutic modalities. aCompleted
fellowship. bWalter cAsim Mandal, PhD, former Postdoctoral Fellow COLLABORATORS Rafael Caruso, MD, Ophthalmic and Visual Function Branch,
NEI, Andrea Gropman,
MD, Neurogenetics Branch, NINDS, Sandra L. Hofmann, MD, PhD,
Brenda Klaunberg,
DVM, Mouse Imaging Facility, NINDS, Alan Koretsky,
PhD, Laboratory of Functional and Molecular Imaging, NINDS, Jeeva Munasinghe, PhD, Mouse Imaging Facility, NINDS, N. Pattabiraman,
PhD, Gary Silverman, MD, Yongping Yang,
PhD, Mouse Cancer Genetics Program, NCI, Frederick, MD For
further information, contact mukherja@mail.nih.gov |
|
