Danielle Crippen

Coupling endoplasmic reticulum stress to the cell-death program: a novel HSP90-independent role for the small chaperone protein p23

The endoplasmic reticulum (ER) is the principal organelle for the biosynthesis of proteins, steroids and many lipids, and is highly sensitive to alterations in its environment. Perturbation of Ca2+ homeostasis, elevated secretory protein synthesis, deprivation of glucose or other sugars, altered glycosylation and/or the accumulation of misfolded proteins may all result in ER stress, and prolonged ER stress triggers cell death. Studies from multiple laboratories have identified the roles of several ER stress-induced cell-death modulators and effectors through the use of biochemical, pharmacological and genetic tools. In the present work, we describe the role of p23, a small chaperone protein, in preventing ER stress-induced cell death. p23 is a highly conserved chaperone protein that modulates HSP90 activity and is also a component of the steroid receptors. p23 is cleaved during ER stress-induced cell death; this cleavage, which occurs close to the carboxy-terminus, requires caspase-3 and/or caspase-7, but not caspase-8. Blockage of the caspase cleavage site of p23 was associated with decreased cell death induced by ER stress. Immunodepletion of p23 or inhibition of p23 expression by siRNA resulted in enhancement of ER stress-induced cell death. While p23 co-immunoprecipitated with the BH3-only protein PUMA (p53-upregulated modulator of apoptosis) in untreated cells, prolonged ER stress disrupted this interaction. The results define a protective role for p23, and provide further support for a model in which ER stress is coupled to the mitochondrial intrinsic apoptotic pathway through the activities of BH3 family proteins.

Loss of intestinal nuclei and intestinal integrity in aging C. elegans

The roundworm C. elegans is widely used as an aging model, with hundreds of genes identified that modulate aging (Kaeberlein et al., 2002. Mech. Ageing Dev.123, 1115–1119). The development and bodyplan of the 959 cells comprising the adult have been well described and established for more than 25 years ( Sulston & Horvitz, 1977 . Dev. Biol.56, 110–156; Sulston et al., 1983. Dev. Biol.100, 64–119.). However, morphological changes with age in this optically transparent animal are less well understood, with only a handful of studies investigating the pathobiology of aging. Age‐related changes in muscle ( Herndon et al., 2002 . Nature419, 808–814), neurons ( Herndon et al., 2002 ), intestine and yolk granules ( Garigan et al., 2002 . Genetics161, 1101–1112; Herndon et al., 2002 ), nuclear architecture ( Haithcock et al., 2005 . Proc. Natl Acad. Sci. USA102, 16690–16695), tail nuclei ( Golden et al., 2007 . Aging Cell6, 179–188), and the germline ( Golden et al., 2007 ) have been observed via a variety of traditional relatively low‐throughput methods. We report here a number of novel approaches to study the pathobiology of aging C. elegans. We combined histological staining of serial‐sectioned tissues, transmission electron microscopy, and confocal microscopy with 3D volumetric reconstructions and characterized age‐related morphological changes in multiple wild‐type individuals at different ages. This enabled us to identify several novel pathologies with age in the C. elegans intestine, including the loss of critical nuclei, the degradation of intestinal microvilli, changes in the size, shape, and cytoplasmic contents of the intestine, and altered morphologies caused by ingested bacteria. The three‐dimensional models we have created of tissues and cellular components from multiple individuals of different ages represent a unique resource to demonstrate global heterogeneity of a multicellular organism.

Enzyme Replacement in a Human Model of Mucopolysaccharidosis IVA In Vitro and Its Biodistribution in the Cartilage of Wild Type Mice

Mucopolysaccharidosis IVA (MPS IVA; Morquio A syndrome) is a lysosomal storage disorder caused by deficiency of N-acetylgalactosamine-6-sulfatase (GALNS), an enzyme that degrades keratan sulfate (KS). Currently no therapy for MPS IVA is available. We produced recombinant human (rh)GALNS as a potential enzyme replacement therapy for MPS IVA. Chinese hamster ovary cells stably overexpressing GALNS and sulfatase modifying factor-1 were used to produce active (∼2 U/mg) and pure (≥97%) rhGALNS. The recombinant enzyme was phosphorylated and was dose-dependently taken up by mannose-6-phosphate receptor (Kuptake = 2.5 nM), thereby restoring enzyme activity in MPS IVA fibroblasts. In the absence of an animal model with a skeletal phenotype, we established chondrocytes isolated from two MPS IVA patients as a disease model in vitro. MPS IVA chondrocyte GALNS activity was not detectable and the cells exhibited KS storage up to 11-fold higher than unaffected chondrocytes. MPS IVA chondrocytes internalized rhGALNS into lysosomes, resulting in normalization of enzyme activity and decrease in KS storage. rhGALNS treatment also modulated gene expression, increasing expression of chondrogenic genes Collagen II, Collagen X, Aggrecan and Sox9 and decreasing abnormal expression of Collagen I. Intravenous administration of rhGALNS resulted in biodistribution throughout all layers of the heart valve and the entire thickness of the growth plate in wild-type mice. We show that enzyme replacement therapy with recombinant human GALNS results in clearance of keratan sulfate accumulation, and that such treatment ameliorates aberrant gene expression in human chondrocytes in vitro. Penetration of the therapeutic enzyme throughout poorly vascularized, but clinically relevant tissues, including growth plate cartilage and heart valve, as well as macrophages and hepatocytes in wild-type mouse, further supports development of rhGALNS as enzyme replacement therapy for MPS IVA.

Higher-order nuclear organization in growth arrest of human mammary epithelial cells: a novel role for telomere-associated protein TIN2

Nuclear organization, such as the formation of specific nuclear subdomains, is generally thought to be involved in the control of cellular phenotype; however, there are relatively few specific examples of how mammalian nuclei organize during radical changes in phenotype, such as those occurring during differentiation and growth arrest. Using human mammary epithelial cells in which growth arrest is essential for morphological differentiation, we show that the arrest of cell proliferation is accompanied by a reorganization of the telomere-associated protein, TIN2, into one to three large nuclear subdomains. The large TIN2 domains do not contain telomeres and occur concomitant with the continued presence of TIN2 at telomeres. The TIN2 domains were sensitive to DNase, but not RNase, occurred frequently, but not exclusively near nucleoli, and overlapped often with dense domains containing heterochromatin protein 1γ. Expression of truncated forms of TIN2 simultaneously prevented the formation of TIN2 domains and relaxed the stringent morphogenesis-induced growth arrest in human mammary epithelial cells. Here we show that a novel extra-telomeric organization of TIN2 is associated with the control of cell proliferation and identify TIN2 as an important regulator of mammary epithelial differentiation.

Identification of new modulators and protein alterations in non‐apoptotic programmed cell death

This study describes the first proteomic analysis of paraptosis—a non‐apoptotic form of programmed cell death. As with apoptosis, the first description of paraptosis was based on morphological criteria. Since there are no known markers for paraptosis, the purpose of this study was to dissect changes in the proteome profile occurring during paraptosis. Using one‐ and two‐dimensional SDS–PAGE, Western analysis, and mass spectrometry, we show that during paraptosis, alterations occur mainly in cytoskeletal proteins, signal transduction proteins, mitochondrial proteins, and some metabolic proteins. We also report the identification of: (1) a paraptosis inhibitor, phosphatidylethanolamine binding protein (PEBP‐1), and (2) a candidate mediator of paraptosis, prohibitin. Identification of specific paraptotic changes will ultimately lead to tools to detect this type of programmed cell death in in vivo systems and allow for its further characterization. J. Cell. Biochem. 111: 1401–1412, 2010.

C-Terminal Cleavage of the Amyloid-β Protein Precursor at Asp664: A Switch Associated with Alzheimer's Disease

In addition to the proteolytic cleavages that give rise to amyloid-beta (Abeta), the amyloid-beta protein precursor (AbetaPP) is cleaved at Asp664 intracytoplasmically. This cleavage releases a cytotoxic peptide, APP-C31, removes AbetaPP-interaction motifs required for signaling and internalization, and is required for the generation of AD-like deficits in a mouse model of the disease. Although we and others had previously shown that Asp664 cleavage of AbetaPP is increased in AD brains, the distribution of the Asp664-cleaved forms of AbetaPP in non-diseased and AD brains at different ages had not been determined. Confirming previous reports, we found that Asp664-cleaved forms of AbetaPP were increased in neuronal cytoplasm and nuclei in early-stage AD brains but were absent in age-matched, non-diseased control brains and in late-stage AD brains. Remarkably, however, Asp664-cleaved AbetaPP was prominent in neuronal somata and in processes in entorhinal cortex and hippocampus of non-diseased human brains at ages <45 years. Our observations suggest that Asp664 cleavage of AbetaPP may be part of the normal proteolytic processing of AbetaPP in young (<45 years) human brain and that this cleavage is down-regulated with normal aging, but is aberrantly increased and altered in location in early AD.

Novel Mediators of Amyloid Precursor Protein Signaling

Multiple recent reports implicate amyloid precursor protein (APP) signaling in the pathogenesis of Alzheimers disease, but the APP-dependent signaling network involved has not been defined. Here, we report a novel consensus sequence for interaction with the PDZ-1 and PDZ-2 domains of the APP-interacting proteins Mint1, Mint2, and Mint3 (X11α, X11β, and X11γ), and multiple novel interactors for these proteins, with the finding that transcriptional coactivators are highly represented among these interactors. Furthermore, we show that Mint3 interaction with a set of the transcriptional coactivators leads to nuclear localization and transactivation, whereas interaction of the same set with Mint1 or Mint2 prevents nuclear localization and transactivation. These results define new mediators of the signal transduction network mediated by APP.

Valosin-Containing Protein Gene Mutations: Cellular Phenotypes Relevant to Neurodegeneration

Previously, we identified valosin-containing protein (VCP) as a mediator of ER stress-induced cell death. Mutations in the VCP gene including R93, R155, and R191 have been described that manifest clinically as hereditary inclusion body myopathy with Paget’s disease of bone and frontotemporal dementia. In addition, other studies have demonstrated that as a consequence of a mutation generated in the second ATP binding domain of VCP (K524A), cells accumulated large cytoplasmic vacuoles and underwent programmed cell death. In order to better understand the biochemical and molecular consequences of the clinically relevant VCP mutations as well as the genetically engineered ATPase-inactive mutant K524A and any relationship these may have to ER stress-induced cell death, we introduced analogous mutations separately and together into the human VCP gene and evaluated their effect on proteasome activity, Huntingtin protein aggregation and ER stress-induced cell death. Our results indicate that the VCP K524A mutant and the triple mutant VCP R93C-R155C-K524A block protein degradation, trigger Huntingtin aggregate formation, and render cells highly susceptible to ER stress-induced cell death as compared to VCPWT or other VCP mutants.

The small chaperone protein p23 and its cleaved product p19 in cellular stress.

The presence of misfolded proteins elicits cellular responses including an endoplasmic reticulum (ER) stress response that may protect cells against the toxic buildup of misfolded proteins. Accumulation of these proteins in excessive amounts, however, overwhelms the “cellular quality control” system and impairs the protective mechanisms designed to promote correct folding and degrade misfolded proteins, ultimately leading to organelle dysfunction and cell death. Studies from multiple laboratories have identified the roles of several ER stress-induced cell death modulators and effectors. Earlier, we reported the role of the small co-chaperone protein p23 in preventing ER stress-induced cell death. p23 undergoes caspase-dependent cleavage to yield a 19-kD product (p19), and mutation of this caspase cleavage site not only blocks the formation of the 19-kD product but also attenuates the ER stress-induced cell death process triggered by various stressors. Thus, a critical question is whether p23 and/or p19 could serve as an in vivo marker for neurodegenerative diseases featuring misfolded proteins and cellular stress. In the present study, we used an antibody that recognizes both p23 and p19 as well as a specific neo-epitope antibody that detects only the p19 fragment. These antibodies were used to detect the presence of both these proteins in cells, primary neurons, brain samples from a mouse model of Alzheimer’s disease (AD), and fixed human AD brain samples. While patients with severe AD did display a consistent reduction in p23 levels, our inability to observe p19 in mouse or human AD brain samples suggests that the usefulness of the p23 neo-epitope antibody is restricted to cells and primary neurons undergoing cellular stress.