On the role of monocytes/macrophages in the pathogenesis of central nervous system lesions in hereditary cystatin C amyloid angiopathy (original) (raw)
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Intracellular accumulation of the amyloidogenic L68Q variant of human cystatin C in NIH/3T3 cells
Molecular Pathology, 1998
Aim-To study the cellular transport of L68Q cystatin C, the cystatin variant causing amyloidosis and brain haemorrhage in patients suVering from hereditary cystatin C amyloid angiopathy (HCCAA). Methods-Expression vectors for wildtype and L68Q cystatin C were constructed and used to transfect mouse NIH/ 3T3 cells. Stable cell clones were isolated after cotransfection with pSV2neo. Clones expressing human wild-type and L68Q cystatin C were compared with respect to secreted cystatin C by enzyme linked immunosorbent assay (ELISA), and for intracellular cystatin C by western blotting and immunofluorescence cytochemistry. Colocalisation studies in cells were performed by double staining with antibodies against human cystatin C and marker proteins for lysosomes, the Golgi apparatus, or the endoplasmic reticulum, and evaluated by confocal microscopy. Results-Concentrations of human cystatin C secreted from transfected NIH/3T3 cells were similar to those secreted from human cells in culture. In general, clones expressing the gene encoding L68Q cystatin C secreted slightly lower amounts of the protein than clones expressing wildtype human cystatin C. Both immunofluorescence cytochemistry and western blotting experiments showed an increased accumulation of cystatin C in cells expressing the gene encoding L68Q cystatin C compared with cells expressing the gene for the wild-type protein. The intracellularly accumulating L68Q cystatin C was insoluble and located mainly in the endoplasmic reticulum. Conclusions-The cellular transport of human cystatin C is impeded by the pathogenic amino acid substitution Leu68→Gln. The resulting intracellular accumulation and increased localised concentration of L68Q cystatin C might be an important event in the molecular pathophysiology of amyloid formation and brain haemorrhage in patients with HCCAA.
The Role of Cystatin C in Cerebral Amyloid Angiopathy and Stroke: Cell Biology and Animal Models
Brain Pathology, 2006
a variant of the cysteine protease inhibitor, cystatin c, forms amyloid deposited in the cerebral vasculature of patients with hereditary cerebral hemorrhage with amyloidosis, icelandic type (hchwa-i), leading to cerebral hemorrhages early in life. however, cystatin c is also implicated in neuronal degenerative diseases in which it does not form the amyloid protein, such as alzheimer disease (ad). accumulating data suggest involvement of cystatin c in the pathogenic processes leading to amyloid deposition in cerebral vasculature and most significantly to cerebral hemorrhage in patients with cerebral amyloid angiopathy (caa). this review focuses on cell culture and animal models used to study the role of cystatin c in these processes. Pathol 2006;16:60-70. 182. Zannis VI, Nicolosi RJ, Jensen E, Breslow JL, Hayes KC (1985) Plasma and hepatic apoE isoproteins of nonhuman primates. Differences in apoE among humans, apes, and New and Old World monkeys. J Lipid Res 26:1421-1430.
Involvement of cystatin C in pathophysiology of CNS diseases
Frontiers in Bioscience, 2008
2. Cystatin C-type cerebral amyloid angiopathy 3. Amyloid fibril formation by cystatin C 4. Concentration in CSF 5. Involvement in AD 6. Involvement in neuronal cell death 7. Conclusions 8. Acknowledgments 9. References
Solubilized cystatin C amyloid is cytotoxic to cultured human cerebrovascular smooth muscle cells
Experimental and Molecular Pathology, 2007
Cerebral amyloid angiopathy (CAA) is characterized by the accumulation of amyloid within arteries of the cerebral cortex and leptomeninges. This condition is age related, especially prevalent in Alzheimer's disease (AD) and the main feature of certain hereditary disorders. The vascular smooth muscle cells (VSMC) appear to play a vital role in the development of CAA and have been found to produce the amyloid β precursor protein (AβPP) and process it to Aβ the major component of most CAA amyloid. Moreover, synthesized Aβ has proven to be toxic to cerebral VSMC in culture possibly explaining the disintegration and disappearance of the muscle cells from affected cerebral blood vessels seen in CAA. An aggressive and extremely rare form of CAA, known as Hereditary Cerebral Hemorrhage With Amyloidosis-Icelandic Type (HCHWA-I), exhibits this withdrawal of VSMC as amyloid accumulates in the vessel wall. However, the amyloid in HCHWA-I is made from a variant of cystatin C (L68Q) instead of the more common Aβ. To evaluate possible cytotoxicity in this condition solubilized cystatin C amyloid extracted from HCHWA-I leptomeninges was applied to cerebral smooth muscle cells in culture and was found to kill the cells.
Journal of Biological Chemistry, 1998
A cystatin C variant with L68Q substitution and a truncation of 10 NH 2-terminal residues is the major constituent of the amyloid deposited in the cerebral vasculature of patients with the Icelandic form of hereditary cerebral hemorrhage with amyloidosis (HCHWA-I). Variant and wild type cystatin C production, processing, secretion, and clearance were studied in human cell lines stably overexpressing the cystatin C genes. Immunoblot and mass spectrometry analyses demonstrated monomeric cystatin C in cell homogenates and culture media. While cystatin C formed concentration-dependent dimers, the HCHWA-I variant dimerized at lower concentrations than the wild type protein. Amino-terminal sequence analysis revealed that the variant and normal proteins produced and secreted are the full-length cystatin C. Pulse-chase experiments demonstrated similar levels of normal and variant cystatin C production and secretion. However, the secreted variant cystatin C exhibited an increased susceptibility to a serine protease in conditioned media and in human cerebrospinal fluid, explaining its depletion from the cerebrospinal fluid of HCHWA-I patients. Thus, the amino acid substitution may induce unstable cystatin C with intact inhibitory activity and predisposition to self-aggregation and amyloid fibril formation.
Codeposition of Cystatin C with Amyloid-β Protein in the Brain of Alzheimer Disease Patients
Journal of Neuropathology and Experimental Neurology, 2001
Immunohistochemical analysis of brains of patients with Alzheimer disease (AD) revealed that the cysteine proteinase inhibitor cystatin C colocalizes with amyloid -protein (A) in parenchymal and vascular amyloid deposits. No evidence of cerebral hemorrhage was observed in any of the brains studied. Immunoelectron microscopy demonstrated dual staining of amyloid fibrils with anti-A and anti-cystatin C antibodies. Cystatin C immunoreactivity was also observed in amyloid deposits in the brain of transgenic mice overexpressing human  amyloid precursor protein. Massive deposition of the variant cystatin C in the cerebral vessels of patients with the Icelandic form of hereditary cerebral hemorrhage with amyloidosis is thought to be responsible for the pathological processes leading to stroke. Anti-cystatin C antibodies strongly labeled pyramidal neurons within cortical layers most prone to amyloid deposition in the brains of AD patients. Immunohistochemistry with antibodies against the carboxyl-terminus of A x-42 showed intracellular immunoreactivity in the same neuronal subpopulation. It remains to be established whether the association of cystatin C to A plays a primary role in amyloidogenesis of AD or is a late event in which the protein is bound to the previously formed A amyloid fibrils.
Cystatin C modulates cerebral β-amyloidosis
Nature Genetics, 2007
The CST3 Thr25 allele of CST3, which encodes cystatin C, leads to reduced cystatin C secretion and conveys susceptibility to Alzheimer's disease. Here we show that overexpression of human cystatin C in brains of APP-transgenic mice reduces cerebral amyloid-b deposition and that cystatin C binds amyloid-b and inhibits its fibril formation. Our results suggest that cystatin C concentrations modulate cerebral amyloidosis risk and provide an opportunity for genetic risk assessment and therapeutic interventions.
Journal of Molecular Neuroscience, 2004
Cystatin C, an inhibitor of cysteine proteases, colocalizes with amyloid β (Aβ) in parenchymal and vascular amyloid deposits in brains of Alzheimer's disease (AD) patients, suggesting that cystatin C has a role in AD. Cystatin C also colocalizes with β amyloid precursor protein (βAPP) in transfected cultured cells. In vitro analysis of the association between the two proteins revealed that binding of cystatin C to full-length βAPP does not affect the level of Aβ secretion. Here we studied the effect of in vivo overexpression of cystatin C on the levels of endogenous brain Aβ. We have generated lines of transgenic mice expressing either wild-type human cystatin C or the Leu68Gln variant that forms amyloid deposits in the cerebral vessels of Icelandic patients with hereditary cerebral hemorrhage, under control sequences of the human cystatin C gene. Western blot analysis of brain homogenates was used to select lines of mice expressing various levels of the transgene. Analysis of Aβ40 and Aβ42 concentrations in the brain showed no difference between transgenic mice and their nontransgenic littermates. Thus, in vivo overexpression of human cystatin C does not affect Aβ levels in mice that do not deposit Aβ.
Neurobiology of Disease, 2001
Cystatin C is an essential secretory cofactor for neurogenesis with potent protease inhibitor activities. Polymorphisms of cystatin C are genetically associated with Alzheimer's disease (AD), and the L68Q mutation causes hereditary cerebral hemorrhage with amyloidosis of the Icelandic type, in which cystatin C and -amyloid are colocalized in cortical blood vessels. To determine whether cystatin C and -amyloid also colocalize in brain amyloid plaques, we analyzed transgenic mice expressing the Swedish APP (SweAPP) mutation. We found high levels of cystatin C in astrocytes surrounding -amyloid plaques, and discrete layers of cystatin C attached to amyloid plaque cores covered by a layer of -amyloid. In addition, cystatin C accumulated in reactive astrocytes throughout the brain, independently of, and before the onset of, amyloid plaque formation. These results show that expression of SweAPP is associated with increased cystatin C in reactive astrocytes, and they suggest an early role of cystatin C in appositional amyloid plaque growth.