The Relationship Between Lymphangiogenesis and Liver Regeneration After Partial Hepatectomy in Cholestatic Mice.

Background: The mechanisms of lymphangiogenesis in the cholestatic liver after partial hepatectomy (PH) remain unclear. We aimed to demonstrate the relationship between lymphangiogenesis and liver regeneration after partial hepatectomy in the cholestatic liver. Methods and Results: C57BL/6 mice were subjected to 70% partial hepatectomy only (PH group, n = 20) and 70% partial hepatectomy with temporary common bile duct (BD) obstruction by clipping (BD+PH group, n = 20). Five mice per group were sacrificed at 1, 3, 5, and 7 days after the procedure. The liver function was examined by blood tests, and the liver regeneration rate was assessed by body weight and liver weight. Immunohistochemical staining of lymphatic vessel endothelial hyaluronan receptor-1 (LYVE-1) showed liver lymphangiogenesis. The gene expression of lymphangiogenesis-associated factors (e.g., vascular endothelial growth factor receptor-3 [VEGFR-3]) was examined by a real-time polymerase chain reaction. The liver function in the BD+PH group was worse than that in the PH group on postoperative day 1 (POD1) (aspartate aminotransferase: 6528 ± 1641 U/L vs. 2741 ± 368 U/L, p < 0.05, alanine aminotransferase: 4160 ± 1255 U/L vs. 2315 ± 357 U/L, total bilirubin: 1.36 ± 1.16 mg/dL vs. 0.09 ± 0.01 mg/dL), and the liver regeneration rate in the BD+PH group was worse on POD7 (4.57% vs. 5.91%, p < 0.05). The LYVE-1 expression in Glisson's capsule peaked on POD5 and POD7 in the PH and BD+PH groups, respectively. The peak gene expression of VEGFR-3 in the BD+PH group was delayed in comparison with the PH group. Conclusions: Lymphangiogenesis after partial hepatectomy in the cholestatic liver was suggested to be delayed due to impaired liver regeneration and the late expression of VEGFR-3.

Contrasting pathology of the stress granule proteins TIA-1 and G3BP in tauopathies.

Stress induces aggregation of RNA-binding proteins to form inclusions, termed stress granules (SGs). Recent evidence suggests that SG proteins also colocalize with neuropathological structures, but whether this occurs in Alzheimer's disease is unknown. We examined the relationship between SG proteins and neuropathology in brain tissue from P301L Tau transgenic mice, as well as in cases of Alzheimer's disease and FTDP-17. The pattern of SG pathology differs dramatically based on the RNA-binding protein examined. SGs positive for T-cell intracellular antigen-1 (TIA-1) or tristetraprolin (TTP) initially do not colocalize with tau pathology, but then merge with tau inclusions as disease severity increases. In contrast, G3BP (ras GAP-binding protein) identifies a novel type of molecular pathology that shows increasing accumulation in neurons with increasing disease severity, but often is not associated with classic markers of tau pathology. TIA-1 and TTP both bind phospho-tau, and TIA-1 overexpression induces formation of inclusions containing phospho-tau. These data suggest that SG formation might stimulate tau pathophysiology. Thus, study of RNA-binding proteins and SG biology highlights novel pathways interacting with the pathophysiology of AD, providing potentially new avenues for identifying diseased neurons and potentially novel mechanisms regulating tau biology.

Induction of autophagy by cystatin C: a mechanism that protects murine primary cortical neurons and neuronal cell lines.

Cystatin C (CysC) expression in the brain is elevated in human patients with epilepsy, in animal models of neurodegenerative conditions, and in response to injury, but whether up-regulated CysC expression is a manifestation of neurodegeneration or a cellular repair response is not understood. This study demonstrates that human CysC is neuroprotective in cultures exposed to cytotoxic challenges, including nutritional-deprivation, colchicine, staurosporine, and oxidative stress. While CysC is a cysteine protease inhibitor, cathepsin B inhibition was not required for the neuroprotective action of CysC. Cells responded to CysC by inducing fully functional autophagy via the mTOR pathway, leading to enhanced proteolytic clearance of autophagy substrates by lysosomes. Neuroprotective effects of CysC were prevented by inhibiting autophagy with beclin 1 siRNA or 3-methyladenine. Our findings show that CysC plays a protective role under conditions of neuronal challenge by inducing autophagy via mTOR inhibition and are consistent with CysC being neuroprotective in neurodegenerative diseases. Thus, modulation of CysC expression has therapeutic implications for stroke, Alzheimer's disease, and other neurodegenerative disorders.

Complexes of amyloid-beta and cystatin C in the human central nervous system.

A role for cystatin C (CysC) in the pathogenesis of Alzheimer's disease (AD) has been suggested by the genetic linkage of a CysC gene (CST3) polymorphism with late-onset AD, the co-localization of CysC with amyloid-beta (Abeta) in AD brains, and binding of CysC to soluble Abeta in vitro and in mouse models of AD. This study investigates the binding between Abeta and CysC in the human central nervous system. While CysC binding to soluble Abeta was observed in AD patients and controls, a SDS-resistant CysC/Abeta complex was detected exclusively in brains of neuropathologically normal controls, but not in AD cases. The association of CysC with Abeta in brain from control individuals and in cerebrospinal fluid reveals an interaction of these two polypeptides in their soluble form. The association between Abeta and CysC prevented Abeta accumulation and fibrillogenesis in experimental systems, arguing that CysC plays a protective role in the pathogenesis of AD in humans and explains why decreases in CysC concentration caused by the CST3 polymorphism or by specific presenilin 2 mutations can lead to the development of the disease. Thus, enhancing CysC expression or modulating CysC binding to Abeta have important disease-modifying effects, suggesting a novel therapeutic intervention for AD.

Brain levels of CDK5 activator p25 are not increased in Alzheimer's or other neurodegenerative diseases with neurofibrillary tangles.

Elevated levels of p25 and constitutive activation of CDK5 have been observed in AD brains. This has led to the hypothesis that increased p25 levels could promote neurofibrillary tangles (NFT) through CDK5-mediated hyperphosphorylation of tau, the principal component of NFTs. We examined p25 immunoreactivity in brains from sporadic and familial AD cases, as well as other neurologic diseases that exhibit NFT, such as Down's syndrome (DS), Pick's disease (Pick), corticobasal degeneration (CBD), progressive supranuclear palsy (PSP), frontotemporal dementia (FTD). Neither the p25 immunoreactivity nor the p25/p35 ratio was elevated in the AD brains or in the other tauopathies (n = 34) compared with controls (n = 11). Although Abeta peptides have been suggested to activate calpain-mediated cleavage of p35 to p25 in cultured neurons, p25 levels in brains of TgCRND8 mice, which express high levels of brain Abeta peptides, were similar to those of non-Tg littermates. Our data suggest that high Abeta levels in brain do not activate p35 proteolysis, and p25 is unlikely to be a causative agent for NFT formation in AD or other tauopathies.

Mature glycosylation and trafficking of nicastrin modulate its binding to presenilins.

Nicastrin is an integral component of the high molecular weight presenilin complexes that control proteolytic processing of the amyloid precursor protein and Notch. We report here that nicastrin is most probably a type 1 transmembrane glycoprotein that is expressed at moderate levels in the brain and in cultured neurons. Immunofluorescence studies demonstrate that nicastrin is localized in the endoplasmic reticulum, Golgi, and a discrete population of vesicles. Glycosidase analyses reveal that endogenous nicastrin undergoes a conventional, trafficking-dependent maturation process. However, when highly expressed in transfected cells, there is a disproportionate accumulation of the endo-beta-N-acetylglucosaminidase H-sensitive, immature form, with no significant increase in the levels of the fully mature species. Immunoprecipitation revealed that presenilin-1 interacts preferentially with mature nicastrin, suggesting that correct trafficking and co-localization of the presenilin complex components are essential for activity. These findings demonstrate that trafficking and post-translational modifications of nicastrin are tightly regulated processes that accompany the assembly of the active presenilin complexes that execute gamma-secretase cleavage. These results also underscore the caveat that simple overexpression of nicastrin in transfected cells may result in the accumulation of large amounts of the immature protein, which is apparently unable to assemble into the active complexes capable of processing amyloid precursor protein and Notch.