Lafora progressive myoclonus epilepsy (Lafora disease; LD) is a fatal autosomal recessive neurodegenerative disorder caused by loss-of-function mutations in either the EPM2A gene, encoding the dual ...specificity phosphatase laforin, or the EPM2B gene, encoding the E3-ubiquitin ligase malin. Previously, we and others have shown that both proteins form a functional complex that regulates glycogen synthesis by a novel mechanism involving ubiquitination and proteasomal degradation of at least two proteins, glycogen synthase and R5/PTG. Since laforin and malin localized at the endoplasmic reticulum (ER) and their regulatory role likely extend to other proteins unrelated to glycogen metabolism, we postulated that their absence may also affect the ER-unfolded protein response pathway.
Here, we demonstrate that siRNA silencing of laforin in Hek293 and SH-SY5Y cells increases their sensitivity to agents triggering ER-stress, which correlates with impairment of the ubiquitin-proteasomal pathway and increased apoptosis. Consistent with these findings, analysis of tissue samples from a LD patient lacking laforin, and from a laforin knockout (Epm2a-/-) mouse model of LD, demonstrates constitutive high expression levels of ER-stress markers BIP/Grp78, CHOP and PDI, among others.
We demonstrate that, in addition to regulating glycogen synthesis, laforin and malin play a role protecting cells from ER-stress, likely contributing to the elimination of unfolded proteins. These data suggest that proteasomal dysfunction and ER-stress play an important role in the pathogenesis of LD, which may offer novel therapeutic approaches for this fatal neurodegenerative disorder.
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DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
The role of complement in C3 glomerulopathy Zipfel, Peter F.; Skerka, Christine; Chen, Qian ...
Molecular immunology,
September 2015, 2015-Sep, 2015-09-00, 20150901, Letnik:
67, Številka:
1
Journal Article
Recenzirano
•C3 glomerulopathy is a complement mediated kidney disease.•It is caused by defective alternative pathway regulation frequently on the level of the C3 convertase.•C3 glomerulopathy has autoimmune and ...genetic causes.•CFHR genes are associated with the disorder.
C3 glomerulopathy describes a spectrum of disorders with glomerular pathology associated with C3 cleavage product deposition and with defective complement action and regulation (Fakhouri et al., 2010; Sethi et al., 2012b). Kidney biopsies from these patients show glomerular accumulation or deposition of C3 cleavage fragments, but no or minor deposition of immunoglobulins (Appel et al., 2005; D’Agati and Bomback, 2012; Servais et al., 2007; Sethi and Fervenza, 2011). At present the current situation asks for a better definition of the underlining disease mechanisms, for precise biomarkers, and for a treatment for this disease. The complement system is a self activating and propelling enzymatic cascade type system in which inactive, soluble plasma components are activated spontaneously and lead into an amplification loop (Zipfel and Skerka, 2009). Activation of the alternative pathway is spontaneous, occurs by default, and cascade progression leads to amplification by complement activators. The system however is self-controlled by multiple regulators and inhibitors, like Factor H that control cascade progression in fluid phase and on surfaces. The activated complement system generates a series of potent effector components and activation products, which damage foreign-, as well as modified self cells, recruit innate immune cells to the site of action, coordinate inflammation and the response of the adaptive immune system in form of B cells and T lymphocytes (Kohl, 2006; Medzhitov and Janeway, 2002; Ogden and Elkon, 2006; Carroll, 2004; Kemper and Atkinson, 2007; Morgan, 1999; Muller-Eberhard, 1986; Ricklin et al., 2010). Complement controls homeostasis and multiple reactions in the vertebrate organism including defense against microbial infections (Diaz-Guillen et al., 1999; Mastellos and Lambris, 2002; Nordahl et al., 2004; Ricklin et al., 2010). In consequence defective control of the spontaneous self amplifying cascade or regulation is associated with numerous human disorders (Ricklin and Lambris, 2007; Skerka and Zipfel, 2008; Zipfel et al., 2006). Understanding the exact action and regulation of this sophisticated homeotic cascade system is relevant to understand disease pathology of various complement associated human disorders. Furthermore this knowledge is relevant for a better diagnosis and appropriate therapy. At present diagnosis of C3 glomerulopathy is primarily based on the kidney biopsy, and histological, immmunohistological and electron microscopical evaluation (D’Agati and Bomback, 2012; Fakhouri et al., 2010; Medjeral-Thomas et al., 2014a,b; Sethi et al., 2012b). The challenge is to define the actual cause of the diverse glomerular changes or damages, to define how C3 deposition results in the reported glomerular changes, the location of the cell damage and the formation of deposits.
Dysregulation of the complement alternative pathway (AP) is a major pathogenic mechanism in atypical hemolytic-uremic syndrome (aHUS). Genetic or acquired defects in factor H (FH), the main AP ...regulator, are major aHUS drivers that associate with a poor prognosis. FH activity has been suggested to be downregulated by homologous FH-related (FHR) proteins, including FHR-3 and FHR-1. Hence, their relative levels in plasma could be disease-relevant. The genes coding for FH, FHR-3, and FHR-1 (
, and
, respectively) are polymorphic and located adjacent to each other on human chromosome 1q31.3. We have previously shown that haplotype
associates with aHUS and reduced FH levels. In this study, we used a specific enzyme-linked immunosorbent assay to quantify FHR-3 in plasma samples from controls and patients with aHUS genotyped for the three known
alleles (
, and
). In the 218 patients carrying at least one copy of
, significant differences between
genotype groups were found, with
patients having the lowest FHR-3 concentration (0.684-1.032 µg/mL),
and
patients presenting intermediate levels (1.437-2.201 µg/mL), and
and
patients showing the highest concentration (2.330-4.056 µg/mL) (
< 0.001). These data indicate that
is a low-expression allele, whereas
, associated with increased risk of aHUS, is a high-expression allele. Our study reveals that the aHUS-risk haplotype
generates twofold more FHR-3 than the non-risk
haplotype. In addition, FHR-3 levels were higher in patients with aHUS than in control individuals with the same
genotype. These data suggest that increased plasma levels of FHR-3, altering the balance between FH and FHR-3, likely impact the FH regulatory functions and contribute to the development of aHUS.
Genetics of atypical hemolytic uremic syndrome (aHUS) Rodríguez de Córdoba, Santiago; Hidalgo, Marta Subías; Pinto, Sheila ...
Seminars in thrombosis and hemostasis,
06/2014, Letnik:
40, Številka:
4
Journal Article
Recenzirano
Odprti dostop
Hemolytic uremic syndrome (HUS) is a rare, life-threatening disease characterized by thrombocytopenia, microangiopathic hemolytic anemia, and acute renal failure. The atypical form of HUS (aHUS), ...representing 5 to 10% of cases, lacks the association with infection by Shiga toxin producing Escherichia coli strains that characterizes the commonest clinical presentation of HUS. In the majority of aHUS cases, the disease results from the complement-mediated damage to the microvascular endothelium because of inherited defects in complement genes or autoantibodies against complement regulatory proteins. Incomplete penetrance of aHUS in carriers of mutations is common to all aHUS-associated complement genes and it is now established that the overall genetic predisposition to aHUS of an individual results from the combination of different inherited factors. Moreover, the patient's genotype influences the clinical evolution, the response to plasma therapies, and the recurrence after transplantation. Here, we describe the genetic component of aHUS, the lessons that we have learned from the functional characterization of the aHUS-associated mutations, and the benefits of a comprehensive genetic analysis of the patients.
The efficiency of the complement system as an innate immune defense mechanism depends on a fine control that restricts its action to pathogens and prevents non-specific damage to host tissues. ...Genetic and functional analyses have shown that this critical control of complement activation may be impaired in atypical hemolytic uremic syndrome (aHUS) patients. Mutations in HF1, MCP or FI have been found in aHUS patients, but incomplete penetrance of the disease in individuals carrying these mutations is relatively frequent and no genetic defect has yet been found in a majority of aHUS patients. We report here the identification of a specific SNP haplotype block, spanning the MCP gene in the regulators of complement activation gene cluster, which is over-represented in aHUS patients and strongly associates with the severity of the disease. Linkage disequilibrium analyses suggest that this SNP haplotype also includes the CR1, DAF and C4BP genes. Initial studies identified two SNPs in the haplotype that influence the transcription activity of the MCP promoter in transient transfection experiments. Notably, the SNP haplotype block was found to be particularly frequent among patients who carry mutations in HF1, MCP or FI. These findings and the identification of aHUS patients carrying mutations in two complement regulatory genes provide an important insight into the etiology of aHUS. Together, they suggest that complement regulatory molecules act as a protein network and that multiple hits, involving plasma- and membrane-associated complement regulatory proteins, are necessary to impair protection to host tissues and to confer significant predisposition to aHUS.
Pregnancy-associated atypical hemolytic uremic syndrome (aHUS) refers to the thrombotic microangiopathy resulting from uncontrolled complement activation during pregnancy or the postpartum period. ...Pregnancy-associated aHUS is a devastating disease for which there is a limited clinical understanding and treatment experience. Here we report a retrospective study to analyze the clinical and prognostic data of 22 cases of pregnancy-associated aHUS from the Spanish aHUS Registry under different treatments. Sixteen patients presented during the first pregnancy and as many as nine patients required hemodialysis at diagnosis. Identification of inherited complement abnormalities explained nine of the 22 cases, with CFH mutations and CFH to CFHR1 gene conversion events being the most prevalent genetic alterations associated with this disorder (66%). In thirteen of the cases, pregnancy complications were sufficient to trigger a thrombotic microangiopathy in the absence of genetic or acquired complement alterations. The postpartum period was the time with highest risk to develop the disease and the group shows an association of cesarean section with pregnancy-associated aHUS. Seventeen patients underwent plasma treatments with a positive renal response in only three cases. In contrast, ten patients received eculizumab with an excellent renal response in all, independent of carrying or not inherited complement abnormalities. Although the cohort is relatively small, the data suggest that pregnancy-associated aHUS is not different from other types of aHUS and suggest the efficacy of eculizumab treatment over plasma therapies. This study may be useful to improve prognosis in this group of aHUS patients.
Lafora disease (LD) is a fatal progressive epilepsy essentially caused by loss‐of‐function mutations in the glycogen phosphatase laforin or the ubiquitin E3 ligase malin. Glycogen in LD is ...hyperphosphorylated and poorly hydrosoluble. It precipitates and accumulates into neurotoxic Lafora bodies (LBs). The leading LD hypothesis that hyperphosphorylation causes the insolubility was recently challenged by the observation that phosphatase‐inactive laforin rescues the laforin‐deficient LD mouse model, apparently through correction of a general autophagy impairment. We were for the first time able to quantify brain glycogen phosphate. We also measured glycogen content and chain lengths, LBs, and autophagy markers in several laforin‐ or malin‐deficient mouse lines expressing phosphatase‐inactive laforin. We find that: (i) in laforin‐deficient mice, phosphatase‐inactive laforin corrects glycogen chain lengths, and not hyperphosphorylation, which leads to correction of glycogen amounts and prevention of LBs; (ii) in malin‐deficient mice, phosphatase‐inactive laforin confers no correction; (iii) general impairment of autophagy is not necessary in LD. We conclude that laforin's principle function is to control glycogen chain lengths, in a malin‐dependent fashion, and that loss of this control underlies LD.
Synopsis
Abnormal glycogen chain length distribution strictly correlates with glycogen accumulation and Lafora body (LB) formation in Lafora disease (LD). Against current hypotheses, neither glycogen hyperphosphorylation nor deficient general autophagy are prerequisites of the disease.
By methodological advances chain length distribution (CLD) and phosphorylation of glycogen were determined in brain tissue confirming that overexpressed wild‐type laforin corrects the molecular phenotype in an LD mouse model.
Phosphatase‐inactive laforin does not correct glycogen hyperphosphorylation in malin‐ and laforin‐deficient mice and prevents abnormal CLD and accumulation of glycogen as well as LB formation.
Prevention of abnormal chain length distribution and accumulation of brain glycogen as well as LB formation by phosphatase‐inactive laforin is malin‐dependent as no rescue occurs in malin‐deficient mice.
General impairment of autophagy is not necessary in LD as markers of autophagic flux are not changed in any of our LD mouse models.
Laforin controls glycogen chain length distribution in a malin‐dependent fashion, and lack of this control leads to abnormal glycogen structure, glycogen accumulation, LB formation, hence to LD.
Abnormal glycogen chain length distribution strictly correlates with glycogen accumulation and Lafora body (LB) formation in Lafora disease (LD). Against current hypotheses, neither glycogen hyperphosphorylation nor deficient general autophagy are prerequisites of the disease.
The complement system is a key component of the host immune response for the recognition and clearance of Streptococcus pneumoniae. In this study, we demonstrate that the amidase LytA, the main ...pneumococcal autolysin, inhibits complement-mediated immunity independently of effects on pneumolysin by a complex process of impaired complement activation, increased binding of complement regulators, and direct degradation of complement C3. The use of human sera depleted of either C1q or factor B confirmed that LytA prevented activation of both the classical and alternative pathways, whereas pneumolysin inhibited only the classical pathway. LytA prevented binding of C1q and the acute-phase protein C-reactive protein to S. pneumoniae, thereby reducing activation of the classical pathway on the bacterial surface. In addition, LytA increased recruitment of the complement downregulators C4BP and factor H to the pneumococcal cell wall and directly cleaved C3b and iC3b to generate degradation products. As a consequence, C3b deposition and phagocytosis increased in the absence of LytA and were markedly enhanced for the lytA ply double mutant, confirming that a combination of LytA and Ply is essential for the establishment of pneumococcal pneumonia and sepsis in a murine model of infection. These data demonstrate that LytA has pleiotropic effects on complement activation, a finding which, in combination with the effects of pneumolysin on complement to assist with pneumococcal complement evasion, confirms a major role of both proteins for the full virulence of the microorganism during septicemia.
The factor H–related protein family (CFHR) is a group of minor plasma proteins genetically and structurally related to complement factor H (fH). Notably, deficiency of CFHR1/CFHR3 associates with ...protection against age-related macular degeneration and with the presence of anti-fH autoantibodies in atypical hemolytic uremic syndrome (aHUS). We have developed a proteomics strategy to analyze the CFHR proteins in plasma samples from controls, patients with aHUS, and patients with type II membranoproliferative glomerulonephritis. Here, we report on the identification of persons carrying novel deficiencies of CFHR1, CFHR3, and CFHR1/CFHR4A, resulting from point mutations in CFHR1 and CFHR3 or from a rearrangement involving CFHR1 and CFHR4. Remarkably, patients with aHUS lacking CFHR1, but not those lacking CFHR3, present anti-fH autoantibodies, suggesting that generation of these antibodies is specifically related to CFHR1 deficiency. We also report the characterization of a novel CFHR1 polymorphism, resulting from a gene conversion event between CFH and CFHR1, which strongly associates with aHUS. The risk allotype CFHR1*B, with greater sequence similarity to fH, may compete with fH, decreasing protection of cellular surfaces against complement damage. In summary, our comprehensive analyses of the CFHR proteins have improved our understanding of these proteins and provided further insights into aHUS pathogenesis.
Atypical hemolytic uremic syndrome (aHUS), a rare form of thrombotic microangiopathy caused by complement pathogenic variants, mainly affects the kidney microvasculature. A retrospective genetic ...analysis in our aHUS cohort (
=513) using multiple ligation probe amplification uncovered nine unrelated patients carrying a genetic abnormality in the complement factor H related 1 gene (
) that originates by recurrent gene conversion events between the
and
genes. The novel
mutants encode an FHR-1 protein with two amino acid substitutions, L290S and A296V, converting the FHR-1 C terminus into that of factor H (FH). Next-generation massive-parallel DNA sequencing (NGS) analysis did not detect these genetic abnormalities. In addition to the
mutant, six patients carried the previously uncharacterized
variant. In functional analyses, the mutant FHR-1 protein strongly competed the binding of FH to cell surfaces, impairing complement regulation, whereas the
polymorphism lacked functional consequences. Carriers of the
mutation presented with severe aHUS during adulthood; 57% of affected women in this cohort presented during the postpartum period. Analyses in patients and unaffected carriers showed that FH plasma levels determined by the nonmutated chromosome modulate disease penetrance. Crucially, in the activated endothelial (HMEC-1) cell assay, reduced FH plasma levels produced by the nonmutated chromosome correlated inversely with impairment of complement regulation, measured as C5b-9 deposition. Our data advance understanding of the genetic complexities underlying aHUS, illustrate the importance of performing functional analysis, and support the use of complementary assays to disclose genetic abnormalities not revealed by current NGS analysis.
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