Friedreich's ataxia (FRDA) is an autosomal recessive, degenerative disease that involves the central and peripheral nervous systems and the heart. A gene, X25, was identified in the critical region for the FRDA locus on chromosome 9q13. This gene encodes a 210-amino acid protein, frataxin, that has homologs in distant species such as Caenorhabditis elegans and yeast. A few FRDA patients were found to have point mutations in X25, but the majority were homozygous for an unstable GAA trinucleotide expansion in the first X25 intron.

Limb-girdle muscular dystrophy type 2D (LGMD 2D) is an autosomal recessive disorder caused by mutations in the α-sarcoglycan gene. To determine how α-sarcoglycan deficiency leads to muscle fiber degeneration, we generated and analyzed α-sarcoglycan– deficient mice. Sgca-null mice developed progressive muscular dystrophy and, in contrast to other animal models for muscular dystrophy, showed ongoing muscle necrosis with age, a hallmark of the human disease. Sgca-null mice also revealed loss of sarcolemmal integrity, elevated serum levels of muscle enzymes, increased muscle masses, and changes in the generation of absolute force. Molecular analysis of Sgca-null mice demonstrated that the absence of α-sarcoglycan resulted in the complete loss of the sarcoglycan complex, sarcospan, and a disruption of α-dystroglycan association with membranes. In contrast, no change in the expression of ε-sarcoglycan (α-sarcoglycan homologue) was observed. Recombinant α-sarcoglycan adenovirus injection into Sgca-deficient muscles restored the sarcoglycan complex and sarcospan to the membrane. We propose that the sarcoglycan–sarcospan complex is requisite for stable association of α-dystroglycan with the sarcolemma. The Sgca-deficient mice will be a valuable model for elucidating the pathogenesis of sarcoglycan deficient limb-girdle muscular dystrophies and for the development of therapeutic strategies for this disease.

beta-Sarcoglycan, a 43 kDa dystrophin-associated glycoprotein, is an integral component of the dystrophin-glycoprotein complex. We have cloned human beta-sarcoglycan cDNA and mapped the beta-sarcoglycan gene to chromosome 4q12. Pericentromeric markers and an intragenic polymorphic CA repeat cosegregated perfectly with autosomal recessive limb-girdle muscular dystrophy in several Amish families. A Thr-to-Arg missense mutation was identified within the beta-sarcoglycan gene that leads to a dramatically reduced expression of beta-sarcoglycan in the sarcolemma and a concomitant loss of adhalin and 35 DAG, which may represent a disruption of a functional subcomplex within the dystrophin-glycoprotein complex. Thus, the beta-sarcoglycan gene is the fifth locus identified (LGMD2E) that is involved in autosomal recessive limb-girdle muscular dystrophy.

Elevated levels of plasma low density lipoprotein (LDL)-cholesterol, leading to familial hypercholesterolemia, are enhanced by mutations in at least three major genes, the LDL receptor (LDLR), its ligand apolipoprotein B, and the proprotein convertase PCSK9. Single point mutations in PCSK9 are associated with either hyperor hypocholesterolemia. Accordingly, PCSK9 is an attractive target for treatment of dyslipidemia. PCSK9 binds the epidermal growth factor domain A (EGF-A) of the LDLR and directs it to endosomes/ lysosomes for destruction. Although the mechanism by which PCSK9 regulates LDLR degradation is not fully resolved, it seems to involve both intracellular and extracellular pathways. Here, we show that clathrin light chain small interfering RNAs that block intracellular trafficking from the trans-Golgi network to lysosomes rapidly increased LDLR levels within HepG2 cells in a PCSK9-dependent fashion without affecting the ability of exogenous PCSK9 to enhance LDLR degradation. In contrast, blocking the extracellular LDLR endocytosis/degradation pathway by a 4-, 6-, or 24-h incubation of cells with Dynasore or an EGF-AB peptide or by knockdown of endogenous autosomal recessive hypercholesterolemia did not significantly affect LDLR levels. The present data from HepG2 cells and mouse primary hepatocytes favor a model whereby depending on the dose and/or incubation period, endogenous PCSK9 enhances the degradation of the LDLR both extraand intracellularly. Therefore, targeting either pathway, or both, would be an effective method to reduce PCSK9 activity in the treatment of hypercholesterolemia and coronary heart disease.

High density lipoprotein (HDL) cholesterol levels are associated with decreased risk of cardiovascular disease, but not all HDL are functionally equivalent. A primary determinant of HDL functional status is the conformational adaptability of its main protein component, apoA-I, an exchangeable apolipoprotein. Chemical modification of apoA-I, as may occur under conditions of inflammation or diabetes, can severely impair HDL function and is associated with the presence of cardiovascular disease. Chemical modification of apoA-I also impairs its ability to exchange on and off HDL, a critical process in reverse cholesterol transport. In this study, we developed a method using electron paramagnetic resonance spectroscopy (EPR) to quantify HDL-apoA-I exchange. Using this approach, we measured the degree of HDL-apoA-I exchange for HDL isolated from rabbits fed a high fat, high cholesterol diet, as well as human subjects with acute coronary syndrome and metabolic syndrome. We observed that HDL-apoA-I exchange was markedly reduced when atherosclerosis was present, or when the subject carries at least one risk factor of cardiovascular disease. These results show that HDL-apoA-I exchange is a clinically relevant measure of HDL function pertinent to cardiovascular disease.

Four types of limb-girdle muscular dystrophy (LGMD) are known to be caused by mutations in distinct sarcoglycan genes. The BIO 14.6 hamster is a model for sarcoglycan-deficient LGMD with a deletion in the delta-sarcoglycan (delta-SG) gene. We investigated the function of the sarcoglycan complex and the feasibility of sarcoglycan gene transfer for LGMD using a recombinant delta-SG adenovirus in the BIO 14.6 hamster. We demonstrate extensive long-term expression of delta-sarcoglycan and rescue of the entire sarcoglycan complex, as well as restored stable association of alpha-dystroglycan with the sarcolemma. Importantly, muscle fibers expressing delta-sarcoglycan lack morphological markers of muscular dystrophy and exhibit restored plasma membrane integrity. In summary, the sarcoglycan complex is requisite for the maintenance of sarcolemmal integrity, and primary mutations in individual sarcoglycan components can be corrected in vivo.

The BIO14.6 hamster is an extensively used animal model of autosomal recessive cardiomyopathy and muscular dystrophy. Recently, a large deletion in the 5' end of the delta-sarcoglycan gene was found to be the primary genetic defect in the hamster. In the present investigation, we studied the effects of the delta-sarcoglycan deletion on transcription, expression, and function of the dystrophin-glycoprotein complex in skeletal and cardiac muscle. We demonstrated that in striated muscle the genetic defect leads to the complete deficiency of delta-sarcoglycan and a concomitant loss of alpha-, beta-, and gamma-sarcoglycan. In addition, absence of the sarcoglycan complex reduced the expression of alpha-dystroglycan in striated muscle fibers. These findings indicated that the primary defect in the BIO14.6 hamster leads to the dissociation of the dystroglycan complex from the sarcoglycan complex and disrupted anchorage of alpha-dystroglycan to the cell surface. Using intravenous injection of Evans blue dye as an in vivo tracer assay, we demonstrated that perturbation of the dystrophin-glycoprotein complex caused extensive fiber damage in skeletal and cardiac muscle of the BIO14.6 hamster. Based on our results, we propose that loss of delta-sarcoglycan results in the impairment of sarcolemmal integrity, finally leading to muscular dystrophy and cardiomyopathy.

PCSK9, a target for the treatment of dyslipidemia, enhances the degradation of the LDL receptor (LDLR) in endosomes/ lysosomes, up-regulating LDL-cholesterol levels. Whereas the targeting and degradation of the PCSK9-LDLR complex are under scrutiny, the roles of the N-and C-terminal domains of PCSK9 are unknown. Although autocatalytic zymogen processing of PCSK9 occurs at Gln 152 2, here we show that human PCSK9 can be further cleaved in its N-terminal prosegment at Arg 46 2 by an endogenous enzyme of insect High Five cells and by a cellular mammalian protease, yielding an ϳ4-fold enhanced activity. Removal of the prosegment acidic stretch resulted in ϳ3-fold higher binding to LDLR in vitro, in >4-fold increased activity on cellular LDLR, and faster cellular internalization in endosome/lysosome-like compartments. Finally, swapping the acidic stretch of PCSK9 with a similar one found in the glycosylphosphatidylinositol-anchored heparin-binding protein 1 does not impair PCSK9 autoprocessing, secretion, or activity and confirmed that the acidic stretch acts as an inhibitor of PCSK9 function. We also show that upon short exposure to pH values 6.5 to 5.5, an ϳ2.5-fold increase in PCSK9 activity on total and cell surface LDLR occurs, and PCSK9 undergoes a second cleavage at Arg 248 , generating a two-chain PCSK9-⌬N 248 . At pH values below 5.5, PCSK9 dissociates from its prosegment and loses its activity. This pH-dependent activation of PCSK9 represents a novel pathway to further activate PCSK9 in acidic endosomes. These data enhance our understanding of the functional role of the acidic prosegment and on the effect of pH in the regulation of PCSK9 activity.Complications resulting from cardiovascular disorders are the main cause of death worldwide. High levels of circulating low density lipoprotein-cholesterol represent a major risk factor that leads to coronary heart disease associated with increased death and morbidity worldwide (1). LDL is constantly cleared by internalization into cells by the LDL receptor (LDLR), 4 which binds and internalizes LDL via its unique apolipoprotein B (apoB) protein. Mutations in LDLR or APOB genes are major causes for the frequent autosomal dominant genetic disorder known as familial hypercholesterolemia (2, 3). More recently, the PCSK9 gene (4), which is highly expressed in liver and small intestine (5), was identified as the third locus associated with familial hypercholesterolemia (6). PCSK9 binds the LDLR and triggers its intracellular degradation in acidic endosomes/lysosomes, resulting in increased circulating LDL-cholesterol (7-10).Following its autocatalytic cleavage, PCSK9 is secreted as a stable noncovalent complex with its 122-amino acid (aa 31-152) N-terminal prosegment (5, 7). This cleavage results in a conformational change (11), which favors the binding of PCSK9 to the EGFA domain of the LDLR (12), with much increased affinity at acidic pH values (11).Overexpression studies in liver suggested that PCSK9 targets the LDLR (9, 13, 14) toward degradation in late endosomes/l...