The expression of autophagy-related proteins within the corpus luteum lifespan in pigs.

Autophagy is a cellular process that involves the degradation of intracellular components. Recent studies suggested a role for autophagy in corpus luteum (CL) regression; however, a complete understanding of its contribution to CL function remains unclear. The present research using porcine CLs obtained from gilts at the early (CL1, n = 5), middle (CL2, n = 5), and late (CL3, n = 5) luteal phase of the estrous cycle aimed to assess the incidence of autophagy during CL development. The stages of collected CLs were verified through morphological analysis and intraluteal progesterone concentration. The presence of autophagosomes was assessed using transmission electron microscopy, and the expression of autophagic markers was examined at mRNA (BECN1 and Lamp1) and protein (Beclin 1, LC3-II, and Lamp 1) levels. Lamp 1 immunolocalization was also performed in luteal tissue. Double-membrane autophagosomes and autophagy-related proteins were found in all examined CLs. Interestingly, there was a greater expression of Beclin 1 (P = 0.005 and P = 0.025) and Lamp 1 (P = 0.009 and P = 0.032) protein in CL3 as compared with CL1 and CL2. In addition, the presence of autolysosomes in CL3 indicated advanced autophagy at that developmental stage. Overall, the occurrence of autophagy throughout CL development and regression suggests it has a role in the regulation of CL lifespan in pigs. In the early and mature CL, autophagy is proposed to promote luteal formation and function, whereas in the late CL, it may participate in luteal regression.

Spinocerebellar ataxia type 7 associated with pigmentary retinal dystrophy.

Spinocerebellar ataxia type 7 (SCA7) is an autosomal-dominant, late-onset, slowly progressive disorder, primarily characterized by gradual loss of motor coordination, resulting from dysfunction and degeneration of the cerebellum and its connecting pathways. The disease is caused by expansion of a CAG trinucleotide repeat within the SCA7 gene, which encodes a polyglutamine tract within a novel protein, termed ataxin-7. The expansion of polyglutamine-encoding CAG repeats in dissimilar genes underlies eight neurodegenerative conditions besides SCA7, including a number of dominant ataxias related to SCA7. Although elongated polyglutamine itself can initiate neuronal dysfunction and death, its toxicity is modulated by the context of the disease proteins, as evidenced by the differing clinical and pathological presentation of the various disorders. In this respect, it is exciting that SCA7 constitutes the only polyglutamine disorder, in which the photoreceptors of the retina are also severely affected, leading to retinal degeneration and blindness. Since the discovery of the SCA7 mutation, numerous studies attempted to pinpoint the molecular mechanisms underlying the unique features of SCA7, particularly the retinal involvement. Here we summarize the clinical, pathological, and genetic aspects of SCA7, and review the current understanding of the pathogenesis of this disorder.

Association of ataxin-7 with the proteasome subunit S4 of the 19S regulatory complex.

Spinocerebellar ataxia type 7 (SCA7) is a neurodegenerative disorder characterized by ataxia and selective neuronal cell loss caused by the expansion of a translated CAG repeat encoding a polyglutamine tract in ataxin-7, the SCA7 gene product. To gain insight into ataxin-7 function and to decipher the molecular mechanisms of neurodegeneration in SCA7, a two-hybrid assay was performed to identify ataxin-7 interacting proteins. Herein, we show that ataxin-7 interacts with the ATPase subunit S4 of the proteasomal 19S regulatory complex. The ataxin-7/S4 association is modulated by the length of the polyglutamine tract whereby S4 shows a stronger association with the wild-type allele of ataxin-7. We demonstrate that endogenous ataxin-7 localizes to discrete nuclear foci that also contain additional components of the proteasomal complex. Immunohistochemical analyses suggest alterations either of the distribution or the levels of S4 immunoreactivity in neurons that degenerate in SCA7 brains. Immunoblot analyses demonstrate reduced levels of S4 in SCA7 cerebella without evident alterations in the levels of other proteasome subunits. These results suggest a role for S4 and ubiquitin-mediated proteasomal proteolysis in the molecular pathogenesis of SCA7.

Genomic organisation of the spinocerebellar ataxia type 7 (SCA7) gene responsible for autosomal dominant cerebellar ataxia with retinal degeneration.

Autosomal dominant cerebellar ataxia with retinal degeneration (ADCA type II) is a progressive neurodegenerative disorder caused by a CAG expansion in the spinocerebellar ataxia 7 (SCA7) gene. Here, we describe the genomic organisation of the human SCA7 gene. The exon-intron boundaries were identified by sequencing plasmid subclones of a P1 artificial chromosome (PAC) clone containing the entire SCA7 gene. We found 13 exons, ranging in size from 69 to 979 bp, with all exon-intron boundaries following the GT-AG rule. The ATG initiation codon at position 554 of the cDNA occurs in exon 3 at position 12 and the coding region extends to the first five codons of exon 13, with the CAG repeat being located in exon 3 starting at codon 30. The intron sizes were determined by long-distance polymerase chain reaction with primers from neighbouring exons and by restriction mapping of the SCA7 PAC clone. The introns varied in size from 233 bp to about 40 kb, resulting in an overall size estimate for the SCA7 gene of 140 kb. Sequence analysis of intron 7 (491 bp) revealed a polymorphic GT/AC repeat, a useful intragenic marker for SCA7 in segregation studies.

Isolation of CAG/CTG repeats from within the chromosome 2p21-p24 locus for autosomal dominant spastic paraplegia (SPG4) by YAC fragmentation.

Pure autosomal dominant spastic paraplegia (SPG) is a genetically heterogeneous neurodegenerative disorder of the central nervous system clinically characterized by progressive spasticity mainly affecting the lower limbs. Three distinct loci have been mapped to chromosomes 14q (SPG3), 2p (SPG4) and 15q (SPG6). In particular, SPG4 families show striking intrafamilial variability suggestive of anticipation and evidence has been provided that CAG/CTG repeat expansions may be involved. To isolate CAG/CTG repeat containing sequences from within the SPG4 candidate region, a novel approach was developed. Fragmentation vectors were assembled allowing direct fragmentation of yeast artificial chromosomes (YACs) with a short (> or = 21 bp) CAG/CTG sequence as the target site for homologous recombination. We used the CAG/CTG YAC fragmentation vectors to isolate CAG/CTG containing sequences from four YACs spanning the SPG4 candidate region between D2S400 and D2S367. A total of four CAG/CTG containing sequences were isolated of which three were novel. However, none of the four CAG/CTG repeats showed expanded alleles in two Belgian SPG4 families. In addition, we showed that the CAG/CTG alleles detected by the repeat expansion detection (RED) method could be fully explained by two polymorphic nonpathogenic CAG/CTG repeats on chromosomes 17 and 18, respectively. Also, the RED expansions in six SPG families could not be explained by amplification of the CAG/CTG repeats at the SPG4 locus. Together, our data do not support the hypothesis of a CAG/CTG repeat expansion as the molecular mechanism underlying SPG4 pathology.

Molecular genetic analysis of autosomal dominant cerebellar ataxia with retinal degeneration (ADCA type II) caused by CAG triplet repeat expansion.

Autosomal dominant cerebellar ataxia with retinal degeneration (ADCAII) was previously mapped by linkage analysis studies to chromosome 3p12-p21.1 (SCA7). Positional cloning efforts have recently identified a novel gene, SCA7 , containing a translated CAG repeat, expanded in SCA7 patients. We cloned the SCA7 gene from a yeast artificial chromosome (YAC) clone contig spanning the SCA7 candidate region. Using a combination of genomic sequencing and cosmid-based exon trapping, two expressed sequence tags were identified. Sequencing of the corresponding cDNA clones and RT-PCR analysis identified the full-length SCA7 cDNA. Together, our sequence data defined the intron/exon boundaries of the first two coding exons of the SCA7 gene, with the first exon containing the expanded CAG repeat. Further, sequence comparison with the published SCA7 cDNA identified one additional putative exon in the 5'-UTR region of the SCA7 gene. The SCA7 gene was mapped on the YAC contig in the 2.5 cM interval between D3S1600 and D3S1287. In one extended Belgian SCA7 pedigree the expanded alleles ranged from 38 to at least 55 repeats with allele lengths being inversely correlated with onset age of ADCAII symptoms. The SCA7 repeats increased in length in successive generations. Normal alleles had from four to 18 repeats, with 10 repeats being the most common allele.

Pure familial spastic paraplegia: clinical and genetic analysis of nine Belgian pedigrees.

We ascertained 9 multigeneration Belgian families with pure dominant spastic paraplegia (SPG) for clinical and genetic studies. Linkage was examined using simple tandem repeat (STR) markers located near the 5 loci for familial SPG on chromosomes Xq28 (SPG1), Xq21.3-q22 (SPG2), 2p21-p24 (SPG4), 14q12-q23 (SPG3) and 15q11.1 (SPG6). Positive linkage results were obtained only for markers at the SPG4 locus mapping the SPG4 gene between D2S400 and D2S367, a region of 4 cM. In order to facilitate the positional cloning of the SPG4 gene, we constructed a contiguous YAC map covering the SPG4 candidate region. Our physical mapping data indicate that the SPG4 gene resides within maximal 5 Mb.