👤 S L Hofmann

Infantile and juvenile neuronal ceroid lipofuscinosis (NCLs) are progressive neurodegenerative disorders of childhood with distinct ages of clinical onset, but with a similar pathological outcome. Infantile and juvenile NCL are inherited in an autosomal recessive manner due to mutations in the CLN1 and CLN3 genes, respectively. Recently developed Cln1- and Cln3-knockout mouse models share similarities in pathology with the respective human disease. Using oligonucleotide arrays we identified reproducible changes in gene expression in the brains of both 10-week-old Cln1- and Cln3-knockout mice as compared to wild-type controls, and confirmed changes in levels of several of the cognate proteins by immunoblotting. Despite the similarities in pathology, the two mutations affect the expression of different, non-overlapping sets of genes. The possible significance of these changes and the pathological mechanisms underlying NCL diseases are discussed. Show less

Spermatogenesis is the process of differentiation of diploid type A spermatogonia to haploid spermatozoa. Several subtypes of A spermatogonia have been characterized in the adult mouse testis. These include A-single (A(s)), A-paired (A(pr)), A-aligned (A(al)), and A1-A4. However, in the immature testis, very little information is available on subtypes and morphological features of type A spermatogonia. Six-day-old mouse testes, fixed either in Bouin solution or 5% glutaraldehyde, were embedded in paraffin and Epon, respectively. Thick sections (approximately 1 microm) of Epon-embedded tissue were stained with toluidine blue and revealed three subtypes of spermatogonia by light microscopy. The smallest spermatogonia (subtype I) appeared as single cells and exhibited a round or oval flattened nucleus with one or two prominent dense nucleoli and a characteristic unstained round and centrally located vacuole. These cells bound toluidine blue more avidly and appeared darker in comparison with the other cell types. Electron microscopy of thin sections (90 nm) revealed a finely granulated chromatin homogeneously distributed in the nucleus and sparse organelles in the cytoplasm. The second subtype of spermatogonia (subtype II) also displayed dark staining but was larger than subtype I; there was no central vacuole in the nucleus and heterochromatin clumps were observed. The largest subtype of spermatogonia (subtype III) showed large heterochromatin clumps and a pale staining nucleus. Intercellular bridges were noted between subtypes II and III. Based on the dye avidity, the three subtypes were classified as dark, transitional, and pale spermatogonia, respectively. Image analyses of 30 different cells of each subtype revealed a decline in gray-scale intensity from subtype I to III. Five-micrometer sections of paraffin-embedded tissue were immunoassayed with an antibody against the glial cell-derived neurotrophic factor family receptor alpha-1 (GFRalpha-1) receptor, a putative marker for undifferentiated spermatogonia, showing positive reaction only in germ cells. The pattern of GFRalpha-1 expression, coupled to the overall morphology of the cells, indicates that at this stage of development, mouse seminiferous tubules contain essentially A(s), A(pr), and possibly A(al) spermatogonia. Thus, the present study indicates the presence of subtypes of type A spermatogonia in the immature mouse testis similar to that described previously in adult monkey and man. Show less

Sertoli cells isolated from 6-day postpartum mouse testes were conditionally immortalized with the simian virus 40 large tumor antigen gene (SV40-LTAg) under the control of a promoter inducible with ponasterone A, an analog of ecdysone. This strategy produced 2 cell lines, which exhibited mixed phenotypes. We first tested the conditional expression of the LTAg gene in the presence or absence of ponasterone A. The results showed that both cell lines expressed LTAg when the inducer was present in the culture media. When ponasterone A was removed, the majority of the cells died. After 60 generations, however, the continued expression of LTAg in the absence of the hormone indicated that unknown changes may have occurred in the genome of the cells. One of the cell lines was further subcloned, resulting in 7 new lines exhibiting a morphology resembling that of Sertoli cells in tissue culture. Reverse transcriptase-polymerase chain reaction (RT-PCR) was performed on RNA collected from each cell line in order to determine which cells were phenotypically similar to Sertoli cells in vivo. All cell lines expressed the products of the Sertoli cell-specific genes stem cell factor (SCF) and sulfated glycoprotein-2 (SGP-2), in addition to alpha-inhibin, GATA-1, and steroidogenic factor-1. Further, the lines express growth and differentiation factors known to act upon germ cells in vivo and in vitro such as leukemia inhibitory factor (LIF), transforming growth factor beta (TGF-beta), and basic fibroblast growth factor (bFGF). Moreover, when used as feeder layers in cocultures, at least 2 of these lines are able to maintain the viability of type A spermatogonia for at least 7 days and to support the first steps of spermatogonial differentiation. Show less

Proximal spinal muscular atrophy (SMA) is a neuromuscular disorder caused by homozygous mutations of the SMN1 gene. SMN1 interacts with multiple proteins with functions in snRNP biogenesis, pre-mRNA splicing and presumably neural transport. SMN2, a nearly identical copy of SMN1, produces predominantly exon 7-skipped transcripts, whereas SMN1 mainly produces full-length transcripts. The SR-like splicing factor Htra2-beta1 facilitates correct splicing of SMN2 exon 7 through direct interaction with an exonic splicing enhancer within exon 7. In rare cases, siblings with identical 5q13-homologues and homozygous absence of SMN1 show variable phenotypes, suggesting that SMA is modified by other factors. By analysing nine SMA discordant families, we demonstrate that in all families unaffected siblings produce significantly higher amounts of SMN, Gemin2, Gemin3, ZPR1 and hnRNP-Q protein in lymphoblastoid cell lines, but not in primary fibroblasts, compared with their affected siblings. Protein p53, an additional SMN-interacting protein, is not subject to an SMN-dependent regulation. Surprisingly, Htra2-beta1 is also regulated by this tissue-specific mechanism. A similar regulation was found in all type I-III SMA patients, although at a different protein level than in discordant families. Thus, our data show that reduced SMN protein levels cause a reduction in the amount of its interacting proteins and of Htra2-beta1 in both discordant and non-discordant SMA families. We provide evidence that an intrinsic SMA modifying factor acts directly on the expression of SMN, thus influencing the SMA phenotype. Further insights into the molecular pathway and the identification of SMA modifying gene(s) may help to find additional targets for a therapy approach. Show less

Thirty-eight mutations and seven polymorphisms have recently been reported in the genes underlying the neuronal ceroid lipofuscinoses (NCLs) including 11 new mutations described here. A total of 114 mutations and 28 polymorphisms have now been described in the five human genes identified which cause NCL. Thirty-eight mutations are recorded for CLN1/PPT; 40 for CLN2/TTP-1, 31 for CLN3, four for CLN5, one for CLN8. Two mutations have been described in animal genes (cln8/mnd, CTSD). All mutations in NCL genes are contained in the NCL Mutation Database (http://www.ucl.ac.uk/NCL). Show less

In the mammalian testis, type A spermatogonia proliferate and differentiate into sperm under the tight control of both endocrine and paracrine factors. In order to study the complex process of spermatogenesis at the molecular level, an in vitro system must be devised in which type A spermatogonia can be cultured for a prolonged period of time. Therefore, cocultures including type A spermatogonia and Sertoli cells, which act as nurse cells to the developing germ cells, are desirable. We have developed a method for the specific isolation of type A spermatogonia using magnetic beads and antibodies that recognize the c-kit receptor or the homophilic adhesion molecule, Ep-CAM. Purified spermatogonia could survive for a period of 25 days when cocultivated on Sertoli cell monolayers. Moreover, we recently established Sertoli cell lines that produce growth factors that are essential for the maintenance of spermatogonia in a proliferative state. Some of these Sertoli cell lines are able to reorganize into tubular structures when cultivated on a layer of Matrigel as extracellular matrix. We show here that type A spermatogonia associate specifically with the Sertoli cell tubules, and are able to replicate their DNA in this environment. Thus, these in vitro culture systems could be used for the long-term culture of primary, nonimmortalized type A spermatogonia. Show less

The neuronal ceroid-lipofuscinoses (Batten disease) are a group of severe neurodegenerative disorders characterized clinically by visual loss, seizures and psychomotor degeneration, and pathologically by loss of neurons and lysosomal accumulation of autofluorescent storage material resembling ageing pigment. To date, eight genetic loci have been identified (CLN1-8). Four CLN genes have been isolated (CLN1, CLN2, CLN3 and CLN5) and their gene products have been characterized. CLN1 is a lysosomal palmitoyl-protein thioesterase (PPT) and CLN2 is a lysosomal pepstatin-insensitive peptidase. CLN3 and CLN5 are proteins with multiple membrane-spanning regions and have no homologies to other proteins that would suggest their function. The CLN3 protein is associated with lysosomal membranes and the intracellular location of the CLN5 protein is unknown. Therefore, there is ample evidence that the neuronal ceroid-lipofuscinoses represent a new class of lysosomal storage disorders. Show less

The infantile form of neuronal ceroid lipofuscinosis (NCL) has been well studied in Finland, where there is a high carrier frequency (1:70) for a single mutation in the causative gene, CLN1, or PPT. We have recently studied a group of 29 NCL subjects in the United States with palmitoyl-protein thioesterase (PPT) deficiency and described 19 different CLN1/PPT mutations in our population. In this report, we present a review of our previous findings, including a more detailed analysis of phenotype-genotype correlations, and present previously unpublished data concerning the clinical manifestations of the disorder in children of families with multiple affected members. Our studies indicate that about half of PPT-deficient patients in the United States are very similar to Finnish infants with INCL, but that a different mutation (R151X) accounts for 40% of U.S. alleles. The Finnish mutation (R122W) is rare in the United States. The other half of U.S. PPT-deficient patients develop symptoms after the age of 2 years, much later than Finnish patients. One common mutation (the "Scottish" allele, T75P) accounts for 13% of alleles and results in a juvenile-onset phenotype that is clinically indistinguishable from JNCL with CLN3 mutations. Other rare mutations were also associated with JNCL phenotypes, such as D79G and G250V. A preliminary expression study of two of these mutant enzymes supports the conclusion that juvenile-onset NCL (JNCL with GROD) is caused by missense mutations in the PPT gene that result in mutated enzymes with residual PPT enzyme activity. Show less