Apoptotic cell death in mouse models of GM2 gangliosidosis and observations on human Tay-Sachs and Sandhoff diseases

Tay-Sachs and Sandhoff diseases are autosomal recessive neurodegenerative diseases resulting from the inability to catabolize GM2 ganglioside by beta-hexosaminidase A (Hex A) due to mutations of the alpha subunit (Tay-Sachs disease) or beta subunit (Sandhoff disease) of Hex A. Hex B (beta beta homodimer) is also defective in Sandhoff disease. We previously developed mouse models of both diseases and showed that Hexa-/- (Tay-Sachs) mice remain asymptomatic to at least 1 year of age while Hexb-/- (Sandhoff) mice succumb to a profound neurodegenerative disease by 4-6 months of age. Here we find that neuron death in Hexb-/- mice is associated with apoptosis occurring throughout the CNS, while Hexa-/- mice were minimally involved at the same age. Studies of autopsy samples of brain and spinal cord from human Tay-Sachs and Sandhoff diseases revealed apoptosis in both instances, in keeping with the severe expression of both diseases. We suggest that neuron death is caused by unscheduled apoptosis, implicating accumulated GM2 ganglioside or a derivative in triggering of the apoptotic cascade.      

Mutation spectrum and genotype-phenotype analyses in Cowden disease and Bannayan-Zonana syndrome, two hamartoma syndromes with germline PTEN mutation

The tumour suppressor gene PTEN , which maps to 10q23.3 and encodes a 403 amino acid dual specificity phosphatase (protein tyrosine phosphatase; PTPase), was shown recently to play a broad role in human malignancy. Somatic PTEN deletions and mutations were observed in sporadic breast, brain, prostate and kidney cancer cell lines and in several primary tumours such as endometrial carcinomas, malignant melanoma and thyroid tumours. In addition, PTEN was identified as the susceptibility gene for two hamartoma syndromes: Cowden disease (CD; MIM 158350) and Bannayan-Zonana (BZS) or Ruvalcaba-Riley-Smith syndrome (MIM 153480). Constitutive DNA from 37 CD families and seven BZS families was screened for germline PTEN mutations. PTEN mutations were identified in 30 of 37 (81%) CD families, including missense and nonsense point mutations, deletions, insertions, a deletion/insertion and splice site mutations. These mutations were scattered over the entire length of PTEN , with the exception of the first, fourth and last exons. A 'hot spot' for PTEN mutation in CD was identified in exon 5 that contains the PTPase core motif, with 13 of 30 (43%) CD mutations identified in this exon. Seven of 30 (23%) were within the core motif, the majority (five of seven) of which were missense mutations, possibly pointing to the functional significance of this region. Germline PTEN mutations were identified in four of seven (57%) BZS families studied. Interestingly, none of these mutations was observed in the PTPase core motif. It is also worthy of note that a single nonsense point mutation, R233X, was observed in the germline DNA from two unrelated CD families and one BZS family. Genotype-phenotype studies were not performed on this small group of BZS families. However, genotype-phenotype analysis inthe group of CD families revealed two possible associations worthy of follow-up in independent analyses. The first was an association noted in the group of CD families with breast disease. A correlation was observed between the presence/absence of a PTEN mutation and the type of breast involvement (unaffected versus benign versus malignant). Specifically and more directly, an association was also observed between the presence of a PTEN mutation and malignant breast disease. Secondly, there appeared to be an interdependent association between mutations upstream and within the PTPase core motif, the core motif containing the majority of missense mutations, and the involvement of all major organ systems (central nervous system, thyroid, breast, skin and gastrointestinal tract). However, these observations would need to be confirmed by studying a larger number of CD families.      

Functional consequences of ROMK mutants linked to antenatal Bartter's syndrome and implications for treatment

The antenatal variant of Bartter's syndrome is an autosomal recessive kidney disease characterized by polyhydramnios, premature delivery, hypokalemic alkalosis and hypercalciuria. It is genetically heterogeneous, having been linked recently to mutations in an ATP- sensitive, renal outer medullary K+channel, ROMK, and earlier to mutations in the Na-K-2Cl co-transporter, NKCC2. We characterized four of the mutations reported in three heterozygous ROMK variants of antenatal Bartter's and found that each expressed a distinct phenotype in Sf9 cells. One mutation expressed normal function and appears to be an allelic polymorphism. The other three mutations produced channels with significantly reduced K+fluxes. However, the mechanisms in each case were different and reflected abnormalities in phosphorylation, proteolytic processing or protein trafficking. The different mechanisms may be important in the design of appropriate therapy for patients with this disease.      

A model of corrective gene transfer in X-linked ichthyosis

Single gene recessive genetic skin disorders offer attractive prototypes for the development of therapeutic cutaneous gene delivery. We have utilized X-linked ichthyosis (XLI), characterized by loss of function of the steroid sulfatase arylsulfatase C (STS), to develop a model of corrective gene delivery to human skin in vivo. A new retroviral expression vector was produced and utilized to effect STS gene transfer to primary keratinocytes from XLI patients. Transduction was associated with restoration of full-length STS protein expression as well as steroid sulfatase enzymatic activity in proportion to the number of proviral integrations in XLI cells. Transduced and uncorrected XLI keratinocytes, along with normal controls, were then grafted onto immunodeficient mice to regenerate full thickness human epidermis. Unmodified XLI keratinocytes regenerated a hyperkeratotic epidermis lacking STS expression with defective skin barrier function, effectively recapitulating the human disease in vivo. Transduced XLI keratinocytes from the same patients, however, regenerated epidermis histologically indistinguishable from that formed by keratinocytes from patients with normal skin. Transduced XLI epidermis demonstrated STS expression in vivo by immunostaining as well as a normalization of histologic appearance at 5 weeks post-grafting. In addition, transduced XLI epidermis demonstrated a return of barrier function parameters to normal. These findings demonstrate corrective gene delivery in human XLI patient skin tissue at both molecular and functional levels and provide a model of human cutaneous gene therapy.      

Fulminant hepatic failure induced oxidative stress in nonsynaptic mitochondria of cerebral cortex in rats

Fulminant hepatic failure (FHF) is a condition with sudden onset of necrosis of hepatocytes and degeneration of liver tissue without any established liver disease. FHF is associated with increased ammonia levels in blood and brain, which is supposed to be neurotoxic, ultimately leading to neuronal death. Evidences from previous studies suggest for mitochondrial dysfunctions under hyperammonemic conditions. In the present investigation, on thioacetamide-induced FHF rat models, studies were undertaken on cerebral nonsynaptic mitochondrial oxidative stress. The results of the present study reveal elevated lipid peroxidation along with reduced total thiol levels in the cerebral cortex mitochondria of experimental animals compared to saline treated control rats. In addition, the enzymatic activities of glutathione peroxidase and glutathione reductase were decreased, with an elevation in Mn-SOD activity. Overall, thioacetamide-induced FHF in rats enhanced the levels of lipid peroxidation coupled with impaired antioxidant defenses in the cerebral nonsynaptic mitochondria.     

Estimation of size of clonal unit for keratinocytes in normal human skin

OBJECTIVE: It has been suggested that keratinocyte (KC) stem cells reside at the epicenter of a clonal population of cells. To estimate the territory or surface area covered by a single stem-cell-derived KC population in human skin, clonal skin maps were created from 3 healthy adult women and from normal skin of a psoriatic patient. DESIGN: Two hundred fifty-eight punch biopsy samples of various sizes (ranging from 2 to 8 mm in diameter) were analyzed for clonality employing X chromosome inactivation patterns at the human androgen receptor gene (HUMARA) locus. DNA was isolated and clonality established by significant decrease of either maternal or paternal X chromosome band patterns following restriction enzyme digestion, polymerase chain reaction amplification, and gel electrophoresis. RESULTS: Fifty-three (41%) of 128 two-mm biopsies were clonal, whereas only 6 (14%) of 43 three-mm, 5 (14%) of 36 four-mm, and 3 (8%) of 35 five-mm biopsies revealed a clonal population of KCs. By contrast, in 5 different biopsies from a psoriatic patient, including 4- or 5-mm sizes, all but 1 were clonal; even an 8-mm biopsy contained a clonal population of KCs. Mantel-Haenszel chi(2) analysis revealed a P value of.001, reflecting a strong trend in probability for presence of a single clone of KCs as related to size of the biopsy sample. By sequentially analyzing 30 contiguous 2-mm biopsy samples within a given strip of skin, 10 clonal domain changes, as reflected in maternal versus paternal switches, were observed. CONCLUSIONS: These results provide direct evidence of a clonal population of KCs in normal and psoriatic lesion-free skin, and indicate that a clonal epidermal unit of KCs frequently can be detected in small biopsies (2 mm), but that in normal skin sampling, overlapping clones are apparently present in larger (ie, 4-5-mm) biopsies, producing nonclonal patterns. The clonal domain of progeny in normal skin has a rather limited territorial boundary (2 mm in diameter). However, in lesion-free skin from a psoriatic patient, there may be clonal expansion of KCs due to perturbation in epidermopoiesis and/or stem cell distribution.     

Mutational analysis of the SOX9 gene in campomelic dysplasia and autosomal sex reversal: lack of genotype/phenotype correlations

It has previously been shown that, in the heterozygous state, mutations in the SOX9 gene cause campomelic dysplasia (CD) and the often associated autosomal XY sex reversal. In 12 CD patients, 10 novel mutations and one recurrent mutation were characterized in one SOX9 allele each, and in one case, no mutation was found. Four missense mutations are all located within the high mobility group (HMG) domain. They either reduce or abolish the DNA-binding ability of the mutant SOX9 proteins. Among the five nonsense and three frameshift mutations identified, two leave the C-terminal transactivation (TA) domain encompassing residues 402-509 of SOX9 partly or almost completely intact. When tested in cell transfection experiments, the recurrent nonsense mutation Y440X, found in two patients who survived for four and more than 9 years, respectively, exhibits some residual transactivation ability. In contrast, a frameshift mutation extending the protein by 70 residues at codon 507, found in a patient who died shortly after birth, showed no transactivation. This is apparently due to instability of the mutant SOX9 protein as demonstrated by Western blotting. Amino acid substitutions and nonsense mutations are found in patients with and without XY sex reversal, indicating that sex reversal in CD is subject to variable penetrance. Finally, none of 18 female patients with XY gonadal dysgenesis (Swyer syndrome) showed an altered SOX9 banding pattern in SSCP assays, providing evidence that SOX9 mutations do not usually result in XY sex reversal without skeletal malformations.      

Distribution of cholinergic neurotransmitter enzymes in the hippocampus and the dentate gyrus of the adult and the developing mouse

The distribution of the cholinergic neutrotransmitter enzyme, acetylcholinesterase, in the hippocampus and the dentate gyrus of the adult and the developing mouse was examined using a histochemical procedure. The pattern of acetylcholinesterase staining in the hippocampal region of the adult mouse closely resembled that reported for the rat by other investigators. Enzyme activity was present predominantly in the neuropil, where it was concentrated in the supra- and the infrapyramidal zones of the hippocampus as well as in the supragranular region and the hilus of the dentate gyrus. In contrast to the adult pattern, during the first week of postnatal development acetyleholinesterase activity appeared to be largely intracellular. Strong staining was observed in the cytoplasm of scattered neurons throughout the neuropil laminae, particularly in the hilus of the dentate gyrus. During the succeeding weeks, the characteristic neuropil reaction developed in a slow and progressive manner. reaching the adult pattern by the end of the third postnatal week. Between the third and the fifth weeks, there was a substantial increase in the staining intensity of the enzyme. As a result of the increased neuropil reaction, acetylcholinesterase-positive cells became less conspicuous after the second postnatal week. The progressive acquisition of staining for acetylcholinesterase in the neuropil of the hippocampus and the dentate gyrus of the mouse during the early postnatal period compared well with the proposed model of development of septohippocampal connections in the rat.The histochemical distribution of choline acetyltransferase in the hippocampus and the dentate gyrus of the adult mouse was also examined. The reaction was largely intracellular, in the cytoplasm of the pyramidal and the granule cells. Neuropil staining was confined to the mossy fibers and their terminals. This distribution profile is in conflict with the localization of this enzyme in the hippocampal region established by other investigators on the basis of microdissection and assay. The significance of the results of choline acetyltransferase histochemistry in relation to methodological problems is discussed.     

Treatment of infants with ureteropelvic junction obstruction: findings from the PURSUIT network

International Urology and Nephrology - Studies based on administrative databases show that infant pyeloplasty is associated with minority race/ethnicity but lack clinical data that may influence...