Raymonde Szargel

The deoxyguanosine kinase gene is mutated in individuals with depleted hepatocerebral mitochondrial DNA.

Mitochondrial DNA (mtDNA)–depletion syndromes (MDS; OMIM 251880) are phenotypically heterogeneous, autosomal-recessive disorders characterized by tissue-specific reduction in mtDNA copy number. Affected individuals with the hepatocerebral form of MDS have early progressive liver failure and neurological abnormalities, hypoglycemia and increased lactate in body fluids. Affected tissues show both decreased activity of the mtDNA-encoded respiratory chain complexes (I, III, IV, V) and mtDNA depletion. We used homozygosity mapping in three kindreds of Druze origin to map the gene causing hepatocerebral MDS to a region of 6.1 cM on chromosome 2p13, between markers D2S291 and D2S2116. This interval encompasses the gene (DGUOK) encoding the mitochondrial deoxyguanosine kinase (dGK). We identified a single-nucleotide deletion (204delA) within the coding region of DGUOK that segregates with the disease in the three kindreds studied. Western-blot analysis did not detect dGK protein in the liver of affected individuals. The main supply of deoxyribonucleotides (dNTPs) for mtDNA synthesis comes from the salvage pathway initiated by dGK and thymidine kinase-2 (TK2). The association of mtDNA depletion with mutated DGUOK suggests that the salvage-pathway enzymes are involved in the maintenance of balanced mitochondrial dNTP pools.

Mutations in SLC19A2 cause thiamine-responsive megaloblastic anaemia associated with diabetes mellitus and deafness.

Thiamine-responsive megaloblastic anaemia (TRMA), also known as Rogers syndrome, is an early onset, autosomal recessive disorder defined by the occurrence of megaloblastic anaemia, diabetes mellitus and sensorineural deafness, responding in varying degrees to thiamine treatment (MIM 249270). We have previously narrowed the TRMA locus from a 16-cM to a 4-cM interval on chromosomal region 1q23.3 (Refs 3, 4) and this region has been further refined to a 1.4-cM interval. Previous studies have suggested that deficiency in a high-affinity thiamine transporter may cause this disorder. Here we identify the TRMA gene by positional cloning. We assembled a P1-derived artificial chromosome (PAC) contig spanning the TRMA candidate region. This clarified the order of genetic markers across the TRMA locus, provided 9 new polymorphic markers and narrowed the locus to an approximately 400-kb region. Mutations in a new gene, SLC19A2, encoding a putative transmembrane protein homologous to the reduced folate carrier proteins, were found in all affected individuals in six TRMA families, suggesting that a defective thiamine transporter protein (THTR-1) may underlie the TRMA syndrome.

Hypotrichosis with juvenile macular dystrophy is caused by a mutation in CDH3, encoding P-cadherin

Congenital hypotrichosis associated with juvenile macular dystrophy (HJMD; MIM601553) is an autosomal recessive disorder of unknown etiology, characterized by hair loss heralding progressive macular degeneration and early blindness. We used homozygosity mapping in four consanguineous families to localize the gene defective in HJMD to 16q22.1. This region contains CDH3, encoding P-cadherin, which is expressed in the retinal pigment epithelium and hair follicles. Mutation analysis shows in all families a common homozygous deletion in exon 8 of CDH3. These results establish the molecular etiology of HJMD and implicate for the first time a cadherin molecule in the pathogenesis of a human hair and retinal disorder.

Monoubiquitylation of α-Synuclein by Seven in Absentia Homolog (SIAH) Promotes Its Aggregation in Dopaminergic Cells

α-Synuclein plays a major role in Parkinson disease. Unraveling the mechanisms of α-synuclein aggregation is essential to understand the formation of Lewy bodies and their involvement in dopaminergic cell death. α-Synuclein is ubiquitylated in Lewy bodies, but the role of α-synuclein ubiquitylation has been mysterious. We now report that the ubiquitin-protein isopeptide ligase seven in absentia homolog (SIAH) directly interacts with and monoubiquitylates α-synuclein and promotes its aggregation in vitro and in vivo, which is toxic to cells. Mass spectrometry analysis demonstrates that SIAH monoubiquitylates α-synuclein at lysines 12, 21, and 23, which were previously shown to be ubiquitylated in Lewy bodies. SIAH ubiquitylates lysines 10, 34, 43, and 96 as well. Suppression of SIAH expression by short hairpin RNA to SIAH-1 and SIAH-2 abolished α-synuclein monoubiquitylation in dopaminergic cells, indicating that endogenous SIAH ubiquitylates α-synuclein. Moreover, SIAH co-immunoprecipitated with α-synuclein from brain extracts. Inhibition of proteasomal, lysosomal, and autophagic pathways, as well as overexpression of a ubiquitin mutant less prone to deubiquitylation, G76A, increased monoubiquitylation of α-synuclein by SIAH. Monoubiquitylation increased the aggregation of α-synuclein in vitro. At the electron microscopy level, monoubiquitylated α-synuclein promoted the formation of massive amounts of amorphous aggregates. Monoubiquitylation also increased α-synuclein aggregation in vivo as observed by increased formation of α-synuclein inclusion bodies within dopaminergic cells. These inclusions are toxic to cells, and their formation was prevented when endogenous SIAH expression was suppressed. Our data suggest that monoubiquitylation represents a possible trigger event for α-synuclein aggregation and Lewy body formation.

Oral health and salivary composition in diabetic patients

The salivary composition and flow rate were examined in 20 patients with insulin-dependent diabetes mellitus (IDDM) and in 19 patients with non-insulin-dependent diabetes mellitus (NIDDM) and compared with 20 healthy controls. Resting and stimulated whole and submandibular saliva was analyzed. Significantly lower resting salivary flow rates were found in the IDDM patients as compared to the NIDDM group. In the IDDM patients potassium concentration in resting saliva was significantly higher compared with healthy controls and in stimulated whole saliva compared with NIDDM patients. No difference in salivary total protein, amylase, lactoferrin, or lysozyme was found among the three groups examined. The IgA concentration of the IDDM patients was significantly higher in whole resting saliva compared with controls and in the submandibular saliva compared with both NIDDM patients and controls. No difference was found between controls and the diabetic patients examined in prevalence of complaint of dry mouth. The salivary flow rates, however, were significantly lower in the three subgroups with dry mouth compared with the subgroups without this complaint. Caries were detected in 100% of the diabetic patients and controls. No correlation was observed between the incidence of caries and any of the salivary parameters examined. A higher prevalence and severity of periodontal disease was detected in the diabetic patients as compared to the controls. A significant positive correlation was found between the gingival index and the concentrations of total protein, albumin, lysozyme, and lactoferrin in whole resting saliva in the three groups examined.

Salivary composition in diabetic patients

Abstract Salivary composition and flow rate were examined in 35 patients with insulin-dependent diabetes mellitus (IDDM) and compared to 31 healthy controls. Significantly lower whole-saliva flow rate was observed in the IDDM patients, but was not correlated with the subjective complaint of xerostomy. Glucose concentration was significantly higher in the parotid saliva of the IDDM patients. Potassium concentration was significantly higher in whole and parotid, resting and stimulated saliva, as was total protein concentration in resting whole and in stimulated parotid saliva of the diabetics. No significant difference between diabetics and healthy controls was found in sodium and lgA concentration or in amylase activity. The significantly higher glucose, lower flow rate, and higher potassium and protein concentrations indicate that salivary glands are affected in IDDM. The subjective complaint of dry mouth, often present in diabetics, while not correlated with salivary flow rate, might reflect qualitative changes in salivary composition and/or altered mucosal perception. Salivary glucose concentration, although significantly higher in the diabetics, was not significantly correlated with serum glucose, preventing the use of saliva for monitoring blood sugar.

Clinical Science Whole-saliva Secretion Rates in Old and Young Healthy Subjects

Resting and stimulated whole-saliva secretion rates were compared in old and young healthy volunteers. The stimulated secretion rate was similar in both age groups, while the resting flow rate was significantly lower in the old females and males as compared with rates in the young.

The salivary flow rate and composition of whole and parotid resting and stimulated saliva in young and old healthy subjects

Resting and stimulated whole and parotid salivary composition and flow rate were examined in 63 healthy volunteers. No significant differences were found between the young and old in secretion rates and salivary concentrations of sodium, potassium, calcium, magnesium, and total protein. The activity of amylase in the resting and stimulated parotid saliva was significantly lower in the old.

Phosphorylation of Parkin by the cyclin-dependent kinase 5 at the linker region modulates its ubiquitin-ligase activity and aggregation.

Mutations in Parkin are responsible for a large percentage of autosomal recessive juvenile parkinsonism cases. Parkin displays ubiquitin-ligase activity and protects against cell death promoted by several insults. Therefore, regulation of Parkin activities is important for understanding the dopaminergic cell death observed in Parkinson disease. We now report that cyclin-dependent kinase 5 (Cdk5) phosphorylates Parkin both in vitro and in vivo. We found that highly specific Cdk5 inhibitors and a dominant negative Cdk5 construct inhibited Parkin phosphorylation, suggesting that a significant portion of Parkin is phosphorylated by Cdk5. Parkin interacts with Cdk5 as observed by co-immunoprecipitation experiments of transfected cells and rat brains. Phosphorylation by Cdk5 decreased the auto-ubiquitylation of Parkin both in vitro and in vivo. We identified Ser-131 located at the linker region of Parkin as the major Cdk5 phosphorylation site. The Cdk5 phosphorylation-deficient S131A Parkin mutant displayed a higher auto-ubiquitylation level and increased ubiquitylation activity toward its substrates synphilin-1 and p38. Additionally, the S131A Parkin mutant more significantly accumulated into inclusions in human dopaminergic cells when compared with the wild-type Parkin. Furthermore, S131A Parkin mutant increased the formation of synphilin-1/α-synuclein inclusions, suggesting that the levels of Parkin phosphorylation and ubiquitylation may modulate the formation of inclusion bodies relevant to the disease. The data indicate that Cdk5 is a new regulator of the Parkin ubiquitin-ligase activity and modulates its ability to accumulate into and modify inclusions. Phosphorylation by Cdk5 may contribute to the accumulation of toxic Parkin substrates and decrease the ability of dopaminergic cells to cope with toxic insults in Parkinson disease.

Composition of whole unstimulated saliva of healthy children: Changes with age

Whole unstimulated saliva was collected from 136 healthy subjects divided into 5 groups according to age: (1) 25 infants, 7-11 months old; (2) 28 toddlers, 2-3 yr old; (3) 28 children, 6-8 yr old; (4) 28 adolescents, 12-14 yr old; (5) 27 adults, 25-63 yr old. The concentrations of Na, K, total protein, IgA and amylase activity were measured. A significant ascending linear correlation with age was found for concentrations of Na, total protein, IgA and amylase activity. There were significant differences between age groups in K and IgA concentrations. Salivary amylase activity was very variable, and a significant difference was found between infants and toddlers only. Salivary composition thus changes significantly during childhood, implying a process of development and maturation of the salivary glands and indicating the need of age-matched controls for the clinical use of saliva.