The GBA p.Trp378Gly mutation is a probable French-Canadian founder mutation causing Gaucher disease and synucleinopathies.

Biallelic GBA mutations cause Gaucher disease (GD), and heterozygous carriers are at risk for synucleinopathies. No founder GBA mutations in French-Canadians are known. GBA was fully sequenced using targeted next generation and Sanger sequencing in French-Canadian Parkinson disease (PD) patients (n = 436), rapid eye movement (REM)-sleep behavior disorder (RBD) patients (n = 189) and controls (n = 891). Haplotype, identity-by-descent (IBD) and principal component analyses (PCA) were performed using single nucleotide polymorphism-chip data. Data on GD patients from Toronto and Montreal were collected from patients' files. A GBA p.Trp378Gly mutation was identified in two RBD and four PD patients (1% of all patients combined), and not in controls. The two RBD patients had converted to DLB within 3 years of their diagnosis. Haplotype, IBD and PCA analysis demonstrated that this mutation is from a single founder. Out of 167 GD patients screened, 15 (9.0%) carried the p.Trp378Gly mutation, all in trans with p.Asn370Ser. Three (20%) of the GD patients with the p.Trp378Gly mutation had developed Parkinsonism, and 11 patients had family history of PD. The p.Trp378Gly mutation is the first French-Canadian founder GBA mutation to be described, which leads to synucleinopathies and to GD type 1 when in compound heterozygosity with p.Asn370Ser.

Parkin maintains mitochondrial levels of the protective Parkinson's disease-related enzyme 17-ß hydroxysteroid dehydrogenase type 10.

Mutations of the PARK2 and PINK1 genes, encoding the cytosolic E3 ubiquitin-protein ligase Parkin and the mitochondrial serine/threonine kinase PINK1, respectively, cause autosomal recessive early-onset Parkinson's disease (PD). Parkin and PINK1 cooperate in a biochemical mitochondrial quality control pathway regulating mitochondrial morphology, dynamics and clearance. This study identifies the multifunctional PD-related mitochondrial matrix enzyme 17-ß hydroxysteroid dehydrogenase type 10 (HSD17B10) as a new Parkin substrate. Parkin overproduction in cells increased mitochondrial HSD17B10 abundance by a mechanism involving ubiquitin chain extension, whereas PARK2 downregulation or deficiency caused mitochondrial HSD17B10 depletion in cells and mice. HSD17B10 levels were also found to be low in the brains of PD patients with PARK2 mutations. Confocal and Förster resonance energy transfer (FRET) microscopy revealed that HSD17B10 recruited Parkin to the translocase of the outer membrane (TOM), close to PINK1, both in functional mitochondria and after the collapse of mitochondrial membrane potential (ΔΨm). PD-causing PARK2 mutations impaired interaction with HSD17B10 and the HSD17B10-dependent mitochondrial translocation of Parkin. HSD17B10 overproduction promoted mitochondrial elongation and mitigated CCCP-induced mitochondrial degradation independently of enzymatic activity. These effects were abolished by overproduction of the fission-promiting dynamin-related protein 1 (Drp1). By contrast, siRNA-mediated HSD17B10 silencing enhanced mitochondrial fission and mitophagy. These findings suggest that the maintenance of appropriate mitochondrial HSD17B10 levels is one of the mechanisms by which Parkin preserves mitochondrial quality. The loss of this protective mechanism may contribute to mitochondrial dysfunction and neuronal degeneration in autosomal recessive PD.

Loss of the Parkinson's disease-linked gene DJ-1 perturbs mitochondrial dynamics.

Growing evidence highlights a role for mitochondrial dysfunction and oxidative stress as underlying contributors to Parkinson's disease (PD) pathogenesis. DJ-1 (PARK7) is a recently identified recessive familial PD gene. Its loss leads to increased susceptibility of neurons to oxidative stress and death. However, its mechanism of action is not fully understood. Presently, we report that DJ-1 deficiency in cell lines, cultured neurons, mouse brain and lymphoblast cells derived from DJ-1 patients display aberrant mitochondrial morphology. We also show that these DJ-1-dependent mitochondrial defects contribute to oxidative stress-induced sensitivity to cell death since reversal of this fragmented mitochondrial phenotype abrogates neuronal cell death. Reactive oxygen species (ROS) appear to play a critical role in the observed defects, as ROS scavengers rescue the phenotype and mitochondria isolated from DJ-1 deficient animals produce more ROS compared with control. Importantly, the aberrant mitochondrial phenotype can be rescued by the expression of Pink1 and Parkin, two PD-linked genes involved in regulating mitochondrial dynamics and quality control. Finally, we show that DJ-1 deficiency leads to altered autophagy in murine and human cells. Our findings define a mechanism by which the DJ-1-dependent mitochondrial defects contribute to the increased sensitivity to oxidative stress-induced cell death that has been previously reported.

Effects of genetic depletion of monoamines on somatosensory cortical development.

Raised levels of serotonin cause alterations in the development of the barrelfield of the primary somatosensory cortex (S1) in rodents. We examined the development of S1 in genetic mouse models in which the levels of serotonin and/or dopamine and noradrenaline are drastically reduced. Mice lacking the vesicular monoamine transporter type 2 (VMAT2 KO) are hypomorphic with rare pups surviving until postnatal day (P) 6. Serotonin, dopamine and noradrenaline are almost undetectable in the brain. In S1 we find that the segregation of thalamocortical axons into whisker patterns is delayed by 1 day and that layer IV granular neurons fail to form normal barrels. Moreover, the growth of cortical layers II-IV is reduced. Despite severe malnutrition, we show that these alterations are not caused by increased cell death in the thalamus or S1. Moreover, the maturation of cortical neurons is not altered as reflected by calcium-binding protein immunolabeling. Mice lacking both VMAT2 and monoamine oxidase type A (MAOA) were generated. VMAT2-MAOA DKO mice are hypomorphic but survive until P13. Increased levels of serotonin but profoundly reduced dopamine and noradrenaline levels are found in the brains. In S1, alterations are similar to those observed in MAOA KO mice: thalamocortical axons and granular neurons failed to form barrels. In addition there is a severe reduction in the thickness of the upper cortical layers as in the VMAT2 KO mice. These results show that monoamines have no instructive effect per se on the formation of thalamocortical patterning in S1. However, monoamines appear to be essential for the normal cytoarchitectonic maturation of the granular (IV) and supragranular cortical layers (II-III). Since developmental cell death and chemoarchitectonic differentiation of these neurons are not modified, it is possible that these alterations result from migration defects and/or from altered synaptic maturation.

Molecular mechanisms of neurotransmitter release.

The release of neurotransmitter from neurons represents one of the pivotal events in synaptic transmission. Neurotransmitters are released from synaptic vesicles in presynaptic neurons in response to neural activity, diffuse across the synaptic cleft, and bind specific receptors in order to bring about changes in postsynaptic neurons. Some of the molecular processes that govern neurotransmitter release are now becoming better understood. The steps involved can be broken down into two partially overlapping presynaptic cycles, the neurotransmitter cycle and the synaptic vesicle cycle. The neurotransmitter cycle involves transmitter biosynthesis, storage, reuptake, and degradation. The synaptic vesicle cycle involves targeting to the nerve terminal, docking, fusion, endocytosis, and recycling. Biochemical and structural studies have yielded important insight into our understanding of each of these two cycles. Further, both pharmacological and genetic interference with either of these cycles results in profound alterations in synaptic transmission and behavior, demonstrating the crucial role of neurotransmitter release.

Vesicular neurotransmitter transport and the presynaptic regulation of quantal size.

Specific transport activities package classical neurotransmitters into secretory vesicles for release by regulated exocytosis, but the proteins responsible for the vesicular transport of neurotransmitters are still being identified. One family of proteins includes vesicular transporters for monoamines and acetylcholine. Genetic manipulation in cells and in mice now shows that changes in the expression of these proteins can alter the amount of neurotransmitter stored per synaptic vesicle, the amount released and behavior. Although the mechanisms responsible for regulating these transporters in vivo remains unknown, recent work has demonstrated the potential for regulation by changes in intrinsic activity and in location. In addition, a recently identified vesicular transporter for GABA defines a novel family of proteins that mediates the packaging of amino acid neurotransmitters.

Genomic structure of the human GT334 (EHOC-1) gene mapping to 21q22.3.

Several inherited diseases have been mapped to the distal tip of human chromosome 21. In our recent efforts to clone candidate genes for some of these disorders, we have assembled a cosmid and BAC contig spanning 770 kb. We have identified expressed sequences from this contig by means of a cDNA hybrid selection scheme. We present here the isolation, cDNA sequence, genomic organization, and polymorphisms analysis of one such expressed sequence, GT334, which had been identified independently and designated EHOC-1. GT334 is split into 23 exons, and spans an estimated 95 kb of genomic DNA. A pseudogene of the histone H2AZ gene has been identified, and maps within the third intron. We have identified an ORF potentially encoding a protein 1259 amino acids in length, longer than that described in the EHOC-1 gene. The GT334 gene was screened for single base pair changes using single-strand conformation polymorphism (SSCP) analysis and we have identified seven sequence variations within this gene. These polymorphisms can be used as markers in the genetic mapping of other diseases localized to this region.

Vesicular transport regulates monoamine storage and release but is not essential for amphetamine action.

To assess the role of exocytotic release in signaling by monoamines, we have disrupted the neuronal vesicular monoamine transporter 2 (VMAT2) gene. VMAT2-/- mice move little, feed poorly, and die within a few days after birth. Monoamine cell groups and their projections are indistinguishable from those of wild-type littermates, but the brains of mutant mice show a drastic reduction in monoamines. Using midbrain cultures from the mutant animals, amphetamine but not depolarization induces dopamine release. In vivo, amphetamine increases movement, promotes feeding, and prolongs the survival of VMAT2-/- animals, indicating that precise, temporally regulated exocytotic release of monoamine is not required for certain complex behaviors. In addition, the brains of VMAT2 heterozygotes contain substantially lower monoamine levels than those of wild-type littermates, and depolarization induces less dopamine release from heterozygous than from wild-type cultures, suggesting that VMAT2 expression regulates monoamine storage and release.

Isolation and characterization of GT335, a novel human gene conserved in Escherichia coli and mapping to 21q22.3.

As part of efforts to identify candidate genes for disorders mapped to 21q22.3, we have constructed a 405-kb cosmid contig encompassing five tightly linked markers mapping to this region. A subset of these cosmids was used to identify cDNA fragments by the method of hybrid selection. We present here the cDNA sequence of one such gene (GT335) mapping to this region. The gene is expressed as a 1.7-kb transcript predominantly in heart and skeletal muscle, potentially displays alternate splicing, and is predicted to encode a protein 268 amino acids in length. GT335 spans an estimated 13 kb of genomic DNA and is split into seven exons. Five of the six introns conform to the GT . . . AG consensus for intronic splice junctions; the sixth contains nonconventional (AT . . . AC) intronic junctions. We screened this gene for single-basepair mutations using single-strand conformation polymorphism and sequence analysis of both cDNA and genomic DNA from a number of unrelated individuals and have identified several sequence variations, two of which cause conservative amino acid substitutions. This gene is well conserved evolutionarily, with homologs identified in zebrafish and Escherichia coli, suggesting that it plays an important role in basic cellular metabolism.

Adenylosuccinate lyase (ADSL) and infantile autism: absence of previously reported point mutation.

Autism is a heterogeneous neuropsychiatric syndrome of unknown etiology. There is evidence that a deficiency in the enzyme adenylosuccinate lyase (ADSL), essential for de novo purine biosynthesis, could be involved in the pathogenesis of certain cases. A point mutation in the ADSL gene, resulting in a predicted serine-to-proline substitution and conferring structural instability to the mutant enzyme, has been reported previously in 3 affected siblings. In order to determine the prevalence of the mutation, we PCR-amplified the exon spanning the site of this mutation from the genomic DNA of patients fulfilling DSM-III-R criteria for autistic disorder. None of the 119 patients tested were found to have this mutation. Furthermore, on preliminary screening using singlestrand conformation polymorphism (SSCP), no novel mutations were detected in the coding sequence of four ADSL exons, spanning approximately 50% of the cDNA. In light of these findings, it appears that mutations in the ADSL gene represent a distinctly uncommon cause of autism.

Hemostatic markers in acute transient ischemic attacks.

BACKGROUND AND PURPOSE: Hemostatic abnormalities have been shown previously in stroke patients. The purpose of this study was to assess the activity of selected parameters of the coagulation system in acute reversible cerebral ischemia. METHODS: We measured fibrinopeptide A, thrombin-antithrombin III, and D-dimer in 36 patients in both the acute (< 7 days) and postacute stage (1 and 3 months) after a transient ischemic attack (TIA). The results were compared with those of 20 asymptomatic patients with a history of remote TIA and 65 age- and sex-matched controls. RESULTS: Mean fibrinopeptide A and thrombin-antithrombin III values were elevated in the acute stage after a TIA (P < .02) compared with levels at 1 month. In contrast, D-dimer was significantly increased at all three times points after the event when compared with remote TIA (P < .05) or control subjects (P < .001). No association could be found between marker levels and clinical outcome or the degree of cervical atherosclerosis as assessed by duplex ultrasonography. CONCLUSIONS: These findings suggest that after acute reversible cerebral ischemia, there is early transient activation of thrombogenesis and ongoing fibrinolysis.

Mapping of the human adenylosuccinate lyase (ADSL) gene to chromosome 22q13.1-->q13.2.

Adenylosuccinate lyase (ADSL) is an essential enzyme involved in de novo purine biosynthesis. A deficiency in ADSL in humans has been shown to predispose to a neurodevelopmental syndrome with autistic features. Using both a somatic cell hybrid mapping panel and fluorescence in situ hybridization, we have precisely localized the human ADSL gene to chromosome 22q13.1-->q13.2.

In situ hybridization evidence for angiotensinogen messenger RNA in the rat proximal tubule. An hypothesis for the intrarenal renin angiotensin system.

We examined angiotensinogen gene expression in rat kidney by in situ hybridization histochemistry. Using a rat cRNA probe to angiotensinogen, we demonstrated angiotensinogen mRNA to be localized predominantly in the proximal renal tubule, with considerably lesser amounts in distal tubular segments and glomerular tufts. Previous studies have localized renin immunoreactivity to the juxtaglomerular cells, glomerular tufts, and proximal tubules. Such findings provide further evidence for a local tissue renin angiotensin system within the kidney which may influence regional function. Based on our data, we hypothesize that a major site of angiotensin production is the proximal tubule. We postulate that angiotensin synthesized in and/or around the proximal tubule may directly modulate tubular transport of sodium, bicarbonate, and water. In addition to the proximal tubule, the specific localization of the renin angiotensin components elsewhere in the kidney would also support the other proposed regional functions of the intrarenal system, including modulation of tubuloglomerular balance.