Synuclein Deficiency Decreases the Efficiency of Dopamine Uptake by Synaptic Vesicles.

In the present study, we analyzed the uptake of radiolabeled dopamine by intact synaptosomes and purified synaptic vesicles isolated from the dorsal striatum of mice with constitutive inactivation of all three synuclein-coding genes and wild-type mice. Synuclein deficiency substantially compromised the uptake of this neurotransmitter by synaptic vesicles but had no effect on synaptosomal dopamine uptake.

Gamma-Carbolines Derivatives As Promising Agents for the Development of Pathogenic Therapy for Proteinopathy.

Uncontrolled protein aggregation, accompanied by the formation of specific inclusions, is a major component of the pathogenesis of many common neurodegenerative diseases known as proteinopathies. The intermediate products of this aggregation are toxic to neurons and may be lethal. The development strategy of pathogenic therapy for proteinopathy is based on the design of drugs capable of both inhibiting proteinopathy progression and increasing the survival of affected neurons. The results of a decade-long research effort at leading Russian and international laboratories have demonstrated that Dimebon (Latrepirdine), as well as a number of its derivatives from a gamma-carboline group, show a strong neuroprotective effect and can modulate the course of a neurodegenerative process in both in vitro and in vivo model systems. The accumulated data indicate that gamma-carbolines are promising compounds for the development of pathogenic therapy for proteinopathies.

Dimebon reduces the levels of aggregated amyloidogenic protein forms in detergent-insoluble fractions in vivo.

Aggregation of proteins liable to assembling into fibrils with subsequent formation of amyloid incorporations is an important component in the pathogenesis of many neurodegenerative diseases. Dimebon, a Russian drug, reduces the content of detergent-insoluble fibrillar forms of synuclein, the main protein component of pathological incorporations in neurons of transgenic mouse strain used in the study.

Deletion of alpha-synuclein decreases impulsivity in mice.

The presynaptic protein alpha-synuclein, associated with Parkinson's Disease (PD), plays a role in dopaminergic neurotransmission and is implicated in impulse control disorders (ICDs) such as drug addiction. In this study we investigated a potential causal relationship between alpha-synuclein and impulsivity, by evaluating differences in motor impulsivity in the 5-choice serial reaction time task (5-CSRTT) in strains of mice that differ in the expression of the alpha-synuclein gene. C57BL/6JOlaHsd mice differ from their C57BL/6J ancestors in possessing a chromosomal deletion resulting in the loss of two genes, snca, encoding alpha-synuclein, and mmrn1, encoding multimerin-1. C57BL/6J mice displayed higher impulsivity (more premature responding) than C57BL/6JOlaHsd mice when the pre-stimulus waiting interval was increased in the 5-CSRTT. In order to ensure that the reduced impulsivity was indeed related to snca, and not adjacent gene deletion, wild type (WT) and mice with targeted deletion of alpha-synuclein (KO) were tested in the 5-CSRTT. Similarly, WT mice were more impulsive than mice with targeted deletion of alpha-synuclein. Interrogation of our ongoing analysis of impulsivity in BXD recombinant inbred mouse lines revealed an association of impulsive responding with levels of alpha-synuclein expression in hippocampus. Expression of beta- and gamma-synuclein, members of the synuclein family that may substitute for alpha-synuclein following its deletion, revealed no differential compensations among the mouse strains. These findings suggest that alpha-synuclein may contribute to impulsivity and potentially, to ICDs which arise in some PD patients treated with dopaminergic medication.

Adaptor protein Ruk1 forms protein-protein complexes with endonuclease activity in HEK293 cells.

The structural and functional organization of the adaptor protein Ruk(1) is characterized by the presence of three SH3-domains at the N-terminus followed by Pro- and Ser-rich sequences and a C-terminal coiled-coil region. Multiple modules in the Ruk(1) structure involved in protein-protein interactions can provide for formation of ligand clusters with varied properties and subcellular location. To study the nature and biological role of such complexes, the recombinant protein Ruk(1) with a Glu-epitope at the C-terminus (Ruk(1) Glu-tagged) was purified from transfected HEK293 cells by affinity chromatography on protein G-Sepharose with covalently conjugated anti-Glu-tag antibodies. By SDS polyacrylamide gel electrophoresis with subsequent staining with silver, a set of minor bands in addition to the 85-kD Ruk(1) Glu-tagged was detected in the purified preparation of the recombinant protein. Proteins with affinity for nucleic acids were also revealed in the Ruk(1) Glu-tagged preparation by retardation of electrophoretic mobility of 32P-labeled oligodeoxyribonucleotides in gel. The Ruk(1) Glu-tagged preparation was also shown to hydrolyze both deoxyribonucleotides and plasmid DNA. ZnCl2 and heparin inhibited the DNAse activity. These findings suggest the presence of DNases associated with the Ruk(1) protein in HEK293 cells. Such complexes were isolated from lysates of HEK293 cells by chromatography on heparin-Sepharose. By elution with 0.5 and 1.0 M NaCl, two fractions with DNase activity and containing proteins with molecular weights of 83, 80, and 72 kD were obtained. The reaction was inhibited by ZnCl2 and heparin, and previous precipitation of Ruk-related proteins with anti-Ruk antibodies resulted in the exhaustion of nuclease activity. By immunoblotting with anti-Ruk antibodies, 83-kD protein immunologically related to the Ruk(1) protein was identified in the fractions. It was concluded that the adaptor protein Ruk(1) forms complexes with endonucleases in HEK293 cells.

Cerd4, third member of the d4 gene family: expression and organization of genomic locus.

Two members of the d4 family of presumptive transcription modulators, neuro-d4 (Neud4) and ubi-d4/Requiem (Req), have been characterized previously. We cloned and characterized the third member of this gene family, cer-d4 (Cerd4), from chicken and mouse cDNA libraries. The expression patterns of Cerd4 gene in both species are similar and more restricted than expression patterns of other two d4 genes. The main sites of Cerd4 expression are retina and cerebellum, where multiple transcripts could be detected. Two major types of Cerd4 proteins are a full-length isoform possessing all domains characteristic to the d4 family and truncated XZ isoform without C-terminal tandem of PHD fingers. The developmental kinetics of expression of these isoforms is different. The intron/exon structure of human Cerd4 gene is similar to that of neuro-d4 and ubi-d4/Requiem genes, but most introns of Cerd4 gene are much larger than the corresponding introns of the other two genes.

Chicken synucleins: cloning and expression in the developing embryo.

Synucleins comprise a family of small intracellular proteins that have recently attracted considerable attention because of their involvement in human diseases. Mutations of alpha-synuclein has been found in several families with hereditary early-onset Parkinson's disease and accumulation of this protein in characteristic cytoplasmic inclusions is a pathohistological hallmark of several neurodegenerative diseases that have been recently classified as 'alpha;-synucleinopathies' (reviewed in Brain Res. Bull. 50 (1999) 465; J. Neurosci. Res. 58 (1999) 120; Philos. Trans. R. Soc. Lond. Biol. Sci. 354 (1999) 1101; Brain Pathol. 9 (1999) 733). Aggregates of beta-synuclein and persyn (gamma-synuclein) also have been found in dystrophic neurites associated with Parkinson's and other neurodegenerative diseases (Proc. Natl. Acad. Sci. USA 96 (1999) 13450; and our unpublished observations). Moreover, persyn has been implicated in malignization of breast tumours (Cancer Res. 57 (1997) 759; Cancer Res. 59 (1999) 742; Hum. Mol. Genet. 7 (1998) 1417). All synucleins have distinct, although overlapping, patterns of expression in the embryonic, postnatal and adult mammalian nervous systems, suggesting important, although still not clear, biological functions in neuronal developing. Chicken embryo is a unique object for developmental studies that allows in vivo manipulations not always possible for mammalian embryos. Studies of synucleins expression in this model system could shed light on their functions in the developing nervous system. We cloned three chicken synucleins from the embryonic neural cDNA libraries and studied their expression in normal chicken embryonic tissues by Northern and in situ hybridization with specific probes. Our results demonstrate that primary structures and expression patterns of synucleins are similar in birds and mammals, suggesting that conserved function of synucleins is important for embryonic development of vertebrates.

Induction of neuronal death by alpha-synuclein.

The molecular and cellular mechanisms underlying neuronal loss in neurodegenerative diseases are unclear. It is generally thought that aggregation of mutated, abnormally modified or abnormally folded proteins leads to the accumulation of extracellular, intracellular or intranuclear deposits that severely compromise cell physiology, leading to the death of the affected neurons. However, there is growing evidence that neuronal apoptosis in the absence of obvious pathological deposits could have a serious impact on the pathogenesis of neurodegenerative diseases. alpha-Synuclein has been implicated in aetiology and pathogenesis of certain neurodegenerative diseases, although the precise role of this protein in neurodegeneration is uncertain. The normal functions of alpha-synuclein and other members of the synuclein family in the development and function of the nervous system also remain elusive. Here we show that overexpression of wild-type and mutant forms of alpha-synuclein in cultured neurons, but not the closely related persyn (gamma-synuclein), causes apoptosis. These findings suggest that abnormalities of alpha-synuclein metabolism could lead to the neuronal loss occurring in certain forms of neurodegeneration before the formation of characteristic pathological lesions.

Negative regulation of PI 3-kinase by Ruk, a novel adaptor protein.

Class I(A) phosphatidylinositol 3-kinase (PI 3-kinase) is a key component of important intracellular signalling cascades. We have identified an adaptor protein, Ruk(l), which forms complexes with the PI 3-kinase holoenzyme in vitro and in vivo. This interaction involves the proline-rich region of Ruk and the SH3 domain of the p85 alpha regulatory subunit of the class I(A) PI 3-kinase. In contrast to many other adaptor proteins that activate PI 3-kinase, interaction with Ruk(l) substantially inhibits the lipid kinase activity of the enzyme. Overexpression of Ruk(l) in cultured primary neurons induces apoptosis, an effect that could be reversed by co-expression of constitutively activated forms of the p110 alpha catalytic subunit of PI 3-kinase or its downstream effector PKB/Akt. Our data provide evidence for the existence of a negative regulator of the PI 3-kinase signalling pathway that is essential for maintaining cellular homeostasis. Structural similarities between Ruk, CIN85 and CD2AP/CMS suggest that these proteins form a novel family of adaptor molecules that are involved in various intracellular signalling pathways.

[Genomic organization of the murine neuro-d4 gene]. / Genomnaia organizatsiia gena neuro-d4 myshi.

As the first step toward obtaining the null mutation or knock out of the neuro-d4 gene, we isolated phages containing fragments of the gene from a mouse genomic library. The nucleotide sequence of a region of the gene more than 10 kb in size was determined.

Mutations in the gene encoding human persyn are not associated with amyotrophic lateral sclerosis or familial Parkinson's disease.

The synucleins are a family of small proteins expressed in nervous tissue, which have been implicated in neurodegeneration. Using single strand conformation polymorphism analysis we screened for polymorphisms and mutations in the gene encoding human persyn, a recently discovered member of the synuclein family, in controls, patients with sporadic or familial amyotrophic lateral sclerosis (ALS) or familial Parkinson's disease (PD). Six polymorphisms in the genomic sequence of persyn were detected; A590C (5' untranslated region), G1943C (exon 3), G2049A (intron 3), T4502C (intron 3), T4552A (exon 4) and C5019T (3' untranslated region). However no associations with disease state were found in our sample group.

Genomic structure and chromosomal localization of the mouse persyn gene.

Synucleins are a family of small intracellular proteins expressed mainly in the nervous system. The involvement of synucleins in neurodegeneration and malignancy has been demonstrated, but the physiological functions of these proteins remain elusive. Further studies including generation of animals with modified persyn expression are necessary to clarify the functions of these proteins and the mechanisms of their involvement in human diseases. We cloned and determined the organization and chromosomal localization of the mouse gene coding for persyn, a member of the synuclein family. The gene is composed of five exons, and its general structure is very similar to that of the human persyn gene. Using fluorescence in situ hybridization, we assigned the persyn gene to the boundary of bands B and C on mouse chromosome 14. We found a fragment of the gene that directs expression of the persyn protein in sensory neurons and could be used for generation of transgenic animals.

Denervation of the skin following section of the inferior alveolar nerve leads to increased NGF accumulation without change in NGF mRNA expression.

Following inferior alveolar nerve section and capping of the nerve to prevent regeneration, amounts of nerve growth factor (NGF) and NGF mRNA have been quantified in the chin skin, a discrete target of the nerve. NGF protein in the target region increases rapidly following nerve section and to levels known to induce sprouting. NGF on the ipsilateral side increased many-fold above unoperated control and the contralateral side also increased above control. Measurement of NGF mRNA levels using quantitative Northern analysis revealed, however, that there was no change in the expression levels of NGF mRNA, indicating that the large increase in protein occurs due to a cessation of transport back to the cell bodies of the innervating neurons.

Developmentally regulated expression of persyn, a member of the synuclein family, in skin.

Synucleins constitute a group of unique, evolutionarily conserved proteins that are expressed predominantly in neurons of the central and peripheral nervous system. Although the normal cellular functions of synucleins are not clear, these proteins have been implicated in various neurodegenerative conditions in humans. We found that persyn, a recently characterized member of the synuclein family, is expressed not only in the nervous system but also in the stratum granulosum of the epidermis of neonatal and adult mice. This finding together with our recent observations that persyn influences neurofilament network integrity in sensory neurons raises the possibility that persyn in skin could be involved in modulation of the keratin network.