Andrei Surguchov

Molecular and cellular biology of synucleins.

Synucleins are small, soluble proteins expressed primarily in neural tissues and certain tumors. The family includes three known proteins: alpha-synuclein, beta-synuclein, and gamma-synuclein. A typical structural feature of synucleins is the presence of a repetitive, degenerative AA motif KTKEGV throughout the first 87 residues and acidic stretches within the C-terminal region. Members of the synuclein family are natively unfolded proteins that are characterized by a high net charge and low hydropathy. The synuclein family recently came into the spotlight when one of its members, alpha-synuclein, was linked both genetically and neuropathologically to Parkinsons disease. It has a role in other neurodegenerative diseases, such as dementia with Lewy bodies, multiple system atrophy, neurodegeneration with brain iron accumulation type 1, and Alzheimers disease. Interestingly, another member of the family, beta-synuclein, possesses antagonistic properties to alpha-synuclein. The third member of the family, gamma-synuclein, is implicated in different types of cancer, some neurodegenerative diseases and ocular pathology. The involvement of synuclein proteins in the etiology of common human diseases has raised exciting questions and is currently the subject of intense investigation.

Conformational diseases: Looking into the eyes

Conformational diseases, a general term comprising more than 40 disorders are caused by the accumulation of unfolded or misfolded proteins. Improper protein folding (misfolding) as well as accrual of unfolded proteins can lead to the formation of disordered (amorphous) or ordered (amyloid fibril) aggregates. The gradual accumulation of protein aggregates and the acceleration of their formation by stress explain the characteristic late or episodic onset of the diseases. The best studied in this group are neurodegenerative diseases and amyloidosis accompanied by the deposition of a specific aggregation-prone proteins or protein fragments and formation of insoluble fibrils. Amyloidogenic protein accumulation often occurs in the brain tissues, e.g. in Alzheimers disease with the deposition of amyloid-beta and Tau, in scrapie and bovine spongiform encephalopathy with the accumulation of prion protein, in Parkinsons disease with the deposition of alpha-synuclein. Other examples of amyloid proteins are transthyretin, immunoglobulin light chain, gelsolin, etc. In addition to the brain, the accumulation of unfolded or misfolded proteins leading to pathology takes place in a wide variety of organs and tissues, including different parts of the eye. The best studied ocular conformational diseases are cataract in the lens and retinitis pigmentosa in the retina, but accumulation of misfolded proteins also occurs in other parts of the eye causing various disorders. Furthermore, ocular manifestation of systemic amyloidosis often causes the deposition of amyloidogenic proteins in different ocular tissues. Here we present the data regarding naturally unfolded and misfolded proteins in eye tissues, their structure-function relationships, and molecular mechanisms underlying their involvement in diseases. We also summarize the etiology of ocular conformational diseases and discuss approaches to their treatment.

Effect of γ-synuclein overexpression on matrix metalloproteinases in retinoblastoma Y79 cells

gamma-Synuclein is a small cytoplasmic protein implicated in neurodegenerative diseases and cancer. However, the mechanism of its involvement in diseases is not clear. We studied the role of gamma-synuclein in the regulation of matrix metalloproteinases in retinoblastoma cell culture. Matrix metalloproteinases play important roles in the remodeling of extracellular matrix implicated in tumor progression and in the neurodegenerative diseases. Western blot and zymography data demonstrated a moderate elevation of matrix metalloproteinases-2 and significant upregulation of matrix metalloproteinases-9 in stable cell lines overexpressing gamma-synuclein. No effect of gamma-synuclein overexpression on matrix metalloproteinases-1 level or activity was found. Chloramphenicol-acetyltransferase assay demonstrated that overexpression of gamma-synuclein increases the efficiency of the matrix metalloproteinases-9 promoter. This increment of promoter activity may be mediated by the AP-1 binding site(s), since point mutations in one of these sites (Pr18 or Pr19) and elimination of the distal AP-1 site (Pr14) reduced the increment of promoter activity.

Protein aggregation in retinal cells and approaches to cell protection.

Summary1.Retinal dystrophies (RD) comprise a group of clinically and genetically heterogeneous retinal disorders, which typically result in the degeneration of photoreceptors followed by the impairment or loss of vision. Although age-related macular degeneration (AMD) and retinitis pigmentosa (RP) are among the most common forms of RD, currently, there is no effective treatment for either disorder.2.Recently, abnormal protein accumulation and aggregation due to protein misfolding and proteasome inhibition have been implicated in the pathogenesis of RD. In this paper we describe effects of several factors on protein aggregation and survival of photoreceptor cells.3.Expression of rhodopsin carrying P23H mutation causes its accumulation in intracellular inclusion bodies in a perinuclear area of photoreceptor cells. β- and γ-synucleins and heat shock protein Hsp-70, but not α-synuclein, protect cultured ocular cells from mutant opsin accumulation. This effect might be explained by their chaperonic activity.4.Knock-out of α- and γ-synucleins does not affect gross retinal morphology, but induces tyrosine hydroxylase in the inner prexiform layer of the retina. Selegiline—a monoamine oxidase inhibitor used for the treatment of Parkinsons disease, reduces apoptosis and increases viability in cultured retinal pigment epithelium cells (APRE-19).5.These results suggest that chaperones and selegiline may be considered promising candidates for the protection of ocular cells from the accumulation of misfolded and aggregated proteins.

Retinal involvement in dementia with Lewy bodies: A clue to hallucinations?

Visual hallucinations are a core feature of dementia with Lewy bodies. Their pathophysiology is not well understood, because neither clinical nor histological data have shown their basic mechanisms. Here, we report the presence of pale inclusions in the outer plexiform layer of the retina in a patient with dementia with Lewy bodies. These inclusions are related to cytoskeletal disorganization of the cones at ultrastructural level and modifications of the immunohistochemical pattern of distribution of synucleins in the retina. These modifications may participate in the visual impairment in dementia with Lewy bodies. Ann Neurol 2003;54:542‐547

Synucleins: Are they two‐edged swords?

The synuclein family consists of three distinct highly homologous genes, α‐synuclein, β‐synuclein, and γ‐synuclein, which have so far been found only in vertebrates. Proteins encoded by these genes are characterized by an acidic C‐terminal region and five or six imperfect repeat motifs (KTKEGV) distributed throughout the highly conserved N‐terminal region. Numerous data demonstrate that synucleins are implicated in two groups of the most devastating human disorders, i.e., neurodegenerative diseases (NDDs) and cancer. Mutations in the α‐synuclein gene are associated with familial forms of Parkinsons disease (PD), and accumulation of α‐synuclein inclusions is a hallmark of this disorder. In breast cancer, increased expression of γ‐synuclein correlates with disease progression. Conversely, some results indicate that the members of the synuclein family may have a protective effect. How might these small proteins combine such controversial properties? We present evidence that synucleins features are basically regulated by two mechanisms, i.e., posttranslational modifications (PTMs) and the level of their expression. We also discuss a new, emerging area of investigation of synucleins, namely, their role in the cell‐to‐cell propagation of pathology.

Interaction of Myocilin with γ-Synuclein Affects Its Secretion and Aggregation

SummaryMutations in the gene encoding human myocilin are associated with some cases of juvenile and early-onset glaucoma. Glaucomatous mutations prevent myocilin from being secreted. The analysis of the defects associated with mutations point to the existence of factor(s) in addition to mutations that might be implicated in the development of glaucoma. In the present paper, we found that interaction of myocilin with one of the members of the synuclein family alters its properties, including its ability to be secreted. Results of immunoprecipitation show that myocilin is a γ-synuclein-interacting protein. Further analysis demonstrated that both myocilin and γ-synuclein are expressed in human TM cells, immortalized rat ganglion (RGC-5) cells, and HT22 hippocampal neurons. According to Western blotting, in addition to monomeric form with molecular weight 17 kDa γ-synuclein is present as higher molecular weight forms (∼35 and 68 KDa), presumably dimer and tetramer. Myocilin and γ-synuclein have partially overlapping perinuclear localization. Dexamethasone upregulates myocilin expression in RGC-5 cells and HT22 hippocampal neurons. We found alterations of myocilin properties as a result of its interaction with γ-synuclein. In cultured cells, γ-synuclein upregulates myocilin expression, inhibits its secretion and prevents the formation of high molecular weight forms of myocilin. Although both α-synuclein and γ-synuclein are expressed in HTM cells, only γ-synuclein interacts with myocilin and alters its properties.We conclude that myocilin and γ-synuclein interact and as a result, myocilins properties are changed. Since myocilin and γ-synuclein have partially overlapping intracellular localization in cell types that are implicated in glaucoma development, their interaction may play an important role in glaucoma.

γ-Synuclein: Cell-Type-Specific Promoter Activity and Binding to Transcription Factors

Abstractγ-Synuclein, also referred to as breast-cancer-specific gene 1, is the third member of the neuronal protein family synuclein. Synucleins attracted the attention of many investigators because of their role in human diseases. γ-Synuclein participates in the pathogenesis of several types of cancer and some neurodegenerative diseases. Its role in tumorigenesis is due to the upregulation of transcription and the effect on downstream targets, including signaling pathways and transcription factors. γ-Synuclein is also expressed in neurons and glial cells, but the regulation of its expression, as well as the mechanism of transition from normal functions to pathology in these cell types, is not studied. Here, we examined how γ-synuclein promoter is regulated in neuronal and glial cells. We also show that γ-synuclein is able to bind directly to several transcription factors. These results are discussed in connection with the implication of γ-synuclein in diseases.

Intracellular Dynamics of Synucleins: "Here, There and Everywhere".

Synucleins are small, soluble proteins expressed primarily in neural tissue and in certain tumors. The synuclein family consists of three members: α-, β-, and γ-synucleins present only in vertebrates. Members of the synuclein family have high sequence identity, especially in the N-terminal regions. The synuclein gene family came into the spotlight, when one of its members, α-synuclein, was found to be associated with Parkinsons disease and other neurodegenerative disorders, whereas γ-synuclein was linked to several forms of cancer. There are a lot of controversy and exciting debates concerning members of the synuclein family, including their normal functions, toxicity, role in pathology, transmission between cells and intracellular localization. Important findings which remain undisputable for many years are synuclein localization in synapses and their role in the regulation of synaptic vesicle trafficking, whereas their presence and function in mitochondria and nucleus is a debated topic. In this review, we present the data on the localization of synucleins in two intracellular organelles: the nucleus and mitochondria.

New α- and γ-synuclein immunopathological lesions in human brain

IntroductionSeveral neurodegenerative diseases are classified as proteopathies as they are associated with the aggregation of misfolded proteins. Synucleinopathies are a group of neurodegenerative disorders associated with abnormal deposition of synucleins. α-Synucleinopathies include Parkinsons disease, dementia with Lewy bodies, and multiple system atrophy. Recently accumulation of another member of the synuclein family- γ–synuclein in neurodegenerative diseases compelled the introduction of the term γ–synucleinopathy. The formation of aggregates and deposits of γ–synuclein is facilitated after its oxidation at methionine 38 (Met38).ResultsSeveral types of intracytoplasmic inclusions containing post-translationally modified α- and γ–synucleins are detected. Oxidized Met38-γ-synuclein forms aberrant inclusions in amygdala and substantia nigra. Double staining revealed colocalization of oxidized-γ-synuclein with α-synuclein in the cytoplasm of neurons. Another type of synuclein positive inclusions in the amygdala of dementia with Lewy bodies patients has the appearance of Lewy bodies. These inclusions are immunoreactive when analyzed with antibodies to α-synuclein phosphorylated on serine 129, as well as with antibodies to oxidized-γ-synuclein. Some of these Lewy bodies have doughnut-like shape with round or elongated shape. The separate immunofluorescent images obtained with individual antibodies specific to oxidized-γ-synuclein and phospho-α-synuclein clearly shows the colocalization of these synuclein isoforms in substantia nigra inclusions. Phospho-α-synuclein is present almost exclusively at the periphery of these structures, whereas oxidized-γ-syn immunoreactivity is also located in the internal parts forming dot-like pattern of staining.We also identified several types of oxidized-γ-syn positive astrocytes with different morphology and examined their immunohistochemical phenotypes. Some of them are compact cells with short processes, others have longer processes. Oxidized-γ-synuclein positive astrocytes may also display mixed morphological and immunocytochemical phenotypes between protoplasmic and fibrous astrocytes.ConclusionsThese results reveal new γ–synuclein positive lesions in human brain. Oxidized-γ-synuclein is colocalized with phospho-α-synuclein in doughnut-like inclusions. Several types of astrocytes with different morphology are immunopositive for oxidized-γ-synuclein.