Pericentrin expression in Down’s syndrome

Down’s syndrome (DS) is the most frequent genetic cause of intellectual disability and is a chromosomal abnormality of chromosome 21 trisomy. The pericentrin gene (PCNT) has sequenced in 21q22.3 inside of the minimal critical region for Down’s syndrome. Alterations of PCNT gene are associated with dwarfism, cardiomyopathy and other pathologies. In this study, we have evaluated the possible differential expression of PCNT mRNA, by qRT-PCR, in peripheral blood leukocytes of DS subjects compared with the normal population. In the present case–control study, PCNT gene expression was increased by 72.72 % in 16 out 22 DS samples compared with normal subjects. Our data suggest that changes in the expression levels of PCNT in DS subjects may be involved into the molecular mechanism of Down’s syndrome.     

Biochemical and morphological consequences of human α-synuclein expression in a mouse α-synuclein null background

A consensus about the functions of human wild-type or mutated α-synuclein (αSYN) is lacking. Both forms of αSYN are implicated in Parkinson's disease, whereas the wild-type form is implicated in substance abuse. Interactions with other cellular proteins and organelles may meditate its functions. We developed a series of congenic mouse lines containing various allele doses or combinations of the human wild-type αSYN (hwαSYN) or a doubly mutated (A30P*A53T) αSYN (hm(2) αSYN) in a C57Bl/6J line spontaneously deleted in mouse αSYN (C57BL/6JOla). Both transgenes had a functional role in the nigrostriatal system, demonstrated by significant elevations in striatal catecholamines, metabolites and the enzyme tyrosine hydroxylase compared with null-mice without a transgene. Consequences occurred when the transgenes were expressed at a fraction of the endogenous level. Hemizygous congenic mice did not exhibit any change in the number or size of dopaminergic neurons in the ventral midbrain at 9 months of age. Human αSYN was predominantly located in neuronal cell bodies, neurites, synapses, and in intraneuronal/intraneuritic aggregates. The hm(2) αSYN transgene resulted in more aggregates and dystrophic neurites than did the hw5 transgene. The hwαSYN transgene resulted in higher expression of two striatal proteins, synaptogamin 7 and UCHL1, compared with the levels of the hm(2) αSYN transgene. These observations suggest that mutations in αSYN may impair specific functional domains, leaving others intact. These lines may be useful for exploring interactions between hαSYN and environmental or genetic risk factors in dopamine-related disorders using a mouse model.     

Parkinson's disease and α-synuclein expression

Genetic studies of Parkinson's disease over the last decade or more have revolutionized our understanding of this condition. α-Synuclein was the first gene to be linked to Parkinson's disease, and is arguably the most important: the protein is the principal constituent of Lewy bodies, and variation at its locus is the major genetic risk factor for sporadic disease. Intriguingly, duplications and triplications of the locus, as well as point mutations, cause familial disease. Therefore, subtle alterations of α-synuclein expression can manifest with a dramatic phenotype. We outline the clinical impact of α-synuclein locus multiplications, and the implications that this has for Parkinson's disease pathogenesis. Finally, we discuss potential strategies for disease-modifying therapies for this currently incurable disorder.     

Decreased α-synuclein expression in the aging mouse substantia nigra

Because of its normal function in synaptic plasticity and pathologic involvement in age-related neurodegenerative diseases, the protein α-synuclein could play an important role in aging processes. Here we compared α-synuclein expression in the substantia nigra and other brain regions of young (2-month-old), middle-aged (10-month-old), and old (20-month-old) mice. Levels of nigral α-synuclein mRNA, as assessed by both in situ hybridization and qPCR, were high in young mice and progressively declined in middle-aged and old animals. This age-dependent mRNA loss was paralleled by a marked reduction of α-synuclein protein; immunoreactivity of midbrain sections stained with an anti-α-synuclein antibody was most robust in 2-month-old mice and weakest in 20-month-old animals. Lowering of nigral α-synuclein could not be explained by a loss of dopaminergic neurons and was relatively specific since no change in β-synuclein mRNA and protein occurred with advancing age. Finally, age-related decreases in α-synuclein were widespread throughout the mouse brain, affecting other regions (e.g., hippocampus) besides the substantia nigra. The data suggest that loss of α-synuclein could contribute to or be a marker of synaptic dysfunction in the aging brain. They also emphasize important differences in α-synuclein expression between rodents and primates since earlier reports have shown a marked elevation of α-synuclein protein in the substantia nigra of older humans and non-human primates.     

Increase expression of α-synuclein in aged human brain associated with neuromelanin accumulation

Although the increased prevalence of Parkinson’s disease (PD) with aging suggests that aging processes predispose dopamine neurons to degeneration, the mechanism involved remains unknown. Dopamine neurons contain significant amounts of neuromelanin, and the amount of neuromelanin increases with aging. In the present study, age-related changes in the number of nigral neurons expressing neuromelanin (NM), α-synuclein, and tyrosine hydroxylase (TH) were stereologically analyzed in the postmortem brains of 28 healthy humans with an age range of 17–84 years. Stereological counting of NM content, α-synuclein content, and TH immunoreactivity revealed significant accumulation of NM and α-synuclein in neurons during the aging process. In cells containing a large amount of NM, α-synuclein-immunoreactive cells in aged individuals outnumbered those of younger individuals. In non-NM cells, the α-synuclein expression profile was similar across age groups. Furthermore, TH-immunoreactive neurons decreased significantly with aging, which was associated with accumulation of NM and α-synuclein. Our results suggest that age related accumulation of NM might induce α-synuclein over-expression and thereby make dopamine neurons more vulnerable to injuries.     

Variable expression of mitochondrial complex IV in the course of nigral intracellular accumulation of α-synuclein

IntroductionParkinson's disease (PD) is a neurodegenerative disease characterized by the deposition of disease-associated α-synuclein, which is thought to follow a sequential distribution in the human brain. Accordingly, α-Synuclein pathology affects the substantia nigra (SN) only in Braak stage 3 out of 6. Moreover, intracellular accumulation of α-synuclein follows maturation from non-ubiquitinated (p62 negative) to ubiquitinated (p62 positive) forms (Lewy bodies). Mitochondrial dysfunction is thought to be a central player in the pathogenesis of PD. It is not clear whether the nigral neurons already show mitochondrial alterations in stages preceding the deposition of α-synuclein in the SN, and how deposition of pre-aggregates or ubiquitinated mature inclusions relate to this.MethodsUsing cell-based morphometric immunohistochemistry we evaluated the volume density of mitochondrial complex-IV (COX-IV) immunoreactivity in SN neurons lacking or showing α-synuclein deposits in non-diseased individuals and those with Lewy body pathology Braak stage <3 lacking nigral α-synuclein pathology and Braak stage >3 with prominent nigral α-synuclein deposition.ResultsIncreased volume density of COX-IV immunoreactivity appears before detectable pathological α-synuclein in nigral neurons. The volume density decreases significantly as pathological pre-aggregates of α-synuclein accumulates in the neurons and remains at a low level in neurons with p62 positive Lewy bodies.ConclusionsCOX-IV expression shows a change before and during accumulation of α-synuclein in the SN underpinning the role of early mitochondrio protective therapy strategies in PD.     

The developmental and aging changes of Down’s syndrome cell adhesion molecule expression in normal and Down’s syndrome brains

We studied the expression of Down’s syndrome cell adhesion molecule (DSCAM) in Down’s syndrome (DS) and control brains, using antisera against peptide fragments of DSCAM. On Western blots of human, mouse and rat brain homogenates, the antisera recognized a product at approximately 200 kDa. In the brain of a 2-year-old patient with DS, Western blotting revealed an overexpression of DSCAM compared to an age-matched control. Immunohistochemistry demonstrated DSCAM in the cerebral and cerebellar white matter of both control and DS subjects, in accordance with the temporal and spatial sequence of myelination. In DS brains, immunoreactivity for DSCAM, compared to that for controls, was enhanced in the Purkinje cells at all ages, and in the cortical neurons during adulthood. In demented DS patients, DSCAM immunoreactivity was observed in the core and periphery of senile plaques. The pattern of DSCAM expression suggests that it may play a role as an adhesion molecule regulating myelination. The overexpression of DSCAM may also play a role in the mental retardation and the precocious dementia of DS patients, although the mechanism of neuronal dysfunction is undetermined. Received: 13 December 1999 / Revised, accepted: 23 February 2000     

Absence of α-synuclein pathology in postencephalitic parkinsonism

Postencephalitic parkinsonism (PEP), a chronic complication of encephalitis lethargica, is a tauopathy characterized by multisystem neuronal loss and gliosis with widespread neurofibrillary lesions composed of both 3- and 4-repeat (3R and 4R) tau isoforms. Previous immunohistochemical studies in a small number of PEP cases demonstrated absence of Lewy bodies as well as the lack of other α-synuclein pathology, classifying PEP as a “pure” tauopathy. Neuropathologic examination of 10 brains with clinico-pathologically verified PEP confirmed widespread neurodegeneration in subcortical and brainstem areas associated with multifocal neurofibrillary pathology comprising both 3R and 4R tau. Very rare β-amyloid deposits were observed in two elderly patients, while Lewy bodies and neurites or any other α-synuclein deposits were completely absent. The causes and molecular background of total absence of α-synuclein pathology in PEP, in contrast to most other tauopathies, remain as unknown as the pathogenesis of PEP.     

Pathological roles of α-synuclein in neurological disorders

Substantial genetic, neuropathological, and biochemical evidence implicates the presynaptic neuronal protein α-synuclein in Parkinson's disease and related Lewy body disorders. How dysregulation of α-synuclein leads to neurodegeneration is, however, unclear. Soluble oligomeric, but not fully fibrillar, α-synuclein is thought to be toxic. The major neuronal target of aberrant α-synuclein might be the synapse. The effects of aberrant α-synuclein might include alteration of calcium homoeostasis or mitochondrial fragmentation and, in turn, mitochondrial dysfunction, which could link α-synuclein dysfunction to recessive and toxin-induced parkinsonism. α-Synuclein also seems to be linked to other genetic forms of Parkinson's disease, such as those linked to mutations in GBA or LRRK2, possibly through common effects on autophagy and lysosomal function. Finally, α-synuclein is physiologically secreted, and this extracellular form could lead to the spread of pathological accumulations and disease progression. Consequently, factors that regulate the levels, post-translational modifications, specific aberrant cellular effects, or secretion of α-synuclein might be targets for therapy.     

cDNA cloning, prokaryotic expression and purification of rat α-synuclein

Objective To clone the cDNA of rat α-Syn gene, investigate its prokaryotic expression and produce purified recombinant rat α-Syn protein. Methods Rat α-Syn cDNA was amplified from the rat brain total RNA by RT-PCR and was cloned into pGEX-4T-1, a prokaryotic expressing vector. The recombinant plasmid containing rat α-Syn gene was transformed into E. Coli BL21 to express a fusion protein with rat α-Syn protein tagged by glutathione-S-transferase (GST). The fusion protein was then cleaved by thrombin during passing through the GST-agarose 4B column to release the recombinant rat α-Syn protein. The recombinant rat α-Syn protein was further purified using Superdex S200 gel filtration. Results DNA sequencing confirmed that the cloned cDNA contained 420 base pairs encoding 140 amino acids, which was identical to the reported amino acid sequence of rat α-Syn. After transformation, the recombinant plasmid pGEX-ra-Syn expressed a soluble protein that was inducible by IPTG. The purified recombinant protein was shown to be single band on SDS-PAGE, with a molecular size of around 18000, which was identical to the reported molecular size of rat α-Syn. Western blot analysis demonstrated that the recombinant protein was recognized by specific antibody against α-Syn. Conclusion The rat α-Syn gene was successfully expressed in prokaryotic expression system and highly purified rat α-Syn recombinant protein was produced.     

Polymorphisms of the α-synuclein promoter: expression analyses and association studies in Parkinson's disease

Summary. Mutations of the α-synuclein gene have shown to be relevant in some rare families with autosomal dominant Parkinson's disease (PD). Furthermore, α-synuclein protein is a major component of the Lewy bodies also in sporadic PD patients. Increased levels of wildtype α-synuclein in the cell leads to increased intracellular hydrogen peroxide levels and causes death of dopaminergic neurons in rat primary culture. Subsequently, oxidative stress has been directly linked with α-synuclein aggregation in vitro. This raises the question whether increased α-synuclein expression might be linked to higher susceptibility to PD and whether α-synuclein promoter polymorphisms are associated with PD. Here, two polymorphisms (−116C>G and −668T>C) of the α-synuclein promoter defining four haplotypes have been characterized in 315 German PD patients. The influence of the four haplotypes on gene expression was studied by CAT reporter gene assays in neuronal SK-N-AS cells. The −668C/−116G haplotype revealed significant higher CAT expression than the −668T/−116G or the −668T/−116C haplotype, respectively. Although the −668C/−116G haplotype was more common in PD patients, this difference was not significant. Received January 8, 2002; accepted June 10, 2002 Published online August 22, 2002 Authors' address: O. Riess, M.D., Department of Medical Genetics, University of Tübingen, Calwerstrasse 7, D-72076 Tübingen, Federal Republic of Germany, e-mail: olaf.riess@med.uni-tuebingen.de     

The Janus face of N-terminal lysines in α-synuclein

Parkinson’s disease(PD)is the second most prevalent progressive neurodegenerative disorder after Alzheimer’s disease.PD usually starts with a tremor in the extremities(usually in the hands)and gradually evolves with other symptoms such as bradykinesia,muscle stiffness,impaired posture,loss of automatic movements or speech changes.These symptoms worsen as the condition progresses and eventually lead to death.     

A comparison of the expression of α-synuclein mRNA in the brain of rats with different levels of alcohol consumption

Recent studies suggest that the presynaptic protein α-synuclein has one of the key positions in the regulation of the functions of the dopamine system and the mechanisms of different addictions, including alcohol addiction. However, the role of α-synuclein at certain stages of addiction formation is still unclear. In particular, on the basis of the data from different studies it is not possible to make an unambiguous conclusion on the direct influence of alcohol on the expression of the α-synuclein gene in the brain and, if it occurs, whether these changes are a neuroadaptive response or, in contrast, one of key links of addiction pathogenesis. The task of the current study was to compare the expression of the α-synuclein gene in the brain of animals that chronically consumed alcohol in a “free-choice?model and had different level of its consumption. From the 60th to 120th days of life, outbred male Wistar rats were maintained in individual cages with two drinking bottles that contained 10% ethanol solution and water. Ethanol consumption was evaluated daily in g/kg body weight and the preference was estimated as the percentage of the total volume of liquid consumed. At an age of 120 days, the animals were decapitated and the midbrain, striatum, hypothalamus, and amygdala were dissected. The levels of expression of α-synuclein mRNA were determined by the real-time quantitative polymerase chain reaction (RT-PCR). Our results indicate that animals with a high level of alcohol consumption (a daily average consumption of ethanol 10.4 ± 0.79 g/kg) had significantly decreased indices of α-synuclein expression in the midbrain (by 45%) and hypothalamus (by 42%) as compared to animals with a permanently low level of consumption (3.6 ± 0.46 g/kg). These changes were found in the brain areas that form the dopaminergic pathways in the brain but not in the areas that are the targets of dopamine neurons. The data we obtained support the hypothesis on the possible “protective?role of α-synuclein in the dopamine-synthesizing brain structures of animals with a low level of alcohol preference and the ability to “control?intake by its maintenance at a constant level.     

Characterization of kinases involved in the phosphorylation of aggregated α-synuclein

α-Synuclein (α-syn) is the major component of pathological inclusions characteristic of several neurodegenerative disorders, such as Parkinson's disease. The major posttranslational modification of α-syn is phosphorylation at S129, and previous studies estimate that approximately 90% of α-syn in proteinaceous, pathological inclusions is phosphorylated at this site. α-Syn can be phosphorylated by polo-like kinases (PLKs) 1-3 and casein kinases (CK) 1 and 2; however, the kinases associated with the hyperphosphorylation of aggregated α-syn are still under debate. Using a high-efficiency cellular model of α-syn aggregate formation, we found that selective inhibitors for CK2 and PLKs each partially inhibited S129 phosphorylation of soluble (nonaggregated) α-syn, but only PLK inhibitors modestly attenuated the phosphorylation of aggregated α-syn. In addition, none of the kinase inhibitors used had a substantial effect on the propensity of α-syn to aggregate. Overexpression of all PLKs promoted robust phosphorylation of soluble α-syn, but none altered the propensity of α-syn to aggregate. Overexpression of only PLK2 increased phosphorylation of aggregated α-syn at S129, which likely is due to increased phosphorylation of soluble α-syn, which then was incorporated into aggregates. Overexpression of PLK1 and treatment with BI2536 resulted in a significant reduction of phosphorylated, aggregated α-syn protein, beyond that of BI2536 treatment alone. These studies suggest that phosphorylation of α-syn is independent of α-syn aggregate formation, that PLK1 is involved in the phosphorylation of aggregated α-syn at S129 in this system, and that mechanisms resulting in hyperphosphorylation of aggregated α-syn appear to be independent of those responsible for the phosphorylation of soluble α-syn.     

Curcumin inhibits aggregation of α-synuclein

Aggregation of amyloid-beta protein (Aβ) is a key pathogenic event in Alzheimer’s disease (AD). Curcumin, a constituent of the Indian spice Turmeric is structurally similar to Congo Red and has been demonstrated to bind Aβ amyloid and prevent further oligomerization of Aβ monomers onto growing amyloid β-sheets. Reasoning that oligomerization kinetics and mechanism of amyloid formation are similar in Parkinson’s disease (PD) and AD, we investigated the effect of curcumin on α-synuclein (AS) protein aggregation. In vitro model of AS aggregation was developed by treatment of purified AS protein (wild-type) with 1 mM Fe³⁺ (Fenton reaction). It was observed that the addition of curcumin inhibited aggregation in a dose-dependent manner and increased AS solubility. The aggregation-inhibiting effect of curcumin was next investigated in cell culture utilizing catecholaminergic SH-SY5Y cell line. A model system was developed in which the red fluorescent protein (DsRed2) was fused with A53T mutant of AS and its aggregation examined under different concentrations of curcumin. To estimate aggregation in an unbiased manner, a protocol was developed in which the images were captured automatically through a high-throughput cell-based screening microscope. The obtained images were processed automatically for aggregates within a defined dimension of 1–6 μm. Greater than 32% decrease in mutant α-synuclein aggregation was observed within 48 h subsequent to curcumin addition. Our data suggest that curcumin inhibits AS oligomerization into higher molecular weight aggregates and therefore should be further explored as a potential therapeutic compound for PD and related disorders. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Neuromelanin enhances the toxicity of α-synuclein in SK-N-SH cells

The key pathological feature of Parkinson's disease (PD) is selective degeneration of the neuromelanin (NM)-pigmented dopaminergic neurons in the substantia nigra (SN). NM, like other risk factors, such as oxidative stress (OS) and α-synuclein (α-syn), is involved in the pathogenesis of PD. But whether or not NM synergizes with α-syn or OS in the pathogenesis of PD remains unexplored. In the present study, we examined the effects of NM on cellular viability, apoptosis and free radical production in α-syn over-expressing human neuroblastoma cell line (SK-N-SH) in the presence or absence of the oxidizer Fenton's Reagent (FR). We showed that NM synergized with FR in suppressing cell viability, and in inducing apoptosis and hydroxyl radical production in all SK-N-SH cell lines. α-Syn over-expressing cells exhibited more pronounced effect, especially the A53T mutation. Our findings suggest that NM synergizes with both OS and α-syn in conferring dopaminergic vulnerability, adding to our understanding of the pathogenesis of PD.     

Accumulation of toxic α-synuclein oligomer within endoplasmic reticulum occurs in α-synucleinopathy in vivo

In Parkinson's disease (PD) and other α-synucleinopathies, prefibrillar α-synuclein (αS) oligomer is implicated in the pathogenesis. However, toxic αS oligomers observed using in vitro systems are not generally seen to be associated with α-synucleinopathy in vivo. Thus, the pathologic significance of αS oligomers to αS neurotoxicity is unknown. Herein, we show that, αS that accumulate within endoplasmic reticulum (ER)/microsome forms toxic oligomers in mouse and human brain with the α-synucleinopathy. In the mouse model of α-synucleinopathy, αS oligomers initially form before the onset of disease and continue to accumulate with the disease progression. Significantly, treatment of αS transgenic mice with Salubrinal, an anti-ER stress compound that delays the onset of disease, reduces ER accumulation of αS oligomers. These results indicate that αS oligomers with toxic conformation accumulate in ER, and αS oligomer-dependent ER stress is pathologically relevant for PD.