Intranuclear ubiquitin-immunopositive structures in human substantia nigra neurons

Marinesco bodies were discovered in substantia nigra neurons of human brain in 1902. The relationships between these intranuclear inclusions and the other structures of the cellular nucleus are still obscure. The aim of this study is to elucidate the morphological and cytochemical peculiarities of intranuclear ubiquitin-immunopositive bodies in the substantia nigra neurons of human brain and to evaluate the interconnections of these peculiarities with nucleolus by means of light microscopy, immunocytochemistry, and confocal laser microscopy. It is found that up to 20% of neurons in substantia nigra of human brain contain ubiquitin-immunopositive Marinesco bodies. These rounded structures are 1–8 μm—more often 2–4 μm—in diameter. Only one-third of them are tightly adjacent to the nucleolus. By a method of silver impregnation of argentophilic proteins associated with nucleolar organizer, the absence was shown of argentophilic proteins, which are characteristic for the nucleolus, in Marinesco bodies. Special ubiquitin-positive substantially smaller structures (less than 1 μm) are revealed in the neurons’ nuclei along with Marinesco bodies. These structures are probably the initial forms in the formation of Marinesco bodies. The existence of two types of ubiquitin-immunopositive intranuclear bodies is revealed by means of confocal microscopy: one has high intensity of immunofluorescence, and the other has low intensity. Heterogeneous distribution of immunopositive product is characteristic of the former. The presence of DNA in Marinesco bodies is detected by using SYTOX Green fluorescent dye. The absence of peripheral heterochromatin zone and weak susceptibility to toluidine blue together with the presence of DNA and the absence of argentophilic proteins suggests substantial structural and chemical differences between Marinesco bodies and nucleoli, which argues against the idea that the detected bodies are modified nucleoli.     

The TrkB-Positive Dopaminergic Neurons are Less Sensitive to MPTP Insult in the Substantia Nigra of Adult C57/BL Mice

Tyrosine kinase receptors TrkB and TrkC mediate neuroprotective effects of the brain-derived neurotrophic factor (BDNF) and neurotrophins in the dopaminergic nigro-striatal system, but it is obscure about their responses or expression changes in the injured substantia nigra under Parkinson’s disease. In present study, immunofluorescence, Fluoro-Jade staining and laser scanning confocal microscopy were applied to investigate distribution and changes of TrkB and TrkC in the dopamine neurons of the substantia nigra by comparison of control and 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) mouse model. It revealed that TrkB and TrkC-immunoreactivities were substantially localized in cytoplasm and cell membrane of the substantia nigra neurons of control adults. While neurons double-labeled with tyrosine hydroxylase (TH)/TrkB, or TH/TrkC were distributed in a large numbers in the substantia nigra of controls, they apparently went down at 36.2–65.7% of normal level, respectively following MPTP insult. In MPTP model, cell apoptosis or degeneration of nigral neurons were confirmed by caspase-3 and Fluoro-Jade staining. More interestingly, TH/TrkB-positive neurons survived more in cell numbers in comparison with that of TH/TrkC-positive ones in the MPTP model. This study has indicated that TrkB-containing dopamine neurons are less sensitive in the substantia nigra of MPTP mouse model, suggesting that specific organization of Trks may be involved in neuronal vulnerability to MPTP insult, and BDNF-TrkB signaling may play more important role in protecting dopamine neurons and exhibit therapeutic potential for Parkinson’s disease.     

Increased Protein Oxidation in Human Substantia Nigra Pars Compacta in Comparison with Basal Ganglia and Prefrontal Cortex Measured with an Improved Dinitrophenylhydrazine Assay

Abstract: The dopaminergic phenotype of neurons in human substantia nigra deteriorates during normal aging, and loss of these neurons is prominent in Parkinson's disease. These degenerative processes are hypothesized to involve oxidative stress. To compare oxidative stress in the nigra and related regions, we measured carbonyl modifications of soluble proteins in postmortem samples of substantia nigra, basal ganglia, and prefrontal cortex from neurologically normal subjects, using an improved 2,4-dinitrophenylhydrazine assay. The protein carbonyl content was found to be about twofold higher in substantia nigra pars compacta than in the other regions. To further analyze this oxidative damage, the distribution of carbonyl groups on soluble proteins was determined by western immunoblot analysis. This method revealed that carbonyl content of the major proteins in each region was linearly dependent on molecular weight. This distribution raises the possibility that protein carbonyl content is controlled by a size-dependent mechanism in vivo. Our results suggest that oxidative stress is elevated in human substantia nigra pars compacta in comparison with other regions and that oxidative damage is higher within the dopaminergic neurons. Elevated oxidative damage may contribute to the degeneration of nigral dopaminergic neurons in aging and in Parkinson's disease.     

Influenza A H3N2 subtype virus NS1 protein targets into the nucleus and binds primarily via its C-terminal NLS2/NoLS to nucleolin and fibrillarin

ABSTRACT: BACKGROUND: Influenza A virus non-structural protein 1 (NS1) is a virulence factor, which is targeted into the cell cytoplasm, nucleus and nucleolus. NS1 is a multi-functional protein that inhibits host cell pre-mRNA processing and counteracts host cell antiviral responses. Previously, we have shown that the NS1 protein of the H3N2 subtype influenza viruses possesses a C-terminal nuclear localization signal (NLS) that also functions as a nucleolar localization signal (NoLS) and targets the protein into the nucleolus. RESULTS: Here, we show that the NS1 protein of the human H3N2 virus subtype interacts in vitro primarily via its C-terminal NLS2/NoLS and to a minor extent via its N-terminal NLS1 with the nucleolar proteins, nucleolin and fibrillarin. Using chimeric green fluorescence protein (GFP)-NS1 fusion constructs, we show that the nucleolar retention of the NS1 protein is determined by its C-terminal NLS2/NoLS in vivo. Confocal laser microscopy analysis shows that the NS1 protein colocalizes with nucleolin in nucleoplasm and nucleolus and with B23 and fibrillarin in the nucleolus of influenza A/Udorn/72 virus-infected A549 cells. Since some viral proteins contain NoLSs, it is likely that viruses have evolved specific nucleolar functions. CONCLUSION: NS1 protein of the human H3N2 virus interacts primarily via the C-terminal NLS2/NoLS and to a minor extent via the N-terminal NLS1 with the main nucleolar proteins, nucleolin, B23 and fibrillarin.     

Formation of a complex between nucleolin and replication protein A after cell stress prevents initiation of DNA replication

We used a biochemical screen to identify nucleolin, a key factor in ribosome biogenesis, as a high-affinity binding partner for the heterotrimeric human replication protein A (hRPA). Binding studies in vitro demonstrated that the two proteins physically interact, with nucleolin using an unusual contact with the small hRPA subunit. Nucleolin significantly inhibited both simian virus 40 (SV-40) origin unwinding and SV-40 DNA replication in vitro, likely by nucleolin preventing hRPA from productive interaction with the SV-40 initiation complex. In vivo, use of epifluorescence and confocal microscopy showed that heat shock caused a dramatic redistribution of nucleolin from the nucleolus to the nucleoplasm. Nucleolin relocalization was concomitant with a tenfold increase in nucleolin-hRPA complex formation. The relocalized nucleolin significantly overlapped with the position of hRPA, but only poorly with sites of ongoing DNA synthesis. We suggest that the induced nucleolin-hRPA interaction signifies a novel mechanism that represses chromosomal replication after cell stress.     

A 23-amino acid motif spanning the basic domain targets zebrafish myogenic regulatory factor myf5 into nucleolus

Myf5 is a nuclear protein and one of the basic helix-loop-helix (bHLH) myogenic factors that play an important role in muscle specification and differentiation. The motif responsible for the nuclear translocation of Myf5 was unknown. Using on-line monitoring of EGFP (enhanced green fluorescent protein)-tagged zebrafish Myf5 translocation, we demonstrated that Myf5-EGFP protein resided in the nucleoplasm and nucleolus of zebrafish fibroblast cell lines (ZEM2S and ZF4), mammalian nonmuscle cell line (COS1), and muscle cell lines (RD and C2C12). In contrast, zebrafish MyoD-EGFP was localized in the nucleus but did not condense in the nucleolus. Using indirect immunofluorescent staining, we determined that zebrafish Myf5 was colocalized with nucleophosmin/B23, a nucleolus protein. Deletion analysis revealed that amino acid residues 60 to 82 (60KRKASTVDRRRAATMRERRRLKK82) of Myf5 were sufficient and necessary for nucleolus targeting. A GST pulldown assay followed by Western analysis showed that nucleolin/C23 could be pulled down specifically by GST-Myf5, but not by GST-MyoD. Based on these findings, we propose that the distinct functions of Myf5 and MyoD may result from their differential binding affinity to nucleolin/C23.     

Parkinson's disease: mechanisms and models

Parkinson's disease (PD) results primarily from the death of dopaminergic neurons in the substantia nigra. Current PD medications treat symptoms; none halt or retard dopaminergic neuron degeneration. The main obstacle to developing neuroprotective therapies is a limited understanding of the key molecular events that provoke neurodegeneration. The discovery of PD genes has led to the hypothesis that misfolding of proteins and dysfunction of the ubiquitin-proteasome pathway are pivotal to PD pathogenesis. Previously implicated culprits in PD neurodegeneration, mitochondrial dysfunction and oxidative stress, may also act in part by causing the accumulation of misfolded proteins, in addition to producing other deleterious events in dopaminergic neurons. Neurotoxin-based models (particularly MPTP) have been important in elucidating the molecular cascade of cell death in dopaminergic neurons. PD models based on the manipulation of PD genes should prove valuable in elucidating important aspects of the disease, such as selective vulnerability of substantia nigra dopaminergic neurons to the degenerative process.     

Distribution of nucleolar proteins B23 and nucleolin during mouse spermatogenesis

The intracellular distribution of nucleolar phosphoproteins B23 and nucleolin was studied during mouse spermatogenesis, a process that is characterized by a progressive reduction of nucleolar activity. Biochemical analyses of isolated germ cell fractions were performed in parallel with the in situ ultrastructural immunolocalization of these two proteins by means of specific antibodies and colloidal gold markers, and by silver staining. RNA blot experiments showed that mRNA for nucleolin progressively decreased during spermatogenesis whereas mRNA for B23 increased in amount during early spermatogenic stages. Immunoblotting confirmed that both proteins were present during early spermatogenesis up to the round spermatid stage and absent from mature sperm. Immunoelectron microscopy revealed that in spermatogonia, leptotene and pachtyene spermatocytes, and in Golgi phase spermatids, B23 and nucleolin were localized in the dense fibrillar component and granular component of the nucleolus but not in the fibrillar centers. In the dense fibrillar residue of the cap phase spermatids, labeling with anti-nucleolin but not with anti-B23 was observed. During nucleolar inactivation, neither of the two polypeptides was dispersed to the nucleoplasm. Silver salts stained the fibrillar centers and dense fibrillar component but not the granular component of the nucleolus. Our results suggest that there is no direct relationship between nucleolar activity and the occurrence of B23 and nucleolin or silver staining. Moreover, we confirm that silver staining and the presence of B23 or nucleolin are not directly related to each other.by M. Trendelenburg     

Alpha-synuclein up-regulation in substantia nigra dopaminergic neurons following administration of the parkinsonian toxin MPTP

Mutations in alpha-synuclein cause a form of familial Parkinson's disease (PD), and wild-type alpha-synuclein is a major component of the intraneuronal inclusions called Lewy bodies, a pathological hallmark of PD. These observations suggest a pathogenic role for alpha-synuclein in PD. Thus far, however, little is known about the importance of alpha-synuclein in the nigral dopaminergic pathway in either normal or pathological situations. Herein, we studied this question by assessing the expression of synuclein-1, the rodent homologue of human alpha-synuclein, in both normal and 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-intoxicated mice. In normal mice, detectable levels of synuclein mRNA and protein were seen in all brain regions studied and especially in ventral midbrain. In the latter, there was a dense synuclein-positive nerve fiber network, which predominated over the substantia nigra, and only few scattered synuclein-positive neurons. After a regimen of MPTP that kills dopaminergic neurons by apoptosis, synuclein mRNA and protein levels were increased significantly in midbrain extracts; the time course of these changes paralleled that of MPTP-induced dopaminergic neurodegeneration. In these MPTP-injected mice, there was also a dramatic increase in the number of synuclein-immunoreactive neurons exclusively in the substantia nigra pars compacta; all synuclein-positive neurons were tyrosine hydroxylase-positive, but none coexpressed apoptotic features. These data indicate that synuclein is highly expressed in the nigrostriatal pathway of normal mice and that it is up-regulated following MPTP-induced injury. In light of the synuclein alterations, it can be suggested that, by targeting this protein, one may modulate MPTP neurotoxicity and, consequently, open new therapeutic avenues for PD.     

Harpagoside attenuates MPTP/MPP⁺ induced dopaminergic neurodegeneration and movement disorder via elevating glial cell line-derived neurotrophic factor

Parkinson's disease is a chronic neurodegenerative movement disorder characterized by the loss of dopaminergic neurons in the substantia nigra pars compacta. New therapeutic approaches aiming at delaying or reversing the neurodegenerative process are under active investigations. In this work, we found that harpagoside, an iridoid purified from the Chinese medicinal herb Scrophularia ningpoensis, could not only prevent but also rescue the dopaminergic neurodegeneration in MPTP/MPP(+) intoxication with promising efficacy. Firstly, in cultured mesencephalic neurons, harpagoside significantly attenuated the loss of TH-positive neuron numbers and the shortening of axonal length. Secondly, in a chronic MPTP mouse model, harpagoside dose-dependently improved the loco-motor ability (rotarod test), increased the TH-positive neuron numbers in the substantia nigra pars compacta (unbiased stereological counting) and increased the striatal DAT density ((125) I-FP-CIT autoradiography). Thirdly, harpagoside markedly elevated the GDNF mRNA and GDNF protein levels in MPTP/MPP(+) lesioned models. However, the protecting effect of harpagoside on the dopaminergic degeneration disappeared when the intrinsic GDNF action was blocked by either the Ret inhibitor PP1 or the neutralizing anti-GDNF antibody. Taken together, we conclude that harpagoside attenuates the dopaminergic neurodegeneration and movement disorder mainly through elevating glial cell line-derived neurotrophic factor.     

Morphofunctional interaction of CART peptide and dopaminergic brain neurons

In Wistar rats, after 6 h of sleep deprivation and subsequent 2 h postdeprivation sleep, we found significant changes in optical density of CART peptide in neurons of nucleus accumbens and hypothalamic nucleus arcuatus as well as in processes coming into substantia nigra from nucleus accumbens. The obtained data revealed unidirectional changes of optical density of CART and tyrosine hydroxylase in the studied structures: a decrease after sleep deprivation (p < 0.05) and, on the contrary, an increase after postdeprivation sleep (p < 0.05). Confocal laser microscopy showed morphological connections of CART and dopaminergic neurons and possible colocalization of these both substances in the same neuron at the postdeprivation sleep. In experiments in vitro, after 1 h of incubation of surviving brain sections from the substantia nigra area in the medium with CART peptide there was revealed a rise of optical density of tyrosine hydroxylase in the substantia nigra pars compacta by 55% (p < 0.05). The obtained data indicate an activating effect of CART peptide on brain dopaminergic neurons and its role as a modulator of their functional activity.     

α-Synuclein overexpression impairs mitochondrial function by associating with adenylate translocator

α-Synuclein (α-syn), a protein involved in the pathogenesis of Parkinson's disease (PD), is known to accumulate in mitochondria, disrupt mitochondrial function. However, the molecular mechanisms that link these pathological responses have not been investigated. In rats overexpressing α-syn in the substantia nigra (SN) through adeno-associated virus (AAV) transduction, about 50% of tyrosine hydroxylase positive neurons were lost after 24 weeks. Overexpression of α-syn was also associated with morphological deformation of mitochondria and depolarization of the mitochondrial membrane potential (ΔΨm). Both co-immunoprecipitation and confocal microscopy demonstrated that mitochondrial α-syn associated with adenylate translocator (ANT), a component of the mitochondrial permeability transition pore (mPTP). The depolarization of ΔΨm was partially reversed in vitro by bongkrekic acid (BKA), an inhibitor of ANT, suggesting that the molecular association between α-syn and ANT facilitated ΔΨm depolarization. Concomitant with α-syn accumulation in mitochondria, abnormal mitochondrial morphology, ΔΨm depolarization, and loss of TH-positive neurons, there was a decrease in apoptosis-inducing factor (AIF) within the mitochondrial matrix, suggesting possible translocation to the cytosol. Our findings suggest that overexpression of α-syn may cause mitochondrial defects in dopaminergic neurons of the substantia nigra through an association with adenylate translocator and activation of mitochondria-dependent cell death pathways. Disruption of normal mitochondrial function may contribute to the loss of dopaminergic neurons in Parkinson's disease.     

Overexpression of alpha-synuclein in rat substantia nigra results in loss of dopaminergic neurons, phosphorylation of alpha-synuclein and activation of caspase-9: resemblance to pathogenetic changes in Parkinson's disease

To elucidate the role of alpha-synuclein in the pathogenesis of Parkinson's disease, both human alpha-synuclein transgenic mice and targeted overexpression of human alpha-synuclein in rat substantia nigra using viral vector-based methods have been studied, however, little is known about the pathogenetic changes of dopaminergic neuron loss. Therefore, it is necessary to address whether the pathogenetic changes in brains with Parkinson's disease are recapitulated in these models. Here, we used the recombinant adeno-associated viral (rAAV) vector system for human alpha-synuclein gene transfer to rat substantia nigra and observed approximately 50% loss of dopaminergic neurons at 13 weeks after infection, which was comparably slower than the progression of neurodegeneration reported in other studies. In the slower progression of neurodegeneration, we identified several important features in common with the pathogenesis of Parkinson's disease, such as phosphorylation of alpha-synuclein at Ser129 and activation of caspase-9. Both findings were also evident in cortical tissues overexpressing alpha-synuclein via rAAV. Our results indicate that overexpression of alpha-synuclein via rAAV apparently recapitulates several important features of brains with Parkinson's disease and dementia with Lewy bodies, and thus alpha-synucleinopathy described here is likely to be an ideal model for the study of the pathogenesis of Parkinson's disease and dementia with Lewy bodies.     

Dopaminergic neuron loss and up-regulation of chaperone protein mRNA induced by targeted over-expression of alpha-synuclein in mouse substantia nigra

Several transgenic mouse lines with altered alpha-synuclein expression have been developed that show a variety of Parkinson's disease-like symptoms without specific loss of dopaminergic neurons. Targeted over-expression of human alpha-synuclein using viral-vector mediated gene delivery into the substantia nigra of rats and non-human primates leads to dopaminergic cell loss and the formation of alpha-synuclein aggregates reminiscent of Lewy bodies. In the context of these recent findings, we used adeno-associated virus (AAV) to over-express wild type human alpha-synuclein in the substantia nigra of mice. We hypothesized that this over-expression would recapitulate pathological hallmarks of Parkinson's disease, creating a mouse model to further characterize the disease pathogenesis. Recombinant AAV expressing alpha-synuclein was stereotaxically injected into the substantia nigra of mice, leading to a 25% reduction of dopaminergic neurons after 24 weeks of transduction. Furthermore, examination of mRNA levels of stress-related proteins using laser capture microdissection and quantitative PCR revealed a positive correlation of Hsp27 expression with the extent of viral transduction at 4 weeks and a positive correlation of Hsp40, Hsp70 and caspase 9 with the extent of viral transduction at 24 weeks. Taken together, our findings suggest that targeted over-expression of alpha-synuclein can induce pathology at the gross anatomical and molecular level in the substantia nigra, providing a mouse model in which upstream changes in Parkinson's disease pathogenesis can be further elucidated.     

How does parkin ligate ubiquitin to Parkinson's disease?

Recessive mutations in the human PARKIN gene are the most common cause of hereditary parkinsonism, which arises from the degeneration of dopaminergic neurons in the substantia nigra. However, the molecular mechanisms by which the loss of parkin causes dopaminergic neurodegeneration are not well understood. Parkin is an enzyme that ubiquitinates several candidate substrate proteins and thereby targets them for proteasomal degradation. Hypothesis-driven searches have led to the discovery of aggregation-prone protein substrates of parkin. Moreover, the enzyme is upregulated when under unfolded protein stress. Thus, loss-of-function mutations of parkin might impair the removal of potentially toxic protein aggregates. However, the limited neuropathological information that is available from parkin-proven patients, as well as the recent knockout of the parkin gene in fruit flies and mice, may indicate a more complex disease mechanism, possibly involving the misfolding of parkin itself or of additional substrates. The risk factors that predispose dopaminergic neurons to degenerate on parkin failure are yet to be identified.     

Nucleolar translocalization of GRA10 of Toxoplasma gondii transfectionally expressed in HeLa cells

Toxoplasma gondii GRA10 expressed as a GFP-GRA10 fusion protein in HeLa cells moved to the nucleoli within the nucleus rapidly and entirely. GRA10 was concentrated specifically in the dense fibrillar component of the nucleolus morphologically by the overlap of GFP-GRA10 transfection image with IFA images by monoclonal antibodies against GRA10 (Tg378), B23 (nucleophosmin) and C23 (nucleolin). The nucleolar translocalization of GRA10 was caused by a putative nucleolar localizing sequence (NoLS) of GRA10. Interaction of GRA10 with TATA-binding protein associated factor 1B (TAF1B) in the yeast two-hybrid technique was confirmed by GST pull-down assay and immunoprecipitation assay. GRA10 and TAF1B were also co-localized in the nucleolus after co-transfection. The nucleolar condensation of GRA10 was affected by actinomycin D. Expressed GFP-GRA10 was evenly distributed over the nucleoplasm and the nucleolar locations remained as hollows in the nucleoplasm under a low dose of actinomycin D. Nucleolar localizing and interacting of GRA10 with TAF1B suggested the participation of GRA10 in rRNA synthesis of host cells to favor the parasitism of T. gondii.