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1.
J Pain Res ; 11: 1181-1190, 2018.
Article in English | MEDLINE | ID: mdl-29950890

ABSTRACT

PURPOSE: Gabapentinoids are currently the mainstay of pharmacological treatments for patients with neuropathic pain. Little is known about the effects of this therapy on the integrity of neuronal networks, especially in patients with an already-damaged nervous system. Since gabapentinoids can worsen cognitive functions and recent studies have shown alterations in the brains of patients with neuropathic pain, it may be possible that these drugs have neurotoxic effects. METHODS: Rat clonal PC12 pheochromocytoma (autonomic) and primary sensory dorsal-root ganglion (DRG) neurons from newborn Wistar rats were employed for this study. To mimic neuronal damage, cells were exposed to cytotoxins using either hydrogen peroxide (H2O2) or vincristine. RESULTS: No direct cytotoxic effects were observed after incubating PC12 cells for 24 hours with increasing concentrations of gabapentin or pregabalin using MTT cytotoxicity assays. Even a 7-day incubation did not cause cellular damage. Furthermore, in preinjured PC12 and DRG neurons, neither gabapentin nor pregabalin prevented or enhanced the cytotoxic effects of H2O2 or vincristine after incubation for 24 hours and 7 days, respectively. Cell morphology and integrity of the cytoskeleton assessed by employing immunostaining of cytoskeletal proteins (α-tubulin, neurofilament L) remained intact and were not altered by gabapentinoids. CONCLUSION: Based on these results, gabapentinoids are unlikely to be neurotoxic in cultured autonomic (PC12) and sensory DRG cells, even when cells are preinjured. These results are of high clinical relevance, as it seems unlikely that the morphological changes recently observed in the brains of neuropathic pain patients are caused or worsened by gabapentinoids.

2.
J Mol Neurosci ; 57(2): 304-13, 2015 Oct.
Article in English | MEDLINE | ID: mdl-26315608

ABSTRACT

Oligodendrocytes, the myelin-forming cells of the central nervous system, play important roles in brain development and maintenance. Activity-dependent neuroprotective protein (ADNP), an early marker essential for brain formation, interacts with microtubule end-binding proteins (EB1, EB2, and EB3). EB1 and EB3 are highly expressed in neurons (axons and dendritic spines, respectively) and EB1 enhancement of neurite outgrowth is attenuated by EB2. ADNP/EB presence in oligodendrocytes has not been studied so far. Here, we measured messenger RNA (mRNA) levels of ADNP and EB1-EB3 in rat brain oligodendrocytes during culture maturation and in rat brains during development (1, 35, and 75 days) in comparison with rat astrocytes, dorsal root ganglion (DRG) neurons, and the oligodendroglia cell lines (OLN-93 cell line, not expressing the microtubule-associated protein (MAP) tau, and OLN-93 cells stably transfected to express various forms of tau). Results showed that all transcripts studied were expressed in oligodendrocytes. ADNP and EB2 mRNA transcript content peaked at the time of oligodendrocyte maturation (5 days in vitro) and was highest in newborn rat brains compared with mature brains. ADNP2 (the only family member of ADNP), and EB1, although expressed in lower quantities, essentially paralleled ADNP and EB2 expression patterns, respectively. EB3 mRNA, peaking upon oligodendrocyte maturation, showed an apparent second peak of expression (10 days in vitro) and increased in the mature rat brain compared with the newborn brain. DRG cells expressed the highest levels of EB3, when compared with oligodendrocyte precursors and with astrocytes but not when compared with mature oligodendrocytes. Mature oligodendrocytes and oligodendrocyte precursors expressed ~30-40-fold more EB2 vs. EB3, and ~4-7-fold vs. ADNP. DRGs expressed ~5-fold more EB2 vs. EB3 and astrocytes showed an in-between (~20-fold) ratio. Only DRGs expressed similar EB1 and EB3 transcript levels, contrasting with oligodendrocyte and astrocytes (~10-30-fold more EB1). Astrocytes expressed more ADNP than DRGs and oligodendrocyte precursor cells (~2-fold) but not compared with mature oligodendrocytes. EB1 and EB3 were previously found to be associated with tau. Immortalized oligodendrocytes showed an intermediate phenotype of mRNA expression compared with oligodendrocyte precursor cells and mature oligodendrocytes with tau transfection reducing overall ADNP and EB expression. In summary, ADNPs and EBs are highly expressed in oligodendrocytes suggesting an impact on myelin formation in health and disease.


Subject(s)
Nerve Tissue Proteins/metabolism , Neurogenesis , Oligodendroglia/metabolism , Animals , Brain/cytology , Brain/growth & development , Cell Line , Cells, Cultured , Ganglia, Spinal/cytology , Ganglia, Spinal/growth & development , Gene Expression Regulation, Developmental , Microtubule-Associated Proteins/metabolism , Myelin Sheath/metabolism , Nerve Tissue Proteins/genetics , Oligodendroglia/cytology , RNA, Messenger/genetics , RNA, Messenger/metabolism , Rats , Rats, Wistar
3.
J Neurochem ; 135(1): 194-205, 2015 Oct.
Article in English | MEDLINE | ID: mdl-26212128

ABSTRACT

α-Synuclein (α-syn)-containing glial cytoplasmic inclusions originating in oligodendrocytes are characteristically observed in multiple system atrophy. The mechanisms of glial cytoplasmic inclusion formation remain rather elusive. α-Syn over-expression, uptake from the environment, oxidative stress or impairment of the proteolytic degradation systems have been discussed. Here, we investigated whether in oligodendrocytes autophagy plays a major role in the degradation and aggregation of endogenously expressed α-syn and of α-syn taken up from the extracellular environment. Furthermore, we studied whether in cells with impaired mitochondria the accumulation and aggregation of exogenously added α-syn is promoted. Using primary cultures of rat brain oligodendrocytes and an oligodendroglial cell line, genetically engineered to express green fluorescent protein-microtubule-associated light chain 3 with or without α-syn to monitor the autophagic flux, we demonstrate that both exogenously applied α-syn and α-syn stably expressed endogenously are effectively degraded by autophagy and do not affect the autophagic flux per se. Mitochondrial impairment with the protonophore carbonyl cyanide 3-chlorophenylhydrazone or 3-nitropropionic acid disturbs the autophagic pathway and leads to the accumulation of exogenously applied α-syn and enhances its propensity to form aggregates intracellularly. Thus, mitochondrial dysfunction and oxidative stress, which occur over time and are significant pathological features in synucleinopathies, have an impact on the autophagic pathway and participate in pathogenesis. Glial cytoplasmic inclusions are characteristically observed in multiple system atrophy, their occurrence might be related to failure in protein degradation systems. Here, we show that in oligodendrocytes autophagy is the major route of α-synuclein degradation which is either endogenously expressed or added exogenously (1, 2). Mitochondrial impairment (3) disturbs the autophagic flux and leads to the accumulation of exogenously applied α-synuclein, and enhances its propensity to form aggregates intracellulary (4).


Subject(s)
Autophagy , Mitochondria/metabolism , Oligodendroglia/metabolism , Oxidative Stress/physiology , alpha-Synuclein/metabolism , Animals , Cells, Cultured , Cytoplasm/metabolism , Multiple System Atrophy/drug therapy , Nerve Tissue Proteins/metabolism , Nitro Compounds/pharmacology , Propionates/pharmacology , Rats
4.
Glia ; 63(9): 1621-35, 2015 Sep.
Article in English | MEDLINE | ID: mdl-25847153

ABSTRACT

Oligodendrocytes, the myelin forming cells of the CNS, are characterized by their numerous membranous extensions, which enwrap neuronal axons and form myelin sheaths. During differentiation oligodendrocytes pass different morphological stages, downregulate the expression of the proteoglycan NG2, and acquire major myelin specific proteins, such as myelin basic proteins (MBP) and proteolipid protein. MBP mRNA is transported in RNA granules along the microtubules (MTs) to the periphery and translated locally. MTs participate in the elaboration and stabilization of the myelin forming extensions and are essential for cellular sorting processes. Their dynamic properties are regulated by microtubule associated proteins (MAPs). The MAP tau is present in oligodendrocytes and involved in the regulation and stabilization of the MT network. To further elucidate the functional significance of tau in oligodendrocytes, we have downregulated tau by siRNA technology and studied the effects on cell differentiation and neuron-glia contact formation. The data show that tau knockdown impairs process outgrowth and leads to a decrease in MBP expression. Furthermore, MBP mRNA transport to distant cellular extensions is impaired and cells remain in the NG2 stage. In myelinating cocultures with dorsal root ganglion neurons, oligodendrocyte precursor cells after tau miR RNA lentiviral knockdown develop into NG2 positive cells with very long and thin processes, contacting axons loosely, but fail to form internodes. This demonstrates that tau is important for MBP mRNA transport and involved in process formation. The disturbance of the balance of tau leads to abnormalities in oligodendrocyte differentiation, neuron-glia contact formation and the early myelination process.


Subject(s)
Myelin Basic Protein/metabolism , Oligodendroglia/metabolism , RNA Transport/physiology , RNA, Messenger/metabolism , tau Proteins/metabolism , Animals , Brain/metabolism , Cell Line , Cells, Cultured , Coculture Techniques , Down-Regulation , Ganglia, Spinal/metabolism , Humans , Microtubules/metabolism , Myelin Sheath/metabolism , Neurons/metabolism , Proto-Oncogene Proteins c-fyn/metabolism , Rats, Wistar , Tubulin/metabolism , tau Proteins/genetics
5.
J Mol Neurosci ; 55(4): 1031-46, 2015 Apr.
Article in English | MEDLINE | ID: mdl-25434725

ABSTRACT

Proteinaceous inclusions in nerve cells and glia are a defining neuropathological hallmark in a variety of neurodegenerative diseases, including Alzheimer's disease, Parkinson's disease, progressive supranuclear palsy (PSP), and corticobasal degeneration (CBD). Their occurrence may be related to malfunctions of the proteolytic degradation systems. In cultured oligodendrocytes, proteasomal inhibition leads to protein aggregate formation resembling coiled bodies, which are characteristic for PSP and CBD. Large protein aggregates are excluded from the proteasome and can only be degraded by autophagy, a lysosomal pathway. Autophagy is a highly selective process, which requires a variety of receptor proteins for ubiquitinated proteins, such as p62 and histone deacetylase 6 (HDAC6). HDAC6 is mainly localized in the cytoplasm, and alpha-tubulin is its major substrate. HDAC6 is considered as a sensor of proteasomal stress; it is involved in the autophagosomal pathway and can mediate the retrograde transport of ubiquitinated proteins along the microtubules. As we have shown recently, HDAC6 is present in oligodendrocytes and its inhibition leads to morphological alterations, microtubule bundling, modulation of acetylation, and phosphorylation of the microtubule-associated protein tau. The present study was undertaken to investigate whether HDAC6 is involved in protein aggregate formation in oligodendrocytes and whether its inhibition modifies the consequences of MG-132-induced inhibition of the ubiquitin proteasome system (UPS). The data show that HDAC6 and acetylated tau are recruited to protein aggregates after proteasomal inhibition. Pharmacological inhibition of HDAC6 by the selective inhibitor tubastatin A (TST) and its small hairpin RNA (shRNA)-mediated downregulation alters the assembly of MG-132-induced compact protein aggregates. After TST treatment, they appear more diffusely dispersed throughout the cytoplasm. This is not a protective means but promotes the onset of apoptotic cell death. Furthermore, the heat shock response is altered, and TST suppresses the MG-132-stimulated induction of HSP70. To test whether the alteration of protein aggregate formation is related to the influence of HDAC6 on the autophagic degradation system, an oligodendroglial cell line, i.e., OLN-93 cells stably expressing green fluorescent protein (GFP)-microtubule associated protein light chain 3 (LC3) and tau, was used. During autophagosome formation, endogenous LC3 is processed to LC3-I, which is then converted to LC3-II. An increase of LC3-II is used as a reliable marker for autophagosome formation and abundance. It is demonstrated that inhibition of HDAC6 leads to the accumulation of LC3-positive autophagosomal vacuoles and an increase in LC3-II immunoreactivity, but the autophagic flux is rather impaired. Hence, the inhibition or dysregulation of HDAC6 contributes to stress responses and pathological processes in oligodendrocytes.


Subject(s)
Autophagy , Histone Deacetylase Inhibitors/pharmacology , Histone Deacetylases/metabolism , Hydroxamic Acids/pharmacology , Indoles/pharmacology , Phagosomes/metabolism , tau Proteins/metabolism , Animals , Cell Line , Cells, Cultured , HSP70 Heat-Shock Proteins/metabolism , Histone Deacetylase 6 , Microtubule-Associated Proteins/metabolism , Oligodendroglia/drug effects , Oligodendroglia/metabolism , Proteasome Endopeptidase Complex/metabolism , Rats , Rats, Wistar
6.
Neurochem Int ; 61(8): 1302-13, 2012 Dec.
Article in English | MEDLINE | ID: mdl-23017599

ABSTRACT

Formaldehyde is a neurotoxic environmental pollutant that can also be produced in the body by certain enzymatic reactions. To test for the potential consequences of an exposure of oligodendrocytes to formaldehyde, we used OLN-93 cells as a model system. Treatment with formaldehyde altered the cellular glutathione (GSH) content of these cells by inducing a rapid time- and concentration-dependent export of GSH. Half-maximal effects were observed for a formaldehyde concentration of about 0.2 mM. While the basal GSH efflux from OLN-93 cells was negligible even when the cellular GSH content was doubled by pre-incubation of the cells with cadmium chloride, the formaldehyde-stimulated export increased almost proportionally to the cellular GSH content. In addition, the stimulated GSH export required the presence of formaldehyde and was almost completely abolished after removal of the aldehyde. Analysis of kinetic parameters of the formaldehyde-induced GSH export revealed similar K(m) and V(max) values of around 100 nmol/mg and 40 nmol/(hmg), respectively, for both OLN-93 cells and cultured astrocytes. The transporter responsible for the formaldehyde-induced GSH export from OLN-93 cells is most likely the multidrug resistance protein 1 (Mrp1), since this transporter is expressed in these cells and since the inhibitor MK571 completely prevented the formaldehyde-induced GSH export. The rapid export of GSH from formaldehyde-treated viable oligodendroglial cells is likely to compromise the cellular antioxidative and detoxification potential which may contribute to the known neurotoxicity of formaldehyde.


Subject(s)
Formaldehyde/pharmacology , Glutathione/metabolism , Oligodendroglia/drug effects , ATP Binding Cassette Transporter, Subfamily B, Member 1/antagonists & inhibitors , ATP Binding Cassette Transporter, Subfamily B, Member 1/metabolism , Animals , Astrocytes/drug effects , Astrocytes/metabolism , Biological Transport, Active/drug effects , Cell Line, Transformed/drug effects , Cell Line, Transformed/metabolism , Cells, Cultured/drug effects , Cells, Cultured/metabolism , Nerve Tissue Proteins/metabolism , Oligodendroglia/metabolism , Primary Cell Culture , Propionates/pharmacology , Quinolines/pharmacology , Rats , Rats, Wistar
7.
Biochim Biophys Acta ; 1823(11): 2057-68, 2012 Nov.
Article in English | MEDLINE | ID: mdl-22565157

ABSTRACT

A pathological hallmark of many neurodegenerative diseases is the aggregation of proteins. Protein aggregate formation may be linked to a failure of the ubiquitin proteasome system (UPS) and/or the autophagy pathway. The UPS involves the ubiquitination of proteins followed by proteasomal degradation. Deubiquitination of target proteins is performed by proteases called deubiquitinating proteins (DUBs). Inhibition of DUBs may lead to the dysregulation of homeostasis and have pathological consequences. To assess the effects of DUB-inhibition, we have used the oligodendroglial cell line, OLN-t40, stably expressing the longest human tau isoform. Cells were incubated with PR-619, a broad-range, reversible inhibitor of ubiquitin isopeptidases. Incubation with PR-619 led to morphological changes, the upregulation of heat shock proteins (HSP), including HSP70 and αB-crystallin, and to protein aggregates near the MTOC, containing ubiquitin, HSPs, and the ubiquitin binding protein p62, which may provide a link between the UPS and autophagy. Thus, inhibition of DUB activity caused stress responses and the formation of protein aggregates resembling pathological inclusions observed in aggregopathies. Furthermore, PR-619 led to the stabilization of the microtubule network, possibly through the modulation of tau phosphorylation, and small tau deposits assembled near the MTOC. Hence, organization and integrity of the cytoskeleton were affected, which is particularly important for the maintenance of the cellular architecture and intracellular transport processes, and essential for the functionality and survival of neural cells. Our data demonstrate that DUB inhibitors provide a useful tool to elucidate the manifold mechanisms of DUB functions in cells and their dysregulation in neurodegenerative diseases. This article is part of a Special Issue entitled: Ubiquitin Drug Discovery and Diagnostics.


Subject(s)
Aminopyridines/pharmacology , Microtubule-Organizing Center/drug effects , Microtubules/drug effects , Oligodendroglia/drug effects , Proteasome Endopeptidase Complex/drug effects , Protein Multimerization/drug effects , Thiocyanates/pharmacology , Ubiquitin Thiolesterase/antagonists & inhibitors , Blotting, Western , Cell Survival/drug effects , Cells, Cultured , Fluorescent Antibody Technique, Indirect , HSP70 Heat-Shock Proteins/metabolism , Humans , Microtubules/metabolism , Oligodendroglia/cytology , Oligodendroglia/metabolism , Phosphorylation/drug effects , Ubiquitin/metabolism , Ubiquitin Thiolesterase/metabolism , Ubiquitination/drug effects , tau Proteins/metabolism
8.
J Mol Neurosci ; 47(2): 256-66, 2012 Jun.
Article in English | MEDLINE | ID: mdl-22411133

ABSTRACT

α-Synuclein-containing glial cytoplasmic inclusions (GCIs) originating in oligodendrocytes are the characteristic hallmark for neuropathological diagnosis of multiple system atrophy (MSA). α-Synuclein can be degraded either by the proteasomal machinery or by autophagy, a lysosomal pathway which involves the formation of autophagosomes. The autophagosome takes up polyubiquitinated proteins via the autophagosomal protein LC3 and the ubiquitin binding protein p62. In the present study, neuropathological examination of seven MSA cases revealed that LC3-immunoreactivity is found to be associated with α-synuclein-positive GCIs. These are also prominently stained by antibodies against p62 and ubiquitin, indicating that the autophagic pathway is upregulated during pathogenesis, which might be due to a persistent downregulation of proteasomal activity. To further address this question in a cellular context, we have investigated whether proteasomal inhibition in cultured rat brain oligodendrocytes promotes the recruitment of LC3 and p62 to protein aggregates. The data show that the autophagic marker LC3-II is upregulated and LC3 is recruited to the growing protein aggregates in cultured oligodendrocytes when the proteasome is impaired. However, aggregated proteins remain in the oligodendroglial cytoplasm and cannot be cleared efficiently. In conclusion, autophagy and the ubiquitin proteasome system are closely connected, and the presence of LC3-positive vesicles in GCIs indicates that macroautophagy participates in MSA pathogenesis.


Subject(s)
Autophagy/physiology , Multiple System Atrophy/pathology , Oligodendroglia/pathology , Adult , Aged , Animals , Cells, Cultured , Female , Humans , Inclusion Bodies/metabolism , Inclusion Bodies/pathology , Male , Middle Aged , Oligodendroglia/metabolism , Rats , alpha-Synuclein/metabolism
9.
J Mol Neurosci ; 43(3): 290-302, 2011 Mar.
Article in English | MEDLINE | ID: mdl-20725866

ABSTRACT

α-Synuclein (α-syn) is the major constituent of Lewy bodies and glial cytoplasmic inclusions which are pathological hallmarks of neurodegenerative disorders like Parkinson's disease or multiple system atrophy (MSA), respectively. It accumulates and aggregates during the pathogenic process, and missense mutations, such as A53T, are increasing its probability of aggregate formation. Furthermore, α-syn interacts with polyunsaturated fatty acids, and this interaction may promote the oligomerization process. To investigate whether membrane lipid modification by docosahexaenoic acid (DHA) modifies the aggregation process of α-syn in oligodendroglial cells, we have used OLN-93 cells stably expressing the human α-syn A53T mutation. Cells were supplemented with DHA (25 µM) for 3 days and then subjected to oxidative stress (OS) exerted by hydrogen peroxide. The data show that modification of the oligodendroglial cell membranes by DHA followed by OS caused the formation of fibrillary α-syn inclusions, a decrease in α-syn solubility, and an increase in phosphorylation at serine 129, which has been suggested to play a proaggregatory role. The aggregates contain αB-crystallin and ubiquitinated proteins and SUMO-1 immunoreactivity. SUMO-1 has been implicated in protein aggregation and identified as a constituent in inclusion bodies in MSA. Hence, membrane lipid modification in oligodendroglial cells promotes the formation of α-syn inclusion bodies resembling protein aggregates in neurodegenerative disease. This effect is not only attributable to the A53T mutation but also is observable in OLN cells expressing wild-type α-syn.


Subject(s)
Docosahexaenoic Acids/metabolism , Inclusion Bodies/metabolism , Membrane Lipids/metabolism , Oligodendroglia/metabolism , Oxidative Stress , SUMO-1 Protein/metabolism , alpha-Synuclein/metabolism , Animals , Cell Line/drug effects , Humans , Hydrogen Peroxide/pharmacology , Membrane Lipids/chemistry , Oligodendroglia/cytology , Oxidants/pharmacology , Rats , SUMO-1 Protein/genetics , alpha-Synuclein/genetics
10.
J Neurochem ; 114(3): 654-65, 2010 Aug.
Article in English | MEDLINE | ID: mdl-20163522

ABSTRACT

Oligodendrocytes are the myelin-forming cells of the CNS and guarantee proper nerve conduction. Sphingosine, one major component of myelin, has recently been identified to activate TRPM3, a member of the melastatin-related subfamily of transient receptor potential (TRP) channels. TRPM3 has been demonstrated to be expressed in brain with unknown cellular distribution. Here, we show for the first time that TRPM3 is expressed in oligodendrocytes in vitro and in vivo. TRPM3 is present during oligodendrocyte differentiation. Immunohistochemistry of adult rat brain slices revealed staining of white matter areas, which co-localized with oligodendrocyte markers. Analysis of the developmental distribution revealed that, prior to myelination, TRPM3 channels are localized on neurons. On oligodendrocytes they are found after the onset of myelination. RT-PCR studies showed that the transcription of TRPM3 splice variants is also developmentally regulated in vitro. Ca(2+) imaging approaches revealed the presence of a sphingosine-induced Ca(2+) entry mechanism in oligodendrocytes - with a pharmacological profile similar to the profile published for heterologously expressed TRPM3. These findings indicate that TRPM3 participates as a Ca(2+)-permeable and sphingosine-activated channel in oligodendrocyte differentiation and CNS myelination.


Subject(s)
Brain/metabolism , Myelin Sheath/physiology , Oligodendroglia/metabolism , Sphingosine/physiology , TRPM Cation Channels/biosynthesis , Alternative Splicing/genetics , Animals , Animals, Newborn , Brain/cytology , Brain/growth & development , Calcium Signaling/genetics , Cell Membrane Permeability/genetics , Homeostasis/genetics , Neuroglia/metabolism , Neuroglia/physiology , Organ Culture Techniques , Rats , Rats, Wistar , TRPM Cation Channels/genetics , TRPM Cation Channels/physiology
11.
J Neurochem ; 113(2): 465-76, 2010 Apr.
Article in English | MEDLINE | ID: mdl-20096089

ABSTRACT

Polyunsaturated fatty acids (PUFA) are highly abundant in brain tissue, and docosahexaenoic acid (DHA) might protect cells from oxidative stress (OS) during inflammation and demyelinating disorders, but also might exert pro-oxidant effects. Here we investigated if PUFA supplements lead to heat shock protein induction, altered cell survival properties and stress responses to OS exerted by hydrogen peroxide in oligodendroglial OLN-93 cells. The data show that supplements of various fatty acids (FA) with 18-22 carbons chain length and 2-6 double bonds led to PUFA enrichment in cellular membranes. Depending on the degree of desaturation, FA-supplements caused the up-regulation of heme oxygenase-1 (HSP32), a stress protein inducible by OS, and an increase in sensitivity to hydrogen peroxide-treatment. DHA, with the highest number of double bonds, was most effective. Co-treatment with DHA and the lipophilic vitamin E analogue alpha-tocopherol, suppressed heme oxygenase-1 up-regulation and cell survival was restored. Analysis of the lipid profile demonstrates that alpha-tocopherol not only has antioxidant capacities, but also directly modified the PUFA profile in cell membranes. Enrichment with higher omega-3, -6 and -9 PUFA and an increase in the biosynthesis rate of very long chain fatty acids, mainly changed the FA profile of ethanolamine and serine phosphoglycerides.


Subject(s)
Fatty Acids, Unsaturated/pharmacology , Heme Oxygenase (Decyclizing)/metabolism , Membrane Lipids/metabolism , Oligodendroglia/cytology , Oxidative Stress/physiology , Up-Regulation/drug effects , Actins/metabolism , Animals , Antioxidants/pharmacology , Cell Death/drug effects , Cell Line, Transformed , Dose-Response Relationship, Drug , Heme Oxygenase (Decyclizing)/genetics , Hot Temperature , Hydrogen Peroxide/pharmacology , Oligodendroglia/drug effects , Oligodendroglia/metabolism , Oxidative Stress/drug effects , Rats , Thiobarbituric Acid Reactive Substances/metabolism , Time Factors , Tubulin/metabolism , Up-Regulation/physiology , alpha-Tocopherol/pharmacology
12.
PLoS One ; 5(1): e8753, 2010 Jan 18.
Article in English | MEDLINE | ID: mdl-20090920

ABSTRACT

BACKGROUND: The accumulation and aggregation of alpha-synuclein in nerve cells and glia are characteristic features of a number of neurodegenerative diseases termed synucleinopathies. alpha-Synuclein is a highly soluble protein which in a nucleation dependent process is capable of self-aggregation. The causes underlying aggregate formation are not yet understood, impairment of the proteolytic degradation systems might be involved. METHODOLOGY/PRINCIPAL FINDINGS: In the present study the possible aggregate clearing effects of the geldanamycin analogue 17-AAG (17-(Allylamino)-17-demethoxygeldanamycin) was investigated. Towards this, an oligodendroglial cell line (OLN-93 cells), stably expressing human alpha-synuclein (A53T mutation) was used. In these cells small punctate aggregates, not staining with thioflavine S, representing prefibrillary aggregates, occur characteristically. Our data demonstrate that 17-AAG attenuated the formation of alpha-synuclein aggregates by stimulating macroautophagy. By blocking the lysosomal compartment with NH(4)Cl the aggregate clearing effects of 17-AAG were abolished and alpha-synuclein deposits were enlarged. Analysis of LC3-II immunoreactivity, which is an indicator of autophagosome formation, further revealed that 17-AAG led to the recruitment of LC3-II and to the formation of LC3 positive puncta. This effect was also observed in cultured oligodendrocytes derived from the brains of newborn rats. Inhibition of macroautophagy by 3-methyladenine prevented 17-AAG induced occurrence of LC3 positive puncta as well as the removal of alpha-synuclein aggregates in OLN-A53T cells. CONCLUSIONS: Our data demonstrate for the first time that 17-AAG not only causes the upregulation of heat shock proteins, but also is an effective inducer of the autophagic pathway by which alpha-synuclein can be removed. Hence geldanamycin derivatives may provide a means to modulate autophagy in neural cells, thereby ameliorating pathogenic aggregate formation and protecting the cells during disease and aging.


Subject(s)
Autophagy , Benzoquinones/pharmacology , Cytoplasm/drug effects , Lactams, Macrocyclic/pharmacology , alpha-Synuclein/metabolism , Animals , Cell Line , Cytoplasm/metabolism , Humans , Mutation , Rats , alpha-Synuclein/genetics
13.
J Mol Neurosci ; 39(1-2): 226-34, 2009 Sep.
Article in English | MEDLINE | ID: mdl-19266322

ABSTRACT

alpha-Synuclein is the major building block of cytoplasmic inclusions in neurodegenerative disorders named synucleinopathies. These inclusion bodies often contain the small heat shock protein alphaB-crystallin and the microtubule-associated protein tau. Oxidative modification of alpha-synuclein has been linked to fibril formation, and alpha-synuclein aggregation may induce the fibrillization of tau. To study alpha-synuclein aggregate formation, we have engineered oligodendroglial cells (OLN-93 cells) to stably express the longest human isoform of tau and wild-type alpha-synuclein or the A53T alpha-synuclein mutation. Under normal growth conditions, small punctuated alpha-synuclein aggregates were formed, which were more abundant in cells expressing the A53T mutation. After exposure to oxidative stress, protein inclusions were enlarged and were positive for thioflavin S, but the solubility of alpha-synuclein was not altered. Oxidative stress followed by proteasomal inhibition caused the occurrence of larger thioflavin S-positive inclusions, immunoreactive for tau and alphaB-crystallin, thus resembling glial cell inclusion bodies. Furthermore, this double stress situation led to a decrease in alpha-synuclein solubility, and alphaB-crystallin and HSP90 were present in the insoluble fraction. The formation and recruitment of tau to thioflavin S-positive protein aggregates in OLN-93 cells only expressing tau in the absence of alpha-synuclein, either after oxidative or proteasomal stress or both, was not observable. The data indicate that oxidatively modified alpha-synuclein is degraded by the proteasome and that it plays a pro-aggregatory role for tau in this cell culture model system.


Subject(s)
Inclusion Bodies/metabolism , Oligodendroglia , Oxidative Stress , alpha-Synuclein/metabolism , tau Proteins/metabolism , Animals , Cysteine Proteinase Inhibitors/metabolism , Humans , Hydrogen Peroxide/metabolism , Hydrolysis , Inclusion Bodies/chemistry , Leupeptins/metabolism , Oligodendroglia/cytology , Oligodendroglia/metabolism , Oxidants/metabolism , Proteasome Endopeptidase Complex/metabolism , Proteasome Inhibitors , Protein Isoforms/genetics , Protein Isoforms/metabolism , alpha-Crystallin B Chain/metabolism , tau Proteins/genetics
14.
Glia ; 57(14): 1566-77, 2009 Nov 01.
Article in English | MEDLINE | ID: mdl-19330846

ABSTRACT

Proteasomal dysfunction has been implicated in neurodegenerative diseases, and molecular chaperones may provide a first line of defence against protein aggregate formation. We have shown before that oligodendrocytes respond to proteasomal inhibition by the onset of apoptotic cell death, whereas astrocytes have a higher capability to cope with stressful conditions that might be causally related to their high constitutive level of HSP25. This study was undertaken to investigate the effects of the proteasomal inhibitor MG-132 on aggregate formation in astrocytes, and to test if HSP25 exerts a protective means. Our data show that upon proteasomal inhibition aggresomes are formed in astrocytes that contain the small HSPs, HSP25 and alpha B-crystallin, and ubiquitinated proteins. HSP expression is induced and HSP25, alpha B-crystallin and ubiquitinated proteins are translocated from the soluble to the detergent-insoluble fraction. Simultaneously, the cytoskeletal organization is disturbed, microfilaments are fragmented, GFAP intermediate filaments and microtubules surround the aggresome, and mitochondria are assembled in these structures. Mitochondria membrane potential, however, stays intact. Aggresome formation and apoptotic cell death do not correlate. After the removal of MG-132, the observed effects are reversible. MG-132 promotes the formation of small oligomers of HSP25, which have been connected to the protection of the microfilament system. Downregulation of HSP25 by siRNA approach causes actin filament breakdown in control cells in the absence of stress stimuli, and sensitizes astrocytes against stress induced by proteasomal inhibition. Hence, HSP25 enables astrocytes to prevent irreversible damage and to recover after removal of the proteasomal inhibitor MG-132.


Subject(s)
Astrocytes/physiology , HSP27 Heat-Shock Proteins/metabolism , Proteasome Inhibitors , Stress, Physiological/physiology , Actins/metabolism , Animals , Apoptosis/drug effects , Astrocytes/cytology , Astrocytes/drug effects , Brain/cytology , Brain/drug effects , Brain/physiology , Cells, Cultured , Cysteine Proteinase Inhibitors/pharmacology , Cytoskeleton/drug effects , HSP27 Heat-Shock Proteins/genetics , Intermediate Filaments/metabolism , Leupeptins/pharmacology , Membrane Potential, Mitochondrial/drug effects , Microtubules/metabolism , Mitochondria/drug effects , RNA, Small Interfering/metabolism , Rats , Rats, Wistar , Ubiquitinated Proteins/metabolism , alpha-Crystallin B Chain/metabolism
15.
Glia ; 56(16): 1736-46, 2008 Dec.
Article in English | MEDLINE | ID: mdl-18563798

ABSTRACT

The tubulin polymerization-promoting protein (TPPP)/p25alpha was identified as a brain specific protein, is associated with microtubules (MTs) in vitro and can promote abnormal MT assembly. Furthermore it has aggregation promoting properties and is a constituent in pathological protein deposits of neurodegenerative diseases. In the brain, TPPP/p25alpha is present in myelinating oligodendrocytes. Here we show, using cultured rat brain oligodendrocytes, that TPPP/p25alpha expression is increasing during development in culture, and particularly in immature cells is associated with the centrosome. MT binding properties in oligodendrocytes are rather low, however, when MTs are disassembled by nocodazole, TPPP/p25alpha accumulates in the perinuclear region. Treatment of oligodendrocytes with the proteasomal inhibitor MG-132 (1 micaroM; 18 h) caused an increase in the amount of TPPP/p25alpha by about 40%, a decrease in its solubility, and led to the appearance of TPPP/p25alpha-positive cytoplasmic inclusions, which stained with thioflavin S and resembled inclusion bodies. Hence, it might be speculated that acute or chronic malfunction of the proteasomal degradation system, leading to the accumulation of aggregation prone proteins and the pro-aggregatory protein TPPP/p25alpha or to the aggregation of TPPP/p25alpha on its own, is causally related to the protein aggregation process in a variety of neurodegenerative diseases.


Subject(s)
Brain/metabolism , Carrier Proteins/metabolism , Nerve Tissue Proteins/metabolism , Oligodendroglia/metabolism , Animals , Brain/growth & development , Carrier Proteins/genetics , Cell Differentiation , Cells, Cultured , Centrosome/metabolism , Enzyme Inhibitors/pharmacology , Gene Expression Regulation, Developmental/genetics , Inclusion Bodies/metabolism , Leupeptins/pharmacology , Nerve Tissue Proteins/genetics , Nocodazole/pharmacology , Peptide Hydrolases/genetics , Peptide Hydrolases/metabolism , Proteasome Endopeptidase Complex/metabolism , Proteasome Inhibitors , Rats , Rats, Wistar , Tubulin/biosynthesis , Tubulin Modulators/pharmacology , Up-Regulation/physiology
16.
Glia ; 53(8): 891-901, 2006 Jun.
Article in English | MEDLINE | ID: mdl-16609961

ABSTRACT

Proteasomal dysfunction has been implicated in neurodegenerative disorders and during aging processes. In frontotemporal dementias, corticobasal degeneration, and progressive supranuclear palsy, oligodendrocytes are specifically damaged. Application of proteasomal inhibitors to cultured oligodendrocytes is associated with apoptotic cell death. The present study was undertaken to investigate the death pathway activated in oligodendrocytes by proteasomal inhibition. Our data show that the proteasomal inhibitor MG-132 causes oxidative stress, as indicated by the upregulation of the small heat shock protein heme oxygenase-1 (HO-1) and the appearance of oxidized proteins. Activation of the mitochondrial pathway was involved in the apoptotic process. Mitochondrial membrane potential was disturbed, and cytochrome c was released from the mitochondria. Concomitantly, death-related caspases 3 and 9 were activated and poly(ADP-ribose)-polymerase cleavage occurred. MG-132-induced cell death, DNA-fragmentation, and caspase activation could be prevented by the broad caspase inhibitor zVAD-fmk. In contrast to oligodendrocytes, cultured astrocytes showed resistance to the treatment with proteasomal inhibitors and did not reveal cytotoxic responses. This was also observed in astrocytes differentiated in the presence of dibutyryl cyclic AMP. Hence, individual cells respond differently to proteasomal inhibition and the therapeutic use of proteasomal inhibitors, e.g. for the treatment of cancer or inflammatory diseases, needs to be carefully evaluated.


Subject(s)
Apoptosis/physiology , Astrocytes/metabolism , Brain/metabolism , Mitochondria/metabolism , Oligodendroglia/metabolism , Proteasome Endopeptidase Complex/metabolism , Amino Acid Chloromethyl Ketones/pharmacology , Animals , Animals, Newborn , Apoptosis/drug effects , Astrocytes/drug effects , Astrocytes/pathology , Brain/physiopathology , Bucladesine/pharmacology , Caspases/drug effects , Caspases/metabolism , Cells, Cultured , Cysteine Proteinase Inhibitors/pharmacology , Cytochromes c/metabolism , Enzyme Inhibitors/pharmacology , Heme Oxygenase-1/drug effects , Heme Oxygenase-1/metabolism , Leupeptins/pharmacology , Mitochondria/drug effects , Mitochondria/pathology , Mitochondrial Membranes/drug effects , Mitochondrial Membranes/metabolism , Neurodegenerative Diseases/metabolism , Neurodegenerative Diseases/physiopathology , Oligodendroglia/drug effects , Oligodendroglia/pathology , Oxidative Stress/drug effects , Oxidative Stress/physiology , Poly(ADP-ribose) Polymerases/drug effects , Poly(ADP-ribose) Polymerases/metabolism , Proteasome Inhibitors , Rats , Rats, Wistar
17.
Am J Pathol ; 168(3): 947-61, 2006 Mar.
Article in English | MEDLINE | ID: mdl-16507910

ABSTRACT

Heat shock proteins (Hsps) facilitate refolding of denatured polypeptides, but there is limited understanding about their roles in neurodegenerative diseases characterized by misfolded proteins. Because Parkinson's disease (PD), dementia with Lewy bodies, and multiple system atrophy are alpha-synucleinopathies characterized by filamentous alpha-synuclein (alpha-syn) inclusions, we assessed which Hsps might be implicated in these disorders by examining human brain samples, transgenic mouse models, and cell culture systems. Light and electron microscopic multiple-label immunohistochemistry showed Hsp90 was the predominant Hsp examined that co-localized with alpha-syn in Lewy bodies, Lewy neurites, and glial cell inclusions and that Hsp90 co-localized with alpha-syn filaments of Lewy bodies in PD. Hsp90 levels were most predominantly increased in PD brains, which correlated with increased levels of insoluble alpha-syn. These alterations in Hsp90 were recapitulated in a transgenic mouse model of PD-like alpha-syn pathologies. Cell culture studies also revealed that alpha-syn co-immunoprecipitated preferentially with Hsp90 and Hsc70 relative to other Hsps, and exposure of cells to proteasome inhibitors resulted in increased levels of Hsp90. These data implicate predominantly Hsp90 in the formation of alpha-syn inclusions in PD and related alpha-synucleinopathies.


Subject(s)
Brain/metabolism , HSP90 Heat-Shock Proteins/metabolism , Inclusion Bodies/metabolism , Neurodegenerative Diseases/metabolism , Ubiquitin/metabolism , alpha-Synuclein/metabolism , Adult , Aged , Aged, 80 and over , Animals , Brain/pathology , Brain Chemistry , Female , HSP90 Heat-Shock Proteins/analysis , Humans , Inclusion Bodies/chemistry , Male , Mice , Mice, Transgenic , Middle Aged , Neurodegenerative Diseases/pathology , Neurons/metabolism , Neurons/pathology , Oligodendroglia/metabolism , Oligodendroglia/pathology , Proteasome Inhibitors , Substantia Nigra/metabolism , Substantia Nigra/pathology , Up-Regulation
18.
J Neurosci ; 24(25): 5748-57, 2004 Jun 23.
Article in English | MEDLINE | ID: mdl-15215297

ABSTRACT

Molecular chaperones and the ubiquitin-proteasome system are participants in the defense against unfolded proteins and provide an effective protein quality control system that is essential for cellular functions and survival. Ubiquitinated tau-positive inclusion bodies containing the small heat shock protein alphaB-crystallin in oligodendrocytes are consistent features of a variety of neurodegenerative diseases, and defects in the proteasome system might contribute to the aggregation process. Oligodendrocytes, the myelin-forming cells of the CNS, are specifically sensitive to stress situations. Here we can show that in cultured rat brain oligodendrocytes proteasomal inhibition by MG-132 or lactacystin caused apoptotic cell death and the induction of heat shock proteins in a time- and concentration-dependent manner. Specifically, alphaB-crystallin was upregulated, and ubiquitinated proteins accumulated. After incubation with MG-132 the tau was dephosphorylated, which enhanced its microtubule-binding capacity. Proteasomal inhibition led to ubiquitination of tau and its association with alphaB-crystallin and to the occurrence of thioflavine S-positive aggregates in the oligodendroglial cytoplasm. These aggregates were positive for tau and also contained ubiquitin and alphaB-crystallin; hence they resembled the glial cytoplasmic inclusions observed in white matter disease and frontotemporal dementias with parkinsonism linked to chromosome 17 (FTDP-17). In summary, the data underscore the specific sensitivity of oligodendrocytes to stress situations and point to a causal relationship of proteasomal impairment and inclusion body formation.


Subject(s)
Acetylcysteine/analogs & derivatives , Heat-Shock Proteins/biosynthesis , Oligodendroglia/metabolism , Proteasome Inhibitors , Ubiquitin/metabolism , tau Proteins/metabolism , Acetylcysteine/pharmacology , Animals , Apoptosis , Brain/cytology , Brain/metabolism , Cells, Cultured , HSP70 Heat-Shock Proteins/biosynthesis , Heat-Shock Proteins/genetics , Inclusion Bodies/metabolism , Leupeptins/pharmacology , Microtubules/metabolism , Oligodendroglia/cytology , Oligodendroglia/drug effects , Oxidative Stress , Phosphorylation , RNA, Messenger/biosynthesis , Rats , Rats, Wistar , alpha-Crystallin B Chain/biosynthesis
19.
Glia ; 46(4): 446-55, 2004 May.
Article in English | MEDLINE | ID: mdl-15095374

ABSTRACT

Oligodendrocytes, the myelin-forming cells of the CNS, are specifically sensitive to oxidative stress and respond by the onset of programmed cell death (PCD). To further unravel the molecular events underlying their enhanced susceptibility, we have investigated whether mitochondrial damage occurs during oxidative stress-induced PCD in cultured rat brain oligodendrocytes. Mitochondria are considered as a central control point of apoptosis, and mitochondrial dysfunction has been linked to neurodegenerative disease. Upon a number of stimuli through the release of cytochrome c, they coordinate caspase activation, causing morphological and biochemical changes associated with PCD. Oxidative stress was exerted by the application of hydrogen peroxide. The data show that hydrogen peroxide-induced apoptosis in oligodendrocytes involves mitochondrial damage and cytochrome c release and is accompanied by the activation of the death-related caspases 3 and 9. Concomitantly, the activation and nuclear translocation of extracellular signal regulated kinases ERK1,2 are observed, which have been implicated to participate in the regulation of cell death and survival. DNA fragmentation could not be attenuated by the ERK1,2 inhibitor PD 98059, indicating that the ERK1,2- pathway in oligodendrocytes may be involved in the initial survival response after exposure to stressful stimuli.


Subject(s)
Apoptosis/physiology , Hydrogen Peroxide/toxicity , Mitochondria/metabolism , Oligodendroglia/drug effects , Oligodendroglia/metabolism , Oxidative Stress/physiology , Animals , Animals, Newborn , Apoptosis/drug effects , Caspases/drug effects , Caspases/metabolism , Causality , Cells, Cultured , Cytochromes c/metabolism , Demyelinating Diseases/metabolism , Enzyme Inhibitors/pharmacology , Mitochondria/drug effects , Mitogen-Activated Protein Kinases/drug effects , Mitogen-Activated Protein Kinases/metabolism , Oxidants/toxicity , Oxidative Phosphorylation/drug effects , Rats , Rats, Wistar , Signal Transduction/drug effects , Signal Transduction/physiology
20.
J Neurosci ; 23(26): 8872-80, 2003 Oct 01.
Article in English | MEDLINE | ID: mdl-14523089

ABSTRACT

Tau-positive inclusions in oligodendrocytes are consistent neuropathological features of corticobasal degeneration, progressive supranuclear palsy, and frontotemporal dementias with Parkinsonism linked to chromosome 17. Here we show by immunohistochemistry that tau-positive oligodendroglial inclusion bodies also contain the small heat-shock protein (HSP) alphaB-crystallin but not HSP70. To study the molecular mechanisms underlying inclusion body formation, we engineered an oligodendroglia cell line (OLN-t40) to overexpress the longest human tau isoform. Treatment of OLN-t40 cells with okadaic acid (OA), an inhibitor of protein phosphatase 2A, caused tau hyperphosphorylation and a decrease in the binding of tau to microtubules. Simultaneously, tau-positive aggregates that also stained with the amyloid-binding dye thioflavin-S as well as with antibodies to tau and alphaB-crystallin were detected. However, they were only transiently expressed and were degraded within 24 hr. When the proteasomal apparatus was inhibited by carbobenzoxy-l-leucyl-l-leucyl-l-leucinal (MG-132) after OA treatment, the aggregates were stabilized and were still detectable after 18 hr in the absence of OA. Incubation with MG-132 alone inhibited tau proteolysis and led to the induction of HSPs, including alphaB-crystallin and to its translocation to the perinuclear region, but did not induce the formation of thioflavin-S-positive aggregates. Hence, although tau hyperphosphorylation induced by protein phosphatase inhibition contributes to pathological aggregate formation, only hyperphosporylation of tau followed by proteasome inhibition leads to stable fibrillary deposits of tau similar to those observed in neurodegenerative diseases.


Subject(s)
Inclusion Bodies/metabolism , Multienzyme Complexes/antagonists & inhibitors , Okadaic Acid/pharmacology , Oligodendroglia/metabolism , tau Proteins/metabolism , Benzothiazoles , Brain/metabolism , Brain/pathology , Cell Line , Cysteine Endopeptidases , Cytoskeleton/metabolism , Enzyme Inhibitors/pharmacology , Fluorescent Dyes , Humans , Immunohistochemistry , Inclusion Bodies/drug effects , Inclusion Bodies/pathology , Leupeptins/pharmacology , Microtubules/metabolism , Oligodendroglia/drug effects , Oligodendroglia/pathology , Phosphoprotein Phosphatases/antagonists & inhibitors , Phosphorylation/drug effects , Proteasome Endopeptidase Complex , Protein Phosphatase 2 , Tauopathies/metabolism , Tauopathies/pathology , Thiazoles , alpha-Crystallin B Chain/metabolism
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