Short-term incubation of gabapentin or pregabalin does not affect chemically induced injury in neuronal cell models in vitro.

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.

HDAC6 inhibition by tubastatin A is protective against oxidative stress in a photoreceptor cell line and restores visual function in a zebrafish model of inherited blindness.

Retinal diseases, such as hereditary retinitis pigmentosa and age-related macular degeneration, are characterized by the progressive loss of photoreceptors. Histone deacetylase 6 (HDAC6) is considered as a stress surveillance factor and a potential target for neuroprotection and regeneration. Overexpression of HDAC6 has been connected to neurodegenerative disorders, and its suppression may provide protection. Here we show that HDAC6 is constitutively present in the mouse retina, and in the cone-like mouse cell line 661W. In 661W cells HDAC6 inhibition by the specific inhibitor tubastatin A (TST) led to the acetylation of α-tubulin, which is a major substrate for HDAC6. After oxidative stress, exerted by hydrogen peroxide, TST promoted cell survival and the upregulation of heat-shock proteins HSP70 and HSP25 by activation of heat-shock transcription factor 1. Furthermore, in response to oxidative stress the redox regulatory protein peroxiredoxin 1 (Prx1) was modulated in 661W cells by HDAC6 inhibition. The peroxide reducing activity of Prx1 is dependent on its acetylation, which is mediated by HDAC6. Pre-incubation with TST prevented the inactivation of Prx1 and its preserved activity may exert protective effects in photoreceptor cells. To determine whether TST treatment has a therapeutic effect on visual function, the dyeucd6 zebrafish model of inherited sight loss was utilized. Zebrafish have developed as a suitable model system for pharmacological testing. In vivo application of TST caused the hyperacetylation of α-tubulin, indicating that HDAC6 is active in this model. Furthermore, TST was sufficient to rescue visual function and retinal morphology. Hence, HDAC6 inhibition and the regulation of peroxiredoxin activity may play a significant role in protecting retinal cells and in particular photoreceptors, which are exposed to high levels of reactive oxygen species derived from oxidative stress-induced injuries.

Higher levels of myelin phospholipids in brains of neuronal α-Synuclein transgenic mice precede myelin loss.

α-Synuclein is a protein involved in the pathogenesis of synucleinopathies, including Parkinson's disease (PD), dementia with Lewy bodies (DLB) and multiple system atrophy (MSA). We investigated the role of neuronal α-Syn in myelin composition and abnormalities. The phospholipid content of purified myelin was determined by 31P NMR in two mouse lines modeling PD, PrP-A53T α-Syn and Thy-1 wt-α-Syn. Significantly higher levels of phospholipids were detected in myelin purified from brains of these α-Syn transgenic mouse models than in control mice. Nevertheless, myelin ultrastructure appeared intact. To further investigate the effect of α-Syn on myelin abnormalities, we systematically analyzed the striatum, a brain region associated with neurodegeneration in PD. An age and disease-dependent loss of myelin basic protein (MBP) signal was detected by immunohistochemistry in striatal striosomes (patches). The age-dependent loss of MBP signal was associated with lower P25α levels in oligodendrocytes. In addition, we found that α-Syn inhibited oligodendrocyte maturation and the formation of membranous sheets in vitro. Based on these results we concluded that neuronal α-Syn is involved in the regulation and/or maintenance of myelin phospholipid. However, axonal hypomyelination in the PD models is evident only in progressive stages of the disease and associated with α-Syn toxicity.

Dimethylsulfoniopropionate Promotes Process Outgrowth in Neural Cells and Exerts Protective Effects against Tropodithietic Acid.

The marine environment harbors a plethora of bioactive substances, including drug candidates of potential value in the field of neuroscience. The present study was undertaken to investigate the effects of dimethylsulfoniopropionate (DMSP), produced by several algae, corals and higher plants, on cells of the mammalian nervous system, i.e., neuronal N2a and OLN-93 cells as model system for nerve cells and glia, respectively. Additionally, the protective capabilities of DMSP were assessed in cells treated with tropodithietic acid (TDA), a marine metabolite produced by several Roseobacter clade bacteria. Both cell lines, N2a and OLN-93, have previously been shown to be a sensitive target for the action of TDA, and cytotoxic effects of TDA have been connected to the induction of oxidative stress. Our data shows that DMSP promotes process outgrowth and microtubule reorganization and bundling, accompanied by an increase in alpha-tubulin acetylation. Furthermore, DMSP was able to prevent the cytotoxic effects exerted by TDA, including the breakdown of the mitochondrial membrane potential, upregulation of heat shock protein Hsp32 and activation of the extracellular signal-regulated kinases 1/2 (ERK1/2). Our study points to the conclusion that DMSP provides an antioxidant defense, not only in algae but also in mammalian neural cells.

Protein aggregate formation in oligodendrocytes: tau and the cytoskeleton at the intersection of neuroprotection and neurodegeneration.

Oligodendrocytes are dependent on an intact, dynamic microtubule (MT) network, which participates in the elaboration and stabilization of myelin forming extensions, and is essential for cellular sorting processes. The microtubule-associated protein tau is constituent of oligodendrocytes. During culture maturation it is developmentally regulated and important for MT stability, MT formation and intracellular trafficking. Downregulation of tau impairs process outgrowth and the transport of myelin basic protein (MBP) mRNA to the cell periphery. Cells fail to differentiate into MBP-expressing, sheet-forming oligodendrocytes. Tau-positive inclusions originating in oligodendrocytes and white matter pathology are prominent in frontotemporal dementias, such as Pick's disease, progressive supranuclear palsy and corticobasal degeneration. An impairment or overload of the proteolytic degradation systems, i.e. the ubiquitin proteasomal system and the lysosomal degradation pathway, has been connected to the formation of protein aggregates. Large protein aggregates are excluded from the proteasome and degraded by autophagy, which is a highly selective process and requires receptor proteins for ubiquitinated proteins, including histone deacetylase 6 (HDAC6). HDAC6 is present in oligodendrocytes, and α-tubulin and tau are substrates of HDAC6. In this review our current knowledge of the role of tau and protein aggregate formation in oligodendrocyte cell culture systems is summarized.

Cytotoxic Effects of Tropodithietic Acid on Mammalian Clonal Cell Lines of Neuronal and Glial Origin.

The marine metabolite tropodithietic acid (TDA), produced by several Roseobacter clade bacteria, is known for its broad antimicrobial activity. TDA is of interest not only as a probiotic in aquaculture, but also because it might be of use as an antibacterial agent in non-marine or non-aquatic environments, and thus the potentially cytotoxic influences on eukaryotic cells need to be evaluated. The present study was undertaken to investigate its effects on cells of the mammalian nervous system, i.e., neuronal N2a cells and OLN-93 cells as model systems for nerve cells and glia. The data show that in both cell lines TDA exerted morphological changes and cytotoxic effects at a concentration of 0.3-0.5 µg/mL (1.4-2.4 µM). Furthermore, TDA caused a breakdown of the mitochondrial membrane potential, the activation of extracellular signal-regulated kinases ERK1/2, and the induction of the small heat shock protein HSP32/HO-1, which is considered as a sensor of oxidative stress. The cytotoxic effects were accompanied by an increase in intracellular Ca(2+)-levels, the disturbance of the microtubule network, and the reorganization of the microfilament system. Hence, mammalian cells are a sensitive target for the action of TDA and react by the activation of a stress response resulting in cell death.

ADNP/ADNP2 expression in oligodendrocytes: implication for myelin-related neurodevelopment.

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.

Mitochondrial impairment and oxidative stress compromise autophagosomal degradation of α-synuclein in oligodendroglial cells.

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).

Inhibition of UCH-L1 in oligodendroglial cells results in microtubule stabilization and prevents α-synuclein aggregate formation by activating the autophagic pathway: implications for multiple system atrophy.

α-Synuclein (α-syn) positive glial cytoplasmic inclusions (GCI) originating in oligodendrocytes (ODC) are a characteristic hallmark in multiple system atrophy (MSA). Their occurrence may be linked to a failure of the ubiquitin proteasome system (UPS) or the autophagic pathway. For proteasomal degradation, proteins need to be covalently modified by ubiquitin, and deubiquitinated by deubiquitinating enzymes (DUBs) before proteolytic degradation is performed. The DUB ubiquitin carboxyl-terminal hydrolase L1 (UCH-L1) is a component of the UPS, it is abundantly expressed in neuronal brain cells and has been connected to Parkinson's disease (PD). It interacts with α-syn and tubulin. The present study was undertaken to investigate whether UCH-L1 is a constituent of ODC, the myelin forming cells of the CNS, and is associated with GCIs in MSA. Furthermore, LDN-57444 (LDN), a specific UCH-L1 inhibitor, was used to analyze its effects on cell morphology, microtubule (MT) organization and the proteolytic degradation system. Towards this an oligodendroglial cell line (OLN cells), stably transfected with α-syn or with α-syn and GFP-LC3, to monitor the autophagic flux, was used. The data show that UCH-L1 is expressed in ODC derived from the brains of newborn rats and colocalizes with α-syn in GCIs of MSA brain sections. LDN treatment had a direct impact on the MT network by affecting tubulin posttranslational modifications, i.e., acetylation and tyrosination. An increase in α-tubulin detyrosination was observed and detyrosinated MT were abundantly recruited to the cellular extensions. Furthermore, small α-syn aggregates, which are constitutively expressed in OLN cells overexpressing α-syn, were abolished, and LDN caused the upregulation of the autophagic pathway. Our data add to the knowledge that the UPS and the autophagy-lysosomal pathway are tightly balanced, and that UCH-L1 and its regulation may play a role in neurodegenerative diseases with oligodendroglia pathology.

Downregulation of the microtubule associated protein tau impairs process outgrowth and myelin basic protein mRNA transport in oligodendrocytes.

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.

Inhibition of HDAC6 modifies tau inclusion body formation and impairs autophagic clearance.

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.

Activation of autophagy by rapamycin does not protect oligodendrocytes against protein aggregate formation and cell death induced by proteasomal inhibition.

Pathological protein inclusions containing the microtubule-associated protein tau, ubiquitin, and a variety of heat shock proteins, originating in oligodendrocytes, are consistent features observed in a number of neurodegenerative diseases. Defects in the proteolytic degradation systems have been associated with protein aggregate formation. The ubiquitin proteasome system (UPS) and autophagy are critically involved in the maintenance of cellular homeostasis and their activities need to be carefully balanced. A genuine crosstalk exists between the UPS and the autophagosomal system, and when the UPS is impaired, autophagy might act as a compensatory mechanism. Autophagy represents a lysosomal degradation system for degrading long-lived proteins and organelles, including damaged mitochondria. As we have shown before, proteasomal impairment by the reversible proteasomal inhibitor MG-132 (carbobenzoxy-L-leucyl-L-leucyl-L-leucinal) in oligodendrocytes leads to protein aggregate formation and apoptotic cell death, caused by activation of caspases and the mitochondrial pathway. The present study was undertaken to elucidate whether upregulation of the autophagic pathway by rapamycin can protect oligodendrocytes and ameliorate the consequences of MG-132-induced protein aggregate formation. The data show that rapamycin attenuated the formation of dense protein aggregates, but did not enhance the survival capability of oligodendrocytes after proteasomal inhibition. After activation of the autophagic pathway in combination with proteasomal inhibition, caspase 3 activation and poly(ADP-ribose) polymerase-1 cleavage were even more pronounced than after proteasomal inhibition alone. Furthermore, rapamycin augmented MG-132-induced activation of extracellular signal-regulated kinases 1 and 2, which are involved in the regulation of cell death and survival. In summary, depending on the cellular context and system, rapamycin may promote cell survival or, under other conditions in concert with apoptosis, may augment cell death, which seems to be the case in oligodendrocytes. Its therapeutic use for neurodegenerative disorders is most likely limited, since long-term administration may impair neuronal survival and specifically damage the myelin forming cells of the CNS.

HDAC6 inhibition results in tau acetylation and modulates tau phosphorylation and degradation in oligodendrocytes.

Histone deacetylase 6 (HDAC6) is a unique member of the HDAC family. It is localized within the cytoplasm and has unique substrate specificities for nonhistone proteins, such as α-tubulin. Furthermore, it plays a major role in protein aggregate formation and recently was demonstrated to interact with the microtubule associated protein tau and tau was identified as a possible substrate for HDAC6 in neurons. This study was undertaken to investigate whether HDAC6 is present in oligodendrocytes and whether it is involved in tubulin and tau acetylation in these cells. We show for the first time that HDAC6 is expressed in cultured rat brain oligodendrocytes. Its inhibition by the specific HDAC6 inhibitor tubastatin A (TST) leads to morphological alterations, microtubule bundling, and tubulin acetylation, and changes in tau-isoform expression and phosphorylation. Furthermore, the microtubule binding activity of tau was reduced. Using the oligodendroglial cell lines OLN-t40 and OLN-t44, which were genetically engineered to express either the longest human tau isoform with four microtubule binding repeats (4R-tau), or the shortest tau isoform with three repeats (3R-tau), respectively, we demonstrate that tau is acetylated by HDAC6 within the 4R-binding domain. Tau acetylation reduced its turnover rate and acetylated tau was degraded slower in these cells. TST and shRNA-mediated knockdown of HDAC6 in oligodendroglia cells caused an increase in pathological hyperphosphorylated tau detectable with the 12E8 antibody. Hence HDAC6 and dysregulation of the deacetylation and acetylation process in oligodendrocytes may contribute to diseases with oligodendroglial pathology.

Oxidative stress promotes uptake, accumulation, and oligomerization of extracellular α-synuclein in oligodendrocytes.

The accumulation and aggregation of α-synuclein (α-Syn) in glial cytoplasmic inclusions originating in oligodendrocytes is a characteristic hallmark of multiple system atrophy, a progressive adult onset neurodegenerative disorder. The origin of α-Syn deposition in oligodendrocytes in multiple system atrophy is still unclear, but the uptake of α-Syn from the environment after neuronal secretion has been discussed. The present study was undertaken to investigate the consequences of α-Syn uptake from the environment in cultured oligodendroglial cells and its localization and potential to form intracellular aggregates in the absence or presence of the microtubule-associated protein tau, which has been demonstrated to act synergistically with α-Syn. Primary rat brain oligodendrocytes and clonal oligodendroglial OLN-93 cells were incubated with human recombinant soluble and pre-aggregated α-Syn. The data show that oligodendrocytes are capable to take up and internalize soluble and pre-aggregated α-Syn from their growth medium. In a time-dependent manner, α-Syn oligomerizes and small intracellular aggregates are formed. These do not exert cytotoxic responses or mitochondrial impairment. Oxidative stress exerted by hydrogen peroxide further promotes α-Syn oligomer formation and leads to an enlargement of the aggregates. This process is not affected or modified by the presence of tau in OLN-93 cells. Furthermore, membrane lipid modification by docosahexaenoic acid promotes α-Syn uptake and oligomerization, indicating that changing the membrane lipid composition and structure contributes to the protein aggregation process and pathological events. Hence, although α-Syn taken up by oligodendrocytes from the environment is not toxic per se, under conditions of oxidative stress, which might occur during chronic disease progression and aging, aggregates are enlarged and eventually may contribute to cytotoxicity and cellular death.

Inclusion body formation, macroautophagy, and the role of HDAC6 in neurodegeneration.

The failure to clear misfolded or aggregated proteins from the cytoplasm of nerve cells and glia is a common pathogenic event in a variety of neurodegenerative disorders. This might be causally related to defects in the major proteolytic systems, i.e., the ubiquitin-proteasomal system and the autophagic pathway. Large protein aggregates and defective organelles are excluded from the proteasome. They can be degraded only by macroautophagy, which is a highly selective process. It requires p62 to act as a bridge connecting ubiquitinated protein aggregates and autophagosomes, and the tubulin deacetylase histone deacetylase 6 (HDAC6). HDAC6 has recently been identified as a constituent in Lewy bodies of Parkinson disease and glial cytoplasmic inclusions of multiple system atrophy. It is considered a sensor of proteasomal inhibition and a cellular stress surveillance factor, and plays a central role in autophagy by controlling the fusion process of autophagosomes with lysosomes. Upon proteasomal inhibition, HDAC6 is relocated and recruited to polyubiquitin-positive aggresomes. Tubulin acetylation is a major consequence of HDAC6 inhibition, and HDAC6 inhibition restores microtubule (MT)-dependent transport mechanisms in neurons. This suggests the involvement of HDAC6 in neurodegenerative diseases. Furthermore, the protein tau seems to be a substrate for HDAC6. Tau acetylation impairs MT assembly and promotes tau fibrillization in vitro. It has been suggested that acetylation and phosphorylation of tau at multiples sites may act synergistically in the pathogenesis of tau fibrillization. In this review, we will survey the process of aggresome formation, macroautophagy and the role of autophagosomal proteins and HDAC6 in inclusion body formation.

Inhibition of protein deubiquitination by PR-619 activates the autophagic pathway in OLN-t40 oligodendroglial cells.

Protein aggregate formation may be the result of an impairment of the protein quality control system, e.g., the ubiquitin proteasome system (UPS) and the lysosomal autophagic pathway. For proteasomal degradation, proteins need to be covalently modified by ubiquitin and deubiquitinated before the substrates are proteolytically degraded. Deubiquitination is performed by a large family of proteases, the deubiquitinating enzymes (DUBs). DUBs display a variety of functions and their inhibition may have pathological consequences. Using the broad specificity DUB inhibitor PR-619 we previously have shown that DUB inhibition leads to an overload of ubiquitinated proteins, to protein aggregate formation and subsequent inhibition of the UPS. This study was undertaken to investigate whether PR-619 modulates autophagic functions to possibly compensate the failure of the proteasomal system. Using the oligodendroglial cell line OLN-t40 and a new oligodendroglial cell line stably expressing GFP-LC3, we show that DUB inhibition leads to the activation of autophagy and to the recruitment of LC3 and of the ubiquitin binding protein p62 to the forming aggresomes without impairing the autophagic flux. Furthermore, PR-619 induced the transport of lysosomes to the forming aggregates in a process requiring an intact microtubule network. Further stimulation of autophagy by rapamycin did not prevent PR-619 aggregate formation but rather exerted cytotoxic effects. Hence, inhibition of DUBs by PR-619 activated the autophagic pathway supporting the hypothesis that the UPS and the autophagy-lysosomal pathway are closely linked together.

FAS-dependent cell death in α-synuclein transgenic oligodendrocyte models of multiple system atrophy.

Multiple system atrophy is a parkinsonian neurodegenerative disorder. It is cytopathologically characterized by accumulation of the protein p25α in cell bodies of oligodendrocytes followed by accumulation of aggregated α-synuclein in so-called glial cytoplasmic inclusions. p25α is a stimulator of α-synuclein aggregation, and coexpression of α-synuclein and p25α in the oligodendroglial OLN-t40-AS cell line causes α-synuclein aggregate-dependent toxicity. In this study, we investigated whether the FAS system is involved in α-synuclein aggregate dependent degeneration in oligodendrocytes and may play a role in multiple system atrophy. Using rat oligodendroglial OLN-t40-AS cells we demonstrate that the cytotoxicity caused by coexpressing α-synuclein and p25α relies on stimulation of the death domain receptor FAS and caspase-8 activation. Using primary oligodendrocytes derived from PLP-α-synuclein transgenic mice we demonstrate that they exist in a sensitized state expressing pro-apoptotic FAS receptor, which makes them sensitive to FAS ligand-mediated apoptosis. Immunoblot analysis shows an increase in FAS in brain extracts from multiple system atrophy cases. Immunohistochemical analysis demonstrated enhanced FAS expression in multiple system atrophy brains notably in oligodendrocytes harboring the earliest stages of glial cytoplasmic inclusion formation. Oligodendroglial FAS expression is an early hallmark of oligodendroglial pathology in multiple system atrophy that mechanistically may be coupled to α-synuclein dependent degeneration and thus represent a potential target for protective intervention.

SUMO-1 is associated with a subset of lysosomes in glial protein aggregate diseases.

Oligodendroglial inclusion bodies characterize a subset of neurodegenerative diseases. Multiple system atrophy (MSA) is characterized by α-synuclein glial cytoplasmic inclusions and progressive supranuclear palsy (PSP) is associated with glial tau inclusions. The ubiquitin homologue, SUMO-1, has been identified in inclusion bodies in MSA, located in discrete sub-domains in α-synuclein-positive inclusions. We investigated SUMO-1 associated with oligodendroglial inclusion bodies in brain tissue from MSA and PSP and in glial cell models. We examined MSA and PSP cases and compared to age-matched normal controls. Fluorescence immunohistochemistry revealed frequent SUMO-1 sub-domains within and surrounding inclusions bodies in both diseases and showed punctate co-localization of SUMO-1 and the lysosomal marker, cathepsin D, in affected brain regions. Cell counting data revealed that 70-75 % of lysosomes in inclusion body-positive oligodendrocytes were SUMO-1-positive consistently across MSA and PSP cases, compared to 20 % in neighbouring inclusion body negative oligodendrocytes and 10 % in normal brain tissue. Hsp90 co-localized with some SUMO-1 puncta. We examined the SUMO-1 status of lysosomes in 1321N1 human glioma cells over-expressing α-synuclein and in immortalized rat oligodendrocyte cells over-expressing the four repeat form of tau following treatment with the proteasome inhibitor, MG132. We also transfected 1321N1 cells with the inherently aggregation-prone huntingtin exon 1 mutant, HttQ74-GFP. Each cell model showed the association of SUMO-1-positive lysosomes around focal cytoplasmic accumulations of α-synuclein, tau or HttQ74-GFP, respectively. Association of SUMO-1 with lysosomes was also detected in glial cells bearing α-synuclein aggregates in a rotenone-lesioned rat model. SUMO-1 labelling of lysosomes showed a major increase between 24 and 48 h post-incubation of 1321N1 cells with MG132 resulting in an increase in a 90 kDa SUMO-1-positive band that was immunopositive for Hsp90 and immunoprecipitated with an anti-SUMO-1 antibody. That SUMO-1 co-localizes with a subset of lysosomes in neurodegenerative diseases with glial protein aggregates and in glial cell culture models of protein aggregation suggests a role for SUMO-1 in lysosome function.