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.

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.

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.

Near-field mapping of plasmonic antennas by multiphoton absorption in poly(methyl methacrylate).

Mapping the optical near-field response around nanoantennas is a challenging yet indispensable task to engineer light-matter interaction at the nanometer scale. Recently, photosensitive molecular probes, which undergo morphological or chemical changes induced by the local optical response of the nanostructures, have been proposed as a handy alternative to more cumbersome optical and electron-based techniques. Here, we report four-photon absorption in poly(methyl methacrylate) (PMMA) as a very promising tool for nanoimaging the optical near-field around nanostructures over a broad range of near-infrared optical wavelengths. The high performance of our approach is demonstrated on single-rod antennas and coupled gap antennas by comparing experimental maps with 3D numerical simulations of the electric near-field intensity.

Vascular endothelial growth factor (VEGF) affects processing of amyloid precursor protein and beta-amyloidogenesis in brain slice cultures derived from transgenic Tg2576 mouse brain.

The up-regulation of the angiogenic vascular endothelial growth factor (VEGF) in brains of Alzheimer patients in close relationship to beta-amyloid (Abeta) plaques, suggests a link of VEGF action and processing of the amyloid precursor protein (APP). To reveal whether VEGF may affect APP processing, brain slices derived from 17-month-old transgenic Tg2576 mice were exposed with 1ng/ml VEGF for 6, 24, and 72h, followed by assessing cytosolic and membrane-bound APP expression, level of both soluble and fibrillar Abeta-peptides, as well as activities of alpha- and beta-secretases in brain slice tissue preparations. Treatment of brain slices with VEGF did not significantly affect the expression level of APP, regardless of the exposure time studied. In contrast, VEGF exposure of brain slices for 6h reduced the formation of soluble, SDS extractable Abeta(1-40) and Abeta(1-42) as compared to brain slice cultures incubated in the absence of any drug, while the fibrillar Abeta peptides did not change significantly. This effect was less pronounced 24h after VEGF exposure, but was no longer detectable when brain slices were exposed by VEGF for 72h, which indicates an adaptive response to chronic VEGF exposure. The VEGF-mediated reduction in Abeta formation was accompanied by a transient decrease in beta-secretase activity peaking 6h after VEGF exposure. To reveal whether the VEGF-induced changes in soluble Abeta-level may be due to actions of VEGF on Abeta fibrillogenesis, the fibrillar status of Abeta was examined using the thioflavin-T binding assay. Incubation of Abeta preparations obtained from Tg2576 mouse brain cortex, in the presence of VEGF slightly decreased the fibrillar content with increasing incubation time up to 72h. The data demonstrate that VEGF may affect APP processing, at least in vitro, suggesting a role of VEGF in the pathogenesis of Alzheimer's disease.