Valproic acid stimulates clusterin expression in human astrocytes: Implications for Alzheimer's disease.

Clusterin is a secreted molecular chaperone, also called apolipoprotein J. Recent genetic studies have demonstrated that clusterin is a significant susceptibility gene for late-onset Alzheimer's disease (AD). Clusterin shares several properties with apolipoprotein E, a well-known risk gene for AD, i.e. they bind to amyloid-beta peptides and are present in neuritic plaques, enhance the clearance of amyloid-beta peptides in brain, and are included in lipid particles and thus regulate cholesterol traffic. Biochemical studies indicate that clusterin can prevent the progress of AD pathogenesis. We have observed earlier that histone deacetylase (HDAC) inhibitors can induce the expression of clusterin in several neuroblastoma and glioma cell lines. Recent studies have revealed that valproic acid, a common and well-tolerated drug for epilepsy and bipolar disorders, is a potent HDAC inhibitor. In this study, we examined whether valproic acid can induce the expression of clusterin in human astrocytes. Our results demonstrated that valproic acid is a potent inducer of clusterin expression and secretion in human astrocytes at the therapeutical concentrations. Another clinically used HDAC inhibitor, the cancer drug, Vorinostat (SAHA, suberoylanilide hydroxamic acid), also robustly stimulated the expression of clusterin in human astrocytes. One could postulate that valproic acid may be able to prevent amyloid-beta aggregation in AD, as observed in transgenic AD mice, by increasing clusterin expression.

Clusterin: a forgotten player in Alzheimer's disease.

Clusterin, also known as apolipoprotein J, is a versatile chaperone molecule which contains several amphipathic and coiled-coil alpha-helices, typical characteristics of small heat shock proteins. In addition, clusterin has three large intrinsic disordered regions, so-called molten globule domains, which can stabilize stressed protein structures. Twenty years ago, it was demonstrated that the expression of clusterin was clearly increased in Alzheimer's disease (AD). Later it was observed that clusterin can bind amyloid-beta peptides and prevent their fibrillization. Clusterin is also involved in the clearance of amyloid-beta peptides and fibrils by binding to megalin receptors and enhancing their endocytosis within glial cells. Clusterin is a complement inhibitor and can suppress complement activation observed in AD. Clusterin is also present in lipoprotein particles and regulates cholesterol and lipid metabolism of brain which is disturbed in AD. Clusterin is a stress-induced chaperone which is normally secreted but in conditions of cellular stress, it can be transported to cytoplasm where it can bind to Bax protein and inhibit neuronal apoptosis. Clusterin can also bind to Smad2/3 proteins and potentiate the neuroprotective TGFbeta signaling. An alternative splicing can produce a variant isoform of clusterin which can be translocated to nuclei where it induces apoptosis. The role of nuclear clusterin in AD needs to be elucidated. We will review here the extensive literature linking clusterin to AD and examine the recent progress in clusterin research with the respect to AD pathology. Though clusterin can be viewed as a multipotent guardian of brain, it is unable to prevent the progressive neuropathology in chronic AD.

Epigenetic regulation of clusterin/apolipoprotein J expression in retinal pigment epithelial cells.

Age-related macular degeneration (AMD) is the leading cause of blindness worldwide. AMD is characterized by the deposition of drusen aggregates under the retinal pigment epithelium (RPE). Clusterin/apo J, a multifunctional secreted chaperone, is one of the major proteins accumulating in drusen deposits. The regulation of clusterin expression is not well characterized but the promoter of clusterin contains a CpG-rich methylation domain. Since aging affects both DNA methylation and histone acetylation status, the epigenetic regulation might have an important role in clusterin/apo J expression. Our purpose was to elucidate whether the induction of DNA hypomethylation with 5-aza-2'-deoxycytidine (AZA) and histone hyperacetylation with trichostatin A (TSA) could affect the clusterin transcription, protein levels, and secretion in retinal pigment epithelial cells. We observed that both TSA and AZA treatments induced a prominent increase in the expression levels of clusterin mRNA and protein in ARPE-19 cells, as well as in the secretion of clusterin protein. Furthermore, valproic acid, an antiepileptic drug and a recently identified inhibitor of histone deacetylases (HDAC), induced a significant increase in clusterin protein expression and secretion in retinal pigment epithelial cells. HDAC inhibitors are characterized as inhibitors of angiogenesis, and clusterin as a complement inhibitor. Our results indicate that epigenetic factors regulate the clusterin expression of RPE cells and thus might affect the pathogenesis of AMD via the inhibition of angiogenesis and inflammation.

Amyloid-beta 1-42 induced endocytosis and clusterin/apoJ protein accumulation in cultured human astrocytes.

Recent studies indicate that astrocytes may be the primary target of secreted amyloid-beta 1-42 peptides, with the neurotoxicity representing a secondary response to astrocytic stress. Our purpose was to clarify the astrocytic stress response induced by amyloid-beta peptides in human and rat astrocytes. Human amyloid-beta 1-42 peptides and fibrils induced the appearance of cytoplasmic vacuoles in normal human astrocytes (NHA) and CCFsttg1 astrocytoma cells. Vacuoles appeared 9-12h after the amyloid-beta exposure and remained present for several days. Rat primary neonatal astrocytes showed similar but less prominent vacuolar response. Human amyloid-beta peptides 1-16, 1-28, 10-20, 17-21 and 25-35 did not cause vacuole formation. Electron microscopic observations revealed large endocytic vacuoles containing fibrillar amyloid material. Stress marker analysis did not show any increase in protein levels of HSP70, HSP90, GRP78 and GRP94. However, the protein level of clusterin/apoJ, a secreted chaperone, was strongly increased both in NHA and CCFsttg1 astrocytes. Endocytic response associated with the accumulation of clusterin/apoJ protein suggests that clusterin/apoJ has a role in the clearance of amyloid-beta peptides.

Characterization of the pro-inflammatory signaling induced by protein acetylation in microglia.

Protein acetylation regulates the extent of inflammatory responses and disturbances in protein acetylation have been proposed to play an important role in inflammatory and neurodegenerative diseases. We have recently observed that histone deacetylase inhibitors, such as trichostatin A (TSA) and SAHA, strongly potentiate the LPS induced inflammatory response in several rat and mouse inflammatory models. Our aim here was to characterise pro-inflammatory signaling mediated via increased protein acetylation and protein phosphorylation in microglial N9 cells. First we observed that TSA induced pro-inflammatory response was independent of the different Toll-like receptors activated, since LPS, flagellin and unmethylated CpG oligonucleotides, equally potentiated IL-6 secretion from N9 microglia. Next we compared the protein acetylation induced potentiation to that induced by okadaic acid, a well-known inducer of pro-inflammatory responses. The time scale of the IL-6 responses showed that the effects of okadaic acid were clearly early-response effects appearing as soon as 6h after exposure, whereas TSA evoked a significant inhibition in IL-6 secretion up to 12h but after that it induced an exponential increase in cytokine and nitric oxide production up to 24h. It seems that okadaic acid induces an early moderate response and TSA a late but exponential potentiation of microglial inflammatory responses. The pro-inflammatory responses of TSA and okadaic acid were both dependent on NF-kappaB signaling but independent on the DNA-binding capacity of nuclear NF-kappaB complexes. Interestingly, we observed that the transactivation of the NF-kappaB-Luc reporter gene was clearly activated during TSA induced pro-inflammatory potentiation. Our studies imply that the potentiation of the inflammatory response by increased acetylation is due to the enhancement of transactivation of NF-kappaB driven inflammatory genes. Our studies on signaling pathways revealed that PI3K inhibitors LY294002 and Wortmannin blocked the TSA induced pro-inflammatory response but surprisingly did not affect the okadaic acid induced response. Furthermore, LY294002 did not inhibit DNA-binding activity of NF-kappaB but still inhibited NF-kappaB-Luc reporter gene transactivation. These results indicate that PI 3-kinase regulates the transactivation efficiency of NF-kappaB-dependent transcription rather than transduction of NF-kappaB signaling.

Induction of clusterin/apoJ expression by histone deacetylase inhibitors in neural cells.

The regulation of clusterin expression is poorly characterized although some regulatory elements have been identified, such as CpG-rich methylation domain. Environmental stress, aging, diet and diseases regulate DNA methylation and protein acetylation status but interestingly, the same insults increase clusterin expression in vivo. Our purpose was to elucidate whether histone deacetylase inhibitors, such as TSA, SAHA and M344, as well as an inhibitor of DNA methylation, 5'-aza-2'-deoxycytidine, could regulate the expression of clusterin in cultured neural cells. We observed that histone deacetylase inhibitors induced the expression of clusterin mRNA and protein in all neural cells studied. The induction of clusterin mRNA was blocked by actinomycin D which indicates that TSA regulates clusterin expression at the transcriptional level. An inhibitor of DNA methylation, 5'-aza-2'-deoxycytidine, itself did not affect the expression of clusterin mRNA but strongly potentiated the TSA-induced expression of clusterin. Proteasomal stress (MG-132 and PI-1 treatments) and apoptotic stress (okadaic acid treatment) did not affect clusterin expression which indicates that the induction of clusterin expression requires more specific inducers than cellular stress in general. Furthermore, LPS did not affect clusterin expression in N9 microglia although activated NF-kappaB signaling and IL-6 expression. CAPE and helenalin, inhibitors of NF-kappaB signalling, did not affect the clusterin mRNA expression either in non-treated or in TSA-treated N9 microglia. These observations suggest that clusterin induction is NF-kappaB-independent and unrelated to the inflammatory response in N9 microglia.

Regulation of microglial inflammatory response by sodium butyrate and short-chain fatty acids.

1. Recent studies have shown that sodium butyrate and other short-chain fatty acids (SCFAs) can prevent inflammation in colon diseases. Our aim was to elucidate whether sodium butyrate and SCFAs regulate the inflammatory responses in different neural inflammation models in cell cultures. 2. Inflammatory responses to LPS-induced microglial activation were recorded by the secretion of nitric oxide (NO) and cytokines IL-6 and TNF-alpha and related to the changes in the DNA-binding activities of NF-kappaB complex. 3. We observed that sodium butyrate is strongly anti-inflammatory against LPS-induced responses in rat primary microglia as well as in hippocampal slice cultures and in neural cocultures of microglial cells, astrocytes and cerebellar granule neurons. 4. In murine N9 microglial cell line, instead, sodium butyrate and other SCFAs (propionate, valerate and caproate) enhanced the LPS-induced inflammatory response. 5. The pretreatment with butyrate before LPS exposure induced an equal or more enhanced response than simultaneous exposure with butyrate and LPS. This indicates that butyrate induces an adaptative response against microglial activation. 6. We also observed that butyrate treatment both in transformed N9 cells and in hippocampal slice cultures downregulates the NF-kappaB-binding capacity induced by LPS stimulation. 7. Our results show that butyrate is anti-inflammatory in primary, brain-derived microglial cells, as observed recently in colon diseases, but proinflammatory in transformed, proliferating N9 microglial cells, which may be related to the anticancer properties of butyrate observed in tumor cells.