Phospho-HDAC6 Gathers Into Protein Aggregates in Parkinson’s Disease and Atypical Parkinsonisms

HDAC6 is a unique histone deacetylase that targets cytoplasmic non-histone proteins and has a specific ubiquitin-binding activity. Both of these activities are required for HDAC6-mediated formation of aggresomes, which contain misfolded proteins that will ultimately be degraded via autophagy. HDAC6 deacetylase activity is increased following phosphorylation on serine 22 (phospho-HDAC6). In human, HDAC6 localizes in neuronal Lewy bodies in Parkinson\u2019s disease (PD) and in oligodendrocytic Papp\u2013Lantos bodies in multiple system atrophy (MSA). However, the expression of phospho-HDAC6 in post-mortem human brains is currently unexplored. Here, we evaluate and compare the distribution of HDAC6 and its phosphorylated form in human brains obtained from patients affected by three forms of parkinsonism: two synucleinopathies (PD and MSA) and a tauopathy (progressive supranuclear palsy, PSP). We find that both HDAC6 and its phosphorylated form localize with pathological protein aggregates, including \u3b1-synuclein-positive Lewy bodies in PD and Papp\u2013Lantos bodies in MSA, and phospho-tau-positive neurofibrillary tangles in PSP. We further find a direct interaction of HDAC6 with \u3b1-synuclein with proximity ligation assay (PLA) in neuronal cell of PD patients. Taken together, our findings suggest that both HDAC6 and phospho-HDAC6 regulate the homeostasis of intra-neuronal proteins in parkinsonism

Activation of M2 muscarinic acetylcholine receptors by orthosteric and dualsteric agonists in glioblastoma cancer stem cells: effects on cell proliferation and drug resistance

The involvement of muscarinic receptors in cancer has been largely documented. While M3 muscarinic receptors results to promote tumor growth and progression, M2 subtype negatively modulates cell growth and survival in different tumor types. Our previous studies demonstrated that M2 orthosteric agonist Arecaidine Propargyl Ester (APE), arrested cell proliferation and induced apoptosis in glioblastoma cell lines and in glioblastoma cancer stem cells (GSC), an undifferentiated GB subpopulation characterized by high chemioresistance. Studies on glioblastoma stable cell lines have demonstrated that low doses of M2 agonist APE were able to counteract the drug resistance for conventional drugs such as doxorubicin (Doxo) and Temozolomide, negatively modulating the drug efflux pump AGCG2. Similarly, in GSCs, the hybrid (orthosteric/allosteric) muscarinic agonist N-8-Iper inhibited cell growth in a dose dependent manner and also impaired cell survival at low doses. The inhibitory effects of the N-8-Iper appeared to be mediated by M2 receptor activation, since they were strongly reduced by co-administration of the selective M2 receptor antagonist methoctramine as well as upon M2 receptor silencing. The dualsteric agonist N8-Iper also decreased the expression of ABC drug efflux pumps (C1 and G2) both as transcript and as protein. Moreover the co-treatment of M2 agonists with low doses of Doxo (6.2 microM) significantly affected cell growth compared with the treatment with Doxo alone. Our data suggest that M2 receptor agonists, decreasing the drug efflux pumps expression, may impair the cancer stem cell chemoresistance, making the tumor cells more responsive to low doses of conventional drugs and reducing the side effects induced by chemotherapy

Selective agonists for M2 muscarinic acetylcholine receptors inhibit cell proliferation and survival in human glioblastoma cells: possible effects on drug resistance

Introduction. The involvement of muscarinic acetylcholine receptors (mAChRs) in cancer has been largely documented. Recently, we have demonstrated that activation of M2AChRs arrests cell proliferation and induces apoptosis in glioblastoma (GB) cell lines and in glioblastoma cancer stem cells (GSC). In the present work, we compared the effects mediated by the preferential activation of M2AChRs, through the M2 agonist arecaidine propargyl ester (APE) and the dualsteric agonist N8-Iper, both in GB cell lines and in GSC, analyzing their antiproliferative and pro-apoptotic effects and their potential use in combined therapy with conventional drugs (i.e., temozolomide). Material and Methods. U251 established cell lines and GSC (GB7 cell line) obtained from human biopsy were used as cell models. MTT assay, trypan blue staining and FACS analysis were used to evaluate cell viability and cell death. The M2 silencing (by siRNA) and pharmacological competition were used to confirm the ability of M2 agonists to selectively bind this receptor subtype. Transcript levels for multidrug efflux pumps (e.g., ATP binding cassette, ABC) were analyzed by Real-Time PCR and Western Blot analysis. Results. In GB7 cells, treatment with the N8-Iper decreased cell proliferation in a time and dose dependent manner as previously demonstrated after APE treatments. FACS analysis has also shown that in GB7 cells N8-Iper caused a significant increase of cell death. The pharmacological competition with M1 and M3 antagonists (pirenzepine and 4-DAMP, respectively) and the M2 antagonist methoctramine indicated that the effects of N8-Iper were dependent on selective activation of the M2AChR subtype. The ability of APE and N-8-Iper to activate M2 receptor populations was also confirmed by CHRM2 siRNA transfection. Moreover, APE and N-8-Iper decreased the mRNA levels and the protein expression of the ABC-G2 pump. Finally, the combined treatment with an M2 agonist and temozolomide indicated a synergic effect of the two drugs in decreasing GB cell survival. Conclusions. Our findings suggest that M2AChR agonists represent a new putative therapeutic tool in glioblastoma treatment. Moreover, the ability of M2 agonists to decrease the ABC-G2 drug efflux pumps expression in GSC cells, coupled with their synergic effect in combination with temozolomide, are indicative of their relevant role in reducing the GB chemoresistance

Selective agonists for M2 muscarinic receptors inhibit cell proliferation and survival in human glioblastoma cells: Possible implications in drug resistance

Introduction. The involvement of muscarinic receptors in cancer has been largely documented. Recently, we have demonstrated that the activation of M2 muscarinic receptors, by the agonist Arecaidine Propargyl Ester (APE), arrests cell proliferation and induces apoptosis in glioblastoma (GB) cell lines. In the present work, we investigated the effects mediated by M2 receptors in glioblastoma cancer stem cells (GSC), an undifferentiated GB subpopulation characterized by high chemioresistance. Material and Methods. GB7 and GB8 cell lines obtained from human biopsies were cultured in Euromed-N supplemented with N2, B27, EGF and FGF. MTT assay and trypan blue staining were used to evaluate cell viability and cell death, respectively. By means of M2 silencing (by siRNA) and pharmacological competition we confirmed the ability of M2 agonists to selectively bind this receptor subtype. Transcript levels for muscarinic receptors and multidrug efflux pumps (e.g. ATP binding cassette, ABC) were analyzed by RT-PCR analysis. Results. Our experiments were performed with the M2 agonist APE, the muscarinic orthosteric superagonist Iperoxo and its related dualsteric agonists P-6-Iper and N-8- Iper. In GB7, treatment with the M2 agonist APE (100 \u3bcM) decreased cell proliferation in a time and dose dependent manner. In GB8 APE induced cell death. Cell proliferation and survival of U251 and U87 cell lines and GSC cells were unaffected by treatment with Iperoxo and P-6-Iper. Conversely, N-8-Iper decreased cell proliferation in a time and dose dependent manner. Also in GB7 cells, N-8-Iper inhibited cell growth and survival also at lower concentration (12.5 \u3bcM). The co-treatment of GB cells with M2 agonists (APE or N-8-iper) and different muscarinic antagonists confirmed that the decreased agonist-induced cell proliferation and survival were dependent on selective activation of M2 receptor. Similarly, the silencing of M2 receptor abolished the M2- mediated agonists effects. Moreover, APE and N-8-Iper decreased, particularly in GB7 cells, the mRNA levels for the ABC drug efflux pumps (C1 and G2). Conclusions. Our data suggest that M2 receptor agonists represent a new relevant tools to investigate glioblastoma-related mechanisms. Furthermore, the ability of M2 agonists to decrease the drug efflux pumps expression, in particular in GSC cells, suggests that they may have a role in reducing the GSC chemoresistance, and make them more responsive to conventional drugs (e.g. temozolomide)