LMK235, a small molecule inhibitor of HDAC4/5, protects dopaminergic neurons against neurotoxin- and α-synuclein-induced degeneration in cellular models of Parkinson's disease

Epigenetic modifications in neurodegenerative disease are under investigation for their roles in disease progression. Alterations in acetylation rates of certain Parkinson's disease (PD)-linked genes have been associated with the pathological progression of this disorder. In light of this, and given the lack of disease-modifying therapies for PD, HDAC inhibitors (HDIs) are under consideration as potential pharmacological agents. The neuroprotective effects of pan-HDACs and some class-specific inhibitors have been tested in in vivo and in vitro models of PD, with varying outcomes. Here we used gene co-expression analysis to identify HDACs that are associated with human dopaminergic (DA) neuron development. We identified HDAC3, HDAC5, HDAC6 and HDAC9 as being highly correlated with the DA markers, SLC6A3 and NR4A2. RT-qPCR revealed that mRNA expression of these HDACs exhibited similar temporal profiles during embryonic mouse midbrain DA (mDA) neuron development. We tested the neuroprotective potential of a number of class-specific small molecule HDIs on human SH-SY5Y cells, using neurite growth as a phenotypic readout of neurotrophic action. Neither the class I-specific HDIs, RGFP109 and RGFP966, nor the HDAC6 inhibitor ACY1215, had significant effects on neurite outgrowth. However, the class IIa HDI, LMK235 (a HDAC4/5 inhibitor), significantly increased histone acetylation and neurite outgrowth. We found that LMK235 increased BMP-Smad-dependent transcription in SH-SY5Y cells and that this was required for its neurite growth-promoting effects on SH-SY5Y cells and on DA neurons in primary cultures of embryonic day (E) 14 rat ventral mesencephalon (VM). These effects were also seen in SH-SY5Y cells transfected with HDAC5 siRNA. Furthermore, LMK235 treatment exerted neuroprotective effects against degeneration induced by the DA neurotoxin 1-methyl-4-phenylpyridinium (MPP+), in both SH-SY5Y cells and cultured DA neurons. Treatment with LMK235 was also neuroprotective against axonal degeneration induced by overexpression of wild-type (WT) or A53T mutant α-synuclein in both SH-SY5Y cells and primary cultures of DA neurons. In summary, these data show the neuroprotective potential of the class IIa HDI, LMK235, in cell models of relevance to PD

Defining the potential of class-IIa histone deacetylases as a therapeutic target for Parkinson’s disease

Parkinson’s disease (PD) is a neurodegenerative disease characterized by early dopaminergic (DA) neuron degeneration, coupled with intracellular aggregation of α-synuclein (α-syn) in Lewy Bodies (LB). Given the lack of a disease-modifying therapy for PD, histone deacetylases (HDACs) have emerged as potential druggable therapeutic targets. The neuroprotective effects of pan-HDAC inhibitors (HDIs) and of some specific HDIs have been tested in both in vitro and in vivo models of PD, showing various outcomes, however which is the best class of HDACs to be targeted for neuroprotection in PD is unclear. In the first experimental chapter, we used gene co-expression analysis to show that HDAC5 and HDAC9 were positively co-expressed with the DA neuronal markers TH, GIRK2 and ALDH1A1 in the human substantia nigra (SN). We then demonstrated that both siRNA against HDAC5 and HDAC9, and the HDI MC1568 which inhibits both HDAC5 and HDAC9, promoted neurite outgrowth in SH-SY5Y cells and DA neurons. Neurite growth was used as a single cell readout of neurotrophic action. Furthermore, MC1568 treatment increased the expression of the neurotrophic factor BMP2, and of its downstream effector SMAD1. A reporter assay revealed that these HDAC5 and HDAC9 siRNAs, or treatment with MC1568, increased BMP-Smad dependent transcription. Furthermore, inhibition of BMP signalling either pharmacologically with dorsomorphin, or by overexpression of Smad4 dominant negative or inhibitory Smad7, abolished the neurite outgrowth-promoting effects of BMP2. Moreover, siRNAs against HDAC5 or HDAC9, but not against HDAC4 or HDAC7, promoted neurite growth in the presence of WT and A53T α-syn. Treatment with MC1568 protected DA neurons from MPP+-induced neurodegeneration in vitro. These findings confirmed HDAC5 and HDAC9 as novel regulators of BMP-Smad neurotrophic factor signalling pathway, making them potential therapeutic targets for PD. In the second experimental chapter, we expanded our gene co-expression analysis and found that HDAC3, HDAC5, HDAC6 and HDAC9 were co-expressed with the DA neuronal markers SLC6A3 and NR4A2 in the human SN. A RT-qPCR confirmed that mRNA of all four HDACs exhibited similar temporal expression profiles during DA neuronal development. We next investigated whether class-specific pharmacological inhibition of these four HDACs could promote neurite growth. We found that inhibition of HDAC1 and HDAC3, or of HDAC3 alone, using RGFP109 or RGFP966 respectively, and inhibition of HDAC6 using ACY1215, did not promote neurite growth or affect survival in SH-SY5Y cells. Conversely, LMK235, which is a class-IIa-specific HDI which mainly inhibits HDAC4 and HDAC5, significantly increased both histone acetylation levels and neurite outgrowth in SH-SY5Y cells. Using a GFP reporter assay, we confirmed that administration of LMK235 increased Smad-dependent transcription. We then showed that inhibition of BMPR1B receptor in both SH-SY5Y cells and primary DA neurons prevented the neurite growth-promoting effects of LMK235. Furthermore, LMK235 was neuroprotective against MPP+ neurotoxic insult, and against wild type (WT) and A53T α-syn overexpression, in both SH-SY5Y cells and DA neurons. These data confirmed the neuroprotective effects of class-IIa HDAC inhibition in vitro. In the third experimental chapter, we investigated the cellular localisation of HDAC5 and the therapeutic potential of the classIIa inhibitor MC1568 in vivo, in 6-hydroxydopamine (6-OHDA) treated in vitro models of PD. We found that 6-OHDA treatment of both SH-SY5Y cells and DA neurons led to nuclear accumulation of HDAC5. This was prevented by PMA, which activates the canonical PKC pathway that is known to cause HDAC5 shuttling out of the nucleus. Having confirmed that MC1568 treatment protected SH-SY5Y cells from 6-OHDA-induced neurodegenration in vitro, we next examined whether peripheral administration (7 daily i.p. injections) of MC1568 was neuroprotective in the intrastriatal 6-OHDA lesion rat model of PD in vivo. Behavioural analysis at 8 and 12 days after lesion surgery showed that MC1568 treatment partially ameliorated 6-OHDA-induced forelimb akinesia. Post-mortem analysis of nigrostriatal integrity showed that MC1568 partially protected striatal dopaminergic terminals and DA neurons in the SN and prevented the increase in microglial numbers in both the striatum (ST) and SN. Finally, we confirmed that 6-OHDA lesion induced HDAC5 nuclear accumulation in DA neurons in the SN, which was prevented by MC1568 treatment. Collectively, these data rationalise the strategy of peripheral administration of MC1568 for neuroprotection in PD. In the fourth and final experimental chapter, we sought to determine whether HDAC5 nuclear accummulation occurred in other in vitro and in vivo models of PD . We verified that there are increased nuclear levels of HDAC5 in SH-SY5Y cells and in DA neurons treated with the neurotoxin MPP+, or in those overexpressing of α-syn or LRRK2 G2019S. We found that combined overexpression of nuclear-restricted HDAC5 and α-syn further increased neurite degeneration. Furthermore, activation of the canonical CaMK/PKC pathway prevented HDAC5 nuclear accumulation and the consequent neurite degeneration. Finally, we demonstrated that HDAC5 nuclear accumulation occurred in DA neurons in the SN, in both the MPTP mouse model and the AAV-αSyn rat model of PD. Collectively, the data presented in this thesis rationalise the future development of strategies focused on HDAC5 inhibition as a potential neuroprotective strategy for PD

Alpha-Linolenic Acid and Cardiovascular Events: A Narrative Review

Cardiovascular diseases (CVDs) represent the leading cause of global mortality with 1.7 million deaths a year. One of the alternative systems to drug therapy to minimize the risk of CVDs is represented by alpha-linolenic acid (ALA), an essential fatty acid of the omega-3 series, known for its cholesterol-lowering effect. The main purpose of this review is to analyze the effects of ALA and investigate the relevant omega-6/omega-3 ratio in order to maintain functionally beneficial effects. Concerning the lipid-lowering preventive effects, ALA may favorably affect the values of LDL-C and triglycerides in both adult and pediatric populations. Furthermore, ALA has shown protective effects against hypertension, contributing to balancing blood pressure through customary diet. According to the 2009 EFSA statement, dietary ALA may contribute to reducing the risk of CVDs, thanks to anti-hypertensive, anti-atherosclerotic and cardioprotective effects

Magnetic resonance advanced imaging analysis in adolescents: cortical thickness study to identify attenuated psychosis syndrome

PurposePsychosis is a symptom common to several mental illnesses and a defining feature of schizophrenia spectrum disorders, whose onset typically occurs in adolescence. Neuroradiological studies have reported evidence of brain structural abnormalities in patients with overt psychosis. However, early identification of brain structural changes in young subjects at risk for developing psychosis (such as those with Attenuated Psychosis Syndrome -APS) is currently lacking.MethodsBrain 3D T-1-weighted and 64 directions diffusion-weighted images were acquired on 55 help-seeking adolescents (12-17 years old) with psychiatric disorders who referred to our Institute. Patients were divided into three groups: non-APS (n = 20), APS (n = 20), and Early-Onset Psychosis (n = 15). Cortical thickness was calculated from T(1)w images, and Tract-Based Spatial Statistics analysis was performed to study the distribution of white matter fractional anisotropy and all diffusivity metrics. A thorough neuropsychological test battery was adopted to investigate cognitive performance in several domains.ResultsIn patients with Attenuated Psychotic Syndrome, the left superior frontal gyrus was significantly thinner compared to patients with non-APS (p = 0.048), and their right medial orbitofrontal cortex thickness was associated with lower working memory scores (p = 0.0025, r = -0.668 for the working memory index and p = 0.001, r = -0.738 for the digit span). Early-Onset Psychosis patients showed thinner left pars triangularis compared to non-APS individuals (p = 0.024), and their left pars orbitalis was associated with impaired performance at the symbol search test (p = 0.005, r = -0.726). No differences in diffusivity along main tracts were found between sub-groups (p > 0.05).ConclusionThis study showed specific associations between structural imaging features and cognitive performance in patients with APS. Characterizing this disorder using neuroimaging could reveal useful information that may aid in the development and evaluation of preventive strategies in these individuals

Hyperparathyroidism, arterial hypertension and aortic stiffness: a possible bidirectional link between the adrenal cortex and the parathyroid glands that causes vascular damage?

n/