Neuroprotective strategies against neurodegeneration in cellular model systems

In the present study we analyzed new neuroprotective therapeutical strategies in PD (Parkinson’s disease) and AD (Alzheimer’s disease). Current therapeutic strategies for treating PD and AD offer mainly transient symptomatic relief but it is still impossible to block the loss of neuron and then the progression of PD and AD. There is considerable consensus that the increased production and/or aggregation of α- synuclein (α-syn) and β-amyloid peptide (Aβ), plays a central role in the pathogenesis of PD, related synucleinopathies and AD. Therefore, we identified antiamyloidogenic compounds and we tested their effect as neuroprotective drug-like molecules against α-syn and β-amyloid cytotoxicity in PC12. Herein, we show that two nitro-catechol compounds (entacapone and tolcapone) and 5 cathecol-containing compounds (dopamine, pyrogallol, gallic acid, caffeic acid and quercetin) with antioxidant and anti-inflammatory properties, are potent inhibitors of α-syn and β-amyloid oligomerization and fibrillization. Subsequently, we show that the inhibition of α-syn and β-amyloid oligomerization and fibrillization is correlated with the neuroprotection of these compounds against the α-syn and β-amyloid-induced cytotoxicity in PC12. Finally, we focused on the study of the neuroprotective role of microglia and on the possibility that the neuroprotection properties of these cells could be use as therapeutical strategy in PD and AD. Here, we have used an in vitro model to demonstrate neuroprotection of a 48 h-microglial conditioned medium (MCM) towards cerebellar granule neurons (CGNs) challenged with the neurotoxin 6-hydroxydopamine (6-OHDA), which induces a Parkinson-like neurodegeneration, with Aβ42, which induces a Alzheimer-like neurodegeneration, and glutamate, involved in the major neurodegenerative diseases. We show that MCM nearly completely protects CGNs from 6-OHDA neurotoxicity, partially from glutamate excitotoxicity but not from Aβ42 toxin

Microglial polarization differentially affects neuronal vulnerability to the β-amyloid protein: Modulation by melatonin

Microglial cells play a central but yet debated role in neuroinflammatory events occurring in Alzheimer's disease (AD). We here explored how microglial features are modulated by melatonin following beta-amyloid (A beta 42)-induced activation and examined the cross-talk with A beta-challenged neuronal cells. Human microglial HMC3 cells were exposed to A beta 42 (200 nM) in the presence of melatonin (MEL; 1 mu M) added since the beginning (MELco) or after a 72 h-exposure to A beta 42 (MELpost). In both conditions, MEL favored an anti-inflammatory activation and rescued SIRT1 and BDNF expression/release. Caspase-1 up-regulation and phospho-ERK induction following a prolonged exposure to A beta 42 were prevented by MEL. In addition, MEL partially restored proteasome function-ality that was altered by long-term A beta 42 treatment, re-establishing both 20S and 26S chymotrypsin-like activity. Differentiated neuronal-like SH-SY5Y cells were exposed to A beta 42 (200 nM for 24 h) in basal medium or in the presence of conditioned medium (CM) collected from microglia exposed for different times to A beta 42 alone or in combination with MELco or MELpost. A beta 42 significantly reduced pre-synaptic proteins synaptophysin and VAMP2 and mean neuritic length. These effects were prevented by CM from anti-inflammatory microglia (A beta 42 for 6 h), or from MELco and MELpost microglia, but the reduction of neuritic length was not rescued when the SIRT1 inhibitor EX527 was added.In conclusion, our data add to the concept that melatonin shows a promising anti-inflammatory action on microglia that is retained even after pro-inflammatory activation, involving modulation of proteasome function and translating into neuroprotective microglial effects

Novel therapeutic strategy for neurodegeneration by blocking Aβ seeding mediated aggregation in models of Alzheimer's disease

Aβ accumulation plays a central role in the pathogenesis of Alzheimer's disease (AD). Recent studies suggest that the process of Aβ nucleated polymerization is essential for Aβ fibril formation, pathology spreading and toxicity. Therefore, targeting this process represents an effective therapeutic strategy to slow or block disease progression. To discover compounds that might interfere with the Aβ seeding capacity, toxicity and pathology spreading, we screened a focused library of FDA-approved drugs in vitro using a seeding polymerization assay and identified small molecule inhibitors that specifically interfered with Aβ seeding-mediated fibril growth and toxicity. Mitoxantrone, bithionol and hexachlorophene were found to be the strongest inhibitors of fibril growth and protected primary cortical neuronal cultures against Aβ-induced toxicity. Next, we assessed the effects of these three inhibitors in vivo in the mThy1-APPtg mouse model of AD (8-month-old mice). We found that mitoxantrone and bithionol, but not hexachlorophene, stabilized diffuse amyloid plaques, reduced the levels of Aβ42 oligomers and ameliorated synapse loss, neuronal damage and astrogliosis. Together, our findings suggest that targeting fibril growth and Aβ seeding capacity constitutes a viable and effective strategy for protecting against neurodegeneration and disease progression in AD

Knockout or inhibition of USP30 protects dopaminergic neurons in a Parkinson’s disease mouse model

Mutations in SNCA, the gene encoding α-synuclein (αSyn), cause familial Parkinson’s disease (PD) and aberrant αSyn is a key pathological hallmark of idiopathic PD. This α-synucleinopathy leads to mitochondrial dysfunction, which may drive dopaminergic neurodegeneration. PARKIN and PINK1, mutated in autosomal recessive PD, regulate the preferential autophagic clearance of dysfunctional mitochondria (“mitophagy”) by inducing ubiquitylation of mitochondrial proteins, a process counteracted by deubiquitylation via USP30. Here we show that loss of USP30 in Usp30 knockout mice protects against behavioral deficits and leads to increased mitophagy, decreased phospho-S129 αSyn, and attenuation of SN dopaminergic neuronal loss induced by αSyn. These observations were recapitulated with a potent, selective, brain-penetrant USP30 inhibitor, MTX115325, with good drug-like properties. These data strongly support further study of USP30 inhibition as a potential disease-modifying therapy for PD.</p

Role of endothelial cell stress in the pathogenesis of chronic heart failure

Endothelial cells are key modulators of diverse physiological processes, and their impaired function is a cause of numerous cardiovascular diseases. Under physiologic condition, the reactive oxygen and nitrogen mediators in endothelia lead to the signal propagation of the initial stimulus, by forming molecules with a longer half-life like hydrogen peroxide. Hydrogen peroxide is the focus of growing attention in endothelial biology, and consequently the enzymes involved in its generation and clearance are viewed as novel mediators of great importance. In particular, among peroxidases, myeloperoxidase is recognized as a key enzyme, capable of impairing intracellular NO reservoirs as well as producing oxidized amino acids such as 3-chlorotyrosine or 3-nitrotyrosine. This process switches the functional pathways from normal signalling to a condition characterized by oxidative and/or nitrosative stress. Understanding the molecular mechanisms involved in these stress responses in endothelium will lead to better therapeutic strategies for oxidative stress-driven cardiovascular diseases

Knockout or inhibition of USP30 protects dopaminergic neurons in a Parkinson’s disease mouse model

Acknowledgements: We thank Dr Laura Parton for project management/co-leadership of MTX115325, providing the foundation for Mission compound studies and MTX115325 development in PD. We also thank Drs. Krutika Joshi and Veronique VanderHorst of BIDMC for their support with slides scanning for TH+ neuronal counting and TH+ fiber densitometry. We thank Matthew Jacobsen for the pathology assessment of mouse livers and Nirav Prakas Patel for early work on mouse ESCs. We would like to thank Prof. Maria Grazia Spillantini for her valuable comments on the manuscript. Research in the D.K.S. lab is funded by an NINDS grant (R21NS109408), the Weston Brain Institute and the Owens Foundation. Research in the G.B. lab is supported by the UK Dementia Research Institute, that receives contributions from UK DRI Ltd, the UK MRC, the Alzheimer’s Society, and Alzheimer’s Research UK as well as a grant from the Romanian Ministry of Research, Innovation and Digitization (no. PNRR-III-C9-2022-I8-66; contract 760114). IGG was funded by a grant from the Medical Research Council, UK (MC_UU_00018/2). Research in the S.P.J. lab is funded by Cancer Research UK Discovery grant (DRCPGM\100005), and ERC Synergy grant DDREAMM (855741). This project has received funding to SJ from CRUK programme grant C6/A11224, C6/A18796 and Wellcome Investigator Award (206388/Z/17/Z) together with core infrastructure funding by Cancer Research UK (C6946/A24843) and Wellcome (WT203144). The Sanger Mouse Genetics Project was supported by the Wellcome Trust (098051). CRUK supports D.J.A.Funder: G.B. lab is supported by the UK Dementia Research Institute, that receives contributions from UK DRI Ltd, the UK MRC, the Alzheimer’s Society, and Alzheimer’s Research UK as well as a grant from the Romanian Ministry of Research, Innovation and Digitization (no. PNRR-III-C9-2022-I8-66; contract 760114).AbstractMutations in SNCA, the gene encoding α-synuclein (αSyn), cause familial Parkinson’s disease (PD) and aberrant αSyn is a key pathological hallmark of idiopathic PD. This α-synucleinopathy leads to mitochondrial dysfunction, which may drive dopaminergic neurodegeneration. PARKIN and PINK1, mutated in autosomal recessive PD, regulate the preferential autophagic clearance of dysfunctional mitochondria (“mitophagy”) by inducing ubiquitylation of mitochondrial proteins, a process counteracted by deubiquitylation via USP30. Here we show that loss of USP30 in Usp30 knockout mice protects against behavioral deficits and leads to increased mitophagy, decreased phospho-S129 αSyn, and attenuation of SN dopaminergic neuronal loss induced by αSyn. These observations were recapitulated with a potent, selective, brain-penetrant USP30 inhibitor, MTX115325, with good drug-like properties. These data strongly support further study of USP30 inhibition as a potential disease-modifying therapy for PD.</jats:p