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Alzheimer's disease (AD), the most common type of dementia, currently represents an extremely challenging and unmet medical need worldwide. Amyloid-β (Aβ) and Tau proteins are prototypical AD hallmarks, as well as validated drug targets. Accumulating evidence now suggests that they synergistically contribute to disease pathogenesis. This could not only help explain negative results from anti-Aβ clinical trials but also indicate that therapies solely directed at one of them may have to be reconsidered. Based on this, herein, we describe the development of a focused library of 2,4-thiazolidinedione (TZD)-based bivalent derivatives as dual Aβ and Tau aggregation inhibitors. The aggregating activity of the 24 synthesized derivatives was tested in intact Escherichia coli cells overexpressing Aβ42 and Tau proteins. We then evaluated their neuronal toxicity and ability to cross the blood-brain barrier (BBB), together with the in vitro interaction with the two isolated proteins. Finally, the most promising (most active, nontoxic, and BBB-permeable) compounds 22 and 23 were tested in vivo, in a Drosophila melanogaster model of AD. The carbazole derivative 22 (20 μM) showed extremely encouraging results, being able to improve both the lifespan and the climbing abilities of Aβ42 expressing flies and generating a better outcome than doxycycline (50 μM). Moreover, 22 proved to be able to decrease Aβ42 aggregates in the brains of the flies. We conclude that bivalent small molecules based on 22 deserve further attention as hits for dual Aβ/Tau aggregation inhibition in AD., This work was conducted by A.E.G during a scholarship at the University of Bologna, Italy, supported by the International Cooperation Program CAPES/ PDSE Process #88881.187586/ 2018-01. O.S. acknowledges the Ministry of Education, Youth and Sports of the Czech Republic (project ERDF no. CZ.02.1.01/0.0/0.0/18_069/0010054). DMT: grant PID2020-118127RB-I00 funded by MCIN/AEI/10.13039/501100011033. M.L.B. and M.B. would also like to acknowledge the University of Bologna (RFO 2019) and the Italian Ministry of Education, Universities and Research (MIUR), for financial support.

Multicellular tumor spheroids are rapidly emerging as an improved in vitro model with respect to more traditional 2D culturing. Microwell culturing is a simple and accessible method for generating a large number of uniformly sized spheroids, but commercially available systems often do not enable researchers to perform complete culturing and analysis pipelines and the mechanical properties of their culture environment are not commonly matching those of the target tissue. We herein report a simple method to obtain custom-designed self-built microwell arrays made of polydimethylsiloxane or agarose for uniform 3D cell structure generation. Such materials can provide an environment of tunable mechanical flexibility. We developed protocols to culture a variety of cancer and non-cancer cell lines in such devices and to perform molecular and imaging characterizations of the spheroid growth, viability, and response to pharmacological treatments. Hundreds of tumor spheroids grow (in scaffolded or scaffold-free conditions) at homogeneous rates and can be harvested at will. Microscopy imaging can be performed in situ during or at the end of the culture. Fluorescence (confocal) microscopy can be performed after in situ staining while retaining the geographic arrangement of spheroids in the plate wells. This platform can enable statistically robust investigations on cancer biology and screening of drug treatments. ispartof: Cell Mol Life Sci vol:80 issue:4 pages:93- ispartof: location:Switzerland status: Published online

68 p.-16 fig.-4 tab.+8 fig. supl.-5 tab. supl., Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease without any effective treatment. Protein TDP-43 is a pathological hallmark of ALS in both sporadic and familiar patients. Post-translational modifications of TDP-43 promote its aggregation in the cytoplasm. Tau-Tubulin kinase (TTBK1) phosphorylates TDP-43 in cellular and animal models; thus, TTBK1 inhibitors emerge as a promising therapeutic strategy for ALS. The design, synthesis, biological evaluation, kinase–ligand complex structure determination, and molecular modeling studies confirmed novel pyrrolopyrimidine derivatives as valuable inhibitors for further development. Moreover, compound 29 revealed good brain penetration in vivo and was able to reduce TDP-43 phosphorylation not only in cell cultures but also in the spinal cord of transgenic TDP-43 mice. A shift to M2 anti-inflammatory microglia was also demonstrated in vivo. Both these activities led to motor neuron preservation in mice, proposing pyrrolopyrimidine 29 as a valuable lead compound for future ALS therapy., This work was supported by Comunidad de Madrid (grant B2017/BMD-3813), the European Social Fund+ (ESF+), MINECO (Grants SAF2016-76693-R to A.M., RTI2018-0988885-B-I00 to E.d.L., and CTQ2015-66313-R to A.M.R.), AIE (RTI2018-099318-B-I00 to D.L., cofunded by the European Regional Development Fund (FEDER)), ISCiii CIBERNED (CB18/05/00040 to A.M., V.P., and A.M.R. and CB06/05/0089 to E.dL.), MECD (FPU16/04466 to V.N.), Cost Action CA15135 “MuTaLig” (COST-STSM-CA15135-37514 to L.M.G.), FONDECYT (Grant 11180604 to D.R.). V.P. has received financial support through the Postdoctoral Junior Leader Fellowship Program (LCF/BQ/PR18/11640007) from “la Caixa” Banking Foundation.

Dementia is a pathological condition characterized by a decline in memory, as well as in other cognitive and social functions. The cellular and molecular mechanisms of brain damage in dementia are not completely understood; however, neuroinflammation is involved. Evidence suggests that chronic inflammation may impair cognitive performance and that dietary protein source may differentially influence this process. Dietary protein source has previously been shown to modify systemic inflammation in mouse models. Thus, we aimed to investigate the effect of chronic dietary protein source substitution in an ageing and dementia male mouse model, the senescence-accelerated mouse-prone 8 (SAMP8) model. We observed that dietary protein source differentially modified memory as shown by inhibitory avoidance testing at 4 months of age. Also, dietary protein source differentially modified neuroinflammation and gliosis in male SAMP8 mice. Our results suggest that chronic dietary protein source substitution may influence brain ageing and memory-related mechanisms in male SAMP8 mice. Moreover, the choice of dietary protein source in mouse diets for experimental purposes may need to be carefully considered when interpreting results.

Hormonal contraceptives prevent ovulation with subsequent reduction in endogenous levels of estradiol, progesterone and its neuroactive metabolite allopregnanolone. These neurosteroids modulate several brain functions, including neuronal plasticity, cognition and memory. We hypothesized that hormonal contraceptives might affect synaptic plasticity, learning and memory, as a consequence of suppressed endogenous hormones levels. Female rats were orally treated with a combination of ethinyl estradiol (EE, 0.020 mg) and levonorgestrel (LNG, 0.060 mg) once daily for four weeks. Decreased hippocampal brain-derived neurotrophic factor (BDNF) levels and altered histone H3 post-translational modifications (PTMs) were observed 14 days after discontinuation from chronic EE-LNG treatment. These effects were not accompanied by alterations in long-term plasticity at glutamatergic synapses, recognition memory in the novel object and novel place location tests, or spatial learning, memory, and behavioral flexibility in the Morris water maze test. Thus, decreased BDNF content does not affect synaptic plasticity and cognitive performance; rather it might be relevant for the occurrence of certain psychiatric symptoms, reported by some women using hormonal contraceptives. These results provide the first evidence of hippocampal epigenetic changes induced by hormonal contraceptives and complement previous studies on the neurobiological actions of hormonal contraceptives; the finding that effects of chronic EE-LNG treatment on BDNF content and histone PTMs are observed 14 days after drug discontinuation warrants further investigation to better understand the implications of such long-term consequences for women's health.

Polypharmacology is a new trend in amyotrophic lateral sclerosis (ALS) therapy and an effective way of addressing a multifactorial etiology involving excitotoxicity, mitochondrial dysfunction, oxidative stress, and microglial activation. Inspired by a reported clinical trial, we converted a riluzole (1)-rasagiline (2) combination into single-molecule multi-target-directed ligands. By a ligand-based approach, the highly structurally integrated hybrids 3-8 were designed and synthesized. Through a target- and phenotypic-based screening pipeline, we identified hit compound 6. It showed monoamine oxidase A (MAO-A) inhibitory activity (IC50 = 6.9 mu M) rationalized by in silico studies as well as in vitro brain permeability. By using neuronal and non-neuronal cell models, including ALS-patient-derived cells, we disclosed for 6 a neuroprotective/neuroinflammatory profile similar to that of the parent compounds and their combination. Furthermore, the unexpected MAO inhibitory activity of 1 (IC50 = 8.7 mu M) might add a piece to the puzzle of its anti-ALS molecular profile.

Microglial cells, the immune cells of the central nervous system (CNS), play a crucial role for the proper brain development and function and in CNS homeostasis. While in physiological conditions, microglia continuously check the state of brain parenchyma, in pathological conditions, microglia can show different activated phenotypes: In the early phases, microglia acquire the M2 phenotype, increasing phagocytosis and releasing neurotrophic and neuroprotective factors. In advanced phases, they acquire the M1 phenotype, becoming neurotoxic and contributing to neurodegeneration. Underlying this phenotypic change, there is a switch in the expression of specific microglial genes, in turn modulated by epigenetic changes, such as DNA methylation, histones post-translational modifications and activity of miRNAs. New roles are attributed to microglial cells, including specific communication with neurons, both through direct cell–cell contact and by release of many different molecules, either directly or indirectly, through extracellular vesicles. In this review, recent findings on the bidirectional interaction between neurons and microglia, in both physiological and pathological conditions, are highlighted, with a focus on the complex field of microglia immunomodulation through epigenetic mechanisms and/or released factors. In addition, advanced technologies used to study these mechanisms, such as microfluidic, 3D culture and in vivo imaging, are presented.

Parkinson’s disease (PD) is a widely spread neurodegenerative disorder caused by both genetic and environmental factors, leading to the appearance of characteristic motor and non-motor symptoms. Activated microglia plays a pivotal role in the pathogenesis of PD, sustaining inflammatory processes at the level of neurodegenerative regions by releasing Reactive Oxygen and Nitrogen Species, exposing neurons to oxidative stress. It has recently been observed that the dysregulation of long non-coding RNAs (lncRNAs) could affect the modulation of microglial phenotype in neurodegenerative diseases. Interestingly, we identified a differential expression of some lncRNAs by comparing transcriptomic data of PD patient's vs control brain samples through a detailed meta-analysis. Our study aims to characterize the expression and function of LINC00520, a lncRNA not previously linked to PD, in several PD models. In particular, we tested its gene expression compared to a control lncRNA, LINC00674, in human neuroblastoma SH-SY5Y cells subjected to 6-OHDA treatment and in human monocytic THP-1 cells differentiated towards microglial fate with PMA and stimulated with LPS. We then enrolled the human microglia cell line HMC3 treated with IFN-γ boosted by glucose, to promote the NF-κB inflammatory pathway, or 6-OHDA. The results show a significant upregulation of LINC00520 both in inflammatory states and in oxidative stress conditions. Moreover, preliminary data of RNAi assays against LINC00520 suggested its involvement in oxidative stress-related pathways. Characterization of ZFLNCG09760, a putative LINC00520 ortholog in zebrafish, as an in vivo model, is currently ongoing.

International audience; A series of multitarget-directed ligands (MTDLs) was designed by functionalizing a pseudo-irreversible butyrylcholinesterase (BChE) inhibitor. The obtained hybrids were investigated in vitro regarding their hBChE and hAChE inhibition, their enzyme kinetics, and their antioxidant physicochemical properties (DPPH, ORAC, metal chelating). In addition, in vitro assays were applied to investigate antioxidant effects using murine hippocampal HT22 cells and immunomodulatory effects on the murine microglial N9 cell line. The MTDLs retained their antioxidative properties compared to the parent antioxidant-moieties in vitro and the inhibition of hBChE was maintained in the submicromolar range. Representative compounds were tested in a pharmacological Alzheimer's disease (AD) mouse model and demonstrated very high efficacy at doses as low as 0.1 mg/kg. The most promising compound was also tested in BChE(-/-) mice and showed reduced efficacy. In vivo neuroprotection by BChE inhibition can be effectively enhanced by incorporation of structurally diverse antioxidant moieties.

To address the multifactorial nature of Alzheimer's Disease (AD), a multi-target-directed ligand approach was herein developed. As a follow-up of our previous studies, a small library of newly designed 2-arylbenzofuran derivatives was evaluated towards cholinesterases and cannabinoid receptors. The two most promising compounds, 8 and 10, were then assessed for their neuroprotective activity and for their ability to modulate the microglial phenotype. Compound 8 emerged as able to fight AD from several directions: it restored the cholinergic system by inhibiting butyrylcholinesterase, showed neuroprotective activity against A beta(1-42) oligomers, was a potent and selective CB2 ligand and had immunomodulatory effects, switching microglia from the pro-inflammatory M1 to the neuroprotective M2 phenotype. Derivative 10 was a potent CB2 inverse agonist with promising immunomodulatory properties and could be considered as a tool for investigating the role of CB2 receptors and for developing potential immunomodulating drugs addressing the endocannabinoid system. (C) 2019 Elsevier Masson SAS. All rights reserved.

[Image: see text] Several evidence pointed out the role of epigenetics in Alzheimer’s disease (AD) revealing strictly relationships between epigenetic and “classical” AD targets. Based on the reported connection among histone deacetylases (HDACs) and glycogen synthase kinase 3β (GSK-3β), herein we present the discovery and the biochemical characterization of the first-in-class hit compound able to exert promising anti-AD effects by modulating the targeted proteins in the low micromolar range of concentration. Compound 11 induces an increase in histone acetylation and a reduction of tau phosphorylation. It is nontoxic and protective against H(2)O(2) and 6-OHDA stimuli in SH-SY5Y and in CGN cell lines, respectively. Moreover, it promotes neurogenesis and displays immunomodulatory effects. Compound 11 shows no lethality in a wt-zebrafish model (

The structures of melatonin and ferulic acid were merged into tertiary amide-based histone deacetylase 6 (HDAC6) inhibitors to develop multi-target-directed inhibitors for neurodegenerative diseases to incorporate antioxidant effects without losing affinity and selectivity at HDAC6. Structure-activity relationships led to compound

Despite Alzheimer’s disease (AD) incidence being projected to increase worldwide, the drugs currently on the market can only mitigate symptoms. Considering the failures of the classical paradigm “one target-one drug-one disease” in delivering effective medications for AD, polypharmacology appears to be a most viable therapeutic strategy. Polypharmacology can involve combinations of multiple drugs and/or single chemical entities modulating multiple targets. Taking inspiration from an ongoing clinical trial, this work aims to convert a promising cromolyn–ibuprofen drug combination into single-molecule “codrugs.” Such codrugs should be able to similarly modulate neuroinflammatory and amyloid pathways, while showing peculiar pros and cons. By exploiting a linking strategy, we designed and synthesized a small set of cromolyn–ibuprofen conjugates (4–6). Preliminary plasma stability and neurotoxicity assays allowed us to select diamide 5 and ethanolamide 6 as promising compounds for further studies. We investigated their immunomodulatory profile in immortalized microglia cells, in vitro anti-aggregating activity towards Aβ42-amyloid self-aggregation, and their cellular neuroprotective effect against Aβ42-induced neurotoxicity. The fact that 6 effectively reduced Aβ-induced neuronal death, prompted its investigation into an in vivo model. Notably, 6 was demonstrated to significantly increase the longevity of Aβ42-expressing Drosophila and to improve fly locomotor performance.

The structures of melatonin and ferulic acid were merged into tertiary amide-based histone deacetylase 6 (HDAC6) inhibitors to develop multi-target-directed inhibitors for neurodegenerative diseases to incorporate antioxidant effects without losing affinity and selectivity at HDAC6. Structure-activity relationships led to compound 10b as a hybrid molecule showing pronounced and selective inhibition of HDAC6 (IC50 = 30.7 nM, > 25-fold selectivity over other subtypes). This compound shows comparable DPPH radical scavenging ability to ferulic acid, comparable ORAC value to melatonin and comparable Cu2+ chelating ability to EDTA. It also lacks neurotoxicity on HT-22 cells, exhibits a pronounced immunomodulatory effect, and is active in vivo showing significantly higher efficacy in an AD mouse model to prevent both Aβ25-35-induced spatial working and long-term memory dysfunction at lower dose (0.3 mg/kg) compared to positive control HDAC6 inhibitor ACY1215 and an equimolar mixture of the three entities ACY1215, melatonin and ferulic acid, suggesting potentially disease-modifying properties.

Since 2002, no clinical candidate against Alzheimer's disease has reached the market; hence, an effective therapy is urgently needed. We followed the so-called "multitarget directed ligand" approach and designed 36 novel tacrine-phenothiazine heterodimers which were in vitro evaluated for their anticholinesterase properties. The assessment of the structure-activity relationships of such derivatives highlighted compound 1dC as a potent and selective acetylcholinesterase inhibitor with IC50 = 8 nM and 1aA as a potent butyrylcholinesterase inhibitor with IC50 = 15 nM. Selected hybrids, namely, 1aC, 1bC, 1cC, 1dC, and 2dC, showed a significant inhibitory activity toward τ(306-336) peptide aggregation with percent inhibition ranging from 50.5 to 62.1%. Likewise, 1dC and 2dC exerted a remarkable ability to inhibit self-induced Aβ1-42 aggregation. Notwithstanding, in vitro studies displayed cytotoxicity toward HepG2 cells and cerebellar granule neurons; no pathophysiological abnormality was observed when 1dC was administered to mice at 14 mg/kg (i.p.). 1dC was also able to permeate to the CNS as shown by in vitro and in vivo models. The maximum brain concentration was close to the IC50 value for acetylcholinesterase inhibition with a relatively slow elimination half-time. 1dC showed an acceptable safety and good pharmacokinetic properties and a multifunctional biological profile.

Our goal was the evaluation of a series of N-1,2,3-triazole-isatin derivatives for multi-target activity which included cholinesterase (ChE) inhibition and β-amyloid (Aβ) peptide anti-aggregation. The compounds have shown considerable promise as butyrylcholinesterase (BuChE) inhibitors. Although the inhibition of eel acetylcholinesterase (eeAChE) was weak, the inhibitions against equine BuChE (eqBuChE) and human BuChE (hBuChE) were more significant with a best inhibition against eqBuChE of 0.46 μM. In some cases, these molecules gave better inhibitions for hBuChE than eqBuChE. For greater insights into their mode of action, molecular docking studies were carried out, followed by STD-NMR validation. In addition, some of these compounds showed weak Aβ anti-aggregation activity. Hepatotoxicity studies showed that they were non-hepatoxic and neurotoxicity studies using neurite outgrowth experiments led to the conclusion that these compounds are only weakly neurotoxic.

Multiple sclerosis (MS) is a complex inflammatory, degenerative, and demyelinating disease of the central nervous system. Although treatments exist, MS cannot be cured by available drugs, which primarily target neuroinflammation. Thus, it is feasible that a well concerted polypharmacological approach able to act at multiple points within the intricate network of inflammation, neurodegeneration, and demyelination/remyelination pathways would succeed where other drugs have failed. Starting from reported beneficial effects of α-linolenic acid (ALA) and valproic acid (VPA) in MS, and by applying a rational strategy, we developed a small set of codrugs obtained by conjugating VPA and ALA through proper linkers. A cellular profiling identified 1 as a polypharmacological tool able not only to modulate microglia polarization, but also to counteract neurodegeneration and demyelination and induce oligodendrocyte precursor cell differentiation, by acting on multiple biochemical and epigenetic pathways.

Aspartate-Glutamate Carrier 1 (AGC1) deficiency is a rare neurological disease caused by mutations in the solute carrier family 25, member 12 (SLC25A12) gene, encoding for the mitochondrial aspartate-glutamate carrier isoform 1 (AGC1), a component of the malate&ndash, aspartate NADH shuttle (MAS), expressed in excitable tissues only. AGC1 deficiency patients are children showing severe hypotonia, arrested psychomotor development, seizures and global hypomyelination. While the effect of AGC1 deficiency in neurons and neuronal function has been deeply studied, little is known about oligodendrocytes and their precursors, the brain cells involved in myelination. Here we studied the effect of AGC1 down-regulation on oligodendrocyte precursor cells (OPCs), using both in vitro and in vivo mouse disease models. In the cell model, we showed that a reduced expression of AGC1 induces a deficit of OPC proliferation leading to their spontaneous and precocious differentiation into oligodendrocytes. Interestingly, this effect seems to be related to a dysregulation in the expression of trophic factors and receptors involved in OPC proliferation/differentiation, such as Platelet-Derived Growth Factor &alpha, (PDGF&alpha, ) and Transforming Growth Factor &beta, s (TGF&beta, s). We also confirmed the OPC reduction in vivo in AGC1-deficent mice, as well as a proliferation deficit in neurospheres from the Subventricular Zone (SVZ) of these animals, thus indicating that AGC1 reduction could affect the proliferation of different brain precursor cells. These data clearly show that AGC1 impairment alters myelination not only by acting on N-acetyl-aspartate production in neurons but also on OPC proliferation and suggest new potential therapeutic targets for the treatment of AGC1 deficiency.

Several evidence pointed out the role of epigenetics in Alzheimer's disease (AD) revealing strictly relationships between epigenetic and "classical" AD targets. Based on the reported connection among histone deacetylases (HDACs) and glycogen synthase kinase 3β (GSK-3β), herein we present the discovery and the biochemical characterization of the first-in-class hit compound able to exert promising anti-AD effects by modulating the targeted proteins in the low micromolar range of concentration. Compound

[Image: see text] Alzheimer’s disease (AD) represents a global problem, with an estimation of the majority of dementia patients in low- and middle-income countries by 2050. Thus, the development of sustainable drugs has attracted much attention in recent years. In light of this, taking inspiration from the HDAC inhibitor vorinostat (1), we develop the first HDAC inhibitors derived from cashew nut shell liquid (CNSL), an inexpensive agro-food waste material. CNSL derivatives 8 and 9 display a HDAC inhibitory profile similar to 1, together with a more promising safety for 9 compared to 1. Moreover, both compounds and particularly 9 were able to effectively modulate glial cell-induced inflammation and to revert the pro-inflammatory phenotype. All these results demonstrate that the use of inexpensive food waste materials could be successfully applied for the development of accessible and sustainable drug candidates for the treatment of AD.

Concerned by the devastating effects of Alzheimer's disease, and the lack of effective drugs, we have carried out the design of a series of tacrine-phenolic heterodimers in order to tackle the multifactorial nature of the disease. Hybridization of both pharmacophores involved the modification of the nature (imino, amino, ether) and the length of the tether, together with the type (hydroxy, methoxy, benzyloxy), number and position of the substituents on the aromatic residue. Title compounds were found to be strong and selective inhibitors of human BuChE (from low nanomolar to subnanomolar range), an enzyme that becomes crucial in the more advanced stages of the disease. The lead compound, bearing an ether-type tether, had an IC50 value of 0.52 nM against human BuChE, and a selectivity index of 323, with an 85-fold increase of activity compared to parent tacrine; key interactions were analysed using molecular modelling. Moreover, it also inhibited the self-aggregation of Aβ42, lacking neurotoxicity up to 5 μM concentration, and showed neuroprotective activity in primary rat neurons in a serum and K+ deprivation model, widely employed for reproducing neuronal injury and senescence. Moreover, low hepatoxicity effects and complete stability under physiological conditions were found for that compound. So, overall, our lead compound can be considered as a promising multitarget-directed ligand against Alzheimer's disease, and a good candidate for developing new drugs.

A combination of tacrine and tryptophan led to the development of a new family of heterodimers as multi-target agents with potential to treat Alzheimer's disease. Based on the in vitro biological profile, compound S-K1035 was found to be the most potent inhibitor of human acetylcholinesterase (hAChE) and human butyrylcholinesterase (hBChE), demonstrating balanced IC50 values of 6.3 and 9.1 nM, respectively. For all the tacrine-tryptophan heterodimers, favorable inhibitory effect on hAChE as well as on hBChE was coined to the optimal spacer length ranging from five to eight carbon atoms between these two pharmacophores. S-K1035 also showed good ability to inhibit Aβ42 self-aggregation (58.6 ± 5.1% at 50 μM) as well as hAChE-induced Aβ40 aggregation (48.3 ± 6.3% at 100 μM). The X-ray crystallographic analysis of TcAChE in complex with S-K1035 pinpointed the utility of the hybridization strategy applied and the structures determined with the two K1035 enantiomers in complex with hBChE could explain the higher inhibition potency of S-K1035. Other in vitro evaluations predicted the ability of S-K1035 to cross blood-brain barrier and to exert a moderate inhibition potency against neuronal nitric oxide synthase. Based on the initial promising biochemical data and a safer in vivo toxicity compared to tacrine, S-K1035 was administered to scopolamine-treated rats being able to dose-dependently revert amnesia.

We have designed and synthesized a series of 14 hybrid molecules out of the cholinesterase (ChE) inhibitor tacrine and a benzimidazole-based human cannabinoid receptor subtype 2 (hCB(2)R) agonist and investigated them in vitro and in vivo. The compounds are potent ChE inhibitors, and for the most promising hybrids, the mechanism of human acetylcholinesterase (hAChE) inhibition as well as their ability to interfere with AChE-induced aggregation of β-amyloid (Aβ), and Aβ self-aggregation was assessed. All hybrids were evaluated for affinity and selectivity for hCB(1)R and hCB(2)R To ensure that the hybrids retained their agonist character, the expression of cAMP-regulated genes was quantified, and potency and efficacy were determined. Additionally, the effects of the hybrids on microglia activation and neuroprotection on HT-22 cells were investigated. The most promising in vitro hybrids showed pronounced neuroprotection in an Alzheimer’s mouse model at low dosage (0.1 mg/kg, i.p.), lacking hepatotoxicity even at high dose (3 mg/kg, i.p.).

INTRODUCTION: The importance of microglia in most neurodegenerative pathologies, from Parkinson's disease to amyotrophic lateral sclerosis and Alzheimer's disease, is increasingly recognized. Until few years ago, microglial activation in pathological conditions was considered dangerous to neurons due to its causing inflammation. Today we know that these glial cells also play a crucial physiological and neuroprotective role, which is altered in neurodegenerative conditions. AREAS COVERED: The neuroinflammatory hypothesis for neurodegenerative diseases has led to the trial of anti-inflammatory agents as therapeutics with largely disappointing results. New information about the physiopathological role of microglia has highlighted the importance of immunomodulation as a potential new therapeutic approach. This review summarizes knowledge on microglia as a potential therapeutic target in the most common neurodegenerative diseases, with focus on compounds directed toward the modulation of microglial immune response through specific molecular pathways. EXPERT OPINION: Here we support the innovative concept of targeting microglial cells by modulating their activity, rather than simply trying to counteract their inflammatory neurotoxicity, as a potential therapeutic approach for neurodegenerative diseases. The advantage of this therapeutic approach could be to reduce neuroinflammation and toxicity, while at the same time strengthening intrinsic neuroprotective properties of microglia and promoting neuroregeneration.

50 p.-5 fig., Overcoming the lack of effective treatments and the continuous clinical trial failures in neurodegenerative drug discovery might require a shift from the prevailing paradigm targeting pathogenesis to the one targeting simultaneously neuroprotection and neuroregeneration. In the studies reported herein, we sought to identify small molecules that might exert neuroprotective and neuroregenerative potential as tools against neurodegenerative diseases. In doing so, we started from the reported neuroprotective/neuroregenerative mechanisms of psychotropic drugs featuring a tricyclic alkylamine scaffold. Thus, we designed a focused-chemical library of 36 entries aimed at exploring the structural requirements for efficient neuroprotective/neuroregenerative cellular activity, without the manifestation of toxicity. To this aim, we developed a synthetic protocol, which overcame the limited applicability of previously reported procedures. Next, we evaluated the synthesized compounds through a phenotypic screening pipeline, based on primary neuronal systems. Phenothiazine 2Bc showed improved neuroregenerative and neuroprotective properties with respect to reference drug desipramine (2Aa). Importantly, we have also shown that 2Bc outperformed currently available drugs in cell models of Alzheimer's and Parkinson's diseases and attenuates microglial activation by reducing iNOS expression., This work was supported by the University of Bologna (GrantRFO 2016). Financial support from MINECO (grant no. BFU2014-57494-R to AVM).

Several findings propose the altered tau protein network as an important target for Alzheimer's disease (AD). Particularly, two points of pharmacological intervention can be envisaged: inhibition of phosphorylating tau kinase GSK-3β and tau aggregation process. On the basis of this consideration and on our interest in multitarget paradigms in AD, we report on the discovery of 2,4-thiazolidinedione derivatives endowed with such a profile. 28 and 30 displayed micromolar IC values toward GSK-3β, together with the capacity of inhibiting AcPHF6 aggregation of 60% and 80% at 10 μM, respectively. In addition, they showed PAMPA-BBB permeability, together with a suitable cellular safety profile. 30 also displayed inhibition of both K18 and full-length tau aggregations. Finally, both compounds were able to improve cell viability in an okadaic acid-induced neurodegeneration cell model. To the best of our knowledge, 28 and 30 are the first balanced, nontoxic, dual-acting compounds hitting tau cascade at two different hubs., This work was supported by the University of Bologna (RFO2015_2016), European Cooperation of Science and Technology (EUCOST) Action CA15135, Ministerio de Economia y Competitividad (CTQ2015-66313-R), Italian Ministry of Health (GR-2013-02355724), MJFF, ALZ, Alzheimer’s Research UK and the Weston Brain Institute (BAND2015). The authors thank E. De Cecco (PhD candidate, SISSA) for providing K18 and 2N4R protein samples for the in vitro assays.

Long INterspersed Element-1 (L1) is a transposable element that can insert copies of itself in new genomic locations causing genomic instability. In somatic cells, L1 retrotransposition activity is usually repressed but somatic L1 retrotransposition has recently been observed during neuronal differentiation. In this study, we evaluate whether L1 elements are differentially active in rat tissues during postnatal development. To this purpose, we quantified L1 in genomic DNA extracted from the olfactory bulb (OB), cerebellum (CE), cortex (CO) and heart (H). Each analysis was repeated on rats aged 7, 21 and 60 days. We found that L1 content in OB and CE tissue was significantly higher than H tissue, in rats of all three ages studied, suggesting that L1 activity could be modulated in postnatal development and neurogenesis.

Amyotrophic lateral sclerosis (ALS) is a fatal motor neuron disease. Mutations in the gene encoding copper/zinc superoxide dismutase-1 (SOD1) are responsible for most familiar cases, but the role of mutant SOD1 protein dysfunction in non-cell autonomous neurodegeneration, especially in relation to microglial activation, is still unclear. Here, we focused our study on microglial cells, which release SOD1 also through exosomes. We observed that in rat primary microglia the overexpression of the most-common SOD1 mutations linked to fALS (G93A and A4V) leads to SOD1 intracellular accumulation, which correlates to autophagy dysfunction and microglial activation. In primary contact co-cultures, fALS mutant SOD1 overexpression by microglial cells appears to be neurotoxic by itself. Treatment with the autophagy-inducer trehalose reduced mutant SOD1 accumulation in microglial cells, decreased microglial activation and abrogated neurotoxicity in the co-culture model. These data suggest that i) the alteration of the autophagic pathway due to mutant SOD1 overexpression is involved in microglial activation and neurotoxicity; ii) the induction of autophagy with trehalose reduces microglial SOD1 accumulation through proteasome degradation and activation, leading to neuroprotection. Our results provide a novel contribution towards better understanding key cellular mechanisms in non-cell autonomous ALS neurodegeneration.

none 13 no Available online 20 October 2017 Purine nucleoside phosphorylase (PNP), a crucial enzyme in purine metabolism which converts ribonucleosides into purine bases, has mainly been found inside glial cells. Since we recently demonstrated that PNP is released from rat C6 glioma cells, we then wondered whether this occurs in normal brain cells. Using rat primary cultures of microglia, astrocytes and cerebellar granule neurons, we found that in basal condition all these cells constitutively released a metabolically active PNP with Km values very similar to those measured in C6 glioma cells. However, the enzyme expression/release was greater in microglia or astrocytes that in neurons. Moreover, we exposed primary brain cell cultures to pro-inflammatory agents such as lipopolysaccharide (LPS) or ATP alone or in combination. LPS alone caused an increased interleukin-1β (IL-1β) secretion mainly from microglia and no modification in the PNP release, even from neurons in which it enhanced cell death. In contrast, ATP administered alone to glial cells at high micromolar concentrations significantly stimulated the release of PNP within 1 h, an effect not modified by LPS presence, whereas IL-1β secretion was stimulated by ATP only in cells primed for 2 h with LPS. In both cases ATP effect was mediated by P2X7 receptor (P2X7R), since it was mimicked by cell exposure to Bz-ATP, an agonist of P2X7R, and blocked by cell pre-treatment with the P2X7R antagonist A438079. Interestingly, ATP-induced PNP release from glial cells partly occurred through the secretion of lysosomal vesicles in the extracellular medium. Thus, during inflammatory cerebral events PNP secretion promoted by extracellular ATP accumulation might concur to control extracellular purine signals. Further studies could elucidate whether, in these conditions, a consensual activity of enzymes downstream of PNP in the purine metabolic cascade avoids accumulation of extracellular purine bases that might concur to brain injury by unusual formation of reactive oxygen species. mixed Peña-Altamira LE, Polazzi E, Giuliani P, Beraudi A, Massenzio F, Mengoni I, Poli A, Zuccarini M, Ciccarelli R, Di Iorio P, Virgili M, Monti B, Caciagli F. Peña-Altamira LE, Polazzi E, Giuliani P, Beraudi A, Massenzio F, Mengoni I, Poli A, Zuccarini M, Ciccarelli R, Di Iorio P, Virgili M, Monti B, Caciagli F.

Purine nucleoside phosphorylase (PNP), a crucial enzyme in purine metabolism which converts ribonucleosides into purine bases, has mainly been found inside glial cells. Since we recently demonstrated that PNP is released from rat C6 glioma cells, we then wondered whether this occurs in normal brain cells. Using rat primary cultures of microglia, astrocytes and cerebellar granule neurons, we found that in basal condition all these cells constitutively released a metabolically active PNP with Km values very similar to those measured in C6 glioma cells. However, the enzyme expression/release was greater in microglia or astrocytes that in neurons. Moreover, we exposed primary brain cell cultures to pro-inflammatory agents such as lipopolysaccharide (LPS) or ATP alone or in combination. LPS alone caused an increased interleukin-1β (IL-1β) secretion mainly from microglia and no modification in the PNP release, even from neurons in which it enhanced cell death. In contrast, ATP administered alone to glial cells at high micromolar concentrations significantly stimulated the release of PNP within 1 h, an effect not modified by LPS presence, whereas IL-1β secretion was stimulated by ATP only in cells primed for 2 h with LPS. In both cases ATP effect was mediated by P2X

The study of microglia, the immune cells of the brain, has experienced a renaissance after the discovery of microglia polarization. In fact, the concept that activated microglia can shift into the M1 pro-inflammatory or M2 neuroprotective phenotypes, depending on brain microenvironment, has completely changed the understanding of microglia in brain aging and neurodegenerative diseases. Microglia polarization is particularly important in aging since an increased inflammatory status of body compartments, including the brain, has been reported in elderly people. In addition, inflammatory markers, mainly derived from activated microglia, are widely present in neurodegenerative diseases. Microglial inflammatory dysfunction, also linked to microglial senescence, has been extensively demonstrated and associated with cognitive impairment in neuropathological conditions related to aging. In fact, microglia polarization is known to influence cognitive function and has therefore become a main player in neurodegenerative diseases leading to dementia. As the life span of human beings increases, so does the prevalence of cognitive dysfunction. Thus, therapeutic strategies aimed to modify microglia polarization are currently being developed. Pharmacological approaches able to shift microglia from M1 pro-inflammatory to M2 neuroprotective phenotype are actually being studied, by acting on many different molecular targets, such as glycogen synthase kinase-3 (GSK3) β, AMP-activated protein kinase (AMPK), histone deacetylases (HDACs), etc. Furthermore, nutritional approaches can also modify microglia polarization and, consequently, impact cognitive function. Several bioactive compounds normally present in foods, such as polyphenols, can have anti-inflammatory effects on microglia. Both pharmacological and nutritional approaches seem to be promising, but still need further development. Here we review recent data on these approaches and propose that their combination could have a synergistic effect to counteract cognitive aging impairment and Alzheimer’s disease (AD) through immunomodulation of microglia polarization, i.e., by driving the shift of activated microglia from the pro-inflammatory M1 to the neuroprotective M2 phenotype.

We have designed a series of tacrine-based homo- and heterodimers that incorporate an antioxidant tether (selenoureido, chalcogenide) as new dual compounds: for the treatment of Alzheimer's disease and as antiproliferative agents. Symmetrical homodimers bearing a dichalcogenide or selenide-based tether, the best compounds in the series, were found to be strong and highly selective electric eel AChE inhibitors, with inhibition constants within the low nanomolar range. This high inhibitory activity was confirmed on recombinant human AChE for the most interesting derivatives. The three most promising homodimers also showed a good inhibitory activity towards amyloid-β self aggregation. The symmetric disulfide derivative bis[5-(1′,2′,3′,4’-tetrahydroacridin-9′-ylamino)pentyl]disulfide (19) showed the best multipotent profile and was not neurotoxic on immortalized mouse cortical neurons even at 50 μM concentration. These results represent an improvement in activity and selectivity compared to parent tacrine, the first marketed drug against Alzheimer's disease. Title compounds also exhibited excellent in vitro antiproliferative activities against a panel of 6 human tumor cell lines, with GI50 values within the submicromolar range for the most potent derivatives (0.12–0.95 μM); such values represent a spectacular increase compared to currently-used chemotherapeutic agents, such as 5-FU (up to 306−fold) and cisplatin (up to 162−fold). Cell cycle experiments indicated the accumulation of cells in the G1 phase of the cycle, a different mechanism than the reported for cisplatin. The breast cancer cell lines turned out to be the most sensitive one of the panel tested. Dirección General de Investigación CTQ2016-78703-P, CTQ2011-28417-C02-01, CEI10/00018 Junta de Andalucía FQM134 Fondo Europeo de Desarrollo Regional 501100008530 EU Research Potential FP7-REGPOT-2012-CT2012-31637-IMBRAIN

Multi-target drug discovery is one of the most followed approaches in the active central nervous system (CNS) therapeutic area, especially in the search for new drugs against Alzheimer's disease (AD). This is because innovative multi-target-directed ligands (MTDLs) could more adequately address the complexity of this pathological condition. In a continuation of our efforts aimed at a new series of anti-AD MTDLs, we combined the structural features of the cholinesterase inhibitor drug tacrine with that of resveratrol, which is known for its purported antioxidant and anti-neuroinflammatory activities. The most interesting hybrid compounds (5, 8, 9 and 12) inhibited human acetylcholinesterase at micromolar concentrations and effectively modulated Aβ self-aggregation in vitro. In addition, 12 showed intriguing anti-inflammatory and immuno-modulatory properties in neuronal and glial AD cell models. Importantly, the MTDL profile is accompanied by high-predicted blood-brain barrier permeability, and low cytotoxicity on primary neurons.

The mitochondrial aspartate-glutamate carrier isoform 1 (AGC1) catalyzes a Ca2+-stimulated export of aspartate to the cytosol in exchange for glutamate, and is a key component of the malate-aspartate shuttle which transfers NADH reducing equivalents from the cytosol to mitochondria. By sustaining the complete glucose oxidation, AGC1 is thought to be important in providing energy for cells, in particular in the CNS and muscle where this protein is mainly expressed. Defects in the AGC1 gene cause AGC1 deficiency, an infantile encephalopathy with delayed myelination and reduced brain N-acetylaspartate (NAA) levels, the precursor of myelin synthesis in the CNS. Here, we show that undifferentiated Neuro2A cells with down-regulated AGC1 display a significant proliferation deficit associated with reduced mitochondrial respiration, and are unable to synthesize NAA properly. In the presence of high glutamine oxidation, cells with reduced AGC1 restore cell proliferation, although oxidative stress increases and NAA synthesis deficit persists. Our data suggest that the cellular energetic deficit due to AGC1 impairment is associated with inappropriate aspartate levels to support neuronal proliferation when glutamine is not used as metabolic substrate, and we propose that delayed myelination in AGC1 deficiency patients could be attributable, at least in part, to neuronal loss combined with lack of NAA synthesis occurring during the nervous system development.

Gene expression is controlled by several epigenetic mechanisms involving post-translational modification of histones (acetylation, phosphorylation and others). These mechanisms in the brain are not only important for normal function but also for the development of pathologies when their derangement does occur. The present review deals with post-translational modifications of histones in two neurodegenerative diseases characterized by different etiology and pathological progression, Huntington's disease and Parkinson's disease. A relatively large body of evidence supports an important role of these mechanisms in Huntington's disease while knowledge of similar mechanisms in Parkinson's disease is at a lower degree of understanding. Starting from available information on pathologies, the present state of possible therapeutic targets is considered and future developments are discussed.