Marco De Amici’s research while affiliated with University of Milan and other places

Dopaminergic neurotransmission is involved in several important brain functions, such as motor control, learning, reward‐motivated behavior, and emotions. Dysfunctions of dopaminergic system may lead to the development of various neurological and psychiatric disorders, like Parkinson's disease, schizophrenia, depression, and addictions. Despite years of sustained research, it is not fully established how dopaminergic neurotransmission governs these important functions through a relatively small number of neurons that release dopamine. Light‐driven neurotechnologies, based on the use of small light‐regulated molecules or overexpression of light‐regulated proteins in neurons, have greatly contributed to the advancement of our understanding of dopaminergic circuits and our ability to control them selectively. Here, we overview the current state‐of‐the‐art of light‐driven control of dopaminergic neurotransmission. While we provide a concise guideline for the readers interested in pharmacological, pharmacogenetic, and optogenetic approaches to modulate dopaminergic neurotransmission, our primary focus is on the usage of photocaged and photo‐switchable small dopaminergic molecules. We argue that photopharmacology, photoswitchable molecules of varied modalities, can be employed in a wide range of experimental paradigms, providing unprecedent insights into the principles of dopaminergic control, and represent the most promising light‐based therapeutic approach for spatiotemporally precise correction of dopamine‐related neural functions and pathologies.

Cholinergic transmission plays a critical role in both the central and peripheral nervous systems, affecting processes such as learning, memory, and inflammation. Conventional cholinergic drugs generally suffer from poor selectivity and temporal precision, leading to undesired effects and limited therapeutic efficacy. Photopharmacology aims to overcome the limitations of traditional drugs using photocleavable or photoswitchable ligands and spatiotemporal patterns of illumination. Spanning from muscarinic and nicotinic modulators to cholinesterase inhibitors, this review explores the development and application of light‐activated compounds as tools for unraveling the role of cholinergic signaling in both physiological and pathological contexts, while also paving the way for innovative phototherapeutic approaches.

The 1,3-dithiolane ring has been recently rehabilitated as a chemical scaffold in drug design. However, for derivatives that are substituted in position 4, the introduction of a chiral center on the heterocycle demands the separation and characterization of the stereoisomers. We report the first chiral resolution and absolute configuration (AC) assignment for (1,4-dithiaspiro[4.5]decan-2-yl)methanol (R/S)-1, a key synthon for dithiolane-based biologically active compounds. Using (semi)preparative enantioselective HPLC, we isolated enantiomeric 1. The AC was assigned by using (+)-1 for the enantioselective synthesis of (+)-BS148, a sigma receptor modulator. An X-ray diffraction analysis established the (R)-configuration of (+)-BS148 and, by extension, of (+)-1. This method provides a reliable approach for preparing enantiopure 1,3-dithiolane scaffolds and establishes reference standards for AC determination of related compounds.

Rare diseases are particular pathological conditions affecting a limited number of people and few drugs are known to be effective as therapeutic treatment. Gaucher disease, caused by a deficiency of the lysosomal enzyme glucocerebrosidase, belongs to this class of disorders, and it is considered the most common among the Lysosomal Storage Diseases. The two main therapeutic approaches are the Enzyme Replacement Therapy (ERT) and the Substrate Reduction Therapy (SRT). ERT, consisting in replacing the defective enzyme by administering a recombinant enzyme, is effective in alleviating the visceral symptoms, hallmarks of the most common subtype of the disease whereas it has no effects when symptoms involve CNS, since the recombinant protein is unable to significantly cross the Blood Brain Barrier. The SRT strategy involves inhibiting glucosylceramide synthase (GCS), the enzyme responsible for the production of the associated storage molecule. The rational design of new inhibitors of GCS has been hampered by the lack of either the crystal structure of the enzyme or an in‐silico model of the active site which could provide important information regarding the interactions of potential inhibitors with the target, but, despite this, interesting results have been obtained and are herein reviewed.

Simple Summary Tumor cells use autophagy as a pro-survival strategy. However, several studies have shown that excessive stimulation of the autophagic process can promote cell death; in this context, autophagy acquires an antitumor effect. To further explore the consequences of the cytotoxic effects induced by M2 muscarinic receptor activation that we have previously described both in glioblastoma multiforme (GBM) stable cell lines and in human GBM cancer stem cells, here we investigated the involvement of autophagy and apoptosis in the cell death engendered by treatment with M2 muscarinic agonists. Moreover, we compared the effects mediated by orthosteric and dualsteric M2 muscarinic agonists in the modulation of these different mechanisms of cell death. Abstract Background: Although autophagy is a pro-survival process of tumor cells, it can stimulate cell death in particular conditions and when differently regulated by specific signals. We previously demonstrated that the selective stimulation of the M2 muscarinic receptor subtype (mAChR) negatively controls cell proliferation and survival and causes oxidative stress and cytotoxic and genotoxic effects in both GBM cell lines and GBM stem cells (GSCs). In this work, we have evaluated whether autophagy was induced as a downstream mechanism of the observed cytotoxic processes induced by M2 mAChR activation by the orthosteric agonist APE or the dualsteric agonist N8-Iper (N8). Methods: To assess the activation of autophagy, we analyzed the expression of LC3B using Western blot analysis and in LC3B-EGFP transfected cell lines. Apoptosis was assessed by measuring the protein expression of Caspases 3 and 9. Results: Our data indicate that activation of M2 mAChR by N8 promotes autophagy in both U251 and GB7 cell lines as suggested by the LC3B-II expression level and analysis of the transfected cells by fluorescence microscopy. Autophagy induction by M2 mAChRs is regulated by the decreased activity of the PI3K/AKT/mTORC1 pathway and upregulated by pAMPK expression. Downstream of autophagy activation, an increase in apoptosis was also observed in both cell lines after treatment with the two M2 agonists. Conclusions: N8 treatment causes autophagy via pAMPK upregulation, followed by apoptosis in both investigated cell lines. In contrast, the absence of autophagy in APE-treated GSC cells seems to indicate that cell death could be triggered by mechanisms alternative to those observed for N8.

Background: Although autophagy is a pro-survival process of tumor cells, in particular conditions and when differently regulated by specific signals it can stimulate cell death. We previously demonstrated that the selective stimulation of the M2 muscarinic receptor subtype (M2 mAChR) negatively controls cell proliferation and survival and causes oxidative stress and cytotoxic and genotoxic effects in both GBM cell lines and GBM stem cells (GSCs). In this work, we have evaluated whether autophagy was induced as a downstream mechanism of the observed cytotoxic processes induced by M2 mAChR activation by the orthosteric agonist APE or the dualsteric agonist N-8-Iper. Methods: To assess the activation of autophagy, we analyzed the expression of LC3B by Western blot analysis and in LC3B-EGFP transfected cell lines. Apoptosis was assessed by Caspases 3 and 9 protein expression. Results: Our data indicate that activation of M2 mAChR by N-8-Iper promotes autophagy in both U251 and GB7 cells lines as suggested by the LC3B-II expression level and analysis of the transfected cells by fluorescence microscopy. Autophagy induction by M2 mAChRs is regulated by the decreased activity of the PI3K/AKT/mTORC1 pathway and upregulated by the pAMPK expression. Downstream autophagy activation, the increase of apoptosis was also observed in both cell lines after treatment with the two M2 agonists. Conclusions: N-8-Iper treatment causes autophagy via pAMPK upregulation, followed by apoptosis in both investigated cell lines. In contrast, the absence of autophagy in APE-treated GSC cells seems to indicate that cell death could be triggered by mechanisms alternative to those observed for N-8-Iper.

The study of nicotinic acetylcholine receptors (nAChRs) has significantly progressed in the last decade, due to a) the improved techniques available for structural studies; b) the identification of ligands interacting at orthosteric and allosteric recognition sites on the nAChR proteins, able to tune channel conformational states; c) the better functional characterization of receptor subtypes/subunits and their therapeutic potential; d) the availability of novel pharmacological agents able to activate or block nicotinic-mediated cholinergic responses with subtype or stoichiometry selectivity. The copious literature on nAChRs is related to the pharmacological profile of new, promising subtype selective derivatives as well as the encouraging preclinical and early clinical evaluation of known ligands. However, recently approved therapeutic derivatives are still missing, and examples of ligands discontinued in advanced CNS clinical trials include drug candidates acting at both neuronal homomeric and heteromeric receptors. In this review, we have selected heteromeric nAChRs as the target and comment on literature reports of the past five years dealing with the discovery of new small molecule ligands or the advanced pharmacological/preclinical investigation of more promising compounds. The results obtained with bifunctional nicotinic ligands and a light-activated ligand as well as the applications of promising radiopharmaceuticals for heteromeric subtypes are also discussed.

Background: Schwann cells (SCs) are glial cells involved in peripheral axon myelination. SCs also play a strategic role after peripheral nerve injury, regulating local inflammation and axon regeneration. Our previous studies demonstrated the presence of cholinergic receptors in SCs. In particular, the α7 nicotinic acetylcholine receptors (nAChRs) are expressed in SCs after peripheral axotomy, suggesting their involvement in the regulation of SC-regenerating properties. To clarify the role that α7 nAChRs may play after peripheral axon damage, in this study we investigated the signal transduction pathways triggered by receptor activation and the effects produced by their activation. Methods: Both ionotropic and metabotropic cholinergic signaling were analyzed by calcium imaging and Western blot analysis, respectively, following α7 nAChR activation. In addition, the expression of c-Jun and α7 nAChRs was evaluated by immunocytochemistry and Western blot analysis. Finally, the cell migration was studied by a wound healing assay. Results: Activation of α7 nAChRs, activated by the selective partial agonist ICH3, did not induce calcium mobilization but positively modulated the PI3K/AKT/mTORC1 axis. Activation of the mTORC1 complex was also supported by the up-regulated expression of its specific p-p70 S6KThr389 target. Moreover, up-regulation of p-AMPKThr172, a negative regulator of myelination, was also observed concomitantly to an increased nuclear accumulation of the transcription factor c-Jun. Cell migration and morphology analyses proved that α7 nAChR activation also promotes SC migration. Conclusions: Our data demonstrate that α7 nAChRs, expressed by SCs only after peripheral axon damage and/or in an inflammatory microenvironment, contribute to improve the SCs regenerating properties. Indeed, α7 nAChR stimulation leads to an upregulation of c-Jun expression and promotes Schwann cell migration by non-canonical pathways involving the mTORC1 activity.