The effects of proteasome on baseline and methamphetamine-dependent dopamine transmission.

Abstract The Ubiquitin Proteasome System (UPS) is a major multi-catalytic machinery, which guarantees cellular proteolysis and turnover. Beyond cytosolic and nuclear cell compartments, the UPS operates at the synapse to modulate neurotransmission and plasticity. In fact, dysregulations of the UPS are linked with early synaptic alterations occurring in a variety of dopamine (DA)-related brain disorders. This is the case of psychiatric conditions such as methamphetamine (METH) addiction. While being an extremely powerful DA releaser, METH impairs UPS activity, which is largely due to DA itself. In turn, pre- and post- synaptic neurons of the DA circuitry show a high vulnerability to UPS inhibition. Thus, alterations of DA transmission and UPS activity are intermingled within a chain of events underlying behavioral alterations produced by METH. These findings, which allow escaping the view of a mere implication of the UPS in protein toxicity-related mechanisms, indicate a more physiological role for the UPS in modulating DA-related behavior. This is seminal for those plasticity mechanisms which underlie overlapping psychiatric disorders such as METH addiction and schizophrenia

Autophagy as a gateway for the effects of methamphetamine: From neurotransmitter release and synaptic plasticity to psychiatric and neurodegenerative disorders.

As a major eukaryotic cell clearing machinery, autophagy grants cell proteostasis, which is key for neurotransmitter release, synaptic plasticity, and neuronal survival. In line with this, besides neuropathological events, autophagy dysfunctions are bound to synaptic alterations that occur in mental disorders, and early on, in neurodegenerative diseases. This is also the case of methamphetamine (METH) abuse, which leads to psychiatric disturbances and neurotoxicity. While consistently altering the autophagy machinery, METH produces behavioral and neurotoxic effects through molecular and biochemical events that can be recapitulated by autophagy blockade. These consist of altered physiological dopamine (DA) release, abnormal stimulation of DA and glutamate receptors, as well as oxidative, excitotoxic, and neuroinflammatory events. Recent molecular insights suggest that METH early impairs the autophagy machinery, though its functional significance remains to be investigated. Here we discuss evidence suggesting that alterations of DA transmission and autophagy are intermingled within a chain of events underlying behavioral alterations and neurodegenerative phenomena produced by METH. Understanding how METH alters the autophagy machinery is expected to provide novel insights into the neurobiology of METH addiction sharing some features with psychiatric disorders and parkinsonism

Methamphetamine persistently increases alpha-synuclein and suppresses gene promoter methylation within striatal neurons

Abstract Methamphetamine (Meth) produces a variety of epigenetic effects in the brain, which are seminal to establish long-lasting alterations in neuronal activity. However, most epigenetic changes were described by measuring the rough amount of either histone acetylation and methylation or direct DNA methylation, without focusing on a specific DNA sequence. This point is key to comprehend Meth-induced phenotypic changes, brain plasticity, addiction and neurodegeneration. In this research paper we analyze the persistence of Meth-induced striatal synucleinopathy at a prolonged time interval of Meth withdrawal. At the same time, Meth-induced alterations, specifically within alpha-synuclein gene (SNCA) or its promoter, were evaluated. We found that exposure to high and/or prolonged doses of Meth, apart from producing nigro-striatal toxicity, determines a long-lasting increase in striatal alpha-synuclein levels. This is consistent along immune-blotting, immune-histochemistry, and electron microscopy. This was neither associated with an increase of SNCA copy number nor with alterations within SNCA sequence. However, we documented persistently demethylation within SNCA promoter, which matches the increase in alpha-synuclein protein. The amount of the native protein, which was measured stoichiometrically within striatal neurons, surpasses the increase reported following SNCA multiplications. Demethylation was remarkable (ten-fold of controls) and steady, even at prolonged time intervals being tested so far (up to 21 days of Meth withdrawal). Similarly alpha-synuclein protein assayed stoichiometrically steadily increased roughly ten-fold of controls. Meth-induced increase of alpha-synuclein was also described within limbic areas. These findings are discussed in the light of Meth-induced epigenetic changes, Meth-induced phenotype alterations, and Meth-induced neurodegeneration

A Sentinel in the Crosstalk Between the Nervous and Immune System: The (Immuno)-Proteasome

The wealth of recent evidence about a bi-directional communication between nerve- and immune- cells revolutionized the traditional concept about the brain as an “immune-privileged” organ while opening novel avenues in the pathophysiology of CNS disorders. In fact, altered communication between the immune and nervous system is emerging as a common hallmark in neuro-developmental, neurodegenerative, and neuro-immunological diseases. At molecular level, the ubiquitin proteasome machinery operates as a sentinel at the crossroad between the immune system and brain. In fact, the standard proteasome and its alternative/inducible counterpart, the immunoproteasome, operate dynamically and coordinately in both nerve- and immune- cells to modulate neurotransmission, oxidative/inflammatory stress response, and immunity. When dysregulations of the proteasome system occur, altered amounts of standard- vs. immune-proteasome subtypes translate into altered communication between neurons, glia, and immune cells. This contributes to neuro-inflammatory pathology in a variety of neurological disorders encompassing Parkinson's, Alzheimer's, and Huntingtin's diseases, brain trauma, epilepsy, and Multiple Sclerosis. In the present review, we analyze those proteasome-dependent molecular interactions which sustain communication between neurons, glia, and brain circulating T-lymphocytes both in baseline and pathological conditions. The evidence here discussed converges in that upregulation of immunoproteasome to the detriment of the standard proteasome, is commonly implicated in the inflammatory- and immune- biology of neurodegeneration. These concepts may foster additional studies investigating the role of immunoproteasome as a potential target in neurodegenerative and neuro-immunological disorders

The Neuroanatomy of the Reticular Nucleus Locus Coeruleus in Alzheimer's Disease.

Alzheimer's Disease (AD) features the accumulation of β-amyloid and Tau aggregates, which deposit as extracellular plaques and intracellular neurofibrillary tangles (NFTs), respectively. Neuronal Tau aggregates may appear early in life, in the absence of clinical symptoms. This occurs in the brainstem reticular formation and mostly within Locus Coeruleus (LC), which is consistently affected during AD. LC is the main source of forebrain norepinephrine (NE) and it modulates a variety of functions including sleep-waking cycle, alertness, synaptic plasticity, and memory. The iso-dendritic nature of LC neurons allows their axons to spread NE throughout the whole forebrain. Likewise, a prion-like hypothesis suggests that Tau aggregates may travel along LC axons to reach out cortical neurons. Despite this timing is compatible with cross-sectional studies, there is no actual evidence for a causal relationship between these events. In the present mini-review, we dedicate special emphasis to those various mechanisms that may link degeneration of LC neurons to the onset of AD pathology. This includes the hypothesis that a damage to LC neurons contributes to the onset of dementia due to a loss of neuroprotective effects or, even the chance that, LC degenerates independently from cortical pathology. At the same time, since LC neurons are lost in a variety of neuropsychiatric disorders we considered which molecular mechanism may render these brainstem neurons so vulnerable

The Effects of Amphetamine and Methamphetamine on the Release of Norepinephrine, Dopamine and Acetylcholine From the Brainstem Reticular Formation

Amphetamine (AMPH) and methamphetamine (METH) are widely abused psychostimulants, which produce a variety of psychomotor, autonomic and neurotoxic effects. The behavioral and neurotoxic effects of both compounds (from now on defined as AMPHs) stem from a fair molecular and anatomical specificity for catecholamine-containing neurons, which are placed in the brainstem reticular formation (RF). In fact, the structural cross-affinity joined with the presence of shared molecular targets between AMPHs and catecholamine provides the basis for a quite selective recruitment of brainstem catecholamine neurons following AMPHs administration. A great amount of investigations, commentary manuscripts and books reported a pivotal role of mesencephalic dopamine (DA)-containing neurons in producing behavioral and neurotoxic effects of AMPHs. Instead, the present review article focuses on catecholamine reticular neurons of the low brainstem. In fact, these nuclei add on DA mesencephalic cells to mediate the effects of AMPHs. Among these, we also include two pontine cholinergic nuclei. Finally, we discuss the conundrum of a mixed neuronal population, which extends from the pons to the periaqueductal gray (PAG). In this way, a number of reticular nuclei beyond classic DA mesencephalic cells are considered to extend the scenario underlying the neurobiology of AMPHs abuse. The mechanistic approach followed here to describe the action of AMPHs within the RF is rooted on the fine anatomy of this region of the brainstem. This is exemplified by a few medullary catecholamine neurons, which play a pivotal role compared with the bulk of peripheral sympathetic neurons in sustaining most of the cardiovascular effects induced by AMPHs

Realdo Colombo in the fifth centenary of his birth

The date of birth of Realdo Colombo is still uncertain. However, 1516 is conventionally credited as the year where he was born in Cremona. Colombo’s life can be divided into three periods, according to the cities where he worked: Padua, Pisa and Rome. A talented anatomist, in Padua Colombo became assistant of Andreas Vesalius in 1541. In 1545 he moved to Pisa at the behest of the Grand Duke Cosimo I de’ Medici. Finally, he was invited in Rome by Pope Paul III and became the physician of many important patients, including Michelangelo Buonarroti. He also performed the autopsy on the body of Saint Ignatius of Loyola. In his unique masterpiece, De re anatomica, consisting of 15 books, Colombo reported original observations. He hoped to have a text illustrated by Michelangelo that would have competed with the fabrica of Vesalius, but that purpose did not realize. Indeed, the unique engraving of the volume, published posthumously in 1559, is the frontispiece. The most important anatomical discovery attributed to Colombo is the original description of the pulmonary circulation, based on hundreds of dissections and vivisections. The Galen’s long-standing doctrine of the blood circulation from the right ventricle to the left ventricle through invisible pores of the interventricular septum was definitively rejected. Although two other figures had already described the pulmonary circulation – the thirteenth century Arabic physician Ibn al-Nafis, in the Commentary on Anatomy in Avicenna’s Canon, and the Spanish philosopher Michael Servetus, in the theological book Christianismi restitutio – Colombo seems to have arrived at his conclusions independently. He also understood the function of the cardiac valves. Colombo’s book had a profound effect on William Harvey, when he prepared his lectures on anatomy for the College of Physicians of London, and was determinant for the publication of his description of the blood circulation in De motu cordis (1628). Other anatomical observations are attributed to Colombo. He corrected previous misconceptions, demonstrating that the right kidney is lower than the left, and showing that the lens is in the anterior chamber of the eye. He recognized anatomical variants, such as the presence of palmaris longus muscle, and described congenital malformations, such as the horseshoe kidney. He also seems to have coined the term “placenta” and claimed to have been the first to describe the clitoris and its function

An innovative strategy to investigate microbial protein modifications in a reliable fast and sensitive way: A therapy oriented proof of concept based on UV-C irradiation of SARS-CoV-2 spike protein

: The characterization of modifications of microbial proteins is of primary importance to dissect pathogen lifecycle mechanisms and could be useful in identifying therapeutic targets. Attempts to solve this issue yielded only partial and non-exhaustive results. We developed a multidisciplinary approach by coupling in vitro infection assay, mass spectrometry (MS), protein 3D modelling, and surface plasma resonance (SPR). As a proof of concept, the effect of low UV-C (273 nm) irradiation on SARS-CoV-2 spike (S) protein was investigated. Following UV-C exposure, MS analysis identified, among other modifications, the disruption of a disulphide bond within the conserved S2 subunit of S protein. Computational analyses revealed that this bond breakage associates with an allosteric effect resulting in the generation of a closed conformation with a reduced ability to bind the ACE2 receptor. The UV-C-induced reduced affinity of S protein for ACE2 was further confirmed by SPR analyses and in vitro infection assays. This comprehensive approach pinpoints the S2 domain of S protein as a potential therapeutic target to prevent SARS-CoV-2 infection. Notably, this workflow could be used to screen a wide variety of microbial protein domains, resulting in a precise molecular fingerprint and providing new insights to adequately address future epidemics

Dopamine Reduces SARS-CoV-2 Replication In Vitro through Downregulation of D2 Receptors and Upregulation of Type-I Interferons

Recent evidence suggests that SARS-CoV-2 hinders immune responses via dopamine (DA)-related mechanisms. Nonetheless, studies addressing the specific role of DA in the frame of SARS-CoV-2 infection are still missing. In the present study, we investigate the role of DA in SARS-CoV-2 replication along with potential links with innate immune pathways in CaLu-3 human epithelial lung cells. We document here for the first time that, besides DA synthetic pathways, SARS-CoV-2 alters the expression of D1 and D2 DA receptors (D1DR, D2DR), while DA administration reduces viral replication. Such an effect occurs at non-toxic, micromolar-range DA doses, which are known to induce receptor desensitization and downregulation. Indeed, the antiviral effects of DA were associated with a robust downregulation of D2DRs both at mRNA and protein levels, while the amount of D1DRs was not significantly affected. While halting SARS-CoV-2 replication, DA, similar to the D2DR agonist quinpirole, upregulates the expression of ISGs and Type-I IFNs, which goes along with the downregulation of various pro-inflammatory mediators. In turn, administration of Type-I IFNs, while dramatically reducing SARS-CoV-2 replication, converges in downregulating D2DRs expression. Besides configuring the CaLu-3 cell line as a suitable model to study SARS-CoV-2-induced alterations at the level of the DA system in the periphery, our findings disclose a previously unappreciated correlation between DA pathways and Type-I IFN response, which may be disrupted by SARS-CoV-2 for host cell invasion and replication

DISSECTING INTERPLAY MECHANISMS BETWEEN THE CELL-CLEARING SYSTEMS AUTOPHAGY AND PROTEASOME IN DRUG OF ABUSE-RELATED NEUROTOXICITY

An interconnection has been documented between the effects of the widely addictive and neurotoxic drug methamphetamine (METH), and the cell-clearing systems autophagy and proteasome, which grant proteostasis to govern both synaptic plasticity and neuronal survival. While consistently altering the autophagy machinery and the ubiquitin-proteasome system (UPS), METH produces multifaceted, and long-lasting effects in the human/animal brain, encompassing psychomotor morbidities such as hyper-locomotion, stereotypies, addiction, psychosis, depression, memory impairment, and altered cortical excitability. All these effects vary over time following reiterated METH exposure and some of them occur as the consequence of neurotoxicity or the onset of behavioral sensitization, which involves mostly the dopamine (DA) mesostriatal, and mesocorticolimbic brain system. METH-induced behavioral sensitization consists of a compulsive pattern of drug-taking behavior, which translates into long-lasting neuronal adaptations making reward and motivation brain systems hypersensitive to drug and drug-associated stimuli. The biochemical bases underlying the effects of METH are largely due to altered synaptic transmission at the level of monoamine, mainly the DA brain system. In fact, as measured by brain dialysis, reiterated METH administration in mice produces dramatic oscillations of extracellular DA, which ranges from high peaks (exceeding by 10-fold baseline levels) to severe deficiency (no detectable extracellular levels) within just a few hours. This surpasses at large the slight oscillations produced by physiological DA release to produce abnormal, pulsatile stimulation of postsynaptic DA receptors (DRs). This involves all types of DA receptors, though a post-synaptic sensitization is triggered mostly by type 1-DRs (DRD1) through non-canonical transduction pathways that alter the responsivity of postsynaptic neurons to sustain METH addiction. This involves reward-related brain areas where DA terminals are most abundant, namely the medium-sized spiny neurons (MSNs) of the ventral striatum, although other limbic and isocortical brain regions are involved as well. From a behavioral perspective, the occurrence of certain features of METH addiction, including craving, relapse, psychotic episodes, and cognitive impairments, makes it reminiscent of psychiatric disorders such as schizophrenia. The sensitizing effects of METH intake in humans, which are bound to the occurrence and relapse of psychotic episodes resembling those occurring in schizophrenic patients, support the use of METH as a valid experimental model of schizophrenia. Exposure to repeated/high doses of METH produces striatal DA depletion, which is due to a loss of striatal DA terminals, and as occasionally documented, of cell bodies within the substantia nigra pars compacta (SNpc), and even the ventral tegmental area (VTA). The neurotoxic effects of METH within DA terminals and cell bodies are consistent with an increased risk to develop neurodegeneration being reminiscent of Parkinson's disease (PD), which is now quite well established in METH abusers. METH toxicity against DA axons and cell bodies is largely related to an increase in DA-related oxidative species, which impair proteostasis and mitochondrial function. In detail, within DA-PC12 cells and nigral cell bodies, METH produces cytoplasmic alterations which also extend to the cytoplasm and nucleus of striatal GABA neurons. These are reminiscent of those occurring in the brain METH abusers, and include massive mitochondrial damage along with neuronal inclusions which stain for proteins that are substrates of the cell-clearing systems autophagy and proteasome, such as ubiquitin, alpha-synuclein, tau, and prion protein. It is fascinating that autophagy and proteasome alterations are bound to both frank proteinopathy and neurotoxicity which may occur during METH administration/intake, and also to the molecular and biochemical events which sustain METH-induced behavioral sensitization. This is in line with evidence that autophagy and the proteasome intermingle with secretory/trafficking pathways to ensure neuronal survival while controlling behavior through the turnover of synaptic components and modulation of neurotransmitters that are implicated in both METH-induced addiction and neurotoxicity, including DA, glutamate (GLUT), and GABA. In turn, abnormal events that are bound to the effects of METH, including alterations of various synaptic proteins (eg, Bassoon, EndophilinA, Rab10, alpha-syn), and the occurrence of non-canonical biochemical pathways (eg, DRD1, PKC, mTOR), do converge in alterations of both neurotransmission and cell-clearing systems. In line with this, compounds which are known to act as cell-clearance activators have been shown to counteract both METH-induced behavioral sensitization and neurotoxicity, though the specific role of autophagy has been poorly investigated. On the other hand, a plethora of experimental evidence has been provided showing that inhibition of either autophagy or the UPS in animal models can disrupt neuronal cell biology and predispose to early behavioral changes including mood disorders and psychotic symptoms, up to neurodegeneration. This suggests that in the brain, rescuing cell-clearing capacity may produce plastic effects which may relate to both behavioral improvements and neuroprotection. Nonetheless, while it is quite well-established that METH impairs the proteasome through oxidative-related damage, controversial results and confounding outcomes still exist on the autophagy status during METH administration. A further level of complexity emerges when considering that autophagy and proteasome do not behave as independent systems. Indeed, a plethora of cross-talk mechanisms exists between autophagy and the UPS, with autophagy modulators influencing UPS activity and vice-versa. What is more, recent evidence indicates a morphological convergence between these two systems within a single cell-compartment, which has been designated as “autophagoproteasome” or “proteaphagy”. However, it remains to be elucidated whether such a unique cell compartment hosting both autophagy and proteasome markers represents a system to clear inactive proteasomes (as postulated for proteaphagy), or it is rather endowed with empowered catalytic activity. The present study is aimed at dissecting the role of the autophagy and proteasome systems, with a focus on their interplay mechanisms, in a model of METH administration, which apart from being an addictive psychostimulant, is a powerful neurotoxin for DA terminals and neurons. This is done in the attempt to correlate the joint contribution of the two cell-clearing systems with the detrimental phenomena which occur during METH administration, which may be relevant for psychiatric and neurodegenerative diseases beyond drug addiction. To finely dissect the dynamics between autophagy and proteasome interplay, an in vitro model of DA-containing PC12 cells administrated with METH was chosen. The occurrence of the “autophagoproteasome” at baseline and following METH treatment was documented and analyzed through stoichiometric immune-gold analysis at transmission electron microscopy (TEM), and confocal microscopy. Co-immunoprecipitation experiments were carried out to analyze potential molecular-binding mechanisms between autophagy and proteasome components. Again this allows detecting within the autophagoproteasome the concurrence of shared protein substrates such as alpha-syn, and the adaptor protein p62, which is known to shuttle ubiquitinated substrates, and also the proteasome itself, within autophagy vacuoles. In the light of recent evidence indicating a role of mTOR in either autophagy or proteasome activity, as well as in autophagoproteasome formation, METH was eventually combined with mTOR inhibitors and activators to analyze autophagoproteasome occurrence and potential correlations with METH-induced cell death or protection. The results show that METH toxicity is correlated with a decreased amount of autophagoproteasomes, as evident by the count of both immuno-gold particles, and immunofluorescent puncta concomitantly staining for the autophagy and proteasome markers LC3 and P20S, respectively. Remarkably, co-immunoprecipitation analyses unraveled the co-occurrence of P20S within LC3-positive immunoprecipitates, suggesting a molecular binding between autophagy and proteasome components. What is more, in these LC3-positive immunoprecipitates containing P20S particles (likely corresponding to the autophagoproteaseome), two key substrates were detected. These include the adaptor protein p62, and alpha-syn. These findings suggest that misfold-prone proteins such -syn, which are massively induced by METH, are likely substrates of the autophagoproteasome, which is instead impaired by METH. Eventually, since mTOR was recently shown to act upstream of the proteasome aside from autophagy, we sought to investigate how mTOR modulators could affect autophagoproteasome formation in correlation with METH-induced toxicity. The mTOR activator asparagine further suppresses the autophagoproteasome meanwhile exacerbating METH-induced toxicity. Contrariwise, the mTOR inhibitor rapamycin rescues the autophagoproteasome while affording protection against METH toxicity. Remarkably, rapamycin administration is able to counteract the massive cell death and autophagoproteasome suppression, which are induced by the combined administration of METH and asparagine. When coupled with evidence that mTOR inhibition potentiates overall UPS activity besides autophagy, this suggests that a concomitant acceleration of catalytic activity may occur to provide neuroprotection within such a unique cell compartment. This is supported by the presence of active P20S proteasomes, and the co-occurrence of alpha-syn and p62, within LC3-immunoprecipitates roughly corresponding to the autophagoproteasome. These data suggest that a potentially synergistic cell-clearing and neuroprotective activity is likely to take place herewith. Considering the role of autophagy and proteasome systems in the modulation of both DA-related behavior and neuronal survival, this appears as a key for both METH-induced behavioral sensitization and neurotoxicity. Serendipitously, we could also disclose the fine mechanisms through which METH impairs the autophagy machinery aside from it hampering the autophagoproteasome. Besides altering the compartmentalization of P20S proteasome with LC3-positive autophagy vacuoles, METH produces a misplacement of LC3 particles from autophagy vacuoles to the cytosol. This challenges the previous concept of a mere engulfment of autophagy compartments by oxidized/altered substrates while providing a novel insight into the mechanisms of action of METH upon the autophagy machinery. In fact, the densely fluorescent LC3 spots that are commonly detected at confocal microscopy following METH, correspond to immature autophagosomes rather than stagnant autophagy vacuoles, since the greatest contribution is provided by LC3 that is stochastically distributed in cytosolic compartments other than autophagy vacuoles. In these same experimental conditions, the effects of the mTOR inhibitor rapamycin, which are demonstrated to be neuroprotective against cell death, rescue the autophagoproteasome while reinstating the vacuolar compartmentalization of LC3. These findings cast the hypothesis that dysfunction in autophagy and proteasome and their synergistic merging may bridge drugs of abuse, psychiatric disease, and neurodegeneration, providing a platform for further experimental clues