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Digital archive of theses discussed at the University of Pisa


Thesis etd-03192021-082349

Thesis type
Tesi di dottorato di ricerca
Thesis title
Academic discipline
Course of study
tutor Prof. Fornai, Francesco
supervisore Prof.ssa Ferrucci, Michela
  • alpha-synuclein
  • autophagoproteasome
  • autophagy
  • drug abuse
  • methamphetamine
  • neurotoxicity
  • proteasome
Graduation session start date
Release date
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.