TOSSICITA’ ED EFFETTI DELLA METANFETAMINA

La metanfetamina (MA) è una sostanza d’abuso che induce una varietà di effetti tossici a livello centrale tra cui ansia, confusione e allucinazioni. L’esposizione alla MA risulta neurotossica per le cellule dopaminergiche del sistema nigro-striatale. Infatti la MA induce un immediato massivo rilascio di dopamina (DA) in striato, che produce stress ossidativo, portando a degenerazione i terminali dopaminergici striatali e alla conseguente riduzione dei livelli di DA in striato. I corpi cellulari della substantia nigra pars compacta (SNpc) vengono distrutti per dosi elevate di MA. Prima di andare incontro a morte nel loro citoplasma è possibile osservare strutture multi lamellari e inclusioni positive per proteine quali l’ubiquitina e l’alfa-sinucleina. Quest’ultima proteina in presenza di specie reattive dell’ossigeno forma aggregati, tossici per la cellula. Inclusioni citoplasmatiche positive per l’-sinucleina e per le proteine del sistema Ubiquitina Proteasoma (UP) sono presenti anche nella malattia degenerativa nota come malattia di Parkinson (MdP) che pertanto viene parzialmente mimata dalla tossicità da MA

A2A receptors and methamphetamine toxicity: a role of adenosine as an endogenous neurotoxin

Adenosine A2A are a class of purinergic receptors largely expressed in dopamine (DA)-rich areas of the central nervous system. In particular, they are abundant within basal ganglia, where they modulate the activity of various neurotransmitters, including DA. Despite the lack of knowledge on their fine physiological mechanisms, it is worth to mention that A2A antagonists prevent neuronal death and dyskinesia in Parkinsonism. Moreover the neuroprotective effects observed after blockade of adenosine A2A receptors in several models of neurotoxicity suggests a toxic effect for endogenous adenosine In the light of these evidences, in the present study, by using in vitro models of DA neurons, we investigated: (i) whether A2A antagonists protect DA containing neurons against methamphetamine (METH); (ii) whether activation of A2A receptors produce neurodegeneration. This was done either using A2A agonist receptor NECA or the endogenous compound adenosine; (iii) whether specific cell mechanisms are involved in these phenomena. We found that A2A antagonists protect DA cells against METH neurotoxicity. Moreover, we found that NECA and adenosine both produced a toxic effects. In the light of the key role of autophagy in modulating the survival of DA neurons we found that A2A antagonists increase, while A2A agonists decrease, autophagy. These results suggest that neuroprotection induced by A2A antagonists may be mediated by enhancement of autophagy. As expected we found that pre-treatment with a non-adenosine related inducer of autophagy produced the same protective effects obtained with A2A antagonists. Our data indicate for the first time, that A2A antagonists are protective in DA neurons against METH. Such an effect appear to be mediated by the enhancement of autophagy. On the other hand we found that activation of A2A receptor produces neurotoxicity. Interestingly these effects was reproduced by administering endogenous adenosine. This suggests that adenosine may produce neurodegeneration by inhibiting the autophagy pathway

Morphological characterization of a single knock out double transgenic mouse model of spinal muscle atrophy

Spinal muscular atrophy (SMA) is a neurogenetic autosomal recessive disorder characterized by degeneration of lower motor neurons associated with muscle atrophy and paralysis. Due to a lack of an in depth knowledge on the molecular mechanisms and fine neuropathology of SMA, validation of appropriate animal models is key in fostering SMA research. Recent studies set up an animal model showing long survival and slow disease progression. This model is knocked out for mouse SMN (Smn−/−) gene and carries a human mutation of the SMN1 gene (SMN1A2G), along with human SMN2 gene. In the present study we used this knockout double transgenic mouse as a SMA III model, to characterize the spinal cord pathology along with motor deficit at prolonged survival times (18 months). This long time interval (i.e. up to 535 days) was never analyzed before especially concerning specific motor tasks. We found that the delayed disease progression was likely to maintain fair motor activity despite a dramatic loss of large motor neurons (44.77%). At this stage, spared motor neurons showed significant cell body enlargement. Moreover, similar to what was described in patients affected by SMA we found neuronal heterotopy in the anterior white matter. Motor neuron degeneration was accompanied by the loss of SMN protein in the spinal cord. In summary, the present study validates over a long time period a SMA III mouse model showing neuropathology reminiscent of human patients and provide a useful experimental model to probe novel therapeutic strategies

Lamina X of the spinal cord in motor neuron disease

A number of plastic events were described in the spinal cord in the course of amyotrophic lateral sclerosis (ALS). These consist of various morphological effects, involving neurons, glia, and inflammatory cells, as well. Among plastic changes, an increase in neuronal progenitor cells (NPC) occurs within ependymal cells layer of lamina X. This stem cell-like activity is known to be weak in baseline conditions but it is known to increase significantly during spinal cord disorders, when it preferentially generates glial cells, due to the strong gliogenic effect of the spinal cord “milieu”. In the present work, we used immunohistochemistry and electron microscopy to analyze cell number within lamina X at the end stage of disease in the G93A mouse model of ALS in baseline conditions and following chronic lithium administration. These cells were identified by using GFAP, bIII-tubulin, NeuN, and calbindin- D28K immunostaining. In the absence of lithium we observed an increase of lamina X cells in ALS mice with a glial phenotype, while in G93A mice treated with lithium these cells differentiate towards neuronal-like phenotype. These effects of lithium are concomitant with slowed disease progression and are reminiscent of the neurogenetic effects described in the sub-ependymal ventricular zone of the hippocampus. The present data confirm the scarce NPC activity in the intact spinal cord which is enhanced by disease conditions; in the presence of chronic lithium, such increased NPCs differentiate towards a neuron-like rather than a glial phenotype

Further steps in the role of autophagy in methamphetamine toxicity

Methamphetamine (METH) abuse is known to cause a variety of disorders Incluing depression and psychosis. METH induces nigrostriatal damage in animal models and in humans consisting of intracellular alterations in nigral DA cell bodies, degeneration of DA terminals and decreased striatal DA levels. Following METH exposure, the number of nigral cell bodies is quite preserved but autophagy-like vacuoles and cytoplamic accumulation of misfolded proteins are observed. The DA-containing PC12 cell lines represent a simple model of METH toxicity and are commonly used in vitro to understand the pathophysiology of DA neurons. We analyzed at morphological level the effects of plasmid-dependent autophagy modulation on METH toxicity in PC12 cell line. We profited from the high number of authophagic like vacuoles induced by low doses of METH in order to isolate cell fraction in which to study their origin, dynamic structure and molecular composition. We found that authophagy-like vacuoles are positive both for autophagy and proteasome markers. The modulation of autophagy via a p62 containing plasmid protected from METH toxicity, while the inhibition of autophagy machinery worsened METH neurotoxicity. The present data substantiate the protective role of the autophagy machinery in METH-induced DA toxicity where the pro-autophagy protein p62 possesses a key role

Ultrastuctural alteration after MET H treatment

Methamphetamine (METH) is an illicit recreational drug known to cause a variety of mental disorders, including anxiety, confusion, and hallucination. Exposure to METH induces nigrostriatal damage in experimental animal models and in humans. METH leads to cellular alterations of the dopamine (DA) system consisting of striatal DA release that produces an oxidative stress, and eventually, causes intracellular alterations in nigral DA cell bodies, degeneration of DA terminals and decreases striatal DA levels. However, the number of DA neurons in the substantia nigra pars compacta seems not to be affected by METH, but their cytoplasm features autophagic-like vacuolization and cytoplasmic accumulation of a-synuclein-, ubiquitin- and parkinpositive inclusion-like bodies. The PC12 cell line, derived from the rat pheochromocytoma, is commonly used as an in vitro model to understand the physiology of central DA neurons. A number of factors contribute to the wide use of PC12 cells: they are not expensive, easy to handle, and mimic many features of central DA neurons. In particular they contain DA and present DA receptor on the external membrane. In light of this evidence using the PC12 cell line we analyzed the ultrastructural alterations induced by METH treatment (0.1-10 mM for 72 h). On the other hand the electron microscopy technique represents the gold standard for the study of the cell death, apoptosis, and the occurrence of autophagy vacuoles. Although the neurotransmitter pattern of PC12 cells is close to DA neurons, ultrastructural morphometry demonstrates that, in baseline conditions, PC12 cells possess very low vesicle density and low catecholamine levels. Again, compartmentalization of secretory elements in PC12 cells is already pronounced in baseline conditions, while it is only slightly affected following catecholamine-releasing stimuli. This low flexibility is caused by the low ability of PC12 cells to compensate for sustained catecholamine release, due both to non-sufficient DA synthesis and poor DA storage mechanisms. Moreover increasing the dose of METH leads to a higher number of apoptotic cells and an higher concentration of autophagy-like vacuoles per cell. Interestingly these vacuoles were immunoreactive for the protein of the autophagy pathway and for a-synuclein. Noteworthy, METH induces mithocondrial alterations consisting in matrix dilution and disrupted cristae. These latter findings pose METH as a robust mitochondrial neurotoxin reminiscent of MPTP and rotenone