Recent studies demonstrated that methamphetamine (METH) produces intracellular bodies which are reminiscent of those occurring during degenerative disorders. In vivo studies demonstrate the occurrence of these morphological alterations both in the dopamine (DA) neurons of the substantia nigra and striatal cells. These consist of neuronal bodies staining for a variety of antigens belonging to the ubiquitinproteasome pathway. The formation of these intracellular bodies both in the substantia nigra and PC12 cells depends on the presence of endogenous DA. In the present study, we analyze the mechanisms which lead to METH-induced intracellular bodies within non-dopaminergic striatal neurons. We found that METH is no longer able to produce inclusions in vivo, in striatal cells, when striatal DA is lost. Similarly, in vitro, in primary striatal cell cultures which do not possess DA, METH administration does not produce inclusions. On the other hand, administration of DA to striatal cell cultures produces neuronal inclusions and cell death, which are both related to the inhibition of the ubiquitin-proteasome system and activation of specific-DA receptors. In line with this, we produced subcellular alterations by administering dopamine agonists. Keywords: dopamine receptors, electron microscopy, methamphetamine, striatal inclusions, ubiquitin, ubiquitin-proteasome system. Recent data indicate that methamphetamine (METH), when administered in vivo to C57 Black mice, produces a-synucleinand ubiquitin-positive intracellular bodies within dopamine (DA) containing neurons of the substantia nigra pars compacta (Fornai et al. 2004a). In keeping with this, a recent study in chronic METH abusers demonstrates the occurrence of analogous ubiquitin-positive inclusions within nigral DA cells (Quan et al. 2005). High amount of these intracellular bodies were reproduced by administering METH to PC12 cell cultures, which were analogous to nigral DA neurons, produced, and release DA (Cubells et al. 1994;Larsen et al. 2002; Fornai et al. 2004a,b). This is in line with the robust effect of METH to increase cytosolic DA, which in turn, binds to parkin, thereby inactivating this protein (LaVoie et al. 2005), or to a-synuclein protofibrils, thereby promoting their precipitation (Conway et al. 2000;Norris et al. 2005).Similar inclusions were described within striatal cells following METH administration in vivo (Fornai et al. 2004a); however, we never explored their fine ultrastructure for the co-localization of ubiquitin and a-synuclein and the biochemical mechanisms, which are responsible for their localization within striatal perikaria. A major point which needs to be considered is that intrinsic striatal perikaria do not contain DA. However, these cell bodies receive a rich dopaminergic innervation arising from cell bodies in the substantia nigra, which is responsible for massive release of striatal extracellular DA following METH administration (Battaglia et al. 2002). This is a critical point, as DA released by the mesostriatal pa...

A 3 adenosine receptor activation has been previously demonstrated to result in both neuroprotective and neurodegenerative effects, depending upon specific pathophysiological conditions. This dual effect may depend on receptor regulation mechanisms that are able to change receptor availability and/or function. In the present study, we investigated desensitization, internalization, and down-regulation of native A 3 adenosine receptors in human astrocytoma cells after exposure to the agonist 2-chloro-N6-(3-iodobenzyl)-N-methyl-5Ј-carbamoyladenosine (Cl-IBMECA). Cl-IBMECA induced a concentrationdependent inhibition of adenylyl cyclase activity with an EC 50 value of 2.9 Ϯ 0.1 nM. The effect was suggested to be mediated by A 3 adenosine receptor subtype by the use of selective adenosine receptor antagonists. Cell treatment with pertussis toxin abolished Cl-IBMECA-mediated inhibition of adenylyl cyclase activity, evidencing an A 3 receptor coupling to inhibitory G protein. Short-term exposure to the agonist Cl-IBMECA (100 nM) caused rapid receptor desensitization, within 15 min. Agonist-induced desensitization was accompanied by receptor internalization: A 3 adenosine receptor internalized with rapid kinetics, within 30 min, after cell exposure to 100 nM Cl-IB-MECA. The localization of A 3 adenosine receptors on the plasma membrane and in intracellular compartments was directly revealed by immunogold electron microscopy. After desensitization, the removal of agonist led to the restoration of A 3 adenosine receptor functioning through receptor recycling to the cell surface within 120 min. Prolonged agonist exposure (1-24 h) resulted in a marked down-regulation of A 3 adenosine receptors that reached 21.9 Ϯ 2.88% of control value after 24 h. After down-regulation, the recovery of receptor functioning was slow (24 h) and associated with the restoration of receptor levels close to control values. In conclusion, our results demonstrated that A 3 receptors, in astrocytoma cells, are regulated after short-and long-term agonist exposure.Actions of adenosine are mediated by four G protein-coupled membrane receptors (GPCRs): A 1 , A 2A , A 2B , and A 3 receptors (Fredholm et al., 1994). Although expressed at very low levels in mammalian brain, the A 3 adenosine receptor (AR) subtype has been implicated in behavioral depression and modulation of ischemic cerebral damage (for review, see von Lubitz, 1999). Through the development of selective A 3 AR agonists (e.g., Cl-IBMECA) and antagonists (e.g., MRS 1191 and MRS 1220) (Kim et al., 1994(Kim et al., , 1996Jacobson et al., 1997), the putative pathophysiological roles of this receptor have became clear. It has been demonstrated that A 3 AR agonists profoundly affect cell survival, by promoting cell protection or cell death, depending upon the cell type and/or agonist concentration (for reviews, see Jacobson, 1998;Jacobson et al., 1999). In human astrocytoma cells (ADF cells), exposure to nanomolar Cl-IBMECA concentrations increased resistance to apoptosis, by a mechanism...

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Amyotrophic lateral sclerosis (ALS) is characterized by massive loss of motor neurons. Data from ALS patients and experimental models indicate that mitochondria are severely damaged within dying or spared motor neurons. Nonetheless, recent data indicate that mitochondrial preservation, although preventing motor neuron loss, fails to prolong lifespan. On the other hand, the damage to motor axons plays a pivotal role in determining both lethality and disease course. Thus, in the present article each motor neuron compartment (cell body, central, and peripheral axons) of G93A SOD-1 mice was studied concerning mitochondrial alterations as well as other intracellular structures. We could confirm the occurrence of ALS-related mitochondrial damage encompassing total swelling, matrix dilution and cristae derangement along with non-pathological variations of mitochondrial size and number. However, these alterations occur to a different extent depending on motor neuron compartment. Lithium, a well-known autophagy inducer, prevents most pathological changes. However, the efficacy of lithium varies depending on which motor neuron compartment is considered. Remarkably, some effects of lithium are also evident in wild type mice. Lithium is effective also in vitro, both in cell lines and primary cell cultures from the ventral spinal cord. In these latter cells autophagy inhibition within motor neurons in vitro reproduced ALS pathology which was reversed by lithium. Muscle and glial cells were analyzed as well. Cell pathology was mostly severe within peripheral axons and muscles of ALS mice. Remarkably, when analyzing motor axons of ALS mice a subtotal clogging of axoplasm was described for the first time, which was modified under the effects of lithium. The effects induced by lithium depend on several mechanisms such as direct mitochondrial protection, induction of mitophagy and mitochondriogenesis. In this study, mitochondriogenesis induced by lithium was confirmed in situ by a novel approach using [2-3H]-adenosine.