Michela Ferrucci

Methamphetamine produces neuronal inclusions in the nigrostriatal system and in PC12 cells

Mice treated with the psychostimulant methamphetamine (MA) showed the appearance of intracellular inclusions in the nucleus of medium sized striatal neurones and cytoplasm of neurones of the substantia nigra pars compacta but not in the frontal cortex. All inclusions contained ubiquitin, the ubiquitin activating enzyme (E1), the ubiquitin protein ligase (E3‐like, parkin), low and high molecular weight heat shock proteins (HSP 40 and HSP 70). Inclusions found in nigral neurones stained for α‐synuclein, a proteic hallmark of Lewy bodies that are frequently observed in Parkinsons disease and other degenerative disorders. However, differing from classic Lewy bodies, MA‐induced neuronal inclusions appeared as multilamellar bodies resembling autophagic granules. Methamphetamine reproduced this effect in cultured PC12 cells, which offered the advantage of a simple cellular model for the study of the molecular determinants of neuronal inclusions. PC12 inclusions, similar to those observed in nigral neurones, were exclusively localized in the cytoplasm and stained for α‐synuclein. Time‐dependent experiments showed that inclusions underwent a progressive fusion of the external membranes and developed an electrodense core. Inhibition of dopamine synthesis by α‐methyl‐p‐tyrosine (αMpT), or administering the antioxidant S‐apomorphine largely attenuated the formation of inclusions in PC12 cells exposed to MA. Inclusions were again observed when αMpT‐treated cells were loaded with l‐DOPA, which restored intracellular dopamine levels.

Suppression of autophagy precipitates neuronal cell death following low doses of methamphetamine

Methamphetamine abuse is toxic to dopaminergic neurons, causing nigrostriatal denervation and striatal dopamine loss. Following methamphetamine exposure, the number of nigral cell bodies is generally preserved, but their cytoplasm features autophagic‐like vacuolization and cytoplasmic accumulation of α‐synuclein‐, ubiquitin‐ and parkin‐positive inclusion‐like bodies. Whether autophagy is epiphenomenal or it plays a role in the mechanism of methamphetamine toxicity and, in the latter case, whether its role consists of counteracting or promoting the neurotoxic effect remains obscure. We investigated the signaling pathway and the significance (protective vs. toxic) of autophagy activation and the convergence of the autophagic and the ubiquitin‐proteasome pathways at the level of the same intracellular bodies in a simple cell model of methamphetamine toxicity. We show that autophagy is rapidly up‐regulated in response to methamphetamine. Confocal fluorescence microscopy and immuno‐electron microscopy studies demonstrated the presence of α‐synuclein aggregates in autophagy‐lysosomal structures in cells exposed to methamphetamine, a condition compatible with cell survival. Inhibition of autophagy either by pharmacologic or genetic manipulation of the class III Phosphatidylinositol‐3 kinase‐mediated signaling prevented the removal of α‐synuclein aggregates and precipitated a bax‐mediated mitochondrial apoptosis pathway.

Autophagy and amyotrophic lateral sclerosis: The multiple roles of lithium.

In a pilot clinical study that we recently published we found that lithium administration slows the progression of Amyotrophic Lateral Sclerosis (ALS) in human patients. This clinical study was published in addition with basic (in vitro) and pre-clinical (in vivo) data demonstrating a defect of autophagy as a final common pathway in the genesis of ALS. In fact, lithium was used as an autophagy inducer. In detailing the protective effects of lithium we found for the first time that this drug stimulates the biogenesis of mitochondria in the central nervous system and, uniquely in the spinal cord, it induces neuronogenesis and neuronal differentiation. In particular, the effects induced by lithium can be summarized as follows: (i) the removal of altered mitochondria and protein aggregates; (ii) the biogenesis of well-structured mitochondria; (iii) the suppression of glial proliferation; (iv) the differentiation of newly formed neurons in the spinal cord towards a specific phenotype. In this addendum we focus on defective autophagy as a “leit motif” in ALS and the old and novel features of lithium which bridge autophagy activation to concomitant effects that may be useful for the treatment of a variety of neurodegenerative disorders. In particular, the biogenesis of mitochondria and the increase of calbindin D 28K-positive neurons, which are likely to support powerful neuroprotection towards autophagy failure, mitochondriopathy, and neuronal loss in the spinal cord. Addendum to: Fornai F, Longone P, Cafaro L, Kastsiuchenka O, Ferrucci M, Manca ML, Lazzeri G, Spalloni A, Bellio N, Lenzi P, Modugno N, Siciliano G, Isidoro C, Murri L, Ruggieri S, Paparelli A. Lithium delays progression of amyotrophic lateral sclerosis. Proc Natl Acad Sci U S A 2008; 105:2052-2057.

A systematic study of brainstem motor nuclei in a mouse model of ALS, the effects of lithium

Transgenic mice expressing the human superoxide dismutase 1 (SOD-1) mutant at position 93 (G93A) develop a phenotype resembling amyotrophic lateral sclerosis (ALS). In fact, G93A mice develop progressive motor deficits which finally lead to motor palsy and death. This is due to the progressive degeneration of motor neurons in the ventral horn of the spinal cord. Although a similar loss is reported for specific cranial motor nuclei, only a few studies so far investigated degeneration in a few brainstem nuclei. We recently reported that chronic lithium administration delays onset and duration of the disease, while reducing degeneration of spinal motor neuron. In the present study, we extended this investigation to all somatic motor nuclei of the brain stem in the G93A mice and we evaluated whether analogous protective effects induced by lithium in the spinal cord were present at the brain stem level. We found that all motor but the oculomotor nuclei were markedly degenerated in G93A mice, and chronic treatment with lithium significantly attenuated neurodegeneration in the trigeminal, facial, ambiguus, and hypoglossal nuclei. Moreover, in the hypoglossal nucleus, we found that recurrent collaterals were markedly lost in G93A mice while they were rescued by chronic lithium administration.

Occurrence of neuronal inclusions combined with increased nigral expression of α-synuclein within dopaminergic neurons following treatment with amphetamine derivatives in mice

In recent years several clinical and research findings have demonstrated the involvement of the presynaptic protein alpha-synuclein in a variety of neurodegenerative disorders which are known as synucleinopathies. Although the function of this protein in the physiology of the cell remains unknown, it is evident that both genetic alterations or a mere overexpression of the native molecule produces a degeneration of nigral dopamine-containing neurons leading to movement disorders, as demonstrated in inherited Parkinsons disease. In the present study, we investigated whether widely abused drugs such as methamphetamine and methylenedioxymethamphetamine (ecstasy), which are known to damage the nigrostriatal dopamine pathway of mice, increase the expression of alpha-synuclein within dopamine neurons of the substantia nigra pars compacta. The results of this study demonstrate that nigrostriatal dopamine denervation and occurrence of intracellular inclusions in nigral neurons produced by amphetamine derivatives are related to increased expression of alpha-synuclein within dopamine neurons of the substantia nigra. This lends substance to the hypothesis that increased amounts of native alpha-synuclein may be per se a detrimental factor for the dopamine neurons.

A damage to locus coeruleus neurons converts sporadic seizures into self-sustaining limbic status epilepticus

Various studies demonstrated that the neurotransmitter norepinephrine (NE) plays a relevant role in modulating seizures; in particular, a powerful effect consists in delaying the kindling of limbic areas such as the amygdala and hippocampus. Given the rich NE innervation of limbic regions, we selected a sensitive trigger area, the anterior piriform cortex, to test whether previous loss of noradrenergic terminals modifies sporadic seizures in rats. The damage to locus coeruleus terminals was produced by using the selective neurotoxin N‐(‐2‐chloroethyl)‐N‐ethyl‐2‐bromobenzylamine (DSP‐4, 60 mg/kg i.p.). In intact rats, bicuculline (a GABA‐A antagonist, 118 pmol) microinfused into this area produced sporadic seizures, while in rats previously injected with DSP‐4, bicuculline determined long‐lasting self‐sustaining status epilepticus. In intact rats, sporadic seizures were accompanied by a marked increase in norepinephrine release in the contralateral piriform cortex, while in locus coeruleus‐lesioned rats this phenomenon was attenuated. While bicuculline‐induced sporadic seizures were prevented by the focal infusion of amino‐7‐phosphonoheptanoic acid (AP‐7, a selective NMDA antagonist), or 1,2,3,4‐tetrahydro‐6‐nitro‐2,3‐dioxo‐benzo[f]quinoxaline‐7‐sulphonamide (NBQX, a selective non‐NMDA antagonist), status epilepticus obtained in norepinephrine‐lesioned rats was insensitive to AP‐7 but was still inhibited by NBQX. By using fluorescent staining for damaged (Fluoro‐Jade B) and intact (DAPI) neurons, as well as cresyl violet, we found that rats undergoing status epilepticus developed neuronal loss in various limbic regions. This study demonstrates a powerful effect of noradrenergic terminals in regulating the onset of limbic status epilepticus and its sensitivity to specific glutamate antagonists.

Abnormal involuntary movements (AIMs) following pulsatile dopaminergic stimulation: severe deterioration and morphological correlates following the loss of locus coeruleus neurons.

Parkinsonian patients are treated with dopamine replacement therapy (typically, intermittent administration of the dopamine precursor L-DOPA); however, this is associated with the onset of abnormal involuntary movements, which seriously impair the quality of life. The molecular mechanisms underlying abnormal involuntary movements represent an intense field of investigation in the area of neurobiology of disease, although their aetiology remains unclear. Apart from the fine cellular mechanisms, the pathways responsible for the generation of abnormal involuntary movements may involve changes in neurotransmitter systems. A potential candidate is noradrenaline, since a severe loss of this neurotransmitter characterizes Parkinsons disease, and noradrenergic drugs produce a symptomatic relief of L-DOPA-induced dyskinesia. In previous studies we found that pulsatile dopamine release, in the absence of the physiological noradrenaline innervation, produces motor alterations and ultrastructural changes within striatal neurons. In the present study we demonstrate that a unilateral damage to the noradrenaline system anticipates the onset and worsens the severity of L-DOPA-induced contralateral abnormal involuntary movements in hemi-parkinsonian rats. Similarly, ubiquitin-positive striatal ultrastructural changes occur in unilaterally dopamine-depleted, noradrenaline-deficient rats following chronic L-DOPA administration. This study confirms a significant impact of the noradrenergic system in the natural history of Parkinsons disease and extends its role to the behavioural and morphological effects taking place during pulsatile dopamine replacement therapy.

Fine ultrastructure and biochemistry of PC12 cells: A comparative approach to understand neurotoxicity

The PC12 cell line is commonly used as a tool to understand the biochemical mechanisms underlying the physiology and degeneration of central dopamine neurons. Despite the broad use of this cell line, there are a number of points differing between PC12 cells and dopamine neurons in vivo which are missed out when translating in vitro data into in vivo systems. This led us to compare the PC12 cells with central dopamine neurons, aiming at those features which are predictors of in vivo physiology and degeneration of central dopamine neurons. We carried out this comparison, either in baseline conditions, following releasing or neurotoxic stimuli (i.e. acute or chronic methamphetamine), to end up with therapeutic agents which are suspected to produce neurotoxicity (l-DOPA). Although the neurotransmitter pattern of PC12 cells is close to dopamine neurons, ultrastructural morphometry demonstrates that, in baseline conditions, PC12 cells possess very low vesicles density, which parallels 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 dopamine synthesis and poor dopamine storage mechanisms. This contrasts markedly with dopamine-containing neurons in vivo lending substance to opposite findings between these compartments concerning the sensitivity to a number of neurotoxins.

Similarities between Methamphetamine Toxicity and Proteasome Inhibition

Abstract: The monoamine neurotoxin methamphetamine (METH) is commonly used as an experimental model for Parkinsons disease (PD). In fact, METH‐induced striatal dopamine (DA) loss is accompanied by damage to striatal nerve endings arising from the substantia nigra. On the other hand, PD is characterized by neuronal inclusions within nigral DA neurons. These inclusions contain a‐synuclein, ubiquitin, and various components of a metabolic pathway named the ubiquitin‐proteasome (UP) system, while mutation of genes coding for various components of the UP system is responsible for inherited forms of PD. In this presentation we demonstrate for the first time the occurrence of neuronal inclusions in vivo in the nigrostriatal system of the mouse following administration of METH. We analyzed, in vivo and in vitro, the shape and the fine structure of these neuronal bodies by using transmission electron microscopy. Immunocytochemical investigation showed that these METH‐induced cytosolic inclusions stain for ubiquitin, a‐synuclein, and UP‐related molecules, thus sharing similar components with Lewy bodies occurring in PD, with an emphasis on enzymes belonging to the UP system. In line with this, blockade of this multicatalytic pathway by the selective inhibitor epoxomycin produced cell inclusions with similar features. Moreover, using a multifaceted pharmacological approach, we could demonstrate the need for endogenous DA in order to form neuronal inclusions.

Protein clearing pathways in ALS

In the present review a large amount of experimental and clinical studies on ALS are discussed in an effort to dissect common pathogenic mechanisms which may provide novel information and potential therapeutic strategies for motor neuron degeneration.Protein clearing systems play a critical role in motor neuron survival during excitotoxic stress, aging and neurodegenerative disorders. Among various mechanisms which clear proteins from the cell recent studies indicate autophagy as the most prominent pathway to promote survival of motor neurons.Autophagy regulates the clearance of damaged mitochondria, endoplasmic reticulum and misfolded proteins in eukaryotic cells. Upon recruitment of the autophagy pathway, an autophagosome is produced and directed towards lysosomal degradation.Here we provide evidence that in both genetic and sporadic amyotrophic lateral sclerosis (ALS, the most common motor neuron disorder) a defect in the autophagy machinery is common. In fact, swollen, disrupted mitochondria and intracellular protein aggregates accumulate within affected motor neurons. These structures localize within double membrane vacuoles, autophagosomes, which typically cluster in perinuclear position. In keeping with this, when using autophagy inhibitors or suppressing autophagy promoting genes, motor symptoms and motor neuron death are accelerated. Conversely stimulation of autophagy alleviates motor neuron degeneration.Therefore, autophagy represents an important target when developing novel treatments in ALS.