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MPTP treatment is a widely validated model to produce experimental parkinsonism. Among different administration protocols, chronic exposure to little amounts of MPTP has been reported to reproduce neuropathological features characterizing dopaminergic neurons in human Parkinson disease [1]. Recent studies by our group have found that MPTP, besides dopaminergic neurons, affects also spinal motor neurons [2], thus bridging, at neuropathological level, Parkinson disease and Amyotrophic Lateral Sclerosis. In the present study we show that chronic MPTP treatment, as well as acute one, is able to induce motor neurons loss in lumbar spinal cord. We have investigated the involvement of autophagy and the occurrence of alterations in alpha-synuclein immunostaining, after MPTP treatment. The activation of autophagic pathway is proved by the significant increase of MAP LC3β in the motor neurons of MPTP treated mice. The same neurons possess altered alpha-synuclein immunostaining, detected by two novel homemade monoclonal antibodies. Motor neuron pathology could depend on direct effect of MPTP, but it can be also due to a remodeling in anatomical connections of motor neurons with descending pathways or intra-spinal networks. Among these, we have particularly investigated the Renshaw cells and the coeruleo-spinal noradrenaline projections, which can intervene in both physiology and survival of motor neurons [3]. Our result shed new light on the physiopathology of motor symptoms in parkinsonism and call for novel pharmacological and neurophysiological approaches to relive motor function in Parkinson disease, through modulation of spinal circuits., Italian Journal of Anatomy and Embryology, Vol 117, No 2 (Supplement) 2012

Motor neuron loss is a feature of amyotrophic lateral sclerosis (ALS), spinal muscle atrophy (SMA) and it is recently described in other neurodegenerative disorders. In humans these disorders vary from dramatically short up to long disease duration. A short disease duration in humans ranges from some months up to 1 or 2 years. In contrast animal models commonly used to evaluate motor neuron disorders are extremely short lasting, where motor impairment lasts about a few weeks. This is the case of the G93A mouse model which is expected to reproduce ALS in humans. Despite commonalities, this condensed time interval is likely to produce different pathological correlates. Therefore, it is not surprising that experimental morphology and therapeutics fail to detect some hallmarks of human disorders in these animal models. This temporal discrepancy is often missed out and genetic background as well as evolution-based structural differences in motor systems are considered as critical determinants instead. The present communication wish to introduce the benefits of a long-lasting animal model of motor neuron disorders. In detail, we had been able to characterize the motor failure, the biochemical abnormalities and the morphological alterations which develop only at prolonged time interval (18 months) using a single knock out double transgenic mouse model of human SMAIII. In these mice we had been able to dissect for the first time morphological alterations typical of human disorders such as motor neuron heterotopy as well as motor neuron loss. This is correlated with a profound loss of the survival motor neuron (SMN) protein. There was a remarkable somatotopic correlation between the specific motor deficit (proximal vs distal limb muscles) and the severity of motor neuron loss in the corresponding pool (medial vs lateral) of motor neurons in the spinal cord. This ideal experimental context provides the chance to evaluate the therapeutic effects of specific drugs administered in the long run ruling out bias due to experimental variability when the drugs are administered only for a few weeks. In this way it is also easy to discern symptomatic vs disease modifying effects. In line with this, we compared the effects of GSK3 beta inhibitors and/or autophagy activators and we found a remarkable induction in the SMN protein which was in line with protection from motor neuron loss and a steady effect counteracting the long term progressive motor deterioration., Italian Journal of Anatomy and Embryology, Vol 117, No 2 (Supplement) 2012

Mutations in the PTEN-induced putative kinase1 (PINK1) represent the second most common cause of autosomal recessive Parkinson’s disease. The PINK1 protein has a mitochondrial localization and interacts with a variety of proteins, including the pro-autophagy protein beclin1 and the ubiquitin-ligase parkin. In particular, PINK1 is able to recruit parkin to the surface of dysfunctional mitochondria, to promote the ubiquitination of several mitochondrial proteins and the subsequent activation of the mitophagy cascade. Aim of this study was to use a dopaminergic cell model and transmission electron microscopy to characterize whether the modulation of PINK1 expression: (i) modifies the number and morphology of mitochondria and of autophagy organelles (autophagosomes); (ii) alters the recruitment of beclin1, parkin and ubiquitin to the mitochondria; (iii) affects cell survival. We used PC12 cells transfected either with the empty vector (pcDNA), or vectors expressing wild type PINK1 (PINK1wt), a pathogenic mutant (PINK1W437X), shRNA against rat PINK1 (shPINK1) or scramble (shSCR). Samples were analyzed both in baseline conditions and following methamphetamine (METH) treatment to provide a neurotoxic, autophagy-dependent stimulation. We showed that, especially upon METH exposure, the modulation of PINK1 levels dramatically affected the morphology and clearance of mitochondria. In fact, the number of abnormal mitochondria was reduced in PINK1wt, while it was significantly increased upon shPINK1 and also, to a lesser extent, in PINK1W437X cells. In keeping with this, mitochondrial ubiquitin clusters and mitochondrial levels of parkin and beclin1 were increased in PINK1wt cells while they were reduced both in PINK1 silenced and PINK1W437X cells. Interestingly, the number of autophagic vacuoles was unaffected by PINK1 modulation in baseline conditions, and was significantly reduced only in cells lacking functional PINK1 and upon METH exposure. All these effects were significantly associated with a modulation of apoptotic cell death. Our data provide robust sub-cellular evidence that PINK1 counteracts neurodegeneration by simultaneously recruiting beclin1, parkin and ubiquitin and thus enhancing the clearance of damaged mitochondria. In the absence of functional PINK1 and upon autophagy stress, we observed a failure of the autophagy system at large, with marked accumulation of dysfunctional mitochondria and dramatic increase in apoptotic cell death., Italian Journal of Anatomy and Embryology, Vol 116, No 1 (Supplement) 2011

Trimethyltin (TMT) is a triorganotin compound which determines neurodegeneration of specific brain areas particularly damaging the limbic system. Organotin compounds are used as heat stabilizers in polyvinyl chloride polymers, industrial and agricultural biocides, and industrial catalysts in chemical reactions. The mechanism underlying TMT selective tissue-specific pattern of cellular damage is not completely known unless a correlation exists between TMT toxicity and the expression of stannin, a highly-conserved protein mainly localized within mitochondria. Ultrastructural studies performed on samples obtained from the brain of humans and rodents intoxicated with TMT describe increased number of lysosomes and big vacuoles suggestive of altered autophagy. Our results show that autophagy inhibitors added to TMT dramatically increased the toxicity of this compound both in hippocampal and cortical neurons. Conversely, autophagy inducers such as rapamycin and lithium prevent the neurotoxicity of TMT. Due to its diverse targets, the action of lithium may be complex and its neuroprotective property is sometimes controversial. Our data show that in acute treatment experiments hippocampal and cortical neurons behave quite differently when challenged with lithium. The neuroprotective effect of lithium acutely administered against TMT in hippocampal but not in cortical neurons can be completely reverted by an excess of inositol and is possibly related to the inactivation of inositol monophosphatase, a key regulator of autophagy. Lithium is also effective in preventing TMT toxicity in astrocyte cell cultures in a range of concentrations of 0.2-4 mM., Italian Journal of Anatomy and Embryology, Vol 116, No 1 (Supplement) 2011

1-methyl, 4-phenyl, 1, 2, 3, 6-tetrahydropiridine (MPTP) is a neurotoxin, widely used to produce experimental models of Parkinson Disease in rodents and primates. Although dopaminergic neurons are the most sensitive to MPTP neurotoxicity, different neuronal subtypes could be affected. In particular, noradrenergic neurons of the Locus Coeruleus may be involved as well as nigral dopaminergic neurons. Moreover, apart from catecholamine-containing nuclei, recent studies indicate that MPTP may produce pathological effects within the spinal cord. This point deserves compelling interest since it suggests, at experimental level, commonalities between Parkinson Disease and Amyotrophic Lateral Sclerosis. For instance, recent reports demonstrate that MPTP activates apoptotic proteins at the level of spinal cord. However, to our knowledge, none of these studies so far analyzed whether motor neuron loss really occurs following MPTP administration. Therefore, in the present study we evaluated the effect of a robust dose of MPTP (20 mg/Kg X3) in the nigro-striatal system and spinal cord. Along with a severe dopaminergic cells loss within the substantia nigra, quantified by stereology and a marked decrease of striatal catecholoamine fibers measured by semi-quantitative densitometry, we found a significant (roughly 30 %) depletion of motor neurons in the lumbar spinal cord of MPTP-treated C57BL/6J mice. At the same level, spared motor neurons often present an altered morphology, being dysmorphic and vacuolated. Furthermore, using four different antibodies (tree monoclonal, one polyclonal), recognizing distinct epitopes in the sequence of alpha-synuclein, we found that alpha-synuclein immunostaining is markedly altered in the spinal cord of MPTP-treated mice. The present data shed new lights on similarities between dopaminergic neurons and spinal motor neurons, while suggesting that MPTP might be a neurotoxin diverse from what originally considered., Italian Journal of Anatomy and Embryology, Vol 116, No 1 (Supplement) 2011

Italian Journal of Anatomy and Embryology, Vol 115, No 1/2 (Supplement) 2010