MANIFESTAZIONI PARALLELE FRA SISTEMA NERVOSO CENTRALE ED ENTERICO NELLA MALATTIA DI PARKINSON

La Malattia di Parkinson (MdP) è una patologia neurodegenerativa che interessa i neuroni dopaminergici della substantia nigra determinando nei pazienti disturbi motori. Alterazioni non motorie interessano diversi organi, in particolare il tratto gastrointestinale. Nel presente studio abbiamo riprodotto sperimentalmente la MdP con la neurotossina 1-metil, 4-fenil, 1,2,3,6,-tetraidropiridina (MPTP). In topi C57BL di nove settimane sono stati valutati gli effetti indotti dal trattamento con MPTP, nella substantia nigra, nello striato e nel duodeno. Su questi animali sono stati condotti test motori (Open field e PaGE test). Una settimana dopo il trattamento con MPTP (5 mg/kg x2/dì, per 3 settimane) il duodeno è stato prelevato ed esaminato con tecniche di istologia, immunoistochimica e immunoblotting per la tirosina idrossilasi (TH) e per l’α-sinucleina. Esperimenti di immunocitochimica sono stati condotti al microscopio elettronico. I test motori hanno mostrato deficit nel PaGE test in tutti gli animali trattati con MPTP, mentre non sono stati trovate differenze tra trattati e controlli sottoposti all’open field test. L’analisi delle sezioni istologiche ha mostrato alcune alterazioni quali una lieve atrofia della mucosa duodenale e alterazioni al livello ghiandolare. Al microscopio elettronico l’orletto a spazzola è apparso discontinuo. In tutti i topi trattati con MPTP risultava ridotta l’immunopositività alla TH sia nella substantia nigra che nello striato, confermando l’effetto neurotossico centrale sui neuroni dopaminergici. A livello del duodeno è risultata ridotta l’immunopositività alla TH. Questo è stato confermato con la tecnica semiquantitativa di immunoblotting. Inoltre, indagini immunologiche hanno evidenziato un aumento dell’α-sinucleina promosso dal trattamento con MPTP, sia a livello centrale che a livello periferico. L’immunopositività all’α-sinucleina è stata evidenziata, inoltre, mediante indagini in microscopia elettronica. Il presente studio mostra che il trattamento con MPTP induce alterazioni a livello del duodeno in maniera analoga a quanto avviene nel sistema dopaminergico nella substantia nigra e nello striato. Inoltre, questo modello sperimentale di MdP riproduce le alterazioni gastrointestinali osservate nei pazienti parkinsoniani. Questi risultati danno sostanza al concetto che il sistema nervoso enterico agisca come un “secondo cervello”

Parallelism between central and enteric nervous system damage in experimental parkinsonism

Parkinson’s disease (PD) is a neurodegenerative condition which affects dopaminergic neurons of the substantia nigra (SN), leading to a movement disorder. Non motor alterations occur in several viscera, in particular the gastrointestinal tract. In 9-week old C57BL mice we examined the effects of the parkinsonism-inducing neurotoxin 1-methyl, 4-phenyl, 1,2,3,6,-tetrahydropyridine (MPTP, administered either acutely or chronically) in SN and striatum, as well as in duodenum. Motor tests (open field and PaGE) were performed. One week after treatment with MPTP (acute: 20 mg/KgX3, 2h apart or chonic: 5 mg/kg x2/die, for 3 weeks), histological investigations, immunohistochemistry and immunoblotting for tyrosine hydroxylase (TH), and α-synuclein (α-syn) were carried out. Immunocytochemical investigations were analyzed under electron microscopy. Motor tests showed a failure of the PaGE test in all MPTP-treated animals, whereas no difference was found in open field test in comparison with controls. Analysis of histological sections showed some alterations consisting of slight atrophy of duodenal mucosa and glandular disarrangement only after chronic treatment. Under electron microscopy the brush border appeared discontinuous. In all MPTP-administered mice, TH immunopositivity was reduced in SN and striatum, confirming its central dopaminergic neurotoxicity. At duodenal level, TH immunostaining was lost following all MPTP treatments with a slight variation in chronic compared with acute administrations. This was confirmed by semiquantitative immunoblotting. Moreover, α-syn immunostaining was enhanced by MPTP treatment but this was way more evident following chronic administration both at central and peripheral level. Following chronic treatment α-Syn immunopositive structures were investigated under electron microscopy. Our study shows that chronic more than acute administration of MPTP induces alterations at duodenal level reminiscent of dopaminergic damage in SN and striatum. Moreover, this experimental model of parkinsonism features gastrointestinal dysfunction observed in PD patients. These findings lend substance to the concept of the enteric nervous system as a double brain which recapitulates and is an ancestry of the central nervous system

Morphological effects of chronic excitotoxicity depend on autophagy activation failure

It is well known that exposure to excitatory amino acid in specific experimental conditions might produce a defect in the autophagy pathway. Such an effect was observed in motor neurons exposed chronically to glutamate agonists. In particular, it is well known that glutamate induces motor neuron death and this is supposed to play a key role in the physiopathology of motor neuron loss in amyotrophic lateral sclerosis (ALS). Similarly, a defective recruitment of autophagy was recently documented in ALS. In the present study, we used primary motor neuron cultures to analyzed whether AMPA receptor stimulation via kainic acid produces activation of autophagy and whether this is defective compared with what it is required to rescue motor neurons. In particular we found that exposure of motor neurons to kainic acid produces intracellular alterations associated with defective autophagy. The ultrastructural alterations consist of increased motor neurons size, damaged mitochondria, protein accumulation, large cytoplasmic vacuoles placed in perinuclear positions These cellular alterations is reminiscent of ALS, which in turn is characterized by a defective autophagy. Once we confirmed that excitotoxicity recruits autophagy, which remains defective to clear altered proteins and organelles, we provided a pharmacological stimulation of such a pathway in order to ameliorate motor neuron survival. In this experimental conditions, We observe that pharmacological activation of the autophagy machinery is able to counteract kainic acid-mediated motor neuron damage

Sub-cellular motor neuron analysis in a 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. The disease course including onset and severity depends by reduced amounts of the survival motor neuron (SMN) protein. Such a protein is increased when the enzyme glycogen synthase kinase-3beta (GSK3beta) is inhibited. In the present study we used a knockout double transgenic mouse (Smn−/−; SMN1A2G; SMN2) modelling SMAIII to dissect the spinal cord pathology at ultrastructural analysis at prolonged survival time (18 months). We analysed the subcellular structure of spinal cord motor neurons both in baseline conditions and following the administration of a GSK3beta inhibitor. We found that motor neurons increased their diameter confirming our previous light microscopy data. The amount of immunogold labelled SMN particles was dramatically reduced in the whole cell body incuding nucleus and cytoplasm. Remarkably, at nuclear level we could detect marked reduction of the SMN protein with Cajal-like bodies thus mimicking the human disease. In mice receiving long-term lithium administration the level of the SMN protein were massively increase way more than other SMAIII mice and significantly exceeding the levels counted in controls. When compared with control mice administered long-term lithium SMN levels in SMA III mice were overlapping with healthy animals, at large. The effects of lithium on ultrastructural morphology of motor neurons extended to the preservation of mitochondrial compartment which was slightly affected in motor neurons from SMA III mice. These data confirm the essential role of GSK3beta inhibition in increasing the amount of the SMN protein and provide a novel action for an old drug which increases SMN level exceeding any other compound tested so far in this motor neuron pathology. At the same time the beneficial effects of lithium on mitochondrial morphology are confirmed. As an appendix to the present study we wish to mention the ubiquitous nature of these effects which were replicated in non-motor neuron cell lines. Apart from the significance in cell biology this latter observation provide the basis to analyze the effects of a lithium treatment on affected patients using peripheral or skin-derived cell cultures. This work was supported by an educational grant from CUCC

Mascagni’s bicentenary: the “prince of anatomists” in Pisa.

The bicentenary of the death of Paolo Mascagni is the occasion to celebrate an important anatomist who strongly took in the spirit of the Enlightenment. Born in Pomarance (Pisa), Mascagni spent the first part of his life in the University of Siena, where he had many interests, including chemistry, mineralogy, agriculture, and botany. However, thanks to his mentor Pietro Tabarrani, the anatomical research became his main field of studies. He won great fame with the publication of important masterpieces dedicated to the first complete description and illustration of the lymphatic system and became president of the Sienese “Accademia dei Fisiocritici”. After meeting with that success, Mascagni conceived the idea to realize an ambitious dream of the anatomists: a complete and life-sized illustration of the human body. Nevertheless, political events delayed his purposes and upset his life. During the French occupation of Tuscany in 1799, he embraced the case of Jacobinism and had the appointment to manage education and culture in the new government of the city. After a few months, because of the defeat of the French army, he fell into disgrace spending about eight months in prison. Finally, at the end of 1800 Mascagni was rehabilitated and left the insecure and trouble reminiscing Siena to move to Pisa, where he was named professor of anatomy at the university on January 1st 1801. But the troubles were not over: again, Mascagni was politically undesired and accused to be irreligious. The embarrassing situation was overcome with a compromise solution: the role of professor in Pisa was maintained, but Mascagni was obliged to teach anatomy in the Santa Maria Nuova Hospital in Florence. The short stay in Pisa was very prolific. Besides the anatomical lessons, and the interesting meeting with Georges Cuvier, Mascagni was finally concerned with the preparation of the anatomical tables. Some evidences indicate that he lived on the second floor of the historical Agostini’s Palace on the Lungarno and in a laboratory prepared at the last floor of the same building his pupils coloured the anatomical drawings. The masterpiece was published posthumously in 1823-1831 in nine annual issues by the Pisan printer Nicola Capurro on the initiative of Vaccà-Berlinghieri, Barzellotti and Rosini, bearing the right title of “Anatomia Universa”. The 44 tables represent four front and back dissection layers of the life-sized human body and several detailed viscera. The monumental work also included black and white extra tables and a Latin textbook. Art and science are in debt to Mascagni’s contribution to modern anatomy iconography