Aβ1-42 monomers or oligomers have different effects on autophagy and apoptosis.

Title: Role of JNK in autophagic cell death in the cingulate cortex in a KA-induced rat model of epilepsy.

Authors: A. Vercelli, M. Tropiano, G. Spigolon, C. Bonny*.

Neuroscience Institute Cavalieri Ottolenghi (NICO), Department of Neuroscience, University of Turin, Medical School Regione Gonzole 10, 10043 Orbassano (Italy) and *Division of Molecular Genetics, CHUV, Lausanne (Switzerland)

We have previously shown that the c-Jun N-terminal kinase (JNK) is involved in apoptotic cell death in the rat hippocampus in kainic acid (KA)-induced epileptic seizures and that JNK blockade by a cell penetrating specific inhibitor (D-JNKI-1) partially prevents neuronal cell death. Here we studied the role of autophagy and of apoptosis in neuronal cell death in the cingulate cortex in the same rats, and the effects of JNK inhibition.

Briefly, epileptic seizures were induced by intraperitoneal (i.p.) injection of 15 mg ⁄ kg KA (Tocris Bioscience). One hour after the injection, rats showed the first symptoms (immobility, facial myoclonus and head nodding), and only animals that reached the fourth and fifth stages of the Racine scale were included in the study. To minimize suffering and prevent mortality, 2 h following the onset a single i.p. injection of 4 mg ⁄ kg diazepam (Valium) blocked epileptic seizures within 30 min of administration. D-JNKI1 (DJ 0.3 mg ⁄ kg,) was injected i.p. 2 h following KA injection in the KA-DJ ⁄ 1d and KA-DJ ⁄ 5d groups. Rats were killed one and five days following KA injection, and perfused through the ascending aorta with saline followed by fixative. A series of cryostat section for each animal was Nissl-stained and neuronal counts were performed with StereoInvestigator software (MBF). Other sections were immunoreacted: i) for P-c-Jun as a marker for JNK activity as its elective target associated with microtubule-associated protein-2 (MAP2) (to confirm that JNK was involved in neuronal death); ii) for glial fibrillary acidic protein (GFAP) (to evaluate astrogliosis); iii) for Beclin-1 and LAMP-1 (as markers of autophagic processes); iv) for activated caspase 3 (as marker of apoptosis).

In Nissl-stained sections, an increase in necrotic cell death was found in KA-treated rats, both at 1 and 5 days, and neuronal density decreased by 40% at 1 day and by 70% at 5 days. Following D-JNKI-1 treatment, necrotic profiles almost disappeared at 1 day. Neuronal density was fully prevented by JNK inhibition at 1 day, and showed a 15% decrease at 5 days. C-Jun immunoreactivity colocalized with MAP2-immunoreactivity, showing that JNK was activated in neurons. Markers of autophagy were present in KA-treated rats and were almost absent following its inhibition. On the contrary, neither in Nissl-stained sections nor in activated caspase 3 immunoreacted sections apoptotic markers were found. In parallel, there was a striking astrogliosis in KA-treated rats, partially prevented by JNK inhibition.

Therefore, we show that in the cingulate cortex of rats KA-induced epileptic seizures induce a marked neuronal death, mostly due to necrotic and autophagic phenomena. This death is correlated with JNK activation: in fact, JNK inhibition almost completely prevents neuronal death and appearance of autophagic markers.Supported by MIUR grants to AV.

Allograft transplantation OF ganglionic EMINENCE embryonic STEM CELLs IN a RAT model of HUNTINGTON’S DiSEASE

M. Tropiano, M. Boido, A. Vercelli.

Neuroscience Institute Cavalieri Ottolenghi (NICO), Department of Neuroscience, University of Turin, Regione Gonzole 10, 10043 Orbassano (Italy)

AIMS: Huntington’s disease (HD) is a progressive neurodegenerative disorder caused by huntingtin gene mutation. This leads to a progressive atrophy of the basal ganglia. The neurodegenerative process selectively affects the GABAergic medium sized spiny neurons, that project from the striatum to the globus pallidus and nucleus subthalamicus.

METHODS: To reproduce the striatal neuropathological features of HD, quinolinic acid (QA) represents the most used glutamate agonist in both rodent and primate models of HD. Here, we performed an ipsilateral intrastriatal infusion of 240 nmol QA in Sprague-Dawley male rats (weight 250-300g). This induced an evident rotational behavior of the animal. To assess potential restorative therapies, one week after injury, a group of rats received 100,000 EGFP-positive embryonic (E14) neural cells obtained from the murine medial ganglionic eminence. To reduce the immune response induced by graft, animals received 10mg/kg cyclosporine A. Fifteen days after QA administration, animals were sacrificed and brains dissected.

RESULTS: By Nissl staining, we analyzed brain parenchyma and we measured the ipsilateral ventricle volume by Neurolucida software: its size was markedly increased (about 23%) compared to the contralateral one. Additionally we observed a massive astrogliosis (highlighted by GFAP-immunofluorescence) and a strong microglial activation (IBA-immunofluorescence). Seven days after transplantation, we found a great number of injected surviving undifferentiated cells in the host tissue.

CONCLUSIONS: Although these are preliminary observations, we are currently extending our studies to better evaluate transplanted cell survival/differentiation, and to study their role within striatum and their integration into striatal circuitries.

NICO Seminars:

Laura Sacerdote "Esempi di contributi matematici e statistici allo studio delle neuroscienze" -15/2/2013-

Paolo Malatesta "In vivo modeling hogh grade gliomas" -22/2/2013-

Andrea Graziani "From the gut to the muscle an beyond: new ghrelin functions" -22/3/2013-

Caterina Guiot e Mauro Prato "Therapeutic potential of oxigen loaded nanobubbles in hypoxia-associated deseases -5/4/2013

NICO Lectures; Alvarez-Buylla "Adult Neuronal Stem Cells are specificied in Microdomains" -3/5/2013-

Enzo Terreno "Molecular and cellular imaging: from mice to clinical applications" -12/5/2013-

Tommaso Fellin "Optical dissection of the excitatory circuits controlling recurrent network activity i nthe neocortex" / Serena Bovetti "Scanless two-photon calcium imaging at millisecond temporal resolution in vivo" -24/5/2013

Martina Amanzio "The unaware of executive dysfunction: role of cingulate cortex" -12/7/2013-

NICO Lectures: Elena Cattaneo "Hungtington tra staminali ed evoluzione" -27/9/2013-

Salgado-Araujo "The cerebellar landscape of drug addiction" -18/10/2013-

Gilberto Fisone "Motor and Cognitive dysfunction in Parkinson's disease: modelling and mechanism" -28/10/2013-

Leonardo Chelazzi "Neurocognitive mechanisms of visual selective attention" -8/11/13-

Cinthia Farina "Peripheral and Central processes in multiple sclerosis" -14/2/2014-

Umberto Dianzani "Vaccinazione inversa nella terapia della Sclerosi Multipla" -21/3/2014-

Matteo Caleo "Mechanism of cortical plasticity: from the visual to the motor cortex" - 4/4/2014- 

Fabrizio Pirri  "Nanotools for Neuroscience" -12/6/2014-

"The role of the transcription factor OTX2 in embryonic pluripotent stem cells (ESCS and EPISCS)" A. Simeone, Acting director, Institute of Genetics and Biophysics, Università di Napoli. MBC, Torino. January 16, 2014

"International Stereology Course" S. Geuna, Dipartimento di Scienze cliniche e biologiche, Università di Torino. January 27-28, 2014

"Genetica della Corea di Huntington" E. Cattaneo, Department of Bioscience, Center for stem cells research, Università di Milano. MBC, Torino. February 11, 2014,

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PhD Neuroscience Seminars

Di Francesco Mattia "Skeletal muscle derived stem cells (skMDSCS) differentiate into cardiac pacemaker-like cells: characterization and therapeutic potential" -17/5/2013-

"Awake Brain Surgery: Aspetti clinici e di ricerca -14/6/2013-

Fiorenzo Conti "Novel mechanisms for synaptic homeostasis" -13/2/2014-

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SSST Seminars

Michael Hausser "From Neuronal network to behaviour" -16/5/2013-

Sir Patrick Bateson "Plasticità, Robustezza, Sviluppo ed Evoluzione" -18/11/13-

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Seminari Dipartimento di Matematica

"Workshop on computational neuroscience" -4/10/2013-

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CTSF Seminars

Bruno Quarta "Pensare e Sviluppare un progetto di ricerca: qualche suggerimento pratico" -18/7/2013-

"Essere giovani protagonisti in H2020: Il CV e l'opportunità di finanziamento attraverso la mobilità internazionale" -2/7/2014-

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