Ongoing PhD Research Projects

Started 2014

Started 2013

Started 2012

Started 2010

Started 2014

PhD Students are required to outline their research project in the course of their first year. Thus some research projects abstracts for students enrolled on 2014 are not yet available while the abstracts already available here below are subject to little changes.

Cinzia Caterino
Functional in vivo studies of novel aging-related genes expressed in the stem cell niche
Supervisor: Alessandro Cellerino
Abstract:
Renata Cerna
Long-lasting analgesic effect of anti-TrkA and anti-NGF antibodies in neuropathic pain

 

Supervisors: Antonino Cattaneo, Mara D’Onofrio (at EBRI Rome)
Abstract:

Chronic pain (CP) is a major unmet medical need and more effective safe drugs are badly needed. Among the most common forms of CP is neuropathic pain (NP) associated with nerve injury in the peripheral or central nervous system. There is no effective treatment for these highly prevalent disabling disease states and current treatments (NSAIDs and opioids) cause serious side-effects.

NGF is a key player in the onset and progression of CP syndromes. Consistently, genetic mutations in genes encoding NGF or its receptor TrkA cause severe congenital insensitivity to pain in patients. Inhibition of this target system could therefore lead to a new class of painkillers. Therapeutic anti-NGF antibodies have been clinically tested in patients, demonstrating a remarkably effective analgesia in patients suffering from osteoarthritis and low back pain. However, safety concerns, stemming from those trials, need to be addressed and understood, to fully exploit the therapeutic analgesic potential of this target system.

The PAINCAGE Project investigates the role of the NGF system in pain mechanisms focusing at different levels of pain transmission and perception, from peripheral sensory terminals to the brain.

In our laboratory we work with a model of neuropathic pain induced in mice by chronic constriction injury.

We perform a transcriptome profiling of anti-TrkA and anti-NGF antibodies effects in different regions of pain processing pathways (dorsal root ganglia, spinal cord, anterior cingulate cortex) with a focus on molecular mechanisms that might be involved in long-lasting analgesic effects of both antibodies.

Marco Fantini
Intracellular antibody selection against α-synuclein
Supervisors: Antonino Cattaneo, Simonetta Lisi
Abstract:
Bruno Enrique Pinto
Brain aging and depolarizing GABA
Supervisor: Laura Cancedda (IIT Genova)
Abstract:
Lucia Rota
Molecular mechanisms of neurodegeneration in α-synucleinopathies: α-synuclein and its aggregation
Supervisor: Emanuela Colla
Abstract:
  • Cell model (in vitro): We are developing a cell line inducible for α-synuclein, which we will characterize investigating post-translational modifications and structure of α-synuclein through Western blot, Dot blot and Immunofluorescence analyses. For this purpose, we will also develop two novel biosensors to monitor α-synuclein aggregation in living cell: the former exploit CFP and YFP generating a FRET signal, the latter exploit the SPLIT GFP strategy generating fluorescent emission. Both biosensors will be targeted in the cytoplasm and in the endoplasmic reticulum (ER), thought to be critical in the initial formation of α-synuclein aggregates from previous data. Once characterized the cell model and selected a new colony without basal induction as a proper control, we will evaluate the effect of α-synuclein aggregates in terms of cytotoxicity and ER stress, which we expect to be limited by the treatment with Salubrinol, known inhibitor of oxidative stress in the ER.
  • Animal model (in vivo): We will characterize the intestine motility in A53T mice (animal model for α-synucleinopathies already available and used in our laboratory) with behavioral tests, before the onset of the parkinson-like phenotype. We will perform Immunohistochemistry and Western blot analyses in the same mice, to investigate the presence of α-synuclein aggregates at the level of the intestine. As for the in vitro model, we will treat our animals with Salubrinol so to evaluate its effect on the formation of aggregates and thus on the intestine motility. The uncover of the pathological phenotype already at the peripheral level should give a good timing for preventing its spreading at the central level.
Giacomo Siano
In vivo study of Tau spreading in zebrafish and mouse models by Conformational Sensitive Tau sensor
Supervisor: Antonino Cattaneo, Cristina Di Primio
Abstract:
Elena Tantillo
Human and mouse glioma: Transcriptomic and electrophysiological changes in peritumoral cortical areas
Supervisor:
Abstract:

Started 2013

Francesco Gobbo
RNA-mediated subcellular delivery of optogenetic probes in neurons
Supervisor: Antonino Cattaneo
Abstract:

Neurons are highly polarized cells with locally distributed computation, which is paralleled by asymmetric protein distribution. RNA transport and local expression play a major role in shaping neuron patterning. How this is achieved in neurons, however, remains largely unknown, making the research field prolific and very dynamic. The aim of the project is to select and characterize non-coding RNA sequences that mediate the transport and regulate the competence for translation of the mature transcript.

The main goals are (i) the definition of subcellular patterns by local translation, and (ii) confine the expression of proteins to activated synapses. The latter mirrors the concept of “synaptic tagging”, thus directly addressing the problem of synapse potentiation and its role in neuron activity and in memory formation and recall. We routinely employ primary neuron cultures, and have developed a dual reporter system for direct imaging of transcripts and their protein product. Currently, we are evaluating the performance of dendritic and axonal targeting elements derived from prototypical neural transcripts (e.g. CaMKIIa, MAP2, IMPA1) and their strength in driving out-of-soma translation. In parallel, we are using different combinations of physiological and synthetic RNA sequences in both 5’ and 3’UTRs to restrict protein expression to specific synapses in an activity-dependent way.

Debora Napoli
Experience-dependent DNA methylation regulates plasticity in the developing visual cortex
Supervisor:
Abstract:
Marco Terrigno
Study of cortical lamination and pattering using embryonic stem cell derived cortical neurons
Supervisor:
Abstract:

Started 2012

Caterina Rizzi
Role of Nerve Growth Factor in microglia cells: Relevance for Alzheimer Disease
Supervisors: Antonino Cattaneo, Simona Capsoni
Abstract:

Alterations in the physiological actions of the neurotrophin Nerve Growth Factor (NGF) in the CNS and in the regulation of its complex homeostasis have been supposed to be involved in Alzheimer’s disease (AD). Indeed, our laboratory demonstrated that the selective neutralization of mature NGF in the adult brain of the AD11 mouse model leads to a progressive neurodegeneration reminiscent of AD. The AD11 mouse model shows an early dysregulation of the expression of mRNAs related to neuroinflammation.

Microglia cells are the principal mediators in neuroinflammatory states, and we tested the hypothesis that NGF might be involved in regulating the physiological actions of these cells. We focused our attention on the effects of NGF in the primary microglia. We demonstrated the presence of NGF receptors TrkA and p75NTR and their signaling.

Microglia cells are known to phagocyte beta-amyloid peptides. We found that NGF significantly increases the ability of primary microglia to phagocyte soluble and aggregated beta amyloid42. Through microArray analysis, we also observed the modulation of genes involved in abeta degradation. This matter will be further investigated in future research.

We conclude that NGF plays a relevant role in modulating the physiological state of microglia cells.

Giovanna Testa
Role of NGF and TrkA mutations in hereditary sensory and autonomic neuropathies.
Supervisors: Antonino Cattaneo, Simona Capsoni
Abstract:

The Hereditary Sensory and Autonomic Neuropathies (HSAN) IV and V are rare life−threatening autosomal recessive disorders characterized by a complete lack of pain sensation. HSAN IV is clinically characterized by total lack of reactions to painful stimuli leading to self−mutilation, burn injuries, multiple fractures, and neuropathic joints; it is caused by mutations in the TrkA gene. HSAN V causes a selective congenital loss of pain and temperature sensation leading to painless fractures, bone necrosis, osteochondritis, and neuropathic joint destructions; it is associated to mutations in the NGF gene. Thus, both syndromes are related to impairment in the NGF neurotrophin signaling. It is noteworthy that while HSAN IV patients suffer from severe mental  retardation, HSAN V patients are cognitively normal.

The aim of my project is to study the mechanisms at the basis of the differentially altered NGF signalling in these two diseases. To this purpose, I have contributed to generate two knock-in mouse lines in which the murine NGF or TrkA coding sequences are replaced by the corresponding human coding sequences, in their mutated or wild-type forms.

I am currently characterizing the phenotype of these transgenic mice, by using a combination of molecular, neuroanatomical and behavioral analyses, to elucidate the pathogenetic mechanisms at the basis of HSAN IV and V.

Started 2010

Nicola Maria Carucci
NGF and Astrocytes in neurodegenerative disease models 
Supervisors: Antonino Cattaneo; Simona Capsoni
Abstract:

Astrocytes release NGF and also display TrkA and p75NTR NGF receptors, but the role of the NGF ligand-receptor system on astrocytes has not been extensively investigated. To investigate the impact of interfering with NGF bioactivity and NGF/proNGF equilibrium in astrocyte welfare, we analyzed these cells in AD11 anti-NGF mice (Ruberti et al., 2000), which express antiNGF antibodies selectively binding mature NGF with respect to proNGF, and in mice over-expressing an uncleavable form of proNGF (Tiveron et al., 2013). We show that deprivation of the mature form of NGF in AD11 mice or the over-expression of proNGF induces, in an age-dependent manner, early and marked changes in astrocytic morphology, as determined by glial fibrillary acid protein (GFAP) and S100 immunohistochemistry, followed by confocal microscopy and reconstruction of astrocyte structure, to quantitatively evaluate their morphology. The observed phenotype can be defined as asthenic astrocytes, with few long processes and a shrinkage of the soma.

The alterations of astrocyte morphology observed in vivo in anti NGF AD11 or in TgproNGF#72 mice were reproduced by culturing wild type mouse astrocytes in vitro with mAb αD11 anti NGF antibodies, at concentrations when both NGF and proNGF are blocked, but not at lower anti NGF antibody concentrations, when only mature NGF is bound.

The administration of NGF to cultured astrocytes, from wild type mice, does not cause a significant TrkA phosphorylation or changes in morphology but, surprisingly, the neutralization of NGF with mAb αD11 anti-NGF causes a strong and fast paradoxical phosphorylation of TrkA receptors. Phosphorylated TrkA receptors are located inside the cell, while the membrane TrkA receptors do not appear to be involved in this phenomenon. This anti NGF-induced phosphorylation is followed by Plcγ activation and causes a robust change in mRNA transcriptome of astrocytes at 8, 24 and 48 hours after αD11 administration and, later, by changes in cell morphology. The fast response of astrocytes to NGF depletion (but not to NGF itself) is also observed by a strong induction of calcium waves. We assume that the paradoxical activation of TrkA and the ensuing dramatic morphological alteration of astrocytes determined by anti NGF antibodies is due to the interruption of an autocrine or paracrine ligand-receptor loop. We are now employing four different ER-localized intracellular antibodies in order to dissect the role of TrkA, p75NTR, NGF and proNGF in the pTrkA paradoxical activation observed inside the cell. Thus, we show that the astrocyte welfare is extremely sensitive to the overall homeostasis of the NGF system. Thus, in normal conditions, astrocytes are surprisingly deaf to NGF. On the other hand, we posit that astrocytes can sense the NGF/proNGF balance levels outside the cell, in a very sensitive way and transduce the information with an unexpected and paradoxical fast TrkA activation response. The functional consequences of this homeostatic response to the rupture of the NGF/proNGF equilibrium, in responding to injuries in the brain, remain to be ascertained.