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ABSTRACTS/TEMATICHE DI RICERCA DEL CORSO DI DOTTORATO IN BIOLOGIA MOLECOLARE E CELLULARE - XXXI CICLO 1. Bertoni Giovanni Molecular mechanisms of host-pathogen interaction: role of small RNAs in the regulation of virulence in the opportunistic pathogen Pseudomonas aeruginosa /Meccanismi molecolari dell’interazione ospite-patogeno: ruolo dei picoli RNA nella regolazione della virulenza nel patogeno opportunistico Pseudomonas aeruginosa 2. Biffo Stefano Effects of mutant eukaryotic Initiation Factor 6 (eIF6 Ser235Ala ) on tumor progression /Effetti di mutazioni nell’eukaryotic initiation Factor 6 (eIF6 Ser235Ala ) sulla progressione tumorale 3. Bolognesi Martino Acid sensing ion channels (ASICs): validated targets in multiple sclerosis /Canali acido-sensibili (ASICs): target confermati nella sclerosi multipla 4. Briani Federica Development of new tools to fight bacterial infections /Sviluppo di nuovi strumenti per la lotta alle infezioni batteriche 5. Cappelletti Graziella Molecular architecture of neuronal microtubules in health and disease: unravelling the contribution of parkin/Architettura molecolare dei microtubuli neuronali in situazioni fisiologiche e patologiche: determinazione del contributo di parkin 6. Cattaneo Elena Stem cells for studies of Huntington’s Disease/Cellule staminali per lo studio della Corea di Huntington 7. Colombo Lucia Epigenetic regulation of ovule development in changing environmental conditions /Regolazione epigenetica dello sviluppo dell’ovulo in condizioni ambientali variabili 8. Costa Alex Functional characterization of the Arabidopsis thaliana GLR3.7 (glutamate-like receptors) /Caratterizzazione funzionale del recettore del glutammato GLR3.7 di Arabidopsis thaliana 9. Fornara Fabio Heading North! The genetics of rice adaptation to high latitudes /Verso nord! La genetica dell’adattamento del riso alle latitudini elevate 10. Gissi Carmela Evolutionary dynamics of nuclear genes involved in replication and trascription of the mitochondrial genome in Tunicata/Dinamica evolutiva di geni nucleari implicate nella replicazione e trascrizione del genoma itocondriale nei Tunicati 11. Guerrini Luisa Analysis of the molecular mechanisms at the basis of thalidomide-induced limb defects in zebra fish embryos /Analisi dei meccanismi molecolari alla base dei di fettidi sviluppo indotti dalla talidomide negli embrioni di zebra fish 12. Kater Martin Rice Yield Increase by Altering Inflorescence Architecture / Aumento della produzione di riso attraverso la modifica della struttura dell'infiorescenza 13. Lazzaro Federico Role of the ubiquitin ligases Dma1 and Dma2 in the DNA Double Strand Breaks response/Ruolo delle ubiquitina ligasi Dma1 e Dma2 nella risposta cellulare alle rotture a doppio filamento del DNA 14. Mantovani Roberto NF-YA in Embryonic Stem cells/NF-YA nelle cellule staminali embrionali
Transcript

ABSTRACTS/TEMATICHE DI RICERCA DEL CORSO DI DOTTORATO

IN BIOLOGIA MOLECOLARE E CELLULARE - XXXI CICLO

1. Bertoni Giovanni Molecular mechanisms of host-pathogen interaction: role of small RNAs in the regulation of virulence in the opportunistic pathogen Pseudomonas aeruginosa /Meccanismi molecolari dell’interazione ospite-patogeno: ruolo dei picoli RNA nella regolazione della virulenza nel patogeno opportunistico Pseudomonas aeruginosa

2. Biffo Stefano Effects of mutant eukaryotic Initiation Factor 6 (eIF6Ser235Ala) on tumor progression /Effetti di mutazioni nell’eukaryotic initiation Factor 6 (eIF6Ser235Ala) sulla progressione tumorale

3. Bolognesi Martino Acid sensing ion channels (ASICs): validated targets in multiple sclerosis /Canali acido-sensibili (ASICs): target confermati nella sclerosi multipla

4. Briani Federica Development of new tools to fight bacterial infections /Sviluppo di nuovi strumenti per la lotta alle infezioni batteriche

5. Cappelletti Graziella Molecular architecture of neuronal microtubules in health and disease: unravelling the contribution of parkin/Architettura molecolare dei microtubuli neuronali in situazioni fisiologiche e patologiche: determinazione del contributo di parkin

6. Cattaneo Elena Stem cells for studies of Huntington’s Disease/Cellule staminali per lo studio della Corea di Huntington

7. Colombo Lucia Epigenetic regulation of ovule development in changing environmental conditions /Regolazione epigenetica dello sviluppo dell’ovulo in condizioni ambientali variabili

8. Costa Alex Functional characterization of the Arabidopsis thaliana GLR3.7 (glutamate-like receptors) /Caratterizzazione funzionale del recettore del glutammato GLR3.7 di Arabidopsis thaliana

9. Fornara Fabio Heading North! The genetics of rice adaptation to high latitudes

/Verso nord! La genetica dell’adattamento del riso alle latitudini elevate

10. Gissi Carmela Evolutionary dynamics of nuclear genes involved in replication and trascription of the mitochondrial genome in Tunicata/Dinamica evolutiva di geni nucleari implicate nella replicazione e trascrizione del genoma itocondriale nei Tunicati

11. Guerrini Luisa Analysis of the molecular mechanisms at the basis of thalidomide-induced limb defects in zebra fish embryos /Analisi dei meccanismi molecolari alla base dei di fettidi sviluppo indotti dalla talidomide negli embrioni di zebra fish

12. Kater Martin Rice Yield Increase by Altering Inflorescence Architecture / Aumento della produzione di riso attraverso la modifica della struttura dell'infiorescenza

13. Lazzaro Federico Role of the ubiquitin ligases Dma1 and Dma2 in the DNA Double Strand Breaks response/Ruolo delle ubiquitina ligasi Dma1 e Dma2 nella risposta cellulare alle rotture a doppio filamento del DNA

14. Mantovani Roberto NF-YA in Embryonic Stem cells/NF-YA nelle cellule staminali embrionali

15. Messina Graziella Vessel associated progenitor cells in the cell based Therapy of the Cystic Fibrosis lung disease/Progenitori cellulari associati ai vasi nella terapia cellulare della Fibrosi Cistica

16. Moroni Anna The modulation of HCN channels by intracellular molecules/Modulazione dei canali HCN da parte di molecole intracellulari

17. Moroni Anna – Thiel Gerhard Learning by building: constructing a voltage-gated ion channel from individual modular components/Learning by building: assemblaggio di un canale ionico regolato dal voltaggio a partire da component modulari individuali

18. Muzi Falconi Marco Characterization of the role of Exo1 and translesion DNA polymerases in processing UV-damaged DNA/Caratterizzazione del ruolo di Exo1 e delle DNA polimerasi translesione nel processamento del DNA danneggiato da UV

19. Nardini Marco Structural analysis of transcription factor/DNA complexes /Analisi strutturale di complessi tra fattori trascrizionali e DNA

20. Pellicioli Achille Unravelling the role of the SLX4 pathway in preserving genome integrity and preventing cancer developmentRuolo del pathway di SLX4 nel preservare l’integrità del genoma e nella prevenzione dello sviluppo dei tumori

21. Pesaresi Paolo Organelle-Nucleus Communication Elements: An approach to uncover the

molecular details of the retrograde signaling pathway/Comunicazione organello-nucleo: approcci

molecolari per individuare i dettagli della via di segnalazione retrograda

22. Petroni Katia Role of anthocyanin-enriched diet on cardioprotection/Ruolo di una dieta arricchita in antocianine nella cardioprotezione

23. Ricagno Stefano Towards the understanding of light chains cardiotoxicity, a biochemical and

structural investigation/Verso la comprensione della cardiotossicità delle catene leggere, un’analisi biochimica e strutturale

24. Soldà Giulia Identification of genetics and molecular bases of inherited sensorineural hearing loss by whole-exome sequencing/Identificazione delle basi genetiche e molecolari della perdita di udito sensorineurale mediante sequenziamento dell’esoma

25. Tonelli Chiara Adaptomics. Exploring the natural variation of complex traits in plants through QTL and eQTL dissection /Adattomica. Studio della variabilità dei tratti complessi nelle piante tramite identificazione di QTLs ed eQTLs

26. Zuccato Chiara Huntington’s Disease: biological aspects and pathogenesis

/Corea di Huntington: aspetti biologici e patogenesi

Project leader: GIOVANNI BERTONI ([email protected])

Location: Department of Biosciences

RESEARCH PROJECT SUMMARY

Molecular mechanisms of host-pathogen interaction: role of small RNAs in the regulation of virulence in the opportunistic pathogen Pseudomonas aeruginosa

This project will focus on the study of molecular mechanisms of pathogen-host interaction related to lung diseases caused by Pseudomonas aeruginosa that the Infectious Diseases Society of America (IDSA) lists as one of the six most dangerous bacteria, named “ESKAPE” pathogens, because they effectively escape the effects of antibacterial drugs. Lung infections caused by P. aeruginosa usually affect patients, belonging to all age groups, who are immunocompromised, or who have defective mucociliary clearance, previous epithelial injury or foreign body placement. Lung infections by P. aeruginosa can present as a spectrum of clinical entities from a rapidly fatal pneumonia in a neutropenic patient to a multi-decade bronchitis in patients with cystic fibrosis (CF). The connection between CF and P. aeruginosa is very strong. In fact, persistent P. aeruginosa infections have clearly been linked to a more rapid decline of lung function leading to a greater morbidity and mortality in CF patients.

The lifestyle of P. aeruginosa is that of a widespread free-living bacterium that can act as the most versatile bacterial pathogen studied to date. To act as pathogen, P. aeruginosa orchestrates a wide array of virulence traits to interact with host. Its pathogenic strategy is to cope with host nutrient limitation, to manipulate and damage host cells and to exploit an arsenal of defence mechanisms. However, it is clear that P. aeruginosa does not express all its pathogenic functions at all times. Remarkably, the most important aspect of P. aeruginosa versatility is its ability to quickly adapt to a new environment and manage to persist. A waterborne free environment and the airway epithelium represent very different challenges to a bacterium and the ability to modulate gene expression rapidly for adapting to the harsh host conditions requires a complex network of environmental sensors, signal transduction devices, and regulators. Cues such as the shift from room to body temperature, nutrients, pH, iron limitation, and anaerobic conditions of the thick CF mucus all trigger a cascade of events to equip P. aeruginosa to survive in the airways. If necessary, P. aeruginosa can also turn virulence factors off at later stages of infection to prevent immune recognition.

Thus, P. aeruginosa is endowed with a complex regulatory network that dynamically controls the production of virulence factors enabling survival, proliferation/persistence and host damage after airways infection. In other bacterial pathogens, small RNAs (sRNAs), which are akin to eukaryotic microRNAs in their ability to modulate stability and/or translation of multiple target mRNAs, are now recognized as important components of the regulatory networks involved in the pathogen-host interaction. On the contrary, there is a gap of knowledge on the role of sRNAs in the orchestration of the interaction with human airways underlying the broad range of lung diseases caused by P. aeruginosa. This projects aims to fill this gap focusing on three P. aeruginosa sRNAs recently identified and characterized in our lab. These sRNAs were shown to respond to relevant host cues and to take part to the post-transcriptional regulation of key P. aeruginosa virulence traits. Therefore, they are expected to play the role of regulatory mediators of the P. aeruginosa/airways interaction. To unravel this role, specific aims of this project will be: i) the evaluation of how either knock-out and overexpression of these three sRNAs impact P. aeruginosa pathogenicity and host response, both at early interaction stages with airway cell cultured-based infections models, and during lung infection maintenance and development with murine models; ii) the analysis of sRNAs expression in a panel of clinical P. aeruginosa strains isolated from lung infections; iii) the screen for P. aeruginosa virulence functions regulated by these sRNAs through an innovative approach of quantitative proteomics; iv) the study of the molecular interactions between the sRNAs and the novel identified virulence targets.

The achievements of this project have the potential to foster the development of innovative antipseudomonal strategies. In fact, the study of the functional roles of sRNAs in airways/P. aeruginosa interaction can provide the fundamental knowledge for development of next-generation antibiotics that are not expected to select for resistance since their low impact on bacterial fitness per se.

Project leader: Prof. Stefano Biffo ([email protected])

Location: Molecular Histology and Cell Growth Unit, National Institute of Molecular Genetics "Romeo e Enrica

Invernizzi" - INGM, via Francesco Sforza 35, 20122 Milano

RESEARCH PROJECT SUMMARY

Effects of mutant eukaryotic Initiation Factor 6 (eIF6Ser235Ala

) on tumor progression.

Ribosomes are the “machines” for protein synthesis: they are assembled in the nucleolus and later exported to the

cytoplasm, where they are engaged in translation. Regulating ribosome biogenesis and translation is crucial in

tumorigenesis1,2

. eIF6 (eukaryotic Initiation Factor 6) plays an essential role in both processes: it is necessary for the

maturation of 60S ribosomal subunits in the nucleus and it regulates the availability of 60S in the cytoplasm,

controlling active 80S complex formation at the initiation phase of translation. In the context of initiation, the

activation of eIF6 is driven by the RACK1/PKCβ axis. 60S-bound eIF6 keeps the 60S and 40S ribosome subunits

separated and inhibits translation. PKCβII kinase is recruited by the scaffold protein RACK1 and it is able to

phoshporylate eIF6 on Ser235, leading to eIF6 release from the mature 60S. This event allows the 80S complex

assembly, and the initiation of protein synthesis. Mutation of eIF6 in the PKCβII consensus phosphosite Ser235 in Ala

reduces the rate of translation, suggesting a role for this residue in eIF6 functional activity3,4,5,6

. In humans, eIF6 is

highly expressed in different carcinomas and its overexpression is associated with tumor stage7,8

. Furthermore, eIF6

has been identified as one of 21 essential genes amplified in highly proliferative luminal-subtype human breast

cancer9. eIF6 phosphorylation on Ser235 has been reported in several tumor cells. Previous studies on mice revealed a

strong correlation between eIF6 protein levels, its phospho-Ser235-dependent activity and tumorigenesis. In

particular: i) the protective effect from tumor development in conditions of heterozygosity for eIF6 was observed in a

mouse model of Myc-induced lymphomagenesis (Eμ-Myc transgenic mice); ii) primary fibroblasts transduced with

mutant eIF6S235A

show resistance to oncogenic transformation in vitro and reduced growth in vivo10

. These

observations suggest that eIF6 phosphorylation is involved in tumor development and progression.

The significance of the phosphorylation on Ser235 residue is unknown in vivo. To address this issue we are developing

a Conditional KI eIF6S235A mouse model. First, we are generating mice overexpressing the eIF6 mutant in all tissues in

order to understand whether this point mutation is lethal; second, we will cross KI het mice (if KI omozygotes are

lethal) with Eμ-Myc mice to detect the role of eIF6 phosphorylation on Ser235 in tumorigenesis. These studies, in vivo,

could explain the role of eIF6 phosphorylation and the regulation of its activity as a new potential therapeutical target

in tumor development.

1. Ruggero, D. "Translational control in cancer etiology." Cold Spring Harb Perspect Biol 5(2). 2. Silvera, D., S. C. Formenti, et al. "Translational control in cancer." Nat Rev Cancer 10(4): 254-66. 3. Ceci, M., C. Gaviraghi, et al. (2003). "Release of eIF6 (p27BBP) from the 60S subunit allows 80S ribosome

assembly." Nature 426(6966): 579-84. 4. Brina, D., A. Miluzio, et al. "eIF6 anti-association activity is required for ribosome biogenesis, translational

control and tumor progression." Biochim Biophys Acta”, 2014 Sep 22. 5. Brina, D., S. Grosso, et al. "Translational control by 80S formation and 60S availability: the central role of eIF6,

a rate limiting factor in cell cycle progression and tumorigenesis." Cell Cycle 10(20): 3441-6. 6. Gandin, V., A. Miluzio, et al. (2008). "Eukaryotic initiation factor 6 is rate-limiting in translation, growth and

transformation." Nature 455(7213): 684-8. 7. Sanvito, F., F. Vivoli, et al. (2000). "Expression of a highly conserved protein, p27BBP, during the progression

of human colorectal cancer." Cancer Res 60(3): 510-6. 8. Rosso, P., G. Cortesina, et al. (2004). "Overexpression of p27BBP in head and neck carcinomas and their

lymph node metastases." Head Neck 26(5): 408-17. 9. Gatza, M. L., G. O. Silva, et al. "An integrated genomics approach identifies drivers of proliferation in luminal-

subtype human breast cancer." Nat Genet 46(10): 1051-9. 10. Miluzio, A., A. Beugnet, et al. "Impairment of cytoplasmic eIF6 activity restricts lymphomagenesis and tumor

progression without affecting normal growth." Cancer Cell 19(6): 765-75.

Project leader: [MARTINO BOLOGNESI ([email protected])]

Location: [Department of Biosciences, Via Celoria 26, 20133 Milano]

RESEARCH PROJECT SUMMARY

Acid sensing ion channels (ASICs): validated targets in multiple sclerosis

Extracellular acidosis affects several neuroinflammatory/neurodegenerative disorders including Multiple Sclerosis

(MS). In physiological conditions, the extracellular pH levels are maintained at 7.4 through various H+ transporting

mechanisms. However, during inflammation such levels are persistently reduced, causing long-lasting acidosis in large

regions of the central nervous system (CNS).

Acid Sensing Ion Channels (ASICs) are voltage independent proton gated sodium channels expressed by neurons of

the CNS. In mammals, four genes encode at least six different ASIC isoforms, although CNS neurons preferentially

express Asic1a and ASIC2 a and b isoforms. Pharmacological inhibition of ASICs in animal models of MS as well as in

patients elicits some neuroprotective effects, reducing demyelination and axonal damage in animals and restraining

rates of whole-brain atrophy in patients. ASICs inhibition was obtained with amiloride, a diuretic drug that additionally

inhibits epithelial sodium channels (ENaC), the sodium-hydrogen exchanger 1 (NHE1), Na+/Ca

2+ exchanger and

voltage-gated Ca2+

channels.

Thus, the poor ion channel specificity of amiloride may cause some difficulties in the interpretation of the results due

to possible off-target mechanisms acting in parallel with ASICs inhibition.

Considering the crucial role of ASICs in MS, we recently started the structure-based design and synthesis of novel

ASICs inhibitors inspired by known diarylamidines. Among them, diminazene (DA) is the most potent small-molecule

inhibitor of ASIC channels. Our structure-based approach preliminarily led to the synthesis of a small library of

proprietary DA analogs, showing higher inhibitory activity with respect to DA.

We plan to extend our multi-disciplinary investigation on the molecular properties of ASIC channels, with the main

aim to determine the molecular features needed to design potent, isoform-selective, brain-penetrant DA analogs.

Accordingly, we will solve the crystal structure of the extracellular domain of murine ASIC1a and ASIC2a in order to

analyse the interaction with DA-related compounds.

Practical lab activities will be performed mainly at the Department of Biosciences, nevertheless some experiments will

be executed at Ospedale San Raffaele. Such activities, which will be the basis for the targeted studies, but also the

training grounds for the candidate are: recombinant protein production (both in E.Coli and baculovirus systems) and

purification, biophysical studies (e.g. dynamic light scattering, spectroscopies), biochemical assays (e.g. test, on ASIC

channels expressing CHO-K1 cells, the new molecules generated; functional profiling by electrophysiology -multi-

electrode arrays, Patch clamp- of selectivity, toxicity and inhibitory effects exerted by the ASICs blockers on neurons),

protein crystal growth, X-ray diffraction and crystallographic analyses, in silico simulation studies (molecular dynamics,

docking of small molecules). Rationally-driven modification of DA analogues will greatly benefit from the structural

analysis of their interaction with different ASIC channels and in particular with ASIC1a and 2a that are most abundant

in the brain. Currently, only the crystal structure of the chicken ASIC1a homotrimeric channel in different opening

states is available. The crystallization of mammalian ASIC1a and ASIC2a will be helpful to identify the DA binding site(s)

and to start characterizing its inhibition mechanism. Crystallographic analysis together with in silico docking and

molecular dynamics simulations will be instrumental for the rational design of DA analogues, able to bind and inhibit

both isoforms with higher affinity and selectivity.

Project leader: FEDERICA BRIANI ([email protected]) Location: Dept. of Biosciences

RESEARCH PROJECT SUMMARY

Development of new tools to fight bacterial infections

The alarming increase of bacterial resistance to known antibiotics poses a global threat to public health and demands a continuous effort to develop new therapeutic strategies. Particularly worrisome is the paucity of new drugs active against Gram negative bacteria in pharmaceutical companies' development pipelines

1.

We will address this problem through two complementary strategies:

1) the characterization of new drug candidates found in an "intelligent" target-specific screening that we have recently developed. In particular, we will characterize i) the antibacterial activity of the candidate molecules against different bacterial strains; ii) the mechanism of action and the actual molecular target(s). This analysis will be instrumental to drive a future lead optimization process.

2) to explore bacteriophages as anti-Pseudomonas agents. Phage therapy has been applied mainly in Eastern Europe, and only in recent years has garnered the attention of the Western world

2. Bacteriophages may help in the treatment

of acute and chronic P. aeruginosa infections, against which many currently-in-use antibiotics are not effective. We have recently isolated several P. aeruginosa-specific phages from an environmental source. Our project will aim at characterizing such phages and their anti-pseudomonal activity.

1. Payne, D. J.; Gwynn, M. N.; Holmes, D. J.; et al. Drugs for Bad Bugs: Confronting the Challenges of Antibacterial Discovery. Nat. Rev. Drug Discov. 2007, 6, 29–40. 2. Viertel, Ritter, Horz. (2014) Viruses versus bacteria-novel approaches to phage therapy as a tool against multidrug-resistant pathogens. J Antimicrob Chemother. 69(9):2326-36.

Project leader: Graziella Cappelletti ([email protected])

Department of Biosciences, University of Milan

RESEARCH PROJECT SUMMARY

Molecular architecture of neuronal microtubules in health and disease: unravelling the contribution of parkin.

Neurons are a striking example of cells in which microtubules are essential to achieve a high degree of morphological

and functional complexity. Neuronal microtubules display different orientation and dynamics in axons and dendrites,

and interact with a plethora of specific associated proteins. In addition, the incorporation of tubulin isotypes and post-

translational modifications of tubulin are selectively combined and distributed among different subcellular

compartments, thus generating a tubulin code, that might regulate basic as well as higher-order neuronal functions.

The goal of the present project is to investigate if and how parkin, a well-known E3 ubiquitin ligase, contributes to

regulate the molecular architecture of neuronal microtubules in physiological and pathological context. Notably,

parkin mutations are associated to autosomal recessive Parkinson’s disease. Although previous evidence links parkin

deficiency to microtubule destabilization in neurons (Cappelletti G. et al., Biochem Soc Trans, 2015), the interaction of

parkin with microtubules remains a poorly investigated aspect of parkin biology. This project aims to shed light on

whether the effects evoked by parkin in cells are due to the direct interaction of the protein with tubulin/microtubules

by using a set of complementary in vitro and in cell strategies.

In the first part of the work, the PhD fellow will evaluate the impact of wild type and mutated parkin on microtubule

assembly, structure, stability and dynamics in vitro. The second aim is to analyse the interplay between wt/mutated

parkin and microtubule system in primary neuronal cultures from embryo midbrain of Parkin KO mice. Wt, truncated

and point mutated parkin will be overexpressed in primary neurons and their impact on microtubule dynamics and

subsets of post-translationally modified tubulin will be investigated as previously reported (Cartelli et al., J

Neurochem, 2010). Finally, advanced analyses of parkin/tubulin interaction in fixed and live cells will be performed by

two single-molecule imaging techniques (FRET or dSTORM).

If successful, the outcome of this research will lead to a mechanistic insight into the interaction of parkin with

microtubules and to a better understanding of its physiological and pathogenic functions.

Project leader: Elena Cattaneo ([email protected]) Location: Department of Biosciences and INGM, Via Sforza 35 Milano

RESEARCH PROJECT SUMMARY

Stem cells for studies of Huntington’s Disease

Huntington’s disease (HD) is an autosomal-dominant, progressive neurodegenerative disorder that usually onsets in midlife. It is characterized by motor, cognitive, and psychiatric symptoms. Once symptomatic, patients are rapidly disabled and require increasing multidisciplinary care. Current treatments fail to produce significant relief to the patients therefore HD results as a tremendous burden for medical, social, and family resources. The symptoms and the progression of HD can be linked to its neuropathology, which is characterized by loss of specific neuronal populations in many brain regions. Several studies have shown that medium spiny neurons (MSN) are severely affected. MSN are inhibitory projection neurons and are the primary source of striatal projections. The laboratory is actively involved in international research programs aiming at deriving specific and robust differentiation protocols for the generation of MSNs. Most recently we have developed a protocol to obtain such neurons from human embryonic stem (hES) or from induced pluripotent stem cells (hiPS) using a defined in vitro neural induction system and quantitative assessment tools (Delli Carri et al., Development 2013). Moreover, we are using stem cells to study the normal fuction of huntingtin along evolution (Lo Sardo et al., Nat Neuroci 2012). In this project, by the combination of multiple approaches we aim to develop strategies to further improve the recovery and quality of fully functional human MSNs from hES/hiPS cells with the goal of using them to study huntingtin function in human cells and for future transplantation studies in HD. We will further explore the use of additional morphogenes/active molecules to increase frequency of authentic MSNs during differentiation and apply these to doxycycline-inducible hES lines that over-express critical combinations of transcription factors (TFs) known to be important for striatal specification and differentiation. Moreover, we plan to use recently developed genome editing tools to modify ad hoc endogenous gene expression by either insertion of fluorescent reporters to use as hallmarks for differentiation or to modulate and/or knock out gene of interest for gain/loss-of-function studies. Quality of the neurons obtained at the end of the differentiation protocol will be verified by a convergence of features such as expression of neuronal markers as well as neurochemical and bioelectrical properties. We will also use Cas9-mediated knock-out strategies to inactivate the HD gene and produce new lines expressing huntingtin protein with mutation in critical aminoacidic sites. In conclusion this project aims at (i) developing new hES cell lines over-expressing critical striatal TFs; (ii) characterizing the identity of the neural progenitors and post-mitotic neurons derived from differentiation studies; (iii) generating and characterizing genome edited-PS cell lines and HD hiPS lines; (iv) using the cells to study huntingtin biology and for transplantation studies.

Project leader: Lucia Colombo

Location: Dipartimento di Bioscienze

RESEARCH PROJECT SUMMARY

Epigenetic regulation of ovule development in changing environmental conditions

The understanding of the genetic control of organ formation and differentiation are one of the most challenging aspect of the developmental biology. In this project we combine the study of a very fascinating process with its possible application for improving plant yield.

Ovule formation is the central process in plant reproduction. Ovules are formed from meristematic tissue, the placenta, by the action and interaction of plant hormones and transcriptional regulators. Within the ovule double fertilization occurs and this organ subsequently develops to form the seed. Despite it has been reported that abiotic stresses, such as high temperature and drought, limit seed yield more than any other factors, there are surprisingly large gaps in the basic understanding how environmental conditions impact on ovule development.

Experimental data strongly support a role for epigenetic factors in regulating the expression of the gene set that control ovule development. Indeed, mutations in genes encoding histone modification factors have resulted in altered ovule development compare to wild type. In this regard, the study of the dynamics of epigenetic regulation of key genes involved in ovule development may well be an appropriate starting point for biotechnological modification of the stress response of the reproductive system and consequently be of considerable agricultural importance. This

project proposal integrates an innovative combination of single cell techniques and ‘-omics technologies’ to address fundamental unanswered questions directed to elucidating the complex genetic and epigenetic networks controlling ovule development under both standard conditions and in response to environmental stress

Project leader: Alex Costa e-mail: [email protected]

Location: Department of Biosciences

RESEARCH PROJECT SUMMARY

Functional characterization of the Arabidopsis thaliana GLR3.7 (glutamate-like receptors) Ionotropic glutamate receptors (iGluRs or GLRs) are ligand-gated cation channels that mediate

neurotransmission in animal nervous systems. Homologous proteins in plants (20 members in Arabidopsis, Lacombe et al., 2001 Science) have been implicated in root development, ion transport and several metabolic and signaling pathways. A recent work demonstrates the involvement of two members of the plant GLR family, GLR3.3 and GLR3.6, in long-distance wound signaling (Mousavi et al., 2013 Nature). Moreover, another member of this family, the GLR1.2 specifically expressed in pollen, is crucial for the generation of the Ca

2+ tip gradient and thus ultimately for the proper

pollen tube growth and fertility (Michard et al., 2011, Science). Analyses of animal iGluRs by heterologous expression system such as Xenopus oocytes have shown that these channels have varying conductances for Na

+, K

+ and Ca

2+. They

were therefore classified as non-selective cation channels (NSCC). Studies conducted thus far suggest that also plant iGluRs are NSCCs. Importantly, in a recent study by Vincill et al. (2012) it has been shown that in HEK cells the GLR3.4 of Arabidopsis thaliana is an amino acid gated channel selective to Ca

2+ and capable of inducing cytosolic Ca

2+ peaks in

response to asparagine, glycine or serine. A second work using a different heterologous expression system demonstrated that another Arabidopsis iGluR homolog, AtGLR1.4, functions as a ligand-gated, nonselective, Ca

2+-

permeable cation channel that responds to an even broader range of amino acids, none of which are agonists of animal iGluRs (Tapken et al., 2013 Sci Signal). Altogether these results point out Arabidopsis iGluRs as potential candidates to be Ca

2+ permeable channels involved in different aspects of plant physiology.

In our lab we are currently interested in the Arabidopsis GLR3.7 isoform. In order to study the GLR3.7 role in Ca

2+ signaling events or generally define its functional role we have followed a reverse genetic approach (through the

isolation of a T-DNA insertion lines) and we combined it with a molecular imaging approach (by using the genetically encoded Ca

2+ probe Cameleon). Importantly, we have found that the glr3.7 mutant shows altered amino acids-

induced Ca2+

transients in root tip cells, demonstrating its role in mediating plasma membrane Ca2+

fluxes. Moreover, the glr3.7 mutant shows a strong root hair phenotype with lack of the tip Ca

2+ gradient and consequent loss of cell

polarity. The typical tip Ca2+

oscillations are also affected in the glr3.7 as revealed by the Ca2+

imaging analysis carried out with the SPIM-FRET setup (Costa et al., 2013 PlosOne). Thanks to an established collaboration with several labs (Lisbon, Muenster and Montpellier) we are now trying to define if the GLR3.7 does really function as a channel. Preliminary data support the hypothesis that the GLR3.7 is not a functional channel per se but it could act as a silent regulatory subunit by its participating to the formation of heteromeric channels with other members of the iGluR family (e.g. GLR3.3 and GLR3.6). Last but not least, we have clear indications that GLR3.7 may interact with and be phosophorylated by the CIPK26 kinase, which activity is dependent by the CBL1/9 Ca

2+ sensors, hence allowing to

hypothesize a feedback control of the heteromeric channel activity by Ca2+

. Strictly, the PhD student will carry out a project aimed at define the functional role of the GLR3.7 in the root

hair growth by pursuing three main objectives: 1) identification of the GLR3.7 partners (e.g. GLR3.3 and GLR3.6) in the formation of a functional heteromeric channel; 2) understanding of the role of phosphorylation in the regulation of the channel complex; 3) identification of the amino acids residues, targets of CIPK26/CBL1 phosphorylation, in order to pursue a phosphomimetic in planta analysis.

Project leader: Fabio Fornara ([email protected], http://users.unimi.it/fornaralab/)

Location: Department of Biosciences and greenhouses of the Botanical Garden Città Studi

RESEARCH PROJECT SUMMARY

Heading North! The genetics of rice adaptation to high latitudes

Rice is a tropical plant that has been artificially adapted to grow in several temperate areas of the world, including northern Italy. Flowering (also called “heading” in cereals) starts when specific proteins, encoded by Heading Date 3a (Hd3a) and Rice Flowering Locus T1 (RFT1) are produced in the leaves and then move to the shoot apical meristem. Transcription of Hd3a and RFT1 is under control of a plethora of genes, most of them acting as transcriptional repressors. Varieties adapted to higher latitudes frequently harbour mutations in such repressors, thus releasing expression of Hd3a and RFT1 and allowing flowering to take place at the appropriate time of the year. Among the floral repressors that have been identified to date, Heading date 1 (Hd1), Ghd8 (OsNF-YB11) and PRR37 encode the strongest repressors. Unpublished data from our laboratory indicate that dimers formed between Ghd8 and OsNF-YC subunits can recruit Hd1 or PRR37 in a trimeric NF-Y complex in yeast. These data suggest the existence of an NF-Y complex in rice where interchangeable subunits act to repress flowering under LD. The PhD student will join this project and address the genetics of the complex in rice plants by assaying the interactions between putative components of the complex and generating mutants in specific sub domains of each protein using novel genome-editing technologies. We will address the following questions: - Could more CCT domain proteins interact with the Ghd8 - OsNF-YC dimer? Besides PRR37 and Hd1, other CCT domain proteins, including PRR73, PRR59 and PRR95 represent suitable candidates to test this hypothesis. - Is the effect of mutations in PRR genes additive on flowering? Higher order mutant plants will be generated, in which Hd1 and PRR proteins will be mutated simultaneously in different combinations. This work will greatly improve our understanding of the regulatory mechanisms of flowering in rice and possibly demonstrate that adaptation of rice to European environments has been enabled by artificial selection acting on NF-Y complex components.

Project leader: Carmela Gissi, [email protected]

Location: Dipartimento di Bioscienze, Università degli Studi di Milano

RESEARCH PROJECT SUMMARY

Evolutionary dynamics of nuclear genes involved in replication and transcription of the mitochondrial genome in Tunicata

The mitochondrial genome (mtDNA) of Metazoa is the molecule of choice in animal phylogenetic

reconstructions but is also regarded as a model system for studying the processes governing the evolution

of an entire genome (Gissi et al. 2008). As peculiarity, this genome is characterized by co-evolution with the

nuclear genome, in fact the biogenesis and maintenance of mitochondria depends on tightly regulated

interactions between the nuclear and mt genetic systems (Garesse and Vallejo 2001; Cannino et al. 2007).

For example, the mtDNA of metazoans encodes only for some subunits of the respiratory complexes and

for few components of the mt protein synthesis machinery, while the overwhelming majority of mt

proteins are encoded by the nucleus, including those involved in replication, transcription and repair of the

mtDNA as well as in the formation of the mt nucleoid. In general, we can expect that these nuclear-

encoded mt proteins will evolve in different way depending on the details of the mtDNA organization and

functionality in the different taxa, and then on the overall mtDNA evolutionary trends. At present, the

mtDNA has been completely sequenced in more than 2000 metazoan species belonging to the most diverse

phyla, from sponges to mammals. Interestingly, among Chordata, the mtDNA of vertebrates shows low

evolutionary rate and almost frozen structural and compositional features, while the mtDNA of Tunicata,

the sister taxon of vertebrates, is characterized by fast nucleotide substitution rate, hypervariability of the

gene order (with genes nevertheless all located on the same strand), apparent absence of a major

regulatory region for transcription and replication, and strong variability of base composition and

asymmetry (Gissi et al. 2010; Rubinstein et al. 2013).

The aim of this project is to study the evolutionary dynamics of the above-mentioned nuclear-

encoded gene categories of tunicates, in comparisons to representative of vertebrates and amphioxus (the

only representative of Cephalochordata). These data will help to predict which proteins/protein-regions are

mainly responsible of the differences observed between Tunicata, Vertebrata and Cephalochordata in the

mt genome organization and functionality. This study will allow the candidate to participate to new

genome and transcriptome projects of tunicate species.

References

Cannino G, Di Liegro CM, Rinaldi AM (2007) Nuclear-mitochondrial interaction. Mitochondrion. 7: 359-366. Epub 2007 Aug 2002.

Garesse R, Vallejo CG (2001) Animal mitochondrial biogenesis and function: a regulatory cross-talk between two genomes. Gene. 263: 1-16.

Gissi C, Iannelli F, Pesole G (2008) Evolution of the mitochondrial genome of Metazoa as exemplified by comparison of congeneric species. Heredity 101: 301-320

Gissi C, Pesole G, Mastrototaro F, Iannelli F, Guida V, Griggio F (2010) Hypervariability of ascidian mitochondrial gene order: exposing the myth of deuterostome organelle genome stability. Mol Biol Evol. 27: 211-215.

Rubinstein ND, Feldstein T, Shenkar N, Botero-Castro F, Griggio F, Mastrototaro F, Delsuc F, Douzery EJ, Gissi C, Huchon D (2013) Deep sequencing of mixed total DNA without barcodes allows efficient assembly of highly plastic ascidian mitochondrial genomes. Genome Biol Evol. 5: 1185-1199

Project leader: Luisa Guerrini

Location: Department of Biosciences

RESEARCH PROJECT SUMMARY

Analysis of the molecular mechanisms at the basis of thalidomide-induced limb defects in zebra fish embryos

Severe developmental malformations were detected in human feti in the 1950s, when women used the anti nausea and sedative drug thalidomide in the first trimester of pregnancy (1, 2).

The striking similarities between the phenotypic abnormalities of babies born from mothers exposed to thalidomide during pregnancy and patients affected by syndromes associated to mutations in the p63 gene (3), prompted us to verify whether p63 could be a molecular target for the thalidomide drug. Our results indicate that the Np63 and

Np63 proteins, but not Np63 and p53, are degraded through the proteasome upon thalidomide exposure in several human cell lines expressing either the endogenous or the transfected p63 proteins. By serial deletions analysis, we identified serine 383 in p63 as necessary for thalidomide mediated degradation of p63, since mutation of S383 to alanine abolished thalidomide action on p63. GSK3 kinase is responsible for S383 phosphorylation since the use of a specific GSK3 inhibitor also abolished thalidomide action on p63 (4 and Lopardo et al. in preparation).

Thalidomide has been recently been shown to have teratogenic effects also in the zebra fish (5); we have evidences that thalidomide modulated p63 protein levels also in vivo in developing Zebra fish embryos, with concomitant teratogenic limb defects.

The aim of the project will be to dissect the molecular pathways altered by thalidomide treatment at the basis of the observed limb defects, using zebrafish as animal model.

Project leader: Prof. Martin Kater ([email protected]) Location: Dipartimento di Bioscienze

RESEARCH PROJECT SUMMARY

Rice Yield Increase by Altering Inflorescence Architecture

Rice is one of the most important crops for feeding the world. Rice yield increase over the next 25 years is of enormous importance to meet the demand of a rapidly growing world population. This rise in yield will have to be sustainable and without increase of cultivated land. This project focuses on improving rice yield through altering the inflorescence structure. The inflorescence or panicle has a main axis on which primary branches develop. From the primary branches the secondary branches arise from which the spikelets are born. The number of branches varies

between rice varieties and by that the number of seeds that develop on a panicle. Panicle branching is therefore an important character for rice crop improvement.

Very little is known about the genetic control of panicle branching. Recently, Yoshida et al. (2013) identified a regulator of rice panicle branching called TAWAWA1, however the molecular mechanism is still far from understood. We have in the frame of the French-Italian EVOREPRICE project isolated by laser micro-dissection microscopy isolated the apical, primary and secondary branch meristems of rice inflorescences. This material has been used for RNA extraction and next generation sequencing analysis. The transcriptomes have been analysed and key genes putatively involved in regulating branching have been identified (publication in preparation). These will be subjected to functional analysis using molecular and genetic tools. For making mutants we will use the latest genome editing approaches.

Literature:

- Yoshida et al. (2013). TAWAWA1, a regulator of rice inflorescence architecture, functions through the suppression of

meristem phase transition. Proc Natl Acad Sci U S A 110, 767-772.

- Mantegazza, et al (2014). Gene Coexpression Patterns During Early Development of the Native Arabidopsis

Reproductive Meristem: Novel candidate developmental regulators and patterns of functional redundancy. Plant J.

doi: 10.1111/tpj.12585. [Epub ahead of print].

Project leader: LAZZARO FEDERICO ([email protected])

Location: Dipartimento di Bioscienze

RESEARCH PROJECT SUMMARY

Role of the ubiquitin ligases Dma1 and Dma2 in the DNA Double Strand Breaks response. Double strand breaks (DSBs) are among the most cytotoxic form of DNA damage since they can give rise to

chromosomal rearrangements which are hallmarks of cancer cells. To counteract such challenge, eukaryotic cells developed a complex response globally known as DNA damage response (DDR) which comprehends the activation of the DNA damage checkpoint cascade and of specific DSBs repair pathways. In this project we want to identify and characterize two possible new actors of the DSBs DDR: the two FHA-RING E3 ubiquitin ligases Dma1 and Dma2, orthologes of metazoan RNF8.

Preliminary results show that the loss of the two paralogs makes cells sensitive to the DSBs-inducing agent bleocin, but the sensitivity is not due to a DNA damage checkpoint defect. Rather, in the absence of the two ubiquitin ligases the checkpoint cascade is hyperactivated.

In this project we will analyze and characterize the mechanisms involved in DSB repair and we will try to identify the target involved in this new pathway.

Chahwan R, Gravel S, Matsusaka T, Jackson SP. Dma/RNF8 proteins are evolutionarily conserved E3 ubiquitin ligases

that target septins. Cell Cycle. 2013 Mar 15;12(6):1000-8. doi: 10.4161/cc.23947

Ciccia A, Elledge SJ. The DNA damage response: making it safe to play with knives. Mol Cell. 2010 Oct 22;40(2):179-

204. doi: 10.1016/j.molcel.2010.09.019. Review. PubMed PMID: 20965415

Fraschini R, Bilotta D, Lucchini G, Piatti S. Functional characterization of Dma1 and Dma2, the budding yeast homologues of Schizosaccharomyces pombe Dma1 and human Chfr. Mol Biol Cell. 2004 Aug;15(8):3796-810

Granata M, Panigada D, Galati E, Lazzaro F, Pellicioli A, Plevani P, Muzi-Falconi M. To trim or not to trim: progression

and control of DSB end resection. Cell Cycle. 2013 Jun 15;12(12):1848-60. doi: 10.4161/cc.25042

Kolas NK, Chapman JR, Nakada S, Ylanko J, Chahwan R, Sweeney FD, Panier S, Mendez M, Wildenhain J, Thomson TM, Pelletier L, Jackson SP, Durocher D. Orchestration of the DNA-damage response by the RNF8 ubiquitin ligase. Science. 2007 Dec 7;318(5856):1637-40. Epub 2007 Nov 15

Raspelli E, Cassani C, Lucchini G, Fraschini R. Budding yeast Dma1 and Dma2 participate in regulation of Swe1 levels

and localization. Mol Biol Cell. 2011 Jul 1;22(13):2185-97.

Project leader: Roberto Mantovani ([email protected])

Location: Dipartimento di Bioscienze

RESEARCH PROJECT SUMMARY

NF-YA in Embryonic Stem cells.

Regenerative medicine has taken the center stage in medical sciences since the discovery of embryonic stem cells (ES).

ES cells express “stemness” genes, many of which code for transcription factors. NF-Y is a trimeric CCAAT-binding

factor, composed of NF-YA, NF-YB and NF-YC (1). The CCAAT box is often present in promoters of genes overexpressed

in different types of cancer, and it is believed that NF-Y plays an important role in mediating high levels of expression

(2). We showed that one of the splicing isoform of NF-YA plays a crucial role in maintaining the mouse ES stemness

potential (2, 3). The mechanisms are related to the capacity to connect with the circuitry of stem cells transcription

factors and their regulated genes. In general, two splicing isoforms -long and short- are produced from the NF-YA

locus, and their expression is apparently quite regulated. They differ in 28 AA in the Q-rich transcriptional activation

domain. Somewhat surprisingly, it has recently emerged that the two isoforms have different, often opposing roles in

important cellular processes.

The aim of the project will be to investigate the mechanistic role of NF-YA isoforms in ES cells. The expansion of the

stem cells compartment(s) has been associated to NF-YAs, and we have preliminary data indicating that NF-YAl is

involved in early differentiation decisions. The exact role of NF-Ys in stemness is most likely related to pioneering of

important ES regulators (Sox2, Nanog, KLF4) to their genomic site. In general, we have a wealth of data on the major

NF-Y partners on the genome, derived from ENCODE 85). We will evaluate the interplay of ES TFs by ChIP-Seq and by

analysis of profilings in mouse ES cells in which the isoform is overexpressed. In parallel, we will inactivate NF-Y

isoforms in human iPS, which are equivalent to human ES cells. The expected results are a better understanding of the

molecular mechanisms that lead to differentiation or the expansion of the stem cells pools, as well as the interplay

between NF-Y and other TFs on common targets.

1) Nardini M., Gnesutta N., Donati G., Gatta R, Forni C., Fossati A., Vonrhein C., Moras D., Romier C., Bolognesi M., Mantovani R. Cell, 152, 132-143 (2013).

2) Dolfini D. and Mantovani R. Targeting the Y/CCAAT box in cancer: YB-1 or NF-Y? Cell Death and Differentiation, 20, 676-685 (2013).

3) Dolfini D, Minuzzo M, Pavesi G, Mantovani R. Stem Cells. 30, 2450-9 (2012).

4) Oldfield AJ, Yang P, Conway AE, Cinghu S, Freudenberg JM, Yellaboina S, Jothi R. Mol Cell. 55:708-22 (2014).

5) Fleming JD, Pavesi G, Benatti P, Imbriano C, Mantovani R, Struhl K. Genome Res. 23:1195-209. (2013)

Project leader: Prof. Graziella Messina ([email protected])

Location: Department of Biosciences

RESEARCH PROJECT SUMMARY

Vessel associated progenitor cells in the cell based Therapy of the Cystic Fibrosis lung disease

Cystic Fibrosis (CF) is the most common autosomal recessive genetic disorder in Caucasian population. It is caused by mutations in the gene that encodes for the cystic fibrosis transmembrane conductance regulator (CFTR) protein, which functions mainly as a cAMP-dependent chloride ion channel and is primarily expressed in the apical membrane of secretory epithelia. Lung disease, characterized by airway obstruction, inflammation and chronic bacterial infection is the leading cause of death. At variance with some pharmacological approaches, no one efficacious gene and cell-based therapy have been proved to date. We are developing a cell therapy based on transplantation of mouse mesoangioblasts (mMABs) in mouse models of CF. MABs are vessels-associated progenitor cells that we demonstrated to be able to cross the vessel wall upon intra-arterial injection, undergo skeletal muscle

differentiation and rescue skeletal muscle dystrophy in mice and dogs. Our preliminary results show that MABs engraft lung, tracheal and intestinal epithelium up to 2 months in healthy, wt mice. In F508del CFTR mice this engraftment persists up to 6 months from a single mMAB injection and reduces local inflammation. As additional evidences supporting the use of mMABs in CF, we observed that they express, in vitro, the typical epithelial marker E-cadherin and functional CFTR channel; moreover MAB injection leads to a partial rescue in the expression of the fully glycosylated CFTR protein in F508del CFTR mice up to 4 months from their single transplantation. Finally, mMABs can also engraft nasal epithelium of KOCftr

tm1UNC mice, restoring some CFTR-dependent chloride secretions up to 4 months

and express the epithelial marker E-cadherin, once engrafted in the respiratory epithelia of transplanted mice, thus making these cells eligible for a cell-based therapy in CF. In collaboration with the European Institute of Cystic Fibrosis in Milan and the University of Yale, New Haven (CT), this project will develop by testing the therapeutic properties of mMABs following systemic transplantation in F508del CFTR

and KOCftr

tm1UNC mouse models.. Functional rescue of CFTR together with a reduction of the hyper-inflammatory

response and rescue of the different signs of the disease will be evaluated. Moreover, MAB capability to differentiate in different epithelial cells and /or to participate to the epithelial stem cell niche and the mechanisms driving MAB towards an epithelial fate will be also studied. References: Vezzali et al., Mesoangioblasts - vessel associated progenitor cells- engraft epithelial tissues and express functional CFTR channel: prospects and promise for a cell therapy for Cystic Fibrosis. Manuscript in preparation Sueblinvong, V. and Weiss, D. J. Stem cells and cell therapy approaches in lung biology and diseases. Transl Res 156, 188-205, 2010 Project leader: ANNA MORONI [email protected],

Location: Dept. of Biosciences

RESEARCH PROJECT SUMMARY

The modulation of HCN channels by intracellular molecules

Hyperpolarization-activated cyclic nucleotide-gated (HCN) cation channels are encoded by the HCN1-4 gene family and have four subtypes that are variably distributed in heart and brain [1]. These channels are activated upon hyperpolarization of membrane potential and conduct an inward, excitatory current Ih in the nervous system. Ih controls relevant cellular functions, such as rhythmic firing, dendritic excitability and presynaptic neurotransmitter release [2]. HCN channels are the targets of a number of cellular signals that finely regulate their trafficking and electrical activity (gating). In this project, we will study how the intracellular signals, especially interacting proteins such as TRIP8b [3] and small ligands such as cyclic nucleotide and cyclic dinucleotides [4], regulate function and surface expression of HCN channels, and subsequently provide a clue to study dysregulation of HCN channels in pathological conditions. [1] Santoro B, Tibbs GR. The HCN gene family: molecular basis of the hyperpolarization-activated pacemaker channels. Ann N Y Acad Sci. 1999 Apr 30;868:741-64. [2] Biel M, Wahl-Schott C, Michalakis S, Zong X. Hyperpolarization-activated cation channels: from genes to function. Physiol Rev. 2009 Jul;89(3):847-85. [3] Saponaro A, Pauleta SR, Cantini F, Matzapetakis M, Hammann C, Donadoni C, Hu L, Thiel G, Banci L, Santoro B, Moroni A. Structural basis for the mutual antagonism of cAMP and TRIP8b in regulating HCN channel function. Proc Natl Acad Sci U S A. 2014 Oct 7;111(40):14577-82. [4] Lolicato M, Bucchi A, Arrigoni C, Zucca S, Nardini M, Schroeder I, Simmons K, Aquila M, DiFrancesco D, Bolognesi M, Schwede F, Kashin D, Fishwick CW, Johnson AP, Thiel G, Moroni A. Cyclic dinucleotides bind the C-linker of HCN4 to control channel cAMP responsiveness. Nat Chem Biol. 2014 Jun;10(6):457-62.

Project leaders: ANNA MORONI [email protected],

GERHARD THIEL [email protected]

Location: Department of Biosciences, Department of Biology, University of Milan, Italy

Department of Biology, TU-Darmstadt, Darmstadt, Germany

RESEARCH PROJECT SUMMARY

Learning by building: constructing a voltage-gated ion channel from individual modular components

Voltage dependent K+ channels (Kv) are modular proteins; they are composed of a channel pore, a voltage sensor

donain and a linker, which connects the latter two parts. In spite of detailed structural information available on these

important proteins, there are still many open questions on the mechanism by which the voltage sensor movement

results in an opening of the pore. Here we propose a new approach for tackling the basic mechanism of

electromechanically coupling between pore and voltage sensor in Kv channels. It is based on the successful

construction of a synthetic Kv channel from unrelated elements, a voltage independent miniature K+ channel pore and

the voltage sensor of the phosphatase from the tunicate Ciona intestinalis. By means of a “learning-by-building”

approach, we will construct and characterize new synthetic channels by systematically varying the structural and/or

functional characteristics of the pore, the voltage sensor and the linker respectively. The directed molecular design of

the synthetic channels will be guided by theoretical studies; coarse-grained modeling approaches will be employed in

the context of experimental data to understand the electromechanical coupling between the voltage sensor and the

pore as well as the fold requirements of the linker, which connects these elements for channel gating. Taken together,

experimental and theoretical studies will elucidate the “engineerability” of the linker and the mechano-structural

coupling between sensor and gates. The native combination of these defined elements in synthetic channels will

provide information on i) the impact of the linker in electromechanical coupling ii) the importance of the sensor

domain for inward versus outward rectification iii) the impact of the voltage sensor on gating mechanisms, which are

inherent in the pore.

Project leader: Marco Muzi Falconi ([email protected])

Location: [Department of BioScience]

RESEARCH PROJECT SUMMARY

Characterization of the role of EXO1 and Y-family DNA polymerases in processing UV-damaged DNA

Genomes of all living organisms are under constant attack from endogenous and exogenous agents that may

lead to genome instability.UV light produces, mainly, photoproducts on DNA named CPDs and 6-4PPs responsible for the pathological effects of sun light. In healthy individuals, Nucleotide Excision Repair (NER) removes DNA helix distorting lesions, such as UV-induced damages. Mutations in NER genes cause the onset of human pathologies like: XP, CS and TTD. The principal symptom common to all these diseases is the strong sensitivity to UV. Surveillance mechanisms, known as DNA damage checkpoints, are activated when cells sense the damage, leading to block or delay in cell cycle progression, ensuring removal of the lesions

1. DNA damage checkpoints are activated after UV

irradiation in all phases of the cell cycle. On the contrary, NER yeast mutant strains and human fibroblasts derived from patients do not activate the checkpoint

2–4. Recently, we and other demonstrated the involvement of EXO1

nuclease and Polk in processing a subset of lesions outside S-phase. Indeed, NER and EXO1 defective cells are not able to properly activate DNA damage response

5,6.

Polk belongs to the Y-family of DNA polymerases, which include also Poli, Polh , a set of specialized

enzymes that can bypass DNA lesions on the template strand due to their enlarged active site (TLS). Polh very

efficient in bypassing across CPDs, indeed, XP-V patients are mutated in the corresponding gene. Poli efficiently

accommodates 6-4PPs and Polk downstream of UV-induced bulky lesions 7. Moreover, Polk is involved in the repair of ICLs, that consist of a covalent bond between the two DNA filaments

8.

One of the first reactions in ICL removal requires NER, which cleaves one filament upstream and downstream of the

ICL. This evidence suggests that Polκ, Poli and Polh could play a role in the repair synthesis step in the EXO1-

dependent pathway downstream of NER in response to UV.

Previously, we proposed that EXO1 role after UV might be to process regions of DNA containing “difficult” lesions that

may correspond to Closely Opposing Lesions (COLs), close to each other on the opposite strand. Intriguingly, COLs can

be seen as lesions that resemble ICL from a structural point of view.

Therefore, it would be of great interest establishing if and where, at a genome-wide level, COLs are present and

detailing at a molecular level how are they processed and repaired.

1. Harper, J. W. & Elledge, S. J. The DNA damage response: ten years after. Mol Cell 28, 739–745 (2007).

2. Marti, T. M., Hefner, E., Feeney, L., Natale, V. & Cleaver, J. E. H2AX phosphorylation within the G1 phase after UV irradiation depends on nucleotide excision repair and not DNA double-strand breaks. Proc Natl Acad Sci U S A 103, 9891–9896 (2006).

3. Giannattasio, M., Lazzaro, F., Longhese, M. P., Plevani, P. & Muzi-Falconi, M. Physical and functional interactions between nucleotide excision repair and DNA damage checkpoint. EMBO J 23, 429–438 (2004).

4. Marini, F. et al. DNA nucleotide excision repair-dependent signaling to checkpoint activation. Proc Natl Acad Sci U S A 103, 17325–17330 (2006).

5. Giannattasio, M. et al. Exo1 competes with repair synthesis, converts NER intermediates to long ssDNA gaps, and promotes checkpoint activation. Mol Cell 40, 50–62 (2010).

6. Sertic, S. et al. Human exonuclease 1 connects nucleotide excision repair (NER) processing with checkpoint activation in response to UV irradiation. Proc. Natl. Acad. Sci. U. S. A. 108, 13647–13652 (2011).

7. Sale, J. E., Lehmann, A. R. & Woodgate, R. Y-family DNA polymerases and their role in tolerance of cellular DNA damage. Nat. Publ. Gr. 13, 141–152 (2012).

8. Williams, H. L., Gottesman, M. E. & Gautier, J. Replication-Independent Repair of DNA Interstrand Crosslinks. Mol. Cell 47, 140–147 (2012).

Project leader: Marco Nardini ([email protected])

Location: Department of Biosciences

RESEARCH PROJECT SUMMARY

Structural analysis of transcription factor/DNA complexes

One of the key issues in biology is how the genetic information is transferred to biological functions. Binding

of transcription factors (TFs) to discrete sequences in gene promoters and enhancers, is crucial to the process, which

needs to interface with chromatin, whose fundamental unit is the nucleosome, formed by core histones wrapped by

146 bp of DNA. Binding of TFs entails the recruitment of histone modifying and chromatin remodeling machines, thus

helping to define the chromatin status (“euchromatin” vs “heterochromatin”). TFs fall in essentially two categories:

(i) “pioneer” TFs, with intrinsic chromatin association capacity; (ii) "activating" TFs, binding to a favorable chromatin

landscape pre-set by pioneers.

In this context, the present PhD project focus on NF-Y, a histone-like TF that binds and activates the CCAAT

box [1], and on MYC, a proto-oncogene that binds the E-box (5’-CACGTG-3’), whose altered expression transforms

cells [2]. NF-Y and MYC are deemed to be paradigms of pioneer and activating factors, respectively, and indeed they

were shown to interact directly. Furthermore, the availability of the 3D structures for both NF-Y and MYC [3, 4] makes

both TFs potential targets for development of anti-cancer drugs. The present PhD project will be carried out in the

Nardini’s (structural biology) lab as a continuation of an ongoing research project that led to the successful

determination of the X-ray structure of the NF-Y in complex with its target DNA [3]. The project will pursue analyses of

NF-Y in complex with DNA containing multiple CCAAT boxes, and the NF-Y/MYC/MAX-DNA complex, both by X-ray

crystallography and solution scattering methods (Small/Wide Angle X-ray Scattering, SAXS/WAXS). The SAXS/WAXS

experiments, being performed on solution samples, are always practicable, provided that the sample is sufficiently

pure and monodisperse. For the X-ray crystallography approach, the Nardini lab experience in growing protein-DNA

complex crystals will be crucial for the achievement of this step: several E-box and CCAAT-containing fragments will be

designed and tested in an approach that proved successful for the NF-Y/CCAAT complex [3].

The 3D structure of the NF-Y/DNA complex, will also allow to search rationally for potential inhibitors. As a

part of the PhD project, inhibitor/NF-Y docking simulations will be carried out to screen virtual chemical libraries of

low molecular weight compounds, searching for inhibitors of the NF-Y quaternary assembly and of its DNA-binding

capacity. X-ray crystallography will be then applied to characterize the structure of the complexes between NF-Y and

the best inhibitors. A similar approach will be eventually applied to interfere with the interactions between NF-Y and

MYC/MAX. Potential inhibitors selected through the in silico approaches will be cross-validated in vitro through

Thermal shift and electrophoretic mobility shift assay (EMSA) experiments, and in cells by ChIPs.

[1] Dolfini D, Gatta R, Mantovani R. Crit Rev Biochem Mol Biol. (2012) 47: 29-49.

[2] Prendergast GC, Lawe D, and Ziff EB. Cell (1991) 65: 395-407.

[3] Nardini M, Gnesutta N, Donati G, Gatta R et al. Cell (2013) 152: 132-143.

[4] Nair SK, Burley SK. Cell (2003) 112: 193-205.

Project leader: Achille Pellicioli ([email protected])

Location: Dipartimento di Bioscienze, Via Celoria 26 20133 Milano

RESEARCH PROJECT SUMMARY

Unravelling the role of the SLX4 pathway in preserving genome integrity and preventing cancer development

Chromosomes maintenance and stability are essential goals for all the organisms in order to transfer the correct

genetic information to the progeny. Double Strand Breaks (DSBs) are deleterious lesions that can be a serious threat

for the cell. In fact, defects in DSBs repair leads to chromosomes instability and tumorigenesis, and DSBs are

frequently accumulated in several genetic disorders and senescent cells. These lesions are processed by several

nucleases, leading to the formation of a 3’ end single strand DNA (ssDNA) filament, through a finely regulated process

called DSB resection. This process can be divided in an initial step orchestrated by the Mre11 complex together with

CtIP/Sae2 and a later, processive, step dependent on Exo1 and Bloom helicase/Sgs1. Mutations in the corresponding

human orthologs of Mre11, Sae2 and Sgs1 lead to severe disorders (ataxia telangiectasia-like, Seckel, Jawad and

Bloom syndromes), characterized by genomic instability and cancer predisposition (Jackson, S. P. & Bartek, J. (2009)

Nature 461, 1071-1078).

DSB resection allows the recruitment onto the lesion of both the checkpoint and the recombination factors. In our

laboratory it has been demonstrated that the checkpoint factor Rad9 (53BP1 in human) binds near the lesion and

counteracts the resection process, limiting the formation of ssDNA (Lazzaro, F. et al. (2008) EMBO J 27, 1502-1512 and

Ferrari, M. et al. (2015) PLoS Genet 11: e1004928). A similar inhibitory role in DSB resection has been recently shown

for 53BP1 in human cells. Interestingly, down-regulation of 53BP1 restores homologous recombination and DSB repair

in cells with mutations in the breast cancer gene BRCA1 (Zimmermann, M. et al. (2013) Science 339: 700-704).

Therefore, the studying of the regulation of the DSB resection is fundamental to understand why defects in this

process lead to chromosome rearrangements and cancer. Importantly, we recently found that the Slx4 protein

counteracts Rad9 binding near a DSB in yeast, promoting both the inactivation of the DNA damage checkpoint and the

DSB resection and repair (Manuscript in preparation).

SLX4 is functionally highly conserved from yeast to humans and participates in many different DNA repair pathways

such as resolving replication fork blocks, homologous recombination and inter-strand crosslink repair. The main

function of SLX4 is to act as a scaffold for several nucleases involved in different steps of DSB repair. Furthermore,

SLX4 was recently shown to be a component of the Fanconi anemia pathway (FA), a rare recessive disorder

characterized by chromosomal instability, increased cancer susceptibility, developmental of abnormalities, bone

marrow failure, and childhood cancers (Kim (2014) Molecules and Cells doi.org/10.14348/molcells.2014.0118).

The PhD student will investigate the role of the Slx4 pathway in DBS repair and in the maintenance of genomic

stability in different human cell lines, already available in our laboratory. Particularly useful to characterize SLX4 role in

checkpoint activation and inactivation and DSB repair will be cell lines derived from FANCP patients. He/She will also

study the involvement of SLX4 and 53BP1 in the repair of DNA damages caused by camptothecin, a topoisomerase-

aborting agent, which is highly used in chemotherapy. Furthermore, He/She will set up specific screening to identify

novel genes involved in DSB repair.

Project leader: Paolo Pesaresi, [email protected]

Location: Department of Biosciences

RESEARCH PROJECT SUMMARY

Organelle-Nucleus Communication Elements: An approach to uncover the molecular details of the retrograde

signaling pathway

More than 3 billion years ago biology developed the capacity to efficiently capture solar energy and use it to

power the synthesis of organic molecules. This photosynthetic process set into motion an unprecedented explosion in

biological activity allowing life to prosper and diversify on an enormous scale. At the heart of the reaction is the

chloroplast where light energy and CO2 are converted into the organic molecules of biomass. Chloroplast originates

from an endosymbiontic cyanobacterium, that has retained a minimal genetic machinery, and genes for a small

number of polypeptides. In consequence, the majority of chloroplast proteins are encoded in the nucleus, translated

in the cytosol and imported into the organelle, whereas the few resident genes mainly encode components of the

gene expression system itself and of the photosynthetic apparatus. Hence, chloroplast ribosomes or photosystems are

mosaics-multiprotein complexes made up of nucleus encoded and plastid encoded subunits. This implies the existence

of mechanisms that coordinate gene expression in the different compartments, with the aim to optimize the

photosynthesis performance and plant production under the different physiological and environmental conditions.

Despite the existence of these regulatory mechanisms is well established, only a limited number of factors involved in

the signalling pathways are known, nowadays.

During this PhD project, we wish to explore in depth the molecular mechanisms responsible of the “Retrograde”

signaling pathway, focusing on two major aspects: (i) the chloroplast sensors able to perceive the developmental and

physiological state of the organelle; (ii) the transcription factors responsible to re-orchestrate the nuclear gene

expression according to the functional and developmental needs of the chloroplast.

References:

[1] Zybailov B, et al. PLoS One (2008) 3: e1994.

[2] Pesaresi P., Schneider A., Kleine T., Leister D. Curr Opin Plant Biol (2007) 10: 600-606.

[3] Larkin R.M., Alonso J.M., Ecker J.R., Chory J. Science (2003) 299: 902-906.

[4] Mochizuki N., Brusslan J.A., Larkin R., Nagatani A., Chory J., Proc Natl Acad Sci U S A (2001) 98:

2053-2058.

[5] Susek R.E., Ausubel F.M., Chory J. Cell (1993) 74: 787-799.

[6] Strand A., Asami T., Alonso J., Ecker J.R., Chory J. Nature (2003) 421: 79-83.

[7] Mochizuki N., Tanaka R., Tanaka A., Masuda T., Nagatani A. Proc Natl Acad Sci U S A (2008) 105:

15184-15189.

Project leader: KATIA PETRONI ([email protected])

Location: Department of BioSciences

RESEARCH PROJECT SUMMARY

Role of anthocyanin-enriched diet on cardioprotection

Dietary flavonoids have received considerable attention since epidemiological studies have suggested that

regular consumption of flavonoid-rich foods or beverages is associated with a decreased risk of cardiovascular

mortality [1–3], attributed primarily to their antioxidant properties and by modulating cell signaling and metabolic

pathways. Among the different classes of flavonoids, anthocyanins are the most recognized, visible members, that

contribute to the cardioprotection. In these last years, recent studies have suggested that dietary flavonoids, and

more specifically regular anthocyanin consumption, induce a state of myocardial resistance evidenced by a reduced

infarct size following regional ischemia and reperfusion [4] that is related, at least in part, to an improvement in the

antioxidant defenses of the heart (i.e. cardiac glutathione). Moreover, there are increasing evidences that seem to

confirm that many biological effects of anthocyanins are related not only to their antioxidant properties but also to

their ability to modulate mammalian cell signaling pathways. For instance, recent studies in rats have shown that an

anthocyanin-rich diet modulate the metabolism of (n-3) PUFA and to induce a marked increase in plasma EPA and

DHA, fatty acids known to be protective against heart disease complication [5,6].

Aim of this research proposal is to study the cardioprotective effects that an anthocyanin-enriched diet has on

the myocardial muscle and also its role in the prevention of drug cardiotoxicity such as in the case of many antitumor

drugs. With this aim, we will investigate as a dietary strategy whether using functional foods, as anthocyanin-rich corn,

can have muscle protective properties and can reduce the incidence and prognosis of myopathies [6-7].

The project will be divided in three different tasks, including i) the role of dietary anthocyanins from corn in the

prevention of cardiotoxicity induced by chemotherapic agents, ii), to investigate the effects of dietary anthocyanins on

specific microRNAs involved in cardiac regeneration and aging, iii) to establish the molecular mechanism underpinning

the cardioprotective action of anthocyanins in murine cardiomyocytes.

With these activities, we expect to contribute to the understanding of how and why anthocyanins contribute to

promote cardioprotection.

References [1] Lancet 342:1007-11, 1993.

[2] BMJ 312: 478-81, 1996

[3] Am J Clin Nutr 85:895-909, 2007.

[4] FEBS J 273:2077-2099, 2006.

[5] J Nutr 138:747-752, 2008.

[6] J Nutr 141:37-41, 2011.

[7] Lancet 374:1849-56, 2009.

Project leader: [Stefano Ricagno ([email protected])]

Location: [Department of Biosciences]

RESEARCH PROJECT SUMMARY

Towards the understanding of light chains cardiotoxicity, a biochemical and structural investigation

Light chain amyloidosis (AL) is caused by the presence and aggregation of monoclonal immunoglobulin light chains (LC) prone to misfolding. AL is the most common systemic amyloidosis with an incidence of 10 patients / million per year. Approximately 75% of the patients suffers from heart impairment, which is the leading cause of death in this disease. The biochemical basis of LC cardiotoxicity is not known, making it impossible to develop drug therapies blocking tissue injury. This project aims to clarify the molecular determinants underlying LC cardiotoxicity, by a comparative study of cardiotoxic LC (CT-LC) and non-toxic LC (NT-LC). A set of CT-LC and NT-LC will be expressed, purified and crystallized; the structural analysis of CT-LC and of NT-LC using X-ray crystallography will be conducted aiming to pinpoint the specific structural characteristics of the LC, particularly at the hypervariable regions, which are specific and different in each LC. Another key issue is protein dynamics and fold flexibility, which are often at the base of protein aggregation and protein toxicity. Protein crystallography provides only partial information on dynamics therefore a second structural technique will be employed.

The same set of LCs will be produced as isotopically labeled material and in collaboration with the solid state NMR European facility in Lyon (France) such LCs will be analyzed to evaluate the bulk dynamics and flexibility of CT-LC versus NT-LC.

This investigation is expected to clarify the molecular basis of AL proteotoxicity leading the way towards a rational drug design against LC cardiotoxic aggregation.

Project leader: Giulia Soldà ([email protected])

Location: Department of Biomedical Sciences, Humanitas University, via Manzoni 113, 20089 Rozzano, Milano, Italy

RESEARCH PROJECT SUMMARY

Identification of genetics and molecular bases of inherited sensorineural hearing loss by whole-exome sequencing Inherited nonsyndromic sensorineural hearing loss (NSHL) shows an extremely high genetic heterogeneity, with

more than 70 genes already identified, and many others still to be discovered (http://hereditaryhearingloss.org). We are currently applying whole-exome sequencing (WES) as a cost- and time-effective strategy to search for

pathogenic variants underlying deafness. Indeed, this technique has already proven to be helpful for the discovery of novel genes/mutations responsible for NSHL [1]. Twelve NSHL families, with a clear recessive (10 families) or

dominant (2 families) inheritance pattern and at least two affected individuals, have been already selected for WES; additional families are being recruited. The WES of the first 31 patients has already been performed [2].

The proposed PhD project will involve a combination of both in-silico and wet-lab approaches in order to: 1) Develop data analysis pipelines to efficiently detect and prioritize candidate variants; 2) Functionally characterize by in-vitro and in-vivo studies novel genes/mutations. The data analysis will include, among others, the implementation of the detection of splicing mutations, indels,

and structural variants. Putative pathogenic mutations identified by WES will be tested to evaluate their segregation with the disease within the probands’ families and their recurrence in sporadic and familial NSHL cases. In this frame, the availability of a large non-syndromic deafness series (about 1300 individuals) of patients/families will be a key resource in the validation step, to screen for the identified mutations and to search for additional genetic defects in the candidate genes pointed out by WES. An in-house database of all variants identified by WES in a wide (>3000) cohort of Italian subjects is also available to the study, to identify population-specific polymorphisms.

We are currently analyzing the putative pathogenic role of novel variations in two candidate NSHL-causing gene, both at the mRNA and at the protein level, by expression experiments in eukaryotic cell lines. In addition, the function of these newly identified genes in the auditory system is being tested in zebrafish, by adopting the CRISPR-Cas technology to selectively and stably inactivate the gene of interest [3], thus allowing a life-long analysis of its roles in ear development and homeostasis. Similar approaches will be adopted for the characterization of additional genes/mutations derived by WES data analysis. References:

1. Vona B, Nanda I, Hofrichter MA, Shehata-Dieler W, Haaf T. Non-syndromic hearing loss gene identification: A brief history and glimpse into the future. Mol Cell Probes. 2015, in press. doi:10.1016/j.mcp.2015.03.008.

2. Robusto M, Fang M, Asselta R, Castorina P, Previtali SC, Caccia S, Benzoni E, De Cristofaro R, Yu C, Cesarani A, Liu X, Li W, Primignani P, Ambrosetti U, Xu X, Duga S, Soldà G. The expanding spectrum of PRPS1-associated phenotypes: three novel mutations segregating with X-linked hearing loss and mild peripheral neuropathy. Eur J Hum Genet. 2015;23:766-73.

3. Hwang WY, Fu Y, Reyon D, Maeder ML, Tsai SQ, Sander JD, Peterson RT, Yeh JR, Joung JK. 2013. Efficient genome editing in zebrafish using a CRISPR-Cas system. Nat Biotechnol 31:227-9.

Project leader: [Chiara Tonelli ([email protected])

Location: Dept. Biosciences

RESEARCH PROJECT SUMMARY

Adaptomics. Exploring the natural variation of complex traits in plants through QTL and eQTL dissection

Plants respond to environmental changes by modifying their developmental program, including flowering time and inflorescence architecture. The genetic basis of these adaptive responses is complex, although the florigen genes, encoding a class of short and long range signaling proteins, play a major role in these processes. Florigen genes have been implicated in the control of different aspects of plant vegetative development, including growth regulation and guard cell movements/transpiration. Thus, variations in florigen accumulation could account for the diverse modes of developmental reprograming under drought stress observed in plants. The question arises as to how florigen genes are regulated, temporally and spatially under varying growth conditions. Here we will use Maize (and in part Arabidopsis) to comprehensively relate different developmental adaptations (i.e. via putative effects on growth or on the duration of the vegetative phase) to patterns of florigen gene accumulation in maize germplasm.

In short day (SD) plants, namely tropical maize lines, the florigen gene ZCN8 is upregulated under SD but not under long day (LD). Conversely, in temperate maize, ZCN8 displays diurnal variations, independent of photoperiod. We have screened a panel of Maize lines (about 320) for different developmental and physiological traits including flowering and growth under two watering conditions (i.e. normal watering and reduced watering). A dense genotyping of these lines is being made available to us in the form of SNPs (250k) or Genotyping by Sequencing (GBS) data. Combining this information will allow identification of QTLs of the studied phenotypic variables. A second aspect of this project relates to the mapping of the loci controlling the different patterns of ZCN8 accumulation in this Maize population. This would be a standard QTL analysis, but performed on ZCN8 transcript amount. Comparison with the two dataset will identify co-locations of eQTLs of ZCN8 with QTLs of flowering time, leaf growth, biomass, control of transpiration and water use efficiency.

Project leader: Chiara Zuccato ([email protected]) Location: Department of Biosciences and INGM, Via Sforza 35 Milano

RESEARCH PROJECT SUMMARY

Huntington’s Disease: biological aspects and pathogenesis

The Huntington's disease gene, encoding for huntingtin (HTT) protein, can be traced back through 800 million year of evolution to before the protostome-deuterostome divergence. HTT appears to have originally exerted non-neuronal functions and only later, during deuterostome evolution, acquired more specific activities that are important for central nervous system (CNS) formation and function (Zuccato et al., Physiol Rev, 2010). The HD gene contains a polymorphic tri-nucleotide CAG repeat that is translated into polyglutamine amino acid (polyQ) residues in HTT. When this polyQ stretch, at the 18 aminoacid (aa) position of the protein, expands to over 39 residues, HD occurs, a fatal, genetically dominant, neurodegenerative disease. We are currently investigating the biology of the HD gene and our aim is to re-construct the history of HTT along evolution. Here, we will also explore the relevance of different molecular and cellular dysfunctions previously identified in the lab in order to identify those that significantly contribute to HD pathogenesis. In particular we will focus on reduced transcription of genes controlled by REST/NRSF factor, including BDNF (Zuccato et al., The Journal of Neuroscience, 2007; Conforti et al., Gene Therapy, 2012) and reduced synthesis of cholesterol in the HD brain (Valenza et al., The Journal of Neuroscience, 2005; Leoni et al., Brain 2008; Valenza et al., The Journal of Neuroscience, 2010, Valenza et al., Cell death and differentiation 2015). More recently, we have started investigating a new target (ADAM10/NCadherin) that has emerged from our evolutionary study (Lo Sardo and Zuccato et al., Nat Neurosci. 2012), which is critical for excitatory synaptic circuitries affected in HD.


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