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Area 3: Molecular genetics of eukaryotes Role of metalloproteinases and their tissue inhibitors in the regulation of neurogenesis and gliogenesis from neural stem/progenitor cells Emanuele Cacci Department of Biology and Biotechnologies "Charles Darwin" ℡: +39 06 49912206 - 06 4991 2371 - @: [email protected] Neural progenitor (NP) cells continuously generate new neurons and glia in the adult brain. Regulation of adult neurogenesis involves a wide spectrum of signals from the microenvironment, the so-called neurogenic niche. The identification of intrinsic and extrinsic factors implicated in the modulation of neurogenesis is crucial to define strategies to successfully manipulate the neurogenic and gliogenic potential of NPs. Microglial cells are crucial components of the niche and they exert either beneficial or detrimental effects on neurogenesis, depending on their activation state and ranges of mediators produced (Cacci et al., 2005; Cacci et al., 2008; Ajmone Cat et al., 2010). Acute exposure to the prototypical activating agent lipopolysaccharide confers antineurogenic properties upon microglial cells. We have recently shown that docosahexaenoic acid (DHA), a long-chain polyunsatured fatty acid (L-PUFA) with potent immunomodulatory properties, exerts, through the modulation of microglial function, a proneurogenic effect (Ajmone Cat et al., 2011). Among extrinsic factors, matrix metalloproteinases (MMPs) and their natural tissue inhibitors (TIMPs) are emerging as contributors to neurogenesis modulation. In addition, MMPs are multifaceted contributors to neuroinflammation. Particularly MMP-2 and MMP-9 have been extensively studied in the brain, where they are found expressed at low levels under physiological conditions and up-regulated in various pathological conditions. MMPs and TIMPs are expressed in NPs and other brain cell types, including microglia. Noteworthy, MMPs and TIMPs regulate cytokine production in microglia in autocrine/paracrine manner and, vice versa, cytokines appear to be potent regulators of microglial MMP production. We explored whether MMPs modulate NP properties. We found that treatment of NPs with the clinically tested broad-spectrum MMP inhibitor Marimastat profoundly affected NP differentiation fate. Marimastat treatment allowed for enrichment in neuronal cells, inducing NPs to generate a higher percentage of neurons and a lower percentage of astrocytes, possibly affecting NP commitment. Consistently with its pro-neurogenic effect, Marimastat early down-regulated the expression of Notch target genes, such as HES1 and HES5. Interestingly, a potent inhibitor of the Notch signalling (DAPT), increased NP differentiation into neurons with an efficiency comparable to that of Marimastat and its administration in combination with Marimastat increased slightly, although significantly, neuronal differentiation. These data suggest that the early Marimastat-mediated inhibition of Notch signalling supports NP differentiation. 53 Area 3: Molecular genetics of eukaryotes Since MMP-2 and MMP-9 are the main MMPs in the brain, we speculated that the MMP inhibitor effects on NPs were mediated through MMP-2 and/or MMP-9 inhibition. To clarify this point we investigated the expression profile of MMP-2 and MMP-9 in NPs. MMP-2 and MMP-9 profiling on proliferating and differentiating NPs revealed that MMP-9 was not expressed, whereas MMP-2 increased in the medium as proMMP-2 during differentiation, although its active form was not detectable. MMP-2 silencing or administration of recombinant active MMP-2 demonstrated that MMP-2 does not affect NP neuronal differentiation, nor it is involved in Marimastat pro-neurogenic effect. We also found that TIMP-2 is expressed in NPs and increases during late differentiation, mainly as a consequence of astrocyte generation. Endogenous TIMP-2 did not modulate NP neurogenic potential; however the pro-neurogenic action of Marimastat was at least partially mediated by TIMP-2, as demonstrated by silencing experiments. Our data exclude a major involvement of MMP-2 and MMP-9 in the regulation of basal NP differentiation, but highlight the ability of TIMP-2 to act as a key effector of the pro-neurogenic response to an inducing stimulus, such as Marimastat. We also addressed the molecular mechanisms involved in the regulation of NP properties. We focused our attention on the RE1-silencing transcription factor (REST), a master regulator of neuronal genes that can induce neuronal differentiation in NPs. To clarify which genes are transcriptionally repressed by REST we have used an adenovirus carrying the dominant negative of REST (DN:REST), or shRNA against REST. Analysis of the gene expression profile, including the whole spectrum of MMPs and TIMPs were performed by microarray. Combination of microarray and Chip sequencing analysis identified, among many other genes, two MMPs either directly (MMP-24) or indirectly (ADAM19ts) regulated by REST. We propose these MMPs as candidate for regulating NP differentiation, possibly cooperating with TIMP-2 in mediating the pro-neurogenic effects of Marimastat. Publications Ajmone-Cat M, Salvatori M, De Simone R, Mancini M, Biagioni S, Bernardo A, Cacci E, Minghetti L. Docosahexaenoic acid modulates inflammatory and antineurogenic functions of activated microglial cells. J Neurosci Res 2011 Epub. doi: 10.1002/jnr.22783. Research Group Collaborations Stefano Biagioni, professor; Tonino Anelli, research fellow; Pasquale Caramanica, PhD student. Ferdinando Mannello, Gaetana A. Tonti, Università Carlo Bo, Urbino; Luisa Minghetti, Maria Antonietta Ajmone-Cat, Istituto Superiore di Sanità, Roma; Bukley Noel, King’s College, London, UK. 54