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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.
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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.
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