< Back to previous page
Project
The functional role of MMP-3 in brain plasticity in response to visual deficits.
The brain is able to change its functional and anatomical organization in response
to environmental changes and this ability is termed neuroplasticity. The visual
cortex, the area in the brain that receives visual input, is undoubtedly one of the
best and most studied areas of the brain for understanding cortical plasticity and
development. However, the molecular mechanisms governing cortical plasticity
are stillelusive. Matrix metalloproteinases (MMPs) are Zn2+-dependent
endopeptidases considered to be essential for normal brain development and
neuroplasticity by modulating extracellular matrix proteins, receptors, adhesion
molecules, growth factors and cytoskeletal proteins. Specifically, MMP-3 has
recently been implicated in synaptic plasticity, hippocampus-dependent learning
and neuronal development and migration in the cerebellum. However, the
function of this enzyme in the neocortexis understudied. Therefore, we
explored the phenotypical characteristics of the neuronal architecture and
the capacity for experience-dependent cortical plasticity in the visual cortex
of adult MMP-3-deficient (MMP-3-/-) mice. GolgiCox stainings revealed a
significant reduction in apical dendritic length, spine length and an increased
numberof apical obliques for layer V pyramidal neurons in the visual cortex of
adult MMP-3-/- mice compared to wildtype (WT) animals. To assess the effect
of MMP-3 deficiency on cortical plasticity, we monocularly enucleated (ME)
adult MMP-3-/- mice and analyzed the reactivation of the contralateral visual
cortex seven weeks post-ME. In contrast to previous results in C57Bl/6J adult
mice, activity remained confined to the binocular zone and did not expand
into the monocular regions indicative for an aberrant open-eye potentiation.
Permanent hypoactivity in the monocular cortex lateral and medial to V1 also
indicated a lack of cross-modal plasticity. These observations demonstrate
that genetic inactivation of MMP-3 has profound effects on the structural
integrity and plasticity response of the visual cortex of adult mice. To further
assess the molecular changes governing the MMP-3-/- cortical phenotype, we
performed Western analysis on different neurofilament protein (NF) subunits
and collapsin response mediator proteins (CRMP). The former are markers for
a healthy cytoskeleton and determine the shape and architecture of neurons
whereas CRMPs aremainly involved in regulating neurite outgrowth through
microtubule polymerization. A significant upregulation of both phosphorylated
andnon-phosphorylated NF-high, phosphorylated NF-medium, NF-low and
α-internexin was detected in the visual cortex of MMP-3-/- mice compared
with WT. These results suggest that an altered stoichiometry of NF subunits is
related to the truncated neuronal architecture observed in MMP-3-/- mice. In
addition, the expression level of CRMP-5 was significantly elevated in MMP-3-/-
samples whereas the levels of CRMP-1, -2, -3 and -4 did not differ compared with
WT mice. Recentliterature indicates that overexpression of CRMP-5 negatively
regulates dendritic outgrowth by reducing the number of mitochondria through
increased autophagy and mitophagy. Therefore, we assesed the expression level
of a marker for autophagy, the microtubule-associated protein1 light chain 3
(LC3-II) and found a significant increase in MMP-3-/- samples. The expression
levels of mitochondrial fusion proteinmitofusin-2 (Mfn-2) and dynamin-related
protein 1 (Drp1), a mitochondrial fission protein, did not differ, indicating that
the mitochondrial fusion and fission balance is not altered in the visual cortex
ofMMP-3-/- mice. Taken together, an altered NF composition, overexpression
of CRMP-5 and a possible upregulation of autophagy underlie the structural
phenotype in the visual cortex of MMP-3-/- mice.
To assess the acute role of MMPs in ME-induced visual cortex plasticity
we performed Western analysis for MMP-3 to reveal fluctuations in MMP-3
expression level associated with post-ME survival time in P45 and P120 mice.
This approach revealed significant differences in proMMP-3 expression especially
in P45 mice both in the medial monocular and binocular zone whereas in P120
mice, enucleation did not induce large effects on proMMP-3 expression. This
suggests that MMP-3 ispotentially necessary to inhibit cross-modal plasticity
as normally seen in P45 mice. However, no active form of MMP-3 was visible
using Western analysis and therefore we tried to optimize a fluorescent activity
assay to study MMP-3 proteolysis in visual cortex samples. However, when
performing this assay on samples of MMP-3-/- mice, a fluorescent signal in the
same range as P45 and P120 ME C57Bl/6J samples was measured, rendering this
technique unreliable. To extend the analysis of ME-induced cortical plasticity
to other MMPs, we focused on the gelatinases (MMP-2 and MMP-9) because
their function in synaptic plasticity is well established. Using a combination of
Western analysis and gelatin zymography we revealed no significant differences in
MMP-2 expression in all conditions studied, suggesting that this enzyme does not
play a major role in ME-induced open-eye potentiation orcross-modal plasticity.
No suitable antibodies for MMP-9 were available and gelatin zymography did
not reveal detectable levels of MMP-9 proteolytic activity, possibly due to
sample preparation issues. To conclude, further research is needed to evaluate
the activity of different MMPs in the mouse visual cortex and this will provide
a basis for future experiments for pharmacological intervention to establish a
causal relationship between MMP function and ME-induced cortical plasticity.
to environmental changes and this ability is termed neuroplasticity. The visual
cortex, the area in the brain that receives visual input, is undoubtedly one of the
best and most studied areas of the brain for understanding cortical plasticity and
development. However, the molecular mechanisms governing cortical plasticity
are stillelusive. Matrix metalloproteinases (MMPs) are Zn2+-dependent
endopeptidases considered to be essential for normal brain development and
neuroplasticity by modulating extracellular matrix proteins, receptors, adhesion
molecules, growth factors and cytoskeletal proteins. Specifically, MMP-3 has
recently been implicated in synaptic plasticity, hippocampus-dependent learning
and neuronal development and migration in the cerebellum. However, the
function of this enzyme in the neocortexis understudied. Therefore, we
explored the phenotypical characteristics of the neuronal architecture and
the capacity for experience-dependent cortical plasticity in the visual cortex
of adult MMP-3-deficient (MMP-3-/-) mice. GolgiCox stainings revealed a
significant reduction in apical dendritic length, spine length and an increased
numberof apical obliques for layer V pyramidal neurons in the visual cortex of
adult MMP-3-/- mice compared to wildtype (WT) animals. To assess the effect
of MMP-3 deficiency on cortical plasticity, we monocularly enucleated (ME)
adult MMP-3-/- mice and analyzed the reactivation of the contralateral visual
cortex seven weeks post-ME. In contrast to previous results in C57Bl/6J adult
mice, activity remained confined to the binocular zone and did not expand
into the monocular regions indicative for an aberrant open-eye potentiation.
Permanent hypoactivity in the monocular cortex lateral and medial to V1 also
indicated a lack of cross-modal plasticity. These observations demonstrate
that genetic inactivation of MMP-3 has profound effects on the structural
integrity and plasticity response of the visual cortex of adult mice. To further
assess the molecular changes governing the MMP-3-/- cortical phenotype, we
performed Western analysis on different neurofilament protein (NF) subunits
and collapsin response mediator proteins (CRMP). The former are markers for
a healthy cytoskeleton and determine the shape and architecture of neurons
whereas CRMPs aremainly involved in regulating neurite outgrowth through
microtubule polymerization. A significant upregulation of both phosphorylated
andnon-phosphorylated NF-high, phosphorylated NF-medium, NF-low and
α-internexin was detected in the visual cortex of MMP-3-/- mice compared
with WT. These results suggest that an altered stoichiometry of NF subunits is
related to the truncated neuronal architecture observed in MMP-3-/- mice. In
addition, the expression level of CRMP-5 was significantly elevated in MMP-3-/-
samples whereas the levels of CRMP-1, -2, -3 and -4 did not differ compared with
WT mice. Recentliterature indicates that overexpression of CRMP-5 negatively
regulates dendritic outgrowth by reducing the number of mitochondria through
increased autophagy and mitophagy. Therefore, we assesed the expression level
of a marker for autophagy, the microtubule-associated protein1 light chain 3
(LC3-II) and found a significant increase in MMP-3-/- samples. The expression
levels of mitochondrial fusion proteinmitofusin-2 (Mfn-2) and dynamin-related
protein 1 (Drp1), a mitochondrial fission protein, did not differ, indicating that
the mitochondrial fusion and fission balance is not altered in the visual cortex
ofMMP-3-/- mice. Taken together, an altered NF composition, overexpression
of CRMP-5 and a possible upregulation of autophagy underlie the structural
phenotype in the visual cortex of MMP-3-/- mice.
To assess the acute role of MMPs in ME-induced visual cortex plasticity
we performed Western analysis for MMP-3 to reveal fluctuations in MMP-3
expression level associated with post-ME survival time in P45 and P120 mice.
This approach revealed significant differences in proMMP-3 expression especially
in P45 mice both in the medial monocular and binocular zone whereas in P120
mice, enucleation did not induce large effects on proMMP-3 expression. This
suggests that MMP-3 ispotentially necessary to inhibit cross-modal plasticity
as normally seen in P45 mice. However, no active form of MMP-3 was visible
using Western analysis and therefore we tried to optimize a fluorescent activity
assay to study MMP-3 proteolysis in visual cortex samples. However, when
performing this assay on samples of MMP-3-/- mice, a fluorescent signal in the
same range as P45 and P120 ME C57Bl/6J samples was measured, rendering this
technique unreliable. To extend the analysis of ME-induced cortical plasticity
to other MMPs, we focused on the gelatinases (MMP-2 and MMP-9) because
their function in synaptic plasticity is well established. Using a combination of
Western analysis and gelatin zymography we revealed no significant differences in
MMP-2 expression in all conditions studied, suggesting that this enzyme does not
play a major role in ME-induced open-eye potentiation orcross-modal plasticity.
No suitable antibodies for MMP-9 were available and gelatin zymography did
not reveal detectable levels of MMP-9 proteolytic activity, possibly due to
sample preparation issues. To conclude, further research is needed to evaluate
the activity of different MMPs in the mouse visual cortex and this will provide
a basis for future experiments for pharmacological intervention to establish a
causal relationship between MMP function and ME-induced cortical plasticity.
Date:1 Oct 2010 → 31 Dec 2014
Keywords:Matrix metalloproteinases, Visual cortex, Mouse, Brain, Plasticity
Disciplines:Neurosciences, Biological and physiological psychology, Cognitive science and intelligent systems, Developmental psychology and ageing
Project type:PhD project