1,721,203 research outputs found
Nieuwe inzichten in de functie van matrix metalloproteïnase-2 tijdens de cerebellaire ontwikkeling in de muis (mus musculus)
No full textMatrix metalloproteinases (MMPs) are Zn2+-dependentproteolytic enzymes that may play a central role in a variety of physiologicaland pathological conditions, due to a broad variety of substrates, ranging fromextracellular matrix molecules, growth factors, cell adhesion molecules andeven intracellular targets. Also in the central nervous system (CNS), MMP functionis attributed to diverse functions, with increasing attention for its role ine.g. CNS development, synaptic plasticity and functional repair. MMP-2, ahighly investigated MMP family member, has been suggested to affect cerebellardevelopment, defined by a precisely coordinated sequence of cell proliferation,apoptosis, differentiation, migration and synaptogenesis. However, in contrastto some other MMPs (e.g. MMP-9 andMMP-3), the role and the working mechanisms of this individual MMP incerebellar morphogenesis remained to be elucidated. In this work, we could demonstrate a highly MMP-2 specific spatiotemporalexpression pattern in the developing cerebellum. Using real-time PCR, weobserved a peak in mRNA expression at postnatal day 3 (P3), followed by a decreasein expression after the first postnatal week. Immunohistochemistry revealedhigh MMP-2 protein expression in the matrix of the superficial externalgranular layer (EGL), mainly around P3, coinciding with a period of high granulecell (GC) precursor proliferation. MMP-2 protein levels were also high in the Purkinjecell (PC) cytoplasm and emerging dendrites, from P3 on and until adulthood. No gross abnormalities in the number and global pattern of thecerebellar lobes were found in mice deficient for MMP-2 (MMP-2-/-).However, further detailed histomorphometric analyses on MMP-2-/- andwild-type (WT) cerebella at various postnatal and adult stages unveiled, fromP4 on, a decreased EGL thickness in MMP-2-/- brains, as compared toWT cerebella. Additional analyses revealed that this was caused by a transient reductionin GC number in the proliferative EGL of MMP-2-/- pups, due to anearly increase in GC cell cycle length (30% longer S-phase in MMP-2-/-GCs) and a concomitant decrease in proliferation rate. In addition, wedocumented a delay in GC radial migration through the molecular layer in MMP-2-/-cerebella. However, based on in vitromigration assays, we assumed the delayed migration to occur in response to thedefective GCP proliferation in MMP-2-/- mice. Based on marked MMP-2 protein levels observed in cerebellar PCs, we alsoperformed a range of morphometric analyses on MMP-2-/- and WT PCs atvarious developmental stages. Despite a normal embryonic PC proliferation andsubsequent PC layer formation, postnatal and adult MMP-2-/- micedisplayed severe PC malformations, defined by a reduced PC dendritic tree, smallercell bodies, and an altered width/height of the primary dendrite. The disturbedPC morphogenesis coincided with a temporarily impaired excitatory afferentinput from climbing fibers and parallel fibers. Importantly, the developmentalPC abnormalities might relate to a disturbed adult PC functionality, suggestedfrom an altered spine appearance (i.e.higher spine density and shorter spines) in adult MMP-2-/- PCs andmild abnormalities in cerebellar-related motor performance in adult MMP-2-/-animals, as compared to WT littermates. To identify putative downstream targets and pathways contributing to theobserved cerebellar abnormalities, 2-dimensional difference gel electrophoresiswas performed on P3 MMP-2-/- and WT cerebellar homogenates. From alist of differential proteins, we identified and confirmed glyceraldehyde-3-phosphatedehydrogenase (GAPDH) and collapsin response mediator protein (CRMP1) as proteinsthat might possibly underlie an MMP-2 dependent cerebellar development. In conclusion, using arange of in vivo, ex vivo and in vitro experiments in MMP-2-/- and WT mice, we wereable to identify the specific involvement of MMP-2 in the histogenesis of themouse cerebellar cortex and could shed light on putative downstream targets. Assuch, we emphasized the underestimated beneficial role of MMPs, and MMP-2 inparticular, in CNS development. <w:latentstyles deflockedstate="false" defunhidewhenused="true" <w:lsdexception locked="false" priority="0" semihidden="false" <w:lsdexception locked="false" priority="9" semihidden="false" <w:lsdexception locked="false" priority="10" semihidden="false" <w:lsdexception locked="false" priority="11" semihidden="false" <w:lsdexception locked="false" priority="22" semihidden="false" <w:lsdexception locked="false" priority="20" semihidden="false" <w:lsdexception locked="false" priority="1" semihidden="false" <w:lsdexception locked="false" priority="1" semihidden="false" <w:lsdexception locked="false" priority="60" semihidden="false" <w:lsdexception locked="false" priority="61" semihidden="false" <w:lsdexception locked="false" priority="62" semihidden="false" <w:lsdexception locked="false" priority="63" semihidden="false" <w:lsdexception locked="false" priority="64" 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Acute neuroinflammatie en gliale reactiviteit om neurale circuits te herstellen: Het ontrafelen van de rol van inflammatoire mediatoren tijdens optische zenuwregeneratie in de zebravis
No full textBrain injury and neurodegenerative disorders such as Alzheimer's and Parkinson's disease, multiple sclerosis or glaucoma represent a growing social and economic problem and affect an increasing number of people in our aging society. Neurotrauma and degeneration drastically diminish life quality and lead to severe impairments, largely because the central nervous system (CNS) of adult mammals has only a limited capacity to replace or repair damaged neurons and subsequent axonal regeneration – the latter being the focus of this study.
Following CNS injury, resident microglia and infiltrating myeloid cells dominate within the pathological environment and exert diverse effects, which depend on the specific context of the injury and can be both beneficial or detrimental to the regenerative outcome. Recent innovative insights into the dichotomous role of neuroinflammation have sprouted the idea that directing and instructing the inflammatory machinery may be a better therapeutic objective than suppressing it. Yet, little is known about how inflammation contributes to successful axonal regeneration in the adult CNS, and also the molecules and pathways connecting the two processes remain largely elusive.
In sharp contrast to mammals, zebrafish retain robust regenerative capacities into adulthood, including in the CNS. Importantly, despite these apparent differences in the regenerative ability between mammals and fish, the molecular mechanisms underlying CNS regeneration show remarkable phylogenetic conservation. Hence, zebrafish form a powerful model for comparative studies that aim at the identification of novel pro-regenerative molecules. In both mammals and zebrafish, the retinofugal system is one of the most studied CNS regeneration model systems. It forms an integral part of the CNS, encompassing the neural retina, optic nerve and visual target areas in the brain, and offers the advantage of its unique accessibility and well-described morphology and function, which are highly conserved across vertebrates. Furthermore, the axons of the optic nerve originate from a single neuronal cell population, the retinal ganglion cells (RGCs).
In this PhD dissertation, we exploited the robust regenerative capacity of the zebrafish optic nerve to investigate the mechanisms that underlie successful axonal regeneration, thereby focusing on the role of acute neuroinflammation.
Firstly, we aimed at the detailed characterization of the inflammatory response upon optic nerve crush (ONC) in zebrafish. We showed that optic nerve damage induces a timed induction and resolution of inflammatory cells throughout the retinofugal system. In contrast to mammals, the inflammatory reaction is rather moderate in zebrafish, and does not become chronic. Additionally, we revealed a restricted and transient reactivation of retinal Müller glia during optic nerve regeneration.
Subsequently, we sought to investigate the contribution of acute neuroinflammation to the regenerative process, by modulating the immune response during optic nerve regeneration. In a first approach, we studied the effects of inflammatory stimulation. We showed that robust ocular inflammation in zebrafish can be achieved via intravitreal injection of the yeast cell wall extract zymosan, while the lipopeptide Pam3Cys has only a modest effect. In addition, we demonstrated that zymosan, but not Pam3Cys, elevates the retinal cytokine expression, indicative of the increased inflammatory response. More importantly, inflammatory stimulation was found to accelerate optic nerve regeneration, reminiscent of previous findings in rodent models. Systemic immunosuppression on the other hand, achieved by administration of dex to the fish’ water, significantly impeded the regenerative response upon ONC. Additionally, we aimed to obtain local immunosuppression via intravitreal injection of dexamethasone (dex) or clodronate liposomes. Although this confined treatment resulted in an efficient depletion of microglia/macrophages in the uninjured retina, we were unable to achieve continued immunosuppression after ONC. Instead, our treatment paradigm provoked inflammatory stimulation, which was confirmed by increased retinal cytokine expression. Most likely, exposure to the immunosuppressive drugs caused sensitization of microglia/macrophages, thereby triggering an exaggerated inflammatory reaction upon ONC. Accordingly, we observed an accelerated regenerative response. Taken together, our data clearly point towards a beneficial role of acute inflammation during zebrafish optic nerve regeneration.
Furthermore, we disclosed that inflammatory stimulation induces proliferative gliosis of Müller glia in the zebrafish retina, a finding that has not been described before. Our data are suggestive for crosstalk between innate immune cells and Müller glia, and it is conceivable that zebrafish Müller glia mediate an important part of the beneficial effect of inflammatory stimulation on optic nerve regeneration, similar to their mammalian counterparts. Yet, further study is needed to elucidate the underlying molecular cues and signaling pathways, as well as the respective contributions of inflammatory cells and Müller glia to the spontaneous regenerative process in teleost fish.
Altogether, this dissertation shed new light on the role of acute neuroinflammatory responses in CNS axonal regrowth, and advanced our understanding of the mechanisms that underlie successful optic nerve regeneration in zebrafish. As such, we hope that this work may generate new conceptual insights that contribute to the development of novel therapeutics that can reverse neurodegeneration in the mammalian CNS.status: Publishe
Peptiden en proteasen als modulatoren van neurietuitgroei: inzichten vanuit ex vivo en in vivo muismodellen voor de studie van axonale regeneratie van retinale ganglioncellen
No full textDue to the increasing life expectancy, CNS injury and neurodegenerative diseases, such as Alzheimer's and Parkinson’s disease, age-related macular degeneration and glaucoma, are more prevalent than ever before. The accompanying CNS damage leads to a severe deterioration of life quality. Current pharmacological therapies are able to attenuate the disease progression and reduce the symptoms, however restoration of neuronal function remains largely elusive, not least due to the lack of regenerative capacity in the adult mammalian CNS. A deeper understanding of underlying processes and identification of moleculesnbsp;are involved in CNS regeneration will lead the way to novel therapies to restore these CNS pathologies and improve the quality of life for many patients.
Efforts to identify novel compounds for regenerative treatments are limited by a gap between initial results from high-throughput in vitro screening models and their (pre-)clinical validation. In contrast to cells, tissue explant cultures closely resemble the in vivo situation, making them ideal to study the effects of molecules in a neuro-glial network. We therefore developed a retinal explant model from neonatal mice to determine the effect of compounds on neurite outgrowth. During the development of this model, an automated analysis of neurite outgrowth was established and neurite outgrowth was shown to be independent from the region within the retina that the explant was originating from.
In order to validate our ex vivo findings, an in vivo mouse model was established to study axonal regeneration of retinal ganglion cells after optic nerve injury. Within this regenerative optic nerve crush (RONC) model, microscopic visualization of regenerating axons was optimized.
In search for new modulators of axonal outgrowth, we used the ex vivo retinal explant model to screen a peptide library, developed by the Functional Genomics and Proteomics research group. From the 26 peptides tested, we identified 3 axon-outgrowth stimulating and 3 inhibiting peptides. In a second phase, the most promising peptide, P318, was further characterized to unravel its mode of action. Our first findings suggest that P318 stimulates neurite outgrowth via glial cells Despite its ability to stimulate neurite outgrowth, we also found that administration of the peptide induces cell death in retinal explants. Further research is required to unravel the different underlying mechanisms.
In a parallel study, we used the retinal explant model to study the effect of matrix metalloproteinases (MMPs) on neurite outgrowth. MMPs are zinc dependent endoproteinases that cleave extracellular matrix proteins and other signaling molecules. Previous studies of our lab have shown that both MMP-2 and MT1-MMP deficiency results in an impaired neurite outgrowth in neonatal mouse retinal explants. Using the retinal explant model, we were now able tonbsp;that the observed diminished outgrowth in MMP-2 deficient explants can be rescued bynbsp;of exogenous MMP-2. Furthermore, our studies revealed that MT1-MMP is implicated in neurite outgrowth via the activation of pro-MMP-2. Finally, we investigated the role of MMP-2 during axonal regeneration in the in vivo RONC model. In this pilot study, a reduced axonal regeneration was observed in MMP-2 deficient mice. Our preliminary results confirm the ex vivo data, namely that MMP-2 is involved in axonal regeneration.
Within this study, the ex vivo retinal explant and in vivo RONC model were successfully established to study axonal outgrowth and regeneration. These models were used tonbsp;the role of the bio-active peptide P318 and of MMP-2/MT1-MMP during neurite outgrowth and/or axonal regeneration. These results could form a first step in the development of novel regenerative therapies in the adult CNS.status: Publishe
ROCK Inhibition as a Versatile Strategy for the Treatment of Glaucoma
No full textGlaucoma is a chronic optic neuropathy which is characterized by the loss of retinal ganglio n cells, resulting in irreversible visual field lo ss. It is the most important cause of irreversible¨blindness worldwide and with aging of the populat ion the number of glaucoma patients is still expan ding. Mostly the appearance of this disease i s correlated with an increased intraocular pressur e (IOP), due to an imbalance between aqueous humor ¨production and outflow. Currently, lowering IOP i s the main treatment modality for glaucoma. A ¨sustained reduction of IOP can be achieved with m edical therapy, laser treatment or surgical i nterventions. First choice treatment for glau coma is medical therapy with IOP-lowering eye drop s. Despite the fact that there are several cl asses of glaucoma medications available, none ¨of them directly targets the trabecular meshwork, ¨which represents the main outflow route in t he human eye. Experimental evidence indicate s that the Rho kinase (ROCK) pathway is direc tly involved in the regulation of trabecular outflow. As such, targeting this pathway could pot entially lead to an improved IOP control for the m edical treatment of glaucoma.
Therefore, in the first part of this PhD projec t we investigated the IOP-lowering potential of AM A0076, a novel locally acting ROCK inhibitor. We d emonstrated that AMA0076 is a potent ROCK inhibito r with an IOP-lowering efficacy comparable to a re ference ROCK inhibitor, Y-39983. While the efficac y profile of ROCK inhibitors is promising, they ar e associated with significant ocular hyperemia due to conjunctival vasodilatation. Howeve r, due to its local mode of action AMA0076 induces ¨almost no conjunctival hyperemia in contrast to Y -39983. As such, AMA0076 clearly has an improved t olerability profile with respect to hyperemia comp ared to other ROCK inhibitors. We also compar ed the IOP-lowering effect of AMA0076 to prostagla ndin analogues (PGAs) because these are the most i mportant treatment modality in the medicinal treatment of glaucoma. Our results revealed that the IO P-lowering efficacy of AMA0076 is equivalent¨ to latanoprost in ocular normotensive rabbits . In a rabbit model of ocular hypertension, A MA0076 was even more potent in preventing IOP elev ation compared to PGAs (bimatoprost and latanopros t).
Taken together, these r esults indicate that ROCK inhibitors, and esp ecially AMA0076, may be promising new candidates a s novel IOP-lowering agents for the treatment glau coma.
Surgical interv ention is an important treatment modality for glau coma when other IOP-lowering measures (medical or¨ laser) are inadequate. However, there is a high ri sk of surgical failure due to excessive subco njunctival wound healing, causing obstruction of t he filtration channel by scar tissue. While the in troduction of antimitotics tremendously improved s urgical success, this has come at the cost of an i ncreased risk at postoperative complications due t o their nonspecific mechanism of action. Hence, th ere is an urgent need for novel, more specific and ¨safer agents to prevent glaucoma filtration failu re and improve surgery outcome. In the secon d part of this project, we investigated the effect ¨of ROCK inhibitor AMA0526 on the wound healing re sponse after glaucoma filtration surgery and its s ubsequent effect on surgical outcome. Our in v itro results showed that the ROCK inhibitor,¨ AMA0526, inhibited proliferation of tenon fib roblasts and fibroblast-to-myofibroblast diff erentiation. Using a rabbit model of glaucoma filtration surgery, we subsequently showed that p ostoperative treatment with the ROCK inhibitor sig nificantly improved glaucoma filtration surgery ou tcome. ROCK inhibition reduced postoperative infla mmation, angiogenesis and fibrosis. These results suggest that ROCK inhibitors are involved at¨ different levels in the process of wound healing a nd therefore may be considered as useful agents to ¨improve the success rate of glaucoma filtration surgery.
Evidence from large scale¨ clinical trials indicated that lowering IOP does n ot prevent glaucomatous progression in all patient s and that progression can continue despite e ffective IOP-lowering. These inadequacies in our c urrent treatment paradigm of this still blinding d isease have encouraged research to investigate neu roprotection as an alternative treatment strategy¨ for glaucoma. However, no neuroprotective therapy¨ is currently available to halt glaucomatous damage . Therefore, in the last part of this project we e laborately reviewed the high potential of the ¨Rho-ROCK pathway as a potential neuroprotective t arget for future management of glaucoma disease pr ogression. Our review indicates that there is ¨increasing evidence that ROCK inhibition can ¨promote RGC survival, and axon regeneration, demo nstrating the neuroprotective potential of ROCK in hibition for the treatment of glaucoma.
In conclusion, in this dissertation w e demonstrated that AMA0076 is a potent ROCK inhib itor with strong IOP-lowering capacity that does n ot induce distinct hyperemia. Secondly, we also sh owed that ROCK inhibition may serve as anti-s carring strategy after glaucoma filtration surgery ¨due to its inhibitory effect on inflammation , angiogenesis and fibrosis. Furthermore, the Rho- ROCK pathway is involved in optic nerve neuroprote ction. Earlier research showed that inactivat ion of ROCK increases survival and axon regen eration of RGCs. Considering the IOP lowering, ant i-scarring and neuroprotective properties of ROCK inhibitors, targeting the Rho-ROCK pathway wi th selective inhibitors may be a versatile and att ractive treatment option for glaucoma.status: Publishe
Glaucoom en de hersenen: een nieuwe benadering ter bescherming van de retinale ganglioncellen
No full textSummary PhD thesis Eline Dekeyster
Glaucoma refers to a group of optic neuropathies, all characterized by progressive degeneration of retinal ganglion cells (RGCs) - the cells that send visual information from the eye to the brain - resulting in a gradual loss of vision. Ocular hypertension (OHT) is considered one of the major risk factors for development and progression of glaucoma, and thus lowering eye pressure through topical application of hypotensive eye drops or surgery, is the cornerstone of glaucoma therapy today. Unfortunately, a number of patients do not benefit from such treatments, and although it generally slows down disease progression, controlling eye pressure does not really halt RGC degeneration. This stresses the need for development of new therapies aimed at long‑term retinal neuroprotection and preservation of vision. Within this context, this PhD project intends to contribute to the knowledge on the pathological mechanisms underlying glaucomatous RGC death and to highlight possible novel strategies towards RGC protection.
The first part of this thesis was dedicated to the optimization and characterization of two mouse models of glaucoma: the OHT model and the normotensive optic nerve crush (ONC) model, each focusing on different aspects of the disease.
Next, a closer look was taken at the link between glaucoma and the brain. Although long considered purely an eye disease, glaucoma is increasingly becoming recognized as a disease of the entire visual system. Using the OHT model, the effect of elevated eye pressure on the central visual brain areas was evaluated. Reduced RGC synapse density, retinotopically correlated to degeneration of RGC soma, and intensive but transient astroglial reactivity were observed in the main subcortical RGC projection area, which for mice is the superior colliculus (SC). Furthermore, diminished neuronal activity in the primary visual cortex extending to specific extrastriate areas was seen early after OHT induction. Complete recovery of cortical activity over time demonstrated the capacity of the adult visual cortex to functionally reorganize in an attempt to adapt to glaucomatous RGC degeneration. Our results uncovered for the first time effects on visual cortex activity patterns in a murine OHT model and, yet again, highlight the importance of including the brain in glaucoma research.
For their survival, RGCs depend on neurotrophic factors, e.g. neurotrophins, produced locally in the eye as well as in the projection areas in the brain, from where they are retrogradely transported along the RGC axons towards the cell bodies in the retina. According to the neurotrophin deprivation hypothesis, diminished retrograde delivery of neurotrophic support during an early stage of glaucoma pathogenesis is one of the main triggers that induce apoptotic signaling in RGCs. Therefore, interfering with neurotrophic signaling seems an attractive tool to achieve neuroprotection. In this PhD project, the well-known neurotrophin brain-derived neurotrophic factor (BDNF) was chosen as the lead molecule to study the role of neurotrophic factors in glaucoma. The levels of BDNF and its high-affinity receptor, tropomyosin receptor kinase B (TrkB), were examined in the retina and SC of mice subjected to OHT or ONC. Both models differentially influenced BDNF and TrkB levels. Defining a specific role for BDNF signaling within glaucoma pathology remains difficult as various studies using a variety of animal models of glaucoma have yielded unique results, including those presented within this manuscript.
In line with the hypothesis of deprived neurotrophic support being an important contributor to glaucomatous RGC death, exogenous neurotrophin administration to the eye has been shown to reduce loss of RGCs; however, the neuroprotective effect was mostly transient and insufficient for sustained RGC survival. Therefore, we hypothesized that treatment at the level of extraretinal neurotrophin sources in the brain might be beneficial, as target-derived neurotrophins are likely to induce signaling pathways that diverge from local neurotrophin signaling. Brain-directed treatment was approached in two ways: 1) viral vector-mediated upregulation of BDNF in the SC was used to boost retrograde delivery of BDNF to the retina; 2) a more broad strategy to optogenetically increase neuronal activity in the SC was implemented to enhance production of a whole spectrum of endogenous neurotrophic factors. In light of these hypotheses, viral vector technology was optimized for use in the mouse SC.
Although the previously reported temporary neuroprotective effect of intravitreally-delivered recombinant BDNF was confirmed, viral vector-induced overexpression of BDNF in the SC did not lead to protection of the RGCs in our glaucoma models. This unfortunate result most likely resulted from decreased neurotrophin responsiveness upon vector mediated BDNF overexpression. Regarding the second strategy employing SC manipulation to aim for RGC protection, optogenetic methods were introduced and validated. The basic principle underlying optogenetics is the introduction of genes, e.g. through viral vector technology, encoding light-sensitive ion channels or opsins into cells, rendering them responsive to light of a specific wavelength. For the first time, the application of an opsin with specifically slow kinetic properties, the stabilized step-function opsin (SSFO), was validated for use in the mouse SC. A setup for chronic optogenetic stimulation in awake, freely moving animals, was developed, and shown to effectively stimulate collicular activity. Two consecutive behavioral responses were observed upon unilateral SC stimulation, a short escape response, followed by prolonged pursuit-like behavior.
In conclusion, a large component of this thesis was dedicated to method optimization, such as the two mouse models of glaucoma, viral vector technology, and a setup for in vivo optogenetic stimulation. These methods will importantly contribute to future research within the research group. Furthermore, new findings on the role of higher order visual brain centers in glaucoma pathogenesis were discovered. Although we were not able to deliver a proof-of-concept for the neurotrophin deprivation hypothesis, important insights concerning the complexity of neurotrophic factor treatments were highlighted.status: Publishe
Rol van schildklierhormonen in retinotectale axonregeneratie bij de adulte zebravis
No full textInjury to the adult mammalian central nervous system (CNS) leads to detrimental consequences due to its poor regenerative capacity. Thyroid hormones (THs), well known for their key function in CNS development and maturation, have recently emerged as molecules that can influence regeneration. Several studies have found exogenous THs to be beneficial for peripheral nervous system regeneration. Conversely in adult CNS, studies on the role of THs in in vivo regeneration remain scarce. Since the zebrafish optic nerve (ON) is an easily accessible part of CNS and regenerates spontaneously upon trauma, it is widely studied to identify important molecules during CNS regeneration. We therefore investigated the effect of lowering T3 signaling on the regeneration of the ON following crush in adult zebrafish. In addressing the issue we first showed that the reinnervation of the optic tectum (OT) was accelerated when fish were exposed to iopanoic acid (IOP), a compound lowering T3 availability, or to the TH receptor ß antagonist- methylsulfonylnitrobenzoate. At 7 dpi there was a clear increase in the biocytin labeled area in the OT following anterograde tracing of the retinal ganglion cell axons as well as an increased immunostaining of Gap43, a protein expressed in outgrowing axons. Next we showed that the increased reinnervation of OT was attenuated when IOP-treated fish were co-treated with exogenous T3 confirming that increased OT reinnervation was indeed specifically due to decreased T3 signaling.In search for the mechanism behind the observed response, we first looked at the retinal ganglion cells in the retina. ON crush induced very limited cell death and proliferation at the level of the retina in both control and anti-thyroid drug treated groups. While the mRNA expression level of regeneration markers such as gap43, tuba1, socs3b and snap25a changed as expected and klf9 and bdnf did not change following crush, lowering T3 signaling did not influence the pattern. We therefore found no obvious effect of TH inhibition at the level of the retina.We did however find a correlation between the accelerated OT reinnervation and a more rapid resolution of the microglia/MΦs in the ON and the OT of IOP-treated fish. Beside the presence of reactive astrocytes in the injured ON of control and IOP-treted groups, preliminary data also revealed thepresence of reactive astrocytes in the OT of IOP-treated fish at an early time point after injury, which is 4 days after injury. Finally, we could also show an increased myelin staining in the OT of the treated group. Nevertheless these findings need further investigation to fully decipher their relevance.Taken together these data indicate that lowering T3 signaling accelerates retinal ganglion cell axon extension and OT reinnervation following ON crush in adult zebrafish and that an altered inflammatory response may be involved in this process. The next step is trying to further elucidate the underlying molecules and mechanisms and use this knowledge in further research aimed at improving regeneration in the mammalian CNS.status: Publishe
Matrix metalloproteinases als modulators van retinale dendritische hermodellering en axonal regeneratie in het beschadigd zebravis retinotectaal circuit. Primaire inzichten in een antagonistische interactie tussen dendritische en axonale hergroei na aanbreng van een optisch zenuwletsel ,,
No full textDysfunction of the central nervous system (CNS) after injury or in neurodegenerative diseases increasingly impairs life quality in our aging society. Also optic neuropathies, like glaucoma, are becoming increasingly prevalent in our elderly population, affecting over 60 million people worldwide. They are characterized by retinal ganglion cell (RGC) axonal degeneration and death, ultimately resulting in irreversible blindness because adult mammals lack the capacity to repair/regrow damaged axons in the CNS. Although combinatorial approaches indicate that it might be possible to repair the injured mammalian optic nerve (ON), they have not yet induced substantial visual recovery, nor are they, up to now, suitable for clinical applications. Therefore, comparative studies focus on the spontaneously regenerating adult CNS of zebrafish, which proves to be a valuable research model to investigate regenerative strategies of damaged neurons and to identify novel pro-regenerative molecules. Notably, a recent transcriptome profiling study performed on regenerating adult zebrafish eyes at various time-points after optic nerve crush (ONC), pinpointed several potential pro-regenerative molecules, under which four matrix metalloproteinases (mmp-2, -9, -13a, -14). While MMPs have been implicated in axonal outgrowth during CNS development and are known to be upregulated during vertebrate CNS repair, their impact on in vivo CNS regeneration remains undocumented. Within this context, this PhD project then also intended to unravel the role of Mmp-2, -9, -13a and -14 in ON regeneration in adult zebrafish subjected to ONC.
The first part of this thesis was dedicated to an in-depth characterization of RGC survival, axonal regrowth/extension and primary visual recovery in the zebrafish ONC model.
Next, we obtained first in vivo insights in a contributory role for MMPs in RGC axonal regrowth as retinal broad-spectrum MMP inhibition seriously attenuated optic tectum reinnervation by regenerating RGC axons without influencing RGC survival. Moreover, combined use of Western Blotting and immunostainings revealed a phase-dependent expression pattern for each MMP in the retina after ONC, primarily implying Mmp-2 and -13a in RGC axonal regrowth.
As such, via utilization of several loss-of-function approaches to interfere with the functioning of one specific MMP, e.g. pharmaceutical MMP inhibition, morpholino and genome editing technology, we were able to identify Mmp-2 and -13a as in vivo regulators of RGC axonal regeneration in adult zebrafish after ONC. Notably, since zebrafish lack an inhibitory environment, these data suggest a novel, neuron intrinsic, function for multiple MMPs in RGC axonal regrowth in addition to their role in breaking down environmental barriers as deduced from mammalian studies. The future identification of the underlying MMP substrates by means of quantitative proteomics and subsequent validation of promising candidate molecules in fish and mice might then also unravel new pro-regenerative molecules for the injured mammalian CNS.
While being an important part of the neuronal circuitry, the response of dendrites upon induction of axonal regeneration in mammals is almost unattended. Notably, the few publications that do discuss this issue, indicate that dendritic morphology is seriously influenced by the pro-regenerative molecule used to trigger axonal regrowth, ranging from a reduction to an increase in size and complexity. As the eventual recovery of sight after ON injury will equally depend on the proper restoration of dendritic structure, we then also characterized the inherent response of dendrites during successful RGC axonal regeneration in adult zebrafish. Remarkably, both temporal expression analyses of synaptic and dendritic markers, as well as morphometric analysis of inner plexiform layer thickness, indicated that regeneration-competent injured neurons repeat the developmental order of neurite growth, where axogenesis is primary to dendritogenesis.
Finally, a last part of this work focused on the elucidation of a potential role of MMPs in retinal dendritic remodeling in adult zebrafish after ONC. Both retinal broad- and narrow-spectrum Mmp(-2) inhibition seemingly prevented RGC dendrites from shrinking upon ON injury. Intriguingly, in both conditions, a disturbance of dendritic retraction occurred concurrently with a reduced RGC axonal regrowth, thus indicative of a potential antagonistic interplay between dendritic remodeling and axonal regrowth after ONC. In addition, due the consecutive progress of axonal and dendritic growth during development and regeneration, and based on a publication discussing TRAK1/2 driven polarized mitochondrial transport in neurons respectively regulating axonal and dendritic outgrowth, we hypothesized that an energetic trade-off might lie at the base of this apparent inter-dependency. As such, future experiments should interfere with mitochondrial trafficking in zebrafish RGC dendrites and analyze the effect on RGC axonal regeneration. If these data would result in a proof-of-concept, i.e. dendritic remodeling serving as fuel for axonal regeneration, we envision a major shift in the research focus of the neuroregenerative research field and the potential uncovering of various novel therapeutic targets.status: Publishe
Matrix metalloproteinase-3: een voorwaarde voor structurele en functionele plasticiteit in de visuele cortex van de muis
No full textThe brain is able to change its functional and anatomical organization in responseto environmental changes and this ability is termed neuroplasticity. The visualcortex, the area in the brain that receives visual input, is undoubtedly one of thebest and most studied areas of the brain for understanding cortical plasticity anddevelopment. However, the molecular mechanisms governing cortical plasticityare still elusive. Matrix metalloproteinases (MMPs) are Zn2+-dependentendopeptidases considered to be essential for normal brain development andneuroplasticity by modulating extracellular matrix proteins, receptors, adhesionmolecules, growth factors and cytoskeletal proteins. Specifically, MMP-3 hasrecently been implicated in synaptic plasticity, hippocampus-dependent learningand neuronal development and migration in the cerebellum. However, thefunction of this enzyme in the neocortex is understudied. Therefore, weexplored the phenotypical characteristics of the neuronal architecture andthe capacity for experience-dependent cortical plasticity in the visual cortexof adult MMP-3-deficient (MMP-3-/-) mice. GolgiCox stainings revealed asignificant reduction in apical dendritic length, spine length and an increasednumber of apical obliques for layer V pyramidal neurons in the visual cortex ofadult MMP-3-/- mice compared to wildtype (WT) animals. To assess the effectof MMP-3 deficiency on cortical plasticity, we monocularly enucleated (ME)adult MMP-3-/- mice and analyzed the reactivation of the contralateral visualcortex seven weeks post-ME. In contrast to previous results in C57Bl/6J adultmice, activity remained confined to the binocular zone and did not expandinto 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 demonstratethat genetic inactivation of MMP-3 has profound effects on the structuralintegrity and plasticity response of the visual cortex of adult mice. To furtherassess the molecular changes governing the MMP-3-/- cortical phenotype, weperformed Western analysis on different neurofilament protein (NF) subunitsand collapsin response mediator proteins (CRMP). The former are markers fora healthy cytoskeleton and determine the shape and architecture of neuronswhereas CRMPs are mainly involved in regulating neurite outgrowth throughmicrotubule polymerization. A significant upregulation of both phosphorylatedand non-phosphorylated NF-high, phosphorylated NF-medium, NF-low andalfa-internexin was detected in the visual cortex of MMP-3-/- mice comparedwith WT. These results suggest that an altered stoichiometry of NF subunits isrelated to the truncated neuronal architecture observed in MMP-3-/- mice. Inaddition, 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 withWT mice. Recent literature indicates that overexpression of CRMP-5 negativelyregulates dendritic outgrowth by reducing the number of mitochondria throughincreased autophagy and mitophagy. Therefore, we assesed the expression levelof a marker for autophagy, the microtubule-associated protein 1 light chain 3(LC3-II) and found a significant increase in MMP-3-/- samples. The expressionlevels of mitochondrial fusion protein mitofusin-2 (Mfn-2) and dynamin-relatedprotein 1 (Drp1), a mitochondrial fission protein, did not differ, indicating thatthe mitochondrial fusion and fission balance is not altered in the visual cortexof MMP-3-/- mice. Taken together, an altered NF composition, overexpressionof CRMP-5 and a possible upregulation of autophagy underlie the structuralphenotype in the visual cortex of MMP-3-/- mice. To assess the acute role of MMPs in ME-induced visual cortex plasticitywe performed Western analysis for MMP-3 to reveal fluctuations in MMP-3expression level associated with post-ME survival time in P45 and P120 mice.This approach revealed significant differences in proMMP-3 expression especiallyin P45 mice both in the medial monocular and binocular zone whereas in P120mice, enucleation did not induce large effects on proMMP-3 expression. Thissuggests that MMP-3 is potentially necessary to inhibit cross-modal plasticity as normally seen in P45 mice. However, no active form of MMP-3 was visibleusing Western analysis and therefore we tried to optimize a fluorescent activityassay to study MMP-3 proteolysis in visual cortex samples. However, whenperforming this assay on samples of MMP-3-/- mice, a fluorescent signal in thesame range as P45 and P120 ME C57Bl/6J samples was measured, rendering thistechnique unreliable. To extend the analysis of ME-induced cortical plasticityto other MMPs, we focused on the gelatinases (MMP-2 and MMP-9) becausetheir function in synaptic plasticity is well established. Using a combination ofWestern analysis and gelatin zymography we revealed no significant differences inMMP-2 expression in all conditions studied, suggesting that this enzyme does notplay a major role in ME-induced open-eye potentiation or cross-modal plasticity.No suitable antibodies for MMP-9 were available and gelatin zymography didnot reveal detectable levels of MMP-9 proteolytic activity, possibly due tosample preparation issues. To conclude, further research is needed to evaluatethe activity of different MMPs in the mouse visual cortex and this will providea basis for future experiments for pharmacological intervention to establish acausal relationship between MMP function and ME-induced cortical plasticity.status: Publishe
Matrix metalloproteinasen in glaucoom: doelwitten voor nieuwe therapieën?
No full textMatrix metalloproteinases (MMPs), a family of Zn2+-dependent proteases, were originally named after their ability to cleave and remodel the extracellular matrix (ECM), however, their substrate repertoire has proven to be much broader, comprising other proteinases, growth factors, signaling molecules, cell surface receptors, and even intracellular targets. By proteolytic cleavage, MMPs modify the structure and activity of these substrates, and add a complex extra dimension of biological control. As a result, MMPs are important regulatory nodes in the protease web, a complex network of interactions that regulates protease activities and determines the functional state of the proteome and cell activity. Deregulated MMP activity is a common characteristic of many diseases, including neurodegenerative disorders such as glaucoma, multiple sclerosis, Huntington’s disease, Alzheimer’s disease, Parkinson’s disease, etc. Nevertheless, despite their detrimental impact during central nervous system (CNS) pathology, there is ample evidence corroborating MMPs as fine regulators of CNS physiology, and well-balanced MMP activity is instrumental to CNS development, plasticity and repair.
Multiple studies in glaucoma patients and in animal models of spontaneous and experimentally induced glaucoma, reported changes in the expression and activity of several MMPs in the retina, optic nerve, aqueous humor and trabecular meshwork. These data have led to the hypothesis that MMPs might be involved in glaucoma onset and/or disease progression. However, reports are conflicting and research aiming at providing a clear definition of their causative role is lacking. With this work, we intend to contribute to a better understanding of the role of MMPs during glaucoma pathogenesis and to the development of much-needed novel therapeutic approaches to treat this optic neuropathy. Indeed, with more than 65 million patients worldwide, and still on the increase, glaucoma is and will be a major concern for ophthalmologists today and in the future. Current baroprotective therapies allow to reduce intraocular pressure in the majority of glaucoma patients and to halt -yet not reverse- disease progression. Nevertheless, a sizeable percentage of patients do not benefit from this therapeutic approach, and, as no alternatives are available, experience progressive vision loss.
Four MMPs, namely MMP-2, MMP-3, MMP-9 and MT1-MMP, are the subject of different experimental approaches, aiming to answer the central question: “Can we identify MMPs as novel targets for the development of baroprotective, neuroprotective and/or regenerative therapeutic strategies for the treatment of glaucoma?”status: Publishe
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