A proteomic approach to understand MMP?3?driven developmental processes in the postnatal cerebellum: Chaperonin CCT6A and MAP kinase as contributing factors

Matrix metalloproteinase?3 (MMP?3) deficiency in mice was previously reported to result in a transiently retarded granule cell migration at postnatal day 8 (P8) and a sustained disturbed arborization of Purkinje cell dendrites from P8 on, concomitant with a delayed synapse formation between granule cells and Purkinje cells and resulting in mild deficits in motor performance in adult animals. However, the molecular mechanisms by which MMP?3 contributes to proper development of the cerebellar cortex during the first postnatal weeks remains unknown. In this study, we used a functional proteomics approach to investigate alterations in protein expression in postnatal cerebella of wild?type versus MMP?3 deficient mice, and to further elucidate MMP?3?dependent pathways and downstream targets in vivo. At P8, two?dimensional difference gel electrophoresis and mass spectrometry identified 20 unique proteins with a different expression between the two genotypes. Subsequent “Ingenuity Pathway Analysis” and Western blotting indicate that the chaperonin containing T?complex polypeptide 1, subunit 6A and the MAP kinase signaling pathway play a key role in the MMP?3?dependent regulation of neurite outgrowth and neuronal migration in the developing brain

Molecular activity during cortical map reorganization in the visual cortex of the cat (Felis catus).

Het is een vaststaand feit dat volwassen zoogdierhersenen zich structure el en functioneel aanpassen in antwoord op sensorische veranderingen. Zo lokken centrale retinale letsels een plasticiteitsafhankelijke retinoto pische reorganisatie uit in de primaire visuele cortex. Deze functionele reorganisatie lijkt op meerdere tijdsafhankelijke mechanismen gebaseerd te zijn. Wij hebben de korte- en lange-termijn effecten op de volledige omvang van de primaire evenals vijf hogere orde visuele areas onderzoch t. Dit onderzoek heeft bijgedragen tot een beter inzicht in adulte plastici teit-gemedieerde mechanismen binnen de hersenen van zoogdieren. Wij hebb en onbetwistbaar bewezen dat de ervaringsafhankelijke topografische reor ganisatie gepaard gaat met spatio-temporeel gereguleerde veranderingen i n activiteitsbalans en in eiwitexpressie. Een eerste belangrijk deel van onze studie bestond uit de analyse van de gewijzigde neuronale activiteit in de visuele cortex na het aanbrengen van binoculaire retinale letsels. Wij hebben verscheidene technieken toe gepast om de expressie van de moleculaire activiteitsmerkers c-fos en zi f268 te bepalen en deze aanpak leverde een betrouwbaar beeld van het cor ticale activiteitsniveau. De verschillende datasets kwamen goed met elka ar overeen. De resultaten toonden systematisch aan dat het induceren van de retinale letsels initieel een activiteitsdaling veroorzaakt in de pr imaire corticale letselprojectie zone (LPZ), wat gevolgd wordt door een tijdsafhankelijk herstel ter hoogte van de rand van de LPZ. Bovendien we rd de perifere visuele cortex mee beïnvloed door de centrale retinale le tsels. We hebben dus bewezen dat perifere cortex niet als normale cortex kan beschouwd worden. In area 17 hebben wij respectievelijk langs de dorso-ventrale en de post erior-anterior as van de hersenen een reorganisatie over 2.5 tot 6 mm op gemeten. Het corticale reorganisatie proces trad proportioneel op vermit s langs de twee hersenenassen het corticale herstel in functie van visue le graden gelijke afmetingen behelsde. Door middel van immuncytochemie hebben wij voorts getoond wat het effect is van binoculaire retinale letsels op de cellulaire en laminaire distr ibutie van Fos. Wij namen waar dat de verschillende corticale lagen diff erentieel bijdroegen aan het reorganisatie proces. En de veranderingen i n Fos-expressie konden voornamelijk worden toegeschreven aan laag-specif ieke wijzigingen in Fos eiwitproductie van individuele neuronen en minde r aan modificaties in het percentage Fos-immunopositieve kernen. Parallel aan de activiteitsveranderingen in primaire visuele area 17, ve rtoonden vijf hogere orde visuele areas, als reactie op de visuele depri vatie, ook een gewijzigd corticaal activiteitsniveau. Vooral, area PMLS en area 7 bleken cruciaal te zijn bij de plasticiteitsafhankelijke reorg anisatie, gezien deze twee visuele areas een duidelijke hyperactiviteit vertoonden. De real-time PCR resultaten wezen er dus op dat naast intra- corticale ook inter-corticale interacties een essentiële rol zouden kunn en spelen in de corticale reorganisatie. Een ander hoofdstuk van dit werk was gericht op het effect van de centra le retinale letsels op de eiwitexpressie in gans area 17. Dieren met cen trale retinale letsels vertoonden onmiskenbaar zowel regio-specifieke al s overlevingstijd-afhankelijke veranderingen van de proteine expressis i n de primaire visuele cortex. Dankzij het gebruik van twee-dimensionale differentiële gelelektroforese hebben wij met succes 103 differentiële e iwitten geïdentificeerd, die als potentiële moleculaire plasticiteitsmed iators zouden kunnen functioneren. Analoog aan de veranderingen in corticale activiteit, was perifere area 17 op proteïne niveau wederom te onderscheiden van normale cortex. Wij h ebben de bevestigingsexperimenten op perifere area 17 geconcentreerd, om deze bevinding extra kracht bij te zetten. Western blotting voor Heat shock cognate protein 70 and Protein phosphatase 1b heeft inderdaad b evestigd dat de expressie van deze twee proteïnen in perifere area 17 do or de inductie van centrale retinale letsels specifiek werd beïnvloed. De verkregen resultaten wezen erop dat ervaringsafhankelijke corticale r eorganisatie gebaseerd is op verschillende mechanismen en gekenmerkt wor dt door een complex netwerk van verscheidene, interagerende proteïnen, d ie betrokken zijn bij diverse signaaltransductiecascades. Gezien het fei t dat endocytose/exocytose processen sterk gecorreleerd zijn met LTP en neuriet-uitgroei, suggereren wij dat de regulatie van deze enocytose/exo cytose balans mogelijk een van de hoofdmechanismen is voor de topografis che map reorganisatie. Tenslotte kaartten we aan hoe de plasticiteitsafhankelijke cortica le reorganisatie uiteindelijk niet tot normalizatie leidt maar eerder de meest optimale reactivatiepatronen in de visuele cortex teweeg brengt d ie de nieuwe functionaliteit helpen ondersteunen. Op lange termijn konde n wij inderdaad nog aanhoudende veranderingen in corticale activiteit en proteïne expressie onderscheiden. Wij suggereren dat de sensorische man ipulatie in eerste instantie een onevenwicht in de corticale processen v eroorzaakt. Omwille van dit onevenwicht zouden processen die betrekking hebben op plasticiteit aanleiding geven tot corticale reorganisatie. Uit eindelijk zal de sensorische cortex een nieuw stabiele balans introducer en om via de lange-termijn aanpassingen het behoud van de nieuw verworve n eigenschappen te garanderen.nrpages: 188status: publishe

Immunolocalization of Dynamin I Protein in Projection Neurons of the Visual System of the Adult Cat

We here report on the immunolocalization of Dynamin I (Dyn I) in neurons of the visual system of the cat. The lateral geniculate nucleus (LGN) complex displayed abundant Dyn I immunoreactivity in typical relay cells of the X-, Y- and W-pathway. The superficial and deep layers of the superior colliculus were also populated by Dyn I-immunoreactive projection neurons of the W- and Y-cell system. In primary visual areas 17 and 18, many densely packed layer VI neurons were intensely stained. A clear Dyn I signal was also demonstrated in pyramidal neurons of supragranular layers II and III, while layer IV displayed low Dyn I immunoreactivity. Additionally, area 18 displayed larger border pyramidal neurons in layer III compared to area 17. Generally, Dyn I was localized to the cell body and dendrites of neurons, to the neuropil and sometimes also to axon bundles. Typically, the Dyn I signal was not always uniformly distributed within the somatodendritic compartment. Based on its widespread distribution mainly in projection neurons Dyn I may play a fundamental role in mature neurons of different cortical and subcortical structures of the adult mammalian brain

Effect of binocular retinal lesions on CRMP2 and CRMP4 but not Dyn I and Syt I expression in adult cat area 17

Removal of retinal input from a restricted region of adult cat visual cortex leads to a substantial reorganization of the retinotopy within the sensory-deprived cortical lesion projection zone (LPZ). Still little is known about the molecular mechanisms underlying this cortical map reorganization. We chose two members of the collapsin response mediator protein (CRMP) family, CRMP2 and CRMP4, because of their involvement in neurite growth, and compared gene and protein expression levels between normal control and reorganizing visual cortex upon induction of central retinal lesions. Parallel analysis of Dynamin I (Dyn I) and Synaptotagmin I (Syt I), two molecules implicated in the exocytosis-endocytosis cycle, was performed because changes in neurotransmitter release have been implicated in cortical plasticity. Western blotting and real-time polymerase chain reaction revealed a clear time-dependent effect of retinal lesioning on CRMP2 and CRMP4 expression, with maximal impact 2 weeks post-lesion. Altered CRMP levels were not a direct consequence of decreased visual activity in the LPZ as complete surgical removal of retinal input to one hemisphere had no effect on CRMP2 or CRMP4 expression. Thus, CRMP expression is correlated to cortical reorganization following partial deafferentation of adult visual cortex. In contrast, Dyn I and Syt I were not influenced and thereby do not promote exocytosis-endocytosis cycle modifications in adult cat cortical plasticity.status: publishe

Influence of binocular competition on the expression profiles of CRMP2, CRMP4, Dyn I, and Syt I in developing cat visual cortex

The visual cortex is vulnerable to changes in visual input, especially during the critical period when numerous molecules drive the refinement of the circuitry. From a list of potential actors identified in a recent proteomics study, we selected 2 collapsin response mediator proteins (CRMP2/CRMP4) and 2 synaptic proteins, Dynamin I (Dyn I) and Synaptotagmin I (Syt I), for in-depth analysis of their developmental expression profile in cat visual cortex. CRMP2 and CRMP4 levels were high early in life and clearly declined toward adulthood. In contrast, Dyn I expression levels progressively augmented during maturation. Syt I showed low levels at eye opening and in adults, high levels around the peak of the critical period, and maximal levels at juvenile age. We further determined a role for each molecule in ocular dominance plasticity. CRMP2 and Syt I levels decreased in area 17 upon monocular deprivation, whereas CRMP4 and Dyn I levels remained unaffected. In contrast, binocular removal of pattern vision had no influence on CRMP2 and Syt I expression in kitten area 17. This study illustrates that not the loss of quality of vision through visual deprivation, but disruption of normal binocular visual experience is crucial to induce the observed molecular changes.status: publishe

Immunolocalization of Dynamin I protein in projection neurons of the visual system of the adult cat

© the authors, licensee Libertas Academica Ltd. We here report on the immunolocalization of Dynamin I (Dyn I) in neurons of the visual system of the cat. The lateral geniculate nucleus (LGN) complex displayed abundant Dyn I immunoreactivity in typical relay cells of the X-, Y- and W-pathway. The superficial and deep layers of the superior colliculus were also populated by Dyn I-immunoreactive projection neurons of the W- and Y-cell system. In primary visual areas 17 and 18, many densely packed layer VI neurons were intensely stained. A clear Dyn I signal was also demonstrated in pyramidal neurons of supragranular layers II and III, while layer IV displayed low Dyn I immunoreactivity. Additionally, area 18 displayed larger border pyramidal neurons in layer III compared to area 17. Generally, Dyn I was localized to the cell body and dendrites of neurons, to the neuropil and sometimes also to axon bundles. Typically, the Dyn I signal was not always uniformly distributed within the somatodendritic compartment. Based on its widespread distribution mainly in projection neurons Dyn I may play a fundamental role in mature neurons of different cortical and subcortical structures of the adult mammalian brain.status: publishe

Local cell proliferation upon enucleation in Direct Retinal Brain Targets in the Visual system of the Adult Mouse

In this study we used incorporation of the DNA synthesis marker 5-bromo-2′-deoxyuridine or BrdU to visualize cell proliferation in the visual system of the adult mouse as a response to monocular enucleation. We detected new BrdU-labeled cells in different subcortical retinal target regions and we established a specific time frame in which this cell proliferation occurred. By performing immunofluorescent double stainings for BrdU and different vascular (glucose transporter type 1, collagen type IV), glial (thymosin β 4 , glial fibrillary acidic protein) and neuronal (Neuronal Nuclei, doublecortin) markers, we identified these proliferating cells as activated microglia. Additional immunohistochemical stainings for thymosin β 4 and glial fibrillary acidic protein also revealed reactive astrocytes in the different retinorecipient nuclei and allowed us to delineate a time frame for microglial and astroglial activation. A PCR array experiment further showed increased levels of cytokines, chemokines, growth factors and enzymes that play an important role in microglial-astroglial communication during the glial activation process in response to the deafferentation

Local cell proliferation upon enucleation in direct retinal brain targets in the visual system of the adult mouse

© the author(s), publisher and licensee Libertas Academica Ltd. In this study we used incorporation of the DNA synthesis marker 5-bromo-2'-deoxyuridine or BrdU to visualize cell proliferation in the visual system of the adult mouse as a response to monocular enucleation. We detected new BrdU-labeled cells in different subcortical retinal target regions and we established a specific time frame in which this cell proliferation occurred. By performing immunofluorescent double stainings for BrdU and different vascular (glucose transporter type 1, collagen type IV), glial (thymosin β4, glial fibrillary acidic protein) and neuronal (Neuronal Nuclei, doublecortin) markers, we identified these proliferating cells as activated microglia. Additional immunohistochemical stainings for thymosin β4 and glial fibrillary acidic protein also revealed reactive astrocytes in the different retinorecipient nuclei and allowed us to delineate a time frame for microglial and astroglial activation. A PCR array experiment further showed increased levels of cytokines, chemokines, growth factors and enzymes that play an important role in microglial-astroglial communication during the glial activation process in response to the deafferentation.status: publishe

Retinal lesions induce layer-specific Fos expression changes in cat area 17

Quantitative analysis of the neuronal activity marker Fos revealed activity-dependent and lamina-specific changes in adult cat area 17, 14 days to 1 month after the induction of central retinal lesions. The supra- and infragranular layers were clearly differently engaged in the response to the visual deprivation, both inside and outside the lesion projection zone. The center of the LPZ exhibited an activity decrease in the extragranular layers, which was mainly reflected by an intracellular down-regulation of Fos rather than a decline in the number of Fos-immunoreactive nuclei. Interestingly, the infragranular layers displayed more Fos-immunoreactive neurons in experimental animals. This recruitment of an additional population of Fos expressing neurons in the subcortically projecting infragranular layers might have a protective function against neurodegeneration in the direct retinal target structures.status: publishe