The demise of retinal ganglion cells (RGCs) is characteristic of diseases of the retina such as glaucoma and diabetic or ischemic retinopathies. Pigment epithelium-derived factor (PEDF) is a multifunctional secreted protein that mediates neuroprotection and inhibition of angiogenesis in the retina. We have studied expression and regulation of two of several receptors for PEDF, patatin-like phospholipase 2 gene product/PEDF-R and laminin receptor (LR), in serum-starved RGC under normoxia and hypoxia and investigated their involvement in the survival of retinal neuronal cells. We show that PEDF-R and LR are co-expressed in RGC and R28 retinal precursor cells. Expression of both receptors was enhanced in the presence of complex secretions from retinal glial (Müller) cells and upregulated by VEGF and under hypoxic conditions. PEDF-R- and LR-knocked-down cells demonstrated a markedly attenuated expression of anti-apoptotic Bcl-2 family members (Bcl-2, Bcl-xL) and neuroprotective mediators (PEDF, VEGF, BDNF) suggesting that both PEDF-R and LR mediate pro-survival effects of PEDF on RGC. While this study does not provide evidence for a differential survival-promoting influence of either PEDF-R or LR, it nevertheless highlights the importance of both PEDF receptors for the viability of retinal neurons.

Bergmann Gliazellen reagieren auf elektrische Stimulation der Parallelfasern in Ihrem Soma Kalziumsignalen. Diese Signale sind sensitiv gegenüber den Antagonisten TTX, kalziumfreie Lösung und Cadmium. Ein Antagonist spannungaktivierter Kaliumkanäle, der synaptische Ereignisse verlängert, führt zu erheblich verstärkten Antworten. Um Kalziumantworten in Bergmann Gliazellsomata auszulösen ist eine erheblich stärkere elektrische Stimulation notwendig, als man für Kalziumantworten im Ausläufer der Zellen benötigt Es wurde eine Methode entwickelt, Raum-Zeit-Bilder von Kalziumantworten in Bergmann Gliazellausläufern auf elektrische Stimulation zu erzeugen. Die Darstellungen zeigen zeitlich und räumlich eingeschränkte Reaktionen auf teilen der Ausläufer. Beispiele für das Fortschreiten einer Kalziumwelle vom Ausläufer in das Zellsoma (oder umgekehrt) ließen sich nicht finden. Die räumlich Ausdehnung der beobachteten Signale unterstützt das in Grosche et al. (Grosche, et al, 1999) entwickelte Konzept von Mikrodomänen in Bergmann Gliazellausläufern. Die gemessenen Kalziumsignale sind nicht von den Kalziumspeichern des endoplasmatischen Retikulums abhängig. Dies schließt die Beteiligung G-Protein gekoppelter Rezeporen, die unter Aktivierung von endoplasmatischen IP3-Rezeptoren zu zytosolischen Kalziumsignalen führen, an der Generierung diese Signale aus. Die Beteiligung der Neurotransmitter Adrenalin, Noradrenalin, Histamin, Endothelin, Adenosin-tri-Phosphat und des Neurotransmitters Glutamat auf dem Wege der Aktivierung metabotroper Glutamatrezeptoren an der Detektion stimulationsvermittelter Kalziumsignale wurde über die Applikation spezifischer Antagonisten ausgeschlossen. Die Rolle von Glutamattransportern konnte nicht abschließend geklärt werden, da der Antagonist bei einer alleinigen Applikation zu einer Reduktion des Signals um 20 % führt. Bei einer Applikation mit geleertem endoplasmatischen Retikulum führt er zu einer Steigerung der Signalamplitude um 31.9 %. Die somatischen Kalziumantworten auf elektrische Stimula tion ließen sich zu einem Prozentsatz von 23.7 % durch den Antagonisten CNQX für ionotrope Glutamatrezeptoren vom AMPA/Kainat-Typ blockieren. In Bergmann Gliazellen sind diese Rezeptoren anders als in Neuronen kalziumpermeabel (Müller et al, 1992), da diese Zelle nicht die Untereinheit GluR2 des Kanals exprimieren. Es wurde eine funktionelle Bedeutung dieser Genexpression in der Detektion synaptischer Aktivität nachgewiesen. / Bergmann glia cells react to electrical stimulation of parallel fibers with calcium signals in their somata. These signals are sensitive against antagonists of neuronal activity, namly TTX calcium free solution and cadmium. An antagonist of voltage activated potassium channels, which is able to prolong synaptic events, leads to increased calcium responses. To elicit calcium responses in Bergmann glia somata much stronger stimulation compared to these required to elicit calcium responses in Bergmann glia processes is needed. A method was developed to get space-time images of calcium responses to electrical responses in Bergmann glia processes. The images show reactions restricted in time and space in parts of the processes. Examples of processing of calcium waves from the processes to the somata (or the other way around) could not be found. The special expansion of the observed signals supports the concept of micro domains as developed by Grosche et al. (Grosche, et al, 1999). Calcium signals measured do not d epend on the calcium stores of the endoplasmatic reticulum. This excludes the involvment of G-protein coupled receptors, which lead to cytosolic calcium signals via IP3-receptors, in the generation of these calcium signals. The involvment of the neurotransmitter adrenalin, noradrenalin, histamin, endothelin and adenosin-tri-phosphate in the generation of stimulation induced calcium signals was additionally excluded by the application of specific antagonists. The roll of glutamate transporter could not be fully clarified. The application of the antagonist l-PDC alone leads to a reduction of 20% of the signals amplitude, while the application in presence of emptied endoplasmatic calcium stores leads to an increase by 31.9%. Somatic calcium responses to electrical stimulation were able to be blocked by 23.7 % using the antagonist CNQX specific for ionotrope glutamate receptors of the AMPA/Kainat-Typ. In Bergmann glia cells these receptors differ from the AMPA/Kainat-Typ receptors on neuron with regard to their c alcium permeability (Müller et al, 1992) as result of leak in expression of the GluR2 subunit. A functionel involvment of this specific gene expression in the detection of synaptic activity by Bergmann glia cells was proved.

As funções fisiológicas da proteína prion (PrPc) estão sob ampla investigação e caracterização, especialmente as funções associadas ao desenvolvimento cerebral. Destaca-se que a associação de PrPc com Stress Inducible Protein 1 (STI1), induz neuritogênese e neuroproteção via proteína cinase extracelular reguladora (ERK) e proteína cinase dependente de AMPc (PKA) respectivamente. O presente estudo avaliou como a expressão de PrP cem astrócitos pode modular a interação neurônioglia e o papel de STI1 como um fator autócrino em astrócitos. PrPc modula a interação neurônio-glia, a produção de fatores tróficos solúveis e a organização da laminina secretada na matriz extracelular pelos astrócitos. Desta forma, a expressão de PrP ctanto em astrócitos quanto em neurônios é essencial para a neuritogênese e sobrevivência neuronal. O papel autócrino de STI1 em astrócitos também foi demonstrado. A interação PrPc-STI1 previne a morte celular por ativação da via de PKA, e ativa a diferenciação astrocitária, de uma forma protoplasmática para uma fibrosa pela indução de ERK1/2. De acordo com estes resultados, um menor grau de diferenciação é encontrado em camundongos deficientes para PrPc. Estes apresentam uma expresão reduzida GFAP (proteína fibrilar acídica glial) e aumentada de vimentina e nestina em comparação com aqueles derivados de animais tipo-selvagem. STI1 promove ainda parada da proliferação astrocitária ativando a via de PKC de maneira independente de PrPc. O mecanismo pelo qual STI1 é secretada por astrócitos também foi avaliado e verificou-se que este é independente da via clássica mediada pelo complexo de Golgi. STI1 secretada é encontrada numa forma solúvel e em outra associada a componentes lipídicos e foram caracterizados por microscopia eletrônica como vesículas que variam entre 20-200nm. Dentre as vias de secreção não clássicas dependentes de lipídeos, a via de \"shedding\" de membrana foi descartada visto que STI1 não é secretada em associação com lipoproteínas. STI1 está presente em frações positivas para o receptor de transferrina, Hsp70, Hsp90 e PrPc, sugerindo composição exossomal. Esses resultados indicam que STI1 pode ser classificada como um fator trófico que associado ao seu \"receptor\" ou \"co-receptor\", PrPc, modula a sobrevivência e diferenciação tanto de neurônios quanto de astrócitos / The physiological functions of PrPc are under intense investigation and characterization, particularly those associated with brain development. In neurons, the association of PrPc with its ligand, STI1, induces neuritogenesis and neuroprotection via ERK and PKA signaling pathways, respectively. The present study evaluated whether PrPc expression in astrocytes modulates neuron-glia crosstalk and the autocrine role of STI1 in astrocytes. PrPc modulates neuron-glia interaction, the production and secretion of soluble factors, and the organization of the laminin in the extracellular matrix. PrPc expression in neurons and astrocytes is essential to neuritogenesis and neuronal survival. The autocrine role of STI1 in astrocytes was also demonstrated. The PrPc-STI1 interaction prevents cell death in a PKA-dependent manner, and induces astrocyte differentiation, from a flat to a process-bearing morphology in an ERK1/2 dependent manner. We showed that PrPccnull astrocytes presented a slower rate of astrocyte maturation than wild-type ones, with reduced expression of GFAP and increased vimentin and nestin expression. STI1 inhibited proliferation of both wild-type and PrPCnull astrocytes in a PKC-dependent manner. The mechanisms by which STI1 can be secreted by astrocytes was avaliated and we demonstrated that this secretion is independent on the classical secretory pathway mediated by the Golgi apparatus. Secreted STI1 is found in a soluble form and associated with lipidic compartments and we characterized by electron microscopy as vesicles that range from 20-200nm. Among the non-classical lipid-dependent secretory pathways, STI1 secretion by shedding was ruled out since STI1 was not secreted with lipoprotein fractions. On the other hand, STI1 is present in fractions that are positive for transferrin receptor, Hsp70, Hsp90 and PrPc, suggesting an exosome identity. Taken together, these data indicate that STI1 acts as a neurotrophic factor whose activity is dependent on the expression of PrP c at the neuronal surface, modulating differentiation and survival of both neurons and astrocytes

As funções fisiológicas da proteína prion (PrPc) estão sob ampla investigação e caracterização, especialmente as funções associadas ao desenvolvimento cerebral. Destaca-se que a associação de PrPc com Stress Inducible Protein 1 (STI1), induz neuritogênese e neuroproteção via proteína cinase extracelular reguladora (ERK) e proteína cinase dependente de AMPc (PKA) respectivamente. O presente estudo avaliou como a expressão de PrP cem astrócitos pode modular a interação neurônioglia e o papel de STI1 como um fator autócrino em astrócitos. PrPc modula a interação neurônio-glia, a produção de fatores tróficos solúveis e a organização da laminina secretada na matriz extracelular pelos astrócitos. Desta forma, a expressão de PrP ctanto em astrócitos quanto em neurônios é essencial para a neuritogênese e sobrevivência neuronal. O papel autócrino de STI1 em astrócitos também foi demonstrado. A interação PrPc-STI1 previne a morte celular por ativação da via de PKA, e ativa a diferenciação astrocitária, de uma forma protoplasmática para uma fibrosa pela indução de ERK1/2. De acordo com estes resultados, um menor grau de diferenciação é encontrado em camundongos deficientes para PrPc. Estes apresentam uma expresão reduzida GFAP (proteína fibrilar acídica glial) e aumentada de vimentina e nestina em comparação com aqueles derivados de animais tipo-selvagem. STI1 promove ainda parada da proliferação astrocitária ativando a via de PKC de maneira independente de PrPc. O mecanismo pelo qual STI1 é secretada por astrócitos também foi avaliado e verificou-se que este é independente da via clássica mediada pelo complexo de Golgi. STI1 secretada é encontrada numa forma solúvel e em outra associada a componentes lipídicos e foram caracterizados por microscopia eletrônica como vesículas que variam entre 20-200nm. Dentre as vias de secreção não clássicas dependentes de lipídeos, a via de \"shedding\" de membrana foi descartada visto que STI1 não é secretada em associação com lipoproteínas. STI1 está presente em frações positivas para o receptor de transferrina, Hsp70, Hsp90 e PrPc, sugerindo composição exossomal. Esses resultados indicam que STI1 pode ser classificada como um fator trófico que associado ao seu \"receptor\" ou \"co-receptor\", PrPc, modula a sobrevivência e diferenciação tanto de neurônios quanto de astrócitos / The physiological functions of PrPc are under intense investigation and characterization, particularly those associated with brain development. In neurons, the association of PrPc with its ligand, STI1, induces neuritogenesis and neuroprotection via ERK and PKA signaling pathways, respectively. The present study evaluated whether PrPc expression in astrocytes modulates neuron-glia crosstalk and the autocrine role of STI1 in astrocytes. PrPc modulates neuron-glia interaction, the production and secretion of soluble factors, and the organization of the laminin in the extracellular matrix. PrPc expression in neurons and astrocytes is essential to neuritogenesis and neuronal survival. The autocrine role of STI1 in astrocytes was also demonstrated. The PrPc-STI1 interaction prevents cell death in a PKA-dependent manner, and induces astrocyte differentiation, from a flat to a process-bearing morphology in an ERK1/2 dependent manner. We showed that PrPccnull astrocytes presented a slower rate of astrocyte maturation than wild-type ones, with reduced expression of GFAP and increased vimentin and nestin expression. STI1 inhibited proliferation of both wild-type and PrPCnull astrocytes in a PKC-dependent manner. The mechanisms by which STI1 can be secreted by astrocytes was avaliated and we demonstrated that this secretion is independent on the classical secretory pathway mediated by the Golgi apparatus. Secreted STI1 is found in a soluble form and associated with lipidic compartments and we characterized by electron microscopy as vesicles that range from 20-200nm. Among the non-classical lipid-dependent secretory pathways, STI1 secretion by shedding was ruled out since STI1 was not secreted with lipoprotein fractions. On the other hand, STI1 is present in fractions that are positive for transferrin receptor, Hsp70, Hsp90 and PrPc, suggesting an exosome identity. Taken together, these data indicate that STI1 acts as a neurotrophic factor whose activity is dependent on the expression of PrP c at the neuronal surface, modulating differentiation and survival of both neurons and astrocytes