16 research outputs found
Exploring a role for a Par3/CaMKII protein complex in photoreceptor cell polarity and ciliogenesis
Cell polarity is an essential property of adult neurons, which rely on asymmetric distribution of receptors and transmitters for proper signal propagation and cell function. In the retina, loss of photoreceptor (PR) polarity can lead to retinal dystrophies such as Leber Congenital Amaurosis, but the molecular mechanisms involved in regulating PR polarity remain unclear. A highly conserved protein complex involved in the establishment of cell polarity from C. elegans to mammals is the Par complex. Localized at the subapical region of polarized cells, it is composed of the âpartitioning defectiveâ PDZ domain-containing proteins Par3/Par6 and the atypical protein kinase C (aPKC). Although extensively studied in epithelial cells, the role of the Par complex in mammalian neurons remains poorly understood. Our unpublished results indicate that conditional inactivation (cKO) of Par3 in the developing retina interferes with the polarized growth of the photosensitive cilium at the apical tip of PR cells, eventually leading to PR degeneration. To uncover how Par3 might regulate ciliogenesis in PR cells, we immunoprecipitated Par3 from mouse retinal extracts and carried out mass spectrometry analysis. We found a cluster of calcium/calmodulin-dependent protein kinase II (CaMKII) proteins as potential Par3-interacting partners in the retina. CaMKII is one of the most abundant proteins found in the central nervous system, where it constitutes 1-2% of total proteins. While extensive studies have demonstrated the importance of CaMKII in long-term potentiation (LTP), long term depression (LTD) and dendrite arborisation, its role in cell polarity remains unknown. Using tagged versions of Par3 and CaMKIID, we validated their interaction in vivo and in vitro by co-immunoprecipitation. Interestingly, we found that CaMKIID localizes to the ciliary region of PRs, suggesting that Par3 might recruit CaMKIID at the apical membrane of PR cells, where it could be involved in ciliogenesis. To explore this hypothesis, we investigated whether dominant-negative or constitutively active forms of CaMKIID could impact cilia formation in PRs. Interestingly, overexpression of both mutant forms of CaMKIID during PR development resulted in shortening of the photosensitive cilia (outer segments), similar to what we observed in Par3 cKO retinas. This study suggests that a CaMKIID/Par3 protein complex regulates the establishment of PR cell polarity, raising the possibility that this complex may be generally involved in controlling neuronal polarity throughout the nervous system.Le traitement et la propagation de lâinformation nerveuse repose sur une distribution
asymĂ©trique de rĂ©cepteurs et dâĂ©metteurs Ă la surface de chaque neurone. Ce cloisonnement en
domaines sous-cellulaires distincts est également appelé polarité cellulaire. Dans la rétine, la
perte de polarité des photorécepteurs peut entraßner des dystrophies rétiniennes telle que
l'amaurose congénitale de Leber, mais les mécanismes moléculaires impliqués restent flous. Un
complexe protéique impliqué dans l'établissement de la polarité cellulaire, hautement conservé
de C. elegans aux mammifÚres, est le complexe PAR. Localisé au niveau de la région sous-apicale
des cellules polarisées, le coeur de ce complexe est constitué des protéines de la famille
partitioning defective Par3 / Par6 et de la protéine kinase C atypique aPKC. Bien que largement
étudié dans les cellules épithéliales, le rÎle du complexe Par dans les neurones de mammifÚres
reste mal compris. Nos résultats indiquent que l'inactivation conditionnelle (cKO) de Par3 dans la
rétine de souris en développement interfÚre avec la croissance polarisée du cil photosensible à la
pointe apicale des cellules photoréceptrices (PR), conduisant finalement à une dégénérescence
des PRs. Pour découvrir comment Par3 pourrait réguler la ciliogenÚse des PRs, nous avons
immunoprécipité Par3 à partir d'extraits rétiniens de souris et effectué une analyse par
spectrométrie de masse. Nous avons trouvé un ensemble de protéines appartenant à la famille
des calcium-calmoduline-dépendantes de la protéine kinase II (CaMKII) comme partenaires
potentiels de Par3 dans la rétine. Les CaMKII figurent parmi les protéines les plus abondantes du
systĂšme nerveux central oĂč elles constituent 1 Ă 2% des protĂ©ines totales. Alors que des Ă©tudes
approfondies ont dĂ©montrĂ© l'importance de CaMKII dans la potentialisation et la dĂ©pression Ă
long terme (LTP et LTD), et l'arborisation des dendrites, son rÎle dans la polarité cellulaire reste
inconnu. En utilisant des versions étiquetées de Par3 et CaMKIID, nous avons validé leur
interaction in vivo et in vitro par co-immunoprécipitation. Nous avons mis en évidence une
localisation de CaMKIID dans la région ciliaire des PR, suggérant que Par3 pourrait recruter
CaMKIID Ă la membrane apicale des cellules PR, oĂč il pourrait ĂȘtre impliquĂ© dans la ciliogenĂšse.
Pour explorer cette hypothÚse, nous avons étudié si les formes dominantes négatives ou
constitutivement actives de CaMKIID pouvaient avoir un impact sur la formation des cils des PRs.
vii
La surexpression des deux formes mutantes au cours du développement des PRs a entrainé un
raccourcissement des segments externes, semblable à ce que nous avons observé dans les rétines
Par3 cKO. Cette étude montre qu'un complexe de protéines CaMKIID / Par3 pourrait réguler
lâĂ©tablissement et le maintien de polaritĂ© des PRs, suggĂ©rant lâimplication ce complexe dans le
contrĂŽle de la polaritĂ© neuronale de lâensemble du systĂšme nerveux central
Lysosomal dysfunction disrupts presynaptic maintenance and restoration of presynaptic function prevents neurodegeneration in lysosomal storage diseases
Lysosomal storage disorders (LSDs) are inherited diseases characterized by lysosomal dysfunction and often showing a neurodegenerative course. There is no cure to treat the central nervous system in LSDs. Moreover, the mechanisms driving neuronal degeneration in these pathological conditions remain largely unknown. By studying mouse models of LSDs, we found that neurodegeneration develops progressively with profound alterations in presynaptic structure and function. In these models, impaired lysosomal activity causes massive perikaryal accumulation of insoluble α-synuclein and increased proteasomal degradation of cysteine string protein α (CSPα). As a result, the availability of both α-synuclein and CSPα at nerve terminals strongly decreases, thus inhibiting soluble NSF attachment receptor (SNARE) complex assembly and synaptic vesicle recycling. Aberrant presynaptic SNARE phenotype is recapitulated in mice with genetic ablation of one allele of both CSPα and α-synuclein. The overexpression of CSPα in the brain of a mouse model of mucopolysaccharidosis type IIIA, a severe form of LSD, efficiently re-established SNARE complex assembly, thereby ameliorating presynaptic function, attenuating neurodegenerative signs, and prolonging survival. Our data show that neurodegenerative processes associated with lysosomal dysfunction may be presynaptically initiated by a concomitant reduction in α-synuclein and CSPα levels at nerve terminals. They also demonstrate that neurodegeneration in LSDs can be slowed down by re-establishing presynaptic functions, thus identifying synapse maintenance as a novel potentially druggable target for brain treatment in LSDs
miR-181a/b downregulation exerts a protective action on mitochondrial disease models.
Mitochondrial diseases (MDs) are a heterogeneous group of devastating and often fatal disorders due to defective oxidative phosphorylation. Despite the recent advances in mitochondrial medicine, effective therapies are still not available for these conditions. Here, we demonstrate that the microRNAs miR-181a and miR-181b (miR-181a/b) regulate key genes involved in mitochondrial biogenesis and function and that downregulation of these miRNAs enhances mitochondrial turnover in the retina through the coordinated activation of mitochondrial biogenesis and mitophagy. We thus tested the effect of miR-181a/b inactivation in different animal models of MDs, such as microphthalmia with linear skin lesions and Leber\u27s hereditary optic neuropathy. We found that miR-181a/b downregulation strongly protects retinal neurons from cell death and significantly ameliorates the disease phenotype in all tested models. Altogether, our results demonstrate that miR-181a/b regulate mitochondrial homeostasis and that these miRNAs may be effective gene-independent therapeutic targets for MDs characterized by neuronal degeneration
miR-181a/b downregulation exerts a protective action on mitochondrial disease models.
Mitochondrial diseases (MDs) are a heterogeneous group of devastating and often fatal disorders due to defective oxidative phosphorylation. Despite the recent advances in mitochondrial medicine, effective therapies are still not available for these conditions. Here, we demonstrate that the microRNAs miR-181a and miR-181b (miR-181a/b) regulate key genes involved in mitochondrial biogenesis and function and that downregulation of these miRNAs enhances mitochondrial turnover in the retina through the coordinated activation of mitochondrial biogenesis and mitophagy. We thus tested the effect of miR-181a/b inactivation in different animal models of MDs, such as microphthalmia with linear skin lesions and Leber's hereditary optic neuropathy. We found that miR-181a/b downregulation strongly protects retinal neurons from cell death and significantly ameliorates the disease phenotype in all tested models. Altogether, our results demonstrate that miR-181a/b regulate mitochondrial homeostasis and that these miRNAs may be effective gene-independent therapeutic targets for MDs characterized by neuronal degeneration.Italian Fondazione Telethon (grant no. TGM16YGM02 to S. Ban, the Fondazione Roma (grant no. RPâ201300000009 to S. Ban)) and the AFMâTelethon (grant no. 20685 to B.F.). A.I. received an Umberto Veronesi Fellowship. This research was carried out in the frame of Programme STAR, financially supported by UniNA and Compagnia di San Paolo (Bando STAR, 16âCSPâUNINAâ048, to A.I)
CREB3L1-mediated functional and structural adaptation of the secretory pathway in hormone-stimulated thyroid cells
Many secretory cells increase the synthesis and secretion of cargo proteins in response to specific stimuli. How cells couple increased cargo load with a coordinate rise in secretory capacity to ensure efficient transport is not well understood. We used thyroid cells stimulated with thyrotropin (TSH) to demonstrate a coordinate increase in the production of thyroid-specific cargo proteins and ER-Golgi transport factors, and a parallel expansion of the Golgi complex. TSH also increased expression of the CREB3L1 transcription factor, which alone caused amplified transport factor levels and Golgi enlargement. Furthermore, CREB3L1 potentiated the TSH-induced increase in Golgi volume. A dominant-negative CREB3L1 construct hampered the ability of TSH to induce Golgi expansion, implying that this transcription factor contributes to Golgi expansion. Our findings support a model in which CREB3L1 acts as a downstream effector of TSH to regulate the expression of cargo proteins, and simultaneously increases the synthesis of transport factors and the expansion of the Golgi to synchronize the rise in cargo load with the amplified capacity of the secretory pathway.Fil: GarcĂa, Iris Alejandra. Consejo Nacional de Investigaciones CientĂficas y TĂ©cnicas. Centro CientĂfico TecnolĂłgico CĂłrdoba. Centro de Investigaciones en BioquĂmica ClĂnica e InmunologĂa; Argentina. Universidad Nacional de CĂłrdoba. Facultad de Cs.quĂmicas. Departamento de BioquĂmica; ArgentinaFil: Torres Demichelis, Vanina Andrea. Consejo Nacional de Investigaciones CientĂficas y TĂ©cnicas. Centro CientĂfico TecnolĂłgico CĂłrdoba. Centro de Investigaciones en BioquĂmica ClĂnica e InmunologĂa; Argentina. Universidad Nacional de CĂłrdoba. Facultad de Cs.quĂmicas. Departamento de BioquĂmica; ArgentinaFil: Viale, Diego Luis. Universidad Nacional de San MartĂn. Escuela de Ciencia y TecnologĂa; Argentina. Consejo Nacional de Investigaciones CientĂficas y TĂ©cnicas; ArgentinaFil: Di Giusto, Pablo. Universidad Nacional de CĂłrdoba. Facultad de Cs.quĂmicas. Departamento de BioquĂmica; Argentina. Consejo Nacional de Investigaciones CientĂficas y TĂ©cnicas. Centro CientĂfico TecnolĂłgico CĂłrdoba. Centro de Investigaciones en BioquĂmica ClĂnica e InmunologĂa; ArgentinaFil: Ezhova, Yulia. Telethon Institute of Genetics and Medicine; ItaliaFil: Polishchuk, Roman S.. Telethon Institute of Genetics and Medicine; ItaliaFil: Sampieri, Luciana. Consejo Nacional de Investigaciones CientĂficas y TĂ©cnicas. Centro CientĂfico TecnolĂłgico CĂłrdoba. Centro de Investigaciones en BioquĂmica ClĂnica e InmunologĂa; Argentina. Universidad Nacional de CĂłrdoba. Facultad de Ciencias QuĂmicas. Departamento de BioquĂmica ClĂnica; ArgentinaFil: Martinez, HernĂĄn. Consejo Nacional de Investigaciones CientĂficas y TĂ©cnicas. Centro CientĂfico TecnolĂłgico CĂłrdoba. Centro de Investigaciones en BioquĂmica ClĂnica e InmunologĂa; Argentina. Universidad Nacional de CĂłrdoba. Facultad de Ciencias QuĂmicas. Departamento de BioquĂmica ClĂnica; ArgentinaFil: Sztul, Elizabeth. University of Alabama at Birmingham; Estados UnidosFil: Alvarez, Cecilia Ines. Consejo Nacional de Investigaciones CientĂficas y TĂ©cnicas. Centro CientĂfico TecnolĂłgico CĂłrdoba. Centro de Investigaciones en BioquĂmica ClĂnica e InmunologĂa; Argentina. Universidad de Buenos Aires. Facultad de Farmacia y BioquĂmica. Departamento de BioquĂmica ClĂnica; Argentin
Ouabain-Induced Gene Expression Changes in Human iPSC-Derived Neuron Culture Expressing Dopamine and cAMP-Regulated Phosphoprotein 32 and GABA Receptors
Cardiotonic steroids (CTS) are specific inhibitors and endogenous ligands of a key enzyme in the CNSâthe Na+, K+-ATPase, which maintains and creates an ion gradient on the plasma membrane of neurons. CTS cause the activation of various signaling cascades and changes in gene expression in neurons and other cell types. It is known that intracerebroventricular injection of cardiotonic steroid ouabain causes mania-like behavior in rodents, in part due to activation of dopamine-related signaling cascades in the dopamine and cAMP-regulated phosphoprotein 32 (DARPP-32) expressing medium spiny neurons in the striatum. Dopaminergic projections in the striatum innervate these GABAergic medium spiny neurons. The objective of this study was to assess changes in the expression of all genes in human iPSC-derived expressing DARPP-32 and GABA receptors neurons under the influence of ouabain. We noted a large number of statistically significant upregulated and downregulated genes after a 16-h incubation with non-toxic concentration (30 nM) of ouabain. These changes in the transcriptional activity were accomplished with activation of MAP-kinase ERK1/2 and transcriptional factor cAMP response element-binding protein (CREB). Thus, it can be concluded that 30 nM ouabain incubated for 16 h with human iPSC-derived expressing DARPP-32 and GABA receptors neurons activates genes associated with neuronal maturation and synapse formation, by increasing the expression of genes associated with translation, vesicular transport, and increased electron transport chain function. At the same time, the expression of genes associated with proliferation, migration, and early development of neurons decreases. These data indicate that non-toxic concentrations of ouabain may induce neuronal maturation, neurite growth, and increased synaptogenesis in dopamine-receptive GABAergic neurons, suggesting formation of plasticity and the establishment of new neuronal junctions
Ouabain-Induced Gene Expression Changes in Human iPSC-Derived Neuron Culture Expressing Dopamine and cAMP-Regulated Phosphoprotein 32 and GABA Receptors
Cardiotonic steroids (CTS) are specific inhibitors and endogenous ligands of a key enzyme in the CNSâthe Na+, K+-ATPase, which maintains and creates an ion gradient on the plasma membrane of neurons. CTS cause the activation of various signaling cascades and changes in gene expression in neurons and other cell types. It is known that intracerebroventricular injection of cardiotonic steroid ouabain causes mania-like behavior in rodents, in part due to activation of dopamine-related signaling cascades in the dopamine and cAMP-regulated phosphoprotein 32 (DARPP-32) expressing medium spiny neurons in the striatum. Dopaminergic projections in the striatum innervate these GABAergic medium spiny neurons. The objective of this study was to assess changes in the expression of all genes in human iPSC-derived expressing DARPP-32 and GABA receptors neurons under the influence of ouabain. We noted a large number of statistically significant upregulated and downregulated genes after a 16-h incubation with non-toxic concentration (30 nM) of ouabain. These changes in the transcriptional activity were accomplished with activation of MAP-kinase ERK1/2 and transcriptional factor cAMP response element-binding protein (CREB). Thus, it can be concluded that 30 nM ouabain incubated for 16 h with human iPSC-derived expressing DARPP-32 and GABA receptors neurons activates genes associated with neuronal maturation and synapse formation, by increasing the expression of genes associated with translation, vesicular transport, and increased electron transport chain function. At the same time, the expression of genes associated with proliferation, migration, and early development of neurons decreases. These data indicate that non-toxic concentrations of ouabain may induce neuronal maturation, neurite growth, and increased synaptogenesis in dopamine-receptive GABAergic neurons, suggesting formation of plasticity and the establishment of new neuronal junctions
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The Amyloid Inhibitor CLR01 Relieves Autophagy and Ameliorates Neuropathology in a Severe Lysosomal Storage Disease.
Lysosomal storage diseases (LSDs) are inherited disorders caused by lysosomal deficiencies and characterized by dysfunction of the autophagy-lysosomal pathway (ALP) often associated with neurodegeneration. No cure is currently available to treat neuropathology in LSDs. By studying a mouse model of mucopolysaccharidosis (MPS) type IIIA, one of the most common and severe forms of LSDs, we found that multiple amyloid proteins including α-synuclein, prion protein (PrP), Tau, and amyloid ÎČ progressively aggregate in the brain. The amyloid deposits mostly build up in neuronal cell bodies concomitantly with neurodegeneration. Treating MPS-IIIA mice with CLR01, a "molecular tweezer" that acts as a broad-spectrum inhibitor of amyloid protein self-assembly reduced lysosomal enlargement and re-activates autophagy flux. Restoration of the ALP was associated with reduced neuroinflammation and amelioration of memory deficits. Together, these data provide evidence that brain deposition of amyloid proteins plays a gain of neurotoxic function in a severe LSD by affecting the ALP and identify CLR01 as new potent drug candidate for MPS-IIIA and likely for other LSDs