Loss of Synaptic Connectivity, Particularly in Second Order Neurons Is a Key Feature of Diabetic Retinal Neuropathy in the Ins2Akita Mouse

Retinal neurodegeneration is a key component of diabetic retinopathy (DR), although the detailed neuronal damage remains ill-defined. Recent evidence suggests that in addition to amacrine and ganglion cell, diabetes may also impact on other retinal neurons. In this study, we examined retinal degenerative changes in Ins2^Akita diabetic mice. In scotopic electroretinograms (ERG), b-wave and oscillatory potentials were severely impaired in 9-month old Ins2^Akita mice. Despite no obvious pathology in fundoscopic examination, optical coherence tomography (OCT) revealed a progressive thinning of the retina from 3 months onwards. Cone but not rod photoreceptor loss was observed in 3-month-old diabetic mice. Severe impairment of synaptic connectivity at the outer plexiform layer (OPL) was detected in 9-month old Ins2Akita mice. Specifically, photoreceptor presynaptic ribbons were reduced by 25% and postsynaptic boutons by 70%, although the density of horizontal, rod- and cone-bipolar cells remained similar to non-diabetic controls. Significant reductions in GABAergic and glycinergic amacrine cells and Brn3a+ retinal ganglion cells were also observed in 9-month old Ins2^Akita mice. In conclusion, the Ins2^Akita mouse develops cone photoreceptor degeneration and the impairment of synaptic connectivity at the OPL, predominately resulting from the loss of postsynaptic terminal boutons. Our findings suggest that the Ins2^Akita mouse is a good model to study diabetic retinal neuropathy.

USP17 is required for clathrin mediated endocytosis of epidermal growth factor receptor

Previously we have shown that expression of the deubiquitinating enzyme USP17 is required for cell proliferation and motility. More recently we reported that USP17 deubiquitinates RCE1 isoform 2 and thus regulates the processing of 'CaaX' motif proteins. Here we now show that USP17 expression is induced by epidermal growth factor and that USP17 expression is required for clathrin mediated endocytosis of epidermal growth factor receptor. In addition, we show that USP17 is required for the endocytosis of transferrin, an archetypal substrate for clathrin mediated endocytosis, and that USP17 depletion impedes plasma membrane recruitment of the machinery required for clathrin mediated endocytosis. Thus, our data reveal that USP17 is necessary for epidermal growth factor receptor and transferrin endocytosis via clathrin coated pits, indicate this is mediated via the regulation of the recruitment of the components of the endocytosis machinery and suggest USP17 may play a general role in receptor endocytosis.

Clathrin is spindle-associated but not essential for mitosis

BACKGROUND: Clathrin is a multimeric protein involved in vesicle coat assembly. Recently clathrin distribution was reported to change during the cell cycle and was found to associate with the mitotic spindle. Here we test whether the recruitment of clathrin to the spindle is indicative of a critical functional contribution to mitosis.

METHODOLOGY/PRINCIPAL FINDINGS: Previously a chicken pre-B lymphoma cell line (DKO-R) was developed in which the endogenous clathrin heavy chain alleles were replaced with the human clathrin heavy chain under the control of a tetracycline-regulatable promoter. Receptor-mediated and fluid-phase endocytosis were significantly inhibited in this line following clathrin knockout, and we used this to explore the significance of clathrin heavy chain expression for cell cycle progression. We confirmed using confocal microscopy that clathrin colocalised with tubulin at mitotic spindles. Using a propidium iodide flow cytometric assay we found no statistical difference in the cell cycle distribution of the knockout cells versus the wild-type. Additionally, we showed that the ploidy and the recovery kinetics following cell cycle arrest with nocodazole were unchanged by repressing clathrin heavy chain expression.

CONCLUSIONS/SIGNIFICANCE: We conclude that the association of clathrin with the mitotic spindle and the contribution of clathrin to endocytosis are evolutionarily conserved. However we find that the contribution of clathrin to mitosis is less robust and dependent on cellular context. In other cell-lines silencing RNA has been used by others to knockdown clathrin expression resulting in an increase in the mitotic index of the cells. We show an effect on the G2/M phase population of clathrin knockdown in HEK293 cells but show that repressing clathrin expression in the DKO-R cell-line has no effect on the size of this population. Consequently this work highlights the need for a more detailed molecular understanding of the recruitment and function of clathrin at the spindle, since the localisation but not the impact of clathrin on mitosis appears to be robust in plants, mammalian and chicken B-cells.

The interplay between clathrin-coated vesicles and cell signalling

Internalization of cargo proteins and lipids at the cell surface occurs in both a constitutive and signal-regulated manner through clathrin-mediated and other endocytic pathways. Clathrin-coated vesicle formation is a principal uptake route in response to signalling events. Protein-lipid and protein-protein interactions control both the targeting of signalling molecules and their binding partners to membrane compartments and the assembly of clathrin coats. An emerging aspect of membrane trafficking research is now addressing how signalling cascades and vesicle coat assembly and subsequently disassembly are integrated.

Role of the AP2 beta-appendage hub in recruiting partners for clathrin-coated vesicle assembly

Adaptor protein complex 2 alpha and beta-appendage domains act as hubs for the assembly of accessory protein networks involved in clathrin-coated vesicle formation. We identify a large repertoire of beta-appendage interactors by mass spectrometry. These interact with two distinct ligand interaction sites on the beta-appendage (the "top" and "side" sites) that bind motifs distinct from those previously identified on the alpha-appendage. We solved the structure of the beta-appendage with a peptide from the accessory protein Eps15 bound to the side site and with a peptide from the accessory cargo adaptor beta-arrestin bound to the top site. We show that accessory proteins can bind simultaneously to multiple appendages, allowing these to cooperate in enhancing ligand avidities that appear to be irreversible in vitro. We now propose that clathrin, which interacts with the beta-appendage, achieves ligand displacement in vivo by self-polymerisation as the coated pit matures. This changes the interaction environment from liquid-phase, affinity-driven interactions, to interactions driven by solid-phase stability ("matricity"). Accessory proteins that interact solely with the appendages are thereby displaced to areas of the coated pit where clathrin has not yet polymerised. However, proteins such as beta-arrestin (non-visual arrestin) and autosomal recessive hypercholesterolemia protein, which have direct clathrin interactions, will remain in the coated pits with their interacting receptors.

COP and clathrin-coated vesicle budding:different pathways, common approaches

Vesicle and tubule transport containers move proteins and lipids from one membrane system to another. Newly forming transport containers frequently have electron-dense coats. Coats coordinate the accumulation of cargo and sculpt the membrane. Recent advances have shown that components of both COP1 and clathrin-adaptor coats share the same structure and the same motif-based cargo recognition and accessory factor recruitment mechanisms, which leads to insights on conserved aspects of coat recruitment, polymerisation and membrane deformation. These themes point to the way in which evolutionarily conserved features underpin these diverse pathways.

EpsinR an AP1/clathrin interacting protein involved in vesicle trafficking

EpsinR is a clathrin-coated vesicle (CCV) enriched 70-kD protein that binds to phosphatidylinositol-4-phosphate, clathrin, and the gamma appendage domain of the adaptor protein complex 1 (AP1). In cells, its distribution overlaps with the perinuclear pool of clathrin and AP1 adaptors. Overexpression disrupts the CCV-dependent trafficking of cathepsin D from the trans-Golgi network to lysosomes and the incorporation of mannose-6-phosphate receptors into CCVs. These biochemical and cell biological data point to a role for epsinR in AP1/clathrin budding events in the cell, just as epsin1 is involved in the budding of AP2 CCVs. Furthermore, we show that two gamma appendage domains can simultaneously bind to epsinR with affinities of 0.7 and 45 microM, respectively. Thus, potentially, two AP1 complexes can bind to one epsinR. This high affinity binding allowed us to identify a consensus binding motif of the form DFxDF, which we also find in gamma-synergin and use to predict that an uncharacterized EF-hand-containing protein will be a new gamma binding partner.

The synapsin cycle:a view from the synaptic endocytic zone

Although the synapsin phosphoproteins were discovered more than 30 years ago and are known to play important roles in neurotransmitter release and synaptogenesis, a complete picture of their functions within the nerve terminal is lacking. It has been shown that these proteins play an important role in the clustering of synaptic vesicles (SVs) at active zones and function as modulators of synaptic strength by acting at both pre- and postdocking levels. Recent studies have demonstrated that synapsins migrate to the endocytic zone of central synapses during neurotransmitter release, which suggests that there are additional functions for these proteins in SV recycling.

Identification of protein complexes in Alzheimer's disease

A Doença de Alzheimer (AD) é a maior doença neurodegenerativa a nível mundial, e a principal causa de demência na população idosa. O processamento da proteína precursora de amilóide (APP) pelas β- e g- secretases origina o peptídeo Aβ, que agrega em oligómeros neurotóxicos e em placas senis. Estes são eventos-chave na patogénese da DA que levam à rutura da neurotransmissão sináptica, morte neuronal e inflamação neuronal do hipocampo e córtex cerebral, causando perda de memória disfunção cognitiva geral. Apesar dos grandes avanços no conhecimento do papel do processamento da APP na DA, a sua função fisiológica ainda não foi totalmente elucidada. Os mapas de interações proteína-proteína (PPI) humanos têm desempenhado um papel importante na investigação biomédica, em particular no estudo de vias de sinalização e de doenças humanas. O método dois-híbrido em levedura (YTH) consiste numa plataforma para a produção rápida de redes de PPI em larga-escala. Neste trabalho foram realizados vários rastreios YTH com o objetivo de identificar proteínas específicas de cérebro humano que interagissem com a APP, ou com o seu domínio intracelular (AICD), tanto o tipo selvagem como com os mutantes Y687F, que mimetizam o estado desfosforilado do resíduo Tyr-687. De facto, a endocitose da APP e a produção de Aβ estão dependentes do estado de fosforilação da Tyr-687. Os rastreios YTH permitiram assim obter de redes proteínas que interagem com a APP, utilizando como “isco” a APP, APPY687F e AICDY687F. Os clones positivos foram isolados e identificados através de sequenciação do cDNA. A maior parte dos clones identificados, 118, correspondia a sequências que codificam para proteínas conhecidas, resultando em 31 proteínas distintas. A análise de proteómica funcional das proteínas identificadas neste estudo e em dois projetos anteriores (AICDY687E, que mimetiza a fosforilação, e AICD tipo selvagem), permitiram avaliar a relevância da fosforilação da Tyr-687. Três clones provenientes do rastreio YTH com a APPY687F foram identificados como um novo transcrito da proteína Fe65, resultante de splicing alternativo, a Fe65E3a (GenBank Accession: EF103274), que codifica para a isoforma p60Fe65. A p60Fe65 está enriquecida no cérebro e os seus níveis aumentam durante a diferenciação neuronal de células PC12, evidenciando o potencial papel que poderá desempenhar na patologia da DA. A RanBP9 é uma proteína nuclear e citoplasmática envolvida em diversas vias de sinalização celulares. Neste trabalho caracterizou-se a nova interação entre a RanBP9 e o AICD, que pode ser regulada pela fosforilação da Tyr-687. Adicionalmente, foi identificada uma nova interação entre a RanBP9 e a acetiltransferase de histonas Tip60. Demonstrou-se ainda que a RanBP9 tem um efeito de regulação inibitório na transcrição mediada por AICD, através da interação com a Tip60, afastando o AICD dos locais de transcrição ativos. O estudo do interactoma da APP/AICD, modelado pela fosforilação da Tyr-687, revela que a APP poderá estar envolvida em novas vias celulares, contribuindo não só para o conhecimento do papel fisiológico da APP, como também auxilia a revelar as vias que levam à agregação de Aβ e neurodegeneração. A potencial relevância deste trabalho relaciona-se com a descoberta de algumas interações proteicas/vias de sinalização que podem que podem ser relevantes para o desenvolvimento de novas estratégias terapêuticas na DA.

Synaptic dysfunction in early encephalopathies : from genes to function

Tese de doutoramento, Medicina (Neurologia), Universidade de Lisboa, Faculdade de Medicina, 2015

Molecular mechanisms controlling synaptic recruitment of GluA4 subunit-containing AMPAreceptors

Synaptic recruitment of AMPA receptors (AMPARs) represents a key postsynaptic mechanism driving functional development and maturation of glutamatergic synapses. At immature hippocampal synapses, PKA-driven synaptic insertion of GluA4 is the predominant mechanism for synaptic reinforcement. However, the physiological significance and molecular determinants of this developmentally restricted form of plasticity are not known. Here we show that PKA activation leads to insertion of GluA4 to synaptic sites with initially weak or silent AMPAR-mediated transmission. This effect depends on a novel mechanism involving the extreme C-terminal end of GluA4, which interacts with the membrane proximal region of the C-terminal domain to control GluA4 trafficking. In the absence of GluA4, strengthening of AMPAR-mediated transmission during postnatal development was significantly delayed. These data suggest that the GluA4-mediated activation of silent synapses is a critical mechanism facilitating the functional maturation of glutamatergic circuitry during the critical period of experience-dependent fine-tuning.

Behavioral and synaptic circuit analysis in models of neuropsychiatric disorders: Dissecting the in vivo role of the postsynaptic density proteins nArgBP2 and Shank3 using genetic engineered mice

Dissertation presented to obtain the Ph.D degree in Biology

Immunogold Detection of L-glutamate and D-serine in Small Synaptic-Like Microvesicles in Adult Hippocampal Astrocytes.

Glutamate and the N-methyl-D-aspartate receptor ligand D-serine are putative gliotransmitters. Here, we show by immunogold cytochemistry of the adult hippocampus that glutamate and D-serine accumulate in synaptic-like microvesicles (SLMVs) in the perisynaptic processes of astrocytes. The estimated concentration of fixed glutamate in the astrocytic SLMVs is comparable to that in synaptic vesicles of excitatory nerve terminals (∼45 and ∼55 mM, respectively), whereas the D-serine level is about 6 mM. The vesicles are organized in small spaced clusters located near the astrocytic plasma membrane. Endoplasmic reticulum is regularly found in close vicinity to SLMVs, suggesting that astrocytes contain functional nanodomains, where a local Ca(2+) increase can trigger release of glutamate and/or D-serine.

Etude des propriétés dynamiques de la sécrétion régulée des vésicules glutamatergiques des astrocytes

RESUME GRAND PUBLICLe cerveau est composé de différents types cellulaires, dont les neurones et les astrocytes. Faute de moyens pour les observer, les astrocytes sont très longtemps restés dans l'ombre alors que les neurones, bénéficiant des outils ad hoc pour être stimulés et étudiés, ont fait l'objet de toutes les attentions. Le développement de l'imagerie cellulaire et des outils fluorescents ont permis d'observer ces cellules non électriquement excitables et d'obtenir des informations qui laissent penser que ces cellules sont loin d'être passives et participent activement au fonctionnement cérébral. Cette participation au fonctionnement cérébral se fait en partie par le biais de la libération de substances neuro-actives (appellées gliotransmetteurs) que les astrocytes libèrent à proximité des synapses permettant ainsi de moduler le fonctionnement neuronal. Cette libération de gliotransmetteurs est principalement causée par l'activité neuronale que les astrocytes sont capables de sentir. Néanmoins, nous savons encore peu de chose sur les propriétés précises de la libération des gliotransmetteurs. Comprendre les propriétés spatio-temporelles de cette libération est essentiel pour comprendre le mode de communication de ces cellules et leur implication dans la transmission de l'information cérébrale. En utilisant des outils fluorescents récemment développés et en combinant différentes techniques d'imagerie cellulaire, nous avons pu obtenir des informations très précises sur la libération de ces gliotransmetteurs par les astrocytes. Nous avons ainsi confirmé que cette libération était un processus très rapide et qu'elle était contrôlée par des augmentations de calcium locales et rapides. Nous avons également décrit une organisation complexe de la machinerie supportant la libération des gliotransmetteurs. Cette organisation complexe semble être à la base de la libération extrêmement rapide des gliotransmetteurs. Cette rapidité de libération et cette complexité structurelle semblent indiquer que les astrocytes sont des cellules particulièrement adaptées à une communication rapide et qu'elles peuvent, au même titre que les neurones dont elles seraient les partenaires légitimes, participer à la transmission et à l'intégration de l'information cérébrale.RESUMEDe petites vésicules, les « SLMVs » ou « Synaptic Like MicroVesicles », exprimant des transporteurs vésiculaires du glutamate (VGluTs) et libérant du glutamate par exocytose régulée, ont récemment été décrites dans les astrocytes en culture et in situ. Néanmoins, nous savons peu de chose sur les propriétés précises de la sécrétion de ces SLMVs. Contrairement aux neurones, le couplage stimulussécrétion des astrocytes n'est pas basé sur l'ouverture des canaux calciques membranaires mais nécessite l'intervention de seconds messagers et la libération du calcium par le reticulum endoplasmique (RE). Comprendre les propriétés spatio-temporelles de la sécrétion astrocytaire est essentiel pour comprendre le mode de communication de ces cellules et leur implication dans la transmission de l'information cérébrale. Nous avons utilisé des outils fluorescents récemment développés pour étudier le recyclage des vésicules synaptiques glutamatergiques comme les colorants styryles et la pHluorin afin de pouvoir suivre la sécrétion des SLMVs à l'échelle de la cellule mais également à l'échelle des évènements. L'utilisation combinée de l'épifluorescence et de la fluorescence à onde évanescente nous a permis d'obtenir une résolution temporelle et spatiale sans précédent. Ainsi avons-nous confirmé que la sécrétion régulée des astrocytes était un processus très rapide (de l'ordre de quelques centaines de millisecondes). Nous avons découvert que cette sécrétion est contrôlée par des augmentations de calcium locales et rapides. Nous avons également décrit des compartiments cytosoliques délimités par le RE à proximité de la membrane plasmique et contenant les SLMVs. Cette organisation semble être à la base du couplage rapide entre l'activation des GPCRs et la sécrétion. L'existence de compartiments subcellulaires indépendants permettant de contenir les messagers intracellulaires et de limiter leur diffusion semble compenser de manière efficace la nonexcitabilité électrique des astrocytes. Par ailleurs, l'existence des différents pools de vésicules recrutés séquentiellement et fusionnant selon des modalités distinctes ainsi que l'existence de mécanismes permettant le renouvellement de ces pools lors de la stimulation suggèrent que les astrocytes peuvent faire face à une stimulation soutenue de leur sécrétion. Ces données suggèrent que la libération de gliotransmetteurs par exocytose régulée n'est pas seulement une propriété des astrocytes en culture mais bien le résultat d'une forte spécialisation de ces cellules pour la sécrétion. La rapidité de cette sécrétion donne aux astrocytes toutes les compétences pour pouvoir intervenir de manière active dans la transmission et l'intégration de l'information.ABSTRACTRecently, astrocytic synaptic like microvesicles (SLMVs), that express vesicular glutamate transporters (VGluTs) and are able to release glutamate by Ca2+-dependent regulated exocytosis, have been described both in tissue and in cultured astrocytes. Nevertheless, little is known about the specific properties of regulated secretion in astrocytes. Important differences may exist between astrocytic and neuronal exocytosis, starting from the fact that stimulus-secretion coupling in astrocytes is voltage independent, mediated by G-protein-coupled receptors and the release of Ca2+ from internal stores. Elucidating the spatiotemporal properties of astrocytic exo-endocytosis is, therefore, of primary importance for understanding the mode of communication of these cells and their role in brain signaling. We took advantage of fluorescent tools recently developed for studying recycling of glutamatergic vesicles at synapses like styryl dyes and pHluorin in order to follow exocytosis and endocytosis of SLMVs at the level of the entire cell or at the level of single event. We combined epifluorescence and total internal reflection fluorescence imaging to investigate, with unprecedented temporal and spatial resolution, the events underlying the stimulus-secretion in astrocytes. We confirmed that exo-endocytosis process in astrocytes proceeds with a time course on the millisecond time scale. We discovered that SLMVs exocytosis is controlled by local and fast Ca2+ elevations; indeed submicrometer cytosolic compartments delimited by endoplasmic reticulum (ER) tubuli reaching beneath the plasma membrane and containing SLMVs. Such complex organization seems to support the fast stimulus-secretion coupling reported here. Independent subcellular compartments formed by ER, SLMVs and plasma membrane containing intracellular messengers and limiting their diffusion seem to compensate efficiently the non-electrical excitability of astrocytes. Moreover, the existence of two pools of SLMVs which are sequentially recruited suggests a compensatory mechanisms allowing the refill of SLMVs and supporting exocytosis process over a wide range of multiple stimuli. These data suggest that regulated secretion is not only a feature of cultured astrocytes but results from a strong specialization of these cells. The rapidity of secretion demonstrates that astrocytes are able to actively participate in brain information transmission and processing.

Dysregulated ADAM10-Mediated Processing of APP during a Critical Time Window Leads to Synaptic Deficits in Fragile X Syndrome.

The Fragile X mental retardation protein (FMRP) regulates neuronal RNA metabolism, and its absence or mutations leads to the Fragile X syndrome (FXS). The β-amyloid precursor protein (APP) is involved in Alzheimer's disease, plays a role in synapse formation, and is upregulated in intellectual disabilities. Here, we show that during mouse synaptogenesis and in human FXS fibroblasts, a dual dysregulation of APP and the α-secretase ADAM10 leads to the production of an excess of soluble APPα (sAPPα). In FXS, sAPPα signals through the metabotropic receptor that, activating the MAP kinase pathway, leads to synaptic and behavioral deficits. Modulation of ADAM10 activity in FXS reduces sAPPα levels, restoring translational control, synaptic morphology, and behavioral plasticity. Thus, proper control of ADAM10-mediated APP processing during a specific developmental postnatal stage is crucial for healthy spine formation and function(s). Downregulation of ADAM10 activity at synapses may be an effective strategy for ameliorating FXS phenotypes.