Biology subjects

Pampliega, O.

Publications and source records attributed to Pampliega, O..

5 recordsLinked to original sources

bioRxiv2024

ANKRD55 interacts with an IFT-B-like complex in microglia

IntroductionSNPs associated with genome-wide risk for multiple sclerosis (MS) modulate expression of ankyrin repeat domain protein 55 (ANKRD55). The function of ANKRD55 is not well understood. A role for ANKRD55 in ciliar transport in multiciliated cells has been reported. To gain deeper insight in how ANKRD55 may modulate neuro-inflammatory parameters, we identified the ANKRD55 interactomes from human neuroblastoma, astrocytic, microglial and monocytic cell lines. MethodsCell lines were transfected with synthetic ANKRD55 RNA in conjunction with nanoparticles. ANKRD55 interactomes were determined by affinity purification coupled to mass spectrometry (AP-MS) and analyzed bioinformatically. Results were validated and interpreted using confocal immunofluorescence microscopy, RNAseq transcriptomics, and a visible immunoprecipitation assay (VIP). ResultsShared among the interactomes were the 14-3-3 isoforms 14-3-3{eta} and 14-3-3{beta}{eta}. Unique to the microglial interactome were eight proteins belonging to the intraflagellar transport complex B (IFT-B). The IFT-B complex is known to mediate anterograde protein trafficking from the base to the tip of cilia. The dimer IFT46-IFT56 was identified as the minimum entity of IFT-B needed to support interaction with ANKRD55. To verify whether ANKRD55 is a ciliar transport protein, we induced ciliogenesis by serum starvation. Primary ARL13B+ cilia could be induced in the astrocytic and neuroblastoma, but not microglial, cell lines. By confocal microscopy, ANKRD55 was not detectable in these cilia but was enriched at the basal body. In the microglial cell line, ANKRD55 and IFT-B components were enriched at the centrosome. In two human primary myeloid cell models, monocyte-derived microglia (MoMG) and monocyte-derived dendritic cells (MoDC), we were able to recapitulate the co-localization of ANKRD55 and IFT81 at the centrosome. DiscussionOur work shows that an ANKRD55 - IFT-B-like complex is assembled in microglial cells. Together with the finding that ANKRD55 was not detected in primary cilia, the results suggest that ANKRD55 is associated with an IFT-B pathway that can operate independent of ciliogenesis.

immunology↗

bioRxiv2022

ASTROCYTE AUTOPHAGY RESPONSE UPON NEURONAL CILIA LOSS IN THE AGING BRAIN

Primary cilia are microtubule-based signaling organelles present in the plasma membrane of most cell types, including mature astrocytes and neurons. However, little is known about the role of this organelle in the mature brain. Data from our lab show that neuronal primary cilia (nPC) is required for soluble amyloid beta oligomer signaling and modulation of autophagy, and that these events are age dependent. Here, we hypothesize that astrocytes react to the loss of nPC and that aging might impact these events. For that purpose, we have characterized morphological changes in astrocytes as well as in the cilium and autophagy of these cells in brain tissue from young and old mice with impaired PC in neurons. Our results show that upon loss of PC in neurons astrocytes become reactive and reduce their lysosomal capacity, an effect that is reinforced with aging. Moreover, aging reduced the pool of ciliated astrocytes, which might impact their ability to react to extracellular events. Overall, our data suggest that the PC might act an intermediary in the communication between astrocytes and neurons. Highlights of the paperO_LIAstrocytes become reactive upon loss of primary cilia in neurons, which is reinforced during aging. C_LIO_LIAstrocytes in the old brain are less ciliated. C_LIO_LILoss of neuronal primary cilia decreases lysosomal capacity in astrocytes in age-dependent manner. C_LI

neuroscience↗

bioRxiv2022

Multiple Ciliary Localization Signals Control INPP5E Ciliary Targeting

Primary cilia are sensory membrane protrusions whose dysfunction causes diseases named ciliopathies. INPP5E is a ciliary phosphoinositide phosphatase mutated in ciliopathies like Joubert syndrome. INPP5E regulates numerous ciliary functions, such as cilium stability, trafficking, signaling, or exovesicle release. Despite its key ciliary roles, how INPP5E accumulates in cilia remains poorly understood. Herein, we show that INPP5E ciliary targeting requires its folded catalytic domain and is controlled by four ciliary localization signals (CLSs), the first two of which we newly discover: LLxPIR motif (CLS1), W383 (CLS2), FDRxLYL motif (CLS3) and CaaX box (CLS4). We answer two long-standing questions in the field. First, partial redundancy between CLS1 and CLS4 explains why CLS4 is dispensable for ciliary targeting. Second, the essential need for CLS2 on the catalytic domain surface clarifies why CLS3 and CLS4 are together insufficient for ciliary accumulation. Furthermore, we reveal that some Joubert syndrome mutations in INPP5E catalytic domain affect its ciliary targeting, and shed light on the mechanisms of action of each CLS. Thus, we find that CLS2 and CLS3 promote interaction with TULP3 and ARL13B, while downregulating CEP164 binding. On the other hand, CLS4 recruits PDE6D, RPGR and ARL13B, and cooperates with CLS1 in ATG16L1 binding. Lastly, we show INPP5E immune synapse targeting is CLS-independent. Altogether, we reveal unusual complexity in INPP5E ciliary targeting mechanisms, likely reflecting its multiple key roles in ciliary biology.

cell biology↗

bioRxiv2021

Amyloid beta oligomers modulate neuronal autophagy through the primary cilium

The major neurodegenerative diseases, like Alzheimers disease (AD), accumulate neuropathogenic proteins that compromise autophagic function. In AD, autophagy contributes to intracellular APP processing and amyloid beta (A{beta}) generation by mutant presenilin-1 (PS1). However, how extracellular soluble A{beta} oligomers (A{beta}o) impact intracellular autophagy is not well understood. The primary cilium (PC), a signaling organelle on the surface of mature neurons and glia, is able to bind A{beta}. Since PC signaling pathways knowingly modify autophagy in non-brain cells, we here investigated the role of neuronal PC in the modulation of autophagy during acute extracellular A{beta}o overload. Our results show that, in vivo, recombinant A{beta}o require the presence of neuronal PC to modulate early autophagy and to induce the accumulation of autophagic vacuoles in an age-dependent manner. We show that activated Akt mediates these effects in an age-dependent manner, and that ciliary p75NTR receptor is required to block autophagy by A{beta}o. These findings demonstrate that neuronal PC in the adult brain participates in the deleterious effects mediated by soluble A{beta}o. The PC should therefore be considered as a target organelle to modulate autophagy for the treatment of neurodegenerative diseases. HighlightsO_LIA{beta}o requires the neuronal PC to impair learning in young and old mice. C_LIO_LIAutophagy in whole hippocampus differs from autophagy response in hippocampal neurons. C_LIO_LIA{beta}o induce autophagolysosome accumulation through primary cilia- and age-dependent Akt phosphorylation. C_LI

neuroscience↗

bioRxiv2019

LUZP1, a novel regulator of primary cilia and the actin cytoskeleton, is altered in Townes-Brocks Syndrome

Primary cilia are sensory organelles that are crucial for cell signaling during development and organ homeostasis. Cilia arise from the centrosome and their formation is governed by numerous regulatory factors. We show that the leucine-zipper protein LUZP1 localizes to the pericentriolar material and actin cytoskeleton. Using TurboID proximity labeling and pulldowns, LUZP1 associates with factors linked to centrosome and actin filaments. Loss of LUZP1 reduces F-actin levels, facilitating ciliogenesis and altering Sonic Hedgehog signaling, pointing to a key role in the cytoskeleton-cilia interdependency. Moreover, we show that LUZP1 interacts with a truncated form of the transcription factor SALL1 that causes Townes-Brocks Syndrome. TBS is characterized by digit, heart and kidney malformations and is linked in part to defective cilia. Truncated SALL1 increases the ubiquitin proteasome-mediated degradation of LUZP1. Alteration of LUZP1 levels may be a contributing factor to TBS, suggesting possible therapies using modulators of cilia and cytoskeletal function.

cell biology↗