Defective proteostasis is a hallmark of aging cells and tissues. Among the different components of the proteostasis network, in this study, we focus on a selective form of autophagy known as chaperone-mediated autophagy (CMA), and we set out to understand its physiological role in the retina. Using mice deficient for CMA [knockout for lysosome-associated membrane protein type 2A (Lamp2A)], we have found that CMA blockade leads to impaired visual function, altered retinal proteostasis, and photoreceptor cell death. Conversely, mice that overexpress human LAMP2A show higher resistance to chemically induced photoreceptor degeneration and slower visual function decline. We found a similar protective effect against retinal degeneration upon pharmacological activation of CMA. To start elucidating the mechanisms behind CMA's protective role in the retina, we used comparative proteomics and found elevated levels of enzymes related with glucose metabolism in CMA-deficient retinas that phenocopy those observed in old mice retinas. Overall, our results highlight a cytoprotective role for CMA in retina, in part through proteostatic regulation of enzymes involved in glucose metabolism, and support the feasibility of pharmacologically upregulating CMA against retinal degeneration.

Using constitutive GRF1/2 knockout mice, we showed previously that GRF2 is a key regulator of nuclear migration in retinal cone photoreceptors. To evaluate the functional relevance of that cellular process for two putative targets of the GEF activity of GRF2 (RAC1 and CDC42), here we compared the structural and functional retinal phenotypes resulting from conditional targeting of RAC1 or CDC42 in the cone photoreceptors of constitutive GRF2KO and GRF2WT mice. We observed that single RAC1 disruption did not cause any obvious morphological or physiological changes in the retinas of GRF2WT mice, and did not modify either the phenotypic alterations previously described in the retinal photoreceptor layer of GRF2KO mice. In contrast, the single ablation of CDC42 in the cone photoreceptors of GRF2WT mice resulted in clear alterations of nuclear movement that, unlike those of the GRF2KO retinas, were not accompanied by electrophysiological defects or slow, progressive cone cell degeneration. On the other hand, the concomitant disruption of GRF2 and CDC42 in the cone photoreceptors resulted, somewhat surprisingly, in a normalized pattern of nuclear positioning/movement, similar to that physiologically observed in GRF2WT mice, along with worsened patterns of electrophysiological responses and faster rates of cell death/disappearance than those previously recorded in single GRF2KO cone cells. Interestingly, the increased rates of cone cell apoptosis/death observed in single GRF2KO and double-knockout GRF2KO/CDC42KO retinas correlated with the electron microscopic detection of significant ultrastructural alterations (flattening) of their retinal ribbon synapses that were not otherwise observed at all in single-knockout CDC42KO retinas. Our observations identify GRF2 and CDC42 (but not RAC1) as key regulators of retinal processes controlling cone photoreceptor nuclear positioning and survival, and support the notion of GRF2 loss-of-function mutations as potential drivers of cone retinal dystrophies.

The insulin-degrading enzyme (IDE) is a zinc-dependent metalloendopeptidase that belongs to the M16A metalloprotease family. IDE is markedly expressed in the brain, where it is particularly relevant due to its in vitro amyloid beta (Aβ)-degrading activity. The subcellular localization of IDE, a paramount aspect to understand how this enzyme can perform its proteolytic functions in vivo, remains highly controversial. In this work, we addressed IDE subcellular localization from an evolutionary perspective. Phylogenetic analyses based on protein sequence and gene and protein structure were performed. An in silico analysis of IDE signal peptide suggests an evolutionary shift in IDE exportation at the prokaryote/eukaryote divide. Subcellular localization experiments in microglia revealed that IDE is mostly cytosolic. Furthermore, IDE associates to membranes by their cytoplasmatic side and further partitions between raft and non-raft domains. When stimulated, microglia change into a secretory active state, produces numerous multivesicular bodies and IDE associates with their membranes. The subsequent inward budding of such membranes internalizes IDE in intraluminal vesicles, which later allows IDE to be exported outside the cells in small extracellular vesicles. We further demonstrate that such an IDE exportation mechanism is regulated by stimuli relevant for microglia in physiological conditions and upon aging and neurodegeneration.

Apicobasal polarity is a hallmark of retinal pigment epithelium cells and is required to perform their functions; however, the precise roles of the different proteins that execute polarity are still poorly understood. Here, we have studied the expression and location of Scribble, the core member of the polarity basal protein complex in epithelial-derived cells, in human and mouse RPE cells in both control and pathological conditions. We found that Scribble specifically localizes at the basolateral membrane of mouse and human RPE cells. In addition, we observed an increase in the expression of Scribble during human RPE development in culture, while it acquires a well-defined basolateral pattern as this process is completed. Finally, the expression and location of Scribble were analyzed in human RPE cells in experimental conditions that mimic the toxic environment suffered by these cells during AMD development and found an increase in Scribble expression in cells that develop a pathological phenotype, suggesting that the protein could be altered in cells under stress conditions, as occurs in AMD. Together, our results demonstrate, for the first time, that Scribble is expressed in both human and mouse RPE and is localized at the basolateral membrane in mature cells. Furthermore, Scribble shows impaired expression and location in RPE cells in pathological conditions, suggesting a possible role for this protein in the development of pathologies, such as AMD.

Synaptic loss, neuronal death, and circuit remodeling are common features of central nervous system neurodegenerative disorders. Retinitis pigmentosa (RP), the leading cause of inherited blindness, is a group of retinal dystrophies characterized by photoreceptor dysfunction and death. The insulin receptor, a key controller of metabolism, also regulates neuronal survival and synaptic formation, maintenance, and activity. Indeed, deficient insulin receptor signaling has been implicated in several brain neurodegenerative pathologies. We present evidence linking impaired insulin receptor signaling with RP. We describe a selective decrease in the levels of the insulin receptor and its downstream effector phospho-S6 in retinal horizontal cell terminals in the rd10 mouse model of RP, as well as aberrant synapses between rod photoreceptors and the postsynaptic terminals of horizontal and bipolar cells. A gene therapy strategy to induce sustained proinsulin, the insulin precursor, production restored retinal insulin receptor signaling, by increasing S6 phosphorylation, without peripheral metabolic consequences. Moreover, proinsulin preserved photoreceptor synaptic connectivity and prolonged visual function in electroretinogram and optomotor tests. These findings point to a disease-modifying role of insulin receptor and support the therapeutic potential of proinsulin in retinitis pigmentosa.

Apicobasal polarity is essential for epithelial cell function, yet the roles of different proteins in its completion is not fully understood. Here, we have studied the role of the polarity protein, CRB2, in human retinal pigment epithelial (RPE) cells during polarization in vitro, and in mature murine RPE cells in vivo. After establishing a simplified protocol for the culture of human fetal RPE cells, we studied the temporal sequence of the expression and localization of polarity and cell junction proteins during polarization in these epithelial cells. We found that CRB2 plays a key role in tight junction maintenance as well as in cell cycle arrest. In addition, our studies in vivo show that the knockdown of CRB2 in the RPE affects to the distribution of different apical polarity proteins and results in perturbed retinal homeostasis, manifested by the invasion of activated microglial cells into the subretinal space. Together our results demonstrate that CRB2 is a key protein for the development and maintenance of a polarized epithelium.

Autophagy is a physiological process by which various damaged or non-essential cytosolic components are recycled, contributing to cell survival under stress conditions. In cancer, autophagy can have antitumor or protumor effects depending on the developmental stage. Here, we use Western blotting, immunochemistry, and transmission electron microscopy to demonstrate that the antitumor peptide TAT-Cx43266-283, a c-Src inhibitor, blocks autophagic flux in glioblastoma stem cells (GSCs) under basal and nutrient-deprived conditions. Upon nutrient deprivation, GSCs acquired a dormant-like phenotype that was disrupted by inhibition of autophagy with TAT-Cx43266-283 or chloroquine (a classic autophagy inhibitor), leading to GSC death. Remarkably, dasatinib, a clinically available c-Src inhibitor, could not replicate TAT-Cx43266-283 effect on dormant GSCs, revealing for the first time the possible involvement of pathways other than c-Src in TAT-Cx43266-283 effect. TAT-Cx43266-283 exerts an antitumor effect both in nutrient-complete and nutrient-deprived environments, which constitutes an advantage over chloroquine and dasatinib, whose effects depend on nutrient environment. Finally, our analysis of the levels of autophagy-related proteins in healthy and glioma donors suggests that autophagy is upregulated in glioblastoma, further supporting the interest in inhibiting this process in the most aggressive brain tumor and the potential use of TAT-Cx43266-283 as a therapy for this type of cancer.

BackgroundGlioblastoma is the most aggressive primary brain tumour and has a very poor prognosis. Inhibition of c-Src activity in glioblastoma stem cells (GSCs, responsible for glioblastoma lethality) and primary glioblastoma cells by the peptide TAT-Cx43266–283 reduces tumorigenicity, and boosts survival in preclinical models. Because c-Src can modulate cell metabolism and several reports revealed poor clinical efficacy of various antitumoral drugs due to metabolic rewiring in cancer cells, here we explored the inhibition of advantageous GSC metabolic plasticity by the c-Src inhibitor TAT-Cx43266-283.MethodsMetabolic impairment induced by the c-Src inhibitor TAT-Cx43266-283 in vitro was assessed by fluorometry, western blotting, immunofluorescence, qPCR, enzyme activity assays, electron microscopy, Seahorse analysis, time-lapse imaging, siRNA, and MTT assays. Protein expression in tumours from a xenograft orthotopic glioblastoma mouse model was evaluated by immunofluorescence.FindingsTAT-Cx43266–283 decreased glucose uptake in human GSCs and reduced oxidative phosphorylation without a compensatory increase in glycolysis, with no effect on brain cell metabolism, including rat neurons, human and rat astrocytes, and human neural stem cells. TAT-Cx43266-283 impaired metabolic plasticity, reducing GSC growth and survival under different nutrient environments. Finally, GSCs intracranially implanted with TAT-Cx43266–283 showed decreased levels of important metabolic targets for cancer therapy, such as hexokinase-2 and GLUT-3.InterpretationThe reduced ability of TAT-Cx43266-283–treated GSCs to survive in metabolically challenging settings, such as those with restricted nutrient availability or the ever-changing in vivo environment, allows us to conclude that the advantageous metabolic plasticity of GSCs can be therapeutically exploited through the specific and cell-selective inhibition of c-Src by TAT-Cx43266-283.FundingSpanish Ministerio de Economía y Competitividad (FEDER BFU2015-70040-R and FEDER RTI2018-099873-B-I00), Fundación Ramón Areces. Fellowships from the Junta de Castilla y León, European Social Fund, Ministerio de Ciencia and Asociación Española Contra el Cáncer (AECC).

Millions of individuals worldwide suffer from impaired vision, a condition with multiple origins that often impinge upon the light sensing cells of the retina, the photoreceptors, affecting their integrity. The molecular components contributing to this integrity are however not yet fully understood. Here we have asked whether Secreted Frizzled Related Protein 1 (SFRP1) may be one of such factors. SFRP1 has a context-dependent function as modulator of Wnt signalling or of the proteolytic activity of A Disintegrin And Metalloproteases (ADAM) 10, a main regulator of neural cell-cell communication. We report that in Sfrp1 -/- mice, the outer limiting membrane (OLM) is discontinuous and the photoreceptors disorganized and more prone to light-induced damage. Sfrp1 loss significantly enhances the effect of the Rpe65 Leu450Leu genetic variant -present in the mouse genetic background- which confers sensitivity to light-induced stress. These alterations worsen with age, affect visual function and are associated to an increased proteolysis of Protocadherin 21 (PCDH21), localized at the photoreceptor outer segment, and N-cadherin, an OLM component. We thus propose that SFRP1 contributes to photoreceptor fitness with a mechanism that involves the maintenance of OLM integrity. These conclusions are discussed in view of the broader implication of SFRP1 in neurodegeneration and aging.

By cryolesion of the scleral-corneal perimeter of the eye of a teleost we eliminated both the marginal growth ring and the immature portions adjacent to the neural retina. Electron microscopy revealed necrotic destruction of the whole of the germinative zone and a large number of apoptotic cells in neighbouring zones. In the degenerative processes that follow the lesion, at least three types of cells are involved: microglial cells, pigmentary cells and extravascular heterophilic granulocytes. Both microglia and the pigmentary cells act as phagocytes and participate in the elimination of debris from the lesioned area. Heterophilic granulocytes probably collaborate in the modification of the environment for the subsequent regeneration. During the degenerative processes after the lesion, all three cell types participate in the transformation of the damaged retinal zone into a favourable medium for later regeneration of the neural tissue.

Extracellular vesicle (EV)-mediated glia-to-neuron communication has been recognized in a growing number of physiological and pathological situations. They transport complex sets of molecules that can be beneficial or detrimental for the receiving cell. As in other areas of biology, their analysis is revolutionizing the field of neuroscience, since fundamental signaling processes are being re-evaluated, and applications for neurodegenerative disease therapies have emerged. Using human astrocytic and differentiated neuronal cell lines, we demonstrate that a classical neuroprotective protein, Apolipoprotein D (ApoD), expressed by glial cells and known to promote functional integrity and survival of neurons, is exclusively transported by EVs from astrocytes to neurons, where it gets internalized. Indeed, we demonstrate that conditioned media derived from ApoD-knock-out (KO) astrocytes exert only a partial autocrine protection from oxidative stress (OS) challenges, and that EVs are required for ApoD-positive astrocytic cell line derived medium to exert full neuroprotection. When subfractionation of EVs is performed, ApoD is revealed as a very specific marker of the exosome-containing fractions. These discoveries help us reframe our understanding of the neuroprotective role of this lipid binding protein and open up new research avenues to explore the use of systemically administered ApoD-loaded exosomes that can cross the blood-brain barrier to treat neurodegenerative diseases.

AbstractGlioblastoma is the most aggressive primary brain cancer, with a median survival of 1 to 2 years1. These tumours contain glioma stem cells (GSCs), which are highly tumorigenic, resistant to conventional therapies2, 3, and exhibit metabolic plasticity to adapt to challenging environments4, 5. GSCs can be specifically targeted by a short cell-penetrating peptide based on connexin43 (Cx43) (TAT-Cx43266-283) that reduces tumour growth and increases survival in preclinical models6via c-Src inhibition7. Because several reports revealed poor clinical efficacy of various antitumoral drugs due to metabolic rewiring in cancer cells8–10, we investigated the effect of TAT-Cx43266-283on GSC metabolism and metabolic plasticity. Here we show that TAT-Cx43266-283decreases GSC glucose uptake and oxidative phosphorylation without a compensatory increase in glycolysis, with no effect on neuron or astrocyte metabolism. GSC changes were mediated by decreased hexokinase (HK) activity and aberrant mitochondrial localization, ultrastructure and function. Moreover, TAT-Cx43266-283reduced GSC growth and survival under different nutrient availability conditions by impairing the metabolic plasticity needed to exploit glucose as an energy source in the absence of other nutrients. Finally, GSCs intracranially implanted into mice together with TAT-Cx43266-283showed decreased levels of important targets for cancer therapy, such as HK-211, 12and glucose transporter 3 (GLUT-3)13, evidencing the reduced ability of treated GSCs to survive in challenging environments. Our results confirm the value of TAT-Cx43266-283for glioma therapy alone or in combination with therapies whose resistance relies on metabolic adaptation. More importantly, these results allow us to conclude that the advantageous metabolic plasticity of GSCs is a targetable vulnerability in malignant gliomas.

Glioblastoma is the most aggressive primary brain cancer, with a median survival of 1 to 2 years[1][1]. These tumours contain glioma stem cells (GSCs), which are highly tumorigenic, resistant to conventional therapies[2][2], [3][3], and exhibit metabolic plasticity to adapt to challenging environments[4][4], [5][5]. GSCs can be specifically targeted by a short cell-penetrating peptide based on connexin43 (Cx43) (TAT-Cx43266-283) that reduces tumour growth and increases survival in preclinical models[6][6] via c-Src inhibition[7][7]. Because several reports revealed poor clinical efficacy of various antitumoral drugs due to metabolic rewiring in cancer cells[8][8]–[10][9], we investigated the effect of TAT-Cx43266-283 on GSC metabolism and metabolic plasticity. Here we show that TAT-Cx43266-283 decreases GSC glucose uptake and oxidative phosphorylation without a compensatory increase in glycolysis, with no effect on neuron or astrocyte metabolism. GSC changes were mediated by decreased hexokinase (HK) activity and aberrant mitochondrial localization, ultrastructure and function. Moreover, TAT-Cx43266-283 reduced GSC growth and survival under different nutrient availability conditions by impairing the metabolic plasticity needed to exploit glucose as an energy source in the absence of other nutrients. Finally, GSCs intracranially implanted into mice together with TAT-Cx43266-283 showed decreased levels of important targets for cancer therapy, such as HK-2[11][10], [12][11] and glucose transporter 3 (GLUT-3)[13][12], evidencing the reduced ability of treated GSCs to survive in challenging environments. Our results confirm the value of TAT-Cx43266-283 for glioma therapy alone or in combination with therapies whose resistance relies on metabolic adaptation. More importantly, these results allow us to conclude that the advantageous metabolic plasticity of GSCs is a targetable vulnerability in malignant gliomas. [1]: #ref-1 [2]: #ref-2 [3]: #ref-3 [4]: #ref-4 [5]: #ref-5 [6]: #ref-6 [7]: #ref-7 [8]: #ref-8 [9]: #ref-10 [10]: #ref-11 [11]: #ref-12 [12]: #ref-13

Introduction The homologous, ubiquitously expressed, members (Sos1 and Sos2) of the Sos family of RasGEFs participate in multiple signalling pathways but their specific cellular functions are not clearly defined yet. Material and methods Using a tamoxifen (4OHT)-inducible, conditional Sos1 null mutation, here we generated and analysed wild type (WT), single Sos1-KO, constitutive Sos2-KO and double Sos1/2-DKO primary mouse embryonic fibroblasts (MEFs) in an effort to ascertain the functional specificity or redundancy of Sos1 and Sos2 at the cellular level. Results and discussions Sos1-KO and Sos1/2-DKO MEFs exhibited distinct flat morphology, enlarged cell perimeter and altered cytoskeletal organisation that were not observed in WT and Sos2 KO counterparts. Sos1-KO and Sos1/2-DKO MEFs also displayed significant accumulation of cytoplasmic bodies identified as autophagosomes containing degraded mitochondria by means of electron microscopy and specific markers. Consistent with a mitophagic phenotype, in vivo labelling using specific fluorophores revealed increased levels of mitochondrial oxidative stress in the Sos1-KO and the Sos1/2-DKO cells as compared to Sos2-KO or WT MEFs. Treatment of the MEF cultures with antioxidants such as GSH and NAC corrected the altered perimeter size and proliferative rate of Sos1-KO and Sos1/2-DKO MEFs to levels similar to those of WT and Sos2-KO, but not recover oxidative stress. Furthermore, treatment with the specific mitochondrial superoxide scavenger mitoTEMPO recovered endogenous redox-homeostasis in Sos1-KO and Sos1/2-DKO to normal levels. Analysis of MEFs concomitantly loaded with MitoTracker Green (Δψ-independent) and MitoTracker Red (Δψ-dependent) showed a significantly increased of dysfunctional mitochondria in the Sos1/2-DKO MEFs in comparison to all other genotypes. Finally, Seahorse-based measurements of mitochondrial parameters showed that basal and maximal mitochondrial respiration, spare respiratory capacity, and ATP production, were also significantly decreased in Sos1/2-DKO cells as compared to MEFs of all the other genotypes. Conclusion Our data uncover a direct mechanistic link between Sos RasGEF proteins and the control of mitochondrial oxidative stress and respiration. Dysfunctional mitochondria are a key element in a variety of serious diseases, including cancer, and thus a promising therapeutic target. This work was supported by grants PI16/02137 (FIS) and CIBERONC (ONCG19//2017) from ISCIII; SA043U16 from FEDER-JCyL; FS/35–2017 from FSB and AECC, Spain.

Acquisition of cell polarization is essential for the performance of crucial functions, like a successful secretion and appropriate cell signaling in many tissues, and it depends on the correct functioning of polarity proteins, including the Crumbs complex. The CRB proteins, CRB1, CRB2 and CRB3, identified in mammals, are expressed in epithelial-derived tissues like brain, kidney and retina. CRB2 has a ubiquitous expression and has been detected in embryonic brain tissue; but currently there is no data regarding its localization in the adult brain. In our study, we characterized the presence of CRB2 in adult mice brain, where it is particularly enriched in cortex, hippocampus, hypothalamus and cerebellum. Double immunofluorescence analysis confirmed that CRB2 is a neuron-specific protein, present in both soma and projections where colocalizes with certain populations of exocytic and endocytic vesicles and with other members of the Crumbs complex. Finally, in the cortex of CRB1rd8 mutant mice that contain a mutation in the Crb1 gene generating a truncated CRB1 protein, there is an abnormal increase in the expression levels of the CRB2 protein which suggests a possible compensatory mechanism for the malfunction of CRB1 in this mutant background.

Environmental insults such as oxidative stress can damage cell membranes. Lysosomes are particularly sensitive to membrane permeabilization since their function depends on intraluminal acidic pH and requires stable membrane-dependent proton gradients. Among the catalog of oxidative stress-responsive genes is the Lipocalin Apolipoprotein D (ApoD), an extracellular lipid binding protein endowed with antioxidant capacity. Within the nervous system, cell types in the defense frontline, such as astrocytes, secrete ApoD to help neurons cope with the challenge. The protecting role of ApoD is known from cellular to organism level, and many of its downstream effects, including optimization of autophagy upon neuro-degeneration, have been described. However, we still cannot assign a cellular mechanism to ApoD gene that explains how this protection is accomplished. Here we perform a comprehensive analysis of ApoD intracellular traffic and demonstrate its role in lysosomal pH homeostasis upon paraquat-induced oxidative stress. By combining single-lysosome in vivo pH measurements with immunodetection, we demonstrate that ApoD is endocytosed and targeted to a subset of vulnerable lysosomes in a stress-dependent manner. ApoD is functionally stable in this acidic environment, and its presence is sufficient and necessary for lysosomes to recover from oxidation-induced alkalinization, both in astrocytes and neurons. This function is accomplished by preventing lysosomal membrane permeabilization. Two lysosomal-dependent biological processes, myelin phagocytosis by astrocytes and optimization of neurodegeneration-triggered autophagy in a Drosophila in vivo model, require ApoD-related Lipocalins. Our results uncover a previously unknown biological function of ApoD, member of the finely regulated and evolutionary conserved gene family of extracellular Lipocalins. They set a lipoprotein-mediated regulation of lysosomal membrane integrity as a new mechanism at the hub of many cellular functions, critical for the outcome of a wide variety of neurodegenerative diseases. These results open therapeutic opportunities by providing a route of entry and a repair mechanism for lysosomes in pathological situations.