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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.

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

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.

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 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.

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.

To compact the extracellular sides of myelin, an important transition must take place: from membrane sliding, while building the wraps, to membrane adhesion and water exclusion. Removal of the negatively charged glycocalyx becomes the limiting factor in such transition. What is required to initiate this membrane‐zipping process? Knocking‐out the Lipocalin Apolipoprotein D (ApoD), essential for lysosomal functional integrity in glial cells, results in a specific defect in myelin extracellular leaflet compaction in peripheral and central nervous system, which results in reduced conduction velocity and suboptimal behavioral outputs: motor learning is compromised. Myelination initiation, growth, intracellular leaflet compaction, myelin thickness or internodal length remain unaltered. Lack of ApoD specifically modifies Plp and P0 protein expression, but not Mbp or Mag. Late in myelin maturation period, ApoD affects lipogenic and growth‐related, but not stress‐responsive, signaling pathways. Without ApoD, the sialylated glycocalyx is maintained and ganglioside content remains high. In peripheral nervous system, Neu3 membrane sialidase and lysosomal Neu1 are coordinately expressed with ApoD in subsets of Schwann cells. ApoD‐KO myelin becomes depleted of Neu3 and enriched in Fyn, a kinase with pivotal roles in transducing axon‐derived signals into myelin properties. In the absence of ApoD, partial permeabilization of lysosomes alters Neu1 location as well. Exogenous ApoD rescues ApoD‐KO hypersialylated glycocalyx in astrocytes, demonstrating that ApoD is necessary and sufficient to control glycocalyx composition in glial cells. By ensuring lysosomal functional integrity and adequate subcellular location of effector and regulatory proteins, ApoD guarantees the glycolipid recycling and glycocalyx removal required to complete myelin compaction.

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 neurodegeneration, 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 functi

Using a 4-hydroxytamoxifen (4OHT)-inducible, conditional Sos1-null mutation, we analyzed wild-type (WT), single Sos1-KO, Sos2-KO and double Sos1/2 KO primary mouse embryonic fibroblasts (MEF) with an aim at evaluating the functional specificity or redundancy of the Sos1 and Sos2 alleles at the cellular level. The 4OHT-induced Sos1-KO and Sos1/2-DKO MEFs exhibited distinct flat morphology, enlarged cell perimeter and altered cytoskeletal organization that were not observed in the WT and Sos2-KO counterparts. The Sos1-KO and Sos1/2-DKO MEFs also displayed significant accumulation, in comparison with WT and Sos2-KO MEFs, of cytoplasmic vesicular bodies identified as autophagosomes containing degraded mitochondria by means of electron microscopy and specific markers. Cellular proliferation and migration were impaired in Sos1-KO and Sos1/2-DKO MEFs in comparison with WT and Sos2-KO MEFs, whereas cell adhesion was only impaired upon depletion of both Sos isoforms. RasGTP formation was practically absent in Sos1/2-DKO MEFs as compared with the other genotypes and extracellular signal-regulated kinase phosphorylation showed only significant reduction after combined Sos1/2 depletion. Consistent with a mitophagic phenotype, in vivo labeling with specific fluorophores uncovered increased levels of oxidative stress (elevated intracellular reactive oxygen species and mitochondrial superoxide and loss of mitochondrial membrane potential) in the Sos1-KO and the Sos1/2-DKO cells as compared with Sos2-KO and WT MEFs. Interestingly, treatment of the MEF cultures with antioxidants corrected the altered phenotypes of Sos1-KO and Sos1/2-DKO MEFs by restoring their altered perimeter size and proliferative rate to levels similar to those of WT and Sos2-KO MEFs. Our data uncover a direct mechanistic link between Sos1 and control of intracellular oxidative stress, and demonstrate functional prevalence of Sos1 over Sos2 with regards to cellular proliferation and viability.

Detailed immunocytochemical analyses comparing wild-type (WT), GRF1-knockout (KO), GRF2-KO and GRF1/2 double-knockout (DKO) mouse retinas uncovered the specific accumulation of misplaced, 'ectopic' cone photoreceptor nuclei in the photoreceptor segment (PS) area of retinas from GRF2-KO and GRF1/2-DKO, but not of WT or GRF1-KO mice. Localization of ectopic nuclei in the PS area of GRF2-depleted retinas occurred postnatally and peaked between postnatal day (P)11 and P15. Mechanistically, the generation of this phenotype involved disruption of the outer limiting membrane and intrusion into the PS layer by cone nuclei displaying significant perinuclear accumulation of signaling molecules known to participate in nuclear migration and cytoskeletal reorganization, such as PAR3, PAR6 and activated, phosphorylated forms of PAK, MLC2 and VASP. Electroretinographic recordings showed specific impairment of cone-mediated retinal function in GRF2-KO and GRF1/2-DKO retinas compared with WT controls. These data identify defective cone nuclear migration as a novel phenotype in mouse retinas lacking GRF2 and support a crucial role of GRF2 in control of the nuclear migration processes required for proper postnatal development and function of retinal cone photoreceptors.

The CRB proteins CRB1, CRB2 and CRB3 are members of the cell polarity complex Crumbs in mammals that together with Scribble and Par complexes stablish the polarity of a variety of cell types. Although many members of the Crumbs complex proteins are expressed in the retinal pigment epithelium (RPE), and even though the mRNA of CRB2 has been detected in ARPE-19 cells and in the RPE/Choroid, to date no CRB protein has yet been found in this tissue. To investigate this possibility, we generated an antibody that specifically recognize the mouse CRB2 protein, and we demonstrate the expression of CRB2 in mouse RPE. Confocal analysis shows that CRB2 is restricted to the apicolateral membrane of RPE cells, and more precisely, in the tight junctions. Our study identified CRB2 as the member of the CRB protein family that is present together with the rest of the components of the Crumbs complex in the RPE apico-lateral cell membrane. Considering that the functions of CRB proteins are decisive in the establishment and maintenance of cell-cell junctions in several epithelial-derived cell types, we believe that these findings are a relevant starting point for unraveling the functions that CRB2 might perform in the RPE.