High-throughput microcontact printing of proteins in microwell cell culture plates.

Microcontact printing (MCP) is used to pattern a surface with a specific compound, allowing the spatially restricted response of cells to be assayed as they encounter a molecule of interest. MCP is a relatively low-cost and accessible technique that uses commercially available reagents and common cell culture equipment. However, it can be technically challenging, slow, and incompatible with microwell cell culture plates that are widely used for screening and other applications. Here, we describe a novel protocol using medical biopsy punches to transfer patterns into standard 96-well plates via polydimethylsiloxane (PDMS) cutouts. We demonstrate that this method can be used to deposit patterns of poly-D-lysine (PDL) into the microwells of glass-bottom plates. As a proof-of-concept, we show that cultured rodent glial cells preferentially grow and extend processes on the pattern. This method will allow larger scale MCP experiments in which different patterns, proteins, or other factors can be assayed in parallel.•Biopsy punches enable both cutting out small circular stamps and plunging them into tissue culture microwells to transfer proteins.•Compared to standard MCP, this method offers a more rapid workflow to pattern proteins onto substrates, and allows use of microwell plates that permits larger-scale experiments.

Acetylcholine synergizes with netrin-1 to drive persistent firing in the entorhinal cortex.

The ability of the mammalian brain to maintain spatial representations of external or internal information for short periods of time has been associated with sustained neuronal spiking and reverberatory neural network activity in the medial entorhinal cortex. Here, we show that conditional genetic deletion of netrin-1 or the netrin receptor deleted-in-colorectal cancer (DCC) from forebrain excitatory neurons leads to deficits in short-term spatial memory. We then demonstrate that conditional deletion of either netrin-1 or DCC inhibits cholinergic persistent firing and show that cholinergic activation of muscarinic receptors expressed by entorhinal cortical neurons promotes persistent firing by recruiting DCC to the plasma membrane. Together, these findings indicate that normal short-term spatial memory function requires the synergistic actions of acetylcholine and netrin-1.

Mechanisms of metabolic stress induced cell death of human oligodendrocytes: relevance for progressive multiple sclerosis.

Oligodendrocyte (OL) injury and loss are central features of evolving lesions in multiple sclerosis. Potential causative mechanisms of OL loss include metabolic stress within the lesion microenvironment. Here we use the injury response of primary human OLs (hOLs) to metabolic stress (reduced glucose/nutrients) in vitro to help define the basis for the in situ features of OLs in cases of MS. Under metabolic stress in vitro, we detected reduction in ATP levels per cell that precede changes in survival. Autophagy was initially activated, although ATP levels were not altered by inhibitors (chloroquine) or activators (Torin-1). Prolonged stress resulted in autophagy failure, documented by non-fusion of autophagosomes and lysosomes. Consistent with our in vitro results, we detected higher expression of LC3, a marker of autophagosomes in OLs, in MS lesions compared to controls. Both in vitro and in situ, we observe a reduction in nuclear size of remaining OLs. Prolonged stress resulted in increased ROS and cleavage of spectrin, a target of Ca2+-dependent proteases. Cell death was however not prevented by inhibitors of ferroptosis or MPT-driven necrosis, the regulated cell death (RCD) pathways most likely to be activated by metabolic stress. hOLs have decreased expression of VDAC1, VDAC2, and of genes regulating iron accumulation and cyclophilin. RNA sequencing analyses did not identify activation of these RCD pathways in vitro or in MS cases. We conclude that this distinct response of hOLs, including resistance to RCD, reflects the combined impact of autophagy failure, increased ROS, and calcium influx, resulting in metabolic collapse and degeneration of cellular structural integrity. Defining the basis of OL injury and death provides guidance for development of neuro-protective strategies.

Somatotopy of Mouse Spinothalamic Innervation and the Localization of a Noxious Stimulus Requires Deleted in Colorectal Carcinoma Expression by Phox2a Neurons.

Anterolateral system (AS) neurons transmit pain signals from the spinal cord to the brain. Their morphology, anatomy, and physiological properties have been extensively characterized and suggest that specific AS neurons and their brain targets are concerned with the discriminatory aspects of noxious stimuli, such as their location or intensity, and their motivational/emotive dimension. Among the recently unraveled molecular markers of AS neurons is the developmentally expressed transcription factor Phox2a, providing us with the opportunity to selectively disrupt the embryonic wiring of AS neurons to gain insights into the logic of their adult function. As mice with a spinal-cord-specific loss of the netrin-1 receptor deleted in colorectal carcinoma (DCC) have increased AS neuron innervation of ipsilateral brain targets and defective noxious stimulus localization or topognosis, we generated mice of either sex carrying a deletion of Dcc in Phox2a neurons. Such DccPhox2a mice displayed impaired topognosis along the rostrocaudal axis but with little effect on left-right discrimination and normal aversive responses. Anatomical tracing experiments in DccPhox2a mice revealed defective targeting of cervical and lumbar AS axons within the thalamus. Furthermore, genetic labeling of AS axons revealed their expression of DCC on their arrival in the brain, at a time when many of their target neurons are being born and express Ntn1 Our experiments suggest a postcommissural crossing function for netrin-1:DCC signaling during the formation of somatotopically ordered maps and are consistent with a discriminatory function of some of the Phox2a AS neurons.SIGNIFICANCE STATEMENT How nociceptive (pain) signals are relayed from the body to the brain remains an important question relevant to our understanding of the basic physiology of pain perception. Previous studies have demonstrated that the AS is a main effector of this function. It is composed of AS neurons located in the spinal cord that receive signals from nociceptive sensory neurons that detect noxious stimuli. In this study, we generate a genetic miswiring of mouse AS neurons that results in a decreased ability to perceive the location of a painful stimulus. The precise nature of this defect sheds light on the function of different kinds of AS neurons and how pain information may be organized.

Diverse injury responses of human oligodendrocyte to mediators implicated in multiple sclerosis.

Early multiple sclerosis lesions feature relative preservation of oligodendrocyte cell bodies with dying back retraction of their myelinating processes. Cell loss occurs with disease progression. Putative injury mediators include metabolic stress (low glucose/nutrient), pro-inflammatory mediators (interferon γ and tumour necrosis factor α), and excitotoxins (glutamate). Our objective was to compare the impact of these disease relevant mediators on the injury responses of human mature oligodendrocytes. In the current study, we determined the effects of these mediators on process extension and survival of human brain derived mature oligodendrocytes in vitro and used bulk RNA sequencing to identify distinct effector mechanisms that underlie the responses. All mediators induced significant process retraction of the oligodendrocytes in dissociated cell culture. Only metabolic stress (low glucose/nutrient) conditions resulted in delayed (4-6 days) non-apoptotic cell death. Metabolic effects were associated with induction of the integrated stress response, which can be protective or contribute to cell injury dependent on its level and duration of activation. Addition of Sephin1, an agonist of the integrated stress response induced process retraction under control conditions and further enhanced retraction under metabolic stress conditions. The antagonist ISRIB restored process outgrowth under stress conditions, and if added to already stressed cells, reduced delayed cell death and prolonged the period in which recovery could occur. Inflammatory cytokine functional effects were associated with activation of multiple signalling pathways (including Jak/Stat-1) that regulate process outgrowth, without integrated stress response induction. Glutamate application produced limited transcriptional changes suggesting a contribution of effects directly on cell processes. Our comparative studies indicate the need to consider both the specific injury mediators and the distinct cellular mechanisms of responses to them by human oligodendrocytes to identify effective neuroprotective therapies for multiple sclerosis.

The netrin-1 receptor DCC promotes the survival of a subpopulation of midbrain dopaminergic neurons: Relevance for ageing and Parkinson's disease.

Mechanisms that determine the survival of midbrain dopaminergic (mDA) neurons in the adult central nervous system (CNS) are not fully understood. Netrins are a family of secreted proteins that are essential for normal neural development. In the mature CNS, mDA neurons express particularly high levels of netrin-1 and its receptor Deleted in Colorectal Cancer (DCC). Recent findings indicate that overexpressing netrin-1 protects mDA neurons in animal models of Parkinson's disease (PD), with a proposed pro-apoptotic dependence function for DCC that triggers cell death in the absence of a ligand. Here, we sought to determine if DCC expression influences mDA neuron survival in young adult and ageing mice. To circumvent the perinatal lethality of DCC null mice, we selectively deleted DCC from mDA neurons utilizing DATcre /loxP gene-targeting and examined neuronal survival in adult and aged animals. Reduced numbers of mDA neurons were detected in the substantia nigra pars compacta (SNc) of young adult DATcre /DCCfl/fl mice, with further reduction in aged DATcre /DCCfl/fl animals. In contrast to young adults, aged mice also exhibited a gene dosage effect, with fewer SNc mDA neurons in DCC heterozygotes (DATcre /DCCfl/wt ). Notably, loss of mDA neurons in the SN was not uniform. Neuronal loss in the SN was limited to ventral tier mDA neurons, while mDA neurons in the dorsal tier of the SN, which resist degeneration in PD, were spared from the effect of DCC deletion in both young and aged mice. In the ventral tegmental area (VTA), young adult mice with conditional deletion of DCC had normal mDA neuronal numbers, while significant loss occurred in aged DATcre /DCCfl/fl and DATcre /DCCfl/wt mice compared to age-matched wild-type mice. Our results indicate that expression of DCC is required for the survival of subpopulations of mDA neurons and may be relevant to the degenerative processes in PD.

Dendritic Polyglycerol Amine: An Enhanced Substrate to Support Long-Term Neural Cell Culture.

Long-term stable cell culture is a critical tool to better understand cell function. Most adherent cell culture models require a polymer substrate coating of poly-lysine or poly-ornithine for the cells to adhere and survive. However, polypeptide-based substrates are degraded by proteolysis and it remains a challenge to maintain healthy cell cultures for extended periods of time. Here, we report the development of an enhanced cell culture substrate based on a coating of dendritic polyglycerol amine (dPGA), a non-protein macromolecular biomimetic of poly-lysine, to promote the adhesion and survival of neurons in cell culture. We show that this new polymer coating provides enhanced survival, differentiation and long-term stability for cultures of primary neurons or neurons derived from human induced pluripotent stem cells (hiPSCs). Atomic force microscopy analysis provides evidence that greater nanoscale roughness contributes to the enhanced capacity of dPGA-coated surfaces to support cells in culture. We conclude that dPGA is a cytocompatible, functionally superior, easy to use, low cost and highly stable alternative to poly-cationic polymer cell culture substrate coatings such as poly-lysine and poly-ornithine. Summary statementHere, we describe a novel dendritic polyglycerol amine-based substrate coating, demonstrating superior performance compared to current polymer coatings for long-term culture of primary neurons and neurons derived from induced pluripotent stem cells.

Age-related injury responses of human oligodendrocytes to metabolic insults: link to BCL-2 and autophagy pathways.

Myelin destruction and oligodendrocyte (OL) death consequent to metabolic stress is a feature of CNS disorders across the age spectrum. Using cells derived from surgically resected tissue, we demonstrate that young (

Mitochondrial dynamics and bioenergetics regulated by netrin-1 in oligodendrocytes.

Mitochondria are dynamic organelles that produce energy and molecular precursors that are essential for myelin synthesis. Unlike in neurons, mitochondria in oligodendrocytes increase intracellular movement in response to glutamatergic activation and are more susceptible to oxidative stress than in astrocytes or microglia. The signaling pathways that regulate these cell type-specific mitochondrial responses in oligodendrocytes are not understood. Here, we visualized mitochondria migrating through thin cytoplasmic channels crossing myelin basic protein-positive compacted membranes and localized within paranodal loop cytoplasm. We hypothesized that local extracellular enrichment of netrin-1 might regulate the recruitment and function of paranodal proteins and organelles, including mitochondria. We identified rapid recruitment of mitochondria and paranodal proteins, including neurofascin 155 (NF155) and the netrin receptor deleted in colorectal carcinoma (DCC), to sites of contact between oligodendrocytes and netrin-1-coated microbeads in vitro. We provide evidence that Src-family kinase activation and Rho-associated protein kinase (ROCK) inhibition downstream of netrin-1 induces mitochondrial elongation, hyperpolarization of the mitochondrial inner membrane, and increases glycolysis. Our findings identify a signaling mechanism in oligodendrocytes that is sufficient to locally recruit paranodal proteins and regulate the subcellular localization, morphology, and function of mitochondria.

Effects of Biotin on survival, ensheathment, and ATP production by oligodendrocyte lineage cells in vitro.

Mechanisms implicated in disease progression in multiple sclerosis include continued oligodendrocyte (OL)/myelin injury and failure of myelin repair. Underlying causes include metabolic stress with resultant energy deficiency. Biotin is a cofactor for carboxylases involved in ATP production that impact myelin production by promoting fatty acid synthesis. Here, we investigate the effects of high dose Biotin (MD1003) on the functional properties of post-natal rat derived oligodendrocyte progenitor cells (OPCs). A2B5 positive OPCs were assessed using an in vitro injury assay, culturing cells in either DFM (DMEM/F12+N1) or "stress media" (no glucose (NG)-DMEM), with Biotin added over a range from 2.5 to 250 µg/ml, and cell viability determined after 24 hrs. Biotin reduced the increase in OPC cell death in the NG condition. In nanofiber myelination assays, biotin increased the percentage of ensheathing cells, the number of ensheathed segments per cell, and length of ensheathed segments. In dispersed cell culture, Biotin also significantly increased ATP production, assessed using a Seahorse bio-analyzer. For most assays, the positive effects of Biotin were observed at the higher end of the dose-response analysis. We conclude that Biotin, in vitro, protects OL lineage cells from metabolic injury, enhances myelin-like ensheathment, and is associated with increased ATP production.

Pre- and post-synaptic roles for DCC in memory consolidation in the adult mouse hippocampus.

The receptor deleted in colorectal cancer (DCC) and its ligand netrin-1 are essential for axon guidance during development and are expressed by neurons in the mature brain. Netrin-1 recruits GluA1-containing α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors (AMPARs) and is critical for long-term potentiation (LTP) at CA3-CA1 hippocampal Schaffer collateral synapses, while conditional DCC deletion from glutamatergic neurons impairs hippocampal-dependent spatial memory and severely disrupts LTP induction. DCC co-fractionates with the detergent-resistant component of postsynaptic density, yet is enriched in axonal growth cones that differentiate into presynaptic terminals during development. Specific presynaptic and postsynaptic contributions of DCC to the function of mature neural circuits have yet to be identified. Employing hippocampal subregion-specific conditional deletion of DCC, we show that DCC loss from CA1 hippocampal pyramidal neurons resulted in deficits in spatial memory, increased resting membrane potential, abnormal dendritic spine morphology, weaker spontaneous excitatory postsynaptic activity, and reduced levels of postsynaptic adaptor and signaling proteins; however, the capacity to induce LTP remained intact. In contrast, deletion of DCC from CA3 neurons did not induce detectable changes in the intrinsic electrophysiological properties of CA1 pyramidal neurons, but impaired performance on the novel object place recognition task as well as compromised excitatory synaptic transmission and LTP at Schaffer collateral synapses. Together, these findings reveal specific pre- and post-synaptic contributions of DCC to hippocampal synaptic plasticity underlying spatial memory.

Immunology of oligodendrocyte precursor cells in vivo and in vitro.

Remyelination following myelin/oligodendrocyte injury in the central nervous system (CNS) is dependent on oligodendrocyte progenitor cells (OPCs) migrating into lesion sites, differentiating into myelinating oligodendrocytes (OLs), and ensheathing axons. Experimental models indicate that robust OPC-dependent remyelination can occur in the CNS; in contrast, histologic and imaging studies of lesions in the human disease multiple sclerosis (MS) indicate the variable extent of this response, which is particularly limited in more chronic MS lesions. Immune-mediated mechanisms can contribute either positively or negatively to the presence and functional responses of OPCs. This review addresses i) the molecular signature and functional properties of OPCs in the adult human brain; ii) the status (presence and function) of OPCs in MS lesions; iii) experimental models and in vitro data highlighting the contribution of adaptive and innate immune constituents to OPC injury and remyelination; and iv) effects of MS-directed immunotherapies on OPCs, either directly or indirectly via effects on specific immune constituents.

Activity-Dependent Netrin-1 Secretion Drives Synaptic Insertion of GluA1-Containing AMPA Receptors in the Hippocampus.

Dynamic trafficking of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid glutamate receptors (AMPARs) to synapses is critical for activity-dependent synaptic plasticity underlying learning and memory, but the identity of key molecular effectors remains elusive. Here, we demonstrate that membrane depolarization and N-methyl-D-aspartate receptor (NMDAR) activation triggers secretion of the chemotropic guidance cue netrin-1 from dendrites. Using selective genetic deletion, we show that netrin-1 expression by excitatory neurons is required for NMDAR-dependent long-term potentiation (LTP) in the adult hippocampus. Furthermore, we demonstrate that application of exogenous netrin-1 is sufficient to trigger the potentiation of excitatory glutamatergic transmission at hippocampal Schaffer collateral synapses via Ca2+-dependent recruitment of GluA1-containing AMPARs, promoting the maturation of immature or nascent synapses. These findings identify a central role for activity-dependent release of netrin-1 as a critical effector of synaptic plasticity in the adult hippocampus.

Distinct age and differentiation-state dependent metabolic profiles of oligodendrocytes under optimal and stress conditions.

Within the microenvironment of multiple sclerosis lesions, oligodendrocytes are subject to metabolic stress reflecting effects of focal ischemia and inflammation. Previous studies have shown that under optimal conditions in vitro, the respiratory activity of human adult brain-derived oligodendrocytes is lower and more predominantly glycolytic compared to oligodendrocytes differentiated in vitro from post natal rat brain oligodendrocyte progenitor cells. In response to sub-lethal metabolic stress, adult human oligodendrocytes reduce overall energy production rate impacting the capacity to maintain myelination. Here, we directly compare the metabolic profiles of oligodendrocytes derived from adult rat brain with oligodendrocytes newly differentiated in vitro from oligodendrocyte progenitor cells obtained from the post natal rat brain, under both optimal culture and metabolic stress (low/no glucose) conditions. Oxygen consumption and extracellular acidification rates were measured using a Seahorse extracellular flux analyzer. Our findings indicate that under optimal conditions, adult rat oligodendrocytes preferentially use glycolysis whereas newly differentiated post natal rat oligodendrocytes, and the oligodendrocyte progenitor cells from which they are derived, mainly utilize oxidative phosphorylation to produce ATP. Metabolic stress increases the rate of ATP production via oxidative phosphorylation and significantly reduces glycolysis in adult oligodendrocytes. The rate of ATP production was relatively unchanged in newly differentiated post natal oligodendrocytes under these stress conditions, while it was significantly reduced in oligodendrocyte progenitor cells. Our study indicates that both age and maturation influence the metabolic profile under optimal and stressed conditions, emphasizing the need to consider these variables for in vitro studies that aim to model adult human disease.

Spatially Selective Dissection of Signal Transduction in Neurons Grown on Netrin-1 Printed Nanoarrays via Segmented Fluorescence Fluctuation Analysis.

Axonal growth cones extend during neural development in response to precise distributions of extracellular cues. Deleted in colorectal cancer (DCC), a receptor for the chemotropic guidance cue netrin-1, directs F-actin reorganization, and is essential for mammalian neural development. To elucidate how the extracellular distribution of netrin-1 influences the distribution of DCC and F-actin within axonal growth cones, we patterned nanoarrays of substrate bound netrin-1 using lift-off nanocontact printing. The distribution of DCC and F-actin in embryonic rat cortical neuron growth cones was then imaged using total internal reflection fluorescence (TIRF) microscopy. Fluorescence fluctuation analysis via image cross-correlation spectroscopy (ICCS) was applied to extract the molecular density and aggregation state of DCC and F-actin, identifying the fraction of DCC and F-actin colocalizing with the patterned netrin-1 substrate. ICCS measurement of spatially segmented images based on the substrate nanodot patterns revealed distinct molecular distributions of F-actin and DCC in regions directly overlying the nanodots compared to over the reference surface surrounding the nanodots. Quantifiable variations between the populations of DCC and F-actin on and off the nanodots reveal specific responses to the printed protein substrate. We report that nanodots of substrate-bound netrin-1 locally recruit and aggregate DCC and direct F-actin organization. These effects were blocked by tetanus toxin, consistent with netrin-1 locally recruiting DCC to the plasma membrane via a VAMP2-dependent mechanism. Our findings demonstrate the utility of segmented ICCS image analysis, combined with precisely patterned immobilized ligands, to reveal local receptor distribution and signaling within specialized subcellular compartments.

A novel function of TBK1 as a target of Cdon in oligodendrocyte differentiation and myelination.

During central nervous system development, oligodendrocyte progenitors elaborate multiple branched processes to contact axons and initiate myelination. Using cultured primary rat oligodendrocytes (OLGs), we have recently demonstrated that a cell surface protein belonging to the immunoglobulin superfamily, cell adhesion molecule-related, down-regulated by oncogenes (Cdon), is important in initiating OLG differentiation and axon myelination by promoting the formation of branched cellular processes; however, the molecular mechanism by which Cdon regulates OLG differentiation is not known. Here, using Cdon immunoprecipitation (IP) and liquid chromatography-tandem mass spectrometry analysis, we identified serine/threonine kinase TANK-binding kinase 1 (TBK1) as a candidate novel target of Cdon. We confirmed this interaction using co-IP and immunofluorescence with TBK1 antibodies, showing that TBK1 partly co-localizes with Cdon along cellular processes in puncta-like structures. We show that TBK1 is expressed throughout OLG differentiation, and surprisingly, that levels of phosphorylated TBK1 (ser172) increase during OLG maturation, while total levels of TBK1 protein decrease. To investigate function, TBK1 expression was knocked down using siRNA in OLG primary cultures, reducing protein levels by 69%. Two myelin-specific proteins, myelin basic protein and myelin-associated glycoprotein, were similarly reduced when examined at day 2 and day 4 of OLG differentiation. Reduced Cdon or TBK1 expression also decreased Akt phosphorylation at Threonine 308 in OLG. Our findings provide evidence that a Cdon-TBK1 complex is associated with Akt phosphorylation and early OLG differentiation.

FLIM FRET Visualization of Cdc42 Activation by Netrin-1 in Embryonic Spinal Commissural Neuron Growth Cones.

Netrin-1 is an essential extracellular chemoattractant that signals through its receptor DCC to guide commissural axon extension in the embryonic spinal cord. DCC directs the organization of F-actin in growth cones by activating an intracellular protein complex that includes the Rho GTPase Cdc42, a critical regulator of cell polarity and directional migration. To address the spatial distribution of signaling events downstream of netrin-1, we expressed the FRET biosensor Raichu-Cdc42 in cultured embryonic rat spinal commissural neurons. Using FLIM-FRET imaging we detected rapid activation of Cdc42 in neuronal growth cones following application of netrin-1. Investigating the signaling mechanisms that control Cdc42 activation by netrin-1, we demonstrate that netrin-1 rapidly enriches DCC at the leading edge of commissural neuron growth cones and that netrin-1 induced activation of Cdc42 in the growth cone is blocked by inhibiting src family kinase signaling. These findings reveal the activation of Cdc42 in embryonic spinal commissural axon growth cones and support the conclusion that src family kinase activation downstream of DCC is required for Cdc42 activation by netrin-1.

The oligodendrocyte-specific antibody 'CC1' binds Quaking 7.

The mouse monoclonal antibody marketed as anti-adenomatous polyposis coli clone CC1, often referred to as CC1, is the antibody most commonly used to specifically label mature oligodendrocytes without labeling myelin. Previous studies have shown that despite being raised against adenomatous polyposis coli, this antibody binds another unknown antigen. We show that the CC1 antibody binds Quaking 7, an RNA-binding protein that is highly up-regulated in myelinating oligodendrocytes in the central nervous system. The monoclonal antibody anti-adenomatous polyposis coli (APC) clone CC1, is the antibody most commonly used to specifically label the cell bodies of mature oligodendrocytes. Despite being raised against APC, previous studies showed this antibody binds another unknown antigen. We show that the CC1 antibody binds Quaking (QKI) 7, an RNA-binding protein which is highly up-regulated in myelinating oligodendrocytes.

Truncated netrin-1 contributes to pathological vascular permeability in diabetic retinopathy.

Diabetic retinopathy (DR) is a major complication of diabetes and a leading cause of blindness in the working-age population. Impaired blood-retinal barrier function leads to macular edema that is closely associated with the deterioration of central vision. We previously demonstrated that the neuronal guidance cue netrin-1 activates a program of reparative angiogenesis in microglia within the ischemic retina. Here, we provide evidence in both vitreous humor of diabetic patients and in retina of a murine model of diabetes that netrin-1 is metabolized into a bioactive fragment corresponding to domains VI and V of the full-length molecule. In contrast to the protective effects of full-length netrin-1 on retinal microvasculature, the VI-V fragment promoted vascular permeability through the uncoordinated 5B (UNC5B) receptor. The collagenase matrix metalloprotease 9 (MMP-9), which is increased in patients with diabetic macular edema, was capable of cleaving netrin-1 into the VI-V fragment. Thus, MMP-9 may release netrin-1 fragments from the extracellular matrix and facilitate diffusion. Nonspecific inhibition of collagenases or selective inhibition of MMP-9 decreased pathological vascular permeability in a murine model of diabetic retinal edema. This study reveals that netrin-1 degradation products are capable of modulating vascular permeability, suggesting that these fragments are of potential therapeutic interest for the treatment of DR.

Netrin-1-Regulated Distribution of UNC5B and DCC in Live Cells Revealed by TICCS.

Netrins are secreted proteins that direct cell migration and adhesion during development. Netrin-1 binds its receptors deleted in colorectal cancer (DCC) and the UNC5 homologs (UNC5A-D) to activate downstream signaling that ultimately directs cytoskeletal reorganization. To investigate how netrin-1 regulates the dynamic distribution of DCC and UNC5 homologs, we applied fluorescence confocal and total internal reflection fluorescence microscopy, and sliding window temporal image cross correlation spectroscopy, to measure time profiles of the plasma membrane distribution, aggregation state, and interaction fractions of fluorescently tagged netrin receptors expressed in HEK293T cells. Our measurements reveal changes in receptor aggregation that are consistent with netrin-1-induced recruitment of DCC-enhanced green fluorescent protein (EGFP) from intracellular vesicles to the plasma membrane. Netrin-1 also induced colocalization of coexpressed full-length DCC-EGFP with DCC-T-mCherry, a putative DCC dominant negative that replaces the DCC intracellular domain with mCherry, consistent with netrin-1-induced receptor oligomerization, but with no change in aggregation state with time, providing evidence that signaling via the DCC intracellular domain triggers DCC recruitment to the plasma membrane. UNC5B expressed alone was also recruited by netrin-1 to the plasma membrane. Coexpressed DCC and UNC5 homologs are proposed to form a heteromeric netrin-receptor complex to mediate a chemorepellent response. Application of temporal image cross correlation spectroscopy to image series of cells coexpressing UNC5B-mCherry and DCC-EGFP revealed a netrin-1-induced increase in colocalization, with both receptors recruited to the plasma membrane from preexisting clusters, consistent with vesicular recruitment and receptor heterooligomerization. Plasma membrane recruitment of DCC or UNC5B was blocked by application of the netrin-1 VI-V peptide, which fails to activate chemoattraction, or by pharmacological block of Src family kinase signaling, consistent with receptor recruitment requiring netrin-1-activated signaling. Our findings reveal a mechanism activated by netrin-1 that recruits DCC and UNC5B to the plasma membrane.