Loss of GDE2 leads to complex behavioral changes including memory impairment.

BACKGROUND: Alzheimer's disease (AD) and amyotrophic lateral sclerosis/frontotemporal dementia (ALS/FTD) are debilitating neurodegenerative diseases for which there are currently no cures. Familial cases with known genetic causes make up less than 10% of these diseases, and little is known about the underlying mechanisms that contribute to sporadic disease. Accordingly, it is important to expand investigations into possible pathways that may contribute to disease pathophysiology. Glycerophosphodiester phosphodiesterase 2 (GDE2 or GDPD5) is a membrane-bound enzyme that acts at the cell surface to cleave the glycosylphosphatidylinositol (GPI)-anchor that tethers distinct proteins to the membrane. GDE2 abnormally accumulates in intracellular compartments in the brain of patients with AD, ALS, and ALS/FTD, indicative of GDE2 dysfunction. Mice lacking GDE2 (Gde2KO) show neurodegenerative changes such as neuronal loss, reduced synaptic proteins and synapse loss, and increased Aß deposition, raising the possibility that GDE2 disruption in disease might contribute to disease pathophysiology. However, the effect of GDE2 loss on behavioral function and learning/memory has not been characterized. RESULTS: Here, we show that GDE2 is expressed throughout the adult mouse brain in areas including the cortex, hippocampus, habenula, thalamus, and amygdala. Gde2KO and WT mice were tested in a set of behavioral tasks between 7 and 16 months of age. Compared to WT, Gde2KO mice display moderate hyperactivity that becomes more pronounced with age across a variety of behavioral tests assessing novelty-induced exploratory activity. Additionally, Gde2KO mice show reduced startle response, with females showing additional defects in prepulse inhibition. No changes in anxiety-associated behaviors were found, but Gde2KOs show reduced sociability. Notably, aged Gde2KO mice demonstrate impaired short/long-term spatial memory and cued fear memory/secondary contextual fear acquisition. CONCLUSIONS: Taken together, these observations suggest that loss of GDE2 leads to behavioral deficits, some of which are seen in neurodegenerative disease models, implying that loss of GDE2 may be an important contributor to phenotypes associated with neurodegeneration.

An independent regulator of global release pathways in astrocytes generates a subtype of extracellular vesicles required for postsynaptic function.

Extracellular vesicles (EVs) are heterogeneous in size, composition, and function. We show that the six-transmembrane protein glycerophosphodiester phosphodiesterase 3 (GDE3) regulates actin remodeling, a global EV biogenic pathway, to release an EV subtype with distinct functions. GDE3 is necessary and sufficient for releasing EVs containing annexin A1 and GDE3 from the plasma membrane via Wiskott-Aldrich syndrome protein family member 3 (WAVE3), a major regulator of actin dynamics. GDE3 is expressed in astrocytes but not neurons, yet mice lacking GDE3 [Gde3 knockout (KO)] have decreased miniature excitatory postsynaptic current (mEPSC) amplitudes in hippocampal CA1 neurons. EVs from cultured wild-type astrocytes restore mEPSC amplitudes in Gde3 KOs, while EVs from Gde3 KO astrocytes or astrocytes inhibited for WAVE3 actin branching activity do not. Thus, GDE3-WAVE3 is a nonredundant astrocytic pathway that remodels actin to release a functionally distinct EV subtype, supporting the concept that independent regulation of global EV release pathways differentially regulates EV signaling within the cellular EV landscape.

The distribution and function of GDE2, a regulator of spinal motor neuron survival, are disrupted in Amyotrophic Lateral Sclerosis.

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease that affects the viability of upper and lower motor neurons. Current options for treatment are limited, necessitating deeper understanding of the mechanisms underlying ALS pathogenesis. Glycerophosphodiester phosphodiesterase 2 (GDE2 or GDPD5) is a six-transmembrane protein that acts on the cell surface to cleave the glycosylphosphatidylinositol (GPI)-anchor that tethers some proteins to the membrane. GDE2 is required for the survival of spinal motor neurons but whether GDE2 neuroprotective activity is disrupted in ALS is not known. We utilized a combination of mouse models and patient post-mortem samples to evaluate GDE2 functionality in ALS. Haplogenetic reduction of GDE2 exacerbated motor neuron degeneration and loss in SOD1G93A mice but not in control SOD1WT transgenic animals, indicating that GDE2 neuroprotective function is diminished in the context of SOD1G93A. In tissue samples from patients with ALS, total levels of GDE2 protein were equivalent to healthy controls; however, membrane levels of GDE2 were substantially reduced. Indeed, GDE2 was found to aberrantly accumulate in intracellular compartments of ALS motor cortex, consistent with a disruption of GDE2 function at the cell surface. Supporting the impairment of GDE2 activity in ALS, tandem-mass-tag mass spectrometry revealed a pronounced reduction of GPI-anchored proteins released into the CSF of patients with ALS compared with control patients. Taken together, this study provides cellular and biochemical evidence that GDE2 distribution and activity is disrupted in ALS, supporting the notion that the failure of GDE2-dependent neuroprotective pathways contributes to neurodegeneration and motor neuron loss in disease. These observations highlight the dysregulation of GPI-anchored protein pathways as candidate mediators of disease onset and progression and accordingly, provide new insight into the mechanisms underlying ALS pathogenesis.

GDE2-RECK controls ADAM10 α-secretase-mediated cleavage of amyloid precursor protein.

A disintegrin and metalloprotease 10 (ADAM10) is the α-secretase for amyloid precursor protein (APP). ADAM10 cleaves APP to generate neuroprotective soluble APPα (sAPPα), which precludes the generation of Aß, a defining feature of Alzheimer's disease (AD) pathophysiology. Reduced ADAM10 activity is implicated in AD, but the mechanisms mediating ADAM10 modulation are unclear. We find that the plasma membrane enzyme glycerophosphodiester phosphodiesterase 2 (GDE2) stimulates ADAM10 APP cleavage by shedding and inactivating reversion-inducing cysteine-rich protein with Kazal motifs (RECK), a glycosylphosphatidylinositol (GPI)-anchored inhibitor of ADAM10. In AD, membrane-tethered RECK is highly elevated and GDE2 is abnormally sequestered inside neurons. Genetic ablation of GDE2 phenocopies increased membrane RECK in AD, which is causal for reduced sAPPα, increased Aß, and synaptic protein loss. RECK reduction restores the balance of APP processing and rescues synaptic protein deficits. These studies identify GDE2 control of RECK surface activity as essential for ADAM10 α-secretase function and physiological APP processing. Moreover, our results suggest the involvement of the GDE2-RECK-ADAM10 pathway in AD pathophysiology and highlight RECK as a potential target for therapeutic development.

GDE2 expression in oligodendroglia regulates the pace of oligodendrocyte maturation.

BACKGROUND: Oligodendrocytes generate specialized lipid-rich sheaths called myelin that wrap axons and facilitate the rapid, saltatory transmission of action potentials. Extrinsic signals and surface-mediated pathways coordinate oligodendrocyte development to ensure appropriate axonal myelination, but the mechanisms involved are not fully understood. Glycerophosphodiester phosphodiesterase 2 (GDE2 or GDPD5) is a six-transmembrane enzyme that regulates the activity of surface glycosylphosphatidylinositol (GPI)-anchored proteins by cleavage of the GPI-anchor. GDE2 is expressed in neurons where it promotes oligodendrocyte maturation through the release of neuronally-derived soluble factors. GDE2 is also expressed in oligodendrocytes but the function of oligodendroglial GDE2 is not known. RESULTS: Using Cre-lox technology, we generated mice that lack GDE2 expression in oligodendrocytes (O-Gde2KO). O-Gde2KOs show normal production and proliferation of oligodendrocyte precursor cells. However, oligodendrocyte maturation is accelerated leading to the robust increase of myelin proteins and increased myelination during development. These in vivo observations are recapitulated in vitro using purified primary oligodendrocytes, supporting cell-autonomous functions for GDE2 in oligodendrocyte maturation. CONCLUSIONS: These studies reveal that oligodendroglial GDE2 expression is required for controlling the pace of oligodendrocyte maturation. Thus, the cell-type specific expression of GDE2 is important for the coordination of oligodendrocyte maturation and axonal myelination during neural development.

GDE2-Dependent Activation of Canonical Wnt Signaling in Neurons Regulates Oligodendrocyte Maturation.

Neurons and oligodendrocytes communicate to regulate oligodendrocyte development and ensure appropriate axonal myelination. Here, we show that Glycerophosphodiester phosphodiesterase 2 (GDE2) signaling underlies a neuronal pathway that promotes oligodendrocyte maturation through the release of soluble neuronally derived factors. Mice lacking global or neuronal GDE2 expression have reduced mature oligodendrocytes and myelin proteins but retain normal numbers of oligodendrocyte precursor cells (OPCs). Wild-type (WT) OPCs cultured in conditioned medium (CM) from Gde2-null (Gde2KO) neurons exhibit delayed maturation, recapitulating in vivo phenotypes. Gde2KO neurons show robust reduction in canonical Wnt signaling, and genetic activation of Wnt signaling in Gde2KO neurons rescues in vivo and in vitro oligodendrocyte maturation. Phosphacan, a known stimulant of oligodendrocyte maturation, is reduced in CM from Gde2KO neurons but is restored when Wnt signaling is activated. These studies identify GDE2 control of Wnt signaling as a neuronal pathway that signals to oligodendroglia to promote oligodendrocyte maturation.

GDE3 regulates oligodendrocyte precursor proliferation via release of soluble CNTFRα.

Oligodendrocyte development is tightly controlled by extrinsic signals; however, mechanisms that modulate cellular responses to these factors remain unclear. Six-transmembrane glycerophosphodiester phosphodiesterases (GDEs) are emerging as central regulators of cellular differentiation via their ability to shed glycosylphosphatidylinositol (GPI)-anchored proteins from the cell surface. We show here that GDE3 controls the pace of oligodendrocyte generation by negatively regulating oligodendrocyte precursor cell (OPC) proliferation. GDE3 inhibits OPC proliferation by stimulating ciliary neurotrophic factor (CNTF)-mediated signaling through release of CNTFRα, the ligand-binding component of the CNTF-receptor multiprotein complex, which can function as a soluble factor to activate CNTF signaling. GDE3 releases soluble CNTFRα by GPI-anchor cleavage from the plasma membrane and from extracellular vesicles (EVs) after co-recruitment of CNTFRα in EVs. These studies uncover new physiological roles for GDE3 in gliogenesis and identify GDE3 as a key regulator of CNTF-dependent regulation of OPC proliferation through release of CNTFRα.

Epithelial retinoic acid receptor ß regulates serum amyloid A expression and vitamin A-dependent intestinal immunity.

Vitamin A is a dietary component that is essential for the development of intestinal immunity. Vitamin A is absorbed and converted to its bioactive derivatives retinol and retinoic acid by the intestinal epithelium, yet little is known about how epithelial cells regulate vitamin A-dependent intestinal immunity. Here we show that epithelial cell expression of the transcription factor retinoic acid receptor ß (RARß) is essential for vitamin A-dependent intestinal immunity. Epithelial RARß activated vitamin A-dependent expression of serum amyloid A (SAA) proteins by binding directly to Saa promoters. In accordance with the known role of SAAs in regulating Th17 cell effector function, epithelial RARß promoted IL-17 production by intestinal Th17 cells. More broadly, epithelial RARß was required for the development of key vitamin A-dependent adaptive immune responses, including CD4+ T-cell homing to the intestine and the development of IgA-producing intestinal B cells. Our findings provide insight into how the intestinal epithelium senses dietary vitamin A status to regulate adaptive immunity, and highlight the role of epithelial cells in regulating intestinal immunity in response to diet.

Transcription factor mechanisms guiding motor neuron differentiation and diversification.

The embryonic generation of motor neurons is a complex process involving progenitor patterning, fate specification, differentiation, and maturation. Throughout this progression, the differential expression of transcription factors has served as our road map for the eventual cell fate of nascent motor neurons. Recent findings from in vivo and in vitro models of motor neuron development have expanded our understanding of how transcription factors govern motor neuron identity and their individual regulatory mechanisms. With the advent of next generation sequencing approaches, researchers now have unprecedented access to the gene regulatory dynamics involved in motor neuron development and are uncovering new connections linking neurodevelopment and neurodegenerative disease.

GDE2 is essential for neuronal survival in the postnatal mammalian spinal cord.

BACKGROUND: Glycerophosphodiester phosphodiesterase 2 (GDE2) is a six-transmembrane protein that cleaves glycosylphosphatidylinositol (GPI) anchors to regulate GPI-anchored protein activity at the cell surface. In the developing spinal cord, GDE2 utilizes its enzymatic function to regulate the production of specific classes of motor neurons and interneurons; however, GDE2's roles beyond embryonic neurogenesis have yet to be defined. METHOD: Using a panel of histological, immunohistochemical, electrophysiological, behavioral, and biochemistry techniques, we characterized the postnatal Gde2 -/- mouse for evidence of degenerative neuropathology. A conditional deletion of Gde2 was used to study the temporal requirements for GDE2 in neuronal survival. Biochemical approaches identified deficits in the processing of GPI-anchored GDE2 substrates in the SOD1 G93A mouse model of familial Amyotrophic Lateral Sclerosis that shows robust motor neuron degeneration. RESULTS: Here we show that GDE2 expression continues postnatally, and adult mice lacking GDE2 exhibit a slow, progressive neuronal degeneration with pathologies similar to human neurodegenerative disease. Early phenotypes include vacuolization, microgliosis, cytoskeletal accumulation, and lipofuscin deposition followed by astrogliosis and cell death. Remaining motor neurons exhibit peripheral motor unit restructuring causing behavioral motor deficits. Genetic ablation of GDE2 after embryonic neurogenesis is complete still elicits degenerative pathology, signifying that GDE2's requirement for neuronal survival is distinct from its involvement in neuronal differentiation. Unbiased screens identify impaired processing of Glypican 4 and 6 in Gde2 null animals, and Glypican release is markedly reduced in SOD1 G93A mice. CONCLUSIONS: This study identifies a novel function for GDE2 in neuronal survival and implicates deregulated GPI-anchored protein activity in pathways mediating neurodegeneration. These findings provide new molecular insight for neuropathologies found in multiple disease settings, and raise the possibility of GDE2 hypofunctionality as a component of neurodegenerative disease.

Transcription Factor Hand-offs "Enhance" Motor Neuron Differentiation.

Rhee et al. (2016) in this issue of Neuron and Velasco et al. (2016) in Cell Stem Cell find that the activity of transcription factors binding sequentially to a series of transient early and late enhancers directs gene expression that is essential for motor neuron differentiation and function.

Real-Time Imaging Reveals Properties of Glutamate-Induced Arc/Arg 3.1 Translation in Neuronal Dendrites.

The immediate early gene Arc (also Arg3.1) produces rapid changes in synaptic properties that are linked to de novo translation. Here we develop a novel translation reporter that exploits the rapid maturation and "flash" kinetics of Gaussia luciferase (Gluc) to visualize Arc translation. Following glutamate stimulation, discrete Arc-Gluc bioluminescent flashes representing sites of de novo translation are detected within 15 s at distributed sites in dendrites, but not spines. Flashes are episodic, lasting ∼20 s, and may be unitary or repeated at ∼minute intervals at the same sites. Analysis of flash amplitudes suggests they represent the quantal product of one or more polyribosomes, while inter-flash intervals appear random, suggesting they arise from a stochastic process. Surprisingly, glutamate-induced translation is dependent on Arc open reading frame. Combined observations support a model in which stalled ribosomes are reactivated to rapidly generate Arc protein.

Neuronal differentiation is associated with a redox-regulated increase of copper flow to the secretory pathway.

Brain development requires a fine-tuned copper homoeostasis. Copper deficiency or excess results in severe neuro-pathologies. We demonstrate that upon neuronal differentiation, cellular demand for copper increases, especially within the secretory pathway. Copper flow to this compartment is facilitated through transcriptional and metabolic regulation. Quantitative real-time imaging revealed a gradual change in the oxidation state of cytosolic glutathione upon neuronal differentiation. Transition from a broad range of redox states to a uniformly reducing cytosol facilitates reduction of the copper chaperone Atox1, liberating its metal-binding site. Concomitantly, expression of Atox1 and its partner, a copper transporter ATP7A, is upregulated. These events produce a higher flux of copper through the secretory pathway that balances copper in the cytosol and increases supply of the cofactor to copper-dependent enzymes, expression of which is elevated in differentiated neurons. Direct link between glutathione oxidation and copper compartmentalization allows for rapid metabolic adjustments essential for normal neuronal function.

Prdx4 is a compartment-specific H2O2 sensor that regulates neurogenesis by controlling surface expression of GDE2.

Neural progenitors and terminally differentiated neurons show distinct redox profiles, suggesting that coupled-redox cascades regulate the initiation and progression of neuronal differentiation. Discrete cellular compartments have different redox environments and how they contribute to differentiation is unclear. Here we show that Prdx4, an endoplasmic reticulum (ER) enzyme that metabolizes H2O2, acts as a tunable regulator of neurogenesis via its compartmentalized thiol-oxidative function. Prdx4 ablation causes premature motor neuron differentiation and progenitor depletion, leading to imbalances in subtype-specific motor neurons. GDE2, a six-transmembrane protein that induces differentiation by downregulating Notch signalling through surface cleavage of GPI-anchored proteins, is targeted by Prdx4 oxidative activity. Prdx4 dimers generated by H2O2 metabolism oxidize two cysteine residues within the GDE2 enzymatic domain, which blocks GDE2 trafficking to the plasma membrane and prevents GDE2 neurogeneic function. Thus, Prdx4 oxidative activity acts as a sensor to directly couple neuronal differentiation with redox environments in the ER.

Semaphorin 6B acts as a receptor in post-crossing commissural axon guidance.

Semaphorins are a large family of axon guidance molecules that are known primarily as ligands for plexins and neuropilins. Although class-6 semaphorins are transmembrane proteins, they have been implicated as ligands in different aspects of neural development, including neural crest cell migration, axon guidance and cerebellar development. However, the specific spatial and temporal expression of semaphorin 6B (Sema6B) in chick commissural neurons suggested a receptor role in axon guidance at the spinal cord midline. Indeed, in the absence of Sema6B, post-crossing commissural axons lacked an instructive signal directing them rostrally along the contralateral floorplate border, resulting in stalling at the exit site or even caudal turns. Truncated Sema6B lacking the intracellular domain was unable to rescue the loss-of-function phenotype, confirming a receptor function of Sema6B. In support of this, we demonstrate that Sema6B binds to floorplate-derived plexin A2 (PlxnA2) for navigation at the midline, whereas a cis-interaction between PlxnA2 and Sema6B on pre-crossing commissural axons may regulate the responsiveness of axons to floorplate-derived cues.

Activated retinoid receptors are required for the migration and fate maintenance of subsets of cortical neurons.

Layer-specific cortical neurons are essential components of local, intracortical and subcortical circuits and are specified by complex signaling pathways acting on cortical progenitors. However, whether extrinsic signals contribute to postmitotic cortical neuronal development is unclear. Here we show in mice that retinoic acid (RA) receptors are activated in newly born migrating cortical neurons indicative of endogenous RA in the cortex. Disruption of RA signaling in postmitotic neurons by dominant-negative retinoid receptor RAR403 expression specifically delays late-born cortical neuron migration in vivo. Moreover, prospective layer V-III neurons that express RAR403 fail to maintain their fates and instead acquire characteristics of layer II neurons. This latter phenotype is rescued by active forms of ß-catenin at central and caudal but not rostral cortical regions. Taken together, these observations suggest that RA signaling pathways operate postmitotically to regulate the onset of radial migration and to consolidate regional differences in cortical neuronal identity.

GDE2 promotes neurogenesis by glycosylphosphatidylinositol-anchor cleavage of RECK.

The six-transmembrane protein glycerophosphodiester phosphodiesterase 2 (GDE2) induces spinal motor neuron differentiation by inhibiting Notch signaling in adjacent motor neuron progenitors. GDE2 function requires activity of its extracellular domain that shares homology with glycerophosphodiester phosphodiesterases (GDPDs). GDPDs metabolize glycerophosphodiesters into glycerol-3-phosphate and corresponding alcohols, but whether GDE2 inhibits Notch signaling by this mechanism is unclear. Here, we show that GDE2, unlike classical GDPDs, cleaves glycosylphosphatidylinositol (GPI) anchors. GDE2 GDPD activity inactivates the Notch activator RECK (reversion-inducing cysteine-rich protein with kazal motifs) by releasing it from the membrane through GPI-anchor cleavage. RECK release disinhibits ADAM (a disintegrin and metalloproteinase) protease-dependent shedding of the Notch ligand Delta-like 1 (Dll1), leading to Notch inactivation. This study identifies a previously unrecognized mechanism to initiate neurogenesis that involves GDE2-mediated surface cleavage of GPI-anchored targets to inhibit Dll1-Notch signaling.

Gde2 regulates cortical neuronal identity by controlling the timing of cortical progenitor differentiation.

The mammalian cortex is a multilaminar structure consisting of specialized layer-specific neurons that form complex circuits throughout the brain and spinal cord. These neurons are generated in a defined sequence dictated by their birthdate such that early-born neurons settle in deep cortical layers whereas late-born neurons populate more superficial layers. Cortical neuronal birthdate is partly controlled by an intrinsic clock-type mechanism; however, the role of extrinsic factors in the temporal control of cell-cycle exit is less clear. Here, we show that Gde2, a six-transmembrane protein that induces spinal neuronal differentiation, is expressed in the developing cortex throughout cortical neurogenesis. In the absence of Gde2, cortical progenitors fail to exit the cell cycle on time, remain cycling, accumulate and exit the cell cycle en masse towards the end of the neurogenic period. These dynamic changes in cell-cycle progression cause deficits and delays in deep-layer neuronal differentiation and robust increases in superficial neuronal numbers. Gde2(-/-) cortices show elevated levels of Notch signaling coincident with when progenitors fail to differentiate, suggesting that abnormal Notch activation retains cells in a proliferative phase that biases them to superficial fates. However, no change in Notch signaling is observed at the time of increased cell-cycle exit. These observations define a key role for Gde2 in controlling cortical neuronal fates by regulating the timing of neurogenesis, and show that loss of Gde2 uncovers additional mechanisms that trigger remaining neuronal progenitors to differentiate at the end of the neurogenic period.

A retinoic acid--rich tumor microenvironment provides clonal survival cues for tumor-specific CD8(+) T cells.

While vitamin A has been implicated in host resistance to infectious disease, little is known about the role of vitamin A and its active metabolite, retinoic acid (RA) in host defenses against cancer. Here, we show that local RA production within the tumor microenvironment (TME) is increased up to 5-fold as compared with naïve surrounding tissue, with a commensurate increase in RA signaling to regionally infiltrating tumor-reactive T cells. Conditional disruption of RA signaling in CD8(+) T cells using a dominant negative retinoic acid receptor α (dnRARα) established that RA signaling is required for tumor-specific CD8(+) T-cell expansion/accumulation and protective antitumor immunity. In vivo analysis of antigen-specific CD8(+) T-cell responses revealed that early T-cell expansion was RA-independent; however, late T-cell expansion and clonal accumulation was suppressed strongly in the absence of RA signaling. Our findings indicate that RA function is essential for the survival of tumor-reactive CD8(+) T cells within the TME.

GDE2 regulates subtype-specific motor neuron generation through inhibition of Notch signaling.

The specification of spinal interneuron and motor neuron identities initiates within progenitor cells, while motor neuron subtype diversification is regulated by hierarchical transcriptional programs implemented postmitotically. Here we find that mice lacking GDE2, a six-transmembrane protein that triggers motor neuron generation, exhibit selective losses of distinct motor neuron subtypes, specifically in defined subsets of limb-innervating motor pools that correlate with the loss of force-generating alpha motor neurons. Mechanistically, GDE2 is expressed by postmitotic motor neurons but utilizes extracellular glycerophosphodiester phosphodiesterase activity to induce motor neuron generation by inhibiting Notch signaling in neighboring motor neuron progenitors. Thus, neuronal GDE2 controls motor neuron subtype diversity through a non-cell-autonomous feedback mechanism that directly regulates progenitor cell differentiation, implying that subtype specification initiates within motor neuron progenitor populations prior to their differentiation into postmitotic motor neurons.