ESCRT disruption provides evidence against transsynaptic signaling functions for extracellular vesicles.

Extracellular vesicles (EVs) are released by many cell types including neurons, carrying cargoes involved in signaling and disease. It is unclear whether EVs promote intercellular signaling or serve primarily to dispose of unwanted materials. We show that loss of multivesicular endosome-generating ESCRT (endosomal sorting complex required for transport) machinery disrupts release of EV cargoes from Drosophila motor neurons. Surprisingly, ESCRT depletion does not affect the signaling activities of the EV cargo Synaptotagmin-4 (Syt4) and disrupts only some signaling activities of the EV cargo Evenness Interrupted (Evi). Thus, these cargoes may not require intercellular transfer via EVs, and instead may be conventionally secreted or function cell autonomously in the neuron. We find that EVs are phagocytosed by glia and muscles, and that ESCRT disruption causes compensatory autophagy in presynaptic neurons, suggesting that EVs are one of several redundant mechanisms to remove cargoes from synapses. Our results suggest that synaptic EV release serves primarily as a proteostatic mechanism for certain cargoes.

Local regulation of extracellular vesicle traffic by the synaptic endocytic machinery.

Neuronal extracellular vesicles (EVs) are locally released from presynaptic terminals, carrying cargoes critical for intercellular signaling and disease. EVs are derived from endosomes, but it is unknown how these cargoes are directed to the EV pathway rather than for conventional endolysosomal degradation. Here, we find that endocytic machinery plays an unexpected role in maintaining a release-competent pool of EV cargoes at synapses. Endocytic mutants, including nervous wreck (nwk), shibire/dynamin, and AP-2, unexpectedly exhibit local presynaptic depletion specifically of EV cargoes. Accordingly, nwk mutants phenocopy synaptic plasticity defects associated with loss of the EV cargo synaptotagmin-4 (Syt4) and suppress lethality upon overexpression of the EV cargo amyloid precursor protein (APP). These EV defects are genetically separable from canonical endocytic functions in synaptic vesicle recycling and synaptic growth. Endocytic machinery opposes the endosomal retromer complex to regulate EV cargo levels and acts upstream of synaptic cargo removal by retrograde axonal transport. Our data suggest a novel molecular mechanism that locally promotes cargo loading into synaptic EVs.

Opposing functions for retromer and Rab11 in extracellular vesicle traffic at presynaptic terminals.

Neuronal extracellular vesicles (EVs) play important roles in intercellular communication and pathogenic protein propagation in neurological disease. However, it remains unclear how cargoes are selectively packaged into neuronal EVs. Here, we show that loss of the endosomal retromer complex leads to accumulation of EV cargoes including amyloid precursor protein (APP), synaptotagmin-4 (Syt4), and neuroglian (Nrg) at Drosophila motor neuron presynaptic terminals, resulting in increased release of these cargoes in EVs. By systematically exploring known retromer-dependent trafficking mechanisms, we show that EV regulation is separable from several previously identified roles of neuronal retromer. Conversely, mutations in rab11 and rab4, regulators of endosome-plasma membrane recycling, cause reduced EV cargo levels, and rab11 suppresses cargo accumulation in retromer mutants. Thus, EV traffic reflects a balance between Rab4/Rab11 recycling and retromer-dependent removal from EV precursor compartments. Our data shed light on previous studies implicating Rab11 and retromer in competing pathways in Alzheimer's disease, and suggest that misregulated EV traffic may be an underlying defect.

Photoaffinity Labeling and Quantitative Chemical Proteomics Identify LXRß as the Functional Target of Enhancers of Astrocytic apoE.

Utilizing a phenotypic screen, we identified chemical matter that increased astrocytic apoE secretion in vitro. We designed a clickable photoaffinity probe based on a pyrrolidine lead compound and carried out probe-based quantitative chemical proteomics in human astrocytoma CCF-STTG1 cells to identify liver x receptor ß (LXRß) as the target. Binding of the small molecule ligand stabilized LXRß, as shown by cellular thermal shift assay (CETSA). In addition, we identified a probe-modified peptide by mass spectrometry and proposed a model where the photoaffinity probe is bound in the ligand-binding pocket of LXRß. Taken together, our findings demonstrated that the lead chemical matter bound directly to LXRß, and our results highlight the power of chemical proteomic approaches to identify the target of a phenotypic screening hit. Additionally, the LXR photoaffinity probe and lead compound described herein may serve as valuable tools to further evaluate the LXR pathway.

Cellular Specificity of NF-κB Function in the Nervous System.

Nuclear Factor Kappa B (NF-κB) is a ubiquitously expressed transcription factor with key functions in a wide array of biological systems. While the role of NF-κB in processes, such as host immunity and oncogenesis has been more clearly defined, an understanding of the basic functions of NF-κB in the nervous system has lagged behind. The vast cell-type heterogeneity within the central nervous system (CNS) and the interplay between cell-type specific roles of NF-κB contributes to the complexity of understanding NF-κB functions in the brain. In this review, we will focus on the emerging understanding of cell-autonomous regulation of NF-κB signaling as well as the non-cell-autonomous functional impacts of NF-κB activation in the mammalian nervous system. We will focus on recent work which is unlocking the pleiotropic roles of NF-κB in neurons and glial cells (including astrocytes and microglia). Normal physiology as well as disorders of the CNS in which NF-κB signaling has been implicated will be discussed with reference to the lens of cell-type specific responses.

Class I HDAC inhibition is a novel pathway for regulating astrocytic apoE secretion.

Despite the important role of apolipoprotein E (apoE) secretion from astrocytes in brain lipid metabolism and the strong association of apoE4, one of the human apoE isoforms, with sporadic and late onset forms of Alzheimer's disease (AD) little is known about the regulation of astrocytic apoE. Utilizing annotated chemical libraries and a phenotypic screening strategy that measured apoE secretion from a human astrocytoma cell line, inhibition of pan class I histone deacetylases (HDACs) was identified as a mechanism to increase apoE secretion. Knocking down select HDAC family members alone or in combination revealed that inhibition of the class I HDAC family was responsible for enhancing apoE secretion. Knocking down LXRα and LXRß genes revealed that the increase in astrocytic apoE in response to HDAC inhibition occurred via an LXR-independent pathway. Collectively, these data suggest that pan class I HDAC inhibition is a novel pathway for regulating astrocytic apoE secretion.

Targeting of NF-κB to Dendritic Spines Is Required for Synaptic Signaling and Spine Development.

Long-term forms of brain plasticity share a requirement for changes in gene expression induced by neuronal activity. Mechanisms that determine how the distinct and overlapping functions of multiple activity-responsive transcription factors, including nuclear factor κB (NF-κB), give rise to stimulus-appropriate neuronal responses remain unclear. We report that the p65/RelA subunit of NF-κB confers subcellular enrichment at neuronal dendritic spines and engineer a p65 mutant that lacks spine enrichment (p65ΔSE) but retains inherent transcriptional activity equivalent to wild-type p65. Wild-type p65 or p65ΔSE both rescue NF-κB-dependent gene expression in p65-deficient murine hippocampal neurons responding to diffuse (PMA/ionomycin) stimulation. In contrast, neurons lacking spine-enriched NF-κB are selectively impaired in NF-κB-dependent gene expression induced by elevated excitatory synaptic stimulation (bicuculline or glycine). We used the setting of excitatory synaptic activity during development that produces NF-κB-dependent growth of dendritic spines to test physiological function of spine-enriched NF-κB in an activity-dependent response. Expression of wild-type p65, but not p65ΔSE, is capable of rescuing spine density to normal levels in p65-deficient pyramidal neurons. Collectively, these data reveal that spatial localization in dendritic spines contributes unique capacities to the NF-κB transcription factor in synaptic activity-dependent responses.SIGNIFICANCE STATEMENT Extensive research has established a model in which the regulation of neuronal gene expression enables enduring forms of plasticity and learning. However, mechanisms imparting stimulus specificity to gene regulation, ensuring biologically appropriate responses, remain incompletely understood. NF-κB is a potent transcription factor with evolutionarily conserved functions in learning and the growth of excitatory synaptic contacts. Neuronal NF-κB is localized in both synapse and somatic compartments, but whether the synaptic pool of NF-κB has discrete functions is unknown. This study reveals that NF-κB enriched in dendritic spines (the postsynaptic sites of excitatory contacts) is selectively required for NF-κB activation by synaptic stimulation and normal dendritic spine development. These results support spatial localization at synapses as a key variable mediating selective stimulus-response coupling.

A requirement for nuclear factor-kappaB in developmental and plasticity-associated synaptogenesis.

Structural plasticity of dendritic spines and synapses is a fundamental mechanism governing neuronal circuits and may form an enduring basis for information storage in the brain. We find that the p65 subunit of the nuclear factor-κB (NF-κB) transcription factor, which is required for learning and memory, controls excitatory synapse and dendritic spine formation and morphology in murine hippocampal neurons. Endogenous NF-κB activity is elevated by excitatory transmission during periods of rapid spine and synapse development. During in vitro synaptogenesis, NF-κB enhances dendritic spine and excitatory synapse density and loss of endogenous p65 decreases spine density and spine head volume. Cell-autonomous function of NF-κB within the postsynaptic neuron is sufficient to regulate the formation of both presynaptic and postsynaptic elements. During synapse development in vivo, loss of NF-κB similarly reduces spine density and also diminishes the amplitude of synaptic responses. In contrast, after developmental synaptogenesis has plateaued, endogenous NF-κB activity is low and p65 deficiency no longer attenuates basal spine density. Instead, NF-κB in mature neurons is activated by stimuli that induce demand for new synapses, including estrogen and short-term bicuculline, and is essential for upregulating spine density in response to these stimuli. p65 is enriched in dendritic spines making local protein-protein interactions possible; however, the effects of NF-κB on spine density require transcription and the NF-κB-dependent regulation of PSD-95, a critical postsynaptic component. Collectively, our data define a distinct role for NF-κB in imparting transcriptional regulation required for the induction of changes to, but not maintenance of, excitatory synapse and spine density.

Characterization of the core elements of the NF-κB signaling pathway of the sea anemone Nematostella vectensis.

The sea anemone Nematostella vectensis is the leading developmental and genomic model for the phylum Cnidaria, which includes anemones, hydras, jellyfish, and corals. In insects and vertebrates, the NF-κB pathway is required for cellular and organismal responses to various stresses, including pathogens and chemicals, as well as for several developmental processes. Herein, we have characterized proteins that comprise the core NF-κB pathway in Nematostella, including homologs of NF-κB, IκB, Bcl-3, and IκB kinase (IKK). We show that N. vectensis NF-κB (Nv-NF-κB) can bind to κB sites and activate transcription of reporter genes containing multimeric κB sites or the Nv-IκB promoter. Both Nv-IκB and Nv-Bcl-3 interact with Nv-NF-κB and block its ability to activate reporter gene expression. Nv-IKK is most similar to human IKKε/TBK kinases and, in vitro, can phosphorylate Ser47 of Nv-IκB. Nv-NF-κB is expressed in a subset of ectodermal cells in juvenile and adult Nematostella anemones. A bioinformatic analysis suggests that homologs of many mammalian NF-κB target genes are targets for Nv-NF-κB, including genes involved in apoptosis and responses to organic compounds and endogenous stimuli. These results indicate that NF-κB pathway proteins in Nematostella are similar to their vertebrate homologs, and these results also provide a framework for understanding the evolutionary origins of NF-κB signaling.