Role of the caspase-8/RIPK3 axis in Alzheimer's disease pathogenesis and Aß-induced NLRP3 inflammasome activation.

The molecular mediators of cell death and inflammation in Alzheimer's disease (AD) have yet to be fully elucidated. Caspase-8 is a critical regulator of several cell death and inflammatory pathways; however, its role in AD pathogenesis has not yet been examined in detail. In the absence of caspase-8, mice are embryonic lethal due to excessive receptor interacting protein kinase 3-dependent (RIPK3-dependent) necroptosis. Compound RIPK3 and caspase-8 mutants rescue embryonic lethality, which we leveraged to examine the roles of these pathways in an amyloid ß-mediated (Aß-mediated) mouse model of AD. We found that combined deletion of caspase-8 and RIPK3, but not RIPK3 alone, led to diminished Aß deposition and microgliosis in the mouse model of AD carrying human presenilin 1 and amyloid precursor protein with 5 familial AD mutations (5xFAD). Despite its well-known role in cell death, caspase-8 did not appear to affect cell loss in the 5xFAD model. In contrast, we found that caspase-8 was a critical regulator of Aß-driven inflammasome gene expression and IL-1ß release. Interestingly, loss of RIPK3 had only a modest effect on disease progression, suggesting that inhibition of necroptosis or RIPK3-mediated cytokine pathways is not critical during midstages of Aß amyloidosis. These findings suggest that therapeutics targeting caspase-8 may represent a novel strategy to limit Aß amyloidosis and neuroinflammation in AD.

Transcytosis of TrkA leads to diversification of dendritic signaling endosomes.

The development of the peripheral nervous system relies on long-distance signaling from target organs back to the soma. In sympathetic neurons, this long-distance signaling is mediated by target derived Nerve Growth Factor (NGF) interacting with its axonal receptor, TrkA. This ligand receptor complex internalizes into what is commonly referred to as the signaling endosome which is transported retrogradely to the soma and dendrites to mediate survival signaling and synapse formation, respectively. The molecular identity of signaling endosomes in dendrites has not yet been determined. Here, we perform a detailed analysis of TrkA endosomal compartments and trafficking patterns. We find that signaling endosomes are not uniform but molecularly diversified into Rab7 (late endosome) and Rab11 (recycling endosome) populations in axons and dendrites in vitro and in the soma in vivo. Surprisingly, TrkA-NGF signaling endosomes in dendrites undergo dynamic trafficking events, including putative fusion and fission. Overall, we find that signaling endosomes do not remain as a singular endosomal subtype but instead exist in multiple populations that undergo dynamic endosomal trafficking events. These dynamic events might drive functional diversification of the signaling endosome.

Temporally restricted death and the role of p75NTR as a survival receptor in the developing sensory nervous system.

The peripheral somatosensory system overproduces neurons early in development followed by a period of cell death during final target innervation. The decision to survive or die in somatosensory neurons of the dorsal root ganglion (DRG) is mediated by target-derived neurotrophic factors and their cognate receptors. Subsets of peripheral somatosensory neurons can be crudely defined by the neurotrophic receptors that they express: peptidergic nociceptors (TrkA+), nonpeptidergic nociceptors (Ret+), mechanoreceptors (Ret+ or TrkB+), and proprioceptors (TrkC+). A direct comparison of early developmental timing between these subsets has not been performed. Here we characterized the accumulation and death of TrkA, B, C, and Ret+ neurons in the DRG as a function of developmental time. We find that TrkB, TrkC, and Ret-expressing neurons in the DRG complete developmental cell death prior to TrkA-expressing neurons. Given the broadly defined roles of the neurotrophin receptor p75NTR in augmenting neurotrophic signaling in sensory neurons, we investigated its role in supporting the survival of these distinct subpopulations. We find that TrkA+, TrkB+, and TrkC+ sensory neuron subpopulations require p75NTR for survival, but proliferating progenitors do not. These data demonstrate how diverging sensory neurons undergo successive waves of cell death and how p75NTR represses the magnitude, but not developmental window of this culling. © 2018 Wiley Periodicals, Inc. Develop Neurobiol 78: 701-717, 2018.

Delineating neurotrophin-3 dependent signaling pathways underlying sympathetic axon growth along intermediate targets.

Postganglionic sympathetic neurons detect vascular derived neurotrophin 3 (NT3) via the axonally expressed receptor tyrosine kinase, TrkA, to promote chemo-attraction along intermediate targets. Once axons arrive to their final target, a structurally related neurotrophic factor, nerve growth factor (NGF), also acts through TrkA to promote final target innervation. Does TrkA signal differently at these different locales? We previously found that Coronin-1 is upregulated in sympathetic neurons upon exposure to NGF, thereby endowing the NGF-TrkA complex with new signaling capabilities (i.e. calcium signaling), which dampens axon growth and branching. Based on the notion that axons do not express functional levels of Coronin-1 prior to final target innervation, we developed an in vitro model for axon growth and branching along intermediate targets using Coro1a-/- neurons grown in NT3. We found that, similar to NGF-TrkA, NT3-TrkA is capable of inducing MAPK and PI3K in the presence or absence of Coronin-1. However, unlike NGF, NT3 does not induce calcium release from intracellular stores. Using a combination of pharmacology, knockout neurons and in vitro functional assays, we suggest that the NT3-TrkA complex uses Ras/MAPK and/or PI3K-AKT signaling to induce axon growth and inhibit axon branching along intermediate targets. However, in the presence of Coronin-1, these signaling pathways lose their ability to impact NT3 dependent axon growth or branching. This is consistent with a role for Coronin-1 as a molecular switch for axon behavior and suggests that Coronin-1 suppresses NT3 dependent axon behavior.

TNFa/TNFR2 signaling is required for glial ensheathment at the dorsal root entry zone.

Somatosensory information from the periphery is routed to the spinal cord through centrally-projecting sensory axons that cross into the central nervous system (CNS) via the dorsal root entry zone (DREZ). The glial cells that ensheath these axons ensure rapid propagation of this information. Despite the importance of this glial-axon arrangement, how this afferent nerve is assembled during development is unknown. Using in vivo, time-lapse imaging we show that as centrally-projecting pioneer axons from dorsal root ganglia (DRG) enter the spinal cord, they initiate expression of the cytokine TNFalpha. This induction coincides with ensheathment of these axons by associated glia via a TNF receptor 2 (TNFR2)-mediated process. This work identifies a signaling cascade that mediates peripheral glial-axon interactions and it functions to ensure that DRG afferent projections are ensheathed after pioneer axons complete their navigation, which promotes efficient somatosensory neural function.

Death Receptor 6 Promotes Wallerian Degeneration in Peripheral Axons.

Axon degeneration during development is required to sculpt a functional nervous system and is also a hallmark of pathological insult, such as injury [1, 2]. Despite similar morphological characteristics, very little overlap in molecular mechanisms has been reported between pathological and developmental degeneration [3-5]. In the peripheral nervous system (PNS), developmental axon pruning relies on receptor-mediated extrinsic degeneration mechanisms to determine which axons are maintained or degenerated [5-7]. Receptors have not been implicated in Wallerian axon degeneration; instead, axon autonomous, intrinsic mechanisms are thought to be the primary driver for this type of axon disintegration [8-10]. Here we survey the role of neuronally expressed, paralogous tumor necrosis factor receptor super family (TNFRSF) members in Wallerian degeneration. We find that an orphan receptor, death receptor 6 (DR6), is required to drive axon degeneration after axotomy in sympathetic and sensory neurons cultured in microfluidic devices. We sought to validate these in vitro findings in vivo using a transected sciatic nerve model. Consistent with the in vitro findings, DR6-/- animals displayed preserved axons up to 4 weeks after injury. In contrast to phenotypes observed in Wlds and Sarm1-/- mice, preserved axons in DR6-/- animals display profound myelin remodeling. This indicates that deterioration of axons and myelin after axotomy are mechanistically distinct processes. Finally, we find that JNK signaling after injury requires DR6, suggesting a link between this novel extrinsic pathway and the axon autonomous, intrinsic pathways that have become established for Wallerian degeneration.

Coronin-1 and calcium signaling governs sympathetic final target innervation.

Development of a functional peripheral nervous system requires axons to rapidly innervate and arborize into final target organs and then slow but not halt their growth to establish stable connections while keeping pace with organ growth. Here we examine the role of the NGF-TrkA effector protein, Coronin-1, on postganglionic sympathetic neuron final target innervation. In the absence of Coronin-1 we find that NGF-TrkA-PI3K signaling drives robust axon growth and branching in part by suppressing GSK3ß. In contrast, the presence of Coronin-1 (wild-type neurons) suppresses but does not halt NGF-TrkA-dependent growth and branching. This relative suppression in axon growth behaviors is due to Coronin-1-dependent calcium release via PLC-γ1 signaling, which releases PI3K-dependent suppression of GSK3ß. Finally, we demonstrate that Coro1a(-/-) mice display sympathetic axon overgrowth and overbranching phenotypes in the developing heart. Together with previous work demonstrating the Coronin-1 expression is NGF dependent, this work suggests that periods before and after NGF-TrkA-induced Coronin-1 expression (and likely other factors) defines two distinct axon growth states, which are critical for proper circuit formation in the sympathetic nervous system.

TNF-α/TNFR1 signaling is required for the development and function of primary nociceptors.

Primary nociceptors relay painful touch information from the periphery to the spinal cord. Although it is established that signals generated by receptor tyrosine kinases TrkA and Ret coordinate the development of distinct nociceptive circuits, mechanisms modulating TrkA or Ret pathways in developing nociceptors are unknown. We have identified tumor necrosis factor (TNF) receptor 1 (TNFR1) as a critical modifier of TrkA and Ret signaling in peptidergic and nonpeptidergic nociceptors. Specifically, TrkA+ peptidergic nociceptors require TNF-α-TNFR1 forward signaling to suppress nerve growth factor (NGF)-mediated neurite growth, survival, excitability, and differentiation. Conversely, TNFR1-TNF-α reverse signaling augments the neurite growth and excitability of Ret+ nonpeptidergic nociceptors. The developmental and functional nociceptive defects associated with loss of TNFR1 signaling manifest behaviorally as lower pain thresholds caused by increased sensitivity to NGF. Thus, TNFR1 exerts a dual role in nociceptor information processing by suppressing TrkA and enhancing Ret signaling in peptidergic and nonpeptidergic nociceptors, respectively.

Coronin-1 is a neurotrophin endosomal effector that is required for developmental competition for survival.

Retrograde communication from axonal targets to neuronal cell bodies is critical for both the development and function of the nervous system. Much progress has been made in recent years linking long-distance, retrograde signaling to a signaling endosome, yet the mechanisms governing the trafficking and signaling of these endosomes remain mostly uncharacterized. Here we report that in mouse sympathetic neurons, the target-derived nerve growth factor (NGF)-tropomyosin-related kinase type 1 (TrkA, also called Ntrk1) signaling endosome, on arrival at the cell body, induces the expression and recruitment of a new effector protein known as Coronin-1 (also called Coro1a). In the absence of Coronin-1, the NGF-TrkA signaling endosome fuses to lysosomes sixfold to tenfold faster than when Coronin-1 is intact. We also define a new Coronin-1-dependent trafficking event in which signaling endosomes recycle and re-internalize on arrival at the cell body. Beyond influencing endosomal trafficking, Coronin-1 is also required for several NGF-TrkA-dependent signaling events, including calcium release, calcineurin activation and phosphorylation of cAMP responsive element binding protein (CREB). These results establish Coronin-1 as an essential component of a feedback loop that mediates NGF-TrkA endosome stability, recycling and signaling as a critical mechanism governing developmental competition for survival.

Trk activation of the ERK1/2 kinase pathway stimulates intermediate chain phosphorylation and recruits cytoplasmic dynein to signaling endosomes for retrograde axonal transport.

The retrograde transport of Trk-containing endosomes from the axon to the cell body by cytoplasmic dynein is necessary for axonal and neuronal survival. We investigated the recruitment of dynein to signaling endosomes in rat embryonic neurons and PC12 cells. We identified a novel phosphoserine on the dynein intermediate chains (ICs), and we observed a time-dependent neurotrophin-stimulated increase in intermediate chain phosphorylation on this site in both cell types. Pharmacological studies, overexpression of constitutively active MAP kinase kinase, and an in vitro assay with recombinant proteins demonstrated that the intermediate chains are phosphorylated by the MAP kinase ERK1/2, extracellular signal-regulated kinase, a major downstream effector of Trk. Live cell imaging with fluorescently tagged IC mutants demonstrated that the dephosphomimic mutants had significantly reduced colocalization with Trk and Rab7, but not a mitochondrial marker. The phosphorylated intermediate chains were enriched on immunoaffinity-purified Trk-containing organelles. Inhibition of ERK reduced the amount of phospho-IC and the total amount of dynein that copurified with the signaling endosomes. In addition, inhibition of ERK1/2 reduced the motility of Rab7- and TrkB-containing endosomes and the extent of their colocalization with dynein in axons. NGF-dependent survival of sympathetic neurons was significantly reduced by the overexpression of the dephosphomimic mutant IC-1B-S80A, but not WT IC-1B, further demonstrating the functional significance of phosphorylation on this site. These results demonstrate that neurotrophin binding to Trk initiates the recruitment of cytoplasmic dynein to signaling endosomes through ERK1/2 phosphorylation of intermediate chains for their subsequent retrograde transport in axons.

Long-distance control of synapse assembly by target-derived NGF.

We report a role for long-distance retrograde neurotrophin signaling in the establishment of synapses in the sympathetic nervous system. Target-derived NGF is both necessary and sufficient for formation of postsynaptic specializations on dendrites of sympathetic neurons. This, in turn, is a prerequisite for formation of presynaptic specializations, but not preganglionic axonal ingrowth from the spinal cord into sympathetic ganglia. We also find that NGF-TrkA signaling endosomes travel from distal axons to cell bodies and dendrites where they promote PSD clustering. Furthermore, the p75 neurotrophin receptor restricts PSD formation, suggesting an important role for antagonistic NGF-TrkA and p75 signaling pathways during retrograde control of synapse establishment. Thus, in addition to defining the appropriate number of sympathetic neurons that survive the period of developmental cell death, target-derived NGF also exerts control over the degree of connectivity between the spinal cord and sympathetic ganglia through retrograde control of synapse assembly.

A model for neuronal competition during development.

We report that developmental competition between sympathetic neurons for survival is critically dependent on a sensitization process initiated by target innervation and mediated by a series of feedback loops. Target-derived nerve growth factor (NGF) promoted expression of its own receptor TrkA in mouse and rat neurons and prolonged TrkA-mediated signals. NGF also controlled expression of brain-derived neurotrophic factor and neurotrophin-4, which, through the receptor p75, can kill neighboring neurons with low retrograde NGF-TrkA signaling whereas neurons with high NGF-TrkA signaling are protected. Perturbation of any of these feedback loops disrupts the dynamics of competition. We suggest that three target-initiated events are essential for rapid and robust competition between neurons: sensitization, paracrine apoptotic signaling, and protection from such effects.

Cross-species annotation of basic leucine zipper factor interactions: Insight into the evolution of closed interaction networks.

Dimeric basic leucine zipper (bZIP) factors constitute one of the most important classes of enhancer-type transcription factors. In vertebrates, bZIP factors are involved in many cellular processes, including cell survival, learning and memory, cancer progression, lipid metabolism, and a variety of developmental processes. These factors have the ability to homodimerize and heterodimerize in a specific and predictable manner, resulting in hundreds of dimers with unique effects on transcription. In recent years, several studies have described dimerization preferences for bZIP factors from different species, including Homo sapiens, Drosophila melanogaster, Arabidopsis thaliana, and Saccharomyces cerevisiae. Here, these findings are summarized as novel, graphical representations of closed, interacting protein networks. These representations combine phylogenetic information, DNA-binding properties, and dimerization preference. Beyond summarizing bZIP dimerization preferences within selected species, we have included annotation for a solitary bZIP factor found in the primitive eukaryote, Giardia lamblia, a possible evolutionary precursor to the complex networks of bZIP factors encoded by other genomes. Finally, we discuss the fundamental similarities and differences between dimerization networks within the context of bZIP factor evolution.

Phosphorylation of BATF regulates DNA binding: a novel mechanism for AP-1 (activator protein-1) regulation.

BATF is a member of the AP-1 (activator protein-1) family of bZIP (basic leucine zipper) transcription factors that form transcriptionally inhibitory, DNA binding heterodimers with Jun proteins. In the present study, we demonstrate that BATF is phosphorylated in vivo on multiple serine and threonine residues and at least one tyrosine residue. Reverse-polarity PAGE revealed that serine-43 and threonine-48 within the DNA binding domain of BATF are phosphorylated. To model phosphorylation of the BATF DNA binding domain, serine-43 was replaced by an aspartate residue. BATF(S43D) retains the ability to dimerize with Jun proteins in vitro and in vivo, and the BATF(S43D):Jun heterodimer localizes properly to the nucleus of cells. Interestingly, BATF(S43D) functions like wild-type BATF to reduce AP-1-mediated gene transcription, despite the observed inability of the BATF(S43D):Jun heterodimer to bind DNA. These data demonstrate that phosphorylation of serine-43 converts BATF from a DNA binding into a non-DNA binding inhibitor of AP-1 activity. Given that 40% of mammalian bZIP transcription factors contain a residue analogous to serine-43 of BATF in their DNA binding domains, the phosphorylation event described here represents a mechanism that is potentially applicable to the regulation of many bZIP proteins.

EBNA2 and activated Notch induce expression of BATF.

The immortalization of human B lymphocytes by Epstein-Barr virus (EBV) requires the virus-encoded transactivator EBNA2 and the products of both viral and cellular genes which serve as EBNA2 targets. In this study, we identified BATF as a cellular gene that is up-regulated dramatically within 24 h following the infection of established and primary human B cells with EBV. The transactivation of BATF is mediated by EBNA2 in a B-cell-specific manner and is duplicated in non-EBV-infected B cells by the expression of mammalian Notch proteins. In contrast to other target genes activated by EBNA2, the BATF gene encodes a member of the AP-1 family of transcription factors that functions as a negative regulator of AP-1 activity and as an antagonist of cell growth. A potential role for BATF in promoting EBV latency is supported by studies in which BATF was shown to negatively impact the expression of a BZLF1 reporter gene and to reduce the frequency of lytic replication in latently infected cells. The identification of BATF as a cellular target of EBV provides important new information on how programs of viral and cellular gene expression may be coordinated to promote viral latency and control lytic-cycle entry.

BATF transgenic mice reveal a role for activator protein-1 in NKT cell development.

The importance of regulated AP-1 activity during T cell development was assessed using transgenic mice overexpressing BATF, a basic leucine zipper transcription factor and an AP-1 inhibitor. BATF transgenic animals possess normal thymic cellularity and all major T cell subsets, but show impaired thymocyte proliferation in vitro and no induction of IL-2, IL-4, IL-5, IL-10, and IL-13 expression. Since NKT cells are largely responsible for cytokine production in the thymus, this population was examined by detection of the V alpha 14-J alpha 281 TCR, flow cytometry of NK1.1(+) TCR beta(+) cells, and analysis of cytokine production by heat-stable Ag(low) thymocytes and peripheral NKT cells stimulated in vivo. Results show a severe under-representation of NKT cells in BATF transgenic animals, providing the first evidence that the precise control of AP-1-mediated transcription is critical for the proper emergence of thymus-derived NKT cells in the mouse.