Neuronal Polarity Research Articles

PAR3-PAR6-atypical PKC polarity complex proteins in neuronal polarization.

Polarity is a fundamental feature of cells. Protein complexes, including the PAR3-PAR6-aPKC complex, have conserved roles in establishing polarity across a number of eukaryotic cell types. In neurons, polarity is evident as distinct axonal versus dendritic domains. The PAR3, PAR6, and aPKC proteins also play important roles in neuronal polarization. During this process, either aPKC kinase activity, the assembly of the PAR3-PAR6-aPKC complex or the localization of these proteins is regulated downstream of a number of signaling pathways. In turn, the PAR3, PAR6, and aPKC proteins control various effector molecules to establish neuronal polarity. Herein, we discuss the many signaling mechanisms and effector functions that have been linked to PAR3, PAR6, and aPKC during the establishment of neuronal polarity.

Canonical TGF-β Signaling Negatively Regulates Neuronal Morphogenesis through TGIF/Smad Complex-Mediated CRMP2 Suppression.

Functional neuronal connectivity requires proper neuronal morphogenesis and its dysregulation causes neurodevelopmental diseases. Transforming growth factor-β (TGF-β) family cytokines play pivotal roles in development, but little is known about their contribution to morphological development of neurons. Here we show that the Smad-dependent canonical signaling of TGF-β family cytokines negatively regulates neuronal morphogenesis during brain development. Mechanistically, activated Smads form a complex with transcriptional repressor TG-interacting factor (TGIF), and downregulate the expression of a neuronal polarity regulator, collapsin response mediator protein 2. We also demonstrate that TGF-β family signaling inhibits neurite elongation of human induced pluripotent stem cell-derived neurons. Furthermore, the expression of TGF-β receptor 1, Smad4, or TGIF, which have mutations found in patients with neurodevelopmental disorders, disrupted neuronal morphogenesis in both mouse (male and female) and human (female) neurons. Together, these findings suggest that the regulation of neuronal morphogenesis by an evolutionarily conserved function of TGF-β signaling is involved in the pathogenesis of neurodevelopmental diseases.SIGNIFICANCE STATEMENT Canonical transforming growth factor-β (TGF-β) signaling plays a crucial role in multiple organ development, including brain, and mutations in components of the signaling pathway associated with several human developmental disorders. In this study, we found that Smads/TG-interacting factor-dependent canonical TGF-β signaling regulates neuronal morphogenesis through the suppression of collapsin response mediator protein-2 (CRMP2) expression during brain development, and that function of this signaling is evolutionarily conserved in the mammalian brain. Mutations in canonical TGF-β signaling factors identified in patients with neurodevelopmental disorders disrupt the morphological development of neurons. Thus, our results suggest that proper control of TGF-β/Smads/CRMP2 signaling pathways is critical for the precise execution of neuronal morphogenesis, whose impairment eventually results in neurodevelopmental disorders.

Hypergravity selectively augments neuronal in vitro differentiation

Disturbed neuronal connectivity is the ultimate cause of disability in individuals with neurological disease including spinal cord injury, head trauma, and stroke. Functional neurological recovery is limited through an unfavorable balance between neuronal regrowth and glia scar formation. Neuronal growth requires dynamic cytoskeletal protein rearrangements. Because hypergravity stabilizes microtubules while de‐stabilizing actin filaments, we hypothesized that experimental hypergravity would shift the balance between neuronal and astroglial growth in vitro.We exposed murine primary hippocampal neurons during different developmental stages to 2g using the DLR hypergravity platform. The platform unlike commercial laboratory centrifuges models physiological hypergravity and allows for cell cultivation and live‐cell imaging. We assessed neuritogenesis, neuronal polarization and maturation processes including synaptogenesis and synaptic integration in mature neural networks. Moreover, we studied primary astrocytes to shed light on their role during glial scar formation at neural lesion sites.Exposure of hippocampal neurons to 24h of 2g hypergravity increased neurite number by 30% and neurite projection length by 20% compared to 1g. At later developmental stages, mature synaptic contacts were formed under hypergravity conditions. In contrast, astrocytes showed decreases in cell spreading and lamellipodial protrusions with hypergravity.We conclude that experimental hypergravity ameliorates neuronal cell growth and synaptic contacts while halting astrocyte spreading and migration in vivo. Given the importance of this balance for neuronal regeneration in human neurological disease, we will now study the underlying mechanisms in more detail using, both, DLR hypergravity and clinostat‐induced hypogravity platforms.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Elavl3 regulates neuronal polarity through the alternative splicing of an embryo-specific exon in AnkyrinG

Alternative splicing of RNAs diversifies the functionalities of proteins, and it is optimized for each cell type and each developmental stage. nElavl (composed of Elavl2, Elavl3, and Elavl4) proteins are the RNA-binding proteins that is specifically expressed in neurons, regulate the alternative splicing of target RNAs, and promote neuronal differentiation and maturation. Recent studies revealed that Elavl3 knockout (Elavl3−/−) mice completely lost the expression of nElavl proteins in the Purkinje cells and exhibited cerebellar dysfunction. Here, we found that the alternative splicing of AnkyrinG exon 34 was misregulated in the cerebella of Elavl3−/− mice. AnkyrinG is an essential factor for the formation of neuronal polarity and is required for normal neuronal functions. We revealed that exon 34 of AnkyrinG was normally included in immature neurons and was mostly excluded in mature neurons; however, it was included in the cerebella of Elavl3−/− mice even in adulthood. In the Purkinje cells of adult Elavl3−/− mice, the length of the AnkyrinG-positive region shortened and somatic organelles leaked into the axons. These results suggested that exon 34 of AnkyrinG is an embryonic-stage-preferential exon that should be excluded from mature neurons and that Elavl3 regulates neuronal polarity through alternative splicing of this exon.

Asymmetric, nano-textured surfaces influence neuron viability and polarity.

Three dimensional, nanostructured surfaces have attracted considerable attention in biomedical research since they have proven to represent a powerful platform to influence cell fate. In particular, nanorods and nanopillars possess great potential for the control of cell adhesion and differentiation, gene and biomolecule delivery, optical and electrical stimulation and recording, as well as cell patterning. Here, we investigate the influence of asymmetric poly(dichloro-p-xylene) (PPX) columnar films on the adhesion and maturation of cortical neurons. We show that nanostructured films with dense, inclined polymer columns can support viable primary neuronal culture. The cell-nanostructure interface is characterized showing a minimal cell penetration but strong adhesion on the surface. Moreover, we quantify the influence of the nano-textured surface on the neural development (soma size, neuritogenesis, and polarity) in comparison to a planar PPX sample. We demonstrate that the nanostructures facilitates an enhancement in neurite branching as well as elongation of axons and growth cones. Furthermore, we show for the first time that the asymmetric orientation of polymeric nanocolumns strongly influences the initiation direction of the axon formation. These results evidence that 3D nano-topographies can significantly change neural development and can be used to engineer axon elongation. © 2018 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 106A: 1634-1645, 2018.

Cdc42 Regulates Neuronal Polarity during Cerebellar Axon Formation and Glial-Guided Migration

SummaryCNS cortical histogenesis depends on polarity signaling pathways that regulate cell adhesion and motility. Here we report that conditional deletion of the Rho GTPase Cdc42 in cerebellar granule cell precursors (GCPs) results in abnormalities in cerebellar foliation revealed by iDISCO clearing methodology, a loss of columnar organization of proliferating GCPs in the external germinal layer (EGL), disordered parallel fiber organization in the molecular layer (ML), and a failure to extend a leading process and form a neuron-glial junction during migration along Bergmann glia (BG). Notably, GCPs lacking Cdc42 had a multi-polar morphology and slowed migration rate. In addition, secondary defects occurred in BG development and organization, especially in the lateral cerebellar hemispheres. By phosphoproteomic analysis, affected Cdc42 targets included regulators of the cytoskeleton, cell adhesion and polarity. Thus, Cdc42 signaling pathways are critical regulators of GCP polarity and the formation of neuron-glial junctions during cerebellar development.

Intracellular amyloid beta expression leads to dysregulation of the mitogen-activated protein kinase and bone morphogenetic protein-2 signaling axis.

Alzheimer’s disease (AD) is a neurodegenerative syndrome classically depicted by the parenchymal accumulation of extracellular amyloid beta plaques. However, recent findings suggest intraneuronal amyloid beta (iAβ1–42) accumulation precedes extracellular deposition. Furthermore, the pathologic increase in iAβ1–42 has been implicated in dysregulation of cellular mechanisms critically important in axonal transport. Owing to neuronal cell polarity, retrograde and anterograde axonal transport are essential trafficking mechanism necessary to convey membrane bound neurotransmitters, neurotrophins, and endosomes between soma and synaptic interfaces. Although iAβ1–42 disruption of axonal transport has been implicated in dysregulation of neuronal synaptic transmission, the role of iAβ1–42 and its influence on signal transduction involving the mitogen-activated protein kinase (MAPK) and morphogenetic signaling axis are unknown. Our biochemical characterization of intracellular amyloid beta accumulation on MAPK and morphogenetic signaling have revealed increased iAβ1–42 expression leads to significant reduction in ERK 1/2 phosphorylation and increased bone morphogenetic protein 2 dependent Smad 1/5/8 phosphorylation. Furthermore, rescue of iAβ1–42 mediated attenuation of MAPK signaling can be accomplished with the small molecule PLX4032 as a downstream enhancer of the MAPK pathway. Consequently, our observations regarding the dysregulation of these gatekeepers of neuronal viability may have important implications in understanding the iAβ1–42 mediated effects observed in AD.

The Development of Neuronal Polarity: A Retrospective View.

In 1988, Carlos Dotti, Chris Sullivan, and I published a paper on the establishment of polarity by hippocampal neurons in culture, which continues to be frequently cited 30 years later (Dotti et al., 1988). By following individual neurons from the time of plating until they had formed well developed axonal and dendritic arbors, we identified the five stages of development that lead to the mature expression of neuronal polarity. We were surprised to find that, before axon formation, the cells pass through a multipolar phase, in which several, apparently identical short neurites undergo periods of extension and retraction. Then one of these neurites begins a period of prolonged growth, becoming the definitive axon; the remaining neurites subsequently become dendrites. This observation suggested that any of the initial neurites were capable of becoming axons, a hypothesis confirmed by later work. In this Progressions article, I will try to recall the circumstances that led to this work, recapture some of the challenges we faced in conducting these experiments, and consider why some of today's neuroscientists still find this paper relevant.

Elavl3 is essential for the maintenance of Purkinje neuron axons

Neuronal Elav-like (nElavl or neuronal Hu) proteins are RNA-binding proteins that regulate RNA stability and alternative splicing, which are associated with axonal and synaptic structures. nElavl proteins promote the differentiation and maturation of neurons via their regulation of RNA. The functions of nElavl in mature neurons are not fully understood, although Elavl3 is highly expressed in the adult brain. Furthermore, possible associations between nElavl genes and several neurodegenerative diseases have been reported. We investigated the relationship between nElavl functions and neuronal degeneration using Elavl3−/− mice. Elavl3−/− mice exhibited slowly progressive motor deficits leading to severe cerebellar ataxia, and axons of Elavl3−/− Purkinje cells were swollen (spheroid formation), followed by the disruption of synaptic formation of axonal terminals. Deficit in axonal transport and abnormalities in neuronal polarity was observed in Elavl3−/− Purkinje cells. These results suggest that nElavl proteins are crucial for the maintenance of axonal homeostasis in mature neurons. Moreover, Elavl3−/− mice are unique animal models that constantly develop slowly progressive axonal degeneration. Therefore, studies of Elavl3−/− mice will provide new insight regarding axonal degenerative processes.

Lineage specific transcription factors and epigenetic regulators mediate TGFβ-dependent enhancer activation.

During neurogenesis, dynamic developmental cues, transcription factors and histone modifying enzymes regulate the gene expression programs by modulating the activity of neural-specific enhancers. How transient developmental signals coordinate transcription factor recruitment to enhancers and to which extent chromatin modifiers contribute to enhancer activity is starting to be uncovered. Here, we take advantage of neural stem cells as a model to unravel the mechanisms underlying neural enhancer activation in response to the TGFβ signaling. Genome-wide experiments demonstrate that the proneural factor ASCL1 assists SMAD3 in the binding to a subset of enhancers. Once located at the enhancers, SMAD3 recruits the histone demethylase JMJD3 and the remodeling factor CHD8, creating the appropriate chromatin landscape to allow enhancer transcription and posterior gene activation. Finally, to analyze the phenotypical traits owed to cis-regulatory regions, we use CRISPR–Cas9 technology to demonstrate that the TGFβ-responsive Neurog2 enhancer is essential for proper neuronal polarization.

A Minimal Coarse-Grained Molecular Dynamics Model of Axon Plasma Membrane with its Implication on the Diffusion Behavior of Axon Membrane Proteins

The axon initial segment (AIS) in the neuron has two crucial functions: maintaining the polarity of neurons and initiation of action potentials. One extraordinary feature of the AIS is the separation of somatodendritic and axonal domains which was confirmed by measuring the trajectory of beads attached to various axonal membrane proteins that there exists a diffusion barrier in the AIS restricting the motion of membrane proteins and lipids. According to the “fences and pickets” model, the periodic cytoskeleton and the highly concentrated transmembrane proteins in the AIS are capable of hindering the diffusion of other proteins and lipids through steric interaction and hydrodynamic friction effect. In this work, we established the first coarse-grained molecular dynamics model of axon plasma membrane which includes both axon membrane skeleton and axonal membranes to investigate the diffusion behavior of membrane proteins and lipids in the AIS from a mechanics aspect. First, we found that transmembrane proteins and proteins in the inner lipid leaflet are confined within the areas between the actin rings. We suspected and proved from the computational model that the size of actin associated protein junctions, which connected to the lipid bilayers may restrict the motion of proteins in the outer leaflet. Then we showed that the spectrin-lipid attraction level will affect the circumferential mobility of membrane proteins. Finally, the highly dense membrane proteins slowed down the motion of all membrane proteins even the lipids. We conclude that actin rings and spectrin filaments restrict the motion of membrane proteins along and around the axon, respectively. However, the main mechanism forming the diffusion barrier in the AIS is the accumulation of concentrated transmembrane proteins.

Regulation of neuronal development and function by ROS.

Reactive oxygen species (ROS) have long been studied as destructive agents in the context of nervous system ageing, disease and degeneration. Their roles as signalling molecules under normal physiological conditions is less well understood. Recent studies have provided ample evidence of ROS‐regulating neuronal development and function, from the establishment of neuronal polarity to growth cone pathfinding; from the regulation of connectivity and synaptic transmission to the tuning of neuronal networks. Appreciation of the varied processes that are subject to regulation by ROS might help us understand how changes in ROS metabolism and buffering could progressively impact on neuronal networks with age and disease.

How to navigate counter dogmatic research findings.

In the early 1980s, we had observed that neuronal polarity varies depending on the respective topological origins of co‐cultured neurons and non‐neuronal cells (astrocytes). Following publication of these findings in 1984 (Denis‐Donini et al , 1984), we speculated that homeoprotein transcription factors (HPs) may regulate neuronal geometry, in analogy with the well‐established role of homeogenes (encoding HPs) in the regulation of organ shape in accordance with their position along the body anterior–posterior axis. In other words, we hypothesized that common mechanisms might contribute both to cell and to organ shapes, a pretty daring hypothesis at the time. To test it, we took advantage of the transient disruption of neuronal membranes during tissue dissociation, which allows for the passive internalization of proteins. Based on the high conservation of HDs, we anticipated, and indeed verified experimentally, that the HD Antennapedia could chase a variety of endogenous HPs off their cognate genomic binding sites. Changes in cell‐shape did happen thus validating the hypothesis that HPs could regulate neuronal shape. However, to rule out that this result was an artifact of the excess of HD added during tissue dissociation, the HD was added into the culture medium after cell plating. Indeed, the same morphogenetic changes took place, suggesting an experimental artifact. In a “just in case” experiment, we tagged the HD with fluorescein isothiocyanate to follow its distribution. This led to the rather astonishing discovery that the HD added to the culture medium was internalized by live, undisrupted neurons. We established that the internalization required the conserved 3rd helix of the homeodomain. Furthermore, this surprising finding turned out to be a general feature and we could show that a number of HPs signal directly between cells in vitro and in vivo by transferring as proteins between the cells (Fig 1). Although only about a dozen of …

Faulty neuronal determination and cell polarization are reverted by modulating HD early phenotypes

Increasing evidence suggests that early neurodevelopmental defects in Huntington's disease (HD) patients could contribute to the later adult neurodegenerative phenotype. Here, by using HD-derived induced pluripotent stem cell lines, we report that early telencephalic induction and late neural identity are affected in cortical and striatal populations. We show that a large CAG expansion causes complete failure of the neuro-ectodermal acquisition, while cells carrying shorter CAGs repeats show gross abnormalities in neural rosette formation as well as disrupted cytoarchitecture in cortical organoids. Gene-expression analysis showed that control organoid overlapped with mature human fetal cortical areas, while HD organoids correlated with the immature ventricular zone/subventricular zone. We also report that defects in neuroectoderm and rosette formation could be rescued by molecular and pharmacological approaches leading to a recovery of striatal identity. These results show that mutant huntingtin precludes normal neuronal fate acquisition and highlights a possible connection between mutant huntingtin and abnormal neural development in HD.

Oxygen-Tension and the VHL-Hif1α Pathway Comprise a Developmental Switch Controlling the Onset of Neuronal Polarization and Germinal Zone Exit

Postnatal brain circuit assembly is driven by temporally regulated intrinsic and cell-extrinsic cues that organize neurogenesis, migration, and axo-dendritic specification in post-mitotic neurons. Despite studies implicating cell polarity as an intrinsic organizer of morphogenetic events, the nature of cues instructing neuron polarization and their coupling during postnatal development is unclear. We found a link between oxygen tension, which rises after birth, the von Hippel– Lindau(VHL)–hypoxia-inducible factor 1α (Hif1α) pathway, and the polarization regulating the maturation of post-mitotic cerebellar granule neurons (CGNs). At postnatal stages with low GZ vascularization, Hif1α modulates CGN-progenitor cell-cycle exit. Unexpectedly, the VHL-Hif1α pathway also delays the timing of CGN differentiation, germinal zone exit, and migration initiation by regulating newborn neuron niche interactions via transcriptional repression of the partitioning-defective(Pard) complex. As vascularization proceeds, these inhibitory mechanisms recede, implicating oxygen fluctuation as a developmental switch regulating neuronal polarization and a key shaper of the nascent steps of circuit formation.

Perturbation of canonical and non-canonical BMP signaling affects migration, polarity and dendritogenesis of mouse cortical neurons.

Bone morphogenetic protein (BMP) signaling has been implicated in the regulation of patterning of the forebrain and as a regulator of neurogenesis and gliogenesis in the mammalian cortex. However, its role in other aspects of cortical development in vivo remains unexplored. We hypothesized that BMP signaling might regulate additional processes during the development of cortical neurons after observing active BMP signaling in a spatiotemporally dynamic pattern in the mouse cortex. Our investigation revealed that BMP signaling specifically regulates the migration, polarity and the dendritic morphology of upper layer cortical neurons born at E15.5. On further dissection of the role of canonical and non-canonical BMP signaling in each of these processes, we found that migration of these neurons is regulated by both pathways. Their polarity, however, appears to be affected more strongly by canonical BMP signaling, whereas dendritic branch formation appears to be somewhat more strongly affected by LIMK-mediated non-canonical BMP signaling.

Structural Insights into the Altering Function of CRMP2 by Phosphorylation.

Collapsin response mediator protein 2 (CRMP2) regulates neuronal polarity by controlling microtubule dynamics. CRMP2 activity is regulated by semaphorin-induced phosphorylation at the C-terminal tail domain. Unphosphorylated CRMP2 induces effective axonal microtubule formation to give the axonal characteristics to a neurite, whereas phosphorylated CRMP2 leads to the apparently opposite effect, growth cone collapse. We have recently characterized the structural detail of CRMP2-induced axonal microtubule formation (Niwa et al. (2017) Sci. Rep., 7: 10681). CRMP2 forms the hetero-trimer with GTP-tubulin to induce effective axonal microtubule formation in the future axon. Phosphorylation of CRMP2 has been reported to decrease the affinity between CRMP2 and the microtubule, albeit the molecular mechanisms of how the phosphorylation of CRMP2 changes the structure to achieve distinct effects from unphosphorylated CRMP2 is not well understood. Here we performed a series of biochemical and structural analyses of phospho-mimic CRMP2. Phosphorylation of CRMP2 undergoes small conformational changes at the C-terminal tail with shifting the surface charge, which not only alters the interactions within the CRMP2 tetramer but also alters the interactions with GTP-tubulin. Consequently, phospho-mimic CRMP2 fails to form a hetero-trimer with GTP-tubulin, thus losing the ability to establish and maintain the axonal microtubules.Key words: CRMP2, phosphorylation, microtubule, axon, crystal structure.

Tubulin carboxypeptidase identity revealed

Cytoskeleton Enzymes of the α-tubulin detyrosination/tyrosination cycle create landmarks on microtubules that are essential for their multiple cellular functions and are altered in disease. Tubulin carboxypeptidases (TCPs) responsible for detyrosination have remained elusive for 40 years (see the Perspective by Akhmanova and Maiato). Aillaud et al. identified vasohibins as enzymes that perform the TCP function and found that their small interacting partner SBVP was essential for their activity. Vasohibin/SVBP complexes were involved in neuron polarization and brain cortex development. The authors also developed an inhibitor targeting this family of enzymes. Using a completely different strategy, Nieuwenhuis et al. also showed that vasohibins can remove the C-terminal tyrosine of α-tubulin. Science , this issue p. [1448][1], p. [1453][2]; see also p. [1381][3] [1]: /lookup/doi/10.1126/science.aao4165 [2]: /lookup/doi/10.1126/science.aao5676 [3]: /lookup/doi/10.1126/science.aar3895

Lentivirus Mediating FGF13 Enhances Axon Regeneration after Spinal Cord Injury by Stabilizing Microtubule and Improving Mitochondrial Function.

Fibroblast growth factor 13 (FGF13), a nonsecretory protein of the FGF family, plays a crucial role in developing cortical neurons by stabilizing the microtubule. In previous studies, we showed that regulation of microtubule dynamics was instrumental for both growth cone initiation and for promoting regrowth of injured axon. However, the expression and effect of FGF13 in spinal cord or after spinal cord injury (SCI) remains undefined. Here, we demonstrated a role of FGF13 in regulating microtubule dynamics and in enhancing axon regeneration after SCI. Administration of FGF13 not only promoted neuronal polarization, axon formation, and growth cone initiation in vitro, but it also facilitated functional recovery following SCI. In addition, we found that upregulation of FGF13 in primary cortical neurons was accompanied by enhanced mitochondrial function, which is essential for axon regeneration. Our study has defined a novel mechanism underlying the beneficial effect of FGF13 on axon regeneration, pointing out that FGF13 may serve as a potential candidate for treating SCI or other central nervous system (CNS) injury.

Co-expression network analysis of lncRNAs and mRNAs in OPTN-silenced cells.

The aim of the present study was to reveal the role of long noncoding RNAs(lncRNAs) in the regulation of the pathogenesis of optineurin(OPTN)-silenced cells. The microarray data set GSE12452 was re-annotated using the non-coding RNA function annotation server to identify differentially expressed lncRNAs. Weighted correlation network analysis was used to construct an lncRNA-lncRNA co-expression network and identify co-expression modules. Three OPTN small interfering RNAs (siRNAs) were transiently transfected into HeLa cells. Reverse transcription‑quantitative polymerase chain reaction(RT-qPCR) and western blot analysis were used to detect OPTN expression and select the most effective OPTNsiRNA to construct stably transfected cells. RT-qPCR was used to quantify the identified lncRNAs in the OPTN-silenced cells. The potential functions of these modules were explored by the functional enrichment of the corresponding co-expressed genes. A total of 3,495lncRNAs were re-annotated. Of these, matrix metalloprotease12 andRP11-169D4.1 were upregulated, and RP1-212P9.2 was downregulated. The results of the RT-qPCR analysis of RP1-212P9.2 andRP11‑169D4.1 were consistent with the re-annotated data in the OPTN‑silenced cells. GeneOntology analyses indicated that the biological functions of the mRNAs co-expressed with these lncRNAs were associated with gene product regulation, and neuronal migration, polarity and differentiation. In addition, Kyoto Encyclopedia of Genes and Genomes analysis indicated that the two validated lncRNAs were associated with the transforming growth factor-β signaling pathway and the apoptosis pathway, respectively. In conclusion, the abnormal lncRNAs identified in OPTN-silenced cells indicate that lncRNAs may contribute to the molecular pathogenesis of OPTN-associated diseases.