Enhanced axonal regeneration of ALS patient iPSC-derived motor neurons harboring SOD1A4V mutation.

Amyotrophic lateral sclerosis (ALS) is a devastating neurodegenerative disease, characterized by degeneration of upper and lower motor neurons that leads to muscle weakness, paralysis, and death, but the effects of disease-causing mutations on axonal outgrowth of neurons derived from human induced pluripotent stem cells (iPSC)-derived motor neurons (hiPSC-MN) are poorly understood. The use of hiPSC-MN is a promising tool to develop more relevant models for target identification and drug development in ALS research, but questions remain concerning the effects of distinct disease-causing mutations on axon regeneration. Mutations in superoxide dismutase 1 (SOD1) were the first to be discovered in ALS patients. Here, we investigated the effect of the SOD1A4V mutation on axonal regeneration of hiPSC-MNs, utilizing compartmentalized microfluidic devices, which are powerful tools for studying hiPSC-MN distal axons. Surprisingly, SOD1+/A4V hiPSC-MNs regenerated axons more quickly following axotomy than those expressing the native form of SOD1. Though initial axon regrowth was not significantly different following axotomy, enhanced regeneration was apparent at later time points, indicating an increased rate of outgrowth. This regeneration model could be used to identify factors that enhance the rate of human axon regeneration.

Downregulation of SF3B2 protects CNS neurons in models of multiple sclerosis.

OBJECTIVE: Neurodegeneration induced by inflammatory stress in multiple sclerosis (MS) leads to long-term neurological disabilities that are not amenable to current immunomodulatory therapies. METHODS AND RESULTS: Here, we report that neuronal downregulation of Splicing factor 3b subunit 2 (SF3B2), a component of U2 small nuclear ribonucleoprotein (snRNP), preserves retinal ganglion cell (RGC) survival and axonal integrity in experimental autoimmune encephalomyelitis (EAE)-induced mice. By employing an in vitro system recapitulating the inflammatory environment of MS lesion, we show that when SF3B2 levels are downregulated, cell viability and axon integrity are preserved in cortical neurons against inflammatory toxicity. Notably, knockdown of SF3B2 suppresses the expression of injury-response and necroptosis genes and prevents activation of Sterile Alpha and TIR Motif Containing 1 (Sarm1), a key enzyme that mediates programmed axon degeneration. INTERPRETATION: Together, these findings suggest that the downregulation of SF3B2 is a novel potential therapeutic target to prevent secondary neurodegeneration in MS.

A Variant Allele in Varicella-Zoster Virus Glycoprotein B Selected during Production of the Varicella Vaccine Contributes to Its Attenuation.

Attenuation of the live varicella Oka vaccine (vOka) has been attributed to mutations in the genome acquired during cell culture passage of pOka (parent strain); however, the precise mechanisms of attenuation remain unknown. Comparative sequence analyses of several vaccine batches showed that over 100 single-nucleotide polymorphisms (SNPs) are conserved across all vaccine batches; 6 SNPs are nearly fixed, suggesting that these SNPs are responsible for attenuation. By contrast, prior analysis of chimeric vOka and pOka recombinants indicates that loci other than these six SNPs contribute to attenuation. Here, we report that pOka consists of a heterogenous population of virus sequences with two nearly equally represented bases, guanine (G) or adenine (A), at nucleotide 2096 of the ORF31 coding sequence, which encodes glycoprotein B (gB) resulting in arginine (R) or glutamine (Q), respectively, at amino acid 699 of gB. By contrast, 2096A/699Q is dominant in vOka (>99.98%). gB699Q/gH/gL showed significantly less fusion activity than gB699R/gH/gL in a cell-based fusion assay. Recombinant pOka with gB669Q (rpOka_gB699Q) had a similar growth phenotype as vOka during lytic infection in cell culture including human primary skin cells; however, rpOka_gB699R showed a growth phenotype similar to pOka. rpOka_gB699R entered neurons from axonal terminals more efficiently than rpOka_gB699Q in the presence of cell membrane-derived vesicles containing gB. Strikingly, when a mixture of pOka with both alleles equally represented was used to infect human neurons from axon terminals, pOka with gB699R was dominant for virus entry. These results identify a variant allele in gB that contributes to attenuation of vOka. IMPORTANCE The live-attenuated varicella vaccine has reduced the burden of chickenpox. Despite its development in 1974, the molecular basis for its attenuation is still not well understood. Since the live-attenuated varicella vaccine is the only licensed human herpesvirus vaccine that prevents primary disease, it is important to understand the mechanism for its attenuation. Here we identify that a variant allele in glycoprotein B (gB) selected during generation of the varicella vaccine contributes to its attenuation. This variant is impaired for fusion, virus entry into neurons from nerve terminals, and replication in human skin cells. Identification of a variant allele in gB, one of the essential herpesvirus core genes, that contributes to its attenuation may provide insights that assist in the development of other herpesvirus vaccines.

Protective effects of NAMPT or MAPK inhibitors and NaR on Wallerian degeneration of mammalian axons.

Wallerian degeneration (WD) is a conserved axonal self-destruction program implicated in several neurological diseases. WD is driven by the degradation of the NAD+ synthesizing enzyme NMNAT2, the buildup of its substrate NMN, and the activation of the NAD+ degrading SARM1, eventually leading to axonal fragmentation. The regulation and amenability of these events to therapeutic interventions remain unclear. Here we explored pharmacological strategies that modulate NMN and NAD+ metabolism, namely the inhibition of the NMN-synthesizing enzyme NAMPT, activation of the nicotinic acid riboside (NaR) salvage pathway and inhibition of the NMNAT2-degrading DLK MAPK pathway in an axotomy model in vitro. Results show that NAMPT and DLK inhibition cause a significant but time-dependent delay of WD. These time-dependent effects are related to NMNAT2 degradation and changes in NMN and NAD+ levels. Supplementation of NAMPT inhibition with NaR has an enhanced effect that does not depend on timing of intervention and leads to robust protection up to 4 days. Additional DLK inhibition extends this even further to 6 days. Metabolite analyses reveal complex effects indicating that NAMPT and MAPK inhibition act by reducing NMN levels, ameliorating NAD+ loss and suppressing SARM1 activity. Finally, the axonal NAD+/NMN ratio is highly predictive of cADPR levels, extending previous cell-free evidence on the allosteric regulation of SARM1. Our findings establish a window of axon protection extending several hours following injury. Moreover, we show prolonged protection by mixed treatments combining MAPK and NAMPT inhibition that proceed via complex effects on NAD+ metabolism and inhibition of SARM1.

Spatial transcriptomics reveals a role for sensory nerves in preserving cranial suture patency through modulation of BMP/TGF-ß signaling.

The patterning and ossification of the mammalian skeleton requires the coordinated actions of both intrinsic bone morphogens and extrinsic neurovascular signals, which function in a temporal and spatial fashion to control mesenchymal progenitor cell (MPC) fate. Here, we show the genetic inhibition of tropomyosin receptor kinase A (TrkA) sensory nerve innervation of the developing cranium results in premature calvarial suture closure, associated with a decrease in suture MPC proliferation and increased mineralization. In vitro, axons from peripheral afferent neurons derived from dorsal root ganglions (DRGs) of wild-type mice induce MPC proliferation in a spatially restricted manner via a soluble factor when cocultured in microfluidic chambers. Comparative spatial transcriptomic analysis of the cranial sutures in vivo confirmed a positive association between sensory axons and proliferative MPCs. SpatialTime analysis across the developing suture revealed regional-specific alterations in bone morphogenetic protein (BMP) and TGF-ß signaling pathway transcripts in response to TrkA inhibition. RNA sequencing of DRG cell bodies, following direct, axonal coculture with MPCs, confirmed the alterations in BMP/TGF-ß signaling pathway transcripts. Among these, the BMP inhibitor follistatin-like 1 (FSTL1) replicated key features of the neural-to-bone influence, including mitogenic and anti-osteogenic effects via the inhibition of BMP/TGF-ß signaling. Taken together, our results demonstrate that sensory nerve-derived signals, including FSTL1, function to coordinate cranial bone patterning by regulating MPC proliferation and differentiation in the suture mesenchyme.

Nectin-1 Is an Entry Mediator for Varicella-Zoster Virus Infection of Human Neurons.

Varicella-zoster virus (VZV) maintains lifelong latency in neurons following initial infection and can subsequently be reactivated to result in herpes zoster or severe neurological manifestations such as encephalitis. Mechanisms of VZV neuropathogenesis have been challenging to study due to the strict human tropism of the virus. Although neuronal entry mediators of other herpesviruses, including herpes simplex virus, have been identified, little is known regarding how VZV enters neurons. Here, we utilize a human stem cell-based neuronal model to characterize cellular factors that mediate entry. Through transcriptional profiling of infected cells, we identify the cell adhesion molecule nectin-1 as a candidate mediator of VZV entry. Nectin-1 is highly expressed in the cell bodies and axons of neurons. Either knockdown of endogenous nectin-1 or incubation with soluble forms of nectin-1 produced in mammalian cells results in a marked decrease in infectivity of neurons. Notably, while addition of soluble nectin-1 during viral infection inhibits infectivity, addition after infection has no effect on infectivity. Ectopic expression of human nectin-1 in a cell line resistant to productive VZV infection confers susceptibility to infection. In summary, we have identified nectin-1 as a neuronal entry mediator of VZV. IMPORTANCE Varicella-zoster virus (VZV) causes chickenpox, gains access to neurons during primary infection where it resides lifelong, and can later be reactivated. Reactivation is associated with shingles and postherpetic neuralgia, as well as with severe neurologic complications, including vasculitis and encephalitis. Although the varicella vaccine substantially decreases morbidity and mortality associated with primary infection, the vaccine cannot prevent the development of neuronal latency, and vaccinated populations are still at risk for reactivation. Furthermore, immunocompromised individuals are at higher risk for VZV reactivation and associated complications. Little is known regarding how VZV enters neurons. Here, we identify nectin-1 as an entry mediator of VZV in human neurons. Identification of nectin-1 as a neuronal VZV entry mediator could lead to improved treatments and preventative measures to reduce VZV related morbidity and mortality.

Varicella-zoster virus VLT-ORF63 fusion transcript induces broad viral gene expression during reactivation from neuronal latency.

Varicella-zoster virus (VZV) establishes lifelong neuronal latency in most humans world-wide, reactivating in one-third to cause herpes zoster and occasionally chronic pain. How VZV establishes, maintains and reactivates from latency is largely unknown. VZV transcription during latency is restricted to the latency-associated transcript (VLT) and RNA 63 (encoding ORF63) in naturally VZV-infected human trigeminal ganglia (TG). While significantly more abundant, VLT levels positively correlated with RNA 63 suggesting co-regulated transcription during latency. Here, we identify VLT-ORF63 fusion transcripts and confirm VLT-ORF63, but not RNA 63, expression in human TG neurons. During in vitro latency, VLT is transcribed, whereas VLT-ORF63 expression is induced by reactivation stimuli. One isoform of VLT-ORF63, encoding a fusion protein combining VLT and ORF63 proteins, induces broad viral gene transcription. Collectively, our findings show that VZV expresses a unique set of VLT-ORF63 transcripts, potentially involved in the transition from latency to lytic VZV infection.

iPSCs from people with MS can differentiate into oligodendrocytes in a homeostatic but not an inflammatory milieu.

Multiple sclerosis (MS) is an inflammatory and demyelinating disease of the central nervous system (CNS) that results in variable severities of neurodegeneration. The understanding of MS has been limited by the inaccessibility of the affected cells and the lengthy timeframe of disease development. However, recent advances in stem cell technology have facilitated the bypassing of some of these challenges. Towards gaining a greater understanding of the innate potential of stem cells from people with varying degrees of disability, we generated induced pluripotent stem cells (iPSCs) from peripheral blood mononuclear cells derived from stable and progressive MS patients, and then further differentiated them into oligodendrocyte (OL) lineage cells. We analyzed differentiation under both homeostatic and inflammatory conditions via sustained exposure to low-dose interferon gamma (IFNγ), a prominent cytokine in MS. We found that all iPSC lines differentiated into mature myelinating OLs, but chronic exposure to IFNγ dramatically inhibited differentiation in both MS groups, particularly if exposure was initiated during the pre-progenitor stage. Low-dose IFNγ was not toxic but led to an early upregulation of interferon response genes in OPCs followed by an apparent redirection in lineage commitment from OL to a neuron-like phenotype in a significant portion of the treated cells. Our results reveal that a chronic low-grade inflammatory environment may have profound effects on the efficacy of regenerative therapies.

Quantifying the Local Mechanical Properties of Cells in a Fibrous Three-Dimensional Microenvironment.

Measurements of the mechanical response of biological cells are critical for understanding injury and disease, for developing diagnostic tools, and for computational models in mechanobiology. Although it is well known that cells are sensitive to the topography of their microenvironment, the current paradigm in mechanical testing of adherent cells is mostly limited to specimens grown on flat two-dimensional substrates. In this study, we introduce a technique in which cellular indentation via optical trapping is performed on cells at a high spatial resolution to obtain their regional mechanical properties while they exist in a more favorable three-dimensional microenvironment. We combine our approach with nonlinear contact mechanics theory to consider the effects of a large deformation. This allows us to probe length scales that are relevant for obtaining overall cell stiffness values. The experimental results herein provide the hyperelastic material properties at both high (∼100 s-1) and low (∼1-10 s-1) strain rates of murine central nervous system glial cells. The limitations due to possible misalignment of the indenter in the three-dimensional space are examined using a computational model.

Preferential and Increased Uptake of Hydroxyl-Terminated PAMAM Dendrimers by Activated Microglia in Rabbit Brain Mixed Glial Culture.

Polyamidoamine (PAMAM) dendrimers are multifunctional nanoparticles with tunable physicochemical features, making them promising candidates for targeted drug delivery in the central nervous system (CNS). Systemically administered dendrimers have been shown to localize in activated glial cells, which mediate neuroinflammation in the CNS. These dendrimers delivered drugs specifically to activated microglia, producing significant neurological improvements in multiple brain injury models, including in a neonatal rabbit model of cerebral palsy. To gain further insight into the mechanism of dendrimer cell uptake, we utilized an in vitro model of primary glial cells isolated from newborn rabbits to assess the differences in hydroxyl-terminated generation 4 PAMAM dendrimer (D4-OH) uptake by activated and non-activated glial cells. We used fluorescently-labelled D4-OH (D-Cy5) as a tool for investigating the mechanism of dendrimer uptake. D4-OH PAMAM dendrimer uptake was determined by fluorescence quantification using confocal microscopy and flow cytometry. Our results indicate that although microglial cells in the mixed cell population demonstrate early uptake of dendrimers in this in vitro system, activated microglia take up more dendrimer compared to resting microglia. Astrocytes showed delayed and limited uptake. We also illustrated the differences in mechanism of uptake between resting and activated microglia using different pathway inhibitors. Both resting and activated microglia primarily employed endocytotic pathways, which are enhanced in activated microglial cells. Additionally, we demonstrated that hydroxyl terminated dendrimers are taken up by primary microglia using other mechanisms including pinocytosis, caveolae, and aquaporin channels for dendrimer uptake.

Human Embryonic Stem Cell-Derived Neurons Are Highly Permissive for Varicella-Zoster Virus Lytic Infection.

Varicella-zoster virus (VZV) is highly cell associated when grown in culture and has a much higher (4,000- to 20,000-fold increased) particle-to-PFU ratio in vitro than herpes simplex virus (HSV). In contrast, VZV is highly infectious in vivo by airborne transmission. Neurons are major targets for VZV in vivo; in neurons, the virus can establish latency and reactivate to produce infectious virus. Using neurons derived from human embryonic stem cells (hESC) and cell-free wild-type (WT) VZV, we demonstrated that neurons are nearly 100 times more permissive for WT VZV infection than very-early-passage human embryonic lung cells or MRC-5 diploid human fibroblasts, the cells used for vaccine production or virus isolation. The peak titers achieved after infection were ∼10-fold higher in human neurons than in MRC-5 cells, and the viral genome copy number-to-PFU ratio for VZV in human neurons was 500, compared with 50,000 for MRC-5 cells. Thus, VZV may not necessarily have a higher particle-to-PFU ratio than other herpesviruses; instead, the cells previously used to propagate virus in vitro may have been suboptimal. Furthermore, based on electron microscopy, neurons infected with VZV produced fewer defective or incomplete viral particles than MRC-5 cells. Our data suggest that neurons derived from hESC may have advantages compared to other cells for studies of VZV pathogenesis, for obtaining stocks of virus with high titers, and for isolating VZV from clinical specimens.IMPORTANCE Varicella-zoster virus (VZV) causes chickenpox and shingles. Cell-free VZV has been difficult to obtain, both for in vitro studies and for vaccine production. While numerous cells lines have been tested for their ability to produce high titers of VZV, the number of total virus particles relative to the number of viral particles that can form plaques in culture has been reported to be extremely high relative to that in other viruses. We show that VZV grows to much higher titers in human neurons than in other cell types in vitro and that the number of total virus genomes relative to the number of viral particles that can form plaques in culture is much lower in human neurons than other cultured cells. These findings indicate that human neurons may be useful for studying VZV in vitro, for growing preparations of virus with high titers, and for isolating the virus from human samples.

Role of the JNK Pathway in Varicella-Zoster Virus Lytic Infection and Reactivation.

Mechanisms of neuronal infection by varicella-zoster virus (VZV) have been challenging to study due to the relatively strict human tropism of the virus and the paucity of tractable experimental models. Cellular mitogen-activated protein kinases (MAPKs) have been shown to play a role in VZV infection of nonneuronal cells, with distinct consequences for infectivity in different cell types. Here, we utilize several human neuronal culture systems to investigate the role of one such MAPK, the c-Jun N-terminal kinase (JNK), in VZV lytic infection and reactivation. We find that the JNK pathway is specifically activated following infection of human embryonic stem cell-derived neurons and that this activation of JNK is essential for efficient viral protein expression and replication. Inhibition of the JNK pathway blocked viral replication in a manner distinct from that of acyclovir, and an acyclovir-resistant VZV isolate was as sensitive to the effects of JNK inhibition as an acyclovir-sensitive VZV isolate in neurons. Moreover, in a microfluidic-based human neuronal model of viral latency and reactivation, we found that inhibition of the JNK pathway resulted in a marked reduction in reactivation of VZV. Finally, we utilized a novel technique to efficiently generate cells expressing markers of human sensory neurons from neural crest cells and established a critical role for the JNK pathway in infection of these cells. In summary, the JNK pathway plays an important role in lytic infection and reactivation of VZV in physiologically relevant cell types and may provide an alternative target for antiviral therapy.IMPORTANCE Varicella-zoster virus (VZV) has infected over 90% of people worldwide. While primary infection leads to the typically self-limiting condition of chickenpox, the virus can remain dormant in the nervous system and may reactivate later in life, leading to shingles or inflammatory diseases of the nervous system and eye with potentially severe consequences. Here, we take advantage of newer stem cell-based technologies to study the mechanisms by which VZV infects human neurons. We find that the c-Jun N-terminal kinase (JNK) pathway is activated by VZV infection and that blockade of this pathway limits lytic replication (as occurs during primary infection). In addition, JNK inhibition limits viral reactivation, exhibiting parallels with herpes simplex virus reactivation. The identification of the role of the JNK pathway in VZV infection of neurons reveals potential avenues for the development of alternate antiviral drugs.

NGF-TrkA Signaling by Sensory Nerves Coordinates the Vascularization and Ossification of Developing Endochondral Bone.

Developing tissues dictate the amount and type of innervation they require by secreting neurotrophins, which promote neuronal survival by activating distinct tyrosine kinase receptors. Here, we show that nerve growth factor (NGF) signaling through neurotrophic tyrosine kinase receptor type 1 (TrkA) directs innervation of the developing mouse femur to promote vascularization and osteoprogenitor lineage progression. At the start of primary ossification, TrkA-positive axons were observed at perichondrial bone surfaces, coincident with NGF expression in cells adjacent to centers of incipient ossification. Inactivation of TrkA signaling during embryogenesis in TrkA(F592A) mice impaired innervation, delayed vascular invasion of the primary and secondary ossification centers, decreased numbers of Osx-expressing osteoprogenitors, and decreased femoral length and volume. These same phenotypic abnormalities were observed in mice following tamoxifen-induced disruption of NGF in Col2-expressing perichondrial osteochondral progenitors. We conclude that NGF serves as a skeletal neurotrophin to promote sensory innervation of developing long bones, a process critical for normal primary and secondary ossification.

In vitro system using human neurons demonstrates that varicella-zoster vaccine virus is impaired for reactivation, but not latency.

Varicella-zoster virus (VZV) establishes latency in human sensory and cranial nerve ganglia during primary infection (varicella), and the virus can reactivate and cause zoster after primary infection. The mechanism of how the virus establishes and maintains latency and how it reactivates is poorly understood, largely due to the lack of robust models. We found that axonal infection of neurons derived from hESCs in a microfluidic device with cell-free parental Oka (POka) VZV resulted in latent infection with inability to detect several viral mRNAs by reverse transcriptase-quantitative PCR, no production of infectious virus, and maintenance of the viral DNA genome in endless configuration, consistent with an episome configuration. With deep sequencing, however, multiple viral mRNAs were detected. Treatment of the latently infected neurons with Ab to NGF resulted in production of infectious virus in about 25% of the latently infected cultures. Axonal infection of neurons with vaccine Oka (VOka) VZV resulted in a latent infection similar to infection with POka; however, in contrast to POka, VOka-infected neurons were markedly impaired for reactivation after treatment with Ab to NGF. In addition, viral transcription was markedly reduced in neurons latently infected with VOka compared with POka. Our in vitro system recapitulates both VZV latency and reactivation in vivo and may be used to study viral vaccines for their ability to establish latency and reactivate.

Changes in Neurofilament and Microtubule Distribution following Focal Axon Compression.

Although a number of cytoskeletal derangements have been described in the setting of traumatic axonal injury (TAI), little is known of early structural changes that may serve to initiate a cascade of further axonal degeneration. Recent work by the authors has examined conformational changes in cytoskeletal constituents of neuronal axons undergoing traumatic axonal injury (TAI) following focal compression through confocal imaging data taken in vitro and in situ. The present study uses electron microscopy to understand and quantify in vitro alterations in the ultrastructural composition of microtubules and neurofilaments within neuronal axons of rats following focal compression. Standard transmission electron microscopy processing methods are used to identify microtubules, while neurofilament identification is performed using antibody labeling through gold nanoparticles. The number, density, and spacing of microtubules and neurofilaments are quantified for specimens in sham Control and Crushed groups with fixation at <1 min following load. Our results indicate that the axon caliber dependency known to exist for microtubule and neurofilament metrics extends to axons undergoing TAI, with the exception of neurofilament spacing, which appears to remain constant across all Crushed axon diameters. Confidence interval comparisons between Control and Crushed cytoskeletal measures suggests early changes in the neurofilament spatial distributions within axons undergoing TAI may precede microtubule changes in response to applied loads. This may serve as a trigger for further secondary damage to the axon, representing a key insight into the temporal aspects of cytoskeletal degeneration at the component level, and suggests the rapid removal of neurofilament sidearms as one possible mechanism.

In vitro and in situ visualization of cytoskeletal deformation under load: traumatic axonal injury.

It is difficult to obtain insight into the mechanisms occurring within live cells during mechanical loading, because this complex environment is dynamic and evolving. This is a particular challenge from a subcellular mechanics perspective, where temporal and spatial information on the evolving cytoskeletal structures is required under loading. Using fluorescently labeled proteins, we visualize 3-dimensional live subcellular cytoskeletal populations under mechanical loading using a high-resolution confocal microscope. The mechanical forces are determined using a computational (finite element) model that is validated by integrating instrumentation into the testing platform. Transfected microtubules and neurofilaments of E17 rat neuronal axons are imaged before, during, and after loading. Comparisons between unloaded and loaded live cells demonstrate both spatial and temporal changes for cytoskeletal populations within the imaged volume. NF signal decreases by 24%, yet the microtubule signal exhibits no significant change 20-35 s after loading. Transmission electron microscopy assesses cytoskeletal structure spatial distribution for undeformed and deformed axons. While cytoskeletal degeneration occurs at prolonged time intervals following loads, our data provides insights into real time cytoskeletal evolution occurring in situ. Our findings suggest that, for axons undergoing traumatic injury in response to applied mechanical loads, changes at the substructural level of neurofilaments may precede microtubule rupture and degeneration.

Toll-like receptor 4 deficiency impairs microglial phagocytosis of degenerating axons.

Microglia are rapidly activated in the central nervous system (CNS) in response to a variety of injuries, including inflammation, trauma, and stroke. In addition to modulation of the innate immune response, a key function of microglia is the phagocytosis of dying cells and cellular debris, which can facilitate recovery. Despite emerging evidence that axonal debris can pose a barrier to regeneration of new axons in the CNS, little is known of the cellular and molecular mechanisms that underlie clearance of degenerating CNS axons. We utilize a custom micropatterned microfluidic system that enables robust microglial-axon co-culture to explore the role of Toll-like receptors (TLRs) in microglial phagocytosis of degenerating axons. We find that pharmacologic and genetic disruption of TLR4 blocks induction of the Type-1 interferon response and inhibits phagocytosis of axon debris in vitro. Moreover, TLR4-dependent microglial clearance of unmyelinated axon debris facilitates axon outgrowth. In vivo, microglial phagocytosis of CNS axons undergoing Wallerian degeneration in a dorsal root axotomy model is impaired in adult mice in which TLR4 has been deleted. Since purinergic receptors can influence TLR4-mediated signaling, we also explored a role for the microglia P2 receptors and found that the P2X7R contributes to microglial clearance of degenerating axons. Overall, we identify TLR4 as a key player in axonal debris clearance by microglia, thus creating a more permissive environment for axonal outgrowth. Our findings have significant implications for the development of protective and regenerative strategies for the many inflammatory, traumatic, and neurodegenerative conditions characterized by CNS axon degeneration.

Curcumin protects axons from degeneration in the setting of local neuroinflammation.

Axon degeneration is a hallmark of several central nervous system (CNS) disorders, including multiple sclerosis (MS), Alzheimer's disease (AD) and Parkinson's disease (PD). Previous neuroprotective approaches have mainly focused on reversal or prevention of neuronal cell body degeneration or death. However, experimental evidence suggests that mechanisms of axon degeneration may differ from cell death mechanisms, and that therapeutic agents that protect cell bodies may not protect axons. Moreover, axon degeneration underlies neurologic disability and may, in some cases, represent an important initial step that leads to neuronal death. Here, we develop a novel quantitative microfluidic-based methodology to assess mechanisms of axon degeneration caused by local neuroinflammation. We find that LPS-stimulated microglia release soluble factors that, when applied locally to axons, result in axon degeneration. This local axon degeneration is mediated by microglial MyD88/p38 MAPK signaling and concomitant production of nitric oxide (NO). Intra-axonal mechanisms of degeneration involve JNK phosphorylation. Curcumin, a compound with both anti-oxidant and JNK inhibitory properties, specifically protects axons, but not neuronal cell bodies, from NO-mediated degeneration. Overall, our platform provides mechanistic insights into local axon degeneration, identifies curcumin as a novel axon protectant in the setting of neuroinflammation, and allows for ready screening of axon protective drugs.

Toll/interleukin-1 receptor domain-containing adapter inducing interferon-ß mediates microglial phagocytosis of degenerating axons.

Following CNS injury, microglial phagocytosis of damaged endogenous tissue is thought to play an important role in recovery and regeneration. Previous work has focused on delineating mechanisms of clearance of neurons and myelin. Little, however, is known of the mechanisms underlying phagocytosis of axon debris. We have developed a novel microfluidic platform that enables coculture of microglia with bundles of CNS axons to investigate mechanisms of microglial phagocytosis of axons. Using this platform, we find that axon degeneration results in the induction of type-1 interferon genes within microglia. Pharmacologic and genetic disruption of Toll/interleukin-1 receptor domain-containing adapter inducing interferon-ß (TRIF), a Toll-like receptor adapter protein, blocks induction of the interferon response and inhibits microglial phagocytosis of axon debris in vitro. In vivo, microglial phagocytosis of axons following dorsal root axotomy is impaired in mice in which TRIF has been genetically deleted. Furthermore, we identify the p38 mitogen-activated protein kinase (MAPK) cascade as a signaling pathway downstream of TRIF following axon degeneration and find that inhibition of p38 MAPK by SB203580 (4-(4-fluorophenyl)-2-(4-methylsulfinylphenyl)-5-(4-pyridyl)-1H-imidazole) also blocked clearance of axon debris. Finally, we find that TRIF-dependent microglial clearance of unmyelinated axon debris facilitates axon outgrowth. Overall, we provide evidence that TRIF-mediated signaling plays an unexpected role in axonal debris clearance by microglia, thereby facilitating a more permissive environment for axonal outgrowth. Our study has significant implications for the development of novel regenerative and restorative strategies for the many traumatic, neuroinflammatory, and neurodegenerative conditions characterized by CNS axon degeneration.

Valve-based microfluidic compression platform: single axon injury and regrowth.

We describe a novel valve-based microfluidic axon injury micro-compression (AIM) platform that enables focal and graded compression of micron-scale segments of single central nervous system (CNS) axons. The device utilizes independently controlled "push-down" injury pads that descend upon pressure application and contact underlying axonal processes. Regulated compressed gas is input into the AIM system and pressure levels are modulated to specify the level of injury. Finite element modeling (FEM) is used to quantitatively characterize device performance and parameterize the extent of axonal injury by estimating the forces applied between the injury pad and glass substrate. In doing so, injuries are normalized across experiments to overcome small variations in device geometry. The AIM platform permits, for the first time, observation of axon deformation prior to, during, and immediately after focal mechanical injury. Single axons acutely compressed (~5 s) under varying compressive loads (0-250 kPa) were observed through phase time-lapse microscopy for up to 12 h post injury. Under mild injury conditions (< 55 kPa) ~73% of axons continued to grow, while at moderate (55-95 kPa) levels of injury, the number of growing axons dramatically reduced to 8%. At severe levels of injury (> 95 kPa), virtually all axons were instantaneously transected and nearly half (~46%) of these axons were able to regrow within the imaging period in the absence of exogenous stimulating factors.