Topoisomerase I inhibition and peripheral nerve injury induce DNA breaks and ATF3-associated axon regeneration in sensory neurons.

Although axonal damage induces rapid changes in gene expression in primary sensory neurons, it remains unclear how this process is initiated. The transcription factor ATF3, one of the earliest genes responding to nerve injury, regulates expression of downstream genes that enable axon regeneration. By exploiting ATF3 reporter systems, we identify topoisomerase inhibitors as ATF3 inducers, including camptothecin. Camptothecin increases ATF3 expression and promotes neurite outgrowth in sensory neurons in vitro and enhances axonal regeneration after sciatic nerve crush in vivo. Given the action of topoisomerases in producing DNA breaks, we determine that they do occur immediately after nerve damage at the ATF3 gene locus in injured sensory neurons and are further increased after camptothecin exposure. Formation of DNA breaks in injured sensory neurons and enhancement of it pharmacologically may contribute to the initiation of those transcriptional changes required for peripheral nerve regeneration.

Probing Synaptic Signaling with Optogenetic Stimulation and Genetically Encoded Calcium Reporters.

Optogenetics provides a powerful approach for investigating neuronal electrophysiology at the scale required for drug discovery applications. Probing synaptic function with high throughput using optogenetics requires robust tools that enable both precise stimulation of and facile readout of synaptic activity. Here we describe two functional assays to achieve this end: (1) a pre-synaptic calcium assay that utilizes the channelrhodopsin, CheRiff, patterned optogenetic stimulus, and the pre-synaptically targeted calcium reporter jRGECO1a to monitor pre-synaptic changes in calcium influx and (2) a synaptic transmission assay in which CheRiff and cytosolic jRGECO1a are expressed in non-overlapping sets of neurons, enabling pre-synaptic stimulation and post-synaptic readout of activity. This chapter describes the methodology and practical considerations for implementation of these two assays.

Transcriptional Reprogramming of Distinct Peripheral Sensory Neuron Subtypes after Axonal Injury.

Primary somatosensory neurons are specialized to transmit specific types of sensory information through differences in cell size, myelination, and the expression of distinct receptors and ion channels, which together define their transcriptional and functional identity. By profiling sensory ganglia at single-cell resolution, we find that all somatosensory neuronal subtypes undergo a similar transcriptional response to peripheral nerve injury that both promotes axonal regeneration and suppresses cell identity. This transcriptional reprogramming, which is not observed in non-neuronal cells, resolves over a similar time course as target reinnervation and is associated with the restoration of original cell identity. Injury-induced transcriptional reprogramming requires ATF3, a transcription factor that is induced rapidly after injury and necessary for axonal regeneration and functional recovery. Our findings suggest that transcription factors induced early after peripheral nerve injury confer the cellular plasticity required for sensory neurons to transform into a regenerative state.

Macrophages Expressing GALC Improve Peripheral Krabbe Disease by a Mechanism Independent of Cross-Correction.

Many therapies for lysosomal storage disorders rely on cross-correction of lysosomal enzymes. In globoid cell leukodystrophy (GLD), mutations in GALC cause psychosine accumulation, inducing demyelination, a neuroinflammatory "globoid" reaction and neurodegeneration. The efficiency of GALC cross-correction in vivo, the role of the GALC substrate galactosylceramide, and the origin of psychosine are poorly understood. Using a novel GLD model, we show that cross-correction does not occur efficiently in vivo and that Galc-deficient Schwann cells autonomously produce psychosine. Furthermore, macrophages require GALC to degrade myelin, as Galc-deficient macrophages are transformed into globoid cells by exposure to galactosylceramide and produce a more severe GLD phenotype. Finally, hematopoietic stem cell transplantation in patients reduces globoid cells in nerves, suggesting that the phagocytic response of healthy macrophages, rather than cross-correction, contributes to the therapeutic effect. Thus, GLD may be caused by at least two mechanisms: psychosine-induced demyelination and secondary neuroinflammation from galactosylceramide storage in macrophages.

Recurrent SMARCB1 Mutations Reveal a Nucleosome Acidic Patch Interaction Site That Potentiates mSWI/SNF Complex Chromatin Remodeling.

Mammalian switch/sucrose non-fermentable (mSWI/SNF) complexes are multi-component machines that remodel chromatin architecture. Dissection of the subunit- and domain-specific contributions to complex activities is needed to advance mechanistic understanding. Here, we examine the molecular, structural, and genome-wide regulatory consequences of recurrent, single-residue mutations in the putative coiled-coil C-terminal domain (CTD) of the SMARCB1 (BAF47) subunit, which cause the intellectual disability disorder Coffin-Siris syndrome (CSS), and are recurrently found in cancers. We find that the SMARCB1 CTD contains a basic α helix that binds directly to the nucleosome acidic patch and that all CSS-associated mutations disrupt this binding. Furthermore, these mutations abrogate mSWI/SNF-mediated nucleosome remodeling activity and enhancer DNA accessibility without changes in genome-wide complex localization. Finally, heterozygous CSS-associated SMARCB1 mutations result in dominant gene regulatory and morphologic changes during iPSC-neuronal differentiation. These studies unmask an evolutionarily conserved structural role for the SMARCB1 CTD that is perturbed in human disease.

Enhanced Neuronal Regeneration in the CAST/Ei Mouse Strain Is Linked to Expression of Differentiation Markers after Injury.

Peripheral nerve regeneration after injury requires a broad program of transcriptional changes. We investigated the basis for the enhanced nerve regenerative capacity of the CAST/Ei mouse strain relative to C57BL/6 mice. RNA sequencing of dorsal root ganglia (DRG) showed a CAST/Ei-specific upregulation of Ascl1 after injury. Ascl1 overexpression in DRG neurons of C57BL/6 mice enhanced their neurite outgrowth. Ascl1 is regulated by miR-7048-3p, which is downregulated in CAST/Ei mice. Inhibition of miR-7048-3p enhances neurite outgrowth. Following injury, CAST/Ei neurons largely retained their mature neuronal profile as determined by single-cell RNA- seq, whereas the C57BL/6 neurons acquired an immature profile. These findings suggest that one facet of the enhanced regenerative phenotype is preservation of neuronal identity in response to injury.

GRPR/PI3Kγ: Partners in Central Transmission of Itch.

The gastrin-releasing peptide (GRP) and its receptor (GRPR) are important components of itch transmission. Upstream, but not downstream, aspects of GRPR signaling have been investigated extensively. We hypothesize that GRPR signals in part through the PI3Kγ/Akt pathway. We used pharmacological, electrophysiological, and behavioral approaches to further evaluate GRPR downstream signaling pathways. Our data show that GRP directly activates small-size capsaicin-sensitive DRG neurons, an effect that translates into transient calcium flux and membrane depolarization (∼ 20 mV). GRPR activation also induces Akt phosphorylation, a proxy for PI3Kγ activity, in ex vivo naive mouse spinal cords and in GRPR transiently expressing HEK293 cells. The intrathecal injection of GRP led to intense scratching, an effect largely reduced by either GRPR antagonists or PI3Kγ inhibitor. Scratching behavior was also induced by the intrathecal injection of an Akt activator. In a dry skin model of itch, we show that GRPR blockade or PI3Kγ inhibition reversed the scratching behavior. Altogether, these findings are highly suggestive that GRPR is expressed by the central terminals of DRG nociceptive afferents, which transmit itch via the PI3Kγ/Akt pathway. SIGNIFICANCE STATEMENT: Itch is the most common symptom of the skin and is related to noncutaneous diseases. It severely impairs patients' quality of life when it becomes chronic and there is no specific or effective available therapy, mainly because itch pathophysiology is not completely elucidated. Our findings indicate that the enzyme PI3Kγ is a key central mediator of itch transmission. Therefore, we suggest PI3Kγ as an attractive target for the development of new anti-pruritic drugs. With this study, we take a step forward in our understanding of the mechanisms underlying the central transmission of itch sensation.

The Stress-Induced Atf3-Gelsolin Cascade Underlies Dendritic Spine Deficits in Neuronal Models of Tuberous Sclerosis Complex.

Hyperactivation of the mechanistic target of rapamycin (mTOR) kinase, as a result of loss-of-function mutations in tuberous sclerosis complex 1 (TSC1) or TSC2 genes, causes protein synthesis dysregulation, increased cell size, and aberrant neuronal connectivity. Dysregulated synthesis of synaptic proteins has been implicated in the pathophysiology of autism spectrum disorder (ASD) associated with TSC and fragile X syndrome. However, cell type-specific translational profiles in these disease models remain to be investigated. Here, we used high-fidelity and unbiased Translating Ribosome Affinity Purification (TRAP) methodology to purify ribosome-associated mRNAs and identified translational alterations in a rat neuronal culture model of TSC. We find that expression of many stress and/or activity-dependent proteins is highly induced while some synaptic proteins are repressed. Importantly, transcripts for the activating transcription factor-3 (Atf3) and mitochondrial uncoupling protein-2 (Ucp2) are highly induced in Tsc2-deficient neurons, as well as in a neuron-specific Tsc1 conditional knock-out mouse model, and show differential responses to the mTOR inhibitor rapamycin. Gelsolin, a known target of Atf3 transcriptional activity, is also upregulated. shRNA-mediated block of Atf3 induction suppresses expression of gelsolin, an actin-severing protein, and rescues spine deficits found in Tsc2-deficient neurons. Together, our data demonstrate that a cell-autonomous program consisting of a stress-induced Atf3-gelsolin cascade affects the change in dendritic spine morphology following mTOR hyperactivation. This previously unidentified molecular cascade could be a therapeutic target for treating mTORopathies. SIGNIFICANCE STATEMENT: Tuberous sclerosis complex (TSC) is a genetic disease associated with epilepsy and autism. Dysregulated protein synthesis has been implicated as a cause of this disease. However, cell type-specific translational profiles that are aberrant in this disease are unknown. Here we show that expression of many stress and/or activity-dependent proteins is highly induced while some synaptic proteins are repressed in neurons missing the Tsc2 gene expression. Identification of genes whose translation is abnormal in TSC may provide insights to previously unidentified therapeutic targets.

Stimulation of Proliferation and Migration of Mouse Macrophages by Type B CpG-ODNs Is F-Spondin and IL-1Ra Dependent.

Macrophage proliferation and migration are important for many facets of immune response. Here we showed that stimulation of macrophages with type B CpG oligodeoxynucleotides (CpG-B ODNs) such as CpG-ODN 1668 increased the production of anti-inflammatory cytokine interleukin 1 receptor antagonist (IL-1Ra) in a TLR9- and MyD88-dependent manner. The CpG-B ODNs-induced IL-1Ra increased macrophage migration and promoted macrophage proliferation by down-regulating the expression of a cell cycle negative regulator, p27 to increase cell population in the S phase. The induction of IL-1Ra by CpG-B ODNs was F-spondin dependent. Knockdown of F-spondin and IL-1Ra decreased CpG-B ODNs-induced macrophage migration whereas overexpression of IL-1Ra increased migration of those cells. These findings demonstrated novel roles for F-spondin and IL-1Ra in CpG-B ODNs-mediated cell proliferation and migration of macrophages.

Diminished Schwann cell repair responses underlie age-associated impaired axonal regeneration.

The regenerative capacity of the peripheral nervous system declines with age. Why this occurs, however, is unknown. We demonstrate that 24-month-old mice exhibit an impairment of functional recovery after nerve injury compared to 2-month-old animals. We find no difference in the intrinsic growth capacity between aged and young sensory neurons in vitro or in their ability to activate growth-associated transcriptional programs after injury. Instead, using age-mismatched nerve transplants in vivo, we show that the extent of functional recovery depends on the age of the nerve graft, and not the age of the host. Molecular interrogation of the sciatic nerve reveals that aged Schwann cells (SCs) fail to rapidly activate a transcriptional repair program after injury. Functionally, aged SCs exhibit impaired dedifferentiation, myelin clearance, and macrophage recruitment. These results suggest that the age-associated decline in axonal regeneration results from diminished Schwann cell plasticity, leading to slower myelin clearance.

3'poly-G-tailed ODNs inhibit F-spondin to induce cell death and neurite retraction in rat embryonic neurons.

The effects and mechanism of action of oligodeoxyribonucleotides containing CpG motif (CpG-ODNs) on neuron cells are largely unexamined. Here, we found that CpG-A ODNs but not other types of CpG-ODNs induced neurite retraction and cell apoptosis of rat embryonic neurons in a TLR9-independent manner. These effects of CpG-A ODNs were primarily due to the poly-guanosine at the 3' terminus (3'G-ODNs). Pull-down analysis showed that 3'G-ODNs associated with transcription factor Y-BOX1 (YB-1) to facilitate the translocation of YB-1 into the nucleus via the nuclear localizing sequence of YB-1. YB-1 then interacted with the promoter of F-spondin directly at -45 and -1,375 sites as demonstrated by chromatin immunoprecipitation (ChIP) analysis. Binding of YB-1 to F-spondin promoter resulted in downregulation of F-spondin expression. Overexpression of F-spondin rescued the cell death and neurite retraction induced by 3'G-ODNs in embryonic neuron cells. Taken together, these findings suggest that 3'G-ODNs enhance nucleus YB-1 to inhibit F-spondin leading to cell death and neurite retraction of embryonic neuron cells.

Recombinant VP1, an Akt inhibitor, suppresses progression of hepatocellular carcinoma by inducing apoptosis and modulation of CCL2 production.

BACKGROUND: The application of viral elements in tumor therapy is one facet of cancer research. Recombinant capsid protein VP1 (rVP1) of foot-and-mouth disease virus has previously been demonstrated to induce apoptosis in cancer cell lines. Here, we aim to further investigate its apoptotic mechanism and possible anti-metastatic effect in murine models of hepatocellular carcinoma (HCC), one of the most common human cancers worldwide. METHODOLOGY/PRINCIPAL FINDINGS: Treatment with rVP1 inhibited cell proliferation in two murine HCC cell lines, BNL and Hepa1-6, with IC50 values in the range of 0.1-0.2 µM. rVP1 also induced apoptosis in these cells, which was mediated by Akt deactivation and dissociation of Ku70-Bax, and resulted in conformational changes and mitochondrial translocation of Bax, leading to the activation of caspases-9, -3 and -7. Treatment with 0.025 µM rVP1, which did not affect the viability of normal hepatocytes, suppressed cell migration and invasion via attenuating CCL2 production. The production of CCL2 was modulated by Akt-dependent NF-κB activation that was decreased after rVP1 treatment. The in vivo antitumor effects of rVP1 were assessed in both subcutaneous and orthotopic mouse models of HCC in immune-competent BALB/c mice. Intratumoral delivery of rVP1 inhibited subcutaneous tumor growth as a result of increased apoptosis. Intravenous administration of rVP1 in an orthotopic HCC model suppressed tumor growth, inhibited intra-hepatic metastasis, and prolonged survival. Furthermore, a decrease in the serum level of CCL2 was observed in rVP1-treated mice. CONCLUSIONS/SIGNIFICANCE: The data presented herein suggest that, via inhibiting Akt phosphorylation, rVP1 suppresses the growth, migration, and invasion of murine HCC cells by inducing apoptosis and attenuating CCL2 production both in vitro and in vivo. Recombinant protein VP1 thus has the potential to be developed as a new therapeutic agent for HCC.

CpG-ODNs induces up-regulated expression of chemokine CCL9 in mouse macrophages and microglia.

Unmethylated CpG oligodeoxynucleotides (CpG-ODNs) interact with Toll-like receptor (TLR) 9 to activate macrophage/microglia in central nervous system (CNS). Here, we investigated the potential involvement of the chemokine CCL9 and its receptor CCR1 in the effects of CpG-ODNs on macrophage/microglial cells. CpG-ODNs enhanced the expression of TLR9 mRNA of RAW264.7 macrophage and BV2 microglia cells time dependently. The expression of CCL9 of macrophages/microglia showed different responsiveness upon stimulation with a variety of CpG-ODN sequences. The CpG-ODNs-mediated induction of CCL9 was TLR9/MyD88 dependent and associated with activation of stress kinases, particularly ERK, p38 MAPK and PI3K. The expression of CCR1 was also significantly increased by CpG-ODNs that increased CCL9 expression. These results reveal the potential involvement of CCL9 and CCR1 in regulation of macrophage and microglial cells by CpG-ODNs and may help improving our understanding about the role of the chemokine/chemokine receptor pairs in macrophage/microglia under physiologic and pathologic conditions.

F-spondin plays a critical role in murine neuroblastoma survival by maintaining IL-6 expression.

F-spondin is associated with the regulation of axonal growth and the development of the nervous system. Its mechanism of action, however, is not clearly understood. In this study, we found that murine neuroblastoma Neuro-2a cells expressed a significant level of IL-6, but only trace amounts of IL-12, tumor necrosis factor alpha and nitric oxide. Knock-down of F-spondin mRNA in murine neuroblastoma NB41A3 and Neuro-2a cells using small interfering RNAs led to decreased IL-6 levels along with lower resistance to serum starvation and cytotoxic amyloid beta(1-42) (Abeta(1-42)) peptide. Restoring decline of F-spondin or IL-6 induced by F-spondin knock-down through adding exogenous F-spondin, IL-6 or over-expressing F-spondin reversed the cell death induced by Abeta(1-42) peptide or serum starvation. The decrease of IL-6 level was positively correlated with decrease of NF-kappaB and inhibition of p38 mitogen-activated protein kinase (MAPK). Over-expressing MEKK, a kinase activator of the p38 MAPK pathway, increased IL-6 production, restored the decrease of p38 induced by F-spondin knock-down, and rescued the cells from death caused by Abeta(1-42) peptide. Taken together, these results suggest that F-spondin may play a critical role in murine neuroblastoma survival under adverse conditions by maintaining IL-6 level via a MEKK/p38 MAPK/NF-kappaB-dependent pathway.

Study on the porcinophilic foot-and-mouth disease virus I. production and characterization of monoclonal antibodies against VP1.

Monoclonal antibodies (MAbs) reported here were produced against the porcinophilic foot-and-mouth disease virus (FMDV) that caused the devastating swine disease on 1997 in Taiwan. A panel (25) of MAbs were found to react with VP1 of O/Taiwan/97 (O/97) by ELISA with various potencies. The biological identities of these VP1 reacting MAbs, such as neutralization activity, isotype and capability to distinguish between two serotype O FMDVs, O/97 and O/Taiwan/KM1/99 (O/99), were further analyzed. Eleven out of the total eighteen O/97 neutralizing MAbs were able to neutralize heterologous O/99. Eight O/97 neutralizing and five non-neutralizing MAbs could differentiate two serotype O FMDVs by immunofluorescence assay (IFA) implied that these thirteen MAbs recognized O/97 specific epitope(s). Furthermore, reactivities of the VP1 reacting MAbs with a 29 amino acids synthetic peptide (P29) representing the betaG-betaH loop of VP1 were analyzed by ELISA and fourteen were found positive. MAb clone Q10E-3 reacting strongest with VP1 and P29, neutralizing both but not differentiating two serotype O viruses suggested that the antibody binding site might involve the RGD motif and its C terminal conserved region on betaG-betaH loop. MAbs with diverse characters presented in this study were the first raised against porcinophilic FMDV. The complete set of MAbs may be used for further studies of vaccine, diagnostic methods, prophylaxis, etiological and immunological researches on FMDV.

DNA vaccination against foot-and-mouth disease via electroporation: study of molecular approaches for enhancing VP1 antigenicity.

BACKGROUND: Foot-and-mouth disease virus (FMDV) affects susceptible livestock animals and causes disastrous economic impact. Immunization with plasmid expressing VP1 that contains the major antigenic epitope(s) of FMDV as cytoplasmic protein (cVP1) failed to elicit full protection against FMDV challenge. MATERIALS AND METHODS: In this study, mice were immunized via electroporation with four cDNA expression vectors that were constructed to express VP1 of FMDV, as cytoplasmic (cVP1), secreted (sVP1), membrane-anchored (mVP1) or capsid precursor protein (P1), respectively, to evaluate whether expression of VP1 in specific subcellular compartment(s) would result in better immune responses. RESULTS: Electroporation enhanced immune responses to vectors expressing cVP1 or P1 and expedited the immune responses to vectors expressing sVP1 or mVP1. Immunization of mice via electroporation with mVP1 cDNA was better than sVP1 or cVP1 cDNA in eliciting neutralizing antibodies and viral clearance protection. Vaccination with P1 cDNA, nonetheless, yielded the best immune responses and protection among all four cDNAs that we tested. CONCLUSIONS: These results suggest that the antigenicity of a VP1 DNA vaccine can be significantly enhanced by altering the cellular localization of the VP1 antigen. Electroporation is a useful tool for enhancing the immune responses of vectors expressing VP1 or P1. By mimicking FMDV more closely than that of transgenic VP1 and eliciting immune responses favorably toward Th2, transgenic P1 may induce more neutralizing antibodies and better protection against FMDV challenge.

IL-19 induced Th2 cytokines and was up-regulated in asthma patients.

IL-19 belongs to the IL-10 family, which includes IL-10, IL-19, IL-20, IL-22, melanoma differentiation-associated gene-7 (IL-24), and AK155 (IL-26). IL-10 has been shown to inhibit allergen-induced airway hyperreactivity and inflammation. To determine whether IL-19 was also associated with asthma, we used ELISA to analyze the serum level of IL-19 in patients with asthma and found that their serum IL-19 levels were twice those of healthy controls. Patients with a high level of IL-19 also had high levels of IL-4 and IL-13. In a dust mite-induced murine model of asthma, we found that IL-19 level in asthmatic BALB/cJ mice was also twice that of healthy control mice. IL-19 transcript was also induced in the lungs of asthmatic mice. Electroporation i.m. of the IL-19 gene into healthy mice up-regulated IL-4 and IL-5, but not IL-13. However, IL-19 up-regulated IL-13 in asthmatic mice. In vitro, IL-19 induced IL-4, IL-5, IL-10, and IL-13 production by activated T cells. Activation of T cells was required for induction of IL-13 because IL-19 did not induce IL-13 production on nonstimulated T cells. Taken together, these results demonstrated that IL-19 up-regulates Th2 cytokines on activated T cells and might play an important role in the pathogenesis of asthma.