Adeno-Associated Viral Vector-Delivered Pannexin-1 Mimetic Peptide Alleviates Airway Inflammation in an Allergen-Sensitized Mouse Model.

Asthma is a chronic inflammatory disease around the world. Extracellular adenosine triphosphate works as a dangerous signal in responding to cellular stress, irritation, or inflammation. It has also been reported its association with the pathogenicity in asthma, with increased level in lungs of asthmatics. Pannexin-1 is one of the routes that contributes to the release of adenosine triphosphate form intracellular to extracellular. The aim of this study was to apply pannexin-1 peptide antagonist 10Panx1 into adeno-associated viral (AAV) vectors on ovalbumin (OVA)-induced asthmatic mouse model. The results demonstrated that this treatment was able to reduce the adenosine triphosphate level in bronchoalveolar lavage fluid and downregulate the major relevant to the symptom of asthma attack, airway hyperresponsiveness to methacholine. The histological data also gave a positive support with decreased tissue remodeling and mucus deposition. Other asthmatic related features, including eosinophilic inflammation and OVA-specific T helper type 2 responses, were also decreased by the treatment. Beyond the index of inflammation, the proportion of effector and regulatory T cells was examined to survey the potential mechanism behind. The data provided a slightly downregulated pattern in lung GATA3+ CD4 T cells. However, an upregulated population of CD25+FoxP3+ CD4 T cells was seen in spleens. These data suggested that exogeneous expression of 10Panx1 peptide was potential to alleviated asthmatic airway inflammation, and this therapeutic effect might be from 10Panx1-mediated disruption of T cell activation or differentiation. Collectively, AAV vector-mediated 10Panx1 expression could be a naval therapy option to develop.

Degradation of neurodegenerative disease-associated TDP-43 aggregates and oligomers via a proteolysis-targeting chimera.

BACKGROUND: Amyotrophic lateral sclerosis (ALS) associated with TAR DNA-binding protein 43 (TDP-43) aggregation has been considered as a lethal and progressive motor neuron disease. Recent studies have shown that both C-terminal TDP-43 (C-TDP-43) aggregates and oligomers were neurotoxic and pathologic agents in ALS and frontotemporal lobar degeneration (FTLD). However, misfolding protein has long been considered as an undruggable target by applying conventional inhibitors, agonists, or antagonists. To provide this unmet medical need, we aim to degrade these misfolding proteins by designing a series of proteolysis targeting chimeras (PROTACs) against C-TDP-43. METHODS: By applying filter trap assay, western blotting, and microscopy imaging, the degradation efficiency of C-TDP-43 aggregates was studied in Neuro-2a cells overexpressing eGFP-C-TDP-43 or mCherry-C-TDP-43. The cell viability was characterized by alarmarBlue assay. The beneficial and disaggregating effects of TDP-43 PROTAC were examined with the YFP-C-TDP-43 transgenic C. elegans by motility assay and confocal microscopy. The impact of TDP-43 PROTAC on C-TDP-43 oligomeric intermediates was monitored by fluorescence lifetime imaging microscopy and size exclusion chromatography in the Neuro-2a cells co-expressing eGFP-C-TDP-43 and mCherry-C-TDP-43. RESULTS: Four PROTACs with different linker lengths were synthesized and characterized. Among these chimeras, PROTAC 2 decreased C-TDP-43 aggregates and relieved C-TDP-43-induced cytotoxicity in Neuro-2a cells without affecting endogenous TDP-43. We showed that PROTAC 2 bound to C-TDP-43 aggregates and E3 ligase to initiate ubiquitination and proteolytic degradation. By applying advanced microscopy, it was further shown that PROTAC 2 decreased the compactness and population of C-TDP-43 oligomers. In addition to cellular model, PROTAC 2 also improved the motility of transgenic C. elegans by reducing the C-TDP-43 aggregates in the nervous system. CONCLUSIONS: Our study demonstrated the dual-targeting capacity of the newly-designed PROTAC 2 against both C-TDP-43 aggregates and oligomers to reduce their neurotoxicity, which shed light on the potential drug development for ALS as well as other neurodegenerative diseases.

Modulating ILC2 function for treatment of type 2 airway diseases.

Type 2 airway diseases including chronic rhinosinusitis, allergic rhinitis, and asthma remain a major health concern. These disorders are largely characterized by an uncontrolled type 2 immune response with elevated cytokines of IL-4, IL-5 and IL-13, eosinophilic inflammation, goblet cell hyperplasia as well as tissue remodeling. In the last few decades, critical potential roles of innate lymphoid cells (ILCs) in type 2 human diseases have emerged. Unlike their lymphocyte counterpart T cells, ILCs lack antigen-specific receptors and are largely tissue resident. Specifically, group 2 innate lymphoid cells (ILC2s) respond to airway epithelium-derived alarmins (TSLP, IL-33) and secrete high levels of type 2 cytokines. ILC2 responses can bypass the activation of T cells as well as develop corticosteroid-resistance. Currently approved biologics targeting the alarmin thymic stromal lymphopoietin (TSLP) or the IL-4/IL-13 receptor may reduce ILC2 activation, though novel treatments of type 2 airway diseases remain needed. In this review, we briefly discuss the pathogenesis of ILC2-mediated airway diseases followed by their current and potential treatments.

Polyglutamine-Specific Gold Nanoparticle Complex Alleviates Mutant Huntingtin-Induced Toxicity.

Huntington's disease (HD) belongs to protein misfolding disorders associated with polyglutamine (polyQ)-rich mutant huntingtin (mHtt) protein inclusions. Currently, it is indicated that the aggregation of polyQ-rich mHtt participates in neuronal toxicity and dysfunction. Here, we designed and synthesized a polyglutamine-specific gold nanoparticle (AuNP) complex, which specifically targeted mHtt and alleviated its toxicity. The polyglutamine-specific AuNPs were prepared by decorating the surface of AuNPs with an amphiphilic peptide (JLD1) consisting of both polyglutamine-binding sequences and negatively charged sequences. By applying the polyQ aggregation model system, we demonstrated that AuNPs-JLD1 dissociated the fibrillary aggregates from the polyQ peptide and reduced its ß-sheet content in a concentration-dependent manner. By further integrating polyethyleneimine (PEI) onto AuNPs-JLD1, we generated a complex (AuNPs-JLD1-PEI). We showed that this complex could penetrate cells, bind to cytosolic mHtt proteins, dissociate mHtt inclusions, reduce mHtt oligomers, and ameliorate mHtt-induced toxicity. AuNPs-JLD1-PEI was also able to be transported to the brain and improved the functional deterioration in the HD Drosophila larva model. Our results revealed the feasibility of combining AuNPs, JLD1s, and cell-penetrating polymers against mHtt protein aggregation and oligomerization, which hinted on the early therapeutic strategies against HD.

Reducing Lung ATP Levels and Alleviating Asthmatic Airway Inflammation through Adeno-Associated Viral Vector-Mediated CD39 Expression.

Asthma is a chronic respiratory inflammatory disease. Patients usually suffer long-term symptoms and high medical expenses. Extracellular ATP (eATP) has been identified as a danger signal in innate immunity and serves as a potent inflammatory mediator for asthma. Hydrolyzing eATP in lungs might be a potential approach to alleviate asthmatic inflammation. Recombinant adeno-associated virus (rAAV) vectors that contain tissue-specific cap protein have been demonstrated to efficiently transfer exogenous genes into the lung tissues. To test anti-inflammation efficacy of rAAV-mediated CD39 gene transfer, rAAV-CD39 was generated and applied to OVA-mediated asthmatic mice. BALB/c mice were sensitized intraperitoneally and challenged intratracheally with OVA and treated with rAAV-CD39. At the end of procedure, some inflammatory features were examined. rAAV-CD39 treatment downregulated the levels of pulmonary eATP by the rescued expression of CD39. Several asthmatic features, such as airway hyperresponsiveness, eosinophilia, mucin deposition, and IL-5/IL-13 production in the lungs were decreased in the rAAV-CD39-treated mice. Reduced IL-5/IL-13 production and increased frequency of CD4+FoxP3+ regulatory T cells were detected in draining lymph nodes of rAAV-CD39 treated mice. This evidence suggested that rAAV-mediated CD39 gene transfer attenuated the asthmatic airway inflammation locally. The results suggest that rAAV-CD39 might have therapeutic potential for asthma.

Nanoscopic investigation of C9orf72 poly-GA oligomers on nuclear membrane disruption by a photoinducible platform.

Glycine-alanine dipeptide repeats (GA DPRs) translated from the mutated C9orf72 gene have recently been correlated with amyotrophic lateral sclerosis (ALS). While GA DPRs aggregates have been suggested as amyloid, the biophysical features and cytotoxicity of GA DPRs oligomers has not been explored due to its unstable nature. In this study, we develop a photoinducible platform based on methoxynitrobenzene chemistry to enrich GA DPRs that allows monitoring the oligomerization process of GA DPRs in cells. By applying advanced microscopies, we examined the GA DPRs oligomerization process nanoscopically in a time-dependent manner. We provided direct evidences to demonstrate GA DPRs oligomers rather than nanofibrils disrupt nuclear membrane. Moreover, we found GA DPRs hamper nucleocytoplasmic transport in cells and cause cytosolic retention of TAR DNA-binding protein 43 in cortical neurons. Our results highlight the toxicity of GA DPRs oligomers, which is a key step toward elucidating the pathological roles of C9orf72 DPRs.

Blocking pannexin1 reduces airway inflammation in a murine model of asthma.

Stressed or injured cells release ATP into the extracellular milieu via the pannexin1 (Panx1) channels, which is the basis of inflammation in a variety of conditions, including allergic lung inflammation. Although the role of Panx1 in mediating inflammation has been well established, the role of its mimetic peptide, 10Panx1, which inhibits ATP release from Panx1 channels, in allergic asthma remains understudied. The aim of this study was to evaluate the effects of using 10Panx1 to inhibit Panx1 channel in a murine model of ovalbumin (OVA)-induced asthma. We demonstrate that blockade of Panx1 significantly attenuated goblet cell hyperplasia and inflammatory cell infiltration into the lungs of OVA-sensitized mice. Inhibition of Panx1 also reduced the total and eosinophil cell numbers in the bronchoalveolar lavage fluid (BALF) and reduced expression of CCL11 and CCL2 in lung tissues from mice. Moreover, we detected lower levels of IL-5 and IL-13 in the culture supernatant of OVA-restimulated splenocytes from 10Panx1-treated mice. Collectively, our findings suggest that Panx1 inhibition of allergen-mediated lung inflammation has the potential to suppress allergic responses in asthma.

Actin waves transport RanGTP to the neurite tip to regulate non-centrosomal microtubules in neurons.

Microtubules (MTs) are the most abundant cytoskeleton in neurons, and control multiple facets of their development. While the MT-organizing center (MTOC) in mitotic cells is typically located at the centrosome, the MTOC in neurons switches to non-centrosomal sites. A handful of cellular components have been shown to promote non-centrosomal MT (ncMT) formation in neurons, yet the regulation mechanism remains unknown. Here, we demonstrate that the small GTPase Ran is a key regulator of ncMTs in neurons. Using an optogenetic tool that enables light-induced local production of RanGTP, we demonstrate that RanGTP promotes ncMT plus-end growth along the neurite. Additionally, we discovered that actin waves drive the anterograde transport of RanGTP. Pharmacological disruption of actin waves abolishes the enrichment of RanGTP and reduces growing ncMT plus-ends at the neurite tip. These observations identify a novel regulation mechanism for ncMTs and pinpoint an indirect connection between the actin and MT cytoskeletons in neurons.

Photocontrollable Probe Spatiotemporally Induces Neurotoxic Fibrillar Aggregates and Impairs Nucleocytoplasmic Trafficking.

The abnormal assembly of misfolded proteins into neurotoxic aggregates is the hallmark associated with neurodegenerative diseases. Herein, we establish a photocontrollable platform to trigger amyloidogenesis to recapitulate the pathogenesis of amyotrophic lateral sclerosis (ALS) by applying a chemically engineered probe as a "switch" in live cells. This probe is composed of an amyloidogenic peptide from TDP-43, a photolabile linker, a polycationic sequence both to mask amyloidogenicity and for cell penetration, and a fluorophore for visualization. The photocontrollable probe can self-assemble into a spherical vesicle but rapidly develops massive nanofibrils with amyloid properties upon photoactivation. The photoinduced in vitro fibrillization process is characterized by biophysical techniques. In cellular experiments, this cell-penetrable vesicle was retained in the cytoplasm, seeded the mislocalized endogenous TDP-43 into aggregates upon irradiation, and consequently initiated apoptosis. In addition, this photocontrollable vesicle interfered with nucleocytoplasmic protein transport and triggered cortical neuron degeneration. Our developed strategy provides in vitro and in vivo spatiotemporal control of neurotoxic fibrillar aggregate formation, which can be readily applied in the studies of protein misfolding, aggregation-induced protein mislocalization, and amyloid-induced pathogenesis in different diseases.

Ran-dependent TPX2 activation promotes acentrosomal microtubule nucleation in neurons.

The microtubule (MT) cytoskeleton is essential for the formation of morphologically appropriate neurons. The existence of the acentrosomal MT organizing center in neurons has been proposed but its identity remained elusive. Here we provide evidence showing that TPX2 is an important component of this acentrosomal MT organizing center. First, neurite elongation is compromised in TPX2-depleted neurons. In addition, TPX2 localizes to the centrosome and along the neurite shaft bound to MTs. Depleting TPX2 decreases MT formation frequency specifically at the tip and the base of the neurite, and these correlate precisely with the regions where active GTP-bound Ran proteins are enriched. Furthermore, overexpressing the downstream effector of Ran, importin, compromises MT formation and neuronal morphogenesis. Finally, applying a Ran-importin signaling interfering compound phenocopies the effect of TPX2 depletion on MT dynamics. Together, these data suggest a model in which Ran-dependent TPX2 activation promotes acentrosomal MT nucleation in neurons.

The modulation of Th2 immune pathway in the immunosuppressive effect of human umbilical cord mesenchymal stem cells in a murine asthmatic model.

BACKGROUND: Asthma is a chronic airway inflammatory disease that has a high prevalence nowadays, and seeking the means of relieving asthmatic symptoms is now an issue with increased importance. While mesenchymal stem cells have been demonstrated to display immunomodulatory effects, the effect of fetus-type mesenchymal stem cells (MSCs) on asthmatic symptoms in vivo have not been reported to date. METHODS: Female BALB/c mice at 8 weeks of age were sensitized by ovalbumin, and MSCs derived from Wharton's jelly of human umbilical cord mesenchymal stem cells (hUCMSCs) were injected into the asthmatic mice. Airway hyper-responsiveness, lung eosinophil infiltration, cytokine level in splenocyte cultures and serum immunoglobulin level were measured. Enzyme-linked immunosorbent assay was used to determine cytokine and immunoglobulin levels. RESULTS: This current study demonstrated that hUCMSCs attenuated both lung lymphocyte and eosinophil infiltration, and significantly decreased the concentration of Th2 cytokines interleukin-5 in splenocyte cultures. CONCLUSIONS: Human umbilical cord mesenchymal stem cells have the advantage of being easily harvested non-invasively and are capable of rapid proliferation, therefore an ideal material for stem cell-based immune therapies. The current study showed that fetal-type MSCs were able to suppress asthmatic symptoms efficiently, and its immunomodulatory effect resulted primarily from suppressing the Th2 pathway in the animal model. This study suggested that hUCMSCs could be an ideal candidate for cell-based therapies of asthma.

The effect of fluorescent nanodiamonds on neuronal survival and morphogenesis.

Nanodiamond (ND) has emerged as a promising carbon nanomaterial for therapeutic applications. In previous studies, ND has been reported to have outstanding biocompatibility and high uptake rate in various cell types. ND containing nitrogen-vacancy centers exhibit fluorescence property is called fluorescent nanodiamond (FND), and has been applied for bio-labeling agent. However, the influence and application of FND on the nervous system remain elusive. In order to study the compatibility of FND on the nervous system, neurons treated with FNDs in vitro and in vivo were examined. FND did not induce cytotoxicity in primary neurons from either central (CNS) or peripheral nervous system (PNS); neither did intracranial injection of FND affect animal behavior. The neuronal uptake of FNDs was confirmed using flow cytometry and confocal microscopy. However, FND caused a concentration-dependent decrease in neurite length in both CNS and PNS neurons. Time-lapse live cell imaging showed that the reduction of neurite length was due to the spatial hindrance of FND on advancing axonal growth cone. These findings demonstrate that FNDs exhibit low neuronal toxicity but interfere with neuronal morphogenesis, and should be taken into consideration when applications involve actively growing neurites (e.g. nerve regeneration).

Microtubule-associated type II protein kinase A is important for neurite elongation.

Neuritogenesis is a process through which neurons generate their widespread axon and dendrites. The microtubule cytoskeleton plays crucial roles throughout neuritogenesis. Our previous study indicated that the amount of type II protein kinase A (PKA) on microtubules significantly increased upon neuronal differentiation and neuritogenesis. While the overall pool of PKA has been shown to participate in various neuronal processes, the function of microtubule-associated PKA during neuritogenesis remains largely unknown. First, we showed that PKA localized to microtubule-based region in different neurons. Since PKA is essential for various cellular functions, globally inhibiting PKA activity will causes a wide variety of phenotypes in neurons. To examine the function of microtubule-associated PKA without changing the total PKA level, we utilized the neuron-specific PKA anchoring protein MAP2. Overexpressing the dominant negative MAP2 construct that binds to type II PKA but cannot bind to the microtubule cytoskeleton in dissociated hippocampal neurons removed PKA from microtubules and resulted in compromised neurite elongation. In addition, we demonstrated that the association of PKA with microtubules can also enhance cell protrusion using the non-neuronal P19 cells. Overexpressing a MAP2 deletion construct which does not target PKA to the microtubule cytoskeleton caused non-neuronal cells to generate shorter cell protrusions than control cells overexpressing wild-type MAP2 that anchors PKA to microtubules. Finally, we demonstrated that the ability of microtubule-associated PKA to promote protrusion elongation was independent of MAP2 phosphorylation. This suggests other proteins in close proximity to the microtubule cytoskeleton are involved in this process.

Application of aurintricarboxylic acid for the adherence of mouse P19 neurons and primary hippocampal neurons to noncoated surface in serum-free culture.

Dissociated primary neuron culture has been the most widely used model systems for neuroscience research. Most of these primary neurons are cultured on adhesion matrix-coated surface to provide a proper environment for cell anchorage under serum-free conditions. In this study, we provide an alternative technique to promote the adhesions of these neurons using aurintricarboxylic acid (ATA), a nonpeptide compound, without surface manipulations. We first demonstrated that ATA could promote Chinese hamster ovary cell attachment and proliferation in serum-free medium in a dosage-dependent manner. We later showed that ATA significantly enhanced the attachment of the retinoic acid differentiated P19 mouse embryonal carcinoma (P19) neurons, with an optimal concentration around 30 µg/mL. A similar result was seen in primary hippocampal neurons, with an optimal ATA concentration around 15 µg/mL. Further morphological assessments revealed that the average neurite length and neuronal polarization were almost identical to that obtained using a conventional method with poly-L-lysine surface. The advantages of using the ATA treatment technique for immunochemical analysis are discussed.

An inverted method for culturing dissociated mouse hippocampal neurons.

Dissociated hippocampal neuron culture has long been the model system of choice for many neuroscientists. The ability to culture dissociated hippocampal neurons from genetically modified mice provides an invaluable tool for studying many neuronal processes. In this study, we established a novel method to culture dissociated hippocampal neurons from embryonic and neonatal mice. Dissociated neurons were cultured in a microchamber between the glass coverslip and the plastic cell container without the use of glial feeder cells. Our method significantly simplifies the preparation while produces healthy and long-lived neuronal cultures that are difficult to achieve without the use of feeder cells.