Annemarie Shibata

Myelin-associated glycoprotein inhibits neurite/axon growth and causes growth cone collapse

We have previously shown that myelin‐associated glycoprotein (MAG) inhibits neurite growth from a neuronal cell line. In this study we show that 60% of axonal growth cones of postnatal day 1 hippocampal neurons collapsed when they encountered polystyrene beads coated with recombinant MAG (rMAG). Such collapse was not observed with denatured rMAG. Neurite growth from rat embryonic hippocampal and neonatal cerebellar neurons was also inhibited about 80% on tissue culture substrates coated with rMAG. To investigate further the inhibitory activity of MAG in myelin, we purified myelin from MAG‐deficient mice and separated octylglucoside extracts of myelin by diethylaminoethyl (DEAE) ion‐exchange chromatography. Although there was no significant difference in neurite growth on myelin purified from MAG‐/‐ and MAG+/+ mice, differences were observed in the fractionated material. The major inhibitory peak that is associated with MAG in normal mice was significantly reduced in MAG‐deficient mice. These results suggest that although MAG contributes significantly to axon growth inhibition associated with myelin, its lack in MAG‐deficient mice is masked by other non‐MAG inhibitors. Axon regeneration in these mice was also examined after thoracic lesions of the corticospinal tracts. A very small number of anterogradely labeled axons extended up to 13.2 mm past the lesion in MAG‐/‐ mice. Although there is some enhancement of axon generation, the poor growth after spinal cord injury in MAG‐/‐ mice may be due to the presence of other non‐MAG inhibitors. The in vitro studies, however, provide the first evidence that MAG modulates growth cone behavior and inhibits neurite growth by causing growth cone collapse.

Combination antiretroviral drugs in PLGA nanoparticle for HIV-1

BackgroundCombination antiretroviral (AR) therapy continues to be the mainstay for HIV treatment. However, antiretroviral drug nonadherence can lead to the development of resistance and treatment failure. We have designed nanoparticles (NP) that contain three AR drugs and characterized the size, shape, and surface charge. Additionally, we investigated the in vitro release of the AR drugs from the NP using peripheral blood mononuclear cells (PBMCs).MethodsPoly-(lactic-co-glycolic acid) (PLGA) nanoparticles (NPs) containing ritonavir (RTV), lopinavir (LPV), and efavirenz (EFV) were fabricated using multiple emulsion-solvent evaporation procedure. The nanoparticles were characterized by electron microscopy and zeta potential for size, shape, and charge. The intracellular concentration of AR drugs was determined over 28 days from NPs incubated with PBMCs. Macrophages were imaged by fluorescent microscopy and flow cytometry after incubation with fluorescent NPs. Finally, macrophage cytotoxicity was determined by MTT assay.ResultsNanoparticle size averaged 262 ± 83.9 nm and zeta potential -11.4 ± 2.4. AR loading averaged 4% (w/v). Antiretroviral drug levels were determined in PBMCs after 100 μg of NP in 75 μL PBS was added to media. Intracellular peak AR levels from NPs (day 4) were RTV 2.5 ± 1.1; LPV 4.1 ± 2.0; and EFV 10.6 ± 2.7 μg and continued until day 28 (all AR ≥ 0.9 μg). Free drugs (25 μg of each drug in 25 μL ethanol) added to PBMCs served as control were eliminated by 2 days. Fluorescence microscopy and flow cytometry demonstrated phagocytosis of NP into monocytes-derived macrophages (MDMs). Cellular MTT assay performed on MDMs demonstrated that NPs are not significantly cytotoxic.ConclusionThese results demonstrated AR NPs could be fabricated containing three antiretroviral drugs (RTV, LPV, EFV). Sustained release of AR from PLGA NP show high drug levels in PBMCs until day 28 without cytotoxicity.

Antiretroviral release from poly(dl-lactide-co-glycolide) nanoparticles in mice

OBJECTIVES Free ritonavir, lopinavir and efavirenz injected intraperitoneally were compared with antiretroviral (AR) nanoparticles (NPs). METHODS This is a prospective study in BALB/c mice comparing the pharmacokinetics of free drugs with AR NPs. All animals received free drugs or AR NPs (20 mg/kg) in PBS. In vitro replication assays were used for determination of the anti-HIV efficacy of NP formulations. At specific times (free drugs 0.08, 0.125, 0.25, 0.33, 1, 2 and 3 days; AR NPs 0.125, 0.33, 1, 2, 4, 7, 14, 21, 28, 35 and 42 days) mice were euthanized and serum and organs were harvested for determination of AR concentrations by HPLC. Single treatment of monocyte-derived macrophages (MDMs) infected with HIV-1(ada) compared AR NPs (0.005-0.05 mg/mL) with free efavirenz or lopinavir/ritonavir (0.01-0.1 mg/mL), blank NPs and controls. Results are presented as means ± SEM. RESULTS Serum free AR drug concentrations peaked 4 h post-injection (ritonavir 3.9 ± 3.05, lopinavir 3.4 ± 2.5 and efavirenz 1.8 ± 0.63 µg/mL) and were eliminated by 72 h. Poly(dl-lactide-co-glycolide) NP animals had detectable ritonavir, lopinavir and efavirenz concentrations in all tissues for 28 days. Treatment of MDMs with AR NPs resulted in sustained inhibition of HIV-1(ada) replication. CONCLUSIONS AR drug concentrations from NPs are sustained for 28 days in vivo and anti-HIV inhibition was comparable to that of free drugs in vitro and could be a sustained treatment for delivery of AR drugs.

Development and evaluation of a thermosensitive vaginal gel containing raltegravir + efavirenz loaded nanoparticles for HIV prophylaxis

The objective of this investigation was to develop a thermosensitive vaginal gel containing raltegravir+efavirenz loaded PLGA nanoparticles (RAL+EFV-NPs) for pre-exposure prophylaxis of HIV. RAL+EFV-NPs were fabricated using a modified emulsion-solvent evaporation method and characterized for size and zeta potential. The average size and surface charge of RAL+EFV-NP were 81.8±6.4 nm and -23.18±7.18 mV respectively. The average encapsulation efficiency of raltegravir and efavirenz was 55.5% and 98.2% respectively. Thermosensitive vaginal gel containing RAL+EFV-NPs was successfully prepared using a combination of Pluronic F127 (20% w/v) and Pluronic F68 (1% w/v). Incorporation RAL+EFV-NPs in the gel did not result in nanoparticle aggregation and RAL+EFV-NPs containing gel showed thermogelation at 32.5°C. The RAL+EFV-NPs were evaluated for inhibition of HIV-1(NL4-3) using TZM-bl indicator cells. The EC(90) of RAL+EFV-NPs was lower than raltegravir+efavirenz (RAL+EFV) solution but did not reach significance. Compared to control HeLa cells without any treatment, RAL+EFV-NPs or blank gel were not cytotoxic for 14 days in vitro. The intracellular levels of efavirenz in RAL+EFV-NPs treated HeLa cells were above the EC(90) for 14 days whereas raltegravir intracellular concentrations were eliminated within 6 days. Transwell experiments of NPs-in-gel demonstrated rapid transfer of fluorescent nanoparticles from the gel and uptake in HeLa cells within 30 min. These data demonstrate the potential of antiretroviral NP-embedded vagina gels for long-term vaginal pre-exposure prophylaxis of heterosexual HIV-1 transmission.

The role of neuroD as a differentiation factor in the mammalian retina

NeuroD, a vertebrate homolog of Drosophila atonal gene, plays an important role in the differentiation of neuronal precursors (Lee et al., 1995). We have investigated whether NeuroD subserves a similar function in mammalian retinal neurogenesis. Expression of NeuroD is detected in successive stages of retinal neurogenesis and is associated with a differentiating population of retinal cells. The association of NeuroD predominantly with postmitotic precursors in early as well as late neurogenesis suggests that NeuroD expression plays an important role in the terminal differentiation of retinal neurons. This notion is supported by observations that overexpression of NeuroD during late neurogenesis promotes premature differentiation of late-born neurons, rod photoreceptors, and bipolar cells, and that NeuroD can interact specifically with the E-box element in the proximal promoter of the phenotype-specific gene, opsin.

LincRNA-Cox2 Promotes Late Inflammatory Gene Transcription in Macrophages through Modulating SWI/SNF-Mediated Chromatin Remodeling

Long intergenic noncoding RNAs (lincRNAs) are long noncoding transcripts (>200 nt) from the intergenic regions of annotated protein-coding genes. One of the most highly induced lincRNAs in macrophages upon TLR ligation is lincRNA-Cox2, which was recently shown to mediate the activation and repression of distinct classes of immune genes in innate immune cells. We report that lincRNA-Cox2, located at chromosome 1 proximal to the PG-endoperoxide synthase 2 (Ptgs2/Cox2) gene, is an early-primary inflammatory gene controlled by NF-κB signaling in murine macrophages. Functionally, lincRNA-Cox2 is required for the transcription of NF-κB–regulated late-primary inflammatory response genes stimulated by bacterial LPS. Specifically, lincRNA-Cox2 is assembled into the switch/sucrose nonfermentable (SWI/SNF) complex in cells after LPS stimulation. This resulting lincRNA-Cox2/SWI/SNF complex can modulate the assembly of NF-κB subunits to the SWI/SNF complex, and ultimately, SWI/SNF-associated chromatin remodeling and transactivation of the late-primary inflammatory-response genes in macrophages in response to microbial challenge. Therefore, our data indicate a new regulatory role for NF-κB–induced lincRNA-Cox2 as a coactivator of NF-κB for the transcription of late-primary response genes in innate immune cells through modulation of epigenetic chromatin remodeling.

Nanoformulations of Rilpivirine for Topical Pericoital and Systemic Coitus-Independent Administration Efficiently Prevent HIV Transmission

Vaginal HIV transmission accounts for the majority of new infections worldwide. Currently, multiple efforts to prevent HIV transmission are based on pre-exposure prophylaxis with various antiretroviral drugs. Here, we describe two novel nanoformulations of the reverse transcriptase inhibitor rilpivirine for pericoital and coitus-independent HIV prevention. Topically applied rilpivirine, encapsulated in PLGA nanoparticles, was delivered in a thermosensitive gel, which becomes solid at body temperature. PLGA nanoparticles with encapsulated rilpivirine coated the reproductive tract and offered significant protection to BLT humanized mice from a vaginal high-dose HIV-1 challenge. A different nanosuspension of crystalline rilpivirine (RPV LA), administered intramuscularly, protected BLT mice from a single vaginal high-dose HIV-1 challenge one week after drug administration. Using transmitted/founder viruses, which were previously shown to establish de novo infection in humans, we demonstrated that RPV LA offers significant protection from two consecutive high-dose HIV-1 challenges one and four weeks after drug administration. In this experiment, we also showed that, in certain cases, even in the presence of drug, HIV infection could occur without overt or detectable systemic replication until levels of drug were reduced. We also showed that infection in the presence of drug can result in acquisition of multiple viruses after subsequent exposures. These observations have important implications for the implementation of long-acting antiretroviral formulations for HIV prevention. They provide first evidence that occult infections can occur, despite the presence of sustained levels of antiretroviral drugs. Together, our results demonstrate that topically- or systemically administered rilpivirine offers significant coitus-dependent or coitus-independent protection from HIV infection.

Peripheral nerve induces macrophage neurotrophic activities: regulation of neuronal process outgrowth, intracellular signaling and synaptic function

Rat cortical neurons cultured in conditioned media from human monocyte-derived macrophages (MDM) show increased neuronal protein synthesis, neurite outgrowth, mitogen-activating protein kinase activity, and synaptic function. Neurotrophic properties of human MDM-conditioned media are significantly enhanced by human peripheral nerve and to a more limited extent by CD40 ligand pre-stimulation. Such positive effects of MDM secretions on neuronal function parallel the secretion of brain-derived neurotrophic factor (BDNF). MDM activation cues may serve to balance toxic activities produced during neurodegenerative diseases and thus, under certain circumstances, mitigate neuronal degeneration.

A long noncoding RNA, lincRNA-Tnfaip3, acts as a coregulator of NF-κB to modulate inflammatory gene transcription in mouse macrophages

Long intergenic noncoding RNAs (lincRNAs) are long noncoding transcripts (>200 nt) from the intergenic regions of annotated protein‐coding genes. We report here that the lincRNA gene lincRNA‐Tnfaip3, located at mouse chromosome 10 proximal to the tumor necrosis factor α‐induced protein 3 (Tnfaip3) gene, is an early‐primary response gene controlled by nuclear factor‐κB (NF‐κB) signaling in murine macrophages. Functionally, lincRNA‐ Tnfaip3 appears to mediate both the activation and repression of distinct classes of inflammatory genes in macrophages. Specifically, induction of lincRNA‐Tnfaip3 is required for the transactivation of NF‐κB‐regulated inflammatory genes in response to bacterial LPSs stimulation. LincRNA‐Tnfaip3 physically interacts with the high‐mobility group box 1 (Hmgb1), assembling a NF‐κB/Hmgb1/lincRNA‐Tnfaip3 complex in macrophages after LPS stimulation. This resultant NF‐κB/Hmgb1/lincRNA‐Tnfaip3 complex can modulate Hmgb1‐associated histone modifications and, ultimately, transactivation of inflammatory genes in mouse macrophages in response to microbial challenge. Therefore, our data indicate a new regulatory role of NF‐κB‐induced lincRNA‐Tnfaip3 to act as a coactivator of NF‐κB for the transcription of inflammatory genes in innate immune cells through modulation of epigenetic chromatin remodeling.—Ma, S., Ming, Z., Gong, A.‐Y., Wang, Y., Chen, X., Hu, G., Zhou, R., Shibata, A., Swanson, P. C., Chen, X.‐M. A long noncoding RNA, LincRNA‐Tnfaip3, acts as a coregulator of NF‐κB to modulate inflammatory gene transcription in mouse macrophages. FASEB J. 31, 1215–1225 (2017). www.fasebj.org

The unique and shared properties of neuronal growth cones that enable navigation and specific pathfinding

The neuronal growth cone has as one of its primary functions the role of guiding the elongating neurite to its appropriate target. The events during pathfinding require that the neuronal growth cone responds to particular molecules in the environment by making appropriate changes in the behavior of the growth cone. This paper discusses the role of intracellular calcium as a signalling system mediating these responses. The specific roles of the growth cone filopodia in acting as antennae in advance of the motile growth cone are discussed in the context of calcium mediated pathfinding events.