Unraveling the enigma: housekeeping gene Ugt1a7c as a universal biomarker for microglia.

Background: Microglia, brain resident macrophages, play multiple roles in maintaining homeostasis, including immunity, surveillance, and protecting the central nervous system through their distinct activation processes. Identifying all types of microglia-driven populations is crucial due to the presence of various phenotypes that differ based on developmental stages or activation states. During embryonic development, the E8.5 yolk sac contains erythromyeloid progenitors that go through different growth phases, eventually resulting in the formation of microglia. In addition, microglia are present in neurological diseases as a diverse population. So far, no individual biomarker for microglia has been discovered that can accurately identify and monitor their development and attributes. Summary: Here, we highlight the newly defined biomarker of mouse microglia, UGT1A7C, which exhibits superior stability in expression during microglia development and activation compared to other known microglia biomarkers. The UGT1A7C sensing chemical probe labels all microglia in the 3xTG AD mouse model. The expression of Ugt1a7c is stable during development, with only a 4-fold variation, while other microglia biomarkers, such as Csf1r and Cx3cr1, exhibit at least a 10-fold difference. The UGT1A7C expression remains constant throughout its lifespan. In addition, the expression and activity of UGT1A7C are the same in response to different types of inflammatory activators' treatment in vitro. Conclusion: We propose employing UGT1A7C as the representative biomarker for microglia, irrespective of their developmental state, age, or activation status. Using UGT1A7C can reduce the requirement for using multiple biomarkers, enhance the precision of microglia analysis, and even be utilized as a standard for gene/protein expression.

Theranostics application of tumor-initiating cell probe TiY in non-small cell lung cancer.

Background: Tumor-initiating cells (TIC) often elude conventional cancer treatment, which results in metastasis and cancer relapse. Recently, studies have begun to focus on the TIC population in tumors to provide better therapeutic options. Previously, we have reported the successful development of a TIC-specific probe TiY with the binding target as vimentin. While a low concentration of TiY showed a TIC visualization, at a high concentration, TiY induced selective toxicity onto TIC in vitro. In this study, we aim to assess TiY's applicability in theranostics purposes, from in vivo visualization to therapeutic effect toward TIC, in cancer mouse models. Methods: We performed cell experiments with the TIC line model derived from resected primary non-small cell lung cancer (NSCLC) patient tumor. The animal model studies were conducted in mice of NSCLC patient-derived xenograft (PDX). TiY was intravenously delivered into the mice models at different concentrations to assess its in vivo TIC-selective staining and therapeutic effect. Results: We demonstrated the TIC-selective identification and therapeutic effect of TiY in animal models. TiY treatment induced a significant ablation of the TIC population in the tumor, and further molecular study elucidated that the mechanism of TiY is through vimentin dynamics in TIC. Conclusion: The results underscore the applicability of TiY for cancer treatment by selectively targeting soluble vimentin in TIC.

LRRK2 decreases microglial actin dynamics by filamentous actin depolymerization and Rac1 inhibition.

An active actin dynamic is a crucial feature of brain microglia. Here we report that LRRK2, a primary familial Parkinson's disease-associated gene, negatively regulates microglia's actin dynamics. LRRK2 depolymerized filamentous actin (F-actin) by directly binding to it or inhibiting microglia's Rac-PAK signaling. LRRK2 knockdown resulted in a reduced ruffle and enhanced lamellipodia formation of ADP-activated microglia, altering the microglia's physiological activity to vigorous migration toward damaged cells. These results suggest that LRRK2 is a negative regulator for the controlled actin dynamics in microglia.

A brief review of non-invasive brain imaging technologies and the near-infrared optical bioimaging.

Brain disorders seriously affect life quality. Therefore, non-invasive neuroimaging has received attention to monitoring and early diagnosing neural disorders to prevent their progress to a severe level. This short review briefly describes the current MRI and PET/CT techniques developed for non-invasive neuroimaging and the future direction of optical imaging techniques to achieve higher resolution and specificity using the second near-infrared (NIR-II) region of wavelength with organic molecules.

Target identification of mouse stem cell probe CDy1 as ALDH2 and Abcb1b through live-cell affinity-matrix and ABC CRISPRa library.

CDy1 is a powerful tool to distingusih embryonic stem cells for reprogramming studies and regeneration medicine. However, the stem cell selectivity mechanism of CDy1 has not been fully understood. Here, we report ALDH2 and ABCB1 as the molecular targets of CDy1, elucidated by live-cell affinity-matrix and ABC transporter CRISPRa library screening. The two unique orthogonal mechanisms provide the potential of multi-demensional cellular distinction of specific cell types.

A Near-Infrared Probe Tracks and Treats Lung Tumor Initiating Cells by Targeting HMOX2.

Tumor initiating cells (TIC) are resistant to conventional anticancer therapy and associated with metastasis and relapse in cancer. Although various TIC markers and their antibodies have been proposed, it is limited to the use of antibodies for in vivo imaging or treatment of TIC. In this study, we discovered heme oxygenase 2 (HMOX2) as a novel biomarker for TIC and developed a selective small molecule probe TiNIR (tumor initiating cell probe with near infrared). TiNIR detects and enriches the functionally active TIC in human lung tumors, and through the photoacoustic property, TiNIR also visualizes lung TIC in the patient-derived xenograft (PDX) model. Furthermore, we demonstrate that TiNIR inhibits tumor growth by blocking the function of HMOX2, resulting in significantly increased survival rates of the cancer model mice. The novel therapeutic target HMOX2 and its fluorescent ligand TiNIR will open a new path for the molecular level of lung TIC diagnosis and treatment.

Visualizing Microglia with a Fluorescence Turn-On Ugt1a7c Substrate.

Microglia, the brain-resident macrophage, are involved in brain development and contribute to the progression of neural disorders. Despite the importance of microglia, imaging of live microglia at a cellular resolution has been limited to transgenic mice. Efforts have therefore been dedicated to developing new methods for microglia detection and imaging. Using a thorough structure-activity relationships study, we developed CDr20, a high-performance fluorogenic chemical probe that enables the visualization of microglia both in vitro and in vivo. Using a genome-scale CRISPR-Cas9 knockout screen, the UDP-glucuronosyltransferase Ugt1a7c was identified as the target of CDr20. The glucuronidation of CDr20 by Ugt1a7c in microglia produces fluorescence.

Development of a Universal Fluorescent Probe for Gram-Positive Bacteria.

The rapid and sensitive classification of bacteria is the first step of bacterial community research and the treatment of infection. Herein, a fluorescent probe BacGO is presented, which shows the best universal selectivity for Gram-positive bacteria among known probes with a minimum staining procedure for sample detection and enrichment of the live bacteria. BacGO could also be used to assess of the Gram status in the bacterial community from wastewater sludge. Furthermore, BacGO could sensitively and selectively detect a Gram-positive bacterial infection, not only in vitro but also using an in vivo keratitis mouse model. BacGO provides an unprecedented research tool for the study of dynamic bacterial communities and for clinical application.

Imaging inflammation using an activated macrophage probe with Slc18b1 as the activation-selective gating target.

Activated macrophages have the potential to be ideal targets for imaging inflammation. However, probe selectivity over non-activated macrophages and probe delivery to target tissue have been challenging. Here, we report a small molecule probe specific for activated macrophages, called CDg16, and demonstrate its application to visualizing inflammatory atherosclerotic plaques in vivo. Through a systematic transporter screen using a CRISPR activation library, we identify the orphan transporter Slc18b1/SLC18B1 as the gating target of CDg16.

A Fluorescent Probe for Neural Stem/Progenitor Cells with High Differentiation Capability into Neurons.

Selection of a specific neural stem/progenitor cells (NSPCs) has attracted broad attention in regenerative medicine for neurological disorders. Here, we report a fluorescent probe, CDg13, and its application for isolating strong neurogenic NSPCs. In comparison to the NSPCs isolated by other biomarkers, CDg13-stained NSPCs showed higher capability to differentiate into neurons. Target identification revealed that the fluorescence intensity of the probe within cells is inversely proportional to the expression levels of mouse and human Abcg2 transporters. These findings suggest that low Abcg2 expression is a biomarker for neurogenic NSPCs in mouse brain. Furthermore, CDg13 can be used to isolate Abcg2low cells from heterogeneous cell populations.

Interplay between Leucine-Rich Repeat Kinase 2 (LRRK2) and p62/SQSTM-1 in Selective Autophagy.

The deposit of polyubiquitinated aggregates has been implicated in the pathophysiology of Parkinson's disease (PD), and growing evidence indicates that selective autophagy plays a critical role in the clearance of ubiquitin-positive protein aggregates by autophagosomes. The selective autophagic receptor p62/SQSTM-1, which associates directly with both ubiquitin and LC3, transports ubiquitin conjugates to autophagosomes for degradation. Leucine-rich repeat kinase 2 (LRRK2), a PD-associated protein kinase, is tightly controlled by autophagy-lysosome degradation as well as by the ubiquitin-proteasome pathway. However, little is known about the degradation of ubiquitinated LRRK2 via selective autophagy. In the present study, we found that p62/SQSTM-1 physically interacts with LRRK2 as a selective autophagic receptor. The overexpression of p62 leads to the robust degradation of LRRK2 through the autophagy-lysosome pathway. In addition, LRRK2 indirectly regulates Ser351 and Ser403 phosphorylation of p62. Of particular interest, the interaction between phosphorylated p62 and Keap1 is reduced by LRRK2 overexpression. Therefore, we propose that the interplay between LRRK2 and p62 may contribute to the pathophysiological function and homeostasis of LRRK2 protein.

Boronic Acid: A Bio-Inspired Strategy To Increase the Sensitivity and Selectivity of Fluorescent NADH Probe.

Fluorescent probes have emerged as an essential tool in the molecular recognition events in biological systems; however, due to the complex structures of certain biomolecules, it remains a challenge to design small-molecule fluorescent probes with high sensitivity and selectivity. Inspired by the enzyme-catalyzed reaction between biomolecule and probe, we present a novel combination-reaction two-step sensing strategy to improve sensitivity and selectivity. Based on this strategy, we successfully prepared a turn-on fluorescent reduced nicotinamide adenine dinucleotide (NADH) probe, in which boronic acid was introduced to bind with NADH and subsequently accelerate the sensing process. This probe shows remarkably improved sensitivity (detection limit: 0.084 µM) and selectivity to NADH in the absence of any enzymes. In order to improve the practicality, the boronic acid was further modified to change the measurement conditions from alkalescent (pH 9.5) to physiological environment (pH 7.4). Utilizing these probes, we not only accurately quantified the NADH weight in a health care product but also evaluated intracellular NADH levels in live cell imaging. Thus, these bio-inspired fluorescent probes offer excellent tools for elucidating the roles of NADH in biological systems as well as a practical strategy to develop future sensitive and selective probes for complicated biomolecules.

A Multisite-Binding Switchable Fluorescent Probe for Monitoring Mitochondrial ATP Level Fluctuation in Live Cells.

Adenosine triphosphate (ATP), commonly produced in mitochondria, is required by almost all the living organisms; thus fluorescent probes for monitoring mitochondrial ATP levels fluctuation are essential and highly desired. Herein, we report a multisite-binding switchable fluorescent probe, ATP-Red 1, which selectively and rapidly responds to intracellular concentrations of ATP. Live-cell imaging indicated that ATP-Red 1 mainly localized to mitochondria with good biocompatibility and membrane penetration. In particular, with the help of ATP-Red 1, we successfully observed not only the decreased mitochondrial ATP levels in the presence of KCN and starvation state, but also the increased mitochondrial ATP levels in the early stage of cell apoptosis. These results indicate that ATP-Red 1 is a useful tool for investigating ATP-relevant biological processes.

LRRK2 G2019S mutation attenuates microglial motility by inhibiting focal adhesion kinase.

In response to brain injury, microglia rapidly extend processes that isolate lesion sites and protect the brain from further injury. Here we report that microglia carrying a pathogenic mutation in the Parkinson's disease (PD)-associated gene, G2019S-LRRK2 (GS-Tg microglia), show retarded ADP-induced motility and delayed isolation of injury, compared with non-Tg microglia. Conversely, LRRK2 knockdown microglia are highly motile compared with control cells. In our functional assays, LRRK2 binds to focal adhesion kinase (FAK) and phosphorylates its Thr-X-Arg/Lys (TXR/K) motif(s), eventually attenuating FAK activity marked by decreased pY397 phosphorylation (pY397). GS-LRRK2 decreases the levels of pY397 in the brain, microglia and HEK cells. In addition, treatment with an inhibitor of LRRK2 kinase restores pY397 levels, decreased pTXR levels and rescued motility of GS-Tg microglia. These results collectively suggest that G2019S mutation of LRRK2 may contribute to the development of PD by inhibiting microglial response to brain injury.

High-efficiency in vitro and in vivo detection of Zn2+ by dye-assembled upconversion nanoparticles.

Development of highly sensitive and selective sensing systems of divalent zinc ion (Zn(2+)) in organisms has been a growing interest in the past decades owing to its pivotal role in cellular metabolism, apoptosis, and neurotransmission. Herein, we report the rational design and synthesis of a Zn(2+) fluorescent-based probe by assembling lanthanide-doped upconversion nanoparticles (UCNPs) with chromophores. Specifically, upconversion luminescence (UCL) can be effectively quenched by the chromophores on the surface of nanoparticles via a fluorescence resonant energy transfer (FRET) process and subsequently recovered upon the addition of Zn(2+), thus allowing for quantitative monitoring of Zn(2+). Importantly, the sensing system enables detection of Zn(2+) in real biological samples. We demonstrate that this chromophore-UCNP nanosystem is capable of implementing an efficient in vitro and in vivo detection of Zn(2+) in mouse brain slice with Alzheimer's disease and zebrafish, respectively.

Neural stem cell isolation from the whole mouse brain using the novel FABP7-binding fluorescent dye, CDr3.

Methods for the isolation of live neural stem cells from the brain are limited due to the lack of well-defined cell surface markers and tools to detect intracellular markers. To date most methods depend on the labeling of extracellular markers using antibodies, with intracellular markers remaining inaccessible in live cells. Using a novel intracellular protein FABP7 (Fatty Acid Binding Protein-7) selective fluorescent chemical probe CDr3, we have successfully isolated high FABP7 expressing cells from the embryonic and adult mouse brains. These cells are capable of forming neurospheres in culture, express neural stem cell marker genes and differentiate into neurons, astrocytes and oligodendrocytes. Characterization of cells sorted with Aldefluor or antibodies against CD133 or SSEA-1 showed that the cells isolated by CDr3 exhibit a phenotype distinct from the cells sorted with conventional methods. FABP7 labeling with CDr3 represents a novel method for rapid isolation of neural stem cells based on the expression of a single intracellular marker.

PINK1 Deficiency Enhances Inflammatory Cytokine Release from Acutely Prepared Brain Slices.

Parkinson's disease (PD) is the second most common neurodegenerative motor disease caused by degeneration of dopaminergic neurons in the substantia nigra. Because brain inflammation has been considered a risk factor for PD, we analyzed whether PTEN induced putative kinase 1 (PINK1), an autosomal recessive familial PD gene, regulates brain inflammation during injury states. Using acutely prepared cortical slices to mimic injury, we analyzed expression of the pro-inflammatory cytokines tumor necrosis factor-α, interleukin (IL)-1ß, and IL-6 at the mRNA and protein levels. Both mRNA and protein expression of these cytokines was higher at 6-24 h after slicing in PINK1 knockout (KO) slices compared to that in wild-type (WT) slices. In serial experiments to understand the signaling pathways that increase inflammatory responses in KO slices, we found that IκB degradation was enhanced but Akt phosphorylation decreased in KO slices compared to those in WT slices. In further experiments, an inhibitor of PI3K (LY294002) upstream of Akt increased expression of pro-inflammatory cytokines. Taken together, these results suggest that PINK1 deficiency enhance brain inflammation through reduced Akt activation and enhanced IκB degradation in response to brain injury.

PINK1 deficiency attenuates astrocyte proliferation through mitochondrial dysfunction, reduced AKT and increased p38 MAPK activation, and downregulation of EGFR.

PINK1 (PTEN induced putative kinase 1), a familial Parkinson's disease (PD)-related gene, is expressed in astrocytes, but little is known about its role in this cell type. Here, we found that astrocytes cultured from PINK1-knockout (KO) mice exhibit defective proliferative responses to epidermal growth factor (EGF) and fetal bovine serum. In PINK1-KO astrocytes, basal and EGF-induced p38 activation (phosphorylation) were increased whereas EGF receptor (EGFR) expression and AKT activation were decreased. p38 inhibition (SB203580) or knockdown with small interfering RNA (siRNA) rescued EGFR expression and AKT activation in PINK1-KO astrocytes. Proliferation defects in PINK1-KO astrocytes appeared to be linked to mitochondrial defects, manifesting as decreased mitochondrial mass and membrane potential, increased intracellular reactive oxygen species level, decreased glucose-uptake capacity, and decreased ATP production. Mitochondrial toxin (oligomycin) and a glucose-uptake inhibitor (phloretin) mimicked the PINK1-deficiency phenotype, decreasing astrocyte proliferation, EGFR expression and AKT activation, and increasing p38 activation. In addition, the proliferation defect in PINK1-KO astrocytes resulted in a delay in the wound healing process. Taken together, these results suggest that PINK1 deficiency causes astrocytes dysfunction, which may contribute to the development of PD due to delayed astrocytes-mediated repair of microenvironment in the brain.

Spatial and temporal correlation in progressive degeneration of neurons and astrocytes in contusion-induced spinal cord injury.

BACKGROUND: Traumatic spinal cord injury (SCI) causes acute neuronal death followed by delayed secondary neuronal damage. However, little is known about how microenvironment regulating cells such as microglia, astrocytes, and blood inflammatory cells behave in early SCI states and how they contribute to delayed neuronal death. METHODS: We analyzed the behavior of neurons and microenvironment regulating cells using a contusion-induced SCI model, examining early (3-6 h) to late times (14 d) after the injury. RESULTS: At the penumbra region close to the damaged core (P1) neurons and astrocytes underwent death in a similar spatial and temporal pattern: both neurons and astrocytes died in the medial and ventral regions of the gray matter between 12 to 24 h after SCI. Furthermore, mRNA and protein levels of transporters of glutamate (GLT-1) and potassium (Kir4.1), functional markers of astrocytes, decreased at about the times that delayed neuronal death occurred. However, at P1 region, ramified Iba-1+ resident microglia died earlier (3 to 6 h) than neurons (12 to 24 h), and at the penumbra region farther from the damaged core (P2), neurons were healthy where microglia were morphologically activated. In addition, round Iba-1/CD45-double positive monocyte-like cells appeared after neurons had died, and expressed phagocytic markers, including mannose receptors, but rarely expressed proinflammatory mediators. CONCLUSION: Loss of astrocyte function may be more critical for delayed neuronal death than microglial activation and monocyte infiltration.

Impaired inflammatory responses in murine Lrrk2-knockdown brain microglia.

LRRK2, a Parkinson's disease associated gene, is highly expressed in microglia in addition to neurons; however, its function in microglia has not been evaluated. Using Lrrk2 knockdown (Lrrk2-KD) murine microglia prepared by lentiviral-mediated transfer of Lrrk2-specific small inhibitory hairpin RNA (shRNA), we found that Lrrk2 deficiency attenuated lipopolysaccharide (LPS)-induced mRNA and/or protein expression of inducible nitric oxide synthase, TNF-α, IL-1ß and IL-6. LPS-induced phosphorylation of p38 mitogen-activated protein kinase and stimulation of NF-κB-responsive luciferase reporter activity was also decreased in Lrrk2-KD cells. Interestingly, the decrease in NF-κB transcriptional activity measured by luciferase assays appeared to reflect increased binding of the inhibitory NF-κB homodimer, p50/p50, to DNA. In LPS-responsive HEK293T cells, overexpression of the human LRRK2 pathologic, kinase-active mutant G2019S increased basal and LPS-induced levels of phosphorylated p38 and JNK, whereas wild-type and other pathologic (R1441C and G2385R) or artificial kinase-dead (D1994A) LRRK2 mutants either enhanced or did not change basal and LPS-induced p38 and JNK phosphorylation levels. However, wild-type LRRK2 and all LRRK2 mutant variants equally enhanced NF-κB transcriptional activity. Taken together, these results suggest that LRRK2 is a positive regulator of inflammation in murine microglia, and LRRK2 mutations may alter the microenvironment of the brain to favor neuroinflammation.