Inflammatory Responses Are Not Sufficient to Cause Delayed Neuronal Death in ATP-Induced Acute Brain Injury

BACKGROUND: Brain inflammation is accompanied by brain injury. However, it is controversial whether inflammatory responses are harmful or beneficial to neurons. Because many studies have been performed using cultured microglia and neurons, it has not been possible to assess the influence of multiple cell types and diverse factors that dynamically and continuously change in vivo. Furthermore, behavior of microglia and other inflammatory cells could have been overlooked since most studies have focused on neuronal death. Therefore, it is essential to analyze the precise roles of microglia and brain inflammation in the injured brain, and determine their contribution to neuronal damage in vivo from the onset of injury. METHODS AND FINDINGS: Acute neuronal damage was induced by stereotaxic injection of ATP into the substantia nigra pars compacta (SNpc) and the cortex of the rat brain. Inflammatory responses and their effects on neuronal damage were investigated by immunohistochemistry, electron microscopy, quantitative RT-PCR, and stereological counting, etc. ATP acutely caused death of microglia as well as neurons in a similar area within 3 h. We defined as the core region the area where both TH(+) and Iba-1(+) cells acutely died, and as the penumbra the area surrounding the core where Iba-1(+) cells showed activated morphology. In the penumbra region, morphologically activated microglia arranged around the injury sites. Monocytes filled the damaged core after neurons and microglia died. Interestingly, neither activated microglia nor monocytes expressed iNOS, a major neurotoxic inflammatory mediator. Monocytes rather expressed CD68, a marker of phagocytic activity. Importantly, the total number of dopaminergic neurons in the SNpc at 3 h (∼80% of that in the contralateral side) did not decrease further at 7 d. Similarly, in the cortex, ATP-induced neuron-damage area detected at 3 h did not increase for up to 7 d. CONCLUSIONS: Different cellular components (microglia, astrocytes, monocytes, and neutrophils) and different factors (proinflammatory and neurotrophic) could be produced in inflammatory processes depending on the nature of the injury. The results in this study suggest that the inflammatory responses of microglia and monocytes in response to ATP-induced acute injury could not be neurotoxic

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.

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

10.1021/ja5115248Journal of the American Chemical Society13762336-234

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

10.1016/j.scr.2013.09.002Stem Cell Research1131314-132

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-1β 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

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

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

Abstract 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.</p

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 Abcg2(low) cells from heterogeneous cell populations.112sciescopu