Huifang Lou

Microglial migration mediated by ATP-induced ATP release from lysosomes

Microglia are highly motile cells that act as the main form of active immune defense in the central nervous system. Attracted by factors released from damaged cells, microglia are recruited towards the damaged or infected site, where they are involved in degenerative and regenerative responses and phagocytotic clearance of cell debris. ATP release from damaged neural tissues has been suggested to mediate the rapid extension of microglial process towards the site of injury. However, the mechanisms of the long-range migration of microglia remain to be clarified. Here, we found that lysosomes in microglia contain abundant ATP and exhibit Ca2+-dependent exocytosis in response to various stimuli. By establishing an efficient in vitro chemotaxis assay, we demonstrated that endogenously-released ATP from microglia triggered by local microinjection of ATPγS is critical for the long-range chemotaxis of microglia, a response that was significantly inhibited in microglia treated with an agent inducing lysosome osmodialysis or in cells derived from mice deficient in Rab 27a (ashen mice), a small GTPase required for the trafficking and exocytosis of secretory lysosomes. These results suggest that microglia respond to extracellular ATP by releasing ATP themselves through lysosomal exocytosis, thereby providing a positive feedback mechanism to generate a long-range extracellular signal for attracting distant microglia to migrate towards and accumulate at the site of injury.

P2Y4 Receptor-Mediated Pinocytosis Contributes to Amyloid Beta-Induced Self-Uptake by Microglia

ABSTRACT Brain disturbances, like injuries or aberrant protein deposits, evoke nucleotide release or leakage from cells, leading to microglial chemotaxis and ingestion. Recent studies have identified P2Y12 purinergic receptors as triggers for microglial chemotaxis and P2Y6 receptors as mediators for phagocytosis. However, pinocytosis, known as the internalization of fluid-phase materials, has received much less attention. We found that ATP efficiently triggered pinocytosis in microglia. Pharmacological analysis and knockdown experiments demonstrated the involvement of P2Y4 receptors and the phosphatidylinositol 3-kinase/Akt cascade in the nucleotide-induced pinocytosis. Further evidence indicated that soluble amyloid beta peptide 1-42 induced self-uptake in microglia through pinocytosis, a process involving activation of P2Y4 receptors by autocrine ATP signaling. Our results demonstrate a previously unknown function of ATP as a “drink me” signal for microglia and P2Y4 receptors as a potential therapeutic target for the treatment of Alzheimers disease.

Astrocytes contribute to synapse elimination via type 2 inositol 1,4,5-trisphosphate receptor-dependent release of ATP

Selective elimination of unwanted synapses is vital for the precise formation of neuronal circuits during development, but the underlying mechanisms remain unclear. Using inositol 1,4,5-trisphosphate receptor type 2 knockout (Itpr2−/−) mice to specifically disturb somatic Ca2+ signaling in astrocytes, we showed that developmental elimination of the ventral posteromedial nucleus relay synapse was impaired. Interestingly, intracerebroventricular injection of ATP, but not adenosine, rescued the deficit in synapse elimination in Itpr2−/− mice. Further studies showed that developmental synapse elimination was also impaired in P2ry1−/− mice and was not rescued by ATP, indicating a possible role of purinergic signaling. This hypothesis was confirmed by MRS-2365, a selective P2Y1 agonist, could also rescue the deficient of synapse elimination in Itpr2−/− mice. Our results uncovered a novel mechanism suggesting that astrocytes release ATP in an IP3R2-dependent manner to regulate synapse elimination. DOI: http://dx.doi.org/10.7554/eLife.15043.001

Laterodorsal tegmentum interneuron subtypes oppositely regulate olfactory cue-induced innate fear.

Innate fear has a critical role in survival of animals. Unlike conditioned fear, the neuronal circuitry underlying innate fear is largely unknown. We found that the laterodorsal tegmentum (LDT) and lateral habenula (LHb) are specifically activated by the mouse predator odorant trimethylthiazoline (TMT). Using optogenetics to selectively stimulate GABAergic neurons in the LDT immediately produced fear-like responses (freezing, accelerated heart rate and increased serum corticosterone), whereas prolonged stimulation caused anxiety-like behaviors. Notably, although selective stimulation of parvalbumin (PV)-positive interneurons similarly induced fear-like responses, stimulation of somatostatin-positive interneurons or inhibition of PV neurons in the LDT suppressed TMT-induced fear-like responses without affecting conditioned fear. Finally, activation of LHb glutamatergic inputs to LDT interneurons was sufficient to generate fear-like responses. Thus, the LHb-LDT pathway is important for regulating olfactory cue–induced innate fear. Our results provide a potential target for therapeutic intervention for anxiety disorder.

β-Catenin is critical for cerebellar foliation and lamination

The cerebellum has a conserved foliation pattern and a well-organized layered structure. The process of foliation and lamination begins around birth. β-catenin is a downstream molecule of Wnt signaling pathway, which plays a critical role in tissue organization. Lack of β-catenin at early embryonic stages leads to either prenatal or neonatal death, therefore it has been difficult to resolve its role in cerebellar foliation and lamination. Here we used GFAP-Cre to ablate β-catenin in neuronal cells of the cerebellum after embryonic day 12.5, and found an unexpected role of β-catenin in determination of the foliation pattern. In the mutant mice, the positions of fissure formation were changed, and the meninges were improperly incorporated into fissures. At later stages, some lobules were formed by Purkinje cells remaining in deep regions of the cerebellum and the laminar structure was dramatically altered. Our results suggest that β-catenin is critical for cerebellar foliation and lamination. We also found a non cell-autonomous role of β-catenin in some developmental properties of major cerebellar cell types during specific stages.

A Novel Size-Based Sorting Mechanism of Pinocytic Luminal Cargoes in Microglia

Microglia are the resident immune cells in the CNS and play diverse roles in the maintenance of CNS homeostasis. Recent studies have shown that microglia continually survey the CNS microenvironment and scavenge cell debris and aberrant proteins by phagocytosis and pinocytosis, and that reactive microglia are capable to present antigens to T cells and initiate immune responses. However, how microglia process the endocytosed contents and evoke an immune response remain unclear. Here we report that a size-dependent selective transport of small soluble contents from the pinosomal lumen into lysosomes is critical for the antigen processing in microglia. Using fluorescent probes and water-soluble magnetic nanobeads of defined sizes, we showed in cultured rodent microglia, and in a cell-free reconstructed system that pinocytosed proteins become degraded immediately following pinocytosis and the resulting peptides are selectively delivered to major histocompatibility complex class II (MHC-II) containing lysosomes, whereas undegraded proteins are retained in the pinosomal lumen. This early size-based sorting of pinosomal contents relied on the formation of transient tunnel between pinosomes and lysosomes in a Rab7- and dynamin II-dependent manner, which allowed the small contents to pass through but restricted large ones. Inhibition of the size-based sorting markedly reduced proliferation and cytokine release of cocultured CD4+ T cells, indicating that the size-based sorting is required for efficient antigen presentation by microglial cells. Together, these findings reveal a novel early sorting mechanism for pinosomal luminal contents in microglial cells, which may explain how microglia efficiently process protein antigens and evoke an immune response.

Alternative Splicing of Disabled-1 Controls Multipolar-to-Bipolar Transition of Migrating Neurons in the Neocortex

Abstract Multipolar‐to‐bipolar transition (MBT) is crucial for the neuronal migration and positioning in the neocortex. Reelin‐Disabled‐1 (Dab1) signaling plays a pivotal role in neuronal migration, yet how Dab1 coordinatively regulates downstream molecules to affect MBT remains unclear. We have previously found that alternative splicing produces multiple Dab1 isoforms with different tyrosine motifs and differential ability to recruit downstream effectors. Here, we report that splicing of Dab1 exons 7 and 8 and 9bc dynamically regulates the inclusion and activities of Dab1 tyrosine motifs in the neocortex. By in utero electroporation, we show that expression of Dab1 isoforms missing exons 7 and 8 or retaining exons 9bc in WT neurons resulted in neuronal migration defects with attenuated Dab1 tyrosine phosphorylation, disrupted leading process extension, and disorientated multipolar neurons in the multipolar accumulation zone. Introducing the canonical Dab1 form, but not those missing exons 7 and 8 or retaining exons 9bc, into Dab1‐deficient neurons promoted MBT and rescued neuronal migration defects, suggesting that alternative splicing of Dab1 modulates the tyrosine motif switch and mediates MBT of cortical neurons. Our study reveals a critical mechanism by which Dab1 alternative splicing coordinately controls MBT and neuronal migration in a spatiotemporal manner.

Na+–Ca2+ exchanger mediates ChR2-induced [Ca2+]i elevation in astrocytes

Intracellular Ca(2+) elevation resulting from different Ca(2+) pathways may play different roles in astrocyte functions. Channelrhodopsin-2 (ChR2), a light-gated cation channel, has been used to selectively stimulate astrocytes by inducing intracellular Ca(2+) ([Ca(2+)]i) elevation, but the exact underlying mechanism is still unclear. We found that in the absence of extracellular Ca(2+), light stimulation failed to induce [Ca(2+)]i elevation in astrocytes expressed ChR2. Pharmacological experiments excluded the involvement of Ca(2+)-induced Ca(2+) release from intracellular stores. Further experiments demonstrated that the ChR2-induced [Ca(2+)]i elevation was mainly mediated by reversal of the Na(+)-Ca(2+) exchanger following Na(+) influx through ChR2 channels. Since intracellular Na(+) homeostasis plays important roles in astrocytes, including the modulation of [Ca(2+)]i, neurotransmitter uptake and cell metabolism, our results indicate that ChR2 is a good candidate which could be used for mimicking the intracellular Na(+) disturbance in astrocytes that occurs in various physiological and pathological processes including the uptake of neurotransmitters and ischemia, as well as the activities of various cation channels, ion exchangers, and pumps.

Analysis of microglial migration by a micropipette assay.

Microglial cells have important roles in maintaining brain homeostasis, and they are implicated in multiple brain diseases. There is currently interest in investigating microglial migration that results in cell accumulation at focal sites of injury. Here we describe a protocol for rapidly triggering and monitoring microglial migration by using a micropipette assay. This protocol is an adaptation of the axon turning assay using microglial cells. Chemoattractants released from the micropipette tip produce a chemotactic gradient that induces robust microglial migration. In combination with microscopic imaging, this assay allows simultaneous recording of cell movement and subcellular compartment trafficking, along with quantitative analysis. The actual handling time for the assay takes ∼2–3 h in total. The protocol is simple, inexpensive and convenient to set up, and it can be adopted to examine cell migration in multiple cell types, including cancer cells with a wide range of chemical signals.

A Novel Method to Image Macropinocytosis in vivo

Here we described an experimental protocol for in vivo imaging of macropinocytosis and subsequent intracellular events. By microinjection, we delivered fluorescence dextrans together with or without ATPγS into transparent Drosophila melanogaster embryos. Using a confocal microscope for live imaging, we monitored the generation of dextran-positive macropinosomes and subsequent intracellular events. Our protocol provides a continent and reliable way for investigating macropinocytosis and its underlying mechanisms, especially when combined with genetic strategies.