Fu-De Huang

Besides degradation, lysosomes can also carry molecules for secretion out of the cell, such as ATP and cytokines, during unconventional secretion. Phosphatidylinositols and their metabolizing enzymes play important roles in the sorting and trafficking of lysosomal materials through the trans-Golgi network. The present study reveals a new function of phosphatidylinositol kinase-III alpha in the 'kiss-and-run' fusion of lysosomes at the plasma membrane to release ATP from microglia.

Amyloid β (Aβ) oligomer-induced aberrant neurotransmitter release is proposed to be a crucial early event leading to synapse dysfunction in Alzheimer’s disease (AD). In the present study, we report that the release probability (Pr) at the synapse between the Schaffer collateral (SC) and CA1 pyramidal neurons is significantly reduced at an early stage in mouse models of AD with elevated Aβ production. High nanomolar synthetic oligomeric Aβ₄₂ also suppresses Pr at the SC-CA1 synapse in wild-type mice. This Aβ-induced suppression of Pr is mainly due to an mGluR5-mediated depletion of phosphatidylinositol-4,5-bisphosphate (PIP₂) in axons. Selectively inhibiting Aβ-induced PIP₂ hydrolysis in the CA3 region of the hippocampus strongly prevents oligomeric Aβ-induced suppression of Pr at the SC-CA1 synapse and rescues synaptic and spatial learning and memory deficits in APP/PS1 mice. These results first reveal the presynaptic mGluR5-PIP₂ pathway whereby oligomeric Aβ induces early synaptic deficits in AD.

Stroke is a leading cause of death worldwide. Up to one thousand potential drugs or interventions have been developed to treat stroke, out of which similar to 160 have gone on to clinical trials. However, none of them has been successful. New insights into the molecular and cellular mechanisms of ischemia-induced injury are needed for discovering new therapeutic targets. Recently, Drosophila has been used to uncover new hypoxia-related genes. In this study, we describe an efficient and reliable assay with a sophisticated apparatus for studying the effects of oxygen deprivation on flies. Using this assay, wild-type flies were exposed to an anoxic environment for varying lengths of time, then the cumulative death rate and mobility recovery were systematically analyzed. We found that anoxia for over one hour caused lethality. The cumulative death rate on day 5 after anoxia was linearly and positively correlated with the duration of anoxia, and reached 50% when the duration was 2.5 h-3 h. We also found that the mobility recovery in normoxia was slow, as the climbing ability remained largely unchanged 4 h-6 h after 2.5-h of anoxia. We suggest that 2.5 h-3 h of anoxia and 4 h-6 h of recovery before mobility analysis are appropriate for future use of the anoxia assay.

The cerebrospinal fluid (CSF) is a valuable body fluid for analysis in neuroscience research. It is one of the fluids in closest contact with the central nervous system and thus, can be used to analyze the diseased state of the brain or spinal cord without directly accessing these tissues. However, in mice it is difficult to obtain from the cisterna magna due to its closeness to blood vessels, which often contaminate samples. The area for CSF collection in mice is also difficult to dissect to and often only small samples are obtained (maximum of 5-7 µL or less). This protocol describes in detail a technique that improves on current methods of collection to minimize contamination from blood and allow for the abundant collection of CSF (on average 10-15 µL can be collected). This technique can be used with other dissection methods for tissue collection from mice, as it does not impact any tissues during CSF extraction. Thus, the brain and spinal cord are not affected with this technique and remain intact. With greater CSF sample collection and purity, more analyses can be used with this fluid to further aid neuroscience research and better understand diseases affecting the brain and spinal cord.

Phosphoinositides and their metabolizing enzymes are involved in Aβ₄₂metabolism and Alzheimer's disease pathogenesis. In yeast and mammals, Eighty-five requiring 3 (EFR3), whoseDrosophilahomolog is Rolling Blackout (RBO), forms a plasma membrane-localized protein complex with phosphatidylinositol-4-kinase Type IIIα (PI4KIIIα) and a scaffold protein to tightly control the level of plasmalemmal phosphatidylinositol-4-phosphate (PI₄P). Here, we report that RBO binds toDrosophilaPI4KIIIα, and that in an Aβ₄₂-expressingDrosophilamodel, separate genetic reduction of PI4KIIIα and RBO, or pharmacological inhibition of PI4KIIIα ameliorated synaptic transmission deficit, climbing ability decline, premature death, and reduced neuronal accumulation of Aβ₄₂. Moreover, we found that RBO-PI4KIIIa downregulation increased neuronal Aβ₄₂release and that PI4P facilitated the assembly or oligomerization of Aβ₄₂in/on liposomes. These results indicate that RBO-PI4KIIIa downregulation facilitates neuronal Aβ₄₂release and consequently reduces neuronal Aβ₄₂accumulation likely via decreasing Aβ₄₂assembly in/on plasma membrane. This study suggests the RBO-PI4KIIIα complex as a potential therapeutic target and PI4KIIIα inhibitors as drug candidates for Alzheimer's disease treatment.SIGNIFICANCE STATEMENTPhosphoinositides and their metabolizing enzymes are involved in Aβ₄₂metabolism and Alzheimer's disease pathogenesis. Here, in an Aβ₄₂-expressingDrosophilamodel, we discovered and studied the beneficial role of downregulating RBO or its interacting protein PI4KIIIα—a protein that tightly controls the plasmalemmal level of PI₄P—against the defects caused by Aβ₄₂expression. Mechanistically, RBO-PI4KIIIα downregulation reduced neuronal Aβ₄₂accumulation, and interestingly increased neuronal Aβ₄₂release. This study suggests the RBO-PI4KIIIα complex as a novel therapeutic target, and PI4KIIIα inhibitors as new drug candidates.

Sensorineural hearing loss (SNHL) is caused by an irreversible impairment of cochlear hair cells and subsequent progressive degeneration of spiral ganglion neurons (SGNs). Eighty-five requiring 3 (Efr3) is a plasma membrane protein conserved from yeast to human, and knockout of Efr3a was reported to facilitate the survival of hippocampal newborn neurons in adult mice. Previously, we found Efr3a expression in the auditory neural pathway is upregulated soon after the destruction of hair cells. Here we conducted a time-course analysis of drug-caused damage to hearing ability, hair cells and SGNs in Efr3a knocking down mice (Efr3a-/+, Efr3a KD) and their wild type littermates. Functional examination showed that both groups of mice suffered from serious hearing loss with a higher level of severity in WT mice. Morphologic observation following drugs administration showed that both WT and Efr3a KD mice went through progressive loss of hair cells and SGNs, in association with degenerative changes in the perikarya, intracellular organells, cell body conformation in SGNs, and the changes of SGNs in WT mice were more severe than in Efr3a KD mice. These beneficial effects of Efr3a KD could be ascribed to an increase in the expression of some neurotrophic factors and their receptors in Efr3a KD mice. Our results indicate that Efr3a insufficiency suppresses drug-caused SNHL neurodegeneration in association with an increase in the expression of some neurotrophic factors and their receptors, which may be targeted in the treatment of neurodegeneration.