Efr3b is essential for social recognition by modulating the excitability of CA2 pyramidal neurons.

CA2 pyramidal neurons (PNs) are associated with social behaviors. The mechanisms, however, remain to be fully investigated. Here, we report that Efr3b, a protein essential for phospholipid metabolism at the plasma membrane, is widely expressed in the brain, especially in the hippocampal CA2/CA3 areas. To assess the functional significance of Efr3b in the brain, we generated Efr3bf/f mice and crossed them with Nestin-cre mice to delete Efr3b specifically in the brain. We find that Efr3b deficiency in the brain leads to deficits of social novelty recognition and hypoexcitability of CA2 PNs. We then knocked down the expression of Efr3b specifically in CA2 PNs of C57BL/6J mice, and our results showed that reducing Efr3b in CA2 PNs also resulted in deficits of social novelty recognition and hypoexcitability of CA2 PNs. More interestingly, restoring the expression of Efr3b in CA2 PNs enhances their excitability and improves social novelty recognition in Efr3b-deficient mice. Furthermore, direct activation of CA2 PNs with chemogenetics improves social behaviors in Efr3b-deficient mice. Together, our data suggest that Efr3b is essential for social novelty by modulating the excitability of CA2 PNs.

Hyccin/FAM126A deficiency reduces glial enrichment and axonal sheath, which are rescued by overexpression of a plasma membrane-targeting PI4KIIIα in Drosophila.

Hyccin/FAM126A mutations are linked to hypomyelination and congenital cataract disease (HCC), but whether and how Hyccin/FAM126A deficiency causes hypomyelination remains undetermined. This study shows Hyccin/FAM126A expression was necessary for the expression of other components of the PI4KIIIα complex in Drosophila. Knockdown of Hyccin/FAM126A in glia reduced the enrichment of glial cells, disrupted axonal sheaths and visual ability in the visual system, and these defects could be fully rescued by overexpressing either human FAM126A or FAM126B, and partially rescued by overexpressing a plasma membrane-targeting recombinant mouse PI4KIIIα. Additionally, PI4KIIIα knockdown in glia phenocopied Hyccin/FAM126A knockdown, and this was partially rescued by overexpressing the recombinant PI4KIIIα, but not human FAM126A or FAM126B. This study establishes an animal model of HCC and indicates that Hyccin/FAM126A plays an essential role in glial enrichment and axonal sheath in a cell-autonomous manner in the visual system via controlling the expression and stabilization of the PI4KIIIα complex at the plasma membrane.

Downregulation of RBO-PI4KIIIα Facilitates Aß42 Secretion and Ameliorates Neural Deficits in Aß42-Expressing Drosophila.

Phosphoinositides and their metabolizing enzymes are involved in Aß42 metabolism and Alzheimer's disease pathogenesis. In yeast and mammals, Eighty-five requiring 3 (EFR3), whose Drosophila homolog 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 to Drosophila PI4KIIIα, and that in an Aß42-expressing Drosophila model, 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 Aß42 Moreover, we found that RBO-PI4KIIIa downregulation increased neuronal Aß42 release and that PI4P facilitated the assembly or oligomerization of Aß42 in/on liposomes. These results indicate that RBO-PI4KIIIa downregulation facilitates neuronal Aß42 release and consequently reduces neuronal Aß42 accumulation likely via decreasing Aß42 assembly 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 STATEMENT Phosphoinositides and their metabolizing enzymes are involved in Aß42 metabolism and Alzheimer's disease pathogenesis. Here, in an Aß42-expressing Drosophila model, 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 Aß42 expression. Mechanistically, RBO-PI4KIIIα downregulation reduced neuronal Aß42 accumulation, and interestingly increased neuronal Aß42 release. This study suggests the RBO-PI4KIIIα complex as a novel therapeutic target, and PI4KIIIα inhibitors as new drug candidates.

Brain-specific ablation of Efr3a promotes adult hippocampal neurogenesis via the brain-derived neurotrophic factor pathway.

Efr3 is a newly identified plasma membrane protein and plays an important role in the phosphoinositide metabolism on the plasma membrane. However, although it is highly expressed in the brain, the functional significance of Efr3 in the brain is not clear. In the present study, we generated Efr3af/f mice and then crossed them with Nestin-Cre mice to delete Efr3a, one of the Efr3 isoforms, specifically in the brain. We found that brain-specific ablation of Efr3a promoted adult hippocampal neurogenesis by increasing survival and maturation of newborn neurons without affecting their dendritic tree morphology. Moreover, the brain-derived neurotrophic factor (BDNF)-tropomyosin-related kinase B (TrkB) signaling pathway was significantly enhanced in the hippocampus of Efr3a-deficient mice, as reflected by increased expression of BDNF, TrkB, and the downstream molecules, including phospho-MAPK and phospho-Akt. Furthermore, the number of TUNEL+ cells was decreased in the subgranular zone of dentate gyrus in Efr3a-deficient mice compared with that of control mice. Our data suggest that brain-specific deletion of Efr3a could promote adult hippocampal neurogenesis, presumably by upregulating the expression of BDNF and its receptor, TrkB, and therefore provide new insight into the roles of Efr3 in the brain.-Qian, Q., Liu, Q., Zhou, D., Pan, H., Liu, Z., He, F., Ji, S., Wang, D., Bao, W., Liu, X., Liu, Z., Zhang, H., Zhang, X., Zhang, L., Wang, M., Xu, Y., Huang, F., Luo, B., Sun B. Brain-specific ablation of Efr3a promotes adult hippocampal neurogenesis via the brain-derived neurotrophic factor pathway.

The use of MMSE and MoCA in patients with acute ischemic stroke in clinical.

BACKGROUND: The Mini-Mental State Examination (MMSE) and the Montreal Cognitive Assessment (MoCA) are brief cognitive screening tools that have been developed for the screening of patients with Mild Cognitive Impairment. METHODS: A total of 105 patients were included in this study, aged 53-89 years, with acute ischemic stroke admitted to hospital and fell into two groups: stroke patients with cognitive impairment (SCI) and controls with no cognitive impairment (n-SCI). The patient's characteristics are collected and regression analyses were performed to predict cognitive impairments. We use MMSE and MoCA assessment as prognostic indices for cognitive impairments of patient's with stroke. OBJECTIVES: Our aim was to examine the effectiveness of the MMSE and MoCA in screening cognitive impairments. MAIN RESULTS: There were significant difference among the two groups in the prevalence of diabetes mellitus (p < 0.05) and intracranial atherosclerosis (p < 0.05). A linear regression determined that the age, diabetes, intracranial atherosclerosis predicted the cognitive impairments. The ROC results for MoCA with an AUC of 0.882 and the corresponding results for MMSE show a similar AUC of 0.839. CONCLUSION: Neuropsychological performance of stroke patients was influenced by biological and demographic variables: age, diabetes and intracranial atherosclerosis. The MoCA and MMSE are both reliable assessments for the diagnosis of cognitive impairment after stroke.

Age-dependent alterations in the presynaptic active zone in a Drosophila model of Alzheimer's disease.

The accumulation of beta amyloid (Aß) can cause synaptic impairments, but the characteristics and mechanisms of the synaptic impairment induced by the accumulation of Aß in Alzheimer's disease (AD) remain unclear. In identified single neurons in a newly developed Drosophila AD model, in which Aß accumulates intraneuronally, we found an age-dependent reduction in the synaptic vesicle release probability that was associated with a decrease in the density of presynaptic calcium channel clusters and an increase in the presynaptic and postsynaptic contact length. Moreover, these alterations occurred in the absence of presynaptic bouton loss. In addition, we found that Aß expression also produced an age-dependent decrease in the amount of Bruchpilot (Brp), which plays an important role in controlling Ca(2+) channel clustering and synaptic vesicle release in the presynaptic active zone. Our study indicates that the chronic accumulation of intraneuronal Aß can induce functional and structural changes in the presynaptic active zone prior to a loss of presynaptic buttons in the same neuron.

Effects of tau on Aß-induced synaptic damage in a Drosophila model of Alzheimer's disease.

OBJECTIVES: The etiology and pathologic mechanism underlying Alzheimer's disease (AD) are not clear. This study determined the effects of tau on amyloid-beta peptide(Aß)-induced synaptic damages in a Drosophila model of AD. METHODS: Galactose-regulated upstream promoter element 4(Gal4) and an upstream active sequence system was used to establish four kinds of Aß transgenic Drosophila models of AD. Behavioral evaluation and immunohistochemical localization were performed in Aß transgenic Drosophila models. Tau mutants were introduced into arctic mutant Aß1-42 (arctic mutant Aß [Aßarc]) Drosophila. The P{Gal4}A307 Drosophila strain was used as a control group; 12 strains were obtained to determine the effects of tau with or without Aßarc. Electrophysiologic records of the tau mutant groups were created. RESULTS: The flight and crawling ability of Aß transgenic Drosophila were gradually weakened compared to the control group, and the life span was significantly shorter than the control group. Aß was specifically expressed in the Drosophila giant fiber pathway and further accumulated in neuronal cell bodies based on immunohistochemistry. The percentage of the excitatory junctional potential (EJP) response in transgenic Drosophila expressing Aßarc was significantly decreased, which was approximately 40% lower than the control group. The tau deletion mutation alleviated the synaptic transmission disorder caused by Aß and improved the viability of Drosophila. CONCLUSION: The tau deletion mutation significantly improved the synaptic damage caused by Aß, and tau protein played an indispensable role in the synaptic dysfunction caused by Aß, suggesting that Aß and tau have close interactions in the pathogenesis of AD.

Expression of beta-amyloid induced age-dependent presynaptic and axonal changes in Drosophila.

Alzheimer's disease (AD) is attributable to synapse dysfunction and loss, but the nature and progression of the presynaptic structural and functional changes in AD are essentially unknown. We expressed wild-type or arctic form of beta amyloid(1-42) (Abeta) in a small group of neurons in the adult fly and performed extensive time course analysis of the function and structure of both axon and presynaptic terminals at the identified single-neuron level. Abeta accumulated intracellularly and induced a range of age-dependent changes, including depletion of presynaptic mitochondria, slowdown of bi-directional transports of axonal mitochondria, decreased synaptic vesicles, increased large vacuoles, and elevated synaptic fatigue. These structural and functional synaptic changes correlated with age-dependent deficit in motor behavior. All these alterations were accelerated in flies expressing the arctic form of Abeta. The depletion of presynaptic mitochondria was the earliest detected phenotype and was not caused by the change in axonal transport of mitochondria. Moreover, axonal mitochondria exhibited a dramatic reduction in number but a significant increase in size in aged Abeta-expressing flies, indicating a global depletion of mitochondria in the neuron and an impairment of mitochondria fission. These results suggest that Abeta accumulation depletes presynaptic and axonal mitochondria, leading to other presynaptic deficits.

Overexpression of human MRP1 in neurons causes resistance to antiepileptic drugs in Drosophila seizure mutants.

Multidrug resistance-associated protein 1 (MRP1), an efflux multidrug transporter, was shown to be elevated in both glia and neurons in seizure focus in refractory epilepsy patients. Up-regulation of MRP1 and other multidrug transporters in perivascular astrocytes was suggested to cause resistance to antiepileptic drugs (AEDs) by reducing the concentration of AEDs at the epileptogenic areas. However, it is not known whether the up-regulation of MRP1 in neurons can cause resistance to AEDs, such as sodium phenytoin (PHT) and valproic acid (VPA). PHT inhibits voltage-gated sodium channel (VGSC) by occluding it, but whether PHT enters the channel through its inner or outer pore is not known. The authors overexpressed human MRP1 protein only in neurons in a Drosophila genetic seizure model, bang senseless (bss) mutants. The authors found that overexpression of MRP1 blocked the attenuation of the seizure behavior of bss mutants by acute and chronic application of PHT, and by chronic application of VPA. Conversely, overexpression of MRP1 in neurons increased the tolerance of bss flies to high-dosage PHT and VPA. Thus, up-regulation of MRP1 expression only in neurons causes resistance to AED in seizure flies. Moreover, the current data suggest that PHT enters VGSC through its inner pore.

Inhibition of phosphatidylinositol kinase-III alpha induces or facilitates lysosome exocytosis from microglia.

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.

Altered Expression Profile of Phosphatidylinositols in Erythrocytes of Alzheimer's Disease and Amnestic Mild Cognitive Impairment Patients.

BACKGROUND: Studies have demonstrated that the levels of phospholipids, including phosphatidylinositols (PIs), were decreased in Alzheimer's disease (AD) brain, presenting as a potential biomarker for AD. The plasma phospholipids levels have also been discovered to predict the conversion of cognitively normal elderly adults to amnestic mild cognitive impairment (aMCI) or demented patients. OBJECTIVE: To investigate the expression profile of PIs in erythrocytes of AD and aMCI patients, which would serve as a blood-based method to distinguish AD and aMCI patients from normal controls (NC). METHODS: In this study, we used anion-exchange high-performance liquid chromatography to analyze PIs alterations in erythrocytes from a total of 86 prospectively recruited subjects (including 24 NC, 21 aMCI patients, and 41 AD patients). RESULTS: We found that the levels of PI40 : 4, PI3/5P, and PI(3,4)P2 in aMCI patients, and the levels of PI4P, PI(3,4)P2, and PI3/5P in AD patients were significantly decreased compared to NC. The changed expression profile of PIs could effectively discriminate AD and aMCI patients from NC (AUC = 0.964, 0.938, respectively). CONCLUSION: The altered expression profile of erythrocytes PIs might be a potential blood-based biomarker for AD and aMCI. This alteration of PIs probably reflected the impaired deformability and oxygen-carrying capacity of erythrocytes in AD and aMCI patients.

Amyloid ß oligomers suppress excitatory transmitter release via presynaptic depletion of phosphatidylinositol-4,5-bisphosphate.

Amyloid ß (Aß) 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 Aß production. High nanomolar synthetic oligomeric Aß42 also suppresses Pr at the SC-CA1 synapse in wild-type mice. This Aß-induced suppression of Pr is mainly due to an mGluR5-mediated depletion of phosphatidylinositol-4,5-bisphosphate (PIP2) in axons. Selectively inhibiting Aß-induced PIP2 hydrolysis in the CA3 region of the hippocampus strongly prevents oligomeric Aß-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 Aß induces early synaptic deficits in AD.

Rolling blackout, a newly identified PIP2-DAG pathway lipase required for Drosophila phototransduction.

The rolling blackout (rbo) gene encodes an integral plasma membrane lipase required for Drosophila phototransduction. Photoreceptors are enriched for the RBO protein, and temperature-sensitive rbo mutants show reversible elimination of phototransduction within minutes, demonstrating an acute requirement for the protein. The block is activity dependent, indicating that the action of RBO is use dependent. Conditional rbo mutants show activity-dependent depletion of diacylglycerol and concomitant accumulation of phosphatidylinositol phosphate and phosphatidylinositol 4,5-bisphosphate within minutes of induction, suggesting rapid downregulation of phospholipase C (PLC) activity. The RBO requirement identifies an essential regulatory step in G-protein-coupled, PLC-dependent inositol lipid signaling mediating activation of TRP and TRPL channels during phototransduction.

An Efficient and Reliable Assay for Investigating the Effects of Hypoxia/Anoxia on Drosophila.

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 ~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.

An Improved Method for Collection of Cerebrospinal Fluid from Anesthetized Mice.

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.

TTC7 and Hyccin Regulate Neuronal Aß42 Accumulation and its Associated Neural Deficits in Aß42-Expressing Drosophila.

Neuronal amyloid-ß (Aß) accumulation plays an important role in the pathogenesis of Alzheimer's disease (AD). The conformation and toxicity of Aß are regulated by lipids on the plasma membrane. Previously, we found downregulation of Rolling Blackout (RBO) or phosphatidylinositol-4-kinase type IIIα (PI4KIIIα) reduces neuronal Aß accumulation and associated neural deficits in a Drosophila model expressing Aß42. In mammals, the homologs of RBO and PI4KIIIα were reported to form a plasma membrane-localized complex with a scaffold protein TTC7 and cytosolic protein Hyccin/FAM126A to tightly control the plasmalemmal level of phosphatidylinositol-4-phosphate. Here, we show genetic downregulation of Drosophila TTC7 and Hyccin also reduces neuronal Aß accumulation and associated synaptic and motor defects as well as premature death in Aß42-expressing flies, while overexpression of TTC7 and Hyccin produced the opposite effect. These results, together with our previous study, demonstrate that RBO/TTC7/PI4KIIIα/Hyccin regulate neuronal Aß accumulation and associated neural deficits in the Drosophila model, further supporting the RBO/Efr3-PI4KIIIα complex as a potential therapeutic target for AD.

Rolling blackout is required for synaptic vesicle exocytosis.

Rolling blackout (RBO) is a putative transmembrane lipase required for phospholipase C-dependent phosphatidylinositol 4,5-bisphosphate-diacylglycerol signaling in Drosophila neurons. Conditional temperature-sensitive (TS) rbo mutants display complete, reversible paralysis within minutes, demonstrating that RBO is acutely required for movement. RBO protein is localized predominantly in presynaptic boutons at neuromuscular junction (NMJ) synapses and throughout central synaptic neuropil, and rbo TS mutants display a complete, reversible block of both central and peripheral synaptic transmission within minutes. This phenotype appears limited to adults, because larval NMJs do not manifest the acute blockade. Electron microscopy of adult rbo TS mutant boutons reveals an increase in total synaptic vesicle (SV) content, with a concomitant shrinkage of presynaptic bouton size and an accumulation of docked SVs at presynaptic active zones within minutes. Genetic tests reveal a synergistic interaction between rbo and syntaxin1A TS mutants, suggesting that RBO is required in the mechanism of N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE)-mediated SV exocytosis, or in a parallel pathway necessary for SV fusion. The rbo TS mutation does not detectably alter SNARE complex assembly, suggesting a downstream requirement in SV fusion. We conclude that RBO plays an essential role in neurotransmitter release, downstream of SV docking, likely mediating SV fusion.

An Automated Rapid Iterative Negative Geotaxis Assay for Analyzing Adult Climbing Behavior in a Drosophila Model of Neurodegeneration.

Neurodegenerative diseases are frequently associated with a progressive loss of movement ability, reduced life span, and age-dependent neurodegeneration. To understand the mechanism of these cellular events, and their causal relationships with each other, Drosophila melanogaster, with its sophisticated genetic tools and diverse behavioral features, are used as disease models for assessing neurodegenerative phenotypes. Here we describe a high-throughput method to analyze Drosophila adult negative geotaxis behavior, as an indication for possible motor defects associated with neurodegeneration. An automated machine is designed and developed to drive fly synchronization using an initial electric impulse, later allowing the recording of negative geotaxis behavior over a course of secs to mins. Images from the digitally recorded video are then processed with the self-designed RflyDetection software for statistical data manipulation. Different from the manually controlled negative geotaxis assay based on single fly, this precise, fast, and high-throughput protocol allows data acquisition from more than hundreds of flies simultaneously, providing an efficient approach to advance our understanding in the underlying mechanism of locomotor deficits associated with neurodegeneration.

SH2B1 is Involved in the Accumulation of Amyloid-ß42 in Alzheimer's Disease.

Alzheimer's disease (AD) is characterized by deficits in learning and memory abilities, as well as pathological changes of amyloid-ß (Aß) plaque and neurofibrillary tangle formation in the brain. Insulin has been identified as a modulator of the neuronal pathways involved in learning and memory, and is also implicated as a modulator of Aß and tau metabolism. Disrupted insulin signaling pathways are evident in AD patients and it is understood that type 2 diabetes can increase the risk of developing AD, suggesting a possible link between metabolic disorders and neurodegeneration. SH2B1 is a key protein in the insulin signaling pathway involved in regulating the activity of the insulin receptor. To further identify the role of the insulin signaling pathway in the pathology of AD, SH2B (dSH2B homologue in flies) in neurons was partially knocked out or overexpressed in an AD Drosophila model expressing Aß42. Partial knockout of neuronal SH2B in the Aß42-expressing Drosophila had a detrimental effect on mobility and neurotransmission, and increased levels and intraneuronal accumulation of Aß42, as assessed by ELISA and immunostaining. Alternatively, partial overexpression of neuronal SH2B in the Aß42-expressing Drosophila improved lifespan, mobility, and neurotransmission, as well as decreased levels and intraneuronal accumulation of Aß42. Thus, SH2B1 may be an upstream modulator of Aß metabolism, acting to inhibit Aß accumulation, and has a role in the pathogenesis of AD. SH2B1 may therefore have potential as a therapeutic target for this common form of dementia.