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.

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.

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.

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 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.

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.

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.

Automated rapid iterative negative geotaxis assay and its use in a genetic screen for modifiers of Aß(42)-induced locomotor decline in Drosophila.

The negative-geotaxis climbing assay is used to efficiently study aging and neurodegeneration in Drosophila. To make it suitable for large-scale study, a method called the rapid iterative negative geotaxis (RING) assay has been established by simultaneously photographing the climbing of multiple groups of flies when they are manually tapped down in test tubes. Here, we automated the assay by using a well-controlled electric motor to drive the tapping, and a homemade program to analyze the climbing height of flies. Using the automated RING (aRING) assay, we found that the climbing ability of a strain of wild-type flies, males in particular, declined rapidly before day 21 after eclosion, but slowly from day 21 to 35. We also found that the expression of arctic mutant Aß42 accelerated the age-dependent decline in the climbing ability of flies. Moreover, using aRING, we examined the effect of third chromosome deficiencies on the accelerated locomotor decline in Aß42-expressing flies, and isolated 7 suppressors and 15 enhancers.

Intraneuronal accumulation of Aß42 induces age-dependent slowing of neuronal transmission in Drosophila.

Beta amyloid (Aß42)-induced dysfunction and loss of synapses are believed to be major underlying mechanisms for the progressive loss of learning and memory abilities in Alzheimer's disease (AD). The vast majority of investigations on AD-related synaptic impairment focus on synaptic plasticity, especially the decline of long-term potentiation of synaptic transmission caused by extracellular Aß42. Changes in other aspects of synaptic and neuronal functions are less studied or undiscovered. Here, we report that intraneuronal accumulation of Aß42 induced an age-dependent slowing of neuronal transmission along pathways involving multiple synapses.

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.

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.

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.

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.

Hippocampal theta state in relation to formalin nociception.

In the present study using extracellular electrophysiological recording techniques, we explored the temporal characteristics of hippocampal theta activation in relation to formalin nociception. Results indicate that, compared to hind paw injection of saline, formalin injection in behaving rat evoked biphasic increase in duration of dorsal CA1 theta. Such an increase broadly paralleled animal biphasic behavioral activation, especially lick and moment-to-moment agitated behaviors. Correspondingly, theta-modulated cell firing was observed following formalin injection in anesthetized rat. The formalin-induced theta activation in behaving rat was most marked during peak of theta activation in the 2nd theta state (11-40 min post-injection) comprising 73% of the time in the 5 min block. An increase in theta peak frequency was also observed with respect to pre-injection control. However, the peak of theta in the 2nd theta state mostly preceded the peak of lick and flinch of the affected paw. In the 41-60 min, following formalin injection while the animals displayed robust nociceptive flinching and lifting, the theta activity approached control levels. Furthermore, the theta peak frequency at peak of theta was higher than the corresponding values of sustained theta observed in correlation with the nociceptive behaviors; in contrast, high frequency theta rhythm was observed during formalin-induced other moment-to-moment agitated behaviors. These findings favor the notion that in the formalin model the theta state of the hippocampus reflects a neural drive that is dissociated from the duration of nociceptive experience and is not selective to the typical nociceptive indices of lick, flinch, and lift of the injured paw.

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.