AIMP2 accumulation in brain leads to cognitive deficits and blood secretion in Parkinson's disease.

BACKGROUND: Propagation of neuronal α-synuclein aggregate pathology to the cortex and hippocampus correlates with cognitive impairment in Parkinson's disease (PD) dementia and dementia with Lewy body disease. Previously, we showed accumulation of the parkin substrate aminoacyl-tRNA synthetase interacting multifunctional protein-2 (AIMP2) in the temporal lobe of postmortem brains of patients with advanced PD. However, the potential pathological role of AIMP2 accumulation in the cognitive dysfunction of patients with PD remains unknown. METHODS: We performed immunofluorescence imaging to examine cellular distribution and accumulation of AIMP2 in brains of conditional AIMP2 transgenic mice and postmortem PD patients. The pathological role of AIMP2 was investigated in the AIMP2 transgenic mice by assessing Nissl-stained neuron counting in the hippocampal area and Barnes maze to determine cognitive functions. Potential secretion and cellular uptake of AIMP2 was monitored by dot blot analysis and immunofluorescence. The utility of AIMP2 as a new PD biomarker was evaluated by dot blot and ELISA measurement of plasma AIMP2 collected from PD patients and healthy control followed by ROC curve analysis. RESULTS: We demonstrated that AIMP2 is toxic to the dentate gyrus neurons of the hippocampus and that conditional AIMP2 transgenic mice develop progressive cognitive impairment. Moreover, we found that neuronal AIMP2 expression levels correlated with the brain endothelial expression of AIMP2 in both AIMP2 transgenic mice and in the postmortem brains of patients with PD. AIMP2, when accumulated, was released from the neuronal cell line SH-SY5Y cells. Secreted AIMP2 was taken up by human umbilical vein endothelial cells. Consistent with the fact that AIMP2 can be released into the extracellular space, we showed that AIMP2 transgenic mice have higher levels of plasma AIMP2. Finally, ELISA-based assessment of AIMP2 in plasma samples from patients with PD and controls, and subsequent ROC curve analysis proved that high plasma AIMP2 expression could serve as a reliable molecular biomarker for PD diagnosis. CONCLUSIONS: The pathological role in the hippocampus and the cell-to-cell transmissibility of AIMP2 provide new therapeutic avenues for PD treatment, and plasma AIMP2 combined with α-synuclein may improve the accuracy of PD diagnosis in the early stages.

Alteration of Neural Network and Hippocampal Slice Activation through Exosomes Derived from 5XFAD Nasal Lavage Fluid.

Exosomes, key mediators of intercellular transmission of pathogenic proteins, such as amyloid-beta and tau, significantly influence the progression and exacerbation of Alzheimer's disease (AD) pathology. Present in a variety of biological fluids, including cerebrospinal fluid, blood, saliva, and nasal lavage fluid (NLF), exosomes underscore their potential as integral mediators of AD pathology. By serving as vehicles for disease-specific molecules, exosomes could unveil valuable insights into disease identification and progression. This study emphasizes the imperative to investigate the impacts of exosomes on neural networks to enhance our comprehension of intracerebral neuronal communication and its implications for neurological disorders like AD. After harvesting exosomes derived from NLF of 5XFAD mice, we utilized a high-density multielectrode array (HD-MEA) system, the novel technology enabling concurrent recordings from thousands of neurons in primary cortical neuron cultures and organotypic hippocampal slices. The ensuing results revealed a surge in neuronal firing rates and disoriented neural connectivity, reflecting the effects provoked by pathological amyloid-beta oligomer treatment. The local field potentials in the exosome-treated hippocampal brain slices also exhibited aberrant rhythmicity, along with an elevated level of current source density. While this research is an initial exploration, it highlights the potential of exosomes in modulating neural networks under AD conditions and endorses the HD-MEA as an efficacious tool for exosome studies.

Curcumin Alters Neural Plasticity and Viability of Intact Hippocampal Circuits and Attenuates Behavioral Despair and COX-2 Expression in Chronically Stressed Rats.

Curcumin is a major diarylheptanoid component of Curcuma longa with traditional usage for anxiety and depression. It has been known for the anti-inflammatory, antistress, and neurotropic effects. Here we examined curcumin effect in neural plasticity and cell viability. 60-channel multielectrode array was applied on organotypic hippocampal slice cultures (OHSCs) to monitor the effect of 10 µM curcumin in long-term depression (LTD) through low-frequency stimulation (LFS) to the Schaffer collaterals and commissural pathways. Cell viability was assayed by propidium iodide uptake test in OHSCs. In addition, the influence of oral curcumin administration on rat behavior was assessed with the forced swim test (FST). Finally, protein expression levels of brain-derived neurotrophic factor (BDNF) and cyclooxygenase-2 (COX-2) were measured by Western blot in chronically stressed rats. Our results demonstrated that 10 µM curcumin attenuated LTD and reduced cell death. It also recovered the behavior immobility of FST, rescued the attenuated BDNF expression, and inhibited the enhancement of COX-2 expression in stressed animals. These findings indicate that curcumin can enhance postsynaptic electrical reactivity and cell viability in intact neural circuits with antidepressant-like effects, possibly through the upregulation of BDNF and reduction of inflammatory factors in the brain.

Acute rosmarinic acid treatment enhances long-term potentiation, BDNF and GluR-2 protein expression, and cell survival rate against scopolamine challenge in rat organotypic hippocampal slice cultures.

BACKGROUND: Rosmarinic acid (RA) is a polyphenolic ester of caffeic acid and is commonly found in the Nepetoideae subfamily of flowering mint plants. Because RA has previously exhibited antioxidant, neuroprotective, and antidepressant-like effects, we evaluated its influences on cellular functions in neuronal cultures. OBJECTIVE: To elucidate possible mechanisms of RA, we investigated the influences of acute RA administration on long-term potentiation (LTP), plasticity-related protein expression, and scopolamine-induced cell death in organotypic hippocampal slice cultures. METHODS: LTP analysis in organotypic hippocampal slice cultures (OHSCs) was carried out with various ion channel blockers, such as AP5 (10 µM), CNQX (10 µM), niflumic acid (100 µM), and scopolamine (300 µM) in response to RA (1, 10 or 100 µg/mL) treatment. Protein expression and cell death assays in the presence of scopolamine were examined to observe the effects of RA. For LTP analysis, baseline field excitatory postsynaptic potentials (fEPSPs) were recorded in CA1 by a 60-channel multielectrode array (MEA) every min for 40 min before 15 min of high-frequency stimulation (HFS) to the Schaffer collaterals and commissural pathways, followed by a successive 50 min of recording. For protein expression measurements, anti-BDNF and anti-GluR2 antibodies were used for Western blotting assays in whole-hippocampal tissue homogenate. Finally, for cell death assays, OHSCs were exposed to a culture medium containing propidium iodide (PI) for 24 or 48 h, followed by the assessment of cell death by fluorescent image analysis of PI uptake. RESULTS: and discussion: Our results indicate that RA treatment enhances fEPSPs following HFS in CA1 synapses at 1 and 10 µg/ml RA, an effect that was inhibited by CNQX and NFA but not by AP5. RA treatment also increases the expression of BDNF and GluR-2 proteins and prevents cell death of scopolamine-exposed OHSCs. Our results suggest the possibility that rosmarinic acid can enhance neural plasticity by modulating glutamatergic signaling pathways, as well as providing neuroprotection with reduced cholinergic activity.

Intracellular chloride concentration of the mouse vomeronasal neuron.

BACKGROUND: The vomeronasal organ (VNO) is specialized in detecting pheromone and heterospecific cues in the environment. Recent studies demonstrate the involvement of multiple ion channels in VNO signal transduction, including the calcium-activated chloride channels (CACCs). Opening of CACCs appears to result in activation of VNO neuron through outflow of Cl(-) ions. However, the intracellular Cl(-) concentration remains undetermined. RESULTS: We used the chloride ion quenching dye, MQAE, to measure the intracellular Cl(-) concentration of VNO neuron in live VNO slices. The resting Cl(-) concentration in the VNO neurons is measured at 84.73 mM. Urine activation of the VNO neurons causes a drop in Cl(-) concentration, consistent with the notion of an efflux of Cl(-) to depolarize the cells. Similar observation is made for VNO neurons from mice with deletion of the transient receptor potential canonical channel 2 (TRPC2), which have a resting Cl(-) concentrations at 81 mM. CONCLUSIONS: The VNO neurons rest at high intracellular Cl(-) concentration, which can lead to depolarization of the cell when chloride channels open. These results also provide additional support of TRPC2-independent pathway of VNO activation.

Distinct signals conveyed by pheromone concentrations to the mouse vomeronasal organ.

In mammalian species, detection of pheromone cues by the vomeronasal organ (VNO) at different concentrations can elicit distinct behavioral responses and endocrine changes. It is not well understood how concentration-dependent activation of the VNO impacts innate behaviors. In this study, we find that, when mice investigate the urogenital areas of a conspecific animal, the urinary pheromones can reach the VNO at a concentration of approximately 1% of that in urine. At this level, urinary pheromones elicit responses from a subset of cells that are tuned to sex-specific cues and provide unambiguous identification of the sex and strain of animals. In contrast, low concentrations of urine do not activate these cells. Strikingly, we find a population of neurons that is only activated by low concentrations of urine. The properties of these neurons are not found in neurons responding to putative single-compound pheromones. Additional analyses show that these neurons are masked by high-concentration pheromones. Thus, an antagonistic interaction in natural pheromones results in the activation of distinct populations of cells at different concentrations. The differential activation is likely to trigger different downstream circuitry and underlies the concentration-dependent pheromone perception.

Efficient Mitochondrial Genome Editing by CRISPR/Cas9.

The Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas9 system has been widely used for nuclear DNA editing to generate mutations or correct specific disease alleles. Despite its flexible application, it has not been determined if CRISPR/Cas9, originally identified as a bacterial defense system against virus, can be targeted to mitochondria for mtDNA editing. Here, we show that regular FLAG-Cas9 can localize to mitochondria to edit mitochondrial DNA with sgRNAs targeting specific loci of the mitochondrial genome. Expression of FLAG-Cas9 together with gRNA targeting Cox1 and Cox3 leads to cleavage of the specific mtDNA loci. In addition, we observed disruption of mitochondrial protein homeostasis following mtDNA truncation or cleavage by CRISPR/Cas9. To overcome nonspecific distribution of FLAG-Cas9, we also created a mitochondria-targeted Cas9 (mitoCas9). This new version of Cas9 localizes only to mitochondria; together with expression of gRNA targeting mtDNA, there is specific cleavage of mtDNA. MitoCas9-induced reduction of mtDNA and its transcription leads to mitochondrial membrane potential disruption and cell growth inhibition. This mitoCas9 could be applied to edit mtDNA together with gRNA expression vectors without affecting genomic DNA. In this brief study, we demonstrate that mtDNA editing is possible using CRISPR/Cas9. Moreover, our development of mitoCas9 with specific localization to the mitochondria should facilitate its application for mitochondrial genome editing.

Emerging roles of TRPA1 in sensation of oxidative stress and its implications in defense and danger.

Transient receptor potential ankyrin subtype 1 (TRPA1) is a well-known ion channel that play a central role for pain sensation. In the peripheral sensory nerve terminals innervating the body tegument or organs, TRPA1 detects and is activated by diverse harmful environmental and internal stimuli. The TRPA1 activation results in neuronal firing, which finally sends a warning signal to our brain. However, sensitization or sustained activation of TRPA1 often causes plastic changes both in the neural pathway and in the peripheral tissues, leading to a pathologic state in tissue health and pain mediation. Recently, a unique covalent detection mode for reactive biological attacks was uncovered in the sensory mechanisms of TRPA1. Notably, the pool of the newly found reactive stimulators for TRPA1 includes oxidative stress. Here, we overview the nature of this interaction, and try to find biological meanings of the participation of such a rapid ionotrophic component in disease exacerbations. Acutely, its relatively rapid response can be understood in terms of efficiency for avoiding harmful milieu as quickly as possible, as implicated in the raison d'etre of the pain mechanism. Nonetheless, complex situations in a chronic disease progress may occur. As well, multiple interplays with known molecules on the redox defense mechanism are anticipated. At a therapeutic angle, how to control TRPA1 for promoting body's defensive potential will be a practical question but remains to be answered. Future investigations will likely give more detailed insights to understand the roles and target validity of TRPA1.

Paradoxical contribution of SK3 and GIRK channels to the activation of mouse vomeronasal organ.

The vomeronasal organ (VNO) is essential for intraspecies communication in many terrestrial vertebrates. The ionic mechanisms of VNO activation remain unclear. We found that the calcium-activated potassium channel SK3 and the G protein-activated potassium channel GIRK are part of an independent pathway for VNO activation. In slice preparations, the potassium channels attenuated inward currents carried by TRPC2 and calcium-activated chloride channels (CACCs). In intact tissue preparations, paradoxically, the potassium channels enhanced urine-evoked inward currents. This discrepancy resulted from the loss of a high concentration of lumenal potassium, which enabled the influx of potassium ions to depolarize the VNO neurons in vivo. Both Sk3 (also known as Kcnn3) and Girk1 (also known as Kcnj3) homozygous null mice showed deficits in mating and aggressive behaviors, and the deficiencies in Sk3(-/-) mice were exacerbated by Trpc2 knockout. Our results suggest that VNO activation is mediated by TRPC2, CACCs and two potassium channels, all of which contributed to the in vivo depolarization of VNO neurons.

Requirement of calcium-activated chloride channels in the activation of mouse vomeronasal neurons.

In terrestrial vertebrates, the vomeronasal organ (VNO) detects and transduces pheromone signals. VNO activation is thought to be mediated by the transient receptor potential C2 (TRPC2) channel. The aberrant behavioural phenotypes observed in TRPC2-/- mice are generally attributed to the lost VNO function. Recently, calcium-activated chloride channels have been shown to contribute to VNO activation. Here we show that CACCs can be activated in VNO slice preparations from the TRPC2-/- mice and this activation is blocked by pharmacological agents that inhibit intracellular Ca(2+) release. Urine-evoked Cl(-) current is sufficient to drive spiking changes in VNO neurons from both wild-type (WT) and TRPC2-/- mice. Moreover, blocking Cl(-) conductance essentially abolishes VNO activation in WT neurons. These results suggest a TRPC2-independent signalling pathway in the VNO and the requirement of calcium-activated chloride channels currents to mediate pheromone activation. Our data further suggest that TRPC2-/- mice retain partial VNO function.

Imaging neuronal responses in slice preparations of vomeronasal organ expressing a genetically encoded calcium sensor.

The vomeronasal organ (VNO) detects chemosensory signals that carry information about the social, sexual and reproductive status of the individuals within the same species. These intraspecies signals, the pheromones, as well as signals from some predators, activate the vomeronasal sensory neurons (VSNs) with high levels of specificity and sensitivity. At least three distinct families of G-protein coupled receptors, V1R, V2R and FPR, are expressed in VNO neurons to mediate the detection of the chemosensory cues. To understand how pheromone information is encoded by the VNO, it is critical to analyze the response profiles of individual VSNs to various stimuli and identify the specific receptors that mediate these responses. The neuroepithelia of VNO are enclosed in a pair of vomer bones. The semi-blind tubular structure of VNO has one open end (the vomeronasal duct) connecting to the nasal cavity. VSNs extend their dendrites to the lumen part of the VNO, where the pheromone cues are in contact with the receptors expressed at the dendritic knobs. The cell bodies of the VSNs form pseudo-stratified layers with V1R and V2R expressed in the apical and basal layers respectively. Several techniques have been utilized to monitor responses of VSNs to sensory stimuli. Among these techniques, acute slice preparation offers several advantages. First, compared to dissociated VSNs, slice preparations maintain the neurons in their native morphology and the dendrites of the cells stay relatively intact. Second, the cell bodies of the VSNs are easily accessible in coronal slice of the VNO to allow electrophysiology studies and imaging experiments as compared to whole epithelium and whole-mount preparations. Third, this method can be combined with molecular cloning techniques to allow receptor identification. Sensory stimulation elicits strong Ca2+ influx in VSNs that is indicative of receptor activation. We thus develop transgenic mice that express G-CaMP2 in the olfactory sensory neurons, including the VSNs. The sensitivity and the genetic nature of the probe greatly facilitate Ca2+ imaging experiments. This method has eliminated the dye loading process used in previous studies. We also employ a ligand delivery system that enables application of various stimuli to the VNO slices. The combination of the two techniques allows us to monitor multiple neurons simultaneously in response to large numbers of stimuli. Finally, we have established a semi-automated analysis pipeline to assist image processing.

Encoding gender and individual information in the mouse vomeronasal organ.

The mammalian vomeronasal organ detects complex chemical signals that convey information about gender, strain, and the social and reproductive status of an individual. How these signals are encoded is poorly understood. We developed transgenic mice expressing the calcium indicator G-CaMP2 and analyzed population responses of vomeronasal neurons to urine from individual animals. A substantial portion of cells was activated by either male or female urine, but only a small population of cells responded exclusively to gender-specific cues shared across strains and individuals. Female cues activated more cells and were subject to more complex hormonal regulations than male cues. In contrast to gender, strain and individual information was encoded by the combinatorial activation of neurons such that urine from different individuals activated distinctive cell populations.