ABSTRACT
Due to the increased morbidity and mortality by fungal infections and the emergence of severe antifungal resistance, there is an urgent need for new antifungal agents. Here, we screened for antifungal activity in our in-house library through the minimum inhibitory concentration test and derived two hit compounds with moderate antifungal activities. The hit compounds' antifungal activities and drug-like properties were optimized by substituting various aryl ring, alkyl chain, and methyl groups. Among the optimized compounds, 22h was the most promising candidate with good drug-like properties and exhibited potent fast-acting fungicidal antifungal effects against various fungal pathogens and synergistic antifungal activities with some known antifungal drugs. Additionally, 22h was further confirmed to disturb fungal cell wall integrity by activating multiple cell wall integrity pathways. Furthermore, 22h exerted significant antifungal efficacy in both the subcutaneous infection mouse model and ex vivo human nail infection model.
Subject(s)
Antifungal Agents/therapeutic use , Fungi/drug effects , Mycoses/drug therapy , Animals , Antifungal Agents/pharmacokinetics , Antifungal Agents/pharmacology , Antifungal Agents/toxicity , Cell Wall/drug effects , Drug Evaluation, Preclinical , Drug Synergism , Female , Humans , Male , Mice , Microbial Sensitivity Tests , Mycoses/microbiology , Rats, Sprague-DawleyABSTRACT
Sensory discrimination is essential for survival. However, how sensory information is finely controlled in the brain is not well defined. Here, we show that astrocytes control tactile acuity via tonic inhibition in the thalamus. Mechanistically, diamine oxidase (DAO) and the subsequent aldehyde dehydrogenase 1a1 (Aldh1a1) convert putrescine into GABA, which is released via Best1. The GABA from astrocytes inhibits synaptically evoked firing at the lemniscal synapses to fine-tune the dynamic range of the stimulation-response relationship, the precision of spike timing, and tactile discrimination. Our findings reveal a novel role of astrocytes in the control of sensory acuity through tonic GABA release.
Subject(s)
Astrocytes/physiology , Neural Inhibition/physiology , Thalamus/physiology , Touch Perception/physiology , gamma-Aminobutyric Acid/physiology , Aldehyde Dehydrogenase 1 Family/metabolism , Amine Oxidase (Copper-Containing)/metabolism , Animals , Astrocytes/metabolism , Astrocytes/ultrastructure , Bestrophins/biosynthesis , Bestrophins/genetics , Female , GABA Antagonists , Immunohistochemistry , Inhibitory Postsynaptic Potentials/physiology , Macrolides/pharmacology , Male , Mice , Mice, Knockout , Microscopy, Electron , Neurons/metabolism , Neurons/physiology , Patch-Clamp Techniques , Picrotoxin/pharmacology , Primary Cell Culture , Pyridazines/pharmacology , RNA, Small Interfering/pharmacology , Retinal Dehydrogenase/metabolism , gamma-Aminobutyric Acid/biosynthesis , gamma-Aminobutyric Acid/pharmacologyABSTRACT
Sleep abnormality often accompanies the impairment of cognitive function. Both rapid eye movement (REM) and non-REM (NREM) sleep have associated with improved memory performance. However, the role of composition in NREM sleep, consisting of light and deep NREM, for memory formation is not fully understood. We investigated how the dynamics of NREM sleep states influence memory consolidation. Thalamocortical (TC) neuron-specific phospholipase C ß4 (PLCß4) knockout (KO) increased the total duration of NREM sleep, consisting of destabilized light NREM and stabilized deep NREM. Surprisingly, the longer NREM sleep did not improve memory consolidation but rather impaired it in TC-specific PLCß4 KO mice. Memory function was positively correlated with the stability of light NREM and spindle activity occurring in maintained light NREM period. Our study suggests that a single molecule, PLCß4, in TC neurons is critical for tuning the NREM sleep states and thus affects sleep-dependent memory formation.
Subject(s)
Memory Consolidation/physiology , Memory Disorders/enzymology , Nerve Tissue Proteins/physiology , Phospholipase C beta/physiology , Sleep Stages/physiology , Thalamus/enzymology , Animals , Cerebral Cortex/enzymology , Conditioning, Classical/physiology , Delta Rhythm/physiology , Electroencephalography , Electromyography , Exons/genetics , Exploratory Behavior , Fear/physiology , Male , Memory Disorders/physiopathology , Mice , Mice, Knockout , Mice, Transgenic , Nerve Tissue Proteins/deficiency , Neurons/enzymology , Phospholipase C beta/deficiency , Recognition, Psychology , Sequence Deletion , Sleep, Slow-Wave/physiology , Time FactorsABSTRACT
The real-time selective detection of disease-related markers in blood using biosensors has great potential for use in the early diagnosis of diseases and infections. However, this potential has not been realized thus far due to difficulties in interfacing the sensor with blood and achieving transparent circuits that are essential for detecting of target markers (e.g., protein, ions, etc.) in a complex blood environment. Herein, we demonstrate the real-time detection of a specific protein and ion in blood without a skin incision. Complementary metal-oxide-semiconductor technology was used to fabricate silicon micropillar array (SiMPA) electrodes with a height greater than 600 µm, and the surface of the SiMPA electrodes was functionalized with a self-assembling artificial peptide (SAP) as a receptor for target markers in blood, i.e., cholera toxin (CTX) and mercury(II) ions (Hg). The detection of CTX was investigated in both in vitro (phosphate-buffered saline and human blood serum, HBO model) and in vivo (mouse model) modes via impedance analysis. In the in vivo mode, the SiMPA pierces the skin, comes into contact with the blood system, and creates comprehensive circuits that include all the elements such as electrodes, blood, and receptors. The SiMPA achieves electrically transparent circuits and, thus, can selectively detect CTX in the blood in real time with a high sensitivity of 50 pM and 5 nM in the in vitro and in vivo modes, respectively. Mercury(II) ions can also be detected in both the in vitro and the in vivo modes by changing the SAP. The results illustrate that a robust sensor that can detect a variety of molecular species in the blood system in real time that will be helpful for the early diagnosis of disease and infections.
Subject(s)
Biomarkers/blood , Biosensing Techniques , Cholera Toxin/isolation & purification , Mercury/isolation & purification , Animals , Blood Proteins/chemistry , Blood Proteins/isolation & purification , Cholera Toxin/blood , Humans , Limit of Detection , Mercury/blood , Mice , Semiconductors , Silicon/chemistryABSTRACT
The transition from wakefulness to a nonrapid eye movement (NREM) sleep state at the onset of sleep involves a transition from low-voltage, high-frequency irregular electroencephalography (EEG) waveforms to large-amplitude, low-frequency EEG waveforms accompanying synchronized oscillatory activity in the thalamocortical circuit. The thalamocortical circuit consists of reciprocal connections between the thalamus and cortex. The cortex sends strong excitatory feedback to the thalamus, however the function of which is unclear. In this study, we investigated the role of the thalamic metabotropic glutamate receptor 1 (mGluR1)-phospholipase C ß4 (PLCß4) pathway in sleep control in PLCß4-deficient (PLCß4-/-) mice. The thalamic mGluR1-PLCß4 pathway contains synapses that receive corticothalamic inputs. In PLCß4-/- mice, the transition from wakefulness to the NREM sleep state was stimulated, and the NREM sleep state was stabilized, which resulted in increased NREM sleep. The power density of delta (δ) waves increased in parallel with the increased NREM sleep. These sleep phenotypes in PLCß4-/- mice were consistent in TC-restricted PLCß4 knockdown mice. Moreover, in vitro intrathalamic oscillations were greatly enhanced in the PLCß4-/- slices. The results of our study showed that thalamic mGluR1-PLCß4 pathway was critical in controlling sleep architecture.
Subject(s)
Phospholipase C beta/metabolism , Receptors, Metabotropic Glutamate/metabolism , Sleep/physiology , Thalamus/metabolism , Animals , Cerebral Cortex/physiology , Delta Rhythm/physiology , Mice, Inbred C57BL , Mice, Knockout , Phospholipase C beta/deficiency , Thalamus/physiologyABSTRACT
Neuronal firing patterns, which are crucial for determining the nature of encoded information, have been widely studied; however, the molecular identity and cellular mechanisms of spike-frequency adaptation are still not fully understood. Here we show that spike-frequency adaptation in thalamocortical (TC) neurons is mediated by the Ca2+-activated Cl- channel (CACC) anoctamin-2 (ANO2). Knockdown of ANO2 in TC neurons results in significantly reduced spike-frequency adaptation along with increased tonic spiking. Moreover, thalamus-specific knockdown of ANO2 increases visceral pain responses. These results indicate that ANO2 contributes to reductions in spike generation in highly activated TC neurons and thereby restricts persistent information transmission.
Subject(s)
Anoctamins/metabolism , Calcium/pharmacology , Sensory Receptor Cells/physiology , Thalamus/physiology , Adenoviridae , Animals , Anoctamins/genetics , Bestrophins/genetics , Bestrophins/metabolism , Female , Gene Expression Regulation/drug effects , Gene Knockdown Techniques , HEK293 Cells , Humans , Male , Mice , Mice, Inbred BALB C , NIH 3T3 Cells , Patch-Clamp Techniques , ortho-Aminobenzoates/pharmacologyABSTRACT
Protein tyrosine phosphatases (PTPs) serve as key negative-feedback regulators of mitogen-activated protein kinase (MAPK) signaling cascades. However, their roles and regulatory mechanisms in human fungal pathogens remain elusive. In this study, we characterized the functions of two PTPs, Ptp1 and Ptp2, in Cryptococcus neoformans, which causes fatal meningoencephalitis. PTP1 and PTP2 were found to be stress-inducible genes, which were controlled by the MAPK Hog1 and the transcription factor Atf1. Ptp2 suppressed the hyperphosphorylation of Hog1 and was involved in mediating vegetative growth, sexual differentiation, stress responses, antifungal drug resistance, and virulence factor regulation through the negative-feedback loop of the HOG pathway. In contrast, Ptp1 was not essential for Hog1 regulation, despite its Hog1-dependent induction. However, in the absence of Ptp2, Ptp1 served as a complementary PTP to control some stress responses. In differentiation, Ptp1 acted as a negative regulator, but in a Hog1- and Cpk1-independent manner. Additionally, Ptp1 and Ptp2 localized to the cytosol but were enriched in the nucleus during the stress response, affecting the transient nuclear localization of Hog1. Finally, Ptp1 and Ptp2 played minor and major roles, respectively, in the virulence of C. neoformans. Taken together, our data suggested that PTPs could be exploited as novel antifungal targets.
Subject(s)
Cryptococcus neoformans/enzymology , Fungal Proteins/metabolism , Protein Tyrosine Phosphatases/metabolism , Virulence/genetics , Active Transport, Cell Nucleus , Animals , Base Sequence , Cell Nucleus/metabolism , Cryptococcus neoformans/genetics , Cryptococcus neoformans/growth & development , Cryptococcus neoformans/pathogenicity , Female , Fungal Proteins/chemistry , Fungal Proteins/genetics , Genes, Fungal , MAP Kinase Signaling System , Mice , Molecular Sequence Data , Protein Tyrosine Phosphatases/chemistry , Protein Tyrosine Phosphatases/genetics , Stress, Physiological , Transcription Factors/metabolismABSTRACT
Absence epilepsy accompanies the paroxysmal oscillations in the thalamocortical circuit referred as spike and wave discharges (SWDs). Low-threshold burst firing mediated by T-type Ca(2+) channels highly expressed in both inhibitory thalamic reticular nuclei (TRN) and excitatory thalamocortical (TC) neurons has been correlated with the generation of SWDs. A generally accepted view has been that rhythmic burst firing mediated by T-type channels in both TRN and TC neurons are equally critical in the generation of thalamocortical oscillations during sleep rhythms and SWDs. This review examined recent studies on the T-type channels in absence epilepsy which leads to an idea that even though both TRN and TC nuclei are required for thalamocortical oscillations, the contributions of T-type channels to TRN and TC neurons are not equal in the genesis of sleep spindles and SWDs. Accumulating evidence revealed a crucial role of TC T-type channels in SWD generation. However, the role of TRN T-type channels in SWD generation remains controversial. Therefore, a deeper understanding of the functional consequences of modulating each T-type channel subtype could guide the development of therapeutic tools for absence seizures while minimizing side effects on physiological thalamocortical oscillations.
Subject(s)
Action Potentials/physiology , Calcium Channels, T-Type/physiology , Epilepsy, Absence/physiopathology , Thalamus/physiopathology , Animals , Electroencephalography/trends , Epilepsy, Absence/diagnosis , Epilepsy, Absence/genetics , Humans , Mutation/physiologyABSTRACT
T-type Ca(2+) channels in thalamocortical (TC) neurons have long been considered to play a critical role in the genesis of sleep spindles, one of several TC oscillations. A classical model for TC oscillations states that reciprocal interaction between synaptically connected GABAergic thalamic reticular nucleus (TRN) neurons and glutamatergic TC neurons generates oscillations through T-type channel-mediated low-threshold burst firings of neurons in the two nuclei. These oscillations are then transmitted from TC neurons to cortical neurons, contributing to the network of TC oscillations. Unexpectedly, however, we found that both WT and KO mice for CaV3.1, the gene for T-type Ca(2+) channels in TC neurons, exhibit typical waxing-and-waning sleep spindle waves at a similar occurrence and with similar amplitudes and episode durations during non-rapid eye movement sleep. Single-unit recording in parallel with electroencephalography in vivo confirmed a complete lack of burst firing in the mutant TC neurons. Of particular interest, the tonic spike frequency in TC neurons was significantly increased during spindle periods compared with nonspindle periods in both genotypes. In contrast, no significant change in burst firing frequency between spindle and nonspindle periods was noted in the WT mice. Furthermore, spindle-like oscillations were readily generated within intrathalamic circuits composed solely of TRN and TC neurons in vitro in both the KO mutant and WT mice. Our findings call into question the essential role of low-threshold burst firings in TC neurons and suggest that tonic firing is important for the generation and propagation of spindle oscillations in the TC circuit.
Subject(s)
Brain Waves/physiology , Models, Neurological , Neurons/metabolism , Periodicity , Sleep/physiology , Thalamus/metabolism , Animals , Calcium Channels, T-Type/genetics , Electroencephalography , Mice , Mice, KnockoutABSTRACT
Ryanodine receptors (RyRs) are highly conductive intracellular Ca(2+) release channels which are widely expressed in the CNS. They rapidly increase the intracellular Ca(2+) concentrations in neuronal cells in response to Ca(2+) influx through voltage-gated Ca(2+) channels. A previous study reported that RyRs were expressed in thalamocortical (TC) neurons, but their physiological function has remained elusive. Here, we show that the activation of RyRs in TC neurons in mice decreases their tonic firing rate while blocking them induces the opposite response. Furthermore, activation of RyRs in ventroposteriomedial/ventroposteriolateral nuclei reduces the behavioral responses to inflammatory pain and blocking them increases the responses. This study highlights the importance of the intracellular Ca(2+) release via RyRs in controlling the excitability of TC neurons and in inflammatory pain signal processing in the thalamus.
Subject(s)
Neurons/physiology , Pain/physiopathology , Ryanodine Receptor Calcium Release Channel/physiology , Thalamus/physiopathology , Action Potentials , Animals , Calcium Channel Agonists/pharmacology , Calcium Channel Blockers/pharmacology , Inflammation/physiopathology , Male , Mice , Pain Measurement , Thalamic Nuclei/physiopathologyABSTRACT
Absence seizures are characterized by cortical spike-wave discharges (SWDs) on electroencephalography, often accompanied by a shift in the firing pattern of thalamocortical (TC) neurons from tonic to burst firing driven by T-type Ca(2+) currents. We recently demonstrated that the phospholipase C beta4 (PLCbeta4) pathway tunes the firing mode of TC neurons via the simultaneous regulation of T- and L-type Ca(2+) currents, which prompted us to investigate the contribution of TC firing modes to absence seizures. PLCbeta4-deficient TC neurons were readily shifted to the oscillatory burst firing mode after a slight hyperpolarization of membrane potential. TC-limited knockdown as well as whole-animal knockout of PLCbeta4 induced spontaneous SWDs with simultaneous behavioral arrests and increased the susceptibility to drug-induced SWDs, indicating that the deletion of thalamic PLCbeta4 leads to the genesis of absence seizures. The SWDs were effectively suppressed by thalamic infusion of a T-type, but not an L-type, Ca(2+) channel blocker. These results reveal a primary role of TC neurons in the genesis of absence seizures and provide strong evidence that an alteration of the firing property of TC neurons is sufficient to generate absence seizures. Our study presents PLCbeta4-deficient mice as a potential animal model for absence seizures.
Subject(s)
Epilepsy, Absence/enzymology , Phospholipase C beta/physiology , Thalamus/physiopathology , Animals , Calcium Channel Blockers/pharmacology , Calcium Channels, T-Type/drug effects , Electroencephalography , Enzyme Activators/pharmacology , Epilepsy, Absence/physiopathology , GABA Agonists/pharmacology , Gene Silencing , Membrane Potentials , Mice , Mice, Knockout , Phospholipase C beta/genetics , Thalamus/enzymologyABSTRACT
Two firing modes of thalamocortical (TC) neurons, tonic and burst firings, are thought to reflect the divergent states of sensory signal transmission from the thalamus to the cortex. However, the behavioral consequences of changes in the thalamic firing between the two modes have not been well demonstrated. Moreover, although the firing modes of TC neurons are known to be affected by corticothalamic inputs via thalamic metabotropic glutamate receptor type 1 (mGluR1)-phospholipase C beta4 (PLCbeta4) pathway, its molecular mechanisms have not been well elucidated. We addressed these questions using PLCbeta4-deficient mice, which show decreased visceral pain responses. We demonstrate that burst and tonic firings of TC neurons are concomitantly regulated by PLCbeta4 pathway. Blocking of this pathway by the mutation simultaneously increases bursting and decreases tonic firing of TC neurons through concurrent upregulation of T- and L-type Ca(2+) currents. The mice with increased bursting and decreased tonic firing of TC neurons showed reduced visceral pain responses. Furthermore, we show that modulation of the Ca(2+) channels or protein kinase C (PKC), a downstream molecule of PLCbeta4, altered the firing modes of TC neurons and pain responses in the predicted ways. Our data demonstrate the molecular mechanism and behavioral consequences of altered firing modes of TC neurons in relaying the visceral pain signals. Our study also highlights the thalamic PLCbeta4-PKC pathway as a "molecular switch" for the firing modes of TC neurons and thus for pain sensory gating.
Subject(s)
Action Potentials/genetics , Calcium Channels/metabolism , Neurons/metabolism , Pain/metabolism , Phospholipase C beta/genetics , Thalamus/metabolism , Animals , Calcium Channels, L-Type/metabolism , Calcium Channels, T-Type/metabolism , Male , Mice , Mice, Inbred C57BL , Mice, Knockout , Neural Inhibition/genetics , Pain/genetics , Pain/physiopathology , Pain Threshold/physiology , Protein Kinase C/metabolism , Receptors, Metabotropic Glutamate/metabolism , Signal Transduction/genetics , Synaptic Transmission/geneticsABSTRACT
Low-voltage-activated calcium channels, also known as T-type calcium channels, are widely expressed in various types of neurons. In contrast to high-voltage-activated calcium channels which can be activated by a strong depolarization of membrane potential, T-type channels can be activated by a weak depolarization near the resting membrane potential once deinactivated by hyperpolarization, and therefore can regulate the excitability and electroresponsiveness of neurons under physiological conditions near resting states. Recently, the molecular diversity and functional multiplicity of T-type channels have been demonstrated through molecular genetic studies coupled with physiological and behavioral analysis. Understanding the functional consequences of modulation of each subtype of these channels in vivo could point to the right direction for developing therapeutic tools for relevant diseases.