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Communications Biology Jul 2024The highly complex structure of the brain requires an approach that can unravel its connectivity. Using volume electron microscopy and a dedicated software we can trace...
The highly complex structure of the brain requires an approach that can unravel its connectivity. Using volume electron microscopy and a dedicated software we can trace and measure all nerve fibers present within different samples of brain tissue. With this software tool, individual dendrites and axons are traced, obtaining a simplified "skeleton" of each fiber, which is linked to its corresponding synaptic contacts. The result is an intricate meshwork of axons and dendrites interconnected by a cloud of synaptic junctions. To test this methodology, we apply it to the stratum radiatum of the hippocampus and layers 1 and 3 of the somatosensory cortex of the mouse. We find that nerve fibers are densely packed in the neuropil, reaching up to 9 kilometers per cubic mm. We obtain the number of synapses, the number and lengths of dendrites and axons, the linear densities of synapses established by dendrites and axons, and their location on dendritic spines and shafts. The quantitative data obtained through this method enable us to identify subtle traits and differences in the synaptic organization of the samples, which might have been overlooked in a qualitative analysis.
Topics: Animals; Mice; Microscopy, Electron; Nerve Fibers; Synapses; Axons; Dendrites; Brain; Somatosensory Cortex; Mice, Inbred C57BL; Male; Software; Hippocampus; Volume Electron Microscopy
PubMed: 38951162
DOI: 10.1038/s42003-024-06491-0 -
Zhonghua Nei Ke Za Zhi Jul 2024To investigate the clinical and electrophysiological characteristics of patients with amyotrophic lateral sclerosis (ALS) with positive repetitive nerve stimulation...
To investigate the clinical and electrophysiological characteristics of patients with amyotrophic lateral sclerosis (ALS) with positive repetitive nerve stimulation (RNS) test results on the accessory nerve and negative needle electromyography (EMG) test results on the sternocleidomastoid with the goal to enrich the knowledge of disease progression in patients with ALS. The clinical data of 612 patients diagnosed with ALS at the Neurology Department of the First Medical Center, Chinese PLA General Hospital from June 2016 to August 2022 were collected. In total, 267 cases had undergone EMG tests on the sternocleidomastoid following a positive 3 Hz RNS test result on the accessory nerve, who were selected as the study subjects. The differences in clinical indicators were compared between RNS (+)/EMG (-) group and RNS (+)/EMG (+) group. A binomial distribution model with multiple variables was built to quantitatively analyze the major factors and their effects. At the initial visit, 15.8% of patients with ALS were 3 Hz RNS (+) on the accessory nerve and EMG (-) on the ipsilateral sternocleidomastoid, accounting for 36.3% of RNS (+) patients. The decremental range of the 3 Hz RNS test delivered to the accessory nerve in these patients [-14% (-19%, -12%)] was lower than that in patients with RNS (+)/EMG (+) [-17% (-23%, -13%)] (<0.05), while the ratio of upper limb onset (64.9%) and non-definite diagnosis (28.9%) were higher [54.7% and 13.5% for patients with RNS (+)/EMG (+), <0.05]. Furthermore, the Revised Amyotrophic Lateral Sclerosis Functional Rating Scale (ALSFRS-R) score [40 (37, 42)], body mass index (BMI) [23.8 (22.0, 25.4) kg/m] and forced vital capacity (FVC) [92.8% (76.6%, 103.8%)] were higher in patients with RNS(+)/EMG(+) (<0.05). The multivariate model suggested that, in patients with RNS (+)/EMG (-), the ratio of upper limb onset to lower limb onset was 1.04, while that of upper limb onset to bulbar onset was 2.02, and that of lower limb onset to bulbar onset was 1.94. The ratio of non-definite ALS to definite ALS was 1.13. The ALSFRS-R score, BMI, and FVC had a protective contribution to the electrophysiological function of the motor neurons. The ratio of the effect size of the ALSFRS-R or BMI to that of FVC was 3.37 and 1.14, respectively. Patients with ALS that were 3 Hz RNS (+) on the accessory nerve and EMG (-) on the ipsilateral sternocleidomastoid had a smaller decremental range of the compound muscle action potential amplitude, and a higher proportion of upper limb onset and non-definite ALS. A higher ALSFRS-R score, BMI, and FVC have a protective effect to the electrophysiological function of motor neurons. The effect size of the ALSFRS-R score is the largest, followed by BMI and FVC.
Topics: Humans; Amyotrophic Lateral Sclerosis; Electromyography; Motor Neurons; Neuromuscular Junction; Electric Stimulation; Accessory Nerve; Male; Female; Middle Aged
PubMed: 38951089
DOI: 10.3760/cma.j.cn112138-20230811-00049 -
The Journal of Neuroscience : the... Jul 2024At chemical synapses, voltage-gated Ca-channels (VGCCs) translate electrical signals into a trigger for synaptic vesicle (SV) fusion. VGCCs and the Ca microdomains they...
At chemical synapses, voltage-gated Ca-channels (VGCCs) translate electrical signals into a trigger for synaptic vesicle (SV) fusion. VGCCs and the Ca microdomains they elicit must be located precisely to primed SVs, to evoke rapid transmitter release. Localization is mediated by Rab3 interacting molecule (RIM) and RIM-binding proteins (RIM-BPs), which interact and bind to the C-terminus of the CaV2 VGCC α-subunit. We studied this machinery at the mixed cholinergic/GABAergic neuromuscular junction (NMJ) of hermaphrodites. mutants had mild synaptic defects, through loosening the anchoring of UNC-2/CaV2 and delaying the onset of SV fusion. UNC-10/RIM deletion much more severely affected transmission. Even though postsynaptic depolarization was reduced, mutants had increased cholinergic (but reduced GABAergic) transmission, to compensate for the delayed release. This did not occur when the excitation-inhibition balance was altered by removing GABA transmission. Further analyses of GABA defective mutants and GABA or GABA receptor deletions, as well as cholinergic rescue of RIMB-1, emphasized that GABA neurons may be more affected than cholinergic neurons. Thus RIMB-1 function differentially affects excitation/inhibition balance in the different motor neurons, and RIMB-1 thus may differentially regulate transmission in mixed circuits. Untethering the UNC-2/CaV2 channel by removing its C-terminal PDZ ligand exacerbated the defects, and similar phenotypes resulted from acute degradation of the CaV2 β-subunit CCB-1. Therefore, untethering of the CaV2 complex is as severe as its elimination, yet does not abolish transmission, likely due to compensation by CaV1. Thus, robustness and flexibility of synaptic transmission emerges from VGCC regulation. The machinery for chemical synaptic transmission is organized in a precise spatial arrangement in order to enable efficient and temporally accurate coupling of action potentials with the rise of the Ca concentration through CaV2 P/Q-type voltage gated Ca channels. This triggers the fusion of synaptic vesicles with the plasma membrane and the release of transmitters. Here, we analyzed the molecular and functional interplay of proteins of the active zone scaffold, RIM and RIM-binding protein (RIMB-1), with the CaV2 channel in the neuromuscular junction, a tripartite synapse with cholinergic and GABAergic neuronal input. Our work shows a differential requirement of RIMB-1 in cholinergic vs. GABAergic neurons, that affects the regulation of excitation-inhibition balance at circuit, cellular and ultrastructural levels.
PubMed: 38951038
DOI: 10.1523/JNEUROSCI.0535-22.2024 -
Learning & Memory (Cold Spring Harbor,... Jun 2024How does repeated stimulation of mechanoafferents affect feeding motor neurons? Monosynaptic connections from a mechanoafferent population in the buccal ganglia to five...
How does repeated stimulation of mechanoafferents affect feeding motor neurons? Monosynaptic connections from a mechanoafferent population in the buccal ganglia to five motor followers with different functions were examined during repeated stimulus trains. The mechanoafferents produced both fast and slow synaptic outputs, which could be excitatory or inhibitory. In contrast, other mechanoafferents produce only fast excitation on their followers. In addition, patterns of synaptic connections were different to the different motor followers. Some followers received both fast excitation and fast inhibition, whereas others received exclusively fast excitation. All followers showed strong decreases in fast postsynaptic potential (PSP) amplitude within a stimulus train. Fast and slow synaptic connections were of net opposite signs in some followers but not in others. For one follower, synaptic contacts were not uniform from all subareas of the mechanoafferent cluster. Differences in properties of the buccal ganglia mechanoafferents and other mechanoafferents may arise because the buccal ganglia neurons innervate the interior of the feeding apparatus, rather than an external surface, and connect to motor neurons for muscles with different motor functions. Fast connection patterns suggest that these synapses may be activated when food slips, biasing the musculature to release food. The largest slow inhibitory synaptic PSPs may contribute to a delay in the onset of the next behavior. Additional functions are also possible.
Topics: Animals; Aplysia; Motor Neurons; Ganglia, Invertebrate; Feeding Behavior; Mechanoreceptors; Synapses; Physical Stimulation
PubMed: 38950976
DOI: 10.1101/lm.053880.123 -
Cellular Signalling Jun 2024Diabetes-associated periodontitis (DP) presents severe inflammation and resistance to periodontal conventional treatment, presenting a significant challenge in clinical...
Diabetes-associated periodontitis (DP) presents severe inflammation and resistance to periodontal conventional treatment, presenting a significant challenge in clinical management. In this study, we investigated the underlying mechanism driving the hyperinflammatory response in gingival epithelial cells (GECs) of DP patients. Our findings indicate that lysosomal dysfunction under high glucose conditions leads to the blockage of autophagy flux, exacerbating inflammatory response in GECs. Single-cell RNA sequencing and immunohistochemistry analyses of clinical gingival epithelia revealed dysregulation in the lysosome pathway characterized by reduced levels of lysosome-associated membrane glycoprotein 2 (LAMP2) and V-type proton ATPase 16 kDa proteolipid subunit c (ATP6V0C) in subjects with DP. In vitro stimulation of human gingival epithelial cells (HGECs) with a hyperglycemic microenvironment showed elevated release of proinflammatory cytokines, compromised lysosomal acidity and blocked autophagy. Moreover, HGECs with deficiency in ATP6V0C demonstrated impaired autophagy and heightened inflammatory response, mirroring the effects of high glucose stimulation. Proteomic analysis of acetylation modifications identified altered acetylation levels in 28 autophagy-lysosome pathway-related proteins and 37 sites in HGECs subjected to high glucose stimulation or siATP6V0C. Overall, our finding highlights the pivotal role of lysosome impairment in autophagy obstruction in DP and suggests a potential impact of altered acetylation of relevant proteins on the interplay between lysosome dysfunction and autophagy blockage. These insights may pave the way for the development of effective therapeutic strategies against DP.
PubMed: 38950874
DOI: 10.1016/j.cellsig.2024.111273 -
European Journal of Pharmacology Jun 2024Cucurbitacin B (CuB) is a compound found in plants like Cucurbitaceae that has shown promise in fighting cancer, particularly in lung cancer. However, the specific...
Cucurbitacin B (CuB) is a compound found in plants like Cucurbitaceae that has shown promise in fighting cancer, particularly in lung cancer. However, the specific impact of CuB on ferroptosis and how it works in lung cancer cells has not been fully understood. Our research has discovered that CuB can effectively slow down the growth of non-small cell lung cancer (NSCLC) cells. Even in small amounts, it was able to inhibit the growth of various NSCLC cell lines. This inhibitory effect was reversed when ferroptosis inhibitors DFO, Lip-1 and Fer-1 were introduced. CuB was found to increase the levels of reactive oxygen species (ROS), lipid ROS, MDA, and ferrous ions within H358 lung cancer cells, leading to a decrease in GSH, mitochondrial membrane potential (MMP) and changes in ferroptosis-related proteins in a dose-dependent manner. These findings were also confirmed in A549 lung cancer cells. In A549 cells, different concentrations of CuB induced the accumulation of intracellular lipid ROS, ferrous ions and changes in ferroptosis-related indicators in a concentration-dependent manner. Meanwhile, the cytotoxic effect induced by CuB in A549 cells was counteracted by ferroptosis inhibitors DFO and Fer-1. Through network pharmacology, we identified potential targets related to ferroptosis in NSCLC cells treated with CuB, with STAT3 targets showing high scores. Further experiments using molecular docking and cell thermal shift assay (CETSA) revealed that CuB interacts with the STAT3 protein. Western blot and immunofluorescence staining demonstrated that CuB inhibits the phosphorylation of STAT3 (P-STAT3) in H358 cells. Silencing STAT3 enhanced CuB-induced accumulation of lipid ROS and iron ions, as well as the expression of ferroptosis-related proteins. On the other hand, overexpression of STAT3 reversed the effects of CuB-induced ferroptosis. The results indicate that CuB has the capability to suppress STAT3 activation, resulting in ferroptosis, and could be a promising treatment choice for NSCLC.
PubMed: 38950838
DOI: 10.1016/j.ejphar.2024.176805 -
Biophysical Reports Jun 2024In vitro assays of ion transport are an essential tool for understanding molecular mechanisms associated with ATP-dependent pumps. Because ion transport is generally...
In vitro assays of ion transport are an essential tool for understanding molecular mechanisms associated with ATP-dependent pumps. Because ion transport is generally electrogenic, principles of electrophysiology are applicable, but conventional tools like patch clamp are ineffective due to relatively low turnover rates of the pumps. Instead, assays have been developed to measure either voltage or current generated by transport activity of a population of molecules either in cell-derived membrane fragments or after reconstituting purified protein into proteoliposomes. In order to understand the nuances of these assays and to characterize effects of various operational parameters, we have developed a numerical model to simulate data produced by two relevant assays: fluorescence from voltage sensitive dyes and current recorded by capacitive coupling on solid supported membranes. Parameters of the model, which has been implemented in Python, are described along with underlying principles of the computational algorithm. Experimental data from KdpFABC, a K pump associated with P-type ATPases, are presented and model parameters have been adjusted to mimic these data. In addition, effects of key parameters such as non-selective leak conductance and turnover rate are demonstrated. Finally, simulated data are used to illustrate the effects of capacitive coupling on measured current and to compare alternative methods for quantification of raw data.
PubMed: 38950825
DOI: 10.1016/j.bpr.2024.100169 -
Journal of Molecular and Cellular... Jun 2024Pathological cardiac hypertrophy is considered one of the independent risk factors for heart failure, with a rather complex pathogenic machinery. Sorting nexins (SNXs),...
BACKGROUNDS
Pathological cardiac hypertrophy is considered one of the independent risk factors for heart failure, with a rather complex pathogenic machinery. Sorting nexins (SNXs), denoting a diverse family of cytoplasmic- and membrane-associated phosphoinositide-binding proteins, act as a pharmacological target against specific cardiovascular diseases including heart failure. Family member SNX5 was reported to play a pivotal role in a variety of biological processes. However, contribution of SNX5 to the development of cardiac hypertrophy, remains unclear.
METHODS
Mice underwent transverse aortic constriction (TAC) to induce cardiac hypertrophy and simulate pathological conditions. TAC model was validated using echocardiography and histological staining. Expression of SNX5 was assessed by western blotting. Then, SNX5 was delivered through intravenous administration of an adeno-associated virus serotype 9 carrying cTnT promoter (AAV9-cTnT-SNX5) to achieve SNX5 cardiac-specific overexpression. To assess the impact of SNX5, morphological analysis, echocardiography, histological staining, hypertrophic biomarkers, and cardiomyocyte contraction were evaluated. To unravel potential molecular events associated with SNX5, interactome analysis, fluorescence co-localization, and membrane protein profile were evaluated.
RESULTS
Our results revealed significant downregulated protein level of SNX5 in TAC-induced hypertrophic hearts in mice. Interestingly, cardiac-specific overexpression of SNX5 improved cardiac function, with enhanced left ventricular ejection fraction, fraction shortening, as well as reduced cardiac fibrosis. Mechanistically, SNX5 directly bound to Rab11a, increasing membrane accumulation of Rab11a (a Rab GTPase). Afterwards, this intricate molecular interaction upregulated the membrane content of low-density lipoprotein receptor-related protein 6 (LRP6), a key regulator against cardiac hypertrophy. Our comprehensive assessment of siRab11a expression in HL-1 cells revealed its role in antagonism of LRP6 membrane accumulation under SNX5 overexpression.
CONCLUSIONS
This study revealed that binding of SNX5 with LRP6 triggers their membrane translocation through Rab11a assisting, defending against cardiac remodeling and cardiac dysfunction under pressure overload. These findings provide new insights into the previously unrecognized role of SNX5 in the progression of cardiac hypertrophy.
PubMed: 38950816
DOI: 10.1016/j.yjmcc.2024.06.009 -
Chemosphere Jun 2024Recently, phytoremediation has been regarded as a green and environment friendly technique to treat metals contaminated soils. Thus, in this study, pot experiments were...
Recently, phytoremediation has been regarded as a green and environment friendly technique to treat metals contaminated soils. Thus, in this study, pot experiments were designed to investigate the combine effects of biochar and magnesium (MPs) to purify cadmium (Cd)-contaminated soils by Medicago sativa L. (alfalfa). The results showed that the combined use of biochar and Mg significantly increased the accumulation of Cd and promoted the transport of Cd from root to shoot in alfalfa, simultaneously. Importantly, the combined use of biochar and Mg could increase the accumulation of Cd in shoot and whole plant (shoot + root) of alfalfa up-to 59.1% and 23.1%, respectively. Moreover, the enhancement mechanism can be analyzed from several aspects. Firstly, the photosynthesis was enhanced, which was beneficial to plant growth. The product of photosynthesis provided energy for uptake and transport of Cd. Meanwhile, its transport in phloem could promote the transport of Cd. Secondly, the enhancement of antioxidant capacity of alfalfa effectively protected the membrane structure of alfalfa, which indicated that Cd could enter alfalfa from the channel on the cell membrane. Lastly, the chemical form of Cd and microbial community structure in soil were changed. Overall, these changes reduced the Cd toxicity in soil, enhanced the resistance capability of alfalfa, increased the Cd uptake by alfalfa and promoted the growth of alfalfa. Thus, the obtained results suggested that the combined use of biochar and Mg is an effective approach to enhance phytoremediation performance for purifying Cd-contaminated soils.
PubMed: 38950747
DOI: 10.1016/j.chemosphere.2024.142737 -
Neuropharmacology Jun 2024Vasogenic brain edema, a potentially life-threatening consequence following an acute ischemic stroke, is a major clinical problem. This research aims to explore the...
Vasogenic brain edema, a potentially life-threatening consequence following an acute ischemic stroke, is a major clinical problem. This research aims to explore the therapeutic benefits of nimodipine, a calcium channel blocker, in mitigating vasogenic cerebral edema and preserving blood-brain barrier (BBB) function in an ischemic stroke rat model. In this research, animals underwent the induction of ischemic stroke via a 60-minute blockage of the middle cerebral artery and treated with a nonhypotensive dose of nimodipine (1 mg/kg/day) for a duration of five days. The wet/dry method was employed to identify cerebral edema, and the Evans blue dye extravasation technique was used to assess the permeability of the BBB. Furthermore, immunofluorescence staining was utilized to assess the protein expression levels of matrix metalloproteinase-9 (MMP-9) and intercellular adhesion molecule-1 (ICAM-1). The study also examined mitochondrial function by evaluating mitochondrial swelling, succinate dehydrogenase (SDH) activity, the collapse of mitochondrial membrane potential (MMP), and the generation of reactive oxygen species (ROS). Post-stroke administration of nimodipine led to a significant decrease in cerebral edema and maintained the integrity of the BBB. The protective effects observed were associated with a reduction in cell apoptosis as well as decreased expression of MMP-9 and ICAM-1. Furthermore, nimodipine was observed to reduce mitochondrial swelling and ROS levels while simultaneously restoring MMP and SDH activity. These results suggest that nimodipine may reduce cerebral edema and BBB breakdown caused by ischemia/reperfusion. This effect is potentially mediated through the reduction of MMP-9 and ICAM-1 levels and the enhancement of mitochondrial function.
PubMed: 38950691
DOI: 10.1016/j.neuropharm.2024.110054