The levels and distribution of amyloid deposits in the brain does not correlate well with Alzheimer's disease (AD) progression. Therefore, it is likely that amyloid precursor protein and its ...proteolytic fragments other than amyloid β (Aβ) contribute to the onset of AD.
We developed a sensitive assay adapted to the detection of C99, the direct precursor of β-amyloid. Three postmortem groups were studied: control with normal and stable cognition; patients with moderate AD, and individuals with severe AD. The amount of C99 and Aβ was quantified and correlated with the severity of AD.
C99 accumulates in vulnerable neurons, and its levels correlate with the degree of cognitive impairment in patients suffering from AD. In contrast, Aβ levels are increased in both vulnerable and resistant brain areas.
These results raise the possibility that C99, rather than Aβ plaques, is responsible for the death of nerve cells in AD.
Mammalian inner hair cells (IHCs) transduce sound into depolarization and transmitter release. Big conductance and voltage‐ and Ca2+‐activated K+ (BK) channels are responsible for fast membrane ...repolarization and small time constants of mature IHCs. For unknown reasons, they activate at around ‐75 mV with a voltage of half‐maximum activation (Vhalf) of ‐50 mV although being largely insensitive to Ca2+ influx. Ca2+‐independent activation of BK channels was observed by others in heterologous expression systems if γ subunits leucine‐rich repeat‐containing protein (LRRC)26 (γ1) and LRRC52 (γ2) were coexpressed with the pore‐forming BKα subunit, which shifted Vhalf by ‐140 and ‐100 mV, respectively. Using nested PCR, we consistently detected transcripts for LRRC52 but not for LRRC26 in IHCs of 3‐wk‐old mice. Confocal immunohistochemistry showed synchronous up‐regulation of LRRC52 protein with BKα at the onset of hearing. Colocalization of LRRC52 protein and BKα at the IHC neck within ≤40 nm was specified using an in situ proximity ligation assay. Mice deficient for the voltage‐gated Cav1.3 Ca2+ channel encoded by Cacna1d do not express BKα protein. LRRC52 protein was neither expressed in IHCs of BKα nor in IHCs of Cav1.3 knockout mice. Together, LRRC52 is a γ2 subunit of BK channel complexes and is a strong candidate for causing the Ca2+‐independent activation of BK currents at negative membrane potentials in mouse IHCs.—Lang, I., Jung, M., Niemeyer, B. A., Ruth, P., Engel, J. Expression of the LRRC52 γ subunit (γ2) may provide Ca2+‐independent activation of BK currents in mouse inner hair cells. FASEB J. 33, 11721‐11734 (2019). www.fasebj.org
Tumor‐derived exosomal proteins have emerged as promising biomarkers for cancer diagnosis, but the quantitation accuracy is hindered by large numbers of normal cell‐derived exosomes. Herein, we ...developed a dual‐target‐specific aptamer recognition activated in situ connection system on exosome membrane combined with droplet digital PCR (ddPCR) (TRACER) for quantitation of tumor‐derived exosomal PD‐L1 (Exo‐PD‐L1). Leveraging the high binding affinity of aptamers, excellent selectivity of dual‐aptamer recognition, and the high sensitivity of ddPCR, this method exhibits significant sensitivity and selectivity for tracing tumor‐derived Exo‐PD‐L1 in a wash‐free manner. Due to the excellent sensitivity, the level of tumor‐derived Exo‐PD‐L1 detected by TRACER can distinguish cancer patients from healthy donors, and for the first time was identified as a more reliable tumor diagnostic marker than total Exo‐PD‐L1. The TRACER strategy holds great potential for converting exosomes into reliable clinical indicators and exploring the biological functions of exosomes.
We developed a dual‐target‐specific aptamer recognition system combined with droplet digital PCR for precise quantitative analysis of exosomal PD‐L1 (Exo‐PD‐L1). This method can distinguish tumor‐derived from non‐tumor‐derived Exo‐PD‐L1, holding great potential for the analysis of exosome subtypes and offering unprecedented opportunities for the study of the biological functions of exosomes and their conversion into reliable clinical indicators.
N6-methyladenosine (m6A) modification as the most prevalent mammalian RNA internal modification has been considered as the invasive biomarkers in clinical diagnosis and biological mechanism ...researches. It is still challenged to explore m6A functions due to technical limitations on base- and location-resolved m6A modification. Herein, we firstly proposed a sequence-spot bispecific photoelectrochemical (PEC) strategy based on in situ hybridization mediated proximity ligation assay for m6A RNA characterization with high sensitivity and accuracy. Firstly, the target m6A methylated RNA could be transferred to the exposed cohesive terminus of H1 based on the special self-designed auxiliary proximity ligation assay (PLA) with sequence-spot bispecific recognition. The exposed cohesive terminus of H1 could furtherly trigger the next catalytic hairpin assembly (CHA) amplification and in situ exponential nonlinear hyperbranched hybridization chain reaction for highly sensitive monitoring of m6A methylated RNA. Compared with conventional technologies, the proposed sequence-spot bispecific PEC strategy for m6A methylation of special RNA based on proximity ligation-triggered in situ nHCR performed improved sensitivity and selectivity with a detection limit of 53 fM, providing new insights into highly sensitive monitoring m6A methylation of RNA in bioassay, disease diagnosis and RNA mechanism.
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•Sequence-Site bispecific recognition provide a more accurate and sensitive way to detect N6-methyladenosine modification.•Photoelectrochemistry performed with in situ hybridization mediated proximity linkage analysis.•In situ nonlinear hyperbranched hybridization chain reaction realizes high signal amplification.
Diacylglycerol lipase α (DAGLα), a major biosynthetic enzyme for endogenous cannabinoid signaling, has emerged as a risk gene in multiple psychiatric disorders. However, its role in the regulation of ...dendritic spine plasticity is unclear.
DAGLα wild-type or point mutants were overexpressed in primary cortical neurons or human embryonic kidney 293T cells. The effects of mutated variants on interaction, dendritic spine morphology, and dynamics were examined by proximity ligation assay or fluorescence recovery after photobleaching. Behavioral tests and immunohistochemistry were performed with ankyrin-G conditional knockout and wild-type male mice.
DAGLα modulated dendritic spine size and density, but the effects of changes in its protein level versus enzymatic activity were different, implicating either a 2-arachidonoylglycerol (2-AG)–dependent or –independent mechanism. The 2-AG–independent effects were mediated by the interaction of DAGLα with ankyrin-G, a multifunctional scaffold protein implicated in psychiatric disorders. Using superresolution microscopy, we observed that they colocalized in distinct nanodomains, which correlated with spine size. In situ proximity ligation assay combined with structured illumination microscopy revealed that DAGLα phosphorylation upon forskolin treatment enhanced the interaction with ankyrin-G in spines, leading to increased spine size and decreased DAGLα surface diffusion. Ankyrin-G conditional knockout mice showed significantly decreased DAGLα-positive neurons in the forebrain. In mice, ankyrin-G was required for forskolin-dependent reversal of depression-related behavior.
Taken together, ANK3 and DAGLA, both neuropsychiatric disorder genes, interact in a complex to regulate spine morphology. These data reveal novel synaptic signaling mechanisms and potential therapeutic avenues.
ABSTRACT
5‐Lipoxygenase (5‐LO) catalyzes the initial steps in the biosynthesis of proinflammatory leukotrienes. Upon cell activation, 5‐LO translocates to the nuclear membrane where arachidonic acid ...is transferred by 5‐LO‐activating protein (FLAP) to 5‐LO for metabolism. Although previous data indicate association of 5‐LO with FLAP, the in situ assembly of native 5‐LO/FLAP complexes remains elusive. Here, we show time‐resolved 5‐LO/FLAP colocalization by immunofluorescence microscopy and in situ 5‐LO/FLAP interaction by proximity ligation assay at the nuclear membrane of Ca2+‐ionophore A23187‐activated human monocytes and neutrophils in relation to 5‐LO activity. Although 5‐LO translocation and product formation is completed within 1.5‐3 min, 5‐LO/FLAP interaction is delayed and proceeds up to 30 min. Though monocytes and neutrophils contain comparable amounts of 5‐LO protein, neutrophils produce 3‐5 times higher levels of 5‐LO products due to prolonged activity, accompanied by delayed 5‐LO nuclear membrane translocation. Arachidonic acid seemingly acts as adaptor for 5‐LO/FLAP assembly, whereas FLAP inhibitors (MK886, 100 nM; BAY X 1005, 3 μM) disrupt the complex. We conclude that FLAP may regulate 5‐LO activity in 2 ways: first by inducing an initial flexible association for efficient 5‐LO product synthesis, followed by the formation of a tight 5‐LO/FLAP complex that terminates 5‐LO activity.—Gerstmeier, J., Weinigel, C., Rummler, S., Rådmark, O., Werz, O., Garscha, U. Time‐resolved in situ assembly of the leukotriene‐synthetic 5‐lipoxygenase/5‐lipoxygenase‐activating protein complex in blood leukocytes. FASEB J. 30, 276‐285 (2016). www.fasebj.org
Inositol 1,4,5-trisphosphate (IP3) receptors (IP3Rs) allow extracellular stimuli to redistribute Ca2+ from the ER to cytosol or other organelles. We show, using small interfering RNA (siRNA) and ...vacuolar H+-ATPase (V-ATPase) inhibitors, that lysosomes sequester Ca2+ released by all IP3R subtypes, but not Ca2+ entering cells through store-operated Ca2+ entry (SOCE). A low-affinity Ca2+ sensor targeted to lysosomal membranes reports large, local increases in cytosolic Ca2+ during IP3-evoked Ca2+ release, but not during SOCE. Most lysosomes associate with endoplasmic reticulum (ER) and dwell at regions populated by IP3R clusters, but IP3Rs do not assemble ER-lysosome contacts. Increasing lysosomal pH does not immediately prevent Ca2+ uptake, but it causes lysosomes to slowly redistribute and enlarge, reduces their association with IP3Rs, and disrupts Ca2+ exchange with ER. In a “piston-like” fashion, ER concentrates cytosolic Ca2+ and delivers it, through large-conductance IP3Rs, to a low-affinity lysosomal uptake system. The involvement of IP3Rs allows extracellular stimuli to regulate Ca2+ exchange between the ER and lysosomes.
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•IP3 receptors (IP3Rs) selectively deliver Ca2+ to lysosomes•Lysosomes associate preferentially with clusters of IP3Rs in ER membranes•Low lysosomal pH maintains the IP3R-lysosome contacts required for Ca2+ uptake•ER and its Ca2+ channels deliver Ca2+ to low-affinity lysosomal transporters
Ca2+ exchanges between ER and lysosomes regulate cytosolic Ca2+ signals and lysosome behavior. Atakpa et al. show that clusters of IP3 receptors populate ER-lysosome contact sites and facilitate local delivery of Ca2+ from the ER to lysosomes.
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•Using Proximity Ligation Assay we have demonstrated the direct interaction of BDNF with its receptor, Trkβ, in the rat cingulate cortex.•A significant increase in BDNF-Trkβ ...interaction was observed shortly (3 h) after both acute and repeated administration of an antidepressant drug, imipramine (IMI).•None of the treatments with IMI induced alterations in the level of mRNA encoding BDNF and Trkβ.•Acute IMI administration induces a strong pro-cognitive effect, assessed by the NOR test.
Given the important role of brain-derived neurotrophic factor (BDNF)-mediated Trkβ signalling in the mechanism of action of antidepressants (ADs), we examined ligand–receptor interactions in the rat cingulate cortex using a proximity ligation assay (PLA) in response to acute and repeated administration of imipramine (IMI), followed by various drug-free periods. Both the acute and chronic administration of IMI increased the BDNF-Trkβ interaction observed 3 h after drug administration. Withdrawal of IMI for 72 h or 7 days did not alter BDNF-Trkβ interaction. A significant reduction in this interaction after chronic IMI administration followed by 21 drug-free days was observed, but it returned to the control value when a new dose of IMI was given after this time. The level of mRNA encoding BDNF or Trkβ did not change in the experimental groups of animals, so one can conclude that alterations in the BDNF-Trkβ interaction depend not on acute vs. repeated treatment with IMI but on the presence of the drug in the body. This effect correlates well with the strong pro-cognitive effect of acute IMI, assessed by the novel object recognition (NOR) test.
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