Crystal Violet Selectively Detects Aß Oligomers but Not Fibrils In Vitro and in Alzheimer's Disease Brain Tissue.

Deposition of extracellular Amyloid Beta (Aß) and intracellular tau fibrils in post-mortem brains remains the only way to conclusively confirm cases of Alzheimer's Disease (AD). Substantial evidence, though, implicates small globular oligomers instead of fibrils as relevant biomarkers of, and critical contributors to, the clinical symptoms of AD. Efforts to verify and utilize amyloid oligomers as AD biomarkers in vivo have been limited by the near-exclusive dependence on conformation-selective antibodies for oligomer detection. While antibodies have yielded critical evidence for the role of both Aß and tau oligomers in AD, they are not suitable for imaging amyloid oligomers in vivo. Therefore, it would be desirable to identify a set of oligomer-selective small molecules for subsequent development into Positron Emission Tomography (PET) probes. Using a kinetics-based screening assay, we confirm that the triarylmethane dye Crystal Violet (CV) is oligomer-selective for Aß42 oligomers (AßOs) grown under near-physiological solution conditions in vitro. In postmortem brains of an AD mouse model and human AD patients, we demonstrate that A11 antibody-positive oligomers but not Thioflavin S (ThioS)-positive fibrils colocalize with CV staining, confirming in vitro results. Therefore, our kinetic screen represents a robust approach for identifying new classes of small molecules as candidates for oligomer-selective dyes (OSDs). Such OSDs, in turn, provide promising starting points for the development of PET probes for pre-mortem imaging of oligomer deposits in humans.

The use of bubble charts in analyzing the global second-stage cesarean delivery rates: a systematic review.

OBJECTIVE: This study aimed to systematically review the worldwide second-stage cesarean delivery rate concerning pre-second-stage cesarean delivery and assisted vaginal birth rates. DATA SOURCES: PubMed, Medline Ovid, EBSCOhost, Embase, Scopus, and Google Scholar were queried from inception to February 2023, with the following terms: "full dilatation," "second stage," and "cesarean," with their word variations. Furthermore, an additional cohort of 353,434 cases from our recently published study was included. STUDY ELIGIBILITY CRITERIA: Only original studies that provided sufficient information on the number of pre-second-stage cesarean deliveries, second-stage cesarean deliveries, and vaginal births were included for the calculation of different modes of delivery. Systemic reviews, meta-analyses, or case reports were excluded. METHODS: Study identification and data extraction were independently performed by 2 authors. Selected studies were categorized on the basis of parity, study period, and geographic regions for comparison. RESULTS: A total of 25 studies were included. The overall pre-second-stage cesarean delivery rate, the second-stage cesarean delivery rate, and the second-stage cesarean delivery-to-assisted vaginal birth ratio were 17.94%, 2.65%, and 0.19, respectively. Only 5 studies described singleton, term, cephalic presenting pregnancies of nulliparous women, and their second-stage cesarean delivery rates were significantly higher than those studies with cohorts of all parity groups (4.50% vs 0.83%; P<.05). In addition, the second-stage cesarean delivery rate showed a secular increase across 2009 (0.70% vs 1.05%; P<.05). Moreover, it was the highest among African studies (5.14%) but the lowest among studies from East Asia and South Asia (0.94%). The distributions of second-stage cesarean delivery rates of individual studies and subgroups were shown with that of pre-second-stage cesarean delivery and assisted vaginal birth using the bubble chart. CONCLUSION: The overall worldwide pre-second-stage cesarean delivery rate was 17.94%, the second-stage cesarean delivery rate was 2.65%, and the second-stage cesarean delivery-to-assisted vaginal birth ratio was 0.19. The African studies had the highest second-stage cesarean delivery rate (5.14%) and second-stage cesarean delivery-to-assisted vaginal birth ratio (1.88), whereas the studies from East Asia and South Asia were opposite (0.94% and 0.11, respectively).

The use of bubble charts in analyzing second stage cesarean delivery rates.

BACKGROUND: The incidence of second stage cesarean delivery has been rising globally because of the failure or the anticipated difficulty of performing instrumental delivery. Yet, the best way to interpret the figure and its optimal rate remain to be determined. This is because it is strongly influenced by the practice of other 2 modes of birth, namely cesarean delivery performed before reaching the second stage and assisted vaginal birth during the second stage. In this regard, a bubble chart that can display 3-dimensional data through its x-axis, y-axis, and the size of each plot (presented as a bubble) may be a suitable method to evaluate the relationship between the rates of these 3 modes of births. OBJECTIVE: This study aimed to conduct an epidemiologic study on the incidence of second stage cesarean deliveries rates among >300,000 singleton term births in 10 years from 8 obstetrical units and to compare their second stage cesarean delivery rates in relation to their pre-second stage cesarean delivery rates and assisted vaginal birth rates using a bubble chart. STUDY DESIGN: The territory-wide birth data collected between 2009 and 2018 from all 8 public obstetrical units (labelled as A to H) were reviewed. The inclusion criteria were all singleton pregnancies with cephalic presentation that were delivered at term (≥37 weeks' gestation). Pre-second stage cesarean delivery rate was defined as all elective cesarean deliveries and those emergency cesarean deliveries that occurred before full cervical dilatation was achieved as a proportion of the total number of births. The second stage cesarean delivery rate and assisted vaginal birth rate were calculated according to the respective mode of delivery as a proportion of the number of cases that reached full cervical dilatation. The rates of these 3 modes of births were compared among the parity groups and among the 8 units. Using a bubble chart, each unit's second stage cesarean delivery rate (y-axis) was plotted against its pre-second stage cesarean delivery rate (x-axis) as a bubble. Each unit's second stage cesarean delivery to assisted vaginal birth ratio was represented by the size of the bubble. RESULTS: During the study period, a total of 353,434 singleton cephalic presenting term pregnancies were delivered in the 8 units, and 180,496 (51.1%) were from nulliparous mothers. When compared with the multiparous group, the nulliparous group had a significantly lower pre-second stage cesarean delivery rate (18.58% vs 21.26%; P<.001) but a higher second stage cesarean delivery rate (0.79% vs 0.22%; P<.001) and a higher assisted vaginal birth rate (17.61% vs 3.58%; P<.001). Using the bubble of their averages as a reference point in the bubble chart, the 8 units' bubbles were clustered into 5 regions indicating their differences in practice: unit B and unit H were close to the average in the center. Unit A and unit F were at the upper right corner with a higher pre-second stage cesarean delivery rate and second stage cesarean delivery rate. Unit D and unit E were at the opposite end. Unit C was at the upper left corner with a low pre-second stage cesarean delivery rate but a high second stage cesarean delivery rate, whereas unit G was at the opposite end. Unit C and unit G were also in the extremes in terms of pre-second stage cesarean delivery to assisted vaginal birth ratio (0.09 and 0.01, respectively). Although some units seemed to have very similar second stage cesarean delivery rates, their obstetrical practices were differentiated by the bubble chart. CONCLUSION: The second stage cesarean delivery rate must be evaluated in the context of the rates of pre-second stage cesarean delivery and assisted vaginal birth. A bubble chart is a useful method for analyzing the relationship among these 3 variables to differentiate the obstetrical practice between different units.

The multifaceted functions of ß-arrestins and their therapeutic potential in neurodegenerative diseases.

Arrestins are multifunctional proteins that regulate G-protein-coupled receptor (GPCR) desensitization, signaling, and internalization. The arrestin family consists of four subtypes: visual arrestin1, ß-arrestin1, ß-arrestin2, and visual arrestin-4. Recent studies have revealed the multifunctional roles of ß-arrestins beyond GPCR signaling, including scaffolding and adapter functions, and physically interacting with non-GPCR receptors. Increasing evidence suggests that ß-arrestins are involved in the pathogenesis of a variety of neurodegenerative diseases, including Alzheimer's disease (AD), frontotemporal dementia (FTD), and Parkinson's disease (PD). ß-arrestins physically interact with γ-secretase, leading to increased production and accumulation of amyloid-beta in AD. Furthermore, ß-arrestin oligomers inhibit the autophagy cargo receptor p62/SQSTM1, resulting in tau accumulation and aggregation in FTD. In PD, ß-arrestins are upregulated in postmortem brain tissue and an MPTP model, and the ß2AR regulates SNCA gene expression. In this review, we aim to provide an overview of ß-arrestin1 and ß-arrestin2, and describe their physiological functions and roles in neurodegenerative diseases. The multifaceted roles of ß-arrestins and their involvement in neurodegenerative diseases suggest that they may serve as promising therapeutic targets.

Disruption of Mitophagy Flux through the PARL-PINK1 Pathway by CHCHD10 Mutations or CHCHD10 Depletion.

Coiled-coil-helix-coiled-coil-helix domain-containing 10 (CHCHD10) is a nuclear-encoded mitochondrial protein which is primarily mutated in the spectrum of familial and sporadic amyotrophic lateral sclerosis (ALS)-frontotemporal dementia (FTD). Endogenous CHCHD10 levels decline in the brains of ALS-FTD patients, and the CHCHD10S59L mutation in Drosophila induces dominant toxicity together with PTEN-induced kinase 1 (PINK1), a protein critical for the induction of mitophagy. However, whether and how CHCHD10 variants regulate mitophagy flux in the mammalian brain is unknown. Here, we demonstrate through in vivo and in vitro models, as well as human FTD brain tissue, that ALS/FTD-linked CHCHD10 mutations (R15L and S59L) impair mitophagy flux and mitochondrial Parkin recruitment, whereas wild-type CHCHD10 (CHCHD10WT) normally enhances these measures. Specifically, we show that CHCHD10R15L and CHCHD10S59L mutations reduce PINK1 levels by increasing PARL activity, whereas CHCHD10WT produces the opposite results through its stronger interaction with PARL, suppressing its activity. Importantly, we also demonstrate that FTD brains with TAR DNA-binding protein-43 (TDP-43) pathology demonstrate disruption of the PARL-PINK1 pathway and that experimentally impairing mitophagy promotes TDP-43 aggregation. Thus, we provide herein new insights into the regulation of mitophagy and TDP-43 aggregation in the mammalian brain through the CHCHD10-PARL-PINK1 pathway.

Slingshot homolog-1-mediated Nrf2 sequestration tips the balance from neuroprotection to neurodegeneration in Alzheimer's disease.

Oxidative damage in the brain is one of the earliest drivers of pathology in Alzheimer's disease (AD) and related dementias, both preceding and exacerbating clinical symptoms. In response to oxidative stress, nuclear factor erythroid 2-related factor 2 (Nrf2) is normally activated to protect the brain from oxidative damage. However, Nrf2-mediated defense against oxidative stress declines in AD, rendering the brain increasingly vulnerable to oxidative damage. Although this phenomenon has long been recognized, its mechanistic basis has been a mystery. Here, we demonstrate through in vitro and in vivo models, as well as human AD brain tissue, that Slingshot homolog-1 (SSH1) drives this effect by acting as a counterweight to neuroprotective Nrf2 in response to oxidative stress and disease. Specifically, oxidative stress-activated SSH1 suppresses nuclear Nrf2 signaling by sequestering Nrf2 complexes on actin filaments and augmenting Kelch-like ECH-associated protein 1 (Keap1)-Nrf2 interaction, independently of SSH1 phosphatase activity. We also show that Ssh1 elimination in AD models increases Nrf2 activation, which mitigates tau and amyloid-ß accumulation and protects against oxidative injury, neuroinflammation, and neurodegeneration. Furthermore, loss of Ssh1 preserves normal synaptic function and transcriptomic patterns in tauP301S mice. Importantly, we also show that human AD brains exhibit highly elevated interactions of Nrf2 with both SSH1 and Keap1. Thus, we demonstrate here a unique mode of Nrf2 blockade that occurs through SSH1, which drives oxidative damage and ensuing pathogenesis in AD. Strategies to inhibit SSH1-mediated Nrf2 suppression while preserving normal SSH1 catalytic function may provide new neuroprotective therapies for AD and related dementias.

A Par3/LIM Kinase/Cofilin Pathway Mediates Human Airway Smooth Muscle Relaxation by TAS2R14.

TAS2Rs (bitter taste receptors) are GPCRs (G protein-coupled receptors) expressed on human airway smooth muscle (HASM) cells; when activated by receptor agonists they evoke marked airway relaxation. In both taste and HASM cells, TAS2Rs activate a canonical Gßγ-mediated stimulation of Ca2+ release from intracellular stores by activation of PLCß (phospholipase Cß). Alone, this [Ca2+]i signaling does not readily account for relaxation, particularly since bronchoconstrictive agonists acting at Gq-coupled receptors also increase [Ca2+]i. We established that TAS2R14 activation in HASM promotes relaxation through F-actin (filamentous actin) severing. This destabilization of actin was from agonist-promoted activation (dephosphorylation) of cofilin, which was pertussis toxin sensitive. Cofilin dephosphorylation was due to TAS2R-mediated deactivation of LIM domain kinase. The link between early receptor action and the distal cofilin dephosphorylation was found to be the polarity protein partitioning defective 3 (Par3), a known binding partner with PLCß that inhibits LIM kinase. The physiologic relevance of this pathway was assessed using knock-downs of cofilin and Par3 in HASM cells and in human precision-cut lung slices. Relaxation by TAS2R14 agonists was ablated with knock-down of either protein as assessed by magnetic twisting cytometry in isolated cells or intact airways in the slices. Blocking [Ca2+]i release by TAS2R14 inhibited agonist-promoted cofilin dephosphorylation, confirming a role for [Ca2+]i in actin-modifying pathways. These results further elucidate the mechanistic basis of TAS2R-mediated HASM relaxation and point toward nodal points that may act as asthma or chronic obstructive pulmonary disease response modifiers or additional targets for novel bronchodilators.

Slingshot homolog-1 mediates the secretion of small extracellular vesicles containing misfolded proteins by regulating autophagy cargo receptors and actin dynamics.

Increasing evidence indicates that the accumulation misfolded proteins in Alzheimer's disease (AD) arises from clearance defects in the autophagy-lysosome pathway. Misfolded proteins such as Aß and tau are secreted in small extracellular vesicles (i.e., exosomes) and are propagated from cell to cell in part through secreted small extracellular vesicles (sEVs). Recent studies suggest that autophagic activity and exosome secretion are coregulated events, and multiple autophagy-related proteins are found in sEVs, including the cargo receptors Sqstm1/p62 and optineurin. However, whether and how autophagy cargo receptors per se regulate the secretion of sEVs is unknown. Moreover, despite the prominent role of actin dynamics in secretory vesicle release, its role in EV secretion is unknown. In this study, we leveraged the dual axes of Slingshot Homolog-1 (SSH1), which inhibits Sqstm1/p62-mediated autophagy and activates cofilin-mediated actin dynamics, to study the regulation of sEV secretion. Here we show that cargo receptors Sqstm1/p62 and optineurin inhibit sEV secretion, an activity that requires their ability to bind ubiquitinated cargo. Conversely, SSH1 increases sEV secretion by dephosphorylating Sqstm1/p62 at pSer403, the phospho-residue that allows Sqstm1/p62 to bind ubiquitinated cargo. In addition, increasing actin dynamics through the SSH1-cofilin activation pathway also increases sEV secretion, which is mimicked by latrunculin B treatment. Finally, Aß42 oligomers and mutant tau increase sEV secretion and are physically associated with secreted sEVs. These findings suggest that increasing cargo receptor engagement with autophagic cargo and reducing actin dynamics (i.e., SSH1 inhibition) represents an attractive strategy to promote misfolded protein degradation while reducing sEV-mediated cell to cell spread of pathology.

Pathological characterization of a novel mouse model expressing the PD-linked CHCHD2-T61I mutation.

Coiled-coil-helix-coiled-coil-helix domain containing 2 (CHCHD2) is a mitochondrial protein that plays important roles in cristae structure, oxidative phosphorylation and apoptosis. Multiple mutations in CHCHD2 have been associated with Lewy body disorders (LBDs), such as Parkinson's disease (PD) and dementia with Lewy bodies, with the CHCHD2-T61I mutation being the most widely studied. However, at present, only CHCHD2 knockout or CHCHD2/CHCHD10 double knockout mouse models have been investigated. They do not recapitulate the pathology seen in patients with CHCHD2 mutations. We generated the first transgenic mouse model expressing the human PD-linked CHCHD2-T61I mutation driven by the mPrP promoter. We show that CHCHD2-T61I Tg mice exhibit perinuclear mitochondrial aggregates, neuroinflammation, and have impaired long-term synaptic plasticity associated with synaptic dysfunction. Dopaminergic neurodegeneration, a hallmark of PD, is also observed along with α-synuclein pathology. Significant motor dysfunction is seen with no changes in learning and memory at 1 year of age. A minor proportion of the CHCHD2-T61I Tg mice (~10%) show a severe motor phenotype consistent with human Pisa Syndrome, an atypical PD phenotype. Unbiased proteomics analysis reveals surprising increases in many insoluble proteins predominantly originating from mitochondria and perturbing multiple canonical biological pathways as assessed by ingenuity pathway analysis, including neurodegenerative disease-associated proteins such as tau, cofilin, SOD1 and DJ-1. Overall, CHCHD2-T61I Tg mice exhibit pathological and motor changes associated with LBDs, indicating that this model successfully captures phenotypes seen in human LBD patients with CHCHD2 mutations and demonstrates changes in neurodegenerative disease-associated proteins, which delineates relevant pathological pathways for further investigation.

ß-arrestin1 promotes tauopathy by transducing GPCR signaling, disrupting microtubules and autophagy.

G protein-coupled receptors (GPCRs) have been shown to play integral roles in Alzheimer's disease pathogenesis. However, it is unclear how diverse GPCRs similarly affect Aß and tau pathogenesis. GPCRs share a common mechanism of action via the ß-arrestin scaffolding signaling complexes, which not only serve to desensitize GPCRs by internalization, but also mediate multiple downstream signaling events. As signaling via the GPCRs, ß2-adrenergic receptor (ß2AR), and metabotropic glutamate receptor 2 (mGluR2) promotes hyperphosphorylation of tau, we hypothesized that ß-arrestin1 represents a point of convergence for such pathogenic activities. Here, we report that ß-arrestins are not only essential for ß2AR and mGluR2-mediated increase in pathogenic tau but also show that ß-arrestin1 levels are increased in brains of Frontotemporal lobar degeneration (FTLD-tau) patients. Increased ß-arrestin1 in turn drives the accumulation of pathogenic tau, whereas reduced ARRB1 alleviates tauopathy and rescues impaired synaptic plasticity and cognitive impairments in PS19 mice. Biochemical and cellular studies show that ß-arrestin1 drives tauopathy by destabilizing microtubules and impeding p62/SQSTM1 autophagy flux by interfering with p62 body formation, which promotes pathogenic tau accumulation.

Mitochondrial CHCHD2: Disease-Associated Mutations, Physiological Functions, and Current Animal Models.

Rare mutations in the mitochondrial protein coiled-coil-helix-coiled-coil-helix domain containing 2 (CHCHD2) are associated with Parkinson's disease (PD) and other Lewy body disorders. CHCHD2 is a bi-organellar mediator of oxidative phosphorylation, playing crucial roles in regulating electron flow in the mitochondrial electron transport chain and acting as a nuclear transcription factor for a cytochrome c oxidase subunit (COX4I2) and itself in response to hypoxic stress. CHCHD2 also regulates cell migration and differentiation, mitochondrial cristae structure, and apoptosis. In this review, we summarize the known disease-associated mutations of CHCHD2 in Asian and Caucasian populations, the physiological functions of CHCHD2, how CHCHD2 mutations contribute to α-synuclein pathology, and current animal models of CHCHD2. Further, we discuss the necessity of continued investigation into the divergent functions of CHCHD2 and CHCHD10 to determine how mutations in these similar mitochondrial proteins contribute to different neurodegenerative diseases.

SSH1 impedes SQSTM1/p62 flux and MAPT/Tau clearance independent of CFL (cofilin) activation.

Accumulation of toxic protein assemblies and damaged mitochondria are key features of neurodegenerative diseases, which arise in large part from clearance defects in the Macroautophagy/autophagy-lysosome system. The autophagy cargo receptor SQSTM1/p62 plays a major role in the clearance of ubiquitinated cargo through Ser403 phosphorylation by multiple kinases. However, no phosphatase is known to physiologically dephosphorylate SQSTM1 on this activating residue. RNAi-mediated knockdown and overexpression experiments using genetically encoded fluorescent reporters and defined mutant constructs in cell lines, primary neurons, and brains show that SSH1, the canonical CFL (cofilin) phosphatase, mediates the dephosphorylation of phospho-Ser403-SQSTM1, thereby impairing SQSTM1 flux and phospho-MAPT/tau clearance. The inhibitory action of SSH1 on SQSTM1 is fully dependent on SQSTM1 Ser403 phosphorylation status and is separable from SSH1-mediated CFL activation. These findings reveal a unique action of SSH1 on SQSTM1 independent of CFL and implicate an inhibitory role of SSH1 in SQSTM1-mediated clearance of autophagic cargo, including phospho-MAPT/tau. Abbreviations: AAV: adeno-associated virus; Aß42O: amyloid ß1-42 oligomers; AD: Alzheimer disease; CA3: cornu Ammonis 3; CSNK2/CK2: casein kinase 2; FCCP: 2-[2-[4-(trifluoromethoxy)phenyl]hydrazinylidene]-propanedinitrile; FTLD: frontotemporal lobar degeneration; GFP: green fluorescent protein; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; SQSTM1/p62: sequestosome-1; PLA: proximity ligation assay; RFP: red fluorescent protein; RIPA: radioimmunoprecipitation assay; shRNA: short hairpin RNA; siRNA: small interfering RNA; Ser403: Serine403; SSH1: slingshot protein phosphatase 1; TBK1: TANK-binding kinase 1; ULK: unc-51 like kinase 1.

ß-Arrestin2 oligomers impair the clearance of pathological tau and increase tau aggregates.

Multiple G protein-coupled receptors (GPCRs) are targets in the treatment of dementia, and the arrestins are common to their signaling. ß-Arrestin2 was significantly increased in brains of patients with frontotemporal lobar degeneration (FTLD-tau), a disease second to Alzheimer's as a cause of dementia. Genetic loss and overexpression experiments using genetically encoded reporters and defined mutant constructs in vitro, and in cell lines, primary neurons, and tau P301S mice crossed with ß-arrestin2-/- mice, show that ß-arrestin2 stabilizes pathogenic tau and promotes tau aggregation. Cell and mouse models of FTLD showed this to be maladaptive, fueling a positive feedback cycle of enhanced neuronal tau via non-GPCR mechanisms. Genetic ablation of ß-arrestin2 markedly ablates tau pathology and rescues synaptic plasticity defects in tau P301S transgenic mice. Atomic force microscopy and cellular studies revealed that oligomerized, but not monomeric, ß-arrestin2 increases tau by inhibiting self-interaction of the autophagy cargo receptor p62/SQSTM1, impeding p62 autophagy flux. Hence, reduction of oligomerized ß-arrestin2 with virus encoding ß-arrestin2 mutants acting as dominant-negatives markedly reduces tau-laden neurofibrillary tangles in FTLD mice in vivo. Reducing ß-arrestin2 oligomeric status represents a new strategy to alleviate tau pathology in FTLD and related tauopathies.

Activated cofilin exacerbates tau pathology by impairing tau-mediated microtubule dynamics.

Alzheimer's disease (AD) is a progressive neurodegenerative disorder and the most common form of dementia. While the accumulation of Aß is pivotal to the etiology of AD, both the microtubule-associated protein tau (MAPT) and the F-actin severing protein cofilin are necessary for the deleterious effects of Aß. However, the molecular link between tau and cofilin remains unclear. In this study, we found that cofilin competes with tau for direct microtubule binding in vitro, in cells, and in vivo, which inhibits tau-induced microtubule assembly. Genetic reduction of cofilin mitigates tauopathy and synaptic defects in Tau-P301S mice and movement deficits in tau transgenic C. elegans. The pathogenic effects of cofilin are selectively mediated by activated cofilin, as active but not inactive cofilin selectively interacts with tubulin, destabilizes microtubules, and promotes tauopathy. These results therefore indicate that activated cofilin plays an essential intermediary role in neurotoxic signaling that promotes tauopathy.