Brain Lysates Research Articles

Can Magnoflorine Improve Memory? Immunohistochemical Studies on Parvalbumin Immunoreactive Neurons and Fibers of Mice Hippocampus.

Background/Objectives: We assessed the influence of long-term injection of magnoflorine (MAG) on memory acquisition in mice for the first time. Methods: This isoquinoline alkaloid that belongs to the aporphines was isolated from the roots of Berberis vulgaris by centrifugal partition chromatography (CPC) using a biphasic solvent system composed of chloroform: methanol: water in the ratio 4:3:3 (v/v/v) with 20 mM of hydrochloric acid and triethylamine, within 64 min. Results: Our results indicated that long-term injection of MAG 20 mg/kg dose improve the long-term memory acquisition in mice that were evaluated in the passive avoidance (PA) test with no toxicity records. The analysis of brain lysates and animal plasma by HPLC-ESI-QTOF-MS/MS showed the ability of the compound to cross the blood-brain barrier, and an elevated level of phosphatidylcholine PC (14:1(9Z)/14:1(9Z)) with the molecular formula of C36H69NO8P was observed in both treated groups with 10 mg/kg and 20 mg/kg MAG in comparison to the control group. Conclusions: This phenomenon may explain MAG's cognition-enhancing properties as the PC may induce the synthesis and strengthening of neuronal cells. Also, the 7-day-long administration of MAG at 10 mg/kg and 20 mg/kg increased the mean number of parvalbumin (PV)-IR neurons in the hippocampus. Statistically, the largest PV-IR neurons were observed at the 20 mg/kg dose, which may indicate a potential effect of MAG on Ca2+ metabolism. However, no statistical differences were observed in the mean number of PV-IR nerve fibers in both doses of MAG, regardless of the hippocampal fields. This positive effect of MAG on hippocampal neurons provides further support for the neuroprotective effect of this alkaloid.

PTau pathology in the retina of TAU58 mice: association with ganglion cell degeneration and implications on seeding and propagation of pTau from human brain lysates

The accumulation of abnormal phosphorylated Tau protein (pTau) in neurons of the brain is a pathological hallmark of Alzheimer’s disease (AD). PTau pathology also occurs in the retina of AD cases. Accordingly, questions arise whether retinal pTau can act as a potential seed for inducing cerebral pTau pathology and whether retinal pTau pathology causes degeneration of retinal neurons. To address these questions, we (1) characterized pTau pathology in the retina of TAU58 mice, (2) determined the impact of pTau pathology on retinal ganglion cell density, and (3) used this mouse model to test whether brain lysates from AD and/or non-AD control cases induce seeding in the retina and/or propagation into the brain. TAU58 mice developed retinal pTau pathology at 6 months of age, increasing in severity and extent with age. TAU58 mice showed reduced retinal ganglion cell density compared to wild-type mice, which declined with age and pTau pathology progression. Brain lysates from non-AD Braak neurofibrillary tangle (NFT) stage I controls increased retinal pTau pathology after subretinal injection compared to phosphate-buffered saline (PBS) but did not accelerate pTau pathology in the brain. In contrast, subretinally injected AD brain lysates accelerated pTau pathology in the retina and the contralateral superior colliculus. Subretinal injection of AD brain lysates, but not of non-AD brain, induced in this context a neuroinflammatory response in the retina and in the contralateral primary visual cortex. These results lead to the following conclusions: (1) Brain lysates from AD and non-AD sources can accelerate tauopathy within the retina. (2) The anterograde propagation of pTau pathology from the retina to the brain can be triggered by subretinal injections of AD brain lysates. (3) Such subretinal injections also provoke a neuroinflammatory response in both the retina and the visual cortex. (4) The accumulation of retinal pTau is associated with the degeneration of the involved ganglion cells, indicating that retinal tauopathy might contribute to vision impairment in the elderly and underscore the retina’s potential role in spreading tau pathology to the brain.

Nitro‐oxidation of BKCa mediates neuro‐ and micro‐vascular dysfunction in female 5x‐FAD mice

AbstractBackgroundCerebral microvascular dysfunction and nitro‐oxidative stress are present in patients with Alzheimer’s disease (AD) and may contribute to disease progression and severity. A pro‐nitro‐oxidative environment can lead to post‐translational modifications of ion channels central to microvascular regulation in the brain, including the large conductance Ca2+‐activated K+ channels (BKCa). Nitro‐oxidative modulation of BKCa can resulting in decreased activity and vascular hyper‐contractility, thus compromising neurovascular regulation. We hypothesized that nitro‐oxidation of vascular BKCa function blunts micro‐ and neuro‐vascular responses in 5x‐FAD mice.MethodsSix‐months‐old female 5x‐FAD and wild‐type (WT) littermates were used. Brain lysates were used to quantify BKCa nitro‐oxidation. Cerebral arteries lysates were used to assess BK subunit expression by qPCR. A different set of cerebral arteries were used for patch clamp electrophysiology assessment of BKCa currents, ex vivo pressure myography and quantification of calcium (Ca2+) sparks in smooth muscle cells (SMC) by spinning‐disk confocal microscopy. Basal cerebral perfusion and neurovascular coupling were assessed by laser speckle contrast imaging. Differences between means were tested using t‐tests and considered statistically significant when P<0.05.ResultsWe observed that BKCa S‐nitrosylation, a proxy for nitro‐oxidative modification, was increased in the brain of AD patients and in 5x‐FAD females. In functional experiments, we observed that BKCa currents were lower in cerebral artery SMC of 5x‐FAD than in WT. As a consequence, microvascular reactivity to BKCa blockade (iberiotoxin, 30 nM) was blunted in 5x‐FAD, suggesting lower basal BKCa activity. This reduction in microvascular BKCa activity was independent of SMC Ca2+ sparks or BKCa mRNA expression. We then performed rescue experiments by incubating membrane patches and pressurized arteries with the reducing agent dithiothreitol (DTT, 10 µM), and observed that DTT restored BKCa currents and sensitivity to iberiotoxin in cerebral artery SMC from 5x‐FAD. In vivo experiments showed that basal perfusion to the frontal cortex and neurovascular coupling were lower in 5x‐FAD when compared to WT littermates.ConclusionThese data suggest that excessive nitro‐oxidation of BKCa mediates neuro‐ and micro‐vascular impairments in female 5x‐FAD mice, and that this process is reversible and can be rescued by reducing agents.Funding: National Institutes on Aging and Alzheimer’s Association

Optimizing Ultrasensitive RT‐QuiC assay on AD tau seed‐monomer reactions

AbstractBackgroundAlzheimer’s disease (AD) is pathologically defined by the presence of extracellular Aβ plaque and intracellular tau inclusions. Emerging evidence shows that tau aggregates contain pathogenic bioactivities of templating monomeric tau into filamentous fibrils and propagating through cells. Based on these findings, assays have been developed to detect minute amounts of pathogenic tau in human samples. With great potential, it remains unclear about the sensitivity and fidelity of tau fibrillization due to the presence of pathogenic tau species.MethodsTo that end, our study focuses on optimizing the Real‐Time Quaking‐Induced Conversion (RT‐QuiC) assay for its potential of detecting and amplifying insoluble tau seeds derived from AD patient. To this end, we utilized recombinant tau monomers t306 and k12, 2R and 3R tau peptide respectively known for their rapid fibrillization and high amplification signals in previous studies. More specifically, we optimized the RT‐QuiC assay using both crude and purified brain lysates from AD and control cases.ResultsThe specificity of the RT‐QuiC assay were successfully reproduced by multiple cases of AD and control brain lysates. Moreover, we tested the RT‐QuiC assay with purified AD tau seeds and assessed the sensitivity of the assays based on the amount of insoluble tau. To further interrogate the system, we also evaluated the impact of reaction buffer on the ThT‐based measurement of tau fibrillization. Our results show that the original RT‐QuiC assay can sensitively detect insoluble AD tau seeds at the nM level in our hands. The optimized RT‐QuiC assay could differentiate better between AD‐seeded reactions and controls with an improved sensitivity up to fM level, with a better separation between AD and control and faster reaction rate. The assay showed a clear dose‐dependent response till pM range.ConclusionUpon optimization, the RT‐QuiC assay is both ultrasensitive and specific to AD tau fibrillization. Our findings offer insights into the fibrillization mechanism of tau aggregates and expand our understanding of pathogenic tau seeds in AD. By optimizing the assay to more accessible patient samples in the future, namely CSF and plasma, the RT‐QuiC can serve as a potent diagnostic tool for AD tau progressions.

Seeding-competent TDP-43 persists in human patient and mouse muscle.

TAR DNA binding protein 43 (TDP-43) is an RNA binding protein that accumulates as aggregates in the central nervous systems of some patients with neurodegenerative diseases. However, TDP-43 aggregation is also a sensitive and specific pathologic feature found in a family of degenerative muscle diseases termed inclusion body myopathy. TDP-43 aggregates from amyotrophic lateral sclerosis (ALS) and frontotemporal dementia brain lysates may serve as self-templating aggregate seeds in vitro and in vivo, supporting a prion-like spread from cell to cell. Whether a similar process occurs in patient muscle is not clear. We developed a mouse model of inducible, muscle-specific cytoplasmic localized TDP-43. These mice develop muscle weakness with robust accumulation of insoluble and phosphorylated sarcoplasmic TDP-43, leading to eosinophilic inclusions, altered proteostasis, and changes in TDP-43-related RNA processing that resolve with the removal of doxycycline. Skeletal muscle lysates from these mice also have seeding-competent TDP-43, as determined by a FRET-based biosensor, that persists for weeks upon resolution of TDP-43 aggregate pathology. Human muscle biopsies with TDP-43 pathology also contain TDP-43 aggregate seeds. Using lysates from muscle biopsies of patients with sporadic inclusion body myositis (IBM), immune-mediated necrotizing myopathy (IMNM), and ALS, we found that TDP-43 seeding capacity was specific to IBM. TDP-43 seeding capacity anticorrelated with TDP-43 aggregate and vacuole abundance. These data support that TDP-43 aggregate seeds are present in IBM skeletal muscle and represent a unique TDP-43 pathogenic species not previously appreciated in human muscle disease.

Dihydrosamidin: the basic khellactone ester derived from Phlojodicarpus komarovii and its impact on neurotrophic factors, energy and antioxidant metabolism after rat cerebral ischemia–reperfusion injury

Dihydrosamidin (DHS), a khellactone 3′-O-isovaleroyl-4′-O-acetyl ester, is naturally found in Apiaceae plants yet remains underexplored in biomedical research. Employing HPLC-PDA-MS analysis, the profiling of coumarins in Phlojodicarpus komarovii revealed its significant presence, reaching DHS concentrations of 95 mg/g. DHS administering at a dosage of 80 mg/kg during bilateral transient occlusion of the common carotid artery in Wistar rats prevented neuronal death and decreased neuron-specific enolase levels in the blood serum, increases neurotrophic factors and vascular endothelial growth factor A levels in brain lysate. DHS influenced energy metabolism by reducing the lactate, enhancing the activity of pyruvate kinase and increasing the activities of NADH dehydrogenase and succinate dehydrogenase in brain cells. DHS reduced levels of malondialdehyde and increased activities of superoxide dismutase, catalase, glutathione reductase, glutathione peroxidase, along with increased levels of reduced glutathione in brain homogenate. Thus, DHS administration promotes neuroplasticity, modulates glycolysis and oxidative phosphorylation and enhances antioxidant defences.

High-molecular-weight oligomer tau (HMWoTau) species are dramatically increased in Braak-stage dependent manner in the frontal lobe of human brains, demonstrated by a novel oligomer Tau ELISA with a mouse monoclonal antibody (APNmAb005).

Disease-specific oligomers Tau assay system is anticipated in Alzheimer disease (AD) to elucidate their etiological roles. We developed a highly sensitive and selective ELISA for high-molecular-weight oligomer tau (HMWoTau) with LLOQ of 0.3 pg/well for the first time, using a novel mouse monoclonal antibody APNmAb005. The target molecule was identified as HMWoTau with circa 2000 kD as a minimum size and the more oligomerized species (>5000 kD), in combination analysis with Size-Exclusion-Chromatography and Sucrose-Density-Gradient-Centrifugation for both recombinant human (rh) Tau-derived aggregates and AD brain-lysates in PBS(-). HMWoTau was labeled by Thioflavin S and visualized as a homogeneous globular particle (about 30 nm in diameter) by two different technologies of atomic force microscopy and dSTORM-Nanoimager. Specific quantitation was also confirmed by immune-absorption, rhHMWoTau-spiked, and cross-reactivity studies. APNmAb005 failed to detect the HMWoTau signal by treatment with DTT/SDS under no influence on the pan-tau antibody, indicating its conformation-specific recognition. APNmAb005-ELISA showed AD-specific and statistically significant ELISA signals from 1 ng brain lysate protein/well. Analysis of the frontal neocortex (N = 40, Braak stage I-VI) by ELISA revealed the detection-limit levels of HMWoTau species at stage I-III, and drastic and statistically significant increases at stage V/VI (AD). By contrast, total Tau and p181 Tau showed 1/4-1/5 levels of AD even at Stage I, while both tau species also showed a statistically significant increase in AD. In sum, our novel APNmAb005-ELISA clarified the disease-specific increase in HMWoTau species and will be useful for not only further etiological elucidation but also the potential diagnostics in AD and relevant tauopathy.

Hyperphosphorylated tau targeting human serum albumin Fusion protein as therapeutics for Alzheimer’s diseases

IntroductionNeurofibrillary tangles (NFTs) are composed of hyperphosphorylated forms of microtubule-associated protein tau (Tau), which is responsible for neurodegeneration in Alzheimer’s disease (AD). The hippocampal region has been a major focus of AD research because the deposits of phosphorylated tau protein in these regions are correlated with early memory deficits. Despite extensive studies, therapeutic strategies to reduce tau hyperphosphorylation and NFTs deposition remain unclear. AL04, a recently developed recombinant fusion protein comprising Cystatin C, human serum albumin, and a novel blood brain barrier (BBB) penetrating peptide, is currently under investigation. Previous studies have demonstrated its effectiveness in reducing amyloid beta plaques in AD mouse model. MethodsIn this study, we investigated the effects of AL04 on lowering hyperphosphorylated tau and NFTs in JNPL3 mouse model harboring human tau-P301L mutation. 3-month-old female mice intraperitoneally received AL04 (5 mg/kg) or PBS treatment every other week for 24 weeks. We used confocal microscopy and western blot to visualize and analyze changes in hyperphosphorylated tau Ser202/Thr205 labeled with AT8 antibody in the brain. ResultsWe found that the AL04 treatment decreases hyperphosphorylated tau at PP2A-sensitive epitope Ser202/Thr205 in the hippocampus of the brain. In the brain lysates of AL04 treated mice, we observed the reduction of I2PP2A, inhibitor of PP2A, and the induction of autophagy receptor proteins such as SQSTM-1/p62 and OPTN. ConclusionOur data suggests that AL04 can be used as an AD prophylactic/therapeutic agent as it lowers the hyperphosphorylated tau by downregulating I2PP2A. We also propose that AL04 can induce the degradation of hyperphosphorylated tau aggregates through the upregulation of the autophagy pathway.

Plasma p-tau212 as a biomarker of sporadic and Down Syndrome Alzheimer's disease.

All individuals with Down Syndrome (DS) will develop full-blown Alzheimeŕs disease (AD) pathology by age 40, decades before the occurrence of sporadic late-onset AD. Understanding this strong biological relation between age and AD pathology risk in DS is important to accelerate diagnostics, disease monitoring, and treatment. Several genes encoded in chromosome 21 including dual-specificity tyrosine phosphorylation-regulated kinase 1A (DYRK1A) have been proven to contribute to the pathology. A recently validated plasma immunoassay to measure tau phosphorylation at threonine-212 (p-tau212) has very high diagnostic accuracy in detecting AD. P-tau212 is also very sensitive to DYRK1A phosphorylation and is increased in DSAD brain lysates. Here, we assessed the potential of this biomarker in DSAD and sporadic AD. Using Simoa technology, we tested p-tau212 and p-tau181 (n=245 for plasma, n=114 matching cerebrospinal fluid (CSF) samples). We used AUC-ROC to examine diagnostic performance and the DeLong test to compare the AUC-ROC differences between methods. Spearman correlation is used to examine correlations. Fold changes relative to median levels were calculated for their respective asymptomatic groups. ANCOVA followed by Tukey posthoc test was used to calculate differences across groups. LOESS was used to determine the temporality of plasma biomarker changes. We have confirmed that p-tau212 has extremely high accuracy in detecting AD-related changes in euploid controls. For the DS population, we observed a strong correlation between plasma and CSF p-tau212 (r=0.867; p<0.001). In prodromal DS (pDS) and dementia DS (dDS), we observed significantly elevated levels of p-tau212 in reference to asymptomatic DS (aDS). The diagnostic accuracy to differentiate between aDS and dDS was AUC=0.91 and AUC = 0.86 in discriminating between DS amyloid positive and amyloid negative participants. Plasma p-tau212 started increasing approximately when people became amyloid PET-positive. We have confirmed that the levels of plasma p-tau212 are increased in the DS population and sporadic AD cases including prodromal and MCI states. Plasma p-tau212 might have utility for theragnostic, monitoring therapy efficacy, and as a target engagement biomarker in clinical trials both in sporadic and DSAD.

Unveiling the structural proteome of an Alzheimer's disease rat brain model.

Studying native protein structures at near-atomic resolution in a crowded environment presents challenges. Consequently, understanding the structural intricacies of proteins within pathologically affected tissues often relies on mass spectrometry and proteomic analysis. Here, we utilized cryoelectron microscopy (cryo-EM) and the Build and Retrieve (BaR) method to investigate protein complexes' structural characteristics such as post-translational modification, active site occupancy, and arrested conformational state in Alzheimer's disease (AD) using brain lysate from a rat model (TgF344-AD). Our findings reveal novel insights into the architecture of these complexes, corroborated through mass spectrometry analysis. Interestingly, it has been shown that the dysfunction of these protein complexes extends beyond AD, implicating them in cancer, as well as other neurodegenerative disorders such as Parkinson's disease, Huntington's disease, and schizophrenia. By elucidating these structural details, our work not only enhances our understanding of disease pathology but also suggests new avenues for future approaches in therapeutic intervention.

Endothelial Neurogranin Regulates Blood-Brain Barrier Permeability via Modulation of the AKT Pathway.

Neurogranin (Ng) expression is a biomarker for Alzheimer's disease. A loss of brain Ng and an increase in CSF Ng positively correlate with cognitive decline. Ng is known to regulate neuronal calcium-calmodulin binding and synaptic plasticity, which are critical for learning/memory. Interestingly, we discovered that Ng is also expressed in mouse and human blood-brain barrier (BBB). However, the role of Ng expression in brain vasculature remains largely undefined. In this study, we investigated the role of Ng expression on neurovascular structure and function using Ng null mice and human cerebral microvascular endothelial (hCMEC/D3) cells. We performed brain clearing and immunolabeling of blood vessels from whole brains and brain slices. Deletion of Ng significantly decreases neurovascular density in mice. Using in vivo permeability assays, we found increased neurovascular permeability in Ng null mice. We also observed significant changes in the expression of tight junction proteins using western blot and immunofluorescent staining. To identify the molecular pathways involved, we carried out label-free proteomics on brain lysates from endothelial-specific Ng knockout mice. Ingenuity Pathway Analysis indicated that the AKT pathway is attenuated in the vasculature of endothelial-specific Ng knockout mice. To validate these in vivo findings, we pharmacologically manipulated AKT signaling in hCMEC/D3 cells and observed that inhibition of AKT activation causes increased permeability. Our results indicate that the loss of Ng expression alters neurovascular structure and permeability, potentially contributing to neurological dysfunction. Therefore, modulating Ng expression in the BBB may offer a novel therapeutic approach for Alzheimer's disease.

OPALIN is an LGI1 receptor promoting oligodendrocyte differentiation

Leucine-rich glioma-inactivated protein 1 (LGI1), a secretory protein in the brain, plays a critical role in myelination; dysfunction of this protein leads to hypomyelination and white matter abnormalities (WMAs). Here, we hypothesized that LGI1 may regulate myelination through binding to an unidentified receptor on the membrane of oligodendrocytes (OLs). To search for this hypothetic receptor, we analyzed LGI1 binding proteins through LGI1-3 × FLAG affinity chromatography with mouse brain lysates followed by mass spectrometry. An OL-specific membrane protein, the oligodendrocytic myelin paranodal and inner loop protein (OPALIN), was identified. Conditional knockout (cKO) of OPALIN in the OL lineage caused hypomyelination and WMAs, phenocopying LGI1 deficiency in mice. Biochemical analysis revealed the downregulation of Sox10 and Olig2, transcription factors critical for OL differentiation, further confirming the impaired OL maturation in Opalin cKO mice. Moreover, virus-mediated re-expression of OPALIN successfully restored myelination in Opalin cKO mice. In contrast, re-expression of LGI1-unbound OPALIN_K23A/D26A failed to reverse the hypomyelination phenotype. In conclusion, our study demonstrated that OPALIN on the OL membrane serves as an LGI1 receptor, highlighting the importance of the LGI1/OPALIN complex in orchestrating OL differentiation and myelination.

Rab27b promotes lysosomal function and alpha-synuclein clearance in neurons.

Alpha-synuclein (αsyn) is the key pathogenic protein implicated in synucleinopathies including Parkinson's Disease (PD) and Dementia with Lewy Bodies (DLB). In these diseases, αsyn is thought to spread between cells where it accumulates and induces pathology; however, mechanisms that drive its propagation or aggregation are poorly understood. We have previously reported that the small GTPase Rab27b is elevated in human PD and DLB and that it can mediate the autophagic clearance and toxicity of αsyn in a paracrine αsyn cell culture neuronal model. Here, we expanded our previous work and further characterized a role for Rab27b in neuronal lysosomal processing and αsyn clearance. We found that Rab27b KD in this αsyn inducible neuronal model resulted in lysosomal dysfunction and increased αsyn levels in lysosomes. Similar lysosomal proteolytic defects and enzymatic dysfunction were observed in both primary neuronal cultures and brain lysates from Rab27b knockout (KO) mice. αSyn aggregation was exacerbated in Rab27b KO neurons upon treatment with αsyn preformed fibrils. We found no changes in lysosomal counts or lysosomal pH in either model, but we did identify defects in acidic vesicle trafficking in Rab27b KO primary neurons which may drive lysosomal dysfunction and promote αsyn aggregation. Rab27b OE enhanced lysosomal activity and reduced insoluble αsyn accumulation. Finally we found elevated Rab27b levels in human postmortem incidental Lewy Body Disease (iLBD) subjects relative to healthy controls. These data suggest a role for Rab27b in neuronal lysosomal activity and identify it as a potential therapeutic target in synucleinopathies.

A multiverse of α-synuclein: investigation of prion strain properties with carboxyl-terminal truncation specific antibodies in animal models

Synucleinopathies are a group of neurodegenerative disorders characterized by the presence of misfolded α-Synuclein (αSyn) in the brain. These conditions manifest with diverse clinical and pathophysiological characteristics. This disease diversity is hypothesized to be driven by αSyn strains with differing biophysical properties, potentially influencing prion-type propagation and consequentially the progression of illness. Previously, we investigated this hypothesis by injecting brain lysate (seeds) from deceased individuals with various synucleinopathies or human recombinant αSyn preformed fibrils (PFFs) into transgenic mice overexpressing either wild type or A53T human αSyn. In the studies herein, we expanded on these experiments, utilizing a panel of antibodies specific for the major carboxyl-terminally truncated forms of αSyn (αSynΔC). These modified forms of αSyn are found enriched in human disease brains to inform on potential strain-specific proteolytic patterns. With monoclonal antibodies specific for human αSyn cleaved at residues 103, 114, 122, 125, and 129, we demonstrate that multiple system atrophy (MSA) seeds and PFFs induce differing neuroanatomical spread of αSyn pathology associated with host specific profiles. Overall, αSyn cleaved at residue 103 was most widely present in the induced pathological inclusions. Furthermore, αSynΔC-positive inclusions were present in astrocytes, but more frequently in activated microglia, with patterns dependent on host and inoculum. These findings support the hypothesis that synucleinopathy heterogeneity might stem from αSyn strains with unique biochemical properties that include proteolytic processing, which could result in dominant strain properties.

Heme oxidation of NO-receptor soluble guanylyl cyclase is an important contributor to cerebrovascular and cognitive dysfunctions

Introduction: Decreased cerebral blood flow (CBF) is one of the major hemodynamic alterations leading to neurodegeneration and age-related cognitive decline. Nitric Oxide (NO)-dependent vasomotor reactivity is central in regulating cerebrovascular hemodynamics and adequate brain blood perfusion. NO receptor Soluble Guanylyl Cyclase (sGC) is the primary mediator of NO-dependent vasodilation. Oxidative stress, often persisting in diseased vasculature, renders sGC insensitive to NO and impairs the NO signaling. The objective of our study was to investigate the role of sGC oxidation in regulation CBF and brain function. Hypothesis: Oxidative stress disrupts NO/cGMP signaling in brain by decreasing sGC activity via oxidation of sGC heme. Approach: To assess causal effect of sGC oxidation on NO signaling function we administered ODQ, a sGC-specific heme oxidizing agent, to simulate long-term effect of sGC oxidation in cerebral vasculature. Two groups of young wild-type male and female mice (4 months old C57BL/6) were treated intraperitoneally (IP) twice a week for one month with ODQ (20 mg/kg) or a solvent as control. Changes in cerebrovascular reactivity and memory function were assessed and compared in treated and control groups. Using a non-invasive pulsed Doppler ultrasound-based system we assessed the changes in blood flow velocity (PVF, % of baseline) in the mouse middle cerebral artery (MCA) of male and female groups. MCA vasodilation was induced by bolus IP injection of NO-donor sodium nitroprusside (SNP) or BAY 58-2667, an allosteric NO-independent sGC activator. Memory function was assessed by the Novel Object Recognition Task (NORT, RI, recognition index) in all experimental groups. Western blotting and densitometry assessed the expression of major sGC subunits (α1, α2 and β1) in cleared brain lysates from ODQ-treated and control groups. Results: We observed a significantly blunted vasodilative response to SNP (1 mg/kg) in ODQ-treated mice vs controls of both genders (PVF, max response at 2 min post-injection, males 73.9 ± 10.4% (n=7/7) vs 58.5 ± 8.6 %, p=0.01; females (n=6/6) 77.4 ± 10.6 % vs 61.9 ± 5.4 %, p=0.01). Changes in response to NO-donor were more pronounced in males than females. The difference in BAY 58-2667 response between ODQ-treated and control groups was not statistically significant. ODQ significantly reduced the long-term memory in males but not female, according to NORT (RI, males (n=7/7) 0.58 ± 0.13 vs 0.69 ± 0.05; p=0.01; females (n=6/6) ODQ 0.59 ± 0.17 vs 0.73 ± 0.1, p=0.13). No statistically significant difference in expression of sGC subunits between control and ODQ-treated (n= 4/4) of male and female groups was determined. Conclusions: ODQ treatment significantly impairs vasodilation response to NO-donor but not to NO-independent sGC activator in mice MCA. ODQ treatment negatively affects long-term memory function in male, but not female mice. ODQ treatment does not significantly affect the expression of sGC subunits. Together our data suggest that the oxidation of sGC heme moiety impairs the regulation of cerebral blood flow of both genders and impairs long-term memory function gender-specifically in young mice. This research was supported by National Institute of Health grants RAG071999A (Iraida Sharina), HL139838, and American Heart Association 23TPA1070711 (Emil Martin). This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Clinical and Pathological Features of FTDP-17 with MAPT p.K298_H299insQ Mutation.

MAPT is a causative gene in frontotemporal dementia with parkinsonism linked to chromosome 17 (FTDP-17), a hereditary degenerative disease with various clinical manifestations, including progressive supranuclear palsy, corticobasal syndrome, Parkinson's disease, and frontotemporal dementia. To analyze genetically, biochemically, and pathologically multiple members of two families who exhibited various phenotypes of the disease. Genetic analysis included linkage analysis, homozygosity haplotyping, and exome sequencing. We conducted tau protein microtubule polymerization assay, heparin-induced tau aggregation, and western blotting with brain lysate from an autopsy case. We also evaluated abnormal tau aggregation by using anti-tau antibody and PM-PBB3. We identified a variant, c.896_897insACA, p.K298_H299insQ, in the MAPT gene of affected patients. Similar to previous reports, most patients presented with atypical parkinsonism. Biochemical analysis revealed that the mutant tau protein had a reduced ability to polymerize microtubules and formed abnormal fibrous aggregates. Pathological study revealed frontotemporal lobe atrophy, midbrain atrophy, depigmentation of the substantia nigra, and four-repeat tau-positive inclusions in the hippocampus, brainstem, and spinal cord neurons. The inclusion bodies also stained positively with PM-PBB3. This study confirmed that the insACA mutation caused FTDP-17. The affected patients showed symptoms resembling Parkinson's disease initially and symptoms of progressive supranuclear palsy later. Despite the initial clinical diagnosis of frontotemporal dementia in the autopsy case, the spread of lesions could explain the process of progressive supranuclear palsy. The study of more cases in the future will help clarify the common pathogenesis of MAPT mutations or specific pathogeneses of each mutation.

Rational design of structure-based vaccines targeting misfolded alpha-synuclein conformers of Parkinson's disease and related disorders.

Synucleinopathies, including Parkinson's disease (PD), multiple system atrophy (MSA), and dementia with Lewy bodies (DLB), are neurodegenerative disorders caused by the accumulation of misfolded alpha-synuclein protein. Developing effective vaccines against synucleinopathies is challenging due to the difficulty of stimulating an immune-specific response against alpha-synuclein without causing harmful autoimmune reactions, selectively targeting only pathological forms of alpha-synuclein. Previous attempts using linear peptides and epitopes without control of the antigen structure failed in clinical trials. The immune system was unable to distinguish between native alpha-synuclein and its amyloid form. The prion domain of the fungal HET-s protein was selected as a scaffold to introduce select epitopes from the surface of alpha-synuclein fibrils. Four vaccine candidates were generated by introducing specific amino acid substitutions onto the surface of the scaffold protein. The approach successfully mimicked the stacking of the parallel in-register beta-sheet structure seen in alpha-synuclein fibrils. All vaccine candidates induced substantial levels of IgG antibodies that recognized pathological alpha-synuclein fibrils derived from a synucleinopathy mouse model. Furthermore, the antisera recognized pathological alpha-synuclein aggregates in brain lysates from patients who died from DLB, MSA, or PD, but did not recognize linear alpha-synuclein peptides. Our approach, based on the rational design of vaccines using the structure of alpha-synuclein amyloid fibrils and strict control over the exposed antigen structure used for immunization, as well as the ability to mimic aggregated alpha-synuclein, provides a promising avenue toward developing effective vaccines against alpha-synuclein fibrils.

Deregulation of mitochondrial reverse electron transport alters the metabolism of reactive oxygen species and NAD+/NADH and presents a therapeutic target in Alzheimer’s disease

Aim: Oxidative stress and NAD+/NADH imbalance caused by alterations in reactive oxygen species (ROS) and NAD(H) metabolism are pathological features associated with normal aging and age-related diseases including Alzheimer’s disease (AD). How abnormalities in ROS and NAD(H) metabolism occur under these pathological conditions is not well understood, nor is it known whether they are mechanistically linked and can be therapeutically targeted together. The aim of this study is to identify the cause of aberrant ROS and NAD(H) metabolism and test its role in the pathogenesis of AD. Methods: Reverse electron transport (RET) along mitochondrial complex I can occur under certain thermodynamic conditions, leading to excessive ROS generation and NAD+ conversion to NADH, and thus lowered NAD+/NADH ratio. Brain samples from AD patients and mouse AD models were used to assess the status of RET by measuring ROS and NAD+/NADH ratio in brain lysates and purified mitochondria respiring under RET conditions. A small molecule RET inhibitor was used to treat APP(swe)/PS1(deltaE9) and 5xFAD mouse models and human induced pluripotent stem cell (iPSC)-derived neuronal model of AD. Effects on behavior and AD-related neuropathology were examined. The biochemical mechanism underlying RET alteration was examined by protein-protein interaction studies. Results: RET is aberrantly activated in transgenic AD mouse brains and in individuals with AD. Pharmacological inhibition of RET reduced amyloid burden and neuroinflammation and rescued cognitive and behavioral deficits in the APP(swe)/PS1(deltaE9) and 5xFAD mouse models. In human AD iPSC-derived neurons, RET inhibition reduced amyloid aggregation, tau hyperphosphorylation, and early endosomal defects. Mechanistically, the AD-associated amyloid precursor protein C-terminal fragment (APP.C99) was found to interact with complex I proteins to promote RET. Conclusion: RET is aberrantly activated in AD, causing altered ROS and NAD+/NADH metabolism. Pharmacological inhibition of RET is beneficial in mouse and human iPSC models of AD. RET activation represents a key pathological driver and a rational therapeutic target for AD and possibly other age-related neurodegenerative diseases.

In need of a specific antibody against the oxytocin receptor for neuropsychiatric research: A KO validation study

Antibodies are one of the most utilized tools in biomedical research. However, few of them are rigorously evaluated, as there are no accepted guidelines or standardized methods for determining their validity before commercialization. Often, an antibody is considered validated if it detects a band by Western blot of the expected molecular weight and, in some cases, if blocking peptides result in loss of staining. Neither of these approaches are unquestionable proof of target specificity. Since the oxytocin receptor has recently become a popular target in neuropsychiatric research, the need for specific antibodies to be used in brain has arisen. In this work, we have tested the specificity of six commercially available oxytocin receptor antibodies, indicated by the manufacturers to be suitable for Western blot and with an available image showing the correct size band (45-55 KDa). Antibodies were first tested by Western blot in brain lysates of wild-type and oxytocin receptor knockout mice. Uterus tissue was also tested as control for putative differential tissue specificity. In brain, the six tested antibodies lacked target specificity, as both wild-type and receptor knockout samples resulted in a similar staining pattern, including the expected 45-55 KDa band. Five of the six antibodies detected a selective band in uterus (which disappeared in knockout tissue). These five specific antibodies were also tested for immunohistochemistry in uterus, where only one was specific. However, when the uterine-specific antibody was tested in brain tissue, it lacked specificity. In conclusion, none of the six tested commercial antibodies are suitable to detect oxytocin receptor in brain by either Western blot or immunohistochemistry, although some do specifically detect it in uterus. The present work highlights the need to develop standardized antibody validation methods, including a proper negative control, in order to grant quality and reproducibility of the generated data.

Parallel Metabolomics and Lipidomics of a PSMA/GCPII Deficient Mouse Model Reveal Alteration of NAAG Levels and Brain Lipid Composition.

Glutamate carboxypeptidase II (GCPII, also known as PSMA or FOLH1) is responsible for the cleavage of N-acetyl-aspartyl-glutamate (NAAG) to N-acetyl-aspartate and glutamate in the central nervous system and facilitates the intestinal absorption of folate by processing dietary folyl-poly-γ-glutamate in the small intestine. The physiological function of GCPII in other organs like kidneys is still not known. GCPII inhibitors are neuroprotective in various conditions (e.g., ischemic brain injury) in vivo; however, their utilization as potential drug candidates has not been investigated in regard to not yet known GCPII activities. To explore the GCPII role and possible side effects of GCPII inhibitors, we performed parallel metabolomic and lipidomic analysis of the cerebrospinal fluid (CSF), urine, plasma, and brain tissue of mice with varying degrees of GCPII deficiency (fully deficient in Folh1, -/-; one allele deficient in Folh1, +/-; and wild type, +/+). Multivariate analysis of metabolites showed no significant differences between wild-type and GCPII-deficient mice (except for NAAG), although changes were observed between the sex and age. NAAG levels were statistically significantly increased in the CSF, urine, and plasma of GCPII-deficient mice. However, no difference in NAAG concentrations was found in the whole brain lysate likely because GCPII, as an extracellular enzyme, can affect only extracellular and not intracellular NAAG concentrations. Regarding the lipidome, the most pronounced genotype-linked changes were found in the brain tissue. In brains of GCPII-deficient mice, we observed statistically significant enrichment in phosphatidylcholine-based lipids and reduction of sphingolipids and phosphatidylethanolamine plasmalogens. We hypothesize that the alteration of the NAA-NAAG axis by absent GCPII activity affected myelin composition. In summary, the absence of GCPII and thus similarly its inhibition do not have detrimental effects on metabolism, with just minor changes in the brain lipidome.