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Results for “Pathology”

Search indexed bioRxiv preprints in genomics, neuroscience, cell biology and bioinformatics. Read source abstracts and check manuscript versions; preprints are not peer reviewed.

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At least 883 records · Page 49Linked to original sources

bioRxiv2021

Nebulized delivery of a broadly neutralizing SARS-CoV-2 RBD-specific nanobody prevents clinical, virological and pathological disease in a Syrian hamster model of COVID-19

There remains an unmet need for globally deployable, low-cost therapeutics for the ongoing severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic. Previously, we reported on the isolation and in vitro characterization of a potent single-domain nanobody, NIH-CoVnb-112, specific for the receptor binding domain (RBD) of SARS-CoV-2. Here, we report on the molecular basis for the observed broad in vitro neutralization capability of NIH-CoVnb-112 against variant SARS-CoV-2 pseudoviruses, including the currently dominant Delta variant. The structure of NIH-CoVnb-112 bound to SARS-CoV-2 RBD reveals a large contact surface area overlapping the angiotensin converting enzyme 2 (ACE2) binding site, which is largely unencumbered by the common RBD mutations. In an in vivo pilot study, we demonstrate effective reductions in weight loss, viral burden, and lung pathology in a Syrian hamster model of COVID-19 following nebulized delivery of NIH-CoVnb-112. These findings support the further development of NIH-CoVnb-112 as a potential adjunct preventative therapeutic for the treatment of SARS-CoV-2 infection.

biochemistry↗

bioRxiv2021

Orally administered epeleuton inhibits SARS-CoV-2 viral load, replication and pathology in the Syrian Hamster model

Early treatment of patients with confirmed COVID-19 presenting mild symptoms can reduce the number that progress to more severe disease and require hospitalization. Considering the potential for the development of drug resistance to existing therapies and the emergence of new SARS-CoV-2 variants, there is a need for an expanded armamentarium of treatment options for COVID-19. Epeleuton is a novel orally administered second-generation n-3 fatty acid with potential direct antiviral and immunomodulatory actions, and a favourable clinical safety profile. In this study we show that epeleuton inhibits SARS-CoV-2 infectious viral load, replication and disease pathology in the lungs and upper airways in the Syrian hamster model of SARS-CoV-2 infection. These data support the potential utility of epeleuton in the early treatment and prevention of SARS-CoV-2 infection. Clinical trials are needed to evaluate the efficacy of epeleuton as an outpatient treatment and prevention of COVID-19.

microbiology↗

bioRxiv2021

DeepMed: A unified, modular pipeline for end-to-end deep learning in computational pathology

The interpretation of digitized histopathology images has been transformed thanks to artificial intelligence (AI). End-to-end AI algorithms can infer high-level features directly from raw image data, extending the capabilities of human experts. In particular, AI can predict tumor subtypes, genetic mutations and gene expression directly from hematoxylin and eosin (H&E) stained pathology slides. However, existing end-to-end AI workflows are poorly standardized and not easily adaptable to new tasks. Here, we introduce DeepMed, a Python library for predicting any high-level attribute directly from histopathological whole slide images alone, or from images coupled with additional meta-data (https://github.com/KatherLab/deepmed). Unlike earlier computational pipelines, DeepMed is highly developer-friendly: its structure is modular and separates preprocessing, training, deployment, statistics, and visualization in such a way that any one of these processes can be altered without affecting the others. Also, DeepMed scales easily from local use on laptop computers to multi-GPU clusters in cloud computing services and therefore can be used for teaching, prototyping and for large-scale applications. Finally, DeepMed is user-friendly and allows researchers to easily test multiple hypotheses in a single dataset (via cross-validation) or in multiple datasets (via external validation). Here, we demonstrate and document DeepMeds abilities to predict molecular alterations, histopathological subtypes and molecular features from routine histopathology images, using a large benchmark dataset which we release publicly. In summary, DeepMed is a fully integrated and broadly applicable end-to-end AI pipeline for the biomedical research community.

cancer biology↗

bioRxiv2021

Mapping prion pathology in mice using quantitative imaging: an MRI study

Human prion diseases are fatal neurodegenerative disorders which cause cognitive impairment and neurological deficits. Additional measures of tissue status are necessary for improving the sensitivity and specificity of clinical diagnosis as in many cases clinical forms of prion disease are commonly mistaken for other forms of dementia. To that effect, we developed a set of quantitative magnetic resonance-based tools, including magnetic resonance spectroscopy (MRS), magnetization transfer ratio (MTR) and quantitative T1 and T2 imaging to study the course of the disease in an animal model of prion disease. Using in vivo MTR, significant changes were detected in the cortex and thalamus of late-stage prion -infected mice as compared to littermates. In addition, we found a significant increase of MTR in thalamus and cortex of 80 dpi healthy mice when compared with 160 dpi healthy mice suggestive of changes occurring during the development of the brain. Using quantitative T2 mapping, significantly higher values were measured in thalamus of prion mice at all stages of the disease (T2=40ms) while T1 was found to be significantly higher in cortex (T1=1.89s) and hippocampus, albeit only in late-stage prion mice as compared to aged-matched controls (T1=1.67s). Using quantitative MRS significant changes were detected in glutamate (Glu) and myo-inositol (Ins) at all stages of prion disease when compared with the control group. NAA, Cr, Lactate and Lipids were only found to be significantly different at early and late stages of the disease while Taurine (Tau) was only significantly increased in the asymptomatic stage without any significant change at early and late stages of the disease. These changes in MRI and MRS signals, which precede clinical signs of disease, could provide insights into the pathogenesis of this disease and may enable early detection of pathology.

biophysics↗

bioRxiv2021

Capturing a crucial 'disorder-to-order transition' at the heart of the coronavirus molecular pathology - triggered by highly persistent, interchangeable salt-bridges

The COVID-19 origin debate has greatly been influenced by Genome comparison studies of late, revealing the seemingly sudden emergence of the Furin-Like Cleavage Site at the S1/S2 junction of the SARS-CoV-2 Spike (FLCSSpike) containing its 681PRRAR685 motif, absent in other related respiratory viruses. Being the rate-limiting (i.e., the slowest) step, the host Furin cleavage is instrumental in the abrupt increase in transmissibility in COVID-19, compared to earlier onsets of respiratory viral diseases. In such a context, the current paper entraps a disorder-to-order transition of the FLCSSpike (concomitant to an entropy arrest) upon binding to Furin. The interaction clearly seems to be optimized for a more efficient proteolytic cleavage in SARS-CoV-2. The study further shows the formation of dynamically interchangeable and persistent networks of salt-bridges at the Spike-Furin interface in SARS-CoV-2 involving the three arginines (R682, R683, R685) of the FLCSSpike with several anionic residues (E230, E236, D259, D264, D306) coming from Furin, strategically distributed around its catalytic triad. Multiplicity and structural degeneracy of plausible salt-bridge network archetypes seems the other key characteristic features of the Spike-Furin binding in SARS-CoV-2 allowing the system to breathe - a trademark of protein disorder transitions. Interestingly, with respect to the homologous interaction in SARS-CoV (2002/2003) taken as a baseline, the Spike-Furin binding events generally in the coronavirus lineage seems to have a preference for ionic bond formation, even with lesser number of cationic residues at their potentially polybasic FLCSSpike patches. The interaction energies are suggestive of a characteristic metastabilities attributed to Spike-Furin interactions generally to the coronavirus lineage - which appears to be favorable for proteolytic cleavages targeted at flexible protein loops. T he current findings not only offer novel mechanistic insights into the coronavirus molecular pathology and evolution but also add substantially to the existing theories of proteolytic cleavages.

biophysics↗

bioRxiv2022

Seed-competent tau monomer initiates pathology in PS19 tauopathy mice

Tau aggregation into ordered assemblies causes myriad neurodegenerative tauopathies. We previously reported that tau monomer exists in either inert (Mi) or seed-competent (Ms) conformational ensembles, and that Ms encodes strains, which are biologically active, self-propagating assemblies. We have previously isolated Ms from tauopathy brains, but it is unknown if disease begins with Ms formation followed by fibril assembly, or if Ms derives from fibrils and is an epiphenomenon. Consequently, we studied a tauopathy mouse model (PS19) that expresses full-length human (1N4R) tau containing a disease-associated mutation (P301S). Using tau repeat domain biosensor cells, we detected insoluble tau seeding activity at 2 months. We found insoluble tau protein assemblies by immunoblot at 3 months. We next immunopurified monomer from mice aged 1-6 weeks using size exclusion chromatography. We detected soluble seeding activity at 4 weeks, before insoluble material or larger assemblies, with assemblies ranging from n=1-3 tau units. By 5 and 6 weeks, large soluble assemblies had formed. This indicated the first detectable pathological forms of tau were Ms. We next tested for post-translational modifications of tau monomer from 1-6 weeks. We detected no phosphorylation unique to Ms in PS19 or Alzheimers disease brain. We conclude that tauopathy begins with formation of Ms monomer, whose activity is phosphorylation-independent. Ms self-assembles to form oligomers before it forms insoluble fibrils. The conversion of tau monomer from Mi to Ms thus constitutes the first detectable step in the initiation of tauopathy in this mouse model, with obvious implications for origins of disease in humans.

neuroscience↗

bioRxiv2022

Model-based hearing-restoration strategies for cochlear synaptopathy pathologies

It is well known that ageing and noise exposure are important causes of sensorineural hearing loss, and can result in damage of the outer hair cells or other structures of the inner ear, including synaptic damage to the auditory nerve (AN), i.e., cochlear synaptopathy (CS). Despite the suspected high prevalence of CS among people with self-reported hearing difficulties but seemingly normal hearing, conventional hearing-aid algorithms do not compensate for the functional deficits associated with CS. Here, we present and evaluate a number of auditory signal-processing strategies designed to maximally restore AN coding for listeners with CS pathologies. We evaluated our algorithms in subjects with and without suspected age-related CS to assess whether physiological and behavioural markers associated with CS can be improved. Our data show that after applying our algorithms, envelope-following responses and perceptual amplitude-modulation sensitivity were consistently enhanced in both young and older listeners. Speech-in-noise intelligibility showed small improvements after processing but mostly for young normal-hearing participants, with median improvements of up to 8.3%. Since our hearing-enhancement strategies were designed to optimally drive the AN fibres, they were able to improve temporal-envelope processing for listeners both with and without suspected CS. Our proposed algorithms can be rapidly executed and can thus extend the application range of current hearing aids and hearables, while leaving sound amplification unaffected.

bioengineering↗

bioRxiv2022

3D printed bioreactor enabling the physiological culture and the study of pathological processes in large vessels.

Routine cardiovascular interventions such as balloon angioplasty, result in vascular activation and remodeling, often requiring re-intervention. 2D in vitro culture models and small animal experiments have enabled the discovery of important molecular and cellular pathways involved in this process, however the clinical translation of these results is often underwhelming. There is a critical need for an ex vivo model representative of the human vascular physiology and encompassing the complexity of the vascular wall and the physical forces regulating its function. Vascular bioreactors for ex vivo culture of large vessels are viable alternatives, but their custom-made design and insufficient characterization often hinders the reproducibility of the experiments. The objective of the study was to design and validate a novel 3D printed cost-efficient and versatile perfusion system, capable of sustaining the viability and functionality of large porcine arteries for 7 days and enabling monitoring of post-injury remodeling. MultiJet Fusion 3D printing technology was used to engineer the EasyFlow insert, converting a conventional 50 ml centrifuge tube into a mini bioreactor. Porcine carotid arteries either left untreated or injured with a conventional angioplasty balloon, were cultured under pulsatile flow for up to 7 days. Pressure, heart rate, medium viscosity and shear conditions were adjusted to represent the typical arterial physiology. Tissue viability, cell activation and matrix remodeling were analyzed by immunohistochemistry, and vascular function was monitored by duplex ultrasound. Physiological blood flow conditions in the EasyFlow bioreactor preserved endothelial coverage and smooth muscle organization and extracellular matrix structure in the vessel wall, as compared to static culture. Injured arteries presented hallmarks of early remodeling, such as intimal denudation, smooth muscle cell disarray and media/adventitia activation in flow culture. Duplex ultrasound confirmed physiological hemodynamic conditions, dose-dependent vasodilator response to nitroglycerin in untreated vessels and impaired dilator response in angioplastied vessels. We here validate a low-cost, robust and reproducible system to study vascular physiopathology, laying the basis for future investigations into the pathological remodeling of blood vessels and creating a platform to test novel therapies and devices ex vivo, in a patient relevant system.

bioengineering↗

bioRxiv2022

Sequential increase of PHGDH expression with Alzheimer's pathology and symptoms

We report consistent increases in phosphoglycerate dehydrogenase (PHGDH) expression in mouse models of Alzheimers disease (AD) (3xTg-AD) and related tauopathy (PS19), particularly in hippocampal astrocytes. Human single-cell RNA-sequencing data reveal a sequential increase of PHGDH expression in people with no, early, and late AD pathology, which is corroborated by protein mass spectrometry and immunohistochemical analyses of three independent cohorts. A sequential increase of PHGDH expression also correlates with increasing clinical AD symptoms and worsening cognitive decline in patients. The consistent increase of PHGDH expression in six AD cohorts (Mayo, ROSMAP, Mount Sinai, Baltimore, Amsterdam, and UCSD/UCI) corroborates with the recent report of PHGDH extracellular RNA in blood plasma as a candidate biomarker for early diagnosis of AD and offers a caution to the suggested use of L-serine as a potential therapy of AD.

neuroscience↗

bioRxiv2022

Increased vulnerability of Purkinje cells in the posterior cerebellum of SCA1 mice is associated with molecular and cellular alterations related to disease pathology

Regional heterogeneity of neurons and glia is a key feature of the brain, yet the effect of disease on heterogeneity and its relationship with selective neuronal vulnerability remains poorly understood. Using region-specific RNA sequencing, we identified a large number of differentially expressed genes (DEGs) across distinct regions of the cerebellar cortex, supporting the notable intrinsic regional transcriptional heterogeneity of the healthy cerebellum. Further, we used fiber photometry to identify regional physiological differences in the activity of Purkinje cells (PCs) during self-motivated, unrestrained walking and non-walking states. In the inherited cerebellar neurodegenerative disease Spinocerebellar ataxia type 1 (SCA1), patients exhibit preferential degeneration of the posterior cerebellum, suggesting regionally selective vulnerability. We demonstrated that in a mouse model of SCA1 the Purkinje cells and glia residing in the posterior vermis of cerebellum also undergo earlier and more severe pathology. Intriguingly, the intrinsic transcriptional heterogeneity of anterior and posterior cerebellum seen in healthy mice was diminished in SCA1 mice. This disruption was also demonstrated via fiber photometry, where we found notable impacts in PC activity in the posterior cerebellum as well as loss of regional differences in PC activity during self-motivated, unrestrained walking, and non-walking states in SCA1 mice. Our findings indicate regionally distinct mechanisms of pathogenesis across cerebellar regions that result in reduced intracerebellar heterogeneity.

neuroscience↗

bioRxiv2022

Astrocyte-targeted gene delivery of interleukin 2 specifically increases brain-resident regulatory T cell numbers and protects against pathological neuroinflammation

The ability of immune-modulating biologics to prevent and reverse pathology has transformed recent clinical practice. Full utility in the neuroinflammation space, however, requires identification of both effective targets for local immune-modulation and a delivery system capable of crossing the blood-brain-barrier. The recent identification and characterization of a small population of regulatory T cells (Tregs) resident in the brain presents one such potential therapeutic target. Here we identified brain IL2 levels as a limiting factor for brain-resident Tregs. We developed a gene-delivery approach for astrocytes, with a small-molecule on-switch to allow temporal control, and enhanced production in reactive astrocytes to spatially-direct delivery to inflammatory sites. Mice with brain-specific IL2 delivery were protected from traumatic brain injury, stroke and multiple sclerosis models, without impacting the peripheral immune system. These results validate brain-specific IL2 gene-delivery as effective protection against neuroinflammation, and provide a versatile platform for delivery of diverse biologics to neuroinflammatory patients.

immunology↗

bioRxiv2022

Hsp multichaperone complex buffers pathologically modified Tau

Alzheimers disease is a neurodegenerative disorder in which misfolding and aggregation of pathologically modified Tau is critical for neuronal dysfunction and degeneration. The two central chaperones Hsp70 and Hsp90 coordinate protein homeostasis, but the nature of the interaction of Tau with the Hsp70/Hsp90 machinery has remained enigmatic. Here we show that Tau is a high-affinity substrate of the human Hsp70/Hsp90 machinery. Complex formation involves extensive intermolecular contacts, blocks Tau aggregation and depends on Taus aggregation-prone repeat region. The Hsp90 co-chaperone p23 directly binds Tau and stabilizes the multichaperone/substrate complex, whereas the E3 ubiquitin-protein ligase CHIP efficiently disassembles the machinery targeting Tau to proteasomal degradation. Because phosphorylated Tau binds the Hsp70/Hsp90 machinery but is not recognized by Hsp90 alone, the data establish the Hsp70/Hsp90 multichaperone complex as a critical regulator of Tau in neurodegenerative diseases.

biophysics↗

bioRxiv2022

Single-neuron bursts encode pathological oscillations in Parkinson's disease and essential tremor

Deep brain stimulation procedures offer an invaluable opportunity to study disease through intracranial recordings from awake patients. Herein, we address the relationship between singleneuron and aggregate-level (local field potential; LFP) activities in the subthalamic nucleus (STN) and thalamic ventral intermediate nucleus (Vim) of patients with Parkinsons disease (n=19) and essential tremor (n=16), respectively. Both disorders have been characterized by pathologically elevated LFP oscillations, as well as an increased tendency for neuronal bursting. Our findings suggest that periodic single-neuron bursts encode both pathophysiological beta (13-33Hz; STN) and tremor (4-10Hz; Vim) LFP oscillations, evidenced by strong time-frequency and phase-coupling relationships between the bursting and LFP signals. Spiking activity occurring outside of bursts had no relationship to the LFP. In STN, bursting activity most commonly preceded the LFP oscillation, suggesting that neuronal bursting generated within STN may give rise to an aggregate-level LFP oscillation. In Vim, LFP oscillations most commonly preceded bursting activity, suggesting that neuronal firing may be entrained by periodic afferent inputs. In both STN and Vim, the phasecoupling relationship between LFP and high-frequency oscillation (HFO) signals closely resembled the relationships between the LFP and single-neuron bursting. This suggests that periodic singleneuron bursting is likely representative of a higher spatial and temporal resolution readout of periodic increases in the amplitude of HFOs, which themselves may be a higher resolution readout of aggregate-level LFP oscillations. Overall, our results may reconcile "rate" and "oscillation" models of Parkinsons disease and shed light onto the single-neuron basis and origin of pathophysiological oscillations in movement disorders. SignificanceIn surgical patients with Parkinsons disease and essential tremor, we leverage intracranial recordings to establish a link between pathophysiological phenomena across various scales of observation (spatio-temporal resolutions). We provide insights and reconcile theories about aberrant neurocircuit phenomena which underly theses debilitating, medically refractory movement disorders. Furthermore, our connectivity analyses between single-neuron and local field potential activities may shed light on the origin of the deleterious neural oscillations underlying these disorders. Ultimately, our findings may aid in the development or investigation of targeted therapies to address or correct underlying neurocircuit dysfunction, which can include neuropharmaceuticals, but also novel neuromodulatory strategies like closed-loop deep brain stimulation targeting pathophysiological oscillations and phase-dependent stimulation methods seeking to stimulate "at the right time/phase."

neuroscience↗

bioRxiv2022

Prime-pull immunization of mice with a BcfA-adjuvanted vaccine elicits mucosal immunity and prevents SARS CoV-2 infection and pathology

Vaccines against SARS-CoV-2 that induce mucosal immunity capable of preventing infection and disease remain urgently needed. We show that intramuscular priming of mice with an alum and BcfA-adjuvanted Spike subunit vaccine, followed by a BcfA-adjuvanted mucosal booster, generated Th17 polarized tissue resident CD4+ T cells, and mucosal and serum antibodies. The serum antibodies efficiently neutralized SARS-CoV-2 and its Delta variant, suggesting cross-protection against a recent variant of concern (VOC). Immunization with this heterologous vaccine prevented weight loss following challenge with mouse-adapted SARS-CoV-2 and reduced viral replication in the nose and lungs. Histopathology showed a strong leukocyte and polymorphonuclear (PMN) cell infiltrate without epithelial damage in mice immunized with BcfA-containing vaccines. In contrast, viral load was not reduced in the upper respiratory tract of IL-17 knockout mice immunized with the same formulation, suggesting that the Th17 polarized T cell responses are critical for protection. We show that vaccines adjuvanted with alum and BcfA, delivered through a heterologous prime-pull regimen, protect against SARS-CoV-2 infection without causing enhanced respiratory disease. SIGNIFICANCEThere remains a need for SARS CoV-2 booster vaccines that generate mucosal immunity and prevent transmission. We show that systemic priming followed by a mucosal booster with a BcfA-adjuvanted subunit vaccine generates neutralizing antibodies and Th17 polarized systemic and tissue-resident immune responses that provide sterilizing immunity against wildtype SARS CoV-2, and a variant of concern. Importantly, in contrast to alum alone, the addition of BcfA prevents respiratory pathology. These results suggest that a BcfA-adjuvanted mucosal booster may elicit mucosal immunity in individuals previously immunized systemically with approved vaccines. This foundational study in mice sets the stage for testing our vaccine regimen in larger animal models as a booster vaccine.

immunology↗

bioRxiv2022

moRphology - dEep Learning Imaging Cells (RELIC) - to Differentiate Between Normal and Pathological Kidney Exfoliated Cells

Chronic kidney disease (CKD) is characterised by progressive loss of kidney function leading to kidney failure. Significant kidney damage can occur before symptoms are detected. Currently, kidney tissue biopsy is the gold standard for evaluation of renal damage and CKD severity. This study explores how to precisely quantify morphology characteristics of kidney cells exfoliated into urine, with a view to establish a future urine-based non-invasive diagnostic for CKD. We report the development of a novel deep learning method, which was able to discover a RELIC (moRphology dEep Learning Imaging Cells) signature that can differentiate between kidney cells exfoliated in human urine of and CKD patients with varying degree of kidney damage and non-CKD controls. Exfoliated proximal tubule cells (PTCs) originating from kidneys were isolated from the urine of patients with different levels of kidney damage using previously published methods. An advanced combination of artificial intelligence techniques, deep learning, swarm intelligence, and discriminative analysis was used to discover a RELIC signature in brightfield microscopy images of exfoliated PTCs. Kidney damage in the study subjects was characterised by assessing kidney tissues obtained through a nephrectomy or kidney biopsy. A special deep learning algorithm was developed and trained to create a predictive tool. The algorithm was then used to analyse data from patients with normal and fibrotic kidneys. Data were then classified according to different groups (healthy or fibrosis) and clustering of the training and validation cells was determined for model validation. We developed a novel deep learning method, to obtain RELIC signatures and identify specific deep morphological features which can be used to differentiate urinary PTC cells shed by people with CKD (confirmed by tissue histology obtained from an invasive kidney biopsy) from those without CKD, with a discriminatory accuracy of 82%. We identified a RELIC signature which can be used on a collection of bright field images of exfoliated urinary PTCs to create a predictive tool and differentiate between normal and pathological kidney cells. This study, for the first time, provides a proof of concept that urinary exfoliated tubule cells in patients with kidney fibrosis and healthy controls differ in appearance (morphology) as observed under a basic brightfield microscope. The results suggest that morphological signatures of exfoliated PTCs have the potential to serve as a non-invasive marker of kidney fibrosis.

bioinformatics↗

bioRxiv2022

Selection of Single Domain Anti-Transferrin Receptor Antibodies for Blood-Brain Barrier Transcytosis Using a Neurotensin Based Assay and Histological Assessment of Target Engagement in a Mouse Model of Alzheimer's Disease-Related Amyloid-Beta Pathology

The blood-brain barrier (BBB) presents a major obstacle in developing specific diagnostic imaging agents for many neurological disorders. In this study we aimed to generate single domain anti-mouse transferrin receptor antibodies (anti-mTfR VHHs) to mediate BBB transcytosis as components of novel MRI molecular contrast imaging agents. Anti-mTfR VHHs were produced by immunizing a llama with mTfR, generation of a VHH phage display library, immunopanning, and in vitro characterization of candidates. Site directed mutagenesis was used to generate additional variants. VHH fusions with neurotensin (NT) allowed rapid, hypothermia-based screening for VHH-mediated BBB transcytosis in wild-type mice. One anti-mTfR VHH variant was fused with an anti-amyloid-beta (A{beta}) VHH dimer and labeled with fluorescent dye for direct assessment of in vivo target engagement in a mouse model of AD-related A{beta} plaque pathology. An anti-mTfR VHH called M1 and variants had binding affinities to mTfR of <1nM to 1.52nM. The affinity of the VHH binding to mTfR correlated with the efficiency of the VHH-NT induced hypothermia effects after intravenous injection of 600 nmol/kg body weight, ranging from undetectable for nonbinding mutants to -6{degrees}C for the best mutants. The anti-mTfR VHH variant M1P96H with the strongest hypothermia effect was fused to the anti-A{beta} VHH dimer and labeled with Alexa647; the dye-labeled VHH fusion construct still bound both mTfR and A{beta} plaques. However, after intravenous injection at 600 nmol/kg body weight into APP/PS1 transgenic mice, there was no detectible labeling of plaques above control levels. Thus, NT-induced hypothermia did not correlate with direct target engagement in cortex. There was a surprising dissociation between NT-induced hypothermia, presumably mediated by hypothalamus, and direct engagement with A{beta}-plaques in cortex. Alternative methods to assess anti-mTfR VHH BBB transcytosis will need to be developed for anti-mTfR VHH screening and the development of novel MRI molecular contrast agents.

neuroscience↗

bioRxiv2022

Evolution avoids a pathological stabilizing interaction in the immune protein S100A9

Stability constrains evolution. While much is known about constraints on destabilizing mutations, less is known about the constraints on stabilizing mutations. We recently identified a mutation in the innate immune protein S100A9 that provides insight into such constraints. When introduced into human S100A9, M63F simultaneously increases the stability of the protein and disrupts its natural ability to activate Toll-like receptor 4. Using chemical denaturation, we found that M63F stabilizes a calcium-bound conformation of hS100A9. We then used NMR to solve the structure of the mutant protein, revealing that the mutation distorts the hydrophobic binding surface of hS100A9, explaining its deleterious effect on function. Hydrogen deuterium exchange (HDX) experiments revealed stabilization of the region around M63F in the structure, notably Phe37. In the structure of the M63F mutant, the Phe37 and Phe63 sidechains are in contact, plausibly forming an edge-face {nu}-stack. Mutating Phe37 to Leu abolished the stabilizing effect of M63F as probed by both chemical denaturation and HDX. It also restored the biological activity of S100A9 disrupted by M63F. These findings reveal that Phe63 creates a "molecular staple" with Phe37 that stabilizes a non-functional conformation of the protein, thus disrupting function. Using a bioinformatic analysis, we found that S100A9 proteins from different organisms rarely have Phe at both positions 37 and 63, suggesting that avoiding a pathological stabilizing interaction indeed constrains S100A9 evolution. This work highlights an important evolutionary constraint on stabilizing mutations: they must avoid inappropriately stabilizing non-functional protein conformations. SIGNIFICANCE STATEMENTProtein stability constrains protein evolution. While much is known about evolutionary constraints on destabilizing mutations, less is known about the constraints on stabilizing mutations. We recently found a mutation to an innate immune protein that increases its stability but disrupts its function. Here we show, through careful biophysical and functional studies, that this mutation stabilizes a nonfunctional form of the protein through a direct interaction with another amino acid. We find that specific amino acids can be tolerated at each of the interacting positions individually, but not at both simultaneously. This pattern has been conserved over millions of years of evolution. Our work highlights an underappreciated evolutionary constraint on stabilizing mutations: they must avoid inappropriately stabilizing non-functional protein conformations.

biophysics↗

bioRxiv2022

APOE modulates microglial immunometabolism in response to age, amyloid pathology, and inflammatory challenge

The E4 allele of Apolipoprotein E (APOE) is associated with both metabolic dysfunction and a heightened pro-inflammatory response - two findings that may be intrinsically linked through the concept of immunometabolism. Here, we combined bulk, single-cell, and spatial transcriptomics with cell-specific and spatially resolved metabolic analyses to systematically address the role of APOE across age, neuroinflammation, and AD pathology. RNAseq highlighted immunometabolic changes across the APOE4 glial transcriptome, specifically in subsets of metabolically distinct microglia enriched in the E4 brain during aging or following an inflammatory challenge. E4 microglia display increased Hif1 expression, a disrupted TCA cycle, and are inherently pro-glycolytic, while spatial transcriptomics and MALDI mass spectrometry imaging highlight an E4-specific response to amyloid that is characterized by widespread alterations in lipid metabolism. Taken together, our findings emphasize a central role for APOE in regulating microglial immunometabolism.

neuroscience↗