Intercellular Signaling Pathways as Therapeutic Targets for Vascular Dementia Repair.

Vascular dementia (VaD) is a white matter ischemic disease and the second-leading cause of dementia, with no direct therapy. Within the lesion site, cell-cell interactions dictate the trajectory towards disease progression or repair. To elucidate the underlying intercellular signaling pathways, a VaD mouse model was developed for transcriptomic and functional studies. The mouse VaD transcriptome was integrated with a human VaD snRNA-Seq dataset. A custom-made database encompassing 4053 human and 2032 mouse ligand-receptor (L-R) interactions identified significantly altered pathways shared between human and mouse VaD. Two intercellular L-R systems, Serpine2-Lrp1 and CD39-A3AR, were selected for mechanistic study as both the ligand and receptor were dysregulated in VaD. Decreased Seprine2 expression enhances OPC differentiation in VaD repair. A clinically relevant drug that reverses the loss of CD39-A3AR function promotes tissue and behavioral recovery in the VaD model. This study presents novel intercellular signaling targets and may open new avenues for VaD therapies.

HIV and COVID-19: two pandemics with significant (but different) central nervous system complications.

Human immunodeficiency virus (HIV) and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) cause significant neurologic disease. Central nervous system (CNS) involvement of HIV has been extensively studied, with well-documented invasion of HIV into the brain in the initial stage of infection, while the acute effects of SARS-CoV-2 in the brain are unclear. Neuropathologic features of active HIV infection in the brain are well characterized whereas neuropathologic findings in acute COVID-19 are largely non-specific. On the other hand, neuropathologic substrates of chronic dysfunction in both infections, as HIV-associated neurocognitive disorders (HAND) and post-COVID conditions (PCC)/long COVID are unknown. Thus far, neuropathologic studies on patients with HAND in the era of combined antiretroviral therapy have been inconclusive, and autopsy studies on patients diagnosed with PCC have yet to be published. Further longitudinal, multidisciplinary studies on patients with HAND and PCC and neuropathologic studies in comparison to controls are warranted to help elucidate the mechanisms of CNS dysfunction in both conditions.

Structure-based design of nanobodies that inhibit seeding of Alzheimer's patient-extracted tau fibrils.

Despite much effort, antibody therapies for Alzheimer's disease (AD) have shown limited efficacy. Challenges to the rational design of effective antibodies include the difficulty of achieving specific affinity to critical targets, poor expression, and antibody aggregation caused by buried charges and unstructured loops. To overcome these challenges, we grafted previously determined sequences of fibril-capping amyloid inhibitors onto a camel heavy chain antibody scaffold. These sequences were designed to cap fibrils of tau, known to form the neurofibrillary tangles of AD, thereby preventing fibril elongation. The nanobodies grafted with capping inhibitors blocked tau aggregation in biosensor cells seeded with postmortem brain extracts from AD and progressive supranuclear palsy (PSP) patients. The tau capping nanobody inhibitors also blocked seeding by recombinant tau oligomers. Another challenge to the design of effective antibodies is their poor blood-brain barrier (BBB) penetration. In this study, we also designed a bispecific nanobody composed of a nanobody that targets a receptor on the BBB and a tau capping nanobody inhibitor, conjoined by a flexible linker. We provide evidence that the bispecific nanobody improved BBB penetration over the tau capping inhibitor alone after intravenous administration in mice. Our results suggest that the design of synthetic antibodies that target sequences that drive protein aggregation may be a promising approach to inhibit the prion-like seeding of tau and other proteins involved in AD and related proteinopathies.

Persistent animal identification leveraging non-visual markers.

Our objective is to locate and provide a unique identifier for each mouse in a cluttered home-cage environment through time, as a precursor to automated behaviour recognition for biological research. This is a very challenging problem due to (i) the lack of distinguishing visual features for each mouse, and (ii) the close confines of the scene with constant occlusion, making standard visual tracking approaches unusable. However, a coarse estimate of each mouse's location is available from a unique RFID implant, so there is the potential to optimally combine information from (weak) tracking with coarse information on identity. To achieve our objective, we make the following key contributions: (a) the formulation of the object identification problem as an assignment problem (solved using Integer Linear Programming), (b) a novel probabilistic model of the affinity between tracklets and RFID data, and (c) a curated dataset with per-frame BB and regularly spaced ground-truth annotations for evaluating the models. The latter is a crucial part of the model, as it provides a principled probabilistic treatment of object detections given coarse localisation. Our approach achieves 77% accuracy on this animal identification problem, and is able to reject spurious detections when the animals are hidden.

Learning fast and fine-grained detection of amyloid neuropathologies from coarse-grained expert labels.

Precise, scalable, and quantitative evaluation of whole slide images is crucial in neuropathology. We release a deep learning model for rapid object detection and precise information on the identification, locality, and counts of cored plaques and cerebral amyloid angiopathy (CAA). We trained this object detector using a repurposed image-tile dataset without any human-drawn bounding boxes. We evaluated the detector on a new manually-annotated dataset of whole slide images (WSIs) from three institutions, four staining procedures, and four human experts. The detector matched the cohort of neuropathology experts, achieving 0.64 (model) vs. 0.64 (cohort) average precision (AP) for cored plaques and 0.75 vs. 0.51 AP for CAAs at a 0.5 IOU threshold. It provided count and locality predictions that approximately correlated with gold-standard human CERAD-like WSI scoring (p = 0.07 ± 0.10). The openly-available model can quickly score WSIs in minutes without a GPU on a standard workstation.

Hyaline protoplasmic astrocytopathy in epilepsy.

Hyaline protoplasmic astrocytopathy (HPA) describes a rare histologic finding of eosinophilic, hyaline cytoplasmic inclusions in astrocytes, predominantly in the cerebral cortex. It has mainly been observed in children and adults with a history of developmental delay and epilepsy, frequently with focal cortical dysplasia (FCD), but the nature and significance of these inclusions are unclear. In this study, we review the clinical and pathologic features of HPA and characterize the inclusions and brain tissue in which they are seen in surgical resection specimens from five patients with intractable epilepsy and HPA compared to five patients with intractable epilepsy without HPA using immunohistochemistry for filamin A, previously shown to label these inclusions, and a variety of astrocytic markers including aldehyde dehydrogenase 1 family member L1 (ALDH1L1), SRY-Box Transcription Factor 9 (SOX9), and glutamate transporter 1/excitatory amino acid transporter 2 (GLT-1/EAAT2) proteins. The inclusions were positive for ALDH1L1 with increased ALDH1L1 expression in areas of gliosis. SOX9 was also positive in the inclusions, although to a lesser intensity than the astrocyte nuclei. Filamin A labeled the inclusions but also labeled reactive astrocytes in a subset of patients. The immunoreactivity of the inclusions for various astrocytic markers and filamin A as well as the positivity of filamin A in reactive astrocytes raise the possibility that these astrocytic inclusions may be the result of an uncommon reactive or degenerative phenomenon.

Inference and Learning for Generative Capsule Models.

Capsule networks (see Hinton et al., 2018) aim to encode knowledge of and reason about the relationship between an object and its parts. In this letter, we specify a generative model for such data and derive a variational algorithm for inferring the transformation of each model object in a scene and the assignments of observed parts to the objects. We derive a learning algorithm for the object models, based on variational expectation maximization (Jordan et al., 1999). We also study an alternative inference algorithm based on the RANSAC method of Fischler and Bolles (1981). We apply these inference methods to data generated from multiple geometric objects like squares and triangles ("constellations") and data from a parts-based model of faces. Recent work by Kosiorek et al. (2019) has used amortized inference via stacked capsule autoencoders to tackle this problem; our results show that we significantly outperform them where we can make comparisons (on the constellations data).

Small molecules disaggregate alpha-synuclein and prevent seeding from patient brain-derived fibrils.

The amyloid aggregation of alpha-synuclein within the brain is associated with the pathogenesis of Parkinson's disease (PD) and other related synucleinopathies, including multiple system atrophy (MSA). Alpha-synuclein aggregates are a major therapeutic target for treatment of these diseases. We identify two small molecules capable of disassembling preformed alpha-synuclein fibrils. The compounds, termed CNS-11 and CNS-11g, disaggregate recombinant alpha-synuclein fibrils in vitro, prevent the intracellular seeded aggregation of alpha-synuclein fibrils, and mitigate alpha-synuclein fibril cytotoxicity in neuronal cells. Furthermore, we demonstrate that both compounds disassemble fibrils extracted from MSA patient brains and prevent their intracellular seeding. They also reduce in vivo alpha-synuclein aggregates in C. elegans. Both compounds also penetrate brain tissue in mice. A molecular dynamics-based computational model suggests the compounds may exert their disaggregating effects on the N terminus of the fibril core. These compounds appear to be promising therapeutic leads for targeting alpha-synuclein for the treatment of synucleinopathies.

Frequency and types of alternative breeding strategies employed by nesting American black ducks in North Carolina.

Although most birds are considered to be at least partially monogamous, molecular evidence continues to uncover that many species can have multiple sexual mates. Many species of Waterfowl (Order Anseriformes) consistently deploy alternative breeding strategies, and although cavity nesting species have been well studied, few attempts to understand rates of alternative breeding strategies exist in the Anatini tribe. Here, we assay mitochondrial DNA and thousands of nuclear markers across 20 broods of American black ducks (Anas rubripes; "black duck") that included 19 females and 172 offspring to study population structure as well as types and rates of secondary breeding strategies in coastal North Carolina. First, we report high levels of relatedness among nesting black ducks and offspring and while 17 (of 19) females were of pure black duck descent, three were found to be black duck x mallard (A. platyrhynchos) hybrids. Next, we evaluated for mismatched mitochondrial DNA and paternity identities across each female's clutch to determine types and frequency of alternative or secondary breeding strategies. Although we report that nest parasitism occurred in two nests, 37% (7 of 19) of the sampled nests were multi-paternal as a result of extra-pair copulation. In addition to being part of a mix of strategies used to increase fecundity by successfully breeding females, we posit nest densities providing easier alternative mate access for males also explains high rates of extra-pair copulation among our sampled black ducks. Ultimately, however, while some proportion of females of many species engage in forms of secondary breeding strategies, we conclude that the decision to do so appears to be seasonally flexible for each individual.

Post-translational modifications of soluble α-synuclein regulate the amplification of pathological α-synuclein.

Cell-to-cell transmission and subsequent amplification of pathological proteins promote neurodegenerative disease progression. Most research on this has focused on pathological protein seeds, but how their normal counterparts, which are converted to pathological forms during transmission, regulate transmission is less understood. Here we show in cultured cells that phosphorylation of soluble, nonpathological α-synuclein (α-Syn) at previously identified sites dramatically affects the amplification of pathological α-Syn, which underlies Parkinson's disease and other α-synucleinopathies, in a conformation- and phosphorylation site-specific manner. We performed LC-MS/MS analyses on soluble α-Syn purified from Parkinson's disease and other α-synucleinopathies, identifying many new α-Syn post-translational modifications (PTMs). In addition to phosphorylation, acetylation of soluble α-Syn also modified pathological α-Syn transmission in a site- and conformation-specific manner. Moreover, phosphorylation of soluble α-Syn could modulate the seeding properties of pathological α-Syn. Our study represents the first systematic analysis how of soluble α-Syn PTMs affect the spreading and amplification of pathological α-Syn, which may affect disease progression.

Learning fast and fine-grained detection of amyloid neuropathologies from coarse-grained expert labels.

Precise, scalable, and quantitative evaluation of whole slide images is crucial in neuropathology. We release a deep learning model for rapid object detection and precise information on the identification, locality, and counts of cored plaques and cerebral amyloid angiopathies (CAAs). We trained this object detector using a repurposed image-tile dataset without any human-drawn bounding boxes. We evaluated the detector on a new manually-annotated dataset of whole slide images (WSIs) from three institutions, four staining procedures, and four human experts. The detector matched the cohort of neuropathology experts, achieving 0.64 (model) vs. 0.64 (cohort) average precision (AP) for cored plaques and 0.75 vs. 0.51 AP for CAAs at a 0.5 IOU threshold. It provided count and locality predictions that correlated with gold-standard CERAD-like WSI scoring (p=0.07± 0.10). The openly-available model can quickly score WSIs in minutes without a GPU on a standard workstation.

Neuropathology of microbleeds in cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL).

Cerebral microbleeds (CMBs) detected on magnetic resonance imaging are common in patients with cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL). The neuropathologic correlates of CMBs are unclear. In this study, we characterized findings relevant to CMBs in autopsy brain tissue of 8 patients with genetically confirmed CADASIL and 10 controls within the age range of the CADASIL patients by assessing the distribution and extent of hemosiderin/iron deposits including perivascular hemosiderin leakage (PVH), capillary hemosiderin deposits, and parenchymal iron deposits (PID) in the frontal cortex and white matter, basal ganglia and cerebellum. We also characterized infarcts, vessel wall thickening, and severity of vascular smooth muscle cell degeneration. CADASIL subjects had a significant increase in hemosiderin/iron deposits compared with controls. This increase was principally seen with PID. Hemosiderin/iron deposits were seen in the majority of CADASIL subjects in all brain areas. PVH was most pronounced in the frontal white matter and basal ganglia around small to medium sized arterioles, with no predilection for the vicinity of vessels with severe vascular changes or infarcts. CADASIL subjects have increased brain hemosiderin/iron deposits but these do not occur in a periarteriolar distribution. Pathogenesis of these lesions remains uncertain.

IL-17/CXCL5 signaling within the oligovascular niche mediates human and mouse white matter injury.

Cerebral small vessel disease and brain white matter injury are worsened by cardiovascular risk factors including obesity. Molecular pathways in cerebral endothelial cells activated by chronic cerebrovascular risk factors alter cell-cell signaling, blocking endogenous and post-ischemic white matter repair. Using cell-specific translating ribosome affinity purification (RiboTag) in white matter endothelia and oligodendrocyte progenitor cells (OPCs), we identify a coordinated interleukin-chemokine signaling cascade within the oligovascular niche of subcortical white matter that is triggered by diet-induced obesity (DIO). DIO induces interleukin-17B (IL-17B) signaling that acts on the cerebral endothelia through IL-17Rb to increase both circulating and local endothelial expression of CXCL5. In white matter endothelia, CXCL5 promotes the association of OPCs with the vasculature and triggers OPC gene expression programs regulating cell migration through chemokine signaling. Targeted blockade of IL-17B reduced vessel-associated OPCs by reducing endothelial CXCL5 expression. In multiple human cohorts, blood levels of CXCL5 function as a diagnostic and prognostic biomarker of vascular cognitive impairment.

Underwater hearing in sea ducks with applications for reducing gillnet bycatch through acoustic deterrence.

As diving foragers, sea ducks are vulnerable to underwater anthropogenic activity, including ships, underwater construction, seismic surveys and gillnet fisheries. Bycatch in gillnets is a contributing source of mortality for sea ducks, killing hundreds of thousands of individuals annually. We researched underwater hearing in sea duck species to increase knowledge of underwater avian acoustic sensitivity and to assist with possible development of gillnet bycatch mitigation strategies that include auditory deterrent devices. We used both psychoacoustic and electrophysiological techniques to investigate underwater duck hearing in several species including the long-tailed duck (Clangula hyemalis), surf scoter (Melanitta perspicillata) and common eider (Somateria mollissima). Psychoacoustic results demonstrated that all species tested share a common range of maximum auditory sensitivity of 1.0-3.0 kHz, with the long-tailed ducks and common eiders at the high end of that range (2.96 kHz), and surf scoters at the low end (1.0 kHz). In addition, our electrophysiological results from 4 surf scoters and 2 long-tailed ducks, while only tested at 0.5, 1 and 2 kHz, generally agree with the audiogram shape from our psychoacoustic testing. The results from this study are applicable to the development of effective acoustic deterrent devices or pingers in the 2-3 kHz range to deter sea ducks from anthropogenic threats.

Structure-based discovery of small molecules that disaggregate Alzheimer's disease tissue derived tau fibrils in vitro.

Alzheimer's disease (AD) is the consequence of neuronal death and brain atrophy associated with the aggregation of protein tau into fibrils. Thus disaggregation of tau fibrils could be a therapeutic approach to AD. The small molecule EGCG, abundant in green tea, has long been known to disaggregate tau and other amyloid fibrils, but EGCG has poor drug-like properties, failing to fully penetrate the brain. Here we have cryogenically trapped an intermediate of brain-extracted tau fibrils on the kinetic pathway to EGCG-induced disaggregation and have determined its cryoEM structure. The structure reveals that EGCG molecules stack in polar clefts between the paired helical protofilaments that pathologically define AD. Treating the EGCG binding position as a pharmacophore, we computationally screened thousands of drug-like compounds for compatibility for the pharmacophore, discovering several that experimentally disaggregate brain-derived tau fibrils in vitro. This work suggests the potential of structure-based, small-molecule drug discovery for amyloid diseases.

On Suspicious Coincidences and Pointwise Mutual Information.

Barlow (1985) hypothesized that the co-occurrence of two events A and B is "suspicious" if P(A,B)≫P(A)P(B). We first review classical measures of association for 2 × 2 contingency tables, including Yule's Y (Yule, 1912), which depends only on the odds ratio λ and is independent of the marginal probabilities of the table. We then discuss the mutual information (MI) and pointwise mutual information (PMI), which depend on the ratio P(A,B)/P(A)P(B), as measures of association. We show that once the effect of the marginals is removed, MI and PMI behave similarly to Y as functions of λ. The pointwise mutual information is used extensively in some research communities for flagging suspicious coincidences. We discuss the pros and cons of using it in this way, bearing in mind the sensitivity of the PMI to the marginals, with increased scores for sparser events.

Neuropathologic Findings in Elderly HIV-Positive Individuals.

The elderly HIV-positive population is growing due to the widespread use of combination antiretroviral therapy (cART), but the effects of longstanding HIV infection on brain aging are unknown. A significant proportion of HIV-positive individuals develop HIV-associated neurocognitive disorder (HAND) even on cART, but the pathogenesis of HAND is unknown. Although neuroinflammation is postulated to play an important role in aging and neurodegenerative diseases such as Alzheimer disease (AD), it is unclear whether HIV accelerates aging or increases the risk for AD. We examined the brains of 9 elderly HIV-positive subjects on cART without co-infection by hepatitis C virus compared to 7 elderly HIV-negative subjects. Microglial and astrocyte activation and AD pathologic change in association with systemic comorbidities and neurocognitive assessment were evaluated. There was no difference in microglial or astrocyte activation between our HIV-positive and HIV-negative cohorts. One HIV-positive subject and 2 HIV-negative subjects demonstrated significant amyloid deposition, predominantly in the form of diffuse senile plaques, but these individuals were cognitively normal. Neurofibrillary tangles were sparse in the HIV-positive cohort. There was a high prevalence of cardiovascular comorbidities in all subjects. These findings suggest that multiple factors likely contribute to aging and cognitive impairment in elderly HIV-positive individuals on cART.

A lesser scaup (Aythya affinis) naturally infected with Eurasian 2.3.4.4 highly pathogenic H5N1 avian influenza virus: Movement ecology and host factors.

Despite the recognized role of wild waterfowl in the potential dispersal and transmission of highly pathogenic avian influenza (HPAI) virus, little is known about how infection affects these birds. This lack of information limits our ability to estimate viral spread in the event of an HPAI outbreak, thereby limiting our abilities to estimate and communicate risk. Here, we present telemetry data from a wild Lesser Scaup (Aythya affinis), captured during a separate ecology study in the Chesapeake Bay, Maryland. This bird tested positive for infection with clade 2.3.4.4 HPAI virus of the A/goose/Guangdong/1/1996 (Gs/GD) H5N1 lineage (results received post-release) during the 2021-2022 ongoing outbreaks in North America. While the infected bird was somewhat lighter than other adult males surgically implanted with transmitters (790 g, x̅ = 868 g, n = 11), it showed no clinical signs of infection at capture, during surgery, nor upon release. The bird died 3 days later-pathology undetermined as the specimen was not able to be recovered. Analysis of movement data within the 3-day window showed that the infected individual's maximum and average hourly movements (3894.3 and 428.8 m, respectively) were noticeably lower than noninfected conspecifics tagged and released the same day (x̅ = 21,594.5 and 1097.9 m, respectively; n = 4). We identified four instances where the infected bird had close contact (fixes located within 25 m and 15 min) with another marked bird during this time. Collectively, these data suggest that the HPAI-positive bird observed in this study may have been shedding virus for some period prior to death, with opportunities for direct bird-to-bird or environmental transmission. Although limited by low sample size and proximity to the time of tagging, we hope that these data will provide useful information as managers continue to respond to this ongoing outbreak event.

Nest parasitism, promiscuity, and relatedness among wood ducks.

Nest parasitism is a common reproductive strategy used by many species of cavity nesting birds. Among these, the wood duck (Aix sponsa) is known to have evolved very specific strategies of when and whom to parasitize that is often based on population and/or environmental queues. Here, we investigated the genetic relationship of two female wood ducks competing over an artificial nesting box in Delaware, including the continued incubation of one female despite the death and body remains of the other female throughout the incubation process. We test whether such an extreme case of nest parasitism can be explained by relatedness, egg lineage composition, or a combination of other factors. To do so, we extracted genomic DNA from blood and tissue of the females, as well as chorioallantoic membranes of all viable and inviable eggs. Subsequently, we assessed relatedness among females and eggs based on hundreds of nuclear loci and the mitochondrial control region. We concluded that (1) the two incubating females were entirely unrelated, (2) the single clutch is in fact represented by a minimum of four unrelated females, and (3) a single female can lay eggs sired by different males. The latter finding is the first direct evidence for successful extra-pair copulation in wood ducks. With decreasing costs and increasing effectiveness, genomic methods have the potential to provide important insights into more complex ecological and evolutionary tactics of such populations.