Selective targeting and modulation of plaque associated microglia via systemic hydroxyl dendrimer administration in an Alzheimer's disease mouse model.

BACKGROUND: In Alzheimer's disease (AD), microglia surround extracellular plaques and mount a sustained inflammatory response, contributing to the pathogenesis of the disease. Identifying approaches to specifically target plaque-associated microglia (PAMs) without interfering in the homeostatic functions of non-plaque associated microglia would afford a powerful tool and potential therapeutic avenue. METHODS: Here, we demonstrated that a systemically administered nanomedicine, hydroxyl dendrimers (HDs), can cross the blood brain barrier and are preferentially taken up by PAMs in a mouse model of AD. As proof of principle, to demonstrate biological effects in PAM function, we treated the 5xFAD mouse model of amyloidosis for 4 weeks via systemic administration (ip, 2x weekly) of HDs conjugated to a colony stimulating factor-1 receptor (CSF1R) inhibitor (D-45113). RESULTS: Treatment resulted in significant reductions in amyloid-beta (Aß) and a stark reduction in the number of microglia and microglia-plaque association in the subiculum and somatosensory cortex, as well as a downregulation in microglial, inflammatory, and synaptic gene expression compared to vehicle treated 5xFAD mice. CONCLUSIONS: This study demonstrates that systemic administration of a dendranib may be utilized to target and modulate PAMs.

Sex-specific associations between AD genotype and the microbiome of human amyloid beta knock-in (hAß-KI) mice.

INTRODUCTION: Emerging evidence links changes in the gut microbiome to late-onset Alzheimer's disease (LOAD), necessitating examination of AD mouse models with consideration of the microbiome. METHODS: We used shotgun metagenomics and untargeted metabolomics to study the human amyloid beta knock-in (hAß-KI) murine model for LOAD compared to both wild-type (WT) mice and a model for early-onset AD (3xTg-AD). RESULTS: Eighteen-month female (but not male) hAß-KI microbiomes were distinct from WT microbiomes, with AD genotype accounting for 18% of the variance by permutational multivariate analysis of variance (PERMANOVA). Metabolomic diversity differences were observed in females, however no individual metabolites were differentially abundant. hAß-KI mice microbiomes were distinguishable from 3xTg-AD animals (81% accuracy by random forest modeling), with separation primarily driven by Romboutsia ilealis and Turicibacter species. Microbiomes were highly cage specific, with cage assignment accounting for more than 40% of the PERMANOVA variance between the groups. DISCUSSION: These findings highlight a sex-dependent variation in the microbiomes of hAß-KI mice and underscore the importance of considering the microbiome when designing studies that use murine models for AD. HIGHLIGHTS: Microbial diversity and the abundance of several species differed in human amyloid beta knock-in (hAß-KI) females but not males. Correlations to Alzheimer's disease (AD) genotype were stronger for the microbiome than the metabolome. Microbiomes from hAß-KI mice were distinct from 3xTg-AD mice. Cage effects accounted for most of the variance in the microbiome and metabolome.

The Abca7V1613M variant reduces Aß generation, plaque load, and neuronal damage.

BACKGROUND: Variants in ABCA7, a member of the ABC transporter superfamily, have been associated with increased risk for developing late onset Alzheimer's disease (LOAD). METHODS: CRISPR-Cas9 was used to generate an Abca7V1613M variant in mice, modeling the homologous human ABCA7V1599M variant, and extensive characterization was performed. RESULTS: Abca7V1613M microglia show differential gene expression profiles upon lipopolysaccharide challenge and increased phagocytic capacity. Homozygous Abca7V1613M mice display elevated circulating cholesterol and altered brain lipid composition. When crossed with 5xFAD mice, homozygous Abca7V1613M mice display fewer Thioflavin S-positive plaques, decreased amyloid beta (Aß) peptides, and altered amyloid precursor protein processing and trafficking. They also exhibit reduced Aß-associated inflammation, gliosis, and neuronal damage. DISCUSSION: Overall, homozygosity for the Abca7V1613M variant influences phagocytosis, response to inflammation, lipid metabolism, Aß pathology, and neuronal damage in mice. This variant may confer a gain of function and offer a protective effect against Alzheimer's disease-related pathology. HIGHLIGHTS: ABCA7 recognized as a top 10 risk gene for developing Alzheimer's disease. Loss of function mutations result in increased risk for LOAD. V1613M variant reduces amyloid beta plaque burden in 5xFAD mice. V1613M variant modulates APP processing and trafficking in 5xFAD mice. V1613M variant reduces amyloid beta-associated damage in 5xFAD mice.

Genetic diversity promotes resilience in a mouse model of Alzheimer's disease.

INTRODUCTION: Alzheimer's disease (AD) is a neurodegenerative disorder with multifactorial etiology, including genetic factors that play a significant role in disease risk and resilience. However, the role of genetic diversity in preclinical AD studies has received limited attention. METHODS: We crossed five Collaborative Cross strains with 5xFAD C57BL/6J female mice to generate F1 mice with and without the 5xFAD transgene. Amyloid plaque pathology, microglial and astrocytic responses, neurofilament light chain levels, and gene expression were assessed at various ages. RESULTS: Genetic diversity significantly impacts AD-related pathology. Hybrid strains showed resistance to amyloid plaque formation and neuronal damage. Transcriptome diversity was maintained across ages and sexes, with observable strain-specific variations in AD-related phenotypes. Comparative gene expression analysis indicated correlations between mouse strains and human AD. DISCUSSION: Increasing genetic diversity promotes resilience to AD-related pathogenesis, relative to an inbred C57BL/6J background, reinforcing the importance of genetic diversity in uncovering resilience in the development of AD. HIGHLIGHTS: Genetic diversity's impact on AD in mice was explored. Diverse F1 mouse strains were used for AD study, via the Collaborative Cross. Strain-specific variations in AD pathology, glia, and transcription were found. Strains resilient to plaque formation and plasma neurofilament light chain (NfL) increases were identified. Correlations with human AD transcriptomics were observed.

BIN1K358R suppresses glial response to plaques in mouse model of Alzheimer's disease.

INTRODUCTION: The BIN1 coding variant rs138047593 (K358R) is linked to Late-Onset Alzheimer's Disease (LOAD) via targeted exome sequencing. METHODS: To elucidate the functional consequences of this rare coding variant on brain amyloidosis and neuroinflammation, we generated BIN1K358R knock-in mice using CRISPR/Cas9 technology. These mice were subsequently bred with 5xFAD transgenic mice, which serve as a model for Alzheimer's pathology. RESULTS: The presence of the BIN1K358R variant leads to increased cerebral amyloid deposition, with a dampened response of astrocytes and oligodendrocytes, but not microglia, at both the cellular and transcriptional levels. This correlates with decreased neurofilament light chain in both plasma and brain tissue. Synaptic densities are significantly increased in both wild-type and 5xFAD backgrounds homozygous for the BIN1K358R variant. DISCUSSION: The BIN1 K358R variant modulates amyloid pathology in 5xFAD mice, attenuates the astrocytic and oligodendrocytic responses to amyloid plaques, decreases damage markers, and elevates synaptic densities. HIGHLIGHTS: BIN1 rs138047593 (K358R) coding variant is associated with increased risk of LOAD. BIN1 K358R variant increases amyloid plaque load in 12-month-old 5xFAD mice. BIN1 K358R variant dampens astrocytic and oligodendrocytic response to plaques. BIN1 K358R variant decreases neuronal damage in 5xFAD mice. BIN1 K358R upregulates synaptic densities and modulates synaptic transmission.

A non-human primate model for human norovirus infection.

Norovirus infection can cause gastrointestinal disease in humans. Development of therapies and vaccines against norovirus have been limited by the lack of a suitable and reliable animal model. Here we established rhesus macaques as an animal model for human norovirus infection. We show that rhesus macaques are susceptible to oral infection with human noroviruses from two different genogroups. Variation in duration of virus shedding (days to weeks) between animals, evolution of the virus over the time of infection, induction of virus-specific adaptive immune responses, susceptibility to reinfection and preferential replication of norovirus in the jejunum of rhesus macaques was similar to infection reported in humans. We found minor pathological signs and changes in epithelial cell surface glycosylation patterns in the small intestine during infection. Detection of viral protein and RNA in intestinal biopsies confirmed the presence of the virus in chromogranin A-expressing epithelial cells, as it does in humans. Thus, rhesus macaques are a promising non-human primate model to evaluate vaccines and therapeutics against norovirus disease.

Single cell spatial transcriptomics reveals distinct patterns of dysregulation in non-neuronal and neuronal cells induced by the Trem2R47H Alzheimer's risk gene mutation.

INTRODUCTION: The R47H missense mutation of the TREM2 gene is a strong risk factor for development of Alzheimer's Disease. We investigate cell-type-specific spatial transcriptomic changes induced by the Trem2R47H mutation to determine the impacts of this mutation on transcriptional dysregulation. METHODS: We profiled 15 mouse brain sections consisting of wild-type, Trem2R47H, 5xFAD and Trem2R47H; 5xFAD genotypes using MERFISH spatial transcriptomics. Single-cell spatial transcriptomics and neuropathology data were analyzed using our custom pipeline to identify plaque and Trem2R47H induced transcriptomic dysregulation. RESULTS: The Trem2R47H mutation induced consistent upregulation of Bdnf and Ntrk2 across many cortical excitatory neuron types, independent of amyloid pathology. Spatial investigation of genotype enriched subclusters identified spatially localized neuronal subpopulations reduced in 5xFAD and Trem2R47H; 5xFAD mice. CONCLUSION: Spatial transcriptomics analysis identifies glial and neuronal transcriptomic alterations induced independently by 5xFAD and Trem2R47H mutations, impacting inflammatory responses in microglia and astrocytes, and activity and BDNF signaling in neurons.

Microglia influence immune responses and restrict neurologic disease in response to central nervous system infection by a neurotropic murine coronavirus.

Intracranial (i.c.) inoculation of susceptible mice with a glial-tropic strain of mouse hepatitis virus (JHMV), a murine coronavirus, results in an acute encephalomyelitis followed by viral persistence in white matter tracts accompanied by chronic neuroinflammation and demyelination. Microglia serve numerous functions including maintenance of the healthy central nervous system (CNS) and are among the first responders to injury or infection. More recently, studies have demonstrated that microglia aid in tailoring innate and adaptive immune responses following infection by neurotropic viruses including flaviviruses, herpesviruses, and picornaviruses. These findings have emphasized an important role for microglia in host defense against these viral pathogens. In addition, microglia are also critical in optimizing immune-mediated control of JHMV replication within the CNS while restricting the severity of demyelination and enhancing remyelination. This review will highlight our current understanding of the molecular and cellular mechanisms by which microglia aid in host defense, limit neurologic disease, and promote repair following CNS infection by a neurotropic murine coronavirus.

Microglia promote anti-tumour immunity and suppress breast cancer brain metastasis.

Breast cancer brain metastasis (BCBM) is a lethal disease with no effective treatments. Prior work has shown that brain cancers and metastases are densely infiltrated with anti-inflammatory, protumourigenic tumour-associated macrophages, but the role of brain-resident microglia remains controversial because they are challenging to discriminate from other tumour-associated macrophages. Using single-cell RNA sequencing, genetic and humanized mouse models, we specifically identify microglia and find that they play a distinct pro-inflammatory and tumour-suppressive role in BCBM. Animals lacking microglia show increased metastasis, decreased survival and reduced natural killer and T cell responses, showing that microglia are critical to promote anti-tumour immunity to suppress BCBM. We find that the pro-inflammatory response is conserved in human microglia, and markers of their response are associated with better prognosis in patients with BCBM. These findings establish an important role for microglia in anti-tumour immunity and highlight them as a potential immunotherapy target for brain metastasis.

A single nanobody neutralizes multiple epochally evolving human noroviruses by modulating capsid plasticity.

Acute gastroenteritis caused by human noroviruses (HuNoVs) is a significant global health and economic burden and is without licensed vaccines or antiviral drugs. The GII.4 HuNoV causes most epidemics worldwide. This virus undergoes epochal evolution with periodic emergence of variants with new antigenic profiles and altered specificity for histo-blood group antigens (HBGA), the determinants of cell attachment and susceptibility, hampering the development of immunotherapeutics. Here, we show that a llama-derived nanobody M4 neutralizes multiple GII.4 variants with high potency in human intestinal enteroids. The crystal structure of M4 complexed with the protruding domain of the GII.4 capsid protein VP1 revealed a conserved epitope, away from the HBGA binding site, fully accessible only when VP1 transitions to a "raised" conformation in the capsid. Together with dynamic light scattering and electron microscopy of the GII.4 VLPs, our studies suggest a mechanism in which M4 accesses the epitope by altering the conformational dynamics of the capsid and triggering its disassembly to neutralize GII.4 infection.

COVID-19 and the impact on Alzheimer's disease pathology.

Coronavirus disease 2019 (COVID-19) has rapidly escalated into a global pandemic that primarily affects older and immunocompromised individuals due to underlying clinical conditions and suppressed immune responses. Furthermore, COVID-19 patients exhibit a spectrum of neurological symptoms, indicating that COVID-19 can affect the brain in a variety of manners. Many studies, past and recent, suggest a connection between viral infections and an increased risk of neurodegeneration, raising concerns about the neurological effects of COVID-19 and the possibility that it may contribute to Alzheimer's disease (AD) onset or worsen already existing AD pathology through inflammatory processes given that both COVID-19 and AD share pathological features and risk factors. This leads us to question whether COVID-19 is a risk factor for AD and how these two conditions might influence each other. Considering the extensive reach of the COVID-19 pandemic and the devastating impact of the ongoing AD pandemic, their combined effects could have significant public health consequences worldwide.

Norovirus evolves as one or more distinct clonal populations in immunocompromised hosts.

Noroviruses are a major cause of acute gastroenteritis worldwide and can establish chronic infection in immunocompromised individuals. To investigate the mechanisms of norovirus evolution during chronic infection, we selected seven representative patients from a National Institutes of Health study cohort who sustained norovirus infection for periods ranging from 73 to 1,492 days. Six patients shed viruses belonging to a single genotype (GII.2[PNA], GII.4 New Orleans[P4], GII.4 Den Haag[P4], GII.3[P21], GII.6[P7], or GII.14[P7]) over the period examined, while one patient sequentially shed two genotypes (GII.6[P7] followed by GII.4 Sydney[P31]). Norovirus genomes from consecutive stool samples were sequenced at high resolution (>3,300 reads/nucleotide position) using the Illumina platform and subjected to bioinformatics analysis. Norovirus sequences could be resolved into one or more discrete clonal RNA genomes that persisted within these patients over time. Phylogenetic analyses inferred that clonal populations originated from a single founder virus and not by reinfection with community strains. Estimated evolutionary rates of clonal populations during persistent infection were similar to those of noroviruses from acute infection in the global database, suggesting that inherently higher RNA-dependent polymerase error rates were not associated with the ability to persist. The high-resolution analysis of norovirus diversity and evolution at the population level described here should allow a better understanding of adaptive mutations sustained during chronic infection. IMPORTANCE Noroviruses are an important cause of chronic diarrhea in patients with compromised immune systems. Presently, there are no effective therapies to clear the virus, which can persist for years in the intestinal tract. The goal of our study was to develop a better understanding of the norovirus strains that are associated with these long-term infections. With the remarkable diversity of norovirus strains detected in the immunocompromised patient cohort we studied, it appears that most, if not all, noroviruses circulating in nature may have the capacity to establish a chronic infection when a person is unable to mount an effective immune response. Our work is the most comprehensive genetic data set generated to date in which near full-length genomes from noroviruses associated with chronic infection were analyzed by high-resolution next-generation sequencing. Analysis of this data set led to our discovery that certain patients in our cohort were shedding noroviruses that could be subdivided into distinct haplotypes or populations of viruses that were co-evolving independently. The ability to track haplotypes of noroviruses during chronic infection will allow us to fine-tune our understanding of how the virus adapts and maintains itself in the human host, and how selective pressures such as antiviral drugs can affect these distinct populations.

Spatial and single-nucleus transcriptomic analysis of genetic and sporadic forms of Alzheimer's Disease.

The pathogenesis of Alzheimer's disease (AD) depends on environmental and heritable factors, with remarkable differences evident between individuals at the molecular level. Here we present a transcriptomic survey of AD using spatial transcriptomics (ST) and single-nucleus RNA-seq in cortical samples from early-stage AD, late-stage AD, and AD in Down Syndrome (AD in DS) donors. Studying AD in DS provides an opportunity to enhance our understanding of the AD transcriptome, potentially bridging the gap between genetic mouse models and sporadic AD. Our analysis revealed spatial and cell-type specific changes in disease, with broad similarities in these changes between sAD and AD in DS. We performed additional ST experiments in a disease timecourse of 5xFAD and wildtype mice to facilitate cross-species comparisons. Finally, amyloid plaque and fibril imaging in the same tissue samples used for ST enabled us to directly link changes in gene expression with accumulation and spread of pathology.

Performance and risks of ChatGPT used in drug information: an exploratory real-world analysis.

OBJECTIVES: To investigate the performance and risk associated with the usage of Chat Generative Pre-trained Transformer (ChatGPT) to answer drug-related questions. METHODS: A sample of 50 drug-related questions were consecutively collected and entered in the artificial intelligence software application ChatGPT. Answers were documented and rated in a standardised consensus process by six senior hospital pharmacists in the domains content (correct, incomplete, false), patient management (possible, insufficient, not possible) and risk (no risk, low risk, high risk). As reference, answers were researched in adherence to the German guideline of drug information and stratified in four categories according to the sources used. In addition, the reproducibility of ChatGPT's answers was analysed by entering three questions at different timepoints repeatedly (day 1, day 2, week 2, week 3). RESULTS: Overall, only 13 of 50 answers provided correct content and had enough information to initiate management with no risk of patient harm. The majority of answers were either false (38%, n=19) or had partly correct content (36%, n=18) and no references were provided. A high risk of patient harm was likely in 26% (n=13) of the cases and risk was judged low for 28% (n=14) of the cases. In all high-risk cases, actions could have been initiated based on the provided information. The answers of ChatGPT varied over time when entered repeatedly and only three out of 12 answers were identical, showing no reproducibility to low reproducibility. CONCLUSION: In a real-world sample of 50 drug-related questions, ChatGPT answered the majority of questions wrong or partly wrong. The use of artificial intelligence applications in drug information is not possible as long as barriers like wrong content, missing references and reproducibility remain.

Ablation of microglia following infection of the central nervous system with a neurotropic murine coronavirus infection leads to increased demyelination and impaired remyelination.

Intracranial inoculation of susceptible mice with a glial-tropic strain of mouse hepatitis virus (JHMV), a murine coronavirus, results in an acute encephalomyelitis followed by viral persistence in white matter tracts accompanied by chronic neuroinflammation and demyelination. Microglia are the resident immune cell of the central nervous system (CNS) and are considered important in regulating events associated with neuroinflammation as well as influencing both white matter damage and remyelination. To better understand mechanisms by which microglia contribute to these immune-mediated events, JHMV-infected mice with established demyelination were treated with the small molecular inhibitor of colony stimulating factor 1 receptor (CSF1R), PLX5622, to deplete microglia. Treatment with PLX5622 did not affect viral replication within the CNS yet the severity of demyelination was increased and remyelination impaired compared to control mice. Gene expression analysis revealed that targeting microglia resulted in altered expression of genes associated with immune cell activation and phagocytosis of myelin debris. These findings indicate that microglia are not critical in viral surveillance in persistently JHMV-infected mice yet restrict white matter damage and remyelination, in part, by influencing phagocytosis of myelin debris.

A Trem2R47H mouse model without cryptic splicing drives age- and disease-dependent tissue damage and synaptic loss in response to plaques.

BACKGROUND: The TREM2 R47H variant is one of the strongest genetic risk factors for late-onset Alzheimer's Disease (AD). Unfortunately, many current Trem2 R47H mouse models are associated with cryptic mRNA splicing of the mutant allele that produces a confounding reduction in protein product. To overcome this issue, we developed the Trem2R47H NSS (Normal Splice Site) mouse model in which the Trem2 allele is expressed at a similar level to the wild-type Trem2 allele without evidence of cryptic splicing products. METHODS: Trem2R47H NSS mice were treated with the demyelinating agent cuprizone, or crossed with the 5xFAD mouse model of amyloidosis, to explore the impact of the TREM2 R47H variant on inflammatory responses to demyelination, plaque development, and the brain's response to plaques. RESULTS: Trem2R47H NSS mice display an appropriate inflammatory response to cuprizone challenge, and do not recapitulate the null allele in terms of impeded inflammatory responses to demyelination. Utilizing the 5xFAD mouse model, we report age- and disease-dependent changes in Trem2R47H NSS mice in response to development of AD-like pathology. At an early (4-month-old) disease stage, hemizygous 5xFAD/homozygous Trem2R47H NSS (5xFAD/Trem2R47H NSS) mice have reduced size and number of microglia that display impaired interaction with plaques compared to microglia in age-matched 5xFAD hemizygous controls. This is associated with a suppressed inflammatory response but increased dystrophic neurites and axonal damage as measured by plasma neurofilament light chain (NfL) level. Homozygosity for Trem2R47H NSS suppressed LTP deficits and loss of presynaptic puncta caused by the 5xFAD transgene array in 4-month-old mice. At a more advanced (12-month-old) disease stage 5xFAD/Trem2R47H NSS mice no longer display impaired plaque-microglia interaction or suppressed inflammatory gene expression, although NfL levels remain elevated, and a unique interferon-related gene expression signature is seen. Twelve-month old Trem2R47H NSS mice also display LTP deficits and postsynaptic loss. CONCLUSIONS: The Trem2R47H NSS mouse is a valuable model that can be used to investigate age-dependent effects of the AD-risk R47H mutation on TREM2 and microglial function including its effects on plaque development, microglial-plaque interaction, production of a unique interferon signature and associated tissue damage.

Mucosal and systemic neutralizing antibodies to norovirus induced in infant mice orally inoculated with recombinant rotaviruses.

Rotaviruses (RVs) preferentially replicate in the small intestine and frequently cause severe diarrheal disease, and the following enteric infection generally induces variable levels of protective systemic and mucosal immune responses in humans and other animals. Rhesus rotavirus (RRV) is a simian RV that was previously used as a human RV vaccine and has been extensively studied in mice. Although RRV replicates poorly in the suckling mouse intestine, infection induces a robust and protective antibody response. The recent availability of plasmid only-based RV reverse genetics systems has enabled the generation of recombinant RVs expressing foreign proteins. However, recombinant RVs have not yet been experimentally tested as potential vaccine vectors to immunize against other gastrointestinal pathogens in vivo. This is a newly available opportunity because several live-attenuated RV vaccines are already widely administered to infants and young children worldwide. To explore the feasibility of using RV as a dual vaccine vector, we rescued replication-competent recombinant RRVs harboring bicistronic gene segment 7 that encodes the native RV nonstructural protein 3 (NSP3) protein and a human norovirus (HuNoV) VP1 protein or P domain from the predominant genotype GII.4. The rescued viruses expressed HuNoV VP1 or P protein in infected cells in vitro and elicited systemic and local antibody responses to HuNoV and RRV following oral infection of suckling mice. Serum IgG and fecal IgA from infected suckling mice bound to and neutralized both RRV and HuNoV. These findings have encouraging practical implications for the design of RV-based next-generation multivalent enteric vaccines to target HuNoV and other human enteric pathogens.