Cognitive decline, Aß pathology, and blood-brain barrier function in aged 5xFAD mice.

BACKGROUND: Patients with Alzheimer's disease (AD) develop blood-brain barrier dysfunction to varying degrees. How aging impacts Aß pathology, blood-brain barrier function, and cognitive decline in AD remains largely unknown. In this study, we used 5xFAD mice to investigate changes in Aß levels, barrier function, and cognitive decline over time. METHODS: 5xFAD and wild-type (WT) mice were aged between 9.5 and 15.5 months and tested for spatial learning and reference memory with the Morris Water Maze (MWM). After behavior testing, mice were implanted with acute cranial windows and intravenously injected with fluorescent-labeled dextrans to assess their in vivo distribution in the brain by two-photon microscopy. Images were processed and segmented to obtain intravascular intensity, extravascular intensity, and vessel diameters as a measure of barrier integrity. Mice were sacrificed after in vivo imaging to isolate brain and plasma for measuring Aß levels. The effect of age and genotype were evaluated for each assay using generalized or cumulative-linked logistic mixed-level modeling and model selection by Akaike Information Criterion (AICc). Pairwise comparisons were used to identify outcome differences between the two groups. RESULTS: 5xFAD mice displayed spatial memory deficits compared to age-matched WT mice in the MWM assay, which worsened with age. Memory impairment was evident in 5xFAD mice by 2-threefold higher escape latencies, twofold greater cumulative distances until they reach the platform, and twice as frequent use of repetitive search strategies in the pool when compared with age-matched WT mice. Presence of the rd1 allele worsened MWM performance in 5xFAD mice at all ages but did not alter the rate of learning or probe trial outcomes. 9.5-month-old 15.5-month-old 5xFAD mice had twofold higher brain Aß40 and Aß42 levels (p < 0.001) and 2.5-fold higher (p = 0.007) plasma Aß40 levels compared to 9.5-month-old 5xFAD mice. Image analysis showed that vessel diameters and intra- and extravascular dextran intensities were not significantly different in 9.5- and 15.5-month-old 5xFAD mice compared to age-matched WT mice. CONCLUSION: 5xFAD mice continue to develop spatial memory deficits and increased Aß brain levels while aging. Given in vivo MP imaging limitations, further investigation with smaller molecular weight markers combined with advanced imaging techniques would be needed to reliably assess subtle differences in barrier integrity in aged mice.

A high-throughput single-cell RNA expression profiling method identifies human pericyte markers.

AIMS: We sought to identify and optimise a universally available histological marker for pericytes in the human brain. Such a marker could be a useful tool for researchers. Further, identifying a gene expressed relatively specifically in human pericytes could provide new insights into the biological functions of this fascinating cell type. METHODS: We analysed single-cell RNA expression profiles derived from different human and mouse brain regions using a high-throughput and low-cost single-cell transcriptome sequencing method called EasySci. Through this analysis, we were able to identify specific gene markers for pericytes, some of which had not been previously characterised. We then used commercially (and therefore universally) available antibodies to immunolabel the pericyte-specific gene products in formalin-fixed paraffin-embedded (FFPE) human brains and also performed immunoblots to determine whether appropriately sized proteins were recognised. RESULTS: In the EasySci data sets, highly pericyte-enriched expression was notable for SLC6A12 and SLC19A1. Antibodies against these proteins recognised bands of approximately the correct size in immunoblots of human brain extracts. Following optimisation of the immunohistochemical technique, staining for both antibodies was strongly positive in small blood vessels and was far more effective than a PDGFRB antibody at staining pericyte-like cells in FFPE human brain sections. In an exploratory sample of other human organs (kidney, lung, liver, muscle), immunohistochemistry did not show the same pericyte-like pattern of staining. CONCLUSIONS: The SLC6A12 antibody was well suited for labelling pericytes in human FFPE brain sections, based on the combined results of single-cell RNA-seq analyses, immunoblots and immunohistochemical studies.

Brain barriers virtual: an interim solution or future opportunity?

BACKGROUND: Scientific conferences are vital communication events for scientists in academia, industry, and government agencies. In the brain barriers research field, several international conferences exist that allow researchers to present data, share knowledge, and discuss novel ideas and concepts. These meetings are critical platforms for researchers to connect and exchange breakthrough findings on a regular basis. Due to the worldwide COVID-19 pandemic, all in-person meetings were canceled in 2020. In response, we launched the Brain Barriers Virtual 2020 (BBV2020) seminar series, the first stand-in virtual event for the brain barriers field, to offer scientists a virtual platform to present their work. Here we report the aggregate attendance information on two in-person meetings compared with BBV2020 and comment on the utility of the virtual platform. METHODS: The BBV2020 seminar series was hosted on a Zoom webinar platform and was free of cost for participants. Using registration- and Zoom-based data from the BBV2020 virtual seminar series and survey data collected from BBV2020 participants, we analyzed attendance trends, global reach, participation based on career stage, and engagement of BBV2020. We compared these data with those from two previous in-person conferences, a BBB meeting held in 2018 and CVB 2019. RESULTS: We found that BBV2020 seminar participation steadily decreased over the course of the series. In contrast, live participation was consistently above 100 attendees and recording views were above 200 views per seminar. We also found that participants valued BBV2020 as a supplement during the COVID-19 pandemic in 2020. Based on one post-BBV2020 survey, the majority of participants indicated that they would prefer in-person meetings but would welcome a virtual component to future in-person meetings. Compared to in-person meetings, BBV2020 enabled participation from a broad range of career stages and was attended by scientists in academic, industry, and government agencies from a wide range of countries worldwide. CONCLUSIONS: Our findings suggest that a virtual event such as the BBV2020 seminar series provides easy access to science for researchers across all career stages around the globe. However, we recognize that limitations exist. Regardless, such a virtual event could be a valuable tool for the brain barriers community to reach and engage scientists worldwide to further grow the brain barriers research field in the future.

Blood-brain barrier leakage in Alzheimer's disease: From discovery to clinical relevance.

Alzheimer's disease (AD) is the most common form of dementia. AD brain pathology starts decades before the onset of clinical symptoms. One early pathological hallmark is blood-brain barrier dysfunction characterized by barrier leakage and associated with cognitive decline. In this review, we summarize the existing literature on the extent and clinical relevance of barrier leakage in AD. First, we focus on AD animal models and their susceptibility to barrier leakage based on age and genetic background. Second, we re-examine barrier dysfunction in clinical and postmortem studies, summarize changes that lead to barrier leakage in patients and highlight the clinical relevance of barrier leakage in AD. Third, we summarize signaling mechanisms that link barrier leakage to neurodegeneration and cognitive decline in AD. Finally, we discuss clinical relevance and potential therapeutic strategies and provide future perspectives on investigating barrier leakage in AD. Identifying mechanistic steps underlying barrier leakage has the potential to unravel new targets that can be used to develop novel therapeutic strategies to repair barrier leakage and slow cognitive decline in AD and AD-related dementias.

Apolipoprotein E genotype-dependent nutrigenetic effects to prebiotic inulin for modulating systemic metabolism and neuroprotection in mice via gut-brain axis.

OBJECTIVE: The goal of the study was to identify the potential nutrigenetic effects to inulin, a prebiotic fiber, in mice with different human apolipoprotein E (APOE) genetic variants. Specifically, we compared responses to inulin for the potential modulation of the systemic metabolism and neuroprotection via gut-brain axis in mice with human APOE ϵ3 and ϵ4 alleles. METHOD: We performed experiments with young mice expressing the human APOE3 (E3FAD mice and APOE4 gene (E4FAD mice). We fed mice with either inulin or control diet for 16 weeks starting from 3 months of age. We determined gut microbiome diversity and composition using16s rRNA sequencing, systemic metabolism using in vivo MRI and metabolomics, and blood-brain barrier (BBB) tight junction expression using Western blot. RESULTS: In both E3FAD and E4FAD mice, inulin altered the alpha and beta diversity of the gut microbiome, increased beneficial taxa of bacteria and elevated cecal short chain fatty acid and hippocampal scyllo-inositol. E3FAD mice had altered metabolism related to tryptophan and tyrosine, while E4FAD mice had changes in the tricarboxylic acid cycle, pentose phosphate pathway, and bile acids. Differences were found in levels of brain metabolites related to oxidative stress, and levels of Claudin-1 and Claudin-5 BBB tight junction expression. DISCUSSION: We found that inulin had many similar beneficial effects in the gut and brain for both E3FAD and E4FAD mice, which may be protective for brain functions and reduce risk for neurodegeneration. . E3FAD and E4FAD mice also had distinct responses in several metabolic pathways, suggesting an APOE-dependent nutrigenetic effects in modulating systemic metabolism and neuroprotection.

Intranasal administration of the chemotherapeutic perillyl alcohol results in selective delivery to the cerebrospinal fluid in rats.

Perillyl alcohol (POH) has been extensively studied for the treatment of peripheral and primary brain tumors. The intranasal route of administration has been preferred for dosing POH in early-stage clinical trials associated with promising outcomes in primary brain cancer. However, it is unclear how intranasal POH targets brain tumors in these patients. Multiple studies indicate that intranasally applied large molecules may enter the brain and cerebrospinal fluid (CSF) through direct olfactory and trigeminal nerve-associated pathways originating in the nasal mucosa that bypass the blood-brain barrier. It is unknown whether POH, a small molecule subject to extensive nasal metabolism and systemic absorption, may also undergo direct transport to brain or CSF from the nasal mucosa. Here, we compared CSF and plasma concentrations of POH and its metabolite, perillic acid (PA), following intranasal or intravascular POH application. Samples were collected over 70 min and assayed by high-performance liquid chromatography. Intranasal administration resulted in tenfold higher CSF-to-plasma ratios for POH and tenfold higher CSF levels for PA compared to equal dose intravascular administration. Our preclinical results demonstrate POH undergoes direct transport from the nasal mucosa to the CSF, a finding with potential significance for its efficacy as an intranasal chemotherapeutic for brain cancer.

Passive Immunotherapies for Central Nervous System Disorders: Current Delivery Challenges and New Approaches.

Passive immunotherapy, i.e., the administration of exogenous antibodies that recognize a specific target antigen, has gained significant momentum as a potential treatment strategy for several central nervous system (CNS) disorders, including Alzheimer's disease, Parkinson's disease, Huntington's disease, and brain cancer, among others. Advances in antibody engineering to create therapeutic antibody fragments or antibody conjugates have introduced new strategies that may also be applied to treat CNS disorders. However, drug delivery to the CNS for antibodies and other macromolecules has thus far proven challenging, due in large part to the blood-brain barrier and blood-cerebrospinal fluid barriers that greatly restrict transport of peripherally administered molecules from the systemic circulation into the CNS. Here, we summarize the various passive immunotherapy approaches under study for the treatment of CNS disorders, with a primary focus on disease-specific and target site-specific challenges to drug delivery and new, cutting edge methods.

Delivery of immunoglobulin G antibodies to the rat nervous system following intranasal administration: Distribution, dose-response, and mechanisms of delivery.

The intranasal route has been hypothesized to circumvent the blood-brain and blood-cerebrospinal fluid barriers, allowing entry into the brain via extracellular pathways along olfactory and trigeminal nerves and the perivascular spaces (PVS) of cerebral blood vessels. We investigated the potential of the intranasal route to non-invasively deliver antibodies to the brain 30 min following administration by characterizing distribution, dose-response, and mechanisms of antibody transport to and within the brain after administering non-targeted radiolabeled or fluorescently-labeled full length immunoglobulin G (IgG) to normal adult female rats. Intranasal [125I]-IgG consistently yielded highest concentrations in the olfactory bulbs, trigeminal nerves, and leptomeningeal blood vessels with their associated PVS. Intranasal delivery also resulted in significantly higher [125I]-IgG concentrations in the CNS than systemic (intra-arterial) delivery for doses producing similar endpoint blood concentrations. Importantly, CNS targeting significantly increased with increasing dose only with intranasal administration, yielding brain concentrations that ranged from the low-to-mid picomolar range with tracer dosing (50 µg) up to the low nanomolar range at higher doses (1 mg and 2.5 mg). Finally, intranasal pre-treatment with a previously identified nasal permeation enhancer, matrix metalloproteinase-9, significantly improved intranasal [125I]-IgG delivery to multiple brain regions and further allowed us to elucidate IgG transport pathways extending from the nasal epithelia into the brain using fluorescence microscopy. The results show that it may be feasible to achieve therapeutic levels of IgG in the CNS, particularly at higher intranasal doses, and clarify the likely cranial nerve and perivascular distribution pathways taken by antibodies to reach the brain from the nasal mucosae.