Organotypic Brain Slice Cultures Research Articles

Potential of ex vivo organotypic slice cultures in neuro-oncology.

Over recent decades, in vitro and in vivo models have significantly advanced brain cancer research; however, each presents distinct challenges for accurately mimicking in situ conditions. In response, organotypic slice cultures have emerged as a promising model recapitulating precisely specific in vivo phenotypes through an ex vivo approach. Ex vivo organotypic brain slice models can integrate biological relevance and patient-specific variability early in drug discovery, thereby aiming for more precise treatment stratification. However, the challenges of obtaining representative fresh brain tissue, ensuring reproducibility, and maintaining essential central nervous system (CNS)-specific conditions reflecting the in situ situation over time have limited the direct application of ex vivo organotypic slice cultures in robust clinical trials. In this review, we explore the benefits and possible limitations of ex vivo organotypic brain slice cultures in neuro-oncological research. Additionally, we share insights from clinical experts in neuro-oncology on how to overcome these current limitations and improve the practical application of organotypic brain slice cultures beyond academic research.

The isoflavone puerarin promotes generation of human iPSC-derived pre-oligodendrocytes and enhances endogenous remyelination in rodent models.

Puerarin, a natural isoflavone, is commonly used as a Chinese herbal medicine for the treatment of various cardiovascular and neurological disorders. It has been found to be neuroprotective via TrK-PI3K/Akt pathway, which is associated with anti-inflammatory and antioxidant effects. Myelin damage in diseases such as multiple sclerosis (MS) and ischemia induces activation of endogenous oligodendrocyte progenitor cells (OPC) and subsequent remyelination by newly formed oligodendrocytes. It has been shown that human-induced pluripotent stem cells (hiPSC)-derived OPCs promote remyelination when transplanted to the brains of disease models. Here, we ask whether and how puerarin is beneficial to the generation of hiPSC-derived OPCs and oligodendrocytes, and to the endogenous remyelination in mouse demyelination model. Our results show that puerarin increases the proportion of O4+ pre-oligodendrocytes differentiated from iPSC-derived neural stem cells. Invitro, puerarin increases proliferation of rat OPCs and enhances mitochondrial activity. Treatment of puerarin at progenitor stage increases the yielding of differentiated oligodendrocytes. In rat organotypic brain slice culture, puerarin promotes both myelination and remyelination. Invivo, puerarin increases oligodendrocyte repopulation during remyelination in mouse spinal cord following lysolethicin-induced demyelination. Our findings suggest that puerarin promotes oligodendrocyte lineage progression and myelin repair, with a potential to be developed into therapeutic agent for neurological diseases associated with myelin damage.

From Organotypic Mouse Brain Slices to Human Alzheimer’s Plasma Biomarkers: A Focus on Nerve Fiber Outgrowth

Alzheimer’s disease (AD) is a neurodegenerative disease characterized by memory loss and progressive deterioration of cognitive functions. Being able to identify reliable biomarkers in easily available body fluids such as blood plasma is vital for the disease. To achieve this, we used a technique that applied human plasma to organotypic brain slice culture via microcontact printing. After a 2-week culture period, we performed immunolabeling for neurofilament and myelin oligodendrocyte glycoprotein (MOG) to visualize newly formed nerve fibers and oligodendrocytes. There was no significant change in the number of new nerve fibers in the AD plasma group compared to the healthy control group, while the length of the produced fibers significantly decreased. A significant increase in the number of MOG+ dots around these new fibers was detected in the patient group. According to our hypothesis, there are factors in the plasma of AD patients that affect the growth of new nerve fibers, which also affect the oligodendrocytes. Based on these findings, we selected the most promising plasma samples and conducted mass spectrometry using a differential approach and we identified three putative biomarkers: aldehyde-dehydrogenase 1A1, alpha-synuclein and protein S100-A4. Our method represents a novel and innovative approach for translating research findings from mouse models to human applications.

NLRP3 inflammasome activation and pyroptosis are dispensable for tau pathology.

Neuroinflammation is widely recognized as a key factor in the pathogenesis of Alzheimer's disease (AD), alongside ß-amyloid deposition and the formation of neurofibrillary tangles. The NLR family pyrin domain containing 3 (NLRP3) inflammasome, part of the innate immune system, has been implicated in the neuropathology of both preclinical amyloid and tau transgenic models. Activation of the NLRP3 pathway involves an initial priming step, which increases the expression of Nlrp3 and interleukin (IL)-1β, followed by the assembly of the NLRP3 inflammasome complex, comprising NLRP3, ASC, and caspase-1. This assembly leads to the proteolytic maturation of the pro-inflammatory cytokines IL-1β and IL-18. Additionally, the NLRP3 inflammasome induces Gasdermin D (GSDMD) cleavage, forming membrane pores through which IL-1β and IL-18 are secreted. Inhibition of NLRP3 has been shown to enhance plaque clearance by modulating microglial activation. Furthermore, blocking NLRP3 in tau transgenic mice has been found to reduce tau phosphorylation by affecting the activity of certain tau kinases and phosphatases. In this study, organotypic brain slice cultures from P301S transgenic mice were treated with lipopolysaccharide (LPS) plus nigericin as a positive control or exposed to tau seeds (K18) to evaluate NLRP3 inflammasome activation. The effect of tau seeding on NLRP3 activity was further examined using Meso Scale Discovery (MSD) assays to measure IL1β secretion levels in the presence and absence of NLRP3 inhibitors. The role of NLRP3 activity was investigated in full-body Nlrp3 knockout mice crossbred with the tau transgenic P301S model. Additionally, full-body and microglia-selective Gsdmd knockout mice were crossbred with P301S mice, and tau pathology and neurodegeneration were evaluated at early and late stages of the disease using immunohistochemistry and biochemical assays. Activation of the NLRP3 pathway was observed in the mouse organotypic slice culture (OSC) model following stimulation with LPS and nigericin or exposure to tau seeds. However, Nlrp3 deficiency did not mitigate tauopathy or neurodegeneration in P301S mice in vivo, showing only a minor effect on plasma neurofilament (NF-L) levels. Consistently, Gsdmd deficiency did not alter tau pathology in P301S mice. Furthermore, neither full-body nor microglia-selective Gsdmd deletion had an impact on neuronal pathology or the release of pro-inflammatory cytokines. The absence of key components of the NLRP3 inflammasome pathway did not yield a beneficial effect on tau pathology or neurodegeneration in the preclinical Tau-P301S mouse model of AD. Nonetheless, organotypic slice cultures could serve as a valuable ex vivo mechanistic model for evaluating NLRP3 pathway activation and pharmacological inhibitors.

Neurovirulence of Usutu virus in human fetal organotypic brain slice cultures partially resembles Zika and West Nile virus

Usutu (USUV), West Nile (WNV), and Zika virus (ZIKV) are neurotropic arthropod-borne viruses (arboviruses) that cause severe neurological disease in humans. However, USUV-associated neurological disease is rare, suggesting a block in entry to or infection of the brain. We determined the replication, cell tropism and neurovirulence of these arboviruses in human brain tissue using a well-characterized human fetal organotypic brain slice culture model. Furthermore, we assessed the efficacy of interferon-β and 2′C-methyl-cytidine, a synthetic nucleoside analogue, in restricting viral replication. All three arboviruses replicated within the brain slices, with WNV reaching the highest titers, and all primarily infected neuronal cells. USUV- and WNV-infected cells exhibited a shrunken morphology, not associated with detectable cell death. Pre-treatment with interferon-β inhibited replication of all arboviruses, while 2′C-methyl-cytidine reduced only USUV and ZIKV titers. Collectively, USUV can infect human brain tissue, showing similarities in tropism and neurovirulence as WNV and ZIKV. These data suggest that a blockade to infection of the human brain may not be the explanation for the low clinical incidence of USUV-associated neurological disease. However, USUV replicated more slowly and to lower titers than WNV, which could help to explain the reduced severity of neurological disease resulting from USUV infection.

Temporal dynamics of neocortical development in organotypic mouse brain cultures: a comprehensive analysis.

Murine organotypic brain slice cultures have been widely used in neuroscientific research and are offering the opportunity to study neuronal function under normal and disease conditions. Despite the broad application, the mechanisms governing the maturation of immature cortical circuits in vitro are not well understood. In this study, we present a detailed investigation into the development of the neocortex in vitro. Using a holistic approach, we studied organotypic whole hemisphere brain slice cultures from postnatal mice and tracked the development of the somatosensory area over a 5-wk period. Our analysis revealed the maturation of passive and active intrinsic properties of pyramidal cells together with their morphology, closely resembling in vivo development. Detailed multielectrode array (MEA) electrophysiological assessments and RNA expression profiling demonstrated stable network properties by 2 wk in culture, followed by the transition of spontaneous activity toward more complex patterns including high-frequency oscillations. However, culturing weeks 4 and 5 exhibited increased variability and initial signs of neuronal loss, highlighting the importance of considering developmental stages in experimental design. This comprehensive characterization is vital for understanding the temporal dynamics of the neocortical development in vitro, with implications for neuroscientific research methodologies, particularly in the investigation of diseases such as epilepsy and other neurodevelopmental disorders.NEW & NOTEWORTHY The development of the mouse neocortex in vitro mimics the in vivo development. Mouse brain cultures can serve as a model system for cortical development for the first 2 wk in vitro and as a model system for the adult cortex from 2 to 4 wk in vitro. Mouse organotypic brain slice cultures develop high-frequency network oscillations at γ frequency after 2 wk in vitro. Mouse brain cultures exhibit increased heterogeneity and variability after 4 wk in culture.

Auto-loaded TRAIL-exosomes derived from induced neural stem cells for brain cancer therapy

Transdifferentiation (TD), a somatic cell reprogramming process that eliminates pluripotent intermediates, creates cells that are ideal for personalized anti-cancer therapy. Here, we provide the first evidence that extracellular vesicles (EVs) from TD-derived induced neural stem cells (Exo-iNSCs) are an efficacious treatment strategy for brain cancer. We found that genetically engineered iNSCs generated EVs loaded with the tumoricidal gene product TRAIL at nearly twice the rate of their parental fibroblasts, and TRAIL produced by iNSCs was naturally loaded into the lumen of EVs and arrayed across their outer membrane (Exo-iNSC-TRAIL). Uptake studies in ex vivo organotypic brain slice cultures showed that Exo-iNSC-TRAIL selectively accumulates within tumor foci, and co-culture assays demonstrated that Exo-iNSC-TRAIL killed metastatic and primary brain cancer cells more effectively than free TRAIL. In an orthotopic mouse model of brain cancer, Exo-iNSC-TRAIL reduced breast-to-brain tumor xenografts by approximately 3000-fold compared to treatment with free TRAIL, with all Exo-iNSC-TRAIL treated animals surviving through 90 days post-treatment. In additional in vivo testing against aggressive U87 and invasive GBM8 glioblastoma tumors, Exo-iNSC-TRAIL also induced a statistically significant increase in survival. These studies establish a novel, easily generated, stable, tumor-targeted EV to efficaciously treat multiple forms of brain cancer.

MODL-13. ORGANOTYPIC BRAIN SLICE CULTURE PLATFORM AS A MODEL OF PEDIATRIC LOW GRADE GLIOMAS

Abstract BACKGROUND Pediatric low grade glioma (pLGG), the most common subgroup of central nervous system tumors in children, suffers from a paucity of models that support pLGG biological discovery, preclinical target identification, and drug screening studies. METHODS We propose the use of an organotypic brain slice culture (OBSC) platform as a novel model in which we are able to reliably support fresh, zero-passage pLGG patient tumor tissue for preclinical testing. We performed individualized drug screening with the frequently used up-front agents: carboplatin, vincristine, trametinib, and dabrafenib. RESULTS We have collected and established on OBSCs 100% (3/3) pLGGs since 2023. With these three patient samples, we have applied our quantitative drug screening algorithm, which combines tumor killing efficacy with healthy tissue toxicity, and yielded resultant drug sensitivity scores (DSS) to quantify patient-specific treatment efficacies. CONCLUSIONS Overall, these early results suggest that the OBSC platform may serve as a novel and reliable model for pLGG, filling a critical void in representative models for this common and often highly morbid pediatric tumor.

Coenzyme A protects against ferroptosis via CoAlation of thioredoxin reductase 2.

The Cystine-xCT transporter-Glutathione (GSH)-GPX4 axis is the canonical pathway to protect against ferroptosis. While not required for ferroptosis-inducing compounds (FINs) targeting GPX4, FINs targeting the xCT transporter require mitochondria and its lipid peroxidation to trigger ferroptosis. However, the mechanism underlying the difference between these FINs is still unknown. Given that cysteine is also required for coenzyme A (CoA) biosynthesis, here we show that CoA supplementation specifically prevents ferroptosis induced by xCT inhibitors but not GPX4 inhibitors. We find that, auranofin, a thioredoxin reductase inhibitor, abolishes the protective effect of CoA. We also find that CoA availability determines the enzymatic activity of thioredoxin reductase, but not thioredoxin. Importantly, the mitochondrial thioredoxin system, but not the cytosolic thioredoxin system, determines CoA-mediated ferroptosis inhibition. Our data show that the CoA regulates the in vitro enzymatic activity of mitochondrial thioredoxin reductase (TXNRD2) by covalently modifying the thiol group of cysteine (CoAlation) on Cys-483. Replacing Cys-483 with alanine on TXNRD2 abolishes its in vitro enzymatic activity and ability to protect cells from ferroptosis. Targeting xCT to limit cysteine import and, therefore, CoA biosynthesis reduced CoAlation on TXNRD2, an effect that was rescued by CoA supplementation. Furthermore, the fibroblasts from patients with disrupted CoA metabolism demonstrate increased mitochondrial lipid peroxidation. In organotypic brain slice cultures, inhibition of CoA biosynthesis leads to an oxidized thioredoxin system, mitochondrial lipid peroxidation, and loss in cell viability, which were all rescued by ferrostatin-1. These findings identify CoA-mediated post-translation modification to regulate the thioredoxin system as an alternative ferroptosis protection pathway with potential clinical relevance for patients with disrupted CoA metabolism.

Human post-mortem organotypic brain slice cultures: a tool to study pathomechanisms and test therapies

Human brain experimental models recapitulating age- and disease-related characteristics are lacking. There is urgent need for human-specific tools that model the complex molecular and cellular interplay between different cell types to assess underlying disease mechanisms and test therapies. Here we present an adapted ex vivo organotypic slice culture method using human post-mortem brain tissue cultured at an air-liquid interface to also study brain white matter. We assessed whether these human post-mortem brain slices recapitulate the in vivo neuropathology and if they are suitable for pathophysiological, experimental and pre-clinical treatment development purposes, specifically regarding leukodystrophies. Human post-mortem brain tissue and cerebrospinal fluid were obtained from control, psychiatric and leukodystrophy donors. Slices were cultured up to six weeks, in culture medium with or without human cerebrospinal fluid. Human post-mortem organotypic brain slice cultures remained viable for at least six weeks ex vivo and maintained tissue structure and diversity of (neural) cell types. Supplementation with cerebrospinal fluid could improve slice recovery. Patient-derived organotypic slice cultures recapitulated and maintained known in vivo neuropathology. The cultures also showed physiologic multicellular responses to lysolecithin-induced demyelination ex vivo, indicating their suitability to study intrinsic repair mechanisms upon injury. The slice cultures were applicable for various experimental studies, as multi-electrode neuronal recordings. Finally, the cultures showed successful cell-type dependent transduction with gene therapy vectors. These human post-mortem organotypic brain slice cultures represent an adapted ex vivo model suitable for multifaceted studies of brain disease mechanisms, boosting translation from human ex vivo to in vivo. This model also allows for assessing potential treatment options, including gene therapy applications. Human post-mortem brain slice cultures are thus a valuable tool in preclinical research to study the pathomechanisms of a wide variety of brain diseases in living human tissue.

Auto-loaded TRAIL-exosomes derived from induced neural stem cells for brain cancer therapy.

Transdifferentiation (TD), a somatic cell reprogramming process that eliminates pluripotent intermediates, creates cells that are ideal for personalized anti-cancer therapy. Here, we provide the first evidence that extracellular vesicles (EVs) from TD-derived induced neural stem cells (Exo-iNSCs) are an efficacious treatment strategy for brain cancer. We found that genetically engineered iNSCs generated EVs loaded with the tumoricidal gene product TRAIL at nearly twice the rate as their parental fibroblasts, and the TRAIL produced by iNSCs were naturally loaded into the lumen of EVs and arrayed across their outer membrane (Exo-iNSC-TRAIL). Uptake studies in ex vivo organotypic brain slice cultures showed Exo-iNSC-TRAIL selectively accumulates within tumor foci, and co-culture assays showed that Exo-iNSC-TRAIL killed metastatic and primary brain cancer cells more effectively than free TRAIL. In an orthotopic mouse model of brain cancer, Exo-iNSC-TRAIL reduced breast-to-brain tumor xenografts around 3000-fold greater than treatment with free TRAIL, with all Exo-iNSC-TRAIL treated animals surviving through 90 days post-treatment. In additional in vivo testing against aggressive U87 and invasive GBM8 glioblastoma tumors, Exo-iNSC-TRAIL also induced a statistically significant increase in survival. These studies establish a new easily generated, stable, tumor-targeted EV to efficaciously treat multiple forms of brain cancer.

Herpes simplex virus infection induces necroptosis of neurons and astrocytes in human fetal organotypic brain slice cultures.

Herpes simplex virus (HSV) encephalitis (HSE) is a serious and potentially life-threatening disease, affecting both adults and newborns. Progress in understanding the virus and host factors involved in neonatal HSE has been hampered by the limitations of current brain models that do not fully recapitulate the tissue structure and cell composition of the developing human brain in health and disease. Here, we developed a human fetal organotypic brain slice culture (hfOBSC) model and determined its value in mimicking the HSE neuropathology in vitro. Cell viability and tissues integrity were determined by lactate dehydrogenase release in supernatant and immunohistological (IHC) analyses. Brain slices were infected with green fluorescent protein (GFP-) expressing HSV-1 and HSV-2. Virus replication and spread were determined by confocal microscopy, PCR and virus culture. Expression of pro-inflammatory cytokines and chemokines were detected by PCR. Cell tropism and HSV-induced neuropathology were determined by IHC analysis. Finally, the in situ data of HSV-infected hfOBSC were compared to the neuropathology detected in human HSE brain sections. Slicing and serum-free culture conditions were optimized to maintain the viability and tissue architecture of ex vivo human fetal brain slices for at least 14days at 37°C in a CO2 incubator. The hfOBSC supported productive HSV-1 and HSV-2 infection, involving predominantly infection of neurons and astrocytes, leading to expression of pro-inflammatory cytokines and chemokines. Both viruses induced programmed cell death-especially necroptosis-in infected brain slices at later time points after infection. The virus spread, cell tropism and role of programmed cell death in HSV-induced cell death resembled the neuropathology of HSE. We developed a novel human brain culture model in which the viability of the major brain-resident cells-including neurons, microglia, astrocytes and oligodendrocytes-and the tissue architecture is maintained for at least 2weeks in vitro under serum-free culture conditions. The close resemblance of cell tropism, spread and neurovirulence of HSV-1 and HSV-2 in the hfOBSC model with the neuropathological features of human HSE cases underscores its potential to detail the pathophysiology of other neurotropic viruses and as preclinical model to test novel therapeutic interventions.

Human organotypic brain slice cultures: a detailed and improved protocol for preparation and long-term maintenance

The investigation of the human brain at cellular and microcircuit level remains challenging due to the fragile viability of neuronal tissue, inter- and intra-variability of the samples and limited availability of human brain material. Especially brain slices have proven to be an excellent source to investigate brain physiology and disease at cellular and small network level, overcoming the temporal limits of acute slices. Here we provide a revised, detailed protocol of the production and in-depth knowledge on long-term culturing of such human organotypic brain slice cultures for research purposes. We highlight the critical pitfalls of the culturing process of the human brain tissue and present exemplary results on viral expression, single-cell Patch-Clamp recordings, as well as multi-electrode array recordings as readouts for culture viability, enabling the use of organotypic brain slice cultures of these valuable tissue samples for basic neuroscience and disease modeling (Fig. 1).

P-tau Ser356 is associated with Alzheimer’s disease pathology and is lowered in brain slice cultures using the NUAK inhibitor WZ4003

Tau hyperphosphorylation and aggregation is a common feature of many dementia-causing neurodegenerative diseases. Tau can be phosphorylated at up to 85 different sites, and there is increasing interest in whether tau phosphorylation at specific epitopes, by specific kinases, plays an important role in disease progression. The AMP-activated protein kinase (AMPK)-related enzyme NUAK1 has been identified as a potential mediator of tau pathology, whereby NUAK1-mediated phosphorylation of tau at Ser356 prevents the degradation of tau by the proteasome, further exacerbating tau hyperphosphorylation and accumulation. This study provides a detailed characterisation of the association of p-tau Ser356 with progression of Alzheimer’s disease pathology, identifying a Braak stage-dependent increase in p-tau Ser356 protein levels and an almost ubiquitous presence in neurofibrillary tangles. We also demonstrate, using sub-diffraction-limit resolution array tomography imaging, that p-tau Ser356 co-localises with synapses in AD postmortem brain tissue, increasing evidence that this form of tau may play important roles in AD progression. To assess the potential impacts of pharmacological NUAK inhibition in an ex vivo system that retains multiple cell types and brain-relevant neuronal architecture, we treated postnatal mouse organotypic brain slice cultures from wildtype or APP/PS1 littermates with the commercially available NUAK1/2 inhibitor WZ4003. Whilst there were no genotype-specific effects, we found that WZ4003 results in a culture-phase-dependent loss of total tau and p-tau Ser356, which corresponds with a reduction in neuronal and synaptic proteins. By contrast, application of WZ4003 to live human brain slice cultures results in a specific lowering of p-tau Ser356, alongside increased neuronal tubulin protein. This work identifies differential responses of postnatal mouse organotypic brain slice cultures and adult human brain slice cultures to NUAK1 inhibition that will be important to consider in future work developing tau-targeting therapeutics for human disease.

Effects of corticosterone and amyloid beta on the corticosteroid receptors in organotypic brain slice cultures

Effects of corticosterone and amyloid beta on the corticosteroid receptors in organotypic brain slice cultures

Early Age- and Sex-Dependent Regulation of Astrocyte-Mediated Glutamatergic Synapse Elimination in the Rat Prefrontal Cortex: Establishing an Organotypic Brain Slice Culture Investigating Tool.

Clinical and pre-clinical studies of neuropsychiatric (NP) disorders show altered astrocyte properties and synaptic networks. These are refined during early postnatal developmental (PND) stages. Thus, investigating early brain maturational trajectories is essential to understand NP disorders. However, animal experiments are highly time-/resource-consuming, thereby calling for alternative methodological approaches. The function of MEGF10 in astrocyte-mediated synapse elimination (pruning) is crucial to refine neuronal networks during development and adulthood. To investigate the impact of MEGF10 during PND in the rat prefrontal cortex (PFC) and its putative role in brain disorders, we established and validated an organotypic brain slice culture (OBSC) system. Using Western blot, we characterized the expression of MEGF10 and the synaptic markers synaptophysin and PSD95 in the cortex of developing pups. We then combined immunofluorescent-immunohistochemistry with Imaris-supported 3D analysis to compare age- and sex-dependent astrocyte-mediated pruning within the PFC in pups and OBSCs. We thereby validated this system to investigate age-dependent astrocyte-mediated changes in pruning during PND. However, further optimizations are required to use OBSCs for revealing sex-dependent differences. In conclusion, OBSCs offer a valid alternative to study physiological astrocyte-mediated synaptic remodeling during PND and might be exploited to investigate the pathomechanisms of brain disorders with aberrant synaptic development.

New in vitro tools to investigate the role of microglia in neurodegeneration

AbstractBackgroundHuman induced pluripotent stem cells (iPSCs) have transpired as an attractive tool to investigate neurological diseases. And while immune cells, and in particular microglia, have emerged as important players in many neurological and in particular in neurodegenerative diseases, it remains a challenge to fully recapitulate an in vivo‐like phenotype ex vivo. This is due to the fact that microglial identity is shaped by the neural tissue environment.MethodWe have now developed a chimeric brain slice culture system, where we engraft iPSC‐derived microglia onto mouse (and human) organotypic brain slice cultures. Brain slice cultures offer the advantage that many aspects of the complex cytoarchitecture of the mature/aged living brain, including cortical layering, columnar organization and electrophysiological properties can be preserved over several weeks.ResultWe found that iPSC‐derived microglial precursors integrate and differentiate well in the brain slice cultures, with morphology, network characteristics and functional responses reminiscent of human microglia. Gene expression profiling of iPSC‐derived microglia revealed enhanced maturation over time. Upon induction of neurodegenerative disease pathology in these slice cultures, the iPSC‐derived microglia show transcriptional changes converging towards those observed in patients with neurodegenerative disease pathology.ConclusionThis system is a novel cellularly complex tool allowing to dissect the dynamics and molecular mechanisms of microglial genetic risk factors of many neurological diseases, while preserving experimental amenability to test therapeutic approaches in a human(ized) system.

Modulation of cannabinoid receptor‐2 alters brain clearance of human tau

AbstractBackgroundMultiple studies have demonstrated a strong neuroinflammatory response in the presence of Tau pathology, which is strongly associated with the clinical symptoms and cognitive decline found in Alzheimer’s disease (AD). The correlation between AD progression and tau pathologies, rather than Ab accumulation, suggests that targeting pathological tau may be a more effective therapeutic approach. Microglia have been implicated in tauopathies as they may contribute to tau phosphorylation and aggregation and the uptake and spread of seed‐competent tau to healthy neurons due to dysfunctional degradation. Cannabinoid type 2 receptors (CB2) are highly expressed in immune cells and upregulated in activated microglia under conditions of neurologic disease, such as AD. The pharmacological modulation of CB2 have demonstrated a reduction in inflammation and plaque deposition, suggesting a role of CB2 on the immune system to influence AD‐related pathologies and phenotypes. However, there are limited findings on the CB2 regulation of tau accumulation.MethodWe have generated organotypic brain slice cultures from CB2‐EGFP/f/f mice and transduced with rAAV‐P301L/S320F‐human tau, which previously has shown to accumulate total tau and develop insoluble tau species. Slices were treated with CB2‐selective compounds and evaluated for tau pathology and microglial phenotypes using real‐time imaging and biochemical analyses.ResultPrevious investigation in our lab of a synucleinopathy animal model revealed the capacity of CB2 signaling to alter the function of immune cells and reduce accumulation of phosphorylated human alpha‐synuclein. Furthermore, we have determined that treatment with a CB2 inverse agonist will increase phagocytosis of pHrodo E. coli bioparticles in microglia BV2 cultures stimulated by LPS. Our preliminary work in brain slices over‐expressing mutant human tau show a trend toward reduced ratio of phosphorylated tau (Ser202/Thr205) to total tau in the soluble protein fraction (p = 0.0549) in slices treated with CB2 inverse agonist SR144528 compared to vehicle. We are repeating the experiment and performing further evaluations in organotypic brain slice cultures from CB2‐KO mice.ConclusionThese studies will advance our understanding of the role of brain CB2 in pathologic tau removal and potentially highlight CB2 as a therapeutic target against tauopathies such as AD.

Hyper‐sensitive interferon response in a mouse model of Alzheimer’s disease‐Down syndrome

AbstractBackgroundDown syndrome (DS) is caused by trisomy of chromosome 21 (Hsa21) and leads to an elevated risk of early‐onset Alzheimer’s disease. People with DS have a perturbed immune system including an over‐activated interferon response, which is likely due to four genes encoding interferon receptors being located on Hsa21 (1). How this affects inflammation in the brain is not well understood. In this study, we investigate if an extra copy of these genes is sufficient to elevate the interferon response in the brain of mouse models of DS, using in vivo and ex vivo approaches.MethodsThe Dp1Tyb (MGI:5703798) mouse has three‐copies of ∼148 Hsa21 orthologous genes, while the Dp2Tyb (MGI:5703800) mouse has a sub‐region of ∼32 of those Hsa21 orthologous genes in three‐copies, including the four interferon receptor genes. Organotypic brain slice cultures (OBSCs) were prepared from P7‐P9 pups and treated with interferon‐β (24 hours). Slices were collected for western blotting, qPCR, or immunohistochemistry, and media for CXCL10 ELISA.Dp2Tyb mice were crossed with AppNL‐G‐F (MGI:5637817) mice to generate four genotypes, to determine if the amyloid‐β interferon response is elevated by the additional copy of the Dp2Tyb region. Mice were tested in the light‐dark box and marble burying test to assay the effect on anxiety. Amyloid accumulation and interferon response in the brain will be determined by immunofluorescence, MSD amyloid‐β assay and qPCR.ResultsBoth Dp1Tyb and Dp2Tyb OBSCs had a hypersensitive response to interferon‐β treatment, with higher expression of interferon‐stimulated genes in lysed slices and higher levels of CXCL10 protein secreted into the media.ConclusionBoth DS preclinical models displayed a hypersensitive interferon response, indicating that interferon hypersensitivity may occur in the brain of individuals with DS and is likely due to having three‐copies of the interferon receptor genes encoded on Hsa21. Amyloid pathology has been shown to induce an interferon response (2,3). How interferon hypersensitivity in individuals with Down syndrome affects the neuroinflammatory response to amyloid pathology is unknown. To investigate this, we have crossed the Dp2Tyb model with the AppNL‐G‐F mouse model of amyloid pathology.

Evaluating dynamic autophagic responses to tau inclusions

AbstractBackgroundAlzheimer’s disease and other tauopathies are characterised by the accumulation of misfolded microtubule‐associated protein tau in intracellular inclusions. We recently showed that newly formed tau inclusions are dynamic structures with a slower, but constant turnover of aggregated tau in murine organotypic brain slice cultures (BSCs) (Croft et al., 2021). Clearance by autophagy or the proteasomal machinery are indicated to be the key mechanisms involved in pathogenic protein clearance. However, the current tools to measure these processes have received mixed review, and in particular struggle to capture these processes in a continuous manner.MethodsTo dynamically assess autophagy in the presence of tau inclusions we have repurposed the autophagic flux sensor (GFP‐LC3‐RFP∆G‐LC3) to be delivered by rAAV to specific central nervous system (CNS) cell types in BSCs. This probe delivers a cytosolic internal control so autophagic flux can be estimated by calculating the GFP/RFP ratio. When utilized in conjunction with live imaging, the rate of autophagic flux can be measured over time in the same CNS cells expressing pathological or physiological tau. The rate of autophagic flux can also be measured in response to established/novel compounds or genetic manipulation.ResultsWe have successfully developed and characterized the use of rAAV‐GFP‐LC3‐RFP∆G‐LC3 specifically in neurons and astrocytes in BSCs and are expanding its use to other CNS cell types. We have validated its use to sense autophagic flux whilst pharmacologically enhancing and inhibiting autophagy in BSCs. We are now using this sensor to determine dynamic autophagic processes in the presence of soluble tau and aggregated tau and its response to other manipulations.ConclusionsMethods to understand autophagic processes have traditionally only provided snapshots over time. With this tool we can now dynamically assess autophagy in ex vivo BSCs and characterize changes in response to tau and other pathologies. Overall, understanding the mechanisms that enable tau turnover may guide therapies for Alzheimer’s disease and other neurodegenerative conditions where tau plays a role. Importantly, we will also begin to understand whether enhancing tau inclusion clearance may be beneficial or detrimental to cell health.