Accelerating the in vitro emulation of Alzheimer's disease-associated phenotypes using a novel 3D blood-brain barrier neurosphere co-culture model.

High failure rates in clinical trials for neurodegenerative disorders such as Alzheimer's disease have been linked to an insufficient predictive validity of current animal-based disease models. This has created an increasing demand for alternative, human-based models capable of emulating key pathological phenotypes in vitro. Here, a three-dimensional Alzheimer's disease model was developed using a compartmentalized microfluidic device that combines a self-assembled microvascular network of the human blood-brain barrier with neurospheres derived from Alzheimer's disease-specific neural progenitor cells. To shorten microfluidic co-culture times, neurospheres were pre-differentiated for 21 days to express Alzheimer's disease-specific pathological phenotypes prior to the introduction into the microfluidic device. In agreement with post-mortem studies and Alzheimer's disease in vivo models, after 7 days of co-culture with pre-differentiated Alzheimer's disease-specific neurospheres, the three-dimensional blood-brain barrier network exhibited significant changes in barrier permeability and morphology. Furthermore, vascular networks in co-culture with Alzheimer's disease-specific microtissues displayed localized ß-amyloid deposition. Thus, by interconnecting a microvascular network of the blood-brain barrier with pre-differentiated neurospheres the presented model holds immense potential for replicating key neurovascular phenotypes of neurodegenerative disorders in vitro.

Infiltrating CD8+ T cells exacerbate Alzheimer's disease pathology in a 3D human neuroimmune axis model.

Brain infiltration of peripheral immune cells and their interactions with brain-resident cells may contribute to Alzheimer's disease (AD) pathology. To examine these interactions, in the present study we developed a three-dimensional human neuroimmune axis model comprising stem cell-derived neurons, astrocytes and microglia, together with peripheral immune cells. We observed an increase in the number of T cells (but not B cells) and monocytes selectively infiltrating into AD relative to control cultures. Infiltration of CD8+ T cells into AD cultures led to increased microglial activation, neuroinflammation and neurodegeneration. Using single-cell RNA-sequencing, we identified that infiltration of T cells into AD cultures led to induction of interferon-γ and neuroinflammatory pathways in glial cells. We found key roles for the C-X-C motif chemokine ligand 10 (CXCL10) and its receptor, CXCR3, in regulating T cell infiltration and neuronal damage in AD cultures. This human neuroimmune axis model is a useful tool to study the effects of peripheral immune cells in brain disease.

Endothelium and Subendothelial Matrix Mechanics Modulate Cancer Cell Transendothelial Migration.

Cancer cell extravasation, a key step in the metastatic cascade, involves cancer cell arrest on the endothelium, transendothelial migration (TEM), followed by the invasion into the subendothelial extracellular matrix (ECM) of distant tissues. While cancer research has mostly focused on the biomechanical interactions between tumor cells (TCs) and ECM, particularly at the primary tumor site, very little is known about the mechanical properties of endothelial cells and the subendothelial ECM and how they contribute to the extravasation process. Here, an integrated experimental and theoretical framework is developed to investigate the mechanical crosstalk between TCs, endothelium and subendothelial ECM during in vitro cancer cell extravasation. It is found that cancer cell actin-rich protrusions generate complex push-pull forces to initiate and drive TEM, while transmigration success also relies on the forces generated by the endothelium. Consequently, mechanical properties of the subendothelial ECM and endothelial actomyosin contractility that mediate the endothelial forces also impact the endothelium's resistance to cancer cell transmigration. These results indicate that mechanical features of distant tissues, including force interactions between the endothelium and the subendothelial ECM, are key determinants of metastatic organotropism.

The neuroimmune axis of Alzheimer's disease.

Alzheimer's disease (AD) is a genetically complex and heterogeneous disorder with multifaceted neuropathological features, including ß-amyloid plaques, neurofibrillary tangles, and neuroinflammation. Over the past decade, emerging evidence has implicated both beneficial and pathological roles for innate immune genes and immune cells, including peripheral immune cells such as T cells, which can infiltrate the brain and either ameliorate or exacerbate AD neuropathogenesis. These findings support a neuroimmune axis of AD, in which the interplay of adaptive and innate immune systems inside and outside the brain critically impacts the etiology and pathogenesis of AD. In this review, we discuss the complexities of AD neuropathology at the levels of genetics and cellular physiology, highlighting immune signaling pathways and genes associated with AD risk and interactions among both innate and adaptive immune cells in the AD brain. We emphasize the role of peripheral immune cells in AD and the mechanisms by which immune cells, such as T cells and monocytes, influence AD neuropathology, including microglial clearance of amyloid-ß peptide, the key component of ß-amyloid plaque cores, pro-inflammatory and cytotoxic activity of microglia, astrogliosis, and their interactions with the brain vasculature. Finally, we review the challenges and outlook for establishing immune-based therapies for treating and preventing AD.

Neurotechnological Approaches to the Diagnosis and Treatment of Alzheimer's Disease.

Alzheimer's disease (AD) is the most common cause of dementia in the elderly, clinically defined by progressive cognitive decline and pathologically, by brain atrophy, neuroinflammation, and accumulation of extracellular amyloid plaques and intracellular neurofibrillary tangles. Neurotechnological approaches, including optogenetics and deep brain stimulation, have exploded as new tools for not only the study of the brain but also for application in the treatment of neurological diseases. Here, we review the current state of AD therapeutics and recent advancements in both invasive and non-invasive neurotechnologies that can be used to ameliorate AD pathology, including neurostimulation via optogenetics, photobiomodulation, electrical stimulation, ultrasound stimulation, and magnetic neurostimulation, as well as nanotechnologies employing nanovectors, magnetic nanoparticles, and quantum dots. We also discuss the current challenges in developing these neurotechnological tools and the prospects for implementing them in the treatment of AD and other neurodegenerative diseases.

Microfluidic separation of axonal and somal compartments of neural progenitor cells differentiated in a 3D matrix.

This protocol describes the differentiation of human neural progenitor cells (hNPCs) in a microfluidic device containing a thin 3D matrix with two separate chambers, enabling a cleaner separation between axons and soma/bulk neurons. We have used this technique to study how mitochondria-associated ER membranes (MAMs) regulate the generation of somal and axonal amyloid ß (Aß) in FAD hNPCs, a cellular model of Alzheimer's disease. This protocol also details the quantification of Aß molecules and isolation of pure axons via axotomy. For complete details on the use and execution of this profile, please refer to Bhattacharyya et al. (2021).

Astrocytic interleukin-3 programs microglia and limits Alzheimer's disease.

Communication within the glial cell ecosystem is essential for neuronal and brain health1-3. The influence of glial cells on the accumulation and clearance of ß-amyloid (Aß) and neurofibrillary tau in the brains of individuals with Alzheimer's disease (AD) is poorly understood, despite growing awareness that these are therapeutically important interactions4,5. Here we show, in humans and mice, that astrocyte-sourced interleukin-3 (IL-3) programs microglia to ameliorate the pathology of AD. Upon recognition of Aß deposits, microglia increase their expression of IL-3Rα-the specific receptor for IL-3 (also known as CD123)-making them responsive to IL-3. Astrocytes constitutively produce IL-3, which elicits transcriptional, morphological, and functional programming of microglia to endow them with an acute immune response program, enhanced motility, and the capacity to cluster and clear aggregates of Aß and tau. These changes restrict AD pathology and cognitive decline. Our findings identify IL-3 as a key mediator of astrocyte-microglia cross-talk and a node for therapeutic intervention in AD.

Patterning of interconnected human brain spheroids.

Brain spheroids are emerging as valuable in vitro models that are accelerating the pace of research in various diseases. For Alzheimer's disease (AD) research, these models are enhanced using genetically engineered human neural progenitor cells and novel cell culture methods. However, despite these advances, it remains challenging to study the progression of AD in vitro as well as the propagation of pathogenic amyloid-ß (Aß) and tau tangles between diseased and healthy neurons using the brain spheroids model. To address this need, we designed a microfluidic system of connected microwells for arranging two types of brain spheroids in complex patterns and enabling the formation of thick bundles of neurites between the brain spheroids and the accumulation of pathogenic Aß within the spheroids.

Axonal generation of amyloid-ß from palmitoylated APP in mitochondria-associated endoplasmic reticulum membranes.

Axonal generation of Alzheimer's disease (AD)-associated amyloid-ß (Aß) plays a key role in AD neuropathology, but the cellular mechanisms involved in its release have remained elusive. We previously reported that palmitoylated APP (palAPP) partitions to lipid rafts where it serves as a preferred substrate for ß-secretase. Mitochondria-associated endoplasmic reticulum (ER) membranes (MAMs) are cholesterol-rich lipid rafts that are upregulated in AD. Here, we show that downregulating MAM assembly by either RNA silencing or pharmacological modulation of the MAM-resident sigma1 receptor (S1R) leads to attenuated ß-secretase cleavage of palAPP. Upregulation of MAMs promotes trafficking of palAPP to the cell surface, ß-secretase cleavage, and Aß generation. We develop a microfluidic device and use it to show that MAM levels alter Aß generation specifically in neuronal processes and axons, but not in cell bodies. These data suggest therapeutic strategies for reducing axonal release of Aß and attenuating ß-amyloid pathology in AD.

A Synergistic Engineering Approach to Build Human Brain Spheroids.

Self-assembling brain spheroids derived from human stem cells closely emulate the tangled connectivity of the human brain, recapitulate aspects of organized tissue structure, and are relatively easy to manipulate compared to other existing three-dimensional (3D) cellular models. However, current platforms generate heterogeneously sized and short-lived spheroids, which do not robustly and reproducibly model human brain development and diseases. Here, we present a method to generate large-scale arrays of homogeneously sized 3D brain spheroids derived from human-induced pluripotent stem cells (hiPSCs) or immortalized neural progenitor cells to recapitulate Alzheimer's disease (AD) pathology in vitro. When embedded in extracellular matrix, these brain spheroids develop extensive outward projection of neurites and form networks, which are mediated by thick bundles of dendrites. This array facilitates cost-effective, high-throughput drug screening and mechanistic studies to better understand human brain development and neurodegenerative conditions, such as AD .

The interplay between microglial states and major risk factors in Alzheimer's disease through the eyes of single-cell RNA-sequencing: beyond black and white.

Microglia constitute ~10-20% of glial cells in the adult human brain. They are the resident phagocytic immune cells of the central nervous system and play an integral role as first responders during inflammation. Microglia are commonly classified as "HM" (homeostatic), "M1" (classically activated proinflammatory), or "M2" (alternatively activated). Multiple single-cell RNA-sequencing studies suggest that this discrete classification system does not accurately and fully capture the vast heterogeneity of microglial states in the brain. In fact, a recent single-cell RNA-sequencing study showed that microglia exist along a continuous spectrum of states. This spectrum spans heterogeneous populations of homeostatic and neuropathology-associated microglia in both healthy and Alzheimer's disease (AD) mouse brains. Major risk factors, such as sex, age, and genes, modulate microglial states, suggesting that shifts along the trajectory might play a causal role in AD pathogenesis. This study provides important insight into the cellular mechanisms of AD and underlines the potential of novel cell-based therapies for AD.

Beyond the sleep-amyloid interactions in Alzheimer's disease pathogenesis.

Cognitive impairment in older adults is associated with sleep and circadian rhythm disturbances. Numerous studies have linked disrupted sleep and circadian rhythms with amyloid-ß (Aß), a key pathological hallmark in Alzheimer's disease (AD). While previous evidence suggests that Aß initiates AD pathogenesis, tau, another major hallmark of AD, seems to drive neurodegeneration. Recent studies imply that sleep-wake cycles affect brain tau more significantly than Aß levels, leading to accelerated AD progression and cognitive decline. The study of sleep disturbances in AD is shedding light on our understanding of the mechanism underlying sleep disturbances in AD and dementia.

Tau reduction in the presence of amyloid-ß prevents tau pathology and neuronal death in vivo.

Several studies have now supported the use of a tau lowering agent as a possible therapy in the treatment of tauopathy disorders, including Alzheimer's disease. In human Alzheimer's disease, however, concurrent amyloid-ß deposition appears to synergize and accelerate tau pathological changes. Thus far, tau reduction strategies that have been tested in vivo have been examined in the setting of tau pathology without confounding amyloid-ß deposition. To determine whether reducing total human tau expression in a transgenic model where there is concurrent amyloid-ß plaque formation can still reduce tau pathology and protect against neuronal loss, we have taken advantage of the regulatable tau transgene in APP/PS1 × rTg4510 mice. These mice develop both neurofibrillary tangles as well as amyloid-ß plaques throughout the cortex and hippocampus. By suppressing human tau expression for 6 months in the APP/PS1 × rTg4510 mice using doxycycline, AT8 tau pathology, bioactivity, and astrogliosis were reduced, though importantly to a lesser extent than lowering tau in the rTg4510 alone mice. Based on non-denaturing gels and proteinase K digestions, the remaining tau aggregates in the presence of amyloid-ß exhibit a longer-lived aggregate conformation. Nonetheless, lowering the expression of the human tau transgene was sufficient to equally ameliorate thioflavin-S positive tangles and prevent neuronal loss equally well in both the APP/PS1 × rTg4510 mice and the rTg4510 cohort. Together, these results suggest that, although amyloid-ß stabilizes tau aggregates, lowering total tau levels is still an effective strategy for the treatment of tau pathology and neuronal loss even in the presence of amyloid-ß deposition.

Human Neurospheroid Arrays for In Vitro Studies of Alzheimer's Disease.

Neurospheroids are commonly used for in vitro disease modeling and drug screening. However, the heterogeneity in size of the neurospheroids mixtures available through current methods limits their utility when employed for basic mechanistic studies of neurodegenerative diseases or screening for new interventions. Here, we generate neurospheroids from immortalized neural progenitor cells and human induced pluripotent stem cells that are uniform in size, into large-scale arrays. In proof of concept experiments, we validate the neurospheroids array as a sensitive and robust tool for screening compounds over extended time. We show that when suspended in three-dimensional extracellular matrix up to several weeks, the stem cell-derived neurospheroids display extensive neurite outgrowth and extend thick bundles of dendrites outward. We also cultivate genetically-engineered stem cell-derived neurospheroids with familial Alzheimer's disease mutations for eight weeks in our microarray system. Interestingly, we observed robust accumulation of amyloid-ß and phosphorylated tau, key hallmarks of Alzheimer's disease. Overall, our in vitro model for engineering neurospheroid arrays is a valuable tool for studying complex neurodegenerative diseases and accelerating drug discovery.

Three-Dimensional Models of the Human Brain Development and Diseases.

Deciphering the human brain pathophysiology remains one of the greatest challenges of the 21st century. Neurological disorders represent a significant proportion of diseases burden; however, the complexity of the brain physiology makes it challenging to model its diseases. Simple in vitro models have been very useful for precise measurements in controled conditions. However, existing models are limited in their ability to replicate complex interactions between various cells in the brain. Studying human brain requires sophisticated models to reconstitute the tangled architecture and functions of brain cells. Recently, advances in the development of three-dimensional (3D) brain cell culture models have begun to recapitulate various aspects of the human brain physiology in vitro and replicate basic disease processes of Alzheimer's disease, amyotrophic lateral sclerosis, and microcephaly. In this review, we discuss the progress, advantages, limitations, and future directions of 3D cell culture systems for modeling the human brain development and diseases.

Trapped Chromatin Fibers Damage Flowing Red Blood Cells.

Neutrophils are the most abundant white blood cells in the circulation and serve antimicrobial functions. One of their antimicrobial mechanisms involves the release of neutrophil extracellular traps (NETs), long chromatin fibers decorated with antimicrobial granular proteins that contribute to the elimination of pathogens. However, the release of NETs has also been associated with disease processes. While recent research has focused on biochemical reactions catalyzed by NETs, significantly less is known about the mechanical effect of NETs in circulation. Here, microfluidic devices and biophysical models are employed to study the consequences of the interactions between NETs trapped in channels and red blood cells (RBCs) flowing in blood over the NETs. It has been found that the RBCs can be deformed and ruptured after interactions with NETs, generating RBC fragments. Significant increases in the number of RBC fragments have also been found in the circulation of patients with conditions in which NETs have been demonstrated to be present in circulation, including sepsis and kidney transplant. Further studies will probe the potential utility of RBC fragments in the diagnostic, monitoring, and treatment of diseases associated with the presence of NETs in circulation.

Mechanically switchable polymer fibers for sensing in biological conditions.

The area of in vivo sensing using optical fibers commonly uses materials such as silica and polymethyl methacrylate, both of which possess much higher modulus than human tissue. The mechanical mismatch between materials and living tissue has been seen to cause higher levels of glial encapsulation, scarring, and inflammation, leading to failure of the implanted medical device. We present the use of a fiber made from polyvinyl alcohol (PVA) for use as an implantable sensor as it is an easy to work with functionalized polymer that undergoes a transition from rigid to soft when introduced to water. This ability to switch from stiff to soft reduces the severity of the immune response. The fabricated PVA fibers labeled with fluorescein for sensing applications showed excellent response to various stimuli while exhibiting mechanical switchability. For the dry fibers, a tensile storage modulus of 4700 MPa was measured, which fell sharply to 145 MPa upon wetting. The fibers showed excellent response to changing pH levels, producing values that were detectable in a range consistent with those seen in the literature and in proposed applications. The results show that these mechanically switchable fibers are a viable option for future sensing applications.

Influence of resveratrol release on the tissue response to mechanically adaptive cortical implants.

The stability and longevity of recordings obtained from intracortical microelectrodes continues to remain an area of concern for neural interfacing applications. The limited longevity of microelectrode performance has been associated with the integrity of the blood brain barrier (BBB) and the neuroinflammatory response to the microelectrode. Here, we report the investigation of an additive approach that targets both mechanical and chemical factors believed to contribute to chronic BBB instability and the neuroinflammatory response associated with implanted intracortical microelectrodes. The implants investigated were based on a mechanically adaptive, compliant nanocomposite (NC), which reduces the tissue response and tissue strain. This material was doped with various concentrations of the antioxidant resveratrol with the objective of local and rapid delivery. In vitro analysis of resveratrol release, antioxidant activity, and cytotoxicity suggested that a resveratrol content of 0.01% was optimal for in vivo assessment. Thus, probes made from the neat NC reference and probes containing resveratrol (NC Res) were implanted into the cortical tissue of rats for up to sixteen weeks. Histochemical analysis suggested that at three days post-implantation, neither materials nor therapeutic approaches (independently or in combination) could alter the initial wound healing response. However, at two weeks post-implantation, the NC Res implant showed a reduction in activated microglia/macrophages and improvement in neuron density at the tissue-implant interface when compared to the neat NC reference. However, sixteen weeks post-implantation, when the antioxidant was exhausted, NC Res and the neat NC reference exhibited similar tissue responses. The data show that NC Res provides short-term, short-lived benefits due to the antioxidant release, and a long-term reduction in neuroinflammation on account of is mechanical adaptive, compliant nature. Together, these results demonstrate that local delivery of resveratrol can provide an additive advantage by providing a consistent reduction in the tissue response.

Progress towards biocompatible intracortical microelectrodes for neural interfacing applications.

To ensure long-term consistent neural recordings, next-generation intracortical microelectrodes are being developed with an increased emphasis on reducing the neuro-inflammatory response. The increased emphasis stems from the improved understanding of the multifaceted role that inflammation may play in disrupting both biologic and abiologic components of the overall neural interface circuit. To combat neuro-inflammation and improve recording quality, the field is actively progressing from traditional inorganic materials towards approaches that either minimizes the microelectrode footprint or that incorporate compliant materials, bioactive molecules, conducting polymers or nanomaterials. However, the immune-privileged cortical tissue introduces an added complexity compared to other biomedical applications that remains to be fully understood. This review provides a comprehensive reflection on the current understanding of the key failure modes that may impact intracortical microelectrode performance. In addition, a detailed overview of the current status of various materials-based approaches that have gained interest for neural interfacing applications is presented, and key challenges that remain to be overcome are discussed. Finally, we present our vision on the future directions of materials-based treatments to improve intracortical microelectrodes for neural interfacing.

Reinforcing Poly(ethylene) with Cellulose Nanocrystals.

The fabrication of nanocomposites of low-density polyethylene (LDPE), one of the world's most widely used polymers, and cellulose nanocrystals (CNCs), which represent the world's most abundant bio-based nanofiller, is reported. While the hydrophobic polymer and the hydrophilic filler seem to be intrinsically incompatible, this article shows that it is possible to kinetically trap homogeneous nanocomposites by a templating approach. An organogel is first prepared by exchanging the solvent of an aqueous CNC dispersion against acetone, impregnating the resulting organogel, in which the CNCs form a percolating network with a hot LDPE solution in toluene, and compression-molding the resulting materials into thin films. At a filler content of 7.6% v/v, the resulting materials display a three- to four-fold increase in strength and stiffness compared with the neat LDPE, which confirms that the CNC network could be largely maintained. It is also possible to reprocess these nanocomposites and dilute them with LDPE using conventional melt-processing techniques.