Brain-Targeted Liposomes Loaded with Monoclonal Antibodies Reduce Alpha-Synuclein Aggregation and Improve Behavioral Symptoms in Parkinson's Disease.

Monoclonal antibodies (mAbs) hold promise in treating Parkinson's disease (PD), although poor delivery to the brain hinders their therapeutic application. In the current study, it is demonstrated that brain-targeted liposomes (BTL) enhance the delivery of mAbs across the blood-brain-barrier (BBB) and into neurons, thereby allowing the intracellular and extracellular treatment of the PD brain. BTL are decorated with transferrin to improve brain targeting through overexpressed transferrin-receptors on the BBB during PD. BTL are loaded with SynO4, a mAb that inhibits alpha-synuclein (AS) aggregation, a pathological hallmark of PD. It is shown that 100-nm BTL cross human BBB models intact and are taken up by primary neurons. Within neurons, SynO4 is released from the nanoparticles and bound to its target, thereby reducing AS aggregation, and enhancing neuronal viability. In vivo, intravenous BTL administration results in a sevenfold increase in mAbs in brain cells, decreasing AS aggregation and neuroinflammation. Treatment with BTL also improve behavioral motor function and learning ability in mice, with a favorable safety profile. Accordingly, targeted nanotechnologies offer a valuable platform for drug delivery to treat brain neurodegeneration.

Unique features of the arterial blood-brain barrier.

CNS vasculature differs from vascular networks of peripheral organs by its ability to tightly control selective material exchange across capillary barriers. Capillary permeability is mostly defined by unique cellular components of the endothelium. While capillaries are extensively investigated, the barrier properties of larger vessels are understudied. Here, we investigate barrier properties of CNS arterial walls. Using tracer challenges and various imaging modalities, we discovered that at the mouse cortex, the arterial barrier does not reside at the classical level of the endothelium. The arterial wall's unique permeability acts bi-directionally; CSF substances travel along the glymphatic path and can penetrate from the peri-vascular space through arteriolar walls towards the lumen. We found that caveolae vesicles in arteriole endothelial are functional transcytosis machinery components, and that a similar mechanism is evident in the human brain. Our discoveries highlight vascular heterogeneity investigations as a potent approach to uncover new barrier mechanisms.

P450 oxidoreductase regulates barrier maturation by mediating retinoic acid metabolism in a model of the human BBB.

The blood-brain barrier (BBB) selectively regulates the entry of molecules into the central nervous system (CNS). A crosstalk between brain microvascular endothelial cells (BMECs) and resident CNS cells promotes the acquisition of functional tight junctions (TJs). Retinoic acid (RA), a key signaling molecule during embryonic development, is used to enhance in vitro BBB models' functional barrier properties. However, its physiological relevance and affected pathways are not fully understood. P450 oxidoreductase (POR) regulates the enzymatic activity of microsomal cytochromes. POR-deficient (PORD) patients display impaired steroid homeostasis and cognitive disabilities. Here, we used both patient-specific POR-deficient and CRISPR-Cas9-mediated POR-depleted induced pluripotent stem cell (iPSC)-derived BMECs (iBMECs) to study the role of POR in the acquisition of functional barrier properties. We demonstrate that POR regulates cellular RA homeostasis and that POR deficiency leads to the accumulation of RA within iBMECs, resulting in the impaired acquisition of TJs and, consequently, to dysfunctional development of barrier properties.

Nano-scale architecture of blood-brain barrier tight-junctions.

Tight junctions (TJs) between blood-brain barrier (BBB) endothelial cells construct a robust physical barrier, whose damage underlies BBB dysfunctions related to several neurodegenerative diseases. What makes these highly specialized BBB-TJs extremely restrictive remains unknown. Here, we use super-resolution microscopy (dSTORM) to uncover new structural and functional properties of BBB TJs. Focusing on three major components, Nano-scale resolution revealed sparse (occludin) vs. clustered (ZO1/claudin-5) molecular architecture. In mouse development, permeable TJs become first restrictive to large molecules, and only later to small molecules, with claudin-5 proteins arrangement compacting during this maturation process. Mechanistically, we reveal that ZO1 clustering is independent of claudin-5 in vivo. In contrast to accepted knowledge, we found that in the developmental context, total levels of claudin-5 inversely correlate with TJ functionality. Our super-resolution studies provide a unique perspective of BBB TJs and open new directions for understanding TJ functionality in biological barriers, ultimately enabling restoration in disease or modulation for drug delivery.

Hippocampal neural stem cells facilitate access from circulation via apical cytoplasmic processes.

Blood vessels (BVs) are considered an integral component of neural stem cells (NSCs) niches. NSCs in the dentate gyrus (DG(have enigmatic elaborated apical cellular processes that are associated with BVs. Whether this contact serves as a mechanism for delivering circulating molecules is not known. Here we uncovered a previously unrecognized communication route allowing exclusive direct access of blood-borne substances to hippocampal NSCs. BBB-impermeable fluorescent tracer injected transcardially to mice is selectively uptaken by DG NSCs within a minute, via the vessel-associated apical processes. These processes, measured >30 nm in diameter, establish direct membrane-to-membrane contact with endothelial cells in specialized areas of irregular endothelial basement membrane and enriched with vesicular activity. Doxorubicin, a brain-impermeable chemotherapeutic agent, is also readily and selectively uptaken by NSCs and reduces their proliferation, which might explain its problematic anti-neurogenic or cognitive side-effect. The newly-discovered NSC-BV communication route explains how circulatory neurogenic mediators are 'sensed' by NSCs.

Angiomodulin (IGFBP7) is a cerebral specific angiocrine factor, but is probably not a blood-brain barrier inducer.

BACKGROUND: Several secreted factors have been identified as drivers of cerebral vasculature development and inducers of blood-brain barrier (BBB) differentiation. Vascular endothelial growth factor A (VEGF-A) is central for driving cerebral angiogenesis and Wnt family factors (Wnt7a, Wnt7b and norrin) are central for induction and maintenance of barrier properties. Expressed by developing neural tissue (neuron and glia progenitors), they influence the formation of central nervous system (CNS) vascular networks. Another type of factors are tissue-specific paracrine factors produced by endothelial cells (ECs), also known as 'angiocrine' factors, that provide instructive signals to regulate homeostatic and regenerative processes. Very little is known about CNS angiocrine factors and their role in BBB development. Angiomodulin (AGM) was reported to be expressed by developing vasculature and by pathological tumor vasculature. Here we investigated AGM in the developing CNS and its function as a potential BBB inducer. METHODS: We analyzed microarray data to identify potential angiocrine factors specifically expressed at early stages of barrier formation. We then tested AGM expression with immunofluorescence and real-time PCR in various organs during development, post-natal and in adults. Permeability induction with recombinant proteins (Miles assay) was used to test potential interaction of AGM with VEGF-A. RESULTS: Several angiocrine factors are differentially expressed by CNS ECs and AGM is a prominent CNS-specific angiocrine candidate. Contrary to previous reports, we found that AGM protein expression is specific to developing CNS endothelium and not to highly angiogenic developing vasculature in general. In skin vasculature we found that AGM antagonizes VEGF-A-induced vascular hyperpermeability. Finally, CNS AGM expression is not specific to BBB vasculature and AGM is highly expressed in non-BBB choroid-plexus vasculature. CONCLUSIONS: We propose AGM as a developmental CNS vascular-specific marker. AGM is not a pan-endothelial marker, nor a general marker for developing angiogenic vasculature. Thus, AGM induction in the developing CNS might be distinct from its induction in pathology. While AGM is able to antagonize VEGF-A-induced vascular hyperpermeability in the skin, its high expression levels in non-BBB CNS vasculature does not support its potential role as a BBB inducer. Further investigation including loss-of-function approaches might elucidate AGM function in the developing CNS.

Systemic microbial TLR2 agonists induce neurodegeneration in Alzheimer's disease mice.

BACKGROUND: Accumulating data suggest a central role for brain microglia in mediating cortical neuronal death in Alzheimer's disease (AD), and for Toll-like receptor 2 (TLR2) in their toxic activation. Amyloid deposition in preclinical AD is associated with microglial activation but not directly with neurodegeneration. We examined in transgenic 5xFAD mice the hypothesis that systemic TLR2 agonists, derived from common infectious agents, may accelerate neurodegeneration in AD. METHODS: Microbial wall-derived TLR2 agonists zymosan and lipoteichoic acid were administered intraperitoneally or intracerebroventricularly to 7-month-old wild-type or 5xFAD mice. Immunofluorescent stainings were used to quantify cortical neurons and evaluate tissue reaction. Microglial activation was assessed using functional assays, RNA expression, and FACS analysis. RESULTS: Repeated low-dose systemic administration of zymosan or lipoteichoic acid killed cortical neurons in 5xFAD mice but not in wild-type mice. Direct CNS delivery of a selective TLR2 antagonist blocked the neurotoxicity of systemically administered zymosan, indicating that CNS TLR2 mediates this effect. Systemically administered zymosan crossed the disrupted blood-brain barrier in 5xFAD mice and entered brain parenchyma. By intracerebroventricular delivery, we found a dose- and exposure time-dependent acute neurotoxic effect of the microbial TLR2 agonist, killing cortical neurons. 5xFAD mice exhibited significantly increased vulnerability to TLR2 agonist-induced neuronal loss as compared to wild-type mice. Microbial TLR2-induced neurodegeneration was abolished by inhibiting microglia. The vulnerability of 5xFAD mice brains was mediated by an increase in number and neurotoxic phenotype of TLR2-expressing microglia. CONCLUSIONS: We suggest that repeated exposure to microbial TLR2 agonists may facilitate neurodegeneration in AD by their microglial-mediated toxicity to the hyper-vulnerable environment of the AD brain.

Mitochondrial Transfer Ameliorates Cognitive Deficits, Neuronal Loss, and Gliosis in Alzheimer's Disease Mice.

Pathogenesis of neurodegenerative diseases involves dysfunction of mitochondria, one of the most important cell organelles in the brain, with its most prominent roles in producing energy and regulating cellular metabolism. Here we investigated the effect of transferring active intact mitochondria as a potential therapy for Alzheimer's disease (AD), in order to correct as many mitochondrial functions as possible, rather than a mono-drug related therapy. For this purpose, AD-mice (amyloid-ß intracerebroventricularly injected) were treated intravenously (IV) with fresh human isolated mitochondria. One to two weeks later, a significantly better cognitive performance was noticed in the mitochondria treated AD-mice relative to vehicle treated AD-mice, approaching the performance of non-AD mice. We also detected a significant decrease in neuronal loss and reduced gliosis in the hippocampus of treated mice relative to untreated AD-mice. An amelioration of the mitochondrial dysfunction in brain was noticed by the increase of citrate-synthase and cytochrome c oxidase activities relative to untreated AD-mice, reaching activity levels of non-AD-mice. Increased mitochondrial activity was also detected in the liver of mitochondria treated mice. No treatment-related toxicity was noted. Thus, IV mitochondrial transfer may possibly offer a novel therapeutic approach for AD.

Tertiary lymphoid structures in the choroid plexus in neuropsychiatric lupus.

The central nervous system manifestations of systemic lupus erythematosus (SLE) remain poorly understood. Given the well-defined role of autoantibodies in other lupus manifestations, extensive work has gone into the identification of neuropathic autoantibodies. However, attempts to translate these findings to patients with SLE have yielded mixed results. We used the MRL/MpJ-Faslpr/lpr mouse, a well-established, spontaneous model of SLE, to establish the immune effectors responsible for brain disease. Transcriptomic analysis of the MRL/MpJ-Faslpr/lpr choroid plexus revealed an expression signature driving tertiary lymphoid structure formation, including chemokines related to stromal reorganization and lymphocyte compartmentalization. Additionally, transcriptional profiles indicated various stages of lymphocyte activation and germinal center formation. The extensive choroid plexus infiltrate present in MRL/MpJ-Faslpr/lpr mice with overt neurobehavioral deficits included locally proliferating B and T cells, intercellular interactions between lymphocytes and antigen-presenting cells, as well as evidence for in situ somatic hypermutation and class switch recombination. Furthermore, the choroid plexus was a site for trafficking lymphocytes into the brain. Finally, histological evaluation in human lupus patients with neuropsychiatric manifestations revealed increased leukocyte migration through the choroid plexus. These studies identify a potential new pathway underlying neuropsychiatric lupus and support tertiary lymphoid structure formation in the choroid plexus as a novel mechanism of brain-immune interfacing.

Leptin receptor deficiency induces early, transient and hyperglycaemia-independent blood-brain barrier dysfunction.

Diabetes mellitus (DM) significantly increases susceptibility to central nervous system (CNS) pathologies, including stroke, vascular dementia, cognitive deficits and Alzheimer's disease. Previous studies (mostly using the streptozotocin model) suggested that blood-brain barrier (BBB) disruption is involved in these conditions. Here, we examined the integrity of brain capillaries and BBB permeability in Leprdb/db obesity-related diabetic mice. Surprisingly, significant BBB leakage was observed only in young mice at the pre-hyperglycemic stage. Thorough examination of barrier permeability at later diabetic stages showed no evidence for significant BBB leakage during the hyperglycemic state. Electron microscopy imaging of mice with short-term hyperglycaemia supported normal BBB permeability but indicated other stress-related changes in capillary ultrastructure, such as mitochondrial degeneration. Based on our study with this mouse genetic model of obesity-related DM, we suggest that previously reported hyperglycaemia-induced BBB leakage is most likely not the underlying mechanism of DM-related CNS pathologies. Finally we propose that BBB hyper-permeability might be an early and transient phenomenon while stress-related endothelial pathologies do correlate with a short-term diabetic state.

Mechanisms of neuropsychiatric lupus: The relative roles of the blood-cerebrospinal fluid barrier versus blood-brain barrier.

The pathogenesis of neuropsychiatric lupus (NPSLE) is believed to include the entry of circulating neuropathic antibodies to the brain via a pathologically permeable blood-brain barrier (BBB). Nevertheless, direct evidence of BBB pathology or mechanisms underlying BBB dysfunction is missing. Here, we examined BBB integrity in an established NPSLE mouse model (MRL/faslpr/lpr). Surprisingly, challenging the barrier with various exogenous tracers demonstrated insignificant changes in BBB permeability. Furthermore, electron microscopy showed no ultrastructure changes supporting hyper-permeability. However, we found that abnormal function of the blood-cerebrospinal fluid barrier (BCSFB) in the choroid plexus underlies brain exposure to neuropathic antibodies. Considerable intrathecal lymphocyte infiltration likely occurs through the BCSFB, accompanied by epithelial hyper-permeability to antibodies. Our results challenge the commonly held view of BBB disruption in NPSLE, supporting a shift in focus to BCSFB dysfunction as a causative factor in the disease.

Joint volumetric extraction and enhancement of vasculature from low-SNR 3-D fluorescence microscopy images.

To simultaneously overcome the challenges imposed by the nature of optical imaging characterized by a range of artifacts including space-varying signal to noise ratio (SNR), scattered light, and non-uniform illumination, we developed a novel method that segments the 3-D vasculature directly from original fluorescence microscopy images eliminating the need for employing pre- and post-processing steps such as noise removal and segmentation refinement as used with the majority of segmentation techniques. Our method comprises two initialization and constrained recovery and enhancement stages. The initialization approach is fully automated using features derived from bi-scale statistical measures and produces seed points robust to non-uniform illumination, low SNR, and local structural variations. This algorithm achieves the goal of segmentation via design of an iterative approach that extracts the structure through voting of feature vectors formed by distance, local intensity gradient, and median measures. Qualitative and quantitative analysis of the experimental results obtained from synthetic and real data prove the effcacy of this method in comparison to the state-of-the-art enhancing-segmenting methods. The algorithmic simplicity, freedom from having a priori probabilistic information about the noise, and structural definition gives this algorithm a wide potential range of applications where i.e. structural complexity significantly complicates the segmentation problem.

The blood brain barrier and neuropsychiatric lupus: new perspectives in light of advances in understanding the neuroimmune interface.

Experts have previously postulated a linkage between lupus associated vascular pathology and abnormal brain barriers in the immunopathogenesis of neuropsychiatric lupus. Nevertheless, there are some discrepancies between the experimental evidence, or its interpretation, and the working hypotheses prevalent in this field; specifically, that a primary contributor to neuropsychiatric disease in lupus is permeabilization of the blood brain barrier. In this commonly held view, any contribution of the other known brain barriers, including the blood-cerebrospinal fluid and meningeal barriers, is mostly excluded from the discussion. In this review we will shed light on some of the blood brain barrier hypotheses and try to trace their roots. In addition, we will suggest new research directions to allow for confirmation of alternative interpretations of the experimental evidence linking the pathology of intra-cerebral vasculature to the pathogenesis of neuropsychiatric lupus.

Blood-Brain Barrier Permeability Is Regulated by Lipid Transport-Dependent Suppression of Caveolae-Mediated Transcytosis.

The blood-brain barrier (BBB) provides a constant homeostatic brain environment that is essential for proper neural function. An unusually low rate of vesicular transport (transcytosis) has been identified as one of the two unique properties of CNS endothelial cells, relative to peripheral endothelial cells, that maintain the restrictive quality of the BBB. However, it is not known how this low rate of transcytosis is achieved. Here we provide a mechanism whereby the regulation of CNS endothelial cell lipid composition specifically inhibits the caveolae-mediated transcytotic route readily used in the periphery. An unbiased lipidomic analysis reveals significant differences in endothelial cell lipid signatures from the CNS and periphery, which underlie a suppression of caveolae vesicle formation and trafficking in brain endothelial cells. Furthermore, lipids transported by Mfsd2a establish a unique lipid environment that inhibits caveolae vesicle formation in CNS endothelial cells to suppress transcytosis and ensure BBB integrity.

Vessel maturation schedule determines vulnerability to neuronal injuries of prematurity.

Premature birth is a major risk factor for multiple brain pathologies, notably periventricular leukomalacia (PVL), which is distinguished by bilateral necrosis of neural tissue around the ventricles and a sequela of neurological disturbances. The 2 hallmarks of brain pathologies of prematurity are a restricted gestational window of vulnerability and confinement of injury to a specific cerebral region. Here, we examined the proposition that both of these features are determined by the state of blood vessel immaturity. We developed a murine genetic model that allows for inducible and reversible VEGF blockade during brain development. Using this system, we determined that cerebral vessels mature in a centrifugal, wave-like fashion that results in sequential acquisition of a functional blood-brain barrier and exit from a VEGF-dependent phase, with periventricular vessels being the last to mature. This developmental program permitted selective ablation of periventricular vessels via episodic VEGF blockade within a specific, vulnerable gestational window. Enforced collapse of ganglionic eminence vessels and resultant periventricular neural apoptosis resulted in a PVL-like phenotype that recapitulates the primary periventricular lesion, ventricular enlargement, and the secondary cortical deficit in out-migrating GABAergic inhibitory interneurons. These findings provide an animal model that reproduces the temporal and spatial specificities of PVL and indicate that damage to VEGF-dependent, immature periventricular vessels contributes to PVL development.

The molecular, cellular, and morphological components of blood-brain barrier development during embryogenesis.

The blood brain barrier (BBB) is a hallmark of blood vessels in the brain and functions to protect the brain from unwanted blood born materials, support the unique metabolic needs of the brain, and define a stable environment crucial for brain homeostasis. The temporal profile of BBB development was long debated until recent studies produced convincing evidence demonstrating that the BBB is established and functional during embryogenesis. Here we review research focused on the molecular, cellular and morphological characteristics of BBB development. Our review discusses the precise temporal profile of BBB formation, the development of endothelial cell ultrastructure and the molecular components that provide sealing and transporting properties, the molecular pathways involved in the induction of BBB specific endothelial cell differentiation, the signaling pathways driving developmental angiogenesis versus barrier-genesis, and finally the contribution of other cell types to BBB formation. We examine aspects of BBB development that are still unresolved while highlighting research tools that could provide new insight to answer these open questions.

A novel method for identifying a graph-based representation of 3-D microvascular networks from fluorescence microscopy image stacks.

A novel approach to determine the global topological structure of a microvasculature network from noisy and low-resolution fluorescence microscopy data that does not require the detailed segmentation of the vessel structure is proposed here. The method is most appropriate for problems where the tortuosity of the network is relatively low and proceeds by directly computing a piecewise linear approximation to the vasculature skeleton through the construction of a graph in three dimensions whose edges represent the skeletal approximation and vertices are located at Critical Points (CPs) on the microvasculature. The CPs are defined as vessel junctions or locations of relatively large curvature along the centerline of a vessel. Our method consists of two phases. First, we provide a CP detection technique that, for junctions in particular, does not require any a priori geometric information such as direction or degree. Second, connectivity between detected nodes is determined via the solution of a Binary Integer Program (BIP) whose variables determine whether a potential edge between nodes is or is not included in the final graph. The utility function in this problem reflects both intensity-based and structural information along the path connecting the two nodes. Qualitative and quantitative results confirm the usefulness and accuracy of this method. This approach provides a mean of correctly capturing the connectivity patterns in vessels that are missed by more traditional segmentation and binarization schemes because of imperfections in the images which manifest as dim or broken vessels.

Sensory-related neural activity regulates the structure of vascular networks in the cerebral cortex.

Neurovascular interactions are essential for proper brain function. While the effect of neural activity on cerebral blood flow has been extensively studied, whether or not neural activity influences vascular patterning remains elusive. Here, we demonstrate that neural activity promotes the formation of vascular networks in the early postnatal mouse barrel cortex. Using a combination of genetics, imaging, and computational tools to allow simultaneous analysis of neuronal and vascular components, we found that vascular density and branching were decreased in the barrel cortex when sensory input was reduced by either a complete deafferentation, a genetic impairment of neurotransmitter release at thalamocortical synapses, or a selective reduction of sensory-related neural activity by whisker plucking. In contrast, enhancement of neural activity by whisker stimulation led to an increase in vascular density and branching. The finding that neural activity is necessary and sufficient to trigger alterations of vascular networks reveals an important feature of neurovascular interactions.

Fgd5 identifies hematopoietic stem cells in the murine bone marrow.

Hematopoietic stem cells (HSCs) are the best-characterized tissue-specific stem cells, yet experimental study of HSCs remains challenging, as they are exceedingly rare and methods to purify them are cumbersome. Moreover, genetic tools for specifically investigating HSC biology are lacking. To address this we sought to identify genes uniquely expressed in HSCs within the hematopoietic system and to develop a reporter strain that specifically labels them. Using microarray profiling we identified several genes with HSC-restricted expression. Generation of mice with targeted reporter knock-in/knock-out alleles of one such gene, Fgd5, revealed that though Fgd5 was required for embryonic development, it was not required for definitive hematopoiesis or HSC function. Fgd5 reporter expression near exclusively labeled cells that expressed markers consistent with HSCs. Bone marrow cells isolated based solely on Fgd5 reporter signal showed potent HSC activity that was comparable to stringently purified HSCs. The labeled fraction of the Fgd5 reporter mice contained all HSC activity, and HSC-specific labeling was retained after transplantation. Derivation of next generation mice bearing an Fgd5-CreERT2 allele allowed tamoxifen-inducible deletion of a conditional allele specifically in HSCs. In summary, reporter expression from the Fgd5 locus permits identification and purification of HSCs based on single-color fluorescence.

An image-based RNAi screen identifies SH3BP1 as a key effector of Semaphorin 3E-PlexinD1 signaling.

Extracellular signals have to be precisely interpreted intracellularly and translated into diverse cellular behaviors often mediated by cytoskeletal changes. Semaphorins are one of the largest families of guidance cues and play a critical role in many systems. However, how different cell types translate extracellular semaphorin binding into intracellular signaling remains unclear. Here we developed and performed a novel image-based genome-wide functional RNAi screen for downstream signaling molecules that convert the interaction between Semaphorin 3E (Sema3E) and PlexinD1 into cellular behaviors. One of the genes identified in this screen is a RhoGAP protein, SH3-domain binding protein 1 (SH3BP1). We demonstrate that SH3BP1 mediates Sema3E-induced cell collapse through interaction with PlexinD1 and regulation of Ras-related C3 botulinum toxin substrate 1 (Rac1) activity. The identification and characterization of SH3BP1 as a novel downstream effector of Sema3E-PlexinD1 provides an explanation for how extracellular signals are translated into cytoskeletal changes and unique cell behavior, but also lays the foundation for characterizing other genes identified from our screen to obtain a more complete picture of plexin signaling.