Distinct molecular profiles of skull bone marrow in health and neurological disorders.

The bone marrow in the skull is important for shaping immune responses in the brain and meninges, but its molecular makeup among bones and relevance in human diseases remain unclear. Here, we show that the mouse skull has the most distinct transcriptomic profile compared with other bones in states of health and injury, characterized by a late-stage neutrophil phenotype. In humans, proteome analysis reveals that the skull marrow is the most distinct, with differentially expressed neutrophil-related pathways and a unique synaptic protein signature. 3D imaging demonstrates the structural and cellular details of human skull-meninges connections (SMCs) compared with veins. Last, using translocator protein positron emission tomography (TSPO-PET) imaging, we show that the skull bone marrow reflects inflammatory brain responses with a disease-specific spatial distribution in patients with various neurological disorders. The unique molecular profile and anatomical and functional connections of the skull show its potential as a site for diagnosing, monitoring, and treating brain diseases.

Neuroimmune cardiovascular interfaces control atherosclerosis.

Atherosclerotic plaques develop in the inner intimal layer of arteries and can cause heart attacks and strokes1. As plaques lack innervation, the effects of neuronal control on atherosclerosis remain unclear. However, the immune system responds to plaques by forming leukocyte infiltrates in the outer connective tissue coat of arteries (the adventitia)2-6. Here, because the peripheral nervous system uses the adventitia as its principal conduit to reach distant targets7-9, we postulated that the peripheral nervous system may directly interact with diseased arteries. Unexpectedly, widespread neuroimmune cardiovascular interfaces (NICIs) arose in mouse and human atherosclerosis-diseased adventitia segments showed expanded axon networks, including growth cones at axon endings near immune cells and media smooth muscle cells. Mouse NICIs established a structural artery-brain circuit (ABC): abdominal adventitia nociceptive afferents10-14 entered the central nervous system through spinal cord T6-T13 dorsal root ganglia and were traced to higher brain regions, including the parabrachial and central amygdala neurons; and sympathetic efferent neurons projected from medullary and hypothalamic neurons to the adventitia through spinal intermediolateral neurons and both coeliac and sympathetic chain ganglia. Moreover, ABC peripheral nervous system components were activated: splenic sympathetic and coeliac vagus nerve activities increased in parallel to disease progression, whereas coeliac ganglionectomy led to the disintegration of adventitial NICIs, reduced disease progression and enhanced plaque stability. Thus, the peripheral nervous system uses NICIs to assemble a structural ABC, and therapeutic intervention in the ABC attenuates atherosclerosis.

Tissue Clearing Approaches in Atherosclerosis.

Recent advances in cardiovascular research have led to a more comprehensive understanding of molecular mechanisms of atherosclerosis. It has become apparent that the disease involves three layers of the arterial wall: the intima, the media, and a connective tissue coat termed the adventitia. It is also now appreciated that arteries are surrounded by adipose and neuronal tissues. In addition, adjacent to and within the adventitia, arteries are embedded in a loose connective tissue containing blood vessels (vasa vasora) and lymph vessels, artery-draining lymph nodes and components of the peripheral nervous system, including periarterial nerves and ganglia. During atherogenesis, each of these tissues undergoes marked structural and cellular alterations. We propose that a better understanding of these cell-cell and cell-tissue interactions may considerably advance our understanding of cardiovascular disease pathogenesis. Methods to acquire subcellular optical access to the intact tissues surrounding healthy and diseased arteries are urgently needed to achieve these aims. Tissue clearing is a landmark next-generation, three-dimensional (3D) microscopy technique that allows to image large-scale hitherto inaccessible intact deep tissue compartments. It allows for detailed reconstructions of arteries by a combination of labelling, clearing, advanced microscopies and other imaging and data-analysis tools. Here, we describe two distinct tissue clearing protocols; solvent-based modified three-dimensional imaging of solvent-cleared organs (3DISCO) clearing and another using aqueous-based 2,2'-thiodiethanol (TDE) clearing, both of which complement each other.

An ocular glymphatic clearance system removes ß-amyloid from the rodent eye.

Despite high metabolic activity, the retina and optic nerve head lack traditional lymphatic drainage. We here identified an ocular glymphatic clearance route for fluid and wastes via the proximal optic nerve in rodents. ß-amyloid (Aß) was cleared from the retina and vitreous via a pathway dependent on glial water channel aquaporin-4 (AQP4) and driven by the ocular-cranial pressure difference. After traversing the lamina barrier, intra-axonal Aß was cleared via the perivenous space and subsequently drained to lymphatic vessels. Light-induced pupil constriction enhanced efflux, whereas atropine or raising intracranial pressure blocked efflux. In two distinct murine models of glaucoma, Aß leaked from the eye via defects in the lamina barrier instead of directional axonal efflux. The results suggest that, in rodents, the removal of fluid and metabolites from the intraocular space occurs through a glymphatic pathway that might be impaired in glaucoma.

Aquaporin 1 and the Na+/K+/2Cl- cotransporter 1 are present in the leptomeningeal vasculature of the adult rodent central nervous system.

BACKGROUND: The classical view of cerebrospinal fluid (CSF) production posits the choroid plexus as its major source. Although previous studies indicate that part of CSF production occurs in the subarachnoid space (SAS), the mechanisms underlying extra-choroidal CSF production remain elusive. We here investigated the distributions of aquaporin 1 (AQP1) and Na+/K+/2Cl- cotransporter 1 (NKCC1), key proteins for choroidal CSF production, in the adult rodent brain and spinal cord. METHODS: We have accessed AQP1 distribution in the intact brain using uDISCO tissue clearing technique and by Western blot. AQP1 and NKCC1 cellular localization were accessed by immunohistochemistry in brain and spinal cord obtained from adult rodents. Imaging was performed using light-sheet, confocal and bright field light microscopy. RESULTS: We determined that AQP1 is widely distributed in the leptomeningeal vasculature of the intact brain and that its glycosylated isoform is the most prominent in different brain regions. Moreover, AQP1 and NKCC1 show specific distributions in the smooth muscle cell layer of penetrating arterioles and veins in the brain and spinal cord, and in the endothelia of capillaries and venules, restricted to the SAS vasculature. CONCLUSIONS: Our results shed light on the molecular framework that may underlie extra-choroidal CSF production and we propose that AQP1 and NKCC1 within the leptomeningeal vasculature, specifically at the capillary level, are poised to play a role in CSF production throughout the central nervous system.

Deep Learning Reveals Cancer Metastasis and Therapeutic Antibody Targeting in the Entire Body.

Reliable detection of disseminated tumor cells and of the biodistribution of tumor-targeting therapeutic antibodies within the entire body has long been needed to better understand and treat cancer metastasis. Here, we developed an integrated pipeline for automated quantification of cancer metastases and therapeutic antibody targeting, named DeepMACT. First, we enhanced the fluorescent signal of cancer cells more than 100-fold by applying the vDISCO method to image metastasis in transparent mice. Second, we developed deep learning algorithms for automated quantification of metastases with an accuracy matching human expert manual annotation. Deep learning-based quantification in 5 different metastatic cancer models including breast, lung, and pancreatic cancer with distinct organotropisms allowed us to systematically analyze features such as size, shape, spatial distribution, and the degree to which metastases are targeted by a therapeutic monoclonal antibody in entire mice. DeepMACT can thus considerably improve the discovery of effective antibody-based therapeutics at the pre-clinical stage. VIDEO ABSTRACT.

Corrigendum: A Novel RNA Editing Sensor Tool and a Specific Agonist Determine Neuronal Protein Expression of RNA-Edited Glycine Receptors and Identify a Genomic APOBEC1 Dimorphism as a New Genetic Risk Factor of Epilepsy.

[This corrects the article DOI: 10.3389/fnmol.2017.00439.].

Panoptic imaging of transparent mice reveals whole-body neuronal projections and skull-meninges connections.

Analysis of entire transparent rodent bodies after clearing could provide holistic biological information in health and disease, but reliable imaging and quantification of fluorescent protein signals deep inside the tissues has remained a challenge. Here, we developed vDISCO, a pressure-driven, nanobody-based whole-body immunolabeling technology to enhance the signal of fluorescent proteins by up to two orders of magnitude. This allowed us to image and quantify subcellular details through bones, skin and highly autofluorescent tissues of intact transparent mice. For the first time, we visualized whole-body neuronal projections in adult mice. We assessed CNS trauma effects in the whole body and found degeneration of peripheral nerve terminals in the torso. Furthermore, vDISCO revealed short vascular connections between skull marrow and brain meninges, which were filled with immune cells upon stroke. Thus, our new approach enables unbiased comprehensive studies of the interactions between the nervous system and the rest of the body.

Presence of Inhibitory Glycinergic Transmission in Medium Spiny Neurons in the Nucleus Accumbens.

It is believed that the rewarding actions of drugs are mediated by dysregulation of the mesolimbic dopaminergic system leading to increased levels of dopamine in the nucleus accumbens (nAc). It is widely recognized that GABAergic transmission is critical for neuronal inhibition within nAc. However, it is currently unknown if medium spiny neurons (MSNs) also receive inhibition by means of glycinergic synaptic inputs. We used a combination of proteomic and electrophysiology studies to characterize the presence of glycinergic input into MSNs from nAc demonstrating the presence of glycine transmission into nAc. In D1 MSNs, we found low frequency glycinergic miniature inhibitory postsynaptic currents (mIPSCs) which were blocked by 1 µM strychnine (STN), insensitive to low (10, 50 mM) and high (100 mM) ethanol (EtOH) concentrations, but sensitive to 30 µM propofol. Optogenetic experiments confirmed the existence of STN-sensitive glycinergic IPSCs and suggest a contribution of GABA and glycine neurotransmitters to the IPSCs in nAc. The study reveals the presence of glycinergic transmission in a non-spinal region and opens the possibility of a novel mechanism for the regulation of the reward pathway.

A Novel RNA Editing Sensor Tool and a Specific Agonist Determine Neuronal Protein Expression of RNA-Edited Glycine Receptors and Identify a Genomic APOBEC1 Dimorphism as a New Genetic Risk Factor of Epilepsy.

C-to-U RNA editing of glycine receptors (GlyR) can play an important role in disease progression of temporal lobe epilepsy (TLE) as it may contribute in a neuron type-specific way to neuropsychiatric symptoms of the disease. It is therefore necessary to develop tools that allow identification of neuron types that express RNA-edited GlyR protein. In this study, we identify NH4 as agonist of C-to-U RNA edited GlyRs. Furthermore, we generated a new molecular C-to-U RNA editing sensor tool that detects Apobec-1- dependent RNA editing in HEPG2 cells and rat primary hippocampal neurons. Using this sensor combined with NH4 application, we were able to identify C-to-U RNA editing-competent neurons and expression of C-to-U RNA-edited GlyR protein in neurons. Bioinformatic analysis of 1,000 Genome Project Phase 3 allele frequencies coding for human Apobec-1 80M and 80I variants showed differences between populations, and the results revealed a preference of the 80I variant to generate RNA-edited GlyR protein. Finally, we established a new PCR-based restriction fragment length polymorphism (RFLP) approach to profile mRNA expression with regard to the genetic APOBEC1 dimorphism of patients with intractable temporal lobe epilepsy (iTLE) and found that the patients fall into two groups. Patients with expression of the Apobec-1 80I variant mostly suffered from simple or complex partial seizures, whereas patients with 80M expression exhibited secondarily generalized seizure activity. Thus, our method allows the characterization of Apobec-1 80M and 80l variants in the brain and provides a new way to epidemiologically and semiologically classify iTLE according to the two different APOBEC1 alleles. Together, these results demonstrate Apobec-1-dependent expression of RNA-edited GlyR protein in neurons and identify the APOBEC1 80I/M-coding alleles as new genetic risk factors for iTLE patients.

Expression Patterns of Extracellular Matrix Proteins during Posterior Commissure Development.

Extracellular matrix (ECM) molecules are pivotal for central nervous system (CNS) development, facilitating cell migration, axonal growth, myelination, dendritic spine formation, and synaptic plasticity, among other processes. During axon guidance, the ECM not only acts as a permissive or non-permissive substrate for navigating axons, but also modulates the effects of classical guidance cues, such as netrin or Eph/ephrin family members. Despite being highly important, little is known about the expression of ECM molecules during CNS development. Therefore, this study assessed the molecular expression patterns of tenascin, HNK-1, laminin, fibronectin, perlecan, decorin, and osteopontin along chick embryo prosomere 1 during posterior commissure development. The posterior commissure is the first transversal axonal tract of the embryonic vertebrate brain. Located in the dorso-caudal portion of prosomere 1, posterior commissure axons primarily arise from the neurons of basal pretectal nuclei that run dorsally to the roof plate midline, where some turn toward the ipsilateral side. Expressional analysis of ECM molecules in this area these revealed to be highly arranged, and molecule interactions with axon fascicles suggested involvement in processes other than structural support. In particular, tenascin and the HNK-1 epitope extended in ventro-dorsal columns and enclosed axons during navigation to the roof plate. Laminin and osteopontin were expressed in the midline, very close to axons that at this point must decide between extending to the contralateral side or turning to the ipsilateral side. Finally, fibronectin, decorin, and perlecan appeared unrelated to axonal pathfinding in this region and were instead restricted to the external limiting membrane. In summary, the present report provides evidence for an intricate expression of different extracellular molecules that may cooperate in guiding posterior commissure axons.

Potentiation of Gamma Aminobutyric Acid Receptors (GABAAR) by Ethanol: How Are Inhibitory Receptors Affected?

In recent years there has been an increase in the understanding of ethanol actions on the type A γ-aminobutyric acid chloride channel (GABAAR), a member of the pentameric ligand gated ion channels (pLGICs). However, the mechanism by which ethanol potentiates the complex is still not fully understood and a number of publications have shown contradictory results. Thus many questions still remain unresolved requiring further studies for a better comprehension of this effect. The present review concentrates on the involvement of GABAAR in the acute actions of ethanol and specifically focuses on the immediate, direct or indirect, synaptic and extra-synaptic modulatory effects. To elaborate on the immediate, direct modulation of GABAAR by acute ethanol exposure, electrophysiological studies investigating the importance of different subunits, and data from receptor mutants will be examined. We will also discuss the nature of the putative binding sites for ethanol based on structural data obtained from other members of the pLGICs family. Finally, we will briefly highlight the glycine gated chloride channel (GlyR), another member of the pLGIC family, as a suitable target for the development of new pharmacological tools.

Intracellular glycine receptor function facilitates glioma formation in vivo.

The neuronal function of Cys-loop neurotransmitter receptors is established; however, their role in non-neuronal cells is poorly defined. As brain tumors are enriched in the neurotransmitter glycine, we studied the expression and function of glycine receptors (GlyRs) in glioma cells. Human brain tumor biopsies selectively expressed the GlyR α1 and α3 subunits, which have nuclear localization signals (NLSs). The mouse glioma cell line GL261 expressed GlyR α1, and knockdown of GlyR α1 protein expression impaired the self-renewal capacity and tumorigenicity of GL261 glioma cells, as shown by a neurosphere assay and GL261 cell inoculation in vivo, respectively. We furthermore showed that the pronounced tumorigenic effect of GlyR α1 relies on a new intracellular signaling function that depends on the NLS region in the large cytosolic loop and impacts on GL261 glioma cell gene regulation. Stable expression of GlyR α1 and α3 loops rescued the self-renewal capacity of GlyR α1 knockdown cells, which demonstrates their functional equivalence. The new intracellular signaling function identified here goes beyond the well-established role of GlyRs as neuronal ligand-gated ion channels and defines NLS-containing GlyRs as new potential targets for brain tumor therapies.

Direct binding of GABAA receptor ß2 and ß3 subunits to gephyrin.

GABAergic transmission is essential to brain function, and a large repertoire of GABA type A receptor (GABA(A) R) subunits is at a neuron's disposition to serve this function. The glycine receptor (GlyR)-associated protein gephyrin has been shown to be essential for the clustering of a subset of GABA(A) R. Despite recent progress in the field of gephyrin-dependent mechanisms of postsynaptic GABA(A) R stabilisation, the role of gephyrin in synaptic GABA(A) R localisation has remained a complex matter with many open questions. Here, we analysed comparatively the interaction of purified rat gephyrin and mouse brain gephyrin with the large cytoplasmic loops of GABA(A) R α1, α2, ß2 and ß3 subunits. Binding affinities were determined using surface plasmon resonance spectroscopy, and showed an ~ 20-fold lower affinity of the ß2 loop to gephyrin as compared to the GlyR ß loop-gephyrin interaction. We also probed in vivo binding in primary cortical neurons by the well-established use of chimaeras of GlyR α1 that harbour respective gephyrin-binding motifs derived from the different GABA(A) R subunits. These studies identify a novel gephyrin-binding motif in GABA(A) R ß2 and ß3 large cytoplasmic loops.

Irregular RNA splicing curtails postsynaptic gephyrin in the cornu ammonis of patients with epilepsy.

Anomalous hippocampal inhibition is involved in temporal lobe epilepsy, and reduced gephyrin immunoreactivity in the temporal lobe epilepsy hippocampus has been reported recently. However, the mechanisms responsible for curtailing postsynaptic gephyrin scaffolds are poorly understood. Here, we have investigated gephyrin expression in the hippocampus of patients with intractable temporal lobe epilepsy. Immunohistochemical and western blot analyses revealed irregular gephyrin expression in the cornu ammonis of patients with temporal lobe epilepsy and four abnormally spliced gephyrins lacking several exons in their G-domains were isolated. Identified temporal lobe epilepsy gephyrins have oligomerization deficits and they curtail hippocampal postsynaptic gephyrin and GABA(A) receptor α2 while interacting with regularly spliced gephyrins. We found that cellular stress (alkalosis and hyperthermia) induces exon skipping in gephyrin messenger RNA, which is responsible for curtailed postsynaptic gephyrin and GABA(A) receptor α2 scaffolds. Accordingly, we did not obtain evidence for gephyrin gene mutations in patients with temporal lobe epilepsy. Cellular stress such as alkalosis, for example arising from seizure activity, could thus facilitate the development of temporal lobe epilepsy by reducing GABA(A) receptor α2-mediated hippocampal synaptic transmission selectively in the cornu ammonis.

Glycine Receptors Caught between Genome and Proteome - Functional Implications of RNA Editing and Splicing.

Information processing in the brain requires a delicate balance between excitation and inhibition. Glycine receptors (GlyR) are involved in inhibitory mechanisms mainly at a synaptic level, but potential novel roles for these receptors recently emerged due to the discovery of posttranscriptional processing. GLR transcripts are edited through enzymatic modification of a single nucleotide leading to amino acid substitution within the neurotransmitter binding domain. RNA editing produces gain-of-function receptors well suited for generation and maintenance of tonic inhibition of neuronal excitability. As neuronal activity deprivation in early stages of development or in epileptic tissue is detrimental to neurons and because RNA editing of GlyR is up-regulated in temporal lobe epilepsy patients with a severe course of disease a pathophysiological role of these receptors emerges. This review contains a state-of-the-art discussion of (patho)physiological implications of GlyR RNA editing.

Splice-specific roles of glycine receptor alpha3 in the hippocampus.

Glycine receptor (GlyR) alpha3 is involved in vision, and processing of acoustic and nociceptive signals, and RNA editing of GLRA3 transcripts was associated with hippocampal pathophysiology of mesial temporal lobe epilepsy (TLE). However, neither the role of GlyR alpha3 splicing in hippocampal neurons nor the expression of splice variants have yet been elucidated. We report here that the long (L) splice variant of GlyR alpha3 predominates in the brain of rodents. Cellular analysis using primary hippocampal neurons and hippocampus cryosections revealed preferential association of synaptic alpha3L clusters with glutamatergic nerve endings in strata granulare and pyramidale. In primary hippocampal neurons GlyR alpha3L clusters also preferred glutamatergic nerve endings while alpha3K was mainly in a diffuse state. Co-expression of GlyR beta subunit with alpha3L or alpha3K produced heteromeric receptor clusters and favoured their association with GABAergic terminals. However, heteromeric alpha3L was still more efficient than heteromeric alpha3K in associating with glutamatergic nerve endings. To give physiological relevance to these results we have finally analysed GlyR alpha3 splicing in human hippocampus obtained from patients with intractable TLE. As up-regulation of alpha3K occurred at the expense of alpha3L in TLE patients with a severe course of disease and a high degree of hippocampal damage, our results again involve post-transcriptional processing of GLRA3 transcripts in the pathophysiology of TLE.

Translational regulation of the human achaete-scute homologue-1 by fragile X mental retardation protein.

Fragile X syndrome is a common inherited cause of mental retardation that results from loss or mutation of the fragile X mental retardation protein (FMRP). In this study, we identified the mRNA of the basic helix-loop-helix transcription factor human achaete-scute homologue-1 (hASH1 or ASCL1), which is required for normal development of the nervous system and has been implicated in the formation of neuroendocrine tumors, as a new FMRP target. Using a double-immunofluorescent staining technique we detected an overlapping pattern of both proteins in the hippocampus, temporal cortex, subventricular zone, and cerebellum of newborn rats. Forced expression of FMRP and gene silencing by small interference RNA transfection revealed a positive correlation between the cellular protein levels of FMRP and hASH1. A luciferase reporter construct containing the 5'-untranslated region of hASH1 mRNA was activated by the full-length FMRP, but not by naturally occurring truncated FMR proteins, in transient co-transfections. The responsible cis-element was mapped by UV-cross-linking experiments and reporter mutagenesis assays to a (U)(10) sequence located in the 5'-untranslated region of the hASH1 mRNA. Sucrose density gradient centrifugation revealed that hASH1 transcripts were translocated into a translationally active polysomal fraction upon transient transfection of HEK293 cells with FMRP, thus indicating translational activation of hASH1 mRNA. In conclusion, we identified hASH1 as a novel downstream target of FMRP. Improved translation efficiency of hASH1 mRNA by FMRP may represent an important regulatory switch in neuronal differentiation.