Adipo-Clear: A Tissue Clearing Method for Three-Dimensional Imaging of Adipose Tissue

Adipose tissue plays a central role in energy homeostasis and thermoregulation. It is composed of different types of adipocytes, as well as adipocyte ...

The Rockefeller University

Axon degeneration is widespread both in neurodegenerative disease and in normal neural development, but the molecular pathways regulating these degenerative processes and the extent to which they are distinct or overlapping remain incompletely understood. We report that calpastatin, an inhibitor of calcium-activated proteases of the calpain family, functions as a key endogenous regulator of axon degeneration. Calpastatin depletion was observed in degenerating axons after physical injury, and maintaining calpastatin inhibited degeneration of transected axons in vitro and in the optic nerve in vivo. Calpastatin depletion also occurred in a caspase-dependent manner in trophic factor-deprived sensory axons and was required for this in vitro model of developmental degeneration. In vivo, calpastatin regulated the normal pruning of retinal ganglion cell axons in their target field. These findings identify calpastatin as a key checkpoint for axonal survival after injury and during development, and demonstrate downstream convergence of these distinct pathways of axon degeneration. 

Regulation of axon degeneration after injury and in development by the endogenous calpain inhibitor calpastatin.

In the developing brain, initial neuronal projections are formed through extensive growth and branching of developing axons, but many branches are later pruned to sculpt the mature pattern of connections. Despite its widespread occurrence, the mechanisms controlling pruning remain incompletely characterized. Based on pharmacological and biochemical analysis in vitro and initial genetic analysis in vivo, prior studies implicated a pathway involving binding of the Amyloid Precursor Protein (APP) to Death Receptor 6 (DR6) and activation of a downstream caspase cascade in axonal pruning. Here, we further test their involvement in pruning in vivo and their mechanism of action through extensive genetic and biochemical analysis. Genetic deletion of DR6 was previously shown to impair pruning of retinal axons in vivo. We show that genetic deletion of APP similarly impairs pruning of retinal axons in vivo and provide evidence that APP and DR6 act cell autonomously and in the same pathway to control pruning. Prior analysis had suggested that β-secretase cleavage of APP and binding of an N-terminal fragment of APP to DR6 is required for their actions, but further genetic and biochemical analysis reveals that β-secretase activity is not required and that high-affinity binding to DR6 requires a more C-terminal portion of the APP ectodomain. These results provide direct support for the model that APP and DR6 function cell autonomously and in the same pathway to control pruning in vivo and raise the possibility of alternate mechanisms for how APP and DR6 control pruning. 

Genetic analysis reveals that amyloid precursor protein and death receptor 6 function in the same pathway to control axonal pruning independent of β-secretase.

Netrins are secreted proteins that regulate axon guidance and neuronal migration. Deleted in colorectal cancer (DCC) is a well-established netrin-1 receptor mediating attractive responses. We provide evidence that its close relative neogenin is also a functional netrin-1 receptor that acts with DCC to mediate guidance in vivo. We determined the structures of a functional netrin-1 region, alone and in complexes with neogenin or DCC. Netrin-1 has a rigid elongated structure containing two receptor-binding sites at opposite ends through which it brings together receptor molecules. The ligand/receptor complexes reveal two distinct architectures: a 2:2 heterotetramer and a continuous ligand/receptor assembly. The differences result from different lengths of the linker connecting receptor domains fibronectin type III domain 4 (FN4) and FN5, which differs among DCC and neogenin splice variants, providing a basis for diverse signaling outcomes. 

Neural migration. Structures of netrin-1 bound to two receptors provide insight into its axon guidance mechanism.

The visualization of molecularly labeled structures within large intact tissues in three dimensions is an area of intense focus. We describe a simple, rapid, and inexpensive method, iDISCO, that permits whole-mount immunolabeling with volume imaging of large cleared samples ranging from perinatal mouse embryos to adult organs, such as brains or kidneys. iDISCO is modeled on classical histology techniques, facilitating translation of section staining assays to intact tissues, as evidenced by compatibility with 28 antibodies to both endogenous antigens and transgenic reporters like GFP. When applied to degenerating neurons, iDISCO revealed unexpected variability in number of apoptotic neurons within individual sensory ganglia despite tight control of total number in all ganglia. It also permitted imaging of single degenerating axons in adult brain and the first visualization of cleaved Caspase-3 in degenerating embryonic sensory axons in vivo, even single axons. iDISCO enables facile volume imaging of immunolabeled structures in complex tissues. PAPERCLIP:

iDISCO: a simple, rapid method to immunolabel large tissue samples for volume imaging.

Development of neuronal circuits is controlled by evolutionarily conserved axon guidance molecules, including Slits, the repulsive ligands for roundabout (Robo) receptors, and Netrin-1, which mediates attraction through the DCC receptor. We discovered that the Robo3 receptor fundamentally changed its mechanism of action during mammalian evolution. Unlike other Robo receptors, mammalian Robo3 is not a high-affinity receptor for Slits because of specific substitutions in the first immunoglobulin domain. Instead, Netrin-1 selectively triggers phosphorylation of mammalian Robo3 via Src kinases. Robo3 does not bind Netrin-1 directly but interacts with DCC. Netrin-1 fails to attract pontine neurons lacking Robo3, and attraction can be restored in Robo3(-/-) mice by expression of mammalian, but not nonmammalian, Robo3. We propose that Robo3 evolution was key to sculpting the mammalian brain by converting a receptor for Slit repulsion into one that both silences Slit repulsion and potentiates Netrin attraction. 

Signaling switch of the axon guidance receptor Robo3 during vertebrate evolution.

Axonal death disrupts functional connectivity of neural circuits and is a critical feature of many neurodegenerative disorders. Pathological axon degeneration often occurs independently of known programmed death pathways, but the underlying molecular mechanisms remain largely unknown. Using traumatic injury as a model, we systematically investigate mitogen-activated protein kinase (MAPK) families and delineate a MAPK cascade that represents the early degenerative response to axonal injury. The adaptor protein Sarm1 is required for activation of this MAPK cascade, and this Sarm1-MAPK pathway disrupts axonal energy homeostasis, leading to ATP depletion before physical breakdown of damaged axons. The protective cytoNmnat1/Wld(s) protein inhibits activation of this MAPK cascade. Further, MKK4, a key component in the Sarm1-MAPK pathway, is antagonized by AKT signaling, which modulates the degenerative response by limiting activation of downstream JNK signaling. Our results reveal a regulatory mechanism that integrates distinct signals to instruct pathological axon degeneration. 

Pathological axonal death through a MAPK cascade that triggers a local energy deficit.

Commissural axon guidance depends on a myriad of cues expressed by intermediate targets. Secreted semaphorins signal through neuropilin-2/plexin-A1 receptor complexes on post-crossing commissural axons to mediate floor plate repulsion in the mouse spinal cord. Here, we show that neuropilin-2/plexin-A1 are also coexpressed on commissural axons prior to midline crossing and can mediate precrossing semaphorin-induced repulsion in vitro. How premature semaphorin-induced repulsion of precrossing axons is suppressed in vivo is not known. We discovered that a novel source of floor plate-derived, but not axon-derived, neuropilin-2 is required for precrossing axon pathfinding. Floor plate-specific deletion of neuropilin-2 significantly reduces the presence of precrossing axons in the ventral spinal cord, which can be rescued by inhibiting plexin-A1 signaling in vivo. Our results show that floor plate-derived neuropilin-2 is developmentally regulated, functioning as a molecular sink to sequester semaphorins, preventing premature repulsion of precrossing axons prior to subsequent down-regulation, and allowing for semaphorin-mediated repulsion of post-crossing axons. 

Floor plate-derived neuropilin-2 functions as a secreted semaphorin sink to facilitate commissural axon midline crossing.

Actin, spectrin, and associated molecules form a periodic, submembrane cytoskeleton in the axons of neurons. For a better understanding of this membrane-associated periodic skeleton (MPS), it is important to address how prevalent this structure is in different neuronal types, different subcellular compartments, and across different animal species. Here, we investigated the organization of spectrin in a variety of neuronal- and glial-cell types. We observed the presence of MPS in all of the tested neuronal types cultured from mouse central and peripheral nervous systems, including excitatory and inhibitory neurons from several brain regions, as well as sensory and motor neurons. Quantitative analyses show that MPS is preferentially formed in axons in all neuronal types tested here: Spectrin shows a long-range, periodic distribution throughout all axons but appears periodic only in a small fraction of dendrites, typically in the form of isolated patches in subregions of these dendrites. As in dendrites, we also observed patches of periodic spectrin structures in a small fraction of glial-cell processes in four types of glial cells cultured from rodent tissues. Interestingly, despite its strong presence in the axonal shaft, MPS is disrupted in most presynaptic boutons but is present in an appreciable fraction of dendritic spine necks, including some projecting from dendrites where such a periodic structure is not observed in the shaft. Finally, we found that spectrin is capable of adopting a similar periodic organization in neurons of a variety of animal species, including Caenorhabditis elegans, Drosophila, Gallus gallus, Mus musculus, and Homo sapiens.

Prevalent presence of periodic actin-spectrin-based membrane skeleton in a broad range of neuronal cell types and animal species.

Understanding how neural information is processed in physiological and pathological states would benefit from precise detection, localization, and quantification of the activity of all neurons across the entire brain, which has not, to date, been achieved in the mammalian brain. We introduce a pipeline for high-speed acquisition of brain activity at cellular resolution through profiling immediate early gene expression using immunostaining and light-sheet fluorescence imaging, followed by automated mapping and analysis of activity by an open-source software program we term ClearMap. We validate the pipeline first by analysis of brain regions activated in response to haloperidol. Next, we report new cortical regions downstream of whisker-evoked sensory processing during active exploration. Last, we combine activity mapping with axon tracing to uncover new brain regions differentially activated during parenting behavior. This pipeline is widely applicable to different experimental paradigms, including animal species for which transgenic activity reporters are not readily available. 

Mapping of Brain Activity by Automated Volume Analysis of Immediate Early Genes.

The brain is highly sensitive to proper fuel availability as evidenced by the rapid decline in neuronal function during ischemic attacks and acute severe hypoglycemia. We previously showed that sustained presynaptic function requires activity-driven glycolysis. Here, we provide strong evidence that during action potential (AP) firing, nerve terminals rely on the glucose transporter GLUT4 as a glycolytic regulatory system to meet the activity-driven increase in energy demands. Activity at synapses triggers insertion of GLUT4 into the axonal plasma membrane driven by activation of the metabolic sensor AMP kinase. Furthermore, we show that genetic ablation of GLUT4 leads to an arrest of synaptic vesicle recycling during sustained AP firing, similar to what is observed during acute glucose deprivation. The reliance on this biochemical regulatory system for "exercising" synapses is reminiscent of that occurring in exercising muscle to sustain cellular function and identifies nerve terminals as critical sites of proper metabolic control.

GLUT4 Mobilization Supports Energetic Demands of Active Synapses.

Considerable progress has been made in converting human pluripotent stem cells (hPSCs) into functional neurons. However, the protracted timing of human neuron specification and functional maturation remains a key challenge that hampers the routine application of hPSC-derived lineages in disease modeling and regenerative medicine. Using a combinatorial small-molecule screen, we previously identified conditions to rapidly differentiate hPSCs into peripheral sensory neurons. Here we generalize the approach to central nervous system (CNS) fates by developing a small-molecule approach for accelerated induction of early-born cortical neurons. Combinatorial application of six pathway inhibitors induces post-mitotic cortical neurons with functional electrophysiological properties by day 16 of differentiation, in the absence of glial cell co-culture. The resulting neurons, transplanted at 8 d of differentiation into the postnatal mouse cortex, are functional and establish long-distance projections, as shown using iDISCO whole-brain imaging. Accelerated differentiation into cortical neuron fates should facilitate hPSC-based strategies for disease modeling and cell therapy in CNS disorders.

Combined small-molecule inhibition accelerates the derivation of functional cortical neurons from human pluripotent stem cells.

While the cell-intrinsic pathways governing beige adipocyte development and phenotype have been increasingly delineated, comparatively little is known about how beige adipocytes interact with other cell types in fat. Here, we introduce a whole-tissue clearing method for adipose that permits immunolabeling and three-dimensional profiling of structures including thermogenic adipocytes and sympathetic innervation. We found that tissue architecture and sympathetic innervation differ significantly between subcutaneous and visceral depots. Subcutaneous fat demonstrates prominent regional variation in beige fat biogenesis with localization of UCP1 beige adipocytes to areas with dense sympathetic neurites. We present evidence that the density of sympathetic projections is dependent on PRDM16 in adipocytes, providing another potential mechanism underlying the metabolic benefits mediated by PRDM16. This powerful imaging tool highlights the interaction of tissue components during beige fat biogenesis and reveals a previously undescribed mode of regulation of the sympathetic nervous system by adipocytes.

Three-Dimensional Adipose Tissue Imaging Reveals Regional Variation in Beige Fat Biogenesis and PRDM16-Dependent Sympathetic Neurite Density.

During brainstem development, newborn neurons originating from the rhombic lip embark on exceptionally long migrations to generate nuclei important for audition, movement, and respiration. Along the way, this highly motile population passes several cranial nerves yet remains confined to the CNS. We found that Ntn1 accumulates beneath the pial surface separating the CNS from the PNS, with gaps at nerve entry sites. In mice null for Ntn1 or its receptor DCC, hindbrain neurons enter cranial nerves and migrate into the periphery. CNS neurons also escape when Ntn1 is selectively lost from the sub-pial region (SPR), and conversely, expression of Ntn1 throughout the mutant hindbrain can prevent their departure. These findings identify a permissive role for Ntn1 in maintaining the CNS-PNS boundary. We propose that Ntn1 confines rhombic lip-derived neurons by providing a preferred substrate for tangentially migrating neurons in the SPR, preventing their entry into nerve roots.

Netrin-1 Confines Rhombic Lip-Derived Neurons to the CNS.

Recent studies have identified a specialized subset of CD31endomucin (CD31EMCN) vascular endothelium that positively regulates bone formation. However, it remains unclear how CD31EMCN endothelium levels are coupled to anabolic bone formation. Mice with an osteoblast-specific deletion of Shn3, which have markedly elevated bone formation, demonstrated an increase in CD31EMCN endothelium. Transcriptomic analysis identified SLIT3 as an osteoblast-derived, SHN3-regulated proangiogenic factor. Genetic deletion of Slit3 reduced skeletal CD31EMCN endothelium, resulted in low bone mass because of impaired bone formation and partially reversed the high bone mass phenotype of Shn3 mice. This coupling between osteoblasts and CD31EMCN endothelium is essential for bone healing, as shown by defective fracture repair in SLIT3-mutant mice and enhanced fracture repair in SHN3-mutant mice. Finally, administration of recombinant SLIT3 both enhanced bone fracture healing and counteracted bone loss in a mouse model of postmenopausal osteoporosis. Thus, drugs that target the SLIT3 pathway may represent a new approach for vascular-targeted osteoanabolic therapy to treat bone loss.

Targeting skeletal endothelium to ameliorate bone loss.

CNS cortical histogenesis depends on polarity signaling pathways that regulate cell adhesion and motility. Here we report that conditional deletion of the Rho GTPase  in cerebellar granule cell precursors (GCPs) results in abnormalities in cerebellar foliation revealed by iDISCO clearing methodology, a loss of columnar organization of proliferating GCPs in the external germinal layer (EGL), disordered parallel fiber organization in the molecular layer (ML), and a failure to extend a leading process and form a neuron-glial junction during migration along Bergmann glia (BG). Notably, GCPs lacking  had a multi-polar morphology and slowed migration rate. In addition, secondary defects occurred in BG development and organization, especially in the lateral cerebellar hemispheres. By phosphoproteomic analysis, affected Cdc42 targets included regulators of the cytoskeleton, cell adhesion and polarity. Thus, Cdc42 signaling pathways are critical regulators of GCP polarity and the formation of neuron-glial junctions during cerebellar development.

Zhuhao Wu

Laboratory of Brain Development and Repair,
Laboratory of Molecular Genetics

The Rockefeller University

Adipo-Clear: A Tissue Clearing Method for Three-Dimensional Imaging of Adipose Tissue

Zhuhao Wu

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}Laboratory of Brain Development and Repair,Laboratory of Molecular Genetics

The Rockefeller University

Adipo-Clear: A Tissue Clearing Method for Three-Dimensional Imaging of Adipose Tissue

Laboratory of Brain Development and Repair,
Laboratory of Molecular Genetics