Midline Assembloids Reveal Regulators of Human Axon Guidance.

Organizers are specialized cell populations that orchestrate cell patterning and axon guidance in the developing nervous system. Although non-human models have led to fundamental discoveries about the organization of the nervous system midline by the floor plate, an experimental model of human floor plate would enable broader insights into regulation of human neurodevelopment and midline connectivity. Here, we have developed stem cell-derived organoids resembling human floor plate (hFpO) and assembled them with spinal cord organoids (hSpO) to generate midline assembloids (hMA). We demonstrate that hFpO promote Sonic hedgehog-dependent ventral patterning of human spinal progenitors and Netrin-dependent guidance of human commissural axons, paralleling non-human models. To investigate evolutionary-divergent midline regulators, we profiled the hFpO secretome and identified 27 evolutionarily divergent genes between human and mouse. Utilizing the hMA platform, we targeted these candidates in an arrayed CRISPR knockout screen and reveal that GALNT2 , a gene involved in O-linked glycosylation, impairs floor plate-mediated guidance of commissural axons in humans. This novel platform extends prior axon guidance discoveries into human-specific neurobiology with implications for mechanisms of nervous system evolution and neurodevelopmental disorders.

Blockage of MLKL prevents myelin damage in experimental diabetic neuropathy.

SignificanceDiabetic neuropathy is a commonly occurring complication of diabetes that affects hundreds of millions of patients worldwide. Patients suffering from diabetic neuropathy experience abnormal sensations and have damage in their peripheral nerve axons as well as myelin, a tightly packed Schwann cell sheath that wraps around axons to provide insulation and increases electrical conductivity along the nerve fibers. The molecular events underlying myelin damage in diabetic neuropathy are largely unknown, and there is no efficacious treatment for the disease. The current study, using a diabetic mouse model and human patient nerve samples, uncovered a molecular mechanism underlying myelin sheath damage in diabetic neuropathy and provides a potential treatment strategy for the disease.

Flotillin-mediated endocytosis and ALIX-syntenin-1-mediated exocytosis protect the cell membrane from damage caused by necroptosis.

Necroptosis is a form of regulated necrosis that is implicated in various human diseases including Alzheimer's disease. Necroptosis requires the translocation of the pseudokinase MLKL from the cytosol to the plasma membrane after its phosphorylation by the kinase RIPK3. Using protein cross-linking followed by affinity purification, we detected the lipid raft-associated proteins flotillin-1 and flotillin-2 and the ESCRT-associated proteins ALIX and syntenin-1 in membrane-localized MLKL immunoprecipitates. Phosphorylated MLKL was removed from membranes through either flotillin-mediated endocytosis followed by lysosomal degradation or ALIX-syntenin-1-mediated exocytosis. Thus, cells undergoing necroptosis need to overcome these independent suppressive mechanisms before plasma membrane disruption can occur.

RIP1 kinase inhibitor halts the progression of an immune-induced demyelination disease at the stage of monocyte elevation.

Demyelination in the central nervous system (CNS) underlies many human diseases, including multiple sclerosis (MS). We report here the findings of our study of the CNS demyelination process using immune-induced [experimental autoimmune encephalomyelitis (EAE)] and chemical-induced [cuprizone (CPZ)] mouse models of demyelination. We found that necroptosis, a receptor-interacting protein 3 (RIP3) kinase and its substrate mixed lineage kinase domain-like protein (MLKL)-dependent cell death program, played no role in the demyelination process, whereas the MLKL-dependent, RIP3-independent function of MLKL in the demyelination process initially discovered in the peripheral nervous system in response to nerve injury, also functions in demyelination in the CNS in these models. Moreover, a receptor-interacting protein 1 (RIP1) kinase inhibitor, RIPA-56, blocked disease progression in the EAE-induced model but showed no effect in the CPZ-induced model. It does so most likely at a step of monocyte elevation downstream of T cell activation and myelin-specific antibody generation, although upstream of breakdown of the blood-brain barrier. RIP1-kinase dead knock-in mice shared a similar result as mice treated with the RIP1 inhibitor. These results indicate that RIP1 kinase inhibitor is a potential therapeutic agent for immune-mediated demyelination diseases that works by prevention of monocyte elevation, a function previously unknown for RIP1 kinase.

Mixed Lineage Kinase Domain-like Protein MLKL Breaks Down Myelin following Nerve Injury.

Successful regeneration of severed peripheral nerves requires the breakdown and subsequent clearance of myelin, tightly packed membrane sheaths of Schwann cells that protect nerve fibers and harbor nerve growth-inhibitory proteins. How Schwann cells initiate myelin breakdown in response to injury is still largely unknown. Here we report that, following sciatic nerve injury, MLKL, a pseudokinase known to rupture cell membranes during necroptotic cell death, is induced and targets the myelin sheath membrane of Schwann cells to promote myelin breakdown. The function of MLKL in disrupting myelin sheaths requires injury-induced phosphorylation of serine 441, an activation signal distinct from the necroptosis-inducing phosphorylation by RIP3 kinase. Mice with Mlkl specifically knocked out in Schwann cells showed delayed myelin sheath breakdown. Lack of MLKL reduced nerve regeneration following injury, whereas overexpression of MLKL accelerated myelin breakdown and promoted the regeneration of axons.

Discovery of Highly Potent 2-Sulfonyl-Pyrimidinyl Derivatives for Apoptosis Inhibition and Ischemia Treatment.

A series of 2-sulfonyl-pyrimidinyl derivatives was developed as apoptosis inhibitors. These represent the first class of apoptosis inhibitors that function through stabilizing mitochondrial respiratory complex II. Starting from a phenotypic screen hit with micromolar activity, we optimized the cellular apoptosis inhibition activity of 2-sulfonyl-pyrimidinyl derivatives to picomolar level (compound 42, also named as TC9-305). The therapeutic potential of these new apoptosis inhibitors was further demonstrated by their neuroprotective effect on an ischemic animal model.

A Small Molecule That Protects the Integrity of the Electron Transfer Chain Blocks the Mitochondrial Apoptotic Pathway.

In response to apoptotic stimuli, mitochondria in mammalian cells release cytochrome c and other apoptogenic proteins, leading to the subsequent activation of caspases and apoptotic cell death. This process is promoted by the pro-apoptotic members of the Bcl-2 family of proteins, such as Bim and Bax, which, respectively, initiate and execute cytochrome c release from the mitochondria. Here we report the discovery of a small molecule that efficiently blocks Bim-induced apoptosis after Bax is activated on the mitochondria. The cellular target of this small molecule was identified to be the succinate dehydrogenase subunit B (SDHB) protein of complex II of the mitochondrial electron transfer chain (ETC). The molecule protects the integrity of the ETC and allows treated cells to continue to proliferate after apoptosis induction. Moreover, this molecule blocked dopaminergic neuron death and reversed Parkinson-like behavior in a rat model of Parkinson's disease.

Broadening of cortical inhibition mediates developmental sharpening of orientation selectivity.

Orientation selectivity (OS) of visual cortical neurons is progressively sharpened during development. However, synaptic circuit mechanisms underlying the OS sharpening remain unclear. In the current study, in vivo whole-cell voltage-clamp recordings from layer 4 excitatory neurons in the developing mouse primary visual cortex revealed changes of orientation tuning profiles of their excitatory and inhibitory inputs during a post-eye-opening period when OS of their spiking responses becomes sharpened. In addition to a parallel strengthening of excitation and inhibition during this developmental period, the orientation tuning of excitatory inputs keeps relatively constant, whereas the tuning of inhibitory inputs is broadened, and becomes significantly broader than that of excitatory inputs. Neuron modeling and dynamic-clamp recording demonstrated that this developmental broadening of the inhibitory tuning is sufficient for sharpening OS. Depriving visual experience by dark rearing impedes the normal developmental strengthening of excitation, but a similar broadening of inhibitory tuning, likely caused by a nonselective strengthening of inhibitory connections, results in the apparently normal OS sharpening in excitatory neurons. Our results thus provide the first demonstration that an inhibitory synaptic mechanism can primarily mediate the functional refinement of cortical neurons.

Broad inhibition sharpens orientation selectivity by expanding input dynamic range in mouse simple cells.

Orientation selectivity (OS) is an emergent property in the primary visual cortex (V1). How OS arises from synaptic circuits remains unsolved. Here, in vivo whole-cell recordings in the mouse V1 revealed that simple cells received broadly tuned excitation and even more broadly tuned inhibition. Excitation and inhibition shared a similar orientation preference and temporally overlapped substantially. Neuron modeling and dynamic-clamp recording further revealed that excitatory inputs alone would result in membrane potential responses with significantly attenuated selectivity, due to a saturating input-output function of the membrane filtering. Inhibition ameliorated the attenuation of excitatory selectivity by expanding the input dynamic range and caused additional sharpening of output responses beyond unselectively suppressing responses at all orientations. This "blur-sharpening" effect allows selectivity conveyed by excitatory inputs to be better expressed, which may be a general mechanism underlying the generation of feature-selective responses in the face of strong excitatory inputs that are weakly biased.

Brain natriuretic peptide modulates the delayed rectifier outward K(+) current and promotes the proliferation of mouse Schwann cells.

Brain natriuretic peptide (BNP) may act as a neuromodulator via its associated receptors (natriuretic peptide receptors, NPRs) in the central nervous system (CNS), but few studies have reported its activity in the peripheral nervous system (PNS). In this study, we observed that BNP increased the tetraethylammonium chloride (TEA)-sensitive delayed rectifier outward potassium current (I(K)) in mouse Schwann cells (SCs) using whole-cell recording techniques. At concentrations of 1-100 nM, BNP reversibly activated I(K) in a dose-dependent manner, with modulating its steady-state activation and inactivation properties. The effect of BNP on I(K) was abolished by preincubation with the specific antagonist of NPR-A, and could not be mimicked by application of NPR-C agonist. These results were supported by immunocytochemical findings indicating that NPR-A was expressed in SCs. The application of 8-Br-guanosine 3',5'-monophosphate (8-Br-cGMP) mimicked the effect of BNP on I(K), but BNP was unable to further increase I(K) after the application of cyclic guanosine monophosphate (cGMP). Genistein blocked I(K) and also completely eliminated the effects of BNP and cGMP on I(K). The selective K(V)2.1 subunit blocker, Jingzhaotoxin-III (JZTX-III), reduced I(K) amplitude by 30%, but did not abolish the increase effect of BNP on I(K) amplitude. In addition, BNP significantly stimulated SCs proliferation and this effect could be partly inhibited by TEA. Together these results suggest that BNP modulated I(K) probably via cGMP- and tyrosine kinase-dependent pathways by activation of NPR-A. This effect of BNP on I(K) in SCs might partly explain its effect on cell proliferation.