ARID1B controls transcriptional programs of axon projection in an organoid model of the human corpus callosum.

Mutations in ARID1B, a member of the mSWI/SNF complex, cause severe neurodevelopmental phenotypes with elusive mechanisms in humans. The most common structural abnormality in the brain of ARID1B patients is agenesis of the corpus callosum (ACC), characterized by the absence of an interhemispheric white matter tract that connects distant cortical regions. Here, we find that neurons expressing SATB2, a determinant of callosal projection neuron (CPN) identity, show impaired maturation in ARID1B+/- neural organoids. Molecularly, a reduction in chromatin accessibility of genomic regions targeted by TCF-like, NFI-like, and ARID-like transcription factors drives the differential expression of genes required for corpus callosum (CC) development. Through an in vitro model of the CC tract, we demonstrate that this transcriptional dysregulation impairs the formation of long-range axonal projections, causing structural underconnectivity. Our study uncovers new functions of the mSWI/SNF during human corticogenesis, identifying cell-autonomous axonogenesis defects in SATB2+ neurons as a cause of ACC in ARID1B patients.

In vitro modeling of the human dopaminergic system using spatially arranged ventral midbrain-striatum-cortex assembloids.

Ventral midbrain dopaminergic neurons project to the striatum as well as the cortex and are involved in movement control and reward-related cognition. In Parkinson's disease, nigrostriatal midbrain dopaminergic neurons degenerate and cause typical Parkinson's disease motor-related impairments, while the dysfunction of mesocorticolimbic midbrain dopaminergic neurons is implicated in addiction and neuropsychiatric disorders. Study of the development and selective neurodegeneration of the human dopaminergic system, however, has been limited due to the lack of an appropriate model and access to human material. Here, we have developed a human in vitro model that recapitulates key aspects of dopaminergic innervation of the striatum and cortex. These spatially arranged ventral midbrain-striatum-cortical organoids (MISCOs) can be used to study dopaminergic neuron maturation, innervation and function with implications for cell therapy and addiction research. We detail protocols for growing ventral midbrain, striatal and cortical organoids and describe how they fuse in a linear manner when placed in custom embedding molds. We report the formation of functional long-range dopaminergic connections to striatal and cortical tissues in MISCOs, and show that injected, ventral midbrain-patterned progenitors can mature and innervate the tissue. Using these assembloids, we examine dopaminergic circuit perturbations and show that chronic cocaine treatment causes long-lasting morphological, functional and transcriptional changes that persist upon drug withdrawal. Thus, our method opens new avenues to investigate human dopaminergic cell transplantation and circuitry reconstruction as well as the effect of drugs on the human dopaminergic system.

Identification of a population of peripheral sensory neurons that regulates blood pressure.

The vasculature is innervated by a network of peripheral afferents that sense and regulate blood flow. Here, we describe a system of non-peptidergic sensory neurons with cell bodies in the spinal ganglia that regulate vascular tone in the distal arteries. We identify a population of mechanosensitive neurons, marked by tropomyosin receptor kinase C (TrkC) and tyrosine hydroxylase in the dorsal root ganglia, which projects to blood vessels. Local stimulation of TrkC neurons decreases vessel diameter and blood flow, whereas systemic activation increases systolic blood pressure and heart rate variability via the sympathetic nervous system. Ablation of the neurons provokes variability in local blood flow, leading to a reduction in systolic blood pressure, increased heart rate variability, and ultimately lethality within 48 h. Thus, a population of TrkC+ sensory neurons forms part of a sensory-feedback mechanism that maintains cardiovascular homeostasis through the autonomic nervous system.

Intraarticular Adeno-Associated Virus Serotype AAV-PHP.S-Mediated Chemogenetic Targeting of Knee-Innervating Dorsal Root Ganglion Neurons Alleviates Inflammatory Pain in Mice.

OBJECTIVE: Joint pain is the major clinical symptom of arthritis that affects millions of people. Controlling the excitability of knee-innervating dorsal root ganglion (DRG) neurons (knee neurons) could potentially provide pain relief. We undertook this study to evaluate whether the newly engineered adeno-associated virus (AAV) serotype, AAV-PHP.S, can deliver functional artificial receptors to control knee neuron excitability following intraarticular knee injection. METHODS: The AAV-PHP.S virus, packaged with dTomato fluorescent protein and either excitatory (Gq ) or inhibitory (Gi ) designer receptors exclusively activated by designer drugs (DREADDs), was injected into the knee joints of adult mice. Labeling of DRG neurons with AAV-PHP.S from the knee was evaluated using immunohistochemistry. The functionality of Gq - and Gi -DREADDs was evaluated using whole-cell patch clamp electrophysiology on acutely cultured DRG neurons. Pain behavior in mice was assessed using a digging assay, dynamic weight bearing, and rotarod performance, before and after intraperitoneal administration of the DREADD activator, Compound 21. RESULTS: We showed that AAV-PHP.S can deliver functional genes into ~7% of lumbar DRG neurons when injected into the knee joint in a similar manner to the well-established retrograde tracer, fast blue. Short-term activation of AAV-PHP.S-delivered Gq -DREADD increased excitability of knee neurons in vitro (P = 0.02 by unpaired t-test), without inducing overt pain in mice when activated in vivo. By contrast, in vivo Gi -DREADD activation alleviated digging deficits induced by Freund's complete adjuvant-mediated knee inflammation (P = 0.0002 by repeated-measures analysis of variance [ANOVA] followed by Holm-Sidak multiple comparisons test). A concomitant decrease in knee neuron excitability was observed in vitro (P = 0.005 by ANOVA followed by Holm-Sidak multiple comparisons test). CONCLUSION: We describe an AAV-mediated chemogenetic approach to specifically control joint pain, which may be utilized in translational arthritic pain research.

A ligand-based system for receptor-specific delivery of proteins.

Gene delivery using vector or viral-based methods is often limited by technical and safety barriers. A promising alternative that circumvents these shortcomings is the direct delivery of proteins into cells. Here we introduce a non-viral, ligand-mediated protein delivery system capable of selectively targeting primary skin cells in-vivo. Using orthologous self-labelling tags and chemical cross-linkers, we conjugate large proteins to ligands that bind their natural receptors on the surface of keratinocytes. Targeted CRE-mediated recombination was achieved by delivery of ligand cross-linked CRE protein to the skin of transgenic reporter mice, but was absent in mice lacking the ligand's cell surface receptor. We further show that ligands mediate the intracellular delivery of Cas9 allowing for CRISPR-mediated gene editing in the skin more efficiently than adeno-associated viral gene delivery. Thus, a ligand-based system enables the effective and receptor-specific delivery of large proteins and may be applied to the treatment of skin-related genetic diseases.

Nerve growth factor-mediated photoablation of nociceptors reduces pain behavior in mice.

Nerve growth factor (NGF) and its receptors TrkA and p75 play a key role in the development and function of peripheral nociceptive neurons. Here, we describe novel technology to selectively photoablate TrkA-positive nociceptors through delivery of a phototoxic agent coupled to an engineered NGF ligand and subsequent near-infrared illumination. We demonstrate that this approach allows for on demand and localized reversal of pain behaviors in mouse models of acute, inflammatory, neuropathic, and joint pain. To target peripheral nociceptors, we generated a SNAP-tagged NGF derivative NGF that binds to TrkA/p75 receptors but does not provoke signaling in TrkA-positive cells or elicit pain behaviors in mice. NGF was coupled to the photosensitizer IRDye700DX phthalocyanine (IR700) and injected subcutaneously. After near-infrared illumination of the injected area, behavioral responses to nociceptive mechanical and sustained thermal stimuli, but not innocuous stimuli, were substantially reduced. Similarly, in models of inflammatory, osteoarthritic, and neuropathic pain, mechanical hypersensitivity was abolished for 3 weeks after a single treatment regime. We demonstrate that this loss of pain behavior coincides with the retraction of neurons from the skin which then reinnervate the epidermis after 3 weeks corresponding with the return of mechanical hypersensitivity. Thus NGF-mediated photoablation is a minimally invasive approach to reversibly silence nociceptor input from the periphery, and control pain and hypersensitivity to mechanical stimuli.