transplantation-chemogenetically-engineered-cortical-interneuron

Transplantation of Chemogenetically Engineered Cortical Interneuron Progenitors into Early Postnatal Mouse Brains

Neuronal development is regulated by a complex combination of environmental and genetic factors. Assessing the relative contribution of each component is ...

The Francis Crick Institute

The enteric nervous system (ENS) coordinates the major functions of the gastrointestinal tract. Its development takes place within a constantly changing environment which, after birth, culminates in the establishment of a complex gut microbiota. How such changes affect ENS development and its subsequent function throughout life is an emerging field of study that holds great interest but which is inadequately explored thus far. In this addendum, we discuss our recent findings showing that a component of the ENS, the enteric glial cell network that resides in the gut lamina propria, develops after birth and parallels the evolution of the gut microbiota. Importantly, this network was found to be malleable throughout life by incorporating new cells that arrive from the area of the gut wall in a process of directional movement which was controlled by the lumen gut microbiota. Finally, we postulate on the roles of the intestinal epithelium and the immune system as potential intermediaries between gut microbiota and ENS responses.

The gut microbiota keeps enteric glial cells on the move; prospective roles of the gut epithelium and immune system.

Over the last 20 years, there has been increasing focus on the development of novel stem cell based therapies for the treatment of disorders and diseases affecting the enteric nervous system (ENS) of the gastrointestinal tract (so-called enteric neuropathies). Here, the idea is that ENS progenitor/stem cells could be transplanted into the gut wall to replace the damaged or absent neurons and glia of the ENS. This White Paper sets out experts' views on the commonly used methods and approaches to identify, isolate, purify, expand and optimize ENS stem cells, transplant them into the bowel, and assess transplant success, including restoration of gut function. We also highlight obstacles that must be overcome in order to progress from successful preclinical studies in animal models to ENS stem cell therapies in the clinic.

White paper on guidelines concerning enteric nervous system stem cell therapy for enteric neuropathies.

The gastrointestinal (GI) tract is essential for the absorption of nutrients, induction of mucosal and systemic immune responses, and maintenance of a healthy gut microbiota. Key aspects of gastrointestinal physiology are controlled by the enteric nervous system (ENS), which is composed of neurons and glial cells. The ENS is exposed to and interacts with the outer (microbiota, metabolites, and nutrients) and inner (immune cells and stromal cells) microenvironment of the gut. Although the cellular blueprint of the ENS is mostly in place by birth, the functional maturation of intestinal neural networks is completed within the microenvironment of the postnatal gut, under the influence of gut microbiota and the mucosal immune system. Recent studies have shown the importance of molecular interactions among microbiota, enteric neurons, and immune cells for GI homeostasis. In addition to its role in GI physiology, the ENS has been associated with the pathogenesis of neurodegenerative disorders, such as Parkinson's disease, raising the possibility that microbiota-ENS interactions could offer a viable strategy for influencing the course of brain diseases. Here, we discuss recent advances on the role of microbiota and the immune system on the development and homeostasis of the ENS, a key relay station along the gut-brain axis.

The Effect of Microbiota and the Immune System on the Development and Organization of the Enteric Nervous System.

In vertebrate organisms, the neural crest (NC) gives rise to multipotential and highly migratory progenitors which are distributed throughout the embryo and generate, among other structures, the peripheral nervous system, including the intrinsic neuroglial networks of the gut, i.e. the enteric nervous system (ENS). The majority of enteric neurons and glia originate from vagal NC-derived progenitors which invade the foregut mesenchyme and migrate rostro-caudally to colonise the entire length of the gut. Although the migratory behaviour of NC cells has been studied extensively, it remains unclear how their properties and response to microenvironment change as they navigate through complex cellular terrains to reach their target embryonic sites.

Geminin prevents DNA damage in vagal neural crest cells to ensure normal enteric neurogenesis.

Hirschsprung disease (HSCR) is characterized by absence of enteric neurons from the distal colon and severe intestinal dysmotility. To understand the pathophysiology and genetics of HSCR we developed a unique zebrafish model that allows combined genetic, developmental and in vivo physiological studies. We show that ret mutant zebrafish exhibit cellular, physiological and genetic features of HSCR, including absence of intestinal neurons, reduced peristalsis, and varying phenotype expressivity in the heterozygous state. We perform live imaging experiments using a UAS-GAL4 binary genetic system to drive fluorescent protein expression in ENS progenitors. We demonstrate that ENS progenitors migrate at reduced speed in ret heterozygous embryos, without changes in proliferation or survival, establishing this as a principal pathogenic mechanism for distal aganglionosis. We show, using live imaging of actual intestinal movements, that intestinal motility is severely compromised in ret mutants, and partially impaired in ret heterozygous larvae, and establish a clear correlation between neuron position and organised intestinal motility. We exploited the partially penetrant ret heterozygous phenotype as a sensitised background to test the influence of a candidate modifier gene. We generated mapk10 loss-of-function mutants, which show reduced numbers of enteric neurons. Significantly, we show that introduction of mapk10 mutations into ret heterozygotes enhanced the ENS deficit, supporting MAPK10 as a HSCR susceptibility locus. Our studies demonstrate that ret heterozygous zebrafish is a sensitized model, with many significant advantages over existing murine models, to explore the pathophysiology and complex genetics of HSCR.

A Novel Zebrafish ret Heterozygous Model of Hirschsprung Disease Identifies a Functional Role for mapk10 as a Modifier of Enteric Nervous System Phenotype Severity.

Interactions between the nervous system and immune system are required for organ function and homeostasis. Evidence suggests that enteric neurons and intestinal immune cells share common regulatory mechanisms and can coordinate their responses to developmental challenges and environmental aggressions. These discoveries shed light on the physiology of system interactions and open novel perspectives for therapy designs that target underappreciated neurological-immunological commonalities. Here we highlight findings that address the importance of neuroimmune cell units (NICUs) in intestinal development, homeostasis and disease.

Neuroimmune regulation during intestinal development and homeostasis.

The enteric nervous system (ENS) is essential for digestive function and gut homeostasis. Here we show that the amorphous neuroglia networks of the mouse ENS are composed of overlapping clonal units founded by postmigratory neural crest-derived progenitors. The spatial configuration of ENS clones depends on proliferation-driven local interactions of ENS progenitors with lineally unrelated neuroectodermal cells, the ordered colonization of the serosa-mucosa axis by clonal descendants, and gut expansion. Single-cell transcriptomics and mutagenesis analysis delineated dynamic molecular states of ENS progenitors and identified RET as a regulator of neurogenic commitment. Clonally related enteric neurons exhibit synchronous activity in response to network stimulation. Thus, lineage relationships underpin the organization of the peripheral nervous system.

Lineage-dependent spatial and functional organization of the mammalian enteric nervous system.

Multiple populations of wake-promoting neurons have been characterized in mammals, but few sleep-promoting neurons have been identified. Wake-promoting cell types include hypocretin and GABA (γ-aminobutyric-acid)-releasing neurons of the lateral hypothalamus, which promote the transition to wakefulness from non-rapid eye movement (NREM) and rapid eye movement (REM) sleep. Here we show that a subset of GABAergic neurons in the mouse ventral zona incerta, which express the LIM homeodomain factor Lhx6 and are activated by sleep pressure, both directly inhibit wake-active hypocretin and GABAergic cells in the lateral hypothalamus and receive inputs from multiple sleep-wake-regulating neurons. Conditional deletion of Lhx6 from the developing diencephalon leads to decreases in both NREM and REM sleep. Furthermore, selective activation and inhibition of Lhx6-positive neurons in the ventral zona incerta bidirectionally regulate sleep time in adult mice, in part through hypocretin-dependent mechanisms. These studies identify a GABAergic subpopulation of neurons in the ventral zona incerta that promote sleep.

Lhx6-positive GABA-releasing neurons of the zona incerta promote sleep.

Cortical networks are composed of excitatory projection neurons and inhibitory interneurons. Finding the right balance between the two is important for controlling overall cortical excitation and network dynamics. However, it is unclear how the correct number of cortical interneurons (CIs) is established in the mammalian forebrain. CIs are generated in excess from basal forebrain progenitors, and their final numbers are adjusted via an intrinsically determined program of apoptosis that takes place during an early postnatal window. Here, we provide evidence that the extent of CI apoptosis during this critical period is plastic and cell-type specific and can be reduced in a cell-autonomous manner by acute increases in neuronal activity. We propose that the physiological state of the emerging neural network controls the activity levels of local CIs to modulate their numbers in a homeostatic manner.

Modulation of Apoptosis Controls Inhibitory Interneuron Number in the Cortex.

The mammalian cortex consists of two main neuronal types: the principal excitatory pyramidal neurons (PNs) and the inhibitory interneurons (INs). The interplay between these two neuronal populations - which drive excitation and inhibition (E/I balance), respectively - is crucial for controlling the overall activity in the brain. A number of neurological and psychiatric disorders have been associated with changes in E/I balance. It is not surprising, therefore, that neural networks employ several different mechanisms to maintain their firing rates at a stable level, collectively referred as homeostatic forms of plasticity. Here, we share our views on how the size of IN populations may provide an early homeostatic checkpoint for controlling brain activity. In a recent paper published in , we demonstrate that the extent of IN apoptosis during a critical early postnatal period is plastic, cell type specific, and can be reduced in a cell-autonomous manner by acute increases in neuronal activity. We propose that a critical interplay between the physiological state of the network and its cellular units fine-tunes the size of IN populations with the aim of stabilizing network activity.

Homeostatic Regulation of Interneuron Apoptosis During Cortical Development.

Coordination of gastrointestinal function relies on joint efforts of enteric neurons and glia, whose crosstalk is vital for the integration of their activity. To investigate the signaling mechanisms and to delineate the spatial aspects of enteric neuron-to-glia communication within enteric ganglia we developed a method to stimulate single enteric neurons while monitoring the activity of neighboring enteric glial cells. We combined cytosolic calcium uncaging of individual enteric neurons with calcium imaging of enteric glial cells expressing a genetically encoded calcium indicator and demonstrate that enteric neurons signal to enteric glial cells through pannexins using paracrine purinergic pathways. Sparse labeling of enteric neurons and high-resolution analysis of the structural relation between neuronal cell bodies, varicose release sites and enteric glia uncovered that this form of neuron-to-glia communication is contained between the cell body of an enteric neuron and its surrounding enteric glial cells. Our results reveal the spatial and functional foundation of neuro-glia units as an operational cellular assembly in the enteric nervous system.

Article	Year
The gut microbiota keeps enteric glial cells on the move; prospective roles of the gut epithelium and immune system. Gut microbes\| PubMed ID: 26558327	2015
White paper on guidelines concerning enteric nervous system stem cell therapy for enteric neuropathies. Developmental biology\| PubMed ID: 27059883	2016
The Effect of Microbiota and the Immune System on the Development and Organization of the Enteric Nervous System. Gastroenterology\| PubMed ID: 27521479	2016
Geminin prevents DNA damage in vagal neural crest cells to ensure normal enteric neurogenesis. BMC biology\| PubMed ID: 27776507	2016
A Novel Zebrafish ret Heterozygous Model of Hirschsprung Disease Identifies a Functional Role for mapk10 as a Modifier of Enteric Nervous System Phenotype Severity. PLoS genetics\| PubMed ID: 27902697	2016
Neuroimmune regulation during intestinal development and homeostasis. Nature immunology\| PubMed ID: 28092371	2017
Lineage-dependent spatial and functional organization of the mammalian enteric nervous system. Science (New York, N.Y.)\| PubMed ID: 28522527	2017
Lhx6-positive GABA-releasing neurons of the zona incerta promote sleep. Nature\| PubMed ID: 28847002	2017
Modulation of Apoptosis Controls Inhibitory Interneuron Number in the Cortex. Cell reports\| PubMed ID: 29444425	2018
Homeostatic Regulation of Interneuron Apoptosis During Cortical Development. Journal of experimental neuroscience\| PubMed ID: 30013387	2018
Structurally defined signaling in neuro-glia units in the enteric nervous system. Glia\| PubMed ID: 30730592	2019

Vassilis Pachnis

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