Reverse engineering neuron type-specific and type-orthogonal splicing-regulatory networks using single-cell transcriptomes.

Cell type-specific alternative splicing (AS) enables differential gene isoform expression between diverse neuron types with distinct identities and functions. Current studies linking individual RNA-binding proteins (RBPs) to AS in a few neuron types underscore the need for holistic modeling. Here, we use network reverse engineering to derive a map of the neuron type-specific AS regulatory landscape from 133 mouse neocortical cell types defined by single-cell transcriptomes. This approach reliably inferred the regulons of 350 RBPs and their cell type-specific activities. Our analysis revealed driving factors delineating neuronal identities, among which we validated Elavl2 as a key RBP for MGE-specific splicing in GABAergic interneurons using an in vitro ESC differentiation system. We also identified a module of exons and candidate regulators specific for long- and short-projection neurons across multiple neuronal classes. This study provides a resource for elucidating splicing regulatory programs that drive neuronal molecular diversity, including those that do not align with gene expression-based classifications.

Multidimensional analysis of cortical interneuron synaptic features reveals underlying synaptic heterogeneity.

Cortical interneurons represent a diverse set of neuronal subtypes characterized in part by their striking degree of synaptic specificity. However, little is known about the extent of synaptic diversity because of the lack of unbiased methods to extract synaptic features among interneuron subtypes. Here, we develop an approach to aggregate image features from fluorescent confocal images of interneuron synapses and their post-synaptic targets, in order to characterize the heterogeneity of synapses at fine scale. We started by training a model that recognizes pre- and post-synaptic compartments and then determines the target of each genetically-identified interneuron synapse in vitro and in vivo. Our model extracts hundreds of spatial and intensity features from each analyzed synapse, constructing a multidimensional data set, consisting of millions of synapses, which allowed us to perform an unsupervised analysis on this dataset, uncovering novel synaptic subgroups. The subgroups were spatially distributed in a highly structured manner that revealed the local underlying topology of the postsynaptic environment. Dendrite-targeting subgroups were clustered onto subdomains of the dendrite along the proximal to distal axis. Soma-targeting subgroups were enriched onto different postsynaptic cell types. We also find that the two main subclasses of interneurons, basket cells and somatostatin interneurons, utilize distinct strategies to enact inhibitory coverage. Thus, our analysis of multidimensional synaptic features establishes a conceptual framework for studying interneuron synaptic diversity.

St18 specifies globus pallidus projection neuron identity in MGE lineage.

The medial ganglionic eminence (MGE) produces both locally-projecting interneurons, which migrate long distances to structures such as the cortex as well as projection neurons that occupy subcortical nuclei. Little is known about what regulates the migratory behavior and axonal projections of these two broad classes of neurons. We find that St18 regulates the migration and morphology of MGE neurons in vitro. Further, genetic loss-of-function of St18 in mice reveals a reduction in projection neurons of the globus pallidus pars externa. St18 functions by influencing cell fate in MGE lineages as we observe a large expansion of nascent cortical interneurons at the expense of putative GPe neurons in St18 null embryos. Downstream of St18, we identified Cbx7, a component of Polycomb repressor complex 1, and find that it is essential for projection neuron-like migration but not morphology. Thus, we identify St18 as a key regulator of projection neuron vs. interneuron identity.

Vascular-derived SPARC and SerpinE1 regulate interneuron tangential migration and accelerate functional maturation of human stem cell-derived interneurons.

Cortical interneurons establish inhibitory microcircuits throughout the neocortex and their dysfunction has been implicated in epilepsy and neuropsychiatric diseases. Developmentally, interneurons migrate from a distal progenitor domain in order to populate the neocortex - a process that occurs at a slower rate in humans than in mice. In this study, we sought to identify factors that regulate the rate of interneuron maturation across the two species. Using embryonic mouse development as a model system, we found that the process of initiating interneuron migration is regulated by blood vessels of the medial ganglionic eminence (MGE), an interneuron progenitor domain. We identified two endothelial cell-derived paracrine factors, SPARC and SerpinE1, that enhance interneuron migration in mouse MGE explants and organotypic cultures. Moreover, pre-treatment of human stem cell-derived interneurons (hSC-interneurons) with SPARC and SerpinE1 prior to transplantation into neonatal mouse cortex enhanced their migration and morphological elaboration in the host cortex. Further, SPARC and SerpinE1-treated hSC-interneurons also exhibited more mature electrophysiological characteristics compared to controls. Overall, our studies suggest a critical role for CNS vasculature in regulating interneuron developmental maturation in both mice and humans.

Alcohol reduces the activity of somatostatin interneurons in the mouse prefrontal cortex: A neural basis for its disinhibitory effect?

The prefrontal cortex (PFC) is involved in executive ("top-down") control of behavior and its function is especially susceptible to the effects of alcohol, leading to behavioral disinhibition that is associated with alterations in decision making, response inhibition, social anxiety and working memory. The circuitry of the PFC involves a complex interplay between pyramidal neurons (PNs) and several subclasses of inhibitory interneurons (INs), including somatostatin (SST)-expressing INs. Using in vivo calcium imaging, we showed that alcohol dose-dependently altered network activity in layers 2/3 of the prelimbic subregion of the mouse PFC. Low doses of alcohol (1 g/kg, intraperitoneal, i.p.) caused moderate activation of SST INs and weak inhibition of PNs. At moderate to high doses, alcohol (2-3 g/kg) strongly inhibited the activity of SST INs in vivo, and this effect may result in disinhibition, as the activity of a subpopulation of PNs was simultaneously enhanced. In contrast, recordings in brain slices using ex vivo electrophysiology revealed no direct effect of alcohol on the excitability of either SST INs or PNs over a range of concentrations (20 and 50 mM) consistent with the blood alcohol levels reached in the in vivo experiments. This dose-dependent effect of alcohol on SST INs in vivo may reveal a neural basis for the disinhibitory effect of alcohol in the PFC mediated by other neurons within or external to the PFC circuitry.

Photoactivatable Cre recombinase 3.0 for in vivo mouse applications.

Optogenetic genome engineering tools enable spatiotemporal control of gene expression and provide new insight into biological function. Here, we report the new version of genetically encoded photoactivatable (PA) Cre recombinase, PA-Cre 3.0. To improve PA-Cre technology, we compare light-dimerization tools and optimize for mammalian expression using a CAG promoter, Magnets, and 2A self-cleaving peptide. To prevent background recombination caused by the high sequence similarity in the dimerization domains, we modify the codons for mouse gene targeting and viral production. Overall, these modifications significantly reduce dark leak activity and improve blue-light induction developing our new version, PA-Cre 3.0. As a resource, we have generated and validated AAV-PA-Cre 3.0 as well as two mouse lines that can conditionally express PA-Cre 3.0. Together these new tools will facilitate further biological and biomedical research.

Loss of Clustered Protocadherin Diversity Alters the Spatial Distribution of Cortical Interneurons in Mice.

Cortical interneurons (cINs) are locally projecting inhibitory neurons that are distributed throughout the cortex. Due to their relatively limited range of influence, their arrangement in the cortex is critical to their function. cINs achieve this arrangement through a process of tangential and radial migration and apoptosis during development. In this study, we investigated the role of clustered protocadherins (cPcdhs) in establishing the spatial patterning of cINs through the use of genetic cPcdh knockout mice. cPcdhs are expressed in cINs and are known to play key functions in cell spacing and cell survival, but their role in cINs is poorly understood. Using spatial statistical analysis, we found that the 2 main subclasses of cINs, parvalbumin-expressing and somatostatin-expressing (SST) cINs, are nonrandomly spaced within subclass but randomly with respect to each other. We also found that the relative laminar distribution of each subclass was distinctly altered in whole α- or ß-cluster mutants. Examination of perinatal time points revealed that the mutant phenotypes emerged relatively late, suggesting that cPcdhs may be acting during cIN morphological elaboration and synaptogenesis. We then analyzed an isoform-specific knockout for pcdh-αc2 and found that it recapitulated the α-cluster knockout but only in SST cells, suggesting that subtype-specific expression of cPcdh isoforms may help govern subtype-specific spatial distribution.

Non-canonical Wnt Signaling through Ryk Regulates the Generation of Somatostatin- and Parvalbumin-Expressing Cortical Interneurons.

GABAergic interneurons have many important functions in cortical circuitry, a reflection of their cell diversity. The developmental origins of this diversity are poorly understood. Here, we identify rostral-caudal regionality in Wnt exposure within the interneuron progenitor zone delineating the specification of the two main interneuron subclasses. Caudally situated medial ganglionic eminence (MGE) progenitors receive high levels of Wnt signaling and give rise to somatostatin (SST)-expressing cortical interneurons. By contrast, parvalbumin (PV)-expressing basket cells originate mostly from the rostral MGE, where Wnt signaling is attenuated. Interestingly, rather than canonical signaling through ß-catenin, signaling via the non-canonical Wnt receptor Ryk regulates interneuron cell-fate specification in vivo and in vitro. Indeed, gain of function of Ryk intracellular domain signaling regulates SST and PV fate in a dose-dependent manner, suggesting that Ryk signaling acts in a graded fashion. These data reveal an important role for non-canonical Wnt-Ryk signaling in establishing the correct ratios of cortical interneuron subtypes.

A modular gain-of-function approach to generate cortical interneuron subtypes from ES cells.

Whereas past work indicates that cortical interneurons (cINs) can be generically produced from stem cells, generating large numbers of specific subtypes of this population has remained elusive. This reflects an information gap in our understanding of the transcriptional programs required for different interneuron subtypes. Here, we have utilized the directed differentiation of stem cells into specific subpopulations of cortical interneurons as a means to identify some of these missing factors. To establish this approach, we utilized two factors known to be required for the generation of cINs, Nkx2-1 and Dlx2. As predicted, their regulated transient expression greatly improved the differentiation efficiency and specificity over baseline. We extended upon this "cIN-primed" model in order to establish a modular system whereby a third transcription factor could be systematically introduced. Using this approach, we identified Lmo3 and Pou3f4 as genes that can augment the differentiation and/or subtype specificity of cINs in vitro.

Cortex shatters the glass ceiling.

Recreating developmental structures in vitro has been a primary challenge for stem cell biologists. Recent studies in Cell Stem Cell (Eiraku et al., 2008) and Nature (Gaspard et al., 2008) demonstrate that embryonic stem cells can recapitulate early cortical development, enabling them to generate specific cortical subtypes.

SPARC from olfactory ensheathing cells stimulates Schwann cells to promote neurite outgrowth and enhances spinal cord repair.

Olfactory ensheathing cells (OECs) transplanted into the lesioned CNS can stimulate reportedly different degrees of regeneration, remyelination, and functional recovery, but little is known about the mechanisms OECs may use to stimulate endogenous repair. Here, we used a functional proteomic approach, isotope-coded affinity tagging and mass spectrometry, to identify active components of the OEC secreteome that differentially stimulate outgrowth. SPARC (secreted protein acidic rich in cysteine) (osteonectin) was identified as an OEC-derived matricellular protein that can indirectly enhance the ability of Schwann cells to stimulate dorsal root ganglion outgrowth in vitro. SPARC stimulates Schwann cell-mediated outgrowth by cooperative signal with laminin-1 and transforming growth factor beta. Furthermore, when SPARC-null OECs were transplanted into lesioned rat spinal cord, the absence of OEC-secreted SPARC results in an attenuation of outgrowth of specific subsets of sensory and supraspinal axons and changes the pattern of macrophage infiltration in response to the transplanted cells. These data provide the first evidence for a role for SPARC in modulating different aspects of CNS repair and indicate that SPARC can change the activation state of endogenous Schwann cells, resulting in the promotion of outgrowth in vitro, and in vivo.

A re-assessment of the consequences of delayed transplantation of olfactory lamina propria following complete spinal cord transection in rats.

This study is part of the NIH "Facilities of Research-Spinal Cord Injury" contract to support independent replication of published studies. We repeated a study reporting that delayed transplantation of olfactory lamina propria (OLP) into the site of a complete spinal cord transection led to significant improvement in hindlimb motor function and induced axon regeneration. Adult female rats received complete spinal cord transections at T10. Thirty days post-injury, pieces of OLP, which contains olfactory ensheathing cells (OECs), or respiratory lamina propria (RLP), which should not contain OECs, were placed into the transection site. Hindlimb motor function was tested using the BBB scale from day 1 post-injury through 10 weeks following transplantation. To assess axonal regeneration across the transection site, Fluorogold was injected into the distal segment, and the distribution of 5HT-containing axons was assessed using immunostaining. BBB analyses revealed no significant recovery after OLP transplantation and no significant differences between OLP vs. RLP transplant groups. Fluorogold injections into caudal segments did not lead to retrograde labeling in any animals. Immunostaining for 5HT revealed that a few 5HT-labeled axons extended into both RLP and OLP transplants and a few 5HT-labeled axons were present in sections caudal to the injury in 2 animals that received OLP transplants and 1 animal that received RLP transplants. Our results indicate that, although OLP transplants may stimulate regeneration under some circumstances, the effect is not so robust as to reliably overcome the hostile setting created by a complete transection paradigm.

Peripheral olfactory ensheathing cells reduce scar and cavity formation and promote regeneration after spinal cord injury.

Bridging of a lesion site and minimizing local damage to create an environment permissive for regeneration are both primary components of a successful strategy to repair spinal cord injury (SCI). Olfactory ensheathing cells (OECs) are prime candidates for autologous transplantation to bridge this gap, but little is known currently about their mechanism of action. In addition, OECs from the accessible lamina propria (LP) of the olfactory mucosa are a more viable source in humans but have yet to be tested for their ability to promote regeneration in established SCI models. Here, mouse LP-OECs expressing green fluorescent protein (GFP) transplanted directly into both rat and mouse dorsolateral spinal cord lesion sites demonstrate limited migration but interact with host astrocytes to develop a new transitional zone at the lesion border. LP-OECs also promote extensive migration of host Schwann cells into the central nervous system repair zone and stimulate angiogenesis to provide a biological scaffold for repair. This novel environment created by transplanted and host glia within the spinal cord inhibits cavity and scar formation and promotes extensive sprouting of multiple sensory and motor axons into and through the lesion site. Sixty days after rat SCI, serotonin- and tyrosine hydroxylase-positive axons sprouted across the lesion into the distal cord, although axotomized rubrospinal axons did not. Thus, even in a xenotransplant paradigm, LP-OECs work collaboratively with host glial cells to create an environment to ameliorate local damage and simultaneously promote a regenerative response in multiple axonal populations.

PACAP is present in the olfactory system and evokes calcium transients in olfactory receptor neurons.

Pituitary adenylate cyclase activating peptide (PACAP), a neuroregulatory peptide, is found in germinative regions of the CNS, including the olfactory bulb, throughout adulthood. We show that 1) PACAP immunoreactivity is also present in the neonatal mouse and adult mouse and rat olfactory epithelium, 2) PACAP expression pattern differs between neonatal and adult mice, and 3) PACAP is produced by olfactory ensheathing cells. PACAP may thus be a key factor in the uniquely supportive role of olfactory ensheathing cells in regeneration of neurons from olfactory epithelium and lesioned spinal cord. Using calcium imaging, we demonstrated physiological responses to PACAP in both neonatal and adult olfactory receptor neurons (ORNs). We propose that PACAP plays an important role in normal turnover of ORNs by providing neurotrophic support during development and regeneration and neuroprotective support of mature neurons.

Olfactory ensheathing cells of the lamina propria in vivo and in vitro.

Olfactory ensheathing cells (OECs) continuously support the regeneration of olfactory receptor neurons (ORNs). In addition, OECs promote regeneration of neurons within the CNS in a number of transplantation paradigms, but details of exactly how they support regeneration remain elusive. The majority of studies using OECs to promote regeneration have thus far focused on understanding the cell biology of OECs purified from the olfactory bulb (OB). Here we show that a population of OECs similar to those obtained from the OB is present in the lamina propria (LP) beneath the olfactory epithelium (OE). These OECs are the first glial cells encountered by the axons of developing ORNs as they exit the OE and display distinct and variable expression of p75, S100beta, GFAP, and O4, characteristic markers of bulb OECs. Once purified in vitro, they display Schwann cell-like and astrocyte-like properties and expand rapidly. In addition to resembling OB-OECs, LP-OECs also express a unique combination of developmentally important proteins-CD 44, beta1 integrin, P200, Notch 3, NG2, VEGF, and PACAP and CREB binding protein (CBP/p300)-not previously reported in OB-OECs. These data suggest that LP-OECs, like OB-OECs, are a developmentally distinct class of glia that are capable of both immature and mature function, depending on environmental stimuli, within the adult nervous system.