Nf2/Merlin Controls Spinal Cord Neural Progenitor Function in a Rac1/ErbB2-Dependent Manner

Objective

Individuals with the neurofibromatosis type 2 (NF2) cancer predisposition syndrome develop spinal cord glial tumors (ependymomas) that likely originate from neural progenitor cells. Whereas many spinal ependymomas exhibit indolent behavior, the only treatment option for clinically symptomatic tumors is surgery. In this regard, medical therapies are unfortunately lacking due to an incomplete understanding of the critical growth control pathways that govern the function of spinal cord (SC) neural progenitor cells (NPCs).

Methods

To identify potential therapeutic targets for these tumors, we leveraged primary mouse Nf2-deficient spinal cord neural progenitor cells.

Results

We demonstrate that the Nf2 protein, merlin, negatively regulates spinal neural progenitor cell survival and glial differentiation in an ErbB2-dependent manner, and that NF2-associated spinal ependymomas exhibit increased ErbB2 activation. Moreover, we show that Nf2-deficient SC NPC ErbB2 activation results from Rac1-mediated ErbB2 retention at the plasma membrane.

Significance

Collectively, these findings establish ErbB2 as a potential rational therapeutic target for NF2-associated spinal ependymoma.     

Two Notch ligands, Dll1 and Jag1, are differently restricted in their range of action to control neurogenesis in the mammalian spinal cord

Background

Notch signalling regulates neuronal differentiation in the vertebrate nervous system. In addition to a widespread function in maintaining neural progenitors, Notch signalling has also been involved in specific neuronal fate decisions. These functions are likely mediated by distinct Notch ligands, which show restricted expression patterns in the developing nervous system. Two ligands, in particular, are expressed in non-overlapping complementary domains of the embryonic spinal cord, with Jag1 being restricted to the V1 and dI6 progenitor domains, while Dll1 is expressed in the remaining domains. However, the specific contribution of different ligands to regulate neurogenesis in vertebrate embryos is still poorly understood.

Methodology/Principal Findings

In this work, we investigated the role of Jag1 and Dll1 during spinal cord neurogenesis, using conditional knockout mice where the two genes are deleted in the neuroepithelium, singly or in combination. Our analysis showed that Jag1 deletion leads to a modest increase in V1 interneurons, while dI6 neurogenesis was unaltered. This mild Jag1 phenotype contrasts with the strong neurogenic phenotype detected in Dll1 mutants and led us to hypothesize that neighbouring Dll1-expressing cells signal to V1 and dI6 progenitors and restore neurogenesis in the absence of Jag1. Analysis of double Dll1;Jag1 mutant embryos revealed a stronger increase in V1-derived interneurons and overproduction of dI6 interneurons. In the presence of a functional Dll1 allele, V1 neurogenesis is restored to the levels detected in single Jag1 mutants, while dI6 neurogenesis returns to normal, thereby confirming that Dll1-mediated signalling compensates for Jag1 deletion in V1 and dI6 domains.

Conclusions/Significance

Our results reveal that Dll1 and Jag1 are functionally equivalent in controlling the rate of neurogenesis within their expression domains. However, Jag1 can only activate Notch signalling within the V1 and dI6 domains, whereas Dll1 can signal to neural progenitors both inside and outside its domains of expression.     

Hilar GABAergic interneuron activity controls spatial learning and memory retrieval

Background

Although extensive research has demonstrated the importance of excitatory granule neurons in the dentate gyrus of the hippocampus in normal learning and memory and in the pathogenesis of amnesia in Alzheimer''s disease (AD), the role of hilar GABAergic inhibitory interneurons, which control the granule neuron activity, remains unclear.

Methodology and Principal Findings

We explored the function of hilar GABAergic interneurons in spatial learning and memory by inhibiting their activity through Cre-dependent viral expression of enhanced halorhodopsin (eNpHR3.0)—a light-driven chloride pump. Hilar GABAergic interneuron-specific expression of eNpHR3.0 was achieved by bilaterally injecting adeno-associated virus containing a double-floxed inverted open-reading frame encoding eNpHR3.0 into the hilus of the dentate gyrus of mice expressing Cre recombinase under the control of an enhancer specific for GABAergic interneurons. In vitro and in vivo illumination with a yellow laser elicited inhibition of hilar GABAergic interneurons and consequent activation of dentate granule neurons, without affecting pyramidal neurons in the CA3 and CA1 regions of the hippocampus. We found that optogenetic inhibition of hilar GABAergic interneuron activity impaired spatial learning and memory retrieval, without affecting memory retention, as determined in the Morris water maze test. Importantly, optogenetic inhibition of hilar GABAergic interneuron activity did not alter short-term working memory, motor coordination, or exploratory activity.

Conclusions and Significance

Our findings establish a critical role for hilar GABAergic interneuron activity in controlling spatial learning and memory retrieval and provide evidence for the potential contribution of GABAergic interneuron impairment to the pathogenesis of amnesia in AD.     

Single collateral reconstructions reveal distinct phases of corticospinal remodeling after spinal cord injury

Background

Injuries to the spinal cord often result in severe functional deficits that, in case of incomplete injuries, can be partially compensated by axonal remodeling. The corticospinal tract (CST), for example, responds to a thoracic transection with the formation of an intraspinal detour circuit. The key step for the formation of the detour circuit is the sprouting of new CST collaterals in the cervical spinal cord that contact local interneurons. How individual collaterals are formed and refined over time is incompletely understood.

Methodology/Principal Findings

We traced the hindlimb corticospinal tract at different timepoints after lesion to show that cervical collateral formation is initiated in the first 10 days. These collaterals can then persist for at least 24 weeks. Interestingly, both major and minor CST components contribute to the formation of persistent CST collaterals. We then developed an approach to label single CST collaterals based on viral gene transfer of the Cre recombinase to a small number of cortical projection neurons in Thy1-STP-YFP or Thy1-Brainbow mice. Reconstruction and analysis of single collaterals for up to 12 weeks after lesion revealed that CST remodeling evolves in 3 phases. Collateral growth is initiated in the first 10 days after lesion. Between 10 days and 3–4 weeks after lesion elongated and highly branched collaterals form in the gray matter, the complexity of which depends on the CST component they originate from. Finally, between 3–4 weeks and 12 weeks after lesion the size of CST collaterals remains largely unchanged, while the pattern of their contacts onto interneurons matures.

Conclusions/Significance

This study provides a comprehensive anatomical analysis of CST reorganization after injury and reveals that CST remodeling occurs in distinct phases. Our results and techniques should facilitate future efforts to unravel the mechanisms that govern CST remodeling and to promote functional recovery after spinal cord injury.     

Nogo-66 promotes the differentiation of neural progenitors into astroglial lineage cells through mTOR-STAT3 pathway

Background

Neural stem/progenitor cells (NPCs) can differentiate into neurons, astrocytes and oligodendrocytes. NPCs are considered valuable for the cell therapy of injuries in the central nervous system (CNS). However, when NPCs are transplanted into the adult mammalian spinal cord, they mostly differentiate into glial lineage. The same results have been observed for endogenous NPCs during spinal cord injury. However, little is known about the mechanism of such fate decision of NPCs.

Methodology/Principal Findings

In the present study, we have found that myelin protein and Nogo-66 promoted the differentiation of NPCs into glial lineage. NgR and mTOR-Stat3 pathway were involved in this process. Releasing NgR from cell membranes or blocking mTOR-STAT3 could rescue the enhanced glial differentiation by Nogo-66.

Conclusions/Significance

These results revealed a novel function of Nogo-66 in the fate decision of NPCs. This discovery could have profound impact on the understanding of CNS development and could improve the therapy of CNS injuries.     

A novel conserved evx1 enhancer links spinal interneuron morphology and cis-regulation from fish to mammals

Cerebellar GABAergic interneurons and glia originate from progenitors that delaminate from the ventricular neuroepithelium and proliferate in the prospective white matter. Even though this population of progenitor cells is multipotent as a whole, clonal analysis indicates that different lineages are already separated during postnatal development and little is known about the mechanisms that regulate the specification and differentiation of these cerebellar types at earlier stages. Here, we investigate the role of Ascl1 in the development of inhibitory interneurons and glial cells in the cerebellum. This gene is expressed by maturing oligodendrocytes and GABAergic interneurons and is required for the production of appropriate quantities of these cells, which are severely reduced in Ascl1^−/− mouse cerebella. Nevertheless, the two lineages are not related and the majority of oligodendrocytes populating the developing cerebellum actually derive from extracerebellar sources. Targeted electroporation of Ascl1-expression vectors to ventricular neuroepithelium progenitors enhances the production of interneurons and completely suppresses astrocytic differentiation, whereas loss of Ascl1 function has opposite effects on both cell types. Our results indicate that Ascl1 directs ventricular neuroepithelium progenitors towards inhibitory interneuron fate and restricts their ability to differentiate along the astroglial lineage.     

Brainstem and spinal cord circuitry regulating REM sleep and muscle atonia

Background

Previous work has suggested, but not demonstrated directly, a critical role for both glutamatergic and GABAergic neurons of the pontine tegmentum in the regulation of rapid eye movement (REM) sleep.

Methodology/Principal Findings

To determine the in vivo roles of these fast-acting neurotransmitters in putative REM pontine circuits, we injected an adeno-associated viral vector expressing Cre recombinase (AAV-Cre) into mice harboring lox-P modified alleles of either the vesicular glutamate transporter 2 (VGLUT2) or vesicular GABA-glycine transporter (VGAT) genes. Our results show that glutamatergic neurons of the sublaterodorsal nucleus (SLD) and glycinergic/GABAergic interneurons of the spinal ventral horn contribute to REM atonia, whereas a separate population of glutamatergic neurons in the caudal laterodorsal tegmental nucleus (cLDT) and SLD are important for REM sleep generation. Our results further suggest that presynaptic GABA release in the cLDT-SLD, ventrolateral periaqueductal gray matter (vlPAG) and lateral pontine tegmentum (LPT) are not critically involved in REM sleep control.

Conclusions/Significance

These findings reveal the critical and divergent in vivo role of pontine glutamate and spinal cord GABA/glycine in the regulation of REM sleep and atonia and suggest a possible etiological basis for REM sleep behavior disorder (RBD).     

The Use of PRV-Bartha to Define Premotor Inputs to Lumbar Motoneurons in the Neonatal Spinal Cord of the Mouse

Background

The neonatal mouse has become a model system for studying the locomotor function of the lumbar spinal cord. However, information about the synaptic connectivity within the governing neural network remains scarce. A neurotropic pseudorabies virus (PRV) Bartha has been used to map neuronal connectivity in other parts of the nervous system, due to its ability to travel trans-neuronally. Its use in spinal circuits regulating locomotion has been limited and no study has defined the time course of labelling for neurons known to project monosynaptically to motoneurons.

Methodology/Principal Findings

Here we investigated the ability of PRV Bartha, expressing green and/or red fluorescence, to label spinal neurons projecting monosynaptically to motoneurons of two principal hindlimb muscles, the tibialis anterior (TA) and gastrocnemius (GC). As revealed by combined immunocytochemistry and confocal microscopy, 24–32 h after the viral muscle injection the label was restricted to the motoneuron pool while at 32–40 h the fluorescence was seen in interneurons throughout the medial and lateral ventral grey matter. Two classes of ipsilateral interneurons known to project monosynaptically to motoneurons (Renshaw cells and cells of origin of C-terminals) were consistently labeled at 40 h post-injection but also a group in the ventral grey matter contralaterally. Our results suggest that the labeling of last order interneurons occurred 8–12 h after motoneuron labeling and we presume this is the time taken by the virus to cross one synapse, to travel retrogradely and to replicate in the labeled cells.

Conclusions/Significance

The study establishes the time window for virally - labelling monosynaptic projections to lumbar motoneurons following viral injection into hindlimb muscles. Moreover, it provides a good foundation for intracellular targeting of the labeled neurons in future physiological studies and better understanding the functional organization of the lumbar neural networks.     

A new pharmacological role for donepezil: attenuation of morphine-induced tolerance and apoptosis in rat central nervous system

Background

Tolerance to the analgesic effect of opioids is a pharmacological phenomenon that occurs after their prolonged administration. It has been shown that morphine-induced tolerance is associated with apoptosis in the central nervous system and neuroprotective agents which prevented apoptosis signaling could attenuate tolerance to the analgesic effects. On the other hand donepezil, an acetylcholinesterase inhibitor, has been reported to have neuroprotective effects. Therefore in this study, the effect of systemic administration of donepezil on morphine-induced tolerance and apoptosis in the rat cerebral cortex and lumbar spinal cord was evaluated. Various groups of rats received morphine (ip) and different doses of donepezil (0, 0.5, 1, 1.5 mg/kg/day). Nociception was assessed using tail flick apparatus. Tail flick latency was recorded when the rat shook its tail. For apoptosis assay other groups of rats received the above treatment and apoptosis was evaluated by in situ terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end-labeling (TUNEL) method.

Results

The results showed that administration of donepezil (0.5, 1, 1.5 mg/kg, ip) delayed the morphine tolerance for 9, 12 and 17 days, respectively. Furthermore pretreatment injection of donepezil attenuated the number of apoptotic cells in the cerebral cortex and lumbar spinal cord compared to the control group.

Conclusion

In conclusion, we found that systemic administration of donepezil attenuated morphine-induced tolerance and apoptosis in the rat cerebral cortex and lumbar spinal cord.     

Mutation of pescadillo disrupts oligodendrocyte formation in zebrafish

Background

In vertebrates, the myelin sheath is essential for efficient propagation of action potentials along the axon shaft. Oligodendrocytes are the cells of the central nervous system that create myelin sheaths. During embryogenesis, ventral neural tube precursors give rise to oligodendrocyte progenitor cells, which divide and migrate throughout the central nervous system. This study aimed to investigate mechanisms that regulate oligodendrocyte progenitor cell formation.

Methodology/Principal Findings

By conducting a mutagenesis screen in transgenic zebrafish, we identified a mutation, designated vu166, by an apparent reduction in the number of oligodendrocyte progenitor cells in the dorsal spinal cord. We subsequently determined that vu166 is an allele of pescadillo, a gene known to play a role in ribosome biogenesis and cell proliferation. We found that pescadillo function is required for both the proper number of oligodendrocyte progenitors to form, by regulating cell cycle progression, and for normal levels of myelin gene expression.

Conclusions/Significance

Our data provide evidence that neural precursors require pes function to progress through the cell cycle and produce oligodendrocyte progenitor cells and for oligodendrocyte differentiation.     

Biophysical characteristics reveal neural stem cell differentiation potential

Background

Distinguishing human neural stem/progenitor cell (huNSPC) populations that will predominantly generate neurons from those that produce glia is currently hampered by a lack of sufficient cell type-specific surface markers predictive of fate potential. This limits investigation of lineage-biased progenitors and their potential use as therapeutic agents. A live-cell biophysical and label-free measure of fate potential would solve this problem by obviating the need for specific cell surface markers.

Methodology/Principal Findings

We used dielectrophoresis (DEP) to analyze the biophysical, specifically electrophysiological, properties of cortical human and mouse NSPCs that vary in differentiation potential. Our data demonstrate that the electrophysiological property membrane capacitance inversely correlates with the neurogenic potential of NSPCs. Furthermore, as huNSPCs are continually passaged they decrease neuron generation and increase membrane capacitance, confirming that this parameter dynamically predicts and negatively correlates with neurogenic potential. In contrast, differences in membrane conductance between NSPCs do not consistently correlate with the ability of the cells to generate neurons. DEP crossover frequency, which is a quantitative measure of cell behavior in DEP, directly correlates with neuron generation of NSPCs, indicating a potential mechanism to separate stem cells biased to particular differentiated cell fates.

Conclusions/Significance

We show here that whole cell membrane capacitance, but not membrane conductance, reflects and predicts the neurogenic potential of human and mouse NSPCs. Stem cell biophysical characteristics therefore provide a completely novel and quantitative measure of stem cell fate potential and a label-free means to identify neuron- or glial-biased progenitors.     

Distribution and characterization of progenitor cells within the human filum terminale

Background

Filum terminale (FT) is a structure that is intimately associated with conus medullaris, the most caudal part of the spinal cord. It is well documented that certain regions of the adult human central nervous system contains undifferentiated, progenitor cells or multipotent precursors. The primary objective of this study was to describe the distribution and progenitor features of this cell population in humans, and to confirm their ability to differentiate within the neuroectodermal lineage.

Methodology/Principal Findings

We demonstrate that neural stem/progenitor cells are present in FT obtained from patients treated for tethered cord. When human or rat FT-derived cells were cultured in defined medium, they proliferated and formed neurospheres in 13 out of 21 individuals. Cells expressing Sox2 and Musashi-1 were found to outline the central canal, and also to be distributed in islets throughout the whole FT. Following plating, the cells developed antigen profiles characteristic of astrocytes (GFAP) and neurons (β-III-tubulin). Addition of PDGF-BB directed the cells towards a neuronal fate. Moreover, the cells obtained from young donors shows higher capacity for proliferation and are easier to expand than cells derived from older donors.

Conclusion/Significance

The identification of bona fide neural progenitor cells in FT suggests a possible role for progenitor cells in this extension of conus medullaris and may provide an additional source of such cells for possible therapeutic purposes.Filum terminale, human, progenitor cells, neuron, astrocytes, spinal cord.     

A spatial bias for the origins of interneuron subgroups within the medial ganglionic eminence

Although it is well established that the ventral telencephalon is the primary source of GABAergic cortical interneurons in rodents, little is known about the specification of specific interneuron subtypes. It is also unclear whether the potential to achieve a given fate is established at their place of origin or by signals received during their migration to or during their maturation within the cerebral cortex. Using both in vivo and in vitro transplantation techniques, we find that two major interneuron subgroups have largely distinct origins within the MGE. Somatostatin (SST)-expressing interneurons are primarily generated within the dorsal MGE, while parvalbumin (PV)-expressing interneurons primarily originate from the ventral MGE. In addition, we show that significant heterogeneity exists between gene expression patterns in the dorsal and ventral MGE. These results suggest that, like the spinal cord, neuronal fate determination in the ventral telencephalon is largely the result of spatially segregated, molecularly distinct microdomains arranged on the dorsal-ventral axis.     

Histological and Functional Benefit Following Transplantation of Motor Neuron Progenitors to the Injured Rat Spinal Cord

Background

Motor neuron loss is characteristic of cervical spinal cord injury (SCI) and contributes to functional deficit.

Methodology/Principal Findings

In order to investigate the amenability of the injured adult spinal cord to motor neuron differentiation, we transplanted spinal cord injured animals with a high purity population of human motor neuron progenitors (hMNP) derived from human embryonic stem cells (hESCs). In vitro, hMNPs displayed characteristic motor neuron-specific markers, a typical electrophysiological profile, functionally innervated human or rodent muscle, and secreted physiologically active growth factors that caused neurite branching and neuronal survival. hMNP transplantation into cervical SCI sites in adult rats resulted in suppression of intracellular signaling pathways associated with SCI pathogenesis, which correlated with greater endogenous neuronal survival and neurite branching. These neurotrophic effects were accompanied by significantly enhanced performance on all parameters of the balance beam task, as compared to controls. Interestingly, hMNP transplantation resulted in survival, differentiation, and site-specific integration of hMNPs distal to the SCI site within ventral horns, but hMNPs near the SCI site reverted to a neuronal progenitor state, suggesting an environmental deficiency for neuronal maturation associated with SCI.

Conclusions/Significance

These findings underscore the barriers imposed on neuronal differentiation of transplanted cells by the gliogenic nature of the injured spinal cord, and the physiological relevance of transplant-derived neurotrophic support to functional recovery.     

Gbx2 and Fgf8 are sequentially required for formation of the midbrain-hindbrain compartment boundary

In vertebrates, the common expression border of two homeobox genes, Otx2 and Gbx2, demarcates the prospective midbrain-hindbrain border (MHB) in the neural plate at the end of gastrulation. The presence of a compartment boundary at the MHB has been demonstrated, but the mechanism and timing of its formation remain unclear. We show by genetic inducible fate mapping using a Gbx2(CreER) knock-in mouse line that descendants of Gbx2(+) cells as early as embryonic day (E) 7.5 do not cross the MHB. Without Gbx2, hindbrain-born cells abnormally populate the entire midbrain, demonstrating that Gbx2 is essential for specifying hindbrain fate. Gbx2(+) and Otx2(+) cells segregate from each other, suggesting that mutually exclusive expression of Otx2 and Gbx2 in midbrain and hindbrain progenitors is responsible for cell sorting in establishing the MHB. The MHB organizer gene Fgf8, which is expressed as a sharp transverse band immediately posterior to the lineage boundary at the MHB, is crucial in maintaining the lineage-restricted boundary after E7.5. Partial deletion of Fgf8 disrupts MHB lineage separation. Activation of FGF pathways has a cell-autonomous effect on cell sorting in midbrain progenitors. Therefore, Fgf8 from the MHB may signal the nearby mesencephalic cells to impart distinct cell surface characteristics or induce local cell-cell signaling, which consequently prevents cell movements across the MHB. Our findings reveal the distinct function of Gbx2 and Fgf8 in a stepwise process in the development of the compartment boundary at the MHB and that Fgf8, in addition to its organizer function, plays a crucial role in maintaining the lineage boundary at the MHB by restricting cell movement.     

Time-lapse imaging of the dynamics of CNS glial-axonal interactions in vitro and ex vivo

Background

Myelination is an exquisite and dynamic example of heterologous cell-cell interaction, which consists of the concentric wrapping of multiple layers of oligodendrocyte membrane around neuronal axons. Understanding the mechanism by which oligodendrocytes ensheath axons may bring us closer to designing strategies to promote remyelination in demyelinating diseases. The main aim of this study was to follow glial-axonal interactions over time both in vitro and ex vivo to visualize the various stages of myelination.

Methodology/Principal Findings

We took two approaches to follow myelination over time: i) time-lapse imaging of mixed CNS myelinating cultures generated from mouse spinal cord to which exogenous GFP-labelled murine cells were added, and ii) ex vivo imaging of the spinal cord of shiverer (Mbp mutant) mice, transplanted with GFP-labelled murine neurospheres. We demonstrate that oligodendrocyte-axonal interactions are dynamic events with continuous retraction and extension of oligodendroglial processes. Using cytoplasmic and membrane-GFP labelled cells to examine different components of the myelin-like sheath, we provide evidence from time-lapse fluorescence microscopy and confocal microscopy that the oligodendrocytes'' cytoplasm-filled processes initially spiral around the axon in a corkscrew-like manner. This is followed subsequently by focal expansion of the corkscrew process to form short cuffs, which then extend longitudinally along the axons. We predict from this model that these spiral cuffs must extend over each other first before extending to form internodes of myelin.

Conclusion

These experiments show the feasibility of visualizing the dynamics of glial-axonal interaction during myelination over time. Moreover, these approaches complement each other with the in vitro approach allowing visualization of an entire internodal length of myelin and the ex vivo approach validating the in vitro data.     

Gene Expression in the Spinal Cord in Female Lewis Rats with Experimental Autoimmune Encephalomyelitis Induced with Myelin Basic Protein

Background

Experimental autoimmune encephalomyelitis (EAE), the best available model of multiple sclerosis, can be induced in different animal strains using immunization with central nervous system antigens. EAE is associated with inflammation and demyelination of the nervous system. Micro-array can be used to investigate gene expression and biological pathways that are altered during disease. There are few studies of the changes in gene expression in EAE, and these have mostly been done in a chronic mouse EAE model. EAE induced in the Lewis with myelin basic protein (MBP-EAE) is well characterised, making it an ideal candidate for the analysis of gene expression in this disease model.

Methodology/Principal Findings

MBP-EAE was induced in female Lewis rats by inoculation with MBP and adjuvants. Total RNA was extracted from the spinal cords and used for micro-array analysis using AffimetrixGeneChip Rat Exon 1.0 ST Arrays. Gene expression in the spinal cords was compared between healthy female rats and female rats with MBP-EAE. Gene expression in the spinal cord of rats with MBP-EAE differed from that in the spinal cord of normal rats, and there was regulation of pathways involved with immune function and nervous system function. For selected genes the change in expression was confirmed with real-time PCR.

Conclusions/Significance

EAE leads to modulation of gene expression in the spinal cord. We have identified the genes that are most significantly regulated in MBP-EAE in the Lewis rat and produced a profile of gene expression in the spinal cord at the peak of disease.