En1 is necessary for survival of neurons in the ventral nuclei of the lateral lemniscus.

The ventral nuclei of the lateral lemniscus (VNLL) are part of the central auditory system thought to participate in temporal sound processing. While the timing and location of VNLL neurogenesis have been determined, the genetic factors that regulate VNLL neuron development are unknown. Here, we use genetic fate-mapping techniques to demonstrate that all glycinergic and glycinergic/GABAergic VNLL neurons derive from a cellular lineage that expresses the homeobox transcription factor Engrailed 1 (En1). We also show that En1 deletion does not affect migration or adoption of a neuronal cell fate but does lead to VNLL neuron death during development. Furthermore, En1 deletion blocks expression of the transcription factor FoxP1 in a subset of VNLL neurons. Together, these data identify En1 as a gene important for VNLL neuron development and survival. © 2016 Wiley Periodicals, Inc. Develop Neurobiol 76: 1266-1274, 2016.

En1 directs superior olivary complex neuron positioning, survival, and expression of FoxP1.

Little is known about the genetic pathways and transcription factors that control development and maturation of central auditory neurons. En1, a gene expressed by a subset of developing and mature superior olivary complex (SOC) cells, encodes a homeodomain transcription factor important for neuronal development in the midbrain, cerebellum, hindbrain and spinal cord. Using genetic fate-mapping techniques, we show that all En1-lineal cells in the SOC are neurons and that these neurons are glycinergic, cholinergic and GABAergic in neurotransmitter phenotype. En1 deletion does not interfere with specification or neural fate of these cells, but does cause aberrant positioning and subsequent death of all En1-lineal SOC neurons by early postnatal ages. En1-null cells also fail to express the transcription factor FoxP1, suggesting that FoxP1 lies downstream of En1. Our data define important roles for En1 in the development and maturation of a diverse group of brainstem auditory neurons.

Development of glycinergic innervation to the murine LSO and SPN in the presence and absence of the MNTB.

Neurons in the superior olivary complex (SOC) integrate excitatory and inhibitory inputs to localize sounds in space. The majority of these inhibitory inputs have been thought to arise within the SOC from the medial nucleus of the trapezoid body (MNTB). However, recent work demonstrates that glycinergic innervation of the SOC persists in Egr2; En1(CKO) mice that lack MNTB neurons, suggesting that there are other sources of this innervation (Jalabi et al., 2013). To study the development of MNTB- and non-MNTB-derived glycinergic SOC innervation, we compared immunostaining patterns of glycine transporter 2 (GlyT2) at several postnatal ages in control and Egr2; En1(CKO) mice. GlyT2 immunostaining was present at birth (P0) in controls and reached adult levels by P7 in the superior paraolivary nucleus (SPN) and by P12 in the lateral superior olive (LSO). In Egr2; En1(CKO) mice, glycinergic innervation of the LSO developed at a similar rate but was delayed by one week in the SPN. Conversely, consistent reductions in the number of GlyT2(+) boutons located on LSO somata were seen at all ages in Egr2; En1(CKO) mice, while these numbers reached control levels in the SPN by adulthood. Dendritic localization of GlyT2+ boutons was unaltered in both the LSO and SPN of adult Egr2; En1(CKO) mice. On the postsynaptic side, adult Egr2; En1(CKO) mice had reduced glycine receptor α1 (GlyRα1) expression in the LSO but normal levels in the SPN. GlyRα2 was not expressed by LSO or SPN neurons in either genotype. These findings contribute important information for understanding the development of MNTB- and non-MNTB-derived glycinergic pathways to the mouse SOC.

Microglial displacement of inhibitory synapses provides neuroprotection in the adult brain.

Microglia actively survey the brain microenvironment and play essential roles in sculpting synaptic connections during brain development. While microglial functions in the adult brain are less clear, activated microglia can closely appose neuronal cell bodies and displace axosomatic presynaptic terminals. Microglia-mediated stripping of presynaptic terminals is considered neuroprotective, but the cellular and molecular mechanisms are poorly defined. Using 3D electron microscopy, we demonstrate that activated microglia displace inhibitory presynaptic terminals from cortical neurons in adult mice. Electrophysiological recordings further establish that the reduction in inhibitory GABAergic synapses increased synchronized firing of cortical neurons in γ-frequency band. Increased neuronal activity results in the calcium-mediated activation of CaM kinase IV, phosphorylation of CREB, increased expression of antiapoptotic and neurotrophic molecules and reduced apoptosis of cortical neurons following injury. These results indicate that activated microglia can protect the adult brain by migrating to inhibitory synapses and displacing them from cortical neurons.

Sound localization ability and glycinergic innervation of the superior olivary complex persist after genetic deletion of the medial nucleus of the trapezoid body.

The medial nucleus of the trapezoid body (MNTB) in the superior olivary complex (SOC) is an inhibitory hub considered critical for binaural sound localization. We show that genetic ablation of MNTB neurons in mice only subtly affects this ability by prolonging the minimum time required to detect shifts in sound location. Furthermore, glycinergic innervation of the SOC is maintained without an MNTB, consistent with the existence of parallel inhibitory inputs. These findings redefine the role of MNTB in sound localization and suggest that the inhibitory network is more complex than previously thought.

Lipopolysaccharide-induced microglial activation and neuroprotection against experimental brain injury is independent of hematogenous TLR4.

Intraperitoneal injection of the Gram-negative bacterial endotoxin lipopolysaccharide (LPS) elicits a rapid innate immune response. While this systemic inflammatory response can be destructive, tolerable low doses of LPS render the brain transiently resistant to subsequent injuries. However, the mechanism by which microglia respond to LPS stimulation and participate in subsequent neuroprotection has not been documented. In this study, we first established a novel LPS treatment paradigm where mice were injected intraperitoneally with 1.0 mg/kg LPS for four consecutive days to globally activate CNS microglia. By using a reciprocal bone marrow transplantation procedure between wild-type and Toll-like receptor 4 (TLR4) mutant mice, we demonstrated that the presence of LPS receptor (TLR4) is not required on hematogenous immune cells but is required on cells that are not replaced by bone marrow transplantation, such as vascular endothelia and microglia, to transduce microglial activation and neuroprotection. Furthermore, we showed that activated microglia physically ensheathe cortical projection neurons, which have reduced axosomatic inhibitory synapses from the neuronal perikarya. In line with previous reports that inhibitory synapse reduction protects neurons from degeneration and injury, we show here that neuronal cell death and lesion volumes are significantly reduced in LPS-treated animals following experimental brain injury. Together, our results suggest that activated microglia participate in neuroprotection and that this neuroprotection is likely achieved through reduction of inhibitory axosomatic synapses. The therapeutic significance of these findings rests not only in identifying neuroprotective functions of microglia, but also in establishing the CNS location of TLR4 activation.

Preconditioning paradigms and pathways in the brain.

Preconditioning is a phenomenon in which the brain protects itself against future injury by adapting to low doses of noxious insults. Preconditioning stimuli include ischemia, low doses of endotoxin, hypoxia, hypothermia and hyperthermia, cortical spreading depression, anesthetics, and 3-nitropropionic acid, among others. Understanding of the mechanisms underlying preconditioning has been elusive, but NMDA receptor activation, nitric oxide, inflammatory cytokines, and suppression of the innate immune system appear to have a role. Elucidation of the endogenous cell survival pathways involved in preconditioning has significant clinical implications for preventing neuronal damage in susceptible patients.

Evidence for synaptic stripping by cortical microglia.

Recent studies have described significant demyelination and microglial activation in the cerebral cortex of brains from multiple sclerosis patients. To date, however, experimental models of cortical demyelination or cortical inflammation have not been extensively studied. In this report we describe focal cortical inflammation induced by stereotaxic injection of killed bacteria (BCG), followed 1 month later by subcutaneous injection of the same antigen, a protocol that overcomes the immune privilege of the cortex. Intracerebral BCG injection produced focal microglial activation at the injection site (termed acute lesion). Ten days after peripheral challenge (termed immune-mediated lesion), larger areas and higher densities of activated microglia were found near the injection site. In both paradigms, activated microglia and/or their processes closely apposed neuronal perikarya and apical dendrites. In the immune-mediated lesions, approximately 45% of the axosomatic synapses was displaced by activated microglia. Upon activation, therefore, cortical microglial migrate to and strip synapses from neuronal perikarya. Since neuronal pathology was not a feature of either the acute or immune-mediated lesion, synaptic stripping by activated microglia may have neuroprotective consequences.

Recovery of myelin after induction of oligodendrocyte cell death in postnatal brain.

A transgenic mouse line (Oligo-TTK) was established to monitor oligodendrocyte cell death and myelin formation in the CNS. The expression of a conditionally toxic gene, the herpes simplex virus-1 thymidine kinase (HSV1-TK), was made under control of the MBP (myelin basic protein) gene promoter. A truncated form of the HSV1-TK (TTK) gene was used to avoid both bystander effect resulting from leaking in thymidine kinase activity and sterility in transgenic males observed in previous transgenic mice. The transgene was expressed in the CNS with a restricted localization in oligodendrocytes. Oligodendrocyte proliferation and myelin formation are therefore tightly controlled experimentally by administration of ganciclovir (GCV) via the induction of oligodendrocyte cell death. The most severe and irreversible hypomyelination was obtained when GCV was given daily from postnatal day 1 (P1) to P30. Oligodendrocyte plasticity and myelin recovery were analyzed in another phenotype generated by GCV treatment from P1 to P15. In this model, after dysmyelination, an apparent normal behavior was restored with no visible pathological symptoms by P30. Proliferating cells, which may be implicated in myelin repair in this model, are detected primarily in myelin tracts expressing the oligodendrocyte phenotype. Therefore, the endogenous potential of oligodendrocytes to remyelinate was clearly demonstrated in the mice of this study.

New insights on neuronal alterations in jimpy mutant brain.

In spite of abundant data on oligodendrocyte abnormalities in dysmyelinated jimpy brain, little is known about the axonal damage and the expression of neuronal genes. Recent findings indicate that Nogo-A, oligodendrocyte-myelin glycoprotein (OMgp), and myelin-associated glycoprotein (MAG) inhibit axonal growth by binding a common receptor, the Nogo-A receptor (NgR)-p75 complex. In order to evaluate neuronal modifications in the absence of myelin and in the presence of abnormal oligodendrocytes at different developmental stages, the expression of these inhibitory proteins and their receptors was investigated in jimpy mutant brain. Despite the decrease in oligodendrocyte number at P15 and P25 in jimpy, Nogo-A and OMgp mRNA levels are not significantly different compared with control, suggesting an overexpression of neuronal Nogo-A and OMgp in mutant. Double immunolabeling for Nogo-A and neurofilaments shows strong axonal staining of Nogo-A in jimpy and its down-regulation in oligodendrocytes. The current data raise questions about functions of Nogo-A other than neurite growth inhibition in the CNS. No significant changes in NgR mRNA levels were observed in jimpy, where the increase in p75 level can be correlated with the cell death of oligodendrocytes. In the paranodal region, the cell adhesion molecule neurofascin glial isoform NFN155 mRNA level is reduced by 40% whereas neuronal form NFN186 is up-regulated. These results may explain the failure of paranodal region organization, even with normal level of CASPR (paranodin) mRNA detected in jimpy brain.

Detection of oligodendrocytes in tissue sections using PCR synthesis of digoxigenin-labeled probes.

Oligodendrocytes, the myelin-forming cells in the central nervous system, were visualized with excellent resolution at the light microscopic level using in situ hybridization (ISH). Digoxigenin (Dig)-tagged probes were synthesized and efficiently labeled by PCR. Specific probes to myelin genes were made by RT from brain total RNAs, followed by PCR with designed specific primers in the presence of Dig-11-dUTP. Probes specific to proteolipid protein (PLP), PLP and its isoform DM20 (PLP/DM20), and myelin oligodendrocyte glycoprotein (MOG) were synthesized and labeled. ISH was then applied on vibratomed tissue sections from mouse brains. Despite a low expression of MOG-specific and PLP-specific mRNAs in adult and newborn mouse brains, an oligodendrocyte population was detected. The specificity of Dig-labeled probes was confirmed with the double labeling of carbonic anhydrase II (CA II) and glial fibrillary acidic protein (GFAP) immunocytochemistry and ISH. This versatile and easy method for synthesis and labeling of specific probes to oligodendrocytes can be also applied to detect many other mRNAs in the nervous system and in other tissues.

Trafficking of PLP/DM20 and cAMP signaling in immortalized jimpy oligodendrocytes.

The synthesis, transport, and insertion of jimpy proteolipid protein and DM20 were studied in normal (158N) and jimpy (158JP) immortalized oligodendrocyte lines. Four different expression vectors encoding fusion proteins composed of native PLP and DM20 or jimpy PLP or DM20 were linked to enhanced green fluorescent protein (EGFP). All four transfected fusion proteins had similar distributions in the cell bodies and processes of the two cell types. Both normal and jimpy PLP-EGFP and DM20-EGFP were detected in both cell lines as far as 200 microM from the cell body, indicating synthesis and transport of mutated PLP and DM20 toward the plasma membrane. Immunocytochemistry of fixed normal and jimpy cells with the O10 antibody, which recognizes a conformationally sensitive PLP/DM20 epitope, confirmed that normal and jimpy PLP and DM20 were transported to the plasma membrane. Live staining of normal and jimpy cells transiently transfected with the native PLP showed positive staining, indicating PLP was correctly inserted into the membrane of both normal and jimpy oligodendrocytes. However, live staining of normal and jimpy cells transiently transfected with jimpy PLP showed no positive staining, indicating the mutated protein is abnormally inserted into the plasma membrane. Electrophysiological recordings of the resting membrane potential measured in the whole cell mode of the patch-clamp technique showed the absence of a developmentally regulated negative shift in the membrane potential in jimpy cells compared to normal native or immortalized oligodendrocytes. Treatment of 158N cells and native oligodendrocytes with dibutyryl cAMP (dbcAMP) caused morphological and biochemical differentiation, but failed to do so in 158JP cells, suggesting an abnormal signaling pathway in jimpy. The defect in cAMP signaling in jimpy oligodendrocytes was associated with the suppression of increase in mRNA level of the inducible cAMP early repressor (ICER). When the jimpy oligodendrocyte line was transfected with normal PLP or DM20 and exposed to dbcAMP, the cells failed to differentiate. This finding suggests that improper insertion of jimpy protein into the plasma membrane alters the membrane in such a way that certain signaling pathways are permanently altered. The abnormal insertion of jimpy PLP/DM20 into the plasma membrane may be the basis for the lack of cell signaling and abnormal resting potential in jimpy oligodendrocytes.