The opposing roles of the transcription factor E2A and its antagonist Id3 that orchestrate and enforce the naive fate of T cells.

It is established that the transcription factor E2A and its antagonist Id3 modulate the checkpoints consisting of the precursor to the T cell antigen receptor (pre-TCR) and the TCR. Here we demonstrate that Id3 expression was higher beyond the pre-TCR checkpoint, remained high in naive T cells and showed a bimodal pattern in the effector-memory population. We show how E2A promoted T lineage specification and how pre-TCR-mediated signaling affected E2A genome-wide occupancy. Thymi in Id3-deficient mice had aberrant development of effector-memory cells, higher expression of the chemokine receptor CXCR5 and the transcriptional repressor Bcl-6 and, unexpectedly, T cell-B cell conjugates and B cell follicles. Collectively, our data show how E2A acted globally to orchestrate development into the T lineage and that Id3 antagonized E2A activity beyond the pre-TCR checkpoint to enforce the naive fate of T cells.

The transcriptional regulators Id2 and Id3 control the formation of distinct memory CD8+ T cell subsets.

During infection, naive CD8(+) T cells differentiate into effector cells, which are armed to eliminate pathogens, and memory cells, which are poised to protect against reinfection. The transcriptional program that regulates terminal differentiation into short-lived effector-memory versus long-lived memory cells is not clearly defined. Through the use of mice expressing reporters for the DNA-binding inhibitors Id2 and Id3, we identified Id3(hi) precursors of long-lived memory cells before the peak of T cell population expansion or upregulation of cell-surface receptors that indicate memory potential. Deficiency in Id2 or Id3 resulted in loss of distinct CD8(+) effector and memory populations, which demonstrated unique roles for these inhibitors of E-protein transcription factors. Furthermore, cytokines altered the expression of Id2 and Id3 differently, which provides insight into how external cues influence gene expression.

Conditional Lpar1 gene targeting identifies cell types mediating neuropathic pain.

LPA1 is one of six known receptors (LPA1-6) for lysophosphatidic acid (LPA). Constitutive Lpar1 null mutant mice have been instrumental in identifying roles for LPA-LPA1 signaling in neurobiological processes, brain development, and behavior, as well as modeling human neurological diseases like neuropathic pain. Constitutive Lpar1 null mutant mice are protected from partial sciatic nerve ligation (PSNL)-induced neuropathic pain, however, the cell types that are functionally responsible for mediating this protective effect are unknown. Here, we report the generation of an Lpar1flox/flox conditional null mutant mouse that allows for cre-mediated conditional deletion, combined with a PSNL pain model. Lpar1flox/flox mice were crossed with cre transgenic lines driven by neural gene promoters for nestin (all neural cells), synapsin (neurons), or P0 (Schwann cells). CD11b-cre transgenic mice were also used to delete Lpar1 in microglia. PSNL-initiated pain responses were reduced following cre-mediated Lpar1 deletion with all three neural promoters as well as the CD11b promoter, supporting involvement of Schwann cells, central and/or peripheral neurons, and microglia in mediating pain. Interestingly, rescue responses were nonidentical, implicating distinct roles for Lpar1-expressing cell types. Our results with a new Lpar1 conditional mouse mutant expand an understanding of LPA1 signaling in the PSNL model of neuropathic pain.

Submegabase copy number variations arise during cerebral cortical neurogenesis as revealed by single-cell whole-genome sequencing.

Somatic copy number variations (CNVs) exist in the brain, but their genesis, prevalence, forms, and biological impact remain unclear, even within experimentally tractable animal models. We combined a transposase-based amplification (TbA) methodology for single-cell whole-genome sequencing with a bioinformatic approach for filtering unreliable CNVs (FUnC), developed from machine learning trained on lymphocyte V(D)J recombination. TbA-FUnC offered superior genomic coverage and removed >90% of false-positive CNV calls, allowing extensive examination of submegabase CNVs from over 500 cells throughout the neurogenic period of cerebral cortical development in Mus musculus Thousands of previously undocumented CNVs were identified. Half were less than 1 Mb in size, with deletions 4× more common than amplification events, and were randomly distributed throughout the genome. However, CNV prevalence during embryonic cortical development was nonrandom, peaking at midneurogenesis with levels triple those found at younger ages before falling to intermediate quantities. These data identify pervasive small and large CNVs as early contributors to neural genomic mosaicism, producing genomically diverse cellular building blocks that form the highly organized, mature brain.

Targeted deletion of LPA5 identifies novel roles for lysophosphatidic acid signaling in development of neuropathic pain.

Lysophosphatidic acid (LPA) is a bioactive lipid that serves as an extracellular signaling molecule acting through cognate G protein-coupled receptors designated LPA(1-6) that mediate a wide range of both normal and pathological effects. Previously, LPA(1), a G(αi)-coupled receptor (which also couples to other G(α) proteins) to reduce cAMP, was shown to be essential for the initiation of neuropathic pain in the partial sciatic nerve ligation (PSNL) mouse model. Subsequent gene expression studies identified LPA(5), a G(α12/13)- and G(q)-coupled receptor that increases cAMP, in a subset of dorsal root ganglion neurons and also within neurons of the spinal cord dorsal horn in a pattern complementing, yet distinct from LPA(1), suggesting its possible involvement in neuropathic pain. We therefore generated an Lpar5 null mutant by targeted deletion followed by PSNL challenge. Homozygous null mutants did not show obvious base-line phenotypic defects. However, following PSNL, LPA(5)-deficient mice were protected from developing neuropathic pain. They also showed reduced phosphorylated cAMP response element-binding protein expression within neurons of the dorsal horn despite continued up-regulation of the characteristic pain-related markers Caα(2)δ(1) and glial fibrillary acidic protein, results that were distinct from those previously observed for LPA(1) deletion. These data expand the influences of LPA signaling in neuropathic pain through a second LPA receptor subtype, LPA(5), involving a mechanistically distinct downstream signaling pathway compared with LPA(1).

Lysophosphatidic acid (LPA) and its receptor, LPA1 , influence embryonic schwann cell migration, myelination, and cell-to-axon segregation.

Schwann cell (SC) migration is an important step preceding myelination and remyelination in the peripheral nervous system, and can be promoted by peptide factors like neuregulins. Here we present evidence that a lipid factor, lysophosphatidic acid (LPA), influences both SC migration and peripheral myelination through its cognate G protein-coupled receptor (GPCR) known as LPA1 . Ultrastructural analyses of peripheral nerves in mouse null-mutants for LPA1 showed delayed SC-to-axon segregation, polyaxonal myelination by single SCs, and thinner myelin sheaths. In primary cultures, LPA promoted SC migration through LPA1 , while analysis of conditioned media from purified dorsal root ganglia neurons using HPLC/MS supported the production of LPA by these neurons. The heterotrimeric G-alpha protein, Gαi , and the small GTPase, Rac1, were identified as important downstream signaling components of LPA1 . These results identify receptor mediated LPA signaling between neurons and SCs that promote SC migration and contribute to the normal development of peripheral nerves through effects on SC-axon segregation and myelination.

Suppression of E-protein activity interferes with the development of BCR-ABL-mediated myeloproliferative disease.

E-proteins are a class of helix-loop-helix (HLH) proteins, which play multiple roles throughout lymphoid development. The DNA binding activities of the E-proteins are regulated by a distinct class of antagonistic HLH proteins, named Id1-4. Here we demonstrate that Id2 deficient mice in a C57BL/6 genetic background exhibit increased cellularity in the granulocyte/myeloid progenitor compartment and show significantly higher numbers of maturing neutrophils. Within 6 months of age, Id2 deficient mice succumbed from overwhelming granulocytosis. The disease closely mimicked the distinctive features of human chronic myeloid leukemia: leukocytosis with maturing neutrophils, splenomegaly, hepatomegaly, and myeloid infiltration into peripheral tissues, including spleen, liver, and lungs. Strikingly, forced Id2 expression in murine bone marrow cells substantially delayed the onset of myeloproliferative disease (MPD). Collectively, these studies show that suppression of E-protein activity interferes with the development of BCR-ABL-mediated MPD.

Receptor editing and marginal zone B cell development are regulated by the helix-loop-helix protein, E2A.

Previous studies have indicated that the E2A gene products are required to initiate B lineage development. Here, we demonstrate that E2A(+/-) B cells that express an autoreactive B cell receptor fail to mature due in part to an inability to activate secondary immunoglobulin (Ig) light chain gene rearrangement. Both RAG1/2 gene expression and RS deletion are severely defective in E2A(+/-) mice. Additionally, we demonstrate that E2A(+/-) mice show an increase in the proportion of marginal zone B cells with a concomitant decrease in the proportion of follicular B cells. In contrast, Id3-deficient splenocytes show a decline in the proportion of marginal zone B cells. Based on these observations, we propose that E-protein activity regulates secondary Ig gene rearrangement at the immature B cell stage and contributes to cell fate determination of marginal zone B cells. Additionally, we propose a model in which E-proteins enforce the developmental checkpoint at the immature B cell stage.

Dimethyl fumarate inhibits integrin α4 expression in multiple sclerosis models.

Dimethyl fumarate is an orally bioavailable compound for the treatment of multiple sclerosis and psoriasis. A mechanism involving nuclear factor erythroid 2-like 2 activation has been proposed to account for its efficacy in multiple sclerosis. Here, we report that dimethyl fumarate inhibits expression of integrin α4 on circulating lymphocytes in experimental autoimmune encephalomyelitis mice and also on activated human Jurkat T cells in a manner distinct from nuclear factor erythroid 2-like 2 activation. Our results offer an alternative mechanism for the efficacy of dimethyl fumarate in multiple sclerosis.

Genomic mosaicism with increased amyloid precursor protein (APP) gene copy number in single neurons from sporadic Alzheimer's disease brains.

Previous reports have shown that individual neurons of the brain can display somatic genomic mosaicism of unknown function. In this study, we report altered genomic mosaicism in single, sporadic Alzheimer's disease (AD) neurons characterized by increases in DNA content and amyloid precursor protein (APP) gene copy number. AD cortical nuclei displayed large variability with average DNA content increases of ~8% over non-diseased controls that were unrelated to trisomy 21. Two independent single-cell copy number analyses identified amplifications at the APP locus. The use of single-cell qPCR identified up to 12 copies of APP in sampled neurons. Peptide nucleic acid (PNA) probes targeting APP, combined with super-resolution microscopy detected primarily single fluorescent signals of variable intensity that paralleled single-cell qPCR analyses. These data identify somatic genomic changes in single neurons, affecting known and unknown loci, which are increased in sporadic AD, and further indicate functionality for genomic mosaicism in the CNS.

Lysophosphatidic acid signaling may initiate fetal hydrocephalus.

Fetal hydrocephalus (FH), characterized by the accumulation of cerebrospinal fluid, an enlarged head, and neurological dysfunction, is one of the most common neurological disorders of newborns. Although the etiology of FH remains unclear, it is associated with intracranial hemorrhage. Here, we report that lysophosphatidic acid (LPA), a blood-borne lipid that activates signaling through heterotrimeric guanosine 5'-triphosphate-binding protein (G protein)-coupled receptors, provides a molecular explanation for FH associated with hemorrhage. A mouse model of intracranial hemorrhage in which the brains of mouse embryos were exposed to blood or LPA resulted in development of FH. FH development was dependent on the expression of the LPA(1) receptor by neural progenitor cells. Administration of an LPA(1) receptor antagonist blocked development of FH. These findings implicate the LPA signaling pathway in the etiology of FH and suggest new potential targets for developing new treatments for FH.

Neuronal DNA content variation (DCV) with regional and individual differences in the human brain.

It is widely assumed that the human brain contains genetically identical cells through which postgenomic mechanisms contribute to its enormous diversity and complexity. The relatively recent identification of neural cells throughout the neuraxis showing somatically generated mosaic aneuploidy indicates that the vertebrate brain can be genomically heterogeneous (Rehen et al. [2001] Proc. Natl. Acad. Sci. U. S. A. 98:13361-13366; Rehen et al. [2005] J. Neurosci. 25:2176-2180; Yurov et al. [2007] PLoS ONE:e558; Westra et al. [2008] J. Comp. Neurol. 507:1944-1951). The extent of human neural aneuploidy is currently unknown because of technically limited sample sizes, but is reported to be small (Iourov et al. [2006] Int. Rev. Cytol. 249:143-191). During efforts to interrogate larger cell populations by using DNA content analyses, a surprising result was obtained: human frontal cortex brain cells were found to display "DNA content variation (DCV)" characterized by an increased range of DNA content both in cell populations and within single cells. On average, DNA content increased by approximately 250 megabases, often representing a substantial fraction of cells within a given sample. DCV within individual human brains showed regional variation, with increased prevalence in the frontal cortex and less variation in the cerebellum. Further, DCV varied between individual brains. These results identify DCV as a new feature of the human brain, encompassing and further extending genomic alterations produced by aneuploidy, which may contribute to neural diversity in normal and pathophysiological states, altered functions of normal and disease-linked genes, and differences among individuals.