Usp9x-deficiency disrupts the morphological development of the postnatal hippocampal dentate gyrus.

Within the adult mammalian brain, neurogenesis persists within two main discrete locations, the subventricular zone lining the lateral ventricles, and the hippocampal dentate gyrus. Neurogenesis within the adult dentate gyrus contributes to learning and memory, and deficiencies in neurogenesis have been linked to cognitive decline. Neural stem cells within the adult dentate gyrus reside within the subgranular zone (SGZ), and proteins intrinsic to stem cells, and factors within the niche microenvironment, are critical determinants for development and maintenance of this structure. Our understanding of the repertoire of these factors, however, remains limited. The deubiquitylating enzyme USP9X has recently emerged as a mediator of neural stem cell identity. Furthermore, mice lacking Usp9x exhibit a striking reduction in the overall size of the adult dentate gyrus. Here we reveal that the development of the postnatal SGZ is abnormal in mice lacking Usp9x. Usp9x conditional knockout mice exhibit a smaller hippocampus and shortened dentate gyrus blades from as early as P7. Moreover, the analysis of cellular populations within the dentate gyrus revealed reduced stem cell, neuroblast and neuronal numbers and abnormal neuroblast morphology. Collectively, these findings highlight the critical role played by USP9X in the normal morphological development of the postnatal dentate gyrus.

NFIX regulates neural progenitor cell differentiation during hippocampal morphogenesis.

Neural progenitor cells have the ability to give rise to neurons and glia in the embryonic, postnatal and adult brain. During development, the program regulating whether these cells divide and self-renew or exit the cell cycle and differentiate is tightly controlled, and imbalances to the normal trajectory of this process can lead to severe functional consequences. However, our understanding of the molecular regulation of these fundamental events remains limited. Moreover, processes underpinning development of the postnatal neurogenic niches within the cortex remain poorly defined. Here, we demonstrate that Nuclear factor one X (NFIX) is expressed by neural progenitor cells within the embryonic hippocampus, and that progenitor cell differentiation is delayed within Nfix(-/-) mice. Moreover, we reveal that the morphology of the dentate gyrus in postnatal Nfix(-/-) mice is abnormal, with fewer subgranular zone neural progenitor cells being generated in the absence of this transcription factor. Mechanistically, we demonstrate that the progenitor cell maintenance factor Sry-related HMG box 9 (SOX9) is upregulated in the hippocampus of Nfix(-/-) mice and demonstrate that NFIX can repress Sox9 promoter-driven transcription. Collectively, our findings demonstrate that NFIX plays a central role in hippocampal morphogenesis, regulating the formation of neuronal and glial populations within this structure.

Nuclear factor one X regulates Bobby sox during development of the mouse forebrain.

The transcription factor nuclear factor one X (NFIX) plays a central role during the development of the neocortex and hippocampus, through the activation of astrocyte-specific gene expression and the repression of progenitor-specific pathways. However, our understanding of transcriptional targets of NFIX during cortical development remains limited. Here, we identify the transcription factor Bobby sox (Bbx) as a target for NFI-mediated transcriptional control. BBX is expressed within ventricular zone progenitor cells within the developing neocortex and hippocampus, and its expression is upregulated in Nfix (-/-) mice. Moreover, we reveal that NFIX can repress Bbx promoter-driven expression. Collectively, these data suggest that Bbx is a downstream target of NFIX during development of the forebrain.

Heterozygosity for nuclear factor one x affects hippocampal-dependent behaviour in mice.

Identification of the genes that regulate the development and subsequent functioning of the hippocampus is pivotal to understanding the role of this cortical structure in learning and memory. One group of genes that has been shown to be critical for the early development of the hippocampus is the Nuclear factor one (Nfi) family, which encodes four site-specific transcription factors, NFIA, NFIB, NFIC and NFIX. In mice lacking Nfia, Nfib or Nfix, aspects of early hippocampal development, including neurogenesis within the dentate gyrus, are delayed. However, due to the perinatal lethality of these mice, it is not clear whether this hippocampal phenotype persists to adulthood and affects hippocampal-dependent behaviour. To address this we examined the hippocampal phenotype of mice heterozygous for Nfix (Nfix (+/-)), which survive to adulthood. We found that Nfix (+/-) mice had reduced expression of NFIX throughout the brain, including the hippocampus, and that early hippocampal development in these mice was disrupted, producing a phenotype intermediate to that of wild-type mice and Nfix(-/-) mice. The abnormal hippocampal morphology of Nfix (+/-) mice persisted to adulthood, and these mice displayed a specific performance deficit in the Morris water maze learning and memory task. These findings demonstrate that the level of Nfix expression during development and within the adult is essential for the function of the hippocampus during learning and memory.

Tolerance induction with gene-modified stem cells and immune-preserving conditioning in primed mice: restricting antigen to differentiated antigen-presenting cells permits efficacy.

Bone marrow (BM) or hematopoietic stem cell (HSC) transplantation is used as curative therapy for hematologic malignancies. Incorporation of gene therapy to drive tolerogenic expression of antigens is a promising strategy to overcome the limited long-term efficacy of autologous HSC transplantation for autoimmune diseases. HSC engraftment and tolerance induction is readily achieved after myeloablative or immune-depleting conditioning regardless of the cellular compartment in which antigen is expressed. It is unclear whether the efficiency of engraftment and tolerance induction is influenced by targeting antigen to specific cellular compartments. This is particularly important when using clinically feasible low-intensity conditioning aimed at preserving infectious immunity in individuals where immunologic memory exists to the autoantigen to be expressed. Here we demonstrate that, under immune-preserving conditions, confining expression of a transgenically expressed antigen to dendritic cells permits stable, long-term engraftment of genetically modified BM even when recipients are immune to the expressed antigen. In contrast, broader expression within the hematopoietic compartment leads to graft rejection and therapeutic failure because of antigen expression in HSCs. These findings are relevant to the clinical application of genetically engineered HSCs and provide evidence that careful selection of promoters for HSC-mediated gene therapy is important, particularly where tolerance is sought under immune-preserving conditions.

Steady-state antigen-expressing dendritic cells terminate CD4+ memory T-cell responses.

CD4(+) T cells are important effectors of inflammation and tissue destruction in many diseases of immune dysregulation. As memory T cells develop early during the preclinical stages of autoimmune and inflammatory diseases, immunotherapeutic approaches to treatment of these diseases, once established, must include the means to terminate memory T-cell responses. Traditionally, it has been considered that, due to their terminally differentiated nature, memory T cells are resistant to tolerance induction, although emerging evidence indicates that some immunotherapeutic approaches can terminate memory T-cell responses. Here, we demonstrate that CD4(+) memory T-cell responses can be terminated when cognate antigen is transgenically expressed in steady-state DC. Transfer of in-vitro-generated CD4(+) memory T cells establishes, in nontransgenic recipients, a stable and readily recalled memory response to cognate antigen. In contrast, upon transfer to mice expressing cognate antigen targeted to DC, memory CD4(+) T cells undergo a phase of limited proliferation followed by substantial deletion, and recall responses are effectively silenced. This finding is important in understanding how to effectively apply immunotherapy to ongoing T-cell-mediated autoimmune and inflammatory diseases.

Triggering of proteinase-activated receptor 4 leads to joint pain and inflammation in mice.

OBJECTIVE: To investigate the role of proteinase-activated receptor 4 (PAR-4) in mediating joint inflammation and pain in mice. METHODS: Knee joint blood flow, edema, and pain sensitivity (as induced by thermal and mechanical stimuli) were assessed in C57BL/6 mice following intraarticular injection of either the selective PAR-4 agonist AYPGKF-NH(2) or the inactive control peptide YAPGKF-NH(2). The mechanism of action of AYPGKF-NH(2) was examined by pretreatment of each mouse with either the PAR-4 antagonist pepducin P4pal-10 or the bradykinin antagonist HOE 140. Finally, the role of PAR-4 in mediating joint inflammation was tested by pretreating mice with acutely inflamed knees with pepducin P4pal-10. RESULTS: PAR-4 activation caused a long-lasting increase in joint blood flow and edema formation, which was not seen following injection of the control peptide. The PAR-4-activating peptide was also found to be pronociceptive in the joint, where it enhanced sensitivity to a noxious thermal stimulus and caused mechanical allodynia and hyperalgesia. The proinflammatory and pronociceptive effects of AYPGKF-NH(2) could be inhibited by pepducin P4pal-10 and HOE 140. Finally, pepducin P4pal-10 ameliorated the clinical and physiologic signs of acute joint inflammation. CONCLUSION: This study demonstrates that local activation of PAR-4 leads to proinflammatory changes in the knee joint that are dependent on the kallikrein-kinin system. We also show for the first time that PARs are involved in the modulation of joint pain, with PAR-4 being pronociceptive in this tissue. Thus, blockade of articular PAR-4 may be a useful means of controlling joint inflammation and pain.