Role of Adult-Born Versus Preexisting Neurons Born at P0 in Olfactory Perception in a Complex Olfactory Environment in Mice.

Olfactory perceptual learning is defined as an improvement in the discrimination of perceptually close odorants after passive exposure to these odorants. In mice, simple olfactory perceptual learning involving the discrimination of two odorants depends on an increased number of adult-born neurons in the olfactory bulb, which refines the bulbar output. However, the olfactory environment is complex, raising the question of the adjustment of the bulbar network to multiple discrimination challenges. Perceptual learning of 1 to 6 pairs of similar odorants led to discrimination of all learned odor pairs. Increasing complexity did not increase adult-born neuron survival but enhanced the number of adult-born neurons responding to learned odorants and their spine density. Moreover, only complex learning induced morphological changes in neurons of the granule cell layer born during the first day of life (P0). Selective optogenetic inactivation of either population confirmed functional involvement of adult-born neurons regardless of the enrichment complexity, while preexisting neurons were required for complex discrimination only.

Transcriptional regulation of CRMP5 controls neurite outgrowth through Sox5.

Transcriptional regulation of proteins involved in neuronal polarity is a key process that underlies the ability of neurons to transfer information in the central nervous system. The Collapsin Response Mediator Protein (CRMP) family is best known for its role in neurite outgrowth regulation conducting to neuronal polarity and axonal guidance, including CRMP5 that drives dendrite differentiation. Although CRMP5 is able to control dendritic development, the regulation of its expression remains poorly understood. Here we identify a Sox5 consensus binding sequence in the putative promoter sequence upstream of the CRMP5 gene. By luciferase assays we show that Sox5 increases CRMP5 promoter activity, but not if the putative Sox5 binding site is mutated. We demonstrate that Sox5 can physically bind to the CRMP5 promoter DNA in gel mobility shift and chromatin immunoprecipitation assays. Using a combination of real-time RT-PCR and quantitative immunocytochemistry, we provide further evidence for a Sox5-dependent upregulation of CRMP5 transcription and protein expression in N1E115 cells: a commonly used cell line model for neuronal differentiation. Furthermore, we report that increasing Sox5 levels in this neuronal cell line inhibits neurite outgrowth. This inhibition requires CRMP5 because CRMP5 knockdown prevents the Sox5-dependent effect. We confirm the physiological relevance of the Sox5-CRMP5 pathway in the regulation of neurite outgrowth using mouse primary hippocampal neurons. These findings identify Sox5 as a critical modulator of neurite outgrowth through the selective activation of CRMP5 expression.

Collapsin response-mediator protein 5 (CRMP5) phosphorylation at threonine 516 regulates neurite outgrowth inhibition.

The collapsin response-mediator proteins (CRMPs) are multifunctional proteins highly expressed during brain development but down-regulated in the adult brain. They are involved in axon guidance and neurite outgrowth signalling. Among these, the intensively studied CRMP2 has been identified as an important actor in axon outgrowth, this activity being correlated with the reorganisation of cytoskeletal proteins via the phosphorylation state of CRMP2. Another member, CRMP5, restricts the growth-promotional effects of CRMP2 by inhibiting dendrite outgrowth at early developmental stages. This inhibition occurs when CRMP5 binds to tubulin and the microtubule-associated protein MAP2, but the role of CRMP5 phosphorylation is still unknown. Here, we have studied the role of CRMP5 phosphorylation by mutational analysis. Using non-phosphorylatable truncated constructs of CRMP5 we have demonstrated that, among the four previously identified CRMP5 phosphorylation sites (T509, T514, T516 and S534), only the phosphorylation at T516 residue was needed for neurite outgrowth inhibition in PC12 cells and in cultured C57BL/6J mouse hippocampal neurons. Indeed, the expression of the CRMP5 non-phosphorylated form induced a loss of function of CRMP5 and the mutant mimicking the phosphorylated form induced the growth inhibition function seen in wildtype CRMP5. The T516 phosphorylation was achieved by the glycogen synthase kinase-3ß (GSK-3ß), which can phosphorylate the wildtype protein but not the non-phosphorylatable mutant. Furthermore, we have shown that T516 phosphorylation is essential for the tubulin-binding property of CRMP5. Therefore, CRMP5-induced growth inhibition is dependent on T516 phosphorylation through the GSK-3ß pathway. The findings provide new insights into the mechanisms underlying neurite outgrowth.

Human T lymphotropic virus type 1 increases T lymphocyte migration by recruiting the cytoskeleton organizer CRMP2.

Recruitment of virus-infected T lymphocytes into the CNS is an essential step in the development of virus-associated neuroinflammatory diseases, notably myelopathy induced by retrovirus human T leukemia virus-1 (HTLV-1). We have recently shown the key role of collapsin response mediator protein 2 (CRMP2), a phosphoprotein involved in cytoskeleton rearrangement, in the control of human lymphocyte migration and in brain targeting in animal models of virus-induced neuroinflammation. Using lymphocytes cloned from infected patients and chronically infected T cells, we found that HTLV-1 affects CRMP2 activity, resulting in an increased migratory potential. Elevated CRMP2 expression accompanies a higher phosphorylation level of CRMP2 and its more pronounced adhesion to tubulin and actin. CRMP2 forms, a full length and a shorter, cleaved one, are also affected. Tax transfection and extinction strategies show the involvement of this viral protein in enhanced full-length and active CRMP2, resulting in prominent migratory rate. A role for other viral proteins in CRMP2 phosphorylation is suspected. Full-length CRMP2 confers a migratory advantage possibly by preempting the negative effect of short CRMP2 we observe on T lymphocyte migration. In addition, HTLV-1-induced migration seems, in part, supported by the ability of infected cell to increase the proteosomal degradation of short CRMP2. Finally, gene expression in CD69(+) cells selected from patients suggests that HTLV-1 has the capacity to influence the CRMP2/PI3K/Akt axis thus to positively control cytoskeleton organization and lymphocyte migration. Our data provide an additional clue to understanding the infiltration of HTLV-1-infected lymphocytes into various tissues and suggest that the regulation of CRMP2 activity by virus infection is a novel aspect of neuroinflammation.

Identification of a new CRMP5 isoform present in the nucleus of cancer cells and enhancing their proliferation.

Collapsin Response Mediator Protein 5 (CRMP5) belongs to a family of five cytosolic proteins highly expressed in the developing nervous system but downregulated in the adult brain. When expressed at the adult stage, CRMP5 is involved in neurological disorders. Indeed, CRMP5 is found expressed in cancer cells of some brain tumors, such as glioblastoma, or in small cell lung cancer causing paraneoplastic neurological syndromes as a result of cancer-induced auto-immune processes. Nevertheless, its role in cancer pathology is still obscure. Here, we show a new short isoform, derived from C-terminal processing of CRMP5, presenting a nuclear localization both in human glioblastoma, and in cancer cell lines (H69, GL15). By mutational analysis, we demonstrate that nuclear translocation occurs via nuclear localization signal (NLS), where the essential residue for nuclear location is K391. Direct CRMP5/ tubulin interaction, previously shown during brain development, does not occur for cytosolic CRMP5 in pathological conditions, leading to the suggestion that in cancer cells CRMP5 is not sequestered in the cytosol; therefore it may undergo C-terminal truncation allowing the exposure of the NLS for active translocation. Moreover, we show that the function associated with the CRMP5 nuclear targeting is an increase of cell proliferation activity.

Pallidin function in Drosophila surface glia regulates sleep and is dependent on amino acid availability.

The Pallidin protein is a central subunit of a multimeric complex called biogenesis of lysosome-related organelles complex 1 (BLOC1) that regulates specific endosomal functions and has been linked to schizophrenia. We show here that downregulation of Pallidin and other members of BLOC1 in the surface glia, the Drosophila equivalent of the blood-brain barrier, reduces and delays nighttime sleep in a circadian-clock-dependent manner. In agreement with BLOC1 involvement in amino acid transport, downregulation of the large neutral amino acid transporter 1 (LAT1)-like transporters JhI-21 and mnd, as well as of TOR (target of rapamycin) amino acid signaling, phenocopy Pallidin knockdown. Furthermore, supplementing food with leucine normalizes the sleep/wake phenotypes of Pallidin downregulation, and we identify a role for Pallidin in the subcellular trafficking of JhI-21. Finally, we provide evidence that Pallidin in surface glia is required for GABAergic neuronal activity. These data identify a BLOC1 function linking essential amino acid availability and GABAergic sleep/wake regulation.

Clinical interpretation of variants identified in RNU4ATAC, a non-coding spliceosomal gene.

Biallelic variants in RNU4ATAC, a non-coding gene transcribed into the minor spliceosome component U4atac snRNA, are responsible for three rare recessive developmental diseases, namely Taybi-Linder/MOPD1, Roifman and Lowry-Wood syndromes. Next-generation sequencing of clinically heterogeneous cohorts (children with either a suspected genetic disorder or a congenital microcephaly) recently identified mutations in this gene, illustrating how profoundly these technologies are modifying genetic testing and assessment. As RNU4ATAC has a single non-coding exon, the bioinformatic prediction algorithms assessing the effect of sequence variants on splicing or protein function are irrelevant, which makes variant interpretation challenging to molecular diagnostic laboratories. In order to facilitate and improve clinical diagnostic assessment and genetic counseling, we present i) an update of the previously reported RNU4ATAC mutations and an analysis of the genetic variations affecting this gene using the Genome Aggregation Database (gnomAD) resource; ii) the pathogenicity prediction performances of scores computed based on an RNA structure prediction tool and of those produced by the Combined Annotation Dependent Depletion tool for the 285 RNU4ATAC variants identified in patients or in large-scale sequencing projects; iii) a method, based on a cellular assay, that allows to measure the effect of RNU4ATAC variants on splicing efficiency of a minor (U12-type) reporter intron. Lastly, the concordance of bioinformatic predictions and cellular assay results was investigated.

ERRα Expression in Bone Metastases Leads to an Exacerbated Antitumor Immune Response.

Bone is the most common metastatic site for breast cancer. Although the estrogen-related receptor alpha (ERRα) has been implicated in breast cancer cell dissemination to the bone from the primary tumor, its role after tumor cell anchorage in the bone microenvironment remains elusive. Here, we reveal that ERRα inhibits the progression of bone metastases of breast cancer cells by increasing the immune activity of the bone microenvironment. Overexpression of ERRα in breast cancer bone metastases induced expression of chemokines CCL17 and CCL20 and repressed production of TGFß3. Subsequently, CD8+ T lymphocytes recruited to bone metastases escaped TGFß signaling control and were endowed with exacerbated cytotoxic features, resulting in significant reduction in metastases. The clinical relevance of our findings in mice was confirmed in over 240 patients with breast cancer. Thus, this study reveals that ERRα regulates immune properties in the bone microenvironment that contributes to decreasing metastatic growth. SIGNIFICANCE: This study places ERRα at the interplay between the immune response and bone metastases of breast cancer, highlighting a potential target for intervention in advanced disease.

Topographical representation of odor hedonics in the olfactory bulb.

Hedonic value is a dominant aspect of olfactory perception. Using optogenetic manipulation in freely behaving mice paired with immediate early gene mapping, we demonstrate that hedonic information is represented along the antero-posterior axis of the ventral olfactory bulb. Using this representation, we show that the degree of attractiveness of odors can be bidirectionally modulated by local manipulation of the olfactory bulb's neural networks in freely behaving mice.

Estrogen related receptor alpha in castration-resistant prostate cancer cells promotes tumor progression in bone.

Bone metastases are one of the main complications of prostate cancer and they are incurable. We investigated whether and how estrogen receptor-related receptor alpha (ERRα) is involved in bone tumor progression associated with advanced prostate cancer. By meta-analysis, we first found that ERRα expression is correlated with castration-resistant prostate cancer (CRPC), the hallmark of progressive disease. We then analyzed tumor cell progression and the associated signaling pathways in gain-of-function/loss-of-function CRPC models in vivo and in vitro. Increased levels of ERRα in tumor cells led to rapid tumor progression, with both bone destruction and formation, and direct impacts on osteoclasts and osteoblasts. VEGF-A, WNT5A and TGFß1 were upregulated by ERRα in tumor cells and all of these factors also significantly and positively correlated withERRα expression in CRPC patient specimens. Finally, high levels of ERRα in tumor cells stimulated the pro-metastatic factor periostin expression in the stroma, suggesting that ERRα regulates the tumor stromal cell microenvironment to enhance tumor progression. Taken together, our data demonstrate that ERRα is a regulator of CRPC cell progression in bone. Therefore, inhibiting ERRα may constitute a new therapeutic strategy for prostate cancer skeletal-related events.

Why Optogenetics Needs in Vivo Neurochemistry.

In neuroscience, the consequences of optogenetic manipulation are often studied using in vivo electrophysiology and by observing behavioral changes induced by light stimulation in genetically targeted rodents. In contrast, reports on the in vivo neurochemical effects of optogenetic stimulation are scarce despite the improving quality of analytical techniques available to monitor biochemical compounds involved in neurotransmission. This intriguing lack of neurochemical information suggests the existence of unknown or misunderstood factors hampering the expected rise of a novel specialty putatively be termed "neurochemical optogenetics".

Human corticotropin-releasing factor type 1 receptor analysis with white blood cells mRNAs and DNA.

The human corticotropin-releasing factor receptor type 1 (hCRF-R1) functional transcript is mainly expressed in the anterior pituitary corticotrophs, a tissue usually not available for clinical investigation. Splice variants translated into defective isoforms of the receptor have been described in few peripheral tissues. The aim of this work was to determine whether peripheral white blood cells from healthy individuals, an accessible tissue for clinical investigation, were suitable for the analysis of the hCRF-R1 transcript and gene. We report that: (i) specific amplification of the hCRF-R1 transcript from peripheral white blood cells mRNAs is feasible; (ii) this transcript is similar to the functional transcript; (iii) the draft sequence of chromosome 17 and unrelated sequences allow direct sequencing of all 14 exons of the gene, adjacent splice sites and related branch points. In conclusion, these approaches would be suitable for studies in patients having isolated secondary glucocorticoids deficiency to implicate the hCRH-R1 in the etiology of the disease.

Insight into the role of CRMP2 (collapsin response mediator protein 2) in T lymphocyte migration: the particular context of virus infection.

Lymphocyte migration into the central nervous system is a critical step in the physiopathology of a variety of neurological diseases, including multiple sclerosis and virus-induced neuroinflammation. To better understand the molecular mechanisms involved in cells migration, we focused our studies on collapsin response mediator proteins (CRMPs), a group of phosphoproteins that mediate neural cell motility. There is now evidence that collapsin response mediator protein 2 (CRMP2) plays critical roles in the polarization (uropod formation) of T lymphocytes and their subsequent migration. CRMP2 was known to respond to semaphorin, ephrin and neurotrophin signaling in neurons. The link between the chemokine CXCL12, CRMP2 activity and cell migration has been demonstrated in T lymphocytes. These observations and comparisons of the activity of CRMPs in immune and non-immmune cells are summarized here. The ability of a human retrovirus to enhance lymphocyte migration through the modulation of CRMP2 activity is also discussed. In conclusion, viruses have the ability to manipulate the lymphocyte motility machinery, intensifying neural tissue invasion in infected patients.

A 7.1 kbp beta-myosin heavy chain promoter, efficient for green fluorescent protein expression, probably induces lethality when overexpressing a mutated transforming growth factor-beta type II receptor in transgenic mice.

The roles of transforming growth factor-beta (TGFbeta) in heart or skeletal muscle development and physiology are still the subject of controversies. Our aim was to block, in transgenic mice, the TGFbeta signalling pathway by a dominant negative mutant of the TGFbeta type II receptor fused to the enhanced green fluorescent protein (TbetaRII-KR-EGFP) under the control of a 7.1 kbp mouse beta-myosin heavy chain (betaMHC) promoter to investigate the roles of TGFbeta in the heart and slow skeletal muscles. First, we generated two transgenic lines overexpressing EGFP under the control of the 7.1 kbp betaMHC promoter. In embryos, EGFP was detectable as early as 7.5 days post coitum. In embryos, newborns and adults, EGFP was expressed mainly in the cardiac ventricles and in slow skeletal muscles. EGFP expression was intense in the bladder but weak in the intestines. In contrast to the endogenous betaMHC promoter, the activity of the 7.1 kbp betaMHC promoter in the transgene was not repressed after birth and remained high in adult transgenic mice. We obtained two founders with the transgene comprising the TbetaRII-KR-EGFP sequence under the control of the 7.1 kbp betaMHC promoter. These founders were generated at a very low frequency and expressed barely detectable levels of TbetaRII-KR-EGFP mRNA. Our failure to obtain transgenic lines overexpressing the dominant negative receptor suggests that the blocking of the TGFbeta signalling pathway in the heart and slow skeletal muscles could be embryonically lethal. To conclude, the 7.1 kbp betaMHC promoter directs high levels of transgene expression in the cardiac ventricles and in slow skeletal muscles of the mouse. Analysis of the consequences of the blocking of the TGFbeta signalling pathway in the heart will require the use of tissue specific means of conditional gene invalidation.