Differences in Doublecortin Immunoreactivity and Protein Levels in the Hippocampal Dentate Gyrus Between Adult and Aged Dogs

Doublecortin (DCX), a microtubule-associated protein, specifically expresses in neuronal precursors. This protein has been used as a marker for neuronal precursors and neurogenesis. In the present study, we observed differences in DCX immunoreactivity and its protein levels in the hippocampal dentate gyrus between adult and aged dogs. In the adult dog, DCX immunoreactive cells with well-stained processes were detected in the subgranular zone of the dentate gyrus. Numbers of DCX immunoreactive cells in the dentate gyrus of the aged dog were significantly decreased compared to those in the adult dog. DCX immunoreactive cells in both adult and aged dog did not show NeuN (a marker for mature neurons) immunoreactivity. NeuN immunoreactivity in the aged dog was poor compared to that in the adult dog. DCX protein level in the aged dentate gyrus was decreased by 80% compared to that in the adult dog. These results suggest that the reduction of DCX in the aged hippocampal dentate gyrus may be involved in some neural deficits related to the hippocampus.     

Phosphorylation of doublecortin by protein kinase A orchestrates microtubule and actin dynamics to promote neuronal progenitor cell migration

Doublecortin (DCX) is a microtubule-associated protein that is specifically expressed in neuronal cells. Genetic mutation of DCX causes lissencephaly disease. Although the abnormal cortical lamination in lissencephaly is thought to be attributable to neuronal cell migration defects, the regulatory mechanisms governing interactions between DCX and cytoskeleton in the migration of neuronal progenitor cells remain obscure. In this study we found that the G(s) and protein kinase A (PKA) signal elicited by pituitary adenylate cyclase-activating polypeptide promotes neuronal progenitor cells migration. Stimulation of G(s)-PKA signaling prevented microtubule bundling and induced the dissociation of DCX from microtubules in cells. PKA phosphorylated DCX at Ser-47, and the phospho-mimicking mutant DCX-S47E promoted cell migration. Activation of PKA and DCX-S47E induced lamellipodium formation. Pituitary adenylate cyclase-activating polypeptide and DCX-S47E stimulated the activation of Rac1, and DCX-S47E interacted with Asef2, a guanine nucleotide exchange factor for Rac1. Our data reveal a dual reciprocal role for DCX phosphorylation in the regulation of microtubule and actin dynamics that is indispensable for proper brain lamination.     

New GABAergic interneurons in the adult neocortex and striatum are generated from different precursors

Ongoing neurogenesis in the adult mammalian dentate gyrus and olfactory bulb is generally accepted, but its existence in other adult brain regions is highly controversial. We labeled newly born cells in adult rats with the S-phase marker bromodeoxyuridine (BrdU) and used neuronal markers to characterize new cells at different time points after cell division. In the neocortex and striatum, we found BrdU-labeled cells that expressed each of the eight neuronal markers. Their size as well as staining for gamma-aminobutyric acid (GABA), glutamic acid decarboxylase 67, calretinin and/or calbindin, suggest that new neurons in both regions are GABAergic interneurons. BrdU and doublecortin-immunoreactive (BrdU+/DCX+) cells were seen within the striatum, suggesting migration of immature neurons from the subventricular zone. Surprisingly, no DCX+ cells were found within the neocortex. NG2 immunoreactivity in some new neocortical neurons suggested that they may instead be generated from the NG2+ precursors that reside within the cortex itself.     

Phagocytic activity of neuronal progenitors regulates adult neurogenesis

Whereas thousands of new neurons are generated daily during adult life, only a fraction of them survive and become part of neural circuits; the rest die, and their corpses are presumably cleared by resident phagocytes. How the dying neurons are removed and how such clearance influences neurogenesis are not well understood. Here, we identify an unexpected phagocytic role for the doublecortin (DCX)-positive neuronal progenitor cells during adult neurogenesis. Our in vivo and ex vivo studies demonstrate that DCX(+) cells comprise a significant phagocytic population within the neurogenic zones. Intracellular engulfment protein ELMO1, which promotes Rac activation downstream of phagocytic receptors, was required for phagocytosis by DCX(+) cells. Disruption of engulfment in vivo genetically (in Elmo1-null mice) or pharmacologically (in wild-type mice) led to reduced uptake by DCX(+) cells, accumulation of apoptotic nuclei in the neurogenic niches and impaired neurogenesis. Collectively, these findings indicate a paradigm wherein DCX(+) neuronal precursors also serve as phagocytes, and that their phagocytic activity critically contributes to neurogenesis in the adult brain.     

Developmental patterns of doublecortin expression and white matter neuron density in the postnatal primate prefrontal cortex and schizophrenia

Postnatal neurogenesis occurs in the subventricular zone and dentate gyrus, and evidence suggests that new neurons may be present in additional regions of the mature primate brain, including the prefrontal cortex (PFC). Addition of new neurons to the PFC implies local generation of neurons or migration from areas such as the subventricular zone. We examined the putative contribution of new, migrating neurons to postnatal cortical development by determining the density of neurons in white matter subjacent to the cortex and measuring expression of doublecortin (DCX), a microtubule-associated protein involved in neuronal migration, in humans and rhesus macaques. We found a striking decline in DCX expression (human and macaque) and density of white matter neurons (humans) during infancy, consistent with the arrival of new neurons in the early postnatal cortex. Considering the expansion of the brain during this time, the decline in white matter neuron density does not necessarily indicate reduced total numbers of white matter neurons in early postnatal life. Furthermore, numerous cells in the white matter and deep grey matter were positive for the migration-associated glycoprotein polysialiated-neuronal cell adhesion molecule and GAD65/67, suggesting that immature migrating neurons in the adult may be GABAergic. We also examined DCX mRNA in the PFC of adult schizophrenia patients (n?=?37) and matched controls (n?=?37) and did not find any difference in DCX mRNA expression. However, we report a negative correlation between DCX mRNA expression and white matter neuron density in adult schizophrenia patients, in contrast to a positive correlation in human development where DCX mRNA and white matter neuron density are higher earlier in life. Accumulation of neurons in the white matter in schizophrenia would be congruent with a negative correlation between DCX mRNA and white matter neuron density and support the hypothesis of a migration deficit in schizophrenia.     

Identification and cloning of a fur regulatory gene in Yersinia pestis.

Yersinia pestis is one of many microorganisms responding to environmental iron concentrations by regulating the synthesis of proteins and an iron transport system(s). In a number of bacteria, expression of iron uptake systems and other virulence determinants is controlled by the Fur regulatory protein. DNA hybridization analysis revealed that both pigmented and nonpigmented cells of Y. pestis possess a DNA locus homologous to the Escherichia coli fur gene. Introduction of a Fur-regulated beta-galactosidase reporter gene into Y. pestis KIM resulted in iron-responsive beta-galactosidase activity, indicating that Y. pestis KIM expresses a functional Fur regulatory protein. A cloned 1.9-kb ClaI fragment of Y. pestis chromosomal DNA hybridized specifically to the fur gene of E. coli. The coding region of the E. coli fur gene hybridized to a 1.1-kb region at one end of the cloned Y. pestis fragment. The failure of this clone to complement an E. coli fur mutant suggests that the 1.9-kb clone does not contain a functional promoter. Subcloning of this fragment into an inducible expression vector restored Fur regulation in an E. coli fur mutant. In addition, a larger 4.8-kb Y. pestis clone containing the putative promoter region complemented the Fur- phenotype. These results suggest that Y. pestis possesses a functional Fur regulatory protein capable of interacting with the E. coli Fur system. In Y. pestis Fur may regulate the expression of iron transport systems and other virulence factors in response to iron limitation in the environment. Possible candidates for Fur regulation in Y. pestis include genes involved in ferric iron transport as well as hemin, heme/hemopexin, heme/albumin, ferritin, hemoglobin, and hemoglobin/haptoglobin utilization.     

Disclosure of a pro-apoptotic glyceraldehyde-3-phosphate dehydrogenase promoter: anti-dementia drugs depress its activation in apoptosis

Overexpression and subsequent nuclear accumulation of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is involved in neuronal apoptosis induced by several stimuli in which GAPDH antisense oligonucleotides specifically block the increment (2 approximately 3 fold) of GAPDH mRNA contents occurring prior to neuronal death. However, these agents do not affect the basal, constitutive mRNA contents. This suggests that there may be distinct gene regulations for GAPDH mRNA expression. Herein, we cloned two types of promoter regions upstream of this gene; viz., #104 (1.02-kb) and #302 (2.46-kb). These fragments were inserted into the pGL3 luciferase reporter system and transiently transfected into cultured cerebellar neurons undergoing cytosine arabinonucleoside-induced apoptosis. The functional analysis of these constructs revealed that #104, but not #302, increased luciferase activity in response to the apoptotic stimulus. Deletion and replacement mutation analysis of the #104 fragment disclosed the promoter core harbored between the 154-bp and 84-bp domains (3.5-fold activity of the control). Furthermore, anti-dementia drugs (such as Cognex and Aricept) markedly depress the expression of this pro-apoptotic GAPDH promoter activity. Interestingly, immunocytochemical examination of human post-mortem materials from patients with Alzheimer's disease revealed nuclear aggregated GAPDH in neurons of the affected brain regions, implying an association with apoptotic cell death. The current findings indicate that induction of the pro-apoptotic protein GAPDH is genetically regulated at the level of promoter activation, and this protein may be an important molecular target for developing anti-apoptotic therapeutic agents in certain neurological illnesses.     

Identification of a critical control element directing expression of the muscle-specific transcription factor MRF4 in the mouse embryo

Skeletal muscle development in the vertebrate embryo critically depends on the myogenic regulatory factors (MRFs) including MRF4 and Myf5. Both genes exhibit distinct expression patterns during mouse embryogenesis, although they are genetically closely linked with multiple regulatory elements dispersed throughout the common gene locus. MRF4 has a biphasic expression profile, first in somites and later in foetal skeletal muscles. Here, we demonstrate by transgenic analysis that elements within a 7.5-kb promoter fragment of the MRF4 gene are sufficient to drive the embryonic wave of expression very similar to the endogenous gene in somites of mouse embryos. In contrast, a 3-kb fragment of the proximal promoter fails to support expression in the myotome, suggesting that essential cis-acting elements are located between -7.5 and -3 kb upstream of MRF4. Further analysis of this sequence delimits an essential region between -6.6 and -5.6 kb that together with the 3-kb promoter fragment directs transgene expression in the epaxial myotome of all somites during the appropriate developmental period. These data provide evidence that the partly overlapping expression patterns of Mrf4 and Myf5 in somites are controlled by distinct regulatory elements. We also show that 11.4 kb sequence upstream of MRF4, including the promoter and the somitic control region identified in this study, is not sufficient to elicit target specificity towards the strong Myf5 (-58/-48 kb) enhancer, suggesting that additional yet unidentified elements are necessary to convey promoter selectivity and protect the MRF4 gene from this enhancer.     

Adult neurogenesis in the short-lived teleost Nothobranchius furzeri: localization of neurogenic niches, molecular characterization and effects of aging

We studied adult neurogenesis in the short‐lived annual fish Nothobranchius furzeri and quantified the effects of aging on the mitotic activity of the neuronal progenitors and the expression of glial fibrillary acid protein (GFAP) in the radial glia. The distribution of neurogenic niches is substantially similar to that of zebrafish and adult stem cells generate neurons, which persist in the adult brain. As opposed to zebrafish, however, the N. furzeri genome contains a doublecortin (DCX) gene. Doublecortin is transiently expressed by newly generated neurons in the telencephalon and optic tectum (OT). We also analyzed the expression of the microRNA miR‐9 and miR‐124 and found that they have complementary expression domains: miR‐9 is expressed in the neurogenic niches of the telencephalon and the radial glia of the OT, while miR‐124 is expressed in differentiated neurons. The main finding of this paper is the demonstration of an age‐dependent decay in adult neurogenesis. Using unbiased stereological estimates of cell numbers, we detected an almost fivefold decrease in the number of mitotically active cells in the OT between young and old age. This reduced mitotic activity is paralleled by a reduction in DCX labeling. Finally, we detected a dramatic up‐regulation of GFAP in the radial glia of the aged brain. This up‐regulation is not paralleled by a similar up‐regulation of S100B and Musashi‐1, two other markers of the radial glia. In summary, the brain of N. furzeri replicates two typical hallmarks of mammalian aging: gliosis and reduced adult neurogenesis.