A powerful transgenic tool for fate mapping and functional analysis of newly generated neurons

Background: Lack of appropriate tools and techniques to study fate and functional integration of newly generated neurons has so far hindered understanding of neurogenesis' relevance under physiological and pathological conditions. Current analyses are either dependent on mitotic labeling, for example BrdU-incorporation or retroviral infection, or on the detection of transient immature neuronal markers. Here, we report a transgenic mouse model (DCX CreERT2) for time-resolved fate analysis of newly generated neurons. This model is based on the expression of a tamoxifen-inducible Cre recombinase under the control of a doublecortin (DCX) promoter, which is specific for immature neuronal cells in the CNS. Results: In the DCX-CreERT2 transgenic mice, expression of CreERT2 was restricted to DCX+ cells. In the CNS of transgenic embryos and adult DCX-CreERT2 mice, tamoxifen administration caused the transient translocation of CreERT2 to the nucleus, allowing for the recombination of loxP-flanked sequences. In our system, tamoxifen administration at E14.5 resulted in reporter gene activation throughout the developing CNS of transgenic embryos. In the adult CNS, neurogenic regions were the primary sites of tamoxifen-induced reporter gene activation. In addition, reporter expression could also be detected outside of neurogenic regions in cells physiologically expressing DCX (e. g. piriform cortex, corpus callosum, hypothalamus). Four weeks after recombination, the vast majority of reporter-expressing cells were found to co-express NeuN, revealing the neuronal fate of DCX+ cells upon maturation. Conclusions: This first validation demonstrates that our new DCX-CreERT2 transgenic mouse model constitutes a powerful tool to investigate neurogenesis, migration and their long-term fate of neuronal precursors. Moreover, it allows for a targeted activation or deletion of specific genes in neuronal precursors and will thereby contribute to unravel the molecular mechanisms controlling neurogenesis

Institutional analysis in a digital era: mechanisms and methods to understand emerging fields

Walter Powell, Achim Oberg, Valeska Korff, Carrie Oelberger and Karina Kloos deal with the processes and mechanisms of organizational change and field transformation. On the one hand, this is a classical topic of neo-institutional theory and research, and the authors make use of an impressive array of knowledge from previous studies here. On the other hand, and based on that intellectual history, as the authors call it, they conduct a highly innovative study by focusing on new organizational forms and field transformation in the nonprofit sector. To underline innovativeness, the authors have developed a web crawler in order to determine change by analyzing organizations, websites and their references to other organizations through hyperlinks. By doing so, they identify the diversity and dynamics of organizational fields whose boundaries are becoming increasingly porous

The endemisation of schistosomiasis in Porto de Galinhas, Pernambuco, Brazil, 10 years after the first epidemic outbreak

In 2000, after heavy rains and floods in Porto de Galinhas, Pernambuco, Brazil, an outbreak of schistosomiasis was recorded, of which 62.2% (412 cases) were of the acute clinical form. Between 2001-2009, occasional findings of Biomphalaria snails parasitised by Schistosoma mansoni indicated that disease transmission was still occurring. This motivated a new epidemiological survey between August-December 2010 to provide an update of the occurrence of this health hazard and to investigate the process of disease endemisation at this locality. This survey gathered parasitological, clinical and malacological data. The results of this survey, compared with data from the year 2000 survey, showed the following: (i) over these 10 years, there were declines in the total percentage of cases and the percentage of acute forms, (ii) the acute clinical form now represents 23.3% in contrast with the 62.2% detected in 2000 and (iii) the current prevalence of schistosomiasis is 15.7%, while in 2000 32.1% of the individuals were diagnosed as parasitised. Today, the chronic clinical form represents 76.7% of the total number of cases diagnosed, thus showing that over the 10-year period the occurrences of clinical forms became inverted. These findings, together with visual observation of insalubrious environmental conditions, indicate that schistosomiasis has become endemic in Porto de Galinhas

Expression analysis of <i>Lrrk2</i> mRNA in the forebrain and midbrain of postnatal mice.

<p>ISH for <i>Lrrk2</i> mRNA in coronal sections of forebrain (left part) and midbrain (right part) from postnatal day 7, postnatal day 21 and adult mice. Note that expression of <i>Lrrk2</i> is highly dynamic in the postnatal forebrain. While the expression level in the striatum and the olfactory tubercle seem to increase dramatically during development, the <i>Lrrk2</i> level in cortex remain rather unchanged (<b>A,C,E</b>). On the level of the midbrain, ISH signals for <i>Lrrk2</i> augment considerably in the hippocampus and cortex, while the level in midbrain structures like the Substantia nigra pars compacta (white arrows, SNc) remain quite low (<b>B,D,F</b>). Abbreviations: Cc, corpus callosum; Cx, cortex; Cp, choroid plexus (white arrowhead in B’); Cs, superior colliculus; Hi, hippocampus; Ot, olfactory tubercle; Pc, piriform cortex; Pn, parafascicular nucleus; Rn, red nucleus; Se, septum; SNc, SN pars compacta; SNr, SN pars reticulata; St, striatum. Scale bars represent 1 mm.</p

Comparative expression analysis of <i>Lrrk1</i> and <i>Lrrk2</i> mRNA in the brain of adult mice.

<p>ISH for <i>Lrrk1</i> (top part) and <i>Lrrk2</i> (bottom part) mRNA in sections from P21 (<b>A–D</b>) and adult mice (<b>E</b>). Note that <i>Lrrk1</i> mRNA is barely detectable in the adult mouse brain and only visible in the non-neuronal meninges (white arrow) and the olfactory bulb (white arrowhead) (<b>A</b>). A detailed view onto the adult olfactory bulb depicts the solely neuronal expression of <i>Lrrk1</i> in the mitral cell layer (<b>B</b>). Specificity of the <i>Lrrk1</i> signals were verified by using the corresponding sence-probe as negative control (<b>C</b>). In contrast, strong <i>Lrrk2</i> expression can be detected in various regions throughout the postnatal (<b>D</b>) and adult CNS (<b>E</b>). Abbreviations: An, anterior olfactory nucleus; Bs, brain stem; Cb, cerebellum; Cc, corpus callosum; Cm, motor cortex; Co, cortex; Cp, choroid plexus; Cs, somatosensory cortex; Cv, visual cortex; Gl, glomerular layer; Gr, granual layer; Hi, hippocampus; Me, meninges; Mi, mitral layer; Ob, olfactory bulb; Ot, olfactory tubercle; Pn, parafascicular nucleus; Pl, plexiform layer; Po, pons; SNc, substantia nigra pars compacta; St, striatum; Th, thalamus; Lv, lateral ventricle. Scale bars represent 1 mm (A, D, E) and 500 µm (B).</p

Cellular expression analysis of <i>Lrrk2</i> mRNA in the striatum of adult mice.

<p>(<b>A</b>) High magnification of a representative brightfield ISH image using radioactive-labeled <i>Lrrk2</i>-specific riboprobes and counterstained by cresyl violet: <i>Lrrk2</i> mRNA is predominantly expressed in neurons (black arrows, <i>Lrrk2</i>-positive neurons; white arrows, <i>Lrrk2</i>-negative neurons; black arrowheads, <i>Lrrk2</i>-positive glia; white arrowheads, <i>Lrrk2</i>-negative glia). (<b>B, C</b>) Representative images of double <i>in situ</i>−/Immunohistochemistry (ISH/IHC) in the medial part of the putamen: ISH for <i>Lrrk2</i> using DIG-labeled riboprobes (non-fluorescent black precipitate) followed by IHC stainings (green) for the two main dopamine receptors D1 (DRD1a) and D2 (DRD2). (<b>D, E</b>) Quantification of the <i>Lrrk2</i>/DRD1a and <i>Lrrk2</i>/DRD2 ISH/IHC stainings in the striatum revealed 36% <i>Lrrk2</i>-positive, 25% DRD1a-positive and 39% double positive cells. In case of the DRD2 <i>in situ</i>−/Immunohistochemistry, 38% <i>Lrrk2</i>-positive, 24% DRD2-positive and 37% double positive cells could be detected. Scale bar represents 25 µm.</p

Comparative expression analysis of <i>Lrrk1</i> and <i>Lrrk2</i> mRNA during embryogenesis.

<p>ISH for <i>Lrrk1</i> (left part) and <i>Lrrk2</i> (right part) mRNA in sections from E10, E12.5 and E15.5 embryos. For each embryo, a brightfield image (e.g. A, for anatomical orientation) and a darkfield image (e.g. A’, ISH signals in white) are shown. (<b>A–D</b>) At E10, small hotspots of <i>Lrrk1</i> expression were observed only in the cephalic mesenchyme (Cm in A+B), the palate and the optic stalk (Oc in B), while <i>Lrrk2</i> mRNA was found basically in the urogenital ridge (Ur in C, arrow). Note that at this stage of gestation expression of <i>Lrrk1</i> and <i>Lrrk2</i> was virtually absent from the developing CNS (A–D). (<b>E,F</b>) At E12.5, additional spots for <i>Lrrk1</i> expression were visible in the epithelia of the nose and mouth (Nc in E) and for <i>Lrrk2</i> expression in the choroid plexus (Cp in F, arrow) and developing pituitary gland (Pi in F). (<b>G,H</b>) Around E15.5, <i>Lrrk1</i> and <i>Lrrk2</i> expression became stronger in several organs of the embryos (G+H) including liver (Li), kidney (Ki), lung (Lu) and heart (Pe) as well as in the choroid plexus (Cp, arrows). Ao, Aorta; Ba, branchial arch; Cm, cephalic mesenchyme; Cx, cortex; Cp, choroid plexus; Da, dorsal aorta; Dg, dorsal root ganglia; Fb, forebrain; Ge, ganglionic eminence; Gu, gut; Hb, hindbrain; Hl, hind limb; Id, intervertebral disc; In, incisive; Jl, lower jaw; Ki, kidney; Lb, limb but; Li, liver; Lu, lung; Mb, midbrain; Nc, nasal cavity; Oc, optic cup; Os, optic stalk; Pa, palate; Pe, pericardium; Rp, Rathke’s pouch; Sp, spinal cord; Ur, urogenital ridge; V4, fourth ventricle; Vl, lateral ventricle. Orientation of sections: A+C, coronal; B, horizontal; D–H, sagittal. Scale bars represent 500 µm in A–D, 1 mm in E–H.</p

Loss of Parkin or PINK1 Function Increases Drp1-dependent Mitochondrial Fragmentation*

Loss-of-function mutations in the parkin gene (PARK2) and PINK1 gene (PARK6) are associated with autosomal recessive parkinsonism. PINK1 deficiency was recently linked to mitochondrial pathology in human cells and Drosophila melanogaster, which can be rescued by parkin, suggesting that both genes play a role in maintaining mitochondrial integrity. Here we demonstrate that an acute down-regulation of parkin in human SH-SY5Y cells severely affects mitochondrial morphology and function, a phenotype comparable with that induced by PINK1 deficiency. Alterations in both mitochondrial morphology and ATP production caused by either parkin or PINK1 loss of function could be rescued by the mitochondrial fusion proteins Mfn2 and OPA1 or by a dominant negative mutant of the fission protein Drp1. Both parkin and PINK1 were able to suppress mitochondrial fragmentation induced by Drp1. Moreover, in Drp1-deficient cells the parkin/PINK1 knockdown phenotype did not occur, indicating that mitochondrial alterations observed in parkin- or PINK1-deficient cells are associated with an increase in mitochondrial fission. Notably, mitochondrial fragmentation is an early phenomenon upon PINK1/parkin silencing that also occurs in primary mouse neurons and Drosophila S2 cells. We propose that the discrepant findings in adult flies can be explained by the time of phenotype analysis and suggest that in mammals different strategies may have evolved to cope with dysfunctional mitochondria