Cell Mol Neurobiol. 2015 Jan;35(1):57-70. doi: 10.1007/s10571-014-0145-7.

Time course of spinal doublecortin expression in developing rat and porcine spinal cord: implication in in vivo neural precursor grafting studies.

Juhasova J, Juhas S, Hruska-Plochan M, Dolezalova D, Holubova M, Strnadel J, Marsala S, Motlik J, Marsala M.
  • Laboratory of Cell Regeneration and Plasticity, Institute of Animal Physiology and Genetics, AS CR, v.v.i., Rumburska 89, 27721, Libechov, Czech Republic.

 

Abstract

Expression of doublecortin (DCX), a 43-53 kDa microtubule binding protein, is frequently used as (i) an early neuronal marker to identify the stage of neuronal maturation of in vivo grafted neuronal precursors (NSCs), and (ii) a neuronal fate marker transiently expressed by immature neurons during development. Reliable identification of the origin of DCX-immunoreactive cells (i.e., host vs. graft) requires detailed spatial and temporal mapping of endogenous DCX expression at graft-targeted brain or spinal cord regions. Accordingly, in the present study, we analyzed (i) the time course of DCX expression in pre- and postnatal rat and porcine spinal cord, and (ii) the DCX expression in spinally grafted porcine-induced pluripotent stem cells (iPS)-derived NSCs and human embryonic stem cell (ES)-derived NSCs. In addition, complementary temporospatial GFAP expression study in porcine spinal cord was also performed. In 21-day-old rat fetuses, an intense DCX immunoreactivity distributed between the dorsal horn (DH) and ventral horn was seen and was still present in the DH neurons on postnatal day 20. In animals older than 8 weeks, no DCX immunoreactivity was seen at any spinal cord laminae. In contrast to rat, in porcine spinal cord (gestational period 113-114 days), DCX was only expressed during the pre-natal period (up to 100 days) but was no longer present in newborn piglets or in adult animals. Immunohistochemical analysis was confirmed with a comparable expression profile by western blot analysis. Contrary, the expression of porcine GFAP started within 70-80 days of the pre-natal period. Spinally grafted porcine iPS-NSCs and human ES-NSCs showed clear DCX expression at 3-4 weeks postgrafting. These data indicate that in spinal grafting studies which employ postnatal or adult porcine models, the expression of DCX can be used as a reliable marker of grafted neurons. In contrast, if grafted neurons are to be analyzed during the first 4 postnatal weeks in the rat spinal cord, additional markers or grafted cell-specific labeling techniques need to be employed to reliably identify grafted early postmitotic neurons and to differentiate the DCX expression from the neurons of the host.

PMID: 25487013

 

 

Supplements:

Developmental study of CNPase expression during fetal and postnatal period in the miniature pig spinal cord

 

INTRODUCTION

CNPase is a unique enzyme (the enzyme that metabolizes 2′,3′-cAMP to 2′-AMP) predominantly presented in oligodendrocytes and Schwann cells that form myelin in the nervous system. Developmentally, this enzyme appears early in oligodendrocytes, earlier than most other myelin proteins. The enzyme itself exists in two forms in most species with molecular masses of 48 (CNP2) and 46 kD (CNP1) (1). Since the enzyme is a myelin- associated enzyme, it is of considerable interest in the study of disease and disorders in which myelin is affected, such as sclerosis multiplex, subacute sclerosing panencephalitis, acquired immunodeficiency with CNS involvement, peripheral neuropathies, etc. (2–4). Moreover, in the brain, the deficiency in CNPase leads to increased susceptibility to brain injury and neurological diseases (5). Another important use is the identification of oligodendrocyte progenitor cells, very early in prenatal and postnatal period development (6). The goal of our experiments was to study an expression of CNPase in the miniature pig spinal cord at fetal and postnatal stages.

 

METHODS

Sample preparation and Cryosectioning

General anesthesia of pigs (no fetal stages) was induced by intramuscular application TKX mixture (Tiletamine 100 mg, Ketamine 10% 3ml, Xylazine 2% 3 ml) at a dose of 2 ml per 20 kg of body weight. We perfused the animals with heparinized ice-cold PBS and followed by 4% paraphormaldehyde (PFA) in PBS (pH 7.4). Whole body perfusion (no 40 and 60D fetuses, only PFA fixation) was done through the aorta (fetal stages) or left ventricle (postnatal stages). Spinal cords were dissected out, post-fixed in the same fixative solution overnight and cryoprotected by soaking sequentially in 10%, 20% and 30% sucrose at 4° C. Transverse sections of lumbar, thoracic and cervical spinal cord or brain were cut on freezing microtome (CM1850; Leica Microsystems) at 40 μm thicknesses, and stored in PBS at 4° C.

Immunofluorescence

For double immunostaining of minipig spinal cords or brains, a nonspecific binding was blocked for 1 hr at RT with 1% bovine serum albumin (Albumin bovine Fraction V; 11922; SERVA) in 0.2 % Triton-X PBS. Free-floating sections were sequentially probed with anti-CNPase antibody (mouse monoclonal; 1:250; C5922; Sigma) in 0.2 % Triton-X PBS for 72 hr at 4° C. Afterwards we incubated the slides with secondary antibody (Alexa Fluor® 488 goat anti-mouse IgG (H+L); A-11001; 1:500; Alexa Fluor® 555 goat anti-mouse IgG; A-21424; 1:500; Invitrogen) in 0.2 % Triton-X PBS for 1.5 hr at room temperature. Next the sections were analyzed and photographed on epifluorescence microscope (AX70; Olympus) and confocal microscope (TCS SP5; Leica Microsystems).

Western Blot

Miniature pig spinal cords and brain without PFA fixation were cryo-sectioned. Collection of fifty sections (10μm) were lysed for 30 minutes using lysis buffer containing 50mM Tris (pH 7.4) (5429.3; Roth), 250mM NaCl (3957.2; Roth), 5mM EDTA (E5134; Sigma), 50mM NaF (S-1504; Sigma), 1mM Na3VO4 (S6508; Sigma) in 1% Triton® X-100 (T8532; Sigma) with protease inhibitor cocktail tablets (Complete Mini, EDTA-free; 11836170001; Roche) and 1mM phenylmethylsulphonyl fluoride (PMSF; 837091; Roche). Sonications of all samples was performed in cold water bath for 5 minutes and followed by centrifugation at 10 000 g at 4oC for 20 min. Total protein levels were determined by BCA Protein Assay Kit (#23225, ThermoScientific) and 15 micrograms of each sample was loaded on the 10% gel acrylamide gels together with sample buffer (125 mM Tris-HCl, 4% SDS, 20% glycerol, 10% 2-mercaptoethanol, 0.004% bromophenol blue) and distilled water. After polyacrylamide gel electrophoresis the proteins were transferred to nitrocellulose membranes using semidry blotting system. The membranes were blocked with 5% nonfat dry milk (NFDM) in tris-buffered saline with 0.5% Tween 20 (TBS-T). The membranes were incubated with the primary antibody mouse monoclonal antibody anti-CNPase (Sigma, C5922) diluted 1:500 in 5% NFDM in TBS-T overnight on roller at 4°C. After washing with TBS-T buffer the membranes was consequently incubated in donkey anti mouse Ab HRP conjugate, (Jacksson Lab) diluted 1:10 000 in 5% NFDM in TBS-T one hour at RT with gentle shaking. SuperSignal West Pico Chemiluminescent Substrate (34077, Pierce) detection system was used for visualization. Western blotting signal was quantified by determining the grey values of given bands using the ImageJ software.

 

RESULTS

40 days old fetal stage – no CNPase activity in any part of spinal cord detected by immunofluorescence or Western blotting (Fig. 1A and 2).

60 – 80 days old fetal stage – in miniature pig spinal cord the CNPase activity started closely before 70 embryonal day and possess rostro-caudal pattern from cervical to lumbal spinal cord (Fig. 2). By immunofluorescence we detected weak CNPase positivity in the dorsal and lateral horn of miniature pig spinal cord only (Fig. 1B – C).

100 – 110 days old fetal stage – the immunofluorescence detection showed that the CNPase activity was present at the whole gray matter (weak white matter signal) of miniature pig spinal cord (Fig. 1D – D´´).

Newborn piglet – 9months old pig – the Western blotting revealed strong CNPase activity in the all miniature pig spinal cord segments (Fig. 2). The CNPase positivity was present at the whole gray matter (weak white matter signal – 1 day old pig – Fig. 1E – E´) and also white matter (> 3 month old pig – Fig. 1F – G´) of miniature pig spinal cord.

 

 

fig1-1Fig. 1.

A. No CNPase (green) immunoreactivity in the porcine fetal spinal cord at 40 day (only artefacts – lumbal).

B. Distribution of CNPase (green) immunoreactivity in the porcine fetal spinal cord at 60 day (lumbal). Starting CNPase positivity in the neuropil of dorsal horn, the rest spinal cord is without CNPase expression.

C. Slight CNPase (green) positivity in dorsal and lateral horn of miniature pig spinal cord at 80 fetal day (lumbal). There was no expression in ventral horn.

D-D´´. Intense CNPase (green) positivity in the whole gray matter (no present in white matter) of fetal spinal cord at 100 day (lumbal). Dorsal (D), lateral (D´) and ventral (D´´) horn.

E-E´. Similar to 100 day old miniature pig fetus the CNPase (green) expression was intensively present in gray matter (no white matter) of newborn piglet spinal cord (lumbal).

F-F´´´´ The massive CNPase (green) positivity in the whole gray matter of lumbal spinal cord (3 month old pig). The white matter showed lower CNPase positivity in comparison with gray matter. F´ (white matter), F´´ (central canal), F´´´ (dorsal horn), F´´´´ (ventral horn) – higher magnification of boxed areas from F image.

G-G´. The massive CNPase (red) expression in the gray (G´- ventral horn) and also in the white (G) matter of lumbal spinal cord (9 month old pig).

All images – Dapi positive nuclei (blue).

 

 

CONCLUSION

The study showed that CNPase expression, mimicking the oligodendrocyte differentiation and early myelinization process, in the miniature pig spinal cord, started closely before 70 day of fetal period, predominantly at the cervical spinal cord, proceeding towards thoracic and lumbosacral level at later developmental stages up to adulthood. On the other side GFAP expression, as a marker of astrocytes and ependymal cells, was present in all spinal cord segments (cervical, thoracic and lumbal) at the same fetal time (70D) yet.

 

ACKNOWLEDGEMENT

This study was supported by the National Programme of Sustainability, project number LO1609 (Czech Ministry of Education, Youth and Sports), Center PIGMOD (Pig Model of Disease), Ministry of Heath of the Czech republic, grant 15-25813A, the Norwegian Financial Mechanism 2009– 2014 and the Ministry of Education, Youth and Sports under Project Contract no. MSMT 28477/ 2014 HUNTINGTON 7F14308 and RVO: 67985904. All rights reserved.

 

 

Fig.2Fig. 2.

Western blot of different parts of miniature pig spinal cords. No CNPase specific bands were detected at 40 days old miniature pig fetal stage. At first it´s expression was detected in the next fetal stage (70D), but there missed a bands of lumbal spinal cord and the thoracal bands was smaller than cervical one. At the next fetal stage (100D) and postnatal period we detected significant bands of CNPase in all parts of spinal cords.

 

References

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