P1‐073: NEURITIN ATTENUATES IMPAIRMENTS IN COGNITIVE FUNCTION IN ALZHEIMER'S DISEASE

Field: Neuroscience (Life Sciences)

Neuritin, also known as candidate plasticity gene 15 (CPG 15), encodes a small, extracellular glycosylphosphatidylinositol (GPI)-anchored cell surface protein that was first identified in a screen for activity-regulated genes induced by kainate stimulated seizure in the rat dentate gyrus. Neuritin is critical for dendritic outgrowth, maturation, and axonal regeneration. Its gene is located within the 6p2424-p25 interval on chromosome 6 . Three cyclic AMP responsive elements were identified in the promoter region of the gene, suggesting that neuritin expression is mediated by cyclic AMP responsive element binding protein (CREB). An in vivo study showed an important role for CREB in activity-dependent neuritin expression in the barrel cortex of control and CREB a, D -knockout mice. After eliciting receptive field plasticity by whisker trimming, neuritin expression increases in the barrel corresponding to the spared whisker. However, in CREB mutant mice, neuritin expression is not induced to the same extent as in wild-type (wt) littermates, indicating that CREB is necessary for neuritin regulation during receptive field plasticity in the adult cortex. [a1] Viral-mediated expression of neuritin A-PCR mediated neuritin cDNA was subcloned into IRES-EGFP lentiviral vector. Viral particles were prepared and concentrated by MACROGEN (Seoul, South Korea). The viral particle from IRES-EGFP-neuritin or EGFP-lentivirus was bilaterally injected into the dentate gyrus of the hippocampus of 13 month-old wt, Tg2576 mice or 7 week-old SD rats, at 4.4 mm posterior to the bregma (AP), 1.1mm lateral to the midline (ML), 8.0 mm ventral to the surface of the skull (DV), according to the Brain Atlas. Morris water maze test The water maze apparatus, which is a circular pool (140 cm in diameter, 45 cm high) was filled with 21-23 °C opaque water using dry milk powder to water and located in a laboratory that contained prominent extra-maze cues. Animals are required to find a submerged platform in the pool using the spatial cues. The three starting points were changed daily. Spatial training consisted of 5 sessions, (3 trials per session per day) during which the platform was left in the same position. In each training session, the latency to escape on to the hidden platform was recorded. 24 h later at final train session, the escape platform was removed, and each mouse was allowed to swim for 60 sec in the maze as a probe test. The times of the mice stayed in each zone was recorded. Data collection was automated by a video image motion analyzer (Ethovision, Noldus Information Technology h.v., Netherlands). Quantitative real-time RT-PCR (qRT-PCR)Total RNA was extracted from the tissues using a RNA purification kit (Quiagen, Hilden, Germany) and 0.5 μg of RNA was processed for cDNA synthesis using oligo (dT)20 primers and SuperScript III reverse transcriptase (Invitrogen, CA, USA). cDNA was then amplified on the 7500 Fast Real-Time PCR system (Applied Biosystems, CA, USA), employing the ΔΔCt method with SYBR Green I (Roche, CA, USA). Primers used here are as follows; neurutin forward: 5'-CGAGA GAGAC ACCAG GAGCA-3' ; reverse: 5'-GGGCG AAAGA TATGT GGGAT-3', GAPDH forward; 5'-ACCACCTGGAGAAGGCTGG-3', reverse; 5'-CTCAGTGTAGCCCAGGATGC-3. Primary cortical neuron culture and transfection The cerebral cortex was dissected from an embryonic day 18 Sprague-Dawley (SD) rat embryo and dissociated by gentle triturate. Cells were plated on poly-D-lysine (Sigma, MO, USA)-coated plates and cultured in B27-supplemented medium (Gibco-Invitrogen, Carlsbad, CA) for 7 days. Cells were transiently transfected with 8 m g of neurutin subcloned in flag-IRES-EGFP vector, which was a generous gift from Dr. Nedivi at Massachusetts Institute of Technology, 9 m l CaCl 2, 58 m l DW and 75 m l 2 X Hank's buffered solution (Invitrogen, Carlsbad, CA) in 1ml of growing medium according to the manufacturer's instruction. At 48h post-transfection, the efficiencies of the transient transfections of plasmids were confirmed by the expression of GFP fluorescence. Fluorescence microscopic observation was performed using Olympus IX50 (Tokyo, Japan). Western blotting Protein was separated in SDS/PAGE gel, electrophoresed at 20∼100 m g of protein/lane, and transferred onto a nitrocellulose membrane (Amersham Pharmacia, Buckinghamshire, UK). The protein blot was probed with anti-synaptophysin (Chemicon international, Temecula, CA) detected using horseradish peroxidase-conjugated secondary antibody (GE Healthcare AB, Sweden). Immunoreactive bands were visualized using an enhanced chemiluminescence system (ECL plus ; GE Healthcare AB, Sweden). BrdU staining BrdU (50mg/kg/day, Sigma) was injected intraperitoneally for 6 days into the SD rats with viral-mediated expression of EGFP or neuritin. H ippocampal regions were dissected from the brains isolated from the rats and processed for immunohistochemistry. Immunohistochemistry Fixed brains in 10% neutral buffered formalin for 48 h were dehydrated and embedded in paraffin. Before immunostaining, slides were deparaffinized by oven heating and immersion in xylene. After dehydration through graded alcohols to water, a primary antibody was revealed by incubating the cells for overnight with Cy3-conjugated secondary antibody (Molecular Probes, CA, USA). After three washes with permeabilization buffer and one wash with TBS, slices were mounted on microscope slides in mounting medium (DAKO, CA, USA). Frozen virus-injected wt, Tg2576 or SD rat brain block were sectioned 30 μm and placed into vial for free floating immunohistochemistry. Brain section inactivated endogenous peroxidase and incubated 2N HCl for 30 min at 37 °C. They were blocked with 5% bovine serum albumin (BSA) in TBS with TritonX-100 and incubated primary and secondary antibody. Then they washed 3 times with TBS and placed onto glass slide to mount. Confocal microscopic observation was performed using Zeiss LSM510 (Jena, Germany). Fetal neural stem cell culture Fetal neural stem cell culture was performed as previously described [17]. Cells were isolated by mechanical dissociation in MEM (minimum essential medium) supplemented with 1M HEPES and 1% penicillin/streptomysin (pH 7.2), and 10 7 cells were cultured in a T-75 flask. The generation of neural spheres derived from fetal neural stem cells was performed as previously described [18] for immunocytochemistry analysis, we dissociated the neurospheres, and subcultured the fetal neural stem cells (5 x 10 4 cells/well) in 24-well plates that contained 12 mm glass coverslips (Marienfeld, Lauda-Konigshofen, Germany) pre-coated with laminin (Roche, Mannheim, Germany) at a concentration of 20 μg/ml in Minimum Essential Medium with 1M HEPES and penicillin/streptomycin. The cells were incubated in the differentiation medium without growth factors (EGF and bFGF) for 5 days and analyzed with immunocytochemistry Immunocytochemistry Cells were fixed in 4% paraformaldehyde for 30 min at room temperature and were them washed with phosphate buffered saline (PBS), and with PBS containing 0.05% Tween 20 for permeabilization. Next, cells were blocked wit blocking buffer (3% normal goat serum, 2% BSA and 3% Triton-X 100 in PBS) for 1h at room temperature. After washing, cells were incubated in PBS containing antibodies against mouse anti-Tuj-1 (1:100) and rabbit GFAP (1:200) for 2 h at room temperature. After the 2 h incubation, cells were washed with PBS, and then cells were incubated in PBS containing Alexa Flour 488 or 546- conjugates secondary antibodies and 4'.6-diamidino-2-phenylindole (DAPI,1 μ M) 1h at room temperature. Cells were then observed under a confocal microscope (Olympus FluoView 1000) Statistical Analysis Data are expressed as the means ± SEM values. One-way ANOVA and student's t -test (SPSS, IL, USA) were used to determine statistical significance where p<0.05 was considered to be significant. 1)Neutin is down-regulated in the brains of AD patients 2)Neutin attenuates learning and memory impairments in Tg2576 mice 3)Neuritin regulates neurogenesis in the dentate gyrus 4)Neuritin increases the neuronal population during the differentiation of neural stem cells Here we demonstrate that the expression of neuritin was significantly down-regulated in the hippocampus and cerebral cortex of AD patients compared to age-matched control subjects. We also demonstrate that viral-mediated expression of neuritin in the dentate gyrus of Tg2576 mice harboring the Swedish double mutated human APP695 gene attenuated the deficits in learning and memory.It was also found that the expression of neuritin up-regulates protein level of synaptophysin, a presynaptic marker, in primary hippocampal neuron cultures. In addition, NeuN/BrdU staining was up-regulated in the hippocampal region of the Sprawue-Dawley (SD) rats expressing neuritin. Furthermore, neuritin increased the neural differentiation in fetal neural stem cells. Taken together, our results suggest that neuritin possesses a therapeutic potential for AD by upregulating synaptogenesis and neurogenesis in brains.