Neuritin Attenuates Cognitive Function Impairments in Tg2576 Mouse Model of Alzheimer's Disease

<div><p>Neuritin, also known as CPG15, is a neurotrophic factor that was initially discovered in a screen to identify genes involved in activity-dependent synaptic plasticity. Neuritin plays multiple roles in the process of neural development and synaptic plasticity, although its binding receptor(s) and downstream signaling effectors remain unclear. In this study, we found that the cortical and hippocampal expression of neuritin is reduced in the brains of Alzheimer's disease (AD) patients and demonstrated that viral-mediated expression of neuritin in the dentate gyrus of 13-month-old Tg2576 mice, an AD animal model, attenuated a deficit in learning and memory as assessed by a Morris water maze test. We also found that neuritin restored the reduction in dendritic spine density and the maturity of individual spines in primary hippocampal neuron cultures prepared from Tg2576 mice. It was also shown that viral-mediated expression of neuritin in the dentate gyrus of 7-week-old Sprague-Dawley rats increased neurogenesis in the hippocampus. Taken together, our results demonstrate that neuritin restores the reduction in dendritic spine density and the maturity of individual spines in primary hippocampal neurons from Tg2576 neurons, and also attenuates cognitive function deficits in Tg2576 mouse model of AD, suggesting that neuritin possesses a therapeutic potential for AD.</p></div

Neuritin increases synaptophysin expression in rat primary neuron cultures.

<p>Primary cultured cortical neurons were transfected at DIV 10 with EGFP or neuritin subcloned in a flag-IRES-EGFP vector by employing a calcium phosphate method. 3 days after transfection, the cells were lysed and Western blotting analysis was performed with an anti-synaptophysin antibody. β-actin was used as a loading control. Densitometric analysis was also performed. Neuritin transfection significantly increased synaptophysin expression levels by approximately 3.6-fold (*<i>p</i><0.05, Student's <i>t</i>-test). The data are presented as the mean ±SEM (n = 5 dishes from 5 independent cultures for each group).</p

Neuritin is down-regulated in human AD brains.

<p>(A) Neuritin mRNA levels in the cortex (medial frontal gyrus) and hippocampus of age-matched control subjects (69, 79 and 80 years old) and AD patients (67, 75, 81 and 87 years old) were evaluated by qRT-PCR. Neuritin mRNA levels in the cortex and hippocampus of the AD brains were significantly lower than those of the age-matched control subjects. The relative ratio of neuritin to GAPDH mRNA in the AD patients was 15.10±6.28% compared to control subjects in cortical regions and 69.16±5.95% compared to the control subjects in the hippocampus (***<i>p</i><0.001 and *<i>p</i><0.05, respectively, Student's <i>t</i>-test). Data are presented as the mean ± SEM. (B) Human brain tissues embedded in paraffin were cut into 4 µm-thick coronal sections and were used to perform immunohistochemistry with an anti-neuritin antibody (red) and DAPI (blue) staining. The fluorescent images were collected from the dentate gyrus, the CA1 and CA3 regions of the hippocampus on an LSM 510 confocal microscope (Zeiss). The black and white images merged with DAPI staining are also shown. The results are representative of 3 human AD brains and 3 age-matched control brains. Scale bars represent 100 µm.</p

Neuritin up-regulates neurogenesis in the dentate gyrus of 7-week-old SD rats.

<p>(A) A experimental scheme is shown. 7-week-old male SD rats were injected with EGFP- or neuritin-lentiviral particles in the dentate gyrus of the hippocampus using a stereotaxic apparatus (n = 6 for each group). BrdU was intraperitoneally administered for 6 days. (B) After 2 weeks, GFP expression was confirmed by confocal laser scanning microscopy. The scale bars indicate 200 µm (low-scaled panel) and 50 µm (magnified panel). (C) Brain sections were stained with anti-GFP (green), anti-BrdU (red), and anti-NeuN (purple) antibodies. Orthogonal analysis of the staining was conducted by confocal microscopy. (D) Cells stained with BrdU antibody were counted. (E) Staining with anti-GFP and anti-BrdU antibodies was performed and counted. (F) Staining with anti-BrdU, anti-NeuN and anti-GFP antibodies was performed and counted. The results were normalized as percentage ratios to the staining in EGFP expressing rats. Rats with neuritin expression showed an increase in BrdU/GFP expressing cells by 330.77±130.78% compared to the rats with EGFP expression. Quantitative analysis of triple staining with BrdU/NeuN/GFP antibodies showed that neuritin expression induced a significant increase of newly generated neuronal cells in the dentate gyrus by 338.46±123.08% compared to EGFP-injection (*<i>p</i><0.05, Student's <i>t</i>-test).</p

Neuritin restores the reduction in the dendritic spine density and the maturity of individual spines in Tg2576 neurons.

<p>(A) Representative images of dendrites and spines of rabies virus encoding EGFP-infected neurons are shown in the designated conditions. Scale bar represents 10 µm. (B) A summary histogram shows mean numbers of spines. Neuritin peptide (150 ng/ml) was added as a treatment for 3 days, and the dendritic spine density was then analyzed. The reduction in dendritic spine density in Tg2576 neurons (32.57±2.80/100 µm) was significantly increased to match the normal control levels in wt neurons (56.75±1.95/100 µm). (C) Proportion of spines (type I, II, III and IV) were analyzed by their morphology. Tg2576 neurons revealed a significant decrease particularly in type II spines (mushroom type; mature spines with a large head, >0.6 µm, and a short neck), but yielded a slightly higher density of type I spines (stubby type; less mature spines, relatively small spine head, <0.6 µm, and short neck, <0.3 µm) compared to the other groups. (D) A summary histogram showing mean numbers of each type of spine in the designated conditions (*<i>p</i><0.05, ***<i>p</i><0.001, one-way ANOVA followed by <i>post hoc</i> Bonferroni test).</p