Neuritin Attenuates Cognitive Function Impairments in Tg2576 Mouse Model of Alzheimer’s Disease Yoori Choi1., Kihwan Lee1,2., Junghwa Ryu1., Hyoun Geun Kim3, A Young Jeong3, Ran-Sook Woo4, Jun-Ho Lee5, Jin Won Hyun6, Seokyung Hahn7, Joung-Hun Kim3*, Hye-Sun Kim1,8* 1 Department of Pharmacology and Biomedical Sciences, Neuroscience Research Institute, College of Medicine, Seoul National University, Seoul, Republic of Korea, 2 National Research Laboratory for Pain, Dental Research Institute and Department of Neurobiology and Physiology, School of Dentistry, Seoul National University, Seoul, Republic of Korea, 3 Department of Life Science, Pohang University of Science and Technology (POSTECH), Pohang, Republic of Korea, 4 Department of Anatomy and Neuroscience, School of Medicine, Eulji University, Daejeon, Republic of Korea, 5 Department of Emergency Medical Technology, Daejeon University, Daejeon, Republic of Korea, 6 Department of Biochemistry, School of Medicine, Jeju National University, Jeju, Republic of Korea, 7 Medical Research Collaborating Center, Seoul National University Hospital, Seoul, Republic of Korea, 8 Seoul National University Bundang Hospital, Seoul National University College of Medicine, Sungnam, Republic of Korea Abstract 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. Citation: Choi Y, Lee K, Ryu J, Kim HG, Jeong AY, et al. (2014) Neuritin Attenuates Cognitive Function Impairments in Tg2576 Mouse Model of Alzheimer’s Disease. PLoS ONE 9(8): e104121. doi:10.1371/journal.pone.0104121 Editor: Robert Blum, University of Wurzburg, Germany Received December 5, 2013; Accepted July 11, 2014; Published August 7, 2014 Copyright: ß 2014 Choi et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This study was financially supported by grants from the Korea Healthcare Technology R&D Project (A111230) of Ministry for Health, Welfare and Family Affairs of the Republic of Korea (A111284), by the National Research Foundation of Korea (NRF)funded by the Ministry of Education, Science and Technology, grant number 2011-0021866, and in part by Seoul National University Bundang Hospital Research Fund (02-2009-025). Thank you again for the opportunity to resubmit our manuscript. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * Email: [email protected] (J-HK); [email protected] (H-SK) . These authors contributed equally to this work. Introduction binding protein (CREB). An in vivo study showed an important role for CREB in activity-dependent neuritin expression in the Alzheimer’s disease (AD) is the most common form of dementia barrel cortex of control and CREB a, D-knockout mice. After and is characterized by two neuropathological hallmarks, neuritic eliciting receptive field plasticity by whisker trimming, the neuritin plaques and neurofibrillary tangles. AD primarily targets synapses, expression level increases in the barrel corresponding to the spared and synaptic loss and dysfunction have been reported to be well whisker. However, in CREB mutant mice, neuritin expression is correlated with cognitive dysfunction in AD [1–3]Immunoreac- not induced to the same extent as in wild-type (wt) littermates, tivity against synaptophysin, a presynaptic marker protein, is indicating that CREB is necessary for neuritin regulation during reduced by approximately 25% in the brains of patients with mild receptive field plasticity in the adult cortex [13]. AD [4]. During developmental stages, neuritin expression has been Neuritin, also known as candidate plasticity gene 15 (CPG 15), shown to be continuously increased from embryonic day 12 to encodes a small, extracellular glycosylphosphatidylinositol (GPI)- adulthood in the cortex and hippocampus [6]. In early embryonic anchored cell surface protein [5,6] that was first identified in a development, neuritin is expressed in multiple brain regions and screen for activity-regulated genes induced by kainate stimulated acts as a survival factor for neural progenitors and differentiated seizure in the rat dentate gyrus [7]. Neuritin is known as a critical neurons [8,14]. Later in development, neuritin promotes the regulator for dendritic outgrowth, maturation, and axonal growth and stabilization of axonal and dendritic arbors with regeneration [6,8–11]. Its gene is located within the 6p2424-p25 synapse formation and maturation [8,10,15]. interval on chromosome 6 [12]. 3 cyclic AMP responsive elements Here we demonstrate that the expression of neuritin was were identified in the promoter region of the gene, suggesting that significantly decreased in the hippocampus and cerebral cortex of neuritin expression is mediated by cyclic AMP responsive element AD patients compared to age-matched control subjects. When PLOS ONE | www.plosone.org 1 August 2014 | Volume 9 | Issue 8 | e104121 Neuritin in Alzheimer’s Disease transiently expressed in primary cortical neuron cultures, neuritin particles from IRES-EGFP or neuritin were bilaterally injected increased the level of synaptophysin, a presynaptic marker, which into the dentate gyrus of the hippocampus of 13-month-old wt and represents an increase in synaptogenesis. Importantly, lentiviral- Tg2576 mice 2.1 mm posterior to the bregma (AP), 1.8 mm mediated expression of neuritin in the dentate gyrus of Tg2576 lateral to the midline (ML) and 1.2 mm ventral to the surface of mice harboring the Swedish double mutated human APP695 gene the skull (DV) or unilaterally injected into the dentate gyrus of the attenuated the deficits in learning and memory. In addition, hippocampus of 7-week-old SD rats 3.5 mm posterior to the neuritin rescued the reduction in the dendritic spine density and bregma (AP), 2.0 mm lateral to the midline (ML) and 4.0 mm the numbers of mushroom-typed mature spines in primary ventral to the surface of the skull (DV), according to the Brain hippocampal neuron cultures from Tg2576 mice. We also found Atlas. that BrdU/NeuN staining increased in the hippocampal region of the Sprague-Dawley (SD) rats expressing neuritin. Collectively, Morris water maze test neuritin possesses a therapeutic potential for neurodegenerative The water maze apparatus, which is a circular pool (140 cm in diseases such as AD by upregulating synaptogenesis and neuro- diameter, 45 cm high), was filled with a 21–23uC water/dry milk genesis in brains. powder mixture to create an opaque setting and located in a laboratory that contained prominent extra-maze cues. The Materials and Methods animals are required to find a submerged platform in the pool Reagents using the spatial cues. The paradigm consisted of 3 trials per day Antibodies were purchased as follows: anti-NeuN (Millipore, over 5 consecutive days in which the mice were placed at one of 3 MA, USA), anti-BrdU (Abcam, Cambridge, UK), anti-neuritin different start positions for each trial and given 60 seconds to (R&D systems, MI, USA), anti- beta-actin (anti-b- actin, Santa locate the hidden platform. The latency time to escape onto the Cruz, CA, USA), anti-synaptophysin (Millipore, MA, USA), anti- hidden platform was recorded. 24 h after the last day of spatial glial fibrillary acidic protein (anti-GFAP, Dakocytomation, training, the probe test was carried out by removing the platform Glostrup, Denmark) antibodies. Neuritin peptide was purchased and allowing each mouse to swim freely for 60 s. The amount of from Abcam. time that each mouse spent swimming in each quadrant was recorded. Data collection was automated by a video image motion Human AD brains analyzer (Ethovision, Noldus Information Technology h.v., Netherlands). Frozen and paraffin-embedded tissues of from 69 to 98 years old-AD and age-matched control subjects were obtained from the Netherlands Brain Bank (http://www.brainbank.nl/about-us/the- Quantitative real-time RT-PCR (qRT-PCR) nbb/). AD tissues were diagnosed with neuropathological evidence Total RNA was extracted from tissue using an RNA purification using the criteria for Braak & Braak stage V or VI. The kit (Qiagen, Hilden, Germany) and 0.5 mg of RNA was processed neuropathological diagnosis for non-demented controls was the for cDNA synthesis using oligo (dT)20 primers and SuperScript III neuropathological criteria for Braak & Braak stage 0 or I. To reverse transcriptase (Invitrogen, CA, USA). The cDNA was then measure the levels of neuritin mRNA in the cortex and amplified using a 7500 Fast Real-Time PCR system (Applied hippocampus, brain tissues from 3 age-matched control subjects Biosystems, CA, USA), employing the DDCt method with SYBR (69, 79 and 80 years old) and 4 AD patients (67, 75, 81 and 87 Green I (Roche, CA, USA).