Neurology International 2014; volume 6:5388

Highly expressed genes within to memory is the optogenetic work of Deisseroth and colleagues,5 who were able to Correspondence: Michael Andrew Meyer, hippocampal sector CA1: immediately and reversibly silence excitatory Department of Neurology, Sisters Hospital, 2157 implications for the physiology gluatamatergic CA1 electrical activity using a Main Street, Buffalo, NY 14214, USA. of memory stereotactically implanted light sensitive Tel.: +1.716.862.2750. lentiviral vector encoding eNpHR3.1 fused in- E-mail: [email protected] Michael A. Meyer frame to enhanced yellow fluorescent protein (eNpHR3.1- EYFP) under control of the calci- Key words: memory, hippocampus, CA1, gene, atlas. Department of Neurology, Sisters um/calmodulin-dependent protein kinase IIa Hospital, Buffalo, NY, USA (CaMKIIa) promoter which is selective and Conflict of interests: the author declares no specific for glutamate neurons. The authors potential conflict of interests. concluded that contextual fear memory recall, even weeks after training, can be reversibly Received for publication: 9 March 2014. Abstract abolished by temporally precise optogenetic Accepted for publication: 7 April 2014. inhibition of CA1 and found that long-term memory retrieval normally depends on the hip- This work is licensed under a Creative Commons As the CA1 sector has been implicated to Attribution NonCommercial 3.0 License (CC BY- play a key role in memory formation, a dedicat- pocampus but can adaptively shift to alternate NC 3.0). ed search for highly expressed genes within structures […] we confirm the remote- this region was made from an on-line atlas of timescale importance of the anterior cingulate ©Copyright M.A. Meyer, 2014 gene expression within the mouse brain cortex (ACC) and implicate CA1 in ACC Licensee PAGEPress, Italy (GENSAT). From a data base of 1013 genes, 16 recruitment for remote recall. Neurology International 2014; 6:5388 were identified that had selective localization Clearly, CA1 is the anatomic focus for memo- doi:10.4081/ni.2014.5388 of gene expression within the CA1 region, and ry investigations as neuronal activity from CA1 included Angpt2, ARHGEF6, CCK, Cntnap1, is critical in memory formation and retrieval. DRD3, EMP1, Epha2, Itm2b, Lrrtm2, Mdk, However, the biochemical events leading to the PNMT, Ppm1e, Ppp2r2d, RASGRP1, Slitrk5, and CA1 memory trace remains elusive and there- only with attention focused on finding consistently Sstr4. Of the 16 identified, the most selective fore became the physiologic focus of this inves- strong patterns of CA1 expression that were and intense localization for both adult and tigation to identify those genes which are selec- found not only in adult but at the P7 stage as post-natal day 7 was noted for ARHGEF6, which tively and intensely expressed within the CA1 well. The original parasagittal images of the 16 is known to be linked to non-syndromic mental region. By searching a mouse brain histologicaluse selected genes were enlarged to show just the retardation, and has also been localized to den- expression atlas of 1013 genes, a set of 16 genes CA1 region as shown in Figure 1, with Kyte & dritic spines. Further research on the role was found that met these criteria, with the most Doolittle hydropathy plots made for the gene’s played by ARHGEF6 in memory formation is selectively intense expression found for the amino acid sequence (http://web.expasy.org/ strongly advocated ARHGEF6 gene at both the post-natal day 7 and adult stage. protscale/) and shown along the left and right hand outer panels of Figure 1.

Introduction Materials and Methods Results and Discussion Although the anatomy of new memory forma- tion within the brain has been clarified, with The Gene Expression Nervous System Atlas the anatomic focus narrowed down to the CA1 (GENSAT) project, was the main resource for High activity was selectively localized to CA1 region of the hippocampus, the physiologic and this on-line atlas search.6 Details on the tech- for the following 16 genes: Angpt2, ARHGEF6, biochemical mechanisms that make this hap- nical aspects of the GENSAT project for tissue CCK, Cntnap1, DRD3, EMP1, Epha2, Itm2b, pen within the CA1 region remain elusive. processing of EGFP transgenic mice, as well as Lrrtm2, Mdk, PNMT, Ppm1e, Ppp2r2d, RAS- Confirming basic science studies in animals histochemical details are found at: http://www. GRP1, Slitrk5, and Sstr4 (Figure 1). that CA1 is critical to new memory formation,Non-commercial a gensat.org/HistologyProtocol.pdf. Selectively intense localization CA1 was human autopsy study published in 1986 linked As outlined in the histology core protocol, noted for Angpt2, which is otherwise known as selective post-hypoxic damage to CA1 with clin- transgenic mice were kept until adulthood at angiopoietin 2. Staining was found within the ically evident memory impairments; as noted by 42 days, and then sacrificed for histologic pro- neuropil as well as CA1 cell bodies, but was the authors, the affected patient showed cessing, sectioning and EGFP immunolabelling absent elsewhere in the brain except for the marked anterograde amnesia, little if any retro- with rabbit IgG anti-GFP followed by immunos- mild localization to the molecular layer of the grade amnesia, and showed no signs of cognitive taining with a secondary antibody (Biotin con- cerebellum. Angpt2 is a protein that antago- impairment other than memory.1 Subsequent jugated anti-rabbit IgG). nizes the action of angiopoietin 1 (ANGPT1) high resolution magnetic resonance imaging Using the original GENSAT on-line data and endothelial TEK tyrosine kinase (TIE-2, (MRI) studies in Alzheimer’s disease confirm base, all 1013 gene entries were inspected for TEK) and interferes with the ability of ANGPT1 the impact that CA1 atrophy has on memory histochemical evidence of selectively intense to remodel vessels partly due to its’ ability to performance;2 two other studies on transient localization of activity to the CA1 region in induce endothelial cell apoptosis. Not much is global amnesia (TGA) patients with MR defined sagittal sections from adult mice; diamino ben- known about the action of Angpt2 within the hippocampal ischemic events clearly show how zadine (DAB) was the chromogen and brain; however, high-frequency stimulation of focal damage to CA1 disrupts not only place parasagittal section number 6 was used for the ventrolateral thalamus over 14 days was memory but time dependent memory features uniform comparison of genes (Figure 1); sim- found to modulate hippocampal gene expres- including autobiographical memory.3,4 ilar sections of mouse brain at post-natal day 7 sion, with Angpt2 being particularly affected.7 One of the most elegant studies linking CA1 (P7) were also individually reviewed as well CCK displayed selectively intense CA1 stain-

[page 32] [Neurology International 2014; 6:5388] Article ing for the neuropil as well as the cell body that it is needed for proper targeting of the of the blood-brain barrier. As noted by Bangsow layer with some staining noted for the frontal contactin-associated protein (Caspr/paran- et al., Epithelial Membrane Protein 1 expres- cortex and nearby entorhinal cortex. The odin) towards the synapse. Additional findings sion was transiently induced in laser-capture octapeptide sulfated form of Cholecystokinin were that contactin is needed for the proper microdissected rat brain vessels after a 20-min (CCK-8S) is the most common form of CCK distribution of the receptor protein tyrosine global cerebral ischemia, in parallel with the within the brain and has been found to phosphatase beta (RPTPbeta)/phosphacan. loss of occludin immunoreactivity, suggesting increase filopodia and spine density at in vitro The investigators concluded that contactin a possible role in blood-brain barrier alter- day 7, 14 and 21 in hippocampal neurons of not plays a selective role in synaptic plasticity and ations in acute stroke.12 However, its potential only wild-type mice but also in double trans- identify PPF and LTD, but not LTP (long-term role in hippocampus related memory forma- genic mice with mutations for the amyloid pre- potentiation), as contactin-dependent processes. tion remains elusive. cursor protein and presenilin (APP/PS1); As dopamine is a key neurotransmitter with- Ephrin receptor type A2 (EPHA2) was also CI988, an antagonist of CCK-2 receptor, in the brain, it is very important to note that the identified in this search for CA1 proteins. inhibits this effect of CCK-8S.8 The memory- D3 receptor subtype displayed selectively Ephrin and related receptor proteins are enhancing effects of CCK have been reviewed intense CA1 localization. The DRD3 dopamine known to be linked to developmental events, by Hadjiivanova et al.9 receptor D3 is linked to G proteins that inhibit especially for neuronal tissue, as well as to With regards to selectively intense CA1 adenyl cycles, and displays genetic variations affect axon guidance, synaptic plasticity, and to localization of CNTNAP1 (contactin associated linked to essential tremor. Mice lacking promote long-term potentiation. As shown by protein 1, also known as P190; CASPR; NRXN4; dopamine D2 and D3 receptors have spatial Thundyil et al.13 with a mouse middle cerebral CNTNAP) it is important to note that Murai et working memory deficits: studies by Glickstein artery occlusion model for stroke, neurological al.10 have reported a novel role for glyco- et al.11 have shown that mice lacking dopamine deficit scores and brain infarct volumes were sylphosphatidyl inositol (GPI)-anchored con- D2 and D3 receptors have spatial working mem- significantly less in EphA2(-/-) mice compared tactin in hippocampal CA1 synaptic plasticity. ory deficits in addition to reduced c-fos respons- with controls, leading the investigators to con- These investigators found that contactin selec- es to D(1) agonist stimulation. clude that the data provide the first evidence tively supports paired-pulse facilitation (PPF) Another CA1 protein found in this search is that the EphA2 receptor directly contributes to and NMDA (N-methyl-D-aspartate) receptor- the epithelial membrane protein 1 (EMP1), blood-brain barrier damage and neuronal death dependent long-term depression (LTD) and which is felt to be a novel tight junction protein followingonly ischemic stroke. However, the role use

Non-commercial

Figure 1. Sixteen highly expressed genes within adult mouse CA1 hippocampus are shown above, with hydropathy plots for the expressed protein shown as well.

[Neurology International 2014; 6:5388] [page 33] Article which Epha2 may play in hippocampus mediat- showed that they had working memory deficits drites, where it appeared to be associated with ed memory formation remains unknown. that could be found at 4 weeks after birth, as microtubules.22 One of the most interesting proteins identi- revealed by Y-maze testing as well as the ele- Another gene that seems to be strongly fied in this search was integral membrane pro- vated plus-maze test; they concluded that MdK expressed within CA1 with prominent pyrami- tein 2B (ITM2B, also known as BRI; other was an important regulator of hippocampal dal cell staining as well as in the neuropil is aliases include FBD; ABRI; BRI2; E25B; E3-16; post-natal development. the neuron-specific transmembrane protein, imBRI2; BRICD2B). ITM2B is a transmem- Of particular interest was finding the epi- SLIT and NTRK-like protein-5 (Slitrk5). Not brane protein which after C-terminus process- nephrine synthesis enzyme, PNMT, is selec- much is known about this gene with respect to ing by proteases, releases a small secreted pep- tively expressed within CA1 in addition to brain physiology, but a gene knockout model tide that blocks the deposition of beta-amyloid. other patterns of intense expression within shows that the deficiency leads to obsessive- Mutations which extend the length of the C- the dorsal medulla and mild activity within the compulsive like behaviors in mice, with exces- terminus end that then result in a larger size thalamus. To investigate the selective impact sive self-grooming observed along with hight- to the secreted peptide, have been found to of epinephrine on arousal and fear-related ened anxiety type behaviors, which seems to cause two neurodegenerative diseases: famil- memory retrieval, Toth et al. studied PNMT be relieved by the selective serotonin reuptake ial British dementia (FBD) and familial knock-out mice unable to synthesize epineph- inhibitor fluoxetine;23 the investigators feel Danish dementia (FDD). As reported by rine and found that they had impaired condi- that Slitrk5 acts as molecule at corticostriatal Tamayev et al.14 for their mouse model of FBD, tioned fear response and startle reactivity.19 synapses. However, little is known overall the British mutation drastically reduces expres- Although the Ca (2+)/calmodulin-depend- about Slitrk5 and it’s possible role in hip- sion of mature BRI2 in both KI (knock-in) mice ent protein kinase phosphatase-N (otherwise pocampal physiology. and human FBD brains. This deficit is associat- known as PPM1E or POPX1) was found in this A particularly interesting finding in this ed with severe hippocampal memory deficits in study to be selectively expressed within the study is detection of the Somatostatin receptor FBD(KI) mice. Remarkably, these animals CA1 region in an intense fashion, relatively lit- subtype 4 (SSTR4) as showing selectively showed no cerebral amyloidosis and tauopathy. tle is known about this enzyme. PPM1E intense expression within the CA1 region of Bri2(+/-) mice present memory deficits similar appears to be a protein phosphatase that the hippocampus. Studies by Gastambide et to those in FBD(KI) animals. Collectively, specifically dephosphorylates and regulates al.24 found that this gene is involved in memo- these results indicate that the British BRI2 multifunctional calcium and Calmodulin-regu- ry formation,only as intra-hippocampal injections mutation underlies abnormal memory due to lated kinases but unique in that it localizes the of the SSTR4 agonist (L-803,087) dramatically loss of BRI2 function and independently of nucleus (hence, -N). Ishida et al.20 found that impaired place memory formation in a dose- histopathological alterations typically evident by using gene knockdown studies in zebrafish, dependent manner yet agonists to in advanced neurodegenerative disease. they were able to conclude that this enzymeuseSomatostatin receptor types 1, 2, or 3 had no Selectively intense staining of the CA1 neu- was essential for the early development of the effect. Other studies by Bito et al.25 concluded ropil was found for LRRTM2 (leucine rich brain and spinal cord. As the pattern of expres- that […] hippocampal functions of somato- repeat transmembrane neuronal 2) in addition sion for PPM1E was fairly intense and specific statin might be mediated through diverse but to mild reactivity within the molecular layer of for CA1, further research on the functional role selective second messenger systems activated the dentate gyrus as well as the CA3 stratum this enzyme may play in hippocampal physiol- via SSTR4 and reveal an unsuspected coupling lucidum. This protein is particularly relevant to ogy overall with specific inquiry to any possible of a neuronal SSTR subtype to a mitogenic sig- the CA1 role in memory in that it is known to role in memory formation. naling pathway. be a key regulator of excitatory synapse devel- On a possibly related note, another poorly The most interesting finding of all in this opment and function. The studies of de Wit et studied protein phosphatase known as study is detection of ARHGEF6 as being selec- al. have shown that LRRTM2 interacts with PPP2R2D (alias B-Delta, or B55-Delta) also tively expressed within CA1 in an intense Neurexin1 and regulates excitatory synapse showed strong and selective CA1 staining, yet manner for not only adult mice, but also for formation,15 whereas Soler-Llavina et al.16 relatively little Is known about it make func- those examined at post-natal day 7. This gene found that the protein was essential for main- tional correlations for the hippocampus. Of encodes the guanine nucleotide exchange fac- tenance of long-term potentiation of mature note, Hui-Feng Chen et al.21 estimated overall tor PIX/Cool-2 for the Rho GTPases Rac1 and synapses on adult CA1 pyramidal neurons. that Protein phosphatase 2A (PP2A) accounts Cdc42, and activates Rho proteins by mediat- Clinical correlates include a study by for up to 1% of cellular protein, and that certain ing the exchange of GDP for GTP; this is a crit- Kleffmann et al.,17 who studied a patientNon-commercial with functional variants in haplotypes and nuclear ical function as Rho GTPases appears to act as a small and restricted 5q31 microdeletion and factor 1 may regulate the transcription of the important molecular switches by alternating associated intellectual disability/developmen- regulatory subunit gene PPP2R2D (protein between an inactive, GDP-bound state and the tal delay (ID/DD); they concluded […] phosphatase 2, regulatory subunit B, delta). physiologically active form where GTP is LRRTM2 as the most promising candidate gene Especially high levels of the Ras guanyl bound. As mutations in five different X-linked for ID/DD due to its expression pattern, func- nucleotide-releasing protein (RasGRP) was genes that encode proteins that interact with tion as a key regulator of excitatory develop- found here in this study for the CA1 region. Rho GTPases (FGD1, OPHN1, PAK3, ARHGEF9 ment, and interaction with Neurexin 1. This protein is a Ras guanyl nucleotide as well as ARHGEF6) are linked to intellectual Midkine (MdK) is a growth factor involved exchange factor that is expressed prominently disability,26 Ramakers et al.27 investigated in the development and repair of numerous in the hippocampus, but also within the den- ARHGEF6 knock-out mice and found that they types of tissues, especially neural tissue. Aside drites and cell bodies of fronto-parietal pyram- exhibited impaired spatial and complex learn- from a fairly prominent localization to the idal neurons which can be traced to extend ing and had diminished behavioral control molecular layer of the cerebellum, this search down into the internal capsule and beyond. when subjected to mildly stressful situations. identified selectively intense localization to Apparently, RasGRP can activate the Ras signal Furthermore, these ARHGEF6 knock-out mice the CA1 region of the hippocampus. Although pathway in response to changes in diacylglyc- showed an overall loss in spine synapses with- knockout mice without the MdK gene encoding erol and possibly calcium; electron microscopy in the hippocampus; corresponding electro- for the Midkine protein showed no gross found RasGRP immunoreactivity in hippocam- physiological changes in CA1 neurons were abnormalities, the study of Nakamura et al.18 pal pyramidal neuronal cell bodies and den- also seen with reduced early-phase long-term

[page 34] [Neurology International 2014; 6:5388] Article potentiation and increased long-term depres- 3. Bartsch T, Schönfeld R, Müller FJ, et al. microdeletions: definition of a critical sion. It therefore appears likely that ARHGEF6 Focal lesions of human hippocampal CA1 region and analysis of LRRTM2, a candi- may play a critical role in memory formation, neurons in transient global amnesia impair date gene for intellectual disability. Mol and further research is clearly needed. place memory. Science 2010;328:1412-5. Syndromol 2012;3:68-75. 4. Bartsch T, Döhring J, Rohr A, et al. CA1 18. Nakamura E, Kadomatsu K, Yuasa S, et al. neurons in the human hippocampus are Disruption of the midkine gene (Mdk) critical for autobiographical memory, men- resulted in altered expression of a calcium Conclusions tal time travel, and autonoetic conscious- binding protein in the hippocampus of ness. Proc Natl Acad Sci USA 2011;108: infant mice and their abnormal behaviour. Although a prior study by Lein et al.28 17562-7. Genes Cells 1998;3:811-22. attempted to identify genes selectively 5. Goshen I, Brodsky M, Prakash R, et al. 19. Toth M, Ziegler M, Sun P, et al. Impaired expressed within CA1 and neighboring sectors Dynamics of retrieval strategies for conditioned fear response and startle reac- of the hippocampus, it was limited by making remote memories. Cell 2011;147:678-89. tivity in epinephrine-deficient mice. the initial search through micro-array technolo- 6. Gong S, Zheng C, Doughty ML, et al. A Behav Pharmacol 2013;24:1-9. gy followed by in-situ hybridization (ISH) analy- gene expression atlas of the central nerv- 20. Ishida A, Tsumura K, Oue M, et al. An active sis of the more intensely expressed genes. This ous system based on bacterial artificial C-terminally truncated form of Ca initial micro-array approach followed by ISH chromosomes. Nature 2003;425:917-25. (2+)/calmodulin-dependent protein kinase found genes displaying restricted expression 7. Kádár E, Lim LW, Carreras G, et al. High- phosphatase-N (CaMKP-N/PPM1E). Biomed within CA1 to include Nov and Bhlhb2 with frequency stimulation of the ventrolateral Res Int 2013;2013:134813. genes displaying enriched expression in CA1 thalamus regulates gene expression in 21. Chen HF, Lin LN, Chen YX, et al. being Kl, Tmsb10, Etv1, Stac. Dusp1, AA591428, hippocampus, motor cortex and caudate- Identification and functional analysis of Egr1, and Pbx3. The current study of the GEN- putamen. Brain Res 2011;1391:1-13. variant haplotypes in the 5’-flanking SAT atlas under discussion here was conducted 8. Zhang LL, Wei XF, Zhang YH, et al. CCK-8S region of protein phosphatase 2A-B gene. with remarkably different methods, and individ- increased the filopodia and spines density PLoS One 2012;7:e35524. ually inspected the histological features of gene in cultured hippocampal neurons of 22. Pierret P, Vallée A, Mechawar N, et al. APP/PS1 and wild-type mice. Neurosci Lett only expression directly amongst 1013 genes as the Cellular and subcellular localization of Ras initial discriminating factor rather than micro- 2013;542:47-52. guanyl nucleotide-releasing protein in the array signal analysis. 9. Hadjiivanova C, Belcheva S, Belcheva I. rat hippocampus. Neuroscience 2001;108: Hopefully, this study has provided a more Cholecystokinin and learning and memory 381-90. comprehensive analysis to find those genes processes. Acta Physiol Pharmacoluse Bulg 23. Shmelkov SV, Hormigo A, Jing D, et al. which have physiologic relevance to learning 2003;27:83-8. Slitrk5 deficiency impairs corticostriatal and memory; in this regard, a surprising num- 10. Murai KK, Misner D, Ranscht B. Contactin circuitry and leads to obsessive-compul- ber of genes were found out of the group of 16 supports synaptic plasticity associated sive-like behaviors in mice. Nat Med identified that already have known links to with hippocampal long-term depression 2010;16:598-602. memory, and includes ARHGEF6, SSRT4, PNMT, but not potentiation. Curr Biol 2002; 24. Gastambide F, Viollet C, Lepousez G, et al. MdK, LRRTM2, ITM2B, DRD3, CNTNAP1 and 12:181-90. Hippocampal SSTR4 somatostatin recep- CCK-8. Of particular interest is ARHGEF6, 11. Glickstein SB, Hof PR, Schmauss C. Mice which has been tied to the important role that lacking dopamine D2 and D3 receptors tors control the selection of memory dendritic spines play in hippocampal mediated have spatial working memory deficits. J strategies. Psychopharmacology (Berl) memory formation.26 As ARHGEF6 is an activa- Neurosci 2002;22:5619-29. 2009;202:153-63. tor of Rho GTPases that regulate the actin 12. Bangsow T, Baumann E, Bangsow C, et al. 25. Bito H, Mori M, Sakanaka C, et al. cytoskeleton, it appears natural for it be able to The epithelial membrane protein 1 is a Functional coupling of SSTR4, a major hip- influence dendritic spine morphogenesis; novel tight junction protein of the blood- pocampal somatostatin receptor, to adeny- mutations within the ARHGEF6 gene also has brain barrier. J Cereb Blood Flow Metab late cyclase inhibition, arachidonate been linked to mental retardation.27 Although 2008;28:1249-60. release and activation of the mitogen-acti- ITM2B and LRRTM2 also appear to have impor- 13. Thundyil J, Manzanero S, Pavlovski D, et vated protein kinase cascade. J Biol Chem tant roles in memory, more researchNon-commercial is clearly al. Evidence that the EphA2 receptor exac- 1994;269:12722-30. needed on the role which ARHGEF6 plays in hip- erbates ischemic brain injury. PLoS One 26. Nodé-Langlois R, Muller D, Boda B. pocampal physiology. 2013;8:e53528. Sequential implication of the mental retar- 14. Tamayev R, Giliberto L, Li W, et al. Memory dation proteins ARHGEF6 and PAK3 in deficits due to familial British dementia spine morphogenesis. J Cell Sci 2006;119: BRI2 mutation are caused by loss of BRI2 4986-93. References function rather than amyloidosis. J 27. Ramakers GJ, Wolfer D, Rosenberger G, et Neurosci 2010;30:14915-24. al. Dysregulation of Rho GTPases in the 1. Zola-Morgan S, Squire LR, Amaral DG. 15. de Wit J, Sylwestrak E, O’Sullivan ML, et al. Pix/Arhgef6 mouse model of X-linked Human amnesia and the medial temporal LRRTM2 interacts with Neurexin1 and intellectual disability is paralleled by region: enduring memory impairment fol- regulates excitatory synapse formation. impaired structural and synaptic plasticity lowing a bilateral lesion limited to field Neuron 2009;64:799-806. and cognitive deficits. Hum Mol Genet CA1 of the hippocampus. J Neurosci 16. Soler-Llavina GJ, Arstikaitis P, Morishita 2012;21:268-86. 1986;6:2950-67. W, et al. Leucine-rich repeat transmem- 28. Lein ES, Zhao X, Gage FH. Defining a 2. Kerchner GA, Deutsch GK, Zeineh M, et al. brane proteins are essential for mainte- molecular atlas of the hippocampus using Hippocampal CA1 apical neuropil atrophy nance of long-term potentiation. Neuron DNA microarrays and high-throughput in and memory performance in Alzheimer’s 2013;79:439-46. situ hybridization. J Neurosci 2004;24: disease. Neuroimage 2012;63:194-202. 17. Kleffmann W, Zink AM, Lee JA, et al. 5q31 3879-89.

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