A Functional Mouse Retroposed Gene
Rps23r1 Reduces Alzheimer’s b-Amyloid
Levels and Tau Phosphorylation
Yun-wu Zhang,1,2,8Shijie Liu,2,8Xue Zhang,2,8Wu-Bo Li,3,8Yaomin Chen,2Xiumei Huang,1,2Liangwu Sun,2Wenjie Luo,4
William J. Netzer,4Richard Threadgill,3Gordon Wiegand,3Ruishan Wang,1,2Stanley N. Cohen,5Paul Greengard,4
Francesca-Fang Liao,2,7Limin Li,3,6,* and Huaxi Xu2,*
1Institute for Biomedical Research and Fujian Provincial Key Laboratory of Neurodegenerative Disease and Aging Research,
Xiamen University, Xiamen 361005, China
2Neurodegenerative Disease Research Program, Burnham Institute for Medical Research, La Jolla, CA 92037, USA
3Functional Genetics, Inc., Gaithersburg, MD 20878, USA
4Laboratory of Molecular and Cellular Neuroscience, The Rockefeller University, New York, NY 10065, USA
5Department of Genetics, Stanford University School of Medicine, Stanford, CA 94305, USA
6Department of Pathology, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences & Peking Union Medical College,
Beijing 100005, China
7Department of Pharmacology, University of Tennessee Health Science Center College of Medicine, Memphis, TN 38163, USA
8These authors contributed equally to this work
*Correspondence: email@example.com (L.L.), firstname.lastname@example.org (H.X.)
Senile plaques consisting of b-amyloid (Ab) and
neurofibrillary tangles composed of hyperphos-
phorylated tau are major pathological hallmarks of
Alzheimer’s disease (AD). Elucidation of factors that
modulate Ab generation and tau hyperphosphoryla-
tion is crucial for AD intervention. Here, we identify
a mouse gene Rps23r1 that originated through retro-
position of ribosomal protein S23. We demonstrate
that RPS23R1 protein reduces the levels of Ab and
tau phosphorylation by interacting with adenylate
cyclases to activate cAMP/PKA and thus inhibit
GSK-3 activity. The function of Rps23r1 is demon-
strated in cells of various species including human,
and in transgenic mice overexpressing RPS23R1.
Furthermore, the AD-like pathologies of triple trans-
genic AD mice were improved and levels of synaptic
maker proteins increased after crossing them with
Rps23r1 transgenic mice. Our studies reveal a new
target/pathway for regulating AD pathologies and
uncover a retrogene and its role in regulating protein
Alzheimer’s disease (AD) is featured by extracellular neuritic
plaques, intracellular neurofibrillary tangles (NFTs), synaptic
(Tanzi and Bertram, 2005). Neuritic plaques are composed of
aggregates of heterogeneous b-amyloid (Ab) peptides, which
are derived from b-amyloid precursor protein (APP) through
sequential cleavages by b-secretase (BACE1) and the g-secre-
tase complex (consisting of at least four components: presenilin,
nicastrin, APH-1, and PEN-2) (Cole and Vassar, 2007; De
Strooper, 2003; Zhang and Xu, 2007). Multiple lines of evidence
suggest that overproduction/aggregation of Ab in the brain is
a causative factor for AD pathogenesis (Hardy and Selkoe,
2002). NFTs are composed of hyperphosphorylated microtu-
bule-associated protein tau (Buee et al., 2000; Lee et al., 2001).
Numerous studies have shown that pathogenic APP metabo-
lism/Ab generation and tau phosphorylation are highly regulated
via various signal transduction pathways, e.g., protein kinases
and phosphatases (Buxbaum et al., 1994; Fang et al., 2000;
Xu et al., 1996) and steroid and peptide hormones (Gasparini
et al., 2001; Xu et al., 1998). Among these regulatory pathways,
glycogen synthase kinase-3 (GSK-3, a and b isoforms),
a serine/threonine kinase essential for a variety of cellular func-
tions including cell adhesion, cell division, transcription (Frame
and Cohen, 2001), has been demonstrated to regulate both Ab
generation and tau phosphorylation (Flaherty et al., 2000; Phiel
et al., 2003). This unique feature renders manipulation of GSK-3
activity an attractive therapeutic approach for AD (Frame and
Cohen, 2001; Martinez et al., 2002; Medina and Castro, 2008).
Hence identification of new genes involved in these processes
will be instrumental in developing novel AD therapeutics.
The creation of genetic novelty by the formation of new genes
has an important role in evolution. New genes can originate
through different mechanisms that include exon shuffling, gene
duplication, gene fusion/fission, mobile element integration,
lateral gene transfer, and retroposition (Long et al., 2003).
Retroposition is a process in which a parental mRNA is reverse-
transcribed and inserted into the organism’s genome, creating
duplicate genes in new genomic positions (Hollis et al., 1982;
Karin and Richards, 1982; Ueda et al., 1982). Although these
intronless retroposed gene copies commonly lack the regulatory
elements of parental genes and thus routinely have been classi-
fied as processed pseudogenes (Jeffs and Ashburner, 1991;
posed gene copies can recruit regulatory elements as well as
328 Neuron 64, 328–340, November 12, 2009 ª2009 Elsevier Inc.
protein-encoding sequences at or near the retroposition site and
mann et al., 2009; Long et al., 2003; Vinckenbosch et al., 2006).
Nevertheless, studies to elucidate the functions of these newly
originated genes, especially the functions related to diseases,
are limited (Kaessmann et al., 2009; Vinckenbosch et al., 2006).
Random homozygous gene perturbation (RHGP; previously
called random homozygous knockout, RHKO) is a genome-
wide genetic approach that identifies genes based on their
biological functions (Li and Cohen, 1996; Liu et al., 1999,
2000a, 2000b). The design of RHGP enables either reduced or
tion site of the gene search vector cassette that contains a regu-
lated promoter. This strategy has been used successfully to
identify genes whose functional homozygous inactivation leads
to reversible tumorigenesis (Li and Cohen, 1996; Liu et al.,
1999, 2000a, 2000b) or altered sensitivity to chemotherapeutic
agents (Lih et al., 2006).
Here,usingtheRHGP approach,weidentified ageneRps23r1
that originated through retroposition of the mouse ribosomal
protein S23 (Rps23) mRNA. The Rps23r1 gene is reversely tran-
scribed relative to its parental gene, expressing a structurally
unrelated yet functional protein RPS23R1. More importantly,
we demonstrated both in vitro and in vivo that overexpression
of the RPS23R1 protein decreases the levels of Ab and tau
phosphorylation and increases synaptic marker proteins in AD
transgenic mice by inhibiting GSK-3 activity via the adenylate
cyclase/ protein kinase A (PKA) pathway.
Genome-wide Screening for Genes that Regulates Ab
It has been shown that reduction of Ab levels is accompanied
by cell-surface accumulation of APP bCTF (the product of
b-cleavage and immediate substrate for g-cleavage), which is
readily detectable in cells deficient in PS1 (Chen et al., 2000),
and these cells can be identified using an antibody specifically
recognizing the N terminus of APP bCTF (FCA18) (Ancolio
et al., 1999). Based on this observation, we adapted RHGP as
a high throughput screen to search for genes that regulate Ab
We modified the original vector pLLGSV (Li and Cohen, 1996)
to increase efficiency of retroviral integration and gene recovery.
The new RHGP gene search vector contains modified LTRs and
utilizes the Cre-LoxP mediated recombination to minimize
promoter interference in provirus and to facilitate genomic
DNA cloning (Figure 1A). This vector was transfected into
Phoenix-Ampho cells for viral packaging. Harvested retrovirus
was used to infect mouse neuroblastoma N2a cells stably
expressing the human APP Swedish mutation (N2aSwe). After
random insertion, the provirus (Figure 1B) expressed Cre recom-
binase for recognition and recombination of the two LoxP
sites located in the 50LTR and 30LTR, respectively, generating
the final integrated provirus (Figure 1C). A tetracycline regulated
promoter (TRE-CMV) promoter in the final integrated provirus
transcription into flanking chromosomal gene that can either
overexpress, when TRE-CMV is in the same orientation, or
suppress (by expressing antisense transcripts) when TRE-CMV
is in the opposite orientation relative to the flanking gene.
Moreover, transcription of the tetracycline-regulated (tet-off)
transactivator was reversed in the presence of tetracycline (or
Therefore, we first acquired N2aSwe cells with stable integra-
tion of the RHGP search vector by puromycin selection. These
FCA18 antibody followed by multiple rounds of FACS sorting
to enrich for cells showing surface APP bCTF accumulation.
Less than 0.1% of cells showing bCTF accumulation after first
Figure 1. Genetic Screening Using Random
Homozygous Gene Perturbation (RHGP)
(A) The new RHGP gene search vector has a tetra-
cycline-regulated element (TRE) regulated CMV
promoter, which drives expression of the puro-
mycin N-acetyl-transferase gene (Pac), a plasmid
replication origin and a chloramphenicol resis-
tance marker (Ori-CAT), and a LoxP site in the
30LTR. In addition, there is a Cre recombinase
gene (Cre) between the 50LTR and the 30LTR.
(B) The initial provirus randomly inserted into chro-
mosomes of mammalian cells upon retroviral
(C) The final integrated provirus after the expres-
sion of the Cre recombinase in the initial provirus,
which mediates DNA recombination at the loxP
(D)Strategy forscreening forAb-reducinggenes in
N2aSwe cells with RHGP vector integration.
Rps23r1 Ameliorates AD-like Pathologies
Neuron 64, 328–340, November 12, 2009 ª2009 Elsevier Inc. 329
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