Hum Genet (2012) 131:535563

DOI 10.1007/s00439-011-1119-1

REVIEW PAPER

Modeling human neurodegenerative diseases

in transgenic systems
Miguel A. Gama Sosa Rita De Gasperi
Gregory A. Elder

Received: 13 October 2011 / Accepted: 23 November 2011 / Published online: 14 December 2011
Springer-Verlag (outside the USA) 2011

Abstract Transgenic systems are widely used to study

the cellular and molecular basis of human neurodegenerative diseases. A wide variety of model organisms have been
utilized, including bacteria (Escherichia coli), plants (Arabidopsis thaliana), nematodes (Caenorhabditis elegans),
arthropods (Drosophila melanogaster), Wsh (zebraWsh,
Danio rerio), rodents (mouse, Mus musculus and rat, Rattus
norvegicus) as well as non-human primates (rhesus monkey, Macaca mulatta). These transgenic systems have enormous value for understanding the pathophysiological basis
of these disorders and have, in some cases, been instrumental in the development of therapeutic approaches to treat
these conditions. In this review, we discuss the most commonly used model organisms and the methodologies available for the preparation of transgenic organisms. Moreover,
we provide selected examples of the use of these technolo-

M. A. Gama Sosa (&) R. De Gasperi

Research and Development Service, 3F-20, James J. Peters
Department of Veterans AVairs Medical Center,
130 West Kingsbridge Road, Bronx, NY 10468, USA
e-mail: miguel.gama-sosa@mssm.edu
M. A. Gama Sosa R. De Gasperi G. A. Elder
Department of Psychiatry, Mount Sinai School of Medicine
of New York University, New York, NY, USA
M. A. Gama Sosa R. De Gasperi G. A. Elder
The Friedman Brain Institute, Mount Sinai School of Medicine
of New York University, New York, NY, USA
G. A. Elder
Neurology Service, James J. Peters Department of Veterans
AVairs Medical Center, Bronx, NY, USA
G. A. Elder
Department of Neurology, Mount Sinai School of Medicine
of New York University, New York, NY, USA

gies for the preparation of transgenic animal models of neurodegenerative diseases, including Alzheimers disease
(AD), frontotemporal lobar degeneration (FTLD), amyotrophic lateral sclerosis (ALS), Huntingtons disease (HD) and
Parkinsons disease (PD) and discuss the application of
these technologies to AD as an example of how transgenic
modeling has aVected the study of human neurodegenerative diseases.

Introduction
Inherited neurodegenerative diseases are a heterogeneous
group of genetic disorders characterized by loss of neuronal
structure and function, and are generally accompanied by
neuronal loss. These diseases may result directly from
degeneration of particular neuronal populations or indirectly from alterations in glial support cells. The particular
topological pattern of brain involvement determines the
speciWc clinical manifestations of each disease. Several of
these diseases are characterized by an accumulation of
abnormal or aggregated proteins or other biological materials either extracellularly or within neurons. These accumulations take various forms and result in the neuritic plaques
or neuroWbrillary tangles in Alzheimers disease (AD),
Lewy bodies in Parkinsons disease (PD), glycogen and
polyglucosan bodies in Lafora disease, or glycolipids and
complex carbohydrates in the lysosomal storage diseases.
As with other genetic disorders, once a pattern of inheritance has been identiWed, the chromosomal loci can be
determined by linkage studies. Sequencing of putative loci
within members of an established pedigree can be used to
identify the speciWc genetic changes associated with the
disease. This process has been greatly facilitated by recent
advances in DNA sequencing technology (Mardis 2011),

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which now allow the sequencing of more than 600 gigabases per run at dramatically reduced costs ($1,000 USD
per genome). Once a causative mutation has been identiWed, transgenic systems can be generated to model the
human disease or aspects of the altered gene function.
Transgenic models provide further conWrmation of the
genetic basis for the disease and aid in identiWcation of cellular and molecular mechanisms responsible for disease
phenotypes. They also provide models in which the eYcacy
and side eVects of potential treatments can be evaluated.
Various transgenic systems of bacterial, plant, and animal
origin are currently used to model diVerent aspects of
human inherited neurodegenerative diseases. These systems vary in terms of ease of manipulation and phylogenetic relatedness to humans, but have all been useful for the
study of neurodegenerative diseases.

Bacterial transgenic systems

Because of their relatively simple genetics and the straightforward methodologies for introducing genetic modiWcations, bacteria were the Wrst organisms to be genetically
altered in the laboratory. Although bacteria are far removed
from humans phylogenetically, bacterial systems have been
useful for modeling particular aspects of human neurodegenerative conditions. One particular example is the production of pathogenic prion proteins (PrP), where attributes
of pathogenic PrP were induced in Escherichia coli
(E. coli) expressing recombinant murine PrP. The PrP generated was protease resistant and aggregated into a self-perpetuating state that induced classic prion pathology when
inoculated into brains of mice (Wang et al. 2010). Similarly, a yeast prion protein was converted into an infectious
prion protein in E. coli (Garrity et al. 2010). These systems
provide unique experimental systems for studying the spontaneous conversion of normal to pathogenic PrP and also
provide direct evidence supporting the prion hypothesis.

Plant transgenic systems

Plant somatic cells are totipotent, and can regenerate from
single cells grown in vitro. Therefore, genetic modiWcations
made in somatic cells develop into adult plants in which all
cells contain the desired alteration. DNA can be introduced
into plant cells by a variety of techniques, including treatment with polyethylene glycol (PEG), electroporation,
microinjection, biolistic transformation, and the use of
Agrobacterium, plant viruses and liposomes as DNA carriers (Ziemienowicz 2010).
Although multicellular, plants are only distantly related
to humans. Nonetheless, several insights have been derived

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from plant studies in which genes related to human neurodegeneration have been examined. Indeed, an intersection
between the Welds of neuroscience and plant starch metabolism helped reveal the molecular defects involved in Lafora
disease (Gentry et al. 2007, 2009; Niittyla et al. 2006).
Lafora disease is an autosomal recessive disorder that produces a syndrome of progressive myoclonic epilepsy and is
pathologically characterized by accumulation of insoluble
glucan (Lafora bodies). Based on human studies, two genes
were known to be involved in the disease: EMP2A, a gene
coding for the phosphatase laforin, which is known to contain a canonical dual speciWcity phosphatase (DSP) and a
carbohydrate binding module (CBM), and EMP2B, a gene
coding for malin, which functions as an E3 ubiquitin ligase
that binds, ubiquitinates, and promotes degradation of laforin. Niittyla et al. discovered a gene in plants (Arabidopsis
thaliana) that they called starch excess 4 (SEX4), which, as
with laforin, contained DSP and CBM domains. Strikingly,
in plants, mutations in SEX4 resulted in an increase in
insoluble glucans similar to those found in Lafora disease
patients. Using A. thaliana transgenic plants, SEX4 was
shown to have the same biochemical properties as laforin
(glucan binding and phosphatase activity) and laforin was
found to be a functional equivalent of SEX4, as human
laforin could rescue the phenotype in SEX4 mutated plants
(Gentry et al. 2007, 2009; Vander Kooi et al. 2010).
Transgenic plants have also been used to study the function of genes associated with familial Alzheimers disease
(FAD) and trinucleotide repeat disorders. The presenilins
(presenilin 1 and 2) are transmembrane proteins that, when
mutated, are the most common genetic cause of FAD. Presenilins form part of the -secretase complex, which cleaves
a range of substrate proteins in animals, including notch and
the amyloid precursor protein (APP). However, presenilins
have long been suspected to have -secretase-independent
functions. Interestingly, plants produce presenilin (psn)
despite lacking homologs for notch or APP. Transgenic systems using the moss bryophyta Physcomitrella patens have
been used to investigate the -secretase-dependent and -independent functions of presenilin in a setting where there is no
interference from eVects of presenilins on notch or APP processing. Interestingly, using a catalytically inactive psn
mutant, a -secretase-independent function of P. patents
presenilin was found to be involved in proper cytoskeletal
function, including organelle transport and cell polarity, and
human presenilin 1 rescued abnormal growth and light
responses in P. patents knock-out plants (Pppsn) (Khandelwal et al. 2007). Furthermore, P. patents psn was able to
revert cell proliferation abnormalities in mouse embryonic
Wbroblasts. Recently genetic defects in plants caused by triplet repeat expansions (TTC/GAA) in the isopropyl malate
isomerase large subunit 1 gene have been studied in wild
type and transgenic Arabidopsis thaliana (Sureshkumar et al.

Hum Genet (2012) 131:535563

2009). As in humans, the triplet GAA repeat expansions

cause a reduction in gene activity that may severely impair
strain growth and development.

Animal transgenic systems

Despite the successes in bacteria and plants, transgenic
modeling of human neurodegenerative disorders has been
primarily conducted in animals. Indeed, the goal of reproducing a phenotype akin to the broader clinical manifestations of the human disorders has only been achieved in
animals. Animal systems allow for testing of potential therapeutic strategies and investigation of disease progression
in a manner not possible in humans. Transgenic models of
neurodegenerative disorders have been produced in a variety of invertebrate and vertebrate species.
Among invertebrate systems the arthropod fruit Xy Drosophila melanogaster and the nematode worm Caenorhabditis
elegans have been the most widely used (Jeibmann and Paulus
2009; Link 2001; Lu and Vogel 2009; Thompson and Marsh
2003). One central appeal of invertebrate models is the power
of the genetic tools available, including the ability to perform
forward genetic screens to identify genes that modulate disease development. Furthermore, these models enable the rapid
production and analysis of transgenic lines expressing variants
of disease-associated proteins (Link 2001). The major disadvantage of organisms like D. melanogaster or C. elegans is
that they lack factors, such as immune function and myelination that are critical components in many vertebrate pathological processes. Anatomic divergence may also preclude the
direct modeling of some processes in invertebrate models
(Jeibmann and Paulus 2009; Link 2001).
Caenorhabditis elegans The nematode C. elegans is a
transparent roundworm. One of its great appeals as a model
system is its simplicity (Muller and Grossniklaus 2010).
C. elegans contains fewer than 1,000 cells that divide in a
stereotypical manner in which the lineage of every cell can
be traced back to the egg. The worms are easy to grow
and display all the hallmarks of a multicellular organism,
including a complex organ structure and social, sexual, and
learning behaviors. Adult C. elegans hermaphrodites contain only 302 neurons, and the complete pattern of synaptic
connections has been reconstructed by serial electron
microscopy. Neuronal classes include chemosensory,
mechanosensory, and thermosensory types. Their identities
are known with such precision that seventy-Wve motor neurons are known to innervate the muscles of the body wall
(excluding the head) and of these, 56 are cholinergic and 19
are GABAergic. C. elegans larvae contain four serotonergic and eight dopaminergic neurons. Many of the proteins
involved in formation, traYcking, and release of synaptic
vesicles are highly conserved with those found in mam-

537

mals. Because the organism is transparent throughout its

life cycle, fusion proteins containing green Xuorescent protein (GFP) have been extensively used to visualize speciWc
neurons and synapses in vivo. In some instances, neuronal
death has been directly observed in living worms by the
appearance of vacuolated neurons. More generally, GFP
tagging of speciWc neuronal populations allows degeneration and neuronal cell loss to be observed throughout life
(Teschendorf and Link 2009).
Human disease-associated proteins can be expressed in
C. elegans using transgenic cassettes employing a C. elegansspeciWc promoter and transgenic cDNA. Targeted neuronal
expression can be accomplished using pan-neuronal promoters (including unc-119, snb-1, aex-3, rgef-1) or speciWc
neuronal subtype promoters (such as mec-7, mechanosensory neurons; osm-1, chemosensory neurons; dat-1, dopaminergic neurons) (Teschendorf and Link 2009). Body wall
muscle-speciWc promoters, such as unc-54 and myo-3, also
exist. These promoters often produce representative expression patterns; although, important exceptions have been
observed. Also, vector-free DNA constructs appear to
improve transgene expression (Etchberger and Hobert 2008).
Moreover, transgenic organisms have been generated by
recombining large fosmids with the galK selectable marker
(Zhang et al. 2008, 2011b). DNA is injected into either
maturing oocytes or, more commonly, into the syncytial
gonad, which targets the hermaphrodite germline. The coinjection of a selectable marker, such as rol-6, which alters
cell morphology, or GFP allows easy identiWcation of transgenic organisms. More recently, a selection system using
puromycin has been developed for rapid isolation of transgenic worms (Semple et al. 2010).
Comprehensive databases of C. elegans promoters that
are active in the nervous system can be found at http://chinook.uoregon.edu/promoters.html and http://www.wormbase.org. A detailed description of genetic screening in this
organism has also been described (Jorgensen and Mango
2002). To capture genuine spatial and temporal expression
patterns, investigators have modiWed large segments of
DNA in bacterial artiWcial chromosomes (BACs) or fosmids that have then been used to generate transgenic lines
of C. elegans (Zhang et al. 2008, 2011b; Dolphin and Hope
2006; Sarov et al. 2006). Using locus-speciWc zinc-Wnger
nucleases (ZFN), speciWc knockouts for the ben-1 and rex-1
genes have been created in C. elegans (Reyon et al. 2011;
Wood et al. 2011). Furthermore, the FLP-FRT conditional
recombination system has been used for gene activation in
this organism (Davis et al. 2008).
Drosophila melanogaster The fruit Xy has been widely
used to model both normal biological functions of genes
linked to neurodegenerative diseases and to determine
mechanisms by which mutations lead to neuronal dysfunction and cell death (Bonini and Fortini 2003; Muqit and

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Fig. 1 Basic Gal4/UAS System. The Saccharomyces cerevisiae transactivator Gal4, produced from a driver regulatory cassette, binds as a
homodimer to a 17-bp (CGG-N11-CCG) upstream activating sequence
(UAS) on the target transgene cassette. Gal4 homodimers activate the

target transgene by binding to four tandemly repeated UAS sequences

(UAS4) placed upstream of a minimal promoter (TATA) (Lewandoski
2001)

Feany 2002). Drosophila develops through embryo, larval,

pupal, and adult stages. The adult Xy brain and the complex
eye have been extensively used to study neurodegenerative
diseases (Jeibmann and Paulus 2009).
The embryonic CNS in Drosophila contains both neurons and glial cells. Neurons build commissures in close
association with midline glial cells. CNS glial cells can be
classiWed as midline or lateral glia, and surface glial cells
form a continuous covering of the CNS and peripheral
nerves. Drosophila glia can be further divided into diVerent
classes, including peripheral and exit glial cells, subperineural glial cells which enclose the CNS, and channel glial
cells which line the channels in the ventral nerve cord. The
larval brain is composed of two hemispheres and the subesophageal ganglion. In the adult, association areas necessary for olfactory learning and memory (mushroom bodies)
contain about 2,500 neurons that have small densely packed
cell bodies known as Kenyon cells. Neurons in the Xy
visual system develop in a stereotypical manner. Eye development begins with the diVerentiation of the R8 photoreceptor neurons at uniformly spaced positions. These
neurons signal neighboring cells to develop into ommatidia
(unit eyes). The sequential diVerentiation of the other
seven-photoreceptor types (R1R7) then follows. Each R8
neuron recruits one of each cell type such that seven photoreceptors eventually cluster around each R8 neuron (Jeibmann and Paulus 2009).
The power of Drosophila lies in the ability to dissect cellular signaling pathways by performing large-scale unbiased genetic screens that make no assumptions about what
kind of molecules are involved. Forward genetic screens
(from mutant phenotype to causative gene) using chemical
mutagenesis, insertional mutagenesis, or RNA interference
(RNAi) have been used to identify mutations associated
with neurodegeneration. For example, Drosophila mutants
in the bubblegum, swisscheese, and drop-dead loci were
identiWed by screening for Xies with reduced life span and
then analyzing their brain pathology. Some of these genes
have mammalian homologs that also cause neurodegenera-

tion when mutated, indicating that this approach can identify conserved genes that are essential for maintaining
nervous system integrity across distant organisms (Lu and
Vogel 2009; Roman 2004). Dominant genes associated
with neurodegenerative diseases frequently code for proteins that can misfold and aggregate. One example is -synuclein, which is found in the Lewy bodies characteristic of
Parkinsons disease. Similar protein misfolding has been
observed when -synuclein was expressed in transgenic
Drosophila (Feany and Bender 2000; Auluck et al. 2002).
The use of P elements allows the number of transgene
copies to be increased in almost any genetic background.
These vectors are most commonly used to generate hypermorphs by overexpressing a gene of interest or to complement loss of function mutations. For example, the Gal4/
UAS binary system has been used to overexpress many
proteins (Fig. 1; DuVy 2002). This system involves two
transgenic cassettes with one harboring the yeast Gal4 transcription factor under the control of deWned promoter/
enhancer sequences or enhancer detector P elements and
the other containing an upstream activating sequence
(UAS) responder with the gene of interest cloned downstream of a Gal4 responsive promoter. Hormone-inducible
Gal4 systems based on the Gal4-estrogen receptor
(Gal4ER) or the Gal4-progesterone receptor fusion proteins
have also been described (Jones 2009). These systems can
activate transcription from UAS responders after feeding
Xies either -estradiol or the anti-progestin RU486.
In a related approach, the Tet-On tetracycline/doxycycline inducible system (Fig. 2) combines a modiWed version
of the inducible tetracycline-responsive transactivator
(rtTA-M2) with the Gal4 system (Stebbins et al. 2001;
Stebbins and Yin 2001). A Tet-OV system in which transcription is negatively regulated by the presence of tetracycline or doxycycline combined with the GAL/UAS system
has also been described (Stebbins et al. 2001). More
recently, a Tet-OV system using a transposon (piggyBac)based vector has been developed for gene targeting studies
(Hara et al. 2008). The use of deWned promoters, such as

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539

Fig. 2 Schematic representation of tetracycline-regulated systems: Tet-OV and Tet-On (Stieger et al. 2009)

torso-like tsl, which targets border cells and a subset of posterior cells of the follicle, as well as reverse polarity repo
(glial), embryonal lethal abnormal vision elav (pan-neuronal), Glass Multimer Reporter (GMR, eye-speciWc) and
mhc (pan-muscular), allow spatial expression patterns to be
controlled (Jeibmann and Paulus 2009).
Conditional control of transgene expression can be
achieved through the use of the Saccharomyces cerevisiae
FLP/FRT and the P1-bacteriophage Cre/loxP recombination systems (Fig. 3; Bischof and Basler 2008). These systems rely on the capacity of site-speciWc FLP or Cre
recombinases to recognize and bind to their recognition
sequences and catalyze recombination between FRT and
LoxP sites, respectively. These systems (mainly FLP) have
been extensively used to generate genetic mosaics, chromosomal rearrangements, and site-speciWc mutagenesis (Horn
and Handler 2005; Oberstein et al. 2005; Siegal and Hartl
2000; St Johnston 2002). Streptomyces phage C31-mediated transgenic systems that produce speciWc targeted integration have also been described. These systems are based
on the site-speciWc C31 integrase, which mediates
sequence-directed, irreversible, and highly eYcient integration between a bacterial attachment site (attB) and a phage

attachment site (attP). C31-based systems include Pacman, which is a BAC transgenic platform that allows targeted insertion of large DNA fragments into speciWc
locations (Venken et al. 2006), and the FlyC31 system
(Bateman et al. 2006). Zinc-Wnger nuclease (ZFN) technology has also been used for gene targeting in Drosophila
(Beumer et al. 2006, 2008; Reyon et al. 2011), and RNAi
technology is routinely used for genetic downregulation
and genomic screens (Mohr et al. 2010). Neurodegenerative disorders associated with loss of function mutations
can be modeled by targeting endogenous Drosophila genes
through homologous recombination, transposon-mediated
mutagenesis, or transgenic RNAi. Importantly, all of these
allow for the generation of genetic knockouts (Roman
2004). A comprehensive database of Drosophila genetics
and genomics can be found at http://flybase.org.
ZebraWsh (Danio rerio) The zebraWsh Danio rerio has
experienced a dramatic rise in popularity as an experimental organism. This low-cost vertebrate system has a close
evolutionary relationship with mammals, yet combines the
power of forward and reverse genetic screens otherwise
found only in invertebrates. Despite the evolutionary divergence of teleost Wsh over more than 400 million years,

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Fig. 3 Genomic engineering

using Cre/Flp-mediated DNA
recombination. Cre or Flp catalyze recombination between two
directly repeated loxP or FRT
sequences (triangles), respectively. In the example, a region
B is deleted which may lead to
gene inactivation or cause activation of a downstream gene if
region B contains sequences
that block transcription

zebraWsh are more closely related to humans than invertebrates. This organism, originally from the river Ganges in
India, is a common aquarium Wsh that is easy to maintain
and breed. Another major advantage of zebraWsh is the
ease of imaging due to its transparency and external
development.
An adult female can lay 200500 eggs every 10 days and
the eggs are fertilized externally. The larvae have adult features after 72 h, are autonomous after 4 days, and are fertile
by 3 months of age. In addition, although there are some
notable diVerences in structure and scale between the
zebraWsh and human brain, the overall organization shows
many similarities. SpeciWcally, zebraWsh have a fore-, midand hindbrain including a diencephalon, telencephalon and
cerebellum. Furthermore, the peripheral nervous system
has both motor and sensory components. ZebraWsh exhibit
many higher order behaviors including memory, conditioned responses, and social behaviors like schooling (Lieschke and Currie 2007). SpeciWc regions of zebraWsh brain
are strikingly conserved with the human counterparts, making zebraWsh an excellent organism to model human neurodegenerative conditions. Moreover, the advanced optical
imaging techniques available in zebraWsh research allow

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sophisticated anatomic correlations to be drawn between

brain structure and the development of complex behavioral
traits (Cheng et al. 2011).
Many approaches to generating transgenic zebraWsh are
currently available, including microinjection or electroporation of plasmid DNA or transgenic cassettes into the cytoplasm of fertilized eggs. Use of the I-Sce1 meganuclease
and the Tol2 transposon has greatly increased eYciency of
transgenesis and decreased mosaicism (Kawakami et al.
2000; Sager et al. 2010; Thermes et al. 2002). Other techniques include sperm-mediated gene transfer, pseudotyped
retroviral infection, retroviral infection of in vitro-cultured
sperm, and BAC transgenesis (Gama Sosa et al. 2010a;
Sager et al. 2010). The ability to regulate gene expression
in a cell type-speciWc and temporally restricted manner has
been achieved using Gal4/UAS (Halpern et al. 2008) and
Tet-inducible systems (Huang et al. 2011c; Knopf et al.
2010). The Gal4 driver in the zebraWsh Gal4/UAS system
contains an attenuated version of the herpes simplex virus
(HSV) VP16 trans-activation domain. Using these modiWed
Gal4-Vp16 constructs, driver lines are being generated to
mirror expression patterns of endogenous genes in order to
target desired temporal and tissue-speciWc transgenic

Hum Genet (2012) 131:535563

expression (Asakawa et al. 2008; Distel et al. 2009; Sager

et al. 2010). A database of currently available zebraWsh
Gal4 lines can be found at http://www.zfin.org.
More recently, a mifepristone-inducible LexPR system
has been developed in zebraWsh, which can be induced at
any stage of the life cycle by administration of mifepristone
in the water. This system utilizes a transgene derived
recombinant transcription factor (LexPR) consisting of the
DNA-binding domain of the bacterial LexA repressor coupled to a truncated ligand-binding domain of the human
progesterone receptor with an activation domain of the
human NF-B/p65 protein. The gene of interest is placed
under the control of an operator-promoter sequence
(LexOP) containing four ColE1 operator sequences fused
to a minimal 35S promoter from the cauliXower mosaic
virus (CaMV). Upon binding of the progesterone antagonist
mifepristone (RU-486) to the progesterone receptor LDB,
the LexPR transactivator binds to the operator sequences
and induces gene transcription (Emelyanov and Parinov
2008). The Cre/loxP (Hans et al. 2009; Langenau et al.
2005; Liu et al. 2008b; Pan et al. 2005; Thummel et al.
2005) and FLP/FRT (Wong et al. 2011) site-speciWc
recombination systems have also been applied to conditional mutagenesis in zebraWsh (Deiters and Yoder 2006).
A well-established gene knockdown technology in
zebraWsh uses morpholinos, which are synthetic oligonucleotides of approximately 25 bases that hybridize speciWcally
to complementary sequences of mRNA to disrupt translation initiation or splicing (Bill et al. 2009; Deiters and
Yoder 2006). Morpholinos have standard nucleic acid bases
that are linked to morpholine rings instead of ribose or
deoxyribose. The morpholine rings are attached to nonionic phosphorodiamidate groups instead of phosphates.
Morpholinos can be microinjected into the cytoplasm of
single cell stage embryos or into the yolk of up to 16-cell
stage embryos. Morpholinos can diVuse into cells until the
16-cell stage when a membrane forms between the yolk and
the cells that will form the embryo proper. Another eVective approach for targeted gene knockdown in zebraWsh is
the use of short interfering RNAs (siRNAs) (Kelly and
Hurlstone 2011).
The lack of a gene targeting technology equivalent to
homologous recombination in embryonic stem (ES) cells
prevents the range of gene targeting in zebraWsh that can be
achieved in the mouse. However, using locus-speciWc
ZFNs, speciWc gene knockouts are routinely created in
zebraWsh (Amacher 2008; Ekker 2008; Remy et al. 2010).
Gene knockouts are produced by constructing chimeric
ZFNs consisting of a DNA-binding zinc-Wnger domain and
a cleavage domain of a restriction endonuclease (FokI).
Each DNA-binding zinc-Wnger domain is engineered to
confer speciWcity for a particular triplet of DNA base pairs.
The DNA-binding domains of individual ZFNs typically

541

contain between three and six individual zinc-Wnger

repeats. Gene knockouts are generated by injecting ZFNs
directly into the embryo (Reyon et al. 2011).
More recently, a novel technology for gene targeting in
zebraWsh has been developed based on the use of transcription activator-like (TAL) eVectors. TAL eVectors are a family of virulence factors produced by the bacteria
Xanthomonas, which bind to speciWc promoter sequences
in the host plant to regulate genes aVecting bacterial growth
(Boch and Bonas 2010; Romer et al. 2010). To create a
technology that allows genome editing, a TAL eVector
DNA-binding domain that recognizes single nucleotides is
fused to a FokI cleavage domain (TAL eVector nucleases,
TALENs) (Boch et al. 2009; Moscou and Bogdanove 2009;
Voytas and Joung 2009; Zhang et al. 2011a). The use of
customized TALENs has been successful for targeting the
tnikb and dip2a loci in zebraWsh (Huang et al. 2011b).
Mutant orthologues of human disease loci in zebraWsh
have been generated on a large scale using N-ethyl-N-nitrosourea (ENU) chemical mutagenesis as well as by insertional mutagenesis by retroviral insertion (Amsterdam et al.
2004; Gaiano et al. 1996; Golling et al. 2002; Lieschke and
Currie 2007) or by the use of transposons (Sleeping Beauty
SB, Tol2 or Ds/Ac systems) (Choo et al. 2006; Emelyanov
et al. 2006; Ivics et al. 1997; Kawakami 2005; Kawakami
et al. 1998). Mutant genes involved in human disease have
also been identiWed using a reverse genetic approach called
targeting-induced local lesions in genomes (TILLING) that
combines ENU mutagenesis in the generation of an F1
mutant zebraWsh library with heteroduplex DNA screening
of pooled DNA by Cel I endonuclease screening or direct
DNA sequencing (Draper et al. 2004; Moens et al. 2008;
Wienholds et al. 2003). Genes of interest obtained through
this process can be found at http://www.sanger.ac.uk/
Projects/D_rerio/mutres/ and http://webapps.fhcrc.org/science/tilling/index.php. Genetically mapped retroviral insertions in zebraWsh orthologues of human disease genes also
exist, and these mutant genes are available commercially
(Znomics). Information on transposon insertions can be
obtained through the International Gene Trap Consortium
at http://www.genetrap.org/.
Mouse (Mus musculus) The mouse was the Wrst organism in which transgenic technology and gene targeting
became widely available. Despite the notable advances that
have occurred in other species, the mouse remains the most
widely used system for modeling human neurodegenerative
diseases. The mouse is closely related to humans through
the evolutionary radiation of Epitheria about 75 million
years ago, which resulted in the generation of rodents and
primates. The general outline of development is similar in
mice and humans, and there are syntenic relationships
between mouse and human genes across much of the
genome. The availability of inbred strains allows studies to

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be performed on genetically homogeneous backgrounds.

Other advantages of the mouse include its relatively low
cost, short generation time (18.521 days, depending on the
strain), the high environmental control that is possible, and
the availability of well-developed technologies for genome
manipulation that allow virtually any disease-associated
genetic alteration in humans to be introduced into the
mouse. In addition, techniques for forward genetic mutagenesis screens, complex behavioral analysis, and in vivo
imaging are well developed in the mouse (Muller and
Grossniklaus 2010).
Transgenic models relevant to Alzheimers disease
(Crews et al. 2010; Elder et al. 2010b; Gotz and Ittner
2008; Harvey et al. 2011; Trancikova et al. 2011), Parkinsons disease (Bezard and Przedborski 2011; Crabtree and
Zhang 2011; Harvey et al. 2011; Skaper and Giusti 2010;
Trancikova et al. 2011), Huntingtons disease (Harvey et al.
2011; Skaper and Giusti 2010; Trancikova et al. 2011),
amyotrophic lateral sclerosis (ALS) (Harvey et al. 2011;
Trancikova et al. 2011), schizophrenia (Arguello and
Gogos 2010; Jaaro-Peled 2009; Young et al. 2010), childhood-onset psychiatric disorders (Robertson and Feng
2011), lysosomal storage disorders (Haskins et al. 2006;
Suzuki and Mansson 1998), bipolar disorder (Kato et al.
2007), depression (Snyder et al. 2011; Urani and Gass
2003), and genetic predisposition to drug addiction
(Changeux 2010; Sora et al. 2010) have all been created in
the mouse. These models have been used to develop novel
therapeutic strategies. For example, pharmacological chaperone therapy by active-site-speciWc chaperones has proved
promising for the treatment of some forms of Fabrys disease (deWciency of -galactosidase A, -gal A) by treating a
transgenic mouse expressing mutant -gal A (R301Q) with
the competitive inhibitor 1-deoxygalactonojirimycin
(Khanna et al. 2010; Germain and Fan 2009). Moreover,
for some diseases, preclinical testing in the mouse is virtually regarded as a necessary step before proceeding to
human clinical trials.
Various strategies have been developed to prepare transgenic mice. The most common involves injection of a
transgenic DNA cassette into the male pronucleus of a fertilized mouse egg followed by its implantation into a pseudopregnant mother. Usually a cDNA or other cassette
containing the sequences to be expressed are positioned
downstream of a heterologous promoter that confers spatial
and temporal expression. The inclusion of upstream Kozak
sequences allows for ribosomal recognition of the initial
ATG site, while introns placed at the 5 or 3 end of the
transgene allow for splicing, which makes the mRNA more
stable and favors translocation from the nucleus to the cytoplasm. A termination signal containing a poly (A)-addition
sequence also is placed at the 3 end to improve transgene
expression.

123

Hum Genet (2012) 131:535563

Generally, restricted transgene expression in mature neurons can be achieved with pan neuronal promoters, such as
the tubulin 1, platelet-derived growth factor B-chain
(PDGF-) (Liu et al. 2004), neuron-speciWc enolase (NSE)
(Sakimura et al. 1987; Wen et al. 2004), neuroWlament L
(NFL) (Beaudet et al. 1993), or the microtubule-associated
protein 2 (MAP2). Regionally restricted neuronal expression
can be achieved with promoters like calmodulin kinase II
(CamKII), that drives forebrain speciWc neuronal expression,
while expression in interneurons can be driven with the Glutamic acid decarboxylase 1 (GAD1) promoter (Ma et al.
2006). Pan-neural transgene expression has also been
achieved with the murine thymocyte antigen promoter
(Thy1.2) (Luthi et al. 1997; van der Putten et al. 2000) and
with the prion protein (PrP) promoter (Borchelt et al. 1996;
Xu et al. 2010). Transgene expression in neural progenitor
cells can be achieved with the neural speciWc enhancer from
the second intron of the nestin gene in combination with a
minimal promoter or the native nestin promoter (Zimmerman
et al. 1994). The doublecortin promoter (Dcx) is transiently
active in diVerentiating and migrating neurons in the embryo
and adult (Walker et al. 2007; Piens et al. 2010). AstrocytespeciWc transgene expression can be driven by the glial Wbrillary acidic protein (GFAP) promoter (Miura et al. 1990;
Brenner et al. 1994; Zhuo et al. 1997). The use of the human
GFAP promoter also results in transgenic expression in
radial glial cells during development, despite the fact that the
endogenous murine GFAP promoter is not active in radial
glia. Oligodendrocyte-speciWc promoters include myelin
basic protein (MBP), proteolipid protein (PLP), and 23cyclic nucleotide 3-phosphodiesterase (CNPase). Mammalian promoter databases and software tools can be found at
http://rulai.cshl.edu.
Transgenes typically integrate randomly as concatemers
of 70100 kb in length. In some instances, local chromatin eVects may result in transgene silencing or altered
expression due to eVects from local enhancer/promoter elements (Gaszner and Felsenfeld 2006), which can be pronounced in brain (Elder et al. 1994). Local chromatin
eVects can be often avoided by use of insulator sequences
that establish genomic barriers, which block interactions
between adjacent chromatin domains and protect DNA
sequences from eVects of distal and neighboring genetic
elements (Gaszner and Felsenfeld 2006; Giraldo et al.
2003; Houdebine 2010). Local chromatin eVects can also
be largely avoided with the use of P1 bacteriophage artiWcial chromosomes (PACs), bacterial artiWcial chromosomes
(BACs), and yeast artiWcial chromosomes (YACs) (Montoliu et al. 1993) that permit the cloning of large genomic
regions usually containing all necessary regulatory elements for proper temporal and spatial expression (Giraldo
and Montoliu 2001). PACs and BACs can include up to
350-kb inserts, while YACs can accommodate more than

Hum Genet (2012) 131:535563

1 Mb. Relatively simple technologies have been developed

for introduction of genetic modiWcations into artiWcial chromosomes through recombination (recombineering or retroWtting). This technology has, for example, been used to
perform widespread expression pattern mapping of genes
expressed in the CNS using BACs retroWtted with GFP
(GENSAT project, http://www.gensat.org).
Smaller transgenes (<7.5 kb) can be cloned into recombinant retroviral or lentiviral vectors and the infectious particles can be used to infect zona pellucida-free embryos.
Alternatively, infectious viruses can be injected directly
into the perivitelline space of the embryo or used to infect
cultured ES cells. Intracytoplasmic sperm injection (ICSI)
of DNA-loaded sperm cells has also proven eVective for
production of transgenic mice, particularly when large
DNA constructs, such as sub-megabase sized artiWcial
chromosomes are used (Moreira et al. 2004, 2006). With
this approach, foreign DNA is loaded onto freeze-thawed
spermatozoa in vitro, and individual sperm heads are
injected into metaphase II oocytes. This methodology has
for example been used to prepare transgenic mice harboring
the human amyloid precursor protein (APP) by YAC transgenesis (Moreira et al. 2007).
A major step in the establishment of the mouse as the
dominant system for modeling human neurodegenerative
diseases was the development of gene targeting strategies
(Cheah and Behringer 2001; Joyner and Guillemot 1994).
Although primarily used to generate gene knockouts, gene
targeting through homologous recombination also permits
introduction of point mutations, deletions, insertions, inversions, or translocations into ES cells. After selection, chimeric mice are generated by injecting genetically modiWed
ES cells into mouse blastocysts. Historically, most ES cell
lines in widespread use were derived from the 129/Sv
strain, which contains the wild-type alleles of the albino,
pink-eyed dilution, and agouti coat-color loci. By injecting
modiWed 129 ES cells into recipient blastocysts from
C57BL/6 donors, which carry a recessive black coat color,
chimeric mice can be readily identiWed by their agouti coat
color. Upon breeding of the chimeric mice, lines of genetically modiWed mice can be established. Because strain
background may have signiWcant eVects on phenotype,
mutant lines are frequently backcrossed to other inbred
strains (usually C57BL/6). Unfortunately, even with the use
of speed congenics, backcrossing can require over a year to
place the targeted mutation on a C57BL/6 background. A
more direct approach would be to use ES cells derived from
the C57BL/6 strain. However, historically, C57BL/6 ES
cell lines have been regarded as diYcult to establish and
maintain, although in recent years their use has become
more common, and some commercial suppliers now routinely generate gene-targeted mice on the C57BL/6 background.

543

While not as widely used as gene targeting in ES cells,

gene knockouts in the mouse can also be generated using
ZFNs (Carbery et al. 2010; Cui et al. 2011; Goldberg et al.
2010; Li et al. 2011a; Meyer et al. 2010). Random generation of point mutations with ENU or random gene interruptions with gene traps through mobile DNA elements,
including class I retrotransposons like LINE1 (L1) (Ostertag et al. 2007; An et al. 2006) and class II transposons like
Sleeping Beauty (SB) (Izsvak and Ivics 2005; Yae et al.
2006; Largaespada 2009b; Takeda et al. 2008), are generating large comprehensive resources of mutant mouse strains
(Carlson and Largaespada 2005; Largaespada 2009a). More
recently, the DNA transposon piggyBac from the cabbage
looper moth Trichoplusia ni has been used as both a mutagenic tool (Chew et al. 2011; Rad et al. 2010; Wang et al.
2008) and as a method for mobilizing large pieces of DNA
(100 kb) within the mouse genome (Li et al. 2011b).
The ENU mutagenesis approach involves the selection
of a phenotypic trait of interest followed by identiWcation of
the genetic locus or loci that control that trait (Justice et al.
1999). This approach, usually coordinated in large consortiums or centers, involves phenotyping F1 mutant mice
to identify traits of interest with genetic loci that can be
subsequently determined by gene mapping approaches.
Recessive phenotypes require outcrossing and backcrossing
to recover mutations in the homozygous state. Random SB
transposon mutagenesis usually includes a transposon cassette containing a poly-A gene trap composed of an internal
promoter, a reporter gene like GFP, and a splice donor. If
the gene trap integrates into a transcription unit in proper
orientation, the reporter gene will be activated marking the
active locus. Linker-mediated PCR can be used to determine transposon integration site (Largaespada 2009a).
Because ZFN and TALEN-mediated technologies generate
speciWc gene knockouts, these approaches are largely
replacing the use of mobile DNA elements and ENU for
genome-wide mutagenesis.
Constitutive and conditional knockdown of gene expression has also been accomplished with RNAi technology
(Gao and Zhang 2007). In this technique, expression vectors are designed to contain sense and antisense regions that
are complementary to a selected mRNA target. The resulting transcripts, which have a stem-loop structure, can fold
back into short hairpin (<30 bp) RNAs (shRNAs) that are
processed by the Dicer ribonuclease into siRNAs. The
eYciency of gene knockdown can be as high as 90% or
greater. In one study, a scalable system for studying gene
function in mice was developed using a combination of
Xuorescence-coupled miR30-based shRNAs with high
eYciency ES cell targeting (Premsrirut et al. 2011).
A potential drawback of the gene knockout approach is
the embryonic lethality of many null mutations, which precludes study of gene function in the adult. To circumvent

123

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Hum Genet (2012) 131:535563

Fig. 4 Flp recombinase-mediated cassette exchange (RMCE). Flp

recombinase catalyzes the reciprocal recombination between two identical short Flp recombinase targets (FRTs) present in the target locus
and in the exchange plasmid. The wild-type FRT recognition consists

of two 13-bp inverted repeats that Xank an 8-bp core sequence (red).
The availability of FRT mutants permits the exchange of genetic cassettes for the targeted integration of a desired transgene

these problems, conditional recombination systems, such as

the Cre-loxP (Nagy 2000; Miller 2011; Wang 2009) and
Flp-FRT systems described above, allow for the location
and timing of gene inactivation to be closely regulated. An
ever-expanding number of mouse Cre deleter lines have
been created (http://nagy.mshri.on.ca/cre_new/). Other
conditional systems, such as the D6 phage Dre-rox and
those using novel yeast site-speciWc recombinases (B3 and
KD) that allow genome manipulation in vivo, have also
been developed (Anastassiadis et al. 2009; Nern et al.
2011).
The tamoxifen (RU486)-inducible conditional system
uses a chimeric Cre protein fused to a mutated nuclear
estrogen receptor that no longer responds to natural estrogen but which can be activated with synthetic RU486
(Metzger et al. 1995; Garcia and Mills 2002; Hayashi and
McMahon 2002). This system permits temporal and tissue
or cell type-speciWc gene inactivation within a time frame
dictated by the investigator, rather than determined by time
of onset and pattern of a speciWc promoters expression.
Cre recombination has also been used to activate gene
expression in brain neurons by including strong translational stop sequences Xanked by loxP sites between a promoter and the regulated gene (De Gasperi et al. 2008). Cre
can be used to delete megabase regions and mediate trans-

locations between non-homologous mammalian chromosomes at very high eYciencies (Brault et al. 2007; Horev
et al. 2011; Wirth et al. 2007). In comparison to Cre, Flp
has been considered less eYcient partly due to an unsuitable optimum temperature in mammalian cells. However, a
thermostable Flpe variant has been identiWed (Buchholz
et al. 1998; Ringrose et al. 1998). The Flp/FRT system is
widely used for recombinase-mediated cassette exchange
(RMCE) to generate knock-in mutations in speciWc genes
in the mouse genome (Fig. 4) (Roebroek et al. 2011). Also
the Cre/loxP system has been successfully used for RMCE
(Liu et al. 2006).
Because Cre and Flp excision/recombination is an irreversible event, they are not useful when the ability to
switch a gene on and oV is desirable. Thus, a variety of
inducible/regulatable systems has been developed. The
most commonly used of these in transgenic mice are the
Tet-regulated systems (Fig. 2; Stieger et al. 2009). The TetOV system uses a chimeric tetracycline transactivator (tTA)
protein made by fusing the E. coli tetracycline repressor
(TetR) with the HSV transcriptional activator VP16 protein. tTA binds to the tet-O operator and activates a CMV
minimal promoter coupled to the tet-O operator (tetresponse element, TRE) activating the transgene of interest.
Tetracycline or its more stable analog doxycycline

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Hum Genet (2012) 131:535563

(Dox) bind to tTA preventing it from binding to the TRE,

thus preventing transactivation of the transgene (Gossen
and Bujard 1992). To circumvent the necessity of continuous doxycycline administration to maintain genes in the
oV state, the Tet-On system was developed. The Tet-On
system is based on the observation that mutations within
the chimeric tTA activator protein (rtTA) can reverse its
behavior so that, rather than spontaneously binding to the
TRE, it does so only in the presence of tet or doxycycline
(Urlinger et al. 2000).
A variation on the Tet system known as the Kruppelassociated box (KRAB) Tet-On system also permits tight
regulation of transgene expression by doxycycline. This
system takes advantage of the repressor activity of a fusion
protein tTRKRAB made of the KRAB domain, found in
many vertebrate transcriptional regulators (such as human
Kox1 or mouse Kid-1), and the tetracycline repressor
(tetR). The tTRKRAB-mediated repression of cellular Pol
II or Pol III promoters juxtaposed to tet operator (tetO)
sequences can be reversibly controlled by doxycycline
(Deuschle et al. 1995; Szulc et al. 2006). A database containing tTA and rtTA eVector mice, Tet-O reporter strains,
and Cre-inducible rtTA strains can be found at http://
www.zmg.uni-mainz.de/tetmouse/.
A tet-inducible shRNA gene knockdown system has
been developed for use in ES cells, which uses recombinase-mediated cassette exchange (RMCE) through Flpmediated recombination in the Rosa26 locus (Seibler et al.
2007). The exchange vector carries the shRNA under the
control of the pol III H1-tetO promoter, a codon-optimized
itetR gene under the control of the chicken -actin promoter and a truncated neoR gene for positive clonal selection after successful RMCE. Transgenic animals are then
prepared from the recombinant ES cells (Seibler et al.
2007). Other inducible systems that have been successfully
used in transgenic mice include the lac-inducible system
(Cronin et al. 2001) as well as insect- and mammaliansteroid inducible systems (Albanese et al. 2002; Graham
2002).
Rat (Rattus norvegicus) Rats have a lifespan of 2.5
3.5 years and a gestation time of 1922 days. This rodent
exhibits many physiological and morphological characteristics similar to those of humans. Historically, neuroscientists have preferred rats to mice as model systems and,
indeed, many aspects of rat pharmacology and physiology
are regarded as more relevant to humans and easier to
observe than in mice (Abbott 2004; Bugos et al. 2009). Furthermore, rats generally exhibit a more robust capacity for
learning in behavioral tasks. Although, virtually all the
technologies used for generating transgenic mice should be
applicable to the rat in theory, transgenic technologies are
markedly less available in rats. For example, while core
facilities for generation of transgenic mice are common in

545

major academic research centers, these facilities have only

recently become more available for rats. In addition, rat ES
cells have been diYcult to establish and maintain although
rat ES cells for gene targeting by homologous recombination have been recently described (Tong et al. 2010, 2011).
Moreover, random mutagenesis by ENU (Zan et al. 2003),
mobile DNA elements, such as the piggyBac (Jang and
Behringer 2007) and Sleeping beauty (SB) DNA transposons (Ivics et al. 2011; Izsvak et al. 2010), ZFNs (Cui et al.
2011; Geurts et al. 2009), and TALENs (Christian et al.
2010; Tesson et al. 2011) have permitted the generation of
knock out rats that mimic human diseases (Huang et al.
2011a). A database of mutant rats generated by mobile
DNA technology can be found at http://www.knockoutrat.org/.

Other transgenic organisms

Transgenic technology has been developed in a variety of
other mammalian species. For example, human disease
models have been prepared in transgenic rabbits (Oryctolagus cuniculus) (Bosze et al. 2003; Liang et al. 2006), and
commercial entities have been established solely dedicated to the production of transgenic rabbits. Transgenic
models of Alzheimers disease have been created in pigs
(Sus domestica) using nuclear transplantation via cell
fusion (Kragh et al. 2009), and ZFNs have been used to
generate biallelic knockout pigs (Hauschild et al. 2011).
More recently, it was shown in transgenic pigs prepared
by somatic cell nuclear transplantation (SCNT) that
expression of the Huntingtons disease protein results in
neuronal apoptosis (Yang et al. 2010). A transgenic Huntingtons disease model in rhesus monkeys (Macaca mulatta) has been prepared by lentiviral injection into the
oocyte of polyglutamine-expanded Huntingtin (HTT) followed by fertilization by ICSI (Yang et al. 2008). Transgenic rhesus monkeys have also been generated using a
simian immunodeWciency virus (SIV)-based vector to
infect early-cleavage stage embryos (Niu et al. 2010). A
major obstacle to generating transgenic non-human primates is the low eYciency of assisted reproductive technologies in producing oocytes and embryos suitable for
genetic engineering as well as the technical challenges of
embryonic stem cell derivation and cloning. In addition,
the high cost of production and maintenance of nonhuman primates and other large animals prevents their
wider use in research.
EVorts to develop transgenic models for neurodegenerative disorders are being made in other species as well. For
example, human APP695 has been expressed in the ascidian
non-vertebrate chordate Ciona intestinalis, where it was
observed to be processed into A peptides in the nervous

123

546

system resulting in formation of amyloid plaques and

altered larval behavior (Virata and Zeller 2010). Clearly,
eVorts to develop models of human neurodegenerative disease can be expected to continue in traditional as well as
non-traditional species.

Applications to age-related human inherited

neurodegenerative diseases
Human neurodegenerative diseases take various forms.
Among the most common are age-related neurodegenerative disorders, such as Alzheimers disease (Selkoe
2011), Parkinsons disease (Shulman et al. 2011), Huntingtons disease (Finkbeiner 2011), the frontotemporal
dementias (Ferrari et al. 2011; Seelaar et al. 2011), and
ALS (Ferrari et al. 2011). In each of these disorders,
there is a selective vulnerability of distinct neuronal
populations to the neurodegenerative process, and the
clinical manifestations of each disorder reXect the distinctive subpopulations of neurons involved. For example, loss of dopaminergic neurons is responsible for the
extrapyramidal features characteristic of Parkinsons
disease, while loss of motor neurons causes the weakness and atrophy characteristic of ALS. Another
common feature of human neurodegenerative disorders
is the accumulation of protein aggregates. These accumulations can take various forms. For example, in
Alzheimers disease, accumulation of -amyloid results
in extracellular neuritic plaques and aggregates of
abnormally phosphorylated forms of tau protein predominate in intracellular neuroWbrillary tangles. In ALS,
characteristic intracellular accumulations of cytoskeletal
elements are also seen, but the aggregates are composed primarily of neuroWlament proteins. In contrast, the Lewy bodies characteristic of Parkinsons disease and related disorders
contain aggregates of -synuclein.
With the exception of Huntingtons disease, these disorders are largely sporadic, and although a number of
genetic risk factors have been identiWed, the disorders
can be traced to a single inherited gene in only a small
number of cases. However, despite their rarity, it is
likely that the genetic forms of these disorders target
common pathways that are also aVected in the sporadic
diseases. In addition, because transgenic modeling
requires a genetic lesion or at least a hypothesis that can
be modeled genetically, the mutations found in these
familial cases have formed the basis for most of the
transgenic modeling that has been conducted in animals.
In Table 1, selected examples are presented of transgenic models related to hereditary neurodegenerative
diseases that were created with the transgenic systems
discussed in this review.

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Hum Genet (2012) 131:535563

Transgenic modeling and the example of Alzheimers

disease
While it is beyond the scope of this review to discuss the
relevance of all the models listed in Table 1 to their respective disorders the case of Alzheimers disease illustrates
many of the issues common across disorders. AD may in
many ways be regarded as an ideal disease for modeling in
transgenic animals. In humans it has a well-recognized
pathology consisting of senile plaques and neuroWbrillary
tangles (NFTs). The major constituents of these lesions are
well deWned. In the case of plaques being the -amyloid
(A) peptide, which is derived from processing of the
larger amyloid precursor protein (APP) that itself belongs
to the APP-related protein family. By contrast hyperphosphorylated forms of tau are the major constituents of NFTs.
AD also has other well-recognized pathological features
including neuronal and synaptic loss, dystrophic neurites,
reactive astrocytes and activated microglia. There is in
addition a well-deWned behavioral phenotype.
EVorts at modeling AD have been conducted in all the
transgenic systems described in Table 1 based in large part
on the amyloid cascade hypothesis which postulates that
shunting of APP processing toward longer more neurotoxic
A42 species sets oV a chain of pathological events (Selkoe
2001, 2011). Mutations in three genes including the amyloid precursor protein (APP) as well as two related proteins, presenlin-1 and presenlin-2, have been identiWed as
causing familial Alzheimers disease (FAD) providing the
genetic lesions needed for a transgenic model. However,
characteristics of the individual organisms have dictated
that diVerent strategies be used to model AD in diVerent
organisms.
In C. elegans there is a single APP-related gene, apl-1
(Ewald and Li 2010). Knockout of apl-1 causes lethality
during early larval development while overexpression
causes defects in brood size, movement and reduces viability. A functional domain homologous to the Fe65 binding
domain within the cytoplasmic tail of mammalian APP
exists in apl-1 and a C. elegans ortholog of Fe65 (FEH-1)
exists. Several orthologues to the mammalian - secretases
can be found in C. elegans although no -secretase activity
has been clearly identiWed. C. elegans contains an active
-secretase complex and like mammalian systems has multiple presenilins. Indeed work with the C. elegans presenilin SEL-12 provided the Wrst insights into the role of
presenilins in mediating Notch signaling (Ewald and Li
2010). However, apl-1 lacks an A sequence and thus does
not produce A peptide. As such to establish C. elegans
models of AD, investigators have introduced various forms
of transgenic human A. In muscle cells, human A42 has
been expressed using an A sequence fused to an artiWcial
signal sequence designed to allow the extracellular release

Hum Genet (2012) 131:535563

547

Table 1 Transgenic technologies used in the preparation of animal models of neurodegenerative diseases
Disease

Strategy

Pathology and other Wndings

Reference

Caenorhabditis elegans
AD

Pan neuronal expression of human A42

DeWcits in odorant preference-associated

learning behavior, serotonin-controlled
behaviors, experience-dependent
learning, and egg laying

(Dosanjh et al. 2010)

AD

Body wall muscle expression

of human A42

Accumulation of amyloid deposits with

Wbrillary structure accompanied by
progressive paralysis

(Link 1995)

AD

Inducible pan-neuronal
expression of human A42

Neuronal amyloid deposits associated with

subtle phenotypic changes

(Wu et al. 2006)

FTLD

Expression in dopaminergic
neurons of wt or A53T
mutant -synuclein

Accumulation of insoluble phosphorylated

tau, age-dependent neurodegeneration
and neuronal loss, uncoordinated
movement, and greater toxicity in worms
expressing the FTLD-17 mutant tau than
the wt form

(Kraemer et al. 2003)

FTLD/ALS

Pan neuronal expression

of wt human TDP-43

Motor neuron dysfunction, abnormal

motor neuron synapses, and
uncoordinated phenotype

(Ash et al. 2010)

PD

Expression in dopaminergic neurons

of wt or A53T mutant -synuclein

Degeneration and loss of dopaminergic

neurons including dendritic atrophy
and accumulation of -synuclein in perikarya

(Lakso et al. 2003;

Settivari et al. 2009;
Kuwahara et al. 2006;
Harrington et al. 2010;
Settivari et al. 2009)

HD

Expression of the polyglutamine repeat

HT-Q150 in sensory neurons

Age-dependent neuronal dysfunction

(Faber et al. 1999)

HD

Genome wide RNAi screen for modiWers

of YFP-polyQ aggregation

IdentiWcation of large set of genes

involved in protein folding or
degradation that increase polyQ
aggregation when their expression
is knocked down

(Nollen et al. 2004)

Fruit Fly Drosophila melanogaster

AD

gmr-GAL4/UAS-driven triple transgenic

Xies expressing human wild-type APP,
human BACE and wild-type
Drosophila presenilin (dPsn)
or dPsn with point mutations
corresponding to human FAD mutations
in photoreceptor cells of the compound eye

Elevations of both human A40 and A42

peptides; thioXavin S stained amyloid
plaques in the retina and age-dependent
retinal photoreceptor degeneration that
was enhanced by the presence of FAD
related dPsn mutations and suppressed
by genetic reduction of dPsn or by the
treatment with  or -secretase inhibitors

(Greeve et al. 2004)

AD

Pan-neural GAL4-UAS-driven
expression of human 42

Decreased life span, slower locomotion,

altered tapping reXexes and grooming
routines, and age-dependent learning
defects; amyloid deposition associated
with neurodegeneration

(Finelli et al. 2004;

Iijima et al. 2004;
Crowther et al. 2005;
Iijima et al. 2008)

FTLD

Pan-neural GAL4-UAS driven

expression of wt and R406W tau

Accumulation of abnormally
phosphorylated tau without
neuroWbrillary tangles, progressive
neurodegeneration, and early death.
Tau-induced pathology more pronounced
with the FTLD-associated mutation

(Wittmann et al. 2001)

ALS

Motor neuron and photoreceptor

GAL4-UAS driven
expression of wt and mutant hSOD1

Neither loss of motor neurons nor retinal

degeneration was observed, although
motor neuron expression of either wt or
mutant hSOD1 led to progressive
motor dysfunction. Earlier attempts at
expressing hSOD1 in motor neurons
increased longevity and could rescue
the shortened lifespan of a dSod null mutant

(Elia et al. 1999;

Parkes et al. 1998;
Watson et al. 2008)

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Hum Genet (2012) 131:535563

Table 1 continued
Strategy

Pathology and other Wndings

Reference

PD

Pan-neuronal and photoreceptor

GAL4-UAS driven expression of
-synuclein (wt and mutants
A30P and A53T)

Overexpression of wt and mutant forms of

-synuclein causes dopaminergic
degeneration with progressive loss of
dopaminergic neurons and locomotor
dysfunction. Neuronal inclusions reminiscent
of Lewy bodies were found in the cytoplasm

(Feany and Bender 2000)

HD

Photoreceptor neuronal-speciWc
expression of human huntingtin,
2, 75 or 120Q repeats

Neuronal degeneration for which severity

and age of onset correlate with repeat length.
Nuclear localization of huntingtin preceding
neuronal degeneration

(Jackson et al. 1998)

HD

FRT/FLP-mediated deletion
of the huntingtin locus

Removal of the Drosophila HTT homolog, htt,

(Zhang et al. 2009)
results in reduced mobility, long-term survival,
and modulation of adult axonal terminal complexity.
Htt deletion also accelerates the neurodegenerative
phenotype associated with the HD-Q93
polyglutamine repeat associated with HD

Disease

ZebraWsh Danio rerio

AD

Antisense morpholino gene knock-down

of presenilin (psen1 and psen2)
and APP (appa and appb) orthologues

Knockdown of both psen1 and psen2 causes

hydrocephalus and reduced melanin
pigmentation. Psen1 MO also exhibit
somite defects, altered myo D expression,
a short tail, smaller head, and defective
brain development including reduced
branching of trigeminal neurons Loss
of appa and appb function resulted
in reduced body length and defective
convergent-extension movements
during gastrulation

(Campbell et al. 2006;

Groth et al. 2002;
Newman et al. 2009;
Nornes et al. 2008, 2009;
van Tijn et al. 2011)

FTLD ZebraWsh enolase-2 promoter driving

neuronal expression of human
4-repeat tau

Expression found throughout the CNS;

accumulations of tau occur in axons
and neuronal soma in the retina, brain,
and spinal cord

(Bai et al. 2007; Xi et al. 2011)

FTLD Pan-neuronal Huc2 promoter

induced GAL4-VP16
transactivation of UAS Xanked by
Tau P301L (3) and DsRed (5)
in early neuronal cells

Red Xuorescent cells can be seen expressing

tau in embryos. Mutant tau-expressing
embryos exhibit a transient motor phenotype
caused by delayed outgrowth of motor axons
as well as behavioral deWcits in escape response.
Transgenic animals have hyperphosphorylated
tau, tangle-like inclusions, and neurodegeneration
in the spinal cord

(Paquet et al. 2009, 2010)

ALS

In vitro transcribed RNAs of TDP-43

(TARDBP wt and mutants G348C,
A382T, A315T) were injected into 14
cell stage blastulae

Preferential vulnerability of motor neurons

to toxicity of TDP-43 mutants; shorter
motor neuron axons, premature and
excessive branching, and swimming deWcits

(Kabashi et al. 2010)

HD

Rhodopsin promoter driven expression

of EGFP-tagged huntingtin exon 1 with
71Q expanded polyglutamine repeat
(EGFP-HDQ71) in rod photoreceptors

Aggregates of EGFP-HDQ71
fusion protein and loss of rhodopsin expression
associated with rod photoreceptor degeneration

(Williams et al. 2008)

Age-dependent brain amyloid deposition,

dystrophic neurites, reactive astrocytes,
and activated microglia near plaques; l
earning deWcits. Pathology has
earlier-onset in the Swedish/Indiana
APP double mutant

(Calhoun et al. 1999;

Chishti et al. 2001;
Games et al. 1995;
Hsiao et al. 1996)

Mouse Mus musculus

AD

Mouse models generated by pronuclear

injection of mutant APP cDNAs
resulting in pan-neuronal
expression of mutant APPs
associated with FAD
(Indiana, Swedish and Swedish +
Indiana mutants)

123

Hum Genet (2012) 131:535563

549

Table 1 continued
Strategy

Pathology and other Wndings

AD

Pronuclear injection of PS1 FAD

mutations using platelet-derived growth
factor (PDGF), thy-1, neuron-speciWc
enolase (NSE) promoters and others

Age-dependent neuronal and synaptic loss,

(DuV et al. 1996;
increased lipid and protein peroxidation,
Elder et al. 2010b;
and increased sensitivity to excitotoxic
Wen et al. 2004;
injury; impaired hippocampal neurogenesis Wen et al. 2002)
in adult mice; A elevated in brain but
no plaque deposition

AD

Transgenesis with human BAC harboring

the wt PS1 and a retroWtted BAC
harboring the M146 V FAD mutation

Age-dependent brain vascular pathology

(Gama Sosa et al. 2010b)
associated with the expression of the
human PS1 harboring the M416 V
mutation. Transgenic expression human wt
and mutant PS1 mimics entirely
that of the endogenous murine presenilin

AD

Transgenesis with a YAC harboring the

entire 400-kb human APP gene.
The 650-kb YAC was introduced by l
ipid-mediated transfection into ES cells
that were then used to generate chimeric mice

Resulting transgenic animals express

human APP mRNA and protein at
levels comparable to endogenous
APP. No brain amyloid deposition
was observed

(Lamb et al. 1993)

AD

APP Swedish mutation associated with

FAD introduced into the mouse APP gene.
In addition, the three amino acid diVerences
between mouse and human APPs in
the A region were humanized

FAD mutation expressed at physiological

levels and mice produce human A;
increased A production but no amyloid
plaques or neuropathology

(Reaume et al. 1996)

AD

Flp-mediated RMCE harboring a

Tet-inducible GSAP RNAi transgenic cassette

Doxycycline-induction of gamma-secretase
activating protein (GSAP) RNAi in
double transgenic animals harboring
the APPswe and PS19 mutant
alleles reduced amyloid plaque load

(He et al. 2010;

Seibler et al. 2007)

AD

Tet-OV inducible system for APP695

High-level APP expression rapidly
expression in forebrain neurons. Transgenic
induces amyloid pathology. Doxycyline
mice harboring the mouse/human chimeric
administration inhibits 95% of APP
APP695 with the Swedish (KM570/571NL)
expression and reduces A production
to levels found in non-transgenic mice,
and Indiana (V617F) FAD mutations under the
halting the progression of
control of a tet-responsive (tet-O) promoter were
amyloid pathology
bred to animals expressing tTA under
control of the CamK2a promoter

Disease

Reference

(Jankowsky et al. 2005)

AD/FTLD Pronuclear injection of transgenes containing

Thy 1.2 driven APP Swe FAD and tau P301L
FTLD mutations were co-injected into oocytes
from homozygous PS1 M146 V knock-in mice
to generate 3xTg mice

Elevated A 40 and 42, age-dependent

parenchymal amyloid plaques, and
tau pathology development along
with synaptic dysfunction and
behavioral deWcits

(Oddo et al. 2003)

FTLD

Development of progressive motor

neuron disease; tangle-like
accumulations containing tau in
brain and spinal cord with gliosis
and neuronal loss including 50% loss
of anterior horn motor neurons

(Lewis et al. 2000)

Pronuclear injection of PrP-driven four repeat

human tau containing the P301L mutation
associated with FTDP-17

FTLD/ALS Pan-neuronal constitutive overexpression

of TDP-43 or mutant TDP-43 (A315T)
via pronuclear injection of transgenic
cassettes using PrP and Thy1.2 promoters

Expression of TDP-43 wt and mutant

(Gendron and Petrucelli 2011;
proteins results in variable pathological
Shan et al. 2010;
phenotypes depending on the animal model Stallings et al. 2010;
Wegorzewska and
Baloh 2011;
Wegorzewska et al. 2009;
Wils et al. 2010;
Xu et al. 2010)

123

550

Hum Genet (2012) 131:535563

Table 1 continued
Strategy

Pathology and other Wndings

Reference

FTLD/ALS

Forebrain-targeted Tet-OV conditional expression

of TDP-43. Bigenic mice were created with
cassettes harboring the Camk2a promoter
driving the expression of Tet-OV tTA and
the tetracycline-responsive promoter to
drive hTDP-43 (tetO-hTDP). hTDP-43 was
preferentially induced (in the absence of
doxycycline) in forebrain neurons

Selective degeneration of hippocampal

dentate gyrus and neocortical neurons
along with dysregulation and loss of
endogenous TDP-43 and presence of
rare phosphorylated and ubiquitinated
TDP-43 inclusions in aVected neurons.
Transgenic mice develop a motor
phenotype associated with spasticity

(Igaz et al. 2011)

ALS

Pronuclear injection of a human SOD1

transgene containing the G93A
substitution associated with familial ALS

Hemizygous transgenic mice develop

motor neuron disease associated with
progressive limb paralysis and
death by 56 months of age

(Gurney et al. 1994)

ALS/AD

CD11b-TKmt-30 mice contain a transgenic

cassette in which the HSV-1 TKmt-30
gene is expressed under the control of
the CD11b promoter, which directs high
levels of expression in macrophages.
Proliferating microglial cells in the brain
can be selectively eliminated by
ganciclovir treatment

After ganciclovir treatment, selective

ablation of proliferating microglial
cells occurs. Ablation of proliferating
microglia does not aVect motor neuron
degeneration in a mouse model of
ALS but causes increased amyloid
deposition in a mouse model of AD,
suggesting that microglia play a
critical role in restricting AD
associated senile plaque formation

(Dziennis et al. 1995;

Gowing et al. 2008;
Simard et al. 2006)

PD

Transgenesis with a BAC harboring the dopamine

transporter (Slc6a3) gene retroWtted with
a FLAG-tagged park2 truncated mutant
(Q311X) in exon 2

Mutant parkin-expressing animals exhibit

age-dependent dopaminergic neuron
degeneration and accumulation
of -synuclein in substantia
nigra (SN) neurons

(Lu et al. 2009)

PD

Transgenesis with retroWtted BAC

constructs containing the entire
FLAG-tagged wt or mutant (G2019S)
sequence of the leucine-rich repeat
kinase 2 (LRRK2) gene

Transgenic LRRK2-wt mice had elevated

striatal dopamine (DA) release with
unaltered DA uptake or tissue content,
mice were hyperactive and showed
enhanced performance in motor
function tests. LRRK2-G2019S transgenics
showed an age-dependent decrease
in striatal DA content, decreased striatal
DA release and uptake. LRRK2-G2019S
overexpression was not associated with
loss of dopaminergic neurons in
substantia nigra or degeneration of
nigrostriatal terminals by 12 months

(Li et al. 2010)

PD

Conditional knockout of the 19S RP

subunit, Psmc1 gene involved in 26S
proteasomal function in forebrain/substantia
nigra neurons using the Cre/loxP system.
Because Psmc1 null mice are embryonic
lethal, Psmc1 conditional knock-out mice
were generated by introducing loxP sites
in introns 1 and 3 in ES cells. Spatial ablation
of Psmc1 was performed by crossing the
Xoxed transgenic mice (Psmc1X/X) with a Cre
deleter strain expressing Cre recombinase
under the control of either the Camk2a
promoter or the tyrosine hydroxylase locus

Neurodegeneration, ubiquitin, and

-synuclein pathology with Lewy-like
inclusions apparent in Psmc1X/X Cre
animals. Inclusion bodies found
in mitochondria

(Bedford et al. 2008)

PD

Selective Tet-On inducible monoamine oxidase

B (MAO-B) in astrocytes. Two transgenic
cassettes were injected by pronuclear injection.
One harbored the MAO-B cDNA under the
control of a bidirectional Tet-inducible promoter
(also regulating the expression of  galactosidase)
and the other driving expression of the
tet-responsive reverse transactivator, rtTA
under the control of the mouse GFAP promoter

Dox-treatment induces hMAO-B and

-galactosidase in GFAP+ cells causing
altered spontaneous locomotor activity
associated with progressive degeneration
of TH+ SN neurons and reduced striatal DA

(Mallajosyula
et al. 2008)

Disease

123

Hum Genet (2012) 131:535563

551

Table 1 continued
Strategy

Pathology and other Wndings

PD

Human -synuclein (wild-type or mutant A53T

and A30P) cDNAs were included in
a transgenic cassette harboring the promoter,
5 intronic and 3 untranslated sequences
of the murine prion protein gene

Transgenic expression of A53T -synuclein

(Lee et al. 2002)
(but not the wild-type nor the A30P proteins)
resulted in the development of adult onset
neurodegenerative disease with a
progressive motor dysfunction together
with accumulations of -synuclein and ubiquitin

HD

Pronuclear injection of a 1.9-kb transgenic

containing 1 kb of 5 UTR
(promoter sequences), Htt exon 1
carrying expanded CAG repeats
(130 units) and the Wrst
262 bp of intron 1

Transgenic mice (R6/2) manifest a

rapidly progressive disease similar to
the juvenile form of HD in humans.
Overt behavioral symptoms apparent
by 5-6 weeks of age. Motor and learning
impairments are present. Limb
clasping, weight loss, and death occur
by 15 weeks. Nuclear inclusions present
particularly in the striatum and
CA1 region of the hippocampus

(Carter et al. 1999;

Davies et al. 1997;
Lione et al. 1999;
Mangiarini et al. 1996)

HD

YAC transgenesis with full-length normal

and mutant (72 and 128 repeat) human Htt

Mutant Htt results in abnormal behavior

and causes selective degeneration of
medium-sized spiny neurons in the lateral
striatum. An earlier and more severe
pathology found in YAC 128 transgenic
mice expressing longer CAG repeats.
Progressive cognitive dysfunction
and mood disturbance precede motor
abnormalities. Selective atrophy and
neuronal loss occurs in the striatum
and cortex of YAC128 transgenics

(Hodgson et al. 1999;

Slow et al. 2003)

HD

Knock-in mutant Htt using homologous

recombination in ES cells

Expression of mutant Htt in its native

genomic context. Presence of nuclear
staining and microaggregates early in
the course of the disease. Early overt
behavioral changes seen and strength
of the phenotype generally correlated
with the number of CAG repeats (200)

(Heng et al. 2009;

Levine et al. 2004;
Lin et al. 2001;
Raymond et al. 2011;
Shelbourne et al. 1999;
Wheeler et al. 2000;
White et al. 1997)

HD

BAC transgenesis with full-length

mutant Htt with 97 CAG repeats

Transgenic mice (BACHD) exhibit progressive

motor deWcits associated with later
neuropathology in striatum and cortex.
Pathology occurs without evidence
of aggregated mutant Htt

(Gray et al. 2008)

Disease

Reference

Rat Rattus norvegicus

AD

Neuronal expression of normal and mutant

APP under the control of either synapsin-1,
PDGF or ubiquitin C (UbC)
promoters. Pronuclear
injection of transgenic cassettes

No -amyloid plaques unless two independent

FAD mutant transgenic lines are bred
to double homozygosity. High-level
expression and the presence of an
FAD mutation necessary to induce
age-dependent plaque deposition

(Bugos et al. 2009; Clarke

et al. 2007; Echeverria
et al. 2004; Agca et al.
2008; Flood et al. 2009;
Folkesson et al. 2007;
Liu et al. 2008a;
Ruiz-Opazo et al. 2004)

AD

Pronuclear injection of a cassette harboring the

hAPP751 cDNA containing the Swedish
and Indiana mutations under the control
of the Thy1.2 promoter

Pan-neuronal expression of human

APP751 was found. Homozygous
transgenic animals produce extracellular
A deposits by 6 months of age associated
with glial activation and dystrophic neurites.
Cognitive functions are altered
as early as 3 months of age

(Leon et al. 2010)

Constitutive expression of the mutant

TDP-43 causes early postnatal death
in transgenic rats. Founders develop
weakness in the limbs and do not
survive past 29 days

(Zhou et al. 2010)

FTLD/ALS Transgenesis with a 22-kb normal and

retroWtted-mutant (M337 V) human
TDP-43 gene extracted from a BAC

123

552

Hum Genet (2012) 131:535563

Table 1 continued
Disease
FTLD/ALS

Strategy

Pathology and other Wndings

Reference

Tet-OV inducible system for TDP-43 expression.

tTA protein expression controlled by a CMV
enhancer fused to the chicken  actin promoter (CAG).
Normal and mutant TDP-43 cDNAs were expressed
under the control of a tTA-dependent promoter
constructed by the juxtaposition of multiple t
etracycline-responsive elements (TRE, tet-O)
upstream from the minimal CMV promoter

Overexpression of mutant TDP-43 but

not wt protein induce progressive
widespread neurodegeneration
aVecting predominantly the motor
system with denervation atrophy
of skeletal muscles

(Zhou et al. 2009, 2010)

of A3-42 after cleavage (Link 1995). Expression of this

human A42 minigene in muscle cells resulted in progressive paralysis and staining of muscle cells with a human
A42 speciWc antibody revealed A42 deposits that reacted
with Congo red and thioXavin S. Ultrastructural studies,
however, revealed that the deposits were intracytoplasmic
rather than extracellular as are amyloid plaques found in
AD (Ewald and Li 2010). Pan-neuronal expression of
human A42 has also been associated with amyloid deposits and deWcits in odorant preference-associated learning
behavior, serotonin-controlled behaviors, experiencedependent learning and egg laying behavior (Dosanjh et al.
2010). Transgenic C. elegans has also been used to investigate how speciWc residues within human A aVect Wbril
formation (Ewald and Li 2010).
Models such as those developed in C. elegans may be
questioned for their reliance on the isolated overexpression
of a non-native protein that is not naturally processed. Overexpression of APP in Downs syndrome is associated with
AD pathology in humans (Zigman et al. 1996) and families
have been identiWed in which a duplication of the APP gene
leads to FAD presumably due to overexpression of wild type
APP (Kasuga et al. 2009; Sleegers et al. 2006). Thus, there
is a precedent for APP overexpression leading to AD in
humans. Yet there is no evidence for APP overexpression in
the most common sporadic forms of AD. The lack of extracellular amyloid deposits can also be seen as a limitation of
these models. On the other hand current models of the amyloid hypothesis postulate that soluble forms of oligomeric
A rather than plaque amyloid are the critical toxic species
(Lublin and Gandy 2010) and indeed after only several
hours of A induction in C. elegans, paralysis occurs without detectable A deposits (Drake et al. 2003). Toxicity has
been correlated with levels of oligomeric A (Wu et al.
2006), making it plausible that the changes are physiologically relevant. Induction of oxidative stress has long been
postulated as one of the key steps leading to injury in the
amyloid cascade and transgenic worms expressing human
A42 exhibit signs of oxidative stress prior to deposition of
A (Drake et al. 2003), suggesting that soluble A42 may
induce oxidative stress in vivo in C. elegans.

123

Like C. elegans, Drosophila has a single homolog of

APP. Flies deWcient for Drosophila APP (dAPPL) exhibit
behavioral abnormalities that can be rescued with a human
APP transgene and dAPPL has also been implicated in neurite outgrowth and synapse formation (Iijima et al. 2008).
Drosophila also contains orthologs of the  and -secretases although -secretase activity in Drosophila is low. In
addition, while dAPPL shares about 30% amino acid identity with the human protein, like C. elegans apl-1 it lacks a
homologous A region although it has been suggested that
an equivalent A-peptide exists that upon cleavage can
generate a neurotoxic peptide capable of generating amyloid deposits (Carmine-Simmen et al. 2009).
In transgenic Drosophila human A peptides have been
introduced either by overexpressing full-length human APP
or human A peptides. To create a partially humanized system Greeve et al. (2004) engineered triple transgenic Xies
that express human wild type APP, human BACE and also
overexpresses wild-type Drosophila presenilin (dPsn) or
dPsn with point mutations corresponding to human FAD
mutations. The lines produced exhibited modest elevations
of A40 and A42 peptides. Triple transgenic Xies developed thioXavin S stained amyloid plaques in the retina and
exhibited an age-dependent degeneration of the photoreceptors that was enhanced in Xies expressing the FAD
related dPsn mutations. This phenotype could be suppressed by genetically reducing endogenous Drosophila
dPsn or by the treatment with  or -secretase inhibitors.
Others have taken the approach used in C. elegans of
introducing isolated human A peptides fused to an N-terminal signal peptide directly into neurons (Crowther et al.
2005; Finelli et al. 2004; Iijima et al. 2004, 2008). Both
A40 and A42 peptides accumulate during aging in the Xy
brain, but only A42 forms amyloid deposits in brain and
retina (Finelli et al. 2004; Iijima et al. 2004). Age-dependent short-term memory defects are detected in both A40
and A42 expressing Xies but at later ages, A42, but not
A40, induces locomotor deWcits, as well as causes an agedependent neurodegeneration and premature death. These
models have also been associated with intraneuronal accumulation of A42 (Iijima et al. 2004, 2008).

Hum Genet (2012) 131:535563

As with similar approaches used in C. elegans, strategies

that rely on the isolated overexpression of a non-native protein may be considered artiWcial. Indeed as discussed below
overexpression strategies even in mammalian systems may
be questioned as artiWcial. Organisms like D. melanogaster
or C. elegans also lack a mammalian like immune system
which is thought to play a role in the development of AD
pathology. Yet if the essential tenets of the amyloid hypothesis are correct then whether overexpressed or not any system that exhibits a robust amyloid dependent pathology
may be suitable to model disease pathogenesis or search for
disease modiWers. Retinal toxicity for example has been
widely used as a phenotypic readout for screens in Drosophila aimed at identifying genetic modiWers of neurodegenerative disease.
Transgenic modeling has been most aggressively pursued in mice and transgenic mice now exist that mimic
nearly the full range of AD-related pathology (Table 1;
Elder et al. 2010a). These models have suggested new
insights into the pathophysiology of AD and have been the
source of novel therapeutic approaches, most notably the
development of immunotherapy (Wisniewski and Boutajangout 2010). These models, however, raise a number of
issues as well. Firstly, like the models in C. elegans and
Drosophila it is clear that their success has depended on the
overexpression of APP transgenes containing FAD-associated mutations at levels that are not physiological. Indeed,
despite the fact that a number of APP wild transgenic lines
have been created only one transgenic mouse line overexpressing wild-type human APP has been reported as
developing amyloid plaques (Herzig et al. 2004) and for
plaque pathology to develop it seems to require the presence of an APP FAD.
In this context it is interesting to compare APP FAD
over-expression models to APP knock-in mice. For example, the APP Swedish mutation (associated with FAD) has
been introduced into the mouse APP gene (Reaume et al.
1996). The three amino acid diVerences between mouse and
human APPs in the A region were humanized such that
these mice produce human A. These mice should represent the most natural model of human FAD in the mouse.
Yet despite increased A production, no amyloid plaques
or neuropathology have been observed to develop.
The models have also been troubled by the diYculty of
producing the full spectrum of AD pathology including
neuronal loss. For example neither of the commonly studied PDAPP nor Tg2576 mice exhibit neuronal loss despite
having extensive amyloid deposition (Irizarry et al. 1997a,
b). More substantial neuronal loss has been reported in
mice expressing multiple PS1 and APP mutations (Casas
et al. 2004; Oakley et al. 2006; Schmitz et al. 2004) and
gross neuronal loss has been reported in mice expressing
three APP and two PS1 FAD mutations (Oakley et al.

553

2006). Thus, neuronal loss can be induced in the mouse but

only it would seem by combining multiple mutations that
individually are suYcient to cause disease in humans.
Another problem with the models has been the general
diYculty of recreating the characteristic cytoskeletal
pathology found in AD (Elder et al. 2010a). Various suggestions have been made for the diYculty of inducing neuroWbrillary tangle-like lesions in the mouse including
diVerences between human and mouse tau, although the
reasons remain to be fully explained. Recently mice that
exhibit NTF-like lesions and plaques have been produced
by combining FAD mutations with mutant forms of tau
associated with FTLD. In the 3xTg-AD model Oddo et al.
(2003) generated triple transgenic mice by co-injecting two
transgenes containing the FAD APP Swedish mutation and
the P301L mutations associate with FTLD into fertilized
eggs harvested from PS1M146V knock-in mice. The resulting transgenic lines thus expressed mutations in APP and
tau from exogenous transgenes combined with a PS1 FAD
mutation from the endogenous mouse gene. With aging
these mice exhibited increased A40 and 42 levels. Intraneuronal A accumulated and the mice developed amyloid
plaques and NFT-like lesions that immunostained with
antibodies to conformationally altered tau epitopes. Functionally, 3xTg-AD mice developed age-dependent synaptic
dysfunction including altered long-term potentiation and
deWcits in spatial memory (Billings et al. 2005; Oddo et al.
2003).
The 3xTg-AD model thus exhibits a broader spectrum of
AD pathology with the much-sought-after plaque and tangle pathology. However, they represent a composite of two
distinct diseases that do not naturally occur together.
Plaque development is almost certainly driven by the APP
and PS1 FAD mutations while tangle-like pathology is
driven by the tau mutations. Indeed in 3xTg-AD mice, the
pathologies arise in spatially distinct patterns with A
deposition starting in neocortex and appearing later in hippocampus, while tau pathology progresses in the opposite
direction, beginning in hippocampus and appearing later in
neocortex (Oddo et al. 2003). The two pathologies do
appear, however, to interact. Intracerebral injections of
anti-A antibodies into the hippocampus of 3xTg-AD mice
not only reduce A accumulation but cause clearance of
early stage tau pathology (Oddo et al. 2004).
Due to the lack of a plaque pathology, mice expressing
only presenlin FAD mutations have been less studied than
APP or APP/PS FAD mutant transgenic mice which
develop robust plaque pathology (Elder et al. 2010a). Why
presenilin FAD mutant transgenic mice do not develop a
more AD-like pathology is unclear given the potency of
especially the presenlin-1 FAD mutations in humans, which
typically induce disease at younger ages than APP FAD
mutations (Bertram and Tanzi 2004a, b; Elder et al. 2010b).

123

554

Why single transgenic PS1 and PS2 mice fail to develop

plaque pathology is not entirely clear, but may relate to the
generally lower levels of A42 found in single PS transgenics compared to APP overexpressing lines as well as the
lack of elevation of A40 in PS transgenics. It may also
relate to diVering aggregation properties of mouse and
human A (Jankowsky et al. 2007). Yet despite the lack of
a plaque and tangle pathology presenilin FAD mutant mice
exhibit a wide range of anatomic, electrophysiological and
biochemical changes that mimic many of the features of
AD (Elder et al. 2010b). Rat models while becoming more
available appear to suVer the same limitations as models in
the mouse requiring high-level expression of an APP FAD
mutation to induce signiWcant plaque pathology (Table 1;
Bugos et al. 2009; Clarke et al. 2007; Echeverria et al.
2004; Agca et al. 2008; Flood et al. 2009; Folkesson et al.
2007; Leon et al. 2010; Liu et al. 2008a; Ruiz-Opazo et al.
2004).

Hum Genet (2012) 131:535563

its orthologues protect against neurodegeneration in both

invertebrate and mammalian models of neurodegenerative
disease including Alzheimers disease and ALS (Gan and
Tang 2010; Kim et al. 2007; Pallas et al. 2009). In a transgenic mouse model of ALS, deletion of p66Shc ameliorates
mitochondrial dysfunction and delays disease onset as well
as improves motor performance and prolongs survival
(Pesaresi et al. 2011). Inhibition of mTOR by rapamycin
abolishes cognitive deWcits and reduces -amyloid levels in
a mouse model of Alzheimers disease (Spilman et al.
2010). Circadian rhythms generate oscillations in physiology
and behavior. Circadian clock gene expression is often
altered in human disease states and transgenic mice with
mutations in clock genes develop premature aging (Khapre
et al. 2010). Circadian dysfunctions are found in transgenic
mouse models of AD (Wisor et al. 2005; Sterniczuk et al.
2010), PD (Kudo et al. 2011a) and HD (Kudo et al. 2011b).
These and many other factors related to how organisms age
may inXuence the phenotypic expression of a neurodegenerative process in a transgenic model.

The factor of aging in transgenic models

of neurodegeneration
Conclusions
Many factors may aVect the ability of transgenic organisms
to model human diseases. These include obvious anatomical and morphological diVerences between humans and
model organisms as well as possibly diVerences in critical
proteins such as tau or APP. Another factor, however, is the
process of aging itself. All age-related neurodegenerative
diseases develop in the context of aging. Aging as a comorbid process is believed to be the result of the gradual
loss of metabolic and genetic assets necessary to preserve
the integrity and functionality of all cellular components. It
has been assumed (Minois et al. 2010) that senescence is
under the control of many genes (Ljubuncic and Reznick
2009), and is aVected by accumulation of deleterious
somatic mutations (Best 2009). The factor of pleiotrophic
antagonism has also been invoked whereby certain gene
functions that may be beneWcial in early life may be deleterious as the organism ages (Williams 1957).
Due to diVerences in cellular turnover, metabolic rates,
life span, oxidative stress, diet and other environmental factors as well as species-speciWc genetic factors, aging may
be modeled insuYciently in relation to development of neurodegenerative diseases in transgenic animal models. Nevertheless, it is clear that aging inXuences considerably the
development of neurodegenerative processes. Over activation or disruption of key lifespan determinant pathways
such as SIR2/SIRT1 (NAD-dependent class III histone
deacetylases), p66Shc (66-kDa isoform of the growth factor
adapter Shc) and mTOR (mammalian target of rapamycin)
clearly aVect the development of neurodegenerative diseases in model organisms. For example, SIR2/SIRT1 and

123

Advances in the genetic tools available across a wide range

of species have made transgenic modeling more versatile
than ever before. While the mouse remains the most widely
used model system, signiWcant eVorts have been made in a
variety of other species. In particular, recent advances in
the tools available for genetic manipulation in the rat have
lead to increased interest in this species as a model system.
These models have improved understanding of disease
pathogenesis and, in some cases, have lead to new therapeutic approaches. They will continue to play central roles
for years to come in both preclinical testing and as tools for
developing insight into the biological basis of human neurodegenerative diseases.
Acknowledgments Supported by the Alzheimers Association
(IIRG-07-57318), Department of Veterans AVairs (1 I01 BX00034201) and the National Institute on Aging (P50 AG005138).

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