Neuroscience Research 75 (2013) 6–12
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Neuroscience Research
journal homepage: www.elsevier.com/locate/neures
Review article
Channelrhodopsins—Their potential in gene therapy for neurological disorders
Zhi-Gang Jia,c
, Toru Ishizukaa,c
, Hiromu Yawoa,b,c,∗
a
Department of Developmental Biology and Neuroscience, Tohoku University Graduate School of Life Sciences, Sendai 980-8577, Japan
b
Center for Neuroscience, Tohoku University Graduate School of Medicine, Sendai 980-8575, Japan
c
JST, CREST, Sendai 980-8577, Japan
a r t i c l e i n f o
Article history:
Received 27 June 2012
Received in revised form 18 August 2012
Accepted 10 September 2012
Available online 28 September 2012
Keywords:
Channelrhodopsin
Halorhodopsin
Optogenetics
Neural dysfunction
Neurological disorders
Gene delivery
Primates
Optics
Parkinson disease
Deep brain stimulation (DBS)
Epilepsy
Retinis pigmentosa (RP)
Age-related macular degeneration (AMD)
Retinal prosthesis
Spinal cord injury (SCI)
adeno-associated virus (AAV)
a b s t r a c t
Recently, channelrhodopsins (ChRs) have begun to be used to manipulate the neuronal activity, since they
can be targeted to specific neurons or neural circuits using genetic methods. To advance the potential
applications in the investigation and treatment of neurological disorders, the following types of basic
research should receive extensive financial support. The spectral and kinetic properties of ChRs should be
optimized according to the application by generating variants of ChRs or exploring new rhodopsins from
other species. These ChRs should be targeted to the specific types of neurons involved in the neurological
disorders through a gene expression system using cell- or tissue-specific promoters/enhancers as well
as gene delivery systems with modified virus vectors. The methods have to be developed to apply the
genes of interest with safety and long-term effectiveness. Sophisticated opto-electrical devices should be
developed. Appropriate primate animal model systems should be established to minimize the structural
differences between small animals such as rodents and human beings. In this paper, we will review
the current progress in the basic research concerned with the potential clinical application of ChRs and
discuss the future directions of research on ChRs so that they could be applied for human welfare.
© 2012 Elsevier Ireland Ltd and the Japan Neuroscience Society. All rights reserved.
Contents
1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7
2. Current strategies to treat neurological disorders. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7
3. Molecular biology of ChRs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7
4. The potential of application of ChRs to neurological disorders . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7
4.1. Retinal prosthesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7
4.2. Spinal cord injury. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8
4.3. Parkinson’s diseases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8
5. Perspectives . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8
5.1. ChRs variants and new rhodopsins from other species. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8
5.2. The target specificity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9
Abbreviations: AAV, adeno-associated virus; AMD, age-related macular degeneration; ArchT, archaerhodopsin-T from Halorubrum strain TP009; ChR, channelrhodopsin;
ChR1, channelrhodopsin-1; ChR2, channelrhodopsin-2; ChRFR, ChR-fast receiver; ChRWR, ChR-wide receiver; ChRGR, ChR-green receiver; DBS, deep brain stimulation; GPi,
globus pallidus interna; IgG, immunoglobulin G; L-DOPA, L-3, 4-dihydroxyphenylalanine; LED, light-emitting diode; LV, lentiviral vector; Mac, Leptosphaeria maculans opsin;
MChR1, channelrhodopsin-1 from Mesostigma viride; NpHR, halorhodopsin from Natronomonas pharaonis; PD, Parkinson’s disease; PPN, pedunculopontine nucleus; RO, raphe
obscurus; RP, retinitis pigmentosa; SCI, spinal cord injury; SFO, step-fuction opsin; SSFO, stabilized step-function opsin; STN, subthalamic nucleus; TM, transmembrane;
VChR1, channelrhodopsin-1 from Vovox cateri.
∗ Corresponding author at: Tohoku University Graduate School of Life Sciences, Developmental Biology and Neuroscience, 2-1-1 Katahira, Aoba-ku, Sendai 980-8577,
Japan.
E-mail address: yawo-hiromu@m.tohoku.ac.jp (H. Yawo).
0168-0102/$ – see front matter © 2012 Elsevier Ireland Ltd and the Japan Neuroscience Society. All rights reserved.
http://dx.doi.org/10.1016/j.neures.2012.09.004
Z.-G. Ji et al. / Neuroscience Research 75 (2013) 6–12 7
5.3. Safety and long-term effectiveness . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9
5.4. Optimized light delivery . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9
5.5. The development of non-human primate models. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10
Acknowledgments. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10
1. Introduction
The brain is a network consisting of billions of neurons and
their synapses. It is the signal transference from one neuron to
another that enables one to feel, move, learn and think. When
there is a dysfunction in this system, various neurological diseases
can arise. To date, various approaches have been developed
to treat people for neurological diseases such as pharmacological,
surgical and gene therapies. These methods have improved
patients’ quality of life, but with some disadvantages. When channelrhodpsins
(ChRs) were identified from the Chlamydomonas
genome by several groups about ten years ago (Nagel et al., 2002,
2003; Sineshchekov et al., 2002; Suzuki et al., 2003), an innovative
method referred to as “optogenetics” was developed (Deisseroth
et al., 2006; Deisseroth, 2011), which has the potential of leading
to new strategies for investigating and treating neurological diseases.
In this review, we will discuss potential clinical applications
and future research on ChR-based gene therapies for neurological
diseases.
2. Current strategies to treat neurological disorders
To date, neurological disorders are commonly treated with
pharmacological and surgical approaches, and attempts have been
made using gene therapies, as well. Although often effective, they
also have shortcomings that need to be overcome. Pharmacological
therapy is frequently accompanied by side effects because of
the low specificity of the drugs for their target receptors and the
undesired spread. For example L-DOPA, one of the most effective
drugs currently prescribed for Parkinson’s disease (PD), is
frequently accompanied by adverse effects such as motor fluctuations
and dyskinesia (Rascol et al., 2000; Olanow et al., 2006).
These side effects make long term treatment difficult, although
recent reports suggest that L-DOPA-induced dyskinesia could be
attenuated if L-DOPA is combined with a catechol-O-methyl transferase
inhibitor (entacapone) or a monoamine oxidase inhibitor
B (selegiline or rasagiline) (Rascol et al., 2002; Miyasaki, 2006).
Deep brain stimulation (DBS) is one of the surgical therapies for
the treatment of neurological disorders. In the case of PD, DBS
has been used to target various regions such as the subthalamic
nucleus (STN), globus pallidus interna (GPi), and pedunculopontine
nucleus (PPN). Although it relieves patients from some symptoms
of PD, the basic mechanisms of DBS remain unclear, though several
hypotheses have been described (Limousin et al., 1998; Vitek,
2002; Kringelbach et al., 2007; Montgomery and Gale, 2008). With
the development of molecular and genetic techniques, numerous
genes that are responsible for various neurological disorders have
been identified. However, the mechanisms of neurological disorders
are complex and do not always involve only a single gene.
For example, epilepsy, which is caused by an imbalance between
excitatory and inhibitory neurons, is derived from various genetic
abnormalities such as SCN1A (Escayg et al., 2000; Catterall et al.,
2010), SCN1B (Wallace et al., 1998), KCNQ2 (Singh et al., 1998;
Su et al., 2011), and GABAA receptor subunit genes (Macdonald
et al., 2010). Therefore, gene therapy for epilepsy is still a challenge.
However, as ChR2 came to be used to manipulate neural activity in
neuroscience research, it has become a candidate for overcoming
these limitations.
3. Molecular biology of ChRs
Chlamydomonas reinhardtii is one of unicellular green algae
that senses light through an eyespot apparatus. Under electron
microscopy, the eyespot is an organelle composed of a plasma
membrane, a specialized chloroplast membrane and two layers
of highly ordered carotenoid-rich granules. Each granular layer is
subtended by a thylakoid membrane. ChRs including ChR1 and
ChR2, are generally considered to be localized in the plasma membrane
in apposition with the carotenoid granules (Lamb et al.,
1999; Suzuki et al., 2003). They are light-activated cation channels
and mediate the light-dependent behavior of the algae. ChRs are
the members of the microbial (archaeal, type-I) rhodopsin family
and consist of a seven-pass transmembrane (TM) apoprotein
and a retinal which covalently binds to it. The photoisomerization
of all-trans-retinal to 13-cis configuration is coupled to conformational
changes of the apoprotein which gate the channel structure
to become open to the permeation of cations such as H+, Na+, and K+.
In other words, the light signal is converted into an electrical one by
ChRs (Hegemann, 2008). Previously, several groups independently
reported that neurons could be endowed with photosensitivity by
genetically introducing ChR2 (Boyden et al., 2005; Li et al., 2005;
Ishizuka et al., 2006). In doing so, one can manipulate the activity
of neurons either in vitro or in vivo by light with high spatial resolution
and millisecond-order precision (Boyden et al., 2005; Ishizuka
et al., 2006). Neuronal activity can also be negatively regulated by
light (Zhang et al., 2011) if the neurons are engineered to express
either a Cl−-transporting rhodopsin, e.g. NpHR from Natronomonas
pharaonis (Han and Boyden, 2007; Zhang et al., 2007) or an H+transporting
rhodopsin, e.g. archaerhodopsin-3 from Halorubrum
sodomense (Arch), archaerhodopsin from Halorubrum strain TP009
(ArchT) and the Leptosphaeria maculans opsin (Mac) (Chow et al.,
2010). These properties can enable ChR2 and other rhodopsins to
be used for manipulating neural circuits and treating neurological
diseases. Numerous reports have suggested that optogenetics
would have the advantages of safety and targeting specificity when
applied to the clinical field.
4. The potential of application of ChRs to neurological
disorders
4.1. Retinal prosthesis
Vision is one of the major sources of information about the
world. Generally, light entering the eye is absorbed by the photoreceptor
cells, the rods and cones, which are the major receptor
cells directly sensitive to light. In the photoreceptor cells, light signals
are transduced into electrical signals. Then, the information
is transmitted through bipolar cells directly or indirectly to retinal
ganglion neurons, which generate action potentials. Finally, the
signal is sent to the brain by the axons of retinal ganglion cells
for further processing, generating visual perception of the external
world. However, in cases of retinal degenerative diseases, such
as retinitis pigmentosa (RP) and age-related macular degeneration
(AMD), the patients are seriously impaired in their every-day life.
Although light-sensing photoreceptor cells are degenerated in the
patients with RP or AMD, other downstream retinal neurons, such
as bipolar cells and retinal ganglion cells, are still preserved, with
8 Z.-G. Ji et al. / Neuroscience Research 75 (2013) 6–12
the function of their optic nerve projections being largely maintained
(Humayun et al., 1999). If the remaining neurons could be
made light sensitive, the quality of life of such patients could be
remarkably improved. Bi et al. expressed ChR2 in inner retinal ganglion
neurons of rd1/rd1 mice, one of the RP animal models, and
demonstrated that the mice became sensitive to blue light with
the expression of ChR2 (Bi et al., 2006). Meanwhile, Tomita et al.
independently delivered ChR2 genes into ganglion neurons of RCS
rats, another model of RP, using AAV vector, and demonstrated the
recovery of visual-evoked potentials in these rats (Tomita et al.,
2007). These pioneering studies using ChR2 open the possibility of
treating the loss/weakness of vision with this new strategy. Using
a transgenic rat, which expressed ChR2 in retinal ganglion neurons,
the optomotor responses, such as moving its head to track
objects made up by blue light stripes over a black background,
were shown to remain even after the induction of degeneration of
photoreceptor cells with toxic light exposure (Tomita et al., 2009)
although there remain some debates (Thyagarajan et al., 2010).
Lagali et al. expressed ChR2 specifically in the ON-bipolar cells
through the mGluR6 promoter. Their behavioral experiment also
showed the restoration of vision (Lagali et al., 2008). Recently, the
safety and long-term effectiveness in multiple mouse models of
loss/weakness of vision have been demonstrated (Sugano et al.,
2011; Doroudchi et al., 2011). More importantly, exiting news from
Wayne State University reported that RetroSense Therapeutics (a
biotechnology company that is developing a game-changing gene
therapy to restore vision in patients suffering from loss/weakness
of vision) signed a license agreement with Pan’s group (Wayne State
University) for novel gene-therapy approaches to vision (Navitsky,
2012), which will be an excellent step for ChR-based retinal prosthesis
in the clinical setting.
4.2. Spinal cord injury
Spinal cord injury (SCI) includes any damage or trauma to the
spinal cord. With acute SCI, such patients can lose the peripheral
sense and/or the ability to move the body, in addition to other
symptoms such as dysfunction of bladder and bowel, and respiratory
insufficiency. Approximately 300,000 people with SCI
live in the US (100,000 patients in Japan), and there are about
12,000 new patients every year (Woo et al., 2011). In 2005, Li et al.
employed ChR2 to control spinal cord neural activity and offered
the possibility that the symptoms of spinal cord injury could be
attenuated using ChR2 (Li et al., 2005). Thereafter, as one of the key
papers using ChR2 in the treatment for SCI, the report of Alilain
et al. (2008) should be noted. They made surgical hemi-section of
the spinal cord at the left C2 level in a rat model of SCI, resulting in
hemiplegia. As a result, the rats breathed with difficulty due to the
half-paralyzed diaphragm. After introducing ChR2 in the phrenic
nucleus, blue light illumination triggered the activity of phrenic
nerves in the paralyzed diaphragm in a manner synchronous with
the respiratory rhythm of the unparalyzed side. More recently,
Kiehn’s group generated a transgenic mouse line that expressed
ChR2 in the hindbrain and spinal cord under the control of the
VGluT2 promoter. They were able to control locomotor-like
activity of the mice with appropriate left-right and flexor-extensor
alternation through blue light illumination. In addition, when they
activated glutamatergic neurons in the hindbrain, the neurons
provided a direct command that could activate the spinal locomotor
network (Hägglund et al., 2010). Guyenet’s group employed
PRSx8 promoter to express ChR2 selectively in Phox2b-containing
neurons, which might be central respiratory chemoreceptors. They
firstly showed the crucial evidence that Phox2b-containing neurons
play an important role in central respiratory chemoreception
(Abbott et al., 2009, 2011). By expressing ChR2 selectively in raphe
obscurus (RO) serotonergic neurons, they also showed that RO
serotonergic neurons potentiate the central respiratory chemoreflex
but do not appear to have a central respiratory chemoreceptor
function (Depuy et al., 2011). All these suggested that ChR2-based
gene therapy could open up a new strategy for helping SCI patients
to walk and respire without external assistance.
4.3. Parkinson’s diseases
Parkinson’s disease (PD) is one of the most common neurodegenerative
disorders affecting mainly the elderly population. It is
characterized by the loss of dopaminergic neurons in the substantia
nigra, resulting in motor impairments as well as non-motoric
symptoms (Thomas and Beal, 2007). Recently, the neural circuitry
involved in PD was optogenetically investigated by Gradinaru et al.
(2009). They utilized enhanced NpHR (eNpHR) or ChR2, respectively
to inhibit or activate the excitatory neurons in STN of
hemiparkinsonism rats the substantia nigra of which had been
lesioned by a dose of 6-hydoxydopamine. However, neither the
eNpHR-mediated inhibition nor the ChR2 mediated excitation
of STN neurons relieved the behavioral abnormality of hemiparkinsonism
rats, suggesting STN excitatory neurons would not
be the targets of DBS. However, using the hemi-parkinsonism
mice that expressed ChR2 exclusively in the afferent fibers but
not in the cell bodies of STN, they found that high frequency
photo-stimulation relieved but low frequency photo-stimulation
worsened the PD symptoms. Thus their results suggested the possible
involvement of STN afferents in DBS, which is consistent
with previous reports that DBS may activate adjacent fiber pathways
(Xu et al., 2008). Within the striatum of the basal ganglia,
medium spiny neurons can be categorized into at least two types:
D1 receptor-expressing (D1) and D2 expressing (D2) neurons. In the
classical basal ganglia circuitry, the activation of D1 neurons facilitates
movement, whereas that of D2 inhibits movement, and thus
they have been termed “direct pathway” and “indirect pathway”
neurons, respectively (Albin et al., 1989; Alexander and Crutcher,
1990). However, this idea has never been experimentally tested.
Recently Kravitz et al. utilized the Cre-loxP system to express ChR2
in only the direct pathway (D1) or indirect pathway (D2). They
presented the first experimental evidence for the classical. More
importantly, they could regulate the parkinsonism symptoms by
optogenetically activating the D1 neurons (Kravitz et al., 2010).
Optogenetics also appeared promising for the investigation and
possible treatment of other neurological disorders such as epilepsy
(Tønnesen et al., 2009), schizophreniza (Peled, 2011; Maher and
LoTurco, 2012), and autism (Tye and Deisseroth, 2012).
Taken together, all these studies suggested that ChR-based
therapy could lead to exciting and promising treatments for neurological
disorders.
5. Perspectives
Although ChR-based gene therapy appears promising, for clinical
applications, further research concerning the following will
be necessary: (1) the development of new ChR variants with various
advantages; (2) target selectivity; (3) safety and long-term
effectiveness; (4) optimized light delivery systems and (5) the
development of non-human primate animal models.
5.1. ChRs variants and new rhodopsins from other species
ChR2 has unique advantages for gene therapy in neurological
diseases. However, its clinical application would be limited by several
disadvantages such as strong current desensitization, small
current amplitude and blue light preference (Nagel et al., 2003;
Ishizuka et al., 2006). Gene therapy would be facilitated by variants
Z.-G. Ji et al. / Neuroscience Research 75 (2013) 6–12 9
of ChRs with various properties generated by targeted mutagenesis,
by TM helix shuffling, or by both in combination. Among these,
those including the TM1 and 2 of ChR1, for example, ChRFR, ChRWR
and ChIEF, generally showed reduced desensitization, improved
folding/membrane expression and enhanced photocurrent (Wang
et al., 2009; Lin et al., 2009; Yawo, 2009). In addition, ChRGR, a
derivative of ChR1 whose TM6 and C-terminal subdomain of TM7
were replaced by the counterparts from ChR2, showed similar
wavelength sensitivity as ChR1, but exhibited not only enhanced
photocurrents but also almost negligible desensitization (Wen
et al., 2010).
The variants derived from the targeted mutation of ChR2
showed certain improvements in various properties. For example,
ChR2-T159C exhibited improved membrane protein expression
and the largest photocurrent of all available ChR variants (Berndt
et al., 2011). ChR2 step-function opsins (SFOs) such as ChR2-C128T,
ChR2-C128A, ChR2-C128S and ChR2-D156A, showed extended
inward current flowing after the turning off of blue light (Berndt
et al., 2009; Bamann et al., 2010). SFOs should be very useful for
clinical experiments because of their long-term excitation, reduced
light requirements, faster activation and inactivation by light of
longer wavelength (Berndt et al., 2009; Yizhar et al., 2011; Tye and
Deisseroth, 2012). More recently, stabilized step-function opsins
(SSFOs), a double mutant of ChR2, C128S/D156A, was reported to
be more sensitive to light and could be activated even in brain tissue
as deep as 3 mm (Yizhar et al., 2011).
The counterparts of channelrhodopsin from other species such
as VChR1 isolated from Vovox cateri and MChR1 from Mesostigma
viride showed maximal action spectrum at 520 and 528 nm, respectively
(Zhang et al., 2008; Govorunova et al., 2011). Activation of
ChRs with light of longer wavelength is preferable to minimize light
scattering and absorption by tissues, while reducing the photocytotoxicity
to the cells. Excitation with red-shifted wavelength is
also preferable to avoid strong pupil constriction when applied for
retinal prosthesis (Busskamp and Roska, 2011).
We believe more suitable ChR variants will be produced, that,
with the aid of structural studies (Kato et al., 2012), will accelerate
the development of gene therapy for neurological diseases.
5.2. The target specificity
One of advantages of ChR-based gene therapy is that the gene
expression can be regulated in a cell-type-specific manner. For
example, the mGluR6 promoter has been employed to drive the
ChR2 gene to be expressed specifically in ON-bipolar cells of degenerated
retinas in a Pde6b (rd1) mice model (Lagali et al., 2008).
The CaMKII␣ and PRSx8 promoters allow ChR2 to be expressed
exclusively in excitatory neurons and Phox2b-containing neurons,
respectively (Abbott et al., 2009; Gradinaru et al., 2009). Thus
the challenge for future research will be to identify new promoters/enhancers
that drive ChR2 gene expression in specific neurons
or tissue. To deliver the gene using viral vectors, the cell-specific
promoters/enhancers should be small in their size because of the
packaging capacity of the virus. For example, the maximal packaging
capacity of adeno-associated viruses (AAVs) is 4.1–4.9 kb
(Dong et al., 1996), although it was increased in some AAV mutants
(Grieger and Samulski, 2005; Allocca et al., 2008). To increase
the packaging capacity, studies on developing dual vector expression
of the transgene have appeared promising. On the basis of
the property of AAV, Duan et al. developed a dual-vector system
with which they coinfected the reporter vector containing
the transgene and the driver vector encoding multiple enhancer
sequences (Duan et al., 2000). Several reports suggested that the
limited packaging capacity has been overcome by the two-vector
system (Duan et al., 2001; Goncalves et al., 2005; Palfi et al.,
2012).
Alternatively, ChR2 could be expressed in a specific type of
neuron by relying on the tropism of the viral vectors. Previous
studies reported that targeting a gene to a specific cell type can be
made by modifying the tropism of viruses, such as the adenovirus
(AD) and the AAV (Nicklin et al., 2005; Betley and Sternson, 2011).
Based on different capsid proteins, which are responsible for the
tropism of viruses, various serotypes of AAV and mutations have
been identified (Rutledge et al., 1998; Daya and Berns, 2008; PetrsSilva
et al., 2009). Some of them have been used for gene targeting.
For example, in the visual system, AAV2/2 has been demonstrated
to drive gene expression specifically in retinal ganglion cells and
optic nerve fibers after intra-vitreal delivery (Auricchio et al., 2001).
In contrast, AAV6 is favored exclusively by astrocytes (Betley and
Sternson, 2011), and ShH10, a new AAV variant, mainly by Müller
cells (Klimczak et al., 2009). Another promising way to target ChRs
to specific neurons is employing antibody-displaying viral vectors,
which not only target the transgene to a specific cell type but also
overcome the limited packaging capacity of traditional viral vectors.
Recently, Konno et al. reported that they employed the ZZ
Sindbis virus vector to specifically infect pyramidal neurons in CA1
and granule cells in the dentate gyrus in an antibody-specific manner
in mice (Konno et al., 2011). Also, Ried et al. inserted the Z34C,
an immunoglobulin G (IgG) binding domain of protein A, into the
AAV2 capsid and made an AAV2-Z34C vector in order to target the
vectors to specific cell surface receptors (Ried et al., 2002).
5.3. Safety and long-term effectiveness
Safety and long-term effectiveness are important issues to be
solved for gene therapy. The genes of interest have been delivered
using either viral vectors or non-viral vectors. Because of
the rapidity of experimental implementation, most of the animal
experiments of ChR optogenetics have been carried out using virus
vectors such as AAVs and lentiviral vectors (LVs). AAV-mediated
gene delivery has the advantages of wide tissue tropism, efficient
transduction of non-dividing cells and long-term effectiveness.
Over 100 variants with diverse cell tropisms have been identified
and some of them have been used in the clinical field (Daya and
Berns, 2008). It has been reported that AAV-mediated gene expression
is safe and effective in the primate brain for at least 8 years
(Hadaczek et al., 2010). LV-mediated gene delivery has the advantages
of stable gene integration in both non-dividing and dividing
cells as well as long-term expression. Numerous animal experiments
have shown the safety and long-term effectiveness of these
virus vectors for delivering ChR2. However, one has to address the
issue of determining the viral titers that are both safe and effective
before applying viral vectors for ChR-based gene therapy in a clinical
setting, since the issue is still unsolved even for experiments
using animal models. Ideally, the dose of virus should be minimal
to avoid the immune response of host. To this end, expression cassettes
have been used to express higher levels of the transgene with
lower doses of the vector. The expression cassette is the part of the
vector DNA that is used for cloning and transformation. It usually
includes three parts: promoter sequence, open reading frame and
3 -untranslated region. One example is the self-complementary
AAV vector which has been demonstrated to significantly enhance
the expression level of the transgene with minimal reduction of the
packaging capacity (McCarty et al., 2001; McCarty, 2008; Koilkonda
et al., 2009).
5.4. Optimized light delivery
To deliver light to the targeted neurons or regions, several issues
have to be addressed. For example, what are the optical properties
of different tissues in the human brain? What is a more suitable
light source, laser or LED? What are the optimal devices to deliver
10 Z.-G. Ji et al. / Neuroscience Research 75 (2013) 6–12
the light to the experimental or therapeutic system? The former
two issues have been extensively reviewed previously (Carter and
de Lecea, 2011; Yizhar et al., 2011). Here, we highlight the recent
advances regarding the light delivery system.
At present, in vivo, most experiments have been performed with
light sources such as LED and laser that are connected to the targeting
regions through an optic cable. The optic cables and the large
LED or laser are inconvenient for experiments with free-moving
animals or to deliver the light as a medical treatment. Therefore,
one challenge is to invent smart wireless implants. Recently, prototypes
of wireless lighting devices have been developed (Iwai et al.,
2011; Wentz et al., 2011). Iwai et al. (2011) implanted a batterypowered
fiber optics system in the primary motor cortex of freely
moving transgenic mice that express ChR2 in the layer V neurons
and showed that the device could remotely induced a behavior.
Wentz et al. (2011) made a head-borne device powered by a lowstrength
oscillating magnetic field, thus, successfully reduced its
weight to as little as 2 g. They employed this device to control the
activities of the ChR2-expressing pyramidal cells in the motor cortex
of freely-moving transgenic mice. Such wireless devices could
become smaller and more powerful with the development of optoelectronics
if only the problem of cost could be solved.
Previously, most the devices delivered monochromatic light on
a single spot. However, it has become increasingly necessary to
develop multicolored and spatio-temporally patterned irradiating
devices for both basic research and clinical applications. For example,
to control the ON-OFF responses in the retina, one needs to
deliver different wavelengths to activate depolarizing rhodopsins
(eg, ChR2) and hyperpolarizing rhodopsins (eg, NpHR) (Zhang et al.,
2009). Alternative irradiation of blue and yellow light is necessary
to exploit the full potential of SFO and SSFO, which are expected
to be applied clinically as well as for basic experiments (Berndt
et al., 2009; Yizhar et al., 2011; Tye and Deisseroth, 2012). It is also
important to stimulate multiple sites in a spatially patterned fashion
for the research of some neurological disorders such as epilepsy.
Previously, an implantable probe having multiple waveguides was
devised to deliver different wavelengths of light to multiple sites
(Zorzos et al., 2010). The probe consisted of a parallel array of
waveguide cores each of which conducts light from a laser at either
473 or 632 nm. Recently, an image projector was applied to deliver
multicolored light with a pre-programmed spatio-temporal pattern
(Sakai et al., 2012; Stirman et al., 2012). Using these systems,
the depolarizing rhodopsins (e.g. ChR2) and the hyperpolarizing
rhodopsins (e.g. ArchT and Mac) can be differentially activated.
These devices, in combination with microendoscopy technique
(Hayashi et al., 2012), would enable one to deliver multicolored
and patterned light in the brain of in vivo animals.
In the future, the researchers may devise light-emitting
nanoparticles that could be controlled and charged by magnetic
fields or near-infrared light to generate specific wavelengths of
light. If successful, it would no longer be necessary to consider the
problem of inserting many optic fibers into the brain.
5.5. The development of non-human primate models
The current investigations of ChR optogenetics have been extensively
promoted using rat and mouse model systems, and only
a limited number of studies have been done using non-human
primates (Han et al., 2009; Diester et al., 2011; Gerits et al.,
2012). Although the rodent models are convenient, gene therapy
investigations in rodents have not always predicted the results
of higher-order models (Gagliardi and Bunnell, 2009). One of the
reasons may be the difference in the size of the brain between
rodents and primates. For example, the human brain is 650–700
times greater in the weight than the rodent brain (Mink et al.,
1981). Such size differences have to be taken into consideration
to determine the titer of the virus vector to guarantee the safety
and effectiveness in the clinical setting. Secondly, the nervous system
of rodents is structurally different from those of primates. In
the case of the eye, different from human beings and non-human
primates, rodents usually lack the fovea. Thus, before the introduction
of ChR-based gene therapy to the clinical field, it would be
necessary to evaluate the safety and effectiveness in non-human
primates that have anatomical and electrophysiological properties
relatively similar to those of a human being. There would be
additional benefits with non-human primates as an experimental
model system. For example, although blind rats or mice can
become sensitive to light with the retinal expression of ChRs, it
remains unclear whether they can correctly integrate the signals
generated by ChRs to recognize form and color. This question could
be addressed by experiments using non-human primates with the
appropriate training paradigms. Recently, the safety and effectiveness
of ChR2 have been investigated using non-human primates
(Diester et al., 2011), but the long-term stability of ChR2 expression
in the non-human primate brains remains to be tested.
Acknowledgments
We are grateful to Drs. H. Onodera and H. Tomita for reviewing
the manuscript and to B. Bell for language assistance. This work was
supported by a Grant-in-Aid for Scientific Research on Innovative
Areas “Mesoscopic Neurocircuitry” (No. 23115501) of the Ministry
of Education, Culture, Sports, Science and Technology (MEXT) of
Japan, Global COE Program (Basic & Translational Research Center
for Global Brain Science), MEXT and the Program for Promotion of
Fundamental Studies in Health Sciences of the National Institute of
Biomedical Innovation (NIBIO).
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