Synthetic small molecules that induce neurogenesis in skeletal muscle

Article abstract of DOI:10.1021/ja072817z

Neurons are not regenerated effectively, and their injury causes neurodegenerative diseases. These diseases may be treated by the transplantation of neural stem cells. However, ethical and technical issues restrict cell therapies using neural stem cells.

Full text of DOI:10.1021/ja072817z

Published on Web 07/10/2007
Synthetic Small Molecules that Induce Neurogenesis in Skeletal Muscle
Darren R. Williams, Myung-Ryul Lee, Young-Ah Song, Sung-Kyun Ko, Gun-Hee Kim, and Injae Shin*
Department of Chemistry, Yonsei UniVersity, Seoul 120-749, Korea
Received April 23, 2007; E-mail: injae@yonsei.ac.kr
Neurodegenerative diseases such as stroke, Parkinson’s, and
Alzheimer’s diseases result from the loss of neurons and are
prevalent throughout the world. Recent advances in stem cell
biology offer the prospect of a new therapeutic approach for the
treatment of these diseases.1 However, this approach requires a
sufficient source of stem cells, a precise control over differentiation,
a suppression of host rejection against allogenic cells, and the
prevention of tumor formation by undifferentiated cells.2 These
technical problems as well as ethical issues concerning the use of
discarded embryos restrict stem-cell-based therapies. Adult stem
cells have the potential to differentiate into several cell types and
Figure 1. Structures of (a) an imidazole library (see Supporting Information
thus could be also used for cell therapies.3 However, the adult stem
for substituents R1-R4), (b) FM1-43, and (c) neurodazine (Nz).
cells have reduced efficiency to grow and differentiate into various
types of cells compared to embryonic stem cells.
differentiated neurons in the presence of a high extracellular
A more convenient and attractive approach for the generation
concentration of KCl when the synaptic vesicles are recycled back
of neurons is the use of small molecules that induce the neuronal
into the neurons after depolarization. Thus, our screening method
differentiation of easily available cells or tissues. Generation of
using FM1-43 allows active compounds inducing neurogenesis to
neurogenic cells from non-stem cells using a small molecule inducer
be rapidly identified by observing the fluorescent intensity of treated
can avoid the above-mentioned problems that arise from stem cells
cells. Associated neurite outgrowth of cells treated with neurogen-
therapies. Retinoic acid (RA) is a widely used small molecule for
the induction of neuronal differentiation.4 However, this molecule
also has other biological activities, such as the regulation of
embryogenesis, the development of bone, and the maintenance of
epithelium.5 Recently, synthetic small molecules that induce neu-
ronal differentiation were also developed.6 These molecules induce
the conversion of pluripotent murine embryonal carcinoma cells
and embryonic stem cells to neurogenic cells. However, more
interesting small molecules would be those with the capacity to
induce neurogenesis in easily available cells or tissues, such as
myoblasts and muscle tissues. Herein, we describe the first small
molecule with neurogenesis-inducing activity in non-pluripotent
myoblasts and the cells derived from skeletal muscle.
Various imidazole derivatives show a broad range of bioactivities,
such as antineoplastic, immunosuppressive, and anti-inflammatory
activities.7 Thus, we synthesized an imidazole library with diverse
substituents at the positions of 1, 2, 4, or 5 on a solid support to
identify active compounds inducing interesting differentiation in
cells, such as neurogenesis (Figure 1a).8 Amine-conjugated dieth-
ylene gycol was inserted into this library for the facile preparation.
We could obtain about 300 imidazole derivatives with more than
70% purity (based on HPLC analysis).
C2C12 myoblasts have the advantage of being well-characterized
and possess the potential to be differentiated into non-muscle cell
types, such as adipocytes and osteoblasts.9 In addition, these cells
were converted to a physiologically active neuronal phenotype by
genetic manipulation, such as disruption of the function of a single
genetic element.10 Therefore, C2C12 cells were chosen for primary
screening. We adopted a high-throughput screening approach to
select imidazole-based chemical inducers of neuronal differentiation
of these cells. We utilized the fluorescent dye FM1-43 (Figure 1b),
which is employed to detect depolarization-induced synaptic vesicle
recycling that is a neuron-specific property.11 This dye enters the
esis-inducing agents was further examined by light microscopy.
About 300 imidazole derivatives (3 µM) were incubated with a
relatively low density of C2C12 myoblasts (104 cells/mL) in culture
media supplemented with 2% fetal bovine serum to suppress the
onset of terminal myogenesis. Fluorescence analysis showed that
four compounds exhibited relatively high depolarization-induced
fluorescent intensity in comparison with untreated cells. In addition,
phenotypic analysis using light microscopy indicated that these
compounds induced the development of membrane outgrowth
resembling a prominent axonal development. To confirm the
neurogenesis-inducing effect of these compounds in C2C12 myo-
blasts, four active compounds without a diethylene gycol linker
were resynthesized in solution. On the basis of our neurogenesis
activity test using FM1-43, neurodazine (Nz) exhibited the highest
fluorescence intensity after treatment with FM1-43 and was selected
as a neurogenesis-inducing agent for further studies (Figure 1c).
To ascertain that the C2C12 cells differentiated by treatment with
Nz indeed have neurogenic properties, we performed immunocy-
tochemical and Western blot analyses with antibodies against
neuron-specific proteins, such as neuron-specific âIII tubulin (NST,
TuJ1), neuron-specific enolase (NSE), neurofilament 200 (NF200),
GAP-43 (growth associated protein-43, a marker of axon develop-
ment), choline acetyltransferase (CAT), microtubule-associated
protein 2 (MAP-2), and synapsin. These analyses showed that Nz
induced the expression of neuron-specific markers in treated C2C12
cells (Figure 2). We also investigated synaptic vesicle recycling of
the cells differentiated by Nz as a function of external K+
concentration since synaptic vesicle recycling is a neuron-specific
property. Synaptic vesicle recycling detected by FM1-43 became
apparent in the cells derived from Nz-treated C2C12 myoblasts
when the concentration of KCl approached 90 mM (see Supporting
Information). The degree of synaptic vesicle recycling was similar
to that displayed by the cells derived from rat PC12 cells treated
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10.1021/ja072817z CCC: $37.00 © 2007 American Chemical Society

C O M M U N I C A T I O N S
Figure 3. Neurogenesis of human skeletal muscle fibers by Nz. (a) Single
muscle fibers isolated from the abductor hallucis muscle and (b-c)
immunocytochemical analysis of mononucleates and satellite cells after
treatment with 2 µM Nz for 7 days (bar ) 50 µm). (d) Synaptic vesicle
recycling of mononucleates after treatment with 2 µM Nz for 7 days using
FM1-43 (error ) SD).
Figure 2. Neurogenesis of C2C12 myoblasts by Nz. C2C12 cells were
treated (a) without and (b-h) with 2 µM Nz for 7 days. The upregulation
of neuron-specific markers in Nz-treated C2C12 cells is visualized by
immunocytochemistry (bar ) 50 µm) (see Supporting Information for
Western blot analysis).
is known to be involved in inducing neuronal differentiation in rat
PC12 and neuroepithelial cells,15 was upregulated by Nz. However,
the expression of key regulators of myogenesis, such as MyoD,
Myf-5, and myogenin,16 remained unchanged during the early stages
of Nz treatment, suggesting that C2C12 cells develop a neuronal
phenotype without suppressing their own potential to differentiate
into muscle fibers under permissive conditions, such as fusion with
neighboring cells in culture.
with 100 ng/mL nerve growth factor (a well-characterized model
of neurogenesis).12 On the basis of immunocytochemical analysis
and FM1-43 studies, 40-50% myoblasts were converted to a
neuronal phenotype by Nz.
The conversion of differentiated myotubes to neurogenic cells
is an attractive prospect because it would suggest that neurogenic
cells can be derived from adult skeletal muscle fibers. The
differentiation of C2C12 myoblasts into myotubes is an in vitro
model of skeletal muscle myogenesis. Since C2C12 myotubes are
not directly converted to neurogenic cells by Nz, we attempted a
two-step procedure to differentiate myotubes into neurogenic cells.
Myotubes differentiated from C2C12 myoblasts were first treated
with 10 µM myoservin to induce cellularization of myotubes.13
Mononucleates isolated from cellularized myotubes were then
incubated with 2 µM Nz for 7 days in the absence of myoseverin.
The differentiated cells were confirmed to have neurogenic proper-
ties by immunocytochemical and Western blot analyses of neuron-
specific protein expression and synaptic vesicle recycling studies
using FM1-43 (see Supporting Information). These studies show
that the Nz-treated mononucleates derived from C2C12 myotubes
possess a similar neurogenic potential to Nz-treated C2C12 myo-
blasts.
The results obtained from experiments using mouse myotubes
prompted us to test if Nz can be used to generate neurogenic cells
from human skeletal muscle. For these studies, single muscle fibers
were isolated from the abductor hallucis muscle.14 The single muscle
fibers were treated with 15 µM myoseverin for 20 h in order to
obtain mononucleates. The isolated mononucleates were further
incubated with 2 µM Nz for 7 days. Alternatively, the single muscle
fibers were cultured to produce satellite cells which are quiescent,
mononuclear muscle cells residing between the sarcolemma and
basement membrane of the muscle fibers. The satellite cells were
treated with 2 µM Nz for 7 days. Mononucleates and the satellite
cells treated with Nz developed a neuronal phenotype on the basis
of immunocytochemical analysis and synaptic vesicle recycling
studies (Figure 3).
In conclusion, we have developed the first small molecule that
can induce neurogenesis of non-pluripotent myoblasts and the cells
derived from mature, human skeletal muscle. These studies build
upon recent research illustrating the value of chemical approaches
for providing tools that differentiate lineage-committed cells into
other cell types.9c
Acknowledgment. This work was supported by a grant of the
NRL program (MOST/KOSEF). S.-K.K. and G.-H.K. thank the BK
21 program (KRF).
Supporting Information Available: Preparation of a library and
cell experiments. This material is available free of charge via the Internet
at http://pubs.acs.org.
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