Nikita Chaudhri Faculty Mentor: Dr. Vidya Chandrasekaran The Role of Sonic Hedgehog on Dendritic Growth in Sympathetic Neurons Abstract Dendrites are the parts of a neuron that receive signals from other neurons and transmit these signals down the cell body. Previous studies have observed that dendrites retract in cases of neurodegenerative disease, neuronal injury, stress, and aging. Therefore, it is necessary to identify and understand the molecules that could potentially rebuild the dendritic arbor of a neuron in order to restore its function. In this study, we explored a new player, Sonic Hedgehog (Shh), for its importance in the dendritic growth pathway of sympathetic neurons. Shh has been shown to have many neuronal effects in many different systems, such as setting up the neural crest and the identity of spinal neurons early in embryonic development. This study shows that Shh is also needed for dendritic growth at later stages of development. Our data indicates that cells treated with Shh had an increase in the number of dendrites and growth of dendritic arbor of neuronal cells compared to untreated cells. This effect was specific to dendrites, did not affect cell survival, and was mediated by the Ptc and Smo signaling pathway. 1. Introduction Dendrites are important parts of the neuronal cell that are responsible for receiving signals from other neurons. Across the nervous system, there is a huge diversity in the branching of dendrites, or dendritic trees. The extent of these dendritic trees depends on the number of neurons a given neuronal cell communicates with (1). For example, sensory neurons communicate with a couple of cells and have one dendrite. On the other hand, Purkinje neurons
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Nikita Chaudhri
Faculty Mentor: Dr. Vidya Chandrasekaran
The Role of Sonic Hedgehog on Dendritic Growth in Sympathetic Neurons
Abstract
Dendrites are the parts of a neuron that receive signals from other neurons and transmit
these signals down the cell body. Previous studies have observed that dendrites retract in cases
of neurodegenerative disease, neuronal injury, stress, and aging. Therefore, it is necessary to
identify and understand the molecules that could potentially rebuild the dendritic arbor of a
neuron in order to restore its function. In this study, we explored a new player, Sonic Hedgehog
(Shh), for its importance in the dendritic growth pathway of sympathetic neurons. Shh has been
shown to have many neuronal effects in many different systems, such as setting up the neural
crest and the identity of spinal neurons early in embryonic development. This study shows that
Shh is also needed for dendritic growth at later stages of development. Our data indicates that
cells treated with Shh had an increase in the number of dendrites and growth of dendritic arbor of
neuronal cells compared to untreated cells. This effect was specific to dendrites, did not affect
cell survival, and was mediated by the Ptc and Smo signaling pathway.
1. Introduction
Dendrites are important parts of the neuronal cell that are responsible for receiving
signals from other neurons. Across the nervous system, there is a huge diversity in the
branching of dendrites, or dendritic trees. The extent of these dendritic trees depends on the
number of neurons a given neuronal cell communicates with (1). For example, sensory neurons
communicate with a couple of cells and have one dendrite. On the other hand, Purkinje neurons
in the cerebellum communicate with over 200,000 cells and have an elaborate dendritic arbor
(2). The diversity of neurons and the establishment of their arbor take place during embryonic
development (3). An understanding of the molecules controlling dendritic growth and
retraction will provide a better understanding of how this diversity is generated.
In addition to embryonic development, dendritic growth and retraction play an important
role in neuronal injury when dendrites pull back and can no longer receive information from
other neurons in the body. Dendritic retraction associated with neuronal damage is observed in
many neurodegenerative diseases such as Multiple Sclerosis, Alzheimer’s disease, and
Parkinson’s disease (4). Thus, knowing all the molecules that control dendritic growth will
provide a better understanding of how to induce this growth and design therapies for
neurodegeneration and neuronal injury. This study focuses on one family of molecules, the Shh
family, to explore its effects on dendritic growth.
Shh has been shown to be important in many aspects of neuronal development in
embryos. Early in development, Shh is needed for establishing the identity of spinal neurons (5).
Later during development, Shh seems to be needed for maintaining the identity of interneurons
in the ventral telencephalon and setting up the nigrostriatal circuit (6, 7). Furthermore, Shh is
needed in axon guidance, neurite outgrowth, and synapse formation in dopaminergic, retinal, and
hippocampal neurons in the central nervous system (8, 9). These effects of Shh seem to be
mediated through its normal pathway, which involve Patched (Ptc) and Smoothened (Smo)
receptors and Gli proteins (9). Shh has a role throughout embryonic development and well into
postnatal development in many cell types. However, in the peripheral nervous system, Shh is
known to be important for early patterning of the ganglia and neural crest induction, which form
the precursors of sympathetic neurons (10, 11) with no studies showing the role of Shh at later
stages of embryonic development. One of the key processes that occur late in embryonic
development is the growth of dendrites in these sympathetic neurons (12). Therefore, the
purpose of this research is to look for the effects of Shh on dendritic growth in sympathetic
neurons at later stages of development.
2. Materials and Methods
2.1 Materials
E21 rats and BMP-7 were gifted by Dr. Pamela Lein’s Lab at UC Davis Department of
Molecular Biosciences. Recombinant Shh-N was obtained from R&D Systems (Minneapolis,
MN). Ptc Antibody and Smo Antibody were obtained from Abcam (Cambridge, MA). NGF
(125mg/ml) was obtained from Harlan Bioproducts. Prionex (10% solution) was obtained from
Millipore (Billerica, MA). Cyclopamine and SMI-32 antibody were obtained from Calbiochem
(Billerica, MA). All other supplies were obtained from Invitrogen (Grand Island, NY).
2.2 Tissue Culture
Sympathetic neurons were dissociated from the superior cervical ganglia (SCG) of E21
perinatal rats according to previously described methods (13). Cells were plated on 24-well
showing nearly as many dendrites as the control (1.10 ± 0.12). This suggests that the induction
of dendritic growth by Shh was mediated through a pathway involving Smo receptor.
3.5 Shh does not affect the nuclear translocation of Smad-1 proteins
Since Shh is working through its normal signaling pathway, and the effect of BMP-7 and
Shh together does not seem to be additive, we looked at how the BMP and Shh pathways interact
with each other during dendritic growth. In the BMP signaling pathway, BMP family members
bind to BMP receptors (BMPRI and BMPRII). When BMP binds, the receptors phosphorylate
the Smad-1 or Smad-5 proteins. The phosphorylated Smads leave the receptor and combine with
Smad-4 downstream. These Smad-1/Smad-4 and Smad-5/Smad-4 complexes enter the nucleus
and turn transcription on or off at specific genes (18). Therefore, the nuclear translocation of
phosphorylated Smad-1 protein was used to determine if Shh induces the BMP-7 signaling
pathway. Neurons exposed to control media showed Smad-1 staining in the cytoplasm, with no
staining in the nucleus (Fig. 8A). Neurons treated with BMP-7 showed nuclear staining (Fig.
8B). However, the neurons treated with Shh at 1 µg/mL did not show nuclear staining for
phosphorylated Smad-1, as evidenced by the staining in the cytoplasm and the dark, unstained
nucleus of the cell (Fig. 8C), indicating that Smad-1 translocation was unaffected by the presence
of Shh.
3.5 Shh pathway is downstream from the BMP-7 signaling pathway in sympathetic
neurons
Since Shh does not seem to be influencing the BMP pathway, we looked at whether the
BMP-7 pathway was inducing the Shh pathway by treating neurons with both cyclopamine (100
nM) in conjunction with BMP-7 (5 ng/mL). Compared to BMP-7 alone, which showed 2.12 ±
0.10 dendrites per cell (Fig. 10), cells with both cyclopamine and BMP-7 showed a decrease in
dendritic growth with 1.24 ± 0.01 dendrites per cell (Fig. 9D, 10). These results show that
blocking Shh signaling resulted in an inhibition of BMP-7 induced dendritic growth.
Furthermore, there was a higher amount of Smo protein (86 kDa) in control lysates
compared to lysates from BMP-7 treated cells, suggesting that Smo receptor many be a potential
point of interaction between the two pathways.
4. Discussion
Our results show that Sonic Hedgehog induces dendritic growth in sympathetic neurons.
This study is the first study showing an effect of this family on dendritic growth. Shh, however,
is a mild inducer of dendritic growth in these neurons. While the average number of dendrites in
Shh treated and control neurons varied between different experiments (Fig. 2B and 7), the same
general trends were seen. The average number of dendrites was always higher in neurons that
had been treated with Shh compared to neurons under control conditions. Variability in the
number of dendrites can be due to factors such as cell plate density, maturity of rat pups upon
dissection, and inherent differences between superior cervical ganglia in individual rat pups.
One possible reason for the mild induction of dendritic growth by Shh was the
concentration of Shh used in this study. In this study, the dendritic growth observed with the
Shh dose curve was still increasing at 1 µg/mL and has not yet leveled off at its maximum
concentration (Fig. 3). It would therefore be interesting to look at the effects of higher
concentrations of Shh to see if we can induce more dendrites and potentiate dendritic growth in
the presence of submaximal concentrations of BMP-7. Furthermore, our data indicates that there
is an endogenous concentration of Shh in sympathetic neurons in vitro with the absence of glial
cells. Therefore, it would be interesting to look at whether glial cells also produce Shh to see if
the endogenous concentration of Shh is actually greater in vivo thus having a greater effect on
dendritic growth in the animal.
In addition, this study shows that Ptc and Smo receptors are present in sympathetic
neurons, and the inhibition of Smo reduces Shh’s effects on dendritic growth. This indicates that
Shh is working through its normal signaling pathway to induce dendritic growth in sympathetic
neurons. Our data also shows that BMP is inducing the Smo receptor levels, however, this may
be due to the fact that Smo is also located in the dendrites, which are extended in the presence of
BMP. Therefore, to further confirm if BMP is inducing the Shh pathway, it would valuable to
look at the location of Gli, the downstream signaling component in the Shh pathway, to see if
BMP is inducing the Shh pathway downstream in the neuron.
Previous studies have shown that Shh and BMP interact in other neuronal systems. Shh
and BMP work against each other in the patterning of the frontonasal process during
development (19). On the other hand, both earlier BMP signaling and later Hh signaling are
needed to induce satb2 expression in the palate and jaw (20). In our study, BMP induces the Shh
signaling pathway but Shh does not affect the BMP signaling pathway because the translocation
of Smad-1, the downstream signaling component in the BMP pathway, was unaffected by the
presence of Shh. Therefore, it would be interesting to look at the interaction between Gli and
Smad-1 to help understand how these two pathways specifically interact at the downstream level
to control the growth of dendrites. Furthermore, studies have identified specific genes that are
regulated by BMP-7 in sympathetic neurons during primary dendritic growth (16). Therefore, it
would be helpful to determine if these BMP-7 regulated genes are also targets of the Shh
pathway, which would show if these two molecules are interacting at a transcriptional level.
In summation, this study shows that Shh induces dendritic growth, Shh and its signaling
components are present in sympathetic neurons, and the Shh signaling pathway is induced by
BMP-7. These discoveries provide a more complete understanding of how the dendritic arbor of
a sympathetic neuron is generated. Such an understanding can shed light on developing
treatments to recreate the dendritic arbor of damaged neurons that have lost their dendrites and,
therefore, their function. Such damaged neurons are seen in patients with neurodegenerative
diseases and neuronal injuries. Therefore, this study provides relevant information to help
develop treatments for neurodegenerative disease and neuronal injury.
Following the elimination of glial cells, cultures of sympathetic neurons from E21 rat
pups were treated for 5 days with either control medium (A, D), BMP-7 at 5 ng/mL (B, E), or
with Shh at 1 µg/mL (C, D). Panels A-C are the phase contrast images under 20X magnification
of the cells D-F that are the fluorescent images immunostained with phosphorylated
neurofilament antibody (SMI-32) against dendritic proteins. A and D are control, B and E are
BMP-7 (5 ng/mL), and C and F are Shh (1 µg/mL) treated cells.
(B) (A) (C)
(D)
(E) (F)
Figure 1: The effect of Shh on dendritic growth
0
50
100
150
200
250
Control Shh 0.5 μg/mL BMP-7 5ng/mL Shh 0.5 μg/mL+BMP-7 5ng/mL
Total Dendritic Arbor (μm)
Treatment
The Effect of Shh on Dendritic Arbor
Dendritic Arbor (μm)
Figure 2: The effect of Shh on dendritic growth
0
0.5
1
1.5
2
2.5
3
3.5
4
4.5
Control Shh - N 0.5 μg/mL
BMP-7 5 ng/mL
Shh -N 0.5 μg/mL + BMP-7 5
ng/mL
BMP-7 50 ng/mL
Shh -N 0.5 μg/mL + BMP-7 50
ng/mL
Number of dendrites/cell
Treatment
The Effect of Shh on Dendritic Growth
(A)
(B)
*
*
Following the elimination of glial cells, cultures of sympathetic neurons from E21 rat pups were treated under conditions for 5 days. The neurons were immunostained with phosphorylated neurofilament antibody (SMI-32) against dendritic proteins. Cells were counted using microscopy under UV light with a secondary fluorescent antibody. The dendritic arbor of the cells was quantified using Image J. The changes in the number of dendrites per cell are shown in (A) and the changes to the dendritic arbor are shown in (B). The data are expressed as mean ± SEM (N ≈ 100) for panel A and dendritic arbor size per cell (N ≈ 50) for panel B. * Denotes treatments that are statistically significant to control as deduced by ANOVA followed by Tukey’s Test (p < 0.05).
Following the elimination of glial cells, the sympathetic neurons from E21 SCG treated
with Shh at increasing concentrations ranging from 0.03 µg/mL to 1 µg/mL for 5 days. The
number of dendrites per cells was measured at different doses after 5 days of treatment. The
graph shows the number of dendrites per cell versus the concentration of Shh. The data is
represented as mean ± SEM.
Shh Dose Curve
Figure 3: The effect of Shh on dendritic growth is dose dependent
Following the elimination of glial cells, sympathetic neurons were treated with control
medium, BMP-7 (1 ng/mL), Shh (1 µg/mL), and BMP-7 and Shh together for five days. Then,
cells were treated with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) for
two hours and then lysed with DMSO. The absorbance of the lysed cells was measured at 562
nm using Microplate Manager software and Benchmark Reader. The data is represented as mean
± SEM (n = 2). Data was analyzed with a one-way ANOVA followed by Tukey’s Test showing