1. Identification of an axonal targeting
sequence in the survival motor neuron
protein: does a break in axonal targeting
trigger neuronal apoptosis?
1203048
2. Identification of an axonal targeting sequence in the survival
motor neuron protein: does a break in axonal targeting trigger
neuronal apoptosis?
Word Count: 6050
Student Number: 1203048
University of Warwick
2015
3. Table of Contents
Abstract……………………………………………………………...………………….1
1. Introduction………………………………………..………………………………...3
2. Materials and Methods……………………………………………………………..9
2.1. cDNA Constructs…………………………………...………………….…9
2.2. Co-Immunoprecipitation…………………………………………….……9
2.3. Cell Culture……………………………………...……………………….10
2.4. Transient Expression Studies…………………………..……………...10
2.5. Live Cell Imaging…………………………………….………………….10
2.6. Microscopy………………………………………………….……………10
2.7. Statistical Analysis………………………………………………………11
3. Results……………………………………………...………………………………12
3.1. SMN236 co-localises with Gemin3 to axonal granules……………..12
3.2. ATGs containing SMN travel in a bidirectional manner……………..14
3.3. ATGs containing SMN236 exhibit significantly similar anterograde
and retrograde movements to ATGs containing FL-SMN…………20
4. Discussion………………………………………………………………….………22
Acknowledgments……………………………………………………………………28
References……………………………………………………………………………28
4. 1
Abstract
Spinal muscular atrophy (SMA) is a common autosomal recessive disease
amongst children and is characterised by the degeneration of alpha-motor
neurons of the spinal cord. The disease is caused by mutations in the survival
motor neuron (SMN) gene, where a reduction in SMN protein has been linked to
a higher severity of SMA. Although the protein is expressed in all somatic cells,
the specific vulnerability of the motor neurons has yet to be accounted for.
Aside from its well-characterised role in snRNP assembly, SMN has been
suggested to play a role in the transport of RNA within axons that is required for
neuronal outgrowth and maintaining the dynamic interface of the neuromuscular
junction. Within neurons, SMN has been shown to localise with Gemin3 to
granules that show rapid bidirectional movement. 25 neuroblastoma (SH-SY5Y)
cells were differentiated using retinoic acid and transfected with truncated SMN
containing only exons 2, 3 and 6 (SMN236). The localisation of SMN into
axonal granules and the level of co-localisation with Gemin3 were then
calculated. SMN236 and Gemin3 were fluorescently tagged with enhanced
green fluorescent protein (EGFP) and TRITC respectively. The movement of 10
axonal transport granules (ATGs) containing SMN236 were also measured
under live cell imaging microscopy. Velocity was calculated as distance moved
per second (μm/s) where positive values represent anterograde movement, and
negative values represent retrograde movement. All statistical analyses were
performed using SPSS v22.0 to evaluate the data against full length (FL)-SMN.
The aim of the experiment was to determine whether truncated SMN236 was
able also to form sub-complexes with Gemin3 and co-localise to granules within
the axon. In addition, the ability for granules containing SMN236 to exhibit
dynamic movements along the axon was investigated. It was found that in
differentiated neuroblastoma cells, the mean percentage of co-localisation with
Gemin3 of SMN236 (57.2%, p=0.00) was significantly similar to that of FL-SMN
5. 2
(62.6%, p=0.00). Furthermore, dynamic ATGs containing FL-SMN and ATGs
containing SMN236 migrated along axons in a significantly similar bidirectional
manner: both exhibiting a range of anterograde (p=0.642) and retrograde
(p=0.601) movements. These findings strongly suggest an axonal targeting
sequence is located within exons 2, 3 and/or 6 of SMN. By finding the axonal
targeting sequence, the role SMN plays in the transportation of mRNA along
axons can be further characterised. As a result, the specific mechanism of
neuronal apoptosis and the pathology of SMA may be better understood.
Clinically, this will be integral to finding a successful treatment method.
6. 3
1. Introduction
Spinal Muscular Atrophy (SMA) is an autosomal recessive disease that
results in the loss of alpha-motor neurons of the spinal cord. It is the leading
genetic cause of infant death, with an incidence rate of 1 in 10,000, and a
carrier frequency of 1 in 501. The different forms of SMA can be classified into
three clinical subgroups depending on the age of onset of symptoms and
severity of motor impairment1. These are termed type I, II and III respectively,
and are all caused by mutations in the SMA-determining gene: the survival
motor neuron (SMN) gene2.
As its name suggests, SMA is characterised by atrophy and the weakening of
skeletal muscles. The most common and severe form is known as type I SMA,
which accounts for approximately 50% of diagnosed patients3. It is
distinguished by the onset of disease occurring before 6 months of age and
death ensuing by 2 years of age. Typically, patients have no control of head
movement and are unable to sit without support. Death is most commonly
caused by respiratory failure or infection due to weak intercostal muscles and
bulbar denervation, which subsequently decreases airway protection. Type II is
of intermediate severity whereby patients have a lifespan of up to adolescence,
with an age of onset between 7-18 months. Patients are able to maintain a
sitting position unaided, but few are able to stand without support in the form of
leg braces. The mildest form of the disease is type III, occurring after 18 months
of age. Patients are able to live a regular lifespan and show a wide variety of
symptoms. These include muscle weakness and joint overuse, but unlike the
other two classifications, all major motor milestones are reached.
Regardless of the severity of SMA, the disease is caused by deleterious
mutations of the SMN gene2. In humans, the gene has been mapped to
7. 4
chromosome 5q13 which contains a 500kb inverted duplication2,4,5. The gene
exists as multiple copies: one SMN1 found in the telomeric region, and several
copies of SMN2 in the centromeric region2. The two only differ by 5 nucleotides
but they both encode identical proteins: the 294 amino acid SMN protein2.
However, only homozygous mutations in SMN1 result in the development of
SMA. Consequently, SMN1 is identified as the ‘disease-determining gene’
where 98% of patients with SMA have homozygous deletions, mutations or
rearrangements in this telomeric copy2. On the other hand, mutations in the
near-identical SMN2 do not cause SMA. SMN2 is designated as the ‘disease-
modifying gene’ with all SMA patients retaining at least one copy. A higher copy
number of SMN2 has been found to correlate with a lower severity of SMA6-8.
This is due to the lower levels of functional protein that SMN2 is able to
produce.
The SMN gene contains 9 exons and 8 introns, however SMN2 undergoes
alternative splicing to primarily produce truncated SMN protein9,10. SMN2 has a
significant C to T transition at codon 280 within exon 7 that results in the
omission of exon 7 during the splicing process10,11. Mutations resulting in exon
skipping are a common mechanism in genetic diseases. However in the
unusual case of SMA, this nucleotide transition is translationally silent. Lorson
et al. and other research groups suggest that this change in nucleotide at
position 6 of exon 7 (exon7+6) disrupts an exonic splicing enhancer (ESE)
dependent on the SR protein splicing factor, ASF/FS210-12. The recognition of
the exon by the ESE is required for promoting its inclusion. However, Kashima
et al. propose that the T nucleotide of exon7+6 on SMN2 in fact binds hnRNP
A1 protein with higher affinity in order to disrupt the splicing process13,14. hnRNP
A1 is an exonic splicing silencer (ESS) that inhibits the splicing of the exon. The
exact mechanism of the splicing of exon 7 in SMN2 is still under debate. Yet
nonetheless, it is evident that SMN2 primarily produces truncated protein,
SMNΔ7. This is less stable than the full length SMN protein and is, most
importantly, non-functional11. Therefore, the significantly reduced levels of
functioning SMN protein produced by SMN2 cannot fully compensate for the
loss of protein otherwise produced by SMN1 and is ultimately the cause of
SMA.
8. 5
The exact role of SMN is still yet to be determined. However the vast multiplicity
of binding partners it is able to interact with suggests that SMN is involved in
various cellular processes. SMN localises in nuclear bodies called ‘gems’ that
are distinct from coiled (Cajal) bodies7. Cajal bodies are conserved structures
found in the nucleus of plant and animal cells that contain components required
for metabolism of small nuclear ribonucleoprotein particles (snRNPs)15. These
are the principal components of the splicesome, which are involved in exon
recognition and ligation during the splicing process16. As well as being closely
associated with Cajal bodies, Liu et al. found that gems (Gemini of Cajal bodies)
also exhibit similar properties and responses to changes in metabolic
conditions, and are therefore suggested to have a similar role in pre-mRNA
processing17. Furthermore, during foetal development, gems and Cajal bodies
are found to localise separately, but co-localisation of the two appears to
increase with age18. SMN, being so closely associated with snRNPs, has
consequently been found to play an essential role in the formation and
assembly of the splicesome19. Each snRNP is made up of a uridine-rich snRNP
(U1, U2, U4, U5 and U6), a set of seven Smith class (Sm) core proteins (B/B',
D1, D2, D3, E, F, and G), as well as other snRNP-specific proteins20. These
proteins are first transcribed in the nucleus as precursor snRNAs, and are
subsequently exported into the cytoplasm20. Here, SMN, as part of a complex
with eight other factors including Gemins 2-7, is necessary and sufficient for the
ATP-dependent binding of these Sm proteins to U snRNAs20,21. This SMN-
bound complex is then imported back into the nucleus where it dissociates, and
mature snRNPs assemble into the splicesome complex for pre-mRNA
processing22. These findings indicate that SMN plays a crucial role as a
molecular chaperone for these snRNPs; the insufficient levels of this activity in
patients with SMA provide a molecular cause for the disease. This is further
evidenced by deletions of both SMN1 and SMN2 in humans being
embryonically lethal. Consequently, the SMN gene can be described as a
housekeeping gene. Indeed, inactivation of SMN in mice, that have only one
copy of the SMN gene (Smn), led to extensive cell death during embryonic
development23.
9. 6
SMN protein is ubiquitously expressed in all somatic cells and much work has
been dedicated to characterising its role in snRNP assembly. However, it is only
the alpha-motor neurons of the spinal cord that are affected in SMA, and this
specificity is still largely unclear. It may be due, in part, to the relatively higher
levels of SMN protein expressed in the central nervous system7. Additionally,
the specific localisation of SMN in ribonucleoprotein granules within the neurites
of motor neurons is thought to provide some insight for this specificity24. These
dynamic granules, from hereon termed axonal transport granules (ATGs), are
actively transported in a bidirectional manner dependent on microtubules and
microfilaments of the cytoskeleton24-26. SMN has been found to co-localise in
these ATGs with several other factors, including ribosomal RNA (rRNA),
Gemin2 and Gemin3, heterogenous nuclear ribonucleoprotein (hnRNP) R/Q,
and zipcode binding protein 1(ZBP1)24,25,27,28. However, the most remarkable
discovery was that these ATGs do not contain the Sm protein components
necessary for snRNP biogenesis, which in turn suggests an independent role
for SMN within axons of neurons29. hnRNPs are RNA-binding proteins that are
involved in a diversity of functions, including the processing and transport of
RNAs30. Specifically, hnRNP R and Q interact with SMN through the arginine-
glycine (RG)-rich regions within their C-terminals27. Furthermore, hnRNP R
interacts with β-actin mRNA and enhances its localisation in neurites27.
Suppression of hnRNP R has been shown to reduce axon growth in the spinal
motor neurons of developing zebra fish embryos31. Furthermore, ZBP1 is
another binding partner of SMN within neurite granules and also interacts with
β-actin mRNA. It binds to the 54-nucleotide zipcode, found in the 3’
untranslated region (UTR), of β-actin mRNA to aid its localisation and
translation in neurons. Thus, the interactions of SMN with these factors in
axonal granules strongly suggest a role for SMN in the axonal transport of
mRNA (particularly the subcellular trafficking of β-actin mRNA). This less
characterised function of SMN may provide explanations for the specific
vulnerability of motor neurons in SMA.
The transportation of mRNA in granules along microtubules of the cytoskeleton
in living neurons provides a method for efficient site-specific translation, for
example at neuronal synapses32. This can be a useful delivery method to avoid
10. 7
inappropriate interactions of the translated proteins, as well as being a more
efficient method compared to transporting fully-folded proteins from cell body to
growth cones that may be up to one meter apart. In particular, the active
transport of β-actin mRNA - one of the most structural components of the
cytoskeleton - along axons allows its translation into protein for dendrite
outgrowth when appropriate and necessary to maintain the integrity of the
cytoskeleton. Furthermore, this method for the localisation and translation of
mRNA in growth cones has been linked to axon guidance and growth cone
motility32. Therefore the role SMN has been suggested to play in the transport
of mRNA is indirectly involved in modulating the growth of axons and growth
cones of neurons. Evidence of this can be seen in Smn-deficient motor neurons
of mice, where correlations between reduced axon growth and reduced β-actin
protein in axons and growth cones were observed33. Conversely, the
overexpression of SMN or hnRNP R was shown to promote neurite outgrowth in
PC12 cells in mice33. Ultimately, the active transport of an array of mRNAs and
its localised translation is required to ensure that the dynamic interface at the
neuromuscular junction is maintained, allowing successful communication at the
synaptic terminal. The characteristic weakening of skeletal muscles in SMA
patients, therefore, may be accounted for by a reduction in the SMN-dependent
production of β-actin-based filopodia at the growth cones of motor neurons.
Consequently, it is of great therapeutic value to further understand the
mechanism behind the trafficking of β-actin and the role SMN has to play, in
order to recover the loss of motor neurons in SMA patients. To do so, the
mechanism by which SMN is targeted to these ATGs in neuronal processes
needs to be investigated further.
Several functional domains of SMN has been identified: a nucleic acid-binding
site encoded by exon 2, a self-association site encoded by exon 2b, a Tudor
domain encoded by exon 3 that interacts with the RG-motifs within binding
partners, a polyproline-rich domain encoded by exon 5 and another self-
association site containing several YG elements encoded by exon 634-38. An
increasing number of studies have suggested that the self-associating property
of SMN is important for its function. Indeed, severity of SMA has been seen to
correlate with the ability for SMN to self-oligomerize38. In addition, the removal
11. 8
of exon 7 impairs the self-association of SMN via exon 635. Lorson et al. found
that this in turn alters the biological function of SMN35. The importance of SMN
oligomerization is further evidenced by the formation of non-functional
monomeric SMN and protein instability when self-association is inhibitied39. It is
also worthy of noting that a 9 amino acid sequence, PAKKNKSQK, in exon 2b
was identified to play an integral role in SMN cellular targeting, specifically to
Cajal bodies39. Thus, it is feasible to hypothesise that SMN may also contain an
intrinsic signal for targeting to ATGs within neurons, aiding its function in RNA
transport.
The data presented in this report was generated by the Young laboratory group
and was used to identify an axonal targeting sequence within SMN. The aim of
the experiments was to identify an internal axonal targeting sequence within
SMN to further understand SMA pathology. The ability of truncated SMN
containing only exons 2, 3 and 6 (SMN236) to form sub-complexes with Gemin3
and co-localise to granules within differentiated SH-SY5Y cells was
investigated, as well as the dynamic movements. It was found that, similar to
endogenous full-length (FL)-SMN, truncated SMN236 efficiently localises with
Gemin3 into axonal granules. The granules also migrate along axons of these
neuron-like cells in the same bidirectional fashion regardless of the length of
SMN contained within them. This strongly suggests that the information
encoded by exons 2, 3, and 6 contains the sequence required for the targeting
of SMN into ATGs. Clinically, not only do these findings help enhance our
knowledge on the role of SMN, but the pathology of SMA, as well as providing
necessary advancements required to develop successful treatment methods.
12. 9
2. Materials and Methods
The following experiments were carried out at the Young laboratory and the
subsequent data produced were analysed for this report.
2.1. cDNA Constructs
SMN236 constructs were cloned in a stepwise manner into the pEGFP (BD
Bioscience) mammalian expression vector using primers specific for exons 2, 3
and 6 only. The template cDNAs were amplified by PCR from carrier pEGFP-C3
vectors using Pfu polymerase and 35 cycles of 92°C 45 seconds denature,
52°C 45 seconds anneal, 68°C 4 minutes 30 seconds extension. Post PCR, 10
μl of amplified product were loaded with 2 μl DNA loading dye on a 1% w/v
agarose-TBE gel to ensure the correct size fragment has been amplified.
2.2. Co-Immunoprecitation
Neuroblastoma cells (SH-SY5Y) were transfected with GFP-tagged FL-SMN
and SMN236 using Amaxa Nucleofector device and reagents, specifying
programme G-004 as the optimised conditions to give high efficiency, as
recommended by the manufacturer. Cells were washed 3x in pre-warmed PBS
then pelleted. Cells were lysed using RIPA buffer and sonicated. Anti-GFP
antibody (Clontech) was incubated with the cell lysate overnight at 4°C with
rotation. Protein G dynabeads were added to bind the anti-GFP antibody and
incubated at 4°C for 4 hours with rotation. Beads were then washed 4x in RIPA
buffer and protein eluted by addition of 2x SDS sample buffer. Beads were
boiled for 10 minutes at 100°C and loaded onto a 10% SDS PAGE. Semi-dry
western blot was performed and blots developed using Pierce Femto developer
on X-ray film.
13. 10
2.3. Cell Culture
SH-SY5Y cells were grown in Dulbecco’s Modified Eagle’s Medium (DMEM)
supplemented with 10% Foetal Calf Serum (FCS) and 1% each of Penicillin and
Streptomycin in 5% CO2 at 37°C.
2.4. Transient Expression Studies
SH-SY5Y cells were transfected using Amaxa Neucleofector and immediately
post-transfection, cells were re-suspended in 500 μl of DMEM and differentiated
with 10 μM of retinoic acid. For each reaction 1.5 ml of re-suspended cells were
transferred to a 1.5 ml microcentrifuge tube and centrifuged at 200 x g for 10
minutes. The supernatant was discarded and the cell pellet re-suspended in
100 ml of DMEM. 2 μg of DNA was added to the cells and transferred to an
amaxa cuvette. Cells were electroporated using programme I 13. Upon
completion 500 ml DMEM was added. The cells were then transferred to a
microcentrifuge tube. 500 ml of cells was split over 6 wells of a 6-well dish
containing 2 ml DMEM and 10 μM retinoic acid with a coverslip. This was
incubated at 37oC 5% CO2 incubator for 48 hours to allow differentiation to
occur.
Fixed cells were incubated with Gemin3 mouse monoclonal antibody (C-5): sc-
374373 (Santa Cruz Biotech) followed by incubation with a TRITC-conjugate
anti-mouse antibody. Cell nuclei were counterstained with 4’, 6’-diamidino-2-
phenylindole (DAPI).
2.5. Live Cell Imaging
SH-SY5Y cells were transfected using Amaxa Neucleofector device and
reagents according to manufacturers instructions and cultured on
FluoroDishes™ in DMEM supplemented with 10% FCS and 10 μM retinoic acid
for 48 hours.
2.6. Microscopy
Fluorescence microscopy was performed using a Nikon TE 2000-U microscope
and a Hamamatsou camera. Images were acquired by taking Z-stacks and de-
convolved using Openlab version 4.0.1 (Improvision). The images were then
14. 11
processed using Photoshop 5.5 (Adobe). Live cell microscopy was performed
on sealed dishes that were transferred to a heated microscope stage and time
lapse images were acquired using Openlab version 4.0.1 (Improvision). Cells
were imaged at exposure rates (between 0.6 and 1 second depending on
magnification used) to ensure that granules in distal regions were clearly visible
for a total of 30 frames, each frame 2 seconds in length.
2.7. Statistical Analysis
The data generated was statistically analysed using SPSS Statistics (version
22, IMB SPSS Statistics) and graphs were subsequently produced using
GraphPad Prism (version 6.0f, GraphPad Software Inc.). Descriptive statistical
analysis was run to determine the mean, standard error of mean (SEM),
standard deviation and kurtosis of data on the localisation of SMN. The
kurtoses of the data indicated a normal distribution and therefore, subsequent
parametric independent-samples t tests were run to compare the means and
determine their significance.
Descriptive statistical analysis was then repeated on the live cell imaging data
to determine the mean, SEM, standard deviations, interquartile ranges and
kurtosis of anterograde velocities, then retrograde velocities of 10 ATGs
containing SMN. Again, the kurtoses of the data indicated a normal distribution
and parametric tests (ANOVA, Turkey HSD) were run to compare the means.
The mean anterograde velocities of each ATG were combined and descriptive
statistics were run to determine mean, SEM and standard deviation for
anterograde transport of ATGs containing FL-SMN in comparison to ATGs
containing SMN236. The same was repeated for retrograde transport and
subsequent parametric independent-samples t tests were run to compare the
means and determine their significance.
15. 12
3. Results
3.1. SMN236 co-localises with Gemin3 to axonal granules
Endogenous SMN has been previously shown to co-localise with Gemin3 to
both stationary and dynamic granules in the axons of primary cells28. To
determine if SMN236 is also able to form sub-complexes with Gemin3 that co-
localises to axonal granules, SMN236 was cloned into pEGFP-C3 vectors and
overexpressed in differentiating SH-SY5Y cells. The cells were then treated with
retinoic acid to differentiate into neuron-like cells. After ensuring the plasmid
DNA was transfected to the correct position, the neurite lengths of the treated
cells were measured (data not shown). This clarified that retinoic acid was
causing the differentiation and that there are no significant differences in neurite
length. GFP alone was used as a control to show GFP was not the cause of
transfection or growth of the neurite.
Fig. 1 shows that fluorescently tagged FL-SMN, SMN236 and Gemin3 were
efficiently targeted to independent granules in fixed SHSY5Y cells. Axonal
granules containing SMN only were stained green and granules containing
Gemin3 only were stained red. GFP-tagged FL-SMN and SMN236 also both
localised to the same granules as Gemin3, indicated by yellow staining (figs. 1A
and 1B). After counting the number of differently stained granules in 25 different
cells, independent FL-SMN was found in 26.160 (±2.134 SEM) granules;
independent Gemin3 was found in 24.520 (±3.005 SEM) granules; and both FL-
SMN and Gemin3 were found in 84.800 (±5.449 SEM) granules (figs. 1B and
1C). FL-SMN showed a mean percentage of co-localisation with Gemin3 of
62.6% (p=0.00) (fig. 1).
16. 13
Figure 1. Both GFP-FL-SMN and GFP-SMN236 co-localise with TRITC-
Gemin3 in axonal granules. SHSY5Y cells transfected with 2 μg EGFP-
SMN cDNA were plated in 10% feotal calf serum and retinoic acid (10 μM)
supplemented with DMEM and differentiated for 48 h. Cells were
counterstained with TRITC-Gemin3 using Gemin3 antibody (C-5): sc-374373
(Santa Cruz Biotech). (A) Bodies stained for GFP-SMN only are green,
bodies stained for TRITC-Gemin3 only are red and bodies stained for both
GFP-SMN and TRITC-Gemin3 are yellow. For both FL-SMN and SMN236
the counts were repeated in 25 neuroblastoma cells. (B) The mean counts
for the bodies containing different proteins are recorded. The standard error
of mean (SEM) for each value is shown (error bars). (C) The table presents
the calculated mean counts, SEMs and standard deviations for the
localisation of GFP-SMN (FL-SMN or SMN236) and TRITC-Gemin3 and their
co-localisation in axonal granules. Significant analysis shows that both GFP-
FL-SMN and GFP-SMN236 are co-localised with TRITC-Gemin3 to axonal
granules (p < 0.05).
Ind
SM
N
G
em
in3
SM
N
+G
3
0
50
100
150
Protein Identified in Granule
MeanCount
Localisation of SMN and Gemin3
in Axonal Granules
FL-SMN
SMN236
A.
B.
C.
17. 14
The experiments were repeated with GFP-tagged SMN236 and the number of
stained bodies was counted again in 25 different cells. Independent SMN236
was found in 23.880 (±2.005 SEM) granules; independent Gemin3 was found in
31.200 (±2.401 SEM) granules; and both SMN236 and Gemin3 were found in
73.560 (±3.781 SEM) granules (figs. 1B and 1C). SMN236 shows a mean
percentage of co-localisation with Gemin3 of 57.2% (p=0.00). Analysis of the
data shows that the significantly increased levels of localisation of GFP-tagged
SMN (both FL-SMN and SMN236) with Gemin3 to the same axonal granules
are non-random events (p < 0.05). Therefore, resembling endogenous SMN,
SMN236 is also able to form sub-complexes with Gemin3 that co-localise to
granules together within the axons of neurons.
3.2. ATGs containing SMN travel in a bidirectional manner
SH-SY5Y cells have previously been demonstrated as a functional working
model used to study intracellular transport in real time28. Endogenous GFP-
tagged SMN has also been shown to localise to dynamic bodies26,27. The
previous experiments using fixed cell microscopy could not provide information
on the movement of the granules, thus live cell imaging was used to track the
movements of axonal granules in real time. The results in fig. 2 show the
dynamic nature of ATGs containing GFP tagged FL-SMN. The velocities of 10
ATGs were measured for 30 frames to calculate the distance covered per
second (μm/s) (figs. 2 and 3). Positive velocities represent anterograde
movement whereas negative velocities represent retrograde movement of
granules. Fig. 2 shows that each ATG moves independently and each displays
both anterograde and retrograde movements in varying velocities. Anterograde
movement for ATGs containing GFP-tagged FL-SMN ranges from 0.047 μm/s
(figs. 2J and 3J) to 5.639 μm/s (p=0.504) (figs. 2H and 3H), whilst retrograde
Figure 2. (page 15) GFP-tagged FL-SMN localises to dynamic axonal
transport granules (ATGs). SH-SY5Y cells transfected with 2 μg EGFP-SMN
(FL-SMN) cDNA cultured in RPMI-1640 phenol red free media supplemented
with 10% foetal calf serum and 10 μM retinoic acid for 48 h. 10 ATGs were
followed using live cell imaging for 30 frames (2 seconds) and the distance
covered per frame divided by 2 represents velocity (μm/s). Positive velocities
represent anterograde movements, and negative velocities represent
retrograde movement of the ATG. Each ATG exhibits movements in a
bidirectional manner.
19. 16
A. B.
C. D.
E. F.
G. H.
I. J.
Figure 3. Tables showing velocities of granules containing GFP-tagged
FL-SMN. SH-SY5Y cells were transfected with GFP-tagged FL-SMN cDNA
using Amaxa Neucleofector device and reagents and cultured on
FluoroDishes™ in DMEM supplemented with 10% foetal calf serum and 10 μM
retinoic acid for 48 hours. Cells were imaged for a total of 30 frames, each
frame 2 seconds in length. The tables show the velocities of 10 granules (A-J)
containing GFP-tagged FL-SMN followed by live cell imaging. Positive
velocities represent anterograde movements and negative velocities represent
retrograde movements.
20. 17
movement ranges from -0.010 μm/s (figs. 2J and 3J) to -9.600 μm/s (p=0.835)
(figs. 2E and 3E). Fig. 3 shows the net velocities of each individual ATG
containing GFP-tagged FL-SMN. 8 of the 10 ATGs measured, exhibited net
anterograde movement ranging from 15.253 μm/s (fig. 3A) to 0.230 μm/s (fig.
3J). Only 2 ATGs exhibited net retrograde movements of -3.156 μm/s (fig. 3I)
and -3.807 μm/s (fig. 3H).
The results in fig. 4 and 5 show the dynamic nature of ATGs containing GFP-
tagged SMN236. Again, the velocities of a total of 10 ATGs were measured to
show anterograde and retrograde movement. Anterograde movement of ATGs
containing GFP-tagged SMN236 ranges from 0.009 μm/s (figs. 4H and 5H) to
7.290 μm/s (p=0.277) (figs. 4D and 5D). The retrograde movement of these
ATGs range from -0.021 μm/s (figs. 4J and 5J) to -7.651 μm/s (p=0.01) (figs. 4D
and 5D). Minor significances between means were observed between the
retrograde movements of ATG1 and ATG4 (p=0.000); ATG2 and ATG4
(p=0.009); and ATG4 and ATG6 (p=0.003). Fig. 5 shows the net velocities of
each individual ATG containing GFP-tagged SMN236: these have a greater
range than ATGs containing FL-SMN. Only 4 of the 10 ATGs exhibited net
anterograde movement – ranging from 12.685 μm/s (fig. 5A) to 22.030 μm/s
(fig. 5F). Conversely, 6 of the 10 ATGs exhibited net retrograde movement –
ranging from -1.263 μm/s (fig. 5I) to -17.293 μm/s (fig. 5D).
The data demonstrates that both ATGs containing FL-SMN and ATGs
containing SMN236 migrate along axons in a bidirectional manner. It is evident
that there is no regular pattern for the movement of ATGs as both show a wide
range of anterograde and retrograde movements.
Figure 4. (page 18) GFP-tagged SMN236 localises to dynamic axonal
transport granules (ATGs). SHSY5Y cells transfected with 2 μg GFP-
SMN236 cDNA cultured in RPMI-1640 phenol red free media supplemented
with 10% FCS and 10 μM retinoic acid for 48 h. 10 ATGs were followed using
live cell imaging for 30 frames (2 seconds) and the distance covered per frame
divided by 2 represents velocity (μm/s). Positive velocities represent
anterograde movements, and negative velocities represent retrograde
movement of the ATG. Each ATG exhibits movements in a bidirectional
manner.
22. 19
A. B.
C. D.
E.
G.
I.
F.
H.
J.
Figure 5. Tables showing velocities of granules containing GFP-tagged
SMN236. SH-SY5Y cells were transfected with EGFP-tagged SMN236 cDNA
using Amaxa Neucleofector device and reagents and cultured on
FluoroDishes™ in DMEM supplemented with 10% foetal calf serum and 10
μM retinoic acid for 48 h. Cells were imaged for a total of 30 frames, each
frame 2 seconds in length. The table’s shows the velocities of 10 granules (A-
J) containing GFP-tagged SMN236 followed by live cell imaging. Positive
velocities represent anterograde movements and negative velocities
represent retrograde movements.
23. 20
3.3. ATGs containing SMN236 exhibit significantly similar anterograde
and retrograde movements to ATGs containing FL-SMN
The mean anterograde and retrograde velocity for each ATG were calculated
and the combined means of the 10 ATGs were measured and are displayed in
fig. 6. The mean anterograde velocity for ATGs containing FL-SMN was 1.607
μm/s (±0.083 SEM), and ATGs containing SMN236 was 1.681 μm/s (±0.131
SEM; p=0.642) (figs. 6A and 6C). The mean retrograde velocity for ATGs
containing FL-SMN was -1.452 μm/s (±0.091 SEM) and ATGs containing
SMN236 was -1.575 μm/s (±0.212 SEM; p=0.601) (figs. 6B and 6D). The
standard deviations from the means are also displayed as error bars in fig. 6A
and 6B.
Figs. 6C and 6D show box and whisker plots for the mean velocities of ATGs. In
comparison to the bar charts displayed in figs. 6A and 6B, these display the
interquartile ranges of the data. It is possible, then, to compare the spread of
data as well as the means. The interquartile range of the anterograde velocities
for ATGs containing FL-SMN was 0.528 μm/s (range: 1.337 - 2.004) (figs. 6C
and 6E), whereas for ATGs containing SMN235 was 0.456 μm/s (range: 0.997
– 2.535) (figs. 6C and 6E). Conversely, the interquartile range of the retrograde
velocities for ATGs containing FL-SMN was 0.470 μm/s (range: -1.900 – -1.011)
(figs. 6D and 6E), whereas for ATGs containing SMN236 was 0.999 μm/s
(range: -2.956 – -0.719) (figs. 6D and 6E). Significant analysis of the data
indicates that ATGs containing SMN236 and ATGs containing FL-SMN showed
no considerable differences in anterograde and retrograde movements, and are
therefore significantly similar (p > 0.05) (fig. 6E).
24. 21
FL
SM
N
236
SM
N
0.0
0.5
1.0
1.5
2.0
2.5
SMN Length
MeanVelocity(µm.s)
Anterograde Transport
A.
FL
SM
N
236
SM
N
-2.5
-2.0
-1.5
-1.0
-0.5
0.0
SMN Length
MeanVelocity(µm.s)
Retrograde Transport
B.
FL
SM
N
236
SM
N
0.0
0.5
1.0
1.5
2.0
2.5
3.0
SMN Length
MeanVelocity(µm.s)
Anterograde TransportC.
FL
SM
N
236
SM
N
-4
-3
-2
-1
0
SMN Length
MeanVelocity(µm.s)
Retrograde TransportD.
Figure 6. Axonal transport granules (ATGs) containing SMN236 exhibits
similar anterograde and retrograde movement to ATGs containing FL-
SMN. SH-SY5Y cells were transfected with EGFP-tagged SMN236 cDNA
using Amaxa Neucleofector device and reagents and cultured on
FluoroDishes™ in DMEM supplemented with 10% foetal calf serum and 10
μM retinoic acid for 48 h. Cells were imaged for a total of 30 frames, each
frame 2 seconds in length. The velocities of 10 ATGs containing FL-SMN,
and another 10 containing SMN236, were measured as distance covered per
second (μm/s). The combined mean anterograde (A and C) and retrograde
(B and D) velocities were calculated for the 10 ATGs. Standard deviations
from the mean are also displayed as error bars (A and B). Interquartile
ranges and mean transport velocity of ATGs (C and D) show ATGs
containing SMN236 exhibits extremely similar anterograde and retrograde
movement to ATGs containing FL-SMN. (E) Significant analysis shows the
movements of granules containing FL-SMN or SMN236 are significantly
similar (p < 0.05).
E.
25. 22
4. Discussion
The subcellular targeting of SMN has previously been reported to be
dependent on the regions encoded by exons 2, 3 and 6 of SMN39. Specifically,
a 9 amino acid sequence has been identified in exon 2b to play an integral role
for targeting SMN to Cajal bodies within somatic cells39. In addition, Todd et al.
showed that Gemin3 forms sub-complexes with SMN that localises to neurite
granules26. Consequently, the data presented here suggests that the sequence
for targeting SMN specifically to granules within axons of neurons is also
contained within exons 2, 3, and 6 of SMN. The data shows that fluorescently
tagged SMN236 is also able to co-localise with Gemin3 to axonal granules in
differentiated SH-SY5Y neuroblastoma cells (fig. 1). The SH-SY5Y cell line has
previously been demonstrated to be a suitable model for studying intracellular
transport in real time26. It is possible, therefore, to infer the results from these
experiments in the context of neurons in vivo. Furthermore, the differentiation
and growth of neurites were measured to ensure comparisons could be made
effectively between cells. FL-SMN was shown to have a mean percentage of
co-localisation with Gemin3 of 62.6%, and SMN236 with a mean percentage of
co-localisation with Gemin3 of 57.2% (fig. 1). Statistical analysis shows that
SMN236 behaves in a significantly similar fashion to FL-SMN in co-localising
with Gemin3 to the same granules. This suggests the axonal targeting
sequence is found within SMN236. To find the minimal sequence required for
ATG targeting, it is necessary to repeat the experiments systemically using
exons 2, 3 and 6 individually.
However, it is important to note that endogenous SMN is also found within the
differentiated SH-SY5Y cells. Therefore, due to the exons 2b and 6 encoded
self-association sites, it may be possible that SMN236 associates with
endogenous SMN35,38. The co-localisation observed between SMN236 and
26. 23
Gemin3 may in fact, be due to the interaction between Gemin3 and
endogenous SMN. It is therefore necessary to determine whether the Gemin3
binding site is within exons 2, 3 and 6 of SMN in order to fully understand the
co-localisation mechanism. Further experiments using small interfering (si)RNA
to knockdown endogenous SMN is required to allow this. This will reveal
whether SMN236 can truly co-localise efficiently with Gemin3 to ATGs in the
absence of FL-SMN.
Additionally, the live cell imaging data demonstrates that ATGs containing either
FL-SMN or SMN236 in SH-SY5Y cells exhibit significantly similar anterograde
and retrograde movements (figs. 2-6). Although there were some minor
significances calculated between mean retrograde movements of ATGs
containing SMN236, these were extremely small and thus may be accounted for
through experimental errors. It has previously been found that SMN and
Gemin3 form sub-complexes that localise to both stationary and dynamic
neurite granules26. However, the previously described co-localisation studies on
fixed cells are unable to determine between stationary and dynamic granules.
Thus, one can only infer that due to the significant levels of co-localisation with
SMN236, Gemin3 can also form sub-complexes with SMN236 which can
localise to dynamic ATGs. Nevertheless, until co-transfection experiments can
run parallel with live cell imaging to ensure the ATGs moving in a bidirectional
manner contain both SMN236 and Gemin3, we cannot conclude that Gemin3 is
present in these dynamic ATGs in addition with SMN236.
The findings reported here are consistent with previous studies that show SMN-
containing granules exhibiting rapid, bidirectional movements within neuronal
axons26,28. SMN-granule movement has also been shown to be dependent on
microtubules and microfilaments of the cytoskeleton for the delivery of RNA and
other molecules to neurite terminals28. Although not specifically demonstrated
here, it is likely that the bidirectional movement observed in ATGs containing
SMN236 is also dependent on the cytoskeleton of neurons, due to the
significantly similar movements observed when compared with ATGs containing
FL-SMN. 8 out of the 10 ATGs containing FL-SMN measured travelled in a net
anterograde direction, whereas only 4 of 10 ATGs containing SMN236 did the
27. 24
same (figs. 3 and 5). Also, ATGs containing SMN236 showed a slightly wider
range of velocites, as opposed to the more structured, rapid form of movements
displayed by ATGs containing FL-SMN (figs. 2-5). From these initial results, it
appears that SMN236 may be missing a transportation signal that indicates
which overall direction the granule is required to move towards. The control of
this movement is essential for the efficient transport of the components of the
granules towards the neurite terminals. Therefore, it is necessary to investigate
this further in order to fully understand the mechanism by which these granules
travel towards neurite outgrowths.
Due to its interactions with rRNA, hnRNPs and ZBP1, SMN has been
suggested to play a considerable role in the delivery of β-actin for maintenance
of the growth cones24,27,28. Correlations have also been observed between
levels of SMN and the transport of β-actin mRNA, and have shown the ability to
promote neurite outgrowth in motor neurons of mice33. Consequently, a
reduction of SMN is thought to reduce the growth of neurites, interfering with the
dynamic nature of the neuromuscular junction. This results in motor neuronal
apoptosis and SMA pathology and so replenishing SMN levels at the
neuromuscular junction may provide a method to reduce symptom severity.
Therefore, the extensive similarities observed between the localisation and
transport of SMN236 with that of FL-SMN may provide insight into the potential
therapeutic benefits of delivering SMN236 to patients with SMA, by fulfilling its
role in delivering β-actin mRNA.
Additionally, exon 2 of SMN encodes a nucleic acid binding site, and exon 3
encodes the Tudor domain that interacts with a common RG-motif found in
several SMN-binding partners34,36. As well as being able to migrate along axons
in a similar fashion to FL-SMN, SMN236 can continue to interact with the
required binding partners involved in its suggested role of RNA axonal
transport. In particular, hnRNP R contains RG-rich regions in its C-terminal to
mediate interactions with exon 3 of SMN; it also interacts with β-actin
mRNA30,31. SMN236 can therefore potentially replace endogenous SMN in the
delivery of β-actin mRNA to stimulate regular axon growth within motor neurons
of SMA patients and preventing further degeneration.
28. 25
Currently, there have been no successful developments for therapies to treat
SMA. Nevertheless, the most promising advances have been aimed at either
redirecting SMN2 splicing to increase SMN protein levels, or viral delivery of the
SMN transgene40,41. From studies using mouse models, the gene replacement
therapy method - using scAAV9 expressing FL-SMN cDNA - has provided the
most extensive improvements42. However, sudden inexplicable deaths are
common following the delivery of the gene42. Therefore, it is essential to
develop novel approaches in the delivery of SMN protein. Investigations have
recently used cell-penetrating peptides to deliver protein directly to the cells of
the central nervous system43. The TAT peptide was studied due to its ability to
penetrate the blood brain barrier, however it has proved inefficient in improving
cell viability compared to controls43. Nevertheless, delivering protein using cell-
penetrating peptides requires further investigation to fully understand its
therapeutic potentials, as repeated delivery of smaller peptides will provide a
much less complicated treatment method in comparison to entire gene
replacement therapy. The three exons in SMN236 encode only a 168 amino
acid peptide sequence, which is significantly shorter than the original 294 amino
acid FL-SMN protein2. Thus, using SMN236 as a therapeutic option as opposed
to FL-SMN has its benefits in the ease of rapid delivery of the relatively smaller
peptide.
In spite of this, it is vital for one to consider the consequences of removing the
remaining exons. It has become evident that exon 7 of SMN has a critical role in
modulating protein function, localisation and oligomerization. Indeed, the well-
characterised effect of the splicing of exon 7 in SMN2 has detrimental effects,
resulting in only approximately 10% of functioning protein11. The localisation of
SMN within the cytoplasm has also been found to be dependent on a sequence
within the C-terminus of exon 726. Reduced neurite outgrowth and abnormal
accumulation of SMN in the nucleus was also observed with overexpression of
SMNΔ726. Moreover, the removal of exon 7 has been shown to reduce the
ability of SMN to oligomerize and subsequently alter the biological function of
SMN, indicating an importance for SMN oligomerization38. Furthermore, exon 5
is also thought to be required for efficient self-oligomerization in vitro39. A critical
concentration of oligomerization competent molecules in motor neurons has
29. 26
been suggested to be required for SMN function, due to the direct correlation
between severity of SMA and oligomerization of SMN protein7,8. This is thought
to be because of the generation of high-affinity binding sites for Sm proteins
created by SMN oligomers44. In light of these observations, and further, with
regards to implications for future studies, it would be beneficial to investigate
how efficiently SMN236 functions in comparison to endogenous SMN through
its ability to self-associate as well as interact with its numerous binding partners.
Only then can the full potentials of SMN236 as a peptide treatment be
determined.
SMA is characterised by the degeneration of motor neurons of the spinal cord,
however, there has been emerging evidence that suggests an involvement for
additional peripheral organs in some severe cases. A correlation between the
loss of SMN and neuromuscular dysfunction has been well established and
SMN is hypothesised to be involved in the transportation of mRNAs to the
neuromuscular junction necessary for neurite growth and development45. Thus,
one might suppose restoring SMN levels in neurons would prevent neuronal
apoptosis and reduce symptom severity. Despite this, restoration of SMN
protein specifically in the motor neurons or muscles of SMA mice models has
found to have little therapeutic benefits46. It is therefore important to fully
understand the pathology of SMA in order to develop successful treatment
methods. Patients with severe type I SMA often die prematurely before the age
of 2 years due to various complications and the effects of the disease on the
peripheral organs are usually unseen. However, recent clinical assessments on
patients with type I SMA surviving under assisted ventilation has shown cardiac
and autonomic nervous system defects, as well as damage to other organs
such as the brain, lungs, pancreas and liver in some severe cases46.
Considering SMN is ubiquitously expressed in all cell types, it is not surprising
that other tissues are also affected by the extreme loss of SMN in patients with
severe SMA. This also indicates that not only is the role of SMN with neurons
important when understanding SMA, but the well-characterised role of snRNP
assembly is also affected. With these new findings, it is important to investigate
the effects of reduced levels of SMN on all the peripheral organs in detail, to
30. 27
establish the most effective therapeutic approaches and the reduction of future
complications.
In conclusion, the data presented here suggests that SMN236 contains an
internal sequence within for the targeting of the protein to transport granules
along neurites of neuroblastoma cells. Granules containing SMN236 appears
to migrate along the axon-like differentiations of SH-SY5Y cells in a similar
bidirectional fashion as FL-SMN. Despite this, the full potentials of SMN236
have yet to be determined, although, as a small peptide, it may be possible to
replace the suggested role of endogenous SMN as an mRNA transporter along
axons. Specifically due to maintaining the ability to interact with hnRNP R via
exon 3, the delivery of β-actin mRNA to growth cones ensures the dynamic
interface at the neuromuscular junction is maintained. Whilst the loss of this
interface due to neuronal apoptosis following extensive loss of SMN protein is
thought to be one of the leading causes of the degeneration of motor neurons i n
patients with SMA, emerging evidence suggests the ubiquitous role of snRNP
assembly also plays a part in SMA pathology46. Therefore, the results
presented here indicate the potential for SMN236 to be used as a therapeutic
method for replenishing SMN levels in SMA patients. Despite this, many
questions remain unanswered: how functional is SMN236? What are the
consequences of removing exons 1, 4, 5, 7, and 8? And most importantly, due
to the increasing amount of evidence on the involvement of additional
peripheral organs, what are the additional consequences to decreasing SMN
levels in SMA? It is clear that much more research needs to be done to fully
understand the role of SMN within multiple cell types and to pinpoint the
malfunction that is causing SMA. Only then can successful therapeutic methods
be developed.
31. 28
Acknowledgements
The experimental results presented here were provided by Adrian G. Todd,
Robert Morse and Philip J. Young. I would like to thank them for generating the
data and allowing its use as part of this project.
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