1

Supporting Information

Supplementary Materials and Methods

Materials. Reagents were from Sigma-Aldrich unless otherwise specified. AlexaFluor555-

maleimide, Texas Red-dextran (3,000 Da) and AlexaFluor488 α-bungarotoxin were from

Invitrogen. The ChAT antibody was from Millipore and anti-myelin basic protein (MBP)

from Roche.

Animals. Transgenic mice carrying a human wild-type (WT) SOD1 (B6SJL-

Tg[SOD1]2Gur/J) or mutant SOD1G93A (TgN[SOD1-G93A]1Gur) gene were obtained from

Jackson Laboratories. Colonies were maintained by breeding male heterozygous carriers with

female (C57BL/6 x SJL) F1 hybrids. Mice were identified by genotyping for the human

SOD1 transgene using DNA extracted from tail snips (1). Female thy1-mitoCFP-S

(MitoMouse) (2) were bred with male SOD1G93A to generate double heterozygous mice.

Animals were housed in a controlled temperature and humidity environment and maintained

on a 12 h light/dark cycle with access to food and water provided ad libitum. All experiments

were carried out under license from the UK Home Office in accordance with the Animals

(Scientific Procedures) Act 1986 and following approval from the Cancer Research UK and

Institute of Neurology Ethical Review Committees.

Neuronal cultures. Primary MN cultures were prepared from E12.5 WT mouse embryos as

previously described (3). Cultures from 5 d in vitro were incubated with 40 nM HC555 for 30

min at 37oC, washed twice in Dulbecco’s modified Eagle’s medium, without phenol red,

supplemented with 30 mM HEPES-NaOH, pH 7.4, and imaged using time-lapse confocal

microscopy (4, 5).

2

DRG neurons were isolated from adult SOD1G93A mice at 4 different disease stages: pre-

symptomatic, early symptomatic, symptomatic and late symptomatic and plated onto poly-L-

lysine and laminin-coated MatTek dishes (MatTek Inc) and cultured as previously described

(6). After 2 d in vitro, DRG neurons were incubated with 40 nM HC555 and 50 nM

Mitotracker Green for 30 min at 37˚C. Cells were washed twice in Dulbecco’s modified

Eagle’s medium (without phenol red, supplemented with 30 mM HEPES-NaOH, pH 7.4) and

imaged using time-lapse confocal microscopy (4, 5).

Histochemistry and immunofluorescence. To assess the distribution of HC555 at the NMJ

and its uptake into sciatic nerve axons, this probe was injected into the TA and GC muscles.

In selected cases, a sciatic nerve ligation at the mid-thigh level was performed before the

intramuscular injection. 6 h later (or 3, 5 and 8 h in the absence of nerve ligation), the animals

were transcardially perfused and the muscles and sciatic nerves removed, post-fixed and

cryoprotected prior to serial sectioning (muscles, 30 µm; sciatic nerve, 10 µm). Specimens

were allowed to dry and incubated in AlexaFluor488-α bungarotoxin (1:200 in distilled

water) for 1 h. Alternatively, confocal images of transverse sections of sciatic nerve were

taken and the number of axons containing HC555 that counterstained with the ChAT antibody

(1:250) were calculated as a percentage of the total number of axons. Myelin sheaths were

immunostained for MBP (1:500).

Silver cholinesterase staining. This technique, which combines the use of potassium

ferricyanide for the staining of cholinesterase at NMJs and silver impregnation (modified

from 7), allowed the detection of motor axons and nerve terminals and was used to monitor

the percentage of denervation. Muscles were removed and immediately fixed in a slightly

stretched position using buffered-formaldehyde (4% formaldehyde, 1% CaCl2, 0.5% MgCl2

6H2O, 0.1% CdCl2 2.5H2O) in veronal-acetate buffer (pH 6.45) for 6 h. Subsequently,

3

muscles were incubated in 10% sucrose overnight at 4˚C. Longitudinal sections (40 µm

thick) were then cut on a cryomicrotome and held in distilled water on ice for 10 min,

followed by incubation in an acetylthiocholine iodide solution (1.6 mM acetylthiocholine

iodide in 0.06 M maleate buffer (pH 6.0), containing 5 mM sodium citrate, 3 mM copper

sulphate, 0.5 mM potassium ferricyanide and 0.4 M sucrose) for 20 min on ice. Sections were

then rinsed in distilled water for 30 s and immersed in potassium ferricyanide (7.3 mM in

distilled water) for 10 min at room temperature to allow an iodide precipitate to develop.

Sections were washed three times in distilled water for 5 min, immersed in absolute ethanol

for 1 h then washed in distilled water twice for 15 min. Sections were then placed in a silver

solution (0.1% CaCO3, 0.05% CuSO4 5H2O, 10% AgNO3 in distilled water) for 35 min at

37˚C to visualise the axons. After a rinse in distilled water, sections were immersed in a

reducer solution (1% hydroquinone, 10% Na2SO3 in distilled water), rinsed again and

mounted onto slides coated with gelatin. Over 500 end plates were examined and quantified

for each muscle.

MN survival. MN survival was monitored on serial sections (20 µm) of lumbar spinal cord

(L2-L6) were cut on a cryostat and stained with cresyl fast violet, a Nissl stain (8). The

number of surviving MNs within the sciatic motor pool of each ventral horn was assessed by

counting the number of Nissl-stained MNs in every third spinal cord section (n=60). This

method avoided counting the same cell twice in consecutive sections. Morphological criteria

were also employed, so that only large polygonal neurons in which the nucleolus was clearly

visible at high magnification were included in the counts. This method of counting has

previously been used to assess MN survival (9). To assess the extent of MN degeneration,

mean MN survival in the sciatic motor pool of SOD1G93A mice was compared to the number

surviving in WT littermates and SOD1WT mice.

4

Uptake by lumbar MNs and DRG neurons. Following each in vivo imaging experiment,

mice were terminally anaesthetized with sodium pentobarbitone and perfused transcardially

with 4% paraformaldehyde (PFA; TAAB Ltd) in 77 mM Na2HPO4, 23 mM NaH2PO4. The

TA and GC muscles, the imaged sciatic nerve and the L2-L6 section of the spinal cord were

removed, post-fixed for 4 h and then cryoprotected in 30% sucrose in PBS at 4˚C. Serial

sections (20 µm) of the L2-L6 region of the spinal column were cut and unstained sections

were used to examine HC555 localization in lumbar MNs and DRGs using confocal

microscopy. Sections were excited using the 543 nm laser line and emitted fluorescence

collected using the 560-615 nm bandpass filter.

TrkB antibody studies. The sciatic nerve was ligated at the mid-thigh level of both WT and

SOD1G93A mice at 75 d of age. Subsequently 8 µl of Alexafluor555-conjugated TrkB

antibody (Millipore) together with BDNF was injected into the TA and GC muscles.

Approximately 6 h later the sciatic nerve was gently squeezed and the extruded axoplasm

snap frozen. TrkB antibody was detected by western blotting using a horseradish peroxidase-

conjugated swine anti-rabbit antibody (Dako) and quantified by ImageJ. Data were

normalised for GAPDH levels using a horseradish peroxidase-conjugated anti-GAPDH rabbit

polyclonal antibody (Cell Signaling).

Statistical analysis. The parameters in Fig. 1-5, S2-S5 and S7 {AREA, SD, mean speed},

their standard errors and confidence intervals were estimated using non-linear regression (10,

11) for each sample separately, regressing the relative frequencies against the lower bound of

the speed bin, fitting against the model: Freq=(AREA/(SD*(2*pi)^0.5))*exp(-0.5*((speed-

mean speed)/SD)^2). The parameter 'mean speed', along with its standard error, were used in

two-tailed z-tests to infer the equivalence or otherwise the location of the peaks amongst the

pairwise comparisons. The p-values were then corrected using the conservative Bonferroni

5

correction method. An omnibus F-test of the 'mean speed' across all the groups was passed at

an alpha of 0.05 (p<0.0001 F=173.6 with 6:329 degrees of freedom – the p-value could not

be determined to more precision than this inequality by our software). R2 statistics of the non-

linear regression were all found to be >0.9, and residuals were visually checked, and

determined to be free of any systematic effects that would invalidate our approach.

The estimated values of the mean speed parameter, for samples labelled WT or early

symptomatic SOD1G93A, were then used to build a linear discriminant classifier (12) to find

the boundary between the two classes, and then predict the classes of the unknown samples

(pre-symptomatic data, both 25 d and 36 d SOD1G93A mice) by seeing on which side of the

mean speed boundary they lie. The difference between WT and early symptomatic SOD1G93A

mean speeds were tested using two slightly different approaches. In the 'Separate' strategy,

each of the peaks was summarised (as before) using non-linear regression to extract the

modal speeds, and the n=5 (WT), n=6 (early symptomatic SOD1G93A) replicates in each

group were subjected to a α=0.05 two-tailed Student's t-test, which passed with p=1.23e-15.

Graphs didn't provide any visual evidence that the requirement for normality of group means

was violated. In the 'Together' approach, we estimated two curves through the dataset, one

through the WT replicates (n=5), and one through the early symptomatic SOD1G93A replicates

(n=6) with non-linear regression, and an F-test at α=0.5 indicated a significant difference in

the modal speeds of the two groups (p=6.53e-75, F=471.6 on 1:494 degrees of freedom).

Thus both approaches found a significant difference between WT and early symptomatic

SOD1G93A mean speeds at α=0.05. Calculations were performed using GraphPad Prism

version 4.03 for Windows (GraphPad Software).

Statistical analysis of MN counts, TrkB transport, muscle denervation and mitochondrial

pausing were performed using an unpaired two-tailed Student’s t-test with equal variance

6

using Kaleidagraph or SigmaStat (2.03; SyStat Software) and the normality of the data was

assessed by Sigma Stat. Significance for all statistical analysis was set at p<0.05.

References

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3. Bilsland LG, Nirmalananthan N, Yip J, Greensmith L, Duchen MR (2008) Expression

of mutant SOD1 in astrocytes induces functional deficits in motoneuron

mitochondria. J Neurochem 107:1271-1283.

4. Lalli G, Gschmeissner S, Schiavo G (2003) Myosin Va and microtubule-based motors

are required for fast axonal retrograde transport of tetanus toxin in motor neurons. J

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5. Kieran D, et al. (2005) A mutation in dynein rescues axonal transport defects and

extends the life span of ALS mice. J Cell Biol 169:561-567.

6. Deinhardt K, et al. (2006) Rab5 and Rab7 control endocytic sorting along the axonal

retrograde transport pathway. Neuron 52:293-305.

7. Namba T, Nakamura T, Grob D (1967) Staining for nerve fiber and cholinesterase

activity in fresh frozen sections. Am J Clin Pathol 47:74-77.

8. Culling CFA (1963) Handbook of Histopathological Techniques. 2nd Ed. pp 362-363.

9. Bilsland LG, et al. (2006) Increasing cannabinoid levels by pharmacological and

genetic manipulation delay disease progression in SOD1 mice. Faseb J 20:1003-

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10. Motulsky H, Christopoulos A (2004) Fitting models to biological data using linear

and nonlinear regression. A practical guide to curve fitting Oxford University Press,

New York.

11. Bates DM, Watts DG (1980) Non linear regression analysis and its applications.

Wiley, New York.

12. Fisher RA (1936) The use of multiple measurements in the taxonomic problems. Ann

Eugenics 7:179-188.

13. Chiu AY, et al. (1995) Age-dependent penetrance of disease in a transgenic mouse

model of familial amyotrophic lateral sclerosis. Mol Cell Neurosci 6:349-362.

14. Mourelatos Z, Gonatas NK, Stieber A, Gurney ME, Dal Canto MC (1996) The Golgi

apparatus of spinal cord motor neurons in transgenic mice expressing mutant Cu,Zn

superoxide dismutase becomes fragmented in early, preclinical stages of the disease.

Proc Natl Acad Sci U S A 93:5472-5477.

15. Pun S, Santos AF, Saxena S, Xu L, Caroni P (2006) Selective vulnerability and

pruning of phasic motoneuron axons in motoneuron disease alleviated by CNTF. Nat

Neurosci 9:408-419.

16. Hegedus J, Putman CT, Gordon T (2007) Time course of preferential motor unit loss

in the SOD1(G93A) mouse model of amyotrophic lateral sclerosis. Neurobiol Dis

28:154-164.

17. Tu PH, et al. (1997) Selective degeneration fo Purkinje cells with Lewy body-like

inclusions in aged NFHLACZ transgenic mice. J Neurosci 17:1064-1074.

18. Johnston JA, Dalton MJ, Gurney ME, Kopito RR (2000) Formation of high molecular

weight complexes of mutant Cu, Zn-superoxide dismutase in a mouse model for

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19. Fischer LR, et al. (2004) Amyotrophic lateral sclerosis is a distal axonopathy:

evidence in mice and man. Exp Neurol 185:232-240.

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low sprouting competence in motoneuron diseases. J Neurosci 20:2534-2542.

Supplementary Figure Legends

Figure S1: Internalization of HC555 by motor axons. Following intramuscular injection,

HC555 accumulated at the NMJ within 3 h (A; in red), and then declined (B, 5 h; C, 8 h; scale

bar, 5 µm) due to uptake by axons (arrowhead) in the sciatic nerve (D; dashed box has been

magnified; scale bar, 10 µm). The motor end plate has been counterstained with

AlexaFluor488 α-bungarotoxin (A-D; in green).

Figure S2: The rate of retrograde transport of HC555 was not influenced by age. HC555 was

injected intramuscular into female WT mice of varying ages. Following a recovery period,

the mice were anaesthetized, the sciatic nerve exposed and retrograde transport imaged.

Figure S3: Kinetic analysis of HC555 retrograde transport. To control for the effects of

SOD1G93A overexpression, WT littermates and SOD1WT mice were used as controls (A-C).

No significant difference in MN survival was found between WT and SOD1WT mice (A;

p>0.05; n=3). Quantitative analysis of retrograde transport (B) however, indicated that

transport of HC555 was significantly faster in SOD1WT mice (200 carriers; 30 axons; n=3)

compared to WT mice (258 carriers; 44 axons; n=5). This increase in speed in SOD1WT mice

was due to a higher percentage of fast carriers (C). Displacement of HC555 positive carriers

tracked during the course of a representative experiment from WT (D) and SOD1G93A mice at

pre-symptomatic (E) and early symptomatic (F) stages of disease. The start of tracking for

9

each carrier, which is displayed by a single dotted line, was set to time = 0. The slope of the

line is proportional to the speed of transport. (G) HC555 accumulated at the NMJ (labeled

with AlexaFluor488 α-bungarotoxin in merged image; asterisks) and was taken up into

innervating axons (arrowhead; see also Fig. S1) of SOD1G93A mice at the late symptomatic

stage of disease. In the sciatic nerve, HC555 was found in axons co-labeled for ChAT,

indicating that they were motor axons (H; arrowheads). In addition, a punctate HC555 signal

was detected in MN cell bodies located in the spinal cord of late symptomatic mice (I). Scale

bars, 20 µm. Error bars represent S.E.M..

Figure S4: Retrograde transport was unaffected in DRG neurons from SOD1G93A mice.

HC555 was internalized by sensory neurons projecting in the sciatic nerve (A). Following

intramuscular injection, sections (20 µm) of the spinal column revealed accumulations of

HC555 in the DRG somas (arrowheads; scale bar, 50 µm). (B) DRGs were removed from

adult SOD1G93A mice at varying stages of disease and maintained in culture. Kinetic analysis

of HC555 transport revealed that there was no significant difference in the rates of transport in

SOD1G93A DRG neurons (56–235 carriers; 10-29 axons; n ≥ 2 independent cultures) at any

disease stage examined, compared to WT.

Figure S5: The onset of retrograde transport deficits in pre-symptomatic SOD1G93A mice. (A)

Analysis of the rate of retrograde transport in pre-symptomatic SOD1G93A mice at 36 d

following intramuscular injection, identified two distinct disease-stage sub-populations; one

(grey circles; 140 carriers; 30 axons) characterized by a peak speed of retrograde HC555

carriers statistically indistinguishable from that of WT animals (black; 258 carriers; 44 axons)

and another pool (cyan; 204 carriers; 22 axons) in which the peak speed did not differ

significantly from early symptomatic SOD1G93A mice (red; 193 carriers; 39 axons). This

suggests that this pre-symptomatic stage represents the approximate age at which retrograde

10

transport deficits became evident in SOD1G93A mice. Indeed, analysis of retrograde transport

at an earlier stage of disease (25 d; purple), revealed peak speeds that did not differ

significantly from WT. Error bars represent S.E. of the mean speed. (B) Speed distribution

curves of HC555 transport for WT and SOD1G93A mice at 25 d (164 carriers; 24 axons). Error

bars represent S.E.M..

Figure S6: Muscle denervation in SOD1G93A mice at 36 d of age. Fixed muscle sections from

36 d old WT and SOD1G93A mice underwent silver cholinesterase staining to label the motor

end plates and the innervating axons. Muscle sections from WT mice; scale bar, 100 µm (A)

and 25 µm (B). The number of denervated motor end plates was quantified and displayed in

(C). There was no significant difference in the innervation of NMJ between WT and

SOD1G93A mice at a pre-symptomatic stage. 7.0±0.9% (n=6 muscles) and 7.5±1.1% (n=6

muscles) of NMJs were denervated in WT EDL and soleus muscles, respectively, whereas in

SOD1G93A mice the relative values were 9.0±0.7% (n=6 muscles; EDL) and 9.4±1.4 (n=6

muscles; soleus). Error bars represent S.E.M.; n ≥ 500 end plates from at least 4 mice.

Figure S7: Axonal transport of mitochondria was unaffected in DRG neurons isolated from

SOD1G93A mice. DRG neurons were removed from both WT and SOD1G93A mice at 36 d and

maintained in culture. Cultures were loaded with Mitotracker Green (50 nM) for 30 min at

37˚C. Axonal transport of Mitotracker was monitored by time-lapse confocal microscopy.

Quantitative kinetic analysis revealed that there was no significant difference in the rates of

transport of mitochondria in either the anterograde (a) or retrograde (b) direction in DRG

neurons from SOD1G93A mice (in green) compared to WT (in black). Error bars represent

S.E.M..

11

Table legend

Table S1: Summary of the key pathological signs in SOD1G93A mice during disease

progression. This table attempts to correlate our observed changes in axonal retrograde

transport and MN survival (in bold) with the documented selective vulnerabilities of different

MN pools and with overt signs of disease seen in SOD1G93A mice. FF, fast, fatiguable; FR,

fast, resistant.

Movies

Movie 1: Retrograde transport of HC555 in a single axon in the intact sciatic nerve of WT

mice. HC555 was injected intramuscularly into WT mice. After a recovery period, the mouse

was terminally anaesthetized and the sciatic nerve exposed. Retrograde transport of HC555

was observed in the direction of the spinal cord (to the left) within individual axons of the

intact sciatic nerve. The video consists of 100 frames and is played at 5 frames/s. The image

size is 71.6 µm x 12.7 µm.

Movie 2: Retrograde transport of HC555 in DRG axons. This video shows a DRG axon that

has been incubated with 40 nM HC555 for 30 min at 37˚C and imaged by time-lapse

microscopy. This video consists of 100 frames and is played at 5 frames/s. The image size is

70.0 µm x 12.1 µm.

Table S1. Key pathological signs in SOD1G93A mice during disease progression

Disease stage

Disease signs

MN pathology

Axonal transport

Pre-symptomatic (36±0.95 d)

No overt disease signs.

Ultrastructural pathology including abnormal mitochondria, Golgi fragmentation and vacuolation (13, 14).

No MN loss.

Synaptic vesicle stalling (35-38 d).

Loss of synaptic vesicle markers in FF MNs (38-46 d)(15).

Deficit in retrograde transport of HC555 (this work).

Deficit in slow anterograde and fast axonal transport (5, 15).

Early symptomatic

(74±1.4 d) Loss of tetanic muscle force in fast muscles (16).

Loss of approximately 23% of lumbar MNs.

FF innervated muscle fibres have become denervated. Onset of denervation of FR innervated muscle fibres (15)

Protein aggregation and neurofilament inclusions in MNs (17, 18)

Greater deficit in retrograde transport of HC555 as a consequence of vulnerable fast MN degeneration (this work).

Symptomatic (95±1.4 d)

Fine hindlimb tremor, muscle spasticity and hyperreflexia (13).

Further loss of muscle force/grip strength.

Decline in constant rotarod performance (19)

Loss of approximately 39% of lumbar MNs.

Innervation by fast MNs predominantly lost. Compensatory sprouting of slow MNs to reinnervate previously fast NMJs (15).

Deficit in retrograde transport of HC555, but less severe than at early-symptomatic stage due to death of fast MNs and apparent compensation by slow, relatively unaffected MNs, and DRGs (this work).

Late symptomatic

(113±3.5 d)

Weight loss.

Hindlimb paralysis and muscle atrophy (13).

Death at approximately 130 d (1).

Loss of approximately 56% of lumbar MNs.

Denervation of soleus muscle innervated by slow MNs (20).

Deficit in retrograde transport of HC555 as at the symptomatic disease stage (95 d; this work).

α-bungarotoxin HC555 MergeA

B

C

HC555

Merge

3 h

5 h

8 h

Figure S1 - Bilsland et al.

Dα-bungarotoxin

Rel

ativ

e fre

quen

cy

Speed (μm/s)-0.4 0 0.4 0.8 1.2 1.6 2.0 2.4 2.8 3.2

38 d WT81 d WT101 d WT125 d WT

0

0.02

0.04

0.06

0.08

0.1

0.12

0.14

0.16

Figure S2 Bilsland et al.

Speed (μm/s)

Rel

ativ

e fre

quen

cy

-0.4 0 0.4 0.8 1.2 1.6 2.0 2.40

0.05

0.10

0.15

0.20

0.25

0.30

WT

Pre-symptomatic SOD1G93A

Early symptomatic SOD1G93A

Symptomatic SOD1G93A

Late symptomatic SOD1G93A

PhaseHC555

DRGs

A

B

Figure S4 - Bilsland et al.

Figure S5 - Bilsland et al.

WT

(25 d)

Pre-sym

ptomati

c

Early s

ympto

matic

(36 d)

(74 d)

Pre-sym

ptomati

c

1.2

0.8

1.0

1.4

1.6

Spee

d (µ

m/s

)

0

0.02

0.04

0.06

0.08

0.10

0.12

0.14

WTSOD1G93A

-0.4 0 0.4 0.8 1.2 1.6 2.0 2.4 2.8 3.2

Rel

ativ

e fre

quen

cy

Speed (μm/s)

0.16

A

B

EDL SOL

WT

2

4

6

8

10

12

0

% o

f den

erva

tion

CA

B

Figure S6 - Bilsland et al

SOD1G93A

Figure S7 - Bilsland et al

A

B

0 0.4 0.8 1.2 1.6 2.0 2.4Speed (μm/s)

0

0.1

0.2

0.3

0.4

Rel

ativ

e fre

quen

cy

WTSOD1G93A

- 0.4

Rel

ativ

e fre

quen

cy

0

0.1

0.2

0.3

0.4

WTSOD1G93A

0 0.4 0.8 1.2 1.6 2.0 2.4- 0.4

Retrograde transport

Anterograde transport

Mitochondrial speed

Speed (μm/s)