A role for motoneuron subtype-selective ER stress in disease … · 2009-11-23 · Supplementary figures Supplementary Figure 1: Early widespread alterations in gene expression unrelated

A role for motoneuron subtype-selective ER stress in disease manifestations

of FALS miceSmita Saxena, Erik Cabuy and Pico Caroni

Nature Neuroscience: doi:10.1038/nn.2297

Supplementary figures

Supplementary Figure 1:

Early widespread alterations in gene expression unrelated to selective axonal pathology in SOD1(G93A)

mice.

a) Comparable numbers of genes altered in VUL and RES MNs at P32. Values are numbers of gene probes with

raw expression values differing from wildtype values by a factor larger than 2 (p<0.01).

b) Early alterations in gene expression in cells involved (MC: primary motor cortex, layerV) and not known to

be involved (GC: dentate gyrus granule cells; SOLssc: sub-synaptic cytoplasm in soleus muscle) in disease.

Comparisons as in (a).

c) Gene expression alterations in SOD1(G93A) mice grouped according to cell functions. Analysis based on

Ingenuity software. Data from 3 mice each; the analysis is based on net changes (up or down) in transcript

levels; -log(p): probability that a particular cell function is affected in the disease model (see Methods).

d) Gene expression alterations in SOD1(G93A) mice grouped according to signaling pathways. Analysis as in

(c).



Increased BiP and Pi-eIF2α immunoreactivity in same large ventral horn cells of G93A-fast mice.

White lines: partial outline of ventral spinal cord gray matter. All panels images were acquired and processed in

the same way.

Bars: 50 (lower panels) and 100 µm.



Second subpopulation of MNs with gradually increasing BiP signals in G93A-fast mice.

Left: immunocytochemistry of ventral horn for ER protein ER01α, showing higher numbers of brightly labeled

cells at P60, compared to P38. Analysis as described for Fig. 3b. Note how the second subpopulation (MN-

high2) is detectable from P38 (but not P32) on, and includes about 40% of the MNs in the lumbar spinal cord.

Bar: 100 µm.



Absence of detectable ER stress or ubiquitin accumulations in MNs of mice overexpressing wt-hSOD1.

Upper row: high-level expression of hSOD1 in spinal cord cells of tg-hSOD1 mouse. Left panels:

immunocytochemistry for hSOD1 and BiP; low magnification view of one half spinal cord, with central canal on

the right. Right panels: higher-magnification views of ventral horn section included in white boxes. Note high

hSOD1 signals in large ventral horn spinal MNs, and absence of BiP upregulation in those cells.

Lower row: absence of ubiquitin accumulations in ventral horn cells of tg-hSOD1 mice at P35 and P180.

Transgenic mice at P280 exhibited slightly elevated ubiquitin signals, which were however much weaker than

those detected in G93A-fast mice at P30.

Bars: 50 µm.



Patterns of ubiquitin accumulations in G93A-slow spinal cord.

a) Upregulation of MN ubiquitin signal at P105. Note accumulation of signal in dotted patterns filling up cell

body cytosol and proximal dendrites, and barely detectable signals in wildtype mice. Also note absence of

microglia ubiquitin signal in the absence of nerve crush (compare to (b)).

b) Upregulation of ubiquitin signal in G93A-slow mice upon peripheral nerve crush. Unless indicated otherwise,

the peripheral nerve was crushed at P70, and mice were analyzed at P77. Note dramatic upregulation in CD11b-

positive microglia surrounding MNs in G93A-slow mice (upper two panels from left; lower panels on the left).

In addition, prominent ubiquitin signal was widely distributed within the cytosol of lesioned MNs (ipsilateral),

whereas contralateral MNs only exhibited perinuclear ubiquitin-positive dots. Upper right panel: treatment with

Salubrinal before and throughout crush experiment did not noticeably affect ubiquitin accumulations in MNs or

microglia. Lower right panel: upon crush-recrush experiment (first crush at P60, second crush at P70) ubiquitin

accumulation in MN cell bodies was substantially more prominent than upon a single crush; also note how

microglia signals have decreased 14 days after the second crush.

Bars: 20 µm.



Accumulation patterns of Ubiquitin and Pi-eIF2α in the brain of G93A-fast mice.

Upper row: Accumulation patterns at the level of individual cells. Note how Pi-eIF2α signals are largely absent

at P85, and how a subset of neurons in layerV motor cortex accumulate both signals at P125. Many hippocampal

CA3a pyramidal neurons accumulate Pi-eIF2α, but not ubiquitin signals at P125. Lower panels, left: at P65, a

subset of motor cortex neurons, including a fraction of layerV pyramidal neurons, accumulate high ubiquitin

signals, but no detectable Pi-eIF2α signal. Lower panels, right: at P125, ubquitin signals have mostly vanished;

Pi-eIF2α signal mainly accumulates in CA1 pyramidal neurons, and in layerV pyramidal neurons (arrows).

Bars: 30 µm.



UPR and UPS genes in RES MNs of G85R mice as a function of age.

Upregulation of UPR and stress genes, and downregulation of UPS genes in G85R RES MNs at P160, but not

P130.



Schematic summary of how increasing cell-selective ER stress relates to microglia activation and

denervation in VUL and RES MNs of FALS mice.

BiP levels in VUL (red) and RES MNs (blue (FR); violet (S)), and ubiquitin levels in VUL+RES MNs (green)

are plotted as a function of age. As indicated in the summary on the right, the mutant SOD1 models of FALS

differ in the rates of ER stress progression in VUL MNs, and in the onset times of ubiquitin accumulation in

VUL+RES MNs, but exhibit similar progression patterns beyond the transition to UPR in VUL MNs (dashed

line).



Mild widespread upregulation of Pi-eIF2α upon treatment with Salubrinal.

The panels show spinal cord sections labeled for Pi-eIF2α (left: low-magnification views of entire spinal cord

sections; right: higher magnification of panel on the left, including central canal region). G93A-slow mice were

subjected to sciatic nerve crush at P70, and analyzed 7 days later; crush side indicated by „ipsilateral“. Upper

rows: mice treated i.p. with vehicle; lower rows: mice treated i.p. with Salubrinal from P65 to P76. The samples

and images were treated in the same way, and acquired at high sensitivity to reveal weak signals. Note how Pi-

eIF2α signal is generally upregulated in the Salubrinal-treated mouse, validating its use in our experiments as an

inhibitor of Pi-eIF2α dephosphorylation. Also note how Salubrinal prevented the dramatic upregulation of Pi-

eIF2α signal in a subset of lesioned MNs of G93A-slow mice. The faint contralateral MN signal is comparable

to signals detected in wildtype mice (not shown). When Salubrinal was applied into muscle, there was no

widespread enhancement of Pi-eIF2α signals in the spinal cord (not shown).

Bar: 100 µm.



Salubrinal treatment started after UPR transition reverts and delays disease manifestations.

Schematic: protocol of experiments described in the figure. The durations of the SAL treatment is indicated by

the bar; the downward arrows indicates the age at which mice were analyzed.Values in the presence of SAL

(ipsilateral, third columns) are compared to those on the contralateral side (second columns), and to those (first

columns) in untreated mutant mice at an age selected by subtracting the approximate duration of the SAL

treatment from the age at the analysis. Details as in Fig. 6c; data from 3 mice per treatment.

Bars: 50 µm.



CNTF alleviates ER stress in VUL MNs of G93A mice.

a) CNTF applications from P20 to P28 suppress early upregulation of BiP immunoreactivity in VUL MNs of

G93A-fast mice. Values are the means and standard errors from 3 mice (50 cells each).

b) CNTF applications from P28 to P37 prevent UPR and microglia activation in G93A-fast mice. For the data

shown in the photographs, CNTF was applied from P28 to P40, and ventral horns were analyzed at P41 for

ERO1α immunoreactivity. For the quantitative analysis, all values are means and standard errors from 3 mice

(50 cells each). Gene profiling values at P38 (highlighted in the gray boxes) are from ipsilateral (+CNTF) and

contralateral spinal cords of the same mice. The BiP, Pi-eIF2α and CD11b microglia activation data are from

three additional groups of 3 mice each. Note how CNTF suppressed the upregulation of BiP and Pi-eIF2α

immunoreactivity, and of UPR/stress genes, but prevented the downregulation of stress management pathways

associated with the UPR transition.

Bars: 50 µm.



Chronic microglia activation enhances ubiquitin, but not Pi-eIF2a in G93A-slow MNs.

Mice were treated with Poly-I:C or vehicle (i.p.) every third day from P30 to P96, and analyzed at P100. Note

how the chronic treatment produced a dramatic enlargement of microglia in G93A-slow, and a much milder

response in wildtype mice. The increased CD11b signal in mutant mice was accompanied by an increase in

ubiquitin, but no Pi-eIF2α signal in G93A-slow MNs. All panels are pairs of double-labelings for CD11b and

either ubiquitin or Pi-eIF2α. The quantitative analysis is as described for Fig. 4a.

Bar: 100 µm.



Model of how increasing ER stress selectively in VUL MNs influences disease manifestations.

The model has two main implications about the process underlying disease manifestations in FALS mice. First,

α-MNs in the spinal cord are preferentially affected by mutant SOD1, as revealed by ubiquitin accumulations in

VUL and RES MNs, before a UPR in VUL MNs. Shadings of gray in the background suggest age-related

increase in mutant-related load on MNs. Second, selectively vulnerable neurons have a critical role in

influencing disease manifestations. The colours of the VUL and RES MN ovals indicate levels of ER stress in

the subtypes of MNs. Hypothetical roles of activated mutant microglia (and possibly additional local non-

neuronal cells) are suggested by arrows with question marks.


Supplementary methods

Retrograde labeling of Motoneurons

Mice were anaesthetized using a 1:1.5µg/gm body weight mix of ketamine and rompun

(xylazine, PROVET AG, Switzerland), and maintained at 36.9 0C during the surgical process.

All experimental procedures were approved by the Animal Committee of FMI, and the

Veterinary office of Basel. Hind limbs were epilated, and an incision was made into the skin,

to expose the muscle of interest. RITC-Dextrane (Invitrogen Corp., Carlsbad, CA 92008) was

either 3,000 MW (D-3308) or 10, 000 MW (fluororuby D-1817, lysine fixable), and was

injected locally (6mg/ml in PBS-0.1% BSA; 5-10 µl volume) into lateral gastrocnemius

(subcompartment-l1) or soleus muscle. The incision was sutured using Silkam® USP 6 (B.

Braun Medical AG, Switzerland).

Mice were sacrificed 2-3 days after tracer application (4 days for mice older than P50). Spinal

cords were then rapidly collected, embedded in Tissue-Tek OCT Compound (Zoeterwoude,

The Netherlands), placed at –20°C for 1 h, and then stored at –80°C. Upon cryostat sectioning

(14 µm), all sections were mounted on membrane slides (MMI, Glattbrugg, Switzerland), and

dehydrated in 95% ethanol for 15s. Upon air-drying, single motoneurons were immediately

microdissected using a MMI CellCut laser dissection microscope (MMI, Glattbrugg,

Switzerland). Criteria for motoneuron selection included a diameter of greater than 20 µm and

an identifiable nucleus. Approximately 25-30 cell sections, representing 10-11 cells, were

collected per tube.

Where indicated, CNTF rat recombinant protein (Sigma Aldrich) was reconstituted at a

concentration of 1 µg/ml in 0.1% PBS-BSA, and triceps surae muscle was injected every

alternate day with 80-100 µl of the working solution6. Salubrinal (Alexis Biochemicals) was

reconstituted at a concentration of 2.6 mM in PBS, 0.1% BSA, with 10% DMSO, and 80-100

µl of the working solution were injected every day (P14-P21; P30-P160) or every alternate

day (P22-P32; P39-P50) into triceps surae (Fig. 6c); for all other experiments, Salubrinal was

applied intraperitoneally. Poly-I:C (Sigma) was reconstituted in physiological saline and

administered intraperitoneally every third day at a concentration of 1mg/kg body weight.

RNA isolation, amplification and microarray profiling

Laser-dissected cells were immediately covered with 50 µl extraction buffer (Arcturus, USA),

left to lyse on the lid of the tube at RT for at least 15 minutes, and stored at -80°C. Total

cellular RNA was isolated with the PicoPure RNA isolation kit (Arcturus, USA), with minor


modifications. The optional DNase treatment step to remove potential genomic DNA

contamination was omitted and the centrifugation times during washing were reduced to 30

seconds.

All RNA samples were processed in batches of maximally 4 times three replicates (12

samples in total). Preparation of in vitro transcription (IVT) products was performed

according to Affymetrix protocols (GeneChip Expression Analysis Technical manual, Rev.

5), with minor modifications. The fragmented cRNA (15 µg) was used in a hybridization

cocktail containing spiked controls (Affymetrix). A total of 200 µl of this hybridization

cocktail was hybridized at 45°C for 16 hours to GeneChip Mouse Genome 430 2.0 Arrays

(Affymetrix). Following hybridization, the arrays were processed using a GeneChip Fluidics

Station 400 according to recommended protocols (FS450_0001, Affymetrix). Fluorescent

images of arrays were captured using the GeneChip Scanner 3000 7G (Affymetrix), and

image data were acquired and analyzed using the GeneChip Operating Software (GCOS,

Affymetrix). Average present call values were about 50%.

Statistical Analysis

Expression values and detection p-values were calculated using the GC-RMA implementation

found in Genedata's (Basel, Switzerland) Refiner array 4.1 package. Expression values were

quantile normalized and median scaled to a value of 20 in Genedata's Analyst 4.1 package.

Gene expression changes were evaluated upon removal of any gene with a detection p-value >

0.04 (Absent) in all conditions.

A statistical power analysis was performed to determine the minimum fold difference we

could consider with a power of 0.8 or better. Based on this, we found that our experiment was

able to reliably detect fold changes greater than 1.5. To further reduce the number of

genes in the analysis, we performed either a 1.5 or 2 fold filter on the genes present in at least

one condition and constructed a union list of these genes (this reduces the false discovery

implications of subsequent statistical analyses). This list was then used to identify statistically

significant effects in the data set using an N-way ANOVA (p<0.01). False discovery rate was

estimated using a Benjamini and Hochberg correction. Genes passing these filters were then

subjected to SOM clustering and pathway analysis using Ingenuity's Pathway Suite.

Immunocytochemistry

Mice were perfused with 4% PFA, lumbar level 3-5 (L3-L5) spinal cord or brain were

isolated, and kept overnight at 4 0C in 30% sucrose. After embedding, 50 µm cryostat sections


were post fixed with 4% PFA for 10 min, followed by PBS washes. Antibodies used for

immunocytochemistry were: rabbit-anti Ero1-Lα, 1:20; rabbit-anti Pi-eIF2α (Ser 51) 1:150;

rabbit-anti BiP, 1:100; and rabbit-anti PERK, 1:100 (Cell Signaling Technology, Inc,

Danvers, MA 01923 ); mouse anti-Ubiquitin 1:100 (Zymed); mouse anti Mono- and

Polyubiquitinylated Conjugates (clone FK) 1:150 (Biomol); mouse-anti KDEL Grp78, Grp94

(BiP), 1:1000 (Stressgen, Ann Arbor, MI 48108 ); rabbit-anti ATF4, 1:600 (abcam); rabbit-

anti VAchT, 1:1000 (Chemicon International, Temecula, CA); rat-anti CD11b, 1:300

(Serotec, Kidlington, OX5 1GE); rabbit-anti ATF3 (H90), 1:200 (Santa Cruz Biotechnology,

Inc, Santa Cruz, CA. 95060); rabbit-anti-synaptophysin, 1:200 (Dako, Glostrup, DK); rabbit-

anti human SOD1, 1:500 (Sigma). Antibodies were applied in PBS-3%BSA -0.3% Triton X

100, and incubated overnight at 40C. Sections were then briefly washed with PBS and

incubated for 60 min at RT with goat-anti-rabbit (Alexa 488, Invitrogen) or goat-anti-mouse

(Alexa 546, Invitrogen). ATF3 stainings were done on freshly embedded 50 µm cryostat

tissue sections fixed with 4% PFA for 10min. Pi-eIF2α stainings were carried out on 50 µm

floating sections in the presence of 1mM Na-Vanadate. For muscle labelings, sections were

processed as described above, and acetylcholine receptors at the synapse were visualized with

FITC-α-Bungarotoxin (1:1000; Invitrogen). Confocal images were acquired using an

Olympus Fluoview (Olympus, Tokyo) microscope, fitted with a 20X air objective, or an LSM

Meta (Carl Zeiss AG) microscope, fitted with a 40X oil objective. Images were processed

using Imaris software.

For the analysis of BiP (or Pi-eIF2α, or Ubiquitin) labeling intensities, data were acquired

with identical confocal settings, ensuring that signals at the brightest cells were not saturated,

and that background levels outside clusters were still detectable. Images were then analyzed

quantitatively using Image-Pro 5 software (Media Cybernetics). Signal intensities were

acquired within areas inside cells, excluding areas lacking signal; background levels outside

cells were subtracted from these values.

Further analyses

For sciatic nerve crush experiments, the nerve was exposed at the level of the femur and

biceps femoris, and crushed with No.5 jeweler forceps for 30 seconds. Recrushes were

applied 10 days later, at a position slightly distal from the previous crush.

For muscle force measurement, mice were initially handled for 4 days, and then trained with

grids for further 4 days. The test consisted in lifting twice grids of 40g, then 30g, 20g, and 10g

for a maximum duration of 30 sec each. All grid lifting trials were interrupted by 15 sec


pauses. Only the longest time was retained for each weight, and muscle force values were the

sum of the longest times for each grid.

Supplementary Video 1:

P132 G93A-fast mouse treated with vehicle from P30 on (this mouse will die at P136).

Supplementary Video 2:

P132 G93A-fast mouse treated with Salubrinal from P30 on (this mouse will die at P163).


Documents

A role for motoneuron subtype-selective ER stress in disease … · 2009-11-23 · Supplementary figures Supplementary Figure 1: Early widespread alterations in gene expression unrelated