www.sciencemag.org/content/348/6233/453/suppl/DC1

Supplementary Materials for

SARM1 activation triggers axon degeneration locally via NAD+ destruction

Josiah Gerdts, E. J. Brace, Yo Sasaki, Aaron DiAntonio, Jeffrey Milbrandt*

*Corresponding author. E-mail: jmilbrandt@wustl.edu

Published 24 April 2015, Science 348, 453 (2015) DOI: 10.1126/science.1258366

This PDF file includes:

Materials and Methods Supplementary Text Figs. S1 to S9 References (24–27)

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Materials and Methods

Cell culture: Culture of DRG neurons in 96-well and 24-well plates was

performed as described (24). For microfluidic chamber experiments, 103 DRG neurons

were grow in Axis AX15010TC chambers (Millipore) following manufacturer

instructions. Immunostaining of DRG axons with anti-beta Tubulin antibody was

performed as described (24). For SARMps

cleavage analysis, ~2000 DRG neurons were

seeded in 24 well plates and infected on DIV 1 with SARMps

Frb-Ntev-C virus and Fkbp-

Ctev virus; after 100 nM rapa treatment for the indicated times, cell lysates were

harvested for western blot analysis. Parp1-/- and Cd38-/- neurons were from embryos

obtained by crossing Parp1/Cd38 double heterozygous mice (13). Sarm1-/- neurons were

obtained from Sarm1-/- as described (6).

HEK293T cells were maintained in DMEM containing 10% FBS and 2mM L-

Glutamine (DFG) and passaged by suspending in 0.05% trypsin. For production of HTir

cell line, 106 HEK293T cells were infected with lentiviral particles (Frb-sTIR-

3xFlag_IRES-Cerulean + Fkbp-sTIR-Gfp) at low MOI in the presence of 8 µg/ml

polybrene. After 48 hrs, this pool was split into a 384 well plate at single cell density.

After two weeks, wells containing uniformly GFP/Cerulean positive cells were selected

for amplification. HTir cells stably expressing shRNA targeting PARG were created by

infecting cells with sh-PARG lentivirus and selection of a polyclonal population by

puromycin treatment (1 ug/ml; 2 passages). Mouse embryonic fibroblasts were obtained

from E12.5 mouse tissue and maintained in DFG and experiments were performed when

growth was stabilized (> 10 passages). 106 MEF cells were infected with lentiviral

particles followed by puromycin selection for > 2 passages. AP20187 (also called "BB

Homodimerizer", Clontech) was used at 50 nM, rapamycin (Sigma) was used at 100 nM,

FK866 (Sigma) was used at 10 nM, Nicotinamide Riboside (Chromadex) was used 1 at 1

mM, Olaparib (Selleckchem) was used at 100 nM, and XAV939 (Sigma) was used at 100

nM.

Axon Isolation for Metabolite Measurement: Embryonic DRG neurons from

embryonic day 12.5 mice were grown in dense clusters in 6-well plates coated with

PDL/Laminin. Custom silicone molding was used to restrict cell dispersion during the

first 12 hours in culture, after which the molding was removed and axons grew radially

from the dense clusters for seven days. At the start of the experiment, a scalpel was used

to sever all axons extending beyond the perimeter of the soma cluster, and the cell bodies

were subsequently removed by pipetting. After zero, 3, or 4 hours post transection, the

wells were rinsed 2x with cold PBS and metabolites were collected in 0.2 ml 1M HClO4.

Protein from the HClO4 precipitate (see HPLC) and well surface were collected in 0.5 ml

0.1M KOH, and protein was quantified using the BCA assay (Pierce). Empty wells were

used to subtract protein attributable to media and well coating.

Sciatic nerve transection: Sarm1-/- knockout (6) or wild-type C57Bl/6 mice (8-10

weeks old) were anesthetized with 2,2,2 Tribromoethanol (Sigma) at a dose of 400

mg/kg. The right side of Sciatic nerve was exposed and transected at the sciatic notch. At

30 hours after axotomy, both left (control) and right (transected) side of the sciatic nerve

were dissected out and placed in ice cold PBS. Extra tissues including muscle, fat cells,

and epineurium were removed from dissected sciatic nerve and weighed and then flash

frozen in the liquid nitrogen prior to metabolite extraction. For morphologic analysis of

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nerves, sciatic nerve segments were fixed with 3% glutaraldehyde and incubated

overnight at 4°C with 1% osmium tetroxide in phosphate buffer. Specimens were

dehydrated and embedded in epoxy. Nerve cross sections (0.4 µm) were stained with 1%

toluidene blue and morphology was analyzed using light microscopy.

Fly stocks. Drosophila melanogaster were raised on standard fly food at 25°C.

The following strains were used in this study: Canton S (wild type), m12-Gal4 (single

motor axon resolution), ppk-Gal4 (single sensory axon resolution), UAS-cyto-

mNMNAT1-mcherry. The transgenic lines: UAS-SAM-TIR-Venus, UAS-SAM-TIRmut

-

Venus (alanine substitution of residues 697-704 in the TIR domain), and UAS-FkbpF36V

-

TIR-Venus are identical to constructs described above with the exception that they were

cloned into pUAST and injected (Best Gene Inc.) into Drosophila embryos to create

transgenic lines. Male and female larvae were used for experiments.

NMNAT protection of axonal degeneration in Drosophila. Transgenic UAS-

FkbpF36V

-TIR-Venus, mCD8GFP flies either expressing or not expressing UAS-cyto-

mNMNAT under the control of M12-Gal4 were grown for four day on normal food at

25°C. Larva were moved to soft food containing 10 micromolar AP20187 for 30 hours

to induce dimerization of sTIR domains before animals were dissected. The Drosophila

nerve degeneration score was computed as reported (25). Briefly, axons were scored by

researchers blind to genotype using a scale ranging from completely continuous (0%), to

continuous with varicosities (25%), to partially discontinuous (50%), to mostly

fragmented with a few segments of continuity (75%), to completely fragmented (100%).

Immunocytochemistry: Larval filet preps were fixed in in 4% paraformaldehyde

in PBS for 20 minutes at room temperature. Blocking and staining were performed in

PBS + 0.1% Triton X-100 +5% goat serum. Cy3 conjugated goat anti-HRP (1:1000,

Jackson ImmunoResearch) and alexa488 conjugated anti-GFP (1:1000, Life

Technologies). All samples were mounted and imaged in 70% glycerol containing

Vectashield.

Imaging and Analysis: Images of Drosophila 3rd

instar larva were acquired on a

Nikon Eclipse C1 Confocal microscope using 60X oil objectives. Images shown are

maximal Z-projections of confocal stacks. Samples for each experiment were processed

simultaneously and used identical confocal gain settings.

Molecular cloning: All mammalian expression plasmids were derived from the

lentiviral FCIV vector (4) that contains a human ubiquitin promoter and IRES-driven

Venus fluorescent protein. The full coding sequences of SARMps

-Cerulean, SARMps

Frb-

Ntev-Cerulean, Fkbp-Ctev, Frb-sTIR-Flag, Fkbp-sTIR-GFP, FkbpF36V

sTIR-Cerulean,

FkbpF36V

-MYD88TIR-Cerulean, FkbpF36V

-TLR4TIR-Cerulean, and FkbpF36V

-Tnkp-

Cerulean are provided in genbank format in the Supplemental Sequences section.

SARMps

-Cerulean was created by PCR amplifying human SARM1 in two fragments

(AA1-554, AA559-724) with an overlap sequence encoding the amino acid sequence

G(559)-ENLYFQS-G(559) and these fragments were inserted with a C-terminal Cerulean

fusion into the FCIV vector lacking IRES-Venus. SARMps

-Frb-Ntev-Cerulean was

created by cutting SARMps

-Cerulean with BstXI and inserting Frb-Ntev in frame. Full

length TEV protease (S219V; Addgene #8835) was subcloned into FCIV and included a

C-terminal Flag tag. Frb and Fkbp were PCR amplified from Frb-Nluc (Addgene

#31181) and Cluc-Fkbp (Addgene #31184) from the laboratory of David Piwnica-

Worms. The Ntev domain was subcloned from TEV and included AA: 1-118. Ctev

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included AA: 119-241. These were fused to the C-termini of Frb and Fkbp with flexible

linkers. Similarly Frb and Fkbp were fused to the SARM1 TIR domain (AA560-724)

with flexible linkers. Frb-sTIR contained a C-terminal 3xFlag tag and was inserted into

FCIV with Venus replaced with Cerulean (FCIC). Fkbp-sTIR contained a C-terminal

GFP fusion tag. The FkbpF36V

variant was synthesized as a Gene Block (IDT) and fused

to sTIR-Cerulean. Human TLR4 TIR and human MYD88 TIR were synthesized as Gene

Block fragments (IDT) and fused between FkbpF36V

and Cerulean in a similar manner as

sTIR. DNA encoding the PARP domain of human Tankyrase 1 (identified as Tnkp) was

synthesized and fused to an N-terminal FkbpF36V

and C-terminal Cerulean fluorescent

protein. Mouse Nmnat1 (cytoplasmic variant) and Nampt were described (26).

mitoDSRed was described (6). Sh-PARG (NM_003631.1-2843s1c1) was from Sigma.

SARMps inactivation by split TEV protease: Lentiviral stocks of SARMps

(with

C-terminal Frb-Ntev-Cerulean) and Fkbp-Ctev were prepared separately as described

below, and DRG neurons in 96- or 24-well plates were infected with both viruses 1 day

after dissection. Expression of SARMps

was verified in >90% of cells as evident by

Cerulean fluorescence. TEV complementation was initiated by rapamycin addition (100

nM) at varied intervals relative to axon severing. The following considerations are noted

for readers interested in split TEV as a tool to control signaling: We note that co-

expression of these proteins leads to a small but detectable amount of SARMps

cleavage

in the absence of rapamycin (as evident in the western blot in Figure 1); however, this

minor degree of unstimulated cleavage does not affect the axon degeneration phenotype.

We thus conclude that the system is well-suited for the experiments performed. We also

note that a separately-evaluated split TEV system using three expressed proteins (Frb-

Ntev, Fkbp-Ctev, and SARMps

) resulted in measurable SARMps

cleavage but a rate

below that required for the experiment presented. The system presented in Figure 1 that

involved direct attachment of Frb-Ntev to SARMps

was found to accelerate cleavage

kinetics.

Lentiviral preparation and transduction was performed as described (6). Briefly,

HEK293T cells were cotransfected with lentiviral vectors and 48 hours later media

containing lentiviral particles (106-10

7 particles/ml) was collected. Lentiviral particles

were pelleted with the LentiX Concentrator reagent (Clontech) and resuspended in 1/10

volume PBS, then frozen at -80 deg until use. Lentiviral particles were added directly to

neuronal media 5 days prior to experimentation.

Western Blotting: Cell lysates were resolved by LDS-PAGE, transferred to

nitrocellulose, and blocked with 5% milk in PBS with 0.1% TritonX100. Primary

antibodies were rabbit anti-GFP (Life Technologies), mouse anti-alpha Tubulin (Sigma),

and rabbit anti-Poly ADP-Ribose (Trevigen). These were diluted 1:1000 in SuperBlock

reagent (Thermo) and incubated overnight. Horseradish peroxidase-conjugated secondary

antibodies (1:1000) were detected by luminol/peroxide assay (Thermo).

Electroporation: 5x106 HTir cells were trypsinized and resuspended in 0.15 ml

HEPES-buffered saline (pH 7.4) containing 1 mM ATP and 5mM NAD+ with either 1

mM NaAD or 1 microCi 14

C-NAD+ (Perkin Elmer). These suspensions were kept on ice

for 5 minutes prior to- and 5 minutes following electroporation in 2 mm cuvettes using a

BioRAD GenePulser II. A single 290V / 125 uF pulse was delivered with 100 Ohm

parallel resistance. The cells were transferred to microcentrifuge tubes and washed twice

in 500 ul cold DMEM and then each sample was divided equally into 2 tubes containing

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300 ul DMEM and warmed to 37 deg on an incubation block. In these split samples, 100

nM rapamycin was added to one tube and the other served as a control. After incubation

for the indicated times, the cells were pelleted, rinsed with cold PBS, and flash frozen

prior to metabolite extraction.

Thin Layer Chromatography: Flash-frozen cell pellets were extracted by the

methanol:chloroform phase separation method. 0.15 ml 2:1 methanol:chloroform was

added, the samples were sonicated, and an additional 0.15 ml 1:1 water:chloroform was

added. The aqueous fraction was collected and completely dried in a vacuum centrifuge.

Metabolites were resuspended in 10 ul H2O and 0.7 ul was loaded onto glass-backed

silica TLC plates (Analtech). Plates were developed in a mobile phase consisting of 70:30

ethanol:ammonium acetate (1M, pH 5) and exposed on a phosphorimager plate for 36

hrs. To extract nicotinamide from DMEM media, 0.1 ml of media was collected and

completely dried in a vacuum centrifuge. The resin was incubated with 0.1 ml acetonitrile

for 15 m at room temperature, and the supernatant was collected, dried, and resuspended

in 10 ul H2O for loading onto TLC plates. This method was found to efficiently extract

nicotinamide with minimal transfer of salts. 14

C-Nicotinamide standard was prepared by

digestion of 14

C-NAD+ with purified ADP-Ribosyl Cyclase enzyme from Aplysia

californica (Sigma).

Axon degeneration measurement: For axotomy experiments, DRG neurons were

grown in 96-well spot cultures, in which cells are clustered to permit severing of axons

and imaging of distal axons (24). Axons were manually severed using a blade. NGF

deprivation was performed as described (24). For quantification of axon degeneration, six

brightfield images per well of axons were acquired from 96-well plates using an Operetta

High Content Image system (Perkin Elmer). These images were processed using ImageJ

software (NIH) with the “Degeneration Index” (DI) algorithm (13), which reports axon

degeneration as the fractional area fragmented axons. Higher DI values correspond to

more extensive axon degeneration. The mean DI value per well (=n) was used for

statistical comparisons and data summary.

Cell death quantification: DRG neuron cell viability was quantified using the

Ethidium Homodimer (EH) stain (botium). EH (20 uM) and nuclear stain Hoechst-33342

(Life Technologies) were added directly to cell culture media and fluorescence images

were acquired using the Operetta High Content Imaging System with Ex/Em=570/615

(EH) and Ex/Em=380/445 (Hoechst). Fluorescence + brightfield overlays were generated

using Matlab software and Ethidium Homodimer positive cells were manually counted

(50-100 / well) from randomized blinded images. The fraction of EH positive cells per

well (=n) was used for statistical comparisons and data summary.

HPLC: For HPLC analysis of adherent cells, cell culture vessels were placed on

ice and rinsed 2x with cold PBS and quenched with 1M perchloric acid (PCA) (100-200

ul). Frozen sciatic nerve samples were transferred into 1.5ml tube containing 0.2 ml of

ice cold 0.5M HClO4 and immediately dissociated by sonication. After 10 minutes on

ice, PCA supernatants were collected and spun (10,000xg 5 min) to remove cell debris

and the supernatants were neutralized by mixing with 1/3 volume 3M K2CO3.

Neutralized extracts were diluted in H2O (1:5) for HPLC loading. For protein

measurement, PCA precipitates were dissolved in 0.25 N NaOH and protein was

quantified using the BCA method for normalization of HPLC measurements. For

extracellular Nam measurement, 106 adherent HTir cells were rinsed 2x with warm PBS

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and maintained in 0.5 ml PBS with or without 100 nM rapamycin for 10 minutes. The

PBS was then collected, dried by vacuum, then processed with PCA/K2CO3 as above

and diluted with H2O to 0.5 ml for HPLC loading. Metabolite extracts were resolved on a

reverse phase Supelcosil LC-18-T column (Supelco) using a Shimadzu LC20AT HPLC

system using a 0.5M potassium phosphate : methanol gradient mobile phase (27).

Integrated absorbance was calculated from absorbance traces using retention time

windows defined by internal NAD+/ATP/Nam standards, which were run with each

experiment. Standard curves were used to quantify concentrations for each sample. These

protein-normalized metabolite values were further normalized to mean control values to

produce the normalized measurements reported in figures. For sciatic nerve studies,

NAD+ and ATP levels are reported in moles per gram tissue based on the mass of the

isolated nerve segment.

Fluorescence measurements: Images of live DRG neurons were acquired with the

Operetta high-content imaging system (Perkin Elmer) using the following filter settings:

1) Hoechst-33342 (Sigma) Ex/Em = 380/445; 2) Fluo-4 (Life Technologies) Ex/Em =

475/525; 3) TMRM (Life Technologies) Ex/Em = 570/615. Images were processed with

Matlab (Mathworks) and ImageJ (NIH). 50-100 cells per field were identified by

segmentation of the Hoechst channel and non-overlapping cell masks were defined by

dilating the nuclear masks constrained by neighboring cell masks. Per-cell fluorescence

intensity was calculated as the median pixel intensity from each cell mask, and the mean

of the per-cell fluorescence per well was used for quantification in figures.

Statistics and data presentation: Sample n was defined as the number of biological

specimens (cell culture wells, nerves, or cell suspensions) that were manipulated and

measured independently. Sample n was at least 4 for each experiment, and all

experiments were performed at least 3 times. One-way analysis of variance (ANOVA)

comparisons with Tukey multiple comparison adjustment were performed for multiple

groups and unpaired t-tests were used for individual comparisons. Figures show the

results of a single representative experiment. Unless otherwise noted, error bars represent

SEM. All data analyses were done with Matlab (Mathworks) and Spotfire (Tibco).

Supplementary Text

Sequences: LOCUS SARMpsC 2907 bp DNA linear 03-JUL-2014 FEATURES Location/Qualifiers misc_feature 1..1662 /note="SARM1 1-554" misc_feature 1663..1683 /note="TEV site" misc_feature 1684..2181 /note="SARM1 AA559-724" misc_feature 2191..2907 /note="Cerulean" ORIGIN 1 ATGGTCCTGA CGCTGCTTCT CTCCGCCTAC AAGCTGTGTC GCTTCTTCGC CATGTCGGGC 61 CCACGGCCGG GCGCCGAGCG GCTGGCGGTG CCTGGGCCAG ATGGGGGCGG TGGCACGGGC 121 CCATGGTGGG CTGCGGGTGG CCGCGGGCCC CGCGAAGTGT CGCCGGGGGC AGGCACCGAG 181 GTGCAGGACG CCCTGGAGCG CGCGCTGCCG GAGCTGCAGC AGGCCTTGTC CGCGCTGAAG 241 CAGGCGGGCG GCGCGCGGGC CGTGGGCGCC GGCCTGGCCG AGGTCTTCCA ACTGGTGGAG 301 GAGGCCTGGC TGCTGCCGGC CGTGGGCCGC GAGGTAGCCC AGGGTCTGTG CGACGCCATC 361 CGCCTCGATG GCGGCCTCGA CCTGCTGTTG CGGCTGCTGC AGGCGCCGGA GTTGGAGACG 421 CGTGTGCAGG CCGCGCGCCT GCTGGAGCAG ATCCTGGTGG CTGAGAACCG AGACCGCGTG 481 GCGCGCATTG GGCTGGGCGT GATCCTGAAC CTGGCGAAGG AACGCGAACC CGTAGAGCTG 541 GCGCGGAGCG TGGCAGGCAT CTTGGAGCAC ATGTTCAAGC ATTCGGAGGA GACATGCCAG 601 AGGCTGGTGG CGGCCGGCGG CCTGGACGCG GTGCTGTATT GGTGCCGCCG CACGGACCCC 661 GCGCTGCTGC GCCACTGCGC GCTGGCGCTG GGCAACTGCG CGCTGCACGG GGGCCAGGCG

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721 GTGCAGCGAC GCATGGTAGA GAAGCGCGCA GCCGAGTGGC TCTTCCCGCT CGCCTTCTCC 781 AAGGAGGACG AGCTGCTTCG GCTGCACGCC TGCCTCGCAG TAGCGGTGTT GGCGACTAAC 841 AAGGAGGTGG AGCGCGAGGT GGAGCGCTCG GGCACGCTGG CGCTCGTGGA GCCGCTTGTG 901 GCCTCGCTGG ACCCTGGCCG CTTCGCCCGC TGTCTGGTGG ACGCCAGCGA CACAAGCCAG 961 GGCCGCGGGC CCGACGACCT GCAGCGCCTC GTGCCGTTGC TCGACTCTAA CCGCTTGGAG 1021 GCGCAGTGCA TCGGGGCTTT CTACCTCTGC GCCGAGGCTG CCATCAAGAG CCTGCAAGGC 1081 AAGACCAAGG TGTTCAGCGA CATCGGCGCC ATCCAGAGCC TGAAACGCCT GGTTTCCTAC 1141 TCTACCAATG GCACTAAGTC GGCGCTGGCC AAGCGCGCGC TGCGCCTGCT GGGCGAGGAG 1201 GTGCCACGGC CCATCCTGCC CTCCGTGCCC AGCTGGAAGG AGGCCGAGGT TCAGACGTGG 1261 CTGCAGCAGA TCGGTTTCTC CAAGTACTGC GAGAGCTTCC GGGAGCAGCA GGTGGATGGC 1321 GACCTGCTTC TGCGGCTCAC GGAGGAGGAA CTCCAGACCG ACCTGGGCAT GAAATCGGGC 1381 ATCACCCGCA AGAGGTTCTT TAGGGAGCTC ACGGAGCTCA AGACCTTCGC CAACTATTCT 1441 ACGTGCGACC GCAGCAACCT GGCGGACTGG CTGGGCAGCC TGGACCCGCG CTTCCGCCAG 1501 TACACCTACG GCCTGGTCAG CTGCGGCCTG GACCGCTCCC TGCTGCACCG CGTGTCTGAG 1561 CAGCAGCTGC TGGAAGACTG CGGCATCCAC CTGGGCGTGC ACCGCGCCCG CATCCTCACG 1621 GCGGCCAGAG AAATGCTACA CTCCCCGCTG CCCTGTACTG GTGAGAATCT GTACTTTCAG 1681 TCAGGGGACA CTCCAGATGT CTTCATCAGC TACCGCCGGA ACTCAGGTTC CCAGCTGGCC 1741 AGTCTCCTGA AGGTGCACCT GCAGCTGCAT GGCTTCAGTG TCTTCATTGA TGTGGAGAAG 1801 CTGGAAGCAG GCAAGTTCGA GGACAAACTC ATCCAGAGTG TCATGGGTGC CCGCAACTTT 1861 GTGTTGGTGC TATCACCTGG AGCACTGGAC AAGTGCATGC AAGACCATGA CTGCAAGGAT 1921 TGGGTGCATA AGGAGATTGT GACTGCTTTA AGCTGCGGCA AGAACATTGT GCCCATCATT 1981 GATGGCTTCG AGTGGCCTGA GCCCCAGGTC CTGCCTGAGG ACATGCAGGC TGTGCTTACT 2041 TTCAACGGTA TCAAGTGGTC CCACGAATAC CAGGAGGCCA CCATTGAGAA GATCATCCGC 2101 TTCCTGCAGG GCCGCTCCTC CCGGGACTCA TCTGCAGGCT CTGACACCAG TTTGGAGGGT 2161 GCTGCACCCA TGGGTCCAAC Cgccacaacc ATGGTGAGCA AGGGCGAGGA GCTGTTCACC 2221 GGGGTGGTGC CCATCCTGGT CGAGCTGGAC GGCGACGTAA ACGGCCACAA GTTCAGCGTG 2281 TCCGGCGAGG GCGAGGGCGA TGCCACCTAC GGCAAGCTGA CCCTGAAGTT CATCTGCACC 2341 ACCGGCAAGC TGCCCGTGCC CTGGCCCACC CTCGTGACCA CCCTGACCTG GGGCGTGCAG 2401 TGCTTCGCCC GCTACCCCGA CCACATGAAG CAGCACGACT TCTTCAAGTC CGCCATGCCC 2461 GAAGGCTACG TCCAGGAGCG CACCATCTTC TTCAAGGACG ACGGCAACTA CAAGACCCGC 2521 GCCGAGGTGA AGTTCGAGGG CGACACCCTG GTGAACCGCA TCGAGCTGAA GGGCATCGAC 2581 TTCAAGGAGG ACGGCAACAT CCTGGGGCAC AAGCTGGAGT ACAACGCCAT CAGCGACAAC 2641 GTCTATATCA CCGCCGACAA GCAGAAGAAC GGCATCAAGG CCAACTTCAA GATCCGCCAC 2701 AACATCGAGG ACGGCAGCGT GCAGCTCGCC GACCACTACC AGCAGAACAC CCCCATCGGC 2761 GACGGCCCCG TGCTGCTGCC CGACAACCAC TACCTGAGCA CCCAGTCCAA GCTGAGCAAA 2821 GACCCCAACG AGAAGCGCGA TCACATGGTC CTGCTGGAGT TCGTGACCGC CGCCGGGATC 2881 ACTCTCGGCA TGGACGAGCT GTACAAG // LOCUS SARMpsFrbNtevC 3597 bp DNA linear 03-JUL-2014 FEATURES Location/Qualifiers misc_feature 1..1662 /note="SARM1 1-554" misc_feature 1663..1683 /note="TEV site" misc_feature 1684..2181 /note="SARM1 AA559-724" misc_feature 2194..2463 /note="FRB" misc_feature 2485..2514 /note="Linker" misc_feature 2881..3597 /note="Cerulean" misc_feature 2515..2868 /note="TevN" ORIGIN 1 ATGGTCCTGA CGCTGCTTCT CTCCGCCTAC AAGCTGTGTC GCTTCTTCGC CATGTCGGGC 61 CCACGGCCGG GCGCCGAGCG GCTGGCGGTG CCTGGGCCAG ATGGGGGCGG TGGCACGGGC 121 CCATGGTGGG CTGCGGGTGG CCGCGGGCCC CGCGAAGTGT CGCCGGGGGC AGGCACCGAG 181 GTGCAGGACG CCCTGGAGCG CGCGCTGCCG GAGCTGCAGC AGGCCTTGTC CGCGCTGAAG 241 CAGGCGGGCG GCGCGCGGGC CGTGGGCGCC GGCCTGGCCG AGGTCTTCCA ACTGGTGGAG 301 GAGGCCTGGC TGCTGCCGGC CGTGGGCCGC GAGGTAGCCC AGGGTCTGTG CGACGCCATC 361 CGCCTCGATG GCGGCCTCGA CCTGCTGTTG CGGCTGCTGC AGGCGCCGGA GTTGGAGACG 421 CGTGTGCAGG CCGCGCGCCT GCTGGAGCAG ATCCTGGTGG CTGAGAACCG AGACCGCGTG 481 GCGCGCATTG GGCTGGGCGT GATCCTGAAC CTGGCGAAGG AACGCGAACC CGTAGAGCTG 541 GCGCGGAGCG TGGCAGGCAT CTTGGAGCAC ATGTTCAAGC ATTCGGAGGA GACATGCCAG 601 AGGCTGGTGG CGGCCGGCGG CCTGGACGCG GTGCTGTATT GGTGCCGCCG CACGGACCCC 661 GCGCTGCTGC GCCACTGCGC GCTGGCGCTG GGCAACTGCG CGCTGCACGG GGGCCAGGCG 721 GTGCAGCGAC GCATGGTAGA GAAGCGCGCA GCCGAGTGGC TCTTCCCGCT CGCCTTCTCC 781 AAGGAGGACG AGCTGCTTCG GCTGCACGCC TGCCTCGCAG TAGCGGTGTT GGCGACTAAC 841 AAGGAGGTGG AGCGCGAGGT GGAGCGCTCG GGCACGCTGG CGCTCGTGGA GCCGCTTGTG 901 GCCTCGCTGG ACCCTGGCCG CTTCGCCCGC TGTCTGGTGG ACGCCAGCGA CACAAGCCAG 961 GGCCGCGGGC CCGACGACCT GCAGCGCCTC GTGCCGTTGC TCGACTCTAA CCGCTTGGAG 1021 GCGCAGTGCA TCGGGGCTTT CTACCTCTGC GCCGAGGCTG CCATCAAGAG CCTGCAAGGC 1081 AAGACCAAGG TGTTCAGCGA CATCGGCGCC ATCCAGAGCC TGAAACGCCT GGTTTCCTAC 1141 TCTACCAATG GCACTAAGTC GGCGCTGGCC AAGCGCGCGC TGCGCCTGCT GGGCGAGGAG 1201 GTGCCACGGC CCATCCTGCC CTCCGTGCCC AGCTGGAAGG AGGCCGAGGT TCAGACGTGG 1261 CTGCAGCAGA TCGGTTTCTC CAAGTACTGC GAGAGCTTCC GGGAGCAGCA GGTGGATGGC 1321 GACCTGCTTC TGCGGCTCAC GGAGGAGGAA CTCCAGACCG ACCTGGGCAT GAAATCGGGC 1381 ATCACCCGCA AGAGGTTCTT TAGGGAGCTC ACGGAGCTCA AGACCTTCGC CAACTATTCT 1441 ACGTGCGACC GCAGCAACCT GGCGGACTGG CTGGGCAGCC TGGACCCGCG CTTCCGCCAG

8

1501 TACACCTACG GCCTGGTCAG CTGCGGCCTG GACCGCTCCC TGCTGCACCG CGTGTCTGAG 1561 CAGCAGCTGC TGGAAGACTG CGGCATCCAC CTGGGCGTGC ACCGCGCCCG CATCCTCACG 1621 GCGGCCAGAG AAATGCTACA CTCCCCGCTG CCCTGTACTG GTGAGAATCT GTACTTTCAG 1681 TCAGGGGACA CTCCAGATGT CTTCATCAGC TACCGCCGGA ACTCAGGTTC CCAGCTGGCC 1741 AGTCTCCTGA AGGTGCACCT GCAGCTGCAT GGCTTCAGTG TCTTCATTGA TGTGGAGAAG 1801 CTGGAAGCAG GCAAGTTCGA GGACAAACTC ATCCAGAGTG TCATGGGTGC CCGCAACTTT 1861 GTGTTGGTGC TATCACCTGG AGCACTGGAC AAGTGCATGC AAGACCATGA CTGCAAGGAT 1921 TGGGTGCATA AGGAGATTGT GACTGCTTTA AGCTGCGGCA AGAACATTGT GCCCATCATT 1981 GATGGCTTCG AGTGGCCTGA GCCCCAGGTC CTGCCTGAGG ACATGCAGGC TGTGCTTACT 2041 TTCAACGGTA TCAAGTGGTC CCACGAATAC CAGGAGGCCA CCATTGAGAA GATCATCCGC 2101 TTCCTGCAGG GCCGCTCCTC CCGGGACTCA TCTGCAGGCT CTGACACCAG TTTGGAGGGT 2161 GCTGCACCCA TGGGTCCAAC CGCCACAACC ATGGAGATGT GGCATGAAGG CCTGGAAGAG 2221 GCATCTCGTT TGTACTTTGG GGAAAGGAAC GTGAAAGGCA TGTTTGAGGT GCTGGAGCCC 2281 TTGCATGCTA TGATGGAACG GGGCCCCCAG ACTCTGAAGG AAACATCCTT TAATCAGGCC 2341 TATGGTCGAG ATTTAATGGA GGCCCAAGAG TGGTGCAGGA AGTACATGAA ATCAGGGAAT 2401 GTCAAGGACC TCACCCAAGC CTGGGACCTC TATTATCATG TGTTCCGACG AATCTCAAAG 2461 CAGCAGATCT CGTACGCGTC CCGGGGCGGT GGCTCATCTG GCGGAGGTGC TAGCATGGAA 2521 AGCTTGTTTA AGGGGCCGCG TGATTACAAC CCGATATCGA GCACCATTTG TCATTTGACG 2581 AATGAATCTG ATGGGCACAC AACATCGTTG TATGGTATTG GATTTGGTCC CTTCATCATT 2641 ACAAACAAGC ACTTGTTTAG AAGAAATAAT GGAACACTGT TGGTCCAATC ACTACATGGT 2701 GTATTCAAGG TCAAGAACAC CACGACTTTG CAACAACACC TCATTGATGG GAGGGACATG 2761 ATAATTATTC GCATGCCTAA GGATTTCCCA CCATTTCCTC AAAAGCTGAA ATTTAGAGAG 2821 CCACAAAGGG AAGAGCGCAT ATGTCTTGTG ACAACCAACT TCCAAACTAC CGGTTCAACC 2881 ATGGTGAGCA AGGGCGAGGA GCTGTTCACC GGGGTGGTGC CCATCCTGGT CGAGCTGGAC 2941 GGCGACGTAA ACGGCCACAA GTTCAGCGTG TCCGGCGAGG GCGAGGGCGA TGCCACCTAC 3001 GGCAAGCTGA CCCTGAAGTT CATCTGCACC ACCGGCAAGC TGCCCGTGCC CTGGCCCACC 3061 CTCGTGACCA CCCTGACCTG GGGCGTGCAG TGCTTCGCCC GCTACCCCGA CCACATGAAG 3121 CAGCACGACT TCTTCAAGTC CGCCATGCCC GAAGGCTACG TCCAGGAGCG CACCATCTTC 3181 TTCAAGGACG ACGGCAACTA CAAGACCCGC GCCGAGGTGA AGTTCGAGGG CGACACCCTG 3241 GTGAACCGCA TCGAGCTGAA GGGCATCGAC TTCAAGGAGG ACGGCAACAT CCTGGGGCAC 3301 AAGCTGGAGT ACAACGCCAT CAGCGACAAC GTCTATATCA CCGCCGACAA GCAGAAGAAC 3361 GGCATCAAGG CCAACTTCAA GATCCGCCAC AACATCGAGG ACGGCAGCGT GCAGCTCGCC 3421 GACCACTACC AGCAGAACAC CCCCATCGGC GACGGCCCCG TGCTGCTGCC CGACAACCAC 3481 TACCTGAGCA CCCAGTCCAA GCTGAGCAAA GACCCCAACG AGAAGCGCGA TCACATGGTC 3541 CTGCTGGAGT TCGTGACCGC CGCCGGGATC ACTCTCGGCA TGGACGAGCT GTACAAG // LOCUS Frb-Ntev 681 bp DNA linear 03-JUL-2014 FEATURES Location/Qualifiers misc_feature 1..273 /note="FRB" misc_feature 274..318 /note="Linker" misc_feature 325..678 /note="NTev" ORIGIN 1 atgGAGATGT GGCATGAAGG CCTGGAAGAG GCATCTCGTT TGTACTTTGG GGAAAGGAAC 61 GTGAAAGGCA TGTTTGAGGT GCTGGAGCCC TTGCATGCTA TGATGGAACG GGGCCCCCAG 121 ACTCTGAAGG AAACATCCTT TAATCAGGCC TATGGTCGAG ATTTAATGGA GGCCCAAGAG 181 TGGTGCAGGA AGTACATGAA ATCAGGGAAT GTCAAGGACC TCACCCAAGC CTGGGACCTC 241 TATTATCATG TGTTCCGACG AATCTCAAAG CAGCAGATCT CGTACGCGTC CCGGGGCGGT 301 GGCTCATCTG GCGGAGGTgc tagcATGGAA AGCTTGTTTA AGGGGCCGCG TGATTACAAC 361 CCGATATCGA GCACCATTTG TCATTTGACG AATGAATCTG ATGGGCACAC AACATCGTTG 421 TATGGTATTG GATTTGGTCC CTTCATCATT ACAAACAAGC ACTTGTTTAG AAGAAATAAT 481 GGAACACTGT TGGTCCAATC ACTACATGGT GTATTCAAGG TCAAGAACAC CACGACTTTG 541 CAACAACACC TCATTGATGG GAGGGACATG ATAATTATTC GCATGCCTAA GGATTTCCCA 601 CCATTTCCTC AAAAGCTGAA ATTTAGAGAG CCACAAAGGG AAGAGCGCAT ATGTCTTGTG 661 ACAACCAACT TCCAAACTTA A // LOCUS Fkbp-Ctev 738 bp DNA linear 03-JUL-2014 FEATURES Location/Qualifiers misc_feature 1..321 /note="Fkbp1a" misc_feature 337..360 /note="Linker" misc_feature 367..735 /note="CTev" ORIGIN 1 GGAGTGCAGG TGGAAACCAT CTCCCCAGGA GACGGGCGCA CCTTCCCCAA GCGCGGCCAG 61 ACCTGCGTGG TGCACTACAC CGGGATGCTT GAAGATGGAA AGAAATTTGA TTCCTCCCGG 121 GACAGAAACA AGCCCTTTAA GTTTATGCTA GGCAAGCAGG AGGTGATCCG AGGCTGGGAA 181 GAAGGGGTTG CCCAGATGAG TGTGGGTCAG AGAGCCAAAC TGACTATATC TCCAGATTAT 241 GCCTATGGTG CCACTGGGCA CCCAGGCATC ATCCCACCAC ATGCCACTCT CGTCTTCGAT 301 GTGGAGCTTC TAAAACTGGA AtcGTACGCG TCCCGGGGCG GTGGCTCATC TGGCGGAGGT 361 gctagcAAGA GCATGTCTAG CATGGTGTCA GACACTAGTT GCACATTCCC TTCATCTGAT 421 GGCATATTCT GGAAGCATTG GATTCAAACC AAGGATGGGC AGTGTGGCAG TCCATTAGTA 481 TCAACTAGAG ATGGGTTCAT TGTTGGTATA CACTCAGCAT CGAATTTCAC CAACACAAAC 541 AATTATTTCA CAAGCGTGCC GAAAAACTTC ATGGAATTGT TGACAAATCA GGAGGCGCAG 601 CAGTGGGTTA GTGGTTGGCG ATTAAATGCT GACTCAGTAT TGTGGGGGGG CCATAAAGTT 661 TTCATGGTGA AACCTGAAGA GCCTTTTCAG CCAGTTAAGG AAGCGACTCA ACTCATGAAT

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721 CGTCGTCGCC GTCGCTAA // LOCUS Frb-sTIR-3xF 885 bp DNA linear 01-JUL-2014 FEATURES Location/Qualifiers misc_feature 1..273 /note="FRB" misc_feature 274..318 /note="Linker" misc_feature 319..738 /note="TIR" misc_feature 811..885 /note="Flag" source 1..885 /dnas_title="Frb-sTIR-3xF" misc_feature 319..807 /note="SARM1 561-724" ORIGIN 1 atgGAGATGT GGCATGAAGG CCTGGAAGAG GCATCTCGTT TGTACTTTGG GGAAAGGAAC 61 GTGAAAGGCA TGTTTGAGGT GCTGGAGCCC TTGCATGCTA TGATGGAACG GGGCCCCCAG 121 ACTCTGAAGG AAACATCCTT TAATCAGGCC TATGGTCGAG ATTTAATGGA GGCCCAAGAG 181 TGGTGCAGGA AGTACATGAA ATCAGGGAAT GTCAAGGACC TCACCCAAGC CTGGGACCTC 241 TATTATCATG TGTTCCGACG AATCTCAAAG CAGCAGATCT CGTACGCGTC CCGGGGCGGT 301 GGCTCATCTG GCGGAGGTac tccagatgtc ttcatcagct accgccggaa ctcaggttcc 361 cagctggcca gtctcctgaa ggtgcacctg cagctgcatg gcttcagtgt cttcattgat 421 gtggagaagc tggaagcagg caagttcgag gacaaactca tccagagtgt catgggtgcc 481 cgcaactttg tgttggtgct atcacctgga gcactggaca agtgcatgca agaccatgac 541 tgcaaggatt gggtgcataa ggagattgtg actgctttaa gctgcggcaa gaacattgtg 601 cccatcattg atggcttcga gtggcctgag ccccaggtcc tgcctgagga catgcaggct 661 gtgcttactt tcaacggtat caagtggtcc cacgaatacc aggaggccac cattgagaag 721 atcatccgct tcctgcaggg ccgctcctcc cgggactcat ctgcaggctc tgacaccagt 781 ttggagggtg ctgcACCCAT GGGTCCACCG GACTATAAAG ATGATGATGA TAAGGACTAT 841 AAAGATGATG ATGATAAGGA CTATAAAGAT GATGATGATA AGTAA // LOCUS Fkbp-sTIR-GFP 1584 bp DNA linear 01-JUL-2014 FEATURES Location/Qualifiers misc_feature 364..783 /note="TIR" misc_feature 4..324 /note="Fkbp1a" misc_feature 865..1581 /note="GFP" misc_feature 364..855 /note="SARM1 AA561-724" misc_feature 340..363 /note="Linker" ORIGIN 1 atgGGAGTGC AGGTGGAAAC CATCTCCCCA GGAGACGGGC GCACCTTCCC CAAGCGCGGC 61 CAGACCTGCG TGGTGCACTA CACCGGGATG CTTGAAGATG GAAAGAAATT TGATTCCTCC 121 CGGGACAGAA ACAAGCCCTT TAAGTTTATG CTAGGCAAGC AGGAGGTGAT CCGAGGCTGG 181 GAAGAAGGGG TTGCCCAGAT GAGTGTGGGT CAGAGAGCCA AACTGACTAT ATCTCCAGAT 241 TATGCCTATG GTGCCACTGG GCACCCAGGC ATCATCCCAC CACATGCCAC TCTCGTCTTC 301 GATGTGGAGC TTCTAAAACT GGAAtcGTAC GCGTCCCGGG GCGGTGGCTC ATCTGGCGGA 361 GGTactccag atgtcttcat cagctaccgc cggaactcag gttcccagct ggccagtctc 421 ctgaaggtgc acctgcagct gcatggcttc agtgtcttca ttgatgtgga gaagctggaa 481 gcaggcaagt tcgaggacaa actcatccag agtgtcatgg gtgcccgcaa ctttgtgttg 541 gtgctatcac ctggagcact ggacaagtgc atgcaagacc atgactgcaa ggattgggtg 601 cataaggaga ttgtgactgc tttaagctgc ggcaagaaca ttgtgcccat cattgatggc 661 ttcgagtggc ctgagcccca ggtcctgcct gaggacatgc aggctgtgct tactttcaac 721 ggtatcaagt ggtcccacga ataccaggag gccaccattg agaagatcat ccgcttcctg 781 cagggccgct cctcccggga ctcatctgca ggctctgaca ccagtttgga gggtgctgca 841 cccatgggtc caaccgccac aaccATGGTG AGCAAGGGCG AGGAGCTGTT CACCGGGGTG 901 GTGCCCATCC TGGTCGAGCT GGACGGCGAC GTAAACGGCC ACAAGTTCAG CGTGTCCGGC 961 GAGGGCGAGG GCGATGCCAC CTACGGCAAG CTGACCCTGA AGTTCATCTG CACCACCGGC 1021 AAGCTGCCCG TGCCCTGGCC CACCCTCGTG ACCACCCTGA CCTACGGCGT GCAGTGCTTC 1081 AGCCGCTACC CCGACCACAT GAAGCAGCAC GACTTCTTCA AGTCCGCCAT GCCCGAAGGC 1141 TACGTCCAGG AGCGCACCAT CTTCTTCAAG GACGACGGCA ACTACAAGAC CCGCGCCGAG 1201 GTGAAGTTCG AGGGCGACAC CCTGGTGAAC CGCATCGAGC TGAAGGGCAT CGACTTCAAG 1261 GAGGACGGCA ACATCCTGGG GCACAAGCTG GAGTACAACT ACAACAGCCA CAACGTCTAT 1321 ATCATGGCCG ACAAGCAGAA GAACGGCATC AAGGTGAACT TCAAGATCCG CCACAACATC 1381 GAGGACGGCA GCGTGCAGCT CGCCGACCAC TACCAGCAGA ACACCCCCAT CGGCGACGGC 1441 CCCGTGCTGC TGCCCGACAA CCACTACCTG AGCACCCAGT CCGCCCTGAG CAAAGACCCC 1501 AACGAGAAGC GCGATCACAT GGTCCTGCTG GAGTTCGTGA CCGCCGCCGG GATCACTCTC 1561 GGCATGGACG AGCTGTACAA Gtaa // LOCUS Fkbp_F36V-TLR4TIR 1590 bp DNA linear 03-JUL-2014 FEATURES Location/Qualifiers misc_feature 1..333 /label=Fkbp(F36V)

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misc_feature 334..372 /note="Linker" misc_feature 373..792 /note="sTIR" misc_feature 874..1590 /note="Cerulean" misc_feature 373..864 /note="SARM1 AA561-724" ORIGIN 1 atggcttcta gaggagtgca ggtggagact atctccccag gagacgggcg caccttcccc 61 aagcgcggcc agacctgcgt ggtgcactac accgggatgc ttgaagatgg aaagaaagtt 121 gattcctccc gggacagaaa caagcccttt aagtttatgc taggcaagca ggaggtgatc 181 cgaggctggg aagaaggggt tgcccagatg agtgtgggtc agagagccaa actgactata 241 tctccagatt atgcctatgg tgctactggg cacccaggca tcatcccacc acatgccact 301 ctcgtcttcg atgtggagct tctaaaactg gaaTCGTACG CGTCCCGGGG CGGTGGCTCA 361 TCTGGCGGAG GTactccaga tgtcttcatc agctaccgcc ggaactcagg ttcccagctg 421 gccagtctcc tgaaggtgca cctgcagctg catggcttca gtgtcttcat tgatgtggag 481 aagctggaag caggcaagtt cgaggacaaa ctcatccaga gtgtcatggg tgcccgcaac 541 tttgtgttgg tgctatcacc tggagcactg gacaagtgca tgcaagacca tgactgcaag 601 gattgggtgc ataaggagat tgtgactgct ttaagctgcg gcaagaacat tgtgcccatc 661 attgatggct tcgagtggcc tgagccccag gtcctgcctg aggacatgca ggctgtgctt 721 actttcaacg gtatcaagtg gtcccacgaa taccaggagg ccaccattga gaagatcatc 781 cgcttcctgc agggccgctc ctcccgggac tcatctgcag gctctgacac cagtttggag 841 ggtgctgcac ccatgggtcc aaccgccaca accATGGTGA GCAAGGGCGA GGAGCTGTTC 901 ACCGGGGTGG TGCCCATCCT GGTCGAGCTG GACGGCGACG TAAACGGCCA CAAGTTCAGC 961 GTGTCCGGCG AGGGCGAGGG CGATGCCACC TACGGCAAGC TGACCCTGAA GTTCATCTGC 1021 ACCACCGGCA AGCTGCCCGT GCCCTGGCCC ACCCTCGTGA CCACCCTGAC CTGGGGCGTG 1081 CAGTGCTTCG CCCGCTACCC CGACCACATG AAGCAGCACG ACTTCTTCAA GTCCGCCATG 1141 CCCGAAGGCT ACGTCCAGGA GCGCACCATC TTCTTCAAGG ACGACGGCAA CTACAAGACC 1201 CGCGCCGAGG TGAAGTTCGA GGGCGACACC CTGGTGAACC GCATCGAGCT GAAGGGCATC 1261 GACTTCAAGG AGGACGGCAA CATCCTGGGG CACAAGCTGG AGTACAACGC CATCAGCGAC 1321 AACGTCTATA TCACCGCCGA CAAGCAGAAG AACGGCATCA AGGCCAACTT CAAGATCCGC 1381 CACAACATCG AGGACGGCAG CGTGCAGCTC GCCGACCACT ACCAGCAGAA CACCCCCATC 1441 GGCGACGGCC CCGTGCTGCT GCCCGACAAC CACTACCTGA GCACCCAGTC CAAGCTGAGC 1501 AAAGACCCCA ACGAGAAGCG CGATCACATG GTCCTGCTGG AGTTCGTGAC CGCCGCCGGG 1561 ATCACTCTCG GCATGGACGA GCTGTACAAG // LOCUS Fkbp_F36V-TLR4TIR 1644 bp DNA linear 03-JUL-2014 FEATURES Location/Qualifiers misc_feature 871..918 /note="2xFlag" misc_feature 379..792 /note="TIR domain" misc_feature 379..870 /note="TLR4" misc_feature 928..1644 /note="Cerulean" misc_feature 1..333 /label=Fkbp(F36V) misc_feature 334..378 /note="Linker" ORIGIN 1 atggcttcta gaggagtgca ggtggagact atctccccag gagacgggcg caccttcccc 61 aagcgcggcc agacctgcgt ggtgcactac accgggatgc ttgaagatgg aaagaaagtt 121 gattcctccc gggacagaaa caagcccttt aagtttatgc taggcaagca ggaggtgatc 181 cgaggctggg aagaaggggt tgcccagatg agtgtgggtc agagagccaa actgactata 241 tctccagatt atgcctatgg tgctactggg cacccaggca tcatcccacc acatgccact 301 ctcgtcttcg atgtggagct tctaaaactg gaaTCGTACG CGTCCCGGGG CGGTGGCTCA 361 TCTGGCGGAG GTGCTAGCGC CTTTGTTATC TACTCAAGCC AGGATGAGGA CTGGGTAAGG 421 AATGAGCTAG TAAAGAATTT AGAAGAAGGG GTGCCTCCAT TTCAGCTCTG CCTTCACTAC 481 AGAGACTTTA TTCCCGGTGT GGCCATTGCT GCCAACATCA TCCATGAAGG TTTCCATAAA 541 AGCCGAAAGG TGATTGTTGT GGTGTCCCAG CACTTCATCC AGAGCCGCTG GTGTATCTTT 601 GAATATGAGA TTGCTCAGAC CTGGCAGTTT CTGAGCAGTC GTGCTGGTAT CATCTTCATT 661 GTCCTGCAGA AGGTGGAGAA GACCCTGCTC AGGCAGCAGG TGGAGCTGTA CCGCCTTCTC 721 AGCAGGAACA CTTACCTGGA GTGGGAGGAC AGTGTCCTGG GGCGGCACAT CTTCTGGAGA 781 CGACTCAGAA AAGCCCTGCT GGATGGTAAA TCATGGAATC CAGAAGGAAC AGTGGGTACA 841 GGATGCAATT GGCAGGAAGC AACATCTATC GACTATAAAG AcGATGAcGA TAAGGAtTAc 901 AAgGATGAcG AcGAcAAagc cacaaccATG GTGAGCAAGG GCGAGGAGCT GTTCACCGGG 961 GTGGTGCCCA TCCTGGTCGA GCTGGACGGC GACGTAAACG GCCACAAGTT CAGCGTGTCC 1021 GGCGAGGGCG AGGGCGATGC CACCTACGGC AAGCTGACCC TGAAGTTCAT CTGCACCACC 1081 GGCAAGCTGC CCGTGCCCTG GCCCACCCTC GTGACCACCC TGACCTGGGG CGTGCAGTGC 1141 TTCGCCCGCT ACCCCGACCA CATGAAGCAG CACGACTTCT TCAAGTCCGC CATGCCCGAA 1201 GGCTACGTCC AGGAGCGCAC CATCTTCTTC AAGGACGACG GCAACTACAA GACCCGCGCC 1261 GAGGTGAAGT TCGAGGGCGA CACCCTGGTG AACCGCATCG AGCTGAAGGG CATCGACTTC 1321 AAGGAGGACG GCAACATCCT GGGGCACAAG CTGGAGTACA ACGCCATCAG CGACAACGTC 1381 TATATCACCG CCGACAAGCA GAAGAACGGC ATCAAGGCCA ACTTCAAGAT CCGCCACAAC 1441 ATCGAGGACG GCAGCGTGCA GCTCGCCGAC CACTACCAGC AGAACACCCC CATCGGCGAC 1501 GGCCCCGTGC TGCTGCCCGA CAACCACTAC CTGAGCACCC AGTCCAAGCT GAGCAAAGAC 1561 CCCAACGAGA AGCGCGATCA CATGGTCCTG CTGGAGTTCG TGACCGCCGC CGGGATCACT 1621 CTCGGCATGG ACGAGCTGTA CAAG

11

// LOCUS Fkbp_F36V-MYD88T 1551 bp DNA linear 03-JUL-2014 FEATURES Location/Qualifiers misc_feature 835..1551 /note="Cerulean" misc_feature 1..333 /label=Fkbp(F36V) misc_feature 334..378 /note="Linker" misc_feature 814..834 /note="Linker" misc_feature 379..813 /note="Myd88" ORIGIN 1 atggcttcta gaggagtgca ggtggagact atctccccag gagacgggcg caccttcccc 61 aagcgcggcc agacctgcgt ggtgcactac accgggatgc ttgaagatgg aaagaaagtt 121 gattcctccc gggacagaaa caagcccttt aagtttatgc taggcaagca ggaggtgatc 181 cgaggctggg aagaaggggt tgcccagatg agtgtgggtc agagagccaa actgactata 241 tctccagatt atgcctatgg tgctactggg cacccaggca tcatcccacc acatgccact 301 ctcgtcttcg atgtggagct tctaaaactg gaaTCGTACG CGTCCCGGGG CGGTGGCTCA 361 TCTGGCGGAG GTGCTAGCCG TTTCGATGCC TTCATCTGct aTTGCCCCAG CGACATCCAG 421 TTTGTGCAGG AGATGATCCG GCAACTGGAA CAGACAAACT ATCGACTGAA GTTGTGTGTG 481 TCTGACCGCG ATGTCCTGCC TGGCACCTGT GTCTGGTCTA TTGCTAGTGA GCTCATCGAA 541 AAGAGGTTGG CTAGAAGGCC ACGGGGTGGG TGCCGCCGGA TGGTGGTGGT TGTCTCTGAT 601 GATTACCTGC AGAGCAAGGA ATGTGACTTC CAGACCAAAT TTGCACTCAG CCTCTCTCCA 661 GGTGCCCATC AGAAGCGACT GATCCCCATC AAGTACAAGG CAATGAAGAA AGAGTTCCCC 721 AGCATCCTGA GGTTCATCAC TGTCTGCGAC TACACCAACC CCTGCACCAA ATCTTGGTTC 781 TGGACTCGCC TTGCCAAGGC CTTGTCCCTG CCCggaGGCT CATCTGGAGG aaccATGGTG 841 AGCAAGGGCG AGGAGCTGTT CACCGGGGTG GTGCCCATCC TGGTCGAGCT GGACGGCGAC 901 GTAAACGGCC ACAAGTTCAG CGTGTCCGGC GAGGGCGAGG GCGATGCCAC CTACGGCAAG 961 CTGACCCTGA AGTTCATCTG CACCACCGGC AAGCTGCCCG TGCCCTGGCC CACCCTCGTG 1021 ACCACCCTGA CCTGGGGCGT GCAGTGCTTC GCCCGCTACC CCGACCACAT GAAGCAGCAC 1081 GACTTCTTCA AGTCCGCCAT GCCCGAAGGC TACGTCCAGG AGCGCACCAT CTTCTTCAAG 1141 GACGACGGCA ACTACAAGAC CCGCGCCGAG GTGAAGTTCG AGGGCGACAC CCTGGTGAAC 1201 CGCATCGAGC TGAAGGGCAT CGACTTCAAG GAGGACGGCA ACATCCTGGG GCACAAGCTG 1261 GAGTACAACG CCATCAGCGA CAACGTCTAT ATCACCGCCG ACAAGCAGAA GAACGGCATC 1321 AAGGCCAACT TCAAGATCCG CCACAACATC GAGGACGGCA GCGTGCAGCT CGCCGACCAC 1381 TACCAGCAGA ACACCCCCAT CGGCGACGGC CCCGTGCTGC TGCCCGACAA CCACTACCTG 1441 AGCACCCAGT CCAAGCTGAG CAAAGACCCC AACGAGAAGC GCGATCACAT GGTCCTGCTG 1501 GAGTTCGTGA CCGCCGCCGG GATCACTCTC GGCATGGACG AGCTGTACAA G // LOCUS FKBP_F36V-Tnkp-Cerulean 1815 bp DNA linear 08-OCT-2014 FEATURES Location/Qualifiers misc_feature 325..369 /note="Linker" misc_feature 370..1089 /note="Tnks1 parp domain" misc_feature 4..324 /label=FkbpF36V misc_feature 1099..1815 /note="Cerulean fluorescent protein" source 1..1815 /dnas_title="REF FhTnksCer" ORIGIN 1 atgggagtgc aggtggagac tatctcccca ggagacgggc gcaccttccc caagcgcggc 61 cagacctgcg tggtgcacta caccgggatg cttgaagatg gaaagaaagt tgattcctcc 121 cgggacagaa acaagccctt taagtttatg ctaggcaagc aggaggtgat ccgaggctgg 181 gaagaagggg ttgcccagat gagtgtgggt cagagagcca aactgactat atctccagat 241 tatgcctatg gtgctactgg gcacccaggc atcatcccac cacatgccac tctcgtcttc 301 gatgtggagc ttctaaaact ggaaTCGTAC GCGTCCCGGG GCGGTGGCTC ATCTGGCGGA 361 GGTGCTAGCG GTGGACAACA AGGCACCAAT CCTTATTTGA CTTTTCACTG TGTTAATCAG 421 GGAACGATTT TGCTGGATCT TGCTCCAGAA GATAAAGAAT ATCAGTCAGT GGAAGAAGAG 481 ATGCAAAGTA CTATTCGAGA ACACAGAGAT GGTGGTAATG CTGGCGGCAT CTTCAACAGA 541 TACAATGTCA TTCGAATcCA AAAAGTTGTC AACAAGAAGT TGAGGGAGCG GTTCTGCCAC 601 CGACAGAAGG AAGTGTCTGA GGAGAATCAC AACCATCACA ATGAGCGCAT GTTGTTTCAT 661 GGTTCTCCTT TCATTAATGC CATTATTaAT AAAGGGTTTG ATGAGCGACA TGCATACATA 721 GGAGGAATGT TTGGGGCCGG GATTTATTTT GCTGAAAACT CCTCAAAAAG CAACCAATAT 781 GTTTATGGAA TTGGAGGAGG AACAGGCTGC CCTACACACA AGGACAGGTC ATGCTATATA 841 TGTCACAGAC AAATGCTCTT CTGTAGAGTG ACCCTTGGGA AATCCTTTCT GCAGTTTAGC 901 ACtATGAAAA TGGCCCACGC GCCTCCAGGG CACCACTCAG TCATTGGTAG ACCGAGCGTC 961 AATGGGCTGG CATATGCTGA ATATGTCATC TACAGAGGAG AACAGGCATA CCCAGAGTAT 1021 CTTATCACTT ACCAGATCAT GAAGCCAGAA GCCCCTTCCC AGACCGCAAC AGCCGCAGAG 1081 CAGAAGACCg ccacaaccAT GGTGAGCAAG GGCGAGGAGC TGTTCACCGG GGTGGTGCCC 1141 ATCCTGGTCG AGCTGGACGG CGACGTAAAC GGCCACAAGT TCAGCGTGTC CGGCGAGGGC 1201 GAGGGCGATG CCACCTACGG CAAGCTGACC CTGAAGTTCA TCTGCACCAC CGGCAAGCTG 1261 CCCGTGCCCT GGCCCACCCT CGTGACCACC CTGACCTGGG GCGTGCAGTG CTTCGCCCGC 1321 TACCCCGACC ACATGAAGCA GCACGACTTC TTCAAGTCCG CCATGCCCGA AGGCTACGTC 1381 CAGGAGCGCA CCATCTTCTT CAAGGACGAC GGCAACTACA AGACCCGCGC CGAGGTGAAG 1441 TTCGAGGGCG ACACCCTGGT GAACCGCATC GAGCTGAAGG GCATCGACTT CAAGGAGGAC

12

1501 GGCAACATCC TGGGGCACAA GCTGGAGTAC AACGCCATCA GCGACAACGT CTATATCACC 1561 GCCGACAAGC AGAAGAACGG CATCAAGGCC AACTTCAAGA TCCGCCACAA CATCGAGGAC 1621 GGCAGCGTGC AGCTCGCCGA CCACTACCAG CAGAACACCC CCATCGGCGA CGGCCCCGTG 1681 CTGCTGCCCG ACAACCACTA CCTGAGCACC CAGTCCAAGC TGAGCAAAGA CCCCAACGAG 1741 AAGCGCGATC ACATGGTCCT GCTGGAGTTC GTGACCGCCG CCGGGATCAC TCTCGGCATG 1801 GACGAGCTGT ACAAG //

13

Fig. S1: Diagram of SARMps

. Full length human SARM1 (green) contains an N-terminus

/ armadillo repeat region (NTerm/Arm), two tandem SAM domains (SAMx2), and a TIR

domain. TEV protease consensus sequence ENLYFQS was inserted into human SARM1

between residues G554 and G559 which reside between the SAM and TIR domains. TEV

cleavage of this peptide occurs within the consensus sequence (arrowheads). The C-

terminus of SARMps

was fused to the rapamycin binding domain Frb, followed by a

flexible linker, N-terminal split TEV protease (N-Tev), and the GFP fluorescent protein

variant cerulean.

14

Fig. S2: SARM

ps inactivation by TEV protease. A) SARM

ps cleavage was evident in

DRG neurons after thirty minutes of split TEV complementation (rapamycin addition).

Micrographs show SARMps

and mitochondrial marker mitoDSRed localization in DRG

neurons. Full-length SARMps

colocalized with mitoDSRed due to N-terminal

mitochondrial association (6), but cleaved SARMps

redistributed to a diffuse cytosolic

pattern observed at 30 minutes after rapamycin treatment. Scale bar = 50 micrometers. B)

Wild-type neurons showed extensive axon degeneration in response to severing that was

unaffected by lentiviral TEV protease expression. shRNA targeting Sarm1 (sh-Sarm1)

suppressed axon degeneration and this suppression was lost upon co-expression of human

SARMps

(RNAi resistant). TEV abolished the function of SARMps

: coexpression of TEV

with SARMps

failed to restore injury-induced axon degeneration elicited by severing.

Phase-contrast images corresponding to bars 4 and 5 are shown (right panel). Error bars =

SEM; * p < 0.01; one-way analysis of variance (ANOVA) with Tukey’s post-hoc test.

Scale bars = 50 micrometers.

15

Figure S3: SAM-TIR induces axon loss in sensory neurons in vivo. The segmental

nerves of third instar larva are stained with anti-HRP antibody (red) and single sensory

axons are labeled by the expression of UAS-mCD8:GFP (green) due to the ppk-Gal4

driver. Sensory neurons expressing ppk-Gal4 express mCD8:GFP and either the UAS-

SAM-TIR or UAS-SAM-TIRmut

transgene. Axons from ppk expressing sensory neurons

were completely absent in animals expressing SAM-TIR, but not those expressing SAM-

TIRmut

. Nerves were scored for the absence or presence of GFP labeled axons (SAM-

TIR: 0 of 49 nerves; SAM-TIRmut

: 47 of 47; χ2=96; p<0.001); scale bar = 20

micrometers. Loss of sensory neuron cell bodies was also quantified: the two most

ventral cell bodies of ppk expressing neurons from each A2–A5 hemisegment were

scored for presence of GFP labeled cell bodies (GFP: 95 of 96; SAM-TIR: 0 of 96; SAM-

TIRmut

: 96 of 96 nerves; χ2=284; p<0.001).

16

17

Fig. S4: Timecourse measurements of neuronal stress and degeneration following sTIR

dimerization by AP20187. All cells expressed lentiviral FkbpF36V

-sTIR. A) Axon

degeneration was detectable within 1.5 hours following sTIR dimerization and continued

through 12 hours. B) Neuronal death measured by Ethidium Homodimer (EH) exclusion

was detectable within 6 hours following sTIR dimerization and continued through 24

hours. C-E) DRG neurons exhibited rapid loss of mitochondrial membrane potential and

increased intracellular calcium following sTIR dimerization. C) Images of DRG neurons

stained with TMRM (mitochondrial potential marker), Fluo-4 (intracellular calcium

marker), or Ethidium Homodimer (cell death marker) acquired at the indicated times

following sTIR dimerization (AP20187). Hoechst-33342 (blue) shows neuronal cell

bodies. Within 1.5 hrs, TMRM signal dissipated and Fluo-4 signal increased, while

Ethidium Homodimer appeared later; scale bar = 50 micrometers. D) Quantification of

mean Fluo-4 fluorescence normalized to untreated controls. E) Quantification of mean

TMRM fluorescence normalized to untreated controls. Error bars in A and B = SEM.

Error bars in D and E show standard deviations. * p < 0.01; one-way analysis of variance

(ANOVA) with Tukey’s post-hoc test.

18

Fig S5: ATP loss and morphologic changes in isolated axons and sciatic nerves. A) In

cultured sensory neurons, axonal ATP declined in a SARM1-dependent manner

following injury. B) Sciatic nerve ATP levels trended lower but were not statistically

different from baseline at 30 hours post-axotomy (wild-type n=5; t-test p=0.078). C)

Sciatic nerves were morphologically intact 30 hours after nerve transection. Sciatic

nerves distal to transected site from wild-type and Sarm1-/- animals were collected at

zero and 30 hours following nerve transection. Scale bar = 25 micrometers. Signs of

demyelination, macrophage invasion or axonal loss were not apparent after 30 hours

post-axotomy in wild-type or Sarm1-/- sciatic nerves. Error bars = SEM; * p < 0.01; one-

way analysis of variance (ANOVA) with Tukey’s post-hoc test.

19

Fig. S6: Effect of sTIR dimerization and NAD+ biosynthesis on HTir cell viability. A) In

HTir cells, sTIR dimerization induced toxicity quantified by ATP loss, which was

blocked by NR (1 mM) and increased by Nampt inhibition (FK = FK866 10 nM). B)

HTir cells showed morphologic changes following sTIR dimerization (12 hours; 100 nM

rapamycin), including retraction of processes and bleb formation. These changes were

blocked when NAD+ synthesis was augmented with 1 mM Nicotinamide Riboside (NR)

supplementation. Conversely, blocking NAD+ synthesis with FK866 (10 nM)

exacerbated morphologic changes induced by sTIR dimerization, while FK866 alone was

not toxic. Both NR and FK866 were added simultaneously with rapamycin. Scale bar =

50 micrometers. Error bars = SEM; * p < 0.01; one-way analysis of variance (ANOVA)

with Tukey’s post-hoc test.

20

Fig. S7: Effect of Tnkp dimerization on PAR formation, NAD loss, and energetic failure.

A) Tnkp dimerization induced Poly ADP-Ribose (PAR) formation. HEK293T cells

stably expressing FkbpF36V

-Tnkp and treated with AP20187 or 1 mM H2O2 (positive

control) showed PAR accumulation. Tankyrase inhibitor XAV939 (100 nM) blocked

Tnkp-induced PAR formation whereas NR (1 mM) supplementation did not. PAR signal

was observed primarily at molecular weights corresponding to PARP1 (116 kDa) or

FkbpF36V

-Tnkp-Cerulean (67 kDa), suggesting PAR attachment occured primarily on

peptides proximal to the activated PARP domains. B) In HEK293T cells stably

expressing FkbpF36V

-Tnkp, Tnkp dimerization by AP20187 induced NAD+ loss that was

more rapid than that induced by NAD+ synthesis inhibitor FK866 (10 nM). C) NAD+

depletion by Tnkp dimerization + FK866 induces ATP loss within 8 hours that was fully

blocked by Tankyrase inhibitor XAV939 or NR supplementation (1 mM); Error bars =

SEM; * p < 0.01; one-way analysis of variance (ANOVA) with Tukey’s post-hoc test.

21

Fig. S8: Nicotinamide (Nam) release from cells upon SARM1 TIR dimerization. A) Cells

were washed twice with warm PBS to remove Nam, and sTIR was dimerized with 100

nM rapamycin (rapa) for 10 minutes in PBS. Extracellular Nam quantified by HPLC was

increased following sTIR dimerization; * p < 0.01 unpaired t test. B) Representative

HPLC traces. Nam standard is shown (black; 5 uM). Following ten minutes rapamycin

treatment HPLC analysis of extracellular buffer showed a Nam peak (red) that was not

observed in the absence of rapamycin (blue).

22

Fig. S9: SARM1 TIR-induced axon degeneration, neuronal death, and NAD+ loss were

unaffected by genetic ablation of Parp1 or Cd38. A) DRG neurons cultured from

littermate Parp1-/-, Cd38-/-, and control mice (double heterozygous) showed similar axon

degeneration in response to SARM1 TIR dimerization (AP20187) measured at 12 hours.

B) Neuronal cell death evaluated by Ethidium Homodimer positivity was also observed

in all genotypes in response to SARM1 TIR dimerization; measured at 24 hours. C)

Mouse embryonic fibroblasts (MEFs) derived from wild type (control), Parp1-/-, and

Cd38-/- mice showed NAD+ depletion following 30 minutes sTIR dimerization with

AP20187. NAD+ was normalized by sample protein content and then to control (bar 1).

Error bars = SEM; * p < 0.01; one-way analysis of variance (ANOVA) with Tukey’s

post-hoc test.

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