DOI: 10.5061/dryad.x0k6djhvb
title: Data from: An Elongator mouse model of ALS spotlights TDP-43 in the motor neuron nucleolus

description: Dysfunction of the Elongator complex is associated with amyotrophic
 lateral sclerosis (ALS). To investigate the potential mechanisms via which
 Elongator contributes to ALS, we generated conditional knockout mouse
 models in which either Elp1 or Elp3 is selectively ablated in
 cholinergic neurons including alpha motor neurons of the spinal cord.
 These mice exhibit numerous ALS phenotypes including a progressive loss of
 motor strength, motor neuron degeneration, and denervation of the
 neuromuscular junction. To interrogate the molecular mechanisms that
 contribute to motor neuron cell death in this model, we examined multiple
 disease pathways including the expression of TDP-43, a protein whose
 mislocalization and aggregation are associated with both familial and
 sporadic forms of ALS. Surprisingly, we see robust expression of TDP-43 in
 the nucleolus of motor neurons from control mice and clearing of TDP-43
 from nucleoli in the conditional knockout. Further analysis of the
 nucleolar marker fibrillarin indicates that motor neurons in Elp
 conditional knockout mice exhibit nucleolar shrinkage, an indicator of
 nucleolar stress. Although the presence of TDP-43 in the nucleus is well
 characterized, a function for TDP-43 in the nucleolus, the nuclear
 subdomain where ribosome biogenesis takes place, has not been previously
 described. Thus, this study directly links dysfunction of the Elongator
 complex with TDP-43 clearing and nucleolar disruption, two hallmark
 cellular pathologies of ALS.  
description: Mice All experiments with animals were
 performed according to the National Institutes of Health Guide for Care
 and Use of Laboratory Animals and were approved by the Montana State
 University Institutional Animal Care and Use Committee.  Additionally, all
 experiments involving live mice were conducted according to ARRIVE
 guidelines 2.0 where applicable.   Sample sizes were based on pilot
 studies and sample sizes were calculated using a statistical power of
 80%.  <em>Ikbkaptm1a(KOMP)Wtsi</em>− ‘knockout first’ mice
 containing a frt-flanked <em>LacZ</em> <em>Elp1
 </em>(previously known as <em>Ikbkap</em>) reporter that
 disrupts <em>Elp1</em> expression before the
 <em>LoxP</em> flanked 4th exon were obtained from the
 International Knockout Mouse Consortium.  This strain was generated on a
 C57Bl/6j background and has been previously described
 <sup>26</sup>. <em>Elp1 </em>CKO mice
 (<em>Chat-Cre; Elp1<sup>LoxP/LoxP</sup></em>) were
 generated by crosses between <em>Chat-Cre; Elp1<sup>+/LoxP
 </sup></em>X<em> Elp1<sup>LoxP/LoxP</sup>;
 Chat-GFP/GFP </em>mice.  A humane endpoint for <em>Elp1
 </em>CKO males was defined by a mass &lt; 55%
 of their control counterparts combined with a hindlimb clasping score of
 three and constant fasciculations when moving about the home cage.  A
 humane endpoint for <em>Elp1 </em>CKO females was defined by a
 weight &lt; 60% of their control counterparts
 combined with a hindlimb clasping score of three and constant
 fasciculations when moving about the home cage.  The following strains
 were purchased from the Jackson Laboratory:
 <em>ROSA<sup>mT-mG</sup></em>, stock no. 007576;
 <em>Chat-Cre, </em>stock no. 006410; <em>Chat-GFP,
 </em>stock no. 007902.  <em>Elp3<sup>Lox</sup>
 </em>mice have been described previously <sup>36</sup>. 
   Floxed <em>Elp3 </em>mice were generated
 on a 129 SvJ background.  <em>Elp3 </em>CKO mice were
 generated through the same genetic strategy as <em>Elp1
 </em>CKO mice (<em>Chat-Cre; Elp3<sup>+/LoxP
 </sup></em>X<em>
 Elp3<sup>LoxP/LoxP</sup></em>) except that the
 <em>Chat-GFP </em>allele was not incorporated into
 the<em> Elp3<sup>LoxP/LoxP</sup></em> strain.  To
 visualize cholinergic neurons in <em>Elp3 </em>CKO mice and
 controls, an anti-ChAT antibody was used (see below).
 Hindlimb clasping scoring Hindlimb clasping
 was scored using a previously published scoring
 system<sup>30</sup>.  Briefly, the mouse is suspended by the
 tail and hindlimb position is observed for 10 seconds.  If hindlimbs are
 consistently splayed outward, away from the abdomen, the mouse is assigned
 a score of 0. If one hindlimb is retracted toward the abdomen for more
 than 50% of the time suspended, it receives a score of 1. If both
 hindlimbs are partially retracted toward the abdomen for more than 50% of
 the time suspended, it receives a score of 2. If both hindlimbs are
 entirely retracted and touching the abdomen for more than 50% of the time
 suspended, it receives a score of 3. PaGE
 testing Paw grip endurance (PaGE) testing as a measure
 of motor function was performed as described in Weydt et al.
 (2003)<sup>49</sup>, and was modified slightly.  Briefly, each
 mouse was placed on a wire lid from a conventional rodent housing cage;
 the lid was gently shaken to induce gripping and turned upside down
 (180°). The latency until the mouse released both hind limbs was measured
 in seconds. Each mouse was tested five times with an arbitrary maximμmof
 60 s, and the longest latency to fall or release both hind limbs was
 recorded.  Immunofluorescence (IF)
 All washing, blocking, secondary antibody, and post-fixation
 steps were performed at room temperature. All other steps were performed
 at 4°C unless stated otherwise.  Following fixation, tissues were rinsed
 in PBS, cryoprotected through a series of sucrose solutions in PBS (15%,
 30%), incubated for 2 h in a 1:1 mixture of 30% sucrose and optimal
 cutting temperature (OCT) compound (Tissue-Tek,Torrance, CA), followed by
 2 h in OCT.  Tissues were then embedded in OCT and frozen in a dry ice
 ethanol bath.  For immunostaining, slides were bathed in TBS
 (tris-buffered saline) for 10 min, followed by NGS block (10% normal goat
 serum, 1% glycine, 0.4% Triton X-100 in 30mM Tris, 150mM NaCl) for 1 h,
 and overnight incubation in primary antibody (in NGS block). Slides were
 then rinsed in NGS block, incubated in Alexa Fluor secondary antibody
 (1:2,000 in NGS block) for 1 h, rinsed in 3:1 TBS:NGS block, and mounted
 in Prolong Antifade Diamond (Invitrogen, La Jolla, CA). Control and
 experimental embryos were cryosectioned on the same day and sections were
 incubated in primary antibody on the same day that they were sectioned.
 For antigen retrieval, slides were incubated in citrate buffer at 95°C for
 10 min following sectioning. Antibodies
 Primary antibodies included the following:  GFP (Abcam, ab13970,
 1:1000), ELP1 (Abnova PAB12857, 1:800, knockout validated<sup>26,
 50</sup>.  ELP3 (Proteintech, 17016-1-AP, 1:200, knockout validated
 here using <em>Chat-Cre; Elp3<sup>LoxP/LoxP
 </sup></em>mice), NeuN (Proteintech, 26975-1-AP, 1:4000, see
 the manufacturer’s website for validation details), TDP-43 RRM2
 (Proteintech, 10782-2-AP, 2 ug/ml, knockout validated).  *Note: although
 the manufacturer’s website describes this antibody as recognizing the
 TDP-43 N-terminus, Tsuji et al., 2012<sup>38</sup>, showed the
 antibody recognizes amino acids 203-209 of human TDP-43 which corresponds
 to the second RNA recognition motif. TDP-43 C-terminus (Proteintech,
 12892-1-AP, 2 ug/ml, knockout validated), Fibrillarin (Novus Biologicals,
 NB300-269, 1:500, knockdown validated), ChAT (Millipore Sigma AB144P,
 1:100, see the manufacturer’s website for validation details).  Secondary
 antibodies used were Alexa Fluor goat anti-rabbit 488, goat anti-mouse
 568, donkey anti-chick 488, donkey anti-goat 568 (Invitrogen,
 1:2,000).  Immunohistochemistry LacZ
 staining was performed as previously described<sup>51</sup>. 
 Briefly, slides with tissue sections (20 um) were incubated for three
 hours in 1 ml of X-gal solution (1 mg/ml X-gal, 5mM potassiμmferricyanide,
 5mM potassiμmforrocyanide, 2mM MgCl2, 0.25% Triton X-100 in PBS) at 30
 degrees C.  Tissue sections were then fixed in 1 ml of 4% paraformaldehyde
 for 10 min, followed by three washes in TBS.  To combine with IF, tissue
 sections were then incubated in NGS block followed by the application of
 primary antibody as above. Alpha motor neuron
 quantification The <em>Chat-GFP </em>allele
 was included in all <em>Elp1 </em>CKO experiments where alpha
 motor neurons were quantified or measured.  Only cells in the ventral horn
 (Fig. S6) that were GFP-positive and NeuN-positive were selected for
 further analysis<sup>34</sup>.  For adult animals (6 weeks),
 spinal cords were removed from both control (<em>Chat-Cre;
 Elp1<sup>+/LoxP</sup></em>) and CKO (<em>Chat-Cre;
 Elp1<sup>LoxP/LoxP</sup></em>) animals via hydraulic
 extrusion<sup>52</sup>.  The lumbar enlargement was isolated
 and fixed for 2.5 h at 4°C in 4% paraformaldehyde in PBS.   Sixty 16 μm
 sections were trimmed to reach the L5/L4 level.  Every other 16 μm section
 was then collected, filling one slide (approximately 48 sections).  IF was
 performed as described above using anti-GFP and anti-NeuN antibodies, and
 the field of view (40X) containing the highest number of GFP-positive
 neurons in the ventral horn (lamina IX, Fig. S6) was photographed.  For
 control mice, sections to be photographed were selected randomly using a
 random number generator.  For the CKO, given the reduced number of large
 motor neurons, all sections were photographed, and the field of view (40X)
 was positioned to include the largest possible number of large motor
 neurons.  The number of alpha motor neurons (GFP-positive and
 NeuN-positive) per ventral horn were counted and their areas measured
 using ImageJ.  Averages were then calculated for the 10 sections with the
 largest number of alpha motor neurons (GFP-positive, NeuN-positive, and an
 area &gt; 440 μm<sup>2</sup>) present
 per section.  The same approach was used to quantify the number of
 GFP-positive, NeuN-positive neurons with an area less than 440
 μm<sup>2</sup>.  For E18.5, embryos were decapitated and fixed
 in 4% paraformaldehyde in PBS for 2.25 hours.  Sections were cut at 16 μm
 and every other section was collected from the mid to upper lumbar axial
 level, filling two slides.  Every other section was photographed, and the
 number of alpha motor neurons in a single hemisphere (the hemisphere with
 the highest number of alpha motor neurons) counted per section and an
 average calculated for the 12 sections with the largest number of alpha
 motor neurons present per section. Neuromuscular
 junction analysis The <em>Chat-GFP
 </em>allele was included in both control (<em>Chat-Cre;
 Elp1<sup>+/LoxP</sup></em>) and CKO (<em>Chat-Cre;
 Elp1<sup>LoxP/LoxP</sup></em>) animals to visualize
 innervation of the NMJ.  Anterior tibialis muscles were dissected from
 female controls and CKOs and fixed for 20 min in 4% paraformaldehyde in
 PBS at 4°C.  The muscles were then worked up for cryosectioning as
 described above for spinal cords.  Sections were cut at a thickness of 25
 μm and every other section collected, filling two to three slides for a
 total of 60 sections.  IF was performed as described above using an
 anti-GFP antibody to visualize innervation of the NMJ.  Alpha bungarotoxin
 555 (Molecular Probes, #B160; 1 μg/ml) was included at the secondary
 antibody step.  Sections to be analyzed were selected randomly using a
 random number generator.  NMJs were also randomly selected by generating a
 number between 1 and 10 and counting to that NMJ starting from the top
 right corner of the section.  50 NMJs were analyzed per animal and tallied
 as being either fully innervated (GFP pattern matches the pattern of alpha
 bungarotoxin), or not fully innervated (GFP either partially or totally
 missing).  Anterior tibialis muscles from three control and three
 experimental animals were analyzed per time point.
 TDP-43 and Fibrillarin corrected fluorescence and area
 analysis Nu area in Fig. 4j corresponds to the entire
 nucleus (including the nucleolus).  Fluorescence levels per unit area were
 calculated as follows: No = (No corrected total fluorescence (CTF) / No
 area); Nu = (Nu CTF – No CTF) / (Nu area – No area);  Cytoplasm = (total
 cell CTF – Nu CTF) / (total cell area – Nu area).  For TDP-43 and
 fibrillarin fluorescence measurements, spinal cords from control
 (<em>Chat-Cre; Elp1<sup>+/LoxP</sup></em>) and CKO
 (<em>Chat-Cre; Elp1<sup>LoxP/LoxP</sup></em>) were
 collected, processed for cryosectioning, and sectioned as described above
 (alpha motor neuron quantification).  IF was performed as described above
 using either anti-TDP-43 and anti-GFP primary antibodies, or
 anti-fibrillarin and anti-GFP antibodies.  Antigen retrieval was included
 for fibrillarin.  For controls, a random number generator was used to
 randomly select sections.  The field of view in the spinal cord ventral
 horn (lamina IX, Fig. S6) (40X) with the highest density of GFP-positive
 neurons was photographed.  For CKOs, given the reduced number of surviving
 motor neurons, all GFP+ cells in the ventral horn were photographed.
 Identical exposure, gain, and offset settings were used for both control
 and experimental images. Next, for both controls and CKOs, ImageJ was used
 to measure the area of photographed, GFP+ cells.  In cells with a soma
 area &gt; 440 μm2 and that contained a whole intact
 nucleus via DAPI staining, TDP-43 or fibrillarin areas and fluorescence
 levels were measured using Image J processing and analysis software (https://imagej.nih.gov/ij/).  A minimum of 20 cells were analyzed per spinal cord for controls and between 8 and 20 for the CKOs given the reduced number of alpha motor neurons with soma areas &gt; 440 μm<sup>2</sup> in the CKO spinal cords.  A minimum of three spinal cords were analyzed per genotype and the exact number of spinal cords for each analysis is shown on the individual bar graphs in Fig. 4. Microscopy Images were captured using a Nikon TE200 inverted microscope equipped with a QImaging QICAM 12-bit Mono Fast 1394 Cooled camera and SPOT software. Identical exposure times, gain, and offset settings were used to capture control and experimental images.
description: # Data from: An Elongator mouse model of ALS spotlights TDP-43 in the
 motor neuron nucleolus Dataset DOI:
 [10.5061/dryad.x0k6djhvb](10.5061/dryad.x0k6djhvb) ## Description of the
 data and file structure Dysfunction of Elongator is associated with
 amyotrophic lateral sclerosis (ALS).  To investigate this relationship, we
 generated mouse models in which *Elp1* or* Elp3* is selectively ablated in
 alpha motor neurons of the spinal cord. These mice exhibit a progressive
 loss of motor strength and motor neuron degeneration. To interrogate the
 molecular mechanisms that contribute to motor neuron cell death in this
 model, we examined multiple disease pathways including the expression of
 TDP-43 whose aggregation is associated with the human disease. 
 Importantly, we document TDP-43’s robust presence in the nucleolus of
 wild-type motor neurons and its clearance from both the nucleus and the
 nucleolus of motor neurons in *Elp* conditional knockout mice.  Thus, this
 study directly links dysfunction of Elongator with nucleolar disruption
 and TDP-43 clearing, two hallmark cellular pathologies of ALS.   ### Files
 and variables #### File: Images_motor_neuron_counts.zip
 **Description:** Files contain images of motor neurons in the spinal cord
 lumbar enlargement (ventral horn region) of 6-week-old male and female
 control and *Elp1* conditional knockout mice. Controls are designated with
 a C (i.e. C1) and conditional knockouts are designated with CKO (i.e.
 CKO1). Motor neurons are labeled with NeuN (no identifier in the file
 name) and Chat-GFP (labeled with GFP in the file name). Quantification of
 motor neurons in these images is available as Supplementary Data 2 in the
 associated publication. #### File: Images_ELP3_in_Elp3.zip
 **Description:** Files contain images of motor neurons in the spinal cord
 lumbar enlargement (ventral horn region) of 2-week-old male control and
 *Elp3 *conditional knockout mice. Controls are designated with a C (i.e.
 C1281) and conditional knockouts are designated with CKO (i.e. CKO1278).
 Motor neurons are labeled with CHAT (labeled with CHAT in the file name)
 and ELP3 (no identifier in the file name). Quantification of ELP3 levels
 in these images can be found in
 "ELP3_in_Elp3_2wk_males_Source_Data.xlsx". Image numbers
 correspond to "Image #" (Column A) in this source data file.
 #### File: Images_TDP43.zip **Description:** Files contain images of motor
 neurons in the spinal cord lumbar enlargement (ventral horn region) of
 2-week-old and 6-week-old female control and *Elp1 *conditional knockout
 mice. Controls are designated with a C (i.e. 10C) and conditional
 knockouts are designated with CKO (i.e. 12CKO). Motor neurons are labeled
 with TDP43 (no identifier in file name) and Chat-GFP (GFP in file name).
 Quantification of TDP43 fluorescence levels is available as Supplementary
 Data 3 in the associated publication. #### File: Images_ELP3_in_Elp1.zip
 **Description:** Files contain images of motor neurons in the spinal cord
 lumbar enlargement (ventral horn region) of 6-week-old female control and
 *Elp1* conditional knockout mice. Motor neurons are labeled with Chat-GFP
 (GFP in file name) and ELP3 (no identifier in file name). Quantification
 of ELP3 levels in these images can be found in
 "ELP3_in_Elp1_6wk_females_Source_Data.xlsx". Image numbers
 correspond to "Image #" (Column A) in this source data file.
 #### File: Elp1_Hindlimb_Clasping_and_Muscle_Mass_Source_Data.xlsx
 **Description:** File contains hindlimb clasping and muscle mass source
 data for Figure S2a,b in the associated publication ##### Variables * C =
 control mouse (i.e. C1) * CKO = conditional knockout mouse (i.e. CKO1) *
 Std Dev = standard deviation * Std Error = standard error * Gastroc =
 gastrocnemius muscle * Soleus = soleus muscle * TA = anterior tibialis
 muscle * mg = milligram #### File:
 ELP3_in_Elp1_6wk_females_Source_Data.xlsx **Description:** File contains
 source data for Figure S2c in the associated publication (quantification
 of ELP3 fluorescence in 6-week-old female *Elp1 *control and conditional
 knockout mice). ##### Variables * C = control mouse (i.e. C1) * CKO =
 conditional knockout mouse (i.e. CKO1) * GFP = Green fluorescent protein
 expression driven by the Chat promoter * Cell Areas were measured using
 ImageJ and are shown in arbitrary units. * Cell Mean = the mean gray value
 of the cell being measured. Cell means were measured using ImageJ and are
 shown in arbitrary units. * Cell Int Den = the integrated density of the
 cell being measured. Cell integrated densities were measured using ImageJ
 and are shown in arbitrary units. * BG = background mean gray value.
 Background mean gray values were measured using ImageJ and are shown in
 arbitrary units. * CTCF = corrected total cell fluorescence. CTCF values
 were calculated using the formula: CTCF = Cell Int Den - (Cell Area X BG
 Mean). * #### File: ELP3_in_Elp3_2wk_males_Source_Data.xlsx
 **Description:** File contains source data for Figure S3g in the
 associated publication (quantification of ELP3 fluorescence in 2-week-old
 male *Elp3 *control and conditional knockout mice). ##### Variables * C =
 control mouse (i.e. 1281C) * CKO = conditional knockout mouse (i.e.
 1278CKO) * Cell Areas were measured using ImageJ and are shown in
 arbitrary units. * Cell Mean = the mean gray value of the cell being
 measured. Cell means were measured using ImageJ and are shown in arbitrary
 units. * Cell Int Den = the integrated density of the cell being measured.
 Cell integrated densities were measured using ImageJ and are shown in
 arbitrary units. * BG = background mean gray value. Background mean gray
 values were measured using ImageJ and are shown in arbitrary units. * CTCF
 = corrected total cell fluorescence. CTCF values were calculated using the
 formula: CTCF = Cell Int Den - (Cell Area X BG Mean). ## Code/software All
 images are TIFF and can be viewed with any basic image viewing software,
 including Windows Photos and Preview on Mac. Image editing software like
 Adobe Photoshop can also be used. Source data files can be viewed in
 Excel.