Stage-dependent remodeling of projections to motor cortex in ALS mouse model revealed by a new variant retrograde-AAV9
- University of Ulm, Germany
- University Hospital Heidelberg, Germany
- University of Kiel, Germany
Figures
Figure 1
AAV9-SLR is a new AAV variant with pronounced retrograde infectivity.
AAV9-SLR, AAV9-NSS and AAV9-RGD variants (or WT-AAV9 and WT-AAV2) were injected in dorsal striatum (N = 3) and local infection rate (B), retrograde infection to Substantia Nigra (C) and Motor Cortex …
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Figure 1—source data 1
Detailed statistic concerning AAV variants infectivity (Figure 1).
- https://doi.org/10.7554/eLife.36892.003
Figure 2 with 2 supplements
Projections to primary and secondary motor cortex in P20 wild-type.
(A) Experimental design depicting injection site in pMO and relative projecting neurons. (B) List of the regions projecting to primary MO (pMO) identified by injection of AAV9-SLR in pMO of WT mice …
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Figure 2—source data 1
Detailed statistic concerning projecting neurons to primary motor cortex (pMO) and secondary motor cortex (sMO) in WT animals(P20) traced via AAV9-SLR injection.
Numbers are expressed in term of total neuronal count.
- https://doi.org/10.7554/eLife.36892.009
Figure 2—figure supplement 1
Projections to primary and secondary motor cortex in P20 wild-type.
(A) Bar chart listing the number of neurons for each region projecting to primary motor cortex (pMO) identified by injection of AAV9-SLR in pMO of WT mice. Values are expressed in term of total …
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Figure 2—figure supplement 1—source data 1
Detailed statistic concerning projecting neurons to primary motor cortex (pMO) and secondary motor cortex (sMO) in WT animals (P20) traced via AAV9-SLR injection.
Total count was normalized for the volume of the injection site and contribution from each brain region is reported in term of percentage.
- https://doi.org/10.7554/eLife.36892.006
Figure 2—figure supplement 2
Projection from thalamic nuclei to primary and secondary motor cortex in WT at P20.
(A) Distribution of neurons projecting to primary motor cortex from individual thalamic nuclei with values expressed in terms of total numbers. Detailed statistic provided in Supplementary file 1h. …
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Figure 2—figure supplement 2—source data 1
Detailed statistic concerning input from thalamic nuclei to primary motor cortex (pMO) and secondary motor cortex (sMO) in WT animals(P20) traced via AAV9-SLR injection.
Numbers are reported in term of total neuronal count, normalized neuronal count for the volume of the injection site and contribution from each nucleus reported in term of percentage.
- https://doi.org/10.7554/eLife.36892.008
Figure 3 with 1 supplement
Increased projections to primary motor cortex in mSOD at pre-symptomatic stage (P20) from ipsilateral SS and contralateral sMO.
(A) Experimental design depicting injection site in pMO and relative projecting neurons. (B) Number of neurons, normalized for injection site volume, projecting to primary motor cortex in WT (N = 6) …
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Figure 3—source data 1
Detailed statistic concerning projecting neurons to primary motor cortex (pMO) in WT vs mSOD animals(P20) traced via AAV9-SLR injection.
Total count was normalized for the volume of the injection site. Discrimination for each cortical layer is reported in term of percentage.
- https://doi.org/10.7554/eLife.36892.013
Figure 3—figure supplement 1
Increased projections to primary motor cortex in mSOD at pre-symptomatic stage (P20) from ipsilateral SS and contralateral sMO.
(A) Number of neurons projecting to primary motor cortex in WT (N = 6) and mSOD (N = 3). Significant increase in the number of neurons projecting to pMO from SS. No other structure, among the 28 …
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Figure 3—figure supplement 1—source data 1
Detailed statistic concerning projecting neurons to primary motor cortex (pMO) in WT vs mSOD animals (P20) traced via AAV9-SLR injection.
Discrimination for each cortical layer is reported. Numbers are expressed in term of total neuronal count.
- https://doi.org/10.7554/eLife.36892.012
Figure 4 with 1 supplement
Increased projections to primary motor cortex in mSOD at pre-symptomatic stage (P20) is specific for motor network and is confirmed via the retrograde tracer choleratoxin.
(A) Experimental design depicting injection site in pMO via choleratoxin (CTb) with two regions that have been investigated for retrograde infection (SS and cMOp). (B) Normalized (for injection site …
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Figure 4—source data 1
Total number of neurons projecting to primary motor cortex, WT vs mSOD via Choleratoxin-b injection.
CTb + retrogradely labeled projections from SS and cMOp to pMO. Total number of neurons projecting to visual network (V1) from LGN and MO in WT and mSOD. Numbers expressed as total neuronal count and as normalized neuronal count for the volume of the injection site.
- https://doi.org/10.7554/eLife.36892.016
Figure 4—figure supplement 1
Increased projections to primary motor cortex in mSOD at pre-symptomatic stage (P20) is specific for motor network and is confirmed via the retrograde tracer choleratoxin.
(A) Total number of neurons projecting to primary motor cortex, WT (N = 3) vs mSOD (N = 3), via Choleratoxin-b injection. CTb + retrogradely labelled projections from SS and cMOp to pMO. Increased …
Figure 5
No change in projections to secondary motor cortex, at pre-symptomatic stage (P20).
(A) Number of neurons, normalized for injection site volume, projecting to secondary motor cortex (sMO) in WT (N = 3) and mSOD (N = 3). No statistically significant differences in input to sMO were …
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Figure 5—source data 1
Detailed statistic concerning projecting neurons to secondary motor cortex (sMO) in WT vs mSOD animals (P20) traced via AAV9-SLR injection.
Total neuronal count count is reported, together with its normalization for the volume of the injection site.
- https://doi.org/10.7554/eLife.36892.018
Figure 6 with 1 supplement
Progressive changes in projections to pMO in disease progression.
(A) Projection neurons were mapped at P20, P60 and P90 timepoints; fold change of normalized total number of projecting neurons of mSOD vs WT was plotted for the three stages of disease progression. …
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Figure 6—source data 1
Detailed statistic concerning projecting neurons to primary motor cortex (pMO) in WT vs mSOD animals, traced via AAV9-SLR injection, during disease progression.
Three time points have been investigated: P20, P60 and P90. Total neuronal count is reported together with its normalization for the volume of the injection site. Moreover fold change for each time-point normalized over respective WT is provided.
- https://doi.org/10.7554/eLife.36892.022
Figure 6—figure supplement 1
No neuronal loss in pMO of mSOD animal (P90).
(A) Bar chart comparing NeuN+ neurons in primary motor cortex of adult mSOD animals (P90). No difference has been detected (WT: 1087 ± 128 vs mSOD 1087 ± 236), proving that changes in number of …
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Figure 6—figure supplement 1—source data 1
Detailed statistic concerning the number of NeuN +neurons in the motor cortex of adult mSOD animals (P90).
- https://doi.org/10.7554/eLife.36892.021
Figure 7
Pyramidal neurons projecting to pMO display simultaneous spines loss on basal dendrites during disease progression independently of misfolded SOD accumulation.
(A) Spine density on pyramidal neurons projecting to primary at P20; each dot representing average spine density for one single neuron. Spine density was comparable in pyramidal neurons projecting …
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Figure 7—source data 1
Detailed statistic concerning spine density on pyramidal neurons projecting to primary motor cortex during disease progression (P20, P60, P90).
- https://doi.org/10.7554/eLife.36892.024
Figure 8
misfSOD expression does not trigger network remodeling nor loss of spines in basal dendrites of connected pyramidal neurons.
(A) Experimental design depicting injection site in pMO via choleratoxin (CTb) with two regions that have been investigated for misfSOD immunostaining intensity (SS and AUD). (B) Comparison of …
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Figure 8—source data 1
Detailed statistic concerning comparison of misfSOD expression between neurons CTb + and CTb- in layer II/III and layer V of SS and AUD in mSOD.
Comparison of misfSOD expression between neurons CTb + and CTb- in layer V of contralateral SS in mSOD. Spine density analysis on two differential neuronal populations, misfSOD- and misfSOD+, projecting from SS to primary motor cortex at P60.
- https://doi.org/10.7554/eLife.36892.026
Figure 9
Functional connectivity alterations in human ALS.
(A) BOLD synchronization illustrated as coronal (upper panel) and axial connectivity (lower) heat maps showing voxel-wise Fisher’s r-to-z transformed correlation coefficients (thresholded for |z(r)|≥…
Tables
Key resources table
| Reagent type (species) or resource | Designation | Source of reference | Identifiers/RRIDS | Additional information |
|---|---|---|---|---|
| Antibody | Anti-Red Fluorescece Protein (RFP) | Rockland | 600-401-379/ RRID: AB_828390 | (1:1000) |
| Antibody | DAPI | Invitrogen | D1306 | (1:1000) |
| Antibody | Anti-Tyrosin- Hydroxilase | Sigma | T2928/ RRID: AB_477569 | (1:4000) |
| Antibody | Anti-misfolded SOD1 (B8H10) | MediMabs | MM-0070/ RRID:AB_10015296 | (1:1000) |
| Antibody | Donkey anti rabbit Alexa-568 | Life Technologies | A10042 | (1:500) |
| Antibody | Anti NeuN | Millipore | MAB377/ RRID:AB_2298772 | (1:100) |
| Antibody | Donkey anti-mouse Alexa-488 | Life Technologies | A21202 | (1:500) |
| Strain, strain background (Mus musculus) | B6SJL-Tg(SOD1 *G93A)1Gur/J | Jackson Laboratories | RRID:IMSR_JAX:002726 | high-copy, henceforth mSOD |
| Strain, strain background (Mus musculus) | B6.Cg-Gt(ROSA) 26Sortm6/ (CAG-ZsGreen)Hze/J | Jackson Laboratories | 007906 | henceforth ZsGreen-ROSA26 |
| Strain, strain background (Mus musculus) | B6.Cg-Gt(ROSA) 26Sortm6/(CAG-Td Tomato)Hze/J | Jackson Laboratories | 007914 | henceforth tdTomato-ROSA26 |
| Chemical compound, drug | Phenol Red Solution | Sigma‐Aldrich | P0290 | |
| Chemical compound, drug | Protease inhibitor mix | Serva | 39101.03 | |
| Chemical compound, drug | Polyethylenimine (PEI) | Polysciences | 23966 | |
| Chemical compound, drug | Optiprep | Progen | 1114542 | |
| Chemical compound, drug | Buprenorphine | Reckitt Benckiser | ||
| Chemical compound, drug | Meloxicam | Böhringer Ingelheim | ||
| Chemical compound, drug | Ketamine 10% | WDT | ||
| Chemical compound, drug | Rompun 2% (Xylazin) | Bayer | ||
| Recombinant DNA reagent | AAV9-RGDLRVS -CMV-Cre | Varadi et al., 2012 | ||
| Recombinant DNA reagent | AAV9-SLRSPPS -CMV-Cre | Varadi et al., 2012 | ||
| Recombinant DNA reagent | AAV9-NSSRFTP -CMV-Cre | Varadi et al., 2012 | ||
| Recombinant DNA reagent | WT-AAV2- CMV-Cre | Werfel et al., 2014 | ||
| Recombinant DNA reagent | WT-AAV9-CMV-Cre | Werfel et al., 2014 | ||
| Peptide, recombinant protein | Alexa-488-conjugated cholera toxin B | Invitrogen | C34775 | |
| Peptide, recombinant protein | Alexa-647-conjugated cholera toxin B | Invitrogen | C34778 |
Table 1
Subject demographics and clinical characterization.
https://doi.org/10.7554/eLife.36892.028| Healthy controls | ALS, all, T0 | p-value | |
|---|---|---|---|
| Subjects (number) | 28 | 71 | NA |
| Gender (male:female) | 15:13 | 39:32 | 1.000* |
| Age (years) | 54.8 (±12.9), 22.4—75.7 | 58.4 (±13.7), 19.7—85.1 | 0.228† |
| Duration of disease (month) | NA | 19.2 (±17.6), 2.6—84.7 | NA |
| Age of onset (years) | NA | 56.8 (±13.9), 19.2—84.6 | NA |
| ALSFRS-R‡ | NA | 40 (±5), 24—48 | NA |
| Rate of disease progression§ (1/month) | NA | 0.8 (±1.2), 0.0—7.8 | NA |
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Data are shown as mean (±std), min—max. All values were computed using the MATLAB (The Mathworks Inc, Natick, MA) based ‘Statistics Toolbox’.
*Fisher’s exact test refers to comparison between all ALS patients and healthy controls.
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†Two-sample unpaired t-test assuming unequal variances refers to comparison between all ALS patients and healthy controls.
‡ALSFRS-R, revised ALS Functional Rating Scale (maximum score 48, falling with increasing physical impairment).
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§Rate of disease progression computed as (48 - ALSFRS-R)/(disease duration)(Menke et al., 2014). NA, not applicable.
Additional files
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Supplementary file 1
(a) AAV variants injected in dorsal striatum (DS) and analyzed for their local infectivity ability. Number of neurons are normalized over AAV9- WT and expressed in terms of percentage. (b) AAV variants injected in dorsal striatum (DS) and analyzed for their retrograde infection ability to substantia nigra (SNr). Number of neurons are normalized over AAV9- WT and expressed in terms of percentage. (c) AAV variants injected in dorsal striatum (DS) and analyzed for their retrograde infection ability to motor cortex (MO). Number of neurons are normalized over AAV9- WT and expressed in terms of percentage. (d) AAV variants injected in primary visual cortex (V1) and analyzed for their retrograde infection ability to lateral geniculate nucleus (LGN). Number of neurons are normalized over AAV9- WT and expressed in terms of percentage. (e) Input to primary motor cortex (pMO) and secondary motor cortex (sMO) in WT animals (P20) traced via AAV9-SLR injection. Numbers are expressed in term of total neuronal count. (f) Input to primary motor cortex (pMO) and secondary motor cortex (sMO) in WT animals(P20) traced via AAV9-SLR injection. Neuronal count normalized for the volume of the injection site. (g) Input to primary motor cortex (pMO) and secondary motor cortex (sMO) in WT animals (P20) traced via AAV9-SLR injection. Total count was normalized for the volume of the injection site and contribution from each brain region is reported in term of percentage. (i) Input from thalamic nuclei to primary motor cortex (pMO) and secondary motor cortex (sMO) in WT animals(P20) traced via AAV9-SLR injection. Neuronal count normalized for the volume of the injection site. (j) Input from thalamic nuclei to primary motor cortex (pMO) and secondary motor cortex (sMO) in WT animals(P20) traced via AAV9-SLR injection. Total count was normalized for the volume of the injection site and contribution from each nucleus is reported in term of percentage.
- https://doi.org/10.7554/eLife.36892.029
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Supplementary file 2
(a) Input to primary motor cortex in WT and mSOD animals (P20) traced via AAV9-SLR injection.Analysis of total number of neurons is reported. Numbers are expressed in term of total neuronal count. (b) Input to primary motor cortex in WT and mSOD animals (P20) traced via AAV9-SLR injection. Neuronal count normalized for the volume of the injection site. (c) Input from cortical layers (ipsilateral SS and ipsilateral AUD) to primary motor cortex in WT and mSOD animals(P20) traced via AAV9-SLR injection. Numbers are expressed in term of total neuronal count. (c) Input from cortical layers (ipsilateral SS and ipsilateral AUD) to primary motor cortex in WT and mSOD animals (P20) traced via AAV9-SLR injection. Neuronal count normalized for the volume of the injection site. (e) Input from cortical layers (ipsilateral SS and ipsilateral AUD) to primary motor cortex in WT and mSOD animals (P20) traced via AAV9-SLR injection. Total neuronal count was normalized for the volume of the injection site, values are expressed in terms of percentage. (f) Input to primary motor cortex in WT and mSOD animals (P20) traced via choleratoxin (CTb) injection. Numbers are expressed in term of total neuronal count. (g) Input to primary motor cortex in WT and mSOD animals (P20) traced via choleratoxin (CTb) injection. Neuronal count normalized for the volume of the injection site. (h) Input to primary visual cortex (V1) in WT and mSOD animals (P20) traced via AAV9-SLR injection. Numbers are expressed in term of total neuronal count. (i) Input to primary visual cortex (V1) in WT and mSOD animals (P20) traced via AAV9-SLR injection. Neuronal count normalized for the volume of the injection site. (j) Input to secondary motor cortex in WT and mSOD animals (P20) traced via AAV9-SLR injection. Analysis of total number of neurons is reported. Numbers are expressed in term of total neuronal count. (k) Input to secondary motor cortex in WT and mSOD animals (P20) traced via AAV9-SLR injection. Neuronal count normalized for the volume of the injection site.
- https://doi.org/10.7554/eLife.36892.030
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Supplementary file 3
(a) Input to primary motor cortex in mSOD animals traced via AAV9-SLR injection during disease progression: early pre- symptomatic(P20), intermediate (P60) and later stage (P90). Total neuronal count was normalized for the volume of injections ite, numbers expressed in term of fold change over the relative WT. (b) Spine density analysis on basal dendrites (10 µm stretch) of cortical pyramidal neurons projecting to pMO. Comparison between WT and earlypre-symptomatic mice (P20). Tracing via AAV9-SLR injected in pMO. (c) Spine density analysis on basal dendrites (10 µm stretch) of cortical pyramidal neurons projecting to pMO. Comparison between WT and intermediate stage mice (P60). Tracing via AAV9-SLR injected in pMO. (d) Spine density analysis on basal dendrites (10 µm stretch) of cortical pyramidal neurons projecting to pMO. Comparison between WT and later stage mice (P90). Tracing via AAV9-SLR injected in pMO. (e) Analysis for misfSOD intensity in cortical projecting neurons to primary motor cortex in mSOD pre-symptomatic mice (P20). Tracing via choleratoxin (CTb). (f) Analysis for misfSOD intensity in projecting neurons to SS in mSOD mice (P50). Tracing via choleratoxin (CTb). (g) Spine density analysis on basal dendrites (10 µm stretch) of cortical pyramidal neurons projecting to primary motor cortex. Comparison between misfSOD- and misfSOD +neurons of mSOD mice (P60). Tracing via AAV9-SLR injected in pMO.
- https://doi.org/10.7554/eLife.36892.031
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Transparent reporting form
- https://doi.org/10.7554/eLife.36892.032
