An optogenetic cell therapy to restore control of target muscles in an aggressive mouse model of amyotrophic lateral sclerosis
- Department of Neuromuscular Diseases, UCL Queen Square Institute of Neurology, University College London, United Kingdom
- UCL Queen Square Motor Neuron Disease Centre, UCL Queen Square Institute of Neurology, University College London, United Kingdom
- Department of Electronic and Electrical Engineering, University College London, United Kingdom
Figures
Figure 1
Engrafted allogeneic ChR2+ motor neuron can survive and robustly reinnervate target muscles in SOD1G93A mice but occurs rarely.
(A) Confocal tile-scan of a longitudinal section of the triceps surae muscle from a 135d SOD1G93A mouse, 45d post-engraftment, showing a rare example of graft survival, in the absence of …
Figure 2 with 1 supplement
FK506 enables long-term survival of engrafted ChR2+ motor neurons but inhibits muscle reinnervation and exacerbates disease progression in SOD1G93A mice.
(A) Representative confocal image showing that GFP/YFP+ axons are able to project within intramuscular branches, following intraneural engraftment of ChR2+ motor neurons and daily immunosuppression …
Figure 2—figure supplement 1
FK506 severely affects body mass in most SOD1G93A but not wild-type mice.
(A) Longitudinal body mass analysis of individual SOD1G93A mice (n = 18) treated with FK506 (5mg/kg/d), spanning different age ranges, reveals that FK506 prevents body mass increase in the majority …
Figure 3 with 1 supplement
FK506 causes peripheral nerve axonopathy in SOD1G93A and wild-type mice.
(A) Representative examples of wild-type (top) and SOD1G93A (bottom) sciatic nerve transverse sections, showing common peroneal nerve (CPN) and tibial nerve (TN), labeled for total axons (βIII …
Figure 3—figure supplement 1
FK506 moderately reduces total sciatic nerve axon counts in wild-type mice but loss of total and motor axons is not observed in SOD1G93A mice when all axon calibers are grouped.
Automated analysis of total (i.e. sensory and motor) axon numbers reveals that daily treatment with FK506 causes a significant reduction of total axon counts in wild-type tibial nerve (TN) and …
Figure 4 with 1 supplement
Transient H57-597 mAb treatment confers complete ChR2+ motor neuron allograft survival and, importantly, allows robust triceps surae muscle reinnervation up until late-stage disease in SOD1G93A mice.
(A) 3D reconstruction of 67 individual tile-scans, acquired from serial sections from an entire triceps surae muscle, from a 135d SOD1G93A mouse following engraftment of ChR2+ motor neurons at 95d …
Figure 4—figure supplement 1
Subtype identity of engrafted ChR2+ motor neurons does not affect the maximum contractile response of the targeted muscle to acute optical stimulation in SOD1G93A mice.
(A) Fast-firing motor neurons (produced using a 7-day differentiation protocol thus labeled as ‘7DD’) or slow-firing ChR2+ motor neurons (produced using a 5-day differentiation protocol thus labeled …
Figure 5 with 6 supplements
Daily optical stimulation training (OST) of post-symptom onset SOD1G93A mice engrafted with ChR2+ motor neurons, significantly enhances contractile response to optical stimulation.
(A) Schematic indicating intraneural engraftment site in distal tibial nerve and reinnervated triceps surae (TS) muscle, along with stimulation device (top inset) implantation site and subcutaneous …
Figure 5—figure supplement 1
An existing implantable device underwent minor modifications to improve suitability for optical stimulation training experiments.
(A) Modifications to the original PCB design and (B) encapsulation method, conferred long-term functionality of the wireless stimulation following in vivo implantation. (C) Incorporation of a radio …
Figure 5—figure supplement 2
Light power recording and stimulation pattern recordings used to elicit acute optical nerve stimulation (ONS) throughout study.
(A) Low temporal resolution recording show electrical TTL trigger pulses (green; top), used to activate an LED stimulator, and light power recordings measured using a digital light meter; note: LED …
Figure 5—figure supplement 3
Optimization of optical nerve stimulation (ONS) pulse width to evoke maximum twitch contractile force.
Isometric muscle tension force was recorded in response to 5 repeated ONS pulses, with varying pulse width from 1ms – 25ms (A); higher temporal resolution images of ONS evoked muscle twitch …
Figure 5—figure supplement 4
Optimization of optical nerve stimulation (ONS) pulse pattern to evoke maximum tetanic contractile force.
Repetitive tetanic contractions following delivery of custom designed 25Hz ONS pulse patterns, in a ChR2+ motor neuron engrafted SOD1G93A mouse; pulse width varied from 5 to 20ms and pulse interval …
Figure 5—figure supplement 5
Daily optical stimulation training (OST) in SOD1G93A mice does not affect muscle contractile characteristics in response to acute optical nerve stimulation (ONS).
Automated peak analysis of brief twitch contractions elicited by acute optical stimulation at the experimental end-point revealed that OST did not significantly alter any of the following muscle …
Figure 5—figure supplement 6
Comparison of optical nerve stimulation (ONS) versus electrical nerve stimulation (ENS) in late-stage SOD1G93A mice shows that supramaximal ENS still induces stronger contractile force, even after optical stimulation training (OST).
(A) Comparison of maximal twitch and tetanic force values, acquired from the same TS muscle in each animal in response to either supramaximal ONS or ENS stimuli delivered at specified pulse …
Figure 6 with 1 supplement
Optical stimulation training prevents atrophy of muscle fibres that have been reinnervated by ChR2+ motor neurons in late-stage SOD1G93A mice.
(A) Confocal tile-scan showing a single longitudinal section through the triceps surae muscle of 135d SOD1G93A mouse (35d post-engraftment), following daily OST; endplates (labeled with αBTx) …
Figure 6—figure supplement 1
Daily optical stimulation training (OST) appears to enhance the extent of innervation of end-plates by engrafted ChR2+ motor neurons.
Confocal images showing examples of intramuscular nerves and end-plates, within the triceps surae muscle, innervated by engrafted ChR2+ motor neurons in late-stage SOD1G93A mice (n = 3) that had …
Videos
Video 1
3D reconstruction of innervated endplates from SOD1G93A mice in the absence of immunosuppression.
Video 2
FK506 facilitates graft survival but allows intraneural tumour formation that causes severe motor dysfunction.
Video 3
3D reconstruction of an entire triceps surae muscle group from a late-stage SOD1G93A mouse, after ChR2+ motor neuron engraftment showing extent of reinnervation.
Video 4
3D reconstruction of individual endplates (red) reinnervated by engrafted ChR2+ motor neuron (green) in a 135d SOD1G93A mouse (35d post-engraftment) in combination with transient H57-597 mAb treatment.
Video 5
Daily optical stimulation training significantly enhances elicited muscle force in SOD1G93A mice.
Video 6
3D visualization of longitudinal muscle section from an engrafted SOD1G93A mouse along with ‘dCALMS’ muscle fiber analysis technique.
Tables
Appendix 1—table 1
Single Nucleotide Polymorphism (SNP) analysis of Clone #C9G and control mESC lines to confirm genetic background strain.
SNP analysis confirmed that mESC Clone #C9G, used in this study, originated from a different genetic background compared to host mice (C57Bl/6J background strain). C57BL/6J mESCs and HBG3 mESCs were …
| Conformity of Sample to Reference Strain Allelic Profile | |||||
|---|---|---|---|---|---|
| Sample ID-Code | Reference | # Called | Call Rate | Percent Match | Percent Het |
| 001-Clone C9G mESCs | 129S1SvImJ | 382 | 99.5% | 97.5% | 0.3% |
| 002-C57BL/6 J mESCs | B6J | 383 | 99.7% | 99.7% | 0.0% |
| 003-Clone HBG3 mESCs | B6J | 377 | 98.2% | 62.5% | 10.9% |
Appendix 1—table 2
Summary of data from mice treated with FK506, including graft outcome (where applicable).
List of experimental mice, including genotype, sex, age at start and end of experiment, initial and final body mass and % change (values that decreased or failed to increase are highlighted in red). …
| ID | Genotype | Sex | Age (g) | Body mass (g) | δ BM (%) | Onset (days) | Tumor | ||
|---|---|---|---|---|---|---|---|---|---|
| Start | End | Initial | Final | ||||||
| 14.1 c | Wild-Type | M | 71 | 93 | 24.8 | 26.3 | 106.0 | - | * |
| 14.1 h | Wild-Type | F | 71 | 98 | 19.1 | 20.5 | 107.3 | 82 | - |
| 14.1 a | SOD1G93A | M | 71 | 100 | 26.5 | 25.5 | 96.2 | 82 | ** |
| 14.1b | SOD1G93A | M | 71 | 101 | 25.6 | 26 | 101.6 | 82 | ** |
| 14.1 g | SOD1G93A | F | 71 | 98 | 19.4 | 19.1 | 98.5 | 80 | * |
| 14B.1a | SOD1G93A | M | 57 | 83 | 25.2 | 21 | 83.3 | 69 | *** |
| 14B.1b | SOD1G93A | M | 57 | 101 | 26.3 | 27.2 | 103.4 | 70 | ** |
| 14B.1c | SOD1G93A | M | 92 | 121 | 28.2 | 28.4 | 100.7 | 101 | not recorded |
| 14B.2a | SOD1G93A | F | 90 | 124 | 21.1 | 22.9 | 108.5 | 104 | not recorded |
| 14B.2b | SOD1G93A | F | 90 | 124 | 20.8 | 22.3 | 107.2 | 104 | not recorded |
| 14B.2c | SOD1G93A | F | 90 | 127 | 20.2 | 22.2 | 109.9 | 104 | not recorded |
| 14B.2d | SOD1G93A | F | 90 | 120 | 21.9 | 22.2 | 101.4 | 104 | not recorded |
| 14B.2e | SOD1G93A | F | 90 | 117 | 22.4 | 20.7 | 92.4 | 104 | *** |
| 13B.1a | SOD1G93A | M | 90 | 113 | 24.2 | 21.1 | 87.2 | 101 | ** |
| 13B.2b | SOD1G93A | M | 85 | 112 | 23.8 | 23.5 | 98.7 | 101 | ** |
| Animals listed below underwent FK506 treatment in the absence of intraneural engraftment | |||||||||
| 20.1d | Wild-Type | F | 101 | 131 | 19.2 | 20.95 | 109.1 | Not applicable | |
| 20.1 f | Wild-Type | F | 101 | 131 | 21.3 | 23.3 | 109.4 | ||
| 20.1 h | Wild-Type | F | 101 | 131 | 18.1 | 21.2 | 117.1 | ||
| 33.1e | Wild-Type | F | 95 | 128 | 21.1 | 22.5 | 106.6 | ||
| 33.1 f | Wild-Type | F | 95 | 128 | 22 | 22.5 | 102.3 | ||
| 33.1 g | Wild-Type | F | 95 | 128 | 21.3 | 21 | 98.6 | ||
| 33.1b | Wild-Type | M | 95 | 107† | 26.7 | 24 | 89.9 | ||
| 20.1e | SOD1G93A | F | 101 | 131 | 17.5 | 19.8 | 113.1 | ||
| 20.1 g | SOD1G93A | F | 101 | 131 | 17 | 21.2 | 124.7 | ||
| 33.1 a | SOD1G93A | M | 95 | 111† | 25.8 | 23 | 89.1 | ||
| 33.1 c | SOD1G93A | M | 95 | 128 | 24.7 | 25.8 | 104.5 | ||
| 33.1d | SOD1G93A | M | 95 | 128 | 23.2 | 23.2 | 100.0 | ||
Appendix 1—table 3
Summary of data following in vivo engraftment of ChR2+ motor neurons in SOD1G93A mice.
Table shows a full list of all experimental animals reported in the optical stimulation section of this study, including animal ID, sex, age at start and end of study period, duration of graft, body …
| 2*ID | 2*Sex | Age (g) | 2*Duration (d) | BM (g) | 2*Δ BM (%) | 2*Max Force (g) | ||
|---|---|---|---|---|---|---|---|---|
| Start | End | Start | End | |||||
| Fast-firing Motor neurons (derived from 7DD dissociated EBs pretreated with MMC) | ||||||||
| 21.1 a | M | 103 | 139 | 36 | 25.2 | 23 | 91.3 | 0.39 |
| 21.1e | M | 103 | 140 | 37 | 23.3 | 21.7 | 93.1 | 0.33 |
| 25A1b | M | 118 | 137 | 19 | 28.3 | 23.2 | 82.0 | 0.55 |
| 25 A.1d | M | 118 | 137 | 19 | 27 | 23.2 | 85.9 | 0.54 |
| 24.2 a | M | 117 | 137 | 20 | 27.2 | 23.6 | 86.8 | 0.57 |
| 25B.1b | M | 109 | 135 | 26 | 24.2 | 21 | 86.8 | 0.47 |
| 25 C.1a | M | 101 | 137 | 36 | 26.3 | 24.5 | 93.2 | 0.88 |
| 21.1 f | F | 103 | 140 | 37 | 20.8 | 19 | 91.3 | 0.43 |
| 22.1d | F | 100 | 120 | 20 | 20.5 | 19.5 | 95.1 | 1.10 |
| 22.1e | F | 100 | 121 | 21 | 23.4 | 20.4 | 87.2 | - |
| 24.2d | F | 114 | 137 | 23 | 19.5 | 18.2 | 93.3 | 0.50 |
| 25B.1c | F | 109 | 135 | 26 | 19.8 | 19.2 | 97.0 | 1.04 |
| 25 C.1d | F | 100 | 122 | 22 | 18.5 | 18.6 | 100.5 | 1.11 |
| 25 C.1f | F | 100 | 138 | 38 | 20.2 | 18.5 | 91.6 | 1.48 |
| Average: | 106.8±7.2 | 133.9±7.2 | 27.1±7.8 | 23.2±0.7 | 21.0±0.9 | 91.1±1.6 | 0.72±0.1 | |
| Slow-firing Motor neurons (derived from 5DD dissociated EBs pretreated with MMC) - untrained | ||||||||
| 28.2d | F | 97 | 142 | 45 | 20.3 | 18.4 | 90.6 | 0.57 |
| 28.2 f | F | 97 | 142 | 45 | 20.2 | 17.8 | 88.1 | 0.63 |
| 28.1b | F | 104 | 136 | 32 | 22 | 19.3 | 87.7 | 0.33 |
| 26.1 a | M | 104 | 143 | 39 | 24.8 | 22.3 | 89.9 | 0.24 |
| 28.2b | M | 97 | 128 | 31 | 26.2 | 23.1 | 88.2 | 1.26 |
| 31.1 c | M | 95 | 137 | 42 | 28 | 23.9 | 85.4 | 0.64 |
| 1.1 a | M | 90 | 125 | 35 | 25.9 | 26 | 100.4 | 1.31 |
| 1.1b | M | 90 | 126 | 36 | 28.9 | 30.1 | 104.2 | 2.24 |
| 35–1 c | M | 93 | 124 | 31 | 26.4 | 25.9 | 98.1 | 0.22 |
| 35-1b | M | 93 | 130 | 37 | 29 | 28.3 | 97.6 | 0.97 |
| 35-1e | M | 93 | 130 | 37 | 23.7 | 24 | 101.3 | 0.46 |
| 40–1 c | M | 95 | 133 | 38 | 24 | 23 | 95.8 | - |
| Average: | 95.7±4.6 | 133±6.9 | 37.3±4.8 | 25.0±0.7 | 23.5±0.9 | 93.9±1.7 | 0.81±0.18 | |
| Slow-firing Motor neurons (derived from 5DD dissociated EBs pretreated with MMC)+optical stimulation training | ||||||||
| 28.2 c | M | 97 | 128 | 31 | 25.6 | 24.4 | 95.3 | 8.96 |
| 1.1 c | M | 90 | 121 | 31 | 23.4 | 22.6 | 96.6 | 6.81 |
| 35-1d | M | 93 | 131 | 38 | 27.4 | 28 | 102.2 | 8.69 |
| 40–1 a | M | 95 | 132 | 37 | 25.1 | 24.2 | 96.4 | 3.60 |
| 38–1 c | F | 93 | 136 | 43 | 20.2 | 19.8 | 98.0 | 10.67 |
| 38.1e | F | 95 | 142 | 47 | 22.4 | 19 | 84.8 | 8.73 |
| 45.1 c | F | 96 | 137 | 41 | 21.6 | 21.4 | 99.1 | 4.99 |
| Average: | 94.1±2.3 | 132.4±6.8 | 38.3±6.0 | 23.7±1.0 | 22.8±1.2 | 96.1±2.2 | 7.49±0.94 | |
