Functional and cellular effects of retrogradely delivered constitutively active Ras homolog enriched in brain, in coordination with and independent of robot rehabilitation and subthreshold optogenetics

Jennifer Dawn Pastorino

doi:10.17918/00011231

Adult spinal cord injury (SCI) causes severe tissue loss and neuronal degradation, often leading to loss of motor and sensory function. Previous regenerative strategies have targeted the mammalian target of rapamycin (mTOR) pathway through Phosphatase and TENsin homolog (PTEN) silencing, promoting axonal growth but also inducing adverse effects such as tumor formation, seizures, and neuronal hypertrophy. To overcome the limitations of a full t9/t10 transection injury, we explored constitutively active Rheb (CaRheb) as a direct activator of mTOR signaling. Using a retrogradely transported adeno-associated virus, we targeted descending corticospinal neurons to promote regeneration while avoiding hypertrophy. Soma hypertrophy may hinder the recruitment process, following the Size Principle, where the decreased resistance in the postsynaptic cell requires increased synaptic input. Therefore, smaller cells would get recruited as opposed to the larger cells that have regenerated. To enhance recovery, we combined this approach with two therapies: subthreshold optogenetic stimulation to support cortical plasticity, and a six-week robotic rehabilitation paradigm to reinforce functional improvements. We hypothesize that functional kinematic scores will be significantly higher, there is more body weight support percentage, and that animals will exhibit collapse in function less frequently when receiving retrograde CaRheb and Robot Rehabilitation. Furthermore, that each measure will significantly improve with subthreshold optogenetics added. We also hypothesize no significant difference between cell soma size between any animal group. Animals receiving retrograde CaRheb and robotic rehabilitation exhibited significant locomotor improvements without evidence of soma hypertrophy, tumorigenesis, or seizures. While the addition of subthreshold optogenetic stimulation did not further enhance functional outcomes, co-transfection with CaRheb and Channelrhodopsin-2 (ChR2) moderately, but significantly increased pyramidal soma size in cage rest rats, an effect mitigated by robot rehabilitation. Overall, these findings demonstrate that retrograde CaRheb delivery combined with robotic rehabilitation effectively promotes functional recovery, and potentially regeneration, after SCI while minimizing adverse effects associated with PTEN suppression. In contrast, cortical optogenetic stimulation did not provide added benefit in this model, suggesting a ceiling effect reached with CaRheb and Robot Rehabilitation. We discuss here the significant functional improvements of animals receiving retrograde CaRheb in combination with Robot Rehabilitation, without hypertrophy of cells. Results from this study support the interest in various regenerative therapies to improve functional outcomes, and initiate interest in future directions of tracing, electrophysical properties, and sholl analysis of cells transfected with CaRheb. The outcomes presented here will further knowledge of mTOR-mediated regeneration, supporting the option of retrograde CaRheb in functional recovery when combined with Robot Rehabilitation.

Functional and cellular effects of retrogradely delivered constitutively active Ras homolog enriched in brain, in coordination with and independent of robot rehabilitation and subthreshold optogenetics

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Functional and cellular effects of retrogradely delivered constitutively active Ras homolog enriched in brain, in coordination with and independent of robot rehabilitation and subthreshold optogenetics

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