Neurons in the CNS lose regenerative potential with maturity, leading to minimal corticospinal tract (CST) axon regrowth after spinal cord injury (SCI). In young rodents, knockdown of PTEN, which antagonizes PI3K signaling by hydrolyzing PIP3, promotes axon regeneration following SCI. However, this effect diminishes in adults, potentially due to lower PI3K activation leading to reduced PIP3. This study explores whether increased PIP3 generation can promote long-distance regeneration in adults. We used a hyperactive PI3K, PI3Kδ (PIK3CD), to boost PIP3 levels in mature cortical neurons and assessed CST regeneration after SCI. Adult rats received AAV1-PIK3CD and AAV1-eGFP, or AAV1-eGFP alone, in the sensorimotor cortex concurrent with a C4 dorsal SCI. Transduced neurons showed increased pS6 levels, indicating elevated PI3K/Akt/mTOR signaling. CST regeneration, confirmed with retrograde tracing, was evaluated up to 16 weeks post injury. At 12 weeks, ∼100 axons were present at lesion sites, doubling to 200 by 16 weeks, with regeneration indices of 0.1 and 0.2, respectively. Behavioral tests showed significant improvements in paw reaching, grip strength, and ladder-rung walking in PIK3CD-treated rats, corroborated by electrophysiological recordings of cord dorsum potentials and distal flexor muscle electromyography. Thus, PI3Kδ upregulation in adult cortical neurons enhances axonal regeneration and functional recovery post SCI.

Center for Advanced Preclinical Imaging 1st Faculty of Medicine Charles University Salmovska 3 120 00 Prague Czech Republic

Institute of Experimental Medicine CAS Department of Auditory Neuroscience Videnska 1083 142 20 Prague Czech Republic

Institute of Experimental Medicine CAS Department of Neuroregeneration Videnska 1083 142 20 Prague Czech Republic

Institute of Experimental Medicine CAS Department of Neuroregeneration Videnska 1083 142 20 Prague Czech Republic; Institute of Biology and Ecology Faculty of Science P J Safarik University in Kosice Srobarova 2 Kosice 041 54 Slovak Republic

John van Geest Centre for Brain Repair Department of Clinical Neurosciences Cambridge Institute for Medical Research University of Cambridge Cambridge CB2 0XY UK

John van Geest Centre for Brain Repair Department of Clinical Neurosciences Cambridge Institute for Medical Research University of Cambridge Cambridge CB2 0XY UK; Institute of Experimental Medicine CAS Centre for Reconstructive Neuroscience Videnska 1083 14220 Prague Czech Republic

Kings College London Wolfson Sensory Pain and Regeneration Centre Guy's Campus London Bridge London SE1 1UL UK

Netherlands Institute for Neuroscience Meibergdreef 47 1105 BA Amsterdam the Netherlands

School of Biomedical Sciences University of Leeds Leeds LS2 9JT UK; Institute of Experimental Medicine CAS Centre for Reconstructive Neuroscience Videnska 1083 14220 Prague Czech Republic

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