• Cell reprogramming

Motor deficits caused by stroke represent one of the crucial causes of disability worldwide. Around 70-80% of patients survive the ischemic attack, but over 50% of stroke survivors experience a persistent disability in skilled movements of the upper extremities. Since rehabilitation is not that effective at re-acquiring motor function, new regenerative and plasticizing treatments are strongly needed.
One recent and appealing strategy to stimulate recovery from stroke is the replacement of the lost-nervous tissue with neurons obtained by direct reprogramming of endogenous reactive astrocytes resident in the perilesional area. Since these cells are able to proliferate in response to a brain injury spontaneously, they represent an eligible candidate to be converted into neurons.
In this thesis, I tested the effect of using reprogramming transcription factors to directly convert reactive astrocytes into new neurons after a focal cortical ischemic injury in the primary motor cortex.
I used a genetically modified strain of C57BL6J mice, namely GFAP-Cre mice, in which the Cre recombinase protein is expressed under the control of the Glial Fibrillary Acidic Protein (GFAP) promoter. The reprogramming and reporter (green fluorescent protein, GFP) genes were delivered in the mouse motor cortex via injection of flexed Adeno-associated viruses (AAVs), allowing the expression only in glial cells expressing Cre recombinase. 3 days after the photothrombotic lesion in the Caudal forelimb area (CFA), the neurogenic determinants were administered in the perilesional cortical area. 60 days after the injection, a remarkable percentage of newly generated neurons among the total GFP-positive (originally astrocytes) cells was detected.
To evaluate the effect of reprogrammed neurons on motor recovery, I performed two different behavioral tests: the cylinder test and the gridwalk test. Reprogramming protocol was combined with rehabilitation which may induce a significant enhancement of motor function in ischemic mice. To assess this hypothesis, GFAP-Cre mice were trained to perform a retraction task on the M-Platform, a robotic device developed to study the neural correlates of post-stroke rehabilitation. Data from both tests showed a significant motor function recovery in animals that received the reprogramming factors and physical rehabilitation compared to controls that only performed physical rehabilitation.
Furthermore, I ran an immunohistochemical analysis to investigate the astrocytic origin and quantify the pool of cells that ideally converted into newborn neurons. The ideal target corresponds to cells that constitutively express the green fluorescent protein, suggesting an astrocytic origin, which is also positive for neuronal markers.
In conclusion, direct reprogramming of endogenous astrocytes in the mouse motor cortex gives rise to a new neuronal population that hopefully, as supported by previous studies, acquires a motor identity, integrates into the host tissue, and supports the recovery of function of the affected forelimb.