Characterizing the Mechanism of Young Bone Marrow-derived Microglia-like Cells on Restoring Cognitive Function in the Aged Brain and After Ischemic Injury

Neurodegeneration in the aged population is the leading cause of death, disability, and impaired quality of life worldwide. Among the many proposed mechanisms for neurodegenerative diseases, emerging evidence indicate microglial deterioration to be at the center of this. Here, microglia in the aged brain become primed and acquire a pro-inflammatory phenotype marked by increased secretion of inflammatory chemokines and cytokines. This in turn can lead to synaptic pruning, neuronal death, and cognitive impairments. In this work I propose a novel approach to address this important issue: bone marrow (BM) reconstitution with young stem cell antigen-1 (Sca-1)-expressing hematopoietic stem cells (HSCs) for the introduction of healthy blood borne-derived microglia-like cells into the aged brain for attenuation of age- and injury-induced loss of neuronal and cognitive function. My findings reveal that transplanted young HSCs repopulate the aged recipient BM and home to the brain to ultimately give rise to several thousand microglia-like cells in regions of the brain involved in neurogenesis, learning and memory, reward circuitry, and emotional regulation. Multicolor flow cytometry revealed these cells to be distinct from brain-resident microglia, while immunofluorescence staining demonstrated young and old BM-derived microglia-like cells to be positive for markers associated with anti- and pro-inflammatory phenotypes, respectively. These BM-derived microglia-like cells were seen to populate the hippocampus 1- and 3-months post-reconstruction, effectively reversing age- and ischemic injury-induced loss of novel object recognition and spatial memory and learning. The presence of these cells was significantly diminished from the hippocampus 3-months post-reconstitution; however the benefits in behavior, synaptic plasticity, and dendritic spine density in pyramidal neurons persisted. Selective BM-derived microglia-like cell depletion at 1-month post-reconstitution using generated Cx3cr1CreER/+R26iDTR/+ mice revealed the necessity of these cells as there was a loss-of-function on behavioral performance. RNA and protein screening on ex vivo microglia-like cells revealed significant upregulation in brain-derived neurotrophic factor (BDNF) and proteins involved in cytoskeletal remodeling. Blocking BDNF binding in reconstituted animals 1-month post-transplantation using the antagonist, ANA-12, abolished functional improvements and dendritic spine arborization. In summary, I demonstrate the sufficiency and necessity of young, but not old, BM-derived microglia-like cells on restoring cognitive capacity in aged recipients.Ph.D