Myelination is a quintessential part of the developing nervous system. The Macklin lab has investigated the mechanisms of mTOR signaling in oligodendrocyte differentation for many years. This project has been a collaborative project with Dr. Teresa Wood's laboratory at Rutgers University. These studies are being continued, and there are currently has several projects investigating developmental myelination mechanisms. 

1. Regional differences in mTOR signaling regulating myelination in brain relative to spinal cord. Loss of Raptor (mTORC1) signaling impacts spinal cord myelination dramatically, while loss of Rictor (mTORC2) signaling primarily impacts myelination in corpus callosum. Investigations on the impact of these signaling pathways on cytoskeletal organization are underway. 

2. Initialization of myelin wrapping. Previous studies in zebrafish demonstrated that oligodendrocytes sample an axon several times before committing to ensheathment (Almeda et al., 2023). We are now further investigating potential mechanisms driving oligodendrocyte wrapping of axons, including endocytosis and exocytosis, using both in vitro neuronal-oligodendrocyte co-cultures and in vivo systems.

3. Activity dependent myelination in development. Neuronal activity is known to mediate myelination in adulthood; however its effects during development are not well understood. We are using DREADDs to inhibit or activate cortical neuronal activity, and studying the subsequent effects on oligodendrocyte differentiation and myelination during early development. This project utilizes in vitro neuronal-oligodendrocyte co-cultures and in vivo model systems.

Intrathecal B-cell production of antibodies is a hallmark of MS diagnosis, but the pathogenic role of these antibodies remains unknown. In collaboration with Drs. Jeffrey Bennett and Gregory Owens at CU Anschutz, we are studying how myelin-binding human recombinant antibodies from MS patient cerebrospinal fluid affect oligodendrocytes, demyelination, and remyelination using in vitro, ex vivo, and in vivo models. 

In vitro, we are investigating direct effects of MS recombinant antibody on oligodendrocyte differentation, maturation, and myelin production. 

Ex vivo, we use MS recombinant antibody plus human complement to demyelinate cerebellar slice cultures. With this controlled system, we can study mechanisms of demyelination and remyelination with myriad manipulations such as continued antibody exposure or microglial loss. 

We are also developing an in vivo model of antibody-mediated demyelination. Direct injection of the MS recombinant antibody plus human complement into the corpus callosum generates a transient demyelinating lesion that can be used to further investigate the in vivo implications of our in vitro and ex vivo studies. 

Stroke induces robust cell death and demyelination in adults; however, juveniles are somewhat protected from stroke-mediated damage (Ahrendsen et al., 2016). The juvenile brain is in an active state of myelination, while the adult brain has low levels of active myelination. This difference in myelination status could be driving juvenile protection from stroke. 

In vivo, we are using the MCAO stroke model (in collaboration with Drs. Paco Herson and Nidia Quillinan) to investigate whether actively myelinating adult mice (transgenic mice expressing constitutively expressing AKT in oligodendrocytes, Flores et al., PMID:18614687) are protected from stroke-induced damage as actively myelinating juvenile mice are. 

In vitro, we are investigating signaling pathways associated with stroke response in primary oligodendrocyte cell cultures subjected to oxidative damage via oxygen deprivation.