NEURONAL GROWTH PROGRAM

Neurons in cortex adjacent to the infarct can sprout new connections. This process is remarkable in cortex in the adult brain, as in most cases the connections of adult cortex are static. Sprouting neurons within the adult cortex activate neuronal growth-associated genes during phases of axonal sprouting after stroke.  However, the specific molecular growth program, or regeneration transcriptome, has until recently not CTb labeled neurons in cortexbeen determined. This is in large part because the neurons that form a new connection after stroke are small in number and imbedded within cortical circuits in which most neurons do not engage in axonal sprouting. In order to identify the molecular growth program after stroke, the neurons that actually utilize this program must be isolated and studied. How does one do this? In other models of nerve regeneration, the regenerating neurons can be isolated with relative ease, such as in the dorsal root ganglion or the retina for the regenerating optic nerve. In cortex this is not possible without selectively labeling sprouting neurons after stroke. The Carmichael lab developed a way to do selectively label sprouting neurons after stroke with sequential injections of axonal tracers. The figure at right shows a low power view of neurons back-labeled in cortex after stroke by sequential injections of Alexa 488-CTb (green) at the time of stroke, and Alexa647-CTb 21 days later (red). Red-only cells have establishec a connection with the injection site that was not present at the time of the stroke.

 These sprouting neurons can be selectively isolated with laser capture microdissection and their gene expression profile compared to adjacent neurons that do not sprout. We did this with neurons at different stages of the sprouting process and in aged and young adult animals. This last approach is important because stroke is a disease predominantly of aged humans (greater than 65 years old) and this is the translational goal of stroke neural repair studies. The figure below shows a photomicrograph taken from the laser capture miicroscope of a tissue section in which CTb647 was injected at the time of the stroke and CTb-488 was injected 21 days later in an aged animal. The arrow shows a CTb-488 only neuron that was cut out with laser capture. The arrowheads show double labeled neurons that were cut out (not shown) and used for an mRNA source in a comparison gene expression analysis to sprouting neurons (Li et al. Nature Neurosciece 2010).

 Laser Capture Aged Sprouting Neurons

Gene expression analysis of sprouting neurons after stroke produced the regeneration transcriptome of an adult cortical neuron as it varies by time during the regeneration process, and by age. Mechanistic studies of select molecules in this regeneration transcriptome confirm their role in post-stroke axonal sprouting. Several interesting features have come out of this data set. Sprouting neurons activate gene systems that regulate chromatin structure (ATRX) and thus function at an epigenetic level to allow transcriptional changes in cassettes of regeneration-associated genes. Aged sprouting neurons paradoxically upregulate receptors for growth collapsing proteins (EphA4). Neurons that sprout new connections after stroke are dependent on growth factor support for extended periods after the stroke, and will die if this is not available. This last point is seen with IGF-1. Aged sprouting neurons, or the tissue immediately surrounding them, express a conjointly regulated molecular network related to IGF-1 at day 7 after stroke. This network is then no longer regulated at day 21 after stroke. This data indicate that sprouting neurons express an IGF-1 regulated gene network for a several week period after stroke that is essential to survival of the sprouting neurons.

IGF1 network 7d after stroke

A network of genes related to IGF-1, spanning cell membrane interactions to nuclear transcriptional control, is induced by stroke in sprouting neurons inthe aged brain at 7 days after stroke.

IGF1 network 21d after stroke

This same network of genes is no longer differentially regulated in sprouging neurons at day 21 after stroke. 

 

Future studies in this project will determine the role of additional genes in the regeneration transcriptome, how to harness them for a neural repair therapy, and the interaction of regeneration-associated genes with behavioral activity after stroke.