CJH332H1 Lecture Notes - Lecture 17: Cholera Toxin, Axon, Atrx
Lecture 17: Neural Stem Cell Transformation
What happens to repair in aging?
• Age related sprouting transcriptome provides molecular control of axonal sprouting after stroke
• Looking at single cell changes in most labs now (cellular molecular neuroscience)
• Transcriptome (older technique) to examine individual cells
What happens in aging?
• Adult rai ad CN“ orall does’t repair itself easil as it does in younger brains
• However, interestingly, after stroke, there appears to be an increase in axonal sprouting in the adult brain but
doesnt seem to lead to functional recovery as seen in younger animals
• This becomes important as there are very few therapeutic interventions that can be performed after stroke
• Molecular studies of axonal sprouting in CNS injury have focused mostly on proteins that inhibit the formation of
new connections, such as myelin-associated proteins and glial extracellular matrix molecules (Lingo1 and Nogo
receptor-1 and proteoglycans)
- Identify genes that we can exogenously turn on
CNS overview – lack of regeneration
• Damaging peripheral neurons (leaving SC also PNS) can regenerate
itself – slower in aging but still can repair itself
- After axonal injury, distal debris/downstream of injury can be
eliminated by different macrophages
- We do’t hae ihiitor oleules to ihiit this ao fro
growing out again – can regenerate
• Damaging CNS does not repair itself – macrophages not present to clear debris, and has inhibitory molecules
that inhibit axonal regeneration (nogo, MAG, OGmp) and connecting with targets, astrocytes secrete factors
that physically prevent (reactive astroglial scar) axonal growth/crossing
- Nogo is released from oligodendrocytes after their injury
Main elements of this paper
• Present paper by Li et al, develop method to selectively label sprouting neurons in peri-infarct (penumbra)
cortex after stroke
• Genetic profiles of sprouting neurons are compared to non-sprouting neurons from the same rat, from the same
brain region which were exposed to the same stroke conditions
• They were able to identify a sprouting transcriptome after stroke that changed with time after injury, differed
between young and aged rats and mice, and contained molecules that had different mechanistic roles in the
process of brain reorganization after stroke
Paper summary overview
• Cortex connects to neuronal cell body in layer II and III which makes
synaptic connections to other groups of neurons in the same layer
(glutamatergic)
• Deeper cortical neuronal cell body (layer V) sends their projections to
other groups of neurons deeper within different brain structures
• Causing damage/stroke (area of infarct), all the neurons in the damaged area all die (no opportunity to repair)
- Within penumbral areas, there are cell bodies that might re-sprout (more likely to regain functional
connectivity and survive)
find more resources at oneclass.com
find more resources at oneclass.com
• Spontaneous growth of new horizontal connections in layers II/III and over distances of up to several millimeters
• Aged rates upregulated transcriptional regulator ATRX and insulin-like growth factor (IGF-1)
• Also upregulated receptor components for the neurite growth inhibitor NogoA, NgR1 and Lingo1
• Stroke was produced in the whisker (barrel) field of the somatosensory cortex in both young adult and aged rats,
which induces a region of axonal sprouting in peri-infarct sensorimotor cortex
• To label neurons that sprout and established new projection pattern in peri-infarct cortex, authors injected two
fluorescent conjugates of the tracer cholera toxin B (CTB) into an adjacent, non-stroked sensorimotor cortex
• CTB back-labels neurons that project to the injection site – it is a retrograde tracer (axons to neuronal cell body)
- This shows that sprouting occurs in living tissue (not after the fact)
- Labels the neuron that the axonal injection was made to
Rapid, retrograde transport of CTB
• Layer 2-3 projections will cross midbrain to opposite side (callosal projection neurons)
- Injection of CTB on right will have neurons light up on the other side
Stroke, injections and follow up
• Stroke was produced by permanently occluding two anterior branches of the distal MCA over the parietal cortex
(all the neurons at the occlusion will die)
• Grey zone shows the area of stroke induced cell death of neurons
• Green represent nearby healthy somatosensory cortex – can peripheral regions functionally recover/regenerate
• Inject CTB in the nearby regions of the grey zone (penumbra) at the time of stroke and then again at either 7d or
21d after stroke
- Labeling with different colors to show which axons were there at the beginning of stroke and which are new
ones (to compensate the loss of axonal connections due to stroke)
Labeling and laser capture
• Young adult sprouting neurons and sprouting neurons from an aged brain
• Left column – neurons labeled by the first tracer injection (CTB in red)
• Middle column – neurons labeled by second tracer injection (CTB in green)
- These have adapted to stroke, looking like they are regenerating
• Right column – neurons seen after laser capture of a tracer 2 (only neuron)
- Cut out the tissue with ONE cell body into the container to use for genome sequencing – great precision
(only neurons regenerating and only ONE cell)
• Neurons with double label (non-sprouting neurons; arrowheads) or labeled by CTB-Alexa 647 (tracer 2-only,
sprouting neurons; arrows) were separately laser captured in peri-infarct cortex
• Neurons that were double labeled from the two separate (initial and later) tracer injections were those that did
not change their projection to the injection site afters stroke (non-sprouting group)
• Neurons that took up only the second tracer were those that did not originally have an axonal projection to the
injection site at the time of the injection of the first tracer (time of stroke) and thus established a new projection
pattern after stroke (sprouting group)
• Compare the gene expression/RNA profiles of the non-sprouting vs sprouting groups
Genetic profiles
• Whole genome expression analysis was used to identify the distinct transcriptional profile of a sprouting neuron
in peri-infarct cortex after stroke
• Found that the majority of differential gene regulation in axonal sprouting after stroke had taken place by day 7
(under week) and also found that more genes were differentially expressed in younger than in older animals
find more resources at oneclass.com
find more resources at oneclass.com
Document Summary
What happens to repair in aging: age related sprouting transcriptome provides molecular control of axonal sprouting after stroke, transcriptome (older technique) to examine individual cells. Looking at single cell changes in most labs now (cellular molecular neuroscience) Identify genes that we can exogenously turn on. Cns overview lack of regeneration: damaging peripheral neurons (leaving sc also pns) can regenerate itself slower in aging but still can repair itself. After axonal injury, distal debris/downstream of injury can be eliminated by different macrophages. Nogo is released from oligodendrocytes after their injury. This shows that sprouting occurs in living tissue (not after the fact) Labels the neuron that the axonal injection was made to. Layer 2-3 projections will cross midbrain to opposite side (callosal projection neurons) Injection of ctb on right will have neurons light up on the other side. Inject ctb in the nearby regions of the grey zone (penumbra) at the time of stroke and then again at either 7d or.
