Proposal Concepts

Question 1

General question

Answer 1

Researchers do not fully understand the underlying mechanisms that determine differences in intrinsic regenerative abilities across different neuron types (Ertürk et al., 2007).

Question 2

PDK1

Answer 2

regulates AKT pathway, regulates insulin and growth factor kinases, neurogenesis, neuronal migration

Through its phosphorylation targets, which include protein kinase B (Akt) and protein kinase C, PDK1 plays an essential role in cell survival, proliferation, and migration (Kim et al., 2021). The kinase has also been linked to the DLK/c-Jun pathway, known to be involved in neuronal homeostasis, apoptosis, and axon degeneration (Ghosh et al., 2011).

Question 3

Specific question

Answer 3

We are interested in understanding how PDK1 activity and regulation promote growth cone or retraction bulb formation in axons with differential regenerative abilities.

Question 4

Experimental approach broadly

Answer 4

we will use neurons from the CNS (retinal ganglion cells (RGCs)) that do not have regenerative capabilities and neurons from the PNS (dorsal root ganglion cells (DRGs)) that have regenerative capabilities. We will employ transgene constructs with GFP combined with different elements of the PDK1 transcript to elucidate PDK1 translational regulation (upstream regulators) and use different genetic and imaging techniques, including CRISPR, fluorescence microscopy, and Western blots, to study downstream pathways.

Question 5

Aim 1

Answer 5

Our first aim is to determine how PDK1 regulates microtubule polymerisation and destabilization post-injury.

Question 6

Aim 1 model

Answer 6

Model: PDK1 activates (either directly or by some intermediate) GSK3b which deactivates by phosphorylation CRMP2, which normally stabilizes MTs by preventing detyrosination but is inhibited when it is phosphorylation.

Question 7

Aim 1 Rationale

Answer 7

However, the exact molecular pathways and mechanisms that underlie the process by which MTs are destabilized is a controversial field. One molecule of interest is the GSK3β, which has been implicated in microtubule stability and changes in axonal regenerative abilities (Akram et al., 2022). Interestingly, the exact role of GSK3β in axon growth initiation is not completely understood due to contradicting reports as to whether GSK3 inhibition promotes or prevents axon regeneration. It has been postulated that its role may actually be dependent on physiological parameters, including type of neuron and growth environment (Diekmann & Fischer, 2015).

Honed in on the GSK3β/CRMP2 pathway. GSK3β has been shown to be upregulated in retinal ganglion cells (RGCs), a type of CNS neuron that doesn’t normally regenerate (Leibinger et al., 2017). GSK3β acts upstream of CRMP2, which, when active, binds to tubulin heterodimers to promote MT organization and stability (Akram et al., 2022). GSK3β phosphorylates CRMP2 in an inhibitory fashion, which prevents CRPM2 from exerting its MT stabilizing abilities and as a result, reduces the ability of these neurons to regenerate their axons.

Given that PDK1 may act upstream of GSK3β and that lower PDK1 levels and lower GSK3β levels are indicative of stronger regenerative potential, we hypothesize a model where PDK1 acts upstream of GSK3β to have some effect on axon regeneration.