Plasmalogens regulate the AKT-ULK1 signaling pathway to control the position of the axon initial segment.

da Silva FT, Granadeiro LS, Bessa-Noto D, Luz LL, Safronov BV, and Brites P. (2021) Plasmalogens regulate the AKT-ULK1 signaling pathway to control the position of the axon initial segment. Progress in Neurobiology

The axon initial segment (AIS) is an area on a neuron after the axon hillock and found close to the cell body. The AIS is enriched in ion channels and has an intricate cytoskeleton allowing it to generate action potentials. Action potentials are a change in membrane potential causing an impulse to be sent down an axon, alerting the axonal terminal to release neurotransmitters and signal other neurons. The location and size of the AIS is very important and it has been shown that injury, epilepsy, and some disease states can alter these. Plasmalogens are a class of lipids that contain a vinyl ether bond at sn-1 and this causes these lipids to have unique characteristics. This double bond is able to scavenge radial oxygen species giving plasmalogens anti-oxidative properties, they are crucial for membrane structure and organization, and aid vesicular fusion. When people are unable to synthesize plasmalogens, it results in rhizomelic chondrodysplasia punctata, a very rare disease characterized by developmental defects, shortened proximal bones, cataracts, epilepsy, spasticity, and cognitive impairment. In addition, a reduction in plasmalogens has been associated with Alzheimer’s disease, Parkinson’s disease, and multiple sclerosis. The exact role of lipid composition in AIS placement is unknown, but as plasmalogens are highly abundant in neurons da Silva et al investigated this relationship using the glyceronephosphate O-acyltransferase (Gnpat) knockout mouse model.

To determine if plasmalogens influenced the positioning of the AIS on a neuron, hippocampal neurons from wild-type mice and Gnpat KO mice were cultured and immunofluorescence of AIS markers such as ankyrin-G (AnkG), pan-Nav (for voltage-gated sodium channels), ADAM22 (a disintegrin and metalloproteinase domain-containing protein 22), and FGF14 (fibroblast growth factor 14) were analyzed. A difference in location was detected with the Gnpat KO neurons having a more distal shift in location of the AIS on the axons. The length of the AIS was not different between wild-type and Gnpat KO neurons, but the Gnpat KO neurons had a wider range of distribution with the AIS found over 10 mm away from the cell body while AIS on wild-type neurons were found much closer to the cell body. As alkyl glycerols are precursors in the plasmalogen biosynthetic pathway, batyl alcohol (BA; a naturally occurring 18:0 alkyl glycerol) treatment was found to partially recover the AIS positioning in Gnpat KO neurons, while 1-O-tetradecyl glycerol (TDG) caused a complete restoration of AIS location in these cells although not naturally found in animals. This was also seen in Gnpat mice when treated with BA or TDG with BA causing marginal benefits, but TDG restored plasmalogen levels to that seen in wild-type mice as well as central nervous system (CNS) pathology.

It was clear that plasmalogens had a role in the location of AIS, but to determine if this had functional effects, neuron excitability was studied. Patch clamp recordings of CA1 pyramidal neurons in Gnpat KO mice had normal resting potential but impaired excitability through increased thresholds and requiring more current to generate action potentials and smaller overshoots. As well, when compared to wild-type neurons, Gnpat KO neurons had fewer action potentials, indicating a reduced level of excitability.

It was demonstrated that a disruption in calcium, another essential component of cell signaling, was not involved, therefore da Silva et al wanted to determine if the AKT (protein kinase B) signaling pathway was responsible for regulating AIS positioning in neurons. AKT phosphorylation was analyzed in hippocampal neuron cultures and the Gnpat KO cells showed lower levels of AKT phosphorylation at Ser 473 and Thr308 compared to wild-type cells. An AKT activator, SC79, was used to investigate if reactivation of the AKT pathway could produce normal AIS position, and this was found to be successful in the Gnpat KO neurons with the positioning returning to that seen in wild-type cells. Different AKT downstream targets were analyzed to further elucidate the pathway allowing AKT to control AIS positioning by treating wild-type neurons with rapamycin and a similar AIS location shift as that seen in Gnpat KO cells was achieved. This implicated the AKT-mTORC1 pathway in AIS positioning. To confirm this finding they looked at two mTORC1 substrates that are active when AKT activation is impaired (4E binding proteain 1 (4E-BP1) and unc-51-like kinase 1 (ULK1)). To mimic the effect of plasmalogen deficiency in Gnpat KO neurons, either 4E-BP1 or ULK1 were inhibited and only suppression of unc-51-like kinase 1 (ULK1) was able to recover AIS positioning. These results indicate that in plasmalogen deficiency, the AKT-ULK1 pathway has an essential role in the location of AIS.

da Silva et al demonstrated the critical role that plasmalogens have on AIS position on axons and neuronal excitability. This was confirmed when AIS positioning was returned to close to the cell body after treatment with plasmalogen precursors, especially TDG. They also demonstrated the mechanism that a plasmalogen deficiency influences in these neurons. Through many tests it was narrowed down to the AKT-ULK1 pathway altering the position of AIS in Gnpat KO neurons. This work also provided data for the functional effects of this location shift as Gnpat KO neurons demonstrated reduced excitability. Although many roles of plasmalogens have been well characterized, it is still unclear how exactly a plasmalogen reduction or deficiency results in the cognitive decline seen in AD or the deficits in RCDP. This work could provide part of the explanation for the cognitive effects of changes in plasmalogen levels since this impaired excitability would cause impairments in signaling. The work described by da Silva et al was all in vitro, therefore the effect of supplementation in vivo will be necessary to confirm these effects of plasmalogens in a living animal. In addition, further work into the pathways that are regulated by plasmalogens and the downstream effects on cellular morphology could provide many answers for the relationship between cognitive ability and plasmalogen level.