Poster AA1 - Apolipoprotein E3 Improves Acetylcholine Receptor Expression in Skeletal Muscle

Introduction: A healthy motor endplate is densely packed with acetylcholine receptors (AChRs) responsible for mediating muscle activation from the motor nerve. However, peripheral nerve injuries result in gradual loss of the endplate, also known as fragmentation. Denervation and subsequent fragmentation are common in muscle injuries such as lacerations and car crashes. In both cases, fragmentation occurs parallel to atrophy which concurrently result in decreased patient quality of life. However, fragmentation is recoverable if the nerve is surgically reconnected within the 18-month regenerative window. Despite successful recovery, most patients will still face long term functional deficits due to dampened neurotransmission caused by fragmented remodeling. Our long-term goal is to create a treatment that preserves the motor endplate by supplementing the muscle with pro-synaptic signaling that will slow the fragmentation process. The aim of the present study was to evaluate the role of Apolipoprotein E (Apoe) in synaptogenesis and investigate its ability to activate AChR clustering.

Materials and

Methods: For our in vitro co-culture study, C2C12 myoblasts were plated onto a Corning TCPS 24-well plate at 10,000 cells/cm2 in α-MEM 10% FBS 1% P/S media cultured at 37C 5% CO2. NSC34 motor neurons were plated in parallel on Corning transwell membranes at 10,000 cells/cm2 in DMEM 10% FBS 1% P/S media cultured at 37C 5% CO2. Co-culture began once myoblasts reached 100% confluence. The cells were allowed to co-culture for 3 days in Neurobasal media 4 mM L-glutamine 1% P/S. After 3 days, RNA was collected and RT-qPCR was used to measure levels of myogenic and synaptic markers. Next, we measured Apoe, agrin, Nrg1, and acetylcholine signaling by measuring these factors from the transwell co-culture released into media and contained in the cell lysates using ELISAs. Results were normalized with DNA content measured with PicoGreen DNA quantification. Conditioned media cultures were normalized with total DNA per well. Transwell co-culture were normalized to combined C2C12 and NSC34 DNA. We also tested whether Apoe3 or Apoe4 were involved in myogenesis and motor end plate development. Treatment with exogenous Apoe 3 or 4 started when the C2C12 myoblasts reached 100% confluence then were dosed every 24 hours for 6 days then collected on day 7. The Apoe proteins were sourced from Acro Biosystems with poly-his tags. C2C12 cells received either Apoe3 or Apoe4 at 10-7, 10-8, or 10-9 molarity mixed in with the α-MEM full media (n=6/group), RNA was isolated, and our full gene panel were tested.

Results, Conclusions, and Discussions:

Results: C2C12 myoblasts co-cultured with NSC-34 motor neurons increased myogenic markers and acetylcholine receptor γ and ε levels with a concomitant increase in Apoe. These results were correlated with α-bungarotoxin staining that revealed increased clusters of dispersed AChRs in myoblasts treated with NSC-34 cells. The ELISA data showed Apoe levels increased in NSC cell lysate whereas secretion increased in co-culture. The effect of treatment different Apoe isoforms, and determined that proliferation in C2C12 myotubes did not change and was not dose dependent. Similarly, myogenic markers Myh1, Myog, and MyoD did not change between doses or isoform of Apoe. However, Apoe3 had a dose dependent effect on Chrng expression with no effect of Apoe4 on Chrng expression. The expression for Lrp4, a known receptor for Apoe, was unaffected by both treatments. No changes in synaptic expression were measured in Apoe4 treated C2C12 cells.

Discussion: After peripheral nerve injury, the complex communication between motor nerves and muscles deteriorates, leading to gradual changes in motor endplate structure that weaken muscle functional, even if nerve reconnection is successful. Our present data shows an increased levels of Apoe during muscle-nerve co-cultures, suggesting its involvement in synaptic development potentially through interaction with Lrp4. These results correlate with increased synaptogenesis indicated by increased bungarotoxin staining. Additionally, our previous myoblast time-course study showed evidence of intrinsic expression of Apoe in C2C12 cells during myogenesis which drove our decision to treat with exogenous Apoe3. Our treatment with exogenous Apoe3, but not Apoe4, enhanced acetylcholine receptor expression suggesting increased AChR clustering. Future studies will further confirm these results using bungarotoxin staining and inhibitor studies. These data suggest Apoe3 may be an important signaling protein for synaptogenesis and requires further study to understand its role.

Conclusions: Understanding the complex muscle-nerve signaling pathways responsible for motor endplate formation and protection is an important step to improve patient outcomes following denervation injuries. Our data show Apoe may play a role in this process and may have a potential therapeutic effect to protect motor endplates.

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