APOE4, Age & Sex Regulate Respiratory Plasticity Elicited By Acute Intermittent Hypercapnic-Hypoxia

Abstract

Rationale: Acute intermittent hypoxia (AIH) is a promising strategy to induce functional motor recovery following chronic spinal cord injuries and neurodegenerative diseases. Although significant results are obtained, human AIH trials report considerable inter-individual response variability.

Objectives: Identify individual factors ( e.g. , genetics, age, and sex) that determine response magnitude of healthy adults to an optimized AIH protocol, acute intermittent hypercapnic-hypoxia (AIHH).

Methods: Associations of individual factors with the magnitude of AIHH (15, 1-min O ₂ =9.5%, CO ₂ =5% episodes) induced changes in diaphragm motor-evoked potential amplitude (MEP) and inspiratory mouth occlusion pressures (P _0.1 ) were evaluated in 17 healthy individuals (age=27±5 years) compared to Sham. Single nucleotide polymorphisms (SNPs) in genes linked with mechanisms of AIH induced phrenic motor plasticity ( BDNF, HTR _2A , TPH ₂ , MAOA, NTRK ₂ ) and neuronal plasticity (apolipoprotein E, APOE ) were tested. Variations in AIHH induced plasticity with age and sex were also analyzed. Additional experiments in humanized ( h ) ApoE knock-in rats were performed to test causality.

Results: AIHH-induced changes in diaphragm MEP amplitudes were lower in individuals heterozygous for APOE ₄ ( i.e., APOE _3/4 ) allele versus other APOE genotypes (p=0.048). No significant differences were observed between any other SNPs investigated, notably BDNFval/met ( all p>0.05 ). Males exhibited a greater diaphragm MEP enhancement versus females, regardless of age (p=0.004). Age was inversely related with change in P _0.1 within the limited age range studied (p=0.007). In hApoE ₄ knock-in rats, AIHH-induced phrenic motor plasticity was significantly lower than hApoE ₃ controls (p<0.05).

Conclusions: APOE ₄ genotype, sex and age are important biological determinants of AIHH-induced respiratory motor plasticity in healthy adults.

Addition to knowledge base: Acute intermittent hypoxia (AIH) is a novel rehabilitation strategy to induce functional recovery of respiratory and non-respiratory motor systems in people with chronic spinal cord injury and/or neurodegenerative diseases. Since most AIH trials report considerable inter-individual variability in AIH outcomes, we investigated factors that potentially undermine the response to an optimized AIH protocol, acute intermittent hypercapnic-hypoxia (AIHH), in healthy humans. We demonstrate that genetics (particularly the lipid transporter, APOE ), age and sex are important biological determinants of AIHH-induced respiratory motor plasticity.

Figure 1:. Conceptual diagram depicting cell signaling mechanisms (and candidate biomarker genes) for acute intermittent hypercapnic-hypoxia (AIHH) induced respiratory motor plasticity.

The panel of SNPs with a population prevalence of >10% were tested for association with reduced AIHH-induced plasticity in humans. These include 6 SNPs in genes involved in AIH cell signaling: (1) raphe chemosensitive cells (PHOX2B), (2) serotonin precursors in the central nervous system (tryptophan hydroxylase-2, TPH-2), (3) serotonin clearance enzyme (monoamine oxidase A, MAOA), (4) serotonin-2A receptors (HTR2A), (5) brain-derived neurotrophic factor (BDNF) and (6) TrkB receptors (NTRK2). A seventh dysfunctional SNP in neuroplasticity related gene, APOE (APOE4), was also tested for association.

Figure 2:. Relative (%-change from baseline) changes in diaphragm motor-evoked potential (MEP) amplitudes and mouth occlusion pressure (P0.1) in individuals with BDNFval/met (panels A and B) and APOE3/4 (panels C and D) SNP.

No associations were observed between individuals with BDNFval/met and the change in MEP amplitudes (panel A) or P0.1 (panel B). Individuals with dysfunctional APOE3/4 allele were associated with a significantly lower AIHH-induced change in MEP amplitude (t=−2.28, p=0.048, panel C). However, no association between APOE3/4 and AIHH-induced P0.1 responses were observed (panel D). ∆=change. *p<0.05. Results expressed as mean ± SD. = participant (S6) was identified as the most influential point (Cook’s D >4) in the %-change in diaphragm MEP amplitudes, therefore, the data was not included in group analyses.

Figure 3:. AIHH elicits phrenic long-term facilitation in hApoE3 but not hApoE4 knock-in rats.

Panel A shows average traces of phrenic nerve amplitude for hApoE3 (n=4; gray) and hApoE4 (n=3; black) knock-in rats, *p<0.050 vs baseline. Panel B phrenic burst amplitude (%-change from baseline) in hApoE3 (gray circles) and hApoE4 (black circles) rats, +p<0.005 versus hApoE4. ∆=change. Results expressed as mean ± SD.

Figure 4:. Relationship between age and sex on the magnitude (%-change from baseline) of change in diaphragm motor-evoked potential amplitudes (MEP, panels A and B), and mouth occlusion pressure in 0.1 seconds (P0.1, panels C and D) following AIHH.

No association between age and the magnitude of change in diaphragm MEP amplitudes was observed (panel A). Regardless of age, males (black line, panel B) had significantly greater responses in MEP amplitudes versus females (gray line, panel B). The magnitude of change in P0.1 reduced significantly with age (panel C); however, the decline was more pronounced in males (r=−0.73, p=0.036, black line, panel D) versus females (r=−0.29, p=0.480, gray line, panel D). ∆=change. *p<0.05. Results expressed as mean ± SD. = participant (S6) was identified as the most influential point (Cook’s D >4) in the %-change in diaphragm MEP amplitudes, therefore, the data was not included in group analyses.