Involvement of ace in T.castaneum apoptosis
Before studying the role of AChE in the neuronal apoptosis of T. castaneum we explored the possibility of multiple splice variants from the two ace genes. While ace-1 includes only two exons making alternative transcripts rather unlikely, ace-2 consists of seven exons carrying the potential for multiple different splice variants (Fig. 1A). We designed primers spanning central regions of various pairs of ace-2 exons (indicated in Fig. 1A) in order to detect potential splice variants in the present transcripts. Transcripts were analysed in brains of untreated pupae and brains of pupae after 24 h exposure to hypoxia (< 0,3% O2). RT-PCR analysis revealed no alternative splicing products of ace-2, neither in normoxic control nor in hypoxia-treated pupae (Fig. 1B). All detected PCR products included the exon that was interspersed between the two exons targeted by the primers. Hence, all PCR products were clearly larger than expected if the sandwiched exon was spliced out (size of potential PCR product from alternate transcript indicated by yellow boxes in Fig. 1B). The results are in line with the existence of only one transcript that includes all seven exons in normal and hypoxia-challenged T. castaneum brains.
We previously demonstrated that pharmacological inhibition of AChE rescues primary cultured locust neurons from hypoxia-induced apoptosis . Following a similar protocol, primary neuron cultures from T. castaneum were exposed to hypoxic conditions (< 0,3% O2) for 36 h. Hypoxia-exposure reduced the median relative survival of cultured neurons (0,8) in comparison to normoxic control cultures (normalized to 1.0; Fig. 1C). Hypoxia-induced cell death was completely prevented in the presence of 10 µM of the two AChE inhibitors neostigmine bromide (NSB; median relative survival 0,98) and territrem B (TRB; median relative survival 1,1). Neuron survival in hypoxia was significantly increased by both AChE inhibitors compared to untreated hypoxic cultures reaching the same level as the normoxic control cultures.
Expression of Tc-ace-1 and Tc-ace-2 under apoptogenic conditions was studied by qPCR in brains of T. castaneum following hypoxia-exposure (< 0,3% oxygen) of pupae for 24 and 36 hours. 24 h hypoxia significantly increases transcript levels of both Tc-ace-1 (2,36 fold ± 0,8 Stdv) and ace-2 (1,47 fold ± 0,6 Stdv) compared to brains of control animals in normoxic atmosphere (Fig. 1D). Prolonging the hypoxic period to 36 h reduced high expression levels detected after 24 h. While Tc-ace-1 expression remained significantly elevated (1,37 fold ± 0,3 Stdv), Tc-ace-2 transcript levels were no longer different from controls kept under normoxic conditions (1,02 fold ± 0,4 Stdv). The results presented in Figs. 1C and 1D indicate a pro-apoptotic involvement of both T. castaneum ace genes in hypoxia-induced neuronal apoptosis.
In order to assess the individual contributions of Tc-ace-1 and Tc-ace-2 to hypoxia-induced apoptosis in T. castaneum we inhibited the production of the respective AChE proteins by RNA interference in primary cultured brain neurons before subjecting them to hypoxia (< 0,3% O2; 36 h). Neuron survival was compared between normoxic control cultures, hypoxia-exposed cultures and hypoxia-exposed cultures after dsRNA-mediated knockdown of either Tc-ace-1 or Tc-ace-2 expression. For each ace gene two dsRNA fragments that target non-overlapping regions of the respective transcript, were designed and knockdown was induced by soaking RNAi as described previously (Hahn et al., 2017; Knorr et al., 2020). Figure 2 depicts data from experiments with the respective fragment 1 to knock down Tc-ace-1 and Tc-ace-2 expression (Data from experiments with fragment 2 are provided in the supplement (Fig. 5 supporting information).
Hypoxia significantly reduced relative neuron survival compared with normoxic control cultures in both experimental series (Fig. 2A: median relative survival 0,80; Fig. 2B: median relative survival 0,78). Knock down of Tc-ace-1 with fragment 1 significantly increased relative neuron survival in hypoxia-exposed cultures (median relative survival 0.97), however without reaching survival levels in normoxic control cultures (Fig. 2A). Knock down of Tc-ace-1 expression with fragment 2 elevated median relative neuron survival in hypoxia-exposed cultures from 0,77 to 0,82 (not significant, Fig. 5A supporting information). RNAi-mediated suppression of Tc-ace-2 expression with fragment 1 significantly increased neuron survival in hypoxia-exposed cultures (median relative survival 0.89) (Fig. 2B). Similar results were obtained after knock down of Tc-ace-2 expression with fragment 2 which increased median neuron survival in hypoxia from 0,84 to 0,94 (Fig. 5B; supporting information). However, interference with Tc-ace-2 expression with either fragment was not sufficient to increase cell survival in hypoxia to the levels of normoxic cultures. In summary, dsRNA-mediated interference with Tc-ace-1 and Tc-ace-2 expression for five days prior to hypoxia-exposure partially rescues T. castaneum primary neurons from hypoxia-induced apoptosis.
rhEpo prevents hypoxia-induced apoptosis and elevated ace expression
Previous studies reported anti-apoptotic effects of rhEpo on locust and beetle neurons [45, 46] whereas AChE was associated with pro-apoptotic activity (; this study). In order to evaluate a potential convergence of these pro- and anti-apoptotic pathways we combined rhEpo and AChE-inhibitor treatment of hypoxia-exposed neurons and studied potential regulatory effects of rhEpo on ace expression in both L. migratoria and T. castaneum primary neuron cultures.
Hypoxia-induced apoptosis of locust neurons was completely prevented by 33,3 ng/ml rhEpo, 10 µM NSB and combined treatment with rhEpo and NSB (Fig. 3A). Relative neuron survival was statistically similar in rhEpo-, NSB- and rhEpo/NSB-treated cultures indicating no additive effects of the beneficial compounds following combined application. The same hypoxic treatment (< 0,3% O2; 36 h) that caused apoptotic death of primary cultured locust neurons elevated the expression of ace-1 transcript (1,47 fold ± 0,3 Stdv) compared to normoxic control cultures (Fig. 3B). Lm-ace-1 expression was normalized to 18s rRNA and gapdh which were not affected by hypoxia-exposure. Elevated ace-1 expression was prevented by neuroprotective concentration of rhEpo, indicating a link of Epo-mediated neuroprotection with the suppression of pro-apoptotic AChE expression during apoptogenic stress (Fig. 3B). Since sequence information about L. migratoria ace-2 is not available, expression of Lm-ace-2 could not be analysed.
In primary neuron cultures of T. castaneum hypoxia-induced apoptosis was prevented by 0,8 ng/ml rhEpo and by 10 µM NSB (Fig. 3C). Combined treatment with the same concentrations of rhEpo and NSB also increased relative neuron survival in hypoxia-exposed cultures (from 0,81 to 0,96 median relative survival) to the level of normoxic control cultures, but this increase did not reach significance level. Hypoxia (< 0,3% O2; 36 h) increased the expression of Tc-ace-1 (1,2 fold ± 0,2 Stdv) and ace-2 (1,33 fold ± 0,3 Stdv) transcripts in T. castaneum neurons (Fig. 3D). Tc-ace gene expression was normalized to rps3 and rps18 whose abundance remained stable during the hypoxic period. Treatment of hypoxia-exposed cultures with neuroprotective concentration of rhEpo prevented the increase of Tc-ace-1 expression (1,05 fold ± 0,2 Stdv compared with normoxic control cultures) but not the increase of Tc-ace-2 expression (1,2 ± 0,1 Stdv) (Fig. 3D). Thus, while hypoxia induces apoptosis and elevated expression of Tc-ace-1 and Tc-ace-2 in T. castaneum neurons, Epo-mediated neuroprotection correlates with suppressed ace-1 expression, suggesting that elevated Tc-ace-2 transcript levels alone are not sufficient to drive apoptosis.
Previous studies reported optimum-type dose-response curves for Epo-mediated protection of mammalian and insect neurons [46, 53, 55–57]. So far, no mechanistic explanation for toxic effects of high Epo concentrations mediated via homodimeric EpoR or alternative Epo receptors has been provided. In this context, we compared ace expression in L. migratoria and T. castaneum primary neuron cultures following exposure to previously established neuroprotective and toxic concentrations of rhEpo. Serum was removed from culture media after three days in vitro as a mild apoptogenic stimulus before neurons were stimulated with rhEpo for 48 h starting on day five in vitro. Stimulation of locust neurons with neuroprotective concentrations of rhEpo (33,3 ng/ml) had no impact on Lm-ace-1 expression while toxic concentrations of rhEpo (333 ng/ml) significantly increased ace-1 transcript levels (2,3 fold ± 0,8 Stdv) compared to untreated controls (Fig. 4A). Lm-ace-1 expression was normalized to 18s rRNA and gapdh that were not affected by rhEpo. In T. castaneum neurons neuroprotective concentrations of rhEpo (0,8 ng/ml) reduced the expression of both Tc-ace-1 (0,72 fold ± 0,1 Stdv) and Tc-ace-2 (0,69 fold ± 0,1 Stdv) compared with untreated control cultures (Fig. 4B). Toxic concentrations of rhEpo (8 ng/ml) affected the expression of the two ace genes differentially, leading to increased Tc-ace-1 (1,33 fold ± 0,3 Stdv) and decreased Tc-ace-2 (0,71 fold ± 0,2 Stdv) transcript levels compared with untreated controls (Fig. 4B). Tc-ace gene expression was normalized to rps3 and rps18 whose abundance was not altered by rhEpo stimulation. Thus, toxic concentrations of rhEpo elevate the expression of pro-apoptotic ace-1 in both locust and beetle neurons.