Effects of Combining Biofactors on Bioenergetic Parameters, Aβ Levels and Survival in Alzheimer Model Organisms

Increased amyloid beta (Aβ) levels and mitochondrial dysfunction (MD) in the human brain characterize Alzheimer disease (AD). Folic acid, magnesium and vitamin B6 are essential micro-nutrients that may provide neuroprotection. Bioenergetic parameters and amyloid precursor protein (APP) processing products were investigated in vitro in human neuroblastoma SH-SY5Y-APP695 cells, expressing neuronal APP, and in vivo, in the invertebrate Caenorhabditis elegans (CL2006 & GMC101) expressing muscular APP. Model organisms were incubated with either folic acid and magnesium-orotate (ID63) or folic acid, magnesium-orotate and vitamin B6 (ID64) in different concentrations. ID63 and ID64 reduced Aβ, soluble alpha APP (sAPPα), and lactate levels in SH-SY5Y-APP695 cells. The latter might be explained by enhanced expression of lactate dehydrogenase (LDHA). Micronutrient combinations had no effects on mitochondrial parameters in SH-SY5Y-APP695 cells. ID64 showed a significant life-prolonging effect in C. elegans CL2006. Incubation of GMC101 with ID63 significantly lowered Aβ aggregation. Both combinations significantly reduced paralysis and thus improved the phenotype in GMC101. Thus, the combinations of the tested biofactors are effective in pre-clinical models of AD by interfering with Aβ related pathways and glycolysis.


Introduction
At present, 50 million people are suffering from Alzheimer's disease (AD) and this number will rise to approximately 152 million in 2050 [1]. Unfortunately, there is no cure for AD yet. Approved drugs only treat symptoms [2]. There are several hypotheses regarding the etiology of Alzheimer's disease, but the causes of the disease are unknown. Previous research has focused on amyloid and tau, which has not yet led to major breakthroughs. Therefore, there is a trend towards multifactorial treatments and, among other things, energy metabolism with regard to mitochondrial functions. Two of the hallmarks of AD are mitochondrial dysfunction (MD) [3] and overproduction of beta-amyloid (Aβ) [4]. The first signs of beginning MD are a reduction of glucose consumption [5] and a reduced activity of key enzymes of the oxidative metabolism [6,7]. Almost all mitochondrial functions are impaired in AD [8,9]. The limited function of the electron transport chain (ETC) is the reason for the decrease in complexes IV and I. This results in a decreased mitochondrial membrane potential (MMP) and ATP production [10]. Another important characteristic of AD is that Aβ is cleaved of from a much larger amyloid precursor protein (APP) [11]. APP is cleaved via two pathways, a non-amyloid and an amyloidogenic pathway. The APP is spliced by the different types of protease, namely α-, βand γ-secretase [11,12]. Depending on which protease cleaves the APP, Aβ peptides are produced. The α-protease cleaves the APP closer to the membrane, resulting in a shorter fragment in the membrane, which is then further cleaved by the γ-protease to a non-amyloidogenic product. However, when β-protease cleaves the protein, larger fragments are produced, which are then cleaved by The present work investigated the effects of different B vitamins and MgOr on MD and the processing of APP in SH-SY5Y-APP 695 cells a cellular model of early AD. Furthermore, the effects of the substances were tested in CL2006 and GMC101, both invertebrate models of AD.

General Overview of Tests and Results
In Table 1 below, all tests and results are listed to provide a general overview of the subsequent tests and results. Here, the substance under investigation is shown against the control. For more detailed insights, the results are described in the respective chapters.
To investigate the effect on the Aβ 1-40 production, SH-SY5Y-APP 695 cells were in-  (Figure 1). ID63 had a significant lowering effect on the Aβ 1-40 level (p > 0.0001). The ID63 combination even had an over additive effect compared to the single substances MgOr (p = 0.0028) and Fol (p > 0.0001). The ID64 combination had a significantly decreasing effect on the Aβ levels compared to the control (p > 0.0001). Furthermore, ID64 had a significantly reducing effect in comparison to the single substances MgOr (p = 0.0497), Fol (p > 0.0001) and Vit B6 (p > 0.0001).

Aβ 1-42 Production
To study the production of Aβ 1-42 , SH-SY5Y-APP 695 cells were incubated for 24 h with ID63, ID64 or the single compounds ( Figure 2). In comparison to the control, the single compounds MgOr (p = 0.0024) and folic acid (p = 0.0004), as well as the combination ID 64 (p = 0.0039) had a significant lowering effect on Aβ 1-42 levels, while the combination ID63 had a slight reducing effect on the Aβ 1-42 levels, although not a significant one. ID64 also showed significantly lower Aβ 1-42 levels in comparison to B6 (p = 0.0009). However, folic acid alone, reduced the levels to a higher extent than any combinations. ID63, ID64 or the single compounds ( Figure 2). In comparison to the control, the single compounds MgOr (p = 0.0024) and folic acid (p = 0.0004), as well as the combination ID 64 (p = 0.0039) had a significant lowering effect on Aβ1-42 levels, while the combination ID63 had a slight reducing effect on the Aβ1-42 levels, although not a significant one. ID64 also showed significantly lower Aβ1-42 levels in comparison to B6 (p = 0.0009). However, folic acid alone, reduced the levels to a higher extent than any combinations.

sAPPα and sAPPβ Level
The α-and β-secretase cleaving products of APP, sAPPα ( Figure 3A) and sAPPβ (Figure 3B), respectively, were determined after incubation with either ID63 or ID64 for 24 h. Figure 4A shows that ID63 had a significantly lowering effect on the sAPPα (p = 0.0214) compared to the control. ID64 had an even greater effect on the reduction of sAPPα fragments (p > 0.0001). In contrast to the sAPPα fragment production, the sAPPβ fragments ( Figure 3B) were lowered compared to the control though not significantly. ID64 had a

sAPPα and sAPPβ Level
The αand β-secretase cleaving products of APP, sAPPα ( Figure 3A) and sAPPβ ( Figure 3B), respectively, were determined after incubation with either ID63 or ID64 for 24 h. Figure 4A shows that ID63 had a significantly lowering effect on the sAPPα (p = 0.0214) compared to the control. ID64 had an even greater effect on the reduction of sAPPα fragments (p > 0.0001). In contrast to the sAPPα fragment production, the sAPPβ fragments ( Figure 3B) were lowered compared to the control though not significantly. ID64 had a greater effect than ID63. It should be noted that basal levels of sAPPβ were approximately one hundredfold lower compared with sAPPα ( Figure 3).

Effect on the Mitochondrial Function
To investigate the effect of ID63 and ID64 on mitochondrial function, we incubated SH-SY5Y-APP 695 cells for 24 h with ID63 or ID64. Respiration under O 2 consumption through the respiratory chain builds up the mitochondrial membrane potential, which allows ATP to be generated with the help of ATP synthase. First, we measured the ATP level after incubation with ID63 or ID64. Afterwards, the MMP was examined as well as the O 2 consumption and citrate synthase activity ( Figure 4).
Neither ID63 nor ID64 had an increased effect on the ATP level ( Figure 5A,B) or an effect on the MMP level ( Figure 4C,D). ID63 had a slightly increasing effect on the complex activity of complex I, II and IV compared to the control ( Figure 4E). In contrast, ID64 had a slightly decreasing effect on the complex activity of complex II and IV ( Figure 4F). Whereas ID63 had no effect on the citrate synthase activity compared with the control (Figure 4G), and ID64 even had a slightly decreasing effect compared with the control ( Figure 4H). However, none of these effects is statistically significant.
OR PEER REVIEW 6 of 23 greater effect than ID63. It should be noted that basal levels of sAPPβ were approximately one hundredfold lower compared with sAPPα ( Figure 3).

Lactate and Pyruvate Level
To investigate if the glycolysis is affected by ID63 or ID64, the lactate and pyruvate levels were measured. As seen in Figure 6, only lactate is significantly reduced by ID63 (p = 0.0328) and ID64 (p > 0.0001) compared to the control while pyruvate levels were not influenced. The ratio of lactate/pyruvate was significantly affected by ID64 (p = 0.0057) ( Figure 5C).  Neither ID63 nor ID64 had an increased effect on the ATP level ( Figure 5A,B) or an effect on the MMP level ( Figure 4C,D). ID63 had a slightly increasing effect on the complex activity of complex I, II and IV compared to the control ( Figure 4E). In contrast, ID64 had a slightly decreasing effect on the complex activity of complex II and IV ( Figure 4F). Whereas ID63 had no effect on the citrate synthase activity compared with the control ( Figure 4G), and ID64 even had a slightly decreasing effect compared with the control ( Figure 4H). However, none of these effects is statistically significant.

Lactate and Pyruvate Level
To investigate if the glycolysis is affected by ID63 or ID64, the lactate and pyruvate levels were measured. As seen in Figure 6, only lactate is significantly reduced by ID63 (p = 0.0328) and ID64 (p > 0.0001) compared to the control while pyruvate levels were not influenced. The ratio of lactate/pyruvate was significantly affected by ID64 (p = 0.0057) ( Figure 5C).

qPCR
To investigate the molecular basis of altered lactate and pyruvate levels, the gene expression of pyruvate dehydrogenase kinase 1 (PDK1) and lactate dehydrogenase A (LDHA) were examined after 24 h incubation using qRT-PCR. ID63 and ID64 had no significant effect on PDK1 gene expression compared to the control ( Figure 6). Both combinations increased LDHA mRNA levels ( Figure 6B), with ID64 (p = 0.0148) showing a significant increase in gene expression.

Effect on the Lifespan of C. elegans in Heat Stress Survival Assay
To test the effect of the combinations in vivo two invertebrate AD-models were used. C. elegans CL2006 were incubated with the same compounds but in different concentrations. The single compounds Fol 50 µM (p > 0.0001), Vit B6 100 µM (p = 0.0397) and MgOr

qPCR
To investigate the molecular basis of altered lactate and pyruvate levels, the gene expression of pyruvate dehydrogenase kinase 1 (PDK1) and lactate dehydrogenase A (LDHA) were examined after 24 h incubation using qRT-PCR. ID63 and ID64 had no significant effect on PDK1 gene expression compared to the control ( Figure 6). Both combinations increased LDHA mRNA levels ( Figure 6B), with ID64 (p = 0.0148) showing a significant increase in gene expression.

Effect on the Lifespan of C. elegans in Heat Stress Survival Assay
To test the effect of the combinations in vivo two invertebrate AD-models were used. C. elegans CL2006 were incubated with the same compounds but in different concentrations. The single compounds Fol 50 µM (p > 0.0001), Vit B6 100 µM (p = 0.0397) and MgOr 100 µM (p = 0.001) had a significant life-extending effect compared to the control (see Figure 7A,B). As shown in Figure 7A, the ID63 worm extended lifespan of CL2006 compared to the control by trend (p = 0.0502), whhereas in Figure 7B, it can be seen that ID64 worm had a significant life-prolonging effect (p = 0.0002) compared to the control. Subsequently, the mean survival of the nematodes after the incubations of the combinations and single substances was assessed. Thereby Figure 7C shows that ID63 worm (p = 0.0196), Fol 50 µM (p < 0.0001), MgOr 100 µM (p = 0.0004), ID64 worm (p < 0.0001) and Vit B6 (p = 0.0107) had a significant increasing effect on the mean survival of the nematodes compared to the control. Folic acid alone was numerically more effective than the combinations or the other single compounds.

Effect on the Paralysis
To investigate the effect of combining biofactors on paralysis induced by Aβ, C. elegans GMC101 were incubated for 24 h at 25 • C with either ID63 worm or ID64 worm . Both combinations (p = 0.0243 for ID63 worm and p = 0.0149 for ID64 worm ) were able to significantly decrease the paralysis induced by Aβ (Figure 8). Thus, the phenotype of this AD-worm was significantly enhanced by both biofactor combinations to a comparable extent.

Effect on the paralysis
To investigate the effect of combining biofactors on paralysis induced by Aβ, C. elegans GMC101 were incubated for 24 h at 25 °C with either ID63worm or ID64worm. Both combinations (p = 0.0243 for ID63worm and p = 0.0149 for ID64worm) were able to significantly decrease the paralysis induced by Aβ (Figure 8). Thus, the phenotype of this AD-worm was significantly enhanced by both biofactor combinations to a comparable extent.

Aβ1-42 Production in GMC101
To study the production of Aβ1-42 in nematodes, GMC101 was incubated for 24 h at 25 °C with both combinations and then the Aβ level was examined. There was no effect on the Aβ1-42 levels compared to the control (Figure 9). Since Aβ1-40 is not produced in GMC101 this amyloid peptide was not investigated.

Aβ 1-42 Production in GMC101
To study the production of Aβ 1-42 in nematodes, GMC101 was incubated for 24 h at 25 • C with both combinations and then the Aβ level was examined. There was no effect on the Aβ 1-42 levels compared to the control (Figure 9). Since Aβ 1-40 is not produced in GMC101 this amyloid peptide was not investigated.

Aβ1-42 Aggregation
To investigate the aggregation of Aβ in nematodes, GMC101 was incubated at 25 °C for 24 h and stained with thioflavine (ThT), which labels β-sheet structures of aggregated peptides. There was a significant reduction of Aβ-aggr by the combination ID63worm (p = 0.0324) compared to the control, whereas ID64worm was without an effect ( Figure 10).

Aβ 1-42 Aggregation
To investigate the aggregation of Aβ in nematodes, GMC101 was incubated at 25 • C for 24 h and stained with thioflavine (ThT), which labels β-sheet structures of aggregated peptides. There was a significant reduction of Aβ-aggr by the combination ID63 worm (p = 0.0324) compared to the control, whereas ID64 worm was without an effect ( Figure 10). Figure 9. Effect of ID63 and ID64 in transgene nematodes GMC101 compared to the controls on the Aβ1-42 level after 48 h incubation. Neither ID63 nor ID64 did not lead to a significant alteration of Aβ1-42. N = 8. Aβ1-42 levels were adjusted to the protein content. N = 8. Mean ± SEM. Student's t-test. ID63worm = Fol 50 µM and MgOr 100 µM; ID64worm Fol 50 µM, MgOr 100 µM and Vit B6 100 µM.

Aβ1-42 Aggregation
To investigate the aggregation of Aβ in nematodes, GMC101 was incubated at 25 °C for 24 h and stained with thioflavine (ThT), which labels β-sheet structures of aggregated peptides. There was a significant reduction of Aβ-aggr by the combination ID63worm (p = 0.0324) compared to the control, whereas ID64worm was without an effect ( Figure 10).

Discussion
In the present work, we examined the effect of MgOr, Fol and Vit B6 in different combinations on bioenergetic parameters including mitochondrial function and glycolysis, as well as Aβ production in cellular and invertebrate models of AD. We wanted to create a combination product that achieves an optimal result by combining several biofactors. The hit compounds used in the current study were selected after a screening of seven substances of interest whose concentrations used in the experiments were based on known literature values [37,[43][44][45][46][47]. We selected the concentrations because they were mostly tested in our cell model, related to AD or cell survival and they were used in relative physiological concentrations. This resulted in the combinations ID63 and ID64. The combinations ID63 and ID64 significantly reduced Aβ 1-40 levels in SH-SY5Y-APP 695 cells compared to the control (Figure 1). Even when compared to the individual components of the combinations, ID63 was able to show an over additively reducing effect on Aβ levels. Similar results were obtained by Li et al. who found a dose-dependent decrease of Aβ 1-40 levels by incubation with Fol, modulating DNA methyltransferase activity [48]. In a study with AD patients, an intervention with Fol significantly reduced Aβ levels and increased the concentration of s-adenosyl methionine (SAM) [49]. Low SAM levels are a risk factor for AD, whereby incubation with SAM led to a decrease in Aβ levels in SK-N-SH cells [50]. Fol and Vit B6 are essential for the SAM cycle [51]. It seems that in our SH-SY5Y model the additional administration of Vit B6 shows no additional effect on Aβ 1-40 levels. Furthermore, lower Mg 2+ are associated with the occurrence of AD which negatively affects brain energy metabolism [52]. In addition, low Mg levels are also negatively correlated with the occurrence of Aβ 1-40 and Aβ 1-42 [53]. As a result, an administration of Mg 2+ leads to a decrease in Aβ levels of N2a-APP cells [37]. After the incubation with ID63, SH-SY5Y-APP 695 cells showed reduced levels of Aβ 1-42 compared to the control, although not significantly. In contrast, the incubation with ID64 showed a significant reduction of Aβ 1-42 ( Figure 2) compared to the control. Administration of a Fol-rich diet significantly reduced Aβ 1-42 levels in APP/PS1 mice compared to the standard diet [54]. Similarly, the incubation with Mg significantly decreased Aβ 1-42 production in Na2 neuroblastoma cells and transgenic mice [37,55,56]. In our work, we could not reproduce these described effects shown despite the administration of both substances in SH-SY5Y-APP 695 cells. However, it seems that Vit B6 has a crucial role in the reduction of Aβ 1-42 levels (ID64) even if Vit B6 alone showed no effects. In contrast, it did not provide any benefit at the Aβ 1-40 level ( Figure 1). Next, we examined sAPPα and -β levels after incubation with ID63 and ID64. The reducing effects on Aβ 1-42 levels by ID64 in SH-SY5Y-APP 695 cells could not be confirmed in transgene nematodes GMC101. On the one hand, incubation decreased the sAPPα levels significantly compared to the control ( Figure 3A). On the other hand, both ID63 and ID64 had a reducing but not significant effect on the sAPPβ levels compared to the control ( Figure 3B). Whereas it should be noted that the concentration of sAPPβ is 100 times lower than sAPPα. Studies showed that the concentration of sAPPα is generally higher than that of sAPPβ [57,58]. An application of Mg 2+ increased the amount of sAPPα in APP/PS1 transgenic mice and simultaneously decreased the concentration of sAPPβ [37]. The results shown in this study [37], where both sAPPα and -β were decreased, could not be reproduced in our work. We assume that this was due to a general decrease in APP production, since the application of Fol can reduce APP processing [59,60], which in turn can affect sAPPα and -β, resulting in generally lowered levels.
Next, we tested ID63 and ID64 on MD in SH-SY5Y-APP 695 cells. SH-SY5Y-APP 695 cells show reduced ATP level, MMP and O 2 consumption compared to their non-transfected SH-SY5Y-MOCK cells [61]. Mg and Fol are vital compounds for enzymes and ATP production in cells [31,33,36]. A deficit leads to the reduced production of ATP [31,34]. In our case, which is not a deficit model, we did not observe any improvement in ATP level, MPP, O 2 consumption or citrate synthase activity in SH-SY5Y-APP 695 cells (Figure 4). In a study by Viel et al. using a similar cocktail consisting of some of our compounds, the mitochondrial complex activity in a transgenic rat model was increased to that of wild type rats [28]. This effect is not found in our case, possibly due to the additional substances contained in the cocktail, which had been the decisive factor here.
It has been shown that in the brains of AD patients there is a switch from aerobic respiration to glycolytic metabolism [62,63]. This is accompanied by an increase in lactate and pyruvate values [64,65], which results from insufficient utilization in oxidative phosphorylation [66]. Both biofactor combinations used in our investigations were able to significantly reduce the lactate values compared to the untreated control cells. The switch to increased oxidative phosphorylation could not be shown in our work, because there was no effect on ATP, MMP, OXPHOS or citrate synthase activity in the respiratory chain. To investigate the impact of the two combinations on the glycolytic genes, mRNA levels of PDK1 and LDHA were determined ( Figure 6). There was an increase in the expression of PDK1 by both combinations compared to the control. PDK1 phosphorylates pyruvate dehydrogenase and inactivates it. This may lead to a reverse transport of pyruvate from the mitochondrion into the cytosol, where it is used for glycolytic energy production via lactate [67,68]. Both combinations were able to increase the expression of LDHA, while ID64 did so significantly. LDHA converts pyruvate to lactate and vice versa [23,69], although the expression was increased there were decreased lactate levels with both ID63 and ID64 ( Figure 5). By upregulating PDK1 and LDHA expression, the cell may reduce the effects of Aβ toxicity and ROS production by shifting from mitochondrial to glycolytic energy production [23,69]. Both combinations appear to support this process, resulting in an upregulation of expression relative to control.
Based on the results obtained, especially those related to Aβ, we wanted to test our compounds in another model of AD. For this purpose, we adjusted the concentrations and tested them in two invertebrate models of AD. CL2006 and GMC101 both express human Aβ in their muscle cells. While CL2006 produces Aβ continuously at 20 • C [70], GMC101 needs a temperature shift to 25 • C to initiate Aβ production [71]. Phenotypically, both are identified like wild-type N2 [72]. ID63 worm had no significant life-prolonging effect on CL2006 compared to the control. Whereas the single substances and ID64 worm had a significant effect on the lifespan (Figure 7A,B). In contrast, all tested combinations and single substances, have a significantly increased effect on mean survival ( Figure 7C). CL2006 continuously produces Aβ, which has a toxic effect on the lifespan [70,73]. In particular, it has effects on DAF-16, which is expressed in the nucleus during stress and has an effect on life span extension [74]. DAF-16 is notable for being responsible for activating genes involved in longevity, lipogenesis, heat shock survival and oxidative stress responses [75,76], homologs being found in C. elegans, humans and mice [77]. It was demonstrated that incubation with Fol extended the lifespan by increasing the expression of DAF-16 [78]. It seems that the substances used have a similar effect on lifespan, whereas the single substance Fol seems to be superior. Furthermore, it was investigated whether the substances can reduce the toxic effect caused by Aβ. For this purpose, it was examined whether the paralysis in GMC101 changes because of the administration (Figure 8). Both combinations were able to reduce the paralyzes, indicating the significantly reduced toxicity of Aβ. Similar effects have been shown with other substances, which reduced the paralysis and thus the toxicity of Aβ [73,[79][80][81]. The reduced toxicity could be a consequence of the reduction of Aβ levels in C. elegans. It could be shown that the administration of single substances leads to a reduction of the Aβ levels [37,48,50,54,55]. To verify this, the Aβ 1-42 levels were determined in GMC101 (Figure 9). Here, we found no significant effect in reduced Aβ level after incubation with both combinations. It should be noted here that the effects on Aβ 1-42 were also relatively moderate in the cells (see above). Aβ 1-40 was not measured in GMC101 because the worm does not produce these peptides. To examine the aggregation of Aβ after incubation with both combinations, samples were stained with ThT, which labels Aβ-structures in proteins ( Figure 10). A significant reduction in Aβ-aggr was observed after incubation with ID63 worm . The combination with additional vitamin B6 did not reduce aggregation further. A similar effect was found in a study by Yu et al., in which the aggregation of Aβ in mice was reduced by incubation with magnesium [82].
It remains to be considered that there are both advantages and disadvantages to administering the substances as a single or combination preparation. Combined administration could lead to synergistic effects that enhance the positive effects and thus lead to an additive effect, as shown in the Aβ 1-40 values Figure 1. However, it cannot be ruled out that negative effects may occur when more than one substance is administered, as the substances may influence each other. Complex formation or competition for transport systems into the cell could occur. Especially when moving from an in vitro to an in vivo experiment. In order to exclude these effects, further experiments will have to be carried out in the future.

Cell Culture
The cultivation of SY5Y cells was performed in 75 cm 2 cell culture flasks under sterile conditions. To ensure optimal growth, cells were split several times per week once a cell density of approximately 70-80% was reached. All cells were cultured in an incubator at 37 • C and 5% CO 2 in DMEM supplemented with 10% (v/v) heat-inactivated fetal calf serum, 60 µg/mL streptomycin, 0.3 mg/mL hygromycin, 1% MEM non-essential amino acids, and 1 mM sodium pyruvate 1%, 60 units/mL penicillin. SH-SY5Y cells were stably transfected with DNA constructs harboring human wild-type APP 695 (APP 695 ) and were kindly donated by A. Eckert (Basel, Switzerland) (for details; please refer to [83]).

ATP Measurement
A bioluminescence assay was used to determine the ATP levels, which is based on the production of light from ATP and luciferin in the presence of luciferase. The test was performed using the ATPlite Luminescence Assay System (PerkinElmer, Rodgau, Germany) according to the previously published protocol [84].

MMP Measurement
Mitochondrial membrane potential (MMP) was measured using the fluorescence dye rhodamine-123 (R123). Cells were incubated for 15 min with 0.4 µM R123 and centrifuged at 750× g for 5 min before being washed with Hank's Balanced Salt Solution (HBSS) buffer supplemented with Mg 2+ , Ca 2+, and HEPES. The cells were suspended with fresh HBSS before they were evaluated by measuring the R123 fluorescence. The excitation wavelength was set to 490 nm and the emission wavelength to 535 nm witch CLARIOstar (BMG Labtech, Ortenberg, Germany).

Cellular Respiration
Respiration in SH-SY5Y 695 cells was assessed using an Oxygraph-2k (Oroboros, Innsbruck, Austria) and DatLab 7.0.0.2. The cells were treated according to a complex protocol developed by Dr. Erich Gnaiger [85]. They were incubated with different substrates, inhibitors and uncouplers. First, cells were washed with PBS (containing potassium chloride 26.6 mM, potassium phosphate monobasic 14.705 mM, sodium chloride 1379.31 mM and sodium phosphate dibasic 80.59 mM) and scraped into mitochondrial respiration medium (MiRO5) developed by Oroboros [85]. Afterwards, the cells were centrifuged, resuspended in MiRO5, and diluted to 10 6 cells/mL. After 2 mL of cell suspension was added to each chamber and endogenous respiration was stabilized, the cells were treated with digitonin (10 µg/10 6 cells) to permeabilize the membrane, leaving the outer and inner mitochondrial membrane intact. OXPHOS was measured by adding the complex I and II substrates malate (2 mM), glutamate (10 mM) and ADP (2 mM), followed by succinate (10 mM). Gradual addition of carbonyl cyanide-4-before it is evaluated by measuring the R123 fluorescence (trifluoromethoxy) phenylhydrazone showed the maximum capacity of the electron transfer system. Rotenon (0.1 mM) was added to measure the activity of complex II. To investigate the leak respiration, oligomycin (2 µL/mL) was injected. To inhabitation of complex III to determined residual oxygen consumption, antimycin A (2.5 µM) was added. This value was subtracted from all respiratory states. Adding N measured N, N , N -tetramethyl-p-phenylenediamine (0.5 mM) and ascorbate (2 mM) the activity of complex IV. To measure the sodium autoxidation rate, azide (≥100 mM) was added. Afterwards, complex IV respiration was corrected by subtracting the autoxidation rate of azid. NaOH served as control.

Citrate Synthase Activity
Cell samples from respirometry measurements were frozen and stored at −80 • C for the determination of citrate synthase activity. Samples were thawed while the reaction mix (0.1 mM 5,5 -dithiol-bis-(2-nitrobenzoic acid) (DTNB), 0.5 mM oxaloacetate, 50 µM EDTA, 0.31 mM acetyl coenzyme A, 5 mM triethanolamine hydrochloride, and 0.1 M Tris-HCl) was mixed and heated at 30 • C for 5 min. Afterwards, 40 µL of samples were submitted in triplets and mixed with 110 µL of the reaction mix. The absorption was measured at 412 nm.

Protein Quantification
Protein content was determined using a Pierce TM Protein Assay Kit (Thermo Fisher Scientific, Waltham, MA, USA) according to the manufacturer's instructions. Bovine serum albumin was used as a standard.

Quantification of Human Soluble Amyloid Precursor Protein α (sAPPα)
The sample preparation was the same according to the Aβ 1-40 quantification. The sAPPα levels were determined using the Human Soluble Amyloid Precursor Protein α (sAPPα) ELISA Kit (Cusabio, Wuhan, China). The process was carried out according to the manufacturer's instructions. Levels were normalized to the protein content.

Quantification of Human Soluble Amyloid Precursor Protein β (sAPPβ)
The sample preparation was the same according to the Aβ 1-40 quantification. The sAPPβ levels were determined using the Human Soluble Amyloid Precursor Protein β (sAPPβ) ELISA Kit (BT LAB, Zhejiang, China). The process was carried out according to the manufacturer's instructions. Levels were normalized to the protein content.

Pyruvate and Lactate Content
Frozen cells, which were previously harvested and incubated for 24 h, were thawed at room temperature. Pyruvate and lactate concentrations were assessed using a pyruvate assay kit (MAK071, Sigma Aldrich, Darmstadt, Germany) and a lactate assay kit (MAK064, Sigma Aldrich, Darmstadt, Germany) according to the manufacturer's instructions.

Real-Time qRT-PCR
To isolate RNA, we used the RNeasy Mini Kit (Qiagen, Hilden, Germany) according to the manufacturer's guidelines. Nanodrop TM 2000c spectrometer (Thermo Fisher Scientific, Waltham, MA, USA) was used to quantify RNA. The TURBO DNA-free TM kit was used according to the manufacturer's instructions (Thermo Fisher Scientific, Waltham, MA, USA) to remove residual genomic DNA. cDNA was synthesized from 1 µg total RNA using an iScript cDNA Synthesis Kit (Bio-Rad, Munich, Germany). qRT-PCR was conducted using a CfX 96 Connect™ system (Bio-Rad, Munich, Germany). All used primers are listed in Table 2. The cDNA aliquots were analyzed in triplicate and diluted 1:10 with RNase-free water (Qiagen, Hilden, Germany). PCR cycling conditions were as follows: initial denaturation for 3 min at 95 • C, followed by 45 cycles at 95 • C for 10 s, 58 • C for 30 s (or 56 • C for 45 s, depending on the primer), and 72 • C for 29 s. Expression was analyzed with −(2∆∆Cq) using Bio-Rad CfX manager software. To normalize the values a factor was calculated based on the geometric mean of the levels of multiple control genes of ß-actin (ACTB), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and phosphoglycerate kinase 1 (PGK1) according to the MIQE guidelines [86]. RNA free water served as an assay control to exclude impurities. 4.14. Nematode and Bacterial Strain C. elegans strains were obtained from the Caenorhabditis Genetics Center (University of Minnesota, MN, USA) and included CL2006 [Punc-54::human A-beta 3-42; pRF4 (rol-6(su1006))] and GMC101 [(Punc-54::A-beta::unc-54 3Prime UTR; Pmtl2::GFP)]. The strain GMC101 expresses the full-length human Aβ 1-42 peptide in body-wall muscle cells that aggregates in vivo. Shifting L4 or young adult animals from 20 • C to 25 • C could induce the expression of Aβ and cause paralysis. The strain CL2006 constitutively expressed Aβ when cultured at 20 • C.
Nematodes were maintained on nematode growth medium (NGM) agar plates seeded with the bacterial E. coli strain OP50. According to standard protocols, the seeded plates were stored at 20 • C [87]. Synchronous populations were generated for all experiments by using a standard bleaching protocol [88].

Cultivation and Treatment
Post-bleaching generated larvae were washed twice in M9-buffer and the number of larvae in 10 µL were adjusted to 10 larvae. Afterward, the synchronized larvae were raised in cell culture flasks (Sarstedt, Nümbrecht, Germany) in either an amount of 1000 or 5000 nematodes, depending on the experiments. OP50-NGM was added to the flasks as a standardized source of food. The larvae were maintained under shaking at 20 • C until they reached young adulthood within 3 days. The micronutrients were dissolved in advance in M9 buffer. For each micronutrient observed in this study, we generated a series of concentrations as follows. Folic acid (Fol) 50 µM, magnesium orotate (MgOr) 0.1 mM (together ID63 worm ) and Fol 50 µM, MgOr 0.1 mM and vitamin B6 (Vit B6) 100 µM (together ID64 worm ). After reaching adulthood (48 h before the experiment), the micronutrients were added to the flasks. Pure M9-buffer was used as a control. Then, 24 h before the experiment amyloid aggregation was proceeded by upshifting young adult GMC101 from 20 • C to 25 • C.

Paralyze Assay
Cell culture flasks containing approximately 1000 adult amyloid beta producing nematodes were incubated for several hours to achieve Aβ induced paralysis (GMC101 24 h at 25 • C). On a NGM Agar Plate, 25 nematodes were placed and by physically touching with a platinum tip the paralysis status was recorded. Nematodes which normally act after being touched by the wire are recognized as "not paralyzed" whereas uncoordinated movements or just head movements were recorded as "paralyzed".

Heat-Stress Survival Assay
After 48 h of incubation of CL2006 with the mentioned effectors, the time till death was determined using a microplate thermotolerance assay [89]. In preparation, the nematodes were washed out of the flasks with M9-buffer into 15 mL tubes followed by 3 additional washing steps. Each well of a black 384-well low-volume microtiter plate (Greiner Bio-One, Frickenhausen, Germany) was prefilled with 6.5 µL M9-buffer/Tween ® 20 (1% v/v). In the following step, one nematode in 1 µL M9-buffer was transferred and immersed in the well under a stereomicroscope (Breukhoven Microscope systems, Netherlands). SYTOX™ Green (Life Technologies, Karlsruhe, Germany) in a final concentration of 1 µM was added to reach a final volume of 15 µL in the well. SYTOX™ Green creates a fluorescent signal after binding to DNA. The plates were sealed with a Rotilab sealing film (Greiner Bio-One, Frickenhausen, Germany). The heat-shock was applied and the fluorescence was measured every 30 min for 17 h at 37 • C following the protocol previously described [90]. The excitation was set at 485 nm and the emission was detected at 538 nm.

ThT Dying of Aβ Aggregates
Detection and quantification of Aβ aggregates (Aβ-aggr) in GMC101 were performed using the fluorescent dye thioflavin T (ThT) according to a previously described method with minor modifications [91]. Synchronized and heat incubated nematodes were washed out of the cell culture flasks with M9-buffer/Tween ® 20 (1% v/v) and separated from larvae. After centrifugation, 200 µL of a thick pellet of nematodes was transferred into a microcentrifuge tube and were frozen in liquid nitrogen. Samples were thawed with 500 µL of PBS including proteinase inhibitor. Afterward, the samples were homogenized with a sonifier 3 × 20 s on ice. Protein contents in the homogenate were assessed according to the Pierce™ BCA Protein Assay Kit (Thermo Fisher Scientific, Waltham, MA, USA). Bovine serum albumin was used as a standard. Finally, fluorescence was measured in a black 96-well plate by adding 1 mM ThT (final concentration 20 mM). The volume in each well was 100 µL by adding M9. To determine the fluorescence of ThT, samples were measured by excitation at 440 nm and emission at 482 nm.

Statistics
Unless otherwise stated, values are presented as mean ± standard error of the mean (SEM). Statistical analyses were performed by applying one-way ANOVA with Tukey's multiple comparison post-hock test, log-rank (Mantel-Cox) test and student's unpaired t-test (Prism 9.1 GraphPad Software, San Diego, CA, USA). Statistical significance was defined for p values ns = not significant, * p < 0.05, ** p < 0.01, *** p < 0.001 and **** p < 0.0001.

Conclusions
In the present study, we reported that different combinations of folic acid, magnesium orotate, and vitamin B6 had significant effects on glycolysis, Aβ production, and Aβ aggregation in SH-SY5Y-APP 695 cells and C. elegans. The phenotype of the in vivo model was significantly improved, highlighting the potential of the tested biofactor combinations as candidate therapeutics in AD. However, since the data did not consistently show a benefit of either combination, this study does not allow a clear statement as to whether vitamin B6 is required in addition to the combination of folic acid and magnesium orotate.

Data Availability Statement:
The data presented in this study are available on request from the corresponding author.