International Journal o  f
Molecular Sciences
Article
Effects of Combining Biofactors on Bioenergetic Parameters,
Aβ Levels and Survival in Alzheimer Model Organisms
Lukas Babylon, Fabian Schmitt, Yannik Franke, Tim Hubert and Gunter P. Eckert *
Biomedical Research Center Seltersberg (BFS), Laboratory for Nutrition in Prevention and Therapy,
Institute of Nutritional Sciences, Justus Liebig University Giessen, Schubertstr. 81, 35392 Giessen, Germany
* Correspondence: eckert@uni-giessen.de
Abstract: 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.
Citation: Babylon, L.; Schmitt, F.; Keywords: Alzheimer disease; mitochondria; mitochondria dysfunction; folic acid; vitamin B6;
Franke, Y.; Hubert, T.; Eckert, G.P.
magnesium-orotate; amyloid beta; C. elegans; biofactor
Effects of Combining Biofactors on
Bioenergetic Parameters, Aβ Levels
and Survival in Alzheimer Model
Organisms. Int. J. Mol. Sci. 2022, 23,
8670. https://doi.org/10.3390/ 1. Introduction
ijms23158670 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
Academic Editor: Anna Atlante
AD yet. Approved drugs only treat symptoms [2]. There are several hypotheses regarding
Received: 7 July 2022 the etiology of Alzheimer’s disease, but the causes of the disease are unknown. Previous
Accepted: 2 August 2022 research has focused on amyloid and tau, which has not yet led to major breakthroughs.
Published: 4 August 2022 Therefore, there is a trend towards multifactorial treatments and, among other things,
Publisher’s Note: MDPI stays neutral energy metabolism with regard to mitochondrial functions. Two of the hallmarks of AD are
with regard to jurisdictional claims in mitochondrial dysfunction (MD) [3] and overproduction of beta-amyloid (Aβ) [4]. The first
published maps and institutional affil- signs of beginning MD are a reduction of glucose consumption [5] and a reduced activity
iations. 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
Copyright: © 2022 by the authors. AD is that Aβ is cleaved of from a much larger amyloid precursor protein (APP) [11]. APP
Licensee MDPI, Basel, Switzerland. is cleaved via two pathways, a non-amyloid and an amyloidogenic pathway. The APP is
This article is an open access article spliced by the different types of protease, namely α-, β- and γ-secretase [11,12]. Depending
distributed under the terms and on which protease cleaves the APP, Aβ peptides are produced. The α-protease cleaves the
conditions of the Creative Commons APP closer to the membrane, resulting in a shorter fragment in the membrane, which is
Attribution (CC BY) license (https:// then further cleaved by the γ-protease to a non-amyloidogenic product. However, when
creativecommons.org/licenses/by/
β-protease cleaves the protein, larger fragments are produced, which are then cleaved by
4.0/).
Int. J. Mol. Sci. 2022, 23, 8670. https://doi.org/10.3390/ijms23158670 https://www.mdpi.com/journal/ijms
Int. J. Mol. Sci. 2022, 23, 8670 2 of 21
γ-protease to form different large Aβ proteins [12] including Aβ1–40 and Aβ1–42 which may
be the main triggers for AD [2,11,12]. It appears that Aβ1–42 has the greater neurotoxic
potential compared to the Aβ1–40 form. Furthermore, Aβ1–42 tends to aggregate more,
which can lead to plaque formation. In addition, the relationship between Aβ1–40 and
Aβ1–42 is important, as the two influence each other [13–15]. There is evidence that sAPPα
and sAPPβ share some properties [16], where sAPPα can be neuroprotective, whereas
sAPPβ lacks most of the neuroprotective properties and has a rather negative effect [17].
Another early sign of AD is impaired glucose metabolisation which leads to MD and an
increase in oxidant production [18,19]. The glycolysis pathway represents a way to ensure
sufficient energy production and bypass Aβ induced impairment of mitochondria [20,21].
The enzymes pyruvate dehydrogenase kinase 1 (PDK1) and LDHA are of interest here.
PDK1 phosphoylates pyruvate dehydrogenase and inactivates it. LDHA converts pyruvate
into lactate. Both are markers of aerobic glycolysis. If a change occurs here, conclusions can
be drawn about energy production [22,23].
There is evidence that specific biofactors [24], which are defined as substances required
by the body for its normal physiological functioning and/ or with health-beneficial and/
or disease-preventive biological activities, may interfere with pathophysiological processes
leading to AD [25–27]. A cocktail containing some of our tested compounds had a positive
effect on AD symptoms in a TgF344-AD rat model. This could raise the mitochondrial
function of the transgenic rats to the level of the wild-type rats [28]. Here, folic acid,
magnesium-orotate and vitamin B6 in different combinations were tested in cellular and
an invertebrate model of AD. The synthetically produced water-soluble folic acid, which
consists of pterin, p-aminobenzoic acid and L-glutamic acid, belongs to the vitamin B
complexes. Folate (also known as vitamin B9) is used as an umbrella term for the various
derivatives of tetrahydrofolate (THF), with the synthetically produced form being referred
to as folic acid [29,30]. Folate deficiency impairs DNA as well as mtDNA synthesis and
stability and causes oxidative stress in the form of ROS, which, as already listed, is also
associated with AD pathogenesis. In this context, neuronal impairment and increased cell
death occur in AD. In addition, a deficiency of folate leads to a decrease in the methylation
of enzymes and promoter regions of genes that are presumably also involved in AD
pathogenesis [31,32]. Magnesium-orotate (MgOr), which is very poorly soluble in water, is
the magnesium salt of orotic acid. As a source of magnesium (Mg), MgOr is used for the oral
treatment of Mg deficiency. Orotic acid is a key intermediate in the biosynthetic pathway of
pyrimidines and improves energy status by stimulating, among other things, the synthesis
of glycogen and ATP [33]. Mg2+ is the fourth most abundant mineral as well as the second
most abundant intracellular divalent cation in the human body and acts as a cofactor [34–36].
Mg is involved in protein synthesis, cellular energy production and storage, reproduction,
DNA and RNA synthesis, and mitochondrial membrane potential [35]. Mg is also involved
in the maintenance of physiological nerve and muscle function, cardiac excitability, and
neuromuscular conduction [35,36]. Several pathological mechanisms in AD are discussed
on which Mg might have a positive influence. Mg appears to inhibit the activity of γ-
secretase and the proinflammatory TNF-α (tumor necrosis factor α) produced by microglia.
Mg also inhibited IL-1β (interleukin-1β) and Aβ-induced, which all together induced
inflammation. In addition, Mg has been reported to decrease the influx of Aβ across
the blood-brain barrier [34]. High Mg concentrations have been shown to promote APP
processing towards α-secretase due to the upregulation of transcription factors such as
CREB [37]. Mg deficiency may be a risk factor for ADs and that possible supplementation
may be a potentially valuable adjunct treatment for AD [38]. The water soluble vitamin
B6 (Vit B6) is used as an umbrella term for various derivates from which pyridoxine is the
most common form. It is an enzymatic cofactor required for more than 140 biochemical
reactions, including transaminations, α-decarboxylations and replacement reactions [39].
Through the application of Vit B6, the oxidative stress induced by Aβ could be inhibited [40].
Furthermore, Vit B6 reduces the plasma levels of Aβ [41] and prevents the grey matter
atrophy related to AD [42].
Int. J. Mol. Sci. 2022, 23, 8670 3 of 21
The present work investigated the effects of different B vitamins and MgOr on MD and
the processing of APP in SH-SY5Y-APP695 cells a cellular model of early AD. Furthermore,
the effects of the substances were tested in CL2006 and GMC101, both invertebrate models
of AD.
2. Results
2.1. 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.
Table 1. General overview of all tests and results of all biofactors. Combinations ID63 and ID64 and
the single substances compared to the control.
ID63 vs. CTR ID64 vs. CTR MgOr vs. CTR Fol vs. CTR Vit B6 vs. CTR
Aβ1–40 Significant lower Significant lower Significant lower Significant lower No significant change
Aβ1–42 No significant change Significant lower Significant lower Significant lower Significant lower
sAPPα Significant lower Significant lower Not tested Not tested Not tested
sAPPβ No significant change No significant change Not tested Not tested Not tested
ATP level No significant change No significant change Not tested Not tested Not tested
MMP level No significant change No significant change Not tested Not tested Not tested
Respiration No significant change No significant change Not tested Not tested Not tested
Citrate synthase activity No significant change No significant change Not tested Not tested Not tested
Lactate level Significant lower Significant lower Not tested Not tested Not tested
Pyruvate level No significant change No significant change Not tested Not tested Not tested
Lactate/Pyruvate Ratio No significant change Significant lower Not tested Not tested Not tested
Gen expression
PDK1 No significant change No significant change Not tested Not tested Not tested
Gen expression
LDHA No significant change Significant higher Not tested Not tested Not tested
Lifespan C. elegans in % No significant change Significant higher Significanthigher Significant higher Significant higher
Mean survival C. elegans Significant higher Significant higher Significanthigher Significant higher Significant higher
Paralysis C. elegans Significant lower Significant lower Not tested Not tested Not tested
Aβ1–42 C. elegans No significant change No significant change Not tested Not tested Not tested
Aβ1–42 aggreation
C. elegans Significant lower No significant change Not tested Not tested Not tested
2.2. Aβ1–40 Production
First, we tested different concentrations of biofactors on Aβ1–40 production in SH-SY5Y-
APP695 cells. Cells were incubated for 24 h with zinc orotate (ZnO), magnesium-orotate
(MgO), benfotiamine (vitamin B1), folic acid (Fol), cholecalciferol (Vit D3), cobalamin (Vit
B12), and pyridoxine (Vit B6) to select possible hit substances for further experiments (data
not shown). Potential hit substances were identified, from which finally Fol 10 µM, MgOr
200 µM and Vit B6 100 nM turned out to be the most promising ones, which we applied in
two different combinations.
To investigate the effect on the Aβ1–40 production, SH-SY5Y-APP695 cells were in-
cubated with both combinations (MgOr 200 µM & Fol 10 µM = ID63//MgOr 200 µM &
Fol 10 µM & Vit B6 100 nM = ID64) and the single compounds for 24 h (Figure 1). ID63
had a significant lowering effect on the Aβ1–40 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
Int. J. Mol. Sci. 2022, 23, x FOR PEER REVIEW 4 of 23 
 
orotate (MgO), benfotiamine (vitamin B1), folic acid (Fol), cholecalciferol (Vit D3), cobal-
amin (Vit B12), and pyridoxine (Vit B6) to select possible hit substances for further exper-
iments (data not shown). Potential hit substances were identified, from which finally Fol 
10 µM, MgOr 200 µM and Vit B6 100 nM turned out to be the most promising ones, which 
we applied in two different combinations. 
To investigate the effect on the Aβ1–40 production, SH-SY5Y-APP695 cells were incu-
bated with both combinations (MgOr 200 µM & Fol 10 µM = ID63//MgOr 200 µM & Fol 
10 µM & Vit B6 100 nM = ID64) and the single compounds for 24 h (Figure 1). ID63 had a 
significant lowering effect on the Aβ1–40 level (p > 0.0001). The ID63 combination even had 
Int. J. Mol. Sci. 2022, 23, 8670 an over additive effect compared to the single substances MgOr (p = 0.0028) and Fol 4(po f>2 1
0.0001). The ID64 combination had a significantly decreasing effect on the Aβ levels com-
pared to the control (p > 0.0001). Furthermore, ID64 had a significantly reducing effect in 
colemvpelasrcisoomnp taor etdhet ositnhgeleco snutbroslta(pnc>es0 .M00g0O1)r.  F(pu r=t h0e.r0m49o7r)e, ,FIDol6 (4ph >a d0.a00s0ig1n) iafincdan Vtliyt rBe6d u(pc in> g
0.e0f0fe0c1t).i n comparison to the single substances MgOr (p = 0.0497), Fol (p > 0.0001) and Vit B6
(p > 0.0001).
 
Figure 1. Effect of ID63 and ID64 in SH-SY5Y cells compared to the control or their single
Fisguubrseta 1. Eff
695
nces oenctt hofe IAD63 anldev IeDl 6a4ft eirn 2S4Hh-SinYc5uYb6a9t5i coenl.lsN co=m6.pAared tol ethveel scownetrreola dorju tshβ β teedir tsointhgelep sroutbe-in
stances on the Aβ 1–1–40 lev4e0l after 24 h incubation. N = 6. Aβ 1–41–40 lev0els were adjusted to the protein 
content. Significance was determined by Student’s unpaired t-test and one-way ANOVA. + significant
against control; * significant against ID63; # significant against ID64. * p < 0.05, **** p < 0.0001,
#### p < 0.0001 and + p < 0.05, ++++ p < 0.0001. Data are displayed as the mean ± SEM. ID63 = 200 µM
 MgOr and 10 µM Fol; ID64 = 200 µM MgOr, 10 µM Fol and 100 nM Vit B6.
2.3. Aβ1–42 Production
To study the production of Aβ1–42, SH-SY5Y-APP695 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 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.
Int. J. Mol. Sci. 2022, 23, x FOR PEER REVIEW 5 of 23 
 
content. Significance was determined by Student’s unpaired t-test and one-way ANOVA. + signifi-
cant against control; * significant against ID63; # significant against ID64. * p < 0.05, **** p < 0.0001, 
#### p < 0.0001 and + p < 0.05, ++++ p < 0.0001. Data are displayed as the mean ± SEM. ID63 = 200 µM 
MgOr and 10 µM Fol; ID64 = 200 µM MgOr, 10 µM Fol and 100 nM Vit B6. 
2.3. Aβ1–42 Production 
To study the production of Aβ1–42, SH-SY5Y-APP695 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 Aβ1–42 levels, while the combination ID63 
had a slight reducing effect on the Aβ1–42 levels, although not a significant one. ID64 also 
Int. J. Mol. Sci. 2022, 23, 8670 showed significantly lower Aβ1–42 levels in comparison to B6 (p = 0.0009). However, fo5loicf  21
acid alone, reduced the levels to a higher extent than any combinations. 
 
FigFuigrue r2e. 2E.ffeEcftf eocft IoDf6I3D a6n3da InDd64ID in6 4SHin-SSYH5-YSY5695 cYe6ll9s5 ccoemllspacroemdp taor ethde tcoonthtreocl oonr ttrhoel ior rsitnhgelier ssuinbg- le
stasnucbessta onnc etshoen Athβe Aβle1v–e42l laefvteerl afte1–42  24 hr  2i4nchuibnactuiobnat. ioNn .=N 6.= A6β. Aβl1e–v42ellsev1–42  welesrwe eardejuasdtjeuds tetod tthoet hperpotreoitne in
cocnotnentetn. St.igSnigifnicifiacnacnec we awsa dsedteertmerimneinde bdyb SytuSdtuednet’nst ’usnupnapiraeidre td-tte-stet satnadn odnoen-we-awya AyNAONVOAV.A *.* *p* <p 0<.001.0 1
anadn *d*** *p* <p 0<.0001.0. 0D1a. taD aartae darisepdlaisypelda yaes dthaes mtheeanm ±e SaEnM±. SIDEM63. =I D20603 µ=M2 0M0gµOMr aMngdO 1r0 aµnMd F1o0l;µ IMD6F4o l;
= 2ID006 4µM=  2M00gµOMr, 1M0 gµOMr, F1o0lµ aMndF 1o0l0a ndM1 V00it nBM6. Vit B6.
2.42.. 4s.AsPAPPαP aαnadn sdAsPAPPβP LβeLveevl el
ThTeh αe- αa-ndan βd-sβec-rseetcarseet acsleeacvlienagv pinrgodpurcotds uocf tAs PoPf ,A sAPPP,PsαA (PFPigαur(eF i3gAu)r ean3dA s)AaPnPdβs (AFiPgP-β
ur(eF i3gBu)r,e re3sBp),ercetsivpeelcyt,i vweleyr,ew deerteerdmetienremdi naeftdera fitnecruibnacutiboant iwonithw eitihtheeitrh IeDr6I3D o63r IoDr 6ID4 6fo4rf o2r42 h4. h.
FiFgiugruer 4eA4 Ashsohwows tshtaht aItDI6D36 h3ahda da saigsinginfiicfiacnatnlyt llyowloewrienrgin egffeefcfet cotno tnhteh seAsPAPPαP α(p (=p 0=.002.01241) 4)
cocmompapraerde dto ttohet hceonctornotl.r oIDl.64ID h6a4d haand evaenn egvreenategrr eeaffteecrt eofnfe tchteo rnedthuectrioend uocft sioAnPPoαf  sfAraPgP-α
mfernatgsm (pe n>t s0(.0p0>010).0. 0In01 c).oInntrcaosnt ttroa stthteo sthAePsPAαP fPrαagfmraegnmt epnrtoprod(Figure 3B) were lowered compared to the control thougdhucnt
uiocnti,o tnh,et hseAsPAPPPβ fragmentsot significantlβy. fIrDag6m4 ehnatds a
(Fgigreuartee r3Bef)f ewcet rteh alnowIDer6e3d.  Ictosmhopualrdedb eton othteed ctohnattrboal stahloluevgehl snooft ssAigPnPificwanetrleya. pIDpr6o4β x himada tael y
one hundredfold lower compared with sAPPα (Figure 3).
2.5. Effect on the Mitochondrial Function
 To investigate the effect of ID63 and ID64 on mitochondrial function, we incubated
SH-SY5Y-APP695 cells for 24 h with ID63 or ID64. Respiration under O2 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 O2 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),
Int. J. Mol. Sci. 2022, 23, x FOR PEER REVIEW 6 of 23 
 
Int. J. Mol. Sci. 2022, 23, 8670 6 of 21
greater effect than ID63. It should be noted that basal levels of sAPPβ were approximately 
one hundredfold lowanedr cIDo6m4 pevaernehda dwaitshli gshAtlPy Pdeαc r(eFasiginugreeff e3c)t. compared with the control (Figure 4H).
However, none of these effects is statistically significant.
Figure 3. Effect of the incubation with ID63 or ID64 on the human soluble amyloid precursor p rotein
α (sAPPα) and β (sAPPβ) after 24 h of incubation. (A) sAPPα level of SH-SY5Y cells after the
Figure 3. Effect of the ininccuubbaattiioonnw withitIhD 6ID3 o6r3I Do6r4 IDco6m4p aorned thtoet hheucmonatrno ls. o(Blu) bsAleP Paβmleyvleoliodf pSHrec
6u95-SY5rYsor cpelrlos taeftien 695 r
α (sAPPα) and β (sAPtPheβi)n caufbtaetrio n24w ihth oIDf 6i3nocruIDb6a4ticoonm.p a(Ared) tsoAthPePcoαn tlreovl. eNl =o6f. SsAHP-PSlYev5eYls6w95e creelaldsj uastfetdert otthhee 
incubation with ID63 oprr oIDtei6n4c ocnotmentp. aSrigendifi tcoa ntcheew caosndterteorlm. i(nBe)d sbAy PonPeβ-w laeyvAeNl oOfV SAH. *-pSY< 50.Y0569a5n cde*l*l*s* apf<te0r.0 t0h01e. 
incubation with ID63 oDr aItDa a6r4e  dciospmlapyeadreasdt hteom theaen c±oSnEtMro. lI.D N63 == 260.0 sµAMPMPg lOevr aenlsd 1w0eµrMe Faodl;jIuDs6t4e=d 2t0o0 µthMeM pgrOor-,
tein content. Significan1c0eµ wMaFso ldanedte1r0m0 ninMeVdit bBy6. one-way ANOVA. * p < 0.05 and **** p < 0.0001. Data 
are displayed as the m2e.a6.nL ±ac tSaEteMan.d IPDy6ru3v =at e2L0e0v eµl M MgOr and 10 µM Fol; ID64 = 200 µM MgOr, 10 
µM Fol and 100 nM Vit B6.T o 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).
 
Int. J. Mol. Sci. 2022, 23, x FOR PEER REVIEW 7 of 23 
 Int. J. Mol. Sci. 2022, 23, 8670 7 of 21
 
Figure 4. ATP level, MMP level, respiration and citrate synthase activity of SH-SY5Y-APP cells
Figure 4. ATP level, MMP level, respiration and citrate synthase activity of SH-SY5Y-APP 69695 c5ells 
incinucbuabteadte dfofro r242 4hh wwitihth IIDD6633 oorr IIDD6644.. ((AA)) AATTPPl elevveel lo of fS HSH-S-YSY5Y5-YA-PAPP6P95 ceclellslsin695  icnucbuabtaetdedw withitIhD I6D36a3n d
an(dB )(BA)T APTlePv elelvoefli nocf uinbacutibonatwiointh wIDith6 4IDco6m4 pcaormedpatoretdh etoc otnhter oclo.nCtreollls. tCreealltse dtrweaittehdc ewllitchu lcteulrle cmuletudriue m
mesedrivuemd saesrvcoedn taros lc(o1n0t0r%ol) (.1N00=%1).2 N. ( =C )12M. (MCP) MleMvePl  olefv2el× o1f 025 ×S 1H0-5 SSYH5-YS-YA5PYP-A69P5 Pce69l5l sceilnlsc uinbcautebdatwedit h
wiItDh6 I3Da6n3d an(Dd) (MD)M MPMlePv elel voefli nocf uinbcautiboantiwonit hwIiDth6 I4Dc6o4m cpoamrepdartoedt htoe cthoen tcronl.trNol=. N16 =.  (1E6). R(Ee)s pRiersaptiioran-of
tioSnH o-Sf YS5HY-SAYP5PY6-9A5 PcePll6s95i nceclulsb aitnecduwbaitehdI Dw6i3tha nIDd 6(F3) aincdu b(Fa)t iionncuwbiathtioIDn6 w4 citohm IpDa6r4e dcotomtphaerceodn troo lt.hSeH -
coSnYtr5oYl-. ASHPP-S69Y55cYe-lAlsPaPd6j9u5 scteeldls taodijnutsetrenda ttoio innatleurnnaittsio(nIUal) uonf ictist r(aIUte) soyfn ctihtraastee ascytnivthitays. eN ac=ti1v5i.ty(G. N)  C= i1tr5a. te
(Gs)y Cnitthraastee saycntitvhiatyseo afcStiHvi-tSyY o5fY S-AHP-SPY5Yc-AelPlsPi6n95c cuebllast iendcuwbiathtedID w63itha nIDd6(3H a)nCdi t(rHat)e Csiytrnatthea synth695 se actaisvei ty
acitnivciutyb aitnecduwbaittehdI Dw6i4thc oIDm6p4a croedmtpoacroendt rtoo lc. oNnt=ro1l2. .NS i=g n12ifi. cSaignnceifwicaans cdee wterams dineetderbmyinSteudd beyn tS’stuudnepnati’rse d
untp-taeisrte.dD ta-ttaesat.r eDdaitsap alraey eddisapslatyheedm aesa tnhe± mSeEaMn .±I DSE6M3 =. I2D0603 =M 20M0 gµOM MgOr and 10 µM Fol; ID64 = µ r and 10 µM Fol; ID64 = 200 µM
200 µM MgOr, 10 µM Fol and 100 nM Vit B6. Leak I (G + M) = leak respiration with glutamate and 
maMlagtOe;r O, 1X0PµHMOSF o=l oaxnidda1t0iv0en pMhoVspithBo6r.yLlaetaioknI s(yGst+emM; )E=TCle a=k elreecstproirna ttiroannswpiotrht gchluatianm; Laetaeka InId (Ommayla)t e;
= lOeaXkP rHesOpSir=atoioxnid watiitvhe oplyhgoospmhyocriynl.a tion system; ETC = electron transport chain; Leak II (Omy) = leak
respiration with olygomycin.
 
Int. J. Mol. Sci. 2022, 23, x FOR PEER REVIEW 8 of 23 
 
2.5. Effect on the Mitochondrial Function 
To investigate the effect of ID63 and ID64 on mitochondrial function, we incubated 
SH-SY5Y-APP695 cells for 24 h with ID63 or ID64. Respiration under O2 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 O2 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 
Int. J. Mol. Sci. 2022, 23, 8670 (Figure 4G), and ID64 even had a slightly decreasing effect compared with the con8torof 2l 1
(Figure 4H). However, none of these effects is statistically significant. 
 
Figure 5. Effect of the incubation with ID63 or ID64 on lactate and pyruvate level after 24 h of
Fiingcuurbea 5ti. oEnf.fe(cAt )oLf atchtea tiencleuvbealtioofnS Hw-iSthY I5DY63 ocre lIlDs 6a4ft oern tlhaectiantceu abnadti opnyrwuivthatIeD l6ev3eolr aIfDte6r4 2c4o hm opfa irnecdut-o
btahteiocno.n (tAro) lL. a(Bct)aPtey rleuvvealt eofl eSvHe-lSoYf5SYH69-5S cYe5l
6l95Ys aftecre ltlhsea fitnecrutbhaetiionncu wbiatthio InDw63i tohr IIDD6634o croImDp6a4rceodm top athreed 
control. (B) Pyruvate level of SH-SY5Y 695695 cells after the incubation with ID63 or ID64 compared to 
to the control. (C) Lactate to p
Int. J. Mol. Sci. 2022, 23, x FOR PEER REthVeIE cWon trol. (C) Lactate to pyruva
ytreu rvaatitoe. rNat i=o 6. . NLe=ve6l.s wLeevree lasdwjuesrteeda dtoju tshtee dprtootethine cpornotteenint9. 
c
 S
o
oifg
n
 2n
teint.
Signifi 3 
f-
 icance wcaansc edewtearsmdienteedrm biyn eodnbey-woanye -AwNayOAVNAO. *V Ap .<* 0p.0<50, .*0*5 ,p* *< p0<.001. 0a1ndan *d***** *p* p< <0.00.000010.1 D. Dataat aaarere 
ddisispplalayyeedd aass tthhee meeaann ± SSEEM.. IIDD6633 == 220000 µµM MggOOrr aanndd1 100µ µMMF Fool;lI; DID6464= =2 02000µ MµMM MgOgrO, 1r0, 1µ0M µMFo l
Faonl dan1d0 010n0M nVMit VBi6t .B6. 
2.6. 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). 
Figure 6. Effect of the incubation with ID63 or ID64 on the gene expression after 2 4 h of incubation.
Fig(Aur)eG 6.e nEeffeecxtp orfe tshsieo incoufbpaytirounv watiethd IeDh6y3d oror gIDen6a4s oenk tihnea sgeen1e( ePxDpKre1s)sioofnS aHft-eSrY 254Y h6 9o5f cinelclusbaafttieornt. he
(A)in Gcuenbea teioxnprwesitshioInD o6f3 poyrrIuDv6a4tec odmehpyadrerdogteontahseec koinntarosel. 1( B(P )
DGKen1e) oexf pSrHes-SsiYo5nYo6f95l caecltlast eafdteerh tyhder oingceun-ase
batAio(nL DwHithA I)Do6f 3S Hor- SIYD56Y4 comcepllasraefdt etrot thheei co695 ncnutbraotli.o (nBw) GitehnIeD e6x3porresIDsi6o4n coofm lapcatareted dtoehthyedrcoognetrnoals. eN A= 8.
(LDSiHgnAifi) coaf nScHe -wSYas5Yde69t5e crmellisn eadftebry tohne ei-nwcuaybaAtiNonO VwAit.h* IpD<630 .o0r5 .IDD6a4ta caorme dpiasrpelda ytoed thaes tchoentmroela. nN± = S8E. M.
Significance was determined by one-way ANOVA. * p < 0.05. Data are displayed as the mean ± SEM. 
ID6ID3 6=3 2=002 0µ0Mµ MMgMOgrO anr dan 1d0 1µ0Mµ MFolF; oIlD; I6D4 6=4 2=002 0µ0Mµ MMgMOgrO, 1r0,  1µ0Mµ MFoFl oalnadn 1d001 0n0Mn MViVt Bit6B. 6.
2.7. qPCR
2.7. qPCTRo investigate the molecular basis of altered lactate and pyruvate levels, the gene
expTroe sinsivoenstoigfaptey rtuhvea mteodleechuyladrr obgaesnisa soef kalitnearseed 1la(cPtaDtKe 1a)ndan pdylraucvtaattee dleevheylsd, rtohgee ngeansee A
ex(pLrDesHsiAon) wofe rpeyreuxvaamtein dedehayfdterrog2e4nhasien ckuinbaasteio 1n (uPsDinKg1)q RaTn-dP ClaRct.aItDe 6d3ehaynddroIDge6n4ahsea dAn o
(LDsiHgnAifi) cwaenrtee efxfeacmt ionnedP aDftKe1r 2g4e hn einecxupbraetsisoino nuscinomg qpRarTe-dPCtoR.t IhDe6c3o anntrdo IlD(F64ig huarde  n6)o. siBgo-th
nifciocamnbt ienfafteicotn osnin PcDreKas1e gdeLnDe HexAprmesRsNioAn cleovmelpsa(rFeidgu troe t6hBe) ,cownitthroIlD (F64ig(upr=e 60).0. 1B4o8t)hs hcoomwbini-g a
nastiiognsifi icnacnretainsecdre LasDeHinAg menReNexAp lrevsseilos n(F. igure 6B), with ID64 (p = 0.0148) showing a sig-
nificant increase in gene expression. 
2.8. 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 concentra-
tions. 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 Fig-
ure 7A,B). As shown in Figure 7A, the ID63worm extended lifespan of CL2006 compared to 
the control by trend (p = 0.0502), whhereas in Figure 7B, it can be seen that ID64worm 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 sub-
stances was assessed. Thereby Figure 7C shows that ID63worm (p = 0.0196), Fol 50 µM (p < 
0.0001), MgOr 100 µM (p = 0.0004), ID64worm (p < 0.0001) and Vit B6 (p = 0.0107) had a 
significant increasing effect on the mean survival of the nematodes compared to the con-
trol. Folic acid alone was numerically more effective than the combinations or the other 
single compounds. 
 
Int. J. Mol. Sci. 2022, 23, 8670 9 of 21
2.8. 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 ID63worm extended lifespan of CL2006 compared to the control
by trend (p = 0.0502), whhereas in Figure 7B, it can be seen that ID64worm 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 ID63worm (p = 0.0196), Fol 50 µM (p < 0.0001),
MgOr 100 µM (p = 0.0004), ID64worm (p < 0.0001) and Vit B6 (p = 0.0107) had a significant
Int. J. Mol. Sci. 2022, 23, x FOR PEER RE
 iVnIEcWr easing effect on the mean survival of the nema10t oof d23 es compared to the control. Folic acid
alone was numerically more effective than the combinations or the other single compounds.
 
FigFuirge 7u. Trhee 7lif.esTpahn eunldiefre hseapt-astnressu onf Cd. elergahnse aaftter- strteratemsesnt owfithC e.itheelre IgDa63nwsorma, IfDt6e4rwortmr eatment with either ID63worm, ID64worm
oro threirt hsinegle substances in CL2006. (A) The single substances lead to an increase in heat-stress resistance ofi trhes nienmgatloedess aultbhosutgah nthce ecosmbiinatCionL o2f t0h0em6 d. oe(sA no)t hTavhee a ssiginifgicalnet ismupabcts. tances lead to an increase in heat-stress
(Br) eAfsteirs ctoamnbicnaetioonf wtithh veitanmeinm B6a, thteo rdesuelstinag ltrtehatmoeuntg lehadts htoe a scigonimficabnti inncaretaisoe inn hoeaft-them does not have a significant impact.
(B) After combination with vitamin B6, the resulting treatment leads to a significant increase in heat-
 stress resistance. For heat-stress experiments, the survival was assessed according to the penetration
of SYTOX Green nucleic acid stain into dead cells. N > 60. log-rank (MantelCox) test. (C) Mean
Survival of C. elegans after treatment with either ID63worm, ID64worm or their single substances in
CL2006. Significance was determined by one-way ANOVA. p * < 0.05, p ** < 0.01, p *** < 0.001
and p **** < 0.0001. ID63worm = Fol 50 µM and MgOr 100 µM; ID64worm Fol 50 µM, MgOr 100 µM and
Vit B6 100 µM.
Int. J. Mol. Sci. 2022, 23, x FOR PEER REVIEW 11 of 23 
 
stress resistance. For heat-stress experiments, the survival was assessed according to the penetration 
Int. J. Mol. Sci. 2022 23 of SYTOX Green nucleic acid stain into dead cells. N > 60. log-rank (MantelCox) test. (C) Mean , , 8670 Survival of C. elegans after treatment with either ID63worm, ID64worm or their single substances in 10 of 21
CL2006. Significance was determined by one-way ANOVA. p * < 0.05, p ** < 0.01, p *** < 0.001 and p 
**** < 0.0001. ID63worm = Fol 50 µM and MgOr 100 µM; ID64worm Fol 50 µM, MgOr 100 µM and Vit B6 
100 µM. 
2.9. Effect on the Paralysis
2.9. EffeTcot onin thvee psatriaglyasitse the effect of combining biofactors on paralysis induced by Aβ, C.
elegTaon isnGveMstiCga1te0 1thew eeffreecti nofc cuobmabtiendingfo brio2fa4cthoras to2n5 pa◦rCalywsiist hindeuitchede rbyID Aβ, C. ele-gans GMC101 were incubated for 24 h at 25 °C with either ID63worm or ID64 63 or ID64 . Bothworm. Bothw coormm- worm
bcinoamtiobnisn a(pt i=o n0.s02(4p3= fo0r. 0ID24633wforomr aInDd 6p3 =w 0o.r0m14a9n fodr pID=604w.0or1m4) 9wfeorer aIDble6 4tow osirgmn)ifwicaenrtelya ble to significantly
ddecerceraesea sthee tphaeraplyasrisa ilnydsuisceidn bdyu Aceβd (FbigyurAe β8). (TFhiugsu, rthee8 p)h. eTnhotuyps,e tohf ethpish AeDn-owtoyrpme of this AD-worm
wwasa ssigsnigifniciafinctlayn etnlhyanecnehda bnyc beodthb byiobfaocttohr cboimofbaincatotiorncso tmo ab cionmatpiaornabslet oexatecnot. mparable extent.
 
FFigiugrue r8e. T8h. eT Ahβe iAndβuciendd puacrealdyspisa arfatelry striesatamfteenrt wtrietha temitheenr tIDw6i3twhorme iotrh IeDr6I4Dwo6rm3 iwn oGrmMCo1r0I1D. 64worm in GMC101.
BBotoht thhet fheeedifnege dofi bnogtho trfeabtmotehnt tcroemabtimnaetinotnsc loeamdsb tion saigtinoifnicsanlte raedduscttioons oifg Anβifi incdaunctedr peadrualc-tion of Aβ induced
ysis after incubation of the nematodes for 24 h at 25 °C. N = 4. Mean ± SEM. On◦e-way ANOVA with 
Tpuakerya’lsy csoims paafrtiesorni pnocsut bhoact itoesnt. po f* <t h0.e05n IeDm63awotrom d= eFsol f5o0r µ2M4 ahnda Mt g2O5r 10C0. µNM; =ID644.worMm Feoal n ± SEM. One-way
5A0 µNMO, VMAgOwr 1it0h0 µTMuk aenyd’ Vs ict oBm6 1p00a rµiMso. n post hoc test. p * < 0.05 ID63worm = Fol 50 µM and MgOr 100 µM;
ID64worm Fol 50 µM, MgOr 100 µM and Vit B6 100 µM.2.10. Aβ1–42 Production in GMC101 
2.10T.oA stβu1d–y4 2thPe rpordouductcitoionn ionf GAβM1–4C2 i1n0 n1ematodes, 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 TAoβstud1–42 levyelst hceomppraordedu ctoti othne ocofnAtroβl 1(–F4i2guinre n9e).m Siantcoe dAeβs, GisM nCot1 p0r1odwucaesd iinnc ubated for 24 h at1–40 
G2M5C◦1C01w thiitsh amboyltohidc poemptbidien wataiso nost iannvedsttighaetnedt. he Aβ level was examined. There was no effect
Int. J. Mol. Sci. 2022, 23, x FOR PEER REoVI nEWt he Aβ1–42 levels compared to the control (Figure 9).
12S oifn 2c3 e Aβ1–40 is not produced in
GMC101 this amyloid peptide was not investigated.
 
 
FigFuirge u9.r Eeff9ec.t oEff IfDe6c3t aondf ID646 i3n taranndsgeInDe 6n4emiantotdreas nGsMgCe1n01e conmepmaraedto tod tehes cGonMtroCls 1on0 1thec ompared to the controls on the
Aβ1–42 level after 48 h incubation. Neither ID63 nor ID64 did not lead to a significant alteration of 
AβA1–β42. 1N– 4=2 8.l Aeβv1e–42l leavfetles rwe4r8e ahdjuinstecdu tbo athtei oprno.teNin ceointthenetr. NI D= 86. 3Mneaon r± SIDEM6. 4Stdudiedntn’s ot-ttelset. ad to a significant alteration of
IDA63βwo1rm– =4 2Fo. l N50 µ=M8 a.ndA Mβg1O–r4 2100le µvMe; lIsD6w4weorrme Foal d50j uµMst,e MdgOtor 1t0h0 eµMp arnodt eViitn B6c o10n0 tµeMn. t. N = 8. Mean ± SEM. Student’s
2.1t-1t.e Asβt.1–4I2D A6g3grwegoartmion= Fol 50 µM and MgOr 100 µM; ID64worm Fol 50 µM, MgOr 100 µM and Vit B6 100 µM.
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). 
 
Figure 10. The effect of either ID63worm or ID64worm on the accumulation of aggregated Aβ (Aβ-aggr) 
in GMC101 after 48 h incubation. The treatment of the nematodes with ID63 leads to a significant 
 
Int. J. Mol. Sci. 2022, 23, x FOR PEER REVIEW 12 of 23 
 
 
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. 
Int. J. Mol. Sci. 2022, 23, 8670 ID63worm = Fol 50 µM and MgOr 100 µM; ID64worm Fol 50 µM, MgOr 100 µM and Vit B6 100 µM11.o f 21
2.11. Aβ1–42 Aggregation 
2.11.TAoβ i1n–v42esAtiggarteeg athtieo naggregation of Aβ in nematodes, GMC101 was incubated at 25 °C 
for 24T ho ainnvde ssttiaginateedt hweitahg tghrieogfalatvioinneo (fTAhβT)i,n wnheimcha tloabdeelss, βG-MshCee1t0 s1trwuacstuinrecsu obfa taegdgraetg2a5te◦dC 
pfoerpt2i4dehsa. nTdhesrtea iwneads wa istihgnthifiiocflanavt irneed(uTchtiTo)n,  wofh Aicβh-laagbgerl sbβy- sthhee ectosmtrbuicntautrieosno IfDa6g3gwroermg a(pte =d 
0p.0ep32ti4d) ecso.mTphaerreed wtoa tshae csoignntrifiolc,a wnthereredausc ItDio6n4woofrmA wβa-as gwgirthboyutth aen ceoffmecbti n(FaitgiounreI 1D06).3 worm
(p = 0.0324) compared to the control, whereas ID64worm was without an effect (Figure 10).
 
FFiigguurree 1100.. TThhee eeffffeecctt ooff eeiitthheerr IIDD6633worm oorr IIDD6644wor mono nthteh eacaccucmumuulaltaitoionn oof faaggggrreeggaatetedd AAβ (Aworm worm β (Aββ-a-aggggrr)) 
iinn GGMCC110011 aafftteerr 4488 hh iinnccuubbaattiioonn.. TThhee ttrreeaattmeenntt ooff tthhee nneemaattooddeess wwiitthh IIDD6633 lleeaaddss ttoo aa ssiiggnniiffiiccaanntt 
reduction of accumulated Aβ. Whereby the treatment with ID64 combination did not lead to an
alteration of Aβ levels; N = 8; mean ± SEM; one-way ANOVA with Tukey’s comparison post hoc
test. * p < 0.05. ID63
 worm
= Fol 50 µM and MgOr 100 µM; ID64worm Fol 50 µM, MgOr 100 µM and
Vit B6 100 µM.
3. 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 [37,43–47]. 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 Aβ1–40 levels in SH-SY5Y-APP695 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
Aβ1–40 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 Aβ1–40 levels.
Furthermore, lower Mg2+ 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 Aβ1–40 and Aβ1–42 [53]. As a result, an administration of Mg2+
Int. J. Mol. Sci. 2022, 23, 8670 12 of 21
leads to a decrease in Aβ levels of N2a-APP cells [37]. After the incubation with ID63,
SH-SY5Y-APP695 cells showed reduced levels of Aβ1–42 compared to the control, although
not significantly. In contrast, the incubation with ID64 showed a significant reduction of
Aβ1–42 (Figure 2) compared to the control. Administration of a Fol-rich diet significantly
reduced Aβ1–42 levels in APP/PS1 mice compared to the standard diet [54]. Similarly, the
incubation with Mg significantly decreased Aβ1–42 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-APP695 cells. However,
it seems that Vit B6 has a crucial role in the reduction of Aβ1–42 levels (ID64) even if
Vit B6 alone showed no effects. In contrast, it did not provide any benefit at the Aβ1–40
level (Figure 1). Next, we examined sAPPα and -β levels after incubation with ID63 and
ID64. The reducing effects on Aβ1–42 levels by ID64 in SH-SY5Y-APP695 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 Mg2+ 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-APP695 cells. SH-SY5Y-APP695 cells
show reduced ATP level, MMP and O2 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, O2
consumption or citrate synthase activity in SH-SY5Y-APP695 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 lac-
tate 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], al-
though 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
Int. J. Mol. Sci. 2022, 23, 8670 13 of 21
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]. ID63worm had no significant life-prolonging
effect on CL2006 compared to the control. Whereas the single substances and ID64worm
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 [73,79–81]. 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 Aβ1–42
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 Aβ1–42 were also relatively moderate in the cells (see above). Aβ1–40 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 ID63worm. 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 admin-
istration could lead to synergistic effects that enhance the positive effects and thus lead
to an additive effect, as shown in the Aβ1–40 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.
4. Materials and Methods
4.1. Cell Culture
The cultivation of SY5Y cells was performed in 75 cm2 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% CO2 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 APP695 (APP695) and were
kindly donated by A. Eckert (Basel, Switzerland) (for details; please refer to [83]).
Int. J. Mol. Sci. 2022, 23, 8670 14 of 21
4.2. Cell Treatment
First of all, cells were incubated for 24 h with different concentrations of zinc orotate
(ZnOr), magnesium-orotate (MgOr), benfotiamine (vitamin B1), folic acid (Fol), cholecalcif-
erol (Vit D3), cobalamin (Vit B12), and pyridoxine (Vit B6) for possible hit substances. After
the hit substances were determined, cells were incubated with 200 µM magnesium-orotate
(MgOr) and folic acid 10 µM (Fol) (combination ID63) or 200 µM MgOr, 10 µM folic acid
and 100 nM Vit B6 (combination ID64). We have derived the concentrations used here from
the literature [37,43–47]. The solution medium NaOH diluted 1:8 with water served as a
control. The ratio had the best dissolving properties without being toxic to the cells.
4.3. 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].
4.4. 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 Mg2+, Ca2+, 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).
4.5. Cellular Respiration
Respiration in SH-SY5Y695 cells was assessed using an Oxygraph-2k (Oroboros, Inns-
bruck, 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, in-
hibitors 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 106 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/106 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.
4.6. 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)
Int. J. Mol. Sci. 2022, 23, 8670 15 of 21
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.
4.7. Aβ1–40 Measurement
After 24 h incubation, the Aβ1–40 levels were determined in SH-SY5Y-APP695 cells
using HTRF Amyloid-Beta 1–40 kit (Cisbio, Codolet, France). The protocol was the same as
described earlier [61]. Aβ concentrations were normalized against protein content.
4.8. Aβ1–42 Measurement
After 24 h incubation, the Aβ1–42 levels were determined in SH-SY5Y-APP695 cells
and nematodes GMC101 using the Human-Aβ1–42 ELISA Kit (InvitrogenTM, Waltham,
MA, USA). The protocol was performed according to the manufacturer’s instructions. Aβ
concentrations were normalized against protein content.
4.9. Protein Quantification
Protein content was determined using a PierceTM Protein Assay Kit (Thermo Fisher
Scientific, Waltham, MA, USA) according to the manufacturer’s instructions. Bovine serum
albumin was used as a standard.
4.10. Quantification of Human Soluble Amyloid Precursor Protein α (sAPPα)
The sample preparation was the same according to the Aβ1–40 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.
4.11. Quantification of Human Soluble Amyloid Precursor Protein β (sAPPβ)
The sample preparation was the same according to the Aβ1–40 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.
4.12. 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.
4.13. Real-Time qRT-PCR
To isolate RNA, we used the RNeasy Mini Kit (Qiagen, Hilden, Germany) according to
the manufacturer’s guidelines. NanodropTM 2000c spectrometer (Thermo Fisher Scientific,
Waltham, MA, USA) was used to quantify RNA. The TURBO DNA-freeTM 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.
Int. J. Mol. Sci. 2022, 23, 8670 16 of 21
Table 2. Oligonucleotide primer sequences, product sizes, and primer concentrations for quantitative
real-time PCR.
Primer Sequence Manufacturer Product Size Concentration (nM)
ß-Actin (ACTB) 5′-ggacttcgagcaagagatgg-3′ Biomol, Hamburg,
NM_001101.2 5′-agcactgtgttggcgtacag-3′ Germany 234 200
Glyceraldehyde-3-
phosphate dehydrogenase 5′-gagtcaacggatttggtcgt-3′ Biomol, Hamburg,
(GAPDH) 5′-ttgattttggagggatctcg-3′ Germany 238 200
NM_002046.2
Phosphoglycerate kinase 1 ′
(PGK1) 5 -ctgtgggggtatttgaatgg-3
′ Biomol, Hamburg,
NM_000291.2 5
′-cttccaggagctccaaa-3′ Germany 198 200
Pyruvate dehydrogenase
kinase, isozyme 1 5′-atacggatcagaaaccgaca-3′ Biomol, Hamburg,
(PDK1) 5′-cagacgcctagcattttcat-3′ Germany 291 100
NM_002610
Human lactate
dehydrogenase A like 6B 5′-ggtgtccctttgaaggatct-3′ Biomol, Hamburg,
(LDHA) 5′-tgcagtcacttctttgtgga-3′ Germany 87 400
NM_033195
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 Aβ1–42 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].
4.15. 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 ID63worm) and Fol 50 µM, MgOr 0.1 mM and vitamin B6 (Vit B6) 100 µM (together
ID64worm). 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.
4.16. 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
Int. J. Mol. Sci. 2022, 23, 8670 17 of 21
after being touched by the wire are recognized as “not paralyzed” whereas uncoordinated
movements or just head movements were recorded as “paralyzed”.
4.17. 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.
4.18. 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.
4.19. 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.
5. 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-APP695 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.
Author Contributions: Investigation, L.B., F.S., Y.F. and T.H.; Supervision, G.P.E.; Writing—original
draft, L.B. All authors have read and agreed to the published version of the manuscript.
Funding: This research received grants from the Wörwag Pharma Böblingen, Germany (#82540149).
Institutional Review Board Statement: Not applicable.
Int. J. Mol. Sci. 2022, 23, 8670 18 of 21
Informed Consent Statement: Not applicable.
Data Availability Statement: The data presented in this study are available on request from the
corresponding author.
Acknowledgments: We would like to thank the Wörwag Pharma GmbH & Co. KG and the research
team for their support of our research.
Conflicts of Interest: The authors declare that they have no conflict of interest.
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