SUL-138 rescues memory and LTP deficits and reduces Aβ plaque load in APP/PS1 mice
We hypothesized that supporting mitochondrial bioenergetics is sufficient to restore synaptic plasticity in the hippocampus of APP/PS1 mice. To test this, we used the 6-chromanol SUL-138, which enhances the efficiency of mitochondria complex 1 (type I NADH dehydrogenase) and complex 4 (cytochrome c oxidase) of the electron transport chain, reducing ROS and increasing ATP production (Fig. 1A) [23]. APP/PS1 mice and wildtype littermates were treated orally with SUL-138 or vehicle for three months (between 3 and 6 months of age) and conditioned fear memory and hippocampal long-term potentiation (LTP) were subsequently measured (Fig. 1B). SUL-138 plasma concentrations demonstrated high oral uptake (Supplementary Table 1). SUL-138 treatment increased LTP in the CA1 region of the hippocampus, particularly during the maintenance phase at 30-60 min after LTP induction, both in wildtype (p = 0.0463, t = 1.762, df = 21, Student’s t-test) and APP/PS1 mice (p = 0.0421, t = 1.842, df = 16, Student’s t-test) (Fig. 1C–E, S1). Conditioned fear memory was impaired in APP/PS1 mice compared to wildtype at 6 months of age (Fig. 1F, p = 0.0453, t = 2.033, df = 80, one-way ANOVA, post hoc Fisher’s LSD), as observed in previous studies [28, 29]. In APP/PS1 mice, SUL-138 treatment rescued contextual fear memory to wildtype levels (p = 0.0261, t = 2.267, df = 80, one-way ANOVA, post hoc Fisher’s LSD). Interestingly, SUL-138 treatment also increased contextual fear memory in wildtype mice (p = 0.0022, t = 3.162, df = 80, one-way ANOVA, post hoc Fisher’s LSD). None of the groups showed substantial freezing in a novel context and basal locomotor activity was the same in SUL-138-treated and vehicle-treated wildtype and APP/PS1 mice (WT VEH vs WT SUL: p = 0.8922, t = 0.1359, df = 79 and p = 0.8918, t = 0.1365, df = 79; APP VEH vs APP SUL: p = 0.2462, t = 1.523, df = 79 and p = 0.2468, t = 1.522, df = 79 ; one-way ANOVA; Figure S2).
Next, we assessed the effect of three months of SUL-138 treatment on a key pathological hallmark of AD, the formation of amyloid plaques, in the hippocampus of APP/PS1 mice (Fig. 1G/H, S3). SUL-138 treatment decreased both number (by 54%) and size (by 30%) of plaques compared to vehicle treated APP/PS1 mice (p = 0.0203 and p = 0.0042, Student’s t-test; average of the 4 hippocampi of 2 slices/animal) (Fig. 2B–D, S3A&B). This decrease in plaque load seemed not to be due to alterations in amyloid clearance as we did not observe changes in GFAP or Iba1 expression upon SUL-138 treatment (Figure S4). Taken together, these data show that SUL-138 increases synaptic plasticity and memory in mice to a level that is sufficient to rescue impairments in APP/PS1 mice and is able to prevent pathological amyloid accumulation.
SUL-138 treatment alters protein levels in hippocampal synapse-enriched P2 fractions
We subsequently investigated the effects of SUL-138 treatment on hippocampal protein expression to uncover potential mechanisms underlying enhanced plasticity and reduced amyloid plaque load. We used mass spectrometry to quantify protein levels in hippocampal P2 fractions, enriched for synapses, followed by differential expression analysis (DEA; see methods, Supplementary dataset 1) in vehicle (VEH) or SUL-138 (SUL) treated APP/PS1 (APP) and wildtype (WT) mice. The total number of detected proteins amounted 7846 (Fig. 2A). Differential protein expression was determined for selected pairwise comparisons; APP VEH vs. WT VEH to detect protein dysregulation due to disease and APP SUL vs. APP VEH and WT SUL vs. WT VEH to identify effects of SUL-138 on protein expression under healthy conditions (Fig. 2B). After multiple testing correction, 549 differentially expressed proteins were identified in the APP VEH vs. WT VEH comparison (FDR, q ≤ 0.05). After SUL-138 treatment in APP/PS1 mice, 151 proteins were differentially expressed (APP SUL vs. APP VEH; FDR, q ≤ 0.05), of which 66 (44%) overlap with the APP VEH vs. WT VEH comparison, indicating a potential rescue of APP/PS1-altered protein expression. In SUL-138-treated wildtype mice, 77 differentially expressed proteins were identified (WT SUL vs. WT VEH; FDR, q ≤ 0.05), of which 22 (28%) overlap with the APP SUL vs. APP VEH comparison, indicating that the number proteins affected by SUL-138 treatment is substantially smaller and partially differs in wildtype mice compared to APP/PS1 mice.
Protein dysregulation in APP/PS1 mice
Figure 2C shows the protein dysregulation in the hippocampus due to disease (APP/PS1 mice compared to wildtype controls - APP VEH vs. WT VEH). Of all differentially regulated proteins, most are downregulated in APP/PS1 mice (365/549; FDR, q ≤ 0.05) and cellular component (CC) GO enrichment analysis [30] revealed many synaptic terms, e.g., “glutamatergic synapse,” “postsynaptic density,” and also “intermediate filament” (Fig. 2E), whereas biological process (BP) terms point to “neuron projection morphogenesis,” “transsynaptic signaling,” and “actin filament organization” (supplementary dataset 2, Figure S5A, lower panel). Focusing on the 184 significantly upregulated proteins (FDR q ≤ 0.05), CC enrichment analysis reveals enrichment of, e.g., “mitochondrial matrix” and “chaperone complex” (Fig. 2D) and “protein folding” and “nucleotide metabolic process” (supplementary dataset 3). In addition, BP terms such as “amyloid beta formation,” “amyloid precursor protein catabolic process,” and “amyloid fibril formation” are enriched, as is expected in this mouse model (Figure S5A, upper panel).
In total, 15.2% of measured known AD-associated proteins (22 of 138 measured UniProt “Alzheimer human” proteins [31] and 5 out of the 18 measured GWAS gene-encoded proteins [32, 33]; 27.8%) are significantly altered in APP VEH mice. These are APBA1, APOE, APP, BIN1, CLU, CTSD, DBN1, DNM1L, GFAP, GPC1, HSD17B10, IDE, LMNA, LMNB1, MAP2, NPEPPS, OGT, PRNP, PSEN1, SNCB, SOD2, and SYNPO for UniProt and APH1B, APP, APOE, BIN1, and CLU for GWAS (Figure S6A). These findings confirm the AD-like molecular phenotype of APP/PS1 mice.
Effects of SUL-138 treatment in APP/PS1 mice
Figure 2F shows hippocampal protein regulation as a result of 3 months of SUL-138 treatment in APP/PS1 mice (APP SUL vs. APP VEH). Most proteins (109/151) are upregulated in APP/PS1 mice after SUL-138 treatment and CC enrichment analysis shows that these proteins belong to, e.g., “supramolecular complex,” “postsynaptic cytoskeleton,” and “intermediate filament” and to the BP terms “cytoskeleton organization,” “fatty acid oxidation,” and “carboxylic acid metabolic process” (Fig. 2G and S5B, upper panel; supplementary dataset 4). Downregulated proteins (42/151) show enrichment of “presynapse,” “synaptic membrane,” and “presynaptic cytosol” (Fig. 2H and S3B, lower panel; supplementary dataset 5), which include the AD-associated proteins APP, CLU, MAP2, and serine/threonine-protein kinases A, B, and G.
Overall, 66 of 151 significantly SUL-138-regulated proteins overlap with proteins affected in APP/PS1 mice (Fig. 2B, F). Of these 66 proteins, 56 (10.2% of all 549 APP/PS1-affected proteins) are significantly rescued, i.e., regulated in the opposite direction (blue vs. red highlight), of which enrichment analyses revealed that they belong to, e.g., “supramolecular fiber,” “axon,” and “intermediate filament cytoskeleton” and to the BP terms “neuron projection morphogenesis,” “supramolecular fiber organization,” and “intermediate filament−based process” (Figure S7A&B; supplementary dataset 6). Furthermore, SUL-138 treatment of APP/PS1 mice induced an opposite direction of regulation of the AD-associated proteins APP, CLU, IDE, MAP2, LMNB1, LMNA, and GFAP (Figure S6B) compared to the APP VEH vs. WT VEH contrast (Figure S6A), indicating that SUL-138 treatment partially precludes AD-associated protein expression. In addition, SUL-138 treatment significantly affected the levels of the AD-associated proteins ACE, GOT1, RBM25, and RGS6, even though they were not significantly changed in APP/PS1 mice.
Taken together, these data show that SUL-138 partially prevents AD-associated protein dysregulation in APP-PS1 mice and restores the levels of several postsynaptic cytoskeletal proteins. GO enrichment analyses further points to mitochondrial metabolic processes as possible targets of SUL-138 in APP/PS1 mice.
Effects of SUL-138 treatment in wildtype mice
Figure 2I shows the hippocampal proteome changes in SUL-138-treated wildtype mice. SUL-138 changed the levels of 77 proteins in wildtype animals which is less than the 151 regulated proteins in APP/PS1 mice (FDR, q ≤ 0.05). Differentially expressed proteins were mostly upregulated (60/77), with a significant CC GO term enrichment for “axon,” “mitochondrial matrix,” and “intermediate filament cytoskeleton” (Fig. 2J). BP enrichment analysis revealed “mitochondrial fatty acid beta oxidation” as the most enriched process (supplementary dataset 7, Figure S5C, upper panel). Downregulated proteins (17/77) showed no significant CC enrichment and BP enrichment for “glycogen catabolic process” and interestingly “amyloid beta clearance” and “amyloid beta metabolic processes,” due to a significant decrease in the levels of, e.g., APOE and IDE in wildtype mice (supplementary dataset 8, Figure S5C, lower panel). Of the 77 regulated proteins affected by SUL-138 treatment in wildtype mice, 29 overlap with dysregulated proteins in APP/PS1 mice (APP VEH vs. WT VEH; red and blue highlight in Fig. 2C). Eight AD-associated proteins were regulated by SUL-138 in wildtype mice: ACE, APOE, CTSD, DBN1, IDE, LMNA, PPP3CA, and SNCA (Figure S6C). Taken together, these data show that SUL-138, independent of genotype, regulates AD-associated proteins, proteins involved in metabolism and cytoskeleton proteins.
SUL-138 does not have a major effect on synaptic protein expression
The synaptic signature of the GO enrichment analyses together with the effects of SUL-138 on synaptic plasticity (Fig. 1C–E) prompted us to investigate synaptic protein expression in more detail using in-depth synaptic functional annotation (SynGO, Fig. 3), a dedicated and curated synapse ontology database [34]. In total, 166 regulated proteins in the disease model (APP VEH vs. WT VEH) were annotated in SynGO, while SUL-138 treatment altered 44 synaptic proteins in APP/PS1 mice (APP SUL vs. APP VEH). Furthermore, SUL-138 treatment of wildtype mice led to regulation of 23 synaptic proteins (WT SUL vs. WT VEH; Fig. 3A). The SynGO annotation shows that downregulated proteins in APP/PS1 mice contain a wide spectrum of synaptic proteins belonging to both the pre- and the postsynaptic domain (Fig. 3B, Figure S8A, Supplementary dataset 9). Upregulated proteins show some enrichment of presynaptic proteins, in particular endocytic zone and synaptic vesicle proteins, including several AD-associated proteins, i.e., APP, APBA1, CTSD, BIN1, DNM1L, OGT, PSEN1, and SNCB.
SUL-138 treatment in APP/PS1 mice had a very modest effect on postsynaptic protein expression, increasing the levels of several actin and intermediate filament cytoskeletal proteins, specifically INA, NEFH, NEFL, NEFM and MYH10, MYO6, and SYNE, and decreasing the levels of some presynaptic cytosol and membrane proteins, which were primarily AD-associated proteins again, i.e., APP, CLU, MAP2, and GPC4, and serine/threonine-protein kinases A, B, and G (Fig. 3C and S8B, Supplementary dataset 10).
In wildtype mice, SUL-138-induced changes in protein levels were not associated with any specific SynGO terms (Fig. 3D and S8C, Supplementary dataset 11). Together, these data suggest that the increase in synaptic plasticity by SUL-138 treatment is not due to specific changes in synaptic protein levels other than a few actin and intermediate filament proteins.
SUL-138 differentially affects the mitochondrial proteome in APP/PS1 and wildtype mice
As our primary GO analyses pointed to mitochondrial metabolic process being dysregulated in APP/PS1 mice as well as being regulated by SUL-138 treatment in both APP/PS1 and wildtype mice, we next performed an in-depth analysis of mitochondrial protein expression using MitoXplorer, a tool that specifically enables the functional annotation of mitochondrial proteins [35]. In each of the three comparisons, a substantial number of MitoXplorer-annotated mitochondrial proteins were significantly differentially expressed (97 for APP VEH vs. WT VEH, 29 for APP SUL vs. APP VEH and 18 for WT SUL vs. WT VEH; FDR, q ≤ 0.05) (Fig. 4A). The APP/PS1 genotype induced a significant enrichment of proteins involved in amino acid metabolism (APP VEH vs. WT VEH). In contrast, SUL-138 treatment induced a significant enrichment for fatty acid degradation (FAD) and fatty acid beta oxidation (FAO) in both APP/PS1 and WT mice (APP SUL vs. APP VEH and WT SUL vs. WT VEH) (Fig. 4B; supplementary dataset 12), suggesting that SUL-138 shifts energy substrate usage.
Examining in more detail mitochondrial protein dysregulation in APP/PS1 mice (APP VEH vs. WT VEH) and the effect of SUL-138 (APP SUL vs. WT VEH), we plotted the fold changes of all mitochondrial proteins that are significantly changed in at least one comparison (FDR, q ≤ 0.05) in a pairs contrast graph (Fig. 4C), showing a specific upregulation of several mitochondrial processes in APP/PS1 mice, which is further enhanced by SUL-138. The upregulation of these proteins does not seem to be due to an overall increase in mitochondrial abundance as it is not observed for all mitochondrial proteins (inset in Fig. 4C). Thus, rather than restoring mitochondrial protein expression in APP/PS1 mice, SUL-138 selectively enhances the expression of proteins involved in specific mitochondrial pathways, in particular the amino acid metabolism, FAD and FAO and glycolysis pathways. When the effects of SUL-138 in APP/PS1 mice (APP SUL vs. APP VEH) and in wildtype mice (WT SUL vs. WT VEH) are compared in the same way, a clear and interesting separation between SUL-138 effects can be observed (Fig. 4D). Proteins involved in FAD and FAO are more strongly upregulated by SUL-138 in wildtype mice, whereas upregulation of glycolysis and amino acid metabolism proteins is more pronounced in APP/PS1 mice. Figure 4E, G (& S9) summarizes the changes in mitochondrial metabolic pathways in APP/PS1 mice and as a result of SUL-138 treatment. Whereas proteins involved in FAD and FAO are upregulated by SUL-138 in both wildtype and APP/PS1 mice, upregulation of glycolysis and amino acid metabolism proteins is only observed in APP/PS1 mice. In addition to the general effects of SUL-138 on FAD and FAO, electron transfer flavoprotein subunit alpha and beta (ETFA and B), which transfer electrons from FAD and FAO to the ubiquinone pool of the oxidative phosphorylation complex in the electron transport chain, are upregulated in both APP/PS1 and wildtype mice. Together, these results indicate that SUL-138 treatment shifts mitochondrial energy production to FAD and FAO in general and increases glycolysis, amino acid metabolism, and oxidative phosphorylation in APP/PS1 mice specifically.