MT biogenesis is altered in 5xFAD mice
The MT transcription factor A (TFAM) is a nuclear protein that plays a key role in MT DNA replication. TFAM upregulation is linked to increased MT DNA, suggesting that the protein helps coordinate the nuclear and MT genomes for MT biogenesis (31). Sirt3 is a MT deacetylase that promotes MT biogenesis by deacetylating TFAM and enhancing its activity (32, 33). To measure MT biogenesis in 5xFAD mice, we quantified TFAM and Sirt3 expression in hippocampal MT using Western blot analysis. Male 5xFAD mice at 2 months-of-age had reduced TFAM levels and Sirt3 was trending towards significance, but female mice showed no detectable difference compared to the controls (Fig. 1). At 4 months-of-age, TFAM expression was increased in females and decreased in males. Sirt3 levels were reduced in females with a trend towards reduction in males. At 6 months-of-age, TFAM and Sirt3 levels were significantly decreased in 5xFAD females, with males also exhibiting a significant reduction in Sirt3. Surprisingly, these changes were not observed in 8-months-old male mice, although Sirt3 remained reduced in 5xFAD females. These results suggest an early defect in MT biogenesis, with the appearance of that defect first in male mice.
MT fission:fusion balance is disturbed in 5xFAD mice
We searched for changes in fission:fusion proteins using hippocampal MT using the same procedures. The MT fusion protein, MFN2, was dramatically reduced in females at 2 months-of-age (Fig. 2), becoming ~5.5 fold lower than in controls by 8 months. Males failed to show a significant decrease until 6 months-of-age. The fission protein, MFF, remained relatively unaffected except for the ratio between the phosphorylated and dephosphorylated form at 6 and 8 months-of-age (Sup Fig. 1). FIS1, showed decreased expression in both sexes at 4 months-of-age (Fig. 2). The expression of MTP18, a novel regulator of MT fission, was significantly depressed in both 5xFAD sexes across all ages (Fig. 2). Another fission marker, DRP1, showed decreased expression in females at 2 months-of-age. A significant reduction in DRP1 levels in males was observed by 4 months-of-age. The most striking result is that MTP18 is reduced in both sexes at all ages.
Blockade in 5xFAD mitophagy
Mitophagy is critical in regulating MT quality control and compromised mitophagy has been implicated in the pathogenesis of AD (34). To probe the induction of mitophagy in 5xFAD mice, we monitored the conversion of soluble microtubule-associated protein 1A/1B-light chain 3B (LC3BI) to LC3BII, a lipid bound form found predominantly in autophagosomal membranes. Specifically, we quantified the levels of LC3B-II and the LC3BII/I ratio in the MT fraction isolated from the hippocampus of 2, 4, 6, and 8 months old B6/SJL and 5xFAD mice. A significant increase in LC3BII levels was observed starting at 4 months-of-age in 5xFAD females and at 6 months-of-age in males (Fig. 3). Intriguingly, both 5xFAD females and males showed a significant increase in LC3BII/I ratio at 2 months-of-age, and this ratio remained elevated at 4, 6, and 8 months-of-age (Fig. 3). Our observations are consistent with the previous studies showing LC3BII recruitment to MT in the 5xFAD and 3xTg models of AD (35, 36). Together, elevated LC3BII levels and enhanced LC3BI to II conversion indicate increased mitophagy induction in 5xFAD mice.
To measure the progression of mitophagy, we quantified the mitophagy adaptor SQSTM1/p62 which is degraded after mitophagy is induced. Remarkably, the levels of SQSTM1/p62 in the MT fraction remained unchanged in both the sexes at all ages except for 6-month-old females, which showed a significant increase (Fig. 3). These results suggest that the progression of normal mitophagy is blocked in the 5xFAD mice. To test this possibility, we measured the levels of OPTN, another mitophagy receptor critically involved in the elimination of damaged MT (37). The levels of OPTN remained unchanged in the females at 2 and 4 months-of-age but increased significantly at 6 and 8 months-of-age (Fig. 3). Males exhibited a completely different pattern, being increased as early as 2 months-of-age and remaining significantly elevated. Thus, the enhanced LC3B-II accumulation and unaltered/increased p62 and OPTN levels indicate that even though there is increased mitophagy initiation in the 5xFAD mice, the subsequent steps in the mitophagy pathway are blocked.
We also measured the levels of FUNDC1, an outer MT membrane (OMM) residing protein that acts as a major mitophagy receptor and promotes hypoxia induced MT clearance independent of the PINK1/Parkin pathway (36, 38, 39), which is discussed below. Strikingly, our results showed a significant decrease in the levels of FUNDC1 in the MT fraction starting at 2 months-of-age in both females and males (Sup Fig. 1). This suggests a defect in the initiation of receptor-mediated mitophagy. Our results indicate that in 5xFAD mice, the PINK1/Parkin-independent mitophagy pathways are compromised very early in both sexes.
Defective PINK1/Parkin-dependent mitophagy
The PINK1 (PTEN-induced kinase1)/Parkin cascade has recently been identified as the major pathway for quality control mitophagy in neuronal cells (40, 41). Thus, we tested PINK1 expression levels in the hippocampal MT fraction. PINK1 levels began declining at 2 months-of-age in females and 4 months-of-age in males (Fig. 4). Reduced PINK1 should cause a decreased recruitment of Parkin to the OMM. We found that Parkin levels also declined as early as 2 months-of-age (Fig. 4). This observation was consistent in both the sexes.
Parkin levels on the MT could be reduced from defective translocation. Therefore, we examined the levels of Beclin (BECN1), a component of the PI3 kinase complex that recruits Parkin to damaged/depolarized MT (42). Interestingly, BECN1 levels start declining at 4 months-of-age in females and remain low (Fig. 4). In males, BECN1 levels were reduced at 2 and 4, but not at 6 and 8 months-of-age, suggesting that, compensatory mechanisms come into play to restore the BECN1 levels in older males, and that reduced Parkin on MT may not be completely dependent on BECN1 levels.
MT calcium uptake is defective
Calcium signaling is perturbed in AD, which affects key enzymes and several organelles including MT as reported earlier in studies of mouse AD models and human post-mortem brains (43, 44). To address the relationship between calcium dysregulation and MT dysfunction, we measured protein levels for key regulators of MT calcium import, MICU1 and MCU.
MCU is a Ca2+ activated MT calcium uniporter present on the inner membrane of MT. MT calcium uptake 1, MICU1, is a component of the regulatory complex along with MICU2 and EMRE, which regulates the activity of MCU (45). Our results indicated a significant reduction in MICU1 and MCU protein expression at 2 months-of-age in 5xFAD males but not in females (Fig. 5). However, this depressed expression level did not extend to 4-month-old males. Reduced MCU expression was evident in females beginning from 4 months-of-age, which continued until 8 months-of-age, MICU1 was depressed in 5xFAD females at 6 and 8 months-of-age. Both proteins were reduced in 5xFAD males at 6 months-of-age, but this reduction was not detected at 8 months. Overall, the deficit in MICU1 and MCU protein abundance was consistent across all ages in 5xFAD females but not in 5xFAD males.
Early defects in MT bioenergetics
Impaired MT biogenesis, MT dynamics, and mitophagy would be expected to result in decreased oxidative phosphorylation and impaired MT respiratory efficiency. To test this, the MT oxygen consumption rate (OCR) was measured in freshly isolated MT from mice of both genotypes using the Seahorse XFe96 extracellular flux analyzer. Purified MT were energized by pyruvate and malate as respiratory substrates and MT respiratory parameters including basal respiration, ATP production, maximal respiratory capacity, and spare respiratory capacity (SRC) were quantified. We detected no significant differences in basal respiration using MT isolated from any age or sex (Fig. 6). No significant differences in ATP-coupled OCR were found across age and sex (Fig. 6). In contrast, maximal respiratory capacity appeared to be compromised in both males and females at all ages (Fig. 6). SRC is a measure of the ability of the MT to meet the demands for extra energy, beyond the basal level, in response to stressed conditions. SRC was depressed at all ages in female 5xFAD mice. Males also showed depressed SRC at most ages. These results indicate that abnormal MT bioenergetics emerge early in 5xFAD mice and that the pronounced bioenergetic parameters include maximal respiration and SRC.
ATP production is not compromised
Prior studies have reported abnormalities in the MT ETC complexes due to AD pathology (46), and the alpha chain of ATP synthase – a subunit of complex V – has been detected in the neurofibrillary tangles that form in degenerating neurons of AD patients (47). In addition, reduced ATP levels were previously reported in AD postmortem brains and 5xFAD mice (48, 49), which is consistent with the expected impact of MT degeneration and loss of ATP synthase activity. Considering these reports, our surprising results showing normal ATP-coupled OCR in the 5xFAD model (Fig. 6) prompted us to measure ATP generation directly, rather than using the proxy of OCR.
We utilized a luminescence based real-time ATP assay. MT isolated from the brains of these mice revealed no change in ATP production in 2-month-old 5xFAD females, whereas male mice showed a slight but significant increase in ATP (Fig. 7). Both 4- and 6-month-old female and male 5xFAD mice exhibited significantly increased ATP compared to control mice (Fig. 7). This increase persisted in 8-month-old males but was lost in females of the same age (Fig. 7). These results suggest a modest increase in MT ATP production in the 5xFAD mice, perhaps from compensatory pathways, but no decrease in MT ATP generation at least until 8 months-of-age.