Detection of mitochondrial activity and amyloid deposition in SAMP10 mice
In this study, we showed that the SUVRs of 18F-BCPP-EF and 11C-PiB in the cerebral cortex were significantly higher in 15-week-old SAMP10 mice than in control mice, and that these SUVRs were inversely correlated with each other in 15-week-old SAMP10 mice (Figs. 1 and 2). To our knowledge, this is the first study to report a change in mitochondrial activity in SAMP10 mice on in vivo PET imaging, highlighting elevated mitochondrial activity at an early stage in SAMP10 mice, comparable to the early stage of AD spectrum disorder in humans. Although many researchers have investigated frontal lobe atrophy and Aβ-deposition using SAMP10 mice older than 7 months, we here used younger mice to analyze early molecular changes at the beginning of neurodegeneration. Aβ-deposition was not obvious at 5 weeks-of-age, but we did observe an elevation in the SUVR of 11C-PiB in 15-week-old SAMP10 mice. As the SUVR of 18F-BCPP-EF, which reflects oxidative phosphorylation in mitochondria, was also increased at the same stage (Figs. 1 and 2), it is probable that metabolism had increased to compensate for an energy loss in neurons, which happens with mitochondrial dysfunction in the pathological condition of Aβ-deposition in the AD brain. Although the pathological process is different, this compensatory theory is in line with previous reports of elevated dopamine synthesis in early Parkinson's disease [21, 22]. Of course, in a later stage of disease, reduced energy production would occur in association with more serious pathological events, as neurodegeneration progresses [23, 24].
A previous metabolomic study showed a difference in the metabolic pathway between MCI and AD patients [25], with the level of pyruvate in cerebrospinal fluid being significantly higher in MCI patients than in cognitively normal (CN) individuals and AD patients. It is also reported that gene expression of complex I-V subunits in the electron transport chain is elevated in MCI compared with AD and age-matched CN individuals [26]. Thus, the increase in mitochondrial activity in 15-week-old SAMP10 mice in the current study may reflect these changes in energy metabolism found in the state of senescence to MCI.
A Possible Key Player: Glial Cells
A possible key event that might be at play in the inverse correlation between Aβ deposition and mitochondrial activity is neuroinflammation, specifically neuroprotective glial activation. Aβ accumulation affects neurons and neuroinflammatory cells, and upregulation of IL-1β and IFN-γ in 3-month-old SAMP10 mice, and IL-6 in later-stage SAMP10 mice, have been observed [27]. In our recent study, neuroprotective microglia were more dominant than neuroinflammatory microglia in the early stage of neurodegeneration in SAMP10 mice [12]. We showed that elevation of mitochondrial energy metabolism, or oxidative phosphorylation, occurred according to the progression of neurodegeneration (Figs. 1 and 2A). Although the high energy requirements of neurons means that they form a major contribution to the oxidative metabolism of the brain, glial cells are also responsible for some of the oxidative metabolism. The oxidative metabolism in microglia changes as symptoms progress. While induction of M1-type inflammatory microglia by lipopolysaccharide leads to a reduction of mitochondrial oxygen consumption and lactate production, these reductions are not caused by IL-4/IL-13, inducers of M2-type protective microglia [28]. This indicates the occurrence of higher metabolism in M2 type microglia. When protective microglia are dominant in the early stage of neurodegeneration, oxidative phosphorylation in mitochondria remains at a high level. However, as the number of protective microglia decreases and neuroinflammatory microglia become prominent with the progression of Aβ deposition in later stages, the oxidative phosphorylation activity in microglia will be reduced by mitochondrial dysfunction, concomitant with that in neurons [28].
Interaction Of Amyloid And Neuronal Mitochondria
One of the mechanisms by which amyloid leads to mitochondrial dysfunction is the transportation of APP into mitochondria [29]. Pre-sequence protein (PreP), a processing enzyme that recognizes mitochondrial-targeting signal peptides and cleaves after protein import, can degrade Aβ in the mitochondria [30]. Interestingly, the proteolytic activity of PreP is decreased in the AD brain [31]. Aβ can be localized to the inner mitochondrial membrane [32], and constituents of the γ-secretase complex, such as nicastrin, APH-1, PEN-2, and presenilin-1, which function in APP processing, are also localized within the mitochondria-associated membrane [33, 34]. It has been reported that mitochondrial dysfunction and neurodegeneration occur in model mice with deleted HtrA2, a serine protease that interacts with Aβ, APP, and presenilin-1 within the intermembrane space [35]. These pieces of evidence suggest that Aβ and Aβ-related enzymes are linked with failure of mitochondrial function.
Immunohistochemical Confirmation
Our immunohistochemical analyses presented in Figs. 3 to 5 show that in 15-week-old SAMP10 mice, mitochondrial ATPB, a key enzyme for ATP production, was mainly present in neurons, although some was present in microglia, and slightly elevated levels were also present in reactive/perivascular astrocytes. Because the population of neurons was most abundant in the cerebral cortex and upregulation of ATPB in neuron was prominent compared to astrocytes and microglia, the primary contributor to the higher SUVR of 18F-BCPP-EF in the 15-week-old SAMP10 mice was considered to be neurons. As the number of GFAP + reactive astrocytes in the cortex had increased (data not shown) [17], and specific CB2 + protective microglia were activated at this early stage in the SAMP10 mice [12], we speculate that the extent of the polarized neuroinflammatory responses (neurotoxic or neuroprotective) of these glial cells would be of relevance to future neuronal degeneration. The protective cytokines (including neurotrophic factors) that are released from microglia exposed to neuropathic substances such as Aβ might stimulate neurons to supply more glucose and glutamine from perivascular astrocytes. These supplies may enable neurons to survive by temporarily increasing energy production.
The blood-brain barrier breakdown caused by pericyte dysfunction and impairment of platelet-derived growth factor receptor-β (PDGFRβ) signaling have recently been attracting attention as pathological features of AD. Pericytes are involved in the efflux of accumulated Aβ in the brain. Originally, we thought that pericyte activity might be elevated at an early stage of neurodegeneration; however, in 15-week-old SAMP10 mice, there was no increase in the immunostaining level of ATPB in pericytes, while adjacent astrocytes contained a higher level of ATPB (Supplementary Fig. 2). In this relatively early stage animal model, the contribution of pericytes to accumulation of Aβ in the brain parenchyma may be minimal. Indeed, the number of pericytes starts to decrease after 4 months-of-age in 5xFAD mice [18].
We also observed elevation of TREM2 (an important protein for clearance of Aβ) in microglia in 15-week-old SAMP10 mice (Fig. 6). TREM2 is a key player in the switching of microglia from a homeostatic state to a disease-associated state. Interestingly, soluble TREM2 in cerebrospinal fluid is higher in Aβ + Tau + MCI patients than in CN individuals [36]. Furthermore, TREM2 expression in mononuclear cells in the peripheral blood of MCI patients, especially those likely to convert to AD, was significantly higher than in CN individuals [37]. TREM2 activates the mTOR pathway that regulates mitochondrial energy production by promoting the synthesis of mitochondrial proteins, including components of MC-1 and MC-5 [38]. Therefore, the correlation between mitochondrial activity and TREM2 expression is reasonable.