Synaptosomal Mitochondrial Dysfunction in 5xFAD Mouse Model of Alzheimer's Disease

Abstract

Brain mitochondrial dysfunction is hallmark pathology of Alzheimer's disease (AD). Recently, the role of synaptosomal mitochondrial dysfunction in the development of synaptic injury in AD has received increasing attention. Synaptosomal mitochondria are a subgroup of neuronal mitochondria specifically locating at synapses. They play an essential role in fueling synaptic functions by providing energy on the site; and their defects may lead to synaptic failure, which is an early and pronounced pathology in AD. In our previous studies we have determined early synaptosomal mitochondrial dysfunction in an AD animal model (J20 line) overexpressing human Amyloid beta (Aβ), the key mediator of AD. In view of the limitations of J20 line mice in representing the full aspects of amyloidopathy in AD cases, we employed 5xFAD mice which are thought to be a desirable paradigm of amyloidopathy as seen in AD subjects. In addition, we have also examined the status of synaptosomal mitochondrial dynamics as well as Parkin-mediated mitophagy which have not been previously investigated in this mouse model. In comparison to nontransgenic (nonTg mice), 5xFAD mice demonstrated prominent synaptosomal mitochondrial dysfunction. Moreover, synaptosomal mitochondria from the AD mouse model displayed imbalanced mitochondrial dynamics towards fission along with activated Parkin and LC3BII recruitment correlating to spatial learning & memory impairments in 5xFAD mice in an age-dependent manner. These results suggest that synaptosomal mitochondrial deficits are primary pathology in Aβ-rich environments and further confirm the relevance of synaptosomal mitochondrial deficits to the development of AD.

Fig 1. Aβ accumulation in synaptosomal mitochondria from 5xFAD mice at 4 (young) and 9 (old) months old.

(A) Age-dependent brain Aβ1–40 and 1–42 deposition in 5xFAD mice. *, # P<0.05 vs the counterpart in young 5xFAD mice. (B) Age-dependent Aβ1–40 accumulation in synaptosomal and nonsynaptosomal mitochondria from 5xFAD mice. (C) Age-dependent Aβ1–42 accumulation in synaptosomal and nonsynaptosomal mitochondria from 5xFAD mice. N = 7 young mice and 8 old mice. (D) Aβ deposits in neurons, neuronal mitochondria and extracellular area in 5xFAD mice at 8 months old. Panels I shows the staining of Aβ (red), NISSL (blue) and F1FO ATP synthase β subunit (green). Panel II, III and IV represent three-dimension reconstructions of the F1FO ATP synthase β subunit staining, Aβ staining, and merged images, representatively. Scale bars = 20 nm.

Fig 2. Impaired synaptosomal mitochondrial respiration in 5xFAD mice.

(A) Mitochondrial RCRs of synaptosomal and nonsynaptosomal mitochondria from young and old 5xFAD and nonTg mice. *P<0.05 vs nonsynaptosomal counterpart. #P<0.05 vs other groups. (B) State III and Sate IV oxygen consumption rates of synaptosomal mitochondria from young and old nonTg and 5xFAD mice. *P<0.05 vs synaptosomal mitochondria from old 5xFAD mice. (C) State III and Sate IV oxygen consumption rates of nonsynaptosomal mitochondria from young and old nonTg and 5xFAD mice. *P<0.05 vs State III oxygen consumption rates of all the other groups. N = 3–4 mice of each group. (D) ATP production of synaptosomal and nonsynaptosomal mitochondria from young and old 5xFAD and nonTg mice. *P<0.05 vs all the other fractions. (E) Brain ATP levels in young and old nonTg and 5xFAD mice. *P<0.05 vs nonTg counterpart; #P<0.05 vs all the other groups. N = 6–7 mice of each group.

Fig 3. Altered mitochondrial fusion and fusion protein levels in synaptosomal mitochondria from 5xFAD mice.

Synaptosomal and nonsynaptosomal mitochondria from 4 (Young) and 9 (Old) months old nonTg and 5xFAD mice were subjected to the immunoblots for the levels of MFN2 (A1 synaptosomal mitochondria; A2 nonsynaptosomal mitochondria), OPA1 (B1 synaptosomal mitochondria; B2 nonsynaptosomal mitochondria) and Dlp1 translocation (C1 synaptosomal mitochondria; C2 nonsynaptosomal mitochondria). (D1) and (D2) show representative immunoreactive bands from synaptosomal and nonsymaptosomal mitochondria, respectively. Mitochondrial TOM40 was used to determine the loading amount. N = 5–9 mice per group.

Fig 4. Decreased axonal mitochondrial length in cultured 5xFAD mouse neurons.

(A) 5xFAD mouse neurons demonstrated significantly decreased axonal mitochondrial length in comparison to nonTg neurons. The lower panel are representative images. Scale bar = 5 μm. (B) Cumulative data for axonal mitochondrial length showing a leftward shift in 5xFAD mouse neurons. N = 8–12 neurons from each group.

Fig 5. Increased Parkin translocation and LC3BII recruitment in synaptosomal mitochondria from 5xFAD mice.

Synaptosomal mitochondria and cortices from young and old nonTg and 5xFAD mice were subjected to the immunoblots for the levels of synaptosomal mitochondrial recruited Parkin (A1), the levels of Parkin in cortex (A2), and the levels of synaptosomal mitochondria-recruited LC3BII (B). The left panels show the analyzed results in fold increase and the right panels are representative immunoblot bands. Mitochondrial TOM40 and β-actin were used as the loading controls for mitochondrial fractions and cortex homogenate, respectively. N = 4–6 mice per group.

Fig 6. Age-dependent spatial reference learning and memory impairments of 5xFAD mice.

5xFAD mice demonstrated impaired learning ability to locate the hidden platform in an age-dependent manner (A). *P<0.05 vs other groups. (B) 5xFAD mice had compromised function in spatial reference memory in an age-dependent manner. *P<0.05 vs other groups. (C) Mice in different groups didn't show significant change in their swimming speed. N = 5–6 mice of each group.