Intracellular amyloid β oligomers impair organelle transport and induce dendritic spine loss in primary neurons

Abstract

Introduction: Synaptic dysfunction and intracellular transport defects are early events in Alzheimer's disease (AD). Extracellular amyloid β (Aβ) oligomers cause spine alterations and impede the transport of proteins and organelles such as brain-derived neurotrophic factor (BDNF) and mitochondria that are required for synaptic function. Meanwhile, intraneuronal accumulation of Aβ precedes its extracellular deposition and is also associated with synaptic dysfunction in AD. However, the links between intracellular Aβ, spine alteration, and mechanisms that support synaptic maintenance such as organelle trafficking are poorly understood.

Results: We compared the effects of wild-type and Osaka (E693Δ)-mutant amyloid precursor proteins: the former secretes Aβ into extracellular space and the latter accumulates Aβ oligomers within cells. First we investigated the effects of intracellular Aβ oligomers on dendritic spines in primary neurons and their tau-dependency using tau knockout neurons. We found that intracellular Aβ oligomers caused a reduction in mushroom, or mature spines, independently of tau. We also found that intracellular Aβ oligomers significantly impaired the intracellular transport of BDNF, mitochondria, and recycling endosomes: cargoes essential for synaptic maintenance. A reduction in BDNF transport by intracellular Aβ oligomers was also observed in tau knockout neurons.

Conclusions: Our findings indicate that intracellular Aβ oligomers likely contribute to early synaptic pathology in AD and argue against the consensus that Aβ-induced spine loss and transport defects require tau.

Fig. 1

APP expression and Aβ oligomer accumulation in APP-transfected neurons. Mouse primary neurons were transfected with two plasmid vectors to simultaneously express APP and GFP. a Cells were stained with DAPI and human APP-specific 6E10 antibody (red). All GFP-positive neurons expressed human APP. Scale bar, 200 μm. b Cell homogenates were subjected to Western blot with 6E10 and anti-actin antibodies. c No significant difference in the levels of human APP expression between APP_WT- and APP_OSK-transfectants. AU, arbitrary unit. d Cells were stained with 6E10 (red) or Aβ oligomer-specific 11A1 antibody (red). Only APP_OSK-expressing neurons accumulated abundant iAβOs. Scale bar, 30 μm

Fig. 2

Spine alteration in APP_OSK-expressing neurons. a Mouse primary neurons were doubly transfected with APP and GFP. Scale bar, 30 μm. Lower panels, enlarged views of the dendrites surrounded with a square. b Individual spines on dendrites of GFP-positive neurons were classified into the four types: mushroom, stubby, thin, and filopodia-like protrusions. Compared with mock-transfectants, APP_WT-expressing cells showed a small increase the number of total and mushroom-type spines with no significant changes in other types. In contrast, APP_OSK-expressing cells exhibited a significant reduction in the number of total and mushroom-type spines, but no significant changes in other types

Fig. 3

Effects of extracellular Aβ on spines. Synthetic wild-type (WT) or Osaka-mutant (OSK) Aβ42 peptides were added into culture media of GFP-expressing neurons at various concentrations. a Before adding the peptides, Aβ oligomer formation in the peptide solutions were examined by Western blot with 6E10 antibody. Aβ WT peptide showed mainly monomers and faintly dimers, whereas Aβ OSK peptide exhibited abundant monomers as well as dimers and trimers and faintly tetramers and 12-mers. b, c After a 2-day culture in the presence of Aβ peptides, spine densities and types were measured. Aβ WT peptide (b) increased total and mushroom-type spines at physiological, low concentrations from 100 pM to 400 pM. In contrast, Aβ OSK peptide (c) did not affect spine morphology at those concentrations. However, both Aβ WT and Aβ OSK peptides significantly decreased total and mushroom-type spines at higher concentrations: at 25 nM of Aβ WT peptide and over 1 nM of Aβ OSK peptide

Fig. 4

Spine alteration in APP_OSK-expressing tau knockout neurons. a Primary neurons were prepared from tau knockout mice and doubly transfected with APP and GFP. Scale bar, 30 μm. Lower panels, enlarged views of the dendrites surrounded with a square. b Spine density and morphology in GFP-positive neurons were analyzed. APP_WT-expressing cells showed no differences in the number of total and each type spine compared to mock-transfectants. In contrast, APP_OSK-expressing cells exhibited a significant reduction in the number of total and mushroom-type spines with no significant changes in other types. c, d Comparison of spine alteration between wild-type (Fig. 2) and tau knockout neurons expressing APP_OSK. c There were no differences in the density of mushroom-type and total spines between wild-type and tau knockout neurons. d APP_OSK expression reduced the spine density in both wild-type and tau knockout neurons: No differences in the reduction of mushroom-type (p = 0.4835) and total spines (p = 0.9702) between these neurons

Fig. 5

Axonal and dendritic transport of BDNF is impaired in APP_OSK-expressing neurons. Wild-type mouse primary neurons were doubly transfected with APP-EGFP and BDNF-mRFP. Transport of BDNF in living neurons was recorded for 25 s, and those in 100-μm segments of the axon (a) and 45-μm segments of the dendrite (c) were analyzed. Axons and dendrites were initially identified based on morphology and confirmed retrospectively by immunostaining MAP2. b Compared with mock-transfectants, both APP_WT- and APP_OSK-expressing cells showed a decrease of bidirectional transport of BDNF in axons, but the differences were significant only in APP_OSK-expressing cells. d Similarly, a decrease of bidirectional transport of BDNF in dendrites was observed in both APP_WT- and APP_OSK-expressing cells, but significant reduction was observed only for total event in APP_OSK-expressing cells

Fig. 6

Axonal and dendritic transport of BDNF is impaired in APP_OSK-expressing tau knockout neurons. Tau knockout mouse primary neurons were doubly transfected with APP-EGFP and BDNF-mRFP. Transport of BDNF in living neurons was recorded for 25 s, and those in 100-μm segments of the axon (a) and 45-μm segments of the dendrite (c) were analyzed. b Compared with mock-transfectants, APP_WT-expressing cells showed no differences in BDNF transport in axons. In contrast, APP_OSK-expressing cells showed a significant decrease of anterograde, retrograde, and total axonal flux of BDNF. d In dendrites, both APP_WT- and APP_OSK-expressing cells showed a decrease of BDNF transport, but the differences were significant only in APP_OSK-expressing cells

Fig. 7

Impaired axonal transport and aberrant dendritic distribution of mitochondria in APP_OSK-expressing neurons. Rat primary neurons were triply transfected with APP, Mt-eYFP, and BFP. a Transport of mitochondria in living neurons was recorded for 300 s, and those in 100-μm segments of the axon were analyzed. b Compared with mock-transfectants, bidirectional transport of mitochondria in axons was reduced in APP_OSK-, but not APP_WT-, expressing cells. c In dendrites, mitochondria were evenly distributed along the dendritic shafts in mock-transfectants (a-c) and APP_WT-expressing cells (d-f). In contrast, mitochondria in dendrites of APP_OSK-expressing cells (g-i) were of shorter length and unevenly distributed. Scale bar, 20 μm

Fig. 8

Impaired dendritic transport of recycling endosomes in APP_OSK-expressing neurons. Rat primary neurons were triply transfected with APP, TfR-GFP, and BFP. a Transports of TfR in living neurons were recorded for 100 s, and those in 45-μm segments of the dendrite were analyzed. b Compared with mock-transfectants, bidirectional transport of TfR-positive vesicles in dendrites was reduced in APP_OSK-, but not APP_WT-, expressing cells