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. 2014 Nov;71:53-61.
doi: 10.1016/j.nbd.2014.07.011. Epub 2014 Aug 1.

Intracellular Amyloid and the Neuronal Origin of Alzheimer Neuritic Plaques

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Free PMC article

Intracellular Amyloid and the Neuronal Origin of Alzheimer Neuritic Plaques

Anna Pensalfini et al. Neurobiol Dis. .
Free PMC article

Abstract

Genetic analysis of familial forms of Alzheimer's disease (AD) causally links the proteolytic processing of the amyloid precursor protein (APP) and AD. However, the specific type of amyloid and mechanisms of amyloid pathogenesis remain unclear. We conducted a detailed analysis of intracellular amyloid with an aggregation specific conformation dependent monoclonal antibody, M78, raised against fibrillar Aß42. M78 immunoreactivity colocalizes with Aß and the carboxyl terminus of APP (APP-CTF) immunoreactivities in perinuclear compartments at intermediate times in 10month 3XTg-AD mice, indicating that this represents misfolded and aggregated protein rather than normally folded APP. At 12months, M78 immunoreactivity also accumulates in the nucleus. Neuritic plaques at 12months display the same spatial organization of centrally colocalized M78, diffuse chromatin and neuronal nuclear NeuN staining surrounded by peripheral M78 and APP-CTF immunoreactivity as observed in neurons, indicating that neuritic plaques arise from degenerating neurons with intracellular amyloid immunoreactivity. The same staining pattern was observed in neuritic plaques in human AD brains, showing elevated intracellular M78 immunoreactivity at intermediate stages of amyloid pathology (Braak A and B) compared to no amyloid pathology and late stage amyloid pathology (Braak 0 and C, respectively). These results indicate that intraneuronal protein aggregation and amyloid accumulation is an early event in AD and that neuritic plaques are initiated by the degeneration and death of neurons by a mechanism that may be related to the formation of extracellular traps by neutrophils.

Keywords: Alzheimer; Intracellular amyloid; Neuritic plaques; Nuclear pathology.

Figures

Fig. 1
Fig. 1
Characterization of M78 specificity. (a) Epitope mapping. M78 recognizes a discontinuous epitope in Aß consisting of 8-11, 18-24 and 26-32 (shown in red). (b) Kinetics of the appearance of M78 immunoreactivity during the aggregation of Aß42, α-synuclein and IAPP. Top strip: Aß42 samples stained with Ponceau S as a loading control. No immunoreactivity is detected immediately after dilution of monomer stock solutions in buffer. Bottom 3 strips: M78 immunoreactivity is observed for all 3 samples at a time of 1-2 days of incubation, which is coincident with fibril formation. (c) Western blotting of α-synuclein, IAPP and Aß42 samples after 3 days of incubation. M78 stains high molecular weight bands of α-synuclein, IAPP and Aß aggregates and a range of smaller sizes of Aß aggregates down to approximately the size of dimer. (d) Thermal denaturation of M78 epitope on western blots. After thermal denaturation, the 17 kDa tetrameric band disappears, while the staining of the high molecular weight material at the top of the gel increases slightly.
Fig. 2
Fig. 2
M78 labels intracellular amyloid and nuclei at intermediate times in 3XTg-AD mice. (a) At 3 mo, a subset of neurons exhibit elevated perinuclear 6E10 staining, but no M78 immunoreactivity is observed. (b) At 10 mo, M78 immunoreactivity (red) is primarily perinuclear and colocalized with 6E10 (green) in neurons. Nuclei are labeled with DAPI (blue). (c) At 12 mo, the M78 staining is primarily nuclear and is surrounded by 6E10 and M78 immunoreactivity in neurons. (d) At 14 mo, M78 staining is primarily restricted to plaques that stain weakly with 6E10. (e) No 6E10 or M78 staining is observed in 14 mo wild type brain. (a-c, e), CA1. (d), stratum oriens adjacent to CA1. (f). Triple label confocal immunofluorescence micrographs of 12 mo 3XTg-AD subiculum labeled with DAPI (blue), M78 (red) and anti-APPCTF antiserum (green). Arrows point to diffuse DAPI DNA staining that colocalizes with M78 immunoreactivity in the center of the neuritic plaques. The dystrophic neurites surrounding the plaque core are strongly positive for both M78 and APP-CTF. (g) The core of the M78+ neuritic plaques (green) also contains NeuN immunoreactivity (red) (arrows). The intensity of the DAPI image is enhanced compared to the merged image to show the details of the diffuse chromatin staining. Bar = 20 μm.
Fig. 3
Fig. 3
Intranuclear M78 immunoreactivity in human brain and correlation with plaque pathology. (a, b) Adjacent sections from the Broadman's B11 region were stained with M78 (a) or 6E10, (b). Bar = 100 μm. (c-e) Triple label immunofluorescence staining with M78 (red) and (c) NeuN (green), (d) myelin oligodendrocyte glycoprotein (green), or (e) glial fibrilliary acidic protein (GFAP), (green) and chromatin (DAPI, blue). Arrows point to co-localization of M78 and specific cell markers. Bar = 20 μm. (f), Double label immunofluorescence staining with M78 (red) and 6E10 (green). The arrows point to cells with neuronal morphology that stain for both M78 and 6E10. Bar = 20 μm. (g) M78 nuclear immunoreactivity correlates with intermediate stages of plaque area deposition (stage A and B) in the frontal cortex of subjects from the ADRC cohort. (h) Average counts per mm2 of M78 positive nuclei in the frontal cortex of the combined ADRC and The 90+ study subjects are plotted against the OC positive plaque area fraction. The bars indicate the values of mean ± SEM. (i) Triple color immunofluorescence labeling of neuritic plaques with M78 (red), anti APP-CTF (green) and DAPI (blue). Arrows point to neuritic plaques containing a central core of diffuse DAPI fluorescence and M78 immunoreactivity with dystrophic neurites that stain for APP-CTF. The intensity of the DAPI image is enhanced compared to the merged image to show the details of the diffuse chromatin staining. Bar = 20 μm.
Fig.4
Fig.4
Model for neuritic plaque development from neurons with accumulated intracellular and intranuclear amyloid. Step 1: An initial perturbation of intracellular APP metabolism within a single neuron leads to the early accumulation and aggregation of Aß, longer Aß containing fragments and APP-CTFs in a perinuclear localization. This perturbation may arise spontaneously due to an imbalance between the levels of secreted versus intracellularly retained Aß/APP or it could be initiated by the uptake of extracellular oligomeric Aß seeds by a prion-like mechanism. Step 2: misfolded Aß and APP CTFs accumulate in perinuclear lysosomal/autophagic vesicles without being degraded and within the nucleus. Step 3: the continuing build-up of perinuclear amyloid aggregates causes filling and distension of “dystrophic neurites” around the M78 positive nucleus. Step 4: Intracellular amyloid accumulation causes the demise and lysis of the neuron followed by infiltration of astrocytes and microglia that clear the cell debris and digest the protease sensitive parts of APP, leaving behind the protease resistant amyloid core.
Fig.5
Fig.5
Model for an “alternative” amyloid hypothesis. BACE-mediated cleavage of APP results in the generation of soluble sAPPß and the transmembrane APP-CTF99. The next step in the amyloidogenic pathway consists of the γ-secretase-mediated cleavage of APP-CTF99. The high processivity of wild type γ-secretase releases soluble, short, “good” Aß species in the extracellular space and AICD in the cytosol. If the γ-secretase processivity is lost because of FAD-linked PS mutations or γ-secretase inhibition, the long Aß species and APP-CTF99 aggregate intracellularly and accumulate because of their intrinsic resistance to degradation, initiating the pathogenic mechanisms leading to cell death and formation of neuritic plaques.

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