Introduction
In the previous installment, we reviewed the latest research progress on APOE4‑expressing mice in the context of Alzheimer’s disease. Recently, a groundbreaking paper has emerged, suggesting that the pathogenic mechanisms underlying AD may be driven by alternative factors. In this issue, we will explore these findings together.
As everyone knows, the prevailing pathogenic mechanism of Alzheimer’s disease (AD) in the field is β‑amyloid deposition, also referred to as senile plaques. On June 2 this year, an article titled “Faulty autolysosome acidification in Alzheimer’s disease mouse models induces autophagic build-up of Aβ in neurons, yielding senile plaques” generated considerable attention, suggesting that the etiological factors underlying AD may extend beyond β‑amyloid alone.
The researchers stated:
The pathogenesis of Alzheimer’s disease involves impaired acidification of lysosomes in neuronal autophagy.
Autophagy is the primary pathway for lysosomal degradation, eliminating aberrant proteins, aggregates, and damaged organelles in response to disease and cellular stress. In the major autophagy–lysosome pathway (ALP), autophagosomes (APs) are formed; upon fusion with lysosomes or endolysosomes, these APs give rise to autolysosomes (ALs).
Lysosomes contain numerous enzymes, such as various cathepsins and other acid hydrolases. vATPase, which is a proton pump that hydrolyzes ATP and acidifies the lumen of the AL.
Therefore, when lysosomal acidification is impaired, autophagy becomes dysfunctional, leading to the accumulation of numerous proteins that should be degraded. Among these accumulated proteins are β‑amyloid and its precursor protein.
As insoluble proteins accumulate to ever greater levels, eventually causing the lysosome to rupture, the lysosome’s myriad hydrolytic enzymes and the accumulated proteins are released into the cytoplasm. When the cell membrane also breaks down, large aggregates of deposited proteins emerge throughout the brain—these are the amyloid plaques seen on brain imaging.
Some have described amyloid plaques as the remnants of neurons and β‑amyloid protein.
To identify and detect ALP deficiency in AD mice, the researchers employed AD model mice and utilized a neuron-specific tandem fluorescent marker (Thy-1 mRFP-eGFP-LC3, using TRGL mice). They found that, in all five AD model mouse lines, deficits in vATPase activity and autophagic dysfunction had already emerged within neuronal populations prior to the deposition of extracellular β‑amyloid.
Detection of ALP dysfunction in the body
Researchers used red–green tandem fluorescent labeling to mark autophagosomes in neurons and blue fluorescence to label lysosomes. Initially, autophagosomes appear yellow (a merged red–green fluorescence); upon fusion with lysosomes and maturation, the acidic environment of the lysosome and the influence of the blue fluorescence cause the fluorescence to shift from yellow to red and ultimately to purple.
When the lysosome’s acidification function is impaired and enzyme activity is insufficient, the merged fluorescence will not exhibit the characteristic shift from red to purple; instead, it will appear as white fluorescence. (Figure 1)
These color contrasts allow for clear detection of detailed changes in ALP activity within the mouse.
AL underacidification occurs prior to amyloid-beta deposition.
The researchers crossed TRGL mice with Tg2576 mice (an Alzheimer’s disease model), and the resulting F1 offspring began to develop β‑amyloid plaques starting at 10–12 months of age. At 1.6 months, the ALP pattern in the Tg2576/TRGL hybrid was indistinguishable from that of the TRGL single‑cross strain (Extended Data Figure 1a); however, by 5 months, more than 90% of the perinuclear regions in layers III–V of the neocortex had acquired yellow fluorescent AVs in addition to acidified ALs (Extended Data Figure 1a). This suggests that… Amyloid plaques appear in the brain at least five months earlier.
Expand Figure 1a
The researchers also found that vATPase enzyme activity was similarly reduced in the brains of two other AD mouse models—5xFAD mice—occurring at an earlier time point (Extended Figure 1d).
Expand 1D
APP-βCTF/Aβ accumulates in pa-AL during the early stages of the disease.
In AD, APP‑βCTF and Aβ first accumulate within cells, followed by extracellular deposition of β‑amyloid. By 5 months of age, 40% of neurons in the III–V layers of the neocortex of Tg2576/TRGL mice harbor Aβ/APP‑βCTF‑positive plaques (Fig. 3a). Based on co‑immunolabeling with CTSD and four‑fluorophore imaging (Fig. 3a, arrows; see also Fig. 3b), nearly all of these plaques are pa‑AL (88.6 ± 2.4%).
A large accumulation of pa-AL in progressively damaged neurons.
In 10-month-old Tg2576/TRGL mice, a subpopulation of neocortical neurons (layers III–V) begins to accumulate enlarged pa-ALs in large numbers, causing the plasma membrane to bulge outward (Fig. 4a). Most of the affected perinuclear AVs are positive for the ly‑marker upon IHF analysis, indicating that they are pa-ALs (Fig. 4d and Extended Data Fig. 3b), which reflects severe defects in AL maturation and acidification.
The same autophagic neurodegenerative pattern was observed across five distinct AD mouse models, including those with accelerated neuropathological onset (5xFAD, TgCRND8, and PSAPP) as well as those with delayed onset (Tg2576 and app51—a model exhibiting an unusually late onset expressing hAPPwt) (Fig. 5, 4e). In 5xFAD/TRGL mice, robust ALP disruption and neuronal degeneration were evident at an early stage, beginning around 2 months of age depending on sex (Extended Data Fig. 3–e).
Generalized neurodegeneration is a distinct form of neurodegeneration in Alzheimer’s disease.
Using confocal imaging, we observed that autophagic vacuoles (AVs) are directly derived from… PANTHOS The perinuclear cytoplasm of the neuron extends outward through a conical neck toward the perinuclear center (Fig. 5a), forming a corona‑like structure; neurons exhibiting this morphology are referred to as “corona neurons.”
Figure 5a
ANTHOS neurons are the primary source of amyloid plaques.
In 5xFAD/TRGL mice, prior to the emergence of β‑amyloid plaques, Aβ and APP‑βCTF selectively accumulate in perinuclear astrocytes (Figure 6a). The transition of neurons toward the PANTHOS phenotype is accompanied by a sustained increase in perinuclear Aβ/APP‑βCTF immunoreactivity. Co‑labeling these PANTHOS neurons with DAPI and an anti‑β‑amyloid antibody (4G8) reveals that, at the most severely affected perinuclear core, there is a 4G8‑positive halo surrounded by a DAPI‑positive nuclear remnant (Figure 6b).
In the later stages of PANTHOS, as more β‑amyloid protein accumulates in the center, DAPI fluorescence gradually diminishes (Extended Data Fig. 6d).
Furthermore, in the cortical PANTHOS lesions of 2.7‑month‑old 5xFAD/TRGL mice, DAPI‑positive nuclear signals were detected in 91.4 ± 1.29% of neurons (n = 6; two sections per mouse), including condensed or fragmented/diffuse signals localized around the nucleus (Fig. 7c). In older mice (6 months), despite severe glial infiltration and neurodegeneration, 67.8% of PANTHOS neurons still exhibited DAPI‑positive nuclear signals (Fig. 7d). This percentage may be an underestimate, as immunoelectron microscopy using the nuclear marker KDM1/LSD1 revealed residual nuclear material even after loss of nuclear integrity.
Figure 7
The transition from fully nucleated PANTHOS neurons to more advanced stages characterized by the disappearance of DAPI‑positive nuclei, accompanied by glial cell infiltration, may reflect a loss of cellular integrity and conversion into extracellular plaques.
Lysosomal permeabilization promotes neuronal cell death.
Compared with littermate WT mice, cytoplasmic and membrane–vesicle fractionation analyses in 6-month-old 5xFAD mice revealed a significant increase in the levels of lysosomal enzymes in the brain cytoplasm (Figure 7e). Lysosomal enzyme leakage was detectable in the brains of 6-month-old animals but not in younger brains (2.7 months), at which stage fewer neurons were affected.
In the AD model, ANTHOS neurons evolve into senile plaques.
To characterize the progression of PANTHOS neuronal pathology into mature plaques, we immunolabeled PANTHOS with thioflavin S (Thio‑S) to detect dense core senile plaques (Fig. 8a and Extended Data Fig. 7b,c). In quantitative analyses at 2.2 months of age in 5xFAD/TRGL mice, half of the PANTHOS‑positive cells were Thio‑S–positive; by 6 months of age, over 95% of PANTHOS‑positive cells were Thio‑S–positive (Fig. 8a, panel).
In aged 5xFAD mice, when neighboring PANTHOS neurons coalesce into a larger structure—shown in Figures 8c, A1, and A2—and incorporate multiple thio‑S–positive dense cores (Figure 8d), PANTHOS lesions frequently expand into larger amyloid plaques. Within these growing lesions, newly recruited PANTHOS neurons remain identifiable (Extended Data Figure 8a, arrows); however, the loss of integrity of the original PANTHOS neurons and their adjacent neighbors, coupled with the clearance of other cellular debris, gives rise to a persistent central core enriched in β‑amyloid, ultimately forming an enlarged extracellular dense core characteristic of senile plaques (Figures 8b, A3, and A4).
Among the plaques formed in five distinct Alzheimer’s disease models, PANTHOS neurons constitute the vast majority. Subsequent microglial and astrocytic infiltration of PANTHOS neurons heralds eventual cell death, converting intraneuronal amyloid pathology into extracellular amyloid plaques.
In Alzheimer’s disease, the formation of β‑amyloid plaques is generally thought to result from the extracellular deposition of Aβ‑derived β‑amyloid protein, which in turn triggers secondary neurodegeneration and neuronal cell death.
In contrast, researchers have demonstrated in various AD models that extracellular plaques primarily arise from the intraneuronal accumulation of β‑amyloid within membranous tubules, giving rise to a concentrated amyloid “core” within individual intact PANTHOS neurons, which subsequently undergoes degeneration to form classical senile plaques. This “inside‑out” process is consistent with and supports the hypothesis put forward by many investigators.
These findings demonstrate that lysosomal acidification dysfunction is a pathogenic mechanism underlying genetic and metabolic disorders associated with neurodegenerative diseases.
Evidence from researchers indicates that the PANTHOS cascade in AD models can be significantly alleviated through pharmacological approaches that target lysosomal pH defects.
References:
https://doi.org/10.1038/s41593-022-01084-8
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