In Alzheimer’s disease, defective autophagy seeds Aβ fibrils around the nuclei of neurons, which then burst, leaving amyloid plaques in their wake. This is according to researchers led by Ralph Nixon and Ju-Hyun Lee of New York University, Orangeburg. In the June 2 Nature Neuroscience, they reported that in five mouse models of amyloidosis, poorly acidified lysosomes stuffed with Aβ distort the plasma membranes of neurons and form toxic rosettes around their nuclei. Dubbed PANTHOS, these flower-shaped blebs seed Aβ fibrils and leak proteases into the cytoplasm. The neurons eventually burst, leaving behind amyloid cores that tally with plaque load. PANTHOS, aka poisonous anthos, from the Greek for flower, occurs in AD brain tissue as well.
- Aβ accumulates in lysosomes when they fail to acidify.
- As the vesicles swell, they hijack Golgi and ER membranes.
- Perinuclear rosettes of Aβ fibrils then form.
- When neurons burst, the rosettes coalesce into amyloid plaques.
“This meticulously conducted work is a tour de force,” said George Perry, University of Texas, San Antonio. “It changes the paradigm by clearly showing that amyloid accumulation occurs within neurons and that aggregates come out after the neurons degenerate or die.”
Charles Glabe, University of California, Irvine, agreed. “This inside-out view has largely been ignored for at least 30 years, while the outside-in paradigm has dominated,” he told Alzforum. Glabe and others had previously proposed that intracellular Aβ kills neurons and creates plaques in the brains of people with mild cognitive impairment, though how this happened was a mystery (Mar 2013 conference news; Gouras et al., 2000; D’Andrea et al., 2001). “The issue of whether Aβ pathology starts from the inside-out, or outside-in, is one of the most important questions that needed to be resolved,” he said.
Nixon and colleagues discovered PANTHOS when studying the role of the autophagy/lysosomal system in Alzheimer’s. Scientists have become increasingly interested in the role of this degradation pathway in AD and other neurodegenerative diseases, but have been stymied tracking changes in autophagic lysosome in the brain. To overcome this, first author Lee created transgenic animals expressing the autophagy marker LC3 coupled with two dyes: one that fluoresces red when lysosomes are acidic, and the other that emits green light only when the pH creeps above 6.0. Lysosomes that fail to acidify appear yellow. A Thy-1 promoter ensured that only neurons made this tandem fluorescent LC3 reporter (tfLC3).
Lee crossed these tfLC3 mice with five models of amyloidosis: TgCRND8, PS/APP, 5xFAD, Tg2576, and APP51 (Herzig et al., 2004). The first three begin to develop amyloid plaques by about 5 months, but for Tg2576 and APP51 they form at about 12 and 20 months, respectively.
Using confocal microscopy, the scientists measured fluorescence within cortical slices from the transgenic mice months before they developed plaques. Compared to tfLC3 controls, transgenics had four times as many poorly acidified autolysosomes. “The most striking finding was that autolysosomal pH in neurons was increased long before the accumulation of amyloid plaques,” Tim Sargeant, South Australian Health and Medical Research Institute, Adelaide, wrote.
The authors thought a faulty vATPase was to blame. These pump protons into lysosomes, and their activity fell 35 percent before plaques first appeared and by half by the time they had become abundant.
When the lysosome pH stays high, proteases cannot clear cellular debris, such as Aβ. Indeed, 40 percent of cortical neurons from young, pre-plaque mice contained puncta of β-CTF/Aβ. Almost all were within the poorly acidified autolysosomes. Lee told Alzforum that the vesicles also included short, partially formed fibrils of Aβ.
As the mice aged, the autophagic process backed up. Swollen vesicles around the nucleus bulged the plasma membrane, morphing the cell body into a PANTHOS shape with distended lysosome petals around the nucleus (image below). These neurons appeared in all five mouse models. Zhenyu Yue, Icahn School of Medicine at Mount Sinai, New York, was surprised by the extreme extent that autolysosomes packed into the PANTHOS petals. “It suggests massive upregulation and impairment of autophagy,” he told Alzforum.
Deadly Flower. The PANTHOS structure, comprising a nuclear center (black) surrounded by blebs of poorly acidified autolysosomes (yellow) forms in Tg2576 (left), 5xFAD (second), TgCRND8 (middle), PS/APP (fourth), and APP51 (right) mice. [Courtesy of Lee et al., Nature Neuroscience, 2022.]
To get a better look at this phenomenon, the scientists homed in on cortical neurons from 5xFAD/LC3 mice. In 2.5-month-old animals that were just developing plaques, Aβ fibrils accrued within the failing autolysosomes and in tubules surrounding the nucleus. Lee said immunostaining showed that these tubules formed from the endoplasmic reticulum membrane. Autophagic vesicles can syphon from the ER to support their own swelling membranes (Uemura et al., 2014; Hayashi-Nishino et al., 2010; Axe et al., 2008). The authors reasoned that this happens in the AD mice. Alas, the autophagosomes floundered, leaving the tubules to linger and fill with Aβ fibrils.
Why has nobody detected these perinuclear Aβ fibrils before? Nixon said that without three-dimensional reconstruction of the neuron, it would be extremely difficult to tell if the fibrils lay within or outside the cell. “Only after correlating fluorescence and electron microscopy images of the same PANTHOS cell did we realize that the tubular structures are within an intact cell,” he told Alzforum (see three-dimensional reconstruction in movie below).
Flower Power. In mouse brain tissue, correlative light-electron microscopy, which images a sample with light and an electron beam simultaneously, renders a three-dimensional image of one PANTHOS neuron with a nuclear center (false-colored blue) surrounded by swollen autolysosome petals (red). The rest of the neuron is colored yellow. [Courtesy of Lee et al., Nature Neuroscience, 2022.]
As the PANTHOS neurons deteriorated, nearly all contained a dense amyloid core. In 2.5-month-old mice, half of the PANTHOS neurons were thioflavin S-positive, while by 6 months almost all were, by which time the animals had full-blown plaque pathology. “PANTHOS neurons look like plaques, even though they’re intact cells,” Nixon said. Remarkably, the scientists noticed that some Thio-S-positive PANTHOS neurons had burst, and their contents seemed to mingle with debris from neighboring cells to create a large amyloid plaque with multiple dense-cores (image below).
Perry and Gunnar Gouras, Lund University, Sweden, emphasized that, while some plaques may form from neuronal soma this way, others arise from dystrophic neurites and synapses (reviewed by Gouras et al., 2013). “Synapses are affected early in AD, and we have seen synaptic endosomes as the earliest sites of APP β-CTF/Aβ damage in AD that leads to early synaptic dysfunction, massive autophagic vacuole accumulation, and neuron death,” he wrote (full comment below). Still, Lee found that in mice, at least, the majority of plaques formed via PANTHOS. Using the 3D6 antibody to label plaques, Lee found that all the antibody binding was accounted for in PANTHOS lesions.
Do plaques form through PANTHOS in people, too? Analysis of prefrontal cortex tissue from three adults who had died at Braak stage II revealed the same perinuclear autophagic lysosomes and amyloid fibrils in some neurons, though the authors did not quantify how many. Nixon said that work is ongoing.
In all, these findings suggest that, months before plaques develop in mice, intraneuronal Aβ accumulates in faulty lysosomes, and that this distorts and kills neurons, leaving behind amyloid plaques (image below). “This study provides strong support that lysosome dysfunction is an early, causal, and, most importantly, pathogenic process in Alzheimer’s disease,” Rick Livesey, University College London, wrote (full comment below)..
PANTHOS Progression. This model shows how poorly acidified autolysosomes (purple) gather in neuron somas (left), pushing out the cell membrane to form PANTHOS petals (middle). Lysosomes begin leaking proteases (pink) and Aβ fibrils (gray squiggles) aggregate in lysosomal tubules around the nucleus. The overstuffed neuron collides with nearby PANTHOS cells and ruptures (right), recruiting glia to turn the amyloid debris into larger plaques. [Courtesy of Lee et al., Nature Neuroscience, 2022.]
In older mice, microglia swarmed PANTHOS neurons that had burst, and the authors think that these glia help to mop up diffuse Aβ and help package it into the dense core plaques, as has been reported previously (Apr 2021 news; May 2016 news). They also believe that correcting the lysosomal pH deficit might prevent these downstream pathologies. “It would make more sense for therapeutics to target the autophagy-lysosome pathway (the cause) rather than solely amyloid plaques (the effect), as is the case with current clinical trials that use Aβ immunotherapy by itself,” Sargeant wrote. Glabe agreed. “If the plaque is the tombstone marker for neuritic neurons, then removing it won’t raise the dead,” he said.
In preliminary experiments, Lee is trying to rescue lysosomal function by giving 5xFAD mice isoproterenol. He recently found that this β-adrenergic agonist, used to treat heart rhythm problems, promotes lysosome acidification and function in AD patient-derived fibroblasts (Lee et al., 2020). So far, he has found that animals treated with isoproterenol have fewer PANTHOS neurons.—Chelsea Weidman Burke
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