Neurons crowded with distended endosomes are a hallmark of Alzheimer’s disease, but does this malfunction drive the neurodegenerative cascade? A new transgenic mouse, described last November 24 in Cell Reports, supports the possibility. Researchers led by Ralph Nixon at New York University describe how overexpression of Rab5 in neurons caused endosomes to swell and build up. It also caused other AD-like features as the mice aged, including synaptic deficits, tau hyperphosphorylation, and withering cholinergic neurons. This chain of events unfolded in the absence of Aβ accumulation, suggesting the endosomal malfunctions were sufficient to bring on key characteristics of AD.
- Expressing human Rab5 in neurons causes endosomes to swell.
- The transgenic mice have synaptic deficits and hyperphosphorylated tau.
- Cholinergic neurons and fibers degenerate; memories fade.
“This paper provides additional support to implicate endosomal trafficking in AD, and shows this pathway is a convergence point for both APP-dependent and APP-independent mechanisms of disease,” commented Scott Small of Columbia University in New York (full comment below).
“The studies … suggest that endosomal dysfunction is a contributor to both the prodromal and the neurodegenerative phases of AD, the latter of which has commonly been attributed to the consequences of one or more of extracellular Aβ accumulation, amyloid plaque deposition, and tau aggregation,” wrote Rick Livesey and Christy Hung of University College London. “Their findings also open up new avenues to explore whether Rab5 overactivation and its signaling effectors might be potential targets for therapeutic interventions against AD or other neurodegenerative conditions involving endosomal-lysosomal dysfunction.” (Full comment below.)
Endosomal abnormalities emerge early in Alzheimer’s, prior to overt accumulation of Aβ (Cataldo et al., 2000). The endosomal swellings have been tied to the β-CTF fragment of amyloid precursor protein (APP). The product of BACE1 cleavage, this fragment has been implicated in the recruitment of Rab5 to endosomes, where the small GTPase kicks on and fuels endocytosis (Jul 2015 news). The β-CTF-Rab5 pathway has been blamed for endosomal dysfunction in cell culture models of familial AD, as well as in a mouse model of Down’s syndrome, which expresses an extra copy of APP (Aug 2019 news; Jiang et al., 2019; Alam et al., 2017).
Might endosomal malfunctions drive neurodegeneration, even when Aβ is not accumulating? To address this question, first author Anna Pensalfini and colleagues generated a mouse that overexpresses a Myc-tagged version of human Rab5 in neurons. The mice overexpressed Rab5 by about 2.5-fold compared to wild-type mice, on par with the elevated levels of the GTPase in the AD brain (Ginsberg et al., 2010). Using multiple approaches, the researchers reported that this overexpression of Rab5 also led to its activation—the transgenic mice had twice the amount of GTP-bound Rab5 lodged in their neuronal endosomes as did their wild-type counterparts.
As the authors describe it, this pathologically active (PA) Rab5 ratcheted up endocytosis in the hippocampus and cortex. In 6- to 8-month-old PA-Rab5 mice, swollen endosomes took up twice as much space within neurons as they did in neurons from wild-type. The endosomes crowded neuronal cell bodies and dendrites, resembling the endosomal pathology seen in the human Alzheimer’s brain. The endosomal dysfunction did not significantly affect levels of APP metabolites—including β-CTF and Aβ peptides.
Endosome Invasion. Endosomes in human Rab5-expressing neurons (red) took up twice as much space as those in wild-type neurons (left) with only mouse Rab5 (green). [Courtesy of Pensalfini et al., Cell Reports, 2020.]
Besides endosomal distension, what else happened? The researchers found that the excess Rab5 triggered synaptic snafus. In hippocampal brain slices from 6-month-old mice, both long-term potentiation and depression—the yin and yang of synaptic sensitivity that makes synapses so plastic—were derailed. The researchers found fewer AMPA receptors on neuronal cell membranes, probably because the revved-up endocytosis snatched them from the surface. The investigators detected all manner of defects in dendritic spines. Depending on the region in question, spines were either fewer or shorter—morphological changes that have been observed in AD mouse models and in the human Alzheimer’s brain.
Rab5 overexpression messed with AKT, a kinase required for pro-survival signaling. In so doing, Rab5 switched on GSK-3β, a kinase downstream of AKT that is known to phosphorylate tau. And sure enough, the PA-Rab5 mice accumulated hyperphosphorylated tau in different regions of the cortex. As measured by immunofluorescence using two different tau antibodies, PHF1S396/404 and CP13S202, the researchers spotted Rab5+ swollen endosomes co-localized with phospho-tau within neurons.
Tussles without Tangles. In human Rab5-expressing mice, phospho-tau (red) accumulates in cortical neuron endosomes (left), where it tussles with Rab5 (green, center). Right panel shows both. [Courtesy of Pensalfini et al., 2020.]
Did Rab5 lead to neurodegeneration? In the PA-Rab5 mice, the researchers detected a gradual loss of basal forebrain cholinergic neurons as gauged by staining for choline acetyltransferase (ChAT), starting at 7 months of age. They monitored these neurons because they are the first to die in people with AD, which could be because they rely on endosome-mediated, retrograde transport of growth factors made in the hippocampus. Cholinergic fibers withered, and both their cell bodies and dendrites choked up with swollen endosomes. All the while, endosomal dysfunction and other signs of cellular distress, including autophagic structures crammed into dendrites and synaptic terminals, manifested in hippocampal regions that receive cholinergic input. Together, the findings suggest that the endosomal dysfunction triggered by Rab5 overexpression dismantled key cholinergic circuitry.
The PA-Rab5 mice also showed signs of memory loss. At 6 months, compared to wild-type, they were less adept at distinguishing between novel and familiar objects.
Overall, the findings suggest that Rab5 overactivation leads to several characteristics of AD, including synaptic dysfunction, tau hyperphosphorylation, and degeneration of the cholinergic system, Nixon said.
“Although numerous in-vitro experiments have indirectly linked hyperactivation of Rab5 to cellular pathology in AD, this is the first in-vivo study that has established a causal effect of Rab5-endosomal dysfunction on AD-like cellular pathology, and synaptic and behavioral deficits in mice,” wrote Chengbiao Wu of the University of California, San Diego. “This study is timely and consistent with the fact that many newly identified AD risk factors are involved in regulating endocytic trafficking.”
Nixon stressed that in the context of AD, β-CTF and likely a complex mix of other factors are responsible for stoking Rab5. William Mobley of the University of California, San Diego, called the quality of the work impressive. He also noted that this transgenic model does not recapitulate what happens in AD or DS, where Rab5 is disproportionately activated while its expression remains normal. Mobley thinks it’s unclear how Rab5 becomes activated in disease.
Mobley is investigating if Aβ itself triggers an uptick in β-CTF, setting off a feedback loop that promotes Rab5 overactivation and endosomal dysfunction. In the PA-Rab5 mouse, Rab5 is constitutively overactivated, rendering the model unable to address what activates the GTPase in AD. Mobley also wondered how Rab5 overexpression affects other parts of the endolysosomal system, noting that defects in lysosomal digestion, which are also known to occur in AD and DS, may also damage neurons.
For other scientists’ perspective on the data, see comments below.
The findings do not rule out that Aβ contributes to endolysosomal dysfunction, Nixon stressed, noting that later parts of the pathway, including lysosomal digestion, likely play a role in disease. He thinks Rab5 makes a promising therapeutic target for AD and related diseases. A recent trial of neflamapimod, an inhibitor of the p38 MAP kinase that overactivates Rab5, reported a benefit in dementia with Lewy bodies, and is still on the table as an investigational AD therapeutic. That said, Nixon believes BACE inhibitors, which reduce levels of β-CTF as well as Aβ, remain an obvious choice. Though these inhibitors had side effects in multiple clinical trials, lower doses would suffice to reduce β-CTF enough to halt downstream endosomal dysfunction (Dec 2020 news).—Jessica Shugart
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- New Data from Past BACE Inhibitor Trials Shed Light on Side Effects
Research Models Citations
- Cataldo AM, Peterhoff CM, Troncoso JC, Gomez-Isla T, Hyman BT, Nixon RA. Endocytic pathway abnormalities precede amyloid beta deposition in sporadic Alzheimer's disease and Down syndrome: differential effects of APOE genotype and presenilin mutations. Am J Pathol. 2000 Jul;157(1):277-86. PubMed.
- Jiang Y, Sato Y, Im E, Berg M, Bordi M, Darji S, Kumar A, Mohan PS, Bandyopadhyay U, Diaz A, Cuervo AM, Nixon RA. Lysosomal Dysfunction in Down Syndrome Is APP-Dependent and Mediated by APP-βCTF (C99). J Neurosci. 2019 Jul 3;39(27):5255-5268. Epub 2019 May 1 PubMed.
- Alam J, Jiang Y, Nixon R. Antagonism of p38 MAPK Alpha (p38α) Reverses APP-Induced Endosomal Abnormalities and Improves Lysosomal Function in Down Syndrome Fibroblasts. Alzheimer's & Dementia Supplement, July 2017
- Ginsberg SD, Alldred MJ, Counts SE, Cataldo AM, Neve RL, Jiang Y, Wuu J, Chao MV, Mufson EJ, Nixon RA, Che S. Microarray analysis of hippocampal CA1 neurons implicates early endosomal dysfunction during Alzheimer's disease progression. Biol Psychiatry. 2010 Nov 15;68(10):885-93. Epub 2010 Jul 23 PubMed.
- Germann UA, Alam JJ. P38α MAPK Signaling-A Robust Therapeutic Target for Rab5-Mediated Neurodegenerative Disease. Int J Mol Sci. 2020 Jul 31;21(15) PubMed.
- Pensalfini A, Kim S, Subbanna S, Bleiwas C, Goulbourne CN, Stavrides PH, Jiang Y, Lee JH, Darji S, Pawlik M, Huo C, Peddy J, Berg MJ, Smiley JF, Basavarajappa BS, Nixon RA. Endosomal Dysfunction Induced by Directly Overactivating Rab5 Recapitulates Prodromal and Neurodegenerative Features of Alzheimer's Disease. Cell Rep. 2020 Nov 24;33(8):108420. PubMed.