Endocytosis is implicated in Alzheimer’s pathogenesis, because the continuous swallowing up of proteins from the cell surface and packaging them into little organelles brings the amyloid precursor protein (APP) into contact with the secretases that chop it and produce Aβ. How this changes as cells age, though, and how genetic risk factors for Alzheimer’s influence it, is still unclear. Data presented at the14th International Conference on Alzheimer’s and Parkinson’s Diseases, held March 27–31 in Lisbon, Portugal, suggest that as cells get older, the endocytosis of APP speeds up, giving it more access to its cleaving enzymes and increasing its processing. Other presentations showed that over- or underexpressing certain endosomal factors brings on symptoms of disease, suggesting that too much or too little endocytosis could kick off intracellular disease before pathology accumulates.

  • Endocytosis of APP picks up with aging.
  • Overexpressing Rab5 to stimulate endocytosis phenocopies AD.
  • BIN1 knockouts have trouble releasing synaptic vesicles.

Faster with Age
Many scientists believe that sluggish Aβ clearance in aging is a cause for late-onset AD (LOAD) (e.g., Saido and Leissring, 2012; Aug 2016 conference news). This stands in contrast to the increased Aβ production responsible for autosomal-dominant forms of the disease caused by mutations in APP or γ-secretase. Could a similar revving up of APP processing and Aβ production also come into play in LOAD, and if so, does endocytosis have something to do with it?

To find out, first author Tatiana Burrinha and colleagues in the lab of Claudia Almeida, University Institute of Lisbon, Portugal, cultured primary embryonic cortical neurons from wild-type mice and aged them. These neurons undergo three differentiation stages in a dish: They develop axons and dendrites after a week, reach peak maturation at 21 days, and exhibit signs of aging at 28 days, including synapse loss and increased free radicals. The researchers compared 21-day-old mature neurons with 28-day-old ones to look for differences in Aβ levels or APP processing.

More APP with Age. As cultured neurons get older, their neurites endocytose more APP, leading to more Aβ production. [Image courtesy of Tatiana Burrinha.]

In the old neurons, immunofluorescence using anti-Aβ42 antibody 12F4 revealed 50 percent more intracellular Aβ42 specifically in neurites. There, early endosomes were larger than in younger neurites. Western blots with the anti-APP APPY188 antibody indicated that intracellular APP C-terminal fragments (CTFs) doubled in aged cells compared with mature cells, but full-length APP remained the same, suggesting its expression did not change. Old cells had half the number of pre- and postsynaptic markers as 21-day-old cells, and a γ-secretase inhibitor rescued about 40 percent of that deficit. Together the results hinted that the trafficking of APP changes with aging, and this harms synapses.

To see if these observations held in vivo, the researchers compared the levels of processed APP fragments between 18-month aged wild-type mice and 6-month-old young adults. According to western blots on cortical homogenates, α- and β-CTF fragments doubled in the aged animals relative to young adults. Levels of the EEA1 marker of early endosomes tripled, while the amount of η–CTF rose as well. Together, the data suggest that APP processing goes up with aging in vivo, said Almeida.

That synapses decline in these experiments suggested to the authors that the age-related increase in Aβ production is a cause of synapse loss in age-related disease. It may add to synaptoxicity caused by previously reported defects in the clearance of Aβ42, said Almeida (Iwata et al., 2002; Solé-Domènech et al., 2016Elahy et al., 2015). 

Almeida’s new data fit with enlarged endosomes reported in postmortem AD patients by the late Anne Cataldo (Cataldo et al., 1997; Cataldo et al., 2000; April 2009 news). 

Almeida’s research doesn’t point to a specific drug target yet, but could in the future. “Once we know how APP endocytosis is increased with neuronal aging we may develop an inhibitor that does not interfere with synaptic vesicle endocytosis,” said Almeida. “This drug could potentially prevent amyloid accumulation and synaptic decline with aging.”

Gopal Thinakaran, University of Chicago, cautioned that data from aged cells in a dish might not represent what is going on in the aging brain. Lotta Agholme, University of Gothenburg, Sweden, echoed his concern. “Just neurons in a dish is a tricky thing, because you don’t have the whole brain. If you don’t have Alzheimer’s-related processes going on, it’s hard to find a good model,” Ralph Nixon, New York University, said. “Aging in vivo is more involved than neurons that have been in a dish for weeks.”

That said, the in vivo data is a step toward validating the result, the commentators agreed. Nixon lauded that Almeida analyzed APP processing fragments besides Aβ. He believes β-CTFs are toxic in their own right. At AD/PD, Eunju Im from Nixon’s lab showed that the elevated levels of β-CTFs in fibroblasts from people with Down’s syndrome and human APP-expressing N2a cells interfered with lysosomal degradation, impeding assembly of the complex on the lysosome surface necessary to render these organelles sufficiently acidic. Elevating β-CTFs in 2N human fibroblasts by inhibiting γ-secretase led to similar deficits.

Mice Model Endosomal Problems
Nixon presented postdoctoral fellow Anna Pensalfini’s data on a new mouse model of neuronal Rab5 overactivation that leads to increased endocytosis and enlarged endosomes in AD. Rab5 is a GTPase that helps initiate endocytosis and fuse the early endosomes that make sorting and signaling endosomes. APP’s β-CTF binds and activates Rab5, hyperactivating it in AD. However, in this model, Rab5 was expressed and activated to comparable levels as in AD and Down’s syndrome, without added APP fragments.

At 7 months, Rab5T mice developed upregulated endocytosis, enlarged endosomes, enhanced long-term depression, reduced long-term potentiation, shorter, sparser dendritic spines in the hippocampus, and tau hyperphosphorylation. The mice floundered on the novel object recognition test and their basal forebrain cholinergic neurons degenerated at 11 months.

“This argues strongly that endocytosis specifically as activated by Rab5 is capable of triggering a cascade of biological events that phenocopy synaptic and cholinergic deficits seen in AD,” Nixon told Alzforum.

For his part, Thinakaran introduced a conditional BIN1 knockout mouse. BIN1 is a common risk factor for Alzheimer’s disease, according to genome-wide association studies. Scientists don’t know how BIN1 normally functions in the brain. Some suspect it regulates BACE trafficking, and too little of it appears to hasten tau propagation (Nov 2015 news; Oct 2016 news). Earlier this year, Thinakaran’s lab reported that reduced BIN1 expression affected neither Aβ levels nor BACE1 localization (Andrew et al., 2019). 

In Lisbon, Thinakaran reported that synaptic transmission was weaker in hippocampal slices of mice lacking BIN1 in excitatory neurons than controls. And while high-frequency stimulation depleted vesicles in controls within five to 10 seconds, BIN1-less neurons sustained synaptic vesicle release for far longer. Using super-resolution microscopy, postdoctoral fellow Pierre De Rossi saw that without BIN1, SNARE proteins that normally facilitate fusion of plasma membranes and vesicle release were disorganized. Using three-dimensional reconstruction of CA3 to CA1 synapses, the researchers saw more synaptic vesicles either docked or hanging around in the reserve pool, which suggests fewer were successfully released. In addition, the cKO neurons were endocytosing more and making more vesicles. The mice struggled to find the hidden platform in the Morris water maze.

“Different lines of data are consistent with each other,” Thinakaran told Alzforum. “They all point to a role for BIN1 in presynaptic vesicle release that has a profound impact on memory consolidation.” Changes in BIN1 localization or levels could affect synaptic transmission and cognition independent of any pathology, he added.—Gwyneth Dickey Zakaib

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References

News Citations

  1. Refining Models of Amyloid Accumulation in Alzheimer’s Disease
  2. Anne Cataldo, 57, Autophagy Researcher Known for Generous Collegiality
  3. Alzheimer’s GWAS Hits Point to Endosomes, Synapses
  4. Lack of BIN1 Sows Tau Trouble for Neurons

Paper Citations

  1. . Proteolytic Degradation of Amyloid β-Protein. Cold Spring Harb Perspect Med. 2012 Jun;2(6):a006379. PubMed.
  2. . Region-specific reduction of A beta-degrading endopeptidase, neprilysin, in mouse hippocampus upon aging. J Neurosci Res. 2002 Nov 1;70(3):493-500. PubMed.
  3. . The endocytic pathway in microglia during health, aging and Alzheimer's disease. Ageing Res Rev. 2016 Dec;32:89-103. Epub 2016 Jul 12 PubMed.
  4. . Blood-brain barrier dysfunction developed during normal aging is associated with inflammation and loss of tight junctions but not with leukocyte recruitment. Immun Ageing. 2015;12:2. Epub 2015 Mar 7 PubMed.
  5. . Increased neuronal endocytosis and protease delivery to early endosomes in sporadic Alzheimer's disease: neuropathologic evidence for a mechanism of increased beta-amyloidogenesis. J Neurosci. 1997 Aug 15;17(16):6142-51. PubMed.
  6. . 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.
  7. . Reduction of the expression of the late-onset Alzheimer's disease (AD) risk-factor BIN1 does not affect amyloid pathology in an AD mouse model. J Biol Chem. 2019 Mar 22;294(12):4477-4487. Epub 2019 Jan 28 PubMed.

Further Reading

Papers

  1. . A novel role for the late-onset Alzheimer's disease (LOAD)-associated protein Bin1 in regulating postsynaptic trafficking and glutamatergic signaling. Mol Psychiatry. 2019 Apr 9; PubMed.
  2. . Reduction of the expression of the late-onset Alzheimer's disease (AD) risk-factor BIN1 does not affect amyloid pathology in an AD mouse model. J Biol Chem. 2019 Mar 22;294(12):4477-4487. Epub 2019 Jan 28 PubMed.

Primary Papers

  1. . Neuronal aging potentiates beta-amyloid generation via amyloid precursor protein endocytosis. bioRχiv. April 23, 2019