Deacidification of endolysosomes by neuronal aging drives synapse loss.

Previously, we found that age-dependent accumulation of beta-amyloid is not sufficient to cause synaptic decline. Late-endocytic organelles (LEOs) may be driving synaptic decline as lysosomes (Lys) are a target of cellular aging and relevant for synapses. We found that LAMP1-positive LEOs increased in size and number and accumulated near synapses in aged neurons and brains. LEOs' distal accumulation might relate to the increased anterograde movement in aged neurons. Dissecting the LEOs, we found that late-endosomes accumulated while there are fewer terminal Lys in aged neurites, but not in the cell body. The most abundant LEOs were degradative Lys or endolysosomes (ELys), especially in neurites. ELys activity was reduced because of acidification defects, supported by the reduction in v-ATPase subunit V0a1 with aging. Increasing the acidification of aged ELys recovered degradation and reverted synaptic decline, while alkalinization or v-ATPase inhibition, mimicked age-dependent Lys and synapse dysfunction. We identify ELys deacidification as a neuronal mechanism of age-dependent synapse loss. Our findings suggest that future therapeutic strategies to address endolysosomal defects might be able to delay age-related synaptic decline.

Aging impact on amyloid precursor protein neuronal trafficking.

Neurons live a lifetime. Neuronal aging may increase the risk of Alzheimer's disease. How does neuronal membrane trafficking maintain synapse function during aging? In the normal aged brain, intraneuronal beta-amyloid (Aß) accumulates without Alzheimer's disease mutations or risk variants. However, do changes with neuronal aging potentiate Aß accumulation? We reviewed the membrane trafficking of the amyloid precursor protein in neurons and highlighted its importance in Aß production. Importantly, we reviewed the evidence supporting the impact of aging on neuronal membrane trafficking, APP processing, and consequently Aß production. Dissecting the molecular regulators of APP trafficking during neuronal aging is required to identify strategies to delay synaptic decline and protect from Alzheimer's disease.

Alzheimer's disease BIN1 coding variants increase intracellular Aß levels by interfering with BACE1 recycling.

Genetic studies have identified BIN1 as the second most important risk locus associated with late-onset Alzheimer's disease (LOAD). However, it is unclear how mutation of this locus mechanistically promotes Alzheimer's disease (AD) pathology. Here we show the consequences of two coding variants in BIN1 (rs754834233 and rs138047593), both in terms of intracellular beta-amyloid (iAbeta) accumulation and early endosome enlargement, two interrelated early cytopathological AD phenotypes, supporting their association with LOAD risk. We previously found that Bin1 deficiency potentiates iAbeta production by enabling BACE1 cleavage of the amyloid precursor protein in enlarged early endosomes due to decreased BACE1 recycling. Here, we discovered that the expression of the two LOAD mutant forms of Bin1 does not rescue the iAbeta accumulation and early endosome enlargement induced by Bin1 knockdown and recovered by wild-type Bin1. Moreover, the overexpression of Bin1 mutants, but not wild-type Bin1, increased the iAbeta42 fragment by reducing the recycling of BACE1, which accumulated in early endosomes, recapitulating the phenotype of Bin1 knockdown. We showed that the mutations in Bin1 reduced its interaction with BACE1. The endocytic recycling of transferrin was similarly affected, indicating that Bin1 is a general regulator of endocytic recycling. These data demonstrate that the LOAD-coding variants in Bin1 lead to a loss of function in endocytic recycling, which may be an early causal mechanism of LOAD.

Upregulation of APP endocytosis by neuronal aging drives amyloid-dependent synapse loss.

Neuronal aging increases the risk of late-onset Alzheimer's disease. During normal aging, synapses decline, and ß-amyloid (Aß) accumulates intraneuronally. However, little is known about the underlying cell biological mechanisms. We studied neuronal aging using normal-aged brain and aged mouse primary neurons that accumulate lysosomal lipofuscin and show synapse loss. We identified the upregulation of amyloid precursor protein (APP) endocytosis as a neuronal aging mechanism that potentiates APP processing and Aß production in vitro and in vivo. The increased APP endocytosis may contribute to the early endosome enlargement observed in the aged brain. Mechanistically, we showed that clathrin-dependent APP endocytosis requires F-actin and that clathrin and endocytic F-actin increase with neuronal aging. Finally, Aß production inhibition reverts synaptic decline in aged neurons, whereas Aß accumulation, promoted by endocytosis upregulation in younger neurons, recapitulates aging-related synapse decline. Overall, we identify APP endocytosis upregulation as a potential mechanism of neuronal aging and, thus, a novel target to prevent late-onset Alzheimer's disease. This article has an associated First Person interview with the first author of the paper.

Impact of late-onset Alzheimer's genetic risk factors on beta-amyloid endocytic production.

The increased production of the 42 aminoacids long beta-amyloid (Aß42) peptide has been established as a causal mechanism of the familial early onset Alzheimer's disease (AD). In contrast, the causal mechanisms of the late-onset AD (LOAD), that affects most AD patients, remain to be established. Indeed, Aß42 accumulation has been detected more than 30 years before diagnosis. Thus, the mechanisms that control Aß accumulation in LOAD likely go awry long before pathogenesis becomes detectable. Early on, APOE4 was identified as the biggest genetic risk factor for LOAD. However, since APOE4 is not present in all LOAD patients, genome-wide association studies of thousands of LOAD patients were undertaken to identify other genetic variants that could explain the development of LOAD. PICALM, BIN1, CD2AP, SORL1, and PLD3 are now with APOE4 among the identified genes at highest risk in LOAD that have been implicated in Aß42 production. Recent evidence indicates that the regulation of the endocytic trafficking of the amyloid precursor protein (APP) and/or its secretases to and from sorting endosomes is determinant for Aß42 production. Thus, here, we will review the described mechanisms, whereby these genetic risk factors can contribute to the enhanced endocytic production of Aß42. Dissecting causal LOAD mechanisms of Aß42 accumulation, underlying the contribution of each genetic risk factor, will be required to identify therapeutic targets for novel personalized preventive strategies.

A Novel Protocol to Quantitatively Measure the Endocytic Trafficking of Amyloid Precursor Protein (APP) in Polarized Primary Neurons with Sub-cellular Resolution.

Alzheimer's disease's established primary trigger is ß-amyloid (Aß) (Mucke and Selkoe, 2012). The amyloid precursor protein (APP) endocytosis is required for Aß generation at early endosomes (Rajendran and Annaert, 2012). APP retention at endosomes depends on its sorting for degradation in lysosomes ( Haass et al., 1992 ; Morel et al., 2013 ; Edgar et al., 2015 ; Ubelmann et al., 2017 ). The following endocytosis assay has been optimized to assess the amyloid precursor protein (APP) endocytosis and degradation by live murine cortical primary neurons ( Ubelmann et al., 2017 ).

Measuring the Endocytic Recycling of Amyloid Precursor Protein (APP) in Neuro2a Cells.

The established primary trigger of Alzheimer's disease's is ß-amyloid (Aß) (Mucke and Selkoe, 2012). Amyloid precursor protein (APP) endocytosis is required for Aß generation at early endosomes (Rajendran and Annaert, 2012). APP retention at endosomes also depends on its recycling back to the plasma membrane ( Koo et al., 1996 ; Ubelmann et al., 2017 ). The following recycling assay has been optimized to assess APP recycling by live murine Neuro2a cells, a neuroblastoma cell line ( Ubelmann et al., 2017 ).

Bin1 and CD2AP polarise the endocytic generation of beta-amyloid.

The mechanisms driving pathological beta-amyloid (Aß) generation in late-onset Alzheimer's disease (AD) are unclear. Two late-onset AD risk factors, Bin1 and CD2AP, are regulators of endocytic trafficking, but it is unclear how their endocytic function regulates Aß generation in neurons. We identify a novel neuron-specific polarisation of Aß generation controlled by Bin1 and CD2AP We discover that Bin1 and CD2AP control Aß generation in axonal and dendritic early endosomes, respectively. Both Bin1 loss of function and CD2AP loss of function raise Aß generation by increasing APP and BACE1 convergence in early endosomes, however via distinct sorting events. When Bin1 levels are reduced, BACE1 is trapped in tubules of early endosomes and fails to recycle in axons. When CD2AP levels are reduced, APP is trapped at the limiting membrane of early endosomes and fails to be sorted for degradation in dendrites. Hence, Bin1 and CD2AP keep APP and BACE1 apart in early endosomes by distinct mechanisms in axon and dendrites. Individuals carrying variants of either factor would slowly accumulate Aß in neurons increasing the risk for late-onset AD.