X11α haploinsufficiency enhances Aβ amyloid deposition in Alzheimer's disease transgenic mice

The neuronal adaptor protein X11α/mint-1/APBA-1 binds to the cytoplasmic domain of the amyloid precursor protein (APP) to modulate its trafficking and metabolism. We investigated the consequences of reducing X11α in a mouse model of Alzheimer's disease (AD). We crossed hAPPswe/PS-1ΔE9 transgenic (AD tg) mice with X11α heterozygous knockout mice in which X11α expression is reduced by approximately 50%. The APP C-terminal fragments C99 and C83, as well as soluble Aβ40 and Aβ42, were increased significantly in brain of X11α haploinsufficient mice. Aβ/amyloid plaque burden also increased significantly in the hippocampus and cortex of one year old AD tg/X11α (+/−) mice compared to AD tg mice. In contrast, the levels of sAPPα and sAPPβ were not altered significantly in AD tg/X11α (+/−) mice. The increased neuropathological indices of AD in mice expressing reduced X11α suggest a normal suppressor role for X11α on CNS Aβ/amyloid deposition.     

Cytoplasmic gelsolin increases mitochondrial activity and reduces Aβ burden in a mouse model of Alzheimer's disease

Accumulation of amyloid-β (Aβ) peptides is thought to be a critical event in the pathology of Alzheimer's disease (AD), because they induce multiple neurotoxic effects, including mitochondrial dysfunction and apoptotic cell death. Therefore the reduction of Aβ is considered a primary therapeutic target. Gelsolin, an Aβ binding protein, has been shown to inhibit apoptosis, although the underlying mechanism is unclear. To clarify these effects, we manipulated cytoplasmic gelsolin levels through viral-directed overexpression in the brain of APP/Ps1 transgenic mice. We observed that gelsolin reduces brain Aβ burden in the APP/Ps1 mice, possibly by enhancing Aβ clearance via megalin. The reduction in brain Aβ levels was accompanied by an inhibition of nitric oxide production and cell death, not only in the choroid plexus but also in the cerebral cortex. Notably, overexpressed gelsolin restored the impaired mitochondrial activity in the APP/Ps1 mice, resulting in the increase of cytochrome c oxidase activity. By contrast, RNA interference to block gelsolin expression, confirmed that cytoplasmic gelsolin acts as a modulator of brain Aβ levels and its neurotoxic effects. We conclude that gelsolin might prevent brain amyloidosis and Aβ-induced apoptotic mitochondrial changes. These findings make cytoplasmic gelsolin a potential therapeutic strategy in AD.     

Nordihydroguaiaretic acid protects hippocampal neurons against amyloid β-peptide toxicity, and attenuates free radical and calcium accumulation

Recent findings indicate that amyloid β-peptide (Aβ) can be neurotoxic by a mechanism involving an increase in the concentration of intracellular free Ca²⁺ ([Ca²⁺]_i) and the generation of free radicals. In the present study, the lipoxygenase inhibitor/antioxidant nordihydroguaiaretic acid (NDGA) protected cultured rat hippocampal neurons against the toxicity of Aβ in a concentration-dependent manner. Measurements of cellular oxidation (using the oxidation-sensitive dye 2,7-dichlorofluorescin) and intracellular free Ca²⁺ levels (using the Ca²⁺ indicator dye fura-2), showed that NDGA suppressed Aβ-induced accumulation of reactive oxygen species (ROS) and Ca²⁺; Ca²⁺ responses to glutamate were also suppressed by NDGA. NDGA prevented neuronal injury and accumulation of ROS induced by iron, indicating a role for NDGA as an antioxidant in NDGA-mediated neuroprotection. Another lipoxygenase inhibitor (AA861) also protected against Aβ and iron toxicity whereas the the 5-lipoxygenase-activating protein inhibitor L655,238 and the cyclooxygenase inhibitor indomethacin were ineffective. These findings suggest that NDGA can interupt a neurodegenerative pathway relevant to the pathophysiology of Alzheimer's disease.     

Insulin protects against amyloid β-peptide toxicity in brain mitochondria of diabetic rats

This study compared the status of brain mitochondria isolated from 12-week streptozotocin (STZ)-diabetic rats versus STZ-diabetic animals treated with insulin during a period of 4 weeks. Brain mitochondria isolated from 12-week citrate (vehicle)-treated rats were used as control. For that purpose, several mitochondrial parameters were evaluated: respiratory indexes (respiratory control ratio (RCR) and ADP/O ratio), transmembrane potential (ΔΨm), repolarization lag phase, repolarization level, ATP, glutathione and coenzyme Q (CoQ) contents, production of H₂O₂, ATPase activity, and the capacity of mitochondria to accumulate Ca²⁺. Furthermore, the effect of Aβ_1–40 was also analyzed.We observed that STZ-induced diabetes promoted a significant decrease in mitochondrial CoQ9, ATPase activity, and a lower capacity of mitochondria to accumulate Ca²⁺ when compared with control and insulin-treated diabetic rats. The presence of 4 μM Aβ_1–40 induced a significant decrease in RCR in the three groups of rats. However, this peptide induced a significant increase in the repolarization lag phase and a significant decrease in the repolarization level in control and diabetic animals without insulin treatment. Furthermore, this peptide exacerbated significantly the production of H₂O₂ in STZ-diabetic rats, this effect being avoided by insulin treatment.Our data show that although diabetes induces some alterations in brain mitochondrial activity, those alterations do not interfere significantly with mitochondria functional efficiency. Similarly, insulin does not affect basal mitochondria function. However, in the presence of amyloid β-peptide, insulin seems to prevent the decline in mitochondrial oxidative phosphorylation efficiency and avoids an increase in oxidative stress, improving or preserving the function of neurons under adverse conditions, such as Alzheimer's disease.     

The release of calcium from the endoplasmic reticulum induced by amyloid-beta and prion peptides activates the mitochondrial apoptotic pathway

In this study, we analyzed whether ER Ca²⁺ release, induced by amyloid-β (Aβ) and prion (PrP) peptides activates the mitochondrial-mediated apoptotic pathway. In cortical neurons, addition of the synthetic Aβ1–40 or PrP106–126 peptides depletes ER Ca²⁺ content, leading to cytosolic Ca²⁺ overload. The Ca²⁺ released through ryanodine (RyR) and inositol 1,4,5-trisphosphate (IP₃R) receptors was shown to be involved in the loss of mitochondrial membrane potential, Bax translocation to mitochondria and apoptotic death. Our data further demonstrate that Ca²⁺ released from the ER leads to the depletion of endogenous GSH levels and accumulation of reactive oxygen species, which were also involved in the depolarization of the mitochondrial membrane. These results illustrate that the early Aβ- and PrP -induced perturbation of ER Ca²⁺ homeostasis affects mitochondrial function, activating the mitochondrial-mediated apoptotic pathway and help to clarify the mechanism implicated in neuronal death that occurs in AD and PrD.     

Presenilin/γ-Secretase Activity Is Located in Acidic Compartments of Live Neurons

Presenilin (PSEN)/γ-secretase is a protease complex responsible for the proteolytic processing of numerous substrates. These substrates include the amyloid precursor protein (APP), the cleavage of which by γ-secretase results in the production of β-amyloid (Aβ) peptides. However, exactly where within the neuron γ-secretase processes APP C99 to generate Aβ and APP intracellular domain (AICD) is still not fully understood. Here, we employ novel Förster resonance energy transfer (FRET)-based multiplexed imaging assays to directly “visualize” the subcellular compartment(s) in which γ-secretase primarily cleaves C99 in mouse cortex primary neurons (from both male and female embryos). Our results demonstrate that γ-secretase processes C99 mainly in LysoTracker-positive low-pH compartments. Using a new immunostaining protocol which distinguishes Aβ from C99, we also show that intracellular Aβ is significantly accumulated in the same subcellular loci. Furthermore, we found functional correlation between the endo-lysosomal pH and cellular γ-secretase activity. Taken together, our findings are consistent with Aβ being produced from C99 by γ-secretase within acidic compartments such as lysosomes and late endosomes in living neurons.SIGNIFICANCE STATEMENT Alzheimer''s disease (AD) genetics and histopathology highlight the importance of amyloid precursor protein (APP) processing by γ-secretase in pathogenesis. For the first time, this study has enabled us to directly “visualize” that γ-secretase processes C99 mainly in acidic compartments such as late endosomes and lysosomes in live neurons. Furthermore, we uncovered that intracellular β-amyloid (Aβ) is significantly accumulated in the same subcellular loci. Emerging evidence proposes the great importance of the endo-lysosomal pathway in mechanisms of misfolded proteins propagation (e.g., Tau, α-Syn). Therefore, the predominant processing of C99 and enrichment of Aβ in late endosomes and lysosomes may be critical events in the molecular cascade leading to AD.     

Distribution of β-amyloid and amyloid precursor protein in the brain of spawning (senescent) salmon: a natural, brain-aging model

Brain amyloid precursor protein (APP), a normal constituent of neurons, glial cells and cerebrospinal fluid, has several proposed functions (e.g., in neuronal growth and survival). It appears, however, that altered processing of APP is an initial or downstream step in the neuropathology of brain aging, Alzheimer's disease (AD), and Down's syndrome (DS). Some studies suggest that proteolytic cleavage of APP, producing β-amyloid (Aβ_1–42), could have neurotoxic or neuroprotective effects. In this study, we utilized antibodies to human APP₆₉₅ and Aβ_1–42, and Congo red staining, to search for amyloid deposition in the brain of semelparous spawning kokanee salmon (Oncorhynchus nerka kennerlyi). Intracellular APP₆₉₅ immunoreactivity (APP-ir) was observed in brain regions involved in gustation (glomerulosus complex), olfaction (putative hippocampus, olfactory bulb), vision (optic tectum), the stress response (nucleus preopticus and nucleus lateralis tuberis), reproductive behavior (nucleus preopticus magnocellularis, nucleus preopticus periventricularis, ventral telencephalon), and coordination (cerebellum). Intra- and extra-neuronal Aβ_1–42 immunoreactivity (Aβ-ir) were present in all APP-ir regions except the nucleus lateralis tuberis and Purkinje cells of the cerebellum (coordination). Thus, the relationship between APP and Aβ deposition during brain aging could shed light on the processing of APP into Aβ, neurodegeneration, and possible protection of neurons that are functioning in spawning but senescent salmon. Pacific salmon, with their predictable and synchronized life history, could provide research options not available with the existing models for studies of brain aging and amyloidosis.     

Amyloid precursor protein and membrane phospholipids in primary cortical neurons increase with development, or after exposure to nerve growth factor or Aβ1–40

We examined the relationships between membrane phospholipid levels, the secretion and expression of the amyloid precursor protein (APP), and the responses of both to nerve growth factor (NGF), Aβ_1–40 or Aβ_40–1 in developing cortical neurons cultured from rat embryos. Neuronal membrane phospholipid levels per cell, and phosphatidylcholine, phosphatidylserine, phosphatidylinositol and phosphatidylethanolamine increased individually between the first and seventh days of culturing. The amounts of APP holoprotein and APP mRNAs in the cells, as well as the amounts of soluble APP (APPs) secreted by them, also increased during neuronal development in vitro. The increases in APPs exceeded the increases in APP which, in turn, exceed those in phospholipid levels. The levels of APP holoprotein, but not of phospholipids, increased when neurons were grown in a choline-free medium, suggesting that increases in APP are not sufficient to stimulate changes in membrane phospholipids. Treatment of neuron cultures for four days with NGF or Aβ_1–40, but not with Aβ_40–1, dose-dependently increased membrane phospholipids, tau and GAP-43, as well as APP holoprotein and secreted APPs. These results indicate that agents, like NGF or Aβ_1–40, which enhance membrane phospholipid levels may promote neurite formation, APP expression and APPs secretion in primary neuronal cultures.     

New Mouse Model of Alzheimer’s

Amyloid β-peptide (Aβ) accumulation is a key characteristic of Alzheimer’s disease (AD); therefore, mouse models of AD exhibiting Aβ pathology are valuable tools for unraveling disease mechanisms. However, the overexpression of Aβ precursor protein (APP) used in previous mouse models may cause Aβ-independent artifacts that influence data interpretation. To circumvent these problems, we used an APP knock-in (KI) strategy to introduce mutations to the mouse APP gene to develop a new generation of AD mouse models. These new models, termed APP^NL-F and APP^NL-G-F, have endogenous APP levels and develop robust Aβ amyloidosis, which induce synaptic degeneration and memory impairments. Thus, we suggest that these novel APP KI mice will serve as important tools to elucidate molecular mechanisms of AD.     

Dysregulation of cellular calcium homeostasis in Alzheimer’s disease

Calcium is one of the most important intracellular messengers in the brain, being essential for neuronal development, synaptic transmission and plasticity, and the regulation of various metabolic pathways. The findings reviewed in the present article suggest that calcium also plays a prominent role in the pathogenesis of Alzheimer’s disease (AD). Associations between the pathological hallmarks of AD (neurofibrillary tangles [NFT] and amyloid plaques) and perturbed cellular calcium homeostasis have been established in studies of patients, and in animal and cell culture models of AD. Studies of the effects of mutations in the β-amyloid precursor protein (APP) and presenilins on neuronal plasticity and survival have provided insight into the molecular cascades that result in synaptic dysfunction and neuronal degeneration in AD. Central to the neurodegenerative process is the inability of neurons to properly regulate intracellular calcium levels. Increased levels of amyloid β-peptide (Aβ) induce oxidative stress, which impairs cellular ion homeostasis and energy metabolism and renders neurons vulnerable to apoptosis and excitotoxicity. Subtoxic levels of Aβ may induce synaptic dysfunction by impairing multiple signal transduction pathways. Presenilin mutations perturb calcium homeostasis in the endoplasmic reticulum in a way that sensitizes neurons to apoptosis and excitotoxicity; links between aberrant calcium regulation and altered APP processing are emerging. Environmental risk factors for AD are being identified and may include high calorie diets, folic acid insufficiency, and a low level of intellectual activity (bad habits); in each case, the environmental factor impacts on neuronal calcium homeostasis. Low calorie diets and intellectual activity may guard against AD by stimulating production of neurotrophic factors and chaperone proteins. The emerging picture of the cell and molecular biology of AD is revealing novel preventative and therapeutic strategies for eradicating this growing epidemic of the elderly.     

LRP1 modulates APP trafficking along early compartments of the secretory pathway

The amyloid beta peptide (Aβ) is a central player in Alzheimer's disease (AD) pathology. Aβ liberation depends on APP cleavage by β- and γ-secretases. The low density lipoprotein receptor related protein 1 (LRP1) was shown to mediate APP processing at multiple steps. Newly synthesized LRP1 can interact with APP, implying an interaction between these two proteins early in the secretory pathway. We wanted to investigate whether LRP1 mediates APP trafficking along the secretory pathway, and, if so, whether it affects APP processing. Indeed, the early trafficking of APP within the secretory pathway is strongly influenced by its interaction with the C-terminal domain of LRP1. The LRP1-construct expressing an ER-retention motif, LRP-CT KKAA, had the capacity to retard APP traffic to early secretory compartments. In addition, we provide evidence that APP metabolism occurs in close conjunction with LRP1 trafficking, highlighting a new role of lipoprotein receptors in neurodegenerative diseases.     

Toll-like receptor-4 mediates neuronal apoptosis induced by amyloid beta-peptide and the membrane lipid peroxidation product 4-hydroxynonenal

The innate immune system senses the invasion of pathogenic microorganisms and tissue injury through Toll-like receptors (TLR), a mechanism thought to be limited to immune cells. We recently found that neurons express several TLRs, and that the levels of TLR2 and TLR4 are increased in neurons in response to energy deprivation. Here we report that TLR4 expression increases in neurons when exposed to amyloid β-peptide (Aβ1–42) or the lipid peroxidation product 4-hydroxynonenal (HNE). Neuronal apoptosis triggered by Aβ and HNE was mediated by jun N-terminal kinase (JNK); neurons from TLR4 mutant mice exhibited reduced JNK and caspase-3 activation and were protected against apoptosis induced by Aβ and HNE. Levels of TLR4 were decreased in inferior parietal cortex tissue specimens from end-stage AD patients compared to aged-matched control subjects, possibly as the result of loss of neurons expressing TLR4. Our findings suggest that TLR4 signaling increases the vulnerability of neurons to Aβ and oxidative stress in AD, and identify TLR4 as a potential therapeutic target for AD.     

A novel GSK-3β inhibitor reduces Alzheimer's pathology and rescues neuronal loss in vivo

Amyloid deposits, neurofibrillary tangles, and neuronal cell death in selectively vulnerable brain regions are the chief hallmarks in Alzheimer's (AD) brains. Glycogen synthase kinase-3 (GSK-3) is one of the key kinases required for AD-type abnormal hyperphosphorylation of tau, which is believed to be a critical event in neurofibrillary tangle formation. GSK-3 has also been recently implicated in amyloid precursor protein (APP) processing/Aβ production, apoptotic cell death, and learning and memory. Thus, GSK-3 inhibition represents a very attractive drug target in AD and other neurodegenerative disorders. To investigate whether GSK-3 inhibition can reduce amyloid and tau pathologies, neuronal cell death and memory deficits in vivo, double transgenic mice coexpressing human mutant APP and tau were treated with a novel non-ATP competitive GSK-3β inhibitor, NP12. Treatment with this thiadiazolidinone compound resulted in lower levels of tau phosphorylation, decreased amyloid deposition and plaque-associated astrocytic proliferation, protection of neurons in the entorhinal cortex and CA1 hippocampal subfield against cell death, and prevention of memory deficits in this transgenic mouse model. These results show that this novel GSK-3 inhibitor has a dual impact on amyloid and tau alterations and, perhaps even more important, on neuronal survival in vivo further suggesting that GSK-3 is a relevant therapeutic target in AD.     

Unbalanced Regulation of α7 nAChRs by Ly6h and NACHO Contributes to Neurotoxicity in Alzheimer's Disease

α7 nicotinic acetylcholine receptors (nAChRs) are widely expressed in the brain where they promote fast cholinergic synaptic transmission and serve important neuromodulatory functions. However, their high permeability to Ca²⁺ also predisposes them to contribute to disease states. Here, using transfected HEK-tsa cells and primary cultured hippocampal neurons from male and female rats, we demonstrate that two proteins called Ly6h and NACHO compete for access to α7 subunits, operating together but in opposition to maintain α7 assembly and activity within a narrow range that is optimal for neuronal function and viability. Using mixed gender human temporal cortex and cultured hippocampal neurons from rats we further show that this balance is perturbed during Alzheimer''s disease (AD) because of amyloid β (Aβ)-driven reduction in Ly6h, with severe reduction leading to increased phosphorylated tau and α7-mediated neurotoxicity. Ly6h release into human CSF is also correlated with AD severity. Thus, Ly6h links cholinergic signaling, Aβ and phosphorylated tau and may serve as a novel marker for AD progression.SIGNIFICANCE STATEMENT One of the earliest and most persistent hypotheses regarding Alzheimer''s disease (AD) attributes cognitive impairment to loss of cholinergic signaling. More recently, interest has focused on crucial roles for amyloid β (Aβ) and phosphorylated tau in Alzheimer''s pathogenesis. Here, we demonstrate that these elements are linked by Ly6h and its counterpart, NACHO, functioning in opposition to maintain assembly of nicotinic acetylcholine receptors (nAChRs) within the physiological range. Our data suggests that Aβ shifts the balance away from Ly6h and toward NACHO, resulting in increased assembly of Ca²⁺-permeable nAChRs and thus a conversion of basal cholinergic to neurotoxic signaling.     

Co-expression of β-amyloid precursor protein (βAPP) and apolipoprotein E in cell culture: analysis of βAPP processing

Apolipoprotein E (ApoE) is the major genetic risk factor for Alzheimer's disease (AD). The ApoE4 allele is associated with earlier disease onset and greater cerebral deposition of the amyloid beta peptide (Aβ), the major constituent of senile (amyloid) plaques. The molecular mechanism underlying these effects of ApoE4 remains unclear; ApoE alleles could have different influences on Aβ production, extracellular aggregation, or clearance. Because the missense mutations on chromosomes 14 and 21 that cause familial forms of AD appear to lead to increased secretion of Aβ, it is important to determine whether ApoE4 has a similar effect. Here, we have examined the effects of all three ApoE alleles on the processing of βAPP and the secretion of Aβ in intact cells. We established neural (HS683 human glioma) and non-neural (Chinese hamster ovary) cell culture systems that constitutively secrete both ApoE and Aβ at concentrations like those in human cerebrospinal fluid. βAPP metabolites, generated in the presence of each ApoE allele, were analysed and quantified by two methods: immunoprecipitation and phosphorimaging, and ELISA. We detected no consistent allele-specific effects of ApoE on βAPP processing in either cell type. Our data suggest that the higher amyloid burden found in AD subjects expressing ApoE4 is not due to increased amyloidogenic processing of βAPP, in contrast to findings in AD linked to chromosome 14 or 21. These co-expressing cell lines will be useful in the further search for the effects of ApoE on Aβ aggregation or clearance under physiologically relevant conditions.     

Overexpression of wild-type human APP in mice causes cognitive deficits and pathological features unrelated to Aβ levels

Transgenic mice expressing mutant human amyloid precursor protein (APP) develop an age-dependent amyloid pathology and memory deficits, but no overt neuronal loss. Here, in mice overexpressing wild-type human APP (hAPP_wt) we found an early memory impairment, particularly in the water maze and to a lesser extent in the object recognition task, but β-amyloid peptide (Aβ₄₂) was barely detectable in the hippocampus. In these mice, hAPP processing was basically non-amyloidogenic, with high levels of APP carboxy-terminal fragments, C83 and APP intracellular domain. A tau pathology with an early increase in the levels of phosphorylated tau in the hippocampus, a likely consequence of enhanced ERK1/2 activation, was also observed. Furthermore, these mice presented a loss of synapse-associated proteins: PSD95, AMPA and NMDA receptor subunits and phosphorylated CaMKII. Importantly, signs of neurodegeneration were found in the hippocampal CA1 subfield and in the entorhinal cortex that were associated to a marked loss of MAP2 immunoreactivity. Conversely, in mice expressing mutant hAPP, high levels of Aβ₄₂ were found in the hippocampus, but no signs of neurodegeneration were apparent. The results support the notion of Aβ-independent pathogenic pathways in Alzheimer's disease.     

Deposition of Alzheimer’s vascular amyloid-β is associated with decreased expression of brain -3-hydroxyacyl-coenzyme A dehydrogenase (ERAB)

-3-hydroxyacyl-coenzyme A dehydrogenase type II (HADH) was described as an endoplasmic reticulum amyloid β-peptide-binding protein (ERAB), which enhances Aβ toxicity, and accumulates in neurons in Alzheimer’s disease (AD). Hence, HADH/ERAB was suggested to mediate the amyloid-induced neurodegeneration. We estimated the in vivo interactions of HADH and Aβ in an immunocytochemical study of ten Alzheimer’s disease and seven normal brains using five monoclonal HADH-specific antibodies. We found no HADH in amyloid plaques or vascular amyloid. The neuronal expression of HADH was not correlated with the severity of amyloid load in neuropil. HADH was expressed in vascular smooth muscle cells in young and old controls and in amyloid-free blood vessels in AD cases, but little or no HADH was in smooth muscle cells in arteries with amyloid deposits. The putative intracellular interaction between HADH and Aβ in amyloid-producing cells was further studied in vascular smooth muscle cells isolated from brain blood vessels with amyloid-β angiopathy — the cells that were shown previously to accumulate Aβ intracellularly [‘Research advances in Alzheimer’s disease and related disorders’ (1995) 747; Brain Res. 676 (1995) 225; Neurosci. Lett. 183 (1995) 120]. HADH had a mitochondrial localization and did not co-localize with an endoplasmic reticulum marker. Cells that accumulated Aβ were those with low expression of HADH and the proteins did not co-localize. Explanation of the association between low levels of HADH and deposition of Aβ by brain smooth muscle cells requires further studies.     

In vivo Ca2+ imaging of astrocytic microdomains reveals a critical role of the amyloid precursor protein for mitochondria

The investigation of amyloid precursor protein (APP) has been mainly confined to its neuronal functions, whereas very little is known about its physiological role in astrocytes. Astrocytes exhibit a particular morphology with slender extensions protruding from somata and primary branches. Along these fine extensions, spontaneous calcium transients occur in spatially restricted microdomains. Within these microdomains mitochondria are responsible for local energy supply and Ca²⁺ buffering. Using two-photon in vivo Ca²⁺ imaging, we report a significant decrease in the density of active microdomains, frequency of spontaneous Ca²⁺ transients and slower Ca²⁺ kinetics in mice lacking APP. Mechanistically, these changes could be potentially linked to mitochondrial malfunction as our in vivo and in vitro data revealed severe, APP-dependent structural mitochondrial fragmentation in astrocytes. Functionally, such mitochondria exhibited prolonged kinetics and morphology dependent signal size of ATP-induced Ca²⁺ transients. Our results highlight a prominent role of APP in the modulation of Ca²⁺ activity in astrocytic microdomains whose precise functioning is crucial for the reinforcement and modulation of synaptic function. This study provides novel insights in APP physiological functions which are important for the understanding of the effects of drugs validated in Alzheimer's disease treatment that affect the function of APP.     

Amyloidogenic Processing of Amyloid Precursor Protein Drives Stretch-Induced Disruption of Axonal Transport in hiPSC-Derived Neurons

Traumatic brain injury (TBI) results in disrupted brain function following impact from an external force and is a risk factor for sporadic Alzheimer''s disease (AD). Although neurologic symptoms triggered by mild traumatic brain injuries (mTBI), the most common form of TBI, typically resolve rapidly, even an isolated mTBI event can increase the risk to develop AD. Aberrant accumulation of amyloid β peptide (Aβ), a cleaved fragment of amyloid precursor protein (APP), is a key pathologic outcome designating the progression of AD following mTBI and has also been linked to impaired axonal transport. However, relationships among mTBI, amyloidogenesis, and axonal transport remain unclear, in part because of the dearth of human models to study the neuronal response following mTBI. Here, we implemented a custom-microfabricated device to deform neurons derived from human-induced pluripotent stem cells, derived from a cognitively unimpaired male individual, to mimic the mild stretch experienced by neurons during mTBI. Although no cell lethality or cytoskeletal disruptions were observed, mild stretch was sufficient to stimulate rapid amyloidogenic processing of APP. This processing led to abrupt cessation of APP axonal transport and progressive formation of aberrant axonal accumulations that contained APP, its processing machinery, and amyloidogenic fragments. Consistent with this sequence of events, stretch-induced defects were abrogated by reducing amyloidogenesis either pharmacologically or genetically. In sum, we have uncovered a novel and manipulable stretch-induced amyloidogenic pathway directly responsible for APP axonal transport dysregulation. Our findings may help to understand and ultimately mitigate the risk of developing AD following mTBI.SIGNIFICANCE STATEMENT Mild traumatic brain injury is a risk factor for sporadic Alzheimer''s disease (AD). Increased amyloid β peptide generation after injury may drive this risk. Here, by using a custom-built device to impose mild stretch to human neurons, we found that stretch triggers amyloid precursor protein (APP) cleavage, and thus amyloid β peptide generation, consequently disrupting APP axonal transport. Compellingly, protecting APP from cleavage was sufficient to spare axonal transport dysregulation and the consequent aberrant axonal accumulation of APP. Supporting such protective mechanism, the expression of the AD-protective APP^A673T genetic variant conferred protection against stretch-induced APP axonal transport phenotypes. Our data reveal potential subcellular pathways contributing to the development of AD-associated phenotypes following mild traumatic brain injury, and putative strategies for intervening in these pathways.     

Mitochondrial dysfunction and accumulation of the β-secretase-cleaved C-terminal fragment of APP in Alzheimer's disease transgenic mice

Mitochondrial dysfunction is an early feature of Alzheimer's disease (AD) and may play an important role in the pathogenesis of disease. Emerging evidence indicates that amyloid-β (Aβ) peptides enter mitochondria and may thereby disrupt mitochondrial function in brains of AD patients and transgenic model mice. However, it remains to be determined whether the β-cleaved C-terminal fragment (C99), another neurotoxic fragment of amyloid precursor protein (APP), may accumulate in mitochondria of neurons affected by AD. Using immunoblotting, digitonin fractionation and immunofluorescence labeling techniques, we found that C99 is targeted to mitochondria, in particular, to the mitoplast (i.e., inner membrane and matrix compartments) in brains of AD transgenic mice (5XFAD model). Furthermore, full-length APP (fl-APP) was also identified in mitochondrial fractions of 5XFAD mice. Remarkably, partial deletion of the β-site APP-cleaving enzyme 1 (BACE1^+/−) almost completely abolished mitochondrial targeting of C99 and fl-APP in 5XFAD mice at 6 months of age. However, substantial amounts of C99 and fl-APP accumulation remained in mitochondria of 12-month-old BACE1^+/−·5XFAD mouse brains. Consistent with these changes in mitochondrial C99/fl-APP levels, BACE1^+/− deletion age-dependently rescued mitochondrial dysfunction in 5XFAD mice, as assessed by cytochrome c release from mitochondria, reduced redox or complex activities and oxidative DNA damage. Moreover, BACE1^+/− deletion also improved memory deficits as tested by the spontaneous alternation Y-maze task in 5XFAD mice at 6 months but not at 12 months of age. Taken together, our findings suggest that mitochondrial accumulation of C99 and fl-APP may occur through BACE1-dependent mechanisms and contribute to inducing mitochondrial dysfunction and cognitive impairments associated with AD.