Novel Arf1 Inhibitors Drive Cancer Stem Cell Aging and Potentiate Anti-Tumor Immunity.

The small G protein Arf1 has been identified as playing a selective role in supporting cancer stem cells (CSCs), making it an attractive target for cancer therapy. However, the current Arf1 inhibitors have limited translational potential due to their high toxicity and low specificity. In this study, two new potent small-molecule inhibitors of Arf1, identified as DU101 and DU102, for cancer therapy are introduced. Preclinical tumor models demonstrate that these inhibitors triggered a cascade of aging in CSCs and enhance anti-tumor immunity in mouse cancer and PDX models. Through single-cell sequencing, the remodeling of the tumor immune microenvironment induced by these new Arf1 inhibitors is analyzed and an increase in tumor-associated CD8+ CD4+ double-positive T (DPT) cells is identified. These DPT cells exhibit superior features of active CD8 single-positive T cells and a higher percentage of TCF1+PD-1+, characteristic of stem-like T cells. The frequency of tumor-infiltrating stem-like DPT cells correlates with better disease-free survival (DFS) in cancer patients, indicating that these inhibitors may offer a novel cancer immunotherapy strategy by converting the cold tumor immune microenvironment into a hot one, thus expanding the potential for immunotherapy in cancer patients.

Erratum: Long noncoding RNAs heat shock RNA omega nucleates TBPH and promotes intestinal stem cell differentiation upon heat shock.

[This corrects the article DOI: 10.1016/j.isci.2024.109732.].

Long noncoding RNAs heat shock RNA omega nucleates TBPH and promotes intestinal stem cell differentiation upon heat shock.

In Drosophila, long noncoding RNA Hsrω rapidly assembles membraneless organelle omega speckles under heat shock with unknown biological function. Here, we identified the distribution of omega speckles in multiple tissues of adult Drosophila melanogaster and found that they were selectively distributed in differentiated enterocytes but not in the intestinal stem cells of the midgut. We mimicked the high expression level of Hsrω via overexpression or intense heat shock and demonstrated that the assembly of omega speckles nucleates TBPH for the induction of ISC differentiation. Additionally, we found that heat shock stress promoted cell differentiation, which is conserved in mammalian cells through paraspeckles, resulting in large puncta of TDP-43 (a homolog of TBPH) with less mobility and the differentiation of human induced pluripotent stem cells. Overall, our findings confirm the role of Hsrω and omega speckles in the development of intestinal cells and provide new prospects for the establishment of stem cell differentiation strategies.

Recording γ-secretase activity in living mouse brains.

γ-Secretase plays a pivotal role in the central nervous system. Our recent development of genetically encoded Forster resonance energy transfer (FRET)-based biosensors has enabled the spatiotemporal recording of γ-secretase activity on a cell-by-cell basis in live neurons in culture. Nevertheless, how γ-secretase activity is regulated in vivo remains unclear. Here we employ the near-infrared (NIR) C99 720-670 biosensor and NIR confocal microscopy to quantitatively record γ-secretase activity in individual neurons in living mouse brains. Intriguingly, we uncovered that γ-secretase activity may influence the activity of γ-secretase in neighboring neurons, suggesting a potential "cell non-autonomous" regulation of γ-secretase in mouse brains. Given that γ-secretase plays critical roles in important biological events and various diseases, our new assay in vivo would become a new platform that enables dissecting the essential roles of γ-secretase in normal health and diseases.

Real-time imaging of mitochondrial redox reveals increased mitochondrial oxidative stress associated with amyloid ß aggregates in vivo in a mouse model of Alzheimer's disease.

BACKGROUND: Reactive oxidative stress is a critical player in the amyloid beta (Aß) toxicity that contributes to neurodegeneration in Alzheimer's disease (AD). Damaged mitochondria are one of the main sources of reactive oxygen species and accumulate in Aß plaque-associated dystrophic neurites in the AD brain. Although Aß causes neuronal mitochondria reactive oxidative stress in vitro, this has never been directly observed in vivo in the living mouse brain. Here, we tested for the first time whether Aß plaques and soluble Aß oligomers induce mitochondrial oxidative stress in surrounding neurons in vivo, and whether this neurotoxic effect can be abrogated using mitochondrial-targeted antioxidants. METHODS: We expressed a genetically encoded fluorescent ratiometric mitochondria-targeted reporter of oxidative stress in mouse models of the disease and performed intravital multiphoton microscopy of neuronal mitochondria and Aß plaques. RESULTS: For the first time, we demonstrated by direct observation in the living mouse brain exacerbated mitochondrial oxidative stress in neurons after both Aß plaque deposition and direct application of soluble oligomeric Aß onto the brain, and determined the most likely pathological sequence of events leading to oxidative stress in vivo. Oxidative stress could be inhibited by both blocking calcium influx into mitochondria and treating with the mitochondria-targeted antioxidant SS31. Remarkably, the latter ameliorated plaque-associated dystrophic neurites without impacting Aß plaque burden. CONCLUSIONS: Considering these results, combination of mitochondria-targeted compounds with other anti-amyloid beta or anti-tau therapies hold promise as neuroprotective drugs for the prevention and/or treatment of AD.

Sleep restoration by optogenetic targeting of GABAergic neurons reprograms microglia and ameliorates pathological phenotypes in an Alzheimer's disease model.

BACKGROUND: Alzheimer's disease (AD) patients exhibit memory disruptions and profound sleep disturbances, including disruption of deep non-rapid eye movement (NREM) sleep. Slow-wave activity (SWA) is a major restorative feature of NREM sleep and is important for memory consolidation. METHODS: We generated a mouse model where GABAergic interneurons could be targeted in the presence of APPswe/PS1dE9 (APP) amyloidosis, APP-GAD-Cre mice. An electroencephalography (EEG) / electromyography (EMG) telemetry system was used to monitor sleep disruptions in these animals. Optogenetic stimulation of GABAergic interneurons in the anterior cortex targeted with channelrhodopsin-2 (ChR2) allowed us to examine the role GABAergic interneurons play in sleep deficits. We also examined the effect of optogenetic stimulation on amyloid plaques, neuronal calcium as well as sleep-dependent memory consolidation. In addition, microglial morphological features and functions were assessed using confocal microscopy and flow cytometry. Finally, we performed sleep deprivation during optogenetic stimulation to investigate whether sleep restoration was necessary to slow AD progression. RESULTS: APP-GAD-Cre mice exhibited impairments in sleep architecture including decreased time spent in NREM sleep, decreased delta power, and increased sleep fragmentation compared to nontransgenic (NTG) NTG-GAD-Cre mice. Optogenetic stimulation of cortical GABAergic interneurons increased SWA and rescued sleep impairments in APP-GAD-Cre animals. Furthermore, it slowed AD progression by reducing amyloid deposition, normalizing neuronal calcium homeostasis, and improving memory function. These changes were accompanied by increased numbers and a morphological transformation of microglia, elevated phagocytic marker expression, and enhanced amyloid ß (Aß) phagocytic activity of microglia. Sleep was necessary for amelioration of pathophysiological phenotypes in APP-GAD-Cre mice. CONCLUSIONS: In summary, our study shows that optogenetic targeting of GABAergic interneurons rescues sleep, which then ameliorates neuropathological as well as behavioral deficits by increasing clearance of Aß by microglia in an AD mouse model.

Arf1 Ablation in Colorectal Cancer Cells Activates a Super Signal Complex in DC to Enhance Anti-Tumor Immunity.

The anti-tumor immune response relies on interactions among tumor cells and immune cells. However, the molecular mechanisms by which tumor cells regulate DCs as well as DCs regulate T cells remain enigmatic. Here, the authors identify a super signaling complex in DCs that mediates the Arf1-ablation-induced anti-tumor immunity. They find that the Arf1-ablated tumor cells release OxLDL, HMGB1, and genomic DNA, which together bound to a coreceptor complex of CD36/TLR2/TLR6 on DC surface. The complex then is internalized into the Rab7-marked endosome in DCs, and further joined by components of the NF-κB, NLRP3 inflammasome and cGAS-STING triple pathways to form a super signal complex for producing different cytokines, which together promote CD8+ T cell tumor infiltration, cross-priming and stemness. Blockage of the HMGB1-gDNA complex or reducing expression in each member of the coreceptors or the cGAS/STING pathway prevents production of the cytokines. Moreover, depletion of the type I IFNs and IL-1ß cytokines abrogate tumor regression in mice bearing the Arf1-ablated tumor cells. These findings reveal a new molecular mechanism by which dying tumor cells releasing several factors to activate the triple pathways in DC for producing multiple cytokines to simultaneously promote DC activation, T cell infiltration, cross-priming and stemness.

Optogenetic targeting of astrocytes restores slow brain rhythm function and slows Alzheimer's disease pathology.

Patients with Alzheimer's disease (AD) exhibit non-rapid eye movement (NREM) sleep disturbances in addition to memory deficits. Disruption of NREM slow waves occurs early in the disease progression and is recapitulated in transgenic mouse models of beta-amyloidosis. However, the mechanisms underlying slow-wave disruptions remain unknown. Because astrocytes contribute to slow-wave activity, we used multiphoton microscopy and optogenetics to investigate whether they contribute to slow-wave disruptions in APP/PS1 mice. The power but not the frequency of astrocytic calcium transients was reduced in APP/PS1 mice compared to nontransgenic controls. Optogenetic activation of astrocytes at the endogenous frequency of slow waves restored slow-wave power, reduced amyloid deposition, prevented neuronal calcium elevations, and improved memory performance. Our findings revealed malfunction of the astrocytic network driving slow-wave disruptions. Thus, targeting astrocytes to restore circuit activity underlying sleep and memory disruptions in AD could ameliorate disease progression.

ARF1 maintains intestinal homeostasis by modulating gut microbiota and stem cell function.

AIMS: The small GTPase protein ARF1 has been shown to be involved in the lipolysis pathway and to selectively kill stem cells in Drosophila melanogaster. However, the role of ARF1 in mammalian intestinal homeostasis remains elusive. This study aimed to explore the role of ARF1 in intestinal epithelial cells (IECs) and reveal the possible mechanism. MATERIALS AND METHODS: IEC-specific ARF1 deletion mouse model was used to evaluate the role of ARF1 in intestine. Immunohistochemistry and immunofluorescence analyses were performed to detect specific cell type markers, and intestinal organoids were cultured to assess intestinal stem cell (ISC) proliferation and differentiation. Fluorescence in situ hybridization, 16S rRNA-seq analysis, and antibiotic treatments were conducted to elucidate the role of gut microbes in ARF1-mediated intestinal function and the underlying mechanism. Colitis was induced in control and ARF1-deficient mice by dextran sulfate sodium (DSS). RNA-seq was performed to elucidate the transcriptomic changes after ARF1 deletion. KEY FINDINGS: ARF1 was essential for ISC proliferation and differentiation. Loss of ARF1 increased susceptibility to DSS-induced colitis and gut microbial dysbiosis. Gut microbiota depletion by antibiotics could rescue the intestinal abnormalities to a certain extent. Furthermore, RNA-seq analysis revealed alterations in multiple metabolic pathways. SIGNIFICANCE: This work is the first to elucidate the essential role of ARF1 in regulating gut homeostasis, and provides novel insights into the pathogenesis of intestinal diseases and potential therapeutic targets.

Optogenetic Targeting of Astrocytes Restores Slow Brain Rhythm Function and Slows Alzheimer's Disease Pathology.

Patients with Alzheimer's disease (AD) exhibit non-rapid eye movement (NREM) sleep disturbances in addition to memory deficits. Disruption of NREM slow waves occurs early in the disease progression and is recapitulated in transgenic mouse models of beta-amyloidosis. However, the mechanisms underlying slow-wave disruptions remain unknown. Because astrocytes contribute to slow-wave activity, we used multiphoton microscopy and optogenetics to investigate whether they contribute to slow-wave disruptions in APP mice. The power but not the frequency of astrocytic calcium transients was reduced in APP mice compared to nontransgenic controls. Optogenetic activation of astrocytes at the endogenous frequency of slow waves restored slow-wave power, reduced amyloid deposition, prevented neuronal calcium elevations, and improved memory performance. Our findings revealed malfunction of the astrocytic network driving slow-wave disruptions. Thus, targeting astrocytes to restore circuit activity underlying sleep and memory disruptions in AD could ameliorate disease progression.

Neuronally Derived Soluble Abeta Evokes Cell-Wide Astrocytic Calcium Dysregulation in Absence of Amyloid Plaques in Vivo.

The key pathologic entities driving the destruction of synaptic function and integrity during the evolution of Alzheimer's disease (AD) remain elusive. Astrocytes are structurally and functionally integrated within synaptic and vascular circuitry and use calcium-based physiology to modulate basal synaptic transmission, vascular dynamics, and neurovascular coupling, which are central to AD pathogenesis. We used high-resolution multiphoton imaging to quantify all endogenous calcium signaling arising spontaneously throughout astrocytic somata, primary processes, fine processes, and capillary endfeet in the brain of awake APP/PS1 transgenic mice (11 male and 6 female mice). Endogenous calcium signaling within capillary endfeet, while surprisingly as active as astrocytic fine processes, was reduced ∼50% in the brain of awake APP/PS1 mice. Cortical astrocytes, in the presence of amyloid plaques in awake APP/PS1 mice, had a cell-wide increase in intracellular calcium associated with an increased frequency, amplitude, and duration of spontaneous calcium signaling. The cell-wide astrocytic calcium dysregulation was not directly related to distance to amyloid plaques. We could re-create the cell-wide intracellular calcium dysregulation in the absence of amyloid plaques following acute exposure to neuronally derived soluble Abeta from Tg2576 transgenic mice, in the living brain of male C57/Bl6 mice. Our findings highlight a role for astrocytic calcium pathophysiology in soluble-Abeta mediated neurodegenerative processes in AD. Additionally, therapeutic strategies aiming to protect astrocytic calcium physiology from soluble Abeta-mediated toxicity may need to pharmacologically enhance calcium signaling within the hypoactive capillary endfeet while reducing the hyperactivity of spontaneous calcium signaling throughout the rest of the astrocyte.SIGNIFICANCE STATEMENT Astrocytic calcium signaling is functionally involved in central pathologic processes of Alzheimer's disease. We quantified endogenous calcium signaling arising spontaneously in the brain of awake APP/PS1 mice, as general anesthesia suppressed astrocytic calcium signaling. Cell-wide astrocytic calcium dysregulation was not related to distance to amyloid plaques but mediated in part by neuronally derived soluble Abeta, supporting a role for astrocytes in soluble-Abeta mediated neurodegeneration. Spontaneous calcium signaling is largely compartmentalized and capillary endfeet were as active as fine processes but hypoactive in the presence of amyloid plaques, while the rest of the astrocyte became hyperactive. The cell-wide calcium pathophysiology in astrocytes may require a combination therapeutic strategy for hypoactive endfeet and astrocytic hyperactivity.

Near-infrared fluorescence lifetime imaging of amyloid-ß aggregates and tau fibrils through the intact skull of mice.

Non-invasive methods for the in vivo detection of hallmarks of Alzheimer's disease can facilitate the study of the progression of the disease in mouse models and may enable its earlier diagnosis in humans. Here we show that the zwitterionic heptamethine fluorophore ZW800-1C, which has peak excitation and emission wavelengths in the near-infrared optical window, binds in vivo and at high contrast to amyloid-ß deposits and to neurofibrillary tangles, and allows for the microscopic imaging of amyloid-ß and tau aggregates through the intact skull of mice. In transgenic mouse models of Alzheimer's disease, we compare the performance of ZW800-1C with that of the two spectrally similar heptamethine fluorophores ZW800-1A and indocyanine green, and show that ZW800-1C undergoes a longer fluorescence-lifetime shift when bound to amyloid-ß and tau aggregates than when circulating in blood vessels. ZW800-1C may prove advantageous for tracking the proteinic aggregates in rodent models of amyloid-ß and tau pathologies.

Spontaneous vasomotion propagates along pial arterioles in the awake mouse brain like stimulus-evoked vascular reactivity.

Sensory stimulation evokes a local, vasodilation-mediated blood flow increase to the activated brain region, which is referred to as functional hyperemia. Spontaneous vasomotion is a change in arteriolar diameter that occurs without sensory stimulation, at low frequency (∼0.1 Hz). These vessel diameter changes are a driving force for perivascular soluble waste clearance, the failure of which has been implicated in neurodegenerative disease. Stimulus-evoked vascular reactivity is known to propagate along penetrating arterioles to pial arterioles, but it is unclear whether spontaneous vasomotion propagates similarly. We therefore imaged both stimulus-evoked and spontaneous changes in pial arteriole diameter in awake, head-fixed mice with 2-photon microscopy. By cross-correlating different regions of interest (ROIs) along the length of imaged arterioles, we assessed vasomotion propagation. We found that both during rest and during visual stimulation, one-third of the arterioles showed significant propagation (i.e., a wave), with a median (interquartile range) wave speed of 405 (323) µm/s at rest and 345 (177) µm/s during stimulation. In a second group of mice, with GCaMP expression in their vascular smooth muscle cells, we also found spontaneous propagation of calcium signaling along pial arterioles. In summary, we demonstrate that spontaneous vasomotion propagates along pial arterioles like stimulus-evoked vascular reactivity.

A neuron-immune circuit regulates neurodegeneration in the hindbrain and spinal cord of Arf1-ablated mice.

Neuroimmune connections have been revealed to play a central role in neurodegenerative diseases (NDs). However, the mechanisms that link the central nervous system (CNS) and peripheral immune cells are still mostly unknown. We recently found that specific ablation of the Arf1 gene in hindbrain and spinal cord neurons promoted NDs through activating the NLRP3 inflammasome in microglia via peroxided lipids and adenosine triphosphate (ATP) releasing. Here, we demonstrate that IL-1ß with elevated chemokines in the neuronal Arf1-ablated mouse hindbrain and spinal cord recruited and activated γδ T cells in meninges. The activated γδ T cells then secreted IFN-γ that entered into parenchyma to activate the microglia-A1 astrocyte-C3-neuronal C3aR neurotoxic pathway. Remarkably, the neurodegenerative phenotypes of the neuronal Arf1-ablated mice were strongly ameliorated by IFN-γ or C3 knockout. Finally, we show that the Arf1-reduction-induced neuroimmune-IFN-γ-gliosis pathway exists in human NDs, particularly in amyotrophic lateral sclerosis and multiple sclerosis. Together, our results uncover a previously unknown mechanism that links the CNS and peripheral immune cells to promote neurodegeneration.

Reduced excitatory neuron activity and interneuron-type-specific deficits in a mouse model of Alzheimer's disease.

Alzheimer's disease (AD) is characterized by progressive memory loss and cognitive decline. These impairments correlate with early alterations in neuronal network activity in AD patients. Disruptions in the activity of individual neurons have been reported in mouse models of amyloidosis. However, the impact of amyloid pathology on the spontaneous activity of distinct neuronal types remains unexplored in vivo. Here we use in vivo calcium imaging with multiphoton microscopy to monitor and compare the activity of excitatory and two types of inhibitory interneurons in the cortices of APP/PS1 and control mice under isoflurane anesthesia. We also determine the relationship between amyloid accumulation and the deficits in spontaneous activity in APP/PS1 mice. We show that somatostatin-expressing (SOM) interneurons are hyperactive, while parvalbumin-expressing interneurons are hypoactive in APP/PS1 mice. Only SOM interneuron hyperactivity correlated with proximity to amyloid plaque. These inhibitory deficits were accompanied by decreased excitatory neuron activity in APP/PS1 mice. Our study identifies cell-specific neuronal firing deficits in APP/PS1 mice driven by amyloid pathology. These findings highlight the importance of addressing the complexity of neuron-specific deficits to ameliorate circuit dysfunction in Alzheimer's disease.

Disruption of the lipolysis pathway results in stem cell death through a sterile immunity-like pathway in adult Drosophila.

We previously showed that the Arf1-mediated lipolysis pathway sustains stem cells and cancer stem cells (CSCs); its ablation resulted in necrosis of stem cells and CSCs, which further triggers a systemic antitumor immune response. Here we show that knocking down Arf1 in intestinal stem cells (ISCs) causes metabolic stress, which promotes the expression and translocation of ISC-produced damage-associated molecular patterns (DAMPs; Pretaporter [Prtp] and calreticulin [Calr]). DAMPs regulate macroglobulin complement-related (Mcr) expression and secretion. The secreted Mcr influences the expression and localization of enterocyte (EC)-produced Draper (Drpr) and LRP1 receptors (pattern recognition receptors [PRRs]) to activate autophagy in ECs for ATP production. The secreted ATP possibly feeds back to kill ISCs by activating inflammasome-like pyroptosis. We identify an evolutionarily conserved pathway that sustains stem cells and CSCs, and its ablation results in an immunogenic cascade that promotes death of stem cells and CSCs as well as antitumor immunity.

An MST4-pß-CateninThr40 Signaling Axis Controls Intestinal Stem Cell and Tumorigenesis.

Elevated Wnt/ß-catenin signaling has been commonly associated with tumorigenesis especially colorectal cancer (CRC). Here, an MST4-pß-cateninThr40 signaling axis essential for intestinal stem cell (ISC) homeostasis and CRC development is uncovered. In response to Wnt3a stimulation, the kinase MST4 directly phosphorylates ß-catenin at Thr40 to block its Ser33 phosphorylation by GSK3ß. Thus, MST4 mediates an active process that prevents ß-catenin from binding to and being degraded by ß-TrCP, leading to accumulation and full activation of ß-catenin. Depletion of MST4 causes loss of ISCs and inhibits CRC growth. Mice bearing either MST4T178E mutation with constitutive kinase activity or ß-cateninT40D mutation mimicking MST4-mediated phosphorylation show overly increased ISCs/CSCs and exacerbates CRC. Furthermore, the MST4-pß-cateninThr40 axis is upregulated and correlated with poor prognosis of human CRC. Collectively, this work establishes a previously undefined machinery for ß-catenin activation, and further reveals its function in stem cell and tumor biology, opening new opportunities for targeted therapy of CRC.

Neuronal accumulation of peroxidated lipids promotes demyelination and neurodegeneration through the activation of the microglial NLRP3 inflammasome.

Peroxidated lipids accumulate in the presence of reactive oxygen species and are linked to neurodegenerative diseases. Here we find that neuronal ablation of ARF1, a small GTPase important for lipid homeostasis, promoted accumulation of peroxidated lipids, lipid droplets and ATP in the mouse brain and led to neuroinflammation, demyelination and neurodegeneration, mainly in the spinal cord and hindbrain. Ablation of ARF1 in cultured primary neurons led to an increase in peroxidated lipids in co-cultured microglia, activation of the microglial NLRP3 inflammasome and release of inflammatory cytokines in an Apolipoprotein E-dependent manner. Deleting the Nlrp3 gene rescued the neurodegenerative phenotypes in the neuronal Arf1-ablated mice. We also observed a reduction in ARF1 in human brain tissue from patients with amyotrophic lateral sclerosis and multiple sclerosis. Together, our results uncover a previously unrecognized role of peroxidated lipids released from damaged neurons in activation of a neurotoxic microglial NLRP3 pathway that may play a role in human neurodegeneration.

A Novel NIR-FRET Biosensor for Reporting PS/γ-Secretase Activity in Live Cells.

Presenilin (PS)/γ-secretase plays a pivotal role in essential cellular events via proteolytic processing of transmembrane proteins that include APP and Notch receptors. However, how PS/γ-secretase activity is spatiotemporally regulated by other molecular and cellular factors and how the changes in PS/γ-secretase activity influence signaling pathways in live cells are poorly understood. These questions could be addressed by engineering a new tool that enables multiplexed imaging of PS/γ-secretase activity and additional cellular events in real-time. Here, we report the development of a near-infrared (NIR) FRET-based PS/γ-secretase biosensor, C99 720-670 probe, which incorporates an immediate PS/γ-secretase substrate APP C99 with miRFP670 and miRFP720 as the donor and acceptor fluorescent proteins, respectively. Extensive validation demonstrates that the C99 720-670 biosensor enables quantitative monitoring of endogenous PS/γ-secretase activity on a cell-by-cell basis in live cells (720/670 ratio: 2.47 ± 0.66 (vehicle) vs. 3.02 ± 1.17 (DAPT), ** p < 0.01). Importantly, the C99 720-670 and the previously developed APP C99 YPet-Turquoise-GL (C99 Y-T) biosensors simultaneously report PS/γ-secretase activity. This evidences the compatibility of the C99 720-670 biosensor with cyan (CFP)-yellow fluorescent protein (YFP)-based FRET biosensors for reporting other essential cellular events. Multiplexed imaging using the novel NIR biosensor C99 720-670 would open a new avenue to better understand the regulation and consequences of changes in PS/γ-secretase activity.

Increased mitochondrial calcium levels associated with neuronal death in a mouse model of Alzheimer's disease.

Mitochondria contribute to shape intraneuronal Ca2+ signals. Excessive Ca2+ taken up by mitochondria could lead to cell death. Amyloid beta (Aß) causes cytosolic Ca2+ overload, but the effects of Aß on mitochondrial Ca2+ levels in Alzheimer's disease (AD) remain unclear. Using a ratiometric Ca2+ indicator targeted to neuronal mitochondria and intravital multiphoton microscopy, we find increased mitochondrial Ca2+ levels associated with plaque deposition and neuronal death in a transgenic mouse model of cerebral ß-amyloidosis. Naturally secreted soluble Aß applied onto the healthy brain increases Ca2+ concentration in mitochondria, which is prevented by blockage of the mitochondrial calcium uniporter. RNA-sequencing from post-mortem AD human brains shows downregulation in the expression of mitochondrial influx Ca2+ transporter genes, but upregulation in the genes related to mitochondrial Ca2+ efflux pathways, suggesting a counteracting effect to avoid Ca2+ overload. We propose lowering neuronal mitochondrial Ca2+ by inhibiting the mitochondrial Ca2+ uniporter as a novel potential therapeutic target against AD.