High mitochondrial calcium levels precede neuronal death in vivo in Alzheimer’s disease

MITOCHONDRIAL CALCIUM OVERLOAD AFTER PLAQUE DEPOSITION

Ca²⁺ is essential for the normal function of mitochondrial activity, and therefore Ca²⁺ levels are tightly regulated in mitochondria. However, excessive levels of Ca²⁺ in mitochondria, i.e. mitochondrial Ca²⁺ overload, can lead to an increase of reactive oxidative stress (ROS) species, decrease of ATP production and eventually to cell death via apoptosis. Cytosolic Ca²⁺ homeostasis is impaired in AD, as evidenced by hyperactive neurons close to amyloid plaques or high cytosolic Ca²⁺ in transgenic mice with plaque deposition. But, is mitochondrial Ca²⁺ homeostasis also impaired in vivo in AD? Are Ca²⁺ levels increased or diminished in the neurons? In vitro studies suggested that mitochondrial Ca²⁺ homeostasis was altered in the presence of Aβ oligomers (Aβo), but these questions remained unresolved in vivo. To address this issue, we used a transgenic mouse model of cerebral amyloidosis, APP/PS1 – a model that develops amyloid plaques comparable to those from human patients starting at around four months of age – and intravital imaging with multiphoton microscopy. To measure Ca²⁺ concentration in mitochondria, we used a ratiometric Ca²⁺ reporter (Yellow Cameleon, YC) targeted to neuronal mitochondria (hSyn.2mt.YC3.6), that we injected as adenovirus (AAV) in the somatosensory cortex of the mice. This ratiometric reporter, based on Förster resonance energy transfer (FRET), allows for quantitative measurement of absolute Ca²⁺ concentration and direct comparison between cells and compartments. We observed mitochondrial Ca²⁺ overload in the neurons of old APP/PS1 transgenic mice, once plaque deposition had started, unlike the young transgenic mice (pre-pathology). Transgenic mice showed twice as many mitochondria with high Ca²⁺ compared to the wild-type (Wt) mice. This difference was found in both somas and neurites. Other studies have previously reported no changes in cytosolic Ca²⁺ or neuronal function before plaque deposition, supporting the notion that mitochondrial Ca²⁺ overload in neurons follows, rather than precedes, amyloid plaque deposition. Figure 1 shows an example of in vivo multiphoton microscopy images of mitochondria in neurons in APP/PS1 mouse and in Wt mouse after transduction of the AAV. We did not find any correlation between mitochondrial Ca²⁺ overload and the distance to plaques, suggesting that diffusible soluble Aβ species (Aβ oligomers, Aβo), rather than individual plaques, are responsible for the mitochondrial Ca²⁺ increase in the neurons of the transgenic mouse.

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FIGURE 1: In vivo multiphoton microscopy images of mitochondrial Ca2+ in neurons in the Wt and APP/PS1 mice. Mice were injected with AAV.hSyn.2mtYC3.6 and a cranial window was implanted. Mitochondrial Ca2+ was later evaluated with multiphoton microscopy. Pictures are representative of old wild-type (Wt, top) and transgenic mice (APP/PS1 Tg, bottom). The field of view shows the AAV transduction in neuronal mitochondria (green) and the blood vessels labeled with fluorescent Dextran (red). Pseudocolor images represent the color coded mitochondrial Ca2+ concentrations for the corresponding field of view images. Arrow points to a neuron with mitochondrial Ca2+ overload. Scale bar represents 20 μm.

SOLUBLE Aβ OLIGOMERS INCREASE MITOCHONDRIAL CALCIUM IN NEURONS VIA THE MCU

To confirm that soluble Aβ species drive the mitochondrial Ca²⁺ increase in neurons, we topically applied naturally secreted Aβo (obtained from primary cortical neurons isolated from transgenic embryos, referred to as transgenic conditioned media, TgCM) at concentrations similar to those found in the human AD brain directly onto the surface of the living brain of a Wt naïve mouse (C57BL/6J) expressing the adenovirus hSyn.2mtYC3.6. Application of TgCM transiently increased mitochondrial Ca²⁺concentration in the neurons, whereas Wt conditioned media (obtained from Wt littermates) or Aβ-immunodepleted TgCM did not. Neurites were more susceptible to this increase than somas. These results confirmed our previous observations in the APP/PS1 transgenic mouse, and implicated soluble Aβo in the mitochondrial Ca²⁺ dyshomeostasis in vivo. After returning to normal mitochondrial Ca²⁺ levels, we observed that mitochondrial shape and size were altered – mitochondria were smaller and rounded –, suggesting that the toxic effects of Aβo in vivo were not only restricted to mitochondrial Ca²⁺, but also to mitochondrial morphology. Additionally, this suggests that the change in size and shape is a secondary event after a mitochondrial Ca²⁺ increase. It is believed that mitochondrial fragmentation precedes neuronal cell death, which establishes a connection with mitochondrial Ca²⁺. Interestingly, the APP/PS1 transgenic mouse also exhibits fragmented and swollen mitochondria once the pathology is present, and the brains from AD patients show impaired balance of mitochondrial fission and fusion proteins.

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How does Aβ induce mitochondrial Ca²⁺ uptake? And is it possible to inhibit it in order to prevent or decrease the mitochondrial Ca²⁺ overload? The mitochondrial Ca²⁺ uniporter (MCU) complex is the main pathway for mitochondrial Ca²⁺ influx. However, the mechanism by which Aβ induces a Ca²⁺ increase in neuronal mitochondria in vivo was not previously known. Various hypotheses proposed that Aβ could create pores in membranes, form a Ca²⁺-permeable pore itself or act on native channels. In this study, we demonstrated that blocking the MCU complex in vivo with Ru360 – a specific blocker of the channel – prevented the mitochondrial Ca²⁺ uptake elicited by TgCM. To confirm that Ru360 application did not render neurons insensitive to Aβ or blocked the Ca²⁺ influx channels of the plasma membrane, we measured the cytosolic Ca²⁺ influx induced by Aβo with the same Ca²⁺ reporter (YC) targeted to the cytosol of neurons (CBA.YC3.6). We observed that Ru360 did not interfere with the rise in the cytosolic Ca²⁺ induced by Aβ. These results suggest that MCU is required for the increase in mitochondrial Ca²⁺ induced by Aβ in vivo, and points to MCU as a potential target candidate for AD.

HIGH MITOCHONDRIAL CALCIUM PRECEDES NEURONAL DEATH IN VIVO

What are the implications for increased neuronal mitochondrial Ca²⁺ in the in vivo brain? We observed that in the APP/PS1 transgenic mouse, the small fraction of neurons with elevated mitochondrial Ca²⁺ in the soma died within 24 hours. Their nuclei became condensed (a sign of apoptosis) and the AAV fluorescence in the soma was lost. This effect did not happen in cells with normal mitochondrial Ca²⁺ levels. Rapid neuronal death was previously observed in the APP/PS1 model following increased oxidative stress in the cytosol. Our in vitro data showed that, in addition, soluble Aβ triggered opening of the mitochondrial permeability transition pore (mPTP), a decrease of the mitochondrial membrane potential and activation of caspases, events that were secondary to the increase in mitochondrial Ca²⁺. All these findings together link high mitochondrial Ca²⁺, oxidative stress and neuronal cell death via apoptosis in vivo in AD.

MITOCHONDRIAL CALCIUM DYSREGULATION IN THE HUMAN ALZHEIMER’S BRAIN

Lastly, is mitochondrial Ca²⁺ homeostasis impaired in the human Alzheimer’s brain? Because direct analysis of mitochondrial Ca²⁺ levels in the human brain is not feasible, could we evaluate the proteins or channels involved in mitochondrial Ca²⁺ transport in the human brain? And if we can, to what extend are they affected? Using data publicly available at the AMP-AD (Accelerating Medicines Partnership-Alzheimer’s disease) knowledge portal, we evaluated the differential expression of genes encoding proteins involved in the mitochondrial Ca²⁺ transport. Ca²⁺ influx into mitochondria is mainly led by the MCU complex, which includes the MCU pore, the regulatory subunits MICU1 and MICu2, the essential regulator EMRE, and other proteins such as MCUb and MICU3. Ca²⁺ efflux from mitochondria is primarily controlled by the Na⁺/Ca²⁺ exchanger (NCLX). We compared the expression of the genes encoding these proteins between AD subjects and control individuals using microarray and RNA-seq datasets from the MSBB (Mount Sinai Brain Bank) and the ROSMAP (Religious Orders Study and Memory and Aging Project) cohorts. We observed that the MCU and all subunits of the complex – Ca²⁺ influx – were downregulated in AD compared with control subjects, whereas NCLX – Ca²⁺ efflux – was the only one upregulated. These results suggest a dysregulation in the human genes encoding for mitochondrial Ca²⁺ transport, and imply a counteracting effect to avoid mitochondrial Ca²⁺ overload in the neurons in the AD human brain.

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This study helped clarify the understanding of the dynamics of mitochondrial Ca²⁺ in AD. It suggests that Aβ aggregates (particularly soluble Aβ) increase mitochondrial Ca²⁺ in AD via the MCU in vivo, an effect that is linked to neuronal cell death via apoptosis (Figure 2). We demonstrated that the extent of mitochondrial Ca²⁺ alterations are spatially restricted in the living brain and highlight the need for imaging studies that can probe these anatomically specific effects. However, this work highlights other questions that remain unresolved. Does mitochondrial Ca²⁺ elevation occur concomitantly with plaque formation? Is it possible to observe mitochondrial morphological abnormalities and altered dynamics (fission, fusion and transport) induced by Aβ in vivo? Does tau have any effect on mitochondria, both in terms of Ca²⁺ abnormalities and motility? Additionally, our studies point to MCU as a possible therapeutic target for AD and other neurodegenerative diseases in which mitochondrial Ca²⁺ is impaired, however, further efforts will be needed to unveil the clinical relevance of our findings.

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FIGURE 2: Major effects of Aβ aggregates on mitochondrial Ca2+ homeostasis in neurons. In physiological conditions, mitochondria take up Ca2+ from the cytosol via the MCU to produce ATP among other functions. In AD, Aβ aggregates (particularly soluble Aβ) increase mitochondrial Ca2+ via the MCU in vivo. This leads to mitochondrial Ca2+ overload and activates mPTP and caspases, which can trigger neuronal cell death via apoptosis. Blocking the MCU with Ru360 inhibits mitochondrial Ca2+ uptake, pointing to the MCU as a potential target for therapeutics aimed at preventing or reversing the progression of AD.