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Accumulation of amyloid β-peptide (Aβ) is a driver of Alzheimer’s disease (AD). Amyloid precursor protein (App) knock-in mouse models recapitulate AD-associated Aβ pathology, allowing elucidation of downstream effects of Aβ accumulation and their temporal appearance upon disease progression. Here we have investigated the sequential onset of AD-like pathologies in AppNL-Fand AppNL-G-Fknock-in mice by time-course transcriptome analysis of hippocampus, a region severely affected in AD. Strikingly, energy metabolism emerged as one of the most significantly altered pathways already at an early stage of pathology. Functional experiments in isolated mitochondria from hippocampus of both AppNL-Fand AppNL-G-Fmice confirmed an upregulation of oxidative phosphorylation driven by the activity of mitochondrial complexes I, IV and V, associated with higher susceptibility to oxidative damage and Ca2+-overload. Upon increasing pathologies, the brain shifts to a state of hypometabolism with reduced abundancy of mitochondria in presynaptic terminals. These late-stage mice also displayed enlarged presynaptic areas associated with abnormal accumulation of synaptic vesicles and autophagosomes, the latter ultimately leading to local autophagy impairment in the synapses. In summary, we report that Aβ-induced pathways in Appknock-in mouse models recapitulate key pathologies observed in AD brain, and our data herein adds a comprehensive understanding of the pathologies including dysregulated metabolism and synapses and their timewise appearance to find new therapeutic approaches for AD.

Neurodegenerative diseases are a spectrum of chronic, debilitating disorders characterised by the progressive degeneration and death of neurons. Mitochondrial dysfunction has been implicated in most neurodegenerative diseases, but in many instances it is unclear whether such dysfunction is a cause or an effect of the underlying pathology, and whether it represents a viable therapeutic target. It is therefore imperative to utilise and optimise cellular models and experimental techniques appropriate to determine the contribution of mitochondrial dysfunction to neurodegenerative disease phenotypes. In this consensus article, we collate details on and discuss pitfalls of existing experimental approaches to assess mitochondrial function in in vitro cellular models of neurodegenerative diseases, including specific protocols for the measurement of oxygen consumption rate in primary neuron cultures, and single-neuron, time-lapse fluorescence imaging of the mitochondrial membrane potential and mitochondrial NAD(P)H. As part of the Cellular Bioenergetics of Neurodegenerative Diseases (CeBioND) consortium (www.cebiond.org), we are performing cross-disease analyses to identify common and distinct molecular mechanisms involved in mitochondrial bioenergetic dysfunction in cellular models of Alzheimer’s, Parkinson’s, and Huntington’s diseases. Here we provide detailed guidelines and protocols as standardised across the five collaborating laboratories of the CeBioND consortium, with additional contributions from other experts in the field.

We studied effects of the familial Alzheimer's disease presenilin 1 (PS1) exon 9 deletion (PS1-ΔE9) mutation on basal and carbachol-stimulated phosphoinositide (PI) hydrolysis and intracellular Ca2+concentrations ([Ca2+]i) in human SH-SY5Y neuroblastoma cells. We demonstrate that PS1-ΔE9 cells have an enhanced basal PI hydrolysis and [Ca2+]ias compared with both wild type PS1 (PS1-WT) and nontransfected (NT) cells. Both were reversed by the phospholipase C (PLC) inhibitor neomycin. The PS1-ΔE9-related high basal [Ca2+]iwas also reversed by xestospongin C confirming that this effect was inositol trisphosphate receptor-mediated. Carbachol gave a greater stimulation of [Ca2+]iin PS1-ΔE9 cells that took longer to return to basal as compared with responses seen in NT and PS1-WT cells. This long tail-off effect seen in PS1-ΔE9 cells after carbachol stimulation was reversed by xestospongin C and dantrolene, suggesting that it was mediated by inositol trisphosphate receptor and ryanodine receptor amplification of Ca2+. Ruthenium red only reduced carbachol peak elevations of [Ca2+]iin NT and PS1-WT cells and not in PS1-ΔE9 cells. No significant between cell type differences were seen for basal and carbachol-stimulated [Ca2+]iwith either ryanodine or the endoplasmic reticulum Ca2+ATPase inhibitor cyclopiazonic acid. Immunostaining experiments revealed that for all the cell types PS1 is present at the plasma membrane and co-localizes with N-cadherin, a component of the cell-cell adhesion complex. Immunoblotting of cell extracts for PLC-β1 showed that, compared with NT and PS1-WT cells, the PS1-ΔE9 transfectants gave a relative increase in levels of the calpain generated N-terminal fragment (100 kDa) over full-length (150 kDa) PLC-β1. Our results suggest that the PS1-ΔE9 mutation causes upstream changes in PI signaling with enhanced basal PLC activity as a primary effect that leads to a higher [Ca2+]i. This may provide a novel mechanism by which the PS1-ΔE9 mutation sensitizes cells to apoptotic stimuli and enhanced amyloid β generation.

Mutations in the amyloid precursor protein, presenilin 1 and presenilin 2 genes are believed to cause early onset familial Alzheimer's disease by increasing the production of more amyloidogenic forms of the β-amyloid peptide (so called Aβ42). However, the phenotypes of familial Alzheimer's disease mutations are more complex than can be explained only by effects on amyloid precursor protein processing and β-amyloid production. This review summarises alternative ideas suggesting that familial Alzheimer's disease mutations cause disease by altering cellular signalling systems to increase the vulnerability of neurons to die by apoptosis.

Apoptotic machinery designed for cell's organized self‐destruction involve different systems of proteases which cleave vital proteins and disassemble nuclear and cytoplasmic structures, committing the cell to death. The most studied apoptotic proteolytic system is the caspase family, but calpains and the proteasome could play important roles as well. Alzheimer's disease associated presenilins showed to be a substrate for such proteolytic systems, being processed early in several apoptotic models, and recent data suggest that alternative presenilin fragments could regulate cell survival. Mutations in genes encoding presenilins proved to sensitize neurons to apoptosis by different mechanisms e.g. increased caspase‐3 activation, oxyradicals production and calcium signaling dysregulation. Here we review the data involving presenilins in apoptosis and discuss a possible role of presenilins in the regulation of apoptotic biochemical machinery.

Cytokine production during type I insulin-dependent diabetes mellitus has been linked to alterations in β-cell function such as inhibition of glucose-stimulated insulin secretion. This and other adverse effects of cytokines, including interleukin-1β (IL-1β) involve the induction of nitric oxide synthase, with production of nitric oxide. Here, we show that IL-1β induces apoptosis in a pancreatic β-cell line, RINm5F cells. Cells treated with IL-1β underwent DNA fragmentation, nuclear condensation, and apoptotic body formation. The production of nitric oxide preceded the appearance of these typical features of apoptosis. Inhibition of the nitric oxide synthase activity by NG-monomethyl-L-arginine prevented IL-1β-induced nitric oxide generation and apoptotic cell killing. These results show that—besides the known alterations in β-cell function—IL-1β-induced nitric oxide production activates the cell death program.

We have previously reported that glutamate can trigger a succession of necrosis and apoptosis in cerebellar granule cells (CGC). Since specific blockers of the N- methyl- d- aspartate(NMDA) receptor channel prevented both types of cell death, the role of Ca 2+-dependent processes in the initiation of glutamate toxicity was further investigated. We examined the possible involvement of mitochondria and the role of the Ca 2+/calmodulin-regulated protein phosphatase, calcineurin, in the development of either type of cell death. Cyclosporin A and the more selective calcineurin inhibitor, FK-506, prevented the development of both early necrosis and delayed apoptosis. In addition, cyclosporin A prevented the collapse of mitochondrial membrane potential observed during the exposure to glutamate and the concomitant necrotic phase. When CsA was added immediately after glutamate removal, it also prevented delayed apoptosis of neurons that had survived the necrotic phase. Altogether, these results suggest the involvement of calcineurin and a role for mitochondrial deenergization as early signals in neuronal apoptosis induced by glutamate.

Various forms of cell death induced by the glutamate receptor agonist, α-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA), were analyzed by determining the capacity of cultured cerebellar granule cells to metabolize 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) into formazan, by measuring the leakage of lactate dehydrogenase (LDH), by using confocal microscopy to visualize propidium iodide staining of apoptotic nuclei, and by using field inversion gel electrophoresis (FIGE) for the detection of AMPA-produced cleavage of DNA into high molecular-weight fragments (50 kbp). All these measures indicated that stimulation of AMPA receptors may be involved in the neurotoxic effects of glutamate, and that AMPA-induced neurotoxicity in cerebellar granule cells display morphologically distinct features of both necrotic and apoptotic modes of cell death. In agreement with previous observations, a blockade of AMPA receptor desensitization was necessary to unmask AMPA-induced functional responses in cultured cerebellar granule neurons in vitro. Microfluorimetric measurements of free cytoplasmic calcium concentrations ([Ca2+]i) in single cerebellar neurons revealed that AMPA neurotoxicity was accompanied by a pronounced elevation of [Ca2+]i. Our current results add further evidence to the notion that glutamate-induced neurotoxicity in cerebellar granule cells is mediated not only through NMDA receptors but also through a direct activation of AMPA receptor-regulated cation channels.

DURING ischaemic brain injury, glutamate accumulation with overstimulation of postsynaptic glutamate receptors and intracellular Ca2+overload lead to neuronal death. We have shown previously that delayed neuronal death in cultures of cerebellar granule cells (CGCs) exposed to glutamate occurs by apoptosis. Here, we report that lamin cleavage and dissolution of the microtubule network precede chromatin fragmentation in glutamate-induced CGC apoptosis. Like other events that characterize excitotoxic cell death, degradation of lamins, β-tubulin and disruption of microtubule architecture is inhibited by the NMDA-receptor antagonist MK-801. Our findings suggest that cleavage of key cytoskeletal elements is an important step in glutamate- induced neuronal apoptosis.

Nitric oxide (NO) is a diffusible messenger involved in several patho‐physiological processes including immune‐mediated cytotoxicity and neural cell killing. NO or the products of its redox chemistry can cause DNA damage and activate subsequent lethal reactions including energy depletion and cell necrosis. However, regardless of whether it is endogenously produced in response to cytokines, or generated by chemical breakdown of donor molecules, NO can also induce apoptosis in different systems. Here, we report that NO generation in response to a cytokine induced NO‐synthase or by NO donors stimulates the expression of the tumor suppressor gene, p53, in RAW 264.7 macrophages or pancreatic RINm5F cells prior to apoptosis. NO‐synthase inhibitors such as NG‐monomethyl‐l‐arginine prevent the inducible NO generation as well as p53 expression and apoptosis. Since p53 expression is linked to apoptosis in some cells exposed to DNA damaging agents, we suggest that NO‐induced apoptosis in these cell systems is the consequence of DNA damage and subsequent expression of this tumor suppressor gene.

To investigate the involvement of proteases in apoptosis, rat thymocytes were treated with the glucocorticoid hormone methylprednisolone or the topoisomerase II inhibitor etoposide in the presence of selective substrate inhibitors of either interleukin-1β-converting enzyme (ICE), (Z-Val-Ala-Asp-chloromethylketone, VADcmk) or Ca²⁺-regulated serine protease (Suc-Ala-Ala-Pro-Phe-chloromethylketone, AAPFcmk). VADcmk protected from lamin proteolysis, chromatin fragmentation, cell shrinkage, and formation of apoptotic nuclei in both methylprednisolone- and etoposide-treated thymocytes when present during the initiation of the apoptotic process. AAPFcmk prevented lamin breakdown, chromatin fragmentation, and apoptotic morphological changes in thymocytes treated with methylprednisolone, but not with etoposide. Both MPS- and etoposide-treated thymocytes exhibited enhanced ICE-like protease activity which was maximal 1 h after treatment. This increase in proteolytic activity was blocked by VADcmk, but not AAPFcmk. Our findings suggest that ICE-like protease activity is critically involved in the early phase of both methylprednisolone- and etoposide-induced apoptosis in thymocytes, whereas the Ca²⁺-regulated serine protease is an obligatory component of the proteolytic cascade in methylprednisolone-induced apoptosis. Copyright 1995, 1999 Academic Press

Nitric oxide (NO) is a diffusible messenger involved in several patho-physiological processes including immune-mediated cytotoxicity and neural cell killing. NO or the products of its redox chemistry can cause DNA damage and activate subsequent lethal reactions including energy depletion and cell necrosis. However, regardless of whether it is endogenously produced in response to cytokines, or generated by chemical breakdown of donor molecules, NO can also induce apoptosis in different systems. Here, we report that NO generation in response to a cytokine induced NO-synthase or by NO donors stimulates the expression of the tumor suppressor gene, p53, in RAW 264.7 macrophages or pancreatic RINm5F cells prior to apoptosis. NO-synthase inhibitors such as NG-monomethyl- l-arginine prevent the inducible NO generation as well as p53 expression and apoptosis. Since p53 expression is linked to apoptosis in some cells exposed to DNA damaging agents, we suggest that NO-induced apoptosis in these cell systems is the consequence of DNA damage and subsequent expression of this tumor suppressor gene.

We have previously reported that glutamate can trigger a succession of necrosis and apoptosis in cerebellar granule cells (CGC). Since specific blockers of the (NMDA) receptor channel prevented both types of cell death, the role of Ca2+‐dependent processes in the initiation of glutamate toxicity was further investigated. We examined the possible involvement of mitochondria and the role of the Ca2+/calmodulin‐regulated protein phosphatase, calcineurin, in the development of either type of cell death. Cyclosporin A and the more selective calcineurin inhibitor, FK‐506, prevented the development of both early necrosis and delayed apoptosis. In addition, cyclosporin A prevented the collapse of mitochondrial membrane potential observed during the exposure to glutamate and the concomitant necrotic phase. When CsA was added immediately after glutamate removal, it also prevented delayed apoptosis of neurons that had survived the necrotic phase. Altogether, these results suggest the involvement of calcineurin and a role for mitochondrial deenergization as early signals in neuronal apoptosis induced by glutamate.