Alzheimer's disease is the most common type of dementia. A hallmark of this condition is progressive loss of memory, accompanied by a buildup of hard clumps of protein between the brain cells. These protein clumps, known as amyloid plaques, are a key focus of research into Alzheimer's disease. They are likely to be toxic to brain cells, but their role in the development and progression of the disease is not yet known.

Though the cause of Alzheimer's disease remains unclear, an inherited form of the disease may hold some clues. Mutations in genes for proteins called presenilins cause an earlier onset form of Alzheimer's disease, in which symptoms can develop in people who are in their 40s or 50s. The presenilin proteins appear in a cell structure called the endoplasmic reticulum, which plays many roles in the normal activities of a cell. Among other things, this structure stores and releases calcium ions, and cells use these ions to send and process many signals.

The cell's energy-producing powerhouses, the mitochondria, use calcium to boost their metabolic activity. This allows them to make more energy for the cell, but in the process they also make damaging byproducts. These byproducts include oxygen-containing chemicals, known as reactive oxygen species (ROS), which react strongly with other molecules. While low levels of ROS are a normal part of cell activity, if the levels get too high, these chemicals can attack and damage structures within the cell.

Untangling the effects of amyloid plaques and presenilins on brain cells in humans is challenging. But, a nematode worm called Caenorhabditis elegans does not form plaques, making it possible to look at presenilins on their own. Previous work in these worms has shown that presenilin mutations affect the endoplasmic reticulum and change the appearance of mitochondria. Here, Sarasija et al. extend this work to find out more about the effects presenilin mutations have on living cells.

Presenilin mutations in young adult worms increased the amount of calcium released by the endoplasmic reticulum. This increased the activity of the mitochondria and caused ROS levels to rise to damaging levels. This caused stress inside the cells, and the worms started to show early signs damage to their nervous systems. Mutations that decreased the movement of calcium from the endoplasmic reticulum to the mitochondria helped to prevent the damage. Treating the mitochondria with antioxidants to mop up the extra ROS also protected the cells.

This kind of damage to brain cells did not depend on amyloid plaques. Whilst the plaques are likely to be toxic, these new findings highlights the role that other chemical and biological processes might play in Alzheimer's disease. Further work to reveal the underlying cause of Alzheimer's disease may lead to new therapies to treat this condition in the future.

In all metazoans, mitochondria are essential organelles and mitochondrial dysfunction is frequently observed in neurodegenerative diseases and aging. While mitochondria have a well-established role in ATP generation, they also have a vital role in Ca²⁺ homeostasis. Several studies have shown that release of endoplasmic reticulum (ER) Ca²⁺ results in the elevation of mitochondrial Ca²⁺ levels which in turn stimulates metabolic activity of the mitochondria (Das and Harris, 1990; Glancy and Balaban, 2012; Hansford and Zorov, 1998; McCormack and Denton, 1993; Mildaziene et al., 1995; Wernette et al., 1981). Thus, insults or deregulated signaling between the ER and mitochondria can cause mitochondrial dysfunction and affect cellular fitness.

Alzheimer’s disease (AD) is the leading cause of dementia in the elderly and accounts for between 60–80% of all cases of dementia. Familial Alzheimer’s disease (FAD) is a subset of AD where there is a genetic predisposition to the disease as a result of mutations predominantly in the presenilin genes, PSEN1 and PSEN2 (ALZFORUM, 2016). PSENs are ~50 kDa multipass transmembrane proteins that primarily localize to the ER (Bezprozvanny and Mattson, 2008) and are enriched in the ER membrane associated with mitochondria (Area-Gomez et al., 2009). Notably, PSENs are the critical aspartyl protease component of the gamma-secretase complex, a multi-subunit protease that resides in cellular membranes. Gamma-secretase is involved in the cleavage of amyloid precursor protein (APP) into beta amyloid (Abeta) peptides, and in the cleavage and activation of several other transmembrane proteins, including Notch (Beel and Sanders, 2008). Dominating the study of AD pathogenesis is the amyloid hypothesis which ascertains that altered PSEN function leads to an increase in the toxic Abeta42 peptide, and the eventual accumulation of amyloid plaques in the brain leads to neurodegeneration and dementia observed in AD patients (Hardy, 2006). However, it is noteworthy that while amyloid plaques serve as a histopathological hallmark of AD, there is no correlation between plaque load and the severity of dementia, and postmortem analyses of some AD patients with exaggerated cognitive decline have shown a lack of plaque formation (Giannakopoulos et al., 2003; Guillozet et al., 2003; Terry et al., 1991). Also, using neuroimaging techniques, extensive plaque formation has been observed in people with no cognitive impairment (Nordberg, 2008; Villemagne et al., 2008). Concomitantly, there is a large body of work that implicates exacerbated ER Ca²⁺ release as the causative agent in AD pathogenesis (Bandara et al., 2013; Chan et al., 2000; Cheung et al., 2008; Green et al., 2008; Leissring et al., 1999; Stutzmann et al., 2004; Tu et al., 2006). Moreover, there is evidence of enhanced ER-mitochondria crosstalk in cells from FAD patients with mutations in PSEN1, PSEN2, and APP, as well as patients with sporadic AD (Area-Gomez et al., 2012) and enhanced ER to mitochondrial Ca²⁺ transfer is observed in cells expressing FAD-mutant PSEN2 (Zampese et al., 2011). Additionally, reactive oxygen-mediated oxidative damage has been observed in brains of AD patients (Lovell and Markesbery, 2007; Lovell et al., 2011) and an increase in hydrogen peroxide levels is observed in AD mice models even prior to the appearance of plaques (Manczak et al., 2006). However, despite decades of research, a clear connection between these seemingly disparate observations does not exist that explains the pathogenesis of AD.

In C. elegans, disruption of the PSEN ortholog, SEL-12, leads to ER Ca²⁺ dysregulation that causes mitochondrial disorganization and reduced organismal health (Sarasija and Norman, 2015). Here, we examine whether these mitochondrial defects observed in sel-12 mutants lead to mitochondrial metabolic defects that can result in neuronal dysfunction. From these analyses, we find that sel-12 mutations result in increased mitochondrial Ca²⁺ concentration, accelerated oxidative phosphorylation (OXPHOS), elevated reactive oxygen species (ROS) generation and oxidative stress mediated neurodegeneration. Remarkably, we demonstrate that reducing ER to mitochondrial Ca²⁺ transfer in sel-12 mutants can normalize mitochondrial Ca²⁺ levels and function, reduce the levels of ROS and suppress neurodegeneration. Moreover, we demonstrate similar mitochondrial dysfunction in fibroblasts isolated from FAD patients. Lastly, we show that treating sel-12 mutants with a mitochondria-targeted antioxidant suppresses the neurodegenerative phenotypes observed in sel-12 mutants. Since C. elegans do not encode an Abeta peptide (Daigle and Li, 1993; McColl et al., 2012), our data indicates that neurodegeneration occurs in presenilin mutants by faulty ER to mitochondria Ca²⁺ transfer leading to mitochondrial metabolic dysfunction, completely independent of Abeta signaling, and consummates in the rise of mitochondrial ROS generation to detrimental levels. Therefore, our study provides a comprehensive delineation of AD pathogenesis in an intact animal model and its importance is underscored by the fact that we provide evidence that Abeta signaling does not appear necessary for neurodegeneration.

Mutations in the genes encoding PSEN1 and PSEN2 are the most frequent cause of FAD. Despite the identification of PSEN involvement in AD over 20 years ago, the mechanism causing neurodegeneration in FAD patients has remained elusive. In this study, we provide evidence that mutations in the gene encoding a C. elegans PSEN homolog, sel-12 result in ER to mitochondria Ca²⁺ signaling defects, elevated mitochondrial Ca²⁺ levels, mitochondrial structural disorganization, dysfunction of mitochondrial metabolism and subsequent oxidative stress mediated neurodegeneration. We also demonstrate that the structural and functional neurodegeneration observed in sel-12 mutants are independent of the gamma-secretase activity of SEL-12 and are a result of mitochondria generated superoxide that arise due to increased ER to mitochondria Ca²⁺ signaling resulting in amplified OXPHOS. Moreover, we show that skin fibroblasts isolated from FAD patients with mutations in PSEN1 show a similar mitochondrial phenotype as observed in C. elegans sel-12 mutants, demonstrating a conserved role of PSEN in mitochondrial regulation. While dysregulation in ER Ca²⁺ signaling (Chan et al., 2000; Cheung et al., 2008; Green et al., 2008; Leissring et al., 1999; Stutzmann et al., 2004), mitochondrial dysfunction (Area-Gomez et al., 2012; Kipanyula et al., 2012; Zampese et al., 2011), and deleterious effects of ROS have been observed separately in various in-vivo and in-vitro models of AD, here we provide evidence that link these observations and demonstrate the central role played by mitochondrial metabolism in AD pathogenesis using an intact animal model of AD where neurodegeneration arises due to altered ER to mitochondrial Ca²⁺ signaling, in the absence of Abeta peptides.

In order to maintain neuronal function and health, optimal mitochondrial activity is critical to provide energy and concomitantly manage ROS production. Furthermore, mitochondria have a critical role in buffering Ca²⁺ and Ca²⁺ in turn modulates mitochondrial activity. Indeed, increased mitochondrial Ca²⁺ aids in the allosteric activation of several TCA cycle enzymes, including pyruvate dehydrogenase α-ketoglutarate dehydrogenase, and isocitrate dehydrogenase (McCormack and Denton, 1993), stimulates the ATP synthase (complex V) (Das and Harris, 1990), α-glycerophosphate dehydrogenase (McCormack and Denton, 1993; Wernette et al., 1981) and the adenine nucleotide translocase (ANT) (McCormack and Denton, 1993; Mildaziene et al., 1995). Thus, the increase in mitochondrial Ca²⁺ concentration results in the coordinated upregulation of the TCA cycle and OXPHOS machinery, allowing for increased oxygen consumption, and ATP and ROS production. Here, we discover that mutations in sel-12 lead to deregulated ER and mitochondrial Ca²⁺ signaling resulting in increased mitochondrial Ca²⁺ levels, which promotes mitochondrial metabolic activity. Additionally, we provide evidence that this elevated metabolic activity elevates ROS levels in sel-12 mutants and the deleterious effects of ROS results in the emergence of structural and functional phenotypes of neurodegeneration.

Upon reducing ER Ca²⁺ release or mitochondrial Ca²⁺ uptake, the mitochondria of sel-12 mutants are no longer disorganized and show normal OCR, and the high levels of ROS and neurodegenerative phenotypes observed in sel-12 mutants are suppressed. These data indicate that SEL-12 activity is required for normal ER to mitochondrial Ca²⁺ signaling, proper mitochondrial function and mitochondrial superoxide maintenance. Consistent with PSENs having a role in ER Ca²⁺ regulation, several studies in a variety of PSEN FAD models as well as tissue samples isolated from AD patients have shown that loss of PSEN function leads to perturbed ER Ca²⁺ signaling (Chan et al., 2000; Green et al., 2008; Ito et al., 1994; Leissring et al., 1999; Smith et al., 2005; Tu et al., 2006). Furthermore, PSENs are predominantly ER membrane proteins that are enriched in the mitochondria-associated ER membrane (Area-Gomez et al., 2009). The mitochondria-associated ER membrane is a highly active subcompartment of the ER that is physically connected to the mitochondria and is critical for several metabolic functions, such as Ca²⁺ homeostasis, cholesterol metabolism and the synthesis and transfer of phospholipids between the ER and mitochondria. Importantly, elevated ER to mitochondrial contact and crosstalk has been observed in PSEN1 and PSEN2 knockout cells, in cells expressing FAD mutant PSEN2, and in skin fibroblasts from FAD and sporadic AD patients (Area-Gomez et al., 2012; Filadi et al., 2016; Kipanyula et al., 2012; Zampese et al., 2011). Although the functional importance of these observations has not been resolved, our analyses of skin fibroblasts isolated from FAD patients, indicates that there is an up regulation of OXPHOS, ATP production and ROS generation that can be reduced by inhibiting mitochondrial Ca²⁺ uptake, similar to what we observe in sel-12 mutants. We also demonstrate that in sel-12 mutants neurodegeneration is caused by elevated ER to mitochondrial transfer of Ca²⁺. Taken together, these data present an inclusive model that indicates that PSEN function on the ER membrane is required for normal ER Ca²⁺ transfer to the mitochondria and in the absence of optimal PSEN function, excessive Ca²⁺ is transferred to the mitochondria leading to mitochondrial dysfunction and an elevation of mitochondrial generated superoxide radicals that promote neurodegeneration (Figure 9D). However, recent analyses of PSEN1/2 knockout mouse embryonic fibroblasts (MEFs), unlike our studies, have shown reduced mitochondrial activity suggesting PSEN function is vital for mitochondrial function (Contino et al., 2017; Pera et al., 2017). Significantly, one of these studies demonstrated that the reduction of mitochondrial function in PSEN1/2 KO MEFs could be rescued by PSEN2 and not PSEN1, implicating a role of PSEN2 in mitochondrial function in MEFs (Contino et al., 2017). While the reduced mitochondrial activity might appear to be at odds with our study, a recent analysis of astrocytes derived from iPSCs isolated from skin cells from patients with PSEN1 mutations also showed elevated OCR and ROS production, which could be rescued by CRISPR/Cas9 repair of the PSEN1 mutation (Oksanen et al., 2017). Thus, illustrating an important role of PSEN1 in the regulation of mitochondrial activity in astrocytes similar to SEL-12 in C. elegans. Also of note, we did observe in day eight vs. day one adult sel-12 mutants a drastic reduction of mitochondrial activity in comparison to aged matched wild type animals indicating that in aged animals mitochondria function becomes impaired in sel-12 mutants, consistent with the decreased mitochondrial function as a result of PSEN mutations (Figure 1—figure supplement 1C,D). The impairment of mitochondrial function with age in sel-12(ty11) animals could be a result of increased mitochondrial activity resulting in ROS-mediated oxidative damage at earlier stages of adulthood.

The best-studied function of PSEN is its role as the proteolytic component of the gamma-secretase complex and its cleavage of APP and Notch. Gamma-secretase mediated cleavage is required for the activation of Notch signaling and emphasizes the critical role of PSEN in mediating Notch function (De Strooper et al., 1997). Also, it is the gamma-secretase mediated cleavage of APP that leads to the generation of Abeta peptides of various lengths, including the toxic Abeta42 (Bezprozvanny and Mattson, 2008; Hardy, 2006). Here, we provide genetic and pharmacological evidence that gamma-secretase activity of SEL-12 is not required for neurodegenerative phenotypes observed in sel-12 mutants. While we are not able to rescue Notch signaling defects using a sel-12 protease dead genetic construct, we are able to rescue the mechanosensory behavioral defects observed in sel-12 null mutants (Figure 4D, Figure 4—figure supplement 1A-D). Furthermore, using a gamma-secretase inhibitor, which abolished Notch signaling, we do not recapitulate the neurodegenerative phenotypes observed in sel-12 mutants (Figure 4B,C). Consistently, our analyses of Notch mutants also did not reveal mechanosensory defects (Figure 4A). These results demonstrate that the role of SEL-12 in the regulation of mitochondrial function and nervous system fitness does not require gamma-secretase activity or Notch signaling.

In the last two decades, research into AD therapeutics has relied heavily on the amyloid hypothesis, which holds the toxic Abeta peptides and their aggregation to form plaques responsible for AD pathogenesis. However, the exact role Abeta peptides have in AD is not clear, which is further emphasized by the failure of phase III trials of anti-Abeta therapies like the gamma-secretase inhibitor, semagacestat (Doody et al., 2013) and solanezumab, a monoclonal antibody targeting amyloid plaques (Le Couteur et al., 2016). This suggests the need to shift focus from Abeta peptides to more proximal causes of AD. Therefore, C. elegans provides a unique system for investigating the mechanisms underlying AD. Besides the usual strengths (e.g. rapid growth cycle, genetic manipulation, cell biological approaches, simple nervous system), C. elegans encodes a single APP-like protein, apl-1. Unlike APP, APL-1, like its APL-1 mammalian homologs (APLP1 and ALPL2), lack the Abeta peptide sequence. Furthermore, APL-1 lacks the beta-secretase recognition sequences and the C. elegans genome does not encode a beta-secretase; therefore, it is unlikely that C. elegans produce Abeta peptides (Daigle and Li, 1993; McColl et al., 2012) and indeed, no Abeta peptides have been detected in C. elegans (McColl et al., 2012). Thus, C. elegans provides a model system that can explore the mechanism underlying AD without the confounding role of Abeta. Excitingly, utilizing this experimental system, we find that in the absence of Abeta, mutations in PSEN result in deregulated ER to mitochondria Ca²⁺ signaling that causes elevated mitochondrial Ca²⁺ which hyperactivates mitochondrial respiration leading to the accumulation of superoxides and the subsequent reduction of neuronal cell function and health.

In spite of our findings indicating that the role of SEL-12 in neurodegeneration is independent of Abeta accumulation, due to the fact that worms do not express Abeta, some toxic effects of Abeta accumulation have been demonstrated in C. elegans. Overexpression of human Abeta1-42 results in chemosensory defects, locomotor defects (Ahmad and Ebert, 2017; Dosanjh et al., 2010; Fong et al., 2016; Wu et al., 2006) and touch defects (Figure 5A). Moreover, similar to the drastic reduction of OXPHOS we observe in older adult sel-12 mutants compared to age matched wild type animals (Figure 1—figure supplement 1C,D), recent studies have shown that overexpression of human Abeta1-42 in the nervous system or body wall muscle results in the reduction of OXPHOS (Fong et al., 2016; Sorrentino et al., 2017). However, there are some important distinctions between overexpression of human Abeta1-42 and mutations in sel-12. Although overexpression of human Abeta1-42 in the nervous system disrupts touch response similar to sel-12 mutants (Figure 5A), it fails to result in hallmark sel-12 mutation defects such as axonal morphology phenotypes, disorganized mitochondria, and excessive calcium loading (Figure 5B–E). Therefore, the mechanism of Abeta1-42 overexpression appears distinct from presenilin mutations. These important findings suggest that an initial insult in FAD, since C. elegans does not produce Abeta peptides, is elevated ROS production from the mitochondria due to an increase in ER Ca²⁺ transfer. Thus, Abeta accumulation may be a secondary, albeit critical, component to the pathogenesis of the disease.

Source data files have been provided for all figures.

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Author details

1. Shaarika Sarasija

   Department of Regenerative and Cancer Cell Biology, Albany Medical College, Albany, United States

   Contribution

   Conceptualization, Data curation, Formal analysis, Validation, Investigation, Visualization, Methodology, Writing—original draft, Writing—review and editing

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0002-3610-2178

2. Jocelyn T Laboy

   Department of Regenerative and Cancer Cell Biology, Albany Medical College, Albany, United States

   Contribution

   Resources, Data curation, Methodology

   Competing interests

   No competing interests declared

3. Zahra Ashkavand

   Department of Regenerative and Cancer Cell Biology, Albany Medical College, Albany, United States

   Contribution

   Data curation, Formal analysis, Investigation

   Competing interests

   No competing interests declared

4. Jennifer Bonner

   Department of Biology, Skidmore College, Saratoga Springs, United States

   Contribution

   Resources, Formal analysis, Investigation, Methodology

   Competing interests

   No competing interests declared

5. Yi Tang

   Department of Regenerative and Cancer Cell Biology, Albany Medical College, Albany, United States

   Contribution

   Resources, Methodology

   Competing interests

   No competing interests declared

6. Kenneth R Norman

   Department of Regenerative and Cancer Cell Biology, Albany Medical College, Albany, United States

   Contribution

   Conceptualization, Resources, Data curation, Formal analysis, Supervision, Funding acquisition, Investigation, Visualization, Methodology, Writing—original draft, Project administration, Writing—review and editing

   For correspondence

   normank@mail.amc.edu

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0002-0773-9073

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