Endoplasmic Reticulum in Disease States

#### **Chapter 4**

## Mitochondria-Endoplasmic Reticulum Interaction in Central Neurons

*Liliya Kushnireva and Eduard Korkotian*

#### **Abstract**

The proteins presenilin-1/2 play a key role in the interactions between mitochondria and the endoplasmic reticulum at synaptic contacts of central neurons. Several novel observations suggest that mutations in presenilin-1 lead to an abnormal energy state, an early sign of neurodegeneration and Alzheimer's disease. Recent studies suggest that in the postsynaptic region, calcium stores are widely represented in the spine apparatus, which is located in a strategically important compartment - the neck of mature mushroom-shaped dendritic spines. Moreover, in the dendritic shaft area, at the base of the spines, one finds oblong mitochondrial clusters supplying the postsynaptic area and the local protein synthesis with ATP. Calcium signals, generated by the postsynaptic membranes, affect both calcium release from local stores through ryanodine channels and the uptake based on store-operated calcium entry. The entire complex of nanoscale signaling most likely determines the production of ATP. Violation of the functional relationship between mitochondria and reticular calcium depots can lead to disruption of signaling pathways that stimulate ATP production at the stages of increased activity of individual synapses. In this chapter, we will present the signaling mechanisms of interaction between mitochondria, spine clusters, and calcium nano-stores in postsynaptic area.

**Keywords:** calcium, ATP, neurodegeneration, presenilin, mitochondria, calcium store, spine apparatus

#### **1. Introduction**

The endoplasmic reticulum (ER) is the largest intracellular organelle in neurons. It ranges from the nuclear membrane through the axon to presynaptic terminals, and through all dendritic arbors, penetrating into some dendritic spines in the form of thin smooth tubules or their extensions such as spine apparatus [1]. Internally differentiated into smooth (sER) and rough (ribosomal, rER), the ER performs many cellular functions, including the synthesis and transport of essential intracellular molecules. However, the most enigmatic and least explored function of the ER is the storage and transmission of Ca2+ signals from a small compartment called the spine apparatus (SA), which is located mainly in mature, mushroom-type dendritic

spines [2]. This is a small multilayer lamellar structure, sometimes connected to the sER in the dendritic shaft by a thin tube passing through the neck of the spine [3]. Localization of the spine apparatus depends on synaptic activity [2]. About 80% of the large dendritic spines have a spacious SA, while only 20% of the small thin (immature) spines contain it [3] since more often the sER network can reach only the neck of the spine or be completely absent [4]. The cytoplasm of the spine has been shown to be composed of actin and actin-regulating proteins that are longitudinally located in the neck of the spine and organized into a dense lattice surrounding the sER, or SA in the head of the spine [5], whose marker is the actin-modulating protein synaptopodin (SP) [6, 7]. There are contradictions in studies involving electron microscopy (EM), which do not allow unequivocal answers to the question of whether the SA is an autonomous structure derived from the ER, indicating the final stage of the "maturity" of the spine, and all other inclusions of the ER in the spine are only transitional stages, or SA formation occurs independently of inclusion of a continuous ER compartment into the spine, and in such a case, they may sometimes coexist [3, 4, 8–12]. Nonetheless, there is a growing number of studies in which the spine apparatus is mentioned as one of the main players in the regulation of synaptic plasticity and learning and memory mechanisms. [7, 11, 13–17].

#### **2. Spine apparatus functions**

Calcium deposition in the postsynaptic terminal underlies synaptic transmission, driving a wide range of synaptic plasticity mechanisms for efficient learning and memory processing. Excitatory or inhibitory inputs lead to a differentiated local increase of calcium in dendritic spines, which functions as units of biophysical and biochemical computations in the neuron, regulating their duration and distribution [18]. The volume of the SA, also considered an ER store, affects the temporal dynamics of Ca2+, its distribution to neighboring dendrite sites, and binding to numerous Ca2+ − gated signaling pathways [2]. The volume of the spine apparatus positively correlates with the total volume of the spine [6], with the size of the spine head (which is indirectly confirmed in experiments with SP) [19] and may increase due to the activity of N-methyl-d-aspartate receptors (NMDAR) [2]. Activation of postsynaptic NMDARs by glutamate in CA1 hippocampal neurons triggers the activation of ryanodine receptors (RyR) and Ca2+ − induced release of Ca2+ (CICR) from the store. This release of Ca2+ occurs and is often limited to the head of the spine [2], however, it is able to spread further along the dendrite, penetrating into adjacent spines, especially in young neurons [20]. In young (P8-P17) tissues, hippocampal postsynaptic RyRs CA3-CA1 mediate a propagating Ca2+ signal from active synapses, triggered by NMDAR-mediated Ca2+ influx into the dendrite and adjacent coactive synapses, lowering their induction threshold for plasticity [21]. Immunological experiments show colocalization of SP and RyR in calcium stores [19]. Modeling indicates that RyRs are likely located on the SA at the base of the spine neck and their activation is triggered by the binding of two calcium ions, which move from the head to the neck inside the spine cytoplasm and generate calcium flow from the SA down into the dendritic shaft, which also is verified experimentally [22]. RyR – mediated calciuminduced calcium release (CICR) from stores can lead to either long-term potentiation (LTP) or long-term depression (LTD), depending on the pattern of synaptic activity. The mechanisms of LTP and LTD are the basis of synaptic plasticity and require an increase in postsynaptic Ca2+ concentration ([Ca2+]i). Caffeine, which releases

#### *Mitochondria-Endoplasmic Reticulum Interaction in Central Neurons DOI: http://dx.doi.org/10.5772/intechopen.105738*

Ca2+ from stores, increases the number of active α-amino-3-hydroxy-5-methyl-4 isoxazolepropionic acid receptors (AMPARs) in hippocampal cultures, thus enhancing the function of synapses [19] and increasing LTP induction in hippocampal slices [23, 24]. Pharmacological blockade of CICR by RyR impairs LTP induction at hippocampal synapses [2, 11, 25]. In addition to LTP, RyRs also promote LTD, and their genetic ablation inhibits low-frequency stimulation-induced LTD in the same area of the brain [19]. In addition, it has been shown that glutamate-induced growth of new spines in the cortex is also mediated by the release of Ca2+ from calcium stores [26].

SP-positive spines demonstrate stronger responses to glutamate uncaging than SP-negative ones. In addition, SP mediates the accumulation of the GluR1 subunit of the AMPA receptor in the heads of spines [19]. Adult mice have more mature spines containing SA and a larger area of SP inclusions than the young. Hippocampal neurons in SP knockout (SPKO) young mice lack SA and decrease LTP, do not solve cognitive tasks, and lose spatial memory. However, with age, SPKO mice develop compensatory mechanisms for LTP recovery. They demonstrate increased excitability and expression of an activity-regulated cytoskeleton-associated protein (Arc), encoded by a member of the immediate-early gene (IEG) family *ARC*, in hippocampal dentate gyrus (DG) granule cells after exposure to a novel environment. Arc mRNA and protein are known to accumulate in dendritic regions that receive high-frequency synaptic inputs [27]. Learning or stress-induced Arc accumulation in dendrites is critical for plasticity and memory consolidation [28]. DG is an area required for spatial learning processes, which in SPKO mice activates more cells than in mice with a normal genotype. Since the intracellular concentration of Ca2+ regulates excitability through the activation of calcium-gated K<sup>+</sup> − currents, a decrease in calcium availability as a result of the absence of SP and calcium stores can cause increased excitability in the SPKO brain. Therefore, it is important to compensate for the decrease in [Ca2+]i in postsynaptic sites for synaptic plasticity [29].

In addition to RyR, the release of calcium from ER stores is enhanced by activation of inositol triphosphate receptors (IP3R), which are enriched in dendritic branches [19]. Activation of type I metabotropic glutamate receptors (mGluR) on the plasma membrane (PM) causes an increase in the concentration of inositol triphosphate via phospholipase C (PLC). This, in turn, enhances the release of Ca2+ from the stores into the cytoplasm by stimulating the IP3R on the ER membrane. IP3R activation can propagate the calcium wave along the dendritic segment or be limited to postsynaptic microdomains, depending on the ambient level of inositol triphosphate in the cytoplasm. IP3R is able to be co-activated by intracellular Ca2+ and these two mechanisms, RyR-mediated CICR and IP3R-induced release of calcium from the stores, are capable of mutual reinforcement [30]. Thus, the SP -associated Ca2+ − stores in the form of a SA play an important role in the storage and regulation of Ca2+ secretions needed for neuronal plasticity.

In ER stores, Ca2+ is bound to calcium-binding proteins (CBPs), such as calnexin and calreticulin. Each CBP binds to many Ca2+ ions in a low affinity and highcapacity manner. When the Ca2+ store is open, exporters can easily separate Ca2+ from the CBP. The store also maintains the concentration of free Ca2+, which determines the driving force for the release of Ca2+. To compensate for the depletion of the Ca2+ pool, its accumulation in the ER is carried out through the use of Ca2+ pumps of the Ca2+-ATPase family (SERCA) together with a store-operated calcium entry (SOCE) via store-operated calcium channels (SOCs). The functioning of SOCs depends on the Ca2+ concentration inside the store and calreticulin, which accounts for nearly half of total ER Ca2+ binding, acts not only as a Ca2+ buffer but also as an important

chaperone and regulator of SERCA pumps. Calcium depletion from ER stores is determined by stromal interaction molecules (STIMs), which are diffusely distributed throughout the resting ER. When the storage is empty, STIM transmembrane calcium sensors accumulate on the membrane of the depleted ER store, closest to PM, where they activate the voltage-independent calcium release-activated protein (Orai), providing an influx of Ca2+ into the store so as to replenish it (**Figure 1**) [30]. Two homologs of STIM, STIM1 and STIM2, are found in neural tissue but appear to be associated with different functions in developing and mature neurons, although both are associated with the Orai channel. STIM1 clusters predominate in young cells, they are more mobile, and their movement along dendrites triggers local Ca2+ transitions, while STIM2 clusters are active in mature neurons, are more dispersed, and much less mobile. STIM1 plays an important role in the formation and functional maturation of filopodia and growth cones in young cells, and STIM2 binds to SOC under conditions of Ca2+ deficiency, restoring local [Ca2+]i levels and moving into active dendritic spines [31]. Inhibition of SERCA pumps by thapsigargin results in slow Ca2+ leak from the ER, stimulating STIM1 oligomerization and formation of STIM1/Orai1 complexes, and antagonists of STIM/Orai dependent SOCE reduce LTP in hippocampal neurons. Synaptic activity is critical for maintaining the morphology of mature dendritic spines, as blockade of synaptic activity by tetrodotoxin (TTX) causes STIM-associated increases in spontaneous calcium transients and a decrease in the proportion of mature spines versus the proportion of immature filopodium [31, 32].

The STIM/Orai complex is also studied in the context of the development of neurodegenerative diseases, in particular Alzheimer's disease (AD). STIM1 and STIM2 are involved in maintaining Ca2+ homeostasis in neurons and are involved in the production of beta-amyloid peptide (Aβ), which accumulates in AD. Overexpression of these proteins can initiate pathological activation or deactivation of SOCE-dependent mechanisms, disrupting synaptic transmission and thus stimulating neurodegenerative mechanisms [31, 33].

#### **3. Structural plasticity of spines**

LTP and LTD are associated with an increase and decrease in spine volume, respectively. Similar to functional plasticity, structural plasticity also requires Ca2+ influx through postsynaptic NMDARs, activation of Ca2+/calmodulin-dependent protein kinase II (CaMKII) for recruiting GluR subunits, small guanosine triphosphatases (GTPase) and actin polymerization [34]. What specific interactions are required in local targeting of mRNA and protein synthesis to increase the morphology of active spines? To answer this question, the synaptic tagging and capture (STC) hypothesis was proposed: LTP induction creates a "tag" in potentiated synapses that can capture plasticity-related proteins (PRPs), including Homer protein homolog 1a (Homer1a) and Arс. Homer1a, a postsynaptic scaffold protein, is recruited from the soma into the stimulated spine. A synaptic tag can be a temporary morpho-functional state of the synapse, which is represented by a complex of proteins in interaction with the structures of the actin cytoskeleton. For example, LTP is known to induce the formation of a stable pool of F-actin that can act as a synaptic tag. However, this tag is also found in unstimulated spines. So, after LTP, Arc accumulates in unstimulated spines and is excluded from potentiated ones. The amount of synaptic Arc is negatively correlated with the amount of surface GluA1 at the synapses and promotes AMPAR endocytosis. It is likely that this reverse synaptic labeling helps maintain the synaptic

#### *Mitochondria-Endoplasmic Reticulum Interaction in Central Neurons DOI: http://dx.doi.org/10.5772/intechopen.105738*

weight contrast between active and inactive spines in areas of high synaptic plasticity such as the hippocampus [27, 34]. This mechanism is especially attractive when the spines are close to each other and are part of the same functional synaptic cluster. It is known that morpho-functional changes correlate among neighboring spines. Decrease in the LTP induction threshold decays along ~10 μm of the dendritic branch, and in young neurons, the repeated release of glutamate from individual spikes reduces the induction threshold in the surrounding area to several minutes [34]. We hypothesize that intra-cluster differentiation of synapses is essential for structural plasticity. For example, after LTP induction, the activity of GTPases Ras and Rho extends to ~5–10 μm along the dendrite with the ability to penetrate into neighboring spines. In addition, single spike activity can also trigger molecular signaling involving calcineurin, IP3R, and metabotropic glutamate receptor 1 (mGluR1), which act on adjacent spines [35]. It is possible that Arc-dependent depotentiation of non-target spines helps de-tag neighboring spines for accurate PRP capture.

The mechanism linking local synaptic events in individual spines and signals entering the nucleus includes such transcriptional regulators as cAMP response element-binding protein (CREB) and synapto-nuclear protein messenger *Jacob*, which are translocated into the nucleus in response to synaptic activity. Upon NMDAR activation, *Jacob* is phosphorylated and dissociated from the spines, translocating to the nucleus in an importin-dependent manner. The presence of phosphorylated *Jacob* in the nucleus increases CREB phosphorylation, inducing the expression of CREdependent genes that are involved in the generation of the protein tag only in potentiated synapses [34]. However, such a tag is likely not so much a complex of protein molecules as a short-term reconfiguration of the spine cytoskeleton due to CaMKII activity, including restructuring of postsynaptic density (PSD) and the number of AMPAR. During LTP, the autophosphorylated form of CaMKII remains active in the PSD even after the Ca2+ concentration returns to baseline levels and such a tagged synapse is able to capture PRP. Such synapse stabilizes its new structural conformation before the tagging state disappears, and thus will retain a change in its synaptic efficiency. Inhibition of CaMKII autophosphorylation disrupts the tag, interfering with structural, but not early functional plasticity [36].

If we also turn to SA as a potential tag of functionally improved synapses, like Ostroff et al., who have shown that the presence of SA in large spines correlates with the presence of polyribosomes in their heads after learning. This may be an indicator of a high degree of spatial specificity of translation in dendrites. Accumulation of polyribosomes in the heads of large spines reflects the enhancement of translation during learning, suggesting a link between structural plasticity and memory consolidation [37].

#### **4. The role of mitochondria**

The development, maturation, and maintenance of functioning synapses require the maintenance of ionic balance, proteostasis, and modification of synaptic proteomes at the expense of a significant amount of energy [38]. Of great importance is the local specificity of these processes for plasticity, which has an advantage in comparison with the generalized delivery of proteins from the soma. To meet global and local energy needs, cells regulate mitochondrial movement, fission, fusion, and "parking" mitochondria in every area of the cell. Neuronal mitochondria play a crucial role in maintaining synaptic functions, in particular, by providing local

translation both under basic conditions and during plasticity processes [38–40]. Rangaraju et.al from Schuman's laboratory used adenosine triphosphate (ATP) inhibitors, local inhibition of mitochondrial compartments, and visualization of newly synthesized proteins, to show that local translation is provided by local mitochondrial clusters. Translation of proteins necessary for morphological plasticity, going on from mRNAs, localized near synapses. Glutamate-inducted LTP was not established in areas containing nonfunctioning mitochondria, preventing the morphological changes characteristic of this mechanism. Also, in mitochondria-free regions, there was a significant decrease in protein synthesis compared to stimulated regions with functional mitochondria, although at basal levels of neuronal activity, the energy needs of local translation are adequately met by global levels of ATP available in the dendrite or ATP produced by neighboring functioning mitochondria [38].

Fluctuations in cytosolic calcium, glucose and ATP levels, synaptic activity, neurotransmitters, and growth factors are able to regulate positioning, mitochondrial transport, and dynamics. Moving these organelles to energy-demanding sites, such as synapses, dendritic spines and axons [41], is essential for their functioning. Mitochondrial morphology differentiates not only depending on cell type but also on localization in a particular cellular compartment and can change with age [42]. *In vitro* and *in vivo* imaging of axonal mitochondria often shows them to be short, while dendritic mitochondria have a more elongated morphology and often show greater complexity than axonal [43]. This complexity can be expressed at least in terms of length or volume and tendency to form clusters of mitochondria in the form of tubes, often overlapping with each other in dendritic shafts. For example, in the experiments of Faitg and others, the average volume of individual mitochondria in hippocampal neurons varied between 0.11 and 1 μm3 , while axonal mitochondria had an average volume of 0.12–0.27 μm3 and did not reach values >1 μm3 ; somatic mitochondria occupied intermediate values (0.14–0.16 μm3 ) [42]. In another study with cortical neurons, the length of dendritic mitochondria varied from 0.52 to 13.28 μm, while the length of axonal mitochondria was much smaller from 0.3 to 1.13 μm [43]. More than 50% of dendritic extensions are filled with mitochondria, but less than in axons 1% are filled [38, 43]. Another study compared mitochondria in the presynaptic bouton and in the postsynaptic dendrites of the hippocampus. The area of presynaptic mitochondria was almost half, on average, 0.077 μm<sup>2</sup> versus postsynaptic 0.146 μm<sup>2</sup> . In addition, presynaptic mitochondria were significantly darker, i.e., had a higher electron density than postsynaptic [44]. It is possible that the elongated morphology of mitochondria in dendrites provides them with a bioenergetic advantage [45], while an increase in density indicates their greatest activity. Tubular mitochondrial stretches in dendrites are observed in hippocampal neurons, the average length of which sometimes even exceeds 10 − 30um in length [38, 46]. These may be called a mitochondrial cluster, since such a structure more likely represents individual mitochondria undergoing longitudinal outer (OMM) and inner mitochondrial membranes (IMM) fusion [47, 48], because normal elliptical mitochondria are smaller under normal conditions (see numbers above). Such clusters can be cleaved by mitochondrial uncouplers such as FCCP or CCCP [49]. Clustering of mitochondria increases their functional stability, optimizing the use of the local mitochondrial pool in cell compartments under basic conditions and during activity. Fusion and fission, the direction of movement and parking of mitochondria in certain areas of neuronal processes are of great importance for meeting the changing energy needs of various areas of the cell.

#### **5. Mitochondrial movement and parking**

In neurons, the movement of mitochondria is realized by their attachment to microtubules and it depends on their polarity [50]. In axons, microtubules have a distinct polarity, so that anterograde transport of mitochondria along (+)-terminal microtubules move them toward the growth cone or presynaptic end of the axon using kinesin family proteins (KIFs), while retrograde transport along (−)-terminal microtubules use motor protein complex of dynein, directing mitochondria toward the soma. In dendrites, microtubules can show mixed polarity so that KIFs and dynein motors can drive cargo transport in dendrites either anterograde or retrograde depending on microtubule polarity [50]. There are also reports that the movement of mitochondria over short distances in areas rich in actin cytoskeletons, such as the axon growth cone, presynaptic terminal, and sometimes in large dendritic spines observed in the cortex and hippocampus, is carried out with the help of myosin motors [46, 50]. The share of movement mitochondria varies from 5 to 20 to 35–45% of the mitochondrial pool in a culture of hippocampal neurons, and mitochondrial movement occurs more intensely in axons than in dendrites, where mitochondria can often be relatively immobile both in synaptic and non-synaptic areas. Movement increases during blocking of activity by TTX in all outgrowths, while induced glutamate excitotoxicity induces a persistent increase in [Ca2+]i, slowing down movement and promoting mitochondrial rounding in all neuronal outgrowths [45]. Under physiological conditions, Ca2+ influx occurs in areas of high metabolic demand, such as axon terminals and complexes of postsynaptic structures, where mitochondria tend to accumulate. According to various data, from 36% to ∼50% of presynaptic axon terminals of hippocampal neurons contain mitochondria [38, 45]. Sustained elevation of [Ca2+] i levels in some areas of the cell may be a marker of local activity or ATP deficiency, which will recruit and retain mitochondria to stop in these areas, thus balancing ATP production with local needs [51]. Numerous signaling pathways have been elucidated that are regulated by Ca2+, modulating mobility or inducing arrest of mitochondrial pools. Mitochondrial microtubule-based mobility is mediated by KIF Ca2+-dependent manner, and the OMM-anchored mitochondrial Rho GTPase 1 (Miro1) is a Ca2+ sensor (because it contains two helix-loop-helix EF-hand Ca2+-binding motifs) for mitochondrial localization in synapses, and its association with PTEN-induced kinase 1 (PINK1) and recruitment of the Parkin protein is necessary for mitochondrial motility stops [50, 52, 53]. Mitochondrial arrest during neuronal activity is produced by synapsereleased glutamate, which activates NMDA receptors and induces Ca 2+ − influx, which binds to Miro1. The miro1-mediated mitochondrial arrest may recruit passing mitochondria to active synapses where ATP and calcium buffering requirements would be higher [53]. In a culture of rat hippocampal neurons, perfusion of 30 μM glutamate (with 1 μM glycine to activate NMDA receptors) for 10 min reduced the number of moving mitochondria by 95% in the presence of extracellular calcium ([Ca2+]o), but only a 28% decrease in movement was observed with absence of [Ca2+]o. In the same study, it was demonstrated that moving mitochondria were stopped in areas that were positive for the synaptic marker synaptophysin and synaptic vesicle protein 2 (SV2), and the distribution of mitochondria in dendrites to the nearest synapse was reduced after the application of glutamate. All of this indicates that mitochondria are recruited to synaptic zones during activity [53]. In addition to these mechanisms, a positive correlation is also found between anterograde mitochondrial transport in axons and electrical potential through IMM (MtMP, Ψm), increasing ATP production. Thus, the

maximum rate of ATP production in isolated mitochondria approximately doubles with an increase in Ψm for every 10 mV [54]. Mitochondria with high membrane potential also tend to accumulate at synapses [55]. It can be concluded that the motility and spatial distribution of mitochondria for ATP production can be dynamically regulated by local [Ca2+]i.

### **6. The role of mitochondria in Ca2+ buffering and signaling**

Mitochondria play an important role in Ca2+ signaling in neurons. They have a buffering capacity for Ca2+ binding that arises from their highly hyperpolarized membrane. Membrane potential values of functioning mitochondria in a culture of rat cortical neurons vary within ≃100 – ≃160 mV [54]. MtMP allows rapid transfer of cytosolic Ca2+ across the IMM along its electrochemical gradient into the organelle by the mitochondrial calcium uniporter (MCU), despite the last low affinity for Ca2+ ions. The mitochondrial matrix accumulates high levels of Ca2+ both during global Ca2+ signals and in response to a moderate local increase in [Ca2+]i [56]. This uptake is balanced by Ca2+ efflux through the Na+ /Ca2+ antiporter or, more rarely, through the mitochondrial permeability transition pore (mPTP), these pathways are also sensitive to mitochondrial membrane depolarization [56, 57]. Small transient mitochondrial depolarizations reflect mitochondrial buffering activity in high [Ca2+] i micro-domains. From studies in isolated mitochondria, uptake of 17 μM Ca2+ caused a 2–3 mV mitochondrial depolarization [58], while a much greater loss of MtMP accompanies opening of mPTP, nonselective pores in the inner mitochondrial membrane. Through these any molecule weighing less than 1500 da can penetrate, which can lead to an increase in osmotic pressure, swelling of mitochondria and subsequent rupture of the OMM, or cause depletion of matrix substances, incl. and Ca2+ [55]. Temporary opening of the pore can be caused by Ca2+ overload and is characterized by uncoupling of the oxidative phosphorylation chain, resulting in a decrease in ATP production, but is reversible upon restoration of cellular homeostasis. In contrast, the permanent opening of mPTP triggers mitochondria-mediated apoptosis due to the release of cytochrome C [30, 57]. Mitochondria are able to buffer about 75 μM of Ca2+ before it is released, and this point of maximum buffering seems to depend on the rate of Ca2+ uptake. Rapid mitochondrial depolarization also leads to subsequent calcium release, as shown in experiments with CCCP [59]. Interestingly, the content of Ca2+ in mitochondria is inversely proportional to the rate of mitochondrial movement. Motile mitochondria tend to have a lower intra-mitochondrial Ca2+ signal ([Ca2+]m), however, the [Ca2+]m levels do not affect the direction of movement, and no difference was shown in [Ca2+]m between anterograde and retrograde mitochondrial movement. We have previously said that microtubule-based mitochondrial motility is driven by [Ca2+]i levels, however direct Ca2+ influx into the mitochondrial matrix via the Miro1 mediated MCU is also capable of altering mitochondrial motility. In experiments with mutations in the EF-hand domains of Miro1, mitochondrial movement did not stop, despite an increase in [Ca2+]i levels. Blocking the MCU also allowed mitochondrial movement to be preserved even in the presence of high levels of cytoplasmic [Ca2+] i, but only partially, indicating that [Ca2+]i also makes a significant contribution to mitochondrial mobility. Miro1 can change the level of Ca2+ influx into mitochondria, acting as a [Ca2+]i sensor, similar to STIM ER, influencing the amount of Ca2+ influx into mitochondria and their rate, which can be regulated by the amount of Miro1 protein associated with kinesin motors at a given time, while as soon as the concentration

of [Ca2+]m reaches a critical level, the interaction of Miro1 with kinesin complexes is disintegrated and mitochondria stop. The influx of Ca2+ into mitochondria increases ATP production by activating the tricarboxylic acid cycle, as well as increasing the activity of electron transport chain enzymes and the ATP synthase complex, so that ultimately mitochondrial arrest is beneficial near synaptic areas [60].

#### **7. ER-mitochondria interactions**

Mitochondrial clusters occupy most of the dendritic branches and are closely associated with the ER, especially at the base of the spines (**Figure 1**) [18]. However, such a distribution of ER and mitochondria is not constant. Contacts between mitochondria and the ER along the dendrites enable functional inter-organellar communication and play a central role in the regulation of postsynaptic calcium-signaling, and dysregulation of its communication has been demonstrated in neurodegenerative diseases such as Alzheimer's and Parkinson's diseases [18]. Contact sites between OMM mitochondria and ERs (Mitochondria-ER Contact Sites, or MERCs), constituting about 2–20% of the mitochondrial surface area, were detected using EM studies, then demonstrated in experiments using dimerization-dependent fluorescent proteins. Membrane constituents from a specific set of protein and lipid complexes that form MERC are called mitochondria-associated ER membranes (MAMs). The term MAM is used to describe the results of experiments on the biochemical/functional characterization of isolated contacts between mitochondria and the ER, while the term MERC is used during morpho-functional visualization [61].

MERCs provide a direct route for calcium transfer from the ER to the mitochondria and are required for mitochondrial functions, including ATP production. An increase in MERC surface area increases mitochondrial calcium influx and hence stimulates ATP production [18]. The MERC width is not constitutive and can change depending on the metabolic status of the cell. Excessive expansion, or vice versa, constriction of MERC disrupts the efficient transfer of calcium from the ER to the mitochondria. Ca2+ uptake by the MCU is most effective when the ER membrane faces the OMM at a distance of ≈15 nm, but not more than 30 nm, when calcium will leak and diffuse into the cytoplasm and not less than 10 nm, which is determined by the length of the protruding part of IP3R or RyR [61]. The Ca2+ release from the ER is able to control mitochondrial mobility by locally increasing [Ca2+]i. Inhibition of movement of mitochondria at an optimal distance from active Ca2+ receptor channels from the ER is necessary for mitochondrial calcium buffering, which also serves as a means of stimulating mitochondrial ATP production. Although mitochondria store less Ca2+ than ER, they are important buffers of [Ca2+]i. Voltage-gated anion channels (VDACs) located on the OMM are responsible for the rapid transfer of Ca2+ from the ER to mitochondria, and their function results in the formation of Ca2+-rich microdomains in the mitochondrial intermembrane space [62]. Balance of [Ca2+] m is maintained by influx through the MCU and outflow through the mitochondrial sodium-calcium exchanger (NCLX), but at elevated levels of activity, mitochondria are also able to buffer excess [Ca2+]i due to precipitation inside the matrix in the form of Ca2+ − phosphate [30]. Sigma-1 receptors (σ-1R) found on MAM may influence mitochondrial Ca2+ − signaling. σ-1Rs are able to exhibit chaperone activity, stabilizing IP3R to MAM under conditions when Ca2+ reserves in the ER are depleted, preventing degradation of Ca2+ entry into mitochondria, restoring Ψm and subsequent ATP production (**Figure 1**) [62].

#### **Figure 1.**

*Top. Mature mushroom-shaped dendritic spine contains spine apparatus (SA), which is required for synaptic transmission and plasticity. SERCA pump mediates Ca2+ uptake into SA due to synaptic activity or STIM/ Orai-dependent store-operated calcium entry mechanism. Excitatory input (lightning) activates calcium influx through postsynaptic NMDAR, which triggers calcium-induced calcium release from SA through RyR. This Ca2+ transient spreads along the spine neck toward the dendrite. In turn, this initial transient trigger molecular signaling leads to the release of Ca2+ from IP3Rs, which are localized on the dendrite-running ER. Calcium released from RyR and IP3R may attract Mobile extrasynaptic mitochondria located within 5–10 μm to the base of the spine. Mobile mitochondria move along microtubules driven by a protein of the kinesin family (KIF). Continuous Ca2+ transients at the base of mature spine dock the mitochondrion by dissociating it from microtubule. Retention and accumulation of mitochondria take place in the area of locally elevated calcium around ER. Calcium originated from IP3R covers an area around voltage-gated anion channels (VDAC), the intermembrane space of mitochondria, and then through the mitochondrial calcium uniporter (MCU) moves to the mitochondrial matrix. Stable postsynaptic mitochondria provide ATP-dependent mechanisms of plasticity, the local ribosome-dependent translation of proteins on the base of mRNAs possibly attracted by local Ca2+ gradients. The nearby filopodium does not receive presynaptic inputs and is therefore unable to take part in synaptic plasticity. Bottom. Mature and immature dendritic spines both receive excitatory presynaptic inputs. A more intense postsynaptic response occurs in a mature spine containing both NMDA and AMPA receptors. Synaptic input initiates calcium release from the SA and ER, inducing local translation of plasticity-related proteins (PRPs). PRPs can also penetrate into the adjacent, co-activated but yet immature spine, causing the incorporation of AMPA receptors. A stable postsynaptic mitochondrial cluster sequesters [Ca2+]i, which has been released during activity. This mechanism highly limits calcium distribution along the dendrite, maintaining individual spine autonomy. New mitochondria fuse into a cluster. Their contacts with the ER are maintained by mitofusins (Mfn) 1/2. Sigma-1 receptor (σ-1R) helps to stabilize and attach IP3R to VDAC in mitochondriaassociated ER membranes (MAM). Ca2+ influx through MCU is also mediated by mitochondrial rho GTPase 1 (Miro1), which is able to function as a [Ca2+]m sensor. Miro1 modulates the level of Ca2+ influx into the mitochondrial matrix. It is also involved in MAM - mitochondria stabilization mechanism. The larger volume of the mitochondrial cluster enables the production of higher levels of ATP for efficient PRP production, promoting functional and morphological plasticity. Individual mitochondria are also shown to be able to enter the heads of the mature spines with large head volume and SA, where they possibly feed local translation mechanisms.*

*Mitochondria-Endoplasmic Reticulum Interaction in Central Neurons DOI: http://dx.doi.org/10.5772/intechopen.105738*

Knockout of Sig-1R in hippocampal neurons results in shorter and smaller mitochondria and also causes a decrease in MtMP and release of cytochrome c, which leads to disruption of cytoskeletal networks loss of mature dendritic spines, and formation of immature dendritic spines [63, 64]. Genetic deletion of σ-1R also impairs MAM stability and leads to a decreased number of MERC [65]. A reduced amount of σ-1R has been observed postmortem in the hippocampi of AD patients, and certain σ-1R gene polymorphisms coexist with the well-known risk factor Alzheimer's disease apolipoprotein ε4 (APOE ε4) [66].

Mitochondrial fission and fusion can also be regulated by the ER, for example, by Wnt-5a, a member of the Wingless/integrase (Wnt) secreted glycoprotein family, which induces an increase in Ca2+ efflux from the ER via IP3R and RYR. This, in turn, activates Ca2+-dependent signaling molecules, including CaMKII, protein kinase C (PKC), and calcineurin, which may promote phosphorylation of dynaminrelated protein 1 (Drp1) associated with increased mitochondrial fragmentation [41]. Although at rest, fragmented dendritic mitochondria are less beneficial for energy production, but they are able to increase ATP production at high levels of arousal through higher H2O2 production [67].

#### **8. Functionally and spatially related clusters of spines are served by mitochondria clusters**

Modeling and electrophysiological experiments indicate that neighboring synaptic inputs can sum nonlinearly and turn a dendritic compartment or a cluster of spines into a separate summing unit of activity. The experimentally observed signs of synaptic clustering are expressed in the sharing of synaptic inputs from the same axon by two or more neighboring spines. That is, the formation of synapses and their clustering is a consequence of their relative position with the nearest axon (**Figure 2**) [35]. The presence of clustering is also evidenced by the above mechanisms of transfer of intracellular signaling molecules by diffusion from the stimulated spine to a certain distance to neighboring dendritic spines, which can "learn" a similar pattern of activity. There is also evidence for intercluster competition: LTP induction in multiple spines on the same dendritic segment can cause spine contraction and weakening of the synapses of neighboring, more distant, unstimulated spines [34].

It can be expected that the united cluster of spines should have its own stable energy source in the form of a mitochondrial cluster, which will limit Ca2+ diffusion and ATP production mainly to this region. The calculations by Rangaraju et al. suggest that mitochondrial clusters spanning about 30 μm of dendritic length are capable of local ATP synthesis to provide energy for 30–300 spines (with a uniform distribution of 1–10 spines per μm of dendrite length) [38]. This co-compartmentalization provides an advantage at the time of obtaining simultaneous excitation for several dendritic spines, in which ATP production from one local mitochondrion or ATP levels in the dendrite would be clearly insufficient, and the expectation of ATP influx from neighboring mitochondria would adversely affect time-dependent plasticity and competitiveness given synaptic cluster. High-frequency excitatory input that leads to a significant increase in the local level of [Ca2+]i also require immediate absorption, which is successfully performed in the case of mitochondrial clustering. Could be supplemented to reformulate Hebbian plasticity into: "Spines that are repeatedly active at the same time with the same inputs will tend to become 'associated' so that

activity in one will facilitate activity in the other. Together, they represent a cluster unit of information in the brain and it will be provided by the own mitochondrial cluster."

What are the advantages of such a clustered mitochondrial service structure for a single spine? Modeling shows that ATP availability in dendrites is directly proportional to mitochondrial length [18]. In the case when several spines of the cluster simultaneously receive excitation, it will cause a more extensive total postsynaptic response, and then such a cluster, which has an extended mitochondrial cluster under it, acting, in this case, as a whole, will be provided with more energy. In pathological cases, the breakdown of such a cluster organization can occur: a decrease in the number of mature functional spines, which will lead to disorganization of the synaptic cluster, and a violation of mitochondrial recruitment and mitochondrial clustering will make energy production more chaotic.

Will an individual spine that receives a local signal be "lost" in such a unified structure? It is known that within a cluster, spines may compete for limited resources (**Figure 2**). After LTP induction, there is a marked loss of small spines, which is accompanied by an increase in the remaining spines, so that the total synaptic surface area per dendrite length remains constant [35]. There are hypotheses, the essence of which is that, despite the apparent morphological integrity of the mitochondrial cluster, the limited spaces represented by the cristae compounds create lateral gradients of critical metabolites and macromolecules within the cristae. Thus, in the mitochondria of the brain, the cristae are connected to the surface of the IMM through narrow long tubular sections. Modeling suggests that such compounds may lead to micro-compartmentation within the mitochondria, which may have important functional implications by creating a barrier to the diffusion of molecules between the cristae and intermembrane spaces. Rearrangements of such barriers change the energy output [68], allowing local ATP targeting.

#### **Figure 2.**

*Stylization based on a real image of the mitochondria distribution in the dendritic shaft. Two clusters of spines are united, each to their axonal presynaptic input, and compete for motile extrasynaptic mitochondrion (arrow). The more active cluster of spines attracts extrasynaptic mitochondrion as well as more efficiently stabilizes adjacent mitochondrial cluster due to an increase in ATP demand. Active spines receive higher levels of ATP from their local synaptic mitochondrial, compared to spines that do not receive sufficient synaptic inputs. Therefore, both intercluster and intracluster spine competition can be proposed.*

#### **9. The role of presenilin-1 in interactions between mitochondria and the ER in the neurodegeneration context**

The "MAM hypothesis", for AD suggests that this disease is a consequence of a disruption of ER-mitochondrial interactions, since studies show an increased expression of MAM-associated proteins in the brain of humans and mice with AD, up to the appearance of Aβ plaques, which precedes the more common "amyloid hypothesis". For example, fibroblasts obtained from AD patients whose symptoms include impaired lipid metabolism have elongated, up to more than 200 nm MERC and increased lipid traffic [61]. PS1 and PS2 are two highly homologous isoforms of mammalian presenilin, with mutations associated with about 40% of all known cases of familial AD. Their gamma-secretase activity is enriched in MAM where they affect the processing of amyloid precursor protein (APP) to form Aβ. PS-1 plays a key role in the interactions between mitochondria and the ER in the area of synaptic contacts. Presenilin-2 (PS2) in the presence of the mitochondrial fusion protein mitofusin 2 (Mfn-2) located in OMM associated with mitofusin 1 (Mfn-1) on the ER membrane promotes mitochondrial binding to the ER [30]. Mutant presenilins have also been implicated in disrupting Ca2+ signaling in neurons due to the release of excess amounts of Ca2+ from the stores with the help of RyR and IP3R. RyR expression levels are elevated in cultured neurons expressing mutant PS1 in 3xTg-AD mice starting at a young age [69].

The most interesting and not related to the gamma-secretase activity of presenilins is their participation in the role of channels of passive leakage of Ca2+ ER in the hippocampus, shown in the laboratory of Bezprozvanny [70]. The increase in resting Ca2+ levels observed in PS1-transfected cells may be due to "leak" storage and the fact that the SERCA pump takes longer to pump the leaked Ca2+ back to the store. Calculations predict that under physiological conditions the SERCA pump reaches thermodynamic equilibrium when [Ca2+]ER is 2.4 mM, however, visualization gives different values, in the range of 100–500 μM [Ca2+]ER. The researchers proposed an explanation for this difference as the presenilin-mediated leak of the ER membrane to Ca2+ ions. Then the stationary intraluminal level of Ca2+ in the ER is determined by the balance between its injection with SERCA and passive leak into the cytosol. However, the PS1-M146V mutant was not able to function as a Ca2+ leak channel, which led to an overflow of Ca2+ stores and an increase in the level of released Ca2+ upon IP3R activation [70].

According to an alternative hypothesis, presenilins do not necessarily directly regulate the leakage of calcium ions from cisterns in the endoplasmic reticulum. It is possible that they serve as regulators of the family of ryanodine receptors (RyR1–3), which in turn are responsible for the leakage due to CICR [71]. Several studies have established close proximity and molecular linkage between these proteins on the ER membrane [71, 72]. The deep functional relationship between presenilins and RyR is highlighted by the fact that violation of control or mutation of PSs leads to increased expression of RyR2 and RyR3 [71]. Increased levels of RyR expression are found in models expressing PS1 mutants, possibly as a compensatory mechanism associated with loss of PS leakage function [71]. It has been also suggested that N-termini fragments of presenilin-1 or -2 interact with RyR, significantly increasing its sensitivity to cytosolic calcium and the opening probability of this channel (**Figure 3**) [73]. Mutant presenilin can drastically reduce the RyR gating rate and, consequently, the leakage of calcium ions from the ER depo. However, it is important to mention that RyR leakage is directly dependent on cytosolic calcium levels. Consequently, the leak will be

enhanced at high synaptic activity and in those locations on the dendritic shaft where synapses are located. In numerous studies, it is noted that the production of ATP by mitochondria is closely related to the release of calcium through RyR in the MAM region [74, 75]. It is likely that a drop of calcium release through RyR in the MAM zone due to presenilin mutation does not provide the mitochondria with a sufficient calcium signal to initiate ATP production, even in areas of high synaptic activity. Moreover, PS mutations have been found to overload the ER with calcium ions, which are "locked" inside due to reduced leakage in mutant PSs, particularly in Alzheimer's disease (**Figure 3**) [76, 77].

Postsynaptic areas are among the major consumers of energy, which is used to maintain ionic balance, enzymatic processes, and local protein synthesis. Synapses are associated with large and sharp fluctuations in the levels of calcium ions, which are involved in signaling at the junction of the cytosol, ER, and mitochondria. This signaling is aimed at rapid and fine regulation of ATP levels. The energy supply and trafficking of mitochondrial clusters are vital for maintaining synaptic transmission. Thus, in a study by Du et al. [78], it was found that impairment of functionality, permeability, and trafficking of synaptic mitochondria occurs much earlier than among extrasynaptic mitochondria as well as long before the generation of amyloid plaques in a mouse model of AD. Histopathological hallmarks that occur during brain aging or AD are associated with functional insufficiency of synaptic mitochondria. Damaged mitochondria gradually increase and generalize oxidative stress, synaptic dysfunction, loss of contact, and culminate in neuronal degeneration [79, 80].

#### **Figure 3.**

*PS1, possibly, acts as a channel for passive Ca2+ leakage from SA and/or may increase Ca2+ − induced release of Ca2+ by RyR. The N-terminal fragment of PS1 is proposed to bind directly to RyR, increasing its sensitivity to [Ca2+]i and the probability of channel opening. Calcium ions diffuse toward the synaptic mitochondria, entering the matrix through the VDAC/MCU, where they stimulate ATP production. Mutations of PS1 gene possibly abolish the leak function, leading to an overflow of Ca2+ reserves in SA. Alternatively, mPS1 may drastically reduce the RyR gating rate and restrict leakage of calcium ions from ER store, through yet unknown mechanisms. Significantly lowered calcium levels released from SA are insufficient for local ATP production by the synaptic mitochondria. The observed increase in RyR expression in mutant PS1 may be a compensatory mechanism to increase calcium leakage from the overfilled store in the absence of correct regulation by presenilin.*

Particularly, disruptions of the mitochondrial energy metabolism are associated with proteins responsible for the production of ATP [81]. In all likelihood, it is in disorders of calcium homeostasis between synaptic mitochondrial clusters and regional calcium stores that one should look for the root causes of many, if not all, neurodegenerative pathologies.

#### **10. ER in aging and neurodegeneration**

Even in the absence of neurodegeneration, age-related cognitive decline can be observed in association with the loss of synapses in some most active areas of neocortex and hippocampus. Thus, during normal aging, there is a significant decrease in the density of "thin" small-headed spines, which are usually characterized as highly plastic learning spines, in the pyramidal cells of layer III (the most vulnerable cell population in AD) of the prefrontal cortex [82]. The decrease in density, however, has no effect or only minor effect on mushroom spines with normal aging [9]. Whereas in AD, electron microscopy reveals a pathological decrease in the overall density of spines, a decrease in their size along with the presence of abnormally large protrusions in different areas of the brain, as well as morphological abnormalities of the SA and mitochondria (**Figure 4**) [83].

Possibly that a part of the entire pool of thin plastic spines is enriched with ER due to extensive synaptic activation leading to LTP initiation. In its turn, activation could stabilize spine morphology during LTP processes, and increase the head volume, thus, moving them into the pool of mature spines [84, 85]. The remaining poorly activated group of thin spines that did not receive ER, may be completely eliminated. Moreover, the increased and clustered synaptic inputs of such thin highly plastic spines may partially compensate for the bald spots of the synaptic network and improve test performance in plasticity and cognitive abilities [86]. Expectedly, this requires recruiting more mitochondria through local calcium activity. Calcium dysregulation from ER stores may worsen plasticity indicators, reducing the probability of ER penetration into thin, synaptopodin-negative spines, depriving them of the opportunity to achieve energy and mature, while leaving only the perspective of elimination.

In long-term cultured rat hippocampal neurons (15–21 DIV) that mimic physiological aging, a dramatic decrease in SOCE and Orai1/STIM1 is observed, while calcium content in ER stores and the caffeine-induced Ca2+ release from the ER are elevated on the contrary. A mechanism of age-dependent SOCE suppression may be proposed to explain the loss of mushroom spines in both physiological aging and cognitive decline in AD [14, 87]. Regulation of the mechanism of SOCE by σ-1R is another important factor in the stabilization of large dendritic spines. Thus, instability of large spines in the hippocampus was observed following dysregulation of the normal SOCE mechanism by σ-1R, in several disorders including AD [88].

Impaired transmission of ER-mitochondrial Ca2+ also carries negative physiological consequences for the senescent neurons, since such transmission is necessary to ensure stable ATP flashes from postsynaptic mitochondria upon request. Effective reuptake of Ca2+ leakage from ER drops as a result of mitochondrial depolarization in senescent neurons. As a compensatory mechanism for this, cells may develop an increase in the number of MERC contacts, which paradoxically lead to [Ca2+] m overload, disrupting mitochondrial functions and ATP production, respectively [87]. A similar situation is observed in neurodegeneration when the regulation of Ca2+ leakage from the ER into mitochondria is disrupted by mutant presenilin,

#### **Figure 4.**

*The ubiquitous mobile ER in young neurons penetrates the pool of thin highly plastic spines more often, thus transforming into spine apparatus and stabilizing the spines. Mitochondria are extensively recruited toward the dendritic region under active postsynaptic compartments to contact ER and make a targeted ATP release. During normal aging, mitochondria drop their resting membrane potential and the ability of addressed ATP production. Consequently, most stable mushroom spines recruit more mitochondria to compensate the lack of ATP/energy by expanding the MERK regions. During neurodegeneration, dysfunctional mitochondria are unable to provide sufficient ATP inflow and calcium sequestration, causing degradation of mature spines and overflow of ER stores with calcium, which leads to a general disruption of ER, malfunction, and, finally, the cell death.*

which pathologically overloads the stores with calcium [89]. However, abnormally increased levels of calcium, when released upon activation, may become inefficient and even toxic for mitochondria that "tuned themselves" to poor consumption of Ca2+ in MERC. Such disorganization can be superimposed on the already existing aging-induced functional disorders of MERC/MAM and mitochondrial homeostasis (**Figure 4**).

The multidimensionality of subtle changes in the status of ER stores and their consequences, ER dynamic omnipresence in neurons and close functional relationship with mitochondria as well as other membrane organelles, makes it among the main players in the pathological processes of aging and neurodegeneration.

#### **11. Conclusions**


spent on maintaining ion homeostasis, enzymatic processes, and local protein synthesis.


### **Acknowledgements**

The authors thank prof. Menahem Sehgal for constructive comments during the preparation of the manuscript.

#### **Conflict of interest**

The authors declare no conflict of interest.

*Updates on Endoplasmic Reticulum*

### **Author details**

Liliya Kushnireva and Eduard Korkotian\* Department of Brain Sciences, The Weizmann Institute, Rehovot, Israel

\*Address all correspondence to: eduard.korkotian@weizmann.ac.il

© 2022 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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#### **Chapter 5**

## The Role of Endoplasmic Reticulum Stress and Its Regulation in the Progression of Neurological and Infectious Diseases

*Mary Dover, Michael Kishek, Miranda Eddins, Naneeta Desar, Ketema Paul and Milan Fiala*

#### **Abstract**

The unfolded protein response (UPR) is a cellular mechanism activated by endoplasmic reticulum (ER) stress, which ranges from inhibition of protein synthesis to apoptosis. ER stress is induced in general by aggregated autologous or foreign (e.g. viral) proteins, oxidative stress, mitochondrial dysfunction, disruption of intracellular calcium, or inflammation. In patients with Alzheimer's disease (AD) and amyotrophic lateral sclerosis (ALS), the known stressors are aggregated amyloid-beta and superoxide dismutase (SOD-1), respectively, but autologous DNA released by trauma into the cytoplasm may also be involved in ALS. In HIV-1-associated neurocognitive disorders (HAND), ER stress is induced by HIV-1 and antiretroviral therapy. Additionally, in cases of epilepsy, ER stress has been implicated in neuronal dysfunction. In this chapter, we examine a clinical and immunologic approach to ER stress in the progression of neurological and infectious diseases. In addition, we will briefly discuss emerging treatments including omega fatty acids, progesterone, and DHA, which repair and favorably regulate UPR in some patients with neurological diseases.

**Keywords:** ER stress, UPR, Alzheimer's disease, amyotrophic lateral sclerosis, HIV, epilepsy

#### **1. Introduction**

For decades, researchers in a diverse array of scientific fields have been working to understand the pathogenesis of neurodegenerative diseases including Alzheimer's disease (AD) and amyotrophic lateral sclerosis (ALS). However, there is still much discourse within the scientific community on what exactly causes such diseases to occur. In recent years, the endoplasmic reticulum (ER), has been implicated in the progression of neurological diseases [1] including AD, ALS, epilepsy, and human immunodeficiency virus (HIV)-associated neurocognitive disorder (HAND). To understand how exactly this cellular organelle can lead to such detrimental neurological disorders, a basic understanding of the function of the ER is necessary.

The ER is a cellular organelle that functions to fold proteins correctly following their translation by ribosomes. The ER also plays an integral role in physiological homeostasis, primarily via calcium regulation [2] and protein synthesis [3]. Typically, correctly folded proteins are secreted by the ER and are transported to the Golgi body for further processing and sorting for transport to their eventual destinations. However, ER overload results in protein misfolding and accumulation of unfolded proteins in the ER lumen, which induces ER stress [4]. ER stress signals the cell to enter the survival pathway and initiates an unfolded protein response (UPR) [5] through three ER membrane-associated proteins: inositol requiring enzyme 1 (IRE1), pancreatic ER kinase (PERK), and activating transcription factor-6 (ATF6) [6].

IRE1 (encoded by the *ERN1* gene) is an ER transmembrane receptor sensitive to misfolded protein aggregation in the ER lumen [7]. Activated IRE1 stimulates the translation of the X-box binding protein 1 (XBP1) into a transcription factor, which upregulates ER chaperones and ER-associated degradation (ERAD) [8]. Ideally in a functioning ER, these ERAD elements will assist with the degradation of unfolded proteins within the ER lumen to reduce ER stress.

PERK reduces protein synthesis by inactivation of eukaryotic initiation factor 2α, leading either to a pro-apoptotic pathway or a protective pathway involving chaperones and foldases [9]. Thus, PERK expression will mitigate the additional buildup of proteins within the ER. However, in extreme conditions, the cell suffers apoptosis in response to excessive misfolded protein aggregation [10].

Finally, ATF6 increases the transcription of several ER proteins including chaperones, foldases, and ERAD elements. These ER proteins increase the cell's ability to fold proteins and reduce the load of unfolded proteins in the ER [11].

These ER stress proteins communicate between ER and mitochondria in the region called the mitochondria-associated ER membrane [9]. Activation of all three of these transmembrane proteins is prompted by unfolded protein buildup in the lumen of the ER, which in turn activates the nuclear transcription of ERAD elements (**Figure 1**).

Thus, the role of ER stress in the progression of AD, ALS, epilepsy, and HAND are of rising interest in the scientific community and may serve as a possible explanation

*The Role of Endoplasmic Reticulum Stress and Its Regulation in the Progression of Neurological… DOI: http://dx.doi.org/10.5772/intechopen.105543*

for their development. Due to the increasing evidence implicating ER stress in these diseases, researchers have shifted their focus to studying possible treatments that target the ER to mitigate disease progression. In this chapter, we will discuss the role of ER stress in AD, ALS, epilepsy, and HAND, and review the current therapeutic options for treating ER stress such as omega fatty acids including docosahexaenoic acid (DHA), and progesterone.

#### **2. ER stress and the UPR in Alzheimer's disease**

AD is a progressive neurodegenerative disease characterized by the buildup of the cytotoxic protein aggregates amyloid-beta (Aβ) and fibrillary phospho-tau (P-τ). Within the brain, Aβ accumulates into plaques that can block synaptic function [12], induce ER stress [13], and ultimately cause neuronal apoptosis [14]. These cytotoxic proteins ultimately lead to widespread neurodegeneration leading to cognitive decline [12]. AD can arise via genetic factors in familial AD (fAD) or lifestyle factors in sporadic AD (sAD), but ER stress has been shown to play a role in the progression of both cases.

Many factors including oxidative stress, mitochondrial dysfunction, glutamateinduced neurotoxicity, and imbalance of calcium contribute to ER stress and UPR induction in AD patients [15].

Oxidative stress in the cell often results from the buildup of reactive oxygen species (ROS) from dysfunctional mitochondria [16] and can induce ER stress. In patients with AD, an imbalance in antioxidants can lead to an increase in ROS which can cause widespread cell damage [16]. In normal aging, this antioxidant imbalance is increasingly common and the body is often pushed into a "pro-oxidative state" meaning that the level of ROS is higher than normal [17]. However, in patients with AD, the aggregation of Aβ exacerbates this pro-oxidative state [18]. A 2001 study revealed that Aβ's interaction with Iron within the brain causes increased ROS, and by treating aggregated Aβ with an Iron chelator there is a marked reduction of neural toxicity [18]. Typically, ROS levels are mediated by the ER proteins PERK and ATF6. However, when ROS levels exceed the normal range, PERK and ATF6 are unable to produce enough antioxidants, and the ER enters a state of ER stress [17], exacerbating cellular dysfunction and leading to neuronal apoptosis (**Figure 2**).

Additionally, glutamate has been implicated in the buildup of ROS in the AD brain. When glutamate binds to NMDA receptors, this prompts glutamate-induced neurotoxicity (GNT). GNT causes a major influx of calcium into the cell and prompts the release of ROS from the mitochondria [19]. A buildup of GNT in the AD brain can impair glutamate receptors necessary for metabolism [20], lead to increased production of ROS, and cause an imbalance of cytosolic calcium leading to cell death (**Figure 2**) [19].

However, the exact etiology of AD concerning ER stress is still under investigation. Recent studies have pointed to the buildup of Aβ and P-τ as the cause of the development of ER stress and the UPR [13]. On the other hand, some believe that ER stress arises from increased ROS in the brain, leading to the buildup of Aβ and P-τ, which are released into the brain following cellular apoptosis [21]. Therefore, the sequence of biochemical events in AD is still very much under debate.

Additionally, the presenilin proteins have been implicated in ER-stress-mediated AD pathogenesis. Presenilin 1 (PS1) and presenilin 2 (PS2) are part of the γ-secretase complex which mediates the cleavage of the amyloid-beta precursor protein (APP) [22]. In fAD, mutations in these proteins have been found to alter the amyloid

#### **Figure 2.**

*A scheme depicting the effect of decreased antioxidants, glutamate neurotoxicity (GNT), and amyloid-beta (Aβ) aggregation on the buildup of ROS and ER stress in AD patients. Image created with Biorender.com.*

precursor protein (APP) cleavage process, resulting in higher levels of cytotoxic Aβ [21]. The mutant PS1 affects the ER stress response attributed to the inhibited activation of the ER stress pathways IRE1, PERK, and ATF6. Cells expressing PS1 mutants also display increased Aβ production and increased sensitivity to apoptosis caused by ER stress [21]. Therefore, the damage associated with the PS1 mutant proteins cannot be reversed by chaperones and folding proteins, and apoptosis is the most common outcome. In patients with sAD, mutations in the PS2 gene are also linked to disease progression by downregulation of the UPR pathways [23].

The target genes of the transcription factor XBP1 are also linked to AD. XBP1 affects the expression of at least one of the key proteins in the γ-secretase complex, primarily UBQLN1, the gene coding for ubiquitin, which is a negative regulator of presenilins. UBQLN1 plays a role in the control of APP trafficking. Therefore, in the production of Aβ, reduced expression of XBP1 in AD increases the production of Aβ and causes apoptosis [24].

Thus, the combined effects of a buildup of ROS, mitochondrial dysfunction, GNT, and presenilin mutation exacerbate the effects of normal aging in patients with AD and lead to an increase in ER stress. Thus, ER stress has been shown to play an integral role in the pathogenesis of AD through several pathways.

*The Role of Endoplasmic Reticulum Stress and Its Regulation in the Progression of Neurological… DOI: http://dx.doi.org/10.5772/intechopen.105543*

#### **3. ER stress in epilepsy**

Epilepsy is a neurological disease that involves chronic seizures as well as abnormal brain activity, which causes periods of unusual behavior or sensations [25]. There are different types of epileptic seizures, including generalized, focal, and unknown onset epilepsy, which differ in the area in which seizures occur [26]. The most common type of epilepsy among adults is focal epilepsy known as temporal lobe epilepsy (TLE), however, patients who continue to experience seizures are at risk of other areas of the brain becoming damaged, such as the hippocampus [27].

Current research incriminates ER stress as an important factor in the pathogenesis of epilepsy [25]. Neuronal death after a seizure is caused by apoptotic signaling pathways, of which there have been two gene families identified to be critical. These gene families are the Bcl-2 family proteins and the caspases [27], which have both been linked to ER stress generation [28]. Bcl-2 proteins are apoptosis regulators, and caspases are proteases that cleave certain proteins and enzymes that induce apoptosis. A study on the hippocampus of patients with epilepsy in 2006 revealed that caspases 6, 7, and 9 were higher in patients with epilepsy than in controls and were localized to the ER-containing region of the temporal lobe [27]. Additionally, patients' hippocampal regions contained altered levels of Bcl-2 protein and an increase in both the ER stressrelated motif KDEL and calnexin [27]. KDEL and calnexin are markers for ER stress and were used here to examine levels of ER stress in epilepsy patients [27, 29]. Finally, the researchers showed co-localization of the ER stress marker KDEL and caspases within the epileptic patients' brain [27]. Thus, this study found that apoptotic pathways in epilepsy are directly linked to the ER and can induce ER stress.

The ER stress-related pathways discussed earlier were also abnormal in animal studies. In a rat model of status epilepticus (SE), phosphorylated PERK (p-PERK), phosphorylated eIF2α (p-eIF2α), and C/EBP homologous protein (*CHOP*) [30] were increased. Although the *CHOP* protein was considered as causing cell death, it has been reinterpreted as protective against neuronal death after a seizure [31]. In another study, the hippocampus of mice with status epilepticus (SE) was found to have elevated mRNA levels of spliced XBP1 compared to controls [31], suggesting an activation of the IRE1 branch. Higher XBP1 levels lead to an increase in the expression of chaperones for ER stress reduction. In the same study, it was found that these mice also had increased levels of ATF in the hippocampus [31]. Therefore, ER stress in epilepsy appears to induce both injurious pro-apoptotic and beneficial anti-apoptotic pathways.

#### **4. ER stress in amyotrophic lateral sclerosis**

The neurologic disorder ALS is characterized by its neuropathology demonstrating neuronal apoptosis in the spinal cord and brain, and inflammatory attack of neurons induced by aggregated superoxide dismutase (SOD-1) and other stimuli [32, 33]. The progression of ALS is associated with a gradual decrease in movement, paralysis, and overall weakness [34]. ALS has a sporadic form (sALS) with an unknown cause and a familial form (fALS) [35].

ER stress in individuals with fALS can be triggered by the Cu/Zn SOD-1 protein malfunction. This protein is responsible for degrading ROS, therefore, a mutation in SOD-1 promotes the disease [36]. As previously described, the increase of ROS results

from the increase in GNT and a greater amount of calcium within the cell, damaging both the ER and mitochondria [1]. Disease progression of ALS is identified by mitochondrial dysfunction and a change in the mitochondrial membrane structure due to a release in molecules that initiate apoptosis [21]. Due to the aggregation of mutated SOD1 in the mitochondria of motor neurons, motor skills are adversely affected [35]. The protein previously implicated in neuronal apoptosis in epilepsy, Bcl-2, is also responsible for maintaining the structural integrity of the mitochondrial membrane [37]. When mutated SOD-1 interacts with the Bcl-2 protein in ALS patients, the membrane is weakened. Thus, there is an increase in ROS production due to the mutation of the SOD-1 protein creating an increase in ER stress in individuals with ALS (**Figure 3**) [21].

In ALS individuals, caspase activation is also common [38]. We know from caspase's role in epilepsy that caspases have been implicated in neuronal apoptosis and ER stress genesis [28]. Caspase activation is especially prevalent when the SOD1 mutation is present. In a mouse model of mutant Cu/Zn SOD-1 exhibiting symptoms of ALS, an increase in oxidative stress was related to activation of caspase 12, 9, and 3 in the spinal cord [38]. Caspase 12 is located in the endoplasmic reticulum [39] wherein the cleavage of this enzyme overall increases the oxidative stress further causing an exaggeration of ALS symptoms in the mice. This cleavage of caspase-12 may be a result of the activation of calpain, a calcium-dependent enzyme, in the mice's spinal cord [21].

Thus, SOD-1 aggregates in individuals with ALS exacerbate ROS release and mitochondrial dysfunction, which induces ER stress and leads to eventual neuronal apoptosis.

#### **5. ER stress in human immunodeficiency virus 1**

Human immunodeficiency virus 1 (HIV-1) is the result of acquired immunodeficiency syndrome (AIDS), characterized by the destruction of CD4 T cells [40]. In addition to the dramatic opportunistic infection *Pneumocystis carinii* pneumonia, AIDS is associated with degenerative complications in the brain and the heart, HIV-1

#### *The Role of Endoplasmic Reticulum Stress and Its Regulation in the Progression of Neurological… DOI: http://dx.doi.org/10.5772/intechopen.105543*

encephalitis, and cardiomyopathy, which display inflammatory damage by virusinfected monocyte/macrophages [40]. HIV-1 particles are carried into the brain and the heart in monocytes through interendothelial gaps opened by virus envelope gp120, TNF-α, and the antiretroviral (ART) drug azidothymidine [41]. Because of this, besides encephalitis, the neuropathology of HIV-1 includes subtler HIVassociated neurocognitive disorders (HAND) [42].

ER stress and UPR activation play a large role in the neurodegeneration of AIDS and HIV-1 patients and, thus, are a potential therapeutic target. In HAND, the expression of the three UPR pathways in astrocytes leads to the opening of the mitochondrial permeability transition pore (mPTP) linked to apoptosis [42]. The inflammatory cytokine IL-1β along with ART drugs increases cytosolic calcium and triggers ER stress through upregulation of UPR pathways, mitochondrial depolarization, excitotoxicity, and increased ROS [43]. A recent study in 2020 examined the effects of ART on the ER stress pathway in HAND. The researchers found that HIV along with IL-1β increased the UPR transcripts IRE1, PERK, and ATF6. Another HAND signal, the nucleotide reverse transcriptase inhibitor (NRTI) abacavir, upregulated AEG-1 transcription and regulated calcium signaling and ER quality control by co-localizing with calnexin, an integral calcium-dependent chaperon protein in the ER [43]. Together, IL-1B and abacavir induce increased intracellular calcium (via ER calcium release) in astrocytes to levels comparable to that of the known ER stressor thapsigargin [43]. A prolonged intracellular increase in calcium triggers mitochondrial depolarization and ER stress response, as it increases mitochondrial permeability transition pore (mPTP). The mPTP opening leads to increased ROS as well as calcium-dependent exocytosis of glutamate, which causes GNT and neuronal damage experienced in HAND [43].

Additionally, HIV-1 upregulates the Tat protein, which induces GNT, ER stress, mitochondrial dysfunction, and UPR [44]. Therefore, astrocyte ER stress could act as a therapeutic target for HIV-1 neuronal infection: IL-1β and abacavir induce intracellular calcium dysregulation and mitochondrial dysfunction (mPTP opening), which can lead to apoptosis via triggering ER stress and increase in UPR signaling pathways [43].

#### **6. Therapies for treating ER stress in neurodegenerative patients**

Due to its implications in a variety of neurodegenerative diseases, ER stress and UPR proteins are growing targets for immunotherapy treatment for several neurological disorders. Various lipid-based molecules such as omega-3 fatty acids and progesterone induce neuroprotective effects against ER stress, regulating Aβ-induced neuroinflammation.

Omega-3 fatty acids have been found to modulate UPR counteracting ER stress. In macrophages of patients with AD and mild cognitive impairment (MCI), *in vitro* supplementation with fish-derived ω-3 fatty acids resulted in downregulation of ER stress signature genes *CHOP, DDIT3*, and *CASP3* (caspase-3), which typically promote apoptosis [15]. Further, fatty acid treatment upregulated genes associated with UPR proteins including *IRE1*, *ATF6*, and *ATF4* [15]. Thus, the researchers concluded that the phospho-PERK pathway was heterogeneously affected by omega-3 fatty acids via an increase in immunoenhancement but a decrease in pro-apoptosis, but omega-3 can enhance UPR genes necessary for combating ER stress in patients with AD [15].

Docosahexaenoic acid (DHA), a specific omega-3 fatty acid beneficially responds to the ER stress induced by traumatic brain injury (TBI), which triggers calcium homeostatic disruption. The mechanism by which DHA prevents ER stress is through its protectins, which block ER stress-inducing IP3R-mediated ER Ca2+ depletion [45]. Protectins such as neuroprotection D1 (NPD1), a DHA derivative, upregulate anti-apoptotic factors and downregulate pro-apoptotic factors [45]. In addition, DHA's resolving derivatives also directly decrease the inflammation responses to ER stress by blocking pro-inflammatory cytokines TNF-α and ILβ1 as well as reducing pro-inflammatory mediators prostaglandin E2, thromboxanes, and leukotrienes [45]. DHA administration reduced post-TBI increase of CHOP*-*gene expressing microglia and macrophages [46]. Thereby, DHA as an omega-3 fatty is seen to decrease the triggering and the effects of ER stress through various mechanisms and is, therefore, a potential therapy.

The neurosteroid progesterone has additionally been seen to improve ER stress in its applications in AD astrocytes with the release of IL-1 and TNF-α [47]. In these AD cells, Aβ-induced ER stress and inflammation were mediated by progesterone. Progesterone is associated with the downregulation of pro-inflammatory cytokines which in turn reduces ER stress activation. This effect is further shown through progesterone's attenuation of GRP78 expression, which is implicated in amyloid-betainduced ER stress response [47].

Though therapies targeting neurodegenerative diseases such as ALS or epilepsy have not implicated ER stress as a potential therapeutic target, substantial research on ER stress treatment in AD brains has uncovered omega-3 fatty acids such as DHA and neurosteroid progesterone as potential supplemental therapies for ER stress and neuroinflammation.

#### **7. Conclusions**

ER stress has been implicated in several neurological and infectious diseases, primarily AD, ALS, epilepsy, and HIV-1. In AD, mutant PS1 and PS2 induced amyloid-beta aggregation and an increase in ROS lead to ER stress and neuronal death. In ALS, aggregated SOD-1 increases the buildup of ROS in the cytoplasm, inducing ER stress. Patients with epilepsy have also shown elevated ER stress with increased levels of pro-apoptotic chaperones, yet the exact mechanism is still under investigation. Finally, HIV-1-associated neurocognitive decline leads to a buildup of the pro-inflammatory cytokine IL-1B which induces ER stress and exacerbates neurocognitive decline. The apparent role of ER stress in the progression of these neurological and infectious diseases has prompted scientists to explore treatments targeting ER stress and the UPR. Treatment with omega fatty acids, progesterone, and DHA have shown positive effects on ER stress levels in patients with AD, yet a true treatment has not been developed.

#### **Conflict of interest**

The authors declare no conflict of interest.

*The Role of Endoplasmic Reticulum Stress and Its Regulation in the Progression of Neurological… DOI: http://dx.doi.org/10.5772/intechopen.105543*

#### **Author details**

Mary Dover, Michael Kishek, Miranda Eddins, Naneeta Desar, Ketema Paul and Milan Fiala\* Department of Integrative Physiology and Biology, UCLA School of Life Sciences, Los Angeles, California, USA

\*Address all correspondence to: mfiala@ucla.edu

© 2022 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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*The Role of Endoplasmic Reticulum Stress and Its Regulation in the Progression of Neurological… DOI: http://dx.doi.org/10.5772/intechopen.105543*

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#### **Chapter 6**

## Advanced Glycation End Product Induced Endothelial Dysfunction through ER Stress: Unravelling the Role of Paraoxonase 2

*Ramya Ravi and Bharathidevi Subramaniam Rajesh*

#### **Abstract**

Hyperglycemia accelerates the formation of advanced glycation end products (AGEs). AGEs are a heterogeneous group of compounds generated by non-enzymatic glycation of proteins or lipids with glucose through Amadori rearrangement and its accumulation increases with aging in diabetes. AGEs augments ROS generation, diminishes the antioxidant defense of the cells, decreases mitochondrial membrane potential, ATP production, and elevates the levels of mitochondrial fission protein (Drp1) and mitophagic proteins (Parkin and PTEN) leading to dysfunction of mitochondria. In this chapter, we have discussed how AGEs trigger the endoplasmic reticulum stress and inflammation and mediate endothelial dysfunction in diabetes and also have discussed the role played by endogenous Paraoxonase 2 (PON2) in mitigating endothelial dysfunction by inhibiting the adverse effects of AGE.

**Keywords:** advanced glycation end product, ER stress, mitochondrial fission, paraoxonase 2

#### **1. Introduction**

Diabetes mellitus (DM) is a growing metabolic health problem, and around 346 million people are currently affected by diabetes worldwide, and it is anticipated to double by 2030 [http://www.who.int/mediacentre/factsheets/fs312/en/index.html]. Macro and microvascular complications are the major cause of morbidity and mortality in patients with type 2 diabetes [1]. The important pathology associated with vascular diseases is the formation and accumulation of atherosclerotic plaques leading to the process of atherosclerosis, which ultimately results in the narrowing of the blood vessels [2].

Vascular endothelial cells (ECs) are monolayers of cells that line the inner surface of the luminal vessel wall. It acts as a physical barrier between the bloodstream and the luminal wall [3, 4]. The term endothelial cell dysfunction (ECD) is referred to as loss/dysregulation of EC cells function: such as impairment of the barrier function, reduction of the anti-coagulants, disturbance in the balance between the vasodilation and vasoconstrictions, increased pro-inflammatory response, attachment, and adhesion of leukocytes to

the endothelial cell surface, increase the production of pro-oxidant molecules [5, 6]. These points highlight the importance of ECs in maintaining normal vascular homeostasis.

During endothelial injury, oxidized lipids and protein adducts (advanced glycation end products) accumulate in the arterial walls. Then the circulating monocytes adhere to the endothelial cells that express adhesion molecules, such as vascular adhesion molecule-1 (VCAM-1) and selectins, and then migrate into the sub-endothelial space. These monocytes that infiltrate the arterial wall get differentiated into macrophages, and this macrophage also accumulates oxidized lipids to form foam cells. Then the foam cells attract the T-lymphocytes, which in turn induce the proliferation of smooth muscle cells in arterial walls. The entire process leads to the formation of a lipid-rich atherosclerotic lesion and rupture of this lesion leads to vascular infarction. Additionally, increased platelet aggregation and coagulation are also observed due to impaired nitric oxide generation, free-radical generation from the platelet, and elevated levels of plasminogen activator inhibitor. All these factors contribute to the vascular wall occlusion and further increase the risk of cardiovascular events as shown in **Figure 1** [2, 7].

Numerous studies have demonstrated the existence of an association between elevated AGE levels and cardiovascular disease in DM patients. In line with this, clinical studies reported that CML and pentosidine levels are increased in the progression of the disease and act as a predictor of cardiovascular events [8–14]. A fourfold increase in the incidence of coronary artery disease, a 10-fold increase in peripheral vascular disease and a 3–4-fold higher mortality rate with as much as 75% of diabetics ultimately dying from vascular disease have been reported [15].

Vascular complications are classified into two types, namely macrovascular and microvascular. Wherein the large vessels such as arteries and veins get affected in macrovascular disease and microvascular involves small vessels such as capillaries. Chronic hyperglycemia initiates the production of AGE and turns on diabetic vascular complications through elevated production of reactive oxygen species, which

*Advanced Glycation End Product Induced Endothelial Dysfunction through ER Stress... DOI: http://dx.doi.org/10.5772/intechopen.106018*

instigates various signaling cascades such as Receptor for advanced glycation end product receptor (RAGE) activation, protein kinase C (PKC) and Mitogen activated protein kinases (MAPK) pathways. Macrovascular complications associated with diabetes are arteriosclerotic cardiovascular diseases (ASCVDs) such as coronary heart disease (CHD), peripheral artery disease (PAD), and stroke [2]. Microvascular complications associated with diabetes are diabetic retinopathy (DR), neuropathy, and nephropathy [16, 17] as shown in **Figure 2**.

The pathogenic mechanisms underlying diabetic nephropathy involve the generation of reactive oxygen species (ROS), accumulation of AGEs, and activation of intracellular signaling molecules such as PKC [18, 19]. Studies suggest that increased formation of AGEs in the vitreous may be involved in the development of diabetic retinopathy by inducing the production of interleukins −6 (IL-6) from retinal Müller cells [20]. AGEs interaction RAGE plays an important role in the pathogenesis of DR [21]. In DR, AGEs (both early and late Amadori products) have been localized to vascular cells (endothelial and pericytes), neurons, glia, and also in the vitreous [22, 23]. Pentosidine and Nε-(carboxymethyl)lysine (CML) levels are increased in aqueous, vitreous, and serum of DR patients when compared with non-diabetic controls especially increased in proliferative DR (PDR) patients than non-proliferative DR (NPDR) and referred to as biomarker for microvascular complications [24, 25] with the progression of DR with decreased visual acuity, emphasizing that AGEs are novel biomarkers/risk markers for type 2 DR [26, 27]. In addition to the above AGEs, methylglyoxal derivative hydroimidazolone was also increased in DR patients [28, 29].

AGEs are a heterogeneous group of compounds generated by non-enzymatic glycation of proteins or lipids with glucose through Amadori rearrangement and

**Figure 2.** *AGEs-induced macrovascular and microvascular complications.*

its accumulation increases with aging and in diabetes [30]. The term AGEs has been applied to a broad range of advanced glycation end products such as CML, Nε -(carboxymethyl)hydroxylysine, pyrraline, and pentosidine [31]. Among these, CML is the predominant epitope in the AGE adducts detected in tissue proteins of diabetic patients [32–34]. Its level is found to be increased in serum and aqueous humor of type 2 diabetic patients with retinopathy (DR) [2]. It is also used as a biomarker to predict the progression of different stages of DR. AGEs-stimulated cell response is initiated by its engagement with the receptors present on the cell surface [35]. The most studied AGE receptor is RAGE. Several other AGE receptors identified so far consist of AGE-receptor complex (AGER1/OST-48, AGER2/80 K-H, AGER3/Gal-3), some members of the Toll-like Receptor (TLRs) family (TLR4, TLR2), and scavenging receptors (SRs) family (SR-AI, SR-AII, CD36, LOX-1, FEEL-1, and FEEL-2). The expression of these AGE-activated receptors depends on the cell/tissue type [36].

RAGE and TLRs are well-known Pattern Recognition Receptors (PRRs), which recognize molecules found in pathogens (Pathogen-Associated Molecular Patterns–PAMPs, ex-LPS) or molecules released from damaged or stressed cells such as high mobility group B (HMGB1) and serum amyloid proteins referred as Damage-Associated Molecular patterns (DAMPs) [37]. It is expressed on the surface of various cells, including endothelial, epithelial, and fibroblast mediates multiple signaling pathways such as MAPK kinase and Nuclear factor kappa B (NFĸB), which activate a pro-inflammatory response [38, 39]. Among various TLRs, TLR-2 and 4 are reported to be increased in monocytes of type 1 and type 2 diabetic patients [40, 41] associated with microvascular complications such as retinopathy, nephropathy, and neuropathy [42].

Although various pathways are involved in the pathogenesis of endothelial dysfunction such as activation of the polyol pathway, auto-oxidation of glucose, PKC pathway activation, and formation of AGEs, all these pathways may intersect at several points to increase the complexity of the disease [43]. Among these, increased formations of AGEs (advanced glycation end product) is one of the causes of ECs dysfunction, which has been implicated in the pathogenesis of diabetes-induced vascular complications, as evident by their *in vivo* accumulation as reported in numerous diseases [44]. In this chapter, we would focus on how AGEs induce an inflammatory response, mitochondrial dysfunction, and endothelial dysfunction through ER stress.

#### **2. AGE-induced effect in endothelial cells**

The high glucose-induced "oxidative stress" and "endoplasmic reticulum (ER) stress" of the endothelium may play major roles in the initiation and progression of cardiovascular clinical manifestations in diabetes [45]. While diabetes management has largely focused on the control of hyperglycemia, the rising burden of this disease is mainly correlated to its vascular complications [46]. This is reflected by a type II diabetes differs principally from type I diabetes in that it is accompanied by a period of hyperinsulinemia and is characterized by late as opposed to early onset of hyperglycemia. In type I DM, vascular involvement (through endothelial dysfunction) occurs as a result of metabolic insult/hyperglycemia, while in type II DM, endothelial dysfunction plays a more direct role and is aggravated by, rather than caused by, hyperglycemia [6].

*Advanced Glycation End Product Induced Endothelial Dysfunction through ER Stress... DOI: http://dx.doi.org/10.5772/intechopen.106018*

#### **2.1 AGE and oxidative stress**

One of the best-characterized actions of AGEs on ECs is the induction of ROS [47, 48]. There is considerable evidence to show that AGE induces ROS generation and diminishes the antioxidant defense of the cells [49–51].

Oxidative stress is induced either by the abundant production of ROS or the failure of the antioxidative machinery mechanism. The main source of ROS is the electrons present in the mitochondrial respiratory chain, which results in the formation of superoxide anion (O2-), hydroxyl radical (OH.) hydrogen peroxide (H2O2). AGEs induce mitochondrial dysfunction through the generation of ROS production.

Mitochondria are complex organelle that undergoes complete fusion and division under physiological or pathological conditions. Mitochondrial fission is defined as the division of a mitochondrion from two or more separate mitochondrial compartments, and this process is essential in the distribution of mtDNA during cell division and helps in the removal of damaged mitochondria through mitophagy. Mitochondrial fusion is merging two or more mitochondria. It is regulated by at least three proteins: optic atrophy 1 (OPA1), mitofusin 1 (MFN1), and mitofusin 2 (MFN2), and mitochondrial fission are controlled by dynamin-related protein 1 (DRP1/DLP1/DNM1), fission 1 (FIS1), and mitochondrial fission factor (MFF) [52, 53]. Studies show that in diabetes, mitochondrial fission is increased [54–56]. Diabetic animal model (diabetic-STZ model) study demonstrated that HG alters the mitochondrial respiration and alters glomerular bioenergetics, whereas podocytes isolated from those mice showed fragmented mitochondria [57]. Another study in Sprague-Dawley rats (STZ induced) reported an increase in neuronal pyknosis with increased DRP1 expression in neurons when compared with Normal control (NC) group associating that High glucose (HG) aggravated ischemic brain damage and alteration in mitochondrial dynamics [58]. Very importantly, AGEs such as AGE-BSA promoted mitochondrial fission, loss of membrane potential, and apoptosis through the RAGE pathway, whereas blocking the RAGE signaling reduces the events of mitochondrial abnormalities in endothelial and osteoblastic cells [59, 60]. In a rodent model, AGE-BSA disturbs the mitochondrial respiratory chain and induces mitochondrial pore formation with an increase in RAGE expression suggesting the fact that AGE-induced mitochondrial dysfunction plays a major role in diabetic neuropathy [61]. Proteomics study by tandem mass spectrometry (nanoLC-ESI-ETD MS/MS) revealed that renal tubular cells exposed to HG increased phosphorylation and oxidation of mitochondrial proteins when compared with the NG [62]. CML-BSA, another AGE, produces detrimental effects in diabetic db/db mice inducing mitophagy. In this study, CML-BSA treatment in pancreatic β-cells increased RAGE and ROS with a decrease in membrane potential and ATP production with elevated levels of mitochondrial fission (Drp1) and mitophagic proteins (Parkin and PTEN) supporting the factor that increases the concentration of AGEs damage the β-cells and reduces the insulin cells function by making vulnerable to AGEs-induced damages [63].

Under physiological conditions, low concentrations of ROS are needed to maintain cellular proliferation, migration, and survival [64]. However, during the pathological condition, overproduction of ROS induces deleterious effects on the cells and tissues, and to overcome this, the cellular environment has an antioxidative defense system that is capable of scavenging the ROS. The antioxidant defense system includes low-molecular ROS scavengers, antioxidative enzymes, and degrading or repairing proteins [50]. The low-molecular ROS scavengers include glutathione (GSH), vitamin C, and D. Secondly, the antioxidative enzymes superoxide dismutases (SOD) and catalase, which can convert ROS into less reactive ions. The last part of the defense

system includes the proteasome and protease system, which will degrade the damaged proteins. The impairments in the antioxidant defense system lead to oxidative stress, which eventually activates various cellular signaling pathways.

#### **2.2 Pro-inflammatory response by AGE**

Further, AGEs also increase pro-inflammatory response, by augmenting endoplasmic reticulum (ER) stress and amplifying the angiogenic potential of ECs, which has been observed in a myriad of human diseases such as atherosclerosis, acute/chronic inflammatory diseases, vascular complication, and aging [65–68].

Numerous AGEs-induced vascular diseases display elevated levels of pro-inflammatory cytokine either by directly activating ROS or through engaging with RAGE receptor. AGE-RAGE is a prominent axis that facilitates the activation of NFĸB and MAPK signaling pathways, which subsequently induces the expression of cytokines, chemokines, and adhesion molecules [38, 69]. Elevated levels of AGEs enhanced the release of pro-inflammatory cytokines such as interleukin-6 (IL-6), interleukin-8 (IL-8), tumor necrosis factor (TNF-α), and interferon-beta (IL-1β). RAGE also binds HMGB1, commonly referred to as damage-associated molecules (DAMPs), and acts as an inflammatory mediator and is released into circulation when there is an injury [70]. Recognition of HMGB1 by RAGE induces the NFĸB activation and its downstream cytokines IL-6, IL-8, and TNF-α. [70, 71] Accumulating evidence shows that DAMPs are reported to mediate the pathogenesis of atherosclerosis and diabetic vascular complications.

Another important feature of inflammatory response is the recruitment and attachment of circulating leukocytes to the endothelium, which is the initial stage in the development of atherosclerosis. Increased adhesion of leukocytes to ECs is facilitated by enhanced expression of chemokines such as monocyte chemoattractant protein (MCP-1) and adhesion molecules such as intercellular adhesion molecules (ICAM), vascular cell adhesion molecules (VCAM), and E-selectins. Studies also demonstrate that direct interaction exists between the ECs adhesion molecules and lymphocyte-function-associated antigen 1 (LFA-1), macrophage-1 antigen (Mac-1), and very late antigen-1 (VLA-1), which are the major counter receptors present on the surface of the leukocytes [39, 72]. Abundant studies show that AGEs elevate the expression of adhesion molecules on the surface of the endothelial cells [73, 74].

#### **2.3 AGE increases ER stress**

Numerous studies provided molecular insights, highlighting the functional link existing between endothelial dysfunction and endoplasmic reticulum (ER) stress [75]. ER is the vital organelle that plays important role in protein folding, lipid biosynthesis, and calcium (Ca2+) regulation. When the ER homeostasis is disturbed either due to increased synthesis of protein, accumulation of misfolded proteins, increased oxidative stress, or alteration in calcium load leads to a condition termed as ER stress. It is closely monitored by the evolutionarily conserved quality control system called unfolded protein response (UPR) [75, 76].

The UPR system is activated by three transmembrane proteins: 1) RNA-dependent protein kinase-like ER eukaryotic initiation factor-2α kinase (PERK), 2) inositolrequiring ER-to-nucleus signaling protein 1 (IRE1), and 3) activating transcription factor 6 (ATF6). These three sensors are inactive form when they are bound with glucose-regulated protein kinase-78 (GRP78) and released upon induction of ER

*Advanced Glycation End Product Induced Endothelial Dysfunction through ER Stress... DOI: http://dx.doi.org/10.5772/intechopen.106018*

stress. These sensors resolve the ER stress by 1) activating the induction of UPR genes, which enhances the protein folding; 2) attenuation of protein translation, therefore, it reduces the workload of ER; and 3) activating ER-associated degradation (ERAD) pathway activation, which eliminates the unfolded protein through proteasome degradation pathway [75, 77].

During ER stress, activation of the PERK pathway leads to phosphorylation of the eukaryotic translation initiation factor 2 alpha (eIF2α), resulting in the attenuation of protein translation and activating the ATF4 (activating transcription factor-4). ATF4 subsequently induces the activation of pro-apoptotic mediator CHOP (C/EBPα-homologous protein, also known as GADD153) and its downstream target gene, DNA-damage-inducible protein-34 (GADD34). Activation of IRE1 promotes splicing of the endoribonuclease unconventional splicing of an mRNA encoding the transcription factor X-box-binding protein 1 (XBP1s). XBP1s activation further induces the activation of UPR genes, which either increases the ER folding capacity or intersects with AFT4-CHOP to activate the apoptosis. Another UPR pathway is the activation of AFT6, which translocates to the Golgi apparatus. It alleviates misfolded proteins through activation of the ERAD pathway mediated by ATF6-XBP1as shown in **Figure 3** [75, 76].

Studies reported that under stress conditions, ER show an unusual morphological pattern called ER whorls, which can be used as a biomarker for ER stress [78]. Further compared with the normal cells, DTT treatment (inducer of ER stress) activated the formation of ER whorls, which are seen beneath the plasma membrane, which is in turn imported into the vacuole and proceeds for autophagy [79]. Earlier a study has shown the presence of whorl-like sER in the Leydig cells of STZ-induced mouse model indicating that diabetes can induce morphological changes in ER. Till now there are no studies that have described the effect of AGE on ER morphology. Whereas there are studies that have highlighted the distorted mitochondrial

**Figure 3.** *AGEs-induced ER stress in endothelial cells.*

morphology (mitochondrial fission) seen during mitochondrial fission in diabetic animal models [80, 81].

ER and mitochondria are connected through mitochondrial- associated ER membranes (MAMs), which help in the transfer of Ca2+, ATP, and metabolites [82, 83]. As ER stress magnitudes, it increases the releases of Ca2+ from ER to mitochondria, which leads to the opening of mitochondrial membrane pore and releases of cytochrome C, which further intensifies the ROS production. Taken together, it creates a vicious cycle of ER stress and mitochondrial dysfunction, which progresses toward apoptotic signaling [84].

A vast number of studies link ER stress with inflammatory and oxidative signaling pathways, which play a putative role in the development and progression of endothelial dysfunction. Several lines of evidence support the fact that ER stress acts as a potent inflammatory activator because each of the UPR arms activates NFĸB pathway, which in turn releases an array of inflammatory cytokines IL-6, IL-8, and TNF-α [85, 86]. A lot of *in vivo* and *in vitro* studies also support the notion that AGEs-induced ER stress promotes the activation of ROS, autophagy, pro-inflammatory cytokines response, and apoptosis in most of diabetic vascular complications [85, 87–91].

Although several studies have identified an array of molecular entities and pathways that activate endothelial dysfunction, the cellular processes underlying endothelial dysfunction are majorly oxidative stress and inflammation. Increasing the intake of fruits and vegetables rich in polyphenols and flavonoids (anti-inflammatory and antioxidant properties) protects the endothelium and reduces the risk of cardiovascular complications [92, 93]. Increasing the endogenous antioxidant may pay way to develop a more effective and safer option. One such endogenous molecule with antioxidative, anti-apoptotic, and anti-inflammatory properties is paraoxonase [94]. Decreased serum paraoxonase (PON) activity is seen in both diabetes, and its complications have been reported, which is attributed to its glycation [95, 96]. The human PON enzyme consists of three family members, paraoxonase (PON1), paraoxonase 2 (PON2), and paraoxonase 3 (PON3). These genes are located on the long arm of chromosome no 7 (7q21-22) with nine exons. Based on the structural homology and from the evolutionary point of view, PON2 is reported to be the oldest member of the family followed by PON3 and PON1 [97]. PON1 is HDL associated and secreted in serum, whereas PON2 and PON3 are located intracellularly mainly in the endoplasmic reticulum, mitochondria, and the nuclear membrane and are ubiquitously expressed in most of the tissues including the liver, kidney, intestine, placenta, etc. [98]. Its role has been explored in many cells including epithelial, endothelial, macrophages, and smooth muscle cells.

PON2 is a ubiquitously expressed antioxidant and anti-inflammatory protein, where its expression and regulation during diabetes- induced complication have not been studied so far. In our recent study, we have established in HUVECs that AGE treatment decreases mRNA, protein, and activity of PON2, whereas overexpression of PON2 alleviates the GA and CML-induced oxidative stress, ER stress, and inflammation through NFκB and ERK1/2 phosphorylation and thereby mitigates pro-inflammatory response [99]. Further silencing of PON2 aggravates GA and CML-induced oxidative stress, ER stress, and pro-inflammatory cytokines expression in HUVEC cells. We found that in diabetic retina PON2 expression was significantly downregulated and HRECs treatment with CML increased mitochondrial fission and aggravates the mitochondrial-dependent apoptosis [100]. Conversely, overexpression of PON2 inhibits the JNK1/2-mediated signaling pathway and rescues the cells from mitochondrial fission and apoptosis as shown in **Figure 4** [100].

*Advanced Glycation End Product Induced Endothelial Dysfunction through ER Stress... DOI: http://dx.doi.org/10.5772/intechopen.106018*

#### **Figure 4.**

*Possible mechanistic role of PON2 in mitigating AGEs-induced ER stress, pro-inflammation, and mitochondrial dysfunction in endothelial cells.*

#### **3. Conclusion**

There are a large number of studies that have established the deleterious effects of AGE on various cellular models. In this chapter, we have discussed in detail how AGEs induce mitochondrial dysfunction, inflammation and endothelial dysfunction through augmenting ER stress. We have also highlighted the association between mitochondrial stress and ER stress. The role of antioxidant PON in inhibiting these deleterious effects has also been discussed.

#### **Acknowledgements**

We thank the Council of Scientific and Industrial Research (CSIR-27 (0310)/14- EMR-11) and Indian Council of Medical Research (ICMR-ID no: 2017-0976/CMB-BMS) for providing the funding and the fellowship.

#### **Conflict of interest**

The authors declare no conflict of interest.

*Updates on Endoplasmic Reticulum*

### **Author details**

Ramya Ravi and Bharathidevi Subramaniam Rajesh\* R.S. Mehta Jain Department of Biochemistry and Cell Biology, KBIRVO, Vision Research Foundation, Chennai, India

\*Address all correspondence to: drbarathi@snmail.org

© 2022 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

*Advanced Glycation End Product Induced Endothelial Dysfunction through ER Stress... DOI: http://dx.doi.org/10.5772/intechopen.106018*

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#### **Chapter 7**

## Endoplasmic Reticulum Involvement in Heart Injury: An Overview

*Gaia Favero, Francesca Bonomini, Marzia Gianò and Rita Rezzani*

#### **Abstract**

The Endoplasmic Reticulum (ER) is a multifunctional organelle present in the cytoplasm of the eukaryotic cells. It is involved in many aspects of cellular physiology and it presents important interaction with other cellular organelles. Different physiological and/or pathological factors may alter ER morphology and homeostasis, resulting in the accumulation of a large number of unfolded/misfolded proteins in the ER lumen and so inducing ER stress. Alterations in ER have been found to be related to different disorders. In particular, ER stress is implicated in the development and progression of various heart injuries, such as myocardial infarction, ischemia/ reperfusion, heart failure, diabetic cardiomyopathy, arrhythmias and cardiotoxicity. Furthermore, the efficiency to counteract the ER stress declines significantly during the physiopathological aging process. In this chapter, we present the correlation between the ER and cardiac injury focusing mainly on the aging process and then we report a brief overview of the potential involvement of some bioactive molecules as preventive/therapeutic compounds that can contrast heart disorders through ER modulation.

**Keywords:** aging, arrhythmias, biomolecules, cardiac hypertrophy, heart, endoplasmic reticulum

#### **1. Introduction**

The ER is a complex and multifunctional organelle present in the cytosol of the eukaryotic cells. This organelle is involved in many aspects of cellular physiology such as regulation of calcium, synthesis, transport and folding of protein, synthesis and metabolism of steroids and lipids and, not to be underestimated, it has important interaction with other cellular organelles [1, 2]. The ER is composed of a continuous lipid bilayer that encloses a luminal space [3–6]. It is morphologically characterized by a membrane system with two major shaped domains: the nuclear envelope and the peripheral ER which includes rough leaves and smooth tubules [7]. The nuclear envelope consists of ER membrane wrapped around DNA and various nuclear elements and it is organized in two continuous flat cisternae sheets surrounding the

nucleus, defined outer and inner nuclear membranes [6, 8]; while the peripheral ER presents cisternae and an interconnected nuclear network of tubules [5, 9–11]. The nuclear envelope pores selectively control the transport of molecules inside and outside the nucleus. The inner and outer nuclear membranes are ample flat sheetlike cisternae stacked over each other and separated by the internuclear membrane space. Historically, ER cisternae have been classified as ribosome bound: "rough ER - RER" or ribosome-free: "smooth ER - SER" [10]. The RER presents many ribosomes associated to the membrane surface and extends from the nuclear envelope to the cell periphery, so defining specialized areas for protein synthesis, folding and degradation. Furthermore, the outer membrane of the nuclear envelope may be considered as part of the RER domain because it is physically continuous with the RER membranes [12]. The SER is composed of irregular and convoluted tubules without associated ribosomes. However, it is continuous with the RER and the majority of proteins in this compartment come from the RER domain [13]. SER includes membranes specialized for drug metabolism and steroid synthesis, as well as tubulovesicular elements forming ER exit sites [14]. Differences in membrane curvature ulteriorly differentiate the RER and SER subdomains [11]. The different ER membranes organization in domains or regions observed among different cell types correlates strictly with their functions: cells specialized to the production, storage and secretion of proteins, such as exocrine cells, are rich in RER; whereas, endocrine cells that synthesize steroid hormones and muscle cells are rich in SER [15, 16]. Furthermore, the previously described distribution of RER and SER is clearly observed in hepatocytes, neurons and endocrine cells; however, in some cells there is not a clear distinction between both domains and so tubules with associated ribosomes are mixed with tubules without associated ribosomes.

As previously depicted, the structure of the ER is complex due to the various distinct domains present within one continuous membrane bilayer. These domains are shaped by interactions with the cytoskeleton, by proteins that stabilize membrane shape and by a homotypic fusion machinery that allows the ER membrane to maintain its morphology. The ER also contains domains that control the interaction with other organelles, such as the Golgi apparatus, endosomes, mitochondria, lipid droplets and peroxisomes [9, 10, 17]. The purpose of differently shaped ER domains is still under evaluation to better clarify also the related functions. **Figure 1** schematically reported the main ER specialized domains, the ER compartmentalization with the nuclear membrane and its interaction with cellular organelles.

Different physiological and/or pathological factors may alter ER morphology and homeostasis, resulting in the accumulation of a large number of unfolded or misfolded proteins in the ER lumen and so inducing ER stress (**Figure 2**) [18–20]. Alteration in ER has been found to be related with different disorders [4, 21]; in fact, in various pathologies the morphology/structure of ER was abnormal and various mutation in morphology-regulating proteins have been observed, indicating that morphology and functions of the ER are intrinsically linked. Furthermore, in chronic conditions, a persistent ER stress can induce and exacerbate cellular and tissue senescence accelerating the aging-related process [21, 22]. To counteract ER stress and reduce the synthesis of proteins, cells activate an endogenous adaptive stress response defined Unfolded Protein Reaction (UPR). During this adaptive mechanism, ER showed an increased expression of folding protein and degradation of protein related genes [19, 23]. Therefore, ER stress is an adaptive cell mechanism

*Endoplasmic Reticulum Involvement in Heart Injury: An Overview DOI: http://dx.doi.org/10.5772/intechopen.111772*

#### **Figure 1.**

*Schematic representation of the main endoplasmic reticulum specialized domains, the endoplasmic reticulum compartmentalization with the nuclear membrane and its interaction with cellular organelles. RER: Ribosomefree endoplasmic reticulum; SER: Smooth endoplasmic reticulum.*

#### **Figure 2.**

*Cardiomyocyte senescence's markers and regulation of aging process by endoplasmic reticulum stress. Metabolic alterations, oxidative stress, toxins and viruses can induce endoplasmic reticulum stress and activate the unfolded protein reaction via PERK signaling pathway leading to heart injury. ER: Endoplasmic reticulum; PERK: Protein kinase RNA-activated (PKR)-like ER kinase; UPR: Unfolded protein reaction.*

activated to restore the ER morphology and functions by triggering UPR; whereas the persistence of the ER stress is deleterious and induces cell apoptosis [24–26]. To date many studies are focused on ER (stress) as a possible target strategy to counteract various diseases progression and drug resistance by hitting UPR components and the complex interplay between UPR, autophagy and apoptosis. The UPR may be activated by different sensors: Inositol Requiring Enzyme1 (IRE1), Activating Transcription Factor6 (ATF6) and Protein Kinase RNA-activated (PKR)-like ER Kinase (PERK), leading to transcriptional and translational reprogramming to counteract unfolded proteins accumulation and upregulating the expression of UPR-related proteins and proteins of the autophagy-related pathway [27]. PERK is involved in a pro-survival pathway, but it can also promote apoptosis through the activation of CHOP, the main mediator of apoptosis induced by the UPR [28, 29]. CHOP induces the expression of ER Oxidoreductase 1 alpha (ERO1α), which promotes cell death through the hyperoxidation of ER proteins [30]. CHOP also participates in the regulation of autophagy, self-digestive intracellular process through which cells recycle organelles and damaged or unnecessary proteins [31–33]. Prolong ER stress, oxidative stress and inflammation are typical features of many pathologies, including metabolic diseases and different age-related diseases, such as neurodegenerative disorders, various cancers and cardiovascular diseases [22, 34]. Various studies have demonstrated that damage at myocardial tissue level is characterized by accumulation, at ER level, of ubiquitinated proteins and this accumulation at ER level promotes ER stress which, in turn, induces the release of ER-related apoptotic proteins thus contributing, in a vicious cycle, to heart injury [35–37].

Furthermore, autophagy is a crucial process involved also in the development of cardiovascular diseases [38]. A variety of autophagy proteins are localized at the ER level and originate from mitochondrial-associated ER membrane (ER interaction site with mitochondria) [39]. ER is one of the membrane donors required for the formation of autophagosomes, double-membrane vesicles involved in autophagy [40, 41].

Furthermore, ER stress is progressively increased during the physiopathological aging process also at heart level. The modulation of ER stress and an enhancement of the intensity of UPR adaptive stress response could be valid strategies to prevent/treat different diseases.

In the following paragraphs, we present the main ER alterations correlated with cardiac injury focusing mainly on the physiopathological process of aging and then we report a brief overview of the potential involvement of bioactive molecules as preventive/therapeutic compounds that can contrast heart disorders through ER modulation.

#### **2. Endoplasmic reticulum and heart injury**

Cardiovascular diseases are to date the leading morbidity and mortality burden in the world [42–44]. The molecular mechanism(s) involved in the pathogenetic processes of cardiac disorders are not fully understood. Age-related changes contribute to reduce heart capacity to adapt to different physiological and pathological factors and, in turn, the ability to restore the "normal" morphology and functions is reduced/ impaired [45, 46]. Notably, aging promotes alteration of mitochondrial function, condition strictly linked to increased reactive oxygen species production and oxidative stress (which play a fundamental role in aging-related diseases). In addition, elevated levels of oxidative and nitrosative stress occur in parallel with ER stress [45, 47] and,

#### *Endoplasmic Reticulum Involvement in Heart Injury: An Overview DOI: http://dx.doi.org/10.5772/intechopen.111772*

therefore, aging-induced ER stress may contribute to mitochondrial dysfunction [36]. In the last decade, various studies have reported that ER stress, mitochondrial injury and oxidative stress are involved in the development and progression of a various heart diseases, such as myocardial infarction, cardiac hypertrophy, ischemia/reperfusion, heart failure, diabetic cardiomyopathies, arrhythmias and cardiotoxicity [48–51]. Furthermore, the cell ability to counteract the ER stress and the efficacy of the UPR signaling adaptive response decline significantly during aging [45]. The UPR is, in fact, fundamental for maintaining heart health because cardiomyocytes lack the ability to replicate or efficiently repair themselves, making them deficient in regenerative capability [52]. If ER stress becomes too severe, the UPR signaling adaptive response leads to cell death through apoptosis or autophagy processes [29, 53]. During ER stress, IRE1 binds to Tumor Necrosis Factor-Receptor-Associated Factor 2 (TRAF2) which in turn activates the Apoptosis Signal-Regulating Kinase 1 (ASK1), a Mitogen-Activated Protein (MAP) Kinase Kinase Kinase (MAP3K). The IRE1-TRAF2-ASK1 complex promotes the activation of p38 and c-Jun N-terminal Kinase (JNK), known apoptosisregulating MAP kinases (MAPK) [54]. In particular, JNK and p38 may be activated in response to a variety of cell stresses, including oxidative stress, DNA damage and inflammatory cytokines [55]. Under physiological conditions, glucose-regulated protein (GRP)78, a mediator of the UPR, binds to PERK, IRE1 or ATF6 forming a stable and inactive complex which inhibits the transmission of the downstream signals. When the UPR is promoted, GRP78 disassociates from this complex and binds to unfolded or misfolded proteins, activating downstream cascade reactions and promoting cell death [56–58]. It is fundamental to underline that this adaptive process involves a complex correlation between autophagy and apoptosis, fundamental to determine cell fate in response to ER stress. In fact, autophagy promotes cell survival through degradation of misfolded proteins that have been retained in the ER or induces cell death through the autophagy process [52]. Understanding how to suppress IRE1 activity and related downstream pathway to inhibit ER stress-induced apoptosis and autophagy-related cell death is of fundamental importance. Xue et al. [59] observed that Activin A, a member of the transforming growth factor-beta superfamily, has important neuroprotective effects against ER stress-mediated apoptosis and autophagy by inhibiting the activation of the IRE1-TRAF2-ASK1-JNK/p38 complex *in vitro*. This will be an important starting point for better understanding how to modulate ER stress and related injuries.

Therefore, investigate the ER stress also in aging-related heart injury is actually an important research focus which needs more studies.

For the first time, Sreedhar et al. [60] identified the involvement of ER stressinduced apoptosis in the hearts of SAMP8 mice, known aging mouse model [61, 62], demonstrating that ER is an important factor in cardiac aging. In particular, SAMP8 mice showed an increased p38 expression at heart level, suggesting a relation between MAPK signaling cascade and ER stress. At heart level, the SAMP8 mice presented elevated expression of the pro-apoptotic transcription factor CHOP and caspases, highlighting that p38 induces ER stress-related apoptosis. Currently, a weak/moderate ER stress is considered beneficial for heart function by restoring ER morphology and homeostasis and protecting cardiomyocytes through an adaptive mechanism. On the contrary, a prolonged and severe ER stress promotes cardiomyocyte death [63–65]. Notably, mitochondrial damage and ER stress have been well recognized as important cardiac ischemia/reperfusion injury upstream factors controlling the cardiomyocyte death [20, 66]. Aging decreases the efficacy of the endogenous adaptive response and increases ER-mediated myocardial apoptotic signaling after ischemic/reperfusion injuries [24, 67].

Cardiomyopathy is the most common cause of morbidity and mortality in diabetic patients [68]. Diabetic conditions promote ER stress response by oxidative mechanisms which contribute to eliminate unhealthy cells and lead to diabetic cardiomyopathy development. Such conditions have been shown to promote cardiac hypertrophy, collagen deposition, stiffness and so important cardiac dysfunction. ER stress activation indeed has been observed in both hypertrophic and failing hearts [20, 68, 69]. Accumulating studies have demonstrated that the ER stress response is also involved in the cardiac hypertrophy progression which is typically characterized by increased size of cardiomyocyte, interstitial fibrosis, apoptosis and contractile dysfunction [43, 70]. STING, an ER-resident protein regulating innate immunity, is highly expressed in hearts of patients with dilated cardiomyopathy or hypertrophic cardiomyopathy [71]. In addition, Zhang et al. [71] reported that STING knockout attenuated cardiac hypertrophy induced by aortic banding showing a significant alteration of cardiomyocyte size, ER stress and hypertrophic markers expression. Moreover, STING deletion significantly reduced inflammation and fibrosis at heart level. The authors so concluded that the modulation of STING expression was, at least in part, regulated by ER stress, underling its important contribution in the cardiac hypertrophy progression. Furthermore, Yao et al. reported a fundamental physiological role of AGGF1: it may regulate ER stress signaling and blocking ER stress-induced apoptosis in cardiac hypertrophy [51]. The authors described that ER stress induces the downregulation of AGGF1 level in a mouse model and human patients with heart failure. In detail, AGGF1 regulates ER stress signaling through CHOP pathway and so inhibits ER stress-induced apoptosis. Liu et al. [72] observed that the expression of SOCS3, a mechanical stress inducible gene, is involved in hypertrophic hearts after 2 weeks of transverse aortic constriction, well-established animal model of pressure overload-induced cardiac hypertrophy [72, 73]. Pressure overload promotes ER stress-induced apoptosis of cardiomyocytes, leading to cardiac hypertrophy and heart failure [74]. In detail, the authors observed that SOCS3-GRP78-ER stress signaling promotes the transition from cardiac hypertrophy to heart failure during pressure overload *in vivo*. In fact, prolonged pressure overload significantly decreases SOCS3 expression that, acting as a negative regulator of cardiac hypertrophy, interacts with GRP78 and induces GRP78 ubiquitination and proteasomal degradation so modulating ER stress. Moreover, cardiac-specific SOCS3 knockout mice presented significant cardiac hypertrophy, chamber dilatation and abnormal myofilament calcium sensitivity after pressure overload. Notably, the treatment of mice with 4-phenylbutyric acid, a short chain fatty acid that is clinically used to treat urea cycle disorders, for 4 weeks attenuates ER stress and related downstream pathways targeting GRP78 and so determining inhibition of cardiac hypertrophy and dysfunction.

STING, AGGF1 and SOCS3 may represent new targets not only for cardiovascular diseases, but also for other diseases associated with ER stress.

During aging, the proteostasis network becomes unable to maintain proteostasis and key UPR molecules, such as PERK, are damaged leading to misfolded proteins accumulation within the ER [75–78]. Age-related injury due to protein misfolding, aggregating proteins and dysfunctional UPR sensors has been shown to lead to diabetes mellitus, neurodegeneration, cancer, heart diseases and arrhythmias [75, 77]. Due to the strict morphological and functional link between ER protein and calcium homeostasis, mounting evidences report that prolonged ER stress is correlated with heart arrhythmic risk via perturbed redox status within the ER as well as downregulation of cardiac ion channel proteins. Furthermore, UPR is involved directly and indirectly in proarrhythmic cardiac remodeling through UPR-induced oxidative stress, altered

#### *Endoplasmic Reticulum Involvement in Heart Injury: An Overview DOI: http://dx.doi.org/10.5772/intechopen.111772*

glycosylation and modulation of ion channels involved in excitation-contraction (including ER calcium handling proteins) [72], so ulteriorly correlating ER stress to heart arrhythmias. Importantly, inhibition of PERK may prevent downregulation of these channels, attenuate aberrant electrical remodeling, reduce ventricular arrhythmia inducibility and improve survival after myocardial infarct *in vivo*. Therefore, it is fundamental that cardiomyocytes carefully balance the UPR to safely counteract ER stress and maintain proteostasis [78]. Recently, Nakamura et al. [70] reported that ER stress, activating nuclear factor-kappa B (NF-kB) pathway, promotes ventricular arrhythmia in failing hearts via the cardiac dopamine receptor D1 (D1R) upregulation. D1R is upregulated in cardiomyocytes of failing hearts (in mice and humans) leading to heart failure-associated ventricular arrhythmia. ER stress-induced NF-κB signaling pathway could potentially serve as a target to improve the prognosis of heart failure patients [79]. Moreover, Hamilton et al. [80] recently observed a novel association interacting complex Ryanodine Receptor (RyR2)-Endoplasmic Reticulum protein44 (ERp44) which may be stabilized by Ero1α in cardiac hypertrophy. Cardiac hypertrophy-mediated upregulation of Ero1α results in the removal of ERp44 from the complex, inducing RyR2 dysfunction. This dysfunction increases the risk of calcium-dependent ventricular arrhythmias. The authors, notably, concluded that Ero1α may be a promising target to reduce arrhythmogenesis and to improve cardiac function during hypertrophy and heart failure, without disturbing the finely balanced intra-ER redox environment.

Autophagy is also impaired in arrhythmogenic cardiomyopathy. Pitsch et al. [81] observed, in an animal model of arrhythmogenic cardiomyopathy, signs of increased autophagy prior to structural disease onset in the "normal"-appearing mutant myocardium. In particular, the authors reported, at heart level, numerous autophagyrelated vacuoles and the upregulation of autophagy during onset and progression of the arrhythmogenic cardiomyopathy. Furthermore, ventricles presented elevated expression of the pro-apoptotic ER stress marker CHOP both at disease onset and during chronic disease progression. In addition, reduced Ryr2 mRNA expression together with severe enlarged ER cisternae underlined ER dysfunction.

Furthermore, it has been documented that ER stress is involved also in cardiotoxicity and heart injury that potentially may evolve into heart failure. Interestingly, Ni et al. [82] observed upregulation of ER chaperones in patients with end-stage heart failure. In detail, the authors investigated the expression of various ER stress factors in hearts of 4 patients subjected to heart transplantation who suffered from dilated cardiomyopathy with end-stage heart failure and 9 patients undergoing mitral valve replacement, as well as 4 healthy subjects. This study, interestingly, reported a significant upregulation of the phosphorylated level of PERK together with c-Jun phosphorylation in failing hearts compared with healthy hearts. These results indicated that prolonged ER stress and associated apoptosis are general occurrence in human failing hearts. Similar results were subsequently found in isoproterenol-stimulated cardiomyocytes and in a rat model of heart failure after abdominal aortic constriction and isoproterenol subcutaneous injection. Furthermore, the authors observed also that long-term oral treatment with β-adrenergic receptor blockers inhibits ER stress, correlated to cardiac hypertrophy and heart failure.

#### **3. Possible endoplasmic reticulum modulation against heart injury**

To date, the protective and therapeutic strategies against cardiovascular diseases are not fully efficient and effective especially in elderly people, so amplify the actual knowledge on ER stress involvement in heart injury will be helpful in finding novel drug targets. In the last decade, at this aim, different potential therapeutic biomolecules have been investigated; however, there are still many unresolved questions that need to be debated and investigated in deep.

Different studies have indicated that sirtuin 1, NAD<sup>+</sup> -dependent deacetylases implicated in many aspects of aging process [83], is involved in the regulation of ER stress in cardiomyocytes [84–86]. Hsu et al. [45] hypothesized that sirtuin 1 is able to protect aging heart. In detail, the authors observed a significative contractile dysfunction associated with improved ER stress and oxidative stress in aged sirtuin 1<sup>−</sup>/<sup>−</sup> mouse hearts. The study demonstrated also, *in vitro*, that a sirtuin 1 activator reduced ER stress and myocardial apoptosis induced by oxidative stress; whereas a sirtuin 1 inhibitor reversed the sirtuin 1 protective effect at cardiomyocytes level. Therefore, the development of "drugs" targeting sirtuin 1 could have interesting preventive/ therapeutic potentials against cardiac contractile injury aging-related.

Recently, Monceaux et al. [63] evaluated the ability of ten phenolic phytonutrients (resveratrol, berberine, butein, catechin, ferulic acid, isoliquiritigenin, malvidin, piceatannol, pterostilbene and tyrosol) to modulate ER stress. Interestingly, all these bioactive compounds are able to protect the heart from severe ER stress. Investigating in deep the mechanisms of action of the phytonutrients, the authors reported that ferulic acid, pterostilbene and tyrosol protect cardiomyocytes from severe ER stress by selectively downregulating the PERK pathway of the UPR signaling through sirtuin 1-mediated deacetylation of the translation initiation factor eIF2α (factor which modulates the transcription of UPR target genes, such as ATF4). Interestingly, this study reported that ferulic acid, pterostilbene and tyrosol, by downregulating the PERK/eIF2α/ATF4 pathway, reduce the level of the pro-apoptotic transcription factor CHOP and so limit ER stress-related apoptosis at heart level (**Figure 3**).

Furthermore, Liu et al. [72] evaluated the protective role in reducing the activation of the UPR under ER stress conditions of Wenxin Granules (mix of five drugs: Codonopsis, Rhizoma Polygoni, Panax notoginseng, Amber and Gansong), known traditional Chinese medicine with important effects in the inhibition of myocardial remodeling, modulation of cardiac conduction systems and heart arrhythmias [87, 88]. The authors performed an in vivo study with a rat model of myocardial infarction obtained through the ligation of the anterior descending branch of the left coronary artery and Wenxin Granules were administered intragastrically once per day for two consecutive weeks. In detail, the human daily dose of Wenxin Granules was converted into an equivalent dose for rats (approximately 6 times the human dose for the low-dose group and approximately 12 times the human dose for the highdose group). Compared with the sham operated group, the myocardial infarction model group showed larger hearts and a significant increase of the left ventricular inner diameter; whereas Wenxin Granules improved significantly the morphological myocardial alteration observed in the infarction model group. Furthermore, the myocardial infarction model group presented elevated expression levels of the ER stress proteins GRP78, PERK, ATF6 and XBP1 and, notably, the expression levels of the ER stress pathway proteins were reduced after Wenxin Granules administration. Furthermore, the expression levels of the apoptotic CHOP and Bax in the myocardial infarction model group significantly increased compared with the sham group, whereas the Bcl-2/Bax ratio significantly decreased. Notably, Wenxin Granules were able to improve ventricular remodeling, prevented the excessive ER stress-mediated UPR activation and inhibited myocardial cell apoptosis [72].

*Endoplasmic Reticulum Involvement in Heart Injury: An Overview DOI: http://dx.doi.org/10.5772/intechopen.111772*

#### **Figure 3.**

*Polyphenol cardioprotection's mechanism against endoplasmic reticulum stress. Pterostilbene, tyrosol and ferulic acid protect cardiomyocytes downregulating the PERK signaling pathway mediated by sirtuin 1. This pathway decreases the expression of ATF4 and CHOP inhibiting apoptosis and cardiac dysfunction. Modified from Monceaux et al. [63]. ATF4: Activating transcription factor4; ER: Endoplasmic reticulum; PERK: Protein kinase RNA-activated (PKR)-like ER kinase.*

Recently, Tu et al. [89] analyzed for the first time the effects of fish oil against atrial fibrillation vulnerability by reducing ER stress in a canine model. The atrial fibrillation model animals were obtained using long-term rapid atrial pacing; the animals were fed with a chow supplemented with fish oil (0.6 dietary ω-3 fatty acids/kg/day) and initiated the treatment 1 week before surgery and continued for 4 weeks post-surgery. The fish oil treatment not only reduced myocardial ER stress, but decreased also pro-inflammatory factors, calcium handling-related proteins and reversed the elevated level of CHOP and caspase12 in the atrial fibrillation group, so significantly reduced atrial fibrillation inducibility and duration.

#### **4. Conclusion**

The actual knowledge on the role of ER stress in heart injury are mainly from *in vivo* animal studies, so the understanding of the ER and UPR involvement in human diseases is still limited. Due to that, ER stress plays a central role in heart injury development, ER stress inhibitors could be important for preventive/therapeutic interventions. The development of novel therapeutic/protective approaches modulating the ER stress pathway may reduce the drain of health care costs and resources resulting from the worldwide spread against cardiovascular diseases.

*Updates on Endoplasmic Reticulum*

### **Author details**

Gaia Favero1,2\*, Francesca Bonomini1,2,3, Marzia Gianò1 and Rita Rezzani1,2,3

1 Anatomy and Physiopathology Division, Department of Clinical and Experimental Sciences, University of Brescia, Italy

2 Interdepartmental University Center of Research "Adaptation and Regeneration of Tissues and Organs (ARTO)", University of Brescia, Italy

3 Italian Society of Orofacial Pain (SISDO), Brescia, Italy

\*Address all correspondence to: gaia.favero@unibs.it

© 2023 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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*Endoplasmic Reticulum Involvement in Heart Injury: An Overview DOI: http://dx.doi.org/10.5772/intechopen.111772*

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#### **Chapter 8**

## Targeting Endoplasmic Reticulum and Mitochondrial Dynamics to Combat Triple-Negative Breast Cancer

*Priyanka Menon Kunnel and Bibu John Kariyil*

#### **Abstract**

Triple negative breast cancer (TNBC) is a cancer that is aggressive with short survival rate. In comparison to other breast cancer subtypes, TNBC tumors are bigger, more chemo resistant, highly proliferative, and usually more abundant in stem and immune cells. These modifications are functionally dependent on a highquality endoplasmic reticulum and mitochondrial pool. Endoplasmic reticulum and mitochondrial health are monitored and enhanced on a regular basis via endoplasmic reticulum and mitochondrial dynamics. The role of endoplasmic reticulum and mitochondrial dynamics in tumor growth and metastasis has been highlighted by recent advances in understanding the endoplasmic reticulum and mitochondrial dynamics in TNBC. This chapter examines the current knowledge of endoplasmic reticulum and mitochondrial dynamics in TNBC.

**Keywords:** endoplasmic reticulum dynamics, endoplasmic reticulum transmembrane proteins, mitochondrial dynamics, molecular mechanisms, apoptosis, triple-negative breast cancer

#### **1. Introduction**

Triple-negative breast cancer (TNBC) is a subgroup of breast tumors that does not have estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor 2 (HER2) that are commonly found in breast cancer. TNBC cells undergo significant molecular alterations at the cellular level. They are highly proliferative, more chemo resistant, larger in size, and more aggressive than other types of breast cancer (BC) cells. Compared to other BC subtypes, this subtype has a higher concentration of stem cells and lymphocytes. Porporato et al. reviewed that all stages of cancer development, including proliferation, malignant transformation, and progression, require mitochondria [1]. They not only supply sufficient energy but also protect TNBC cells from excessive amounts of reactive oxygen species (ROS). Reactive oxygen species improve TNBC cells metastatic potential in the early stages of development. High amounts of ROS, on the other hand, cause ROS-induced apoptosis, which is harmful to cancer growth [2]. To survive significant ROS-related damage, TNBC cells must have powerful defensive mechanisms. Mitochondrial dynamics are crucial in maintaining this equilibrium [3].

The endoplasmic reticulum (ER) is an intracellular organelle that is dynamic in both function and structure. It plays a variety of roles in cellular homeostasis, development, and stress tolerance. It is the organelle in charge of protein folding, translocation, and posttranslational modification. Oxidative stress, altered glycosylation, nutrient deprivation, calcium depletion, DNA damage, and energy disturbance are all caused by physiological, biochemical, and pathological stimuli in the ER, resulting in ER stress and the build-up of unfolded or misfolded proteins in the ER. In order to survive, the cells must overcome ER dysfunction and ER stress. Apoptosis can result from unresolved ERS [4].

In TNBC cells, pharmacological aggravation of ER stress causes significant cell death, and this method is even effective in multidrug-resistant forms [5]. The ER stress process has been connected to autophagy, a cellular function that appears to be important for general cell homeostasis, cancer, and chemoresistance. Autophagy and ER stress are linked: several agents that aggravate ER stress cause an increase in autophagic activity; on the other hand, there is evidence that blocking autophagy promotes ER stress [6]. *In vitro* and *in vivo* augmenting of ER stress and simultaneously inhibiting autophagy may result in effective TNBC death [7].

#### **2. Endoplasmic reticulum dynamics and role of ER in TNBC**

#### **2.1 Endoplasmic reticulum dynamics**

ER stress promotes an increase in transcription of p53 unregulated modulator of apoptosis (PUMA), Bcl2-like11 (BIM), BH3-only proteins, and NADPH oxidase activator (NOXA) due to an imbalance between anti- and pro-apoptotic Bcl-2 proteins. ER stress promotes interactions between Bax and PUMA. This results in the release of cytochrome c and caspase-dependent cleavage of p53 resulting in apoptosis [8]. ER stress in tumor cells may restore homeostasis. It also makes the surrounding environment more conducive to tumor survival and proliferation [9]. Malnutrition, hypoxia, pH fluctuations, and poor vascularization are stressful situations that might inhibit tumor cell development and activate the unfolded protein response (UPR). ER stress is caused by both nutritional deficiency [10] in tumor cells and nutrient excess in normal conditions [11]. High proliferation rates of cancer cells necessitate higher ER protein folding, assembly, and transport activities. This might result in physiological ER stress. This response is cytoprotective and plays a role in tumor development as well as adaptation to harsh conditions [12].

Inositol-requiring enzyme 1α (IRE1α), pancreatic ER kinase-like ER kinase (PERK), and activating transcription factor 6 (ATF6) localized in the ER, are the three ER stress signaling branches involved in tumorigenesis. X-box binding protein (XBP1) aids in cancer progression and is increased in breast cancer [13]. The ER resident chaperone calreticulin is found on the cell surface of tumor cells and has been linked to immunogenic cell death and calreticulin localization on tumor cell surfaces. This link could be due to ER stress in tumor cells [14]. ER chaperone and UPR components are over-expressed in breast cancers.

The ER stress response, on the other hand, is also directly implicated in proapoptotic pathways in both UPR-dependent and UPR-independent ways [15]. To *Targeting Endoplasmic Reticulum and Mitochondrial Dynamics to Combat Triple-Negative… DOI: http://dx.doi.org/10.5772/intechopen.106597*

activate IRE1α, the cytosolic domain of IRE1α interacts with the Bax/Bak apoptotic pathway [16]. EI24/PIG8, an ER-localized Bcl2-binding protein, inhibits breast cancer invasiveness via modulating Bcl-2 function [17]. Bim is also involved in the death of MCF-7 cells generated from breast cancer by activating ER stress-induced apoptosis [18]. Another potential anti-tumor technique is to activate the CHOP-GADD34 axis [19]. In ER stress-exposed cells, CHOP causes cell death by increasing protein synthesis and oxidation [20].

#### **2.2 Unfolded protein response**

Depending on the cell state, the UPR can be both cytoprotective and cytotoxic. The UPR's objective is to keep the ER folding environment in check when it is under stress. Tumor cells will die if UPR fails to restore ER equilibrium under prolonged ER stress. In combination with induced tumor dormancy, the UPR can shield tumor cells against apoptosis, allowing for tumor regeneration once favorable conditions have been restored [21].

By dissociating Grp78/binding immunoglobulin protein (Bip), a key chaperone protein, from three membrane-bound ER stress sensors, comprising ATF6, PERK, and IRE1α, cells aim to maintain normal folding processes in the ER [22]. Following the separation of detecting proteins from Grp78/Bip, these sensors are activated in order, with PERK being the first, blocking general protein synthesis by phosphorylating eIF2α [23]. During cellular stress, these activities also cause the transcription factor NF-κB to be inhibited. Another transcription factor triggered by translocation to the Golgi apparatus is ATF6, which is cleaved and the transcription factor in its active form is released to regulate gene expression [24]. Following IRE-1 activation and downregulation, splicing of XBP1 occurs, and the spliced XBP1 protein translocates to the nucleus, where it activates the transcription of chaperones involved in protein folding and secretion or ER-associated protein degradation (ERAD) [25]. Rapid tumor growth and insufficient vascularization occur during carcinogenesis, resulting in microenvironmental stress [26].

#### **2.3 Role of ER in TNBC**

Since cancer cells are constantly dividing, they are affected by lack of nutrition and oxygen, as well as by lack of vascularization. As a result, cancer alters the expression patterns of ER resident proteins. On tumors, ER stress has two effects. It has adaptive meaning in the sense that it promotes tumor growth. Second, it possesses cytotoxic properties that cause apoptosis. Cancer cells activate UPR to adapt to their surroundings, and macrophages secrete cytokines, growth factors, and angiogenic substances to generate more favorable microenvironments for cancer cell proliferation and invasiveness [27]. Cancer cells use NF-κB pathways to promote cyclooxygenase-2 expression during ER stress, which has antiapoptotic effects. It also maintains IL-8 production in human epithelial cells and enhances pro-inflammatory NF-κB activation via CHOP [28].

Apoptosis is caused by a variety of pathways. One such pathway is ER stress. The caspase-12 family of proapoptotic cysteine proteases is linked with the ER membrane and plays a key role in ER stress-induced apoptosis, although it is not activated by non-ER stimuli [29]. Vascular endothelial growth factor (VEGF) promotes endothelial cell proliferation and angiogenesis by increasing Grp78 expression on the endothelial surface. Through mitogen-activated protein kinase (MAPK) signaling, Grp78 knockdown reduces endothelial cell growth [30]. The action of P38MAPK keeps cells

in a G0-like quiescent state [31]. In chicken embryo chorioallantois membrane system and subcutaneous xenograft models, PERK-elf2α also pauses cell development at G0/ G1 and prevents carcinogenesis [32].

#### *2.3.1 Glucose-regulated protein 78/binding immunoglobulin protein*

Grp78, an ER chaperone protein, is one of the cancer cells' most active components and is overexpressed in a variety of malignancies [33]. It is been considered as a chaperone protein that helps cancer cells adapt to hypoxic conditions and as a resistance protein to anti-cancer drugs [34]. In cancer systems, Grp78 affects cell apoptosis, proliferation, invasion, inflammation, and immunity [35]. It has recently been discovered to play a role in carcinogenesis, metastasis, and angiogenesis [36]. Grp78, through physical and functional interactions with BIK in the ER, suppresses BIKmediated apoptosis and confers resistance to estrogen starvation-induced apoptosis in human breast cancer cells [37]. Grp78 is primarily found inside the ER, although it may be translocated to the surface of tumor cells during ER stress [38]. In addition to the ER, some Grp78 is found in the cytosol, nucleus, and mitochondria during ER stress [39]. A noteworthy prospective anticancer treatment is to block Grp78 translocation. Grp78, present on the surface of cell was found in receptor-positive BT474 breast cancer cells but not in triple-negative MDA-MB-468 cells. The absence of ER, PR, and HER2 receptors in this TNBC was linked to Grp78 negative expression on tumor cells of breast cancer [40]. GRP78 expression was also found to be low in TNBC cell lines, such as MDA-MB-231, by others [41]. Grp78 expression was found to be substantially linked to TNBC invasiveness, distant metastasis, and proliferation. Also, patients with Grp78 expression were found to have shorter overall survival (OS) and disease-free survival (DFS). Furthermore, elevated Grp78 expression was linked to disease-free survival (DFS) in TNBC patients [42].

#### **3. Role of ER transmembrane proteins in TNBC**

#### **3.1 Pancreatic endoplasmic reticulum kinase-like endoplasmic reticulum kinase**

PERK/eIF2 regulates tumor initiation and survival, making it easier for cells to adapt to varied circumstances including hypoxia and oxidative stress [43]. Tumor cells multiply quickly, resulting in the creation of new blood vessels and, eventually, a link to the microenvironment and nutrition restriction. Cytotoxic circumstances result from increased demand for glucose and oxygen. Reactive oxygen species (ROS) are produced when the generation of ATP and NADPH in a reducing equivalent form is disrupted. ROS build up in the mitochondria, causing ER stress to activate [44]. A cellular stress sensor in the ER responds to changes in nutritional shortage, which has been linked to cancer. PERK is a trans-ER membrane serine/threonine protein kinase with an ER luminal domain at the N-terminus and a cytoplasmic protein kinase domain at the C-terminus [45]. The transcription factors Nrf2 transcription factor [46] and elF2 are phosphorylated by PERK. The phosphorylation of elF2 suppresses most transcript translation while promoting the translation of a few mRNAs, such as the transcription factor ATF4 [47]. PERK phosphorylates Nrf2, which is then liberated from an inhibiting E3 ligase complex and translocated into the nucleus, where it generates enzymes that reduce intracellular ROS [48]. When hypoxia occurs in tumors, the transcription regulator HIF1 is stabilized, and the whole branch of

*Targeting Endoplasmic Reticulum and Mitochondrial Dynamics to Combat Triple-Negative… DOI: http://dx.doi.org/10.5772/intechopen.106597*

the UPR, namely PERK, is fully activated, resulting in the phosphorylation of elf2, ATF4, and GADD34. Although elf2 phosphorylation reduces overall protein synthesis, ATF4, a transcription factor, is linked to cancer cell growth and survival despite food restriction via amino acid synthesis [49]. TNBC viability and proliferation were reduced *in vivo* by CCT020312, a specific eIF2/PERK activator that activated the PERK/eIF2/ATF4/CHOP pathway while inactivating the AKT/mTOR pathway [50]. In luminal androgen receptor (LAR), TNBC ER stress reduced androgen receptor (AR) expression at the transcriptional level via PERK/eIF2/ATF4 signaling. ATF4 also inhibits AR promoter activity by binding to the promoter region of AR from −2813 to −2486 nt and from −2084 to −1742 nt [51]. In MDA-MB-468 and T47D cell lines, PERK-dependent signaling is involved in tumor initiation and expansion to maintain redox homeostasis and drive tumor growth [52].

#### **3.2 Inositol-requiring enzyme 1**α**/X-box binding protein**

In cells and tissues, an ER transmembrane sensor called IRE1α protects against ER stress. IRE1 is activated during ER stress by autophosphorylation and oligomerization, which causes its endoribonuclease to cleave and start splicing the XBP1 mRNA [53]. IRE1α-dependent mRNA decay (RIDD), which is different from XBP1 splicing, aids in the restoration of ER homeostasis. This is done by targeting mRNAs encoding secretory proteins. IRE1α RNase activity controls the activity of RIDD [54]. In the UPR, the IRE1-XBP1 pathway has also been suggested to play a pro-survival role [53]. The UPR, on the other hand, causes cellular death in the presence of persistent and uncompensated stress. [55]. IRE1-TRAF2-ASK is another pathway that has been proposed. Phosphorylation of IRE1 causes it to bind to tumor-necrosis factor receptor-associated factor 2 (TRAF2) and activate apoptosis signal-regulating kinase (ASK1), resulting in JNK and p38 activation and ER-stressed caused cell death [56]. By directly activating procaspase-4, the IRE1 and TRAF2 pathways are also engaged in mitochondria-independent apoptosis [57]. TNBCs have an elevated basal level of endoplasmic reticulum stress. There is activation of the XBP1 branch of the UPR, a significant cellular stress response system in the tumor microenvironment [58].

#### **3.3 Inositol 1,4,5 triphosphate receptors**

Calcium ions play a key in the development of metastases. The cytosolic Ca2+ concentration increases by a factor of 5 to 10 when Ca2+ channels activate (from 100 nM to 500–1000 nM). Part of this calcium signaling is generated inside the cell by inositol (1,4,5)-triphosphate (IP3), which has three identified subtypes: IP3R1, IP3R2, and IP3R3. Each subtype has its own calcium release signature: IP3R2 produces strong Ca2+ oscillations, IP3R1 produces milder oscillations, while IP3R3 produces monophasic transients [59].

The ER is the cell's principal intracellular Ca2+ storing organelle. Ca2+ is rapidly transported between the two intracellular organelles due to the tight connection between mitochondria and ER membranes [60]. The IP3R, along with the ryanodine receptors, has been identified as the primary ER Ca2+ channel [61]. Massive and longterm Ca2+ overload in the mitochondria can open the permeability transition pore [62]. As a result, proapoptotic and caspase-activating substances in mitochondria, such as cytochrome c, are released into the cytoplasm. By binding to IP3R, cytochrome c in the cytoplasm exacerbates Ca2+ release, avoiding Ca2+ −dependent regulation of the receptor and increasing caspase activation to promote apoptosis [63].

#### **Figure 1.**

*Endoplasmic reticular stress activates the ER stress signaling branches that cause an increase in unfolded protein response. Depending on the cell status, UPR leads to either apoptosis or anti-apoptosis.*

In breast cancer tissue, IP3R3 is the most highly expressed subtype. Furthermore, invasive breast cancer tissue, such as TNBCs, has much higher levels of IP3R3 and IP3R1 expression than non-tumor tissue. Because its expression is linked to the severity of BC, IP3R3 can be regarded as a sign of aggressiveness in BC [59].

#### **3.4 STAT 3**

STAT3 is constitutively activated and overexpressed in TNBC cells. By regulating the expression of its downstream target genes, it contributes to cell survival, cell cycle progression, migration, invasion, anti-apoptosis, chemoresistance, immunosuppression, and stem cell self-renewal and differentiation [64]. STAT3, an oncogenic transcription factor, has been found to be abundant in ER and MAM lately. Reduced ER-mitochondria Ca2+ transport and IP3R3 degradation mediated by the IP3R3/ STAT3 association increase cell resistance to apoptosis in constitutively active STAT3 (**Figure 1**) [65].

#### **4. Mitochondrial dynamics in TNBC**

Mitochondrial dynamics is a mechanism that regulates the quality of the mitochondrial pool. In simple terms, it is a dynamic flow of mitochondria that includes divisions and assembly, allowing for the destruction or repair of malfunctioning parts. Mitochondrial dynamics thus serve as the first line of defense against ROSinduced mitochondrial damage. Given that TNBC cells have a high quantity of ROS, it is assumed that this process is critical for TNBC survival.

Changing mitochondrial dynamics helps cancer cells survive because they regulate a delicate balance between delivering energy and apoptosis. Many cancer tissues, including lung, glioma, neuroblastoma, colorectal, pancreatic, and melanoma, contain fragmented mitochondria [66]. Similarly, mitochondrial fission is significantly enhanced in TNBC clinical samples and is associated with poorer TNBC patient survival. Drp1 was upregulated and Mfn1 was downregulated in TNBC tumor tissues, according to immunohistochemistry (IHC) labeling. The results are further supported by cancer cell labeling and the relative mRNA expression ratio of tumor/

#### *Targeting Endoplasmic Reticulum and Mitochondrial Dynamics to Combat Triple-Negative… DOI: http://dx.doi.org/10.5772/intechopen.106597*

peritumor determined by qRTPCR [67]. Hypoxia also increases Drp1 expression in TNBC MDA-MB-231 cells, but not in ER-positive MCF7 cells [68]. TNBC tumors are more hypoxic than other BC subtypes, therefore this pattern is particularly intriguing [69]. A mutation in the tumor stressor gene TP53 is also found in the majority of TNBC tumors [70].

Increase in fission offers TNBC tumors with an optimal environment for development, given that knockout mice without p53 (Trp53) are able to survive longer in hypoxia environments because tumorgenesis levels are lower than in normoxic situations. Fission has been identified as a critical stage in the release of cytochrome c and, as a result, apoptosis progression. Fission-dependent apoptosis, on the other hand, appears to be context and cell-type dependent. Mitochondrial fission boosted TNBC cell growth *in vitro* and *in vivo* by suppressing apoptosis and enhancing proliferation through a notch-dependent mechanism [71].

#### **4.1 Mitochondrial fusion and fission**

Mitochondria are double membrane organelles consisting of an outer mitochondrial membrane (OMM), an inner mitochondrial membrane (IMM), and an intermembrane space between the two membranes. They are not static organelles, but rather a dynamic pool that is constantly undergoing fusion and fission. This dynamic process is essential for mitochondrial health and cellular adaptation to environmental changes.

Mitochondrial fusion occurs when two mitochondria fuse to form one mitochondrion. Mitofusin 1 (Mfn1) and mitofusin 2 (Mfn2), as well as optic atrophy protein 1 (OPA1) are critical proteins in mitochondrial fusion. Mfn1 and 2 are homodimers and heterodimers that are found near the OMM. The mitofusins are in control of OMM fusion, while OPA1 is in charge of IMM fusion. Furthermore, OPA1 interacts with Mfn2 during fusion, and interruption of this mechanism results in mitochondrial fission. ROS induce mutations in mtDNA, which results in altered respiratory functions in mitochondria. As a result, mitochondrial fusion is critical because it allows for the interchange of metabolites and gene products between fusing mitochondria, improving overall respiratory performance. Similarly, mitochondrial fusion impediment is linked to a reduction in mitochondrial function [72]. Fusion of mitochondrion and promotion of docking is the primary function of mitofusins. The oligomerization of the GTPase domains of MFNs is essential for the tethering of two OMMS, this oligomerization also requires GTP hydrolysis. When GTP is bound and hydrolyzed, the GTPase domains undergo a conformational shift that causes them to oligomerize, allowing the two mitochondria to dock at the two outer membranes and fuse. Ubiquitination, deacetylation, and phosphorylation regulate MFN 1's activity. The extracellular signal-regulated kinase (ERK) phosphorylates Mfnl in the HRI domain, which inhibits mitochondrial fusion and causes death. In situations of glucose deprivation, however, deacetylation of Mfn1 by histone deacetylase 6 (HDAC6) leads to its activation and facilitation of fusion. JNK phosphorylates MFN2 in response to cellular stress, which recruits E3 ubiquitin ligase, which ubiquitinates MFN2 and causes its proteasomal destruction. MFN2 degradation leads to mitochondrial fragmentation and an increase in apoptotic cell death. S-OPA1 and L-OPA1 combination is essential for IMM fusion. Similarly, discovered that L-OPA1 and S-OPA1 collaborate to promote fusion activity in liposomes, resulting in efficient and rapid membrane pore opening. Other research, on the other hand, has found that only L-OPA1 is required to stimulate IMM fusion [73]. A phospholipid cardiolipin, IMM component is required for IMM fusion.

#### **Figure 2.**

*Mfn1 and Mfn2 are the membrane proteins involved in mitochondrial fusion, its downregulation activates TNBC cells. Drp1 is the main membrane protein involved in mitochondrial fission. Its upregulation activates TNBC cells.*

Mitochondrial fission occurs when one mitochondrion splits into two daughter mitochondria. Dynamin-related proteins (DRPs) and cofactors mediate mitochondrial fission. Constriction of mitochondria occurs where ER binds to mitochondria. At the ER–mitochondrial interaction point, Drp1 is recruited to OMM. Drp1 binding site and constriction are indicated by contact between ER tubules and mitochondria. Drp1 binds to the ER binding site on the OMM and interacts with adaptor proteins such as mitochondrial fission factor (MFF), mitochondrial dynamics protein, and cardiolipin (CL) once activated. Drpl undergoes oligomerization when the fission machinery is assembled, forming a ring around the OMM. Drp1, MFF, Mid49/51, and Mdv1-dependent GTP hydrolysis provides energy for scission completion and constriction [73].

Various cellular stresses, ROS, hypoxia, posttranslational modification, etc., regulate mitochondrial dynamics.

When these spatiotemporal events are disrupted, the consequence is either a fragmented network with many small round mitochondria or a hyperfused network with elongated mitochondria (**Figure 2**).

#### **5. Mitochondria-associated ER membranes**

Mitochondria-associated ER membranes (MAMs) function as signaling hubs that regulate ER and mitochondrial activity. They regulate lipid metabolism, Ca2+ homeostasis, mitochondrial function and cell death. Bcl2, p53 tumor suppressor, etc., are the cell-death pathway protein involved in MAM [74]. Also, proteins like Mfn2 and Drp1 are enriched in MAM. Mfn2 is present on both OMM and ER. Mfn2 along with Mfn1 on OMM establish ER–mitochondrial interactions [75]. Rapid Ca2+ exchange between mitochondria and ER also determines mitochondrial bioenergetics and cell fate. An oxidizing enzyme endoplasmic reticulum oxidoreductin 1-α (ERO1-α) is enriched in

*Targeting Endoplasmic Reticulum and Mitochondrial Dynamics to Combat Triple-Negative… DOI: http://dx.doi.org/10.5772/intechopen.106597*

MAM. ERO1-α affects ER redox homeostasis, ER Ca2+ flow, and consequent mitochondrial Ca2+ build-up. It also modulates oxidative folding. ERO1-α expression is correlated with programmed cell death ligand 1 (PD-L1) in TNBC [76]. It is also an indicator of poor prognosis in breast cancer.

When MAMs are disrupted, a variety of cellular processes, such as apoptosis, inflammation, and autophagy, become dysfunctional. These cellular processes are very important in the pathophysiology of TNBC [74].

#### **6. Conclusion**

The significance of the ER stress response and dynamic transformations of mitochondria, which regulate cell fate decisions in carcinogenesis and cancer resistance, is being clarified by increasing evidence. Understanding how these events are controlled from not only a molecular perspective but also a biological perspective is essential to comprehending a variety of human disorders. The search for novel medications to control these occurrences is an ongoing process.

#### **Conflict of interest**

The authors declare no conflict of interest.

#### **Author details**

Priyanka Menon Kunnel and Bibu John Kariyil\* Faculty of Veterinary and Animal Sciences, College of Veterinary and Animal Sciences, Kerala Veterinary and Animal Sciences University, Thrissur, Kerala, India

\*Address all correspondence to: bibujohn@kvasu.ac.in

© 2022 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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### *Edited by Gaia Favero*

This book examines the endoplasmic reticulum and its involvement in the physiopathological processes in plants and humans. It emphasizes how aging promotes morphological, (ultra)structural, physiological, and functional changes in the endoplasmic reticulum. The modulation of endoplasmic reticulum stress and an enhancement of the intensity of the unfolded protein response are potential strategies to prevent/treat different diseases and promote a global healthy state. A thorough understanding of these mechanisms and their role in physiology and pathophysiology conditions is fundamental in the development of new endoplasmic reticulum-targeted therapies.

### *Miroslav Blumenberg, Biochemistry Series Editor*

Published in London, UK

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