Multiple Faces of Dynamin-related Protein 1 and Its Role in Alzheimer’s Disease Pathogenesis

Abstract

Mitochondria play a large role in neuronal function by constantly providing energy, particularly at synapses. Recent studies suggest that amyloid beta (Aβ) and phosphorylated tau interact with the mitochondrial fission protein, dynamin-related protein 1 (Drp1), causing excessive fragmentation of mitochondria and leading to abnormal mitochondrial dynamics and synaptic degeneration in Alzheimer’s disease (AD) neurons. Recent research also revealed Aβ-induced and phosphorylated tau-induced changes in mitochondria, particularly affecting mitochondrial shape, size, distribution and axonal transport in AD neurons. These changes affect mitochondrial health and, in turn, could affect synaptic function and neuronal damage and ultimately leading to memory loss and cognitive impairment in patients with AD. This article highlights recent findings in the role of Drp1 in AD pathogenesis. This article also highlights Drp1 and its relationships to glycogen synthase kinase 3, cyclin-dependent kinase 5, p53, and microRNAs in AD pathogenesis.

Introduction

Mitochondria are double-membrane organelle that plays an important role in producing cellular energy, living up to their name as the powerhouse of cells. Mitochondria constantly undergo fission and fusion to maintain normal cell biological functions [1–3]. Mitochondrial fission is the division of single mitochondrion into two. Mitochondrial fission is mainly controlled by the dynamin-related protein 1 (Drp1). Recent research revealed that various pathological conditions, such as cancer, obesity, diabetes, cardiovascular diseases, alcohol injury, and neurodegenerative disorders such as Huntington’s disease (HD), Alzheimer’s disease (AD), amyotrophic lateral sclerosis and Parkinson’s disease (PD) are associated with impaired balance between mitochondrial fission and fusion, in addition with causal factors [4–17]. Mitochondrial fission plays a phenomenal role in regulating various signaling pathways for cell survival and death mechanisms [18, 19], and it is also required for the bioenergetics function of axonal mitochondria. GTPase Drp1 enzymatic activity is essential for mitochondrial division [4, 6] and increased GTPase Drp1 enzymatic activity causes excessive mitochondrial fragmentation.

Mitochondrial fusion is the union or fusion of two mitochondria into one elongated mitochondrion. Mitochondrial fusion is controlled by mitofusins 1 and 2 (Mfn1, Mfn2) and by optic atrophy 1 (Opa1) [14, 15, 20, 6]. Mitochondrial fusion is important for electrical conductivity, keeping mitochondrial contents intact, and maintaining mitochondrial ATP and neuronal function. It is well established that neuronal mitochondrial half-life is about 2–3 weeks and damaged mitochondria are known to be cleared by mitophagy mechanisms [21]. The purpose of this article is to assess recent advancements of Drp1 research on mitochondria, and also Drp1 relationships to GSK3β, CDK5, p53, and microRNAs in AD pathogenesis.

Drp1 structure

Drp1 is critical for mitochondrial division and is essential for the distribution of mitochondria in axons, dendrites and synapses. Drp1 has been found in the brain, lung, heart, kidney, spleen, liver, hepatocytes testis and fibroblast of humans. Drp1 has also been found in plants, yeast, worms and rodents and other mammals [6]. As shown in Figure 1, the structure of Drp1 contains an N-terminal GTPase domain, a helical domain at the center and a GED domain at the c-terminus. The GTPase domain is highly conserved and reported to involve physiological relevance in [6]. Several researchers have independently sequenced cDNA clones of mammalian Drp1. Based on published studies and NCBI databases, the human Drp1 has been found to have several splice variants: variant 1 consists of 736 amino acids; in variant 2, exon 15 is spliced out; in variant 3, exons 15 and 16 are spliced out and have a total of 699 amino acids; variant 4 has 725 amino acids; variant 5, 710 amino acids; and variant 6, 749 amino acids [23–24]. Drp1 contains a highly conserved GTPase and is involved in various cellular functions. Similar to the human Drp1, the mouse Drp1 has been found in multiple variants: variant 1 consists of 712 amino acids; in variant 2, exon 3 is spliced out; and in variant 3, exons 15 and 16 are spliced out.

Figure 1. Schematic representations of the important domains of human Drp1.

Arrows on the domains are the sites of post-translational modifications. Phosphorylation, SUMOylation, S-Nirosylation were represented with the aid of arrows on Drp1 domains. The GSK3β could be interacted with GED domain of Drp1 at Ser 637 position. All amino acids were numbered with respect to human Drp1 splice variant 1 sequence (longer form).

In yeast, Dnm1p controls mitochondrial morphology and distribution. However, disruption of the Dnm1p gene was found to cause the collapse of the mitochondrial membrane in one side of the cell [25]. The yeast genetics studies have identified several proteins, including Dnm1 as the central component and its receptor Fis1 and adaptors Mdv1, and Caf4. All these proteins are involved to maintain mitochondria structure and function [26–32]. The other 2 anchor proteins Num1 and Mdm36 are unique with dual functions: they connect Dnm1 to mitochondria and they regulate both mitochondrial division and positioning within the cell [33–36].

In additional studies of Dmn1 in Caenorhabditis elegans, overexpression of wild-type Drp1 caused fragmentation of mitochondria, leading to the overproduction of defective mitochondria in neurons. However, defective Drp1 has been found to increase mitochondrial fusion and it might block mitochondrial transport from the cellular body to the cellular axons, dendrites, and nerve terminals [37]. These various experiments shed light on the role of Drp1 in neurons.

Drp1 function

Drp1 is a multi-functional protein. Recent research findings suggest that Drp1 is involved in mitochondrial division, mitochondrial distribution, peroxisomal fragmentation, phosphorylation, SUMOlyation and ubiquitination. Figure 2 depicts functions of Drp1.

Figure 2. Drp1 functions.

Drp1 plays a crucial role in mitochondrial fission, fragmentation and distribution. Drp1 interacts with GSK3β and involved with the functions of synaptic plasticity across the neurons by regulating of phosphorylation of GSK3β. Drp1 also regulate the peroxisomal fragmentation, SUMOylation and ubiquitination in mammalian cells.

Drp1and mitochondrial division

Several lines of research suggest that Drp1 in yeast, C. elegans, and mammals is involved in mitochondrial division [26, 37–41] and that a reduction in Drp1 increases mitochondrial fusion and mitochondrial connectivity [26, 37, 41]. Studies focusing on the structure of Drp1 have found that purified human Drp1 self-assembles into multimeric, ring-like structures with dimensions, similar to those of dynamin multimers. These similarities between dynamin and Drp1 suggest that Drp1wraps around the constriction points of dividing mitochondria, analogous to dynamin collars at the necks of budding vesicles [41]. Other proteins, including fission 1 (Fis1), mitochondrial fission factor (Mff), and mitochondrial dynamics proteins of 49 and 51 kDa (MiD49 and MiD51) have been proposed to recruit and assemble Drp1 at the outer membrane [42, 43].

Studies of Drp1 in mammalian cells have investigated its expression [44]. Findings suggest that normal expression of Drp1 is critical for mitochondrial dynamics, and its mutant Drp1 overexpression (the dominant negative mutant Drp1) is associated with perinuclear clusters of mitochondria and the breakage of inter-mitochondrial connectivity, suggesting that mutant Drp1 overexpression may cause abnormal mitochondrial dynamics in mammalian cells [41]. Researchers have also found significant role of Drp1 and Mfn2 on mitochondrial morphology and cell survival in neuronal cells compared to non-neuronal cells [44]. In detail, knockdown or dominant-negative interference of endogenous Drp1 significantly increased mitochondrial length in both neurons and non-neuronal cells, but caused cell death only in cortical neurons. Conversely, depletion of the fusion protein, Mfn2, but not Mfn1, caused extensive mitochondrial fission and cell death. Thus, Drp1 and Mfn2 in normal cortical neurons not only regulate mitochondrial morphology, but are also required for cell survival [44].

In the last decade, researchers focused on mitochondria associated membranes’ (MAM) role in mitochondrial fission. It is well established that a close interaction between mitochondria and endoplasmic reticulum (ER) that regulates calcium between these organelles [22,45–48]. Recently Area-Gomez and colleagues found elevated MAM functions at the ER-mitochondrial interface, which in turn caused increased crosstalk between mitochondria and ER in AD [49]. Hedskog and colleagues also found in the upregulation of MAM-associated proteins AD mouse models before the appearance of amyloid plaques [50]. Mfn2 and OPA1 mutations causes significant disruption of all the homeostatic mechanisms that operate at MAM interfaces and it is says MAM role of mitochondrial dynamics [51–54]. It is interesting to understand the interactions between ER and mitochondria and how these interactions promote mitochondrial fragmentation in relation to Drp1 in AD, other neurodegenerative diseases and also diseases that involve oxidative stress and mitochondrial dysfunction [56–58]. In Friedman and colleagues proposed model, the ER tubules wrap around a mitochondrial segment and forms constrictions which in turn facilitates the recruitment of DRP1 at this area to complete mitochondrial fission process [55].

However established research studies revealed that, the depletion of mouse endogenous Drp1, caused cell death in cortical neurons and also significantly increased mitochondrial length in non-neuronal and neuronal cells [59–60]. Thus, Drp1 may play an important role not only in regulating mitochondrial morphology and mitochondrial division, but also in affecting cell survival in neurons.

Drp1 and mitochondrial distribution

Drp1 has been found to play an important role in mitochondrial distribution in mammalian cells [61–66]. Several investigators have summarized Drp1 role in mitochondrial distribution [6, 67–68]. Recent studies on Drp1’s role in mitochondrial distribution on AD are summarized below.

In AD neurons, Drp1 has been found to play a role in mitochondrial distribution. The intracellular distribution of mitochondria critically important to neuronal survival since the great morphological complexity and dependency on mitochondria for energy at multiple selective sites make neurons particularly sensitive to perturbations in mitochondrial distribution [69]. The loss of mitochondria from axon terminals leads to synaptic dysfunction in flies [70–72].

To understand mitochondrial distribution in AD neurons, Wang et al. (2009) silenced Drp1, OPA1, Mfn1 and Mfn2, and the overexpression of Fis1 in human neuroblastoma (M17) cells and studied mitochondrial distribution. They found in M17 cells that silenced Drp1, OPA1, Mfn1 and Mfn2 and overexpression of Fis1 conditions caused perinuclear accumulation of mitochondria leaving other, more remote areas of the cell devoid of mitochondria in AD neurons [20].

Using APP mice primary neurons and cellular, molecular and electron microscopy methods, Calkins et al. (2011) studied mitochondrial motility, content and distribution and mitochondrial transport. They found excessive fragmentation of mitochondria and accumulated in the soma of hippocampal neurons in APP neurons relative to wild-type neurons. Further, they also found reduced anterograde moving mitochondria, suggesting that abnormal distribution of mitochondria and synaptic degeneration in neurons that overexpress mutant APP and Aβ [73].

These studies suggest a significant reduction of mitochondrial density and uneven mitochondrial coverage in neuronal processes may lead to synaptic degeneration and neuronal dysfunction in AD neurons.

Drp1 and peroxisomal fragmentation

Several recent studies have revealed that Drp1 is involved in fragmenting peroxisomes in mammalian cells through Fis1 and Mff receptors, which have been found to be receptors for both Drp1 and peroxisomes [42, 60, 74]. In addition, MiD49 and MiD51 have been found to be adaptor proteins for peroxisomes and mitochondria [42], which leads to the question whether Fis1, Mff, MiD49, and MiD51 are related to each other and to Drp1.

Palmer et al. (2013) investigated whether MiD49, MiD51, and Mff have a role in mitochondrial dynamics and to what extent they are involved in Drp1 in mitochondrial fragmentation and function. They found that the overexpression of these adaptor proteins caused the elongation of peroxisomes [42]. Unlike Fis1 and Mff, MiD49 and MiD51 were not targeted to the peroxisomal surface, suggesting that they specifically act to facilitate Drp1-directed fission at mitochondria. Moreover, when MiD49 or MiD51 was targeted to the surface of peroxisomes and lysosomes Drp1 was specifically recruited to these organelles. In addition, the Drp1 recruitment activity of MiD49/51, appeared stronger than that of Mff or Fis1. Based on these findings, Palmer et al. concluded that MiD49 and MiD51 can act independently of Mff and Fis1 in Drp1 recruitment and suggest that they provide specificity to the division of mitochondria [42].

In another study, Wakabayashi et al. (2009) found peroxisomes were elongated in Drp1-null embryonic fibroblasts [60]. Although Drp1 controls the division of mitochondria and peroxisomes, it is likely that the embryonic lethality of Drp1−/− mice results from defects in mitochondrial division rather than peroxisomal defects because mice lacking Pex11β, a peroxisomal protein specifically required for peroxisomal division, die after birth [75]. In another study, Koch et al. (2003) proposed a direct role for Drp1 in peroxisomal fission and in the maintenance of peroxisomal morphology in mammalian cells [74]. Koch et al. (2005) also demonstrated the role of human fission 1 in peroxisomal growth and division; In conclusion, they found hFis1 is needed for targeting peroxisomes and mitochondria; hFis1 was found to promote peroxisome division upon ectopic expression, whereas the silencing of Fis1 with RNA inhibited fission and caused the tubulation of peroxisomes [76].

These studies suggest that Drp1 has a significant role in the fragmentation of peroxisomes and tabulation. However, further research still needed to understand functional and physiological importance of Drp1 in peroxisomal fragmentation, particularly in disease state such as AD.

Drp1 phosphorylation and mitochondrial functional changes

Phosphorylation of Drp1 appears to be involved in mitochondrial fragmentation in both normal and disease states. Reddy [77, 78] has summarized Drp1 phosphorylation sites and their involvement in mitochondrial fragmentation and fusion in neurodegenerative diseases, such as AD and HD.

Drp1 phosphorylation at Ser 656 is essential for maintaining the shape of mitochondria and regulating mitochondrial apoptosis [79]. Drp1 phosphorylation at Ser 637 may alter Drp1 function and mitochondrial morphology in turn it regulates mitochondrial division [80]. Upon the induction of apoptosis, Drp1 translocates from the cytosol to the mitochondrial outermembrane, where it induces mitochondrial division. On the other hand, inhibition of Drp1 by overexpression of a dominant-negative mutant counteracts the conversion to a puncti form mitochondrial phenotype, prevents the loss of the mitochondrial membrane potential and the release of cytochrome c, and reveals a reproducible swelling of mitochondria. In addition, the inhibition of Drp1 has been found to block cell death by implicating mitochondrial fission during apoptosis [81]. These findings suggest that Drp1 activation is crucial for mitochondrial fragmentation, which in turn initiates apoptotic cell death. In contrast, Bax-induced mitochondrial fragmentation is required for the release of cytochrome c, but failed to block mitochondrial fission that is associated with Bax/Bak activation. This suggests that Bax/Bak-initiated remodeling of mitochondrial networks and cytochrome c release is separate events and that the proteins in the Bcl-2 family influence mitochondrial fission-fusion dynamics, independent of apoptosis [82–83]. Interestingly, cristae remodeling also plays an important role in cytochrome c release during intrinsic apoptosis. Drp1 normally located in the cytoplasm but during mitochondrial division it translocate to mitochondrial outermembrane and binds with Fis1 (adaptor to mitochondrial outer membrane). Opa1 (a dynamin-related protein that resides in the inner mitochondrial membrane) has some distinct molecular and genetic functions in regulating mitochondrial dynamics and in cristae remodeling during apoptosis as the cristae is the rich source for cytochrome C. So down regulation of Opa1 not only causes mitochondrial fragmentation but also alters the shape of the cristae [84–85].

These findings suggest that Drp1 activation is necessary for mitochondrial fragmentation and that Drp1 activation occurs as an early event in apoptotic cell death. Cytochrome c release is dependent of activities of Bax and Bak. Bax activation is not associated with mitochondrial outer-membrane permeabilization. Further research is needed to confirm these results and further research is still needed to understand Drp1 phosphorylation in AD.

Drp1 and SUMOylation

Many researchers have sought to determine the role of Drp1 in SUMOylation, and found that SUMOylation proteins interact with Drp1, and that Drp1 in conjunction with these proteins may regulate mitochondrial fission [86–95].

Although SUMOylation is dispensable for development of neural cell types, this regulatory mechanism is necessary for neuronal survival. Fu et al., (2009) found a causal link of SUMO modification enzymes to apoptosis of neural cells, suggesting a new pathogenic mechanism for neurodegeneration [96]. Harder et al. (2004) identified 2 Drp1-interacting proteins (Ubc9 and Sumo1) and demonstrated mitochondrial fragmentation [91]. Analysis of endogenous Drp1 levels indicated that the overexpression of Sumo1 specifically protects Drp1 from degradation. This overexpression may partially account for the excess in mitochondrial fragmentation [91–92]. Figueroa-Romero et al. (2009) investigated Drp1 regulation in conjunction with SUMOylation of small ubiquitin like modifier- SUMO proteins and findings suggest that SUMOylation of Drp1 is linked to the activity cycle and is influenced by Drp1 localization [97]. Zunino, et al. and Braschi et al. (2009) studied Drp1 regulators of SUMOylation proteins and has shown pivotal role in mitochondrial fragmentation. These studies shed the light on interorganellar communication during the cell cycle [93,94].

Further research is needed to understand the role of Drp1 in SUMOylation, particularly in diseases such as AD.

Drp1 and ubiquitination

Drp1 has been reported to ubiquitinate with mitochondrial E3 ubiquitin ligase MARCH5 and to regulate mitochondrial fission [98,99].

To understand the role of Drp1 in ubiquitination, used immunostaining and biochemical methods Karbowski et al. (2007) studied Drp1 association with the MARCH5 protein. They found that Drp1 interferes with the mutant proteins MARCH5, and MARCH5 RNA induces an abnormal elongation and interconnection of mitochondria, suggesting that MARCH5 mutations interfere with mitochondrial division [98]. In another study, Park et al. (2010) studied the connection between decreased MARCH5 protein and mitochondrial connectivity. They found highly interconnected and elongated mitochondria in cells with reduced MARCH5 protein. Based on these observations, they concluded that a lack of MARCH5 results in mitochondrial elongation, which blocks Drp1 activity, in turn promoting cellular senescence and/or an accumulation of Mfn1 in mitochondria [99]. In continuation, Park et al. (2014) also studied MARCH5-mediated quality control on acetylated Mfn1, which facilitates cell survival and mitochondrial homeostasis in mitochondrial stress conditions [100]. Drp1 and mitofusins proteins are crucial for mitochondrial division and fusion. Nakamura et al. [101] studied interaction between Drp1 and MARCH-V by immunoprecipitation studies and demonstrated interaction of MARCH-V with ubiquitinated Drp1 and Mfn2. Their results have shown that MARCH-V plays an important role in mitochondrial dynamics through Drp1 and Mfn2 [101]. Interestingly, mitochondrial dynamics also regulated mitochondrial ubiquitin ligase (MITOL) through MAM and Mfn2. Sugiura et al. (2013) identified K192 in the GTPase domain of Mfn2 as a major ubiquitination site for MITOL. Later, K192R mutation blocked oligomerization event in the presence of GTP. Finally these researchers’ results suggested that MITOL regulates ER tethering (at MAM) to mitochondria by activating Mfn2 via K192 ubiquitination [102].

Overall, these studies suggest that Drp1 is involved with ubiquitination. However, it is unclear the mechanistic role of Drp1 in AD and other neurodegenerative diseases.

Drp1 and its relationship with glycogen synthase kinaseβ in Alzheimer’s disease

Glycogen synthase kinase 3 (GSK3) is a serine/threonine protein kinase. GSK3 is omnipresent in all tissues, from yeast to mammals, but it is primarily present in the brain. GSK3 is a multifunctional serine threonine kinase, exists in 2 paralogs: GSK3α and GSK3β. GSK3α has the molecular weight of 51kDa, and GSK3β, 46kDa (Figure 3).

Figure 3. General overview of GSK3.

GSK3 dysregulation via mitochondria with neurodegenerative diseases such as Alzheimer’s disease, Parkinson’s disease, Schizophrenia and other bipolar disorders. GSK3 also plays a regulatory role in various types of cancers, diabetes and inflammatory diseases.

GSK-3 was first isolated from skeletal muscle in 1980, at which time it was identified as one of 5 enzymes capable of phosphorylating glycogen synthase [103]. GSK3β are almost identical in the catalytic kinase domain but are differentiated in the N-terminal and C-terminal regions [104–106]. The activities of these 2 paralogs are positively regulated by the phosphorylation of Tyr 279 and Tyr 216, and are negatively regulated by the phosphorylation of Ser²¹ and Ser⁹ [107]. Unlike GSK3β, neither physiological functions nor pathological roles of GSK-3α are well understood. GSK3β has been implicated in multiple biological processes, such as apoptosis, cell differentiation, insulin response, embryonic development, neurogenesis, neuronal polarization, neurite outgrowth, and the regulation of axon-dendrite morphology [108]. Increasing evidence suggests that GSK3β dysregulation is implicated in many diseases, including AD [109–113], cancer [114–116], diabetes [117], inflammatory diseases [118], schizophrenia, bipolar disorder, several mood disorders [119], and mitochondrial diseases [120] (Figure 3).

Of the 2 paralogs, GSK3β is more likely involved in AD pathogenesis. In AD patients and animal models, GSK3β was found to be elevated and increased activity of GSK3β is directly linked to increased levels of Aβ production, which was found to be associated with Aβ deposits, formation of tau phosphorylation and neurofibrillary tangles, and synaptic damage [109,111,121–123]. The possible explanation for the elevation of GSK3β in AD is due to the association of Aβ with NMDA receptors, wnt signaling and insulin [124]. Activated GSK-3β was found to cause tau phosphorylation, NFT formation, and neuronal cell death, whereas the blockade of GSK-3β expression (by antisense oligonucleotides) or its activity by lithium or other GSK3β inhibitors was found to inhibit Aβ-induced toxicity and tau phosphorylation [125–127].

There are limited studies have shown association between GSK3β and Drp1. Initially, Hong et al., (1998) demonstrated association between GSK3β and Drp1 [128]. Chen et al., (2000) reported in their functional characterization studies that this Drp1 variant lacks a proline-rich domain on its C-terminal regions and this region was identified as a critical region for interacting with GSK3β [129]. Chou et al. (2012), studied functional consequences of GSK3β-mediated phosphorylation of Drp1 at the Ser693 site in addition to Ser616 and Ser637 located within the GED domain [130]. Phosphorylation of Drp1 at Ser 637 prevents mitochondrial fragmentation; conversely phosphorylation of Drp1 at Ser616 promotes mitochondrial fragmentation. In this study, they clearly demonstrated that GSK3β-mediated phosphorylation at the Ser693 site induces elongated mitochondrial morphology and exhibits an anti-apoptotic effect against H₂O₂-induced apoptosis rather than inducing autophagy [130]. Prevention of mitochondrial fragmentation is regulated by cAMPK and promotion is carried out by CAMK1α (Figures 4 and 5). But, recent consensus would be that CDK1/Cyclin B phosphorylates Drp1 at Ser616 to stimulate mitochondrial fission (131).

Figure 4. Drp1 phosphorylation sites and their involvement in mitochondrial fission and fusion.

(A) Phosphorylation of Drp1 at Ser637 prevents mitochondrial fragmentation; (B) conversely phosphorylation of Drp1 at Ser 616 promotes mitochondrial fragmentation. Prevention of mitochondrial fragmentation is regulated by cAMPK and promotion is carried out by CAMK1α. Drp1 is found in cytosol and cycles on/off mitochondria.

Figure 5. GK3β-induced Drp1 phosphorylation and mitochondrial fragmentation in Alzheimer’s disease.

GSK3β-mediated phosphorylation of Drp1 at the Ser693 site in addition to Ser616 and Ser637 located within the GED domain induces elongated mitochondrial morphology which in turn inhibits mitochondrial fission/fragmentation. Blockage of GSK3β mediated Drp1 rescues Aβ induced neuronal apoptosis and provides protection from AD.

Chou et al., (2012) studied interaction between GSK3β and Drp1 by performing co-immunoprecipitation, yeast 2-hybrid experiments, co-localization experiments and gel overlay assay. In yeast 2-hybrid experiments, authors created different truncated Drp1 fragments for mapping its binding region with GSK3β [130]. They found wild-type and truncated 2, 3, and 5 fragments of Drp1 interact with GSK3β, but not the truncated 1, 4 and 6 fragments. Their findings suggest that Drp1 fragment (634–690) interacts with GSK3β. Further they confirmed these interactions using co-localized studies. Taken together, these findings suggest that Drp1 (634–390) directly interacts with GSK3β [130]. Chou et al., (2012) further found Drp1 Ser693 is a GSK3β phosphorylated site [130]. Details of Drp1 phosphorylated sites by GSK3β were summarized in Table 1.

Table 1.

Summary of Drp1 phosphorylation sites and effects on mitochondrial dynamics

Drp1 Phosphorylation site	Mitochondrial function	Reference	Remarks
Ser600	Promotes Mt. fragmentation	Han et al., 2008 (ref. 216)	Regulated by CAMK1α
Ser637	Promotes Mt. fragmentation	Chou et al., 2012 (ref. 130)	Independent with CAMK1α regulation
Ser616	Promotes Mt. fragmentation	Chou et al., 2012 (ref. 130)	Independent with CAMK1α regulation
Ser616	Promotes Mt. fission	Kashatus et al., 2011 (ref. 131)	Regulated by CDK1/Cyclin B
Ser693	Prevents Mt. fission/fragmentation	Chou et al., 2012 (ref. 130)	Independent with CAMK1α regulation
Ser40/44 (phosphomimic Drp1)	Promotes Mt. fragmentation	Yan et al., 2015 (ref. 1)	GSK3β dependent regulation

Recently, Yan et al. (2015) studied GSK3β dependent Drp1 phosphorylation by using electron microscopy, gene expression analysis, transfection and biochemical methods. These researchers identified Drp1 phosphorylation at Ser40 and Ser44 was found to increase Drp1 GTPase activity and mitochondrial distribution. Moreover, when Yan et al. transfected neurons with Ser40 and Ser44, phosphomimic Drp1 increased mitochondria fragmentation and neurons with increased mitochondrial fragmentation were more vulnerable to Aβ-induced apoptosis [1]. Therefore, the inhibition of GSK3β induced Drp1 phosphorylation, which may be an effective way to protect neurons from Aβ toxicity. Therefore, in this study researchers blocked GSK3β-induced Drp1 phosphorylation with the artificial polypeptide TAT-Drp1-SpS and found significant reduction in Aβ-induced neuronal apoptosis in cultured neurons. The administration of TAT-Drp1-SpS in the Cornu Ammonis 1 region of the hippocampus significantly reduced Aβ burden and rescued the memory deficits in AD transgenic mice [1].

These findings suggest GSK3β plays a pivotal role in mitochondrial fragmentation by Drp1 phosphorylation in AD neurons and it has been suggested that GSK3β inhibitors are potential drug targets to treat patients with AD. However, further research is needed to confirm the interactions between GSKβ and Drp1/Opa1 along with mitochondrial fusion proteins such as mfn1/mfn2 in AD and also other neurodegenerative diseases.

Alzheimer’s disease

Alzheimer’s disease (AD) is a progressive, age-dependent neurodegenerative disease characterized by the progressive decline of memory, cognitive functions, and changes in behavior and personality. The prevalence of AD is high among aged individuals: 13% of individuals 65 years old have AD and 50% of individuals 85 years of age and older have AD [105]. Histopathological examination of AD postmortem brains revealed that the presence of extracellular neuritic plaques, intracellular neurofibrillary tangles and neuronal loss. AD is also associated with the loss of synapses, oxidative stress & mitochondrial structural and functional abnormalities, inflammatory responses, changes in cholinergic neurotransmission, hormonal changes and cell cycle abnormalities [3, 16, 20, 64, 132–146] (Figure 6). Genetic mutations are responsible for causing early onset, familial AD for a small proportion of total AD patients, but the causal factors for the vast majority of late-onset, sporadic AD cases are still largely unknown. Several factors, including aging, lifestyle, diet, environmental exposure, apolipoprotein allele E4 genotype, and several other genetic variants reported to involve in late-onset AD [141–142].

Figure 6. Summary of cellular and molecular changes in the pathogenesis of Alzheimer’s disease.

Alzheimer’s disease is associated with the loss of synapses, oxidative stress, mitochondrial structural and functional changes, inflammatory responses, alterations in cholinergic neurotransmission, hormonal dysregulation and cell cycle abnormalities. All these changes are affected on long-term potentiation which in turn causes long term depression across the neurons, ultimately effecting synaptic transmission.

Mitochondrial oxidative damage and synaptic dysfunction are early events in AD pathogenesis. Increasing evidence suggests that mitochondrial abnormalities are largely involved in AD progression, and pathogenesis, including 1) defective glucose metabolism in AD brains, 2) reduced mitochondrial enzyme activities in AD, 3) mitochondrial DNA defects in AD [147–150], 4) abnormal mitochondrial gene expressions in AD brains [151] and AD transgenic mice [152], abnormal mitochondrial dynamics 3, 20, 64, 73,139,140,153,154].

Oxidative stress and Mitochondrial Dysfunction in Alzheimer’s disease

Several lines of evidence suggest that mitochondrial dysfunction and oxidative stress have been extensively reported in AD pathogenesis:

1. AD postmortem brains - Increased production of free radicals and lipid peroxidation, elevated oxidative DNA protein damages and decreased ATP production in postmortem AD brains relative to brains from non-demented healthy subjects [140, 155–158].

2. AD mouse models - several studies reported increased production of free radicals and lipid peroxidation and reduced levels ATP, cytochrome oxidative enzymatic activity in affected brain regions [66, 155, 159–160].

3. AD cells and primary neurons – Increasing studies found that increased levels of lipid peroxidation and free radical production, and reduced levels of mitochondrial ATP, cell viability and cytochrome oxidase activity in primary neurons from AD transgenic mice [73] and neuronal cells treated with Aβ peptide [20, 64, 139, 159, 160].

These studies strongly suggest that mitochondrial dysfunction and oxidative stress are important features of AD pathogenesis.

Aβ and phosphorylated tau association with mitochondria in Alzheimer’s disease neurons

Increasing evidence suggests that mutant APP and Aβ are associated with mitochondria in AD postmortem brains and also brains of several lines transgenic AβAPP mice [158, 159, 161–174]. This abnormal association of mutant APP and Aβ with mitochondria induces free radical production and causes mitochondrial dysfunction.

Recently, several groups found N-terminal tau (20–22 kDa) is associated with mitochondria [175–177]. A 22–22kDa N-terminal tau is enriched in human mitochondria from cryopreserved synaptosomes of AD brains and that the amount of tau is correlated with pathological synaptic changes and mitochondrial dysfunction in AD neurons [175].

Findings from these studies suggest that abnormal association between Aβ and mitochondria, and tau and mitochondria, causes mitochondrial dysfunction in AD pathogenesis.

Abnormal mitochondrial dynamics in relation to elevated Drp1 in AD

Abnormal mitochondrial dynamics and synaptic damage have been reported as early cellular changes in AD pathogenesis [3, 20, 73, 139, 140, 153, 154, 178].

Using human neuroblastoma (M17) cells that express mutant human APP and/or wild-type APP and molecular & cellular and electron microscopy studies, Zhu’s group [178] have studied mitochondrial dynamics in AD. They found 40% M17 cells overexpressing WT APP (APPwt M17 cells) and more than 80% M17 cells overexpressing APPswe mutant (APPswe M17 cells) displayed alterations in mitochondrial morphology and distribution. Specifically, mitochondria exhibited a fragmented structure and an abnormal distribution accumulating around the perinuclear area [178]. These mitochondrial changes were abolished by treatment with beta-site APP-cleaving enzyme inhibitor IV. From a functional perspective, APP overexpression affected mitochondria at multiple levels, including elevating ROS levels, decreasing mitochondrial membrane potential, and reducing ATP production, and also caused neuronal dysfunction such as differentiation deficiency upon retinoic acid treatment. They also found reduced Drp1 and Opa1 and increased levels of Fis1 in APPwt and APPswe M17 cells. They concluded that overexpression of APP, through amyloid beta production, causes an imbalance of mitochondrial fission/fusion that results in mitochondrial fragmentation and abnormal distribution, which contributes to mitochondrial and neuronal dysfunction [178].

In the next study, the same group [20] studied abnormal mitochondrial dynamics by investigating the changes in the expression of mitochondrial fission and fusion proteins in AD brain. They found oligomeric amyloid-beta-derived diffusible ligands (ADDLs) caused mitochondrial fragmentation and reduced mitochondrial density in neuronal processes. More importantly, ADDL-induced synaptic loss particularly reduced dendritic spines and PSD95 in M17 neurons that correlated with abnormal mitochondrial distribution [20].

More recently, the Reddy laboratory has reported abnormal mitochondrial dynamics (increased fission and decreased fusion), Aβ interaction with Drp1, increased mitochondrial fragmentation and impaired axonal transport and synaptic degeneration [38, 39, 73, 139, 140]. These abnormalities increased with disease progression.

Using electron and confocal microscopy, gene expression analysis, and biochemical methods, Manczak et al., (2010) studied mitochondrial structure and function and neurite outgrowth in mouse neuroblastoma (N2a) cells treated with Aβ peptide 25–35 [139]. They found increased expressions of mitochondrial fission genes and decreased expression of fusion genes in cells treated with Aβ peptide relative to untreated cells [139]. Electron microscopy of the N2a cells incubated with Aβ revealed a significantly increased number of mitochondria, indicating that Aβ fragments mitochondria. Biochemical analysis revealed that function is defective in mitochondria. Neurite outgrowth was significantly decreased in Aβ-incubated N2a cells, indicating that Aβ affects neurite outgrowth [139]. These observations suggest that Aβ impairs mitochondrial dynamics, fragments mitochondria and causes synaptic dysfunction and neurite outgrowth, ultimately leading to neuronal damage.

Using primary neurons from a well-characterized AβPP mice (Tg2576 line), Reddy’s group [73] studied mitochondrial activity, including mitochondrial dynamics (fission-fusion gene expressions and protein levels), mitochondrial morphology and mitochondrial function and total number of motile mitochondria and axonal transport of mitochondria [73]. They also studied the nature of Aβ-induced synaptic alterations, and cell death in primary neurons from AβPP mice. They found increased mRNA levels of fission genes – Drp1 and Fis1 and reduced levels of fusion genes Mfn1, Mfn2 and Opa1, and reduced mRNA levels of synaptic genes in AβPP neurons relative to wild-type neurons, indicating the presence of abnormal mitochondrial dynamics in neurons expressing human mutant APP [73]. Further, significantly reduced total motile mitochondria and anterograde moving mitochondria AβPP neurons compared with wild-type neurons. Transmission electron microscopy revealed a large number of small mitochondria and structurally damaged mitochondria, with broken cristae in AβPP primary neurons. They also found an increased accumulation of oligomeric Aβ and increased apoptotic neuronal death in the primary AβPP neurons relative to the wild-type neurons. Their results revealed an accumulation of intraneuronal oligomeric Aβ, leading to mitochondrial and synaptic deficiencies, and ultimately causing neurodegeneration in AβPP cultures [73].

In their next study, Reddy’s group [140] measured mRNA and protein levels of mitochondrial structural genes in the frontal cortex of patients with early, definite and severe AD and in control subjects [140]. They found increased expressions of the mitochondrial fission genes Drp1 and Fis1 and decreased expression of the mitochondrial fusion genes Mfn1, Mfn2, Opa1 and Tomm40. The matrix gene CypD was up-regulated in AD patients relative to control subjects. Results from their real-time RT-PCR and immunoblotting analyses suggest that abnormal mitochondrial dynamics increase as AD progresses [140].

In 2013, Swerdlow’s group [154] studied bioenergetic function using cytoplasmic hybrid (cybrid) cell lines of AD patients. They transferred mitochondria from MCI, AD and control subject platelets to mtDNA-depleted SH-SY5Y cells. They found mitochondrial fission-fusion balance shifted towards increased fission in the AD and MCI cybrids – in other words, increased levels of Drp1 and Fis1 and reduced levels of Mfn1, Mfn2 and Opa1 in AD cybrids. They also found changes in oxygen consumption, respiratory coupling and glucose utilization in AD and MCI cybrids relative to control cybrids. They conclude reduced bioenergetic function is present during very early AD, is not brain-limited and induces protean retrograde responses that likely have both adaptive and mal-adaptive consequences [154].

Findings these studies suggest that the presence of Aβ-induced abnormal mitochondrial dynamics, reduced total and anterograde moving mitochondria, synaptic damage and defective mitochondrial function in AD neurons.

Drp1 interaction with Aβ and phosphorylated Tau in Alzheimer’s disease

The Reddy Laboratory next studied the relationship between Drp1 and Aβ, and Drp1 and phosphorylated tau in AD neurons [38, 140].

Using co-immunoprecipitation and immunofluorescence analysis, they studied interaction between Drp1 and Aβ. Drp1 immunoprecipitation and immunofluorescence analysis of Aβ antibodies revealed that Drp1 interacts with Aβ monomers and oligomers in AD patients, and these abnormal interactions are increased with disease progression [140].

Further, using co-immunoprecipitation and immunofluorescence analyses and AD postmortem brains and cortical tissues from APP, APP/PS1 and 3XTg.AD mice, they also studied physical interaction between Drp1 and phosphorylated tau. They found physical interaction between Drp1 and phosphorylated tau [38].

Findings from these studies suggest that Drp1 interacts with Aβ and phosphorylated tau, likely leading to excessive mitochondrial fragmentation, and mitochondrial and synaptic deficiencies, ultimately possibly leading to neuronal damage and cognitive decline.

Nakanishi et al., [179] also supports a role for hyperphosphorylated tau in mitochondrial impairment. By overexpression of the postsynaptic protein i.e α1-takusan (via interaction with PSD-95, mitigates oligomeric Aβinduced synaptic loss) inhibits Aβ-induced tau hyperphosphorylation and prevents Aβ-induced mitochondrial fragmentation in cortical and hippocampal cultured neurons.

Treatment designed to reduce the expression of Drp1, Aβ and/or phosphorylated tau may decrease the interaction between Drp1 and phosphorylated tau and the interaction between Drp1 and Aβ, may protect AD neurons from toxic insults of excessive Drp1, Aβ and/or phosphorylated tau [179].

Drp1 knockout mice and neuronal survival

The function of Drp1 was studied in Drp1 knock out mice, neural specific (NS) Drp1 (−/−) and homozygote Drp1 knockout mice. Two groups independently studied the function of Drp1 by creating knockout mouse models [59, 60]. These groups studied synapse formation, generation of synapses, neuronal structure in addition with biochemistry and molecular biology of both heterozygote and homozygote knockout mice.

Ishihara et al., (2009) group found the physiological significance of Drp1-dependent mitochondrial fission in tissue development, especially in brains of Drp1-knockout mice [45]. The cell lines that reduced from Drp1−/− mice has shown highly interconnected and perinuclearly aggregated mitochondria. In striking contrast, primary cultured neuronal cells from ND-Drp1−/− mice has shown not only severe defects in synapse formation but also had a high sensitivity to Ca²⁺-dependent apoptosis [59]. These results suggest that Drp1-dependent mitochondrial fission is dispensable for the function of non-polarized cells, but indispensable for neurons (polarized cells) to maintain the normal spatiotemporal properties of mitochondria that are essential for the responses to Ca²⁺ or synaptic functions. In addition, use of the established cell lines from Drp1−/− mice has clarified role of Bax/Bak-regulated apoptosis via mitochondrial fission. These results confirmed Drp1-dependent mitochondrial fission is not essential for the release of cytochrome c and the subsequent progression of apoptosis, but Drp1 facilitates these processes [59].

Bax/Bak mediated mitochondrial outer-membrane permeabilization to a class of intermembrane space-localized proteins [180, 181] occurring before the release of cytochrome c is not affected by Drp1 knockout, which is consistent with observations in Drp1-knockdown cells [182, 183]. Intriguingly, contrast studies have been reported that a chemical inhibitor of Drp1, mdivi-1, potently blocks the Bax/Bak dependent release of both Smac/Diablo and cytochrome c [184]. It could be speculated that Drp1 affects Bax/Bak activity to modulate structural or conformational changes in the mitochondrial inner membrane through interaction between the fusion/fission machineries of the mitochondrial outer and inner membranes [184].

In another study, Wakabayashi et al., (2009) developed complete and tissue-specific mouse knockouts of Drp1 [60]. These researchers found homozygous deletion of Drp1 lead to embryonic lethality Homozygote knockout Drp1 mice died by embryonic day 11.5, but this embryonic lethality was not likely caused by ATP deprivation, as Drp1-null cells showed normal intracellular ATP levels [60]. Homozygote knockout Drp1 mice embryos also have shown defective in placental development and decreased cardimyocyte beat rates. The timing of Drp1 (−/−) embryonic lethality is similar to the timing of lethality in mice defective in Mfn1, Mfn2, and Opa1, which are all necessary for mitochondrial fusion [185–187]. These observations suggest that proper mitochondrial dynamics are critical for embryonic development in mice.

Drp1 was knocked out in the cerebellum and surrounding regions using brain-specific Drp1KO with En1- Cre recombinase and floxed alleles of Drp1 [60]. Development of the cerebellum was dramatically decreased, and the mice died within 24 h of birth. In Drp1KO cerebella, proliferation of neurons was greatly decreased, and the number of Purkinje cells was dramatically decreased [60]. Similarly, when Drp1 was deleted in a broad region of the brain using Nes-Cre recombinase, brain development was inhibited and many apoptotic cells were observed in the premature, superficial layer neurons and the deep cortical layers [59]. In neurons isolated from Nes-Cre Drp1KO mice, the size of mitochondria was increased and the number was decreased [59]. In particular, synapses lacked mitochondria, and synapse formation was defective in these neurons in culture [59]. These observations are consistent with findings in Drosophila mutants carrying mutations in Drp1 [188]. Drp1 mutant flies failed to distribute mitochondria at the neuromuscular junctions, leading to defects in mobilization of the vesicle reserve pool, likely due to decreased amounts of ATP in this region [188]. As a result, when Drp1 mutants were exposed to high frequency stimulation to induce continuous neurosecretion, the mutants were unable to maintain normal levels of neurotransmission [188]. In mammals, Drp1 may have a more direct role in endocytosis of synaptic vesicles. Drp1 forms protein complexes with components of clathrin-coated vesicles and controls the size of endocytic vesicles in response to synaptic stimulation [189].

Recently, Reddy laboratory [38,39] studied effects of partial Drp1 reduction on mitochondrial function and synaptic activity. They sought to characterize synaptic, dendritic and mitochondrial proteins, and mitochondrial function and GTPase enzymatic activity, in Drp1 heterozygote knockout mice. Interestingly, they found no significant changes in synaptic, dendritic, and mitochondrial proteins in the Drp1 heterozygote knockout mice compared to the wild-type mice. Further, mitochondrial function and GTPase enzymatic activity appeared to be normal. However, H₂O₂ and lipid peroxidation levels were significantly reduced in the Drp1 heterozygote knockout mice compared to the wild-type mice. They speculated these findings suggest that partial Drp1 reduction does not affect mitochondrial and synaptic viability [38, 39].

Overall, these knockout studies revealed that Drp1 is essential for mitochondrial division, mitochondrial distribution, mitochondrial morphogenesis, apoptosis and cell survival.

Drp1 reduction in Alzheimer’s disease mouse models

There are very limited numbers of studies about the research on Drp1 using AD mouse models. Yan et al. (2015) did their research to find the role of Drp1 along with GSK3β in mitochondrial fragmentation in double mutant APPSwe/PSEN1dE9 (APP/PS1) transgenic mice. They used co-immunoprecipitation, phosphorylation assays, GTP assays, TUNEL analysis, electron microscopy, Aβ ELISA, Aβ plaque and behavioral analysis (Morris water test) studies on AD transgenic mice. They found GSK3β plays crucial in role mitochondrial fragmentation for causing Aβ neurotoxicity in AD. They concluded that GSK3β plays the pivotal role in the phosphorylation of Drp1 in mitochondrial fragmentation and thus influence mitochondrial dynamics. This mitochondrial fragmentation causes spatial learning and memory deficits, neuronal apoptosis and Aβ mediated neurotoxicity [1].

To understand the beneficial effects of partial reduction of Drp1 in AD pathogenesis, currently, Reddy laboratory is conducting 2 studies.

In the first study, they crossed Drp1+/− mice with AβPP transgenic mice (Tg2576 line) and created double mutant mice – Drp1+/−xAβPP [SFN, 2015 Program # 131.13/D21]. Using real-time RT-PCR and immunoblotting analyses, we measured mRNA expressions and protein levels of genes related to the mitochondrial dynamics – Drp1 and Fis1 (fission), Mfn1, Mfn2 and Opa1 (fusion), CypD (Matrix), mitochondrial biogenesis - Nrf1, Nrf2, PGC1α and TFAM and synaptic - synaptophysin, PSD95, synapsin 1, synaptobrevin 1, neurogranin, GAP43 and synaptopodin in brain tissues from 6-month-old Drp1+/−, AβPP, Drp1+/−xAβPP and wild-type mice. Using biochemical methods, we also studied mitochondrial function and measured soluble Aβ in brain tissues from all lines of mice in our study. They found decreased mRNA expressions and protein levels of fission genes Drp1 and Fis1 and matrix gene CypD, and increased levels of fusion genes Mfn1, Mfn2 and Opa1, Nrf1, Nrf2, PGC1α, TFAM (biogenesis) and synaptophysin, PSD95, synapsin 1, synaptobrevin 1, neurogranin, GAP43 and synaptopodin (synaptic) in 6-month-old Drp1+/−xAβPP mice relative to AβPP mice. Mitochondrial functional assays revealed that mitochondrial dysfunction is reduced in Drp1+/−xAβPP mice relative to AβPP mice, suggesting that reduced Drp1 enhances mitochondrial function in AD neurons. Sandwich ELISA assay revealed that soluble Aβ levels were significantly reduced in Drp1+/−xAβPP mice relative to AβPP mice, indicating that reduced Drp1 decreases soluble Aβ production in AD progression [SFN, 2015 Program # 131.13/D21].

In the second study, Reddy’s group [SFN, 2015 Program # 131.09/D17] crossed Drp1+/− mice with tau transgenic mice (P301L line) and created double mutant mice – Drp1+/−xTau. Using real-time RT-PCR and immunoblotting analyses, they measured mRNA expressions and protein levels of genes related to the mitochondrial dynamics – Drp1 and Fis1, Mfn1, Mfn2 and Opa1, CypD, mitochondrial biogenesis Nrf1, Nrf2, PGC1α and TFAM and synaptic genes synaptophysin, PSD95, synapsin 1, synaptobrevin 1, neurogranin, GAP43 and synaptopodin in brain tissues from 6-month-old Drp1+/−, Tau, Drp1+/−xTau and wild-type mice. Using biochemical methods, we also studied mitochondrial function and measured soluble and phosphorylated tau in brain tissues from all lines of mice in this study. They found reduced mRNA expressions and protein levels of fission genes Drp1 and Fis1, and increased levels of fusion genes Mfn1, Mfn2 and Opa1, biogenesis genes Nrf1, Nrf2, PGC1α, TFAM, and synaptic genes synaptophysin, PSD95, synapsin 1, synaptobrevin 1, neurogranin, GAP43 and synaptopodin genes in 6-month-old Drp1+/−xTau mice relative to AβPP mice. Mitochondrial functional assays revealed that mitochondrial dysfunction is reduced in Drp1+/−xTau mice relative to tau mice, suggesting that reduced Drp1 enhances mitochondrial function in AD neurons. Measurement of phosphorylated tau studies revealed that soluble and phosphorylated tau levels were significantly reduced in Drp1+/−xTau mice relative to Tau mice, indicating that reduced Drp1 decreases soluble and phosphorylated tau production in AD progression [SFN, 2015 Program # 131.09/D17].

Taken together, these findings suggest that reduced Drp1 protects against Aβ toxicity, phosphorylated tau, mitochondrial dysfunction and synaptic damage in AD. These findings also suggest that partial reduction of Drp1 decreases the production of soluble and phosphorylated tau, reduces mitochondrial dysfunction, and maintains mitochondrial dynamics, enhances mitochondrial biogenesis and synaptic activity. Findings of this study may have implications for the development of Drp1 based therapeutics for patients with AD and other tauopathies.

Drp1 and CDK5 in Alzheimer’s disease

Accumulated Aβ causes hyperactivation of N-methyl-D-aspartate receptors (NMDARs) which triggers downstream pathways such as phosphorylated tau (or p-tau), caspases, a cyclin-dependent kinase (Cdk5)/(Drp1), calcineurin/PP2B, PP2A, GSK-3β. These events cause endocytosis of AMPA receptors (AMPARs) as well as NMDARs through calcium ions (Figure 7). Ultimately, these pathways lead to consequent synaptic dysfunction, mitochondrial dynamics dysregulation, bioenergetics compromise and impaired long-term potentiation, and cognitive decline [190].

Figure 7. GS3β-Drp1-p53-CDK5 interaction in the pathogenesis of Alzheimer’s disease.

Amyloid beta peptide causes hyper activation of N-methyl-D-aspartate receptors, which triggers downstream pathways such as phosphorylated tau, cyclin-dependent kinase 5, dynamin-related protein 1 and GSK-3β. These events cause endocytosis of AMPA receptors as well as NMDARs through calcium ions. These pathways may lead to abnormalities in mitochondrial dynamics and bioenergetics and impaired long-term potentiation, and synaptic dysfunction in Alzheimer’s disease neurons. The p53-dependent apoptotic pathway mobilizes the pro-apoptotic protein Bax, in turn causes mitochondrial fragmentation during apoptosis by forming a complex with Drp1 and Mfn2 at fission sites. As a transcriptional regulator, p53 may also have a direct influence on the expression and function of proteins regulating mitochondrial morphology.

Using cultured primary cortical neurons, Cho et al., (2009) studied found Aβ protein triggered mitochondrial fission, synaptic loss, and neuronal damage, in part via S-nitrosylation of Drp1. Homology modeling of human Drp1 and Quantification of dendritic spine density analysis [191].

Qu et al., (2011 and 2012) studied the role of found that transnitrosylation from Cdk5 to Drp1 would be associated change in Gibbs free energy. According to their biotin-switch data, it has shown that SNO-Drp1 levels are elevated in Cdk5-transfected cells, which cause massive mitochondrial fragmentation compared with control plasmid-transfected cells. These findings suggest that transnitrosylation of NO from Cdk5 to Drp1 might be involved in the pathway to SNO-Cdk5–mediated spine loss, and eventually results in neuronal cell death AD [192, 193].

In cerebrocortical cultures, Molokanova et al. (2014) found extra synaptic NMDAR-mediated increase in NO can produce S-nitrosylation of Drp1 and Cdk5 (by biotin sandwich assay) targets known to contribute to Aβ-induced synaptic damage. In support of a pathological role for S-nitrolyation of Drp1, Drp1 is apparently S-nitrosylated aberrantly because it is found at high levels in postmortem human AD brains but not in control brains [190–191], as well as in peripheral blood lymphocytes of AD but not control patients [194].

All these findings suggest that increased levels of NO associated with aberrant formation of SNO-Cdk5 and SNO-Drp1, leading to the dysregulation of downstream pathways, synaptic dysfunction, and finally neuronal damage and neuronal death in AD. Taken together, these findings also suggest that SNO-Drp1 may represent a potential therapeutic target for protecting neurons and their synapses in AD.

Drp1 and P53 in Alzheimer’s disease

Oxidative stress is the consequence of mitochondrial dysfunction is majorly associated with the pathogenesis of AD, PD, HD, ALS and aging [6, 11, 12, 14, 195, 196]. This oxidative stress is the generation of DNA damages with subsequent induction of the p53 a tumor suppressor protein [197]. Thus, induction of the p53 protein in neurons has been associated for a diverse array of neurological diseases including AD, PD and HD disease, and as well as nervous system injury [197–199]. Numerous lines of evidence suggest that p53 induction is responsible for neuronal damage. Mitochondria change size and shape by undergoing fission and fusion, processes that are orchestrated by a well conserved cellular machinery comprised of dynamin-related GTPases, Drp1 for fission and mitofusins (Mfn1/2), and Opa1 for fusion [38, 39, 63, 77, 78, 200].

Several studies demonstrated that diverse forms of acute neurotoxic stress commonly cause mitochondrial fission and that enhancing the expression of fusion proteins or suppressing Drp1 activity reduces fission as well as neuronal cell death [38, 39, 66, 77, 78, 200, 201]. Despite the likely contribution of p53 to neuronal dysfunction and loss due to oxidative stress associated with neurodegenerative conditions and aging, little progress has been made in characterizing how DNA damage directly alters mitochondrial morphology in compromised neurons. The p53-dependent apoptotic pathway mobilizes the pro-apoptotic protein Bax through the transcriptional activation of BH3 domain-only proteins such as PUMA [202]. In addition to its activity in permeabilizing the outer mitochondrial membrane, Bax promotes mitochondrial fragmentation during apoptosis by forming a complex with Drp1 and Mfn2 at fission sites [203]. As a transcriptional regulator, p53 may also have a direct influence on the expression and function of proteins regulating mitochondrial morphology.

Wang et al. (2013) found that DNA damage which induced by the topoisomerase I inhibitor, camptothecin (CPT), in cultured cortical neurons does not cause fragmented but rather elongated mitochondria, which is, at least in part, due to decreased expression of Drp1 [204]. Overexpression of Drp1/parkin mitigated the mitochondrial elongation and cell death induced by CPT. Staurosporine, a p53-independent apoptotic stress, caused mitochondrial fragmentation as previously reported while CPT treatment of mouse embryonic fibroblasts resulted in mitochondrial fragmentation. Thus, their findings suggest that p53- dependent neuronal cell death is uniquely associated with a net increase in mitochondrial length involving reduced expression of Drp1. The changes in mitochondrial morphology mediated by p53 could affect mitochondrial function, transport and mitophagy in neurons during the course of aging and neurodegenerative disease [204].

In another Study, Guo et al. [205] found that Drp1 binding to p53 induced mitochondria-related necrosis. In contrast, inhibition of Drp1 by Drp1 siRNA reduced necrotic cell death in mouse embryonic fibroblasts exposed to oxidative stress that is due to H₂O₂. In the same study they confirmed Drp1-p53 interactions by IP analysis. Finally they confirmed neuronal death is due mitochondrial damage, which in turn causes Drp1-p53 interactions through MDM2 [205].

All these findings suggest that under mitochondrial dysfunction Drp1 interacts with p53, which may cause aberrant formation of SNO-Cdk5 and SNO-Drp1, leading to the dysregulation of downstream pathways, synaptic dysfunction, and finally neuronal cell death in AD. However there are no concrete studies, which show the interactions of p53 and CDK5 with Drp1 or other mitochondrial division proteins in neuronal cell death in AD.

Drp1 and microRNAs in Alzheimer’s disease

microRNAs (miRNAs) are a large family of conserved small (18–22 nucleotides) non-coding RNAs. These miRNAs plays an important role in the post-transcriptional regulation of gene expression. In mammals, miRNAs are predicted to control the activity of ~50% of all the protein-coding genes [206]. Aβ formation and Aβ deposits in AD are accompanied by alterations in the levels of many distinct classes of miRNA, certain of which target mRNAs encoding proteins involved in AD pathogenesis [207–211]. Several miRNA species such as miR-101 and miR-106, target APP, and their down-regulation in AD flavors an elevated generation of Aβ [207, 210, 212, 213]. Interestingly, several nucleotide polymorphisms associated with AD located in the miRNA-binding region. Recent studies have shown miR29, 15, 107, 181, 146, 9,101,106 plays crucial role in the pathogenesis of AD [214]. A very few studies have shown that miR-30 and miR499 are involved in the regulation of mitochondrial dynamics by Drp1 through p53 and calcineurin [211, 215].

Li et al. [215] found that miR-30 family members are able to inhibit mitochondrial fission and apoptosis in cardiomyocytes. They identified that p53 is a target of miR-30 family members. p53 induces mitochondrial fission by transcriptionally upregulating Drp1 expression. miR-30 can inhibit mitochondrial fission through suppressing the expression of p53 and its downstream target Drp1 [215]. In another study, Wang et al., [211] reported both the α and β isoforms of the calcineurin catalytic subunit are direct targets of miR-499 and that miR-499 inhibits cardiomyocyte apoptosis through its suppression of calcineurin-mediated dephosphorylation of Drp1, thereby decreasing Drp1 accumulation in mitochondria and Drp1-mediated activation of the mitochondrial fission. In these studies, it has shown that Drp1 is regulated through p53. However, till now there are no studies on the role of miRNAs that clearly demonstrates impaired mitochondrial dynamics (either fission or fusion) in the AD or other neurodegenerative diseases pathogenesis [211] (Figure 8).

Figure 8. Role of microRNA30 and microRNA499 in neuronal survival.

Increased free radicals trigger DNA damage response and/or oxidative stress that may elicit p53-dependent apoptotic pathway. p53 mobilizes the pro-apoptotic protein Bax through the transcriptional activation of BH3 domain in proteins such as PUMA that may elevates Drp1 levels, ultimately leading to mitochondrial fragmentation. MicroRNAs, MiR30 and miR499 reduce mitochondrial fission activity by suppressing the expression of p53 and Drp1 and enhance neuronal survival.

Further research is needed to understand precise link between miRNAs and abnormal Drp1 activation, and also physiological relevance of miRNA in mitochondrial dynamics and synaptic damage in AD pathogenesis.

Concluding remarks and future directions

Mitochondrial dysfunction and synaptic damage are early and important cellular changes in AD pathogenesis. Impaired mitochondrial dynamics (increased fission and reduced fusion) is reported in AD neurons. Increasing evidence suggests that balanced mitochondrial dynamics are critically important in the maintenance of mitochondrial shape, distribution, and sizes are very important in normal neuronal function. Recent mitochondrial studies of yeast, worms, rodents and humans have identified several GTPase proteins, including Drp1 (fission), Mfn1, Mfn2, and Opa1 (fusion). Among these, Drp1 is evolutionarily conserved in lower to higher organisms and may have some structural and functional importance in maintaining cell survival and cell death. Recent knockout mice studies of Drp1 suggest that Drp1 is essential for cell survival and mitochondrial division. Recent advancements in molecular, cellular, electron microscopy and confocal and imaging studies revealed that Drp1 is associated with several cellular functions, including mitochondrial fragmentation, phosphorylation and cell death. Mitochondrial fission protein Drp1 interacts with Aβ and phosphorylated tau in AD. Further research is needed to understand the precise mechanisms of Drp1 in fragmenting mitochondria in relation to Aβ and phosphorylated tau. Identification of interacting binding sites between Drp1 soluble Aβ, and Drp1 and soluble Tau, may provide new clues and may help to develop molecular inhibitors to stop and/or reduce abnormal interactions between Drp1 soluble Aβ, and Drp1 and soluble Tau.

Recent research also revealed that Drp1 is associated with GSK3β, CDK5 and p53. However, the physiological relevance of Drp1 association with GSK3β, CDK5 and p53 in AD is not completely understood. Recent research also found that Drp1 is linked to microRNAs in AD pathogenesis. Further research is needed to answer these important questions.

Highlights.

1. Mitochondrial abnormalities play a large role in neuronal in Alzheimer’s disease.

2. Amyloid beta and phosphorylated tau interacts with the Drp1 in Alzheimer’s disease.

3. Mitochondrial shape, size, distribution and axonal transport are altered in Alzheimer’s neurons.

4. Drp1 association with GSK3beta, CDK5 and miRNAs affects neuronal function in Alzheimer’s disease.

Abbreviations

Drp1

Dynamin-related protein1

GSK3β

Glucose synthase kinase 3 beta

CDK5

Cyclin dependent kinase 5

miRNA

micro ribonucleic acid

NMDAR

N-methyl-D-aspartate receptor

AMPAR

α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor

AD

Alzheimer’s Disease

PD

Parkinson’s Disease

HD

Huntington’s Disease

Aβ

β-amyloid

Fis1

Mitochondrial fission 1 protein

Mfn1

Mitochondrial fusin 1

Opa1

Optic Atrophy 1

CypD

cyclophilin D

PSD95

Postsynaptic Density 95

MiD49

Mitochondrial Dynamics protein 49

MiD51

Mitochondrial Dynamics protein 51

Mdiv1

Mitochondrial division inhibitor 1

Num1

Nuclear migration 1

Mdm 36

Mitochondrial distribution morphology 36

Mff

Mitochondrial fission factor

Nrf1

Nuclear Respiratory Factor 1

Nrf2

Nuclear Respiratory Factor 2

PGC1

Peroxisome proliferator-activated receptor gamma coactivator 1

M17

Human Neuroblastoma cells

N2a

Mouse neuroblastoma 2a

CA1

Cornu Ammonis area 1

GTPase

Guanosine-5′-triphosphate hydrolase

GED

Guanosine triphosphate effector domain

Dnm1

Dynamin 1

APP

Amyloid Precursor protein

co-IP

co-Immunoprecipitation

CaMKIα

Ca^2+/Calmodulin protein kinaseIα

Bax

Bcl2 associated X protein

Bak

Bcl2 associated K protein

Bcl2

B-cell leukemia/lymphoma2

PKA

Protein kinase A

PP2A

Phosphatase 2A

ETC

Electron transport chain

MARCH 5

membrane associated RING-CH-5

PEX 11β

Peroxisomal Biogenesis factor 11β

Ubc9

Ubiquitin-conjugating enzyme 9

Sumo 1

small ubiquitin like modifier 1

APPwt

APP wildtype

APPSwe

APP Swedish

AβPP

Amyloid beta Precursor Protein

Tomm40

Translocase of outer mitochondrial membrane 40

mtDNA

mitochondrial DNA

MCI

mild cognitive impairment

PS1

Presenilin 1

IMS

Inter membrane space

Smac

Second Mitochondria-derived Activator of Caspases

DIABLO

Direct IAP-Binding protein with Low PI

ATP

Adenosine triphosphate

TUNEL

Terminal deoxynucleotidyl transferase dUTP nick end labeling

TFAM

Mitochondrial transcription factor A

GAP43

Glial acidic protein 43

ALS

Amyotropic lateral sclerosis

PUMA

p53 upregulated modulator of apoptosis

CPt

Campothecin

siRNA

small interfering RNA

MDM2

mouse double minute 2

ADDLs

amyloid-beta-derived diffusible ligands