Autophagy and Cell Death in Alzheimer’s, Parkinson’s and Prion Diseases

Neurodegenerative brain disorders (NBD) impair brain cells’ proteostasis with the accumulation of normal, mutant, misfolded or unfolded proteins in the endoplasmic reticulum (ER). The increased ER burden of these proteins elicits the unfolded protein response (UPR) and stimulates autophagy (AUT). In the short term, UPR and AUT attenuate ER’s burden. With prolonged ER stress, the UPR changes from supporting cell survival to promoting apoptosis. The failure of the UPR, to meet the increased protein burden, leads to an increase in cytosolic protein accumulation that initially further stimulates AUT. Over time, the accumulated proteins in the cytosol undergo post-translational changes into toxic monomers and oligomers that repress AUT at multiple levels and promote cell death. This review describes the interlinked signalling pathways of AUT, apoptosis and necroptosis and their modulation by Alzheimer’s, Parkinson’s and prion diseases and outlines the pharmacological strategies for targeting AUT, apoptosis and necroptosis signalling pathways.


Proteostasis in neurodegenerative brain disorders (NBD)
Proteostasis integrates synthesis, folding, trafficking and degradation of proteins. It is perturbed in the early stages of neurodegenerative brain disorders (NBD), before clinical manifestations [1][2][3]. Mutant, misfolded or unfolded proteins (P) or increased P production increases the endoplasmic reticulum (ER) protein burden in NBD such as Alzheimer's (AD), Parkinson's (PD) and prion diseases (PrD). This increased ER burden stimulates the unfolded protein response (UPR) and autophagy (AUT). The UPR response to ER stress is dichotomous [4][5][6][7]. During acute ER stress, UPR supports cell survival, by reducing ER's protein folding load and increasing ER's protein folding capacity. With prolonged ER stress, the UPR preferentially represses cell survival and triggers apoptosis. The failure of ER's stress responses (i.e. increased protein folding capacity and enhanced removal of mutant, misfolded or unfolded proteins by the UPR pathway) to attenuate the P burden leads to an increase in cytosolic P accumulation that further stimulates AUT. Over time, these P undergo post-translational changes and produce toxic monomers and a variety of molecules, including copper ions [24,25], and PrP c expression levels correlate with Cu/Zn superoxide dismutase, glutathione reductase and cytochrome c oxidase activities [26]. These observations support the hypothesis that PrP c is (a) an important endogenous scavenger, protecting structural and signalling proteins from oxidation, due to its high number of methionine residues, and (b) vital for the intracellular transport of copper to superoxide dismutase, which is dependent on copper binding for its antioxidant function. Loss of PrP c , due to conversion to PrP Sc and MET-OX-PrP Sc , which do not bind copper and have a reduced antioxidant activity, reduces the cell's intracellular antioxidant and copper transport capacity and precipitates MITO dysfunction, due to an increased oxidation of cytochrome c oxidase and other MITO proteins [27][28][29][30].
AUT is inhibited at the stage of protein digestion (during autolysosome cargo degradation) by the undigestible PTMP and is diverted to the formation of large endocytic vacuoles that rupture and release the undigested PTMP into the cytosol, thus progressively increasing their intracellular concentration. PTMP of AD and PD accelerate microtube cytoskeletal depolarisation, thus blocking autolysosome retrograde trafficking and accelerating loss of neurites, synapses and synaptic transmission [31][32][33][34][35][36][37][38][39]. PTMP inhibition of MITO function leads to (a) a reduced ATP production and an increased MITO release of ROS and Ca 2+ into the cytosol [38,[40][41][42][43][44] and Programmed Cell Death 4 (b) activation of inflammasomes with an increased release of cytokines interleukin 1 (IL1), from microglia, and tumour necrosis factor alpha (TNFα), from astrocytes and neurons, and finally apoptosis or necroptosis [38,[45][46][47][48][49][50][51][52]. Apoptosis or necroptosis of nerve cells and astrocytes releases PTMP and their oligomers into the extracellular space, thus contributing to the spread of inflammation and neurodegenerative disorder in the brain. The physiological process of apoptosis that normally prevents the spill of cell's molecules to the extracellular space is perturbed by the altered proteostasis into a pathological one in NBD. This transformation is sustained by several intracellular processes including the accumulation of undigestible PTMP, increased oxidative stress, and distorted expression of apoptotic proteins [53][54][55][56].
The AUT capacity of brain cells is important in the regulation of immune responses and inflammation that occur in NBD [57,58]. Protein aggregates (aggresomes), present in age-related NBD, activate inflammasomes. Activated inflammasomes lead to a low-grade inflammation associated with a declined autophagic capacity [59]. On the other hand, autophagy attenuation leads to inflammasome precipitated excessive caspase-1 activation and elevated IL-1β secretion in response to lipopolysaccharide (LPS) stimulation [10, 60, 61]. Also, ER stress and inflammation coexist in NBD, for example, in AD, and are intertwined [57]. Chronic neuroinflammation (CNI) develops into a self-damaging process and is an important factor in sustaining NBD including AD, PD and PRD. CNI includes activation of microglia and astrocytes and infiltration of peripheral immune cells. Transient activation of microglia, accompanied by the release of inflammatory cytokines that amplify the inflammatory response by activating and recruiting astrocytes and peripheral immune cells to the brain lesion, ensures the brain's integrity by removing foreign bodies and cell debris. CNI is toxic to neurons due to sustained release of inflammatory cytokines (e.g. ILs 1β and 6, TNFα) and ROS and microglial phagocytosis of neighbouring intact nerve cells, thus contributing to the development and progression of NBD. The progressive loss of neurons further contributes to generation of cell debris and sustains microglial hyperactivation [62].
The detrimental effects of PTMP, sustained inflammation and increased ROS production are further exacerbated by the formation of AUT-resistant soluble Aβ oligomers (AβO) in AD and AUT-resistant αSYN oligomers in PD that further stimulate chronic inflammation and increased cytosolic ROS, contributing to apoptosis or necroptosis of neurons. Therefore, activation of apoptosis or necroptosis in AD, PD or PrD is triggered by a positive feedback loop between chronic inflammation in the brain (to which astrocytes and microglia are the main contributor) and the production of PTMP. In addition to high levels of ROS, the production of PTMP in the cytosol is facilitated by copper ions in AD [63] and by iron ions, dopamine and accumulation of alpha-synuclein (the precursor of oxidised αSYN monomer) in PD [17]. Although chronic brain inflammation contributes to the process of PrP Sc production, it is not necessary to sustain it, since the PrP Sc only needs the PrP c molecules for its propagation [64].
Activation of tumour necrosis factor receptor superfamily (e.g. TLRs or TNFαR) or DNA damage can trigger necroptosis by activation of the Complex I-IIa-IIbphosphorylated pseudokinase mixed lineage kinase domain-like protein (MLKL) signalling pathway; the final steps are (a) RIP3-dependent phosphorylation of MITO proteins PGAM5 and Drp-1 (increasing MITO ROS production); (b) insertion of phosphorylated MLKL into the MITO membrane with the cumulative effects of increased MITO membrane permeability, loss of membrane potential, decreased ATP and increased ROS production [120,[167][168][169]; and (c) phosphorylated MLKL's translocation to the plasma membrane and activation of Ca 2+ influx through plasma membrane channels with concomitant plasma membrane breakdown [169]. Increased cytosolic ROS production inactivates MAP kinase phosphatase 1, enabling sustained activation of phosphorylated JNK; phosphorylated JNK promotes necroptosis by (a) stimulating MLKL phosphorylation and by (b) promoting cytochrome c release from MITO via activation of BID [170,171].
The high-mobility group box protein 1 (HMGB1) is a nuclear protein released by glia and necrotic or hyper-excitatory neurons after inflammasome activation; it activates receptors for advanced glycation end products (RAGE) and the toll-like receptor (TLR) 4 on neurons and microglia [173,174]. When HMGB1 binds to TLR4 on neurons, it phosphorylates MARCKS via MAP kinases and induces neurite degeneration, present in AD [173]. The disulphide form of HMGB1 potentiates the microglia pro-inflammatory response; therefore, repeated releases of HMGB from damaged nerve cells during chronic neuroinflammation in PD and AD could lead to an exacerbated neuroinflammatory response of microglia [175][176][177]. HMGB1, in a rat model of AD, caused (a) inhibition of microglial amyloid β-peptide 42 clearance and enhanced amyloid β-peptide 42 neurotoxicity [178] and (b) dysfunction of microglial amyloid β-peptide 40 phagocytosis [179].
The nuclear factor kappa-light-chain-enhancer of activated B cells protein complex (NF-κB) signalling pathway was repressed in a prion-infected cell line and animal brain tissues as evidenced by a decreased level of transcription factor p65/nuclear factor NF-kappa-B p65 subunit (p65) and downregulation of phosphoinositide 3-kinase (PI3K) and protein kinase B (PKB/Akt) in both experimental models [180]. In AD cell models, the exposure to amyloid β-peptide or amyloid precursor protein induced NF-κB activation [181,182], and inhibition of NF-κB transcriptional activity increased neuronal death in the presence of amyloid β-peptide [183]. NF-κB activation can protect neurons against amyloid β-peptide-induced cell death [184]. Patients with PD have an increased percentage of dopaminergic neurons in the substantia nigra with nuclear p65 immunoreactivity [185]. NF-κB is one of the several factors that regulate Beclin-1 expression; Beclin-1 promotes AUT by stimulating autophagosome formation [186][187][188]. Increased NF-κB activation in the brain, in addition to stimulating AUT, protects nerve cells against NBDs' mediated injury by several mechanisms including increased transcription of MITO antioxidant enzyme manganese superoxide dismutase (MnSOD) and Bcl-xL genes [189].
Sirtuins (SIRTs), NAD + -dependent protein deacetylases, modulate apoptosis and necroptosis [190]. For example, SIRT1 promotes AUT by deacetylation of ATG5, ATG7 and ATG8 [191]. Following TNFα receptor stimulation, SIRT2 promotes the association of RIP1 and RIP3, the subsequent formation of complex II and necroptosis [192]. In animal and cell culture models of AD, SIRT1 reduces neurodegeneration in mouse hippocampus and promotes primary neuronal survival [193]. The reduced SIRT1 mRNA and protein levels are associated with an accumulation of amyloid β-peptide 42 and tau in the brains of AD patients [194].
Tumour protein p53 (p53) modulates AUT and apoptosis. It promotes apoptosis by Bax activation in the cytoplasm; BAX initiates apoptosis by triggering mitochondrial cyt c release and caspase-3 activation [195]. In the nucleus, p53 activates transcription of Bax, PUMA and Noxa [196]. PUMA displaces cytoplasmic p53 from the Bcl-xL-p53 complex, promoting p53 activation of the apoptotic pathway [197]. In the nucleus, p53 also stimulates AUT through transcription activation of ULK1, sestrin1/2 and damage-regulated AUT modulator (DRAM) [198,199]. Indirectly, p53 promotes AUT by mTOR inhibition, via activation of AMP-dependent kinase and tuberous sclerosis (TSC) 1/TSC2 complex pathway [200]. It was suggested that DRAM has a dual role of promoting either AUT-or p53-mediated apoptosis [201]. In a Drosophila model of AD tauopathy, p53 prevented neurodegeneration by increased expression of amphiphysin, clathrin light chain, clathrin heavy chain, RAS oncogene family and synaptotagmin β synaptic genes [202]. p53 levels are significantly increased in brains of patients with AD [203] and are correlated with brain MITO dysfunction [204]. Recently, it was suggested that tau oligomers sequester and downregulate functional phospho-p53 in an AD mouse model and in patients with AD [205].
Ubiquitin-binding protein p62 (p62) modulates cell death switching between apoptosis and necroptosis. In a cell model, p62 promotes either necroptosis, when p62 is associated with the necrosome (i.e. complex II), or apoptosis when the P62necrosome association is blocked [206]. The p62 regulates apoptotic and autophagic processes [207]. P62 mediates AUT degradation by first binding polyubiquitinated proteins with the ubiquitin-associated domain and then to autophagosomes through the LC3-interacting region [208,209]. In response to tumour necrosis factor receptor stimulation, P62 promotes apoptosis by stimulating activation of caspase-8 [210,211]. The levels of p62 are increased in NBD, for example, in PrD [212,213]. Autophagy disposal of aberrant proteins is stimulated by the p62-Keap1-NRF2 signalling pathway [214]. For example, in a mouse model of AD, increased brain p62 expression improved cognition by an autophagy-mediated mechanism that reduced amyloid β-peptide 40/42 levels [215].

Summary of similarities/differences in the mechanistic pathways between selected NBD
Beclin-1, ATG-5, NF-κB, JNK, p53, p62, HMGB1 and ROS are the key signalling molecules that mediate crosstalk among AUT, apoptosis and necroptosis. ATG5 and Beclin-1 in conjunction with ULK-1 and BAX promote AUT by initiating phagophore induction and nucleation steps. Cleavage of ATG-5 and Beclin-1 by calpain, caspase-3 or increased cytosolic free calcium changes their function from stimulating AUT to promoting apoptosis via increased MITO membrane permeability. Cleaved ATG5 inhibits the anti-apoptotic activity of BCL2 and BCL-XL on BAX and BAK, further promoting increased MITO membrane permeability and apoptosis. P53 activation plays a dual role by promoting apoptosis (via activation of PUMA and NOXA) and AUT by ULK1 activation. The JNK signalling kinase blocks the binding of BCL-2 to Beclin-1, thus enabling Beclin-1 to participate in AUT initiation, and also activates the apoptosis-triggering proteins BAX and BAK. Phosphorylated JNK promotes necroptosis by stimulating MLKL phosphorylation and apoptosis by caspase-8 activation. p62 promotes AUT and apoptosis. HMGB-1 is released during AUT, apoptosis and necroptosis, and by inhibiting the cleavage of ATG-5, BAX and Beclin-1 simultaneously promote AUT and inhibit apoptosis. Mild increases in cytosolic ROS act as signalling molecules that promote a physiological balance between AUT, apoptosis and necroptosis, which favour AUT; moderate and high increases in cytosolic ROS concentrations favour apoptosis and necroptosis over AUT. The products of post-translational protein modifications in AD, PD and PrD favour apoptosis and necroptosis over AUT by (a) increasing the activation of apoptosis (e.g. by increasing MITO membrane permeability) and necroptosis, by chronic activation of TRL4 and TNFα receptors [216][217][218][219][220][221][222][223][224][225][226][227][228][229][230][231][232][233][234], (b) promoting moderate to high increases in cytosolic ROS concentrations and (c) attenuating AUT [42,62,[235][236][237][238][239][240]. In contrast to PD and AD, PrP SC -infected cells are more likely to respond with necroptosis and then apoptosis. For example, a significant upregulation of necroptosis signalling molecules phosphorylated MLKL, MLKL and receptor-interacting serine/threonine-protein kinase 3 (RIP3) was measured in the post-mortem cortical brains of patients with various types of human PRD [241].

Pharmacological strategies targeting AUT, apoptosis and necroptosis signalling pathways
At present, most of the studies, devoted to the development of pharmacological interventions for NBD, are focused on the crosstalk of AUT and apoptosis signalling pathways in neurons. Future research should also include development of pharmacological interventions that target other cells involved in the development of NBD, including microglia, astrocytes, endothelial cells and pericytes [242]. The development of pharmacological interventions for NBD should be guided by several key questions: (a) How to modulate the role of AUT from pro-death to pro-survival? (b) How is the information from the crosstalk among AUT, apoptosis and necroptosis integrated? (c) How to modulate the crosstalk among AUT, apoptosis and necroptosis? and (d) How is the information from the crosstalk among AUT, apoptosis and necroptosis (e.g. inflammation-promoting molecules) shared among different cells involved in the development of NBD? [242]. Examples of pharmacological strategies are given below: Pharmacological strategies to ameliorate MITO dysfunction include: (a) Targeting excessive ROS production: (a1) Mercaptamine that increases levels of glutathione in human [78].
(a3) RTA-308 stimulates Nrf2 to enhance the expression of pro-oxidant genes and to repress inflammatory genes in an animal model [244].
(a5) Ceria nanoparticles are ROS scavengers that localise in MITO and suppress neuronal death in an AD mouse model [249].
TNFα signalling pathway is the focus of pharmacological interventions targeting neuroinflammation in NBD with a variety of compounds [57]: (a) serotonin binds to microglial receptors and has anti-inflammatory effects; serotonin treatment reduced TNFα release in cultured primary microglia cells exposed to AβO and in mouse brains infused with AβO and also prevented AD-associated behavioural changes [266]; (b) etanercept, a decoy TNF receptor and IgG1 Fc fusion protein that inhibits the binding of soluble TNF to cell-surface TNF receptors, was evaluated in several clinical trials on patients with AD; no statistically significant results were reported; however, the drug was well tolerated, and largescale trials are expected [57]; and (c) infliximab, a human monoclonal antibody that binds TNFα and was used to treat human auto-immune and inflammatory diseases, prevented eIF2a phosphorylation and long-term memory loss in a mouse model of AD [7,267].

Conclusions
Neurodegenerative brain disorders (NBD) change brain cell proteostasis due to the accumulation of normal, mutant, misfolded or unfolded proteins in the endoplasmic reticulum (ER). The increased ER burden elicits the unfolded protein response (UPR) and stimulates AUT. In the short term, these responses tend to attenuate ER's stress, by reducing the ER's protein load and increasing the ER's folding capacity. In the long term, with prolonged ER stress, the UPR changes from supporting cell survival to promoting apoptosis. The failure of the ER stress response to meet the increased protein burden is reflected in an increased cytosolic protein accumulation that initially further stimulates AUT. Over time, the accumulated proteins in the cytosol undergo post-translational changes into toxic monomers and oligomers that repress AUT at multiple levels and promote either apoptosis or necroptosis. Apoptosis and necroptosis of the affected cells lead to the release of toxic proteins into the surrounding tissue and trigger the response of microglia and astrocytes. Chronic neuroinflammation, sustained by the spread of progressive failure of AUT among brain cells, due to the release of toxic monomers and oligomers from dying cells and their uptake by initially healthy cells and by the persistent activation of microglia and astrocytes by toxic monomers and oligomers, also contributes to nerve apoptosis or necroptosis. The signalling pathways of apoptosis, AUT and necroptosis are interlinked. A better understanding on how chronic neuroinflammation, Alzheimer's, Parkinson's and prion diseases modulate the crosstalk among these signalling pathways could contribute to the development of new therapeutic interventions for these NBD.

Funding
This work was supported by ARRS grant number P3-0171.