Autophagy-Lysosomal Pathway As Potential Therapeutic Target in Parkinson’s Disease

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Abstract: Cellular quality control systems have gained much attention in recent decades. Among these, autophagy is a natural self-preservation mechanism that continuously eliminates toxic cellular components and acts as an anti-aging process. It is vital for cell survival and to preserve homeostasis. Several cell-type-dependent canonical or non-canonical autophagy pathways have been reported showing varying degrees of selectivity with regard to the substrates targeted. Here, we provide an updated review of the autophagy machinery and discuss the role of various forms of autophagy in neurodegenerative diseases, with a particular focus on Parkinson's disease. We describe recent findings that have led to the proposal of therapeutic strategies targeting autophagy to alter the course of Parkinson's disease progression.

Keywords: autophagy; lysosomes; neurodegenerative disease; Parkinson's disease; autoimmunity

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1. Introduction

Although some elements of Parkinson's disease(PD) were described a very long time ago, the first clear medical description of this disease was published in 1817 by James Parkinson [1]. Since then, substantial efforts have been made to understand the underlying pathogenesis and pathological elements of this complex disease, in terms of neuropathological and anatomopathological changes [2-5]. PD is a multifactorial disease with heterogeneous causative factors, including genetic, environmental, molecular, and cellular components. PD is characterized by a broad spectrum of motor and non-motor signs and symptoms. They include rest tremor, bradykinesia, postural instability/unsteady gait, and rigidity, alongside psychiatric disorders, sleep disorders, dysautonomic disorders, pain, anosmia, and cognitive disorders. Motor signs result primarily from the loss of dopaminergic(DA) neurons in the substantia nigra pars compacta (SNpc) and intracellular inclusions of aggregated and misfolded α-synuclein(c-syn), encapsulated or not in Lewy bodies(LB) and Lewy neurites(LN) in the neurons[3,6](Figure 1; see appendix for definition).

The symptoms of PD develop gradually with age. what is a cistanche They can start with a slight tremor in one hand and a feeling of stiffness in the body; bradykinesia is frequent. Recent studies confirm that more than 3% of the general population from 65 years of age are affected by PD. In 5%-10% of cases, however, symptoms of PD appear earlier; this is referred to as young-onset PD(YOPD). Men are 50% more likely to develop PD than women, but the risk for women appears to increase with age.

Figure 1. Neuropathological findings in Parkinson's disease. (A, B) Post-mortem mesencephalon and pons from a control patient(A)and from a patient with PD(B): SN appeared paler in B due to dopaminergic denervation. (C), SN, H&E staining (×250).(D): H&E staining(×250)of LB in a cortical neuron. The black arrow shows an LB. Abbreviations not described in the text: H&E, hematoxylin, and eosin.

The root cause of PD remains largely unknown. bioflavonoids Some cases of PD have been linked to genetic mutations, but clear hereditary causes of this disease are difficult to establish. Indeed, only 15% of patients with PD have a family history of the disease. Some genes have been associated with distinct, typical, or rarer forms of the disease, which include juvenile or adult-onset, early or late, autosomal recessive, dominant, or X-linked forms [4,7-9]. Causative risk factors associated with particular ethnic groups have also been identified. The genes most frequently linked to PD include GBA, LRRK2, PRKN, SNCA, ATP13A2, ATP10B, DI-1,DNAIC6, FBXO7,HTRA2,MAPT, PINK1,PLA2G6,VPS35,and VPS13C[4,7-13]. The majority of these genes encode proteins that are linked either directly or indirectly to quality control mechanisms that are vital in maintaining cell homeostasis, vesicular transport pathways, autophagy processes, and the endo-lysosomal system. Other genetic alterations have also been associated with PD, including epigenetic changes, such as DNA methylation, chromatin remodeling, histone modifications, microRNAs, and long non-coding RNAs [4,14].

2. Pathogenesis and Pathology

Clinicopathological studies reveal the slow progression of PD from the ventrolateral region of the SNpc, with later spread to other brain regions [15]. Clinical symptoms of PD become detectable when the degeneration of the DA neurons progresses within the SNpc. LBs are observed at sites of neuronal damage(Figure 1). In normal physiology, the α-syn deposited in these structures performs central functions in endocytosis; vesicle trafficking; synthesis, storage, and release of dopamine; Ca2+ homeostasis; microtubule dynamics; and other processes [16]. Thus, neuronal activity is completely dependent on α-syn, and also on mitochondrial homeostasis. Although α-syn is predominantly present in cytosolic eosinophilic LBs, it has also been detected in mitochondria, lysosomes, and other organelles in post-mortem PD brains. The presence of LBs in the peripheral, enteric, and central nervous systems(CNS) has been implicated in both motor and non-motor symptoms of PD [17,18]. buy cistanche Point mutations in the α-syn sequence or other pathological insults lead to the formation of oligomers, which may then group together as larger aggregates. These aggregates can alter numerous cellular and molecular pathways in neurons—-in particular involving autophagy and proteasomal processes, such as mitochondrial function, vesicle trafficking, organelle, and protein degradation—-all of which lead to neurodegeneration. Subsequently, as a result of the neurodegeneration, o-syn aggregates are deposited in the SN, where they activate microglia [19]. This uncontrollable activation can generate pro-inflammatory signals [20]which may lead to further neurodegeneration when a critical threshold is reached.

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2.1.Neuropsychiatric Manifestations of PD

No specific test exists to diagnose PD. Consequently, diagnosis is based on medical history, a review of signs and symptoms, and a neurological and physical examination (Box1). Motor signs of PD usually begin around 60 years of age [21], but YOPD is not rare, particularly in some hereditary forms [22]. Unilateral or asymmetric bradykinesia and/or rest tremors are the first symptoms of the disease [23]. Rest tremor is present in relaxed muscles and disappears during action and sleep. It may be increased by mental calculation. Bradykinesia, defined by the slowness of movement and decreased amplitude or speed, leads to difficulties with repetitive movements, micrography, small-step gait, and speech difficulties (hypophonia and dysarthria), which will emerge as the disease evolves. cistanch Rigidity may cause pain and contribute to postural deformity(thoracolumbar spinal flexion). Progression is slow with the bilateral extension of akinesia, tremor, and hypertonia, followed by postural instability, freezing of gait, falls, and in some patients, camptocormia. Some non-motor signs(premotor) may occur several years before the first motor symptoms; these include depression, hyposmia, constipation, or rapid-eye-movement sleep disorders [24]. Anxiety and apathy may be present from the onset of PD, whereas severe dysautonomia (orthostatic hypotension, urinary dysfunction due to detrusor hyperactivity), sleep fragmentation, cognitive disorders(dysexecutive disorders), and hallucinations emerge later, and will contribute to loss of autonomy [24,25].

2.2. Current Treatments for PD and Clinical Management

Symptomatic treatment is the only clinical option currently available [26], with therapies aiming to compensate for the dopaminergic deficit. Dopaminergic drugs(levodopa associated with dopa-decarboxylase inhibitor, dopaminergic agonists, or monoamine oxidase-type B inhibitors), used individually or in poly-therapy regimens, are very efficient during the early stages of the disease. However, management becomes more difficult as the year's progress. Indeed, dopaminergic treatments, which improve motor signs, can have very disabling complications. A wearing-off phenomenon (end of dose failure) and dyskinesia occur after several years of levodopa treatment [27].Impulse control disorders (pathological gambling or shopping, hyper sexuality; [28], hallucinations, or psychosis may also complicate dopaminergic treatments, and are more frequently encountered with dopamine agonists [29]. Other treatments, including catechol-O-methyltransferase inhibitors to treat motor fluctuations or amantadine for dyskinesia [26] can be used later as the disease progresses. Second-line treatment(continuous subcutaneous infusion of apomorphine, continuous jejunal administration of droxidopa gel, bilateral subthalamic nuclear stimulation) is proposed when motor fluctuations and dyskinesia become significant [30]. These treatments aim to achieve stable striatal dopaminergic stimulation but have no impact on disease progression. Furthermore, some axial symptoms(dysarthria, postural instability) are not dopa-sensitive, and clinical management of non-motor symptoms remains difficult [31,32].

Decades of investigations have led to the development of therapeutic strategies, which have undoubtedly improved the quality of life for patients. However, slowing the disease's progression still remains a challenge, and a persistent priority [33], and novel disease-modifying approaches are eagerly awaited [3,34]. Although the functions of the proteasome and of autophagy, including macroautophagy and chaperone-mediated autophagy(CMA), have long been known to contribute to α-syn clearance [35,36l, dysregulation of these processes remains poorly understood in PD. cistanche Australia Several gene mutations and alterations to proteins involved in PD are closely linked to autophagy, particularly mitophagy and autophagy-lysosomal pathways. In this review, we focus on the involvement of autophagy in PD, comment on the major unanswered questions in the field and propose new directions for possible therapeutic interventions targeting autophagy pathways.

3. Autophagy

Autophagy is a major intracellular degradation system by which cytoplasmic materials are delivered to the lysosome for degradation. Based on the route by which content is delivered to lysosomes, several forms of autophagy have been defined. These different forms also have varying degrees of selectivity for targeted cargos (Table 1; Figure 2). The three main types of autophagy processes are macroautophagy, CMA, and microautophagy/EMI. Whatever the delivery route, the main role of these processes is to degrade unwanted mate-rial that is defective, may be toxic, or has been produced in excess, and thus to maintain cell homeostasis.

3.1.The Autophagy Machinery

The mechanisms of autophagy have been thoroughly investigated and were reviewed in detail by numerous authors [61-63]. The general features of the three pathways are presented in Figure 2. Recent advances related to canonical and non-canonical autophagic processes—especially in mammalian systems—have added to our understanding of the mechanisms that may play important roles in neurodegenerative diseases such as PD. Nevertheless, many of the molecular discoveries upon which our current understanding of the regulation of autophagy is based emerged from analyses involving yeast. In cells, the three forms of autophagy coexist and play vital roles in maintaining cellular homeostasis. However, the large majority of results available in this field relate to macroautophagy. This process has been divided into the following steps: nucleation, elongation, autophagosome formation, autophagosome-lysosome fusion, and degradation (Figure2). Each step is finely genetically-regulated and plays its own specific role in maintaining the dynamic nature of the process. For example, a number of conserved autophagy-related proteins act in a hierarchical manner to mediate autophagosome formation. Upon upstream induction, the autophagy machinery comes into contact with the isolation membrane/phagophore. The early origins and definitive complex source endoplasmic reticulum(ER), Golgi complex, endosomes, and mitochondria] of the nascent isolation membrane remains a matter of debate [64]. An ultrastructural study involving electron microscopy experiments confirmed that a specialized subdomain of the ER contributes to phagophore generation [65]. About 40 autophagy-related (ATG)proteins have been identified as involved in this dynamic process, they are hierarchically organized, starting from the initiation of the process and progressing through to the maturation of autophagosomes. These proteins work together in several functional complexes, notably (i)the Unc-51-like kinase 1(ULK1)/ATG1 kinase complex; (ii) the class II phosphatidylinositol (PI)3-kinase complex;(ii)the PI(3)P-binding ATG2-ATG18 complex;(iv)the two conjugation systems(ATG12 conjugation system and microtubule-associated protein 1A/1B-light chain 3(MAP1LC3)/ATG8 conjugation system); and (v) the fusion machinery (Figure 2).

In fact, several so-called ATG proteins have alternative functions beyond autophagy [66]. Thus, for example, MAP1LC3 lipidation (a mechanism that has long been used to assess autophagic activity [67,68])is also involved in non-autophagic cellular mechanisms such as phagocytosis, LAP, micropinocytosis, or viral infection. These processes are known as non-canonical autophagic processes [69]. In these non-canonical processes, the functions of which are still incompletely characterized [70,71] MAP1LC3 conjugates to single mem-branes (single membrane ATG8 conjugation, SMAC), and cytosolic constituents are not delivered to the lysosome [72].

3.2. Neuronal Autophagy Contributes to Neuronal Physiology

There is compelling evidence to support the idea that neuronal autophagy plays a decisive role in several aspects of neuron development and in preserving neuronal activity [73-75]. In post-mitotic cells like neurons, autophagy is especially important for survival and homeostasis, because these cells cannot eliminate accumulated toxic substances and damaged organelles during cell division. Autophagy, as well as the proteasomal system [76], is therefore one of the vital quality control mechanisms that ensure the longevity of neuronal cells. Presynaptic autophagy in the axon terminal is also essential for synaptic maintenance and plasticity [77].

Among nerve cells, only cortical neurons, Purkinje cells, and hypothalamic neurons can increase their autophagosome content upon a stimulus. The precise reasons for this niche mechanism are currently unknown [62,78]. One possible explanation is trivial and related to the fact that, like for some other cell types, measuring autophagy in neurons, especially in the brain, remains challenging [79,80]. Alternatively, because nerve cells are terminally differentiated—with a lower regenerative capacity than other cells-—they are less autophagic. However, studies on brains from autophagy-deficient mice provided evidence that sequestosome-1 (SQSTM1)/p62 protein and polyubiquitinated proteins accumulate in most neuronal cells[81]. In contrast, SQSTM1 deficiency does not result in a complete lack of autophagy. It appears therefore that the autophagosome content depends on the type of cells and the type of stressor.

3.3. Autophagy and Neurodegenerative Diseases

As introduced above, to prevent neuronal and synaptic dysfunction, neurons have evolved mechanisms to remove toxic and defective components and organelles. These mechanisms are essential to maintain a high degree of neurotransmission and the integrity of the functional proteome in neurons. Autophagy is central to this protective system. Age-related functional loss of autophagy makes the neurons more vulnerable to stress and can lead to cell death [82]. Pathological disruption of autophagy pathways can also result in neurodegenerative disorders that may or may not be linked to aging.

Compromised autophagy has been documented in many neurodegenerative diseases, including PD, Alzheimer's disease(AD), Huntington's disease (HD), and amyotrophic lateral sclerosis(ALS)(for comprehensive reviews, see [63.84]). Investigating the mechanisms linking autophagy to these diseases, it has been observed, for example, that mice specifically deficient for Atg5 in neural cells develop progressive deficits in motor function while also accumulating cytoplasmic inclusion bodies in neurons[85]. Similarly, in mice deficient for Atg7, Atg5, or Ambral, ubiquitin was found to accumulate in the CNS, and cytoplasmic inclusions were associated with motor dysfunctions, and neuronal tube defects in mouse embryos [86]. Mutation of genes linked to autophagic processes—for example, SQSTM1, optineurin/OPTN, E3 ubiquitin ligase PARKIN/PRKN, PINK1, TBK1—have also been implicated in many neurodegenerative diseases. In particular, defects in mitophagy that are also seen in organ-specific and systemic inflammatory diseases, have been documented in neurodegenerative diseases [87]. In addition to these genetic mutations, significant alterations to protein expression have been linked to neurodegenerative diseases. For example, abnormal expression of the protein glandular epithelial cell 1 (GABARAPL1/GEC1) has been associated with neurodegenerative diseases [88]. 3.4. Autophagy and Parkinson's Disease

Among the pathological hallmarks of PD are LBs that contain abnormally aggregated α-syn protein. Mutations or triplication of the gene encoding α-syn (SNCA) are rare but are clearly involved in the initiation and progression of PD. Interestingly, any failure affecting one of the components of the degradative process, either directly or indirectly, impairs the other autophagy processes. The ubiquitin-proteasome system (UPS) is known to be the primary degradative pathway for monoubiquitinated α-syn, whereas the macroautophagy pathway degrades deubiquitinated a-syn [89,90]. In PD, therefore, both mitochondria and lysosomes play crucial roles (Figure 3). 3.4.1.Role of Mitophagy in PD

As an energy-producing organelle, the mitochondrion is central to several neurodegenerative diseases, including PD [91-95]. Multiple investigations revealed that genetic mutations associated with PD (e.g, PRKN, PINK1, and others)are also closely linked to mitochondrial defects, including defects in mitophagy (Table 2)[9]. The type of damage caused to the mitochondria naturally depends on the type of a-syn (forming aggregates or not, produced from mutated or native forms of SNCA). Further studies confirmed that o-syn affects the interaction of the mitochondria-associated membrane with the ER. This interaction plays a pivotal role in regulating Ca4+ signaling and apoptosis. In addition, abnormal o-syn interferes with the peroxisome proliferator-activated receptor-gamma coactivator 1-alpha, which plays a crucial role in mitochondrial biogenesis and in apoptosis. Mitochondrial dysfunction with the involvement of factors related to a-syn has been comprehensively discussed elsewhere [9,97,98].

Figure 3. Autophagy impairment in PD. Impaired forms of autophagy have been observed in PD. Genetic mutations of α-syn are linked to impairment of the autophagy process. Many factors, such as genetic factors, defective mitochondrial trafficking, oxidative stress, dysfunctional ATP cycle, deregulated mitochondrial dynamics, and altered mitogenesis perturb healthy mitochondria. Damaged/dysfunctional mitochondria allow PINK1 to recruit PRKN, which in turn activates other essential proteins, such as OPTN and ubiquitin, Rab7, and others thus initiating a quality control process, i.e., mitophagy. The function of the Rab7 is regulated by the TBC1D15/17 (belong to the TBC family with Rab-GAP functions), which also regulates the shaping and target functions of the isolation membrane by cross-linking with Fis1 and MAP1LC3B. The sequential steps of mitophagy are the formation of the phagophore, maturation into the mitoautophagosome, and fusion of the mitoautophagosome with the lysosome. Conventional autophagy also plays an essential role in (both naive and aggregated) α-syn degradation. αx-syn selectively binds to the pathogen-recognition receptor, TLR-4, which activates the downstream signaling pathway following NF-kB activation, to stimulate SQSTM1/p62 production. The SQSTM1 produced binds to the internalized α-syn and initiates the autophagy process. Dysregulation of the autophagy process leads to the accumulation of α-syn alongside SQSTM1. Apart from mitophagy and macroautophagy, CMA also selectively degrades α-syn, which contains a KFERQ-like motif. Selective CMA inhibition or altered CMA functioning affect α-syn degradation. Abbreviations not described in the text: Fill, Mitochondrial fission 1 protein; GAP, GTPase-activating proteins; IKK, IkB kinase; MyD88, myeloid differentiation protein 88; Rab, Ras superfamily of small G proteins; TBC, Tre-2/Bub2/Cdc16; TIRAP, Toll-interleukin 1 receptor adaptor protein.

Defects in the mitophagy pathway, especially PARK2 (PRKN mutations) and PARK6 (PINK1 mutations), have been proposed as a major cause of familial PD. In healthy conditions, PINK1, which localizes to the mitochondrion, is translocated into the mitochondrial inner membrane where it is degraded. Under certain unknown conditions, mitochondria become damaged and lose membrane potential (Figure 3). This leads to PINK1 activation and recruitment of PRKN, which helps to induce mitophagy while acting on other mitochondrial membrane proteins OPTN and nuclear dot protein 52-kDa (NDP52)][62,119-124]. PRKN mutations are the most frequent cause of autosomal recessive YOPD, followed by mutations in PINK1. Alongside its role in mitophagy, PRKN plays an essential role in lipid processing and the ubiquitination of the GTPase Rab7, which regulate lysosomal dynamics [125-128]. PRKN deficiency results in DA neuronal degeneration in mice, and embryonic fibroblasts derived from PINK1-deficient mice show lysosomal dysfunction [129]. In addition, mutations in PINK1 and PRKNlead to defects in the mitophagy process [62]. However, studies have yet to explain why PRKN is not recruited to mitochondria in DA neurons under depolarized conditions [130]. A consequence of mitophagy dysfunctions in neurons is uncontrolled stress (i.e., generation of reactive oxygen species), which causes neuronal cell death. In line with this effect, targeting mitophagy defects may be beneficial in PD. It has been shown, for example, that an inhibitor of the mitochondrial deubiquitinase USP30, which negatively regulates PRKN-mediated mitophagy, selectively increases mitophagy flux, thus it could be of interest for the development of novel therapeutic approaches [131,132].

In addition to the major effect of PINK1 and PRKN mutations, SNCA mutations have been studied in the context of mitophagy. α-syn interacts with the Miro proteins (outer mitochondrial membrane adapter proteins, useful in mitochondrial motility) and interferes with the Miro degradation process, which is an essential step in the mitophagy process[133]. Studies in mice and yeast harboring mutations in SCNA confirmed the role of α-syn in neuronal death, via mitochondrial dysfunction [134,135].

The transcription factor myocyte enhancer factor 2D(MEF2D) is another essential mitochondrial regulator(Table 2). It is a central factor in the transmission of extracellular signals and activation of genetic programs in response to a wide range of stimuli in several cell types, including neurons. MEF2D is a critical regulator of IL-10 gene expression, involved in the negative control of the microglial inflammatory response, and preventing inflammation-mediated cytotoxicity[136]. Reduced MEF2Dexpression has been directly linked to reduced levels of nicotinamide adenine dinucleotide dehydrogenase 6(NADH)a component of mitochondrial complex I. Post-mortem analysis of brain samples from PD patients revealed reduced levels of both MEF2D and NADH [137].

A number of other genetic mutations, including deficiencies in mitochondrial apoptosis-inducing factor(AF)and mitochondrial transcription factor A (TFAM;[138] that perturb endo-lysosomal pathways, also affect mitochondrial physiology and function, leading for example to impaired mitophagy, dysfunctional oxidative phosphorylation, deregulated mitochondrial dynamics, altered mitogenesis, calcium imbalance, altered mitochondrial trafficking, and induction of oxidative stress(Table 2). PRKN-independent autophagy pathways are involved in the selective mitophagy process via receptor-mediated, lipid-mediated, and ubiquitin ligase-mediated pathways [97,139,140]. It is not currently known to what extent these pathways are linked to PD.

This article is extracted from Cells 2021, 10, 3547. https://doi.org/10.3390/cells10123547 https://www.mdpi.com/journal/cells