Conophylline Protects Cells in Cellular Models of Neurodegenerative Diseases by Inducing Mammalian Target of Rapamycin (mTOR)-independent Autophagy

Macroautophagy is a cellular response that leads to the bulk, nonspecific degradation of cytosolic components, including organelles. In recent years, it has been recognized that autophagy is essential for prevention of neurodegenerative diseases, including Parkinson disease (PD) and Huntington disease (HD). Here, we show that conophylline (CNP), a vinca alkaloid, induces autophagy in an mammalian target of rapamycin-independent manner. Using a cellular model of PD, CNP suppressed protein aggregation and protected cells from cell death caused by treatment with 1-methyl-4-phenylpyridinium, a neurotoxin, by inducing autophagy. Moreover, in the HD model, CNP also eliminated mutant huntingtin aggregates. Our findings demonstrate the possible use of CNP as a therapeutic drug for neurodegenerative disorders, including PD and HD.     

Synaptic proteostasis in Parkinson's disease

There are over 7 million people worldwide suffering from Parkinson's disease, and this number will double in the next decade. Causative mutations and risk variants in >20 genes that predominantly act at synapses have been linked to Parkinson's disease. Synaptic defects precede neuronal death. However, we are only now beginning to understand which molecular mechanisms contribute to this synaptic dysfunction. In this review, we discuss recent data demonstrating that Parkinson proteins act centrally to various protein quality control pathways at the synapse, and we argue that disturbed synaptic proteostasis is an early driver of neurodegeneration in Parkinson's disease.     

Autophagic Defects in Aging: Looking for an “Emergency Exit"?

The ability of cells to renew their intracellular components and get rid of undesired or altered molecules decreases with age. Failure of autophagy is considered one of the main reasons for the build up of damaged components in the tissues of old organisms. We have recently shown that, declined activity of chaperone-mediated autophagy, a selective type of autophagy particularly impaired in aging, increases cell’s vulnerability to stressors. This finding supports that, added to its role in cellular clean up, chaperone-mediated autophagy is an essential component of the cellular response to stress. Failure to perform this function with age could underlie the inability of old cells to adapt to stress conditions, and explain the accelerated course of some protein conformational disorders, such as Parkinson’s disease, as affected individuals age.     

Role of Autophagy Pathway in Parkinson’s Disease and Related Genetic Neurological Disorders

The elucidation of the function of the PINK1 protein kinase and Parkin ubiquitin E3 ligase in the elimination of damaged mitochondria by autophagy (mitophagy) has provided unprecedented understanding of the mechanistic pathways underlying Parkinson’s disease (PD). We provide a comprehensive overview of the general importance of autophagy in Parkinson’s disease and related disorders of the central nervous system. This reveals a critical link between autophagy and neurodegenerative and neurodevelopmental disorders and suggests that strategies to modulate mitophagy may have greater relevance in the CNS beyond PD.     

A novel autophagy modulator 6-Bio ameliorates SNCA/α-synuclein toxicity

Parkinson disease (PD) is a life-threatening neurodegenerative movement disorder with unmet therapeutic intervention. We have identified a small molecule autophagy modulator, 6-Bio that shows clearance of toxic SNCA/α-synuclein (a protein implicated in synucleopathies) aggregates in yeast and mammalian cell lines. 6-Bio induces autophagy and dramatically enhances autolysosome formation resulting in SNCA degradation. Importantly, neuroprotective function of 6-Bio as envisaged by immunohistology and behavior analyses in a preclinical model of PD where it induces autophagy in dopaminergic (DAergic) neurons of mice midbrain to clear toxic protein aggregates suggesting that it could be a potential therapeutic candidate for protein conformational disorders.     

The MAPK1/3 pathway is essential for the deregulation of autophagy observed in G2019S LRRK2 mutant fibroblasts

The link between the deregulation of autophagy and cell death processes can be essential in the development of several neurodegenerative diseases, such as Parkinson disease (PD). However, the molecular mechanism of deregulation of this degradative process in PD patients is unknown. The leucine-rich repeat kinase 2 (LRRK2) gene is related to PD and its implication in autophagy regulation has been described. Our recent work shows that the presence of the G2019S LRRK2 mutation, one of the most prevalent in LRRK2, is accompanied by a deregulation of autophagy basal levels dependent on the MAPK1/3 (ERK2/1) pathway.     

The MAPK1/3 pathway is essential for the deregulation of autophagy observed in G2019S LRRK2 mutant fibroblasts

The link between the deregulation of autophagy and cell death processes can be essential in the development of several neurodegenerative diseases, such as Parkinson disease (PD). However, the molecular mechanism of deregulation of this degradative process in PD patients is unknown. The leucine-rich repeat kinase 2 (LRRK2) gene is related to PD and its implication in autophagy regulation has been described. Our recent work shows that the presence of the G2019S LRRK2 mutation, one of the most prevalent in LRRK2, is accompanied by a deregulation of autophagy basal levels dependent on the MAPK1/3 (ERK2/1) pathway.     

Signaling and induction of chaperone-mediated autophagy by the endoplasmic reticulum under stress conditions

Chaperone-mediated autophagy (CMA), a form of selective autophagy, maintains cellular proteostasis in response to diverse stress conditions. Whether and how endoplasmic reticulum (ER) stress triggers CMA remains elusive. In our recent study, we demonstrate that various types of ER stress activate the CMA pathway via an EIF2AK3/PERK-MAP2K4/MKK4-MAPK14/p38-dependent manner. We term this process ERICA for ER stress-induced chaperone-mediated autophagy. This pathway is activated in response to stress associated with Parkinson disease and is required for the viability of the SNc dopaminergic neurons in an animal model of Parkinson disease.     

The UPS and autophagy in chronic neurodegenerative disease: Six of one and half a dozen of the other—Or not?

In the past twenty years, evidence has accumulated to show that ubiquitinated proteins are a consistent feature of the intraneuronal protein aggregates (inclusions) that characterize chronic neurodegenerative disease. These findings may indicate that age-related dysfunction of the 26S proteasome may be central to disease pathogenesis. The aggregate-prone proteins can also be eliminated by autophagy. We have used the Cre-recombinase/loxP genetic approach to ablate the proteasomal psmc1 ATPase gene and deplete 26S proteasomes in neurons in different regions of the brain to mimic neurodegeneration. Deletion of the gene in dopaminergic neurons in the substantia nigra generates a new model of Parkinson’s disease. Ablation of the gene in the forebrain creates the first model of dementia with Lewy bodies. In both neuroanatomical regions, gene ablation causes the formation of Lewy-like inclusions together with extensive neurodegeneration. There is some evidence for neuronal autophagy in areas adjacent to inclusions. The models indicate that neuronal loss in neurodegenerative diseases can be attributed to proteasomal malfunction accompanied by Lewy-like inclusions as seen in dementia with Lewy bodies and Parkinson’s disease.     

Protective Roles for Induction of Autophagy in Multiple Proteinopathies

Many late-onset neurodegenerative diseases, including Parkinson’s disease, tauopathies, Huntington’s disease and forms of spinocerebellar ataxia, are caused by aggregate-prone proteins. Previously we showed that mutant huntingtin is an autophagy substrate and that autophagy induction reduced soluble and aggregated huntingtin and attenuated its toxicity in cell, fly and mouse models of disease. We have recently shown in cell and fly models that autophagy induction may have general protective effects across a range of diseases caused by aggregate-prone intracellular proteins. First, we showed that this strategy reduces the levels of the primary toxin, the aggregate-prone mutant protein. Second, our recent work suggests that autophagy induction may have additional cytoprotective effects by protecting cells against a range of subsequent pro-apoptotic insults.     

Spatiotemporal dynamics of autophagy receptors in selective mitophagy

Damaged mitochondria are turned over through a process of selective autophagy termed mitophagy. In mitophagy, unhealthy mitochondria are recognized and ubiquitinated by Parkinson disease-linked proteins PINK1 and PARK2. The subsequent recruitment of ubiquitin-binding autophagy receptors leads in turn to the sequestration of the damaged organelles into LC3-positive phagophores, precursors to autophagosomes. The precise identity of these receptors and how they are regulated has been the focus of considerable attention. Our recent work uses live-cell imaging to explore the dynamics and regulation of autophagy receptor recruitment. Utilizing multiple paradigms to induce mitochondrial damage, we identified the rapid, 2-step recruitment of autophagy receptors OPTN, CALCOCO2/NDP52, and TAX1BP1. All 3 receptors are recruited to damaged mitochondria with similar kinetics; however, only OPTN is necessary for efficient formation of a phagophore sequestering damaged mitochondria from the cytosol. OPTN is co-recruited to damaged mitochondria along with its upstream kinase TBK1. Depletion of OPTN or TBK1, or expression of amyotrophic lateral sclerosis (ALS)-linked mutations in either protein, interfere with efficient autophagic engulfment of depolarized mitochondria. These observations suggest that insufficient autophagy of damaged mitochondria may contribute to neurodegenerative disease.     

Interplay of endoplasmic reticulum stress and autophagy in neurodegenerative disorders

The common underlying feature of most neurodegenerative diseases such as Alzheimer disease (AD), prion diseases, Parkinson disease (PD), and amyotrophic lateral sclerosis (ALS) involves accumulation of misfolded proteins leading to initiation of endoplasmic reticulum (ER) stress and stimulation of the unfolded protein response (UPR). Additionally, ER stress more recently has been implicated in the pathogenesis of HIV-associated neurocognitive disorders (HAND). Autophagy plays an essential role in the clearance of aggregated toxic proteins and degradation of the damaged organelles. There is evidence that autophagy ameliorates ER stress by eliminating accumulated misfolded proteins. Both abnormal UPR and impaired autophagy have been implicated as a causative mechanism in the development of various neurodegenerative diseases. This review highlights recent advances in the field on the role of ER stress and autophagy in AD, prion diseases, PD, ALS and HAND with the involvement of key signaling pathways in these processes and implications for future development of therapeutic strategies.     

Parkinson's disease: convergence on synaptic homeostasis

Parkinson's disease, the second most common neurodegenerative disorder, affects millions of people globally. There is no cure, and its prevalence will double by 2030. In recent years, numerous causative genes and risk factors for Parkinson's disease have been identified and more than half appear to function at the synapse. Subtle synaptic defects are thought to precede blunt neuronal death, but the mechanisms that are dysfunctional at synapses are only now being unraveled. Here, we review recent work and propose a model where different Parkinson proteins interact in a cell compartment‐specific manner at the synapse where these proteins regulate endocytosis and autophagy. While this field is only recently emerging, the work suggests that the loss of synaptic homeostasis may contribute to neurodegeneration and is a key player in Parkinson's disease.     

Isorhynchophylline, a natural alkaloid, promotes the degradation of alpha-synuclein in neuronal cells via inducing autophagy

Accumulation of α-synuclein (α-syn) in the brain is a pathogenic feature and also a causative factor of Parkinson disease. Isorhynchophylline (IsoRhy) is a major tetracyclic oxindole alkaloid isolated from the Chinese herbal medicine Uncaria rhynchophylla (Miq.)Jacks (Gouteng in Chinese), which has been used for the treatment of neurological diseases in East Asia for centuries. Here we report a novel function of IsoRhy as a neuronal autophagy inducer. IsoRhy induced autophagy in different neuronal cell lines, including N2a, SH-SY5Y and PC12 cells, and also in primary cortical neurons. Furthermore, IsoRhy induced autophagy in the fat bodies of Drosophila. IsoRhy promoted clearance of wild-type, A53T and A30P α-syn monomers, α-syn oligomers and α-syn/synphilin-1 aggresomes in neuronal cells via the autophagy-lysosome pathway. More importantly, IsoRhy was able to decrease the expression levels of wild-type and A53T α-syn protein in differentiated human dopaminergic neurons. Notably, IsoRhy-induced autophagy was independent of the mTOR pathway but dependent on the function of Beclin 1. Taken together, data from this study raise the possibility that oxindole alkaloid derivatives may serve as a means to stimulate autophagy in neuronal cells, thereby exerting preventive and therapeutic values against neurodegenerative diseases such as Parkinson disease by reducing pathogenic protein aggregates in neurons.     

Heat shock response and autophagy—cooperation and control

Protein quality control (proteostasis) depends on constant protein degradation and resynthesis, and is essential for proper homeostasis in systems from single cells to whole organisms. Cells possess several mechanisms and processes to maintain proteostasis. At one end of the spectrum, the heat shock proteins modulate protein folding and repair. At the other end, the proteasome and autophagy as well as other lysosome-dependent systems, function in the degradation of dysfunctional proteins. In this review, we examine how these systems interact to maintain proteostasis. Both the direct cellular data on heat shock control over autophagy and the time course of exercise-associated changes in humans support the model that heat shock response and autophagy are tightly linked. Studying the links between exercise stress and molecular control of proteostasis provides evidence that the heat shock response and autophagy coordinate and undergo sequential activation and downregulation, and that this is essential for proper proteostasis in eukaryotic systems.     

Mitophagy in cells with mtDNA mutations

Despite the emergence of autophagy as a key process for mitochondrial quality control, the existence and persistence of pathogenic mtDNA mutations in human disease suggests that the degradation of dysfunctional mitochondria does not occur widely in vivo. During macroautophagy, a double-membraned cup-shaped structure engulfs cytosolic content. This autophagic vesicle then fuses with lysosomes, allowing hydrolytic enzymes to degrade the contents. Mitochondrial autophagy, or mitophagy, is thought to degrade damaged or nonfunctioning mitochondria specifically. The Parkinson disease-related proteins PINK1 (a mitochondrially localized kinase) and PARK2 (PARKIN, a cytosolically-localized E3 ubiquitin ligase) are essential for targeting mitochondria for mitophagy. Upon chemical uncoupling of the mitochondrial transmembrane potential (Δψ_m), PINK1 located in the mitochondrial outer membrane recruits PARK2 from the cytosol to the mitochondria, followed by delivery of the organelle to the autophagic machinery for degradation.     

Reticulon 3 attenuates the clearance of cytosolic prion aggregates via inhibiting autophagy

Autophagy plays an important role in targeting cellular proteins, protein aggregates and organelles for degradation for cell survival. Autophagy dysfunction has been extensively described in neurodegenerative conditions linked to protein misfolding and aggregation. However, the role of autophagy in the prion disease process is unclear. Here, we show that when expressed in mouse neuroblastoma N2a cells, cytoplasmic PrP (cyPrP) aggregates lead to endoplasmic reticulum stress (ER stress), activation of reticulon 3 (RTN3), impairment of ubiquitin-proteasome system (UPS), induction of autophagy and apoptosis. RTN3 belongs to the reticulon family with the highest expression in the brain and RTN3 is often activated under ER stress. To assess the function of RTN3 in pathological conditions involving cyPrP protein misfolding, we knocked down the expression of RTN3 in cyPrP-transfected cells; unexpectedly, the inhibition of expression of RTN3 enhances the induction of autophagy resulted from cyPrP aggregates, and the process is mediated by the enhanced interaction between Bcl-2 and Beclin1 promoted by RTN3, which enhances Bcl-2-mediated inhibition of Beclin 1-dependent autophagy. Furthermore, down-regulation of RTN3 promoted the clearance of cyPrP aggregates, allowed the activity of the UPS to resume and alleviated ER stress; ultimately, apoptosis due to the cyPrP aggregates was inhibited. Together, these data suggest that RTN3 negatively regulates autophagy to block the clearance of cyPrP aggregates and provide a clue regarding the potential to induce autophagy for the treatment of prion disease and other neurodegenerative diseases such as Parkinson disease (PD), Alzheimer disease (AD) and Huntington disease (HD).     

Neuronal Autophagy: Self-eating or Self-cannibalism in Alzheimer’s Disease

Autophagy is a major intracellular degeneration pathway involved in the elimination and recycling of damaged organelles and long-lived proteins by lysosomes. Many of the pathological factors, which trigger neurodegenerative diseases, can perturb the autophagy activity, which is associated with misfolded protein aggregates accumulation in these disorders. Alzheimer’s disease, the first neurodegenerative disorder between dementias, is characterized by two aggregating proteins, β-amyloid peptide (plaques) and τ-protein (tangles). In Alzheimer’s disease autophagosomes dynamically form along neurites within neuronal cells and in synapses but effective clearance of these structures needs retrograde transportation towards the neuronal soma where there is a major concentration of lysosomes. Maturation of autophago-lysosomes and their retrograde trafficking are perturbed in Alzheimer’s disease, which causes a massive concentration of autophagy elements along degenerating neurites. Transportation system is disturbed along defected microtubules in Alzheimer’s disease brains. τ-protein has been found to control the stability of microtubules, however, phosphorylation of τ-protein or an increase in the total level of τ-protein can cause dysfunction of neuronal cells microtubules. Current evidence has shown that autophagy is developing in Alzheimer’s disease brains because of ineffective degradation of autophagosomes, which hold amyloid precursor protein-rich organelles and secretases important for β-amyloid peptides generation from amyloid precursor. The combination of raised autophagy induction and abnormal clearance of β-amyloid peptide-generating autophagic vacuoles creates circumstances helpful for β-amyloid peptide aggregation and accumulation in Alzheimer’s disease. However, the key role of autophagy in Alzheimer’s disease development is still under consideration today. One point of view suggests that abnormal autophagy induction causes a concentration of autophagic vacuoles rich in amyloid precursor protein, β-amyloid peptide and the elements crucial for its formation, whereas other hypothesis points to marred autophagic clearance or even decrease in autophagic effectiveness playing a role in maturation of Alzheimer’s disease. In this review we present the recent evidence linking autophagy to Alzheimer’s disease and the role of autophagic regulation in the development of full-blown Alzheimer’s disease.     

Mitophagy in cells with mtDNA mutations: being sick is not enough

Despite the emergence of autophagy as a key process for mitochondrial quality control, the existence and persistence of pathogenic mtDNA mutations in human disease suggests that the degradation of dysfunctional mitochondria does not occur widely in vivo. During macroautophagy, a double-membraned cup-shaped structure engulfs cytosolic content. This autophagic vesicle then fuses with lysosomes, allowing hydrolytic enzymes to degrade the contents. Mitochondrial autophagy, or mitophagy, is thought to degrade damaged or nonfunctioning mitochondria specifically. The Parkinson disease-related proteins PINK1 (a mitochondrially localized kinase) and PARK2 (PARKIN, a cytosolically-localized E3 ubiquitin ligase) are essential for targeting mitochondria for mitophagy. Upon chemical uncoupling of the mitochondrial transmembrane potential (Δψ(m)), PINK1 located in the mitochondrial outer membrane recruits PARK2 from the cytosol to the mitochondria, followed by delivery of the organelle to the autophagic machinery for degradation.