Sigma-2 Receptors-From Basic Biology to Therapeutic Target: A Focus on Age-Related Degenerative Diseases.

There is a large unmet medical need to develop disease-modifying treatment options for individuals with age-related degenerative diseases of the central nervous system. The sigma-2 receptor (S2R), encoded by TMEM97, is expressed in brain and retinal cells, and regulates cell functions via its co-receptor progesterone receptor membrane component 1 (PGRMC1), and through other protein-protein interactions. Studies describing functions of S2R involve the manipulation of expression or pharmacological modulation using exogenous small-molecule ligands. These studies demonstrate that S2R modulates key pathways involved in age-related diseases including autophagy, trafficking, oxidative stress, and amyloid-ß and α-synuclein toxicity. Furthermore, S2R modulation can ameliorate functional deficits in cell-based and animal models of disease. This review summarizes the current evidence-based understanding of S2R biology and function, and its potential as a therapeutic target for age-related degenerative diseases of the central nervous system, including Alzheimer's disease, α-synucleinopathies, and dry age-related macular degeneration.

iPSC-Derived Neurons from Patients with POLG Mutations Exhibit Decreased Mitochondrial Content and Dendrite Simplification.

Mutations in POLG, the gene encoding the catalytic subunit of DNA polymerase gamma, result in clinical syndromes characterized by mitochondrial DNA (mtDNA) depletion in affected tissues with variable organ involvement. The brain is one of the most affected organs, and symptoms include intractable seizures, developmental delay, dementia, and ataxia. Patient-derived induced pluripotent stem cells (iPSCs) provide opportunities to explore mechanisms in affected cell types and potential therapeutic strategies. Fibroblasts from two patients were reprogrammed to create new iPSC models of POLG-related mitochondrial diseases. Compared with iPSC-derived control neurons, mtDNA depletion was observed upon differentiation of the POLG-mutated lines to cortical neurons. POLG-mutated neurons exhibited neurite simplification with decreased mitochondrial content, abnormal mitochondrial structure and function, and increased cell death. Expression of the mitochondrial kinase PTEN-induced kinase 1 (PINK1) mRNA was decreased in patient neurons. Overexpression of PINK1 increased mitochondrial content and ATP:ADP ratios in neurites, decreasing cell death and rescuing neuritic complexity. These data indicate an intersection of polymerase gamma and PINK1 pathways that may offer a novel therapeutic option for patients affected by this spectrum of disorders.

Endogenous PTEN-Induced Kinase 1 Regulates Dendritic Architecture and Spinogenesis.

Mutations in PTEN-induced kinase 1 (PINK1) contribute to autosomal recessive Parkinson's disease with cognitive and neuropsychiatric comorbidities. Disturbances in dendritic and spine architecture are hallmarks of neurodegenerative and neuropsychiatric conditions, but little is known of the impact of PINK1 on these structures. We used Pink1 -/- mice to study the role of endogenous PINK1 in regulating dendritic architecture, spine density, and spine maturation. Pink1 -/- cortical neurons of unknown sex showed decreased dendritic arborization, affecting both apical and basal arbors. Dendritic simplification in Pink1 -/- neurons was primarily driven by diminished branching with smaller effects on branch lengths. Pink1 -/- neurons showed reduced spine density with a shift in morphology to favor filopodia at the expense of mushroom spines. Electrophysiology revealed significant reductions in miniature EPSC (mEPSC) frequency in Pink1 -/- neurons, consistent with the observation of decreased spine numbers. Transfecting with human PINK1 rescued changes in dendritic architecture, in thin, stubby, and mushroom spine densities, and in mEPSC frequency. Diminished spine density was also observed in Golgi-Cox stained adult male Pink1 -/- brains. Western blot study of Pink1 -/- brains of either sex revealed reduced phosphorylation of NSFL1 cofactor p47, an indirect target of PINK1. Transfection of Pink1 -/- neurons with a phosphomimetic p47 plasmid rescued dendritic branching and thin/stubby spine density with a partial rescue of mushroom spines, implicating a role for PINK1-regulated p47 phosphorylation in dendrite and spine development. These findings suggest that PINK1-dependent synaptodendritic alterations may contribute to the risk of cognitive and/or neuropsychiatric pathologies observed in PINK1-mutated families.SIGNIFICANCE STATEMENT Loss of PINK1 function has been implicated in both familial and sporadic neurodegenerative diseases. Yet surprisingly little is known of the impact of PINK1 loss on the fine structure of neurons. Neurons receive excitatory synaptic signals along a complex network of projections that form the dendritic tree, largely at tiny protrusions called dendritic spines. We studied cortical neurons and brain tissues from mice lacking PINK1. We discovered that PINK1 deficiency causes striking simplification of dendritic architecture associated with reduced synaptic input and decreased spine density and maturation. These changes are reversed by reintroducing human PINK1 or one of its downstream mediators into PINK1-deficient mouse neurons, indicating a conserved function, whose loss may contribute to neurodegenerative processes.

Excitotoxicity, calcium and mitochondria: a triad in synaptic neurodegeneration.

Glutamate is the most commonly engaged neurotransmitter in the mammalian central nervous system, acting to mediate excitatory neurotransmission. However, high levels of glutamatergic input elicit excitotoxicity, contributing to neuronal cell death following acute brain injuries such as stroke and trauma. While excitotoxic cell death has also been implicated in some neurodegenerative disease models, the role of acute apoptotic cell death remains controversial in the setting of chronic neurodegeneration. Nevertheless, it is clear that excitatory synaptic dysregulation contributes to neurodegeneration, as evidenced by protective effects of partial N-methyl-D-aspartate receptor antagonists. Here, we review evidence for sublethal excitatory injuries in relation to neurodegeneration associated with Parkinson's disease, Alzheimer's disease, amyotrophic lateral sclerosis and Huntington's disease. In contrast to classic excitotoxicity, emerging evidence implicates dysregulation of mitochondrial calcium handling in excitatory post-synaptic neurodegeneration. We discuss mechanisms that regulate mitochondrial calcium uptake and release, the impact of LRRK2, PINK1, Parkin, beta-amyloid and glucocerebrosidase on mitochondrial calcium transporters, and the role of autophagic mitochondrial loss in axodendritic shrinkage. Finally, we discuss strategies for normalizing the flux of calcium into and out of the mitochondrial matrix, thereby preventing mitochondrial calcium toxicity and excitotoxic dendritic loss. While the mechanisms that underlie increased uptake or decreased release of mitochondrial calcium vary in different model systems, a common set of strategies to normalize mitochondrial calcium flux can prevent excitatory mitochondrial toxicity and may be neuroprotective in multiple disease contexts.

Neuronal autophagy and mitophagy in Parkinson's disease.

Autophagy is the process by which cells can selectively or non-selectively remove damaged proteins and organelles. As the cell's main means of sequestering damaged mitochondria for removal, mitophagy is central to cellular function and survival. Research on autophagy and mitochondrial quality control has increased exponentially in relation to the pathogenesis of numerous disease conditions, from cancer and immune diseases to chronic neurodegenerative diseases like Parkinson's disease (PD). Understanding how components of the autophagic/mitophagic machinery are affected during disease, as well as the contextual relationship of autophagy with determining neuronal health and function, is essential to the goal of designing therapies for human disease. In this review, we will summarize key signaling molecules that consign damaged mitochondria for autophagic degradation, describe the relationship of genes linked to PD to autophagy/mitophagy dysfunction, and discuss additional roles of both mitochondrial and cytosolic pools of PTEN-induced kinase 1 (PINK1) in mitochondrial homeostasis, dendritic morphogenesis and inflammation.

Neuronal Preconditioning Requires the Mitophagic Activity of C-terminus of HSC70-Interacting Protein.

The C terminus of HSC70-interacting protein (CHIP, STUB1) is a ubiquitously expressed cytosolic E3-ubiquitin ligase. CHIP-deficient mice exhibit cardiovascular stress and motor dysfunction before premature death. This phenotype is more consistent with animal models in which master regulators of autophagy are affected rather than with the mild phenotype of classic E3-ubiquitin ligase mutants. The cellular and biochemical events that contribute to neurodegeneration and premature aging in CHIP KO models remain poorly understood. Electron and fluorescent microscopy demonstrates that CHIP deficiency is associated with greater numbers of mitochondria, but these organelles are swollen and misshapen. Acute bioenergetic stress triggers CHIP induction and relocalization to mitochondria, where it plays a role in the removal of damaged organelles. This mitochondrial clearance is required for protection following low-level bioenergetic stress in neurons. CHIP expression overlaps with stabilization of the redox stress sensor PTEN-inducible kinase 1 (PINK1) and is associated with increased LC3-mediated mitophagy. Introducing human promoter-driven vectors with mutations in either the E3 ligase or tetracopeptide repeat domains of CHIP in primary neurons derived from CHIP-null animals enhances CHIP accumulation at mitochondria. Exposure to autophagy inhibitors suggests that the increase in mitochondrial CHIP is likely due to diminished clearance of these CHIP-tagged organelles. Proteomic analysis of WT and CHIP KO mouse brains (four male, four female per genotype) reveals proteins essential for maintaining energetic, redox, and mitochondrial homeostasis undergo significant genotype-dependent expression changes. Together, these data support the use of CHIP-deficient animals as a predictive model of age-related degeneration with selective neuronal proteotoxicity and mitochondrial failure.SIGNIFICANCE STATEMENT Mitochondria are recognized as central determinants of neuronal function and survival. We demonstrate that C terminus of HSC70-Interacting Protein (CHIP) is critical for neuronal responses to stress. CHIP upregulation and localization to mitochondria is required for mitochondrial autophagy (mitophagy). Unlike other disease-associated E3 ligases such as Parkin and Mahogunin, CHIP controls homeostatic and stress-induced removal of mitochondria. Although CHIP deletion results in greater numbers of mitochondria, these organelles have distorted inner membranes without clear cristae. Neuronal cultures derived from animals lacking CHIP are more vulnerable to acute injuries and transient loss of CHIP renders neurons incapable of mounting a protective response after low-level stress. Together, these data suggest that CHIP is an essential regulator of mitochondrial number, cell signaling, and survival.

Alterations in the E3 ligases Parkin and CHIP result in unique metabolic signaling defects and mitochondrial quality control issues.

E3 ligases are essential scaffold proteins, facilitating the transfer of ubiquitin from E2 enzymes to lysine residues of client proteins via isopeptide bonds. The specificity of substrate binding and the expression and localization of E3 ligases can, however, endow these proteins with unique features with variable effects on mitochondrial, metabolic and CNS function. By comparing and contrasting two E3 ligases, Parkin and C-terminus of HSC70-Interacting protein (CHIP) we seek to highlight the biophysical properties that may promote mitochondrial dysfunction, acute stress signaling and critical developmental periods to cease in response to mutations in these genes. Encoded by over 600 human genes, RING-finger proteins are the largest class of E3 ligases. Parkin contains three RING finger domains, with R1 and R2 separated by an in-between region (IBR) domain. Loss-of-function mutations in Parkin were identified in patients with early onset Parkinson's disease. CHIP is a member of the Ubox family of E3 ligases. It contains an N-terminal TPR domain and forms unique asymmetric homodimers. While CHIP can substitute for mutated Parkin and enhance survival, CHIP also has unique functions. The differences between these proteins are underscored by the observation that unlike Parkin-deficient animals, CHIP-null animals age prematurely and have significantly impaired motor function. These properties make these E3 ligases appealing targets for clinical intervention. In this work, we discuss how biophysical and metabolic properties of these E3 ligases have driven rapid progress in identifying roles for E3 ligases in development, proteostasis, mitochondrial biology, and cell health, as well as new data about how these proteins alter the CNS proteome.

Silent scaffolds: inhibition OF c-Jun N-terminal kinase 3 activity in cell by dominant-negative arrestin-3 mutant.

We established a new in vivo arrestin-3-JNK3 interaction assay based on bioluminescence resonance energy transfer (BRET) between JNK3-luciferase and Venus-arrestins. We tested the ability of WT arrestin-3 and its 3A mutant that readily binds ß2-adrenergic receptors as well as two mutants impaired in receptor binding, Δ7 and KNC, to directly bind JNK3 and to promote JNK3 phosphorylation in cells. Both receptor binding-deficient mutants interact with JNK3 significantly better than WT and 3A arrestin-3. WT arrestin-3 and Δ7 mutant robustly promoted JNK3 activation, whereas 3A and KNC mutants did not. Thus, receptor binding, JNK3 interaction, and JNK3 activation are three distinct arrestin functions. We found that the KNC mutant, which tightly binds ASK1, MKK4, and JNK3 without facilitating JNK3 phosphorylation, has a dominant-negative effect, competitively decreasing JNK activation by WT arrestin-3. Thus, KNC is a silent scaffold, a novel type of molecular tool for the suppression of MAPK signaling in living cells.