The multifaceted functions of ß-arrestins and their therapeutic potential in neurodegenerative diseases.

Arrestins are multifunctional proteins that regulate G-protein-coupled receptor (GPCR) desensitization, signaling, and internalization. The arrestin family consists of four subtypes: visual arrestin1, ß-arrestin1, ß-arrestin2, and visual arrestin-4. Recent studies have revealed the multifunctional roles of ß-arrestins beyond GPCR signaling, including scaffolding and adapter functions, and physically interacting with non-GPCR receptors. Increasing evidence suggests that ß-arrestins are involved in the pathogenesis of a variety of neurodegenerative diseases, including Alzheimer's disease (AD), frontotemporal dementia (FTD), and Parkinson's disease (PD). ß-arrestins physically interact with γ-secretase, leading to increased production and accumulation of amyloid-beta in AD. Furthermore, ß-arrestin oligomers inhibit the autophagy cargo receptor p62/SQSTM1, resulting in tau accumulation and aggregation in FTD. In PD, ß-arrestins are upregulated in postmortem brain tissue and an MPTP model, and the ß2AR regulates SNCA gene expression. In this review, we aim to provide an overview of ß-arrestin1 and ß-arrestin2, and describe their physiological functions and roles in neurodegenerative diseases. The multifaceted roles of ß-arrestins and their involvement in neurodegenerative diseases suggest that they may serve as promising therapeutic targets.

Disruption of Mitophagy Flux through the PARL-PINK1 Pathway by CHCHD10 Mutations or CHCHD10 Depletion.

Coiled-coil-helix-coiled-coil-helix domain-containing 10 (CHCHD10) is a nuclear-encoded mitochondrial protein which is primarily mutated in the spectrum of familial and sporadic amyotrophic lateral sclerosis (ALS)-frontotemporal dementia (FTD). Endogenous CHCHD10 levels decline in the brains of ALS-FTD patients, and the CHCHD10S59L mutation in Drosophila induces dominant toxicity together with PTEN-induced kinase 1 (PINK1), a protein critical for the induction of mitophagy. However, whether and how CHCHD10 variants regulate mitophagy flux in the mammalian brain is unknown. Here, we demonstrate through in vivo and in vitro models, as well as human FTD brain tissue, that ALS/FTD-linked CHCHD10 mutations (R15L and S59L) impair mitophagy flux and mitochondrial Parkin recruitment, whereas wild-type CHCHD10 (CHCHD10WT) normally enhances these measures. Specifically, we show that CHCHD10R15L and CHCHD10S59L mutations reduce PINK1 levels by increasing PARL activity, whereas CHCHD10WT produces the opposite results through its stronger interaction with PARL, suppressing its activity. Importantly, we also demonstrate that FTD brains with TAR DNA-binding protein-43 (TDP-43) pathology demonstrate disruption of the PARL-PINK1 pathway and that experimentally impairing mitophagy promotes TDP-43 aggregation. Thus, we provide herein new insights into the regulation of mitophagy and TDP-43 aggregation in the mammalian brain through the CHCHD10-PARL-PINK1 pathway.

Slingshot homolog-1-mediated Nrf2 sequestration tips the balance from neuroprotection to neurodegeneration in Alzheimer's disease.

Oxidative damage in the brain is one of the earliest drivers of pathology in Alzheimer's disease (AD) and related dementias, both preceding and exacerbating clinical symptoms. In response to oxidative stress, nuclear factor erythroid 2-related factor 2 (Nrf2) is normally activated to protect the brain from oxidative damage. However, Nrf2-mediated defense against oxidative stress declines in AD, rendering the brain increasingly vulnerable to oxidative damage. Although this phenomenon has long been recognized, its mechanistic basis has been a mystery. Here, we demonstrate through in vitro and in vivo models, as well as human AD brain tissue, that Slingshot homolog-1 (SSH1) drives this effect by acting as a counterweight to neuroprotective Nrf2 in response to oxidative stress and disease. Specifically, oxidative stress-activated SSH1 suppresses nuclear Nrf2 signaling by sequestering Nrf2 complexes on actin filaments and augmenting Kelch-like ECH-associated protein 1 (Keap1)-Nrf2 interaction, independently of SSH1 phosphatase activity. We also show that Ssh1 elimination in AD models increases Nrf2 activation, which mitigates tau and amyloid-ß accumulation and protects against oxidative injury, neuroinflammation, and neurodegeneration. Furthermore, loss of Ssh1 preserves normal synaptic function and transcriptomic patterns in tauP301S mice. Importantly, we also show that human AD brains exhibit highly elevated interactions of Nrf2 with both SSH1 and Keap1. Thus, we demonstrate here a unique mode of Nrf2 blockade that occurs through SSH1, which drives oxidative damage and ensuing pathogenesis in AD. Strategies to inhibit SSH1-mediated Nrf2 suppression while preserving normal SSH1 catalytic function may provide new neuroprotective therapies for AD and related dementias.

A Par3/LIM Kinase/Cofilin Pathway Mediates Human Airway Smooth Muscle Relaxation by TAS2R14.

TAS2Rs (bitter taste receptors) are GPCRs (G protein-coupled receptors) expressed on human airway smooth muscle (HASM) cells; when activated by receptor agonists they evoke marked airway relaxation. In both taste and HASM cells, TAS2Rs activate a canonical Gßγ-mediated stimulation of Ca2+ release from intracellular stores by activation of PLCß (phospholipase Cß). Alone, this [Ca2+]i signaling does not readily account for relaxation, particularly since bronchoconstrictive agonists acting at Gq-coupled receptors also increase [Ca2+]i. We established that TAS2R14 activation in HASM promotes relaxation through F-actin (filamentous actin) severing. This destabilization of actin was from agonist-promoted activation (dephosphorylation) of cofilin, which was pertussis toxin sensitive. Cofilin dephosphorylation was due to TAS2R-mediated deactivation of LIM domain kinase. The link between early receptor action and the distal cofilin dephosphorylation was found to be the polarity protein partitioning defective 3 (Par3), a known binding partner with PLCß that inhibits LIM kinase. The physiologic relevance of this pathway was assessed using knock-downs of cofilin and Par3 in HASM cells and in human precision-cut lung slices. Relaxation by TAS2R14 agonists was ablated with knock-down of either protein as assessed by magnetic twisting cytometry in isolated cells or intact airways in the slices. Blocking [Ca2+]i release by TAS2R14 inhibited agonist-promoted cofilin dephosphorylation, confirming a role for [Ca2+]i in actin-modifying pathways. These results further elucidate the mechanistic basis of TAS2R-mediated HASM relaxation and point toward nodal points that may act as asthma or chronic obstructive pulmonary disease response modifiers or additional targets for novel bronchodilators.

Slingshot homolog-1 mediates the secretion of small extracellular vesicles containing misfolded proteins by regulating autophagy cargo receptors and actin dynamics.

Increasing evidence indicates that the accumulation misfolded proteins in Alzheimer's disease (AD) arises from clearance defects in the autophagy-lysosome pathway. Misfolded proteins such as Aß and tau are secreted in small extracellular vesicles (i.e., exosomes) and are propagated from cell to cell in part through secreted small extracellular vesicles (sEVs). Recent studies suggest that autophagic activity and exosome secretion are coregulated events, and multiple autophagy-related proteins are found in sEVs, including the cargo receptors Sqstm1/p62 and optineurin. However, whether and how autophagy cargo receptors per se regulate the secretion of sEVs is unknown. Moreover, despite the prominent role of actin dynamics in secretory vesicle release, its role in EV secretion is unknown. In this study, we leveraged the dual axes of Slingshot Homolog-1 (SSH1), which inhibits Sqstm1/p62-mediated autophagy and activates cofilin-mediated actin dynamics, to study the regulation of sEV secretion. Here we show that cargo receptors Sqstm1/p62 and optineurin inhibit sEV secretion, an activity that requires their ability to bind ubiquitinated cargo. Conversely, SSH1 increases sEV secretion by dephosphorylating Sqstm1/p62 at pSer403, the phospho-residue that allows Sqstm1/p62 to bind ubiquitinated cargo. In addition, increasing actin dynamics through the SSH1-cofilin activation pathway also increases sEV secretion, which is mimicked by latrunculin B treatment. Finally, Aß42 oligomers and mutant tau increase sEV secretion and are physically associated with secreted sEVs. These findings suggest that increasing cargo receptor engagement with autophagic cargo and reducing actin dynamics (i.e., SSH1 inhibition) represents an attractive strategy to promote misfolded protein degradation while reducing sEV-mediated cell to cell spread of pathology.

Pathological characterization of a novel mouse model expressing the PD-linked CHCHD2-T61I mutation.

Coiled-coil-helix-coiled-coil-helix domain containing 2 (CHCHD2) is a mitochondrial protein that plays important roles in cristae structure, oxidative phosphorylation and apoptosis. Multiple mutations in CHCHD2 have been associated with Lewy body disorders (LBDs), such as Parkinson's disease (PD) and dementia with Lewy bodies, with the CHCHD2-T61I mutation being the most widely studied. However, at present, only CHCHD2 knockout or CHCHD2/CHCHD10 double knockout mouse models have been investigated. They do not recapitulate the pathology seen in patients with CHCHD2 mutations. We generated the first transgenic mouse model expressing the human PD-linked CHCHD2-T61I mutation driven by the mPrP promoter. We show that CHCHD2-T61I Tg mice exhibit perinuclear mitochondrial aggregates, neuroinflammation, and have impaired long-term synaptic plasticity associated with synaptic dysfunction. Dopaminergic neurodegeneration, a hallmark of PD, is also observed along with α-synuclein pathology. Significant motor dysfunction is seen with no changes in learning and memory at 1 year of age. A minor proportion of the CHCHD2-T61I Tg mice (~10%) show a severe motor phenotype consistent with human Pisa Syndrome, an atypical PD phenotype. Unbiased proteomics analysis reveals surprising increases in many insoluble proteins predominantly originating from mitochondria and perturbing multiple canonical biological pathways as assessed by ingenuity pathway analysis, including neurodegenerative disease-associated proteins such as tau, cofilin, SOD1 and DJ-1. Overall, CHCHD2-T61I Tg mice exhibit pathological and motor changes associated with LBDs, indicating that this model successfully captures phenotypes seen in human LBD patients with CHCHD2 mutations and demonstrates changes in neurodegenerative disease-associated proteins, which delineates relevant pathological pathways for further investigation.

ß-arrestin1 promotes tauopathy by transducing GPCR signaling, disrupting microtubules and autophagy.

G protein-coupled receptors (GPCRs) have been shown to play integral roles in Alzheimer's disease pathogenesis. However, it is unclear how diverse GPCRs similarly affect Aß and tau pathogenesis. GPCRs share a common mechanism of action via the ß-arrestin scaffolding signaling complexes, which not only serve to desensitize GPCRs by internalization, but also mediate multiple downstream signaling events. As signaling via the GPCRs, ß2-adrenergic receptor (ß2AR), and metabotropic glutamate receptor 2 (mGluR2) promotes hyperphosphorylation of tau, we hypothesized that ß-arrestin1 represents a point of convergence for such pathogenic activities. Here, we report that ß-arrestins are not only essential for ß2AR and mGluR2-mediated increase in pathogenic tau but also show that ß-arrestin1 levels are increased in brains of Frontotemporal lobar degeneration (FTLD-tau) patients. Increased ß-arrestin1 in turn drives the accumulation of pathogenic tau, whereas reduced ARRB1 alleviates tauopathy and rescues impaired synaptic plasticity and cognitive impairments in PS19 mice. Biochemical and cellular studies show that ß-arrestin1 drives tauopathy by destabilizing microtubules and impeding p62/SQSTM1 autophagy flux by interfering with p62 body formation, which promotes pathogenic tau accumulation.

Mitochondrial CHCHD2: Disease-Associated Mutations, Physiological Functions, and Current Animal Models.

Rare mutations in the mitochondrial protein coiled-coil-helix-coiled-coil-helix domain containing 2 (CHCHD2) are associated with Parkinson's disease (PD) and other Lewy body disorders. CHCHD2 is a bi-organellar mediator of oxidative phosphorylation, playing crucial roles in regulating electron flow in the mitochondrial electron transport chain and acting as a nuclear transcription factor for a cytochrome c oxidase subunit (COX4I2) and itself in response to hypoxic stress. CHCHD2 also regulates cell migration and differentiation, mitochondrial cristae structure, and apoptosis. In this review, we summarize the known disease-associated mutations of CHCHD2 in Asian and Caucasian populations, the physiological functions of CHCHD2, how CHCHD2 mutations contribute to α-synuclein pathology, and current animal models of CHCHD2. Further, we discuss the necessity of continued investigation into the divergent functions of CHCHD2 and CHCHD10 to determine how mutations in these similar mitochondrial proteins contribute to different neurodegenerative diseases.