Integrated proteomics reveals autophagy landscape and an autophagy receptor controlling PKA-RI complex homeostasis in neurons.

Autophagy is a conserved, catabolic process essential for maintaining cellular homeostasis. Malfunctional autophagy contributes to neurodevelopmental and neurodegenerative diseases. However, the exact role and targets of autophagy in human neurons remain elusive. Here we report a systematic investigation of neuronal autophagy targets through integrated proteomics. Deep proteomic profiling of multiple autophagy-deficient lines of human induced neurons, mouse brains, and brain LC3-interactome reveals roles of neuronal autophagy in targeting proteins of multiple cellular organelles/pathways, including endoplasmic reticulum (ER), mitochondria, endosome, Golgi apparatus, synaptic vesicle (SV) for degradation. By combining phosphoproteomics and functional analysis in human and mouse neurons, we uncovered a function of neuronal autophagy in controlling cAMP-PKA and c-FOS-mediated neuronal activity through selective degradation of the protein kinase A - cAMP-binding regulatory (R)-subunit I (PKA-RI) complex. Lack of AKAP11 causes accumulation of the PKA-RI complex in the soma and neurites, demonstrating a constant clearance of PKA-RI complex through AKAP11-mediated degradation in neurons. Our study thus reveals the landscape of autophagy degradation in human neurons and identifies a physiological function of autophagy in controlling homeostasis of PKA-RI complex and specific PKA activity in neurons.

Selective autophagy of AKAP11 activates cAMP/PKA to fuel mitochondrial metabolism and tumor cell growth.

Autophagy is a catabolic pathway that provides self-nourishment and maintenance of cellular homeostasis. Autophagy is a fundamental cell protection pathway through metabolic recycling of various intracellular cargos and supplying the breakdown products. Here, we report an autophagy function in governing cell protection during cellular response to energy crisis through cell metabolic rewiring. We observe a role of selective type of autophagy in direct activation of cyclic AMP protein kinase A (PKA) and rejuvenation of mitochondrial function. Mechanistically, autophagy selectively degrades the inhibitory subunit RI of PKA holoenzyme through A-kinase-anchoring protein (AKAP) 11. AKAP11 acts as an autophagy receptor that recruits RI to autophagosomes via LC3. Glucose starvation induces AKAP11-dependent degradation of RI, resulting in PKA activation that potentiates PKA-cAMP response element-binding signaling, mitochondria respiration, and ATP production in accordance with mitochondrial elongation. AKAP11 deficiency inhibits PKA activation and impairs cell survival upon glucose starvation. Our results thus expand the view of autophagy cytoprotection mechanism by demonstrating selective autophagy in RI degradation and PKA activation that fuels the mitochondrial metabolism and confers cell resistance to glucose deprivation implicated in tumor growth.

Microglia clear neuron-released α-synuclein via selective autophagy and prevent neurodegeneration.

Microglia maintain brain homeostasis by removing neuron-derived components such as myelin and cell debris. The evidence linking microglia to neurodegenerative diseases is growing; however, the precise mechanisms remain poorly understood. Herein, we report a neuroprotective role for microglia in the clearance of neuron-released α-synuclein. Neuronal α-synuclein activates microglia, which in turn engulf α-synuclein into autophagosomes for degradation via selective autophagy (termed synucleinphagy). Synucleinphagy requires the presence of microglial Toll-like receptor 4 (TLR4), which induces transcriptional upregulation of p62/SQSTM1 through the NF-κB signaling pathway. Induction of p62, an autophagy receptor, is necessary for the formation of α-synuclein/ubiquitin-positive puncta that are degraded by autophagy. Finally, disruption of microglial autophagy in mice expressing human α-synuclein promotes the accumulation of misfolded α-synuclein and causes midbrain dopaminergic neuron degeneration. Our study thus identifies a neuroprotective function of microglia in the clearance of α-synuclein via TLR4-NF-κB-p62 mediated synucleinphagy.

ALS-FTLD-linked mutations of SQSTM1/p62 disrupt selective autophagy and NFE2L2/NRF2 anti-oxidative stress pathway.

Macroautophagy (autophagy) is a key catabolic pathway for the maintenance of proteostasis through constant digestion of selective cargoes. The selectivity of autophagy is mediated by autophagy receptors that recognize and recruit cargoes to autophagosomes. SQSTM1/p62 is a prototype autophagy receptor, which is commonly found in protein aggregates associated with major neurodegenerative diseases. While accumulation of SQSTM1 implicates a disturbance of selective autophagy pathway, the pathogenic mechanism that contributes to impaired autophagy degradation remains poorly characterized. Herein we show that amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD)-linked mutations of TBK1 and SQSTM1 disrupt selective autophagy and cause neurotoxicity. Our data demonstrates that proteotoxic stress activates serine/threonine kinase TBK1, which coordinates with autophagy kinase ULK1 to promote concerted phosphorylation of autophagy receptor SQSTM1 at the UBA domain and activation of selective autophagy. In contrast, ALS-FTLD-linked mutations of TBK1 or SQSTM1 reduce SQSTM1 phosphorylation and compromise ubiquitinated cargo binding and clearance. Moreover, disease mutation SQSTM1G427R abolishes phosphorylation of Ser351 and impairs KEAP1-SQSTM1 interaction, thus diminishing NFE2L2/Nrf2-targeted gene expression and increasing TARDBP/TDP-43 associated stress granule formation under oxidative stress. Furthermore, expression of SQSTM1G427R in neurons impairs dendrite morphology and KEAP1-NFE2L2 signaling. Therefore, our results reveal a mechanism whereby pathogenic SQSTM1 mutants inhibit selective autophagy and disrupt NFE2L2 anti-oxidative stress response underlying the neurotoxicity in ALS-FTLD.Abbreviations: ALS: amyotrophic lateral sclerosis; FTLD: frontotemporal lobar degeneration; G3BP1: GTPase-activating protein (SH3 domain) binding protein 1; GSTM1: glutathione S-transferase, mu 1; HMOX/HO-1: Heme oxygenase 1; IP: immunoprecipitation; KEAP1: kelch-like ECH associated protein 1; KI: kinase inactive; KIR: KEAP1 interaction region; KO: knockout; MAP1LC3/LC3: microtubule-associated protein 1 light chain 3; MBP: maltose binding protein; NBR1: NBR1, autophagy cargo receptor; NFE2L2/Nrf2: nuclear factor, erythroid derived 2, like 2; NQO1: NAD(P)H quinone dehydrogenase 1; SQSTM1/p62: sequestosome 1; SOD1: superoxide dismutase 1, soluble; S.S.: serum starvation; TARDBP/TDP-43: TAR DNA binding protein; TBK1: TANK binding kinase 1; UBA: ubiquitin association; ULK1: unc-51 like autophagy activating kinase 1; WT: wild type.

Neuronal vulnerability in Parkinson disease: Should the focus be on axons and synaptic terminals?

While current effective therapies are available for the symptomatic control of PD, treatments to halt the progressive neurodegeneration still do not exist. Loss of dopamine neurons in the SNc and dopamine terminals in the striatum drive the motor features of PD. Multiple lines of research point to several pathways which may contribute to dopaminergic neurodegeneration. These pathways include extensive axonal arborization, mitochondrial dysfunction, dopamine's biochemical properties, abnormal protein accumulation of α-synuclein, defective autophagy and lysosomal degradation, and synaptic impairment. Thus, understanding the essential features and mechanisms of dopaminergic neuronal vulnerability is a major scientific challenge and highlights an outstanding need for fostering effective therapies against neurodegeneration in PD. This article, which arose from the Movement Disorders 2018 Conference, discusses and reviews the possible mechanisms underlying neuronal vulnerability and potential therapeutic approaches in PD. © 2019 International Parkinson and Movement Disorder Society.

Autophagy Receptors and Neurodegenerative Diseases.

Previously thought of as a nonselective digestion process, autophagy is now known to specifically degrade aggregated proteins and damaged cellular organelles through the action of autophagy receptors, which provides cellular quality control and maintains homeostasis. Autophagy receptors recognize and recruit specific cargoes to the autophagosome-lysosome pathway for degradation in ubiquitin-dependent and -independent manners, and their functions (in selective autophagy) are regulated by protein modifications, for example, phosphorylation and ubiquitination. Growing evidence has linked the genetic variants of autophagy receptors to neurodegenerative diseases and multiple experimental systems have validated their roles in modulating the disease process. Here, we review the recent advances in understanding the physiology and pathophysiology of autophagy receptors in selective autophagy, and discuss their potentials as therapeutic targets for neurodegenerative diseases.

Targeted deletion of Kcne2 impairs HCN channel function in mouse thalamocortical circuits.

BACKGROUND: Hyperpolarization-activated, cyclic nucleotide-gated (HCN) channels generate the pacemaking current, I(h), which regulates neuronal excitability, burst firing activity, rhythmogenesis, and synaptic integration. The physiological consequence of HCN activation depends on regulation of channel gating by endogenous modulators and stabilization of the channel complex formed by principal and ancillary subunits. KCNE2 is a voltage-gated potassium channel ancillary subunit that also regulates heterologously expressed HCN channels; whether KCNE2 regulates neuronal HCN channel function is unknown. METHODOLOGY/PRINCIPAL FINDINGS: We investigated the effects of Kcne2 gene deletion on I(h) properties and excitability in ventrobasal (VB) and cortical layer 6 pyramidal neurons using brain slices prepared from Kcne2(+/+) and Kcne2(-/-) mice. Kcne2 deletion shifted the voltage-dependence of I(h) activation to more hyperpolarized potentials, slowed gating kinetics, and decreased I(h) density. Kcne2 deletion was associated with a reduction in whole-brain expression of both HCN1 and HCN2 (but not HCN4), although co-immunoprecipitation from whole-brain lysates failed to detect interaction of KCNE2 with HCN1 or 2. Kcne2 deletion also increased input resistance and temporal summation of subthreshold voltage responses; this increased intrinsic excitability enhanced burst firing in response to 4-aminopyridine. Burst duration increased in corticothalamic, but not thalamocortical, neurons, suggesting enhanced cortical excitatory input to the thalamus; such augmented excitability did not result from changes in glutamate release machinery since miniature EPSC frequency was unaltered in Kcne2(-/-) neurons. CONCLUSIONS/SIGNIFICANCE: Loss of KCNE2 leads to downregulation of HCN channel function associated with increased excitability in neurons in the cortico-thalamo-cortical loop. Such findings further our understanding of the normal physiology of brain circuitry critically involved in cognition and have implications for our understanding of various disorders of consciousness.

The KCNQ1-KCNE2 K⁺ channel is required for adequate thyroid I⁻ uptake.

The KCNQ1 α subunit and the KCNE2 ß subunit form a potassium channel in thyroid epithelial cells. Genetic disruption of KCNQ1-KCNE2 causes hypothyroidism in mice, resulting in cardiac hypertrophy, dwarfism, alopecia, and prenatal mortality. Here, we investigated the mechanistic requirement for KCNQ1-KCNE2 in thyroid hormone biosynthesis, utilizing whole-animal dynamic positron emission tomography. The KCNQ1-specific antagonist (-)-[3R,4S]-chromanol 293B (C293B) significantly impaired thyroid cell I(-) uptake, which is mediated by the Na(+)/I(-) symporter (NIS), in vivo (dSUV/dt: vehicle, 0.028 ± 0.004 min(-1); 10 mg/kg C293B, 0.009 ± 0.006 min(-1)) and in vitro (EC(50): 99 ± 10 µM C293B). Na(+)-dependent nicotinate uptake by SMCT, however, was unaffected. Kcne2 deletion did not alter the balance of free vs. thyroglobulin-bound I(-) in the thyroid (distinguished using ClO(4)(-), a competitive inhibitor of NIS), indicating that KCNQ1-KCNE2 is not required for Duox/TPO-mediated I(-) organification. However, Kcne2 deletion doubled the rate of free I(-) efflux from the thyroid following ClO(4)(-) injection, a NIS-independent process. Thus, KCNQ1-KCNE2 is necessary for adequate thyroid cell I(-) uptake, the most likely explanation being that it is prerequisite for adequate NIS activity.

KCNE2 forms potassium channels with KCNA3 and KCNQ1 in the choroid plexus epithelium.

Cerebrospinal fluid (CSF) is crucial for normal function and mechanical protection of the CNS. The choroid plexus epithelium (CPe) is primarily responsible for secreting CSF and regulating its composition by mechanisms currently not fully understood. Previously, the heteromeric KCNQ1-KCNE2 K(+) channel was functionally linked to epithelial processes including gastric acid secretion and thyroid hormone biosynthesis. Here, using Kcne2(-/-) tissue as a negative control, we found cerebral expression of KCNE2 to be markedly enriched in the CPe apical membrane, where we also discovered expression of KCNQ1. Targeted Kcne2 gene deletion in C57B6 mice increased CPe outward K(+) current 2-fold. The Kcne2 deletion-enhanced portion of the current was inhibited by XE991 (10 µM) and margatoxin (10 µM) but not by dendrotoxin (100 nM), indicating that it arose from augmentation of KCNQ subfamily and KCNA3 but not KCNA1 K(+) channel activity. Kcne2 deletion in C57B6 mice also altered the polarity of CPe KCNQ1 and KCNA3 trafficking, hyperpolarized the CPe membrane by 9 ± 2 mV, and increased CSF [Cl(-)] by 14% compared with wild-type mice. These findings constitute the first report of CPe dysfunction caused by cation channel gene disruption and suggest that KCNE2 influences blood-CSF anion flux by regulating KCNQ1 and KCNA3 in the CPe.

Protein kinase C downregulates I(Ks) by stimulating KCNQ1-KCNE1 potassium channel endocytosis.

BACKGROUND: The slow-activating cardiac repolarization K(+) current (I(Ks)), generated by the KCNQ1-KCNE1 potassium channel complex, is controlled via sympathetic and parasympathetic regulation in vivo. Inherited KCNQ1 and KCNE1 mutations predispose to ventricular fibrillation and sudden death, often triggered by exercise or emotional stress. Protein kinase C (PKC), which is activated by α1 adrenergic receptor stimulation, is known to downregulate I(Ks) via phosphorylation of KCNE1 serine 102, but the underlying mechanism has remained enigmatic. We previously showed that KCNE1 mediates dynamin-dependent endocytosis of KCNQ1-KCNE1 complexes. OBJECTIVE: This study sought to determine the potential role of endocytosis in I(Ks) downregulation by PKC. METHODS: We utilized patch clamping and fluorescence microscopy to study Chinese hamster ovary (CHO) cells coexpressing KCNQ1, KCNE1, and wild-type or dominant-negative mutant (K44A) dynamin 2, and neonatal mouse ventricular myocytes. RESULTS: The PKC activator phorbol 12-myristate 13-acetate (PMA) decreased I(Ks) density by >60% (P < .05) when coexpressed with wild-type dynamin 2 in CHO cells, but had no effect when coexpressed with K44A-dynamin 2. Thus, functional dynamin was required for downregulation of I(Ks) by PKC activation. PMA increased KCNQ1-KCNE1 endocytosis in CHO cells expressing wild-type dynamin 2, but had no effect on KCNQ1-KCNE1 endocytosis in CHO cells expressing K44A-dynamin 2, determined using the Pearson correlation coefficient to quantify endosomal colocalization of KCNQ1 and KCNE1 with internalized fluorescent transferrin. KCNE1-S102A abolished the effect of PMA on I(Ks) currents and endocytosis. Importantly, PMA similarly stimulated endocytosis of endogenous KCNQ1 and KCNE1 in neonatal mouse myocytes. CONCLUSION: PKC activation downregulates I(Ks) by stimulating KCNQ1-KCNE1 channel endocytosis.

Genetic dissection reveals unexpected influence of beta subunits on KCNQ1 K+ channel polarized trafficking in vivo.

Targeted deletion of the Kcne2 potassium channel ß subunit gene ablates gastric acid secretion and predisposes to gastric neoplasia in mice. Here, we discovered that Kcne2 deletion basolaterally reroutes the Kcnq1 α subunit in vivo in parietal cells (PCs), in which the normally apical location of the Kcnq1-Kcne2 channel facilitates its essential role in gastric acid secretion. Quantitative RT-PCR and Western blotting revealed that Kcne2 deletion remodeled fundic Kcne3 (2.9±0.8-fold mRNA increase, n=10; 5.3±0.4-fold protein increase, n=7) but not Kcne1, 4, or 5, and resulted in basolateral Kcnq1-Kcne3 complex formation in Kcne2(-/-) PCs. Concomitant targeted deletion of Kcne3 (creating Kcne2(-/-)Kcne3(-/-) mice) restored PC apical Kcnq1 localization without Kcne1, 4, or 5 remodeling (assessed by quantitative RT-PCR; n=5-10), indicating Kcne3 actively, basolaterally rerouted Kcnq1 in Kcne2(-/-) PCs. Despite this, Kcne3 deletion exacerbated gastric hyperplasia in Kcne2(-/-) mice, and both hypochlorhydria and hyperplasia in Kcne2(+/-) mice, suggesting that Kcne3 up-regulation was beneficial in Kcne2-depleted PCs. The findings reveal, in vivo, Kcne-dependent α subunit polarized trafficking and the existence and consequences of potassium channel ß subunit remodeling.

Cardiac arrhythmia and thyroid dysfunction: a novel genetic link.

Inherited Long QT Syndrome (LQTS), a cardiac arrhythmia that predisposes to the often lethal ventricular fibrillation, is commonly linked to mutations in KCNQ1. The KCNQ1 voltage-gated K(+) channel α subunit passes ventricular myocyte K(+) current that helps bring a timely end to each heart-beat. KCNQ1, like many K(+) channel α subunits, is regulated by KCNE ß subunits, inherited mutations in which also associate with LQTS. KCNQ1 and KCNE mutations are also associated with atrial fibrillation. It has long been known that thyroid status strongly influences cardiac function, and that thyroid dysfunction causes abnormal cardiac structure and rhythm. We recently discovered that KCNQ1 and KCNE2 form a thyroid-stimulating hormone-stimulated K(+) channel in the thyroid that is required for normal thyroid hormone biosynthesis. Here, we review this novel genetic link between cardiac and thyroid physiology and pathology, and its potential influence upon future therapeutic strategies in cardiac and thyroid disease.

Targeted deletion of Kcne2 causes gastritis cystica profunda and gastric neoplasia.

Gastric cancer is the second leading cause of cancer death worldwide. Predisposing factors include achlorhydria, Helicobacter pylori infection, oxyntic atrophy and TFF2-expressing metaplasia. In parietal cells, apical potassium channels comprising the KCNQ1 alpha subunit and the KCNE2 beta subunit provide a K(+) efflux current to facilitate gastric acid secretion by the apical H(+)K(+)ATPase. Accordingly, genetic deletion of murine Kcnq1 or Kcne2 impairs gastric acid secretion. Other evidence has suggested a role for KCNE2 in human gastric cancer cell proliferation, independent of its role in gastric acidification. Here, we demonstrate that 1-year-old Kcne2(-/-) mice in a pathogen-free environment all exhibit a severe gastric preneoplastic phenotype comprising gastritis cystica profunda, 6-fold increased stomach mass, increased Ki67 and nuclear Cyclin D1 expression, and TFF2- and cytokeratin 7-expressing metaplasia. Some Kcne2(-/-) mice also exhibited pyloric polypoid adenomas extending into the duodenum, and neoplastic invasion of thin walled vessels in the sub-mucosa. Finally, analysis of human gastric cancer tissue indicated reduced parietal cell KCNE2 expression. Together with previous findings, the results suggest KCNE2 disruption as a possible risk factor for gastric neoplasia.

Kcne2 deletion uncovers its crucial role in thyroid hormone biosynthesis.

Thyroid dysfunction is a global health concern, causing defects including neurodevelopmental disorders, dwarfism and cardiac arrhythmia. Here, we show that the potassium channel subunits KCNQ1 and KCNE2 form a thyroid-stimulating hormone-stimulated, constitutively active, thyrocyte K+ channel required for normal thyroid hormone biosynthesis. Targeted disruption of Kcne2 in mice impaired thyroid iodide accumulation up to eightfold, impaired maternal milk ejection, halved milk tetraiodothyronine (T4) content and halved litter size. Kcne2-deficient mice had hypothyroidism, dwarfism, alopecia, goiter and cardiac abnormalities including hypertrophy, fibrosis, and reduced fractional shortening. The alopecia, dwarfism and cardiac abnormalities were alleviated by triiodothyronine (T3) and T4 administration to pups, by supplementing dams with T(4) before and after they gave birth or by feeding the pups exclusively from Kcne2+/+ dams; conversely, these symptoms were elicited in Kcne2+/+ pups by feeding exclusively from Kcne2-/- dams. These data provide a new potential therapeutic target for thyroid disorders and raise the possibility of an endocrine component to previously identified KCNE2- and KCNQ1-linked human cardiac arrhythmias.

Targeted deletion of kcne2 impairs ventricular repolarization via disruption of I(K,slow1) and I(to,f).

Mutations in human KCNE2, which encodes the MiRP1 potassium channel ancillary subunit, associate with long QT syndrome (LQTS), a defect in ventricular repolarization. The precise cardiac role of MiRP1 remains controversial, in part, because it has marked functional promiscuity in vitro. Here, we disrupted the murine kcne2 gene to define the role of MiRP1 in murine ventricles. kcne2 disruption prolonged ventricular action potential duration (APD), suggestive of reduced repolarization capacity. Accordingly, kcne2 (-/-) ventricles exhibited a 50% reduction in I(K,slow1), generated by Kv1.5--a previously unknown partner for MiRP1. I(to,f), generated by Kv4 alpha subunits, was also diminished, by approximately 25%. Ventricular MiRP1 protein coimmunoprecipitated with native Kv1.5 and Kv4.2 but not Kv1.4 or Kv4.3. Unexpectedly, kcne2 (-/-) ventricular membrane fractions exhibited 50% less mature Kv1.5 protein than wild type, and disruption of Kv1.5 trafficking to the intercalated discs. Consistent with the reduction in ventricular K(+) currents and prolonged ventricular APD, kcne2 deletion lengthened the QT(c) under sevoflurane anesthesia. Thus, targeted disruption of kcne2 has revealed a novel cardiac partner for MiRP1, a novel role for MiRPs in alpha subunit targeting in vivo, and a role for MiRP1 in murine ventricular repolarization with parallels to that proposed for the human heart.

Voltage-dependent C-type inactivation in a constitutively open K+ channel.

Most voltage-gated potassium (Kv) channels undergo C-type inactivation during sustained depolarization. The voltage dependence and other mechanistic aspects of this process are debated, and difficult to elucidate because of concomitant voltage-dependent activation. Here, we demonstrate that MinK-KCNQ1 (I(Ks)) channels with an S6-domain mutation, F340W in KCNQ1, exhibit constitutive activation but voltage-dependent C-type inactivation. F340W-I(Ks) inactivation was sensitive to extracellular cation concentration and species, and it altered ion selectivity, suggestive of pore constriction. The rate and extent of F340W-I(Ks) inactivation and recovery from inactivation were voltage-dependent with physiologic intracellular ion concentrations, and in the absence or presence of external K(+), with an estimated gating charge, z(i), of approximately 1. Finally, double-mutant channels with a single S4 charge neutralization (R231A,F340W-I(Ks)) exhibited constitutive C-type inactivation. The results suggest that F340W-I(Ks) channels exhibit voltage-dependent C-type inactivation involving S4, without the necessity for voltage-dependent opening, allosteric coupling to voltage-dependent S6 transitions occurring during channel opening, or voltage-dependent changes in ion occupancy. The data also identify F340 as a critical hub for KCNQ1 gating processes and their modulation by MinK, and present a unique system for further mechanistic studies of the role of coupling of C-type inactivation to S4 movement, without contamination from voltage-dependent activation.