Identification of secretory autophagy as a mechanism modulating activity-induced synaptic remodeling.

The ability of neurons to rapidly remodel their synaptic structure and strength in response to neuronal activity is highly conserved across species and crucial for complex brain functions. However, mechanisms required to elicit and coordinate the acute, activity-dependent structural changes across synapses are not well understood, as neurodevelopment and structural plasticity are tightly linked. Here, using an RNAi screen in Drosophila against genes affecting nervous system functions in humans, we uncouple cellular processes important for synaptic plasticity and synapse development. We find mutations associated with neurodegenerative and mental health disorders are 2-times more likely to affect activity-induced synaptic remodeling than synapse development. We report that while both synapse development and activity-induced synaptic remodeling at the fly NMJ require macroautophagy (hereafter referred to as autophagy), bifurcation in the autophagy pathway differentially impacts development and synaptic plasticity. We demonstrate that neuronal activity enhances autophagy activation but diminishes degradative autophagy, thereby driving the pathway towards autophagy-based secretion. Presynaptic knockdown of Snap29, Sec22, or Rab8, proteins implicated in the secretory autophagy pathway, is sufficient to abolish activity-induced synaptic remodeling. This study uncovers secretory autophagy as a transsynaptic signaling mechanism modulating synaptic plasticity.

Minibrain kinase and calcineurin coordinate activity-dependent bulk endocytosis through synaptojanin.

Neurons use multiple modes of endocytosis, including clathrin-mediated endocytosis (CME) and activity-dependent bulk endocytosis (ADBE), during mild and intense neuronal activity, respectively, to maintain stable neurotransmission. While molecular players modulating CME are well characterized, factors regulating ADBE and mechanisms coordinating CME and ADBE activations remain poorly understood. Here we report that Minibrain/DYRK1A (Mnb), a kinase mutated in autism and up-regulated in Down's syndrome, plays a novel role in suppressing ADBE. We demonstrate that Mnb, together with calcineurin, delicately coordinates CME and ADBE by controlling the phosphoinositol phosphatase activity of synaptojanin (Synj) during varying synaptic demands. Functional domain analyses reveal that Synj's 5'-phosphoinositol phosphatase activity suppresses ADBE, while SAC1 activity is required for efficient ADBE. Consequently, Parkinson's disease mutation in Synj's SAC1 domain impairs ADBE. These data identify Mnb and Synj as novel regulators of ADBE and further indicate that CME and ADBE are differentially governed by Synj's dual phosphatase domains.

CUL4-DDB1-CRBN E3 Ubiquitin Ligase Regulates Proteostasis of ClC-2 Chloride Channels: Implication for Aldosteronism and Leukodystrophy.

Voltage-gated ClC-2 channels are essential for chloride homeostasis. Complete knockout of mouse ClC-2 leads to testicular degeneration and neuronal myelin vacuolation. Gain-of-function and loss-of-function mutations in the ClC-2-encoding human CLCN2 gene are linked to the genetic diseases aldosteronism and leukodystrophy, respectively. The protein homeostasis (proteostasis) mechanism of ClC-2 is currently unclear. Here, we aimed to identify the molecular mechanism of endoplasmic reticulum-associated degradation of ClC-2, and to explore the pathophysiological significance of disease-associated anomalous ClC-2 proteostasis. In both heterologous expression system and native neuronal and testicular cells, ClC-2 is subject to significant regulation by cullin-RING E3 ligase-mediated polyubiquitination and proteasomal degradation. The cullin 4 (CUL4)-damage-specific DNA binding protein 1 (DDB1)-cereblon (CRBN) E3 ubiquitin ligase co-exists in the same complex with and promotes the degradation of ClC-2 channels. The CRBN-targeting immunomodulatory drug lenalidomide and the cullin E3 ligase inhibitor MLN4924 promotes and attenuates, respectively, proteasomal degradation of ClC-2. Analyses of disease-related ClC-2 mutants reveal that aldosteronism and leukodystrophy are associated with opposite alterations in ClC-2 proteostasis. Modifying CUL4 E3 ligase activity with lenalidomide and MLN4924 ameliorates disease-associated ClC-2 proteostasis abnormality. Our results highlight the significant role and therapeutic potential of CUL4 E3 ubiquitin ligase in regulating ClC-2 proteostasis.

Defective Gating and Proteostasis of Human ClC-1 Chloride Channel: Molecular Pathophysiology of Myotonia Congenita.

The voltage-dependent ClC-1 chloride channel, whose open probability increases with membrane potential depolarization, belongs to the superfamily of CLC channels/transporters. ClC-1 is almost exclusively expressed in skeletal muscles and is essential for stabilizing the excitability of muscle membranes. Elucidation of the molecular structures of human ClC-1 and several CLC homologs provides important insight to the gating and ion permeation mechanisms of this chloride channel. Mutations in the human CLCN1 gene, which encodes the ClC-1 channel, are associated with a hereditary skeletal muscle disease, myotonia congenita. Most disease-causing CLCN1 mutations lead to loss-of-function phenotypes in the ClC-1 channel and thus increase membrane excitability in skeletal muscles, consequently manifesting as delayed relaxations following voluntary muscle contractions in myotonic subjects. The inheritance pattern of myotonia congenita can be autosomal dominant (Thomsen type) or recessive (Becker type). To date over 200 myotonia-associated ClC-1 mutations have been identified, which are scattered throughout the entire protein sequence. The dominant inheritance pattern of some myotonia mutations may be explained by a dominant-negative effect on ClC-1 channel gating. For many other myotonia mutations, however, no clear relationship can be established between the inheritance pattern and the location of the mutation in the ClC-1 protein. Emerging evidence indicates that the effects of some mutations may entail impaired ClC-1 protein homeostasis (proteostasis). Proteostasis of membrane proteins comprises of biogenesis at the endoplasmic reticulum (ER), trafficking to the surface membrane, and protein turn-over at the plasma membrane. Maintenance of proteostasis requires the coordination of a wide variety of different molecular chaperones and protein quality control factors. A number of regulatory molecules have recently been shown to contribute to post-translational modifications of ClC-1 and play critical roles in the ER quality control, membrane trafficking, and peripheral quality control of this chloride channel. Further illumination of the mechanisms of ClC-1 proteostasis network will enhance our understanding of the molecular pathophysiology of myotonia congenita, and may also bring to light novel therapeutic targets for skeletal muscle dysfunction caused by myotonia and other pathological conditions.

FKBP8 Enhances Protein Stability of the CLC-1 Chloride Channel at the Plasma Membrane.

Mutations in the skeletal muscle-specific CLC-1 chloride channel are associated with the human hereditary disease myotonia congenita. The molecular pathophysiology underlying some of the disease-causing mutations can be ascribed to defective human CLC-1 protein biosynthesis. CLC-1 protein folding is assisted by several molecular chaperones and co-chaperones, including FK506-binding protein 8 (FKBP8). FKBP8 is generally considered an endoplasmic reticulum- and mitochondrion-resident membrane protein, but is not thought to contribute to protein quality control at the cell surface. Herein, we aim to test the hypothesis that FKBP8 may regulate CLC-1 protein at the plasma membrane. Surface biotinylation and subcellular fractionation analyses reveal that a portion of FKBP8 is present at the plasma membrane, and that co-expression with CLC-1 enhances surface localization of FKBP8. Immunoblotting analyses of plasma membrane proteins purified from skeletal muscle further confirm surface localization of FKBP8. Importantly, FKBP8 promotes CLC-1 protein stability at the plasma membrane. Together, our data underscore the importance of FKBP8 in the peripheral quality control of CLC-1 channel.

Ubiquitin Ligase RNF138 Promotes Episodic Ataxia Type 2-Associated Aberrant Degradation of Human Cav2.1 (P/Q-Type) Calcium Channels.

Voltage-gated CaV2.1 channels comprise a pore-forming α1A subunit with auxiliary α2δ and ß subunits. CaV2.1 channels play an essential role in regulating synaptic signaling. Mutations in the human gene encoding the CaV2.1 subunit are associated with the cerebellar disease episodic ataxia type 2 (EA2). Several EA2-causing mutants exhibit impaired protein stability and exert dominant-negative suppression of CaV2.1 wild-type (WT) protein expression via aberrant proteasomal degradation. Here, we set out to delineate the protein degradation mechanism of human CaV2.1 subunit by identifying RNF138, an E3 ubiquitin ligase, as a novel CaV2.1-binding partner. In neurons, RNF138 and CaV2.1 coexist in the same protein complex and display notable subcellular colocalization at presynaptic and postsynaptic regions. Overexpression of RNF138 promotes polyubiquitination and accelerates protein turnover of CaV2.1. Disrupting endogenous RNF138 function with a mutant (RNF138-H36E) or shRNA infection significantly upregulates the CaV2.1 protein level and enhances CaV2.1 protein stability. Disrupting endogenous RNF138 function also effectively rescues the defective protein expression of EA2 mutants, as well as fully reversing EA2 mutant-induced excessive proteasomal degradation of CaV2.1 WT subunits. RNF138-H36E coexpression only partially restores the dominant-negative effect of EA2 mutants on CaV2.1 WT functional expression, which can be attributed to defective membrane trafficking of CaV2.1 WT in the presence of EA2 mutants. We propose that RNF138 plays a critical role in the homeostatic regulation of CaV2.1 protein level and functional expression and that RNF138 serves as the primary E3 ubiquitin ligase promoting EA2-associated aberrant degradation of human CaV2.1 subunits.SIGNIFICANCE STATEMENT Loss-of-function mutations in the human CaV2.1 subunit are linked to episodic ataxia type 2 (EA2), a dominantly inherited disease characterized by paroxysmal attacks of ataxia and nystagmus. EA2-causing mutants may exert dominant-negative effects on the CaV2.1 wild-type subunit via aberrant proteasomal degradation. The molecular nature of the CaV2.1 ubiquitin-proteasome degradation pathway is currently unknown. The present study reports the first identification of an E3 ubiquitin ligase for CaV2.1, RNF138. CaV2.1 protein stability is dynamically regulated by RNF138 and auxiliary α2δ and ß subunits. We provide a proof of concept that protecting the human CaV2.1 subunit from excessive proteasomal degradation with specific interruption of endogenous RNF138 function may partially contribute to the future development of a novel therapeutic strategy for EA2 patients.

Regulation of CLC-1 chloride channel biosynthesis by FKBP8 and Hsp90ß.

Mutations in human CLC-1 chloride channel are associated with the skeletal muscle disorder myotonia congenita. The disease-causing mutant A531V manifests enhanced proteasomal degradation of CLC-1. We recently found that CLC-1 degradation is mediated by cullin 4 ubiquitin ligase complex. It is currently unclear how quality control and protein degradation systems coordinate with each other to process the biosynthesis of CLC-1. Herein we aim to ascertain the molecular nature of the protein quality control system for CLC-1. We identified three CLC-1-interacting proteins that are well-known heat shock protein 90 (Hsp90)-associated co-chaperones: FK506-binding protein 8 (FKBP8), activator of Hsp90 ATPase homolog 1 (Aha1), and Hsp70/Hsp90 organizing protein (HOP). These co-chaperones promote both the protein level and the functional expression of CLC-1 wild-type and A531V mutant. CLC-1 biosynthesis is also facilitated by the molecular chaperones Hsc70 and Hsp90ß. The protein stability of CLC-1 is notably increased by FKBP8 and the Hsp90ß inhibitor 17-allylamino-17-demethoxygeldanamycin (17-AAG) that substantially suppresses cullin 4 expression. We further confirmed that cullin 4 may interact with Hsp90ß and FKBP8. Our data are consistent with the idea that FKBP8 and Hsp90ß play an essential role in the late phase of CLC-1 quality control by dynamically coordinating protein folding and degradation.

The Cullin 4A/B-DDB1-Cereblon E3 Ubiquitin Ligase Complex Mediates the Degradation of CLC-1 Chloride Channels.

Voltage-gated CLC-1 chloride channels play a critical role in controlling the membrane excitability of skeletal muscles. Mutations in human CLC-1 channels have been linked to the hereditary muscle disorder myotonia congenita. We have previously demonstrated that disease-associated CLC-1 A531V mutant protein may fail to pass the endoplasmic reticulum quality control system and display enhanced protein degradation as well as defective membrane trafficking. Currently the molecular basis of protein degradation for CLC-1 channels is virtually unknown. Here we aim to identify the E3 ubiquitin ligase of CLC-1 channels. The protein abundance of CLC-1 was notably enhanced in the presence of MLN4924, a specific inhibitor of cullin-RING E3 ligases. Subsequent investigation with dominant-negative constructs against specific subtypes of cullin-RING E3 ligases suggested that CLC-1 seemed to serve as the substrate for cullin 4A (CUL4A) and 4B (CUL4B). Biochemical examinations further indicated that CUL4A/B, damage-specific DNA binding protein 1 (DDB1), and cereblon (CRBN) appeared to co-exist in the same protein complex with CLC-1. Moreover, suppression of CUL4A/B E3 ligase activity significantly enhanced the functional expression of the A531V mutant. Our data are consistent with the idea that the CUL4A/B-DDB1-CRBN complex catalyses the polyubiquitination and thus controls the degradation of CLC-1 channels.

The punctate localization of rat Eag1 K+ channels is conferred by the proximal post-CNBHD region.

BACKGROUND: In mammals, Eag K+ channels (KV10) are exclusively expressed in the brain and comprise two isoforms: Eag1 (KV10.1) and Eag2 (KV10.2). Despite their wide presence in various regions of the brain, the functional role of Eag K+ channels remains obscure. Here we address this question by characterizing the subcellular localization of rat Eag1 (rEag1) and rat Eag2 (rEag2) in hippocampal neurons, as well as determining the structural basis underlying their different localization patterns. RESULTS: Immunofluorescence analysis of young and mature hippocampal neurons in culture revealed that endogenous rEag1 and rEag2 K+ channels were present in both the dendrosomatic and the axonal compartments. Only rEag1 channels displayed a punctate immunostaining pattern and showed significant co-localization with PSD-95. Subcellular fractionation analysis further demonstrated a distinct enrichment of rEag1 in the synaptosomal fraction. Over-expression of recombinant GFP-tagged Eag constructs in hippocampal neurons also showed a significant punctate localization of rEag1 channels. To identify the protein region dictating the Eag channel subcellular localization pattern, we generated a variety of different chimeric constructs between rEag1 and rEag2. Quantitative studies of neurons over-expressing these GFP-tagged chimeras indicated that punctate localization was conferred by a segment (A723-R807) within the proximal post-cyclic nucleotide-binding homology domain (post-CNBHD) region in the rEag1 carboxyl terminus. CONCLUSIONS: Our findings suggest that Eag1 and Eag2 K+ channels may modulate membrane excitability in both the dendrosomatic and the axonal compartments and that Eag1 may additionally regulate neurotransmitter release and postsynaptic signaling. Furthermore, we present the first evidence showing that the proximal post-CNBHD region seems to govern the Eag K+ channel subcellular localization pattern.

Densin-180 is not a transmembrane protein.

In the central nervous system, densin-180 (densin) is one of the major components of the post-synaptic density (PSD) of excitatory synapses. Through its intricate interaction with various post-synaptic proteins, this scaffold protein may play a key role in synaptic regulation. Initial structural analyses suggest that densin is a transmembrane protein and may participate in cell-adhesion function between pre- and post-synaptic membranes. Whereas recent biochemical and mass spectrometry studies indicate that densin may instead be a membrane-associated protein with no extracellular domain. To further investigate the structural topology of densin, we began with examining the extracellular accessibility of multiple epitopes in densin. We have provided immunofluorescence evidence showing that none of the tested epitope sites in densin was accessible to extracellularly applied antibodies. In addition, both protease digestion and surface biotinylation data failed to affirm the presence of extracellular domain for densin. However, protein extraction experiments indicated that densin exhibited a significant hydrophobic interaction with the cell membrane that was not expected of cytosolic proteins. Our data therefore do not support the transmembrane model, but rather are consistent with the idea that the topology of densin involves the membrane association configuration.