Activity-Dependent Degradation of the Nascentome by the Neuronal Membrane Proteasome.

Activity-dependent changes in neuronal function require coordinated regulation of the protein synthesis and protein degradation machinery to maintain protein homeostasis, critical for proper neuronal function. However, the biochemical evidence for this balance and coordination is largely lacking. Leveraging our recent discovery of a neuronal-specific 20S membrane proteasome complex (NMP), we began exploring how neuronal activity regulates its function. Here, we found that the NMP degrades exclusively a large fraction of ribosome-associated nascent polypeptides that are being newly synthesized during neuronal stimulation. Using deep-coverage and global mass spectrometry, we identified the nascent protein substrates of the NMP, which included products encoding immediate-early genes, such as c-Fos and Npas4. Intriguingly, we found that turnover of nascent polypeptides and not full-length proteins through the NMP occurred independent of canonical ubiquitylation pathways. We propose that these findings generally define a neuronal activity-induced protein homeostasis program of coordinated protein synthesis and degradation through the NMP.

Neuronal membrane proteasomes regulate neuronal circuit activity in vivo and are required for learning-induced behavioral plasticity.

Protein degradation is critical for brain function through processes that remain incompletely understood. Here, we investigated the in vivo function of the 20S neuronal membrane proteasome (NMP) in the brain of Xenopus laevis tadpoles. With biochemistry, immunohistochemistry, and electron microscopy, we demonstrated that NMPs are conserved in the tadpole brain and preferentially degrade neuronal activity-induced newly synthesized proteins in vivo. Using in vivo calcium imaging in the optic tectum, we showed that acute NMP inhibition rapidly increased spontaneous neuronal activity, resulting in hypersynchronization across tectal neurons. At the circuit level, inhibiting NMPs abolished learning-dependent improvement in visuomotor behavior in live animals and caused a significant deterioration in basal behavioral performance following visual training with enhanced visual experience. Our data provide in vivo characterization of NMP functions in the vertebrate nervous system and suggest that NMP-mediated degradation of activity-induced nascent proteins may serve as a homeostatic modulatory mechanism in neurons that is critical for regulating neuronal activity and experience-dependent circuit plasticity.

EphB-mediated degradation of the RhoA GEF Ephexin5 relieves a developmental brake on excitatory synapse formation.

The mechanisms that promote excitatory synapse formation and maturation have been extensively studied. However, the molecular events that limit excitatory synapse development so that synapses form at the right time and place and in the correct numbers are less well understood. We have identified a RhoA guanine nucleotide exchange factor, Ephexin5, which negatively regulates excitatory synapse development until EphrinB binding to the EphB receptor tyrosine kinase triggers Ephexin5 phosphorylation, ubiquitination, and degradation. The degradation of Ephexin5 promotes EphB-dependent excitatory synapse development and is mediated by Ube3A, a ubiquitin ligase that is mutated in the human cognitive disorder Angelman syndrome and duplicated in some forms of Autism Spectrum Disorders (ASDs). These findings suggest that aberrant EphB/Ephexin5 signaling during the development of synapses may contribute to the abnormal cognitive function that occurs in Angelman syndrome and, possibly, ASDs.

Orthogonal approaches required to measure proteasome composition and activity in mammalian brain tissue.

Proteasomes are large macromolecular complexes with multiple distinct catalytic activities that are each vital to human brain health and disease. Despite their importance, standardized approaches to investigate proteasomes have not been universally adapted. Here, we describe pitfalls and define straightforward orthogonal biochemical approaches essential to measure and understand changes in proteasome composition and activity in the mammalian central nervous system. Through our experimentation in the mammalian brain, we determined an abundance of catalytically active proteasomes exist with and without a 19S cap(s), the regulatory particle essential for ubiquitin-dependent degradation. Moreover, we learned that in-cell measurements using activity-based probes (ABPs) are more sensitive in determining the available activity of the 20S proteasome without the 19S cap and in measuring individual catalytic subunit activities of each ß subunit within all neuronal proteasomes. Subsequently, applying these tools to human brain samples, we were surprised to find that post-mortem tissue retained little to no 19S-capped proteasome, regardless of age, sex, or disease state. In comparing brain tissues (parahippocampal gyrus) from patients with Alzheimer's disease (AD) and unaffected individuals, the available 20S proteasome activity was significantly elevated in severe cases of AD, an observation not previously noted. Taken together, our study establishes standardized approaches for the comprehensive investigation of proteasomes in mammalian brain tissue, and we reveal new insight into brain proteasome biology.

Amelogenin phosphorylation regulates tooth enamel formation by stabilizing a transient amorphous mineral precursor.

Dental enamel comprises interwoven arrays of extremely long and narrow crystals of carbonated hydroxyapatite called enamel rods. Amelogenin (AMELX) is the predominant extracellular enamel matrix protein and plays an essential role in enamel formation (amelogenesis). Previously, we have demonstrated that full-length AMELX forms higher-order supramolecular assemblies that regulate ordered mineralization in vitro, as observed in enamel rods. Phosphorylation of the sole AMELX phosphorylation site (Ser-16) in vitro greatly enhances its capacity to stabilize amorphous calcium phosphate (ACP), the first mineral phase formed in developing enamel, and prevents apatitic crystal formation. To test our hypothesis that AMELX phosphorylation is critical for amelogenesis, we generated and characterized a hemizygous knockin (KI) mouse model with a phosphorylation-defective Ser-16 to Ala-16 substitution in AMELX. Using EM analysis, we demonstrate that in the absence of phosphorylated AMELX, KI enamel lacks enamel rods, the hallmark component of mammalian enamel, and, unlike WT enamel, appears to be composed of less organized arrays of shorter crystals oriented normal to the dentinoenamel junction. KI enamel also exhibited hypoplasia and numerous surface defects, whereas heterozygous enamel displayed highly variable mosaic structures with both KI and WT features. Importantly, ACP-to-apatitic crystal transformation occurred significantly faster in KI enamel. Secretory KI ameloblasts also lacked Tomes' processes, consistent with the absence of enamel rods, and underwent progressive cell pathology throughout enamel development. In conclusion, AMELX phosphorylation plays critical mechanistic roles in regulating ACP-phase transformation and enamel crystal growth, and in maintaining ameloblast integrity and function during amelogenesis.

Q&A with Seth S. Margolis and Eric Villalón Landeros.

Cell Reports speaks with Seth S. Margolis and Eric Villalón Landeros about their scientific journeys, experiences, and interests that have led to their recent work in our journal, which demonstrates a role for the neuronal membrane proteasome in modulating the activity of sensory neurons.

Neuronal membrane proteasome-derived peptides modulate NMDAR-dependent neuronal signaling to promote changes in gene expression.

The neuronal membrane proteasome (NMP) degrades intracellular proteins into peptides that are released directly into the extracellular space, whereby they stimulate neurons to promote signaling mechanisms that remain unknown. Here, we demonstrate that neuronal stimulation promotes NMP activity and, subsequently, enhanced production of NMP peptides. We show that these neuronal activity-dependent NMP peptides can rapidly promote N-methyl-D-aspartate receptor (NMDAR)-dependent calcium influx in neurons. This leads to sustained phosphorylation of the well-defined stimulus-induced transcription factor, cyclic AMP response element (CRE)-binding protein (CREB). Downstream of these events, we identified changes to neuronal target genes which included increased expression of immediate early genes (e.g., Fos, Npas4, Egr4) and other genes known to have critical neuroregulatory roles. Further observations led to the discovery that NMP peptide-induced changes in gene expression is dependent on NMDARs and independent of AMPA receptors or voltage-gated sodium channels. These data demonstrate that NMP peptides are endogenous and selective activators of NMDA receptors and act as sufficient and novel stimuli within the context of neuronal activity-dependent signaling. This novel pathway is parallel to classic neuronal activity-dependent programs and points to NMP and its resulting peptides as potential modulators of neuronal function.

The nociceptive activity of peripheral sensory neurons is modulated by the neuronal membrane proteasome.

Proteasomes are critical for peripheral nervous system (PNS) function. Here, we investigate mammalian PNS proteasomes and reveal the presence of the neuronal membrane proteasome (NMP). We show that specific inhibition of the NMP on distal nerve fibers innervating the mouse hind paw leads to reduction in mechanical and pain sensitivity. Through investigating PNS NMPs, we demonstrate their presence on the somata and proximal and distal axons of a subset of dorsal root ganglion (DRG) neurons. Single-cell RNA sequencing experiments reveal that the NMP-expressing DRGs are primarily MrgprA3+ and Cysltr2+. NMP inhibition in DRG cultures leads to cell-autonomous and non-cell-autonomous changes in Ca2+ signaling induced by KCl depolarization, αß-meATP, or the pruritogen histamine. Taken together, these data support a model whereby NMPs are expressed on a subset of somatosensory DRGs to modulate signaling between neurons of distinct sensory modalities and indicate the NMP as a potential target for controlling pain.

Proteasome cap particle regulates synapses.

Deubiquitylation by free 19S proteasome cap particle modulates synaptic transmission.

Aven-dependent activation of ATM following DNA damage.

BACKGROUND: In response to DNA damage, cells undergo either cell-cycle arrest or apoptosis, depending on the extent of damage and the cell's capacity for DNA repair. Cell-cycle arrest induced by double-stranded DNA breaks depends on activation of the ataxia-telangiectasia (ATM) protein kinase, which phosphorylates cell-cycle effectors such as Chk2 and p53 to inhibit cell-cycle progression. ATM is recruited to double-stranded DNA breaks by a complex of sensor proteins, including Mre11/Rad50/Nbs1, resulting in autophosphorylation, monomerization, and activation of ATM kinase. RESULTS: In characterizing Aven protein, a previously reported apoptotic inhibitor, we have found that Aven can function as an ATM activator to inhibit G2/M progression. Aven bound to ATM and Aven overexpressed in cycling Xenopus egg extracts prevented mitotic entry and induced phosphorylation of ATM and its substrates. Immunodepletion of endogenous Aven allowed mitotic entry even in the presence of damaged DNA, and RNAi-mediated knockdown of Aven in human cells prevented autophosphorylation of ATM at an activating site (S1981) in response to DNA damage. Interestingly, Aven is also a substrate of the ATM kinase. Mutation of ATM-mediated phosphorylation sites on Aven reduced its ability to activate ATM, suggesting that Aven activation of ATM after DNA damage is enhanced by ATM-mediated Aven phosphorylation. CONCLUSIONS: These results identify Aven as a new ATM activator and describe a positive feedback loop operating between Aven and ATM. In aggregate, these findings place Aven, a known apoptotic inhibitor, as a critical transducer of the DNA-damage signal.

The proteasome and its role in the nervous system.

Proteasomes are multisubunit complexes that catalyze the majority of protein degradation in mammalian cells to maintain protein homeostasis and influence the regulation of most cellular processes. The proteasome, a multicatalytic protease complex, is a ring-like structure with a narrow pore that exhibits regulated gating, enabling the selective degradation of target proteins into peptide fragments. This process of removing proteins is essential for eliminating proteins that are no longer wanted, such as unfolded or aggregated proteins. This is important for preserving cellular function relevant to brain health and disease. Recently, in the nervous system, specialized proteasomes have been shown to generate peptides with important cellular functions. These discoveries challenge the prevailing notion that proteasomes primarily operate to eliminate proteins and identify signaling-competent proteasomes. This review focuses on the structure, function, and regulation of proteasomes and sheds light on emerging areas of investigation regarding the role of proteasomes in the nervous system.

Deleting a UBE3A substrate rescues impaired hippocampal physiology and learning in Angelman syndrome mice.

In humans, loss-of-function mutations in the UBE3A gene lead to the neurodevelopmental disorder Angelman syndrome (AS). AS patients have severe impairments in speech, learning and memory, and motor coordination, for which there is currently no treatment. In addition, UBE3A is duplicated in > 1-2% of patients with autism spectrum disorders-a further indication of the significant role it plays in brain development. Altered expression of UBE3A, an E3 ubiquitin ligase, is hypothesized to lead to impaired levels of its target proteins, but identifying the contribution of individual UBE3A targets to UBE3A-dependent deficits remains of critical importance. Ephexin5 is a putative UBE3A substrate that has restricted expression early in development, regulates synapse formation during hippocampal development, and is abnormally elevated in AS mice, modeled by maternally-derived Ube3a gene deletion. Here, we report that Ephexin5 can be directly ubiquitylated by UBE3A. Furthermore, removing Ephexin5 from AS mice specifically rescued hippocampus-dependent behaviors, CA1 physiology, and deficits in dendritic spine number. Our findings identify Ephexin5 as a key driver of hippocampal dysfunction and related behavioral deficits in AS mouse models. These results demonstrate the exciting potential of targeting Ephexin5, and possibly other UBE3A substrates, to improve symptoms of AS and other UBE3A-related developmental disorders.

A role for Cdc2- and PP2A-mediated regulation of Emi2 in the maintenance of CSF arrest.

BACKGROUND: Vertebrate oocytes are arrested in metaphase II of meiosis prior to fertilization by cytostatic factor (CSF). CSF enforces a cell-cycle arrest by inhibiting the anaphase-promoting complex (APC), an E3 ubiquitin ligase that targets Cyclin B for degradation. Although Cyclin B synthesis is ongoing during CSF arrest, constant Cyclin B levels are maintained. To achieve this, oocytes allow continuous slow Cyclin B degradation, without eliminating the bulk of Cyclin B, which would induce release from CSF arrest. However, the mechanism that controls this continuous degradation is not understood. RESULTS: We report here the molecular details of a negative feedback loop wherein Cyclin B promotes its own destruction through Cdc2/Cyclin B-mediated phosphorylation and inhibition of the APC inhibitor Emi2. Emi2 bound to the core APC, and this binding was disrupted by Cdc2/Cyclin B, without affecting Emi2 protein stability. Cdc2-mediated phosphorylation of Emi2 was antagonized by PP2A, which could bind to Emi2 and promote Emi2-APC interactions. CONCLUSIONS: Constant Cyclin B levels are maintained during a CSF arrest through the regulation of Emi2 activity. A balance between Cdc2 and PP2A controls Emi2 phosphorylation, which in turn controls the ability of Emi2 to bind to and inhibit the APC. This balance allows proper maintenance of Cyclin B levels and Cdc2 kinase activity during CSF arrest.

A role for PP1 in the Cdc2/Cyclin B-mediated positive feedback activation of Cdc25.

The Cdc25 phosphatase promotes entry into mitosis through the removal of inhibitory phosphorylations on the Cdc2 subunit of the Cdc2/CyclinB complex. During interphase, or after DNA damage, Cdc25 is suppressed by phosphorylation at Ser287 (Xenopus numbering; Ser216 of human Cdc25C) and subsequent binding of the small acidic protein, 14-3-3. As reported recently, at the time of mitotic entry, 14-3-3 protein is removed from Cdc25 and S287 is dephosphorylated by protein phosphatase 1 (PP1). After the initial activation of Cdc25 and consequent derepression of Cdc2/CyclinB, Cdc25 is further activated through a Cdc2-catalyzed positive feedback loop. Although the existence of such a loop has been appreciated for some time, the molecular mechanism for this activation has not been described. We report here that phosphorylation of S285 by Cdc2 greatly enhances recruitment of PP1 to Cdc25, thereby accelerating S287 dephosphorylation and mitotic entry. Moreover, we show that two other previously reported sites of Cdc2-catalyzed phosphorylation on Cdc25 are required for maximal biological activity of Cdc25, but they do not contribute to PP1 regulation and do not act solely through controlling S287 phosphorylation. Therefore, multiple mechanisms, including enhanced recruitment of PP1, are used to promote full activation of Cdc25 at the time of mitotic entry.

Enhanced sensitivity to cytochrome c-induced apoptosis mediated by PHAPI in breast cancer cells.

Apoptotic signaling defects both promote tumorigenesis and confound chemotherapy. Typically, chemotherapeutics stimulate cytochrome c release to the cytoplasm, thereby activating the apoptosome. Although cancer cells can be refractory to cytochrome c release, many malignant cells also exhibit defects in cytochrome c-induced apoptosome activation, further promoting chemotherapeutic resistance. We have found that breast cancer cells display an unusual sensitivity to cytochrome c-induced apoptosis when compared with their normal counterparts. This sensitivity, not observed in other cancers, resulted from enhanced recruitment of caspase-9 to the Apaf-1 caspase recruitment domain. Augmented caspase activation was mediated by PHAPI, which is overexpressed in breast cancers. Furthermore, cytochrome c microinjection into mammary epithelial cells preferentially killed malignant cells, suggesting that this phenomenon might be exploited for chemotherapeutic purposes.

PKCε Inhibits Neuronal Dendritic Spine Development through Dual Phosphorylation of Ephexin5.

Protein kinase C (PKC)-dependent mechanisms promote synaptic function in the mature brain. However, the roles of PKC signaling during synapse development remain largely unknown. Investigating each brain-enriched PKC isoform in early neuronal development, we show that PKCε acutely and specifically reduces the number of dendritic spines, sites of eventual synapse formation on developing dendrites. This PKCε-mediated spine suppression is temporally restricted to immature neurons and mediated through the phosphorylation and activation of Ephexin5, a RhoA guanine nucleotide exchange factor (GEF) and inhibitor of hippocampal synapse formation. Our data suggest that PKCε acts as an early developmental inhibitor of dendritic spine formation, in contrast to its emerging pro-synaptic roles in mature brain function. Moreover, we identify a substrate of PKCε, Ephexin5, whose early-elevated expression in developing neurons may in part explain the mechanism by which PKCε plays seemingly opposing roles that depend on neuronal maturity.

C-terminal regions of the human telomerase catalytic subunit essential for in vivo enzyme activity.

Most human cancer cells are thought to acquire the ability to divide beyond the capacity of normal somatic cells through illegitimately activating the gene hTERT, which encodes the catalytic subunit of telomerase. While telomerase reverse transcriptase (TERT) is conserved in most eukaryotes, mounting evidence suggests that the C terminus of the human protein may have functions unique to higher eukaryotes. To search for domains responsible for such functions, we assayed a panel of tandem substitution mutations encompassing this region of human TERT for in vitro and in vivo functionality. We found four clusters of mutations that inactivated the biochemical and biological functions of telomerase, separated by mutations that had little or no effect on enzyme activity. We also identified a region where mutations generate catalytically active but biologically inert proteins. This C-terminal region that dissociates activities of telomerase (C-DAT) does not appear to be involved in nuclear localization or protein multimerization. Instead, it appears that the C-DAT region is involved in a step of in vivo telomere synthesis after the assembly of a catalytically active enzyme. Intriguingly, all of the described regions reside in a portion of TERT that is dispensable for cellular viability in yeast, arguing for a divergent role of the C terminus in higher eukaryotes.

A mammalian nervous-system-specific plasma membrane proteasome complex that modulates neuronal function.

In the nervous system, rapidly occurring processes such as neuronal transmission and calcium signaling are affected by short-term inhibition of proteasome function. It is unclear how proteasomes are able to acutely regulate such processes, as this action is inconsistent with their canonical role in proteostasis. Here we describe a mammalian nervous-system-specific membrane proteasome complex that directly and rapidly modulates neuronal function by degrading intracellular proteins into extracellular peptides that can stimulate neuronal signaling. This proteasome complex is closely associated with neuronal plasma membranes, exposed to the extracellular space, and catalytically active. Selective inhibition of the membrane proteasome complex by a cell-impermeable proteasome inhibitor blocked the production of extracellular peptides and attenuated neuronal-activity-induced calcium signaling. Moreover, we observed that membrane-proteasome-derived peptides were sufficient to induce neuronal calcium signaling. Our discoveries challenge the prevailing notion that proteasomes function primarily to maintain proteostasis, and highlight a form of neuronal communication that takes place through a membrane proteasome complex.

Reducing expression of synapse-restricting protein Ephexin5 ameliorates Alzheimer's-like impairment in mice.

Accumulation of amyloid-ß (Aß) protein may cause synapse degeneration and cognitive impairment in Alzheimer's disease (AD) by reactivating expression of the developmental synapse repressor protein Ephexin5 (also known as ARHGEF15). Here, we have reported that Aß is sufficient to acutely promote the production of Ephexin5 in mature hippocampal neurons and in mice expressing human amyloid precursor protein (hAPP mice), a model for familial AD that produces high brain levels of Aß. Ephexin5 expression was highly elevated in the hippocampi of human AD patients, indicating its potential relevance to AD. We also observed elevated Ephexin5 expression in the hippocampi of hAPP mice. Removal of Ephexin5 expression eliminated hippocampal dendritic spine loss and rescued AD-associated behavioral deficits in the hAPP mice. Furthermore, selective reduction of Ephexin5 expression using shRNA in the dentate gyrus of presymptomatic adolescent hAPP mice was sufficient to protect these mice from developing cognitive impairment. Thus, pathological elevation of Ephexin5 expression critically drives Aß-induced memory impairment, and strategies aimed at reducing Ephexin5 levels may represent an effective approach to treating AD.