Diminished Neuronal ESCRT-0 Function Exacerbates AMPA Receptor Derangement and Accelerates Prion-Induced Neurodegeneration.

Endolysosomal defects in neurons are central to the pathogenesis of prion and other neurodegenerative disorders. In prion disease, prion oligomers traffic through the multivesicular body (MVB) and are routed for degradation in lysosomes or for release in exosomes, yet how prions impact proteostatic pathways is unclear. We found that prion-affected human and mouse brain showed a marked reduction in Hrs and STAM1 (ESCRT-0), which route ubiquitinated membrane proteins from early endosomes into MVBs. To determine how the reduction in ESCRT-0 impacts prion conversion and cellular toxicity in vivo, we prion-challenged conditional knockout mice (male and female) having Hrs deleted from neurons, astrocytes, or microglia. The neuronal, but not astrocytic or microglial, Hrs-depleted mice showed a shortened survival and an acceleration in synaptic derangements, including an accumulation of ubiquitinated proteins, deregulation of phosphorylated AMPA and metabotropic glutamate receptors, and profoundly altered synaptic structure, all of which occurred later in the prion-infected control mice. Finally, we found that neuronal Hrs (nHrs) depletion increased surface levels of the cellular prion protein, PrPC, which may contribute to the rapidly advancing disease through neurotoxic signaling. Taken together, the reduced Hrs in the prion-affected brain hampers ubiquitinated protein clearance at the synapse, exacerbates postsynaptic glutamate receptor deregulation, and accelerates neurodegeneration.SIGNIFICANCE STATEMENT Prion diseases are rapidly progressive neurodegenerative disorders characterized by prion aggregate spread through the central nervous system. Early disease features include ubiquitinated protein accumulation and synapse loss. Here, we investigate how prion aggregates alter ubiquitinated protein clearance pathways (ESCRT) in mouse and human prion-infected brain, discovering a marked reduction in Hrs. Using a prion-infection mouse model with neuronal Hrs (nHrs) depleted, we show that low neuronal Hrs is detrimental and markedly shortens survival time while accelerating synaptic derangements, including ubiquitinated protein accumulation, indicating that Hrs loss exacerbates prion disease progression. Additionally, Hrs depletion increases the surface distribution of prion protein (PrPC), linked to aggregate-induced neurotoxic signaling, suggesting that Hrs loss in prion disease accelerates disease through enhancing PrPC-mediated neurotoxic signaling.

Inherently Emissive Puromycin Analogues for Live Cell Labelling.

Puromycin derivatives containing an emissive thieno[3,4-d]-pyrimidine core, modified with azetidine and 3,3-difluoroazetidine as Me2 N surrogates, exhibit translation inhibition and bactericidal activity similar to the natural antibiotic. The analogues are capable of cellular puromycylation of nascent peptides, generating emissive products without any follow-up chemistry. The 3,3-difluoroazetidine-containing analogue is shown to fluorescently label newly translated peptides and be visualized in both live and fixed HEK293T cells and rat hippocampal neurons.

Enhanced activity of Alzheimer disease-associated variant of protein kinase Cα drives cognitive decline in a mouse model.

Exquisitely tuned activity of protein kinase C (PKC) isozymes is essential to maintaining cellular homeostasis. Whereas loss-of-function mutations are generally associated with cancer, gain-of-function variants in one isozyme, PKCα, are associated with Alzheimer's disease (AD). Here we show that the enhanced activity of one variant, PKCα M489V, is sufficient to rewire the brain phosphoproteome, drive synaptic degeneration, and impair cognition in a mouse model. This variant causes a modest 30% increase in catalytic activity without altering on/off activation dynamics or stability, underscoring that enhanced catalytic activity is sufficient to drive the biochemical, cellular, and ultimately cognitive effects observed. Analysis of hippocampal neurons from PKCα M489V mice reveals enhanced amyloid-ß-induced synaptic depression and reduced spine density compared to wild-type mice. Behavioral studies reveal that this mutation alone is sufficient to impair cognition, and, when coupled to a mouse model of AD, further accelerates cognitive decline. The druggability of protein kinases positions PKCα as a promising therapeutic target in AD.

Prions induce an early Arc response and a subsequent reduction in mGluR5 in the hippocampus.

Synapse dysfunction and loss are central features of neurodegenerative diseases, caused in part by the accumulation of protein oligomers. Amyloid-ß, tau, prion, and α-synuclein oligomers bind to the cellular prion protein (PrPC), resulting in the activation of macromolecular complexes and signaling at the post-synapse, yet the early signaling events are unclear. Here we sought to determine the early transcript and protein alterations in the hippocampus during the pre-clinical stages of prion disease. We used a transcriptomic approach focused on the early-stage, prion-infected hippocampus of male wild-type mice, and identify immediate early genes, including the synaptic activity response gene, Arc/Arg3.1, as significantly upregulated. In a longitudinal study of male, prion-infected mice, Arc/Arg-3.1 protein was increased early (40% of the incubation period), and by mid-disease (pre-clinical), phosphorylated AMPA receptors (pGluA1-S845) were increased and metabotropic glutamate receptors (mGluR5 dimers) were markedly reduced in the hippocampus. Notably, sporadic Creutzfeldt-Jakob disease (sCJD) post-mortem cortical samples also showed low levels of mGluR5 dimers. Together, these findings suggest that prions trigger an early Arc response, followed by an increase in phosphorylated GluA1 and a reduction in mGluR5 receptors.

Altered Phosphorylation of the Proteasome Subunit Rpt6 Has Minimal Impact on Synaptic Plasticity and Learning.

Dynamic control of protein degradation via the ubiquitin proteasome system (UPS) is thought to play a crucial role in neuronal function and synaptic plasticity. The proteasome subunit Rpt6, an AAA ATPase subunit of the 19S regulatory particle (RP), has emerged as an important site for regulation of 26S proteasome function in neurons. Phosphorylation of Rpt6 on serine 120 (S120) can stimulate the catalytic rate of substrate degradation by the 26S proteasome and this site is targeted by the plasticity-related kinase Ca2+/calmodulin-dependent kinase II (CaMKII), making it an attractive candidate for regulation of proteasome function in neurons. Several in vitro studies have shown that altered Rpt6 S120 phosphorylation can affect the structure and function of synapses. To evaluate the importance of Rpt6 S120 phosphorylation in vivo, we created two mouse models which feature mutations at S120 that block or mimic phosphorylation at this site. We find that peptidase and ATPase activities are upregulated in the phospho-mimetic mutant and downregulated in the phospho-dead mutant [S120 mutated to aspartic acid (S120D) or alanine (S120A), respectively]. Surprisingly, these mutations had no effect on basal synaptic transmission, long-term potentiation (LTP), and dendritic spine dynamics and density in the hippocampus. Furthermore, these mutants displayed no deficits in cued and contextual fear memory. Thus, in a mouse model that blocks or mimics phosphorylation at this site, either compensatory mechanisms negate these effects, or small variations in proteasome activity are not enough to induce significant changes in synaptic structure, plasticity, or behavior.

Biosynthesis of Orthogonal Molecules Using Ferredoxin and Ferredoxin-NADP+ Reductase Systems Enables Genetically Encoded PhyB Optogenetics.

Transplanting metabolic reactions from one species into another has many uses as a research tool with applications ranging from optogenetics to crop production. Ferredoxin (Fd), the enzyme that most often supplies electrons to these reactions, is often overlooked when transplanting enzymes from one species to another because most cells already contain endogenous Fd. However, we have shown that the production of chromophores used in Phytochrome B (PhyB) optogenetics is greatly enhanced in mammalian cells by expressing bacterial and plant Fds with ferredoxin-NADP+ reductases (FNR). We delineated the rate limiting factors and found that the main metabolic precursor, heme, was not the primary limiting factor for producing either the cyanobacterial or plant chromophores, phycocyanobilin or phytochromobilin, respectively. In fact, Fd is limiting, followed by Fd+FNR and finally heme. Using these findings, we optimized the PCB production system and combined it with a tissue penetrating red/far-red sensing PhyB optogenetic gene switch in animal cells. We further characterized this system in several mammalian cell lines using red and far-red light. Importantly, we found that the light-switchable gene system remains active for several hours upon illumination, even with a short light pulse, and requires very small amounts of light for maximal activation. Boosting chromophore production by matching metabolic pathways with specific ferredoxin systems will enable the unparalleled use of the many PhyB optogenetic tools and has broader implications for optimizing synthetic metabolic pathways.

Proteasome phosphorylation regulates cocaine-induced sensitization.

Repeated exposure to cocaine produces structural and functional modifications at synapses from neurons in several brain regions including the nucleus accumbens. These changes are thought to underlie cocaine-induced sensitization. The ubiquitin proteasome system plays a crucial role in the remodeling of synapses and has recently been implicated in addiction-related behavior. The ATPase Rpt6 subunit of the 26S proteasome is phosphorylated by Ca2+/calmodulin-dependent protein kinases II alpha at ser120 which is thought to regulate proteasome activity and distribution in neurons. Here, we demonstrate that Rpt6 phosphorylation is involved in cocaine-induced locomotor sensitization. Cocaine concomitantly increases proteasome activity and Rpt6 S120 phosphorylation in cultured neurons and in various brain regions of wild type mice including the nucleus accumbens and prefrontal cortex. In contrast, cocaine does not increase proteasome activity in Rpt6 phospho-mimetic (ser120Asp) mice. Strikingly, we found a complete absence of cocaine-induced locomotor sensitization in the Rpt6 ser120Asp mice. Together, these findings suggest a critical role for Rpt6 phosphorylation and proteasome function in the regulation cocaine-induced behavioral plasticity.

Guanidinylated Neomycin Conjugation Enhances Intranasal Enzyme Replacement in the Brain.

Iduronidase (IDUA)-deficient mice accumulate glycosaminoglycans in cells and tissues and exhibit many of the same neuropathological symptoms of patients suffering from Mucopolysaccharidosis I. Intravenous enzyme-replacement therapy for Mucopolysaccharidosis I ameliorates glycosaminoglycan storage and many of the somatic aspects of the disease but fails to treat neurological symptoms due to poor transport across the blood-brain barrier. In this study, we examined the delivery of IDUA conjugated to guanidinoneomycin (GNeo), a molecular transporter. GNeo-IDUA and IDUA injected intravenously resulted in reduced hepatic glycosaminoglycan accumulation but had no effect in the brain due to fast clearance from the circulation. In contrast, intranasally administered GNeo-IDUA entered the brain rapidly. Repetitive intranasal treatment with GNeo-IDUA reduced glycosaminoglycan storage, lysosome size and number, and neurodegenerative astrogliosis in the olfactory bulb and primary somatosensory cortex, whereas IDUA was less effective. The enhanced efficacy of GNeo-IDUA was not the result of increased nose-to-brain delivery or enzyme stability, but rather due to more efficient uptake into neurons and astrocytes. GNeo conjugation also enhanced glycosaminoglycan clearance by intranasally delivered sulfamidase to the brain of sulfamidase-deficient mice, a model of Mucopolysaccharidosis IIIA. These findings suggest the general utility of the guanidinoglycoside-based delivery system for restoring missing lysosomal enzymes in the brain.

Phosphorylation of the 19S regulatory particle ATPase subunit, Rpt6, modifies susceptibility to proteotoxic stress and protein aggregation.

The ubiquitin proteasome system (UPS) is a highly conserved and tightly regulated biochemical pathway that degrades the majority of proteins in eukaryotic cells. Importantly, the UPS is responsible for counteracting altered protein homeostasis induced by a variety of proteotoxic stresses. We previously reported that Rpt6, the ATPase subunit of the 19S regulatory particle (RP) of the 26S proteasome, is phosphorylated in mammalian neurons at serine 120 in response to neuronal activity. Furthermore, we found that Rpt6 S120 phosphorylation, which regulates the activity and distribution of proteasomes in neurons, is relevant for proteasome-dependent synaptic remodeling and function. To better understand the role of proteasome phosphorylation, we have constructed models of altered Rpt6 phosphorylation in S. cerevisiae by introducing chromosomal point mutations that prevent or mimic phosphorylation at the conserved serine (S119). We find that mutants which prevent Rpt6 phosphorylation at this site (rpt6-S119A), had increased susceptibility to proteotoxic stress, displayed abnormal morphology and had reduced proteasome activity. Since impaired proteasome function has been linked to the aggregation of toxic proteins including the Huntington's disease (HD) related huntingtin (Htt) protein with expanded polyglutamine repeats, we evaluated the extent of Htt aggregation in our phospho-dead (rpt6-S119A) and phospho-mimetic (rpt6-S119D) mutants. We showed Htt103Q aggregate size to be significantly larger in rpt6-S119A mutants compared to wild-type or rpt6-S119D strains. Furthermore, we observed that phosphorylation of endogenous Rpt6 at S119 is increased in response to various stress conditions. Together, these data suggest that Rpt6 phosphorylation at S119 may play an important function in proteasome-dependent relief of proteotoxic stress that can be critical in protein aggregation pathologies.

Activity-dependent trafficking of lysosomes in dendrites and dendritic spines.

In neurons, lysosomes, which degrade membrane and cytoplasmic components, are thought to primarily reside in somatic and axonal compartments, but there is little understanding of their distribution and function in dendrites. Here, we used conventional and two-photon imaging and electron microscopy to show that lysosomes traffic bidirectionally in dendrites and are present in dendritic spines. We find that lysosome inhibition alters their mobility and also decreases dendritic spine number. Furthermore, perturbing microtubule and actin cytoskeletal dynamics has an inverse relationship on the distribution and motility of lysosomes in dendrites. We also find trafficking of lysosomes is correlated with synaptic α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid-type glutamate receptors. Strikingly, lysosomes traffic to dendritic spines in an activity-dependent manner and can be recruited to individual spines in response to local activation. These data indicate the position of lysosomes is regulated by synaptic activity and thus plays an instructive role in the turnover of synaptic membrane proteins.

Neurodevelopmental Changes in Excitatory Synaptic Structure and Function in the Cerebral Cortex of Sanfilippo Syndrome IIIA Mice.

Sanfilippo syndrome, MPS IIIA-D, results from deficits in lysosomal enzymes that specifically degrade heparan sulfate, a sulfated glycosaminoglycan. The accumulation of heparan sulfate results in neurological symptoms, culminating in extensive neurodegeneration and early death. To study the impact of storage in postnatal neurodevelopment, we examined murine models of MPS IIIA, which lack the enzyme sulfamidase. We show that changes occur in excitatory postsynaptic structure and function in the somatosensory cortex prior to signs of neurodegeneration. These changes coincide with accumulation of heparan sulfate with characteristic non-reducing ends, which is present at birth in the mutant mice. Accumulation of heparan sulfate was also detected in primary cultures of cortical neural cells, especially astrocytes. Accumulation of heparan sulfate in cultured astrocytes corresponded with augmented extracellular heparan sulfate and glypican 4 levels. Heparan sulfate from the cerebral cortex of MPS IIIA mice showed enhanced ability to increase glutamate AMPA receptor subunits at the cell surface of wild type neurons. These data support the idea that abnormalities in heparan sulfate content and distribution contribute to alterations in postsynaptic function. Our findings identify a disease-induced developmental phenotype that temporally overlaps with the onset of behavioral changes in a mouse model of MPS IIIA.

Benzothiazole Amphiphiles Promote the Formation of Dendritic Spines in Primary Hippocampal Neurons.

The majority of excitatory synapses in the brain exist on dendritic spines. Accordingly, the regulation of dendritic spine density in the hippocampus is thought to play a central role in learning and memory. The development of novel methods to control spine density could, therefore, have important implications for treatment of a host of neurodegenerative and developmental cognitive disorders. Herein, we report the design and evaluation of a new class of benzothiazole amphiphiles that exhibit a dose-dependent response leading to an increase in dendritic spine density in primary hippocampal neurons. Cell exposure studies reveal that the increase in spine density can persist for days in the presence of these compounds, but returns to normal spine density levels within 24 h when the compounds are removed, demonstrating the capability to reversibly control spinogenic activity. Time-lapse imaging of dissociated hippocampal neuronal cultures shows that these compounds promote a net increase in spine density through the formation of new spines. Biochemical studies support that promotion of spine formation by these compounds is accompanied by Ras activation. These spinogenic molecules were also capable of inhibiting a suspected mechanism for dendritic spine loss induced by Alzheimer-related aggregated amyloid-ß peptides in primary neurons. Evaluation of this new group of spinogenic agents reveals that they also exhibit relatively low toxicity at concentrations displaying activity. Collectively, these results suggest that small molecules that promote spine formation could be potentially useful for ameliorating cognitive deficiencies associated with spine loss in neurodegenerative diseases such as Alzheimer disease, and may also find use as general cognitive enhancers.

Aß-Induced Synaptic Alterations Require the E3 Ubiquitin Ligase Nedd4-1.

Alzheimer's disease (AD) is a neurodegenerative disease in which patients experience progressive cognitive decline. A wealth of evidence suggests that this cognitive impairment results from synaptic dysfunction in affected brain regions caused by cleavage of amyloid precursor protein into the pathogenic peptide amyloid-ß (Aß). Specifically, it has been shown that Aß decreases surface AMPARs, dendritic spine density, and synaptic strength, and also alters synaptic plasticity. The precise molecular mechanisms by which this occurs remain unclear. Here we demonstrate a role for ubiquitination in Aß-induced synaptic dysfunction in cultured rat neurons. We find that Aß promotes the ubiquitination of AMPARs, as well as the redistribution and recruitment of Nedd4-1, a HECT E3 ubiquitin ligase we previously demonstrated to target AMPARs for ubiquitination and degradation. Strikingly, we show that Nedd4-1 is required for Aß-induced reductions in surface AMPARs, synaptic strength, and dendritic spine density. Our findings, therefore, indicate an important role for Nedd4-1 and ubiquitin in the synaptic alterations induced by Aß. SIGNIFICANCE STATEMENT: Synaptic changes in Alzheimer's disease (AD) include surface AMPAR loss, which can weaken synapses. In a cell culture model of AD, we found that AMPAR loss correlates with increased AMPAR ubiquitination. In addition, the ubiquitin ligase Nedd4-1, known to ubiquitinate AMPARs, is recruited to synapses in response to Aß. Strikingly, reducing Nedd4-1 levels in this model prevented surface AMPAR loss and synaptic weakening. These findings suggest that, in AD, Nedd4-1 may ubiquitinate AMPARs to promote their internalization and weaken synaptic strength, similar to what occurs in Nedd4-1's established role in homeostatic synaptic scaling. This is the first demonstration of Aß-mediated control of a ubiquitin ligase to regulate surface AMPAR expression.

Ubiquitin-dependent trafficking and turnover of ionotropic glutamate receptors.

Changes in synaptic strength underlie the basis of learning and memory and are controlled, in part, by the insertion or removal of AMPA-type glutamate receptors at the postsynaptic membrane of excitatory synapses. Once internalized, these receptors may be recycled back to the plasma membrane by subunit-specific interactions with other proteins or by post-translational modifications such as phosphorylation. Alternatively, these receptors may be targeted for destruction by multiple degradation pathways in the cell. Ubiquitination, another post-translational modification, has recently emerged as a key signal that regulates the recycling and trafficking of glutamate receptors. In this review, we will discuss recent findings on the role of ubiquitination in the trafficking and turnover of ionotropic glutamate receptors and plasticity of excitatory synapses.

Synaptic structure and function are altered by the neddylation inhibitor MLN4924.

The posttranslational modification of proteins by the ubiquitin-like small molecule NEDD8 has previously been shown to be vital in a number of cell signaling pathways. In particular, conjugation of NEDD8 (neddylation) serves to regulate protein ubiquitination through modifications to E3 ubiquitin ligases. Despite the prevalence of NEDD8 in neurons, very little work has been done to characterize the role of this modifier in these cells. Here, we use the recently developed NEDD8 Activating Enzyme (NAE) inhibitor MLN4924 and report evidence of a role for NEDD8 in regulating mammalian excitatory synapses. Application of this drug to dissociated rat hippocampal neurons caused reductions in synaptic strength, surface glutamate receptor levels, dendritic spine width, and spine density, suggesting that neddylation is involved in the maintenance of synapses.

Synaptic strength is bidirectionally controlled by opposing activity-dependent regulation of Nedd4-1 and USP8.

The trafficking of AMPA receptors (AMPARs) to and from synapses is crucial for synaptic plasticity. Previous work has demonstrated that AMPARs undergo activity-dependent ubiquitination by the E3 ubiquitin ligase Nedd4-1, which promotes their internalization and degradation in lysosomes. Here, we define the molecular mechanisms involved in ubiquitination and deubiquitination of AMPARs. We report that Nedd4-1 is rapidly redistributed to dendritic spines in response to AMPAR activation and not in response to NMDA receptor (NMDAR) activation in cultured rat neurons. In contrast, NMDAR activation directly antagonizes Nedd4-1 function by promoting the deubiquitination of AMPARs. We show that NMDAR activation causes the rapid dephosphorylation and activation of the deubiquitinating enzyme (DUB) USP8. Surface AMPAR levels and synaptic strength are inversely regulated by Nedd4-1 and USP8. Strikingly, we show that homeostatic downscaling of synaptic strength is accompanied by an increase and decrease in Nedd4-1 and USP8 protein levels, respectively. Furthermore, we show that Nedd4-1 is required for homeostatic loss of surface AMPARs and downscaling of synaptic strength. This study provides the first mechanistic evidence for rapid and opposing activity-dependent control of a ubiquitin ligase and DUB at mammalian CNS synapses. We propose that the dynamic regulation of these opposing forces is critical in maintaining synapses and scaling them during homeostatic plasticity.

Activity-dependent growth of new dendritic spines is regulated by the proteasome.

Growth of new dendritic spines contributes to experience-dependent circuit plasticity in the cerebral cortex. Yet the signaling mechanisms leading to new spine outgrowth remain poorly defined. Increasing evidence supports that the proteasome is an important mediator of activity-dependent neuronal signaling. We therefore tested the role of the proteasome in activity-dependent spinogenesis. Using pharmacological manipulations, glutamate uncaging, and two-photon imaging of GFP-transfected hippocampal pyramidal neurons, we demonstrate that acute inhibition of the proteasome blocks activity-induced spine outgrowth. Remarkably, mutation of serine 120 to alanine of the Rpt6 proteasomal subunit in individual neurons was sufficient to block activity-induced spine outgrowth. Signaling through NMDA receptors and CaMKII, but not PKA, is required to facilitate spine outgrowth. Moreover, abrogating CaMKII binding to the NMDA receptor abolished activity-induced spinogenesis. Our data support a model in which neural activity facilitates spine outgrowth via an NMDA receptor- and CaMKII-dependent increase in local proteasomal degradation.

Phosphorylation of Rpt6 regulates synaptic strength in hippocampal neurons.

It has become increasingly evident that protein degradation via the ubiquitin proteasome system plays a fundamental role in the development, maintenance and remodeling of synaptic connections in the CNS. We and others have recently described the activity-dependent regulation of proteasome activity and recruitment of proteasomes into spine compartments involving the phosphorylation of the 19S ATPase subunit, Rpt6, by the plasticity kinase Ca(2+)/calmodulin-dependent protein kinase II α (CaMKIIα) (Bingol and Schuman, 2006; Djakovic et al., 2009; Bingol et al, 2010). Here, we investigated the role of Rpt6 phosphorylation on proteasome function and synaptic strength. Utilizing a phospho-specific antibody we verified that Rpt6 is phosphorylated at Serine 120 (S120) by CaMKIIα. In addition, we found that Rpt6 is phosphorylated by CaMKIIα in an activity-dependent manner. Furthermore, we showed that a serine 120 to aspartic acid phospho-mimetic mutant of Rpt6 (S120D) increases its resistance to detergent extraction in rat hippocampal dendrites, indicating phosphorylated Rpt6 may promote the tethering of proteasomes to scaffolds and cytoskeletal components. Expression of Rpt6 S120D decreased miniature EPSC (mEPSC) amplitude, while expression of a phospho-dead mutant (S120A) increased mEPSC amplitude. Surprisingly, homeostatic scaling of mEPSC amplitude produced by chronic application of bicuculline or tetrodotoxin is both mimicked and occluded by altered Rpt6 phosphorylation. Together, these data suggest that CaMKII-dependent phosphorylation of Rpt6 at S120 may be an important regulatory mechanism for proteasome-dependent control of synaptic remodeling in slow homeostatic plasticity.

Differential effects of UCHL1 modulation on alpha-synuclein in PD-like models of alpha-synucleinopathy.

Parkinson's disease (PD) is a progressive neurodegenerative disorder caused by genetic and environmental factors. Abnormal accumulation and aggregation of alpha-synuclein (a-syn) within neurons, and mutations in the a-syn and UCH-L1 genes have been shown to play a role in the pathogenesis of PD. In light of recent reports suggesting an interaction between a-synuclein and UCH-L1, we investigated the effects of UCH-L1 inhibition on a-syn distribution and expression levels in primary neurons and hippocampal tissues derived from non transgenic (non tg) and a-syn over expressing tg mice. We show that suppression of UCH-L1 activity increased a-syn levels in control, non tg neurons, and resulted in a concomitant accumulation of presynaptic a-syn in these neurons. In contrast, blocking UCH-L1 activity in a-syn over expressing neurons decreased a-syn levels, and enhanced its synaptic clearance. In vitro studies verified the LDN-induced inhibition of UCH-L1 had minimal effect on LC3 (a marker of autophagy) in control cells, in cells over expressing a-syn UCH-L1 inhibition resulted in increased LC3 activity. These findings suggest a possible differential role of UCH-L1 function under normal and pathological conditions. Furthermore, in the context of a-syn-induced pathology, modulation of UCH-L1 activity could serve as a therapeutic tool to enhance the autophagy pathway and induce clearance of the observed accumulated/aggregated a-syn species in the PD brain.

Ubiquitin-dependent endocytosis, trafficking and turnover of neuronal membrane proteins.

Extracellular signaling between cells is often transduced via receptors that reside at the cell membrane. In neurons this receptor-mediated signaling can promote a variety of cellular events such as differentiation, axon outgrowth and guidance, and synaptic development and function. Endocytic membrane trafficking of receptors ensures that the strength and duration of an extracellular signal is properly regulated. The covalent modification of membrane proteins by ubiquitin is a key biological mechanism controlling receptor internalization and endocytic sorting to recycling and degradative pathways in many cell types. In this review we highlight recent findings regarding the ubiquitin-dependent trafficking and turnover of receptors in neurons and the implications for neuronal development and function.