Ubiquitin-specific protease 14 (USP14) is one of three proteasome-associated deubiquitinating enzymes that remove ubiquitin from proteasomal substrates prior to their degradation. In vitro evidence suggests that inhibiting USP14's catalytic activity alters the turnover of ubiquitinated proteins by the proteasome, although whether protein degradation is accelerated or delayed seems to be cell-type and substrate specific. For example, combined inhibition of USP14 and the proteasomal deubiquitinating enzyme UCH37 halts protein degradation and promotes apoptosis in multiple myeloma cells, whereas USP14 inhibition alone accelerates the degradation of aggregate-prone proteins in immortalized cell lines. These findings have prompted interest in USP14 as a therapeutic target both inside and outside of the nervous system. However, loss of USP14 in the spontaneously occurring ataxia mouse mutant leads to a dramatic neuromuscular phenotype and early perinatal lethality, suggesting that USP14 inhibition may have adverse consequences in the nervous system. We therefore expressed a catalytically inactive USP14 mutant in the mouse nervous system to determine whether USP14's catalytic activity is required for neuromuscular junction (NMJ) structure and function.

Regulated protein degradation by the proteasome plays an essential role in the enhancement and suppression of signaling pathways in the nervous system. Proteasome-associated factors are pivotal in ensuring appropriate protein degradation, and we have previously demonstrated that alterations in one of these factors, the proteasomal deubiquitinating enzyme ubiquitin-specific protease 14 (Usp14), can lead to proteasome dysfunction and neurological disease. Recent studies in cell culture have shown that Usp14 can also stabilize the expression of over-expressed, disease-associated proteins such as tau and ataxin-3. Using Usp14-deficient ax(J) mice, we investigated if loss of Usp14 results in decreased levels of endogenous tau and ataxin-3 in the nervous system of mice. Although loss of Usp14 did not alter the overall neuronal levels of tau and ataxin-3, we found increased levels of phosphorylated tau that correlated with the onset of axonal varicosities in the Usp14-deficient mice. These changes in tau phosphorylation were accompanied by increased levels of activated phospho-Akt, phosphorylated MAPKs, and inactivated phospho-GSK3β. However, genetic ablation of tau did not alter any of the neurological deficits in the Usp14-deficient mice, demonstrating that increased levels of phosphorylated tau do not necessarily lead to neurological disease. Due to the widespread activation of intracellular signaling pathways induced by the loss of Usp14, a better understanding of the cellular pathways regulated by the proteasome is required before effective proteasomal-based therapies can be used to treat chronic neurological diseases.

Ataxia represents a pathological coordination failure that often involves functional disturbances in cerebellar circuits. Purkinje cells (PCs) characterize the only output neurons of the cerebellar cortex and critically participate in regulating motor coordination. Although different genetic mutations are known that cause ataxia, little is known about the underlying cellular mechanisms. Here we show that a mutated ax(J) gene locus, encoding the ubiquitin-specific protease 14 (Usp14), negatively influences synaptic receptor turnover. Ax(J) mouse mutants, characterized by cerebellar ataxia, display both increased GABA(A) receptor (GABA(A)R) levels at PC surface membranes accompanied by enlarged IPSCs. Accordingly, we identify physical interaction of Usp14 and the GABA(A)R alpha1 subunit. Although other currently unknown changes might be involved, our data show that ubiquitin-dependent GABA(A)R turnover at cerebellar synapses contributes to ax(J)-mediated behavioural impairment.

Endosomal sorting plays a fundamental role in directing neural development. By altering the temporal and spatial distribution of membrane receptors, endosomes regulate signaling pathways that control the differentiation and function of neural cells. Several genes linked to inherited demyelinating peripheral neuropathies, known as Charcot-Marie-Tooth (CMT) disease, encode proteins that directly interact with components of the endosomal sorting complex required for transport (ESCRT). Our previous studies demonstrated that a point mutation in the ESCRT component hepatocyte growth-factor-regulated tyrosine kinase substrate (HGS), an endosomal scaffolding protein that identifies internalized cargo to be sorted by the endosome, causes a peripheral neuropathy in the neurodevelopmentally impaired teetering mice. Here, we constructed a Schwann cell-specific deletion of Hgs to determine the role of endosomal sorting during myelination. Inactivation of HGS in Schwann cells resulted in motor and sensory deficits, slowed nerve conduction velocities, delayed myelination and hypomyelinated axons, all of which occur in demyelinating forms of CMT. Consistent with a delay in Schwann cell maturation, HGS-deficient sciatic nerves displayed increased mRNA levels for several promyelinating genes and decreased mRNA levels for genes that serve as markers of myelinating Schwann cells. Loss of HGS also altered the abundance and activation of the ERBB2/3 receptors, which are essential for Schwann cell development. We therefore hypothesize that HGS plays a critical role in endosomal sorting of the ERBB2/3 receptors during Schwann cell maturation, which further implicates endosomal dysfunction in inherited peripheral neuropathies.SIGNIFICANCE STATEMENT Schwann cells myelinate peripheral axons, and defects in Schwann cell function cause inherited demyelinating peripheral neuropathies known as CMT. Although many CMT-linked mutations are in genes that encode putative endosomal proteins, little is known about the requirements of endosomal sorting during myelination. In this study, we demonstrate that loss of HGS disrupts the endosomal sorting pathway in Schwann cells, resulting in hypomyelination, aberrant myelin sheaths, and impairment of the ERBB2/3 receptor pathway. These findings suggest that defective endosomal trafficking of internalized cell surface receptors may be a common mechanism contributing to demyelinating CMT.

Ubiquitin-specific protease 14 (USP14) is a major deubiquitinating enzyme and a key determinant of neuromuscular junction (NMJ) structure and function. We have previously reported dramatic ubiquitin depletion in the nervous systems of the USP14-deficient ataxia (ax (J) ) mice and demonstrated that transgenic ubiquitin overexpression partially rescues the ax (J) neuromuscular phenotype. However, later work has shown that ubiquitin overexpression does not correct the ax (J) deficits in hippocampal short term plasticity, and that transgenic expression of a catalytically inactive form of USP14 in the nervous system mimics the neuromuscular phenotype observed in the ax (J) mice, but causes a only a modest reduction of free ubiquitin. Instead, increased ubiquitin conjugates and aberrant activation of pJNK are observed in the nervous systems of the USP14 catalytic mutant mice. In this report, we demonstrate that restoring free ubiquitin levels in the USP14 catalytic mutant mice improved NMJ structure and reduced pJNK accumulation in motor neuron terminals, but had a negative impact on measures of NMJ function, such as motor performance and muscle development. Transgenic expression of ubiquitin had a dose-dependent effect on NMJ function in wild type mice: moderate levels of overexpression improved NMJ function while more robust ubiquitin overexpression reduced muscle development and motor coordination. Combined, these results suggest that maintenance of free ubiquitin levels by USP14 contributes to NMJ structure, but that USP14 regulates NMJ function through a separate pathway.

Neurons are particularly vulnerable to perturbations in endo-lysosomal transport, as several neurological disorders are caused by a primary deficit in this pathway. In this report, we used positional cloning to show that the spontaneously occurring neurological mutation teetering (tn) is a single nucleotide substitution in hepatocyte growth factor-regulated tyrosine kinase substrate (Hgs/Hrs), a component of the endosomal sorting complex required for transport (ESCRT). The tn mice exhibit hypokenesis, muscle weakness, reduced muscle size and early perinatal lethality by 5-weeks of age. Although HGS has been suggested to be essential for the sorting of ubiquitinated membrane proteins to the lysosome, there were no alterations in receptor tyrosine kinase levels in the central nervous system, and only a modest decrease in tropomyosin receptor kinase B (TrkB) in the sciatic nerves of the tn mice. Instead, loss of HGS resulted in structural alterations at the neuromuscular junction (NMJ), including swellings and ultra-terminal sprouting at motor axon terminals and an increase in the number of endosomes and multivesicular bodies. These structural changes were accompanied by a reduction in spontaneous and evoked release of acetylcholine, indicating a deficit in neurotransmitter release at the NMJ. These deficits in synaptic transmission were associated with elevated levels of ubiquitinated proteins in the synaptosome fraction. In addition to the deficits in neuronal function, mutation of Hgs resulted in both hypermyelinated and dysmyelinated axons in the tn mice, which supports a growing body of evidence that ESCRTs are required for proper myelination of peripheral nerves. Our results indicate that HGS has multiple roles in the nervous system and demonstrate a previously unanticipated requirement for ESCRTs in the maintenance of synaptic transmission.

Proteasomes, the primary mediators of ubiquitin-protein conjugate degradation, are regulated through complex and poorly understood mechanisms. Here we show that USP14, a proteasome-associated deubiquitinating enzyme, can inhibit the degradation of ubiquitin-protein conjugates both in vitro and in cells. A catalytically inactive variant of USP14 has reduced inhibitory activity, indicating that inhibition is mediated by trimming of the ubiquitin chain on the substrate. A high-throughput screen identified a selective small-molecule inhibitor of the deubiquitinating activity of human USP14. Treatment of cultured cells with this compound enhanced degradation of several proteasome substrates that have been implicated in neurodegenerative disease. USP14 inhibition accelerated the degradation of oxidized proteins and enhanced resistance to oxidative stress. Enhancement of proteasome activity through inhibition of USP14 may offer a strategy to reduce the levels of aberrant proteins in cells under proteotoxic stress.

In this study, we identified and characterized an N-ethyl-N-nitrosourea (ENU) induced mutation in Usp14 (nmf375) that leads to adult-onset neurological disease. The nmf375 mutation causes aberrant splicing of Usp14 mRNA, resulting in a 95% reduction in USP14. We previously showed that loss of USP14 in ataxia (ax (J)) mice results in reduced ubiquitin levels, motor endplate disease, Purkinje cell axonal dystrophy and decreased hippocampal paired pulse facilitation (PPF) during the first 4-6 weeks of life, and early postnatal lethality by two months of age. Although the loss of USP14 is comparable between the nmf375 and ax (J) mice, the nmf375 mice did not exhibit these ax (J) developmental abnormalities. However, by 12 weeks of age the nmf375 mutants present with ubiquitin depletion and motor endplate disease, indicating a continual role for USP14-mediated regulation of ubiquitin pools and neuromuscular junction (NMJ) structure in adult mice. The observation that motor endplate disease was only seen after ubiquitin depletion suggests that the preservation of NMJ structure requires the stable maintenance of synaptic ubiquitin pools. Differences in genetic background were shown to affect ubiquitin expression and dramatically alter the phenotypes caused by USP14 deficiency.

Strategies for enhancing protein degradation have been proposed for treating neurological diseases associated with a decline in proteasome activity. A proteasomal deubiquitinating enzyme that controls substrate entry into proteasomes, ubiquitin-specific protease 14 (USP14), is an attractive candidate for therapies that modulate proteasome activity. This report tests the validity of genetic and pharmacological tools to study USP14's role in regulating protein abundance. Although previous studies implicated USP14 in the degradation of microtubule associate protein tau, tar DNA binding protein, and prion protein, the levels of these proteins were similar in our neurons cultured from wild type and USP14-deficient mice. Neither loss nor over-expression of USP14 affected the levels of these proteins in mice, implying that modifying the amount of USP14 is not sufficient to alter their steady-state levels. However, neuronal over-expression of a catalytic mutant of USP14 showed that manipulating USP14's ubiquitin-hydrolase activity altered the levels of specific proteins in vivo. Although pharmacological inhibitors of USP14's ubiquitin-hydrolase activity reduced microtubule associate protein tau, tar DNA binding protein, and prion protein in culture, the effect was similar in wild type and USP14-deficient neurons, thus impacting their use for specifically evaluating USP14 in a therapeutic manner. While examining how targeting USP14 may affect other proteins in vivo, this report showed that fatty acid synthase, v-rel reticuloendotheliosis viral oncogene homolog, CTNNB1, and synaptosome associated protein 23 are reduced in USP14-deficient mice; however, loss of USP14 differentially altered the levels of these proteins in the liver and brain. As such, it is critical to more thoroughly examine how inhibiting USP14 alters protein abundance to determine if targeting USP14 will be a beneficial strategy for treating neurodegenerative diseases.

Turnover of synaptic vesicle (SV) proteins is vital for the maintenance of healthy and functional synapses. SV protein turnover is driven by neuronal activity in an endosomal sorting complex required for transport (ESCRT)-dependent manner. Here, we characterize a critical step in this process: axonal transport of ESCRT-0 component Hrs, necessary for sorting proteins into the ESCRT pathway and recruiting downstream ESCRT machinery to catalyze multivesicular body (MVB) formation. We find that neuronal activity stimulates the formation of presynaptic endosomes and MVBs, as well as the motility of Hrs+ vesicles in axons and their delivery to SV pools. Hrs+ vesicles co-transport ESCRT-0 component STAM1 and comprise a subset of Rab5+ vesicles, likely representing pro-degradative early endosomes. Furthermore, we identify kinesin motor protein KIF13A as essential for the activity-dependent transport of Hrs to SV pools and the degradation of SV membrane proteins. Together, these data demonstrate a novel activity- and KIF13A-dependent mechanism for mobilizing axonal transport of ESCRT machinery to facilitate the degradation of SV membrane proteins.

Ubiquitin is an essential signaling protein that controls many different cellular processes. While cellular ubiquitin levels normally cycle between pools of free and conjugated ubiquitin, the balance of these ubiquitin pools can be shifted by exposure to a variety of cellular stresses. Altered ubiquitin pools are also observed in several neurological disorders, suggesting that imbalances in ubiquitin homeostasis may contribute to neuronal dysfunction. To examine the effects of increased ubiquitin levels on the mammalian nervous system, we generated transgenic mice that express ubiquitin under the control of the Thy1.2 promoter. While we did not detect global changes in levels of ubiquitin conjugates in the hippocampus, we found that increasing ubiquitin levels reduced AMPA (GRIA1-4) receptor expression without affecting the levels of NMDA (GRIN) or GABAA receptors. Ubiquitin over-expression also negatively impacted hippocampus-dependent learning and memory as well as baseline excitability and synaptic plasticity at hippocampal CA3-CA1 synapses. These changes occurred in a dose-dependent manner in that mice with the highest levels of ubiquitin over-expression had the greatest deficits in synaptic function and were the most impaired in the learning and memory tasks. As chronic elevation of ubiquitin expression in neurons is sufficient to cause changes in synaptic function and cognition, altered ubiquitin homeostasis may be an important contributor to the stress-induced changes observed in neurological disorders.