Scott M. Wilson

Enhancement of proteasome activity by a small-molecule inhibitor of USP14

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.

Dystonia and cerebellar atrophy in Cacna1a null mice lacking P/Q calcium channel activity

P/Q‐type voltage‐dependent calcium channel CACNA1A mutations cause dominantly inherited migraine, episodic ataxia, and cerebellar atrophy in humans and cause recessively inherited ataxia, episodic dyskinesia, cerebellar atrophy, and absence epilepsy in mice. The basis of these species differences and the disease mechanism(s) are not understood. To address this question and to identify required P/Q function in vivo, we created a germline Cacna1a null mutation (designated Cacna1a Fcrtm1) by gene targeting. Null mice develop dystonia and late‐onset cerebellar degeneration in a specific pattern. This indicates a requirement for P/Q function for survival in a subset of cerebellar neurons. Homozygous null mice completely lack P/Q‐type channel activity, and they also lack ω‐CTx‐MVIIC receptors, indicating that a single gene encodes P/Q channel activity. An increase of L‐and N‐type current densities is detected in P/Q‐null granule cells. Heterozygous Cacna1a Fcrtm1/+ mice are phenotypically normal, despite having a 50% reduction in current density, indicating that reduced current density is not itself sufficient to cause the pathophysiology of spontaneous mouse mutants with ataxia and seizures.

Lysosomal enzyme cathepsin D protects against alpha-synuclein aggregation and toxicity

Abstractα-synuclein (α-syn) is a main component of Lewy bodies (LB) that occur in many neurodegenerative diseases, including Parkinsons disease (PD), dementia with LB (DLB) and multi-system atrophy. α-syn mutations or amplifications are responsible for a subset of autosomal dominant familial PD cases, and overexpression causes neurodegeneration and motor disturbances in animals. To investigate mechanisms for α-syn accumulation and toxicity, we studied a mouse model of lysosomal enzyme cathepsin D (CD) deficiency, and found extensive accumulation of endogenous α-syn in neurons without overabundance of α-syn mRNA. In addition to impaired macroautophagy, CD deficiency reduced proteasome activity, suggesting an essential role for lysosomal CD function in regulating multiple proteolytic pathways that are important for α-syn metabolism. Conversely, CD overexpression reduces α-syn aggregation and is neuroprotective against α-syn overexpression-induced cell death in vitro. In a C. elegans model, CD deficiency exacerbates α-syn accumulation while its overexpression is protective against α-syn-induced dopaminergic neurodegeneration. Mutated CD with diminished enzymatic activity or overexpression of cathepsins B (CB) or L (CL) is not protective in the worm model, indicating a unique requirement for enzymatically active CD. Our data identify a conserved CD function in α-syn degradation and identify CD as a novel target for LB disease therapeutics.

Loss of Usp14 results in reduced levels of ubiquitin in ataxia mice

The ataxia (axJ) mutation is a spontaneous recessive mutation that results in reduced expression of ubiquitin‐specific protease 14, Usp14. Mice homozygous for the axJ mutation are retarded for growth and exhibit several behavioral disorders, including a resting tremor and hindlimb paralysis. Although pathological defects appear to be limited to the central nervous system, reduction of Usp14 expression was widespread in the axJ mice. Usp14 co‐fractionated with proteasomes isolated from livers and brains of wild‐type mice. Proteasomes isolated from the axJ brains still possessed deubiquitinating activity and were functionally competent to hydrolyze 20S proteasomal substrates in vitro. However, the levels of monomeric ubiquitin were reduced approximately 35% in most of the axJ tissues examined. These results indicate that Usp14 functions to maintain the cellular levels of monomeric ubiquitin in mammalian cells, and that alterations in the levels of ubiquitin may contribute to neurological disease.

Regulation of Synaptic Structure by Ubiquitin C-Terminal Hydrolase L1

Ubiquitin C-terminal hydrolase L1 (UCH-L1) is a deubiquitinating enzyme that is selectively and abundantly expressed in the brain, and its activity is required for normal synaptic function. Here, we show that UCH-L1 functions in maintaining normal synaptic structure in hippocampal neurons. We found that UCH-L1 activity is rapidly upregulated by NMDA receptor activation, which leads to an increase in the levels of free monomeric ubiquitin. Conversely, pharmacological inhibition of UCH-L1 significantly reduces monomeric ubiquitin levels and causes dramatic alterations in synaptic protein distribution and spine morphology. Inhibition of UCH-L1 activity increases spine size while decreasing spine density. Furthermore, there is a concomitant increase in the size of presynaptic and postsynaptic protein clusters. Interestingly, however, ectopic expression of ubiquitin restores normal synaptic structure in UCH-L1-inhibited neurons. These findings point to a significant role of UCH-L1 in synaptic remodeling, most likely by modulating free monomeric ubiquitin levels in an activity-dependent manner.

The Proteasome-Associated Deubiquitinating Enzyme Usp14 Is Essential for the Maintenance of Synaptic Ubiquitin Levels and the Development of Neuromuscular Junctions

Dysfunction of the ubiquitin proteasome system (UPS) has been implicated in the pathogenesis of many neurological diseases, including Alzheimers, spinocerebellar ataxia, and several motor neuron diseases. Recent research indicates that changes in synaptic transmission may play a critical role in the progression of neurological disease; however, the mechanisms by which the UPS regulates synaptic structure and function have not been well characterized. In this report, we show that Usp14 is indispensable for synaptic development and function at neuromuscular junctions (NMJs). Usp14-deficient axJ mice display a resting tremor, a reduction in muscle mass, and notable hindlimb rigidity without any detectable loss of motor neurons. Instead, loss of Usp14 causes developmental defects at motor neuron endplates. Presynaptic defects include phosphorylated neurofilament accumulations, nerve terminal sprouting, and poor arborization of the motor nerve terminals, whereas postsynaptic acetylcholine receptors display immature plaque-like morphology. These structural changes in the NMJ correlated with ubiquitin loss in the spinal cord and sciatic nerve. Further studies demonstrated that the greatest loss of ubiquitin was found in synaptosomal fractions, suggesting that the endplate swellings may be caused by decreased protein turnover at the synapse. Transgenic restoration of Usp14 in the nervous system corrected the levels of monomeric ubiquitin in the motor neuron circuit and the defects that were observed in the motor endplates and muscles of the axJ mice. These data define a critical role for Usp14 at mammalian synapses and suggest a requirement for local ubiquitin recycling by the proteasome to control the development and function of NMJs.

El Nino drives timing of breeding but not population growth in the song sparrow (Melospiza melodia).

Increasing evidence suggests that climate change affects the timing of breeding in birds, but there is less evidence to show how such changes affect the population dynamics of birds overall. Over the past 43 years, song sparrows (Melospiza melodia) on Mandarte Island, British Columbia, Canada have not shown an advance in breeding date in response to global warming. However, this population did show considerable annual variation in timing of breeding correlated with the El Niño Southern Oscillation. Birds bred earlier in warmer El Niño years and later in colder La Niña years. Early breeding strongly increased reproductive output. However, annual variation in timing of breeding had little effect on population growth, perhaps because the population is strongly regulated by the rate of recruitment by juveniles. The juvenile recruitment rate declined with increasing population density but showed little response to climate. These findings suggest that populations will vary in response to climate change depending on how climate affects the demographic parameters that contribute most to population growth.

Transgenic Rescue of ataxia Mice with Neuronal-Specific Expression of Ubiquitin-Specific Protease 14

The ataxia mutation (axJ) is a recessive neurological mutation that results in reduced growth, ataxia, and hindlimb muscle wasting in mice. The axJ gene encodes ubiquitin-specific protease 14 (Usp14), a deubiquitinating enzyme (DUB) that associates with the proteasome via its ubiquitin-like (Ubl) domain and is involved in processing ubiquitin chains. Analysis of Usp14 gene products demonstrated that Usp14 undergoes alternative pre-mRNA splicing to produce a full-length form of Usp14 that is capable of binding proteasomes and a form that contains a deletion in the Ubl domain. The full-length form of Usp14 is the only form that appears to be reduced in the axJ mice. Transgenic rescue of the axJ mice with neuronal-specific expression of Usp14 demonstrated that the full-length form of Usp14 was sufficient to restore viability and motor system function to the axJ mice. Biochemical analysis showed that the ubiquitin hydrolyase activity of this form of Usp14 is dependent on the presence of proteasomes, and neuronal expression of full-length Usp14 was able to restore the levels of monomeric ubiquitin in the brains of axJ mice. However, the axJ-rescued mice still displayed the Purkinje cell axonal swellings that are seen in the axJ mice, indicating that this cerebellar alteration is not the primary cause of the axJ movement disorders. These results show that the motor defects observed in the axJ mice are attributable to a neuropathic disease rather than to a muscular disorder and suggest that changes in proteasomal function may contribute to neurological dysfunction in the axJ mice.

Ubiquitin Homeostasis Is Critical for Synaptic Development and Function

The ubiquitin-proteasome system (UPS) controls protein abundance and is essential for many aspects of neuronal function. In ataxia (axJ) mice, profound neurological and synaptic defects result from a loss-of-function mutation in the proteasome-associated deubiquitinating enzyme Usp14, which is required for recycling ubiquitin from proteasomal substrates. Here, we show that transgenic complementation of axJ mice with neuronally expressed ubiquitin prevents early postnatal lethality, restores muscle mass, and corrects developmental and functional deficits resulting from the loss of Usp14, demonstrating that ubiquitin deficiency is a major cause of the neurological defects observed in the axJ mice. We also show that proteasome components are normally induced during the first 2 weeks of postnatal development, which coincides with dramatic alterations in polyubiquitin chain formation. These data demonstrate a critical role for ubiquitin homeostasis in synaptic development and function, and show that ubiquitin deficiency may contribute to diseases characterized by synaptic dysfunction.

Differential effects of Usp14 and Uch-L1 on the ubiquitin proteasome system and synaptic activity.

The ubiquitin proteasome pathway has been implicated in the pathogenesis of many neurodegenerative diseases, and alterations in two different deubiquitinating enzymes, Uch-L1 and Usp14, result in neurological phenotypes in mice. We identified a new mutation in Uch-L1 and compared the roles of Uch-L1 and Usp14 in the ubiquitin proteasome system. Deficiencies in either Uch-L1 or Usp14 result in decreased levels of ubiquitin, suggesting that they both regulate ubiquitin stability in the nervous system. However, the effect of ubiquitin depletion on viability and onset of symptoms is more severe in the Usp14-deficient mice, and changes in hippocampal synaptic transmission were only observed in Usp14-deficient mice. In addition, while Usp14 appears to function at the proteasome, Uch-L1 deficiency resulted in up-regulation of lysosomal components, indicating that Uch-L1 and Usp14 may differentially affect the ubiquitin proteasome system and synaptic activity by regulating different pools of ubiquitin in the cell.