RNA-based drug discovery for spinal muscular atrophy: a story of small molecules and antisense oligonucleotides.

INTRODUCTION: Spinal Muscular Atrophy (SMA), the second most prevalent autosomal genetic disease affecting infants, is caused by the lack of SMN1, which encodes a neuron functioning vital protein, SMN. Improving exon 7 splicing in the paralogous gene SMN2, also coding for SMN protein, increases protein production efficiency from SMN2 to overcome the genetic deficit in SMN1. Several molecular mechanisms have been investigated to improve SMN2 functional splicing. AREAS COVERED: This manuscript will cover two of the three mechanistically distinct available treatment options for SMA, both targeting the SMN2 splicing mechanism. The first therapeutic, nusinersen (Spinraza®, 2017), is an antisense oligonucleotide (ASO) targeting the splicing inhibitory sequence in the intron downstream of exon 7 from SMN2, thus increasing exon 7 inclusion. The second drug is a small molecule, risdiplam (Evrysdi®, 2021), that enhances the binding of splice factors and also promotes exon 7 inclusion. Both therapies, albeit through different mechanisms, increase full-length SMN protein expression. EXPERT OPINION: Nusinersen and risdiplam have directly helped SMA patients and families, but they also herald a sea change in drug development for genetic diseases. This piece aims to draw parallels between both development histories; this may help chart the course for future targeted agents.

Development of mAb-based polyglutamine-dependent and polyglutamine length-independent huntingtin quantification assays with cross-site validation.

Huntington's disease (HD) is caused by an expansion of the CAG trinucleotide repeat domain in the huntingtin gene that results in expression of a mutant huntingtin protein (mHTT) containing an expanded polyglutamine tract in the amino terminus. A number of therapeutic approaches that aim to reduce mHTT expression either locally in the CNS or systemically are in clinical development. We have previously described sensitive and selective assays that measure human HTT proteins either in a polyglutamine-independent (detecting both mutant expanded and non-expanded proteins) or in a polyglutamine length-dependent manner (detecting the disease-causing polyglutamine repeats) on the electrochemiluminescence Meso Scale Discovery detection platform. These original assays relied upon polyclonal antibodies. To ensure an accessible and sustainable resource for the HD field, we developed similar assays employing monoclonal antibodies. We demonstrate that these assays have equivalent sensitivity compared to our previous assays through the evaluation of cellular and animal model systems, as well as HD patient biosamples. We also demonstrate cross-site validation of these assays, allowing direct comparison of studies performed in geographically distinct laboratories.

Optimization of Potent and Selective Ataxia Telangiectasia-Mutated Inhibitors Suitable for a Proof-of-Concept Study in Huntington's Disease Models.

Genetic and pharmacological evidence indicates that the reduction of ataxia telangiectasia-mutated (ATM) kinase activity can ameliorate mutant huntingtin (mHTT) toxicity in cellular and animal models of Huntington's disease (HD), suggesting that selective inhibition of ATM could provide a novel clinical intervention to treat HD. Here, we describe the development and characterization of ATM inhibitor molecules to enable in vivo proof-of-concept studies in HD animal models. Starting from previously reported ATM inhibitors, we aimed with few modifications to increase brain exposure by decreasing P-glycoprotein liability while maintaining potency and selectivity. Here, we report brain-penetrant ATM inhibitors that have robust pharmacodynamic (PD) effects consistent with ATM kinase inhibition in the mouse brain and an understandable pharmacokinetic/PD (PK/PD) relationship. Compound 17 engages ATM kinase and shows robust dose-dependent inhibition of X-ray irradiation-induced KAP1 phosphorylation in the mouse brain. Furthermore, compound 17 protects against mHTT (Q73)-induced cytotoxicity in a cortical-striatal cell model of HD.

Identification of regulators of the myofibroblast phenotype of primary dermal fibroblasts from early diffuse systemic sclerosis patients.

Systemic sclerosis (SSc or scleroderma) is an auto-immune disease characterized by skin fibrosis. While primary cells from patients are considered as a unique resource to better understand human disease biology, the effect of in vitro culture on these cells and their evaluation as a platform to identify disease regulators remain poorly characterized. The goal of our studies was to provide insights into the utility of SSc dermal fibroblast primary cells for therapeutic target discovery. The disease phenotypes of freshly isolated and in vitro cultured SSc dermal fibroblasts were characterized using whole transcriptome profiling, alpha smooth muscle actin (ASMA) expression and cell impedance. SSc dermal fibroblasts retained most of the molecular disease phenotype upon in vitro culture for at least four cell culture passages (approximatively 10 cell doublings). We validated an RNA interference high throughput assay that successfully identified genes affecting the myofibroblast phenotype of SSc skin fibroblasts. These genes included MKL1, RHOA and LOXL2 that were previously proposed as therapeutic anti-fibrotic target, and ITGA5, that has been less studied in fibrosis biology and may be a novel potential modifier of SSc fibroblast biology. Together our results demonstrated the value of carefully-phenotyped SSc dermal fibroblasts as a platform for SSc target and drug discovery.

Assessment of p.Phe508del-CFTR functional restoration in pediatric primary cystic fibrosis airway epithelial cells.

BACKGROUND: Mutations in the cystic fibrosis transmembrane regulator (CFTR) gene can reduce function of the CFTR ion channel activity and impair cellular chloride secretion. The gold standard method to assess CFTR function of ion transport using the Ussing chamber requires a high number of airway epithelial cells grown at air-liquid interface, limiting the application of this method for high throughput screening of potential therapeutic compounds in primary airway epithelial cells (pAECs) featuring less common CFTR mutations. This study assessed an alternative approach, using a small scale halide assay that can be adapted for a personalized high throughput setting to analyze CFTR function of pAEC. METHODS: Pediatric pAECs derived from children with CF (pAECCF) were established and expanded as monolayer cultures, before seeding into 96-well plates for the halide assay. Cells were then transduced with an adenoviral construct containing yellow fluorescent protein (eYFP) reporter gene, alone or in combination with either wild-type CFTR (WT-CFTR) or p.Phe508del CFTR. Four days post transduction, cells were stimulated with forskolin and genistein, and assessed for quenching of the eYFP signal following injection of iodide solution into the assay media. RESULTS: Data showed that pAECCF can express eYFP at high efficiency following transduction with the eYFP construct. The halide assay was able to discriminate functional restoration of CFTR in pAECCF treated with either WT-CFTR construct or the positive controls syntaxin 8 and B-cell receptor-associated protein 31 shRNAs. SIGNIFICANCE: The current study demonstrates that the halide assay can be adapted for pediatric pAECCF to evaluate restoration of CFTR function. With the ongoing development of small molecules to modulate the folding and/or activity of various mutated CFTR proteins, this halide assay presents a small-scale personalized screening platform that could assess therapeutic potential of molecules across a broad range of CFTR mutations.

Quantification of huntingtin protein species in Huntington's disease patient leukocytes using optimised electrochemiluminescence immunoassays.

BACKGROUND: Huntington's disease (HD) is an autosomal dominant neurodegenerative condition caused by an expanded CAG repeat in the gene encoding huntingtin (HTT). Optimizing peripheral quantification of huntingtin throughout the course of HD is valuable not only to illuminate the natural history and pathogenesis of disease, but also to detect peripheral effects of drugs in clinical trial. RATIONALE: We previously demonstrated that mutant HTT (mHTT) was significantly elevated in purified HD patient leukocytes compared with controls and that these levels track disease progression. Our present study investigates whether the same result can be achieved with a simpler and more scalable collection technique that is more suitable for clinical trials. METHODS: We collected whole blood at 133 patient visits in two sample sets and generated peripheral blood mononuclear cells (PBMCs). Levels of mHTT, as well as N-, and C-terminal and mid-region huntingtin were measured in the PBMCs using ELISA-based Meso Scale Discovery (MSD) electrochemiluminescence immunoassay platforms, and we evaluated the relationship between different HTT species, disease stage, and brain atrophy on magnetic resonance imaging. CONCLUSIONS: The assays were sensitive and accurate. We confirm our previous findings that mHTT increases with advancing disease stage in patient PBMCs, this time using a simple collection protocol and scalable assay.

A small molecule mitigates hearing loss in a mouse model of Usher syndrome III.

Usher syndrome type III (USH3), characterized by progressive deafness, variable balance disorder and blindness, is caused by destabilizing mutations in the gene encoding the clarin-1 (CLRN1) protein. Here we report a new strategy to mitigate hearing loss associated with a common USH3 mutation CLRN1(N48K) that involves cell-based high-throughput screening of small molecules capable of stabilizing CLRN1(N48K), followed by a secondary screening to eliminate general proteasome inhibitors, and finally an iterative process to optimize structure-activity relationships. This resulted in the identification of BioFocus 844 (BF844). To test the efficacy of BF844, we developed a mouse model that mimicked the progressive hearing loss associated with USH3. BF844 effectively attenuated progressive hearing loss and prevented deafness in this model. Because the CLRN1(N48K) mutation causes both hearing and vision loss, BF844 could in principle prevent both sensory deficiencies in patients with USH3. Moreover, the strategy described here could help identify drugs for other protein-destabilizing monogenic disorders.

Targeting ATM ameliorates mutant Huntingtin toxicity in cell and animal models of Huntington's disease.

Age-related neurodegenerative disorders including Alzheimer's disease and Huntington's disease (HD) consistently show elevated DNA damage, but the relevant molecular pathways in disease pathogenesis remain unclear. One attractive gene is that encoding the ataxia-telangiectasia mutated (ATM) protein, a kinase involved in the DNA damage response, apoptosis, and cellular homeostasis. Loss-of-function mutations in both alleles of ATM cause ataxia-telangiectasia in children, but heterozygous mutation carriers are disease-free. Persistently elevated ATM signaling has been demonstrated in Alzheimer's disease and in mouse models of other neurodegenerative diseases. We show that ATM signaling was consistently elevated in cells derived from HD mice and in brain tissue from HD mice and patients. ATM knockdown protected from toxicities induced by mutant Huntingtin (mHTT) fragments in mammalian cells and in transgenic Drosophila models. By crossing the murine Atm heterozygous null allele onto BACHD mice expressing full-length human mHTT, we show that genetic reduction of Atm gene dosage by one copy ameliorated multiple behavioral deficits and partially improved neuropathology. Small-molecule ATM inhibitors reduced mHTT-induced death of rat striatal neurons and induced pluripotent stem cells derived from HD patients. Our study provides converging genetic and pharmacological evidence that reduction of ATM signaling could ameliorate mHTT toxicity in cellular and animal models of HD, suggesting that ATM may be a useful therapeutic target for HD.

Quantification assays for total and polyglutamine-expanded huntingtin proteins.

The expansion of a CAG trinucleotide repeat in the huntingtin gene, which produces huntingtin protein with an expanded polyglutamine tract, is the cause of Huntington's disease (HD). Recent studies have reported that RNAi suppression of polyglutamine-expanded huntingtin (mutant HTT) in HD animal models can ameliorate disease phenotypes. A key requirement for such preclinical studies, as well as eventual clinical trials, aimed to reduce mutant HTT exposure is a robust method to measure HTT protein levels in select tissues. We have developed several sensitive and selective assays that measure either total human HTT or polyglutamine-expanded human HTT proteins on the electrochemiluminescence Meso Scale Discovery detection platform with an increased dynamic range over other methods. In addition, we have developed an assay to detect endogenous mouse and rat HTT proteins in pre-clinical models of HD to monitor effects on the wild type protein of both allele selective and non-selective interventions. We demonstrate the application of these assays to measure HTT protein in several HD in vitro cellular and in vivo animal model systems as well as in HD patient biosamples. Furthermore, we used purified recombinant HTT proteins as standards to quantitate the absolute amount of HTT protein in such biosamples.

A monoclonal antibody TrkB receptor agonist as a potential therapeutic for Huntington's disease.

Huntington's disease (HD) is a devastating, genetic neurodegenerative disease caused by a tri-nucleotide expansion in exon 1 of the huntingtin gene. HD is clinically characterized by chorea, emotional and psychiatric disturbances and cognitive deficits with later symptoms including rigidity and dementia. Pathologically, the cortico-striatal pathway is severely dysfunctional as reflected by striatal and cortical atrophy in late-stage disease. Brain-derived neurotrophic factor (BDNF) is a neuroprotective, secreted protein that binds with high affinity to the extracellular domain of the tropomyosin-receptor kinase B (TrkB) receptor promoting neuronal cell survival by activating the receptor and down-stream signaling proteins. Reduced cortical BDNF production and transport to the striatum have been implicated in HD pathogenesis; the ability to enhance TrkB signaling using a BDNF mimetic might be beneficial in disease progression, so we explored this as a therapeutic strategy for HD. Using recombinant and native assay formats, we report here the evaluation of TrkB antibodies and a panel of reported small molecule TrkB agonists, and identify the best candidate, from those tested, for in vivo proof of concept studies in transgenic HD models.

Mutant ubiquitin decreases amyloid ß plaque formation in a transgenic mouse model of Alzheimer's disease.

The mutant ubiquitin UBB(+1) is a substrate as well as an inhibitor of the ubiquitin-proteasome system (UPS) and accumulates in the neuropathological hallmarks of Alzheimer's disease (AD). A role for the UPS has been suggested in the generation of amyloid ß (Aß) plaques in AD. To investigate the effect of UBB(+1) expression on amyloid pathology in vivo, we crossed UBB(+1) transgenic mice with a transgenic line expressing AD-associated mutant amyloid precursor protein (APPSwe) and mutant presenilin 1 (PS1dE9), resulting in APPPS1/UBB(+1) triple transgenic mice. In these mice, we determined the Aß levels at 3, 6, 9 and 11 months of age. Surprisingly, we found a significant decrease in Aß deposition in amyloid plaques and levels of soluble Aß(42) in APPPS1/UBB(+1) transgenic mice compared to APPPS1 mice at 6 months of age, without alterations in UBB(+1) protein levels or proteasomal chymotrypsin activity. These lowering effects of UBB(+1) on Aß deposition were transient, as this relative decrease in plaque load was not significant in APPPS1/UBB(+1) mice at 9 and 11 months of age. We also show that APPPS1/UBB(+1) mice exhibit astrogliosis, indicating that they may not be improved functionally compared to APPPS1 mice despite the Aß reduction. The molecular mechanism underlying this decrease in Aß deposition in APPPS1/UBB(+1) mice is more complex than previously assumed because UBB(+1) is also ubiquitinated at K63 opening the possibility of additional effects of UBB(+1) (e.g. kinase activation).

Alzheimer-associated mutant ubiquitin impairs spatial reference memory.

UBB(+1) is a mutant ubiquitin which accumulates in the hallmarks of tauopathies, including Alzheimer's disease. Transgenic mice expressing high levels of neuronal UBB(+1) exhibit moderately decreased proteasome activity and spatial reference memory deficits at 9months of age. In the present study, we characterized the behavioral phenotype of male UBB(+1) transgenic mice at different ages. We show that UBB(+1) transgenic mice displayed an age-related functional decline similar to wild-type littermates, without gross neurological abnormalities or alterations in procedural motor-learning and motor coordination. At 15months of age, a transgene-specific spatial learning deficit was dependent on the period of training in the Morris watermaze. This deficit could be eliminated after additional training. We conclude that the previously reported spatial reference memory deficits of UBB(+1) transgenic mice persist during aging. In addition, our results demonstrate that the subtle defect in spatial reference memory formation, caused by a decrease in forebrain proteasome activity, is a persistent defect and not a structural defect.

Histological and functional benefit following transplantation of motor neuron progenitors to the injured rat spinal cord.

BACKGROUND: Motor neuron loss is characteristic of cervical spinal cord injury (SCI) and contributes to functional deficit. METHODOLOGY/PRINCIPAL FINDINGS: In order to investigate the amenability of the injured adult spinal cord to motor neuron differentiation, we transplanted spinal cord injured animals with a high purity population of human motor neuron progenitors (hMNP) derived from human embryonic stem cells (hESCs). In vitro, hMNPs displayed characteristic motor neuron-specific markers, a typical electrophysiological profile, functionally innervated human or rodent muscle, and secreted physiologically active growth factors that caused neurite branching and neuronal survival. hMNP transplantation into cervical SCI sites in adult rats resulted in suppression of intracellular signaling pathways associated with SCI pathogenesis, which correlated with greater endogenous neuronal survival and neurite branching. These neurotrophic effects were accompanied by significantly enhanced performance on all parameters of the balance beam task, as compared to controls. Interestingly, hMNP transplantation resulted in survival, differentiation, and site-specific integration of hMNPs distal to the SCI site within ventral horns, but hMNPs near the SCI site reverted to a neuronal progenitor state, suggesting an environmental deficiency for neuronal maturation associated with SCI. CONCLUSIONS/SIGNIFICANCE: These findings underscore the barriers imposed on neuronal differentiation of transplanted cells by the gliogenic nature of the injured spinal cord, and the physiological relevance of transplant-derived neurotrophic support to functional recovery.

Low levels of mutant ubiquitin are degraded by the proteasome in vivo.

The ubiquitin-proteasome system fulfills a pivotal role in regulating intracellular protein turnover. Impairment of this system is implicated in the pathogenesis of neurodegenerative diseases characterized by ubiquitin- containing proteinaceous deposits. UBB(+1), a mutant ubiquitin, is one of the proteins accumulating in the neuropathological hallmarks of tauopathies, including Alzheimer's disease, and polyglutamine diseases. In vitro, UBB(+1) properties shift from a proteasomal ubiquitin-fusion degradation substrate at low expression levels to a proteasome inhibitor at high expression levels. Here we report on a novel transgenic mouse line (line 6663) expressing low levels of neuronal UBB(+1). In these mice, UBB(+1) protein is scarcely detectable in the neuronal cell population. Accumulation of UBB(+1) commences only after intracranial infusion of the proteasome inhibitors lactacystin or MG262, showing that, at these low expression levels, the UBB(+1) protein is a substrate for proteasomal degradation in vivo. In addition, accumulation of the protein serves as a reporter for proteasome inhibition. These findings strengthen our proposition that, in healthy brain, UBB(+1) is continuously degraded and disease-related UBB(+1) accumulation serves as an endogenous marker for proteasomal dysfunction. This novel transgenic line can give more insight into the intrinsic properties of UBB(+1) and its role in neurodegenerative disease.

Modest proteasomal inhibition by aberrant ubiquitin exacerbates aggregate formation in a Huntington disease mouse model.

UBB(+1), a mutant form of ubiquitin, is both a substrate and an inhibitor of the proteasome which accumulates in the neuropathological hallmarks of Huntington disease (HD). In vitro, expression of UBB(+1) and mutant huntingtin synergistically increase aggregate formation and polyglutamine induced cell death. We generated a UBB(+1) transgenic mouse line expressing UBB(+1) within the neurons of the striatum. In these mice lentiviral driven expression of expanded huntingtin constructs in the striatum results in a significant increase in neuronal inclusion formation. Although UBB(+1) transgenic mice show neither a decreased lifespan nor apparent neuronal loss, they appear to be more vulnerable to toxic insults like expanded polyglutamine proteins due to a modest proteasome inhibition. These findings underscore the relevance of an efficient ubiquitin-proteasome system in HD.

The alpha7 nicotinic acetylcholine receptor on fibroblast-like synoviocytes and in synovial tissue from rheumatoid arthritis patients: a possible role for a key neurotransmitter in synovial inflammation.

OBJECTIVE: Recent studies have suggested an important role for neurotransmitters as modulators of inflammation. Therefore, we undertook this study to investigate the expression of the alpha7 subunit of the nicotinic acetylcholine receptor (alpha7nAChR) and its function in rheumatoid arthritis (RA). METHODS: The potential role of the alpha7nAChR in modulating proinflammatory cytokine expression in fibroblast-like synoviocytes (FLS) was identified by screening an adenoviral short hairpin RNA (Ad.shRNA) library. An alpha7-specific antibody was used for immunohistochemistry, and fluorescein isothiocyanate-labeled alpha-bungarotoxin, which binds specifically to the alpha7nAChR, was used for immunofluorescence. Gene expression in FLS was determined by quantitative polymerase chain reaction with primers specific for the alpha7nAChR. In addition, we analyzed messenger RNA (mRNA) expression of dupalpha7, a variant alpha7 transcript. Next, we studied the functional role of the alpha7nAChR in RA FLS by examining the effects of alpha7-specific agonists on the production of interleukin-6 (IL-6) and IL-8 by activated FLS. RESULTS: A screen using an Ad.shRNA library against 807 transcripts revealed that a specific alpha7nAChR shRNA potently modulated IL-8 and matrix metalloproteinase expression in FLS. The alpha7nAChR was expressed in the inflamed synovium from RA patients, predominantly in the intimal lining layer. We found alpha7nAChR expression at both the mRNA and protein level in cultured RA FLS. FLS also constitutively expressed dupalpha7 mRNA. Specific alpha7nAChR agonists reduced tumor necrosis factor alpha-induced IL-6 and IL-8 production by FLS. CONCLUSION: The alpha7nAChR and its dupalpha7 variant are expressed in RA synovium, where they may play a critical role in regulating inflammation. Targeting the alpha7nAChR could provide a novel antiinflammatory approach to the treatment of RA.

Intermediate filament transcription in astrocytes is repressed by proteasome inhibition.

Increased expression of the astrocytic intermediate filament protein glial fibrillary acidic protein (GFAP) is a characteristic of astrogliosis. This process occurs in the brain during aging and neurodegeneration and coincides with impairment of the ubiquitin proteasome system. Inhibition of the proteasome impairs protein degradation; therefore, we hypothesized that the increase in GFAP may be the result of impaired proteasomal activity in astrocytes. We investigated the effect of proteasome inhibitors on GFAP expression and other intermediate filament proteins in human astrocytoma cells and in a rat brain model for astrogliosis. Extensive quantitative RT-PCR, immunocytochemistry, and Western blot analysis resulted unexpectedly in a strong decrease of GFAP mRNA to <4% of control levels [Control (DMSO) 100+/-19.2%; proteasome inhibitor (epoxomicin) 3.5+/-1.3%, n=8; P < or = 0.001] and a loss of GFAP protein in astrocytes in vitro. We show that the proteasome alters GFAP promoter activity, possibly mediated by transcription factors as demonstrated by a GFAP promoter-luciferase assay and RT(2) Profiler PCR array for human transcription factors. Most important, we demonstrate that proteasome inhibitors also reduce GFAP and vimentin expression in a rat model for induced astrogliosis in vivo. Therefore, proteasome inhibitors could serve as a potential therapy to modulate astrogliosis associated with CNS injuries and disease.

The orphan G protein-coupled receptor 3 modulates amyloid-beta peptide generation in neurons.

Deposition of the amyloid-beta peptide is a pathological hallmark of Alzheimer's disease. A high-throughput functional genomics screen identified G protein-coupled receptor 3 (GPR3), a constitutively active orphan G protein-coupled receptor, as a modulator of amyloid-beta production. Overexpression of GPR3 stimulated amyloid-beta production, whereas genetic ablation of GPR3 prevented accumulation of the amyloid-beta peptide in vitro and in an Alzheimer's disease mouse model. GPR3 expression led to increased formation and cell-surface localization of the mature gamma-secretase complex in the absence of an effect on Notch processing. GPR3 is highly expressed in areas of the normal human brain implicated in Alzheimer's disease and is elevated in the sporadic Alzheimer's disease brain. Thus, GPR3 represents a potential therapeutic target for the treatment of Alzheimer's disease.

Long-term proteasome dysfunction in the mouse brain by expression of aberrant ubiquitin.

Many neurodegenerative diseases are characterized by deposits of ubiquitinated and aberrant proteins, suggesting a failure of the ubiquitin-proteasome system (UPS). The aberrant ubiquitin UBB(+1) is one of the ubiquitinated proteins accumulating in tauopathies such as Alzheimer's disease (AD) and polyglutamine diseases such as Huntington's disease. We have generated UBB(+1) transgenic mouse lines with post-natal neuronal expression of UBB(+1), resulting in increased levels of ubiquitinated proteins in the cortex. Moreover, by proteomic analysis, we identified expression changes in proteins involved in energy metabolism or organization of the cytoskeleton. These changes show a striking resemblance to the proteomic profiles of both AD brain and several AD mouse models. Moreover, UBB(+1) transgenic mice show a deficit in contextual memory in both water maze and fear conditioning paradigms. Although UBB(+1) partially inhibits the UPS in the cortex, these mice do not have an overt neurological phenotype. These mouse models do not replicate the full spectrum of AD-related changes, yet provide a tool to understand how the UPS is involved in AD pathological changes and in memory formation.

The neuronal ubiquitin-proteasome system: murine models and their neurological phenotype.

The ubiquitin-proteasome system (UPS) is the main intracellular pathway for regulated protein turnover. This system is of vital importance for maintaining cellular homeostasis and is essential for neuronal functioning. It is therefore not surprising that impairment of this system is implicated in the pathogenesis of a variety of diseases, including neurological disorders, which are pathologically characterized by the presence of ubiquitin-positive protein aggregates. A direct correlation between intact neuronal functioning and the UPS is exemplified by a range of transgenic mouse models wherein mutations in components of the UPS lead to a neurodegenerative or neurological phenotype. These models have been proven useful in determining the role of the UPS in the nervous system in health and disease. Furthermore, recently developed in vivo models harboring reporter systems to measure UPS activity could also substantially contribute to understanding the effect of neurodegeneration on UPS function. The role of the UPS in neurodegeneration in vivo is reviewed by discussing the currently available murine models showing a neurological phenotype induced by genetic manipulation of the UPS.