Laura C. Bott

The ubiquitin-proteasome system in neurodegenerative diseases: precipitating factor, yet part of the solution

The ubiquitin-proteasome system (UPS) has been implicated in neurodegenerative diseases based on the presence of deposits consisting of ubiquitylated proteins in affected neurons. It has been postulated that aggregation-prone proteins associated with these disorders, such as α-synuclein, β-amyloid peptide, and polyglutamine proteins, compromise UPS function, and delay the degradation of other proteasome substrates. Many of these substrates play important regulatory roles in signaling, cell cycle progression, or apoptosis, and their inadvertent stabilization due to an overloaded and improperly functioning UPS may thus be responsible for cellular demise in neurodegeneration. Over the past decade, numerous studies have addressed the UPS dysfunction hypothesis using various model systems and techniques that differ in their readout and sensitivity. While an inhibitory effect of some disease proteins on the UPS has been demonstrated, increasing evidence attests that the UPS remains operative in many disease models, which opens new possibilities for treatment. In this review, we will discuss the paradigm shift that repositioned the UPS from being a prime suspect in the pathophysiology of neurodegeneration to an attractive therapeutic target that can be harnessed to accelerate the clearance of disease-linked proteins.

A Conserved Unfoldase Activity for the p97 AAA-ATPase in Proteasomal Degradation

The multifunctional AAA-ATPase p97 is one of the most abundant and conserved proteins in eukaryotic cells. The p97/Npl4/Ufd1 complex dislocates proteins that fail the protein quality control in the endoplasmic reticulum to the cytosol where they are subject to degradation by the ubiquitin/proteasome system. Substrate dislocation depends on the unfoldase activity of p97. Interestingly, p97 is also involved in the degradation of specific soluble proteasome substrates but the exact mode of action of p97 in this process is unclear. Here, we show that both the central pore and ATPase activity of p97 are necessary for the degradation of cytosolic ubiquitin-fusion substrates. Addition of a flexible extended C-terminal peptide to the substrate relieves the requirement for p97. Deletion mapping reveals a conserved length dependency of 20 residues for the peptide, which allows p97-independent degradation to occur. Our results suggest that initiation of unfolding may be more complex than previously anticipated and that the 19S regulatory complex of the proteasome can require preprocessing of highly folded, ubiquitylated substrates by the p97(Ufd1/Npl4) complex. Our data provide an explanation for the observation that p97 is only essential for a subpopulation of soluble substrates and predict that a common characteristic of soluble p97-dependent substrates is the lack of an initiation site to facilitate unfolding by the 26S proteasome.

Insulinlike Growth Factor (IGF)-1 Administration Ameliorates Disease Manifestations in a Mouse Model of Spinal and Bulbar Muscular Atrophy

Spinal and bulbar muscular atrophy is an X-linked motor neuron disease caused by polyglutamine expansion in the androgen receptor. Patients develop slowly progressive proximal muscle weakness, muscle atrophy and fasciculations. Affected individuals often show gynecomastia, testicular atrophy and reduced fertility as a result of mild androgen insensitivity. No effective disease-modifying therapy is currently available for this disease. Our recent studies have demonstrated that insulinlike growth factor (IGF)-1 reduces the mutant androgen receptor toxicity through activation of Akt in vitro, and spinal and bulbar muscular atrophy transgenic mice that also overexpress a noncirculating muscle isoform of IGF-1 have a less severe phenotype. Here we sought to establish the efficacy of daily intraperitoneal injections of mecasermin rinfabate, recombinant human IGF-1 and IGF-1 binding protein 3, in a transgenic mouse model expressing the mutant androgen receptor with an expanded 97 glutamine tract. The study was done in a controlled, randomized, blinded fashion, and, to reflect the clinical settings, the injections were started after the onset of disease manifestations. The treatment resulted in increased Akt phosphorylation and reduced mutant androgen receptor aggregation in muscle. In comparison to vehicle-treated controls, IGF-1–treated transgenic mice showed improved motor performance, attenuated weight loss and increased survival. Our results suggest that peripheral tissue can be targeted to improve the spinal and bulbar muscular atrophy phenotype and indicate that IGF-1 warrants further investigation in clinical trials as a potential treatment for this disease.

Early onset and novel features in a spinal and bulbar muscular atrophy patient with a 68 CAG repeat

Spinal and bulbar muscular atrophy (SBMA) is an X-linked neuromuscular disease caused by a trinucleotide (CAG) repeat expansion in the androgen receptor gene. Patients with SBMA have weakness, atrophy, and fasciculations in the bulbar and extremity muscles. Individuals with CAG repeat lengths greater than 62 have not previously been reported. We evaluated a 29year old SBMA patient with 68 CAGs who had unusually early onset and findings not seen in others with the disease. Analysis of the androgen receptor gene confirmed the repeat length of 68 CAGs in both peripheral blood and fibroblasts. Evaluation of muscle and sensory function showed deficits typical of SBMA, and in addition the patient had manifestations of autonomic dysfunction and abnormal sexual development. These findings extend the known phenotype associated with SBMA and shed new insight into the effects of the mutated androgen receptor.

Stem cell-derived motor neurons from spinal and bulbar muscular atrophy patients.

Spinal and bulbar muscular atrophy (SBMA, Kennedys disease) is a motor neuron disease caused by polyglutamine repeat expansion in the androgen receptor. Although degeneration occurs in the spinal cord and muscle, the exact mechanism is not clear. Induced pluripotent stem cells from spinal and bulbar muscular atrophy patients provide a useful model for understanding the disease mechanism and designing effective therapy. Stem cells were generated from six patients and compared to control lines from three healthy individuals. Motor neurons from four patients were differentiated from stem cells and characterized to understand disease-relevant phenotypes. Stem cells created from patient fibroblasts express less androgen receptor than control cells, but show androgen-dependent stabilization and nuclear translocation. The expanded repeat in several stem cell clones was unstable, with either expansion or contraction. Patient stem cell clones produced a similar number of motor neurons compared to controls, with or without androgen treatment. The stem cell-derived motor neurons had immunoreactivity for HB9, Isl1, ChAT, and SMI-32, and those with the largest repeat expansions were found to have increased acetylated α-tubulin and reduced HDAC6. Reduced HDAC6 was also found in motor neuron cultures from two other patients with shorter repeats. Evaluation of stably transfected mouse cells and SBMA spinal cord showed similar changes in acetylated α-tubulin and HDAC6. Perinuclear lysosomal enrichment, an HDAC6 dependent process, was disrupted in motor neurons from two patients with the longest repeats. SBMA stem cells present new insights into the disease, and the observations of reduced androgen receptor levels, repeat instability, and reduced HDAC6 provide avenues for further investigation of the disease mechanism and development of effective therapy.

Mutation in CPT1C Associated With Pure Autosomal Dominant Spastic Paraplegia

IMPORTANCE The family of genes implicated in hereditary spastic paraplegias (HSPs) is quickly expanding, mostly owing to the widespread availability of next-generation DNA sequencing methods. Nevertheless, a genetic diagnosis remains unavailable for many patients. OBJECTIVE To identify the genetic cause for a novel form of pure autosomal dominant HSP. DESIGN, SETTING, AND PARTICIPANTS We examined and followed up with a family presenting to a tertiary referral center for evaluation of HSP for a decade until August 2014. Whole-exome sequencing was performed in 4 patients from the same family and was integrated with linkage analysis. Sanger sequencing was used to confirm the presence of the candidate variant in the remaining affected and unaffected members of the family and screen the additional patients with HSP. Five affected and 6 unaffected participants from a 3-generation family with pure adult-onset autosomal dominant HSP of unknown genetic origin were included. Additionally, 163 unrelated participants with pure HSP of unknown genetic cause were screened. MAIN OUTCOME AND MEASURE Mutation in the neuronal isoform of carnitine palmitoyl-transferase (CPT1C) gene. RESULTS We identified the nucleotide substitution c.109C>T in exon 3 of CPT1C, which determined the base substitution of an evolutionarily conserved Cys residue for an Arg in the gene product. This variant strictly cosegregated with the disease phenotype and was absent in online single-nucleotide polymorphism databases and in 712 additional exomes of control participants. We showed that CPT1C, which localizes to the endoplasmic reticulum, is expressed in motor neurons and interacts with atlastin-1, an endoplasmic reticulum protein encoded by the ATL1 gene known to be mutated in pure HSPs. The mutation, as indicated by nuclear magnetic resonance spectroscopy studies, alters the protein conformation and reduces the mean (SD) number (213.0 [46.99] vs 81.9 [14.2]; P < .01) and size (0.29 [0.01] vs 0.26 [0.01]; P < .05) of lipid droplets on overexpression in cells. We also observed a reduction of mean (SD) lipid droplets in primary cortical neurons isolated from Cpt1c-/- mice as compared with wild-type mice (1.0 [0.12] vs 0.44 [0.05]; P < .001), suggesting a dominant negative mechanism for the mutation. CONCLUSIONS AND RELEVANCE This study expands the genetics of autosomal dominant HSP and is the first, to our knowledge, to link mutation in CPT1C with a human disease. The association of the CPT1C mutation with changes in lipid droplet biogenesis supports a role for altered lipid-mediated signal transduction in HSP pathogenesis.

Sexual Reassignment Fails to Prevent Kennedy’s Disease

Spinal and bulbar muscular atrophy is caused by polyglutamine expansion in the androgen receptor. As an X-linked disease dependent on androgens, symptoms and findings are only fully manifest in males. Here we describe a 40-year-old male-to-female transgender SBMA patient who developed full disease manifestations despite undetectable levels of androgens. We used cell culture and animal models to show that spironolactone, the anti-androgen she had taken for 15 years, promotes nuclear localization and toxicity of the mutant protein, which may explain the disease manifestations in this patient.

The polyglutamine-expanded androgen receptor responsible for spinal and bulbar muscular atrophy inhibits the APC/CCdh1 ubiquitin ligase complex

Polyglutamine expansion in the androgen receptor (AR) causes spinal and bulbar muscular atrophy (SBMA), an X-linked neuromuscular disease that is fully manifest only in males. It has been suggested that proteins with expanded polyglutamine tracts impair ubiquitin-dependent proteolysis due to their propensity to aggregate, but recent studies indicate that the overall activity of the ubiquitin-proteasome system is preserved in SBMA models. Here we report that AR selectively interferes with the function of the ubiquitin ligase anaphase-promoting complex/cyclosome (APC/C), which, together with its substrate adaptor Cdh1, is critical for cell cycle arrest and neuronal architecture. We show that both wild-type and mutant AR physically interact with the APC/CCdh1 complex in a ligand-dependent fashion without being targeted for proteasomal degradation. Inhibition of APC/CCdh1 by mutant but not wild-type AR in PC12 cells results in enhanced neurite outgrowth which is typically followed by rapid neurite retraction and mitotic entry. Our data indicate a role of AR in neuronal differentiation through regulation of APC/CCdh1 and suggest abnormal cell cycle reactivation as a pathogenic mechanism in SBMA.

The polyglutamine-expanded androgen receptor has increased DNA binding and reduced transcriptional activity

Expansion of a polyglutamine-encoding trinucleotide CAG repeat in the androgen receptor (AR) to more than 37 repeats is responsible for the X-linked neuromuscular disease spinal and bulbar muscular atrophy (SBMA). Here we evaluated the effect of polyglutamine length on AR function in Xenopus oocytes. This allowed us to correlate the nuclear AR concentration to its capacity for specific DNA binding and transcription activation in vivo. AR variants with polyglutamine tracts containing either 25 or 64 residues were expressed in Xenopus oocytes by cytoplasmic injection of the corresponding mRNAs. The intranuclear AR concentration was monitored in isolated nuclei and related to specific DNA binding as well as transcriptional induction from the hormone response element in the mouse mammary tumor virus (MMTV) promoter. The expanded AR with 64 glutamines had increased capacity for specific DNA binding and a reduced capacity for transcriptional induction as related to its DNA binding activity. The possible mechanism behind these polyglutamine-induced alterations in AR function is discussed.

Systemic Delivery of MicroRNA Using Recombinant Adeno-associated Virus Serotype 9 to Treat Neuromuscular Diseases in Rodents

RNA interference via the endogenous miRNA pathway regulates gene expression by controlling protein synthesis through post-transcriptional gene silencing. In recent years, miRNA-mediated gene regulation has shown potential for treatment of neurological disorders caused by a toxic gain of function mechanism. However, efficient delivery to target tissues has limited its application. Here we used a transgenic mouse model for spinal and bulbar muscular atrophy (SBMA), a neuromuscular disease caused by polyglutamine expansion in the androgen receptor (AR), to test gene silencing by a newly identified AR-targeting miRNA, miR-298. We overexpressed miR-298 using a recombinant adeno-associated virus (rAAV) serotype 9 vector to facilitate transduction of non-dividing cells. A single tail-vein injection in SBMA mice induced sustained and widespread overexpression of miR-298 in skeletal muscle and motor neurons and resulted in amelioration of the neuromuscular phenotype in the mice.