Nuclear receptor signaling via NHR-49/MDT-15 regulates stress resilience and proteostasis in response to reproductive and metabolic cues.

The ability to sense and respond to proteotoxic insults declines with age, leaving cells vulnerable to chronic and acute stressors. Reproductive cues modulate this decline in cellular proteostasis to influence organismal stress resilience in Caenorhabditis elegans We previously uncovered a pathway that links the integrity of developing embryos to somatic health in reproductive adults. Here, we show that the nuclear receptor NHR-49, an ortholog of mammalian peroxisome proliferator-activated receptor α (PPARα), regulates stress resilience and proteostasis downstream from embryo integrity and other pathways that influence lipid homeostasis and upstream of HSF-1. Disruption of the vitelline layer of the embryo envelope, which activates a proteostasis-enhancing intertissue pathway in somatic cells, triggers changes in lipid catabolism gene expression that are accompanied by an increase in fat stores. NHR-49, together with its coactivator, MDT-15, contributes to this remodeling of lipid metabolism and is also important for the elevated stress resilience mediated by inhibition of the embryonic vitelline layer. Our findings indicate that NHR-49 also contributes to stress resilience in other pathways known to change lipid homeostasis, including reduced insulin-like signaling and fasting, and that increased NHR-49 activity is sufficient to improve proteostasis and stress resilience in an HSF-1-dependent manner. Together, our results establish NHR-49 as a key regulator that links lipid homeostasis and cellular resilience to proteotoxic stress.

Nuclear receptor signaling via NHR-49/MDT-15 regulates stress resilience and proteostasis in response to reproductive and metabolic cues.

The ability to sense and respond to proteotoxic insults declines with age, leaving cells vulnerable to chronic and acute stressors. Reproductive cues modulate this decline in cellular proteostasis to influence organismal stress resilience in C. elegans. We previously uncovered a pathway that links the integrity of developing embryos to somatic health in reproductive adults. Here, we show that the nuclear receptor NHR-49, a functional homolog of mammalian peroxisome proliferator-activated receptor alpha (PPARα), regulates stress resilience and proteostasis downstream of embryo integrity and other pathways that influence lipid homeostasis, and upstream of HSF-1. Disruption of the vitelline layer of the embryo envelope, which activates a proteostasis-enhancing inter-tissue pathway in somatic tissues, also triggers changes in lipid catabolism gene expression that are accompanied by an increase in fat stores. NHR-49 together with its co-activator MDT-15 contributes to this remodeling of lipid metabolism and is also important for the elevated stress resilience mediated by inhibition of the embryonic vitelline layer as well as by other pathways known to change lipid homeostasis, including reduced insulin-like signaling and fasting. Further, we show that increased NHR-49 activity is sufficient to suppress polyglutamine aggregation and improve stress resilience in an HSF-1-dependent manner. Together, our results establish NHR-49 as a key regulator that links lipid homeostasis and cellular resilience to proteotoxic stress.

Variants in ATP6V0A1 cause progressive myoclonus epilepsy and developmental and epileptic encephalopathy.

The vacuolar H+-ATPase is a large multi-subunit proton pump, composed of an integral membrane V0 domain, involved in proton translocation, and a peripheral V1 domain, catalysing ATP hydrolysis. This complex is widely distributed on the membrane of various subcellular organelles, such as endosomes and lysosomes, and plays a critical role in cellular processes ranging from autophagy to protein trafficking and endocytosis. Variants in ATP6V0A1, the brain-enriched isoform in the V0 domain, have been recently associated with developmental delay and epilepsy in four individuals. Here, we identified 17 individuals from 14 unrelated families with both with new and previously characterized variants in this gene, representing the largest cohort to date. Five affected subjects with biallelic variants in this gene presented with a phenotype of early-onset progressive myoclonus epilepsy with ataxia, while 12 individuals carried de novo missense variants and showed severe developmental and epileptic encephalopathy. The R740Q mutation, which alone accounts for almost 50% of the mutations identified among our cases, leads to failure of lysosomal hydrolysis by directly impairing acidification of the endolysosomal compartment, causing autophagic dysfunction and severe developmental defect in Caenorhabditis elegans. Altogether, our findings further expand the neurological phenotype associated with variants in this gene and provide a direct link with endolysosomal acidification in the pathophysiology of ATP6V0A1-related conditions.

Embryo integrity regulates maternal proteostasis and stress resilience.

The proteostasis network is regulated by transcellular communication to promote health and fitness in metazoans. In Caenorhabditis elegans, signals from the germline initiate the decline of proteostasis and repression of cell stress responses at reproductive maturity, indicating that commitment to reproduction is detrimental to somatic health. Here we show that proteostasis and stress resilience are also regulated by embryo-to-mother communication in reproductive adults. To identify genes that act directly in the reproductive system to regulate somatic proteostasis, we performed a tissue targeted genetic screen for germline modifiers of polyglutamine aggregation in muscle cells. We found that inhibiting the formation of the extracellular vitelline layer of the fertilized embryo inside the uterus suppresses aggregation, improves stress resilience in an HSF-1-dependent manner, and restores the heat-shock response in the somatic tissues of the parent. This pathway relies on DAF-16/FOXO activation in vulval tissues to maintain stress resilience in the mother, suggesting that the integrity of the embryo is monitored by the vulva to detect damage and initiate an organismal protective response. Our findings reveal a previously undescribed transcellular pathway that links the integrity of the developing progeny to proteostasis regulation in the parent.

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.

Shaping proteostasis at the cellular, tissue, and organismal level.

The proteostasis network (PN) regulates protein synthesis, folding, transport, and degradation to maintain proteome integrity and limit the accumulation of protein aggregates, a hallmark of aging and degenerative diseases. In multicellular organisms, the PN is regulated at the cellular, tissue, and systemic level to ensure organismal health and longevity. Here we review these three layers of PN regulation and examine how they collectively maintain cellular homeostasis, achieve cell type-specific proteomes, and coordinate proteostasis across tissues. A precise understanding of these layers of control has important implications for organismal health and could offer new therapeutic approaches for neurodegenerative diseases and other chronic disorders related to PN dysfunction.

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/C(Cdh1) 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/C(Cdh1) complex in a ligand-dependent fashion without being targeted for proteasomal degradation. Inhibition of APC/C(Cdh1) 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/C(Cdh1) and suggest abnormal cell cycle reactivation as a pathogenic mechanism in SBMA.

A small-molecule Nrf1 and Nrf2 activator mitigates polyglutamine toxicity in spinal and bulbar muscular atrophy.

Spinal and bulbar muscular atrophy (SBMA, also known as Kennedy's disease) is one of nine neurodegenerative disorders that are caused by expansion of polyglutamine-encoding CAG repeats. Intracellular accumulation of abnormal proteins in these diseases, a pathological hallmark, is associated with defects in protein homeostasis. Enhancement of the cellular proteostasis capacity with small molecules has therefore emerged as a promising approach to treatment. Here, we characterize a novel curcumin analog, ASC-JM17, as an activator of central pathways controlling protein folding, degradation and oxidative stress resistance. ASC-JM17 acts on Nrf1, Nrf2 and Hsf1 to increase the expression of proteasome subunits, antioxidant enzymes and molecular chaperones. We show that ASC-JM17 ameliorates toxicity of the mutant androgen receptor (AR) responsible for SBMA in cell, fly and mouse models. Knockdown of the Drosophila Nrf1 and Nrf2 ortholog cap 'n' collar isoform-C, but not Hsf1, blocks the protective effect of ASC-JM17 on mutant AR-induced eye degeneration in flies. Our observations indicate that activation of the Nrf1/Nrf2 pathway is a viable option for pharmacological intervention in SBMA and potentially other polyglutamine diseases.

MiR-298 Counteracts Mutant Androgen Receptor Toxicity in Spinal and Bulbar Muscular Atrophy.

Spinal and bulbar muscular atrophy (SBMA) is a currently untreatable adult-onset neuromuscular disease caused by expansion of a polyglutamine repeat in the androgen receptor (AR). In SBMA, as in other polyglutamine diseases, a toxic gain of function in the mutant protein is an important factor in the disease mechanism; therefore, reducing the mutant protein holds promise as an effective treatment strategy. In this work, we evaluated a microRNA (miRNA) to reduce AR expression. From a list of predicted miRNAs that target human AR, we selected microRNA-298 (miR-298) for its ability to downregulate AR mRNA and protein levels when transfected in cells overexpressing wild-type and mutant AR and in SBMA patient-derived fibroblasts. We showed that miR-298 directly binds to the 3'-untranslated region of the human AR transcript, and counteracts AR toxicity in vitro. Intravenous delivery of miR-298 with adeno-associated virus serotype 9 vector resulted in efficient transduction of muscle and spinal cord and amelioration of the disease phenotype in SBMA mice. Our findings support the development of miRNAs as a therapeutic strategy for SBMA and other neurodegenerative disorders caused by toxic proteins.

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.

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.

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, ß-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.

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, Kennedy's 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.

Muscle matters in Kennedy's disease.

Polyglutamine expansion in the androgen receptor causes Kennedy's disease. Two recent reports, Cortes et al. (2014) in this issue of Neuron and Lieberman et al. (2014) in Cell Reports, raise the possibility that targeting expression of the mutant protein in skeletal muscle, instead of the nervous system, may mitigate manifestations of this disorder.

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.

VCP/p97 is essential for maturation of ubiquitin-containing autophagosomes and this function is impaired by mutations that cause IBMPFD.

VCP (VCP/p97) is a ubiquitously expressed member of the AAA(+)-ATPase family of chaperone-like proteins that regulates numerous cellular processes including chromatin decondensation, homotypic membrane fusion and ubiquitin-dependent protein degradation by the proteasome. Mutations in VCP cause a multisystem degenerative disease consisting of inclusion body myopathy, Paget disease of bone, and frontotemporal dementia (IBMPFD). Here we show that VCP is essential for autophagosome maturation. We generated cells stably expressing dual-tagged LC3 (mCherry-EGFP-LC3) which permit monitoring of autophagosome maturation. We determined that VCP deficiency by RNAi-mediated knockdown or overexpression of dominant-negative VCP results in significant accumulation of immature autophagic vesicles, some of which are abnormally large, acidified and exhibit cathepsin B activity. Furthermore, expression of disease-associated VCP mutants (R155H and A232E) also causes this autophagy defect. VCP was found to be essential to autophagosome maturation under basal conditions and in cells challenged by proteasome inhibition, but not in cells challenged by starvation, suggesting that VCP might be selectively required for autophagic degradation of ubiquitinated substrates. Indeed, a high percentage of the accumulated autophagic vesicles contain ubiquitin-positive contents, a feature that is not observed in autophagic vesicles that accumulate following starvation or treatment with Bafilomycin A. Finally, we show accumulation of numerous, large LAMP-1 and LAMP-2-positive vacuoles and accumulation of LC3-II in myoblasts derived from patients with IBMPFD. We conclude that VCP is essential for maturation of ubiquitin-containing autophagosomes and that defect in this function may contribute to IBMPFD pathogenesis.

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.