Differentiation Drives Widespread Rewiring of the Neural Stem Cell Chaperone Network.

Neural stem and progenitor cells (NSPCs) are critical for continued cellular replacement in the adult brain. Lifelong maintenance of a functional NSPC pool necessitates stringent mechanisms to preserve a pristine proteome. We find that the NSPC chaperone network robustly maintains misfolded protein solubility and stress resilience through high levels of the ATP-dependent chaperonin TRiC/CCT. Strikingly, NSPC differentiation rewires the cellular chaperone network, reducing TRiC/CCT levels and inducing those of the ATP-independent small heat shock proteins (sHSPs). This switches the proteostasis strategy in neural progeny cells to promote sequestration of misfolded proteins into protective inclusions. The chaperone network of NSPCs is more effective than that of differentiated cells, leading to improved management of proteotoxic stress and amyloidogenic proteins. However, NSPC proteostasis is impaired by brain aging. The less efficient chaperone network of differentiated neural progeny may contribute to their enhanced susceptibility to neurodegenerative diseases characterized by aberrant protein misfolding and aggregation.

Superresolution Fluorescence Imaging of Mutant Huntingtin Aggregation in Cells.

Fluorescence-based nanoscopy methods (also known as "superresolution" microscopy) have substantially expanded our options to examine the distributions of molecules inside cells with nanometer-scale resolution and molecular specificity. In the biophysical analysis of aggregation-prone misfolded proteins and peptides, this has enabled the visualization of distinct populations of aggregated species such as fibrillar assemblies within intact neuronal cells, well below previous limits of sensitivity and resolution. With the Huntington's disease protein, polyglutamine-expanded mutant huntingtin, as an example, we provide sample preparation and imaging protocols for superresolution microscopy down to the ~30 nm-level.

Delayed emergence of subdiffraction-sized mutant huntingtin fibrils following inclusion body formation.

Aberrant aggregation of improperly folded proteins is the hallmark of several human neurodegenerative disorders, including Huntington's Disease (HD) with autosomal-dominant inheritance. In HD, expansion of the CAG-repeat-encoded polyglutamine (polyQ) stretch beyond ~40 glutamines in huntingtin (Htt) and its N-terminal fragments leads to the formation of large (up to several µm) globular neuronal inclusion bodies (IBs) over time. We report direct observations of aggregating Htt exon 1 in living and fixed cells at enhanced spatial resolution by stimulated emission depletion (STED) microscopy and single-molecule super-resolution optical imaging. Fibrils of Htt exon 1 arise abundantly across the cytosolic compartment and also in neuritic processes only after nucleation and aggregation into a fairly advanced stage of growth of the prominent IB have taken place. Structural characterizations of fibrils by STED show a distinct length cutoff at ~1·5 µm and reveal subsequent coalescence (bundling/piling). Cytosolic fibrils are observed even at late stages in the process, side-by-side with the mature IB. Htt sequestration into the IB, which in neurons has been argued to be a cell-protective phenomenon, thus appears to saturate and over-power the cellular degradation systems and leaves cells vulnerable to further aggregation producing much smaller, potentially toxic, conformational protein species of which the fibrils may be comprised. We further found that exogenous delivery of the apical domain of the chaperonin subunit CCT1 to the cells via the cell medium reduced the aggregation propensity of mutant Htt exon 1 in general, and strongly reduced the occurrence of such late-stage fibrils in particular.

The Copper Metabolism MURR1 domain protein 1 (COMMD1) modulates the aggregation of misfolded protein species in a client-specific manner.

The Copper Metabolism MURR1 domain protein 1 (COMMD1) is a protein involved in multiple cellular pathways, including copper homeostasis, NF-κB and hypoxia signalling. Acting as a scaffold protein, COMMD1 mediates the levels, stability and proteolysis of its substrates (e.g. the copper-transporters ATP7B and ATP7A, RELA and HIF-1α). Recently, we established an interaction between the Cu/Zn superoxide dismutase 1 (SOD1) and COMMD1, resulting in a decreased maturation and activation of SOD1. Mutations in SOD1, associated with the progressive neurodegenerative disorder Amyotrophic Lateral Sclerosis (ALS), cause misfolding and aggregation of the mutant SOD1 (mSOD1) protein. Here, we identify COMMD1 as a novel regulator of misfolded protein aggregation as it enhances the formation of mSOD1 aggregates upon binding. Interestingly, COMMD1 co-localizes to the sites of mSOD1 inclusions and forms high molecular weight complexes in the presence of mSOD1. The effect of COMMD1 on protein aggregation is client-specific as, in contrast to mSOD1, COMMD1 decreases the abundance of mutant Parkin inclusions, associated with Parkinson's disease. Aggregation of a polyglutamine-expanded Huntingtin, causative of Huntington's disease, appears unaltered by COMMD1. Altogether, this study offers new research directions to expand our current knowledge on the mechanisms underlying aggregation disease pathologies.

Sorting out the trash: the spatial nature of eukaryotic protein quality control.

Failure to maintain protein homeostasis is associated with aggregation and cell death, and underies a growing list of pathologies including neurodegenerative diseases, aging, and cancer. Misfolded proteins can be toxic and interfere with normal cellular functions, particularly during proteotoxic stress. Accordingly, molecular chaperones, the ubiquitin-proteasome system (UPS) and autophagy together promote refolding or clearance of misfolded proteins. Here we discuss emerging evidence that the pathways of protein quality control (PQC) are intimately linked to cell architecture, and sequester proteins into spatially and functionally distinct PQC compartments. This sequestration serves a number of functions, including enhancing the efficiency of quality control; clearing the cellular milieu of potentially toxic species and facilitating asymmetric inheritance of damaged proteins to promote rejuvenation of daughter cells.

Spatial sequestration of misfolded proteins by a dynamic chaperone pathway enhances cellular fitness during stress.

The extensive links between proteotoxic stress, protein aggregation and pathologies ranging from ageing to neurodegeneration underscore the importance of understanding how cells manage protein misfolding. Using live-cell imaging, we determine the fate of stress-induced misfolded proteins from their initial appearance until their elimination. Upon denaturation, misfolded proteins are sequestered from the bulk cytoplasm into dynamic endoplasmic reticulum (ER)-associated puncta that move and coalesce into larger structures in an energy-dependent but cytoskeleton-independent manner. These puncta, which we name Q-bodies, concentrate different misfolded and stress-denatured proteins en route to degradation, but do not contain amyloid aggregates, which localize instead to the insoluble protein deposit compartment. Q-body formation and clearance depends on an intact cortical ER and a complex chaperone network that is affected by rapamycin and impaired during chronological ageing. Importantly, Q-body formation enhances cellular fitness during stress. We conclude that spatial sequestration of misfolded proteins in Q-bodies is an early quality control strategy occurring synchronously with degradation to clear the cytoplasm of potentially toxic species.

The copper-transporting capacity of ATP7A mutants associated with Menkes disease is ameliorated by COMMD1 as a result of improved protein expression.

Menkes disease (MD) is an X-linked recessive disorder characterized by copper deficiency resulting in a diminished function of copper-dependent enzymes. Most MD patients die in early childhood, although mild forms of MD have also been described. A diversity of mutations in the gene encoding of the Golgi-resident copper-transporting P(1B)-type ATPase ATP7A underlies MD. To elucidate the molecular consequences of the ATP7A mutations, various mutations in ATP7A associated with distinct phenotypes of MD (L873R, C1000R, N1304S, and A1362D) were analyzed in detail. All mutants studied displayed changes in protein expression and intracellular localization parallel to a dramatic decline in their copper-transporting capacity compared to ATP7A the wild-type. We restored these observed defects in ATP7A mutant proteins by culturing the cells at 30°C, which improves the quality of protein folding, similar to that which as has recently has been demonstrated for misfolded ATP7B, a copper transporter homologous to ATP7A. Further, the effect of the canine copper toxicosis protein COMMD1 on ATP7A function was examined as COMMD1 has been shown to regulate the proteolysis of ATP7B proteins. Interestingly, in addition to adjusted growth temperature, binding of COMMD1 partially restored the expression, subcellular localization, and copper-exporting activities of the ATP7A mutants. However, no effect of pharmacological chaperones was observed. Together, the presented data might provide a new direction for developing therapies to improve the residual exporting activity of unstable ATP7A mutant proteins, and suggests a potential role for COMMD1 in this process.

Liver-specific Commd1 knockout mice are susceptible to hepatic copper accumulation.

Canine copper toxicosis is an autosomal recessive disorder characterized by hepatic copper accumulation resulting in liver fibrosis and eventually cirrhosis. We have identified COMMD1 as the gene underlying copper toxicosis in Bedlington terriers. Although recent studies suggest that COMMD1 regulates hepatic copper export via an interaction with the Wilson disease protein ATP7B, its importance in hepatic copper homeostasis is ill-defined. In this study, we aimed to assess the effect of Commd1 deficiency on hepatic copper metabolism in mice. Liver-specific Commd1 knockout mice (Commd1(Δhep)) were generated and fed either a standard or a copper-enriched diet. Copper homeostasis and liver function were determined in Commd1(Δhep) mice by biochemical and histological analyses, and compared to wild-type littermates. Commd1(Δhep) mice were viable and did not develop an overt phenotype. At six weeks, the liver copper contents was increased up to a 3-fold upon Commd1 deficiency, but declined with age to concentrations similar to those seen in controls. Interestingly, Commd1(Δhep) mice fed a copper-enriched diet progressively accumulated copper in the liver up to a 20-fold increase compared to controls. These copper levels did not result in significant induction of the copper-responsive genes metallothionein I and II, neither was there evidence of biochemical liver injury nor overt liver pathology. The biosynthesis of ceruloplasmin was clearly augmented with age in Commd1(Δhep) mice. Although COMMD1 expression is associated with changes in ATP7B protein stability, no clear correlation between Atp7b levels and copper accumulation in Commd1(Δhep) mice could be detected. Despite the absence of hepatocellular toxicity in Commd1(Δhep) mice, the changes in liver copper displayed several parallels with copper toxicosis in Bedlington terriers. Thus, these results provide the first genetic evidence for COMMD1 to play an essential role in hepatic copper homeostasis and present a valuable mouse model for further understanding of the molecular mechanisms underlying hepatic copper homeostasis.

Cu,Zn superoxide dismutase maturation and activity are regulated by COMMD1.

The maturation and activation of the anti-oxidant Cu,Zn superoxide dismutase (SOD1) are highly regulated processes that require several post-translational modifications. The maturation of SOD1 is initiated by incorporation of zinc and copper ions followed by disulfide oxidation leading to the formation of enzymatically active homodimers. Our present data indicate that homodimer formation is a regulated final step in SOD1 maturation and implicate the recently characterized copper homeostasis protein COMMD1 in this process. COMMD1 interacts with SOD1, and this interaction requires CCS-mediated copper incorporation into SOD1. COMMD1 does not regulate disulfide oxidation of SOD1 but reduces the level of SOD1 homodimers. RNAi-mediated knockdown of COMMD1 expression results in a significant induction of SOD1 activity and a consequent decrease in superoxide anion concentrations, whereas overexpression of COMMD1 exerts exactly the opposite effects. Here, we identify COMMD1 as a novel protein regulating SOD1 activation and associate COMMD1 function with the production of free radicals.

Nuclear-cytosolic transport of COMMD1 regulates NF-kappaB and HIF-1 activity.

Copper metabolism MURR1 domain1 (COMMD1) is a novel inhibitor of the transcription factors NF-kappaB and HIF-1, which play important roles in inflammation and tumor growth, respectively. In this study, we identified two highly conserved nuclear export signals (NESs) in COMMD1 and revealed that these NESs were essential and sufficient to induce maximal nuclear export of COMMD1. Inhibition of CRM1-mediated nuclear export by Leptomycin B resulted in nuclear accumulation of COMMD1. In addition, low oxygen concentrations induced the active export of COMMD1 from the nucleus in a CRM1-dependent manner. Disruption of the NESs in COMMD1 increased the repression of COMMD1 in transcriptional activity of NF-kappaB and HIF-1. In conclusion, these data indicate that COMMD1 undergoes constitutive nucleocytoplasmic transport as a novel mechanism to regulate NF-kappaB and HIF-1 signaling.

Role of transition metals in the pathogenesis of amyotrophic lateral sclerosis.

ALS (amyotrophic lateral sclerosis) is a devastating progressive neurodegenerative disorder resulting in selective degeneration of motor neurons in brain and spinal cord and muscle atrophy. In approx. 2% of all cases, the disease is caused by a mutation in the Cu,Zn-superoxide dismutase (SOD1) gene. The transition metals zinc and copper regulate SOD1 protein stability and activity, and disbalance of the homoeostasis of these metals has therefore been implicated in the pathogenesis of ALS. Recent data strengthen the hypothesis that these transition metals are excellent potential targets to develop an effective therapy for ALS.

Relevance of animal models for understanding mammalian copper homeostasis.

As a trace element, copper has a crucial role in mammalian metabolism, but it can be toxic in excess. The importance of a balanced copper homeostasis is illustrated by several copper-associated disorders in man, such as Menkes and Wilson disease, and in a wide variety of animal models (eg, mice, dogs, and sheep). Proteins involved in controlling copper metabolism have been well studied in yeast and in vitro. Recently, naturally occurring mutants and transgenic mouse models have been used to study the physiologic role of copper transporters in copper homeostasis. We discuss the most common mammalian animal models used to study copper-related diseases, evaluate what these model systems have recently shown about copper metabolism, and discuss the importance of these models for identifying specific and sensitive biomarkers associated with copper status in the near future.