13 research outputs found
Pharmacokinetic Modeling of [11C]GSK-189254, PET Tracer Targeting H3 Receptors, in Rat Brain
[Image: see text] The histamine H(3) receptor has been considered as a target for the treatment of various central nervous system diseases. Positron emission tomography (PET) studies with the radiolabeled potent and selective histamine H(3) receptor antagonist [(11)C]GSK-189254 in rodents could be used to examine the mechanisms of action of novel therapeutic drugs or to assess changes of regional H(3) receptor density in animal models of neurodegenerative disease. [(11)C]GSK-189254 was intravenously administered to healthy Wistar rats (n = 10), and a 60 min dynamic PET scan was carried out. Arterial blood samples were obtained during the scan to generate a metabolite-corrected plasma input function. PET data were analyzed using a one-tissue compartment model (1T2k), irreversible (2T3k) or reversible two-tissue compartment models (2T4k), graphical analysis (Logan and Patlak), reference tissue models (SRTM and SRTM2), and standard uptake values (SUVs). The Akaike information criterion and the standard error of the estimated parameters were used to select the most optimal quantification method. This study demonstrated that the 2T4k model with a fixed blood volume fraction and Logan graphical analysis can best describe the kinetics of [(11)C]GSK-189254 in the rat brain. SUV(40–60) and the reference tissue-based measurements DVR(2T4k), BP(ND)(SRTM), and SUV ratio could also be used as a simplified method to estimate H(3) receptor availability in case blood sampling is not feasible
Targeting DNA topoisomerases or checkpoint kinases results in an overload of chaperone systems, triggering aggregation of a metastable subproteome
A loss of the checkpoint kinase ataxia telangiectasia mutated (ATM) leads to impairments in the DNA damage response, and in humans causes cerebellar neurodegeneration, and an increased risk of cancer. A loss of ATM is also associated with increased protein aggregation. The relevance and characteristics of this aggregation are still incompletely understood. Moreover, it is unclear to what extent other genotoxic conditions can trigger protein aggregation as well. Here, we show that targeting ATM, but also ATR or DNA topoisomerases, results in the widespread aggregation of a metastable, disease-associated subfraction of the proteome. Aggregation-prone model substrates, including Huntingtin exon 1 containing an expanded polyglutamine repeat, aggregate faster under these conditions. This increased aggregation results from an overload of chaperone systems, which lowers the cell-intrinsic threshold for proteins to aggregate. In line with this, we find that inhibition of the HSP70 chaperone system further exacerbates the increased protein aggregation. Moreover, we identify the molecular chaperone HSPB5 as a cell-specific suppressor of it. Our findings reveal that various genotoxic conditions trigger widespread protein aggregation in a manner that is highly reminiscent of the aggregation occurring in situations of proteotoxic stress and in proteinopathies
Binding of the Dual-Action Anti-Parkinsonian Drug AG-0029 to Dopamine D-2 and Histamine H-3 Receptors:A PET Study in Healthy Rats
Introduction: Parkinson's disease (PD) is a neurodegenerative disorder characterized by motor dysfunction and a diverse range of nonmotor symptoms. Functional relationships between the dopaminergic and histaminergic systems suggest that dual-action pharmaceuticals like AG-0029 (D-2/D-3 agonist/H-3 antagonist) could ameliorate both the motor and cognitive symptoms of PD. The current study aimed to demonstrate the interaction of AG-0029 with its intended targets in the mammalian brain using positron emission tomography (PET). Methods: Healthy male Wistar rats were scanned with a small-animal PET camera, using either the dopamine D-2/D-3 receptor ligand [C-11]raclopride or the histamine H-3 receptor ligand [C-11]GSK-189254, before and after treatment with an intravenous, acute, single dose of AG-0029. Dynamic [C-11]raclopride PET data (60 min duration) were analyzed using the simplified reference tissue model 2 (SRTM2) with cerebellum as reference tissue and the nondisplaceable binding potential as the outcome parameter. Data from dynamic [C-11]GSK-189254 scans (60 min duration) with arterial blood sampling were analyzed using Logan graphical analysis with the volume of distribution (V-T) as the outcome parameter. Receptor occupancy was estimated using a Lassen plot. Results: Dopamine D-2/3 receptor occupancies in the striatum were 22.6 +/- 18.0 and 84.0 +/- 3.5% (mean +/- SD) after administration of 0.1 and 1 mg/kg AG-0029, respectively. In several brain regions, the V-T values of [C-11]GSK-189254 were significantly reduced after pretreatment of rats with 1 or 10 mg/kg AG-0029. The H-3 receptor occupancies were 11.9 +/- 8.5 and 40.3 +/- 11.3% for the 1 and 10 mg/kg doses of AG-0029, respectively. Conclusions: Target engagement of AG-0029 as an agonist at dopamine D-2/D-3 receptors and an antagonist at histamine H-3 receptors could be demonstrated in the rat brain with [C-11]raclopride and [C-11]GSK-189254 PET, respectively. The measured occupancy values reflect the previously reported high (subnanomolar) affinity of AG-0029 to D-2/D-3 and moderate (submicromolar) affinity to H-3 receptors
Myopathy associated BAG3 mutations lead to protein aggregation by stalling Hsp70 networks
BAG3 is a multi-domain hub that connects two classes of chaperones, small heat shock proteins (sHSPs) via two isoleucine-proline-valine (IPV) motifs and Hsp70 via a BAG domain.\ua0Mutations in either the IPV or BAG domain of BAG3 cause a dominant form of myopathy, characterized by protein aggregation in both skeletal and cardiac muscle tissues. Surprisingly, for both disease mutants, impaired chaperone binding is not sufficient to explain disease phenotypes. Recombinant mutants are correctly folded, show unaffected Hsp70 binding but are impaired in stimulating Hsp70-dependent client processing. As a consequence, the mutant BAG3 proteins become the node for a dominant gain of function causing aggregation of itself, Hsp70, Hsp70 clients and tiered interactors within the BAG3 interactome. Importantly, genetic and pharmaceutical interference with Hsp70 binding completely reverses stress-induced protein aggregation for both BAG3 mutations. Thus, the gain of function effects of BAG3 mutants act as Achilles heel of the HSP70 machinery
Comparison of intra-organellar chaperone capacity for dealing with stress-induced protein unfolding
Molecular chaperones are essential for cells to prevent that partially unfolded proteins form non-functional, toxic aggregates. This requirement is increased when cells experience protein unfolding stresses and such could affect all compartments in the eukaryotic cell. Whether all organelles are equipped with comparable chaperone capacities is largely unknown, mainly due to the lack of suitable reporters that allow such a comparison. Here we describe the development of fluorescent luciferase reporters that are sorted to various cellular locations (nucleus, cytoplasm, endoplasmic reticulum, and peroxisomes) and that differ minimally in their intrinsic thermal stability properties. When heating living cells, the rate of inactivation was most rapid for the nuclear-targeted luciferase, indicating that the nucleus is the most sensitive organelle toward heat-induced denaturing stress. Post-heat re-activation, however, occurred at equal kinetics irrespective of luciferase localization. Also, induction of thermotolerance by a priming heat treatment, that coordinately up-regulates all heat-inducible chaperones, resulted in a transient heat resistance of the luciferase in all organelles in a comparable manner. Overexpression of the main heat-inducible Hsp70 family member, HspA1A, protected only the cytosolic and nuclear, but not the other luciferases. Together, our data suggest that in each compartment investigated, including the peroxisome in which so far no chaperones could be detected, chaperone machines are present and can be induced with activities similar to those present in the cytosolic/nuclear compartment
The diverse members of the mammalian HSP70 machine show distinct chaperone-like activities
International audienceHumans contain many HSP70/HSPA and HSP40/DNAJ encoding genes, most of which are localized in the cytosol. To test for possible functional differences or/and substrate specificity, we assessed the effect of overexpression of each of these HSPs on refolding of heat-denatured luciferase and on the suppression of aggregation of a non-foldable polyglutamine (poly-Q) expanded Huntingtin fragment. Overexpressed chaperones that suppressed poly-Q aggregation were found not to be able to stimulate luciferase refolding. Inversely, chaperones that supported luciferase refolding were poor suppressors of polyQ aggregation. This was not related to client specificity per se, as the poly-Q aggregation inhibitors often also suppressed heat-induced aggregation of luciferase. Surprisingly, the exclusively heat-inducible HSPA6 lacks both luciferase refolding and poly-Q aggregation-suppressing activities. Furthermore, whereas overexpression of HSPA1A protected cells from heat-induced cell death, overexpression of HSPA6 did not. Inversely, siRNA mediated blocking of HSPA6 did not impair the development of heat-induced thermotolerance. Yet, HSPA6 has a functional substrate-binding domain and possesses intrinsic ATPase activity that is as high as that of the canonical HSPA1A when stimulated by J-proteins. In vitro data suggest that this may be relevant to substrate specificity as purified HSPA6 could not chaperone heat-unfolded luciferase but was able to assist in reactivation of heat-unfolded p53. So, even within the highly sequence-conserved HSPA family, functional differentiation is larger than expected with HSPA6 being an extreme example that may have evolved to maintain specific critical functions under conditions of severe stress
HSPB7 is the most potent polyQ aggregation suppressor within the HSPB family of molecular chaperones
A small number of heat-shock proteins have previously been shown to act protectively on aggregation of several proteins containing an extended polyglutamine (polyQ) stretch, which are linked to a variety of neurodegenerative diseases. A specific subfamily of heat-shock proteins is formed by the HSPB family of molecular chaperones, which comprises 10 members (HSPB1-10, also called small HSP). Several of them are known to act as anti-aggregation proteins in vitro. Whether they also act protectively in cells against polyQ aggregation has so far only been studied for few of them (e. g. HSPB1, HSPB5 and HSPB8). Here, we compared the 10 members of the human HSPB family for their ability to prevent aggregation of disease-associated proteins with an expanded polyQ stretch. HSPB7 was identified as the most active member within the HSPB family. It not only suppressed polyQ aggregation but also prevented polyQ-induced toxicity in cells and its expression reduces eye degeneration in a Drosophila polyQ model. Upon overexpression in cells, HSPB7 was not found in larger oligomeric species when expressed in cells and-unlike HSPB1-it did not improve the refolding of heat-denatured luciferase. The action of HSPB7 was also not dependent on the Hsp70 machine or on proteasomal activity, and HSPB7 overexpression alone did not increase autophagy. However, in ATG5-/- cells that are defective in macroautophagy, the anti-aggregation activity of HSPB7 was substantially reduced. Hence, HSPB7 prevents toxicity of polyQ proteins at an early stage of aggregate formation by a non-canonical mechanism that requires an active autophagy machinery
HSPB7 is the most potent polyQ aggregation suppressor within the HSPB family of molecular chaperones
A small number of heat-shock proteins have previously been shown to act protectively on aggregation of several proteins containing an extended polyglutamine (polyQ) stretch, which are linked to a variety of neurodegenerative diseases. A specific subfamily of heat-shock proteins is formed by the HSPB family of molecular chaperones, which comprises 10 members (HSPB1-10, also called small HSP). Several of them are known to act as anti-aggregation proteins in vitro. Whether they also act protectively in cells against polyQ aggregation has so far only been studied for few of them (e. g. HSPB1, HSPB5 and HSPB8). Here, we compared the 10 members of the human HSPB family for their ability to prevent aggregation of disease-associated proteins with an expanded polyQ stretch. HSPB7 was identified as the most active member within the HSPB family. It not only suppressed polyQ aggregation but also prevented polyQ-induced toxicity in cells and its expression reduces eye degeneration in a Drosophila polyQ model. Upon overexpression in cells, HSPB7 was not found in larger oligomeric species when expressed in cells and-unlike HSPB1-it did not improve the refolding of heat-denatured luciferase. The action of HSPB7 was also not dependent on the Hsp70 machine or on proteasomal activity, and HSPB7 overexpression alone did not increase autophagy. However, in ATG5-/- cells that are defective in macroautophagy, the anti-aggregation activity of HSPB7 was substantially reduced. Hence, HSPB7 prevents toxicity of polyQ proteins at an early stage of aggregate formation by a non-canonical mechanism that requires an active autophagy machinery
The S/T-Rich Motif in the DNAJB6 Chaperone Delays Polyglutamine Aggregation and the Onset of Disease in a Mouse Model
Expanded CAG repeats lead to debilitating neurodegenerative disorders characterized by aggregation of proteins with expanded polyglutamine (polyQ) tracts. The mechanism of aggregation involves primary and secondary nucleation steps. We show how a noncanonical member of the DNAJ-chaperone family, DNAJB6, inhibits the conversion of soluble polyQ peptides into amyloid fibrils, in particular by suppressing primary nucleation. This inhibition is mediated by a serine/threonine-rich region that provides an array of surface-exposed hydroxyl groups that bind to polyQ peptides and may disrupt the formation of the H bonds essential for the stability of amyloid fibrils. Early prevention of polyQ aggregation by DNAJB6 occurs also in cells and leads to delayed neurite retraction even before aggregates are visible. In a mouse model, brain-specific coexpression of DNAJB6 delays polyQ aggregation, relieves symptoms, and prolongs lifespan, pointing to DNAJB6 as a potential target for disease therapy and tool for unraveling early events in the onset of polyQ diseases. Kakkar et al. show that DNAJB6 is a chaperone that inhibits early steps in the formation of polyQ amyloid fibrils. An S/T-rich region in DNAJB6 is crucial for this function. In a polyQ mouse model, the inhibitory effects of DNAJB6 delay disease onset and increase lifespan