Albrecht M. Clement

BAG3 mediates chaperone‐based aggresome‐targeting and selective autophagy of misfolded proteins

Increasing evidence indicates the existence of selective autophagy pathways, but the manner in which substrates are recognized and targeted to the autophagy system is poorly understood. One strategy is transport of a particular substrate to the aggresome, a perinuclear compartment with high autophagic activity. In this paper, we identify a new cellular pathway that uses the specificity of heat‐shock protein 70 (Hsp70) to misfolded proteins as the basis for aggresome‐targeting and autophagic degradation. This pathway is regulated by the stress‐induced co‐chaperone Bcl‐2‐associated athanogene 3 (BAG3), which interacts with the microtubule‐motor dynein and selectively directs Hsp70 substrates to the motor and thereby to the aggresome. Notably, aggresome‐targeting by BAG3 is distinct from previously described mechanisms, as it does not depend on substrate ubiquitination.

The DSD-1 Carbohydrate Epitope Depends on Sulfation, Correlates with Chondroitin Sulfate D Motifs, and Is Sufficient to Promote Neurite Outgrowth

The neural chondroitin sulfate (CS) proteoglycan (PG) DSD-1-PG was originally identified with the monoclonal antibody (mAb) 473HD. It promotes neurite outgrowth of hippocampal neurons when coated as a substrate in the presence of polycations. This effect is inhibited by mAb 473HD that specifically recognizes the DSD-1 epitope. The DSD-1 epitope is also detectable in CS-C and CS-D preparations from shark cartilage but not in other chondroitin sulfates that are structurally related and differ in their sulfation patterns. Non-sulfated DSD-1-PG and chemically desulfated CS-D were not recognized by mAb 473HD, suggesting that the DSD-1 epitope depends on sulfation. It was possible to enrich DSD-1 epitope-bearing carbohydrates and D disaccharide units from CS-C and CS-D preparations on a mAb 473HD affinity matrix. This indicates that the DSD-1 epitope represents a distinct glycosaminoglycan structure containing D units. The analysis of glycosaminoglycan digestion products by high pressure liquid chromatography revealed that DSD-1-PG preparations contain a unique D disaccharide unit as well as an A, a C, and a non-sulfated disaccharide unit. In neurite outgrowth assays with hippocampal neurons, substrate-bound CS-D promoted neurite outgrowth, whereas CS-A, CS-B, or CS-C did not. This effect of CS-D was inhibited by mAb 473HD. DSD-1 epitope-enriched fractions obtained from CS-D and CS-C promoted neurite outgrowth, whereas CS-C had no such effect prior to enrichment on the mAb 473HD matrix. Based on these findings we conclude that the DSD-1 epitope by itself is sufficient to promote neurite outgrowth and that this activity is possibly associated with D motifs.

DSD-1-Proteoglycan Is the Mouse Homolog of Phosphacan and Displays Opposing Effects on Neurite Outgrowth Dependent on Neuronal Lineage

DSD-1-PG is a chondroitin sulfate proteoglycan (CSPG) expressed by glial cells that can promote neurite outgrowth from rat embryonic mesencephalic (E14) and hippocampal (E18) neurons, an activity that is associated with the CS glycosaminoglycans (GAGs). Further characterization of DSD-1-PG has included sequencing of peptides from the core protein and the cloning of the corresponding cDNA using polyclonal antisera against DSD-1-PG to screen phage expression libraries. On the basis of these studies we have identified DSD-1-PG as the mouse homolog of phosphacan, a neural rat CSPG. Monoclonal antibodies 3H1 and 3F8 against carbohydrate residues on rat phosphacan recognize these epitopes on DSD-1-PG. The epitopes of the antibodies, L2/HNK-1 and L5/Lewis-X, which have been implicated in functional interactions, are also found on DSD-1-PG. Although DSD-1-PG has previously been shown to promote neurite outgrowth, its upregulation after stab wounding of the CNS and its localization in regions that are considered boundaries to axonal extension suggested that it may also have inhibitory functions. Neonatal dorsal root ganglion (DRG) explants grown on a rich supportive substrate (laminin) with and without DSD-1-PG were strikingly inhibited by the proteoglycan. The inhibitory effects of DSD-1-PG on the DRG explants were not relieved by removal of the CS GAGs, indicating that this activity is associated with the core glycoprotein. The neurite outgrowth from embryonic hippocampal neurons on laminin was not affected by the addition of DSD-1-PG. This indicates that DSD-1-PG/mouse phosphacan can have opposing effects on the process of neurite outgrowth dependent on neuronal lineage.

Ubiquitin-independent function of optineurin in autophagic clearance of protein aggregates

Summary Aggregation of misfolded proteins and the associated loss of neurons are considered a hallmark of numerous neurodegenerative diseases. Optineurin is present in protein inclusions observed in various neurodegenerative diseases including amyotrophic lateral sclerosis (ALS), Huntingtons disease, Alzheimers disease, Parkinsons disease, Creutzfeld-Jacob disease and Picks disease. Optineurin deletion mutations have also been described in ALS patients. However, the role of optineurin in mechanisms of protein aggregation remains unclear. In this report, we demonstrate that optineurin recognizes various protein aggregates via its C-terminal coiled-coil domain in a ubiquitin-independent manner. We also show that optineurin depletion significantly increases protein aggregation in HeLa cells and that morpholino-silencing of the optineurin ortholog in zebrafish causes the motor axonopathy phenotype similar to a zebrafish model of ALS. A more severe phenotype is observed when optineurin is depleted in zebrafish carrying ALS mutations. Furthermore, TANK1 binding kinase 1 (TBK1) is colocalized with optineurin on protein aggregates and is important in clearance of protein aggregates through the autophagy-lysosome pathway. TBK1 phosphorylates optineurin at serine 177 and regulates its ability to interact with autophagy modifiers. This study provides evidence for a ubiquitin-independent function of optineurin in autophagic clearance of protein aggregates as well as additional relevance for TBK1 as an upstream regulator of the autophagic pathway.

Chondroitin sulfate E promotes neurite outgrowth of rat embryonic day 18 hippocampal neurons

In light of controversial reports concerning the effects of chondroitin sulfates on neurite outgrowth, several glycosaminoglycans belonging to this structural class were compared with regard to their influence on axon formation by embryonic day 18 hippocampal neurons. In these studies, chondroitin sulfate A (CS-A), CS-B and CS-C proved weak or inefficient in the neurite outgrowth promotion assay. As expected, CS-D stimulated both the fraction of neurite bearing neurons and the length of their processes. This effect could be neutralized by the monoclonal antibody (mAb) 473HD. In contrast, CS-E enacted a dramatic promotion of neurite outgrowth. This effect persisted in the presence of mAb 473HD, consistent with the observation that this antibody did not react with CS-E in glycosaminoglycan transfer and blotting techniques. We conclude that CSE contains a novel glycosaminoglycan based neurite outgrowth promoting motif, which is distinct from other known activities.

One Enzyme, Two Functions PON2 PREVENTS MITOCHONDRIAL SUPEROXIDE FORMATION AND APOPTOSIS INDEPENDENT FROM ITS LACTONASE ACTIVITY

The human enzyme paraoxonase-2 (PON2) has two functions, an enzymatic lactonase activity and the reduction of intracellular oxidative stress. As a lactonase, it dominantly hydrolyzes bacterial signaling molecule 3OC12 and may contribute to the defense against pathogenic Pseudomonas aeruginosa. By its anti-oxidative effect, PON2 reduces cellular oxidative damage and influences redox signaling, which promotes cell survival. This may be appreciated but also deleterious given that high PON2 levels reduce atherosclerosis but may stabilize tumor cells. Here we addressed the unknown mechanisms and linkage of PON2 enzymatic and anti-oxidative function. We demonstrate that PON2 indirectly but specifically reduced superoxide release from the inner mitochondrial membrane, irrespective whether resulting from complex I or complex III of the electron transport chain. PON2 left O2̇̄ dismutase activities and cytochrome c expression unaltered, and it did not oxidize O2̇̄ but rather prevented its formation, which implies that PON2 acts by modulating quinones. To analyze linkage to hydrolytic activity, we introduced several point mutations and show that residues His114 and His133 are essential for PON2 activity. Further, we mapped its glycosylation sites and provide evidence that glycosylation, but not a native polymorphism Ser/Cys311, was critical to its activity. Importantly, none of these mutations altered the anti-oxidative/anti-apoptotic function of PON2, demonstrating unrelated activities of the same protein. Collectively, our study provides detailed mechanistic insight into the functions of PON2, which is important for its role in innate immunity, atherosclerosis, and cancer.

Heterodimer formation of wild-type and amyotrophic lateral sclerosis-causing mutant Cu/Zn-superoxide dismutase induces toxicity independent of protein aggregation

Recent studies provide evidence that wild-type Cu/Zn-superoxide dismutase (SOD1(hWT)) might be an important factor in mutant SOD1-mediated amyotrophic lateral sclerosis (ALS). In order to investigate its functional role in the pathogenesis of ALS, we designed fusion proteins of two SOD1 monomers linked by a polypeptide. We demonstrated that wild-type-like mutants, but not SOD1(G85R) homodimers, as well as mutant heterodimers including SOD1(G85R)-SOD1(hWT) display dismutase activity. Mutant homodimers showed an increased aggregation compared with the corresponding heterodimers in cell cultures and transgenic Caenorhabditis elegans, although SOD1(G85R) heterodimers are more toxic in functional assays. Our data show that (i) toxicity of mutant SOD1 is not correlated to its aggregation potential; (ii) dismutase-inactive mutants form dismutase-active heterodimers with SOD1(hWT); (iii) SOD1(hWT) can be converted to contribute to disease by forming active heterodimers. Therefore, we conclude that toxicity of mutant SOD1 is at least partially mediated through heterodimer formation with SOD1(hWT) in vivo and does not correlate with the aggregation potential of individual mutants.

HSF1-Controlled and Age-Associated Chaperone Capacity in Neurons and Muscle Cells of C. elegans

Protein stability under changing conditions is of vital importance for the cell and under the control of a fine-tuned network of molecular chaperones. Aging and age-related neurodegenerative diseases are directly associated with enhanced protein instability. Employing C. elegans expressing GFP-tagged luciferase as a reporter for evaluation of protein stability we show that the chaperoning strategy of body wall muscle cells and neurons is significantly different and that both are differently affected by aging. Muscle cells of young worms are largely resistant to heat stress, which is directly mediated by the stress response controlled through Heat Shock Transcription Factor 1. During recovery following heat stress the ability to refold misfolded proteins is missing. Young neurons are highly susceptible to chronic heat stress, but show a high potency to refold or disaggregate proteins during subsequent recovery. The particular proteome instability in neurons results from a delayed induction of the heat shock response. In aged neurons protein stability is increased during heat stress, whereas muscle cells show enhanced protein instability due to a deteriorated heat shock response. An efficient refolding activity is absent in both aged tissues. These results provide molecular insights into the differential protein stabilization capacity in different tissues and during aging.

Wild-type Cu/Zn superoxide dismutase (SOD1) does not facilitate, but impedes the formation of protein aggregates of amyotrophic lateral sclerosis causing mutant SOD1

Aggregation of Cu/Zn superoxide dismutase (SOD1) is a hallmark of a subset of familial amyotrophic lateral sclerosis (ALS) cases. The expression of wild-type SOD1 [SOD(hWT)] surprisingly exacerbates the phenotype of mutant SOD1 in vivo. Here we studied whether SOD1(hWT) may affect mutant SOD1 aggregation by employing fluorescence microscopy techniques combined with lifetime-based Förster resonance energy transfer (FRET). Only a very minor fraction of SOD1(hWT) was observed in aggregates induced by mutant SOD1(G37R), SOD1(G85R) or SOD1(G93C). Quite in contrast, co-expression of SOD(hWT) reduced the amount of mutant SOD1 in the aggregate fraction. Furthermore, we did not detect endogenous mouse SOD1 in aggregates formed by mutant SOD1 in two distinct mutant SOD1 mouse lines. The hypothesis that SOD1(WT) is able to keep mutant SOD1 variants in a soluble state is supported by the increased presence of heterodimers upon SOD1(hWT) co-expression. Therefore we propose that SOD1(WT) contributes to disease by heterodimerization with mutant SOD1 forms.

RAB3GAP1 and RAB3GAP2 modulate basal and rapamycin-induced autophagy

Macroautophagy is a degradative pathway that sequesters and transports cytosolic cargo in autophagosomes to lysosomes, and its deterioration affects intracellular proteostasis. Membrane dynamics accompanying autophagy are mostly elusive and depend on trafficking processes. RAB GTPase activating proteins (RABGAPs) are important factors for the coordination of cellular vesicle transport systems, and several TBC (TRE2-BUB2-CDC16) domain-containing RABGAPs are associated with autophagy. Employing C. elegans and human primary fibroblasts, we show that RAB3GAP1 and RAB3GAP2, which are components of the TBC domain-free RAB3GAP complex, influence protein aggregation and affect autophagy at basal and rapamycin-induced conditions. Correlating the activity of RAB3GAP1/2 with ATG3 and ATG16L1 and analyzing ATG5 punctate structures, we illustrate that the RAB3GAPs modulate autophagosomal biogenesis. Significant levels of RAB3GAP1/2 colocalize with members of the Atg8 family at lipid droplets, and their autophagy modulatory activity depends on the GTPase-activating activity of RAB3GAP1 but is independent of the RAB GTPase RAB3. Moreover, we analyzed RAB3GAP1/2 in relation to the previously reported suppressive autophagy modulators FEZ1 and FEZ2 and demonstrate that both reciprocally regulate autophagy. In conclusion, we identify RAB3GAP1/2 as novel conserved factors of the autophagy and proteostasis network.