The travel diaries of tetanus and botulinum neurotoxins.

Tetanus (TeNT) and botulinum (BoNT) neurotoxins, the causative agents of tetanus and botulism, respectively, are the most potent toxic molecules known to mankind. This extreme potency is attributed to: i) their specificity for essential components of the neurotransmitter release machinery present at vertebrate synapses, and ii) their high-affinity targeting to motor neurons by binding to polysialogangliosides and protein receptors. Comprising the clostridial neurotoxin family, TeNT and BoNTs engage distinct surface receptors and intracellular sorting pathways in neurons. BoNTs bind to the intraluminal domain of specific synaptic vesicle proteins that are exposed to the extracellular milieu upon exocytosis, and are taken up by synaptic vesicle recycling. A sizeable proportion of BoNT molecules remain at the neuromuscular junction, where their protease moiety is released into the cytoplasm, blocking synaptic transmission and causing flaccid paralysis. In contrast, TeNT undergoes binding to specific components of the basal membrane at the neuromuscular junction, is endocytosed into motor neurons and sorted to axonal signalling endosomes. Following this, TeNT is transported to the soma of motor neurons located in the spinal cord or brainstem, and then transcytosed to inhibitory interneurons, where it blocks synaptic transmission. TeNT-induced impairment of inhibitory input leads to hyperactivity of motor neurons, causing spastic paralysis, which is the hallmark of tetanus. This review examines the molecular mechanisms leading to the entry, sorting and intracellular trafficking of TeNT and BoNTs.

Hsp90 inhibition protects against inherited retinal degeneration.

The molecular chaperone Hsp90 is important for the functional maturation of many client proteins, and inhibitors are in clinical trials for multiple indications in cancer. Hsp90 inhibition activates the heat shock response and can improve viability in a cell model of the P23H misfolding mutation in rhodopsin that causes autosomal dominant retinitis pigmentosa (adRP). Here, we show that a single low dose of the Hsp90 inhibitor HSP990 enhanced visual function and delayed photoreceptor degeneration in a P23H transgenic rat model. This was associated with the induction of heat shock protein expression and reduced rhodopsin aggregation. We then investigated the effect of Hsp90 inhibition on a different type of rod opsin mutant, R135L, which is hyperphosphorylated, binds arrestin and disrupts vesicular traffic. Hsp90 inhibition with 17-AAG reduced the intracellular accumulation of R135L and abolished arrestin binding in cells. Hsf-1(-/-) cells revealed that the effect of 17-AAG on P23H aggregation was dependent on HSF-1, whereas the effect on R135L was HSF-1 independent. Instead, the effect on R135L was mediated by a requirement of Hsp90 for rhodopsin kinase (GRK1) maturation and function. Importantly, Hsp90 inhibition restored R135L rod opsin localization to wild-type (WT) phenotype in vivo in rat retina. Prolonged Hsp90 inhibition with HSP990 in vivo led to a posttranslational reduction in GRK1 and phosphodiesterase (PDE6) protein levels, identifying them as Hsp90 clients. These data suggest that Hsp90 represents a potential therapeutic target for different types of rhodopsin adRP through distinct mechanisms, but also indicate that sustained Hsp90 inhibition might adversely affect visual function.

Molecular chaperone mediated late-stage neuroprotection in the SOD1(G93A) mouse model of amyotrophic lateral sclerosis.

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disorder characterized by the selective loss of motor neurons in the spinal cord, brain stem, and motor cortex. Mutations in superoxide dismutase (SOD1) are associated with familial ALS and lead to SOD1 protein misfolding and aggregation. Here we show that the molecular chaperone, HSJ1 (DNAJB2), mutations in which cause distal hereditary motor neuropathy, can reduce mutant SOD1 aggregation and improve motor neuron survival in mutant SOD1 models of ALS. Overexpression of human HSJ1a (hHSJ1a) in vivo in motor neurons of SOD1(G93A) transgenic mice ameliorated disease. In particular, there was a significant improvement in muscle force, increased motor unit number and enhanced motor neuron survival. hHSJ1a was present in a complex with SOD1(G93A) and led to reduced SOD1 aggregation at late stages of disease progression. We also observed altered ubiquitin immunoreactivity in the double transgenic animals, suggesting that ubiquitin modification might be important for the observed improvements. In a cell model of SOD1(G93A) aggregation, HSJ1a preferentially bound to mutant SOD1, enhanced SOD1 ubiquitylation and reduced SOD1 aggregation in a J-domain and ubiquitin interaction motif (UIM) dependent manner. Collectively, the data suggest that HSJ1a acts on mutant SOD1 through a combination of chaperone, co-chaperone and pro-ubiquitylation activity. These results show that targeting SOD1 protein misfolding and aggregation in vivo can be neuroprotective and suggest that manipulation of DnaJ molecular chaperones might be useful in the treatment of ALS.

The cell stress machinery and retinal degeneration.

Retinal degenerations are a group of clinically and genetically heterogeneous disorders characterised by progressive loss of vision due to neurodegeneration. The retina is a highly specialised tissue with a unique architecture and maintaining homeostasis in all the different retinal cell types is crucial for healthy vision. The retina can be exposed to a variety of environmental insults and stress, including light-induced damage, oxidative stress and inherited mutations that can lead to protein misfolding. Within retinal cells there are different mechanisms to cope with disturbances in proteostasis, such as the heat shock response, the unfolded protein response and autophagy. In this review, we discuss the multiple responses of the retina to different types of stress involved in retinal degenerations, such as retinitis pigmentosa, age-related macular degeneration and glaucoma. Understanding the mechanisms that maintain and re-establish proteostasis in the retina is important for developing new therapeutic approaches to fight blindness.

NUB1 modulation of GSK3ß reduces tau aggregation.

Abnormal phosphorylation of the microtubule-associated protein tau in neurodegenerative disorders, including Alzheimer's disease (AD) and frontotemporal lobar degeneration, is associated with disrupted axonal transport and synaptic dysfunction ultimately manifesting as histopathological lesions of protein aggregates. Glycogen synthase kinase 3ß (GSK3ß) may be critical for the pathological hyperphosphorylation of tau. Here, we examined the role of the proteasome-associated protein Nedd8 ultimate buster 1 (NUB1) in the neuropathogenic phosphorylation and aggregation of tau. We reveal that NUB1 interacted with both tau and GSK3ß to disrupt their interaction, and abolished recruitment of GSK3ß to tau inclusions. Moreover, NUB1 reduced GSK3ß-mediated phosphorylation of tau and aggregation of tau in intracellular inclusions. Strikingly, NUB1 induced GSK3ß degradation. Deletion of the NUB1 ubiquitin-like (UBL) domain did not impair the interaction with tau and GSK3ß, and the ability to suppress the phosphorylation and aggregation of tau was not affected. However, the UBL motif was necessary for GSK3ß degradation. Deletion of the NUB1 ubiquitin-associated (UBA) domain abrogated the ability of NUB1 to interact with and degrade GSK3ß. Moreover, the UBA domain was required to suppress the aggregation of tau. Silencing of NUB1 in cells stabilized endogenous GSK3ß and exacerbated tau phosphorylation. Thus, we propose that NUB1, by regulating GSK3ß levels, modulates tau phosphorylation and aggregation, and is a key player in neurodegeneration associated with tau pathology. Moreover, NUB1 regulation of GSK3ß could modulate numerous signalling pathways in which GSK3ß is a centrally important effector.

BiP prevents rod opsin aggregation.

Mutations in rod opsin-the light-sensitive protein of rod cells-cause retinitis pigmentosa. Many rod opsin mutations lead to protein misfolding, and therefore it is important to understand the role of molecular chaperones in rod opsin biogenesis. We show that BiP (HSPA5) prevents the aggregation of rod opsin. Cleavage of BiP with the subtilase cytotoxin SubAB results in endoplasmic reticulum (ER) retention and ubiquitylation of wild-type (WT) rod opsin (WT-green fluorescent protein [GFP]) at the ER. Fluorescence recovery after photobleaching reveals that WT-GFP is usually mobile in the ER. By contrast, depletion of BiP activity by treatment with SubAB or coexpression of a BiP ATPase mutant, BiP(T37G), decreases WT-GFP mobility to below that of the misfolding P23H mutant of rod opsin (P23H-GFP), which is retained in the ER and can form cytoplasmic ubiquitylated inclusions. SubAB treatment of P23H-GFP-expressing cells decreases the mobility of the mutant protein further and leads to ubiquitylation throughout the ER. Of interest, BiP overexpression increases the mobility of P23H-GFP, suggesting that it can reduce mutant rod opsin aggregation. Therefore inhibition of BiP function results in aggregation of rod opsin in the ER, which suggests that BiP is important for maintaining the solubility of rod opsin in the ER.

Suppression of protein aggregation by chaperone modification of high molecular weight complexes.

Protein misfolding and aggregation are associated with many neurodegenerative diseases, including Huntington's disease. The cellular machinery for maintaining proteostasis includes molecular chaperones that facilitate protein folding and reduce proteotoxicity. Increasing the protein folding capacity of cells through manipulation of DNAJ chaperones has been shown to suppress aggregation and ameliorate polyglutamine toxicity in cells and flies. However, to date these promising findings have not been translated to mammalian models of disease. To address this issue, we developed transgenic mice that over-express the neuronal chaperone HSJ1a (DNAJB2a) and crossed them with the R6/2 mouse model of Huntington's disease. Over-expression of HSJ1a significantly reduced mutant huntingtin aggregation and enhanced solubility. Surprisingly, this was mediated through specific association with K63 ubiquitylated, detergent insoluble, higher order mutant huntingtin assemblies that decreased their ability to nucleate further aggregation. This was dependent on HSJ1a client binding ability, ubiquitin interaction and functional co-operation with HSP70. Importantly, these changes in mutant huntingtin solubility and aggregation led to improved neurological performance in R6/2 mice. These data reveal that prevention of further aggregation of detergent insoluble mutant huntingtin is an additional level of quality control for late stage chaperone-mediated neuroprotection. Furthermore, our findings represent an important proof of principle that DNAJ manipulation is a valid therapeutic approach for intervention in Huntington's disease.

Molecular chaperone-mediated rescue of mitophagy by a Parkin RING1 domain mutant.

Mitochondrial dysfunction is characteristic of many neurodegenerative diseases. The Parkinson's disease-associated ubiquitin-protein ligase, Parkin, is important in the elimination of damaged mitochondria by autophagy (mitophagy) in a multistep process. Here, we show that a Parkin RING domain mutant (C289G) fails to redistribute to damaged mitochondria and cannot induce mitophagy after treatment with the mitochondrial uncoupler carbonyl cyanide m-methylhydrazone, because of protein misfolding and aggregation. Parkin(C289G) aggregation and inclusion formation were suppressed by the neuronal DnaJ/Hsp40 chaperone HSJ1a(DNAJB2a). Importantly, HSJ1a and DNAJB6 also restored mitophagy by promoting the relocation of Parkin(C289G) and the autophagy marker LC3 to depolarized mitochondria. The rescue of Parkin activity and suppression of aggregation were J domain dependent for HSJ1a, suggesting the involvement of Hsp70 in these processes, but were not dependent on the HSJ1a ubiquitin interaction motif. HSJ1a expression did not enhance mitophagy mediated by wild-type Parkin. These data show the potential of molecular chaperones to mediate the functional recovery of Parkin misfolding mutants and to combat deficits associated with Parkin aggregation in Parkinson's disease.

Treatment with extracellular HSP70/HSC70 protein can reduce polyglutamine toxicity and aggregation.

The accumulation of insoluble protein aggregates is a feature of neurodegenerative disease. Overexpression of Heat Shock Protein 70 (HSP70) can protect cells with protein aggregates from apoptosis. Another trait of HSP70 is its ability to cross the plasma membrane. Therefore, we purified a preparation of HSP70/HSC70 from bovine muscle and used it in a model of Huntington's disease. Human neuroblastoma SK-N-SH cells were transfected with huntington exon 1 with short (25) or long (103) CAG trinucleotide repeats coupled to green flourescent protein (GFP). Cells transfected with the long polyCAG repeat had insoluble protein aggregates and died through apoptosis. Biotinylated HSP70/HSC70 incorporated into the culture medium appeared inside the cells within 3-6 h of incubation. This incorporation correlated with a reduction in apoptotic cells by 40-50%. Confocal microscopy revealed that labelled internalized HSP70/HSC70 co-localized with the polyglutamine inclusions. The measurement of the number and size of inclusions showed that HSP70/HSC70 was able to reduce both these parameters. A filter trap assay and immunoblotting demonstrated that the introduction of HSP70/HSC70 also decreased protein aggregation. Together with earlier data on the effects of exogenously administered HSP70/HSC70 on cultured cells and on animals, these data show that preparations based on HSP70 may have some potential as therapies for a variety of neurodegenerative pathologies.