Sergey S. Novoselov

Late stage treatment with arimoclomol delays disease progression and prevents protein aggregation in the SOD1G93A mouse model of ALS

Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disorder characterized by motoneuron degeneration, resulting in muscle paralysis and death, typically within 1–5 years of diagnosis. Although the pathogenesis of ALS remains unclear, there is evidence for the involvement of proteasome dysfunction and heat shock proteins in the disease. We have previously shown that treatment with a co‐inducer of the heat shock response called arimoclomol is effective in the SODG93A mouse model of ALS, delaying disease progression and extending the lifespan of SODG93A mice ( Kieran et al. 2004 ). However, this previous study only examined the effects arimoclomol when treatment was initiated in pre‐ or early symptomatic stages of the disease. Clearly, to be of benefit to the majority of ALS patients, any therapy must be effective after symptom onset. In order to establish whether post‐symptomatic treatment with arimoclomol is effective, in this study we carried out a systematic assessment of different treatment regimes in SODG93A mice. Treatment with arimoclomol from early (75 days) or late (90 days) symptomatic stages significantly improved muscle function. Treatment from 75 days also significantly increased the lifespan of SODG93A mice, although treatment from 90 days has no significant effect on lifespan. The mechanism of action of arimoclomol involves potentiation of the heat shock response, and treatment with arimoclomol increased Hsp70 expression. Interestingly, this up‐regulation in Hsp70 was accompanied by a decrease in the number of ubiquitin‐positive aggregates in the spinal cord of treated SODG93A mice, suggesting that arimoclomol directly effects protein aggregation and degradation.

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 Huntingtons 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.

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.

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.

DnaJB6 Is Present in the Core of Lewy Bodies and Is Highly Up-Regulated in Parkinsonian Astrocytes

DnaJ/Hsp40 chaperones determine the activity of Hsp70s by stabilizing their interaction with substrate proteins. We have predicted, based on the in silico analysis of a brain‐derived whole‐genome transcriptome data set, an increased expression of DnaJ/Hsp40 homologue, subfamily B, member 6 (DnaJB6) in Parkinsons disease (PD; Moran et al. [ 2006 ] Neurogenetics 7:1–11). We now show that DnaJB6 is a novel component of Lewy bodies (LBs) in both PD substantia nigra and PD cortex and that it is strongly up‐regulated in parkinsonian astrocytes. The presence of DnaJB6 in the center of LBs suggests an early and direct involvement of this chaperone in the neuronal disease process associated with PD. The strong concomitant expression of DnaJB6 in astrocytes emphasizes the involvement of glial cells in PD and could indicate a route for therapeutic intervention. Extracellular alpha‐synuclein originating from intravesicular alpha‐synuclein is prone to aggregation and the potential source of extracellular aggregates (Lee [ 2008 ] J. Mol. Neurosci. 34:17–22). The observed strong expression of DnaJB6 by astrocytes could reflect a protective reaction, so reducing the neuronal release of toxic alpha‐synuclein and supporting the astrocyte response in PD might limit the progression of the disease process.

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

Mitochondrial dysfunction is characteristic of many neurodegenerative diseases. The Parkinsons 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 Parkinsons disease.

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.

The heat shock response plays an important role in TDP-43 clearance: evidence for dysfunction in amyotrophic lateral sclerosis

Insoluble TDP-43 inclusions are the pathological hallmark of ALS and tau-negative frontotemporal lobar degeneration. Chen et al. show that the heat shock response (HSR), which regulates chaperone expression, is compromised in an ALS mouse model and in patients. Activation of the HSR clears insoluble TDP-43 and increases cell survival.

The heat-shock response co-inducer arimoclomol protects against retinal degeneration in rhodopsin retinitis pigmentosa

Retinitis pigmentosa (RP) is a group of inherited diseases that cause blindness due to the progressive death of rod and cone photoreceptors in the retina. There are currently no effective treatments for RP. Inherited mutations in rhodopsin, the light-sensing protein of rod photoreceptor cells, are the most common cause of autosomal-dominant RP. The majority of mutations in rhodopsin, including the common P23H substitution, lead to protein misfolding, which is a feature in many neurodegenerative disorders. Previous studies have shown that upregulating molecular chaperone expression can delay disease progression in models of neurodegeneration. Here, we have explored the potential of the heat-shock protein co-inducer arimoclomol to ameliorate rhodopsin RP. In a cell model of P23H rod opsin RP, arimoclomol reduced P23H rod opsin aggregation and improved viability of mutant rhodopsin-expressing cells. In P23H rhodopsin transgenic rat models, pharmacological potentiation of the stress response with arimoclomol improved electroretinogram responses and prolonged photoreceptor survival, as assessed by measuring outer nuclear layer thickness in the retina. Furthermore, treated animal retinae showed improved photoreceptor outer segment structure and reduced rhodopsin aggregation compared with vehicle-treated controls. The heat-shock response (HSR) was activated in P23H retinae, and this was enhanced with arimoclomol treatment. Furthermore, the unfolded protein response (UPR), which is induced in P23H transgenic rats, was also enhanced in the retinae of arimoclomol-treated animals, suggesting that arimoclomol can potentiate the UPR as well as the HSR. These data suggest that pharmacological enhancement of cellular stress responses may be a potential treatment for rhodopsin RP and that arimoclomol could benefit diseases where ER stress is a factor.

BiP prevents rod opsin aggregation

Misfolding mutations in rod opsin cause the blinding disease retinitis pigmentosa. The ER chaperone BiP is required to prevent rod opsin aggregation, and overexpression of BiP can improve mutant rod opsin mobility. This could be important for therapies based on manipulating rod opsin folding and aggregation.