Damian Spencer

The Unfolded Protein Response and the Role of Protein Disulfide Isomerase in Neurodegeneration

The maintenance and regulation of proteostasis is a critical function for post-mitotic neurons and its dysregulation is increasingly implicated in neurodegenerative diseases. Despite having different clinical manifestations, these disorders share similar pathology; an accumulation of misfolded proteins in neurons and subsequent disruption to cellular proteostasis. The endoplasmic reticulum (ER) is an important component of proteostasis, and when the accumulation of misfolded proteins occurs within the ER, this disturbs ER homeostasis, giving rise to ER stress. This triggers the unfolded protein response (UPR), distinct signaling pathways that whilst initially protective, are pro-apoptotic if ER stress is prolonged. ER stress is increasingly implicated in neurodegenerative diseases, and emerging evidence highlights the complexity of the UPR in these disorders, with both protective and detrimental components being described. Protein Disulfide Isomerase (PDI) is an ER chaperone induced during ER stress that is responsible for the formation of disulfide bonds in proteins. Whilst initially considered to be protective, recent studies have revealed unconventional roles for PDI in neurodegenerative diseases, distinct from its normal function in the UPR and the ER, although these mechanisms remain poorly defined. However, specific aspects of PDI function may offer the potential to be exploited therapeutically in the future. This review will focus on the evidence linking ER stress and the UPR to neurodegenerative diseases, with particular emphasis on the emerging functions ascribed to PDI in these conditions.

Caspase-2: controversial killer or checkpoint controller?

The caspases are an evolutionarily conserved family of cysteine proteases, with essential roles in apoptosis or inflammation. Caspase-2 was the second caspase to be cloned and it resembles the prototypical nematode caspase CED-3 more closely than any other mammalian protein. An absence of caspase-2-specific reagents and the subtle phenotype of caspase-2-deficient mice have hampered definition of the physiological role of caspase-2 and identification of factors regulating its activity. Although some data implicate caspase-2 in apoptotic pathways, a link with apoptosis has been less firmly established for caspase-2 than for some other caspases. Emerging evidence suggests that caspase-2 regulates the cell cycle and may act as a tumour suppressor. This article critically reviews the current state of knowledge regarding the biochemistry and biology of this controversial caspase.

Redox Regulation in Amyotrophic Lateral Sclerosis

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease that results from the death of upper and lower motor neurons. Due to a lack of effective treatment, it is imperative to understand the underlying mechanisms and processes involved in disease progression. Regulations in cellular reduction/oxidation (redox) processes are being increasingly implicated in disease. Here we discuss the possible involvement of redox dysregulation in the pathophysiology of ALS, either as a cause of cellular abnormalities or a consequence. We focus on its possible role in oxidative stress, protein misfolding, glutamate excitotoxicity, lipid peroxidation and cholesterol esterification, mitochondrial dysfunction, impaired axonal transport and neurofilament aggregation, autophagic stress, and endoplasmic reticulum (ER) stress. We also speculate that an ER chaperone protein disulphide isomerase (PDI) could play a key role in this dysregulation. PDI is essential for normal protein folding by oxidation and reduction of disulphide bonds, and hence any disruption to this process may have consequences for motor neurons. Addressing the mechanism underlying redox regulation and dysregulation may therefore help to unravel the molecular mechanism involved in ALS.

Extracellular wildtype and mutant SOD1 induces ER-Golgi pathology characteristic of amyotrophic lateral sclerosis in neuronal cells

Amyotrophic lateral sclerosis (ALS) is a fatal and rapidly progressing neurodegenerative disorder and the majority of ALS is sporadic, where misfolding and aggregation of Cu/Zn-superoxide dismutase (SOD1) is a feature shared with familial mutant-SOD1 cases. ALS is characterized by progressive neurospatial spread of pathology among motor neurons, and recently the transfer of extracellular, aggregated mutant SOD1 between cells was demonstrated in culture. However, there is currently no evidence that uptake of SOD1 into cells initiates neurodegenerative pathways reminiscent of ALS pathology. Similarly, whilst dysfunction to the ER–Golgi compartments is increasingly implicated in the pathogenesis of both sporadic and familial ALS, it remains unclear whether misfolded, wildtype SOD1 triggers ER–Golgi dysfunction. In this study we show that both extracellular, native wildtype and mutant SOD1 are taken up by macropinocytosis into neuronal cells. Hence uptake does not depend on SOD1 mutation or misfolding. We also demonstrate that purified mutant SOD1 added exogenously to neuronal cells inhibits protein transport between the ER–Golgi apparatus, leading to Golgi fragmentation, induction of ER stress and apoptotic cell death. Furthermore, we show that extracellular, aggregated, wildtype SOD1 also induces ER–Golgi pathology similar to mutant SOD1, leading to apoptotic cell death. Hence extracellular misfolded wildtype or mutant SOD1 induce dysfunction to ER–Golgi compartments characteristic of ALS in neuronal cells, implicating extracellular SOD1 in the spread of pathology among motor neurons in both sporadic and familial ALS.

Formaldehyde-releasing prodrugs in combination with Adriamycin can overcome cellular drug resistance.

The anticancer drug Adriamycin is widely used in cancer chemotherapy and is classified as a topoisomerase II inhibitor. However, in the presence of formaldehyde, Adriamycin also forms high levels of DNA adducts. In this study, a new series of butyric acid and formaldehyde-releasing drugs related to AN9 (pivaloyloxymethyl butyrate) was assessed for their ability to facilitate Adriamycin-DNA adduct formation in Adriamycin-sensitive and -resistant cell lines (HL60 and HL60/MX2; MES-SA and MES-SA/Dx5). Drugs that released two molar equivalents of formaldehyde per mole of prodrug were superior in their ability to enhance adduct formation compared to those that released one molar equivalent. Adduct formation (as assessed by binding of radiolabeled Adriamycin to genomic DNA) was always lower in the resistant cell lines compared to the sensitive cell lines. However, in growth inhibition experiments, prodrug combinations were able to overcome Adriamycin resistance to varying degrees, and the combination of Adriamycin with selected prodrugs that release two moles of formaldehyde totally overcame resistance in HL60/MX2 cells. These HL60-derived cells express altered levels of topoisomerase II and also express a mutant form of the enzyme. Combinations of Adriamycin with selected prodrugs that release one or two moles of formaldehyde partially overcame P-glycoprotein-mediated resistance in MES-SA/Dx5 cells. Formaldehyde-releasing prodrugs (as single agents) overcame both forms of resistance in the two resistant cell lines, demonstrating that they were not substrates of these resistance mechanisms. Collectively, these results suggest that changing the mechanism via which Adriamycin exerts its anticancer effect by dramatically increasing adduct levels (requiring coadministration of formaldehyde-releasing prodrugs) may be a useful means of cancer treatment, as well as for overcoming Adriamycin-induced resistance.

Detection of labile anthracycline-DNA adducts by real-time PCR.

Barminomycin was employed as a model anthracycline that yields thermally stable drug-DNA adducts. Real-time PCR was utilized for the detection of these barminomycin-DNA adducts at drug levels as low as 100 nM in a cell-free assay system, with the lowest level of detection at approximately 20 nM. By contrast, doxorubicin-DNA adducts are heat labile and their levels were underestimated by conventional real-time PCR unless the DNA denaturation temperature was lowered by the addition of glycerol. Doxorubicin-DNA adduct levels of 5.5 per 10 kb were detected by real-time PCR (in the presence of 24% glycerol) following treatment with 0.5 microM doxorubicin (and 2 mM formaldehyde), considerably more sensitive than that detected by a gene-specific Southern-based procedure. Both the absolute fluorescence intensity in the linear PCR amplification range and the crossing point method provided useful dose-dependent estimates of adduct levels. The time required for a complete real-time PCR analysis of drug-induced adduct levels was approximately 40 min, and this may ultimately provide oncologists with a rapid means with which to monitor drug-DNA adduct levels in patients under treatment with anthracyclines. Responses to these drugs could be quickly and efficiently monitored in patients, thereby facilitating optimization of drug dosages as well as early detection of resistance to these agents.

ERp57 is protective against mutant SOD1-induced cellular pathology in amyotrophic lateral sclerosis

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disorder and mutations in superoxide dismutase 1 (SOD1) account for 20% of familial ALS cases. The aetiology of ALS remains unclear, but protein misfolding, endoplasmic reticulum (ER) stress and neuronal apoptosis are implicated. We previously established that protein disulphide isomerase (PDIA1) is protective against ER stress and apoptosis in neuronal cells expressing mutant SOD1, and recently mutations in PDIA1 and related PDI family member endoplasmic reticulum protein 57 (ERp57/PDIA3), were associated with ALS. Here, we examined whether ERp57 is also protective against mutant SOD1 or whether distinct specificity exists amongst individual PDI family members. Neuronal cells co-expressing SOD1 and ERp57 were examined for inclusion formation, ER stress, ubiquitin proteasome system (UPS) dysfunction and apoptosis. Over-expression of ERp57 inhibited inclusion formation, ER stress, UPS dysfunction and apoptosis, whereas silencing of ERp57 expression enhanced mutant SOD1 inclusion formation, ER stress and toxicity, indicating a protective role for ERp57 against SOD1 misfolding. ERp57 also inhibited the formation of mutant SOD1 inclusions and apoptosis in primary cortical neurons, thus confirming results obtained from cell lines. ERp57 partially co-localized with TAR DNA-binding protein-43 (TDP-43)-positive inclusions in spinal cords from sporadic ALS patients, thus linking ERp57 to protein misfolding in human sporadic disease. Our results therefore imply that ERp57 has a protective role against pathological events induced by mutant SOD1 and they link ERp57 to the misfolding of TDP-43. This study therefore has implications for the design of novel therapeutics based on the activities of the PDI family of proteins.