Damian C. Crowther

Endoplasmic reticulum dysfunction in neurological disease

Endoplasmic reticulum (ER) dysfunction might have an important part to play in a range of neurological disorders, including cerebral ischaemia, sleep apnoea, Alzheimers disease, multiple sclerosis, amyotrophic lateral sclerosis, the prion diseases, and familial encephalopathy with neuroserpin inclusion bodies. Protein misfolding in the ER initiates the well studied unfolded protein response in energy-starved neurons during stroke, which is relevant to the toxic effects of reperfusion. The toxic peptide amyloid β induces ER stress in Alzheimers disease, which leads to activation of similar pathways, whereas the accumulation of polymeric neuroserpin in the neuronal ER triggers a poorly understood ER-overload response. In other neurological disorders, such as Parkinsons and Huntingtons diseases, ER dysfunction is well recognised but the mechanisms by which it contributes to pathogenesis remain unclear. By targeting components of these signalling responses, amelioration of their toxic effects and so the treatment of a range of neurodegenerative disorders might become possible.

ANS binding reveals common features of cytotoxic amyloid species

Oligomeric assemblies formed from a variety of disease-associated peptides and proteins have been strongly associated with toxicity in many neurodegenerative conditions, such as Alzheimers disease. The precise nature of the toxic agents, however, remains still to be established. We show that prefibrillar aggregates of E22G (arctic) variant of the Abeta(1-42) peptide bind strongly to 1-anilinonaphthalene 8-sulfonate and that changes in this property correlate significantly with changes in its cytotoxicity. Moreover, we show that this phenomenon is common to other amyloid systems, such as wild-type Abeta(1-42), the I59T variant of human lysozyme and an SH3 domain. These findings are consistent with a model in which the exposure of hydrophobic surfaces as a result of the aggregation of misfolded species is a crucial and common feature of these pathogenic species.

Systematic In Vivo Analysis of the Intrinsic Determinants of Amyloid β Pathogenicity

Protein aggregation into amyloid fibrils and protofibrillar aggregates is associated with a number of the most common neurodegenerative diseases. We have established, using a computational approach, that knowledge of the primary sequences of proteins is sufficient to predict their in vitro aggregation propensities. Here we demonstrate, using rational mutagenesis of the Aβ42 peptide based on such computational predictions of aggregation propensity, the existence of a strong correlation between the propensity of Aβ42 to form protofibrils and its effect on neuronal dysfunction and degeneration in a Drosophila model of Alzheimer disease. Our findings provide a quantitative description of the molecular basis for the pathogenicity of Aβ and link directly and systematically the intrinsic properties of biomolecules, predicted in silico and confirmed in vitro, to pathogenic events taking place in a living organism.

Protein misfolding and disease: from the test tube to the organism

Protein misfolding is the underlying cause of many highly debilitating disorders ranging from Alzheimers Disease to Cystic Fibrosis. Great strides have been made recently in understanding what causes proteins to misfold, primarily through the use of biophysical and computational techniques that enable systematic and quantitative analysis of the effects of a range of different perturbations in proteins. Correlation of the results of such analyses with observations made in animal models of disease has however been limited by their seemingly irreconcilable differences in methodology and scope. Several recent studies have however begun to overcome this limitation by combining the two approaches. This strategy has made it possible to investigate many of the consequences of protein misfolding in vivo, ranging from disease pathogenesis to epigenetic regulation, in the context of the fundamental physico-chemical principles derived from extensive and highly detailed studies undertaken in vitro.

Fenton chemistry and oxidative stress mediate the toxicity of the β-amyloid peptide in a Drosophila model of Alzheimer’s disease

The mechanism by which aggregates of the β‐amyloid peptide (Aβ) mediate their toxicity is uncertain. We show here that the expression of the 42‐amino‐acid isoform of Aβ (Aβ1–42) changes the expression of genes involved in oxidative stress in a Drosophila model of Alzheimer’s disease. A subsequent genetic screen confirmed the importance of oxidative stress and a molecular dissection of the steps in the cellular metabolism of reactive oxygen species revealed that the iron‐binding protein ferritin and the H2O2 scavenger catalase are the most potent suppressors of the toxicity of wild‐type and Arctic (E22G) Aβ1–42. Likewise, treatment with the iron‐binding compound clioquinol increased the lifespan of flies expressing Arctic Aβ1–42. The effect of iron appears to be mediated by oxidative stress as ferritin heavy chain co‐expression reduced carbonyl levels in Aβ1–42 flies by 65% and restored the survival and locomotion function to normal. This was achieved despite the presence of elevated levels of the Aβ1–42. Taken together, our data show that oxidative stress, probably mediated by the hydroxyl radical and generated by the Fenton reaction, is essential for Aβ1–42 toxicity in vivo and provide strong support for Alzheimer’s disease therapies based on metal chelation.

Iron promotes the toxicity of amyloid beta peptide by impeding its ordered aggregation

We have previously shown that overexpressing subunits of the iron-binding protein ferritin can rescue the toxicity of the amyloid β (Aβ) peptide in our Drosophila model system. These data point to an important pathogenic role for iron in Alzheimer disease. In this study, we have used an iron-selective chelating compound and RNAi-mediated knockdown of endogenous ferritin to further manipulate iron in the brain. We confirm that chelation of iron protects the fly from the harmful effects of Aβ. To understand the pathogenic mechanisms, we have used biophysical techniques to see how iron affects Aβ aggregation. We find that iron slows the progression of the Aβ peptide from an unstructured conformation to the ordered cross-β fibrils that are characteristic of amyloid. Finally, using mammalian cell culture systems, we have shown that iron specifically enhances Aβ toxicity but only if the metal is present throughout the aggregation process. These data support the hypothesis that iron delays the formation of well ordered aggregates of Aβ and so promotes its toxicity in Alzheimer disease.

Endoplasmic reticulum-associated degradation (ERAD) and autophagy cooperate to degrade polymerogenic mutant serpins.

The serpinopathies are a family of diseases characterized by the accumulation of ordered polymers of mutant protein within the endoplasmic reticulum. They are a diverse group including α1-antitrypsin deficiency and the inherited dementia familial encephalopathy with neuroserpin inclusion bodies or FENIB. We have used transient transfection of COS7 cells and mouse embryonic fibroblasts, PC12 cell lines that conditionally express wild type and mutant neuroserpin and fly models of FENIB to assess the cellular handling of wild type and mutant serpins. By using a polymer-specific monoclonal antibody, we show that mutant neuroserpin forms polymers after a delay of at least 30 min and that polymers can be cleared in PC12 cell lines and from the brain in a fly model of FENIB. At steady state, the fractions of intracellular polymerogenic G392E mutant neuroserpin in the monomeric and polymeric states are comparable. Inhibition of the proteasome with MG132 reveals that both mutant neuroserpin and α1-antitrypsin are degraded predominantly by endoplasmic reticulum-associated degradation (ERAD). Pharmacological and genetic inhibitions demonstrate that autophagy is responsible for bulk turnover of wild type and mutant serpins, but can be stimulated by rapamycin to compensate for proteasome inhibition. The significance of these findings to the treatment of serpinopathies is discussed.

Plasma and CSF serpins in Alzheimer disease and dementia with Lewy bodies

Objective: Serine protease inhibitors (serpins), the acute phase reactants and regulators of the proteolytic processing of proteins, have been recognized as potential contributors to the pathogenesis of Alzheimer disease (AD). We measured plasma and CSF levels of serpins in controls and patients with dementia. Methods: Using rocket immunoelectrophoresis, ELISA, and Luminex xMAP technology, we analyzed plasma levels of α1-antichymotrypsin and α1-antitrypsin, and CSF levels of α1-antichymotrypsin, α1-antitrypsin, and neuroserpin along with three standard biomarkers (total tau, tau phosphorylated at threonine-181, and the Aβ1-42) in patients with AD (n = 258), patients with dementia with Lewy bodies (DLB; n = 38), and age-matched controls (n = 37). Results: The level of CSF neuroserpin was significantly higher in AD compared with controls and DLB, whereas CSF α1-antichymotrypsin and α1-antitrypsin were significantly higher in both AD and DLB groups than in controls. Results from logistic regression analyses demonstrate a relationship between higher CSF levels of α1-antichymotrypsin and neuroserpin and increased predicted probability and odds ratios (ORs) of AD (OR 5.3, 95% CI 1.3 to 20.8 and OR 3.3, CI 1.3 to 8.8). Furthermore, a logistic regression model based on CSF α1-antichymotrypsin, neuroserpin, and Aβ1-42 enabled us to discriminate between AD patients and controls with a sensitivity of 94.7% and a specificity of 77.8%. Conclusions: Higher CSF levels of neuroserpin and α1-antichymotrypsin were associated with the clinical diagnosis of Alzheimer disease (AD) and facilitated the diagnostic classification of AD vs controls. CSF serpin levels did not improve the diagnostic classification of AD vs dementia with Lewy bodies. GLOSSARY: AAT = α1-antitrypsin; ACT = α1-antichymotrypsin; AD = Alzheimer disease; ApoE = apolipoprotein E; AUC = area under the curve; BBB = blood-brain barrier; COPD = chronic obstructive pulmonary disease; %CV = coefficients of variation percentage; DLB = dementia with Lewy bodies; IL = interleukin; MMSE = Mini-Mental State Examination; NSAIDs = nonsteroidal anti-inflammatory drugs; OR = odds ratio; P-tau = tau phosphorylated at threonine-181; ROC = receiver operating characteristic; T-tau = total tau.

Mutant neuroserpin (S49P) that causes familial encephalopathy with neuroserpin inclusion bodies is a poor proteinase inhibitor and readily forms polymers in vitro

Familial encephalopathy with neuroserpin inclusion bodies (FENIB) is an autosomal dominant dementia that is characterized by intraneuronal inclusions of mutant neuroserpin. We report here the expression, purification, and characterization of wild-type neuroserpin and neuroserpin containing the S49P mutation that causes FENIB. Wild-type neuroserpin formed SDS-stable complexes with tPA with an association rate constant and K i of 1.2 × 104 m −1 s−1 and 5.8 nm, respectively. In contrast, S49P neuroserpin formed unstable complexes with an association rate constant and K i of 0.3 × 104 m −1 s−1 and 533.3 nm, respectively. An assessment by circular dichroism showed that S49P neuroserpin had a lower melting temperature than wild-type protein (49.9 and 56.6 °C, respectively) and more readily formed loop-sheet polymers under physiological conditions. Neither the wild-type nor S49P neuroserpin accepted the P7-P2 α1-anti-trypsin or P14-P3 antithrombin-reactive loop peptides that have been shown to block polymer formation in other members of the serpin superfamily. Taken together, these data demonstrate that S49P neuroserpin is a poor proteinase inhibitor and readily forms loop-sheet polymers. These findings provide strong support for the role of neuroserpin polymerization in the formation of the intraneuronal inclusions that are characteristic of FENIB.

The intracellular accumulation of polymeric neuroserpin explains the severity of the dementia FENIB

Familial encephalopathy with neuroserpin inclusion bodies (FENIB) is an autosomal dominant dementia that is characterized by the retention of polymers of neuroserpin as inclusions within the endoplasmic reticulum (ER) of neurons. We have developed monoclonal antibodies that detect polymerized neuroserpin and have used COS-7 cells, stably transfected PC12 cell lines and transgenic Drosophila melanogaster to characterize the cellular handling of all four mutant forms of neuroserpin that cause FENIB. We show a direct correlation between the severity of the disease-causing mutation and the accumulation of neuroserpin polymers in cell and fly models of the disease. Moreover, mutant neuroserpin causes locomotor deficits in the fly allowing us to demonstrate a direct link between polymer accumulation and neuronal toxicity.