Protein Misfolding and the Serpinopathies

The serpins are the largest superfamily of protease inhibitors. They are found in almost all branches of life including viruses, prokaryotes and eukaryotes. They inhibit their target protease by a unique mechanism that involves a large conformational transition and the translocation of the enzyme from the upper to the lower pole of the protein. This complex mechanism, and the involvement of serpins in important biological regulatory processes, makes them prone to mutation-related diseases. For example the polymerization of mutant α₁-antitrypsin leads to the accumulation of ordered polymers within the endoplasmic reticulum of hepatocytes in association with cirrhosis. An identical process in the neuron specific serpin, neuroserpin, results in the accumulation of polymers in neurons and the dementia FENIB. In both cases there is a clear correlation between the molecular instability, the rate of polymer formation and the severity of disease. A similar process underlies the hepatic retention and plasma deficiency of antithrombin, C1 inhibitor, α₁-antichymotrypsin and heparin co-factor II. The common mechanism of polymerization has allowed us to group these conditions together as a novel class of disease, the serpinopathies.Key Words: serpins, α1-antitrypsin, neuroserpin, polymerization, dementia, conformational disease, serpinopathiesSerpins (or serine protease inhibitors) are the largest family of protease inhibitors. They have been found in all major branches of life including viruses, prokaryotes and eukaryotes.^1–3 Despite their name there is increasing evidence that serpins can also inhibit other classes of proteases as demonstrated by the viral serpin CrmA and recently by a plant serpin, serpin1.^4,5 They can even play a non-inhibitory role in events as diverse as blood pressure regulation (angiotensinogen), chromatin condensation (MENT), tumor progression (maspin), protein folding (hsp47) and hormone transport (cortisol and thyroxine binding globulin).⁶One of the most important roles of serpins is the regulation of enzymes involved in proteolytic cascades. Among these serpins are α₁-antitrypsin, α₁-antichymotrypsin, C1 inhibitor, antithrombin and plasminogen activator inhibitor-1, which play an important role in the control of proteases involved in the inflammatory, complement, coagulation and fibrinolytic pathways, respectively.^1,3 The serpin superfamily is characterised by more than 30% homology with the archetypal serpin α₁-antitrypsin and conservation of tertiary structure.^7,8 Serpins adopt a metastable conformation composed in most cases of 9 α-helices, three β-sheet (A to C) and an exposed mobile reactive centre loop (RCL). This flexible RCL typically contains 20 residues that act as a pseudo substrate for the target protease (Fig. 1A).^9–15 After formation of a Michaelis complex^16,17 the enzyme cleaves the P1-P1′ bond of the serpin, releasing the P1'' residue and forming an ester bond between the protease and the serpin.^18,19 This is then followed by a dramatic conformational transition from a stressed to relaxed conformation with the enzyme being pulled from the upper to the lower pole of the serpin and the insertion of the reactive loop as an extra strand in β-sheet A.^20–25 As a consequence of this conformational change the thermal stability of the serpin is greatly enhanced. Whereas a typical serpin in its native state exhibits a midpoint of thermal denaturation of around 50–60°C, a cleaved serpin with its RCL fully incorporated into β-sheet A denatures at temperatures >120°C.^9,26,27 Another consequence is the inactivation of the enzyme, stabilised at the acyl-intermediate and unable to proceed further to deacylation of the complex.^24,28 This serpin-protease complex then binds to members of the lipoprotein receptor family and is cleared from the circulation.^29–31Open in a separate windowFigure 1Inhibition of neutrophil elastase by α₁-antitrypsin and the structural basis of polymerization. (A) After docking (left) the neutrophil elastase (grey) is inactivated by movement from the upper to the lower pole of the protein (right). This is associated with the insertion of the RCL (red) as an extra strand into β-sheet A (green). (B) The structure of α₁-antitrypsin is centred on β-sheet A (green) and the mobile reactive centre loop (red). Polymer formation results from the Z variant of α₁-antitrypsin (Glu342Lys at P17; indicated by arrow) or mutations in the shutter domain (blue circle) that open β-sheet A to favour partial loop insertion and the formation of an unstable intermediate (M*). The patent β-sheet A then accepts the loop of another molecule to form a dimer (D), which then extends into polymers (P). The individual molecules of α₁-antitrypsin within the polymer, although identical, are coloured red, yellow and blue for clarity. Figure reproduced with permission from Lomas et al.⁹⁷Despite the evolutionary advantage conferred upon serpins by the remarkable mobility of the native state, their complexity is also their weak point.^19,32 Mutations affecting the serpins can lead to a variety of diseases, resulting from either a gain or loss of function.^6,19 For example mutations can cause aberrant conformational transitions that result in the retention of the serpin within the cell of synthesis. This will lead to either protein overload and death of the cell in which the serpin is synthesised, or disease as a consequence of the resulting plasma deficiency. Such a mechanism underlies diseases as diverse as cirrhosis, thrombosis, angio-oedema, emphysema and dementia. We review here the common mechanism underlying these diseases that we have grouped together as the serpinopathies.^33–35 The aggregation and accumulation of conformationally destabilized proteins is an important feature of many neurodegenerative diseases, including Alzheimer''s and Parkinson''s disease and the spongiform encephalopathies. Indeed we have used the serpinopathies as a paradigm for these other ‘conformational diseases’.³⁶     

Protein Misfolding and the Serpinopathies

The serpins are the largest superfamily of protease inhibitors. They are found in almost all branches of life including viruses, prokaryotes and eukaryotes. They inhibit their target protease by a unique mechanism that involves a large conformational transition and the translocation of the enzyme from the upper to the lower pole of the protein. This complex mechanism, and the involvement of serpins in important biological regulatory processes, make them prone to mutation-related diseases. For example the polymerization of mutant α 1-antitrypsin leads to the accumulation of ordered polymers within the endoplasmic reticulum of hepatocytes in association with cirrhosis. An identical process in the neuron specific serpin, neuroserpin, results in the accumulation of polymers in neurons and the dementia FENIB. In both cases there is a clear correlation between the molecular instability, the rate of polymer formation and the severity of disease. A similar process underlies the hepatic retention and plasma deficiency of antithrombin, C1 inhibitor, α 1-antichymotrypsin and heparin co-factor II. The common mechanism of polymerization has allowed us to group these conditions together as a novel class of disease, the serpinopathies.     

Genetic counseling and the presenile dementias

Abstract

The clinical application of genetic counseling techniques to the presenile dementing illnesses is discussed, using examples. The problems encountered in adult‐onset illness are very different from those seen in illnesses affecting children. Some general guidelines and some specific ones are presented.     

YKL-40 in the brain and cerebrospinal fluid of neurodegenerative dementias

Background

YKL-40 (also known as Chitinase 3-like 1) is a glycoprotein produced by inflammatory, cancer and stem cells. Its physiological role is not completely understood but YKL-40 is elevated in the brain and cerebrospinal fluid (CSF) in several neurological and neurodegenerative diseases associated with inflammatory processes. Yet the precise characterization of YKL-40 in dementia cases is missing.

Methods

In the present study, we comparatively analysed YKL-40 levels in the brain and CSF samples from neurodegenerative dementias of different aetiologies characterized by the presence of cortical pathology and disease-specific neuroinflammatory signatures.

Results

YKL-40 was normally expressed in fibrillar astrocytes in the white matter. Additionally YKL-40 was highly and widely expressed in reactive protoplasmic cortical and perivascular astrocytes, and fibrillar astrocytes in sporadic Creutzfeldt-Jakob disease (sCJD). Elevated YKL-40 levels were also detected in Alzheimer’s disease (AD) but not in dementia with Lewy bodies (DLB). In AD, YKL-40-positive astrocytes were commonly found in clusters, often around β-amyloid plaques, and surrounding vessels with β-amyloid angiopathy; they were also distributed randomly in the cerebral cortex and white matter. YKL-40 overexpression appeared as a pre-clinical event as demonstrated in experimental models of prion diseases and AD pathology.CSF YKL-40 levels were measured in a cohort of 288 individuals, including neurological controls (NC) and patients diagnosed with different types of dementia. Compared to NC, increased YKL-40 levels were detected in sCJD (p < 0.001, AUC = 0.92) and AD (p < 0.001, AUC = 0.77) but not in vascular dementia (VaD) (p > 0.05, AUC = 0.71) or in DLB/Parkinson’s disease dementia (PDD) (p > 0.05, AUC = 0.70). Further, two independent patient cohorts were used to validate the increased CSF YKL-40 levels in sCJD. Additionally, increased YKL-40 levels were found in genetic prion diseases associated with the PRNP-D178N (Fatal Familial Insomnia) and PRNP-E200K mutations.

Conclusions

Our results unequivocally demonstrate that in neurodegenerative dementias, YKL-40 is a disease-specific marker of neuroinflammation showing its highest levels in prion diseases. Therefore, YKL-40 quantification might have a potential for application in the evaluation of therapeutic intervention in dementias with a neuroinflammatory component.

     

Transition metal abnormalities in progressive dementias

Abnormal distributions of transition metals inside the brain are potential diagnostic markers for several central nervous system diseases, including Alzheimer’s disease (AD), Parkinson’s disease, dementia with Lewy bodies (DLB), bipolar disorders and depression. To further explore this possibility, the total concentrations of iron, zinc, copper, manganese, aluminum, chromium and cadmium were measured in post-mortem hippocampus and amygdala tissues taken from AD, DLB and Control patients. A statistically significant near fifty percent reduction in the total copper levels of AD patients was observed in both the hippocampus and amygdala. The statistical power of the hippocampus and amygdala copper analysis was found to be 86 and 74% respectively. No statistically significant deviations in the total metal concentrations were found for zinc, manganese, chromium or aluminum. Iron was found to be increased by 38% in AD amygdala tissues, but was unchanged in AD hippocampus tissues. Accounting for differences in tissue water content, as a function of both tissue type and disease state, revealed more consistencies with previous literature. To aid in the design of future experiments, the effect sizes for all tissue types and metals studied are also presented.     

Stains for the differential diagnosis of degenerative dementias.

Our understanding of the structural substrates underlying the dementia syndrome has been transformed by the introduction of the Gallyas silver stain and the application of immunostains for tau, ubiquitin, and alpha-synuclein. Visualization of sequential changes in Alzheimer's disease and the recognition of a new substrate for dementia and dementia with argyrophilic grains, are two of the advances related to the application of the Gallyas method. The specificity of alpha-synuclein for recognizing Lewy bodies enables the unequivocal diagnosis of dementia with Lewy bodies. The diverse entities that constitute the Pick complex can be identified by applying immunostains for tau and ubiquitin in addition to the Gallyas silver stain.     

Structural and microtubule binding properties of tau mutants of frontotemporal dementias

Several mutations in the gene encoding the microtubule-associated protein tau are responsible for the formation of neurofibrillary inclusions in frontotemporal dementia with Parkinsonism linked to chromosome 17 (FTDP-17). Here we present the high-resolution characterization of the conformational properties of two FTDP-17 mutants of the four-repeat domain of tau, P301L and DeltaK280, and their properties for binding to polyanions and microtubules. Multidimensional NMR spectroscopy shows that the mutations do no lead to a significant increase in the level of beta-structure in their monomeric state, even though the mutations strongly promote beta-structure during aggregation. However, local structural changes are induced in the second repeat. These changes only weakly affect the binding to the polyanion heparin, which promotes paired helical filament formation. The extent of binding to microtubules, however, is strongly decreased. Our results demonstrate that the reversible binding of tau to microtubules involves specific interactions, which are not essential for binding to polyanions.     

Basal forebrain cholinergic system in the dementias: Vulnerability,resilience, and resistance

The basal forebrain cholinergic neurons (BFCN) provide the primary source of cholinergic innervation of the human cerebral cortex. They are involved in the cognitive processes of learning, memory, and attention. These neurons are differentially vulnerable in various neuropathologic entities that cause dementia. This review summarizes the relevance to BFCN of neuropathologic markers associated with dementias, including the plaques and tangles of Alzheimer's disease (AD), the Lewy bodies of diffuse Lewy body disease, the tauopathy of frontotemporal lobar degeneration (FTLD-TAU) and the TDP-43 proteinopathy of FTLD-TDP. Each of these proteinopathies has a different relationship to BFCN and their corticofugal axons. Available evidence points to early and substantial degeneration of the BFCN in AD and diffuse Lewy body disease. In AD, the major neurodegenerative correlate is accumulation of phosphotau in neurofibrillary tangles. However, these neurons are less vulnerable to the tauopathy of FTLD. An intriguing finding is that the intracellular tau of AD causes destruction of the BFCN, whereas that of FTLD does not. This observation has profound implications for exploring the impact of different species of tauopathy on neuronal survival. The proteinopathy of FTLD-TDP shows virtually no abnormal inclusions within the BFCN. Thus, the BFCN are highly vulnerable to the neurodegenerative effects of tauopathy in AD, resilient to the neurodegenerative effect of tauopathy in FTLD and apparently resistant to the emergence of proteinopathy in FTLD-TDP and perhaps also in Pick's disease. Investigations are beginning to shed light on the potential mechanisms of this differential vulnerability and their implications for therapeutic intervention.

     

Solution conformational study of Scyliorhinin I analogues with conformational constraints by two-dimensional NMR and theoretical conformational analysis.

Two analogues of Scyliorhinin I (Scyl), a tachykinin with N-MeLeu in position 8 and a 1,5-disubstituted tetrazole ring between positions 7 and 8, introduced in order to generate local conformational constraints, were synthesized using the solid-phase method. Conformational studies in water and DMSO-d6 were performed on these peptides using a combination of the two-dimensional NMR technique and theoretical conformational analysis. The algorithm of conformational search consisted of the following three stages: (i) extensive global conformational analysis in order to find all low-energy conformations; (ii) calculation of the NOE effects and vicinal coupling constants for each of the low energy conformations; (iii) determining the statistical weights of these conformations by means of a nonlinear least-squares procedure, in order to obtain the best fit of the averaged simulated spectrum to the experimental one. In both solvents the three-dimensional structure of the analogues studied can be interpreted only in terms of an ensemble of multiple conformations. For [MeLeu8]Scyl, the C-terminal 6-10 fragment adopts more rigid structure than the N-terminal one. In the case of the analogue with the tetrazole ring in DMSO-d6 the three-dimensional structure is characterized by two dominant conformers with similar geometry of their backbones. They superimpose especially well (RMSD = 0.28 A) in the 6-9 fragments. All conformers calculated in both solvents superimpose in their C-terminal fragments much better than those of the first analogue. The results obtained indicate that the introduction of the tetrazole ring into the Scyl molecule rigidifies its structure significantly more than that of MeLeu.     

Prevalence of potentially reversible dementias and actual reversibility in a memory clinic cohort

BACKGROUND: Although clinics for the evaluation of cognitive dysfunction have typically emphasized the detection and treatment of the reversible causes of dementia, it remains unclear whether the treatment of such causes results in reversal of the dementia. Therefore, the appropriate work-up for dementia is in dispute. METHODS: A chart review was performed with records from an urban tertiary care referral-based memory clinic. The records for 196 patients with dementia or suspected dementia, seen between October 1991 and December 1993, were examined to determine the prevalence of potentially reversible dementias and whether the cognitive dysfunction improved or resolved after treatment. Data abstracted from the medical charts included demographic information, medication use, presence of depression, and results of neuropsychological tests, blood work and neuroimaging. The clinical diagnosis, the response to treatment, if applicable, and the outcome (mean follow-up period 16 months) were analysed. The recommendations of the 1989 Canadian Consensus Conference on the Assessment of Dementia (CCCAD) on the use of CT were retrospectively applied in each case. RESULTS: Of the 196 patients, 45 (23.0%) had a potentially reversible condition identified by history, physical examination, blood testing or CT; in only 7 (3.6% of the total) did treatment result in improvement or resolution of the dementia. These 7 patients had higher results for the Mini-Mental State examination (mean result 26) and exhibited only mild cognitive deficits. Potentially reversible lesions were found in the CT scans of 6 (3.1%) patients: 4 had normal-pressure hydrocephalus and 2 had a brain tumour. If the CCCAD recommendations had been followed, CT would have been performed in 76 (38.8%) of the patients, and 1 of the 6 patients with a lesion would have been missed. INTERPRETATION: Both potential and actual reversibility of dementia was low in these memory clinic patients. The patients whose condition improved with intervention had early and milder cognitive deficits, which suggests that thorough evaluation of early memory loss is warranted.     

Structured disorder and conformational selection

Traditionally, molecular disorder has been viewed as local or global instability. Molecules or regions displaying disorder have been considered inherently unstructured. The term has been routinely applied to cases for which no atomic coordinates can be derived from crystallized molecules. Yet, even when it appears that the molecules are disordered, prevailing conformations exist, with population times higher than those of all alternate conformations. Disordered molecules are the outcome of rugged energy landscapes away from the native state around the bottom of the funnel. Ruggedness has a biological function, creating a distribution of structured conformers that bind via conformational selection, driving association and multimolecular complex formation, whether chain-linked in folding or unlinked in binding. We classify disordered molecules into two types. The first type possesses a hydrophobic core. Here, even if the native conformation is unstable, it still has a large enough population time, enabling its experimental detection. In the second type, no such hydrophobic core exists. Hence, the native conformations of molecules belonging to this category have shorter population times, hindering their experimental detection. Although there is a continuum of distribution of hydrophobic cores in proteins, an empirical, statistically based hydrophobicity function may be used as a guideline for distinguishing the two disordered molecule types. Furthermore, the two types relate to steps in the protein folding reaction. With respect to protein design, this leads us to propose that engineering-optimized specific electrostatic interactions to avoid electrostatic repulsion would reduce the type I disordered state, driving the molten globule (MG) --> native (N) state. In contrast, for overcoming the type II disordered state, in addition to specific interactions, a stronger hydrophobic core is also indicated, leading to the denatured --> MG --> N state.     

Integrin structures and conformational signaling

Integrins are cell adhesion molecules that play critical roles in development, wound healing, hemostasis, immunity and cancer. Advances in the past two years have shed light on the structural basis for integrin regulation and signaling, especially on how global conformational changes between bent and extended conformations relate to the inter-domain and intra-domain shape shifting that regulates affinity for ligand. The downward movements of the C-terminal helices of the alpha I and beta I domains and the swing-out of the hybrid domain play pivotal roles in integrin conformational signaling. Experiments have also shown that integrins transmit bidirectional signals across the plasma membrane by coupling extracellular conformational change with an unclasping and separation of the alpha and beta transmembrane and cytoplasmic domains.     

Cell toxicity and conformational disease

Numerous disorders, including Alzheimer's, Parkinson's and other late-onset neurodegenerative diseases, arise from the conformationally driven aggregation of individual proteins. Previous focus on just one end-product of such aggregation - extracellular deposits of amyloid - has diverted attention from what is now recognized as being primarily intracellular disease processes. Recent structural findings show how cytotoxicity can result from even minor changes in conformation that do not lead to amyloid formation, as with the accumulation within the endoplasmic reticulum of intact mutant alpha-1-antitrypsin in hepatocytes and of neuroserpin in neurons. Studies in Alzheimer's and other dementias also indicate that the damage occurs at the stage of the initial intermolecular linkages that precede amyloid formation. The challenge now is to determine the detailed mechanisms of this cytotoxicity.     

Complement activation in chromosome 13 dementias. Similarities with Alzheimer's disease

Chromosome 13 dementias, familial British dementia (FBD) and familial Danish dementia (FDD), are associated with neurodegeneration and cerebrovascular amyloidosis, with striking neuropathological similarities to Alzheimer's disease (AD). Despite the structural differences among the amyloid subunits (ABri in FBD, ADan in FDD, and Abeta in AD), these disorders are all characterized by the presence of neurofibrillary tangles and parenchymal and vascular amyloid deposits co-localizing with markers of glial activation, suggestive of local inflammation. Proteins of the complement system and their pro-inflammatory activation products are among the inflammation markers associated with AD lesions. Immunohistochemistry of FBD and FDD brain sections demonstrated the presence of complement activation components of the classical and alternative pathways as well as the neo-epitope of the membrane attack complex. Hemolytic experiments and enzyme-linked immunosorbent assays specific for the activation products iC3b, C4d, Bb, and C5b-9 indicated that ABri and ADan are able to fully activate the complement cascade at levels comparable to those generated by Abeta1-42. ABri and ADan specifically bound C1q with high affinity and formed stable complexes in physiological conditions. Activation proceeds approximately 70-75% through the classical pathway while only approximately 25-30% seems to occur through the alternative pathway. The data suggest that the chronic inflammatory response generated by the amyloid peptides in vivo might be a contributing factor for the pathogenesis of FBD and FDD and, in more general terms, to other neurodegenerative conditions.