Biophysical Aspects of Alzheimer’s Disease: Implications for Pharmaceutical Sciences

An increasing amount of findings suggests that the aggregation of soluble peptides and proteins into amyloid fibrils is a relevant upstream process in the complex cascade of events leading to the pathology of Alzheimer’s disease and several other neurodegenerative disorders. Nevertheless, several aspects of the correlation between the aggregation process and the onset and development of the pathology remain largely elusive. In this context, biophysical and biochemical studies in test tubes have proven extremely powerful in providing quantitative information about the structure and the reactivity of amyloids at the molecular level. In this review we use selected recent examples to illustrate the importance of such biophysical research to complement phenomenological studies based on cellular and molecular biology, and we discuss the implications for pharmaceutical applications associated with Alzheimer’s disease and other neurodegenerative disorders in both academic and industrial contexts.     

Recent structural and computational insights into conformational diseases

Protein aggregation correlates with the development of several deleterious human disorders such as Alzheimer's disease, Parkinson's disease, prion-associated transmissible spongiform encephalopathies and type II diabetes. The polypeptides involved in these disorders may be globular proteins with a defined 3D-structure or natively unfolded proteins in their soluble conformations. In either case, proteins associated with these pathogeneses all aggregate into amyloid fibrils sharing a common structure, in which beta-strands of polypeptide chains are perpendicular to the fibril axis. Because of the prominence of amyloid deposits in many of these diseases, much effort has gone into elucidating the structural basis of protein aggregation. A number of recent experimental and theoretical studies have significantly increased our understanding of the process. On the one hand, solid-state NMR, X-ray crystallography and single molecule methods have provided us with the first high-resolution 3D structures of amyloids, showing that they exhibit conformational plasticity and are able to adopt different stable tertiary folds. On the other hand, several computational approaches have identified regions prone to aggregation in disease-linked polypeptides, predicted the differential aggregation propensities of their genetic variants and simulated the early, crucial steps in protein self-assembly. This review summarizes these findings and their therapeutic relevance, as by uncovering specific structural or sequential targets they may provide us with a means to tackle the debilitating diseases linked to protein aggregation.     

Anti-amyloid compounds protect from silica nanoparticle-induced neurotoxicity in the nematode C. elegans

Identifying nanomaterial-bio-interactions are imperative due to the broad introduction of nanoparticle (NP) applications and their distribution. Here, we demonstrate that silica NPs effect widespread protein aggregation in the soil nematode Caenorhabditis elegans ranging from induction of amyloid in nucleoli of intestinal cells to facilitation of protein aggregation in body wall muscles and axons of neural cells. Proteomic screening revealed that exposure of adult C. elegans with silica NPs promotes segregation of proteins belonging to the gene ontology (GO) group of “protein folding, proteolysis and stress response” to an SDS-resistant aggregome network. Candidate proteins in this group include chaperones, heat shock proteins and subunits of the 26S proteasome which are all decisively involved in protein homeostasis. The pathway of protein homeostasis was validated as a major target of silica NPs by behavioral phenotyping, as inhibitors of amyloid formation rescued NP-induced defects of locomotory patterns and egg laying. The analysis of a reporter worm for serotonergic neural cells revealed that silica NP-induced protein aggregation likewise occurs in axons of HSN neurons, where presynaptic accumulation of serotonin, e.g. disturbed axonal transport reduces the capacity for neurotransmission and egg laying. The results suggest that in C. elegans silica NPs promote a cascade of events including disturbance of protein homeostasis, widespread protein aggregation and inhibition of serotonergic neurotransmission which can be interrupted by compounds preventing amyloid fibrillation.     

Ion channel formation and membrane-linked pathologies of misfolded hydrophobic proteins: the role of dangerous unchaperoned molecules

1. Protein-membrane interaction includes the interaction of proteins with intrinsic receptors and ion transport pathways and with membrane lipids. Several hypothetical interaction models have been reported for peptide-induced membrane destabilization, including hydrophobic clustering, electrostatic interaction, electrostatic followed by hydrophobic interaction, wedge x type incorporation and hydrophobic mismatch. 2. The present review focuses on the hypothesis of protein interaction with lipid membranes of those unchaperoned positively charged and misfolded proteins that have hydrophobic regions. We advance the hypothesis that protein misfolding that leads to the exposure of hydrophobic regions of proteins renders them potentially cytotoxic. Such proteins include prion, amyloid beta protein (AbetaP), amylin, calcitonin, serum amyloid and C-type natriuretic peptides. These proteins have the ability to interact with lipid membranes, thereby inducing membrane damage and cell malfunction. 3. We propose that the most significant mechanism of membrane damage induced by hydrophobic misfolded proteins is mediated via the formation of ion channels. The hydrophobicity based toxicity of several proteins linked to neurodegenerative pathologies is similar to those observed for antibacterial toxins and viral proteins. 4. It is hypothesized that the membrane damage induced by amyloids, antibacterial toxins and viral proteins represents a common mechanism for cell malfunction, which underlies the associated pathologies and cytotoxicity of such proteins.     

The pathological cascade of Alzheimer's disease: the role of inflammation and its therapeutic implications

Alzheimer's disease is a chronic neurodegenerative disease causing progressive impairment of memory and other cognitive functions. A number of sequential events are suggested to be associated with different pathological aspects observed in Alzheimer's disease, the so-called amyloid cascade hypothesis. Mismetabolism of the beta-amyloid precursor protein, as a result of mutations in the amyloid precursor protein gene or as results of impaired cleavage, leads to the formation of nonfibrillar and fibrillar amyloid-beta deposits. Glial cells are attracted to and activated by these amyloid-beta deposits. After activation, these cells secrete inflammatory mediators and reactive oxygen species, which can aggravate the aggregation of amyloid-beta. Some of the products released by activated glial cells, as well as amyloid-beta itself, can induce or promote neurodegeneration. Several mechanisms, such as mitotic reentry, apoptosis and cytoskeletal changes are suggested to be involved in neuronal loss. This review will outline several pathological mechanisms in Alzheimer's disease as well as some means of therapeutic intervention following the amyloid cascade hypothesis.     

Challenges in the development of high protein concentration formulations

Development of formulations for protein drugs requiring high dosing (in the order of mg/kg) may become challenging for solubility limited proteins and for the subcutaneous (SC) route with <1.5 mL allowable administration volume that requires >100 mg/mL protein concentrations. Development of high protein concentration formulations also results in several manufacturing, stability, analytical, and delivery challenges. The high concentrations achieved by small scale approaches used in preformulation studies would have to be confirmed with manufacturing scale processes and with representative materials because of the lability of protein conformation and the propensity to interact with surfaces and solutes which render protein solubilities that are dependent on the process of concentrating. The concentration dependent degradation route of aggregation is the greatest challenge to developing protein formulations at these higher concentrations. In addition to the potential for nonnative protein aggregation and particulate formation, reversible self-association may occur, which contributes to properties such as viscosity that complicates delivery by injection. Higher viscosity also complicates manufacturing of high protein concentrations by filtration approaches. Chromatographic and electrophoretic assays may not accurately determine the non-covalent higher molecular weight forms because of the dilutions that are usually encountered with these techniques. Hence, techniques must be used that allow for direct measurement in the formulation without substantial dilution of the protein. These challenges are summarized in this review.     

Therapeutic approaches against common structural features of toxic oligomers shared by multiple amyloidogenic proteins

Impaired proteostasis is one of the main features of all amyloid diseases, which are associated with the formation of insoluble aggregates from amyloidogenic proteins. The aggregation process can be caused by overproduction or poor clearance of these proteins. However, numerous reports suggest that amyloid oligomers are the most toxic species, rather than insoluble fibrillar material, in Alzheimer's, Parkinson's, and Prion diseases, among others. Although the exact protein that aggregates varies between amyloid disorders, they all share common structural features that can be used as therapeutic targets. In this review, we focus on therapeutic approaches against shared features of toxic oligomeric structures and future directions.     

Visualization of amyloid formation processes on cell membranes: gangliosides as key molecules for the onset of amyloidosis

Deposition of insoluble amyloid fibrils in tissues is a common hallmark of a wide range of human diseases referred to as amyloidoses, including Alzheimer's disease, type II diabetes mellitus. The amyloid deposits cause cell dysfunction, death, and subsequently severe impairment in tissues. Elucidation of amyloid formation mechanisms is essential for prevention of the onset and development of amyloidoses. Accumulated experimental evidence demonstrates that membrane lipids enhance the fibril formation of amyloidogenic proteins. Our group demonstrated that amyloid formation by amyloid β-protein (Aβ) was facilitated by gangliosides in lipid raft-like model membranes. Phosphatidylserine and phosphatidylglycerol were also reported to trigger fibril formation by human islet amyloid polypeptide (hIAPP). However, it is not verified whether the proposed lipid-protein interactions can occur on plasma membranes of live cells. The author developed a method for visualizing amyloid fibrils on live cell membranes and investigated the roles of gangliosides and cholesterol in lipid rafts for amyloid formation. Congo red, an amyloid-specific dye, was found to be a promising compound for staining amyloids in live cells. Aβ was accumulated on cholesterol-dependent ganglioside-rich domains in PC12 neuronal cells in a time- and concentration-dependent manner, leading to cell death. Nerve growth factor-induced differentiation of PC12 cells increased both gangliosides and cholesterol and thereby greatly potentiated the accumulation and cytotoxic effect of Aβ. Amyloid formation by hIAPP was also facilitated by gangliosides in lipid rafts. Membrane lipid compositions, in this case, gangliosides in lipid rafts, actually caused striking change in amyloid formation on cell membranes.     

Congo red, an amyloid-inhibiting compound, alleviates various types of cellular dysfunction triggered by mutant protein kinase cγ that causes spinocerebellar ataxia type 14 (SCA14) by inhibiting oligomerization and aggregation

Several missense mutations in the protein kinase Cγ (γPKC) gene have been found to cause spinocerebellar ataxia type 14 (SCA14), an autosomal dominant neurodegenerative disease. We previously demonstrated that the mutant γPKC found in SCA14 is susceptible to aggregation that induces apoptotic cell death. Congo red is widely used as a histological dye for amyloid detection. Recent evidence has revealed that Congo red has the property to inhibit amyloid oligomers and fibril formation of misfolded proteins. In the present study, we examine whether Congo red inhibits aggregate formation and cytotoxicity of mutant γPKC. Congo red likely inhibits aggregate formation of mutant γPKC – green fluorescent protein (GFP) without affecting its expression level in SH-SY5Y cells. Congo red counteracts the insolubilization of recombinant mutant γPKC, suggesting that the dye inhibits aggregation of mutant γPKC by a direct mechanism. Congo red also inhibits aggregation and oligomerization of mutant γPKC-GFP in primary cultured cerebellar Purkinje cells. Moreover, the dye reverses the improper development of dendrites and inhibits apoptotic cell death in Purkinje cells that express mutant γPKC-GFP. These results indicate that amyloid-inhibiting compounds like Congo red may be novel therapeutics for SCA14.     

Food allergy: predictive testing of food products

Food allergy is a substantial cause of distress in humans. Several biotechnological techniques can be applied to reduce the antigenicity of food proteins to produce for instance hypoallergenic infant formulas. Biotechnological techniques synthesizing new proteins or new biological varieties for applications in food are also available. For such biotechnologically for derived protein products (novel foods), allergenicity may also pose a major concern. For safety reasons, it is of importance to evaluate the residual antigenicity of modified protein products, to screen for possible cross-reactivity to prevent reactions in previously sensitized individuals, and to test for sensitizing properties of new and/or modified protein products. Besides physico-chemical and immunochemical analyses, several in vitro and in vivo bioassays may be applied in studying the antigenic or allergenic properties of (new or modified) food proteins. In this paper, an overview of several available assays and new developments for determining the antigenic or allergenic properties of dietary proteins, as well as their possible applications and limitations is presented. Special attention is paid to the role of the gastro-intestinal tract physiology in food allergy and in the evaluation of the allergenic potential of food proteins and to the possible applications of animal models in food allergy research and in the evaluation of the allergenicity of food proteins.     

Pharmaceutical strategies against amyloidosis: old and new drugs in targeting a "protein misfolding disease"

The group of diseases caused by abnormalities of the process of protein folding and unfolding is rapidly growing and includes diseases caused by loss of function as well as diseases caused by gain of function of misfolded proteins. Amyloidoses are caused by gain of function of certain proteins that lose their native structure and self-assemble into toxic insoluble, extracellular fibrils. This process requires the contribution of multiple factors of which only a few are established, namely the conformational modification of the amyloidogenic protein, protein's post-translational modifications and the co-deposition of glycosaminoglicans and of serum amyloid P component. In parallel with the exponential growth of biochemical data regarding the key events of the fibrillogenic process, several reports have shown that small molecules, through the interaction with either the amyloidogenic proteins or with the common constituents, can modify the kinetics of formation of amyloid fibrils or can facilitate amyloid reabsorption. These small molecules can be classified on the basis of their protein target and mechanism of action, according to the following properties. 1) molecules that stabilize the amyloidogenic protein precursor 2) molecules that prevent fibrillogenesis by acting on partially folded intermediates of the folding process as well as on low molecular weight oligomers populating the initial phase of fibril formation 3) molecules that interact with mature amyloid fibrils and weaken their structural stability 4) molecules that displace fundamental co-factors of the amyloid deposits like glycosaminoglycans and serum amyloid P component and favor the dissolution of the fibrillar aggregate.     

Antimicrobial properties of amyloid peptides

More than two dozen clinical syndromes known as amyloid diseases are characterized by the buildup of extended insoluble fibrillar deposits in tissues. These amorphous Congo red staining deposits known as amyloids exhibit a characteristic green birefringence and cross-β structure. Substantial evidence implicates oligomeric intermediates of amyloids as toxic species in the pathogenesis of these chronic disease states. A growing body of data has suggested that these toxic species form ion channels in cellular membranes causing disruption of calcium homeostasis, membrane depolarization, energy drainage, and in some cases apoptosis. Amyloid peptide channels exhibit a number of common biological properties including the universal U-shape β-strand-turn-β-strand structure, irreversible and spontaneous insertion into membranes, production of large heterogeneous single-channel conductances, relatively poor ion selectivity, inhibition by Congo red, and channel blockade by zinc. Recent evidence has suggested that increased amounts of amyloids not only are toxic to its host target cells but also possess antimicrobial activity. Furthermore, at least one human antimicrobial peptide, protegrin-1, which kills microbes by a channel-forming mechanism, has been shown to possess the ability to form extended amyloid fibrils very similar to those of classic disease-forming amyloids. In this paper, we will review the reported antimicrobial properties of amyloids and the implications of these discoveries for our understanding of amyloid structure and function.     

Protein stabilization by cyclodextrins in the liquid and dried state

Aggregation is arguably the biggest challenge for the development of stable formulations and robust manufacturing processes of therapeutic proteins. In search of novel excipients inhibiting protein aggregation, cyclodextrins and their derivatives have been under examination for use in parenteral protein products since more than 20 years and significant research work has been accomplished highlighting the great potential of cyclodextrins as stabilizers of therapeutic proteins.Oftentimes, the potential of cyclodextrins to inhibit protein aggregation has been attributed to their capability to incorporate hydrophobic residues on aggregation-prone proteins or on their partially unfolded intermediates into the hydrophobic cavity. In addition, also other mechanisms besides or even instead of complex formation play a role in the stabilization mechanism, e.g. non-ionic surfactant-like effects.In this review a comprehensive overview of the available research work on the beneficial use of cyclodextrins and their derivatives in protein formulations, liquid as well as dried, is provided. The mechanisms of stabilization against different kinds of stress conditions, such as thermal or surface-induced, are discussed in detail.     

Antibody therapeutics, antibody engineering, and the merits of protein stability

Antibodies are highly soluble, multidomain proteins that are well suited for biopharmaceutical development; however, engineering antibodies to perform novel activities or to have enhanced clinical utility can have a detrimental effect on their biophysical properties. Various innovative designs, such as single-chain variable fragments (scFvs) and domain antibodies (dAbs), have been utilized to obtain the antigen-binding properties of natural antibodies, while using a minimal amount of the polypeptide sequence of an antibody. These designs can be used for generating diverse antibody libraries to support discovery and optimization and also serve as excellent building blocks for constructing more complex protein therapeutics, such as bispecific antibodies. However, engineered antibody-like proteins, including scFvs, are often unstable and prone to aggregation, compromising both protein production and quality. Research over the past few years has enhanced our understanding of how interdomain interactions within antibodies contribute to protein stability. This knowledge and sustained research to develop methods for modifying antibody fragments to improve stability have begun to have a positive impact on the quality of antibody libraries for discovery purposes and the viability of highly engineered proteins, such as bispecific antibodies, as therapeutics.     

Stability of Protein Pharmaceuticals

Recombinant DNA technology has now made it possible to produce proteins for pharmaceutical applications. Consequently, proteins produced via biotechnology now comprise a significant portion of the drugs currently under development. Isolation, purification, formulation, and delivery of proteins represent significant challenges to pharmaceutical scientists, as proteins possess unique chemical and physical properties. These properties pose difficult stability problems. A summary of both chemical and physical decomposition pathways for proteins is given. Chemical instability can include proteolysis, deamidation, oxidation, racemization, and -elimination. Physical instability refers to processes such as aggregation, precipitation, denaturation, and adsorption to surfaces. Current methodology to stabilize proteins is presented, including additives, excipients, chemical modification, and the use of site-directed mutagenesis to produce a more stable protein species.     

The search for novel avenues for the therapy and prevention of Alzheimer's disease

The prevention and therapy of neurodegenerative disorders in the elderly is one of the greatest challenges facing molecular medicine today. Alzheimer's is an excellent example of a disease being studied by many groups worldwide. Indeed, while many molecular details of this disorder have been elucidated in the last two decades, there are still no strictly causal therapies available. While certain symptomatic pharmacological treatments are frequently employed, current molecular medicine research is focused on central Alzheimer-associated biochemical changes to find the key switch that turns the detrimental Alzheimer process on. Although amyloid beta proteins and tau proteins are the focus of most investigations, intracellular signaling has recently gained a lot of attention as well. Signaling mediated by glycogen synthase kinase is intensively studied since it is a survival factor for neurons, is directly linked to Alzheimer-specific pathobiochemistry, and its activity can be modulated by well-known neuroprotective factors such as the female sex hormone estrogen.     

Entering the era of proteomics in rheumatology

Proteomics, the large-scale analysis of proteins of a given cell or tissue, is a fast-emerging field in biomedical research. It has become clear that proteomic approaches can assist in unravelling complex disease pathways and can help in the discovery of protein drug targets. The recent flood of proteomic data demonstrates the furious digging in the quest for the so-called ‘Holy Grail’. However, at present only a limited number of reports describing proteomic studies in rheumatology have been published. This review highlights some recent advances in the field of proteomic techniques. These new techniques, as well as classic approaches, have potential applications in the field of rheumatology. Some of the proteome studies in rheumatology are directed to the discovery of diagnostic proteins in biological fluids, and others are designed to elucidate the pathophysiology of affected target tissues. As discussed in this review, proteomics is an emerging area in rheumatology and holds great potential in this field. There is little doubt that established and new proteomic tools will lead to landmark discoveries in this field, with applications ranging from diagnostics and therapeutic monitoring to the discovery of new therapeutic targets.     

Towards understanding the structure-function relationship of human amyloid disease

Immunoglobulin light chain (LC) proteins exhibit the greatest sequence variability of all proteins associated with amyloid disease. The hallmark event in amyloidogenesis is a change in the secondary and/or tertiary structure of a normal, soluble protein, that fosters self-aggregation and fibril formation. The structural heterogeneity of light chain proteins has hampered understanding of the precise mechanisms involved in fibril formation. The development of effective therapeutics will be benefited by a fundamental understanding of mechanisms and structural prerequisites which govern amyloidogenesis. This review focuses on light chain (AL) amyloidosis resulting from the aggregation of kappa and lambda LCs. Specifically the thermodynamic and structural data of several WT and mutant amyloidogenic LCs have been carefully examined. Moreover, we discuss the importance of hydrophobic and ionic interactions on amyloidosis by comparing several available three-dimensional structures of amyloidogenic and highly homologous non-amyloidogenic proteins that can be destabilized to become amyloidogenic by site specific mutations.