Targeting protein aggregation for the treatment of degenerative diseases

Abstract

The aggregation of specific proteins is hypothesized to underlie several degenerative diseases, which are collectively known as amyloid disorders. However, the mechanistic connection between the process of protein aggregation and tissue degeneration is not yet fully understood. Here, we review current and emerging strategies to ameliorate aggregation-associated degenerative disorders, with a focus on disease-modifying strategies that prevent the formation of and/or eliminate protein aggregates. Persuasive pharmacological and genetic evidence now supports protein aggregation as the cause of postmitotic tissue dysfunction or loss. However, a more detailed understanding of the factors that trigger and sustain aggregate formation and of the structure-activity relationships underlying proteotoxicity is needed to develop future disease-modifying therapies.

Figure 1. Amyloidogenesis—a process of aggregation influenced by the physical chemistry of the protein as well as cellular and extracellular components

Amyloidogenic proteins associated with degenerative disorders can be subdivided into two categories based on their native structure. Category 1 proteins, such as transthyretin (TTR) and the prion protein (PrP^c), exhibit a well-defined native state three-dimensional structure, whereas category 2 proteins are intrinsically disordered. Both, intrinsically disordered polypeptides generated by endoproteolytic processing of a precursor protein (category 2a), such as Aβ generated by cleavage of the amyloid precursor protein (APP), as well as full-length intrinsically disordered proteins (category 2b), such as tau and α-synuclein, can be amyloidogenic. The critical step in amyloidogenesis is misfolding and aggregation of category 1 proteins or misassembly of category 2 proteins into a spectrum of aggregate structures, including β-sheet-rich structures and amyloid fibrils. The structures associated with the amyloid cascade are depicted along with their hypothesized mechanisms of proteotoxicity (shown on the far right). The ensemble of structures is likely influenced and some may be generated by the biology of the organism, e.g., incomplete degradation of amyloid could afford novel structures, or aggregation on cell membranes could afford aggregate structures that can only form in the presence of certain lipids and/or carbohydrates.

Figure 2. Mechanisms of Protein Aggregation

(A) In a nucleation-dependent polymerization, the initiating step of aggregation involves the formation of a nucleus, a sparsely populated, high energy species that has a conformation that differs from that of the soluble protein. The nucleus is typically rich in β-sheet structure and presumably is oligomeric, although monomeric species have been implicated. Once the nucleus is formed, monomers are added rapidly to the growing non-covalent polymer to generate thermodynamically more stable aggregates that can also act as seeds. Monomer aggregation can proceed very rapidly by the addition of preformed aggregates, or seeds, because nucleation is no longer required in a “seeded” aggregation reaction. (B) In a nucleated conformational conversion, an equilibrium exists between monomers and structurally heterogeneous oligomers, which are typically (but not always) more stable than monomers. Over time, the oligomers are converted into a nucleus. This nucleus then initiates conformational conversion of neighboring monomers comprising the oligomer into amyloid fibers. Seeding can bypass the requirement for a slow nucleated conformational conversion step. (C) In a downhill polymerization, formation of the aggregation-prone misfolded monomer from a natively folded protein is the rate limiting step (not shown). Subsequent addition of monomers to the growing polymer is energetically favorable and leads to amorphous aggregate formation, as well as the generation of cross-β-sheet amyloid fibrils. Seeding does not accelerate the rate of aggregation in a downhill polymerization, at least in transthyretin amyloidogenesis studied largely in vitro.

Figure 3. Therapeutic Strategies to Ameliorate Amyloidosis

A. Gene therapy by liver transplantation to treat Familial Amyloid Polyneuropathy (FAP). Since TTR is largely produced by the liver, liver transplantation was introduced in the 1990s as a therapy for early stage patients with FAP. Wild type (WT) TTR tetramers (depicted with green squares) produced by the donor liver are much less amyloidogenic than the mutant TTR/WT TTR heterodimers (depicted with red and green squares, respectively) produced by the patient's liver before transplant. This approach successfully slows the progression of FAP and extends life span, but requires organ availability, life-long immunosuppression, and is associated with 10% mortality due to the transplant procedure. B. Protein reduction by chemotherapy to treat Light Chain Amyloidosis (AL). The amyloidogenic protein in AL amyloidosis is an immunoglobulin light chain or a fragment thereof overproduced by a plasma cell dyscrasia. Removal of the proliferating cancerous plasma cells producing the amyloidogenic light chain by chemotherapy regimens, with or without autologous stem cell transplantation, is used for patients without major cardiac involvement that are not too sick to tolerate these aggressive regimens, which leads to apparently durable disease remission. C. Anti-inflammatory treatment dramatically lowers acute phase serum amyloid A (SAA) protein production for the treatment of Amyloid A Amyloidosis (AA amyloidosis). Systemic amyloid A amyloidosis is a long-term complication in some patients suffering from chronic infection or chronic inflammation (e.g., chronic inflammatory arthritis). The amyloidogenic protein is derived by proteolysis of SAA, an acute-phase reactant protein transcriptionally upregulated during inflammation. Persistent high concentrations of SAA fragments in plasma above a critical threshold for aggregation can trigger AA deposition. Treatment of the underlying infectious or inflammatory trigger can reverse this type of amyloidosis, if diagnosed early. D. Reduction of pathogenic Aβ levels by secretase inhibition or modulation has therapeutic potential for Alzheimer's disease (AD) and Cerebral β-Amyloid Angiopathy. The amyloid precursor protein (APP) is a transmembrane protein that is constitutively cleaved by enzymes called secretases. Endoproteolytic processing via the non-amyloidogenic pathway, i.e., α-secretase cleavage followed by γ-secretase cleavage (shown on left) generates non-amyloidogenic APP fragments. In contrast, processing by β-secretase and subsequent γ-secretase cleavage produces amyloidogenic Aβ peptides (shown in red on right). Aggregation of Aβ peptides into extracellular amyloid fibrils or plaques is a histopathological hallmark and a diagnostic criterion for Alzheimer's disease. Reduction of Aβ peptide concentration can be achieved by (1) shifting APP processing towards non-amyloidogenic endoproteolysis, or (2) inhibition of β-secretase , or (3) γ-secretase inhibition or modulation. Inhibition of γ-secretase was shown to be feasible experimentally, however, side effects are a concern due to its many substrates. Interestingly, γ-secretase cleavage generates Aβ peptides of various lengths—as a rule of thumb, the longer the peptide, the more amyloidogenic. Modulation of γ-secretase processing of APP to generate shorter, less amyloidogenic Aβ peptides is currently being investigated. E. Gene silencing by siRNA or antisense oligonucleotides for the treatment of Familial Amyloid Polyneuropathy (FAP). Recently published early-stage, small, placebo-controlled clinical trial data has identified small interfering RNAs (siRNA, also RNAi) that lower mutant and WT transthyretin (TTR) protein production in patients with FAP. Analogous clinical data on antisense oligonucleotides that degrade TTR mRNA and thus lower TTR production and secretion into the blood from the liver are promising.

Figure 4. Seeded or Prion-like Protein Aggregate Spreading: Disease Initiation, Progression and Therapeutic Strategies

(A) Recent advances in our understanding of protein aggregation disorders have revealed that several amyloidogenic proteins once aggregated into amyloid fibrils can seed or catalyze the misfolding and/or misassembly of homotypic monomers, leading to accelerated aggregate formation and aggregate spreading from tissue to tissue. This type of mechanism was first proposed for prion disorders. Aggregate spreading by this mechanism is currently termed “templated misfolding” or “seeded aggregation” (see Figure 2). Once an amyloid seed is formed, it can catalyze the misfolding and/or misassembly of a monomeric homotypic protein into growing aggregates. Amyloid fibril fragmentation generates new seeds, and the vicious cycle of template misfolding/aggregation leads to the spreading of amyloid to neighboring and interconnected tissue, assisted by cellular aggregate uptake, which can result in subsequent template misfolding followed by aggregate secretion. This mechanistic paradigm has important therapeutic implications. Most importantly, it argues for an early intervention or preventive treatment before the amyloid spreading cascade progresses out of control. Several therapeutic strategies are possible (shown in the green box), ranging from stopping active aggregation, to removing aggregates, to inhibiting templated misfolding, to stabilizing seeds against fragmentation and/or altering their structure, to preventing the cellular uptake or secretion of aggregates. Early detection of amyloid diseases, which is still problematic, is important for these therapeutic strategies to have maximal impact (B) Amyloid fibrils often adopt an in-register β-sheet arrangement, wherein the strands are oriented perpendicular to the fibril axis. The amyloid fibril serves as a seed or template to incorporate the homotypic monomer into the growing fibril. The same protein can adopt distinct amyloid structures or strains, which can be reliably propagated by template misfolding, and which appear to be associated with different pathogenic phenotypes. Figure adapted from ref 224.

Figure 5. Combining Therapeutic Strategies to Ameliorate Protein-misfolding / Aggregation Diseases

We envision that amyloid diseases featuring degenerative phenotypes will be treated in the future using combinations of drugs exhibiting distinct mechanisms of action. For example, we envision treating the TTR amyloidoses with kinetic stabilizers (tafamidis; already in clinical use) and TTR messenger RNA degradation drugs (currently in clinical trials), or using a kinetic stabilizer in combination with drugs that enhance the capacity of the proteostasis network (currently being developed) to achieve proteome maintenance. Given the myriad proteins that lead to aggregation-associated degenerative diseases, strategies such as proteostasis network adaptation that could be useful for multiple maladies are particularly appealing. The goal is to achieve a fully functional proteome without pathogenic amyloidogenicity.