Aggregation of the human islet amyloid polypeptide (hIAPP) contributes to the development and progression of Type 2 Diabetes (T2D). hIAPP aggregates within a few hours at few micromolar concentration in vitro but exists at millimolar concentrations in vivo. Natively occurring inhibitors of hIAPP aggregation might therefore provide a model for drug design against amyloid formation associated with T2D. Here, we describe the combined ability of low pH, zinc, and insulin to inhibit hIAPP fibrillation. Insulin dose-dependently slows hIAPP aggregation near neutral pH but had less effect on the aggregation kinetics at acidic pH. We determine that insulin alters hIAPP aggregation in two manners. First, insulin diverts the aggregation pathway to large nonfibrillar aggregates with ThT-positive molecular structure, rather than to amyloid fibrils. Second, soluble insulin suppresses hIAPP dimer formation, which is an important early aggregation event. Further, we observe that zinc significantly modulates the inhibition of hIAPP aggregation by insulin. We hypothesize that this effect arose from controlling the oligomeric state of insulin and show that hIAPP interacts more strongly with monomeric than oligomeric insulin.

Tau protein is a microtubule-associated protein that is widely distributed in the central nervous system and maintains and regulates neuronal morphology and function. Tau protein aggregates abnormally and forms neurofibrillary tangles in neurodegenerative diseases, disrupting the structure and function of neurons and leading to neuronal death, which triggers the initiation and progression of neurological disorders. The aggregation of tau protein in neurodegenerative diseases is associated with post-translational modifications, which may affect the hydrophilicity, spatial conformation, and stability of tau protein, promoting tau protein aggregation and the formation of neurofibrillary tangles. Therefore, studying the role of tau protein in neurodegenerative diseases and the mechanism of aberrant aggregation is important for understanding the mechanism of neurodegenerative diseases and finding therapeutic approaches. This review describes the possible mechanisms by which tau protein promotes neurodegenerative diseases, the post-translational modifications of tau protein and associated influencing factors, and the current status of drug discovery and development related to tau protein, which may contribute to the development of new therapeutic approaches to alleviate or treat neurodegenerative diseases.

TAR DNA-binding protein 43 (TDP-43) is an RNA binding protein found within ribonucleoprotein granules tethered to lysosomes via annexin A11. TDP-43 protein forms inclusions in many neurodegenerative diseases including amyotrophic lateral sclerosis (ALS), frontotemporal lobar degeneration with TDP-43 inclusions (FTLD-TDP) and limbic predominant age-related TDP-43 encephalopathy neuropathologic change (LATE-NC). Annexin A11 is also known to form aggregates in ALS cases with pathogenic variants in ANXA11. Annexin A11 aggregation has not been described in sporadic ALS, FTLD-TDP or LATE-NC cases. To explore the relationship between TDP-43 and annexin A11, genetic analysis of 822 autopsy cases was performed to identify rare ANXA11 variants. In addition, an immunohistochemical study of 368 autopsy cases was performed to identify annexin A11 aggregates. Insoluble annexin A11 aggregates which colocalize with TDP-43 inclusions were present in all FTLD-TDP Type C cases. Annexin A11 inclusions were also seen in a small proportion (3-6%) of sporadic and genetic forms of FTLD-TDP types A and B, ALS, and LATE-NC. In addition, we confirm the comingling of annexin A11 and TDP-43 aggregates in an ALS case with the pathogenic ANXA11 p.G38R variant. Finally, we found abundant annexin A11 inclusions as the primary pathologic finding in a case of progressive supranuclear palsy-like frontotemporal dementia with prominent striatal vacuolization due to a novel variant, ANXA11 p.P75S. By immunoblot, FTLD-TDP with annexinopathy and ANXA11 variant cases show accumulation of insoluble ANXA11 including a truncated fragment. These results indicate that annexin A11 forms a diverse and heterogeneous range of aggregates in both sporadic and genetic forms of TDP-43 proteinopathies. In addition, the finding of a primary vacuolar annexinopathy due to ANXA11 p.P75S suggests that annexin A11 aggregation is sufficient to cause neurodegeneration.

RNA binding proteins have emerged as central players in the mechanisms of many neurodegenerative diseases. In particular, a proteinopathy of fused in sarcoma (FUS) is present in some instances of familial Amyotrophic lateral sclerosis (ALS) and about 10% of sporadic Frontotemporal lobar degeneration (FTLD). Here we establish that focal injection of sonicated human FUS fibrils into brains of mice in which ALS-linked mutant or wild-type human FUS replaces endogenous mouse FUS is sufficient to induce focal cytoplasmic mislocalization and aggregation of mutant and wild-type FUS which with time spreads to distal regions of the brain. Human FUS fibril-induced FUS aggregation in the mouse brain of humanized FUS mice is accelerated by an ALS-causing FUS mutant relative to wild-type human FUS. Injection of sonicated human FUS fibrils does not induce FUS aggregation and subsequent spreading after injection into naïve mouse brains containing only mouse FUS, indicating a species barrier to human FUS aggregation and its prion-like spread. Fibril-induced human FUS aggregates recapitulate pathological features of FTLD including increased detergent insolubility of FUS and TAF15 and amyloid-like, cytoplasmic deposits of FUS that accumulate ubiquitin and p62, but not TDP-43. Finally, injection of sonicated FUS fibrils is shown to exacerbate age-dependent cognitive and behavioral deficits from mutant human FUS expression. Thus, focal seeded aggregation of FUS and further propagation through prion-like spread elicits FUS-proteinopathy and FTLD-like disease progression.

Mutations in the protein superoxide dismutase-1 (SOD1) promote its misfolding and aggregation, ultimately causing familial forms of the debilitating neurodegenerative disease amyotrophic lateral sclerosis (ALS). Currently, over 220 (mostly missense) ALS-causing mutations in the SOD1 protein have been identified, indicating that common structural features are responsible for aggregation and toxicity. Using in silico tools, we predicted amyloidogenic regions in the ALS-associated SOD1-G85R mutant, finding seven regions throughout the structure. Introduction of proline residues into β-strands II (I18P) or III (I35P) reduced the aggregation propensity and toxicity of SOD1-G85R in cells, significantly more so than proline mutations in other amyloidogenic regions. The I18P and I35P mutations also reduced the capability of SOD1-G85R to template onto previously formed non-proline mutant SOD1 aggregates as measured by fluorescence recovery after photobleaching. Finally, we found that, while the I18P and I35P mutants are less structurally stable than SOD1-G85R, the proline mutants are less aggregation-prone during proteasome inhibition, and less toxic to cells overall. Our research highlights the importance of a previously underappreciated SOD1 amyloidogenic region in β-strand II (15QGIINF20) to the aggregation and toxicity of SOD1 in ALS mutants, and suggests that β-strands II and III may be good targets for the development of SOD1-associated ALS therapies.

Amyloid-β (Aβ) is one of the amyloidogenic intrinsically disordered proteins (IDPs) that self-assemble to protein aggregates, incurring cell malfunction and cytotoxicity. While Aβ has been known to regulate multiple physiological functions, such as enhancing synaptic functions, aiding in the recovery of the blood-brain barrier/brain injury, and exhibiting tumor suppression/antimicrobial activities, the hydrophobicity of the primary structure promotes pathological aggregations that are closely associated with the onset of Alzheimer\'s disease (AD). Aβ proteins consist of multiple isoforms with 37-43 amino acid residues that are produced by the cleavage of amyloid-β precursor protein (APP). The hydrolytic products of APP are secreted to the extracellular regions of neuronal cells. Aβ 1-42 (Aβ42) and Aβ 1-40 (Aβ40) are dominant isoforms whose significance in AD pathogenesis has been highlighted in numerous studies to understand the molecular mechanism and develop AD diagnosis and therapeutic strategies. In this review, we focus on the differences between Aβ42 and Aβ40 in the molecular mechanism of amyloid aggregations mediated by the two additional residues (Ile41 and Ala42) of Aβ42. The current comprehension of Aβ42 and Aβ40 in AD progression is outlined, together with the structural features of Aβ42/Aβ40 amyloid fibrils, and the aggregation mechanisms of Aβ42/Aβ40. Furthermore, the impact of the heterogeneous distribution of Aβ isoforms during amyloid aggregations is discussed in the system mimicking the coexistence of Aβ42 and Aβ40 in human cerebrospinal fluid (CSF) and plasma. [BMB Reports 2024; 57(6): 263-272].

Which isoforms of apolipoprotein E (apoE) we inherit determine our risk of developing late-onset Alzheimer\'s Disease (AD), but the mechanism underlying this link is poorly understood. In particular, the relevance of direct interactions between apoE and amyloid-β (Aβ) remains controversial. Here, single-molecule imaging shows that all isoforms of apoE associate with Aβ in the early stages of aggregation and then fall away as fibrillation happens. ApoE-Aβ co-aggregates account for ~50% of the mass of diffusible Aβ aggregates detected in the frontal cortices of homozygotes with the higher-risk APOE4 gene. We show how dynamic interactions between apoE and Aβ tune disease-related functions of Aβ aggregates throughout the course of aggregation. Our results connect inherited APOE genotype with the risk of developing AD by demonstrating how, in an isoform- and lipidation-specific way, apoE modulates the aggregation, clearance and toxicity of Aβ. Selectively removing non-lipidated apoE4-Aβ co-aggregates enhances clearance of toxic Aβ by glial cells, and reduces secretion of inflammatory markers and membrane damage, demonstrating a clear path to AD therapeutics.

Alpha-synuclein (αsyn) is an intrinsically disordered protein that aggregates in the brain in several neurodegenerative diseases collectively called synucleinopathies. Phosphorylation of αsyn at serine 129 (PSER129) was considered rare in the healthy human brain but is enriched in pathological αsyn aggregates and is used as a specific marker for disease inclusions. However, recent observations challenge this assumption by demonstrating that PSER129 results from neuronal activity and can be readily detected in the non-diseased mammalian brain. Here, we investigated experimental conditions under which two distinct PSER129 pools, namely endogenous-PSER129 and aggregated-PSER129, could be detected and differentiated in the mammalian brain. Results showed that in the wild-type (WT) mouse brain, perfusion fixation conditions greatly influenced the detection of endogenous-PSER129, with endogenous-PSER129 being nearly undetectable after delayed perfusion fixation (30-min and 1-h postmortem interval). Exposure to anesthetics (e.g., Ketamine or xylazine) before perfusion did not significantly influence endogenous-PSER129 detection or levels. In situ, non-specific phosphatase calf alkaline phosphatase (CIAP) selectively dephosphorylated endogenous-PSER129 while αsyn preformed fibril (PFF)-seeded aggregates and genuine disease aggregates (Lewy pathology and Papp-Lantos bodies in Parkinson\'s disease and multiple systems atrophy brain, respectively) were resistant to CIAP-mediated dephosphorylation. The phosphatase resistance of aggregates was abolished by sample denaturation, and CIAP-resistant PSER129 was closely associated with proteinase K (PK)-resistant αsyn (i.e., a marker of aggregation). CIAP pretreatment allowed for highly specific detection of seeded αsyn aggregates in a mouse model that accumulates non-aggregated-PSER129. We conclude that αsyn aggregates are impervious to phosphatases, and CIAP pretreatment increases detection specificity for aggregated-PSER129, particularly in well-preserved biological samples (e.g., perfusion fixed or flash-frozen mammalian tissues) where there is a high probability of interference from endogenous-PSER129. Our findings have important implications for the mechanism of PSER129-accumulation in the synucleinopathy brain and provide a simple experimental method to differentiate endogenous-from aggregated PSER129.

Though the book\'s journey into The Hidden World of Protein Aggregation has come to an end, the search for knowledge, the development of healthier lives, and the discovery of nature\'s mysteries continue, promising new horizons and discoveries yet to be discovered. The intricacies of protein misfolding and aggregation remain a mystery in cellular biology, despite advances made in unraveling them. In this chapter, we will summarize the specific conclusions from the previous chapters and explore the persistent obstacles and unanswered questions that motivate scientists to pursue exploration of protein misfolding and aggregation.

The main cause of many neurodegenerative diseases and systemic amyloidoses is protein and peptide aggregation and the formation of amyloid fibrils. The study of aggregation mechanisms, the discovery and description of aggregate structures, and a comprehensive understanding of the molecular mechanisms of amyloid formation are of great importance for the diagnostic processes at the molecular level and for the development of therapeutic strategies to counter aggregation-associated disorders. Given that understanding protein misfolding phenomena is directly related to the protein folding process, we will briefly explain the protein folding mechanism and then discuss the important factors involved in protein aggregation. In the following, we review different mechanisms of amyloid formation and finally represent the current knowledge on how amyloid fibrils are formed based on kinetic and thermodynamic factors.