Neurodegenerative Protein Misfolding Diseases Research Articles

Visual detection of misfolded alpha-synuclein and prions via capillary-based quaking-induced conversion assay (Cap-QuIC)

Neurodegenerative protein misfolding diseases impact tens of millions of people worldwide, contributing to millions of deaths and economic hardships across multiple scales. The prevalence of neurodegenerative disease is predicted to greatly increase over the coming decades, yet effective diagnostics for such diseases are limited. Most diagnoses come from the observation of external symptoms in clinical settings, which typically manifest during relatively advanced stages of disease, thus limiting potential therapeutic applications. While progress is being made on biomarker testing, the underlying methods largely rely on fragile and expensive equipment that limits their point-of-care potential, especially in developing countries. Here we present Capillary-based Quaking Induced Conversion (Cap-QuIC) as a visual diagnostic assay based on simple capillary action for the detection of neurodegenerative disease without necessitating expensive and complex capital equipment. We demonstrate that Cap-QuIC has the potential to be a detection tool for a broad range of misfolded proteins by successfully distinguishing misfolded versus healthy proteins associated with Parkinson’s disease (α-synuclein) and Chronic Wasting Disease (prions). Additionally, we show that Cap-QuIC can accurately classify biological tissue samples from wild white-tailed deer infected with Chronic Wasting Disease. Our findings elucidate the underlying mechanism that enables the Cap-QuIC assay to distinguish misfolded protein, highlighting its potential as a diagnostic technology for neurodegenerative diseases.

Transmission-selective muscle pathology induced by the active propagation of mutant huntingtin across the human neuromuscular synapse.

Neuron-to-neuron transmission of aggregation-prone, misfolded proteins may potentially explain the spatiotemporal accumulation of pathological lesions in the brains of patients with neurodegenerative protein-misfolding diseases (PMDs). However, little is known about protein transmission from the central nervous system to the periphery, or how this propagation contributes to PMD pathology. To deepen our understanding of these processes, we established two functional neuromuscular systems derived from human iPSCs. One was suitable for long-term high-throughput live-cell imaging and the other was adapted to a microfluidic system assuring that connectivity between motor neurons and muscle cells was restricted to the neuromuscular junction. We show that the Huntington's disease (HD)-associated mutant HTT exon 1 protein (mHTTEx1) is transmitted from neurons to muscle cells across the human neuromuscular junction. We found that transmission is an active and dynamic process that starts before aggregate formation and is regulated by synaptic activity. We further found that transmitted mHTTEx1 causes HD-relevant pathology at both molecular and functional levels in human muscle cells, even in the presence of the ubiquitous expression of mHTTEx1. In conclusion, we have uncovered a causal link between mHTTEx1 synaptic transmission and HD pathology, highlighting the therapeutic potential of blocking toxic protein transmission in PMDs.

The Purely Thermodynamic Anti-prionic Mode of Action for the Treatment of Neurodegenerative Diseases

Neurodegenerative protein-misfolding diseases, like Alzheimer’s (AD) and Parkinson’s disease (PD), are driven by prion-like self-replicating and propagating protein assemblies of amyloid β (Aβ), α-synuclein, and many more. The conformation these proteins have in the aggregated state is thermodynamically more stable than their physiological monomer conformation, which is often intrinsically disordered. Therefore, we have developed all-D-enantiomeric peptide ligands that bind the monomeric protein of interest with high affinity, thereby stabilizing the physiological intrinsically disordered monomer structure. These ligands are eliminating already existing aggregates by disassembling them into monomers. This purely thermodynamic driven mode of action (MoA) is truly “anti-prionic”, because it is eliminating already existing oligomers and fibrils, thus disrupting prion-like replication and propagation of toxic protein aggregates. The MoA is realized by all-D-enantiomeric peptides that are specific for the target protein.

Trazodone rescues dysregulated synaptic and mitochondrial nascent proteomes in prion neurodegeneration.

The unfolded protein response (UPR) is rapidly gaining momentum as a therapeutic target for protein misfolding neurodegenerative diseases, in which its overactivation results in sustained translational repression leading to synapse loss and neurodegeneration. In mouse models of these disorders, from Alzheimer's to prion disease, modulation of the pathway-including by the licensed drug, trazodone-restores global protein synthesis rates with profound neuroprotective effects. However, the precise nature of the translational impairment, in particular the specific proteins affected in disease, and their response to therapeutic UPR modulation are poorly understood. We used non-canonical amino acid tagging (NCAT) to measure de novo protein synthesis in the brains of prion-diseased mice with and without trazodone treatment, in both whole hippocampus and cell-specifically. During disease the predominant nascent proteome changes occur in synaptic, cytoskeletal and mitochondrial proteins in both hippocampal neurons and astrocytes. Remarkably, trazodone treatment for just 2 weeks largely restored the whole disease nascent proteome in the hippocampus to that of healthy, uninfected mice, predominantly with recovery of proteins involved in synaptic and mitochondrial function. In parallel, trazodone treatment restored the disease-associated decline in synapses and mitochondria and their function to wild-type levels. In conclusion, this study increases our understanding of how translational repression contributes to neurodegeneration through synaptic and mitochondrial toxicity via depletion of key proteins essential for their function. Further, it provides new insights into the neuroprotective mechanisms of trazodone through reversal of this toxicity, relevant for the treatment of neurodegenerative diseases via translational modulation.

The purely thermodynamic anti-prionic mode of action for the treatment of neurodegenerative diseases - realized by target protein specific all-D-peptides.

Neurodegenerative protein-misfolding diseases, like Alzheimer's (AD) and Parkinson's disease (PD), are driven by prion-like self-replicating and propagating protein assemblies of Aβ, α-synuclein, and many more. Therefore, we have developed the so-called anti-prionic mode of action (MoA). It is realized by all-D-enantiomeric peptide ligands that bind the monomeric protein of interest with high affinity, thereby stabilizing the physiological intrinsically disordered monomer structure. We call this purely thermodynamic driven mode of action “anti-prionic”, because it is eliminating already existing prion-like aggregates by disassembling them into non-toxic and functional monomers. I will summarize the current, and yet unpublished, progress in realizing the anti-prionic MoA for the target proteins α-synuclein and Aβ. Briefly, the most promising all-D-enantiomeric ligand for α-synuclein, SVD-1a, disassembles preformed α-synuclein fibrils (PFF) as shown by AFM, DLS and SEC analysis. SPR and NMR demonstrate picomolar affinity of SVD-1a to α-synuclein monomers, while keeping them in their physiological intrinsically disordered conformation. The all-D-enantiomeric ligand for Aβ, RD2, disassembles Aβ oligomers obtained from brain tissue of former AD patients. A clinical phase Ib, double-bind, placebo-controlled study with 20 ATN positive patients treated once daily orally with RD2 or placebo for 4 weeks with an additional 4 weeks follow up period demonstrates good safety and tolerability, and yields interesting results on efficacy. This is valuable information for the scheduled phase II study. I will also acknowledge the many contributors of both developments that are too many to be included here in the abstract. Conclusion: The unique anti-prionic mode of action for the treatment of AD, PD and other protein misfolding diseases is promising.

Itraconazole Confers Cytoprotection Against Neurodegenerative Disease-Associated Abnormal Protein Aggregation.

Cells perform regular maintenance to avoid the accumulation of misfolded proteins. Prolonged accumulation of these proteotoxic inclusions generates potential risk of ageing-related diseases such as neurodegenerative diseases. Therefore, removal of such abnormal aggregates can ensure the re-establishment of proteostasis. Ubiquitin proteasome system (UPS) actively participates in the selective removal of aberrantly folded clients with the help of complex proteasome machinery. However, specific induction of proteasome functions to remove abnormal proteins remains an open challenge. Here, we show that Itraconazole treatment induces proteasome activities and degrades the accumulation of bonafide-misfolded proteins, including heat-denatured luciferase. Exposure of Itraconazole elevates the degradation of neurodegenerative disease-associated proteins, e.g. expanded polyglutamine, mutant SOD1, and mutant α-synuclein. Our results suggest that Itraconazole treatment prevents the accumulation of neurodegenerative disease-linked misfolded proteins and generates cytoprotection. These findings reveal that Itraconazole removes abnormal proteins through sequential proteasomal activation and represents a potential protective therapeutic role against protein-misfolding neurodegenerative diseases.

Compact Luminol Chemiluminophores for In Vivo Detection and Imaging of β-Sheet Protein Aggregates.

Protein aggregation has been found in a wide range of neurodegenerative protein-misfolding diseases. The demand for in vivo technologies to identify protein aggregation is at the leading edge for the pathogenic study, diagnostic development, and therapeutic intervention of these devastating disorders. Herein, we report a series of luminol analogues to construct a facile chemiluminescence (CL)-based approach for in vivo detection and imaging of β-sheet protein aggregates. The synthesized compounds exhibited a distinct chemiluminescent response with long emission wavelengths toward reactive oxygen species under physiological conditions and displayed signal amplification in the presence of β-sheet protein aggregates, including α-synuclein, β-amyloid, and tau. Among them, CyLumi-3 was further evaluated as a chemiluminescent probe in preclinical models. By intravenous administration into the model mice via the tail vein, in vivo CL imaging noninvasively detected the specific CL of the probe targeting the α-synuclein aggregates in the brains of living mice. Based on its structural characteristics, CyLumi-3 can readily interact with α-synuclein aggregates with significantly enhanced fluorescence and can identify α-synuclein aggregates in vivo via distinctive CL amplification, which could pave the way for a more comprehensive understanding of protein aggregation in preclinical studies and would provide new hints for developing small-molecule chemiluminophores for protein aggregates.

Temperature-Dependent Regulation of Proteostasis and Longevity.

Temperature is an important environmental condition that determines the physiology and behavior of all organisms. Animals use different response strategies to adapt and survive fluctuations in ambient temperature. The hermaphrodite Caenorhabditis elegans has a well-studied neuronal network consisting of 302 neurons. The bilateral AFD neurons are the primary thermosensory neurons in the nematode. In addition to regulating thermosensitivity, AFD neurons also coordinate cellular stress responses through systemic mechanisms involving neuroendocrine signaling. Recent studies have examined the effects of temperature on altering various signaling pathways through specific gene expression programs that promote stress resistance and longevity. These studies challenge the proposed theories of temperature-dependent regulation of aging as a passive thermodynamic process. Instead, they provide evidence that aging is a well-defined genetic program. Loss of protein homeostasis (proteostasis) is one of the key hallmarks of aging. Indeed, proteostasis pathways, such as the heat shock response and aggregation of metastable proteins, are also controlled by thermosensory neurons in C. elegans. Prolonged heat stress is thought to play a critical role in the development of neurodegenerative protein misfolding diseases in humans. This review presents the latest evidence on how temperature coordinates proteostasis and aging. It also discusses how studies of poikilothermic organisms can be applied to vertebrates and provides new therapeutic strategies for human disease.

Morphological evolution of hnRNPA1 pathological condensates

Heterogenous nuclear ribonucleoprotein A1 (hnRNPA1), family member of ubiquitously expressed RNA-binding proteins involved in RNA metabolism, has been implicated in the pathogenesis of amyotrophic lateral sclerosis (ALS) and other neurodegenerative protein misfolding diseases. hnRNPA1 and other RNA-binding proteins (e.g., FUS and TDP-43) are recruited to membrane-less organelles such as stress granules, and are known to readily phase separate in vitro, a property that may play roles not just in their biological functions but also pathologic dysfunctions.

Differential Accumulation of Misfolded Prion Strains in Natural Hosts of Prion Diseases.

Prion diseases, also known as transmissible spongiform encephalopathies (TSEs), are a group of neurodegenerative protein misfolding diseases that invariably cause death. TSEs occur when the endogenous cellular prion protein (PrPC) misfolds to form the pathological prion protein (PrPSc), which templates further conversion of PrPC to PrPSc, accumulates, and initiates a cascade of pathologic processes in cells and tissues. Different strains of prion disease within a species are thought to arise from the differential misfolding of the prion protein and have different clinical phenotypes. Different strains of prion disease may also result in differential accumulation of PrPSc in brain regions and tissues of natural hosts. Here, we review differential accumulation that occurs in the retinal ganglion cells, cerebellar cortex and white matter, and plexuses of the enteric nervous system in cattle with bovine spongiform encephalopathy, sheep and goats with scrapie, cervids with chronic wasting disease, and humans with prion diseases. By characterizing TSEs in their natural host, we can better understand the pathogenesis of different prion strains. This information is valuable in the pursuit of evaluating and discovering potential biomarkers and therapeutics for prion diseases.

Copper(II) Binding to PBT2 Differs from That of Other 8-Hydroxyquinoline Chelators: Implications for the Treatment of Neurodegenerative Protein Misfolding Diseases.

PBT2 (5,7-dichloro-2-[(dimethylamino)methyl]-8-hydroxyquinoline) is a small Cu(II)-binding drug that has been investigated in the treatment of neurodegenerative diseases, namely, Alzheimer's disease (AD). PBT2 is thought to be highly effective at crossing the blood-brain barrier and has been proposed to exert anti-Alzheimer's effects through the modulation of metal ion concentrations in the brain, specifically the sequestration of Cu(II) from amyloid plaques. However, despite promising initial results in animal models and in clinical trials where PBT2 was shown to improve cognitive function, larger-scale clinical trials did not find PBT2 to have a significant effect on the amyloid plaque burden compared with controls. We propose that the results of these clinical trials likely point to a more complex mechanism of action for PBT2 other than simple Cu(II) sequestration. To this end, herein we have investigated the solution chemistry of Cu(II) coordination by PBT2 primarily using X-ray absorption spectroscopy (XAS), high-energy-resolution fluorescence-detected XAS, and electron paramagnetic resonance. We propose that a novel bis-PBT2 Cu(II) complex with asymmetric coordination may coexist in solution with a symmetric four-coordinate Cu(II)-bis-PBT2 complex distorted from coplanarity. Additionally, PBT2 is a more flexible ligand than other 8HQs because it can act as both a bidentate and a tridentate ligand as well as coordinate Cu(II) in both 1:1 and 2:1 PBT2/Cu(II) complexes.

Survival of the cheapest: how proteome cost minimization drives evolution.

Darwin's theory of evolution emphasized that positive selection of functional proficiency provides the fitness that ultimately determines the structure of life, a view that has dominated biochemical thinking of enzymes as perfectly optimized for their specific functions. The 20th-century modern synthesis, structural biology, and the central dogma explained the machinery of evolution, and nearly neutral theory explained how selection competes with random fixation dynamics that produce molecular clocks essential e.g. for dating evolutionary histories. However, quantitative proteomics revealed that selection pressures not relating to optimal function play much larger roles than previously thought, acting perhaps most importantly via protein expression levels. This paper first summarizes recent progress in the 21st century toward recovering this universal selection pressure. Then, the paper argues that proteome cost minimization is the dominant, underlying 'non-function' selection pressure controlling most of the evolution of already functionally adapted living systems. A theory of proteome cost minimization is described and argued to have consequences for understanding evolutionary trade-offs, aging, cancer, and neurodegenerative protein-misfolding diseases.

PrPC knockdown by liposome-siRNA-peptide complexes (LSPCs) prolongs survival and normal behavior of prion-infected mice immunotolerant to treatment.

Prion diseases are members of neurodegenerative protein misfolding diseases (NPMDs) that include Alzheimer’s, Parkinson’s and Huntington diseases, amyotrophic lateral sclerosis, tauopathies, traumatic brain injuries, and chronic traumatic encephalopathies. No known therapeutics extend survival or improve quality of life of humans afflicted with prion disease. We and others developed a new approach to NPMD therapy based on reducing the amount of the normal, host-encoded protein available as substrate for misfolding into pathologic forms, using RNA interference, a catabolic pathway that decreases levels of mRNA encoding a particular protein. We developed a therapeutic delivery system consisting of small interfering RNA (siRNA) complexed to liposomes and addressed to the central nervous system using a targeting peptide derived from rabies virus glycoprotein. These liposome-siRNA-peptide complexes (LSPCs) cross the blood-brain barrier and deliver PrP siRNA to neuronal cells to decrease expression of the normal cellular prion protein, PrPC, which acts as a substrate for prion replication. Here we show that LSPCs can extend survival and improve behavior of prion-infected mice that remain immunotolerant to treatment. LSPC treatment may be a viable therapy for prion and other NPMDs that can improve the quality of life of patients at terminal disease stages.

In Situ Temporospatial Characterization of the Glial Response to Prion Infection.

Mammalian transmissible spongiform encephalopathies (TSEs) display marked activation of astrocytes and microglia that precedes neuronal loss. Investigation of clinical parallels between TSEs and other neurodegenerative protein misfolding diseases, such as Alzheimer's disease, has revealed similar patterns of neuroinflammatory responses to the accumulation of self-propagating amyloids. The contribution of glial activation to the progression of protein misfolding diseases is incompletely understood, with evidence for mediation of both protective and deleterious effects. Glial populations are heterogeneously distributed throughout the brain and capable of dynamic transitions along a spectrum of functional activation states between pro- and antiinflammatory polarization extremes. Using a murine model of Rocky Mountain Laboratory scrapie, the neuroinflammatory response to prion infection was characterized by evaluating glial activation across 15 brain regions over time and correlating it to traditional markers of prion neuropathology, including vacuolation and PrPSc deposition. Quantitative immunohistochemistry was used to evaluate glial expression of iNOS and Arg1, markers of classical and alternative glial activation, respectively. The results indicate progressive upregulation of iNOS in microglia and a mixed astrocytic profile featuring iNOS expression in white matter tracts and detection of Arg1-positive populations throughout the brain. These data establish a temporospatial lesion profile for this prion infection model and demonstrate evidence of multiple glial activation states.

The unfolded protein response in neurodegenerative disorders - therapeutic modulation of the PERK pathway.

The unfolded protein response (UPR) is a highly conserved protein quality control mechanism, activated in response to Endoplasmic Reticulum (ER) stress. Signalling is mediated through three branches, PERK, IRE1, and ATF6, respectively, that together provide a coordinated response that contributes to overcoming disrupted proteostasis. PERK branch activation predominantly causes a rapid reduction in global rates of translation, while the IRE1 and ATF6 branch signalling induce a transcriptional response resulting in expression of chaperones and components of the protein degradation machinery. Protein misfolding neurodegenerative diseases show disruption of proteostasis as a biochemical feature. In the brains of animal models of disease and in human post mortem tissue from many of these disorders, markers of UPR induction, particularly, the PERK pathway can be observed in close association with disease progression. Recent research has revealed dysregulated UPR signalling to be a major pathogenic mechanism in neurodegeneration, and that genetic and pharmacological modulation of the PERK pathway results in potent neuroprotection. Targeting aberrant UPR signalling is the focus of new therapeutic strategies, which importantly could be beneficial across the broad spectrum of neurodegenerative diseases.

Multiple system atrophy: genetic risks and alpha-synuclein mutations.

Multiple system atrophy (MSA) is one of the few neurodegenerative disorders where we have a significant understanding of the clinical and pathological manifestations but where the aetiology remains almost completely unknown. Research to overcome this hurdle is gaining momentum through international research collaboration and a series of genetic and molecular discoveries in the last few years, which have advanced our knowledge of this rare synucleinopathy. In MSA, the discovery of α-synuclein pathology and glial cytoplasmic inclusions remain the most significant findings. Families with certain types of α-synuclein mutations develop diseases that mimic MSA, and the spectrum of clinical and pathological features in these families suggests a spectrum of severity, from late-onset Parkinson’s disease to MSA. Nonetheless, controversies persist, such as the role of common α-synuclein variants in MSA and whether this disorder shares a common mechanism of spreading pathology with other protein misfolding neurodegenerative diseases. Here, we review these issues, specifically focusing on α-synuclein mutations.

Fine-tuning PERK signaling for neuroprotection.

Protein translation and folding are tightly controlled processes in all cells, by proteostasis, an important component of which is the unfolded protein response (UPR). During periods of endoplasmic reticulum stress because of protein misfolding, the UPR activates a coordinated response in which the PERK branch activation restricts translation, while a variety of genes involved with protein folding, degradation, chaperone expression and stress responses are induced through signaling of the other branches. Chronic overactivation of the UPR, particularly the PERK branch, is observed in the brains of patients in a number of protein misfolding neurodegenerative diseases, including Alzheimer's, and Parkinson's diseases and the tauopathies. Recently, numerous genetic and pharmacological studies in mice have demonstrated the effectiveness of inhibiting the UPR for eliciting therapeutic benefit and boosting memory. In particular, fine‐tuning the level of PERK inhibition to provide neuroprotection without adverse side effects has emerged as a safe, effective approach. This includes the recent discovery of licensed drugs that can now be repurposed in clinical trials for new human treatments for dementia. This review provides an overview of the links between UPR overactivation and neurodegeneration in protein misfolding disorders. It discusses recent therapeutic approaches targeting this pathway, with a focus on treatments that fine‐tune PERK signaling.

Impact of high iron intake on cognition and neurodegeneration in humans and in animal models: a systematic review.

ContextAccumulation of brain iron is linked to aging and protein-misfolding neurodegenerative diseases. High iron intake may influence important brain health outcomes in later life.ObjectiveThe aim of this systematic review was to examine evidence from animal and human studies of the effects of high iron intake or peripheral iron status on adult cognition, brain aging, and neurodegeneration.Data SourcesMEDLINE, Scopus, CAB s, the Cochrane Central Register of Clinical Trials, and OpenGrey databases were searched.Study SelectionStudies investigating the effect of elevated iron intake at all postnatal life stages in mammalian models and humans on measures of adult brain health were included.Data ExtractionData were extracted and evaluated by two authors independently, with discrepancies resolved by discussion. Neurodegenerative disease diagnosis and/or behavioral/cognitive, biochemical, and brain morphologic findings were used to study the effects of iron intake or peripheral iron status on brain health. Risk of bias was assessed for animal and human studies. PRISMA guidelines for reporting systematic reviews were followed.ResultsThirty-four preclinical and 14 clinical studies were identified from database searches. Thirty-three preclinical studies provided evidence supporting an adverse effect of nutritionally relevant high iron intake in neonates on brain-health-related outcomes in adults. Human studies varied considerably in design, quality, and findings; none investigated the effects of high iron intake in neonates/infants.ConclusionsHuman studies are needed to verify whether dietary iron intake levels used in neonates/infants to prevent iron deficiency have effects on brain aging and neurodegenerative disease outcomes.

Targeting of Disordered Proteins by Small Molecules in Neurodegenerative Diseases.

The formation of protein aggregates and inclusions in the brain and spinal cord is a common neuropathological feature of a number of neurodegenerative diseases including Alzheimer's disease (AD), Parkinson's disease (PD), amyotrophic lateral sclerosis (ALS), and many others. These are commonly referred as neurodegenerative proteinopathies or protein-misfolding diseases. The main characteristic of protein aggregates in these disorders is the fact that they are enriched in amyloid fibrils. Since protein aggregation is considered to play a central role for the onset of neurodegenerative proteinopathies, research is ongoing to develop strategies aimed at preventing or removing protein aggregation in the brain of affected patients. Numerous studies have shown that small molecule-based approaches may be potentially the most promising for halting protein aggregation in neurodegenerative diseases. Indeed, several of these compounds have been found to interact with intrinsically disordered proteins and promote their clearing in experimental models. This notwithstanding, at present small molecule inhibitors still awaits achievements for clinical translation. Hopefully, if we determine whether the formation of insoluble inclusions is effectively neurotoxic and find a valid biomarker to assess their protein aggregation-inhibitory activity in the human central nervous system, the use of small molecule inhibitors will be considered as a cure for neurodegenerative protein-misfolding diseases.

A central role for calcineurin in protein misfolding neurodegenerative diseases.

Accumulation of misfolded/unfolded aggregated proteins in the brain is a hallmark of many neurodegenerative diseases affecting humans and animals. Dysregulation of calcium (Ca2+) and disruption of fast axonal transport (FAT) are early pathological events that lead to loss of synaptic integrity and axonal degeneration in early stages of neurodegenerative diseases. Dysregulated Ca2+ in the brain is triggered by accumulation of misfolded/unfolded aggregated proteins in the endoplasmic reticulum (ER), a major Ca2+ storing organelle, ultimately leading to neuronal dysfunction and apoptosis. Calcineurin (CaN), a Ca2+/calmodulin-dependent serine/threonine phosphatase, has been implicated in T cells activation through the induction of nuclear factor of activated T cells (NFAT). In addition to the involvement of several other signaling cascades, CaN has been shown to play a role in early synaptic dysfunction and neuronal death. Therefore, inhibiting hyperactivated CaN in early stages of disease might be a promising therapeutic strategy for treating patients with protein misfolding diseases. In this review, we briefly summarize the structure of CaN, inhibition mechanisms by which immunosuppressants inhibit CaN, role of CaN in maintaining neuronal and synaptic integrity and homeostasis and the role played by CaN in protein unfolding/misfolding neurodegenerative diseases.