Neurodegenerative Amyloidoses Research Articles

Biophysical Evidence for the Amyloid Formation of a Recombinant Rab2 Isoform of Leishmania donovani.

The most fatal form of Visceral leishmaniasis or kala-azar is caused by the intracellular protozoan parasite Leishmania donovani. The life cycle and the infection pathway of the parasite are regulated by the small GTPase family of Rab proteins. The involvement of Rab proteins in neurodegenerative amyloidosis is implicated in protein misfolding, secretion abnormalities and dysregulation. The inter and intra-cellular shuttlings of Rab proteins are proposed to be aggregation-prone. However, the biophysical unfolding and aggregation of protozoan Rab proteins is limited. Understanding the aggregation mechanisms of Rab protein will determine their physical impact on the disease pathogenesis and individual health. This work investigates the acidic pH-induced unfolding and aggregation of a recombinant Rab2 protein from L. donovani (rLdRab2) using multi-spectroscopic probes. The acidic unfolding of rLdRab2 induced at acidic pH is characterised by intrinsic fluorescence and ANS assay, while aggregation is determined by Thioflavin-T and 90⁰ light scattering assay. Circular dichroism determined the secondary structure of monomers and aggregates. The aggregate morphology was imaged by transmission electron microscopy. rLdRab2 was modelled to be a Rab2 isoform with loose globular packing. The acidinduced unfolding of the protein is a plausible non-two-state process. At pH 2.0, a partially folded intermediate (PFI) state characterised by ~ 30 % structural loss and exposed hydrophobic core was found to accumulate. The PFI state slowly converted into well-developed protofibrils at high protein concentrations demonstrating its amyloidogenic nature. The native state of the protein was also observed to be aggregation-prone at high protein concentrations. However, it formed amorphous aggregation instead of fibrils. To our knowledge, this is the first study to report in vitro amyloid-like behaviour of Rab proteins in L donovani. This study provides a novel opportunity to understand the complete biophysical characteristics of Rab2 protein of the lower eukaryote, L. donovani.

Bioavailability and Health Impact of Ingested Amyloid-like Protein Fibrils and their Link with Inflammatory Status: A Need for More Research?

The use of amyloid-like protein fibrils (ALFs) in food formulations looks very promising in terms of improving techno-functional properties, but raises some concerns in terms of food safety, because of their structural resemblance to disease-related endogenous amyloids. This review focuses on the biological fate and potential health implications of ingested ALF structures in both healthy and predisposed individuals. A comprehensive overview of ALF gastrointestinal digestion, intestinal absorption, and systemic dissemination is provided, in addition to a thorough assessment of potential ALF cross-seeding of endogenous precursor proteins linked to (non)neurodegenerative amyloidosis. In general, this study concludes that the health impact of ALF consumption remains widely understudied and merits additional research efforts to determine the exact extent to which ALF ingestion may influence the general health status.

Plasma and Brain Concentrations of Doxycycline after Single and Repeated Doses in Wild-Type and APP23 Mice.

Repurposing doxycycline for the treatment of amyloidosis has recently been put forward because of the antiaggregating and anti-inflammatory properties of the drug. Most of the investigations of the therapeutic potential of doxycycline for neurodegenerative amyloidosis, e.g., prion and Alzheimer disease (AD), have been carried out in mouse models, but surprisingly no data are available regarding the concentrations reached in the brain after systemic administration. We filled this gap by analyzing the pharmacokinetic profile of doxycycline in plasma and brain after single and repeated intraperitoneal injections of 10 and 100 mg/kg, in wild-type mice and the APP23 mouse model of AD. The main outcomes of our study are: 1) Peak plasma concentrations ranged from 2 to10 μg/ml, superimposable to those in humans; 2) brain-to-plasma ratio was ∼0.2, comparable to the cerebrospinal fluid/serum ratios in humans; 3) brain Cmax 4-6 hours after a single dose was ∼0.5 (10 mg/kg) and ∼5 μM (100 mg/kg). Notably, these concentrations are lower than those required for the drug's antiaggregating properties as observed in cell-free studies, suggesting that other features underlie the positive cognitive effects in AD mice; 4) elimination half-life was shorter than in humans (3-6 vs. 15-30 hours), therefore no significant accumulation was observed in mouse brain following repeated treatments; and 5) there were no differences between doxycycline concentrations in brain areas of age-matched wild-type and APP23 mice. These data are useful for planning preclinical studies with translational validity, and to identify more reliably the mechanism(s) of action underlying the central in vivo effects of doxycycline.

Distinct mechanisms of mutant huntingtin toxicity in different yeast strains.

Expansion of polyglutamine stretches in several proteins causes neurodegenerative amyloidoses, including Huntington disease. In yeast, mutant huntingtin (mHtt) with a stretch of 103 glutamine residues (HttQ103) forms toxic aggregates. A range of yeast strains have been used to elucidate the mechanisms of mHtt toxicity, and have revealed perturbations of various unrelated processes. HttQ103 aggregates can induce aggregation of cellular proteins, many of which contain glutamine/asparagine-rich regions, including Sup35 and Def1. In the strain 74-D694 HttQ103, toxicity is related to aggregation-mediated depletion of soluble Sup35 and its interacting partner Sup45. Def1 was also implicated in mHtt toxicity, since its lack detoxified HttQ103 in another yeast strain, BY4741. Here we show that in BY4742, deletion of DEF1 lowers HttQ103 toxicity and decreases the amount of its polymers, but does not affect copolymerization of Sup35. Furthermore, in contrast to 74-D694, increasing the levels of soluble Sup35 and Sup45 does not alleviate toxicity of HttQ103 in BY4742. These data demonstrate a difference in the mechanisms underlying mHtt toxicity in different yeast strains and suggest that in humans with Huntington disease, neurons of different brain compartments and cells in other tissues can also be damaged by different mechanisms.

A protein polymerization cascade mediates toxicity of non-pathological human huntingtin in yeast.

Several neurodegenerative amyloidoses, including Huntington disease, are caused by expansion of polyglutamine (polyQ) stretches in otherwise unrelated proteins. In a yeast model, an N-terminal fragment of mutant huntingtin with a stretch of 103 glutamine residues aggregates and causes toxicity, while its non-toxic wild type variant with a sequence of 25 glutamines (Htt25Q) does not aggregate. Here, we observed that non-toxic polymers of various proteins with glutamine-rich domains could seed polymerization of Htt25Q, which caused toxicity by seeding polymerization of the glutamine/asparagine-rich Sup35 protein thus depleting the soluble pools of this protein and its interacting partner, Sup45. Importantly, only polymers of Htt25Q, but not of the initial benign polymers, induced Sup35 polymerization, indicating an intermediary role of Htt25Q in cross-seeding Sup35 polymerization. These data provide a novel insight into interactions between amyloidogenic proteins and suggest a possible role for these interactions in the pathogenesis of Huntington and other polyQ diseases.

Association of Skin with the Pathogenesis and Treatment of Neurodegenerative Amyloidosis

Amyloidosis are a large group of conformational diseases characterized by abnormal protein folding and assembly which results in the accumulation of insoluble protein aggregates that may accumulate systemically or locally in certain organs or tissue. In local amyloidosis, amyloid deposits are restricted to a particular organ or tissue. Alzheimer’s, Parkinson’s disease, and amyotrophic lateral sclerosis are some examples of neurodegenerative amyloidosis. Local manifestation of protein aggregation in the skin has also been reported. Brain and skin are highly connected at a physiological and pathological level. Recently several studies demonstrated a strong connection between brain and skin in different amyloid diseases. In the present review, we discuss the relevance of the “brain–skin connection” in different neurodegenerative amyloidosis, not only at the pathological level, but also as a strategy for the treatment of these diseases.

Hsp104 antagonizes α-synuclein aggregation and reduces dopaminergic degeneration in a rat model of Parkinson disease

Parkinson disease (PD) is characterized by dopaminergic neurodegeneration and intracellular inclusions of alpha-synuclein amyloid fibers, which are stable and difficult to dissolve. Whether inclusions are neuroprotective or pathological remains controversial, because prefibrillar oligomers may be more toxic than amyloid inclusions. Thus, whether therapies should target inclusions, preamyloid oligomers, or both is a critically important issue. In yeast, the protein-remodeling factor Hsp104 cooperates with Hsp70 and Hsp40 to dissolve and reactivate aggregated proteins. Metazoans, however, have no Hsp104 ortholog. Here we introduced Hsp104 into a rat PD model. Remarkably, Hsp104 reduced formation of phosphorylated alpha-synuclein inclusions and prevented nigrostriatal dopaminergic neurodegeneration induced by PD-linked alpha-synuclein (A30P). An in vitro assay employing pure proteins revealed that Hsp104 prevented fibrillization of alpha-synuclein and PD-linked variants (A30P, A53T, E46K). Hsp104 coupled ATP hydrolysis to the disassembly of preamyloid oligomers and amyloid fibers composed of alpha-synuclein. Furthermore, the mammalian Hsp70 and Hsp40 chaperones, Hsc70 and Hdj2, enhanced alpha-synuclein fiber disassembly by Hsp104. Hsp104 likely protects dopaminergic neurons by antagonizing toxic alpha-synuclein assemblies and might have therapeutic potential for PD and other neurodegenerative amyloidoses.

Rapid disease development in scrapie-infected mice deficient for CD40 ligand.

The inhibition of CD40-CD40L interaction-mediated signalling was suggested as a therapeutic strategy for the treatment of Alzheimer's disease. Conversely, CD40-deficient neurons were reported to be more vulnerable to stress associated with ageing as well as nerve growth factor-beta and serum withdrawal. We studied the scrapie infection of CD40L-deficient (CD40L(-/-)) mice to see whether ablation of the CD40L gene would be beneficial or detrimental in this model of a neurodegenerative amyloidosis. CD40L(-/-) mice died on average 40 days earlier than wild-type control mice and exhibited a more pronounced vacuolation of the neuropil and an increased microglia activation. The experimental model indicates that a deficiency for CD40L is highly detrimental in prion diseases and reinforces the neuroprotective function of intact CD40-CD40L interactions. The stimulation of neuroprotective pathways may represent a possibility to delay therapeutically the disease onset in prion infections of the central nervous system.