Neuropathological assessment of the olfactory bulb and tract in individuals with COVID-19.

The majority of patients with Parkinson disease (PD) experience a loss in their sense of smell and accumulate insoluble α-synuclein aggregates in their olfactory bulbs (OB). Subjects affected by a SARS-CoV-2-linked illness (COVID-19) also frequently experience hyposmia. We previously postulated that microglial activation as well as α-synuclein and tau misprocessing can occur during host responses following microbial encounters. Using semiquantitative measurements of immunohistochemical signals, we examined OB and olfactory tract specimens collected serially at autopsies between 2020 and 2023. Deceased subjects comprised 50 adults, which included COVID19 + patients (n = 22), individuals with Lewy body disease (e.g., PD; dementia with Lewy bodies (n = 6)), Alzheimer disease (AD; n = 3), and other neurodegenerative disorders (e.g., progressive supranuclear palsy (n = 2); multisystem atrophy (n = 1)). Further, we included neurologically healthy controls (n = 9), and added subjects with an inflammation-rich brain disorder as neurological controls (NCO; n = 7). When probing for microglial and histiocytic reactivity in the anterior olfactory nuclei (AON) by anti-CD68 immunostaining, scores were consistently elevated in NCO and AD cases. In contrast, microglial signals on average were not significantly altered in COVID19 + patients relative to healthy controls, although anti-CD68 reactivity in their OB and tracts declined with progression in age. Mild-to-moderate increases in phospho-α-synuclein and phospho-tau signals were detected in the AON of tauopathy- and synucleinopathy-afflicted brains, respectively, consistent with mixed pathology, as described by others. Lastly, when both sides were available for comparison in our case series, we saw no asymmetry in the degree of pathology of the left versus right OB and tracts. We concluded from our autopsy series that after a fatal course of COVID-19, microscopic changes in the rostral, intracranial portion of the olfactory circuitry -when present- reflected neurodegenerative processes seen elsewhere in the brain. In general, microglial reactivity correlated best with the degree of Alzheimer's-linked tauopathy and declined with progression of age in COVID19 + patients.

Initial assessment of α-synuclein structure in platelets.

Over the last few years data from our group have indicated that α-synuclein is important in development of immune cells as well as potentially erythrocytes and platelets. The latter is important since this protein may work as negative regulator of granule release. Thus, we sought to begin to understand the structure of this protein in platelets. Flow cytometric analysis of this protein using region-specific (N-terminus, central region and C-terminus) monoclonal antibodies was performed. Antibody to the central region gave the strongest shift among all three antibodies, with the C-terminus having intermediate shift and N-terminus minimal shift. Western blotting using the same antibodies showed similar binding of all antibodies to α-synuclein. These results suggest a similar arrangement of this protein in platelets as seen in neurons. Future studies ought to look at the role that each protein region plays in platelets.

Assessment of Cytotoxicity of α-Synuclein in Budding Yeast Using a Spot Growth Assay and Fluorescent Microscopy.

The budding yeast Saccharomyces cerevisiae is a model organism amenable both to genetic analysis and cell biology. Due to these advantages, yeast has provided platforms to examine the properties of pathogenic proteins involved in human diseases. The methods used to examine the cytotoxicity and intracellular localization of α-Synuclein, a human neuronal protein implicated in Parkinson's disease, using yeast have been described herein. These methods are readily accessible to researchers or graduate students unfamiliar with experiments using yeast and applicable to larger scale analyses, such as high-throughput genetic and chemical screenings.

Membrane Interactions of α-Synuclein Revealed by Multiscale Molecular Dynamics Simulations, Markov State Models, and NMR.

α-Synuclein (αS) is a presynaptic protein that binds to cell membranes and is linked to Parkinson's disease (PD). Binding of αS to membranes is a likely first step in the molecular pathophysiology of PD. The αS molecule can adopt multiple conformations, being largely disordered in water, adopting a ß-sheet conformation when present in amyloid fibrils, and forming a dynamic multiplicity of α-helical conformations when bound to lipid bilayers and related membrane-mimetic surfaces. Multiscale molecular dynamics simulations in conjunction with nuclear magnetic resonance (NMR) and cross-linking mass spectrometry (XLMS) measurements are used to explore the interactions of αS with an anionic lipid bilayer. The simulations and NMR measurements together reveal a break in the helical structure of the central non-amyloid-ß component (NAC) region of αS in the vicinity of residues 65-70, which may facilitate subsequent oligomer formation. Coarse-grained simulations of αS starting from the structure of αS when bound to a detergent micelle reveal the overall pattern of protein contacts to anionic lipid bilayers, while subsequent all-atom simulations provide details of conformational changes upon membrane binding. In particular, simulations and NMR data for liposome-bound αS indicate incipient ß-strand formation in the NAC region, which is supported by intramolecular contacts seen via XLMS and simulations. Markov state models based on the all-atom simulations suggest a mechanism of conformational change of membrane-bound αS via a dynamic helix break in the region of residue 65 in the NAC region. The emergent dynamic model of membrane-interacting αS advances our understanding of the mechanism of PD, potentially aiding the design of novel therapeutic approaches.

Inhibition of Orexin/Hypocretin Neurons Ameliorates Elevated Physical Activity and Energy Expenditure in the A53T Mouse Model of Parkinson's Disease.

Aside from the classical motor symptoms, Parkinson's disease also has various non-classical symptoms. Interestingly, orexin neurons, involved in the regulation of exploratory locomotion, spontaneous physical activity, and energy expenditure, are affected in Parkinson's. In this study, we hypothesized that Parkinson's-disease-associated pathology affects orexin neurons and therefore impairs functions they regulate. To test this, we used a transgenic animal model of Parkinson's, the A53T mouse. We measured body composition, exploratory locomotion, spontaneous physical activity, and energy expenditure. Further, we assessed alpha-synuclein accumulation, inflammation, and astrogliosis. Finally, we hypothesized that chemogenetic inhibition of orexin neurons would ameliorate observed impairments in the A53T mice. We showed that aging in A53T mice was accompanied by reductions in fat mass and increases in exploratory locomotion, spontaneous physical activity, and energy expenditure. We detected the presence of alpha-synuclein accumulations in orexin neurons, increased astrogliosis, and microglial activation. Moreover, loss of inhibitory pre-synaptic terminals and a reduced number of orexin cells were observed in A53T mice. As hypothesized, this chemogenetic intervention mitigated the behavioral disturbances induced by Parkinson's disease pathology. This study implicates the involvement of orexin in early Parkinson's-disease-associated impairment of hypothalamic-regulated physiological functions and highlights the importance of orexin neurons in Parkinson's disease symptomology.

Assessment of the Retina of Plp-α-Syn Mice as a Model for Studying Synuclein-Dependent Diseases.

Purpose: Synucleinopathies such as multiple system atrophy (MSA) and Parkinson's disease are associated with a variety of visual symptoms. Functional and morphological retinal aberrations are therefore supposed to be valuable biomarkers for these neurodegenerative diseases. This study examined the retinal morphology and functionality resulting from human α-synuclein (α-Syn) overexpression in the transgenic Plp-α-Syn mouse model. Methods: Immunohistochemistry on retinal sections and whole-mounts was performed on 8- to 11-week-old and 12-month-old Plp-α-Syn mice and C57BL/6N controls. Quantitative RT-PCR experiments were performed to study the expression of endogenous and human α-Syn and tyrosine hydroxylase (TH). We confirmed the presence of human α-Syn in the retina in western blot analyses. Multi-electrode array (MEA) analyses from light-stimulated whole-mounted retinas were used to investigate their functionality. Results: Biochemical and immunohistochemical analyses showed human α-Syn in the retina of Plp-α-Syn mice. We found distinct staining in different retinal cell layers, most abundantly in rod bipolar cells of the peripheral retina. In the periphery, we also observed a trend toward a decline in the number of retinal ganglion cells. The number of TH+ neurons was unaffected in this human α-Syn overexpression model. MEA recordings showed that Plp-α-Syn retinas were functional but exhibited mild alterations in dim light conditions. Conclusions: Together, these findings implicate an impairment of retinal neurons in the Plp-α-Syn mouse. The phenotype partly relates to retinal deficits reported in MSA patients. We further propose the suitability of the Plp-α-Syn retina as a biological model to study synuclein-mediated mechanisms.

Rethinking calcium profiles around single channels: the exponential and periodic calcium nanodomains.

Many fundamental calcium-dependent physiological processes are triggered by high local calcium levels that are established around the sites of calcium entry into the cell (channels). They are dubbed as calcium nanodomains but their exact profiles are still elusive. The concept of calcium nanodomains stems from a linear model of calcium diffusion and is only valid when calcium increases are smaller than the concentration of cytoplasmic buffers. Recent data indicates that much higher calcium levels cause buffer saturation. Therefore, I sought explicit solutions of a nonlinear reaction-diffusion model and found a dichotomous solution. For small fluxes, the steady state calcium profile is quasi-exponential, and when calcium exceeds buffer concentration a spatial periodicity appears. Analytical results are supported by Monte-Carlo simulations. I also imaged 1D- and radial calcium distributions around single α-synuclein channels in cell-free conditions. Measured Ca profiles are consistent with theoretical predictions. I propose that the periodic calcium patterns may well arise under certain conditions and their specific functional role has to be established.

Using a FRET Library with Multiple Probe Pairs To Drive Monte Carlo Simulations of α-Synuclein.

We describe a strategy for experimentally-constraining computational simulations of intrinsically disordered proteins (IDPs), using α-synuclein, an IDP with a central role in Parkinson's disease pathology, as an example. Previously, data from single-molecule Förster Resonance Energy Transfer (FRET) experiments have been effectively utilized to generate experimentally constrained computational models of IDPs. However, the fluorophores required for single-molecule FRET experiments are not amenable to the study of short-range (<30 Å) interactions. Using ensemble FRET measurements allows one to acquire data from probes with multiple distance ranges, which can be used to constrain Monte Carlo simulations in PyRosetta. To appropriately employ ensemble FRET data as constraints, we optimized the shape and weight of constraining potentials to afford ensembles of structures that are consistent with experimental data. We also used this approach to examine the structure of α-synuclein in the presence of the compacting osmolyte trimethylamine-N-oxide. Despite significant compaction imparted by 2 M trimethylamine-N-oxide, the underlying ensemble of α-synuclein remains largely disordered and capable of aggregation, also in agreement with experimental data. These proof-of-concept experiments demonstrate that our modeling protocol enables one to efficiently generate experimentally constrained models of IDPs that incorporate atomic-scale detail, allowing one to study an IDP under a variety of conditions.

Trichloroethylene and Parkinson's Disease: Risk Assessment.

This study was conducted to investigate the mechanism of action and extent of selective dopaminergic neurodegeneration caused by exposure to trichloroethylene (TCE) leading to the endogenous formation of the neurotoxin 1-trichloromethyl-1,2,3,4-tetrahydro-ß-carboline (TaClo) in rodents. Beginning at 3 months of age, male C57BL/6 mice received oral TCE dissolved in vehicle for 8 months. Dopaminergic neuronal loss was assessed by nigral tyrosine hydroxylase (TH) immunoreactivity. Selective dopaminergic neurodegeneration was determined based on histological analysis of non-dopaminergic neurons in the brain. Behavioral assays were evaluated using open field activity and rotarod tests. Mitochondrial complex I activity, oxidative stress markers, and microglial activation were also examined in the substantia nigra. The level of TaClo was detected using HPLC-electrospray ionization tandem mass spectrometry. Dopaminergic neurotoxicity of TaClo was determined in midbrain organotypic cultures from rat pups. Following 8 months of TCE treatment, there was a progressive and selective loss of 50% of the dopaminergic neurons in mouse substantia nigra (SN) and about 50% loss of dopamine and 72% loss of 3,4-dihydroxyphenylacetic acid in the striatum, respectively. In addition, motor deficits, mitochondrial impairment, oxidative stress, and inflammation were measured. TaClo content was quantified in the brain after TCE treatment. In organotypic cultures, TaClo rather than TCE induced dopaminergic neuronal loss, similar to MPP+. TCE exposure may stimulate the endogenous formation of TaClo, which is responsible for dopaminergic neurodegeneration. However, even prolonged administration of TCE was insufficient for producing a greater than 50% loss of nigral dopamine neurons, indicating that additional co-morbid factors would be needed for mimicking the profound loss of dopamine neurons seen in Parkinson's disease.

Dynamic footprint based locomotion sway assessment in α-synucleinopathic mice using Fast Fourier Transform and Low Pass Filter.

BACKGROUND: Sway is a crucial gait characteristic tightly correlated with the risk of falling in patients with Parkinsons disease (PD). So far, the swaying pattern during locomotion has not been investigated in rodent models using the analysis of dynamic footprint recording obtained from the CatWalk gait recording and analysis system. NEW METHODS: We present three methods for describing locomotion sway and apply them to footprint recordings taken from C57BL6/N wild-type mice and two different α-synuclein transgenic PD-relevant mouse models (α-synm-ko, α-synm-koxα-synh-tg). Individual locomotion data were subjected to three different signal processing analytical approaches: the first two methods are based on Fast Fourier Transform (FFT), while the third method uses Low Pass Filters (LPF). These methods use the information associated with the locomotion sway and generate sway-related parameters. RESULTS: The three proposed methods were successfully applied to the footprint recordings taken from all paws as well as from front/hind-paws separately. Nine resulting sway-related parameters were generated and successfully applied to differentiate between the mouse models under study. Namely, α-synucleinopathic mice revealed higher sway and sway itself was significantly higher in the α-synm-koxα-synh-tg mice compared to their wild-type littermates in eight of the nine sway-related parameters. COMPARISON WITH EXISTING METHOD: Previous locomotion sway index computation is based on the estimated center of mass position of mice. CONCLUSIONS: The methods presented in this study provide a sway-related gait characterization. Their application is straightforward and may lead to the identification of gait pattern derived biomarkers in rodent models of PD.

Using Dimensionality Reduction to Systematically Expand Conformational Sampling of Intrinsically Disordered Peptides.

One of the approaches to improve our ability to characterize biologically important processes and to map out an underlying free energy landscape is to direct MD simulations to explore molecular conformational phase space faster. Intrinsically disordered systems with shallow free energy landscapes of a huge number of metastable minima pose a particular challenge in this regard. Both characterization of the often ill-defined conformational states as well as the assessment of the degree of convergence of phase space exploration are problematic. We have used a multidimensional scaling-like embedding (sketch-map) to describe the energetically accessible regions of phase space for a peptide fragment of the intrinsically disordered protein α-synuclein. Using sketch-map coordinates from a short initial simulation, we guided additional MD simulations to efficiently expand sampling of the conformational space. The sketch-map projections are very well suited to detect rare but possibly functionally relevant events, metastable intermediates, and transition states in the vast amount of data.

Multicenter Assessment of Immunohistochemical Methods for Pathological Alpha-Synuclein in Sigmoid Colon of Autopsied Parkinson's Disease and Control Subjects.

BACKGROUND: Conflicting results from studies of Lewy-type α-synucleinopathy (LTS) in colonic biopsies of subjects with Parkinson's disease (PD) prompted a two-part multicenter assessment. The first assessment, now published (Acta Neuropathol Commun 4 : 35, 2016), examined archived colonic biopsies and found that none of the tested methods was adequately sensitive or specific. OBJECTIVE: As the amount of nervous tissue in typical colonic biopsies may be insufficient, and the clinical diagnosis of PD not completely accurate, the objective of the current study was to use instead full-thickness sections of sigmoid colon from autopsy-proven PD and normal subjects. METHODS: Seven different immunohistochemical (IHC) methods were used, employing five different primary antibodies and four different combinations of epitope exposure and signal development protocols. Specific staining was defined as being restricted to morphological features consistent with neuronal elements. Stained slides from each subject were independently categorized as being positive or negative for LTS, and their density semi-quantitatively graded, by four raters blinded to diagnosis. RESULTS: Agreement and mean diagnostic performance varied markedly between raters. With the two most accurate raters, 5 methods achieved diagnostic accuracies of 70% or greater; one method had 100% accuracy and 100% inter-rater agreement. The submucosa had the highest prevalence of pathological LTS staining, followed by the muscularis and mucosa. CONCLUSIONS: The major conclusion of this study is that, when sufficient submucosa and LTS is present, and when specific staining is defined as being consistent with neuronal morphology, adequately-trained raters may reliably distinguish PD colon from control using suitable IHC methods.

Assessment of the degree of asymmetry of pathological features in neurodegenerative diseases. What is the significance for brain banks?

Brain banks allow researchers access to tissue from well-characterised neurodegenerative disease cases. Fixed tissue employed for diagnosis is often not appropriate for research and frozen tissue is therefore made available. Many brain banks use a protocol where half the brain is fixed and half frozen. Recently a study has shown that there can be asymmetry in protein deposition between the hemispheres especially with tau and TDP-43. We aimed to test this hypothesis by prospectively taking bilateral cortical blocks from 30 brains on arrival, and immunostaining to assess the degree of asymmetry. In 6 out 14 cases of AD (Alzheimer's Disease) (Modified Braak Stage V-VI), there was some asymmetrical staining for tau. In 2 cases, there was moderate discrepancy for tau staining between left and right calcarine cortices. However, careful analysis in both these cases revealed discrepancies in tau staining in adjacent regions even on the same side. The α-synuclein staining showed asymmetry in one case only, the Aß showed only mild asymmetry in 3 cases of AD. The TDP-43 pathology appeared symmetrical in the 2 cases of frontotemporal lobar degeneration with motor neurone disease, but there was asymmetry noted when seen in conjunction with AD. In conclusion, there is the potential for asymmetrical pathology in neurodegenerative diseases and caution should be maintained when freezing half and fixing half of the brain in neurodegenerative diseases. Nevertheless, marked variability in staining can also be identified in adjacent cortical areas so there is no guarantee that an alternative strategy would be superior.

Post-mortem assessment of the short and long-term effects of the trophic factor neurturin in patients with α-synucleinopathies.

Substantial interest persists for developing neurotrophic factors to treat neurodegenerative diseases. At the same time, significant progress has been made in implementing gene therapy as a means to provide long-term expression of bioactive neurotrophic factors to targeted sites in the brain. Nonetheless, to date, no double-blind clinical trial has achieved positive results on its primary endpoint despite robust benefits achieved in animal models. A major issue with advancing the field is the paucity of information regarding the expression and effects of neurotrophic factors in human neurodegenerative brain, relative to the well-characterized responses in animal models. To help fill this information void, we examined post-mortem brain tissue from four patients with nigrostriatal degeneration who had participated in clinical trials testing gene delivery of neurturin to the putamen of patients. Each had died of unrelated causes ranging from 1.5-to-3-months (2 Parkinson's disease patients), to 4+-years (1 Parkinson's disease and 1 multiple-system atrophy-parkinsonian type patient) following gene therapy. Quantitative and immunohistochemical evaluation of neurturin, alpha-synuclein, tyrosine hydroxylase (TH) and an oligodendroglia marker (Olig 2) were performed in each brain. Comparable volumes-of-expression of neurturin were seen in the putamen in all cases (~15-22%; mean=18.5%). TH-signal in the putamen was extremely sparse in the shorter-term cases. A 6-fold increase was seen in longer-term cases, but was far less than achieved in animal models of nigrostriatal degeneration with similar or even far less NRTN exposure. Less than 1% of substantia nigra (SN) neurons stained for neurturin in the shorter-term cases. A 15-fold increase was seen in the longer-term cases, but neurturin was still only detected in ~5% of nigral cells. These data provide unique insight into the functional status of advanced, chronic nigrostriatal degeneration in human brain and the response of these neurons to neurotrophic factor stimulation. They demonstrate mild but persistent expression of gene-mediated neurturin over 4-years, with an apparent, time-related amplification of its transport and biological effects, albeit quite weak, and provide unique information to help plan and design future trials.

Control tissue in brain banking: the importance of thorough neuropathological assessment.

Historically, control brain tissue was classified as such mainly by clinical history, and underwent limited neuropathological analysis. Significant progress has been made in recent years with the collection of more extensive clinical information and more specific classifications of neurodegenerative disease, aided by advances in histological processing and increasingly sensitive detection methods. We hypothesised that this may have resulted in certain pathologies previously going unidentified, due to insufficient block sampling and an inadequate range of stains, resulting in the disease not being recognised. We therefore investigated the significance of changes to our own protocols for examining control brain tissue before and after 2007. Control cases that were originally assessed before 2007 were re-assessed using our current staining protocol and antibodies, and compared with age-matched cases post-2007. We found that almost all cases that were originally described as neuropathologically normal displayed some level of pathology after re-analysis, with four cases displaying what we have termed 'major' pathology that previously went unidentified, emphasising on a small scale the importance of accurate neuropathological analysis of control tissue, and highlighting the inherent difficulty of traditionally classifying tissue simply as 'disease' or 'control'. We hope our findings will stimulate debate within the brain banking community, with the eventual aim being standardisation of protocols for assessing controls across brain banks.

Propuesta de reformas a los prestadores públicos de servicios médicos en Chile: “fortaleciendo la opción pública” / A reform proposal to strengthen public health care

Currently, there is no discussion on the need to improve and strengthen the institutional health care modality of FONASA (MAI), the health care system used by the public services net and by most of the population, despite the widely known and long lasting problems such as waiting lists, hospital debt with suppliers, lack of specialists and increasing services purchase transference to the private sector, etc. In a dichotomous sectorial context, such as the one of health’s social security in Chile (the state on one side and the market on the other), points of view are polarized and stances tend to seek refuge within themselves. As a consequence, to protect the public solution is commonly associated with protecting the “status quo”, creating an environment that is reluctant to change. The author proposes a solution based on three basic core ideas, which, if proven effective, can strengthen each other if combined properly. These are: network financing management, governance of health care services in MAI and investments and human resources in networked self-managed institutions. The proposal of these core ideas was done introducing a reality testing that minimizes the politic complexity of their implementation.

α-Synuclein Misfolding Versus Aggregation Relevance to Parkinson's Disease: Critical Assessment and Modeling.

α-Synuclein, an abundant and conserved presynaptic brain protein, is implicated as a critical factor in Parkinson's disease (PD). The aggregation of α-synuclein is believed to be a critical event in the disease process. α-Synuclein is characterized by a remarkable conformational plasticity, adopting different conformations depending on the environment. Therefore, it is classified as an "intrinsically disordered protein." Recently, a debate has challenged the view on the intrinsically disordered behavior of α-synuclein in the cell. It has been proposed that α-synuclein is a stable tetramer with a low propensity for aggregation; however, its destabilization leads to protein misfolding and its aggregation kinetics. In our critical analysis, we discussed about major issues: (i) why α-synuclein conformational behavior does not fit into the normal secondary structural characteristics of proteins, (ii) potential amino acids involved in the complexity of misfolding in α-synuclein that leads to aggregation, and (iii) the role of metals in misfolding and aggregation. To evaluate the above critical issues, we developed bioinformatics models related to secondary and tertiary conformations, Ramachandran plot, free energy change, intrinsic disordered prediction, solvent accessibility, and FoldIndex pattern. To the best of our knowledge, this is a novel critical assessment to understand the misfolding biology of synuclein and its relevance to Parkinson's disease.

Biochemical assessment of precuneus and posterior cingulate gyrus in the context of brain aging and Alzheimer's disease.

Defining the biochemical alterations that occur in the brain during "normal" aging is an important part of understanding the pathophysiology of neurodegenerative diseases and of distinguishing pathological conditions from aging-associated changes. Three groups were selected based on age and on having no evidence of neurological or significant neurodegenerative disease: 1) young adult individuals, average age 26 years (n = 9); 2) middle-aged subjects, average age 59 years (n = 5); 3) oldest-old individuals, average age 93 years (n = 6). Using ELISA and Western blotting methods, we quantified and compared the levels of several key molecules associated with neurodegenerative disease in the precuneus and posterior cingulate gyrus, two brain regions known to exhibit early imaging alterations during the course of Alzheimer's disease. Our experiments revealed that the bioindicators of emerging brain pathology remained steady or decreased with advancing age. One exception was S100B, which significantly increased with age. Along the process of aging, neurofibrillary tangle deposition increased, even in the absence of amyloid deposition, suggesting the presence of amyloid plaques is not obligatory for their development and that limited tangle density is a part of normal aging. Our study complements a previous assessment of neuropathology in oldest-old subjects, and within the limitations of the small number of individuals involved in the present investigation, it adds valuable information to the molecular and structural heterogeneity observed along the course of aging and dementia. This work underscores the need to examine through direct observation how the processes of amyloid deposition unfold or change prior to the earliest phases of dementia emergence.

P2-substituted N-acylprolylpyrrolidine inhibitors of prolyl oligopeptidase: biochemical evaluation, binding mode determination, and assessment in a cellular model of synucleinopathy.

We have investigated the effect of regiospecifically introducing substituents in the P2 part of the typical dipeptide derived basic structure of PREP inhibitors. This hitherto unexplored modification type can be used to improve target affinity, selectivity, and physicochemical parameters in drug discovery programs focusing on PREP inhibitors. Biochemical evaluation of the produced inhibitors identified several substituent types that significantly increase target affinity, thereby reducing the need for an electrophilic "warhead" functionality. Pronounced PREP specificity within the group of Clan SC proteases was generally observed. Omission of the P1 electrophilic function did not affect the overall binding mode of three representative compounds, as studied by X-ray crystallography, while the P2 substituents were demonstrated to be accommodated in a cavity of PREP that, to date, has not been probed by inhibitors. Finally, we report on results of selected inhibitors in a SH-SY5Y cellular model of synucleinopathy and demonstrate a significant antiaggregation effect on α-synuclein.

Distinct phases of free α-synuclein--a Monte Carlo study.

The α-synuclein protein (αS), implicated in Parkinson's disease, shows conformational versatility. It aggregates into ß-sheet-rich fibrils, occurs in helical membrane-bound forms, is disordered as a free monomer, and has recently been suggested to have a folded helical tetramer as its main physiological form. Here, we use implicit solvent all-atom Monte Carlo methods to explore the conformational ensemble sampled by the free αS monomer. We analyze secondary structure propensities, size, and topological properties and compare with existing experimental data. Our study suggests that free αS has two distinct phases. One phase has the expected disordered character. The other phase also shows large conformational variability. However, in this phase, the ß-strand content is substantial, and the backbone fold shows statistical similarities with that in αS fibrils. Presence of this phase is consistent with data from low-temperature experiments. Conversion of disordered αS to this fibril-like form requires the crossing of a rather large apparent free-energy barrier.