Relationship of serum beta-synuclein with blood biomarkers and brain atrophy.

BACKGROUND: Recent data support beta-synuclein as a blood biomarker to study synaptic degeneration in Alzheimer's disease (AD). METHODS: We provide a detailed comparison of serum beta-synuclein immunoprecipitation - mass spectrometry (IP-MS) with the established blood markers phosphorylated tau 181 (p-tau181) (Simoa) and neurofilament light (NfL) (Ella) in the German FTLD consortium cohort (n = 374) and its relation to brain atrophy (magnetic resonance imaging) and cognitive scores. RESULTS: Serum beta-synuclein was increased in AD but not in frontotemporal lobar degeneration (FTLD) syndromes. Beta-synuclein correlated with atrophy in temporal brain structures and was associated with cognitive impairment. Serum p-tau181 showed the most specific changes in AD but the lowest correlation with structural alterations. NfL was elevated in all diseases and correlated with frontal and temporal brain atrophy. DISCUSSION: Serum beta-synuclein changes differ from those of NfL and p-tau181 and are strongly related to AD, most likely reflecting temporal synaptic degeneration. Beta-synuclein can complement the existing panel of blood markers, thereby providing information on synaptic alterations. HIGHLIGHTS: Blood beta-synuclein is increased in Alzheimer's disease (AD) but not in frontotemporal lobar degeneration (FTLD) syndromes. Blood beta-synuclein correlates with temporal brain atrophy in AD. Blood beta-synuclein correlates with cognitive impairment in AD. The pattern of blood beta-synuclein changes in the investigated diseases is different to phosphorylated tau 181 (p-tau181) and neurofilament light (NfL).

Increased Expression of Alpha-, Beta-, and Gamma-Synucleins in Brainstem Regions of a Non-Human Primate Model of Parkinson's Disease.

Parkinson's disease (PD) is characterized by cell loss in the substantia nigra and the presence of alpha-synuclein (α-syn)-containing neuronal Lewy bodies. While α-syn has received major interest in the pathogenesis of PD, the function of beta- and gamma-synucleins (ß-syn and γ-syn, respectively) is not really known. Yet, these proteins are members of the same family and also concentrated in neuronal terminals. The current preclinical study investigated the expression levels of α-, ß-, and γ-synucleins in brainstem regions involved in PD physiopathology. We analyzed synuclein expression in the substantia nigra, raphe nuclei, pedunculopontine nucleus, and locus coeruleus from control and parkinsonian (by MPTP) macaques. MPTP-intoxicated monkeys developed a more or less severe parkinsonian score and were sacrificed after a variable post-MPTP period ranging from 1 to 20 months. The expression of the three synucleins was increased in the substantia nigra after MPTP, and this increase correlates positively, although not very strongly, with cell loss and motor score and not with the time elapsed after intoxication. In the dorsal raphe nucleus, the expression of the three synucleins was also increased, but only α- and γ-Syn are linked to the motor score and associated cell loss. Finally, although no change in synuclein expression was demonstrated in the locus coeruleus after MPTP, we found increased expression levels of γ-Syn, which are only correlated with cell loss in the pedunculopontine nucleus. Altogether, our data suggest that these proteins may play a key role in brainstem regions and mesencephalic tegmentum. Given the involvement of these brain regions in non-motor symptoms of PD, these data also strengthen the relevance of the MPTP macaque model of PD, which exhibits pathological changes beyond nigral DA cell loss and α-synucleinopathy.

Alpha and Beta Synucleins: From Pathophysiology to Clinical Application as Biomarkers.

The synuclein family includes three neuronal proteins, named α-synuclein, ß-synuclein, and γ-synuclein, that have peculiar structural features. α-synuclein is largely known for being a key protein in the pathophysiology of Parkinson's disease (PD) and other synucleinopathies, namely, dementia with Lewy bodies and multisystem atrophy. The role of ß-synuclein and γ-synuclein is less well understood in terms of physiological functions and potential contribution to human diseases. α-synuclein has been investigated extensively in both cerebrospinal fluid (CSF) and blood as a potential biomarker for synucleinopathies. Recently, great attention has been also paid to ß-synuclein, whose CSF and blood levels seem to reflect synaptic damage and neurodegeneration independent of the presence of synucleinopathy. In this review, we aim to provide an overview on the pathophysiological roles of the synucleins. Because γ-synuclein has been poorly investigated in the field of synucleinopathy and its pathophysiological roles are far from being clear, we focus on the interactions between α-synuclein and ß-synuclein in PD. We also discuss the role of α-synuclein and ß-synuclein as potential biomarkers to improve the diagnostic characterization of synucleinopathies, thus highlighting their potential application in clinical trials for disease-modifying therapies. © 2022 The Authors. Movement Disorders published by Wiley Periodicals LLC on behalf of International Parkinson and Movement Disorder Society.

Synuclein Family Members Prevent Membrane Damage by Counteracting α-Synuclein Aggregation.

The 140 amino acid protein α-synuclein (αS) is an intrinsically disordered protein (IDP) with various roles and locations in healthy neurons that plays a key role in Parkinson's disease (PD). Contact with biomembranes can lead to α-helical conformations, but can also act as s seeding event for aggregation and a predominant ß-sheet conformation. In PD patients, αS is found to aggregate in various fibrillary structures, and the shift in aggregation and localization is associated with disease progression. Besides full-length αS, several related polypeptides are present in neurons. The role of many αS-related proteins in the aggregation of αS itself is not fully understood Two of these potential aggregation modifiers are the αS splicing variant αS Δexon3 (Δ3) and the paralog ß-synuclein (ßS). Here, polarized ATR-FTIR spectroscopy was used to study the membrane interaction of these proteins individually and in various combinations. The method allowed a continuous monitoring of both the lipid structure of biomimetic membranes and the aggregation state of αS and related proteins. The use of polarized light also revealed the orientation of secondary structure elements. While αS led to a destruction of the lipid membrane upon membrane-catalyzed aggregation, ßS and Δ3 aggregated significantly less, and they did not harm the membrane. Moreover, the latter proteins reduced the membrane damage triggered by αS. There were no major differences in the membrane interaction for the different synuclein variants. In combination, these observations suggest that the formation of particular protein aggregates is the major driving force for αS-driven membrane damage. The misbalance of αS, ßS, and Δ3 might therefore play a crucial role in neurodegenerative disease.

Targeted Mass Spectrometry Suggests Beta-Synuclein as Synaptic Blood Marker in Alzheimer's Disease.

Synaptic degeneration is a major hallmark of Alzheimer's disease (AD) and the best pathological correlate of cognitive dysfunction. Synaptic markers are therefore a highly desired read-out for patient diagnosis and possible follow-up in clinical trials. Several synaptic markers for AD are described in cerebrospinal fluid (CSF), but studies in blood have failed so far. Using quantitative mass spectrometry (IP-MS, MRM) we observed increased concentrations of the presynaptic protein beta-synuclein (ßSyn) in CSF and blood of AD patients (n = 64, p < 0.01) and confirmed this finding in two validation cohorts (AD: n = 40 and n = 49, controls: n = 44 and n = 25). ßSyn was already increased in patients with mild cognitive impairment (p < 0.01) and was also markedly increased in Creutzfeldt-Jakob disease (CJD; n = 25, p < 0.001) but not behavioral variant frontotemporal dementia (n = 16), dementia with Lewy bodies/Parkinson's disease dementia (n = 13), Parkinson's disease (n = 25), or amyotrophic lateral sclerosis (n = 30). The diagnostic sensitivity and specificity for CJD versus other neurodegenerative diseases was ≥96%. These findings suggest ßSyn as a candidate blood marker for synaptic degeneration that might be used in clinical AD trials and patient follow-up as part of the recently suggested ATN biomarker panel. It can also serve in the differential diagnosis of CJD.

Age-related distribution and potential role of SNCB in topographically different retinal areas of the common marmoset Callithrix jacchus, including the macula.

Evidence of an age-related increase of ß-synuclein (SNCB) in several parts of the visual system including the retina has been reported. SNCB is thought to function as an antagonist of α-synuclein in neurodegenerative diseases, but the exact role of SNCB remains unclear. The presented work studies two different aspects of the onset and role of SNCB in the retinal pigment epithelium (RPE). First, the topographical and intracellular distributions of SNCB in the RPE of non-human marmoset monkey (Callithrix jacchus) were evaluated in paraffin-embedded eyes and RPE whole mounts from different developmental stages (neonatal, adolescent, and adult). Thus, revealed distinct lifetime-related alterations of the topographical and intracellular distributions of SNCB in the primate macula compared to the retinal periphery. Furthermore, the function and influences of SNCB on ARPE-19 cells and primary porcine RPE (ppRPE) cells were characterized by exposing these cells with recombinant SNCB (rSNCB) at different concentrations. Moreover, apoptosis, protein- and mRNA-expression levels of factors of the p53/MDM2 signaling cascade and inflammation- and oxidation-related genes were investigated. The observed dose-depended decreased apoptosis rates together with the PLD2 mediated activation of the p53 pathway promotes senescence-related processes in SNCB exposed common ARPE-19 cells from human origin. Further, increased HMOX1 and NOX4 levels indicate increased oxidative stress and inflammatory responses triggered by SNCB. The obtained differences in the distribution of SNCB in primate RPE together with alterations of cellular functions in rSNCB-exposed RPE cells (e.g., ARPE-19, ppRPE) support SNCB-related effects like inflammatory response and stress-related properties on RPE over lifetime. The possible functional relevance of SNCB in physiological aging converting into a pathophysiological condition should be investigated in further studies.

Interactions between the Intrinsically Disordered Proteins ß-Synuclein and α-Synuclein.

Several intrinsically disordered proteins have been implicated in the process of amyloid fibril formation in neurodegenerative disease, and developing approaches to inhibit the aggregation of these intrinsically disordered proteins is critical for establishing effective therapies against disease progression. The aggregation pathway of the intrinsically disordered protein alpha-synuclein, which is implicated in several neurodegenerative diseases known as synucleinopathies, has been extensively characterized. Less attention has been leveraged on beta-synuclein, a homologous intrinsically disordered protein that co-localizes with alpha-synuclein and is known to delay alpha-synuclein fibril formation. In this review, we focus on beta-synuclein and the molecular-level interactions between alpha-synuclein and beta-synuclein that underlie the delay of fibril formation. We highlight studies that begin to define alpha-synuclein and beta-synuclein interactions at the monomer, oligomer, and surface levels, and suggest that beta-synuclein plays a role in regulation of inhibition at many different stages of alpha-synuclein aggregation.

Sumoylation Protects Against ß-Synuclein Toxicity in Yeast.

Aggregation of α-synuclein (αSyn) plays a central role in the pathogenesis of Parkinson's disease (PD). The budding yeast Saccharomyces cerevisiae serves as reference cell to study the interplay between αSyn misfolding, cytotoxicity and post-translational modifications (PTMs). The synuclein family includes α, ß and γ isoforms. ß-synuclein (ßSyn) and αSyn are found at presynaptic terminals and both proteins are presumably involved in disease pathogenesis. Similar to αSyn, expression of ßSyn leads to growth deficiency and formation of intracellular aggregates in yeast. Co-expression of αSyn and ßSyn exacerbates the cytotoxicity. This suggests an important role of ßSyn homeostasis in PD pathology. We show here that the small ubiquitin-like modifier SUMO is an important determinant of protein stability and ßSyn-induced toxicity in eukaryotic cells. Downregulation of sumoylation in a yeast strain, defective for the SUMO-encoding gene resulted in reduced yeast growth, whereas upregulation of sumoylation rescued growth of yeast cell expressing ßSyn. This corroborates a protective role of the cellular sumoylation machinery against ßSyn-induced toxicity. Upregulation of sumoylation significantly reduced ßSyn aggregate formation. This is an indirect molecular process, which is not directly linked to ßSyn sumoylation because amino acid substitutions in the lysine residues required for ßSyn sumoylation decreased aggregation without changing yeast cellular toxicity. αSyn aggregates are more predominantly degraded by the autophagy/vacuole than by the 26S ubiquitin proteasome system. We demonstrate a vice versa situation for ßSyn, which is mainly degraded in the 26S proteasome. Downregulation of sumoylation significantly compromised the clearance of ßSyn by the 26S proteasome and increased protein stability. This effect is specific, because depletion of functional SUMO did neither affect ßSyn aggregate formation nor its degradation by the autophagy/vacuolar pathway. Our data support that cellular ßSyn toxicity and aggregation do not correlate in their cellular impact as for αSyn but rather represent two distinct independent molecular functions and molecular mechanisms. These insights into the relationship between ßSyn-induced toxicity, aggregate formation and degradation demonstrate a significant distinction between the impact of αSyn compared to ßSyn on eukaryotic cells.

A pH-dependent switch promotes ß-synuclein fibril formation via glutamate residues.

α-Synuclein (αS) is the primary protein associated with Parkinson's disease, and it undergoes aggregation from its intrinsically disordered monomeric form to a cross-ß fibrillar form. The closely related homolog ß-synuclein (ßS) is essentially fibril-resistant under cytoplasmic physiological conditions. Toxic gain-of-function by ßS has been linked to dysfunction, but the aggregation behavior of ßS under altered pH is not well-understood. In this work, we compare fibril formation of αS and ßS at pH 7.3 and mildly acidic pH 5.8, and we demonstrate that pH serves as an on/off switch for ßS fibrillation. Using αS/ßS domain-swapped chimera constructs and single residue substitutions in ßS, we localized the switch to acidic residues in the N-terminal and non-amyloid component domains of ßS. Computational models of ßS fibril structures indicate that key glutamate residues (Glu-31 and Glu-61) in these domains may be sites of pH-sensitive interactions, and variants E31A and E61A show dramatically altered pH sensitivity for fibril formation supporting the importance of these charged side chains in fibril formation of ßS. Our results demonstrate that relatively small changes in pH, which occur frequently in the cytoplasm and in secretory pathways, may induce the formation of ßS fibrils and suggest a complex role for ßS in synuclein cellular homeostasis and Parkinson's disease.

Life-time expression of the proteins peroxiredoxin, beta-synuclein, PARK7/DJ-1, and stathmin in the primary visual and primary somatosensory cortices in rats.

Four distinct proteins are regulated in the aging neuroretina and may be regulated in the cerebral cortex, too: peroxiredoxin, beta-synuclein, PARK[Parkinson disease(autosomal recessive, early onset)]7/DJ-1, and Stathmin. Thus, we performed a comparative analysis of these proteins in the the primary somatosensory cortex (S1) and primary visual cortex (V1) in rats, in order to detect putative common development-, maturation- and age-related changes. The expressions of peroxiredoxin, beta-synuclein, PARK[Parkinson disease (autosomal recessive, early onset)]7/DJ-1, and Stathmin were compared in the newborn, juvenile, adult, and aged S1 and V1. Western blot (WB), quantitative reverse-transcription polymerase chain reaction (qRT-PCR), and immunohistochemistry (IHC) analyses were employed to determine whether the changes identified by proteomics were verifiable at the cellular and molecular levels. All of the proteins were detected in both of the investigated cortical areas. Changes in the expressions of the four proteins were found throughout the life-time of the rats. Peroxiredoxin expression remained unchanged over life-time. Beta-Synuclein expression was massively increased up to the adult stage of life in both the S1 and V1. PARK[Parkinson disease (autosomal recessive, early onset)]7/DJ-1 exhibited a massive up-regulation in both the S1 and V1 at all ages. Stathmin expression was massively down regulated after the neonatal period in both the S1 and V1. The detected protein alterations were analogous to their retinal profiles. This study is the first to provide evidence that peroxiredoxin, beta-synuclein, PARK[Parkinson disease (autosomal recessive, early onset)]7/DJ-1, and Stathmin are associated with postnatal maturation and aging in both the S1 and V1 of rats. These changes may indicate their involvement in key functional pathways and may account for the onset or progression of age-related pathologies.

Identification of potential novel interaction partners of the sodium-activated potassium channels Slick and Slack in mouse brain.

The sodium-activated potassium channels Slick (Slo2.1, KCNT2) and Slack (Slo2.2, KCNT1) are paralogous channels of the Slo family of high-conductance potassium channels. Slick and Slack channels are widely distributed in the mammalian CNS and they play a role in slow afterhyperpolarization, generation of depolarizing afterpotentials and in setting and stabilizing the resting potential. In the present study we used a combined approach of (co)-immunoprecipitation studies, Western blot analysis, double immunofluorescence and mass spectrometric sequencing in order to investigate protein-protein interactions of the Slick and Slack channels. The data strongly suggest that Slick and Slack channels co-assemble into identical cellular complexes. Double immunofluorescence experiments revealed that Slick and Slack channels co-localize in distinct mouse brain regions. Moreover, we identified the small cytoplasmic protein beta-synuclein and the transmembrane protein 263 (TMEM 263) as novel interaction partners of both, native Slick and Slack channels. In addition, the inactive dipeptidyl-peptidase (DPP 10) and the synapse associated protein 102 (SAP 102) were identified as constituents of the native Slick and Slack channel complexes in the mouse brain. This study presents new insights into protein-protein interactions of native Slick and Slack channels in the mouse brain.