Abnormal salivary total and oligomeric alpha-synuclein in Parkinson's disease

In Parkinson’s disease (PD), alpha-synuclein (a-syn) can be detected in biological fluids including saliva. Although previous studies found reduced a-syn total (a-syntotal) concentration in saliva of PD patients, no studies have previously examined salivary a-syn oligomers (a-synolig) concentrations or assessed the correlation between salivary a-syntotal, a-synolig and clinical features in a large cohort of PD patients. Is well known that a-synolig exerts a crucial neurotoxic effect in PD. We collected salivary samples from 60 PD patients and 40 age- and sex-comparable healthy subjects. PD was diagnosed according to the United Kingdom Brain Bank Criteria. Samples of saliva were analyzed by specific anti-a-syn and anti-oligomeric a-syn ELISA kits. A complete clinical evaluation of each patient was performed using MDS-Unified Parkinson's Disease Rating Scale, Beck Depression Inventory, Montreal Cognitive Assessment and Frontal Assessment Battery. Salivary a-syntotal was lower, whereas a-synolig was higher in PD patients than healthy subjects. The a-synolig/a-syntotal ratio was also higher in patients than in healthy subjects. Salivary a-syntotal concentration negatively correlated with that of a-synolig and correlated with several patients’ clinical features. In PD, decreased salivary concentration of a-syntotal may reflect the reduction of a-syn monomers (a-synmon), as well as the formation of insoluble intracellular inclusions and soluble oligomers. The combined detection of a-syntotal and a-synolig in the saliva might help the early diagnosis of P

Characterisation of a type II functionally-deficient variant of alpha-1-antitrypsin discovered in the general population

Lung disease in alpha-1-antitrypsin deficiency (AATD) results from dysregulated proteolytic activity, mainly by neutrophil elastase (HNE), in the lung parenchyma. This is the result of a substantial reduction of circulating alpha-1-antitrypsin (AAT) and the presence in the plasma of inactive polymers of AAT. Moreover, some AAT mutants have reduced intrinsic activity toward HNE, as demonstrated for the common Z mutant, as well as for other rarer variants. Here we report the identification and characterisation of the novel AAT reactive centre loop variant Gly349Arg (p.G373R) present in the ExAC database. This AAT variant is secreted at normal levels in cellular models of AATD but shows a severe reduction in anti-HNE activity. Biochemical and molecular dynamics studies suggest it exhibits unfavourable RCL presentation to cognate proteases and compromised insertion of the RCL into β-sheet A. Identification of a fully dysfunctional AAT mutant that does not show a secretory defect underlines the importance of accurate genotyping of patients with pulmonary AATD manifestations regardless of the presence of normal levels of AAT in the circulation. This subtype of disease is reminiscent of dysfunctional phenotypes in anti-thrombin and C1-inibitor deficiencies so, accordingly, we classify this variant as the first pure functionally-deficient (type II) AATD mutant

Heteropolymerization of α-1-antitrypsin mutants in cell models mimicking heterozygosity

The most common genotype associated with severe α-1-antitrypsin deficiency (AATD) is the Z homozygote. The Z variant (Glu342Lys) of α-1-antitrypsin (AAT) undergoes a conformational change and is retained within the endoplasmic reticulum (ER) of hepatocytes leading to the formation of ordered polymeric chains and inclusion bodies. Accumulation of mutated protein predisposes to cirrhosis whilst plasma AAT deficiency leads to emphysema. Increased risk of liver and lung disease has also been reported in heterozygous subjects who carry Z in association with the milder S allele (Glu264Val) or even with wild-type M. However, it is unknown whether Z AAT can co-polymerize with other AAT variants in vivo. We co-expressed two AAT variants, each modified by a different tag, in cell models that replicate AAT deficiency. We used pull-down assays to investigate interactions between co-expressed variants and showed that Z AAT forms heteropolymers with S and with the rare Mmalton (Phe52del) and Mwurzburg (Pro369Ser) mutants, and to a lesser extent with the wild-type protein. Heteropolymers were recognized by the 2C1 mAb that binds to Z polymers in vivo. There was increased intracellular accumulation of AAT variants when co-expressed with Z AAT, suggesting a dominant negative effect of the Z allele. The molecular interactions between S and Z AAT were confirmed by confocal microscopy showing their colocalization within dilated ER cisternae and by positivity in Proximity Ligation Assays. These results provide the first evidence of intracellular co-polymerization of AAT mutants and contribute to understanding the risk of liver disease in SZ and MZ heterozygotes

A case of remittent C1-inhibitor deficiency

C1-inhibitor is a serine protease inhibitor (serpin) controlling complement and kinin/contactsystem activation. Mutations in C1-inhibitor gene almost consistently result in reduced C1-inhibitor functional level in plasma causing hereditary angioedema, a life-threatening autosomal dominant disorder. Despite a stable defect, the clinical expression of hereditary angioedema is unpredictable, and the molecular mechanism underlyingth is variability remains undisclosed. We report a case of a patient suffering from abdominal pain and presenting markedly reduced C1-inhibitor plasma levels, episodically undergoing spontaneous normalization, car- rying the Arg378Cys missense mutation in the serpin domain. Immunostaining analysis of patient plasma revealed the presence of C1-inhibitor oligomers, together with the occurrence of a SDS stable band that disappeared in reducing conditions, suitable for a disulphide bridged Arg378Cys homodimer. Expression studies in eukaryotic cell lines resulted in a drop in mutant C1-innhibitor secretion compared to wild type and confirmed the plasma observations. Notwithstanding, the purified recombinant proteins behave similarly. Both proteins formed stable covalent complexes with target proteases, and the kinetic of inhibition of the mutant was just slightly diminished, although this reduction increased with temperature.Thus,our findings suggest that the Arg378Cys C1-inhibitor mutant once correctly folded should maintain the wild type functional and structural features and instead it should bear a folding defect, abnormally susceptible to environmental factors, which may occasionally promote protein oligomerization. Moreover it can form a disulphide linked homodimer. Both these processes could account for its variability in plasma levels

Aberrant disulphide bonding contributes to the ER retention of alpha1-antitrypsin deficiency variants

Mutations in alpha1-antitrypsin (AAT) can cause the protein to polymerise and be retained in the endoplasmic reticulum (ER) of hepatocytes. The ensuing systemic AAT deficiency leads to pulmonary emphysema, while intracellular polymers are toxic and cause chronic liver disease. The severity of this process varies considerably between individuals, suggesting the involvement of mechanistic co-factors and potential for therapeutically beneficial interventions. We show in Hepa1.6 cells that the mildly polymerogenic I (Arg39Cys) AAT mutant forms aberrant inter- and intra-molecular disulphide bonds involving the acquired Cys39 and the only cysteine residue in the wild-type (M) sequence (Cys232). Substitution of Cys39 to serine partially restores secretion, showing that disulphide bonding contributes to the intracellular retention of I AAT. Covalent homodimers mediated by inter-Cys232 bonding alone are also observed in cells expressing the common Z and other polymerising AAT variants where conformational behaviour is abnormal, but not in those expressing M AAT. Prevention of such disulphide linkage through the introduction of the Cys232Ser mutation or by treatment of cells with reducing agents increases Z AAT secretion. Our results reveal that disulphide interactions enhance intracellular accumulation of AAT mutants and implicate the oxidative ER state as a pathogenic co-factor. Redox modulation, e.g. by anti-oxidant strategies, may therefore be beneficial in AAT deficiency-associated liver disease

Intermittent C1-Inhibitor Deficiency Associated with Recessive Inheritance: Functional and Structural Insight

C1-inhibitor is a serine protease inhibitor (serpin) controlling complement and contact system activation. Gene mutations result in reduced C1-inhibitor functional plasma level causing hereditary angioedema, a life-threatening disorder. Despite a stable defect, the clinical expression of hereditary angioedema is unpredictable, and the molecular mechanism underlying this variability remains undisclosed. Here we report functional and structural studies on the Arg378Cys C1-inhibitor mutant found in a patient presenting reduced C1-inhibitor levels, episodically undergoing normalization. Expression studies resulted in a drop in mutant C1-innhibitor secretion compared to wild-type. Notwithstanding, the purified proteins had similar features. Thermal denaturation experiments showed a comparable denaturation profile, but the mutant thermal stability decays when tested in conditions reproducing intracellular crowding.Our findings suggest that once correctly folded, the Arg378Cys C1-inhibitor is secreted as an active, although quite unstable, monomer. However, it could bear a folding defect, occasionally promoting protein oligomerization and interfering with the secretion process, thus accounting for its plasma level variability. This defect is exacerbated by the nature of the mutation since the acquired cysteine leads to the formation of non-functional homodimers through inter-molecular disulphide bonding. All the proposed phenomena could be modulated by specific environmental conditions, rendering this mutant exceptionally vulnerable to mild stress

Salivary alpha-synuclein in the diagnosis of parkinson's disease and progressive supranuclear palsy

Introduction: Alpha-synuclein (α-syn) aggregation is the pathological hallmark of Parkinson's Disease (PD). In this study, we measured α-syn total (α-syn total ), oligomeric α-syn (α-syn olig ) and α-syn olig /α-syn total ratio in the saliva of patients affected by PD and in age and sex-matched healthy subjects. We also compared salivary α-syn total measured in PD with those detected in Progressive Supranuclear Palsy (PSP), in order to assess whether salivary α-syn can be used as a biomarker for PD and for the differential diagnosis between PD and PSP. Methods: We studied 100 PD patients, 20 patients affected by PSP and 80 age- and sex-matched healthy subjects. ELISA analysis was performed using two commercial ELISA platforms and a specific ELISA assay for α-syn aggregates. Results: We detected lower α-syn total and higher α-syn olig in PD than in healthy subjects. Conversely in PSP salivary α-syn total concentration was comparable to that measured in healthy subjects. Receiver Operating Characteristic analyses revealed specific cut-off values able to differentiate PD patients from healthy subjects and PSP patients with high sensitivity and specificity. However, there was no significant correlation between clinical and molecular data. Conclusion: Salivary α-syn detection could be a promising and easily accessible biomarker for PD and for the differential diagnosis between PD and PSP

Polymers of Z α1-antitrypsin are secreted in cell models of disease.

Alpha-1 antitrypsin (A1AT) is predominantly synthesised in the liver and secreted into the circulation, where it protects the lungs from the enzyme neutrophil elastase. Mutations in A1AT, particularly the Z mutation, cause A1AT deficiency, characterised by polymerisation and retention of A1AT within hepatocytes, low levels of circulating A1AT, uncontrolled elastase activity and emphysema. Patients also display variable amounts of circulating polymers in their plasma, but it is unknown whether polymers can be secreted from hepatocytes or form in plasma from secreted monomeric Z-A1AT. Here we investigated the origin of extracellular polymers in cell culture models of A1AT deficiency and showed that: all extracellular Z-A1AT contained only mature N-linked glycosylation, which excludes direct release of immature ER proteins upon cell death; polymers still appeared in the culture medium of transfected cells cultured in the presence of a polymerisation blocking antibody (4B12); most of the polymerised Z-A1AT co-localised with BiP but there was also partial co-localisation with proteins of the Golgi apparatus; and that cells co-transfected with tagged versions of Z-A1AT showed immunoprecipitation patterns compatible with intracellular polymerisation only. These results demonstrate the intracellular origin of secreted polymers in vitro, and support that plasma polymers derive at least in part from hepatocyte secretion

A combined panel of salivary biomarkers in de novo Parkinson's Disease

Objective: To investigate molecular biomarkers of a-synuclein and tau aggregation, autophagy, and inflammation in the saliva of de novo Parkinson's disease (PD) patients in comparison to healthy subjects (HS), and to correlate molecular data with clinical features of PD patients, in order to establish whether abnormalities of these parameters are associated with specific clusters of de novo PD patients, and their potential diagnostic power in differentiating PD patients from HS. Methods: We measured total and oligomeric a-synuclein, total-tau and phosphorylated-tau, MAP-LC3beta, and TNFalpha in the saliva of 80 de novo PD patients and 62 HS, using quantitative Enzyme-Linked Immunosorbent Assay analysis. Results: Oligomeric a-synuclein, total-tau, MAP-LC3beta, and TNFalpha levels resulted significantly higher in patients with respect to HS, while no significant differences were detected for total a-synuclein or phosphorylated-tau. Phosphorylated-tau directly correlated with MAP-LC3beta, whereas it inversely correlated with TNFalpha in PD patients. An inverse correlation was detected between MAP-LC3beta and non-motor symptoms severity. Principal Component Analysis showed that molecular and clinical parameters were independent of each other in de novo PD patients. Receiver Operating Characteristic curve analysis reported an accurate diagnostic performance of oligomeric a-synuclein and MAP-LC3beta. The diagnostic accuracy of total a-synuclein increased when it was combined with other salivary biomarkers targeting different molecular pathways. Interpretation: Our study proposes a novel biomarker panel using saliva, a non-invasive biofluid, in de novo PD patients, with implications in understanding the molecular pathways involved in PD pathogenesis and the relevance of different molecular pathways in determining clinical PD subtypes

Salivary a-synuclein in PD patients and healthy subjects.

<p>Salivary a-syn in patients with Parkinson’s disease (PD) and healthy subjects (HS). Each histogram corresponds to the mean concentration of α-syn_total (panel A), α-syn_olig (panel B) and the α-syn_total/α-syn_olig ratio (panel C) in the saliva of patients with PD (black histogram) and HS (white histogram). Vertical bars denote standard deviation. Asterisk denote significant differences. Scatter-plot (panel D) shows the negative correlation between α-syn_total and α-syn_olig in patients with PD.</p