Tamie J. Chilcote

Phosphorylation of Ser-129 Is the Dominant Pathological Modification of α-Synuclein in Familial and Sporadic Lewy Body Disease

A comprehensive, unbiased inventory of synuclein forms present in Lewy bodies from patients with dementia with Lewy bodies was carried out using two-dimensional immunoblot analysis, novel sandwich enzyme-linked immunosorbent assays with modification-specific synuclein antibodies, and mass spectroscopy. The predominant modification of α-synuclein in Lewy bodies is a single phosphorylation at Ser-129. In addition, there is a set of characteristic modifications that are present to a lesser extent, including ubiquitination at Lys residues 12, 21, and 23 and specific truncations at Asp-115, Asp-119, Asn-122, Tyr-133, and Asp-135. No other modifications are detectable by tandem mass spectrometry mapping, except for a ubiquitous N-terminal acetylation. Small amounts of Ser-129 phosphorylated and Asp-119-truncated α-synuclein are present in the soluble fraction of both normal and disease brains, suggesting that these Lewy body-associated forms are produced during normal metabolism of α-synuclein. In contrast, ubiquitination is only detected in Lewy bodies and is primarily present on phosphorylated synuclein; it therefore likely occurs after phosphorylated synuclein has deposited into Lewy bodies. This invariant pattern of specific phosphorylation, truncation, and ubiquitination is also present in the detergent-insoluble fraction of brain from patients with familial Parkinsons disease (synuclein A53T mutation) as well as multiple system atrophy, suggesting a common pathogenic pathway for both genetic and sporadic Lewy body diseases. These observations are most consistent with a model in which preferential accumulation of normally produced Ser-129 phosphorylated α-synuclein is the key event responsible for the formation of Lewy bodies in various Lewy body diseases.

Effects of α-Synuclein Immunization in a Mouse Model of Parkinson’s Disease

Abnormal folding of alpha-synuclein (alpha-syn) is thought to lead to neurodegeneration and the characteristic symptoms of Lewy body disease (LBD). Since previous studies suggest that immunization might be a potential therapy for Alzheimers disease, we hypothesized that immunization with human (h)alpha-syn might have therapeutic effects in LBD. For this purpose, halpha-syn transgenic (tg) mice were vaccinated with halpha-syn. In mice that produced high relative affinity antibodies, there was decreased accumulation of aggregated halpha-syn in neuronal cell bodies and synapses that was associated with reduced neurodegeneration. Furthermore, antibodies produced by immunized mice recognized abnormal halpha-syn associated with the neuronal membrane and promoted the degradation of halpha-syn aggregates, probably via lysosomal pathways. Similar effects were observed with an exogenously applied FITC-tagged halpha-syn antibody. These results suggest that vaccination is effective in reducing neuronal accumulation of halpha-syn aggregates and that further development of this approach might have a potential role in the treatment of LBD.

Nucleation-dependent polymerization is an essential component of amyloid-mediated neuronal cell death

Accumulating evidence suggests that amyloid protein aggregation is pathogenic in many diseases, including Alzheimers disease. However, the mechanisms by which protein aggregation mediates cellular dysfunction and overt cell death are unknown. Recent reports have focused on the potential role of amyloid oligomers or protofibrils as a neurotoxic form of amyloid-β (Aβ) and related amyloid aggregates. Here we describe studies indicating that overt neuronal cell death mediated by Aβ1-40 is critically dependent on ongoing Aβ1-40 polymerization and is not mediated by a single stable species of neurotoxic aggregate. The extent and rate of neuronal cell death can be controlled by conditions that alter the rate of Aβ polymerization. The results presented here indicate that protofibrils and oligomeric forms of Aβ most likely generate neuronal cell death through a nucleation-dependent process rather than acting as direct neurotoxic ligands. These findings bring into question the use of the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide formazan assay (MTT assay) as a reporter of Aβ-mediated neuronal cell death and suggest that diffusible Aβ protofibrils and oligomers more likely mediate subtle alterations of synaptic function and long-term potentiation rather than overt neuronal cell death. These results have been extended to Aβ1-42, the non-Aβ component of Alzheimers disease amyloid plaques, and human amylin, suggesting that nucleation-dependent polymerization is a common mechanism of amyloid-mediated neuronal cell death. Our findings indicate that ongoing amyloid fibrillogenesis may be an essential mechanistic process underlying the pathogenesis associated with protein aggregation in amyloid disorders.

Red Blood Cells Are the Major Source of Alpha-Synuclein in Blood

Background: α-Synuclein has been directly linked to Parkinson’s disease etiology by mutations in and multiplication of its gene that result in a familial form of Parkinson’s disease. α-Synuclein has been detected in blood, and was found to be elevated in the blood of those individuals with the α-synuclein gene multiplication. Objective: A complete analysis of the level of α-synuclein in blood has not been performed. In this report, we determine the quantitative distribution of α-synuclein in the plasma and different cellular fractions of human blood. The levels of α-synuclein in human and mouse blood are compared. Methods: α-Synuclein levels in the different fractions of blood were quantified by a sandwich ELISA with purified recombinant α-synuclein as an assay standard. Samples were further characterized by Western immunoblot analysis. Results: More than 99% of the α-synuclein resides in the red blood cells (RBCs) with less than 1% of the total detected in the plasma, platelets and peripheral blood mononuclear cells. Conclusions: More than 99% of the α-synuclein in human blood is present in the peripheral blood cells, with the remainder in plasma. Fractionation of peripheral blood cells from human blood and quantification of α-synuclein revealed that only a very small amount of the total α-synuclein is present in peripheral blood mononuclear cells, and platelets, with the majority of α-synuclein in blood being present in RBCs. Considering the abundance and fragility of RBCs, α-synuclein levels in these other blood fractions or other bodily fluids such as cerebrospinal fluid may be artificially elevated by contamination with intact or lysed RBCs.

Hyperglycemia-induced cerebral hematoma expansion is mediated by plasma kallikrein

Hyperglycemia is associated with greater hematoma expansion and poor clinical outcomes after intracerebral hemorrhage. We show that cerebral hematoma expansion triggered by intracerebral infusion of autologous blood is greater in diabetic rats and mice compared to nondiabetic controls and that this augmented expansion is ameliorated by plasma kallikrein (PK) inhibition or deficiency. Intracerebral injection of purified PK augmented hematoma expansion in both diabetic and acutely hyperglycemic rats, whereas injection of bradykinin, plasmin or tissue plasminogen activator did not elicit such a response. This response, which occurs rapidly, was prevented by co-injection of the glycoprotein VI agonist convulxin and was mimicked by glycoprotein VI inhibition or deficiency, implicating an effect of PK on inhibiting platelet aggregation. We show that PK inhibits collagen-induced platelet aggregation by binding collagen, a response enhanced by elevated glucose concentrations. The effect of hyperglycemia on hematoma expansion and PK-mediated inhibition of platelet aggregation could be mimicked by infusing mannitol. These findings suggest that hyperglycemia auguments cerebral hematoma expansion by PK-mediated osmotic-sensitive inhibition of hemostasis.

Plasma Kallikrein Mediates Retinal Vascular Dysfunction and Induces Retinal Thickening in Diabetic Rats

OBJECTIVE Plasma kallikrein (PK) has been identified in vitreous fluid obtained from individuals with diabetic retinopathy and has been implicated in contributing to retinal vascular dysfunction. In this report, we examined the effects of PK on retinal vascular functions and thickness in diabetic rats. RESEARCH DESIGN AND METHODS We investigated the effects of a selective PK inhibitor, ASP-440, and C1 inhibitor (C1-INH), the primary physiological inhibitor of PK, on retinal vascular permeability (RVP) and hemodynamics in rats with streptozotocin-induced diabetes. The effect of intravitreal PK injection on retinal thickness was examined by spectral domain optical coherence tomography. RESULTS Systemic continuous administration of ASP-440 for 4 weeks initiated at the time of diabetes onset inhibited RVP by 42% (P = 0.013) and 83% (P < 0.001) at doses of 0.25 and 0.6 mg/kg per day, respectively. Administration of ASP-440 initiated 2 weeks after the onset of diabetes ameliorated both RVP and retinal blood flow abnormalities in diabetic rats measured at 4 weeks’ diabetes duration. Intravitreal injection of C1-INH similarly decreased impaired RVP in rats with 2 weeks’ diabetes duration. Intravitreal injection of PK increased both acute RVP and sustained focal RVP (24 h postinjection) to a greater extent in diabetic rats compared with nondiabetic control rats. Intravitreal injection of PK increased retinal thickness compared with baseline to a greater extent (P = 0.017) in diabetic rats (from 193 ± 10 μm to 223 ± 13 μm) compared with nondiabetic rats (from 182 ± 8 μm to 193 ± 9 μm). CONCLUSIONS These results show that PK contributes to retinal vascular dysfunctions in diabetic rats and that the combination of diabetes and intravitreal injection of PK in rats induces retinal thickening.

Plasma Kallikrein Mediates Angiotensin II Type 1 Receptor–Stimulated Retinal Vascular Permeability

Hypertension is a leading risk factor for the development and progression of diabetic retinopathy and contributes to a variety of other retinal diseases in the absence of diabetes mellitus. Inhibition of the renin-angiotensin system has been shown to provide beneficial effects against diabetic retinopathy, both in the absence and presence of hypertension, suggesting that angiotensin II (Ang II) and the Ang II type 1 receptor may contribute to retinal vascular dysfunction. We investigated the effects of the Ang II type 1 receptor antagonist candesartan on retinal vascular permeability (RVP) in normotensive rats with streptozotocin-induced diabetes mellitus and in rats with Ang II–induced hypertension. We showed that candesartan treatment decreased diabetes mellitus– and Ang II–stimulated RVP by 58% (P<0.05) and 79% (P<0.05), respectively, compared with untreated controls, suggesting that activation of the Ang II type 1 receptor contributes to blood-retinal barrier dysfunction. We found that plasma kallikrein levels are increased in the retina of rats with Ang II–stimulated hypertension and that intravitreal injection of either plasma kallikrein or bradykinin is sufficient to increase RVP. We showed that a novel small molecule inhibitor of plasma kallikrein, 1-benzyl-1H-pyrazole-4-carboxylic acid 4-carbamimidoyl-benzylamide, delivered systemically via a subcutaneous pump, decreased Ang II–stimulated RVP by 70% (P<0.05) and ameliorates Ang II–induced hypertension, measured from the carotid artery by telemetry, but did not reduce Ang II–induced retinal leukostasis. These findings demonstrate that activation of the Ang II type 1 receptor increases RVP and suggest that systemic plasma kallikrein inhibition may provide a new therapeutic approach for ameliorating blood-retinal barrier dysfunction induced by hypertension.