RESUMEN

Synucleinopathies, including Parkinson's disease (PD), multiple system atrophy (MSA), and dementia with Lewy bodies (DLB), are neurodegenerative disorders caused by the accumulation of misfolded alpha-synuclein protein. Developing effective vaccines against synucleinopathies is challenging due to the difficulty of stimulating an immune-specific response against alpha-synuclein without causing harmful autoimmune reactions, selectively targeting only pathological forms of alpha-synuclein. Previous attempts using linear peptides and epitopes without control of the antigen structure failed in clinical trials. The immune system was unable to distinguish between native alpha-synuclein and its amyloid form. The prion domain of the fungal HET-s protein was selected as a scaffold to introduce select epitopes from the surface of alpha-synuclein fibrils. Four vaccine candidates were generated by introducing specific amino acid substitutions onto the surface of the scaffold protein. The approach successfully mimicked the stacking of the parallel in-register beta-sheet structure seen in alpha-synuclein fibrils. All vaccine candidates induced substantial levels of IgG antibodies that recognized pathological alpha-synuclein fibrils derived from a synucleinopathy mouse model. Furthermore, the antisera recognized pathological alpha-synuclein aggregates in brain lysates from patients who died from DLB, MSA, or PD, but did not recognize linear alpha-synuclein peptides. Our approach, based on the rational design of vaccines using the structure of alpha-synuclein amyloid fibrils and strict control over the exposed antigen structure used for immunization, as well as the ability to mimic aggregated alpha-synuclein, provides a promising avenue toward developing effective vaccines against alpha-synuclein fibrils.

RESUMEN

The pathological misfolding and aggregation of soluble α-synuclein into toxic oligomers and insoluble amyloid fibrils causes Parkinson's disease, a progressive age-related neurodegenerative disease for which there is no cure. HET-s is a soluble fungal protein that can form assembled amyloid fibrils in its prion state. We engineered HET-s(218-298) to form four different fibrillar vaccine candidates, each displaying a specific conformational epitope present on the surface of α-synuclein fibrils. Vaccination with these four vaccine candidates prolonged the survival of immunized TgM83+/- mice challenged with α-synuclein fibrils by 8% when injected into the brain to model brain-first Parkinson's disease or by 21% and 22% when injected into the peritoneum or gut wall, respectively, to model body-first Parkinson's disease. Antibodies from fully immunized mice recognized α-synuclein fibrils and brain homogenates from patients with Parkinson's disease, dementia with Lewy bodies and multiple system atrophy. Conformation-specific vaccines that mimic epitopes present only on the surface of pathological fibrils but not on soluble monomers, hold great promise for protection against Parkinson's disease, related synucleinopathies and other amyloidogenic protein misfolding disorders.

Asunto(s)

Ratones Transgénicos , Enfermedad de Parkinson , alfa-Sinucleína , Animales , Enfermedad de Parkinson/inmunología , Enfermedad de Parkinson/patología , Ratones , alfa-Sinucleína/inmunología , alfa-Sinucleína/metabolismo , Humanos , Amiloide/inmunología , Amiloide/metabolismo , Vacunación , Proteínas Fúngicas/inmunología , Encéfalo/patología , Encéfalo/metabolismo , Encéfalo/inmunología , Femenino , Ratones Endogámicos C57BL

RESUMEN

The etiology of Parkinson's disease is poorly understood and is most commonly associated with advancing age, genetic predisposition, or environmental toxins. Epidemiological findings suggest that patients have a higher risk of developing Parkinson's disease after ischemic stroke, but this potential causality lacks mechanistic evidence. We investigated the long-term effects of ischemic stroke on pathogenesis in hemizygous TgM83 mice, which express human α-synuclein with the familial A53T mutation without developing any neuropathology or signs of neurologic disease for more than 600 days. We induced transient focal ischemia by middle cerebral artery occlusion in 2-month-old TgM83+/- mice and monitored their behavior and health status for up to 360 days post surgery. Groups of mice were sacrificed at 14, 30, 90, 180, and 360 days after surgery for neuropathological analysis of their brains. Motor deficits first appeared 6 months after focal ischemia and worsened until 12 months afterward. Immunohistochemical analysis revealed ischemia-induced neuronal loss in the infarct region and astrogliosis and microgliosis indicative of an inflammatory response, which was most pronounced at 14 days post surgery. Infarct volume and inflammation gradually decreased in size and severity until 180 days post surgery. Surprisingly, neuronal loss and inflammation were increased again by 360 days post surgery. These changes were accompanied by a continuous increase in α-synuclein aggregation, its neuronal deposition, and a late loss of dopaminergic neurons in the substantia nigra, which we detected at 360 days post surgery. Control animals that underwent sham surgery without middle cerebral artery occlusion showed no signs of disease or neuropathology. Our results establish a mechanistic link between ischemic stroke and Parkinson's disease and provide an animal model for studying possible interventions.

Asunto(s)

Accidente Cerebrovascular Isquémico , Enfermedad de Parkinson , Animales , Modelos Animales de Enfermedad , Neuronas Dopaminérgicas/patología , Humanos , Infarto de la Arteria Cerebral Media/complicaciones , Inflamación/complicaciones , Ratones , Ratones Transgénicos , Enfermedad de Parkinson/complicaciones , Enfermedad de Parkinson/genética , Enfermedad de Parkinson/patología , alfa-Sinucleína/genética

RESUMEN

Expansion of CAG repeats, which code for the disease-causing polyglutamine protein, is a common feature in polyglutamine diseases. RNA-mediated mechanisms that contribute to neuropathology in polyglutamine diseases are important. RNA-toxicity describes a phenomenon by which the mutant CAG repeat RNA recruits RNA-binding proteins, thereby leading to aberrant function. For example the MID1 protein binds to mutant huntingtin (HTT) RNA, which is linked to Huntington's disease (HD), at its CAG repeat region and induces protein synthesis of mutant protein. But is this mechanism specific to HD or is it a common mechanism in CAG repeat expansion disorders? To answer this question, we have analyzed the interaction between MID1 and three other CAG repeat mRNAs, Ataxin2 (ATXN2), Ataxin3 (ATXN3), and Ataxin7 (ATXN7), that all differ in the sequence flanking the CAG repeat. We show that ATXN2, ATXN3, and ATXN7 bind to MID1 in a CAG repeat length-dependent manner. Furthermore, we show that functionally, in line with what we have previously observed for HTT, the binding of MID1 to ATXN2, ATXN3, and ATXN7 mRNA induces protein synthesis in a repeat length-dependent manner. Our data suggest that regulation of protein translation by the MID1 complex is a common mechanism for CAG repeat containing mRNAs.