Familial Parkinson's point mutation abolishes multiple system atrophy prion replication.
ABSTRACT: In the neurodegenerative disease multiple system atrophy (MSA), ?-synuclein misfolds into a self-templating conformation to become a prion. To compare the biological activity of ?-synuclein prions in MSA and Parkinson's disease (PD), we developed nine ?-synuclein-YFP cell lines expressing point mutations responsible for inherited PD. MSA prions robustly infected wild-type, A30P, and A53T ?-synuclein-YFP cells, but they were unable to replicate in cells expressing the E46K mutation. Coexpression of the A53T and E46K mutations was unable to rescue MSA prion infection in vitro, establishing that MSA ?-synuclein prions are conformationally distinct from the misfolded ?-synuclein in PD patients. This observation may have profound implications for developing treatments for neurodegenerative diseases.
Project description:Prions are proteins that adopt alternative conformations that become self-propagating; the PrP(Sc) prion causes the rare human disorder Creutzfeldt-Jakob disease (CJD). We report here that multiple system atrophy (MSA) is caused by a different human prion composed of the α-synuclein protein. MSA is a slowly evolving disorder characterized by progressive loss of autonomic nervous system function and often signs of parkinsonism; the neuropathological hallmark of MSA is glial cytoplasmic inclusions consisting of filaments of α-synuclein. To determine whether human α-synuclein forms prions, we examined 14 human brain homogenates for transmission to cultured human embryonic kidney (HEK) cells expressing full-length, mutant human α-synuclein fused to yellow fluorescent protein (α-syn140*A53T-YFP) and TgM83(+/-) mice expressing α-synuclein (A53T). The TgM83(+/-) mice that were hemizygous for the mutant transgene did not develop spontaneous illness; in contrast, the TgM83(+/+) mice that were homozygous developed neurological dysfunction. Brain extracts from 14 MSA cases all transmitted neurodegeneration to TgM83(+/-) mice after incubation periods of ∼120 d, which was accompanied by deposition of α-synuclein within neuronal cell bodies and axons. All of the MSA extracts also induced aggregation of α-syn*A53T-YFP in cultured cells, whereas none of six Parkinson's disease (PD) extracts or a control sample did so. Our findings argue that MSA is caused by a unique strain of α-synuclein prions, which is different from the putative prions causing PD and from those causing spontaneous neurodegeneration in TgM83(+/+) mice. Remarkably, α-synuclein is the first new human prion to be identified, to our knowledge, since the discovery a half century ago that CJD was transmissible.
Project description:Previously, we reported that intracranial inoculation of brain homogenate from multiple system atrophy (MSA) patient samples produces neurological disease in the transgenic (Tg) mouse model TgM83+/-, which uses the prion protein promoter to express human ?-synuclein harboring the A53T mutation found in familial Parkinson's disease (PD). In our studies, we inoculated MSA and control patient samples into Tg mice constructed using a P1 artificial chromosome to express wild-type (WT), A30P, and A53T human ?-synuclein on a mouse ?-synuclein knockout background [Tg(SNCA+/+)Nbm, Tg(SNCA*A30P+/+)Nbm, and Tg(SNCA*A53T+/+)Nbm]. In contrast to studies using TgM83+/- mice, motor deficits were not observed by 330-400 days in any of the Tg(SNCA)Nbm mice after inoculation with MSA brain homogenates. However, using a cell-based bioassay to measure ?-synuclein prions, we found brain homogenates from Tg(SNCA*A53T+/+)Nbm mice inoculated with MSA patient samples contained ?-synuclein prions, whereas control mice did not. Moreover, these ?-synuclein aggregates retained the biological and biochemical characteristics of the ?-synuclein prions in MSA patient samples. Intriguingly, Tg(SNCA*A53T+/+)Nbm mice developed ?-synuclein pathology in neurons and astrocytes throughout the limbic system. This finding is in contrast to MSA-inoculated TgM83+/- mice, which develop exclusively neuronal ?-synuclein aggregates in the hindbrain that cause motor deficits with advanced disease. In a crossover experiment, we inoculated TgM83+/- mice with brain homogenate from two MSA patient samples or one control sample first inoculated, or passaged, in Tg(SNCA*A53T+/+)Nbm animals. Additionally, we performed the reverse experiment by inoculating Tg(SNCA*A53T+/+)Nbm mice with brain homogenate from the same two MSA samples and one control sample first passaged in TgM83+/- animals. The TgM83+/- mice inoculated with mouse-passaged MSA developed motor dysfunction and ?-synuclein prions, whereas the mouse-passaged control sample had no effect. Similarly, the mouse-passaged MSA samples induced ?-synuclein prion formation in Tg(SNCA*A53T+/+)Nbm mice, but the mouse-passaged control sample did not. The confirmed transmission of ?-synuclein prions to a second synucleinopathy model and the ability to propagate prions between two distinct mouse lines while retaining strain-specific properties provides compelling evidence that MSA is a prion disease.
Project description:Multiple system atrophy (MSA), a progressive neurodegenerative disease characterized by autonomic dysfunction and motor impairment, is caused by the self-templated misfolding of the protein ?-synuclein. With no treatment currently available, we sought to characterize the spread of ?-synuclein in a transgenic mouse model of MSA prion propagation to support drug discovery programs for synucleinopathies. Brain homogenates from MSA patient samples or mouse-passaged MSA were inoculated either by standard freehand injection or stereotactically into TgM83+/- mice, which express human ?-synuclein with the A53T mutation. Following disease onset, brains from the mice were tested for biologically active ?-synuclein prions using a cell-based assay and examined for ?-synuclein neuropathology. Inoculation studies using homogenates prepared from brain regions lacking detectable ?-synuclein neuropathology transmitted neurological disease to mice. Terminal animals contained similar concentrations of ?-synuclein prions; however, a time-course study where mice were terminated every five days through disease progression revealed that the kinetics of ?-synuclein prion replication in the mice were variable. Stereotactic inoculation into the thalamus reduced variability in disease onset in the mice, although incubation times were consistent with standard inoculations. Using human samples with and without neuropathological lesions, we observed that ?-synuclein prion formation precedes neuropathology in the brain, suggesting that disease in patients is not limited to brain regions containing neuropathological lesions.
Project description:Prions are proteins that adopt alternative conformations, which become self-propagating. Increasing evidence argues that prions feature in the synucleinopathies that include Parkinson's disease, Lewy body dementia, and multiple system atrophy (MSA). Although TgM83(+/+) mice homozygous for a mutant A53T ?-synuclein transgene begin developing CNS dysfunction spontaneously at ?10 mo of age, uninoculated TgM83(+/-) mice (hemizygous for the transgene) remain healthy. To determine whether MSA brains contain ?-synuclein prions, we inoculated the TgM83(+/-) mice with brain homogenates from two pathologically confirmed MSA cases. Inoculated TgM83(+/-) mice developed progressive signs of neurologic disease with an incubation period of ?100 d, whereas the same mice inoculated with brain homogenates from spontaneously ill TgM83(+/+) mice developed neurologic dysfunction in ?210 d. Brains of MSA-inoculated mice exhibited prominent astrocytic gliosis and microglial activation as well as widespread deposits of phosphorylated ?-synuclein that were proteinase K sensitive, detergent insoluble, and formic acid extractable. Our results provide compelling evidence that ?-synuclein aggregates formed in the brains of MSA patients are transmissible and, as such, are prions. The MSA prion represents a unique human pathogen that is lethal upon transmission to Tg mice and as such, is reminiscent of the prion causing kuru, which was transmitted to chimpanzees nearly 5 decades ago.
Project description:Glial cytoplasmic inclusions (GCIs) containing aggregated and hyperphosphorylated α-synuclein are the signature neuropathological hallmark of multiple system atrophy (MSA). Native α-synuclein can adopt a prion conformation that self-propagates and spreads throughout the brain ultimately resulting in neurodegeneration. A growing body of evidence argues that, in addition to oligodendrocytes, astrocytes contain α-synuclein inclusions in MSA and other α-synucleinopathies at advanced stages of disease. To study the role of astrocytes in MSA, we added MSA brain homogenate to primary cultures of astrocytes from transgenic (Tg) mouse lines expressing human α-synuclein. Astrocytes from four Tg lines, expressing either wild-type or mutant (A53T or A30P) human α-synuclein, propagated and accumulated α-synuclein prions. Furthermore, we found that MSA-infected astrocytes formed two morphologically distinct α-synuclein inclusions: filamentous and granular. Both types of cytoplasmic inclusions shared several features characteristic of α-synuclein inclusions in synucleinopathies: hyperphosphorylation preceded by aggregation, ubiquitination, thioflavin S-positivity, and co-localization with p62. Our findings demonstrate that human α-synuclein forms distinct inclusion morphologies and propagates within cultured Tg astrocytes exposed to MSA prions, indicating that α-synuclein expression determines the tropism of inclusion formation in certain cells. Thus, our work may prove useful in elucidating the role of astrocytes in the pathogenic mechanisms that feature in neurodegeneration caused by MSA prions.
Project description:In multiple system atrophy (MSA), progressive neurodegeneration results from the protein ?-synuclein misfolding into a self-templating prion conformation that spreads throughout the brain. MSA prions are transmissible to transgenic (Tg) mice expressing mutated human ?-synuclein (TgM83+/-), inducing neurological disease following intracranial inoculation with brain homogenate from deceased patient samples. Noting the similarities between ?-synuclein prions and PrP scrapie (PrPSc) prions responsible for Creutzfeldt-Jakob disease (CJD), we investigated MSA transmission under conditions known to result in PrPSc transmission. When peripherally exposed to MSA via the peritoneal cavity, hind leg muscle, and tongue, TgM83+/- mice developed neurological signs accompanied by ?-synuclein prions in the brain. Iatrogenic CJD, resulting from PrPSc prion adherence to surgical steel instruments, has been investigated by incubating steel sutures in contaminated brain homogenate before implantation into mouse brain. Mice studied using this model for MSA developed disease, whereas wire incubated in control homogenate had no effect on the animals. Notably, formalin fixation did not inactivate ?-synuclein prions. Formalin-fixed MSA patient samples also transmitted disease to TgM83+/- mice, even after incubating in fixative for 244 months. Finally, at least 10% sarkosyl was found to be the concentration necessary to partially inactivate MSA prions. These results demonstrate the robustness of ?-synuclein prions to denaturation. Moreover, they establish the parallel characteristics between PrPSc and ?-synuclein prions, arguing that clinicians should exercise caution when working with materials that might contain ?-synuclein prions to prevent disease.
Project description:Assembled ?-synuclein in nerve cells and glial cells is the defining pathological feature of neurodegenerative diseases called synucleinopathies. Seeds of ?-synuclein can induce the assembly of monomeric protein. Here, we used sucrose gradient centrifugation and transiently transfected HEK 293T cells to identify the species of ?-synuclein from the brains of homozygous, symptomatic mice transgenic for human mutant A53T ?-synuclein (line M83) that seed aggregation. The most potent fractions contained Sarkosyl-insoluble assemblies enriched in filaments. We also analyzed six cases of idiopathic Parkinson's disease (PD), one case of familial PD, and six cases of multiple system atrophy (MSA) for their ability to induce ?-synuclein aggregation. The MSA samples were more potent than those of idiopathic PD in seeding aggregation. We found that following sucrose gradient centrifugation, the most seed-competent fractions from PD and MSA brains are those that contain Sarkosyl-insoluble ?-synuclein. The fractions differed between PD and MSA, consistent with the presence of distinct conformers of assembled ?-synuclein in these different samples. We conclude that ?-synuclein filaments are the main driving force for amplification and propagation of pathology in synucleinopathies.
Project description:Parkinson's disease (PD) is the second most common neurodegenerative disease. A key pathological feature of PD is Lewy bodies, of which the major protein component is ?-synuclein (?-syn). Human genetic studies have shown that mutations (A53T, A30P, E46K) and multiplication of the ?-syn gene are linked to familial PD. Mice overexpressing the human A53T mutant ?-syn gene develop severe movement disorders. However, the molecular mechanisms of ?-syn toxicity are not well understood. Recently, mitochondrial dysfunction has been linked with multiple neurodegenerative diseases including Parkinson's disease. Here we investigated whether mitochondrial motility, dynamics and respiratory function are affected in primary neurons from a mouse model expressing the human A53T mutation. We found that mitochondrial motility was selectively inhibited in A53T neurons while transport of other organelles was not affected. In addition, A53T expressing neurons showed impairment in mitochondrial membrane potential and mitochondrial respiratory function. Furthermore, we found that rapamycin, an autophagy inducer, rescued the decreased mitochondrial mobility. Taken together, these data demonstrate that A53T ?-syn impairs mitochondrial function and dynamics and the deficit of mitochondrial transport is reversible, providing further understanding of the disease pathogenesis and a potential therapeutic strategy for PD.
Project description:Parkinson disease (PD) is a neurodegenerative disease with motor as well as non-motor signs in the gastrointestinal tract that include dysphagia, gastroparesis, prolonged gastrointestinal transit time, constipation and difficulty with defecation. The gastrointestinal dysfunction commonly precedes the motor symptoms by decades. Most PD is sporadic and of unknown etiology, but a fraction is familial. Among familial forms of PD, a small fraction is caused by missense (A53T, A30P and E46K) and copy number mutations in SNCA which encodes alpha-synuclein, a primary protein constituent of Lewy bodies, the pathognomonic protein aggregates found in neurons in PD. We set out to develop transgenic mice expressing mutant alpha-synuclein (either A53T or A30P) from insertions of an entire human SNCA gene as models for the familial disease. Both the A53T and A30P lines show robust abnormalities in enteric nervous system (ENS) function and synuclein-immunoreactive aggregates in ENS ganglia by 3 months of age. The A53T line also has abnormal motor behavior but neither demonstrates cardiac autonomic abnormalities, olfactory dysfunction, dopaminergic neurotransmitter deficits, Lewy body inclusions or neurodegeneration. These animals recapitulate the early gastrointestinal abnormalities seen in human PD. The animals also serve as an in vivo system in which to investigate therapies for reversing the neurological dysfunction that target alpha-synuclein toxicity at its earliest stages.
Project description:Synucleinopathies [Parkinson's disease (PD), dementia with Lewy bodies (DLB) and multiple system atrophy (MSA)] share filamentous ?-synuclein assemblies in nerve cells and glial cells. We compared the abilities of brain extracts from MSA and PD patients to induce neuronal ?-synuclein assembly and neurodegeneration following intracerebral injection in heterozygous mice transgenic for human mutant A53T ?-synuclein. MSA extracts were more potent than PD extracts in inducing ?-synuclein assembly and in causing neurodegeneration. MSA assemblies were Campbell-Switzer- and Gallyas-silver-positive, whereas PD assemblies were only Campbell-Switzer-positive, in confirmation of previous findings. However, induced ?-synuclein inclusions were invariably Campbell-Switzer-positive and Gallyas-negative, irrespective of whether MSA or PD brain extracts were injected. The ?-synuclein inclusions of non-injected homozygous mice transgenic for A53T ?-synuclein were also Campbell-Switzer-positive and Gallyas-negative. These findings demonstrate that transgene expression and its intracellular environment dominated over the silver staining properties of the conformers of assembled ?-synuclein.