SNCA A30P/A53T Human
Description
Introduction to SNCA A30P/A53T Human
SNCA A30P/A53T Human refers to the human alpha-synuclein protein carrying two specific point mutations: A30P (alanine to proline at position 30) and A53T (alanine to threonine at position 53). The SNCA gene encodes alpha-synuclein, a 14-kDa protein primarily found in neural tissue that plays a central role in several neurodegenerative disorders collectively known as synucleinopathies . While these mutations can occur independently, research models expressing both mutations simultaneously have been developed to better understand their combined effects on protein aggregation, neuronal function, and disease progression.
Alpha-synuclein aggregation constitutes a hallmark of Parkinson's disease (PD) and related neurological disorders. The A30P and A53T mutations are particularly significant as they are associated with familial forms of PD, providing valuable insights into the molecular mechanisms underlying both genetic and sporadic cases of the disease .
The A53T Mutation
The A53T mutation is a missense point mutation where the 53rd amino acid in the alpha-synuclein protein changes from alanine to threonine. At the genetic level, this mutation results from guanine being changed to adenine at position 209 of the SNCA gene (G209A) . The A53T mutation was first documented in families of Italian and Greek descent and has also been identified in a Korean family . Patients carrying this mutation typically experience earlier disease onset and accelerated progression compared to sporadic PD cases .
The A30P Mutation
The A30P mutation similarly represents a missense mutation where the 30th amino acid in the protein sequence changes from alanine to proline. This mutation has been linked to familial early-onset PD, though patients carrying this mutation generally exhibit age of onset and symptoms similar to those of sporadic PD cases .
Aggregation Properties
Table 1: Comparative Characteristics of Alpha-Synuclein Variants
| Characteristic | Wild-Type | A30P | A53T | A30P/A53T |
|---|---|---|---|---|
| Disease Onset | N/A (sporadic cases) | Similar to sporadic PD | Earlier than sporadic PD | Earlier onset |
| Disease Progression | Variable | Similar to sporadic PD | Accelerated | Potentially accelerated |
| Fibril Formation Rate | Baseline | Variable reports | Faster than wild-type | Enhanced |
| Protofibril Formation | Baseline | Enhanced | Highly enhanced | Significantly enhanced |
| Clinical Phenotype | Classic PD | Similar to sporadic PD | More aggressive | Variable reports |
Pathophysiology and Disease Mechanisms
The pathological significance of these mutations stems from their influence on alpha-synuclein's propensity to aggregate and form toxic species. Both mutations promote the formation of protofibrils, which may represent the neurotoxic species in PD pathogenesis . Recent research has shown that A53T has a particularly pronounced effect on fibril amplification, while the A30P variant shows a modest enhancement of this process compared to wild-type protein .
Interestingly, the secondary structure of aggregates formed by these mutant proteins differs significantly. When aggregated in the presence of phospholipids with saturated fatty acids, A30P fibrils possess a significantly higher amount of parallel β-sheet structures compared to both wild-type and A53T variants . These structural differences may contribute to the distinct pathological consequences of each mutation.
The combined A30P/A53T mutations potentially create unique protein conformations and aggregation dynamics that differ from either single mutation alone. In transgenic mouse models, the double mutation has been associated with increased levels of alpha-synuclein oligomers and decreased extracellular dopaminergic markers .
Transgenic Mouse Models
Several transgenic mouse models have been developed to study the effects of alpha-synuclein mutations. The C57BL/6J-Tg(TH-SNCAA30PA53T)39Eric/J strain, also known as the HM2 model, expresses both A30P and A53T mutant human alpha-synuclein under the control of the rat tyrosine hydroxylase (TH) promoter, directing expression specifically to dopaminergic neurons .
Studies on homozygous transgenic mice carrying both A30P and A53T mutations revealed decreased locomotor activity starting from 3 months of age, contrasting with previous reports that showed behavioral deficits only after 7-9 months . These mice also exhibited decreased amphetamine response in locomotor activity and reduced extracellular dopaminergic markers in the striatum and substantia nigra, alongside significantly elevated levels of alpha-synuclein oligomers .
Behavioral and Neurochemical Phenotypes
In terms of neurochemical changes, these mice show an age-dependent decrease in striatal dopamine and its primary metabolites, homovanillic acid (HVA) and 3,4-dihydroxyphenylacetic acid (DOPAC) . They also exhibit reduced tyrosine hydroxylase immunoreactivity in both the striatum and substantia nigra pars compacta, indicating compromised dopaminergic function .
Table 2: Behavioral and Neurochemical Findings in Various Alpha-Synuclein Mouse Models
Comparative Studies of Single and Double Mutants
Direct comparisons between single mutant and double mutant models provide valuable insights into the unique effects of each mutation. Studies comparing A53T, A30P, and A30P/A53T variants have revealed several important differences.
Transgenic mice expressing only the A53T mutation develop adult-onset neurodegenerative disease with progressive motor dysfunction, while mice expressing the A30P mutation or wild-type human alpha-synuclein do not display the same degree of central nervous system dysfunction . This suggests that the A53T mutation confers significantly greater in vivo neurotoxicity compared to other alpha-synuclein variants.
In studies directly comparing all variants, A53T mutation caused consistently reduced motor activity in open field tests and abnormal endurance and coordination on rotarod tests starting from 6 months of age, with deficits becoming more pronounced at 12 and 18 months . In contrast, A30P transgenic mice showed no motor abnormalities at similar time points .
Clinical Implications and Future Directions
Understanding the specific effects of A30P, A53T, and combined A30P/A53T mutations has important implications for developing targeted therapies for PD. The unique structural properties and aggregation dynamics of these variants suggest that patients with different mutations might benefit from distinct therapeutic approaches.
Research on double mutant models continues to evolve, with recent proteomics studies identifying differentially expressed proteins in the substantia nigra pars compacta of A30P/A53T transgenic mice . These studies may help identify novel biomarkers and therapeutic targets for early intervention in PD.
Product Specs
MDVFMKGLSK AKEGVVAAAE KTKQGVAEAP GKTKEGVLYV GSKTKEGVVH GVTTVAEKTK EQVTNVGGAV VTGVTAVAQK TVEGAGSIAA ATGFVKKDQL GKNEEGAPQE GILEDMPVDP DNEAYEMPSE EGYQDYEPEA.
Q&A
What are the A30P and A53T mutations in the SNCA gene?
Both A30P and A53T are missense point mutations in the alpha-synuclein gene (SNCA). The A30P mutation changes the 30th amino acid from alanine to proline, while A53T changes the 53rd amino acid from alanine to threonine. The A53T mutation specifically results from guanine being changed to adenine at position 209 of the SNCA gene (G209A) . These mutations are associated with rare Mendelian forms of familial Parkinson's disease (PD) .
How do these mutations affect alpha-synuclein's structure and function?
Both mutations alter the protein's propensity to aggregate. The A53T mutation particularly accelerates fibrillization in solution compared to wild-type alpha-synuclein . Research indicates that these mutations affect protofibril formation more significantly than fibril elongation . These structural changes impact neuronal homeostasis and may contribute to dopaminergic neuron dysfunction seen in PD models .
What experimental models are available for studying these mutations?
Researchers can utilize several experimental systems:
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Transgenic mouse models expressing human A30P, A53T, or double mutant A30P*A53T alpha-synuclein
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Patient-derived isogenic neuronal cell lines with gene-corrected controls
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In vitro protein aggregation systems with purified recombinant proteins
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Viral vector-mediated expression systems in various neuronal populations
How can researchers generate and validate isogenic control lines for studying SNCA mutations?
Gene editing techniques, particularly CRISPR-Cas9, allow creation of isogenic controls by correcting the mutation in patient-derived cells. The validation process involves:
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Confirming genetic correction through sequencing
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Verifying expression levels of wild-type and mutant alpha-synuclein
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Confirming clonality of derived lines
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Ruling out off-target effects through whole-genome sequencing
Studies like those in search result utilized two clonal gene-corrected isogenic cell lines derived from a p.A30P SNCA patient to identify image-based phenotypes showing impaired neuritic processes .
What neuroimaging and neuroanatomical techniques are most sensitive for detecting alpha-synuclein pathology?
For detecting alpha-synuclein pathology in experimental models, researchers should employ:
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Immunofluorescence with antibodies specific to total, phosphorylated, or oligomeric alpha-synuclein
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Thioflavin S or Congo red staining for detecting amyloid fibrils
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Proximity ligation assays to detect protein-protein interactions
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Electron microscopy for ultrastructural analysis of fibrils and aggregates
In the SNCA-A30P model, researchers successfully used immunofluorescence to detect alpha-synuclein expression patterns, noting that the human transgene expression closely mirrored endogenous gene expression patterns .
How can researchers quantify alpha-synuclein aggregation and differentiate between oligomeric and fibrillar species?
Recommended methodological approaches include:
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Western blotting with non-denaturing conditions to preserve oligomeric species
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Size-exclusion chromatography combined with multi-angle light scattering
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Thioflavin T fluorescence assays for kinetic monitoring of fibril formation
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FRET-based biosensors for detecting conformational changes
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Atomic force microscopy for direct visualization of different species
Studies show the A53T mutation has greater effects on protofibril formation rates than fibril elongation, suggesting oligomeric species may be the primary neurotoxic forms .
How do the A30P and A53T mutations affect dopaminergic neuron function differently?
The differential effects include:
Research with gene-corrected isogenic neurons showed A30P mutation causes impaired neuronal activity, reduced mitochondrial respiration, energy deficits, and vulnerability to rotenone toxicity .
What transcriptional changes are associated with SNCA mutations?
Research with p.A30P mutant neurons revealed significant transcriptional alterations in lipid metabolism pathways . Other documented changes include:
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Altered expression of genes involved in synaptic function
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Dysregulation of mitochondrial and oxidative stress response genes
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Changes in vesicular trafficking and autophagy pathways
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Alterations in neuroinflammatory markers
These transcriptional changes provide insights into disease mechanisms and potential therapeutic targets.
How do SNCA mutations affect adult neurogenesis?
Research on A30P mice has demonstrated:
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Decreased subventricular zone (SVZ) proliferation
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Fewer Mash1+ transit-amplifying SVZ progenitor cells
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Altered olfactory bulb neurogenesis
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Decreased numbers of calbindin+ and calretinin+ periglomerular neurons
The A30P mutation appears to aggravate the effects of Snca loss in the SVZ, with decreased SVZ neurogenesis mirroring findings in Parkinson's disease patients .
What methodologies best capture neurogenesis deficits in SNCA mutant models?
Effective research approaches include:
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BrdU incorporation assays to label dividing cells (identifying BrdU+GFAP+ cells)
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Phospho-histone 3 (PHi3) immunohistochemistry to mark acutely proliferating cells
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Mash1 expression analysis for transit-amplifying progenitors
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TUNEL assays to quantify cell death in neurogenic regions
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Lineage tracing using reporter constructs
Studies of SNCA-A30P mice revealed fewer BrdU+GFAP+ cells in the SVZ compared to controls, indicating the mutation exacerbates effects caused by Snca loss .
What is the timeline of behavioral deficits in SNCA A30P/A53T models?
In double mutated A30P*A53T alpha-synuclein transgenic mice:
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Decreased locomotor activity begins as early as 3 months of age
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This contrasts with previous studies of the same strain that reported deficits starting only after 7-9 months
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Decreased amphetamine response in locomotor activity is observable
The earlier onset of behavioral deficits in homozygous transgenic mice makes this model particularly valuable for studying early PD pathophysiology .
What behavioral tests are most sensitive for detecting early deficits in SNCA mutant models?
Recommended behavioral assessment protocols include:
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Open field test for general locomotor activity
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Rotarod for motor coordination
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Challenging beam traversal for fine motor control
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Amphetamine challenge to assess dopaminergic system integrity
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Novel object recognition for cognitive assessment
The combination of these tests can detect subtle changes before overt neurodegeneration occurs, as demonstrated in A30P*A53T mice showing decreased locomotor activity and amphetamine response .
How can patient-derived neurons with SNCA mutations facilitate drug discovery?
Patient-derived neurons with SNCA mutations provide powerful tools for drug discovery by:
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Offering image-based phenotypic screening platforms
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Enabling high-throughput assessment of compounds that rescue neuritic process deficits
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Providing a system to test compounds that improve mitochondrial function or reduce alpha-synuclein aggregation
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Allowing transcriptional profiling to identify novel therapeutic targets
Research with p.A30P SNCA patient-derived neurons has identified image-based pathological phenotypes that can serve as entry points for future disease-modifying compound screenings and drug discovery strategies .
What are the key differences between animal models and patient-derived cellular models of SNCA mutations?
Understanding these differences is crucial for experimental design:
Both model systems have complementary strengths and limitations that should inform experimental design and interpretation.
How can researchers address variability in alpha-synuclein expression levels between experimental models?
Recommended methodological solutions include:
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Using BAC transgenic models that more accurately reflect endogenous expression patterns
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Employing knock-in approaches rather than random transgene insertion
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Quantifying alpha-synuclein at both mRNA and protein levels across experimental groups
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Creating isogenic controls to isolate mutation effects from expression level effects
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Developing inducible expression systems to control timing and level of expression
Research with SNCA-A30P mice confirmed that the human transgene expression closely mirrored endogenous gene expression patterns, validating this approach .
What are the best approaches to detect and quantify early alpha-synuclein oligomerization in experimental models?
Advanced techniques for early oligomer detection include:
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Proximity ligation assays specific for oligomeric species
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Conformational antibodies that recognize oligomer-specific epitopes
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FRET-based reporters expressed in cells of interest
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Single-molecule approaches like super-resolution microscopy
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Mass spectrometry methods to characterize oligomeric species
Studies report significantly elevated levels of alpha-synuclein oligomers in A30P*A53T mouse models, correlated with decreased extracellular dopaminergic markers in the striatum and substantia nigra .
How can researchers better model the interaction between SNCA mutations and environmental factors?
Methodological approaches should include:
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Exposing SNCA mutant models to relevant environmental toxins (rotenone, paraquat, etc.)
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Studying interactions with aging by analyzing multiple time points
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Investigating gut-brain axis contributions using combined models
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Exploring mitochondrial stressors in genetically susceptible backgrounds
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Developing multi-hit models that combine genetic and environmental factors
Research has already demonstrated increased vulnerability to rotenone in A30P mutant neurons, supporting the value of this approach .
What emerging technologies might advance understanding of SNCA mutation pathophysiology?
Researchers should consider incorporating:
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Single-cell transcriptomics and proteomics to capture cell-specific responses
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Advanced in vivo imaging to monitor alpha-synuclein dynamics in real-time
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Cryo-electron microscopy to determine oligomer and fibril structures at atomic resolution
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iPSC-derived 3D organoid models to capture complex cellular interactions
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AI-driven analysis of morphological and functional phenotypes
These emerging approaches hold promise for addressing fundamental questions about how SNCA mutations contribute to neurodegeneration and identifying novel therapeutic targets.
Product Science Overview
Alpha-synuclein (α-synuclein) is a protein that plays a crucial role in the pathogenesis of several neurodegenerative diseases, collectively known as synucleinopathies. These include Parkinson’s disease (PD), dementia with Lewy bodies (DLB), and multiple system atrophy (MSA). The protein is predominantly expressed in the brain, particularly in the presynaptic terminals of neurons, where it is involved in synaptic vesicle regulation and neurotransmitter release .
Mutations in the α-synuclein gene (SNCA) are linked to familial forms of Parkinson’s disease. Among these, the A30P and A53T mutations are particularly noteworthy. These mutations result in the substitution of alanine with proline at position 30 (A30P) and alanine with threonine at position 53 (A53T) of the α-synuclein protein .
- A30P Mutation: This mutation is associated with a similar age of onset and symptoms as sporadic PD. However, it leads to a reduced speed of movement and an increased paralysis rate in model organisms .
- A53T Mutation: This mutation generally results in an earlier age of onset and accelerated progression of the disease. It significantly decreases both the speed and frequency of body movements and increases the paralysis rate .
The aggregation of α-synuclein is a hallmark of Parkinson’s disease and other synucleinopathies. The A30P and A53T mutations enhance the propensity of α-synuclein to form aggregates, which are toxic to neurons. These aggregates, known as Lewy bodies, disrupt normal cellular functions and lead to neuronal death .
Various model organisms, including transgenic mice and C. elegans, have been used to study the effects of these mutations. For instance, transgenic mice overexpressing human wild-type, A30P, or A53T α-synuclein have shown a high correlation with the toxic gain of function mechanism for α-synuclein pathogenesis . In C. elegans models, the expression of these mutational variants in muscle cells has provided insights into their behavioral and pathological impacts .
Understanding the molecular mechanisms underlying α-synuclein aggregation and toxicity is crucial for developing therapeutic strategies. Research is ongoing to identify compounds that can inhibit α-synuclein aggregation or promote its clearance from neurons. Additionally, gene therapy approaches are being explored to correct or mitigate the effects of these mutations .
