The Anti-Apoptotic Activity of β-Synuclein Mediated via Akt Signaling Is Severely Lost During Prion Infection

TL;DR

This study shows that β-synuclein protects neurons by activating Akt signaling, but this protection is lost during prion infection, offering new insights into neurodegeneration and potential treatments.

Contribution

The novel contribution is identifying β-synuclein's anti-apoptotic role via Akt signaling and its disruption during prion infection.

Findings

β-synuclein and Akt levels decrease at the terminal stage of prion disease in rodent brains.

β-synuclein suppresses apoptosis via Akt signaling, which is partially lost upon Akt knockdown.

β-synuclein overexpression reduces PrP levels and corrects its abnormal distribution in cellular models.

Abstract

Prion diseases are fatal neurodegenerative disorders characterized by profound neuronal damage. Despite evidence supporting a neuroprotective role for β-synuclein (β-syn) in neurodegeneration, its potential functions and mechanisms in prion disease have not been elucidated. To investigate the role of β-syn, we systematically analyzed its alterations in the central nervous system of several prion-infected rodent models and cell models. A series of biochemical, cellular, and immunofluorescence assays were conducted to explore the relationship between β-syn and protein kinase B (Akt) signaling and between β-syn and prion protein (PrP), and its neuroprotective role in prion disease. Student’s t-test was used for statistics. At the terminal stage of prion disease, β-syn and Akt exhibited a parallel and remarkable decrease in rodent brains, contrasting with the slight but significant increase observed at early to middle stages. Dual-stained immunofluorescence assays confirmed that β-syn is localized within NeuN-positive neurons. Further structural and functional analyses revealed a high-affinity molecular interaction between β-syn and Akt, with the N-terminal region of β-syn being essential for binding to Akt1. In a cell model of PrP aggregation, β-syn overexpression suppressed cytochrome c-induced apoptosis, which was demonstrated by decreased levels of cleaved caspase-3. Notably, this anti-apoptotic effect was partially abolished upon Akt knockdown, indicating a dependence on Akt signaling. Moreover, colocalization of β-syn and PrP was observed in rodent brains. Consistently, in cellular models of prion infection and PrP aggregation, β-syn overexpression not only reduced PrP levels but also ameliorated its aberrant histological distribution. Our findings demonstrate that the anti-apoptotic activity of β-syn, mediated via Akt signaling, is severely lost during prion infection, thereby suggesting a mechanism of intrinsic neuronal vulnerability and revealing a novel therapeutic strategy.

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1. Introduction

Prion diseases (PrDs), also known as transmissible spongiform encephalopathies, are a group of rapidly progressive and transmissible neurodegenerative disorders in humans and a species of animals caused by prions [1,2]. To date, there is still a lack of effective therapeutic or prophylactic strategies for PrDs. The core component of prions is the Scrapie prion protein (PrP^Sc^), a pathologically aggregated form of a host normal cellular protein PrP^C^. Progressive accumulation of PrP^Sc^ in the central nervous system (CNS) underlies a cascade of pathological events, including synaptic alterations, neuroinflammation, spongiform changes, neuronal damage, and death [3,4,5]. Axonal degeneration and synaptic loss are well-documented early pathological events in different types of PrDs, such as human Creutzfeldt–Jakob disease (CJD) and various experimental animal models [6,7]. Evidence from human PrDs and multiple experimental models confirms that apoptosis is closely associated with neuronal dysfunction and death [8]. Hence, alleviating apoptosis and protecting synapses may serve as targets for therapeutics.

β-synuclein (β-syn), a member of the synuclein family, is a 14 kDa presynaptic protein in neurons particularly enriched in the regions of neocortex and hippocampus [9]. It exhibits diverse biological functions, e.g., antagonizing the neurotoxicity and aggregation of α-synuclein (α-syn), maintaining the stability of synaptic structure and signal transmission, participating in regulation for neuroinflammation and resisting oxidative stress via broader cellular processes such as apoptosis and protein degradation pathways [10]. Therefore, β-syn may reflect synaptic alterations. This neuroprotective mechanism of β-syn has been proposed to be linked with the Akt signaling pathway [11]. Recently, the aberrant elevations of β-syn in cerebrospinal fluid (CSF), even in peripheral plasma, have become useful biomarkers for the early diagnosis or monitoring of sporadic CJD (sCJD) [12,13]. However, the possible changes of brain β-syn and its anti-apoptotic function during prion infection are still elusive.

In this study, we identified a remarkable decrease in β-syn levels in different brain regions of scrapie-infected mouse and hamster models and prion-infected cell lines. The β-syn signals colocalized well with neurons but not with astrocytes and microglia. In line with reduced brain β-syn levels, Akt expression was decreased in scrapie-infected rodent brains and prion-infected cell lines, with morphological colocalization of the two proteins. The molecular interactions between Akt and β-syn were computationally predicted by AlphaFold 3 and further verified by co-immunoprecipitation (Co-IP) and bio-layer interferometry (BLI). Overexpression of β-syn efficiently mitigated the apoptotic events in the cells transiently expressing Cyto-PrP, which apparently is associated with the Akt signaling pathway.

2. Results

2.1. Pronounced Reduction of β-Syn in Prion-Infected Rodent Brains and Cell Line

β-syn levels were evaluated by Western blots of brain homogenates from the animal models at terminal disease. Compared to the normal controls, brain β-syn signals were significantly weaker in both 139A- and ME7-infected mice (Figure 1A) and 263K-infected hamsters (Figure 1B), as quantified by actin-normalized relative gray values. IFA specific for β-syn revealed markedly reduced signal intensity (red) in the cortical and hippocampal regions of 139A- and ME7-infected mice (Figure 1C), as well as in 263K-infected hamsters (Figure 1D). The integrated optical density (IOD) values of β-syn in the infected animals were significantly lower than those in normal ones. Western blot and IFA analyses of the prion-infected SMB-S15 cell line showed significantly weaker β-syn-specific bands (Figure 1E) and signals (Figure 1F), respectively, compared to the normal SMB-PS cells. Moreover, the levels of SNCB transcripts in the brains of 139A- and ME7-infected mice and in SMB-S15 cells were evaluated by qRT-PCRs, revealing a marked downregulation of β-syn mRNAs in both prion-infected models and cell lines compared to their normal controls (Figure 1G,H).

2.2. Neuronal-Specific β-Syn Expression in Normal and Prion-Infected Rodent Brains

To identify the cell types expressing β-syn in brain tissues, dual-stained IFAs were performed on brain sections from normal and prion-infected mice and hamsters using anti-β-syn paired with biomarkers for neurons (anti-NeuN), astrocytes (anti-GFAP), or microglia (anti-Iba1). Confocal microscopy revealed that, in addition to a marked reduction in β-syn signal (red) in prion-infected animals, β-syn extensively colocalized with NeuN (green) in brain regions such as the cortex and hippocampus of both normal and infected mice (Figure 2A) and hamsters (Figure 2B). In contrast, almost no colocalization was observed between β-syn and GFAP (Figure 2C,D) or between β-syn and Iba1 (Figure 2E,F), indicating that β-syn expression is mainly neuronal while nearly undetectable in astrocytes and microglia.

2.3. Marked Decrease of Akt in Prion-Infected Rodent Brains and Cell Lines

The interplay between β-syn and Akt in neurons has been previously reported [11]. To assess the possible changes in Akt expression during prion infection, various brain homogenates were subjected to Akt-specific Western blots. Significantly reduced brain Akt levels were observed in the preparations of 139A- and ME7-infected mice (Figure 3A) and 263K-infected hamsters (Figure 3B). Similarly, the SMB-S15 cells showed markedly lower Akt expression compared to the SMB-PS cells (Figure 3C). To evaluate the potential morphological colocalization between β-syn and Akt, dual-stained IFAs were performed on brain sections from normal and prion-infected animals. Merged images revealed extensive overlap between β-syn and Akt signals (yellow) within large round or polygonal cells in both normal and infected mice and hamsters (Figure 3D,E), indicating histological colocalization of the two proteins.

2.4. Coincidental Increases in Early to Middle Stage and Decreases in Terminal Stage of Brain β-Syn and Akt During Prion Infection

The potential dynamic changes of brain β-syn and Akt during prion infection were assessed using brain samples from ME7 and 139A infected mice (n = 3) collected on 80, 120, 150 and 180 days post-infection (dpi), and from 263K infected hamsters (n = 3) collected on 20, 40, 60 and 80 dpi. Western blots revealed that both β-syn and Akt signals in the brain homogenates collected in early to middle stages (120 and 150 dpi for ME7 and 139A infected mice, and 40 and 60 dpi for 263K infected hamsters) were slightly stronger than those in the early stage (80 dpi of ME7 and 139A infected mice, and 20 dpi for 263K infected hamsters) and the terminal stage (180 dpi for ME7 and 139A infected mice, and 80 dpi for 263K infected hamsters) (Figure 4A–C). Quantitative assays of the relative gray values of β-syn and Akt bands, after normalization with individual actin, illustrated that these two proteins displayed coincidental alternative patterns, peaking at 150 dpi for ME7 and 139A infected mice and 60 dpi for 263K infected hamsters, and markedly dropping down in the terminal stage with statistical differences (Figure 4D–F).

2.5. Precisely Molecular Interaction Between β-Syn and Akt

To investigate the molecular interaction between β-syn and Akt in vivo, we employed Co-IP assays on brain homogenates from normal and prion-infected mice, using anti-Akt for capture and anti-β-syn for detection. Bands corresponding to β-syn (18 kDa) were detected in the immunoprecipitated complexes but not in the preparations captured by isotype IgG (Figure 5A). Conversely, reciprocal Co-IP assays with anti-β-syn for capture verified the interaction between β-syn and Akt, as a 57-kDa Akt band was detected in the precipitated products (Figure 5B). Subsequently, monolayer 293T cells, in which the endogenous β-syn and Akt were undetectable, were transiently transfected with the recombinant plasmid expressing mouse β-syn or mouse Akt1 separately, or co-transfected with these two plasmids. Western blot confirmed the expression in the respective lysates (Figure 5C, input lanes). Co-IP assays revealed that Akt1 was co-precipitable with β-syn antibody (Figure 5C, middle lanes), and β-syn was detectable in precipitates obtained with anti-Akt1 (Figure 5C, right lanes). Biomolecular interaction analysis using the Octet RED96E system revealed definite binding activity of the fixed biotin-labeled recombinant full-length Akt1 protein with recombinant full-length β-syn protein, showing an apparent dissociation constant (KD) of 1.5 × 10^−9^ M (KD = kd/ka; kd, dissociation rate constant, s^−1^; ka, association rate constant, M^−1^·s^−1^) (Figure 5D). These data provide strong evidence for the molecular interaction between β-syn and Akt1.

Furthermore, the potential binding sites between mouse β-syn and Akt1 were predicted using AlphaFold 3 and visualized with PyMOL. As illustrated in Figure 4E, seven hydrogen bonds shorter than 3.5 Å were predicted within the N-terminal segment of β-syn. Subsequently, the recombinant plasmids for the full-length (1–133) and four truncated mouse β-syn proteins (β-syn 1–61, 1–85, 61–133, and 85–133) were introduced into HEK 293T cells, together with the plasmid for full-length mouse Akt1. Co-IP assays precipitated with anti-Akt1 and blotted with anti-β-syn revealed β-syn-specific bands in the cell lysates expressing β-syn 1–133, 1–61 and 1–85, but not in those of β-syn 61–133 or 85–133 (Figure 5F), strongly indicating that the binding site for Akt1 resides within the N-terminal region of β-syn.

2.6. Anti-Apoptotic Effect of β-Syn via Akt Signaling in Aggregated PrP Cell Model

To investigate the effect of β-syn on apoptosis induced by PrP aggregation, we transiently and separately transfected 293T cells with plasmids expressing wild-type human PrP (PG5), a cytosolic mutated PrP (Cyto) lacking the N-terminal signal peptide and C-terminal GPI anchor (Supplementary Figure S1A), or co-transfected them with the plasmid expressing β-syn (SNCB) (Supplementary Figure S1B). Western blots of the transfected cells showed that the levels of cleaved-caspase 3 were similar in the preparations of control (293T), PG5 and SNCB, but remarkably increased in that of Cyto (Figure 6A). A band of cleaved-caspase 3 was also detected in the cells co-expressing Cyto and SNCB, but with a significantly lower relative gray value than that of the preparation of Cyto alone after quantitative analysis (Figure 6A). Markedly more TUNEL-positive signals (red) were observed by IFA in the cells expressing Cyto, which was substantially attenuated by the co-expression of β-syn (Figure 6B). This suggests that β-syn can antagonize, at least partially, the Cyto-induced apoptosis in 293T cells.

To test whether the anti-apoptotic effect of β-syn depends on Akt1, an shRNA targeting Akt1 (sh-Akt1) was introduced into 293T cells (Supplementary Figure S1C). As shown in Figure 6C, a caspase-specific Western blot revealed that the introduction of sh-Akt1 alone resulted in a markedly high level of cleaved caspase 3 compared to that of β-syn (SNCB). Co-transfection of sh-Akt1 plus SNCB decreased the level of cleaved caspase 3 compared to that expressing sh-Akt1 alone, implying that expressing β-syn partially mitigates the sh-Akt1-induced upregulation of cleaved caspase 3. Introduction of sh-Akt1 into the cells expressing Cyto and β-syn caused higher levels of cleaved caspase 3 in comparison to the cells expressing Cyto and β-syn alone. TUNEL staining of the above cell preparations also illustrated similar profiles (Figure 6D). More TUNEL-positive signals (red) were observed in the sh-Akt1 cells than in the SNCB cells. More TUNEL-positive signals were also identified in the cells co-transfected with sh-Akt1 and SNCB than in SNCB alone, but less than in sh-Akt1 alone. Despite no statistical significance, slightly more TUNEL-positive signals were detected in the cells expressing Cyto and β-syn after the introduction of sh-Akt1. These data highlight that the anti-apoptotic activity of β-syn, including but not limited to Cyto-PrP-induced apoptosis, is likely mediated via the Akt pathway.

2.7. Histological Colocalization of β-Syn and PrP and PrP Downregulation by β-Syn Overexpression

The potential morphological colocalization of β-syn and PrP in prion-infected brains and cells was evaluated by dual-stained IFAs with anti-β-syn and anti-PrP primary antibodies. Amounts of yellow signals were observed in the merged images of the brains of ME7- and 139A-infected mice and 263K-infected hamsters (Figure 7A), as well as in the SMB-S15 cells (Figure 7B). To assess the influence of the overexpression of β-syn on the levels of abnormal PrP, cultured 293T cells were transfected with the plasmid expressing Cyto-PrP with or without the plasmid for β-syn. Western blot showed that the PrP-specific band in the cells co-expressing Cyto-PrP and β-syn was obviously weaker than that of the cells expressing Cyto-PrP only; meanwhile, co-introduction of sh-Akt partially reversed the β-syn-induced downregulation of Cyto-PrP (Figure 7C). IFAs revealed that the PrP signals in the 293T cells expressing Cyto-PrP were significantly reduced when co-transfected with the plasmid for β-syn, while the PrP levels in the cells expressing PG5-PrP did not change remarkably when co-transfected with or without the plasmid for β-syn (Figure 7D). Large amounts of round particles were observed in the enlarged images of the Cyto-PrP cells, but much fewer were observed in the cells co-expressing β-syn (Figure 7D, lower right panels). Furthermore, the β-syn level in the SMB-S15 cells, which permanently removed prion replication by resveratrol (SMB-RES), was estimated by Western blot. Compared to SMB-15 cells, the β-syn bands in the SMB-RES cells were significantly stronger (Figure 7E), indicating a reversion of cellular β-syn levels after the removal of prion propagation. PrP-specific IFAs indicated that overexpression of exogenous β-syn in SMB-S15 cells markedly reduced the intensity of PrP signals (red) (Figure 7F). The small round PrP-specific particles observed in the SMB-S15 cells were infrequently detected in the cells overexpressing β-syn (green) (Figure 7F, lower right panels).

3. Discussion

PrDs are fatal neurodegenerative disorders characterized primarily by fast and progressive neuronal damage and loss, particularly in the late stage of disease. Synaptic alterations in both morphology and function are among the earliest detectable changes in prion pathogenesis and precede the onset of clinical symptoms [14]. As a biomarker of synaptic impairment, decreased β-syn levels in the brain tissue of Alzheimer’s disease (AD) and sCJD patients have been described previously [12,15]. On the contrary, β-syn exhibits a significant rise in the plasma and/or CSF of patients with AD and CJD [16,17]. Massive synaptic degeneration might further induce a release of β-syn from the presynaptic terminals into the extracellular space and biofluids.

Initial investigations into neuronal cell death in prion diseases have identified apoptosis as the predominant mechanism [18]. Although many other mechanisms, such as activated macroautophagy, enhanced mitophagy, and pyroptosis, have been identified in the CNS tissues of human PrDs and prion-infected animal models [19,20,21], extensive apoptosis remains a significant and non-negligible pathological event in PrDs, which is initiated and mediated via numerous signaling pathways [5]. As a protein expressed mainly in CNS tissues, especially in presynaptic terminals and dopaminergic neurons, the neuroprotective functions of β-syn are well documented; these functions operate through multiple mechanisms, e.g., as a molecular chaperone for α-syn, synaptic plasticity and neurotransmitter release, intracellular metal ion homeostasis, protein degradation, anti-apoptosis, etc. [10]. Given the apoptotic pathology in PrDs, the downregulation of brain β-syn in the prion-infected animal models and prion-infected and PrP aggregation cell models in this study provides solid evidence for its robust role in apoptosis in prion pathogenesis.

Several signaling pathways are associated with β-syn anti-apoptosis, such as PI3K/Akt signaling, p53 signaling, Bcl-2 family proteins, mitochondrial functions, and caspase cascades; among them, PI3K/Akt signaling is the most impressive [11,22]. The Akt protein family, also named protein kinase B (PKB), consists of 3 subtypes with molecular weights ranging from 55 to 60 kDa. Akt functions as a core component in the regulation of cell apoptosis by phosphorylating several downstream targeting proteins, including Bad, MDM2, GSK-3β, and Forkhead Box O (FOXO) [23,24,25,26]. In prion-infected rodents, consistent aberrant alterations of brain β-syn and Akt, along with their morphological colocalization in neurons, strongly suggest that the anti-apoptotic effect of β-syn is significantly dependent on Akt signaling. This finding is further supported by evidence from the Cyto-PrP cell model, in which sh-Akt1 antagonized the protective effect, confirming that this function requires Akt signaling during prion infection. β-syn can induce the phosphorylation of Akt at Ser473, which enhances the regulatory ability of Akt on downstream targets [11]. β-syn can also promote the translocation of Akt from cytoplasm to nucleus, enabling it to phosphorylate target proteins in the nucleus, such as p53 and Murine Double Minute 2 (MDM2) [22]. These biological activities are considered largely dependent on the specific binding between these two proteins. In addition to the demonstrated molecular interaction between β-syn and Akt in both native brain tissue and recombinant cellular systems, we report for the first time a high-affinity interaction (KD = 1.5 nM) between these proteins. This finding confirms a specific, functionally significant interaction. Furthermore, we have identified and validated the critical role of the β-syn N-terminus as a key binding site for Akt. Seven predicted strong hydrogen bonds (≤3.5 Å) in the segment from 21 to 51 aa. of β-syn may serve as the molecular basis for this protein-protein interaction. The Akt molecule is a common target for the other members of the synuclein family. In non-small cell lung cancer tissues, γ-synuclein (γ-syn) promotes cancer cell survival and proliferation by binding to the Akt C-terminus and triggering phosphorylation [27]. Wild-type α-syn also shows binding activity with Akt and enhances the membrane localization and solubility of Akt under insulin-like growth factor-1 (IGF-1) stimulation [28]. Although the interacting site within α-syn is speculated to be at its C-terminus, possibly via electrostatic forces, the exact mechanism remains unclear. Further comparative structural and functional analyses between the members of the synuclein family and Akt are warranted.

Another antagonistic role of β-syn against α-syn in Parkinson’s disease (PD), dementia with Lewy bodies (DLB), and multiple system atrophy (MSA) is its function as molecular chaperone, possibly by prevention of misfolding and aggregation of α-syn by forming heterodimers [29]. Despite not being a core component of Amyloid-β peptide (Aβ) plaques or tau tangles in AD, β-syn demonstrates a protective function, and its alteration correlates with the severity of the disease [16,30]. The interaction of β-syn with α-syn suppresses its aggregation [31]. In light of studies demonstrating the high-affinity binding of soluble α-syn aggregates to PrP23–231, it is implied that β-syn may also bind to PrP^C^, potentially disrupting the conversion of PrP^C^ to PrP^Sc^ [32,33]. Our data illustrate clear morphological overlaps between PrP and β-syn in the prion-infected brains and cell model. The cellular level of β-syn is markedly upregulated after removal of prion replication in prion-infected cells. In a prion-infected cell model, β-syn overexpression significantly reduced the levels of both aggregated Cyto-PrP and total PrP. Furthermore, it remodeled the abnormal PrP morphology from small, round (possibly inclusion body-like) particles to a relatively homogeneous intracellular pattern. These data highlight a picture that, besides its anti-apoptosis via Akt signaling, the direct interface of β-syn with the pathological proteins, at least in PD and PrDs, might be another pathway. Similar to the alterations of brain β-syn in PD and AD, the β-syn level also displays a relatively higher level in the early and middle stages and a significant decrease in the late stage in various scrapie-infected animal models. We may propose a transition from active compensation to passive exhaustion of brain β-syn during prion infection that involves gradual losses of its anti-apoptotic function mainly via Akt-singling and direct interface with prions.

In summary, our findings demonstrate that prion-induced downregulation of β-syn compromises a vital anti-apoptotic mechanism mediated through its direct N-terminal interaction with Akt. This discovery not only provides a mechanistic basis for heightened neuronal vulnerability in PrDs but also reveals promising strategies for therapeutic intervention. Several limitations should be acknowledged. Firstly, our study identifies the interaction but does not yet pinpoint the precise molecular mechanism by which β-syn binding regulates Akt activity. Secondly, the upstream signaling events that lead to the transcriptional downregulation of β-syn in PrDs remain entirely unknown. Finally, although our cellular data strongly suggest the in vivo functional significance of this interaction, definitive proof will require future studies using conditional knockout or transgenic animal models infected with prion. Future efforts should focus on the cause of β-syn transcriptional downregulation in PrDs, thereby uncovering new therapeutic targets for related neurodegenerative diseases.

4. Materials and Methods

4.1. Ethics Statement

All animal experiments were approved by the Animal Care and Welfare Committee of the National Institute for Viral Disease Control and Prevention, China CDC, and conducted in accordance with the Regulations on the Management of Laboratory Animals of China.

4.2. Animal Models and Preparation of Brain Homogenates

This study employed archived brain samples derived from rodent-adapted scrapie infection models, specifically comprising C57BL/6 mice infected with either strain 139A or ME7 and Syrian hamsters infected with strain 263K. Strains 139A and 263K are rodent-adapted sheep scrapie strains of the “drowsy” goat lineage passaged in mice and Golden Syrian hamsters, respectively, after goat passage. ME7 is a classic mouse-adapted scrapie strain derived from the first transmission of natural Suffolk sheep scrapie to mice and is the most commonly used strain for serial scrapie passage in mice. The clinical and neuropathological characteristics of these rodent models have been described elsewhere [34]. The average incubation times for 139A- and ME7-infected mice were 183.9 ± 23.1 and 184.2 ± 11.8 days, and that for 263K-infected hamsters was 80.1 ± 5.7 days, respectively. Age-matched healthy animals were used as controls. The animals were randomly allocated to experimental groups by gender and age, with three animals per group, to ensure intergroup balance and minimize baseline confounding variables.

Brain homogenates (10%, w/v) were prepared according to a previously described protocol [35,36]. Briefly, whole brain tissues were homogenized in ice-cold lysis buffer (100 mM NaCl, 10 mM EDTA, 0.5% Nonidet P-40, 0.5% sodium deoxycholate, 10 mM Tris, pH 7.5) with protease inhibitor cocktail set III (Merck, Darmstadt, Germany, Cat. No. 535140). Tissue debris was removed by low-speed centrifugation at 2000× g for 10 min at 4 °C, and the supernatants were collected for subsequent analysis.

4.3. Cell Culture and Preparation of Lysates

The SMB-S15 cell line and its control counterpart, SMB-PS, were obtained from the Roslin Institute, Edinburgh, UK [37]. Both cell lines were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM; Gibco, Waltham, MA, USA, Cat. No. 11965118) supplemented with 10% fetal bovine serum (FBS; Gibco, Waltham, MA, USA, Cat. No. A5256701) and 1% penicillin/streptomycin (Gibco, Waltham, MA, USA, Cat. No. 15140122) and maintained at 33 °C in a humidified atmosphere containing 5% CO_2_. Human embryonic kidney 293T (HEK 293T) cells were cultured under the same conditions but at 37 °C. When the cell culture reached 80% confluency, the samples were harvested for subsequent analysis.

After harvesting, the cells were centrifuged (1600 rpm, 3 min, 4 °C). The resulting pellets were lysed on ice for 30 min using RIPA buffer (Beyotime, Shanghai, China, Cat. No. P0013B) supplemented with protease inhibitors (Merck, Darmstadt, Germany, Cat. No. 535140). Lysates were then cleared by centrifugation (12,000 rpm, 5 min, 4 °C), and the supernatant was collected and stored at −80 °C for further analysis.

4.4. Plasmids and Cell Transfection

Mammalian expression plasmids for human wild-type PrP (PG5-PrP) and mutated PrP (Cyto-PrP), lacking the signal peptide and glycosylphosphatidylinositol (GPI) anchor sequence, were constructed in the vector pcDNA3.1+, as described elsewhere [38]. The recombinant plasmids expressing full-length (β-syn 1–133) and the different truncated mouse β-syn (β-syn 1–61, 1–85, 61–133, and 85–133) were commercially generated by Tsingke Biotechnology Co., Ltd., Beijing, China, based on the reference sequence of mouse SNCB (NP_291088.1). Different lengths of mouse SNCB fragments were separately inserted into the vector pcDNA3.1. Additionally, the plasmid expressing an shRNA targeting mouse Akt1 (5′-TGGCACCTTTATTGGCTACAA-3′) was also constructed into the lentiviral vector pLKO.1-puro by Tsingke Biotechnology Co., Ltd., Beijing, China, according to the reference sequence of mouse Akt1 (NP_033782.1). The full knockdown sequence of Akt is 5′-CCGGTGGCACCTTTATTGGCTACAACTCGAGTTGTAGCCAATAAAGGTGCCATTTTTT-3′.

HEK 293T cells, lacking detectable endogenous PrP protein, were seeded into 6-well plates and transiently transfected with 2.5 μg of individual plasmids at 70–80% confluence using Lipofectamine 3000 (Thermo Fisher Scientific, Waltham, MA, USA, Cat. No. L3000015) according to the manufacturer’s instructions. The cells were harvested for analysis 48 h post-transfection.

4.5. Western Blot

Briefly, 5–10 μL denatured samples (brain homogenates or cell lysates) were separated by 12% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and electroblotted onto nitrocellulose membranes. Thereafter, membranes were blocked using QuickBlock™ Universal Protein-Free Blocking and Antibody Dilution Buffer (Beyotime, Shanghai, China, Cat. No. P0270) at room temperature (RT) for 10 min, followed by overnight incubation with anti-β-syn EP1537Y (1:5000, Abcam, Cambridge, UK, Cat. No. ab76111), anti-Akt 40D4 (1:2000, Cell Signaling Technology, Danvers, MA, USA, Cat. No. 2920S), anti-Akt1 (1:1000, Santa Cruz Biotechnology, Santa Cruz, CA, USA, Cat. No. sc-5298), anti-PrP EP1802Y (1:5000, Abcam, Cambridge, UK, Cat. No. ab52604), anti-Caspase-3 (1:1000, Cell Signaling Technology, Danvers, MA, USA, Cat. No. 9662S), anti-Cleaved Caspase-3 EPR21032 (1:5000, Abcam, Cambridge, UK, Cat. No. ab214430), or β-actin (1:5000, ZSGB-BIO, Beijing, China, Cat. No. TA-09) at 4 °C. After washing with TBST (Tris-buffered saline containing 0.1% Tween 20), membranes were subsequently incubated with individual horseradish peroxidase (HRP)-conjugated secondary antibodies (1:5000, Jackson ImmunoResearch Laboratories, Inc., West Grove, PA, USA, Cat. Nos. 111-035-003 and 115-035-002) at RT for 1 h, and reactive signals were developed using a commercial enhanced chemiluminescence (ECL) kit (PerkinElmer, Waltham, MA, USA, Cat. No. NEL104001EA). Images were acquired using ChemiScope 6000 (Qinxiang Scientific Instrument Co., Ltd., Shanghai, China). ImageJ software (Version 1.54f, National Institutes of Health, Bethesda, MD, USA) was used to determine the gray value of individual target protein bands with automatic background correction, and the relative gray value of the target protein was calculated by normalizing its gray value to that of the internal reference protein (GAPDH/β-actin).

4.6. Co-Immunoprecipitation (Co-IP)

In total, 50 μL of washed Dynabeads^®^ Protein G (Invitrogen, Waltham, MA, USA, Cat. No. 10004D) was mixed with 1 μg of the capture antibody and incubated at 4 °C for 4 h. Then, 100 μL of cell lysate or brain homogenate was added, and the mixture was incubated overnight. Following magnetic separation, the immunocomplexes were washed three times in washing buffer and resolved using SDS-PAGE.

4.7. Immunofluorescence Assay (IFA)

Paraffin tissue sections (5 μm) were thoroughly deparaffinized (2 × 5 min in Xylol and a descending alcohol row). Antigen retrieval was performed by boiling the sections in Citrate Antigen Retrieval Solution (Beyotime, Shanghai, China, Cat. No. P0081) for 20 min. Sections were briefly rinsed in phosphate buffered saline (PBS), permeabilized with 0.3% Triton X-100 (LABLEAD, Beijing, China, Cat. No. 0649) for 30 min, and then blocked for 1 h. Then, the sections were incubated with anti-β-syn (1:200, Abcam, Cambridge, UK, Cat. No. ab76111), anti-NeuN (Neuronal specific nuclear protein, 1:200, Merck, Darmstadt, Germany, Cat. No. MAB377), anti-GFAP (glial fibrillary acidic protein, 1:200, GenTex, Irvine, CA, USA, Cat. No. GTX34759), or anti-iba1 (Ion calcium-binding bridle molecule 1, 1:200, Sigma-Aldrich, St. Louis, MI, USA, Cat. No. SAB2702364) at 4 °C overnight. Following washing, the sections were incubated with Alexa Fluor 488- and Alexa Fluor 568-conjugated anti-rabbit or anti-mouse secondary antibodies at 37 °C for 1 h. After washing, they were mounted in DAPI (Beyotime, C1002) at RT for 20 min. Data acquisition was performed using a Leica Sp8 confocal microscope and Leica Application Suite (LAS AF Lite, Version 5.3.1.29786, Leica Microsystems, Wetzlar, Germany). The IOD of the target fluorescence was automatically measured and normalized to DAPI staining. For fluorescence analysis, three images were analyzed per animal, with thresholding applied prior to the correlation analysis. The colocalization of the two positive signals in IFAs was quantified by Pearson’s correlation coefficient (r) and interpreted as follows: strong (r > 0.8), medium (0.5 < r < 0.8), weak (0.3 < r < 0.5), and no correlation (r < 0.3).

4.8. Quantitative Real-Time PCR (qPCR)

Total RNAs of SMB cells or brain tissues were isolated using the RNeasy Mini Kit (QIAGEN, Hilden, Germany, Cat. No. 74104) and reverse-transcribed into cDNA with Script III RT MasterMix (GeneBetter, Beijing, China, Cat. No. P518-100) following the manufacturers’ protocols. The primer sequences are as follows: Glyceraldehyde-3-Phosphate Dehydrogenase (GAPDH) (forward: 5′-TTTGCAGTGGCAAAGTGGAG-3′; reverse: 5′-GATGGGCTTCCCGTTGATGA-3′) and mouse β-syn (forward: 5′-GGAAGGCGTCCTCTATGTCG-3′; reverse: 5′-CGTGGCTGCTGCAATGTTC-3′). The PCR reaction mixture, prepared according to the protocol for ChamQ Universal SYBR qPCR Master Mix (Vazyme, Nanjing, China, Cat. No. Q711-02), was amplified in triplicate for 40 cycles using the following parameters: 30 s at 95 °C, 30 s at 60 °C, and 60 s at 72 °C. The target gene Ct was normalized to GAPDH using the 2^−ΔΔCt^ method.

4.9. Terminal Deoxynucleotidyl Transferase dUTP Nick End Labeling (TUNEL) Analysis

DNA fragmentation, as an indicator of apoptosis, in the transfected HEK 293T cells was assessed using the TUNEL BrightRed Apoptosis Detection Kit (Vazyme, A113-02) in accordance with the manufacturer’s instructions. DAPI staining was performed to visualize nuclei after the TUNEL reaction. Images were acquired by confocal fluorescence microscopy, and the percentage of TUNEL-positive nuclei was quantified using ImageJ software.

4.10. Biolayer Interferometry Binding Assays

The binding affinities between mouse β-syn and mouse Akt1 were measured using bio-layer interferometry (BLI) on an Octet RED96e system (Sartorius) with streptavidin-coated biosensors. Both E. coli. expressed recombinant full-length mouse β-syn and Akt1 proteins, used in the assay, were purified and obtained from Tsingke Biotechnology Co., Ltd. The assay was performed at RT according to the following four steps: (1) loading Super Streptavidin (SSA) biosensors (18–5057) with 10 µg/mL mouse Akt1 diluted in 0.02% PBST; (2) incubating the biosensors in PBST for 300 s to establish a baseline; (3) associating with two-fold serially diluted β-syn (15.63–500 nM) in PBST; and (4) dissociating in PBST.

4.11. Protein–Protein Interaction Modeling and Analysis

The amino acid sequences of mouse AKT1 (UniProt ID: P31750) and mouse β-synuclein (UniProt ID: Q91ZZ3) were retrieved from Universal Protein Knowledgebase (UniProtKB, https://www.uniprot.org/). Protein–protein complex modeling was performed with AlphaFold3 (https://alphafoldserver.com/). The optimal model was selected based on three criteria: the highest overall model confidence score (>0.7), a low predicted aligned error (PAE < 5 Å) at the protein-protein interface, and a per-residue predicted local distance difference test (pLDDT) > 60 for interfacial residues. These thresholds correspond to high-confidence predictions according to AlphaFold3 assessment metrics.

The selected model was analyzed using the open-source PyMOL (version 3.1.3). The protein–protein interaction interface was defined by selecting all residues that contained any atom within 5.0 Å of any atom from the binding partner, a standard cutoff for defining molecular contacts. Specific hydrogen bonds were identified using a maximum donor–acceptor distance of 3.5 Å, a stringent cutoff commonly used to define strong hydrogen bonds.

4.12. Statistical Analysis

Data analyses were performed using the software GraphPad Prism 8.4.3 and SPSS 25.0 statistical software (IBM, Armonk, NY, USA). Quantitative analysis of the blots in Western blots and the images in IFAs was performed using ImageJ software. For normally distributed data with homogeneous variance, multiple independent groups were compared using a one-way analysis of variance (one-way ANOVA), whereas two-group comparisons were performed using the unpaired Student’s t-test. All experiments were conducted with three independent biological replicates per group, each measured in triplicate to ensure reproducibility and reliability. The final results are presented as mean ± standard deviation (SD). Student’s t test was evaluated for statistical analysis: *: p < 0.05, **: p < 0.01, ***: p < 0.001, ****: p < 0.0001, and NS for not significant.

5. Conclusions

We have described a detailed molecular interaction between β-syn and Akt. The anti-apoptotic activity of β-syn, which is largely mediated by Akt signaling, is severely lost in the terminal stage of prion infection, suggesting a mechanism of intrinsic neuronal vulnerability and highlighting a novel therapeutic strategy.