Advanced human iPSC-based preclinical model for Parkinson's disease with optogenetic alpha-synuclein aggregation.

Human induced pluripotent stem cells (hiPSCs) offer advantages for disease modeling and drug discovery. However, recreating innate cellular pathologies, particularly in late-onset neurodegenerative diseases with accumulated protein aggregates including Parkinson's disease (PD), has been challenging. To overcome this barrier, we developed an optogenetics-assisted α-synuclein (α-syn) aggregation induction system (OASIS) that rapidly induces α-syn aggregates and toxicity in PD hiPSC-midbrain dopaminergic neurons and midbrain organoids. Our OASIS-based primary compound screening with SH-SY5Y cells identified 5 candidates that were secondarily validated with OASIS PD hiPSC-midbrain dopaminergic neurons and midbrain organoids, leading us to finally select BAG956. Furthermore, BAG956 significantly reverses characteristic PD phenotypes in α-syn preformed fibril models in vitro and in vivo by promoting autophagic clearance of pathological α-syn aggregates. Following the FDA Modernization Act 2.0's emphasis on alternative non-animal testing methods, our OASIS can serve as an animal-free preclinical test model (newly termed "nonclinical test") for the synucleinopathy drug development.

Protein Ligation-Assisted Reconstitution of Split HRP Fragments for Facile Production of HRP Fusion Proteins in E.†coli.

Horseradish peroxidase (HRP) is a pivotal biocatalyst for biosensor development and fine chemical synthesis. HRP proteins are mostly extracted and purified from the roots of horseradish because the solubility and productivity of recombinant HRP in bacteria are significantly low. In this study, we investigate the reconstitution system of split HRP fragments to improve its soluble expression levels in E. coli allowing the cost-effective production of bioactive HRPs. To promote the effective association between two HRP fragments (HRPn and HRPc), we exploit SpyTag-SpyCatcher chemistry, a versatile protein coupling method with high affinity and selectivity. Each HRP fragment was genetically fused with SpyTag and SpyCatcher, respectively, exhibiting soluble expression in the E. coli cytoplasm. The engineered split HRPs were effectively and irreversibly reconstituted into a biologically active and stable assembly that can catalyze intrinsic enzymatic reactions. Compared to the chaperone co-expression system, our approach shows that the production yield of soluble HRP is comparable, but the purity of the final product is relatively high. Therefore, our results can be applied to the high-yield production of recombinant HRP variants and other difficult-to-express proteins in bacteria without complex downstream processes.

TRIP12 ubiquitination of glucocerebrosidase contributes to neurodegeneration in Parkinson's disease.

Impairment in glucocerebrosidase (GCase) is strongly associated with the development of Parkinson's disease (PD), yet the regulators responsible for its impairment remain elusive. In this paper, we identify the E3 ligase Thyroid Hormone Receptor Interacting Protein 12 (TRIP12) as a key regulator of GCase. TRIP12 interacts with and ubiquitinates GCase at lysine 293 to control its degradation via ubiquitin proteasomal degradation. Ubiquitinated GCase by TRIP12 leads to its functional impairment through premature degradation and subsequent accumulation of α-synuclein. TRIP12 overexpression causes mitochondrial dysfunction, which is ameliorated by GCase overexpression. Further, conditional TRIP12 knockout in vitro and knockdown in vivo promotes the expression of GCase, which blocks α-synuclein preformed fibrils (α-syn PFFs)-provoked dopaminergic neurodegeneration. Moreover, TRIP12 accumulates in human PD brain and α-synuclein-based mouse models. The identification of TRIP12 as a regulator of GCase provides a new perspective on the molecular mechanisms underlying dysfunctional GCase-driven neurodegeneration in PD.

A Novel, Selective c-Abl Inhibitor, Compound 5, Prevents Neurodegeneration in Parkinson's Disease.

Parkinson's disease (PD) is a progressive neurodegenerative disorder that affects movement. The nonreceptor tyrosine kinase c-Abl has shown a potential role in the progression of PD. As such, c-Abl inhibition is a promising candidate for neuroprotection in PD and α-synucleinopathies. Compound 5 is a newly synthesized blood-brain barrier penetrant c-Abl inhibitor with higher efficacy than existing inhibitors. The objective of the current study was to demonstrate the neuroprotective effects of compound 5 on the α-synuclein preformed fibril (α-syn PFF) mouse model of PD. Compound 5 significantly reduced neurotoxicity, activation of c-Abl, and Lewy body pathology caused by α-syn PFF in cortical neurons. Additionally, compound 5 markedly ameliorated the loss of dopaminergic neurons, c-Abl activation, Lewy body pathology, neuroinflammatory responses, and behavioral deficits induced by α-syn PFF injection in vivo. Taken together, these results suggest that compound 5 could be a pharmaceutical agent to prevent the progression of PD and α-synucleinopathies.

Alpha-Synuclein Preformed Fibrils Induce Cellular Senescence in Parkinson's Disease Models.

Emerging evidence indicates that cellular senescence could be a critical inducing factor for aging-associated neurodegenerative disorders. However, the involvement of cellular senescence remains unclear in Parkinson's disease (PD). To determine this, we assessed the effects of α-synuclein preformed fibrils (α-syn PFF) or 1-methyl-4-phenylpyridinium (MPP+) on changes in cellular senescence markers, employing α-syn PFF treated-dopaminergic N27 cells, primary cortical neurons, astrocytes and microglia and α-syn PFF-injected mouse brain tissues, as well as human PD patient brains. Our results demonstrate that α-syn PFF-induced toxicity reduces the levels of Lamin B1 and HMGB1, both established markers of cellular senescence, in correlation with an increase in the levels of p21, a cell cycle-arrester and senescence marker, in both reactive astrocytes and microglia in mouse brains. Using Western blot and immunohistochemistry, we found these cellular senescence markers in reactive astrocytes as indicated by enlarged cell bodies within GFAP-positive cells and Iba1-positive activated microglia in α-syn PFF injected mouse brains. These results indicate that PFF-induced pathology could lead to astrocyte and/or microglia senescence in PD brains, which may contribute to neuropathology in this model. Targeting senescent cells using senolytics could therefore constitute a viable therapeutic option for the treatment of PD.

Complement and Coagulation Cascades are Potentially Involved in Dopaminergic Neurodegeneration in α-Synuclein-Based Mouse Models of Parkinson's Disease.

Parkinson's disease (PD) is the second most common neurodegenerative disorder that results in motor dysfunction and, eventually, cognitive impairment. α-Synuclein protein is known as a central protein to the pathophysiology of PD, but the underlying pathological mechanism still remains to be elucidated. In an effort to understand how α-synuclein underlies the pathology of PD, various PD mouse models with α-synuclein overexpression have been developed. However, systemic analysis of the brain proteome of those mouse models is lacking. In this study, we established two mouse models of PD by injecting α-synuclein preformed fibrils (PFF) or by inducing overexpression of human A53T α-synuclein to investigate common pathways in the two different types of the PD mouse models. For more accurate quantification of mouse brain proteome, the proteins were quantified using the method of stable isotope labeling with amino acids in mammals . We identified a total of 8355 proteins from the two mouse models; ∼6800 and ∼7200 proteins from α-synuclein PFF-injected mice and human A53T α-synuclein transgenic mice, respectively. Through pathway analysis of the differentially expressed proteins common to both PD mouse models, it was discovered that the complement and coagulation cascade pathways were enriched in the PD mice compared to control animals. Notably, a validation study demonstrated that complement component 3 (C3)-positive astrocytes were increased in the ventral midbrain of the intrastriatal α-synuclein PFF-injected mice and C3 secreted from astrocytes could induce the degeneration of dopaminergic neurons. This is the first study that highlights the significance of the complement and coagulation pathways in the pathogenesis of PD through proteome analyses with two sophisticated mouse models of PD.

Amyloid-like oligomerization of AIMP2 contributes to α-synuclein interaction and Lewy-like inclusion.

Lewy bodies are pathological protein inclusions present in the brain of patients with Parkinson's disease (PD). These inclusions consist mainly of α-synuclein with associated proteins, such as parkin and its substrate aminoacyl transfer RNA synthetase complex-interacting multifunctional protein-2 (AIMP2). Although AIMP2 has been suggested to be toxic to dopamine neurons, its roles in α-synuclein aggregation and PD pathogenesis are largely unknown. Here, we found that AIMP2 exhibits a self-aggregating property. The AIMP2 aggregate serves as a seed to increase α-synuclein aggregation via specific and direct binding to the α-synuclein monomer. The coexpression of AIMP2 and α-synuclein in cell cultures and in vivo resulted in the rapid formation of α-synuclein aggregates with a corresponding increase in toxicity. Moreover, accumulated AIMP2 in mouse brain was largely redistributed to insoluble fractions, correlating with the α-synuclein pathology. Last, we found that α-synuclein preformed fibril (PFF) seeding, adult Parkin deletion, or oxidative stress triggered a redistribution of both AIMP2 and α-synuclein into insoluble fraction in cells and in vivo. Supporting the pathogenic role of AIMP2, AIMP2 knockdown ameliorated the α-synuclein aggregation and dopaminergic cell death in response to PFF or 6-hydroxydopamine treatment. Together, our results suggest that AIMP2 plays a pathological role in the aggregation of α-synuclein in mice. Because AIMP2 insolubility and coaggregation with α-synuclein have been seen in the PD Lewy body, targeting pathologic AIMP2 aggregation might be useful as a therapeutic strategy for neurodegenerative α-synucleinopathies.

Modular protein-DNA hybrid nanostructures as a drug delivery platform.

With the increasing number of identified intracellular drug targets, cytosolic drug delivery has gained much attention. Despite advances in synthetic drug carriers, however, construction of homogeneous and biocompatible nanostructures in a controllable manner still remains a challenge in a translational medicine. Herein, we present the modular design and assembly of functional DNA nanostructures through sequence-specific interactions between zinc-finger proteins (ZnFs) and DNA as a cytosolic drug delivery platform. Three kinds of DNA-binding ZnF domains were genetically fused to various proteins with different biological roles, including targeting moiety, molecular probe, and therapeutic cargo. The engineered ZnFs were employed as distinct functional modules, and incorporated into a designed ZnF-binding sequence of a Y-shaped DNA origami (Y-DNA). The resulting functional Y-DNA nanostructures (FYDN) showed self-assembled superstructures with homogeneous morphology, strong resistance to exonuclease activity and multi-modality. We demonstrated the general utility of our approach by showing efficient cytosolic delivery of PTEN tumour suppressor protein to rescue unregulated kinase signaling in cancer cells with negligible nonspecific cytotoxicity.

Stress-induced changes in social dominance are scaled by AMPA-type glutamate receptor phosphorylation in the medial prefrontal cortex.

The establishment and maintenance of social dominance are critical for social stability and the survival and health of individual animals. Stress lead to depression and a decrease in the social status of depressed persons is a risk factor for suicide. Therefore, we explored the mechanistic and behavioral links among stress, depression, and social dominance and found that mice subjected to chronic restraint stress (CRS), an animal model of stress-induced depression, showed decreased social dominance as measured by a dominance tube test. Importantly, this submissive behavior was occluded by the antidepressant, fluoxetine, a selective serotonin reuptake inhibitor. It is known that social dominance is controlled by synaptic efficacy in the medial prefrontal cortex (mPFC) and that AMPA-type glutamate receptor (AMPA-R) is a key molecule for synaptic efficacy. We found that the phosphorylation on AMPA-R was bidirectionally changed by CRS and fluoxetine in the mPFC of mice with CRS. Moreover, we found a strong correlation between social dominance and AMPA-R phosphorylation that regulates synaptic efficacy by modulating the synaptic targeting of AMPA-R. Our correlational analysis of the behavior and biochemistry of the CRS model suggests that AMPA-R phosphorylation in the mPFC may serve as a biomarker of social dominance related to stress.

LARGE, an intellectual disability-associated protein, regulates AMPA-type glutamate receptor trafficking and memory.

Mutations in the human LARGE gene result in severe intellectual disability and muscular dystrophy. How LARGE mutation leads to intellectual disability, however, is unclear. In our proteomic study, LARGE was found to be a component of the AMPA-type glutamate receptor (AMPA-R) protein complex, a main player for learning and memory in the brain. Here, our functional study of LARGE showed that LARGE at the Golgi apparatus (Golgi) negatively controlled AMPA-R trafficking from the Golgi to the plasma membrane, leading to down-regulated surface and synaptic AMPA-R targeting. In LARGE knockdown mice, long-term potentiation (LTP) was occluded by synaptic AMPA-R overloading, resulting in impaired contextual fear memory. These findings indicate that the fine-tuning of AMPA-R trafficking by LARGE at the Golgi is critical for hippocampus-dependent memory in the brain. Our study thus provides insights into the pathophysiology underlying cognitive deficits in brain disorders associated with intellectual disability.

Neuronal calcium channel α1 subunit interacts with AMPA receptor, increasing its cell surface localisation.

Voltage-activated Ca2+ channels (Cav) play critical roles in excitable cells including neurons. Unlike the well-defined roles of Cav2 for pre-synaptic neurotransmission, the post-synaptic function of Cav2 is unclear. Based on our previous study demonstrating the postsynaptic association of the Cav2 with the AMPA receptor (AMPA-R), in this study we sought to further analyse the Cav2-AMPA-R association. We used a step-by-step dissociation of partially purified native Cav2-AMPA-R complexes and co-immunoprecipitation of the Cav2-AMPA-R complexes expressed in HEK293T cells to demonstrate that the main subunit of Cav, α1, formed a complex with the AMPA-R without the auxiliary subunits ß, α2δ, γ2/3. The α1 subunit increased the cell-surface localisation of the AMPA-R, which could be a post-synaptic function of the Cav2.

Gamma oscillation in functional brain networks is involved in the spontaneous remission of depressive behavior induced by chronic restraint stress in mice.

BACKGROUND: Depression is one of the most prevalent mood disorders, and is known to be associated with abnormal functional connectivity in neural networks of the brain. Interestingly, a significant proportion of patients with depression experience spontaneous remission without any treatment. However, the relationship between electroencephalographic (EEG) functional connectivity and the spontaneous remission in depression remains poorly understood. Here, we investigated regional and network brain activity using EEG signals from a chronic restraint stress (CRS)-induced mouse model of depression. After 1 (CRS1W) or 3 weeks (CRS3W) following the cessation of a 4-week-long CRS, mice were subjected to depression-associated behavioral tasks. EEG signals were obtained from eight cortical regions (frontal, somatosensory, parietal, and visual cortices in each hemisphere). RESULTS: The CRS1W group exhibited behavioral dysfunctions in the open field and forced swim tasks, whereas the CRS3W group displayed normal levels of behaviors in those tasks. In a linear correlation analysis, the CRS1W group exhibited increased correlation coefficient values at all frequency bands (delta, 1.5-4; theta, 4-8; alpha, 8-12; beta, 12-30; gamma, 30-80 Hz) compared with the control group. However, the differences in delta- and gamma-frequency bands between the control and CRS1W groups were no longer observed in the CRS3W group. Persistent brain network homology revealed significantly different functional connectivity between the control and CRS1W groups, and it demonstrated a huge restoration of the decreased distances in the gamma-frequency band for the CRS3W group. Moreover, the CRS3W group displayed a similar strength of connectivity among somatosensory and frontal cortices as the control group. CONCLUSION: A mouse model of CRS-induced depression showed spontaneous behavioral remission of depressive behavior. Using persistent brain network homology analysis of EEG signals from eight cortical regions, we found that restoration of gamma activity at the network level is associated with behavioral remission.

Differential regulation of observational fear and neural oscillations by serotonin and dopamine in the mouse anterior cingulate cortex.

RATIONALE: The aberrant regulation of serotonin (5-HT) and dopamine (DA) in the brain has been implicated in neuropsychiatric disorders associated with marked impairments in empathy, such as schizophrenia and autism. Many psychiatric drugs bind to both types of receptors, and the anterior cingulate cortex (ACC) is known to be centrally involved with empathy. However, the relationship between the 5-HT/DA system in the ACC and empathic behavior is not yet well known. OBJECTIVES: We investigated the role of 5-HT/DA in empathy-like behavior and in the regulation of ACC neural activity. METHODS: An observational fear learning task was conducted following microinjections of 5-HT, DA, 5-HT and DA, methysergide (5-HT receptor antagonist), SCH-23390 (DA D1 receptor antagonist), or haloperidol (DA D2 receptor antagonist) into the mouse ACC. The ACC neural activity influenced by 5-HT and DA was electrophysiologically characterized in vitro and in vivo. RESULTS: The microinjection of haloperidol, but not methysergide or SCH-23390, decreased the fear response of observing mice. The administration of 5-HT and 5-HT and DA together, but not DA alone, reduced the freezing response of observing mice. 5-HT enhanced delta-band activity and reduced alpha- and gamma-band activities in the ACC, whereas DA reduced only alpha-band activity. Based on entropy, reduced complexity of ACC neural activity was observed with 5-HT treatment. CONCLUSIONS: The current results demonstrated that DA D2 receptors in the ACC are required for observational fear learning, whereas increased 5-HT levels disrupt observational fear and alter the regularity of ACC neural oscillations.