Peptide Transporter 1-Mediated Dipeptide Transport Promotes Hepatocellular Carcinoma Metastasis by Activating MAP4K4/G3BP2 Signaling Axis.

Cancer metastasis is the leading cause of mortality in patients with hepatocellular carcinoma (HCC). To meet the rapid malignant growth and transformation, tumor cells dramatically increase the consumption of nutrients, such as amino acids. Peptide transporter 1 (PEPT1), a key transporter for small peptides, has been found to be an effective and energy-saving intracellular source of amino acids that are required for the growth of tumor cells. Here, the role of PEPT1 in HCC metastasis and its underlying mechanisms is explored. PEPT1 is upregulated in HCC cells and tissues, and high PEPT1 expression is associated with poor prognosis in patients with HCC. PEPT1 overexpression dramatically promoted HCC cell migration, invasion, and lung metastasis, whereas its knockdown abolished these effects both in vitro and in vivo. Mechanistic analysis revealed that high PEPT1 expression increased cellular dipeptides in HCC cells that are responsible for activating the MAP4K4/G3BP2 signaling pathway, ultimately facilitating the phosphorylation of G3BP2 at Thr227 and enhancing HCC metastasis. Taken together, these findings suggest that PEPT1 acts as an oncogene in promoting HCC metastasis through dipeptide-induced MAP4K4/G3BP2 signaling and that the PEPT1/MAP4K4/G3BP2 axis can serve as a promising therapeutic target for metastatic HCC.

GDF1 ameliorates cognitive impairment induced by hearing loss.

Hearing loss is associated with an increased risk of Alzheimer disease (AD). However, the mechanisms of hearing loss promoting the onset of AD are poorly understood. Here we show that hearing loss aggravates cognitive impairment in both wild-type mice and mouse models of AD. Embryonic growth/differentiation factor 1 (GDF1) is downregulated in the hippocampus of deaf mice. Knockdown of GDF1 mimics the detrimental effect of hearing loss on cognition, while overexpression of GDF1 in the hippocampus attenuates the cognitive impairment induced by deafness. Strikingly, overexpression of GDF1 also attenuates cognitive impairment in APP/PS1 transgenic mice. GDF1 activates Akt, which phosphorylates asparagine endopeptidase and inhibits asparagine endopeptidase-induced synaptic degeneration and amyloid-ß production. The expression of GDF1 is downregulated by the transcription factor CCAAT-enhancer binding protein-ß. These findings indicate that hearing loss could promote AD pathological changes by inhibiting the GDF1 signaling pathway; thus, GDF1 may represent a therapeutic target for AD.

Q&A with Zhentao Zhang.

In this interview with Zhentao Zhang, we discuss his research focusing on the molecular mechanisms underlying the aggregation of prion-like proteins in neurodegenerative diseases and spotlight his recent work in Cell Reports that shows that a yeast prion protein interacts with tau and facilitates its aggregation.

Protein-protein interactions regulating α-synuclein pathology.

Parkinson's disease (PD) is a neurodegenerative disease characterized by the degeneration of dopaminergic neurons in the substantia nigra pars compacta (SNpc) and the formation of Lewy bodies (LBs). The main proteinaceous component of LBs is aggregated α-synuclein (α-syn). However, the mechanisms underlying α-syn aggregation are not yet fully understood. Converging lines of evidence indicate that, under certain pathological conditions, various proteins can interact with α-syn and regulate its aggregation. Understanding these protein-protein interactions is crucial for unraveling the molecular mechanisms contributing to PD pathogenesis. In this review we provide an overview of the current knowledge on protein-protein interactions that regulate α-syn aggregation. Additionally, we briefly summarize the methods used to investigate the influence of protein-protein interactions on α-syn aggregation and propagation.

GTF2H4 regulates partial EndMT via NF-κB activation through NCOA3 phosphorylation in ischemic diseases.

Partial endothelial-to-mesenchymal transition (EndMT) is an intermediate phenotype observed in endothelial cells (ECs) undergoing a transition toward a mesenchymal state to support neovascularization during (patho)physiological angiogenesis. Here, we investigated the occurrence of partial EndMT in ECs under hypoxic/ischemic conditions and identified general transcription factor IIH subunit 4 (GTF2H4) as a positive regulator of this process. In addition, we discovered that GTF2H4 collaborates with its target protein excision repair cross-complementation group 3 (ERCC3) to co-regulate partial EndMT. Furthermore, by using phosphorylation proteomics and site-directed mutagenesis, we demonstrated that GTF2H4 was involved in the phosphorylation of receptor coactivator 3 (NCOA3) at serine 1330, which promoted the interaction between NCOA3 and p65, resulting in the transcriptional activation of NF-κB and the NF-κB/Snail signaling axis during partial EndMT. In vivo experiments confirmed that GTF2H4 significantly promoted partial EndMT and angiogenesis after ischemic injury. Collectively, our findings reveal that targeting GTF2H4 is promising for tissue repair and offers potential opportunities for treating hypoxic/ischemic diseases.

N-homocysteinylation of DJ-1 promotes neurodegeneration in Parkinson's disease.

DJ-1, also known as Parkinson's disease protein 7 (Park7), is a multifunctional protein that regulates oxidative stress and mitochondrial function. Dysfunction of DJ-1 is implicated in the pathogenesis of Parkinson's disease (PD). Hyperhomocysteinemia is associated with an increased risk of PD. Here we show that homocysteine thiolactone (HTL), a reactive thioester of homocysteine (Hcy), covalently modifies DJ-1 on the lysine 182 (K182) residue in an age-dependent manner. The N-homocysteinylation (N-hcy) of DJ-1 abolishes its neuroprotective effect against oxidative stress and mitochondrial dysfunction, exacerbating cell toxicity. Blocking the N-hcy of DJ-1 restores its protective effect. These results indicate that the N-hcy of DJ-1 abolishes its neuroprotective effect and promotes the progression of PD. Inhibiting the N-hcy of DJ-1 may exert neuroprotective effect against PD.

Bridging integrator 1 fragment accelerates tau aggregation and propagation by enhancing clathrin-mediated endocytosis in mice.

The bridging integrator 1 (BIN1) gene is an important risk locus for late-onset Alzheimer's disease (AD). BIN1 protein has been reported to mediate tau pathology, but the underlying molecular mechanisms remain elusive. Here, we show that neuronal BIN1 is cleaved by the cysteine protease legumain at residues N277 and N288. The legumain-generated BIN1 (1-277) fragment is detected in brain tissues from AD patients and tau P301S transgenic mice. This fragment interacts with tau and accelerates its aggregation. Furthermore, the BIN1 (1-277) fragment promotes the propagation of tau aggregates by enhancing clathrin-mediated endocytosis (CME). Overexpression of the BIN1 (1-277) fragment in tau P301S mice facilitates the propagation of tau pathology, inducing cognitive deficits, while overexpression of mutant BIN1 that blocks its cleavage by legumain halts tau propagation. Furthermore, blocking the cleavage of endogenous BIN1 using the CRISPR/Cas9 gene-editing tool ameliorates tau pathology and behavioral deficits. Our results demonstrate that the legumain-mediated cleavage of BIN1 plays a key role in the progression of tau pathology. Inhibition of legumain-mediated BIN1 cleavage may be a promising therapeutic strategy for treating AD.

Role of sodium taurocholate cotransporting polypeptide (NTCP) in HBV-induced hepatitis: Opportunities for developing novel therapeutics.

Hepatitis B is an infectious disease caused by the HBV virus. It presents a significant challenge for treatment due to its chronic nature and the potential for developing severe complications, including hepatocirrhosis and hepatocellular carcinoma. These complications not only cause physical and psychological distress to patients but also impose substantial economic and social burdens on both individuals and society as a whole. The internalization of HBV relies on endocytosis and necessitates the involvement of various proteins, including heparin sulfate proteoglycans, epidermal growth factor receptors, and NTCP. Among these proteins, NTCP is pivotal in HBV internalization and is primarily located in the liver's basement membrane. As a transporter of bile acids, NTCP also serves as a receptor facilitating HBV entry into cells. Numerous molecules have been identified to thwart HBV infection by stifling NTCP activity, although only a handful exhibit low IC50 values. In this systematic review, our primary focus dwells on the structure and regulation of NTCP, as well as the mechanism involved in HBV internalization. We underscore recent drug breakthroughs that specifically target NTCP to combat HBV infection. By shedding light on these advances, this review contributes novel insights into developing effective anti-HBV medications.

L116 Deletion in CSPα Promotes α-Synuclein Aggregation and Neurodegeneration.

Parkinsonism is a clinical syndrome that is caused by Parkinson's disease (PD) and other neurodegenerative diseases. Here, we report a patient who exhibited progressive parkinsonism, epilepsy, and cognitive impairment and was diagnosed with adult-onset neuronal ceroid lipofuscinoses (ANCLs). The patient carries a mutation (p.Leu116 del) in the DNAJC5 gene that encodes cysteine string protein (CSPα). Since the patient shows typical parkinsonism and loss of dopamine transporter in the striatum, we investigated the effect of wild-type and L116del mutant CSPα on the aggregation of α-synuclein (α-syn) and neurotoxicity in vitro. Overexpression of wild-type CSPα attenuated the phosphorylation, ubiquitination, and aggregation of α-syn induced by α-syn fibrils. Moreover, wild-type CSPα inhibits oxidative stress and cell apoptosis and rescues inefficient SNARE complex formation induced by α-syn fibrils in SH-SY5Y cells. However, these protective effects of CSPα were abolished by the L116del mutation. Collectively, these results indicate that L116 deletion in CSPα promotes α-syn pathology and neurotoxicity. Boosting CSPα may be therapeutically useful for treating synucleinopathies.

Mitsugumin 53 mitigation of ischemia-reperfusion injury in a mouse model.

OBJECTIVE: Primary graft dysfunction is often attributed to ischemia-reperfusion injury, and prevention would be a therapeutic approach to mitigate injury. Mitsugumin 53, a myokine, is a component of the endogenous cell membrane repair machinery. Previously, exogenous administration of recombinant human (recombinant human mitsugumin 53) protein has been shown to mitigate acute lung injury. In this study, we aimed to quantify a therapeutic benefit of recombinant human mitsugumin 53 to mitigate a transplant-relevant model of ischemia-reperfusion injury. METHODS: C57BL/6J mice were subjected to 1 hour of ischemia (via left lung hilar clamp), followed by 24 hours of reperfusion. mg53-/- mice were administered exogenous recombinant human mitsugumin 53 or saline before reperfusion. Tissue, bronchoalveolar lavage, and blood samples were collected at death and used to quantify the extent of lung injury via histology and biochemical assays. RESULTS: Administration of recombinant human mitsugumin 53 showed a significant decrease in an established biometric profile of lung injury as measured by lactate dehydrogenase and endothelin-1 in the bronchoalveolar lavage and plasma. Biochemical markers of apoptosis and pyroptosis (interleukin-1ß and tumor necrosis factor-α) were also significantly mitigated, overall demonstrating recombinant human mitsugumin 53's ability to decrease the inflammatory response of ischemia-reperfusion injury. Exogenous recombinant human mitsugumin 53 administration showed a trend toward decreasing overall cellular infiltrate and neutrophil response. Fluorescent colocalization imaging revealed recombinant human mitsugumin 53 was effectively delivered to the endothelium. CONCLUSIONS: These data demonstrate that recombinant human mitsugumin 53 has the potential to prevent or reverse ischemia-reperfusion injury-mediated lung damage. Although additional studies are needed in wild-type mice to demonstrate efficacy, this work serves as proof-of-concept to indicate the potential therapeutic benefit of mitsugumin 53 administration to mitigate ischemia-reperfusion injury.

Protocol for screening α-synuclein PET tracer candidates in vitro and ex vivo.

Alpha-synuclein (α-Syn) positron emission tomography (PET) imaging is a valuable approach for diagnosing and monitoring synucleinopathies-related diseases, such as Parkinson disease. Here, we present a protocol for screening potential α-Syn PET tracers using in vitro and ex vivo approaches. We describe steps for employing recombinant pre-formed fibrils and conducting screening procedures on neuronal models, mouse models, and patients' brain tissue sections to assess the specificity and selectivity of the candidate compounds. For complete details on the use and execution of this protocol, please refer to Xiang et al. (2023).1.

α-Synuclein pathology from the body to the brain: so many seeds so close to the central soil.

ABSTRACT: α-Synuclein is a protein that mainly exists in the presynaptic terminals. Abnormal folding and accumulation of α-synuclein are found in several neurodegenerative diseases, including Parkinson's disease. Aggregated and highly phosphorylated α-synuclein constitutes the main component of Lewy bodies in the brain, the pathological hallmark of Parkinson's disease. For decades, much attention has been focused on the accumulation of α-synuclein in the brain parenchyma rather than considering Parkinson's disease as a systemic disease. Recent evidence demonstrates that, at least in some patients, the initial α-synuclein pathology originates in the peripheral organs and spreads to the brain. Injection of α-synuclein preformed fibrils into the gastrointestinal tract triggers the gut-to-brain propagation of α-synuclein pathology. However, whether α-synuclein pathology can occur spontaneously in peripheral organs independent of exogenous α-synuclein preformed fibrils or pathological α-synuclein leakage from the central nervous system remains under investigation. In this review, we aimed to summarize the role of peripheral α-synuclein pathology in the pathogenesis of Parkinson's disease. We also discuss the pathways by which α-synuclein pathology spreads from the body to the brain.

The yeast prion protein Sup35 initiates α-synuclein pathology in mouse models of Parkinson's disease.

Parkinson's disease (PD) is characterized by the pathologic aggregation and prion-like propagation of α-synuclein (α-syn). Emerging evidence shows that fungal infections increase the incidence of PD. However, the molecular mechanisms by which fungi promote the onset of PD are poorly understood. Here, we show that nasal infection with Saccharomyces cerevisiae (S. cerevisiae) in α-syn A53T transgenic mice accelerates the aggregation of α-syn. Furthermore, we found that Sup35, a prion protein from S. cerevisiae, is the key factor initiating α-syn pathology induced by S. cerevisiae. Sup35 interacts with α-syn and accelerates its aggregation in vitro. Notably, injection of Sup35 fibrils into the striatum of wild-type mice led to α-syn pathology and PD-like motor impairment. The Sup35-seeded α-syn fibrils showed enhanced seeding activity and neurotoxicity compared with pure α-syn fibrils in vitro and in vivo. Together, these observations indicate that the yeast prion protein Sup35 initiates α-syn pathology in PD.

A novel transgenic mouse line with hippocampus-dominant and inducible expression of truncated human tau.

BACKGROUND: Intraneuronal accumulation of hyperphosphorylated tau is a defining hallmark of Alzheimer's disease (AD). However, mouse models imitating AD-exclusive neuronal tau pathologies are lacking. METHODS: We generated a new tet-on transgenic mouse model expressing truncated human tau N1-368 (termed hTau368), a tau fragment increased in the brains of AD patients and aged mouse brains. Doxycycline (dox) was administered in drinking water to induce hTau368 expression. Immunostaining and Western blotting were performed to measure the tau level. RNA sequencing was performed to evaluate gene expression, and several behavioral tests were conducted to evaluate mouse cognitive functions, emotion and locomotion. RESULTS: Dox treatment for 1-2 months at a young age induced overt and reversible human tau accumulation in the brains of hTau368 transgenic mice, predominantly in the hippocampus. Meanwhile, the transgenic mice exhibited AD-like high level of tau phosphorylation, glial activation, loss of mature neurons, impaired hippocampal neurogenesis, synaptic degeneration and cognitive deficits. CONCLUSIONS: This study developed a well-characterized and easy-to-use tool for the investigations and drug development for AD and other tauopathies.

Cholestanol accelerates α-synuclein aggregation and spreading by activating asparagine endopeptidase.

Cerebrotendinous xanthomatosis (CTX), an autosomal recessive disorder characterized by high levels of cholestanol in the blood and accumulation of cholestanol in multiple tissues, especially the brain, often presents in parkinsonism. However, it remains unknown whether cholestanol plays a role in the pathogenesis of sporadic Parkinson's disease (PD). Here, we show that the levels of serum cholestanol in patients with sporadic PD are higher than those in control participants. Cholestanol activates the protease asparagine endopeptidase (AEP) and induces the fragmentation of α-synuclein (α-syn) and facilitates its aggregation. Furthermore, cholestanol promotes the spreading of α-syn pathology in a mouse model induced by intrastriatal injection of α-syn fibrils. KO of AEP or administration of an AEP inhibitor ameliorates α-syn pathology, degeneration of the nigrostriatal dopaminergic pathway, and PD-like motor symptoms. These results not only indicate that cholestanol contributes to the aggregation and spreading of α-syn by activating AEP but also reveal an opportunity for treating PD with AEP inhibitors.

The role of glycogen synthase kinase 3 beta in neurodegenerative diseases.

Neurodegenerative diseases (NDDs) pose an increasingly prevalent threat to the well-being and survival of elderly individuals worldwide. NDDs include Alzheimer's disease (AD), Parkinson's disease (PD), Huntington's disease (HD), amyotrophic lateral sclerosis (ALS), and so on. They are characterized by progressive loss or dysfunction of neurons in the central or peripheral nervous system and share several cellular and molecular mechanisms, including protein aggregation, mitochondrial dysfunction, gene mutations, and chronic neuroinflammation. Glycogen synthase kinase-3 beta (GSK-3ß) is a serine/threonine kinase that is believed to play a pivotal role in the pathogenesis of NDDs. Here we summarize the structure and physiological functions of GSK3ß and explore its involvement in NDDs. We also discussed its potential as a therapeutic target.

SARS-CoV-2 Spike Protein S1 Domain Accelerates α-Synuclein Phosphorylation and Aggregation in Cellular Models of Synucleinopathy.

The 2019 novel coronavirus disease (COVID-19) is an infectious disease that began to spread globally since 2019. Some COVID-19 patients have neurological complications, such as olfactory disorders and movement disorders, which coincide with the symptoms of Parkinson's disease (PD). Increasing imaging and autopsy evidence supports that the density of dopaminergic neurons in the nigrostriatal pathway is damaged in some COVID-19 patients. However, the underlying mechanism that causes PD-like symptoms remains unclear. PD is an age-related neurodegenerative disease with Lewy bodies (LBs) as its histopathologic feature. The main component of LBs is abnormally aggregated α-synuclein (α-syn). The prion-like propagation of α-syn aggregates plays a key role in the onset and progression of PD. The spike protein (S protein) of SARS-CoV-2 is a heparin-binding protein that mediates the entry of the virus into host cells. Here we found that the S1 domain interacts with α-syn and promotes α-syn aggregation. The S1 domain induces mitochondrial dysfunction, oxidative stress, and cytotoxicity. The S1-seeded α-syn fibrils show enhanced seeding activity and induce synaptic damage and cytotoxicity. Thus, the S1 domain of SARS-CoV-2 promotes the aggregation of α-syn in the cellular model of synucleinopathy and may contribute to the pathogenesis of PD.

The yeast protein Ure2p triggers Tau pathology in a mouse model of tauopathy.

The molecular mechanisms that trigger Tau aggregation in Alzheimer's disease (AD) remain elusive. Fungi, especially Saccharomyces cerevisiae (S. cerevisiae), can be found in brain samples from patients with AD. Here, we show that the yeast protein Ure2p from S. cerevisiae interacts with Tau and facilitates its aggregation. The Ure2p-seeded Tau fibrils are more potent in seeding Tau and causing neurotoxicity in vitro. When injected into the hippocampus of Tau P301S transgenic mice, the Ure2p-seeded Tau fibrils show enhanced seeding activity compared with pure Tau fibrils. Strikingly, intracranial injection of Ure2p fibrils promotes the aggregation of Tau and cognitive impairment in Tau P301S mice. Furthermore, intranasal infection of S. cerevisiae in the nasal cavity of Tau P301S mice accelerates the aggregation of Tau. Together, these observations indicate that the yeast protein Ure2p initiates Tau pathology. Our results provide a conceptual advance that non-mammalian prions may cross-seed mammalian prion-like proteins.

Soluble TREM2 ameliorates tau phosphorylation and cognitive deficits through activating transgelin-2 in Alzheimer's disease.

Triggering receptor expressed on myeloid cells 2 (TREM2) is a transmembrane protein that is predominantly expressed by microglia in the brain. The proteolytic shedding of TREM2 results in the release of soluble TREM2 (sTREM2), which is increased in the cerebrospinal fluid of patients with Alzheimer's disease (AD). It remains unknown whether sTREM2 regulates the pathogenesis of AD. Here we identified transgelin-2 (TG2) expressed on neurons as the receptor for sTREM2. The microglia-derived sTREM2 binds to TG2, induces RhoA phosphorylation at S188, and deactivates the RhoA-ROCK-GSK3ß pathway, ameliorating tau phosphorylation. The sTREM2 (77-89) fragment, which is the minimal active sequence of sTREM2 to activate TG2, mimics the inhibitory effect of sTREM2 on tau phosphorylation. Overexpression of sTREM2 or administration of the active peptide rescues tau pathology and behavioral defects in the tau P301S transgenic mice. Together, these findings demonstrate that the sTREM2-TG2 interaction mediates the cross-talk between microglia and neurons. sTREM2 and its active peptide may be a potential therapeutic intervention for tauopathies including AD.