Cytoplasmic Accumulation Research Articles

Aberrant accumulation of phosphorylated BRCA1 in brainstem-type and cortical-type Lewy bodies in Lewy body disease.

BRCA1 plays important roles in several biological events during the DNA damage response (DDR). We aimed to determine whether cytoplasmic accumulation of BRCA1 or its phosphorylated form, pBRCA1, is specific to cytoplasmic inclusions in tauopathies, or if it also occurs in α-synuclein-positive inclusions in Lewy body disease (LBD). Using brain tissue from pure LBD, LBD with Alzheimer disease (AD) co-pathology (LBD-AD), and control cases, the immunohistochemical distributions of BRCA1, pBRCA1, its binding partner BARD1, and 53BP1 were examined. The results showed that pBRCA1 (Ser1423) and BARD1 accumulated in brainstem-type Lewy bodies (LBs), whereas only pBRCA1 (Ser1423) was present in cortical-type LBs. There was no significant difference in the frequency of pBRCA1 (Ser1423)-positive LBs between the pure LBD and LBD-AD cases. pBRCA1 (Ser1423) was minimally detected in neuronal nuclei in controls and was absent in neuronal nuclei in LBD cases. In control and LBD cases, 53BP1-immunoreactive deposits were present in the neuronal nuclei. Thus, DDR dysfunction due to cytoplasmic sequestration of pBRCA1 (Ser1423) may play a role in LBD pathogenesis. Additionally, the selective accumulation of BARD1 in brainstem-type LBs, but not cortical-type LBs, points to distinct mechanisms in the formation of these inclusion types, offering further insights into LBD pathology.

Amyotrophic lateral sclerosis caused by FUS mutations: advances with broad implications.

Autosomal dominant mutations in the gene encoding the DNA and RNA binding protein FUS are a cause of amyotrophic lateral sclerosis (ALS), and about 0·3-0·9% of patients with ALS are FUS mutation carriers. FUS-mutation-associated ALS (FUS-ALS) is characterised by early onset and rapid progression, compared with other forms of ALS. However, different pathogenic mutations in FUS can result in markedly different age at symptom onset and rate of disease progression. Most FUS mutations disrupt its nuclear localisation, leading to its cytoplasmic accumulation in the CNS. FUS also forms inclusions in around 5% of patients with the related neurodegenerative condition frontotemporal dementia. However, there are key differences between the two diseases at the genetic and neuropathological level, which suggest distinct pathogenic processes. Experimental models have uncovered potential pathogenic mechanisms in FUS-ALS and informed therapeutic strategies that are currently in development, including the silencing of FUS expression using an intrathecally administered antisense oligonucleotide.

AFP shields hepatocellular carcinoma from macrophage phagocytosis by regulating HuR-mediated CD47 translocation in cellular membrane.

Alpha fetoprotein(AFP) overexpression connecting with macrophage dysfunction remain poorly defined. In this study, explore AFP regulates macrophage immunomodulation in hepatocellular carcinoma(HCC) through comprehensive in vitro and in vivo studies. Immunohistochemical and immunofluorescence staining was used to analyze the relativity of AFP and cellular membrane CD47 expression in clinical 30 HCC tissues, and the expression of AFP and CD47 in HCC cells. The intelligent living-cell high-throughput imaging analyzer was applied to dynamically track and image of macrophages to phagocytize HCC cells. The effect of AFP on regulating the level of CD47 in cellular membrane and growth of tumor in vivo was performed by animal experiment. The association of AFP and CD47 in HCC cells was detected by single cell analysis. The present results indicated that AFP upregulated the localization of CD47 on the HCC cell surface. CD47 overexpression stimulates HCC to escape immune surveillance by transmitting "don't eat me" signals to macrophages, lead to inhibit macrophage to phagocytize HCC cells. Mechanistically, the results demonstrated that AFP enhanced CD47 membrane translocation by interacting with Hu-Antigen R(HuR), an RNA-binding protein that regulates mRNA stability and translation. AFP alters the subcellular distribution of HuR, increasing its cytoplasmic accumulation and binding to CD47 transcript. AFP enhanced CD47 membrane translocation by interacting with HuR. These findings proved that AFP could inhibit macrophage to phagocytize HCC cells by upregulating the localization of CD47 on the HCC cell surface. Combination of AFP with CD47 blockade may be a potential therapeutic strategy for HCC treatment.

NAT10 induces mitochondrial dysfunction in lung epithelial cells by acetylating HMGB1 to exacerbate Pseudomonas aeruginosa-induced acute lung injury.

Pseudomonas aeruginosa (PA) is a major pathogen that causes pneumonia and acute lung injury (ALI). Dysregulated NAT10 expression is associated with inflammatory and infectious diseases, but its role in PA-induced ALI remains unclear. A mouse pneumonia model was established by intratracheal injection of PA, and lentivirus-mediated NAT10 interference and HMGB1 overexpression vectors were administered via the tail vein. Lung mechanics, protein content, total cell counts, neutrophil counts, inflammatory factor levels in bronchoalveolar lavage fluid (BALF), and lung bacterial load were assessed 24 h after PA injection. HE staining was performed to evaluate lung structural damage. Intracellular oxidative stress levels in mouse lung epithelial cells (TC-1 cells) were measured by detecting ROS and MDA levels. Mitochondrial function was analyzed by testing the mitochondrial membrane potential, cytoplasmic accumulation of cytochrome C, mtDNA copy number, and ATP production. An N4-acetylcytidine (ac4C)-RNA immunoprecipitation assay was conducted to assess the ac4C level of HMGB1 mRNA. NAT10 deficiency hindered PA infection-induced increases in immune cell infiltration, inflammatory factor levels, bacterial load, and ultimately lung structural and functional damage. However, upregulation of HMGB1 effectively antagonized the protective effects of NAT10 silencing in vivo. NAT10 knockdown suppressed PA-induced oxidative stress, mitochondrial dysfunction, and apoptosis in vitro. Whereas, HMGB1 overexpression reversed the inhibitory effects of NAT10 downregulation on PA-induced TC-1 cell injury. Mechanistically, as an acetyltransferase, NAT10 enhanced HMGB1 mRNA stability and protein expression by promoting HMGB1 mRNA ac4C modification. NAT10 facilitated mitochondrial dysfunction in lung epithelial cells and exacerbated PA-induced ALI by promoting the N4-acetylcytidine of HMGB1 mRNA.

A cellular model of TDP-43 induces phosphorylated TDP-43 aggregation with distinct changes in solubility and autophagy dysregulation.

Amyotrophic lateral sclerosis (ALS) is an incurable neurodegenerative disease that affects neurons in the brain and spinal cord, causing loss of muscle control, and eventually leads to death. Phosphorylated transactive response DNA binding protein-43 (TDP-43) is the major pathological protein in both sporadic and familial ALS, forming cytoplasmic aggregates in over 95% of cases. Of the 10-15% of ALS cases that are familial, mutations in TDP-43 represent about 5% of those with a family history. We have developed an in vitro overexpression model by introducing three familial ALS mutations (A315T, M337V, and S379P) in the TDP-43 (TARDBP) gene which we define as 3X-TDP-43. This overexpression model TDP-43 shows deficits in autophagy flux and colocalization of TDP-43 with stress granules. We also observe a progressive shift of TDP-43 to the cytoplasm in this model. This overexpression model shows a reduction in solubility of phosphorylated TDP-43 from RIPA to urea soluble. Four glycolytic enzymes, phosphoglycerate kinase one (PGK1), aldolase A (ALDOA), enolase 1 (ENO1), and pyruvate dehydrogenase kinase 1 (PDK1) show significant time-dependent decreases in 3X-TDP-43 expressing cells. Shotgun proteomic analysis shows global changes in the importin subunit alpha-1 (KPNA2), heat shock 70 kDa protein 1A (HSPA1A), and protein disulfide-isomerase A3 (PDIA3) expression levels and coimmunoprecipitation reveals that these proteins complex with TDP-43. Overall, these results suggest that the 3X-TDP-43 model may provide new insights into pathophysiology and an avenue for drug screening in vitro for those suffering from ALS and related TDP-43 proteinopathies.

Micropeptide hSPAR regulates glutamine levels and suppresses mammary tumor growth via a TRIM21-P27KIP1-mTOR axis.

mTOR plays a pivotal role in cancer growth control upon amino acid response. Recently, CDK inhibitor P27KIP1 has been reported as a noncanonical inhibitor of mTOR signaling in MEFs, via unclear mechanisms. Here, we find that P27KIP1 degradation via E3 ligase TRIM21 is inhibited by human micropeptide hSPAR through its C-terminus (hSPAR-C), causing P27KIP1's cytoplasmic accumulation in breast cancer cells. Furthermore, hSPAR/hSPAR-C also serves as an inhibitor of glutamine transporter SLC38A2 expression and thereby decreases the cellular glutamine levels specifically in cancer cells. The resultant glutamine deprivation sequentially triggers translocation of cytoplasmic P27KIP1 to lysosomes, where P27KIP1 disrupts the Ragulator complex and suppresses mTORC1 assembly. Administration of hSPAR or hSPAR-C significantly impedes breast cancer cell proliferation and tumor growth in xenograft models. These findings define hSPAR as an intrinsic control factor for cellular glutamine levels and as a novel tumor suppressor inhibiting mTORC1 assembly.

Antagonistic roles by the conserved nuclear poly(A)-binding proteins PABPN1 and ZC3H14 in nuclear RNA surveillance.

Most eukaryotic genomes are transcribed pervasively, thereby producing an array of long non-coding RNAs (lncRNAs) in addition to protein-coding mRNAs. A large fraction of these lncRNAs is targeted by polyadenylation-dependent decay via the poly(A)-binding protein nuclear 1 (PABPN1) and the RNA exosome. Yet, how PABPN1 contributes to nuclear RNA surveillance by facilitating lncRNA turnover by the RNA exosome remains largely unclear. Here, we show that PABPN1 is important for the nuclear retention of polyadenylated lncRNAs, such that PABPN1 loss of function allows target lncRNAs to evade nuclear decay, leading to cytoplasmic accumulation. Interestingly, we found that another nuclear PABP, ZC3H14, functions antagonistically to PABPN1 and the poly(A)-tail exosome targeting (PAXT) connection in the control of nuclear lncRNA turnover. Collectively, our findings disclose the critical interplay between two conserved nuclear PABPs, PABPN1 and ZC3H14, in RNA surveillance via the control of nuclear RNA export.

Microglial double stranded DNA accumulation induced by DNase II deficiency drives neuroinflammation and neurodegeneration

BackgroundDeoxyribonuclease 2 (DNase II) is pivotal in the clearance of cytoplasmic double stranded DNA (dsDNA). Its deficiency incurs DNA accumulation in cytoplasm, which is a hallmark of multiple neurodegenerative diseases. Our previous study showed that neuronal DNase II deficiency drove tau hyperphosphorylation and neurodegeneration (Li et al., Transl Neurodegener 13:39, 2024). Although it has been verified that DNase II participates in type I interferons (IFN-I) mediated autoinflammation and senescence in peripheral systems, the role of microglial DNase II in neuroinflammation and neurodegenerative diseases such as Alzheimer’s disease (AD) is still unknown.MethodsThe levels of microglial DNase II in triple transgenic AD mice (3xTg-AD) were measured by immunohistochemistry. The cognitive performance of microglial DNase II deficient WT and AD mice was determined using the Morris water maze test, Y-maze test, novel object recognition test and open filed test. To investigate the impact of microglial DNase II deficiency on microglial morphology, cyclic GMP-AMP synthase (cGAS)-stimulator of interferon genes (STING) pathway and IFN-I pathway, neuroinflammation, synapses loss, amyloid pathology and tauopathy, the levels of cGAS-STING and IFN-I pathway related protein, gliosis and proinflammatory cytokines, synaptic protein, complement protein, Aβ levels, phosphorylated tau in the brains of the microglial DNase II deficient WT and AD mice were evaluated by immunolabeling, immunoblotting, q-PCR or ELISA.ResultsWe found that the levels of DNase II were significantly decreased in the microglia of 3xTg-AD mice. Microglial DNase II deficiency altered microglial morphology and transcriptional signatures, activated the cGAS-STING and IFN-I pathway, initiated neuroinflammation, led to synapse loss via complement-dependent pathway, increased Aβ levels and tauopathy, and induced cognitive decline.ConclusionsOur study shows the effect of microglial DNase II deficiency and cytoplasmic accumulated dsDNA on neuroinflammation, and reveals the initiatory mechanism of AD pathology, suggesting that DNase II is a potential target for neurodegenerative diseases.

Aberrant splicing in Huntington's disease accompanies disrupted TDP-43 activity and altered m6A RNA modification.

Huntington's disease (HD) is caused by a CAG repeat expansion in the HTT gene, leading to altered gene expression. However, the mechanisms leading to disrupted RNA processing in HD remain unclear. Here we identify TDP-43 and the N6-methyladenosine (m6A) writer protein METTL3 to be upstream regulators of exon skipping in multiple HD systems. Disrupted nuclear localization of TDP-43 and cytoplasmic accumulation of phosphorylated TDP-43 occurs in HD mouse and human brains, with TDP-43 also co-localizing with HTT nuclear aggregate-like bodies distinct from mutant HTT inclusions. The binding of TDP-43 onto RNAs encoding HD-associated differentially expressed and aberrantly spliced genes is decreased. Finally, m6A RNA modification is reduced on RNAs abnormally expressed in the striatum of HD R6/2 mouse brain, including at clustered sites adjacent to TDP-43 binding sites. Our evidence supports TDP-43 loss of function coupled with altered m6A modification as a mechanism underlying alternative splicing in HD.

Virus-mediated delivery of single-chain antibody targeting TDP-43 protects against neuropathology, cognitive impairment and motor deficit caused by chronic cerebral hypoperfusion.

Chronic cerebral hypoperfusion induced by permanent unilateral common carotid artery occlusion in mice was recently found to induce an age-dependent formation of insoluble cytoplasmic TDP-43 aggregates reminiscent of pathological changes found in human vascular dementia. In this model, the gradual deregulation of TDP-43 homeostasis in cortical neurons was associated with marked cognitive and motor deficits. To target the TDP-43-mediated toxicity in this model, we generated an adeno-associated virus vector encoding a single-chain antibody against TDP-43, called scFv-E6, designed for pan-neuronal transduction following intravenous administration. Injected prior to brain hypoperfusion, this tonic virus-mediated delivery of the scFv-E6 antibody reduced formation of cytoplasmic TDP-43 aggregates in cortical neurons, boosted levels of autophagy markers and attenuated microgliosis. Moreover, the novel object recognition and grip strength tests revealed that neuronal expression of scFv-E6 prevented the cognitive impairment and loss of motor performance caused by two-months of cerebral hypoperfusion. The robust protective effects of scFv-E6 antibody in this model suggest a key role of TDP-43 in neuronal damage and symptom phenotypes caused by chronic cerebral hypoperfusion. Accordingly, TDP-43 should be considered as a new therapeutic target in drug development, including antibody approaches, for treatment of vascular dementia.

Arsenic Exposure Induces Neural Cells Senescence and Abnormal Lipid Droplet Accumulation Leading to Social Memory Impairment in Mice.

The long-term harmful effects of arsenic exposure remain one of the important public health issues. The effects of arsenic exposure on the central nervous system, particularly concerning brain structure and function, have been garnering increasing attention. Hence, the aim of this study was to investigate the impact of chronic low-dose arsenic exposure on murine social memory and to elucidate the underlying molecular mechanisms. Male C57BL/6 mice at six months of age were randomly assigned to a control group and three treatment groups with different arsenic concentrations (50, 100, and 200 μg/L), with exposure durations of 30, 90, 180, and 360 days. The five-social memory test and three-chamber social memory test results indicated that chronic low-dose arsenic exposure disrupted social memory in mice. Further analysis revealed that arsenic exposure led to degeneration of neurons within the dorsal CA2 of the hippocampus (dCA2) and the lateral entorhinal cortex (LEC), which are pivotal for the modulation of social memory, and dCA2 neurons demonstrated structural disruptions and cytoplasmic fragmentation. In addition, arsenic exposure induced neurons and glial cells senescence in both dCA2 and LEC, with a particularly pronounced effect in microglia, and worse with dosage increasing of arsenic exposure, correlating with elevated expression levels of p16INK4A, ferritin light chain and the senescence-associated secretory factors TNF-α and IL-1β, and reduced expression of Lamin B1. Moreover, arsenic exposure triggered substantial cytoplasmic lipid droplets accumulation in neurons, astrocytes and microglia, with an upregulation of PLIN2 expression, a protein associated with lipid droplet formation in astrocytes. At the same time, the aberrant accumulation of lipid droplets further aggravated the astrocytes and microglia aging, especially microglia. Additionally, correlation analysis revealed that social memory impairment was negatively correlated with nerve cell senescence and lipid accumulation. Our findings suggest that arsenic exposure induced cellular functional abnormalities by triggering cellular senescence and the accumulation of lipid droplets, thereby exacerbated neuronal degeneration and result in impaired social memory in mice.

The potential mechanism maintaining transactive response DNA binding protein 43 kDa in the mouse stroke model.

The disruption of transactive response DNA binding protein 43 kDa (TDP-43) shuttling leads to the depletion of nuclear localization and the cytoplasmic accumulation of TDP-43. We aimed to evaluate the mechanism underlying the behavior of TDP-43 in ischemic stroke. Adult male C57BL/6 J mice were subjected to 30 or 60 min of transient middle cerebral artery occlusion (tMCAO), and examined at 1, 6, and 24 h post reperfusion. Immunostaining was used to evaluate the expression of TDP-43, G3BP1, HDAC6, and RAD23B. The total and cytoplasmic number of TDP-43-positive cells increased compared with sham operation group and peaked at 6 h post reperfusion after tMCAO. The elevated expression of G3BP1 protein peaked at 6 h after reperfusion and decreased at 24 h after reperfusion in ischemic mice brains. We also observed an increase of expression level of HDAC6 and the number of RAD23B-positive cells increased after tMCAO. RAD23B was colocalized with TDP-43 24 h after tMCAO. We proposed that the formation of stress granules might be involved in the mislocalization of TDP-43, based on an evaluation of G3BP1 and HDAC6. Subsequently, RAD23B, may also contribute to the downstream degradation of mislocalized TDP-43 in mice tMCAO model.

Rapamycin Abrogates Aggregation of Human α-Synuclein Expressed in Fission Yeast via an Autophagy-Independent Mechanism.

Aggregation of alpha-synuclein (α-Syn) is implicated in the pathogenesis of several neurodegenerative disorders, such as Parkinson's disease and Dementia with Lewy bodies, collectively termed synucleinopathies. Thus, tremendous efforts are being made to develop strategies to prevent or inhibit α-Syn aggregation. Here, we genetically engineered fission yeast to express human α-Syn C-terminally fused to green fluorescent protein (GFP) at low and high levels. α-Syn was localized at the cell tips and septa at low-level expression. At high-level expression, α-Syn was observed to form cytoplasmic aggregates. Notably, rapamycin, a natural product that allosterically inhibits the mammalian target of rapamycin (mTOR) by forming a complex with FKBP12, and Torin1, a synthetic mTOR inhibitor that blocks ATP binding to mTOR, markedly reduced the number of cells harboring α-Syn aggregates. These mTOR inhibitors abrogate α-Syn aggregation without affecting α-Syn expression levels. Rapamycin, but not Torin1, failed to reduce α-Syn aggregation in the deletion cells of fkh1+, encoding FKBP12, indicating the requirement of FKBP12 for rapamycin-mediated inhibition of α-Syn aggregation. Importantly, the effect of rapamycin was also observed in the cells lacking atg1+, a key regulator of autophagy. Collectively, rapamycin abrogates human α-Syn aggregation expressed in fission yeast via an autophagy-independent mechanism mediated by FKBP12.

Neuronal cytoplasmic inclusion bodies in the brain of Lagotto Romagnolo dogs: A qualitative and quantitative histologic evaluation.

Four neurologic diseases affect dogs of the Lagotto Romagnolo (LR) breed, namely benign familial juvenile epilepsy (BFJE), vacuolar storage disease, and 2 forms of cerebellar cortical degeneration. Intraneuronal inclusion bodies in cerebellar Purkinje cells were first described in the BFJE phenotype. Upon further characterization of these diseases, similar inclusions were also noted in the brain of LRs used as controls. This study investigated the clinical, histologic, and electron microscopic findings in 23 LRs to determine the nature of their neuronal inclusions and whether the presence, distribution, or number of inclusions is associated with neurologic signs. Electron microscopy of the inclusions revealed a cytoplasmic aggregate without a limiting membrane. The inclusions appeared proteinaceous on histochemical staining and positive on phosphotungstic-acid-hematoxylin (PTAH) stain for proteins rich in basic amino acids. Markers of commonly known proteinopathies of humans (ubiquitin, p62, LC3, α-synuclein, and β-amyloid) were not detected in the inclusions when assessed by immunohistochemistry. The overall presence of inclusion bodies was not significantly associated with the dog's neurologic status. The results show an association between inclusions in the cerebral cortex and an absence of clinical neurologic disease in LRs. There was no significant difference in the quantitative inclusion body burden when compared in LRs with or without neurologic signs. Although PTAH-positive proteinaceous neuronal inclusions are a common finding in LRs regardless of neurologic signs, these inclusions may be a protective response when present in the cerebral cortex.

CK1δ/ε-mediated TDP-43 phosphorylation contributes to early motor neuron disease toxicity in amyotrophic lateral sclerosis

Hyperphosphorylated TDP-43 aggregates in the cytoplasm of motor neurons is a neuropathological signature of amyotrophic lateral sclerosis (ALS). These aggregates have been proposed to possess a toxic disease driving role in ALS pathogenesis and progression, however, the contribution of phosphorylation to TDP-43 aggregation and ALS disease mechanisms remains poorly understood. We’ve previously shown that CK1δ and CK1ε phosphorylate TDP-43 at disease relevant sites, and that genetic reduction and chemical inhibition could reduce phosphorylated TDP-43 (pTDP-43) levels in cellular models. In this study, we advanced our findings into the hTDP-43-ΔNLS in vivo mouse model of ALS and TDP-43 proteinopathy. This mouse model possesses robust disease-relevant features of ALS, including TDP-43 nuclear depletion, cytoplasmic pTDP-43 accumulation, motor behavior deficits, and shortened survival. We tested the effect of homozygous genetic deletion of Csnk1e in the hTDP-43-ΔNLS mouse model and observed a delay in the formation of pTDP-43 without significant ultimate rescue of TDP-43 proteinopathy or disease progression. Homozygous genetic deletion of Csnk1d is lethal in mice, and we were unable to test the role of CK1δ alone. We then targeted both CK1δ and CK1ε kinases by way of CK1δ/ε-selective PF-05236216 inhibitor in the hTDP-43-ΔNLS mouse model, reasoning that inhibiting CK1ε alone would be insufficient as shown by our Csnk1e knockout mouse model study. Treated mice demonstrated reduced TDP-43 phosphorylation, lowered Nf-L levels, and improved survival in the intermediate stages. The soluble TDP-43 may have been more amenable to the inhibitor treatment than insoluble TDP-43. However, the treatments did not result in improved functional measurements or in overall survival. Our results demonstrate that phosphorylation contributes to neuronal toxicity and suggest CK1δ/ε inhibition in combination with other therapies targeting TDP-43 pathology could potentially provide therapeutic benefit in ALS.

Cytoplasmic Aggregates of Splicing Factor Proline-Glutamine Rich Disrupt Nucleocytoplasmic Transport and Induce Persistent Stress Granules.

Splicing factor proline-glutamine rich (SFPQ), a multifunctional RNA-binding protein (RBP), shows cytoplasmic colocalisation with stress granule (SG) markers; however, the causative relationship and mechanism underlying this coalescence of SFPQ aggregates and SGs remain unclear. In this study, we demonstrate that SFPQ lacking its nuclear localisation sequence spontaneously forms cytoplasmic aggregates that abnormally incorporate immature RNA and induce persistent SGs. mRNA profiling showed that SFPQ mislocalisation induced extensive changes in RNA processing, with a subset of alternatively spliced transcripts associated with nucleocytoplasmic transport. Notably, these altered transporters were sequestered into SFPQ aggregates, constituting aberrant protein-RNA complexes. Importantly, suppression of SG nucleation could not block cytoplasmic SFPQ aggregation with immature RNA and nucleocytoplasmic transporters, both of which, however, were moderately ameliorated by the inhibition of alternative splicing or nuclear export. Our results unveil the physiopathological role and mechanism for mislocalised SFPQ in the RNA metabolism, nucleocytoplasmic transport and pathological SGs.

Fatty Acid Synthase (FASN) Inhibitors Suppress Metformin-Induced Fat Accumulation and Apoptosis in H4IIE Hepatocellular Carcinoma Cells.

We previously reported that metformin, a widely prescribed antidiabetic drug, induces the accumulation of triglyceride (TG) together with the apoptotic death of H4IIE via AMP-activated protein kinase (AMPK) in hepatocellular carcinoma (HCC) cells. However, the effect of cytoplasmic fat accumulation on the growth of HCCs remains controversial. Herein, we investigated the effect of fatty acid synthase (FASN) inhibitors on the basal- or metformin-induced changes including the content of cytoplasmic TG and the viability of HCC cells. Cerulenin and C75, inhibitors of FASN, did not significantly affect the basal TG content but dose-dependently suppressed the metformin-induced increase in the cytoplasmic TG content. Metformin-induced apoptosis of H4IIE cells was also significantly reduced by cerulenin and C75. Metformin enhanced the generation of reactive oxygen species which was suppressed by adding cerulenin or T75. Cerulenin also stimulated cell migration, which was suppressed by metformin. However, the degree of suppressive effect of metformin on TG synthesis, apoptosis, and cell migration was much more prominent by the inhibition of AMPK by compound C than cerulenin. In conclusion, our study found that excess fat accumulation is responsible for the apoptosis of H4IIE HCC cells and is informative for designing anti-tumor reagents, especially in HCC.

Ferredoxin 2 is critical for tumor suppression and lipid homeostasis but dispensable for embryonic development.

Ferredoxin 1 and 2 (FDX1/2) constitute an evolutionarily conserved FDX family of iron-sulfur cluster (ISC) containing proteins. FDX1/2 are cognate substrates of ferredoxin reductase (FDXR) and serve as conduits for electron transfer from NADPH to a set of proteins involved in biogenesis of steroids, hemes, ISC and lipoylated proteins. Recently, we showed that Fdx1 is essential for embryonic development and lipid homeostasis. To explore the physiological role of FDX2, we generated Fdx2-deficient mice. Interestingly, we found that unlike Fdx1-null embryos, which were dead at embryonic day 10.5 to 13.5, Fdx2-null mice were viable. We also found that both Fdx2-null and Fdx2-heterozygous mice had a short lifespan and were susceptible to spontaneous tumors and steatohepatitis. Moreover, we found that FDX2-deficiency increased whereas overexpression of FDX2 decreased cytoplasmic accumulation of lipid droplets. Consistently, we found that FDX2 deficiency led to accumulation of cholesterol and triglycerides. Mechanistically, we found that FDX2 deficiency suppressed expression of cholesterol transporter ABCA1 and activated master lipid transcription regulators SREBP1/2, thus leading to altered lipid metabolism. Untargeted lipidomic analysis showed that FDX2 deficiency led to altered biosynthesis of various lipid classes, including cardiolipins, cholesterol, ceramides, triglycerides, and fatty acids. In summary, our findings underscore an indispensable role of FDX2 in tumor suppression and lipid homeostasis at both cellular and organismal levels without being a prerequisite for embryonic development.

Novel fluid biomarker detects TDP‐43 loss of function in individuals at elevated risk of Alzheimer’s disease

AbstractBackgroundTDP‐43 nuclear clearance and cytoplasmic aggregation occur in an estimated 30‐60% of cases of Alzheimer’s disease (AD), but this pathology can currently only be established at autopsy. Nuclear clearance of TDP‐43 leads to inclusion of cryptic exons in pre‐mRNA, some of which are spliced in‐frame and translated into proteins carrying novel cryptic exon‐encoded epitopes. We developed a Meso Scale Discovery (MSD) ELISA against the TDP‐43‐associated cryptic neoepitope within the HDGFL2 protein and found significantly elevated levels of this cryptic neoepitope in biofluids of presymptomatic ALS‐FTD (Irwin et al., Nature Medicine, 2024), indicating that cryptic HDGFL2 could serve as a biomarker for loss of TDP‐43 function. Here, we used our MSD assay to analyze TDP‐43 dysfunction in individuals at increased risk of AD due to family history.MethodA total of 266 CSF samples from 107 individuals (average age = 66.6 years, 40.8% phosphorylated‐tau181/Aβ42 ratio positive) in the Wisconsin Registry for Alzheimer’s Prevention (WRAP) were analyzed for cryptic HDGFL2 by MSD.ResultSixty‐eight samples (25.6%) showed elevated levels of cryptic HDGFL2. These cryptic HDGFL2‐positive samples had a significantly higher proportion of phosphorylated‐tau/Aβ42‐positive cases (64.1%, p = 0.001). Twenty‐nine of the 107 individuals (27.1%) analyzed were positive for cryptic HDGFL2 at least once longitudinally. In 10 of the 29 (34.5%) individuals, cryptic HDGFL2 levels increased across each longitudinal sample collection, while they decreased longitudinally in 13 (44.8%). In six (20.7%), cryptic HDGFL2 levels displayed a peak during the longitudinal sampling. Twenty‐three of the 29 individuals had associated cognitive data. Eight individuals (34.8%) were from the group with increasing cryptic HDGFL2 levels, 11 (47.8%) from the group with decreasing levels, and four (17.4%) from the group displaying a peak. Each of these individuals was cognitively normal, besides one person in the decreasing group with Mild Cognitive Impairment (MCI). In this individual, cryptic HDGFL2 levels were already elevated prior to MCI diagnosis.ConclusionThe proportion of individuals with elevated cryptic HDGFL2 levels corresponds with the expected percentage of AD cases with co‐pathology of TDP‐43. Our findings of TDP‐43 dysfunction in many cognitively normal individuals suggest that TDP‐43 loss of function is an early event in disease that can precede symptom development.

ALS-linked mutant TDP-43 in oligodendrocytes induces oligodendrocyte damage and exacerbates motor dysfunction in mice

Nuclear clearance and cytoplasmic aggregation of TAR DNA-binding protein of 43 kDa (TDP-43) are pathological hallmarks of amyotrophic lateral sclerosis (ALS) and its pathogenic mechanism is mediated by both loss-of-function and gain-of-toxicity of TDP-43. However, the role of TDP-43 gain-of-toxicity in oligodendrocytes remains unclear. To investigate the impact of excess TDP-43 on oligodendrocytes, we established transgenic mice overexpressing the ALS-linked mutant TDP-43M337V in oligodendrocytes through crossbreeding with Mbp-Cre mice. Two-step crossbreeding of floxed TDP-43M337V and Mbp-Cre mice resulted in the heterozygous low-level systemic expression of TDP-43M337V with (Cre-positive) or without (Cre-negative) oligodendrocyte-specific overexpression of TDP-43M337V. Although Cre-negative mice also exhibit subtle motor dysfunction, TDP-43M337V overexpression in oligodendrocytes aggravated clasping signs and gait disturbance accompanied by myelin pallor in the corpus callosum and white matter of the lumbar spinal cord in Cre-positive mice. RNA sequencing analysis of oligodendrocyte lineage cells isolated from whole brains of 12-month-old transgenic mice revealed downregulation of myelinating oligodendrocyte marker genes and cholesterol-related genes crucial for myelination, along with marked upregulation of apoptotic pathway genes. Immunofluorescence staining showed cleaved caspase 3–positive apoptotic oligodendrocytes surrounded by activated microglia and astrocytes in aged transgenic mice. Collectively, our findings demonstrate that an excess amount of ALS-linked mutant TDP-43 expression in oligodendrocytes exacerbates motor dysfunction in mice, likely through oligodendrocyte dysfunction and neuroinflammation. Therefore, targeting oligodendrocyte protection, particularly through ameliorating TDP-43 pathology, could represent a potential therapeutic approach for ALS.