Unconventional Source of Neurotoxic Protein Aggregation from Organelle Off-Target Bax∆2 in Alzheimer's Disease.

Protein aggregates are a hallmark of Alzheimer's disease (AD). Extensive studies have focused on ß-amyloid plaques and Tau tangles. Here, we illustrate a novel source of protein aggregates in AD neurons from organelle off-target proteins. Bax is a mitochondrial pore-forming pro-death protein. What happens to Bax if it fails to target mitochondria? We previously showed that a mitochondrial target-deficient alternatively spliced variant, Bax∆2, formed large cytosolic protein aggregates and triggered caspase 8-mediated cell death. Bax∆2 protein levels were low in most normal organs and the proteins were quickly degraded in cancer. Here, we found that 85% of AD patients had Bax∆2 required alternative splicing. Increased Bax∆2 proteins were mostly accumulated in neurons of AD-susceptible brain regions. Intracellularly, Bax∆2 aggregates distributed independently of Tau tangles. Interestingly, Bax∆2 aggregates triggered the formation of stress granules (SGs), a large protein-RNA complex involved in AD pathogenesis. Although the functional domains required for aggregation and cell death are the same as in cancer cells, Bax∆2 relied on SGs, not caspase 8, for neuronal cell death. These results imply that the aggregation of organelle off-target proteins, such as Bax∆2, broadens the scope of traditional AD pathogenic proteins that contribute to the neuronal stress responses and AD pathogenesis.

Expression profile of the proapoptotic protein Bax in the human brain.

Bax is a well-known universal proapoptotic protein. Bax protein is detected in almost all human organs, and its expression levels can be correlated with disease progression and therapeutic efficacy in certain settings. Interestingly, increasing evidence has shown that mature neuronal cell death is often not typical apoptosis. Most results on the expression of Bax proteins (predominantly Baxα) in the human brain come from disease-oriented studies, and the data on Bax protein expression in the normal brain are limited and lack consistency due to many variable factors. Here, we analyzed Bax RNA and protein expression data from multiple databases and performed immunostaining of over 80 samples from 25 healthy subjects across 7 different brain regions. We found that Bax protein expression was heterogeneous across brain regions and individual subjects. Both neurons and glial cells, such as astrocytes, could be Bax positive, but Bax positivity appeared to be highly selective, even within the same cell type in the same region. Furthermore, Bax proteins could be localized in the cytosol (evenly spread or concentrated to one region), nucleus or nucleolus depending on the cell type. Such variation and distribution in Bax expression suggest that Bax may function differently in the human brain than in other organs.

BAD-mediated neuronal apoptosis and neuroinflammation contribute to Alzheimer's disease pathology.

Alzheimer's disease (AD) is the most common progressive neurodegenerative disease. However, the underlying molecular mechanism is incompletely understood. Here we report that the pro-apoptotic protein BAD as a key regulator for neuronal apoptosis, neuroinflammation and Aß clearance in AD. BAD pro-apoptotic activity is significantly increased in neurons of AD patients and 5XFAD mice. Conversely, genetic disruption of Bad alleles restores spatial learning and memory deficits in 5XFAD mice. Mechanistically, phosphorylation and inactivation of BAD by neurotropic factor-activated Akt is abrogated in neurons under AD condition. Through reactive oxygen species (ROS)-oxidized mitochondrial DNA (mtDNA) axis, BAD also promotes microglial NLRP3 inflammasome activation, thereby skewing microglia toward neuroinflammatory microglia to inhibit microglial phagocytosis of Aß in AD mice. Our results support a model in which BAD contributes to AD pathologies by driving neuronal apoptosis and neuroinflammation but suppressing microglial phagocytosis of Aß, suggesting that BAD is a potential therapeutic target for AD.

BAD inactivation exacerbates rheumatoid arthritis pathology by promoting survival of sublining macrophages.

The resistance of synovial sublining macrophages to apoptosis has a crucial role in joint inflammation and destruction in rheumatoid arthritis (RA). However, the underlying mechanism is incompletely understood. Here we report that inactivation of the pro-apoptotic BCL-2 family protein BAD is essential for survival of synovial sublining macrophage in RA. Genetic disruption of Bad leads to more severe joint inflammation and cartilage and bone damage with reduced apoptosis of synovial sublining macrophages in collagen-induced arthritis (CIA) and TNFα transgenic (TNF-Tg) mouse models. Conversely, Bad3SA/3SA mice, in which BAD can no longer be inactivated by phosphorylation, are protected from collagen-induced arthritis. Mechanistically, phosphorylation-mediated inactivation of BAD specifically protects synovial sublining macrophages from apoptosis in highly inflammatory environment of arthritic joints in CIA and TNF-Tg mice, and in patients with RA, thereby contributing to RA pathology. Our findings put forward a model in which inactivation of BAD confers the apoptosis resistance on synovial sublining macrophages, thereby contributing to the development of arthritis, suggesting that BAD may be a potential therapeutic target for RA.

Ubl4A is critical for mitochondrial fusion process under nutrient deprivation stress.

Mitochondrial fusion and fission are dynamic processes regulated by the cellular microenvironment. Under nutrient starvation conditions, mitochondrial fusion is strengthened for energy conservation. We have previously shown that newborns of Ubl4A-deficient mice were more sensitive to starvation stress with a higher rate of mortality than their wild-type littermates. Ubl4A binds with the actin-related protein Arp2/3 complex to synergize the actin branching process. Here, we showed that deficiency in Ubl4A resulted in mitochondrial fragmentation and apoptosis. A defect in the fusion process was the main cause of the mitochondrial fragmentation and resulted from a shortage of primed Arp2/3 complex pool around the mitochondria in the Ubl4A-deficient cells compared to the wild-type cells. As a result, the mitochondrial fusion process was not undertaken quickly enough to sustain starvation stress-induced cell death. Consequently, fragmented mitochondria lost their membrane integrity and ROS was accumulated to trigger caspase 9-dependent apoptosis before autophagic rescue. Furthermore, the wild-type Ubl4A, but not the Arp2/3-binding deficient mutant, could rescue the starvation-induced mitochondrial fragmentation phenotype. These results suggest that Ubl4A promotes the mitochondrial fusion process via Arp2/3 complex during the initial response to nutrient deprivation for cell survival.

A Structural Model for Bax∆2-Mediated Activation of Caspase 8-Dependent Apoptosis.

Bax∆2 is a pro-apoptotic anti-tumor protein in the Bax family. While most of the Bax family causes cell death by targeting mitochondria, Bax∆2 forms cytosolic aggregates and activates caspase 8-dependent cell death. We previously showed that the Bax∆2 helix α9 is critical for caspase 8 recruitment. However, the interaction between these two proteins at the structural level is unknown. In this in silico study, we performed molecular dynamics (MD) simulations and protein-protein docking on Bax∆2 variants. The results suggest that the Bax∆2 variants have different stable states. Mutating the Baxα mitochondria-targeting signal [L26P/L27P] appears to introduce a kink into helix α1. Protein-protein docking suggests that helices α9 of both wild-type Bax∆2 and Bax∆2 caspase 8 binding-deficient mutant [L164P] can fit in the same caspase 8 binding site, but the mutant is unable to fit as well as wild-type Bax∆2. Together, these data point to a structural basis for explaining Bax∆2 function in caspase 8-dependent cell death.

Immunohistochemical detection of the pro-apoptotic Bax∆2 protein in human tissues.

The pro-apoptotic Bax isoform Bax∆2 was originally discovered in cancer patients with a microsatellite guanine deletion (G8 to G7). This deletion leads to an early stop codon; however, when combined with the alternative splicing of exon 2, the reading frame is restored allowing production of a full-length protein (Bax∆2). Unlike the parental Baxα, Bax∆2 triggers apoptosis through a non-mitochondrial pathway and the expression in human tissues was unknown. Here, we analyzed over 1000 tissue microarray samples from 13 different organs using immunohistochemistry. Bax∆2-positive cells were detected in all examined organs at low rates (1-5%) and mainly scattered throughout the connective tissues. Surprisingly, over 70% of normal colon samples scored high for BaxΔ2-positive staining. Only 7% of malignant colon samples scored high, with most high-grade tumors being negative. A similar pattern was observed in most organs examined. We also showed that both Baxα and Bax∆2 can co-exist in the same cells. Genotyping showed that the majority of Bax∆2-positive normal tissues contain no G7 mutation, but an unexpected high rate of G9 was observed. Although the underlying mechanism remains to be explored, the inverse correlation of Bax∆2 expression with tissue malignancy suggests that it may have a clinical implication in cancer development and treatment.

The C-terminus of Ubl4A is critical for pro-death activity and association with the Arp2/3 complex.

Ubl4A is a small ubiquitin-like protein involved in diverse cellular functions. We have shown that Ubl4A is critical for survival of the starvation-mediated cell death in vivo. The underlying mechanism for this is through interaction with the actin-related protein Arp2/3 complex and promotion of actin branching. Interestingly, "put-back" of Ubl4A to Ubl4A-deficient cells also results in cell death. Removal of the Ubl4A N-terminus significantly enhances its cytotoxicity, indicating that the pro-death activity of Ubl4A is mainly from its C-terminal region. In vitro protein pull-down assays show that the C-terminal region of Ubl4A can directly interact with the Arp2/3 complex. The single point mutation of an aspartic acid to alanine (D122A) in the Ubl4A C-terminus abolishes its ability to bind the Arp2/3 complex. This mutation also destabilizes Ubl4A proteins susceptible to protease degradation. Importantly, ectopic expression of wild-type Ubl4A can induce cell death in colon cancer cells, but such pro-death activity is diminished in the D122A mutant. These data suggest that Ubl4A C-terminus, especially D122, is critical for Ubl4A-Arp2/3 interaction and its pro-death function.

Nanopore label-free detection of single-nucleotide deletion in Baxα/BaxΔ2.

Baxα, a key tumor suppressor gene, will not be expressed correctly as a result of single nucleotide mutation in its microsatellite region; Instead, BaxΔ2, an isoform of Baxα, is often produced. In addition, lack of the exon 2 due to an alternative splicing, BaxΔ2 has the same sequence as Baxα except single base deletion from eight continuous guanines (G8) to G7. Most of the currently available methods for Bax∆2 detection are inefficient and time-consuming, and/or require the use of labels or dyes. In this work, we reported a label-free nanopore sensing strategy to differentiate between Baxα and BaxΔ2 with a DNA polymer as a molecular probe based on alternative spliced sequences. Two DNA molecules were designed to selectively detect Baxα and BaxΔ2, respectively. The method was rapid, accurate, and highly sensitive: picomolar concentrations of target nucleic acids could be detected in minutes. Our developed simple and fast nanopore-based detection strategy is not only useful for distinguishing between Baxα and Bax∆2, but also provides a useful tool for detection of other single-base mutations in genetic diagnosis.

The BH3-only protein BAD mediates TNFα cytotoxicity despite concurrent activation of IKK and NF-κB in septic shock.

The inflammatory cytokine TNFα plays a crucial role in the pathology of many inflammatory and infectious diseases. However, the mechanism underlying TNFα cytotoxicity in these diseases is incompletely understood. Here we report that the pro-apoptotic BCL-2 family member BAD mediates TNFα cytotoxicity despite concurrent activation of IKK and NF-κB in vitro by inducing apoptosis in cultured cells and in vivo by eliciting tissue damage of multiple organs and contributing to mortality in septic shock. At high doses, TNFα significantly inactivates RhoA through activation of the Src-p190GAP pathway, resulting in massive actin stress fiber destabilization, followed by substantial BAD release from the cytoskeleton to the cytosol. Under this condition, activated IKK fails to phosphorylate all cytosolic BAD, allowing translocation of non-phosphorylated BAD to mitochondria to trigger apoptosis. Polymicrobial infection utilizes the same mechanism as high-dose TNFα to elicit apoptosis-associated tissue damage of multiple organs. Consequently, loss of Bad or elimination of BAD pro-apoptotic activity protects mice from tissue damage of multiple organs and reduces mortality rates. Our results support a model in which BAD mediates TNFα cytotoxicity despite concurrent activation of the IKK-NF-κB pathway in cultured mammalian cells and in septic shock.

Detection of pro-apoptotic Bax∆2 proteins in the human cerebellum.

Bax∆2 is a pro-apoptotic protein originally discovered in colon cancer patients with high microsatellite instability. Unlike most pro-apoptotic Bax family members, Bax∆2 mediates cell death through a non-mitochondrial caspase 8-dependent pathway. In the scope of analyzing the distribution of Bax∆2 expression in human tissues, we examined a panel of human brain samples. Here, we report four cerebellar cases in which the subjects had no neurological disorder or disease documented. We found Bax∆2 positive cells scattered in all areas of the cerebellum, but most strikingly concentrated in Purkinje cell bodies and dendrites. Two out the four subjects tested had strong Bax∆2-positive staining in nearly all Purkinje cells; one was mainly negative; and one had various levels of positive staining within the same sample. Further genetic analysis of the Purkinje cell layer, collected by microdissection from two subjects, showed that the samples contained G7 and G9 Bax microsatellite mutations. Both subjects were young and had no diseases reported at the time of death. As the distribution of Bax∆2 is consistent with that known for Baxα, but in a less ubiquitous manner, these results may imply a potential function of Bax∆2 in Purkinje cells.

Label-Free Detection of DNA Mutations by Nanopore Analysis.

Cancers are caused by mutations to genes that regulate cell normal functions. The capability to rapid and reliable detection of specific target gene variations can facilitate early disease detection and diagnosis and also enables personalized treatment of cancer. Most of the currently available methods for DNA mutation detection are time-consuming and/or require the use of labels or sophisticated instruments. In this work, we reported a label-free enzymatic reaction-based nanopore sensing strategy to detect DNA mutations, including base substitution, deletion, and insertion. The method was rapid and highly sensitive with a detection limit of 4.8 nM in a 10 min electrical recording. Furthermore, the nanopore assay could differentiate among perfect match, one mismatch, and two mismatches. In addition, simulated serum samples were successfully analyzed. Our developed nanopore-based DNA mutation detection strategy should find useful application in genetic diagnosis.

BaxΔ2 sensitizes colorectal cancer cells to proteasome inhibitor-induced cell death.

Proteasome inhibitors, such as bortezomib and carfilzomib, are FDA approved for the treatment of hemopoietic cancers, but recent studies have shown their great potential for treatment of solid tumors. BaxΔ2, a unique proapoptotic Bax isoform, promotes non-mitochondrial cell death and sensitizes cancer cells to chemotherapy. However, endogenous BaxΔ2 proteins are unstable and susceptible to proteasomal degradation. Here, we screened a panel of proteasome inhibitors in colorectal cancer cells with different Bax statuses. We found that all proteasome inhibitors tested were able to block BaxΔ2 degradation without affecting the level of Baxα or Bcl-2 proteins. Among the inhibitors tested, only bortezomib and carfilzomib were able to induce differential cell death corresponding to the distinct Bax statuses. BaxΔ2-positive cells had a significantly higher level of cell death at low nanomolar concentrations than Baxα-positive or Bax-negative cells. Furthermore, bortezomib-induced cell death in BaxΔ2-positive cells was predominantly dependent on the caspase 8/3 pathway, consistent with our previous studies. These results imply that BaxΔ2 can selectively sensitize cancer cells to proteasome inhibitors, enhancing their potential to treat colon cancer and other solid tumors.

The functional domains for Bax∆2 aggregate-mediated caspase 8-dependent cell death.

Bax∆2 is a functional pro-apoptotic Bax isoform having alterations in its N-terminus, but sharing the rest of its sequence with Baxα. Bax∆2 is unable to target mitochondria due to the loss of helix α1. Instead, it forms cytosolic aggregates and activates caspase 8. However, the functional domain(s) responsible for BaxΔ2 behavior have remained elusive. Here we show that disruption of helix α1 makes Baxα mimic the behavior of Bax∆2. However, the other alterations in the Bax∆2 N-terminus have no significant impact on aggregation or cell death. We found that the hallmark BH3 domain is necessary but not sufficient for aggregation-mediated cell death. We also noted that the core region shared by Baxα and Bax∆2 is required for the formation of large aggregates, which is essential for BaxΔ2 cytotoxicity. However, aggregation by itself is unable to trigger cell death without the C-terminus. Interestingly, the C-terminal helical conformation, not its primary sequence, appears to be critical for caspase 8 recruitment and activation. As Bax∆2 shares core and C-terminal sequences with most Bax isoforms, our results not only reveal a structural basis for Bax∆2-induced cell death, but also imply an intrinsic potential for aggregate-mediated caspase 8-dependent cell death in other Bax family members.

Rinsing paired-agent model (RPAM) to quantify cell-surface receptor concentrations in topical staining applications of thick tissues.

Conventional molecular assessment of tissue through histology, if adapted to fresh thicker samples, has the potential to enhance cancer detection in surgical margins and monitoring of 3D cell culture molecular environments. However, in thicker samples, substantial background staining is common despite repeated rinsing, which can significantly reduce image contrast. Recently, 'paired-agent' methods-which employ co-administration of a control (untargeted) imaging agent-have been applied to thick-sample staining applications to account for background staining. To date, these methods have included (1) a simple ratiometric method that is relatively insensitive to noise in the data but has accuracy that is dependent on the staining protocol and the characteristics of the sample; and (2) a complex paired-agent kinetic modeling method that is more accurate but is more noise-sensitive and requires a precise serial rinsing protocol. Here, a new simplified mathematical model-the rinsing paired-agent model (RPAM)-is derived and tested that offers a good balance between the previous models, is adaptable to arbitrary rinsing-imaging protocols, and does not require calibration of the imaging system. RPAM is evaluated against previous models and is validated by comparison to estimated concentrations of targeted biomarkers on the surface of 3D cell culture and tumor xenograft models. This work supports the use of RPAM as a preferable model to quantitatively analyze targeted biomarker concentrations in topically stained thick tissues, as it was found to match the accuracy of the complex paired-agent kinetic model while retaining the low noise-sensitivity characteristics of the ratiometric method.

Deficiency in ubiquitin-like protein Ubl4A impairs migration of fibroblasts and macrophages.

Ubiquitin-like protein Ubl4A is a small, multi-functional protein with no ubiquitination activity. We have previously demonstrated that Ubl4A directly interacts with actin-related protein 2/3 complex (Arp2/3) and promotes Arp2/3-dependent actin branching, thereby accelerating plasma membrane translocation of protein kinase Akt upon insulin stimulation. Here, we show that Ubl4A is critical for plasma membrane protrusion and cell migration. Ubl4A, F-actin and Arp2/3 are co-localized at the cell leading edges during wound closure. Knockout of Ubl4A significantly reduces actin-mediated membrane protrusion and delays wound healing by primary mouse embryonic fibroblasts. Consistently, the ability of fibroblasts to migrate out of corneal tissue ex vivo is also impaired in Ubl4A-deficient mice. Furthermore, cell motility, but not phagocytosis, is significantly decreased in Ubl4A-deficient macrophages compared with wild-type controls. These results imply an important role for Ubl4A in cell migration-associated pathophysiological processes.

Ubl4A is required for insulin-induced Akt plasma membrane translocation through promotion of Arp2/3-dependent actin branching.

The serine-threonine kinase Akt is a key regulator of cell proliferation and survival, glucose metabolism, cell mobility, and tumorigenesis. Activation of Akt by extracellular stimuli such as insulin centers on the interaction of Akt with PIP3 on the plasma membrane, where it is subsequently phosphorylated and activated by upstream protein kinases. However, it is not known how Akt is recruited to the plasma membrane upon stimulation. Here we report that ubiquitin-like protein 4A (Ubl4A) plays a crucial role in insulin-induced Akt plasma membrane translocation. Ubl4A knockout newborn mice have defective Akt-dependent glycogen synthesis and increased neonatal mortality. Loss of Ubl4A results in the impairment of insulin-induced Akt translocation to the plasma membrane and activation. Akt binds actin-filaments and colocalizes with actin-related protein 2 and 3 (Arp2/3) complex in the membrane ruffles and lamellipodia. Ubl4A directly interacts with Arp2/3 to accelerate actin branching and networking, allowing Akt to be in close proximity to the plasma membrane for activation upon insulin stimulation. Our finding reveals a new mechanism by which Akt is recruited to the plasma membrane for activation, thereby providing a missing link in Akt signaling.

Detection of Bax Microsatellite Mutations and BaxΔ2 Isoform in Human Buccal Cells.

Loss of the pro-apoptotic Bcl-2 family protein Bax occurs in ~50% of hereditary nonpolyposis colorectal cancer (HNPCC) due to microsatellite instability (MSI). Recently, we found that some of the "Bax-negative" MSI tumor cells contain a functional Bax isoform, BaxΔ2, which sensitizes cells to selective chemotherapeutics. Here we show the detection of Bax microsatellite mutations and expression of BaxΔ2 proteins in human buccal cells. Our study provides a sensitive and non-invasive approach and a potential clinical application in diagnosis and treatment of MSI colon cancer patients.

BaxΔ2 promotes apoptosis through caspase-8 activation in microsatellite-unstable colon cancer.

UNLABELLED: Loss of apoptotic Bax due to microsatellite mutation contributes to tumor development and chemoresistance. Recently, a Bax microsatellite mutation was uncovered in combination with a specific alternative splicing event that could generate a unique Bax isoform (BaxΔ2) in otherwise Bax-negative cells. Like the prototype Baxα, BaxΔ2 is a potent proapoptotic molecule. However, the proapoptotic mechanism and therapeutic implication of BaxΔ2 remain elusive. Here, the isolation and analysis of isogenic subcell lines are described that represent different Bax microsatellite statuses from colorectal cancer. Colon cancer cells harboring Bax microsatellite G7/G7 alleles are capable of producing low levels of endogenous BaxΔ2 transcripts and proteins. Interestingly, BaxΔ2-positive cells are selectively sensitive to a subgroup of chemotherapeutics compared with BaxΔ2-negative cells. Unlike other Bax isoforms, BaxΔ2 recruits caspase-8 into the proximity for activation, and the latter, in turn, activates caspase-3 and apoptosis independent of the mitochondrial pathway. These data suggest that the expression of BaxΔ2 may provide alternative apoptotic and chemotherapeutic advantages for Bax-negative tumors. IMPLICATIONS: "Bax-negative" colorectal tumors expressing a Bax isoform are sensitive to selective chemotherapeutics.

Deacetylation of p53 induces autophagy by suppressing Bmf expression.

Interferon γ (IFN-γ)-induced cell death is mediated by the BH3-only domain protein, Bik, in a p53-independent manner. However, the effect of IFN-γ on p53 and how this affects autophagy have not been reported. The present study demonstrates that IFN-γ down-regulated expression of the BH3 domain-only protein, Bmf, in human and mouse airway epithelial cells in a p53-dependent manner. p53 also suppressed Bmf expression in response to other cell death-stimulating agents, including ultraviolet radiation and histone deacetylase inhibitors. IFN-γ did not affect Bmf messenger RNA half-life but increased nuclear p53 levels and the interaction of p53 with the Bmf promoter. IFN-γ-induced interaction of HDAC1 and p53 resulted in the deacetylation of p53 and suppression of Bmf expression independent of p53's proline-rich domain. Suppression of Bmf facilitated IFN-γ-induced autophagy by reducing the interaction of Beclin-1 and Bcl-2. Furthermore, autophagy was prominent in cultured bmf(-/-) but not in bmf(+/+) cells. Collectively, these observations show that deacetylation of p53 suppresses Bmf expression and facilitates autophagy.