Asian Cohort for Alzheimer's Disease (ACAD) pilot study on genetic and non-genetic risk factors for Alzheimer's disease among Asian Americans and Canadians.

INTRODUCTION: Clinical research in Alzheimer's disease (AD) lacks cohort diversity despite being a global health crisis. The Asian Cohort for Alzheimer's Disease (ACAD) was formed to address underrepresentation of Asians in research, and limited understanding of how genetics and non-genetic/lifestyle factors impact this multi-ethnic population. METHODS: The ACAD started fully recruiting in October 2021 with one central coordination site, eight recruitment sites, and two analysis sites. We developed a comprehensive study protocol for outreach and recruitment, an extensive data collection packet, and a centralized data management system, in English, Chinese, Korean, and Vietnamese. RESULTS: ACAD has recruited 606 participants with an additional 900 expressing interest in enrollment since program inception. DISCUSSION: ACAD's traction indicates the feasibility of recruiting Asians for clinical research to enhance understanding of AD risk factors. ACAD will recruit > 5000 participants to identify genetic and non-genetic/lifestyle AD risk factors, establish blood biomarker levels for AD diagnosis, and facilitate clinical trial readiness. HIGHLIGHTS: The Asian Cohort for Alzheimer's Disease (ACAD) promotes awareness of under-investment in clinical research for Asians. We are recruiting Asian Americans and Canadians for novel insights into Alzheimer's disease. We describe culturally appropriate recruitment strategies and data collection protocol. ACAD addresses challenges of recruitment from heterogeneous Asian subcommunities. We aim to implement a successful recruitment program that enrolls across three Asian subcommunities.

The prediction of Alzheimer's disease through multi-trait genetic modeling.

To better capture the polygenic architecture of Alzheimer's disease (AD), we developed a joint genetic score, MetaGRS. We incorporated genetic variants for AD and 24 other traits from two independent cohorts, NACC (n = 3,174, training set) and UPitt (n = 2,053, validation set). One standard deviation increase in the MetaGRS is associated with about 57% increase in the AD risk [hazard ratio (HR) = 1.577, p = 7.17 E-56], showing little difference from the HR for AD GRS alone (HR = 1.579, p = 1.20E-56), suggesting similar utility of both models. We also conducted APOE-stratified analyses to assess the role of the e4 allele on risk prediction. Similar to that of the combined model, our stratified results did not show a considerable improvement of the MetaGRS. Our study showed that the prediction power of the MetaGRS significantly outperformed that of the reference model without any genetic information, but was effectively equivalent to the prediction power of the AD GRS.

The Beneficial Role of Photobiomodulation in Neurodegenerative Diseases.

Photobiomodulation (PBM), also known as Low-level Laser Therapy (LLLT), involves the use of light from a laser or light-emitting diode (LED) in the treatment of various disorders and it has recently gained increasing interest. Progressive neuronal loss with attendant consequences such as cognitive and/or motor decline characterize neurodegenerative diseases. The available therapeutic drugs have only been able to provide symptomatic relief and may also present with some side effects, thus precluding their use in treatment. Recently, there has been an exponential increase in interest and attention in the use of PBM as a therapy in various neurodegenerative diseases in animal studies. Because of the financial and social burden of neurodegenerative diseases on the sufferers and the need for the discovery of potential therapeutic inventions in their management, it is pertinent to examine the beneficial effects of PBM and the various cellular mechanisms by which it modulates neural activity. Here, we highlight the various ways by which PBM may possess beneficial effects on neural activity and has been reported in various neurodegenerative conditions (Alzheimer's disease, Parkinson's disease, epilepsy, TBI, stroke) with the hope that it may serve as an alternative therapy in the management of neurodegenerative diseases because of the biological side effects associated with drugs currently used in the treatment of neurodegenerative diseases.

Stem Cell Therapy in Spinal Cord Injury-Induced Neurogenic Lower Urinary Tract Dysfunction.

Spinal cord injury (SCI) is a devastating condition that enormously affects an individual's health and quality of life. Neurogenic lower urinary tract dysfunction (NLUTD) is one of the most important sequelae induced by SCI, causing complications including urinary tract infection, renal function deterioration, urinary incontinence, and voiding dysfunction. Current therapeutic methods for SCI-induced NLUTD mainly target on the urinary bladder, but the outcomes are still far from satisfactory. Stem cell therapy has gained increasing attention for years for its ability to rescue the injured spinal cord directly. Stem cell differentiation and their paracrine effects, including exosomes, are the proposed mechanisms to enhance the recovery from SCI. Several animal studies have demonstrated improvement in bladder function using mesenchymal stem cells (MSCs) and neural stem cells (NSCs). Human clinical trials also provide promising results in urodynamic parameters after MSC therapy. However, there is still uncertainty about the ideal treatment window and application protocol for stem cell therapy. Besides, data on the therapeutic effects regarding NSCs and stem cell-derived exosomes in SCI-related NLUTD are scarce. Therefore, there is a pressing need for further well-designed human clinical trials to translate the stem cell therapy into a formal therapeutic option for SCI-induced NLUTD.

TTR (transthyretin) leads the autophagy disaster relief team against TARDBP/TDP-43 proteinopathy.

TTR (transthyretin) strikes a neuroprotective function in the prevention of amyloid-ß (Aß) deposition in Alzheimer disease (AD). Perturbation of the stringently controlled TARDBP/TDP-43 (TAR DNA binding protein) expression gives rise to cytoplasmic aggregation, characterized by TARDBP proteinopathy affiliated with several neurological disorders, including frontotemporal lobar degeneration with TARDBP pathology (FTLD-TDP) and amyotrophic lateral sclerosis/ALS. Proposedly, TTR can maintain cellular proteostasis susceptible to TARDBP aggregates and initiate its removal. Herein, we disclose that TTR upregulated in response to excessive TARDBP causes TARDBP aggregation in FTLD-TDP and co-accumulates with it. Moreover, TTR expression increases with age in FTLD-TDP but shows a downward decline in the elderly. TTR promotes macroautophagy/autophagy activity and facilitates aggregated TARDBP degradation via autophagy. Compellingly, TTR binds to ATF4 and boosts its nuclear import for autophagy upregulation. Therefore, TTR directs autophagy teamwork in bi-directional regulation through enhancing autophagy activity via ATF4 and chaperoning aggregated TARDBP to phagophores for degradation.Abbreviations: Aß: amyloid-ß; AD: Alzheimer disease; ER: endoplasmic reticulum; FTLD-TDP: frontotemporal lobar degeneration with TARDBP pathology; TARDBP/TDP-43: TAR DNA binding protein; TTR: transthyretin; UPR: unfolded protein response.

Transthyretin attenuates TDP-43 proteinopathy by autophagy activation via ATF4 in FTLD-TDP.

TAR DNA-binding protein-43 (TDP-43) proteinopathies are accompanied by the pathological hallmark of cytoplasmic inclusions in the neurodegenerative diseases, including frontal temporal lobar degeneration-TDP and amyotrophic lateral sclerosis. We found that transthyretin accumulates with TDP-43 cytoplasmic inclusions in frontal temporal lobar degeneration-TDP human patients and transgenic mice, in which transthyretin exhibits dramatic expression decline in elderly mice. The upregulation of transthyretin expression was demonstrated to facilitate the clearance of cytoplasmic TDP-43 inclusions through autophagy, in which transthyretin induces autophagy upregulation via ATF4. Of interest, transthyretin upregulated ATF4 expression and promoted ATF4 nuclear import, presenting physical interaction. Neuronal expression of transthyretin in frontal temporal lobar degeneration-TDP mice restored autophagy function and facilitated early soluble TDP-43 aggregates for autophagosome targeting, ameliorating neuropathology and behavioural deficits. Thus, transthyretin conducted two-way regulations by either inducing autophagy activation or escorting TDP-43 aggregates targeted autophagosomes, suggesting that transthyretin is a potential modulator therapy for neurological disorders caused by TDP-43 proteinopathy.

MicroRNAs in Learning and Memory and Their Impact on Alzheimer's Disease.

Learning and memory formation rely on the precise spatiotemporal regulation of gene expression, such as microRNA (miRNA)-associated silencing, to fine-tune gene expression for the induction and maintenance of synaptic plasticity. Much progress has been made in presenting direct evidence of miRNA regulation in learning and memory. Here, we summarize studies that have manipulated miRNA expression using various approaches in rodents, with changes in cognitive performance. Some of these are involved in well-known mechanisms, such as the CREB-dependent signaling pathway, and some of their roles are in fear- and stress-related disorders, particularly cognitive impairment. We also summarize extensive studies on miRNAs correlated with pathogenic tau and amyloid-ß that drive the processes of Alzheimer's disease (AD). Although altered miRNA profiles in human patients with AD and in mouse models have been well studied, little is known about their clinical applications and therapeutics. Studies on miRNAs as biomarkers still show inconsistencies, and more challenges need to be confronted in standardizing blood-based biomarkers for use in AD.

Short-Term Cortical Electrical Stimulation during the Acute Stage of Traumatic Brain Injury Improves Functional Recovery.

Functional restoration is an important issue in the treatment of traumatic brain injury (TBI). Various electrical stimulation devices and protocols have been applied in preclinical studies and have shown therapeutic potential for brain trauma. Short-term invasive cortical electrical stimulation during the acute stage of TBI might be a feasible adjuvant therapy for patients with moderate-to-severe brain injury receiving neurosurgical treatment in the intensive care unit. However, the therapeutic effects of short-term multisession cortical electrical stimulation for brain trauma are not clear. This study explored the therapeutic effects of acute-stage short-term cortical electrical stimulation on TBI. We conducted seven sessions of one-hour cortical electrical stimulation from day 0 to day 6 in rats after brain trauma by controlled cortical impact and then evaluated the functional outcome and histopathological changes. Our data showed that short-term cortical electrical stimulation improved motor coordination, short-term memory, and learning ability and attenuated neurological severity after brain trauma. Lesion volume, apoptosis, and gliosis after brain trauma were reduced, and trauma-induced neurogenesis in the hippocampus for the innate neural reparative response was increased. Our study demonstrated that short-term cortical electrical stimulation applied in the acute stage of traumatic brain injury is a potential adjuvant therapy to improve the recovery of neurological deficits.

Alzheimer's Disease Variant Portal: A Catalog of Genetic Findings for Alzheimer's Disease.

BACKGROUND: Recent Alzheimer's disease (AD) genetics findings from genome-wide association studies (GWAS) span progressively larger and more diverse populations and outcomes. Currently, there is no up-to-date resource providing harmonized and searchable information on all AD genetic associations found by GWAS, nor linking the reported genetic variants and genes with functional and genomic annotations. OBJECTIVE: Create an integrated/harmonized, and literature-derived collection of population-specific AD genetic associations. METHODS: We developed the Alzheimer's Disease Variant Portal (ADVP), an extensive collection of associations curated from >200 GWAS publications from Alzheimer's Disease Genetics Consortium and other consortia. Genetic associations were systematically extracted, harmonized, and annotated from both the genome-wide significant and suggestive loci reported in these publications. To ensure consistent representation of AD genetic findings, all the extracted genetic association information was harmonized across specifically designed publication, variant, and association categories. RESULTS: ADVP V1.0 (February 2021) catalogs 6,990 associations related to disease-risk, expression quantitative traits, endophenotypes, or neuropathology. This extensive harmonization effort led to a catalog containing >900 loci, >1,800 variants, >80 cohorts, and 8 populations. Besides, ADVP provides investigators with a seamless integration of genomic and publicly available functional annotations across multiple databases per harmonized variant and gene records, thus facilitating further understanding and analyses of these genetics findings. CONCLUSION: ADVP is a valuable resource for investigators to quickly and systematically explore high-confidence AD genetic findings and provides insights into population-specific AD genetic architecture. ADVP is continually maintained and enhanced by NIAGADS and is freely accessible at https://advp.niagads.org.

Somatosensory Cortical Electrical Stimulation After Reperfusion Attenuates Ischemia/Reperfusion Injury of Rat Brain.

Objective: Ischemic stroke is an important cause of death and disability worldwide. Early reperfusion by thrombolysis or thrombectomy has improved the outcome of acute ischemic stroke. However, the therapeutic window for reperfusion therapy is narrow, and adjuvant therapy for neuroprotection is demanded. Electrical stimulation (ES) has been reported to be neuroprotective in many neurological diseases. In this study, the neuroprotective effect of early somatosensory cortical ES in the acute stage of ischemia/reperfusion injury was evaluated. Methods: In this study, the rat model of transient middle cerebral artery occlusion was used to explore the neuroprotective effect and underlying mechanisms of direct primary somatosensory (S1) cortex ES with an electric current of 20 Hz, 2 ms biphasic pulse, 100 µA for 30 min, starting at 30 min after reperfusion. Results: These results showed that S1 cortical ES after reperfusion decreased infarction volume and improved functional outcome. The number of activated microglia, astrocytes, and cleaved caspase-3 positive neurons after ischemia/reperfusion injury were reduced, demonstrating that S1 cortical ES alleviates inflammation and apoptosis. Brain-derived neurotrophic factor (BDNF) and phosphoinositide 3-kinase (PI3K)/Akt/mammalian target of rapamycin (mTOR) signaling pathway were upregulated in the penumbra area, suggesting that BDNF/TrkB signals and their downstream PI3K/Akt signaling pathway play roles in ES-related neuroprotection. Conclusion: This study demonstrates that somatosensory cortical ES soon after reperfusion can attenuate ischemia/reperfusion injury and is a promising adjuvant therapy for thrombolytic treatment after acute ischemic stroke. Advanced techniques and devices for high-definition transcranial direct current stimulation still deserve further development in this regard.

Reconcile the debate over protective effects of BCG vaccine against COVID-19.

While awaiting the COVID-19 vaccines, researchers have been actively exploring the effectiveness of existing vaccines against the new virus, among which the BCG vaccine (Bacillus Calmette-Guérin) receives the most attention. While many reports suggest a potential role for BCG immunization in ameliorating SARS-CoV-2 infection, these findings remain controversial. With country-level COVID-19 outbreak data from Johns Hopkins University Coronavirus Resource Center, and BCG program data from World Atlas of BCG Policies and Practices and WHO/UNICE, we estimated a dynamic model to investigate the effect of BCG vaccination across time during the pandemic. Our results reconcile these varying reports regarding protection by BCG against COVID-19 in a variety of clinical scenarios and model specifications. We observe a notable protective effect of the BCG vaccine during the early stage of the pandemic. However, we do not see any strong evidence for protection during the later stages. We also see that a higher proportion of vaccinated young population may confer some level of communal protection against the virus in the early pandemic period, even when the proportion of vaccination in the older population is low. Our results highlight that while BCG may offer some protection against COVID-19, we should be cautious in interpreting the estimated effectiveness as it may vary over time and depend on the age structure of the vaccinated population.

TDP-43 proteinopathy impairs mRNP granule mediated postsynaptic translation and mRNA metabolism.

Background: Local protein synthesis and mRNA metabolism mediated by mRNP granules in the dendrites and the postsynaptic compartment is essential for synaptic remodeling and plasticity in neuronal cells. Dysregulation of these processes caused by TDP-43 proteinopathy leads to neurodegenerative diseases, such as frontotemporal lobar degeneration and amyotrophic lateral sclerosis. Methods: Using biochemical analysis and imaging techniques, including super-resolution microscopy, we provide evidence, for the first time, for the postsynaptic localization of TDP-43 in mammalian synapses and we show that TDP-43 is a component of neuronal mRNP granules. Results: With activity stimulation and various molecular approaches, we further demonstrate activity-dependent mRNP granule dynamics involving disassembly of mRNP granules, release of mRNAs, activation of local protein translation, and the impairment of granule disassembly in cellular, animal and human models of TDP-43 proteinopathy. Conclusion: Our study elucidates the interplay between TDP-43 and neuronal mRNP granules in normal physiology and TDP-43 proteinopathy in the regulation of local protein translation and mRNA metabolism in the postsynaptic compartment.

Therapeutic Zfra4-10 or WWOX7-21 Peptide Induces Complex Formation of WWOX with Selective Protein Targets in Organs that Leads to Cancer Suppression and Spleen Cytotoxic Memory Z Cell Activation In Vivo.

Synthetic Zfra4-10 and WWOX7-21 peptides strongly suppress cancer growth in vivo. Hypothetically, Zfra4-10 binds to the membrane Hyal-2 of spleen Z cells and activates the Hyal-2/WWOX/SMAD4 signaling for cytotoxic Z cell activation to kill cancer cells. Stimulation of membrane WWOX in the signaling complex by a WWOX epitope peptide, WWOX7-21, is likely to activate the signaling. Here, mice receiving Zfra4-10 or WWOX7-21 peptide alone exhibited an increased binding of endogenous tumor suppressor WWOX with ERK, C1qBP, NF-κB, Iba1, p21, CD133, JNK1, COX2, Oct4, and GFAP in the spleen, brain, and/or lung which led to cancer suppression. However, when in combination, Zfra4-10 and WWOX7-21 reduced the binding of WWOX with target proteins and allowed tumor growth in vivo. In addition to Zfra4-10 and WWOX7-21 peptides, stimulating the membrane Hyal-2/WWOX complex with Hyal-2 antibody and sonicated hyaluronan (HAson) induced Z cell activation for killing cancer cells in vivo and in vitro. Mechanistically, Zfra4-10 binds to membrane Hyal-2, induces dephosphorylation of WWOX at pY33 and pY61, and drives Z cell activation for the anticancer response. Thus, Zfra4-10 and WWOX7-21 peptides, HAson, and the Hyal-2 antibody are of therapeutic potential for cancer suppression.

HDAC1 dysregulation induces aberrant cell cycle and DNA damage in progress of TDP-43 proteinopathies.

TAR DNA-binding protein 43 (TDP-43) has been implicated in frontotemporal lobar degeneration with ubiquitin-positive inclusions (FTLD-TDP) and amyotrophic lateral sclerosis. Histone deacetylase 1 (HDAC1) is involved in DNA repair and neuroprotection in numerous neurodegenerative diseases. However, the pathological mechanisms of FTLD-TDP underlying TDP-43 proteinopathies are unclear, and the role of HDAC1 is also poorly understood. Here, we found that aberrant cell cycle activity and DNA damage are important pathogenic factors in FTLD-TDP transgenic (Tg) mice, and we further identified these pathological features in the frontal cortices of patients with FTLD-TDP. TDP-43 proteinopathies contributed to pathogenesis by inducing cytosolic mislocalization of HDAC1 and reducing its activity. Pharmacological recovery of HDAC1 activity in FTLD-TDP Tg mice ameliorated their cognitive and motor impairments, normalized their aberrant cell cycle activity, and attenuated their DNA damage and neuronal loss. Thus, HDAC1 deregulation is involved in the pathogenesis of TDP-43 proteinopathies, and HDAC1 is a potential target for therapeutic interventions in FTLD-TDP.

Lipids and Alzheimer's Disease.

Lipids, as the basic component of cell membranes, play an important role in human health as well as brain function. The brain is highly enriched in lipids, and disruption of lipid homeostasis is related to neurologic disorders as well as neurodegenerative diseases such as Alzheimer's disease (AD). Aging is associated with changes in lipid composition. Alterations of fatty acids at the level of lipid rafts and cerebral lipid peroxidation were found in the early stage of AD. Genetic and environmental factors such as apolipoprotein and lipid transporter carrying status and dietary lipid content are associated with AD. Insight into the connection between lipids and AD is crucial to unraveling the metabolic aspects of this puzzling disease. Recent advances in lipid analytical methodology have led us to gain an in-depth understanding on lipids. As a result, lipidomics have becoming a hot topic of investigation in AD, in order to find biomarkers for disease prediction, diagnosis, and prevention, with the ultimate goal of discovering novel therapeutics.

WWOX Possesses N-Terminal Cell Surface-Exposed Epitopes WWOX7-21 and WWOX7-11 for Signaling Cancer Growth Suppression and Prevention In Vivo.

Membrane hyaluronidase Hyal-2 supports cancer cell growth. Inhibition of Hyal-2 by specific antibody against Hyal-2 or pY216-Hyal-2 leads to cancer growth suppression and prevention in vivo. By immunoelectron microscopy, tumor suppressor WWOX is shown to be anchored, in part, in the cell membrane by Hyal-2. Alternatively, WWOX undergoes self-polymerization and localizes in the cell membrane. Proapoptotic pY33-WWOX binds Hyal-2, and TGF-ß induces internalization of the pY33-WWOX/Hyal-2 complex to the nucleus for causing cell death. In contrast, when pY33 is downregulated and pS14 upregulated in WWOX, pS14-WWOX supports cancer growth in vivo. Here, we investigated whether membrane WWOX receives extracellular signals via surface-exposed epitopes, especially at the S14 area, that signals for cancer growth suppression and prevention. By using a simulated 3-dimentional structure and generated specific antibodies, WWOX epitopes were determined at amino acid #7 to 21 and #286 to 299. Synthetic WWOX7-21 peptide, or truncation to 5-amino acid WWOX7-11, significantly suppressed and prevented the growth and metastasis of melanoma and skin cancer cells in mice. Time-lapse microscopy revealed that WWOX7-21 peptide potently enhanced the explosion and death of 4T1 breast cancer stem cell spheres by ceritinib. This is due to rapid upregulation of proapoptotic pY33-WWOX, downregulation of prosurvival pERK, prompt increases in Ca2+ influx, and disruption of the IkBα/WWOX/ERK prosurvival signaling. In contrast, pS14-WWOX7-21 peptide dramatically increased cancer growth in vivo and protected cancer cells from ceritinib-mediated apoptosis in vitro, due to a prolonged ERK phosphorylation. Further, specific antibody against pS14-WWOX significantly enhanced the ceritinib-induced apoptosis. Together, the N-terminal epitopes WWOX7-21 and WWOX7-11 are potent in blocking cancer growth in vivo. WWOX7-21 and WWOX7-11 peptides and pS14-WWOX antibody are of therapeutic values in suppressing and preventing cancer growth in vivo.

Exercise Alleviates Osteoporosis in Rats with Mild Chronic Kidney Disease by Decreasing Sclerostin Production.

Chronic kidney disease-mineral bone disorder (CKD-MBD), comprising mineral, hormonal, and bone metabolic imbalance, is a major CKD-related issue; it causes osteoporosis prevalence in CKD patients. Osteocyte-derived sclerostin inhibits the osteogenic Wnt/ß-catenin signaling pathway; its levels rise when kidney function declines. Exercise modulates the physiological functions of osteocytes, potentially altering sclerostin production. It may aid bone and mineral electrolyte homeostasis in CKD. Mild CKD was induced in rats by partial nephrectomy. They were divided into: sham (no CKD), CKD, and CKD + exercise (8 weeks of treadmill running) groups. Micro-CT scanning demonstrated that the CKD + exercise-group rats had a higher bone mineral density (BMD) of the spine and femoral metaphysis and higher femoral trabecular bone volume than the CKD-group rats. Bone formation rates were not significantly different. The CKD + exercise-group rats had lower serum sclerostin (157.1 ± 21.1 vs 309 ± 38.1 pg/mL, p < 0.05) and CTX-1 (bone resorption marker) levels. Immunohistochemistry revealed higher tibial ß-catenin concentrations in the CKD + exercise-group rats. Serum FGF-23, intact parathyroid hormone (iPTH), alkaline phosphatase (ALP), calcium, and phosphate levels showed no significant differences between these groups. Thus, exercise improves BMD and bone microstructure in mild CKD by inhibiting sclerostin production, but does not alter serum minerals.

WWOX Phosphorylation, Signaling, and Role in Neurodegeneration.

Homozygous null mutation of tumor suppressor WWOX/Wwox gene leads to severe neural diseases, metabolic disorders and early death in the newborns of humans, mice and rats. WWOX is frequently downregulated in the hippocampi of patients with Alzheimer's disease (AD). In vitro analysis revealed that knockdown of WWOX protein in neuroblastoma cells results in aggregation of TRAPPC6AΔ, TIAF1, amyloid ß, and Tau in a sequential manner. Indeed, TRAPPC6AΔ and TIAF1, but not tau and amyloid ß, aggregates are present in the brains of healthy mid-aged individuals. It is reasonable to assume that very slow activation of a protein aggregation cascade starts sequentially with TRAPPC6AΔ and TIAF1 aggregation at mid-ages, then caspase activation and APP de-phosphorylation and degradation, and final accumulation of amyloid ß and Tau aggregates in the brains at greater than 70 years old. WWOX binds Tau-hyperphosphorylating enzymes (e.g., GSK-3ß) and blocks their functions, thereby supporting neuronal survival and differentiation. As a neuronal protective hormone, 17ß-estradiol (E2) binds WWOX at an NSYK motif in the C-terminal SDR (short-chain alcohol dehydrogenase/reductase) domain. In this review, we discuss how WWOX and E2 block protein aggregation during neurodegeneration, and how a 31-amino-acid zinc finger-like Zfra peptide restores memory loss in mice.

Chasing the signaling run by tri-molecular time-lapse FRET microscopy.

A feasible design is made to measure three protein/protein interactions to visualize signal pathways by time-lapse Förster resonance energy transfer (FRET) microscopy. When interacting proteins are in close proximity, excitation energy is provided to allow the energy flow from the first molecule to excite the second, followed by energy transfer to the third. By phorbol ester/calcium ionophore stimulation, for example, a real-time complex formation of ectopic IκBα/ERK/WWOX occurs as measured by FRET microscopy, indicative of an ongoing functional signaling. Hyaluronan induces membrane Hyal-2 signaling, which allows FRET measurement of the complex formation of ectopic Smad4/WWOX/Hyal-2 for causing bubbling cell death. If ectopic p53 is recruited to replace Hyal-2, the resulting ectopic Smad4/WWOX/p53 complex induces membrane blebbing without cell death. Together, in this perspective review article, we demonstrate the utilization of time-lapse FRET microscopy to visualize the signaling event via the tri-molecular protein complex formation and their biological outcomes. We show an initial two-protein binding to form the driving force to jumpstart the tri-molecular execution for the signal pathway.