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Background Amyloid plaques and neurofibrillary tangles, the molecular lesions that characterize Alzheimer’s disease (AD) and other forms of dementia, are emerging as determinants of proteinopathies ‘beyond the brain’. This study aims to establish tau’s putative pathophysiological mechanistic roles and potential future therapeutic targeting of tau in heart failure (HF). Methods and results A mouse model of tauopathy and human myocardial and brain tissue from patients with HF, AD, and controls was employed in this study. Tau protein expression was examined together with its distribution, and in vitro tau-related pathophysiological mechanisms were identified using a variety of biochemical, imaging, and functional approaches. A novel tau-targeting immunotherapy was tested to explore tau-targeted therapeutic potential in HF. Tau is expressed in normal and diseased human hearts, in contradistinction to the current oft-cited observation that tau is expressed specifically in the brain. Notably, the main cardiac isoform is high-molecular-weight (HMW) tau (also known as big tau), and hyperphosphorylated tau segregates in aggregates in HF and AD hearts. As previously described for amyloid-beta, the tauopathy phenotype in human myocardium is of diastolic dysfunction. Perturbation in the tubulin code, specifically a loss of tyrosinated microtubules, emerged as a potential mechanism of myocardial tauopathy. Monoclonal anti-tau antibody therapy improved myocardial function and clearance of toxic aggregates in mice, supporting tau as a potential target for novel HF immunotherapy. Conclusion The study presents new mechanistic evidence and potential treatment for the brain–heart tauopathy axis in myocardial and brain degenerative diseases and ageing.

Background: Tau oligomers are one of the most toxic species, displaying prion-like strains which have different conformations resulting in different tauopathies. Passive immunotherapy targeting different tau species is a promising therapeutic approach. Age is one of the greatest risk factors; however, most immunotherapy studies are done in young to middle-aged mice tauopathy models, which is not representative of the many clinical trials done with older humans with established tauopathies. Objective: We utilized two different clones of tau oligomer monoclonal antibodies (TOMAs) in aged Htau and JNPL3 mouse models to investigate the potential of passive immunotherapy. Methods: Aged mice received a single intravenous injection of 120 μg/animal of either TOMA1, TOMA3 clones or a non-specific IgG. Their cognitive functions were assessed one-week post-injection using Y-maze and novel object recognition tests. Brain tissues were analyzed using biochemical and immunological assays. Results: TOMA 1 and 3 rescues cognitive phenotypes in aged animals in a mouse model-specific manner, indicative by a reduction in tau oligomers levels. The TOMAs were shown to have strong reactivity with different tau oligomeric species in the different mouse models in vitro and ex vivo. Conclusion: This is the first study testing tau passive immunotherapy in aged animals and supports our previous reports on of the role of oligomeric tau in disease progression further validating the potential of TOMAs to rescue the late-stage disease pathology and phenotype. Moreover, this study suggests that multiple tau oligomeric strains exist in aged animals; therefore, it is of great importance to further characterize these strains.

Accumulation of pathological tau aggregates is a prominent feature in tauopathies that leads during the course of the diseases to neuronal dysfunction before and cell death after. Microglia and astrocytes have been described as playing important roles in synaptic spreading of toxic tau in several neurodegenerative diseases (NDs). Here, we have investigated the immunological and biochemical properties of aggregated tau species in different brain cell types in tau-induced neurodegenerative diseases such as Alzheimer's disease (AD), progressive supranuclear palsy (PSP), and dementia with Lewy bodies (DLB). Additionally, we examined nuclear size, nuclear density, and chromatin compaction in neuronal and glial cells from diseased brain tissues. Microscopic-histological examination was performed using in-house mouse monoclonal antibodies for toxic tau conformers (TTC-M1 and TTC-M2) and tau oligomers (TOMA1-4). By immunohistochemistry and co-immunofluorescence assays using TOMA/TTC-Ms and cell-type specific markers for neurons, astrocytes, and microglia, we observed that TOMA/TTC-Ms were immunoreactive to diverse tau species in different cell types. Analysis of colocalization coefficients indicated an increased pathological tau deposition mainly in the neurons. Western blot analysis of brain homogenates using TOMA/TTC-Ms revealed distinct patterns of tau aggregation in each disease, suggesting that TOMA/TTC-Ms can distinguish between different tau aggregates present in different tauopathies. Additionally, using DAPI staining, we observed that neuronal and astrocytic nuclei had significantly greater nuclear area and increased chromatin compaction in AD cortices compared to non-demented controls. In contrast, reduction in nuclear density/area and more relaxed chromatin was noticed in DLB neurons, astrocytes and microglia and PSP astrocytes and microglia. Cell-type specific tropism of toxic tau species in tauopathies will provide a greater understanding of the involvement of different brain cell types in tau pathology. In this study, we observed that each disease presented cell-type specific nuclear phenotype and tau deposition pattern.

Oligomeric assemblies of tau and the RNA-binding proteins (RBPs) Musashi (MSI) are reported in Alzheimer’s disease (AD). However, the role of MSI and tau interaction in their aggregation process and its effects are nor clearly known in neurodegenerative diseases. Here, we investigated the expression and cellular localization of MSI1 and MSI2 in the brains tissues of Alzheimer’s disease (AD), amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) as well as in the wild-type mice and tau knock-out and P301L tau mouse models. We observed that formation of pathologically relevant protein inclusions was driven by the aberrant interactions between MSI and tau in the nuclei associated with age-dependent extracellular depositions of tau/MSI complexes. Furthermore, tau and MSI interactions induced impairment of nuclear/cytoplasm transport, chromatin remodeling and nuclear lamina formation. Our findings provide mechanistic insight for pathological accumulation of MSI/tau aggregates providing a potential basis for therapeutic interventions in neurodegenerative proteinopathies., The Musashi family of RNA binding proteins are found in an oligomeric state in the brains of patients with Alzheimer’s disease. Here the authors show that Mushashi1 and Musashi2 interact with tau protein in patient tissue and in models of tauopathy.

The pathological aggregation of tau plays an important role in Alzheimer's disease and many other related neurodegenerative diseases, collectively referred to as tauopathies. Recent evidence has demonstrated that tau oligomers, small and soluble prefibrillar aggregates, are highly toxic due to their strong ability to seed tau misfolding and propagate the pathology seen across different neurodegenerative diseases. We previously showed that novel curcumin derivatives affect preformed tau oligomer aggregation pathways by promoting the formation of more aggregated and nontoxic tau aggregates. To further investigate their therapeutic potential, we have extended our studies o disease-relevant brain-derived tau oligomers (BDTOs). Herein, using well-characterized BDTOs, isolated from brain tissues of different tauopathies, including Alzheimer's disease, progressive supranuclear palsy, and dementia with Lewy bodies, we found that curcumin derivatives modulate the aggregation state of BDTOs by reshaping them and rescue neurons from BDTO-associated toxicity. Interestingly, compound CL3 showed an effect on the aggregation pattern of BDTOs from different tauopathies, resulting in the formation of less neurotoxic larger tau aggregates with decreased hydrophobicity and seeding propensity. Our results lay the groundwork for potential investigations of the efficacy and beneficial effects of CL3 and other promising compounds for the treatment of tauopathies. Furthermore, CL3 may aid in the development of tau imaging agent for the detection of tau oligomeric strains and differential diagnosis of the tauopathies, thus enabling earlier interventions.

Background Alzheimer’s disease (AD) is characterized by progressive cognitive decline due to accumulating synaptic insults by toxic oligomers of amyloid beta (AβO) and tau (TauO). There is growing consensus that preventing these oligomers from interacting with synapses might be an effective approach to treat AD. However, recent clinical trial failures suggest low effectiveness of targeting Aβ in late-stage AD. Researchers have redirected their attention toward TauO as the levels of this species increase later in disease pathogenesis. Here we show that AβO and TauO differentially target synapses and affect each other's binding dynamics. Methods Binding of labeled, pre-formed Aβ and tau oligomers onto synaptosomes isolated from the hippocampus and frontal cortex of mouse and postmortem cognitively intact elderly human brains was evaluated using flow-cytometry and western blot analyses. Binding of labeled, pre-formed Aβ and tau oligomers onto mouse primary neurons was assessed using immunofluorescence assay. The synaptic dysfunction was measured by fluorescence analysis of single-synapse long-term potentiation (FASS-LTP) assay. Results We demonstrated that higher TauO concentrations effectively outcompete AβO and become the prevailing synaptic-associated species. Conversely, high concentrations of AβO facilitate synaptic TauO recruitment. Immunofluorescence analyses of mouse primary cortical neurons confirmed differential synaptic binding dynamics of AβO and TauO. Moreover, in vivo experiments using old 3xTgAD mice ICV injected with either AβO or TauO fully supported these findings. Consistent with these observations, FASS-LTP analyses demonstrated that TauO-induced suppression of chemical LTP was exacerbated by AβO. Finally, predigestion with proteinase K abolished the ability of TauO to compete off AβO without affecting the ability of high AβO levels to increase synaptic TauO recruitment. Thus, unlike AβO, TauO effects on synaptosomes are hampered by the absence of protein substrate in the membrane. Conclusions These results introduce the concept that TauO become the main synaptotoxic species at late AD, thus supporting the hypothesis that TauO may be the most effective therapeutic target for clinically manifest AD.

Immunotherapy is one of the strategies to boost natural defenses to fight cancer. Immuno-oncology is an artificial stimulation of the human immune system to recognize and kill selectively neoplastic cells at different stage of transformation. Cancer cells have tumor antigens and the antibody of the immune system, binding them, can detect molecules on their extracellular side of cell membrane. Among these proteins, it is rising in interest and used for early detection of hepatocellular carcinoma (HCC) Glypican-3 (GPC-3) protein. It is a heparan sulfate proteoglycan (HSPG), anchored to the cell membrane of transformed hepatocytes. We investigated its function as key regulator of hepatocytes neoplastic transformation. Noteworthy, GPC-3 protein has been implicated in different pathways from cell growth to cell motility and migration. More recently, GPC-3 has been evaluated as a useful marker for HCC due to its increased expression in the liver during tumorigenesis and its absence in normal liver. Immunotherapy that targets GPC-3 domains and its connected proteins are currently under investigation. These new biomarkers may hold potential for the detection and treatment of HCC and other diseases in which GPC-3 may be overexpressed and/or play a crucial role. This review will summarize the current knowledge regarding the active immunotherapy developed to treat HCC and it will evaluate aspects of GPC-3 (structure and biology) as advantages and potential pitfalls for considering it as a valuable immunotherapeutic target. We also elaborated the current literature with the aim to better understand its biological interactions at a molecular and cellular level to identify alternative or combined targets, due to the existing gap in the literature surrounding GPC-3. The role GPC-3 plays in the hepatocellular carcinoma phenotype can be targeted for a novel immunotherapy strategy that can specify cell-mediated destruction of neoplastic cell that spares normal liver tissue, and it can be exploited as a new serum marker to trend for diagnosis and disease progression measurements. We believe further investigation of its functions and structure, including alternative cellular localizations, is necessary to evaluate GPC-3 as valuable target to cure this cancer.

SUMMARY Aging, pathological tau oligomers (TauO), and chronic inflammation in the brain play a central role in tauopathies, including Alzheimer’s disease (AD) and frontotemporal dementia (FTD). However, the underlying mechanism of TauO-induced aging-related neuroinflammation remains unclear. Here, we show that TauO-associated astrocytes display a senescence-like phenotype in the brains of patients with AD and FTD. TauO exposure triggers astrocyte senescence through high mobility group box 1 (HMGB1) release and inflammatory senescence-associated secretory phenotype (SASP), which mediates paracrine senescence in adjacent cells. HMGB1 release inhibition using ethyl pyruvate (EP) and glycyrrhizic acid (GA) prevents TauO-induced senescence through inhibition of p38-mitogen-activated protein kinase (MAPK) and nuclear factor κB (NF-κB)—the essential signaling pathways for SASP development. Despite the developed tauopathy in 12-month-old hTau mice, EP+GA treatment significantly decreases TauO and senescent cell loads in the brain, reduces neuroinflammation, and thus ameliorates cognitive functions. Collectively, TauO-induced HMGB1 release promotes cellular senescence and neuropathology, which could represent an important common pathomechanism in tauopathies including AD and FTD., In brief Gaikwad et al. demonstrate that TauO-associated astrocytes exhibit senescence-like phenotype in the brain of patients with AD and FTD. They find that HMGB1 release is a crucial event for TauO-induced cellular senescence, tauopathy progression, and cognitive deficits, indicating that HMGB1 release could represent an important common pathomechanism in tauopathies., Graphical Abstract

Proteinaceous aggregates are major hallmarks of several neurodegenerative diseases. Aggregates of post-translationally modified transactive response (TAR)-DNA binding protein 43 (TDP-43) in cytoplasmic inclusion bodies are characteristic features in frontotemporal dementia (FTD) and amyotrophic lateral sclerosis (ALS). Recent studies have also reported TDP-43 aggregation in Alzheimer's disease (AD). TDP-43 is an RNA/DNA binding protein (RBP) mainly present in the nucleus. In addition to several RBPs, TDP-43 has also been reported in stress granules in FTD and ALS pathologies. Despite knowledge of cytoplasmic mislocalization of TDP-43, the cellular effects of TDP-43 aggregates and their cytotoxic mechanism(s) remain to be clarified. We hypothesize that TDP-43 forms oligomeric assemblies that associate with tau, another key protein involved in ALS and FTD. However, no prior studies have investigated the interactions between TDP-43 oligomers and tau. It is therefore important to thoroughly investigate the cross-seeding properties and cellular localization of both TDP-43 and tau oligomers in neurodegenerative diseases. Here, we demonstrate the effect of tau on the cellular localization of TDP-43 in WT and P301L tau-inducible cell models (iHEK) and in WT HEK-293 cells treated exogenously with soluble human recombinant tau oligomers (Exo-rTauO). We observed cytoplasmic TDP-43 accumulation o in the presence of tau in these cell models. We also studied the occurrence of TDP-43 oligomers in AD, ALS, and FTD human brain tissue using novel antibodies generated against TDP-43 oligomers as well as generic TDP-43 antibodies. Finally, we examined the cross-seeding property of AD, ALS, and FTD brain-derived TDP-43 oligomers (BDT43Os) on tau aggregation using biochemical and biophysical assays. Our results allow us to speculate that TDP-43/tau interactions might play a role in AD, ALS, and FTD.

Ubiquitin-modified tau aggregates are abundantly found in human brains diagnosed with Alzheimer's disease (AD) and other tauopathies. Soluble tau oligomers (TauO) are the most neurotoxic tau species that propagate pathology and elicit cognitive deficits, but whether ubiquitination contributes to tau formation and spreading is not fully understood. Here, we observed that K63-linked, but not K48-linked, ubiquitinated TauO accumulated at higher levels in AD brains compared with age-matched controls. Using mass spectrometry analyses, we identified 11 ubiquitinated sites on AD brain-derived TauO (AD TauO). We found that K63-linked TauO are associated with enhanced seeding activity and propagation in human tau-expressing primary neuronal and tau biosensor cells. Additionally, exposure of tau-inducible HEK cells to AD TauO with different ubiquitin linkages (wild type, K48, and K63) resulted in enhanced formation and secretion of K63-linked TauO, which was associated with impaired proteasome and lysosome functions. Multipathway analysis also revealed the involvement of K63-linked TauO in cell survival pathways, which are impaired in AD. Collectively, our study highlights the significance of selective TauO ubiquitination, which could influence tau aggregation, accumulation, and subsequent pathological propagation. The insights gained from this study hold great promise for targeted therapeutic intervention in AD and related tauopathies.

The influence of Alzheimer’s disease (AD) progression and severity on the structural and functional integrity of the cerebral vasculature is well recognized. The retina is an extension of the brain; thus, changes in retinal vascular features may serve as markers of AD cerebrovascular pathologies. However, differentiating normal aging-versus AD-induced retinal vascular changes is unresolved. Therefore, we compared and quantified changes in superficial (SVP), intermediate (IVP), and deep (DVP) retinal vascular plexuses in young, middle-age, and old triple transgenic mouse model of AD (3xT-AD) to the changes that occur in age-matched controls (C57BL/6j). We used immunostaining combined with a novel tissue optical clearing approach along with a computational tool for quantitative analysis of vascular network alterations (vessel length and density) in SVP, IVP, and DVP. All three layers had comparable structural features and densities in young 3xTg-AD and control animals. In controls, IVP and DVP densities decreased with aging (−14% to −32% change from young to old, p < 0.05), while no changes were observed in SVP. In contrast, vascular parameters in the transgenic group decreased in all three layers with aging (−12% to −49% change from young to old, p < 0.05). Furthermore, in the old group, SVP and DVP vascular parameters were lower in the transgenics compared to age-matched controls (p < 0.05). Our analysis demonstrates that normal aging and progression of AD lead to various degrees of vascular alterations in the retina. Specifically, compared to normal aging, changes in vascular features of SVP and DVP regions of the retina are accelerated during AD progression. Considering recent advances in the field of depth-resolved imaging of retinal capillary network and microangiography, noninvasive quantitative monitoring of changes in retinal vascular network parameters of SVP and DVP may serve as markers for diagnosis and staging of Alzheimer’s disease and discriminating AD-induced vascular attenuation from age-related vasculopathy.

Recently, Glypican-3 (GPC3) has been identified as a potential hepatocellular carcinoma (HCC) diagnostic and/or therapeutic target. GPC3 has been found to be up-regulated in HCC and to be absent in normal and cirrhotic liver. As yet, however, the molecular characteristics of GPC3 and its role in HCC cell physiology and development are still undefined. Human hepatocyte cultures were established from 10 HCC patients. Additional liver samples were obtained from 5 patients without cirrhosis and/or HCC. Soft agar colony formation, (co-)immunofluorescence and Western blot assays were used to characterize the hapatocyte cultures. The expression of GPC3 in the hepatocytes was silenced using siRNA, after which, apoptosis, scratch wound migration and transwell invasion assays were performed. We found that in HCC precursor hepatocytes GPC3 is increasingly expressed in different forms and at different locations, i.e., a non-cleaved form (70 kDa) was found to be localized in the cytoplasm while a N-terminal cleaved form (N-GPC3: 40 kDa) was fond to be localized in the cytoplasm and at the extracellular side of hepatocyte membranes. In addition, we found that the non-cleaved form of GPC3 co-localizes with Furin-Convertase in the Golgi apparatus. We also found that, similar to GPC3, Furin-Convertase is expressed in HCC precursor cells, suggesting a role in GPC3 processing. Subsequent siRNA-mediated GPC3 silencing resulted in a temporary inhibition of cell proliferation, migration and ivasion, while inducing apoptosis in transformed hepatocytes. Our data reveal new aspects of the role of GPC3 in early hepatocyte transformation. In addition we conclude that GPC3 may serve as a new HCC immune-therapeutic target.

Aging, pathological tau oligomers (TauO) and chronic inflammation in the brain play a central role in tauopathies, including Alzheimer's disease (AD) and frontotemporal dementia (FTD). However, the underlying mechanism of TauO-induced aging-related neuroinflammation remains unclear. We here show that TauO-associated astrocytes display a senescence-like phenotype in the brains of AD and FTD patients. TauO exposure triggers astrocyte senescence through HMGB1 release and inflammatory senescence-associated secretory phenotype (SASP), which mediate paracrine senescence in adjacent cells. HMGB1 release inhibition using ethyl pyruvate (EP) and glycyrrhizic acid (GA) prevented TauO-induced senescence through inhibition of p38-MAPK and NF-κB– the essential signaling pathways for SASP development. Despite the developed tauopathy in 12-month-old hTau mice, EP+GA treatment significantly decreased TauO and senescent cell loads in the brain, reduced neuroinflammation, and thus ameliorated cognitive functions. Collectively, TauO-induced HMGB1 release promotes cellular senescence and neuropathology, which could represent an important common pathomechanism in tauopathies including AD and FTD.

Tau aggregates propagate in brain cells and transmit to neighboring cells as well as anatomically connected brain regions by prion-like mechanisms. Soluble tau aggregates (tau oligomers) are the most toxic species that initiate neurodegeneration in tauopathies, such as Alzheimer’s disease (AD), progressive supranuclear palsy (PSP), and dementia with Lewy bodies (DLB). Exogenous tau aggregates have been shown to be internalized by brain cells; however, the precise cellular and molecular mechanisms that underlie the internalization of tau oligomers (TauO) remain elusive. Using brain-derived tau oligomers (BDTOs) from AD, PSP, and DLB patients, we investigated neuronal internalization mechanisms of BDTOs, including the heparan sulfate proteoglycan (HSPG)-mediated pathway, clathrin-mediated pathway, and caveolae-mediated pathway. Here, we demonstrated that the HSPG-mediated pathway regulates internalization of BDTOs from AD and DLB, while HSPG-mediated and other alternative pathways are involved in the internalization of PSP-derived tau oligomers. HSPG antagonism significantly reduced the internalization of TauO, prevented tau translocation to the endosomal–lysosomal system, and decreased levels of hyperphosphorylated tau in neurons, the well-known contributor for neurofibrillary tangles (NFT) accumulation, degeneration of neurons, and cognitive decline. Furthermore, siRNA-mediated silencing of heparan sulfate (HS)-synthesizing enzyme, exostosin-2, leads to decreased internalization of BDTOs, prevented tau-induced autophagy–lysosomal pathway impairment, and decreased hyperphosphorylated tau levels. Collectively, these findings suggest that HSPG-mediated endocytosis and exostsin-2 are involved in neuronal internalization of TauO and subsequent tau-dependent neuropathology in AD and DLB.

Soluble tau aggregates have been highlighted in mTBI-induced cellular dysfunction and neurodegeneration. We used an established mTBI model to investigate differences between tau oligomers isolated after single vs. repetitive mTBI. Single and repeated blast tau oligomers expressed distinct strain-indicating characteristics that correlated with the risk of neurodegeneration., Mild traumatic brain injury accounts for the majority of head injuries and has been correlated with neurodegeneration and dementia. While repetitive mild traumatic brain injury is highly correlated to neurodegeneration, the correlation of a single mild traumatic brain injury with neurodegeneration is still unclear. Because tau aggregates are the main form of mild traumatic brain injury induced pathology, toxic forms of tau protein most likely play a role in the development of post-mild traumatic brain injury neurodegeneration. Therefore, it becomes crucial to characterize the properties of soluble tau aggregates in single versus repetitive mild traumatic brain injury. Herein, we isolated tau oligomers from wild-type mice exposed to single or repetitive mild traumatic brain injury and characterized the tau aggregates at functional, biochemical and biophysical levels. We demonstrated that single versus repetitive mild traumatic brain injuries frequencies lead to the formation of different tau oligomeric polymorphisms. These polymorphisms express different long-term potentiation impairment potencies, toxicity potentials, morphologies and strain indicating properties. To our knowledge, this is the first evidence that soluble tau oligomers derived from single versus repetitive mild traumatic brain injuries form distinct polymorphisms that possibly correlate with the risk of neurodegeneration after mild traumatic brain injury., Graphical Abstract Graphical Abstract

The exact mechanisms leading to neurodegeneration in Alzheimer's disease (AD) and other tauopathies are not yet entirely understood. However, it is known that several RNA‐binding proteins (RBPs) form toxic aggregates and also interact with tau in such granules in tauopathies, including AD. The Musashi (MSI) family of RBPs, consisting of two homologues: Musashi1 and Musashi2, have not been extensively investigated in neurodegenerative diseases. Here, using a tau inducible HEK (iHEK) model we investigate whether MSI proteins contribute to the aggregation of toxic tau oligomers (TauO). Wild‐type and mutant P301L tau iHEK cells are used to study the effect of different tau variants on the cellular localization of MSI proteins. Interestingly, we observe that tau co‐localizes with MSI in the cytoplasm and nuclei, altering the nuclear transport of MSI. Furthermore, incremental changes in the size and density of nuclear MSI/tau foci are observed. We also report here that TauO interact with MSI to cause the formation of distinct nuclear aggregates. Moreover, tau/MSI aggregates induce structural changes to LaminB1, leading to nuclear instability. These results illustrate a possible mechanism of neurodegeneration mediated by the aggregation of MSI proteins and TauO, suggesting that MSI plays a critical role in cellular dysfunction.

Alzheimer’s disease (AD) is the most common neurodegenerative disorder associated with structural and functional alterations of brain cells causing progressive deterioration of memory and other cognitive functions. Recent studies demonstrate that several neurodegenerative diseases, including AD exhibit RNA-binding proteins (RBPs) pathologies, including TAR DNA -binding protein (TDP-43), fused in sarcoma (FUS), superoxide dismutase (SOD1) and T-interacting antigen-1 (TIA-1), highlighting the role of RBPs in neurodegeneration. One such group of RBPs, Musashi proteins comprised of MSI1 and MSI2, has been long studied in neurogenesis and cancer biology. Herein, we have investigated the aggregation properties of MSI1 and MSI2 by in vitro assays, their expression and accumulation as well as their possible interactions with other cellular proteins, such as tau in AD pathology. We have performed atomic force microscopy, Western blot, and immunoprecipitation to demonstrate the aggregation properties of recombinant Musashi proteins. Furthermore, we have studied cortical brain sections from AD (N = 4) and age-matched non-demented subjects (N = 4) by Western blot and immunofluorescence microscopy to investigate MSI1 and MSI2 levels and their localization in human brain tissues. Musashi proteins showed in vitro aggregation properties by forming oligomers. We have observed an increase in Musashi proteins levels in AD brain tissues as compared with age-matched non-demented subjects. Moreover, Musashi proteins are observed to form oligomers in the diseased brain tissues. Interestingly, the co-immunofluorescence study has revealed a change in fluorescence pattern of oligomeric Musashi proteins and tau with a high association in the perinuclear area of the cells suggesting changes in function of Musashi proteins. Our data have demonstrated for the first time that MSI1 and MSI2 are present in an oligomeric state in AD brains compared to the age-matched non-demented subjects and that these large assemblies co-localize with tau contributing to the neurodegenerative pathogenesis. Electronic supplementary material The online version of this article (10.1186/s40478-018-0615-0) contains supplementary material, which is available to authorized users.

Hepatocellular carcinoma (HCC) is a cancer with increasing incidence in the USA and high mortality rate. HCC is often difficult to treat due to underlying comorbidities such as cirrhosis. However, the application of loco-regional thermal ablation using radio frequency (RF) and trans-arterial embolization with chemotherapy (TACE) or without (TAE) has shown promising results in the treatment of patients not amenable to surgical resection or liver transplantation. Conventionally, RF and TAE are performed in two separated sessions or two steps and often RF ablation is performed percutaneously. However, no consensus has been reached regarding the ideal interval between the two treatments. In this article, we discuss the feasibility and benefits of a single-step TAE in combination with laparoscopic RF ablation in one operative session. We also present a case where this procedure has been successfully performed demonstrating its feasibility. We suggest that the use of laparoscopic RF ablation in the same surgical session as TAE is feasible and potentially offers several advantages over the two-step process that is usually performed with embolization followed by percutaneous RF with a long time interval. In this article we discuss such advantages.

Hepatocellular carcinoma (HCC) is the most common primary liver cancer. Currently, there is limited knowledge of neoplastic transformation of hepatocytes in HCC. In clinical practice, the high rate of HCC local recurrence suggests the presence of different hepatocyte populations within the liver and particularly in the tumor proximity. The present study investigated primary human hepatocyte cultures obtained from liver specimens of patients affected by cirrhosis and HCC, their proliferation and transformation. Liver samples were obtained from seven HCC cirrhotic patients and from three patients with normal liver (NL). Immediately after surgery, cell outgrowth and primary cultures were obtained from the HCC lesion, the cirrhotic tissue proximal (CP, 1-3 cm) and distal (CD, >5 cm) to the margin of the neoplastic lesion, or from NL. Cells were kept in culture for 16 weeks. Morphologic analyses were performed and proliferation rate of the different cell populations compared over time. Glypican-3, Heppar1, Arginase1 and CD-44 positivity were tested. The degree of invasiveness of cells acquiring neoplastic characteristics was studied with a transwell migration assay. We observed that HCC cells maintained their morphology and unmodified neoplastic characteristics when cultured. Cells isolated from CP, showed a progressive morphologic transformation in HCC-like cells accompanied by modification of markers expression with signs of invasiveness. Absence of HCC contamination in the CP isolates was confirmed. In CD samples some of these characteristics were present and at significantly lower levels. With the present study, we are the first to have identified and describe the existence of human hepatocytes near the cancerous lesion that can transform in HCC in vitro.

Introduction: Primary liver cancer is a significant cause of cancer-related death in both the United States and the world at large. Hepatocellular carcinoma comprises 90% of these primary liver cancers and has numerous known etiologies. Evaluation of these identified etiologies and other traditional risk factors cannot explain the high incidence rates of hepatocellular carcinoma in Texas. Texas is home to the second largest petrochemical industry and agricultural industry in the nation; industrial activity and exposure to pathogenic chemicals have never been assessed as potential links to the state's increased incidence rate of hepatocellular carcinoma. Methods: The association between the county-level concentrations of 4 air pollutants known to be linked to liver cancer, vinyl chloride, arsenic, benzene, and 1,3-butadiene, and hepatocellular carcinoma rates was evaluated using nonparametric generalized additive logistic regression and gamma regression models. Hepatocellular carcinoma incidence rates for 2000-2013 were evaluated in comparison to 1996 and 1999 pollution concentrations and hepatocellular carcinoma rates for the subset of 2006-2013 were evaluated in comparison to 2002 and 2005 pollution concentrations, respectively. Results: The analysis indicates that the relationship between the incidence of liver cancer and air pollution and risk factors is nonlinear. There is a consistent significant positive association between the incidence of liver cancer and hepatitis C prevalence rates (gamma all years, p < 0.05) and vinyl chloride concentrations (logistic 2002 and 2005, p < 0.0001; gamma 2002 and 2005, p < 0.05). Conclusions: This study suggests that vinyl chloride is a significant contributor to the incidence of liver cancer in Texas. The relationship is notably nonlinear. Further, the study supports the association between incidence of liver cancer and prevalence of hepatitis B.

The incidence of hepatocellular carcinoma (HCC), the most common primary liver cancer, is increasing in the US and tripled during the past two decades. The reasons for such phenomenon remain poorly understood. Texas is among continental states with the highest incidence of liver cancer with an annual increment of 5.7%. Established risk factors for HCC include Hepatitis B and C (HBV, HCV) viral infection, alcohol, tobacco and suspected risk factors include obesity and diabetes. While distribution of these risk factors in the state of Texas is similar to the national data and homogeneous, the incidence of HCC in this state is exceptionally higher than the national average and appears to be dishomogeneous in various areas of the state suggesting that other non-recognized risk factors might play a role. No population-based studies are currently available investigating the effect of exposure to Hazardous Air Pollutants (HAPs) as a contributing risk factor for liver cancer. Incidence rate of liver cancer in Texas by counties for the time period between 2002 and 2012 was obtained from the Texas Cancer Registry (TCR). Through Principal Component Analysis (PCA) a subgroup of pollutants, explaining almost all the dataset variability, were identified and used to cluster Texas counties. The analysis generated 4 clusters showing liver cancer rate either higher or lower than national average in association with either high or low levels of HAPs emission in the environment. The study shows that the selected relevant HAPs, 10 among 253 analyzed, produce a significant correlation (P = 0.01–0.05) and some of these have been previously identified as carcinogens. An association between the increased production and consequent exposure to these HAPs and a higher presence of liver cancer in certain counties is suggested. This study provides a new insight on this complex multifactorial disease suggesting that environmental substances might play a role in the etiology of this cancer.

Glypican-3 (GPC-3), a transmembrane heparan sulfate proteoglycan (HSPG), has recently been investigated as a player in tissue-dependent cellular signaling, specifically as a regulator of growth. Noteworthy, the regulatory protein has been implicated in both stimulatory and inhibitory pathways involving cell growth. Initially, GPC-3 was thought to act as a cell cycle regulator, as a loss-of-function mutation in the gene caused a hyper-proliferative state known as Simpson-Golabi-Behmel (SGB) overgrowth syndrome. Additionally, certain cancer types have displayed a downregulation of GPC-3 expression. More recently, the protein has been evaluated as a useful marker for hepatocellular carcinoma (HCC) due to its increased expression in the liver during times of growth. In contrast, the GPC-3 marker is not detectable in normal adult liver. Immunotherapy that targets GPC-3 and its affiliated proteins is under investigation as these new biomarkers may hold potential for the detection and treatment of HCC and other diseases in which GPC-3 may be overexpressed. Studies have reported that an overexpression of GPC-3 in HCC predicts a poorer prognosis. This prognostic value further pushes the question regarding GPC-3's role in the regulation and progression of HCC. This review will summarize the current knowledge regarding the clinical aspects of GPC-3, while also synthesizing the current literature with the aim to better understand this molecule's biological interactions at a molecular level, not only in the liver, but in the rest of the body as well. Due to the existing gap in the literature surrounding GPC-3, we believe further investigation of function, structure and domains, cellular localization, and other subfields is warranted to evaluate the protein as a whole, as well as its part in the study of HCC.

Isolation of hepatocytes from cirrhotic human livers and subsequent primary culture are important new tools for laboratory research and cell-based therapeutics in the study of hepatocellular carcinoma (HCC). Using such techniques, we have previously identified different subpopulations of human hepatocytes and among them one is showing a progressive transformation of hepatocytes in HCC-like cells. We have hypothesized that increasing the distance from the neoplastic lesion might affect hepatocyte function and transformation capacity. However, limited information is available in comparing the growth and proliferation of human hepatocytes obtained from different areas of the same cirrhotic liver in relation to their distance from the HCC lesion. In this study, hepatocytes from 10 patients with cirrhosis and HCC undergoing surgical resections from specimens obtained at a proximal (CP) and distal (CD) distance from the HCC lesion were isolated and placed in primary culture. CP hepatocytes (CP-Hep) were isolated between 1 to 3 cm (leaving at least 1cm margin to avoid cancer cells and/or satellite lesions), while CD hepatocytes (CD-Hep) were isolated from more than 5 cm or from the contralateral-lobe. A statistical model was built to analyze the proliferation rates of these cells and we evaluated expression of HCC markers (Glypican-3 (GPC3), αSmooth Muscle Actin (α-SMA) and PCNA). We observed a significant difference in proliferation and in-vitro growth showing that CP-Hep had a proliferation pattern and rate significantly different than CD-Hep. Based on these data, this model can provide information to predict growth of human hepatocytes in primary culture in relation to their pre-cancerous state with significant differences in the HCC markers expression. This model provides an important innovative tool for in-vitro analysis of HCC.

Triple-negative breast cancer (TNBC) is a clinically aggressive form of breast cancer that is unresponsive to endocrine agents or trastuzumab. TNBC accounts for ~10-20% of all breast cancer cases and represents the form with the poorest prognosis. Patients with TNBC are at higher risk of early recurrence, mainly in the lungs, brain and soft tissue, therefore, there is an urgent need for new therapies. The present study was carried out in MDA-MB-231 cells, where we assessed the role of caspase-8 (casp-8), a critical effector of death receptors, also involved in non‑apoptotic functions. Analysis of casp-8 mRNA and protein levels indicated that they were up-regulated with respect to the normal human mammalian epithelial cells. We demonstrated that silencing of casp-8 by small interfering-RNA, strongly decreased MDA-MB-231 cell growth by delaying G0/G1- to S-phase transition and increasing p21, p27 and hypo-phosphorylated/active form of pRb levels. Surprisingly, casp-8-knockdown, also potently increased both the migratory and metastatic capacity of MDA-MB‑231 cells, as shown by both wound healing and Matrigel assay, and by the expression of a number of related-genes and/or proteins such as VEGFA, C-MYC, CTNNB1, HMGA2, CXCR4, KLF4, VERSICAN V1 and MMP2. Among these, KLF4, a transcriptional factor with a dual role (activator and repressor), seemed to play critical roles. We suggest that in MDA-MB‑231 cells, the endogenous expression of casp-8 might keep the cells perpetually cycling through downregulation of KLF4, the subsequent lowering of p21 and p27, and the inactivation by hyperphosphorylation of pRb. Simultaneously, by lowering the expression of some migratory and invasive genes, casp-8 might restrain the metastatic ability of the cells. Overall, our findings showed that, in MDA-MB-231 cells, casp-8 might play some unusual roles which should be better explored, in order to understand whether it might be identified as a molecular therapeutic target.

A promising therapeutic approach for intestinal failure consists in elongating the intestine with a bio-engineered segment of neo-formed autologous intestine. Using an acellular biologic scaffold (ABS), we, and others, have previously developed an autologous bio-artificial intestinal segment (BIS) that is morphologically similar to normal bowel in rodents. This neo-formed BIS is constructed with the intervention of naïve stem cells that repopulate the scaffold in vivo, and over a period of time, are transformed in different cell populations typical of normal intestinal mucosa. However, no studies are available to demonstrate that such BIS possesses functional absorptive characteristics necessary to render this strategy a possible therapeutic application. The aim of this study was to demonstrate that the BIS generated has functional absorptive capacity. Twenty male August × Copenhagen-Irish (ACI) rats were used for the study. Two-centimeter sections of ABS were transplanted in the anti-mesenteric border of the small bowel. Animals were studied at 4, 8, and 12 weeks post-engraftment. Segments of intestine with preserved vascular supply and containing the BIS were isolated and compared to intestinal segments of same length in sham control animals (n = 10). d-Xylose solution was introduced in the lumen of the intestinal segments and after 2 h, urine and blood were collected to evaluate d-Xylose levels. Quantitative analysis was performed using ELISA. Morphologic, ultrastructural, and indirect functional absorption analyses were also performed. We observed neo-formed intestinal tissue with near-normal mucosa post-implantation as expected from our previously developed model. Functional characteristics such as morphologically normal enterocytes (and other cell types) with presence of brush borders and preserved microvilli by electron microscopy, preserved water, and ion transporters/channels (by aquaporin and cystic fibrosis transmembrane conductance regulator (CFTR)) were also observed. The capacity of BIS containing neo-formed mucosa to increase absorption of d-Xylose in the blood compared to normal intestine was also confirmed. With this study, we demonstrated for the first time that BIS obtained from ABS has functional characteristics of absorption confirming its potential for therapeutic interventions.

Inadequate absorptive surface area poses a great challenge to the patients suffering a variety of intestinal diseases causing short bowel syndrome. To date, these patients are managed with total parenteral nutrition or intestinal transplantation. However, these carry significant morbidity and mortality. Currently, by emergence of tissue engineering, anticipations to utilize an alternative method to increase the intestinal absorptive surface area are increasing. In this paper, we will review the improvements made over time in attempting elongating the intestine with surgical techniques as well as using intestinal bioengineering. Performing sequential intestinal lengthening was the preliminary method applied in humans. However, these methods did not reach widespread use and has limited outcome. Subsequent experimental methods were developed utilizing scaffolds to regenerate intestinal tissue and organoids unit from the intestinal epithelium. Stem cells also have been studied and applied in all types of tissue engineering. Biomaterials were utilized as a structural support for naive cells to produce bio-engineered tissue that can achieve a near-normal anatomical structure. A promising novel approach is the elongation of the intestine with an acellular biologic scaffold to generate a neo-formed intestinal tissue that showed, for the first time, evidence of absorption in vivo. In the large intestine, studies are more focused on regeneration and engineering of sphincters and will be briefly reviewed. From the review of the existing literature, it can be concluded that significant progress has been achieved in these experimental methods but that these now need to be fully translated into a pre-clinical and clinical experimentation to become a future viable therapeutic option.