Clinical and CSF single-cell profiling of post-COVID-19 cognitive impairment.

Natural history and mechanisms for persistent cognitive symptoms ("brain fog") following acute and often mild COVID-19 are unknown. In a large prospective cohort of people who underwent testing a median of 9 months after acute COVID-19 in the New York City/New Jersey area, we found that cognitive dysfunction is common; is not influenced by mood, fatigue, or sleepiness; and is correlated with MRI changes in very few people. In a subgroup that underwent cerebrospinal fluid analysis, there are no changes related to Alzheimer's disease or neurodegeneration. Single-cell gene expression analysis in the cerebrospinal fluid shows findings consistent with monocyte recruitment, chemokine signaling, cellular stress, and suppressed interferon response-especially in myeloid cells. Longitudinal analysis shows slow recovery accompanied by key alterations in inflammatory genes and increased protein levels of CXCL8, CCL3L1, and sTREM2. These findings suggest that the prognosis for brain fog following COVID-19 correlates with myeloid-related chemokine and interferon-responsive genes.

A paracrine circuit of IL-1ß/IL-1R1 between myeloid and tumor cells drives genotype-dependent glioblastoma progression.

Monocytes and monocyte-derived macrophages (MDMs) from blood circulation infiltrate glioblastoma (GBM) and promote growth. Here, we show that PDGFB-driven GBM cells induce the expression of the potent proinflammatory cytokine IL-1ß in MDM, which engages IL-1R1 in tumor cells, activates the NF-κB pathway, and subsequently leads to induction of monocyte chemoattractant proteins (MCPs). Thus, a feedforward paracrine circuit of IL-1ß/IL-1R1 between tumors and MDM creates an interdependence driving PDGFB-driven GBM progression. Genetic loss or locally antagonizing IL-1ß/IL-1R1 leads to reduced MDM infiltration, diminished tumor growth, and reduced exhausted CD8+ T cells and thereby extends the survival of tumor-bearing mice. In contrast to IL-1ß, IL-1α exhibits antitumor effects. Genetic deletion of Il1a/b is associated with decreased recruitment of lymphoid cells and loss-of-interferon signaling in various immune populations and subsets of malignant cells and is associated with decreased survival time of PDGFB-driven tumor-bearing mice. In contrast to PDGFB-driven GBM, Nf1-silenced tumors have a constitutively active NF-κB pathway, which drives the expression of MCPs to recruit monocytes into tumors. These results indicate local antagonism of IL-1ß could be considered as an effective therapy specifically for proneural GBM.

Charting the Next Road Map for CSF Biomarkers in Alzheimer's Disease and Related Dementias.

Clinical prediction of underlying pathologic substrates in people with Alzheimer's disease (AD) dementia or related dementia syndromes (ADRD) has limited accuracy. Etiologic biomarkers - including cerebrospinal fluid (CSF) levels of AD proteins and cerebral amyloid PET imaging - have greatly modernized disease-modifying clinical trials in AD, but their integration into medical practice has been slow. Beyond core CSF AD biomarkers (including beta-amyloid 1-42, total tau, and tau phosphorylated at threonine 181), novel biomarkers have been interrogated in single- and multi-centered studies with uneven rigor. Here, we review early expectations for ideal AD/ADRD biomarkers, assess these goals' future applicability, and propose study designs and performance thresholds for meeting these ideals with a focus on CSF biomarkers. We further propose three new characteristics: equity (oversampling of diverse populations in the design and testing of biomarkers), access (reasonable availability to 80% of people at risk for disease, along with pre- and post-biomarker processes), and reliability (thorough evaluation of pre-analytical and analytical factors influencing measurements and performance). Finally, we urge biomarker scientists to balance the desire and evidence for a biomarker to reflect its namesake function, indulge data- as well as theory-driven associations, re-visit the subset of rigorously measured CSF biomarkers in large datasets (such as Alzheimer's disease neuroimaging initiative), and resist the temptation to favor ease over fail-safe in the development phase. This shift from discovery to application, and from suspended disbelief to cogent ingenuity, should allow the AD/ADRD biomarker field to live up to its billing during the next phase of neurodegenerative disease research.

Melanoma Cell Intrinsic GABAA Receptor Enhancement Potentiates Radiation and Immune Checkpoint Inhibitor Response by Promoting Direct and T Cell-Mediated Antitumor Activity.

PURPOSE: Most patients with metastatic melanoma show variable responses to radiation therapy and do not benefit from immune checkpoint inhibitors. Improved strategies for combination therapy that leverage potential benefits from radiation therapy and immune checkpoint inhibitors are critical. METHODS AND MATERIALS: We analyzed metastatic melanoma tumors in the TCGA cohort for expression of genes coding for subunits of type A γ-aminobutyric acid (GABA) receptor (GABAAR), a chloride ion channel and major inhibitory neurotransmitter receptor. Electrophysiology was used to determine whether melanoma cells possess intrinsic GABAAR activity. Melanoma cell viability studies were conducted to test whether enhancing GABAAR mediated chloride transport using benzodiazepine-impaired viability. A syngeneic melanoma mouse model was used to assay the effect of benzodiazepine on tumor volume and its ability to potentiate radiation therapy or immunotherapy. Treated tumors were analyzed for changes in gene expression by RNA sequencing and presence of tumor-infiltrating lymphocytes by flow cytometry. RESULTS: Genes coding for subunits of GABAARs express functional GABAARs in melanoma cells. By enhancing GABAAR-mediated anion transport, benzodiazepines depolarize melanoma cells and impair their viability. In vivo, benzodiazepine alone reduces tumor growth and potentiates radiation therapy and α-PD-L1 antitumor activity. The combination of benzodiazepine, radiation therapy, and α-PD-L1 results in near complete regression of treated tumors and a potent abscopal effect, mediated by increased infiltration of polyfunctional CD8+ T cells. Treated tumors show expression of cytokine-cytokine receptor interactions and overrepresentation of p53 signaling. CONCLUSIONS: This study identifies an antitumor strategy combining radiation and/or an immune checkpoint inhibitor with modulation of GABAARs in melanoma using benzodiazepine.

A Chimeric Signal Peptide-Galectin-3 Conjugate Induces Glycosylation-Dependent Cancer Cell-Specific Apoptosis.

PURPOSE: Exploitation of altered glycosylation in cancer is a major goal for the design of new cancer therapy. Here, we designed a novel secreted chimeric signal peptide-Galectin-3 conjugate (sGal-3) and investigated its ability to induce cancer-specific cell death by targeting aberrantly N-glycosylated cell surface receptors on cancer cells. EXPERIMENTAL DESIGN: sGal-3 was genetically engineered from Gal-3 by extending its N-terminus with a noncleavable signal peptide from tissue plasminogen activator. sGal-3 killing ability was tested on normal and tumor cells in vitro and its antitumor activity was evaluated in subcutaneous lung cancer and orthotopic malignant glioma models. The mechanism of killing was investigated through assays detecting sGal-3 interaction with specific glycans on the surface of tumor cells and the elicited downstream proapoptotic signaling. RESULTS: We found sGal-3 preferentially binds to ß1 integrin on the surface of tumor cells due to aberrant N-glycosylation resulting from cancer-associated upregulation of several glycosyltransferases. This interaction induces potent cancer-specific death by triggering an oncoglycan-ß1/calpain/caspase-9 proapoptotic signaling cascade. sGal-3 could reduce the growth of subcutaneous lung cancers and malignant gliomas in brain, leading to increased animal survival. CONCLUSIONS: We demonstrate that sGal-3 kills aberrantly glycosylated tumor cells and antagonizes tumor growth through a novel integrin ß1-dependent cell-extrinsic apoptotic pathway. These findings provide proof-of-principle that aberrant N-oncoglycans represent valid cancer targets and support further translation of the chimeric sGal-3 peptide conjugate for cancer therapy.

Convection-enhanced delivery of cetuximab conjugated iron-oxide nanoparticles for treatment of spontaneous canine intracranial gliomas.

Cetuximab conjugated iron-oxide nanoparticles (cetuximab-IONPs) have shown both in-vitro and in-vivo anti-tumor efficacy against gliomas. The purpose of this pilot study was to evaluate the safety and potential efficacy of cetuximab-IONPs for treatment of spontaneously occurring intracranial gliomas in canines after convection-enhanced delivery (CED). The use of CED allowed for direct infusion of the cetuximab-IONPs both intratumorally and peritumorally avoiding the blood brain barrier (BBB) and limiting systemic effects. A total of eight dogs participated in the study and only two developed mild post-operative complications, which resolved with medical therapy. All canines underwent a single CED treatment of the cetuximab-IONPs over 3 days and did not receive any further adjuvant treatments. Volumetric analysis showed a median reduction in tumor size of 54.9% by MRI at 1-month (4-6 weeks) follow-up. Five dogs were euthanized due to recurrence of neurological signs other than seizures, two due to recurrent seizures, and one dog died in his sleep. Median survival time after surgery was 248 days (mean 367 days).

5-Aminolevulinic Acid Guided Sampling of Glioblastoma Microenvironments Identifies Pro-Survival Signaling at Infiltrative Margins.

Glioblastoma (GBM) contains diverse microenvironments with uneven distributions of oncogenic alterations and signaling networks. The diffusely infiltrative properties of GBM result in residual tumor at neurosurgical resection margins, representing the source of relapse in nearly all cases and suggesting that therapeutic efforts should be focused there. To identify signaling networks and potential druggable targets across tumor microenvironments (TMEs), we utilized 5-ALA fluorescence-guided neurosurgical resection and sampling, followed by proteomic analysis of specific TMEs. Reverse phase protein array (RPPA) was performed on 205 proteins isolated from the tumor margin, tumor bulk, and perinecrotic regions of 13 previously untreated, clinically-annotated and genetically-defined high grade gliomas. Differential protein and pathway signatures were established and then validated using western blotting, immunohistochemistry, and comparable TCGA RPPA datasets. We identified 37 proteins differentially expressed across high-grade glioma TMEs. We demonstrate that tumor margins were characterized by pro-survival and anti-apoptotic proteins, whereas perinecrotic regions were enriched for pro-coagulant and DNA damage response proteins. In both our patient cohort and TCGA cases, the data suggest that TMEs possess distinct protein expression profiles that are biologically and therapeutically relevant.

Intraoperative Spectroscopy with Ultrahigh Sensitivity for Image-Guided Surgery of Malignant Brain Tumors.

Intraoperative cancer imaging and fluorescence-guided surgery have attracted considerable interest because fluorescence signals can provide real-time guidance to assist a surgeon in differentiating cancerous and normal tissues. Recent advances have led to the clinical use of a natural fluorophore called protoporphyrin IX (PpIX) for image-guided surgical resection of high-grade brain tumors (glioblastomas). However, traditional fluorescence imaging methods have only limited detection sensitivity and identification accuracy and are unable to detect low-grade or diffuse infiltrating gliomas (DIGs). Here we report a low-cost hand-held spectroscopic device that is capable of ultrasensitive detection of protoporphyrin IX fluorescence in vivo, together with intraoperative spectroscopic data obtained from both animal xenografts and human brain tumor specimens. The results indicate that intraoperative spectroscopy is at least 3 orders of magnitude more sensitive than the current surgical microscopes, allowing ultrasensitive detection of as few as 1000 tumor cells. For detection specificity, intraoperative spectroscopy allows the differentiation of brain tumor cells from normal brain cells with a contrast signal ratio over 100. In vivo animal studies reveal that protoporphyrin IX fluorescence is strongly correlated with both MRI and histological staining, confirming that the fluorescence signals are highly specific to tumor cells. Furthermore, ex vivo spectroscopic studies of excised brain tissues demonstrate that the hand-held spectroscopic device is capable of detecting diffuse tumor margins with low fluorescence contrast that are not detectable with current systems in the operating room. These results open new opportunities for intraoperative detection and fluorescence-guided resection of microscopic and low-grade glioma brain tumors with invasive or diffusive margins.

Radiosensitivity enhancement of radioresistant glioblastoma by epidermal growth factor receptor antibody-conjugated iron-oxide nanoparticles.

The epidermal growth factor receptor deletion variant EGFRvIII is known to be expressed in a subset of patients with glioblastoma (GBM) tumors that enhances tumorigenicity and also accounts for radiation and chemotherapy resistance. Targeting the EGFRvIII deletion mutant may lead to improved GBM therapy and better patient prognosis. Multifunctional magnetic nanoparticles serve as a potential clinical tool that can provide cancer cell targeted drug delivery, imaging, and therapy. Our previous studies have shown that an EGFRvIII-specific antibody and cetuximab (an EGFR- and EGFRvIII-specific antibody), when bioconjugated to IONPs (EGFRvIII-IONPs or cetuximab-IONPs respectively), can simultaneously provide sensitive cancer cell detection by magnetic resonance imaging (MRI) and targeted therapy of experimental GBM. In this study, we investigated whether cetuximab-IONPs can additionally allow for the radiosensitivity enhancement of GBM. Cetuximab-IONPs were used in combination with single (10 Gy × 1) or multiple fractions (10 Gy × 2) of ionizing radiation (IR) for radiosensitization of EGFRvIII-overexpressing human GBM cells in vitro and in vivo after convection-enhanced delivery (CED). A significant GBM antitumor effect was observed in vitro after treatment with cetuximab-IONPs and subsequent single or fractionated IR. A significant increase in overall survival of nude mice implanted with human GBM xenografts was found after treatment by cetuximab-IONP CED and subsequent fractionated IR. Increased DNA double strands breaks (DSBs), as well as increased reactive oxygen species (ROS) formation, were felt to represent the mediators of the observed radiosensitization effect with the combination therapy of IR and cetuximab-IONPs treatment.

Targeted therapy of glioblastoma stem-like cells and tumor non-stem cells using cetuximab-conjugated iron-oxide nanoparticles.

Malignant gliomas remain aggressive and lethal primary brain tumors in adults. The epidermal growth factor receptor (EGFR) is frequently overexpressed in the most common malignant glioma, glioblastoma (GBM), and represents an important therapeutic target. GBM stem-like cells (GSCs) present in tumors are felt to be highly tumorigenic and responsible for tumor recurrence. Multifunctional magnetic iron-oxide nanoparticles (IONPs) can be directly imaged by magnetic resonance imaging (MRI) and designed to therapeutically target cancer cells. The targeting effects of IONPs conjugated to the EGFR inhibitor, cetuximab (cetuximab-IONPs), were determined with EGFR- and EGFRvIII-expressing human GBM neurospheres and GSCs. Transmission electron microscopy revealed cetuximab-IONP GBM cell binding and internalization. Fluorescence microscopy and Prussian blue staining showed increased uptake of cetuximab-IONPs by EGFR- as well as EGFRvIII-expressing GSCs and neurospheres in comparison to cetuximab or free IONPs. Treatment with cetuximab-IONPs resulted in a significant antitumor effect that was greater than with cetuximab alone due to more efficient, CD133-independent cellular targeting and uptake, EGFR signaling alterations, EGFR internalization, and apoptosis induction in EGFR-expressing GSCs and neurospheres. A significant increase in survival was found after cetuximab-IONP convection-enhanced delivery treatment of 3 intracranial rodent GBM models employing human EGFR-expressing GBM xenografts.

Nanotechnology applications for glioblastoma.

Glioblastoma remains one of the most difficult cancers to treat and represents the most common primary malignancy of the brain. Although conventional treatments have found modest success in reducing the initial tumor burden, infiltrating cancer cells beyond the main mass are responsible for tumor recurrence and ultimate patient demise. Targeting residual infiltrating cancer cells requires the development of new treatment strategies. The emerging field of cancer nanotechnology holds promise in the use of multifunctional nanoparticles for imaging and targeted therapy of glioblastoma. This article examines the current state of nanotechnology in the treatment of glioblastoma and directions of further study.

Magnetic nanoparticles: an emerging technology for malignant brain tumor imaging and therapy.

Magnetic nanoparticles (MNPs) represent a promising nanomaterial for the targeted therapy and imaging of malignant brain tumors. Conjugation of peptides or antibodies to the surface of MNPs allows direct targeting of the tumor cell surface and potential disruption of active signaling pathways present in tumor cells. Delivery of nanoparticles to malignant brain tumors represents a formidable challenge due to the presence of the blood-brain barrier and infiltrating cancer cells in the normal brain. Newer strategies permit better delivery of MNPs systemically and by direct convection-enhanced delivery to the brain. Completion of a human clinical trial involving direct injection of MNPs into recurrent malignant brain tumors for thermotherapy has established their feasibility, safety and efficacy in patients. Future translational studies are in progress to understand the promising impact of MNPs in the treatment of malignant brain tumors.

EGFRvIII antibody-conjugated iron oxide nanoparticles for magnetic resonance imaging-guided convection-enhanced delivery and targeted therapy of glioblastoma.

The magnetic nanoparticle has emerged as a potential multifunctional clinical tool that can provide cancer cell detection by magnetic resonance imaging (MRI) contrast enhancement as well as targeted cancer cell therapy. A major barrier in the use of nanotechnology for brain tumor applications is the difficulty in delivering nanoparticles to intracranial tumors. Iron oxide nanoparticles (IONP; 10 nm in core size) conjugated to a purified antibody that selectively binds to the epidermal growth factor receptor (EGFR) deletion mutant (EGFRvIII) present on human glioblastoma multiforme (GBM) cells were used for therapeutic targeting and MRI contrast enhancement of experimental glioblastoma, both in vitro and in vivo, after convection-enhanced delivery (CED). A significant decrease in glioblastoma cell survival was observed after nanoparticle treatment and no toxicity was observed with treatment of human astrocytes (P < 0.001). Lower EGFR phosphorylation was found in glioblastoma cells after EGFRvIIIAb-IONP treatment. Apoptosis was determined to be the mode of cell death after treatment of GBM cells and glioblastoma stem cell-containing neurospheres with EGFRvIIIAb-IONPs. MRI-guided CED of EGFRvIIIAb-IONPs allowed for the initial distribution of magnetic nanoparticles within or adjacent to intracranial human xenograft tumors and continued dispersion days later. A significant increase in animal survival was found after CED of magnetic nanoparticles (P < 0.01) in mice implanted with highly tumorigenic glioblastoma xenografts (U87DeltaEGFRvIII). IONPs conjugated to an antibody specific to the EGFRvIII deletion mutant constitutively expressed by human glioblastoma tumors can provide selective MRI contrast enhancement of tumor cells and targeted therapy of infiltrative glioblastoma cells after CED.

Transcriptional control of the tumor- and hypoxia-marker carbonic anhydrase 9: A one transcription factor (HIF-1) show?

Transcriptional activation by hypoxia is mediated by the hypoxia-inducible factor (HIF) via binding to the hypoxia-responsive element (HRE). Hypoxia in solid tumors associates with poorer outcome of the disease and reliable cellular markers of tumor hypoxia would represent a valuable diagnostic marker and a potential therapeutic target. In this category, carbonic anhydrase IX (CAIX) is one of the most promising candidates. Here, we summarize the knowledge about transcriptional regulation of CA9. The HRE is the central regulatory element in the CA9 promoter, whereas other elements are limited to lesser roles of amplification of signals received at the HRE. The analysis of known mechanisms of activation of CA9 reveals the prominent role of the HIF-1 pathway. Experimental paradigms with uncoupled HIF-1alpha stability and transcriptional activity (pericellular hypoxia, proteasomal inhibitor) provide evidence that CA9 expression monitors transcriptional activity of HIF-1, rather than the abundance of HIF-1alpha. Furthermore, these paradigms could provide a corollary to some of the apparently discordant cases (CAIX+, HIF-1alpha-) or (CAIX-, HIF-1alpha+) observed in vivo. In conclusion, the existing data support the notion that CA9, due to the unique structure of its promoter, is one of the most sensitive endogenous sensors of HIF-1 activity.

Circadian control of the NAD+ salvage pathway by CLOCK-SIRT1.

Many metabolic and physiological processes display circadian oscillations. We have shown that the core circadian regulator, CLOCK, is a histone acetyltransferase whose activity is counterbalanced by the nicotinamide adenine dinucleotide (NAD+)-dependent histone deacetylase SIRT1. Here we show that intracellular NAD+ levels cycle with a 24-hour rhythm, an oscillation driven by the circadian clock. CLOCK:BMAL1 regulates the circadian expression of NAMPT (nicotinamide phosphoribosyltransferase), an enzyme that provides a rate-limiting step in the NAD+ salvage pathway. SIRT1 is recruited to the Nampt promoter and contributes to the circadian synthesis of its own coenzyme. Using the specific inhibitor FK866, we demonstrated that NAMPT is required to modulate circadian gene expression. Our findings in mouse embryo fibroblasts reveal an interlocked transcriptional-enzymatic feedback loop that governs the molecular interplay between cellular metabolism and circadian rhythms.

Chromatin remodeling, metabolism and circadian clocks: the interplay of CLOCK and SIRT1.

Circadian rhythms govern a wide variety of physiological and metabolic functions in almost all organisms. These are controlled by the circadian clock machinery, which is mostly based on transcriptional-translational feedback loops. Importantly, 10-15% of the mammalian transcripts oscillate in a circadian manner. The complex program of gene expression that characterizes circadian physiology is possible through dynamic changes in chromatin transitions. These remodeling events are therefore of great importance to insure the proper timing and extent of circadian regulation. Recent advances in the field have revealed unexpected links between circadian regulators, chromatin remodeling and cellular metabolism. Specifically, the central clock protein CLOCK has HAT enzymatic properties. It directs acetylation of histone H3 and of its dimerization partner BMAL1 at K537, an event essential for circadian function. In addition, the HDAC activity of the NAD(+)-dependent SIRT1 enzyme is regulated in a circadian manner. It has been proposed that SIRT1 functions as an enzymatic rheostat of circadian function, transducing signals originated by cellular metabolites to the circadian clock. Thus, a specialized program of chromatin remodeling appears to be at the core of the circadian machinery.

The NAD+-dependent deacetylase SIRT1 modulates CLOCK-mediated chromatin remodeling and circadian control.

Circadian rhythms govern a large array of metabolic and physiological functions. The central clock protein CLOCK has HAT properties. It directs acetylation of histone H3 and of its dimerization partner BMAL1 at Lys537, an event essential for circadian function. We show that the HDAC activity of the NAD(+)-dependent SIRT1 enzyme is regulated in a circadian manner, correlating with rhythmic acetylation of BMAL1 and H3 Lys9/Lys14 at circadian promoters. SIRT1 associates with CLOCK and is recruited to the CLOCK:BMAL1 chromatin complex at circadian promoters. Genetic ablation of the Sirt1 gene or pharmacological inhibition of SIRT1 activity lead to disturbances in the circadian cycle and in the acetylation of H3 and BMAL1. Finally, using liver-specific SIRT1 mutant mice we show that SIRT1 contributes to circadian control in vivo. We propose that SIRT1 functions as an enzymatic rheostat of circadian function, transducing signals originated by cellular metabolites to the circadian clock.

Regulation of gene expression by hypoxia: integration of the HIF-transduced hypoxic signal at the hypoxia-responsive element.

Cells experiencing lowered O(2) levels (hypoxia) undergo a variety of biological responses in order to adapt to these unfavorable conditions. The master switch, orchestrating the cellular response to low O(2) levels, is the transcription factor, termed hypoxia-inducible factor (HIF). The alpha subunits of HIF are regulated by 2-oxoglutarate-dependent oxygenases that, in the presence of O(2), hydroxylate specific prolyl and asparaginyl residues of HIF-alpha, inducing its proteasome-dependent degradation and repression of transcriptional activity, respectively. Hypoxia inhibits oxygenases, stabilized HIF-alpha translocates to the nucleus, dimerizes with HIF-beta, recruits the coactivators p300/CBP, and induces expression of its transcriptional targets via binding to hypoxia-responsive elements (HREs). HREs are composite regulatory elements, comprising a conserved HIF-binding sequence and a highly variable flanking sequence that modulates the transcriptional response. In summary, the transcriptional response of a cell is the end product of two major functions. The first (trans-acting) is the level of activation of the HIF pathway that depends on regulation of stability and transcriptional activity of the HIF-alpha. The second (cis-acting) comprises the characteristics of endogenous HREs that are determined by the availability of transcription factors cooperating with HIF and/or individual HIF-alpha isoforms.