The Role of Extracellular Vesicles in the Progression of ALS and Their Potential as Biomarkers and Therapeutic Agents with Which to Combat the Disease

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease that impairs motor neuron function, leading to severe muscular atrophy. The non-cell autonomous and heterogeneous nature of the disease has so far hindered attempts to define ALS etiology, leaving the disease incurable and without effective treatments. Recent studies have focused on the pathologic role of intercellular communication between nerve cells to further our understanding of ALS pathophysiology. In this chapter, we summarize recent works investigating the role of extracellular vesicles (EVs) as a means of cellular crosstalk for ALS disease propagation, diagnosis, and treatment. There is growing evidence that EVs secreted by the majority of mammalian cells serve as effective biomolecule carriers to modulate recipient cell behavior. This underscores the need to understand the EV-mediated interplay that occurs within irreversibly degenerating nervous tissue in ALS patients. Additionally, we highlight current gaps in EV-ALS research, especially in terms of the pathologic role and responsibilities of specific EV cargos in diseased cells, specificity issues associated with the use of EVs in ALS diagnosis, and the efficacy of EV-mediated treatments for the restoration of diseased neuromuscular tissue. Finally, we provide suggestions for future EV-ALS research to better understand, diagnose, and cure this inveterate disease

Model for Studying the Physiological Role of Non-cell Autonomous Factors on Neuromodulation

Thesis (Ph.D.)--University of Washington, 2022The intercellular communication in the ventral horn of the spinal cord is essential for the motor function to innervate skeletal muscle. Hence, the progressive pathologies of several neuromuscular diseases (NMDs) are likely to be driven by defects in more than one cellular subtype in the ventral spinal cord. However, current neuro-centric human NMD modeling efforts do not recapitulate the nature of non-cell autonomous neuromodulation occurring upstream of the disease, which significantly limits our pathologic understanding. Therefore, in spite of the recent advancement in developing human induced pluripotent stem cell (hiPSC)-derived neurons, the pathologic mechanism of non-cell autonomous neurodegeneration remains elusive. Moreover, the current differentiation process of iPSC-derived spinal motor neurons does not accurately mirror the transcriptomic trajectories of the native ventral spinal cord development, limiting the application of the differentiated neurons. To address these shortcomings, we studied the role of exogenous physiological inputs on neuromodulation using human iPSC-derived spinal motor neurons, spinal astrocytes, and inflammatory stress factors. We studied the neuroprotective mode of astrocytes in normal tissue and demonstrated that astrocyte-derived extracellular vesicles enhance the longevity and electrophysiological function of immature iPSCneurons. Additionally, we showed that excessive exposure to the pro-inflammatory cytokine interferong, which is secreted from a broad range of immune cells, induces an extensive transcriptomic alteration of iPSC-derived motor neurons toward neurodegeneration, leads to the expression of pathologic hallmarks of ALS. Finally, we assessed the physiological relevance of the iPSC-derived motor neurons we used in this study. We performed a longitudinal transcriptomic analysis of differentiating iPSC-motor neurons at each critical time point of development, to compare it with that of the developing human spinal cord ventral horn. We expect this comprehensive transcriptomic mapping at a single-cell level will provide us invaluable guidance for improving our spinal neuron differentiation strategy. Together, we believe our findings contribute to understanding how the human motor circuit is developing and affected by surrounding factors in normal and diseased conditions, which could lead to developing more reliable human neuromuscular models eventually to identify future therapeutic targets of NMDs

Exploring the Potential for Biomaterials to Improve the Development of Spinal Motor Neurons from Induced Pluripotent Stem Cells

Neuromuscular diseases (NMDs) are primarily caused by progressive degeneration of motor neurons that leads to skeletal muscle denervation. The physiological complexity and cellular heterogeneity of individual motor units make understanding the underlying pathological mechanisms of NMDs difficult. Moreover, the demonstrable species specificity of neuromuscular synapse structure and function underscores the need to develop reliable human models of neuromuscular physiology with which to study disease etiology and test the efficacy of novel therapeutics. In this regard, human-induced pluripotent stem cells (hiPSCs) represent a valuable tool for developing such models. However, the lack of cellular diversity and transcriptomic immaturity of motor neurons derived from iPSCs has so far limited their downstream applications. To address this shortcoming, biomaterials such as 3D biopolymer scaffolds and biocompatible nanoparticles have been investigated for their ability to improve current neuronal differentiation protocols. In this review, we summarize current efforts and limitations associated with the use of functional biomaterials to increase the physiological relevance of stem cell-derived motor neurons. We also suggest potential future directions for research using biomaterials to overcome outstanding issues related to stem cell-based neuromuscular tissue production for use in NMD modeling applications

Physicochemical properties and bioavailability of naturally formulated fat-soluble vitamins extracted from agricultural products for complementary use for natural vitamin supplements

The purpose of the current study was to evaluate the physicochemical properties, digestive stability, storage stability, and intestinal absorption of formulated natural vitamins (FNV) by mixing fat-soluble vitamins extracted from agricultural products with their synthetic vitamin (SYNV) counterparts using a 6 to 4 ratio (w:w, dry weight). The FNV A, D, E, and K were evenly dispersed without crystal growth in the dispersion specifications for the functional tablet foods. The FNV A, D, E, and K had 89, 73, 65, and 36% of the digestive recovery, respectively, which was comparable to that of the SYNV. FNV D, E, and K were retained over 77%, but rapidly decreased to 15% after 6 months during accelerated storage at 25 30 and 35℃. The comparable radical scavenging capacity was found between the FNV and the SYNV. Results from the current study suggest that fat-soluble vitamins extracted from agricultural products could be reasonable complementary use for natural vitamin supplements

A facile and sensitive immunoassay for the detection of alpha-fetoprotein using gold-coated magnetic nanoparticle clusters and dynamic light scattering

A facile and sensitive immunoassay protocol for the detection of alpha-fetoprotein (AFP) was developed using gold-coated iron oxide magnetic nanoclusters and dynamic light scattering (DLS) methods. The increase in the average particle size due to AFP-mediated aggregation was measured using DLS, and the detection limit was better than 0.01 ng mL(-1)

Increased sensitivity of glioma cells to 5-fluorocytosine following photo-chemical internalization enhanced nonviral transfection of the cytosine deaminase suicide gene.

Despite advances in surgery, chemotherapy and radiotherapy, the outcomes of patients with GBM have not significantly improved. Tumor recurrence in the resection margins occurs in more than 80% of cases indicating aggressive treatment modalities, such as gene therapy are warranted. We have examined photochemical internalization (PCI) as a method for the non-viral transfection of the cytosine deaminase (CD) suicide gene into glioma cells. The CD gene encodes an enzyme that can convert the nontoxic antifungal agent, 5-fluorocytosine, into the chemotherapeutic drug, 5-fluorouracil. Multicell tumor spheroids derived from established rat and human glioma cell lines were used as in vitro tumor models. Plasmids containing either the CD gene alone or together with the uracil phosphoribosyl transferase (UPRT) gene combined with the gene carrier protamine sulfate were employed in all experiments.PCI was performed with the photosensitizer AlPcS2a and 670 nm laser irradiance. Protamine sulfate/CD DNA polyplexes proved nontoxic but inefficient transfection agents due to endosomal entrapment. In contrast, PCI mediated CD gene transfection resulted in a significant inhibition of spheroid growth in the presence of, but not in the absence of, 5-FC. Repetitive PCI induced transfection was more efficient at low CD plasmid concentration than single treatment. The results clearly indicate that AlPcS2a-mediated PCI can be used to enhance transfection of a tumor suicide gene such as CD, in malignant glioma cells and cells transfected with both the CD and UPRT genes had a pronounced bystander effect

Astrocyte-derived extracellular vesicles enhance the survival and electrophysiological function of human cortical neurons in vitro.

Neurons derived from human induced pluripotent stem cells (hiPSCs) are powerful tools for modeling neural pathophysiology and preclinical efficacy/toxicity screening of novel therapeutic compounds. However, human neurons cultured in vitro typically do not fully recapitulate the physiology of the human nervous system, especially in terms of exhibiting morphological maturation, longevity, and electrochemical signaling ability comparable to that of adult human neurons. In this study, we investigated the potential for astrocyte-derived extracellular vesicles (EVs) to modulate survival and electrophysiological function of human neurons in vitro. Specifically, we demonstrate that EVs obtained from human astrocytes promote enhanced single cell electrophysiological function and anti-apoptotic behavior in a homogeneous population of human iPSC-derived cortical neurons. Furthermore, EV-proteomic analysis was performed to identify cargo proteins with the potential to promote the physiological enhancement observed. EV cargos were found to include neuroprotective proteins such as heat shock proteins, alpha-synuclein, and lipoprotein receptor-related protein 1 (LRP1), as well as apolipoprotein E (APOE), which negatively regulates neuronal apoptosis, and a peroxidasin homolog that supports neuronal oxidative stress management. Proteins that positively regulate neuronal excitability and synaptic development were also detected, such as potassium channel tetramerization domain containing 12 (KCTD12), glucose-6- phosphate dehydrogenase (G6PD), kinesin family member 5B (KIF5B), spectrin-alpha non-erythrocytic1 (SPTAN1). The remarkable improvements in electrophysiological function and evident inhibition of apoptotic signaling in cultured neurons exposed to these cargos may hold significance for improving preclinical in vitro screening modalities. In addition, our collected data highlight the potential for EV-based therapeutics as a potential class of future clinical treatment for tackling inveterate central and peripheral neuropathies

A rapid and facile method for measuring corrosion rates using dynamic light scattering

A dynamic light scattering (DLS) method was adopted for measuring the corrosion of iron nanoparticles. The average diameter of the nanoparticles in a sodium chloride suspension increased linearly with time as iron oxide layers formed around the nanoparticles. The nanoparticle corrosion rate determined by DLS was found to be almost identical to the value obtained by conventional immersion tests (ASTM G31). The DLS method offers the advantage that measurements may be completed within several hours under natural corrosion conditions whereas the conventional immersion method requires several months. Application of the DLS method to alloy nanoparticles with a variety of chromium compositions showed that the nanoparticle sizes changed nonlinearly over time, and the curves were best fit by a first order exponential function. The first order time constants were found to be linearly related to the corrosion rates determined by ASTM G31

Exploring novel immunotherapy biomarker candidates induced by cancer deformation.

Triple-negative breast cancer (TNBC) demands urgent attention for the development of effective treatment strategies due to its aggressiveness and limited therapeutic options [1]. This research is primarily focused on identifying new biomarkers vital for immunotherapy, with the aim of developing tailored treatments specifically for TNBC, such as those targeting the PD-1/PD-L1 pathway. To achieve this, the study places a strong emphasis on investigating Ig genes, a characteristic of immune checkpoint inhibitors, particularly genes expressing Ig-like domains with altered expression levels induced by "cancer deformation," a condition associated with cancer malignancy. Human cells can express approximately 800 Ig family genes, yet only a few Ig genes, including PD-1 and PD-L1, have been developed into immunotherapy drugs thus far. Therefore, we investigated the Ig genes that were either upregulated or downregulated by the artificial metastatic environment in TNBC cell line. As a result, we confirmed the upregulation of approximately 13 Ig genes and validated them using qPCR. In summary, our study proposes an approach for identifying new biomarkers applicable to future immunotherapies aimed at addressing challenging cases of TNBC where conventional treatments fall short

Optimization of Predictive Performance for the Therapeutic Response Using Iodine Scan-Corrected Serum Thyroglobulin in Patients with Differentiated Thyroid Carcinoma

We investigated whether the performance of serum thyroglobulin (Tg) for response prediction could be improved based on the iodine uptake pattern on the post-therapeutic I-131 whole body scan (RxWBS) and the degree of thyroid tissue damage with radioactive iodine (RAI) therapy. A total of 319 patients with differentiated thyroid carcinoma who underwent total thyroidectomy and RAI therapy were included. Based on the presence/absence of focal uptake at the anterior midline of the neck above the thyroidectomy bed on RxWBS, patients were classified into positive and negative uptake groups. Serum Tg was measured immediately before (D0Tg) and 7 days after RAI therapy (D7Tg). Patients were further categorized into favorable and unfavorable Tg groups based on the prediction of excellent response (ER) using scan-corrected Tg developed through the stepwise combination of D0Tg with ratio Tg (D7Tg/D0Tg). We investigated whether the predictive performance for ER improved with the application of scan-corrected Tg compared to the single Tg cutoff. The combined approach using scan-corrected Tg showed better predictive performance for ER than the single cutoff of D0Tg alone (p < 0.001). Therefore, scan-corrected Tg can be a promising biomarker to predict the therapeutic responses after RAI therapy