a-strategy-to-identify-compounds-that-affect-cell-growth-survival

A Strategy to Identify Compounds that Affect Cell Growth and Survival in Cultured Mammalian Cells at Low-to-Moderate Throughput

Cytotoxicity is a critical parameter that needs to be quantified when studying drugs that may have therapeutic benefits. Because of this, many drug ...

derivation-of-a-human-brain-organoid-with-microglia-development

Derivation of a Human Brain Organoid with Microglia Development

Three-dimensional (3D) brain organoid cultures derived from induced pluripotent stem cells (iPSC) provide an important alternative in vitro tool&#160;for ...

National Institutes of Health (NIH)

National Institutes of Health

Rapid and effective drug discovery for neurodegenerative disease is currently impeded by an inability to source primary neural cells for high-throughput and phenotypic screens. This limitation can be addressed through the use of pluripotent stem cells (PSCs), which can be derived from patient-specific samples and differentiated to neural cells for use in identifying novel compounds for the treatment of neurodegenerative diseases. We have developed an efficient protocol to culture pure populations of neurons, as confirmed by gene expression analysis, in the 96-well format necessary for screens. These differentiated neurons were subjected to viability assays to illustrate their potential in future high-throughput screens. We have also shown that organelles such as nuclei and mitochondria could be live-labeled and visualized through fluorescence, suggesting that we should be able to monitor subcellular phenotypic changes. Neurons derived from a green fluorescent protein-expressing reporter line of PSCs were live-imaged to assess markers of neuronal maturation such as neurite length and co-cultured with astrocytes to demonstrate further maturation. These studies confirm that PSC-derived neurons can be used effectively in viability and functional assays and pave the way for high-throughput screens on neurons derived from patients with neurodegenerative disorders.

Functional screening assays with neurons generated from pluripotent stem cell-derived neural stem cells.

As people are living longer, the prevalance of neurodegenerative diseases continues to rise resulting in huge socio-economic consequences. Despite major advancements in studying the pathophysiology of these diseases and a large number of clinical trials currently there is no effective treatment for these illnesses. All neuroprotective strategies have either failed or have shown only a minimal effect. There has been a major shift in recent years exploring the potential of neuroregenerative approaches. While the concept of using neurotropins for therapeutic purposes has been in existence for many years, new modes of delivery and expression of this family of molecules makes this approach now feasilble. Further neurotropin mimetics and receptor agonists are also being developed. The use of small molecules to induce the expression of neurotropins including repurposing of FDA approved drugs for this approach is another strategy being pursued. In the review we examine these new developments and discuss the potential for such approaches in the context of the pathophysiology of neurodegenerative diseases.

Neurotrophin strategies for neuroprotection: are they sufficient?

Human immunodeficiency virus type-1 (HIV) infection of the central nervous system promotes neuronal injury and apoptosis that culminate in HIV-associated neurocognitive disorders (HAND). Viral proteins, such as transactivator of transcription (Tat), have emerged as leading candidates to explain HIV-mediated neurotoxicity, though the mechanism remains unclear. To determine the effects of Tat, rat cortical neurons were exposed to nanomolar concentrations of Tat for various time points. Within a few hours, Tat induced the production of reactive oxygen species (ROS), and other indices of mitochondrial destabilization. In addition, we observed a significant induction of DNA double-strand breaks (DSBs) by Tat. We next investigated the neuroprotective activity of the pituitary adenylate cyclase-activating polypeptide 27 (PACAP27) against these cardinal features of Tat-induced neurodegeneration. PACAP27 (100 nM) inhibited all Tat-mediated toxic effects including DNA DSBs. Importantly, PACAP27 prevented the induction of neuronal loss induced by Tat. The neuroprotective effect of PACAP27 is correlated with its ability to release the anti-apoptotic chemokine CCL5. Our data support a mechanism of Tat neurotoxicity in which Tat induces mitochondrial destabilization, thus increasing the release of ROS, which causes DNA DSBs leading to cell death. PACAP27, through CCL5, mitigates the effects of Tat-induced neuronal dysfunction, suggesting that PACAP27 could be a new strategy for an adjunct therapy against HIV-associated neurocognitive disorders.

PACAP27 is protective against tat-induced neurotoxicity.

Effective combined antiretroviral therapy (cART) in HIV-infected patients has made HIV a treatable infection; however, debilitating HIV-associated neurocognitive disorders (HAND) can still affect approximately 50% of HIV-infected individuals even under cART. While cART has greatly reduced the prevalence of the most severe form of HAND, milder forms have increased in prevalence, leaving the total proportion of HIV-infected individuals suffering from HAND relatively unchanged. In this study, an in vitro drug screen identified fluconazole and paroxetine as protective compounds against HIV gp120 and Tat neurotoxicity. Using an accelerated, consistent SIV/macaque model of HIV-associated CNS disease, we tested the in vivo neuroprotective capabilities of combination fluconazole/paroxetine (FluPar) treatment. FluPar treatment protected macaques from SIV-induced neurodegeneration, as measured by neurofilament light chain in the CSF, APP accumulation in axons, and CaMKIIα in the frontal cortex, but did not significantly reduce markers of neuroinflammation or plasma or CNS viral loads. Since HIV and SIV neurodegeneration is often attributed to accompanying neuroinflammation, this study provides proof of concept that neuroprotection can be achieved even in the face of ongoing neuroinflammation and viral replication.

Combination fluconazole/paroxetine treatment is neuroprotective despite ongoing neuroinflammation and viral replication in an SIV model of HIV neurological disease.

Human primary neural tissue is a vital component for the quick and simple determination of chemical compound neurotoxicity in vitro. In particular, such tissue would be ideal for high-throughput screens that can be used to identify novel neurotoxic or neurotherapeutic compounds. We have previously established a high-throughput screening platform using human induced pluripotent stem cell (iPSC)-derived neural stem cells (NSCs) and neurons. In this study, we conducted a 2000 compound screen with human NSCs and rat cortical cells to identify compounds that are selectively toxic to each group. Approximately 100 of the tested compounds showed specific toxicity to human NSCs. A secondary screen of a small subset of compounds from the primary screen on human iPSCs, NSC-derived neurons, and fetal astrocytes validated the results from >80% of these compounds with some showing cell specific toxicity. Amongst those compounds were several cardiac glycosides, all of which were selectively toxic to the human cells. As the screen was able to reliably identify neurotoxicants, many with species and cell-type specificity, this study demonstrates the feasibility of this NSC-driven platform for higher-throughput neurotoxicity screens.

Compounds with species and cell type specific toxicity identified in a 2000 compound drug screen of neural stem cells and rat mixed cortical neurons.

The cause of neurodegeneration in progressive forms of multiple sclerosis is unknown. We investigated the impact of specific neuroinflammatory markers on human neurons to identify potential therapeutic targets for neuroprotection against chronic inflammation.

Protease-activated receptor-1 activation by granzyme B causes neurotoxicity that is augmented by interleukin-1β.

When Zika virus emerged as a public health emergency there were no drugs or vaccines approved for its prevention or treatment. We used a high-throughput screen for Zika virus protease inhibitors to identify several inhibitors of Zika virus infection. We expressed the NS2B-NS3 Zika virus protease and conducted a biochemical screen for small-molecule inhibitors. A quantitative structure-activity relationship model was employed to virtually screen ∼138,000 compounds, which increased the identification of active compounds, while decreasing screening time and resources. Candidate inhibitors were validated in several viral infection assays. Small molecules with favorable clinical profiles, especially the five-lipoxygenase-activating protein inhibitor, MK-591, inhibited the Zika virus protease and infection in neural stem cells. Members of the tetracycline family of antibiotics were more potent inhibitors of Zika virus infection than the protease, suggesting they may have multiple mechanisms of action. The most potent tetracycline, methacycline, reduced the amount of Zika virus present in the brain and the severity of Zika virus-induced motor deficits in an immunocompetent mouse model. As Food and Drug Administration-approved drugs, the tetracyclines could be quickly translated to the clinic. The compounds identified through our screening paradigm have the potential to be used as prophylactics for patients traveling to endemic regions or for the treatment of the neurological complications of Zika virus infection.

Therapeutic candidates for the Zika virus identified by a high-throughput screen for Zika protease inhibitors.

Human endogenous retroviruses have been implicated in neurodegenerative diseases including amyotrophic lateral sclerosis (ALS). Expression of human endogenous retrovirus K (HERV-K) subtype HML-2 envelope (Env) in human neuronal cultures and in transgenic mice results in neurotoxicity and neurodegeneration, and mice expressing HML-2 Env display behavioral and neuromuscular characteristics resembling ALS. This study aims to characterize the neurotoxic properties of HML-2 Env.

Human Endogenous Retrovirus K Envelope in Spinal Fluid of Amyotrophic Lateral Sclerosis Is Toxic.

There is a continuing unmet medical need to develop neuroprotective strategies to treat neurodegenerative disorders. To address this need, we screened over 2000 compounds for potential neuroprotective activity in a model of oxidative stress and found that numerous antifungal agents were neuroprotective. Of the identified compounds, fluconazole was further characterized. Fluconazole was able to prevent neurite retraction and cell death in in vitro and in vivo models of toxicity. Fluconazole protected neurons in a concentration-dependent manner and exhibited efficacy against several toxic agents, including 3-nitropropionic acid, N-methyl D-aspartate, 6-hydroxydopamine, and the HIV proteins Tat and gp120. In vivo studies indicated that systemically administered fluconazole was neuroprotective in animals treated with 3-nitropropionic acid and prevented gp120-mediated neuronal loss. In addition to neuroprotection, fluconazole also induced proliferation of neural progenitor cells in vitro and in vivo. Fluconazole mediates these effects through upregulation and signaling via the insulin growth factor-1 receptor which results in decreased cAMP production and increased phosphorylation of Akt. Blockade of the insulin growth factor-1 receptor signaling with the selective inhibitor AG1024 abrogated the effects of fluconazole. Our studies suggest that fluconazole may be an attractive candidate for treatment of neurodegenerative diseases due to its protective properties against several categories of neuronal insults and its ability to spur neural progenitor cell proliferation.

Fluconazole Is Neuroprotective via Interactions with the IGF-1 Receptor.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection can cause a wide range of neurologic complications; however, its neuropenetrance during the acute phase of the illness is unknown.

Detection of SARS-CoV-2 Nucleocapsid and Microvascular Disease in the Brain: A Case Report.

Prenatal Zika virus (ZIKV) infection is a serious global concern as it can lead to brain injury and many serious birth defects, collectively known as congenital Zika syndrome. Brain injury likely results from viral mediated toxicity in neural progenitor cells. Additionally, postnatal ZIKV infections have been linked to neurological complications, yet the mechanisms driving these manifestations are not well understood. Existing data suggest that the ZIKV envelope protein can persist in the central nervous system for extended periods of time, but it is unknown if this protein can independently contribute to neuronal toxicity. Here we find that the ZIKV envelope protein is neurotoxic, leading to overexpression of poly adenosine diphosphate -ribose polymerase 1, which can induce parthanatos. Together, these data suggest that neuronal toxicity resulting from the envelope protein may contribute to the pathogenesis of post-natal ZIKV-related neurologic complications.

Neurotoxic properties of the Zika virus envelope protein.

Human endogenous retroviruses (HERVs) are associated with the pathogenesis of amyotrophic lateral sclerosis (ALS); a disease characterized by motor neuron degeneration and cell death. The HERV-K subtype HML-2 envelope protein (HERV-K Env) is expressed in the brain, spinal cord, and cerebrospinal fluid of people living with ALS and through CD98 receptor-linked interactions causes neurodegeneration. HERV-K Env-induced increases in oxidative stress are implicated in the pathogenesis of ALS, and ferrous iron (Fe) generates reactive oxygen species (ROS). Endolysosome stores of Fe are central to iron trafficking and endolysosome deacidification releases Fe into the cytoplasm. Because HERV-K Env is an arginine-rich protein that is likely endocytosed and arginine is a pH-elevating amino acid, it is important to determine HERV-K Env effects on endolysosome pH and whether HERV-K Env-induced neurotoxicity is downstream of Fe released from endolysosomes. Here, we showed using SH-SY5Y human neuroblastoma cells and primary cultures of human cortical neurons (HCNs, information on age and sex was not available) that HERV-K Env (1) is endocytosed via CD98 receptors, (2) concentration dependently deacidified endolysosomes, (3) decreased endolysosome Fe concentrations, (4) increased cytosolic and mitochondrial Fe and ROS levels, (5) depolarized mitochondrial membrane potential, and (6) induced cell death, effects blocked by an antibody against the CD98 receptor and by the endolysosome iron chelator deferoxamine. Thus, HERV-K Env-induced increases in cytosolic and mitochondrial Fe and ROS as well as cell death appear to be mechanistically caused by HERV-K Env endocytosis, endolysosome deacidification, and endolysosome Fe efflux into the cytoplasm.

ArticleTotal : 2	Year
Publication title	2019
Publication title Cited by 1	2025

Article	Year
Functional screening assays with neurons generated from pluripotent stem cell-derived neural stem cells. Journal of biomolecular screening\| PubMed ID: 24019252	2014
Neurotrophin strategies for neuroprotection: are they sufficient? Journal of neuroimmune pharmacology : the official journal of the Society on NeuroImmune Pharmacology\| PubMed ID: 24609976	2014
PACAP27 is protective against tat-induced neurotoxicity. Journal of molecular neuroscience : MN\| PubMed ID: 24696163	2014
Combination fluconazole/paroxetine treatment is neuroprotective despite ongoing neuroinflammation and viral replication in an SIV model of HIV neurological disease. Journal of neurovirology\| PubMed ID: 25227932	2014
Compounds with species and cell type specific toxicity identified in a 2000 compound drug screen of neural stem cells and rat mixed cortical neurons. Neurotoxicology\| PubMed ID: 25454721	2014
Protease-activated receptor-1 activation by granzyme B causes neurotoxicity that is augmented by interleukin-1β. Journal of neuroinflammation\| PubMed ID: 28655310	2017
Therapeutic candidates for the Zika virus identified by a high-throughput screen for Zika protease inhibitors. Proceedings of the National Academy of Sciences of the United States of America\| PubMed ID: 33229545	2020
Human Endogenous Retrovirus K Envelope in Spinal Fluid of Amyotrophic Lateral Sclerosis Is Toxic. Annals of neurology\| PubMed ID: 35801347	2022
Fluconazole Is Neuroprotective via Interactions with the IGF-1 Receptor. Neurotherapeutics : the journal of the American Society for Experimental NeuroTherapeutics\| PubMed ID: 35831747	2022
Detection of SARS-CoV-2 Nucleocapsid and Microvascular Disease in the Brain: A Case Report. Neurology\| PubMed ID: 36526429	2023
Neurotoxic properties of the Zika virus envelope protein. Experimental neurology\| PubMed ID: 37327963	2023
HERV-K (HML-2) Envelope Protein Induces Mitochondrial Depolarization and Neurotoxicity via Endolysosome Iron Dyshomeostasis. The Journal of neuroscience : the official journal of the Society for Neuroscience\| PubMed ID: 38383499	2024

Joseph P. Steiner

Biography

JoVE Journal Publications

Other Publications