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Synapse loss is the principal cause of cognitive decline in Alzheimer's disease (AD) and related disorders (ADRD). Synapse development depends on the intricate dynamics of the neuronal cytoskeleton. Cofilin, the major protein regulating actin dynamics, can be sequestered into cofilactin rods, intra-neurite bundles of cofilin-saturated actin filaments that can disrupt vesicular trafficking and cause synaptic loss. Rods are a brain pathology in human AD and mouse models of AD and ADRD. Eliminating rods is the focus of this paper. One pathway for rod formation is triggered in ~20% of rodent hippocampal neurons by disease-related factors (e.g., soluble oligomers of Amyloid-ß (Aß)) and requires cellular prion protein (PrPC), active NADPH oxidase (NOX), and cytokine/chemokine receptors (CCRs). FDA-approved antagonists of CXCR4 and CCR5 inhibit Aß-induced rods in both rodent and human neurons with effective concentrations for 50% rod reduction (EC50) of 1-10 nM. Remarkably, two D-amino acid receptor-active peptides (RAP-103 and RAP-310) inhibit Aß-induced rods with an EC50 of ~1 pM in mouse neurons and ~0.1 pM in human neurons. These peptides are analogs of D-Ala-Peptide T-Amide (DAPTA) and share a pentapeptide sequence (TTNYT) antagonistic to several CCR-dependent responses. RAP-103 does not inhibit neuritogenesis or outgrowth even at 1 µM, >106-fold above its EC50. N-terminal methylation, or D-Thr to D-Ser substitution, decreases the rod-inhibiting potency of RAP-103 by 103-fold, suggesting high target specificity. Neither RAP peptide inhibits neuronal rod formation induced by excitotoxic glutamate, but both inhibit rods induced in human neurons by several PrPC/NOX pathway activators (Aß, HIV-gp120 protein, and IL-6). Significantly, RAP-103 completely protects against Aß-induced loss of mature and developing synapses and, at 0.1 nM, reverses rods in both rodent and human neurons (T½ ~ 3 h) even in the continuous presence of Aß. Thus, this orally available, brain-permeable peptide should be highly effective in reducing rod pathology in multifactorial neurological diseases with mixed proteinopathies acting through PrPC/NOX.

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The centrosome governs many pan-cellular processes including cell division, migration, and cilium formation. However, very little is known about its cell type-specific protein composition and the sub-organellar domains where these protein interactions take place. Here, we outline a protocol for the spatial interrogation of the centrosome proteome in human cells, such as those differentiated from induced pluripotent stem cells (iPSCs), through co-immunoprecipitation of protein complexes around selected baits that are known to reside at different structural parts of the centrosome, followed by mass spectrometry. The protocol describes expansion and differentiation of human iPSCs to dorsal forebrain neural progenitors and cortical projection neurons, harvesting and lysis of cells for protein isolation, co-immunoprecipitation with antibodies against selected bait proteins, preparation for mass spectrometry, processing the mass spectrometry output files using MaxQuant software, and statistical analysis using Perseus software to identify the enriched proteins by each bait. Given the large number of cells needed for the isolation of centrosome proteins, this protocol can be scaled up or down by modifying the number of bait proteins and can also be carried out in batches. It can potentially be adapted for other cell types, organelles, and species as well.

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Methodical screening of safe and efficient drug candidate compounds is crucial for drug development. A high-throughput and accurate compound evaluation method targeting the central nervous system can be developed using in vitro neural networks. In particular, an evaluation system based on a human-derived neural network that can act as an alternative to animal experiments is desirable to avoid interspecific differences. A microelectrode array (MEA) is one such evaluation system, and can measure in vitro neural activity; however, studies on compound evaluation criteria and in vitro to in vivo extrapolation are scarce. In this study, we identified the parameters that can eliminate the effects of solvents from neural activity data obtained using MEA allow for accurate compound evaluation. Additionally, we resolved the issue associated with compound evaluation criteria during MEA using principal component analysis by considering the neuronal activity exceeding standard deviation (SD) of the solvent as indicator of seizurogenic potential. Overall, 10 seizurogenic compounds and three negative controls were assessed using MEA-based co-cultured human-induced pluripotent stem cell-derived neurons and astrocytes, and primary rat cortical neurons. In addition, we determined rat cerebrospinal fluid (CSF) concentrations during tremor and convulsion in response to exposure to test compounds. To characterize the in vitro to in vivo extrapolation and species differences, we compared the concentrations at which neuronal activity exceeding the SD range of the solvent was detectable using the MEA system and rat CSF concentration.

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In vivo evaluations of chemicals in neurotoxicity have certain limitations due to the considerable time and cost required, necessity of extrapolation from rodents to humans, and limited information on toxicity mechanisms. To address this issue, the development of in vitro test methods using new approach methodologies (NAMs) is important to evaluate the chemicals in neurotoxicity. Microelectrode array (MEA) allows the assessment of changes in neural network activity caused by compound administration. However, studies on compound evaluation criteria are scarce. In this study, we evaluated the impact of pesticides on neural activity using MEA measurements of human iPSC-derived neurons. A principal component analysis was performed on the electrical physiological parameters obtained by MEA measurements, and the influence of excessive neural activity due to compound addition was defined using the standard deviation of neural activity with solvent addition as the reference. By using known seizurogenic compounds as positive controls for neurotoxicity in MEA and evaluating pesticides with insufficient verification of their neurotoxicity in humans, we demonstrated that these pesticides exhibit neurotoxicity in humans. In conclusion, our data suggest that the neurotoxicity evaluation method in human iPSC neurons using MEA measurements could be one of the in vitro neurotoxicity test methods that could replace animal experiments.

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Secretory pathways within dendrites of neurons have been proposed for local transport of newly synthesized proteins. However, little is known about the dynamics of the local secretory system and whether the organelles are transient or stable structures. Here, we quantify the spatial and dynamic behavior of dendritic Golgi and endosomes during differentiation of human neurons generated from induced pluripotent stem cells (iPSCs). In early neuronal development, before and during migration, the entire Golgi apparatus transiently translocates from the soma into dendrites. In mature neurons, dynamic Golgi elements, containing cis and trans cisternae, are transported from the soma along dendrites, in an actin-dependent process. Dendritic Golgi outposts are dynamic and display bidirectional movement. Similar structures were observed in cerebral organoids. Using the retention using selective hooks (RUSH) system, Golgi resident proteins are transported efficiently into Golgi outposts from the endoplasmic reticulum. This study reveals dynamic, functional Golgi structures in dendrites and a spatial map for investigating dendrite trafficking in human neurons.

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BACKGROUND: Functional network activity is a characteristic for neuronal cells, and the complexity of the network activity represents the necessary substrate to support complex brain functions. Drugs that drastically increase the neuronal network activity may have a potential higher risk for seizures in human. Although there has been some recent considerable progress made using cultures from different types of human-induced pluripotent stem cell (hiPSC) derived neurons, one of the primary limitations is the lack of - or very low - network activity. METHOD: In the present study, we investigated whether the limited neuronal network activity in commercial hiPSC-neurons (CNS.4U®) is capable of detecting drug-induced potential seizure risks. Therefore, we compared the hiPSC-results to those in rat primary neurons with known high neuronal network activity in vitro. RESULTS: Gene expression and electrical activity from in vitro developing neuronal networks were assessed at multiple time-points. Transcriptomes of 7, 28, and 50 days in vitro were analyzed and compared to those from human brain tissues. Data from measurements of electrical activity using multielectrode arrays (MEAs) indicate that neuronal networks matured gradually over time, albeit in hiPSC this developed slower than rat primary cultures. The response of neuronal networks to neuronal active reference drugs modulating glutamatergic, acetylcholinergic and GABAergic pathways could be detected in both hiPSC-neurons and rat primary neurons. However, in comparison, GABAergic responses were limited in hiPSC-neurons. CONCLUSION: Overall, despite a slower network development and lower network activity, CNS.4U® hiPSC-neurons can be used to detect drug induced changes in neuronal network activity, as shown by well-known seizurogenic drugs (affecting e.g., the Glycine receptor and Na+ channel). However, lower sensitivity to GABA antagonists has been observed.

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In a recent study, Tracy, Madero-Pérez, Swaney, et al. observed that Tau interacts with mitochondrial and presynaptic vesicle proteins in human iPSC-derived neurons. Tau with mutations that cause neurodegeneration has decreased mitochondrial interactions. This elegant interactome mapping with supporting human brain data paves the way to a better understanding of Tau pathology and future Tau-directed therapeutics.

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Genomic instability caused by a deficiency in the DNA damage response and repair has been linked to age-related cognitive decline and neurodegenerative diseases. Preventing genomic instability that ultimately leads to neuronal death may provide a broadly effective strategy to protect against multiple potential genotoxic stressors. Recently, the zinc-dependent class I histone deacetylase (HDAC1) has been identified as a critical factor for protecting neurons from deleterious effects of DNA damage in Alzheimer's disease (AD), amyotrophic lateral sclerosis (ALS), and frontotemporal dementia (FTD). Translating these observations to a novel neuroprotective therapy for AD, ALS, and FTD may be advanced by the identification of small molecules capable of increasing the deacetylase activity of HDAC1 selectively over other structurally similar HDACs. Here, we demonstrate that exifone, a drug previously shown to be effective in treating cognitive deficits associated with AD and Parkinson's disease, the molecular mechanism of which has remained poorly understood, potently activates the deacetylase activity of HDAC1. We show that exifone acts as a mixed, nonessential activator of HDAC1 that is capable of binding to both free and substrate-bound enzyme, resulting in an increased relative maximal rate of HDAC1-catalyzed deacetylation. Exifone can directly bind to HDAC1 based upon biolayer interferometry assays with kinetic and selectivity profiling, suggesting that HDAC1 is preferentially targeted compared to other class I HDACs and the kinase CDK5, which have also been implicated in neurodegeneration. Consistent with a mechanism of deacetylase activation intracellularly, the treatment of human induced pluripotent stem cell (iPSC)-derived neuronal cells resulted in globally decreased histone acetylation. Moreover, exifone treatment was neuroprotective in a tauopathy patient iPSC-derived neuronal model subject to oxidative stress. Taken together, these findings reveal exifone as a potent activator of HDAC1-mediated deacetylation, thereby offering a lead for novel therapeutic development aiming to protect genomic integrity in the context of neurodegeneration and aging.

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The Parkinson disease (PD) genetic LRRK2 gain-of-function mutations may relate to the ER pathological changes seen in PD patients at postmortem. Human induced pluripotent stem cell (iPSC)-derived neurons with the PD pathogenic LRRK2 G2019S mutation exhibited neurite collapse when challenged with the ER Ca2+ influx sarco/ER Ca2+-ATPase inhibitor thapsigargin (THP). Baseline ER Ca2+ levels measured with the ER Ca2+ indicator CEPIA-ER were lower in LRRK2 G2019S human neurons, including in differentiated midbrain dopamine neurons in vitro. After THP challenge, PD patient-derived neurons displayed increased Ca2+ influx and decreased intracellular Ca2+ buffering upon membrane depolarization. These effects were reversed following LRRK2 mutation correction by antisense oligonucleotides and gene editing. Gene expression analysis in LRRK2 G2019S neurons identified modified levels of key store-operated Ca2+ entry regulators, with no alterations in ER Ca2+ efflux. These results demonstrate PD gene mutation LRRK2 G2019S ER calcium-dependent pathogenic effects in human neurons.

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Neurons communicate by regulated secretion of chemical signals from synaptic vesicles (SVs) and dense-core vesicles (DCVs). Here, we investigated the maturation of these two secretory pathways in micro-networks of human iPSC-derived neurons. These micro-networks abundantly expressed endogenous SV and DCV markers, including neuropeptides. DCV transport was microtubule dependent, preferentially anterograde in axons, and 2-fold faster in axons than in dendrites. SV and DCV secretion were strictly Ca2+ and SNARE dependent. DCV secretion capacity matured until day in vitro (DIV) 36, with intense stimulation releasing 6% of the total DCV pool, and then plateaued. This efficiency is comparable with mature mouse neurons. In contrast, SV secretion capacity continued to increase until DIV50, with substantial further increase in secretion efficiency and decrease in silent synapses. These data show that the two secretory pathways can be studied in human neurons and that they mature differentially, with DCV secretion reaching maximum efficiency when that of SVs is still low.

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Cyanide is a life-threatening, bioterrorist agent, preventing cellular respiration by inhibiting cytochrome c oxidase, resulting in cardiopulmonary failure, hypoxic brain injury, and death within minutes. However, even after treatment with various antidotes to protect cytochrome oxidase, cyanide intoxication in humans can induce a delayed-onset neurological syndrome that includes symptoms of Parkinsonism. Additional mechanisms are thought to underlie cyanide-induced neuronal damage, including generation of reactive oxygen species. This may account for the fact that antioxidants prevent some aspects of cyanide-induced neuronal damage. Here, as a potential preemptive countermeasure against a bioterrorist attack with cyanide, we tested the CNS protective effect of carnosic acid (CA), a pro-electrophilic compound found in the herb rosemary. CA crosses the blood-brain barrier to up-regulate endogenous antioxidant enzymes via activation of the Nrf2 transcriptional pathway. We demonstrate that CA exerts neuroprotective effects on cyanide-induced brain damage in cultured rodent and human-induced pluripotent stem cell-derived neurons in vitro, and in vivo in various brain areas of a non-Swiss albino mouse model of cyanide poisoning that simulates damage observed in the human brain. Cyanide, a potential bioterrorist agent, can produce a chronic delayed-onset neurological syndrome that includes symptoms of Parkinsonism. Here, cyanide poisoning treated with the proelectrophillic compound carnosic acid, results in reduced neuronal cell death in both in vitro and in vivo models through activation of the Nrf2/ARE transcriptional pathway. Carnosic acid is therefore a potential treatment for the toxic central nervous system (CNS) effects of cyanide poisoning. ARE, antioxidant responsive element; Nrf2 (NFE2L2, Nuclear factor (erythroid-derived 2)-like 2).

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