Ptch2 is a Potential Regulator of Mesenchymal Stem Cells.

Ptch receptors 1 and 2 mediate Hedgehog signaling pivotal for organ development and homeostasis. In contrast to embryonic lethal Ptch1 -/- phenotype, Ptch2 -/- mice display no effect on gross phenotype. In this brief report, we provide evidence of changes in the putative incisor mesenchymal stem cell (MSC) niches that contribute to accelerated incisor growth, as well as intriguing changes in the bones and skin which suggest a role for Ptch2 in the regulation of MSCs and their regenerative potential. We employed histological, immunostaining, and computed tomography (µCT) analyses to analyze morphological differences between Ptch2 -/- and wild-type incisors, long bones, and skins. In vitro CFU and differentiation assays were used to demonstrate the MSC content and differentiation potential of Ptch2 -/- bone marrow stromal cells. Wound healing assay was performed in vivo and in vitro on 8-week-old mice to assess the effect of Ptch2 on the wound closure. Loss of Ptch2 causes increases in the number of putative MSCs in the continuously growing incisor, associated with increased vascularization observed in the tooth mesenchyme and the neurovascular bundle. Increased length and volume of Ptch2 -/- bones is linked with the increased number and augmented in vitro differentiation potential of MSCs in the bone marrow. Dynamic changes in the Ptch2 -/- skin thickness relate to changes in the mesenchymal compartment and impact the wound closure potential. The effects of Ptch2 abrogation on the postnatal MSCs suggest a crucial role for Ptch2 in Hedgehog signaling regulation of the organ regenerative potential.

Neuron-astrocyte transmitophagy is altered in Alzheimer's disease.

Under physiological conditions in vivo astrocytes internalize and degrade neuronal mitochondria in a process called transmitophagy. Mitophagy is widely reported to be impaired in neurodegeneration but it is unknown whether and how transmitophagy is altered in Alzheimer's disease (AD). Here we report that the internalization of neuronal mitochondria is significantly increased in astrocytes isolated from AD mouse brains. We also demonstrate that the degradation of neuronal mitochondria by astrocytes is increased in AD mice at the age of 6 months onwards. Furthermore, we demonstrate for the first time a similar phenomenon between human neurons and AD astrocytes, and in murine hippocampi in vivo. The results suggest the involvement of S100a4 in impaired mitochondrial transfer between neurons and AD astrocytes together with significant increases in the mitophagy regulator and reactive oxygen species in aged AD astrocytes. These findings demonstrate altered neuron-supporting functions of AD astrocytes and provide a starting point for studying the molecular mechanisms of transmitophagy in AD.

Nanoscale geometry determines mechanical biocompatibility of vertically aligned nanofibers.

Vertically aligned carbon nanofibers (VACNFs) are promising material candidates for neural biosensors due to their ability to detect neurotransmitters in physiological concentrations. However, the expected high rigidity of CNFs could induce mechanical mismatch with the brain tissue, eliciting formation of a glial scar around the electrode and thus loss of functionality. We have evaluated mechanical biocompatibility of VACNFs by growing nickel-catalyzed carbon nanofibers of different lengths and inter-fiber distances. Long nanofibers with large inter-fiber distance prevented maturation of focal adhesions, thus constraining cells from obtaining a highly spread morphology that is observed when astrocytes are being contacted with stiff materials commonly used in neural implants. A silicon nanopillar array with 500 nm inter-pillar distance was used to reveal that this inhibition of focal adhesion maturation occurs due to the surface nanoscale geometry, more precisely the inter-fiber distance. Live cell atomic force microscopy was used to confirm astrocytes being significantly softer on the long Ni-CNFs compared to other surfaces, including a soft gelatin hydrogel. We also observed hippocampal neurons to mature and form synaptic contacts when being cultured on both long and short carbon nanofibers, without having to use any adhesive proteins or a glial monoculture, indicating high cytocompatibility of the material also with neuronal population. In contrast, neurons cultured on a planar tetrahedral amorphous carbon sample showed immature neurites and indications of early-stage apoptosis. Our results demonstrate that mechanical biocompatibility of biomaterials is greatly affected by their nanoscale surface geometry, which provides means for controlling how the materials and their mechanical properties are perceived by the cells. STATEMENT OF SIGNIFICANCE: Our research article shows, how nanoscale surface geometry determines mechanical biocompatibility of apparently stiff materials. Specifically, astrocytes were prevented from obtaining highly spread morphology when their adhesion site maturation was inhibited, showing similar morphology on nominally stiff vertically aligned carbon fiber (VACNF) substrates as when being cultured on ultrasoft surfaces. Furthermore, hippocampal neurons matured well and formed synapses on these carbon nanofibers, indicating high biocompatibility of the materials. Interestingly, the same VACNF materials that were used in this study have earlier also been proven to be capable for electrophysiological recordings and sensing neurotransmitters at physiological concentrations with ultra-high sensitivity and selectivity, thus providing a platform for future neural probes or smart culturing surfaces with superior sensing performance and biocompatibility.

Cell Culture Media, Unlike the Presence of Insulin, Affect α-Synuclein Aggregation in Dopaminergic Neurons.

There are several links between insulin resistance and neurodegenerative disorders such as Parkinson's disease. However, the direct influence of insulin signaling on abnormal α-synuclein accumulation-a hallmark of Parkinson's disease-remains poorly explored. To our best knowledge, this work is the first attempt to investigate the direct effects of insulin signaling on pathological α-synuclein accumulation induced by the addition of α-synuclein preformed fibrils in primary dopaminergic neurons. We found that modifying insulin signaling through (1) insulin receptor inhibitor GSK1904529A, (2) SHIP2 inhibitor AS1949490 or (3) PTEN inhibitor VO-OHpic failed to significantly affect α-synuclein aggregation in dopaminergic neurons, in contrast to the aggregation-reducing effects observed after the addition of glial cell line-derived neurotrophic factor. Subsequently, we tested different media formulations, with and without insulin. Again, removal of insulin from cell culturing media showed no effect on α-synuclein accumulation. We observed, however, a reduced α-synuclein aggregation in neurons cultured in neurobasal medium with a B27 supplement, regardless of the presence of insulin, in contrast to DMEM/F12 medium with an N2 supplement. The effects of culture conditions were present only in dopaminergic but not in primary cortical or hippocampal cells, indicating the unique sensitivity of the former. Altogether, our data contravene the direct involvement of insulin signaling in the modulation of α-synuclein aggregation in dopamine neurons. Moreover, we show that the choice of culturing media can significantly affect preformed fibril-induced α-synuclein phosphorylation in a primary dopaminergic cell culture.

Oxidative Stress in Neurodegenerative Diseases.

Oxidative stress is typically reported in neurodegenerative diseases [...].

α-Synuclein binding activity of the plant growth promoter asterubine.

Preventing the aggregation of certain amyloid proteins has the potential to slow down the progression of diseases like Alzheimer's, Parkinson's, and type 2 diabetes mellitus. During a high-throughput screen of 300 Australian marine invertebrate extracts, the extract of the marine sponge Thorectandra sp. 4408 displayed binding activity to the Parkinson's disease-associated protein, α-synuclein. Isolation of the active component led to its identification as the known plant growth promoter asterubine (1). This molecule shares distinct structural similarities with potent amyloid beta aggregation inhibitors tramiprosate (homotaurine) and ALZ-801. Herein we report the isolation, NMR data acquired in DMSO and α-synuclein binding activity of asterubine (1).

α-Synuclein Aggregation Inhibitory Prunolides and a Dibrominated ß-Carboline Sulfamate from the Ascidian Synoicum prunum.

Seven new polyaromatic bis-spiroketal-containing butenolides, the prunolides D-I (4-9) and cis-prunolide C (10), a new dibrominated ß-carboline sulfamate named pityriacitrin C (11), alongside the known prunolides A-C (1-3) were isolated from the Australian colonial ascidian Synoicum prunum. The prunolides D-G (4-7) represent the first asymmetrically brominated prunolides, while cis-prunolide C (10) is the first reported with a cis-configuration about the prunolide's bis-spiroketal core. The prunolides displayed binding activities with the Parkinson's disease-implicated amyloid protein α-synuclein in a mass spectrometry binding assay, while the prunolides (1-5 and 10) were found to significantly inhibit the aggregation (>89.0%) of α-synuclein in a ThT amyloid dye assay. The prunolides A-C (1-3) were also tested for inhibition of pSyn aggregate formation in a primary embryonic mouse midbrain dopamine neuron model with prunolide B (2) displaying statistically significant inhibitory activity at 0.5 µM. The antiplasmodial and antibacterial activities of the isolates were also examined with prunolide C (3) displaying only weak activity against the 3D7 parasite strain of Plasmodium falciparum. Our findings reported herein suggest that the prunolides could provide a novel scaffold for the exploration of future therapeutics aimed at inhibiting amyloid protein aggregation and the treatment of numerous neurodegenerative diseases.

Human-Specific Regulation of Neurotrophic Factors MANF and CDNF by microRNAs.

Mesencephalic astrocyte derived neurotrophic factor (MANF) and cerebral dopamine neurotrophic factor (CDNF) are novel evolutionary conserved trophic factors, which exhibit cytoprotective activity via negative regulation of unfolded protein response (UPR) and inflammation. Despite multiple reports demonstrating detrimental effect of MANF/CDNF downregulation, little is known about the control of their expression. miRNAs-small non-coding RNAs-are important regulators of gene expression. Their dysregulation was demonstrated in multiple pathological processes and their ability to modulate levels of other neurotrophic factors, glial cell line-derived neurotrophic factor (GDNF) and brain-derived neurotrophic factor (BDNF), was previously reported. Here, for the first time we demonstrated direct regulation of MANF and CDNF by miRNAs. Using bioinformatic tools, reporter assay and analysis of endogenous MANF and CDNF, we identified that miR-144 controls MANF expression, and miR-134 and miR-141 downregulate CDNF levels. We also demonstrated that this effect is human-specific and is executed via predicted binding sites of corresponding miRNAs. Finally, we found that miR-382 suppressed hCDNF expression indirectly. In conclusion, we demonstrate for the first time direct regulation of MANF and CDNF expression by specific miRNAs, despite the fact their binding sites are not strongly evolutionary conserved. Furthermore, we demonstrate a functional effect of miR-144 mediated regulation of MANF on ER stress response markers. These findings emphasize that (1) prediction of miRNA targets based on evolutionary conservation may miss biologically meaningful regulatory pairs; and (2) interpretation of miRNA regulatory effects in animal models should be cautiously validated.

Domain-Independent Inhibition of CBP/p300 Attenuates α-Synuclein Aggregation.

Neurodegenerative diseases are associated with failed proteostasis and accumulation of insoluble protein aggregates that compromise neuronal function and survival. In Parkinson's disease, a major pathological finding is Lewy bodies and neurites that are mainly composed of phosphorylated and aggregated α-synuclein and fragments of organelle membranes. Here, we analyzed a series of selective inhibitors acting on multidomain proteins CBP and p300 that contain both lysine acetyltransferase and bromodomains and are responsible for the recognition and enzymatic modification of lysine residues. By using high-affinity inhibitors, A-485, GNE-049, and SGC-CBP30, we explored the role of two closely related proteins, CBP and p300, as promising targets for selective attenuation of α-synuclein aggregation. Our data show that selective CBP/p300 inhibitors may alter the course of pathological α-synuclein accumulation in primary mouse embryonic dopaminergic neurons. Hence, drug-like CBP/p300 inhibitors provide an effective approach for the development of high-affinity drug candidates preventing α-synuclein aggregation via systemic administration.

Cerebral dopamine neurotrophic factor reduces α-synuclein aggregation and propagation and alleviates behavioral alterations in vivo.

A molecular hallmark in Parkinson's disease (PD) pathogenesis are α-synuclein aggregates. Cerebral dopamine neurotrophic factor (CDNF) is an atypical growth factor that is mostly resident in the endoplasmic reticulum but exerts its effects both intracellularly and extracellularly. One of the beneficial effects of CDNF can be protecting neurons from the toxic effects of α-synuclein. Here, we investigated the effects of CDNF on α-synuclein aggregation in vitro and in vivo. We found that CDNF directly interacts with α-synuclein with a KD = 23 ± 6 nM and reduces its auto-association. Using nuclear magnetic resonance (NMR) spectroscopy, we identified interaction sites on the CDNF protein. Remarkably, CDNF reduces the neuronal internalization of α-synuclein fibrils and induces the formation of insoluble phosphorylated α-synuclein inclusions. Intra-striatal CDNF administration alleviates motor deficits in rodents challenged with α-synuclein fibrils, though it did not reduce the number of phosphorylated α-synuclein inclusions in the substantia nigra. CDNF's beneficial effects on rodent behavior appear not to be related to the number of inclusions formed in the current context, and further study of its effects on the aggregation mechanism in vivo are needed. Nonetheless, the interaction of CDNF with α-synuclein, modifying its aggregation, spreading, and associated behavioral alterations, provides novel insights into the potential of CDNF as a therapeutic strategy in PD and other synucleinopathies.

MicroRNAs Regulating Cytoskeleton Dynamics, Endocytosis, and Cell Motility-A Link Between Neurodegeneration and Cancer?

The cytoskeleton is one of the most mobile and complex cell structures. It is involved in cellular transport, cell division, cell shape formation and adaptation in response to extra- and intracellular stimuli, endo- and exocytosis, migration, and invasion. These processes are crucial for normal cellular physiology and are affected in several pathological processes, including neurodegenerative diseases, and cancer. Some proteins, participating in clathrin-mediated endocytosis (CME), play an important role in actin cytoskeleton reorganization, and formation of invadopodia in cancer cells and are also deregulated in neurodegenerative disorders. However, there is still limited information about the factors contributing to the regulation of their expression. MicroRNAs are potent negative regulators of gene expression mediating crosstalk between different cellular pathways in cellular homeostasis and stress responses. These molecules regulate numerous genes involved in neuronal differentiation, plasticity, and degeneration. Growing evidence suggests the role of microRNAs in the regulation of endocytosis, cell motility, and invasiveness. By modulating the levels of such microRNAs, it may be possible to interfere with CME or other processes to normalize their function. In malignancy, the role of microRNAs is undoubtful, and therefore changing their levels can attenuate the carcinogenic process. Here we review the current advances in our understanding of microRNAs regulating actin cytoskeleton dynamics, CME and cell motility with a special focus on neurodegenerative diseases, and cancer. We investigate whether current literature provides an evidence that microRNA-mediated regulation of essential cellular processes, such as CME and cell motility, is conserved in neurons, and cancer cells. We argue that more research effort should be addressed to study the neuron-specific functions on microRNAs. Disease-associated microRNAs affecting essential cellular processes deserve special attention both from the view of fundamental science and as future neurorestorative or anti-cancer therapies.

Detecting Oxidative Stress Biomarkers in Neurodegenerative Disease Models and Patients.

Oxidative stress is prominent in many neurodegenerative diseases. Along with mitochondrial dysfunction and pathological protein aggregation, increased levels of reactive oxygen and nitrogen species, together with impaired antioxidant defense mechanisms, are frequently observed in Alzheimer's, Parkinson's, Huntington's disease and amyotrophic lateral sclerosis. The presence of oxidative stress markers in patients' plasma and cerebrospinal fluid may aid early disease diagnoses, as well as provide clues regarding the efficacy of experimental disease-modifying therapies in clinical trials. In preclinical animal models, the detection and localization of oxidatively damaged lipids, proteins and nucleic acids helps to identify most vulnerable neuronal populations and brain areas, and elucidate the molecular pathways and the timeline of pathology progression. Here, we describe the protocol for the detection of oxidative stress markers using immunohistochemistry on formaldehyde-fixed, paraffin-embedded tissue sections, applicable to the analysis of postmortem samples and tissues from animal models. In addition, we provide a simple method for the detection of malondialdehyde in tissue lysates and body fluids, which is useful for screening and the identification of tissues and structures in the nervous system which are most affected by oxidative stress.

GDNF/RET Signaling Pathway Activation Eliminates Lewy Body Pathology in Midbrain Dopamine Neurons.

BACKGROUND: Parkinson's disease (PD) is associated with proteostasis disturbances and accumulation of misfolded α-synuclein (α-syn), a cytosolic protein present in high concentrations at pre-synaptic neuronal terminals. It is a primary constituent of intracellular protein aggregates known as Lewy neurites or Lewy bodies. Progression of Lewy pathology caused by the prion-like self-templating properties of misfolded α-syn is a characteristic feature in the brains of PD patients. Glial cell line-derived neurotrophic factor (GDNF) promotes survival of mature dopamine (DA) neurons in vitro and in vivo. However, the data on its effect on Lewy pathology is controversial. OBJECTIVES: We studied the effects of GDNF on misfolded α-syn accumulation in DA neurons. METHODS: Lewy pathology progression was modeled by the application of α-syn preformed fibrils in cultured DA neurons and in the adult mice. RESULTS: We discovered that GDNF prevented accumulation of misfolded α-syn in DA neurons in culture and in vivo. These effects were abolished by deletion of receptor tyrosine kinase rearranged during transfection (RET) or by inhibitors of corresponding signaling pathway. Expression of constitutively active RET protected DA neurons from fibril-induced α-syn accumulation. CONCLUSIONS: For the first time, we have shown the neurotrophic factor-mediated protection against the misfolded α-syn propagation in DA neurons, uncovered underlying receptors, and investigated the involved signaling pathways. These results demonstrate that activation of GDNF/RET signaling can be an effective therapeutic approach to prevent Lewy pathology spread at early stages of PD. © 2020 International Parkinson and Movement Disorder Society.

Studying Pre-formed Fibril Induced α-Synuclein Accumulation in Primary Embryonic Mouse Midbrain Dopamine Neurons.

The goal of this protocol is to establish a robust and reproducible model of α-synuclein accumulation in primary dopamine neurons. Combined with immunostaining and unbiased automated image analysis, this model allows for the analysis of the effects of drugs and genetic manipulations on α-synuclein aggregation in neuronal cultures. Primary midbrain cultures provide a reliable source of bona fide embryonic dopamine neurons. In this protocol, the hallmark histopathology of Parkinson's disease, Lewy bodies (LB), is mimicked by the addition of α-synuclein pre-formed fibrils (PFFs) directly to neuronal culture media. Accumulation of endogenous phosphorylated α-synuclein in the soma of dopamine neurons is detected by immunostaining already at 7 days after the PFF addition. In vitro cell culture conditions are also suitable for the application and evaluation of treatments preventing α-synuclein accumulation, such as small molecule drugs and neurotrophic factors, as well as lentivirus vectors for genetic manipulation (e.g., with CRISPR/Cas9). Culturing the neurons in 96 well plates increases the robustness and power of the experimental setups. At the end of the experiment, the cells are fixed with paraformaldehyde for immunocytochemistry and fluorescence microscopy imaging. Multispectral fluorescence images are obtained via automated microscopy of 96 well plates. These data are quantified (e.g., counting the number of phospho-α-synuclein-containing dopamine neurons per well) with the use of free software that provides a platform for unbiased high-content phenotype analysis. PFF-induced modeling of phosphorylated α-synuclein accumulation in primary dopamine neurons provides a reliable tool to study the underlying mechanisms mediating formation and elimination of α-synuclein inclusions, with the opportunity for high-throughput drug screening and cellular phenotype analysis.

Back and to the Future: From Neurotoxin-Induced to Human Parkinson's Disease Models.

Parkinson's disease (PD) is an age-related neurodegenerative disorder characterized by motor symptoms such as tremor, slowness of movement, rigidity, and postural instability, as well as non-motor features like sleep disturbances, loss of ability to smell, depression, constipation, and pain. Motor symptoms are caused by depletion of dopamine in the striatum due to the progressive loss of dopamine neurons in the substantia nigra pars compacta. Approximately 10% of PD cases are familial arising from genetic mutations in α-synuclein, LRRK2, DJ-1, PINK1, parkin, and several other proteins. The majority of PD cases are, however, idiopathic, i.e., having no clear etiology. PD is characterized by progressive accumulation of insoluble inclusions, known as Lewy bodies, mostly composed of α-synuclein and membrane components. The cause of PD is currently attributed to cellular proteostasis deregulation and mitochondrial dysfunction, which are likely interdependent. In addition, neuroinflammation is present in brains of PD patients, but whether it is the cause or consequence of neurodegeneration remains to be studied. Rodents do not develop PD or PD-like motor symptoms spontaneously; however, neurotoxins, genetic mutations, viral vector-mediated transgene expression and, recently, injections of misfolded α-synuclein have been successfully utilized to model certain aspects of the disease. Here, we critically review the advantages and drawbacks of rodent PD models and discuss approaches to advance pre-clinical PD research towards successful disease-modifying therapy. © 2020 The Authors.

Glial cell line-derived neurotrophic factor receptor Rearranged during transfection agonist supports dopamine neurons in Vitro and enhances dopamine release In Vivo.

BACKGROUND: Motor symptoms of Parkinson's disease (PD) are caused by degeneration and progressive loss of nigrostriatal dopamine neurons. Currently, no cure for this disease is available. Existing drugs alleviate PD symptoms but fail to halt neurodegeneration. Glial cell line-derived neurotrophic factor (GDNF) is able to protect and repair dopamine neurons in vitro and in animal models of PD, but the clinical use of GDNF is complicated by its pharmacokinetic properties. The present study aimed to evaluate the neuronal effects of a blood-brain-barrier penetrating small molecule GDNF receptor Rearranged in Transfection agonist, BT13, in the dopamine system. METHODS: We characterized the ability of BT13 to activate RET in immortalized cells, to support the survival of cultured dopamine neurons, to protect cultured dopamine neurons against neurotoxin-induced cell death, to activate intracellular signaling pathways both in vitro and in vivo, and to regulate dopamine release in the mouse striatum as well as BT13's distribution in the brain. RESULTS: BT13 potently activates RET and downstream signaling cascades such as Extracellular Signal Regulated Kinase and AKT in immortalized cells. It supports the survival of cultured dopamine neurons from wild-type but not from RET-knockout mice. BT13 protects cultured dopamine neurons from 6-Hydroxydopamine (6-OHDA) and 1-methyl-4-phenylpyridinium (MPP+ )-induced cell death only if they express RET. In addition, BT13 is absorbed in the brain, activates intracellular signaling cascades in dopamine neurons both in vitro and in vivo, and also stimulates the release of dopamine in the mouse striatum. CONCLUSION: The GDNF receptor RET agonist BT13 demonstrates the potential for further development of novel disease-modifying treatments against PD. © 2019 International Parkinson and Movement Disorder Society.

Engineered antibody-functionalized porous silicon nanoparticles for therapeutic targeting of pro-survival pathway in endogenous neuroblasts after stroke.

Generation of new neurons by utilizing the regenerative potential of adult neural stem cells (NSCs) and neuroblasts is an emerging therapeutic strategy to treat various neurodegenerative diseases, including neuronal loss after stroke. Committed to neuronal lineages, neuroblasts are differentiated from NSCs and have a lower proliferation rate. In stroke the proliferation of the neuroblasts in the neurogenic areas is increased, but the limiting factor for regeneration is the poor survival of migrating neuroblasts. Survival of neuroblasts can be promoted by small molecules; however, new drug delivery methods are needed to specifically target these cells. Herein, to achieve specific targeting, we have engineered biofunctionalized porous silicon nanoparticles (PSi NPs) conjugated with a specific antibody against polysialylated neural cell adhesion molecule (PSA-NCAM). The PSi NPs loaded with a small molecule drug, SC-79, were able to increase the activity of the Akt signaling pathway in doublecortin positive neuroblasts both in cultured cells and in vivo in the rat brain. This study opens up new possibilities to target drug effects to migrating neuroblasts and facilitate differentiation, maturation and survival of developing neurons. The conjugated PSi NPs are a novel tool for future studies to develop new therapeutic strategies aiming at regenerating functional neurocircuitry after stoke.

Interplay between MicroRNAs and Oxidative Stress in Neurodegenerative Diseases.

MicroRNAs are post-transcriptional regulators of gene expression, crucial for neuronal differentiation, survival, and activity. Age-related dysregulation of microRNA biogenesis increases neuronal vulnerability to cellular stress and may contribute to the development and progression of neurodegenerative diseases. All major neurodegenerative disorders are also associated with oxidative stress, which is widely recognized as a potential target for protective therapies. Albeit often considered separately, microRNA networks and oxidative stress are inextricably entwined in neurodegenerative processes. Oxidative stress affects expression levels of multiple microRNAs and, conversely, microRNAs regulate many genes involved in an oxidative stress response. Both oxidative stress and microRNA regulatory networks also influence other processes linked to neurodegeneration, such as mitochondrial dysfunction, deregulation of proteostasis, and increased neuroinflammation, which ultimately lead to neuronal death. Modulating the levels of a relatively small number of microRNAs may therefore alleviate pathological oxidative damage and have neuroprotective activity. Here, we review the role of individual microRNAs in oxidative stress and related pathways in four neurodegenerative conditions: Alzheimer's (AD), Parkinson's (PD), Huntington's (HD) disease, and amyotrophic lateral sclerosis (ALS). We also discuss the problems associated with the use of oversimplified cellular models and highlight perspectives of studying microRNA regulation and oxidative stress in human stem cell-derived neurons.