Lund Human Mesencephalic Cells Research Articles

Novel Proline Transporter Inhibitor (LQFM215) Presents Antipsychotic Effect in Ketamine Model of Schizophrenia.

The glutamatergic hypothesis of schizophrenia suggests a correlation between NMDA receptor hypofunction and negative psychotic symptoms. It has been observed that the expression of the proline transporter (PROT) in the central nervous system (CNS) is associated with glutamatergic neurotransmission, as L-proline has the capacity to activate and modulate AMPA and NMDA receptors. In this study, we aimed to investigate whether inhibition of proline transporters could enhance glutamatergic neurotransmission and potentially exhibit antipsychotic effects in an experimental schizophrenia model. Using molecular dynamics analysis in silico, we validated an innovative PROT inhibitor, LQFM215. We quantified the cytotoxicity of LQFM215 in the Lund human mesencephalic cell line (LUHMES). Subsequently, we employed the ketamine-induced psychosis model to evaluate the antipsychotic potential of the inhibitor, employing behavioral tests including open-field, three-chamber interaction, and prepulse inhibition (PPI). Our results demonstrate that LQFM215, at pharmacologically active concentrations, exhibited negligible neurotoxicity when astrocytes were co-cultured with neurons. In the ketamine-induced psychosis model, LQFM215 effectively reduced hyperlocomotion and enhanced social interaction in a three-chamber social approach task across all administered doses. Moreover, the compound successfully prevented the ketamine-induced disruption of sensorimotor gating in the PPI test at all tested doses. Overall, these findings suggest that PROT inhibition could serve as a potential therapeutic target for managing symptoms of schizophrenia model.

Translocation of Distinct Alpha Synuclein Species from the Nucleus to Neuronal Processes during Neuronal Differentiation.

Alpha synuclein (aSyn) and its aggregation are crucial for the neurodegeneration of Parkinson’s disease (PD). aSyn was initially described in the nucleus and presynaptic nerve terminals. However, the biology of nuclear aSyn and the link of aSyn between subcellular compartments are less understood. Current knowledge suggests the existence of various aSyn species with distinct structural and biochemical properties. Here, we identified a C-terminal-targeting aSyn antibody (Nu-aSyn-C), which has a high immunoaffinity towards aSyn in the nucleus. Comparing the Nu-aSyn-C antibody to aSyn antibodies developed against phosphorylated or aggregated forms, we observed that nuclear aSyn differs from cytosolic aSyn by an increased phosphorylation and assembly level in proliferating cells. Employing Nu-aSyn-C, we characterized aSyn distribution during neuronal differentiation in midbrain dopaminergic neurons (mDANs) derived from human-induced pluripotent stem cells (hiPSCs) and Lund human mesencephalic cells, and in primary rat hippocampal neurons. We detected a specific translocation pattern of aSyn during neuronal differentiation from the nucleus to the soma and finally to neuronal processes. Interestingly, a remarkable shift of Nu-aSyn-C-positive species towards neurites was detected in hiPSC mDANs from a PD patient carrying aSyn gene duplication. Together, our results reveal distinct nuclear and cytosolic aSyn species that redistribute during neuronal differentiation—a process that is altered in PD-derived neurons.

Aging Human Endothelial Cells Affect Neuronal Viability and Function

It is estimated that more than 50% of adults over 80 will develop dementia, including Alzheimer's disease (AD), and the primary risk factor for dementia is aging. Aging provides the substrate for neurodegenerative disease through increased inflammation and cellular dysfunction. Despite the fact that aging is a key risk factor in neurodegenerative disease, preclinical studies frequently model age-related diseases using young rodents. In this study we used replicative senescence, a standard model of cell senescence that permits the study of cells as they progress to senescence to examine the differential effects of senescent vs. non-senescent primary human endothelial cells on a human neuronal cell line. We hypothesize that senescent (Sen) endothelial cells (ECs), which accumulate with aging, create a pro-inflammatory environment that adversely affects viability and function of neurons. First, we validated the replicative senescence process in human-derived ECs by quantifying β-galactosidase activity, a marker for cellular senescence, and the protein expression of Lamin B1, which decreases with senescence. After determining senescence phenotypes by passage number, ECs at varying stages of cellular senescence (EP: early passage, ES: early senescence, LS: late senescence) were co-cultured with LUHMES (Lund Human Mesencephalic cell line - dopamine-like neurons) to assess neuronal cell viability by measuring release of lactate dehydrogenase (LDH) into the culture medium and neurite outgrowth. Nitric oxide and extracellular vesicles released into the culture medium were also evaluated as indicators of an inflammatory environment. Co-culturing ECs with LUHMES decreased LUHMES cell viability as evidenced by an increase in LDH in co-cultures of LUHMES LS EC and by a decreased number of nuclei in LUHMES co-cultured with any of the EC types (EP, ES, LS). Co-culture with any of the EC types increased the area labeled by MAP2 and phospho-Neurofilament in LUHMES at 24h, indicating increased neurite outgrowth. At 48h, increased neurite outgrowth was observed in LUHMES+EP ECs and LUHMES+ES ECs co-cultures but not in the LUHMES + LP ECs. Nitric oxide was not increased by co-culture of LUHMES with any of the EC subtypes. The amount of extracellular vesicles as quantified by measuring the activity of acetylcholinesterase (AChE) was increased in co-cultures compared to LUHMES controls, but did not significantly differ from EC controls. In summary, ES ECs promote neurite outgrowth whereas LS ECs promote neurodegeneration. These findings suggest the importance of EC aging on neuronal cell morphology and viability, both parameters affected in age-related dementia, including Alzheimer's disease.

Assessing Bioenergetic Function in Response to Reactive Oxygen Species in Neural Cells

In this study, we characterized the bioenergetic response of the Lund human mesencephalic (LUHMES) cell line and a mouse astrocyte cell line to oxidative stress. Extracellular hydrogen peroxide (H2O2) levels and bioenergetic response were investigated in these cell lines after exposure to paraquat (PQ), a redox cycling compound that causes oxidative stress in cells. We used extracellular flux analysis to measure mitochondrial function in adherent astrocytes and LUHMES cells. Extracellular H2O2 was measured fluorometrically. H2O2 levels increased in both cell lines after exposure to 5 µM PQ for 18 h; however, the extent of H2O2 increase with astrocytes was significantly lower than that with LUHMES cells (33% vs. 67%). Measurements of basal mitochondrial respiration showed that PQ almost completely eliminated oxygen consumption rate (OCR) in astrocytes and significantly reduced it in LUHMES cells. Notably, OCR in LUHMES cells was higher than that in astrocytes, indicating that neuronal cells maintain higher oxidative metabolism than glial cells, which is also consistent with higher energy demands of the neuronal cells. Moreover, LUHMES cells exhibited a higher amount of adenosine triphosphate (ATP) being produced by oxidative phosphorylation than by glycolysis. In contrast, astrocytes demonstrated a higher glycolytic capacity and glycolytic reserve than LUHMES cells and higher ATP production rate by glycolysis than its production by mitochondrial oxidative metabolism. Collectively, this study showed the differential bioenergetic responses between astrocytes and LUHMES cells in responding to oxidative stress and the findings may provide insights into the mitochondrial reserve capacity in neurons and astrocytes in responding to oxidative stress. (First online: Mar 30, 2021)

C-Jun N-terminal Kinase Mediates Ligand-independent p75NTR Signaling in Mesencephalic Cells Subjected to Oxidative Stress

The p75 neurotrophin receptor (p75NTR) is a multifunctional protein that regulates cellular responses to pathological conditions in specific regions of the nervous system. Activation of p75NTR in certain neuronal populations induces proteolytic processing of the receptor, thereby generating p75NTR fragments that facilitate downstream signaling. Expression of p75NTR has been reported in neurons of the ventral midbrain, but p75NTR signaling mechanisms in such cells are poorly understood. Here, we used Lund Human Mesencephalic cells, a population of neuronal cells derived from the ventral mesencephalon, to evaluate the effects of oxidative stress on p75NTR signaling. Subjection of the cells to oxidative stress resulted in decreased cell-surface localization of p75NTR and intracellular accumulation of p75NTR fragments. Oxidative stress-induced p75NTR processing was reduced by pharmacological inhibition of metalloproteases or γ-secretase, but was unaltered by blockade of the ligand-binding domain of p75NTR. Furthermore, inhibition of c-Jun N-terminal Kinase (JNK) decreased p75NTR cleavage induced by oxidative damage. Altogether, these results support a mechanism of p75NTR activation in which oxidative stress stimulates JNK signaling, thereby facilitating p75NTR processing via a ligand-independent mechanism involving induction of metalloprotease and γ-secretase activity. These findings reveal a novel role for JNK in ligand-independent p75NTR signaling, and, considering the susceptibility of mesencephalic neurons to oxidative damage associated with Parkinson’s disease (PD), merit further investigation into the effects of p75NTR on PD-related neurodegeneration.

Differentiation of ciliated human midbrain-derived LUHMES neurons.

Many human cell types are ciliated, including neural progenitors and differentiated neurons. Ciliopathies are characterized by defective cilia and comprise various disease states, including brain phenotypes, where the underlying biological pathways are largely unknown. Our understanding of neuronal cilia is rudimentary, and an easy-to-maintain, ciliated human neuronal cell model is absent. The Lund human mesencephalic (LUHMES) cell line is a ciliated neuronal cell line derived from human fetal mesencephalon. LUHMES cells can easily be maintained and differentiated into mature, functional neurons within one week. They have a single primary cilium as proliferating progenitor cells and as postmitotic, differentiating neurons. These developmental stages are completely separable within one day of culture condition change. The sonic hedgehog (SHH) signaling pathway is active in differentiating LUHMES neurons. RNA-sequencing timecourse analyses reveal molecular pathways and gene-regulatory networks critical for ciliogenesis and axon outgrowth at the interface between progenitor cell proliferation, polarization and neuronal differentiation. Gene expression dynamics of cultured LUHMES neurons faithfully mimic the corresponding in vivo dynamics of human fetal midbrain. In LUHMES cells, neuronal cilia biology can be investigated from proliferation through differentiation to mature neurons.

Epigenetic Analysis in Human Neurons: Considerations for Disease Modeling in PD.

Parkinson’s disease (PD) is the second most common neurodegenerative disorder next to Alzheimer’s disease. Most PD cases are considered to be sporadic and despite considerable scientific effort, the underlying cause(s) still remain(s) enigmatic. In particular, it is unknown to which extent epigenetic alterations contribute to the pathophysiology of this devastating disorder. This is partly due to the fact that appropriate PD models are not yet available. Moreover, epigenetic patterns and mechanisms are species specific and murine systems reflect only a few of the idiosyncrasies of human neurons. For several years now, patient-specific stem cell-derived neural and non-neural cells have been employed to overcome this limitation allowing the analysis and establishment of humanized disease models for PD. Thus, several studies tried to dissect epigenetic alterations such as aberrant DNA methylation or microRNA patterns using lund human mesencephalic cell lines or neurons derived from (patient-specific) induced pluripotent stem cells. These studies demonstrate that human neurons have the potential to be used as model systems for the study of epigenetic modifications in PD such as characterizing epigenetic changes, correlating epigenetic changes to gene expression alterations and hopefully using these insights for the development of novel therapeutics. However, more research is required to define the epigenetic (age-associated) landscape of human in vitro neurons and compare these to native neurons before they can be established as suitable models for epigenetic studies in PD. In this review, we summarize the knowledge about epigenetic studies performed on human neuronal PD models, and we discuss advantages and current limitations of these (stem cell-derived) neuronal models for the study of epigenetic alterations in PD.

Loss of IGF1R in Human Astrocytes Alters Complex I Activity and Support for Neurons

The insulin/insulin-like growth factor 1 (IGF1) signaling pathways are implicated in longevity and in progression of Alzheimer’s disease. Previously, we showed that insulin-like growth factor 1 receptor (IGF1R) and downstream signaling transcripts are reduced in astrocytes in human brain with progression of Alzheimer’s neuropathology and developed a model of IGF1 signaling impairment in human astrocytes using an IGF1R-specific monoclonal antibody, MAB391. Here, we have established a novel human astrocyte-neuron co-culture system to determine whether loss of astrocytic IGF1R affects their support for neurons. Astrocyte-neuron co-cultures were developed using human primary astrocytes and differentiated Lund Human Mesencephalic Cells (LUHMES). Neurite outgrowth assays, performed to measure astrocytic support for neurons, showed astrocytes provided contact-mediated support for neurite outgrowth. Loss of IGF1R did not affect neurite outgrowth under control conditions but when challenged with hydrogen peroxide IGF1R-impaired astrocytes were less able to protect LUHMES. To determine how loss of IGF1R affects neuronal support MAB391-treated astrocytes were FACS sorted from GFP-LUHMES and their transcriptomic profile was investigated using microarrays. Changes in transcripts involved in astrocyte energy metabolism were identified, particularly NDUFA2 and NDUFB6, which are related to complex I assembly. Loss of complex I activity in MAB391-treated astrocytes validated these findings. In conclusion, reduced IGF1 signaling in astrocytes impairs their support for neurons under conditions of stress and this is associated with defects in the mitochondrial respiratory chain in astrocytes.

Reverse-transcription quantitative PCR directly from cells without RNA extraction and without isothermal reverse-transcription: a 'zero-step' RT-qPCR protocol.

We describe an ultra-rapid and sensitive method to quantify gene expression levels in cultured cells. The procedure is based on reverse-transcription quantitative PCR (RT-qPCR) directly from cells, without RNA extraction and without an isothermal reverse-transcription step. Human neurons (Lund human mesencephalic cells) were lysed at different stages of differentiation, and the lysates were used directly as template for the combined RT-qPCR reaction. We detected a down-regulation of a proliferation marker and an up-regulation of neuronal dopaminergic genes expression. We were able to detect the reference gene target from as few as a single cell, demonstrating the application of the method for efficient amplification from small cell numbers. The data were fully in line with those obtained by the standard two-step RT-qPCR from the extracted total RNA. Our ‘zero-step’ RT-qPCR method proved to be simple and reliable with a total time from cell lysis to the end of the qPCR as short as 1.5 h. It is therefore particularly suitable for RT-qPCRs where large numbers of samples must be handled, or where data are required within short time.

MitoLUHMES: An Engineered Neuronal Cell Line for the Analysis of the Motility of Mitochondria

Perturbations in the transport of mitochondria and their quality control in neuronal cells underlie many types of neurological pathologies, whereas systems enabling convenient analysis of mitochondria behavior in cellular models of neurodegenerative diseases are limited. In this study, we present a modified version of lund human mesencephalic cells, mitoLUHMES, expressing GFP and mitochondrially targeted DsRed2 fluorescent proteins, intended for in vitro analysis of mitochondria trafficking by real-time fluorescence microscopy. This cell line can be easily differentiated into neuronal phenotype and allows us to observe movements of single mitochondria in single cells grown in high-density cultures. We quantified the perturbations in mitochondria morphology and dynamics in cells treated with model neurotoxins: carbonyl cyanide m-chlorophenylhydrazone and 6-hydroxydopamine. For the first time we filmed the processes of fission, fusion, pausing, and reversal of mitochondria movement direction in LUHMES cells. We present a detailed analysis of mitochondria length, velocity, and frequency of movement for static, anterograde, and retrograde motile mitochondria. The observed neurotoxin treatment-mediated decreases in morphological and kinetic parameters of mitochondria provide foundation for the future studies exploiting mitoLUHMES as a new model for neurobiology.

Ferroptosis, a newly characterized form of cell death in Parkinson's disease that is regulated by PKC

Parkinson's disease (PD) is a complex illness characterized by progressive dopaminergic neuronal loss. Several mechanisms associated with the iron-induced death of dopaminergic cells have been described. Ferroptosis is an iron-dependent, regulated cell death process that was recently described in cancer. Our present work show that ferroptosis is an important cell death pathway for dopaminergic neurons. Ferroptosis was characterized in Lund human mesencephalic cells and then confirmed ex vivo (in organotypic slice cultures) and in vivo (in the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine mouse model). Some of the observed characteristics of ferroptosis differed from those reported previously. For example, ferroptosis may be initiated by PKCα activation, which then activates MEK in a RAS-independent manner. The present study is the first to emphasize the importance of ferroptosis dysregulation in PD. In neurodegenerative diseases like PD, iron chelators, Fer-1 derivatives and PKC inhibitors may be strong drug candidates to pharmacologically modulate the ferroptotic signaling cascade.

Cellular models for Parkinson's disease.

Developing new therapeutic strategies for Parkinson's disease requires cellular models. Current models reproduce the two most salient changes found in the brains of patients with Parkinson's disease: The degeneration of dopaminergic neurons and the existence of protein aggregates consisting mainly of α-synuclein. Cultured cells offer many advantages over studying Parkinson's disease directly in patients or in animal models. At the same time, the choice of a specific cellular model entails the requirement to focus on one aspect of the disease while ignoring others. This article is intended for researchers planning to use cellular models for their studies. It describes for commonly used cell types the aspects of Parkinson's disease they model along with technical advantages and disadvantages. It might also be helpful for researchers from other fields consulting literature on cellular models of Parkinson's disease. Important models for the study of dopaminergic neuron degeneration include Lund human mesencephalic cells and primary neurons, and a case is made for the use of non-dopaminergic cells to model pathogenesis of non-motor symptoms of Parkinson's disease. With regard to α-synuclein aggregates, this article describes strategies to induce and measure aggregates with a focus on fluorescent techniques. Cellular models reproduce the two most salient changes of Parkinson's disease, the degeneration of dopaminergic neurons and the existence of α-synuclein aggregates. This article is intended for researchers planning to use cellular models for their studies. It describes for commonly used cell types and treatments the aspects of Parkinson's disease they model along with technical advantages and disadvantages. Furthermore, this article describes strategies to induce and measure aggregates with a focus on fluorescent techniques. This article is part of a special issue on Parkinson disease.

Neuroprotection by Minocycline Caused by Direct and Specific Scavenging of Peroxynitrite

Minocycline prevents oxidative protein modifications and damage in disease models associated with inflammatory glial activation and oxidative stress. Although the drug has been assumed to act by preventing the up-regulation of proinflammatory enzymes, we probed here its direct chemical interaction with reactive oxygen species. The antibiotic did not react with superoxide or (•)NO radicals, but peroxynitrite (PON) was scavenged in the range of ∼1 μm minocycline and below. The interaction of pharmacologically relevant minocycline concentrations with PON was corroborated in several assay systems and significantly exceeded the efficacy of other antibiotics. Minocycline was degraded during the reaction with PON, and the resultant products lacked antioxidant properties. The antioxidant activity of minocycline extended to cellular systems, because it prevented neuronal mitochondrial DNA damage and glutathione depletion. Maintenance of neuronal viability under PON stress was shown to be solely dependent on direct chemical scavenging by minocycline. We chose α-synuclein (ASYN), known from Parkinsonian pathology as a biologically relevant target in chemical and cellular nitration reactions. Submicromolar concentrations of minocycline prevented tyrosine nitration of ASYN by PON. Mass spectrometric analysis revealed that minocycline impeded nitrations more effectively than methionine oxidations and dimerizations of ASYN, which are secondary reactions under PON stress. Thus, PON scavenging at low concentrations is a novel feature of minocycline and may help to explain its pharmacological activity.