Nancy Stanslowsky

The Molecular Basis for Species-specific Activation of Human TRPA1 Protein by Protons Involves Poorly Conserved Residues within Transmembrane Domains 5 and 6

Background: Extracellular acidosis mediates pain and inflammation by activating sensory afferent neurons. Results: Protons activate and sensitize human TRPA1 in a strongly species-specific manner encoded by transmembrane domains 5 and 6. Conclusion: Our data identify TRPA1 as an ion channel likely to mediate acid-induced pain in humans. Significance: Protons are the first known endogenous agonists of TRPA1 with species-specificity for human TRPA1. The surveillance of acid-base homeostasis is concerted by diverse mechanisms, including an activation of sensory afferents. Proton-evoked activation of rodent sensory neurons is mainly mediated by the capsaicin receptor TRPV1 and acid-sensing ion channels. In this study, we demonstrate that extracellular acidosis activates and sensitizes the human irritant receptor TRPA1 (hTRPA1). Proton-evoked membrane currents and calcium influx through hTRPA1 occurred at physiological acidic pH values, were concentration-dependent, and were blocked by the selective TRPA1 antagonist HC030031. Both rodent and rhesus monkey TRPA1 failed to respond to extracellular acidosis, and protons even inhibited rodent TRPA1. Accordingly, mouse dorsal root ganglion neurons lacking TRPV1 only responded to protons when hTRPA1 was expressed heterologously. This species-specific activation of hTRPA1 by protons was reversed in both mouse and rhesus monkey TRPA1 by exchange of distinct residues within transmembrane domains 5 and 6. Furthermore, protons seem to interact with an extracellular interaction site to gate TRPA1 and not via a modification of intracellular N-terminal cysteines known as important interaction sites for electrophilic TRPA1 agonists. Our data suggest that hTRPA1 acts as a sensor for extracellular acidosis in human sensory neurons and should thus be taken into account as a yet unrecognized transduction molecule for proton-evoked pain and inflammation. The species specificity of this property is unique among known endogenous TRPA1 agonists, possibly indicating that evolutionary pressure enforced TRPA1 to inherit the role as an acid sensor in human sensory neurons.

Therapeutic Potential of Mesenchymal Stromal Cells and MSC Conditioned Medium in Amyotrophic Lateral Sclerosis (ALS) - In Vitro Evidence from Primary Motor Neuron Cultures, NSC-34 Cells, Astrocytes and Microglia

Administration of mesenchymal stromal cells (MSC) improves functional outcome in the SOD1G93A mouse model of the degenerative motor neuron disorder amyotrophic lateral sclerosis (ALS) as well as in models of other neurological disorders. We have now investigated the effect of the interaction between MSC and motor neurons (derived from both non-transgenic and mutant SOD1G93A transgenic mice), NSC-34 cells and glial cells (astrocytes, microglia) (derived again from both non-transgenic and mutant SOD1G93A ALS transgenic mice) in vitro. In primary motor neurons, NSC-34 cells and astrocytes, MSC conditioned medium (MSC CM) attenuated staurosporine (STS) - induced apoptosis in a concentration-dependent manner. Studying MSC CM-induced expression of neurotrophic factors in astrocytes and NSC-34 cells, we found that glial cell line-derived neurotrophic factor (GDNF) and ciliary neurotrophic factor (CNTF) gene expression in astrocytes were significantly enhanced by MSC CM, with differential responses of non-transgenic and mutant astrocytes. Expression of Vascular Endothelial Growth Factor (VEGF) in NSC-34 cells was significantly upregulated upon MSC CM-treatment. MSC CM significantly reduced the expression of the cytokines TNFα and IL-6 and iNOS both in transgenic and non-transgenic astrocytes. Gene expression of the neuroprotective chemokine Fractalkine (CX3CL1) was also upregulated in mutant SOD1G93A transgenic astrocytes by MSC CM treatment. Correspondingly, MSC CM increased the respective receptor, CX3CR1, in mutant SOD1G93A transgenic microglia. Our data demonstrate that MSC modulate motor neuronal and glial response to apoptosis and inflammation. MSC therefore represent an interesting candidate for further preclinical and clinical evaluation in ALS.

4‐Aminopyridine Induced Activity Rescues Hypoexcitable Motor Neurons from Amyotrophic Lateral Sclerosis Patient‐Derived Induced Pluripotent Stem Cells

Despite decades of research on amyotrophic lateral sclerosis (ALS), there is only one approved drug, which minimally extends patient survival. Here, we investigated pathophysiological mechanisms underlying ALS using motor neurons (MNs) differentiated from induced pluripotent stem cells (iPSCs) derived from ALS patients carrying mutations in FUS or SOD1. Patient‐derived MNs were less active and excitable compared to healthy controls, due to reduced Na+/K+ ratios in both ALS groups accompanied by elevated potassium channel (FUS) and attenuated sodium channel expression levels (FUS, SOD1). ALS iPSC‐derived MNs showed elevated endoplasmic reticulum stress (ER) levels and increased caspase activation. Treatment with the FDA approved drug 4‐Aminopyridine (4AP) restored ion‐channel imbalances, increased neuronal activity levels and decreased ER stress and caspase activation. This study provides novel pathophysiological data, including a mechanistic explanation for the observed hypoexcitability in patient‐derived MNs and a new therapeutic strategy to provide neuroprotection in MNs affected by ALS. Stem Cells 2016;34:1563–1575

Functional differentiation of midbrain neurons from human cord blood-derived induced pluripotent stem cells

IntroductionHuman induced pluripotent stem cells (hiPSCs) offer great promise for regenerative therapies or in vitro modelling of neurodegenerative disorders like Parkinson’s disease. Currently, widely used cell sources for the generation of hiPSCs are somatic cells obtained from aged individuals. However, a critical issue concerning the potential clinical use of these iPSCs is mutations that accumulate over lifetime and are transferred onto iPSCs during reprogramming which may influence the functionality of cells differentiated from them. The aim of our study was to establish a differentiation strategy to efficiently generate neurons including dopaminergic cells from human cord blood-derived iPSCs (hCBiPSCs) as a juvenescent cell source and prove their functional maturation in vitro.MethodsThe differentiation of hCBiPSCs was initiated by inhibition of transforming growth factor-β and bone morphogenetic protein signaling using the small molecules dorsomorphin and SB 431542 before final maturation was carried out. hCBiPSCs and differentiated neurons were characterized by immunocytochemistry and quantitative real time-polymerase chain reaction. Since functional investigations of hCBiPSC-derived neurons are indispensable prior to clinical applications, we performed detailed analysis of essential ion channel properties using whole-cell patch-clamp recordings and calcium imaging.ResultsA Sox1 and Pax6 positive neuronal progenitor cell population was efficiently induced from hCBiPSCs using a newly established differentiation protocol. Neuronal progenitor cells could be further maturated into dopaminergic neurons expressing tyrosine hydroxylase, the dopamine transporter and engrailed 1. Differentiated hCBiPSCs exhibited voltage-gated ion currents, were able to fire action potentials and displayed synaptic activity indicating synapse formation. Application of the neurotransmitters GABA, glutamate and acetylcholine induced depolarizing calcium signal changes in neuronal cells providing evidence for the excitatory effects of these ligand-gated ion channels during maturation in vitro.ConclusionsThis study demonstrates for the first time that hCBiPSCs can be used as a juvenescent cell source to generate a large number of functional neurons including dopaminergic cells which may serve for the development of novel regenerative treatment strategies.

4‐Aminopyridine Induced Activity Rescues Hypoexcitable Motor Neurons from ALS Patient‐Derived Induced Pluripotent Stem Cells

Despite decades of research on amyotrophic lateral sclerosis (ALS), there is only one approved drug, which minimally extends patient survival. Here, we investigated pathophysiological mechanisms underlying ALS using motor neurons (MNs) differentiated from induced pluripotent stem cells (iPSCs) derived from ALS patients carrying mutations in FUS or SOD1. Patient‐derived MNs were less active and excitable compared to healthy controls, due to reduced Na+/K+ ratios in both ALS groups accompanied by elevated potassium channel (FUS) and attenuated sodium channel expression levels (FUS, SOD1). ALS iPSC‐derived MNs showed elevated endoplasmic reticulum stress (ER) levels and increased caspase activation. Treatment with the FDA approved drug 4‐Aminopyridine (4AP) restored ion‐channel imbalances, increased neuronal activity levels and decreased ER stress and caspase activation. This study provides novel pathophysiological data, including a mechanistic explanation for the observed hypoexcitability in patient‐derived MNs and a new therapeutic strategy to provide neuroprotection in MNs affected by ALS. Stem Cells 2016;34:1563–1575

Impaired DNA damage response signaling by FUS-NLS mutations leads to neurodegeneration and FUS aggregate formation

Amyotrophic lateral sclerosis (ALS) is the most frequent motor neuron disease. Cytoplasmic fused in sarcoma (FUS) aggregates are pathological hallmarks of FUS-ALS. Proper shuttling between the nucleus and cytoplasm is essential for physiological cell function. However, the initial event in the pathophysiology of FUS-ALS remains enigmatic. Using human induced pluripotent stem cell (hiPSCs)-derived motor neurons (MNs), we show that impairment of poly(ADP-ribose) polymerase (PARP)-dependent DNA damage response (DDR) signaling due to mutations in the FUS nuclear localization sequence (NLS) induces additional cytoplasmic FUS mislocalization which in turn results in neurodegeneration and FUS aggregate formation. Our work suggests that a key pathophysiologic event in ALS is upstream of aggregate formation. Targeting DDR signaling could lead to novel therapeutic routes for ameliorating ALS.Abnormal cytoplasmic aggregates of FUS are a hallmark of some forms of amyotrophic lateral sclerosis (ALS). Here, using neurons derived from patients with FUS-ALS, the authors demonstrate that impairment of PARP-dependent DNA damage signaling is an event that occurs upstream of neurodegeneration and cytoplasmic aggregate formation in FUS-ALS.

Plasmid-Based Generation of Induced Neural Stem Cells from Adult Human Fibroblasts

Direct reprogramming from somatic to neural cell types has become an alternative to induced pluripotent stem cells. Most protocols employ viral expression systems, posing the risk of random genomic integration. Recent developments led to plasmid-based protocols, lowering this risk. However, these protocols either relied on continuous presence of a variety of small molecules or were only able to reprogram murine cells. We therefore established a reprogramming protocol based on vectors containing the Epstein-Barr virus (EBV)-derived oriP/EBNA1 as well as the defined expression factors Oct3/4, Sox2, Klf4, L-myc, Lin28, and a small hairpin directed against p53. We employed a defined neural medium in combination with the neurotrophins bFGF, EGF and FGF4 for cultivation without the addition of small molecules. After reprogramming, cells demonstrated a temporary increase in the expression of endogenous Oct3/4. We obtained induced neural stem cells (iNSC) 30 days after transfection. In contrast to previous results, plasmid vectors as well as a residual expression of reprogramming factors remained detectable in all cell lines. Cells showed a robust differentiation into neuronal (72%) and glial cells (9% astrocytes, 6% oligodendrocytes). Despite the temporary increase of pluripotency-associated Oct3/4 expression during reprogramming, we did not detect pluripotent stem cells or non-neural cells in culture (except occasional residual fibroblasts). Neurons showed electrical activity and functional glutamatergic synapses. Our results demonstrate that reprogramming adult human fibroblasts to iNSC by plasmid vectors and basic neural medium without small molecules is possible and feasible. However, a full set of pluripotency-associated transcription factors may indeed result in the acquisition of a transient (at least partial) pluripotent intermediate during reprogramming. In contrast to previous reports, the EBV-based plasmid system remained present and active inside the cells at all time points.

Neuronal Dysfunction in iPSC-Derived Medium Spiny Neurons from Chorea-Acanthocytosis Patients Is Reversed by Src Kinase Inhibition and F-Actin Stabilization

Chorea-acanthocytosis (ChAc) is a fatal neurological disorder characterized by red blood cell acanthocytes and striatal neurodegeneration. Recently, severe cell membrane disturbances based on depolymerized cortical actin and an elevated Lyn kinase activity in erythrocytes from ChAc patients were identified. How this contributes to the mechanism of neurodegeneration is still unknown. To gain insight into the pathophysiology, we established a ChAc patient-derived induced pluripotent stem cell model and an efficient differentiation protocol providing a large population of human striatal medium spiny neurons (MSNs), the main target of neurodegeneration in ChAc. Patient-derived MSNs displayed enhanced neurite outgrowth and ramification, whereas synaptic density was similar to controls. Electrophysiological analysis revealed a pathologically elevated synaptic activity in ChAc MSNs. Treatment with the F-actin stabilizer phallacidin or the Src kinase inhibitor PP2 resulted in the significant reduction of disinhibited synaptic currents to healthy control levels, suggesting a Src kinase- and actin-dependent mechanism. This was underlined by increased G/F-actin ratios and elevated Lyn kinase activity in patient-derived MSNs. These data indicate that F-actin stabilization and Src kinase inhibition represent potential therapeutic targets in ChAc that may restore neuronal function. SIGNIFICANCE STATEMENT Chorea-acanthocytosis (ChAc) is a fatal neurodegenerative disease without a known cure. To gain pathophysiological insight, we newly established a human in vitro model using skin biopsies from ChAc patients to generate disease-specific induced pluripotent stem cells (iPSCs) and developed an efficient iPSC differentiation protocol providing striatal medium spiny neurons. Using patch-clamp electrophysiology, we detected a pathologically enhanced synaptic activity in ChAc neurons. Healthy control levels of synaptic activity could be restored by treatment of ChAc neurons with the F-actin stabilizer phallacidin and the Src kinase inhibitor PP2. Because Src kinases are involved in bridging the membrane to the actin cytoskeleton by membrane protein phosphorylation, our data suggest an actin-dependent mechanism of this dysfunctional phenotype and potential treatment targets in ChAc.

Striatal Transplantation of Human Dopaminergic Neurons Differentiated From Induced Pluripotent Stem Cells Derived From Umbilical Cord Blood Using Lentiviral Reprogramming.

Human induced pluripotent stem cells (hiPSCs) are promising sources for regenerative therapies like the replacement of dopaminergic neurons in Parkinsons disease. They offer an unlimited cell source that can be standardized and optimized to produce applicable cell populations to gain maximal functional recovery. In the present study, human cord blood-derived iPSCs (hCBiPSCs) were differentiated into dopaminergic neurons utilizing two different in vitro protocols for neural induction: (protocol I) by fibroblast growth factor (FGF-2) signaling, (protocol II) by bone morphogenetic protein (BMP)/transforming growth factor (TGF-β) inhibition. After maturation, in vitro increased numbers of tyrosine hydroxylase (TH)-positive neurons (7.4% of total cells) were observed by protocol II compared to 3.5% in protocol I. Furthermore, 3 weeks after transplantation in hemiparkinsonian rats in vivo, a reduced number of undifferentiated proliferating cells was achieved with protocol II. In contrast, proliferation still occurred in protocol I-derived grafts, resulting in tumor-like growth in two out of four animals 3 weeks after transplantation. Protocol II, however, did not increase the number of TH+ cells in the striatal grafts of hemiparkinsonian rats. In conclusion, BMP/TGF-β inhibition was more effective than FGF-2 signaling with regard to dopaminergic induction of hCBiPSCs in vitro and prevented graft overgrowth in vivo.

Functional effects of cannabinoids during dopaminergic specification of human neural precursors derived from induced pluripotent stem cells

Among adolescents cannabis is one of the most widely used illicit drugs. In adolescence brain development continues, characterized by neuronal maturation and synaptic plasticity. The endocannabinoid system plays an important role during brain development by modulating neuronal function and neurogenesis. Changes in endocannabinoid signaling by Δ9‐tetrahydrocannabinol (THC), the psychoactive component of cannabis, might therefore lead to neurobiological changes influencing brain function and behavior. We investigated the functional maturation and dopaminergic specification of human cord blood‐derived induced pluripotent stem cell (hCBiPSC)‐derived small molecule neural precursor cells (smNPCs) after cultivation with the endogenous cannabinoid anandamide (AEA) and the exogenous THC, both potent agonists at the cannabinoid 1 receptor (CB1R). Higher dosages of 10‐μM AEA or THC significantly decreased functionality of neurons, indicated by reduced ion currents and synaptic activity. A lower concentration of 1‐μM THC had no marked effect on neuronal and dopaminergic maturation, while 1‐μM AEA significantly enhanced the frequency of synaptic activity. As there were no significant effects on DNA methylation in promotor regions of genes important for neuronal function, these cannabinoid actions seem to be mediated by another than this epigenetic mechanism. Our data suggest that there are concentration‐dependent actions of cannabinoids on neuronal function in vitro indicating neurotoxic, dysfunctional effects of 10‐μM AEA and THC during human neurogenesis.