CD4+ T cell-associated cytokines induce a chronic pro-inflammatory phenotype in induced pluripotent stem cell-derived midbrain astrocytes.

Astrocytes are mediators of homeostasis but contribute to neuroinflammation in Parkinson's disease (PD). Mounting evidence suggests involvement of peripheral immune cells in PD pathogenesis. Therefore, this study aimed to determine the potential role of peripheral immune secreted cytokines in modulating midbrain astrocyte reactivity. Human iPSC-derived midbrain astrocytes were exposed to 5% and 10% CD4+ T cell conditioned media (CD4CM) for 24 h, 72 h, and 7 days to assess chronic exposure. Additionally, astrocytes were exposed to the Th17 cell cytokine, IL-17A (10 ng/mL), alone and in combination with TNF-α (0.3 ng/mL) to assess potential synergistic effects of both cytokines at 24 h, 72 h, and 7 days. CD4CM induced acute and chronic alterations in midbrain astrocytes. Increased NFκB translocation to the nucleus, increased expression of the pro-inflammatory genes, IL-1ß, CXCL10 at 24 h, C3, LCN2, IL-6 at 24 and 48 h, as well as an increase in their release of pro-inflammatory cytokines IL-6 and CXCL10 at both these time points were observed. A synergistic response to the combination of IL-17A and TNF-α on increasing inflammatory gene expression and cytokine release occurred. IL-17A and TNF-α increased intensity of S100ß at 24 h, decreased nuclear area and increased circularity of astrocytes at 72 h. A synergistic effect on γH2AX intensity at 72 h and an increase in LDH release at 7 days was observed. Our results demonstrate that IL-17A and TNF-α act synergistically, enhancing midbrain astrocyte reactivity to a similar degree as CD4CM. This highlights the importance of the peripheral immune secreted cytokines in increasing the reactivity status of midbrain astrocytes, implicating their role in PD.

Loss of MicroRNA-7 Regulation Leads to α-Synuclein Accumulation and Dopaminergic Neuronal Loss In Vivo.

Abnormal alpha-synuclein (α-synuclein) expression and aggregation is a key characteristic of Parkinson's disease (PD). However, the exact mechanism(s) linking α-synuclein to the other central feature of PD, dopaminergic neuron loss, remains unclear. Therefore, improved cell and in vivo models are needed to investigate the role of α-synuclein in dopaminergic neuron loss. MicroRNA-7 (miR-7) regulates α-synuclein expression by binding to the 3' UTR of the Synuclein Alpha Non A4 Component of Amyloid Precursor (SNCA) gene and inhibiting its translation. We show that miR-7 is decreased in the substantia nigra of patients with PD and, therefore, may play an essential role in the regulation of α-synuclein expression. Furthermore, we have found that lentiviral-mediated expression of miR-7 complementary binding sites to stably induce a loss of miR-7 function results in an increase in α-synuclein expression in vitro and in vivo. We have also shown that depletion of miR-7 using a miR-decoy produces a loss of nigral dopaminergic neurons accompanied by a reduction of striatal dopamine content. These data suggest that miR-7 has an important role in the regulation of α-synuclein and dopamine physiology and may provide a new paradigm to study the pathology of PD.

Enhanced efficacy of the CDNF/MANF family by combined intranigral overexpression in the 6-OHDA rat model of Parkinson's disease.

Cerebral Dopamine Neurotrophic Factor (CDNF) and Mesencephalic Astrocyte-derived Neurotrophic factor (MANF) are members of a recently discovered family of neurotrophic factors (NTFs). Here, we used intranigral or intrastriatal lentiviral vector-mediated expression to evaluate their efficacy at protecting dopaminergic function in the 6-OHDA model of Parkinson's disease (PD). In contrast to the well-studied Glial-Derived Neurotrophic Factor (GDNF), no beneficial effects were demonstrated by striatal overexpression of either protein. Interestingly, nigral overexpression of CDNF decreased amphetamine-induced rotations and increased tyroxine hydroxylase (TH) striatal fiber density but had no effect on numbers of TH(+) cells in the SN. Nigral MANF overexpression had no effect on amphetamine-induced rotations or TH striatal fiber density but resulted in a significant preservation of TH(+) cells. Combined nigral overexpression of both factors led to a robust reduction in amphetamine-induced rotations, greater increase in striatal TH-fiber density and significant protection of TH(+) cells in the SN. We conclude that nigral CDNF and MANF delivery is more efficacious than striatal delivery. This is also the first study to demonstrate that combined NTF can have synergistic effects that result in enhanced neuroprotection, suggesting that multiple NTF delivery may be more efficacious for the treatment of PD than the single NTF approaches attempted so far.

A defect in the retromer accessory protein, SNX27, manifests by infantile myoclonic epilepsy and neurodegeneration.

The composition of the neuronal cell surface dictates synaptic plasticity and thereby cognitive development. This remodeling of the synapses is governed by the endocytic network which internalize transmembrane proteins, then sort them back to the cell surface or carry them to the lysosome for degradation. The multi-protein retromer complex is central to this selection, capturing specific transmembrane proteins and remodeling the cell membrane to form isolated cargo-enriched transport carriers. We investigated a consanguineous family with four patients who presented in infancy with intractable myoclonic epilepsy and lack of psychomotor development. Using exome analysis, we identified a homozygous deleterious mutation in SNX27, which encodes sorting nexin 27, a retromer cargo adaptor. In western analysis of patient fibroblasts, the encoded mutant protein was expressed at an undetectable level when compared with a control sample. The patients' presentation and clinical course recapitulate that reported for the SNX27 knock-out mouse. Since the cargo proteins for SNX27-mediated sorting include subunits of ionotropic glutamate receptors and endosome-to-cell surface synaptic insertion of AMPA receptors is severely perturbed in SNX27(-/-) neurons, it is proposed that at least part of the neurological aberrations observed in the patients is attributed to defective sorting of ionotropic glutamate receptors. SNX27 deficiency is now added to the growing list of neurodegenerative disorders associated with retromer dysfunction.

Human stem cell-derived ventral midbrain astrocytes exhibit a region-specific secretory profile.

This scientific commentary refers to 'Human stem cell-derived astrocytes exhibit region-specific heterogeneity in their secretory profiles', by Clarke et al. (https://doi.org/10.1093/brain/awaa258) in Brain.

Dopamine-induced proliferation of adult neural precursor cells in the mammalian subventricular zone is mediated through EGF.

A reduction in dopaminergic innervation of the subventricular zone (SVZ) is responsible for the impaired proliferation of its resident precursor cells in this region in Parkinson's disease (PD). Here, we show that this effect involves EGF, but not FGF2. In particular, we demonstrate that dopamine increases the proliferation of SVZ-derived cells by releasing EGF in a PKC-dependent manner in vitro and that activation of the EGF receptor (EGFR) is required for this effect. We also show that dopamine selectively expands the GFAP(+) multipotent stem cell population in vitro by promoting their self-renewal. Furthermore, in vivo dopamine depletion leads to a decrease in precursor cell proliferation in the SVZ concomitant with a reduction in local EGF production, which is reversed through the administration of the dopamine precursor levodopa (L-DOPA). Finally, we show that EGFR(+) cells are depleted in the SVZ of human PD patients compared with age-matched controls. We have therefore demonstrated a unique role for EGF as a mediator of dopamine-induced precursor cell proliferation in the SVZ, which has potential implications for future therapies in PD.

Human Brain-Based Models Provide a Powerful Tool for the Advancement of Parkinson's Disease Research and Therapeutic Development.

Parkinson's disease (PD) is the second most common neurodegenerative disease and affects approximately 2-3% of the population over the age of 65. PD is characterised by the loss of dopaminergic neurons from the substantia nigra, leading to debilitating motor symptoms including bradykinesia, tremor, rigidity, and postural instability. PD also results in a host of non-motor symptoms such as cognitive decline, sleep disturbances and depression. Although existing therapies can successfully manage some motor symptoms for several years, there is still no means to halt progression of this severely debilitating disorder. Animal models used to replicate aspects of PD have contributed greatly to our current understanding but do not fully replicate pathological mechanisms as they occur in patients. Because of this, there is now great interest in the use of human brain-based models to help further our understanding of disease processes. Human brain-based models include those derived from embryonic stem cells, patient-derived induced neurons, induced pluripotent stem cells and brain organoids, as well as post-mortem tissue. These models facilitate in vitro analysis of disease mechanisms and it is hoped they will help bridge the existing gap between bench and bedside. This review will discuss the various human brain-based models utilised in PD research today and highlight some of the key breakthroughs they have facilitated. Furthermore, the potential caveats associated with the use of human brain-based models will be detailed.

FGF but not EGF induces phosphorylation of the cAMP response element binding protein in olfactory mucosa-derived cell cultures.

The stem/progenitor cells of the olfactory epithelium are potentially useful cells for autologous cell-based therapy because of their relative accessibility compared to other sources of neural stem cells. However, they have very limited potential to self-renew in vitro under growth factor stimulation compared to central nervous system-derived stem/progenitor cells. Using a sphere-forming assay and immunocytochemistry to identify cells that contained phosphorylated cAMP response element binding protein (pCREB) as an indicator of cell responsiveness to growth factor activation, we found that olfactory-spheres primed with FGF2 responded to FGF2 and EGF stimulation. In contrast, olfactory-spheres primed with EGF failed to respond to FGF2 or EGF stimulation despite the detection of FGFR1 and EGFR and their transcripts. These data demonstrate that FGF2 but not EGF permit the maintenance of a subset of cells responsive to FGF2 and EGF, whereas EGF induces unresponsive to either growth factor possibly via intrinsic mechanisms of regulation.

Twist-1 regulates the miR-199a/214 cluster during development.

MicroRNAs are known to regulate developmental processes but their mechanism of regulation remains largely uncharacterized. We show the transcription factor Twist-1 drives the expression of a 7.9-kb noncoding RNA transcript (from the Dynamin-3 gene intron) that encodes a miR-199a and miR-214 cluster. We also show that knocking down Twist-1 with shRNAs decreased miR-199a/214 levels and that Twist-1 bound an E-Box promoter motif to developmentally regulate the expression of these miRNAs. The expression of HIF-1 (known to mediate Twist-1 transcription), miR-199a and miR-214 was maximal at E12.5 and the miRNAs were expressed specifically in mouse cerebellum, midbrain, nasal process and fore- and hindlimb buds. This study shows the expression of the miR199a/214 cluster is controlled by Twist-1 via an E-Box promoter element and supports a role for these miRNAs as novel intermediates in the pathways controlling the development of specific neural cell populations.

The Pathogenesis of Parkinson's Disease: A Complex Interplay Between Astrocytes, Microglia, and T Lymphocytes?

Parkinson's disease (PD), the second most common neurodegenerative disease, is characterised by the motor symptoms of bradykinesia, rigidity and resting tremor and non-motor symptoms of sleep disturbances, constipation, and depression. Pathological hallmarks include neuroinflammation, degeneration of dopaminergic neurons in the substantia nigra pars compacta, and accumulation of misfolded α-synuclein proteins as intra-cytoplasmic Lewy bodies and neurites. Microglia and astrocytes are essential to maintaining homeostasis within the central nervous system (CNS), including providing protection through the process of gliosis. However, dysregulation of glial cells results in disruption of homeostasis leading to a chronic pro-inflammatory, deleterious environment, implicated in numerous CNS diseases. Recent evidence has demonstrated a role for peripheral immune cells, in particular T lymphocytes in the pathogenesis of PD. These cells infiltrate the CNS, and accumulate in the substantia nigra, where they secrete pro-inflammatory cytokines, stimulate surrounding immune cells, and induce dopaminergic neuronal cell death. Indeed, a greater understanding of the integrated network of communication that exists between glial cells and peripheral immune cells may increase our understanding of disease pathogenesis and hence provide novel therapeutic approaches.

Efficient and Scalable Generation of Human Ventral Midbrain Astrocytes from Human-Induced Pluripotent Stem Cells.

In Parkinson's disease, progressive dysfunction and degeneration of dopamine neurons in the ventral midbrain cause life-changing symptoms. Neuronal degeneration has diverse causes in Parkinson's, including non-cell autonomous mechanisms mediated by astrocytes. Throughout the CNS, astrocytes are essential for neuronal survival and function, as they maintain metabolic homeostasis in the neural environment. Astrocytes interact with the immune cells of the CNS, microglia, to modulate neuroinflammation, which is observed from the earliest stages of Parkinson's, and has a direct impact on the progression of its pathology. In diseases with a chronic neuroinflammatory element, including Parkinson's, astrocytes acquire a neurotoxic phenotype, and thus enhance neurodegeneration. Consequently, astrocytes are a potential therapeutic target to slow or halt disease, but this will require a deeper understanding of their properties and roles in Parkinson's. Accurate models of human ventral midbrain astrocytes for in vitro study are therefore urgently required. We have developed a protocol to generate high purity cultures of ventral midbrain-specific astrocytes (vmAstros) from hiPSCs that can be used for Parkinson's research. vmAstros can be routinely produced from multiple hiPSC lines, and express specific astrocytic and ventral midbrain markers. This protocol is scalable, and thus suitable for high-throughput applications, including for drug screening. Crucially, the hiPSC derived-vmAstros demonstrate immunomodulatory characteristics typical of their in vivo counterparts, enabling mechanistic studies of neuroinflammatory signaling in Parkinson's.

A monolayer hiPSC culture system for autophagy/mitophagy studies in human dopaminergic neurons.

Macroautophagy/autophagy cytoplasmic quality control pathways are required during neural development and are critical for the maintenance of functional neuronal populations in the adult brain. Robust evidence now exists that declining neuronal autophagy pathways contribute to human neurodegenerative diseases, including Parkinson disease (PD). Reliable and relevant human neuronal model systems are therefore needed to understand the biology of disease-vulnerable neural populations, to decipher the underlying causes of neurodegenerative disease, and to develop assays to test therapeutic interventions in vitro. Human induced pluripotent stem cell (hiPSC) neural model systems can meet this demand: they provide a renewable source of material for differentiation into regional neuronal sub-types for functional assays; they can be expanded to provide a platform for screening, and they can potentially be optimized for transplantation/neurorestorative therapy. So far, however, hiPSC differentiation protocols for the generation of ventral midbrain dopaminergic neurons (mDANs) - the predominant neuronal sub-type afflicted in PD - have been somewhat restricted by poor efficiency and/or suitability for functional and/or imaging-based in vitro assays. Here, we describe a reliable, monolayer differentiation protocol for the rapid and reproducible production of high numbers of mDANs from hiPSC in a format that is amenable for autophagy/mitophagy research. We characterize these cells with respect to neuronal differentiation and macroautophagy capability and describe qualitative and quantitative assays for the study of autophagy and mitophagy in these important cells.Abbreviations: AA: ascorbic acid; ATG: autophagy-related; BDNF: brain derived neurotrophic factor; CCCP: carbonyl cyanide m-chlorophenylhydrazone; dbcAMP: dibutyryl cAMP; DAN: dopaminergic neuron; DAPI: 4',6-diamidino-2-phenylindole; DAPT: N-[N-(3,5-difluorophenacetyl)-L-alanyl]-sphenylglycine; DLG4/PSD95: discs large MAGUK scaffold protein 4; DMEM: Dulbecco's modified eagle's medium; EB: embryoid body; ECAR: extracellular acidification rate; EGF: epidermal growth factor; FACS: fluorescence-activated cell sorting; FCCP: arbonyl cyanide p-triflouromethoxyphenylhydrazone; FGF: fibroblast growth factor; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; GDNF: glia cell derived neurotrophic factor; hiPSC: human induced pluripotent stem cell; LAMP2A: lysosomal associated membrane protein 2A; LT-R: LysoTracker Red; MAP1LC3: microtubule associated protein 1 light chain 3; mDAN: midbrain dopaminergic neuron; MEF: mouse embryonic fibroblast; MT-GR: MitoTracker Green; MT-R: MitoTracker Red; NAS2: normal SNCA2; NEM: neuroprogenitor expansion media; NR4A2/NURR1: nuclear receptor subfamily group A member 2; OA: oligomycin and antimycin A; OCR: oxygen consumption rate; PD: Parkinson disease; SHH: sonic hedgehog signaling molecule; SNCA/α-synuclein: synuclein alpha; TH: tyrosine hydroxylase; VTN: vitronectin.

DNA damaging bystander signalling from stem cells, cancer cells and fibroblasts after Cr(VI) exposure and its dependence on telomerase.

The bystander effect is a feature of low dose radiation exposure and is characterized by a signaling process from irradiated cells to non irradiated cells, which causes DNA and chromosome damage in these 'nearest neighbour' cells. Here we show that a low and short dose of Cr(VI) can induce stem cells, cancer cells and fibroblasts to chronically secrete bystander signals, which cause DNA damage in neighboring cells. The Cr(VI) induced bystander signaling depended on the telomerase status of either cell. Telomerase negative fibroblasts were able to receive DNA damaging signals from telomerase positive or negative fibroblasts or telomerase positive cancer cells. However telomerase positive fibroblasts were resistant to signals from Cr(VI) exposed telomerase positive fibroblasts or cancer cells. Human embryonic stem cells, with positive Oct4 staining as a marker of pluripotency, showed no significant increase of DNA damage from adjacent Cr and mitomycin C exposed fibroblasts whilst those cells that were negatively stained did. This selectivity of DNA damaging bystander signaling could be an important consideration in developing therapies against cancer and in the safety and effectiveness of tissue engineering and transplantation using stem cells.

Graft outcomes influenced by co-expression of Pax7 in graft and host tissue.

Cell replacement therapies offer promise in the treatment of neurotrauma and neurodegenerative disorders and have concentrated on the use of primary fetal brain tissue. However, there is a growing promise of using neural stem cells, in which case other factors may be important in their successful engraftment. We therefore investigated whether the co-expression of the major developmental transcription factor (Pax7 in this study) of donor tissue to graft site influences transplant survival and differentiation in the rat midbrain. Neural progenitor cells were prepared from either the Pax7-expressing dorsal (DM) or non-Pax7-expressing ventral mesencephalon (VM) of embryonic EGFP(+/+) rats. Cells were dissociated and grafted into the adult rat superior colliculus (SC) lesioned with quinolinic acid 3 days previously, a time shown to be associated with the up-regulation of Pax7. Grafts were then examined 4 weeks later. Our results suggest the origin of the graft tissue did not alter graft survival in the SC; however, dorsal grafts appear to have a higher incidence of neuronal survival, whereas ventral grafts have a higher incidence of astrocytic survivors.

Induction of A9 dopaminergic neurons from neural stem cells improves motor function in an animal model of Parkinson's disease.

Neural stem cells (NSCs) are widely endorsed as a cell source for replacement strategies in neurodegenerative disease. However, their usefulness is currently limited by the inability to induce specific neurotransmitter phenotypes in these cells. In order to direct dopaminergic neuronal fate, we overexpressed Pitx3 in NSCs that were then exposed to E11 developing ventral mesencephalon (VM) in explant culture. This resulted in a significant potentiation of dopaminergic differentiation of the cells. When transplanted into the 6-hydroxydopamine lesioned Parkinsonian rats, these cografts of VM and Pitx3 overexpressing NSCs resulted in a significant restitution of motor function. In addition, there were greater numbers of Girk2 positive A9 neurons in the periphery of the transplants that were NSC derived. This demonstrates that given the correct signals, NSCs can be induced to become dopaminergic neurons that can differentiate into the correct nigrastriatal phenotype required for the treatment of Parkinson's disease.

Imaging Autophagy in hiPSC-Derived Midbrain Dopaminergic Neuronal Cultures for Parkinson's Disease Research.

To appreciate the positive or negative impact of autophagy during the initiation and progression of human diseases, the isolation or de novo generation of appropriate cell types is required to support focused in vitro assays. In human neurodegenerative diseases such as Parkinson's disease (PD), specific subsets of acutely sensitive neurons become susceptible to stress-associated operational decline and eventual cell death, emphasizing the need for functional studies in those vulnerable groups of neurons. In PD, a class of dopaminergic neurons in the ventral midbrain (mDANs) is affected. To study these, human-induced pluripotent stem cells (hiPSCs) have emerged as a valuable tool, as they enable the establishment and study of mDAN biology in vitro. In this chapter, we describe a stepwise protocol for the generation of mDANs from hiPSCs using a monolayer culture system. We then outline how imaging-based autophagy assessment methodologies can be applied to these neurons, thereby providing a detailed account of the application of imaging-based autophagy assays to human iPSC-derived mDANs.

Secreted factors from olfactory mucosa cells expanded as free-floating spheres increase neurogenesis in olfactory bulb neurosphere cultures.

BACKGROUND: The olfactory epithelium is a neurogenic tissue comprising a population of olfactory receptor neurons that are renewed throughout adulthood by a population of stem and progenitor cells. Because of their relative accessibility compared to intra-cranially located neural stem/progenitor cells, olfactory epithelium stem and progenitor cells make attractive candidates for autologous cell-based therapy. However, olfactory stem and progenitor cells expand very slowly when grown as free-floating spheres (olfactory-spheres) under growth factor stimulation in a neurosphere assay. RESULTS: In order to address whether olfactory mucosa cells extrinsically regulate proliferation and/or differentiation of immature neural cells, we cultured neural progenitor cells derived from mouse neonatal olfactory bulb or subventricular zone (SVZ) in the presence of medium conditioned by olfactory mucosa-derived spheres (olfactory-spheres). Our data demonstrated that olfactory mucosa cells produced soluble factors that affect bulbar neural progenitor cell differentiation but not their proliferation when compared to control media. In addition, olfactory mucosa derived soluble factors increased neurogenesis, especially favouring the generation of non-GABAergic neurons. Olfactory mucosa conditioned medium also contained several factors with neurotrophic/neuroprotective properties. Olfactory-sphere conditioned medium did not affect proliferation or differentiation of SVZ-derived neural progenitors. CONCLUSION: These data suggest that the olfactory mucosa does not contain factors that are inhibitory to neural stem/progenitor cell proliferation but does contain factors that steer differentiation toward neuronal phenotypes. Moreover, they suggest that the poor expansion of olfactory-spheres may be in part due to intrinsic properties of the olfactory epithelial stem/progenitor cell population.

Laminin enhances the growth of human neural stem cells in defined culture media.

BACKGROUND: Human neural stem cells (hNSC) have the potential to provide novel cell-based therapies for neurodegenerative conditions such as multiple sclerosis and Parkinson's disease. In order to realise this goal, protocols need to be developed that allow for large quantities of hNSC to be cultured efficiently. As such, it is important to identify factors which enhance the growth of hNSC. In vivo, stem cells reside in distinct microenvironments or niches that are responsible for the maintenance of stem cell populations. A common feature of niches is the presence of the extracellular matrix molecule, laminin. Therefore, this study investigated the effect of exogenous laminin on hNSC growth. RESULTS: To measure hNSC growth, we established culture conditions using B27-supplemented medium that enable neurospheres to grow from human neural cells plated at clonal densities. Limiting dilution assays confirmed that neurospheres were derived from single cells at these densities. Laminin was found to increase hNSC numbers as measured by this neurosphere formation. The effect of laminin was to augment the proliferation/survival of the hNSC, rather than promoting the undifferentiated state. In agreement, apoptosis was reduced in dissociated neurospheres by laminin in an integrin beta1-dependent manner. CONCLUSION: The addition of laminin to the culture medium enhances the growth of hNSC, and may therefore aid their large-scale production.

Environmental signals regulate lineage choice and temporal maturation of neural stem cells from human embryonic stem cells.

Human embryonic stem cells (hESCs) are a potential source of defined tissue for cell-based therapies in regenerative neurology. In order for this potential to be realized, there is a need for the evaluation of the behaviour of human embryonic stem cell-derived neural stem cells (hES-NSCs) both in the normal and the injured CNS. Using normal tissue and two experimental models, we examined the response of clinically compatible hES-NSCs to physiological and pathological signals. We demonstrate that the phenotypic potential of a multipotent population of hES-NSCs is influenced by these cues both in vitro and in vivo. hES-NSCs display a temporal profile of neurogenic and gliogenic differentiation, with the generation of mature neurons and glia over 4 weeks in vitro, and 20 weeks in the uninjured rodent brain. However, transplantation into the pathological CNS accelerates maturation and polarizes hES-NSC differentiation potential. This study highlights the role of environmental signals in determining both lineage commitment and temporal maturation of human neural stem cells. Controlled manipulation of environmental signals appropriate to the pathological specificity of the targeted disease will be necessary in the design of therapeutic stem cell-based strategies.