Dissecting the non-neuronal cell contribution to Parkinson's disease pathogenesis using induced pluripotent stem cells

Parkinson's disease (PD) is an incurable age-linked neurodegenerative disease with characteristic movement impairments that are caused by the progressive loss of dopamine-containing neurons (DAn) within the substantia nigra pars compacta. It has been suggested that misfolded protein aggregates together with neuroinfammation and glial reactivity, may impact nerve cell function, leading to neurodegeneration and diseases, such as PD. However, not many studies have been able to examine the role of human glial cells in the pathogenesis of PD. With the advent of induced pluripotent stem cell (iPSC) technology, it is now possible to reprogram human somatic cells to pluripotency and to generate viable human patient-specifc DA neurons and glial cells, providing a tremendous opportunity for dissecting cellular and molecular pathological mechanisms occurring at early stages of PD. This reviews will report on recent work using human iPSC and 3D brain organoid models showing that iPSC technology can be used to recapitulate PD-relevant disease-associated phenotypes, including protein aggregation, cell death or loss of neurite complexity and defcient autophagic vacuoles clearance and focus on the recent co-culture systems that are revealing new insights into the complex interactions that occur between diferent brain cell types during neurodegeneration. Consequently, such advances are the key to improve our understanding of PD pathology and generate potential targets for new therapies aimed at curing PD patients

Stable and Efficient Genetic Modification of Cells in the Adult Mouse V-SVZ for the Analysis of Neural Stem Cell Autonomous and Non-autonomous Effects

Relatively quiescent somatic stem cells support life-long cell renewal in most adult tissues. Neural stem cells in the adult mammalian brain are restricted to two specific neurogenic niches: the subgranular zone of the dentate gyrus in the hippocampus and the ventricular-subventricular zone (V-SVZ; also called subependymal zone or SEZ) in the walls of the lateral ventricles. The development of in vivo gene transfer strategies for adult stem cell populations (i.e. those of the mammalian brain) resulting in long-term expression of desired transgenes in the stem cells and their derived progeny is a crucial tool in current biomedical and biotechnological research. Here, a direct in vivo method is presented for the stable genetic modification of adult mouse V-SVZ cells that takes advantage of the cell cycle-independent infection by LVs and the highly specialized cytoarchitecture of the V-SVZ niche. Specifically, the current protocol involves the injection of empty LVs (control) or LVs encoding specific transgene expression cassettes into either the V-SVZ itself, for the in vivo targeting of all types of cells in the niche, or into the lateral ventricle lumen, for the targeting of ependymal cells only. Expression cassettes are then integrated into the genome of the transduced cells and fluorescent proteins, also encoded by the LVs, allow the detection of the transduced cells for the analysis of cell autonomous and non-autonomous, niche-dependent effects in the labeled cells and their progeny

Using iPS Cells toward the Understanding of Parkinson's Disease

Cellular reprogramming of somatic cells to human pluripotent stem cells (iPSC) represents an efficient tool for in vitro modeling of human brain diseases and provides an innovative opportunity in the identification of new therapeutic drugs. Patient-specific iPSC can be differentiated into disease-relevant cell types, including neurons, carrying the genetic background of the donor and enabling de novo generation of human models of genetically complex disorders. Parkinson's disease (PD) is the second most common age-related progressive neurodegenerative disease, which is mainly characterized by nigrostriatal dopaminergic (DA) neuron degeneration and synaptic dysfunction. Recently, the generation of disease-specific iPSC from patients suffering from PD has unveiled a recapitulation of disease-related cell phenotypes, such as abnormal α-synuclein accumulation and alterations in autophagy machinery. The use of patient-specific iPSC has a remarkable potential to uncover novel insights of the disease pathogenesis, which in turn will open new avenues for clinical intervention. This review explores the current Parkinson's disease iPSC-based models highlighting their role in the discovery of new drugs, as well as discussing the most challenging limitations iPSC-models face today

Long-Term Labeling of Hippocampal Neural Stem Cells by a Lentiviral Vector

Using a lentivirus-mediated labeling method, we investigated whether the adult hippocampus retains long-lasting, self-renewing neural stem cells (NSCs). We first showed that a single injection of a lentiviral vector expressing a green fluorescent protein (LV PGK-GFP) into the subgranular zone (SGZ) of the adult hippocampus enabled an efficient, robust, and long-term marking of self-renewing NSCs and their progeny. Interestingly, a subset of labeled cells showed the ability to proliferate multiple times and give rise to Sox2+ cells, clearly suggesting the ability of NSCs to self-renew for an extensive period of time (up to 6 months). In addition, using GFP+ cells isolated from the SGZ of mice that received a LV PGK-GFP injection 3 months earlier, we demonstrated that some GFP+ cells displayed the essential properties of NSCs, such as self-renewal and multipotency. Furthermore, we investigated the plasticity of NSCs in a perforant path transection, which has been shown to induce astrocyte formation in the molecular layer of the hippocampus. Our lentivirus (LV)-mediated labeling study revealed that hippocampal NSCs are not responsible for the burst of astrocyte formation, suggesting that signals released from the injured perforant path did not influence NSC fate determination. Therefore, our studies showed that a gene delivery system using LVs is a unique method to be used for understanding the complex nature of NSCs and may have translational impact in gene therapy by efficiently targeting NSCs

Cyclin A1 is essential for setting the pluripotent state and reducing tumorigenicity of induced pluripotent stem cells

The proper differentiation and threat of cancer rising from the application of induced pluripotent stem (iPS) cells are major bottlenecks in the field and are thought to be inherently linked to the pluripotent nature of iPS cells. To address this question, we have compared iPS cells to embryonic stem cells (ESCs), the gold standard of ground state pluripotency, in search for proteins that may improve pluripotency of iPS cells. We have found that when reprogramming somatic cells toward pluripotency, 1%-5% of proteins of 5 important cell functions are not set to the correct expression levels compared to ESCs, including mainly cell cycle proteins. We have shown that resetting cyclin A1 protein expression of early-passage iPS cells closer to the ground state pluripotent state of mouse ESCs improves the pluripotency and reduces the threat of cancer of iPS cells. This work is a proof of principle that reveals that setting expression of certain proteins correctly during reprogramming is essential for achieving ESC-state pluripotency. This finding would be of immediate help to those researchers in different fields of iPS cell work that specializes in cell cycle, apoptosis, cell adhesion, cell signaling, and cytoskeleton

DIAGNOSIS AND FOLLOW-UP OF COMPLEX CONGENITAL MALFORMATIONS/MENTAL RETARDATION (MRA/MR)

Complex congenital malformations, associated in 30% of cases with mental retardation, recognize different etiologies: environmental causes, mendelian disease, chromosomal abnormalities, imprinted anomalies. Frequently complex congenital disorders are rare diseases. Rare diseases are infrequent pathological conditions (prevalence in the general population of less than 1/2.000 live births1), and often poorly understood. Because of their rarity these morbid conditions often either go undiagnosed or are diagnosed late with a negative impact for both the affected person and the family. The birth prevalence is high (2-4% of all births). The diagnosis is essential to program complex and integrated care interventions (follow-up programs aimed at early detection of any disease associated with different syndromes) and to carry out proper genetic family counseling (risk of recurrence, prenatal diagnosis, detection of heterozygotes etc)

Neural stem cells in the adult olfactory bulb core generate mature neurons in vivo

Although previous studies suggest that neural stem cells (NSCs) exist in the adult olfactory bulb (OB), their location, identity, and capacity to generate mature neurons in vivo has been little explored. Here, we injected enhanced green fluorescent protein (EGFP)-expressing retroviral particles into the OB core of adult mice to label dividing cells and to track the differentiation/maturation of any neurons they might generate. EGFP-labeled cells initially expressed adult NSC markers on days 1 to 3 postinjection (dpi), including Nestin, GLAST, Sox2, Prominin-1, and GFAP. EGFP+ -doublecortin (DCX) cells with a migratory morphology were also detected and their abundance increased over a 7-day period. Furthermore, EGFP-labeled cells progressively became NeuN+ neurons, they acquired neuronal morphologies, and they became immunoreactive for OB neuron subtype markers, the most abundant representing calretinin expressing interneurons. OB-NSCs also generated glial cells, suggesting they could be multipotent in vivo. Significantly, the newly generated neurons established and received synaptic contacts, and they expressed presynaptic proteins and the transcription factor pCREB. By contrast, when the retroviral particles were injected into the subventricular zone (SVZ), nearly all (98%) EGFP+ -cells were postmitotic when they reached the OB core, implying that the vast majority of proliferating cells present in the OB are not derived from the SVZ. Furthermore, we detected slowly dividing label-retaining cells in this region that could correspond to the population of resident NSCs. This is the first time NSCs located in the adult OB core have been shown to generate neurons that incorporate into OB circuits in vivo

Efficient generation of A9 midbrain dopaminergic neurons by lentiviral delivery of LMX1A in human embryonic stem cells and Induced Pluripotent Stem Cells

Human embryonic stem cells (hESC) and induced pluripotent stem cells (iPSC) offer great hope for in vitro modeling of Parkinson's disease (PD), as well as for designing cell-replacement therapies. To realize these opportunities, there is an urgent need to develop efficient protocols for the directed differentiation of hESC/iPSC into dopamine (DA) neurons with the specific characteristics of the cell population lost to PD, i.e., A9-subtype ventral midbrain DA neurons. Here we use lentiviral vectors to drive the expression of LMX1A, which encodes a transcription factor critical for ventral midbrain identity, specifically in neural progenitor cells. We show that clonal lines of hESC engineered to contain one or two copies of this lentiviral vector retain long-term self-renewing ability and pluripotent differentiation capacity. Greater than 60% of all neurons generated from LMX1A-engineered hESC were ventral midbrain DA neurons of the A9 subtype, compared with ∼10% in green fluorescent protein engineered controls, as judged by specific marker expression and functional analyses. Moreover, DA neuron precursors differentiated from LMX1A-engineered hESC were able to survive and differentiate when grafted into the brain of adult mice. Finally, we provide evidence that LMX1A overexpression similarly increases the yield of DA neuron differentiation from human iPSC. Taken together, our data show that stable genetic engineering of hESC/iPSC with lentiviral vectors driving controlled expression of LMX1A is an efficient way to generate enriched populations of human A9-subtype ventral midbrain DA neurons, which should prove useful for modeling PD and may be helpful for designing future cell-replacement strategies

Lysosomal and network alterations in human mucopolysaccharidosis type VII iPSC-derived neural cells

Mucopolysaccharidosis type VII (MPS VII) is a lysosomal storage disease caused by deficient β-glucuronidase (β-gluc) activity. Significantly reduced β-gluc activity leads to accumulation of glycosaminoglycans (GAGs) in many tissues, including the brain. Numerous combinations of mutations in GUSB (the gene that codes for β-gluc) cause a range of neurological features that make disease prognosis and treatment challenging. Currently, there is little understanding of the molecular basis for MPS VII brain anomalies. To identify a neuronal phenotype that could be used to complement genetic analyses, we generated two iPSC clones derived from skin fibroblasts of an MPS VII patient. We found that MPS VII neurons exhibited reduced β-gluc activity and showed previously established disease-associated phenotypes, including GAGs accumulation, expanded endocytic compartments, accumulation of lipofuscin granules, more autophagosomes, and altered lysosome function. Addition of recombinant β-gluc to MPS VII neurons, which mimics enzyme replacement therapy, restored disease-associated phenotypes to levels similar to the healthy control. MPS VII neural cells cultured as 3D neurospheroids showed upregulated GFAP gene expression, which was associated with astrocyte reactivity, and downregulation of GABAergic neuron markers. Spontaneous calcium imaging analysis of MPS VII neurospheroids showed reduced neuronal activity and altered network connectivity in patient-derived neurospheroids compared to a healthy control. These results demonstrate the interplay between reduced β-gluc activity, GAG accumulation and alterations in neuronal activity, and provide a human experimental model for elucidating the bases of MPS VII-associated cognitive defects