Enhanced production of mesencephalic dopaminergic neurons from lineage-restricted human undifferentiated stem cells.

Current differentiation protocols for generating mesencephalic dopaminergic (mesDA) neurons from human pluripotent stem cells result in grafts containing only a small proportion of mesDA neurons when transplanted in vivo. In this study, we develop lineage-restricted undifferentiated stem cells (LR-USCs) from pluripotent stem cells, which enhances their potential for differentiating into caudal midbrain floor plate progenitors and mesDA neurons. Using a ventral midbrain protocol, 69% of LR-USCs become bona fide caudal midbrain floor plate progenitors, compared to only 25% of human embryonic stem cells (hESCs).

Spatially and temporally distinct patterns of expression for VPS10P domain receptors in human cerebral organoids.

Vacuolar protein sorting 10 protein (VPS10P) domain receptors are a unique class of intracellular sorting receptors that emerge as major risk factors associated with psychiatric and neurodegenerative diseases, including bipolar disorders, autism, schizophrenia, as well as Alzheimer's disease and frontotemporal dementia. Yet, the lack of suitable experimental models to study receptor functions in the human brain has hampered elucidation of receptor actions in brain disease. Here, we have adapted protocols using human cerebral organoids to the detailed characterization of VPS10P domain receptor expression during neural development and differentiation, including single-cell RNA sequencing.

Generation of Human iPSCs by Episomal Reprogramming of Skin Fibroblasts and Peripheral Blood Mononuclear Cells.

Human-induced pluripotent stem cells (iPSCs) can be generated from patient-specific somatic cells by forced expression of the transcription factors OCT4, SOX2, KLF4, and c-MYC. Sustained expression of the transgenes during reprogramming is crucial for the successful derivation of iPSCs. Integrating retroviruses have been used to achieve the required prolonged expression; however, issues of undesirable transgene expression in the iPSC-derived cell types post reprogramming can occur.

Rapid generation of regionally specified CNS neurons by sequential patterning and conversion of human induced pluripotent stem cells.

The differentiation of patient-specific induced pluripotent stem cells (iPSCs) into specific neuronal subtypes has been exploited as an approach for modeling a variety of neurological disorders. However, achieving a highly pure population of neurons is challenging when using directed differentiation methods, especially for neuronal subtypes generated by complex and protracted protocols. In this study, we efficiently produced highly pure populations of regionally specified CNS neurons by using a modified NGN2-Puromycin direct conversion protocol.

Generation of an induced pluripotent stem cell line (DANi-011A) from a Parkinson's disease patient with a LRRK2 p.G2019S mutation.

We generated an induced pluripotent stem cell (iPSC) line from fibroblasts of a clinically diagnosed 70 year old female Parkinson's disease (PD) patient heterozygous for a pathogenic missense variant (p.G2019S; c. 6055 G > A) in the leucine-rich repeat kinase 2 (LRRK2) gene by using non-integrating Sendai viruses.

Generation of eight human induced pluripotent stem cell lines from Parkinson's disease patients carrying familial mutations.

We generated eight induced pluripotent stem cell (iPSC) lines from Parkinson's disease (PD) patients with different familial mutations using non-integrating episomal plasmids. All iPSC lines have a normal karyotype, express pluripotent genes including POU5F1, NANOG, and show alkaline phosphatase activity, as well as the ability to differentiate into all three germ layers. These PD iPSC lines can be used for disease modeling to identify PD mechanisms and for the development or stratification of new drugs.

Long-Term Stress Disrupts the Structural and Functional Integrity of GABAergic Neuronal Networks in the Medial Prefrontal Cortex of Rats.

Clinical and experimental data suggest that fronto-cortical GABAergic deficits contribute to the pathophysiology of major depressive disorder (MDD). To further test this hypothesis, we used a well characterized rat model for depression and examined the effect of stress on GABAergic neuron numbers and GABA-mediated synaptic transmission in the medial prefrontal cortex (mPFC) of rats. Adult male Wistar rats were subjected to 9-weeks of chronic mild stress (CMS) and based on their hedonic-anhedonic behavior they were behaviorally phenotyped as being stress-susceptible (anhedonic) or stress-resilient.

A Modified Monomeric Red Fluorescent Protein Reporter for Assessing CRISPR Activity.

Gene editing in human embryonic stem cells (hESCs) has been significantly enhanced by the discovery and development of CRISPR Cas9, a programmable nuclease system that can introduce targeted double-stranded breaks. The system relies on the optimal selection of a sgRNA sequence with low off-targets and high efficiency. We designed an improved monomeric red fluorescent protein reporter, GEmCherry2, for assessing CRISPR Cas9 activity and for optimizing sgRNA.

Central and Peripheral Nervous System Progenitors Derived from Human Pluripotent Stem Cells Reveal a Unique Temporal and Cell-Type Specific Expression of PMCAs.

The P-type ATPases family consists of ion and lipid transporters. Their unique diversity in function and expression is critical for normal development. In this study we investigated human pluripotent stem cells (hPSC) and different neural progenitor states to characterize the expression of the plasma membrane calcium ATPases (PMCAs) during human neural development and in mature mesencephalic dopaminergic (mesDA) neurons.

Neuronal substrates underlying stress resilience and susceptibility in rats.

Background: Stress and stressful life events have repeatedly been shown as causally related to depression. The Chronic Mild Stress rat model is a valid model of stress-induced depression. Like humans, rats display great heterogeneity in their response to stress and adversity.

Multipotent caudal neural progenitors derived from human pluripotent stem cells that give rise to lineages of the central and peripheral nervous system.

The caudal neural plate is a distinct region of the embryo that gives rise to major progenitor lineages of the developing central and peripheral nervous system, including neural crest and floor plate cells. We show that dual inhibition of the glycogen synthase kinase 3β and activin/nodal pathways by small molecules differentiate human pluripotent stem cells (hPSCs) directly into a preneuroepithelial progenitor population we named "caudal neural progenitors" (CNPs). CNPs coexpress caudal neural plate and mesoderm markers, and, share high similarities to embryonic caudal neural plate cells in their lineage differentiation potential.

Ser129D mutant alpha-synuclein induces earlier motor dysfunction while S129A results in distinctive pathology in a rat model of Parkinson's disease.

Alpha-synuclein phosphorylated at serine 129 (S129) is highly elevated in Parkinson's disease patients where it mainly accumulates in the Lewy bodies. Several groups have studied the role of phosphorylation at the S129 in α-synuclein in a rat model for Parkinson's disease using recombinant adeno-associated viral (rAAV) vectors. The results obtained are inconsistent and accordingly the role of S129 phosphorylation in α-synuclein toxicity remains unclear.

Chronic intranasal deferoxamine ameliorates motor defects and pathology in the α-synuclein rAAV Parkinson's model.

Parkinson's disease is characterized by neuronal death in the substantia nigra and the presence of intracellular inclusions of α-synuclein in the Lewy bodies. Several lines of data support a role for iron in Parkinson's disease: iron is present in Lewy bodies, iron accumulates in the dopaminergic neurons in the substantia nigra, and Parkinson's disease is correlated with polymorphisms of several genes implicated in iron metabolism. Furthermore, iron can compromise the solubility of α-synuclein through direct interaction and can induce neurotoxicity in vitro.

α-Synuclein expression is modulated at the translational level by iron.

Several studies have suggested an interaction between α-synuclein protein and iron in Parkinson's disease. The presence of iron together with α-synuclein in Lewy bodies, the increase of iron in the substantia nigra and the correlation between polymorphism of the several genes implicated in iron metabolism and Parkinson's disease, support a role for iron in the neurodegeneration. Analysis of post mortem brains revealed increased amount of insoluble α-synuclein protein despite unchanged/reduced levels of α-synuclein mRNA in Parkinson's disease.

Co-expression of C-terminal truncated alpha-synuclein enhances full-length alpha-synuclein-induced pathology.

Lewy bodies, which are a pathological hallmark of Parkinson's disease, contain insoluble polymers of alpha-synuclein (alphasyn). Among the different modifications that can promote the formation of toxic alphasyn species, C-terminal truncation is among the most abundant alterations in patients with Parkinson's disease. In vitro, C-terminal truncated alphasyn aggregates faster and sub-stoichiometric amounts of C-terminal truncated alphasyn promote aggregation of the full-length alphasyn (alphasynFL) and induce neuronal toxicity.

Microglia acquire distinct activation profiles depending on the degree of alpha-synuclein neuropathology in a rAAV based model of Parkinson's disease.

Post-mortem analysis of brains from Parkinson's disease (PD) patients strongly supports microglia activation and adaptive immunity as factors contributing to disease progression. Such responses may be triggered by alpha-synuclein (alpha-syn), which is known to be the main constituent of the aggregated proteins found in Lewy bodies in the brains of PD patients. To investigate this we used a recombinant viral vector to express human alpha-syn in rat midbrain at levels that induced neuronal pathology either in the absence or the presence of dopaminergic cell death, thereby mimicking early or late stages of the disease.

Alpha-synuclein aggregation and Ser-129 phosphorylation-dependent cell death in oligodendroglial cells.

Multiple system atrophy is a neurodegenerative disorder characterized by accumulation of aggregated Ser-129-phosphorylated alpha-synuclein in oligodendrocytes. p25alpha is an oligodendroglial protein that potently stimulates alpha-synuclein aggregation in vitro. To model multiple system atrophy, we coexpressed human p25alpha and alpha-synuclein in the rat oligodendroglial cell line OLN-93 and observed a cellular response characterized by a fast retraction of microtubules from the cellular processes to the perinuclear region followed by a protracted development of apoptosis.

Endocannabinoids limit metabotropic glutamate 5 receptor-mediated synaptic inhibition of striatal principal neurons.

Synaptic transmission in the striatum is regulated by metabotropic glutamate (mGlu) receptors through pre- and postsynaptic mechanisms. We investigated the involvement of mGlu 1 and 5 receptors in the control of both excitatory and inhibitory transmission in the striatum. The mGlu 1 and 5 receptor agonist 3,5-DHPG failed to affect glutamate transmission, while it caused a biphasic effect on GABA transmission, characterized by early increase and late decrease in the frequency of spontaneous inhibitory postsynaptic currents (sIPSCs) recorded from striatal principal neurons.