Oxygen tension is critical for proliferation of human and murine midbrain-derived neural precursor cells (mNPCs). Lack of hypoxia-inducible factor-1α (HIF1α) impairs midbrain dopaminergic neurogenesis which could be rescued by vascular endothelial growth factor (VEGF) via VEGFR-2 signaling. Here, we conditionally inactivated the VEGFR-2, encoded by the fetal liver kinase 1 (Flk1) gene, in murine NPCs to determine its role in proliferation and survival in vitro as well as survival of dopaminergic neurons in vivo. Flk1 conditional knock-out (Flk1 CKO) mice showed no general brain phenotype. There was no midbrain-specific impairment of NPC proliferation as seen in HIF1α CKO mice. In the substantia nigra (SN) of adult Flk1 CKO mice, nonbiased stereological cell counts revealed no reduction of TH-positive neurons of Flk1 CKO mice compared with control Cre/wt mice (in which the wild-type Flk1 allele is expressed in parallel with the Cre recombinase allele). In conclusion, VEGF receptor signaling seems not to be relevant to the development and survival of substantia nigra dopaminergic neurons within the hypoxia-HIF1α signaling pathway.

Parkinson's disease is characterized by a continuous loss of neurons within the substantia nigra (SN) leading to a depletion of dopamine. Within the adult SN as a non-neurogenic region, cells with mainly oligodendrocytic precursor characteristics, expressing the neuro-glial antigen-2 (NG2) are continuously generated. Proliferation of these cells is altered in animal models of Parkinson's disease (PD). Exercise and environmental enrichment re-increase proliferation of NG2+ cells in PD models, however, a possible mechanistic role of dopamine for this increase is not completely understood. NG2+ cells can differentiate into oligodendrocytes but also into microglia and neurons as observed in vitro suggesting a possible hint for endogenous regenerative capacity of the SN. We investigated the role of dopamine in NG2-generation and differentiation in the adult SN stimulated by physical activity and environmental enrichment.

Neurogenesis occurs constitutively within the periventricular region (PVR) of the lateral ventricles (LV) of the adult mammalian brain. The occurrence of adult neurogenesis within the PVR outside the neurogenic niche of the LV remains controversial, but neural stem cells can be isolated from PVR of the whole ventricular system. The histological basis of this phenomenon including the regional differences of cellular phenotypes within the PVRs is still enigmatic. The occurrence of neurogenesis or manipulable progenitor cells in caudal parts of the adult brain is however one prerequisite for orthotopic regenerative approaches in Parkinson's disease (PD) and other disorders of the midbrain/brainstem. Using quantitative immunohistochemical techniques and electron microscopy, we found a rostro-caudal gradual loss of cellular diversity within the PVR throughout the whole ventricular axis with loss of transit amplifying epidermal growth factor-receptor(+) type C cells in all parts caudal to the LV, a gradual reduction from rostral to caudal of both stem cells (type B cells or astrocytes) without signs of proliferation outside the PVR of the LV as well as neuroblasts-like cells (polysialylated neural cell adhesion molecule [PSA-NCAM](+), but doublecortin negative cells) with a different morphology compared with neuroblasts of the PVR of the LV. Electron microscopy confirmed these immunohistochemical data. The proportion of Nestin(+)/CD24(+) cells and Nestin(+)/S100beta(+) ependymal cells were consecutively increased in the PVR from rostral to caudal, and ultrastructural analysis showed a region-specific morphology with darker cytoplasm with occasional large lipid droplets as well as indented nuclei within the caudal PVRs. The strong correlation of neuroblast-like cells with the number of neurosphere-forming cells suggests that a quiescent subtype of PSA-NCAM(+) cells might be a source of neurosphere-forming cells. We did not find any evidence for neurogenesis or the occurrence of neuroprogenitors within the substantia nigra or other parts of the midbrain/brainstem outside the PVR. Our data provide the histological framework for future studies on orthotopic regenerative approaches in PD by recruiting endogenous predopaminergic progenitors from the midbrain PVR.

The major catecholamines-dopamine (DA) and norepinephrine (NE)-are not only involved in synaptic communication but also act as important trophic factors and might ultimately be involved in mammalian brain development. The catecholaminergic innervation of neurogenic regions of the developing brain and its putative relationship to neurogenesis is thus of pivotal interest. We here determined DA and NE innervation around the ventricular/subventricular zone (VZ/SVZ) bordering the whole ventricular system of the developing mouse brain from embryonic day 14.5 (E14.5), E16.5, and E19.5 until postnatal day zero (P0) by histological evaluation and HPLC with electrochemical detection. We correlated these data with the proliferation capacity of the respective regions by quantification of MCM2+ cells. During development, VZ/SVZ catecholamine levels dramatically increased between E16.5 and P0 with DA levels increasing in forebrain VZ/SVZ bordering the lateral ventricles and NE levels raising in midbrain/hindbrain VZ/SVZ bordering the third ventricle, the aqueduct, and the fourth ventricle. Conversely, proliferating MCM2+ cell counts dropped between E16.5 and E19.5 with a special focus on all VZ/SVZs outside the lateral ventricles. We detected an inverse strong negative correlation of the proliferation capacity in the periventricular neurogenic regions (log-transformed MCM2+ cell counts) with their NE levels (r = -0.932; p < 0.001), but not their DA levels (r = 0.440; p = 0.051) suggesting putative inhibitory effects of NE on cell proliferation within the periventricular regions during mouse brain development. Our data provide the first framework for further demandable studies on the functional importance of catecholamines, particularly NE, in regulating neural stem/progenitor cell proliferation and differentiation during mammalian brain development.

The regulation of adult neural stem or progenitor cell (aNSC) proliferation and differentiation as an interplay of cell-intrinsic and local environmental cues remains in part unclear, impeding their role in putative regenerative therapies. aNSCs with all major properties of NSCs in vitro have been identified in a variety of brain regions beyond the classic neurogenic niches, including the caudal periventricular regions (PVRs) of the midbrain, though active neurogenesis is either limited or merely absent in these regions. To elucidate cell-intrinsic properties of aNSCs from various PVRs, we here examined the proliferation and early differentiation capacity of murine aNSCs from non-neurogenic midbrain PVRs (PVRMB) compared to aNSCs from the neurogenic ventricular-subventricular zone (PVRV-SVZ) 7 days after transplantation into the permissive pro-neurogenic niche of the dentate gyrus (DG) of the hippocampus in mice. An initial in vitro characterization of the transplants displayed very similar characteristics of both aNSC grafts after in vitro expansion with equal capacities of terminal differentiation into astrocytes and Tuj1+ neurons. Upon the allogenic transplantation of the respective aNSCs into the DG, PVRMB grafts showed a significantly lower graft survival and proliferative capacity compared to PVRV-SVZ transplants, whereby the latter are exclusively capable of generating new neurons. Although these differences might be-in part-related to the transplantation procedure and the short-term study design, our data strongly imply important cell-intrinsic differences between aNSCs from neurogenic compared to non-neurogenic PVRs with respect to their neurogenic potential and/or their sensitivity to neurogenic cues.

Chorea acanthocytosis (ChAc), an ultra-rare devastating neurodegenerative disease, is caused by mutations in the VPS13A gene, which encodes for the protein chorein. Affected patients suffer from chorea, orofacial dyskinesia, epilepsy, parkinsonism as well as peripheral neuropathy. Although medium spinal neurons of the striatum are mainly affected, other regions are impaired as well over the course of the disease. Animal studies as well as studies on human erythrocytes suggest Lynkinase inhibition as valuable novel opportunity to treat ChAc. In order to investigate the peripheral neuropathy aspect, we analyzed induced pluripotent stem cell derived midbrain/hindbrain cell cultures from ChAc patients in vitro. We observed dendritic microtubule fragmentation. Furthermore, by using in vitro live cell imaging, we found a reduction in the number of lysosomes and mitochondria, shortened mitochondria, an increase in retrograde transport and hyperpolarization as measured with the fluorescent probe JC-1. Deep phenotyping pointed towards a proximal axonal deterioration as the primary axonal disease phenotype. Interestingly, pharmacological interventions, which proved to be successful in different models of ChAc, were ineffective in treating the observed axonal phenotypes. Our data suggests that treatment of this multifaceted disease might be cell type and/or neuronal subtype specific, and thus necessitates precision medicine in this ultra-rare disease.