Mitochondrial Aging Defects Emerge in Directly Reprogrammed Human Neurons due to Their Metabolic Profile.

Mitochondria are a major target for aging and are instrumental in the age-dependent deterioration of the human brain, but studying mitochondria in aging human neurons has been challenging. Direct fibroblast-to-induced neuron (iN) conversion yields functional neurons that retain important signs of aging, in contrast to iPSC differentiation. Here, we analyzed mitochondrial features in iNs from individuals of different ages. iNs from old donors display decreased oxidative phosphorylation (OXPHOS)-related gene expression, impaired axonal mitochondrial morphologies, lower mitochondrial membrane potentials, reduced energy production, and increased oxidized proteins levels. In contrast, the fibroblasts from which iNs were generated show only mild age-dependent changes, consistent with a metabolic shift from glycolysis-dependent fibroblasts to OXPHOS-dependent iNs. Indeed, OXPHOS-induced old fibroblasts show increased mitochondrial aging features similar to iNs. Our data indicate that iNs are a valuable tool for studying mitochondrial aging and support a bioenergetic explanation for the high susceptibility of the brain to aging.

Proliferative neural stem cells have high endogenous ROS levels that regulate self-renewal and neurogenesis in a PI3K/Akt-dependant manner.

The majority of research on reactive oxygen species (ROS) has focused on their cellular toxicities. Stem cells generally have been thought to maintain low levels of ROS as a protection against these processes. However, recent studies suggest that ROS can also play roles as second messengers, activating normal cellular processes. Here, we investigated ROS function in primary brain-derived neural progenitors. Somewhat surprisingly, we found that proliferative, self-renewing multipotent neural progenitors with the phenotypic characteristics of neural stem cells (NSC) maintained a high ROS status and were highly responsive to ROS stimulation. ROS-mediated enhancements in self-renewal and neurogenesis were dependent on PI3K/Akt signaling. Pharmacological or genetic manipulations that diminished cellular ROS levels also interfered with normal NSC and/or multipotent progenitor function both in vitro and in vivo. This study has identified a redox-mediated regulatory mechanism of NSC function that may have significant implications for brain injury, disease, and repair.

Human embryonic stem cell-derived motor neurons expressing SOD1 mutants exhibit typical signs of motor neuron degeneration linked to ALS.

Human embryonic stem cell (hESC)-derived neurons have the potential to model neurodegenerative disorders. Here, we demonstrate the expression of a mutant gene, superoxide dismutase 1(SOD1), linked to familial amyotrophic lateral sclerosis (ALS) in hESC-derived motor neurons. Green fluorescent protein (GFP) expression under the control of the HB9 enhancer was used to identify SOD1-transfected motor neurons that express human wild-type SOD1 or one of three different mutants (G93A, A4V and I113T) of SOD1. Neurons transfected with mutant SOD1 exhibited reduced cell survival and shortened axonal processes as compared with control-transfected cells, which could survive for 3 weeks or more. The results indicate that hESC-derived cell populations can be directed to express disease-relevant genes and to display characteristics of the disease-specific cell type. These genetically manipulated hESC-derived motor neurons can facilitate and advance the study of disease-specific cellular pathways, and serve as a model system to test new therapeutic approaches.

Roles of insulin and transferrin in neural progenitor survival and proliferation.

Multipotent neural progenitor cells or neural stem cells (NSC) can be propagated in vitro from a variety of sources and have great potential for neural repair. Although it is well known that NSC divide in response to basic fibroblast growth factor (FGF-2) and epidermal growth factor (EGF), cofactors necessary for survival and maintenance of a multipotent potential are still a matter of debate. In the current study, we examined the requirements for NSC proliferation and survival in vitro using the neurosphere culture system. Apotransferrin (TF), along with EGF and FGF-2, was sufficient for the formation of primary neurospheres derived from embryonic rat cortices. The addition of low concentrations of insulin or insulin-like growth factor-1 (IGF-1) enhanced neurosphere size and number and was necessary for continued passaging. Both insulin and IGF-1 acted at low concentrations, suggesting that their effects were mediated by their cognate receptors, both of which were expressed by neurosphere cultures. Sphere-forming progenitors survived for long periods in culture without EGF or FGF-2 when either insulin or IGF-1 was added to the media. Cell cycle analysis determined that surviving progenitors were relatively quiescent during the period without mitogens. Upon the reintroduction of EGF and FGF-2, surviving progenitors gave rise to new spheres that produced largely glial-restricted progeny compared with sister cultures. These data indicate that the neurogenic potential of NSC may be intimately linked to a continuous exposure to mitogens.

Maternal embryonic leucine zipper kinase is a key regulator of the proliferation of malignant brain tumors, including brain tumor stem cells.

Emerging evidence suggests that neural stem cells and brain tumors regulate their proliferation via similar pathways. In a previous study, we demonstrated that maternal embryonic leucine zipper kinase (Melk) is highly expressed in murine neural stem cells and regulates their proliferation. Here we describe how MELK expression is correlated with pathologic grade of brain tumors, and its expression levels are significantly correlated with shorter survival, particularly in younger glioblastoma patients. In normal human astrocytes, MELK is only faintly expressed, and MELK knockdown does not significantly influence their growth, whereas Ras and Akt overexpressing astrocytes have up-regulated MELK expression, and the effect of MELK knockdown is more prominent in these transformed astrocytes. In primary cultures from human glioblastoma and medulloblastoma, MELK knockdown by siRNA results in inhibition of the proliferation and survival of these tumors. Furthermore, we show that MELK siRNA dramatically inhibits proliferation and, to some extent, survival of stem cells isolated from glioblastoma in vitro. These results demonstrate a critical role for MELK in the proliferation of brain tumors, including their stem cells, and suggest that MELK may be a compelling molecular target for treatment of high-grade brain tumors.

Maternal embryonic leucine zipper kinase (MELK) regulates multipotent neural progenitor proliferation.

Maternal embryonic leucine zipper kinase (MELK) was previously identified in a screen for genes enriched in neural progenitors. Here, we demonstrate expression of MELK by progenitors in developing and adult brain and that MELK serves as a marker for self-renewing multipotent neural progenitors (MNPs) in cultures derived from the developing forebrain and in transgenic mice. Overexpression of MELK enhances (whereas knockdown diminishes) the ability to generate neurospheres from MNPs, indicating a function in self-renewal. MELK down-regulation disrupts the production of neurogenic MNP from glial fibrillary acidic protein (GFAP)-positive progenitors in vitro. MELK expression in MNP is cell cycle regulated and inhibition of MELK expression down-regulates the expression of B-myb, which is shown to also mediate MNP proliferation. These findings indicate that MELK is necessary for proliferation of embryonic and postnatal MNP and suggest that it regulates the transition from GFAP-expressing progenitors to rapid amplifying progenitors in the postnatal brain.

Developmental expression of glial fibrillary acidic protein mRNA in mouse forebrain germinal zones--implications for stem cell biology.

Postnatal neural stem cells (NSCs) express the "traditional" astrocyte marker, glial fibrillary acidic protein (GFAP). Here, we analyze the ontogeny of GFAP mRNA in mouse forebrain germinal zones (GZ). On embryonic day 15, mRNA distribution is highly restricted. Subsequently, expression expands to include many cells in the GZ regions adjacent to the cortex and septum but not to the striatum. Double immunostaining for GFAP and nestin did not demonstrate extensive overlap in the GZ of adult rats, suggesting that either few of the GFAP-expressing cells are stem cells, or that nestin is not a reliable marker for stem cells in the adult rat brain. The current findings indicate that while some GFAP-expressing cells in the GZ may be NSCs, most are not likely to function in a neurogenic capacity.

Neural progenitor genes. Germinal zone expression and analysis of genetic overlap in stem cell populations.

The identification of the genes regulating neural progenitor cell (NPC) functions is of great importance to developmental neuroscience and neural repair. Previously, we combined genetic subtraction and microarray analysis to identify genes enriched in neural progenitor cultures. Here, we apply a strategy to further stratify the neural progenitor genes. In situ hybridization demonstrates expression in the central nervous system germinal zones of 54 clones so identified, making them highly relevant for study in brain and neural progenitor development. Using microarray analysis we find 73 genes enriched in three neural stem cell (NSC)-containing populations generated under different conditions. We use the custom microarray to identify 38 "stemness" genes, with enriched expression in the three NSC conditions and present in both embryonic stem cells and hematopoietic stem cells. However, comparison of expression profiles from these stem cell populations indicates that while there is shared gene expression, the amount of genetic overlap is no more than what would be expected by chance, indicating that different stem cells have largely different gene expression patterns. Taken together, these studies identify many genes not previously associated with neural progenitor cell biology and also provide a rational scheme for stratification of microarray data for functional analysis.