Karl J.L. Fernandes

A dermal niche for multipotent adult skin-derived precursor cells

A fundamental question in stem cell research is whether cultured multipotent adult stem cells represent endogenous multipotent precursor cells. Here we address this question, focusing on SKPs, a cultured adult stem cell from the dermis that generates both neural and mesodermal progeny. We show that SKPs derive from endogenous adult dermal precursors that exhibit properties similar to embryonic neural-crest stem cells. We demonstrate that these endogenous SKPs can first be isolated from skin during embryogenesis and that they persist into adulthood, with a niche in the papillae of hair and whisker follicles. Furthermore, lineage analysis indicates that both hair and whisker follicle dermal papillae contain neural-crest-derived cells, and that SKPs from the whisker pad are of neural-crest origin. We propose that SKPs represent an endogenous embryonic precursor cell that arises in peripheral tissues such as skin during development and maintains multipotency into adulthood.

Evidence that Embryonic Neurons Regulate the Onset of Cortical Gliogenesis via Cardiotrophin-1

Precursor cells of the embryonic cortex sequentially generate neurons and then glial cells, but the mechanisms regulating this neurogenic-to-gliogenic transition are unclear. Using cortical precursor cultures, which temporally mimic this in vivo differentiation pattern, we demonstrate that cortical neurons synthesize and secrete the neurotrophic cytokine cardiotrophin-1, which activates the gp130-JAK-STAT pathway and is essential for the timed genesis of astrocytes in vitro. Our data indicate that a similar phenomenon also occurs in vivo. In utero electroporation of neurotrophic cytokines in the environment of embryonic cortical precursors causes premature gliogenesis, while acute perturbation of gp130 in cortical precursors delays the normal timed appearance of astrocytes. Moreover, the neonatal cardiotrophin-1-/- cortex contains fewer astrocytes. Together, these results describe a neural feedback mechanism; newly born neurons produce cardiotrophin-1, which instructs multipotent cortical precursors to generate astrocytes, thereby ensuring that gliogenesis does not occur until neurogenesis is largely complete.

Influence of the axotomy to cell body distance in rat rubrospinal and spinal motoneurons: differential regulation of GAP-43, tubulins, and neurofilament-M.

Axotomized motoneurons regenerate their axons regardless of whether axotomy occurs proximally or distally from their cell bodies. In contrast, regeneration of rubrospinal axons into peripheral nerve grafts has been detected after cervical but not after thoracic injury of the rubrospinal tract. By using in situ hybridization (ISH) combined with reliable retrograde tracing methods, we compared regeneration‐associated gene expression after proximal and distal axotomy in spinal motoneurons versus rubrospinal neurons. Regardless of whether they were axotomized at the iliac crest (proximal) or popliteal fossa (distal), sciatic motoneurons underwent highly pronounced changes in ISH signals for Growth Associated Protein 43 (GAP‐43) (10–20× increase) and neurofilament M (60–85% decrease). In contrast, tubulin ISH signals substantially increased only after proximal axotomy (3–5× increase). To compare these changes in gene expression with those of axotomized rubrospinal neurons, the rubrospinal tract was transected at the cervical (proximal) or thoracic (distal) levels of the spinal cord. Cervically axotomized rubrospinal neurons showed three‐ to fivefold increases in ISH signals for GAP‐43 and tubulins (only transient) and a 75% decrease for neurofilament‐M. In sharp contrast, thoracic axotomy had only marginal effects. After implantation of peripheral nerve transplants into the spinal cord injury sites, retrograde labeling with the sensitive retrograde tracer Fluoro‐Gold identified regenerating rubrospinal neurons only after cervical axotomy. Furthermore, rubrospinal neurons specifically regenerating into the transplants were hypertrophied and expressed high levels of GAP‐43 and tubulins. Taken together, these data support the concept that, even if central nervous system (CNS) axons are presented with a permissive/supportive environment, appropriate cell body responses to injury are a prerequisite for CNS axonal regeneration. J. Comp. Neurol. 414:495–510, 1999.

Analysis of the neurogenic potential of multipotent skin-derived precursors

Multipotent precursors similar to stem cells of the embryonic neural crest (NC) have been identified in several postnatal tissues, and are potentially useful for research and therapeutic purposes. However, their neurogenic potential, including their ability to produce electrophysiologically active neurons, is largely unexplored. We investigated this issue with regard to skin-derived precursors (SKPs), multipotent NC-related precursors isolated from the dermis of skin. SKP cultures follow an appropriate pattern and time-course of neuronal differentiation, with proliferating nestin-expressing SKPs generating post-mitotic neuronal cells that co-express pan-neuronal and peripheral autonomic lineage markers. These SKP-derived neuron-like cells survive and maintain their peripheral phenotype for at least 5 weeks when transplanted into the CNS environment of normal or kainate-injured hippocampal slices. Undifferentiated SKPs retain key neural precursor properties after multi-passage expansion, including growth factor dependence, nestin expression, neurogenic potential, and responsiveness to embryonic neural crest fate determinants. Despite undergoing an apparently appropriate neurogenic process, however, SKP-derived neuron-like cells possess an immature electrophysiological profile. These findings indicate that SKPs retain latent neurogenic properties after residing in a non-neural tissue, but that additional measures will be necessary to promote their differentiation into electrophysiologically active neurons.

Multipotent skin-derived precursors: adult neural crest-related precursors with therapeutic potential

We previously made the surprising finding that cultures of multipotent precursors can be grown from the dermis of neonatal and adult mammalian skin. These skin-derived precursors (SKPs) display multi-lineage differentiation potential, producing both neural and mesodermal progeny in vitro, and are an apparently novel precursor cell type that is distinct from other known precursors within the skin. In this review, we begin by placing these findings within the context of the rapidly evolving stem cell field. We then describe our recent efforts focused on understanding the developmental biology of SKPs, discussing the idea that SKPs are neural crest-related precursors that (i) migrate into the skin during embryogenesis, (ii) persist within a specific dermal niche, and (iii) play a key role in the normal physiology, and potentially pathology, of the skin. We conclude by highlighting some of the therapeutic implications and unresolved questions raised by these studies.

Aging of the subventricular zone neural stem cell niche: evidence for quiescence-associated changes between early and mid-adulthood

Stem cells can exist in either active or quiescent states. In the aging hippocampus, adult neural stem cells (aNSCs) shift into a quiescent state, contributing to age-related reductions in hippocampal neurogenesis. Here, we focused on the subventricular zone (SVZ) stem cell niche of the adult forebrain, asking to what extent quiescence-associated changes in aNSCs are initiated between early and middle-age. Immunohistochemical and label retention experiments revealed that the overall output of the SVZ stem cell system was already highly decreased in middle-aged mice (12-months-old) compared with young adult mice (2-month-old), as measured by reduced marker expression for multiple neural precursor sub-populations and diminished addition of SVZ-derived neuroblasts to the olfactory bulbs (OBs). These changes were associated with significant cytological aberrations within the SVZ niche, including an overall atrophy of the SVZ and accumulation of large lipid droplets within ependymal cells, which are key support cells of the SVZ niche. Importantly, the reduced output of the middle-aged SVZ stem cell system correlated with quiescence-associated changes in middle-aged aNSCs. Specifically, while tissue culture experiments showed that young adult and middle-aged forebrains possessed equal numbers of neurosphere-forming aNSCs, the middle-aged neurospheres exhibited differences in their in vitro properties, and middle-aged aNSCs in vivo divided less frequently. These findings demonstrate that aNSCs begin undergoing quiescence-associated changes between early and mid-adulthood in the mouse SVZ, and serve as a useful framework for further studies aimed at defining the early events involved in aging-associated quiescence of aNSCs.

A transcriptional role for C/EBP β in the neuronal response to axonal injury

Abstract The molecular mechanisms responsible for inducing gene expression following neuronal injury are not well understood. Here, we address this issue by focusing upon C/EBPβ, a transcription factor implicated in cellular injury and regeneration. We show that C/EBPβ mRNA is expressed in neurons throughout the mature brain and that levels of both C/EBPβ mRNA and phosphoprotein are increased in facial motor neurons following axonal injury. To determine the importance of these increases, we examined the regeneration-associated Tα1 α-tubulin gene which contains functional C/EBP binding sites in its promoter. In transgenic mice, expression of a minimal 176 nucleotide Tα1 α-tubulin promoter:nlacZ reporter gene was upregulated in injured facial motor neurons. This injury-induced transcriptional increase was inhibited in C/EBPβ −/− mice. A similar inhibition was observed in C/EBPβ −/− mice that carried a larger 1.1-kb promoter Tα1:nlacZ reporter construct. Moreover, in situ hybridization revealed that the injury-induced upregulation of the endogenous mouse α1 α-tubulin mRNA, and of a second regeneration-associated mRNA, GAP-43, was inhibited in C/EBPβ −/− mice. Thus, C/EBPβ is essential for the neuronal injury response, acting to transcriptionally activate regeneration-associated gene expression.

Widespread deficits in adult neurogenesis precede plaque and tangle formation in the 3xTg mouse model of Alzheimer's disease.

Alzheimer’s disease (AD) affects cognitive modalities that are known to be regulated by adult neurogenesis, such as hippocampal‐ and olfactory‐dependent learning and memory. However, the relationship between AD‐associated pathologies and alterations in adult neurogenesis has remained contentious. In the present study, we performed a detailed investigation of adult neurogenesis in the triple transgenic (3xTg) mouse model of AD, a unique model that generates both amyloid plaques and neurofibrillary tangles, the hallmark pathologies of AD. In both neurogenic niches of the brain, the hippocampal dentate gyrus and forebrain subventricular zone, we found that 3xTg mice had decreased numbers of (i) proliferating cells, (ii) early lineage neural progenitors, and (iii) neuroblasts at middle age (11 months old) and old age (18 months old). These decreases correlated with major reductions in the addition of new neurons to the respective target areas, the dentate granule cell layer and olfactory bulb. Within the subventricular zone niche, cytological alterations were observed that included a selective loss of subependymal cells and the development of large lipid droplets within the ependyma of 3xTg mice, indicative of metabolic changes. Temporally, there was a marked acceleration of age‐related decreases in 3xTg mice, which affected multiple stages of neurogenesis and was clearly apparent prior to the development of amyloid plaques or neurofibrillary tangles. Our findings indicate that AD‐associated mutations suppress neurogenesis early during disease development. This suggests that deficits in adult neurogenesis may mediate premature cognitive decline in AD.

Mammalian Target of Rapamycin Signaling Is a Key Regulator of the Transit-Amplifying Progenitor Pool in the Adult and Aging Forebrain

Adult forebrain neurogenesis is dynamically regulated. Multiple families of niche-derived cues have been implicated in this regulation, but the precise roles of key intracellular signaling pathways remain vaguely defined. Here, we show that mammalian target of rapamycin (mTOR) signaling is pivotal in determining proliferation versus quiescence in the adult forebrain neural stem cell (NSC) niche. Within this niche, mTOR complex-1 (mTORC1) activation displays stage specificity, occurring in transiently amplifying (TA) progenitor cells but not in GFAP+ stem cells. Inhibiting mTORC1 depletes the TA progenitor pool in vivo and suppresses epidermal growth factor (EGF)-induced proliferation within neurosphere cultures. Interestingly, mTORC1 inhibition induces a quiescence-like phenotype that is reversible. Likewise, mTORC1 activity and progenitor proliferation decline within the quiescent NSC niche of the aging brain, while EGF administration reactivates the quiescent niche in an mTORC1-dependent manner. These findings establish fundamental links between mTOR signaling, proliferation, and aging-associated quiescence in the adult forebrain NSC niche.

Untangling the Influences of Voluntary Running, Environmental Complexity, Social Housing and Stress on Adult Hippocampal Neurogenesis

Environmental enrichment (EE) exerts powerful effects on brain physiology, and is widely used as an experimental and therapeutic tool. Typical EE paradigms are multifactorial, incorporating elements of physical exercise, environmental complexity, social interactions and stress, however the specific contributions of these variables have not been separable using conventional housing paradigms. Here, we evaluated the impacts of these individual variables on adult hippocampal neurogenesis by using a novel “Alternating EE” paradigm. For 4 weeks, adult male CD1 mice were alternated daily between two enriched environments; by comparing groups that differed in one of their two environments, the individual and combinatorial effects of EE variables could be resolved. The Alternating EE paradigm revealed that (1) voluntary running for 3 days/week was sufficient to increase both mitotic and post-mitotic stages of hippocampal neurogenesis, confirming the central importance of exercise; (2) a complex environment (comprised of both social interactions and rotated inanimate objects) had no effect on neurogenesis itself, but enhanced depolarization-induced c-Fos expression (attributable to social interactions) and buffered stress-induced plasma corticosterone levels (attributable to inanimate objects); and (3) neither social isolation, group housing, nor chronically increased levels of plasma corticosterone had a prolonged impact on neurogenesis. Mouse strain, handling and type of running apparatus were tested and excluded as potential confounding factors. These findings provide valuable insights into the relative effects of key EE variables on adult neurogenesis, and this “Alternating EE” paradigm represents a useful tool for exploring the contributions of individual EE variables to mechanisms of neural plasticity.