Previous studies have established the subventricular (SVZ) and subgranular (SGZ) zones as sites of neurogenesis in the adult forebrain (Doetsch et al., 1999a; Doetsch, 2003a). Work from our laboratory further indicated that midline structures known as circumventricular organs (CVOs) also serve as adult neural stem cell (NSC) niches (Bennett et al., 2009, 2010). In the quiescent rat brain, NSC proliferation remains low in all of these sites. Therefore, we recently examined whether ischemic stroke injury (MCAO) or sustained intraventricular infusion of the mitogen bFGF could trigger an up-regulation in NSC proliferation, inducing neurogenesis and gliogenesis. Our data show that both stroke and bFGF induce a dramatic and long-lasting (14day) rise in the proliferation (BrdU+) of nestin+Sox2+GFAP+ NSCs capable of differentiating into Olig2+ glial progenitors, GFAP+nestin-astrocyte progenitors and Dcx+ neurons in the SVZ and CVOs. Moreover, because of the upsurge in NSC number, it was possible to detect for the first time several novel stem cell niches along the third (3V) and fourth (4V) ventricles. Importantly, a common feature of all brain niches was a rich vasculature with a blood-brain-barrier (BBB) that was highly permeable to systemically injected sodium fluorescein. These data indicate that stem cell niches are more extensive than once believed and exist at multiple sites along the entire ventricular system, consistent with the potential for widespread neurogenesis and gliogenesis in the adult brain, particularly after injury. We further suggest that because of their leaky BBB, stem cell niches are well-positioned to respond to systemic injury-related cues which may be important for stem-cell mediated brain repair.

Although many laboratories currently use small molecule inhibitors of the BMP (Dorsomorphin/DM) and TGF-β (SB431542/SB) signaling pathways in protocols to generate midbrain dopamine (mDA) neurons from hES and hiPS cells, until now, these substances have not been thought to play a role in the mDA differentiation process. We report here that the transient inhibition of constitutive BMP (pSMADs 1, 5, 8) signaling, either alone or in combination with TGF-β inhibition (pSMADs 2, 3), is critically important in the upstream regulation of Wnt1-Lmx1a signaling in mDA progenitors. We postulate that the mechanism via which DM or DM/SB mediates these effects involves the up-regulation in SMAD-interacting protein 1 (SIP1), which results in greater repression of the Wnt antagonist, secreted frizzled related protein 1 (Sfrp1) in stem cells. Accordingly, knockdown of SIP1 reverses the inductive effects of DM/SB on mDA differentiation while Sfrp1 knockdown/inhibition mimics DM/SB. The rise in Wnt1-Lmx1a levels in SMAD-inhibited cultures is, however, accompanied by a reciprocal down-regulation in SHH-Foxa2 levels leading to the generation of few TH+ neurons that co-express Foxa2. If however, exogenous SHH/FGF8 is added along with SMAD inhibitors, equilibrium in these two important pathways is achieved such that authentic (Lmx1a+Foxa2+TH+) mDA neuron differentiation is promoted while alternate cell fates are suppressed in stem cell cultures. These data indicate that activators/inhibitors of BMP and TGF-β signaling play a critical upstream regulatory role in the mDA differentiation process in human pluripotent stem cells.

Aberrant expression and activation of the cell cycle protein E2F1 in neurons has been implicated in many neurodegenerative diseases. As a transcription factor regulating G1 to S phase progression in proliferative cells, E2F1 is often upregulated and activated in models of neuronal death. However, despite its well studied functions in neuronal death, little is known regarding the role of E2F1 in the mature brain. In the present study, we used a combined approach to study the effect of E2F1 gene disruption on mouse behavior and brain biochemistry. We identified significant age-dependent olfactory and memory related deficits in E2f1 mutant mice. In addition, we found that E2F1 exhibits punctated staining and localizes closely to the synapse. Furthermore, we found a mirroring age-dependent loss of postsynaptic protein-95 in the hippocampus and olfactory bulb as well as a global loss of several other synaptic proteins. Coincidently, E2F1 expression is significantly elevated at the ages in which behavioral and synaptic perturbations were observed. Lastly, we show that deficits in adult neurogenesis persist late in aged E2f1 mutant mice which may partially contribute to the behavior phenotypes. Taken together, our data suggest that the disruption of E2F1 function leads to specific age-dependent behavioral deficits and synaptic perturbations.

Mutations affecting acetylcholine receptors have been causally linked to the development of congenital myasthenic syndromes (CMS) in humans resulting from neuromuscular transmission defects. In an undergraduate Molecular Neurobiology course, the molecular basis of CMS was explored through study of a Caenorhabditis elegans model of the disease. The nicotinic acetylcholine receptor (nAChR), located on the postsynaptic muscle cell membrane, contains a pentameric ring structure comprised of five homologous subunits. In the nematode C. elegans, unc-63 encodes an α subunit of nAChR. UNC-63 is required for the function of nAChR at the neuromuscular junction. Mutations in unc-63 result in defects in locomotion and egg-laying and may be used as models for CMS. Here, we describe the responses of four unc-63 mutants to the cholinesterase inhibitor pyridostigmine bromide (range 0.9-15.6 mM in this study), a treatment for CMS that mitigates deficiencies in cholinergic transmission by elevating synaptic ACh levels. Our results show that 15.6 mM pyridostigmine bromide enhanced mobility in two alleles, depressed mobility in one allele and in N2, while having no effect on the fourth allele. This indicates that while pyridostigmine bromide may be effective at ameliorating symptoms of CMS in certain cases, it may not be a suitable treatment for all individuals due to the diverse etiology of this disease. Students in the Molecular Neurobiology course enhanced their experience in scientific research by conducting an experiment designed to increase understanding of genetic defects of neurological function.

E2F1 is a transcription factor classically known to regulate G/G to S phase progression in the cell cycle. In addition, E2F1 also regulates a wide range of apoptotic genes and thus has been well studied in the context of neuronal death and neurodegenerative diseases. However, its function and regulation in the mature central nervous system are not well understood. Alternative splicing is a well-conserved post-transcriptional mechanism common in cells of the CNS and is necessary to generate diverse functional modifications to RNA or protein products from genes. Heretofore, physiologically significant alternatively spliced E2F1 transcripts have not been reported. In the present study, we report the identification of two novel alternatively spliced E2F1 transcripts: E2F1b, an E2F1 transcript retaining intron 5, and E2F1c, an E2F1 transcript excluding exon 6. These alternatively spliced transcripts are observed in the brain and neural cell types including neurons, astrocytes, and undifferentiated oligodendrocytes. The expression of these E2F1 transcripts is distinct during maturation of primary hippocampal neuroglial cells. Pharmacologically-induced global translation inhibition with cycloheximide, anisomycin or thapsigargin lead to significantly reduced expression of E2F1a, E2F1b and E2F1c. Conversely, increasing neuronal activity by elevating the concentration of potassium chloride selectively increased the expression of E2F1b. Furthermore, experiments expressing these variants in vitro show the transcripts can be translated to generate a protein product. Taken together, our data suggest that the alternatively spliced E2F1 transcript behave differently than the E2F1a transcript, and our results provide a foundation for future investigation of the function of E2F1 splice variants in the CNS.