Temporal regulation of nuclear factor one occupancy by calcineurin/NFAT governs a voltage-sensitive developmental switch in late maturing neurons.

Dendrite and synapse development are critical for establishing appropriate neuronal circuits, and disrupted timing of these events can alter neural connectivity. Using microarrays, we have identified a nuclear factor I (NFI)-regulated temporal switch program linked to dendrite formation in developing mouse cerebellar granule neurons (CGNs). NFI function was required for upregulation of many synapse-related genes as well as downregulation of genes expressed in immature CGNs. Chromatin immunoprecipitation analysis revealed that a central feature of this program was temporally regulated NFI occupancy of late-expressed gene promoters. Developing CGNs undergo a hyperpolarizing shift in membrane potential, and depolarization inhibits their dendritic and synaptic maturation via activation of calcineurin (CaN) (Okazawa et al., 2009). Maintaining immature CGNs in a depolarized state blocked NFI temporal occupancy of late-expressed genes and the NFI switch program via activation of the CaN/nuclear factor of activated T-cells, cytoplasmic (NFATc) pathway and promotion of late-gene occupancy by NFATc4, and these mechanisms inhibited dendritogenesis. Conversely, inhibition of the CaN/NFATc pathway in CGNs maturing under physiological nondepolarizing conditions upregulated the NFI switch program, NFI temporal occupancy, and dendrite formation. NFATc4 occupied the promoters of late-expressed NFI program genes in immature mouse cerebellum, and its binding was temporally downregulated with development. Further, NFI temporal binding and switch gene expression were upregulated in the developing cerebellum of Nfatc4 (-/-) mice. These findings define a novel NFI switch and temporal occupancy program that forms a critical link between membrane potential/CaN and dendritic maturation in CGNs. CaN inhibits the program and NFI occupancy in immature CGNs by promoting NFATc4 binding to late-expressed genes. As maturing CGNs become more hyperpolarized, NFATc4 binding declines leading to onset of NFI temporal binding and the NFI switch program.

Regulation of human RNA polymerase III transcription by DNMT1 and DNMT3a DNA methyltransferases.

The human small nuclear RNA (snRNA) and small cytoplasmic RNA (scRNA) gene families encode diverse non-coding RNAs that influence cellular growth and division. Many snRNA and scRNA genes are related via their compact and yet powerful promoters that support RNA polymerase III transcription. We have utilized the human U6 snRNA gene family to examine the mechanism for regulated transcription of these potent transcription units. Analysis of nine U6 family members showed enriched CpG density within the promoters of actively transcribed loci relative to inert genes, implying a relationship between gene potency and DNA methylation. Indeed, both pharmacological inhibition of DNA methyltransferase (DNMT) activity and the forced diminution of DNMT-1, DNMT-3a, and DNMT-3b by siRNA targeting resulted in increased U6 levels in asynchronously growing MCF7 adenocarcinoma cells. In vitro transcription assays further showed that template methylation impedes U6 transcription by RNA polymerase III. Both DNMT-1 and DNMT-3a were detected at the U6-1 locus by chromatin immunoprecipitation directly linking these factors to RNA polymerase III regulation. Despite this association, the endogenous U6-1 locus was not substantially methylated in actively growing cells. However, both DNMT occupancy and low frequency methylation were correlated with increased Retinoblastoma tumor suppressor (RB) expression, suggesting that the RB status can influence specific epigenetic marks.

Colon cancer epidemiology, race and socioeconomic status: Comparing trends in counties served by an urban hospital in Newark, NJ with overall NJ-state and nation-wide patterns.

PURPOSE: Disparities in colorectal cancer (CRC) trends are linked with socioeconomic status (SES) and race. To better understand the colon cancer trends at our medical center, this study characterizes the racial and socioeconomic profile of the population served by our center to identify modifiable risk factors amenable to interventions. METHODS: Colon cancer data from our center as well as New Jersey (NJ) and United States (US) were obtained from National Cancer Database. Demographic data on race and SES for NJ counties were obtained from public databases that sourced data from the American Community Survey and the US census. We compared the odds of being diagnosed with early-onset and late-stage colon cancer (III or IV), respectively in NJ and US, across different racial groups. We also quantified the association between Social Vulnerability Index (SVI) and age-adjusted CRC mortality in NJ counties, with and without accounting for the racial composition of each county. RESULTS: In 2015, our center recorded higher proportions of late-stage and early-onset colon cancer diagnoses compared to all hospitals in NJ and US. Trends for stage and patient age at diagnosis of colon cancer for NJ and the US (2010-2019) showed that Black, Hispanic, and Asian/Pacific Islander individuals had greater odds of being diagnosed with early-onset (age<50) and late-stage colon cancer (Stage III/IV) when compared to White population. NJ counties served by our center showed an overrepresentation of either Black or Hispanic-Latino populations and reported significant disadvantage in SES. For NJ counties, each 25 percentile increase in social vulnerability was associated with 1.04 times the rate of age-adjusted colorectal cancer death (95 % CI: 1.00-1.07). CONCLUSION: Public data on race and SES of the target population can help identify areas of social disparities at the county-level to guide targeted interventions such as improving healthcare access and screening rates.

Analysis of gene expression changes in the rat hippocampus after deep brain stimulation of the anterior thalamic nucleus.

Deep brain stimulation (DBS) surgery, targeting various regions of the brain such as the basal ganglia, thalamus, and subthalamic regions, is an effective treatment for several movement disorders that have failed to respond to medication. Recent progress in the field of DBS surgery has begun to extend the application of this surgical technique to other conditions as diverse as morbid obesity, depression and obsessive compulsive disorder. Despite these expanding indications, little is known about the underlying physiological mechanisms that facilitate the beneficial effects of DBS surgery. One approach to this question is to perform gene expression analysis in neurons that receive the electrical stimulation. Previous studies have shown that neurogenesis in the rat dentate gyrus is elicited in DBS targeting of the anterior nucleus of the thalamus(1). DBS surgery targeting the ATN is used widely for treatment refractory epilepsy. It is thus of much interest for us to explore the transcriptional changes induced by electrically stimulating the ATN. In this manuscript, we describe our methodologies for stereotactically-guided DBS surgery targeting the ATN in adult male Wistar rats. We also discuss the subsequent steps for tissue dissection, RNA isolation, cDNA preparation and quantitative RT-PCR for measuring gene expression changes. This method could be applied and modified for stimulating the basal ganglia and other regions of the brain commonly clinically targeted. The gene expression study described here assumes a candidate target gene approach for discovering molecular players that could be directing the mechanism for DBS.

Isolation, culture and long-term maintenance of primary mesencephalic dopaminergic neurons from embryonic rodent brains.

Degeneration of mesencephalic dopaminergic (mesDA) neurons is the pathological hallmark of Parkinson's diseae. Study of the biological processes involved in physiological functions and vulnerability and death of these neurons is imparative to understanding the underlying causes and unraveling the cure for this common neurodegenerative disorder. Primary cultures of mesDA neurons provide a tool for investigation of the molecular, biochemical and electrophysiological properties, in order to understand the development, long-term survival and degeneration of these neurons during the course of disease. Here we present a detailed method for the isolation, culturing and maintenance of midbrain dopaminergic neurons from E12.5 mouse (or E14.5 rat) embryos. Optimized cell culture conditions in this protocol result in presence of axonal and dendritic projections, synaptic connections and other neuronal morphological properties, which make the cultures suitable for study of the physiological, cell biological and molecular characteristics of this neuronal population.

Culturing mouse cerebellar granule neurons.

The cerebellum plays an important role in motor control, motor skill acquisition, memory and learning among other brain functions. In rodents, cerebellar development continues after birth, characterized by the maturation of granule neurons. Cerebellar granule neurons (CGNs) are the most abundant neuronal type in the central nervous system, and they provide an excellent model for investigating molecular, -cellular, and physiological mechanisms underlying neuronal development as well as neural circuitry linked to behavior. Here we describe a procedure to isolate and culture CGNs from postnatal day 6 mice. These cultures can be used to examine numerous aspects of CGN differentiation, electrophysiology, and function.