Heather Dziema

microRNA modulation of circadian clock period and entrainment

microRNAs (miRNAs) are a class of small, noncoding RNAs that regulate the stability or translation of mRNA transcripts. Although recent work has implicated miRNAs in development and in disease, the expression and function of miRNAs in the adult mammalian nervous system have not been extensively characterized. Here, we examine the role of two brain-specific miRNAs, miR-219 and miR-132, in modulating the circadian clock located in the suprachiasmatic nucleus. miR-219 is a target of the CLOCK and BMAL1 complex, exhibits robust circadian rhythms of expression, and the in vivo knockdown of miR-219 lengthens the circadian period. miR-132 is induced by photic entrainment cues via a MAPK/CREB-dependent mechanism, modulates clock-gene expression, and attenuates the entraining effects of light. Collectively, these data reveal miRNAs as clock- and light-regulated genes and provide a mechanistic examination of their roles as effectors of pacemaker activity and entrainment.

The ERK/MAP kinase pathway couples light to immediate‐early gene expression in the suprachiasmatic nucleus

Signalling via the p42/44 mitogen‐activated protein kinase (MAPK) pathway has been identified as an intermediate event coupling light to entrainment of the mammalian circadian clock located in the suprachiasmatic nucleus (SCN). Given this observation, it was of interest to determine where within the entrainment process the MAPK pathway was functioning. In this study, we examined the role of the MAPK pathway as a regulator of light‐induced gene expression in the SCN. Towards this end, we characterized the effect pharmacological disruption of the MAPK cascade has on the expression of the immediate‐early genes c‐Fos, JunB and EGR‐1. We report that uncoupling light from MAPK pathway activation attenuated the expression of all three gene products. In the absence of photic stimulation, inhibition of the MAPK pathway did not alter basal gene product expression levels. Light‐induced activation of cAMP response element (CRE)‐dependent transcription, as assessed using a CRE‐LacZ transgenic mouse strain, was also disrupted by blocking MAPK pathway activation. These results reveal that the MAPK cascade functions as one of the first transduction steps leading from light to rapid transcriptional activation, an essential event in the entrainment process. MAPK pathway‐dependent gene expression in the SCN may result, in part, from stimulation of CRE‐dependent transcription.

Circadian regulation of mammalian target of rapamycin signaling in the mouse suprachiasmatic nucleus.

Circadian (24-h) rhythms influence virtually every aspect of mammalian physiology. The main rhythm generation center is located in the suprachiasmatic nucleus (SCN) of the hypothalamus, and work over the past several years has revealed that rhythmic gene transcription and post-translational processes are central to clock timing. In addition, rhythmic translation control has also been implicated in clock timing; however the precise cell signaling pathways that drive this process are not well known. Here we report that a key translation activation cascade, the mammalian target of rapamycin (mTOR) pathway, is under control of the circadian clock in the SCN. Using phosphorylated S6 ribosomal protein (pS6) as a marker of mTOR activity, we show that the mTOR cascade exhibits maximal activity during the subjective day, and minimal activity during the late subjective night. Importantly, expression of S6 was not altered as a function of circadian time. Rhythmic S6 phosphorylation was detected throughout the dorsoventral axis of the SCN, thus suggesting that rhythmic mTOR activity was not restricted to a subset of SCN neurons. Rather, rhythmic pS6 expression appeared to parallel the expression pattern of the clock gene period1 (per1). Using a transgenic per1 reporter gene mouse strain, we found a statistically significant cellular level correlation between pS6 and per1 gene expression over the circadian cycle. Further, photic stimulation triggered a coordinate upregulation of per1 and mTOR activation in a subset of SCN cells. Interestingly, this cellular level correlation between mTOR activity and per1 expression appears to be specific, since a similar expression profile for pS6 and per2 or c-FOS was not detected. Finally, we show that mTOR activity is downstream of the ERK/MAPK signal transduction pathway. Together these data reveal that mTOR pathway activity is under the control of the SCN clock, and suggests that mTOR signaling may contribute to distinct aspects of the molecular clock timing process.

The Molecular Gatekeeper Dexras1 Sculpts the Photic Responsiveness of the Mammalian Circadian Clock

The mammalian master clock, located in the suprachiasmatic nucleus (SCN), is exquisitely sensitive to photic timing cues, but the key molecular events that sculpt both the phasing and magnitude of responsiveness are not understood. Here, we show that the Ras-like G-protein Dexras1 is a critical factor in these processes. Dexras1-deficient mice (dexras1−/−) exhibit a restructured nighttime phase response curve and a loss of gating to photic resetting during the day. Dexras1 affects the photic sensitivity by repressing or activating time-of-day-specific signaling pathways that regulate extracellular signal-regulated kinase (ERK)/mitogen-activated protein kinase (MAPK). During the late night, Dexras1 limits the capacity of pituitary adenylate cyclase (PAC) activating peptide (PACAP)/PAC1 to affect ERK/MAPK, and in the early night, light-induced phase delays, which are mediated predominantly by NMDA receptors, are reduced as reported previously. Daytime photic phase advances are mediated by a novel signaling pathway that does not affect the SCN core but rather stimulates ERK/MAPK in the SCN shell and triggers downregulation of clock protein expression.

Segregation of expression of mPeriod gene homologs in neurons and glia: possible divergent roles of mPeriod1 and mPeriod2 in the brain

The suprachiasmatic nuclei (SCN) of the mammalian hypothalamus function as the master circadian clock, coordinating the timing of diverse cell populations and organ systems. Dysregulation of clock timing is linked to a broad range of human conditions, including obesity, cardiovascular disease and a wide spectrum of neurological disorders. Aberrant regulation of expression of the PERIOD genes has been associated with improper cell division and human cancers, while the autosomal dominant disorder familial advanced sleep phase syndrome has been mapped to a single missense mutation within the critical clock gene hPERIOD2. An essential tool to begin to dissect the inherent molecular timing process is the clock gene reporter. Here, we functionally characterize two new mouse transgenic clock reporters, mPeriod1-Venus and mPeriod2-DsRED. Venus and DsRED are fluorescent proteins that can be used to monitor transcription in individual cells in real-time. Imaging of the SCN revealed oscillations, as well as light inducibility, in Venus and DsRED expression. Rhythmic Venus and DsRED expression was observed in distinct SCN cell populations, suggesting the existence of discrete cellular SCN clocks. Outside of the SCN, mPeriod1-Venus expression was broadly expressed in neuronal and non-neuronal populations. Conversely, mPeriod2-DsRED was expressed in glial populations and progenitor cells of the dentate gyrus; limited expression was detected in neurons. This distinct expression pattern of the two reporters reveals that the central nervous system possesses mechanistically distinct subpopulations of neuronal and non-neuronal cellular clocks. These novel mouse models will facilitate our understanding of clock timing and its role in human diseases.

Investigation of germline GFRA4 mutations and evaluation of the involvement of GFRA1, GFRA2, GFRA3, and GFRA4 sequence variants in Hirschsprung disease

The RET proto-oncogene on 10q11.2, which encodes a receptor tyrosine kinase expressed in neural crest and its derivatives, is the susceptibility gene for multiple endocrine neoplasia type 2 (MEN 2), characterised by medullary thyroid carcinoma, phaeochromocytoma, and hyperparathyroidism, and one of several susceptibility genes for Hirschsprung disease (HSCR).1–4 HSCR is characterised by aganglionosis of the gut resulting from inappropriate and premature apoptosis of the enteric ganglia. Initially, it was believed that approximately 50% of familial HSCR and 30% of isolated HSCR were the result of germline loss of function mutations in the RET proto-oncogene5,6 (reviewed by Eng and Mulligan7). However, these data were obtained with highly selected series of families and patients with HSCR. A population based survey of HSCR cases showed that only 3% of isolated HSCR carried traditional germline RET mutations.8 In the context of these data and the anecdotal observation that a RET codon 45 variant seemed to modify the expression of HSCR in a MEN 2/HSCR family with RET codon 618 mutation,9 we began to examine the polymorphic alleles at codon 45 and the other coding variants as common low penetrance alleles for HSCR susceptibility. Indeed, we found that certain haplotypes or pairs of haplotypes (“genotypes”) comprising specific combinations of RET polymorphic sequence variants were highly associated with isolated HSCR.9–11 These observations were also noted among HSCR populations from elsewhere in the world.12–14 These data implied that RET and/or loci in proximity to it could act as common low penetrance alleles which predisposed to isolated HSCR. There are perhaps seven susceptibility genes for syndromic and non-syndromic HSCR.3,415–25 Among these seven genes, RET is considered a major susceptibility gene for HSCR. RET is an unusual receptor tyrosine kinase in that it requires …

Over-representation of a germline variant in the gene encoding RET co-receptor GFRα-1 but not GFRα-2 or GFRα-3 in cases with sporadic medullary thyroid carcinoma

In contrast to the hereditary form of medullary thyroid carcinoma (MTC), little is known about the etiology of sporadic MTC. Somatic gain-of-function mutations in the RET proto-oncogene, encoding a receptor tyrosine kinase, are found in an average of 40% of sporadic MTC. We analysed 31 sporadic MTC for somatic and germline variants in GFRA1, GFRA2 and GFRA3 which encode the co-receptors of RET. Although there were no somatic mutations in any of the three genes, a sequence variant (−193C>G) in the 5′-UTR of GFRA1 was found in 15% of cases. Three patients were heterozygous (het); another three patients homozygous (hom) for the G variant. The G allele was not observed in 31 race-matched normal controls. Hence, the relative frequency of this variant in sporadic MTC cases and controls differed significantly (P<0.05). Since this variant lies in the 5′ UTR, likely at the transcriptional start site, we analysed for differential expression of GFRα-1 at the transcript and protein levels. At the mRNA level, GFRA1 was over-expressed in tumors harboring the rare variant (P=0.06). The presence of the G polymorphic allele seemed to be associated with increased expression by immunostaining for GFRα-1. Interestingly, cytoplasmic staining was stronger in intensity for het patients and nuclear staining predominant in hom cases. In conclusion, mutation analysis of GFRA1, GFRA2 and GFRA3 revealed over-representation of a rare variant in GFRA1 (−193C>G) in the germline of sporadic MTC cases. Our data suggest that the mechanism is related to over-expression of GFRα-1 and differential subcellular compartmentalization but the precise mechanism as to how it acts as a low penetrance susceptibility allele for the development of sporadic MTC remains to be elucidated.

Evaluation of germline sequence variants of GFRA1, GFRA2, and GFRA3 genes in a cohort of spanish patients with sporadic medullary thyroid cancer

The etiology of sporadic medullary thyroid carcinoma (sMTC) remains elusive. While germline gain-of-function mutations in the RET proto-oncogene cause hereditary MTC, somatic RET mutations have been described in a variable number of sMTC. So far, S836S of RET, is the only variant whose association with sMTC has been found in several European cohorts. Because RET variants seem to be associated with MTC, it is plausible that variants in genes encoding for RET coreceptors may play a role in the pathogenesis of sMTC. Recently, we described two possible low penetrance susceptibility alleles in the gene encoding RET coreceptor GFRalpha1, -193C > G and 537T > C, in a German series of sMTC. In this study, we have genotyped nine polymorphisms within GFRA1-3 genes for 51 Spanish sMTC, and 100 normal controls. Our results show that no statistical signification was found when Spanish sMTC patients were compared to controls. Taken together with the observations in the German sMTC series, the present findings suggest that GFRA1-193C > G and 537T > C could be in linkage disequilibrium with other loci responsible for the disease with a founder effect in Germany. Alternatively, the combined observations might also suggest that, if indeed the polymorphisms are functional, the effect is small.

Mutation analysis of NTRK2 and NTRK3, encoding 2 tyrosine kinase receptors, in sporadic human medullary thyroid carcinoma reveals novel sequence variants

Somatic mutations in the proto‐oncogene RET are found in 25% to 80% of sporadic medullary thyroid carcinomas (MTCs). The significance of somatic RET mutation in MTC initiation and progression, however, remains unknown. Like RET, TRK is a neurotrophic receptor tyrosine kinase. Immunostaining has shown that only a subset of normal C cells expresses Trk family receptors, but in C‐cell hyperplasia, they consistently express NTRK2, with variable expression of NTRK1 and NTRK3. In later stages of MTC, NTRK2 expression was reduced while NTRK3 expression was increased. In the context of these data, we sought to determine whether sequence variants in NTRK2 and NTRK3 are responsible for these differences in protein expression. We determined the genomic structure of NTRK2 and found that it consists of at least 17 exons varying in size from 36 to 306 bp. Mutation analysis of sporadic MTC did not reveal any sequence variants in NTRK2 but did reveal 3 variants in NTRK3, c.573C>T (N191N, exon 5), c.678T>C (N226N, exon 6) and c.1488C>G (A496A, exon 12) occurring among 19 chromosomes (31%), 1 chromosome (2%) and 24 chromosomes (39%), respectively. Corresponding germline also harbored these variants. There was a trend toward excess association of the NTRK3 variant c.1488C>G (A496A) in cases (24/62 chromosomes, 39%) compared to controls (18/62, 29%), but this difference did not reach significance (p > 0.05). The remaining 2 NTRK3 variants occurred with similar frequencies between MTC cases and population‐matched controls (19 vs. 17 and 1 vs. 0, p > 0.05). We conclude that sequence variants in NTRK2 and NTRK3 are not likely to be responsible for large differences in expression at the protein level, but we cannot exclude very low penetrance effects.

Proteomic Profiling of the Epileptic Dentate Gyrus

The development of epilepsy is often associated with marked changes in central nervous system cell structure and function. Along these lines, reactive gliosis and granule cell axonal sprouting within the dentate gyrus of the hippocampus are commonly observed in individuals with temporal lobe epilepsy (TLE). Here we used the pilocarpine model of TLE in mice to screen the proteome and phosphoproteome of the dentate gyrus to identify molecular events that are altered as part of the pathogenic process. Using a two‐dimensional gel electrophoresis‐based approach, followed by liquid chromatography‐tandem mass spectrometry, 24 differentially expressed proteins, including 9 phosphoproteins, were identified. Functionally, these proteins were organized into several classes, including synaptic physiology, cell structure, cell stress, metabolism and energetics. The altered expression of three proteins involved in synaptic physiology, actin, profilin 1 and α‐synuclein was validated by secondary methods. Interestingly, marked changes in protein expression were detected in the supragranular cell region, an area where robust mossy fibers sprouting occurs. Together, these data provide new molecular insights into the altered protein profile of the epileptogenic dentate gyrus and point to potential pathophysiologic mechanisms underlying epileptogenesis.