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P.P. Kale, V. Addepalli / Int. J. D
ells however these studies do not inform about filopodia domi-ated motility as observed in growth cones. In non-neuronal cells,he major actin nucleator, Arp2/3 along with Ena/VASP is thought toenerate filopodia. This does not appear to be the case for neuronss the removal of Arp2/3 does not affect filopodia formation. Datarom our lab suggests that Ena/VASP may not be the only regulatorsf actin dynamics resulting in filopodial structures.
With a view to investigate the role of other actin regulatorsn the growth cone we have begun characterizing other recentlyescribed actin nucleators. Using RNA in situ hybridizations andRT-PCR we found that members of the formin family (Formin 2)nd Cobl are neuronally enriched at stages when significant amountf axonal outgrowth occurs. Preliminary studies in non-neuronalells reveal deregulated cytoskeleton organisation when Formin 2evel is reduced. We are currently using knockdown approaches tovaluate the effect of these proteins on filopodia generation andranlocation rates of neurons. As Ena/VASP members contributeo filopodia generation, it is possible that they collaborate withctin nucleators. Indeed, bioinformatic analysis reveals the pres-nce EVH1-ligand like motifs in Formin 2 across different species.e are evaluating this interaction biochemically and function-
lly. We aim to uncover the molecular players that mediate actinucleation in neuronal growth cones leading to the generation oflopodial structures.
eurogenic effects of rapid action antidepressants
. Nanavaty ∗, S. Marathe, V. Vaidya
Tata Institute of Fundamental Research, India
Adult neurogenesis encompasses the proliferation, survival, dif-erentiation and integration of new born neurons in the neurogeniciches within the adult brain. This process occurs in key neural cir-uits including the hippocampus where it is known to be influencedy diverse factors. The neurogenic hypothesis of depression is basedn the observation that neurogenesis in the adult hippocampusecreases in animal models of depression and increases followinghronic antidepressant treatments. This increase in neurogenesis isbserved following a lag period of 2–3 weeks following the antide-ressant treatment and is comparable with the delay observed inehavioral effects exerted by classical antidepressants. It remains aatter of debate whether combination treatments that exert faster
ehavioral effects influence neurogenesis differently as comparedo the slower acting classical antidepressants. We sought to exam-ne the effects of a faster acting combination treatment of alpha-2drenoceptor antagonist yohimbine and the tricyclic antidepres-ant imipramine on hippocampal neurogenesis. Using Nestin-GFPice expressing GFP under the Nestin promoter that allows us to
istinguish between the various categories of adult progenitors,e find that the combination treatment leads to an increase in theumber of quiescent neural precursors (QNPs) as opposed to classi-al antidepressants which target the amplifying neural precursorsANPs). Strikingly, another faster acting antidepressant treatmentamely electroconvulsive seizure therapy (ECT) also enhances pro-
iferation of the QNPs. To further investigate if the faster actingombination treatment and ECT may exert their action via influenc-ng similar signaling pathways, we have profiled the hippocampalranscriptome using microarray analysis. Our results highlight theossibility that fast acting antidepressants may exert their effectsy acting via distinct signaling pathways ultimately leading to an
ncrease in the number of QNPs.
uroscience 30 (2012) 640–671 645
Qualitative and quantitative characterization of STIL expressionin the developing and adult mouse brain
R. Pavan Kumar ∗, Richa Kapoor, Harshvardhan Bukka, A.N.Shaithilya, Devesh Tewary, Iswariya Sivaprakasam, ShyamalaMani
Centre for Neuroscience, Indian Institute of Science, Bangalore 560012,India
Introduction: Primary autosomal recessive microcephaly(MCPH) is a rare genetic disorder characterized by diminishedbrain volume and mental retardation. Each of the 7 subtypes ofMCPH result from mutations in specific genes, one of which hasrecently been shown to be the STIL gene (MCPH subtype 7). Threedifferent mutations in the STIL gene have been reported in MCPHpatients in India.
SCL/TAL1 interrupting locus gene, or STIL, is an immediate earlygene that is thought to be associated with centrosome function,spindle formation, cell cycle progression and apoptosis. STIL isrequired for mitotic entry and is found to be expressed in pro-liferating cells. In addition, STIL expression is downregulated indifferentiating cells. While the functions of STIL in the cell are stillunder scrutiny, the expression pattern of the gene has not yet beensystematically studied. Studies in STIL have shown that point muta-tions in the gene lead to MCPH.
Methods: In the following study, through in situ hybridizationand quantitative PCR analysis, we sought to examine the temporaland spatial expression of the STIL mRNA in the mouse brain, bothduring development as well as postnatally. Through homologousrecombination we sought to insert the mutations detected in STILinto the stem cells and study how these mutations affect the stemcells.
Results: Through the use of both in situ hybridization using DIG-labelled riboprobes as well as real-time PCR techniques, we see aclear pattern of STIL mRNA expression that is both time-point andlocation specific, thus indicating a characteristic regulation of thegene.
Discussion: Based on our findings, gaining a mechanistic under-standing of STIL function in the context of the cell cycle becomesimportant, and future studies shall aim to explore the consequencesof STIL overexpression and knock-in mutations in mouse embry-onic stem cells to provide us with insights into cell cycle regulationby STIL.
Maternal thyroid hormone critically effect mammalian neo-cortical development by regulating neurogenesis throughintermediate progenitor cells
V. Mohan ∗, G. Rao, L. Rastogi, P. Kumar, M.M. Godbole
Sanjay Gandhi Post Graduate Institute of Medical Science, Lucknow,India
Maternal thyroid hormone (TH) deficiency before the onset offetal thyroid function causes severe neurological deficits. Whetherdeficits result from defective neurogenesis or imperfect migrationremains elusive. To understand the root cause, we investigatedthe effect of maternal TH deficiency in neurogenesis. Pregnantrats were rendered hypothyroid by administering methimazolein drinking water from gestation day 6 and fetal cortices werecollected from embryonic day (E) 14-18. Immunostaining andimmunoblotting results showed that TH deficiency causes adecrease in the expression of both early and late neuronal mark-ers doublecortin (DCX) and neuronal nuclei (NeuN) respectively atE14 as well as E16 indicating defective neurogenesis along with
6 evl Ne
46 P.P. Kale, V. Addepalli / Int. J. D
compensatory increase at E18. To resolve whether this delayn neurogenesis is due to defect in differentiation from progeni-ors, double labeling with the mitotic marker BrdU and cell cycle
arker Ki67 was performed at E16, and cell cycle quitting fractionas analyzed by flow cytometry and 5 folds decrease was seennder hypothyroidism. It could be either from diminished mitoticool of ventricular zone (VZ) resident apical progenitors or subven-ricular zone (SVZ) resident intermediate progenitor cells (IPCs).
apping of mitotic figures by p-Histone3 immunostaining revealedsignificant reduction in the mitotic progenitors in intermediate
one and increase in the VZ progenitors under hypothyroidism,ndicating loss of proliferating IPCs. This was further confirmed
ith the significant decrease in IPCs specific marker Tbr2 underypothyroidism. The loss of indirect neurogenesis by IPCs waslso evident in the reduction of telencephalic thickness underaternal hypothyroidism. Cultured neuronal progenitors showed
xpression TH transporter MCT8, De-iodinase-II and TH receptorR�1 indicating that IPCs can metabolize maternal TH and explainsheir regulation by altered TH status during development. Taken,ogether we describe direct regulation of neurogenesis by maternalH as seen by diminished pool of IPCs during development underypothyroidism.
yclic AMP pathway independent induction of neurite exten-ion and their networking in the motor neuron cell line NSC34
jeet Kumar, Himanshu K. Mishra, Priyanka Dwivedi, Jamuna R.ubramaniam ∗
Department of Biological Sciences and Bioengineering, Indian Institutef Technology, Kanpur 208016, India
-mail address: [email protected] (J.R. Subramaniam).Matrix stromal cells (HUMS) derived from the human umbili-
al cord secrete several neurotrophic factors. The HUMS cells andheir secreted factors are shown to provide some amount of neu-oprotection in the neurodegenerative disease models of mice. Buthe exact mechanism of protection is not well understood. Here, weeport that the HUMS cells secrete six neurotrophic factors, namely,T-3, NGF, BDNF, VEGF, IGF-1 and GDNF(NFs). These NFs present
n the conditioned medium of the HUMS cells induce differentia-ion, neurite extension and neural networking of a motor neuronell line, NSC34. These motor neurons express the tyrosine kinaseeceptors for the above trophic factors (except for BDNF), which arerucial for neurite extension. The tyrosine kinase inhibitor, K252a,rastically reduces CM induced neurite extension. Further, all theTFs need to be neutralized simultaneously with their antibodies
o abrogate neurite extension, proving the flexibility and prudentackup mechanism of the system. Intriguingly, none of the phe-omenon – differentiation, neurite extension or neural networkingequired cAMP second messenger system coupling as evidenced byAMP pathway activator or inhibitor treatment of the NSC34 cellsith or without CM.
solation and characterization of centrosomes from cellsxpressing modulated levels of Stil
. Chatterjee ∗, S. Mani
Centre for Neuroscience, Indian Institute of Science, Bangalore 560012,ndia
Introduction: Primary microcephaly is an autosomal recessiveongenital disorder which results from hypoplasia of the cerebral
uroscience 30 (2012) 640–671
cortex, characterized by a greater than three standard deviationsbelow the age and sex related mean for head circumference. All theproteins linked to this disorder (MCPH1, CDK5RAP2, ASPM, CENPJand STIL) localize to the centrosome, suggesting a role for the cen-trosome in neurogenesis during brain development.
STIL is a mitotic check point gene which controls the transitionfrom G2 to M phase in the cell cycle. It is expressed in all dividingcells and its expression decreases upon differentiation.
An understanding of the structural components of the centro-some and their functions thereof, has been elusive and isolatedcentrosomes are potentially useful for chemical and structuralstudies of the organelle.
Methodology: ESD3 cells were transfected with overexpressionand downregulation vectors for Stil. Centrosome isolation was donefollowing Moudjou and Bornens (1998). It was isolated on a sucrosegradient, which serves to purify them on the basis of sedimentationvelocity. Coomassie Blue staining was done to check the profileof the isolated proteins and the fractions were characterized bystaining with acetylated tubulin antibodies (centriole marker) andgamma tubulin antibodies (PCM marker).
Results: Coomassie Blue staining showed that proteins rangingfrom 10 kDa to 250 kDa had been isolated onto the sucrose cushion.Bands of 48 kDa (gamma tubulin) and 55 kDa (acetylated tubulin)were seen in a few fractions, indicating the successful isolation ofcentrosomes from the cells.
Discussion: Proteomic characterization of centrosomes will helpin greater understanding of its structural components, functionalrelevance and its potential role in cell signalling. A comparisonof centrosomal components, between normal, high and low Stilexpression conditions, will help in delineating the effects of thisgene on centrosomal function and provide clues as to the possiblerole of centrosomes in neurogenesis itself.
Orexinergic neurons modulate REMS by influencing locuscoeruleus neurons
R.C. Choudhary, H.K. Srivastava ∗, B.N. Mallick
School of Life Sciences, Jawaharlal Nehru University, New Delhi, India
Neural mechanism of rapid eye movement sleep (REMS) regu-lation is being actively investigated. Locus coeruleus (LC) REM-OFFneuronal activity reduces upon sleep onset and eventually becomesquiescent through REMS. Activation of orexinergic-neurons inthe perifornical area (Pef) contribute to wakefulness and theirloss is associated with narcolepsy, a REMS-disorder. Althoughorexinergic-neurons innervate virtually the entire brain, nora-drenalin (NA)-ergic neurons in the LC, in particular, receive heaviestprojections. Independent studies have shown that stimulation oforexinergic-neurons and local microinjection of orexin into LCreduced REMS. Thus, although it was suggestive that stimulationof orexinergic-neurons mediated their action possibly by activat-ing LC-neurons, it was unknown if orexinergic-neurons directlyaffected the LC-neurons to modulate REMS, which was investigatedin this study.
Male wistar rats (250–275 g) were stereotaxic surgicallyprepared with implanted electrodes for electrophysiological sleep-wake-REMS recording and with bilateral guide cannulae into LC aswell as Pef for local microinjections. After recovery from surgery,sleep-waking-REMS were recorded in freely moving normallybehaving chronically prepared rats simultaneously with or withoutmicroinjection(s) of chemicals in either LC or Pef or both.
Glutamate stimulation of orexinergic-neurons significantlyincreased waking and decreased sleep; REMS-duration/h wasdecreased but not REMS-frequency/h. The effects however, were