Faculty Bibliography

A wide range of light-activated molecules (photoswitches and phototriggers) have been used to the study of computational properties of an isolated neuron by acting pre and postsynaptically. However, new tools are being pursued to elicit a presynaptic calcium influx that triggers the release of neurotransmitters, most of them based in calcium-permeable Channelrhodopsin-2 mutants. Here we describe a method to control exocytosis of synaptic vesicles through the use of a light-gated glutamate receptor (LiGluR), which has recently been demonstrated that supports secretion by means of calcium influx in chromaffin cells. Expression of LiGluR in hippocampal neurons enables reversible control of neurotransmission with light, and allows modulating the firing rate of the postsynaptic neuron with the wavelength of illumination. This method may be useful for the determination of the complex transfer function of individual synapses.

The formation of the mammalian cortex requires the generation, migration, and differentiation of neurons. The vital role that the microtubule cytoskeleton plays in these cellular processes is reflected by the discovery that mutations in various tubulin isotypes cause different neurodevelopmental diseases, including lissencephaly (TUBA1A), polymicrogyria (TUBA1A, TUBB2B, TUBB3), and an ocular motility disorder (TUBB3). Here, we show that Tubb5 is expressed in neurogenic progenitors in the mouse and that its depletion in vivo perturbs the cell cycle of progenitors and alters the position of migrating neurons. We report the occurrence of three microcephalic patients with structural brain abnormalities harboring de novo mutations in TUBB5 (M299V, V353I, and E401K). These mutant proteins, which affect the chaperone-dependent assembly of tubulin heterodimers in different ways, disrupt neurogenic division and/or migration in vivo. Our results provide insight into the functional repertoire of the tubulin gene family, specifically implicating TUBB5 in embryonic neurogenesis and microcephaly.

Familial dysautonomia (FD) is a rare hereditary disorder caused by mutations within the gene that encodes for I-kappa-B kinase complex associated protein (IKAP). A deficiency of IKAP affects the development of primary sensory neurons including those carrying baroreflex afferent volleys, a feature that explains their characteristic sensory loss and labile blood pressure. This review describes the history, the genotype of FD and the unusual cardiovascular autonomic phenotype of these patients. We outline the main consequences of a failure to receive information from arterial baroreceptors, including the characteristic "autonomic storms" and severe end-organ target damage.

Calcium is important in controlling nuclear gene expression through the activation of multiple signal-transduction pathways in neurons. Compared with other voltage-gated calcium channels, CaV1 channels demonstrate a considerable advantage in signalling to the nucleus. In this review, we summarize the recent progress in elucidating the mechanisms involved. CaV1 channels, already advantaged in their responsiveness to depolarization, trigger communication with the nucleus by attracting colocalized clusters of activated CaMKII (Ca2+/calmodulin-dependent protein kinase II). CaV2 channels lack this ability, but must work at a distance of >1 mum from the CaV1-CaMKII co-clusters, which hampers their relative efficiency for a given rise in bulk [Ca2+]i (intracellular [Ca2+]). Moreover, Ca2+ influx from CaV2 channels is preferentially buffered by the ER (endoplasmic reticulum) and mitochondria, further attenuating their effectiveness in signalling to the nucleus.

Here we review evidence that the reactive oxygen species, hydrogen peroxide (H(2)O(2)), meets the criteria for classification as a neuromodulator through its effects on striatal dopamine (DA) release. This evidence was obtained using fast-scan cyclic voltammetry to detect evoked DA release in striatal slices, along with whole-cell and fluorescence imaging to monitor cellular activity and H(2)O(2) generation in striatal medium spiny neurons (MSNs). The data show that (1) exogenous H(2)O(2) suppresses DA release in dorsal striatum and nucleus accumbens shell and the same effect is seen with elevation of endogenous H(2)O(2) levels; (2) H(2)O(2) is generated downstream from glutamatergic AMPA receptor activation in MSNs, but not DA axons; (3) generation of modulatory H(2)O(2) is activity dependent; (4) H(2)O(2) generated in MSNs diffuses to DA axons to cause transient DA release suppression by activating ATP-sensitive K(+) (K(ATP)) channels on DA axons; and (5) the amplitude of H(2)O(2)-dependent inhibition of DA release is attenuated by enzymatic degradation of H(2)O(2), but the subsecond time course is determined by H(2)O(2) diffusion rate and/or K(ATP)-channel kinetics. In the dorsal striatum, neuromodulatory H(2)O(2) is an intermediate in the regulation of DA release by the classical neurotransmitters glutamate and GABA, as well as other neuromodulators, including cannabinoids. However, modulatory actions of H(2)O(2) occur in other regions and cell types, as well, consistent with the widespread expression of K(ATP) and other H(2)O(2)-sensitive channels throughout the CNS.

Genetically-modified mice mimicking oculodentodigital dysplasia (ODDD), a disease characterized by reduced Cx43-mediated gap junctional intercellular communication, represent an in vivo model to assess the role of Cx43 in mammary gland development and function. We previously reported that severely compromised-Cx43 function delayed mammary gland development and impaired milk ejection in mice that harboured a G60S Cx43 mutant. Surprisingly, there are no definitive reports of lactation defects in ODDD patients. To address this further, we obtained a second mouse model of ODDD expressing an I130T Cx43 mutant to assess if a Cx43 mutant with residual gap junction channel activity would rescue mammary gland development and function. Our findings show that virgin Cx43^I130T/+ mice, distinct from Cx43^G60S/+ mice, develop with similar body weights compared to control, despite having a reduction in the highly phosphorylated species of Cx43 and reduced Cx43 gap junctional plaques. In addition, virgin Cx43^I130T/+ mice exhibit a delay in ductal elongation at four weeks that is not observed by seven weeks. Cx43^I130T/+ mice develop smaller mammary glands at parturition due to reduced proliferation despite similar overall gland architecture. Distinct from Cx43^G60S/+ mice, Cx43^I130T/+ mice adequately produce and deliver milk to pups suggesting milk ejection is unaffected. Thus, these studies suggest that loss-of-function mutants of Cx43 with residual gap junction channel activity can rescue functional defects in the mammary gland and helps to explain the lack of lactation defects associated with ODDD patients

Background: We have previously demonstrated that the cell adhesion protein contactin2 (Cntn2) is enriched in Purkinje cells of the cardiac conduction system (CCS). Objective: Generation of a mouse embryonic stem cell (mESC) reporter line that allows identification of Purkinje-like cardiomyocytes in vitro. Methods and Results: mESC were generated from transgenic mice carrying a BAC Cntn2-eGFP reporter gene and were subsequently transduced with lentivirus coding for a selectable MHCalpha-mCherry cardiomyocyte reporter gene. Immunostaining analysis confirmed that mESC expressed markers of pluripotency (Oct3/4; Klf4) and spontaneously differentiated into cells of all three germ layers in the absence of LIF (alpha- smooth muscle actin; beta-tubulin; alpha-fetoprotein). Spontaneous or serum-free directed cardiac differentiation resulted in generation of double positive, spontaneously beating cardiomyocytes after three weeks. Yield of double positive cells could be increased by adding endocardialderived factors (Nrg1; ET-1). FACS isolated double positive cells were enriched in transcripts of cardiomyocytes (Tbx5; Nkx2.5) and the CCS (Cntn2; Cx40). Action potential recordings of eGFP positive cardiomyocytes demonstrated distinct plateau phase and elongated action potential duration (APD50=79.9+10.4ms, APD90=170.2+17.5ms; n=11) compared with eGFP negative cardiomyocytes (APD50=53.4+9.4ms, APD90=120.6+17.3ms; n=15). Conclusion: We have established a mESC reporter line for the identification of CCS-like cells. This model should be useful for downstream studies of CCS development and pathology. Cntn2 may also be a useful marker of CCS-like cells derived from human ES and/or iPS cells

Dynamic changes of the actin cytoskeleton are instrumental in morphogenetic processes including changes in cell shape and adhesion. Formin proteins regulate actin microfilament assembly and can specifically influence adherens junction formation. Previous studies in our lab have demonstrated remarkable plasticity of formin isoforms during heart development and in vitro cardiomyocyte differentiation. As gap junction stability is dependent on the presences of mechanical junctions we were interested if modulation of cardiac formins influences expression of Cx43 protein and gap junction function. Objective: To investigate the effect of cardiac formin knockdown (KD) on cell-cell contact formation and functional coupling of cardiomyocytes. Methods: Cardiomyocytes were isolated from neonatal rat hearts (NRCM) and cultured as monolayers (d0); NRCM were treated with transfection agent only (TF), control siRNA (Ctr) or formin specific rat siRNAs (Daam1; Fhod1; Fhod3; Dharmacon) (d1); cultures were subjected to high resolution optical mapping or processed for immunofluorescence analysis (d4). Results: KD of Fhod1 or Fhod3 lead to disruption of sarcomers, cell rounding and ultimately resulted in complete dissociation of NRCM. In contrast, Daam1 KD resulted in significant cell elongation without loss of cell-cell contacts (mean cell areas in mum²: 681.8 + 99.1 (Daam1) vs. 594.9 + 67.6 (TF), 564 + 53.3 (Ctr), 455.9 + 47.4 (Fhod1), 339.3 + 14.3 (Fhod3); P: 0.01, ANOVA). As expected, optical mapping data for discontinuous Fhod1 and Fhod3 monolayers were very variable due to areas of complete block of conduction. Optical mapping analysis of Daam1 silenced NRCM demonstrated significant increase in conduction velocity (0.241 + 0.004 m/s; n=4) compared to NRCM treated with TF only (0.197 + 0.010 m/s, n=3) or Ctr (0.207 + 0.005 m/s, n=3; P: 0.003, ANOVA). Average gap junction diameter (0.24 + 0.03 mum (n=489; Daam1) vs. 0.41 + 0.03 mum (n=550; DF), 0.38 + 0.04 mum (n=574; Ctr) P: 0.009, ANOVA), and total !

BACKGROUND: Mutations in either Abeta Precursor protein (APP) or genes that regulate APP processing, such as BRI2/ITM2B and PSEN1/PSEN2, cause familial dementias. Although dementias due to APP/PSEN1/PSEN2 mutations are classified as familial Alzheimer disease (FAD) and those due to mutations in BRI2/ITM2B as British and Danish dementias (FBD, FDD), data suggest that these diseases have a common pathogenesis involving toxic APP metabolites. It was previously shown that FAD mutations in APP and PSENs promote activation of caspases leading to the hypothesis that aberrant caspase activation could participate in AD pathogenesis. RESULTS: Here, we tested whether a similar mechanism applies to the Danish BRI2/ITM2B mutation. We have generated a genetically congruous mouse model of FDD, called FDD(KI), which presents memory and synaptic plasticity deficits. We found that caspase-9 is activated in hippocampal synaptic fractions of FDD(KI) mice and inhibition of caspase-9 activity rescues both synaptic plasticity and memory deficits. CONCLUSION: These data directly implicate caspase-9 in the pathogenesis of Danish dementia and suggest that reducing caspase-9 activity is a valid therapeutic approach to treating human dementias.

Although previous work identified transcription factors crucial for the specification and migration of parvalbumin (PV)-expressing and somatostatin (SST)-expressing interneurons, the intrinsic factors required for the terminal differentiation, connectivity, and survival of these cell types remain uncharacterized. Here we demonstrate that, within subpopulations of cortical interneurons, Satb1 (special AT-rich binding protein) promotes terminal differentiation, connectivity, and survival in interneurons that express PV and SST. We find that conditional removal of Satb1 in mouse interneurons results in the loss of a majority of SST-expressing cells across all cortical layers, as well as some PV-expressing cells in layers IV and VI, by postnatal day 21. SST-expressing cells initially migrate to the cortex in Satb1 mutant mice, but receive reduced levels of afferent input and begin to die during the first postnatal week. Electrophysiological characterization indicates that loss of Satb1 function in interneurons results in a loss of functional inhibition of excitatory principal cells. These data suggest that Satb1 is required for medial ganglionic eminence-derived interneuron differentiation, connectivity, and survival.