Faculty Bibliography

Furoxans (1,2,5-oxadiazole-N-oxides) are thiol-bioactivated NO-mimetics that have not hitherto been studied in the CNS. Incorporation of varied substituents adjacent to the furoxan ring system led to modulation of reactivity toward bioactivation, studied by HPLC-MS/MS analysis of reaction products. Attenuated reactivity unmasked the cytoprotective actions of NO in contrast to the cytotoxic actions of higher NO fluxes reported previously for furoxans. Neuroprotection was observed in primary neuronal cell cultures following oxygen glucose deprivation (OGD). Neuroprotective activity was observed to correlate with thiol-dependent bioactivation to produce NO(2)(-), but not with depletion of free thiol itself. Neuroprotection was abrogated upon cotreatment with a sGC inhibitor, ODQ, thus supporting activation of the NO/sGC/CREB signaling cascade by furoxans. Long-term potentiation (LTP), essential for learning and memory, has been shown to be potentiated by NO signaling, therefore, a peptidomimetic furoxan was tested in hippocampal slices treated with oligomeric amyloid-beta peptide (Abeta) and was shown to restore synaptic function. The novel observation of furoxan activity of potential therapeutic use in the CNS warrants further studies.

A mutation in the BRI2/ITM2b gene causes loss of BRI2 protein leading to familial Danish dementia (FDD). BRI2 deficiency of FDD provokes an increase in amyloid-beta precursor protein (APP) processing since BRI2 is an inhibitor of APP proteolysis, and APP mediates the synaptic/memory deficits in FDD. APP processing is linked to Alzheimer disease (AD) pathogenesis, which is consistent with a common mechanism involving toxic APP metabolites in both dementias. We show that inhibition of APP cleavage by beta-secretase rescues synaptic/memory deficits in a mouse model of FDD. beta-cleavage of APP yields amino-terminal-soluble APPbeta (sAPPbeta) and beta-carboxyl-terminal fragments (beta-CTF). Processing of beta-CTF by gamma-secretase releases amyloid-beta (Abeta), which is assumed to cause AD. However, inhibition of gamma-secretase did not ameliorate synaptic/memory deficits of FDD mice. These results suggest that sAPPbeta and/or beta-CTF, rather than Abeta, are the toxic species causing dementia, and indicate that reducing beta-cleavage of APP is an appropriate therapeutic approach to treating human dementias. Our data and the failures of anti-Abeta therapies in humans advise against targeting gamma-secretase cleavage of APP and/or Abeta.

Learning and memory deficits in Alzheimer's disease (AD) result from synaptic failure and neuronal loss, the latter caused in part by excitotoxicity and oxidative stress. A therapeutic approach is described that uses NO-chimeras directed at restoration of both synaptic function and neuroprotection. 4-Methylthiazole (MZ) derivatives were synthesized, based upon a lead neuroprotective pharmacophore acting in part by GABA(A) receptor potentiation. MZ derivatives were assayed for protection of primary neurons against oxygen-glucose deprivation and excitotoxicity. Selected neuroprotective derivatives were incorporated into NO-chimera prodrugs, coined nomethiazoles. To provide proof of concept for the nomethiazole drug class, selected examples were assayed for restoration of synaptic function in hippocampal slices from AD-transgenic mice, reversal of cognitive deficits, and brain bioavailability of the prodrug and its neuroprotective MZ metabolite. Taken together, the assay data suggest that these chimeric nomethiazoles may be of use in treatment of multiple components of neurodegenerative disorders, such as AD.

Intracranial recording is an important diagnostic method routinely used in a number of neurological monitoring scenarios. In recent years, advancements in such recordings have been extended to include unit activity of an ensemble of neurons. However, a detailed functional characterization of excitatory and inhibitory cells has not been attempted in human neocortex, particularly during the sleep state. Here, we report that such feature discrimination is possible from high-density recordings in the neocortex by using 2D multielectrode arrays. Successful separation of regular-spiking neurons (or bursting cells) from fast-spiking cells resulted in well-defined clusters that each showed unique intrinsic firing properties. The high density of the array, which allowed recording from a large number of cells (up to 90), helped us to identify apparent monosynaptic connections, confirming the excitatory and inhibitory nature of regular-spiking and fast-spiking cells, thus categorized as putative pyramidal cells and interneurons, respectively. Finally, we investigated the dynamics of correlations within each class. A marked exponential decay with distance was observed in the case of excitatory but not for inhibitory cells. Although the amplitude of that decline depended on the timescale at which the correlations were computed, the spatial constant did not. Furthermore, this spatial constant is compatible with the typical size of human columnar organization. These findings provide a detailed characterization of neuronal activity, functional connectivity at the microcircuit level, and the interplay of excitation and inhibition in the human neocortex.

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 !

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

Purpose: Normal neuronal activity is tightly linked to and depends on the increase of blood flow for instantaneous supply of oxygen and glucose. This study is to evaluate whether there are cerebral blood flow (CBF) regulation abnormalities in MS with measurement of cerebrovascular reactivity (CVR) using hypercapnia perfusion MRI. Materials and Methods: Sixteen patients with MS (14 relapsing remitting and 2 secondary progressive) (mean age: 45.1+14.2 years, mean EDSS: 2.9+1.6) and age-matched 13 healthy controls (mean age: 44.5+12.2 years) were recruited for this study. CO2 is a potent vasodilator, and an increase of CO2 tension in blood (referred to as hypercapnia) is known to cause CBF increase. Such CBF changes were measured with a standard pseudo-continuous arterial spin labeling (pCASL) MRI at 3T, with quantitative CBF (ml/min/100g) maps generated during both room air and hypercapnia (mixed 5%CO2, 21%O2, and 74%N2) exposure. The imaging parameters of pCASL include TR/TE=3950/17ms, 52 repetitions, FOV=22cm, in-plane matrix=64x64, slice thickness=5mm, labeling duration=1500ms, postlabeling delay=1230ms, and label location = 84mm below AC-PC line. End-tidal CO2 (EtCO2) was recorded continuously during the scan with a capnograph device and was used as an input function in the analysis. The CVR was calculated as (% change in CBF comparing CO2 inhalation to room-air breathing) divided by (EtCO2 during CO2 inhalation - EtCO2 during room-air breathing). Segmented whole brain grey matter (GM), white matter (WM), and brain parenchymal CVR were calculated for the group analysis. Results: The averaged CVR (%CBF/mmHg EtCO2) showed significant difference for whole brain parenchymal (P=0.009), GM (P=0.008), and WM (P=0.03) between patients (4.74+0.88%, 4.89+1.08%, and 4.73+1.02%) and healthy controls (3.46+1.51%, 3.51+1.47%, and 3.53+1.83%, respectively). There was a significant correlation between brain parenchymal CVR and EDSS (r=-0.69, P=0.007). Whole brain CVR changes correlate with fractional brain p!

Mitochondria divide and fuse continuously, and the balance between these two processes regulates mitochondrial shape. Alterations in mitochondrial dynamics are associated with neurodegenerative diseases. Here we investigate the physiological and cellular functions of mitochondrial division in postmitotic neurons using in vivo and in vitro gene knockout for the mitochondrial division protein Drp1. When mouse Drp1 was deleted in postmitotic Purkinje cells in the cerebellum, mitochondrial tubules elongated due to excess fusion, became large spheres due to oxidative damage, accumulated ubiquitin and mitophagy markers, and lost respiratory function, leading to neurodegeneration. Ubiquitination of mitochondria was independent of the E3 ubiquitin ligase parkin in Purkinje cells lacking Drp1. Treatment with antioxidants rescued mitochondrial swelling and cell death in Drp1KO Purkinje cells. Moreover, hydrogen peroxide converted elongated tubules into large spheres in Drp1KO fibroblasts. Our findings suggest that mitochondrial division serves as a quality control mechanism to suppress oxidative damage and thus promote neuronal survival.