Faculty Bibliography

The increased availability of transgenic mouse models for studying human diseases has shifted the focus of many laboratories from in vitro to in vivo assays. Herein, methods are described to allow investigators to obtain well-preserved mouse tissue to be stained with the standard histological dyes for amyloid, Congo Red, and Thioflavin S. These sections can as well be used for immunohistological procedures that allow detection of tissue amyloid and pre-amyloid, such as those composed of the amyloid-beta peptide, the tau protein, and the islet amyloid polypeptide.

In the last couple of decades, substantial progress has been made in the development of transgenic mouse models developing amyloid-beta deposits and/or neurofibrillary tangles. These mouse models of Alzheimer's disease and related disorders provide an excellent tool for investigating etiology, pathogenic mechanisms, and potential treatments. An essential component of their characterization is a detailed behavioral assessment, which clarifies the functional consequences of these pathologies. We have selected and refined a series of cognitive and sensorimotor tasks that are ideal for studying these models and the efficacy of various treatments.

Fragile X syndrome (FXS) is the most common form of inherited intellectual disability and autism. The protein (FMRP) encoded by the fragile X mental retardation gene (FMR1), is an RNA-binding protein linked to translational control. Recently, in the Fmr1 knockout mouse model of FXS, dysregulated translation initiation signaling was observed. To investigate whether an altered signaling was also a feature of subjects with FXS compared to typical developing controls, we isolated total RNA and translational control proteins from lymphocytes of subjects from both groups (38 FXS and 14 TD). Although we did not observe any difference in the expression level of messenger RNAs (mRNAs) for translational initiation control proteins isolated from participant with FXS, we found increased phosphorylation of the mammalian target of rapamycin (mTOR) substrate, p70 ribosomal subunit 6 kinase1 (S6K1) and of the mTOR regulator, the serine/threonine protein kinase (Akt), in their protein lysates. In addition, we observed increased phosphorylation of the cap binding protein eukaryotic initiation factor 4E (eIF4E) suggesting that protein synthesis is upregulated in FXS. Similar to the findings in lymphocytes, we observed increased phosphorylation of S6K1 in brain tissue from patients with FXS (n = 4) compared to normal age-matched controls (n = 4). Finally, we detected increased expression of the cytoplasmic FMR1-interacting protein 2 (CYFIP2), a known FMRP interactor. This data verify and extend previous findings using lymphocytes for studies of neuropsychiatric disorders and provide evidence that misregulation of mTOR signaling observed in the FXS mouse model also occurs in human FXS and may provide useful biomarkers for designing targeted treatments in FXS.

Reports an error in "Epac2-dependent mobilization of intracellular Ca2+ by glucagon-like peptide-1 receptor agonist exendin-4 is disrupted in beta -cells of phospholipase C-epsilon knockout mice" by Igor Dzhura, Oleg G. Chepurny, Grant G. Kelley, Colin A. Leech, Michael W. Roe, Elvira Dzhura, Parisa Afshari, Sundeep Malik, Michael J. Rindler, Xin Xu, Youming Lu, Alan V. Smrcka and George G. Holz (The Journal of Physiology, 2010[Dec][15], Vol 588[24], 4871-4889). In the original article, there was an error in the Methods section entitled 'Generation of Epac2 knockout mice' on page 4873. The first sentence of that section should read 'Epac2 KO mice with global disruption of RAPGEF4 gene expression (NCBI GeneID 56508) were generated by the Texas A&M Institute for Genomic Medicine through customized service for Dr. Lu at Louisiana State University Health Sciences Center'. (The following abstract of the original article appeared in record 2011-11969-007). Calcium can be mobilized in pancreatic beta -cells via a mechanism of Ca2+-induced Ca2+ release (CICR), and cAMP-elevating agents such as exendin-4 facilitate CICR in beta -cells by activating both protein kinase A and Epac2. Here we provide the first report that a novel phosphoinositide-specific phospholipase C-epsilon (PLC-epsilon ) is expressed in the islets of Langerhans, and that the knockout (KO) of PLC-epsilon gene expression in mice disrupts the action of exendin-4 to facilitate CICR in the beta -cells of these mice. Thus, in the present study, in which wild-type (WT) C57BL/6 mouse beta -cells were loaded with the photolabile Ca2+ chelator NP-EGTA, the UV flash photolysis-catalysed uncaging of Ca2+ generated CICR in only 9% of the beta -cells tested, whereas CICR was generated in 82% of the beta -cells pretreated with exendin-4. This action of exendin-4 to facilitate CICR was reproduced by cAMP analogues that activate protein kinase A(6-Bnz-cAMP-AM)orEpac2 (8-pCPT-2'-O-Me-cAMP-AM)selectively. However, in beta -cells of PLC-epsilon KO mice, and also Epac2 KO mice, these test substances exhibited differential efficacies in the CICR assay such that exendin-4 was partly effective, 6-Bnz-cAMP-AM was fully effective, and 8-pCPT-2'-O-Me-cAMP-AM was without significant effect. Importantly, transduction of PLC-epsilon KO beta -cells with recombinant PLC-epsilon rescued the action of 8-pCPT-2'-O-Me-cAMP-AM to facilitate CICR, whereas a K2150E PLC-epsilon with amutated Ras association (RA) domain, or a H1640L PLC-epsilon that is catalytically dead, were both ineffective. Since 8-pCPT-2'-O-Me-cAMP-AM failed to facilitate CICR in WT beta -cells transduced with a GTPase activating protein (RapGAP) that downregulates Rap activity, the available evidence indicates that a signal transduction 'module' comprised of Epac2, Rap and PLC-epsilon exists in beta -cells, and that the activities of Epac2 and PLC-epsilon are key determinants of CICR in this cell type.

Hemimegalencephaly (HMG) is a developmental brain disorder characterized by an enlarged, malformed cerebral hemisphere, typically causing epilepsy that requires surgical resection. We studied resected HMG tissue to test whether the condition might reflect somatic mutations affecting genes critical to brain development. We found that two out of eight HMG samples showed trisomy of chromosome 1q, which encompasses many genes, including AKT3, a gene known to regulate brain size. A third case showed a known activating mutation in AKT3 (c.49G-->A, creating p.E17K) that was not present in the patient's blood cells. Remarkably, the E17K mutation in AKT3 is exactly paralogous to E17K mutations in AKT1 and AKT2 recently discovered in somatic overgrowth syndromes. We show that AKT3 is the most abundant AKT paralog in the brain during neurogenesis and that phosphorylated AKT is abundant in cortical progenitor cells. Our data suggest that somatic mutations limited to the brain could represent an important cause of complex neurogenetic disease.

CONTEXT: Surgery-induced neuroinflammation has been implicated in the development of postoperative cognitive dysfunction (POCD). OBJECTIVE: To test the hypothesis that meloxicam, a selective cyclooxygenase (COX)-2 inhibitor, preserves postoperative cognitive function and inhibits surgery-induced neuroinflammation in a mouse model. DESIGN: A mouse model of splenectomy-induced inflammation. METHODS: Sixty Swiss Webster male mice (6-8 week old) were randomised into six groups that underwent splenectomy. Animals in groups 1-4 were tested once on day 1, 5, 9 or 14 to determine the time course of delayed transient cognitive dysfunction associated with splenectomy. Animals in groups 5 and 6 were tested once on day 5 or 9 to determine the ability of the NSAID meloxicam to attenuate cognitive dysfunction. INTERVENTION: Animals in groups 1-4 received one dose 500 mul intraperitoneal physiological saline 24 h after splenectomy. Animals in groups 5 and 6 received one dose of intraperitoneal meloxicam (60 mg kg in 500 mul saline) 24 h after splenectomy. MAIN OUTCOME MEASURES: Short-term working memory as determined by Object Recognition Test (ORT) index on days 1, 5, 9 and 14 was the first main outcome. Tomato lectin staining histochemistry of glial cells was assessed on days 1, 5, 9 and 14 as a second main outcome. RESULTS: Compared with day 1 (group 1), the mean ORT indices at day 5 (group 2) and day 9 (group 3) were decreased by 27.5% [95% confidence interval (CI): 0.9 to 54.1%, P = 0.04] and 23.8% (95% CI, 4.3 to 51.9%, P = 0.09), respectively. At day 5 (group 5) and day 9 (group 6), the ORT indices in the meloxicam groups were reduced by 6.6% (95% CI: -11.4 to 24.5%) and 4.3% (95% CI: -25.3 to 34.0). Thus, the administration of meloxicam attenuated the decrease in ORT indices (P = 0.031). Histochemical staining with tomato lectin showed features of microglia activation at day 5 and 9, which was reduced by the administration of meloxicam. CONCLUSION: These findings suggest that COX-2-dependent mechanisms may play a role in the development of POCD. This effect may be dependent on the modulation of glial cell activation.

In the Neurofibromatosis type 1 (NF1) mouse model, lovastatin, used clinically for hypercholesterolemia, improves cognitive dysfunction. While such impairment has been studied in NF1, the neural substrates remain unclear. The aim of this imaging add-on to a Phase 1 open-label trial was to examine the effect of lovastatin on Default Network (DN) resting state functional connectivity (RSFC). Seven children with NF1 (aged 11.9+/-2.2; 1 female) were treated with lovastatin once daily for 12 weeks. A 7-min 3-T echo-planar-imaging scan was collected one day before beginning treatment (off-drug) and the last day of treatment (on-drug) while performing a flanker task. After regressing-out task-associated variance, we used the residual time series as "continuous resting-state data" for RSFC analyses using 11 DN regions of interest. For qualitative comparisons, we included a group of 19 typically developing children (TDC) collected elsewhere. In the on-drug condition, lovastatin increased long-range positive RSFC within DN core regions (i.e., anterior medial prefrontal cortex and posterior cingulate cortex, PCC). In addition, lovastatin produced less diffuse local RSFC in the dorsomedial prefrontal cortex and PCC. The pattern of RSFC observed in the NF1 participants when on-drug closely resembled the RSFC patterns exhibited by the TDC. Lovastatin administration in this open trial regulated anterior-posterior long-range and local RSFC within the DN. These preliminary results are consistent with a role for lovastatin in normalization of developmental processes and with apparent benefits in a mouse NF1 model.

Herpes simplex virus type-1 (HSV-1) establishes a life-long latent infection in peripheral neurons. This latent reservoir is the source of recurrent reactivation events that ensure transmission and contribute to clinical disease. Current antivirals do not impact the latent reservoir and there are no vaccines. While the molecular details of lytic replication are well-characterized, mechanisms controlling latency in neurons remain elusive. Our present understanding of latency is derived from in vivo studies using small animal models, which have been indispensable for defining viral gene requirements and the role of immune responses. However, it is impossible to distinguish specific effects on the virus-neuron relationship from more general consequences of infection mediated by immune or non-neuronal support cells in live animals. In addition, animal experimentation is costly, time-consuming, and limited in terms of available options for manipulating host processes. To overcome these limitations, a neuron-only system is desperately needed that reproduces the in vivo characteristics of latency and reactivation but offers the benefits of tissue culture in terms of homogeneity and accessibility. Here we present an in vitro model utilizing cultured primary sympathetic neurons from rat superior cervical ganglia (SCG) (Figure 1) to study HSV-1 latency and reactivation that fits most if not all of the desired criteria. After eliminating non-neuronal cells, near-homogeneous TrkA(+) neuron cultures are infected with HSV-1 in the presence of acyclovir (ACV) to suppress lytic replication. Following ACV removal, non-productive HSV-1 infections that faithfully exhibit accepted hallmarks of latency are efficiently established. Notably, lytic mRNAs, proteins, and infectious virus become undetectable, even in the absence of selection, but latency-associated transcript (LAT) expression persists in neuronal nuclei. Viral genomes are maintained at an average copy number of 25 per neuron and can be induced to productively replicate by interfering with PI3-Kinase / Akt signaling or the simple withdrawal of nerve growth factor(1). A recombinant HSV-1 encoding EGFP fused to the viral lytic protein Us11 provides a functional, real-time marker for replication resulting from reactivation that is readily quantified. In addition to chemical treatments, genetic methodologies such as RNA-interference or gene delivery via lentiviral vectors can be successfully applied to the system permitting mechanistic studies that are very difficult, if not impossible, in animals. In summary, the SCG-based HSV-1 latency / reactivation system provides a powerful, necessary tool to unravel the molecular mechanisms controlling HSV1 latency and reactivation in neurons, a long standing puzzle in virology whose solution may offer fresh insights into developing new therapies that target the latent herpesvirus reservoir.

Alzheimer's disease (AD) is associated with impaired clearance of beta-amyloid (Abeta) from the brain, a process normally facilitated by apolipoprotein E (apoE). ApoE expression is transcriptionally induced through the action of the nuclear receptors peroxisome proliferator-activated receptor gamma and liver X receptors in coordination with retinoid X receptors (RXRs). Oral administration of the RXR agonist bexarotene to a mouse model of AD resulted in enhanced clearance of soluble Abeta within hours in an apoE-dependent manner. Abeta plaque area was reduced more than 50% within just 72 hours. Furthermore, bexarotene stimulated the rapid reversal of cognitive, social, and olfactory deficits and improved neural circuit function. Thus, RXR activation stimulates physiological Abeta clearance mechanisms, resulting in the rapid reversal of a broad range of Abeta-induced deficits.

Rapid temporal modulation of acoustic signals among several vertebrate lineages has recently been shown to depend on the actions of superfast muscles. We hypothesized that such fast events, known to require synchronous activation of muscle fibers, would rely upon motoneuronal properties adapted to generating a highly synchronous output to sonic muscles. Using intracellular in vivo recordings, we identified a suite of premotor network inputs and intrinsic motoneuronal properties synchronizing the oscillatory-like, simultaneous activation of superfast muscles at high-gamma frequencies in fish. Motoneurons lacked spontaneous activity, firing synchronously only at the frequency of premotor excitatory input. Population-level motoneuronal output generated a spike-like, vocal nerve volley that directly determines muscle contraction rate and, in turn, natural call frequency. In the absence of vocal output, motoneurons showed low excitability and a weak afterhyperpolarization leading to rapid accomodation in firing rate. By contrast, vocal activity was accompanied by a prominent afterhyperpolarization, indicating a dependency on network activity. Local injection of a GABAA receptor antagonist demonstrated the necessity of electrophysiologically- and immunohistochemically-confirmed inhibitory GABAergic input for motoneuronal synchrony and vocalization. Numerous transneuronally labeled motoneurons following single cell neurobiotin injection together with electrophysiological collision experiments confirmed gap junctional coupling, known to contribute to synchronous activity in other neural networks. Motoneuronal synchrony at the premotor input frequency was maintained during differential recruitment of variably sized motoneurons. Differential motoneuron recruitment led, however, to amplitude modulation (AM) of vocal output and, hence, natural call AM. In sum, motoneuronal intrinsic properties, in particular low excitability, predisposed vocal motoneurons to the synchronizing influences of premotor inputs to translate a temporal input code into a coincident and extremely synchronous, but variable amplitude, output code. We propose an analogous suite of neuronal properties as a key innovation underlying similarly rapid acoustic events observed among amphibians, reptiles, birds and mammals.