Faculty Bibliography

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.

ABSTRACT: Angiogenesis is essential for promoting growth and metastasis of solid tumors by ensuring blood supply to the tumor mass. Targeting angiogenesis is therefore an attractive approach to therapeutic intervention of cancer. Tumor angiogenesis is a process that is controlled by a complex network of molecular components including sensors, signaling transducers, and effectors, leading to cellular responses under hypoxic conditions. Positioned at the center of this network are the hypoxia-inducible factors (HIFs). HIF-1 is a major transcription factor that consists of two subunits, HIF-1alpha and HIF-1beta. It mediates transcription of a spectrum of gene targets whose products are essential for mounting hypoxic responses. HIF-1alpha protein level is very low in the normoxic condition but is rapidly elevated under hypoxia. This dramatic change in the cellular HIF-1alpha level is primarily regulated through the proteosome-mediated degradation process. In the past few years, scientific progress has clearly demonstrated that HIF-1alpha phosphorylation is mediated by several families of protein kinases including GSK3beta and ERKs both of which play crucial roles in the regulation of HIF-1alpha stability. Recent research progress has identified that Polo-like kinase 3 (Plk3) phosphorylates HIF-1alpha at two previously unidentified serine residues and that the Plk3-mediated phosphorylation of these residues results in destabilization of HIF-1alpha. Plk3 has also recently been found to phosphorylate and stabilize PTEN phosphatase, a known regulator of HIF-1alpha and tumor angiogenesis. Given the success of targeting protein kinases and tumor angiogenesis in anti-cancer therapies, Plk3 could be a potential molecular target for the development of novel and effective therapeutic agents for cancer treatment.

The evolution of a total synthesis of the exiguamines, two structurally unusual natural products that are highly active inhibitors of indolamine-2,3-dioxygenase (IDO), is described. The ultimately successful strategy involves advanced cross-coupling methodology and features a potentially biosynthetic tautomerization/electrocyclization cascade reaction that forms two heterocycles and installs a quaternary ammonium ion in a single synthetic operation.

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.

Cysteine string protein alpha (CSPalpha), a presynaptic cochaperone for Hsc70, is required for synapse maintenance. Deletion of CSPalpha leads to neuronal dysfunction, synapse loss, and neurodegeneration. We utilized unbiased, systematic proteomics to identify putative CSPalpha protein clients. We found 22 such proteins whose levels are selectively decreased in CSPalpha knockout synapses. Of these putative CSPalpha protein clients, two directly bind to the CSPalpha chaperone complex and are bona fide clients. They are the t-SNARE SNAP-25 and the GTPase dynamin 1, which are necessary for synaptic vesicle fusion and fission, respectively. Using hippocampal cultures, we show that CSPalpha regulates the stability of client proteins and synaptic vesicle number. Our analysis of CSPalpha-dynamin 1 interactions reveals unexpectedly that CSPalpha regulates the polymerization of dynamin 1. CSPalpha, therefore, participates in synaptic vesicle endocytosis and may facilitate exo- and endocytic coupling. These findings advance the understanding of how synapses are functionally and structurally maintained.

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.

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.