Alison Bienemann

Expression of Long-Term Depression Underlies Visual Recognition Memory

The modifications occurring in the brain during learning and memory are still poorly understood but may involve long-lasting changes in synaptic transmission (synaptic plasticity). In perirhinal cortex, a lasting decrement in neuronal responsiveness is associated with visual familiarity discrimination, leading to the hypothesis that long-term depression (LTD)-like synaptic plasticity may underlie recognition memory. LTD relies on internalization of AMPA receptors (AMPARs) through interaction between their GluR2 subunits and AP2, the clathrin adaptor protein required for endocytosis. We demonstrate that a peptide that blocks interactions between GluR2 and AP2 blocks LTD in perirhinal cortex in vitro. Viral transduction of this peptide in perirhinal cortex produced striking deficits in visual recognition memory. Furthermore, there was a deficit of LTD in perirhinal cortex slices from virally transduced, recognition memory-deficient animals. These results suggest that internalization of AMPA receptors, a process critical for the expression of LTD in perirhinal cortex, underlies visual recognition memory.

cAMP Responsive Element-Binding Protein Phosphorylation Is Necessary for Perirhinal Long-Term Potentiation and Recognition Memory

We established the importance of phosphorylation of cAMP responsive element-binding protein (CREB) to both the familiarity discrimination component of long-term recognition memory and plasticity within the perirhinal cortex of the temporal lobe. Adenoviral transduction of perirhinal cortex (and adjacent visual association cortex) with a dominant-negative inhibitor of CREB impaired the preferential exploration of novel over familiar objects at a long (24 h) but not a short (15 min) delay, disrupted the normal reduced activation of perirhinal neurons to familiar compared with novel pictures, and impaired long-term potentiation of synaptic transmission in perirhinal slices. The consistency of these effects across the behavioral, systems, and cellular levels of analysis provides strong evidence for involvement of CREB phosphorylation in synaptic plastic processes within perirhinal cortex necessary for long-term recognition memory.

Long-term replacement of a mutated nonfunctional CNS gene: reversal of hypothalamic diabetes insipidus using an EIAV-based lentiviral vector expressing arginine vasopressin

Due to the complexity of brain function and the difficulty in monitoring alterations in neuronal gene expression, the potential of lentiviral gene therapy vectors to treat disorders of the CNS has been difficult to fully assess. In this study, we have assessed the utility of a third-generation equine infectious anemia virus (EIAV) in the Brattleboro rat model of diabetes insipidus, in which a mutation in the arginine vasopressin (AVP) gene results in the production of nonfunctional mutant AVP precursor protein. Importantly, by using this model it is possible to monitor the success of the gene therapy treatment by noninvasive assays. Injection of an EIAV-CMV-AVP vector into the supraoptic nuclei of the hypothalamus resulted in expression of functional AVP peptide in magnocellular neurons. This was accompanied by a 100% recovery in water homeostasis as assessed by daily water intake, urine production, and urine osmolality lasting for a 1-year measurement period. These data show that a single gene defect leading to a neurological disorder can be corrected with a lentiviral-based strategy. This study highlights the potential of using viral gene therapy for the long-term treatment of disorders of the CNS.

Targeting of tetracycline-regulatable transgene expression specifically to neuronal and glial cell populations using adenoviral vectors

Targeting regulatable transgene expression specifically to neuronal or glial cell populations would facilitate studies of CNS gene function. We have developed the tetracycline (Tet) regulatable adenoviral system by expressing the Tet-off trans-activator (tTA) under the control of the neuronal-specific synapsin I promoter and the well characterized glial-specific glial fibrillary acidic protein (GFAP) promoter. Transfection of primary hippocampal cultures demonstrated that the respective promoters restricted reporter transgene expression exclusively to neuronal or glial populations. Delivery of the vectors into adult rat hippocampus resulted in a similar pattern of cell-specific transgene expression. These novel vectors provide a highly effective means of directing regulated, cell-specific, transgene expression and as such are important tools for investigations of neuronal and glial cell function and advancing gene therapy studies.

An evaluation of the relationships between catheter design and tissue mechanics in achieving high-flow convection-enhanced delivery

Convection-enhanced delivery (CED) is a rational technique for the direct intracranial administration of a range of therapeutic agents. CED critically depends on the use of a catheter with a narrow outer diameter and low infusion rate. Failure to adhere to these requirements can lead to reflux of infusate along the catheter-brain interface and damage at the catheter-tip. In this study we have tested the hypothesis that the relationship between infusion parameters and infusate distribution, including reflux, is critically dependent on the occurrence of tissue damage. The relationship between catheter outer diameter and the extent of blood-brain barrier disruption and subsequent tissue oedema was evaluated following catheter insertion into the striatum of rats. Three patterns of infusate distribution were observed: (1) Reflux restricted to the traumatised tissue around the catheter site. (2) Distribution in the white matter beyond the area of tissue trauma. (3) Widespread distribution in the striatum, which occurred only with catheters of an outer diameter of 0.35 mm or less. Extensive tissue damage occurred with a 0.2mm outer diameter catheter. This damage was completely prevented by rounding the catheter-tip. Infusions into pig brain demonstrated that high-flow CED could be performed in a large brain in both grey and white matter using a 0.2mm outer diameter catheter, with minimal reflux or MRI-evidence of tissue damage. This study demonstrates that by minimising tissue damage from catheter design and insertion, high flow-rate CED can be utilised to distribute therapeutic agents over large volumes of brain within clinically practical timescales.

Multifunctional receptor-targeted nanocomplexes for the delivery of therapeutic nucleic acids to the Brain

Convection enhanced delivery (CED) is a method of direct injection to the brain that can achieve widespread dispersal of therapeutics, including gene therapies, from a single dose. Non-viral, nanocomplexes are of interest as vectors for gene therapy in the brain, but it is essential that administration should achieve maximal dispersal to minimise the number of injections required. We hypothesised that anionic nanocomplexes administered by CED should disperse more widely in rat brains than cationics of similar size, which bind electrostatically to cell-surface anionic moieties such as proteoglycans, limiting their spread. Anionic, receptor-targeted nanocomplexes (RTN) containing a neurotensin-targeting peptide were prepared with plasmid DNA and compared with cationic RTNs for dispersal and transfection efficiency. Both RTNs were labelled with gadolinium for localisation in the brain by MRI and in brain sections by LA-ICP-MS, as well as with rhodamine fluorophore for detection by fluorescence microscopy. MRI distribution studies confirmed that the anionic RTNs dispersed more widely than cationic RTNs, particularly in the corpus callosum. Gene expression levels from anionic formulations were similar to those of cationic RTNs. Thus, anionic RTN formulations can achieve both widespread dispersal and effective gene expression in brains after administration of a single dose by CED.

A phase I trial of carboplatin administered by convection-enhanced delivery to patients with recurrent/progressive glioblastoma multiforme

Glioblastoma multiforme (GBM) is the commonest primary malignant brain tumour in adults. Standard treatment comprises surgery, radiotherapy and chemotherapy; however this condition remains incurable as these tumours are highly invasive and involve critical areas of the brain making it impossible to remove them surgically or cure them with radiotherapy. In the majority of cases the tumour recurs within 2 to 3 cm of the original site of tumour resection. Furthermore, the blood-brain barrier profoundly limits the access of many systemically administered chemotherapeutics to the tumour. Convection-enhanced delivery (CED) is a promising technique of direct intracranial drug delivery involving the implantation of microcatheters into the brain. Carboplatin represents an ideal chemotherapy to administer using this technique as glioblastoma cells are highly sensitive to carboplatin in vitro at concentrations that are not toxic to normal brain in vivo. This protocol describes a single-centre phase I dose-escalation study of carboplatin administered by CED to patients with recurrent or progressive GBM despite full standard treatment. This trial will incorporate 6 cohorts of 3 patients each. Cohorts will be treated in a sequential manner with increasing doses of carboplatin, subject to dose-limiting toxicity not being observed. This protocol should facilitate the identification of the maximum-tolerated infused concentration of carboplatin by CED into the supratentorial brain. This should facilitate the safe application of this technique in a phase II trial, treating patients with GBM, as well as for the treatment of other forms of malignant brain tumours, including metastases.

Long-term transgene expression can be mediated in the brain by adenoviral vectors when powerful neuron-specific promoters are used

Adenoviral (Ad) vectors are one of the most widely used tools for modelling gene therapy strategies. However, they have not been used in long‐term models of neurological disease, as the period of time for which they mediate strong transgene expression is limited and/or variable. In this study we investigated the longevity of transgene expression in the brain when the powerful neuron‐specific Ad‐synapsin (Sy)‐EGFP‐woodchuck hepatitis virus post‐transcriptional regulatory element (WPRE) vector cassette is used at titres that do not elicit an immune response.

Hsp70 suppresses apoptosis in sympathetic neurones by preventing the activation of c-Jun

The anti‐apoptotic effects of heat‐shock protein (Hsp70) were assessed in SCG neurones following nerve growth factor (NGF) withdrawal. The results showed that the virally mediated expression of Hsp70 mirrored the effects of the c‐Jun‐N‐terminal kinase (JNK) binding domain (JBD) of JNK interacting protein (an inhibitor of JNK and c‐Jun activation) and suppressed the phosphorylation of c‐Jun. Preventing c‐Jun transcriptional activity subsequently led to reduced cytochrome c release and prevented caspase activation as indicated by a decrease in poly (ADP‐ribose) polymerase‐1 (PARP) cleavage. Together, these results show that Hsp70 is a highly effective inhibitor of apoptosis in sympathetic neurones and that it mediates this effect primarily by suppressing c‐Jun transcriptional signalling.

PEGylation improves the receptor-mediated transfection efficiency of peptide-targeted, self-assembling, anionic nanocomplexes

Non-viral vector formulations comprise typically complexes of nucleic acids with cationic polymers or lipids. However, for in vivo applications cationic formulations suffer from problems of poor tissue penetration, non-specific binding to cells, interaction with serum proteins and cell adhesion molecules and can lead to inflammatory responses. Anionic formulations may provide a solution to these problems but they have not been developed to the same extent as cationic formulations due to difficulties of nucleic acid packaging and poor transfection efficiency. We have developed novel PEGylated, anionic nanocomplexes containing cationic targeting peptides that act as a bridge between PEGylated anionic liposomes and plasmid DNA. At optimized ratios, the components self-assemble into anionic nanocomplexes with a high packaging efficiency of plasmid DNA. Anionic PEGylated nanocomplexes were resistant to aggregation in serum and transfected cells with a far higher degree of receptor-targeted specificity than their homologous non-PEGylated anionic and cationic counterparts. Gadolinium-labeled, anionic nanoparticles, administered directly to the brain by convection-enhanced delivery displayed improved tissue penetration and dispersal as well as more widespread cellular transfection than cationic formulations. Anionic PEGylated nanocomplexes have widespread potential for in vivo gene therapy due to their targeted transfection efficiency and ability to penetrate tissues.