BACE1 is required for the release of beta-amyloid (A beta) in vivo, and inhibition of BACE1 activity is targeted for reducing A beta generation in Alzheimer's patients. To further our understanding of the safe use of BACE1 inhibitors in human patients, we aimed to study the physiological functions of BACE1 by characterizing BACE1-null mice. Here, we report the finding of spontaneous behavioral seizures in BACE1-null mice. Electroencephalographic recordings revealed abnormal spike-wave discharges in BACE1-null mice, and kainic acid-induced seizures also occurred more frequently in BACE1-null mice compared with their wild-type littermates. Biochemical and morphological studies showed that axonal and surface levels of Na(v)1.2 were significantly elevated in BACE1-null mice, consistent with the increased fast sodium channel current recorded from BACE1-null hippocampal neurons. Patch-clamp recording also showed altered intrinsic firing properties of isolated BACE1-null hippocampal neurons. Furthermore, population spikes were significantly increased in BACE1-null brain slices, indicating hyperexcitability of BACE1-null neurons. Together, our results suggest that increased sodium channel activity contributes to the epileptic behaviors observed in BACE1-null mice. The knowledge from this study is crucial for the development of BACE1 inhibitors for Alzheimer's therapy and to the applicative study of epilepsy.

Background: Amyotrophic lateral sclerosis (ALS) is a devastating disease that is characterized by the progressive loss of motor neurons. Patients with ALS usually die from respiratory failure due to respiratory muscle paralysis. Consequently, therapies aimed at preserving segmental function of the respiratory motor neurons could extend life for these patients. Insulin-like growth factor-I (IGF-I) is known to be a potent survival factor for motor neurons. In this study we induced high levels of IGF-I expression in the cervical spinal cord of hSOD1G93A rats with intraspinal cord (ISC) injections of an adeno-associated virus serotype 2 vector (CERE-130). This approach reduced the extent of motor neuron loss in the treated segments of the spinal cord. However, a corresponding preservation of motor function was observed in male, but not female, hSOD1G93A rats. We conclude that ISC injection of CERE-130 has the potential to protect motor neurons and preserve neuromuscular function in ALS.

Background/Aims: No validated delivery technique exists for accurate, reproducible delivery of biological therapies to discrete spinal cord targets. To address this unmet need, we have constructed a stabilized platform capable of supporting physiologic mapping, through microelectrode recording, and cellular or viral payload delivery to the ventral horn. Methods: A porcine animal model (n = 7) has been chosen based upon the inherent morphologic similarities between the human and porcine spine. Animals underwent physiologic mapping and subsequent microinjection of a green-fluorescent-protein-labeled cell suspension. Sacrifice (t = 3 h) was performed immediately following behavioral assessment. Results: Histologic analysis has supported our ability to achieve localization to the ipsilateral ventral horn in the spinal cord. Complications included death due to malignant hyperthermia (n = 1), hindlimb dysfunction attributable to epidural hematoma (n = 1), and hindlimb dysfunction attributable to cord penetration (n = 2). Conclusions: These results indicate an ability to achieve accurate targeting, but the elevated incidence of neurologic morbidity will require further studies with longer follow-ups that incorporate procedural and equipment modifications that will allow for a reduced number of cord penetrations and will account for observed cardiorespiratory-associated cord movement. These initial results reinforce the challenges of translating biological restorative therapies from small to large animal models and ultimately to humans.

Restraining excitatory neurotransmission within a seizure focus provides a nondestructive treatment strategy for intractable neocortical epilepsy. Clostridial toxin light chain (LC) inhibits synaptic transmission by digesting a critical vesicle-docking protein, synaptobrevin, without directly altering neuronal health. This study tests the treatment efficacy of adenoviral vector delivered LC (AdLC) on a model of seizures in rats induced by motor cortex penicillin (PCN) injection. LC expression significantly reduced electroencephalogram ( EEG) frequency, amplitude, duration, and latency compared to control groups injected with either an adenoviral vector bearing green fluorescent protein (AdGFP) or phosphate buffered solution (PBS). Correspondingly, LC gene expression improved behavioral manifestations including seizure severity and latency. There was no statistical difference in motor function before and after vector administration between treatment and control groups. Histological analysis revealed spatially discrete LC expression with corresponding synaptobrevin depletion in the cortex surrounding the injection site. Thus, vector-mediated LC gene delivery is capable of improving both the EEG and behavioral manifestations of PCN-induced focal neocortical seizures through synaptobrevin depletion.

Clostridial light chain (LC) inhibits synaptic transmission by digesting a vesicle-docking protein, synaptobrevin, without killing neurons. We here report the feasibility of creating a rat hemiparkinsonism model through LC gene expression in the substantia nigra (SN), inhibiting nigrostriatal transmission. 40 adult Sprague Dawley rats were divided into four groups for SN injections of PBS, 6-hydroxydopamine (6-OHDA), or adenoviral vectors for the expression of LC (AdLC), or GFP (AdGFP). Amphetamine and apomorphine induced rotations were assessed before and after SN injection, revealing significant rotational alterations at 8 or 10 days after injection in both AdLC and 6-OHDA but not PBS and AdGFP groups. Induced rotation recovered by one month in AdLC rats but persisted in 6-OHDA rats. Histological analysis of the SN revealed LC and GFP expression with corresponding synaptobrevin depletion in the LC, but not the GFP groups. Tyrosine hydroxylase (TH) and dopamine transporter (DAT) immunohistochemistry (IHC) showed markedly decreased staining in ipsilateral SN and striatum in 6-OHDA but not AdLC or AdGFP rats. Similarly, compared with contralateral, ipsilateral striatal dopamine level only decreased in 6-OHDA but not AdLC, AdGFP, or PBS treated rats. Thus, LC expression induces nigral synaptobrevin depletion with resulting inhibition of nigrostriatal synaptic transmission. Unlike 6-OHDA, LC expression inhibits synaptic activity without killing neurons. This approach, therefore, represents a potentially reversible means of nigrostriatal pathway inhibition as a model for Parkinson's disease. Such a model might facilitate transient and controlled nigral inhibition for studying striatal recovery, dopaminergic re-innervation, and normalization of striatal receptors following the recovery of nigrostriatal transmission.

OBJECTIVE:The recently discovered X-linked inhibitor of apoptosis protein (XIAP) is among the most potent inhibitors of programmed cell death. In the current experiment, we examine the potential of adenoviral XIAP gene delivery to protect neurons of the peripheral nervous system using in vitro models of amyotrophic lateral sclerosis (ALS) and diabetic neuropathy. METHODS:XIAP complementary deoxyribonucleic acid was fused in frame with the green fluorescent protein sequence and cloned into a first generation adenoviral vector. The impact of XIAP gene expression on glutamate-induced apoptosis was measured in the neuronal SH-SY5Y cell line with immunohistochemistry for active caspase-3 and with cell density assays. Next, the effect of XIAP expressing neurons on the survival of uninfected neighboring neurons was measured. Finally, the impact of XIAP gene expression on glutamate-induced apoptosis was assessed in embryonic motor neuron and dorsal root ganglion cultures. RESULTS:XIAP gene expression reduced the percentage of active caspase-3 positive SH-SY5Y neurons and preserved cell density after glutamate exposure. In heterogeneously infected cultures, cells infected with XIAP were protected, but uninfected neighboring cells were not. In primary E15 models, inhibition of proapoptotic effects was demonstrated after glutamate insult in motor neurons and glucose insult in dorsal root ganglion cells. CONCLUSION:XIAP gene delivery through the neurosurgical delivery of viral vectors may provide a means for neuroprotection in ALS and diabetic neuropathy.

Phage display is a promising tool for the screening of peptides with high affinity for specific cells. Here we describe a novel peptide with neuronal affinity isolated from a C7C library. We designed a two-tiered biopanning strategy initially selecting for ganglioside binding and subsequently selecting for binding to PC12 cells. At the completion of biopanning, 54.8% of phage clones bore the identical peptide (Tet.C7C.1). Immunofluorescence confirmed selective binding of this clone to differentiated PC12 cells. Tet.C7C.1 was synthesized and fluorescein conjugated. The synthetic peptide binds neuronal cell lines (SH-SY5Y, NSC-34 and PC12 cells) and tissue (DRG and spinal cord). The C7C structure creates a loop that minimizes the impact of peptide insertion on the confirmation of the recipient protein. Small loop peptides have the ideal characteristics for modification of viral vector capsids without undermining genome packaging. The neuronal binding properties of this peptide may be applied in the development of neurotropic viral vectors.

Background: Clostridial light chain (LC) inhibits synaptic transmission by digesting a vesicle-docking protein, synaptobrevin, without altering neuronal health. We have previously reported focal synaptic inhibition through adenovirus-mediated gene transfer of LC (AdLC) in the central nervous system. We report here the feasibility of creating reversible rat hemiparkinsonism (HPD) model through AdLC injection into the substantia nigra (SN).

Background: Pituitary tumors are the most common primary intracranial neoplasms. Growth hormone (GH) excess can be caused either by direct GH hypersecretion or GH excess secondary to GH- releasing factor (GHRF) hypersecretion. Neuroendocrine cells release transmitters and neuropeptides by calcium-dependent exocytosis of the contents of vesicles. This process requires assembly of SNARE proteins. Tetanus toxin light chain (TeTxLC) is a 50-kDa protein responsible for blockage of neurotransmitter release through digestion of three SNARE proteins (VAMP/synaptobrevin, syntaxin, and SNAP-25). The present study tested the hypothesis that adenoviral delivery of tetanus toxin light chain (Ad-LC) gene would cause inhibition of exocytotic release of GH. Methods: To study the properties of LC gene expression in pituitary tumors, we used GH3 cells from rat pituitary adenoma (ATCC). Following treatment with adenoviral vectors expressing TeTxLC (Ad-LC), GFP (Ad-GFP) or PBS, supernatants were collected at different time-points. For measurement of GH levels, assays were performed with an ultra-sensitive mouse/rat GH ELISA kit (DSLabs). Results: GH ELISA assay revealed a reduction in GH levels in the Ad-LC-treated groups (90% of reduction 96 hours after treatment), but not in the Ad-GFP and PBS groups. Moreover, TUNEL staining did not detect reduced cell density. Conclusions: This study reveals the ability of our Ad-LC to inhibit GH release. Although most pituitary tumors are considered benign, conventional therapies are often unsatisfactory. With the development of safe and targeted controllable vector systems, gene therapy might become a valuable approach for treating neuroendocrine tumors, such as pituitary adenomas.

Background: Adeno-associated viral vector (AAV) mediates long- term and safe gene expression in a variety of tissue types in vitro and in vivo. AAV vector has a wide rang of tissue tropism. However when tissue-specific gene expression is preferred, strategies such as genetic modification of the viral capsid or the inclusion of a tissues-specific promoter in the expression cassette are usually employed. We report here the development of a versatile AAV targeting system to achieve enhanced and specific motor neuron gene delivery.

Background: On the basis of its extremely efficient uptake and delivery to motor neurons, the tetanus toxin might be useful for neurospecific binding. However, the immune response to the toxin due the vaccination against tetanus, represents an obstacle to its clinical applicability. In order to find specific peptide sequences that can mimic binding of C fragment, we isolated a short neurotropic peptide sequence which when inserted into the AAV vector's coat might retarget the vector toward neuronal membranes without hindering its transduction efficiency. We have previously reported the phage display biopanning strategy for isolation of peptides with specific affinity for the trisialoganglioside (GT1b) Clostridial toxin receptor1 This process identified Tet1, a 12 AA peptide with specific and enhanced binding to differentiated pheochromocytoma (PC12) cells, primary motor neurons, and dorsal root ganglion (DRG) cells in vitro. The present experiment assessed the receptor binding characteristics of this peptide as well as its uptake and delivery to spinal motor neurons in vivo.

Top of pageAbstract We have previously demonstrated focal synaptic inhibition through neuronal expression of the light chain (LC) fragment of tetanus toxin in vivo. The transient effects are spatially discrete lending them to application in deep brain nuclei. This experiment examines feasibility in creating a rat model for Parkinsons disease through gene-based synaptic inhibition of the substantia nigra. The present experiment examined the impact of nigral LC expression on apomorphine induced rotations. Methods: Tetanus light chain (LC) was cloned into an adenoviral vector under control of the CMV promoter containing a GFP marker. Next, the impact of unilateral nigral 6-OHDA on striatal dopamine and glutamate synapses was compared to unilateral nigral LC expression. Rats received medial forebrain bundle (MFB) injections of either 4 or 8|[mu]|L of AdTeTxLC, 4|[mu]|L of 6-OHDA or PBS. Apomorphine-induced rotational behavior was assessed using a rotometer weekly for up to 4 weeks. Results: A significant increase in contralateral rotation was observed in the 6-OHDA positive control group and the 8|[mu]|L TeTxLC group, in comparison to the 4|[mu]|L TeTxLC and PBS groups. 6-OHDA animals demonstrated an average of 7.84 rotations per minute (+/|[minus]|0.45 SEM) and rats receiving 8|[mu]|L TeTxLC demonstrated an average of 4.39 rotations per minute (+/|[minus]|0.41 SEM). PBS rats demonstrated an average of 0.325 rotations per minute and rats receiving 4|[mu]|L TeTxLC demonstrated an average of 0.708 rotations per minute. Significance: This initial model proves the feasibility of dopamine depletion through nigral LC expression. Because LC expression inhibits synaptic activity without killing neurons, this approach represents a strategy for transient dopamine depletion. A subsequent experiment will apply an adeno-associated vector containing a Tet-on expression cassette (rAAV.Tet-on.LC). This latter vector will facilitate controlled, transient nigral suppression and will facilitate the study of behavioral recovery and normalization of striatal receptors following the recovery of striatal dopaminergic input. Transient and controlled nigral inhibition may provide a superior model for studying striatal recovery and dopaminergic re-innervation.