Sustained intrathecal therapeutic protein delivery using genetically transduced tissue implants in a freely moving rat model.

Systemic delivery of therapeutic proteins to the central nervous system (CNS) is challenging because of the blood-brain barrier restrictions. Direct intrathecal delivery is possible but does not produce stable concentrations. We are proposing an alternative approach for localized delivery into the CNS based on the Transduced Autologous Restorative Gene Therapy (TARGT) system. This system was previously developed using a gene therapy approach with dermal tissue implants. Lewis rat dermal tissue was transduced to secrete human EPO (hEPO). TARGT viability and function were retained following cryopreservation. Upon implantation into the rat cisterna magna, a mild inflammatory response was observed at the TARGT-brain interface throughout 21-day implantation. hEPO expression was verified immunohistochemically and by secreted levels in cerebrospinal fluid (CSF), serum, and in vitro post explant. Detectable CSF hEPO levels were maintained during the study. Serum hEPO levels were similar to rat and human basal serum levels. In vitro, the highest hEPO concentration was observed on day 1 post-explant culture and then remained constant for over 21days. Prolonged incubation within the cisterna magna had no negative impact on TARGT hEPO secretion. These promising results suggest that TARGTs could be utilized for targeted delivery of therapeutic proteins to the CNS.

Myxobolus arcticus and Parvicapsula minibicornis infections in sockeye salmon Oncorhynchus nerka following downstream migration in British Columbia.

Factors influencing the health of sockeye salmon Oncorhynchus nerka in British Columbia, Canada, are important for fisheries management and conservation. Juvenile salmon originating from the Fraser River were screened for 3 enzootic parasites (Myxobolus arcticus, Parvicapsula minibicornis, Ceratonova shasta) and the bacterium Renibacterium salmoninarum. Fish were collected from the Strait of Georgia in 2010, 2011 and 2012 and genotyped to stock of origin. Trends in infection status were estimated by year, spawning zone and catch area. The annual prevalences of P. minibicornis (n = 1448) were 23.3, 6.5 and 8.1%, and for M. arcticus (n = 1343), annual prevalences were 40.4, 66.3 and 27.4%, respectively. Logistic regression showed that P. minibicornis was most strongly associated with salmon from the lower Fraser River spawning zone and increased with distance caught from the mouth of the Fraser River. In contrast, infection with M. arcticus was most strongly associated with salmon from the middle Fraser River spawning zone, and there was no trend related to distance from the Fraser River. Neither R. salmoninarum nor C. shasta were detected. These observations are discussed in the context of salmon life history and pathogen biology.

A retinoic acid-inducible transgenic marker of sino-atrial development in the mouse heart.

To study the specification of inflow structures in the heart we generated transgenic animals harboring the human alkaline phosphatase (HAP) gene driven by the proximal 840 bp of a quail SMyHC3 promoter. In transgenic mice, the SMyHC3-HAP reporter was expressed in posterior heart precursors at 8.25 dpc, in sinus venosa and in the atrium at 8.5 and 9.0 dpc, and in the atria from 10.5 dpc onwards. SMyHC3-HAP transgene expression overlapped synthesis and endogenous response to retinoic acid (RA) in the heart, as determined by antibodies directed against a key RA synthetic enzyme and by staining of RAREhsplacZ transgenic animals. A single pulse of all-trans RA administered to pregnant mice at 7.5, but not after 8.5, dpc induced cardiac dismorphology, ranging from complete absence of outflow tract and ventricles to hearts with reduced ventricles expressing both SMyHC3-HAP and ventricular markers. Blockade of RA synthesis with disulfiram inhibited RA-induced transcription and produced hearts lacking the atrial chamber. This study defines a novel marker for atrial-restricted transcription in the developing mouse heart. It also suggests that atrial-specific gene expression is controlled by localized synthesis of RA, and that exclusion of RA from ventricular precursors is essential for correct specification of the ventricles.

The E protein CTF4 and acetylcholine receptor expression in development and denervation supersensitivity.

Motor activity blocks the extrasynaptic expression of many genes in skeletal muscle, including those encoding ion channels, receptors, and adhesion molecules. Denervation reinduces transcription throughout the multinucleated myofiber, restoring the developmental pattern of expression, especially of the genes coding for the acetylcholine receptor. A screen for trans-acting factors binding to the enhancer region of the alpha-subunit gene of the acetylcholine receptor identified CTF4, a ubiquitously expressed and alternatively spliced chicken homologue of the human E protein transcription factor HTF4/HEB. Expression of the CTF4 locus closely parallels that of myogenin and acetylcholine receptor during development and maturation of skeletal muscle, but transcription is not similarly regulated by neuronal cues. Alternative splicing within the region encoding the transactivation domain generates two CTF4 isoforms with different tissue distributions, but similar binding affinities for the acetylcholine receptor alpha-subunit enhancer and similar transcriptional potential when complexed to myogenin. Direct injection of a myogenin, but not a MyoD, antisense expression vector into denervated skeletal muscle caused a significant decrease in the transcriptional activation of a depolarization-sensitive reporter gene. Similarly, injection of a CTF4, but less so of an E12, antisense expression vector impaired the denervation response, further implicating the involvement of a myogenin/CTF4 heterodimer in the expression of AChR genes in vivo.

Control of myogenic factor genes by the membrane depolarization/protein kinase C cascade in chick skeletal muscle.

Myogenic factor genes were found to respond differentially to electrical stimulation of denervated chick skeletal muscle. Myogenin gene activity declined rapidly (t1/2: approximately 2 min), comparable to the rate of acetylcholine receptor (AChR) gene inactivation, while other myogenic bHLH genes either lost activity more slowly (MyoD) or not at all (myf5, herculin). Protein kinase C (PKC) is known to couple membrane activity to AChR gene inactivation; myogenin gene transcription was also rapidly blocked by the PKC activator PMA, whereas electrostimulation remained without effect on myogenin gene activity in muscle that was either exposed to the kinase inhibitor staurosporine or chronically treated with PMA to deplete PKC. These results attest to a special role for myogenin in the activation of AChR genes in denervation supersensitivity.

Response of myogenic determination factors to cessation and resumption of electrical activity in skeletal muscle: a possible role for myogenin in denervation supersensitivity.

1. We have prepared probes specific for the chicken myogenic determination genes MyoD, myogenin, myf5, and herculin and have investigated the expression of these genes in response to denervation and acute electrical stimulation in neonate chick muscle, using ribonuclease protection. 2. Upon denervation, herculin mRNA remains essentially unchanged, myf5 transcript levels approximately double, and MyoD message is up-regulated by two- to fivefold. In contrast, the message coding for myogenin, barely detectable in innervated muscle, rises dramatically (approximately 200-fold) on the second day after nerve section; in this respect it resembles acetylcholine receptor (AChR) alpha-, gamma- and delta-subunit mRNAs. Cohybridization experiments reveal that the increase in myogenin mRNA slightly precedes the rise in AChR alpha-subunit message. 3. Electrical stimulation of denervated muscle leads to an immediate decline in myogenin and AChR alpha-subunit mRNAs, with half-lives of less than an hour and approximately 4 hr, respectively; message stability measurements suggest that this is effected through a rapid shutdown of transcription. Messages coding for MyoD, myf5, and herculin decay much more slowly, as a result of slower turnover. 4. Previous experiments have indicated the involvement of a de novo induced (Tsay, H.-J., Neville, C. M., and Schmidt, J., FEBS Lett. 274:69-72, 1990) autocatalytic (Neville, C. M., Schmidt, M., and Schmidt, J., NeuroReport 2:655-657, 1991) transcription factor in the denervation-triggered up-regulation of AChR alpha-subunit expression; the denervation and electrical stimulation experiments reported here are compatible with the notion that myogenin is that factor.

Expression of myogenic factors in skeletal muscle and electric organ of Torpedo californica.

Fish electric organ is a skeletal muscle homolog in which many muscle-specific genes are inhibited while acetylcholine receptor is expressed at high levels. The molecular mechanisms underlying this discoordinate regulation have not yet been explored. We have obtained partial sequences for MyoD, myogenin, and myf5 from Torpedo californica and have measured their mRNAs in several organs, using ribonuclease protection. We have found that MyoD and myf5 are expressed at comparable levels in muscle and electric organ, whereas myogenin transcripts could not be detected in either tissue. Acetylcholine receptor alpha subunit mRNA, on the other hand, is two orders of magnitude more abundant in electric tissue. We conclude that neither the loss of contractile proteins from, nor the enhanced expression of acetylcholine receptor genes in, the differentiating electrocyte is a simple consequence of the abundance of myogenic factor messages.

Protein synthesis is required for the denervation-triggered activation of acetylcholine receptor genes.

The effect of cycloheximide (CHX) on denervation-induced acetylcholine receptor (AChR) expression was investigated in chickens one day after nerve section, using probe excess solution hybridization to quantitate AChR alpha-subunit gene transcript levels and run-on analysis to measure subunit gene activity. The increase in alpha-subunit transcripts that normally follows denervation was prevented when drug treatment was commenced 2 h before or after denervation but was not blocked when CHX administration was begun 6 h after the operation. Drug-induced reduction of transcript levels results from decreased activity of genes coding for the alpha-, delta-, and gamma-subunits; in contrast, the transcription rates of several non-receptor genes are not affected by CHX. The results suggest that the de novo synthesis of a transcriptional activator is required as a mediating event in the signalling pathway linking the plasma membrane and AChR gene expression.