Human CARD12 is a novel CED4/Apaf-1 family member that induces apoptosis.

The CED4/Apaf-1 family of proteins functions as critical regulators of apoptosis and NF-kappaB signaling pathways. A novel human member of this family, called CARD12, was identified that induces apoptosis when expressed in cells. CARD12 is most similar in structure to the CED4/Apaf-1 family member CARD4, and is comprised of an N-terminal caspase recruitment domain (CARD), a central nucleotide-binding site (NBS), and a C-terminal domain of leucine-rich repeats (LRR). The CARD domain of CARD12 interacts selectively with the CARD domain of ASC, a recently identified proapoptotic protein. In addition, CARD12 coprecipitates caspase-1, a caspase that participates in both apoptotic signaling and cytokine processing. CARD12 may assemble with proapoptotic CARD proteins to coordinate the activation of downstream apoptotic and inflammatory signaling pathways.

Assessing viral gene therapy in neuroendocrine models.

A simple method of manipulating neuronal gene expression would greatly facilitate the design of experiments to increase our understanding of and ability to treat diseases of the CNS. However, until recently most transfection methods could only deliver DNA into dividing cells and it was only possible to manipulate neuronal gene expression through the production of transgenic animals. The development of powerful new viral-based gene transfer systems has generated a great deal of research interest in the field of therapeutic gene transfer during the last decade. One of the most powerful and versatile gene delivery systems currently available is the recombinant adenovirus (Ad) vector. These vectors can transfect postmitotic neurons in the CNS, but have not yet been fully evaluated as CNS gene therapy vectors. Brattleboro rats contain a point mutation in the arginine vasopressin (AVP) gene that results in a pathological phenotype characterized by a lack of circulating AVP. This decrease in AVP in turn causes the characteristics signs of diabetes insipidus, with the production of large volumes of dilute urine and a compensatory drinking of large volumes of water (equivalent to the body weight of the rat per day). We have shown that injection of an Ad encoding the arginine vasopressin cDNA into the supraoptic nuclei of the hypothalamus results in the long-term reversal of this pathological phenotype. This was demonstrated by reduced daily water intake and micturition, as well as increased urine osmolality lasting 4 months. The highly characterized Brattleboro rat model of hypothalamic diabetes insipidus, therefore, provides the means to examine noninvasively the efficacy of viral and nonviral gene therapy strategies in the CNS.

Switching transgene expression in the brain using an adenoviral tetracycline-regulatable system.

We have developed a tetracycline-regulatable adenoviral transfection system that mediates efficient long-term transfer of genes into neuronal cells in vivo. This system allows gene expression to be switched on, then off, and back on again simply by administering or removing doxycycline from the animals' drinking water. This regulatable adenoviral vector system should be of value in behavioral studies and in vivo studies of neuronal gene function, and may further the development of effective gene therapy strategies in the brain.

Overexpression of hsp70i facilitates reactivation of intracellular proteins in neurones and protects them from denaturing stress.

Transfection of neurones with an adenoviral vector (Ad) expressing high levels of hsp70i was shown to protect primary hippocampal cultures from heat stress. To investigate one of the molecular mechanisms which may underlie hsp70i's neuroprotective effects we measured luciferase activity in the presence and absence of hsp70i following heat or chemical inactivation. Luciferase activity was recovered to 80% of control levels in the presence of recombinant hsp70i in vitro. Luciferase activity was also maintained in primary hippocampal neurones exposed to a denaturing stress if transfected with Ad-hsp70i. These results support the hypothesis that hsp70i protects neurones from stress by interacting with cytosolic proteins and thereby protecting them from inactivation.

Long-term gene therapy in the CNS: reversal of hypothalamic diabetes insipidus in the Brattleboro rat by using an adenovirus expressing arginine vasopressin.

The ability of adenovirus (Ad) to transfect most cell types efficiently has already resulted in human gene therapy trials involving the systemic administration of adenoviral constructs. However, because of the complexity of brain function and the difficulty in noninvasively monitoring alterations in neuronal gene expression, the potential of Ad gene therapy strategies for treating disorders of the CNS has been difficult to assess. In the present study, we have used an Ad encoding the arginine vasopressin cDNA (AdAVP) in an AVP-deficient animal model of diabetes insipidus (the Brattleboro rat), which allowed us to monitor chronically the success of the gene therapy treatment by noninvasive assays. Injection of AdAVP into the supraoptic nuclei (SON) of the hypothalamus resulted in expression of AVP in magnocellular neurons. This was accompanied by reduced daily water intake and urine volume, as well as increased urine osmolality lasting 4 months. These data show that a single gene defect leading to a neurological disorder can be corrected with an adenovirus-based strategy. This study highlights the potential of using Ad gene therapy for the long-term treatment of disorders of the CNS.

Tetracycline-regulated transgene expression in hippocampal neurones following transfection with adenoviral vectors.

A transfer system that enabled the efficient introduction of transgenes into neurones and the quantitative control of the expressed transgene would greatly facilitate studies into neuronal gene function. To develop such a system we incorporated the tetracycline (Tet)-responsive On/Off regulatory elements into type-5 adenoviral (Ad) vectors. Regulation of transgene expression following transfection was measured by placing the enhanced green fluorescent protein (EGFP) gene upstream of the Tet regulatory element. The results showed that cultures of primary hippocampal cells could be transfected with very high efficiency (<70%) by the AdTet-On and AdTet-Off systems. Following transfection with the AdTet-On system no EGFP-fluorescent cells could be detected until doxycycline was added. The AdTet-Off system showed the reverse transcriptional regulation, in that the addition of Tet caused EGFP fluorescence to be abolished.

Persistent transgene expression in the hypothalamus following stereotaxic delivery of a recombinant adenovirus: suppression of the immune response with cyclosporin.

Replication deficient, recombinant adenoviruses (Ads) have been used successfully to transfect several forebrain and brainstem nuclei, but have yet to be demonstrated as useful vectors for transgene delivery in the structurally diverse and highly vascularised nuclei of the hypothalamus. In the present study we have assessed the ability of an Ad expressing the lac-Z gene to transfect cells of the paraventricular nucleus (PVN) of the hypothalamus in vivo. We show that: (1) we can achieve stable expression of the lacZ gene in cells of the magnocellular PVN for at least 2 months; (2) there were no obvious differences in the level of AVP mRNA in the PVNs injected with Ad compared with those injected with vehicle suggesting that Ad treatment is not disrupting normal cellular function in the injection region; (3) the introduction of Ads results in a limited immune response; (4) systemic treatment with cyclosporin dramatically reduces its magnitude. We conclude that Ad vectors represent useful tools for neuroendocrinological and gene therapeutic studies of the hypothalamus.

The emerging concept of relaxin as a centrally acting peptide hormone with hemodynamic actions.

The novel finding that relaxin has an action on the brain was first published in 1984. Since then, it has been shown that exogenous relaxin affects the release of a number of hypothalamo-pituitary hormones and has a robust pressor action. In this paper, we review the accumulating evidence that relaxin affects the release of oxytocin and vasopressin by an action at the level of the brain. The potential mechanisms of this central action are discussed and the evidence presented for the interaction between relaxin and the forebrain angiotensin-II system. Furthermore, we articulate the possible physiological influences of relaxin on the changes in cardiovascular function that occur during pregnancy.

Brain angiotensin-II partially mediates the effects of relaxin on vasopressin and oxytocin release in anesthetized rats.

Experiments were conducted in anesthetized rats to assess the contribution of the brain angiotensin-II system in the relaxin-induced secretion of vasopressin and oxytocin. Intravenous injection of porcine relaxin (5 micrograms) caused a significant (P < 0.05, by analysis of variance) increase in plasma concentrations of both hormones. Peak concentrations of both vasopressin (75.2 +/- 2.9 pmol/liter) and oxytocin (38.4 +/- 1.2 pmol/liter) were observed 1-2.5 min after relaxin injection. Thereafter, concentrations fell significantly (P < 0.05) but remained elevated for a further 25 minutes. Continuous infusion of a specific angiotensin-II receptor antagonist into the lateral cerebral ventricle did not affect baseline levels of either vasopressin or oxytocin, but did significantly reduce (P < 0.05) the relaxin-induced release of both peptides. A significant (P < 0.05) short term increase in both plasma vasopressin and oxytocin occurred 1 min after injection of 5 micrograms relaxin, iv, in angiotensin-II-antagonized rats, but the concentrations of both neuropeptides were significantly (P < 0.05) lower than those observed in the angiotensin-intact relaxin-treated controls. These data suggest that relaxin may act through the central angiotensin-II system to induce the release of vasopressin and oxytocin.