SUMMARYPurpose: Given the high incidence of refractory epilepsy, novel therapeutic approaches and concepts are urgently needed. To date, viral-mediated delivery and endogenous expression of antisense sequences as a strategy to prevent seizures have received little attention in epilepsy therapy development efforts. Here we validate adenosine kinase (ADK), the astrocyte-based key negative regulator of the brain's endogenous anticonvulsant adenosine, as a potential therapeutic target for antisense-mediated seizure suppression. Methods: We developed adenoassociated virus 8 (AAV8)-based gene therapy vectors to selectively modulate ADK expression in astrocytes. Cell type selectivity was achieved by expressing an Adk-cDNA in sense or antisense orientation under the control of an astrocyte-specific gfaABC1D promoter. Viral vectors where injected into the CA3 of wild-type mice or spontaneously epileptic Adk-tg transgenic mice that overexpress ADK in brain.After virus injection, ADK expression was assessed histologically and biochemically. In addition, intracranial electroencephalography (EEG) recordings were obtained.Key Findings: We demonstrate in wild-type mice that viral overexpression of ADK within astrocytes is sufficient to trigger spontaneous recurrent seizures in the absence of any other epileptogenic event, whereas ADK downregulation via AAV8-mediated RNA interference almost completely abolished spontaneous recurrent seizures in Adk-tg mice.Significance: Our data demonstrate that modulation of astrocytic ADK expression can trigger or prevent seizures, respectively. This is the first study to use an antisense approach to validate ADK as a rational therapeutic target for the treatment of epilepsy and suggests that gene therapies based on the knock down of ADK might be a feasible approach to control seizures in refractory epilepsy.
Oral isotretinoin showed better and earlier response than topical isotretinoin. Oral isotretinoin should definitely be given a trial particularly in cases of multiple facial warts before trying various destructive procedures.
BackgroundAberrant epigenetic silencing plays a major role in cancer formation by inactivating tumor suppressor genes. While the endpoints of aberrant silencing are known, i.e., promoter region DNA methylation and altered histone modifications, the triggers of silencing are not known. We used the tet-off system to test the hypothesis that a transient reduction in gene expression will sensitize a promoter to undergo epigenetic silencing.Methodology/Principal FindingsThe tet responsive promoter (PTRE) was used to drive expression of the selectable human HPRT cDNA in independent transfectants of an Hprt deficient mouse cell line. In this system, high basal HPRT expression is greatly reduced when doxycycline (Dox) is added to the culture medium. Exposure of the PTRE-HPRT transfectants to Dox induced HPRT deficient clones in a time dependent manner. A molecular analysis demonstrated promoter region DNA methylation, loss of histone modifications associated with expression (i.e., H3 lysine 9 and 14 acetylation and lysine 4 methylation), and acquisition of the repressive histone modification H3 lysine 9 methylation. These changes, which are consistent with aberrant epigenetic silencing, were not present in the Dox-treated cultures, with the exception of reduced H3 lysine 14 acetylation. Silenced alleles readily reactivated spontaneously or after treatment of cells with inhibitors of histone deacetylation and/or DNA methylation, but re-silencing of reactivated alleles did not require a new round of Dox exposure. Inhibition of histone deacetylation inhibited both the induction of silencing and re-silencing, whereas inhibition of DNA methylation had no such effect.Conclusions/SignificanceThis study demonstrates that a transient reduction in gene expression triggers a pathway for aberrant silencing in mammalian cells and identifies histone deacetylation as a critical early step in this process. DNA methylation, in contrast, is a secondary step in the silencing pathway under study. A model to explain these observations is offered.
Melasma is a common acquired, chronic hyperpigmentation most commonly occurring over face. Although benign in nature, it causes significant cosmetic deformity leading to great impact on quality of life and social as well as emotional well-being. Autologous platelet-rich plasma (PRP) in pigmentary disorder is a
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