Adeno-associated viral vectors (AAVs) have become popular for gene therapy, given their many advantages, including their reduced inflammatory profile compared with that of other viruses. However, even in areas of immune privilege such as the eye, AAV vectors are capable of eliciting host-cell responses. To investigate the effects of such responses on several ocular cell types, we tested multiple AAV genome structures and capsid types using subretinal injections in mice. Assays of morphology, inflammation, and physiology were performed. Pathological effects on photoreceptors and the retinal pigment epithelium (RPE) were observed. Müller glia and microglia were activated, and the proinflammatory cytokines TNF-α and IL-1β were up-regulated. There was a strong correlation between cis-regulatory sequences and toxicity. AAVs with any one of three broadly active promoters, or an RPE-specific promoter, were toxic, while AAVs with four different photoreceptor-specific promoters were not toxic at the highest doses tested. There was little correlation between toxicity and transgene, capsid type, preparation method, or cellular contaminants within a preparation. The toxic effect was dose-dependent, with the RPE being more sensitive than photoreceptors. Our results suggest that ocular AAV toxicity is associated with certain AAV cis-regulatory sequences and/or their activity and that retinal damage occurs due to responses by the RPE and/or microglia. By applying multiple, sensitive assays of toxicity, AAV vectors can be designed so that they can be used safely at high dose, potentially providing greater therapeutic efficacy.
Nrf2, a transcription factor that regulates the response to oxidative stress, has been shown to rescue cone photoreceptors and slow vision loss in mouse models of retinal degeneration (rd). The retinal pigment epithelium (RPE) is damaged in these models, but whether it also could be rescued by Nrf2 has not been previously examined. We used an adeno-associated virus (AAV) with an RPE-specific (Best1) promoter to overexpress Nrf2 in the RPE of rd mice. Control rd mice showed disruption of the regular array of the RPE, as well as loss of RPE cells. Cones were lost in circumscribed regions within the cone photoreceptor layer. Overexpression of Nrf2 specifically in the RPE was sufficient to rescue the RPE, as well as the disruptions in the cone photoreceptor layer. Electron microscopy showed compromised apical microvilli in control rd mice but showed preserved microvilli in Best1-Nrf2-treated mice. The rd mice treated with Best1-Nrf2 had slightly better visual acuity. Transcriptome profiling showed that Nrf2 upregulates multiple oxidative defense pathways, reversing declines seen in the glutathione pathway in control rd mice. In summary, Nrf2 overexpression in the RPE preserves RPE morphology and survival in rd mice, and it is a potential therapeutic for diseases involving RPE degeneration, including age-related macular degeneration (AMD).
Introduction: Neuroactive steroids and growth hormones have been implicated in stroke and cerebral disorders; an association largely attributed to age-related decline in circulating hormones. Neurosteroid and hormone levels may also be altered by acute neurological insult. This study examined hormone levels in older patients with acute aneurysmal subarachnoid hemorrhage (aSAH) compared to a matched healthy cohort. We hypothesized that neurosteroid and hormone levels would be significantly altered by acute aSAH. Methods: This analysis matched 24 aSAH patients >64 years of age (mn 69.5 years, 50% male, 87% Caucasian) from R01NR018160 with 24 healthy subjects (mn 68.7 years, 50% male, 87% Caucasian) from the IGNITE study R01AG053952. Demographic and clinical data came from the subject, representative, or medical record. Control samples were drawn at the time of study visits. aSAH samples were collected daily from consent-14 days after aSAH. Plasma samples were assayed for neuroactive steroids (estradiol [E2], estrone [E1], testosterone [T], progesterone [pro], androstenedione [andro]) via liquid chromatography–tandem mass spectrometry. Insulin growth factor (IGF1) was assayed via ELISA. Linear mixed model comparison of mean plasma hormone levels in aSAH and matched controls was conducted using SPSS and SAS. Results: Plasma neuroactive steroid and growth hormone levels were significantly different between aSAH patients and the matched cohort. aSAH patients had higher mean plasma E2 (13.8 vs 9.1 pg/mL; p=.06) and E1 (134.5 vs 45.2 pg/mL; p< 0.001) levels than their matched cohort. aSAH patients also had significantly higher mean andro (1596.7 vs 394.3 pg/mL; p< 0.001) and pro (99.8 vs 21.7 pg/mL; p= 0.001) levels than their matched cohort. aSAH patients had significantly lower mean plasma T (809.4 vs 2591.9 pg/mL; p= 0.003) and IGF1 (40.8 vs 54.6 ng/ml; p= 0.007) levels than their matched cohort. Conclusions: Hormone levels have not been well characterized after aSAH. This study of older adults found significantly higher estrogen and estrogen precursors immediately after aSAH while testosterone levels were significantly lower than the matched cohort. IGF1 levels were also significantly lower after aSAH than the matched cohort. The impact of these responses on recovery after aSAH warrants further investigation.
Summary Objective The objective of this study was to evaluate the association of urine clusterin/apolipoprotein J (Apo J) with the development and/or progression of diabetic kidney disease ( DKD ) in type 2 diabetes. Materials and Methods A total of 159 type 2 diabetic patients and 20 nondiabetic subjects with estimated glomerular filtration rate ( eGFR ) ≥60 mL/min/1.73 m 2 were enrolled. The baseline values of urine clusterin and tubular damage markers were measured. The primary outcome was the annual decline rate in eGFR , and secondary outcomes were the development of chronic kidney disease ( CKD ) stage 3 or greater and the persistence/progression of albuminuria. The median follow‐up duration of enrolled patients was 3.0 (1.0‐5.9) years. Results Baseline clusterin levels in urine were significantly increased in type 2 diabetic subjects compared with those of nondiabetic subjects. The levels of urine clusterin had a significant correlation with urine tubular damage markers. A positive correlation between the annual rate of decline in eGFR and urine clusterin after adjusting for clinical confounding factors was detected. Multivariate analysis further indicated that urine clusterin correlated with the development of CKD stage 3 or greater and persistence/progression of albuminuria. In type 2 diabetic subjects with albuminuria, urine clusterin remained associated with the annual decline rate in eGFR and the progression of CKD stage. Conclusions Urine clusterin reflects tubular damage in the early stage of DKD . The increase in urine clusterin along with albuminuria could be an independent predictive marker for the progression of DKD in type 2 diabetes.
Abstract Leptin regulates both feeding and glycaemia primarily through its receptors expressed on agouti-related peptide (AgRP) and pro-opiomelanocortin-expressing (POMC) neurons; however, it is unknown whether activity of these neuronal populations mediates the regulation of these processes. To determine this, we injected Cre-dependent designer receptors exclusively activated by designer drugs (DREADD) viruses into the hypothalamus of normoglycaemic and diabetic AgRP-ires-cre and POMC-cre mice to chemogenetically activate or inhibit these neuronal populations. Despite robust changes in food intake, activation or inhibition of AgRP neurons did not affect glycaemia, while activation caused significant (P = 0.014) impairment in insulin sensitivity. Stimulation of AgRP neurons in diabetic mice reversed leptin’s ability to inhibit feeding but did not counter leptin’s ability to lower blood glucose levels. Notably, the inhibition of POMC neurons stimulated feeding while decreasing glucose levels in normoglycaemic mice. The findings suggest that leptin’s effects on feeding by AgRP neurons are mediated by changes in neuronal firing, while the control of glucose balance by these cells is independent of chemogenetic activation or inhibition. The firing-dependent glucose lowering mechanism within POMC neurons is a potential target for the development of novel anti-diabetic medicines.
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