Protection of nigral dopaminergic neurons by AAV1 transduction with Rheb(S16H) against neurotoxic inflammation in vivo

Abstract

We recently reported that adeno-associated virus serotype 1 (AAV1) transduction of murine nigral dopaminergic (DA) neurons with constitutively active ras homolog enriched in brain with a mutation of serine to histidine at position 16 [Rheb(S16H)] induced the production of neurotrophic factors, resulting in neuroprotective effects on the nigrostriatal DA system in animal models of Parkinson’s disease (PD). To further investigate whether AAV1-Rheb(S16H) transduction has neuroprotective potential against neurotoxic inflammation, which is known to be a potential event related to PD pathogenesis, we examined the effects of Rheb(S16H) expression in nigral DA neurons under a neurotoxic inflammatory environment induced by the endogenous microglial activator prothrombin kringle-2 (pKr-2). Our observations showed that Rheb(S16H) transduction played a role in the neuroprotection of the nigrostriatal DA system against pKr-2-induced neurotoxic inflammation, even though there were similar levels of pro-inflammatory cytokines, such as tumor necrosis factor-alpha (TNF-α) and interleukin-1-beta (IL-1β), in the AAV1-Rheb(S16H)-treated substantia nigra (SN) compared to the SN treated with pKr-2 alone; the neuroprotective effects may be mediated by the activation of neurotrophic signaling pathways following Rheb(S16H) transduction of nigral DA neurons. We conclude that AAV1-Rheb(S16H) transduction of neuronal populations to activate the production of neurotrophic factors and intracellular neurotrophic signaling pathways may offer promise for protecting adult neurons from extracellular neurotoxic inflammation.

Myricitrin Ameliorates Hyperglycemia, Glucose Intolerance, Hepatic Steatosis, and Inflammation in High-Fat Diet/Streptozotocin-Induced Diabetic Mice

“…Pancreatic tissue sections were stained with an anti-insulin antibody and subjected to immunohistochemistry analysis, and the stained area was visualized using an optical microscope (Eclipse E200; Nikon, Tokyo, Japan) at 100× magnification. Densitometric analysis of insulin-positive cells was performed according to previous studies with some modifications [48,49]. The optical density of insulin-positive cells in stained sections was measured using Science Lab 2001 Image Gauge 4.0 (Fuji Film, Tokyo, Japan).…”

Therapeutic Potential of AAV1-Rheb(S16H) Transduction Against Alzheimer’s Disease

“…All vectors used were AAV1 serotype as previously described [15,16]. A plasmid carrying Rheb was purchased from OriGene Technologies (Rockville, MD, USA).…”

“…Animals were transcardially perfused, and fixed brain sections (30-µm-thick) were processed for immunofluorescence staining as previously described [15], with some modifications. Briefly, brain sections were rinsed in PBS and then incubated at 4 • C for 48 h with one of the following pairs: rabbit anti-FLAG (1:3000; Sigma) and mouse anti-neuronal nuclei (NeuN, 1:500; Millipore), rabbit anti-FLAG (1:3000; Sigma) and mouse anti-glial fibrillary acidic protein (GFAP, 1:2000, Millipore), mouse anti-FLAG (1:2000; Sigma) and rabbit anti-ionized calcium-binding adapter molecule 1 (Iba1, 1:2000; Wako Pure Chemical Industries), mouse anti-NeuN (1:500; Millipore) and rabbit anti-BDNF (1:200; Santa Cruz), rabbit anti-GFAP (1:2000, Millipore) and goat anti-tropomyosin receptor kinase B (TrkB, 1:100 R&D Systems), mouse anti-GFAP (1:2000; Millipore) and goat anti-CNTF (1:100, R&D Systems), and mouse anti-NeuN (1:500; Millipore) and goat anti-CNTF receptor α subunit (CNTFRα, 1:100, R&D Systems).…”

“…Western blot analysis was performed as described previously [15,16]. Briefly, the lysates obtained from astrocyte culture and hippocampal tissue were homogenized and centrifuged at 4 • C for 15 min at 14,000 × g. The supernatant was transferred to a fresh tube, and the concentration was determined using a bicinchoninic acid assay kit (BCA assay, Bio-Rad Laboratories, Hercules, CA, USA).…”

“…We have previously reported that the upregulation of NTFs by activation of a mammalian target of rapamycin complex 1 (mTORC1) following adeno-associated virus 1 (AAV1) transduction with a gene encoding a constitutively active human Ras homolog enriched in brain [Rheb(S16H)] protects against neurodegeneration in animal models [9,[15][16][17]. Also, we observed that Rheb(S16H) transduction protects neurons from thrombin-induced neurotoxicity through neuron-glia interactions in rat hippocampus [18].…”