Epitope analysis following active immunization with tau proteins reveals immunogens implicated in tau pathogenesis

Abstract Background Abnormal tau hyperphosphorylation and its accumulation into intra-neuronal neurofibrillary tangles are linked to neurodegeneration in Alzheimer’s disease and similar tauopathies. One strategy to reduce accumulation is through immunization, but the most immunogenic tau epitopes have so far remained unknown. To fill this gap, we immunized mice with recombinant tau to build a map of the most immunogenic tau epitopes. Methods Non-transgenic and rTg4510 tau transgenic mice aged 5 months were immunized with either human wild-type tau (Wt, 4R0N) or P301L tau (4R0N). Each protein was formulated in Quil A adjuvant. Sera and splenocytes of vaccinated mice were collected to assess the humoral and cellular immune responses to tau. We employed a peptide array assay to identify the most effective epitopes. Brain histology was utilized to measure the effects of vaccination on tau pathology and inflammation. Results Humoral immune responses following immunization demonstrated robust antibody titers (up to 1:80,000 endpoint titers) to each tau species in both mice models. The number of IFN-γ producing T cells and their proliferation were also increased in splenocytes from immunized mice, indicating an increased cellular immune response, and tau levels and neuroinflammation were both reduced. We identified five immunogenic motifs within either the N-terminal (9-15 and 21-27 amino acids), proline rich (168-174 and 220-228 amino acids), or the C-terminal regions (427-438 amino acids) of the wild-type and P301L tau protein sequence. Conclusions Our study identifies five previously unknown immunogenic motifs of wild-type and mutated (P301L) tau protein. Immunization with both proteins resulted in reduced tau pathology and neuroinflammation in a tau transgenic model, supporting the efficacy of tau immunotherapy in tauopathy.http://deepblue.lib.umich.edu/bitstream/2027.42/109522/1/12974_2014_Article_152.pd

Sympathetic Overactivity Contributes to the Pathogenesis of Non-alcoholic Fatty Liver Disease During Diet-induced Obesity

Non-alcoholic fatty liver disease (NAFLD) is associated with the development of obesity and is a significant contributor to chronic liver, metabolic, and cardiovascular diseases. We have recently shown that hepatic sympathetic nerve activity is significantly elevated in mice fed a high fat diet (HFD; 33±2 vs. 63±5 spikes/s, normal chow vs. HFD; p\u3c0.05), although the contribution of the sympathetic nervous system to NAFLD pathology remains unclear. Therefore, we tested the hypothesis that sympathetic overactivity contributes to NAFLD during diet-induced obesity. Male C57B1/6 mice were fed a HFD (60% fat) or normal chow (5% fat) for 15 weeks. 6-hydroxydopamine (6-OHDA, 150 mg/kg i.p.) was then administered to selectively destroy sympathetic nerves, or vehicle control (n=4/group), and mice were sacrificed 3 days later. 6-OHDA treatment did not influence body weight (e.g. 41±3 vs. 40±2 g; HFD-vehicle vs. HFD-OHDA; p\u3e0.05) or visceral adipose tissue mass in normal chow or HFD fed animals. However, HFD resulted in significant increases in liver weight (1.0±0.1 vs. 1.8±0.1 g normal chow-vehicle vs. HFD-OHDA; p\u3c0.05) and selective ablation of sympathetic nerves rescued HFD-induced hepatomegaly (1.3±0.2 g; p\u3e0.05 vs. normal chow). In line with this, histological examination (H&E staining) revealed widespread hepatic lipid accumulation in HFD fed mice, which was reduced to normal levels following 6-OHDA administration (figure). Diet-induced obesity also resulted in elevations in plasma glucose (172±13 vs. 249±20 mg/dl; normal chow-vehicle vs. HFD-vehicle; p\u3c0.05), and ablation of sympathetic nerves restored HFD-mediated hyperglycemia (160±7 mg/dl; HFD-OHDA; p\u3e0.05 vs. normal chow). Concomitant with this, 6-OHDA administration in HFD fed animals was associated with a reduction in hepatic mRNA markers of gluconeogenesis (e.g. G6PC 6.6±1.0 vs. 3.2±0.7 fold normal chow-vehicle; HFD-vehicle vs. HFD-OHDA; p\u3c0.05) and lipogenesis (e.g. Srebp-1c 2.0±0.3 vs. 0.8±0.3 fold normal chow-vehicle; HFD-vehicle vs. HFD-OHDA; p\u3c0.05). Collectively, these findings demonstrate that removal of sympathetic nerve activity rescues obesity-induced hepatomegaly, hepatic steatosis and hyperglycemia, independent of an effect on body weight and adiposity. Moreover, this data reveals a novel role for the sympathetic nervous system in HFD-mediated NAFLD and suggest that targeting hepatic sympathetic overactivity may represent a novel therapeutic approach to treat NAFLD

A Synthetic Luciferin Improves In Vivo Bioluminescence Imaging of Gene Expression in Cardiovascular Brain Regions of Mice

Bioluminescence imaging is a powerful tool for in vivo investigation of biological processes. Incorporation of firefly luciferase into whole animals or organ specific areas, combined with exogenous administration of the substrate D-Luciferin, results in light production that can be captured with a charge-coupled device camera. We have demonstrated the utility of in vivo bioluminescence imaging to spatiotemporally monitor gene expression and transcription factor activation in individual cardiovascular brain nuclei during the development of cardiovascular disease. However, D-Luciferin uptake into the brain is low, which may limit the sensitivity of bioluminescence imaging, particularly when considering small changes in gene expression in single central nervous system areas. Therefore, approaches that improve the sensitivity of in vivo bioluminescence imaging are warranted. Here, we tested the hypothesis that a synthetic luciferase substrate, cyclic alkylaminoluciferin (CycLuc1), would be superior to D-Luciferin for in vivo monitoring of gene expression in cardiovascular brain regions. Male C57B1/6 mice (n=4) underwent targeted delivery of an adenovirus encoding the luciferase gene (luc) downstream of the CMV promoter to the subfornical organ, a circumventricular brain region that is critical in the control of the cardiovascular system. Following gene transfer and recovery, D-Luciferin or CycLuc1 were administered in a randomized fashion on 2 separate days and bioluminescence imaging was performed using an IVIS Lumina K system. The substrate dose (150 mg/kg) was similar between conditions and was chosen based on the typical use of this concentration for in vivo imaging with D-Luciferin. Administration of D-Luciferin revealed a bioluminescent signal from the subfornical organ of 3.2±1.2 x 105 photons/s at 10 minutes after substrate administration. In contrast, in the same animals at an equivalent concentration (figure), CycLuc1 injection was associated with a more intense light emission (7.7±2.6 x 105; p=0.06 vs. D-Luciferin) that was approximately 3-fold greater than that found with D-Luciferin (2.9±0.9 fold D-Luciferin). Similarly, at 20 minutes post substrate administration CycLuc1 provided a 3.3±1.1 fold higher bioluminescent signal than D-Luciferin (2.9±1.2 vs. 7.6±2.6 x 105 photons/s; D-Luciferin vs. CycLuc1; p=0.05). These preliminary findings demonstrate that replacing standard D-Luciferin with the synthetic luciferin CycLuc1 improves the sensitivity of bioluminescent detection from individual central nervous system cardiovascular control areas

Insulin receptor signaling in the subfornical organ protects against the development of metabolic syndrome

Metabolic syndrome encompasses a combination of conditions including obesity, diabetes, and hypertension. Brain insulin resistance has emerged as a contributor to the development of metabolic syndrome, although the neural regions involved remain unclear. While most investigations have focused on insulin action in the hypothalamus, recent evidence suggests that the insulin receptor (IR) gene is also expressed in the subfornical organ (SFO); a circumventricular organ well known for cardiovascular/fluid regulation and recently recognized as a metabolic nucleus. We therefore hypothesized that IR signaling in the SFO is involved in metabolic regulation. We first examined protein levels of SFO IR in male C57Bl/6 mice (n=3) using immunohistochemistry, and observed that IR expressing cells are rich in the SFO. Co-immunohistochemistry further revealed heterogeneous cellular expression of the SFO IR, with 11.9 ± 2.2% of IR-ir detected on astrocytes (GFAP), 57.2 ± 2.6% on endothelial cells (TIE2), and 18.3 ± 0.8% on neurons (NeuN). Interestingly, neuronal expression of IR in the SFO was restricted to glutamatergic cells, but absent in GABAergic cells. To test the functional role of SFO IR, we next utilized mice harboring a conditional allele of the IR gene (IRfl/fl), and selectively knocked down the SFO IR via SFO-targeted delivery of an adeno-associated virus encoding Cre-recombinase (AAV-Cre-eGFP; n=4), or control vector (AAV-eGFP; n=3). Both groups remained on normal chow, and metabolic parameters were continuously monitored using indirect calorimetry for 12 weeks. Selective removal of SFO IR did not influence food and water intake, but resulted in a greater increase in body weight (e.g. 12 weeks: 27.9 ± 1.5 vs. 31.4 ± 1.4 g, AAV-eGFP vs. AAV-Cre-eGFP, ANOVA interaction p=0.0005). This was associated with a significantly lower energy expenditure (e.g. 12 week average: 12.5 ± 0.6 vs. 11.7 ± 0.2 kcal/hr/kg, AAV-eGFP vs. AAV-Cre-eGFP, ANOVA interaction p=0.013) and a slight reduction in ambulatory activity in AAV-Cre-eGFP mice relative to controls. Examination of regional adipose tissue also revealed a ~40% increase in overall adiposity following ablation of SFO IR (total adipose: 1.4 ± 0.4 vs. 2.2 ± 0.3 g, AAV-eGFP vs. AAV-Cre-eGFP, p=0.1). Whole body glucose clearance and insulin sensitivity were comparable between groups. These data demonstrate that ablation of SFO IRs under normal diet conditions results in a deleterious metabolic state. Moreover, these findings indicate a tonic metabolic regulatory role for SFO IR, and suggest that impairments in IR signaling in the SFO may contribute to a development of metabolic syndrome

Hypertensive Actions of Long Chain Fatty Acids are Paralleled by Toll-Like Receptor 4 Upregulation and Nuclear Factor-B (NFB) Activation in the Subfornical Organ

Obesity-induced hypertension is characterized by a low-grade inflammatory state along with elevations in circulating long-chain fatty acids (LCFA). Toll-like receptor 4 (TLR4) is a transmembrane receptor that is activated by LCFA, resulting in downstream inflammatory processes. The central nervous system (CNS) is critical for the control of blood pressure, with the circumventricular subfornical (SFO) organ playing a critical role. The contribution of CNS TLR4 in blood pressure regulation, particularly within the SFO, remains unclear. We hypothesized that an SFO LCFA-TLR4-inflammatory pathway is associated with elevations in blood pressure. Adult male C57B1/6 mice were instrumented with radiotelemeters for the measurement of blood pressure and implanted with an intracerebroventricular (ICV) cannula. Following recovery, a LCFA (oleic acid, 30 nmol), or vehicle control, was administered once a day over 2 days. Whereas mean arterial blood pressure was unchanged in control animals, 2-day ICV infusion of LCFA resulted in a pro-hypertensive response (day 2: 95±2 vs. 110±2 mmHg, vehicle vs. LCFA, p\u3c0.05, n=4). Real-time quantitative PCR analysis revealed SFO TLR4 mRNA upregulation following central LCFA administration when compared to vehicle (1.1±0.3 vs. 2.3±0.4 fold vehicle, vehicle vs. LCFA, p\u3c0.05, n=4). In contrast, TLR4 transcript in the paraventricular nucleus of hypothalamus another cardioregulatory region, was not different between groups (1.1±0.2 vs. 1.4±0.3 fold vehicle, vehicle vs. LCFA, p\u3e0.05, n=4). Based on this, we further evaluated TLR4 protein expression in the SFO using immunohistochemical analyses. TLR4-expressing cells were found predominately within the medial to caudal regions of the SFO. Co-immunolabeling indicated that SFO TLR4-expressing cells were exclusively microglia (Iba1 co-localization: 75±4% TLR4 cells, n=3), whereas no co-expression was found on astrocytes or neurons. Given that TLR4 activation results in the activation of the inflammatory transcription factor NFkB, we subsequently assessed LCFA-TLR4 mediated activation of NFkB in the SFO using an in vivo bioluminescence imaging technique. Male C57B1/6 mice (n=2) underwent SFO-targeted injection of an adenovirus encoding firefly luciferase downstream of the NFκB consensus sequence to allow for in vivo imaging of SFO NFκB activity. Two-day ICV LCFA administration resulted in SFO NF-κB activation that was evident within 24 hrs (1.3±0.2 fold baseline) and maintained at 48 hrs (1.3±0.4 fold baseline). These findings indicate that LCFAs act within the CNS to increase arterial blood pressure. Moreover, the hypertensive actions of LCFA are paralleled by SFO TLR4 upregulation and NFκB activation, suggesting that a LCFA-TLR4-NFκB network in this nucleus may contribute to hypertension development, such as during obesity

A Forebrain-Hypothalamic Circuit Mediates Hepatic Steatosis

Non-alcoholic fatty liver disease (NAFLD), characterized by an accumulation of hepatic triglycerides (i.e. steatosis), is a growing health epidemic. We recently demonstrated a role for the brain in NAFLD. In particular, disruptions in the subfornical organ (SFO) - a small circumventricular forebrain nucleus - appear to play a key role in the development of hepatic steatosis. However, the neural network(s) through which the SFO contributes to NAFLD remains unknown. The paraventricular nucleus of the hypothalamus (PVN) is a central regulator of peripheral autonomic and endocrine function, and the SFO has dense excitatory projections to the PVN. Taken together, we hypothesized that activation of excitatory SFO PVN-projecting neurons would result in NAFLD development. We first confirmed that SFO PVN projecting neurons are excitatory by using retrograde viral labeling from the PVN (CAV2-GFP) combined with SFO immunohistochemistry for the excitatory neuronal marker calcium-calmodulindependent kinase II (CAMKII). Nearly 100% of the retrograde tracer co-localized with CAMKII in the SFO (not shown, n=3). Subsequently, we employed an intersectional viral strategy in which a retrograde transported canine adenovirus was targeted to the PVN to allow for expression of Cre-recombinase in SFO PVN-projecting neurons (CAV2-Cre-GFP), combined with SFO-targeted delivery of a Cre-inducible designer receptors engineered against designer drugs (DREADDs) excitatory construct (AAV2-DIO-hM3Gq-mCherry). With this approach, the pharmacological ligand clozapine-N-oxide (CNO; 3 mg/kg i.p.) was administered once daily over 6 days to activate SFO PVN-projecting neurons (n=4). Oil Red O staining of the liver demonstrated that, relative to control animals, 6-day activation of SFO PVN-projecting neurons resulted in a clear development of hepatic steatosis (2.69±0.02 vs. 2.92±0.02 au x 107 , saline vs. CNO, p\u3c0.05). Real time qPCR analysis further indicated that activation of SFO neurons that project to the PVN resulted in a marked upregulation of liver markers of de novo lipogenesis and gluconeogenesis (e.g. DGAT1: 3.6±0.3 fold saline, p\u3c0.05; G6Pase: 2.6±1.0 fold saline, p=0.12). Importantly, these changes occurred independent of differences in body weight (25±1 vs. 25+1 g, saline vs. CNO, p\u3c0.05) and food intake. Collectively, these findings indicate that short-term activation of excitatory SFO PVN-projecting neurons results in a NAFLD phenotype characterized by elevated liver triglycerides and disruptions in liver metabolic markers. Furthermore, these findings suggest that manipulating this forebrain-hypothalamic network in the context of obesity may be a novel approach to target NAFLD

INTRACRANIAL TARGETING OF GLIOBLASTOMA MULTIFORME WITH COLD ATMOSPHERIC PLASMA

Glioblastoma multiforme (GBM) is a highly malignant aggressive neoplasm of the primary central nervous system characterized by rapid growth, extensive angiogenesis, and resistance to current therapies. The median survival is limited to 16-19 months after diagnosis. In this context, GBM treatment strategies remain largely palliative despite the advancement of multi-modal therapies. Thus, it is necessary to develop novel tools that can target proliferating tumor cells and enhance existing therapies. Conventional lasers in medical devices are based on the thermal interaction with tissues, which lead to necrosis and permanent tissue damage. In contrast, cold atmospheric plasma (CAP) has recently emerged as a novel therapeutic approach for targeting of cancerous tissue. Indeed, recent findings suggest that CAP jet interactions with tissue may allow for cell death without necrosis. However, studies to date have been limited primarily to subcutaneous implantation of tumors. While beneficial, this approach does not replicate the complex environment of the brain (i.e. GBM). Here, we developed a novel approach to target CAP to intracranial GBM tumors. This new device, termed μCAP, consists of a Pyrex syringe through which CAP, employing helium gas, is supplied via the implanted endoscopic cannula. We first performed a set of experiments to test the influence of μCAP on normal brain parenchyma. Female athymic Foxn1nu nude mice underwent intracranial μCAP injection to the frontal lobe (15s total). Helium alone was administered as a control. Histological examination (Nissl staining) of the frontal lobe 7 days later revealed a similar number of apoptotic cells surrounding the injection site between μCAP and control treated animals (9±3 vs. 6±1 apoptotic cells/µm2, control vs. μCAP, n=2-3, p\u3e0.05). Similarly, no evidence of glia infiltration at the injection site was apparent. Next, nude mice underwent implantation of U87 glioblastoma cells (105 cells) into the frontal lobe and were simultaneously instrumented with a custom endoscopic cannula. Tumors were allowed to develop for 7 days and mice were then treated intracranially with μCAP (15s total) or helium control. Using in vivo bioluminescence imaging (Figure), the tumor volume in control animals increased nearly 1000% over the course of a week, whereas μCAP treated tumor volumes remained at baseline levels (day 7: 1035±773 vs. 172±107 radiance %baseline, control vs. μCAP, n=3, p\u3e0.05). These findings indicate that CAP has a minimal effect on healthy brain tissue, and further provide the first evidence for the potential of CAP to inhibit intracranial GBM tumor growth