Joseph G. Quinn

Identification of valid reference genes for the normalization of RT qPCR gene expression data in human brain tissue

BackgroundStudies of gene expression in post mortem human brain can contribute to understanding of the pathophysiology of neurodegenerative diseases, including Alzheimers disease (AD), Parkinsons disease (PD) and dementia with Lewy bodies (DLB). Quantitative real-time PCR (RT qPCR) is often used to analyse gene expression. The validity of results obtained using RT qPCR is reliant on accurate data normalization. Reference genes are generally used to normalize RT qPCR data. Given that expression of some commonly used reference genes is altered in certain conditions, this study aimed to establish which reference genes were stably expressed in post mortem brain tissue from individuals with AD, PD or DLB.ResultsThe present study investigated the expression stability of 8 candidate reference genes, (ubiquitin C [UBC], tyrosine-3-monooxygenase [YWHAZ], RNA polymerase II polypeptide [RP II], hydroxymethylbilane synthase [HMBS], TATA box binding protein [TBP], β-2-microglobulin [B2M], glyceraldehyde-3-phosphate dehydrogenase [GAPDH], and succinate dehydrogenase complex-subunit A, [SDHA]) in cerebellum and medial temporal gyrus of 6 AD, 6 PD, 6 DLB subjects, along with 5 matched controls using RT qPCR (TaqMan® Gene Expression Assays). Gene expression stability was analysed using geNorm to rank the candidate genes in order of decreasing stability in each disease group. The optimal number of genes recommended for accurate data normalization in each disease state was determined by pairwise variation analysis.ConclusionThis study identified validated sets of mRNAs which would be appropriate for the normalization of RT qPCR data when studying gene expression in brain tissue of AD, PD, DLB and control subjects.

A bi-directional μ-opioid-opioid connection between the nucleus of the accumbens shell and the central nucleus of the amygdala in the rat

The central nucleus of the amygdala (CeA) and the nucleus of the accumbens shell (NAc) have been shown to be involved in opioid-mediated feeding behavior. The present study examined whether mu-opioid signalling between the CeA and NAc affected feeding. Male Sprague-Dawley rats were fitted with one cannula placed in the CeA and two cannulae placed in the NAc, which allowed for coadministration of the mu-opioid receptor agonist [D-Ala(2), NMe-Phe(4), Gly-ol(5)]-enkephalin (DAMGO) in one site and the opioid antagonist naltrexone (NTX) in the other site. Single injection of DAMGO (2.4 nmol) into the CeA and bilateral injections of DAMGO (2.4 nmol) into the NAc stimulated feeding (P<0.05). The DAMGO-induced increase of food intake following injection into the CeA was decreased by bilateral injection of NTX (13.2 and 26.5 nmol) into the NAc at 2- and 4-h postinjections (P<0.05). In the reverse situation, the DAMGO-induced increase of food intake following injection into the NAc was decreased by injection of NTX (13.2 and 26.5 nmol) into the CeA at 1-, 2-, and 4-h postinjections (P<0.05). These results suggest that a bi-directional mu-opioid-opioid signalling pathway exists between the CeA and the NAc, which influences feeding.

Evidence for a μ-opioid–opioid connection between the paraventricular nucleus and ventral tegmental area in the rat

The paraventricular nucleus (PVN) and the ventral tegmental area (VTA) have been shown to be involved in opioid mediated feeding behavior. The present study examined whether mu-opioid signalling between the PVN and VTA affected feeding behavior. Male Sprague-Dawley rats were cannulated with one cannula placed in the PVN and two cannulae placed in the VTA, which allowed for co-administration of the mu-opioid receptor agonist [D-Ala(2), NMe-Phe(4), Gly-ol(5)]-enkephalin (DAMGO) in one site and the opioid antagonist naltrexone (NTX) in the other site. Bilateral administration of DAMGO (1.2, 2.4 and 4.9 nmol) into the VTA stimulated feeding dose dependently at 2.4 and 4.9 nmol (P<0.05). The DAMGO (2.4 nmol)-induced increase of food intake following injection into the PVN was blocked by bilateral injection of NTX (6.6, 13.2 and 26.5 nmol) into the VTA at 2 and 4 h (P<0.05). In the reverse situation, the DAMGO (2.4 nmol)-induced increase of food intake following injection into the VTA was blocked by injection of NTX (13.2 and 26.5 nmol) into the PVN at 2 and 4 h (P<0.05). The present study suggests that a bidirectional mu-opioid-opioid signalling pathway exists between the PVN and the VTA which influences feeding.

α-Synuclein mRNA and soluble α-synuclein protein levels in post-mortem brain from patients with Parkinson's disease, dementia with Lewy bodies, and Alzheimer's disease

α-Synuclein is a neuronal protein implicated in the etiology of Parkinsons disease (PD) and dementia with Lewy bodies (DLB). Whilst increased α-synuclein expression due to gene duplication or triplication can cause familial PD, previous studies of α-synuclein levels in idiopathic disease have produced conflicting data. We quantified α-synuclein mRNA and soluble protein in five human post-mortem brain regions from four groups of individuals with PD, DLB, Alzheimers disease (AD) and matched controls. α-Synuclein mRNA levels, measured using quantitative real-time PCR, did not differ significantly between groups in any brain regions examined. In contrast, levels of soluble α-synuclein protein, measured by ELISA, were significantly lower in 4 of the 5 regions for patients with DLB, and in 2 of the 5 regions for patients with PD, compared to controls. Soluble α-synuclein protein levels were not significantly different in the AD patients, compared to controls, in 4 of the 5 regions. This study indicates that although levels of soluble α-synuclein protein are lower in DLB and PD, there is no evidence for a corresponding decrease in α-synuclein mRNA levels. This might result from altered translation, or removal of α-synuclein protein from a soluble detectable state, either by turnover or conversion to an insoluble form.

Feeding association between the nucleus of the solitary tract and the ventral tegmental area

Male Sprague-Dawley rats were fitted with two cannulae in the VTA and one cannula in the NTS for co-administration of the micro-opioid receptor agonist DAMGO in one site and the opioid antagonist naltrexone in the other. Injection of DAMGO into the VTA or the NTS stimulated feeding. The increase in food intake after DAMGO injection into the VTA was decreased following injection of naltrexone into the NTS. Furthermore, the increase in food intake after DAMGO injection into the NTS was decreased following injection of naltrexone into the VTA. These results suggest an opioid-mediated feeding association between the VTA and NTS.

Blood: tests used to assess the physiological and immunological properties of blood

The properties of blood and the relative ease of access to which it can be retrieved make it an ideal source to gauge different aspects of homeostasis within an individual, form an accurate diagnosis, and formulate an appropriate treatment regime. Tests used to determine blood parameters such as the erythrocyte sedimentation rate, hemoglobin concentration, hematocrit, bleeding and clotting times, mean corpuscular hemoglobin, mean corpuscular hemoglobin concentration, mean cell volume, and determination of blood groups are routinely used clinically, and deviations outside the normal range can indicate a range of conditions such as anemia, pregnancy, dehydration, overhydration, infectious disease, cancer, thyroid disease, and autoimmune conditions, to mention a few. As these tests can be performed relatively inexpensively and do not require high levels of technical expertise, they are ideally suited for use in the teaching laboratory, enabling undergraduate students to link theory to practice. The practicals described here permit students to examine their own blood and that of their peers and compare these with clinically accepted normal ranges. At the end of the practicals, students are required to answer a number of questions about their findings and to link abnormal values to possible pathological conditions by answering a series of questions based on their findings.

Ultrasound imaging in teaching cardiac physiology

This laboratory session provides hands-on experience for students to visualize the beating human heart with ultrasound imaging. Simple views are obtained from which students can directly measure important cardiac dimensions in systole and diastole. This allows students to derive, from first principles, important measures of cardiac function, such as stroke volume, ejection fraction, and cardiac output. By repeating the measurements from a subject after a brief exercise period, an increase in stroke volume and ejection fraction are easily demonstrable, potentially with or without an increase in left ventricular end-diastolic volume (which indicates preload). Thus, factors that affect cardiac performance can readily be discussed. This activity may be performed as a practical demonstration and visualized using an overhead projector or networked computers, concentrating on using the ultrasound images to teach basic physiological principles. This has proved to be highly popular with students, who reported a significant improvement in their understanding of Frank-Starlings law of the heart with ultrasound imaging.

mRNA Levels of BACE1 and its interacting proteins, RTN3 and PPIL2, correlate in human post mortem brain tissue

β-Site amyloid precursor protein cleaving enzyme (BACE1) is the rate-limiting enzyme for production of Aβ peptides, proposed to drive the pathological changes found in Alzheimers disease (AD). Reticulon 3 (RTN3) is a negative modulator of BACE1 (β-secretase) proteolytic activity, while peptidylprolyl isomerase (cyclophilin)-like 2 (PPIL2) positively regulated BACE1 gene expression in a cell-based assay. This study aimed to analyze RTN3 and PPIL2 mRNA levels in four brain regions from individuals with AD and controls. BACE1 mRNA had been previously quantified in the samples, as had glial fibrillary acidic protein (GFAP) and neuron-specific enolase (NSE), to track changing cell populations in the tissue. mRNA levels in the human post mortem brain tissue were assayed using quantitative real-time polymerase chain reaction (qPCR) and qbase(PLUS), employing validated stably expressed reference genes. No differences in RTN3 or PPIL2 mRNA levels were found in individuals with AD, compared to controls. Both RTN3 and PPIL2 mRNA levels correlated significantly with BACE1 mRNA and all three showed similar disease stage-dependent changes with respect to NSE and GFAP. These findings indicated that the in vitro data demonstrating an effect of PPIL2 on BACE1 expression have functional relevance in vivo. Further research into BACE1-interacting proteins could provide a fruitful approach to the modulation of this protease and consequently Aβ production.