Neurobiology of vital systems

Neurobiology of vital systems

Project 1: Genes and Cellular Stress in PKD

Supervisors: Prof Jacqueline Phillips, Assoc Prof Julie Atkins

Introduction

Polycystic kidney disease is an inherited disease in which multiple cysts form within the kidney, and there is kidney damage, loss of kidney function and the ultimate requirement for dialysis and kidney transplantation.

Our project is investigating how the genes that cause PKD can cause an increase in stress signals that drive cell damage and therefore damage to the kidney. These same processes can also be driven by the build up of toxins in the blood that arise when kidney function declines, and we will test if the combination of the PKD mutation and toxins worsens the stress response in the cell. 

The way we are going to undertake this study is to grow a cell line in a dish that was originally derived from human kidney cells. We will then do two things  - firstly we will introduce mutations that are known to cause PKD into the cells and study markers for cellular stress. We will then take the cells and expose them to toxins that are found in the blood of people with kidney disease and see if the level of the markers, as an indicator of the level of cell stress, increases.

Determining how PKD mutations and toxins lead to cell stress and then cell damage and kidney disease will help us find new ways to limit kidney damage progression in PKD and therefore delay the need for dialysis and ultimately renal transplant.

Hypothesis:

1. Our primary hypothesis is that the genes that cause PKD initiates a cascade of events in the cell that results in impaired mitochondrial and endoplasmic reticulum (ER) activity and increased levels of reactive oxygen species (ROS), altering critical cellular functions that otherwise serve to protect the cell. This will result in a state of increased oxidative stress and potential cell death. 

2. Our secondary hypothesis is that uraemic toxins, which are a by-product of kidney disease, can also drive these pathways and that the combined effect of the mutation and renal disease results in a state of oxidative stress exceeding that due to each factor alone

Aims:

1. Using cell-culture, determine if expression of different PKD gene mutations induce common changes in mitochondrial function, ER stress and increased levels of ROS.

2. Using cell-culture, determine if exposure to a uraemic environment potentiates PKD gene driven changes in mitochondrial function, ER stress and increased levels of ROS.

Research Plan:

Human embryonic kidney (HEK)293T cells will be used for our studies. Cells will be cultured and transfected with wild type or a human disease-causing PKD mutations, using plasmids tagged with enhanced yellow fluorescent protein (EYFP) to allow confirmation of transfection as well as allowing us to localise the location of the protein in the cells. Gene expression will be analysed by western blot and intracellular fluorescence imaging using confocal microscopy. Alternatively, lowering of expression of PKD genes will be undertaken using specific small interfering (si) RNA molecules. Control experiments will include untransfected, vector-transfected cells and the use of scrambled siRNA as appropriate.

Experiments will then be undertaken to assess superoxide production, mitochondrial function (for example measurement of mitochondrial membrane potential) or determination of complex enzyme activities of the mitochondrial oxidative phosphorylation system kinase. Induction of endoplasmic reticulum stress and the unfolded protein response (UPR) will be investigated by western blot analysis and immunohistochemistry

In parallel, cells from each transfected and control cell populations will be exposed to uraemic toxins.

Enquires:
Professor Jacqueline Phillips

Email: jacqueline.phillips@mq.edu.au

Project 2: At the heart of the problem: increased sympathetic drive to the heart in chronic kidney disease patients

Supervisors: Prof Jacqueline Phillips, Assoc Prof Ann Goodchild, Dr Cara Hildreth

Introduction

Chronic kidney disease affects at least 1.7 million Australians, with a further 1 in 3 at risk. For chronic kidney disease patients, the majority of deaths are not related to their diseased kidneys, but rather because their hearts no longer cope. Heart failure, myocardial infarction, abnormal heart rhythms, sudden cardiac death are common consequences of chronic kidney disease.  The one thing that contributes to all these heart-related causes of death is increased sympathetic nerve activity. Yet, because we do not understand why this is elevated in kidney disease patients and how this is impacting on the ability of the heart to function, there is little we can do to treat or prevent this from occurring. This project aims to identify how to equip the heart to cope with the challenges of increased sympathetic nerve activity. Only then can we hope to reduce the lethal cardiovascular burden associated with kidney disease.

Hypothesis:

That the sympathetic nervous system excessively drives heart activity in chronic kidney disease, leading to increased risk of sudden cardiac death

Aims:

To identify what occurs in chronic kidney disease to cause the sympathetic nervous system to exert too much command over the heart. To address this we must identify (1) where the source of the increased sympathetic nervous system control originates and (2) how the heart is responding to this.

Part (1) – where does the increased sympathetic nervous control originate from?

There is a circular relationship between the heart and the sympathetic nervous system, such that nerves project from the heart toward the brain (afferent signals) that influences the activity within the brain (central component) that then changes the nerve output from the brain back to the heart (efferent component).. We will investigate the following aims:

  • Aim 1a: Does increased sympathetic nervous control of the heart result from a change in afferent signalling?
  • Aim 1b: Does increased sympathetic nervous control of the heart result from a change within the central component?
  • Aim 1c: Does increased sympathetic nervous control of the heart result from a change within the efferent component?

Part (2) – how does the heart respond to the sympathetic nervous system?

The sympathetic nervous system is frequently referred to as the ‘fight or flight’ component of the nervous system and with respect to the heart is responsible for increasing how fast and hard it beats. The parasympathetic nervous system counterbalances these effects. This occurs through cross-talk between the two nerves as they enter the heart and how the heart integrates the information it receives. An imbalance resulting in excessive sympathetic nerve activity can lead to heart muscle hypertrophy and increased work. We will study this in the following aims:

  • Aim 2a: Sympathetic parasympathetic cross-talk – is the sympathetic nervous system turning off the parasympathetic nervous system?
  • Aim 2b: Has the heart lost the ability to properly integrate information from the sympathetic and parasympathetic nervous systems?

Research Plan:

Using an animal model we will test the reflexes that control the heart rate and determine which parts of the brain are (i) involved in the reflex and (ii) if the functional control is altered.

We will also examine heart tissue taken from both an animal model and human tissue from kidney disease patients and examine the receptors and transporters that control nervous communication in the heart, looking at both the sympathetic and parasympathetic nervous systems, and how they communicate at the level of the heart.

This will allow us to identify the cause of increased sympathetic nerve activity to the heart and the impact that the increased sympathetic nerve activity is having on the heart

Enquires:
Professor Jacqueline Phillips

Email: jacqueline.phillips@mq.edu.au

Project 3: How can the brain lead to cardiovascular disease?

Supervisors: Prof Jacqueline Phillips, Assoc Prof Ann Goodchild, Dr Cara Hildreth

Introduction

The baroreceptor reflex is essential for survival. In its absence, blood pressure fluctuates too much, placing individuals at an increased risk of death, independent of whether or not the patient has high blood pressure. Baroreflex function is impaired in many diseases, e.g. hypertension, heart failure, Parkinson’s disease, Alzheimer’s disease, depression and, integral to this proposal, chronic kidney disease (CKD). Despite this high incidence within the general population, little is done to directly treat baroreflex dysfunction, placing these patients at unnecessary risk.

For patients with chronic kidney disease, baroreflex dysfunction is associated with progression to renal failure and a greater chance of cardiovascular events, including sudden cardiac death. Baroreflex dysfunction is therefore not a trivial disease feature. But the reality is we are not able to treat baroreflex dysfunction as we still do not understand what causes it. In this project we will identify what mechanisms are accounting for impaired baroreflex function in CKD patients and if mitochondrial dysfunction in the brain plays a role.

Hypothesis:

We hypothesise that damaged/dysfunctional mitochondria place the brain in a stress-like state (oxidative stress) and alter how it responds to the hormone angiotensin II. We will test this in a model of chronic kidney disease with inherent mitochondrial damage and address the following:

1. What is the temporal relationship between mitochondrial damage, oxidative stress and changes to angiotensin II responsiveness in relationship to the onset of baroreflex dysfunction.

2. Does preventing or reversing mitochondrial damage limit baroreflex damage and does this occurs by: (a) Improving how the brain responds to baroreflex inputs and/or (b) Correcting brain activity in regions that can govern processing of the baroreflex?

Aims and Research Plan

To achieve this we will use a rodent model of chronic kidney disease, the Lewis Polycystic Kidney (LPK) rat.

Aim 1. What is the temporal relationship between mitochondrial damage, oxidative stress and changes to angiotensin II responsiveness in relationship to the onset of baroreflex dysfunction.

We will examine how well the baroreflex is functioning using a technique called telemetry. This allows us to measure blood pressure and heart rate in conscious animals. From this, we can use computer algorithms to examine how well the baroreflex is working. This will give us a time span over which baroreflex dysfunction occurs in the two models. We will take 3 time points: early, mid and late relative to the development of baroreflex dysfunction. At each of these time points we will measure the following: the degree of mitochondrial damage, the level of oxidative stress and the amount of angiotensin receptor present, and the functional responses of these receptors. We will focus on two brain regions- the nucleus tractus solitarius (NTS) and the paraventricular nucleus of the hypothalamus (PVN), as these are the site of baroreflex input to the brain and a region that governs baroreflex processing, respectively. 

Aim 2. If preventing or reversing mitochondrial damage can stop baroreflex damage from occurring and if this occurs by:

a. Improving how the brain responds to baroreflex inputs.

We will chronically treat both the LPK with a compound, mitoQ, to improve mitochondrial health in the brain specifically. Under anaesthesia, we will then microinject drugs into the NTS to see if we can alter the ability of the baroreceptor reflex to communicate information. We will block both inhibitory and excitatory neurotransmitter pathways.

b. Correcting brain activity in regions that can govern processing of the baroreflex.

Using the same treatment groups, we will perform microinjections in anaesthetised animals to inhibit the PVN. We are seeking to identify if this brain region is over active and as a consequence suppressing the baroreflex, and if this can be restored with mitoQ treatment. We will verify this by performing a separate series of studies where we will block the actions of the hormone vasopressin in the NTS. 

Enquires:
Professor Jacqueline Phillips

Email: jacqueline.phillips@mq.edu.au

Project 4: Fear, alerting and cardiorespiratory integration

Introduction

Our lab is interested in understanding the neural mechanisms through which arousing stimuli (‘stressors’) evoke changes in blood pressure and breathing. We all know that anxiety and mental anguish cause changes in breathing and heart rate; our lab has recently shown that regions that subconsciouslyprocess visual information can also evoke similar effects. In this project, we will investigate the mechanisms that underlie this surprising interaction between the visual pathway and the circuits that control blood pressure and breathing.

Hypothesis:

1.That light-activated neurons in the deep superior colliculus form connections with brainstem respiratory and cardiovascular neurons, such that their activation results in increases in blood pressure and breathing

2. That this pathway is normally suppressed but can become unmasked in some behavioral states.

Aims:

1. To determine whether brainstem-projecting colliculus neurons are activated by visual, acoustic or tactile stimuli in rodents

2. To determine the sources of inhibitory input that control these neurons in rodents

The Project

This project is a component of a larger ongoing research effort currently underway in the laboratory. We use a combination of traditional (retrograde tracing, drug microinjection) and advanced (optogenetics, conditional trans-synaptic viral tracing) techniques in integrated neuroanatomical, physiological, and behavioural experiments. We are looking for enthusiastic students who have a good basic neuroscience experience and some understanding of the approaches used to probe neural circuits in experimental animals. Please contact simon.mcmullan@mq.edu.au to discuss the project in more detail and arrange an interview.

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