Aquaporins in the brain: from aqueduct to "multi-duct”

The aquaporin channel family was first considered as a family of water channels, however it is now clear that some of these channels are also permeable to small solutes such glycerol, urea and monocarboxylates. In this review, we will consider AQP4 and AQP9 expressed in the rodent brain. AQP4 is present on astrocytic end-feet in contact with brain vessels and could be involved in ionic homeostasis. However, AQP4 may also be involved in cell adhesion. AQP4 expression is highly modified in several brain disorders and it can play a key role in the cerebral edema formation. However, the exact role of AQP4 in edema formation is still debated. Recently, AQP4 has been shown to be also involved in astrocyte migration during glial scar formation. AQP9 is expressed in astrocytes and in catecholaminergic neurons. Two isoforms of AQP9 are expressed in brain cells, the shortest isoform is localized in the inner membrane of mitochondria and the longest in the cell membrane. The level of expression of AQP9 is negatively regulated by high concentrations of insulin. Taken together, these results suggest that AQP9 could be involved in brain energy metabolism. The induction of AQP9 in astrocytes is observed with time after stroke onset suggesting participation in the clearance of excess lactate in the extracellular space. These recent exciting results suggest that AQPs may not only be involved in water homeostasis in the brain but could also participate in other important physiological function

Aquaporins and Neurodegenerative Diseases

Aquaporins (AQPs) are a family of widely distributed membrane-inserted water channel proteins providing a pathway for osmotically-driven water, glycerol, urea or ions transport through cell membranes and mechanisms to control particular aspects of homeostasis. Beside their physiological expression patterns in Central Nervous System (CNS), it is conceivable that AQPs are also abnormally expressed in some pathological conditions interesting CNS (e.g. neurodegenerative diseases) in which preservation of brain homeostasis is at risk

An absence of dystrophin in cerebellar Purkinje cells impairs inhibitory synaptic function in mature dystrophic mice

Duchenne muscular dystrophy (DMD) is a rapidly progressive X-linked recessive disease affecting about 1 in 3500 live male births. It is caused by mutations in the dystrophin gene, which result in the loss of dystrophin or expression of a non-functional truncated protein product. Full-length dystrophin is mainly expressed in muscles and the central nervous system. In addition to the degeneration of skeletal musculature, about one-third of patients with DMD display various degrees of intellectual impairment, commonly found with intelligence quotient (IQ) scores of one standard deviation below (IQ of 85) the normal population mean (IQ of 100). However, the mechanism underlying the cognitive deficits in DMD remains unclear and no effective treatment is available to reverse this condition in the affected individual. Recent studies showed that the life span of DMD patients today has increased from teens to their fourth decades. With longer survival, the quality of life becomes increasing important. Therefore, research on the cognitive aspect of DMD is as important as research on the muscular aspects because improvements in cognitive function will enhance the quality of life for the growing population of adult DMD patients. The aim of this thesis was to investigate the role of dystrophin in the central nervous system of the mdx mouse, a widely accepted murine model for DMD. This study employed the use of animal with different age groups, corresponding to young (3-4 months), adult (11-12 months), and aged (23-26 months). Adult and aged mdx mice are the focus in this study with findings from the older mouse especially valuable as, disease progression in aged mice closely resembling that of DMD. As numerous evidence has shown a high similarity between the specific cognitive dysfunctions seen in DMD (i.e. impaired verbal intelligence) and in patients with cerebellar lesions (i.e. language disorders), this study examined the function of cerebellar Purkinje cells in mdx mice using electrophysiological recording and calcium imaging. Overall, the data presented in this thesis provides new insights into the role of dystrophin in cerebellar Purkinje neurons. The findings suggest that dystrophin is important for normal inhibitory synaptic function, intrinsic electrophysiological properties, and calcium handling of the mature cerebellar Purkinje cells. The consequences of the absence of dystrophin including the altered GABAA receptor clustering and reduced peak amplitude of mIPSCs could be ameliorated when dystrophin was successfully rescued with Pip6f-PMO in an organotypic mdx cerebellar culture. If mdx mice and DMD patients share similar neuropathogenesis, the development of drugs targeting the altered functions in mdx Purkinje cells may serve as a potential therapy in alleviating the cognitive impairments seen in DMD

An approach for the in-vivo characterization of brain and heart inflammation in Duchenne Muscular Dystrophy

Duchenne muscular dystrophy (DMD) is a neuromuscular disorder caused by dystrophin loss—notably within muscles and CNS neurons. DMD presents as cognitive weakness, progressive skeletal and cardiac muscle degeneration until pre-mature death from cardiac or respiratory failure. Innovative therapies improved life expectancy, but this is accompanied by increased late-onset heart failure and emergent cognitive degeneration. Thus, there is an increasing need to both better understand and track disease pathophysiology in the dystrophic heart and brain prior to onset of severe degenerative symptoms. Chronic inflammation is strongly associated with skeletal and cardiac muscle degeneration, however chronic neuroinflammation’s role is largely unknown in DMD despite being prevalent in other neurodegenerative diseases. Considering the well-known consequences of unchecked chronic inflammation, inflammation’s contribution towards multi-organ degeneration must be explored. Thus, this study explored inflammatory marker translocator protein positron emission tomography (TSPO-PET) to evaluate immune cell infiltration within the hearts and brains of DMD murine models. Four DMD and six healthy mice underwent whole-body PET imaging using the TSPO radiotracer [18F]FEPPA. Confirmatory TSPO-immunofluorescence staining of cardiac and neural tissues were also conducted. Our results indicated that DMD mice showed significant elevations in heart and brain [18F]FEPPA activity, which correlated with increased ex-vivo fluorescence intensity. In summary, this study suggests cardiac and neuroinflammation presence in DMD and highlights TSPO-PET’s utility as a tool for in-vivo assessment of inflammation in several organs simultaneously within DMD

Development of non-invasive MRI to measure water permeability across the blood-brain interface

The blood-brain interface (BBI) is a physical and biochemical barrier that protects and maintains healthy brain function. Disruption of the BBI is indicative of the early stages of certain neurodegenerative diseases, such as Alzheimer’s Disease. However, there is currently a lack of sensitive tools available to accurately quantify the early alterations to the integrity of the BBI. This thesis describes the development and implementation of multiple echo time arterial spin labelling (multi-TE ASL) MRI technique in the mouse brain to measure vascular water permeability across the BBI. The technique was implemented in two high-field MRI system to demonstrate the consistency of the imaging protocols and the sensitivity of the measures of BBI water permeability. The multi-TE ASL technique was used to probe the function of aquaporin-4 (AQP4) water channels, which play a key role in the clearance of the deleterious proteins from the brain. This non-invasive technique was able to demonstrate its sensitivity to targeting AQP4 by measuring a 31% slowing of cortical BBI water permeability with the removal of the AQP4 water channels. The technique also measured a 34% slowing in the BBI water permeability in the cerebellum brain region with a reduction of AQP4 channels at the BBI. Finally, the technique measured a 32% increase in cortical BBI permeability to water in a mouse model of ageing. The non-invasive imaging measurements were 7 associated with a 2-fold increase in mRNA expression of pericytes, while other BBI markers such as tight junction proteins were maintained. Overall, this work has demonstrated the scope of novel MRI technique to target changes to BBI water permeability, with potential for clinical translation for the early detection and understanding of neurodegenerative disease

Aquaporins in the brain: from aqueduct to "multi-duct"

The aquaporin channel family was first considered as a family of water channels, however it is now clear that some of these channels are also permeable to small solutes such glycerol, urea and monocarboxylates. In this review, we will consider AQP4 and AQP9 expressed in the rodent brain. AQP4 is present on astrocytic end-feet in contact with brain vessels and could be involved in ionic homeostasis. However, AQP4 may also be involved in cell adhesion. AQP4 expression is highly modified in several brain disorders and it can play a key role in the cerebral edema formation. However, the exact role of AQP4 in edema formation is still debated. Recently, AQP4 has been shown to be also involved in astrocyte migration during glial scar formation. AQP9 is expressed in astrocytes and in catecholaminergic neurons. Two isoforms of AQP9 are expressed in brain cells, the shortest isoform is localized in the inner membrane of mitochondria and the longest in the cell membrane. The level of expression of AQP9 is negatively regulated by high concentrations of insulin. Taken together, these results suggest that AQP9 could be involved in brain energy metabolism. The induction of AQP9 in astrocytes is observed with time after stroke onset suggesting participation in the clearance of excess lactate in the extracellular space. These recent exciting results suggest that AQPs may not only be involved in water homeostasis in the brain but could also participate in other important physiological functions

The central role of aquaporins in the pathophysiology of ischemic stroke

Stroke is a complex and devastating neurological condition with limited treatment options. Brain edema is a serious complication of stroke. Early edema formation can significantly contribute to infarct formation and thus represents a promising target. Aquaporin (AQP) water channels contribute to water homeostasis by regulating water transport and are implicated in several disease pathways. At least 7 AQP subtypes have been identified in the rodent brain and the use of transgenic mice has greatly aided our understanding of their functions. AQP4, the most abundant channel in the brain, is up-regulated around the peri-infarct border in transient cerebral ischemia and AQP4 knockout mice demonstrate significantly reduced cerebral edema and improved neurological outcome. In models of vasogenic edema, brain swelling is more pronounced in AQP4-null mice than wild-type providing strong evidence of the dual role of AQP4 in the formation and resolution of both vasogenic and cytotoxic edema. AQP4 is co-localized with inwardly rectifying K+-channels (Kir4.1) and glial K+ uptake is attenuated in AQP4 knockout mice compared to wild-type, indicating some form of functional interaction. AQP4-null mice also exhibit a reduction in calcium signaling, suggesting that this channel may also be involved in triggering pathological downstream signaling events. Associations with the gap junction protein Cx43 possibly recapitulate its role in edema dissipation within the astroglial syncytium. Other roles ascribed to AQP4 include facilitation of astrocyte migration, glial scar formation, modulation of inflammation and signaling functions. Treatment of ischemic cerebral edema is based on the various mechanisms in which fluid content in different brain compartments can be modified. The identification of modulators and inhibitors of AQP4 offer new therapeutic avenues in the hope of reducing the extent of morbidity and mortality in stroke.peer-reviewe

The role of myocardial membrane proteins and myocardial oedema in postoperative myocardial dysfunction

The vast majority of children undergoing surgical repair of cardiac lesions do spectacularly well. However a significant proportion, ~ 25%, struggle to progress in the early postoperative period and require additional pharmacological and occasionally mechanical circulatory support. All children typically have some degree of postoperative myocardial dysfunction, with the severe spectrum termed the low cardiac output state (LCOS). LCOS is clinically defined as the requirement for new or escalated inotrope therapy, a widened arteriovenous oxygen difference, cardiac arrest or the need for reinstitution of mechanical circulatory support. LCOS is largely responsible for the morbidity and mortality involved in paediatric cardiac surgery. Despite the predictability of LCOS in the initial postoperative hours, the underlying pathophysiology remains unclear. The period of decline in cardiac function that typifies LCOS is temporally associated with the development of oedema in the tissues of the body, including the heart. This relationship between oedema and dysfunction has increasingly become blurred, with a tendency to elevate the temporal association to a causal link. We sought to explore the causes and contributions to myocardial dysfunction in this setting, including the roles of oedema and ischaemia within the heart. In focusing on oedema and ischaemia we also examined the effects of these insults on relevant myocardial membrane proteins, including those that permit rapid water transport – aquaporins (AQPs), and those involved in membrane mechanics – dystrophin, and membrane repair – dysferlin. Experimental settings which enabled the in vitro dissection of these insults and proteins of interest were combined with a clinically accurate in vivo model. This thesis describes a series of thematically linked experiments that examined LCOS, myocardial oedema and the role of various membrane proteins. We performed isolated cardiomyocyte studies, isolated heart studies as well as a clinically relevant large animal (lamb) cardiopulmonary bypass (CPB) model. Across these models we also explored the role of therapeutically protecting myocardial membranes with Poloxamer 188 (P188) and assessed any influence on myocardial function, oedema and membrane proteins. vi The results from these three models suggest that the clinically accepted dogma of a causative link between myocardial oedema and dysfunction overstates the contribution of myocardial oedema to LCOS. We found that ischaemia/reperfusion was of primary importance in causing myocardial dysfunction. Myocardial oedema without ischaemia had a mild and reversible contribution to myocardial dysfunction, but this was minor in comparison to the gross dysfunction attributable to ischaemia. Isolated cardiomyocytes, with induced oedema, functioned well. Whilst ischaemic cardiomyocytes, with less swelling still had severe contractile dysfunction. Isolated hearts, perfused with an oedema inducing crystalloid perfusate developed myocardial oedema and had minimal reversible systolic and diastolic dysfunction. Isolated hearts which experienced global ischaemia had comparable degrees of myocardial oedema, and significantly greater degrees of myocardial dysfunction that increased in severity with increasing duration of ischaemia. In the lamb CPB model, only those lambs which underwent aortic cross clamping and had a period of ischaemia had poor myocardial function. These lambs also had swollen hearts, raised myocardial AQP1 mRNA and reduced membrane dysferlin protein expression. Membrane dystrophin protein expression was not altered, somewhat unexpectedly with CPB with or without ischaemia. Lambs placed on CPB without ischaemia had good myocardial function, minimal oedema and unchanged membrane protein expression during the survival period. In a blinded lamb CPB trial of P188 there were improved haemodynamics and indicies of myocardial function associated with its use. This was also associated with preservation of dysferlin expression and reduced membrane injury. In parallel isolated heart trials of this therapy, there was a reduction in myocardial oedema associated with its use in non-ischaemic experiments. There was also a suggestion of improved diastolic function in ischaemic experiments, but no change in myocardial water content. In conclusion, we have highlighted the primacy of ischaemia/reperfusion over oedema in contributing to LCOS. We have refuted the accepted dogma that myocardial oedema causes significant dysfunction in itself, with important oedema likely to result from ischaemia. We have shown that AQP1 may be involved in the pathogenesis of the capillary leak syndrome. Finally we have hinted at a role for prophylactic P188 in the vii setting of LCOS, possibly highlighting the role of membrane repair in recovery after surgery. Isolated heart trials of P188 further support a non-rheological mechanism of action and also lend support to the causal separation of myocardial oedema and dysfunction. The integral membrane protein dysferlin, rather than dystrophin, is relevant in the setting of LCOS in the current era