Neurturin (NRTN) is a neurotrophic factor required during development for normal cholinergic innervation of the heart, but whether NRTN continues to function in the adult heart is unknown. We have therefore evaluated NRTN expression in adult mouse heart and the association of NRTN receptors with intracardiac cholinergic neurons and nerve fibers. Mapping the regional distribution and density of cholinergic nerves in mouse heart was an integral part of this goal. Analysis of RNA from adult C57BL/6 mouse hearts demonstrated NRTN expression in atrial and ventricular tissue. Virtually all neurons in the cardiac parasympathetic ganglia exhibited the cholinergic phenotype, and over 90% of these cells contained both components of the NRTN receptor, Ret tyrosine kinase and GDNF family receptor α2 (GFRα2). Cholinergic nerve fibers, identified by labeling for the high affinity choline transporter, were abundant in the sinus and atrioventricular nodes, ventricular conducting system, interatrial septum, and much of the right atrium, but less abundant in the left atrium. The right ventricular myocardium contained a low density of cholinergic nerves, which were sparse in other regions of the working ventricular myocardium. Some cholinergic nerves were also associated with coronary vessels. GFRα2 was present in most cholinergic nerve fibers and in Schwann cells and their processes throughout the heart. Some cholinergic nerve fibers, such as those in the sinus node, also exhibited Ret immunoreactivity. These findings provide the first detailed mapping of cholinergic nerves in mouse heart and suggest that the neurotrophic influence of NRTN on cardiac cholinergic innervation continues in mature animals.

Objective: Previous studies have used acetylcholinesterase (AChE) histochemistry to identify cholinergic nerves in the heart, but this enzyme is not a selective marker for cholinergic neurons. This study maps cholinergic innervation of guinea pig heart using a new antibody to the human high-affinity choline transporter (CHT), which is present only in cholinergic nerves. Methods: Immunohistochemistry was used to localize CHTs in frozen and paraffin sections of heart and whole mount preparations of atrial ganglionated nerve plexus. AChE-positive nerve fibers were identified in sections from separate hearts for comparison. Results: Control experiments established that the antibody to human CHT selectively labeled cholinergic neurons in the guinea pig. CHT-immunoreactive nerve fibers and AChE-positive nerves were very abundant in the sinus and AV nodes, bundle of His, and bundle branches. Both markers also delineated a distinct nerve tract in the posterior wall of the right atrium. AChE-positive nerve fibers were more abundant than CHT-immunoreactive nerves in working atrial and ventricular myocardium. CHT-immunoreactive nerves were rarely observed in left ventricular free wall. Both markers were associated with numerous parasympathetic ganglia that were distributed along the posterior atrial walls and within the interatrial septum, including the region of the AV node. Conclusions: Comparison of labeling patterns for CHT and AChE suggests that AChE histochemistry overestimates the density of cholinergic innervation in the heart. The distribution of CHT-immunoreactive nerve fibers and parasympathetic ganglia in the guinea pig heart suggests that heart rate, conduction velocity, and automaticity are precisely regulated by cholinergic innervation. In contrast, the paucity of CHT-immunoreactive nerve fibers in left ventricular myocardium implies that vagal efferent input has little or no direct influence on ventricular contractile function in the guinea pig.

Neurodegenerative diseases are becoming increasingly prevalent due to the ageing population, and are among the major contributors to disability and disease worldwide. The identification of the gene defects responsible for many of these conditions has played a major role in our understanding of the pathogenic processes involved, and provided opportunity to develop targeted treatment strategies. Cholinergic neurotransmission supports a wide range of physiological and behavioural processes and its dysfunction of cholinergic signalling has been associated with a number of disorders, including myasthenias, cardiovascular disease(1), attention-deficit hyperactivity disorder (ADHD) (2), Alzheimer’s disease (ADi), schizophrenia, addiction(3), and depression(4). SLC5A7 encodes the Na+/Cl- dependent, high-affinity choline transporter (CHT) which represents the rate limiting step in acetylcholine (Ach) synthesis and is critical for normal cholinergic signalling. The work in this thesis details two new inherited disorders, caused by distinct pathogenic disease mechanisms, associated with novel SLC5A7 mutations. Chapter three documents the discovery of two autosomal-dominantly acting SLC5A7/CHT mutations associated with adult onset motor neurone disorders. Initially we identified a frameshift mutation that results in premature truncation of the transporter protein in a large Welsh kindred affected with distal hereditary motor neuropathy type VII (dHMN-VII), in which neurodegeneration and muscle paresis is largely restricted to the distal limb muscles and vocal cords. The mutation responsible results in the dominant-negative interference of the mutant molecule with function of the wild type choline transporter, resulting in significantly reduced (although not completely abolished) transporter activity. This finding is further evidenced by the discovery of a second dHMN family associated with a distinct frameshift SLC5A7 mutation indicative of a similar dominant-negative disease mechanism. Together these findings corroborate a dominant-negative disease mechanism arising from C-terminal truncating SLC5A7 mutations associated with dHMN, and provide further insight into the role of aberrant choline transporter function in neurological disease. Chapter four describes N-terminal missense mutations located in the transmembrane spanning region of SLC5A7/CHT, associated with a severe infantile neuromuscular disorder characterised by predominantly central hypotonia and developmental delay. The phenotypic effects of these mutations are likely to result from the near abolition of CHT-mediated choline transport in homozygous individuals, and are in keeping with those observed in CHT knock-out mouse models(5). The development of a mouse model of the human motor neurone disease arising from SLC5A7 frameshift mutations should allow for further investigation of the mechanism by which truncated CHT leads to the dHMN phenotype. Chapter 5 details treatment hypotheses for dHMN, as well as the generation of a patient-specific knock-in mouse model carrying an Slc5a7 mutation orthologous to that identified in dHMN-VII families in chapter 3, and results from preliminary neurological phenotyping of the mouse model. This model will be crucially important for the exploration of treatment options in dHMN-VII motor neurone disease as a prelude to clinical trials in humans.