The role of the dopaminergic neurotrophin growth/differentiation factor-5 in the developing rat ventral midbrain

Growth differentiation factor-5 (GDF-5) is a member of the transforming growth factor-β superfamily, a family of proteins that play diverse roles in many aspects of cell growth, proliferation and differentiation. GDF-5 has also been shown to be a trophic factor for embryonic midbrain dopaminergic neurons in vitro (Krieglstein et al. 1995) and after transplantation to adult rats in vivo (Sullivan et al. 1998). GDF-5 has also been shown to have neuroprotective and neurorestorative effects on adult dopaminergic neurons in the substantia nigra in animal models of Parkinson’s disease (Sullivan et al. 1997, 1999; Hurley et al. 2004). This experimental evidence has lead to GDF-5 being proposed as a neurotrophic factor with potential for use in the treatment of Parkinson’s disease. However, it is not know if GDF-5 is expressed in the brain and whether it plays a role in dopaminergic neuron development. The experiments presented here aim to address these questions. To that end this thesis is divided into five separate studies each addressing a particular question associated with GDF-5 and its expression patterns and roles during the development of the rat midbrain. Expression of the GDF-5 in the developing rat ventral mesencephalon (VM) was found to begin at E12 and peak on E14, the day that dopaminergic neurons undergo terminal differentiation. In the adult rat, GDF-5 was found to be restricted to heart and brain, being expressed in many areas of the brain, including striatum and midbrain. This indicated a role for GDF-5 in the development and maintenance of dopaminergic neurons. The appropriate receptors for GDF-5 (BMPR-II and BMPR-Ib) were found to be expressed at high levels in the rat VM at E14 and BMPR-II expression was demonstrated on dopaminergic neurons in the E13 mouse VM. GDF-5 resulted in a three-fold increase in the numbers of dopaminergic neurons in cultures of E14 rat VM, without affecting the numbers of neurones or total cells. GDF-5 was found to increase the proportion of neurons that were dopaminergic. The numbers of Nurr1-positive cells were not affected by GDF-5 treatment, but GDF-5 did increase the numbers of Nurr1- positive cells that expressed tyrosine hydroxylase (TH). Taken together this data indicated that GDF-5 increases the conversion of Nurr1-positive, TH-negative cells to Nurr1-positive, TH-positive cells. In GDF-5 treated cultures, total neurite length, neurite arborisation and somal area of dopaminergic were all significantly increased compared to control cultures. Thus this study showed that GDF-5 increased the numbers and morphological differentiation of VM dopaminergic neurones in vitro. In order to examine if GDF-5 could induce a dopaminergic phenotype in neural progenitor cells, neurosphere cultures prepared from embryonic rat VM were established. The effect of the gestational age of the donor VM on the proportion of cell types generated from neurospheres from E12, E13 and E14 VM was examined. Dopaminergic neurons could only be generated from neurospheres which were prepared from E12 VM. Thus in subsequent studies the effect of GDF-5 on dopaminergic induction was examined in progentior cell cultures prepared from the E12 rat VM. In primary cultures of E12 rat VM, GDF-5 increased the numbers of TH-positive cells without affecting the proliferation or survival of these cells. In cultures of expanded neural progenitor cells from the E12 rat VM, GDF-5 increased the expression of Nurr1 and TH, an action that was dependent on signalling through the BMPR-Ib receptor. Taken together, these experiments provide evidence that GDF-5 is expressed in the developing rat VM, is involved in both the induction of a dopaminergic phenotype in cells of the VM and in the subsequent morphological development of these dopaminergic neurons

The gut microbiota as a contributing factor to antipsychotic-induced weight gain and metabolic dysfunction

Schizophrenia represents one of the world’s most devastating illnesses due to its often lifelong course and debilitating nature. The treatment of schizophrenia has vastly improved over recent decades with the discovery of several antipsychotic compounds; however these drugs are not without adverse effects that must be addressed to maximize their therapeutic value. Newer, atypical, antipsychotics are associated with a compilation of serious metabolic side effects including weight gain, insulin resistance, fat deposition, glucose dysregulation and ensuing co-morbidities such as type II diabetes mellitus. The mechanisms underlying these side effects remain to be fully elucidated and adequate interventions are lacking. Further understanding of the factors that contribute these side effects is therefore required in order to develop effective adjunctive therapies and to potentially design antipsychotic drugs in the future with reduced impact on the metabolic health of patients. We investigated if the gut microbiota represented a novel mechanism contributing to the metabolic dysfunction associated with atypical antipsychotics. The gut microbiota comprises the bacteria that exist symbiotically within the gastrointestinal tract, and has been shown in recent years to be involved in several aspects of energy balance and metabolism. We have demonstrated that administration of certain antipsychotics in the rat results in an altered microbiota profile and, moreover, that the microbiota is required for the full scale of metabolic dysfunction to occur. We have further shown that specific antibiotics can attenuate certain aspects of olanzapine and risperidone–induced metabolic dysfunction, in particular fat deposition and adipose tissue inflammation. Mechanisms underlying this novel link appear to involve energy utilization via expression of lipogenic genes as well as reduced inflammatory tone. Taken together, these data indicate that the gut microbiota is an important factor involved in the myriad of metabolic complications associated with antipsychotic therapy. Furthermore, these data support the future investigation of microbial-based therapeutics for not only antipsychotic-induced weight gain but also for tackling the global obesity epidemic.

The role of glucocorticoid-induced tumour necrosis factor receptor in developing mouse sympathetic neurons

Hereditary sensory autonomic neuropathy IV (HSAN IV) is an autosomal recessive disorder characterised by inability to feel pain and anhidrosis and is a consequence of defective NGF/TrkA signalling and growth of sensory and sympathetic neurons. Glucocortiocoid-induced tumour necrosis factors receptor (GITR), a transmembrane protein, activated by its specific ligand, GITRL, is well known for its role in the regulation of innate and acquired immune system responses. Recently, GITR was found to be required for NGF-dependant and extracellular signal-related kinase 1/2 (ERK1/2)-induced neurite growth and target innervation in the developing sympathetic nervous system (SNS). Given this novel role of GITR, it is possible that strategies targeting GITR have potential therapeutic benefit in promoting neurite growth in autonomic neuropathies such as HSAN IV. Using P1 mouse SCG neurons as a model, in addition to various SCG cell treatments, knock down models and transfection methods, we investigated whether GITR increases the sensitivity of sympathetic neurons to NGF; the region of GITR required for the enhancement of NGF-promoted growth, the signalling pathways downstream of GITR and how extensively GITR is involved in regulating peripheral innervation of the SNS. Results indicate that the region responsible for the growth promoting effects of GITR lies in its juxtamembrane intracellular region (here termed the growth promoting domain (GPD)) of GITR. The GPD of GITR activates ERK1/2 and inhibits nuclear factor kappa B (NF-κB) in an inverse fashion to provide an optimal cellular growth environment for P1 SCG neurons. While deleting the GPD of GITR had no effect on TrkA expression, constitutive phosphorylation of specific sites in the GPD reduced TrkA expression indicating a possible role for GITR in increasing the sensitivity of SCG neurons to NGF by the regulation of these sites, TrkA expression and subsequent NGF/TrkA binding. GITR appears to be heterogeneously required for NGF-promoted target innervation of SCG neurons in some organs, implying additional factors are involved in extensive NGF-target innervation of the SNS. In conclusion, this study answers basic biological questions regarding the molecular mechanism behind the role of GITR in the development of the SNS, and provides a basis for future research if GITR modulation is to be developed as a strategy for promoting axonal growth.

Preclinical atherosclerosis in rheumatoid arthritis

Introduction: There is accumulating evidence of an increased risk of cardiovascular morbidity and mortality in rheumatoid arthritis patients. A combination of both traditional cardiovascular risks and rheumatoid specific factors appear to be responsible for driving this phenomenon. Rheumatoid arthritis has been an orphan of cardiologists in the past and rheumatologists themselves are not good at CVD screening. Identifying the extent of preclinical atherosclerosis in RA patients will help us to appreciate the magnitude of this serious problem in an Irish population. Methods: We undertook a cross-sectional study of 63 RA patients and 48 OA controls and compared the 2 groups with respect to 1) traditional CV risks factors, 2) serum biomarkers of inflammation, including CRP, TNFα, IL6 and PAI-1, 3) carotid intima-media thickness (cIMT), carotid plaque and ankle-brachial index (ABI) as markers of pre-clinical atherosclerosis, 4) biochemical and ultrasonic measures of endothelial dysfunction and 5) serum and echocardiographic measures of diastolic dysfunction. Within the RA group, we also investigated for associations between markers of inflammation, subclinical atherosclerosis and diastolic dysfunction. Results: Prevalence of traditional CV risks was similar in the RA and OA groups. A number of biomarkers of inflammation were significantly higher in the RA group: CRP, fibrinogen, IL- 2, -4, -6, TNFα. PAI-1, a marker of thrombosis, correlated with disease activity and subclinical atherosclerosis in RA patients. With regard to subclinical atherosclerosis measures, RA patients had a significantly lower ABI than OA patients. Carotid plaque and cIMT readings were similar in RA and OA patients. Assessment of endothelial function revealed that RA patients had significantly higher concentrations of adhesion molecules, in particular sero-positive RA patients and RA smokers. Adhesion molecule concentrations were associated with markers of diastolic dysfunction in RA. Urine PCR, another marker of endothelial dysfunction also correlated with diastolic dysfunction in RA. Assessment of endothelial function with flow mediated dilatation (FMD) found no difference between the RA and OA groups. Disease activity scores in RA patients were associated with endothelial dysfunction, as assessed by FMD. Conclusions: We did not find significant differences in measures of subclinical atherosclerosis, flow mediated dilatation or diastolic function between RA and OA patients. This is most likely in part due to the fact that there is increasing evidence that OA has an inflammatory component to its pathogenesis and is associated with metabolic syndrome and increased CV risk. We reported a significant association between urinary PCR and measures of diastolic dysfunction. Urinary PCR may be a useful screening tool for diastolic dysfunction in RA. The association between RA disease activity and measures of vascular function supports the theory that the excess cardiovascular burden in RA is linked to uncontrolled inflammation.