Characterisation of the role of canonical BMP-Smad 1/5/8 signalling in the development of ventral midbrain dopaminergic neurons

Ventral midbrain (VM) dopaminergic (DA) neurons, which project to the dorsal striatum via the nigrostriatal pathway, are progressively degenerated in Parkinson’s disease (PD). The identification of the instructive factors that regulate midbrain DA neuron development, and the subsequent elucidation of the molecular bases of their effects, is vital. Such an understanding would facilitate the generation of transplantable DA neurons from stem cells and the identification of developmentally-relevant neurotrophic factors, the two most promising therapeutic approaches for PD. Two related members of the bone morphogenetic protein (BMP) family, BMP2 and growth/differentiation factor (GDF) 5, which signal via a canonical Smad 1/5/8 signalling pathway, have been shown to have neurotrophic effects on midbrain DA neurons both in vitro and in vivo, and may function to regulate VM DA neuronal development. However, the molecular (signalling pathway(s)) and cellular (direct neuronal or indirect via glial cells) mechanisms of their effects remain to be elucidated. The present thesis hypothesised that canonical Smad signalling mediates the direct effects of BMP2 and GDF5 on the development of VM DA neurons. By activating, modulating and/or inhibiting various components of the BMP-Smad signalling pathway, this research demonstrated that GDF5- and BMP2-induced neurite outgrowth from midbrain DA neurons is dependent on BMP type I receptor activation of the Smad signalling pathway. The role of glial cell-line derived neurotrophic factor (GDNF)-signalling, dynamin-dependent endocytosis and Smad interacting protein-1 (Sip1) regulation, in the neurotrophic effects of BMP2 and GDF5 were determined. Finally, the in vitro development of VM neural stem cells (NSCs) was characterised, and the ability of GDF5 and BMP2 to induce these VM NSCs towards DA neuronal differentiation was investigated. Taken together, these experiments identify GDF5 and BMP2 as novel regulators of midbrain DA neuronal induction and differentiation, and demonstrate that their effects on DA neurons are mediated by canonical BMPR-Smad signalling

Endocytosis contributes to BMP2-induced Smad signalling and neuronal growth

Bone morphogenetic protein 2 (BMP2) is a neurotrophic factor which induces the growth of midbrain dopaminergic (DA) neurons in vitro and in vivo, and its neurotrophic effects have been shown to be dependent on activation of BMP receptors (BMPRs) and Smad 1/5/8 signalling. However, the precise intracellular cascades that regulate BMP2-BMPR-Smad-signalling-induced neurite growth remain unknown. Endocytosis has been shown to regulate Smad 1/5/8 signalling and differentiation induced by BMPs. However, these studies were carried out in non-neural cells. Indeed, there are scant reports regarding the role of endocytosis in BMP-Smad signalling in neurons. To address this, and to further characterise the mechanisms regulating the neurotrophic effects of BMP2, the present study examined the role of dynamin-dependent endocytosis in BMP2-induced Smad signalling and neurite growth in the SH-SY5Y neuronal cell line. The activation, temporal kinetics and magnitude of Smad 1/5/8 signalling induced by BMP2 were significantly attenuated by dynasore-mediated inhibition of endocytosis in SH-SY5Y cells. Furthermore, BMP2-induced increases in neurite length and neurite branching in SH-SY5Y cells were significantly reduced following inhibition of dynamin-dependent endocytosis using dynasore. This study demonstrates that BMP2-induced Smad signalling and neurite growth is regulated by dynamin-dependent endocytosis in a model of human midbrain dopaminergic neurons

Inhibition of miR-181a promotes midbrain neuronal growth through a Smad1/5-dependent mechanism: implications for Parkinson’s disease

Parkinson’s disease (PD) is the second most common neurodegenerative disease, and is characterized by the progressive degeneration of nigrostriatal dopaminergic (DA) neurons. Current PD treatments are symptomatic, wear off over time and do not protect against DA neuronal loss. Finding a way to re-grow midbrain DA (mDA) neurons is a promising disease-modifying therapeutic strategy for PD. However, reliable biomarkers are required to allow such growth-promoting approaches to be applied early in the disease progression. miR-181a has been shown to be dysregulated in PD patients, and has been identified as a potential biomarker for PD. Despite studies demonstrating the enrichment of miR-181a in the brain, specifically in neurites of postmitotic neurons, the role of miR-181a in mDA neurons remains unknown. Herein, we used cell culture models of human mDA neurons to investigate a potential role for miR-181a in mDA neurons. We used a bioninformatics analysis to identify that miR-181a targets components of the bone morphogenetic protein (BMP) signalling pathway, including the transcription factors Smad1 and Smad5, which we find are expressed by rat mDA neurons and are required for BMP-induced neurite growth. We also found that inhibition of neuronal miR-181a, resulted in increased Smad signalling, and induced neurite growth in SH-SY5Y cells. Finally, using embryonic rat cultures, we demonstrated that miR-181a inhibition induces ventral midbrain (VM) and cortical neuronal growth. These data describe a new role for miR-181a in mDA neurons, and provide proof of principle that miR-181a dysresgulation in PD may alter the activation state of signalling pathways important for neuronal growth in neurons affected in PD

Targeting bone morphogenetic protein signalling in midbrain dopaminergic neurons as a therapeutic approach in Parkinson's disease.

Parkinson's disease (PD) is the second most common neurodegenerative disease, characterized by the degeneration of midbrain dopaminergic (mDA) neurons and their axons, and aggregation of α-synuclein, which leads to motor and late-stage cognitive impairments. As the motor symptoms of PD are caused by the degeneration of a specific population of mDA neurons, PD lends itself to neurotrophic factor therapy. The goal of this therapy is to apply a neurotrophic factor that can slow down, halt or even reverse the progressive degeneration of mDA neurons. While the best known neurotrophic factors are members of the glial cell line-derived neurotrophic factor (GDNF) family, their lack of clinical efficacy to date means that it is important to continue to study other neurotrophic factors. Bone morphogenetic proteins (BMPs) are naturally secreted proteins that play critical roles during nervous system development and in the adult brain. In this review, we provide an overview of the BMP ligands, BMP receptors (BMPRs) and their intracellular signalling effectors, the Smad proteins. We review the available evidence that BMP–Smad signalling pathways play an endogenous role in mDA neuronal survival in vivo, before outlining how exogenous application of BMPs exerts potent effects on mDA neuron survival and axon growth in vitro and in vivo. We discuss the molecular mechanisms that mediate these effects, before highlighting the potential of targeting the downstream effectors of BMP–Smad signalling as a novel neuroprotective approach to slow or stop the degeneration of mDA neurons in PD

Effects of intracerebral neurotrophic factor application on motor symptoms in Parkinson's disease: a systematic review and meta-analysis

Introduction: Neurotrophic factors (NTFs) have been evaluated for neuroprotective effects in Parkinson's disease (PD). However, clinical trials examining the efficacy of intracerebral administration of NTFs on motor symptoms in PD have produced mixed results, and are thus inconclusive. The objective of this systematic review and meta-analysis was to determine the effects of intracerebral NTF application on motor symptoms in people with PD. Methods: We searched PubMed, MEDLINE, EMBASE, and Cochrane from inception through to March 31 2016 for open-label trials and randomized controlled trials (RCTs) which intracerebrally administered NTFs to PD patients, and which performed motor examination of Unified Parkinson's Disease Rating Scale. Results: Eight studies with a total of 223 participants were included. Fixed effects analysis revealed that NTF treatment did not significantly reduce motor symptoms in PD patients compared to placebo controls (P = 0.98). Combining open-label and RCT data, both treatment with NTFs (P < 0.001) and treatment with placebo (P < 0.05) significantly improved motor function in PD patients when compared to predicted symptoms in untreated PD controls. Finally, random effects analysis revealed that NTF-treated PD patients were not significantly likely to improve following intracerebral NTF administration (P = 0.25). Conclusion: In conclusion, intracerebral NTF administration does not improve motor symptoms in PD patients, when compared to placebo-treated controls. These findings may guide therapeutic decisions and inform future research on NTFs and their application in PD

A small molecule activator of p300/CBP histone acetyltransferase promotes survival and neurite growth in a cellular model of Parkinson’s disease

Parkinson’s disease (PD) is a progressive neurodegenerative disease characterised by motor and non-motor symptoms, resulting from the degeneration of nigrostriatal dopaminergic neurons and peripheral autonomic neurons. Given the limited success of neurotrophic factors in clinical trials, there is a need to identify new small molecule drugs and drug targets to develop novel therapeutic strategies to protect all neurons that degenerate in PD. Epigenetic dysregulation has been implicated in neurodegenerative disorders, while targeting histone acetylation is a promising therapeutic avenue for PD. We and others have demonstrated that histone deacetylase inhibitors have neurotrophic effects in experimental models of PD. Activators of histone acetyltransferases (HAT) provide an alternative approach for the selective activation of gene expression, however little is known about the potential of HAT activators as drug therapies for PD. To explore this potential, the present study investigated the neurotrophic effects of CTPB (N-(4-chloro-3-trifluoromethyl-phenyl)-2-ethoxy-6-pentadecyl-benzamide), which is a potent small molecule activator of the histone acetyltransferase p300/CBP, in the SH-SY5Y neuronal cell line. We report that CTPB promoted the survival and neurite growth of the SH-SY5Y cells, and also protected these cells from cell death induced by the neurotoxin 6-hydroxydopamine. This study is the first to investigate the phenotypic effects of the HAT activator CTPB, and to demonstrate that p300/CBP HAT activation has neurotrophic effects in a cellular model of PD

Romidepsin induces caspase-dependent cell death in human neuroblastoma cells

Neuroblastoma is the most common extracranial pediatric solid tumor, arising from the embryonic sympathoadrenal lineage of the neural crest, and is responsible for 15% of childhood cancer deaths. Although survival rates are good for some patients, those children diagnosed with high-risk neuroblastoma have survival rates as low as 35%. Thus, neuroblastoma remains a significant clinical challenge and the development of novel therapeutic strategies is essential. Given that there is widespread epigenetic dysregulation in neuroblastoma, epigenetic pharmacotherapy holds promise as a therapeutic approach. In recent years, histone deacetylase (HDAC) inhibitors, which cause selective activation of gene expression, have been shown to be potent chemotherapeutics for the treatment of a wide range of cancers. Here we examined the ability of the FDA-approved drug Romidepsin, a selective HDAC1/2 inhibitor, to act as a cytotoxic agent in neuroblastoma cells. Treatment with Romidepsin at concentrations in the low nanomolar range induced neuroblastoma cell death through caspase-dependent apoptosis. Romidepsin significantly increased histone acetylation, and significantly enhanced the cytotoxic effects of the cytotoxic agent 6-hydroxydopamine, which has been shown to induce cell death in neuroblastoma cells through increasing reactive oxygen species. Romidepsin was also more potent in MYCN-amplified neuroblastoma cells, which is an important prognostic marker of poor survival. This study has thus demonstrated that the FDA-approved chemotherapeutic drug Romidepsin has a potent caspase-dependent cytotoxic effect on neuroblastoma cells, whose effects enhance cell death induced by other cytotoxins, and suggests that Romidepsin may be a promising chemotherapeutic candidate for the treatment of neuroblastoma

Zeb2 is a negative regulator of midbrain dopaminergic axon growth and target innervation

Neural connectivity requires neuronal differentiation, axon growth, and precise target innervation. Midbrain dopaminergic neurons project via the nigrostriatal pathway to the striatum to regulate voluntary movement. While the specification and differentiation of these neurons have been extensively studied, the molecular mechanisms that regulate midbrain dopaminergic axon growth and target innervation are less clear. Here we show that the transcription factor Zeb2 cell-autonomously represses Smad signalling to limit midbrain dopaminergic axon growth and target innervation. Zeb2 levels are downregulated in the embryonic rodent midbrain during the period of dopaminergic axon growth, when BMP pathway components are upregulated. Experimental knockdown of Zeb2 leads to an increase in BMP-Smad-dependent axon growth. Consequently there is dopaminergic hyperinnervation of the striatum, without an increase in the numbers of midbrain dopaminergic neurons, in conditional Zeb2 (Nestin-Cre based) knockout mice. Therefore, these findings reveal a new mechanism for the regulation of midbrain dopaminergic axon growth during central nervous system development

Association of distinct type 1 bone morphogenetic protein receptors with different molecular pathways and survival outcomes in neuroblastoma

Neuroblastoma (NB) is a paediatric cancer that arises in the sympathetic nervous system. Patients with stage 4 tumours have poor outcomes and 20% of high-risk cases have MYCN amplification. The bone morphogenetic proteins (BMPs) play roles in sympathetic neuritogenesis, by signalling through bone morphogenetic protein receptor (BMPR)2 and either BMPR1A or BMPR1B. Alterations in BMPR2 expression have been reported in NB; it is unknown if the expression of BMPR1A or BMPR1B is altered. We report lower BMPR2 and BMPR1B, and higher BMPR1A, expression in stage 4 and in MYCN-amplified NB. Kaplan–Meier plots showed that high BMPR2 or BMPR1B expression was linked to better survival, while high BMPR1A was linked to worse survival. Gene ontology enrichment and pathway analyses revealed that BMPR2 and BMPR1B co-expressed genes were enriched in those associated with NB differentiation. BMPR1A co-expressed genes were enriched in those associated with cell proliferation. Moreover, the correlation between BMPR2 and BMPR1A was strengthened, while the correlation between BMPR2 and BMPR1B was lost, in MYCN-amplified NB. This suggested that differentiation should decrease BMPR1A and increaseBMPR1Bexpression.Inagreement,nervegrowthfactortreatmentofculturedsympatheticneuronsdecreasedBmpr1aexpressionandincreasedBmpr1bexpression.Overexpression of dominant negative BMPR1B, treatment with a BMPR1B inhibitor and treatment with GDF5, which signals via BMPR1B, showed that BMPR1B signalling is required for optimal neuritogenesis in NB cells, suggesting that loss of BMPR1B may alter neuritogenesis. The present study shows that expression of distinct BMPRs is associated with different survival outcomes in NB

Exposure to hypertensive disorders of pregnancy increases the risk of autism spectrum disorder in affected offspring

There is growing awareness that prenatal adversity may increase the risk of autism spectrum disorder (ASD). Here, we examined the association between hypertensive disorders of pregnancy (HDP) and ASD risk at 7 years of age using the Millennium Cohort Study (MCS), a representative cohort of 13,192 children born in the UK from 2000 to 2001. We also sought to examine cytokine expression in the serum of women with pre-eclampsia, which is the most common HDP, and whether exposure of foetal neurons to this serum could change patterns of neuronal growth. HDP were reported by mothers 9 months post-delivery. ASD was parent reported at age seven, based on a doctor or health care professional’s diagnosis. Weighted logistic regression was used for data analysis, adjusting for several potential confounders including maternal alcohol consumption, education, depression, age, and poverty status. Sensitivity analyses were performed excluding pre-term births, small for gestational age (SGA), and pre-pregnancy hypertension and depression. There was a significant association between HDP and a twofold increased risk of ASD (AOR = 2.10 [95% CI 1.20–3.70]). Excluding preterm births, SGA births, and offspring of women who had pre-pregnancy hypertension or over the age of 40 did not change the results materially. At the cellular level, exposure of foetal cortical neurons to 3% serum isolated from women with an established HDP increased neuronal growth and branching in vitro. These findings indicate that HDP exposure may increase the risk of ASD in the offspring