The Chemical Neuroanatomy and Gene Expression Profile of the Human Subthalamic Nucleus in Normal, Parkinson’s and Huntington’s Disease

Abstract

The subthalamic nucleus (STN) is one of the most critical structures of the basal ganglia. In movement neurodegenerative disorders such as Parkinson’s (PD) and Huntington’s disease (HD), degeneration of upstream signalling basal ganglia structures results in pathophysiological activities in STN neurons. In particular, changes in plasticity of the robust inhibitory GABAergic connection between the globus pallidus externa (GPe) and the STN are known to highly affect neuronal signalling within the STN. These changes impact on the overall clinical movement manifestations of PD and HD. However, the anatomical, neurochemical and gene expression changes occurring within the STN as a result of the upstream disease pathophysiology are poorly understood in humans. These unanswered questions hence formed the basis of this study. This thesis first presents the neuroanatomical and neurochemical organisation of the STN in neurologically normal post-mortem human brains via immunohistochemistry. The expression of neurotransmitter receptors from the GABAergic, dopaminergic and glutamatergic signalling systems were also investigated. These results obtained from the normal brains hence formed an anatomical basis for the following disease specific comparative studies. In both PD and HD brains, changes in the expression of prominent GABAA inhibitory receptor subunits were investigated within the STN and compared to that of the normal human brains. The purpose of this was to investigate the effect cause by the altered GPe-STN connection in PD and HD, within the STN. The study then further investigated the expression of pathological protein inclusions within STN neurons for both disorders, the mutant Huntingtin (mHtt) protein in HD brains and the abnormal phosphorylated form of α-synuclein in PD brains. Finally, the study expanded to investigate gene expression changes within the human STN in PD, using the latest RNA sequencing and nanostring technologies. The results demonstrated that the normal human STN exhibit a heterogeneous distribution of neurons, with the dorsolateral sub-regions showing lower neuronal densities compared to ventromedial sub-regions. The human STN showed strong expression of different neuronal markers including the calcium binding proteins, calretinin (CR) and parvalbumin (PV), and nonphosphorylated neurofilaments (SMI32). The dorsolateral portions of the STN are highly populated with neurons expressing PV whilst the ventromedial portions showed high CR expression. This regional expression difference was not found for SMI32. Co-localisation studies showed that the majority of STN neurons express all 3 neurochemical markers. The expression of various neurotransmitter receptors from the GABAergic, dopaminergic and glutamatergic systems was then investigated. The human STN was found to show high expression of fast-acting inhibitory GABAA receptors, including the α1, β2/3, α3 and γ2 subunits. In terms of localisation, the α1 and β2/3 subunits showed a high degree of co-localisation on neuronal membranes of STN neurons. The α3 and γ2 subunits however, showed an intracellular expression profile within the STN perikarya. Expression of GABAB receptor subunit R1 and R2 were also found in STN neurons. Both GABAB subunits also showed intracellular expression. For the dopaminergic and glutamatergic receptors investigated, the dopamine D1, D2, D4 and D5 receptors, the ionotropic glutamate AMPA receptor GluA2 subunit and the metabotropic glutamate mGluR1/5 receptors were also found to be expressed at low levels on STN neurons. Since the STN receives massive inhibitory GABAergic afferents from the globus pallidus, and the GABAergic GPe-STN connection is highly affected in PD and HD, expression changes of inhibitory GABAA receptor subunits were investigated in both diseases. Overall four GABAA receptor subunits were investigated in PD and HD, the α1, β2/3, α3 and γ2 subunit. In HD, a small but significant 15% reduction of the α3 subunit was found in the ventromedial portion of the STN, compared with controls. No significant changes were detected for the other GABAA receptor subunits. In PD, a significant increase of α1 and β2/3 subunits (12.34% and 9.37%, respectively) was found in the dorsolateral portion of the STN, compared to controls. The study then further investigated the expression of PD and HD specific pathological protein inclusions within the human STN. In HD cases, prominent mHtt aggregates were detected within numerous STN neurons. The inclusions were mainly of the nucleic or cytoplasmic form. In PD cases, the STN showed no expression of pathological α-synuclein aggregates. However, numerous α-synuclein positive fibres were found traversing through the STN. These fibres were also detected in control cases which may reveal novel functions of the α-synuclein protein. The final chapter of this thesis investigated gene expression changes within the human STN in PD by screening the entire human genome in the RNA-seq platform. Overall, 95 differentially expressed genes were detected within the human STN in PD brains with 72 up-regulated and 23 down-regulated genes. The majority of up-regulated genes were found to be involved in neuroinflammatory, immune response and microvasculature homeostatic pathways. The downregulated genes were found to be involved in blood vessel microstructure, dopamine and lipid biosynthesis and structural properties of extracellular matrix proteins. The results hence suggested that in PD, the STN experiences considerable neuroinflammation and infiltration immune response vehicles through a dysfunctional blood brain barrier (BBB). The chronic neuroinflammation may therefore cause breakdown of extracellular brain parenchyma and phospholipid membranes. One PD case used in the study came from a patient who underwent bilateral pallidotomy surgeries to treat PD. Compared to other PD cases, the differential profile of this case are very similar to that of controls, which suggests that pathological gene expression changes may be altered by pallidal surgery. Twenty-six genes were validated from the sequencing experiment in the nanostring platform. Eighteen genes returned with significant gene expression changes similar to RNA-seq results. One of the genes validated by nanostring, the MAFF gene, showed a striking 7.33 fold increase in the PD group compared to control. The massive increase may hence convey neuroprotective mechanisms in STN neurons in PD. In conclusion, the research present in this thesis of the neuroanatomical and neurochemical organisation within the human STN will form an anatomical basis to support future studies on the role of the STN in the pathogenesis of HD and PD in the human brain. The gene candidates identified to have substantial changes within the human STN in PD would also aid future studies intending to discover potential new PD treatments and contribute to possible future therapeutic strategies.