The Distribution of DARPP-32 in the Normal, Parkinson’s and Huntington’s Disease Human Striatum

Abstract

In the human brain, the striatum is a crucial part of the basal ganglia. The basal ganglia are a collection of nuclei that receive information from the cortex, and through a series of inhibitory and excitatory pathways modulates information that feeds into the cortex. These processes result in efficient brain processing, leading to optimal cognitive functioning, moderation of behaviour and personality, and control of movement. The striatum consists of the caudate nucleus, the putamen and the ventral striatum. It receives major projections from the wider cerebral cortex, the substantia nigra and the thalamus. These projections contain a variety of neural information and are integrated together in general functional territories within the striatum. These regions include associative, sensorimotor and limbic territories that are linked to the caudate nucleus, putamen, and ventral striatum in complex ways. The striatum is also subdivided into two neurochemical compartments termed the striosomes and matrix. The striosomes form a three-dimensional interconnected network within the wider striatum and are linked with the processing of especially limbic information whilst the surrounding matrix region is very much involved in sensorimotor processing. The majority of cells within the striatum are medium spiny neurons (MSNs). The traditional marker for these cells in post-mortem human striatum is calbindin, a calcium binding protein. The calbindin positive cells within the striatum are mainly concentrated within the matrix but are relatively sparse in the striosomes. In the mouse, the marker for MSNs is DARPP-32 (dopamine and cAMP-regulated neuronal phosphoprotein, molecular weight 32 kilodaltons), rather than calbindin. DARPP-32 is reported to identify over 90% of MSNs within the mouse brain. However, while the use of DARPP-32 in post-mortem human brain tissue has been limited, a direct comparison of calbindin and DARPP-32 in the human striatum has not been undertaken. This thesis begins by validating the DARPP-32 antibodies of choice and comparing these findings to previous validated DARPP-32 antibodies in the field. This was done with western blotting and immunohistochemical techniques. Immunohistochemical techniques were carried out on human and rat tissue. Interestingly, while DARPP-32 showed a mostly homogenous distribution throughout the rat striatum, high levels of DARPP-32 immunoreactivity were seen amongst the neuropil and cell soma of the striosomes within the normal human striatum. Some DARPP-32 positive cells were also present in the matrix, but not at the same density as in the striosomes. This is different to the representation of calbindin within the normal human striatum. Furthermore, double-labelling fluorescent studies show that there are three MSN populations within the normal striatum: DARPP-32 only positive, calbindin only positive, and DARPP-32 + calbindin colocalised cells. Examination of the GABAA β2,3 subunit profiles on these three cell types potentially indicate that more GABAA subunits are present on cells that show more calbindin immunoreactivity. Together, these are highly novel findings that provide a new understanding of human basal ganglia circuitry. While Parkinson's disease (PD) is traditionally characterised by the degeneration of the substantia nigra (SN), previous pathological reports show that a loss of dopamine is seen in the striatum, particularly in the putamen. Results in this thesis show a significant loss of DARPP-32 immunoreactivity in the putamen and ventral striatum of post-mortem PD human striatum. In Huntington's disease (HD), the MSNs of the striatum are understood to be very susceptible to neurodegeneration. HD cases with motor symptoms showed a decrease in DARPP-32 immunoreactivity in the putamen, while a striosome loss of DARPP-32 was seen in prominently mood symptomatic HD cases. The preferential striosome loss in mood cases reinforces previous findings from our laboratory. In addition, a decrease of the GABAA β2,3 subunits on all three MSN types was observed, however the GABAA β2,3 subunit profiles were still present on the three different MSN types. This thesis on DARPP-32 broadens global understanding of the neurochemistry of the normal human basal ganglia and provides further characterisation of Parkinson's disease and Huntington's disease.