The ultrastructural distribution of alpha-synuclein-like protein in normal mouse brain

Abstract

The synaptic protein alpha-synuclein is found throughout the brain, although its function remains ill-defined. Abnormal accumulations of alpha-synuclein have been recognised to be associated with a number of neurodegenerative diseases, including Alzheimer's disease and Parkinson's disease. Nevertheless, little is known about the precise localisation of this protein within the normal brain, information which might contribute to our understanding of its role in both health and disease. We raised an antibody which recognises both human and murine alpha-synuclein and this was used to study the distribution of the protein in the normal mouse brain. We used morphological characteristics to classify the immunopositive presynaptic elements and their targets. We conclude that the protein is present in synaptic boutons of axons with different neurochemical phenotypes but that it is not present in all synaptic terminals. Furthermore, the protein is present in the terminals of neurons such as the dopaminergic neurons of the substantia nigra and the glutamatergic neurons of the hippocampus, cell types which accumulate alpha-synuclein in disease. Nevertheless alpha-synuclein is also found in terminals of neurons which have not been reported to accumulate the protein in neurodegenerative disorders.

Introduction

The synuclein family includes at least three proteins, each having a characteristic distribution in the nervous system but of unknown function [14], [16]. Synucleins are highly conserved, being present in a range of species [32]. In rat brain, the three forms of synuclein mRNA, synucleins 1, 2 and 3, are generated by differential splicing of the same mRNA transcript. Synuclein 1 corresponds to human α-synuclein [25], a 140 aminoacid, 14 KDa protein which is identical to the non-amyloid component of plaque precursor protein. Found in presynaptic terminals [10], where it co-localizes with synaptophysin [30], α-synuclein plays a role in synaptic vesicle transport [16] within fully mature synapses [30].

The distribution of α-synuclein in humans and rodents appears to be quite similar. In humans, the highest concentrations of α-synuclein mRNA are in the substantia nigra, especially in dopaminergic neurons of the ventral tier [39]. High concentrations are also reported in the dentate granule cells and CA3 regions of the hippocampal formation and in the deep layers of the cortex. Labelling for mRNA is weak in the striatal complex and absent from the globus pallidus. In rodents, high levels of expression of α-synuclein mRNA and protein are reported in the cerebral cortex [1], [18], [32], the substantia nigra [1], [23], [25], and in limbic areas such as the hippocampus and the amygdala [1], [17], [18], [25], [32]. Within these regions in normal tissue, the protein is found principally in small punctate structures although it has also been reported in a perinuclear zone in some neurons [26]. Ultrastructurally, α-synuclein is reported to be present in synaptic boutons forming symmetrical or asymmetrical specializations [30] but it is not clear if these are linked to particular neurotransmitter content.

Recently α-synuclein has been found associated with hallmark profiles in neurodegenerative diseases, including the amyloid plaques of Alzheimer's Disease [18], [47], and Lewy Bodies in Parkinson's Disease [42], [51]. In normal brains the protein exists as a relatively unfolded molecule in presynaptic terminals [48], but under certain pathological conditions it forms fibrils, accumulates and is deposited as characteristic inclusion bodies in the somata of affected neurons [15]. Such inclusions, linked in some cases of Parkinson's Disease to mutations in α-synuclein [36], are also present in diffuse Lewy Body disease and multiple system atrophy [10], [15]. Inclusions are found in particular in the pigmented nuclei of the brainstem, notably the substantia nigra [9], [42], but also in the hippocampal formation, the entorhinal cortex, amygdala and association cortices [7]. What controls the formation of these presumably pathological structures is unknown. Nor is it known whether specific chemical classes of neurons are especially vulnerable.

The appearance of α-synuclein immunoreactive Lewy bodies in Parkinson's Disease may reflect a condition where normal metabolism of α-synuclein and its transport to the terminals are interrupted [15]. Since protein aggregates and Lewy bodies form in dopaminergic neurons [16], [38], [46] it is likely that, under normal conditions, the terminals of nigral dopamine neurons contain α-synuclein [50]. However, if these immunopositive inclusions are also found in cells in cortical regions [4], [5], [42], it might be presumed that α-synuclein is also present in glutamatergic terminals. The question then arises as to whether all terminals that contain α-synuclein are of one or more specific phenotypes. The ultrastructural characteristics of both dopaminergic and glutamatergic synaptic terminals have been well established and these can be distinguished in the electron microscope. Almost all of the regions that display α-synucleinopathies project to the striatum (either dorsal or ventral) making this an ideal area to investigate in the normal animal. We therefore studied the distribution of α-synuclein-immunoreactive profiles at electron microscopic level in normal mice (a) to determine if all synaptic boutons are α synuclein-immunopositive, (b) to examine and compare the morphology of immunoreactive boutons in cortical and subcortical regions and (c) to compare the nature of the synaptic specialisations and morphology of immunoreactive boutons within the striatal complex and substantia nigra.