Distribution and immunohistochemical characterization of torsinA immunoreactivity in rat brain

doi:10.1016/S0006-8993(01)02302-2

Brain Research

Volume 900, Issue 2, 11 May 2001, Pages 348-354

https://doi.org/10.1016/S0006-8993(01)02302-2 Get rights and content

Abstract

A mutation of the DYT1 gene on chromosome 9q34 has recently been identified as the cause of one form of autosomal-dominantly inherited dystonia. TorsinA, the protein product of this gene, has homology with the family of heat shock proteins, and is found in many peripheral tissues and brain regions. We used a polyclonal antibody to torsinA, developed in our laboratory, to systematically examine the regional distribution of torsinA in rat brain. We find that neurons in all examined structures are immunoreactive for this protein. There is intense immunoreactivity in most neuronal nuclei, with slightly less labeling of cytoplasm and proximal processes. Terminals also are labeled, especially in striatum, neocortex and hippocampus. Double-labeling fluorescence immunohistochemistry using antibodies to neurotransmitters and other neurochemical markers demonstrated that the majority of neurons of all studied neurochemical types are immunoreactive for torsinA. Our findings indicate that torsinA is widely distributed in the central nervous system implicating additional, localized factors, perhaps within the basal ganglia, in the development of dystonia. Many other proteins have a similar widespread distribution, including some which have been implicated in other movement disorders and neurodegenerative processes, such as parkin, α-synuclein, ubiquitin and huntingtin. The distribution of torsinA in rat brain as demonstrated by immunohistochemistry contrasts with the results of in situ hybridization studies of torsinA mRNA in human postmortem brain in which a more limited distribution was found.

Introduction

Dystonia is a neurological disorder characterized by involuntary excessive muscle contractions causing twisting movements and abnormal postures, which can result in significant disability. Recently a mutation in the DYT1 gene on chromosome 9q34 was linked to one of the most severe types of inherited dystonia (dystonia musculorum deformans; Oppenheim’s dystonia) [1], [2]. The DYT1 gene encodes for a novel 332-amino-acid protein termed torsinA [3], whose function is not yet known, but may be suggested by its homology to the heat shock family of proteins, which may have a neuroprotective function [3], [4], [5]. We have recently shown that torsinA shares an ATP-binding property with these proteins and that it localizes to the neuronal nucleus and cytoplasm [6].

Using in situ hybridization techniques Augood and colleagues [7], [8] demonstrated that mRNA for torsinA is expressed in a number of regions of human brain. Particularly high levels were seen in hippocampus, cerebellar dentate nucleus, Purkinje cells, pontine nuclei, pedunculopontine nucleus, oculomotor nucleus, substantia nigra pars compacta and throughout the thalamus. Consistently lower levels were found in globus pallidus, subthalamic nucleus and striatum.

Using a polyclonal antibody to human torsinA we have demonstrated that torsinA is found in neurons in many regions of rat and human brain, including neocortex, cerebellum, hippocampus and dopaminergic neurons of the substantia nigra pars compacta, and that it is also present in several peripheral organs [6].

In this communication we provide further details of the distribution of torsinA immunoreactivity in rat brain and describe immunohistochemical characterization of various types of torsinA-containing neurons using double-labeling immunohistochemistry.

Section snippets

Western blotting

Immunoblotting was carried out using rat brain total homogenate. Protein concentrations were determined using a protein assay kit (Sigma, St. Louis, MO, USA). Proteins were resolved on SDS–PAGE and electroblotted to nitrocellulose membrane using 50 mM Tris–HCl buffer (pH 8.4). The blot was incubated overnight with polyclonal antibody against human torsinA and detected using Renaissance Western blot chemiluminescence reagent (NEN Life Science Products, MA, USA).

Immunohistochemistry

Five adult (200 g) female

Western blotting

Western blot of rat brain homogenate confirmed the specificity of the antibody in rat, demonstrating a single band at approximately 38 kD [6] (Fig. 1A).

Immunohistochemistry

As compared with sections incubated with antibody to torsinA (Fig. 1B), with preadsorbed antibody (Fig. 1C), or in the absence of primary antibody (Fig. 1D), there was no specific labeling.

In all regions examined, neurons were immunoreactive for torsinA, with variable labeling intensity (Fig. 2) within and between regions. The resemblance of the

Discussion

This study confirms and extends our previous results [6] of the wide distribution of torsinA throughout the CNS, and demonstrates its presence in the majority of neurons. The presence of torsinA in the nucleus of neurons correlates with the identification of a bipartite nuclear localization sequence [6]. However, in a minority of neurons torsinA is present solely within the cytoplasm. The punctate immunoreactivity of torsinA in the neuropil suggests it is also present in nerve terminals, for

Acknowledgements

This work was supported by the Bachmann-Strauss Dystonia and Parkinson Foundation, Inc. and the National Parkinson Foundation. Confocal laser scanning microscopy was performed at the MSSM-Microscopy Center, supported with funding from an NSF Major Research Instrumentation grant (DBI-9724504). The authors are grateful to Amy Hsu, Paul Good, Ph.D. and Catherine Mytilineou, Ph.D. for methodological discussions.

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Also, the hippocampal neurons of ΔE-torsinA knock-in mice exhibit an increase in the frequency of miniature excitatory postsynaptic currents (mEPSCs, quantal release of glutamate) (Kakazu et al., 2012b), and an acceleration of synaptic vesicle exocytosis both in the absence of stimulation (Kakazu et al., 2012b) and during electrical stimulation (Kakazu et al., 2012a). TorsinA is present in the cell bodies and proximal dendrites of neurons (Shashidharan et al., 2000; Konakova et al., 2001; Walker et al., 2001, 2002; Siegert et al., 2005). It remains unclear about whether the wild-type and ΔE-torsinA are present at neuronal presynaptic structures that include nerve terminals and associated axonal shafts.
Presynaptic functions of the mammalian central neurons are regulated by a network of protein interactions. Synaptic vesicle recycling in and neurotransmitter release from the presynaptic nerve terminals are altered when a glutamate-deleting mutation is present in the torsinA protein (ΔE-torsinA). This mutation is linked with a hereditary form of the movement disorder dystonia known as DYT1 dystonia. Although torsinA expression is prevalent throughout the central nervous system, its subcellular localization – in particular with respect to presynaptic nerve terminals – remains unclear. This information would be useful in narrowing down possible models for how wild-type torsinA affects presynaptic function, as well as the nature of the presynaptic dysfunction that arises in the context of ΔE-torsinA mutation. Here we report on an analysis of the presynaptic localization of torsinA in cultured neurons obtained from a knock-in mouse model of DYT1 dystonia. Primary cultures of neurons were established from heterozygous and homozygous ΔE-torsinA knock-in mice, as well as from their wild-type littermates. Neurons were obtained from the striatum, cerebral cortex and hippocampus of these mice, and were subjected to immunocytochemistry. This analysis revealed the expression of both proteins in the somata and dendrites. However, neither the nerve terminals nor axonal shafts were immunoreactive. These results were confirmed by fluorogram-based quantitation. Our findings indicate that neither the wild-type nor the ΔE-torsinA mutant protein is present at substantial levels in the presynaptic structures of cultured neurons. Thus, the effects of torsinA, in wild-type and mutant forms, appear to influence presynaptic function indirectly, without residing in presynaptic structures.
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Further elucidating the processes involved in glutamate-mediated changes in [Ca2+]c dynamics is expected to help understand the pathophysiology of ΔE-torsinA mutation. In this initial exploratory study, hippocampal neurons were used because of their high expression of torsinA mRNA and protein [1,2,35,40]. Future studies will build on these findings by assessing other CNS regions, especially those more involved in motor control, for similar endophenotypes.
Increased activities of cytoplasmic calcium and the excitatory neurotransmitter glutamate have been independently implicated in dystonia pathophysiology. However, cellular-level evidence linking these two features is not available. Here we show that glutamate-dependent changes in neuronal calcium dynamics occur in a knock-in mouse model of DYT1 dystonia, the most common hereditary form of this disorder. Fluorescence-based analysis of the dynamics of cytoplasmic calcium concentration ([Ca²⁺]_c) in cultured hippocampal neurons shows that electrical stimulation depolarizes the neurons and increases the dendritic [Ca²⁺]_c, which then decays slowly to the pre-stimulus level. Whereas the peak amplitude of [Ca²⁺]_c was not affected, the decay period was prolonged in neurons of heterozygous mice whose genotype reflects the human condition. We found that this effect was blocked by the antagonists of ionotropic glutamate receptors, and confirmed that glutamate receptors are present in these neurons. As the [Ca²⁺]_c is readout and regulator of neuronal excitability, its abnormality represents an important cellular phenotype of dystonia
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Heat shock proteins (Hsp) are a family of chaperones that are both constitutively expressed and induced by different stresses that suppress protein aggregation, and participate in protein refolding and/or degradation. Torsin A, a protein with homology to yeast Hsp104, co-localizes with α-synuclein in LBs and is found in many peripheral tissues and brain regions [354–356]. Other heat shock proteins have also been shown to co-localize with α-synuclein in LBs [357].
Substantial evidence links α-synuclein, a small highly conserved presynaptic protein with unknown function, to both familial and sporadic Parkinson's disease (PD). α-Synuclein has been identified as the major component of Lewy bodies and Lewy neurites, the characteristic proteinaceous deposits that are the hallmarks of PD. α-Synuclein is a typical intrinsically disordered protein, but can adopt a number of different conformational states depending on conditions and cofactors. These include the helical membrane-bound form, a partially-folded state that is a key intermediate in aggregation and fibrillation, various oligomeric species, and fibrillar and amorphous aggregates. The molecular basis of PD appears to be tightly coupled to the aggregation of α-synuclein and the factors that affect its conformation. This review examines the different aggregation states of α-synuclein, the molecular mechanism of its aggregation, and the influence of environmental and genetic factors on this process.
Printor, a novel torsinA-interacting protein implicated in dystonia pathogenesis
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Early onset generalized dystonia (DYT1) is an autosomal dominant neurological disorder caused by deletion of a single glutamate residue (torsinA ΔE) in the C-terminal region of the AAA⁺ (ATPases associated with a variety of cellular activities) protein torsinA. The pathogenic mechanism by which torsinA ΔE mutation leads to dystonia remains unknown. Here we report the identification and characterization of a 628-amino acid novel protein, printor, that interacts with torsinA. Printor co-distributes with torsinA in multiple brain regions and co-localizes with torsinA in the endoplasmic reticulum. Interestingly, printor selectively binds to the ATP-free form but not to the ATP-bound form of torsinA, supporting a role for printor as a cofactor rather than a substrate of torsinA. The interaction of printor with torsinA is completely abolished by the dystonia-associated torsinA ΔE mutation. Our findings suggest that printor is a new component of the DYT1 pathogenic pathway and provide a potential molecular target for therapeutic intervention in dystonia.

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^☆: Also published on the World Wide Web on 2 April 2001.

²: Present address: Allergan Pharmaceuticals, 2525 Dupont Drive, TL-2L, Irvine, CA 92623-9534, USA.

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Interactive reportDistribution and immunohistochemical characterization of torsinA immunoreactivity in rat brain☆

Abstract

Introduction

Section snippets

Western blotting

Immunohistochemistry

Western blotting

Immunohistochemistry

Discussion

Acknowledgements

Neuron

Trends. Biochem. Sci.

Brain Res.

Genomics

Brain Res. Mol. Brain Res.

Trends Neurosci.

Dystonia gene in Ashkenazi Jewish population is located on chromosome 9q32–34 [see comments]

Ann. Neurol.

Interactive report
Distribution and immunohistochemical characterization of torsinA immunoreactivity in rat brain☆