Increased calbindin-D 28k immunoreactivity in striatal projection neurons of R6/2 Huntington’s disease transgenic mice Z. Sun, H.B. Wang, Y.P. Deng, W.L. Lei, J.P. Xie, C.A. Meade, N. Del Mar, D. Goldowitz, and A. Reiner * Department of Anatomy and Neurobiology, The University of Tennessee Health Science Center, 855 Monroe Avenue, Memphis, TN 38163, USA Received 30 March 2005; revised 17 May 2005; accepted 25 May 2005 Available online 28 June 2005 Striatal degeneration in Huntington’s disease (HD) is associated with increases in perikaryal calbindin immunolabeling in yet-surviving striatal projection neurons. Since similar increases have also been observed in surviving striatal projection neurons after intrastriatal injection of the excitotoxin quinolinic acid, the increased calbindin in HD striatum has been interpreted to suggest an excitotoxic process in HD. We used immunolabeling to assess if calbindin is elevated in striatal projection neurons of R6/2 HD transgenic mice. These mice bear exon 1 of the human huntingtin gene with 144 CAG repeats and show some of the neuropathological signs (e.g., neuronal intranuclear inclusions) and clinical traits (e.g., wasting prior to early death) of HD. We found an increased frequency of calbindin-immunoreactive neuro- nal perikarya in the striatum of 6- and 12-week-old R6/2 mice compared to wild-type controls. This increase was most notable in the normally calbindin-poor dorsolateral striatum. We found no significant changes in the total area of striatum occupied by the calbindin-negative striosomes and no consistent changes in striatal calbindin mRNA. The increase in calbindin in R6/2 striatal neurons was thus limited to the matrix compartment, and it may be triggered by increased Ca 2+ entry due to the demonstrated heightened NMDA sensitivity of these neurons. The data further support the similarity of R6/2 mice to HD, and are consistent with the occurrence of an excitotoxic process in striatum in both. D 2005 Elsevier Inc. All rights reserved. Keywords: Huntington’s disease; Calbindin-D 28k ; Immunoreactivity Introduction Huntington’s disease (HD) is an autosomal dominant neuro- degenerative disease characterized by extensive loss of neurons in the striatal part of the basal ganglia (Ross et al., 1996; Vonsattel and DiFiglia, 1998). The HD gene contains a polymorphic trinucleotide CAG repeat in exon 1 that is expanded beyond 36 repeats in HD patients (The Huntington’s Disease Collaborative Research Group, 1993). This gene encodes a protein termed huntingtin, which is widely expressed in nervous system and thought to be a membrane-associated scaffolding protein that contributes to protein – protein interactions mediating diverse intracellular functions, most notably vesicular trafficking (DiFiglia et al., 1995; Takano and Gusella, 2002). The means by which the mutant protein causes preferential damage to striatal projection neurons in HD is uncertain, although it is thought that the process involves an adverse gain of function (Ross, 1997; Reiner et al., 2003). The work of DiFiglia et al. (1997) showing that mutant huntingtin accumulates in the brains of HD patients in ubiquiti- nated aggregates in neuronal nuclei and in dystrophic neurites, which are evident by light microscopy in neurons of cerebral cortex and striatum, led to the suggestion that mutant protein aggregation might be involved in the pathogenic process. It is currently uncertain, however, if the light microscopically evident aggregates themselves are pathogenic or whether it is smaller oligomers of mutant huntingtin that are pathogenic (Saudou et al., 1998; Szebenyi et al., 2003; Arrasate et al., 2004). Calbindin-D 28k (CALB) is a calcium-binding protein found in projection neurons of the matrix compartment of striatum (Gerfen and Wilson, 1996). By buffering Ca 2+ , CALB is thought to spatially and temporally restrict calcium-mediated signaling at sites of Ca 2+ entry stemming from cell membrane depolarization (Morris et al., 1995; Pickel and Heras, 1996), and some authors have suggested its presence may protect striatal projection neurons against excitotoxic injury (DiFiglia et al., 1989; Figueredo- Cardenas et al., 1998). Increased CALB immunoreactivity has been reported in the soma and dendritic spines of surviving striatal projection neurons in Huntington’s disease (HD) patients (Ferrante et al., 1991; Huang et al., 1995). In an experimental HD model in rats created by intrastriatal administration of the NMDA receptor agonist quinolinic acid (QA), a similar finding was observed (Huang et al., 1995). The increase in CALB immunoreactivity in striatal projection neurons in the QA rat model of HD and in HD itself has been suggested to possibly represent a protective response to excess glutamatergic stimulation, from QA in the former and from disturbed glutamatergic corticostriatal trans- mission in the latter (Ferrante et al., 1991; Huang et al., 1995). 0969-9961/$ - see front matter D 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.nbd.2005.05.023 * Corresponding author. Fax: +1 901 448 7193. E-mail address: areiner@utmem.edu (A. Reiner). Available online on ScienceDirect (www.sciencedirect.com). www.elsevier.com/locate/ynbdi Neurobiology of Disease 20 (2005) 907 – 917