NDRG2: a novel Alzheimer\'s disease associated protein

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www.elsevier.com/locate/ynbdi Neurobiology of Disease 16 (2004) 48 – 58

NDRG2: a novel Alzheimer’s disease associated protein Cathy Mitchelmore, a Stine Bu¨chmann-Møller, a Lene Rask, a Mark J. West, b Juan C. Troncoso, c and Niels A. Jensen a,* a

Laboratory of Mammalian Molecular Genetics, The Panum Institute 6.5, University of Copenhagen, 2200 Copenhagen N, Denmark Neurostereology Laboratory, Department of Neurobiology, University of Aarhus, Aarhus, Denmark c Neuropathology Laboratory, Johns Hopkins University Medical School, Baltimore, MD 21205-2196, USA b

Received 6 August 2003; revised 5 December 2003; accepted 8 January 2004 Available online 11 March 2004

Our understanding of the genes involved in Alzheimer’s disease (AD) is incomplete. Using subtractive cloning technology, we discovered that the A/B-hydrolase fold protein gene NDRG2 (NDRG family member 2) is upregulated at both the RNA and protein levels in AD brains. Expression of NDRG2 in affected brains was revealed in (1) cortical pyramidal neurons, (2) senile plaques and (3) cellular processes of dystrophic neurons. Overexpression of two splice variants encoding a long and short NDRG2 isoform in hippocampal pyramidal neurons of transgenic mice resulted in localization of both isoforms to dendritic processes. Taken together, our findings suggest that NDRG2 upregulation is associated with disease pathogenesis in the human brain and provide new insight into the molecular changes that occur in AD. D 2004 Elsevier Inc. All rights reserved. Keywords: NDRG; a/h-Hydrolase; CA1; Pyramidal neurons; Hippocampus; Senile plaques; Dystrophic neurons; Neuronal degeneration; Alzheimer’s disease; Transgenic mice

Introduction Alzheimer’s disease (AD) is a progressive neurodegenerative disorder characterized neuropathologically by an abundance of senile plaques, intracellular accumulation of neurofibrillary tangles in cortical and subcortical brain regions, and by loss of synapses and neurons in specific brain regions (Terry et al., 1999). It results in the loss of memory and other cognitive and behavioral abnormalities, and ultimately leads to the complete incapacitation of the patients. The entorhinal cortex (Gomez-Isla et al., 1996) and the CA1 subregion of the hippocampus (West et al., 1994) are among the first and most profoundly affected brain regions with respect to neuronal loss and AD pathology (Braak and Braak, 1991). The loss of neurons and synapses in these regions compromises the computational capacity as well as the flow of information through the hippocampus and can explain why some of the first symptoms of

AD are related to defects in declarative memory (Eichenbaum, 2001; Morris, 1999). AD can be subdivided into early- and late-onset forms. Earlyonset AD constitutes only 5 – 10% of all AD cases, has an onset around 50 years of age, and in many cases has been associated with specific mutations in the APP, PS1 or PS2 genes (Price and Sisodia, 1998; Rebeck and Hyman, 1999; Sisodia et al., 1999). Late-onset AD, which occurs later at about 65 years of age, is genetically heterogeneous. Several genetic risk factors have been associated with the disorder, including the q4 allele of the apolipoprotein E (APOE) gene (Corder et al., 1993; Farrer et al., 1997; Saunders et al., 1993), mutations affecting the transcriptional activity of the APOE gene (Bullido et al., 1998; Lambert et al., 1998), the alpha-2 macroglobulin gene (Blacker et al., 1998), a low-density lipoprotein receptor-related protein (Kang et al., 1997; Kounnas et al., 1995), and overexpression of the DSCR1 (Adapt78) gene (Ermak et al., 2001). Although lateonset AD is more prevalent than early-onset cases, both forms of the disease are characterized by similar clinical and neuropathological features. The etiology of the disease is poorly understood and preventative, therapeutic and curative strategies are not well developed. In an attempt to better understand the molecular events involved in the disease process, we have initiated a search for novel proteins that are upregulated in the brains of AD patients, based on a subtractive cloning approach. Here we report the discovery of a protein, designated NDRG2 (NDRG family member 2), which is upregulated in late-onset AD brains and is in AD lesions and in the neurons of brain regions that are known to be vulnerable to AD pathology.

Materials and methods Patient material

* Corresponding author. Laboratory of Mammalian Molecular Genetics, The Panum Institute 6.5, University of Copenhagen, Blegdamsvej 3, DK-2200 Copenhagen N, Denmark. Fax: +45-35327701. E-mail address: [email protected] (N.A. Jensen). Available online on ScienceDirect (www.sciencedirect.com.) 0969-9961/$ - see front matter D 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.nbd.2004.01.003

All human material was obtained with informed consent by the Johns Hopkins University Alzheimer’s Disease Research Center (ADRC), or was purchased from commercial sources (BioChain and Clontech) (see Table 1).

C. Mitchelmore et al. / Neurobiology of Disease 16 (2004) 48–58 Table 1 Description of the human material used in this work Case no.

Age (years)

Sex

Race AD status

Experimental usage

Late onset AD 1341a

89

F

W

1367a

87

M

W

1552a

90

M

W

1575a

93

F

W

1600a

91

F

W

1609a

64

M

W

1866a

74

F

Un

CCLS, RT-PCR CCLS, RT-PCR CCLS, RT-PCR CCLS, RT-PCR IH, Western IH, Western IH

Brain RNAb

77

M

Un

Controls HF RNAc

16 – 70

M+F W

Non-AD

1570a 1640a Brain RNAb Fetal Brain RNAb

53 96 28 24 weeks

M F M M

Non-AD Non-AD Non-AD Non-AD

B W Un Un

Earlystage AD Earlystage AD Advancedstage AD Advancedstage AD Advancedstage AD Advancedstage AD Advancedstage AD AD

Northern

CCLS, RT-PCR Western Western Northern Northern

F, female; M, male; W, Caucasian; B, African; Un, unknown; CCLS, chemical cross-linking subtraction; IH, immunohistochemistry; HF, hippocampal formation. a Obtained from the Johns Hopkins University Alzheimer’s Disease Research Center (ADRC), where all subjects have been examined clinically for cognitive abilities using Mini Mental State Examination (MMSE) scores and for AD neuropathology using Braak scores (Braak and Braak, 1991). Early-stage AD subjects had neuropathological lesions with Braak scores III as well as MMSE scores consistent with moderate dementia (i.e., MMSE 20), whereas advanced stage AD subjects had Braak scores VI and severe dementia (i.e., MMSE scores
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