Address correspondence and reprint requests to Stephen D. Ginsberg, Center for Dementia Research, Nathan Kline Institute, New York University School of Medicine, 140 Old Orangeburg Road, Orangeburg, NY, USA. E-mail: firstname.lastname@example.org
Dysfunction of cholinergic basal forebrain (CBF) neurons of the nucleus basalis (NB) is a cardinal feature of Alzheimer's disease (AD) and correlates with cognitive decline. Survival of CBF neurons depends upon binding of nerve growth factor (NGF) with high-affinity (trkA) and low-affinity (p75NTR) neurotrophin receptors produced within CBF neurons. Since trkA and p75NTR protein levels are reduced within CBF neurons of people with mild cognitive impairment (MCI) and mild AD, trkA and/or p75NTR gene expression deficits may drive NB degeneration. Using single cell expression profiling methods coupled with custom-designed cDNA arrays and validation with real-time quantitative PCR (qPCR) and in situ hybridization, individual cholinergic NB neurons displayed a significant down regulation of trkA, trkB, and trkC expression during the progression of AD. An intermediate reduction was observed in MCI, with the greatest decrement in mild to moderate AD as compared to controls. Importantly, trk down regulation is associated with cognitive decline measured by the Global Cognitive Score (GCS) and the Mini-Mental State Examination (MMSE). In contrast, there is a lack of regulation of p75NTR expression. Thus, trk defects may be a molecular marker for the transition from no cognitive impairment (NCI) to MCI, and from MCI to frank AD.
Cholinergic basal forebrain (CBF) neurons of the nucleus basalis (NB) provide the major cholinergic innervation to the cortex and hippocampus, and play a key role in memory and attentional behaviors (Bartus et al. 1982; Mesulam et al. 1983; Baxter and Chiba 1999). CBF neurons undergo selective degeneration including the loss of presynaptic cholinergic enzymes [e.g. choline acetyltransferase (ChAT)] during the later stages of Alzheimer's disease (AD) that is associated with disease duration and degree of cognitive impairment (Wilcock et al. 1982; Bierer et al. 1995). Degeneration of the CBF system suggests that deficits in cortical cholinergic transmission mediated via NB neurons may contribute to the severe cognitive abnormalities seen in advanced AD (Whitehouse et al. 1982; Mufson et al. 2003). Despite intense interest in the cholinobasal cortical projection system, the molecular and cellular mechanisms underlying NB neurodegeneration during the progression of AD remain unclear. Notably, CBF survival appears to require appropriate binding, internalization, and retrograde transport of the prototypic neurotrophin, nerve growth factor (NGF), which is synthesized and secreted by cells in the cortex (Sofroniew et al. 2001; Mufson et al. 2003). NGF delivery attenuates CBF neurodegeneration and improves learning and memory in animal models of neurodegeneration, excitotoxicity, aging, and amyloid toxicity (Tuszynski 2002). Moreover, aged transgenic mice engineered to produce anti-NGF antibodies exhibit CBF neurodegeneration and inclusions that resemble the pathologic hallmarks of AD (Capsoni et al. 2000; Ruberti et al. 2000), indicating the importance of appropriate growth factor synthesis for CBF viability in vivo and the development of AD-like pathology associated with neurotrophin dysregulation.
NGF exerts functional consequences for cholinergic NB neuronal survival by interacting with at least two neurotrophin receptors, the low-affinity pan-neurotrophin receptor p75NTR and the high-affinity NGF-specific receptor tyrosine kinase, trkA (Kaplan and Miller 2000; Teng and Hempstead 2004). TrkB and trkC are also localized to CBF neurons, albeit at lower levels than trkA (Salehi et al. 1996; Mufson et al. 2002). Trk receptors, along with p75NTR, are produced within CBF neurons and transported anterogradely to the cortex where they bind NGF and other members of this family of neurotrophins (Kaplan and Miller 2000; Howe and Mobley 2004).
Defining the molecular and cellular mechanisms underlying the pathophysiological role of NGF receptors in the selective vulnerability of cholinergic neurons of the NB and the progression of dementia remains elusive. Characterizing these mechanisms may lead to the development of rational therapies for the amelioration of CBF cellular degeneration, intervention for clinical symptoms, and early diagnosis of mild cognitive impairment (MCI) and/or AD. To derive cognitively based molecular mechanisms of NGF receptor dysfunction from individual cholinergic NB neurons, tissue samples were harvested post-mortem from cases clinically characterized with no cognitive impairment (NCI), MCI, and AD from the Religious Orders Study (ROS), an ongoing longitudinal clinicopathological study of aging and AD in older Catholic nuns, priests, and brothers (Mufson et al. 2000, 2002; Bennett et al. 2002). Antemortem cognitive measures including the global cognitive score (GCS), comprised of a battery of 19 different neuropsychological tests (Bennett et al. 2002), and the Mini-Mental State Examination (MMSE) were correlated with gene level expression of p75NTR, trkA, trkB, and trkC derived from individual cholinergic NB neurons.
Materials and methods
Clinical and pathological evaluation of ROS subjects
In order to enter the ROS cohort, subjects are judged by an examining neurologist to not have any coexisting clinical or neurologic condition(s) contributing to cognitive impairment. Neuropsychological tests were chosen to measure a range of cognitive abilities with emphasis on those affected by aging and AD. Cognitive testing was performed under the auspices of a trained neuropsychologist, and scores were available within the last year of death. The 19 tests that constitute the GCS are listed in Supplemental Table 1, and they comprised a composite GCS score for each subject in addition to the individual scores on the respective cognitive tests. A board-certified neurologist with expertise in the evaluation of the elderly made a clinical diagnosis for each ROS participant based upon review of all clinical data and physical examination. Subjects were categorized as NCI (n = 12; mean age 81.0 ± 9.1 years), MCI insufficient to meet criteria for dementia (n = 10; 81.9 ± 4.3), or AD (n = 12; 84.5 ± 6.9) (Table 1). Details of the clinical and neuropsychological evaluation for the ROS cohort have been published previously (Mufson et al. 2000, 2002; Bennett et al. 2002). This study was performed in accordance with IRB guidelines administrated by the Rush University Medical Center and the New York University School of Medicine/Nathan Kline Institute.
Table 1. Clinical, demographic, and neuropathological characteristics
At autopsy, one hemisphere was immersion-fixed in a 4% paraformaldehyde solution in 0.1 m phosphate buffer, pH 7.2 for 24 h at 4°C, cryoprotected, and cut frozen at a section thickness of 40 µm (Mufson et al. 2000, 2002; Chu et al. 2001; Counts et al. 2006). From the opposite hemisphere, tissue from cortex, hippocampus, and brainstem structures were harvested and prepared for paraffin embedding. Tissue sections were stained for the visualization of senile plaques and neurofibrillary tangles using thioflavine-S, antibodies directed against paired helical filament (PHF) tau (gift from Peter Davies) and a modified Bielschowsky silver stain (Mufson et al. 2000, 2002; Bennett et al. 2002). Additional sections were stained for Lewy bodies using commercially available antibodies directed against ubiquitin and alpha-synuclein (Mufson et al. 2000, 2002; Bennett et al. 2002). The remaining tissue slabs were frozen at −80°C. A pathological diagnosis was made while the neuropathologist was blinded to the clinical diagnosis. Neuropathological designations were based on the NIA Reagan and CERAD criteria (Mirra et al. 1991; Hyman and Trojanowski 1997). In addition, a Braak score (Braak and Braak 1991) was tabulated for each case. Exclusion criteria included stroke and Parkinson's disease. ApoE genotyping was performed by PCR analysis (Mufson et al. 2000). The majority of AD cases from the ROS cohort used in this study are mild to moderate AD based upon neuropathological and cognitive criteria. End-stage AD subjects were not overly represented in this study. Currently, consensus criteria for the clinical classification of MCI are being developed (Winblad et al. 2004). The present MCI population was defined as subjects with impaired neuropsychological test scores who were not found to have dementia by the examining neurologist (Mufson et al. 2000; Bennett et al. 2002; DeKosky et al. 2002), similar to the criteria used by independent experts in the field to describe subjects who are not cognitively normal but do not meet established criteria for dementia (Petersen 2004; Winblad et al. 2004).
Accession of CBF NB neurons and immunocytochemistry
Acridine orange histofluorescence (Ginsberg et al. 1997, 1998; Mufson et al. 2002) and bioanalysis (Agilent 2100, Palo Alto, CA, USA) (Ginsberg and Che 2002, 2004) were performed on each brain utilized in this study to ensure that high quality RNA was present in tissue sections prior to performing downstream genetic analyses. RNase-free precautions were used throughout the experimental procedures, and solutions were made with 18.2 mega Ohm RNase-free water (Nanopure Diamond, Barnstead, Dubuque, IA, USA).
Tissue sections were processed for p75NTR immunocytochemistry using a monoclonal antibody raised against human p75NTR (Schatteman et al. 1988; Mufson et al. 1989b, 2002; Counts et al. 2004). p75NTR immunoreactivity colocalizes with approximately 95% of all CBF neurons within the human NB (Mufson et al. 1989a,b), and is an excellent phenotypic marker for these cells. Our laboratory group has also identified CBF neurons for microaspiration using neurofilament immunoreactivity, ChAT immunoreactivity, and cresyl violet staining as part of preliminary studies and separate single cell analyses of the basal forebrain (Ginsberg and Che 2002, 2004; Mufson et al. 2002, 2004). CBF neurons selected for microaspiration were localized to the anterior subfield of the NB located ventral to the anterior commissure (Mufson et al. 2002). The anterior NB subfield (Mufson et al. 1989a) can be identified readily under the dissecting microscope by an investigator blinded to case demographics, ensuring the aspiration of CBF neurons.
Immunocytochemistry was performed as described previously (Mufson et al. 1989b, 2002; Counts et al. 2004). Following several rinses in phosphate-buffered saline (PBS, pH 7.2) tissue sections were incubated for 20 min in a Tris-buffered saline (pH 7.4) solution containing 0.1 m sodium periodate (Sigma, St Louis, MO, USA) to inhibit endogenous peroxidase staining. Tissue sections were incubated for 1 h in a PBS solution containing 0.3% Triton X-100, 3% normal horse serum (NHS) and 2% bovine serum albumin (BSA). Primary antibody (monoclonal p75NTR, 1 : 60 000) was applied for 4 h at 22°C with constant agitation. The diluent for the primary antibody contained 0.4% Triton X-100, 1% NHS and 1% BSA. Sections were processed with the ABC kit (Vector Laboratories, Burlingame, CA, USA) and developed in a 0.2 m sodium acetate imidazole buffer (pH 7.4) with 2.5% nickel II sulfate (Sigma), 0.05% 3′ 3′ diaminobenzidine (DAB, Sigma) and 0.005% hydrogen peroxide (pH 7.2) (Chu et al. 2001; Mufson et al. 2002). Immunostained tissue sections were stored in RNase-free PBS at 4°C until neurons were microaspirated for cDNA array analysis within 72 h.
Single cell microaspiration and Terminal Continuation (TC) RNA amplification
Microaspiration and TC RNA amplification procedures have been described in detail elsewhere (Ginsberg and Che 2002, 2004; Che and Ginsberg 2004) and are diagrammed in Fig. 1. Linearity and fidelity of the TC RNA amplification procedure has been published, including the use of CBF neurons as input sources of RNA (Che and Ginsberg 2004; Ginsberg 2005). Moreover, variability between single cell expression profiles and reproducibility of expression levels has been evaluated extensively by our laboratory group and published previously (Che and Ginsberg 2004; Ginsberg and Che 2004; Ginsberg 2005). Briefly, individual p75NTR-immunoreactive NB neurons were microaspirated from paraformaldehyde-fixed 40 µm thick frozen cut sections of the basal forebrain using a micromanipulator and microcontrolled vacuum source (Eppendorf, Westbury, NY, USA) attached to an inverted microscope (E800, Nikon, Japan). The amplification of RNA from individual NB neurons was performed using a new terminal continuation (TC) RNA amplification methodology (Che and Ginsberg 2004; Ginsberg and Che 2004; Ginsberg 2005). The TC RNA amplification protocol is available at http://cdr.rfmh.org/pages/ginsberglabpage.html. Individual, not pooled, CBF neurons were extracted in 250 µL of Trizol reagent (Invitrogen, Carlsbad, CA, USA). RNAs were reverse transcribed in the presence of the poly d(T) primer (10 ng/µL) and TC primer (10 ng/µL) in 1X first strand buffer (Invitrogen), 1 mm dNTPs, 5 mm DTT, 20 U of RNase inhibitor and 5 U reverse transcriptase (Superscript III, Invitrogen). The synthesized single stranded cDNAs were converted into double stranded cDNAs by adding into the reverse transcription reaction the following: 10 mm Tris (pH 8.3), 50 mm KCl, 1.5 mm MgCl2, and 0.5 U RNase H (Invitrogen) in a total volume of 99 µL. Samples were placed in a thermal cycler and second strand synthesis proceeded as follows: RNase H digestion step 37°C, 10 min; denaturation step 95°C, 3 min; annealing step 50°C, 3 min; elongation step 75°C, 30 min 5 U (1 µL) Taq polymerase (PE Biosystems, Foster City, CA, USA) was added to the reaction at the initiation of the denaturation step (i.e. hot start) (Che and Ginsberg 2004). The reaction was terminated with 5 m ammonium acetate. The samples were extracted in phenol:chloroform:isoamyl alcohol (25 : 24 : 1) and ethanol precipitated with 5 µg of linear acrylamide (Ambion, Austin, TX, USA) as a carrier. The solution was centrifuged at 14 000 r.p.m and the pellet washed once with 95% ethanol and air-dried. The cDNAs were resuspended in 20 µL of RNase free H2O and drop dialyzed on 0.025 µm filter membranes (Millipore, Billerica, MA, USA) against 50 mL of 18.2 MegaOhm RNase-free H2O for 2 h. The sample was collected off the dialysis membrane and hybridization probes were synthesized by in vitro transcription using 33P incorporation in 40 mm Tris (pH 7.5), 7 mm MgCl2, 10 mm NaCl, 2 mm spermidine, 5 mm of DTT, 0.5 mm of ATP, GTP, and CTP, 10 µm of cold UTP, 20 U of RNase inhibitor, T7 RNA polymerase (1000 U, Epicentre, Madison, WI, USA), and 40 µCi of 33P-UTP (GE Healthcare, Piscataway, NJ, USA). The reaction was performed at 37°C for 4 h. Radiolabeled TC RNA probes were hybridized to custom-designed cDNA arrays without further purification.
Custom-designed cDNA array platforms and array hybridization
Array platforms consisted of 1 µg of linearized cDNA purified from plasmid preparations adhered to high-density nitrocellulose (Hybond XL, GE Healthcare). Each cDNA and/or expressed sequence-tagged cDNA (EST) was verified by sequence analysis and restriction digestion. cDNA clones and ESTs from mouse, rat, and human were employed. All of the neurotrophin and neurotrophin receptor clones were derived from human sequences. Approximately 220 cDNAs/ESTs were utilized on the current array platform. The majority of genes are represented by one transcript on the array platform. However, several genes have representation at 3′ and 5′ regions, including the high-affinity neurotrophin receptors (trkA, trkB, and trkC) to assess relative expression levels from separate regions of the gene and evaluate potential RNA degradation (Ginsberg et al. 2000). For example, ESTs that encode the tyrosine kinase domain (TK) and extracellular domain (ECD) were employed for trkA, trkB, and trkC.
Arrays were prehybridized (2 h) and hybridized (12 h) in a solution consisting of 6X SSPE, 5X Denhardt's solution, 50% formamide, 0.1% sodium dodecyl sulfate (SDS), and denatured salmon sperm DNA (200 µg/mL) at 42°C in a rotisserie oven (Ginsberg and Che 2002, 2004; Ginsberg 2005). Following hybridization, arrays were washed sequentially in 2X SSC/0.1% SDS, 1X SSC/0.1% SDS and 0.5X SSC/0.1% SDS for 20 min each at 42°C. Arrays were placed in a phosphor screen for 24 h and developed on a phosphor imager (GE Healthcare).
Data collection and statistical analysis
Hybridization signal intensity was quantified by subtracting background using empty vector (pBs). Expression of TC amplified RNA bound to each linearized cDNA (approximately 220 cDNAs/ESTs) was expressed as a proportion of the total hybridization signal intensity of the array (a global normalization approach). Global normalization effectively minimizes variation due to differences in the specific activity of the synthesized TC probe as well as the absolute quantity of probe present (Eberwine et al. 2001; Ginsberg and Che 2004; Ginsberg 2005). Data analyzed in this manner does not allow the absolute quantitation of mRNA levels. However, an expression profile of relative changes in mRNA levels was generated.
Demographic and clinical characteristics (age, sex, years of education, GCS, MMSE, ApoE allele, and PMI) and neuropathologic classifications (Braak score and NIA-Reagan diagnosis) were compared among clinical diagnostic groups by the Kruskal–Wallis test and Fisher's exact test, with Bonferroni-type correction for pairwise comparisons. Expression levels were clustered and displayed using a bioinformatics and graphics software package (GeneLinker Gold, Predictive Patterns, Kingston, ON). As multiple cells were measured in each subject, between-subject versus within-subject (between-cell) variation in gene expression levels was analyzed by variance component analysis and intraclass correlation coefficients, which ranged between 0.4 and 0.6 for p75 NTR, trkA, trkB and trkC. The association between gene expression levels and subject characteristics (diagnostic groups as well as other demographic, clinical, and neuropathological variables) was evaluated via mixed models repeated measures analyses, which accounts for both between-subject and within-subject variation (SAS Institute 1999). In the analyses, we used random intercept, fixed effect covariate, Kenward-Roger denominator degrees of freedom, unstructured covariance structure, and log-transformed gene expression levels (SAS Institute 1999). The level of statistical significance was set at 0.01 (two-sided).
qPCR was performed on unfixed, microdissected frozen tissue at the level of the anterior NB ventral to the decussation of the anterior commissure (n = 7 NCI; n = 6 MCI, and n = 8 AD). Micropunched tissue was also obtained from the caudate nucleus from the same subjects as a control brain region since striatal trkA-immunoreactive neurons are not selectively vulnerable in AD (Boissiere et al. 1997; Mufson et al. 1997, 2000; Chu et al. 2001). Microdissected tissues contain an admixture of cholinergic neurons, non-cholinergic neurons, glia, and vasculature. PCR primers were designed for five genes, p75NTR, trkA, trkB, trkC, and the housekeeping gene glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Due to the high homology of the trk receptors (Shelton et al. 1995), TaqMan hydrolysis probes were designed (trkA, trkB, and trkC) for gene quantification. The p75NTR and GAPDH primer sequences have been used by our group and employ SYBR green dye chemistry as a reporter (Ginsberg and Che 2004, 2005). Primer sequences are reported in Supplemental Table 2. Samples were run on a real-time PCR cycler (7900HT, ABI) as per the manufacturers instructions. Standard curves and cycle threshold (Ct) were measured using standards obtained from total human brain RNA. Relative changes in PCR product synthesis was analyzed by one-way anova with post hoc analysis (Neumann-Keuls test; level of statistical significance was set at 0.05) for individual comparisons. Amplicon specificity was evaluated by subcloning the amplicon products (Zero Blunt, Invitrogen) and performing sequence analysis.
In situ hybridization
Tissue slabs containing the entire NB were immersion fixed in 4% paraformaldehyde and frozen sectioned at a thickness of 40 µm for in situ hybridization histochemistry (Mufson et al. 1996; Chu et al. 2001). Probes were generated against human p75NTR and trkA. Briefly, a p75NTR probe was generated from 1.5 kilobase (kb) cDNA complementary to human p75NTR mRNA (Johnson et al. 1986) and subcloned into pBs (Higgins and Mufson 1989). Transcription was performed in a solution containing 4 mm Tris-HC1 (pH 7.5), 6 mm MgCl2, 2 mm DTT, 5 U RNasin, 400 mm of ATP, GTP, and UTP, 25 mm of 35S-CTP (800 Ci/mmol; GE Healthcare), 2 mg of linearized template and 10 U T7 RNA polymerase. Prehybridization was performed at 56°C for 1 h in a solution consisting of 50% formamide, 0.5 m NaCl, 25 mm Pipes buffer (pH 6.8), 10 mm EDTA, 250 mm DTT, 5X Denhardt's solution, 0.2% SDS, 10% dextran sulfate, and tRNA (500 mg/mL). Subsequent hybridization was performed for 16 h at 56°C followed by stringent washing conditions (Higgins and Mufson 1989; Mufson et al. 1996). Slides were air-dried and exposed to X-ray film (DuPont Cronex 4, MES Services, Fulton, IL, USA) for 24–48 h, dipped into Kodak NTB-2 emulsion (Eastman Kodak, Rochester, NY, USA; diluted 1 : 1 with distilled water), exposed for 4–6 days at 4°C, and developed for autoradiography. Selected slides were counterstained with cresyl violet for cellular visualization. The material presented for p75NTRin situ hybridization was generated from for an earlier study (Mufson et al. 1996).
A trkA cRNA probe was generated from a 545 basepair (bp) fragment of cDNA coding for the ECD of human trkA (Shelton et al. 1995). TrkA cDNA was obtained from post-mortem human brain using PCR and subcloned into pGEM −7Zf(–) vector in the XbaI/BamHI sites (Shelton et al. 1995). This plasmid was linearized with either SacI to serve as a template for T7 RNA polymerase (antisense) or SphI to serve as template for Sp6 RNA polymerase (sense), respectively. In vitro transcription was performed in the presence of ATP, GTP, UTP and biotin-14-CTP, RNasin, transcription buffer, and T7 or Sp6 RNA polymerase for 2 h at 37°C (Chu et al. 2001). The reaction was stopped by the addition of 1 µL of 0.5 EDTA. Biotin-labeled trkA cRNA was purified by ethanol precipitation and resuspended in 30 µL of diethyl pyrocarbonate treated water. Prehybridization was conducted for 2 h at 37°C using hybridization buffer (50% deionized formamide, 10% dextran sulfate, 10% Denhardt's solution, 30% 2X SSC) containing heat denatured torula yeast RNA (0.1 mg/mL) and 0.2% non-fat milk (Chu et al. 2001). Following rinses in 2X SSC, tissue sections of the NB are incubated in hybridization buffer with heat denatured biotinylated trkA probe (1 µg/mL) and torula yeast RNA (0.1 mg/mL) at 41°C for 18 h (Chu et al. 2001). TrkA mRNA was visualized using either autoradiographic labeling or using a biotinylated reaction product visualized by the ABC method (Vector Laboratories) with 0.025% DAB, 1% nickel II sulfate and 0.0025% hydrogen peroxide (Mufson et al. 2000; Chu et al. 2001). The tissue sections examined for trkA in situ hybridization were derived from material used as part of an earlier report (Chu et al. 2001). Control experiments included using sense probes, pretreatment of tissue sections with RNase, and processing the sections without biotinylated secondary antibodies.
A total of 174 single cholinergic NB neurons were analyzed in 34 post-mortem human brains, with an average of 5–6 cells per subject (range 2–11). Subjects in this study were compatible among the three diagnostic groups in age, gender, post-mortem interval (PMI), years of education, and ApoE4 status (Table 1). Post-mortem neuropathologic examination revealed that 50% (6/12) of NCI cases were classified as Braak stages III-IV and none as Braak stages V-VI; 80% (8/10) of MCI cases were classified as Braak stages III-IV and 20% (2/10) as stages V-VI; 34% (4/12) of AD cases were classified as Braak stages III-IV and 58% (7/12) as stages V-VI (Table 1). In addition, 6 NCI and 1 AD cases were identified as Braak stages 0–II (Table 1). Using NIA-Reagan criteria for pathological diagnosis of AD, 73% of NCI and 30% of MCI cases were classified as having a low likelihood of AD, with 27% of NCI and 70% of MCI cases as having an intermediate likelihood of AD. In contrast, the AD cases were classified as having an intermediate (44%) or high likelihood (44%) of AD (Table 1).
Single cholinergic NB cell expression profile analysis using custom-designed cDNA array platforms revealed differential regulation of neurotrophin receptor mRNAs. Other classes of transcripts were evaluated, including synaptic-related markers, glutamatergic neurotransmission, protein phosphatases/kinases, among other gene classes that will comprise a separate report due to the extensive amount of data. Significant down regulation of trkA, trkB, and trkC was observed in individual neurons microaspirated from AD and MCI brains as compared to NCI (Fig. 2) (see Supplemental Results). Moreover, down regulation was found for two separate ESTs for each trk gene (e.g. ESTs targeted to the ECD and TK domain) (Fig. 2). Individual cholinergic NB neurons from MCI brains displayed reduced levels of trkA, trkB, and trkC as compared to NCI (Fig. 2). Within cholinergic NB neurons, several trk ESTs displayed significantly higher expression levels in MCI than AD (e.g. trkAECD, trkBECD, and trkCTK), indicating that MCI cholinergic NB neurons exhibit intermediate levels of trkA, trkB, and trkC compared to NCI and AD. By contrast, no significant differences in relative expression levels for p75NTR were observed across clinical groups (Fig. 2). No changes in p75NTR expression levels were observed in CBF neurons identified by neurofilament immunoreactivity and cresyl violet staining (Ginsberg and Che 2002, 2004), consistent with the present observations. In addition, regulation of ChAT mRNA was not observed across clinical conditions (Fig. 2a). Taken together, these findings indicate a relative selectivity in the alteration of high-affinity neurotrophin receptors within single NB neurons during the prodromal stage of AD. Furthermore, no regulation of GAPDH was observed across clinical groups (Fig. 2a).
Similar to the expression profile analysis shown in Fig. 2, where an intermediate expression level or ‘step down effect’ from NCI to MCI to AD occurred, post-mortem trk gene expression levels from individual cholinergic NB neurons were also related to two antemortem cognitive assessment measures. A significant association was found between decreased trkA (p = 0.0008), trkB (p = 0.0001), and trkC (p = 0.003) levels and lower composite GCS scores (Fig. 3a). By contrast, no relationship was observed between GCS scores and p75NTR expression. Similar results were found for trk array profiles and MMSE scores (Fig. 3b) (see Supplemental Results). These observations were not facilitated by low GCS or MMSE scores observed in more severe AD cases examined in this study. Rather, these data indicate that virtually all of the MCI and AD cases display intermediate and lower expression levels, respectively, that contribute to the overall decrement in expression levels (Fig. 3).
Validation of the results obtained by array analysis was performed by qPCR. Results were reported as mean Ct ± sd. A high Ct level equates to low expression levels. Evaluation of Ct data did not reveal differential regulation of p75NTR expression across NCI, MCI, and AD (Fig. 4a). Similar to the observations reported on the custom-designed arrays, down regulation of trkA (Fig. 4b), trk B (Fig. 4c), and trkC (Fig. 4d) was observed in MCI and AD compared to NCI (see Supplemental Results for Ct values). Moreover, a significant intermediate or ‘step down effect’ between trkA expression levels in MCI and AD was found by qPCR (Fig. 4b), consistent with the custom-designed array results. Due to the low expression levels of both trkB and trkC in the basal forebrain tissue samples assayed by qPCR, an observation consistent with previous reports (Salehi et al. 1996; Mufson et al. 2002), discrimination of potential expression level differences between MCI and AD (as evidenced by trkA qPCR) was not possible. No differences in GAPDH expression levels were found in NCI, MCI, and AD subjects (data not shown). In contrast to the marked down regulation of trkA in NB tissue dissections, no differential regulation of trkA was observed in the striatum obtained from the same case materials, indicating a regional selectivity to the down regulation of trkA (Fig. 4e). Although there is considerable homology between the trkA, trkB, and trkC genes (Shelton et al. 1995), the TaqMan primers used in these qPCR studies demonstrated virtually no cross reactivity between neurotrophin receptors (Fig. 4f).
Array results were further validated in tissue sections of the basal forebrain using in situ hybridization histochemistry. Probes for both p75NTR and trkA predominantly labeled large multipolar NB neurons, with little or no labeling of glial cells or surrounding neuropil. Consistent with results obtained from single cell data acquired on custom-designed cDNA array platforms and regional tissue microdissections for qPCR, down regulation of trkA expression levels was apparent in NB tissue sections from MCI and AD brains in comparison with NCI brains (Fig. 5a–c). Additionally, no significant differences in p75NTR labeling were found across clinical groups (Figs 5d, e).
Creating a molecular fingerprint of single neurons that are selectively vulnerable requires their precise localization within a defined brain region. Therefore, resolution at the level of homogeneous neuronal populations is necessary to create an expression profile for affected cells such as cholinergic NB neurons. Simultaneous quantitative assessment of multiple transcripts by microaspiration, RNA amplification, and custom-designed cDNA microarray analysis provides a paradigm whereby the genetic signature of anatomically defined cells within a specific brain region can be differentiated from neighboring structures (Che and Ginsberg 2004; Ginsberg and Che 2004, 2005). This experimental design allows for rigorous quantitative analyses of vulnerable cell types during the progression of clinical impairment (Galvin and Ginsberg 2004, 2005).
The present study combines custom-designed cDNA array analysis with qPCR and in situ hybridization derived from individual neurons and microdissected regions of the anterior portion of the cholinergic NB and demonstrates that gene expression for the high-affinity neurotrophin receptors trkA, trkB, and trkC is significantly down regulated during the clinical progression of AD. Although a discrepancy in terms of fold difference is observed between the array and qPCR assays, this is a common occurrence due to several potential factors including RNA quantity and sensitivity of the specific platform (Ginsberg 2005; Ginsberg et al. 2006). An important factor is whether or not both methods show a similar direction, i.e. no change, up regulation, or consistent down regulation, as observed in the present study. Specifically, trk receptor expression was reduced in the NB of subjects with MCI compared to NCI, with even further reductions observed in AD. These results suggest that the onset of neurotrophic dysfunction in CBF neurons occurs during the earliest stages of cognitive decline, and that deficits in trk expression are associated with the clinical presentation of the disease. In support of this hypothesis, down regulation of the trk genes correlate with comprehensive (GCS) and individual (MMSE) measures of cognitive decline across the clinical diagnostic groups. In contrast, there is a lack of regulation of p75NTR expression. This is intriguing, as phenotypic silencing of both trkA and p75NTR protein expression has been reported (Mufson et al. 1989b, 2000) in contrast to the stable expression of ChAT gene expression (present study) and ChAT protein (Gilmor et al. 1999) within NB neurons during the prodromal stage of AD. These differential alterations in gene/protein regulation are also reflected in the cortical projection sites of the cholinergic NB neurons. For example, cortical trkA protein expression is decreased, whereas p75NTR protein levels remain stable during the progression of AD (Counts et al. 2004). Since both receptors are produced within NB neurons and anterogradely transported to the cortex, the possibility exists that the transport of trkA and/or the translation to protein is altered as opposed to p75NTR by the disease process. TrkA binding to NGF is a crucial factor for signal transduction associated with cholinergic neuronal survival (Kaplan and Miller 2000), thus reduction of trkA (as well as trkB and trkC) may have important consequences related to cholinergic basocortical dysfunction as well as cognitive decline during the transition from MCI to AD.
Retrograde transport of NGF bound to activated trkA receptors via signaling endosomes appears to be an important mechanism for delivery of NGF signals to target basal forebrain neurons (Howe and Mobley 2004). These binding events result in the retrograde transport of the bound neurotrophin ligand to CBF consumer neurons and the initiation of downstream cellular signal transduction related to cell survival (Counts and Mufson 2005). TrkA protein levels in cholinergic NB neurons are significantly reduced, along with decreased cortical levels early in the progression of AD (Boissiere et al. 1997; Mufson et al. 1997, 2000; Chu et al. 2001). TrkA gene expression levels have been shown to be down regulated in end-stage AD patients, although correlation with antemortem cognitive measures and evaluation of trkB/trkC was not investigated (Mufson et al. 2002). In contrast, the cholinergic phenotype of these neurons as well as cortical ChAT activity are preserved in people with MCI and mild AD compared to the dramatic reduction of these cholinergic markers in late stage AD (Mufson et al. 2003; Counts et al. 2004; Counts and Mufson 2005). Moreover, recent profiling studies indicate that the receptors for the putative cholinergic survival neuropeptide galanin (GALR1, GALR2 and GALR3) are unchanged in NB neurons in prodromal AD (Counts et al. 2006). These findings suggest a phenotypic down regulation of NGF receptors, but not a frank loss, of cholinergic neurons during the prodromal stage of AD. Therefore, a defect in trkA mRNA expression in NB neurons early in the course of AD could impact protein translation, ligand receptor binding, and retrograde transport of NGF leading to cholinergic cellular degeneration and cognitive decline during the course of the disease (Chu et al. 2001; Counts and Mufson 2005).
The persistence of p75NTR protein expression in the cortex in MCI and AD (Counts et al. 2004) may reflect several possible mechanisms. For example, a compensatory response in remaining p75NTR-containing NB neurons may occur to stabilize cortical receptor levels. This is unlikely since there was no change in p75NTR gene expression. Alternatively, a build up of p75NTR protein could occur in the cortex due to a retrograde transport defect or due to the de novo expression of p75NTR within cortical neurons in AD (Mufson and Kordower 1992). Interestingly, deficits in trkA and retrograde transport of NGF to CBF consumer neurons occurs in end stage AD (Mufson et al. 1997), in a segmental trisomy mouse model of Down's syndrome (Cooper et al. 2001), and in aged rats with cognitive impairment (Cooper et al. 1994; De Lacalle et al. 1996). Moreover, aged rats with mild cognitive impairment display a silencing of trkA expression within CBF neurons prior to cholinergic atrophy or loss of trkA containing neurons (Saragovi 2005). These deficits are enhanced in trkA-positive cholinergic neurons in aged rats with severe cognitive impairment (Saragovi 2005), suggesting that trkA down-regulation is associated with cognitive impairment seen in aging as well as AD. In addition, transgenic mice engineered to produce anti-NGF antibodies display AD-like neuropathology within CBF neurons (Capsoni et al. 2000; Ruberti et al. 2000). Taken together, these findings suggest that early defects in trkA receptor expression may be a precursor (and a potential biomarker) to the extensive cell loss observed within the CBF in end stage AD (Whitehouse et al. 1982; Mufson et al. 1989b).
There is evidence to suggest that an imbalance in the ratio of trkA and p75NTR, in part, may lead to cell death by promoting unscheduled cell cycle re-entry and apoptosis (Yoon et al. 1998; Naumann et al. 2002). p75NTR-mediated apoptosis involves the activation of cell cycle regulatory molecules, and a link between aberrant cell cycle re-entry of post-mitotic neurons and apoptosis has been established in CBF neurons in MCI and early AD (Yang et al. 2003). Several studies suggest that proper neurotrophin receptor signaling depends upon interactions with the NGF precursor protein, proNGF. For example, the ratio of proNGF to mature NGF is increased in cortex obtained from MCI and AD subjects and correlates with cognitive decline (Peng et al. 2004). It has been hypothesized that proNGF bound to trkA initiates cell survival activity (Fahnestock et al. 2004), whereas proNGF bound to p75NTR induces apoptosis (Pedraza et al. 2005). Additional findings indicate that the pro-apoptotic effect of p75NTR-mediated proNGF signaling is dependent upon interactions between p75NTR and the neurotensin receptor sortilin (Nykjaer et al. 2004). These data suggest that the ratio of trkA to p75NTR receptors may determine whether neurons survive or degenerate when exposed to NGF or proNGF. Thus, a ∼50% reduction in cortical trkA that occurs at the onset of AD may signify a relative increase in pro-apoptotic p75NTR signaling in cholinergic NB neurons. Interestingly, brain derived neurotrophic factor (BDNF) and its precursor protein proBDNF, which bind to the trkB receptor are significantly decreased in MCI and AD cortex compared to NCI (Peng et al. 2005). These results suggest that multiple defects in neurotrophin receptor expression and neurotrophin signaling play a key role in NB degeneration that may exacerbate functional deficits and lead to advanced pathology including synaptic dysfunction and cognitive decline early in the pathogenesis of AD.
By utilizing state-of-the-art molecular approaches for multiple mRNA assessments in concert with clinicopathological correlations in a range from normal senescence to frank dementia, this study provides unique insights into specific alterations in neurotrophin receptor gene expression within cholinergic NB neurons during the clinical progression of AD. The loss of trk expression in MCI suggests that trk reduction plays a role in the early stages of cholinergic NB cellular dysfunction contributing to cognitive deficits and to the ultimate demise of these neurons in the later stages of AD. Thus, early defects in trk expression may provide markers for the identification of individuals with MCI and/or in the prodromal stage(s) of AD. Interestingly, a phase I clinical trial whereby genetically modified autologous fibroblasts that secrete human NGF were grafted directly into the NB region improved cognition in mild AD patients (Tuszynski et al. 2005). The efficacy of this treatment may involve increased trk expression, which is positively regulated by NGF (Holtzman et al. 1992; Li et al. 1995). Ultimately, genetic fingerprinting of CBF neurons will provide a foundation for the development of novel pharmacotherapeutic intervention(s) to aid in ameliorating or preventing age and disease-related cognitive decline. Taken together, the current single cell gene array observations (validated independently with qPCR and in situ hybridization measures) in post-mortem human tissues may reflect a specific molecular signature underlying cholinergic NB neuronal dysregulation during the early stages of dementia and progressing towards frank AD.
This work was supported by grants from the NIH (AG10161, AG10688, AG14449, AG21661, AG26032, and NS43939), Alzheimer's Association and Illinois Department of Public Health. We are indebted to the altruism and support of the participants in the ROS. A list of participating groups can be found at the website: http://www.rush.edu/rumc/page-R12394.html. We thank Drs David A. Bennett, director of the ROS clinical core, Julie Schneider, director of the ROS neuropathology core, Sue Leurgans for statistical consultation, and Ralph A. Nixon for critical review of the manuscript. We also thank Ms. Irina Elarova, Ms. Shaona Fang, Mr Marc D. Ruben and Dr Nadeem Mohammad for expert technical assistance.