Expression of SORL1 and a novel SORL1 splice variant in normal and Alzheimers disease brain
© Grear et al; licensee BioMed Central Ltd. 2009
Received: 25 March 2009
Accepted: 04 November 2009
Published: 04 November 2009
Variations in sortilin-related receptor (SORL1) expression and function have been implicated in Alzheimers Disease (AD). Here, to gain insights into SORL1, we evaluated SORL1 expression and splicing as a function of AD and AD neuropathology, neural gene expression and a candidate single nucleotide polymorphism (SNP).
To identify SORL1 splice variants, we scanned each of the 46 internal SORL1 exons in human brain RNA samples and readily found SORL1 isoforms that lack exon 2 or exon 19. Quantification in a case-control series of the more abundant isoform lacking exon 2 (delta-2-SORL1), as well as the "full-length" SORL1 (FL-SORL1) isoform containing exon 2 showed that expression of FL-SORL1 was reduced in AD individuals. Moreover, FL-SORL1 was reduced in cognitively intact individuals with significant AD-like neuropathology. In contrast, the expression of the delta-2-SORL1 isoform was similar in AD and non-AD brains. The expression of FL-SORL1 was significantly associated with synaptophysin expression while delta-2-SORL1 was modestly enriched in white matter. Lastly, FL-SORL1 expression was associated with rs661057, a SORL1 intron one SNP that has been associated with AD risk. A linear regression analysis found that rs661057, synaptophysin expression and AD neuropathology were each associated with FL-SORL1 expression.
These results confirm that FL-SORL1 expression declines in AD and with AD-associated neuropathology, suggest that FL-SORL1 declines in cognitively-intact individuals with AD-associated neuropathology, identify a novel SORL1 splice variant that is expressed similarly in AD and non-AD individuals, and provide evidence that an AD-associated SNP is associated with SORL1 expression. Overall, these results contribute to our understanding of SORL1 expression in the human brain.
SORL1 is a mosaic protein consisting of an amino-terminal portion resembling the vacuolar protein sorting-10 (Vps10) receptor family and a carboxyl-terminal portion having attributes of the low-density lipoprotein receptor (LDLR) family. As such, this type-1 transmembrane protein is capable of binding ligands ranging from receptor-associated protein (RAP) to apolipoprotein E (apoE) . Recently, SORL1 has also been associated with AD at several levels. First, SORL1 interacts with amyloid precursor protein in the Golgi and endosomes to reduce production of the amyloid-β (Aβ) peptide, i.e., decreased SORL1 expression results in increased Aβ production in vitro and in a murine model in vivo [2–4]. Second, SORL1 expression is decreased in the neurons of sporadic AD patients [5, 6], consistent with its possible role in contributing to Aβ accumulation. Furthermore, SORL1 expression is not decreased in familial AD, suggesting that diminished SORL1 expression is likely not a consequence of amyloid accumulation . Third, genetic variants within SORL1 have been associated with AD in many case-control series although other studies have failed to achieve significance [8–14]. In summary, SORL1 variants that reduce SORL1 expression or function may increase AD risk by increasing Aβ production.
Alterations in RNA splicing have emerged as a major mechanism of action of functional genetic variants in diseases ranging from frontotemporal dementia to atypical cystic fibrosis to myotonic dystrophy (reviewed in [15, 16]). Here, we report an analysis of SORL1 expression and splicing in AD versus non-AD brain, reporting that the expression of FL-SORL1 but not delta-2-SORL1 is associated with AD and AD neuropathology, synaptophysin expression, and an AD-associated SNP.
Human autopsy tissue
The University of Kentucky AD Center Neuropathology Core generously provided human brain specimens from the anterior cingulate as well as superior and middle temporal gyri. Diagnoses of AD and non-AD were performed at a consensus conference of the AD Center Neuropathology and Clinical Cores and were based upon evaluation of both cognitive status, i.e., Clinical Dementia Rating and Mini-Mental State Examination (MMSE) scores, as well as neuropathology, i.e., Braak stages which rate the extent of neurofibrillary pathology into the neocortex and NIA-Reagan Institute (NIA-RI) neuropathology classification, which includes counts of both neuritic senile plaques and neurofibrillary tangles and provides a likelihood staging of AD neuropathological diagnosis [17, 18]. The age at death for individuals that were cognitively intact, i.e., non-AD, was 82 ± 9 years (mean ± SD, n = 28) while age at death for AD individuals was 82 ± 6 (n = 29). The average post-mortem interval (PMI) for non-AD individuals was 2.8 ± 0.8 hours (mean ± SD, n = 28) while the PMI for AD individuals was similar at 3.4 ± 0.6 hours (n = 29). Non-AD individuals had Mini-Mental State Examination (MMSE) scores of 28.4 ± 1.6 (n = 28). Neuropathologic evaluation by NIA-RI criteria revealed that among the 28 non-AD individuals, a total of 12 had findings consistent with no likelihood of AD (age at death of 79.8 ± 12.0 (mean ± SD)), while another 10 were categorized as having low likelihood (age at death of 84.6 ± 4.9 (n = 10), and six as moderate likelihood (age at death of 85.0 ± 3.5). The postmortem interval for individuals in each class was similar, i.e., 3.3 ± 0.7 (mean ± SD, n = 12), 2.4 ± 0.7 (n = 10), and 2.0 ± 0.4 (n = 6), respectively. Additionally, the 28 non-AD, cognitively intact individuals included 20 individuals that were Braak stages 0-II, while the remaining eight individuals were Braak stages III-V . Neuropathology in AD individuals was robust, i.e., Braak stages were uniformly VI. The temporal lobe samples were prepared to compare SORL1 expression in matched white and gray matter samples. For this study, white and gray matter were carefully dissected from the temporal lobe; the quality of the separation was confirmed subsequently by a comparison of the ratio of mRNAs associated with neurons, astrocytes and oligodendrocytes, i.e., neurofilament-H, GFAP, and myelin basic protein, respectively.
SORL1 PCR primers.
Exon 1 Forward
Exon 5 Reverse
Exon 4 Forward
Exon 8 Reverse
Exon 7 Forward
Exon 11 Reverse
Exon 10 Forward
Exon 15 Reverse
Exon 14 Forward
Exon 18 Reverse
Exon 17 Forward
Exon 21 Reverse
Exon 20 Forward
Exon 24 Reverse
Exon 23 Forward
Exon 29 Reverse
Exon 28 Forward
Exon 32 Reverse
Exon 31 Forward
Exon 35 Reverse
Exon 34 Forward
Exon 40 Reverse
Exon 39 Forward
Exon 43 Reverse
Exon 42 Forward
Exon 46 Reverse
Exon 45 Forward
Exon 48 Reverse
Exon 2 Forward
Exon 1-3 Junction Forward
Exon 4 Reverse
Primers were designed to amplify SORL1 that retained or lacked exon 2 (Table 1). Briefly, PCR product corresponding to FL-SORL1, i.e., the isoforms containing exon 2, was amplified by using a sense forward primer corresponding to sequence within exon 2 and an antisense reverse primer corresponding to sequence within exon 4. To quantify delta-2-SORL1, i.e., the isoform lacking exon 2, a sense primer corresponding to the exon 1-3 junction was used in combination with the exon 4 antisense primer (Table 1). Amplification reactions were 1 μM with respect to each primer, 1× SYBR-green Master Mix (Applied Biosystems) and the equivalent of 20 ng of template cDNA. PCR profiles consisted of a 10 minute pre-incubation period at 95°C followed by 40 cycles of 94°C for 30 s, 60°C for 30 s and 72°C for 30 sec (BioRad Chromo4). Primer specificity was ensured by separating the PCR products via polyacrylamide gel electrophoresis and SYBR-gold staining as well as melting curves at the completion of real-time PCR. Each cDNA sample was quantified in two separate reactions relative to standard curves generated with purified and quantified PCR product standards. The copy numbers of the SORL1 isoforms were normalized relative to the geometric mean of the copy numbers of hypoxanthine-guanine phosphoribosyltransferase and ribosomal protein L13A . DNA samples were genotyped for rs661057 by using unlabeled PCR primers and TaqMan FAM and VIC dye-labeled MGB probes (Applied Biosystems).
The percentage of the delta-2-SORL1 isoform relative to total SORL1 expression was calculated as the normalized copy number of delta-2-SORL1 divided by the normalized total SORL1 copy number. Differences in gene expression as a function of AD or gray matter versus white matter were evaluated by the two sample t-test; differences in expression as a function of Braak stage, NIA-RI likelihood for the neuropathological diagnosis of AD, or rs661057 were analyzed by one-way analysis of variance. The association of SORL1 expression with Braak stage, synaptophysin and/or rs661057 was evaluated by using linear regression analyses (SPSS v. 17).
Expression of SORL1 isoforms in AD versus non-AD brains.
Mean ± SE
1.978 ± 0.136
1.537 ± 0.140
1.935 ± 0.134
1.493 ± 0.138
0.043 ± 0.003
0.044 ± 0.003
2.29 ± 0.13
3.02 ± 0.14
Expression of SORL1 isoforms relative to Braak stage.
Mean ± SE
2.167 ± 0.150
1.507 ± 0.228
1.537 ± 0.140
2.121 ± 0.150
1.470 ± 0.222
1.493 ± 0.138
0.046 ± 0.003
0.037 ± 0.006
0.044 ± 0.003
2.26 ± 0.16
2.48 ± 0.15
3.02 ± 0.14
Expression of SORL1 isoforms in human brain as a function of NIA-RI neuropathology classification.
Mean ± SE
2.023 ± 0.151
1.584 ± 0.128
1.980 ± 0.150
1.540 ± 0.126
0.043 ± 0.003
0.044 ± 0.003
2.25 ± 0.15
2.93 ± 0.13
Expression of SORL1 isoforms in human brain as a function of rs661057 genotype.
Mean ± SE
2.249 ± 0.196
1.383 ± 0.086
2.081 ± 0.295
2.199 ± 0.195
1.344 ± 0.084
2.033 ± 0.292
0.050 ± 0.003
0.039 ± 0.002
0.048 ± 0.007
2.36 ± 0.20
2.88 ± 0.14
2.46 ± 0.28
Estimated Marginal Means for FL-SORL1 Expression.
Mean ± SE
2.058 ± 0.127
1.706 ± 0.109
2.091 ± 0.137
1.557 ± 0.112
1.998 ± 0.194
The primary findings of this report are four-fold. First, the expression of FL-SORL1 and total SORL1 is reduced in the AD brain, confirming prior reports [5, 6]. Second, the expression of FL-SORL1 and total SORL1 is decreased in cognitively intact individuals with moderate AD neuropathology, consistent with the possibility that SORL1 declines early in the disease. Third, we report the identification of novel SORL1 isoforms lacking exons 2 or 19. Quantification of the delta-2-SORL1 isoform reveals that this isoform is expressed at similar levels in AD and non-AD individuals. Lastly, we interpret our data as suggesting a model for SORL1 expression that includes AD neuropathology, synaptophysin expression, and rs661057, an AD-associated SNP. Overall, these results provide insight into variables associated with SORL1 expression and show that exon skipping is a rare event in SORL1 mRNA.
Since the first report of reduced SORL1 expression in AD neurons in 2004 , a predominant theory has emerged that a reduction in functional SORL1 contributes to increased Aβ and, thereby, increased AD risk [2, 6–8]. Here, we confirm prior results showing that SORL1 expression is generally reduced in AD [5, 6]. Additionally, we report that SORL1 expression is reduced in individual that were cognitively intact but yet had moderate AD-like pathology, and hence could represent "preclinical" AD subjects [23, 24]. This result is similar to that of Sager et al.  who reported that SORL1 expression in individuals with mild cognitive impairment (MCI) was quite variable such that SORL1 expression in some MCI individuals was similar to normal individuals while SORL1 expression in other MCI individuals was reduced to levels similar to those seen in AD. Hence, the decreased SORL1 expression that we observed here in cognitively intact individuals with moderate AD neuropathology is suggestive that declines in SORL1 expression presage the onset of AD dementia.
To investigate the role of SORL1 splicing in SORL1 variation, we evaluated each of the SORL1 exons to identify those that are frequently not present within mature SORL1 mRNA. This led to the identification of the delta-2-SORL1 and delta-19-SORL1 isoforms. The former was modestly enriched in white matter relative to gray matter, consistent with its lack of association with synaptophysin expression. The latter was present at levels too low for reliable quantitation in many of the samples, and hence we were unable to compare expression among groups. The function of the proteins encoded by these novel isoforms is not currently known. Delta-2-SORL1 encodes a SORL1 protein variant that lacks amino acids V96-D134. The functional consequences of loss of this portion of the Vps10p domain are not known but could impact sorting properties of the protein. The loss of exon 19 in delta-19-SORL1 introduces a codon frameshift, resulting in an isoform that encodes the normal SORL1 protein until amino acid 857, followed by a novel 29 amino acid sequence and a premature stop codon after amino acid 886 (Figure 2). Hence, this truncated receptor is predicted to contain the intact Vsp10p domain, as well as the first two LDLR class B repeats but lack several LDLR class A repeats, the fibronectin type III domain, the transmembrane domain and the cytosolic tail. Although the function of this truncated, delta-19-encoded soluble receptor is not yet clear, Jacobsen et al. evaluated a SORL1 minireceptor that consisted of the 731 amino-terminal SORL1 amino acids and found that this truncated receptor bound RAP but not apoE . A similar function may be found for delta-19-SORL1. In summation, while the function of protein encoded by delta-2-SORL1 is unclear, we speculate that the protein encoded by delta-19-SORL1 may represent a dominant negative form of SORL1 that binds some SORL1 ligands, but, since it lacks the cytosolic tail, will not modulate their sorting. If either of these SORL1 variants represent loss of function for SORL1, they may contribute to increased Aβ production and, thereby, AD risk.
In evaluating factors that associate with SORL1 expression, we arrived at a model that includes (i) AD-associated neuropathology, as reflected by Braak stage, (ii) neuronal gene expression, as reflected by synaptophysin expression, and (iii) a SORL1 intron 1 SNP, rs661057, that has been associated with AD in at least some series . The association between synaptophysin and SORL1 expression likely reflects neuronal expression of SORL1 . The mechanisms underlying the association between SORL1 expression and Braak stage or rs661057 are unknown. Regarding rs661057, individuals that are heterozygous for this SNP had lower SORL1 expression than individuals homozygous for the major or minor allele. Hence, rs661057 would appear to show homozygote dominance, with an unclear underlying mechanism. Consideration of the association between rs661057 and AD does not clarify this situation (meta-analysis at http://www.alzgene.org/,) because the SNP seems to show a genotype dose dependent association with AD. Evaluation of the widespread reproducibility of rs661057 association with SORL1 expression in other studies will be of interest to the field.
In summary, we confirmed that SORL1 expression is reduced in the AD brain. Moreover, SORL1 expression was decreased as a function of AD-associated neuropathology prior to dementia, suggesting that the SORL1 reduction precedes conversion to AD. The exons of SORL1 were generally included within the mature message with high efficiency, with only exons 2 and 19 being skipped at detectable levels. Delta-2-SORL1 was modestly enriched in white matter and unchanged in AD. Lastly, Braak stage, synaptophysin expression, and rs661057 were all found to be associated with SORL1 expression.
includes low-density lipoprotein receptor
mini-mental state exam
National Institute on Aging
mild cognitive impairment.
The authors gratefully acknowledge tissue supplied by the University of Kentucky Alzheimers Disease Center, which is supported by P30AG028383, as well as NIH for grant support (R01AG026147 to SE, R01AG031784 to GB and P01AG030128 to SE and GB).
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