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Introduction

According to the amyloid cascade hypothesis, there is an imbalance between the production and clearance of amyloid-β (Aβ), leading to the formation of insoluble aggregates and soluble monomers and larger assemblies of Aβ which drive the synaptic loss and neuronal degeneration in the Alzheimer's disease (AD) process (1). Data supporting the view that life-long amyloid precursor protein (APP) overexpression triggers Aβ deposition and neurodegeneration come from studies on Down's syndrome patients and the finding of a duplication of the APP locus in a certain familial form of AD (2,3).

Aβ is produced through the amyloidogenic pathway from APP by proteolytic cleavage involving the two aspartyl proteases β- and γ-secretase generating Aβ1-42 and C-terminal truncated Aβ isoforms ranging from Aβ1-40 down to Aβ1-17 (4,5). In another catabolic APP pathway, β-secretase cleavage in conjunction with α-secretase cleavage result in the release of several short Aβ isoforms (Aβ1-16 down to Aβ1-13) (5) of which Aβ1-16 previously have been shown to be elevated in sporadic AD (SAD) (6) and familial AD (FAD) (7) compared to non-demented controls. Moreover, this pathway is induced upon pharmacological γ-secretase inhibitor treatment of cells, mice, dogs, rhesus monkeys and humans (5,8–11).

The first identified mutation causing FAD was a missense mutation in the APP gene (12). However, most FAD are caused by mutations in the highly homologous genes of the presenilin enzymes that constitute the active site γ-secretase, presenilin 1 (PSEN1) and presenilin 2 (PSEN2) (13,14). These mutations appear to accelerate Aβ plaque formation and have been shown to increase the Aβ1-42/Aβ1-40 ratio in primary fibroblasts and plasma of affected individuals (15–18). Using immunoprecipitation in combination with mass spectrometry, we recently showed that carriers of the FAD-associated PSEN1 A431E mutation have low CSF levels of Aβ1-37, Aβ1-38 and Aβ1-39 (7). Here we investigated the Aβ isoform pattern in the CSF from carriers of the FAD-associated PSEN1 M139T and L286P mutations.

Materials and methods

Study participants

Participants were recruited from the Genetic Counseling Program for Familial Dementias (PICOGEN) at the Hospital Clinic, Barcelona, Spain (19). Eleven subjects from 3 families with 2 PSEN1 mutations (L286P, M139T) (6 mutation carriers and 5 non-carriers) were included in the study (20,21). The L286P mutation causes familial early onset AD at a median age of 40 years and cerebral hematomas in 42% of the affected subjects. The neuropathological studies in this family reveal the typical features of AD associated with severe amyloid angiopathy and ‘cotton-wool plaques’. The M139T mutation causes familial early onset AD at a median age of 46 with typical clinical and neuropathological characteristics of AD associated with intense amyloid angiopathy but no ‘cotton-wool’ plaques or lobar hematomas. Because of the relatively predictable age of onset within a family we calculate for each participant the ‘adjusted age’ as the subject's age relative to the median familial age of onset. Subjects underwent clinical and cognitive evaluation and were classified clinically as asymptomatic if they had normal cognitive evaluation and the Clinical Dementia Rating (CDR) scale was equal to 0; or symptomatic if cognitive performance was more than 1.5 SD below the mean, with respect to age and education level, in any in any cognitive test or CDR >0 (Table I).

Table I Summary of the 11 subjects included in the study.

Table I

Summary of the 11 subjects included in the study.

Subject no.	Carrier	Mutation	CDRsum	CDRtot	MMSE	Adjusted age
1	1	L286P	2	0.5	28	−2.7
2	1	L286P	2.5	0.5	24	2.6
3	1	L286P	5.5	1	24	4.7
4	0	L286P	0	0	30	−5.2
5	0	L286P	0	0	29	−1.2
6	0	L286P	0	0	29	3.8
7	1	M139T	0	0	30	−21.8
8	1	M139T	0	0	30	−13.1
9	1	M139T	0	0	28	−12.5
10	0	M139T	0	0	29	−20.9
11	0	M139T	0	0	29	−10.6

[i] CDRsum, clinical dementia rating scale sum of boxes; CDRtot, clinical dementia rating scale total score; MMSE, mini-mental state examination score.

CSF analysis

Ten milligrams of CSF was obtained in the morning using a 22 gauge Sprotte needle. The CSF was then centrifuged, aliquoted into siliconized polypropylene Eppendorf tubes and frozen at −80˚C within 2 h of being obtained.

The study was approved by the Hospital Clinic Ethics Committee, and all participants gave informed consent to participate in the study, which was conducted according to the provisions of the Helsinki Declaration.

Immunoprecipitation (IP)-mass spectrometry (MS)

IP and MS analyses were conducted as previously described with the monoclonal antibody 6E10 (epitope 4–9, Signet Laboratories Inc., Dedham, MA, USA) and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS, Autoflex, Bruker Daltonics, Bremen, Germany) operating in reflector mode (22). The peak areas were normalized to the sum of the integrated peaks which result in a relative abundance rather than an absolute concentration pattern (6).

Results

Representative CSF Aβ isoform mass spectra from a PSEN1 L286P FAD mutation carrier and a non-carrier are shown in Fig. 1. All isoforms (Aβ1-13, Aβ1-14, Aβ1-15, Aβ1-16, Aβ1-17, Aβ1-18, Aβ1-19, Aβ1-20, Aβ1-30, Aβ1-33, Aβ1-34, Aβ1-37, Aβ1-38, Aβ1-39, Aβ1-40 and Aβ1-42) were reproducibly detected from all subjects and quantified in respect to their relative abundance.

Figure 1 Representative MALDI-TOF spectra showing Aβ isoforms immunoprecipitated from CSF using the antibody 6E10. (A) Representative Aβ isoform pattern in a control individual. (B) Representative Aβ isoform pattern in a carrier of the FAD-associated PSEN1 L286P mutation.

The MS peaks at mass-to-charge ratio 4073.0, 4130.0, 4229.1 and 4512.2, corresponding to Aβ1-37, Aβ1-38, Aβ1-39 and Aβ1-42, respectively, were lower in the PSEN1 L286P FAD mutation carriers (Fig. 2). The relative level of Aβ1-37 was reduced by 31%, Aβ1-38 by 21%, Aβ1-39 by 29% and Aβ1-42 by 81%. In contrast Aβ1-15 and Aβ1-20 were increased by 47 and 34%, respectively, while the other isoforms detected were more or less unaffected.

Figure 2 Scatter plots displaying the distribution of the Aβ isoform pattern in carriers (filled squares) and non-carriers (open squares) of the FAD-associated PSEN1 L286P mutation and carriers (filled circles) and non-carriers (open circles) of the FAD-associated PSEN1 M139T mutation.

In contrast, PSEN1 M139T carriers displayed similar levels as non-carriers of all Aβ isoforms reproducibly detected except Aβ1-42 that was present at 41% higher levels in the group harboring the mutation as compared to non-carriers (Fig. 2).

Discussion

The reduction in Aβ1-42 in the CSF from AD patients is a well-replicated finding thought to reflect the AD pathology with plaques in the brain acting as sinks trapping Aβ1-42 (23). The data presented here on L286P mutation carriers suggests that this specific PSEN1 mutation modulates γ-secretase function, which is reflected by decreased levels of Aβ1-37, Aβ1-38 and Aβ1-39, in a similar manner as the PSEN1 A431E mutation. It has been speculated that the decrease in Aβ1-37, Aβ1-38, Aβ1-39, due to the PSEN1 A431E mutation, indicates that the γ-secretase cleavage site preference has been changed in a disease-promoting manner (7). Similar changes in the levels of the isoforms have also been reported from cells expressing the PSEN1 Δ9 or L166P mutation, or the PSEN2 N141I mutation (15). Thus, modulating the γ-secretase function to boost cleavages at Gly37, Gly38 and Val39 may be an attractive approach to prevent aggregation of Aβ and consequently the formation of plaques.

The increase in Aβ1-20 in L286P mutation carriers is a finding which previously has been described for the PSEN1 A431E mutation (7). Two candidate enzymes which possibly can cleave at this position are insulin-degrading enzyme and BACE 2 (24). However, the underlying molecular mechanism needs to be further investigated.

We could not detect an increase in Aβ1-16 for L286P mutation carriers as previously described for both sporadic and familial AD patients (6,7). On the other hand, an increase by 47% was observed for Aβ1-15. Whether this is a specific finding for this particular mutation needs to be confirmed in a larger patient sample.

The median age at onset for the PSEN1 L286P mutation is 40 years. The patients included in this group had a mean age of 41.5 thus very close to the critical age and they all displayed cognitive decline. In contrast, the mean age in the patient group carrying the PSEN1 M139T mutation was 30.2 years, which is almost 16 years younger than the expected age of onset (Table I). Furthermore, L286P seems to be a more aggressive mutation than M139T with cotton-wool plaques noted in the neuropath vs. no cotton-wool present in the M139T mutation. The A431E mutation also presents a median age of onset of 40 and cotton-wool plaques, suggesting similarities in the biological effects of L286P and A431E different from M139T.

The slightly increased relative levels of Aβ1-42 in the CSF of M139T mutation carriers suggest that this particular mutation starts with an over-production of Aβ very early in life or that these subjects present normal values of Aβ1-42 while L286P mutation carriers present very low levels. Furthermore, the patients harboring the PSEN1 M139T mutation were all cognitively healthy (Table I).

It should be noted that due to the low number of individuals in the groups analyzed, no statistical analysis was performed. Moreover, there are also several non-quantitative aspects of IP-MS, which should be considered, as discussed elsewhere (25). However, the results presented here with decreased CSF levels of Aβ1-37, Aβ1-38 and Aβ1-39 in patients with a PSEN1 L286P mutation are in agreement with previous findings in PSEN1 A431E mutation carriers and suggest that the pathogenic effect of some PSEN1 mutations may be related to impaired ability of the γ-secretase complex to produce C-terminally truncated Aβ peptides that may inhibit Aβ1-42 oligomerization.

In conclusion, the data suggest that IP-MS analysis of the CSF Aβ isoform pattern may function as an additional screening tool to detect certain forms of PSEN1 mutations in patients with a family history indicating FAD. This hypothesis will be further tested on subjects having other PSEN1 mutations.

Acknowledgements

This study was supported by grants from the Swedish Research Council (projects 2006–6227, 2006–2740 and 2006–3505), the Alzheimer's Association (NIRG-08-90356), The Torsten and Ragnar Söderberg Foundation, Demensförbundet, cNEUPRO, the Royal Swedish Academy of Sciences, the Sahlgrenska University Hospital, the Inga-Britt and Arne Lundberg Research Foundation, the Göteborg Medical Society, the Swedish Medical Society, Swedish Brain Power, Stiftelsen Gamla Tjänarinnor, Gun och Bertil Stohnes stiftelse, Åhlén-stiftelsen, Alzheimer Foundation, Sweden and the Spanish Ministry of Science and Technology (FIS080036).

References