Danish Dementia Mice Suggest That Loss of Function and Not the Amyloid Cascade Causes Synaptic Plasticity and Memory Deﬁcits

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Danish dementia mice suggest that loss of function and not the amyloid cascade causes synaptic plasticity and memory deﬁcits

Robert Tamayeva, Shuji Matsudaa, Mauro Fàb, Ottavio Aranciob, and Luciano D’Adamioa,1

aDepartment of Microbiology & Immunology, Albert Einstein College of Medicine, Bronx, NY 10461; and bDepartment of Pathology and Cell Biology, Taub Institute for Research on Alzheimer’s Disease and the Aging Brain, Columbia University, New York, NY 10032

Edited* by Thomas C. Südhof, Stanford University School of Medicine, Palo Alto, CA, and approved October 25, 2010 (received for review August 9, 2010) According to the prevailing “amyloid cascade hypothesis,” genetic cascade pathogenic hypothesis and suggests that other mecha- dementias such as Alzheimer’s disease and familial Danish dementia nisms cause memory loss in Danish patients. (FDD) are caused by amyloid deposits that trigger tauopathy, neuro- Recent evidence has also raised the suggestion that a loss of degeneration, and behavioral/cognitive alterations. To efficiently function mechanism could play a role in dementia. Mutations in reproduce amyloid lesions, murine models of human dementias in- familial AD genes PSEN1/PSEN2 reduce PSEN1/PSEN2 func- variably use transgenic expression systems. However, recent FDD tion (5), and deletion of PSEN1/PSEN2 in mice causes neuro- transgenic models showed that Danish amyloidosis does not cause degeneration, synaptic dysfunction and memory loss (6, 7). memory defects, suggesting that other mechanisms cause Danish Because a loss of function mechanism cannot be uncovered by dementia. We studied an animal knock-in model of FDD (FDDKI/+) a transgenic approach due to the persistence of the endogenous genetically congruous with human cases. FDDKI/+ mice present re- wild type (WT) alleles, we generated a genetically coherent an- duced Bri2 levels, impaired synaptic plasticity and severe hippocam- imal model of FDD (8). As FDD is an autosomal-dominant dis- pal memory deficits. These animals show no cerebral lesions that ease, we studied FDDKI/+ mice carrying one mutant and one WT are reputed characteristics of human dementia, such as tangles or Bri2 allele (Fig. S1B), identical to FDD patients. To advance our Bri2+/− fi amyloid plaques. mice exhibit synaptic and memory de cits understanding of FDD pathogenesis, we analyzed amyloid and similar to FDDKI/+ mice, and memory loss of FDDKI/+ mice is pre- tau pathology, BRI2 protein expression, synaptic plasticity, and vented by expression of WT BRI2, indicating that Danish dementia cognition in FDD mice. is caused by loss of BRI2 function. Together, the data suggest that KI/+ clinical dementia in Danish patients occurs via a loss of function Results mechanism and not as a result of amyloidosis and tauopathy. FDDKI/+ Mice Do Not Present with FDD-Related Neuropathology. We have previously shown an absence of neuronal loss, GFAP+ amyloid-β precursor protein | ITM2b activated astrocytes and ADan amyloid peptide deposits in brain sections of 18-mo-old FDDKI/KI mice (8). Additional hematox- he condition known as Danish dementia (FDD) is due to ylin and eosin (H&E) staining again showed no significant neu- Ta dominant autosomal mutation in the BRI2/ITM2b gene (1). ronal loss in FDD compared with WT littermates (Fig. S2A). KI/+ The BRI2 precursor (immature, imBRI2) is a type-II membrane GFAP and NeuN Western blots of hippocampal lysates also protein that is cleaved at the C terminus by proprotein con- showed no differences between FDDKI/+ and WT mice (Fig. vertase to produce the mature BRI2 protein (mBRI2) and a S2B). Crystal violet and Congo red staining confirmed the ab- 23aa soluble C-terminal fragment (2). FDD is caused by a 10- sence of amyloid formations in these brain sections (Fig. 1A). nucleotide duplication before the stop codon of the BRI2 gene In FDD, Alzheimer’sAβ codeposits with ADan, mainly in vas- (1). The Danish mutant protein (BRI2-ADan) generates a lon- cular and perivascular amyloid lesions (4). However, Aβ immu- A ger C-terminal fragment, ADan (Fig. S1 ), which forms wide- noreactivity was not seen in FDDKI/+ mice (Fig. 1A), whereas it was spread amyloid angiopathy in the small blood vessels and present in an Aβ-depositing transgenic mouse (9). Tau hyper- capillaries of the cerebrum, choroid plexus, cerebellum, spinal phosphorylation is a characteristic of FDD. However, brains of cord, and retina (1). Neuropathological examination of patients FDDKI/+ mice did not show signs of tauopathy (Fig. 1 B and C, and with FDD also shows diffuse brain atrophy with a particularly Fig. S2C). CP13 (specific to phosphorylated Ser202 that detects severe involvement of the cerebellum, cerebral cortex and white pathological tau in both early and advanced stages), PHF1 (which matter, as well as the presence of very thin and almost demye- detects phosphorylated Ser396 or Ser404 as markers of tauopathy linated cranial nerves; neurofibrillary tangles are the major his- in later-stage tangles) as well as two other anti–phospho-tau tological finding in the hippocampus (1). monoclonal antibodies [CP9 (pT231), MC6 (pS235)] (10), did not Vidal et al. have described a transgenic mouse overexpressing reveal differences between FDDKI/+ and WT littermates. Thus, BRI2-ADan, which presents widespread cerebral amyloid angi- the brains of FDDKI/+ mice maintained normal morphology for opathy and parenchymal amyloid deposition of the ADan pep- mice of this age, and were free of amyloid deposits and tauopathy. tide (3). More recently, another FDD transgenic model with ADan deposition (called ADanPP-Tg mice) has been described (4). ADanPP-Tg mice do not show cognitive and memory defects Author contributions: L.D. designed research; R.T., S.M., and L.D. performed research; in the first 12 mo of life despite considerable amyloid load. At an M.F. and O.A. contributed new reagents/analytic tools; R.T., S.M., M.F., O.A., and L.D. analyzed data; and L.D. wrote the paper. older age, ADanPP-Tg mice show deficits with the cue naviga- Conflict of interest statement: L.D. is a cofounder of Remegenix, a biotechnology com- tion task of the Morris water maze associated with an increase of pany that has licensed a patent on the FDDKI mice from the Albert Einstein College of thigmotaxis and passive floating, lower speed, less body weight, Medicine. L.D. is also an inventor on the patent. and an anxiety-related phenotype (4). Thus, although the ADan *This Direct Submission article had a prearranged editor. peptide is believed to cause Danish dementia, the absence of 1To whom correspondence should be addressed. E-mail: [email protected]. fi memory de cits despite massive ADan peptide production and This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. deposition in ADanPP-Tg mice is inconsistent with the amyloid 1073/pnas.1011689107/-/DCSupplemental.

20822–20827 | PNAS | November 30, 2010 | vol. 107 | no. 48 www.pnas.org/cgi/doi/10.1073/pnas.1011689107 Downloaded by guest on September 27, 2021 fronto-temporal region. The pattern seen in human brains is strikingly similar to that seen in transfected cells. Normal brains showed a predominant BRI2 species, whereas FDD brain samples showed two bands (Fig. 2B). The lower band comigrated with the BRI2 band detected in control samples and was reduced in quantity compared with that in controls. The larger form, detect- able only in FDD cases, represented ∼40% of the total BRI2 protein (Fig. 2B). In FDDKI/+ mice, Bri2 levels were also reduced (Fig. 2 C and D). Fractionation of synaptosomes showed that Bri2 was expressed in the endoplasmic reticulum/Golgi fraction (P3), crude (P2) and puriﬁed (LP1) synaptic membranes from WT mice (Fig. 2E), but was substantially reduced in all fractions isolated Fig. 1. No cerebral amyloidosis and tauopathy in FDDKI/+ mice. (A)Aβ- from FDDKI/+ mice (Fig. 2 E and G). These differences are not amyloid is visible in APP/PS1 transgenic mice, used as positive controls, but due to defective transcription/splicing of the mutant allele because not within the FDD brain. (B) Brain lysates from three WT and three KI/+ ∼50% of the Bri2 mRNA coded for the mutant protein in the FDDKI/+ mice are analyzed by immunoblot using anti–phospho-tau (CP13, PHF1, CP9, MC6) and anti–total-tau (DA9) antibodies. (C) Densitometry val- FDDKI/+ brains. Together, the data indicate that the FDD mu- ues show no difference in phospho-tau between the two genotypes. tation compromises formation of BRI2 in vivo.

Synaptotoxic Oligomers Are Not Increased in FDDKI/+ Mice. BRI2 Danish ITM2b/BRI2 Mutation Compromises Formation of Mature BRI2 binds amyloid-β precursor protein (APP) and reduces Aβ pro-

in Both FDD Patients and FDDKI/+ Mice. To determine whether the duction in vivo (11). Analysis of brain samples from 11-mo-old Danish mutation affects the BRI2 protein, we first analyzed animals showed that Aβ40 levels were not significantly changed in transfected cells. In cells transfected with BRI2-ADan, two bands FDDKI/+ mice compared with WT littermates (Fig. 3A), indicating were visible. The upper band, which is the most abundant, corre- that either Aβ production is not increased in FDDKI/+ mice or that sponds to BRI2-ADan, as it is specifically recognized by an anti- compensatory mechanisms (such as Aβ40 degradation or export of body specific for the ADan peptide (1). The lower band is Aβ40 from the CNS to peripheral tissues) maintain homeostatic β recognized only by the antibody against the NH2 terminus of BRI2 levels of A 40 despite increased production. Because it has been and corresponds to mBRI2. mBRI2 was the predominant BRI2 postulated that toxic forms of amyloid proteins are oligomers (12), species observed in cells transfected with WT BRI2 (Fig. 2A). To we tested whether synaptotoxic oligomers, possibly composed of determine whether this altered pattern of expression was also seen Aβ42 and/or ADan peptides, are augmented in FDDKI/+ mice. To for endogenous BRI2, we compared two human FDD with two this end, we used the prefibrillar oligomer-specific antibody A11 to age-matched, normal control human samples isolated from the perform dot blots (13). We found no differences in neurotoxic NEUROSCIENCE

Fig. 2. Reduced synaptic Bri2 in FDDKI/+ mice. (A) HeLa cells were transfected with either WT BRI2 or BRI2-ADan. Only mBRI2 is detected in cells transfected with WT BRI2. In cells transfected with mutant BRI2, BRI2-ADan represents the majority of total BRI2 species. (B) Analysis of brain lysates from either FDD

patients (FDD) or normal controls (con.). (C and D) Bri2 levels are significantly reduced in FDDKI/+ compared with WT control brains (**P < 0.01). In the right panel of C, samples from FDDKI/KI mice show an even more dramatic reduction in Bri2 levels. Longer exposure of this blot (boxed image) shows expression of Bri2 species compatible with imBri2 and BRI2-ADan. (E) Bri2 is present in total lysates (S1), Endoplasmic reticulum/Golgi fraction (P3), crude (P2), and purified −/− synaptic membranes (LP1) of WT animals, whereas in FDDKI/+ mice, Bri2 is lower in all analyzed fractions. *Nonspecific bands also present in Bri2 mice. Proteins (5 μg) were analyzed for each fraction, except for the blots probed with α-SNAP25 and α-LC3 antibodies, where 15 μg proteins was loaded for S1

fraction and 5 μg for the others. (F and G) mBri2 is signiﬁcantly reduced in synaptic membranes of FDDKI/+ mice (***P < 0.003).

Tamayev et al. PNAS | November 30, 2010 | vol. 107 | no. 48 | 20823 Downloaded by guest on September 27, 2021 we investigated synaptic transmission and plasticity using the Schaffer collateral pathway in hippocampal slices from WT and FDDKI/+ mice. Basal synaptic transmission (BST) was de- termined by measuring the slope of the ﬁeld excitatory postsynaptic potential (fEPSP). We found no difference in BST between FDDKI/+ and WT mice (Fig. S3A). Paired-pulse facili- tation (PPF) was also similar in FDDKI/+ and WT mice (Fig. S3B), suggesting that no changes in Ca2+ mobilization or alterations in the probability of neurotransmitter release were driven by the mutation. Long-term potentiation (LTP), a long-lasting form of synaptic plasticity that is associated with learning and memory, was similar in 3-mo-old FDDKI/+ mice and littermates (Fig. 4A). However, LTP was reduced in 11- to 13-mo-old FDDKI/+ mice (Fig. 4B). Thus, the Danish mutation in Bri2 selectively compromises LTP in older mice.

FDDKI/+ Mice Are Cognitively Impaired. The LTP deﬁcits exhibited by FDDKI/+ mice compelled us to test behavior and memory. Spon- taneous alternation and open-ﬁeld studies showed that FDDKI/+ mice have no defects in habituation and locomotor behavior, motor coordination, sedation, risk assessment, and anxiety-like behavior in novel environments (Figs. S4 and S5). A cohort of FDDKI/+ and WT mice was subjected to novel object recognition (NOR), a nonaversive task that relies on the mouse’s natural ex- ploratory behavior, at age 3–4, 5–6, and 7–8 mo. During the training sessions, both FDDKI/+ and WT mice spent the same amount of time exploring two identical objects at all ages (Fig. 5 A, D, and G). The following day, when a novel object was introduced to replace one of the two old objects, 3- to 4-mo-old FDDKI/+ and B – β WT mice preferentially explored the novel object (Fig. 5 ). At 5 6 Fig. 3. A and synaptotoxic oligomers are not increased in FDDKI/+ mice. (A) – Quantitation of mouse Aβ40 in the brains of 11-mo-old mice. (B) Brain lysates and 7 8 mo of age, however, FDDKI/+ spent the same amount of

from four WT and five FDDKI/+ mice are analyzed by dot-blots using prefibrillar time exploring the two objects as if they were both novel to them, oligomer-specific antibody A11. Brain lysates from APP/PS1 transgenic mice are whereas the WT mice still spent more time exploring the novel used as positive controls for A11 staining. (C and D) Densitometry analysis of object (Fig. 5 E and H). A different cohort of FDDKI/+ and WT blots shown in B (C) and of S3 soluble fraction samples (D) (WT being set to 100). littermates was tested at 11 mo of age, verifying the profound defects of FDDKI/+ (Fig. 5 J and K), and confirming that memory is impaired in FDDKI/+ mice upon aging in an ethologically relevant, amyloid oligomer levels between FDDKI/+ mice and WT litter- nonaversive behavioral context. B C mates either in total brain lysates (Fig. 3 and ) or in synaptic Next, the same two cohorts of mice were used in the radial- soluble protein fractions (Fig. 3D). arm water maze (RAWM) task to examine spatial working memory, which depends upon hippocampal function (15) and Long-Term Potentiation in the Schaffer Collateral Pathway Is tests short-term memory, a type of memory that is affected at Compromised in FDDKI/+ Mice. In 1928 Ramon y Cajal predicted early stages of AD. Mice were required to learn and to memorize that dementia results from the weakening of synapses and some the location of a hidden platform in one of the six arms of a maze evidence supports a role for synaptic dysfunction underlying with respect to spatial cues. WT mice were able to acquire (A) subtle memory changes in AD (14). The synaptic localization of and retain (R) memory of the task at all ages. FDDKI/+ mice 3–4 mBri2 suggests that this protein regulates, albeit with a yet-to-be- mo of age performed the task similarly to their WT littermates revealed mechanism, synaptic functions. The depletion of mBri2 (Fig. 5C). At 5–6 mo of age, however, FDDKI/+ mice showed in synaptic membranes of FDDKI/+ mice is consistent with defective retention of the task (Fig. 5F). More striking differ- the hypothesis that FDD begins as a synaptic disease. Thus, ences were seen at 7–8 mo of age, and with the cohort of 11-mo-

Fig. 4. Impaired LTP in FDDKI/+ mice. (A and B) LTP is impaired in FDDKI/+ with increased age. CA1 ﬁeld-excitatory- postsynaptic potentials (fEPSPs) were recorded by placing both the stimulat- ing and the recording electrodes in CA1 stratum radiatum. A 30-min baseline was recorded every minute at an in- tensity that evoked a response of ∼35% of the maximum evoked response. LTP was induced using θ-burst stimulation (10 bursts repeated at 5 Hz, each burst consisting of four pulses at 100 Hz). Responses were recorded for 2 h after tetanization and plotted as percentage

of baseline fEPSP slope. LTP is normal in 3-mo-old animals [F(1,12) = 0.592; P = 0.465] but is impaired in 11- to 13-mo-old FDDKI/+ animals [F(1,16) = 9.65; P = 0.007]. Representative traces 1 min before (thin) and 120 min after (thick) θ-burst stimulation are shown.

20824 | www.pnas.org/cgi/doi/10.1073/pnas.1011689107 Tamayev et al. Downloaded by guest on September 27, 2021 of cerebral amyloidosis and tauopathy, but correlate with synaptic depletion of mBri2. This suggests that clinical dementia occurs independently of pathological lesions in FDD and via a loss of function mechanism. To test for this, we studied FDDKI/+ mice over-expressing human WT BRI2. In these transgenic mice, human BRI2 is under the control of the CaMKII promoter (16), that drives expression of transgenes in the postnatal forebrain (17), which is the area markedly affected by AD. As shown in Fig. 6 A and B, expression of WT BRI2 in the forebrain of FDDKI/+ mice prevents memory deficits due to the Danish mutation in both RAWM and NOR. +/− Next, we analyzed Bri2 mice (16), which express reduced +/− mBri2 levels and do not produce the ADan peptide. Bri2 animals show memory impairments in RAWM and NOR tests similar to FDDKI/+ mice (Fig. 6 C and D). To extend the parallel analysis +/− of FDDKI/+ and Bri2 mice, we used the fear-conditioning learning test (18). In the training phase, the animals were placed in a fear-conditioning box and were exposed to conditioned stimulus (CS, a tone) paired with an unconditioned stimulus (US, a mild foot shock). Then, 24 h later, conditioning was assessed by measuring the absence of all movement except for that necessitated by breathing (“freezing” behavior), in response to the context [contextual fear conditioning, which depends on hippocampal functions (19)] or the CS within a completely different context [cued conditioning, an amygdala-dependent and hippocampus-independent task (19)]. We found no difference in +/− freezing between either FDDKI/+ or Bri2 and their respective WT littermates during the training phase (Fig. 6E). Twenty-four hours later, we found a decrease in the freezing time of both +/− FDDKI/+ and Bri2 mice compared with WT littermates in contextual conditioning (Fig. 6E). Cued fear-conditioning tests identified no difference in freezing behavior among the different genotypes (Fig. 6F), suggesting that the amygdala is not impaired in these mice. These findings indicate that both FDDKI/+ and +/− Bri2 mice have a selective hippocampus-dependent impairment in associative learning. To further analyze the similarity in functional deficits between +/− FDDKI/+ and Bri2 mice, we performed LTP studies and mea- +/− sured Aβ40 and oligomers in Bri2 mice. We found that LTP was impaired (Fig. 6G), whereas basal synaptic transmission was nor- +/− mal (Fig. S7)inBri2 animals. In addition, Aβ40 (Fig. 6H) and oligomers (Fig. 6 I and J) levels were not significantly changed in – +/− Fig. 5. FDDKI/+ mice develop age-dependent memory impairments. (A C)No the brains of Bri2 mice compared with WT littermates. These NEUROSCIENCE – – memory defects in FDDKI/+ mice at 3 4 mo of age (P = 0.64). (D L)FDDKI/+ mice genetic data reaffirm that loss of Bri2 function rather than the fi show a progressive loss of memory as they age. FDDKI/+ mice develop de cits in ADan peptide causes memory deficits in FDD mice. object recognition and spatial working memory as early as 5–6 mo of age. The KI/+ fi – defects progress to a larger de cit in memory at 7 8 mo of age. A different Discussion cohort of mice was used at 11 mo of age, confirming the loss of memory over time. *P < 0.05, **P < 0.005, ***P < 0.0002, ****P < 0.0001. The so-called amyloid cascade hypothesis (20) posits that the ac- cumulation of neurotoxic amyloidogenic peptides triggers tauopathy, neurodegeneration, and cognitive/behavioral changes. Murine models of human dementia invariably use transgenic ex- old mice, when FDD mice showed severe abnormalities in KI/+ pression systems, which are genetically incongruous with the human short-term spatial memory for platform location during both fi I L diseases because the mutant transgenes are expressed in an arti cial acquisition and retention of the task (Fig. 5 and ). This defect quantitative-spatiotemporal manner, to reproduce amyloidosis (3, fi fi was due to a de cit in memory per se and not to de cits in vision, 4, 21). These models, moreover, cannot replicate loss of function, motor coordination, or motivation because testing with the vis- given the persistence of the two endogenous WT mouse alleles. On fi ible platform showed no difference in the time needed to nd the the contrary, KI models (8, 22, 23) are genetically faithful to the platform and swimming speeds between the FDDKI/+ and WT human pathology and can uncover pathological deficits due to loss mice (Fig. S6). In contrast to this early and major memory of gene function, albeit they do not reproduce amyloidosis. – phenotype of FDDKI/+ mice, adult ADanPP-Tg mice (6 12 mo Memory loss has not been described in transgenic mouse of age) show no memory deficits notwithstanding the consider- models of FDD, even if they exhibit considerable amyloidosis (3, able ADan peptide deposition. Deficiencies due to an anxiety 4); these data do not validate the amyloid cascade hypothesis. phenotype and locomotor dysfunction, and not memory loss, are Analyzing memory in the FDDKI/+ model allows us to directly detected in 18- to 20-mo-old ADanPP-Tg mice (4). test the amyloid cascade hypothesis. We show that FDDKI/+ mice present with severe memory impairments already at 5–6moof Loss of Bri2 Causes Synaptic and Cognitive Deficits. Synaptic plas- age, despite the absence of amyloid and tau lesions. Interestingly, ticity and memory deficits in FDDKI/+ mice occur in the absence FBDKI/+ mice present with a similar phenotype (24).

Tamayev et al. PNAS | November 30, 2010 | vol. 107 | no. 48 | 20825 Downloaded by guest on September 27, 2021 Fig. 6. Memory impairment in FDDKI/+ mice is due to loss of Bri2 function. (A) In RAWM tests, 6–7-mo-old FDDKI/+ mice perform signiﬁcantly worse than WT mice [FDDKI/+ vs. WT: (A2) P = 0.0020, (A3) P = 0.0015, (A4) P = 0.0008, (R) P ≤

0.0001]. However, FDDKI/+ mice expressing transgenic WT BRI2 (FDDKI/+/TgBri2) perform equally as well as WT littermates and showing that WT BRI2 prevents the deﬁcit of FDDKI/+ mice [FDDKI/+ vs. FDDKI/+/TgBri2: (A3) P = 0.013, (A4) P = 0.047, (R) P = 0.012]. (B) The same four sets of mice were placed through

the NOR paradigm. WT, Bri2tg, and FDDKI/+/Bri2tg mice had no novel object recognition deﬁcit. FDDKI/+ mice were the only mice in the set that could not recall the old object.

[FDDKI/+ vs. WT: P = 0.0002; FDDKI/+ vs. FDDKI/+/Bri2tg: P = 0.0055; FDDKI/+ vs. Bri2tg: P = 0.0069]. (C) A total of 11 WT and 11 Bri2 haplo-deﬁcient male mice at the age of 6 mo were

subjected to the RAWM task. Similarly to the FDDKI/+, the Bri2 haplo-deﬁcient mice were unable to perform in the RAWM as well as the WT. *P < 0.05, **P < 0.005, ***P < 0.0001, ****P < 0.0001. (D) The same cohort of WT and Bri2 haplo-deﬁcient

mice were subjected to a NOR task. Like FDDKI/+ animals, − Bri2+/ mice were unable to recognize the old object. *P < +/− 0.005. (E) Both FDDKI/+ and Bri2 mice show no difference in freezing during training compared with WT mice (baseline; +/− P = 0.35 for FDDKI/+ vs. WT, P = 0.81 for Bri2 vs. WT). However, contextual fear conditioning performed 24 h after training showed a reduction of freezing responses in both +/− FDDKI/+ and Bri2 mice compared with WT animals (FDDKI/+ − vs. WT, P = 0.0017; P = 0.0075 for Bri2+/ vs. WT). *P < 0.01, +/− **P < 0.005. (F)FDDKI/+, Bri2 mice and WT littermates showed similar freezing during cued conditioning (P = 0.35 +/− for FDDKI/+ vs. WT, P =0.81forBri2 vs. WT). (G) LTP is im- − paired in 9- to 10-mo-old Bri2+/ mice. LTP was induced using θ-burst stimulation (10 bursts repeated at 5 Hz, each one consisting of four pulses at 100 Hz). Responses were recorded for 2 h after tetanization and plotted as percentage of baseline fEPSP slope. LTP is impaired in Bri2+/− animals [F (1,18)=4.747, P = 0.043]. (H) Quantitation of mouse Aβ40 in the brains of 10- to 11-mo-old mice WT and Bri2+/− (n =3)(I) A11 dot-blots analysis of brain lysates from 10- to 11-mo-old − WT and Bri2+/ mice. Three mice of each genotype were analyzed (m1–3) and each sample was spotted three times (s1– 3). (J) Densitometry values showed no difference in preﬁ- brillar oligomers between WT and Bri2+/− mice. Average value for WT samples was arbitrarily equaled to 100.

A clear difference between FDDKI/+ and FDD transgenic mice normal Bri2 function rather than the formation of a toxic (ADan) fi are the levels of mBri2. Although FDDKI/+ mice present low Bri2 peptide, amyloid plaques and/or neuro brillary tangles. levels in total brains and synapses, FDD transgenic mice express The ADan peptide, albeit below detection, is certainly pro- greatly elevated brain BRI2 levels (4). The evidence that human duced as indicated by the fact that a small fraction of Bri2-ADan FDD brains also show a significant reduction in BRI2 indicates undergoes maturation in mice with two mutant copies of the Itm2b/Bri2 that the FDD mice, and not FDD transgenic animals, faithfully gene. Although our data present no evidence sup- KI/+ porting a role for ADan in synaptic plasticity and memory defi- reproduce this trait of FDD patients. These data suggest that loss cits, ADan may set off other clinical symptoms of FDD patients, of mBri2 may cause the memory deficits exhibited by FDDKI/+ Bri2 such as cataracts, deafness and progressive ataxia, that are not mice. In agreement with this hypothesis, we have found that replicated in FDD mice. fi KI/+ haplo-de cient animals show synaptic plasticity and memory Future studies will analyze the mechanism by which the Danish impairments similar to FDDKI/+ mice. Moreover, expression of mutation causes a reduction in mBRI2 levels, clarify the synaptic WT BRI2 in the forebrain of FDDKI/+ mice prevents the de- function(s) of mBri2 and whether deregulation of this function(s) fi fi velopment of memory de cits. These genetic data reaf rm that cause synaptic plasticity and memory deficits in FDDKI/+ mice. memory deficits in FDDKI/+ mice are the consequence of loss of BRI2 is a physiological inhibitor of APP processing and Aβ pro-

20826 | www.pnas.org/cgi/doi/10.1073/pnas.1011689107 Tamayev et al. Downloaded by guest on September 27, 2021 duction (11, 16, 25–28). Although we did not detect alterations in min cleared the homogenates. Antitau antibodies DA9, PHF1, CP13, MC6, and CP9 were a gift of Peter Davies. Aβ or toxic oligomers levels in FDDKI/+ mice, it is possible that synaptotoxic ADan and/or Aβ oligomers may augment in ana- Other Antibodies. The following antibodies were used: α-calnexin (SPA- tomically restricted locations and/or increase transiently during α α− α memory acquisition/consolidation. These increases may not be 865; Stressgen); - tubulin (DM1A; Sigma); -GM130 (G7295; Sigma); α-SNAP25 (MAB331; Chemicon); α-calreticulin (Pierce); α-LC3 (CS#2775); detected by our analysis. It is also possible that the ELISA system β and A11 (Invitrogen). Secondary antibodies conjugated with HRP were and A11 cannot detect the A and/or ADan forms that are re- from Southern Biotechnology. sponsible for the dementia phenotype. Alternatively, memory loss in Danish dementia may be dependent on deregulation of BRI2/ Image Scanning and Analysis. WB images were scanned with Epson perfection APP complexes functions that are unrelated to Aβ or on BRI2 3200 Photo scanner and were analyzed with National Institutes of Health functions not linked to APP. Nevertheless, here we present a ge- ImageJ software. netically faithful murine model of human dementia with synaptic plasticity and memory deficits. Analysis of this model indicates Electrophysiological Analysis. Experiments were performed as previously that amyloid deposition and tangle formation may follow, rather described (29). Details are given in SI Materials and Methods. than cause, clinical dementia in FDD, contrary to what is affirmed by the amyloid cascade hypothesis. Behavioral Analysis. Cognition was tested using RAWM (30), NOR (31), and fear-conditioning tests. Anxiety phenotype and locomotor dysfunction were fi Materials and Methods tested using visible platform, open- eld, and spontaneous alternation (32) tests. Details are provided in SI Materials and Methods. Ethics Statement and Mice. Mice were maintained on a C57BL/6 background for more than 15 generations. Mice were handled according to the Ethical Statistical Analysis. All data are shown as mean ± SEM. Statistical tests in- Guidelines for Treatment of Laboratory of AECOM. The procedures were cluded two-way ANOVA for repeated measures and t test when appropriate. described in animal protocol no. 20040707.

ACKNOWLEDGMENTS. We thank the following individuals: Erhan Ma, Hong Brain Histology and Immunohistochemistry. Samples were prepared as de- Zhang, and Agnieszka Staniszewski for technical assistance; Dr. Susan scribed elsewhere (8). Details are given in SI Materials and Methods. Newbigging MSc, DVM, DVSc, at the Toronto Centre for Phenogenomics; Dr. Peter Davies and Dr. H. Akiyama (Tokyo Institute of Psychiatry) for anti- Bri2 mRNA Analysis: Protein Extract and Synaptosome Preparations. Methods bodies; Drs. Jorge Ghiso and Tamas Revesz (Dementia Research Center, The used for preparation of protein samples from human and murine brains and National Hospital for Neurology and Neurosurgery, London), for human ﬁ cells can be found in SI Materials and Methods. brain samples; Dr. Luca Giliberto for help with the quanti cation of Bri2 mRNA; and Caterina Schemidt for editing the manuscript. This work was supported by Alzheimer’s Association Grant IIRG-09-129984 (to L.D.), Na- Tau Western Blot. Mouse brains were homogenized in 20 mM Hepes/NaOH pH tional Institutes of Health Grants R01AG033007 (to L.D.) and R01NS049442 7.4, 1 mM EDTA, 1 mM EGTA, and 0.25 M sucrose supplemented with (to O.A.), and Training Program in Cellular and Molecular Biology and Ge- phosphatase inhibitors and protease inhibitors. Spinning at 1,000 × g for 10 netics Grant T32 GM007491 (to R.T).

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