Aß43 levels determine the onset of pathological amyloid deposition.

About 2% of Alzheimer's disease (AD) cases have early onset (FAD) and are caused by mutations in either Presenilins (PSEN1/2) or amyloid-ß precursor protein (APP). PSEN1/2 catalyze production of Aß peptides of different length from APP. Aß peptides are the major components of amyloid plaques, a pathological lesion that characterizes AD. Analysis of mechanisms by which PSEN1/2 and APP mutations affect Aß peptide compositions lead to the implication of the absolute or relative increase in Aß42 in amyloid-ß plaques formation. Here, to elucidate the formation of pathogenic Aß cocktails leading to amyloid pathology, we utilized FAD rat knock-in models carrying the Swedish APP (Apps allele) and the PSEN1 L435F (Psen1LF allele) mutations. To accommodate the differences in the pathogenicity of rodent and human Aß, these rat models are genetically engineered to express human Aß species as both the Swedish mutant allele and the WT rat allele (called Apph) have been humanized in the Aß-coding region. Analysis of the eight possible FAD mutant permutations indicates that the CNS levels of Aß43, rather than absolute or relative increases in Aß42, determine the onset of pathological amyloid deposition in FAD knock-in rats. Notably, Aß43 was found in amyloid plaques in late onset AD and mild cognitive impairment cases, suggesting that the mechanisms initiating amyloid pathology in FAD knock-in rat reflect disease mechanisms driving amyloid pathology in late onset AD. This study helps clarifying the molecular determinants initiating amyloid pathology and supports therapeutic interventions targeting Aß43 in AD.

Knock-in rats with homozygous PSEN1L435F Alzheimer mutation are viable and show selective γ-secretase activity loss causing low Aß40/42 and high Aß43.

Familial forms of Alzheimer's disease (FAD) are caused by mutations in the gene encoding amyloid precursor protein, whose processing can result in formation of ß-amyloid (Aß). FAD can also result from mutations in the presenilin 1/2 (PSEN1/2) genes, whose protein products partially compose the γ-secretase complex that cleaves Aß from amyloid precursor protein fragments. Psen1 KO mice and knock-in (KI) mice with homozygous FAD-associated L435F mutations (Psen1LF/LF ) are embryonic and perinatally lethal, precluding a more rigorous examination of the effect of Alzheimer's disease-causing Psen1 mutations on neurodegeneration. Given that the rat is a more suitable model organism with regard to surgical interventions and behavioral testing, we generated a rat KI model of the Psen1LF mutation. In this study, we focused on young Psen1LF rats to determine potential early pathogenic changes caused by this mutation. We found that, unlike Psen1LF/LF mice, Psen1LF/LF rats survive into adulthood despite loss of γ-secretase activity. Consistent with loss of γ-secretase function, Psen1LF/LF rats exhibited low levels of Aß38, Aß40, and Aß42 peptides. In contrast, levels of Aß43, a longer and potentially more amyloidogenic Aß form, were significantly increased in Psen1LF/LF and Psen1LF/w rats. The longer survival of these KI rats affords the opportunity to examine the effect of homozygous Psen1 Alzheimer's disease-associated mutations on neurodegeneration in older animals.

Tuning of Glutamate, But Not GABA, Release by an Intrasynaptic Vesicle APP Domain Whose Function Can Be Modulated by ß- or α-Secretase Cleavage.

APP, whose mutations cause familial Alzheimer's disease (FAD), modulates neurotransmission via interaction of its cytoplasmic tail with the synaptic release machinery. Here we identified an intravesicular domain of APP, called intraluminal SV-APP interacting domain (ISVAID), which interacts with glutamatergic, but not GABAergic, synaptic vesicle proteins. ISVAID contains the ß- and α-secretase cleavage sites of APP: proteomic analysis of the interactome of ISVAID suggests that ß- and α-secretase cleavage of APP cuts inside the interaction domain of ISVAID and destabilizes protein-protein interactions. We have tested the functional significance of the ISVAID and of ß-/α-secretase-processing of APP using various ISVAID-derived peptides in competition experiments on both female and male mouse and rats hippocampal slices. A peptide encompassing the entire ISVAID facilitated glutamate, but not GABA, release acting as dominant negative inhibitor of the functions of this APP domain in acute hippocampal slices. In contrast, peptides representing the product of ß-/α-secretase-processing of ISVAID did not alter excitatory neurotransmitter release. These findings suggest that cleavage of APP by either ß- or α-secretase may inactivate the ISVAID, thereby enhancing glutamate release. Our present data support the notion that APP tunes glutamate release, likely through intravesicular and extravesicular interactions with synaptic vesicle proteins and the neurotransmitter release machinery, and that ß-/α cleavage of APP facilitates the release of excitatory neurotransmitter.SIGNIFICANCE STATEMENT Alzheimer's disease has been linked to mutations in APP. However, the biological function of APP is poorly understood. Here we show that an intravesicular APP domain interacts with the proteins that control the release of glutamate, but not GABA. Interfering with the function of this domain promotes glutamate release. This APP domain contains the sites cleaved by ß- and α-secretases: our data suggest that ß-/α cleavage of APP inactivates this functional APP domain promoting excitatory neurotransmitter release.

ApoE4 upregulates the activity of mitochondria-associated ER membranes.

In addition to the appearance of senile plaques and neurofibrillary tangles, Alzheimer's disease (AD) is characterized by aberrant lipid metabolism and early mitochondrial dysfunction. We recently showed that there was increased functionality of mitochondria-associated endoplasmic reticulum (ER) membranes (MAM), a subdomain of the ER involved in lipid and cholesterol homeostasis, in presenilin-deficient cells and in fibroblasts from familial and sporadic AD patients. Individuals carrying the ε4 allele of apolipoprotein E (ApoE4) are at increased risk for developing AD compared to those carrying ApoE3. While the reason for this increased risk is unknown, we hypothesized that it might be associated with elevated MAM function. Using an astrocyte-conditioned media (ACM) model, we now show that ER-mitochondrial communication and MAM function-as measured by the synthesis of phospholipids and of cholesteryl esters, respectively-are increased significantly in cells treated with ApoE4-containing ACM as compared to those treated with ApoE3-containing ACM. Notably, this effect was seen with lipoprotein-enriched preparations, but not with lipid-free ApoE protein. These data are consistent with a role of upregulated MAM function in the pathogenesis of AD and may help explain, in part, the contribution of ApoE4 as a risk factor in the disease.

Upregulated function of mitochondria-associated ER membranes in Alzheimer disease.

Alzheimer disease (AD) is associated with aberrant processing of the amyloid precursor protein (APP) by γ-secretase, via an unknown mechanism. We recently showed that presenilin-1 and -2, the catalytic components of γ-secretase, and γ-secretase activity itself, are highly enriched in a subcompartment of the endoplasmic reticulum (ER) that is physically and biochemically connected to mitochondria, called mitochondria-associated ER membranes (MAMs). We now show that MAM function and ER-mitochondrial communication-as measured by cholesteryl ester and phospholipid synthesis, respectively-are increased significantly in presenilin-mutant cells and in fibroblasts from patients with both the familial and sporadic forms of AD. We also show that MAM is an intracellular detergent-resistant lipid raft (LR)-like domain, consistent with the known presence of presenilins and γ-secretase activity in rafts. These findings may help explain not only the aberrant APP processing but also a number of other biochemical features of AD, including altered lipid metabolism and calcium homeostasis. We propose that upregulated MAM function at the ER-mitochondrial interface, and increased cross-talk between these two organelles, may play a hitherto unrecognized role in the pathogenesis of AD.

Late-long-term potentiation magnitude, but not Aß levels and amyloid pathology, is associated with behavioral performance in a rat knock-in model of Alzheimer disease.

Cleavage of Amyloid precursor protein by ß- and γ-secretases lead to Aß formation. The widely accepted pathogenic model states that these mutations cause AD via an increase in Aß formation and accumulation of Aß in Amyloid plaques. APP mutations cause early onset familial forms of Alzheimer's disease (FAD) in humans. We generated App-Swedish (Apps ) knock-in rats, which carry a pathogenic APP mutation in the endogenous rat App gene. This mutation increases ß-secretase processing of APP leading to both augmented Aß production and facilitation of glutamate release in Apps/s rats, via a ß-secretase and APP-dependent glutamate release mechanism. Here, we studied 11 to 14-month-old male and female Apps/s rats. To determine whether the Swedish App mutation leads to behavioral deficits, Apps/s knock-in rats were subjected to behavioral analysis using the IntelliCage platform, an automated behavioral testing system. This system allows behavioral assessment in socially housed animals reflecting a more natural, less stress-inducing environment and eliminates experimenter error and bias while increasing precision of measurements. Surprisingly, a spatial discrimination and flexibility task that can reveal deficits in higher order brain function showed that Apps/s females, but not Apps/s male rats, performed significantly worse than same sex controls. Moreover, female control rats performed significantly better than control and Apps/s male rats. The Swedish mutation causes a significant increase in Aß production in 14-month-old animals of both sexes. Yet, male and female Apps/s rats showed no evidence of AD-related amyloid pathology. Finally, Apps/s rats did not show signs of significant neuroinflammation. Given that the APP Swedish mutation causes alterations in glutamate release, we analyzed Long-term potentiation (LTP), a long-lasting form of synaptic plasticity that is a cellular basis for learning and memory. Strikingly, LTP was significantly increased in Apps/s control females compared to both Apps/s sexes and control males. In conclusion, this study shows that behavioral performances are sex and App-genotype dependent. In addition, they are associated with LTP values and not Aß or AD-related pathology. These data, and the failures of anti-Aß therapies in humans, suggest that alternative pathways, such as those leading to LTP dysfunction, should be targeted for disease-modifying AD therapy.

Trem2 Splicing and Expression are Preserved in a Human Aß-producing, Rat Knock-in Model of Trem2-R47H Alzheimer's Risk Variant.

The R47H variant of the Triggering-Receptor-Expressed on Myeloid cells 2 (TREM2) increases the risk of Alzheimer's disease (AD). Mutagenesis of exon 2 in Knock-in (KI) mouse models of the R47H variant introduced a cryptic splice site, leading to nonsense mediated decay. Since haploinsufficiency does not model Trem2-R47H function, a new rat KI model, the Trem2R47H KI rat was created. Human Aß has higher propensity to form toxic Aß species, which are considered the main pathogenic entity in AD, as compared to rodent Aß, the rat Amyloid Precursor Protein (App) gene was mutated to produce human Aß. Trem2 splicing and expression was measured in Trem2R47H KI rat brains and microglia by qualitative and quantitative RT-PCR. Trem2 levels and Trem2 processing was assessed by Western analysis. APP metabolite levels were determined by enzyme-linked immunosorbent assay (ELISA), for Human Aß and soluble APP, and Western analysis, for full length APP, ßCTF and αCTF. Trem2 expression and Trem2 levels are unchanged in Trem2R47H KI rats. The artifactual splicing seen in KI mouse models is not present; additionally, two novel isoforms of rat Trem2 are described. Trem2R47H rat brains have lower human Aß38, sAPPα and sAPPß levels. Thus, Trem2R47H KI rats may prove valuable to define pathogenic mechanisms triggered by the Trem2 R47H variant, including those mediated by toxic species of human Aß peptides.

Opposite changes in APP processing and human Aß levels in rats carrying either a protective or a pathogenic APP mutation.

Cleavage of APP by BACE1/ß-secretase initiates the amyloidogenic cascade leading to Amyloid-ß (Aß) production. α-Secretase initiates the non-amyloidogenic pathway preventing Aß production. Several APP mutations cause familial Alzheimer's disease (AD), while the Icelandic APP mutation near the BACE1-cleavage site protects from sporadic dementia, emphasizing APP's role in dementia pathogenesis. To study APP protective/pathogenic mechanisms, we generated knock-in rats carrying either the protective (Appp) or the pathogenic Swedish mutation (Apps), also located near the BACE1-cleavage site. α-Cleavage is favored over ß-processing in Appp rats. Consequently, non-amyloidogenic and amyloidogenic APP metabolites are increased and decreased, respectively. The reverse APP processing shift occurs in Apps rats. These opposite effects on APP ß/α-processing suggest that protection from and pathogenesis of dementia depend upon combinatorial and opposite alterations in APP metabolism rather than simply on Aß levels. The Icelandic mutation also protects from aging-dependent cognitive decline, suggesting that similar mechanisms underlie physiological cognitive aging.

Microglia TREM2R47H Alzheimer-linked variant enhances excitatory transmission and reduces LTP via increased TNF-α levels.

To study the mechanisms by which the p.R47H variant of the microglia gene and Alzheimer's disease (AD) risk factor TREM2 increases dementia risk, we created Trem2R47H KI rats. Trem2R47H rats were engineered to produce human Aß to define human-Aß-dependent and -independent pathogenic mechanisms triggered by this variant. Interestingly, pre- and peri-adolescent Trem2R47H rats present increased brain concentrations of TNF-α, augmented glutamatergic transmission, suppression of Long-term-Potentiation (LTP), an electrophysiological surrogate of learning and memory, but normal Aß levels. Acute reduction of TNF-α activity with a neutralizing anti-TNF-α antibody occludes the boost in amplitude of glutamatergic transmission and LTP suppression observed in young Trem2R47H/R47H rats. Thus, the microglia-specific pathogenic Trem2 variant boosts glutamatergic neuronal transmission and suppresses LTP by increasing brain TNF-α concentrations, directly linking microglia to neuronal dysfunction. Future studies will determine whether this phenomenon represents an early, Aß-independent pathway that facilitates dementia pathogenesis in humans.

Facilitation of glutamate, but not GABA, release in Familial Alzheimer's APP mutant Knock-in rats with increased ß-cleavage of APP.

Amyloid precursor protein (APP) modulates glutamate release via cytoplasmic and intravesicular interactions with the synaptic vesicle release machinery. The intravesicular domain, called ISVAID, contains the BACE1 cleavage site of APP. We have tested the functional significance of BACE1 processing of APP using App-Swedish (Apps ) knock-in rats, which carry an App mutation that causes familial Alzheimer's disease (FAD) in humans. We show that in Apps rats, ß-cleavage of APP is favored over α-cleavage. Apps rats show facilitated glutamate, but not GABA, release. Our data support the notion that APP tunes glutamate release, and that BACE1 cleavage of the ISVAID segment of APP facilitates this function. We define this phenomenon as BACE1 on APP-dependent glutamate release (BAD-Glu). Unsurprisingly, Apps rats show no evidence of AD-related pathology at 15 days and 3 months of age, indicating that alterations in BAD-Glu are not caused by pathological lesions. The evidence that a pathogenic APP mutation causes an early enhancement of BAD-Glu suggests that alterations of BACE1 processing of APP in glutamatergic synaptic vesicles could contribute to dementia.

The Familial dementia gene ITM2b/BRI2 facilitates glutamate transmission via both presynaptic and postsynaptic mechanisms.

Mutations in the Integral membrane protein 2B (ITM2b/BRI2) gene, which codes for a protein called BRI2, cause familial British and Danish dementia (FBD and FDD). Loss of BRI2 function and/or accumulation of amyloidogenic mutant BRI2-derived peptides have been proposed to mediate FDD and FBD pathogenesis by impairing synaptic Long-term potentiation (LTP). However, the precise site and nature of the synaptic dysfunction remain unknown. Here we use a genetic approach to inactivate Itm2b in either presynaptic (CA3), postsynaptic (CA1) or both (CA3 + CA1) neurons of the hippocampal Schaeffer-collateral pathway in both female and male mice. We show that after CA3 + CA1 Itm2b inactivation, spontaneous glutamate release and AMPAR-mediated responses are decreased, while short-term synaptic facilitation is increased. Moreover, AMPAR-mediated responses are decreased after postsynaptic but not presynaptic deletion of Itm2b. In contrast, the probability of spontaneous glutamate release is decreased, while short-term synaptic facilitation is increased, primarily after presynaptic deletion of Itm2b. Collectively, these results indicate a dual physiological role of Itm2b in the regulation of excitatory synaptic transmission at both presynaptic termini and postsynaptic termini and suggest that presynaptic and postsynaptic dysfunctions may be a pathogenic event leading to dementia and neurodegeneration in FDD and FBD.