Calcineurin/NFAT Signaling in Activated Astrocytes Drives Network Hyperexcitability in Aß-Bearing Mice.

Hyperexcitable neuronal networks are mechanistically linked to the pathologic and clinical features of Alzheimer's disease (AD). Astrocytes are a primary defense against hyperexcitability, but their functional phenotype during AD is poorly understood. Here, we found that activated astrocytes in the 5xFAD mouse model were strongly associated with proteolysis of the protein phosphatase calcineurin (CN) and the elevated expression of the CN-dependent transcription factor nuclear factor of activated T cells 4 (NFAT4). Intrahippocampal injections of adeno-associated virus vectors containing the astrocyte-specific promoter Gfa2 and the NFAT inhibitory peptide VIVIT reduced signs of glutamate-mediated hyperexcitability in 5xFAD mice, measured in vivo with microelectrode arrays and ex vivo brain slices, using whole-cell voltage clamp. VIVIT treatment in 5xFAD mice led to increased expression of the astrocytic glutamate transporter GLT-1 and to attenuated changes in dendrite morphology, synaptic strength, and NMDAR-dependent responses. The results reveal astrocytic CN/NFAT4 as a key pathologic mechanism for driving glutamate dysregulation and neuronal hyperactivity during AD.SIGNIFICANCE STATEMENT Neuronal hyperexcitability and excitotoxicity are increasingly recognized as important mechanisms for neurodegeneration and dementia associated with Alzheimer's disease (AD). Astrocytes are profoundly activated during AD and may lose their capacity to regulate excitotoxic glutamate levels. Here, we show that a highly active calcineurin (CN) phosphatase fragment and its substrate transcription factor, nuclear factor of activated T cells (NFAT4), appear in astrocytes in direct proportion to the extent of astrocyte activation. The blockade of astrocytic CN/NFAT signaling in a common mouse model of AD, using adeno-associated virus vectors normalized glutamate signaling dynamics, increased astrocytic glutamate transporter levels and alleviated multiple signs of neuronal hyperexcitability. The results suggest that astrocyte activation drives hyperexcitability during AD through a mechanism involving aberrant CN/NFAT signaling and impaired glutamate transport.

Vascular cognitive impairment: Modeling a critical neurologic disease in vitro and in vivo.

BACKGROUND: Vascular contributions to cognitive impairment and dementia (VCID) is a complex form of dementia, combining aspects of vascular disease and other forms of dementia, such as Alzheimer's disease. VCID encompasses a wide spectrum of cerebrovascular-driven cognitive impairment, from mild cognitive impairment to fully developed dementia. This disease state is further complicated by metabolic disorders, such as type 2 diabetes and hypertension, and lifestyle factors, like obesity and high fat diets. SCOPE OF REVIEW: This manuscript is meant to both define VCID and review the in vitro and in vivo models of the disease state. This includes in vitro models of the neurovascular unit, models of chronic cerebral hypoperfusion, animals with NOTCH3 mutations as a model of small vessel disease, large animals with cerebral amyloid angiopathy (CAA), and animal models of mixed dementia. MAJOR CONCLUSIONS: Synthetic microvessels are a promising technique to study the neurovascular unit and canines, despite the cost, are an excellent model to study CAA. While there are several good models of individual aspects of VCID, the heterogeneity of the disease states prevents them from being a model of all aspects of the disease. Therefore, VCID needs to be further defined into disease states that exist within this umbrella term. This includes specific guidelines for stroke counts and stroke locations and further categorization of overlapping cerebrovascular and AD pathologies that contribute to dementia. This will allow for better models and a more thorough understanding of how vascular disease contributes to dementia. GENERAL SIGNIFICANCE: VCID is the second most common form of dementia and is expected to increase in coming years. The heterogeneity of VCID makes it difficult to study, but without better definitions and models, VCID presents a major public health problem for our aging population. This article is part of a Special Issue entitled: Vascular Contributions to Cognitive Impairment and Dementia, edited by M. Paul Murphy, Roderick A. Corriveau and Donna M. Wilcock.

Transgenic models of Alzheimer's disease: better utilization of existing models through viral transgenesis.

Animal models have been used for decades in the Alzheimer's disease (AD) research field and have been crucial for the advancement of our understanding of the disease. Most models are based on familial AD mutations of genes involved in the amyloidogenic process, such as the amyloid precursor protein (APP) and presenilin 1 (PS1). Some models also incorporate mutations in tau (MAPT) known to cause frontotemporal dementia, a neurodegenerative disease that shares some elements of neuropathology with AD. While these models are complex, they fail to display pathology that perfectly recapitulates that of the human disease. Unfortunately, this level of pre-existing complexity creates a barrier to the further modification and improvement of these models. However, as the efficacy and safety of viral vectors improves, their use as an alternative to germline genetic modification is becoming a widely used research tool. In this review we discuss how this approach can be used to better utilize common mouse models in AD research. This article is part of a Special Issue entitled: Animal Models of Disease.

Leptin regulates amyloid ß production via the γ-secretase complex.

Alzheimer's disease (AD) is the most common age-related neurodegenerative disease, affecting an estimated 5.3million people in the United States. While many factors likely contribute to AD progression, it is widely accepted that AD is driven by the accumulation of ß-amyloid (Aß), a small, fibrillogenic peptide generated by the sequential proteolysis of the amyloid precursor protein by the ß- and γ-secretases. Though the underlying causes of Aß accumulation in sporadic AD are myriad, it is clear that lifestyle and overall health play a significant role. The adipocyte-derived hormone leptin has varied systemic affects, including neuropeptide release and neuroprotection. A recent study by Lieb et al. (2009) showed that individuals with low plasma leptin levels are at greater risk of developing AD, through unknown mechanisms. In this report, we show that plasma leptin is a strong negative predictor of Aß levels in the mouse brain, supporting a protective role for the hormone in AD onset. We also show that the inhibition of Aß accumulation is due to the downregulation of transcription of the γ-secretase components. On the other hand, ß-secretase expression is either unchanged (BACE1) or increased (BACE2). Finally, we show that only presenilin 1 (PS1) is negatively correlated with plasma leptin at the protein level (p<0.0001). These data are intriguing and may highlight a role for leptin in regulating the onset of amyloid pathology and AD.

Prolonged diet induced obesity has minimal effects towards brain pathology in mouse model of cerebral amyloid angiopathy: implications for studying obesity-brain interactions in mice.

Cerebral amyloid angiopathy (CAA) occurs in nearly every individual with Alzheimer's disease (AD) and Down's syndrome, and is the second largest cause of intracerebral hemorrhage. Mouse models of CAA have demonstrated evidence for increased gliosis contributing to CAA pathology. Nearly two thirds of Americans are overweight or obese, with little known about the effects of obesity on the brain, although increasingly the vasculature appears to be a principle target of obesity effects on the brain. In the current study we describe for the first time whether diet induced obesity (DIO) modulates glial reactivity, amyloid levels, and inflammatory signaling in a mouse model of CAA. In these studies we identify surprisingly that DIO does not significantly increase Aß levels, astrocyte (GFAP) or microglial (IBA-1) gliosis in the CAA mice. However, within the hippocampal gyri a localized increase in reactive microglia were increased in the CA1 and stratum oriens relative to CAA mice on a control diet. DIO was observed to selectively increase IL-6 in CAA mice, with IL-1ß and TNF-α not increased in CAA mice in response to DIO. Taken together, these data show that prolonged DIO has only modest effects towards Aß in a mouse model of CAA, but appears to elevate some localized microglial reactivity within the hippocampal gyri and selective markers of inflammatory signaling. These data are consistent with the majority of the existing literature in other models of Aß pathology, which surprisingly show a mixed profile of DIO effects towards pathological processes in mouse models of neurodegenerative disease. The importance for considering the potential impact of ceiling effects in pathology within mouse models of Aß pathogenesis, and the current experimental limitations for DIO in mice to fully replicate metabolic dysfunction present in human obesity, are discussed. This article is part of a Special Issue entitled: Animal Models of Disease.

Impairment of proteostasis network in Down syndrome prior to the development of Alzheimer's disease neuropathology: redox proteomics analysis of human brain.

DS is the most frequent genetic cause of intellectual disability characterized by the anomalous presence of three copies of chromosome 21. One of the peculiar features of DS is the onset of Alzheimer's disease neuropathology after the age of 40years characterized by deposition of senile plaques and neurofibrillary tangles. Growing studies demonstrated that increased oxidative damage, accumulation of unfolded/damaged protein aggregates and dysfunction of intracellular degradative system are key players in neurodegenerative processes. In this study, redox proteomics approach was used to analyze the frontal cortex from DS subjects under the age of 40 compared with age-matched controls, and proteins found to be increasingly carbonylated were identified. Interestingly, our results showed that oxidative damage targets specifically different components of the intracellular quality control system such as GRP78, UCH-L1, V0-ATPase, cathepsin D and GFAP that couples with decreased activity of the proteasome and autophagosome formation observed. We also reported a slight but consistent increase of Aß 1-42 SDS- and PBS-soluble form and tau phosphorylation in DS versus CTR. We suggest that disturbance in the proteostasis network could contribute to the accumulation of protein aggregates, such as amyloid deposits and NFTs, which occur very early in DS. It is likely that a sub-optimal functioning of degradative systems occur in DS neurons, which in turn provide the basis for further accumulation of toxic protein aggregates. The results of this study suggest that oxidation of protein members of the proteostatis network is an early event in DS and might contribute to neurodegenerative phenomena.

Sex and Sleep Disruption as Contributing Factors in Alzheimer's Disease.

Alzheimer's disease (AD) affects more women than men, with women throughout the menopausal transition potentially being the most under researched and at-risk group. Sleep disruptions, which are an established risk factor for AD, increase in prevalence with normal aging and are exacerbated in women during menopause. Sex differences showing more disrupted sleep patterns and increased AD pathology in women and female animal models have been established in literature, with much emphasis placed on loss of circulating gonadal hormones with age. Interestingly, increases in gonadotropins such as follicle stimulating hormone are emerging to be a major contributor to AD pathogenesis and may also play a role in sleep disruption, perhaps in combination with other lesser studied hormones. Several sleep influencing regions of the brain appear to be affected early in AD progression and some may exhibit sexual dimorphisms that may contribute to increased sleep disruptions in women with age. Additionally, some of the most common sleep disorders, as well as multiple health conditions that impair sleep quality, are more prevalent and more severe in women. These conditions are often comorbid with AD and have bi-directional relationships that contribute synergistically to cognitive decline and neuropathology. The association during aging of increased sleep disruption and sleep disorders, dramatic hormonal changes during and after menopause, and increased AD pathology may be interacting and contributing factors that lead to the increased number of women living with AD.

The amyloid-ß peptide: Guilty as charged?

Recent years have seen both considerable progress and controversy in the Alzheimer's disease (AD) field. After decades of slow to negligible movement towards the development of disease modifying therapies, promising outcomes in recent clinical trials with several monoclonal antibodies targeting various forms of the amyloid-ß (Aß) peptide have at last opened a possible way forward. In fact, at this point multiple anti-Aß therapeutics are close to receiving (or have already received) regulatory approval. Although these outcomes are not without some degree of divisiveness, the fact remains that targeting amyloid for removal has finally shown at least modest efficacy in slowing the otherwise relentless progression of the disease. Although the validation of the long standing amyloid cascade hypothesis would seem to be at hand, what remains is the puzzling issue of why - if Aß indeed causes AD - does removing it from the brain not stop the disease entirely.

Thermoneutral temperature exposure enhances slow-wave sleep with a correlated improvement in amyloid pathology in a triple-transgenic mouse model of Alzheimer's disease.

Accumulation of amyloid-ß (Aß) plays an important role in Alzheimer's disease (AD) pathology. There is growing evidence that disordered sleep may accelerate AD pathology by impeding the physiological clearance of Aß from the brain that occurs in normal sleep. Therapeutic strategies for improving sleep quality may therefore help slow disease progression. It is well documented that the composition and dynamics of sleep are sensitive to ambient temperature. We therefore compared Aß pathology and sleep metrics derived from polysomnography in 12-month-old female 3xTg-AD mice (n = 8) exposed to thermoneutral temperatures during the light period over 4 weeks to those of age- and sex-matched controls (n = 8) that remained at normal housing temperature (22°C) during the same period. The treated group experienced greater proportions of slow wave sleep (SWS)-i.e. epochs of elevated 0.5-2 Hz EEG slow wave activity during non-rapid eye movement (NREM) sleep-compared to controls. Assays performed on mouse brain tissue harvested at the end of the experiment showed that exposure to thermoneutral temperatures significantly reduced levels of DEA-soluble (but not RIPA- or formic acid-soluble) Aß40 and Aß42 in the hippocampus, though not in the cortex. With both groups pooled together and without regard to treatment condition, NREM sleep continuity and any measure of SWS within NREM at the end of the treatment period were inversely correlated with DEA-soluble Aß40 and Aß42 levels, again in the hippocampus but not in the cortex. These findings suggest that experimental manipulation of SWS could offer useful clues into the mechanisms and treatment of AD.

Targeting astrocytes ameliorates neurologic changes in a mouse model of Alzheimer's disease.

Astrocytes are the most abundant cell type in the brain and play a critical role in maintaining healthy nervous tissue. In Alzheimer's disease (AD) and most other neurodegenerative disorders, many astrocytes convert to a chronically "activated" phenotype characterized by morphologic and biochemical changes that appear to compromise protective properties and/or promote harmful neuroinflammatory processes. Activated astrocytes emerge early in the course of AD and become increasingly prominent as clinical and pathological symptoms progress, but few studies have tested the potential of astrocyte-targeted therapeutics in an intact animal model of AD. Here, we used adeno-associated virus (AAV) vectors containing the astrocyte-specific Gfa2 promoter to target hippocampal astrocytes in APP/PS1 mice. AAV-Gfa2 vectors drove the expression of VIVIT, a peptide that interferes with the immune/inflammatory calcineurin/NFAT (nuclear factor of activated T-cells) signaling pathway, shown by our laboratory and others to orchestrate biochemical cascades leading to astrocyte activation. After several months of treatment with Gfa2-VIVIT, APP/PS1 mice exhibited improved cognitive and synaptic function, reduced glial activation, and lower amyloid levels. The results confirm a deleterious role for activated astrocytes in AD and lay the groundwork for exploration of other novel astrocyte-based therapies.

BACE2 expression increases in human neurodegenerative disease.

ß-Secretase, the rate-limiting enzymatic activity in the production of the amyloid-ß (Aß) peptide, is a major target of Alzheimer's disease (AD) therapeutics. There are two forms of the enzyme: ß-site Aß precursor protein cleaving enzyme (BACE) 1 and BACE2. Although BACE1 increases in late-stage AD, little is known about BACE2. We conducted a detailed examination of BACE2 in patients with preclinical to late-stage AD, including amnestic mild cognitive impairment, and age-matched controls, cases of frontotemporal dementia, and Down's syndrome. BACE2 protein and enzymatic activity increased as early as preclinical AD and were found in neurons and astrocytes. Although the levels of total BACE2 mRNA were unchanged, the mRNA for BACE2 splice form C (missing exon 7) increased in parallel with BACE2 protein and activity. BACE1 and BACE2 were strongly correlated with each other at all levels, suggesting that their regulatory mechanisms may be largely shared. BACE2 was also elevated in frontotemporal dementia but not in Down's syndrome, even in patients with substantial Aß deposition. Thus, expression of both forms of ß-secretase are linked and may play a combined role in human neurologic disease. A better understanding of the normal functions of BACE1 and BACE2, and how these change in different disease states, is essential for the future development of AD therapeutics.

Pittsburgh compound B and the postmortem diagnosis of Alzheimer disease.

OBJECTIVE: Deposition of the amyloid-ß (Aß) peptide in neuritic plaques is a requirement for the diagnosis of Alzheimer disease (AD). Although the continued development of in vivo imaging agents such as Pittsburgh compound B (PiB) is promising, the diagnosis of AD is still challenging. This can be partially attributed to our lack of a detailed understanding of the interrelationship between the various pools and species of Aß and other common indices of AD pathology. We hypothesized that recent advances in our ability to accurately measure Aß postmortem (for example, using PiB), could form the basis of a simple means to deliver an accurate AD diagnosis. METHODS: We conducted a comprehensive analysis of the amount of Aß40 and Aß42 in increasingly insoluble fractions, oligomeric Aß, and fibrillar Aß (as defined by PiB binding), as well as plaques (diffuse and neuritic), and neurofibrillary tangles in autopsy specimens from age-matched, cognitively normal controls (n = 23) and AD (n = 22) cases, across multiple brain regions. RESULTS: Both PiB binding and the amount of sodium dodecyl sulfate (SDS)-soluble Aß were able to predict disease status; however, SDS-soluble Aß was a better measure. Oligomeric Aß was not a predictor of disease status. PiB binding was strongly related to plaque count, although diffuse plaques were a stronger correlate than neuritic plaques. INTERPRETATION: Although postmortem PiB binding was somewhat useful in distinguishing AD from control cases, SDS-soluble Aß measured by standard immunoassay was substantially better. These findings have important implications for the development of imaging-based biomarkers of AD.

Biliverdin reductase-A: a novel drug target for atorvastatin in a dog pre-clinical model of Alzheimer disease.

Biliverdin reductase-A (BVR-A) is a pleiotropic enzyme involved in cellular stress responses. It not only transforms biliverdin-IX alpha into the antioxidant bilirubin-IX alpha but through its serine/threonine/tyrosine kinase activity is able to modulate cell signaling networks. BVR-A's involvement in neurodegenerative disorders such as Alzheimer disease (AD) and amnestic mild cognitive impairment was previously described. Statins have been proposed to reduce risk of AD. In this study we evaluated the effect of atorvastatin treatment (80 mg/day for 14.5 months) on BVR-A in the parietal cortex, cerebellum and liver of a well characterized pre-clinical model of AD, the aged beagle. We found that atorvastatin significantly increased BVR-A protein levels, phosphorylation and activity only in parietal cortex. Additionally, we found significant negative correlations between BVR-A and oxidative stress indices, as well as discrimination learning error scores. Furthermore, BVR-A up-regulation and post-translational modifications significantly correlated with ß-secretase protein levels in the brain, suggesting a possible role for BVR-A in Aß formation.

Synergistic effects of long-term antioxidant diet and behavioral enrichment on beta-amyloid load and non-amyloidogenic processing in aged canines.

A long-term intervention (2.69 years) with an antioxidant diet, behavioral enrichment, or the combined treatment preserved and improved cognitive function in aged canines. Although each intervention alone provided cognitive benefits, the combination treatment was additive. We evaluate the hypothesis that antioxidants, enrichment, or the combination intervention reduces age-related beta-amyloid (Abeta) neuropathology, as one mechanism mediating observed functional improvements. Measures assessed were Abeta neuropathology in plaques, biochemically extractable Abeta(40) and Abeta(42) species, soluble oligomeric forms of Abeta, and various proteins in the beta-amyloid precursor protein (APP) processing pathway. The strongest and most consistent effects on Abeta pathology were observed in animals receiving the combined antioxidant and enrichment treatment. Specifically, Abeta plaque load was significantly decreased in several brain regions, soluble Abeta(42) was decreased selectively in the frontal cortex, and a trend for lower Abeta oligomer levels was found in the parietal cortex. Reductions in Abeta may be related to shifted APP processing toward the non-amyloidogenic pathway, because alpha-secretase enzymatic activity was increased in the absence of changes in beta-secretase activity. Although enrichment alone had no significant effects on Abeta, reduced Abeta load and plaque maturation occurred in animals receiving antioxidants as a component of treatment. Abeta measures did not correlate with cognitive performance on any of the six tasks assessed, suggesting that modulation of Abeta alone may be a relatively minor mechanism mediating cognitive benefits of the interventions. Overall, the data indicate that multidomain treatments may be a valuable intervention strategy to reduce neuropathology and improve cognitive function in humans.

Cognitive impairment in humanized APP×PS1 mice is linked to Aß(1-42) and NOX activation.

Cognitive impairment in Alzheimer's disease (AD) is strongly associated with both extensive deposition of amyloid ß peptides and oxidative stress, but the exact role of these indices in the development of dementia is not clear. This study was designed to determine the relationship between cognitive impairment, activation of the free radical producing enzyme NADPH oxidase (NOX), and progressive changes in Aß deposition and solubility in humanized APP×PS1 knock-in mice of increasing age. Data show that cognitive performance and expression of key synaptic proteins were progressively decreased in aging APP×PS1 mice. Likewise, NOX activity and expression of the specific NOX subunit NOX4 were significantly increased in APP×PS1 mice in an age-dependent manner, and NOX activity and cognitive impairment shared a significant linear relationship. Data further show that age-dependent increases in Aß(1-42) had a significant linear relationship with both NOX activity and cognitive performance in APP×PS1 knock-in mice. Collectively, these data show that NOX expression and activity are significantly upregulated with age in this humanized model of Aß pathogenesis, and suggest that NOX-associated redox pathways are intimately linked to both the loss of cognitive function and the deposition of Aß(1-42).

Circulating neprilysin clears brain amyloid.

The use of the peptidase neprilysin (NEP) as a therapeutic for lowering brain amyloid burden is receiving increasing attention. We have previously demonstrated that peripheral expression of NEP on the surface of hindlimb muscle lowers brain amyloid burden in a transgenic mouse model of Alzheimer's disease. In this study we now show that using adeno-associated virus expressing a soluble secreted form of NEP (secNEP-AAV8), NEP secreted into plasma is effective in clearing brain Abeta. Soluble NEP expression in plasma was sustained over the 3-month time period it was measured. Secreted NEP decreased plasma Abeta by 30%, soluble brain Abeta by approximately 28%, insoluble brain Abeta by approximately 55%, and Abeta oligomersby 12%. This secNEP did not change plasma levels of substance P or bradykinin, nor did it alter blood pressure. No NEP was detected in CSF, nor did the AAV virus produce brain expression of NEP. Thus the lowering of brain Abeta was due to plasma NEP which altered blood-brain Abeta transport dynamics. Expressing NEP in plasma provides a convenient way to monitor enzyme activity during the course of its therapeutic testing.

Cognitive decline in Alzheimer's disease is associated with selective changes in calcineurin/NFAT signaling.

Upon activation by calcineurin, the nuclear factor of activated T-cells (NFAT) translocates to the nucleus and guides the transcription of numerous molecules involved in inflammation and Ca(2+) dysregulation, both of which are prominent features of Alzheimer's disease (AD). However, NFAT signaling in AD remains relatively uninvestigated. Using isolated cytosolic and nuclear fractions prepared from rapid-autopsy postmortem human brain tissue, we show that NFATs 1 and 3 shifted to nuclear compartments in the hippocampus at different stages of neuropathology and cognitive decline, whereas NFAT2 remained unchanged. NFAT1 exhibited greater association with isolated nuclear fractions in subjects with mild cognitive impairment (MCI), whereas NFAT3 showed a strong nuclear bias in subjects with severe dementia and AD. Similar to NFAT1, calcineurin-Aalpha also exhibited a nuclear bias in the early stages of cognitive decline. But, unlike NFAT1 and similar to NFAT3, the nuclear bias for calcineurin became more pronounced as cognition worsened. Changes in calcineurin/NFAT3 were directly correlated to soluble amyloid-beta (Abeta((1-42))) levels in postmortem hippocampus, and oligomeric Abeta, in particular, robustly stimulated NFAT activation in primary rat astrocyte cultures. Oligomeric Abeta also caused a significant reduction in excitatory amino acid transporter 2 (EAAT2) protein levels in astrocyte cultures, which was blocked by NFAT inhibition. Moreover, inhibition of astrocytic NFAT activity in mixed cultures ameliorated Abeta-dependent elevations in glutamate and neuronal death. The results suggest that NFAT signaling is selectively altered in AD and may play an important role in driving Abeta-mediated neurodegeneration.

BACE1 and BACE2 enzymatic activities in Alzheimer's disease.

beta-Secretase is the rate limiting enzymatic activity in the production of the amyloid-beta peptide (Abeta) and is thought to be involved in Alzheimer's disease (AD) pathogenesis. Although BACE1 (beta-site APP Cleaving Enzyme 1, EC 3.4.23.46) has received significant attention, the related BACE2 (EC 3.4.23.45) has not. Though BACE2 is also expressed in the brain, its potential role in AD has not been resolved. In this study, we compared the activities of both BACE1 and BACE2, which were isolated from the same samples of frontal cortex from both AD-affected individuals and age-matched controls. BACE1 activity showed a significant positive correlation with the amount of extractable Abeta, and BACE1 protein and activity were significantly increased in AD cases. Unexpectedly, there were substantial total amounts of BACE2 protein and enzymatic activity in the human brain. BACE2 activity did not change significantly in the AD brain, and was not related to Abeta concentration. These data indicate that BACE1 likely accounts for most of the Abeta produced in the human brain, and that BACE2 activity is not a likely contributor. However, as both forms of BACE compete for the same substrate pool, even small changes in BACE2 activity could have consequences for human disease.