Altered Amyloid Precursor Protein Research Articles

The Abca7V1613M variant reduces Aβ generation, plaque load, and neuronal damage.

Variants in ABCA7, a member of the ABC transporter superfamily, have been associated with increased risk for developing late onset Alzheimer's disease (LOAD). CRISPR-Cas9 was used to generate an Abca7V1613M variant in mice, modeling the homologous human ABCA7V1599M variant, and extensive characterization was performed. Abca7V1613M microglia show differential gene expression profiles upon lipopolysaccharide challenge and increased phagocytic capacity. Homozygous Abca7V1613M mice display elevated circulating cholesterol and altered brain lipid composition. When crossed with 5xFAD mice, homozygous Abca7V1613M mice display fewer Thioflavin S-positive plaques, decreased amyloid beta (Aβ) peptides, and altered amyloid precursor protein processing and trafficking. They also exhibit reduced Aβ-associated inflammation, gliosis, and neuronal damage. Overall, homozygosity for the Abca7V1613M variant influences phagocytosis, response to inflammation, lipid metabolism, Aβ pathology, and neuronal damage in mice. This variant may confer a gain of function and offer a protective effect against Alzheimer's disease-related pathology. ABCA7 recognized as a top 10 risk gene for developing Alzheimer's disease. Loss of function mutations result in increased risk for LOAD. V1613M variant reduces amyloid beta plaque burden in 5xFAD mice. V1613M variant modulates APP processing and trafficking in 5xFAD mice. V1613M variant reduces amyloid beta-associated damage in 5xFAD mice.

The Amyloid Precursor Protein Regulates Synaptic Transmission at Medial Perforant Path Synapses.

The perforant path provides the primary cortical excitatory input to the hippocampus. Because of its important role in information processing and coding, entorhinal projections to the dentate gyrus have been studied in considerable detail. Nevertheless, synaptic transmission between individual connected pairs of entorhinal stellate cells and dentate granule cells remains to be characterized. Here, we have used mouse organotypic entorhino-hippocampal tissue cultures of either sex, in which the entorhinal cortex (EC) to dentate granule cell (GC; EC-GC) projection is present, and EC-GC pairs can be studied using whole-cell patch-clamp recordings. By using cultures of wild-type mice, the properties of EC-GC synapses formed by afferents from the lateral and medial entorhinal cortex were compared, and differences in short-term plasticity were identified. As the perforant path is severely affected in Alzheimer's disease, we used tissue cultures of amyloid precursor protein (APP)-deficient mice to examine the role of APP at this synapse. APP deficiency altered excitatory neurotransmission at medial perforant path synapses, which was accompanied by transcriptomic and ultrastructural changes. Moreover, presynaptic but not postsynaptic APP deletion through the local injection of Cre-expressing adeno-associated viruses in conditional APPflox/flox tissue cultures increased the neurotransmission efficacy at perforant path synapses. In summary, these data suggest a physiological role for presynaptic APP at medial perforant path synapses that may be adversely affected under altered APP processing conditions.SIGNIFICANCE STATEMENT The hippocampus receives input from the entorhinal cortex via the perforant path. These projections to hippocampal dentate granule cells are of utmost importance for learning and memory formation. Although there is detailed knowledge about perforant path projections, the functional synaptic properties at the level of individual connected pairs of neurons are not well understood. In this study, we investigated the role of APP in mediating functional properties and transmission rules in individually connected neurons using paired whole-cell patch-clamp recordings and genetic tools in organotypic tissue cultures. Our results show that presynaptic APP expression limits excitatory neurotransmission via the perforant path, which could be compromised in pathologic conditions such as Alzheimer's disease.

Exercise training and BDNF injections alter APP processing enzymes and improve cognition.

Exercise reduces cognitive aging, neurodegeneration, and Alzheimer's disease (AD) risk. Acute exercise reduces the activity of beta-site amyloid precursor protein cleaving enzyme 1 (BACE1), the rate limiting enzyme in the production of Aβ. However, mechanisms mediating these effects remain largely unknown. Work has implicated brain-derived neurotrophic factor (BDNF) in the processing of amyloid precursor protein (APP). BDNF is an exercise-induced neurotrophin known for its role in synaptic plasticity, neurite growth, and neuronal survival. Previously, our lab has shown using an ex vivo model that treatment of the prefrontal cortex with BDNF reduced BACE1 activity, highlighting a BDNF to BACE1 link. The purpose of this research was to examine whether BDNF treatments resulted in similar biochemical adaptations to APP processing as exercise training. Male C57BL6/J mice were assigned into one of four groups (n=12/group): 1) control; 2) exercise training (progressive treadmill training 5 days/week); 3) BDNF (0.5 mg/kg body mass subcutaneous injection 5 days/week); or 4) endurance training and BDNF, for an 8-week intervention. Recognition memory was measured with a novel object recognition test. Serum, the prefrontal cortex, and hippocampus were collected. BDNF improved recognition memory to a similar extent as endurance training. BDNF and exercise decreased BACE1 activity and increased ADAM10 activity in the prefrontal cortex, indicating a shift in APP processing. Our novel results indicate that BDNF exerts similar beneficial effects on cognition and APP processing as exercise. Future evidence-based preventative or therapeutic interventions that increase BDNF and reduce BACE1 will be valuable for populations at risk of AD.

Adult-Onset Deficiency of Mitochondrial Complex III in a Mouse Model of Alzheimer's Disease Decreases Amyloid Beta Plaque Formation.

For decades, mitochondrial dysfunctions and the generation of reactive oxygen species have been proposed to promote the development and progression of the amyloid pathology in Alzheimer's disease, but this association is still debated. It is unclear whether different mitochondrial dysfunctions, such as oxidative phosphorylation deficiency and oxidative stress, are triggers or rather consequences of the formation of amyloid aggregates. Likewise, the role of the different mitochondrial oxidative phosphorylation complexes in Alzheimer's patients' brain remains poorly understood. Previous studies showed that genetic ablation of oxidative phosphorylation enzymes from early age decreased amyloid pathology, which were unexpected results. To better model oxidative phosphorylation defects in aging, we induced the ablation of mitochondrial Complex III (CIIIKO) in forebrain neurons of adult mice with amyloid pathology. We found that mitochondrial Complex III dysfunction in adult neurons induced mild oxidative stress but did not increase amyloid beta accumulation. On the contrary, CIIIKO-AD mice showed decreased plaque number, decreased Aβ42 toxic fragment, and altered amyloid precursor protein clearance pathway. Our results support the hypothesis that mitochondrial dysfunctions alone, caused by oxidative phosphorylation deficiency, is not the cause of amyloid accumulation.

Altered amyloid precursor protein, tau-regulatory proteins, neuronal numbers and behaviour, but no tau pathology, synaptic and inflammatory changes or memory deficits, at 1month following repetitive mild traumatic brain injury.

Repetitive mild traumatic brain injury, commonly experienced following sports injuries, results in various secondary injury processes and is increasingly recognised as a risk factor for the development of neurodegenerative conditions such as chronic traumatic encephalopathy, which is characterised by tau pathology. We aimed to characterise the underlying pathological mechanisms that might contribute to the onset of neurodegeneration and behavioural changes in the less-explored subacute (1-month) period following single or repetitive controlled cortical impact injury (five impacts, 48 h apart) in 12-week-old male and female C57Bl6 mice. We conducted motor and cognitive testing, extensively characterised the status of tau and its regulatory proteins via western blot and quantified neuronal populations using stereology. We report that r-mTBI resulted in neurobehavioural deficits, gait impairments and anxiety-like behaviour at 1 month post-injury, effects not seen following a single injury. R-mTBI caused a significant increase in amyloid precursor protein, an increased trend towards tau phosphorylation and significant changes in kinase/phosphatase proteins that may promote a downstream increase in tau phosphorylation, but no changes in synaptic or neuroinflammatory markers. Lastly, we report neuronal loss in various brain regions following both single and repeat injuries. We demonstrate herein that repeated impacts are required to promote the initiation of a cascade of biochemical events that are consistent with the onset of neurodegeneration subacutely post-injury. Identifying the timeframe in which these changes occur and the pathological mechanisms involved will be crucial for the development of future therapeutics to prevent the onset or mitigate the progression of neurodegeneration following r-mTBI.

Cerebrospinal fluid amyloid precursor protein as a potential biomarker of fatigue in multiple sclerosis: A pilot study

BackgroundFatigue is the major cause of disability in MS. Fatigue has been suggested to be primary, part of the neurological disease; it can also be secondary to other diseases outside the CNS or exist as a separate comorbidity. The only forms of measurement currently available are through subjective standardized questionnaires, which are not able to identify primary MS-related fatigue. Therefore, there is a need for objective biomarkers of fatigue in MS. This study explored the viability of 17 possible biomarkers of primary fatigue in MS. Our chosen biomarker panel represents the function and health of different parts of the CNS. MethodsWe evaluated 31 MS patients and 17 healthy controls using the Fatigue Severity Scale (FSS) and Insomnia Severity Index (ISI). We assessed clinical parameters and collected CSF from all participants to analyze 17 biomarkers, some of which in multiple targeted sequences, reflecting structural and functional changes in the brain. Based on FSS scores, MS was divided into MS-Fatigue (MS-F, FSS ≥ 4) and MS-NoFatigue (MS-NoF, FSS < 4). ResultsMS-F had significantly lower levels of amyloid precursor protein (APP) peptides than MS-NoF (p = 0.005, p = 0.011). The only biomarker correlating with FSS in any group was APP in MS (r = -0.47, -0.52; p = 0.007, 0.002). APP did not correlate with any clinical parameter in MS but correlated with multiple markers. In MS, FSS correlated with the ISI and months since diagnosis. ConclusionAlthough the mechanisms remain unknown, altered APP metabolism in MS seems to be associated with fatigue. APP should be evaluated as a biomarker of the role of structural MS pathology in the development of fatigue in individual MS patients.

The orphan drug dichloroacetate reduces amyloid beta-peptide production whilst promoting non-amyloidogenic proteolysis of the amyloid precursor protein.

The amyloid cascade hypothesis proposes that excessive accumulation of amyloid beta-peptides is the initiating event in Alzheimer's disease. These neurotoxic peptides are generated from the amyloid precursor protein via sequential cleavage by β- and γ-secretases in the 'amyloidogenic' proteolytic pathway. Alternatively, the amyloid precursor protein can be processed via the 'non-amyloidogenic' pathway which, through the action of the α-secretase a disintegrin and metalloproteinase (ADAM) 10, both precludes amyloid beta-peptide formation and has the additional benefit of generating a neuroprotective soluble amyloid precursor protein fragment, sAPPα. In the current study, we investigated whether the orphan drug, dichloroacetate, could alter amyloid precursor protein proteolysis. In SH-SY5Y neuroblastoma cells, dichloroacetate enhanced sAPPα generation whilst inhibiting β-secretase processing of endogenous amyloid precursor protein and the subsequent generation of amyloid beta-peptides. Over-expression of the amyloid precursor protein partly ablated the effect of dichloroacetate on amyloidogenic and non-amyloidogenic processing whilst over-expression of the β-secretase only ablated the effect on amyloidogenic processing. Similar enhancement of ADAM-mediated amyloid precursor protein processing by dichloroacetate was observed in unrelated cell lines and the effect was not exclusive to the amyloid precursor protein as an ADAM substrate, as indicated by dichloroacetate-enhanced proteolysis of the Notch ligand, Jagged1. Despite altering proteolysis of the amyloid precursor protein, dichloroacetate did not significantly affect the expression/activity of α-, β- or γ-secretases. In conclusion, dichloroacetate can inhibit amyloidogenic and promote non-amyloidogenic proteolysis of the amyloid precursor protein. Given the small size and blood-brain-barrier permeability of the drug, further research into its mechanism of action with respect to APP proteolysis may lead to the development of therapies for slowing the progression of Alzheimer's disease.

The Role of Astrocytes in the Cause of Alzheimer's Disease

There are three leading hypotheses about the cause of Alzheimer’s Disease (AD): the cholinergic theory, where there is a loss of cholinergic neurons; the amyloid hypothesis, where there is an abnormal buildup of amyloid plaques; and the neurotrophic unbalance hypothesis, which states that AD-related loss of cholinergic signaling and altered amyloid precursor protein (APP) processing are due to alterations in nerve growth factor (NGF). This would ultimately mean that the loss of cholinergic neurons and a buildup of amyloid plaques are due to NGF alterations. Astrocytes are involved in the production of amyloid-beta, inflammation responses, and nerve growth. Therefore, astrocytes are an essential component of all three AD hypotheses. This paper will discuss various known and hypothesized ways that astrocytes affect the symptoms and possible causes of AD.

ASSOCIATION OF LIPID PROFILES AND COGNITIVE FUNCTION DETERIORATION OF GERIATRIC OUTPATIENTS AT NEUROLOGY CLINIC GUNUNGSITOLI REGIONAL GENERAL HOSPITAL

Background: The increase of life expectancy in Indonesia causes increasing numbers of dementia, mostly caused by Alzheimer's disease (AD). High serum cholesterol level has been suggested as a risk factor for AD. The strongest evidence linking lipid profile with AD provided by previous experimental studies where adding or reducing cholesterol altered amyloid precursor protein (APP) and amyloid ?-protein (A?) levels is the basic of this research.&#x0D; Objective: To determine whether lipid profile is associated with cognitive function deterioration of geriatric outpatients at Neurology Clinic Gunungsitoli Regional General Hospital.&#x0D; Methods: Participants of this cross-sectional study were outpatient geriatric patients at Neurology Clinic Gunungsitoli Regional General Hospital (n=85; mean age, 62.46±5.49 years old). The cognitive state was evaluated using Montreal Cognitive Assessment Indonesian Version (MoCA-INA) and those with MoCA-INA score &lt;24 were considered cognitively declined. Concentrations of serum lipid profile were measured and correlated with cognitive state using Pearson’s correlation. Multiple logistic regression analysis was used to calculate odds ratios (ORs) for cognitive decline.&#x0D; Results: Based on Pearson’s correlation test, high-density lipoprotein (HDL) level had significant strong positive correlation with MoCA-INA score (r=0.876;p=0.000) and triglyceride level had significant strong negative correlation with MoCA-INA score (r=-0.726;p=0.000). Relatively to cognitive decline, ORs for decreased HDL level was 3.19 (95%CI 2.02-4.36) and increased triglyceride level was 2.59 (95%CI 1.29-3.91).&#x0D; Conclusion: There was a significant relationship between decreased HDL-cholesterol level and increased triglyceride level with cognitive decline in geriatric outpatients.

Amyloid precursor protein glycosylation is altered in the brain of patients with Alzheimer\u2019s disease

BackgroundThe amyloid precursor protein (APP) is a transmembrane glycoprotein that undergoes alternative proteolytic processing. Its processing through the amyloidogenic pathway originates a large sAPPβ ectodomain fragment and the β-amyloid peptide, while non-amyloidogenic processing generates sAPPα and shorter non-fibrillar fragments. Hence, measuring sAPPα and sAPPβ has been proposed as a means to identify imbalances between the amyloidogenic/non-amyloidogenic pathways in the brain of Alzheimer’s disease (AD) patients. However, to date, no consistent changes in these proteolytic fragments have been identified in either the brain or cerebrospinal fluid of AD individuals.MethodsIn frontal cortex homogenates from AD patients (n = 7) and non-demented controls (NDC; n = 7), the expression of total APP mRNA and that of the APP isoforms generated by alternative splicing, APP695 and APP containing the Kunitz protease inhibitor (KPI), was analyzed by qRT-PCR using TaqMan and SYBR Green probes. The balance between the amyloidogenic/non-amyloidogenic pathways was examined in western blots estimating the sAPPα and sAPPβ fragments and their membrane-tethered C-terminal fragments CTFα and CTFβ. CHO-PS70 cells, stably over-expressing wild-type human APP, served to evaluate whether Aβ42 peptide treatment results in altered APP glycosylation. We determined the glycosylation pattern of sAPPα and sAPPβ in brain extracts and CHO-PS70 culture media by lectin-binding assays.ResultsIn the cortex of AD patients, we detected an increase in total APP mRNA relative to the controls, due to an increase in both the APP695 and APP-KPI variants. However, the sAPPα or sAPPβ protein levels remained unchanged, as did those of CTFα and CTFβ. We studied the glycosylation of the brain sAPPα and sAPPβ using lectins and pan-specific antibodies to discriminate between the fragments originated from neuronal APP695 and glial/KPI variants. Lectin binding identified differences in the glycosylation of sAPPβ species derived from the APP695 and APP-KPI variants, probably reflecting their distinct cellular origin. Moreover, the lectin-binding pattern differed in the sAPPα and sAPPβ originated from all the variants. Finally, when the lectin-binding pattern was compared between AD and NDC groups, significant differences were evident in sAPPα glycosylation. Lectin binding of the soluble sAPPα and sAPPβ from CHO-PS70 cells were also altered in cells treated with the Aβ peptide.ConclusionOur analysis of the lectin binding to sAPPα and sAPPβ suggests that glycosylation dictates the proteolytic pathway for APP processing. Differences between the demented and controls indicate that changes in glycosylation may influence the generation of the different APP fragments and, consequently, the pathological progression of AD.

Novel presenilin 1 and 2 double knock-out cell line for in vitro validation of PSEN1 and PSEN2 mutations

Mutations in APP (amyloid precursor protein), PSEN1 (presenilin 1) or PSEN2 (presenilin 2) are the main cause of early-onset familial forms of Alzheimer's disease (autosomal dominant AD or ADAD). These genes affect γ-secretase-dependent generation of Amyloid β (Aβ) peptides, the main constituent of amyloid plaques and one of the pathological hallmarks of AD. Evaluation of patients with ADAD includes assessment of family history, clinical presentation, biomarkers, neuropathology when available and DNA sequencing data. These analyses frequently uncover novel variants of unknown significance in ADAD genes. This presents a barrier to recruitment of such individuals into clinical trials, unless a biochemical test can demonstrate that a novel mutation results in altered APP processing in a manner consistent with pathogenicity. Here we describe generation and characterization of a novel presenilin 1 and 2 double knock-out in N2A mouse neuroblastoma cells using CRISPR/Cas9, which results in complete ablation of Aβ production, decreased Pen-2 expression and Nicastrin glycosylation. Because of the absence of background Aβ secretion from endogenous γ-secretases, these cells can be used for validation of PSEN1 and PSEN2 variant effects on production of Aβ or other γ-secretase substrates and for biochemical studies of γ-secretase function using novel variants. We examined several PSEN1 and PSEN2 mutations of known and unknown pathogenicity. Known mutants increased Aβ42/Aβ40 ratio with varying effect on Aβ40, Aβ42, total Aβ levels and Pen-2 expression, which aligns with previous work on these mutants. Our data on novel PSEN1 V142F, G206V and G206D mutations suggest that these mutations underlie the reported clinical observations in ADAD patients. We believe our novel cell line will be valuable for the scientific community for reliable validation of presenilin mutations and helpful in defining their pathogenicity to improve and facilitate evaluation of ADAD patients, particularly in the context of enrollment in clinical trials.

Kinesin light chain-1 serine-460 phosphorylation is altered in Alzheimer\u2019s disease and regulates axonal transport and processing of the amyloid precursor protein

Damage to axonal transport is an early pathogenic event in Alzheimer’s disease. The amyloid precursor protein (APP) is a key axonal transport cargo since disruption to APP transport promotes amyloidogenic processing of APP. Moreover, altered APP processing itself disrupts axonal transport. The mechanisms that regulate axonal transport of APP are therefore directly relevant to Alzheimer’s disease pathogenesis. APP is transported anterogradely through axons on kinesin-1 motors and one route for this transport involves calsyntenin-1, a type-1 membrane spanning protein that acts as a direct ligand for kinesin-1 light chains (KLCs). Thus, loss of calsyntenin-1 disrupts APP axonal transport and promotes amyloidogenic processing of APP. Phosphorylation of KLC1 on serine-460 has been shown to reduce anterograde axonal transport of calsyntenin-1 by inhibiting the KLC1-calsyntenin-1 interaction. Here we demonstrate that in Alzheimer’s disease frontal cortex, KLC1 levels are reduced and the relative levels of KLC1 serine-460 phosphorylation are increased; these changes occur relatively early in the disease process. We also show that a KLC1 serine-460 phosphomimetic mutant inhibits axonal transport of APP in both mammalian neurons in culture and in Drosophila neurons in vivo. Finally, we demonstrate that expression of the KLC1 serine-460 phosphomimetic mutant promotes amyloidogenic processing of APP. Together, these results suggest that increased KLC1 serine-460 phosphorylation contributes to Alzheimer’s disease.

Familial Alzheimer\u2019s disease patient-derived neurons reveal distinct mutation-specific effects on amyloid beta

Familial Alzheimer’s disease (fAD) mutations alter amyloid precursor protein (APP) cleavage by γ-secretase, increasing the proportion of longer amyloidogenic amyloid-β (Aβ) peptides. Using five control induced pluripotent stem cell (iPSC) lines and seven iPSC lines generated from fAD patients, we investigated the effects of mutations on the Aβ secretome in human neurons generated in 2D and 3D. We also analysed matched CSF, post-mortem brain tissue, and iPSCs from the same participant with the APP V717I mutation. All fAD mutation lines demonstrated an increased Aβ42:40 ratio relative to controls, yet displayed varied signatures for Aβ43, Aβ38, and short Aβ fragments. We propose four qualitatively distinct mechanisms behind raised Aβ42:40. (1) APP V717I mutations alter γ-secretase cleavage site preference. Whereas, distinct presenilin 1 (PSEN1) mutations lead to either (2) reduced γ-secretase activity, (3) altered protein stability or (4) reduced PSEN1 maturation, all culminating in reduced γ-secretase carboxypeptidase-like activity. These data support Aβ mechanistic tenets in a human physiological model and substantiate iPSC-neurons for modelling fAD.

Reduced Aβ secretion by human neurons underconditions of strongly increased BACE activity.

The initial step in the amyloidogenic cascade of amyloid precursor protein (APP) processing is catalyzed by beta-site APP-cleaving enzyme (BACE), and this protease has increased activities in affected areas of Alzheimer's disease brains. We hypothesized that altered APP processing, because of augmented BACE activity, would affect the actions of direct and indirect BACE inhibitors. We therefore compared post-mitotic human neurons (LUHMES) with their BACE-overexpressing counterparts (BLUHMES). Although β-cleavage of APP was strongly increased in BLUHMES, they produced less full-length and truncated amyloid beta (Aβ) than LUHMES. Moreover, low concentrations of BACE inhibitors decreased cellular BACE activity as expected, but increased Aβ1-40 levels. Several other approaches to modulate BACE activity led to a similar, apparently paradoxical, behavior. For instance, reduction in intracellular acidification by bepridil increased Aβ production in parallel with decreased BACE activity. In contrast to BLUHMES, the respective control cells (LUHMES or BLUHMES with catalytically inactive BACE) showed conventional pharmacological responses. Other non-canonical neurochemical responses (so-called 'rebound effects') are well-documented for the Aβ pathway, especially for γ-secretase: a partial block of its activity leads to an increased Aβ secretion by some cell types. We therefore compared LUHMES and BLUHMES regarding rebound effects of γ-secretase inhibitors and found an Aβ rise in LUHMES but not in BLUHMES. Thus, different cellular factors are responsible for the γ-secretase- versus BACE-related Aβ rebound. We conclude that increased BACE activity, possibly accompanied by an altered cellular localization pattern, can dramatically influence Aβ generation in human neurons and affect pharmacological responses to secretase inhibitors. OPEN PRACTICES: Open Science: This manuscript was awarded with the Open Materials Badge. For more information see: https://cos.io/our-services/open-science-badges/.

BACE1 Mediates HIV-Associated and Excitotoxic Neuronal Damage Through an APP-Dependent Mechanism.

HIV-associated neurocognitive disorders (HANDs) share common symptoms with Alzheimer's disease (AD), which is characterized by amyloid-β (Aβ) plaques. Plaques are formed by aggregation of Aβ oligomers, which may be the toxic species in AD pathogenesis, and oligomers are generated by cleavage of amyloid precursor protein (APP) by β-site amyloid precursor protein cleaving enzyme 1 (BACE1). BACE1 inhibitors reverse neuronal loss and cognitive decline in animal models of AD. Although studies have also found evidence of altered APP processing in HIV+ patients, it is unknown whether increased BACE1 expression or Aβ oligomer production is a common neuropathological feature of HAND. Moreover, it is unknown whether BACE1 or APP is involved in the excitotoxic, NMDAR-dependent component of HIV-associated neurotoxicity in vitro Herein, we hypothesize that HIV-associated neurotoxicity is mediated by NMDAR-dependent elevation of BACE1 and subsequent altered processing of APP. Supporting this, we observed elevated levels of BACE1 and Aβ oligomers in CNS of male and female HIV+ patients. In a model of HIV-associated neurotoxicity in which rat neurons are treated with supernatants from HIV-infected human monocyte-derived macrophages, we observed NMDAR-dependent elevation of BACE1 protein. NMDA treatment also increased BACE1 and both pharmacological BACE1 inhibition and genetic loss of APP were partially neuroprotective. Moreover, in APP knock-out (APP-/-) mouse neurons, NMDA-induced toxicity was BACE1 independent, indicating that cytotoxicity of BACE1 is dependent upon APP cleavage. Our findings suggest that increased BACE1 and the resultant Aβ oligomer production may contribute to HIV-associated neuropathogenesis and inhibition of BACE1 could have therapeutic potential in HANDs.SIGNIFICANCE STATEMENT HIV-associated neurocognitive disorders (HANDs) represent a range of cognitive impairments affecting ∼50% of HIV+ individuals. The specific causes of HAND are unknown, but evidence suggests that HIV-infected macrophage infiltration into the brain may cause neuronal damage. Herein, we show that neurons treated with conditioned media from HIV-infected macrophages have increased expression of β-site amyloid precursor protein cleaving enzyme 1 (BACE1), a protein implicated in Alzheimer's disease pathogenesis. Moreover, inhibition of BACE1 prevented neuronal loss after conditioned media exposure, but had no effect on HIV-associated neurotoxicity in neurons lacking its cleavage target amyloid precursor protein. We also observed increased BACE1 expression in HIV+ patient brain tissue, confirming the potential relevance of BACE1 as a therapeutic target in HANDs.

SAPPβ and sAPPα increase structural complexity and E/I input ratio in primary hippocampal neurons and alter Ca2+ homeostasis and CREB1-signaling

One major pathophysiological hallmark of Alzheimer's disease (AD) is senile plaques composed of amyloid β (Aβ). In the amyloidogenic pathway, cleavage of the amyloid precursor protein (APP) is shifted towards Aβ production and soluble APPβ (sAPPβ) levels. Aβ is known to impair synaptic function; however, much less is known about the physiological functions of sAPPβ. The neurotrophic properties of sAPPα, derived from the non-amyloidogenic pathway of APP cleavage, are well-established, whereas only a few, conflicting studies on sAPPβ exist. The intracellular pathways of sAPPβ are largely unknown. Since sAPPβ is generated alongside Aβ by β-secretase (BACE1) cleavage, we tested the hypothesis that sAPPβ effects differ from sAPPα effects as a neurotrophic factor. We therefore performed a head-to-head comparison of both mammalian recombinant peptides in developing primary hippocampal neurons (PHN). We found that sAPPα significantly increases axon length (p = 0.0002) and that both sAPPα and sAPPβ increase neurite number (p < 0.0001) of PHN at 7 days in culture (DIV7) but not at DIV4. Moreover, both sAPPα- and sAPPβ-treated neurons showed a higher neuritic complexity in Sholl analysis. The number of glutamatergic synapses (p < 0.0001), as well as layer thickness of postsynaptic densities (PSDs), were significantly increased, and GABAergic synapses decreased upon sAPP overexpression in PHN. Furthermore, we showed that sAPPα enhances ERK and CREB1 phosphorylation upon glutamate stimulation at DIV7, but not DIV4 or DIV14. These neurotrophic effects are further associated with increased glutamate sensitivity and CREB1-signaling. Finally, we found that sAPPα levels are significantly reduced in brain homogenates of AD patients compared to control subjects. Taken together, our data indicate critical stage-dependent roles of sAPPs in the developing glutamatergic system in vitro, which might help to understand deleterious consequences of altered APP shedding in AD patients, beyond Aβ pathophysiology.

The non-peptidic δ-opioid receptor agonist Tan-67 mediates neuroprotection post-ischemically and is associated with altered amyloid precursor protein expression, maturation and processing in mice.

Tan-67 is a selective non-peptidic δ-opioid receptor (DOR) agonist that confers neuroprotection against cerebral ischemia/reperfusion (I/R)-caused neuronal injury in pre-treated animals. In this study, we examined whether post-ischemic administration of Tan-67 in stroke mice is also neuroprotective and whether the treatment affects expression, maturation and processing of the amyloid precursor protein (APP). A focal cerebral I/R model in mice was induced by middle cerebral artery occlusion for 1h and Tan-67 (1.5, 3 or 4.5mg/kg) wasadministered via the tail vein at 1h after reperfusion. Alternatively, naltrindole, a selective DOR antagonist (5mg/kg), was administered 1h before Tan-67 treatment. Our results showed that post-ischemic administration of Tan-67 (3mg/kg or 4.5mg/kg) was neuroprotective as shown by decreased infarct volume and neuronal loss following I/R. Importantly, Tan-67 improved animal survival and neurobehavioral outcomes. Conversely, naltrindole abolished Tan-67 neuroprotection in infarct volume. Tan-67 treatment also increased APP expression, maturation and processing in the ipsilateral penumbral area at 6h but decreased APP expression and maturation in the same brain area at 24h after I/R. Tan-67-induced increase in APP expression was also seen in the ischemic cortex at 24h following I/R. Moreover, Tan-67 attenuated BACE-1 expression, β-secretase activity and the BACE cleavage of APP in the ischemic cortex at 24h after I/R, which was abolished by naltrindole. Our data suggest that Tan-67 is a promising DOR-dependent therapeutic agent for treating I/R-caused disorder and that Tan-67-mediated neuroprotection may be mediated via modulating APP expression, maturation and processing, despite an uncertain causative relationship between the altered APP and the outcomes observed.

Dimerization leads to changes in APP (amyloid precursor protein) trafficking mediated by LRP1 and SorLA.

Proteolytic cleavage of the amyloid precursor protein (APP) by α-, β- and γ-secretases is a determining factor in Alzheimer's disease (AD). Imbalances in the activity of all three enzymes can result in alterations towards pathogenic Aβ production. Proteolysis of APP is strongly linked to its subcellular localization as the secretases involved are distributed in different cellular compartments. APP has been shown to dimerize in cis-orientation, affecting Aβ production. This might be explained by different substrate properties defined by the APP oligomerization state or alternatively by altered APP monomer/dimer localization. We investigated the latter hypothesis using two different APP dimerization systems in HeLa cells. Dimerization caused a decreased localization of APP to the Golgi and at the plasma membrane, whereas the levels in the ER and in endosomes were increased. Furthermore, we observed via live cell imaging and biochemical analyses that APP dimerization affects its interaction with LRP1 and SorLA, suggesting that APP dimerization modulates its interplay with sorting molecules and in turn its localization and processing. Thus, pharmacological approaches targeting APP oligomerization properties might open novel strategies for treatment of AD.

Modelling APOE ɛ3/4 allele-associated sporadic Alzheimer's disease in an induced neuron.

The recent generation of induced neurons by direct lineage conversion holds promise for in vitro modelling of sporadic Alzheimer's disease. Here, we report the generation of induced neuron-based model of sporadic Alzheimer's disease in mice and humans, and used this system to explore the pathogenic mechanisms resulting from the sporadic Alzheimer's disease risk factor apolipoprotein E (APOE) ɛ3/4 allele. We show that mouse and human induced neurons overexpressing mutant amyloid precursor protein in the background of APOE ɛ3/4 allele exhibit altered amyloid precursor protein (APP) processing, abnormally increased production of amyloid-β42 and hyperphosphorylation of tau. Importantly, we demonstrate that APOE ɛ3/4 patient induced neuron culture models can faithfully recapitulate molecular signatures seen in APOE ɛ3/4-associated sporadic Alzheimer's disease patients. Moreover, analysis of the gene network derived from APOE ɛ3/4 patient induced neurons reveals a strong interaction between APOE ɛ3/4 and another Alzheimer's disease risk factor, desmoglein 2 (DSG2). Knockdown of DSG2 in APOE ɛ3/4 induced neurons effectively rescued defective APP processing, demonstrating the functional importance of this interaction. These data provide a direct connection between APOE ɛ3/4 and another Alzheimer's disease susceptibility gene and demonstrate in proof of principle the utility of induced neuron-based modelling of Alzheimer's disease for therapeutic discovery.

Xanomeline derivative EUK1001 attenuates Alzheimer's disease pathology in a triple transgenic mouse model.

Agonists of M1 muscarinic acetylcholine receptors are promising therapeutic agents for the treatment of Alzheimer's disease (AD). An example of one of these agents is xanomeline, which has been a leading candidate, however induces various unwanted adverse effects. 3‑[3‑(3‑florophenyl‑2‑propyn‑1‑ylthio)‑1,2,5‑thiadiazol-4-yl]-1,2,5,6-tetrahydro‑1‑methylpyridine oxalate (EUK1001), a fluorinated derivative of xanomeline, has been demonstrated to attenuate AD‑like neurodegenerative pathology in presenilin‑deficient mice, which has no β‑amyloid (Aβ) pathology. The present study assessed the effect of EUK1001 on the behavioral performance of the 3xTg‑AD model of AD. EUK1001 treatment decreased cognitive deficits in male and female AD mice in the Morris water maze test and novel object recognition tasks. EUK1001 also decreased Aβ42, however not Aβ40 in the cortex and hippocampus of AD mice. EUK1001 may also alter amyloid precursor protein processing to a nonamyloidgenic pathway invitro. These results demonstrate that EUK1001 may ameliorate the cognitive dysfunction of AD mice, possibly by reducing Aβ production. Therefore, EUK1001 may be an effective treatment for AD.