👤 Rebecca E K MacPherson

The accumulation and deposition of amyloid-beta (Aß) peptides is detrimental to neuronal networks and is driven by the cleavage of amyloid precursor protein (APP) by beta-secretase 1 (BACE1). The proteolytic processing of APP is tightly regulated by the opposing activities of BACE1 and ADAM10, with the latter producing a truncated, non-amyloidogenic fragment. Maintaining this balance is critical for normal physiological function, as complete inhibition of BACE1 has proven detrimental owing to the important physiological roles of its many substrates. Brain-derived neurotrophic factor (BDNF), an important mediator of neuronal function and survival, has recently been shown to reduce BACE1 activity in neural tissue, but the mechanism for this remains unknown. Previous research suggests that BACE1 cleavage of APP is favoured at acidic intracellular compartments, whereas non-amyloidogenic processing preferentially occurs at the plasma membrane. Hence, we hypothesized that BDNF alters the subcellular distribution of BACE1, reducing ß-cleavage of APP. Here, we show that acute BDNF treatment of differentiated neural cells (SH-SY5Y) reduced levels of sAPPß, a product of BACE1 cleavage of APP. Using confocal microscopy and quantitative image analysis, we found that this reduction in sAPPß levels is coincident with increased BACE1 localization to the plasma membrane, and a concomitant reduction of BACE1 localization to early endosomes. This effect appears to be independent of clathrin-mediated endocytosis (CME), as inhibition of CME by PitStop2 treatment increased a-cleavage of APP but did not reduce ß-cleavage independent of BDNF treatment. Hence, BDNF may reduce production of Aß by altering BACE1 distribution and decreasing upstream ß-cleavage. Show less

Heat shock proteins (HSPs) are molecular chaperones that play important roles in protein homeostasis, with HSP70 linked to a role in neuroprotection. HSP70 is upregulated in response to various stressors, such as heat therapy (HT), which has been shown to increase brain-derived neurotrophic factor (BDNF) content. BDNF reduces the activity of β-site amyloid precursor protein cleaving enzyme 1 (BACE1), the rate-limiting enzyme responsible for the generation of amyloid-β (Aβ) peptides that form the characteristic Aβ plaques observed in Alzheimer's disease brains. The current pilot study examined whether 4 wk of HT can increase HSP70 and BDNF content (pro and mature forms) in the brain, and alter markers of amyloid precursor protein (APP) processing. Male mice had their core temperature maintained between 37.0 and 38.0°C in Control (CON, Show less

The protease β-secretase (BACE1) plays a crucial role in the formation of amyloid-beta peptides. Here, we present a protocol for real-time quantification of BACE1 activity in brain tissue and cell lysates using a fluorogenic peptide substrate in a 96-well format. We describe steps for reagent and sample preparation, preparing the plate for BACE1 activity, and incubating and reading the plate, followed by quantification and analysis. This protocol is broadly accessible for laboratories studying enzymatic activity under physiological or pathological conditions. For complete details on the use and execution of this protocol, please refer to Baranowski et al. Show less

People with type 2 diabetes (T2D) have a greater risk of developing neurodegenerative diseases, like Alzheimer's disease, in later life. Exogenous ketone supplements containing the ketone body β-hydroxybutyrate (β-OHB) may be a strategy to protect the brain as β-OHB can support cerebral metabolism and promote neuronal plasticity via expression of brain-derived neurotrophic factor (BDNF). Parallel human (ClinicalTrials.gov ID NCT04194450, ClinicalTrials.gov ID NCT05155410) and rodent trials were conducted to characterize the effect of acute and short-term exogenous ketone supplementation on indices of brain health. First, we aimed to investigate the effect of acute and short-term supplementation of exogenous ketone monoester on circulating BDNF and cognition in adults with T2D. There were no effects of ketone supplementation on plasma BDNF or cognition. Second, we aimed to investigate the mechanistic effects of acute and chronic β-OHB supplementation on cortical BDNF content and recognition memory in C57BL/6J mice with and without insulin resistance. Acutely, β-OHB did not alter recognition memory or BDNF content. Similarly, chronic β-OHB supplementation did not alter recognition memory or BDNF content. Collectively, our data demonstrates that ketone supplementation does not elevate BDNF content in humans or mice. Furthermore, our data does not support the involvement of BDNF in the potential cognitive benefits of β-OHB supplementation. Show less

The accumulation and aggregation of beta-amyloid (Aβ) peptides contributes to neuronal dysfunction and death. These Aβ peptides originate from a transmembrane protein known as amyloid precursor protein (APP), which can be processed via two competing pathways. Alpha-secretase (ADAM10) cleavage is thought to be neuroprotective while beta-secretase (BACE1) cleavage results in the production of Aβ. Aerobic exercise reduces BACE1 activity, but the mechanisms involved are unknown though several exercise-induced mediators such as lactate may be involved. The current study examined whether systemic lactate can alter APP processing and BACE1 and ADAM10 activity. Mice were randomly assigned to one of four groups (n = 10 per group): (1) sedentary; (2) lactate-injection (1.0 g kg Show less

Brain-derived neurotrophic factor (BDNF) content and signaling has been identified as one potential regulator of amyloid precursor protein (APP) processing. Recently published work has demonstrated that BDNF reduces BACE1 activity while also elevating the inhibition of GSK3β in the prefrontal cortex of male C57BL/6J mice. These results provide evidence that BDNF alters APP processing by reducing BACE1 activity, which may act through GSK3β inhibition. The purpose of this study was to further explore the role of GSK3β in BDNF-induced regulation on BACE1 activity. We utilized a cell culture and an in vitro activity assay model to pharmacologically target BDNF and GSK3β signaling to confirm its involvement in the BDNF response. Treatment of differentiated SH-SY5Y neuronal cells with 75 ng/mL BDNF resulted in elevated pTrkB content, pAkt content, pGSK3β content, and reduced BACE1 activity. An in vitro BACE1 activity assay utilizing mouse prefrontal cortex (n = 6/group) supplemented with BDNF, BDNF + ANA12 (Trkb antagonist), or BDNF + wortmannin (Akt inhibitor) demonstrated that BDNF reduced BACE1 activity; however, in the presence of TrkB or Akt inhibition, this effect was abolished. An in vitro ADAM10 activity assay utilizing mouse prefrontal cortex (n = 6/group) supplemented with BDNF, BDNF + ANA12 (Trkb antagonist), or BDNF + wortmannin (Akt inhibitor) demonstrated that BDNF did not alter ADAM10 activity. However, inhibiting BDNF signaling reduced ADAM10 activity. Collectively these studies suggest that GSK3β inhibition may be necessary for BDNF-induced reductions in BACE1 activity. These findings will allow for the optimization of future therapeutic strategies by selectively targeting TrkB activation and GSK3β inhibition. Show less

Exercise reduces cognitive aging, neurodegeneration, and Alzheimer's disease (AD) risk. Acute exercise reduces the activity of β-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 ( Show less

Exercise has been shown to be beneficial for individuals with Alzheimer's disease (AD). In rodent models of AD, exercise decreases the amyloidogenic processing of the amyloid precursor protein (APP). Although it remains unclear as to how exercise is promoting this shift away from pathological APP processing, there is emerging evidence that exercise-induced factors released from peripheral tissues may facilitate these alterations in brain APP processing. Interleukin-6 (IL-6) is released from multiple organs into peripheral circulation during exercise and is among the most characterized exerkines. The purpose of this study is to examine whether acute IL-6 can modulate key enzymes responsible for APP processing, namely, a disintegrin and metalloproteinase 10 (ADAM10) and β-site amyloid precursor protein-cleaving enzyme 1 (BACE1), which initiate the nonamyloidogenic and amyloidogenic cascades, respectively. Male 10-wk-old C57BL/6J mice underwent acute treadmill exercise bout or were injected with either IL-6 or a PBS control 15 min prior to tissue collection. ADAM10 and BACE1 enzyme activity, mRNA, and protein expression, as well as downstream markers of both cascades, including soluble APPα (sAPPα) and soluble APPβ (sAPPβ), were examined. Exercise increased circulating IL-6 and brain IL-6 signaling (pSTAT3 and Show less

Aberrant cleavage of the transmembrane protein, amyloid-beta precursor protein (ABPP), results in the overproduction of amyloid-beta (AB) peptides which can form senile plaques in the brain. These plaques can get lodged within synapses and disrupt neuronal communication ultimately leading to rampant neuron death. The rate-limiting enzyme in AB production is beta-site ABPP cleaving enzyme 1 (BACE1). In females, estrogen loss is associated with increases in AB and BACE1 content and activity. Exercise is known to have anti-amyloidogenic effects and may be able to alter BACE1 in cases of ovarian hormone depletion. This study aimed to examine the effects of physical activity on BACE1 in intact and ovariectomized female mice. Female C57BL/6 mice (24 weeks old) underwent bilateral ovariectomy (OVX; n=20) or SHAM surgery (SHAM; n=20). Mice were assigned to one of four groups (n=10/group) for 8 weeks: (1) sham (SHAM), (2) sham with a wheel (SHAM VWR), (3) ovariectomized (OVX), or (4) ovariectomized with a wheel (OVX VWR). Novel object recognition testing demonstrated that OVX mice had a lower percentage of novel object investigation time compared to SHAM. OVX mice also had higher prefrontal cortex BACE1 activity compared to SHAM (p<0.0001), while the OVX+VWR activity was not different from SHAM. Our results demonstrate that voluntary wheel running in an ovariectomized model prevented increases in BACE1 activity, maintained memory recall, and may provide a method of slowing the progression of Alzheimer's disease. Show less

Members of the poly-ADP-ribose polymerase (PARP) family catalyse the ADP-ribosylation of target proteins and are known to play important roles in many cellular processes, including DNA repair, differentiation and transcription. The majority of PARPs exhibit mono-ADP-ribosyltransferase activity rather than PARP activity; however, little is known about their biological activity. In the present study, we report that 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)-inducible poly-ADP-ribose polymerase (TIPARP), mono-ADP-ribosylates and positively regulates liver X receptor α (LXRα) and LXRβ activity. Overexpression of TIPARP enhanced LXR-reporter gene activity. TIPARP knockdown or deletion reduced LXR regulated target gene expression levels in HepG2 cells and in Tiparp(-/-)mouse embryonic fibroblasts (MEFs) respectively. Deletion and mutagenesis studies showed that TIPARP's zinc-finger and catalytic domains were required to enhance LXR activity. Protein interaction studies using TIPARP and LXRα/β peptide arrays revealed that LXRs interacted with an N-terminal sequence (a.a. 209-236) of TIPARP, which also overlapped with a putative co-activator domain of TIPARP (a.a. 200-225). Immunofluorescence studies showed that TIPARP and LXRα or LXRβ co-localized in the nucleus.In vitroribosylation assays provided evidence that TIPARP mono-ADP-ribosylated both LXRα and LXRβ. Co-immunoprecipitation (co-IP) studies revealed that ADP-ribosylase macrodomain 1 (MACROD1), but not MACROD2, interacted with LXRs in a TIPARP-dependent manner. This was complemented by reporter gene studies showing that MACROD1, but not MACROD2, prevented the TIPARP-dependent increase in LXR activity. GW3965-dependent increases in hepatic Srebp1 mRNA and protein expression levels were reduced in Tiparp(-/-)mice compared with Tiparp(+/+)mice. Taken together, these data identify a new mechanism of LXR regulation that involves TIPARP, ADP-ribosylation and MACROD1. Show less

Liver X receptor (LXR)α and LXRβ play key roles in hepatic de novo lipogenesis through their regulation of lipogenic genes, including sterol regulatory element-binding protein (SREBP)-1c and carbohydrate responsive element-binding protein (ChREBP). LXRs activate lipogenic gene transcription in response to feeding, which is believed to be mediated by insulin. We have previously shown that LXRs are targets for glucose-hexosamine-derived O-linked β-N-acetylglucosamine (O-GlcNAc) modification enhancing their ability to regulate SREBP-1c promoter activity in vitro. To elucidate insulin-independent effects of feeding on LXR-mediated lipogenic gene expression in vivo, we subjected control and streptozotocin-treated LXRα/β(+/+) and LXRα/β(-/-) mice to a fasting-refeeding regime. We show that under hyperglycemic and hypoinsulinemic conditions, LXRs maintain their ability to upregulate the expression of glycolytic and lipogenic enzymes, including glucokinase (GK), SREBP-1c, ChREBPα, and the newly identified shorter isoform ChREBPβ. Furthermore, glucose-dependent increases in LXR/retinoid X receptor-regulated luciferase activity driven by the ChREBPα promoter was mediated, at least in part, by O-GlcNAc transferase (OGT) signaling in Huh7 cells. Moreover, we show that LXR and OGT interact and colocalize in the nucleus and that loss of LXRs profoundly reduced nuclear O-GlcNAc signaling and ChREBPα promoter binding activity in vivo. In summary, our study provides evidence that LXRs act as nutrient and glucose metabolic sensors upstream of ChREBP by modulating GK expression, nuclear O-GlcNAc signaling, and ChREBP expression and activity. Show less