Effects Of Amyloid-beta Research Articles

The effect of amyloid beta, membrane, and ER pathways on the fractional behavior of neuronal calcium

Calcium signal transduction is essential for cellular activities such as gene transcription, death, and neuronal plasticity. Dynamical changes in the concentration of calcium have a profound effect on the intracellular activity of neurons. The Caputo fractional reaction-diffusion equation is a useful tool for modeling the intricate biological process involved in calcium concentration regulation. We include the Amyloid Beta, STIM-Orai mechanism, voltage-dependent calcium entry, inositol triphosphate receptor (IPR), endoplasmic reticulum (ER) flux, SERCA pump, and plasma membrane flux in our mathematical model. We use Green's function and Hankel and Laplace integral transforms to solve the membrane flux problem. Our simulations investigate the effects of various factors on the spatiotemporal behavior of calcium levels, with a simulation on the buffers in Alzheimer's disease-affected neurons. We also look at the effects of calcium-binding substances like the S100B protein and BAPTA and EGTA. Our results demonstrate how important the S100B protein Amyloid beta and the STIM-Orai mechanism are, and how important they are to consider when simulating the calcium signaling system. As such, our research indicates that a more realistic and complete model for modeling calcium dynamics may be obtained by using a generalized reaction-diffusion technique.

Effects of Amyloid Beta (Aβ) Oligomers on Blood-Brain Barrier Using a 3D Microfluidic Vasculature-on-a-Chip Model.

The disruption of the blood-brain barrier (BBB) in Alzheimer's Disease (AD) is largely influenced by amyloid beta (Aβ). In this study, we developed a high-throughput microfluidic BBB model devoid of a physical membrane, featuring endothelial cells interacting with an extracellular matrix (ECM). This paper focuses on the impact of varying concentrations of Aβ1-42 oligomers on BBB dysfunction by treating them in the luminal. Our findings reveal a pronounced accumulation of Aβ1-42 oligomers at the BBB, resulting in the disruption of tight junctions and subsequent leakage evidenced by a barrier integrity assay. Additionally, cytotoxicity assessments indicate a concentration-dependent increase in cell death in response to Aβ1-42 oligomers (LC50 ~ 1 μM). This study underscores the utility of our membrane-free vascular chip in elucidating the dysfunction induced by Aβ with respect to the BBB.

Moringa oleifera leaf extract suppresses TIMM23 and NDUFS3 expression and alleviates oxidative stress induced by Aβ1-42 in neuronal cells via activation of Akt.

Oxidative stress plays an important role in Alzheimer's disease (AD) pathogenesis. Moringa oleifera leaf (MOL) extract has been shown to have antioxidant activities. Here, we studied the antioxidative and anti-apoptotic effects of water-soluble MOL extract in an amyloid beta (Aβ)-induced oxidative stress model of AD. The effect of amyloid beta (Aβ)1-42 and MOL extract on differentiated SH-SY5Y cell viability was assessed by MTT assay. Cells were treated with Aβ1-42, MOL extract, or MOL extract followed by Aβ1-42. The mitochondrial membrane potential (ΔΨm) and the reactive oxygen species (ROS) were evaluated by flow cytometry and dihydroethidium (DHE) assay, respectively. Western blotting was used to assess the expression of mitochondrial proteins TIMM23 and NDUFS3, apoptosis-related proteins Bax, Bcl-2, and cleaved caspase-3 along with fluorescence analysis of caspase-3/7, and Akt phosphorylation. MOL extract pretreatment at 25, 50, and 100 μg/mL prevented ΔΨm reduction. At 100-μg/mL, MOL extract decreased TIMM23 and NDUFS3 proteins and DHE signals in Aβ1-42-treated cells. MOL extract pretreatment (25, 50, and 100 μg/mL) also alleviated the apoptosis indicators, including Bax, caspase-3/7 intensity, and cleaved caspase-3, and increased Bcl-2 levels in Aβ1-42-treated cells, consistent with a reduction in the number of apoptotic cells. The protective effects of MOL extract were possibly mediated through Akt activation, evidenced by increased Akt phosphorylation. The neuroprotective effect of MOL extract could be mediated via the activation of Akt, leading to the suppression of oxidative stress and apoptosis in an Aβ1-42 model of AD.

The Effect of Amyloid Beta on Symptoms Caused by Traumatic Brain Injury in Drosophila melanogaster

Traumatic brain injury (TBI) is the cause of one third of all injury-related deaths in the United States alone, and approximately 1.7 million people sustain a TBI each year. Depending on the severity of the, a TBI could lead to sustained damage to cognitive and locomotive abilities. Treatments for TBI have been previously investigated; however, prior research does not include the investigation of amyloid beta protein (Aβ), a protein found to accumulate rapidly after TBI, as having a role in recovery from TBI. This experiment focused on studying the effect of the presence of Aβ on cognitive and locomotor ability in Drosophila melanogaster after TBI. The cognitive and locomotor skills of D. melanogaster were tested through the means of a food-based choice assay and a climbing assay respectively. After data collection, Mann Whitney statistical tests were done between D. melanogaster groups to determine the significance of the results. From the statistical analysis of the results, the following was derived: the presence of Aβ is related to improved locomotion in D. melanogaster after TBI according to a significant difference in assay success rates between D. melanogaster with and without Aβ (p-value < 0.0001), however there was no demonstrated relation between Aβ and improved cognition (p-value = 0.0663). Therefore, the hypothesis regarding the possible neural repair properties of Aβ is partially supported. It can be concluded that while Aβ had no effect on cognition in D. melanogaster after TBI, its presence is directly related to the improvement of locomotion.

Effects of amyloid beta and dopaminergic depletion on perfusion and clinical symptoms.

Although mixed pathologies are common in Alzheimer's disease (AD) and dementia with Lewy bodies (DLB), the effects of amyloid beta and dopaminergic depletion on brain perfusion and clinical symptoms have not been elucidated. In 99 cognitive impairment patients due to AD and/or DLB and 32 controls, 18F-florbetaben (FBB) and dual-phase dopamine transporter (DAT) positron emission tomography (PET) were performed to measure the FBB standardized uptake value ratio (SUVR), striatal DAT uptakes, and brain perfusion. Higher FBB-SUVR and lower ventral striatal DAT uptake were intercorrelated and, respectively, associated with left entorhinal/temporo-parietal-centered hypoperfusion and vermis/hippocampal-centered hyperperfusion, whereas regional perfusion mediated clinical symptoms and cognition. Amyloid beta deposition and striatal dopaminergic depletion contribute to regional perfusion changes, clinical symptoms, and cognition in the spectrum of normal aging and cognitive impairment due to AD and/or LBD. Amyloid beta (Aβ) deposition was associated with ventral striatal dopaminergic depletion. Aβ deposition and dopaminergic depletion correlated with perfusion. Aβ deposition correlated with hypoperfusion centered in the left entorhinal cortex. Dopaminergic depletion correlated with hyperperfusion centered in the vermis. Perfusion mediated the Aβ deposition/dopaminergic depletion's effects on cognition.

A Transient Survival Model of Alteration of Electrophysiological Properties Due to Amyloid Beta Toxicity Based on SH-SY5Y Cell Line.

Accumulation of toxic strands of amyloid beta (AB), which cause neurofibrillary tangles and, ultimately, cell death, is suspected to be the main culprit behind clinical symptoms of Alzheimer's disease. Although the mechanism of cell death due to AB accumulation is well known, the intermediate phase between the start of accumulation and cell death is less known and investigated, partially due to technical challenges in identifying partially affected cells. First, we aimed to establish an in vitro model that would show resilience against AB toxicity. Then we used morphological, molecular and electrophysiological assays to investigate how the characteristics of the surviving cells changed after AB toxicity. To investigate this phase, we used differentiation of SH-SY5Y neuroblastoma stem cells by Retinoic Acid (RA) and Brain Derived Neurotrophic Factor (BDNF) to establish an in vitro model which would be able to demonstrate various levels of resistance to AB toxicity. We utilized fluorescent microscopy and whole cell patch clamp recordings to investigate behavior of the model. We observed significantly higher morphological resilience against AB toxicity in cells which were differentiated by both Retinoic Acid and Brain Derived Neurotrophic Factor compared to Retinoic Acid only. However, the electrophysiological properties of the Retinoic Acid + Brain-Derived Neurotrophic Factor differentiated cells were significantly altered after AB treatment. We established a transient survival model for AB toxicity and observed the effects of AB on transmembrane currents of differentiated neurons.

The effects of amyloid-beta on hippocampal glutamatergic receptor and transporter expression.

The effects of amyloid-beta on hippocampal glutamatergic receptor and transporter expression.

Disruption of Cholinergic Circuits as an Area for Targeted Drug Treatment of Alzheimer's Disease: In Vivo Assessment of Short-Term Plasticity in Rat Brain.

The search for new therapeutics for the treatment of Alzheimer’s disease (AD) is still in progress. Aberrant pathways of synaptic transmission in basal forebrain cholinergic neural circuits are thought to be associated with the progression of AD. However, the effect of amyloid-beta (Aβ) on short-term plasticity (STP) of cholinergic circuits in the nucleus basalis magnocellularis (NBM) is largely unknown. STP assessment in rat brain cholinergic circuitry may indicate a new target for AD cholinergic therapeutics. Thus, we aimed to study in vivo electrophysiological patterns of synaptic activity in NBM-hippocampus and NBM-basolateral amygdala circuits associated with AD-like neurodegeneration. The extracellular single-unit recordings of responses from the hippocampal and basolateral amygdala neurons to high-frequency stimulation (HFS) of the NBM were performed after intracerebroventricular injection of Aβ 25–35. We found that after Aβ 25–35 exposure the number of hippocampal neurons exhibiting inhibitory responses to HFS of NBM is decreased. The reverse tendency was seen in the basolateral amygdala inhibitory neural populations, whereas the number of amygdala neurons with excitatory responses decreased. The low intensity of inhibitory and excitatory responses during HFS and post-stimulus period is probably due to the anomalous basal synaptic transmission and excitability of hippocampal and amygdala neurons. These functional changes were accompanied by structural alteration of hippocampal, amygdala, and NBM neurons. We have thus demonstrated that Aβ 25–35 induces STP disruption in NBM-hippocampus and NBM-basolateral amygdala circuits as manifested by unbalanced excitatory/inhibitory responses and their frequency. The results of this study may contribute to a better understanding of synaptic integrity. We believe that advancing our understanding of in vivo mechanisms of synaptic plasticity disruption in specific neural circuits could lead to effective drug searches for AD treatment.

Effect of receiver shape and volume on the Alzheimer disease for molecular communication via diffusion.

Nano-devices are featured to communicate via molecular interaction, the so-called molecular communication (MC). In MC systems, the information is carried by molecules where the amount of molecules constitutes the level of the signal. In this study, an MC-based system was analysed with different receiver topology and related parameters, such as size, shape, and orientation of receptors on the receiver. Also in the concept of nano-medicine, the effect of amyloid-beta ([inline-formula removed]), which is believed as the main cause of Alzheimer disease, on the successful reception ratio of molecules with the proposed receiver models was investigated. It was demonstrated that the cubic receiver model is superior to sphere one in terms of the correct reception ratio of the molecular signal. A cubic model where its edge (not rotated around the centre) is placed across the transmitter demonstrated a better performance in reducing the effect of [inline-formula removed] as compared to the sphere model while a cubic model where its corner (rotated around the centre) is placed across the transmitter demonstrated a worse performance than the spherical model. From this expression, it may be concluded that with the adjustment of topological system parameters the probability of successful reception ratio in MC may be possible.

Amyloid β oligomers inhibit growth of human cancer cells.

Effects of amyloid beta (Aβ) oligomers on viability and function of cell lines such as NB4 (human acute promyelocytic leukemia), A549 (human lung cancer (adenocarcinomic alveolar basal epithelial tumor)) and MCF-7 (human breast cancer (invasive breast ductal carcinoma)) were investigated. Two types of Aβ oligomers were used in the study. The first type was produced in the presence of oligomerization inhibitor, hexafluoroisopropanol (HFIP). The second type of amyloids was assembled in the absence of the inhibitor. The first type preparation was predominantly populated with dimers and trimers, while the second type contained mostly pentadecamers. These amyloid species exhibited different secondary protein structure with considerable amount of antiparallel β sheet structural elements in HFIP oligomerized Aβ mixtures. The effect of the cell growth inhibition, which was stronger in the case of HFIP Aβ oligomers, was observed for all cell lines. Tests aiming at elucidating the effects of the amyloid species on cell cycles showed little differences between amyloid preparations. This prompts us to conclude that the effect on the cancer cell proliferation rate is less specific to the biological processes developing inside the cells during the proliferation. Therefore, cell growth inhibition may involve interactions with the peripheral parts of the cancer cells, such as a phospholipid membrane, and only in case of the NB4 cells, where accumulation of amyloid species inside the cells was detected, one may imply the opposite. In general, cancer cells were much less susceptible to the damaging effects of amyloid oligomers compared to earlier observations in mixed neuronal cell cultures.

Effect of amyloid beta on ATP-binding cassette transporter expression and activity in porcine brain microvascular endothelial cells

BackgroundDeposition of amyloid-β peptide (Aβ(1–42)) within the brain is characteristic of Alzheimer's disease. Little is known of the effects of Aβ(1–42) on blood-brain barrier (BBB) ATP-binding Cassette (ABC) efflux transporters which influence BBB permeability. The effects of Aβ(1–42) on ABCB1, ABCC5 and ABCG2 activity and expression and pregnane X receptor (PXR) and constitutive androstane receptor (CAR) transcription factors expression were determined in primary porcine brain endothelial cells (PBECs). MethodsThe effect of Aβ(1–42) on transporter activity was determined by measurement of intracellular accumulation of the fluorescent probes calcein (ABCB1), GS–MF (ABCC5) and Hoechst 33342 (ABCG2). Expression of transporters and transcription factors was assessed by Western blotting. ResultsTreatment of PBECs with Aβ(1–42) significantly decreased activity of ABCB1 (Aβ(1–42) at 10 μg/ml, 25 μg/ml and 50 μg/ml), ABCC5 (Aβ(1–42) at 25 μg/ml and 50 μg/ml) and ABCG2 (Aβ(1–42) at 10 μg/ml, 25 μg/ml and 50 μg/ml). Aβ(1–42) also significantly decreased expression of ABCB1 (p < 0.05 at 25 μg/ml and 50 μg/ml), ABCG2 (p < 0.05 at 25 μg/ml and p ≤ 0.001 at 50 μg/ml), ABCC5 (p < 0.05 at 25 μg/ml and 50 μg/ml), PXR (p < 0.05 at 10 μg/ml, 25 μg/ml and 50 μg/ml Aβ(1–42)) and CAR (p < 0.05 at 25 μg/ml and 50 μg/ml Aβ(1–42)). ConclusionAβ(1–42) inhibits multiple ABC transporters and PXR and CAR in PBECs. General significanceAβ(1–42) reduces ABC transporter activity and expression in BBB endothelial cells and has the potential to influence BBB permeability characteristics.

Tau induces blood vessel abnormalities and angiogenesis-related gene expression in P301L transgenic mice and human Alzheimer’s disease

Mixed pathology, with both Alzheimer's disease and vascular abnormalities, is the most common cause of clinical dementia in the elderly. While usually thought to be concurrent diseases, the fact that changes in cerebral blood flow are a prominent early and persistent alteration in Alzheimer's disease raises the possibility that vascular alterations and Alzheimer pathology are more directly linked. Here, we report that aged tau-overexpressing mice develop changes to blood vessels including abnormal, spiraling morphologies; reduced blood vessel diameters; and increased overall blood vessel density in cortex. Blood flow in these vessels was altered, with periods of obstructed flow rarely observed in normal capillaries. These changes were accompanied by cortical atrophy as well as increased expression of angiogenesis-related genes such as Vegfa, Serpine1, and Plau in CD31-positive endothelial cells. Interestingly, mice overexpressing nonmutant forms of tau in the absence of frank neurodegeneration also demonstrated similar changes. Furthermore, many of the genes we observe in mice are also altered in human RNA datasets from Alzheimer patients, particularly in brain regions classically associated with tau pathology such as the temporal lobe and limbic system regions. Together these data indicate that tau pathological changes in neurons can impact brain endothelial cell biology, altering the integrity of the brain's microvasculature.

Synaptic dysfunction in Alzheimer's disease: the effects of amyloid beta on synaptic vesicle dynamics as a novel target for therapeutic intervention.

The most prevalent form of dementia in the elderly is Alzheimer's disease. A significant contributing factor to the progression of the disease appears to be the progressive accumulation of amyloid-β42 (Aβ42), a small hydrophobic peptide. Unfortunately, attempts to develop therapies targeting the accumulation of Aβ42 have not been successful to treat or even slow down the disease. It is possible that this failure is an indication that targeting downstream effects rather than the accumulation of the peptide itself might be a more effective approach. The accumulation of Aβ42 seems to affect various aspects of physiological cell functions. In this review, we provide an overview of the evidence that implicates Aβ42 in synaptic dysfunction, with a focus on how it contributes to defects in synaptic vesicle dynamics and neurotransmitter release. We discuss data that provide new insights on the Aβ42 induced pathology of Alzheimer's disease and a more detailed understanding of its contribution to the synaptic deficiencies that are associated with the early stages of the disease. Although the precise mechanisms that trigger synaptic dysfunction are still under investigation, the available data so far has enabled us to put forward a model that could be used as a guide to generate new therapeutic targets for pharmaceutical intervention.

Poly(ADP-ribose) polymerase-1 regulates microglia mediated decrease of endothelial tight junction integrity

Alzheimer's disease pathology includes, beside neuronal damage, reactive gliosis and reduced blood-brain barrier (BBB) integrity. Microglia are intimately associated with the BBB and upon AD pathology, pro-inflammatory responses of microglia could contribute to BBB damage. To study whether microglia can directly affect BBB integrity, the effects of amyloid beta (Aβ) -stimulated primary murine microglia on co-cultured mouse brain endothelial cells (bEnd3) and murine astrocyte cultures were assessed. We also assessed whether microglial phenotype modulation via poly(ADP-ribose) polymerase-1 (PARP-1) inhibition/ablation can reverse microglial impact on these BBB forming cells.Unstimulated microglia promoted expression of tight junction proteins (TJPs), zonula ocluden-1 (ZO-1) and occludin in co-cultured endothelia cells, whereas Aβ-stimulated microglia reduced endothelial expression of ZO-1 and occludin. Astrocytes co-cultured with microglia showed elevated glial fibrillary acidic protein (GFAP) expression, which was further increased if microglia had been stimulated with Aβ. Aβ induced microglial release of nitric oxide (NO) and tumour necrosis factor alpha (TNFα), which resulted in reduced endothelial expression of TJPs and increased paracellular permeability. Microglial PARP-1 inhibition attenuated these Aβ-induced events. These findings demonstrate that PARP-1 mediated microglial responses (NO and TNFα) can directly reduce BBB integrity by promoting TJP degradation, increasing endothelial cell permeability and inducing astrogliosis. PARP-1 as a modulator of microglial phenotype can prevent microglial BBB damaging events, and thus is a potential therapeutic target.

Alteration of high-density lipoprotein functionality in Alzheimer's disease patients.

The aims of the present study were to determine whether high-density lipoprotein (HDL) functionality-mediated cholesterol efflux is altered in Alzheimer's disease and to investigate the role and effect of amyloid-beta (Aβ) in the regulation of the anti-atherogenic activity of HDL. Eighty-seven elderly subjects were recruited, of whom 27 were healthy, 27 had mild cognitive impairment (MCI), and 33 had mild Alzheimer's disease (mAD). Our results showed that total cholesterol levels are negatively correlated with the Mini-Mental State Examination (MMSE) score (r = -0.2602, p = 0.0182). HDL from the mAD patients was less efficient at mediating cholesterol efflux from J774 macrophages (p < 0.05) than HDL from the healthy subjects and MCI patients. While HDL from the MCI patients was also less efficient at mediating cholesterol efflux than HDL from the healthy subjects, the difference was not significant. Interestingly, the difference between the healthy subjects and the MCI and mAD patients with respect to the capacity of HDL to mediate cholesterol efflux disappeared when ATP-binding cassette transporter A1 (ABCA1)-enriched J774 macrophages were used. HDL fluidity was significantly inversely correlated with the MMSE scores (r = -0.4137, p < 0.009). In vitro measurements of cholesterol efflux using J774 macrophages showed that neither Aβ1-40 nor Aβ1-42 stimulate cholesterol efflux from unenriched J774 macrophages in basal or ABCA1-enriched J774 macrophages.

Injection of specific amyloid-beta oligomers (beta1-40:beta1-42 = 10:1) into rat medial septum impairs memory retention without inducing hippocampal apoptosis

Objectives: Because of the well-known neurochemical interactions between the septum and hippocampus during memory processes, we investigated the effect of amyloid-beta (A-beta) injection into the medial septum (MS) on the behavior in Wistar rats. We also assessed whether the observed effects were functional or due to apoptosis.Methods: Specific A-beta oligomers (beta1-40:beta1-42 = 10:1) were injected into MS for seven consecutive days. Behavior was assessed with the Morris water maze task.Results: Compared with the control group, rats that received A-beta oligomers exhibited significant memory retention impairment (P < 0·05) without apoptosis in the cornu ammonis (CA)1 and CA3 regions of the dorsal hippocampus.Discussion: These data indicate that septal injection of A-beta impairs memory retention, even in the absence of hippocampal apoptosis. This result might bring new insight to spatial memory-related disorders like Alzheimer’s disease (AD).

S14G-humanin restored cellular homeostasis disturbed by amyloid-beta protein.

Humanin is a potential therapeutic agent for Alzheimer's disease, and its derivative, S14G-humanin, is 1 000-fold stronger in its neuroprotective effect against Alzheimer's disease-relevant insults. Al-though effective, the detailed molecular mechanism through which S14G-humanin exerts its effects remains unclear. Data from this study showed that fibrillar amyloid-beta 40 disturbed cellular ho-meostasis through the cell membrane, increasing intracellular calcium, generating reactive oxygen species, and decreasing the mitochondrial membrane potential. S14G-humanin restored these responses. The results suggested that S14G-humanin blocked the effects of amyloid-beta 40 on the neuronal cell membrane, and restored the disturbed cellular homeostasis, thereby exerting a neu-roprotective effect on hippocampal neurons.

Amyloid peptide Aβ40 inhibits aggregation of Aβ42: Evidence from molecular dynamics simulations

Effects of amyloid beta (Aβ) peptide Aβ(40) on secondary structures of Aβ(42) are studied by all-atom simulations using the GROMOS96 43a1 force field with explicit water. It is shown that in the presence of Aβ(40) the beta-content of monomer Aβ(42) is reduced. Since the fibril-prone conformation N∗ of full-length Aβ peptides has the shape of beta strand-loop-beta strand this result suggests that Aβ(40) decreases the probability of observing N∗ of Aβ(42) in monomer state. Based on this and the hypothesis that the higher is the population of N∗ the higher fibril formation rates, one can expect that, in agreement with the recent experiment, Aβ(40) inhibit fibril formation of Aβ(42). It is shown that the presence of Aβ(40) makes the salt bridge D23-K28 and fragment 18-33 of Aβ(42) more flexible providing additional support for this experimental fact. Our estimation of the binding free energy by the molecular mechanics-Poisson-Boltzmann surface area method reveals the inhibition mechanism that Aβ(40) binds to Aβ(42) modifying its morphology.

Effects of amyloid-beta and human amylin on mitochondrial function and estradiol production

Effects of amyloid-beta and human amylin on mitochondrial function and estradiol production

Neurological side effects due to GSK-3 inhibition by chronic lithium or transgenesis can be prevented by blocking NFAT/Fas pathway

cognitive measures it is important to assess the effect of different amyloidbeta conformations on brain network emergent properties, such as memory trace stability. Methods: The functional effects of different amyloid-beta conformations are studied in a well calibrated computer-based mechanistic disease model of the human cortex. The system of 120 biophysically realistic neurons is based upon preclinical physiology data in rodents and primates, but parametrized using human pathology and pharmacology data. The physiology of 29 different CNS targets, such as DA, 5-HT, NE and cholinergic receptors is added. After implementing broad AD pathology from human imaging data, the stability of a memory trace is calibrated with clinical ADASCog readouts of 28 clinical interventions. Results: Dose-responses of various symptomatic treatment effects with dopamine, serotoninergic and cholinergic targets suggest an improvement of the stability at 12 and 26 weeks, but not in more severe disease states. The effect of amyloid-beta is complex, resulting often in an inverse U-shape dose-response. Close inspection reveals that the balance between excitation and inhibition in cortical networks is crucial in maintaining a functional cognitive network. This observation suggests that there is an optimal strategy for each different state of the disease.Conclusions: This humanized translational ‘in silico cognition’ approach in general can become a useful tool in the discovery and development of new therapeutics in AD, by addressing issues such as a different pharmacology of the clinical candidate for human vs rodent targets, off-target effects that mediate functional outcome and unique human metabolites.