Although inflammation in the brain is meant as a defense mechanism against neurotoxic stimuli, increasing evidence suggests that uncontrolled, chronic, and persistent inflammation contributes ...to neurodegeneration. Most neurodegenerative diseases have now been associated with chronic inflammation, including Alzheimer's disease (AD). Whether anti-inflammatory approaches can be used to treat AD, however, is a major unanswered question. We recently demonstrated that monoacylglycerol lipase (MAGL) hydrolyzes endocannabinoids to generate the primary arachidonic acid pool for neuroinflammatory prostaglandins. In this study, we show that genetic inactivation of MAGL attenuates neuroinflammation and lowers amyloid β levels and plaques in an AD mouse model. We also find that pharmacological blockade of MAGL recapitulates the cytokine-lowering effects through reduced prostaglandin production, rather than enhanced endocannabinoid signaling. Our findings thus reveal a role of MAGL in modulating neuroinflammation and amyloidosis in AD etiology and put forth MAGL inhibitors as a potential next-generation strategy for combating AD.
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► Monoacylglycerol lipase (MAGL) is a major contributor to brain arachidonic acid pools ► MAGL mutation decreases neuroinflammation and amyloid β in Alzheimer's disease mouse ► MAGL blockade recapitulates brain prostaglandin and cytokine-lowering effects ► MAGL inhibitors may be a next-generation strategy for combating Alzheimer's disease
Nomura, Samad, and colleagues find that blockade of monoacylglycerol lipase (MAGL) reduces hallmarks of Alzheimer's disease (AD) such as neuroinflammation and accumulation of amyloid β in an AD mouse model. These changes are modulated by decreasing arachidonic acid levels in the brain that, in turn, lower the production of proinflammatory eicosanoids and cytokines. The development of MAGL inhibitors may therefore represent a potential therapeutic approach toward the treatment of AD.
GABABreceptors are heterodimeric G protein-coupled receptors composed of R1 and R2 subunits that mediate slow synaptic inhibition in the brain by activating inwardly rectifying K+channels (GIRKs) and ...inhibiting Ca2+channels. We demonstrate here that GABABreceptors are intimately associated with 5'AMP-dependent protein kinase (AMPK). AMPK acts as a metabolic sensor that is potently activated by increases in 5'AMP concentration that are caused by enhanced metabolic activity, anoxia, or ischemia. AMPK binds the R1 subunit and directly phosphorylates S783 in the R2 subunit to enhance GABABreceptor activation of GIRKs. Phosphorylation of S783 is evident in many brain regions, and is increased dramatically after ischemic injury. Finally, we also reveal that S783 plays a critical role in enhancing neuronal survival after ischemia. Together our results provide evidence of a neuroprotective mechanism, which, under conditions of metabolic stress or after ischemia, increases GABABreceptor function to reduce excitotoxicity and thereby promotes neuronal survival.
The amyloid hypothesis states that a variety of neurotoxic β-amyloid (Aβ) species contribute to the pathogenesis of Alzheimer's disease. Accordingly, a key determinant of disease onset and ...progression is the appropriate balance between Aβ production and clearance. Enzymes responsible for the degradation of Aβ are not well understood, and, thus far, it has not been possible to enhance Aβ catabolism by pharmacological manipulation. We provide evidence that Aβ catabolism is increased after inhibition of plasminogen activator inhibitor-1 (PAI-1) and may constitute a viable therapeutic approach for lowering brain Aβ levels. PAI-1 inhibits the activity of tissue plasminogen activator (tPA), an enzyme that cleaves plasminogen to generate plasmin, a protease that degrades Aβ oligomers and monomers. Because tPA, plasminogen and PAI-1 are expressed in the brain, we tested the hypothesis that inhibitors of PAI-1 will enhance the proteolytic clearance of brain Aβ. Our data demonstrate that PAI-1 inhibitors augment the activity of tPA and plasmin in hippocampus, significantly lower plasma and brain Aβ levels, restore long-term potentiation deficits in hippocampal slices from transgenic Aβ-producing mice, and reverse cognitive deficits in these mice.
Src kinase signaling has been implicated in multiple mechanisms of ischemic injury, including vascular endothelial growth factor (VEGF)-mediated vascular permeability that leads to vasogenic edema, a ...major clinical complication in stroke and brain trauma. Here we report the effects of two novel Src kinase inhibitors, 4-(2,4-dichloro-5-methoxyphenyl)amino-6-methoxy-7-3-(4-methyl-1-piperazinyl)propoxy-3-quinolinecarbonitrile (SKI-606) and 4-(2,4-dichloro-5-methoxyphenyl)amino-6-methoxy-7-4-(4-methypiperazin-1-yl)but-1-ynyl-3-quinolinecarbonitrile (SKS-927), on ischemia-induced brain infarction and short- and long-term neurological deficits. Two well established transient transient middle cerebral artery occlusion (tMCAO) and permanent permanent middle cerebral artery occlusion (pMCAO) focal ischemia models in the rat were used with drug treatments initiated up to 6 h after onset of stroke to mimic the clinical scenario. Brain penetration of Src inhibitors, their effect on blood-brain barrier integrity and VEGF signaling in human endothelial cells were also evaluated. Our results demonstrate that both agents potently block VEGF-mediated signaling in human endothelial cells, penetrate rat brain upon systemic administration, and inhibit postischemic Src activation and vascular leakage. Treatment with SKI-606 or SKS-927 (at the doses of 3-30 mg/kg i.v.) resulted in a dose-dependent reduction in infarct volume and robust protection from neurological impairments even when the therapy was initiated up to 4- to 6-h after tMCAO. Src blockade after pMCAO resulted in accelerated improvement in recovery from motor, sensory, and reflex deficits during a long-term (3 weeks) testing period poststroke. These data demonstrate that the novel Src kinase inhibitors provide effective treatment against ischemic conditions within a clinically relevant therapeutic window and may constitute a viable therapy for acute stroke.
Developing neurons must respond to a wide range of extracellular signals during the process of brain morphogenesis. One mechanism through which immature neurons respond to such signals is by altering ...cellular actin dynamics. A recently discovered link between extracellular signaling events and the actin cytoskeleton is the WASP/WAVE (Wiscott-Aldrich Syndrome protein/WASP-family verprolin-homologous protein) family of proteins. Through a direct interaction with the Arp2/3 (actin-related protein) complex, this family functions to regulate the actin cytoskeleton by mediating signals from cdc42 as well as other small GTPases. To evaluate the role of WASP/WAVE proteins in the process of neuronal morphogenesis, we used a retroviral gene trap to generate a line of mice bearing a disruption in the WAVE1 gene. Using a heterologous reporter gene, we found that WAVE1 expression becomes increasingly restricted to the CNS over the course of development. Homozygous disruption of the WAVE1 gene results in postnatal lethality. In addition, these animals have severe limb weakness, a resting tremor, and notable neuroanatomical malformations without overt histopathology of peripheral organs. We did not detect any alterations in neuronal morphology in vivo or the ability of embryonic neurons to form processes in vitro. Our data indicate that WAVE1, although important for the general development of the CNS, is not essential for the formation and extension of neuritic processes.
Rodent models of focal and global ischemia were used to examine caspase activation. Several readouts were employed on identical tissue to provide correlative measurement of caspase induction, ...activation and enzymatic activity. In a rat focal ischemia model, caspase-3 enzymatic activity, as recorded by DEVD–AMC cleavage, peaked in penumbral cortex at 6–12 h following ischemia, correlating with increases in caspase 3-cleaved substrates of PARP and α-spectrin and subsequent disappearance of caspase-3 zymogen. Although induction of caspases 8 and 2 proteins was detectable as early as 6 h following ischemia, examination of the same tissues for caspase 8 or 2 enzymatic activities did not show significant modulation up to 12 h after ischemic insult. Caspase 9 induction was evident only after 24 h postischemia and did not correlate with elevated LDHD–AMC cleavage. Following global ischemia in gerbils, levels of caspase-3 enzyme activity peaked at 12 h in hippocampal tissue extracts. Cleaved caspase-3 signal was prominent in NeuN-positive layers in the CA1 region 6–12 h following ischemia. Interestingly, strong caspase-3 immunoreactivity was also detected in the subgranular zone of the dentate gyrus, a known region of ischemia-induced neurogenesis. Caspase-3 activation may be responsible for the loss of these cells, thereby hindering the endogenous recovery process.
Mice transgenic for production of excessive or mutant forms of beta-amyloid differ from patients with Alzheimer's disease in the degree of inflammation, oxidative damage, and alteration of ...intermediary metabolism, as well as the paucity or absence of neuronal atrophy and cognitive impairment. Previous observers have suggested that differences in inflammatory response reflect a discrepancy in the state of the locus coeruleus (LC), loss of which is an early change in Alzheimer's disease but which is preserved in the transgenic mice. In this paper, we extend these observations by examining the effects of the LC on markers of oxidative stress and intermediary metabolism. We compare four groups: wild-type or Tg2576 Aβ transgenic mice injected with DSP4 or vehicle. Of greatest interest were metabolites different between ablated and intact transgenics, but not between ablated and intact wild-type animals. The Tg2576_DSP4 mice were distinguished from the other three groups by oxidative stress and altered energy metabolism. These observations provide further support for the hypothesis that Tg2576 Aβ transgenic mice with this ablation may be a more congruent model of Alzheimer's disease than are transgenics with an intact LC.
In recent years, the first generation of β-secretase (BACE1) inhibitors advanced into clinical development for the treatment of Alzheimer’s disease (AD). However, the alignment of drug-like ...properties and selectivity remains a major challenge. Herein, we describe the discovery of a novel class of potent, low clearance, CNS penetrant BACE1 inhibitors represented by thioamidine 5. Further profiling suggested that a high fraction of the metabolism (>95%) was due to CYP2D6, increasing the potential risk for victim-based drug–drug interactions (DDI) and variable exposure in the clinic due to the polymorphic nature of this enzyme. To guide future design, we solved crystal structures of CYP2D6 complexes with substrate 5 and its corresponding metabolic product pyrazole 6, which provided insight into the binding mode and movements between substrate/inhibitor complexes. Guided by the BACE1 and CYP2D6 crystal structures, we designed and synthesized analogues with reduced risk for DDI, central efficacy, and improved hERG therapeutic margins.
A growing subset of β-secretase (BACE1) inhibitors for the treatment of Alzheimer’s disease (AD) utilizes an anilide chemotype that engages a key residue (Gly230) in the BACE1 binding site. Although ...the anilide moiety affords excellent potency, it simultaneously introduces a third hydrogen bond donor that limits brain availability and provides a potential metabolic site leading to the formation of an aniline, a structural motif of prospective safety concern. We report herein an alternative aminomethyl linker that delivers similar potency and improved brain penetration relative to the amide moiety. Optimization of this series identified analogues with an excellent balance of ADME properties and potency; however, potential drug–drug interactions (DDI) were predicted based on CYP 2D6 affinities. Generation and analysis of key BACE1 and CYP 2D6 crystal structures identified strategies to obviate the DDI liability, leading to compound 16, which exhibits robust in vivo efficacy as a BACE1 inhibitor.
A major challenge in the development of β-site amyloid precursor protein cleaving enzyme 1 (BACE1) inhibitors for the treatment of Alzheimer’s disease is the alignment of potency, drug-like ...properties, and selectivity over related aspartyl proteases such as Cathepsin D (CatD) and BACE2. The potential liabilities of inhibiting BACE2 chronically have only recently begun to emerge as BACE2 impacts the processing of the premelanosome protein (PMEL17) and disrupts melanosome morphology resulting in a depigmentation phenotype. Herein, we describe the identification of clinical candidate PF-06751979 (64), which displays excellent brain penetration, potent in vivo efficacy, and broad selectivity over related aspartyl proteases including BACE2. Chronic dosing of 64 for up to 9 months in dog did not reveal any observation of hair coat color (pigmentation) changes and suggests a key differentiator over current BACE1 inhibitors that are nonselective against BACE2 in later stage clinical development.
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