RESUMO
Microglia undergo two-stage activation in neurodegenerative diseases, known as disease-associated microglia (DAM). TREM2 mediates the DAM2 stage transition, but what regulates the first DAM1 stage transition is unknown. We report that glucose dyshomeostasis inhibits DAM1 activation and PKM2 plays a role. As in tumors, PKM2 was aberrantly elevated in both male and female human AD brains, but unlike in tumors, it is expressed as active tetramers, as well as among TREM2+ microglia surrounding plaques in 5XFAD male and female mice. snRNAseq analyses of microglia without Pkm2 in 5XFAD mice revealed significant increases in DAM1 markers in a distinct metabolic cluster, which is enriched in genes for glucose metabolism, DAM1, and AD risk. 5XFAD mice incidentally exhibited a significant reduction in amyloid pathology without microglial Pkm2 Surprisingly, microglia in 5XFAD without Pkm2 exhibited increases in glycolysis and spare respiratory capacity, which correlated with restoration of mitochondrial cristae alterations. In addition, in situ spatial metabolomics of plaque-bearing microglia revealed an increase in respiratory activity. These results together suggest that it is not only glycolytic but also respiratory inputs that are critical to the development of DAM signatures in 5XFAD mice.
Assuntos
Glucose , Homeostase , Camundongos Transgênicos , Microglia , Animais , Microglia/metabolismo , Microglia/patologia , Camundongos , Homeostase/fisiologia , Glucose/metabolismo , Masculino , Feminino , Humanos , Doença de Alzheimer/metabolismo , Doença de Alzheimer/patologia , Doença de Alzheimer/genética , Glicoproteínas de Membrana/metabolismo , Glicoproteínas de Membrana/genética , Receptores Imunológicos/metabolismo , Receptores Imunológicos/genética , Glicólise/fisiologia , Proteínas de Ligação a Hormônio da TireoideRESUMO
An indazole/aza-indazole scaffold was developed as a novel chemotype for JNK3 inhibition. Extensive structure activity relationship (SAR) studies utilizing various in vitro and in vivo assays led to potent and highly selective JNK3 inhibitors with good oral bioavailability and high brain penetration. One lead compound, 29, was a potent and selective JNK3 inhibitor (IC50 = 0.005 µM) that had significant inhibition (>80% at 1 µM) to only JNK3 and JNK2 in a panel profiling of 374 wild-type kinases, had high potency in functional cell-based assays, had high stability in the human liver microsome (t 1/2 = 92 min), and was orally bioavailable and brain penetrant (brain/plasma ratio: 56%). The cocrystal structure of 29 in human JNK3 at a 2.1 Å resolution showed that indazole or aza-indazole-based JNK3 inhibitors demonstrated a type I kinase inhibition/binding.
RESUMO
We report that the neurotrophin receptor p75 contributes to sensory neuron survival through the regulation of cholesterol metabolism in Schwann cells. Selective deletion of p75 in mouse Schwann cells of either sex resulted in a 30% loss of dorsal root ganglia (DRG) neurons and diminished thermal sensitivity. P75 regulates Schwann cell cholesterol biosynthesis in response to BDNF, forming a co-receptor complex with ErbB2 and activating ErbB2-mediated stimulation of sterol regulatory element binding protein 2 (SREBP2), a master regulator of cholesterol synthesis. Schwann cells lacking p75 exhibited decreased activation of SREBP2 and a reduction in 7-dehydrocholesterol (7-DHC) reductase (DHCR7) expression, resulting in accumulation of the neurotoxic intermediate, 7-dehyrocholesterol in the sciatic nerve. Restoration of DHCR7 in p75 null Schwann cells in mice significantly attenuated DRG neuron loss. Together, these results reveal a mechanism by which the disruption of lipid metabolism in glial cells negatively influences sensory neuron survival, which has implications for a wide range of peripheral neuropathies.SIGNIFICANCE STATEMENT Although expressed in Schwann cells, the role of p75 in myelination has remained unresolved in part because of its dual expression in sensory neurons that Schwann cells myelinate. When p75 was deleted selectively among Schwann cells, myelination was minimally affected, while sensory neuron survival was reduced by 30%. The phenotype is mainly due to dysregulation of cholesterol biosynthesis in p75-deficient Schwann cells, leading to an accumulation of neurotoxic cholesterol precursor, 7-dehydrocholesterol (7-DHC). Mechanism-wise, we discovered that in response to BDNF, p75 recruits and activates ErbB2 independently of ErbB3, thereby stimulating the master regulator, sterol regulatory element binding protein 2 (SREBP2). These results together highlight a novel role of p75 in Schwann cells in regulating DRG neuron survival by orchestrating proper cholesterol metabolism.
Assuntos
Receptores de Fator de Crescimento Neural/deficiência , Receptores de Fator de Crescimento Neural/genética , Células de Schwann/metabolismo , Células Receptoras Sensoriais/metabolismo , Animais , Sobrevivência Celular/fisiologia , Células Cultivadas , Feminino , Células HEK293 , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Ratos , Células de Schwann/ultraestrutura , Células Receptoras Sensoriais/ultraestruturaRESUMO
Potent JNK3 isoform selective inhibitors were developed from a thiophenyl-pyrazolourea scaffold. Through structure activity relationship (SAR) studies utilizing enzymatic and cell-based assays, and in vitro and in vivo drug metabolism and pharmacokinetic (DMPK) studies, potent and highly selective JNK3 inhibitors with oral bioavailability and brain penetrant capability were developed. Inhibitor 17 was a potent and isoform selective JNK3 inhibitor (IC50 = 35 nM), had significant inhibition to only JNK3 in a panel profiling of 374 wild-type kinases, had high potency in functional cell-based assays, had high stability in human liver microsome (t 1/2 = 66 min) and a clean CYP-450 inhibition profile, and was orally bioavailable and brain penetrant. Moreover, cocrystal structures of compounds 17 and 27 in human JNK3 were solved at 1.84 Å, which showed that these JNK3 isoform selective inhibitors bound to the ATP pocket, had interactions in both hydrophobic pocket-I and hydrophobic pocket-II.
RESUMO
Loss of bladder control is a challenging outcome facing patients with spinal cord injury (SCI). We report that systemic blocking of pro-nerve growth factor (proNGF) signaling through p75 with a CNS-penetrating small-molecule p75 inhibitor resulted in significant improvement in bladder function after SCI in rodents. The usual hyperreflexia was attenuated with normal bladder pressure, and automatic micturition was acquired weeks earlier than in the controls. The improvement was associated with increased excitatory input to the spinal cord, in particular onto the tyrosine hydroxylase-positive fibers in the dorsal commissure. The drug also had an effect on the bladder itself, as the urothelial hyperplasia and detrusor hypertrophy that accompany SCI were largely prevented. Urothelial cell loss that precedes hyperplasia was dependent on p75 in response to urinary proNGF that is detected after SCI in rodents and humans. Surprisingly, death of urothelial cells and the ensuing hyperplastic response were beneficial to functional recovery. Deleting p75 from the urothelium prevented urothelial death, but resulted in reduction in overall voiding efficiency after SCI. These results unveil a dual role of proNGF/p75 signaling in bladder function under pathological conditions with a CNS effect overriding the peripheral one.
Assuntos
Fator de Crescimento Neural/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Precursores de Proteínas/metabolismo , Receptores de Fator de Crescimento Neural/metabolismo , Transdução de Sinais , Traumatismos da Medula Espinal/metabolismo , Doenças da Bexiga Urinária/metabolismo , Bexiga Urinária/metabolismo , Animais , Feminino , Deleção de Genes , Humanos , Masculino , Camundongos , Camundongos Knockout , Fator de Crescimento Neural/genética , Proteínas do Tecido Nervoso/genética , Precursores de Proteínas/genética , Receptores de Fator de Crescimento Neural/genética , Traumatismos da Medula Espinal/complicações , Traumatismos da Medula Espinal/genética , Traumatismos da Medula Espinal/patologia , Bexiga Urinária/patologia , Doenças da Bexiga Urinária/etiologia , Doenças da Bexiga Urinária/genética , Doenças da Bexiga Urinária/patologia , Urotélio/metabolismo , Urotélio/patologiaRESUMO
p75 is expressed among Purkinje cells in the adult cerebellum, but its function has remained obscure. Here we report that p75 is involved in maintaining the frequency and regularity of spontaneous firing of Purkinje cells. The overall spontaneous firing activity of Purkinje cells was increased in p75(-/-) mice during the phasic firing period due to a longer firing period and accompanying reduction in silence period than in the wild type. We attribute these effects to a reduction in small conductance Ca(2+)-activated potassium (SK) channel activity in Purkinje cells from p75(-/-) mice compared with the wild type littermates. The mechanism by which p75 regulates SK channel activity appears to involve its ability to activate Rac1. In organotypic cultures of cerebellar slices, brain-derived neurotrophic factor increased RacGTP levels by activating p75 but not TrkB. These results correlate with a reduction in RacGTP levels in synaptosome fractions from the p75(-/-) cerebellum, but not in that from the cortex of the same animals, compared with wild type littermates. More importantly, we demonstrate that Rac1 modulates SK channel activity and firing patterns of Purkinje cells. Along with the finding that spine density was reduced in p75(-/-) cerebellum, these data suggest that p75 plays a role in maintaining normalcy of Purkinje cell firing in the cerebellum in part by activating Rac1 in synaptic compartments and modulating SK channels.
Assuntos
Cerebelo/metabolismo , Neuropeptídeos/metabolismo , Células de Purkinje/metabolismo , Receptores de Fator de Crescimento Neural/metabolismo , Proteínas rac1 de Ligação ao GTP/metabolismo , Animais , Eletrofisiologia , Complexo de Golgi/metabolismo , Glicoproteínas de Membrana/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Microscopia Eletrônica , Técnicas de Patch-Clamp , Canais de Potássio/metabolismo , Proteínas Tirosina Quinases/metabolismo , Transdução de Sinais , Canais de Potássio Ativados por Cálcio de Condutância Baixa/metabolismo , Sinaptossomos/metabolismo , Tetraetilamônio/química , Proteínas rac de Ligação ao GTP/metabolismoRESUMO
The lack of effective therapies for spinal cord injury points to the need for identifying novel targets for therapeutic intervention. Here we report that a small molecule, LM11A-31, developed to block proNGF-p75 interaction and p75-mediated cell death crosses the blood-brain barrier efficiently when delivered orally. Administered starting 4 h postinjury, LM11A-31 promotes functional recovery without causing any toxicity or increased pain in a mouse model of spinal contusion injury. In both weight-bearing open-field tests and nonweight-bearing swim tests, LM11A-31 was effective in improving motor function and coordination. Such functional improvement correlated with a >50% increase in the number of surviving oligodendrocytes and myelinated axons. We also demonstrate that LM11A-31 indeed inhibits proNGF-p75 interaction in vivo, thereby curtailing the JNK3-mediated apoptotic cascade. These results thus highlight p75 as a novel therapeutic target for an orally delivered treatment for spinal cord injury.
Assuntos
Isoleucina/análogos & derivados , Morfolinas/uso terapêutico , Bainha de Mielina/metabolismo , Fator de Crescimento Neural/metabolismo , Precursores de Proteínas/metabolismo , Receptor de Fator de Crescimento Neural/efeitos dos fármacos , Receptor de Fator de Crescimento Neural/metabolismo , Traumatismos da Medula Espinal/tratamento farmacológico , Animais , Western Blotting , DNA/genética , Relação Dose-Resposta a Droga , Membro Anterior/fisiologia , Membro Posterior/fisiologia , Hiperalgesia/tratamento farmacológico , Imuno-Histoquímica , Isoleucina/uso terapêutico , Locomoção/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Proteína Quinase 10 Ativada por Mitógeno/metabolismo , Reação em Cadeia da Polimerase , Traumatismos da Medula Espinal/patologia , Natação/fisiologiaRESUMO
Although Aß peptides are causative agents in Alzheimer's disease (AD), the underlying mechanisms are still elusive. We report that Aß42 induces a translational block by activating AMPK, thereby inhibiting the mTOR pathway. This translational block leads to widespread ER stress, which activates JNK3. JNK3 in turn phosphorylates APP at T668, thereby facilitating its endocytosis and subsequent processing. In support, pharmacologically blocking translation results in a significant increase in Aß42 in a JNK3-dependent manner. Thus, JNK3 activation, which is increased in human AD cases and a familial AD (FAD) mouse model, is integral to perpetuating Aß42 production. Concomitantly, deletion of JNK3 from FAD mice results in a dramatic reduction in Aß42 levels and overall plaque loads and increased neuronal number and improved cognition. This reveals AD as a metabolic disease that is under tight control by JNK3.