Irisin reduces amyloid-ß by inducing the release of neprilysin from astrocytes following downregulation of ERK-STAT3 signaling.

A pathological hallmark of Alzheimer's disease (AD) is the deposition of amyloid-ß (Aß) protein in the brain. Physical exercise has been shown to reduce Aß burden in various AD mouse models, but the underlying mechanisms have not been elucidated. Irisin, an exercise-induced hormone, is the secreted form of fibronectin type-III-domain-containing 5 (FNDC5). Here, using a three-dimensional (3D) cell culture model of AD, we show that irisin significantly reduces Aß pathology by increasing astrocytic release of the Aß-degrading enzyme neprilysin (NEP). This is mediated by downregulation of ERK-STAT3 signaling. Finally, we show that integrin αV/ß5 acts as the irisin receptor on astrocytes required for irisin-induced release of astrocytic NEP, leading to clearance of Aß. Our findings reveal for the first time a cellular and molecular mechanism by which exercise-induced irisin attenuates Aß pathology, suggesting a new target pathway for therapies aimed at the prevention and treatment of AD.

Amyloid-ß42/40 ratio drives tau pathology in 3D human neural cell culture models of Alzheimer's disease.

The relationship between amyloid-ß (Aß) species and tau pathology in Alzheimer's disease (AD) is not fully understood. Here, we provide direct evidence that Aß42/40 ratio, not total Aß level, plays a critical role in inducing neurofibrillary tangles (NTFs) in human neurons. Using 3D-differentiated clonal human neural progenitor cells (hNPCs) expressing varying levels of amyloid ß precursor protein (APP) and presenilin 1 (PS1) with AD mutations, we show that pathogenic tau accumulation and aggregation are tightly correlated with Aß42/40 ratio. Roles of Aß42/40 ratio on tau pathology are also confirmed with APP transmembrane domain (TMD) mutant hNPCs, which display differential Aß42/40 ratios without mutant PS1. Moreover, naïve hNPCs co-cultured with APP TMD I45F (high Aß42/40) cells, not with I47F cells (low Aß42/40), develop robust tau pathology in a 3D non-cell autonomous cell culture system. These results emphasize the importance of reducing the Aß42/40 ratio in AD therapy.

BACE1 Regulates Proliferation and Neuronal Differentiation of Newborn Cells in the Adult Hippocampus in Mice.

ß-Site amyloid precursor protein cleaving enzyme 1 (BACE1) is required for the production of ß-amyloid (Aß), one of the major pathogenic molecules of Alzheimer's disease (AD), and is therefore being actively pursued as a drug target for AD. Adult hippocampal neurogenesis (AHN) is a lifelong process that is known to be important for learning and memory and may have the potential to regenerate damaged neural tissue. In this study, we examined whether BACE1 regulates AHN, which holds important implications for its suitability as a drug target in AD. Cohorts of 2-month-old wild-type (BACE1+/+), heterozygous, and homozygous BACE1 knockout mice (BACE1+/- and BACE1-/-, respectively) were injected with 5-bromo-2'-deoxyuridine (BrdU) and sacrificed 1 day later to examine the impact of loss of BACE1 on neural precursor cell (NPC) proliferation in the adult brain. Parallel cohorts of mice were sacrificed 4 weeks after BrdU injection to determine the effects of BACE1 on survival and differentiation of newborn NPCs. We found that NPC proliferation was increased in BACE1-/- mice compared to BACE1+/+ mice, while no difference was observed in NPC survival across genotypes. Differentiation of NPCs to neuronal lineage was impaired in BACE1-/- mice. However, no differences were observed in astrogenesis, the proportion of immature neurons, or the production of oligodendrocytes across genotypes. Importantly, corresponding with a decrease in neuronal differentiation in the absence of a complementary increase in an alternate cell fate, BACE1-/- mice were found to have a pool of undifferentiated NPCs in the hippocampus compared to BACE1+/+ and BACE1+/- mice.

Amino acid-dependent NPRL2 interaction with Raptor determines mTOR Complex 1 activation.

We assign a new function to a tumor suppressor NPRL2 that activates the mTOR complex 1 (mTORC1) activity. The positive regulation of mTORC1 activity by NPRL2 is mediated through NPRL2 interaction with Raptor. While NPRL2 interacts with Rag GTPases, RagD in particular, to interfere with mTORC1 activity in amino acid scarcity, NPRL2 interacts with Raptor in amino acid sufficiency to activate mTORC1. A reciprocal relationship exists between NPRL2 binding to Rag GTPases and Raptor. NPRL2 majorly locates in the lysosomal membranes and has a higher binding affinity to the dominant negative mutant heterodimer of RagA(GDP)/RagD(GTP) that inactivates mTORC1. However, the binding affinity of NPRL2 with Raptor is much less pronounced in cells expressing the dominant negative mutant heterodimer of RagA(GDP)/RagD(GTP) than in cells expressing the dominant positive mutant heterodimer, RagA(GTP)/RagD(GDP). The positive effect of NPRL2 on TORC1 pathway was also evidenced in Drosophila animal model. Here, we propose a 'seesaw' model in which the interactive behavior of NPRL2 with Raptor determines mTORC1 activation by amino acid signaling in animal cells.

Amelioration of behavioral abnormalities in BH(4)-deficient mice by dietary supplementation of tyrosine.

This study reports an amelioration of abnormal motor behaviors in tetrahydrobiopterin (BH4)-deficient Spr (-/-) mice by the dietary supplementation of tyrosine. Since BH4 is an essential cofactor for the conversion of phenylalanine into tyrosine as well as the synthesis of dopamine neurotransmitter within the central nervous system, the levels of tyrosine and dopamine were severely reduced in brains of BH4-deficient Spr (-/-) mice. We found that Spr (-/-) mice display variable 'open-field' behaviors, impaired motor functions on the 'rotating rod', and dystonic 'hind-limb clasping'. In this study, we report that these aberrant motor deficits displayed by Spr (-/-) mice were ameliorated by the therapeutic tyrosine diet for 10 days. This study also suggests that dopamine deficiency in brains of Spr (-/-) mice may not be the biological feature of aberrant motor behaviors associated with BH4 deficiency. Brain levels of dopamine (DA) and its metabolites in Spr (-/-) mice were not substantially increased by the dietary tyrosine therapy. However, we found that mTORC1 activity severely suppressed in brains of Spr (-/-) mice fed a normal diet was restored 10 days after feeding the mice the tyrosine diet. The present study proposes that brain mTORC1 signaling pathway is one of the potential targets in understanding abnormal motor behaviors associated with BH4-deficiency.

Absence of a human DnaJ protein hTid-1S correlates with aberrant actin cytoskeleton organization in lesional psoriatic skin.

The biochemical mechanism by which the human tumorous imaginal disc1(S) (hTid-1(S)) interferes with actin cytoskeleton organization in keratinocytes of human skin epidermis was investigated. We found that hTid-1, specifically hTid-1(S), interacts with MK5, a p38-regulated/activated protein kinase, and inhibits the protein kinase activity of MK5 that phosphorylates heat shock protein HSP27 in cultured HeLa cells. Thus, hTid-1(S) expression inhibits the phosphorylation of HSP27 known to play important roles in F-actin polymerization and actin cytoskeleton organization. The interplay between MK5/HSP27 signaling and hTid-1(S) expression was supported by the inhibition of HSP27 phosphorylation and MK5 activity in HeLa cells in response to hypoxia during which hTid-1(S) expression was down-regulated. We also found that overexpression of hTid-1(S) results in the inhibition of HSP27 phosphorylation, F-actin polymerization, and actin cytoskeleton organization in transduced HaCaT keratinocytes. This study further proposes that the loss of hTid-1(S) expression in the basal layer of skin epidermis correlates with the enhanced HSP27 phosphorylation, keratinocyte hyperproliferation, and excess actin cytoskeleton organization in lesional psoriatic skin.

Autophagy induction by tetrahydrobiopterin deficiency.

Tetrahydrobiopterin (BH4) deficiency is a genetic disorder associated with a variety of metabolic syndromes such as phenylketonuria (PKU). In this article, the signaling pathway by which BH4 deficiency inactivates mTORC1 leading to the activation of the autophagic pathway was studied utilizing BH4-deficient Spr(-/-) mice generated by the knockout of the gene encoding sepiapterin reductase (SR) catalyzing BH4 synthesis. We found that mTORC1 signaling was inactivated and autophagic pathway was activated in tissues from Spr(-/-) mice. This study demonstrates that tyrosine deficiency causes mTORC1 inactivation and subsequent activation of autophagic pathway in Spr(-/-) mice. Therapeutic tyrosine diet completely rescued dwarfism and mTORC1 inhibition but inactivated autophagic pathway in Spr(-/-) mice. Tyrosine-dependent inactivation of mTORC1 was further supported by mTORC1 inactivation in Pah(enu2) mouse model lacking phenylalanine hydroxylase (Pah). NIH3T3 cells grown under the condition of tyrosine restriction exhibited autophagy induction. However, mTORC1 activation by RhebQ64L, a positive regulator of mTORC1, inactivated autophagic pathway in NIH3T3 cells under tyrosine-deficient conditions. In addition, this study first documents mTORC1 inactivation and autophagy induction in PKU patients with BH4 deficiency.

Alkaline stress-induced autophagy is mediated by mTORC1 inactivation.

The activation of autophagic pathway by alkaline stress was investigated. Various types of mammalian cells were subjected to alkaline stress by incubation in bicarbonate buffered media in humidified air containing atmospheric 0.04% CO(2) . The induction of autophagy following alkaline stress was evaluated by assessing the conversion of cytosolic LC3-I into lipidated LC3-II, the accumulation of autophagosomes, and the formation of autolysosomes. Colocalization of GFP-LC3 with endolysosomal marker in HeLa GFP-LC3 cells undergoing autophagic process by alkaline stress further demonstrates that autophagosomes triggered by alkaline stress matures into autolysosomes for the lysosome dependent degradation. We found that the inactivation of mTORC1 is important for the pathway leading to the induction of autophagy by alkaline stress since the expression of RhebQ64L, a constitutive activator of mTORC1, downregulates the induction of autophagy after alkaline stress in transfected human 293T cells. These results imply that activation of autophagic pathway following the inactivation of mTORC1 is important cellular events governing alkaline stress-induced cytotoxicity and clinical symptoms associated with alkalosis.

Notch signal activates hypoxia pathway through HES1-dependent SRC/signal transducers and activators of transcription 3 pathway.

We report a Notch signal-induced pathway that leads to transcriptional activation of HIF1-alpha gene. HeLa/rtTAA/TRE-N1-IC cell line capable of doxycycline-induced expression of human Notch1-IC was established. The induction of Notch signaling activates HIF1-alpha and its target gene expression in HeLa/rtTAA/TRE-N1-IC cells. Notch signaling enhanced signal transducers and activators of transcription 3 (STAT3) phosphorylation required for HIF1-alpha expression. SRC kinase was found to be responsible for the enhanced STAT3 phosphorylation in response to Notch signaling. Activation of SRC/STAT3 pathway by Notch signaling was dependent on the expression of Notch effector HES1 transcription factor. The induction of HES1 enhanced STAT3 phosphorylation at Tyr 705 as well as SRC phosphorylation at Tyr 416 in inducible HeLa/rtTAA/TRE-HES1 cells, which express HES1 in response to doxycycline treatment. However, the treatment of Trichostatin A that interferes with HES1 transcriptional regulation did not affect STAT3 phosphorylation, and the expression of dominant negative HES1 failed to interfere with HES1-dependent SRC/STAT3 pathway. These observations have led us to the conclusion that HES1-dependent activation of SRC/STAT3 pathway is independent of HES1 transcription regulation. This study first reports HES1-dependent SRC/STAT3 pathway that provides a functional link between Notch signaling and hypoxia pathway.