Xiumei Huang

Central Role of Mitofusin 2 in Autophagosome-Lysosome Fusion in Cardiomyocytes

Background: Mitofusin 2 is a mitochondrial outer membrane protein with multiple functions. Results: Mitofusin 2 deficiency in the heart causes autophagosomes to accumulate and leads to cardiac dysfunction. Conclusion: Mitofusin 2 serves as an adaptor protein to mediate the fusion of autophagosomes with lysosomes in the heart. Significance: Mitofusin 2 has a novel and essential role in cardiac autophagic regulation. In the heart, autophagy has been implicated in cardioprotection and ischemia-reperfusion tolerance, and the dysregulation of autophagy is associated with the development of heart failure. Mitochondrial dynamic proteins are profoundly involved in autophagic processes, especially the initiation and formation of autophagosomes, but it is not clear whether they play any role in cardiac autophagy. We previously reported that mitofusin 2 (MFN2), a mitochondrial outer membrane protein, serves as a major determinant of cardiomyocyte apoptosis mediated by oxidative stress. Here, we reveal a novel and essential role of MFN2 in mediating cardiac autophagy. We found that specific deletion of MFN2 in cardiomyocytes caused extensive accumulation of autophagosomes. In particular, the fusion of autophagosomes with lysosomes, a critical step in autophagic degradation, was markedly retarded without altering the formation of autophagosomes and lysosomes in response to ischemia-reperfusion stress. Importantly, MFN2 co-immunoprecipitated with RAB7 in the heart, and starvation further increased it. Knockdown of MFN2 by shRNA prevented, whereas re-expression of MFN2 restored, the autophagosome-lysosome fusion in neonatal cardiomyocytes. Hearts from cardiac-specific MFN2 knock-out mice had abnormal mitochondrial and cellular metabolism and were vulnerable to ischemia-reperfusion challenge. Our study defined a novel and essential role of MFN2 in the cardiac autophagic process by mediating the maturation of autophagy at the phase of autophagosome-lysosome fusion; deficiency of MFN2 caused multiple molecular and functional defects that undermined cardiac reserve and gradually led to cardiac vulnerability and dysfunction.

Alzheimer's β-Secretase (BACE1) Regulates the cAMP/PKA/CREB Pathway Independently of β-Amyloid

β-Amyloid protein (Aβ), the major component of neuritic plaques in Alzheimers disease (AD), is derived from proteolytic cleavages of the amyloid precursor protein (APP) by β-site APP-cleaving enzyme 1 (BACE1) and the γ-secretase complex. BACE1 is the rate-limiting enzyme for Aβ production, and an increase in BACE1 level/activity contributes to the pathogenesis of sporadic AD. In addition to cleaving APP for Aβ generation, BACE1 plays multiple physiological roles including the regulation of synaptic functions. Here, we found that overexpression of BACE1 reduces cAMP response element binding protein (CREB) phosphorylation, protein kinase A (PKA) activity, and cAMP levels, whereas downregulation of BACE1 has the opposite effect. We showed that BACE1s effect is independent of its activity for Aβ production, which is corroborated by the observation that BACE1 transgenic mice have impaired learning/memory in the absence of neurotoxic human Aβ. Furthermore, we demonstrated that BACE1 interacts via its transmembrane domain with adenylate cyclase, resulting in reduction of cellular cAMP levels and thus PKA inactivation and reduced CREB phosphorylation. Our study suggests that in addition to its function as the β-secretase to produce Aβ, BACE1 may contribute to the memory and cognitive deficits typical of AD by regulating the cAMP/PKA/CREB pathway, which is important for memory functions.

Salubrinal attenuates β-amyloid-induced neuronal death and microglial activation by inhibition of the NF-κB pathway

Alzheimers disease (AD) is characterized by the deposition of β-amyloid (Aβ) peptides in the brain, inducing neuronal cell death and microglial activation. Endoplasmic reticulum (ER) stress has been proposed to be a mediator of Aβ neurotoxicity. In this study, we test whether salubrinal, an ER stress inhibitor, can protect against Aβ-mediated neurotoxicity. We show in rat primary cortical neurons and mouse microglial BV-2 cells that short-term treatment with salubrinal attenuates Aβ-induced neuronal death and microglial activation. Remarkably, our results show that salubrinals neuroprotective effects are not due to inhibition of ER stress. Rather, we demonstrate that salubrinal exerts its effects through the inhibition of IκB kinase (IKK) activation, IκB degradation, and the subsequent nuclear factor-kappa B (NF-κB) activation. These results elucidate inhibition of the NF-κB pathway as a new mechanism responsible for the protective effects of salubrinal against Aβ neurotoxicity. This study also suggests that modulation of Aβ-induced NF-κB activation could be a potential therapeutic strategy for Alzheimers disease.

A Functional Mouse Retroposed Gene Rps23r1 Reduces Alzheimer's β-Amyloid Levels and Tau Phosphorylation

Senile plaques consisting of beta-amyloid (Abeta) and neurofibrillary tangles composed of hyperphosphorylated tau are major pathological hallmarks of Alzheimers disease (AD). Elucidation of factors that modulate Abeta generation and tau hyperphosphorylation is crucial for AD intervention. Here, we identify a mouse gene Rps23r1 that originated through retroposition of ribosomal protein S23. We demonstrate that RPS23R1 protein reduces the levels of Abeta and tau phosphorylation by interacting with adenylate cyclases to activate cAMP/PKA and thus inhibit GSK-3 activity. The function of Rps23r1 is demonstrated in cells of various species including human, and in transgenic mice overexpressing RPS23R1. Furthermore, the AD-like pathologies of triple transgenic AD mice were improved and levels of synaptic maker proteins increased after crossing them with Rps23r1 transgenic mice. Our studies reveal a new target/pathway for regulating AD pathologies and uncover a retrogene and its role in regulating protein kinase pathways.

Appoptosin is a novel pro-apoptotic protein and mediates cell death in neurodegeneration

Apoptosis is an essential cellular process in multiple diseases and a major pathway for neuronal death in neurodegeneration. The detailed signaling events/pathways leading to apoptosis, especially in neurons, require further elucidation. Here we identify a β-amyloid precursor protein (APP)-interacting protein, designated as appoptosin, whose levels are upregulated in brain samples from Alzheimers disease and infarct patients, and in rodent stroke models, as well as in neurons treated with β-amyloid (Aβ) and glutamate. We further demonstrate that appoptosin induces reactive oxygen species release and intrinsic caspase-dependent apoptosis. The physiological function of appoptosin is to transport/exchange glycine/5-amino-levulinic acid across the mitochondrial membrane for heme synthesis. Downregulation of appoptosin prevents cell death and caspase activation caused by glutamate or Aβ insults. APP modulates appoptosin-mediated apoptosis through interaction with appoptosin. Our study identifies appoptosin as a crucial player in apoptosis and a novel pro-apoptotic protein involved in neuronal cell death, providing a possible new therapeutic target for neurodegenerative disorders.

The Rps23rg gene family originated through retroposition of the ribosomal protein s23 mRNA and encodes proteins that decrease Alzheimer's β-amyloid level and tau phosphorylation

Retroposition is an important mechanism for gene origination. However, studies to elucidate the functions of new genes originated through retroposition, especially the functions related to diseases, are limited. We recently identified a mouse gene, Rps23 retroposed gene 1 (Rps23rg1), that regulates beta-amyloid (Abeta) level and tau phosphorylation, two major pathological hallmarks of Alzheimers disease (AD), and found that Rps23rg1 originated through retroposition of the mouse ribosomal protein S23 (Rps23) mRNA. Here we show that retroposition of Rps23 mRNA occurred multiple times in different species but only generated another functionally expressed Rps23rg1-homologous gene, Rps23rg2, in mice, whereas humans may not possess functional Rps23rg homologs. Both Rps23rg1 and Rps23rg2 are reversely transcribed relative to the parental Rps23 gene, expressed in various tissues and encode proteins that interact with adenylate cyclases. Similar to the RPS23RG1 protein, RPS23RG2 can upregulate protein kinase A activity to reduce the activity of glycogen synthase kinase-3, Abeta level and tau phosphorylation. However, the effects of RPS23RG2 are weaker than those of RPS23RG1 and such a difference could be attributed to the extra carboxyl-terminal region of RPS23RG2, which may have an inhibitory effect. In addition, we show that the transmembrane domain of RPS23RG1 is important for its function. Together, our results present a new gene family, whose products and associated signaling pathways might prevent mice from developing AD-like pathologies.

Retinoid X receptor-α mediates (R )-flurbiprofen’s effect on the levels of Alzheimer’s β-amyloid

Alzheimer’s disease (AD) is characterized by the formation of extracellular senile plaques in the brain, whose major component is a small peptide called β‐amyloid (Aβ). Long‐term use of non‐steroidal anti‐inflammatory drugs (NSAIDs) has been found beneficial for AD and several reports suggest that NSAIDs reduce the generation of Aβ, especially the more amyloidogenic form Aβ42. However, the exact mechanism underlying NSAIDs’ effect on AD risk remains largely inconclusive and all clinical trials using NSAIDs for AD treatment show negative results so far. Recent studies have shown that some NSAIDs can bind to certain nuclear receptors, suggesting that nuclear receptors may be involved in NSAID’s effect on AD risk. Here we find that (R)‐flurbiprofen, the R‐enantiomer of the racemate NSAID flurbiprofen, can significantly reduce Aβ secretion, but at the same time, increases the level of intracellular Aβ. In addition, we find that a nuclear receptor, retinoid X receptor α (RXRα), can regulate Aβ generation and that down‐regulation of RXRα significantly increases Aβ secretion. We also show that (R)‐flurbiprofen can interfere with the interaction between RXRα and 9‐cis‐retinoid acid, and that 9‐cis‐retinoid acid decreases (R)‐flurbiprofen’s reduction of Aβ secretion. Moreover, the modulation effect of (R)‐flurbiprofen on Aβ is abolished upon RXRα down‐regulation. Together, these results suggest that RXRα can regulate Aβ generation and is also required for (R)‐flurbiprofen‐mediated Aβ generation.

RPS23RG1 reduces Aβ oligomer-induced synaptic and cognitive deficits

Alzheimer’s disease (AD) is the most common form of dementia in the elderly. It is generally believed that β-amyloidogenesis, tau-hyperphosphorylation, and synaptic loss underlie cognitive decline in AD. Rps23rg1, a functional retroposed mouse gene, has been shown to reduce Alzheimer’s β-amyloid (Aβ) production and tau phosphorylation. In this study, we have identified its human homolog, and demonstrated that RPS23RG1 regulates synaptic plasticity, thus counteracting Aβ oligomer (oAβ)-induced cognitive deficits in mice. The level of RPS23RG1 mRNA is significantly lower in the brains of AD compared to non-AD patients, suggesting its potential role in the pathogenesis of the disease. Similar to its mouse counterpart, human RPS23RG1 interacts with adenylate cyclase, activating PKA/CREB, and inhibiting GSK-3. Furthermore, we show that human RPS23RG1 promotes synaptic plasticity and offsets oAβ-induced synaptic loss in a PKA-dependent manner in cultured primary neurons. Overexpression of Rps23rg1 in transgenic mice consistently prevented oAβ-induced PKA inactivation, synaptic deficits, suppression of long-term potentiation, and cognitive impairment as compared to wild type littermates. Our study demonstrates that RPS23RG1 may reduce the occurrence of key elements of AD pathology and enhance synaptic functions to counteract oAβ-induced synaptic and cognitive deficits in AD.