Gene-Dose-Dependent Reduction Fshr Expression Improves Spatial Memory Deficits in Alzheimer's Mice.

Alzheimer's disease (AD) is a major progressive neurodegenerative disorder of the aging population. High post-menopausal levels of the pituitary gonadotropin follicle-stimulating hormone (FSH) are strongly associated with the onset of AD, and we have shown recently that FSH directly activates the hippocampal Fshr to drive AD-like pathology and memory loss in mice. To establish a role for FSH in memory loss, we used female 3xTg;Fshr+/+, 3xTg;Fshr+/- and 3xTg;Fshr-/- mice that were either left unoperated or underwent sham surgery or ovariectomy at 8 weeks of age. Unoperated and sham-operated 3xTg;Fshr-/- mice were implanted with 17ß-estradiol pellets to normalize estradiol levels. Morris Water Maze and Novel Object Recognition behavioral tests were performed to study deficits in spatial and recognition memory, respectively, and to examine the effects of Fshr depletion. 3xTg;Fshr+/+ mice displayed impaired spatial memory at 5 months of age; both the acquisition and retrieval of the memory were ameliorated in 3xTg;Fshr-/- mice and, to a lesser extent, in 3xTg;Fshr+/- mice- -thus documenting a clear gene-dose-dependent prevention of hippocampal-dependent spatial memory impairment. At 5 and 10 months, sham-operated 3xTg;Fshr-/- mice showed better memory performance during the acquasition and/or retrieval phases, suggesting that Fshr deletion prevented the progression of spatial memory deficits with age. However, this prevention was not seen when mice were ovariectomized, except in the 10-month-old 3xTg;Fshr-/- mice. In the Novel Object Recognition test performed at 10 months, all groups of mice, except ovariectomized 3xTg;Fshr-/- mice showed a loss of recognition memory. Consistent with the neurobehavioral data, there was a gene-dose-dependent reduction mainly in the amyloid ß40 isoform in whole brain extracts. Finally, serum FSH levels < 8 ng/mL in 16-month-old APP/PS1 mice were associated with better retrieval of spatial memory. Collectively, the data provide compelling genetic evidence for a protective effect of inhibiting FSH signaling on the progression of spatial and recognition memory deficits in mice, and lay a firm foundation for the use of an FSH-blocking agent for the early prevention of cognitive decline in postmenopausal women.

Inhibition of asparagine endopeptidase (AEP) effectively treats sporadic Alzheimer's disease in mice.

Alzheimer's disease (AD) is a progressive neurodegenerative disease with cognitive dysfunction as its major clinical symptom. However, there is no disease-modifying small molecular medicine to effectively slow down progression of the disease. Here, we show an optimized asparagine endopeptidase (AEP, also known as δ-secretase) inhibitor, #11 A, that displays an orderly in vivo pharmacokinetics/pharmacodynamics (PK/PD) relationship and robustly attenuates AD pathologies in a sporadic AD mouse model. #11 A is brain permeable with great oral bioavailability. It blocks AEP cleavage of APP and Tau dose-dependently, and significantly decreases Aß40 and Aß42 and p-Tau levels in APP/PS1 and Tau P301S mice after oral administration. Notably, #11 A strongly inhibits AEP and prevents mouse APP and Tau fragmentation by AEP, leading to reduction of mouse Aß42 (mAß42), mAß40 and mouse p-Tau181 levels in Thy1-ApoE4/C/EBPß transgenic mice in a dose-dependent manner. Repeated oral administration of #11 A substantially decreases mAß aggregation as validated by Aß PET assay, Tau pathology, neurodegeneration and brain volume reduction, resulting in alleviation of cognitive impairment. Therefore, our results support that #11 A is a disease-modifying preclinical candidate for pharmacologically treating AD.

Cholestanol accelerates α-synuclein aggregation and spreading by activating asparagine endopeptidase.

Cerebrotendinous xanthomatosis (CTX), an autosomal recessive disorder characterized by high levels of cholestanol in the blood and accumulation of cholestanol in multiple tissues, especially the brain, often presents in parkinsonism. However, it remains unknown whether cholestanol plays a role in the pathogenesis of sporadic Parkinson's disease (PD). Here, we show that the levels of serum cholestanol in patients with sporadic PD are higher than those in control participants. Cholestanol activates the protease asparagine endopeptidase (AEP) and induces the fragmentation of α-synuclein (α-syn) and facilitates its aggregation. Furthermore, cholestanol promotes the spreading of α-syn pathology in a mouse model induced by intrastriatal injection of α-syn fibrils. KO of AEP or administration of an AEP inhibitor ameliorates α-syn pathology, degeneration of the nigrostriatal dopaminergic pathway, and PD-like motor symptoms. These results not only indicate that cholestanol contributes to the aggregation and spreading of α-syn by activating AEP but also reveal an opportunity for treating PD with AEP inhibitors.

Spinal cord injury-activated C/EBPß-AEP axis mediates cognitive impairment through APP C586/Tau N368 fragments spreading.

Spinal cord injury (SCI) leads to mental abnormalities such as dementia and depression; however, the molecular mechanism of SCI-induced dementia remains a matter of debate. Asparagine endopeptidase (AEP) mediated dementia by enhancing amyloid plaque and Tau hyperphosphorylation, indicating that it played an important role in neurodegeneration. Here we revealed that SCI stimulated AEP activation in mice with T9 contusion injury. Activated-AEP cleaved APP and Tau, resulting in APP C586 and Tau N368 formations, and consequentially accelerated Aß deposit and Tau hyperphosphorylation, respectively. At 9 months following injury, mice demonstrated a severe deterioration in cognitive-behavioral function, which was corroborated by the presence of accumulated AD-specific pathologies. Surprisingly, activated AEP was found in the brains of mice with spinal cord injury. In contrast, AEP knockout reduced SCI-induced neuronal death and neuroinflammation, resulting in cognitive-behavioral restoration. Interestingly, compared to the full-length proteins, truncated Tau N368 and APP C586 were easier to bind to each other. These AEP-processed fragments can not only be induced to pre-formed fibrils, but also amplified their abilities of spreading and neurotoxicity in vitro. Furthermore, as a critical transcription factor of AEP, C/EBPß was activated in injured spinal cord. Elevated C/EBPß level, as well as microglia population and inflammatory cytokines were also noticed in the cortex and hippocampus of SCI mice. These neuroinflammation pathologies were close related to the amount of Tau N368 and APP C586 in brain. Moreover, administration with the AEP-specific inhibitor, compound #11, was shown to decelerate Aß accumulation, tauopathy and C/EBPß level in both spinal cord and brain of SCI mice. Thus, this study highlights the fact that spinal cord injury is a potential risk factor for dementia, as well as the possibility that C/EBPß-AEP axis may play a role in SCI-induced cognitive impairment.

A TrkB agonist prodrug prevents bone loss via inhibiting asparagine endopeptidase and increasing osteoprotegerin.

Brain-derived neurotrophic factor (BDNF) and its tropomyosin-related kinase B receptor (TrkB) are expressed in human osteoblasts and mediate fracture healing. BDNF/TrkB signaling activates Akt that phosphorylates and inhibits asparagine endopeptidase (AEP), which regulates the differentiation fate of human bone marrow stromal cells (hBMSC) and is altered in postmenopausal osteoporosis. Here we show that R13, a small molecular TrkB receptor agonist prodrug, inhibits AEP and promotes bone formation. Though both receptor activator of nuclear factor kappa-Β ligand (RANK-L) and osteoprotegerin (OPG) induced by ovariectomy (OVX) remain comparable between WT and BDNF+/- mice, R13 treatment significantly elevates OPG in both mice without altering RANKL, blocking trabecular bone loss. Strikingly, both R13 and anti-RANK-L exhibit equivalent therapeutic efficacy. Moreover, OVX increases RANK-L and OPG in WT and AEP KO mice with RANK-L/OPG ratio lower in the latter than the former, attenuating bone turnover. 7,8-DHF, released from R13, activates TrkB and its downstream effector CREB, which is critical for OPG augmentation. Consequently, 7,8-DHF represses C/EBPß/AEP pathway, inhibiting RANK-L-induced RAW264.7 osteoclastogenesis. Therefore, our findings support that R13 exerts its therapeutic efficacy toward osteoporosis via inhibiting AEP and escalating OPG.

A synapsin â cleavage fragment contributes to synaptic dysfunction in Alzheimer's disease.

Synaptic dysfunction is a key feature of Alzheimer's disease (AD). However, the molecular mechanisms underlying synaptic dysfunction remain unclear. Here, we show that synapsin Ⅰ, one of the most important synaptic proteins, is fragmented by the cysteine proteinase asparagine endopeptidase (AEP). AEP cleaves synapsin at N82 in the brains of AD patients and generates the C-terminal synapsin Ⅰ (83-705) fragment. This fragment is abnormally distributed in neurons and induces synaptic dysfunction. Overexpression of AEP in the hippocampus of wild-type mice results in the production of the synapsin Ⅰ (83-705) fragment and induces synaptic dysfunction and cognitive deficits. Moreover, overexpression of the AEP-generated synapsin Ⅰ (83-705) fragment in the hippocampus of tau P301S transgenic mice and wild-type mice promotes synaptic dysfunction and cognitive deficits. These findings suggest a novel mechanism of synaptic dysfunction in AD.

UNC5C Receptor Proteolytic Cleavage by Active AEP Promotes Dopaminergic Neuronal Degeneration in Parkinson's Disease.

Netrin-1 is a chemotropic cue mediating axon growth and neural migration in neuronal development, and its receptors deletion in colorectal cancer and UNC5s act as dependence receptors regulating neuronal apoptosis. Asparagine endopeptidase (AEP) is an age-dependent protease that cuts human alpha-synuclein (α-Syn) at N103 and triggers its aggregation and neurotoxicity. In the current study, it is reported that UNC5C receptor is cleaved by AEP in Parkinson's disease (PD) and facilitates dopaminergic neuronal loss. UNC5C is truncated by active AEP in human α-SNCA transgenic mice in an age-dependent manner or induced by neurotoxin rotenone. Moreover, UNC5C is fragmented by AEP in PD brains, inversely correlated with reduced netrin-1 levels. Netrin-1 deprivation in primary cultures induces AEP and caspase-3 activation, triggering UNC5C proteolytic fragmentation and enhancing neuronal loss. Noticeably, blocking UNC5C cleavage by AEP attenuates netrin-1 deprivation-elicited neuronal death and motor disorders in netrin flox/flox mice. Overexpression of AEP-truncated UNC5C intracellular fragment strongly elicits α-Syn aggregation and dopaminergic loss, locomotor deficits in α-SNCA transgenic mice. Hence, the findings demonstrate that netrin-1 reduction and UNC5C truncation by AEP contribute to PD pathogenesis.

Muscle-generated BDNF (brain derived neurotrophic factor) maintains mitochondrial quality control in female mice.

Mitochondrial remodeling is dysregulated in metabolic diseases but the underlying mechanism is not fully understood. We report here that BDNF (brain derived neurotrophic factor) provokes mitochondrial fission and clearance in skeletal muscle via the PRKAA/AMPK-PINK1-PRKN/Parkin and PRKAA-DNM1L/DRP1-MFF pathways. Depleting Bdnf expression in myotubes reduced fatty acid-induced mitofission and mitophagy, which was associated with mitochondrial elongation and impaired lipid handling. Muscle-specific bdnf knockout (MBKO) mice displayed defective mitofission and mitophagy, and accumulation of dysfunctional mitochondria in the muscle when they were fed with a high-fat diet (HFD). These animals also have exacerbated body weight gain, increased intramyocellular lipid deposition, reduced energy expenditure, poor metabolic flexibility, and more insulin resistance. In contrast, consuming a BDNF mimetic (7,8-dihydroxyflavone) increased mitochondrial content, and enhanced mitofission and mitophagy in the skeletal muscles. Hence, BDNF is an essential myokine to maintain mitochondrial quality and function, and its repression in obesity might contribute to impaired metabolism.Abbreviation: 7,8-DHF: 7,8-dihydroxyflavone; ACACA/ACC: acetyl Coenzyme A carboxylase alpha; ACAD: acyl-Coenzyme A dehydrogenase family; ACADVL: acyl-Coenzyme A dehydrogenase, very long chain; ACOT: acyl-CoA thioesterase; CAMKK2: calcium/calmodulin-dependent protein kinase kinase 2, beta; BDNF: brain derived neurotrophic factor; BNIP3: BCL2/adenovirus E1B interacting protein 3; BNIP3L/NIX: BCL2/adenovirus E1B interacting protein 3-like; CCL2/MCP-1: chemokine (C-C motif) ligand 2; CCL5: chemokine (C-C motif) ligand 5; CNS: central nervous system; CPT1B: carnitine palmitoyltransferase 1b, muscle; Cpt2: carnitine palmitoyltransferase 2; CREB: cAMP responsive element binding protein; DNM1L/DRP1: dynamin 1-like; E2: estrogen; EHHADH: enoyl-CoenzymeA hydratase/3-hydroxyacyl CoenzymeA dehydrogenase; ESR1/ER-alpha: estrogen receptor 1 (alpha); FA: fatty acid; FAO: fatty acid oxidation; FCCP: carbonyl cyanide-4-(trifluoromethoxy)phenylhydrazone; FFA: free fatty acids; FGF21: fibroblast growth factor 21; FUNDC1: FUN14 domain containing 1; HADHA: hydroxyacyl-CoA dehydrogenase trifunctional multienzyme complex subunit alpha; HFD: high-fat diet; iWAT: inguinal white adipose tissues; MAP1LC3A/LC3A: microtubule-associated protein 1 light chain 3 alpha; MBKO; muscle-specific bdnf knockout; IL6/IL-6: interleukin 6; MCEE: methylmalonyl CoA epimerase; MFF: mitochondrial fission factor; NTRK2/TRKB: neurotrophic tyrosine kinase, receptor, type 2; OPTN: optineurin; PA: palmitic acid; PARL: presenilin associated, rhomboid-like; PDH: pyruvate dehydrogenase; PINK1: PTEN induced putative kinase 1; PPARGC1A/PGC-1α: peroxisome proliferative activated receptor, gamma, coactivator 1 alpha; PRKAA/AMPK: protein kinase, AMP-activated, alpha 2 catalytic subunit; ROS: reactive oxygen species; TBK1: TANK-binding kinase 1; TG: triacylglycerides; TNF/TNFα: tumor necrosis factor; TOMM20: translocase of outer mitochondrial membrane 20; ULK1: unc-51 like kinase 1.

Transgenic Mice Expressing Human α-Synuclein 1-103 Fragment as a Novel Model of Parkinson's Disease.

Parkinson's disease (PD) is one of the most common neurodegenerative disorders. However, its cellular and molecular mechanisms still wrap in the mist. This is partially caused by the absence of appropriate animal models mimicking sporadic PD that constitutes the majority of cases. Previously, we reported that a cysteine protease, asparagine endopeptidase (AEP), is activated in an age-dependent manner, and cleaves α-synuclein in the brain of sporadic PD patients. The AEP-derived α-synuclein 1-103 fragment is required for the pathogenesis of PD. Thus, we designed and characterized a novel transgenic mouse line expressing α-synuclein 1-103 (designated N103 mice). This model shows an abundant accumulation of pathological α-synuclein in the central nervous system, loss of dopaminergic neurons in the substantia nigra, and progressive striatal synaptic degeneration. The N103 mice also manifest age-dependent PD-like behavioral impairments. Notably, the mice show weight loss and constipation, which are the common non-motor symptoms in PD. The RNA-sequencing analysis found that the transcriptomics pattern was extensively altered in N103 mice. In conclusion, the N103 mouse line, as a brand-new tool, might provide new insights into PD research.

Inhibition of PHLPP1/2 phosphatases rescues pancreatic ß-cells in diabetes.

Pancreatic ß-cell failure is the key pathogenic element of the complex metabolic deterioration in type 2 diabetes (T2D); its underlying pathomechanism is still elusive. Here, we identify pleckstrin homology domain leucine-rich repeat protein phosphatases 1 and 2 (PHLPP1/2) as phosphatases whose upregulation leads to ß-cell failure in diabetes. PHLPP levels are highly elevated in metabolically stressed human and rodent diabetic ß-cells. Sustained hyper-activation of mechanistic target of rapamycin complex 1 (mTORC1) is the primary mechanism of the PHLPP upregulation linking chronic metabolic stress to ultimate ß-cell death. PHLPPs directly dephosphorylate and regulate activities of ß-cell survival-dependent kinases AKT and MST1, constituting a regulatory triangle loop to control ß-cell apoptosis. Genetic inhibition of PHLPPs markedly improves ß-cell survival and function in experimental models of diabetes in vitro, in vivo, and in primary human T2D islets. Our study presents PHLPPs as targets for functional regenerative therapy of pancreatic ß cells in diabetes.

Targeting both BDNF/TrkB pathway and delta-secretase for treating Alzheimer's disease.

Alzheimer's disease (AD) is the most common dementia, and no disease-modifying therapeutic agents are currently available. BDNF/TrkB signaling is impaired in AD and is associated with prominent delta-secretase (δ-secretase, also known as asparaginyl endopeptidase or legumain) activation, which simultaneously cleaves both APP and Tau and promotes Aß production and neurofibrillary tangles (NFT) pathologies. Here we show that the optimized δ-secretase inhibitor (#11a) or TrkB receptor agonist (CF3CN) robustly blocks δ-secretase activity separately, and their combination synergistically blunts δ-secretase, exhibiting promising therapeutic efficacy in 3xTg AD mouse model. The optimal δ-secretase inhibitor reveals demonstrable brain exposure and oral bioavailability, suppressing APP N585 and Tau N368 cleavage by δ-secretase. Strikingly, CF3CN treatment evidently escalates BDNF levels. Both #11a and CF3CN display strong in vivo PK/PD properties and ability to suppress δ-secretase activity in the brain. Orally administrated CF3CN strongly activates TrkB that triggers active Akt to phosphorylate δ-secretase T322, preventing its proteolytic activation and mitigating AD pathologies. #11a or CF3CN significantly diminishes AD pathogenesis and improves cognitive functions with the combination exhibiting the maximal effect. Thus, our data support that these derivatives are strong pharmaceutical candidates for the treatment of AD.

Gut inflammation triggers C/EBPß/δ-secretase-dependent gut-to-brain propagation of Aß and Tau fibrils in Alzheimer's disease.

Inflammation plays an important role in the pathogenesis of Alzheimer's disease (AD). Some evidence suggests that misfolded protein aggregates found in AD brains may have originated from the gut, but the mechanism underlying this phenomenon is not fully understood. C/EBPß/δ-secretase signaling in the colon was investigated in a 3xTg AD mouse model in an age-dependent manner. We applied chronic administration of 1% dextran sodium sulfate (DSS) to trigger gut leakage or colonic injection of Aß or Tau fibrils or AD patient brain lysates in 3xTg mice and combined it with excision/cutting of the gut-brain connecting vagus nerve (vagotomy), in order to explore the role of the gut-brain axis in the development of AD-like pathologies and to monitor C/EBPß/δ-secretase signaling under those conditions. We found that C/EBPß/δ-secretase signaling is temporally activated in the gut of AD patients and 3xTg mice, initiating formation of Aß and Tau fibrils that spread to the brain. DSS treatment promotes gut leakage and facilitates AD-like pathologies in both the gut and the brain of 3xTg mice in a C/EBPß/δ-secretase-dependent manner. Vagotomy selectively blunts this signaling, attenuates Aß and Tau pathologies, and restores learning and memory. Aß or Tau fibrils or AD patient brain lysates injected into the colon propagate from the gut into the brain via the vagus nerve, triggering AD pathology and cognitive dysfunction. The results indicate that inflammation activates C/EBPß/δ-secretase and initiates AD-associated pathologies in the gut, which are subsequently transmitted to the brain via the vagus nerve.

7,8-Dihydroxyflavone modulates bone formation and resorption and ameliorates ovariectomy-induced osteoporosis.

Imbalances in bone formation and resorption cause osteoporosis. Mounting evidence supports that brain-derived neurotrophic factor (BDNF) implicates in this process. 7,8-Dihydroxyflavone (7,8-DHF), a plant-derived small molecular TrkB agonist, mimics the functions of BDNF. We show that both BDNF and 7,8-DHF promoted the proliferation, osteogenic differentiation, and mineralization of MC3T3-E1 cells. These effects might be attributed to the activation of the Wnt/ß-catenin signaling pathway as the expression of cyclin D1, phosphorylated-glycogen synthase kinase-3ß (p-GSK3ß), ß-catenin, Runx2, Osterix, and osteoprotegerin (OPG) was all significantly up-regulated. Knockdown of ß-catenin restrained the up-regulation of Runx2 and Osterix stimulated by 7,8-DHF. In particular, blocking TrkB by its specific inhibitor K252a suppressed 7,8-DHF-induced osteoblastic proliferation, differentiation, and expression of osteoblastogenic genes. Moreover, BDNF and 7,8-DHF repressed osteoclastic differentiation of RAW264.7 cells. The transcription factor c-fos and osteoclastic genes such as tartrate-resistant acid phosphatase (TRAP), matrix metalloprotein-9 (MMP-9), Adamts5 were inhibited by 7,8-DHF. More importantly, 7,8-DHF attenuated bone loss, improved trabecular microarchitecture, tibial biomechanical properties, and bone biochemical indexes in an ovariectomy (OVX) rat model. The current work highlights the dual regulatory effects that 7,8-DHF exerts on bone remodeling.

C/EBPß/AEP Signaling Regulates the Oxidative Stress in Malignant Cancers, Stimulating the Metastasis.

Solid tumors start as a local disease, but some are capable of metastasizing to the lymph nodes and distant organs. The hypoxic microenvironment, which is critical during cancer development, plays a key role in regulating cancer progression and metastasis. However, the molecular mechanisms mediating the disseminated cancer cell metastasis remain incompletely understood. Here, we show that C/EBPß/AEP signaling that is upregulated in breast cancers mediates oxidative stress and lung metastasis, and inactivation of asparagine endopeptidase (AEP, also known as legumain) robustly regulates breast cancer reactive oxygen species (ROS) and metastasis. AEP, a protease activated in acidic conditions, is overexpressed in numerous types of cancer and promotes metastasis. Employing a breast cancer cell line MDA-MD-231, we show that C/EBPß, an oxidative stress or inflammation-activated transcription factor, and its downstream target AEP mediate ROS production as well as migration and invasion in cancer cells. Deficiency of AEP in the MMTV-PyMT transgenic breast cancer mouse model significantly regulates oxidative stress and suppresses lung metastasis. Administration of an innovative AEP inhibitor substantially mitigates ROS production and cancer metastasis. Hence, our study demonstrates that pharmacologic inhibition of AEP activity might provide a disease-modifying strategy to suppress cancer metastasis.

Neurotrophic signaling deficiency exacerbates environmental risks for Alzheimer's disease pathogenesis.

The molecular mechanism of Alzheimer's disease (AD) pathogenesis remains obscure. Life and/or environmental events, such as traumatic brain injury (TBI), high-fat diet (HFD), and chronic cerebral hypoperfusion (CCH), are proposed exogenous risk factors for AD. BDNF/TrkB, an essential neurotrophic signaling for synaptic plasticity and neuronal survival, are reduced in the aged brain and in AD patients. Here, we show that environmental factors activate C/EBPß, an inflammatory transcription factor, which subsequently up-regulates δ-secretase that simultaneously cleaves both APP and Tau, triggering AD neuropathological changes. These adverse effects are additively exacerbated in BDNF+/- or TrkB+/- mice. Strikingly, TBI provokes both senile plaque deposit and neurofibrillary tangles (NFT) formation in TrkB+/- mice, associated with augmented neuroinflammation and extensive neuronal loss, leading to cognitive deficits. Depletion of C/EBPß inhibits TBI-induced AD-like pathologies in these mice. Remarkably, amyloid aggregates and NFT are tempospatially distributed in TrkB+/- mice brains after TBI, providing insight into their spreading in the progression of AD-like pathologies. Hence, our study revealed the roles of exogenous (TBI, HFD, and CCH) and endogenous (TrkB/BDNF) risk factors in the onset of AD-associated pathologies.

Amphiphysin I cleavage by asparagine endopeptidase leads to tau hyperphosphorylation and synaptic dysfunction.

Neurofibrillary tangles composed of hyperphosphorylated tau and synaptic dysfunction are characteristics of Alzheimer's disease (AD). However, the underlying molecular mechanisms remain poorly understood. Here, we identified Amphiphysin I mediates both tau phosphorylation and synaptic dysfunction in AD. Amphiphysin I is cleaved by a cysteine proteinase asparagine endopeptidase (AEP) at N278 in the brains of AD patients. The amount of AEP-generated N-terminal fragment of Amphiphysin I (1-278) is increased with aging. Amphiphysin I (1-278) inhibits clathrin-mediated endocytosis and induces synaptic dysfunction. Furthermore, Amphiphysin I (1-278) binds p35 and promotes its transition to p25, thus activates CDK5 and enhances tau hyperphosphorylation. Overexpression of Amphiphysin I (1-278) in the hippocampus of Tau P301S mice induces synaptic dysfunction, tau hyperphosphorylation, and cognitive deficits. However, overexpression of the N278A mutant Amphiphysin I, which resists the AEP-mediated cleavage, alleviates the pathological and behavioral defects. These findings suggest a mechanism of tau hyperphosphorylation and synaptic dysfunction in AD.

A γ-adducin cleavage fragment induces neurite deficits and synaptic dysfunction in Alzheimer's disease.

Neurite deficits and synaptic dysfunction contribute to cognitive impairments in Alzheimer's disease (AD). However, the underlying molecular mechanisms remain unclear. Here, we show that γ-adducin, a cytoskeleton-associated protein that assembles the spectrin-actin framework, is cleaved by a lysosomal cysteine proteinase named asparagine endopeptidase (AEP). AEP is upregulated and activated during aging and cleaves γ-adducin at N357, disrupting spectrin-actin assembly. Moreover, γ-adducin (1-357) fragment downregulates the expression of Rac2, leading to defects in neurite outgrowth. Expression of the γ-adducin (1-357) fragment in the hippocampus of tau P301S transgenic mice resulted in significant AD-like pathology and cognitive deficits. In summary, AEP-mediated fragmentation of γ-adducin plays a vital role in AD. Blocking the activity of AEP might be a novel therapeutic target for AD.

A delta-secretase-truncated APP fragment activates CEBPB, mediating Alzheimer's disease pathologies.

Amyloid-ß precursor protein (APP) is sequentially cleaved by secretases and generates amyloid-ß, the major components in senile plaques in Alzheimer's disease. APP is upregulated in human Alzheimer's disease brains. However, the molecular mechanism of how APP contributes to Alzheimer's disease pathogenesis remains incompletely understood. Here we show that truncated APP C586-695 fragment generated by δ-secretase directly binds to CCAAT/enhancer-binding protein beta (CEBPB), an inflammatory transcription factor, and enhances its transcriptional activity, escalating Alzheimer's disease-related gene expression and pathogenesis. The APP C586-695 fragment, but not full-length APP, strongly associates with CEBPB and elicits its nuclear translocation and augments the transcriptional activities on APP itself, MAPT (microtubule-associated protein tau), δ-secretase and inflammatory cytokine mRNA expression, finally triggering Alzheimer's disease pathology and cognitive disorder in a viral overexpression mouse model. Blockade of δ-secretase cleavage of APP by mutating the cleavage sites reduces its stimulatory effect on CEBPB, alleviating amyloid pathology and cognitive dysfunctions. Clearance of APP C586-695 from 5xFAD mice by antibody administration mitigates Alzheimer's disease pathologies and restores cognitive functions. Thus, in addition to the sequestration of amyloid-ß, APP implicates in Alzheimer's disease pathology by activating CEBPB upon δ-secretase cleavage.

Asparagine endopeptidase cleaves synaptojanin 1 and triggers synaptic dysfunction in Parkinson's disease.

Parkinson's disease (PD) is one of the most common neurodegenerative diseases, which is characterized by the loss of dopaminergic neurons in the nigrostriatal pathway. Synaptic dysfunction impairs dopamine turnover and contributes to the degeneration of dopaminergic neurons. However, the molecular mechanisms underlying synaptic dysfunction and dopaminergic neuronal vulnerability in PD are not clear. Here, we report that synaptojanin 1 (SYNJ1), a polyphosphoinositide phosphatase concentrated at nerve terminals, is a substrate of a cysteine proteinase, asparagine endopeptidase (AEP). SYNJ1 is cleaved by the cysteine proteinase AEP at N599 in the brains of PD patients. AEP-mediated cleavage of SYNJ1 disrupts neuronal phosphoinositide homeostasis and causes synaptic dysfunction. Overexpression of the AEP-generated fragments of SYNJ1 triggers synaptic dysfunction and the degeneration of dopaminergic neurons, inducing motor defects in the α-synuclein transgenic mice. Blockage of AEP-mediated cleavage of SYJN1 alleviates the pathological and behavioral defects in a mouse model of PD. Our results demonstrate that the fragmentation of SYNJ1 by AEP mediates synaptic dysfunction and dopaminergic neuronal degeneration in PD.