Association of GLOD4 with Alzheimer's Disease in Humans and Mice.

Background: Glyoxalase domain containing protein 4 (GLOD4), a protein of an unknown function, is associated with Alzheimer's disease (AD). Three GLOD4 isoforms are known. The mechanism underlying GLOD4's association with AD was unknown. Objective: To assess GLOD4's role in the central nervous system by studying GLOD4 isoforms expression in human frontal cerebral cortical tissues from AD patients and in brains of Blmh-/-5xFAD mouse AD model of AD. Methods: GLOD4 protein and mRNA were quantified in human and mouse brains by western blotting and RT-qPCR, respectively. Mouse brain amyloid-ß (Aß) was quantified by western blotting. Behavioral assessments of mice were performed by cognitive/neuromotor testing. Glod4 gene in mouse neuroblastoma N2a-APPswe cells was silenced by RNA interference and Glod4, Aß precursor protein (Aßpp), Atg5, p62, and Lc3 proteins and mRNAs were quantified. Results: GLOD4 mRNA and protein isoforms were downregulated in cortical tissues from AD patients compared to non-AD controls. Glod4 mRNA was downregulated in brains of Blmh-/-5xFAD mice compared to Blmh+/+5xFAD sibling controls, but not in Blmh-/- mice without the 5xFAD transgene compared to Blmh+/+ sibling controls. The 5xFAD transgene downregulated Glod4 mRNA in Blmh-/- mice of both sexes and in Blmh+/+ males but not females. Attenuated Glod4 was associated with elevated Aß and worsened memory/sensorimotor performance in Blmh-/-5xFAD mice. Glod4 depletion in N2a-APPswe cells upregulated AßPP, and downregulated autophagy-related Atg5, p62, and Lc3 genes. Conclusions: These findings suggest that GLOD4 interacts with AßPP and the autophagy pathway, and that disruption of these interactions leads to Aß accumulation and cognitive/neurosensory deficits.

Diagnostic Performance of Cerebrospinal Fluid Neurofilament Light Chain and Soluble Amyloid-ß Protein Precursor ß in the Subcortical Small Vessel Type of Dementia.

BACKGROUND: The subcortical small vessel type of dementia (SSVD) is a common subtype of vascular dementia, but there is a lack of disease-specific cerebrospinal fluid (CSF) biomarkers. OBJECTIVE: We investigated whether CSF concentrations of neurofilament light chain (NFL), soluble amyloid-ß protein precursor α (sAßPPα), sAßPPß, and CSF/serum albumin ratio could separate SSVD from healthy controls, Alzheimer's disease (AD), and mixed dementia (combined AD and SSVD). METHODS: This was a mono-center study of patients with SSVD (n = 38), AD (n = 121), mixed dementia (n = 62), and controls (n = 96). The CSF biomarkers were measured using immunoassays, and their independent contribution to the separation between groups were evaluated using the Wald test. Then, the area under the receiver operating characteristics curve (AUROC) and 95% confidence intervals (CIs) were calculated. RESULTS: Elevated neurofilament light chain (NFL) and decreased sAßPPß independently separated SSVD from controls, and sAßPPß also distinguished SSVD from AD and mixed dementia. The combination of NFL and sAßPPß discriminated SSVD from controls with high accuracy (AUROC 0.903, 95% CI: 0.834-0.972). Additionally, sAßPPß combined with the core AD biomarkers (amyloid-ß42, total tau, and phosphorylated tau181) had a high ability to separate SSVD from AD (AUROC 0.886, 95% CI: 0.830-0.942) and mixed dementia (AUROC 0.903, 95% CI: 0.838-0.968). CONCLUSIONS: The high accuracy of NFL and sAßPPß to separate SSVD from controls supports that SSVD is a specific diagnostic entity. Moreover, SSVD was distinguished from AD and mixed dementia using sAßPPß in combination with the core AD biomarkers.

Iron Responsiveness to Lysosomal Disruption: A Novel Pathway to Alzheimer's Disease.

Familial Alzheimer's disease (fAD) mutations in the amyloid-ß protein precursor (AßPP) enhance brain AßPP C-Terminal Fragment (CTF) levels to inhibit lysosomal v-ATPase. Consequent disrupted acidification of the endolysosomal pathway may trigger brain iron deficiencies and mitochondrial dysfunction. The iron responsive element (IRE) in the 5'Untranslated-region of AßPP mRNA should be factored into this cycle where reduced bioavailable Fe-II would decrease IRE-dependent AßPP translation and levels of APP-CTFß in a cycle to adaptively restore iron homeostasis while increases of transferrin-receptors is evident. In healthy younger individuals, Fe-dependent translational modulation of AßPP is part of the neuroprotective function of sAßPPα with its role in iron transport.

An Alternative View of Familial Alzheimer's Disease Genetics.

Probabilistic and parsimony-based arguments regarding available genetics data are used to propose that Hardy and Higgin's amyloid cascade hypothesis is valid but is commonly interpreted too narrowly to support, incorrectly, the primacy of the amyloid-ß peptide (Aß) in driving Alzheimer's disease pathogenesis. Instead, increased activity of the ßCTF (C99) fragment of AßPP is the critical pathogenic determinant altered by mutations in the APP gene. This model is consistent with the regulation of APP mRNA translation via its 5' iron responsive element. Similar arguments support that the pathological effects of familial Alzheimer's disease mutations in the genes PSEN1 and PSEN2 are not exerted directly via changes in AßPP cleavage to produce different ratios of Aß length. Rather, these mutations likely act through effects on presenilin holoprotein conformation and function, and possibly the formation and stability of multimers of presenilin holoprotein and/or of the γ-secretase complex. All fAD mutations in APP, PSEN1, and PSEN2 likely find unity of pathological mechanism in their actions on endolysosomal acidification and mitochondrial function, with detrimental effects on iron homeostasis and promotion of "pseudo-hypoxia" being of central importance. Aß production is enhanced and distorted by oxidative stress and accumulates due to decreased lysosomal function. It may act as a disease-associated molecular pattern enhancing oxidative stress-driven neuroinflammation during the cognitive phase of the disease.

Deletion of the Homocysteine Thiolactone Detoxifying Enzyme Bleomycin Hydrolase, in Mice, Causes Memory and Neurological Deficits and Worsens Alzheimer's Disease-Related Behavioral and Biochemical Traits in the 5xFAD Model of Alzheimer's Disease.

BACKGROUND: Bleomycin hydrolase (BLMH), a homocysteine (Hcy)-thiolactone detoxifying enzyme, is attenuated in Alzheimer's disease (AD) brains. Blmh loss causes astrogliosis in mice while the loss of histone demethylase Phf8, which controls mTOR signaling, causes neuropathy in mice and humans. OBJECTIVE: To examine how Blmh gene deletion affects the Phf8/H4K20me1/mTOR/autophagy pathway, amyloid-ß (Aß) accumulation, and cognitive/neuromotor performance in mice. METHODS: We generated a new mouse model of AD, the Blmh-/-5xFAD mouse. Behavioral assessments were conducted by cognitive/neuromotor testing. Blmh and Phf8 genes were silenced in mouse neuroblastoma N2a-APPswe cells by RNA interference. mTOR- and autophagy-related proteins, and AßPP were quantified by western blotting and the corresponding mRNAs by RT-qPCR. Aß was quantified by western blotting (brains) and by confocal microscopy (cells). RESULTS: Behavioral testing showed cognitive/neuromotor deficits in Blmh-/- and Blmh-/-5xFAD mice. Phf8 was transcriptionally downregulated in Blmh-/- and Blmh-/-5xFAD brains. H4K20me1, mTOR, phospho-mTOR, and AßPP were upregulated while autophagy markers Becn1, Atg5, and Atg7 were downregulated in Blmh-/- and Blmh-/-5xFAD brains. Aß was elevated in Blmh-/-5xFAD brains. These biochemical changes were recapitulated in Blmh-silenced N2a-APPswe cells, which also showed increased H4K20me1-mTOR promoter binding and impaired autophagy flux (Lc3-I, Lc3-II, p62). Phf8-silencing or treatments with Hcy-thiolactone or N-Hcy-protein, metabolites elevated in Blmh-/- mice, induced biochemical changes in N2a-APPswe cells like those induced by the Blmh-silencing. However, Phf8-silencing elevated Aß without affecting AßPP. CONCLUSIONS: Our findings show that Blmh interacts with AßPP and the Phf8/H4K20me1/mTOR/autophagy pathway, and that disruption of those interactions causes Aß accumulation and cognitive/neuromotor deficits.

PCP4 Promotes Alzheimer's Disease Pathogenesis by Affecting Amyloid-ß Protein Precursor Processing.

BACKGROUND: Down syndrome (DS) is caused by an extra copy of all or part of chromosome 21. The patients with DS develop typical Alzheimer's disease (AD) neuropathology, indicating the role of genes on human chromosome 21 (HSA21) in the pathogenesis of AD. Purkinje cell protein 4 (PCP4), also known as brain-specific protein 19, is a critical gene located on HSA21. However, the role of PCP4 in DS and AD pathogenesis is not clear. OBJECTIVE: To explore the role of PCP4 in amyloid-ß protein precursor (AßPP) processing in AD. METHODS: In this study, we investigated the role of PCP4 in AD progression in vitro and in vivo. In vitro experiments, we overexpressed PCP4 in human Swedish mutant AßPP stable expression or neural cell lines. In vitro experiments, APP23/PS45 double transgenic mice were selected and treated with AAV-PCP4. Multiple topics were detected by western blot, RT-PCR, immunohistochemical and behavioral test. RESULTS: We found that PCP4 expression was altered in AD. PCP4 was overexpressed in APP23/PS45 transgenic mice and PCP4 affected the processing of AßPP. The production of amyloid-ß protein (Aß) was also promoted by PCP4. The upregulation of endogenous AßPP expression and the downregulation of ADAM10 were due to the transcriptional regulation of PCP4. In addition, PCP4 increased Aß deposition and neural plaque formation in the brain, and exuberated learning and memory impairment in transgenic AD model mice. CONCLUSION: Our finding reveals that PCP4 contributes to the pathogenesis of AD by affecting AßPP processing and suggests PCP4 as a novel therapeutic target for AD by targeting Aß pathology.

Age-Associated UBE2O Reduction Promotes Neuronal Death in Alzheimer's Disease.

BACKGROUND: Alzheimer's disease (AD) is the most common neurodegenerative disease leading to dementia in the elderly. Ubiquitin proteasome system (UPS) is critical for protein homeostasis, while the functional decline of UPS with age contributes to the pathogenesis of AD. Ubiquitin-conjugating enzyme E2O (UBE2O), an E2-E3 hybrid enzyme, is a major component of UPS. However, its role in AD pathogenesis has not been fully defined. OBJECTIVE: We aimed to identify the age-associated expression of UBE2O and its role AD pathogenesis. METHODS: Western blot analysis were used to assess expression of UBE2O in organs/tissues and cell lines. Immunofluorescence staining was performed to examine the cellular distribution of UBE2O. Neuronal death was determined by the activity of lactate dehydrogenase. RESULTS: UBE2O is highly expressed in the cortex and hippocampus. It is predominantly expressed in neurons but not in glial cells. The peak expression of UBE2O is at postnatal day 17 and 14 in the cortex and hippocampus, respectively. Moreover its expression is gradually reduced with age. Importantly, UBE2O is significantly reduced in both cortex and hippocampus of AD mice. Consistently, overexpression of amyloid-ß protein precursor (AßPP) with a pathogenic mutation (AßPPswe) for AD reduces the expression of UBE2O and promotes neuronal death, while increased expression of UBE2O rescues AßPPswe-induced neuronal death. CONCLUSION: Our study indicates that age-associated reduction of UBE2O may facilitates neuronal death in AD, while increasing UBE2O expression or activity may be a potential approach for AD treatment by inhibiting neuronal death.

Generation and Characterization of the First Murine Model of Alzheimer's Disease with Mutated AßPP Inserted in a BALB/c Background (C.B6/J-APPswe).

BACKGROUND: Numerous mouse models of Alzheimer's disease (AD) are available, but all suffer from certain limitations, thus prompting further attempts. To date, no one model exists with amyloidopathy in a BALB/c strain. OBJECTIVE: To generate and characterize the C.B6/J-APPswe mouse, a model of AD with a mutated human gene for the amyloid-ß protein precursor (AßPP) inserted in a BALB/c background. METHODS: We analyzed five groups at different ages (3, 6, 9, 12, and 16-18 months) of C.B6/J-APPswe and wild-type mice (50% males and 50% females) for the main hallmarks of AD by western blotting, amyloid-ß (Aß) ELISA, immunocytochemistry, electrophysiology, and behavioral tests. RESULTS: The C.B6/J-APPswe mouse displays early AßPP and Aß production, late amyloid plaques formation, high level of Tau phosphorylation, synaptic deficits (reduced density and functional impairment due to a reduced post-synaptic responsiveness), neurodegeneration caused by apoptosis and necroptosis/necrosis, microgliosis, astrocytic abnormalities, and sex-related differences in explorative behavior, anxiety-like behavior, and spatial long-term and working memories. Social housing is feasible despite the intra-cage aggressiveness of male animals. CONCLUSION: C.B6/J-APPswe mice develop most of the distinctive features of AD and is a suitable model for the study of brain atrophy mechanisms and of the differences between males and females in the onset of cognitive/non-cognitive deficits.

The Amyloid Cascade Hypothesis 2.0: Generalization of the Concept.

Recently, we proposed the Amyloid Cascade Hypothesis 2.0 (ACH2.0), a reformulation of the ACH. In the former, in contrast to the latter, Alzheimer's disease (AD) is driven by intraneuronal amyloid-ß (iAß) and occurs in two stages. In the first, relatively benign stage, Aß protein precursor (AßPP)-derived iAß activates, upon reaching a critical threshold, the AßPP-independent iAß-generating pathway, triggering a devastating second stage resulting in neuronal death. While the ACH2.0 remains aligned with the ACH premise that Aß is toxic, the toxicity is exerted because of intra- rather than extracellular Aß. In this framework, a once-in-a-lifetime-only iAß depletion treatment via transient activation of BACE1 and/or BACE2 (exploiting their Aß-cleaving activities) or by any means appears to be the best therapeutic strategy for AD. Whereas the notion of differentially derived iAß being the principal moving force at both AD stages is both plausible and elegant, a possibility remains that the second AD stage is enabled by an AßPP-derived iAß-activated self-sustaining mechanism producing a yet undefined deleterious "substance X" (sX) which anchors the second AD stage. The present study generalizes the ACH2.0 by incorporating this possibility and shows that, in this scenario, the iAß depletion therapy may be ineffective at symptomatic AD stages but fully retains its preventive potential for both AD and the aging-associated cognitive decline, which is defined in the ACH2.0 framework as the extended first stage of AD.

Early Mitochondrial Defects in the 5xFAD Mouse Model of Alzheimer's Disease.

BACKGROUND: Mitochondrial (MT) dysfunction is a hallmark of Alzheimer's disease (AD). Amyloid-ß protein precursor and amyloid-ß peptides localize to MT and lead to MT dysfunction in familial forms of AD. This dysfunction may trigger subsequent types of pathology. OBJECTIVE: To identify the MT phenotypes that occur early in order to help understand the cascade of AD pathophysiology. METHODS: The 5xFAD mouse model was used to explore the time course of MT pathologies in both sexes. Protein biomarkers for MT dynamics were measured biochemically and MT function was measured using oxygen consumption and ATP assays. RESULTS: We discovered progressive alterations in mitochondrial dynamics (biogenesis, fission, fusion, and mitophagy) and function (O2 consumption, ATP generation, and Ca2+ import) in the hippocampus of 5xFAD mice in both sexes as early as 2 months of age. Thus, mitochondrial dynamics and function become altered at young ages, consistent with an early role for mitochondria in the AD pathological cascade. CONCLUSION: Our study offers the baseline information required to understand the hierarchical relationship between the multiple pathologies that develop in this mouse model and provides early biomarkers for MT dysfunction. This will aid in dissecting the temporal cascade of pathologies, understanding sex-specific differences, and in testing the efficacy of putative mitochondrial therapeutics.

BACE2: A Promising Neuroprotective Candidate for Alzheimer's Disease.

Alzheimer's disease (AD) is the most common cause of dementia that affects millions of predominantly elderly individuals worldwide. Despite intensive research over several decades, controversies still surround the etiology of AD and the disease remains incurable. Meanwhile, new molecular players of the central amyloid cascade hypothesis have emerged and among these is a protease known as ß-site APP cleavage enzyme 2 (BACE2). Unlike BACE1, BACE2 cleaves the amyloid-ß protein precursor within the Aß domain that accordingly prevents the generation of Aß42 peptides, the aggregation of which is commonly regarded as the toxic entity that drives neurodegeneration in AD. Given this non-amyloidogenic role of BACE2, it is attractive to position BACE2 as a therapeutic target for AD. Indeed, several groups including ours have demonstrated a neuroprotective role for BACE2 in AD. In this review, we discuss emerging evidence supporting the ability of BACE2 in mitigating AD-associated pathology in various experimental systems including human pluripotent stem cell-derived cerebral organoid disease models. Alongside this, we also provide an update on the identification of single nucleotide polymorphisms occurring in the BACE2 gene that are linked to increased risk and earlier disease onset in the general population. In particular, we highlight a recently identified point mutation on BACE2 that apparently leads to sporadic early-onset AD. We believe that a better understanding of the role of BACE2 in AD would provide new insights for the development of viable therapeutic strategies for individuals with dementia.

Modulation of Cytosolic Phospholipase A2 as a Potential Therapeutic Strategy for Alzheimer's Disease.

Background: Alzheimer's disease (AD) is a neurodegenerative disorder lacking any curative treatment up to now. Indeed, actual medication given to the patients alleviates only symptoms. The cytosolic phospholipase A2 (cPLA2-IVA) appears as a pivotal player situated at the center of pathological pathways leading to AD and its inhibition could be a promising therapeutic approach. Objective: A cPLA2-IVA inhibiting peptide was identified in the present work, aiming to develop an original therapeutic strategy. Methods: We targeted the cPLA2-IVA using the phage display technology. The hit peptide PLP25 was first validated in vitro (arachidonic acid dosage [AA], cPLA2-IVA cellular translocation) before being tested in vivo. We evaluated spatial memory using the Barnes maze, amyloid deposits by MRI and immunohistochemistry (IHC), and other important biomarkers such as the cPLA2-IVA itself, the NMDA receptor, AßPP and tau by IHC after i.v. injection in APP/PS1 mice. Results: Showing a high affinity for the C2 domain of this enzyme, the peptide PLP25 exhibited an inhibitory effect on cPLA2-IVA activity by blocking its binding to its substrate, resulting in a decreased release of AA. Coupled to a vector peptide (LRPep2) in order to optimize brain access, we showed an improvement of cognitive abilities of APP/PS1 mice, which also exhibited a decreased number of amyloid plaques, a restored expression of cPLA2-IVA, and a favorable effect on NMDA receptor expression and tau protein phosphorylation. Conclusions: cPLA2-IVA inhibition through PLP25 peptide could be a promising therapeutic strategy for AD.

Urokinase-Type Plasminogen Activator Triggers Wingless/Int1-Independent Phosphorylation of the Low-Density Lipoprotein Receptor-Related Protein-6 in Cerebral Cortical Neurons.

BACKGROUND: Urokinase-type plasminogen activator (uPA) is a serine proteinase found in excitatory synapses located in the II/III and V cortical layers. The synaptic release of uPA promotes the formation of synaptic contacts and the repair of synapses damaged by various forms of injury, and its abundance is decreased in the synapse of Alzheimer's disease (AD) patients. Inactivation of the Wingless/Int1 (Wnt)-ß-catenin pathway plays a central role in the pathogenesis of AD. Soluble amyloid-ß (Aß) prevents the phosphorylation of the low-density lipoprotein receptor-related protein-6 (LRP6), and the resultant inactivation of the Wnt-ß-catenin pathway prompts the amyloidogenic processing of the amyloid-ß protein precursor (AßPP) and causes synaptic loss. OBJECTIVE: To study the role of neuronal uPA in the pathogenesis of AD. METHODS: We used in vitro cultures of murine cerebral cortical neurons, a murine neuroblastoma cell line transfected with the APP-695 Swedish mutation (N2asw), and mice deficient on either plasminogen, or uPA, or its receptor (uPAR). RESULTS: We show that uPA activates the Wnt-ß-catenin pathway in cerebral cortical neurons by triggering the phosphorylation of LRP6 via a plasmin-independent mechanism that does not require binding of Wnt ligands (Wnts). Our data indicate that uPA-induced activation of the Wnt-ß-catenin pathway protects the synapse from the harmful effects of soluble Aß and prevents the amyloidogenic processing of AßPP by inhibiting the expression of ß-secretase 1 (BACE1) and the ensuing generation of Aß40 and Aß42 peptides. CONCLUSION: uPA protects the synapse and antagonizes the inhibitory effect of soluble Aß on the Wnt-ß-catenin pathway by providing an alternative pathway for LRP6 phosphorylation and ß-catenin stabilization.

Platelet-Derived Amyloid-ß Protein Precursor as a Biomarker of Alzheimer's Disease.

BACKGROUND: Platelet proteins may be associated with Alzheimer's disease (AD) pathology. OBJECTIVE: To investigate the relationship between platelet proteins and cerebrospinal fluid (CSF) biomarkers of AD and cognition in individuals with memory decline to identify effective screening methods for detecting the early stages of the disease. METHODS: We classified 68 participants with subjective memory decline according to the ATN framework determined by CSF amyloid-ß (A), CSF p-tau (T), and t-tau (N). All participants underwent Mini-Mental State Examination (MMSE) and platelet-related protein content testing. RESULTS: Eighteen participants had normal AD biomarkers (NCs), 24 subjects had non-AD pathologic changes (non-AD), and 26 subjects fell within the Alzheimer's continuum (AD). The platelet amyloid-ß protein precursor (AßPP) ratio in the AD group was significantly lower than in the non-AD and NCs groups, and positively correlated with MMSE scores and CSF amyloid-ß42 level, which could affect MMSE scores through CSF amyloid-ß42. Levels of platelet phosphorylated-tau 231 and ser396/404 phosphorylated tau were elevated in both AD and non-AD compared to NCs. Additionally, the receiver operating characteristic analysis demonstrated that the platelet AßPP ratio was a sensitive identifier for differentiating the AD from NCs (AUC = 0.846) and non-AD (AUC = 0.768). And ser396/404 phosphorylated tau could distinguish AD from NCs. CONCLUSION: Our study was the first to find an association between platelet AßPP ratio and CSF biomarkers of AD, which contribute to the understanding of the peripheral changes in AD. These findings may help to discover potential feasible and effective screening tools for AD.

Mitochondrial Targeting of Amyloid-ß Protein Precursor Intracellular Domain Induces Hippocampal Cell Death via a Mechanism Distinct from Amyloid-ß.

BACKGROUND: Amyloid-ß (Aß) is a principal cleavage product of amyloid-ß protein precursor (AßPP) and is widely recognized as a key pathogenic player in Alzheimer's disease (AD). Yet, there is increasing evidence of a neurotoxic role for the AßPP intracellular domain (AICD) which has been proposed to occur through its nuclear function. Intriguingly, there is a γ-secretase resident at the mitochondria which could produce AICD locally. OBJECTIVE: We examined the potential of AICD to induce neuronal apoptosis when targeted specifically to the mitochondria and compared its mechanism of neurotoxicity to that of Aß. METHODS: We utilized transient transfection of HT22 neuronal cells with bicistronic plasmids coding for DsRed and either empty vector (Ires), Aß, AICD59, or mitochondrial-targeted AICD (mitoAICD) in combination with various inhibitors of pathways involved in apoptosis. RESULTS: AICD induced significant neuronal apoptosis only when targeted to the mitochondria. Apoptosis required functional mitochondria as neither Aß nor mitoAICD induced significant toxicity in cells devoid of mitochondrial DNA. Both glutathione and a Bax inhibitor protected HT22 cells from either peptide. However, inhibition of the mitochondrial permeability transition pore only protected from Aß, while pan-caspase inhibitors uniquely rescued cells from mitoAICD. CONCLUSION: Our results show that AICD displays a novel neurotoxic function when targeted to mitochondria. Moreover, mitoAICD induces apoptosis via a mechanism that is distinct from that of Aß. These findings suggest that AICD produced locally at mitochondria via organelle-specific γ-secretase could act in a synergistic manner with Aß to cause mitochondrial dysfunction and neuronal death in AD.

The Food Additive ß-Caryophyllene Exerts Its Neuroprotective Effects Through the JAK2-STAT3-BACE1 Pathway.

Despite extensive research on Alzheimer's disease (AD), its diagnosis and treatment remain challenging, and no effective therapies are currently available. Amyloid ß (Aß) extracellular plaques and intracellular neurofibrillary tangles are the histological characteristics of AD that have been directly linked to neuropathological events such as synaptic and neuronal cell loss. In this study, we explored whether the "JAK2-STAT3-BACE1" pathway is involved in neuroprotection conferred by the food flavouring agent ß-caryophyllene (BCP). PC-12 cells with overexpressed amyloid-ß protein precursor (APP) were utilised to construct an AD model in vitro, which was then split into four groups, namely control, empty vector, APP overexpression, and BCP (5, 10, and 20 µM). CCK-8 was used to evaluate cell viability, immunofluorescence was utilised to examine synaptic morphology, and quantitative real-time polymerase chain reaction and western blot were used to examine gene and protein expression levels. The relative expression levels of JAK2, STAT3, and BACE1 mRNA in the transfected PC-12 cells were found to be significantly upregulated. The cell morphology altered dramatically 72 h after transfection, becoming rounder, with a decrease in cell number. BCP exhibited the potential to dramatically increase PC-12 cell viability while protecting cell morphology. BCP inhibited APP, JAK2, STAT3, BACE1 mRNA and BACE1 protein overexpression, as well as JAK2 and STAT3 hyperphosphorylation. Molecular docking simulated the docking of BCP with JAK2, STAT3, BACE1, CB2. And JAK2 was found to be the most stable protein. In conclusion, inhibition of the "JAK2-STAT3-BACE1" signalling pathway may be one of the mechanisms through which BCP protects neurons and antagonises Aß's neurotoxicity.

Degradation and inhibition of epigenetic regulatory protein BRD4 exacerbate Alzheimer's disease-related neuropathology in cell models.

Epigenetic regulation plays substantial roles in human pathophysiology, which provides opportunities for intervention in human disorders through the targeting of epigenetic pathways. Recently, emerging evidence from preclinical studies suggested the potential in developing therapeutics of Alzheimer's disease (AD) by targeting bromodomain containing protein 4 (BRD4), an epigenetic regulatory protein. However, further characterization of AD-related pathological events is urgently required. Here, we investigated the effects of pharmacological degradation or inhibition of BRD4 on AD cell models. Interestingly, we found that both degradation and inhibition of BRD4 by ARV-825 and JQ1, respectively, robustly increased the levels of amyloid-beta (Aß), which has been associated with the neuropathology of AD. Subsequently, we characterized the mechanisms by which downregulation of BRD4 increases Aß levels. We found that both degradation and inhibition of BRD4 increased the levels of BACE1, the enzyme responsible for cleavage of the amyloid-beta protein precursor (APP) to generate Aß. Consistent with Aß increase, we also found that downregulation of BRD4 increased AD-related phosphorylated Tau (pTau) protein in our 3D-AD human neural cell culture model. Therefore, our results suggest that downregulation of BRD4 would not be a viable strategy for AD intervention. Collectively, our study not only shows that BRD4 is a novel epigenetic component that regulates BACE1 and Aß levels, but also provides novel and translational insights into the targeting of BRD4 for potential clinical applications.

Prions and Neurodegenerative Diseases: A Focus on Alzheimer's Disease.

Specific protein misfolding and aggregation are mechanisms underlying various neurodegenerative diseases such as prion disease and Alzheimer's disease (AD). The misfolded proteins are involved in prions, amyloid-ß (Aß), tau, and α-synuclein disorders; they share common structural, biological, and biochemical characteristics, as well as similar mechanisms of aggregation and self-propagation. Pathological features of AD include the appearance of plaques consisting of deposition of protein Aß and neurofibrillary tangles formed by the hyperphosphorylated tau protein. Although it is not clear how protein aggregation leads to AD, we are learning that the cellular prion protein (PrPC) plays an important role in the pathogenesis of AD. Herein, we first examined the pathogenesis of prion and AD with a focus on the contribution of PrPC to the development of AD. We analyzed the mechanisms that lead to the formation of a high affinity bond between Aß oligomers (AßOs) and PrPC. Also, we studied the role of PrPC as an AßO receptor that initiates an AßO-induced signal cascade involving mGluR5, Fyn, Pyk2, and eEF2K linking Aß and tau pathologies, resulting in the death of neurons in the central nervous system. Finally, we have described how the PrPC-AßOs interaction can be used as a new potential therapeutic target for the treatment of PrPC-dependent AD.

Anti-Inflammatory Gene Therapy Improves Spatial Memory Performance in a Mouse Model of Alzheimer's Disease.

The immune system plays a critical role in neurodegenerative processes involved in Alzheimer's disease (AD). In this study, a gene-based immunotherapeutic method examined the effects of anti-inflammatory cellular immune response elements (CIREs) in the amyloid-ß protein precursor (AßPP) mouse model. Bi-monthly intramuscular administration, beginning at either 4 or 6 months, and examined at 7.5 through 16 months, with plasmids encoding Interleukin (IL)-10, IL-4, TGF-ß polynucleotides, or a combination thereof, into AßPP mice improved spatial memory performance. This work demonstrates an efficient gene therapy strategy to downregulate neuroinflammation, and possibly prevent or delay cognitive decline in AD.

Synaptic Mitochondria: An Early Target of Amyloid-ß and Tau in Alzheimer's Disease.

Alzheimer's disease (AD) is characterized by cognitive impairment and the presence of neurofibrillary tangles and senile plaques in the brain. Neurofibrillary tangles are composed of hyperphosphorylated tau, while senile plaques are formed by amyloid-ß (Aß) peptide. The amyloid hypothesis proposes that Aß accumulation is primarily responsible for the neurotoxicity in AD. Multiple Aß-mediated toxicity mechanisms have been proposed including mitochondrial dysfunction. However, it is unclear if it precedes Aß accumulation or if is a consequence of it. Aß promotes mitochondrial failure. However, amyloid ß precursor protein (AßPP) could be cleaved in the mitochondria producing Aß peptide. Mitochondrial-produced Aß could interact with newly formed ones or with Aß that enter the mitochondria, which may induce its oligomerization and contribute to further mitochondrial alterations, resulting in a vicious cycle. Another explanation for AD is the tau hypothesis, in which modified tau trigger toxic effects in neurons. Tau induces mitochondrial dysfunction by indirect and apparently by direct mechanisms. In neurons mitochondria are classified as non-synaptic or synaptic according to their localization, where synaptic mitochondrial function is fundamental supporting neurotransmission and hippocampal memory formation. Here, we focus on synaptic mitochondria as a primary target for Aß toxicity and/or formation, generating toxicity at the synapse and contributing to synaptic and memory impairment in AD. We also hypothesize that phospho-tau accumulates in mitochondria and triggers dysfunction. Finally, we discuss that synaptic mitochondrial dysfunction occur in aging and correlates with age-related memory loss. Therefore, synaptic mitochondrial dysfunction could be a predisposing factor for AD or an early marker of its onset.