Antiretroviral Drugs Promote Amyloidogenesis by De-Acidifying Endolysosomes.

Antiretroviral therapeutics (ART) have effectively increased the long-term survival of HIV-1 infected individuals. However, the prevalence of HIV-1 associated neurocognitive disorders (HAND) has increased and so too have clinical manifestations and pathological features of Alzheimer's disease (AD) in people living with HIV-1/AIDS. Although underlying mechanisms are not clear, chronic exposure to ART drugs has been implicated in the development of AD-like symptoms and pathology. ART drugs are categorized according to their mechanism of action in controlling HIV-1 levels. All ART drugs are organic compounds that can be classified as being either weak acids or weak bases, and these physicochemical properties may be of central importance to ART drug-induced AD-like pathology because weak bases accumulate in endolysosomes, weak bases can de-acidify endolysosomes where amyloidogenesis occurs, and endolysosome de-acidification increases amyloid beta (Aß) protein production and decreases Aß degradation. Here, we investigated the effects of ART drugs on endolysosome pH and Aß levels in rat primary cultured neurons. ART drugs that de-acidified endolysosomes increased Aß levels, whereas those that acidified endolysosomes decreased Aß levels. Acidification of endolysosomes with the mucolipin transient receptor potential (TRPML) channel agonist ML-SA1 blocked ART drug-induced increases in Aß levels. Further, ART drug-induced endolysosome de-acidification increased endolysosome sizes; effects that were blocked by ML-SA1-induced endolysosome acidification. These results suggest that ART drug-induced endolysosome de-acidification plays an important role in ART drug-induced amyloidogenesis and that endolysosome acidification might attenuate AD-like pathology in HIV-1 positive people taking ART drugs that de-acidify endolysosomes. Graphical Abstract.

Calcifying Epithelial Odontogenic (Pindborg) Tumor in a Child: A Case Report and Literature Review.

Calcifying epithelial odontogenic tumor (CEOT) is a rare neoplasm, which accounts for < 1% of all odontogenic tumors. CEOT occurs more frequently in adults with a peak incidence in the 5th decade of life and is extremely rare in the pediatric population. We present a case of a 13-year-old girl who was found to have a mandibular CEOT. We summarize the radiological features, pathological findings, clinical management and literature review focusing on this entity in children.

Role of endolysosomes and pH in the pathogenesis and treatment of glioblastoma.

BACKGROUND: Glioblastoma multiforme (GBM) is a Grade IV astrocytoma with an aggressive disease course and a uniformly poor prognosis. Pathologically, GBM is characterized by rapid development of primary tumors, diffuse infiltration into the brain parenchyma, and robust angiogenesis. The treatment options that are limited and largely ineffective include a combination of surgical resection, radiotherapy, and chemotherapy with the alkylating agent temozolomide. RECENT FINDINGS: Similar to many other forms of cancer, the extracellular environment near GBM tumors is acidified. Extracellular acidosis is particularly relevant to tumorgenesis and the concept of tumor cell dormancy because of findings that decreased pH reduces proliferation, increases resistance to apoptosis and autophagy, promotes tumor cell invasion, increases angiogenesis, obscures immune surveillance, and promotes resistance to drug and radio-treatment. Factors known to participate in the acidification process are nutrient starvation, oxidative stress, hypoxia and high levels of anaerobic glycolysis that lead to increases in lactate. Also involved are endosomes and lysosomes (hereafter termed endolysosomes), acidic organelles with highly regulated stores of hydrogen (H+) ions. Endolysosomes contain more than 60 hydrolases as well as about 50 proteins that are known to affect the number, sizes and distribution patterns of these organelles within cells. Recently, vacuolar ATPase (v-ATPase), the main proton pump that is responsible for maintaining the acidic environment in endolysosomes, was identified as a novel therapeutic target for glioblastoma. CONCLUSIONS: Thus, a greater understanding of the role of endolysosomes in regulating cellular and extracellular acidity could result in a better elucidation of GBM pathogenesis and new therapeutic strategies.

Acidifying Endolysosomes Prevented Low-Density Lipoprotein-Induced Amyloidogenesis.

Cholesterol dyshomeostasis has been linked to the pathogenesis of sporadic Alzheimer's disease (AD). In furthering the understanding of mechanisms by which increased levels of circulating cholesterol augments the risk of developing sporadic AD, others and we have reported that low-density lipoprotein (LDL) enters brain parenchyma by disrupting the blood-brain barrier and that endolysosome de-acidification plays a role in LDL-induced amyloidogenesis in neurons. Here, we tested the hypothesis that endolysosome de-acidification was central to amyloid-ß (Aß) generation and that acidifying endolysosomes protects against LDL-induced increases in Aß levels in neurons. We demonstrated that LDL, but not HDL, de-acidified endolysosomes and increased intraneuronal and secreted levels of Aß. ML-SA1, an agonist of endolysosome-resident TRPML1 channels, acidified endolysosomes, and TRPML1 knockdown attenuated ML-SA1-induced endolysosome acidification. ML-SA1 blocked LDL-induced increases in intraneuronal and secreted levels of Aß as well as Aß accumulation in endolysosomes, prevented BACE1 accumulation in endolysosomes, and decreased BACE1 activity levels. LDL downregulated TRPML1 protein levels, and TRPML1 knockdown worsens LDL-induced increases in Aß. Our findings suggest that endolysosome acidification by activating TRPML1 may represent a protective strategy against sporadic AD.

Coexisting tubular adenoma with a neuroendocrine carcinoma of colon allowing early surgical intervention and implicating a shared stem cell origin.

High-grade colonic neuroendocrine carcinomas (NECs) are uncommon but extremely aggressive. Their co-existence with tubular adenoma (TA) has rarely been reported. We present a 68-year-old man who was found on routine colonoscopy to have multiple colorectal TAs and an ulcerated lesion in the ascending colon. Microscopically, a poorly-differentiated invasive carcinoma juxtaposed with a TA was identified. Differential diagnosis included a poorly-differentiated adenocarcinoma, medullary carcinoma, high-grade NEC and lymphoma. The immunohistochemical profile showed positive staining for keratins, synaptophysin and chromogranin but negative for LCA, CDX2, CK7, CK20, TTF-1 and PSA, supporting the NEC diagnosis. Upon subsequent laparoscopic right hemicolectomy, the tumor was identified as a 3.0 cm umbilicated and ulcerated mass with an adjacent TA. Both TA and NEC showed positive staining for ß-catenin indicating a shared colonic origin. The mitotic counts (77/10 high power fields) and a high proliferation rate (75% by Ki-67) corroborated a high-grade stratification. Mutational analysis indicated a wild-type BRAF and KRAS with mismatch repair proficiency. The AJCC (7th edition) pathologic stage is pT3, pN0, pMx. The patient received adjuvant chemotherapy with cisplatin/etoposides for three cycles and will be followed up for a year to detect recurrence. In conclusion, the co-existence of TA with high grade-NEC in our case allowed early identification and intervention of the otherwise asymptomatic but aggressive tumor. In addition, the finding of a high-grade NEC within a large TA in this case suggests a link between the two lesions and could represent a shared stem cell origin.

Caffeine Blocks HIV-1 Tat-Induced Amyloid Beta Production and Tau Phosphorylation.

The increased life expectancy of people living with HIV-1 who are taking effective anti-retroviral therapeutics is now accompanied by increased Alzheimer's disease (AD)-like neurocognitive problems and neuropathological features such as increased levels of amyloid beta (Aß) and phosphorylated tau proteins. Others and we have shown that HIV-1 Tat promotes the development of AD-like pathology. Indeed, HIV-1 Tat once endocytosed into neurons can alter morphological features and functions of endolysosomes as well as increase Aß generation. Caffeine has been shown to have protective actions against AD and based on our recent findings that caffeine can inhibit endocytosis in neurons and can prevent neuronal Aß generation, we tested the hypothesis that caffeine blocks HIV-1 Tat-induced Aß generation and tau phosphorylation. In SH-SY5Y cells over-expressing wild-type amyloid beta precursor protein (AßPP), we demonstrated that HIV-1 Tat significantly increased secreted levels and intracellular levels of Aß as well as cellular protein levels of phosphorylated tau. Caffeine significantly decreased levels of secreted and cellular levels of Aß, and significantly blocked HIV-1 Tat-induced increases in secreted and cellular levels of Aß. Caffeine also blocked HIV-1 Tat-induced increases in cellular levels of phosphorylated tau. Furthermore, caffeine blocked HIV-1 Tat-induced endolysosome dysfunction as indicated by decreased protein levels of vacuolar-ATPase and increased protein levels of cathepsin D. These results further implicate endolysosome dysfunction in the pathogenesis of AD and HAND, and by virtue of its ability to prevent and/or block neuropathological features associated with AD and HAND caffeine might find use as an effective adjunctive therapeutic agent.

Caffeine, Through Adenosine A3 Receptor-Mediated Actions, Suppresses Amyloid-ß Protein Precursor Internalization and Amyloid-ß Generation.

Intraneuronal accumulation and extracellular deposition of amyloid-ß (Aß) protein continues to be implicated in the pathogenesis of Alzheimer's disease (AD), be it familial in origin or sporadic in nature. Aß is generated intracellularly following endocytosis of amyloid-ß protein precursor (AßPP), and, consequently, factors that suppress AßPP internalization may decrease amyloidogenic processing of AßPP. Here we tested the hypothesis that caffeine decreases Aß generation by suppressing AßPP internalization in primary cultured neurons. Caffeine concentration-dependently blocked low-density lipoprotein (LDL) cholesterol internalization and a specific adenosine A3 receptor (A3R) antagonist as well as siRNA knockdown of A3Rs mimicked the effects of caffeine on neuronal internalization of LDL cholesterol. Further implicating A3Rs were findings that a specific A3R agonist increased neuronal internalization of LDL cholesterol. In addition, caffeine as well as siRNA knockdown of A3Rs blocked the ability of LDL cholesterol to increase Aß levels. Furthermore, caffeine blocked LDL cholesterol-induced decreases in AßPP protein levels in neuronal plasma membranes, increased surface expression of AßPP on neurons, and the A3R antagonist as well as siRNA knockdown of A3Rs mimicked the effects of caffeine on AßPP surface expression. Moreover, the A3R agonist decreased neuronal surface expression of AßPP. Our findings suggest that caffeine exerts protective effects against amyloidogenic processing of AßPP at least in part by suppressing A3R-mediated internalization of AßPP.

Altered Cholesterol Intracellular Trafficking and the Development of Pathological Hallmarks of Sporadic AD.

Compared to the rare familial early onset Alzheimer's disease (AD) that results from gene mutations in AbPP and presenilin-1, the pathogenesis of sporadic AD is much more complex and is believed to result from complex interactions between nutritional, environmental, epigenetic and genetic factors. Among those factors, the presence APOE4 is still the single strongest genetic risk factor for sporadic AD. However, the exact underlying mechanism whereby apoE4 contributes to the pathogenesis of sporadic AD remains unclear. Here, we discuss how altered cholesterol intracellular trafficking as a result of apoE4 might contribute to the development of pathological hallmarks of AD including brain deposition of amyloid beta (Ab), neurofibrillary tangles, and synaptic dysfunction.

Acetate reduces PGE2 release and modulates phospholipase and cyclooxygenase levels in neuroglia stimulated with lipopolysaccharide.

Acetate supplementation attenuates neuroglial activation, increases histone and non-histone protein acetylation, reduces pro-inflammatory cytokine expression, and increases IL-4 transcription in rat models of neuroinflammation and Lyme's neuroborreliosis. Because eicosanoid signaling is involved in neuroinflammation, we measured the effect acetate treatment had on phospholipase, cyclooxygenase, and prostaglandin E2 (PGE2) levels in BV-2 microglia and primary astrocytes stimulated with lipopolysaccharide (LPS). In BV-2 microglia, we found that LPS increased the phosphorylation-state of cytosolic phospholipase A2 (cPLA2), reduced the levels of phospholipase C (PLC) ß1, and increased the levels of cyclooxygenase (Cox)-1 and -2. Acetate treatment returned PLCß1 and Cox-1 levels to normal, attenuated the increase in Cox-2, but had no effect on cPLA2 phosphorylation. In primary astrocytes, LPS increased the phosphorylation of cPLA2 and increased the levels of Cox-1 and Cox-2. Acetate treatment in these cells reduced secretory PLA2 IIA and PLCß1 levels as compared to LPS-treatment groups, reversed the increase in cPLA2 phosphorylation, and returned Cox-1 levels to normal. Acetate treatment reduced PGE2 release in astrocytes stimulated with LPS to control levels, but did not alter PGE2 levels in BV-2 microglia. The amount of acetylated H3K9 bound to the promoter regions of Cox-1, Cox-2, IL-1ß and NF-κB p65 genes, but not IL-4 in were increased in BV-2 microglia treated with acetate. These data suggest that acetate treatment can disrupt eicosanoid signaling in neuroglia that may, in part, be the result of altering gene expression due chromatin remodeling as a result of increasing H3K9 acetylation.

Modulation of inflammatory cytokines and mitogen-activated protein kinases by acetate in primary astrocytes.

Acetate supplementation attenuates neuroglia activation in a rat model of neuroinflammation by a mechanism associated with an increase in brain acetyl-CoA, an alteration in histone acetylation, and reduction of interleukin (IL)-1ß expression. We propose that reduced astroglial activation occurs by disrupting astrocyte-derived inflammatory signaling and cytokine release. Using primary astroglial cultures, we found that LPS (0-25 ng/ml, 4 h) increased tumor necrosis factor (TNF-α) and IL-1ß in a concentration-dependent manner, which was reduced by treatment with sodium acetate (12 mM). LPS did not alter H3K9 acetylation or IL-6 levels, whereas acetate treatment increased H3K9 acetylation by 2-fold and decreased basal levels of IL-6 by 2-fold. Acetate treatment attenuated the LPS-induced increase in TNF-α mRNA, but did not reverse the mRNA levels of other pro-inflammatory cytokines. By contrast, LPS decreased TGF-ß1 and IL-4 protein and TGF-ß1 mRNA, all of which was reversed with acetate treatment. Further, we found that acetate treatment completely reversed LPS-induced phosphorylation of MAPK p38 and decreased basal levels of phosphorylated extracellular signal-regulated kinases1/2 (ERK1/2) by 2-fold. Acetate treatment also reversed LPS-elevated NF-κB p65, CCAAT/enhancer-binding protein beta protein levels, and reduced basal levels of phosphorylated NF-κB p65 at serine 536. These results suggest that acetate treatment has a net anti-inflammatory effect in LPS-stimulated astrocytes that is largely associated with a disruption in MAPK and NF-κB signaling.

Acetate reduces microglia inflammatory signaling in vitro.

Acetate supplementation increases brain acetyl-CoA and histone acetylation and reduces lipopolysaccharide (LPS)-induced neuroglial activation and interleukin (IL)-1ß expression in vivo. To determine how acetate imparts these properties, we tested the hypothesis that acetate metabolism reduces inflammatory signaling in microglia. To test this, we measured the effect acetate treatment had on cytokine expression, mitogen-activated protein kinase (MAPK) signaling, histone H3 at lysine 9 acetylation, and alterations of nuclear factor-kappa B (NF-κB) in primary and BV-2 cultured microglia. We found that treatment induced H3K9 hyperacetylation and reversed LPS-induced H3K9 hypoacetylation similar to that found in vivo. LPS also increased IL-1ß, IL-6, and tumor necrosis factor-alpha (TNF-α) mRNA and protein, whereas treatment returned the protein to control levels and only partially attenuated IL-6 mRNA. In contrast, treatment increased mRNA levels of transforming growth factor-ß1 (TGF-ß1) and both IL-4 mRNA and protein. LPS increased p38 MAPK and JNK phosphorylation at 4 and 2-4 h, respectively, whereas treatment reduced p38 MAPK and JNK phosphorylation only at 2 h. In addition, treatment reversed the LPS-induced elevation of NF-κB p65 protein and phosphorylation at serine 468 and induced acetylation at lysine 310. These data suggest that acetate metabolism reduces inflammatory signaling and alters histone and non-histone protein acetylation.

Acetate supplementation modulates brain histone acetylation and decreases interleukin-1ß expression in a rat model of neuroinflammation.

BACKGROUND: Long-term acetate supplementation reduces neuroglial activation and cholinergic cell loss in a rat model of lipopolysaccharide-induced neuroinflammation. Additionally, a single dose of glyceryl triacetate, used to induce acetate supplementation, increases histone H3 and H4 acetylation and inhibits histone deacetylase activity and histone deacetylase-2 expression in normal rat brain. Here, we propose that the therapeutic effect of acetate in reducing neuroglial activation is due to a reversal of lipopolysaccharide-induced changes in histone acetylation and pro-inflammatory cytokine expression. METHODS: In this study, we examined the effect of a 28-day-dosing regimen of glyceryl triacetate, to induce acetate supplementation, on brain histone acetylation and interleukin-1ß expression in a rat model of lipopolysaccharide-induced neuroinflammation. The effect was analyzed using Western blot analysis, quantitative real-time polymerase chain reaction and enzymic histone deacetylase and histone acetyltransferase assays. Statistical analysis was performed using one-way analysis of variance, parametric or nonparametric when appropriate, followed by Tukey's or Dunn's post-hoc test, respectively. RESULTS: We found that long-term acetate supplementation increased the proportion of brain histone H3 acetylated at lysine 9 (H3K9), histone H4 acetylated at lysine 8 and histone H4 acetylated at lysine 16. However, unlike a single dose of glyceryl triacetate, long-term treatment increased histone acetyltransferase activity and had no effect on histone deacetylase activity, with variable effects on brain histone deacetylase class I and II expression. In agreement with this hypothesis, neuroinflammation reduced the proportion of brain H3K9 acetylation by 50%, which was effectively reversed with acetate supplementation. Further, in rats subjected to lipopolysaccharide-induced neuroinflammation, the pro-inflammatory cytokine interleukin-1ß protein and mRNA levels were increased by 1.3- and 10-fold, respectively, and acetate supplementation reduced this expression to control levels. CONCLUSION: Based on these results, we conclude that dietary acetate supplementation attenuates neuroglial activation by effectively reducing pro-inflammatory cytokine expression by a mechanism that may involve a distinct site-specific pattern of histone acetylation and histone deacetylase expression in the brain.

Acetate supplementation increases brain histone acetylation and inhibits histone deacetylase activity and expression.

Acetate supplementation increases brain, heart, and liver acetyl-CoA levels and reduces lipopolysaccharide-induced neuroinflammation. Because intracellular acetyl-CoA can be used to alter histone acetylation-state, using Western blot analysis, we measured the temporal effect that acetate supplementation had on brain and liver histone acetylation following a single oral dose of glyceryl triacetate (6 g/kg). In parallel experiments, we measured the effect that acetate supplementation had on histone deacetylase (HDAC) and histone acetyltransferase (HAT) enzymic activities and the expression levels of HDAC class I and II enzymes using Western blot analysis. We found that acetate supplementation increased the acetylation-state of brain histone H4 at lysine 8 at 2 and 4 h, histone H4 at lysine 16 at 4 and 24 h, and histone H3 at lysine 9 at 4 h following treatment. No changes in other forms of brain or liver H3 and H4 acetylation-state were found at any post-treatment times measured. Enzymic HAT and HDAC assays on brain extracts showed that acetate supplementation had no effect on HAT activity, but significantly inhibited by 2-fold HDAC activity at 2 and 4 h post-treatment. Western blot analysis demonstrated that HDAC 2 levels were decreased at 4 h following treatment. Based on these results, we conclude that acetyl-CoA derived from acetate supplementation increases brain histone acetylation-state by reducing HDAC activity and expression.