Exosomes: A missing link between chronic systemic inflammation and Alzheimer's disease?

Exosomes are potent mediators of physiological and pathological processes. In Alzheimer's disease and inflammatory disorders, due to exosomes' distinctive ability to cross the blood-brain barrier, a bidirectional communication between the periphery and the central nervous system exists. Since exosomes can carry various biochemical molecules, this review investigates the role of exosomes as possible mediators between chronic systemic inflammatory diseases and Alzheimer's disease. Exosomes carry pro-inflammatory molecules generated in the periphery, travel to the central nervous system, and target glial and neuronal cells. Microglia and astrocytes then become activated, initiating chronic neuroinflammation. As the aging brain is more susceptible to such changes, this state of neuroinflammation can stimulate neuropathologies, impair amyloid-beta clearance capabilities, and generate dysregulated microRNAs that alter the expression of genes critical in Alzheimer's disease pathology. These processes, individually and collectively, become significant risk factors for the development of Alzheimer's disease.

Two boron-containing compounds affect the cellular viability of SH-SY5Y cells in an in vitro amyloid-beta toxicity model.

Boron is a naturally occurring trace element found in organic and inorganic complexes. Boron-containing compounds are required for living organisms for diverse metabolic functions, including nitrogen fixation in microorganisms, cell wall stability in plants, and bone and carbohydrate metabolism in animals. The number of studies about the effect of boron in biological model systems is very limited; so far, there has been no study on the correlation between boron and amyloid-beta toxicity. Here, we investigated the possible effects of 2 boron-containing compounds-sodium borate decahydrate and boric acid-against amyloid-beta toxicity. In our in vitro amyloid-beta toxicity model, we showed that these 2 compounds increase the survival of the SH-SY5Y cells. Furthermore, boron in these 2 forms increases the expression of Sirt1, which has protective functions against cellular stress. The compounds also change the expressions of GSK-3α/ß; by doing so, boron may contribute to the stimulation of intracellular prosurvival pathways. This is the first experimental study indicating the prosurvival effect of boron in an amyloid-beta toxicity model.

Melatonin affects the release of exosomes and tau-content in in vitro amyloid-beta toxicity model.

BACKGROUND: Recent studies have been revealed that oxidative damage is the main cause of aging and age-related neurodegenerative diseases like Alzheimer's disease (AD). Melatonin is secreted from the pineal gland and its secretion has been found to be altered in AD. In the last decade the role of exosomes in spreading toxic proteins and inducing the propagation of diseases like AD has been discussed. However, it is not known how melatonin affects the amount of exosomes released from the cells and the content of the exosomes. OBJECTIVE: Herein, we investigated the possible role of melatonin treatment in the releasing of exosomes and exosomal tau content in an in vitro Aß toxicity model. METHOD: SH-SY5Y cell line was used. The optimum concentration of Aß was determined by cell viability and cell proliferation tests. Melatonin (100 µM) was applied before and after Aß application. Total exosomes isolated from cell culture media were immunoprecipitated. The amount of released exosomes and their tau content were analyzed by Western blots. RESULTS: Our data demonstrated for the first time that melatonin treatment clearly affected the amount of released exosomes. It would decrease the amyloid beta load and toxicity by inhibiting exosome release. We also demonstated that melatonin also affected the level of tau carried by exosomes depending on whether melatonin was applied before or after Aß application. CONCLUSION: It is considered that the effect of melatonin in the release of exosomes and exosomal tau content would contribute the development of therapeutic strategies in AD and related disorders.

Minocycline Increases in-vitro Cortical Neuronal Cell Survival after Laser Induced Axotomy.

BACKGROUND: Antibiotic therapies targeting multiple regenerative mechanisms have the potential for neuroprotective effects, but the diversity of experimental strategies and analyses of non-standardised therapeutic trials are challenging. In this respect, there are no cases of successful clinical application of such candidate molecules when it comes to human patients. METHODS: After 24 hours of culturing, three different minocycline (Sigma-Aldrich, M9511, Germany) concentrations (1 µM, 10 µM and 100 µM) were added to the primary cortical neurons 15 minutes before laser axotomy procedure in order to observe protective effect of minocycline in these dosages. RESULTS: Here, we have shown that minocycline exerted a significant neuroprotective effect at 1 and 100µM doses. Beyond confirming the neuroprotective effect of minocycline in a more standardised and advanced in-vitro trauma model, our findings could have important implications for future studies that concentrate on the translational block between animal and human studies. CONCLUSION: Such sophisticated approaches might also help to conquer the influence of humanmade variabilities in critical experimental injury models. To the best of our knowledge, this is the first study showing that minocycline increases in-vitro neuronal cell survival after laser-axotomy.

The Association between HbA1c Levels, Olfactory Memory and Cognition in Normal, Pre-Diabetic and Diabetic Persons.

BACKGROUND AND AIM: Recent data have shown that olfactory dysfunction is strongly related to Alzheimer's Disease (AD) that is often preceded by olfactory deficits suggesting that olfactory dysfunction might represent an early indicator of future cognitive in prediabetes. METHODS: We have applied to a group of normal (n=15), prediabetic (n=16) and type 2 diabetic outpatients (n=15) olfactory testing, 1.5-T MRI scanner and detailed cognitive evaluation including the standard Mini-Mental State Examination (MMSE) form, Short Blessed Test (SBT), Letter Fluency Test (LFT) and the category fluency test with animal, Fruit and Vegetable Naming (CFT). RESULTS: We have shown that Odour Threshold (OT), Discrimination (OD), and Identification (OI) scores and most cognitive test results were significantly different in the prediabetes and diabetes group compared to those in the control group. OD and OT were significantly different between the prediabetes and diabetes group, although the cognitive test results were only significantly different in the prediabetes and diabetes group compared to those in the control group. In evaluating the association between OI, OT, OD scores and specific cognitive tests, we have found, that impaired olfactory identification was the only parameter that correlated significantly with the SBT both in the pre-diabetes and diabetes group. Although spot glucose values were only correlated with OT, HbA1c levels were correlated with OT, OD, and OI, as well as results of the letter fluency test suggesting that HbA1c levels rather than the spot glucose values play a critical role in specific cognitive dysfunction. CONCLUSION: To the best of our knowledge, this is the first prospective study to demonstrate a strong association between olfactory dysfunction and specific memory impairment in a population with prediabetes and diabetes suggesting that impaired olfactory identification might play an important role as a specific predictor of memory decline.

Squalene attenuates the oxidative stress and activates AKT/mTOR pathway against cisplatin-induced kidney damage in mice.

The clinical use of cisplatin, which is a first-line anticancer agent, is highly restricted due to its adverse effects on kidneys that lead to nephrotoxicity. Therefore, some potential reno-protective substances have been used in combination with cisplatin to cope with nephrotoxicity. Due to its high antitumor activity and oxygen-carrying capacity, we investigated the molecular effects of squalene against cisplatin-induced oxidative stress and kidney damage in mice. Single dose of cisplatin (7 mg/kg) was given to male Balb/c mice. Squalene (100 mg/kg/day) was administered orogastrically to mice for 10 days. Following sacrification, molecular alterations were investigated as analysis of the levels of oxidative stress index (OSI), inflammatory cytokines and cell survival-related proteins in addition to histopathological examinations in mice kidney tissue. The level OSI and Interferon-gamma (IFN-γ) decreased in the cisplatin and squalene cotreated mice compared to cisplatin-treated mice. Squalene treatment also increased the activation of protein kinase B (AKT). Furthermore, cisplatin-induced inactivation of mammalian target of rapamycin (mTOR) and histopathological damages were reversed by squalene. It may be suggested that squalene ameliorated the cisplatin-induced histopathological damages in the kidney through activation of AKT/mTOR signaling pathway by regulating the balance of the redox system due to its antioxidative effect.

Role of erythropoietin and its receptor in the development of endometriosis in rats

Objective: Besides its hematopoietic function, erythropoietin (EPO) may protect tissues from degenerative disorders. As such, EPO and its receptors were revealed in nonhematopoietic cells, including stromal and endometrial epithelial cells. However, the role of EPO in endometrial disorders is still unknown. Here, we aimed to examine the role of EPO and its receptor activation in the development of endometriosis in rats. Material and Methods: Animals were treated with EPO, darbepoietin (the synthetic form of EPO) or EPO's receptor activator, methoxy polyethylene glycol-epoetin beta (MIRCERA), after development of endometriosis. Endometriosis was induced by estrogen-administration following surgical attachment of endometrial surface on the inner abdominal wall. Treatments were started 3 weeks after induction of endometriosis and continued for the following 3 weeks. For the analysis of recurrence of endometriosis, additional analyses were conducted 3 weeks after cessation of treatments. Results: As compared with vehicle-treated animals, lesion size was reduced significantly and recurrence of endometriosis was not observed in all treatment groups. Histopathologic examination revealed that EPO and darbepoietin were more effective than MIRCERA- and vehicle-treated animals. Conclusion: Here we provide evidence that EPO is a promising candidate for the treatment of endometriosis. Our histopathologic results in particular indicate that EPO is more effective than its receptor activator MIRCERA in the development endometriosis.

Neonatal Neurodegeneration in Alzheimer's Disease Transgenic Mouse Model.

Alzheimer's disease (AD) is a progressive disorder characterized by a variety of molecular pathologies causing cortical dementia with a prominent memory deficit. Formation of the pathology, which begins decades before the diagnosis of the disease, is highly correlated with the clinical symptoms. Several proteomics studies were performed using animal models to monitor the alterations of the brain tissue proteome at different stages of AD. However, proteome changes in the brain regions of newborn transgenic mouse model have not been investigated yet. To this end, we analyzed protein expression alterations in cortex, hippocampus and cerebellum of transgenic mice carrying five familial AD mutations (5XFAD) at neonatal day-1. Our results indicate a remarkable difference in protein expression profile of newborn 5XFAD brain with region specific variations. Additionally, the proteins, which show similar expression alteration pattern in postmortem human AD brains, were determined. Bioinformatics analysis showed that the molecular alterations were mostly related to the cell morphology, cellular assembly and organization, and neuroinflammation. Moreover, morphological analysis revealed that there is an increase in neurite number of 5XFAD mouse neurons in vitro. We suggest that, molecular alterations in the AD brain exist even at birth, and perhaps the disease is silenced until older ages when the brain becomes vulnerable.

N-acetylcysteine suppresses colistimethate sodium-induced nephrotoxicity via activation of SOD2, eNOS, and MMP3 protein expressions.

OBJECTIVE: To investigate the molecular mechanisms of colistimethate sodium-induced nephrotoxicity and the protective effect of N-acetylcysteine (NAC) against nephrotoxicity. METHODS: Twenty-eight Wistar rats were divided into four groups comprised of control, colistin, NAC, and colistin-NAC co-treatment, respectively. Serum creatinine and urine N-acetyl-ß-d-glucosaminidase (NAG) levels were measured at different time intervals. Histological changes, apoptosis, total oxidant and antioxidant status, and the expression levels of endothelial nitric oxide synthase (eNOS), superoxide dismutase 2 (SOD2), and matrix metalloproteinase 3 (MMP3) were evaluated in renal tissue. RESULTS: In the colistin group, post-treatment creatinine levels were higher than pretreatment levels (p = .001). There was a significant increase in urine NAG level following colistin treatment on day 10, compared to the baseline value and the first day of treatment (p = .001 and .0001, respectively). Urine NAG levels were higher in the colistin group on the 10th day of treatment than in the other groups (p < .01). Colistin treatment increased the apoptosis index and renal histological damage score (RHDS) significantly and these changes were reversed in NAC co-treatment (RHSD and apoptosis index were 45 and 0 for sterile saline group, 29 and 2 for NAC group, 122 and 7 for colistin group, and 66 and 2 for colistin + NAC group). We observed no difference between groups regarding total antioxidant and total oxidant status in the kidneys. The expression levels of eNOS, SOD2, and MMP3 decreased significantly in the kidneys of colistin-treated rats; these changes were reversed in the kidneys of NAC co-treated rats. CONCLUSIONS: N-acetylcysteine prevented colistin-induced nephrotoxicity through activation of expression levels of SOD2, eNOS, and MMP3.

Early Stage Alterations in CA1 Extracellular Region Proteins Indicate Dysregulation of IL6 and Iron Homeostasis in the 5XFAD Alzheimer's Disease Mouse Model.

In recent years, an increasing number of research papers revealed that the compositional and volumetric alterations in the extracellular matrix are the consequences of aging and may be related to Alzheimer's disease (AD). In this study, we aimed to demonstrate the alterations in hippocampal extracellular fluid proteins in vivo using the 5XFAD mouse model. Samples were obtained from hippocampi of 5XFAD mice (n = 6) and their non-transgenic littermates by intracerebral push-pull perfusion technique at 3 months of age, representing the pre-pathological stage of the AD. Proteins in the hippocampal perfusates were analyzed by Ultra Performance Liquid Chromatography-Electrospray Ionization Quadrupole Time-of-Flight Mass Spectrometry (UPLC-ESI-qTOF-MS/MS). 178 proteins were identified and 19 proteins of them were found to be statistically significantly altered (p≤0.05, fold change ≥40%, unique peptide count ≥3) in the hippocampal CA1 extracellular fluid of the 5XFAD mouse model. Ingenuity pathway analysis of the protein expression results identified IL6 as an upstream regulator. The upregulation of IL6 was validated by immunohistochemical staining of the hippocampus and cortex of the 5XFAD mice prior to Aß plaque formation. Furthermore, the iron level in the hippocampus was measured by inductively coupled plasma-mass spectrometry as IL6 is mentioned in several studies to take part in iron homeostasis and inflammation and found to be increased in 5XFAD mice hippocampus. Alterations in extracellular matrix proteins in addition to increasing amount of hippocampal IL6 and iron in the early stages of AD may reveal inflammation-mediated iron dyshomeostasis in the early stages of neurodegeneration.

Therapeutic role of rifampicin in Alzheimer's disease.

Rifampicin exerts significant brain protective functions in multiple experimental models. Here we summarize the underlying mechanisms of the neuroprotective and pro-cognitive effects of rifampicin that are mediated by its anti-inflammatory, anti-tau, anti-amyloid, and cholinergic effects. Beyond suggesting that rifampicin shows strong brain protective effects in preclinical models of Alzheimer's disease, we also provide substantial clinical evidence for the neuroprotective and pro-cognitive effects of rifampicin. Future neuroimaging studies combined with clinical assessment scores are the following steps to be taken in this field of research.

Dose-dependent neuroprotective effect of enoxaparin on cold-induced traumatic brain injury.

Recent evidence exists that enoxaparin can reduce brain injury because of its anticoagulant activity. To investigate the potential therapeutic effect of enoxaparin on cold-induced traumatic brain injury, at 20 minutes after modeling, male BALB/c mouse models of cold-induced traumatic brain injury were intraperitoneally administered 3 and 10 mg/kg enoxaparin or isotonic saline solution. Twenty-four hours later, enoxaparin at 10 mg/kg greatly reduced infarct volume, decreased cell apoptosis in the cortex and obviously increased serum level of total antioxidant status. By contrast, administration of enoxaparin at 3 mg/kg did not lead to these changes. These findings suggest that enoxaparin exhibits neuroprotective effect on cold-induced traumatic brain injury in a dose-dependent manner.

Altered expression of micro-RNA 199a and increased levels of cardiac SIRT1 protein are associated with the occurrence of atrial fibrillation after coronary artery bypass graft surgery.

BACKGROUND: Postoperative atrial fibrillation (POAF) is a potentially life-threatening complication after coronary artery bypass graft (CABG) surgery. The expression of the cardioprotective SIRT1 protein with its antioxidant activity is increased in cardiac tissue of patients suffering from POAF. So far, information is lacking about the relationship between SIRT1 regulating micro RNAs (miRs), SIRT1 protein and the occurrence of POAF. METHODS: A total of 63 patients undergoing CABG were recruited, and biopsies were obtained from the right atrial appendage during cannulation. Postoperative, all patients were rhythm-monitored until discharge and randomized to POAF (n=20) or sinus rhythm (n=43). The expression of the micro RNAs miR-199a and miR-195 was quantified by real-time PCR. SIRT1 protein was detected by western blot analysis. RESULTS: The relative expression of miR-199a in the POAF group was significantly decreased compared to the control group (0.77±0.27 vs. 1.11±0.69, P=.022) Accordingly, SIRT 1 protein was significantly induced in tissue probes of patients with POAF (P<.001). CONCLUSION: Altered expression of the SIRT1 protein regulating miR-199a in human atrial tissue was found to be related to the occurrence of POAF, indicating its usefulness as a biomarker for cardiac surgery management.

The central theme of Parkinson's disease: α-synuclein.

Parkinson's disease (PD) is the second most common neurodegenerative disorder, defined by the presence of resting tremor, muscular rigidity, bradykinesia, and postural instability. PD is characterized by the progressive loss of dopaminergic neurons within the substantia nigra pars compacta of the midbrain. The neuropathological hallmark of the disease is the presence of intracytoplasmic inclusions, called Lewy bodies (LBs) and Lewy neurites (LNs), containing α-synuclein, a small protein which is widely expressed in the brain. The α-synuclein gene, SNCA, is located on chromosome 4q22.1; SNCA-linked PD shows an autosomal dominant inheritance pattern with a relatively early onset age, and it usually progresses rapidly. Three missense mutations, A53T, A30P, and E46K, in addition to gene multiplications of the SNCA have been described so far. Although it is clear that LBs and LNs contain mainly the α-synuclein protein, the mechanism(s) which leads α-synuclein to accumulate needs to be elucidated. The primary question in the molecular pathology of PD is how wild-type α-synuclein aggregates in PD, and which interacting partner(s) plays role(s) in the aggregation process. It is known that dopamine synthesis is a stressfull event, and α-synuclein expression somehow affects the dopamine synthesis. The aberrant interactions of α-synuclein with the proteins in the dopamine synthesis pathway may cause disturbances in cellular mechanisms. The normal physiological folding state of α-synuclein is also important for the understanding of pathological aggregates. Recent studies on the α-synuclein protein and genome-wide association studies of the α-synuclein gene show that PD has a strong genetic component, and both familial and idiopathic PD have a common denominator, α-synuclein, at the molecular level. It is clear that the disease process in Parkinson's disease, as in other neurodegenerative disorders, is very complicated; there can be several different molecular pathways which are responsible for diverse and possibly also unrelated functions inside the neuron, playing roles in PD pathogenesis.

Are alsin and spartin novel interaction partners?

Mutations in ALS2 gene/alsin are associated with recessive forms of motor neuron disorders including Juvenile Amyotrophic Lateral Sclerosis (JALS), Infantile-onset Ascending Hereditary Spastic Paraplegia (IAHSP) and Juvenile Primary Lateral Sclerosis (JPLS). In this study, we show that alsin and another MND-linked protein, spartin are related to each other both at mRNA and protein levels in Neuro2a cells. We observed significant alterations in spartin expression in alsin knock-down conditions. We further found that both proteins colocalize in N2a cells and spartin isoform-a precipitates with alsin in the same protein complex. In the light of these results we suggest that alsin and spartin may interact each other physically.

Alfa-class prefoldin protein UXT is a novel interacting partner of Amyotrophic Lateral Sclerosis 2 (Als2) protein.

Mutations in Als2 gene cause several autosomal recessive forms of motor neuron diseases including Juvenile Amyotrophic Lateral Sclerosis (JALS), Juvenile Primary Lateral Sclerosis (PLSJ) and Infantile-onset Ascending Hereditary Spastic Paralysis (IAHSP). To find novel protein-protein interactions of Als2 protein we performed a yeast two hybrid screen and fished out the Ubiquitously Expressed Transcript (UXT) protein. UXT is a novel gene encoding for an α-class prefoldin type chaperone which acts as a co-activator for various transcriptional factors such as Nf-κB and AR. The interaction between Als2 and UXT was confirmed by co-immunoprecipitation. Co-localization between endogenous Als2 and UXT was mainly found in the cytoplasm of neuronal Neuro2a cells with immunofluorescence microscopy. Cell cycle arrest of Neuro2a cells showed that Als2 and Uxt transcriptional levels are synchronously changing. Our results suggest that Als2 is a binding partner of Uxt and Als2/Uxt interaction could be important for the activation of Nf-κB pathway. These results provides basis for future research to investigate the role of Nf-κB pathway in the development of motor neuron diseases.

Mitochondrial pathology in muscle of a patient with a novel parkin mutation.

Mutations of the parkin gene on chromosome 6 cause early-onset parkinsonism. Myopathy has not been reported to be a feature of this condition. Here we report the muscle biopsy findings of a 53-year-old man with a novel parkin gene mutation (IVS-9-1 deletion). His symptoms were characterized by typical early-onset, dopa-responsive, and slowly progressive parkinsonism. Parkin gene analysis revealed a homozygous IVS-9-1 deletion in the proband and his sibling. The unusual feature was hypertrophy of bilateral thigh muscles in the proband. Muscle biopsy from the biceps brachii muscle showed abundant cytochrome oxidase (COX) (-) fibers. This is the first report on the coexistence of a myopathy with COX deficiency with parkin disease and may shed light on the function of parkin in muscle.