High frequency repetitive transcranial magnetic stimulation improves cognitive performance parameters in patients with Alzheimer's disease - an exploratory pilot study.

BACKGROUND: Currently available medication for Alzheimer's disease (AD) may slows cognitive decline only transitory, but has failed to bring about long term positive effects. For this slowly progressive neurodegenerative disease so far no disease modifying therapy exists. OBJECTIVE: To find out if non-pharmacologic non-ivasive neuromodulatory repetitive transcranial magnetic stimulation (rTMS) may offer a new alternative or an add on therapeutic strategy against loss of cognitive functions. METHODS: In this exploratory intervention study safety and symptom development before and after frontopolar cortex stimulation (FPC) using intermittent theta burst stimulation (iTBS) at 10 subsequent working days was monitored as add-on treatment in 28 consecutive patients with AD. Out of these, 10 randomly selected patients received sham stimulation as a control. In addition, ​serum concentrations of neurotransmitter precursor amino acids, of immune activation and inflammation markers, of brain derived neurotrophic factor (BDNF) as well as of nitrite were measured. RESULTS: Treatment was well tolerated, no serious adverse effects were observed. Improvement of cognition was detected by an increase of Mini Mental State Examination score (MMSE; p<0.01, paired rank test) and also by an increase in a modified repeat address phrase test, part of the 6-item cognitive ​impairment test (p < 0.01). A trend to an increase in the clock drawing test (CDT; p = 0.08) was also found in the verum treated group. Furtheron, in 10 of the AD patients with additional symptoms of depression treated with iTBS, a significant decrease in the HAMD-7 scale (p <0.01) and a trend to lower serum phenylalanine concentrations (p = 0.08) was seen. No changes of the parameters tested were found in the sham treated patients. CONCLUSION: Our preliminary results may indicate that iTBS is effective in the treatment of AD. Also a slight influence of iTBS on the metabolism of phenylalanine was found after 10 iTBS sessions. An impact of iTBS to influence the enzyme phenylalanine hydroxylase (PAH), as found in previous series of treatment resistant depression, could not be seen in this our first observational trial in 10 AD patients with comorbidity of depression. Longer treatment periods for several weeks in a higher number of AD patients with depression could cause more intense and disease modifying effects visible in different neurotransmitter concentrations important in the pathogenesis of AD.

Lack of association between cortical amyloid deposition and glucose metabolism in early stage Alzheimer´s disease patients.

BACKGROUND: Beta amyloid (Aß) causes synaptic dysfunction leading to neuronal death. It is still controversial if the magnitude of Aß deposition correlates with the degree of cognitive impairment. Diagnostic imaging may lead to a better understanding the role of Aß in development of cognitive deficits. The aim of the present study was to investigate if Aß deposition in the corresponding brain region of early stage Alzheimer´s disease (AD) patients, directly correlates to neuronal dysfunction and cognitive impairment indicated by reduced glucose metabolism. PATIENTS AND METHODS: In 30 patients with a clinical phenotype of AD and amyloid positive brain imaging, 2-[18F] fluoro-2-deoxy-d-glucose (FDG) PET/CT was performed. We extracted the average [18F] flutemetamol (Vizamyl) uptake for each of the 16 regions of interest in both hemispheres and computed the standardized uptake value ratio (SUVR) by dividing the Vimazyl intensities by the mean signal of positive and negative control regions. Data were analysed using the R environment for statistical computing and graphics. RESULTS: Any negative correlation between Aß deposition and glucose metabolism in 32 dementia related and corresponding brain regions in AD patients was not found. None of the correlation coefficient values were statistically significant different from zero based on two-sided p- value. CONCLUSIONS: Regional Aß deposition did not correlate negatively with local glucose metabolism in early stage AD patients. Our findings support the role of Aß as a valid biomarker, but does not permit to conclude that Aß is a direct cause for an aberrant brain glucose metabolism and neuronal dysfunction.

Somatostatin Receptor Subtype Expression in Patients with Acromegaly and Complicated Clinical Course.

Somatostatin analogues are considered to be the first line of treatment in acromegaly. Somatostatin analogues of the first generation mainly target the somatostatin receptor (SSTR) subtype 2 and have been proven efficient in the majority of patients with acromegaly. Pasireotide was the first somatostatin analogue also substantially targeting the SSTR subtype 5. An efficient drug for Cushing's disease tailored to suboptimal-responding patients with acromegaly then became available. We immunohistochemically investigated SSTR subtypes expression in pituitary adenomas from operated acromegaly patients with clinical relapse and a complicated clinical course. Patients received pasireotide in the course of their disease. The predictive value of SSTR subtypes immunhistochemical analysis for the therapeutic response is discussed.

Giant silent corticotrope pituitary adenoma in a patient with complicated clinical course.

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The Immunopathogenesis of Alzheimer's Disease Is Related to the Composition of Gut Microbiota.

The microbiota-gut-brain axis plays an important role in the development of neurodegenerative diseases. Commensal and pathogenic enteric bacteria can influence brain and immune system function by the production of lipopolysaccharides and amyloid. Dysbiosis of the intestinal microbiome induces local and consecutively systemic immune-mediated inflammation. Proinflammatory cytokines then trigger neuroinflammation and finally neurodegeneration. Immune-mediated oxidative stress can lead to a deficiency of vitamins and essential micronutrients. Furthermore, the wrong composition of gut microbiota might impair the intake and metabolization of nutrients. In patients with Alzheimer's disease (AD) significant alterations of the gut microbiota have been demonstrated. Standard Western diet, infections, decreased physical activity and chronic stress impact the composition and diversity of gut microbiota. A higher abundancy of "pro-inflammatory" gut microbiota goes along with enhanced systemic inflammation and neuroinflammatory processes. Thus, AD beginning in the gut is closely related to the imbalance of gut microbiota. Modulation of gut microbiota by Mediterranean diet, probiotics and curcumin can slow down cognitive decline and alter the gut microbiome significantly. A multi-domain intervention approach addressing underlying causes of AD (inflammation, infections, metabolic alterations like insulin resistance and nutrient deficiency, stress) appears very promising to reduce or even reverse cognitive decline by exerting positive effects on the gut microbiota.

Increased Acetylation of Histone H4 at Lysine 12 (H4K12) in Monocytes of Transgenic Alzheimer's Mice and in Human Patients.

BACKGROUND: Alzheimer's disease (AD) is a neurodegenerative disorder characterized by amyloid-ß (Aß) plaque formation, tau pathology, neurodegeneration and inflammatory processes. Monocytes are involved in inflammation in AD and are recruited to the diseased brain. Recently it has been shown that aberrant epigenetic processes including acetylation are associated with the development of AD. The aim of the present study was to examine acetylation of histone H4 at lysine 12 (H4K12) in monocytes in two transgenic AD mouse models (the triple transgenic 3xTg and a model overexpressing amyloid-precursor protein APP with the Swedish-Dutch-Iowa mutations), and to compare with monocytes isolated from human patients with mild cognitive impairment (MCI) and AD. METHODS: Mouse and human monocytes were selectively isolated with a positive (PluriSelect) respectively with a negative selection method (Miltenyi). Histones were extracted and acetylation of H4K12 was analyzed by a quantification fluorometric kit. Moreover, monocyte cytokine release was measured and cell death analyzed by FACS using incorporation of 7-AAD. RESULTS: Our data show a significant increase of monocytic H4K12 acetylation in both transgenic AD mouse models early during development of the plaque deposition in the brain. In line with these data we found significantly elevated acetylation of H4K12 in human patients with MCI but not in patients with AD. Further we observed that the monocytes of AD mice and of AD patients were significantly more vulnerable to cell damage (as seen by 7-AAD incorporation in FACS analysis) and displayed an enhanced release of pro-inflammatory cytokines (MIP2 and TNFα). CONCLUSION: Our findings indicate that epigenetic changes in peripheral monocytes are an early event in AD-pathology. Thus H4K12 acetylation may be considered as a novel biomarker for early changes in AD development.

Platelets in the Alzheimer's disease brain: do they play a role in cerebral amyloid angiopathy?

Alzheimer's disease (AD) is characterized by extracellular beta-amyloid plaques and intracellular tau tangles. AD-related pathology is often accompanied by vascular changes. The predominant vascular lesions in AD are cerebral amyloid angiopathy (CAA) and arteriosclerosis. Platelets circulate along the vessel wall responding immediately to vascular injury. The aim of the present study was to explore the presence and migration of platelets (thrombocytes) to sites of small vascular bleedings and/or to beta-amyloid plaques in the brain. We infused fluorescently labeled red PKH26 mouse platelets into transgenic Alzheimer mice overexpressing APP with Swedish/Dutch/Iowa mutations (APP_SDI) and explored if platelets migrate into the brain. Further we studied whether platelets accumulate in the vicinity of ß-amyloid plaques. Our animal data shows that infused platelets are found in the liver and partly in the lung, while in the brain platelets were visible to a minor degree. In mice, we did not observe a significant association of platelets with beta-amyloid plaques or vessels. In the brain of Alzheimer postmortem patients platelets could be detected by immunohistochemistry for CD41 and CD62P, but the majority was found in vessels with or without beta-amyloid load, and only a few single platelets migrated deeper into the brain. Our findings suggest that platelets do not migrate into the brains of Alzheimer disease but are concentrated in brain vessels.

Effects of ethanol on aggregation, serotonin release, and amyloid precursor protein processing in rat and human platelets.

It is known that oxidative stress leads to amyloid precursor protein (APP) dysregulation in platelets. Ethanol (EtOH) is a vascular risk factor and induces oxidative stress. The aim of the present study was thus to investigate whether EtOH affects APP processing in rat and human platelets. Platelets were exposed to 50 mM EtOH with and without 2 mM calcium-chloride (CaCl2) for 20 or 180 minutes at 37°C. Platelet aggregation, serotonin release and APP isoforms 130 and 106/110 kDa were analyzed. As a control, 100 mM H2O2 was tested in rat platelets. Our data show that EtOH alone did not affect any of the analyzed parameters, whereas CaCl2 significantly increased aggregation of rat and human platelets. In addition, CaCl2 alone enhanced serotonin release in rat platelets. EtOH counteracted CaCl2-induced aggregation and serotonin release. In the presence of CaCl2, EtOH reduced the 130 kDa APP isoform in rat and human platelets. In conclusion, this study shows that in the presence of CaCl2, EtOH affects the platelet function and APP processing in rat and human platelets.

Catestatin-like immunoreactivity in the rat eye.

The aim of the study was to investigate the presence and distribution of the chromogranin A-derived peptide catestatin in the rat eye and trigeminal ganglion by immunofluorescence using an antibody which recognizes not only free catestatin but also larger fragments containing the sequence of catestatin. Western blots were performed in an attempt to characterize the immunoreactivities detected by the catestatin antiserum. Sparse immunoreactive nerve fibers were visualized in the corneal stroma, in the chamber angle, in the sphincter muscle but also in association with the dilator muscle, in the stroma of the ciliary body and processes, but dense in the irideal stroma, around blood vessels at the limbus and in the choroid and in cells of the innermost retina representing amacrine cells as identified by colocalization with substance P. Furthermore, catestatin-immunoreactivity was detected in the trigeminal ganglion in small to medium-sized cells and there were abundant catestatin-positive nerve fibers stained throughout the stroma of the ganglion. Double immunofluorescence of catestatin with substance P revealed colocalization both in cells of the trigeminal ganglion as well as in nerve fibers in the choroid. The immunoreactivities are present obviously as free catestatin and/or small-sized catestatin-containing fragments in the retina and ocular nerves but as large processed fragments as well, weak in the retina and more prominent in remaining ocular tissues, possibly in endothelial cells. This indicates that this peptide is a constituent of sensory neurons innervating the rat eye and the presence in amacrine cells in the retina is typical for neuropeptides. Catestatin is biologically highly active and might be of significance in the pathophysiology of the eye.

Intravenous infusion of nerve growth factor-secreting monocytes supports the survival of cholinergic neurons in the nucleus basalis of Meynert in hypercholesterolemia Brown-Norway rats.

The recruitment of monocytes into the brain has been implicated in Alzheimer's disease and recent studies have indicated that monocytes can reduce amyloid plaque burden. Our previous investigations have shown that hypercholesterolemic rats develop cognitive, cholinergic, and blood-brain barrier dysfunction, but do not develop amyloid plaques. This study was designed to evaluate the effects of repeated intravenous (i.v.) infusion (via the dorsal penile vein) of primary monocytes on cognition, the cholinergic system, and cortical cytokine levels in hypercholesterolemia Brown-Norway rats. In addition, we also transduced the monocytes with nerve growth factor (NGF) to evaluate whether these cells could be used to deliver a neuroprotective agent to the brain. Our results indicate that repeated i.v. infused monocytes migrate into the brains of hypercholesterolemic rats; however, this migration does not translate into marked effects on learning. Animals receiving NGF-loaded monocytes demonstrate slightly improved learning and significantly elevated cholinergic neuron staining compared to treatment with monocytes alone. Furthermore, our data indicate that repeated infusion of monocytes does not lead to elevated cytokine secretion, indicating that no inflammatory response is induced. This study provides an experimental attempt to evaluate the effects of blood-derived primary monocytes in hypercholesterolemia rats.

Cholesterol diet counteracts repeated anesthesia/infusion-induced cognitive deficits in male Brown Norway rats.

A variety of cardiovascular and cerebrovascular diseases are associated with alterations in cholesterol levels and metabolism. Moreover, convincing evidence shows that high cholesterol diet can lead to learning and memory impairments. On the other hand, a significant body of research has also demonstrated that learning is improved by elevated dietary cholesterol. Despite these conflicting findings, it is clear that cholesterol plays an important role in these cognitive properties. However, it remains unclear how this blood-brain barrier (BBB)-impenetrable molecule affects the brain and under what circumstances it provides either detrimental or beneficial effects to learning and memory. The aim of this study was to characterize the effects of 5% cholesterol diet on six-month-old inbred Brown Norway rats. More important, we sought to examine the role that cholesterol can play when repeated anesthesia and intravenous infusion disrupts cognitive function. This present study supports previous work showing that enriched cholesterol diet leads to significant alterations in neuroinflammation and BBB disruption. Following repeated anesthesia and intravenous infusion of saline we observe that animals under normal diet conditions exhibit significant deficiencies in spatial learning and cholinergic neuron populations compared to animals under enriched cholesterol diet, which do not show such deficiencies. These findings indicate that cholesterol diet can protect against or counteract anesthesia/infusion-induced cognitive deficits. Ultimately, these results suggest that cholesterol homeostasis serves an important functional role in the brain and that altering this homeostasis can either exert positive or negative effects on cognitive properties.

Effects of oxidative stress on amyloid precursor protein processing in rat and human platelets.

Alzheimer's disease (AD) is the most common neurodegenerative illness affecting the elderly and is characterized by beta-amyloid (Aß) deposition in the brain (plaques) and in microvessels (Aß-angiopathy). The reasons for Aß deposition are not clear, but an impaired clearance of Aß at the blood-brain barrier may be implicated and oxidative stress possibly plays a major role in this process. Platelets are of particular interest, because they contain high levels of the amyloid precursor protein (APP) and in AD an abnormal expression of platelets APP fragments was found. The aim of the present study was to investigate (1) if oxidative stress induced by hydrogen peroxide (H(2)O(2)) affects APP expression in rat and human platelets and (2) to compare the APP changes with platelets of AD patients. In rat platelets, all three fragments of APP (130-110-106 kilo Dalton, kDa) were found. H(2)O(2) (10 mM, 20 minutes) significantly reduced all three fragments in rat platelets, did not affect CD62P-staining and slightly increased the size of actin as seen in the Western blot. The effect was not seen at 1 mM H(2)O(2) and was counteracted by glutathione. Immunohistochemistry for CD62P, CD61, APP and Annexin-V was used to verify the changes at the cellular level. In platelets of young volunteers (age = 33 ± 4 years), 10 mM H(2)O(2) markedly reduced the smaller APP 110 and 106 kDa fragments after 20 minutes. Our data show that platelets of AD patients (age = 80 ± 1 years) had a significant reduced 130 kDa fragment compared to controls (age = 70 ± 2 years). In summary, oxidative stress may account for a dysfunctional processing of APP in rat and human control platelets and possibly in AD patients.

Chronic vascular risk factors (cholesterol, homocysteine, ethanol) impair spatial memory, decline cholinergic neurons and induce blood-brain barrier leakage in rats in vivo.

Epidemiological studies show that vascular risk factors (e.g. atherosclerosis, diabetes, homocysteine, hypertension or cholesterol) may play a role in the development of Alzheimer's disease. Animal models may help to discover the role of vascular risk factors on cognition. In the present project we treated male Sprague Dawley rats with a diet containing homocysteine (hyperhomocysteinemia) or cholesterol (hypercholesterolemia) for 5 months or exposed the rats to ethanol (20% in drinking water) or a combination of cholesterol+ethanol (mix) for 12 months. Our experiments show that all 3 treatments (homocysteine, cholesterol, ethanol) declined spatial memory in the 8-arm radial maze, reduced the number of cholinergic neurons and induced blood-brain barrier leakage in the cortex. Rats treated with cholesterol also displayed markedly enhanced inflammation in the cortex. Levels of amyloid precursor protein, beta-amyloid((1-42)), as well as tau and phospho-tau 181 were significantly enhanced in the cortex of cholesterol-fed rats. A combination of ethanol and cholesterol did not further potentiate the effects on spatial memory, cholinergic neurons and blood-brain barrier leakage. The data suggest that chronic mild vascular risk factors over months induce small lesions of the brain capillaries in the cortex, which may contribute to the development of vascular dementia or also Alzheimer's disease.

GE-25-like immunoreactivity in the rat eye.

This study aimed to investigate the presence and distribution of the chromogranin A-derived peptide GE-25 in the rat eye. The molecular form detected by the GE-25 antiserum was evaluated in the rat trigeminal ganglion, retina and remaining tissues of the rat eye by means of Western blots and the distribution pattern of GE-25-like immunoreactivity was studied in the rat eye and rat trigeminal ganglion by immunofluorescence. One single band of approximately 70kDa was stained in the trigeminal ganglion and retina which represents the uncleaved intact chromogranin A indicating that the proteolytic processing of chromogranin A to GE-25 is limited in these tissues. Sparse GE-25-like immunoreactive nerve fibers were visualized in the corneal stroma, at the limbus around blood vessels, in the sphincter and dilator muscle and stroma of the iris, in the stroma of the ciliary body and ciliary processes and in the stroma and around blood vessels in the choroid. This distribution pattern is characteristic for neuropeptides whereas the presence of immunoreactivity in the corneal endothelium and in Müller glia in the retina is atypical. GE-25-like immunoreactivity was found in small to medium-sized ganglion cells in the rat trigeminal ganglion clearly indicating that the nerve fibers in the rat eye are of sensory origin. The colocalization of GE-25-immunoreactivity with SP-immunoreactivity in the rat ciliary body is in agreement with the presumption of the sensory nature of the innervation of the anterior segment of the eye by GE-25.

Ethanol transiently suppresses choline-acetyltransferase in basal nucleus of Meynert slices.

The cholinergic system plays a major role in learning and cognition and cholinergic neurons appear to be particularly vulnerable to ethanol (EtOH) exposure. There are conflicting results if EtOH directly damages cholinergic neurons. Thus, the aims of the present study were (1) to investigate the effect of different EtOH concentrations on cholinergic neurons in organotypic brain slices of the nucleus basalis of Meynert (nbM) and (2) to study if the most potent cholinotrophic substance nerve growth factor (NGF) or inhibitors of mitogen activated kinase (MAPK) p38- and nitric-oxide synthase (NOS)-pathways may counteract any EtOH effect. Two-week old organotypic rat brain slices of the nbM were exposed to 1-100 mM EtOH for 7 days with or without drugs and the number of choline-acetyltransferase (ChAT)-positive neurons was counted. Our data show that EtOH significantly reduced the number of ChAT-positive neurons with the most potent effect at a concentration of 50 mM EtOH (54±5 neurons per slice, p<0.001), compared to control slices (120±13 neurons per slice). Inhibition of MAPK p38 (SB 203580, 10 µM) and NOS (L-thiocitrulline, 10 µM) counteracted the EtOH-induced decline of cholinergic neurons and NGF protected cholinergic neurons against the EtOH-induced effect. Withdrawal of EtOH resulted in a reversal of cholinergic neurons to nearly controls. In conclusion, EtOH caused a transient decline of cholinergic neurons, possibly involving MAPK p38- and NOS-pathways suggesting that EtOH does not induce direct cell death, but causes a transient downregulation of the cholinergic key enzyme, possibly reflecting a form of EtOH-associated plasticity.

Platelets in psychiatric disorders.

Several parallels exist between platelets and the brain, which make them interesting for studying the neurobiology of psychiatric disorders, such as Alzheimer's disease, depression, schizophrenia and anxiety disorders. Platelets store, secrete and process the amyloid precursor protein which is cleaved into the ß-amyloid (Aß) peptides. The accumulation of Aß in brain (plaques) and vessels (Aß-angiopathy) is a major hallmark in AD. Platelets contain high amounts of serotonin and a dysfunction of the serotoninergic system is involved in the development of several behavior disorders, such as depression, anxiety disorders and self aggressive disturbances. Furthermore, platelets are able to take up dopamine and express various dopamine receptors, which make them to an interesting tool to study the underlying mechanisms of schizophrenia. In summary, platelets are an interesting and easily accessible cell type to study changes related to different psychiatric disorders and platelets proteins may be useful as diagnostic biomarkers for some psychiatric disorders.

Matrix metalloproteinase-2 and epidermal growth factor are decreased in platelets of Alzheimer patients.

Alzheimer's disease (AD) is the most prevalent type of dementia. Despite considerable advances in diagnostic accuracy, diagnostic procedures that are easily accessible are still sorely needed. Blood biomarkers are therefore in the focus of research. Platelets contain a high concentration of the amyloid precursor protein (APP), which has been mentioned as a potentially useful diagnostic marker. The aim of the present study was to analyze various cell adhesion molecules (CAMs), cytokines, growth factors, and matrix metalloproteinases (MMPs) in platelets of AD and mild cognitively impaired (MCI) patients as compared to healthy controls. Our data show a significant decrease in the levels of epidermal growth factor (EGF) and of MMP-2 in platelets of AD patients and decreased levels of MMP-2 in MCI. The APP ratio was slightly but not significantly decreased in AD patients, whereas CD40L and serotonin were unchanged. Our findings demonstrate specific changes in AD platelets. Whether these biomarkers can be established as potential early diagnostic biomarkers for AD remains to be established in longitudinal studies.