Arne Wrede

Screening of innate immune receptors in neurodegenerative diseases: A similar pattern

In Alzheimers disease (AD), Parkinsons disease (PD), dementia with Lewy bodies (DLB) and amyotrophic lateral sclerosis (ALS), neuroinflammatory responses are considered to contribute to neuronal injury. Recently, the innate immune receptors, toll-like receptors (TLRs) and the LPS receptor (CD14) have been related to neurodegeneration. In this study, we systematically assessed the expression of most TLRs and CD14 in AD, PD/DLB and ALS using murine models of these diseases and human post-mortem brain tissues. A common upregulation of TLR2 and CD14 was found in all three animal models. While these two receptors could also be detected in AD patient tissues, they were absent from DLB and ALS tissues. This uniform pattern of innate immune response in animal models of neurodegenerative diseases clearly indicates that this response is part of a non-specific neuroinflammatory effector phase rather than a disease-specific event. The less dynamic disease progression in humans and the location (extracellular versus intracellular) of the aggregated proteins deposits might explain the divergent results seen between animal models and human tissues.

Deletion of Cellular Prion Protein Results in Reduced Akt Activation, Enhanced Postischemic Caspase-3 Activation, and Exacerbation of Ischemic Brain Injury

Background and Purpose— The physiological function of cellular prion protein (PrPc) is not yet understood. Recent findings suggest that PrPc may have neuroprotective properties, and its absence increases susceptibility to neuronal injury. The purpose of this study was to elucidate the role of PrPc in ischemic brain injury in vivo. Methods— PrP knockout (Prnp0/0) and Prnp+/+ wild-type (WT) mice were subjected to 60-minute transient or permanent focal cerebral ischemia followed by infarct volume analysis 24 hours after lesion. To identify effects of PrPc deletion on mechanisms regulating ischemic cell death, expression analysis of several proapoptotic and antiapoptotic proteins was performed at 6 and 24 hours after transient ischemia and in nonischemic controls using Western blot or immunohistochemistry. Results— Prnp0/0 mice displayed significantly increased infarct volumes after both transient or permanent ischemia when compared with WT animals (70.2±23 versus 13.3±4 mm3; 119.8±24 versus 86.4±25 mm3). Expression of phospho-Akt (Ser473) was significantly reduced in Prnp0/0 compared with WT animals both early after ischemia and in sham controls. Furthermore, postischemic caspase-3 activation was significantly enhanced in the basal ganglia and the parietal cortex of Prnp0/0 mice. In contrast, expression of total Akt, Bax, and Bcl-2 did not differ between both groups. Conclusions— These results demonstrate that PrPc deletion impairs the antiapoptotic phosphatidylinositol 3-kinase/Akt pathway by resulting in reduced postischemic phospho-Akt expression, followed by enhanced postischemic caspase-3 activation, and aggravated neuronal injury after transient and permanent cerebral ischemia.

β-amyloid is a substrate of autophagy in sporadic inclusion body myositis

Sporadic Inclusion Body Myositis (sIBM) is the most common acquired muscle disease in patients above 50 years of age. Apart from inflammation in the skeletal muscle, overexpression of amyloid precursor protein (APP) and intracellular accumulation of its proteolytic fragment β‐amyloid play a central role in the pathogenesis of sIBM. In neurodegenerative disorders, similar aggregations of aberrant proteins have recently been shown to be susceptible to autophagic degradation. Therefore, we analyzed macroautophagy of APP in human muscle cell lines and sIBM muscle biopsies.

Choroid plexus transcytosis and exosome shuttling deliver folate into brain parenchyma

Loss of folate receptor-α function is associated with cerebral folate transport deficiency and childhood-onset neurodegeneration. To clarify the mechanism of cerebral folate transport at the blood-cerebrospinal fluid barrier, we investigate the transport of 5-methyltetrahydrofolate in polarized cells. Here we identify folate receptor-α-positive intralumenal vesicles within multivesicular bodies and demonstrate the directional cotransport of human folate receptor-α, and labelled folate from the basolateral to the apical membrane in rat choroid plexus cells. Both the apical medium of folate receptor-α-transfected rat choroid plexus cells and human cerebrospinal fluid contain folate receptor-α-positive exosomes. Loss of folate receptor-α-expressing cerebrospinal fluid exosomes correlates with severely reduced 5-methyltetrahydrofolate concentration, corroborating the importance of the folate receptor-α-mediated folate transport in the cerebrospinal fluid. Intraventricular injections of folate receptor-α-positive and -negative exosomes into mouse brains demonstrate folate receptor-α-dependent delivery of exosomes into the brain parenchyma. Our results unravel a new pathway of folate receptor-α-dependent exosome-mediated folate delivery into the brain parenchyma and opens new avenues for cerebral drug targeting.

Camptocormia in idiopathic Parkinson's disease: A focal myopathy of the paravertebral muscles†

The objective of our study was to describe the clinical features of camptocormia, an involuntary, marked flexion of the thoracolumbar spine in idiopathic Parkinsons disease (PD) and to understand its etiology. In a prospective, cross‐sectional study, we examined 15 patients with PD and camptocormia using laboratory parameters, EMG, muscle magnetic resonance imaging, and biopsy of the paravertebral muscles. The clinical data were compared with a matched control group of PD patients without camptocormia, and the biopsies were compared with muscles from age‐matched autopsies. Almost all the patients (median age, 68.0 years; 7 women) with camptocormia suffered from advanced PD. Camptocormia occurred at a median of 9.0 years after the PD diagnosis. Compared with our clinical control group, back pain was more frequent and less dopa‐sensitive in the patients with camptocormia who suffered more often from additional diseases of the back. On EMG, we found mainly a myopathic pattern. The MRI of the paravertebral muscles showed localized changes ranging from edema with contrast enhancement, which are considered to be early signs, to atrophy and/or fatty degeneration, interpreted as late degenerative changes. Early signs were seen mainly during the first year and degenerative changes after 1.5 years. Biopsies revealed consistently myopathic changes and in some cases fatty degeneration. Clinical or electromyographic features favoring dystonia were absent. Camptocormia is a major disabling, non‐fluctuating and levodopa‐resistant complication of advanced PD. The cause of camptocormia in idiopathic PD is a focal myopathy. Our findings suggest that the myopathy has a progressive course, which finally leads to degeneration of the paravertebral muscles.

Transcranial direct current stimulation induces polarity-specific changes of cortical blood perfusion in the rat.

OBJECTIVE Transcranial direct current stimulation (tDCS) induces changes in cortical excitability and improves hand-motor function in chronic stroke. These effects depend on polarity, duration of stimulation and current intensity applied. Towards evaluating the therapeutic potential of tDCS in acute stroke, we investigated tDCS-effects on cerebral blood flow (CBF) in a tDCS rat model adapted for this purpose. METHODS In a randomised crossover design eight Sprague-Dawley rats received three single cathodal and anodal tDCS for 15 min every other day. At each polarity, current intensities of 25, 50 and 100 μA were applied. CBF was measured prior and after tDCS for at least 30 min with laser Doppler flowmetry (LDF). RESULTS At higher intensities (50 and 100 μA) anodal tDCS increased CBF up to 30 min. At 100 μA CBF was increased by about 25%, at 50 μA by about 18%. In contrast, cathodal tDCS led to a decrease of CBF, likewise depending on the current intensity applied. At 100 μA the effects were about 25% of baseline levels and persisted for at least 30 min. At 25 and 50 μA, baseline-levels were mostly re-established within 30 min. CONCLUSIONS tDCS modulates CBF in a polarity specific way, the extent of modulation depending on the stimulation parameters applied. Because of its polarity-specificity, we assume that CBF-alterations are causally related to tDCS-induced alterations in cortical excitability via neuro-vascular coupling. tDCS may constitute a therapeutic option in acute stroke patients or in patients at risk for vasospasm-induced ischemia after subarachnoid hemorrhage.

Untreated Glioblastoma Multiforme: Increased Myo-inositol and Glutamine Levels in the Contralateral Cerebral Hemisphere at Proton MR Spectroscopy

PURPOSE To use localized in vivo proton magnetic resonance (MR) spectroscopy of the contralateral hemisphere in patients with glioblastoma multiforme (GBM) to detect alterations in cerebral metabolites as potential markers of infiltrating GBM cells. MATERIALS AND METHODS The study was approved by the ethics committee, and written informed consent was obtained. Twenty-two patients with newly diagnosed and untreated GBM underwent in vivo single-voxel short echo time proton MR spectroscopy with a 3-T MR imaging system. Absolute metabolite concentrations in the hemisphere contralateral to the tumor were compared with data from five patients with low-grade gliomas (LGGs) and from a group of 14 age-matched control subjects by using analysis of variance and subsequent t tests or corresponding nonparametric tests. RESULTS In the contralateral hemisphere, MR spectroscopy revealed increased concentrations of myo-inositol and glutamine. Mean myo-inositol levels were significantly increased in patients with GBM (3.6 mmol/L +/- 0.8 [standard deviation]) relative to levels in control subjects (3.1 mmol/L +/- 0.6; P = .03) and tended to be higher relative to levels in patients with LGG (2.7 mmol/L +/- 0.8; P = .09). Mean glutamine concentrations in patients with GBM (3.4 mmol/L +/- 0.9) differed significantly from those in control subjects (2.7 mmol/L +/- 0.7; P = .01); mean concentrations in patients with GBM differed from those in patients with LGG (2.4 mmol/L +/- 0.5; P = .01). There were no significant differences between data in patients with LGG and in control subjects. CONCLUSION Increased concentrations of myo-inositol and glutamine in the contralateral normal-appearing white matter of GBM patients are consistent with mild astrocytosis and suggest the detectability of early neoplastic infiltration by using proton MR spectroscopy in vivo.

Expression of the T-cell chemoattractant chemokine lymphotactin in Crohn's disease.

Recruitment of lymphocytes is a prominent feature of the inflammatory process in Crohns disease (CD). The present study was undertaken to investigate the expression of the novel lymphocyte-specific chemoattractant lymphotactin (Lptn) as a potential regulatory factor for the recruitment of T cells in CD. The expression of Lptn mRNA was quantified in resection specimens of patients with CD in comparison to normal controls without signs of inflammation by real-time quantitative reverse transcriptase-polymerase chain reaction and localized by nonradioactive in situ hybridization. Furthermore, the phenotype of cells expressing Lptn mRNA was characterized. In contrast to normal controls Lptn mRNA was significantly increased in tissue samples affected by CD. Cells expressing Lptn were identified as T cells, mast cells, and unexpectedly dendritic cells. Lptn mRNA was found to be up-regulated on stimulation with phorbol-12-myristate-13-acetate and concanavalin A in T cells isolated from peripheral blood, which could be prevented by dexamethasone, cyclosporine A, and FK506. A similar regulation mechanism could be identified for the Lptn receptor GPR-5 in peripheral T cells. In addition, Lptn mRNA expression could be induced in mature monocyte-derived dendritic cells. The results indicate that local expression of Lptn by activated T cells and to a lesser extent by mast cells and dendritic cells represents a key regulator for lymphocyte trafficking and maintenance of the inflammatory process observed in CD, which might be partly mediated through an autocrine/paracrine pathway of activated T cells.

Accumulation of pathological prion protein PrPSc in the skin of animals with experimental and natural scrapie.

Prion infectivity and its molecular marker, the pathological prion protein PrPSc, accumulate in the central nervous system and often also in lymphoid tissue of animals or humans affected by transmissible spongiform encephalopathies. Recently, PrPSc was found in tissues previously considered not to be invaded by prions (e.g., skeletal muscles). Here, we address the question of whether prions target the skin and show widespread PrPSc deposition in this organ in hamsters perorally or parenterally challenged with scrapie. In hamsters fed with scrapie, PrPSc was detected before the onset of symptoms, but the bulk of skin-associated PrPSc accumulated in the clinical phase. PrPSc was localized in nerve fibres within the skin but not in keratinocytes, and the deposition of PrPSc in skin showed no dependence from the route of infection and lymphotropic dissemination. The data indicated a neurally mediated centrifugal spread of prions to the skin. Furthermore, in a follow-up study, we examined sheep naturally infected with scrapie and detected PrPSc by Western blotting in skin samples from two out of five animals. Our findings point to the skin as a potential reservoir of prions, which should be further investigated in relation to disease transmission.

Nitric oxide stress in sporadic inclusion body myositis muscle fibres: inhibition of inducible nitric oxide synthase prevents interleukin-1β-induced accumulation of β-amyloid and cell death

Sporadic inclusion body myositis is a severely disabling myopathy. The design of effective treatment strategies is hampered by insufficient understanding of the complex disease pathology. Particularly, the nature of interrelationships between inflammatory and degenerative pathomechanisms in sporadic inclusion body myositis has remained elusive. In Alzheimers dementia, accumulation of β-amyloid has been shown to be associated with upregulation of nitric oxide. Using quantitative polymerase chain reaction, an overexpression of inducible nitric oxide synthase was observed in five out of ten patients with sporadic inclusion body myositis, two of eleven with dermatomyositis, three of eight with polymyositis, two of nine with muscular dystrophy and two of ten non-myopathic controls. Immunohistochemistry confirmed protein expression of inducible nitric oxide synthase and demonstrated intracellular nitration of tyrosine, an indicator for intra-fibre production of nitric oxide, in sporadic inclusion body myositis muscle samples, but much less in dermatomyositis or polymyositis, hardly in dystrophic muscle and not in non-myopathic controls. Using fluorescent double-labelling immunohistochemistry, a significant co-localization was observed in sporadic inclusion body myositis muscle between β-amyloid, thioflavine-S and nitrotyrosine. In primary cultures of human myotubes and in myoblasts, exposure to interleukin-1β in combination with interferon-γ induced a robust upregulation of inducible nitric oxide synthase messenger RNA. Using fluorescent detectors of reactive oxygen species and nitric oxide, dichlorofluorescein and diaminofluorescein, respectively, flow cytometry revealed that interleukin-1β combined with interferon-γ induced intracellular production of nitric oxide, which was associated with necrotic cell death in muscle cells. Intracellular nitration of tyrosine was noted, which partly co-localized with amyloid precursor protein, but not with desmin. Pharmacological inhibition of inducible nitric oxide synthase by 1400W reduced intracellular production of nitric oxide and prevented accumulation of β-amyloid, nitration of tyrosine as well as cell death inflicted by interleukin-1β combined with interferon-γ. Collectively, these data suggest that, in skeletal muscle, inducible nitric oxide synthase is a central component of interactions between interleukin-1β and β-amyloid, two of the most relevant molecules in sporadic inclusion body myositis. The data further our understanding of the pathology of sporadic inclusion body myositis and may point to novel treatment strategies.