Diss. (sammanfattning) Stockholm : Karol. inst. / Härtill 7 uppsatser.

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Genome wide association studies have provided evidence for a significant association between ZNF804A (zinc finger protein 804A) - specifically the intronic single nucleotide polymorphism (SNP) rs1344706 - and schizophrenia, but little is known about the function of the gene or the effects of the SNP. By studying post-mortem human brain tissue, I characterised ZNF804A immunoreactivity in adult and foetal human brain and investigated effects of diagnosis and rs1344706 genotype on ZNF804A mRNA and protein expression. Secondly, I looked in a large sample of healthy volunteers (n=922) at the effects of rs1344706 on brain structure using volumetry and voxel based morphometry (VBM). Furthermore, I recruited healthy volunteers who were either homozygous for the risk allele or homozygous for the non-risk allele (n=50). They participated in magnetoencephalography (MEG) and magnetic resonance (MR) sessions in which brain activity was measured during a working memory task, a visual processing task, and rest. Using magnetic resonance spectroscopy, also neurotransmitter levels were assessed. The experiments conducted for this thesis showed for the first time that ZNF804A immunoreactivity can be detected in both foetal and adult human brain and that it is mainly localised to layer III pyramidal cells, with a granular subcellular distribution throughout the cytoplasm. No effect of rs1344706 on mRNA and protein expression was found. In our structural MRI study, rs1344706 did not affect macroscopic brain structure as measured by volumetry and VBM, and given the large sample size, this seems a convincing negative. However, we did find that rs1344706 alters prefrontal-hippocampal connectivity, with increased connectivity being observed in risk homozygotes. Additionally, using MEG, we found an effect of ZNF804A genotype on hippocampal connectivity in the theta band (4-8Hz), with non-risk homozygotes displaying more connectivity. This finding provides a first clue as to the mechanisms that might underlie the previously observed effects of rs1344706 on prefrontal-hippocampal connectivity. Future studies will need to elucidate the actual function of the ZNF804A protein, in order to bridge the gap between the molecular and neuroimaging findings described in this thesis.

<p> This project was undertaken with the aim of studying the distribution of copper, manganese and zinc in brain and to gain same information on the possible presence of soluble metallo-proteins in this tissue. The distribution of copper, manganese and zinc has been determined by neutron activation analysis. Soluble metallo-proteins of these three elements have been identified by the combination of gel chromatography and neutron activation analysis. In addition, in vivo and in vitro radioisotopic labelling techniques have been used occasionally.</p> / Thesis / Doctor of Philosophy (PhD)

Alzheimer’s Disease, also called AD, is a horrible, degenerative brain disease that more than 35 million people over the world have. Today, there is no cure for this disease, only treatments that are temporarily relieving the symptoms. The two proteins that is thought to be the main cause of AD is amyloid β (Aβ) and tau. Previously, people have tried studying Aβ in vivo using green fluorescent protein fusion together with Aβ. However, this is difficult since the aggregation of Aβ will lead to loss of fluorescence. This study aimed to crystallize the fusion protein mNG-A β1-42 and to investigate its properties as a molecular fluorescent Aβ-amyloid specific probe. Dynamic light scattering (DLS) was used to confirm that the majority of the protein was not in the form of soluble aggregates. The DLS experiments were followed by several rounds of crystallization trials. Initial screening and the subsequent narrowing down of potential conditions where mNG-Aβ1-42 could form crystals. Several staining experiments were conducted as well, including staining brain tissue from mouse with both Swedish and Arctic mutation, from human patients with sporadic AD and from human patients with AD with the Arctic mutation. The DLS experiments showed that the protein used in the crystallization experiments mostly consisted of molecular particles of the same radius. However, there was clear evidence of some larger species present that could have been a potential problem for crystallization. Crystallization experiments suggested that PEG 8000 was the most promising precipitant amongst other conditions identified for crystallization of mNG-Aβ1-42. However, the study was ultimately unsuccessful in developing crystals of sufficient high quality for diffraction studies to commence. The staining experiments demonstrated that mNG-Aβ1-42 could bind both by itself and with another amyloid probe, Congo red, and with antibodies in brain tissue from mouse with both Swedish and Arctic mutation, from human patients with sporadic AD and from human patients with AD with the Arctic mutation. In conclusion, several characteristics of mNG-Aβ1-42 were revealed in this study.

Autism spectrum disorder (ASD) now affects 1 in 68 children in the United States. Disorders within this spectrum share hallmark deficits in verbal and nonverbal communication, repetitive behavior, and social interaction. The cause of ASD is still unknown. Even though hundreds of genetic abnormalities have been identified in ASD, these markers account for less than 1% of all ASD cases. Researchers continue to search for pathological markers common to all or most cases of ASD. The research presented in this dissertation used a novel combination of state-of-the-art methods to investigate brain pathology in ASD. Postmortem anterior cingulate cortex (ACC) from ASD and typically developing brain donors was obtained from 2 national brain banks. The ACC was chosen for study because of its documented role in influencing behaviors characteristically disrupted in ASD. An initial study revealed elevated glial fibrillary acidic protein (GFAP) in ACC white matter from ASD brain donors compared to typically developing control donors. Laser capture microdissection was then employed to isolate specific cell populations from the ACC from ASD and control brain donors. Captured cells were used to interrogate potential gene expression abnormalities that may underlie biological mechanisms that contribute behavioral abnormalities of ASD. The expression of 4 genes associated with synaptic function, NTRK2, GRM8, SLC1A1, and GRIP1, were found to be significantly lower in ACC pyramidal neurons from ASD donors when compared to control donors. These expression abnormalities were not observed in ACC glia. Given robust evidence of neuronal and glial pathology in the ACC in ASD, a novel method for whole transcriptome analysis in single cell populations was developed to permit an unbiased analysis of brain cellular pathology in ASD. A list of genes that were differentially expressed, comparing ASD to control donors, was produced for both white matter and pyramidal neuron samples. By examining the ASD brain at the level of its most basic component, the cell, methods were developed that should allow future research to identify common cellular-based pathology of the ASD brain. Such research will increase the likelihood of future development of novel pharmacotherapy for ASD patients.

Abstract available in the Pharmacotherapy: The Journal of Human Pharmacology and Drug Therapy.

Methylphenidate, a psychostimulant used for the treatment of attention deficit hyperactivity disorder and narcolepsy, is administered as a 50:50 racemic mixture, despite the fact that d‐methylphenidate has been shown to have greater pharmacologic activity. This paper presents a validated LC‐MS/MS approach to separation and quantification of methylphenidate enantiomers using a vancomycin column and triethylammonium acetate to enhance the chiral separation. The method is applicable to the monitoring of these enantiomers in mouse brain, with a limit of detection of 0.5 ng/mL and a lower limit of quantification of 7.5 ng/mL.

The symptoms in Idiopathic Adult Hydrocephalus Syndrome (IAHS) – gait disturbance, incontinence, and cognitive deficit – correlate anatomically to neuronal dysfunction in periventricular white matter. The pathophysiology is considered to include a cerebrospinal fluid (CSF) hydrodynamic disturbance, including pressure oscillations (“B waves”), in combination with cerebrovascular disease. IAHS and Subcortical Arteriosclerotic Encephalopathy (SAE) show clinical similarities, which constitutes a diagnostic problem. The aim of this thesis was to investigate biochemical markers in CSF, possibly related to the pathophysiology, and their usefulness in diagnosis, to investigate the effect of ICP changes on glucose supply and metabolism in periventricular deep white matter, and to present criteria for objective, computerised methods for evaluating the content of B waves in an intracranial pressure (ICP) registration. CSF samples from 62 IAHS patients, 26 SAE patients, and 23 controls were analysed for sulfatide, total-tau (T-tau) hyperphosphorylated tau (P-tau), neurofilament protein light (NFL), and beta-amyloid-42 (Aß42). In ten IAHS patients, recordings of ICP, brain tissue oxygen tension (PtiO2), and samplings of brain extracellular fluid from periventricular white matter by way of microdialysis were performed, at rest and during a CSF infusion and tap test. Microdialysis samples were analysed for glucose, lactate, pyruvate, glutamate, glycerol, and urea. Patterns before and after spinal tap were analysed and changes from increasing ICP during the infusion test were described. The long term ICP registration was used to evaluate two computerised methods according to optimal amplitude threshold, monitoring time, and correlation to the manual visual method. In CSF, NFL was elevated in both IAHS and SAE patients, reflecting the axonal damage. In a multinominal logistic regression model, the combined pattern of high NFL, low P-tau and low Aß42 in CSF was shown to be highly predictive in distinguishing between IAHS, SAE and controls. Analysis of microdialysis samples for glucose, lactate, and pyruvate showed, in combination with PtiO2, a pattern of low-grade ischemia. After the spinal tap of CSF, the pattern changed, indicating increased glucose metabolic rate. During the infusion test, there were prompt decreases in the microdialysis values of glucose, lactate and pyruvate during ICP increase, but no sign of hypoxia. The values normalised immediately when ICP was lowered, indicating that the infusion test is not causing damage. One of the computerised methods, with an amplitude threshold set to 1 mm Hg, was shown robust in evaluating B wave content in an ICP registration. At least 5 hours registration time was needed. The highly predictive pattern of biochemical markers in CSF indicates a possibility of identifying simple tests in diagnosing and selecting patients for surgical treatment. The results of microdialysis and PtiO2 indicate low-grade ischemia in the periventricular white matter, which is ameliorated from CSF removal, and that glucose supply and metabolism are sensitive to short-term ICP elevations, thus proposing a link between ICP oscillations and symptoms from neuronal disturbance. A computerised method for evaluation of B waves is a prerequisite for evaluating the impact of pressure oscillations in the pathophysiology of IAHS.