Anne V. Thomas

Dementia in Parkinson disease Functional imaging of cholinergic and dopaminergic pathways

Objective: To assess neurochemical deficits in patients with Parkinson disease (PD) associated dementia (PDD) in vivo. Methods: The authors performed combined PET with N-[11C]-methyl-4-piperidyl acetate (MP4A) and 18F-fluorodopa (FDOPA) for evaluation of cholinergic and dopaminergic transmitter changes in 17 non-demented patients with PD and 10 patients with PDD. Data were compared to 31 age-matched controls by a combined region-of-interest and voxel-based Statistical Parametric Mapping analysis. Results: The striatal FDOPA uptake was significantly decreased in PD and PDD without differences between the groups. The global cortical MP4A binding was severely reduced in PDD (29.7%, p < 0.001 vs controls) and moderately decreased in PD (10.7%, p < 0.01 vs controls). The PDD group had lower parietal MP4A uptake rates than did patients with PD. Frontal and temporo-parietal cortices showed a significant covariance of striatal FDOPA reduction and decreased MP4A binding in patients with PDD. Conclusions: While non-demented patients with Parkinson disease had a moderate cholinergic dysfunction, subjects with Parkinson disease associated dementia (PDD) presented with a severe cholinergic deficit in various cortical regions. The finding of a closely associated striatal FDOPA and cortical MP4A binding reduction suggests a common disease process leading to a complex transmitter deficiency syndrome in PDD.

Imaging in Neurooncology

SummaryImaging in patients with brain tumors aims toward the determination of the localization, extend, type, and malignancy of the tumor. Imaging is being used for primary diagnosis, planning of treatment including placement of stereotaxic biopsy, resection, radiation, guided application of experimental therapeutics, and delineation of tumor from functionally important neuronal tissue. After treatment, imaging is being used to quantify the treatment response and the extent of residual tumor. At follow-up, imaging helps to determine tumor progression and to differentiate recurrent tumor growth from treatment-induced tissue changes, such as radiation necrosis. A variety of complementary imaging methods are currently being used to obtain all the information necessary to achieve the abovementioned goals. Computed tomography and magnetic resonance imaging (MRI) reveal mostly anatomical information on the tumor, whereas magnetic resonance spectroscopy and positron emission tomography (PET) give important information on the metabolic state and molecular events within the tumor. Functional MRI and functional PET, in combination with electrophysiological methods like transcranial magnetic stimulation, are being used to delineate functionally important neuronal tissue, which has to be preserved from treatment-induced damage, as well as to gather information on tumor-induced brain plasticity. In addition, optical imaging devices have been implemented in the past few years for the development of new therapeutics, especially in experimental glioma models. In summary, imaging in patients with brain tumors plays a central role in the management of the disease and in the development of improved imaging-guided therapies.

Use of 11C-methionine PET to monitor the effects of temozolomide chemotherapy in malignant gliomas

PurposeThe purpose of this study was to monitor the metabolic effects of temozolomide (TMZ) chemotherapy in malignant gliomas by means of repeated positron emission tomography (PET) with [11C]methionine (MET).MethodsFifteen patients with histologically proven malignant glioma were treated by TMZ chemotherapy. MET-PET studies were performed before and after the third cycle of TMZ chemotherapy in all patients, and in 12 patients also after the sixth cycle. Gadolinium-enhanced MRI studies were performed in 12 patients before the first and after the sixth cycle. Clinical status was assessed by the modified Rankin scale. Long-term outcome was assessed by calculating the time to progression (TTP) in months.ResultsDecline in MET uptake during therapy corresponded to a stable clinical status. The median TTP was significantly longer in patients with decline in MET uptake than in those with increasing MET uptake (23 vs 3.5 months; p=0.01, log rank test). There was no significant correlation between change in MET uptake and change in contrast enhancement during treatment for all patients.ConclusionThe present data demonstrate that clinical stability, which is often achieved under TMZ chemotherapy of malignant glioma, corresponds to a decline in or stability of tumour amino acid metabolism. Tumour responses can already be demonstrated with MET-PET after three cycles of chemotherapy, and absence of progression at that time indicates a high probability of further stability during the next three cycles. A reduction in MET uptake during TMZ treatment predicts a favourable clinical outcome. Molecular imaging of amino acid uptake by MET-PET offers a new method of measurement of the biological activity of recurrent glioma.

Ubiquilin 1 Modulates Amyloid Precursor Protein Trafficking and Aβ Secretion

Ubiquilin 1 (UBQLN1) is a ubiquitin-like protein, which has been shown to play a central role in regulating the proteasomal degradation of various proteins, including the presenilins. We recently reported that DNA variants in UBQLN1 increase the risk for Alzheimer disease, by influencing expression of this gene in brain. Here we present the first assessment of the effects of UBQLN1 on the metabolism of the amyloid precursor protein (APP). For this purpose, we employed RNA interference to down-regulate UBQLN1 in a variety of neuronal and non-neuronal cell lines. We demonstrate that down-regulation of UBQLN1 accelerates the maturation and intracellular trafficking of APP, while not interfering with α-, β-, or γ-secretase levels or activity. UBQLN1 knockdown increased the ratio of APP mature/immature, increased levels of full-length APP on the cell surface, and enhanced the secretion of sAPP (α- and β-forms). Moreover, UBQLN1 knockdown increased levels of secreted Aβ40 and Aβ42. Finally, employing a fluorescence resonance energy transfer-based assay, we show that UBQLN1 and APP come into close proximity in intact cells, independently of the presence of the presenilins. Collectively, our findings suggest that UBQLN1 may normally serve as a cytoplasmic “gatekeeper” that may control APP trafficking from intracellular compartments to the cell surface. These findings suggest that changes in UBQLN1 steady-state levels affect APP trafficking and processing, thereby influencing the generation of Aβ.

Alzheimer's disease-associated ubiquilin-1 regulates presenilin-1 accumulation and aggresome formation

The Alzheimers disease (AD)‐associated ubiquilin‐1 regulates proteasomal degradation of proteins, including presenilin (PS). PS‐dependent γ‐secretase generates β‐amyloid (Aβ) peptides, which excessively accumulate in AD brain. Here, we have characterized the effects of naturally occurring ubiquilin‐1 transcript variants (TVs) on the levels and subcellular localization of PS1 and other γ‐secretase complex components and subsequent γ‐secretase function in human embryonic kidney 293, human neuroblastoma SH‐SY5Y and mouse primary cortical cells. Full‐length ubiquilin‐1 TV1 and TV3 that lacks the proteasome‐interaction domain increased full‐length PS1 levels as well as induced accumulation of high‐molecular‐weight PS1 and aggresome formation. Accumulated PS1 colocalized with TV1 or TV3 in the aggresomes. Electron microscopy indicated that aggresomes containing TV1 or TV3 were targeted to autophagosomes. TV1‐ and TV3‐expressing cells did not accumulate other unrelated proteasome substrates, suggesting that the increase in PS1 levels was not because of a general impairment of the ubiquitin‐proteasome system. Furthermore, PS1 accumulation and aggresome formation coincided with alterations in Aβ levels, particularly in cells overexpressing TV3. These effects were not related to altered γ‐secretase activity or PS1 binding to TV3. Collectively, our results indicate that specific ubiquilin‐1 TVs can cause PS1 accumulation and aggresome formation, which may impact AD pathogenesis or susceptibility.

Mutations in amyloid precursor protein affect its interactions with presenilin/γ-secretase

Alzheimers disease is characterized by accumulation of toxic beta-amyloid (Abeta) in the brain and neuronal death. Several mutations in presenilin (PS1) and beta-amyloid precursor protein (APP) associate with an increased Abeta(42/40) ratio. Abeta(42), a highly fibrillogenic species, is believed to drive Abeta aggregation. Factors shifting gamma-secretase cleavage of APP to produce Abeta(42) are unclear. We investigate the molecular mechanism underlying altered Abeta(42/40) ratios associated with APP mutations at codon 716 and 717. Using FRET-based fluorescence lifetime imaging to monitor APP-PS1 interactions, we show that I716F and V717I APP mutations increase the proportion of interacting molecules earlier in the secretory pathway, resulting in an increase in Abeta generation. A PS1 conformation assay reveals that, in the presence of mutant APP, PS1 adopts a conformation reminiscent of FAD-associated PS1 mutations, thus influencing APP binding to PS1/gamma-secretase. Mutant APP affects both intracellular location and efficiency of APP-PS1 interactions, thereby changing the Abeta(42/40) ratio.

Interaction between presenilin 1 and ubiquilin 1 as detected by fluorescence lifetime imaging microscopy and a high-throughput fluorescent plate reader

Presenilin 1 (PS1) in its active heterodimeric form is the catalytic center of the γ-secretase complex, an enzymatic activity that cleaves amyloid precursor protein (APP) to produce amyloid β (Aβ). Ubiquilin 1 is a recently described PS1 interacting protein, the overexpression of which increases PS1 holoprotein levels and leads to reduced levels of functionally active PS1 heterodimer. In addition, it has been suggested that splice variants of the UBQLN1 gene are associated with an increased risk of developing Alzheimer disease (AD). However, it is still unclear whether PS1 and ubiquilin 1 interact when expressed at endogenous levels under normal physiological conditions. Here, we employ three novel fluorescence resonance energy transfer-based techniques to investigate the interaction between PS1 and ubiquilin 1 in intact cells. We consistently find that the ubiquilin 1 N terminus is in close proximity to several epitopes on PS1. We show that ubiquilin 1 interacts both with PS1 holoprotein and heterodimer and that the interaction between PS1 and ubiquilin 1 takes place near the cell surface. Furthermore, we show that the PS1-ubiquilin 1 interaction can be detected between endogenous proteins in primary neurons in vitro as well as in brain tissue of healthy controls and Alzheimer disease patients, providing evidence of its physiological relevance.

Signal peptide peptidase (SPP) dimer formation as assessed by fluorescence lifetime imaging microscopy (FLIM) in intact cells

BackgroundSignal peptide peptidase (SPP) is an intramembrane cleaving protease identified by its cleavage of several type II membrane signal peptides. Conservation of intramembrane active site residues demonstrates that SPP, SPP family members, and presenilins (PSs) make up a family of intramembrane cleaving proteases. Because SPP appears to function without additional protein cofactors, the study of SPP may provide structural insights into the mechanism of intramembrane proteolysis by this biomedically important family of proteins. Previous studies have shown that SPP isolated from cells appears to be a homodimer, but some evidence exists that in vitro SPP may be active as a monomer. We have conducted additional experiments to determine if SPP exists as a monomer or dimer in vivo.ResultsFluorescence lifetime imaging microscopy (FLIM) can be is used to determine intra- or intermolecular interactions by fluorescently labeling epitopes on one or two different molecules. If the donor and acceptor fluorophores are less than 10 nm apart, the donor fluorophore lifetime shortens proportionally to the distance between the fluorophores. In this study, we used two types of fluorescence energy transfer (FRET) pairs; cyan fluorescent protein (CFP) with yellow fluorescent protein (YFP) or Alexa 488 with Cy3 to differentially label the NH2- or COOH-termini of SPP molecules. A cell based SPP activity assay was used to show that all tagged SPP proteins are proteolytically active. Using FLIM we were able to show that the donor fluorophore lifetime of the CFP tagged SPP construct in living cells significantly decreases when either a NH2- or COOH-terminally YFP tagged SPP construct is co-transfected, indicating close proximity between two different SPP molecules. These data were then confirmed in cell lines stably co-expressing V5- and FLAG-tagged SPP constructs.ConclusionOur FLIM data strongly suggest dimer formation between two separate SPP proteins. Although the tagged SPP constructs are expressed throughout the cell, SPP dimer detection by FLIM is seen predominantly at or near the plasma membrane.

Novel neuroimaging findings in a patient with cerebral Whipple's disease: a magnetic resonance imaging and positron emission tomography study.

The authors report a 43-year-old patient with histopathologically proven cerebral Whipples disease. Magnetic resonance imaging (MRI) revealed a multilayered left frontal lesion without mass effect, no perifocal brain edema, no contrast enhancement, and a thin shell of fluid signal that presented as an incomplete, open ring. An [11C]methionine positron emission tomography (PET) study showed low uptake below the threshold that is characteristic for brain tumors. In precise co-registration to the MR images, the PET data showed that increased uptake was mainly located in the direct adjacent part of the MRI lesion. The fluid signal on MRI corresponded to the extensive outflow of fluid from the lesion, which was observed during neurosurgical resection, and also to the neuropathological findings. The authors conclude that this cerebral manifestation of Whipples disease made a unique and hitherto undescribed appearance on MRI; uptake pattern of PET amino acid tracer may help in the preoperative distinction of inflammatory from neoplastic lesions.

Visualizing interaction of proteins relevant to Alzheimer’s disease in intact cells

To understand normal function of memory studying models of pathological memory decline is essential. The most common form of dementia leading to memory decline is Alzheimers disease (AD), which is characterized by the presence of neurofibrillary tangles and amyloid plaques in the affected brain regions. Altered production of amyloid beta (Abeta) through sequential cleavage of amyloid precursor protein (APP) by beta- and gamma-secretases seems to be a central event in the molecular pathogenesis of the disease. Thus, the study of the complex interplay of proteins that are involved in or modify Abeta production is very important to gain insight into the pathogenesis of AD. Here, we describe the use of Fluorescence lifetime imaging microscopy (FLIM), a Fluorescence resonance energy transfer (FRET)-based method, to visualize protein-protein-interaction in intact cells, which has proven to be a valuable method in AD research.