RESUMEN
The postsynaptic density (PSD) comprises numerous scaffolding proteins, receptors, and signaling molecules that coordinate synaptic transmission in the brain. Postsynaptic density protein 95 (PSD-95) is a master scaffold protein within the PSD and one of its most abundant proteins and therefore constitutes a very attractive biomarker of PSD function and its pathological changes. Here, we exploit a high-affinity inhibitor of PSD-95, AVLX-144, as a template for developing probes for molecular imaging of the PSD. AVLX-144-based probes were labeled with the radioisotopes fluorine-18 and tritium, as well as a fluorescent tag. Tracer binding showed saturable, displaceable, and uneven distribution in rat brain slices, proving effective in quantitative autoradiography and cell imaging studies. Notably, we observed diminished tracer binding in human post-mortem Parkinson's disease (PD) brain slices, suggesting postsynaptic impairment in PD. We thus offer a suite of translational probes for visualizing and understanding PSD-related pathologies.
Asunto(s)
Encéfalo , Homólogo 4 de la Proteína Discs Large , Densidad Postsináptica , Animales , Humanos , Homólogo 4 de la Proteína Discs Large/metabolismo , Encéfalo/metabolismo , Encéfalo/diagnóstico por imagen , Ratas , Densidad Postsináptica/metabolismo , Imagen Molecular/métodos , Radioisótopos de Flúor/química , Enfermedad de Parkinson/metabolismo , Enfermedad de Parkinson/diagnóstico por imagen , Péptidos/química , Péptidos/metabolismo , Sondas Moleculares/química , Masculino , Autorradiografía , Ratas Sprague-Dawley , Tritio , Piridinas , PirrolidinonasRESUMEN
α-Synuclein (α-syn) accumulates as insoluble amyloid but also forms soluble α-syn oligomers (αSOs), thought to be even more cytotoxic than fibrils. To detect and block the unwanted activities of these αSOs, we have raised 30 monoclonal antibodies (mAbs) against different forms of αSOs, ranging from unmodified αSOs to species stabilized by lipid peroxidation products and polyphenols, αSOs formed by C-terminally truncated α-syn, and multivalent display of α-syn on capsid virus-like particles (cVLPs). While the mAbs generally show a preference for αSOs, they also bind fibrils, but to variable extents. Overall, we observe great diversity in the mAbs' relative affinities for monomers and αSOs, varied requirements for the C-terminal extension of α-syn, and only a modest effect on α-syn fibrillation. Several mAbs show several orders of magnitude preference for αSOs over monomers in in-solution studies, while the commercial antibody MJF14 only bound 10-fold more strongly to αSOs than monomeric α-syn. Gratifyingly, seven mAbs almost completely block αSO permeabilization of membrane vesicles. Five selected mAbs identified α-syn-related pathologies like Lewy bodies (LBs) and Lewy Neurites, as well as Glial Cytoplasmic Inclusions in postmortem brains from people diagnosed for PD, dementia with LBs or multiple system atrophy, although to different extents. Three mAbs were particularly useful for pathological evaluation of postmortem brain human tissue, including early stages of PD. Although there was no straightforward connection between the mAbs' biophysical and immunohistochemical properties, it is encouraging that this comprehensive collection of mAbs able to recognize different aggregated α-syn species in vitro also holds diagnostic potential.
RESUMEN
The locus coeruleus is the major source of noradrenaline to the brain and contributes to a wide range of physiological and cognitive functions including arousal, attention, autonomic control, and adaptive behaviour. Neurodegeneration and pathological aggregation of tau protein in the locus coeruleus are early features of progressive supranuclear palsy (PSP). This pathology is proposed to contribute to the clinical expression of disease, including the PSP Richardson's syndrome. We test the hypothesis that tau pathology and neuronal loss are associated with clinical heterogeneity and severity in PSP.We used immunohistochemistry in post mortem tissues from 31 patients with a clinical diagnosis of PSP (22 with Richardson's syndrome) and 6 control cases. We quantified the presence of hyperphosphorylated tau, the number of pigmented cells indicative of noradrenergic neurons, and the percentage of pigmented neurons with tau-positive inclusions. Ante mortem assessment of clinical severity using the PSP rating scale was available within 1.8 (±0.9) years for 23 patients.We found an average 49% reduction of pigmented neurons in PSP patients relative to controls. The loss of pigmented neurons correlated with disease severity, even after adjusting for disease duration and the interval between clinical assessment and death. The degree of neuronal loss was negatively associated with tau-positive inclusions, with an average of 44% of pigmented neurons displaying tau-inclusions.Degeneration and tau pathology in the locus coeruleus are related to clinical heterogeneity of PSP. The noradrenergic deficit in the locus coeruleus is a candidate target for pharmacological treatment. Recent developments in ultra-high field magnetic resonance imaging to quantify in vivo structural integrity of the locus coeruleus may provide biomarkers for noradrenergic experimental medicines studies in PSP.
Asunto(s)
Neuronas Adrenérgicas/patología , Locus Coeruleus/patología , Parálisis Supranuclear Progresiva/patología , Neuronas Adrenérgicas/metabolismo , Anciano , Anciano de 80 o más Años , Femenino , Humanos , Locus Coeruleus/metabolismo , Masculino , Persona de Mediana Edad , Fosforilación , Índice de Severidad de la Enfermedad , Parálisis Supranuclear Progresiva/metabolismo , Proteínas tau/metabolismoRESUMEN
Untreated human immunodeficiency virus (HIV) depletes its host CD4 cells, ultimately leading to acquired immunodeficiency syndrome (AIDS). In brain, the HIV confines itself to astrocytes and microglia, the resident brain macrophages, but does not infect oligodendrocytes and neurons. Nonetheless, cognitive symptoms associated with HIV and AIDS are attributed to loss of axons and white matter damage. We used design-based stereology to estimate the numbers of neocortical neurons and glial cells (astrocytes, oligodendrocytes, and microglia), in a series of 12 patients dying with AIDS before the era of retroviral treatments, and in 13 age-matched control brains. Relative to the control material, there was a 19% loss of neocortical neuron (p = 0.04) and a 29% reduction of oligodendrocytes (p = 0.003) in the patients with AIDS, whereas astrocyte and microglia numbers did not differ between patients and controls. Furthermore, we saw a 17% reduction in mean hemispheric volume in the AIDS group (p = 0.0015), which was driven by neocortical and white matter loss (p < 0.05), while the archicortex, subcortical gray matter, and ventricular volumes were within normal limits. Our results confirm previous reports of neuronal loss in AIDS. The new finding of oligodendrocyte loss supports the proposal that HIV in the brain provokes demyelination and axonal dysfunction and suggests that remyelination treatment strategies may be beneficial to patients suffering from HIV-associated neurocognitive deficits.