The development of new treatments for essential tremor, the most frequent movement disorder, is limited by a poor understanding of its pathophysiology and the relative paucity of clinicopathological studies. Here, we report a post-mortem decrease in GABA(A) (35% reduction) and GABA(B) (22-31% reduction) receptors in the dentate nucleus of the cerebellum from individuals with essential tremor, compared with controls or individuals with Parkinson's disease, as assessed by receptor-binding autoradiography. Concentrations of GABA(B) receptors in the dentate nucleus were inversely correlated with the duration of essential tremor symptoms (r(2) = 0.44, P < 0.05), suggesting that the loss of GABA(B) receptors follows the progression of the disease. In situ hybridization experiments also revealed a diminution of GABA(B(1a+b)) receptor messenger RNA in essential tremor (↓27%). In contrast, no significant changes of GABA(A) and GABA(B) receptors (protein and messenger RNA), GluN2B receptors, cytochrome oxidase-1 or GABA concentrations were detected in molecular or granular layers of the cerebellar cortex. It is proposed that a decrease in GABA receptors in the dentate nucleus results in disinhibition of cerebellar pacemaker output activity, propagating along the cerebello-thalamo-cortical pathways to generate tremors. Correction of such defective cerebellar GABAergic drive could have a therapeutic effect in essential tremor.
The development of vectors for drug delivery to the central nervous system remains a major pharmaceutical challenge. Here, we have characterized the brain distribution of two monoclonal antibodies (MAbs) targeting the mouse transferrin receptor (TfR) (clones Ri7 and 8D3) compared with control IgGs after intravenous injection into mice. MAbs were fluorolabeled with either Alexa Fluor (AF) dyes 647 or 750. Intravenous injection of Ri7 or 8D3 MAb coupled with AF750 led to higher fluorescence emission in brain homogenates compared with control IgGs, indicating retention in the brain. Fluorescence microscopy analysis revealed that AF647-Ri7 signal was confined to brain cerebrovasculature, colocalizing with an antibody against collagen IV, a marker of basal lamina. Confocal microscopy analysis confirmed the delivery of injected Ri7 MAb into brain endothelial cells using the pericyte marker anti-␣-smooth muscle actin, the endothelial marker CD31, and the collagen IV antibody. No evidence of colocalization was detected with neurons or astrocytes identified using antibodies specific for neuronal nuclei or glial fibrillary acidic protein, respectively. Our data show that anti-TfR vectors injected intravenously readily accumulate into brain capillary endothelial cells, thus displaying strong drugtargeting potential.
The transferrin receptor (TfR) is highly expressed by brain capillary endothelial cells (BCECs) forming the blood–brain barrier (BBB) and is therefore considered as a potential target for brain drug delivery. Monoclonal antibodies binding to the TfR, such as clone Ri7, have been shown to internalize into BCECs in vivo. However, since Alzheimer’s disease (AD) is accompanied by a BBB dysfunction, it raises concerns about whether TfR-mediated transport becomes inefficient during the progression of the disease. Measurements of TfR levels using Western blot analysis in whole homogenates from human post-mortem parietal cortex and hippocampus did not reveal any significant difference between individuals with or without a neuropathological diagnosis of AD (respectively, n = 19 and 22 for the parietal cortex and n = 12 and 14 for hippocampus). Similarly, TfR concentrations in isolated human brain microvessels from parietal cortex were similar between controls and AD cases. TfR levels in isolated murine brain microvessels were not significantly different between groups of 12- and 18-month-old NonTg and 3xTg-AD mice, the latter modeling Aβ and τ neuropathologies. In situ brain perfusion assays were then conducted to measure the brain uptake and internalization of fluorolabeled Ri7 in BCECs upon binding. Consistently, TfR-mediated uptake in BCECs was similar between 3xTg-AD mice and nontransgenic controls (∼0.3 μL·g– 1·s–1) at 12, 18, and 22 months of age. Fluorescence microscopy analysis following intravenous administration of fluorolabeled Ri7 highlighted that the signal from the antibody was widely distributed throughout the cerebral vasculature but not in neurons or astrocytes. Overall, our data suggest that both TfR protein levels and TfR-dependent internalization mechanisms are preserved in the presence of Aβ and τ neuropathologies, supporting the potential of TfR as a vector target for drug delivery into BCECs in AD.
Monoclonal antibodies (mAbs) targeting blood-brain barrier (BBB) transporters are being developed for brain drug targeting. However, brain uptake quantification remains a challenge, particularly for large compounds, and often requires the use of radioactivity. In this work, we adapted an in situ brain perfusion technique for a fluorescent mAb raised against the mouse transferrin receptor (TfR) (clone Ri7). We first confirmed in vitro that the internalization of fluorolabeled Ri7 mAbs is saturable and dependent on the TfR in N2A and bEnd5 cells. We next showed that the brain uptake coefficient (Clup) of 100 μg (∼220 nM) of Ri7 mAbs fluorolabeled with Alexa Fluor 750 (AF750) was 0.27 ± 0.05 μL g(-1) s(-1) after subtraction of values obtained with a control IgG. A linear relationship was observed between the distribution volume VD (μL g(-1)) and the perfusion time (s) over 30-120 s (r(2) = 0.997), confirming the metabolic stability of the AF750-Ri7 mAbs during perfusion. Co-perfusion of increasing quantities of unlabeled Ri7 decreased the AF750-Ri7 Clup down to control IgG levels over 500 nM, consistent with a saturable mechanism. Fluorescence microscopy analysis showed a vascular distribution of perfused AF750-Ri7 in the brain and colocalization with a marker of basal lamina. To our knowledge, this is the first reported use of the in situ brain perfusion technique combined with quantification of compounds labeled with near-infrared fluorophores. Furthermore, this study confirms the accumulation of the antitransferrin receptor Ri7 mAb in the brain of mice through a saturable uptake mechanism.
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