BackgroundBesides the two main classical features of amyloid beta aggregation and tau-containing neurofibrillary tangle deposition, neuroinflammation plays an important yet unclear role in the pathophysiology of Alzheimer’s disease (AD). Microglia are believed to be key mediators of neuroinflammation during AD and responsible for the regulation of brain homeostasis by balancing neurotoxicity and neuroprotective events. We have previously reported evidence that neuritic plaques are derived from dead neurons that have accumulated intraneuronal amyloid and further recruit Iba1-positive cells, which play a role in either neuronal demise or neuritic plaque maturation or both.MethodsTo study the impact of microglia on neuritic plaque development, we treated two-month-old 5XFAD mice with a selective colony stimulation factor 1 receptor (CSF1R) inhibitor, PLX3397, for a period of 3 months, resulting in a significant ablation of microglia. Directly after this treatment, we analyzed the amount of intraneuronal amyloid and neuritic plaques and performed behavioral studies including Y-maze, fear conditioning and elevated plus maze.ResultsWe found that early long-term PLX3397 administration results in a dramatic reduction of both intraneuronal amyloid as well as neuritic plaque deposition. PLX3397 treated young 5XFAD mice also displayed a significant decrease of soluble fibrillar amyloid oligomers in brain lysates, a depletion of soluble pre-fibrillar oligomers in plasma and an improvement in cognitive function measured by fear conditioning tests.ConclusionsOur findings demonstrate that CSF1R signaling, either directly on neurons or mediated by microglia, is crucial for the accumulation of intraneuronal amyloid and formation of neuritic plaques, suggesting that these two events are serially linked in a causal pathway leading to neurodegeneration and neuritic plaque formation. CSF1R inhibitors represent potential preventative or therapeutic approach that target the very earliest stages of the formation of intraneuronal amyloid and neuritic plaques.

The cholinergic and glutamatergic neurotransmission systems are known to be severely disrupted in Alzheimer's disease (AD). GABAergic neurotransmission, in contrast, is generally thought to be well preserved. Evidence from animal models and human postmortem tissue suggest GABAergic remodeling in the AD brain. Nevertheless, there is no information on changes, if any, in the electrophysiological properties of human native GABA receptors as a consequence of AD. To gain such information, we have microtransplanted cell membranes, isolated from temporal cortices of control and AD brains, into Xenopus oocytes, and recorded the electrophysiological activity of the transplanted GABA receptors. We found an age-dependent reduction of GABA currents in the AD brain. This reduction was larger when the AD membranes were obtained from younger subjects. We also found that GABA currents from AD brains have a faster rate of desensitization than those from non-AD brains. Furthermore, GABA receptors from AD brains were slightly, but significantly, less sensitive to GABA than receptors from non-AD brains. The reduction of GABA currents in AD was associated with reductions of mRNA and protein of the principal GABA receptor subunits normally present in the temporal cortex. Pairwise analysis of the transcripts within control and AD groups and analyses of the proportion of GABA receptor subunits revealed down-regulation of α1 and γ2 subunits in AD. In contrast, the proportions of α2, β1, and γ1 transcripts were up-regulated in the AD brains. Our data support a functional remodeling of GABAergic neurotransmission in the human AD brain.neurodegeneration | synaptic mechanism | gephyrin | glutamate receptor A lzheimer's disease (AD) is associated with a widespread loss of synapse density and continuous degeneration of cholinergic and glutamatergic pathways (1). Although disruption of excitatory pathways is broadly accepted, inhibitory GABAergic pathways are generally thought to be well preserved in AD (reviewed in refs. 2, 3). GABA receptors (GABA A Rs) are pentameric complexes formed by combinations of α1-6, β1-3, γ1-2, and δ subunits; the specific combination or stoichiometry of the different GABA A R subunits contributes to their cellular localization, pharmacological profile, and function (4). Early studies of the temporal cortex, an area greatly affected by the neuropathic hallmarks of AD, showed only slight decreases (13-17%) of benzodiazepine binding (2, 5), suggesting a modest decrease of GABA A Rs in AD. However, unspecific binding of benzodiazepines to voltage-dependent anion channels (6) and to mitochondrial translocator proteins (7) that are increased in AD (8) confound the interpretation of results. Lower levels of GABA A Rsubunits α1, α2, α4, δ, and β2 mRNAs in the prefrontal cortex of AD brains (3) and of α1, α5, and β3 mRNAs in the AD hippocampus (2, 9) suggest that some receptors may have an altered functional profile in AD. Our initial experiments, evaluating the feasibility of recording the activity of native receptors fro...

The monoclonal antibodies 6E10 and 4G8 are among the first anti-amyloid monoclonal antibodies against Aβ and the most widely used antibodies in Alzheimer’s disease research. Although the epitopes for 6E10 and 4G8 have been reported to correspond to residues 1–16 and 17–24, a more recent high-resolution mapping approach indicates that 6E10 maps to residues 4–10 while 4G8 maps to residues 18–23. To characterize the binding specificity of both antibodies in greater detail, we used immunoselection of random sequences from phage display library followed by deep sequencing and analysis of resulting patterns from thousands of immunoselected sequences. We found that the minimum sequence required for 6E10 binding is R-x-D with over half (53%) of the immunoselected sequences conforming to this pattern. The vast majority of these sequences contain an H at position x (R-H-D), corresponding to residues 5–7 of the Aβ target sequences, but Y is also permitted at this position in a minority of sequences. For 4G8 we found that the most frequent pattern is F-x-A contained in approximately 30% of the sequences, followed by F-A, L-x(3)-A, L-x-F, and F-F each accounting for approximately 18% of the sequences. The F-x-A motif also occurs in islet amyloid poly peptide which may explain why 4G8 also recognizes amyloid fibrils of this peptide. Immunoselection of random sequences and deep sequencing may also be a facile and efficient means of determining residues critical for antibody binding and validating the specificity of monoclonal antibodies and polyclonal antisera.

No abstract

Autism is a complex disorder that arises from the pervasive action of genetic and epigenetic factors that alter synaptic connectivity of the brain. Although GABA and glutamate receptors seem to be two of those factors, very little is known about the functional properties of the autistic receptors. Autistic tissue samples stored in brain banks usually have relatively long postmortem times, and it is highly desirable to know whether neurotransmitter receptors in such tissues are still functional. Here we demonstrate that native receptors microtransplanted from autistic brains, as well as de novo mRNA-expressed receptors, are still functional and susceptible to detailed electrophysiological characterization even after long postmortem intervals. The opportunity to study the properties of human receptors present in diseased brains not only opens new avenues toward understanding autism and other neurological disorders, but it also makes the microtransplantation method a useful translational system to evaluate and develop novel medicinal drugs.autism ͉ human brain ͉ GABA receptors ͉ glutamate receptors