In the version of this article initially published, the catalog numbers for BoNT A and B were given in the Methods section as T0195 and T5644; the correct numbers are B8776 and B6403. The error has been corrected in the HTML and PDF versions of the article.

Hypermethylation of the FMR1 gene results in decreased expression of FMRP protein which is the underlying cause of Fragile X Syndrome, an incurable neurological disorder characterized by mental retardation, anxiety, epileptic episodes and autism. Disease modifying therapies for Fragile X are thus aimed at treatments increasing FMRP expression levels in the brain. Here we describe the development and characterization of two homogenous immunoassays for quantitative detection of FMRP protein using time-resolved FRET. The assays display high stability and reproducibility and can be used to quantify endogenous FMRP in human fibroblast. Importantly, due to the simplicity of the assay protocol, the method is well suited to be used in high-throughput screening applications to identify compounds or genetic interventions that result in increased FMRP levels in human cells.

Heterologous expression of the functional amyloid beta (Aβ) antibody β1 in the central nervous system was engineered to maximize antibody exposure in the brain and assess the effects on Aβ production and accumulation in these conditions. A single open reading frame encoding the heavy and light chains of β1 linked by the mouth and foot virus peptide 2A was expressed in brain neurons of transgenic mice. Two of the resulting BIN66 transgenic lines were crossed with APP23 mice, which develop severe central amyloidosis. Brain concentrations at steady-state 5 times greater than those found after peripheral β1 administration were obtained. Similar brain and plasma β1 concentrations indicated robust antibody efflux from the brain. In preplaque mice, β1 formed a complex with Aβ that caused a modest Aβ increase in brain and plasma. At 11 months of age, β1 expression reduced amyloid by 97% compared with age-matched APP23 mice. Interference of β1 with β-secretase cleavage of amyloid precursor protein was relatively small. Our data suggest that severely impaired amyloid formation was primarily mediated by a complex of β1 with soluble Aβ, which might have prevented Aβ aggregation or favored transport out of the brain.

Hypermethylation of the fragile X mental retardation 1 gene FMR1 results in decreased expression of FMR1 protein FMRP, which is the underlying cause of Fragile X syndrome – an incurable neurological disorder characterized by mental retardation, anxiety, epileptic episodes and autism. Disease-modifying therapies for Fragile X syndrome are thus aimed at treatments that increase the FMRP expression levels in the brain. We describe the development and characterization of two assays for simple and quantitative detection of FMRP protein.

Background Huntington's disease (HD) is a progressive neurodegenerative disorder caused by a CAG repeat expansion within the huntingtin gene. Mutant huntingtin protein misfolds and accumulates within neurons where it mediates its toxic effects. Promoting mutant huntingtin clearance by activating macroautophagy is one approach for treating Huntington's disease (HD). In this study, we evaluated the mTOR kinase inhibitor and macroautophagy promoting drug everolimus in the R6/2 mouse model of HD. Results Everolimus decreased phosphorylation of the mTOR target protein S6 kinase indicating brain penetration. However, everolimus did not activate brain macroautophagy as measured by LC3B Western blot analysis. Everolimus protected against early declines in motor performance; however, we found no evidence for neuroprotection as determined by brain pathology. In muscle but not brain, everolimus significantly decreased soluble mutant huntingtin levels. Conclusions Our data suggests that beneficial behavioral effects of everolimus in R6/2 mice result primarily from effects on muscle. Even though everolimus significantly modulated its target brain S6 kinase, this did not decrease mutant huntingtin levels or provide neuroprotection.

Background The mutation in Huntington's disease is a polyglutamine expansion near the N-terminus of huntingtin. Huntingtin expressed in immortalized neurons is cleaved near the N-terminus to form N-terminal polypeptides known as cleavage products A and B (cpA and cpB). CpA and cpB with polyglutamine expansion form inclusions in the nucleus and cytoplasm, respectively. The formation of cpA and cpB in primary neurons has not been established and the proteases involved in the formation of these fragments are unknown. Results Delivery of htt cDNA into the mouse striatum using adeno-associated virus or into primary cortical neurons using lentivirus generated cpA and cpB, indicating that neurons in brain and in vitro can form these fragments. A screen of small molecule protease inhibitors introduced to clonal striatal X57 cells and HeLa cells identified compounds that reduced levels of cpA and are inhibitors of the aspartyl proteases cathepsin D and cathepsin E. The most effective compound, P1-N031, is a transition state mimetic for aspartyl proteases. By western blot analysis, cathepsin D was easily detected in clonal striatal X57 cells, mouse brain and primary neurons, whereas cathepsin E was only detectible in clonal striatal X57 cells. In primary neurons, levels of cleavage product A were not changed by the same compounds that were effective in clonal striatal cells or by mRNA silencing to partially reduce levels of cathepsin D. Instead, treating primary neurons with compounds that are known to inhibit gamma secretase activity either indirectly (Imatinib mesylate, Gleevec) or selectively (LY-411,575 or DAPT) reduced levels of cpA. LY-411,575 or DAPT also increased survival of primary neurons expressing endogenous full-length mutant huntingtin. Conclusion We show that cpA and cpB are produced from a larger huntingtin fragment in vivo in mouse brain and in primary neuron cultures. The aspartyl protease involved in forming cpA has cathepsin-D like properties in immortalized neurons and gamma secretase-like properties in primary neurons, suggesting that cell type may be a critical factor that specifies the aspartyl protease responsible for cpA. Since gamma secretase inhibitors were also protective in primary neurons, further study of the role of gamma-secretase activity in HD neurons is justified.

The quantification of cellular proteins is essential for the study of many different biological processes. This study describes an assay for the detection of the intracellular mutant huntingtin, the causative agent of Huntington's disease, with a method that may be generally applicable to other cellular proteins. A small recombinant protein tag that is recognized by a pair of readily available, high-affinity monoclonal antibodies was designed. This tag was then added to an inducible fragment of the mutant huntingtin protein by genetic engineering. We show that it is possible to use time-resolved FRET to detect low intracellular levels of huntingtin by a simple lysis and detection procedure. This assay was then adapted into a homogeneous, miniaturized format suitable for screening in 1536-well plates. The use of time-resolved FRET also permits the assay to be multiplexed with a standard readout of cell toxicity, thus allowing the identification of conditions causing reduction of protein levels simply due to cytotoxicity. The screening results demonstrated that the assay is able to identify compounds that modulate the levels of huntingtin both positively and negatively and that represent valuable starting points for drug discovery programs.