Frequency-modulation stimulated Raman scattering microscopy with an acousto-optic tunable filter.

Stimulated Raman scattering (SRS) microscopy is increasingly employed for highly specific, label-free, and high-speed bioimaging. Despite its benefits, SRS is susceptible to spurious background signals caused by competing effects, which lower the possible imaging contrast and sensitivity. An efficient approach to suppress these undesired background signals is frequency-modulation (FM) SRS, which exploits the competing effects' weak spectral dependence compared to the SRS signal's high spectral specificity. We propose an FM-SRS scheme realized with an acousto-optic tunable filter, which presents a few advantages compared to other solutions presented in the literature. In particular, it can perform automated measurements from the fingerprint to the CH-stretching region of the vibrational spectrum without any manual adjustment of the optical setup. Moreover, it allows simple all-electronic control of the spectral separation and relative intensities of the pair of probed wavenumbers.

TDP-43 loss of function inhibits endosomal trafficking and alters trophic signaling in neurons.

Nuclear clearance of TDP-43 into cytoplasmic aggregates is a key driver of neurodegeneration in amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD), but the mechanisms are unclear. Here, we show that TDP-43 knockdown specifically reduces the number and motility of RAB11-positive recycling endosomes in dendrites, while TDP-43 overexpression has the opposite effect. This is associated with delayed transferrin recycling in TDP-43-knockdown neurons and decreased ß2-transferrin levels in patient CSF Whole proteome quantification identified the upregulation of the ESCRT component VPS4B upon TDP-43 knockdown in neurons. Luciferase reporter assays and chromatin immunoprecipitation suggest that TDP-43 represses VPS4B transcription. Preventing VPS4B upregulation or expression of its functional antagonist ALIX restores trafficking of recycling endosomes. Proteomic analysis revealed the broad reduction in surface expression of key receptors upon TDP-43 knockdown, including ErbB4, the neuregulin 1 receptor. TDP-43 knockdown delays the surface delivery of ErbB4. ErbB4 overexpression, but not neuregulin 1 stimulation, prevents dendrite loss upon TDP-43 knockdown. Thus, impaired recycling of ErbB4 and other receptors to the cell surface may contribute to TDP-43-induced neurodegeneration by blocking trophic signaling.

An optimised version of the secretome protein enrichment with click sugars (SPECS) method leads to enhanced coverage of the secretome.

The secretome, the entirety of all soluble proteins either being secreted or proteolytically released by a cell, plays a key role in inter-cellular communication of multi-cellular organisms. Pathological alterations contribute to diseases such as hypertension, cancer, autoimmune disorders or neurodegenerative diseases. Hence, studying disease-related perturbations of the secretome and the secretome itself covers an important aspect of cellular physiology. We recently developed the secretome protein enrichment with click sugars (SPECS) method that enables the analysis of secretomes of in vitro cell cultures even in the presence of FCS with MS. So far, SPECS facilitated the identification of protease substrates of BACE1, SPPL3 and ADAM10. Though, the SPECS method has already enabled deep insights into secretome biology, we aimed to improve the SPECS protocol to obtain even more information from MS-based secretome analysis and reduce the amount of input material. Here, we optimised the reaction buffer, the pH and replaced Dibenzocyclooctyne (DBCO) PEG12-biotin with the more water-soluble variant DBCO-sulpho-biotin to finally provide an optimised protocol of the recently published SPECS protocol. Overall, the number of quantified glycoproteins and their average sequence coverage was increased by 1.6- and 2.4-fold, respectively. Thus, the opzimised SPECS protocol allows reducing the input material by half without losing information. These improvements make the SPECS method more sensitive and more universal applicable to cell types with limited availability.

Differential transgene expression patterns in Alzheimer mouse models revealed by novel human amyloid precursor protein-specific antibodies.

Alzheimer's disease (AD) is histopathologically characterized by neurodegeneration, the formation of intracellular neurofibrillary tangles and extracellular Aß deposits that derive from proteolytic processing of the amyloid precursor protein (APP). As rodents do not normally develop Aß pathology, various transgenic animal models of AD were designed to overexpress human APP with mutations favouring its amyloidogenic processing. However, these mouse models display tremendous differences in the spatial and temporal appearance of Aß deposits, synaptic dysfunction, neurodegeneration and the manifestation of learning deficits which may be caused by age-related and brain region-specific differences in APP transgene levels. Consequentially, a comparative temporal and regional analysis of the pathological effects of Aß in mouse brains is difficult complicating the validation of therapeutic AD treatment strategies in different mouse models. To date, no antibodies are available that properly discriminate endogenous rodent and transgenic human APP in brains of APP-transgenic animals. Here, we developed and characterized rat monoclonal antibodies by immunohistochemistry and Western blot that detect human but not murine APP in brains of three APP-transgenic mouse and one APP-transgenic rat model. We observed remarkable differences in expression levels and brain region-specific expression of human APP among the investigated transgenic mouse lines. This may explain the differences between APP-transgenic models mentioned above. Furthermore, we provide compelling evidence that our new antibodies specifically detect endogenous human APP in immunocytochemistry, FACS and immunoprecipitation. Hence, we propose these antibodies as standard tool for monitoring expression of endogenous or transfected APP in human cells and APP expression in transgenic animals.