High-throughput microscopy exposes a pharmacological window in which dual leucine zipper kinase inhibition preserves neuronal network connectivity

Abstract

Therapeutic developments for neurodegenerative disorders are redirecting their focus to the mechanisms that contribute to neuronal connectivity and the loss thereof. Using a high-throughput microscopy pipeline that integrates morphological and functional measurements, we found that inhibition of dual leucine zipper kinase (DLK) increased neuronal connectivity in primary cortical cultures. This neuroprotective effect was not only observed in basal conditions but also in cultures depleted from antioxidants and in cultures in which microtubule stability was genetically perturbed. Based on the morphofunctional connectivity signature, we further showed that the effects were limited to a specific dose and time range. Thus, our results illustrate that profiling microscopy images with deep coverage enables sensitive interrogation of neuronal connectivity and allows exposing a pharmacological window for targeted treatments. In doing so, we revealed a broad-spectrum neuroprotective effect of DLK inhibition, which may have relevance to pathological conditions that ar.e associated with compromised neuronal connectivity. Electronic supplementary material The online version of this article (10.1186/s40478-019-0741-3) contains supplementary material, which is available to authorized users.

Systematic quantification of synapses in primary neuronal culture

“…Yet, despite their ample use, the specificity and generalizability of many remains questionable. Therefore, we screened a panel of synapse-targeting antibodies for their labeling specificity on hippocampal and cortical cultures at 14 days in vitro (DIV), a time point at which synaptic connections are well-established (16, 25–27). Targets included proteins of pre- (vesicular, active zone) and postsynaptic (receptor, scaffold) compartments, as well as trans-synaptic adhesion proteins ( Suppl.…”

“…With 20 nm, the synaptic cleft is well below this limit. Thence, a simple approach to detect mature synapses – i.e., synapses that dispose of a clear pre- and postsynaptic compartment – with more certainty consists of quantifying the apparent overlap between pre- and postsynaptic markers (12, 13, 15, 16). We applied this method to synaptic marker antibodies that showed a crisp punctuate staining (evidenced by the ACF criteria defined above) and were raised in different species so that they could be combined in double stainings ( Fig.…”

“…For spot and synapse density quantification, multidimensional images were analyzed using a previously developed image analysis pipeline for Acapella software (PerkinElmer) (16). In brief, nuclei (DAPI) and neurites (MAP2) were segmented based on a user-defined fixed threshold.…”

“…For double stainings and PSD-mVenus experiments, the density of colocalizing spots in 2 fluorescence channels was calculated as those spots for which at least 1 pixel overlaps. We previously determined that measurements were not biased by chromatic aberration or the overlap criterion (16). For colocalization of antibodies with PSD95-mVenus, the percentage of PSD95-mVenus spots that have an overlapping synapse marker spot was measured.…”

“…However, using a single marker to identify synapses is complicated by the presence of non-synaptic ( e.g., vesicular) or degenerate synaptic structures. To focus more specifically on mature synapses, the colocalization between a pre- and postsynaptic marker has been introduced as readout (11–16). Several gene silencing screens have made use of this approach to identify regulators of excitatory and inhibitory synapses (12, 15).…”