Topoisomerase 1 inhibition reversibly impairs synaptic function

Abstract

Topotecan is a topoisomerase 1 (TOP1) inhibitor that is used to treat various forms of cancer. We recently found that topotecan reduces the expression of multiple long genes, including many neuronal genes linked to synapses and autism. However, whether topotecan alters synaptic protein levels and synapse function is currently unknown. Here we report that in primary cortical neurons, topotecan depleted synaptic proteins that are encoded by extremely long genes, including Neurexin-1, Neuroligin-1, Cntnap2, and GABA A β3. Topotecan also suppressed spontaneous network activity without affecting resting membrane potential, action potential threshold, or neuron health. Topotecan strongly suppressed inhibitory neurotransmission via pre-and postsynaptic mechanisms and reduced excitatory neurotransmission. The effects on synaptic protein levels and inhibitory neurotransmission were fully reversible upon drug washout. Collectively, our findings suggest that TOP1 controls the levels of multiple synaptic proteins and is required for normal excitatory and inhibitory synaptic transmission.synapse | topoisomerase | transcription T opoisomerase inhibitors such as topotecan (Hycamtin) are widely used to treat multiple forms of cancer, including brain metastases, ovarian cancer, and small cell lung cancer (1). Topoisomerases resolve DNA supercoiling during cell division and during gene transcription (2). Type I topoisomerases, encoded by Top1, Top3a, and Top3b in mammals, resolve supercoiling by cleaving a single strand of DNA, whereas type II topoisomerases, encoded by Top2a and Top2b, cleave both DNA strands (2). Recently, both types of topoisomerases were associated with neurodevelopment (3-5). For instance, topoisomerase 1 (TOP1) and topoisomerase 2 (TOP2) inhibitors transcriptionally up-regulate the paternal copy of Ubiquitin-protein ligase E3A (Ube3a) (5), a gene that affects synaptic activity and that is deleted or duplicated in distinct neurodevelopmental disorders (Angelman syndrome and autism, respectively) (6, 7). Moreover, a de novo mutation in Top1 and de novo mutations in genes that interact with Top1 and Top3b were identified recently in patients with autism (8, 9), whereas deletion of Top3b increases the risk for schizophrenia and intellectual disability (10, 11). Top2b is also required for axon outgrowth in different regions of the nervous system and for the survival of postmitotic neurons (12-15).TOP1 is localized primarily in the nucleus of postmitotic neurons and is expressed throughout the developing and adult brain (16), suggesting a nuclear function. Indeed, we recently found that topotecan, a selective TOP1 inhibitor, reduced the expression of extremely long genes (>200 kb) in postmitotic neurons by impairing transcription elongation (17). Topotecan and related camptothecin analogs inhibit TOP1 by covalently trapping the enzyme on DNA (2). TOP1 inhibitors also reduce the expression of long genes in cancer cell lines (17-19), revealing a gene length-dependent component to transcription that is common to sev...

Identification of early gene expression changes in primary cultured neurons treated with topoisomerase I poisons

“…Some of these long transcripts suppressed by CPT are linked to autism [25]. In CNs, many of these transcripts suppressed by CPT encode synaptic proteins and, as a result, excitatory and inhibitory neurotransmission is reversibility blocked by CPT [27]. These observations along with our study collectively highlight the importance of TOP1 in transcriptional regulation in neurons and could explain why they are more sensitive to TOP1 poisons like CPT and ActD.…”

Epigenetic Regulation of UBE3A and Roles in Human Neurodevelopmental Disorders

“…Together these results suggest that topetecan acts directly on the upstream PWS SNORD116 locus to reduce transcript levels of the downstream UBE3A-ATS . The effects of topotecan are not specific for only the PWS/AS locus, as topetecan treatment of mouse neurons reduces the level of multiple synaptic genes, including many that are implicated in ASD such as Cntnap2, Nrxn3 , and Cntn5 [85, 86]. Although topotecan may not be a suitable therapy for AS because of this lack of specificity, these studies do establish that a small molecule approach to the regulation of UBE3A in human neurons would be plausible.…”

Gene Length Matters in Neurons

“…These findings suggested that a length-dependent impairment of gene transcription, particularly in neural tissues and neurons where longer genes are overrepresented (Figure 1, 2) (Gabel et al, 2015), might impair synaptic function and contribute to neurodevelopmental disorders. In support of this possibility, topoisomerase inhibition reversibly depletes Neurexin-1 and Neuroligin-1 at the protein level and reversibly impairs synaptic function (Mabb et al., 2014). …”