In this study, we investigated if pyridostatin affects neuronal homeostasis. Pyridostatin decreased neuronal survival, damaged synapses, and promoted the formation of DNA DSBs. We found that, in neurons, pyridostatin downregulated a DNA DSB-repairing protein BRCA1 at the transcriptional level. Interestingly, in an in vitro gel shift experiment, a single–chain antibody raised against the G-quadruplex structures, binds to a synthetic oligonucleotide, which corresponds to the first putative G-quadruplex in the Brca1 gene promoter, indicating that our data are physiologically relevant. Neuronal G-quadruplexes in general and the G-quadruplex-dependent downregulation of transcription in neurons, therefore, might be a novel pathway of brain aging and a new target for mitigating age-associated neurodegeneration.

Chemotherapeutic drugs, such as doxorubicin, accelerate brain aging, weaken brain neuronal networks, and induce cognitive impairments [ 12 ]. Cancer patients who underwent a doxorubicin-based therapy exhibited elevated molecular markers of senescence, such as p16INK4a and ARF [ 13 ]. We demonstrated that doxorubicin induces severe neurotoxicity and DNA double–strand breaks (DSBs) in neurons, and impairs neuronal autophagy [ 14 , 15 ]. Doxorubicin intercalates into the duplex DNA, leading to the eviction of histone proteins from the chromatin [ 16 ]; however, doxorubicin can also interact with the G-quadruplex complexes, potentially stabilizing these structures [ 17 ].

DNA strands containing four stretches of guanine nucleotides are able to form tetra-stranded stable secondary structures called the G-quadruplex. Although the G-quadruplexes have been studied in vitro for years [ 5 ], these structures are clearly important in DNA recombination, replication, telomere maintenance, and regulation of transcription [ 6 , 7 ]. G-quadruplexes have been implicated in the pathogenesis of fragile X syndrome, frontotemporal dementia, and amyotrophic lateral sclerosis as the negative regulators [ 8 ]. Small molecules that stabilize the G-quadruplexes alter gene expression in cancer cells [ 9 ]. Guanine oxidation is enhanced in the genes, which are suppressed in the aged human brain [ 10 ]. Guanine oxidation increases the stability of the G-quadruplex [ 11 ]. However, how the G-quadruplexes regulate neuronal homeostasis is not clear.

Understanding the mechanisms of aging is a problem of paramount importance. Brain aging is a complex phenomenon, and its mechanisms are poorly understood. In physiological aging, as neurons become older, they exhibit changes in synaptic plasticity, gene transcription, and DNA methylation, and are less capable of degrading oxidized material and accumulate lipofuscin. These changes are observed in the absence of significant neurological phenotypes. In unsuccessful neuronal aging, there are multiple dramatic events, including abnormally enhanced DNA damage, accumulation of impaired organelles, and protein aggregates [ 1 , 2 ]. Neurons lose their synapses, processes, and degenerate. These changes are associated with neurological symptoms. It is not always clear why some age successfully and some do not [ 3 , 4 ].

Results

Pyridostatin is toxic for primary neurons The G-quadruplex is a tetra-stranded secondary DNA structure formed by four stretches of guanines (Fig. 1A-C) [6]. Pyridostatin is a very selective G-quadruplex DNA-binding small molecule designed to form a complex with and stabilize G-quadruplex structures [9,18,19]. For cell lines, the drug is cytotoxic, and we, therefore, hypothesized that pyridostatin may be toxic for primary neurons as well. To test this, primary rat cortical cultures were transfected with the mApple construct (a red fluorescent protein). Pyridostatin or vehicle was added, and the mApple-expressing neurons were tracked for several days with an automated microscope. Loss of the red mApple fluorescence is a sensitive marker of neuronal death [20]. This method allows us to track large cohorts of individual neurons over their lifetimes and to sensitively measure their survival with statistical analyses used in clinical medicine [20]. Analyzing when each neuron lost its fluorescence allows us to measure neuronal survival with cumulative hazard statistics. By following neurons over their lifetimes, we can determine if the applied drug contributes positively, negatively, or even neutrally to neuronal fate (Fig. 1D). We found that treatment with pyridostatin substantially increased the risk of neuronal death in a dose-dependent manner (Fig. 1E). Notably, neurons exposed to pyridostatin retracted neurites before death (Fig. 1D), mimicking neurodegenerative processes commonly observed in neurons that express α-synuclein, mutant LRRK2 or mutant huntingtin [21,22]. Figure 1. Pyridostatin is neurotoxic for primary cortical neurons. (A) G-quadruplex is a non-canonical DNA secondary structure. Four guanine molecules (a single guanine is in red) can assemble into a square planar structure. The structure of a G-quadruplex is stabilized by hydrogen bonds between guanines and the interactions with a monovalent cation (Na+ or K+) resided in the central channel. (B) Repetitive guanine-rich DNA or RNA sequences have the potential to form G-quadruplex structures (C). (D) An example of survival analysis. Primary cortical neurons were transfected with mApple (a morphology and viability marker) and tracked with an automated microscope. Images collected every 24 h demonstrate the ability to return to the same field of neurons and to follow them over time. Each image is a montage of non-overlapping images captured in one well of a 24-well plate. Scale bar is 100 μm. A region from the original images at different time points is zoomed in to demonstrate longitudinal single-cell tracking (bottom panel). Red arrows depict two neurons that degenerate before 96 h after transfection. Note that neurites of the neuron 1 retract overtime. Scale bar is 20 μm. (E) Primary cortical neurons were transfected with mApple and treated with a vehicle or with different concentrations of pyridostatin (PDS; 0.01–5 μM). Transfected neurons were tracked with an automated microscope. Risk of death curves demonstrate that pyridostatin is neurotoxic. ***p<0.0001 (log-rank test). N.s., non-significant. Three hundred neurons were analyzed from three independent experiments. (F) Primary cortical neurons were treated with a vehicle (upper panel, control) or with 1 μM pyridostatin overnight (lower panel; PDS), fixed and stained with antibodies against MAP2c (red) and synapsin (green). Scale bar is 10 μm. (G) Quantification of the synapsin fluorescence intensity from (F). ***p<0.0001 (t-test). (H) Quantification of the neurite density from (F). MAP2c staining was used by the algorithm to identify and analyze neurites. ***p<0.001 (t-test). Three hundred neurons were analyzed from two independent experiments.

Neurite retraction in pyridostatin-treated neurons suggests that neurons have lost their synapses as well. Cultures were treated with pyridostatin and fixed before neurons started dying. Expectedly, we found that pyridostatin reduced the density of synapses. Pyridostatin-treated neurons also had less neurites than vehicle-treated neurons (Fig. 1F–H).

Pyridostatin induces DNA DSBs Putative G-quadruplexes are present on average once per 10 kb of the human genome [23]. In addition to inhibiting DNA replication, the pyridostatin–DNA complex may stall DNA polymerase during transcription [19,24]. DNA damage may then occur via the action of endonucleases, through a mechanism of so-called transcription–coupled–repair poisoning [25]. Since pyridostatin induces DNA DSBs in cell lines [9], we hypothesized that pyridostatin may induce DNA damage in primary neurons. To analyze DNA damage in neurons in our lab, we recapitulated a recent single–cell analysis technique based on the expression of a truncated 53BP1 DSB reporter in cell lines [26]. The mApple-53BP1trunc construct was expressed in two cohorts of primary neurons along with GFP, a marker of cell viability and morphology. The first neuronal cohort was treated with a vehicle; the second cohort was treated with pyridostatin. Both neuronal cohorts were then followed longitudinally (Fig. 2A). Expectedly, the mApple-53BP1trunc construct formed puncta in neurons exposed to pyridostatin, reflecting DNA damage (Fig. 2B). Figure 2. Pyridostatin promotes the formation of 53BP1-positive puncta in primary neurons. (A) Primary cortical neurons were transfected with GFP and mApple-53BP1trunc constructs, and then treated with a vehicle (left panel; control) or with 1 μM pyridostatin (right panel; PDS). Neurons were imaged with an automated microscope every 24 h for 3 days. Scale bar is 5 μm. (B) Quantification of the mApple-53BP1trunc puncta index from (A) at different times. The puncta index was estimated by measuring the standard deviation of the 53BP1 fluorescence intensity. Note that 53BP1 puncta index is higher in pyridostatin-treated neurons than control neurons. *p<0.01, **p<0.001, and ***p<0.0001 (t-test). A.u., arbitrary units. Two hundred neurons were analyzed from two independent experiments. (C) Primary cortical neurons were treated with a vehicle (upper panel; control) or with 1 μM pyridostatin (bottom panel; PDS) overnight, fixed, and stained for MAP2c (red), a marker of DNA damage 53BP1 (green), and with the nuclear Hoechst dye (blue). Scale bar is 10 μm. (D) Quantification of the 53BP1 puncta index from (C). Pyridostatin (PDS) increased the 53BP1 puncta index compared to control neurons (cont). ***p<0.0001 (t-test). A.u., arbitrary units. Three hundred neurons were analyzed from three independent experiments.

Since overexpressed proteins sometimes aggregate in primary neurons, we decided to confirm our finding with the endogenous 53BP1 protein. Cultured neurons were treated with pyridostatin, fixed and stained for endogenous 53BP1 and MAP2c, and with the nuclear DAPI dye. Neurons exposed to pyridostatin had punctuated structures of 53BP1 localized to their nuclei (Fig. 2C,D). Phosphorylated histone H2A variant X (γH2A.X) is commonly used as a read-out of DNA DBSs [14]. Therefore, to make sure that γH2A.X is phosphorylated when neurons are treated with pyridostatin, as seen in non-neuronal cells, neuronal cultures were treated with pyridostatin, fixed and stained for γH2A.X and MAP2c, and with the nuclear DAPI dye. Neurons exposed to pyridostatin had more staining of γH2A.X in the neuronal nuclei than control neurons (Fig. 3A,B). Our data suggest that stabilized G-quadruplexes contribute to DNA damage in neuronal cells. Figure 3. G-quadruplex-stabilizing drugs upregulate γH2A.X. (A) Primary cortical neurons were treated with a vehicle (control) or with 1 μM pyridostatin (PDS) or with 200 nM bortezomib (bortezomib) or with 10 μM cisplatin (cisplatin) overnight, fixed, and stained for MAP2c (red), a marker of DNA DSBs phosphorylated histone H2A variant X, γH2A.X (green), and with the nuclear Hoechst dye (blue). Scale bar is 10 μm. (B) The puncta index was estimated by measuring the standard deviation of the γH2A.X fluorescence intensity. Primary cortical neurons were treated with a vehicle (control, cont) or with 1 μM pyridostatin (PDS), or with 200 nM bortezomib (bort), or with 10 μM cisplatin (cisp) overnight, then fixed, and immunostained with antibodies against MAP2c and γH2A.X, and co-stained with the nuclear Hoechst dye (blue). ***p<0.0001 (t-test). N.s., non-significant (p bort =0.1995; p cis =0.8228). A.u., arbitrary units. Four hundred neurons were analyzed from three independent experiments. (C) Primary cortical neurons were treated with a vehicle (control, cont) or with a G-quadruplex stabilizing drug, TmPyP4 (1 μM, overnight), then fixed, immunostained against MAP2c and γH2A.X, and co-stained with the nuclear Hoechst dye (blue). ***p<0.0001 (t-test). A.u., arbitrary units. Four hundred neurons were analyzed from three independent experiments.

Since DNA damage may, in principle, be a general phenomenon in degenerating neurons, we determined if other neurotoxic small molecules, bortezomib and cisplatin, promote DNA DSBs. Bortezomib, a chemotherapy drug that inhibits the ubiquitin-proteasome system (UPS), was used to promote neurodegeneration. Inhibitors of the UPS are highly neurotoxic [27]. Cisplatin, another anti-tumor agent, cross links DNA and, therefore, promotes cytotoxicity [28]. Cisplatin is also highly toxic for neurons [29]. Primary neurons were treated with bortezomib and cisplatin, and then fixed and stained for γH2A.X and MAP2c. Neither bortezomib nor cisplatin induced DNA DSBs (Fig. 3А,B). Our data demonstrate that DNA DSBs are not a general effect induced by cytotoxic agents, and pyridostatin-associated DNA damage likely depends on pyridostatin’s G-quadruplex-stabilizing properties. Do other G-quadruplex-stabilizing drugs induce DNA DSBs? We tested if TmPyP4 [30], а cationic porphyrin, induces accumulation of γH2A.X-positive puncta in cultured primary neurons. Neuronal cultures were treated with TmPyP4 under conditions identical to the pyridostatin experiment, fixed and stained for γH2A.X and MAP2c, and with the nuclear DAPI dye. TmPyP4 stimulated the formation of γH2A.X-positive puncta in neurons (Fig. 3C).

Pyridostatin downregulates BRCA1 in neurons The breast cancer type 1 susceptibility protein, BRCA1, repairs DNA DSBs in non-neuronal cells and neurons. In addition to well-studied BRCA1 dysregulation in cancer cells, BRCA1 protein is downregulated in the brains of patients with Alzheimer's disease. Amyloid-beta, a peptide associated with Alzheimer's disease, downregulates BRCA1 leading to the formation of DNA DSBs [31]. The Brca1 gene in Rattus norvegicus is a long gene that has two known alternatively spliced RNA products. We hypothesized that the Brca1 gene may contain putative G-quadruplexes. We analyzed the rat Brca1 gene sequence with the QGRP (putative quadruplex forming G-rich sequences) mapper and discovered that, in the gene, two putative sequences in the coding sequence can arrange into the G-quadruplexes. Additionally, its introns contain four putative G-quadruplex-forming sequences. There are also two putative G-quadruplex-forming sequences in the Brca1 gene promoter (Fig. 4A). Therefore, pyridostatin may stabilize these putative G-quadruplexes and inhibit the Brca1 gene transcription. Figure 4. Pyridostatin downregulates BRCA1 levels in primary neurons. (A) A scheme of the Brca1 gene and its promoter showing putative G-quadruplex sequences. (B) Primary neurons were treated with a vehicle (control, -) or with different concentrations of pyridostatin (PDS; 1, 2 or 5 μM) overnight. Then neurons were collected, lysed, and samples were processed with western blotting. Note that pyridostatin reduced BRCA1 protein levels in a dose-dependent manner. Actin was used as a loading control. (C) Quantification of BRCA1 protein levels normalized to actin from (B). **p (cont-1 μM) =0.005, **p (cont-2 μΜ) =0.0015, **p (cont-5 μΜ) =0.0043 (ANOVA). N.s., non-significant, p (1 μΜ−2 μΜ) =0.0547, p (1 μΜ−5 μΜ) =0.0854. Results were pooled from five independent experiments. (D) Primary neurons were treated with a vehicle (control, -) or with a G-quadruplex stabilizing drug, TmPyP4 (1 μM), overnight. Neurons were lysed, and samples were processed by western blotting. TmPyP4 reduced BRCA1 protein levels. Actin was used as a loading control. (E) Quantification of BRCA1 protein normalized to actin from (D). *p<0.0276 (t-test). Results were pooled from four independent experiments. (F) Primary neurons were treated with a vehicle (control; -) or with 2 μM pyridostatin (PDS; +) overnight. The expression of Brca1 was determined by qRT-PCR relative to Actin. *p=0.0445 (t-test). Results were pooled from three independent experiments. (G) Primary neurons were treated with a vehicle (control; -) or with 2 μM pyridostatin (PDS; +) overnight. Relative expression of the Actin, Gapdh and Tbp genes was determined by qRT-PCR. *p=0.0112, **p=0.0039 (t-test). N.s., non-significant (p=0.7583). Results were pooled from three independent experiments. (H) Primary neurons were treated with a vehicle (control; -) or with 2 μM pyridostatin (PDS; +) overnight. The expression of Brca1 was determined by qRT-PCR normalized to Tbp. **p=0.0033 (t-test). Results were pooled from three independent reactions.

First, we tested if the levels of the BRCA1 protein are changed in neurons exposed to pyridostatin. Cultured cortical neurons were treated with pyridostatin, and then their extracts were analyzed by western blotting. Remarkably, the levels of the BRCA1 protein were indeed decreased (Fig. 4B,C). Levels of actin in samples were not affected, suggesting that the actin protein itself is not a target of pyridostatin. To confirm this finding, we investigated if TmPyP4 reduces the levels of BRCA1 in neurons. The BRCA1 protein levels were downregulated in TmPyP4-treated neurons as well (Fig. 4D,E). We, therefore, conclude that, in primary neurons, stabilizing G-quadruplex DNA downregulates BRCA1, at least at the protein level. As a result, DNA DSBs can accumulate due to the action of nucleases and to downregulated BRCA1, at least in part. Pyridostatin was designed to selectively bind to G-quadruplex DNA and to stabilize the G-quadruplex structures [9,18,19]. Nevertheless, the drug can theoretically inhibit protein translation or even affect its degradation. To test whether transcription of the Brca1 gene is directly affected, we sought to investigate whether pyridostatin changes BRCA1’s mRNA levels in neuronal cells exposed to the drug. Neuronal cultures were treated with pyridostatin; mRNA was extracted and analyzed. Remarkably, the levels of BRCA1 mRNA were higher in pyridostatin-treated neurons, when normalized to the control actin mRNA (Fig. 4F). An increase in BRCA1 mRNA levels was unexpected, but could reflect a change in the levels of actin mRNA itself in pyridostatin-treated neurons. To test that, we measured the levels of actin mRNA in vehicle-treated neurons and in neurons exposed to pyridostatin, and discovered that, indeed, actin mRNA was downregulated, indicating that pyridostatin targets the Actin gene as well. Interestingly, we did not observe a decrease in the levels of the actin protein (Fig. 4B), which indicates that actin is a long-lived protein. Searching for other loading controls, we tested mRNA levels of glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and TATA-binding protein (TBP). Interestingly, neurons exposed to pyridostatin had reduced levels of GAPDH, but the levels of TBP mRNA remained unchanged (Fig. 4G). Therefore, TBP mRNA was used as loading control, and in this case, BRCA1 mRNA was downregulated in neurons treated to pyridostatin (Fig. 4H). We were surprised that pyridostatin downregulated BRCA1, actin, and GAPDH, but not TBP mRNAs. We, therefore, analyzed the sequences of the Actin, Gapdh and Tbp genes and their promoters with the QGRP mapper. Both Actin’s and Gapdh’s promoters contain two putative G-quadruplex-forming sequences, and Actin contains one putative G-quadruplex sequence. Remarkably, neither Tbp nor its promoter contains a putative G-quadruplex-forming sequence, indicating that our data generated with pyridostatin are physiologically relevant. Future studies will identify more genes regulated by the G-quadruplex.

Overexpressed BRCA1 mitigates pyridostatin-induced DNA damage BRCA1 is required for DNA damage repair. We wondered if ectopically increasing BRCA1 protein levels would attenuate DNA damage associated with the exposure to pyridostatin. To test this, neurons were transfected with a plasmid that encodes GFP or GFP-BRCA1. Neurons were treated with a vehicle or with pyridostatin overnight and then fixed and immunostained with antibodies against γH2A.X or 53BP1 (Fig. 5A–D). The γH2A.X and 53BP1 puncta indexes in GFP-BRCA1-expressing neurons treated with pyridostatin were lower than in GFP-expressing neurons exposed to pyridostatin (Fig 5A–D). These results indicate that ectopically expressed BRCA1 mitigates DNA damage induced by pyridostatin treatment in neurons and demonstrate that BRCA1 is critical for maintaining neuronal genome integrity. Figure 5. Ectopically expressed BRCA1 mitigates DNA damage associated with pyridostatin treatment. (А) Primary cortical neurons were transfected with GFP or with GFP-BRCA1 constructs, and then treated with a vehicle (control, GFP) or with 1 μM pyridostatin (GFP+PDS and GFP-BRCA1+PDS), overnight, fixed, and stained for a marker of DNA DSBs phosphorylated histone H2A variant X, γH2A.X (red). Scale bar is 10 μm. (B) The puncta index was estimated by measuring the standard deviation of the γH2A.X fluorescence intensity from (A). ***p<0.0001 (ANOVA). N.s., non-significant, p=0.243. A.u., arbitrary units. Two hundred neurons were analyzed from two independent experiments. (C) Primary cortical neurons were transfected with GFP or with GFP-BRCA1 constructs, and then treated with a vehicle (control, GFP) or with 1 μM pyridostatin (GFP+PDS, GFP-BRCA1+PDS), overnight, fixed, and stained for a marker of DNA DSBs 53BP1 (red). Scale bar is 10 μm. (D) The puncta index was estimated by measuring the standard deviation of the γH2A.X fluorescence intensity from (C). ***p<0.0001 (ANOVA). N.s., non-significant, p=0.441. A.u., arbitrary units. Two hundred neurons were analyzed from two independent experiments.

