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. Author manuscript; available in PMC: 2026 Feb 1.
Published in final edited form as: Brain Behav Immun. 2024 Nov 13;124:1–8. doi: 10.1016/j.bbi.2024.11.018

Prolonged STAT1 signaling in neurons causes hyperactive behavior

Danielle N Clark 1,2, Shelby V Brown 2, Li Xu 2, Rae-Ling Lee 2, Joey V Ragusa 3, Zhenghao Xu 2, Joshua D Milner 4, Anthony J Filiano 1,2,3,5
PMCID: PMC11745914  NIHMSID: NIHMS2036420  PMID: 39542073

Abstract

The interferon (IFN)-induced STAT1 signaling pathway is a canonical immune pathway that has also been implicated in regulating neuronal activity. The pathway is enriched in brains of individuals with autism spectrum disorder (ASD) and schizophrenia (SZ). Over-activation of the STAT1 pathway causes pathological transcriptional responses, however it is unclear how these responses might translate into behavioral phenotypes. We hypothesized that prolonged STAT1 signaling in neurons would be sufficient to cause behavioral deficits associated with neurodevelopmental disorders. In this study, we developed a novel mouse model with the clinical STAT1 gain-of-function mutation, T385M, in neurons. These mice were hyperactive and displayed neural hypoactivity with less neuron counts in the caudate putamen. Driving the STAT1 gain-of-function mutation exclusively in dopaminergic neurons, which project to the caudate putamen of the dorsal striatum, mimicked some hyperactive behaviors without a reduction of neurons. Moreover, we demonstrated that this phenotype is neuron specific, as mice with prolonged STAT1 signaling in all excitatory or inhibitory neurons or in microglia were not hyperactive. Overall, these findings suggest that STAT1 signaling in neurons is a crucial player in regulating striatal neuron activity and aspects of motor behavior.

Keywords: STAT1, neurons, hyperactivity, caudate putamen, interferons

1. Introduction

Neurodevelopmental disorders such as autism spectrum disorder (ASD) and schizophrenia (SZ) have traditionally been thought of as disorders of the synapse (Gauthier et al., 2011; Sacai et al., 2020; Zaslavsky et al., 2019). However, recent work has uncovered a role for immune dysregulation and neuroinflammation in these disorders. Interestingly, the risk for ASD is increased in children born to mothers who are hospitalized for infection during pregnancy (Atladóttir et al., 2010; Zerbo et al., 2015). Similarly, studies utilizing the maternal immune activation (MIA) model in mice demonstrate that elevated cytokine levels (including type-I IFNs) and inflammation in the developing brain are detrimental to neurodevelopment, and cause ASD- and SZ-like behavioral and cognitive deficits in the offspring (Ben-Yehuda et al., 2020; Malkova et al., 2012; Minakova and Warner, 2018; Pang et al., 2016; Smith et al., 2007). These studies suggest inflammation during early development may contribute to pathophysiology of various neurodevelopmental disorders.

Many recent studies examined transcriptomic data from post-mortem brains of individuals with ASD and SZ and demonstrated that the most highly upregulated genes are immune-related and include those in the IFN response (Gandal et al., 2018; Gupta et al., 2014; Voineagu et al., 2011). The IFN response is a well-defined immune signaling pathway that is activated in the context of viral infections. Upon viral recognition, immune cells produce and release IFNs, cytokines that activate the downstream transcription factor STAT1. IFN-γ activates STAT1 as a homodimer and IFN-α/β (type I IFNs) activate STAT1 as a heterodimer (with STAT2 and IRF9) to regulate the expression of pro-inflammatory genes (Schroder et al., 2004), including genes enriched in ASD brains. Although microglia, the resident immune cell of the CNS, are often suspected as the main targets of IFNs in the brain, we and others have demonstrated that neurons also respond to IFNs and have differential STAT1 activation in response to physiological versus pathological signaling (Clark et al., 2023). Neurons require low physiological levels of IFN-γ for proper development, function, and homeostasis (Filiano et al., 2016; Flood et al., 2019; Janach et al., 2020; Nagakura et al., 2014), but higher pathological levels can cause altered excitability (Vikman et al., 2003; Vikman et al., 2001; Vikman et al., 2005), morphological differences (Wong et al., 2004), and neurotoxicity (Imitola et al., 2023; Mizuno et al., 2008). Human iPSC-derived neuronal progenitor cells (NPCs) and neurons treated with pathological levels of IFN-γ exhibited similar gene dysregulation as in the brains of individuals with ASD (Warre-Cornish et al., 2020). STAT1 also targets neuron-specific genes in IFN-γ-treated HeLa cells (Satoh and Tabunoki, 2013), including NRCAM and SHANK2, which have been identified as risk factors for ASD. Additionally, deleting the IFN-γ receptor or STAT1 in neurons caused social deficits and hyperexcitability (Filiano et al., 2016). Taken together, these data suggest that dysregulated STAT1 signaling in neurons may drive neuronal dysfunction in neurodevelopmental disorders (Clark et al., 2022).

We previously demonstrated that developing neurons exposed to high levels of IFN-γ exhibit prolonged STAT1 signaling and persistent transcriptional changes, including dysregulation of pathways related to neurodevelopment (Clark et al., 2023). In this study, we investigated the effects of prolonged STAT1 signaling by utilizing a clinical heterozygous Stat1T385M gain-off-unction mutation (GOF) (Sampaio et al., 2013) (Scott et al., 2023). Individuals with this somatic mutation suffer from primary immune regulation disorders, but neurological manifestations, such as seizures, attention lapses, and cognitive disability have also been reported as a comorbidity (Toubiana et al., 2016), though no comprehensive studies utilizing systematic neuropsychiatric testing have been conducted. Utilizing various Cre models to induce Stat1T385M in different cell types, we aimed to elucidate which cell types are sensitive to prolonged STAT1 signaling. We found that prolonged STAT1 signaling in neurons, specifically in dopaminergic neurons, was sufficient to cause hyperactive behaviors. This work not only uncovers a pathological readout for STAT1 gain-of-function mutations in the brain but gives of a better understanding how STAT1 generally regulates proper neural activity and behavior.

2. Methods

2.1. Animals

Syn1-Cre (#003966) (Zhu et al., 2001), Vgat1-Cre (#028862) (Vong et al., 2011), Vglut1-Cre (#037512) (Harris et al., 2014), Vglut2-Cre (#028863) (Pauli et al., 2022; Vong et al., 2011), CaMKIIα-Cre (#005359) (Tsien et al., 1996), Dat-Cre (#006660) (Bäckman et al., 2006) and Csf1R-Cre (#029206) (Loschko et al., 2016) mice were originally purchased from The Jackson Laboratory and bred in-house. Stat1T385M flox mice containing the inducible Stat1T358M mutation were generated by Ozgene and generously provided by Drs. Joshua D. Milner and John J. O’Shea (Figure S1A). All mice used in this study were group housed on ventilated racks under standard 12-hour light/dark conditions. Mice were fed a 5053 or 5R53 PicoLab Rodent diet ad libitum and had access to water through a plumbed Lixit system. All behavior experiments were conducted between 9am and 6pm and performed in accordance with Duke University Institutional Animal Care and Use Committee’s policies. Each specific individual behavioral test was conducted at the same time of day for each cohort.

Allen Brain Atlas was used as a reference to confirm which brain regions expressed Cre in each Cre-model (Figure S2A; Available at https://connectivity.brain-map.org/transgenic). Each cell-specific Cre line’s promoter expresses Cre at different stages of development (Figure S2B). The Stat1T385M line was bred by crossing heterozygous Cre (SynI, Dat, Vgat, Vglut1, Csf1R) with a homozygous Stat1T385M, or by crossing homozygous Cre (Vglut2, CaMKIIα) with heterozygous Stat1T385M. Because the SynI-Cre has been reported to spontaneously recombine in the testes, thus causing offspring to inherit a germline floxed allele (Rempe et al., 2006), we only utilized female SynI-Cre mice for breeding. Adult mice (6+ weeks old) were used for all behavior assays.

The Stat1T385M flox mouse has a clinical T385M point mutation in exon 14 of the endogenous Stat1 gene and cDNA encoding exons 13–25 of the wildtype Stat1 allele followed by a stop codon and flanked by loxP sites inserted before the endogenous exon 13 (Figure S1A). In cell types not expressing Cre, the endogenous Stat1 gene is transcribed through exon 12 and then the wildtype exons 13–25 cDNA is transcribed up until the stop codon, resulting in wildtype Stat1 expression. In cells expressing Cre, the gene is recombined at the loxP sites, resulting in the wildtype cDNA insert and stop codon being excised out, and the entire endogenous Stat1 gene with the T385M mutation being transcribed. Recombination was confirmed by genotyping isolated genomic DNA from neurons and microglia after cell sorting. To isolate genomic DNA from neurons, SynI-Cre+ and Cre− Stat1T385M flox mice were first perfused with PBS and brains removed. Brains were placed into ice cold nuclei isolation buffer (25 mM KCl, 5 mM MgCl2, 10 mM Tris-Cl (pH 8.8), 250 mM sucrose, 1mM dithiothreitol (DTT)) and tissues minced into small pieces using spring scissors then a dounce homogenizer as previously described (Chronister et al., 2019). The homogenate was passed through a 40μM strainer then carefully layered on the top of a OptiPrep gradient (MilliporeSigma; cat# D1556). After spinning at 10,000g 40°C for five minutes, the nuclear pellet was recovered. The nuclei were then stained with Draq5 (Thermo Fisher Scientific; cat# 65–0880-92), and Alexa Fluor 488 conjugated anti-NeuN antibody (MilliporeSigma; cat# MAB377X). NeuN positive nuclei were sorted using an Astrios Cell Sorter (Beckman). DNA was extracted using PureLink Genomic DNA mini kit (Thermo Fisher Scientific; cat# K182001) before running PCR and imaging on an agarose gel (Figure S2C). To isolate genomic DNA from microglia, Csf1r-Cre+ and Cre− Stat1T385M flox mice were first perfused with PBS and brains removed. Brains were minced into small pieces with scissors in petri dish, and digested with digestion buffer (HBSS (Cytiva, cat # SH30268.01) with 2 mg/mL papain (MilliporeSigma, cat # P3125), and 50 U/mL DNase-I (MilliporeSigma, cat # 04716728001)). Microglia and the non-microglia flow through were collected using CD11b microbeads (Miltenyi Biotec, cat # 130–093-634) according to the manufacturer’s instructions. DNA was extracted for PCR and run on an agarose gel (Figure S2D). To determine the specificity of the Dat-Cre, Dat-Cre+ and Cre− Stat1T385M flox mice were first perfused with PBS and brains removed. Parts of the cortex and the substantia nigra were microdissected in nuclei isolation buffer prior to FAC sorting nuclei as described above (Figure S2E).

2.2. Open field

Prior to all behavior assays, mice habituated to the behavior room for at least one hour. Mice were handled by the experimenter on at least 3 separate days prior to beginning experiments. Mice were placed into a novel 40.5 cm × 40.5 cm arena and allowed to roam freely for 15 minutes. Mice were tracked and activity was analyzed using TopScan (CleverSys Inc). The area designated “center of arena” was the inner 20 cm × 20 cm (10 cm away from each wall). Arenas were wiped clean with 70% ethanol between mice.

2.3. Marble burying

Clean mouse cages were set up with approximately 7 cm of clean bedding and 20 clean marbles were arranged in 5 rows of 4 on top of the bedding. Mice were placed in the cages and allowed to roam freely for 20 minutes. To analyze the number of marbles buried over time, images of each cage were captured at 5, 10, and 15 minutes without removing the mouse from the cage, and at 20 min after removing the mouse from the cage. Between mice, the top layer of bedding was removed and replaced with fresh bedding and marbles were cleaned in 70% ethanol and then rinsed in water and dried.

2.4. Tail Suspension

Mice were suspended from their tails using laboratory tape for 6 min and were recorded for later video analysis. Mice had 2.5 cm plastic cylinders (cut from 5 mL serological pipettes) placed around their tails below the tape to prevent tail-climbing activity while suspended. Videos were later analyzed, and the time spent mobile was recorded. Time spent immobile was calculated by subtracting the total time spent mobile from the total test time (6 min).

2.5. Grooming

Mice were placed in a clean empty cage and allowed to roam freely for 20 min. The first 10 min were used to habituate the mice, and the following 10 min were recorded for later analysis. Videos were later analyzed, and the total time spent grooming was recorded by an observer blinded to genotype.

2.6. Pole descent

A 61 cm wooden dowel was secured perpendicularly into a Styrofoam base and taped into a clean empty mouse cage. Rubber band “rungs” were placed 3.81 cm apart to provide grip for the mice. Mice were placed at the top of the dowel facing downward and the time it took each mouse to climb to the bottom of the dowel was recorded. Mice were tested 3 times in a row and the average total time of the 3 trials was used.

2.7. Pentylenetetrazole-induced seizures

Mice were injected intraperitoneally with pentylenetetrazole (PTZ) (40mg/kg) as previously described (Filiano et al., 2016), placed into a clean and empty cage, and observed for 20 min. Seizure activity was scored based on a previously published scale (Li et al., 2014).

2.8. Immunohistochemistry

Mice were perfused with 4% paraformaldehyde (PFA) followed by phosphate buffered saline (PBS), and brains were drop fixed in 4% PFA for 24 hr at 4°C. Brains were incubated in 30% sucrose for 48 hr at 4°C then embedded in optimal cutting temperature compound (Tissue-Tek OCT Compound; Sakura Finetek, cat # 4583) and stored at −80°C until tissue processing. Brains were sliced on a cryostat into 30 μM sections and stored in cryopreservative (30% ethylene glycol, 30% glycerol, 40% PBS) until tissue staining. To count neurons, tissue sections were fixed on microscope slides and washed with PBS to remove embedding media, then permeabilized and blocked for 1 hour with PBS containing 10% goat serum and 0.05% Triton X-100. After blocking, sections were incubated in primary antibody (NeuN: 1:1000; Millipore Sigma, cat # MAB377; Tyrosine hydroxylase (TH): 1:1000, Millipore Sigma, cat # AB9702) in PBS containing 2% goat serum overnight. The next day, sections were washed three times with PBS and then incubated with fluorescently conjugated secondary antibody (1:1000) in PBS for 2 hours. Sections were then washed three times with PBS. To stain nuclei, DAPI was added to the second wash. Sections were mounted with Fluoromount-G™ (Thermo Fisher Sci, cat #00-4958-02) under a glass coverslip. Images were collected on an EVOS Cell Imaging System (Thermo Fisher Sci), and brain regions of interest were identified using DAPI by an observer blinded to the genotype of the images. NeuN positive neurons were counted, and width of hippocampal blades were measured using ImageJ. The substantia nigra was identified by TH+ cells and the area measured using ImageJ. To measure c-Fos expression, mice were placed in the marble burying assay, then sacrificed 90 minutes later. Tissues were stained for c-Fos according to a previously published protocol (Palop et al., 2011) using rabbit anti-c-Fos antibody (1:10,000; Cell Signaling Technology, cat # 2250), biotinylated goat anti-rabbit IgG (1:200; Vector Laboratories, cat # BA-1000), Vectastain elite ABC HRP kit (Vector Laboratories, cat # PK-6100), and SigmaFast 3,3’-Diaminobenzidine tablets (Sigma-Aldrich, cat # D4293). Slides were imaged using brightfield with a Zeiss Axio Scan.Z1 slide scanner (Zeiss) and brightness and contrast was adjusted using Zen Blue Software (Zeiss). Images were analyzed using ImageJ (National Institutes of Health).

2.9. Experimental Design and Statistics

Male and female mice were used in all experiments. The researcher performing each behavior assay was blinded to mouse genotype. For data analysis, the researcher was blinded to both genotype and sex. N = number of mice. Each experimental group consisted of mice from multiple litters and littermates were used in all experiments. For c-Fos analysis, the researcher was blinded to both genotype and sex. Counts and total area for both hemispheres of each region were combined and analyzed together; multiple slices were analyzed within a region for a single mouse, total counts and total area were summed across slices to find total counts/area. N = number of mice (only brain slices with high tissue integrity were used). A Two-way ANOVA was used for all behavioral assays and c-Fos experiments, except the marble burying time course, in which a Two-way repeated measures ANOVA was used; in data with missing values, Mixed Effects Model was used. If a significant main effect was observed, appropriate post hoc tests were applied (Sidak’s or Tukey’s). Raw data, statistics, and N’s are included in extended data.

3. Results

3.1. Prolonged STAT1 signaling in neurons caused hyperactive behavior and neuronal hypoactivity.

To test the effects of neuronal STAT1T385M on behavior, we expressed Stat1T385M in all neurons using the SynI-cre which begins expressing Cre around E12.5 (Zhu et al., 2001) (Figure S2). During a 15-minute open field assay, both female and male SynI-Stat1T385M /+ mice travelled greater distance than their Stat1+/+ littermates (Figure 1A). Surprisingly, despite having greater ambulatory distance, SynI-Stat1T385M/+ mice also spent less time in the center of the open field (Figure 1B). These data suggest that SynI-Stat1T385M/+ mice may have hyperactive behavior similar to other mouse lines used to model ASD and attention deficit and hyperactive disorder (ADHD) (Ey et al., 2011; Russell et al., 2005). To investigate this phenotype further, we utilized other behavioral assays designed to test for behaviors related to anxiety or hyperactivity. Mouse models of ASD can spend less time immobile in the tail suspension assay (Silverman et al., 2010), which is used to test for despair-like behavior and resiliency (Cryan et al., 2005). Interestingly, SynI-Stat1T385M/+ mice spent less time immobile compared to Stat1+/+ littermates, likely due to hyperactivity (Figure 1C). We also used the self-grooming assay which is commonly used to test for repetitive behaviors (Warmus et al., 2014) and has been used in some mouse models of ASD (Silverman, 2010). SynI-Stat1T385M/+ mice spent less time grooming compared to Stat1+/+ littermates (Figure 1D), again likely driven by hyperactivity. To test for compulsive and repetitive behavior, we used the marbles burying assay. After 20 minutes, SynI-Stat1T385M/+ mice buried more marbles than their littermates (Figure 1E). Taken altogether, these assays reveal increased movement and activity in SynI-Stat1T385M/+ mice, suggesting that driving the Stat1T385M mutation in all neurons caused hyperactive behavior.

Fig 1. Prolonged STAT1 signaling in neurons caused hyperactive behavior.

Fig 1.

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Male and female Syn1-Stat1T385M/+ mice and Syn1-Stat1+/+ littermates were tested in the (A-B) open field, (C) tail suspension, (D) grooming, and (E) marble burying assays. (A) Open field distance: Two-way ANOVA: main effect of genotype ***p<0.0005. (B) Open field time in center: Two-way ANOVA: main effect of genotype *p<0.05, main effect of sex ***p<0.0005; (C) Tail Suspension: Two-way ANOVA: main effect of genotype *p<0.05; (D) Grooming: Two-way ANOVA: main effect of genotype *p<0.05; (E) Marble burying after 20 min: Two-way ANOVA: main effect of genotype *p<0.05, sex *p<0.05; Marble burying over time: Two-way repeated measures ANOVA: main effect of group p=0.05, time ***p<0.0005, interaction *p<0.05; n = 21–29 per group.

To determine if hyperactive behavior could be attributed to general motor abnormalities, we further assessed motor in the pole descent assay (Venkatraman et al., 2022). There was no difference in the time it took the SynI-Stat1T385M/+ mice to climb down the pole (Figure S3A), suggesting no deficits in general motor skills. Finally, since IFNs can impact neural activity, we measured the susceptibility of SynI-Stat1T385M/+ mice to pentylenetetrazol (PTZ)-induced seizures. We observed no differences in susceptibility nor in the severity of PTZ-induced seizures (Figure S3B, C).

3.2. Prolonged STAT1 signaling in neurons decreased neuron counts and c-Fos expression in the caudate putamen

To identify neuroanatomical correlates for Stat1T385M–induced behaviors, we quantified total neuron counts and c-Fos expression, a neuronal activation marker, in relevant brain regions. We found fewer total neurons in the caudate putamen (Figure 2A), but not in the motor cortex, thalamus, hippocampus, or substantia nigra, of SynI-Stat1T385M/+ mice compared to SynI-Stat1+/+ littermates (Figure S4A). To induce neural activation, we placed SynI-Stat1T385M/+ mice and wildtype littermates in the marble burying assay, then harvested brains 90 min later to quantify c-Fos expression. We found female and male SynI-Stat1T385M/+ mice had decreased c-Fos expression in the caudate putamen (Figure 2B), but not in the motor cortex, thalamus, or hippocampus (Figure S5A–C). These data suggest that a Stat1T385M mutation does not globally affect neurons; however, brain regions such as the caudate putamen are selectively sensitive and decreased neuronal activation in this region is associated with hyperactive behavior observed in SynI-Stat1T385M/+ mice. Further work is needed to determine if prolonged STAT1 activation in specific brain regions are sufficient to cause behavioral changes.

Fig 2. Prolonged STAT1 signaling in neurons caused reduced total neurons and neural hypoactivity in the caudate putamen.

Fig 2.

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(A) Representative images and total counts of neurons (NeuN) in the caudate putamen of SynI-Stat1T385M/+ and SynI-Stat1+/+ littermates. *p<0.05 t-test; n = 5 mice per group. (B) Representative images of c-Fos+ cells in the caudate putamen. Female and male SynI-Stat1T385M /+ and SynI-Stat1+/+ littermates were put through the marble burying assay, then brains were harvested 90 min later and stained for c-Fos. Caudate putamen counts: Two-way ANOVA: main effect of genotype *p<0.05; n = 6–7.

3.3. Prolonged STAT1 signaling in dopaminergic neurons caused some hyperactive behaviors.

Given the hyperactive phenotype and decreased activation of neurons in the caudate putamen, we hypothesized that dopaminergic neurons would be sensitive to the Stat1T385M mutation. Dopaminergic neurons which project to the caudate putamen from the substantia nigra regulate voluntary movement control (Luo and Huang, 2016), making them an attractive candidate for regulating the hyperactive phenotype we observed in the SynI-Stat1T385M/+ mice. To test this, we crossed the Stat1T385M mouse with a Dat-Cre mouse, which expressed Cre in dopaminergic neurons within the substantia nigra, ventral tegmental area, and the retrorubral field as early as E15 (Bäckman et al., 2006) (Figure S2). We observed similar behaviors between the SynI-Stat1T385M/+ and Dat-Stat1T385M/+ mice in the open field and tail suspension assays, with Dat-Stat1T385M/+ mice having increased ambulatory distance during open field and spending less time immobile during tail suspension compared to Dat-Stat1+/+ littermates (Figure 3A–C). However, there were no differences between Dat-Stat1T385M/+ and Dat-Stat1+/+ littermates in grooming or the marble assay (Figure 3D, E). Unlike SynI-Stat1T385M/+ mice, we did not find differences in total neuron counts of Dat-Stat1T385M/+ mice in the caudate putamen (Figure 3F) or other brain regions (Figure S4B). Overall, Dat-Stat1T385M/+ mice shared some behavioral similarities with SynI-Stat1T385M/+ mice, suggesting STAT1 dysregulation in dopaminergic neurons may have a role in promoting certain aspects of hyperactive behavior.

Fig. 3. Prolonged STAT1 signaling in dopaminergic neurons caused some hyperactive behavior.

Fig. 3

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Male and female Dat-Stat1T385M/+ mice and Dat-Stat1+/+ littermates were tested in the (A-B) open field, (C) tail suspension, (D) grooming assays, and (E) marble burying assays. (A) Open field distance: Two-way ANOVA: main effect of genotype **p<0.005. (B) Open field time in center: Two-way ANOVA: main effect of genotype NS; (C) Tail Suspension: Two-way ANOVA: main effect of genotype *p<0.05; (D) Grooming: Two-way ANOVA: NS; (E) Marble burying after 20 min: Two-way ANOVA: NS; Marble burying over time: Two-way repeated measures ANOVA: main effect of time ***p<0.0005; n = 7–14 per group. (F) Representative images and total counts of neurons (NeuN) in the caudate putamen of Dat-Stat1T385M/+ and Dat-Stat1+/+ littermates (n = 5 mice per group).

3.4. Prolonged STAT1 signaling exclusively in inhibitory or excitatory neurons did not cause hyperactive behavior.

To determine if driving Stat1T385M exclusively in inhibitory neurons is sufficient to result in hyperactive behavior, we utilized a Vgat-Cre mouse which induces recombination in GABAergic inhibitory neurons throughout all brain regions by P4 (Vong et al., 2011) (Figure S2). In the open field assay, Vgat-Stat1T385M/+ mice travelled less distance and spent more time in the center of the arena compared to Vgat-Stat1+/+ littermates (Figure S6A, B), which is opposite of what we observed in the SynI-Stat1T385M/+ mice (Figure 1A, B). No difference was observed in the tail suspension assay (Figure S6C), nor in grooming, although male mice groomed more than females independent of genotype (Figure S6D). In the marble burying assay, there were no differences between the groups, however there was an interaction between groups and time, likely driven by female Vgat-Stat1T385M/+ mice which trended towards burying less marbles over time (Fig S6E). These data suggest that driving Stat1T385M in inhibitory neurons has a less robust effect on behavior than driving Stat1T385M in all neurons, with potentially hypoactive behavior in females in the open field assay.

To test whether driving Stat1T385M in excitatory neurons was sufficient to cause hyperactive behavior, we utilized three different excitatory neuron Cre models to cross with the Stat1T385M flox mouse. The first Cre line we tested was Vglut1-Cre, which expressed Cre primarily in the cortex, the hippocampus, and scattered throughout the striatum as early as P4 (Harris et al., 2014) (Figure S2). Although there was no difference in ambulatory distance (Figure S7A), male Vglut1-Stat1T385M/+ mice spent more time in the center of the arena compared to Vglut1-Stat1+/+ littermates, while female Vglut1-Stat1T385M/+ mice trended toward spending less time in the center than Vglut1-Stat1+/+ littermates, though this difference in females was not significant (Figure S7B). There was no effect of Vglut1-Stat1T385M/+ mice in the tail suspension assay (Figure S7C). Male Vglut1-Stat1T385M/+ mice spent less time grooming compared to Vglut1-Stat1+/+ littermates, with no difference observed in the females (Figure S7D). Vglut1-Stat1T385M/+ mice also buried fewer marbles compared to Vglut1-Stat1+/+ littermates over time (Figure S7E). Overall, these mice displayed various behavioral differences, however they did not have robust hyperactivity as observed in SynI-Stat1T385M/+ mice.

Next, we utilized the Vglut2-Cre line, which expressed Cre primarily in the thalamus and lower brainstem regions beginning around E9 (Pauli et al., 2022; Vong et al., 2011) (Figure S2). The only difference between Vglut2-Stat1T385M/+ mice and Vglut2-Stat1+/+ littermates was the amount of time spent in the center of the open field arena, with Vglut2-Stat1T385M/+ mice spending less time in the center (Figure S8).

Finally, we tested mice with Stat1T385M driven in all forebrain excitatory neurons by utilizing the CaMKIIα-Cre line. It is important to note that this model does not express Cre until beginning around P21 (Tsien et al., 1996) (Figure S2) and although the CamKIIα promoter has historically been used as a tool to drive gene expression in excitatory neurons, it should be noted that under some experimental systems, for example using adeno associated virus, this promoter can be active in some inhibitory neurons (Veres et al., 2023). Unlike any of the other Cre-lines used, we observed no differences in any of the assays between the CaMKIIα-Stat1T385M/+ mice and CaMKIIα-Stat1+/+ littermates (Figure S9). Overall, driving Stat1T385M in excitatory neurons did not result in hyperactive behavior using any of the Cre lines.

3.5. Prolonged STAT1 signaling in myeloid cells did not cause hyperactive behavior.

To rule out the possibility that STAT1 dysregulation in microglia plays a role in causing hyperactivity, we utilized a Csf1R-Cre to drive Stat1T385M in myeloid cells, including microglia, beginning around E9 (Loschko et al., 2016) (Figure S2). There were no differences in open field, tail suspension, or grooming (Figure S10A–D). Csf1R-Stat1T385M/+ mice buried more marbles than Csf1R-Stat1+/+ littermates over time, but there was no difference between any group at any time point (Figure S10E). These data suggest that Stat1T385M in myeloid cells does not cause hyperactive behavior (Figure S10).

Discussion

STAT1 signaling has been implicated in various neurodevelopmental disorders, as well as neuronal dysfunction (Clark et al., 2022; Clark et al., 2023; Filiano et al., 2016), however, the cell types and underlying molecular mechanisms are not well understood. Here we demonstrated that utilizing a clinical heterozygous Stat1T385M mutation to drive prolonged STAT1 signaling in neurons, beginning during embryonic development, caused hyperactive behavior and fewer neurons and neural hypoactivity in the caudate putamen. Furthermore, driving Stat1T385M exclusively in dopaminergic neurons partially mimicked this hyperactive phenotype, whereas driving Stat1T385M exclusively in inhibitory or excitatory neurons, or myeloid cells, resulted in few behavioral differences, and was not sufficient to recapitulate the hyperactive behavior. These results suggest that STAT1 signaling has differential functions in different neuron subtypes, and that STAT1 signaling in dopaminergic neurons has a role in regulating behavior and disruption of this signaling may contribute to neurodevelopmental disorders.

The mechanism underlying how prolonged STAT1 signaling can impact neuronal activity and ultimately behavior need to be further explored. Overexpression of IFN-γ in the choroid plexus selectively caused nigrostriatal degeneration, midbrain calcinosis, and Parkinson-like behavior phenotypes (Strickland et al., 2018). This IFN-γ/STAT1 mediated degeneration is selective for nigrostriatal neurons within the substantia nigra, midbrain, and pons, whereas pyramidal neurons in the hippocampus were not affected (Chakrabarty et al., 2011). Similarly, we observed fewer neuron counts and decreased c-Fos positive neurons in SynI-Stat1T385M/+ mice specifically in the caudate putamen, which receives input from dopaminergic neurons in the nigrostriatal pathway (Luo and Huang, 2016). Whether prolonged STAT1 signaling is impacting neural development or promoting neuron loss in the caudate is unknown, but experimentally ablating dopamine D2 receptor–positive neurons (which normally inhibit motor activity) promotes hyperactive behavior (Durieux et al., 2009). Human studies have found that the degree of caudate asymmetry predicted cumulative severity ratings of inattentive behaviors of children with ADHD (Schrimsher et al., 2002) and boys diagnosed with ADHD had smaller caudate volumes compared to typically developing aged matched controls (Carrey et al., 2012). Adults with ADHD exhibited decreased dopamine activity in the caudate (Volkow et al., 2007), further supporting an important role for dopaminergic neurons in the caudate regulating hyperactive motor behavior.

Why dysregulated STAT1 signaling affects neuronal subtypes differently, more specifically dopaminergic neurons, is also unknown. Interestingly, neuronal STAT1 is differentially expressed throughout the brain, with the highest expression occurring in the olfactory bulb, hippocampus, basal ganglia, and Purkinje neuron layers in the cerebellum (Campbell, 2005), suggesting neurons within these regions may be more responsive to IFNs, and more susceptible to dysregulated STAT1 signaling and Stat1T385M. It is also unclear why Stat1T385M in dopaminergic neurons only recapitulates certain hyperactive behaviors that present with Stat1T385M in all neurons. Despite many treatments for hyperactivity having overlap targeting dopaminergic, noradrenergic, and serotonergic systems (Yu et al., 2020), these monoamines have key differences in neuroanatomical locations and play distinct roles in modifying underlying components of hyperactivity (Nikolaus et al., 2022). Notably, these circuits have distinguishable impacts on reward-seeking, attention, motor activation, and impulsivity, and may underscore behavior differences in Dat-Stat1T385M/+ mice and the more global SynI-Stat1T385M/+ and (Hiraide et al., 2013). Therefore, it is possible that the neurons targeted in each of our Cre models have different susceptibility to dysregulated STAT1 signaling, resulting in different effects on behavior.

Another factor to consider is the timing of Cre expression, and therefore induction of dysregulated STAT1 signaling in each of the models (Figure S2). While SynI-Stat1T385M/+ and Dat-Stat1T385M/+ mice were the only models that exhibited hyperactive behavior, others exhibited subtle behavioral differences often in the opposite direction compared to SynI-Stat1T385M/+ mice. Interestingly, all of these models expressed Cre embryonically or neonatally in neurons. Meanwhile, CaMKIIα-Stat1T385M/+ mice, the only Cre model that expressed Cre beginning around weaning, had no behavioral differences. These data suggest that there may be a critical period early in development during which physiological STAT1 signaling in necessary for proper neurodevelopment. Indeed, others have also demonstrated that neuronal STAT1 is important for synapse refinement between P0-P15 (Yasuda et al., 2021), as well as for the homeostatic regulation of visual cortical plasticity (Nagakura et al., 2014). These results further suggest that STAT1 signaling in neurons is crucial during early neurodevelopment and that disrupting physiological signaling can result in long-term effects.

Overall, our data demonstrate a critical role for STAT1 signaling in neurons, particularly dopaminergic neurons, during development in regulating behavior and neural activity. Furthermore, we present a novel mouse model which will be valuable in the efforts to uncover how physiological STAT1 signaling in neurons contributes to proper neurodevelopment, and how aberrant STAT1 signaling early in development, especially in different neuronal subtypes, contributes to neurodevelopmental disorders.

Supplementary Material

1

Fig. S1 Stat1T385M schematic. The Stat1T385M flox mouse has a clinical T385M point mutation in exon 14 of the endogenous Stat1 gene. cDNA encoding exons 13–25 of the wildtype Stat1 allele followed by a stop codon and flanked by loxP sites was inserted before the endogenous exon 13. In cell types not expressing Cre, the endogenous Stat1 gene is transcribed through exon 12 and then the wildtype exons 13–25 cDNA are transcribed up until the stop codon, resulting in wildtype Stat1 expression. In cells expressing Cre, the gene is recombined at the loxP sites, resulting in the wildtype cDNA insert and stop codon being excised out, and the entire endogenous Stat1 gene with the T385M mutation being transcribed.

2

Fig. S2 Timing and location of Cre expression in mouse lines used in behavior experiments. (A) Visualization of brain regions where each Cre is expressed. Allen Brain Atlas was used as a reference to confirm which brain regions expressed Cre in each Cre model. Available at https://connectivity.brain-map.org/transgenic. (B) Age of Cre expression in each line. (C) Representative scatter plots of gating strategies used to FAC sort neurons from Syn1-Stat1T385M/+ mice and genotyping PCR of recombined genomic Stat1 allele from input, isolated neurons (NeuN), or flow through (f.t.). (D) Representative scatter plots of input, MAC sorted microglia (Mg), or flow through (f.t.) from Csf1r-Stat1T385M/+ mice and genotyping PCR. (E) PCR of recombined genomic Stat1 allele of isolated neuronal nuclei from the cortex (CTX) or substantia nigra (SN) of Dat-Stat1T385M/+ mice.

3

Fig. S3 Prolonged STAT1 signaling in neurons did not cause motor deficits or increased susceptibility to seizure. Male and female Syn1-Stat1T385M/+ mice and Syn1-Stat1+/+ littermates were tested in the (A) pole assay, and (B-C) female mice were subjected to PTZ induced seizures and (B) maximum seizure stage and (C) latency to seizure was measured. (A) Pole descent assay: Two-way ANOVA: NS; n = 14–18. (B) Maximum seizure stage: Unpaired T-test: NS; (C) Latency to seizure: Two-way ANOVA: main effect of score*** p<0.0005; n = 11–12.

4

Fig S4 Prolonged STAT1 signaling in neurons did not affect neuron counts in motor cortex, thalamus, hippocampus, or substantia nigra. Cellularity of neurons from (A) SynI-Stat1T385M/+ and (B) Dat-Stat1 T385M/+ mice compared to their littermates n = 4–6 mice per group. NeuN positive neurons were counted in the motor cortex and thalamus. The width of the dentate gyrus and CA3 of the hippocampus was measured. The area of TH+ neurons of the substantia nigra was measured.

5

Fig S5 Prolonged STAT1 signaling in neurons did not affect c-Fos expression in motor cortex, thalamus or hippocampus. Female and male SynI-Stat1T385M/+ and SynI-Stat1+/+ littermates were put through the marble burying assay, then brains were harvested 90 min later and stained for c-Fos. (A) Motor cortex: Two-way ANOVA: NS; n = 6–8. (B) Thalamus: Two-way ANOVA: NS; n = 6–8 (C) Hippocampus: Two-way ANOVA: NS; n = 6–8.

6

Fig S6 Prolonged STAT1 signaling in inhibitory neurons did not cause hyperactive behavior. Male and female Vgat-Stat1T385M/+ mice and Vgat-Stat1+/+ littermates were tested in the (A-B) open field, (C) tail suspension, (D) grooming, and (E) marble burying assays. (A) Open field distance: Two-way ANOVA: main effect of genotype *p<0.05, main effect of sex ***p<0.0005. (B) Open field time in center: Two-way ANOVA: main effect of genotype *p<0.05, main effect of sex *p<0.05. (C) Tail suspension: Two-way ANOVA: NS (D) Grooming: Two-way ANOVA: main effect of sex *p<0.05; (E) Marble burying after 20 minutes: Two-way repeated measures ANOVA: NS; Marble burying over time: Two-way repeated measures ANOVA: main effect of time ***p<0.0005, interaction *p<0.05; n = 13–18 per group.

7

Fig S7 Prolonged STAT1 signaling in excitatory neurons did not cause hyperactive behavior. Male and female Vglut1-Stat1T385M/+ mice and Vglut1-Stat1+/+ littermates were tested in (A-B) open field, (C) tail suspension, (D) grooming, and (E) marble burying assays. (A) Open field distance: ns (B) Open field time in center: Two-way ANOVA: interaction of genotype and sex **p<0.005; (C) Tail suspension: Two-way ANOVA: main effect of sex *p<0.05; (D) Grooming: Two-way ANOVA: main effect of genotype p = 0.06, main effect of sex *p<0.05; interaction p=0.05. (E) Marbles 20 min: Two-way ANOVA: main effect of genotype **p<0.005. Marbles time course: Two-way repeated measures ANOVA: main effect of group** p<0.005, main effect of time*** p<0.0005, interaction*** p<0.0005; post-hoc Sidak’s multiple comparisons test: female Cre-vs female GOF** p<0.005; n = 10–13 per group.

8

Fig S8 Prolonged STAT1 signaling in excitatory neurons did not cause hyperactive behavior. Male and female Vglut2-Stat1T385M/+ mice and Vglut2-Stat1+/+ littermates were tested in the (A-B) open field, (C) tail suspension, (D) grooming, and (E) marble burying assays. (A) Open field distance: ns; (B) Open field time in center: Two-way ANOVA: ns; (C) Tail suspension: Two-way ANOVA: main effect of sex *p<0.05; (D) Grooming: Two-way ANOVA: ns; (D) Marbles 20 min: Two-way repeated measures ANOVA: NS; Marbles over time: Two-way repeated measures ANOVA: main effect of time ***p<0.0005; n = 9–17 per group.

9

Fig S9 Prolonged STAT1 signaling in excitatory neurons did not cause hyperactive behavior. Male and female CaMKIIa-Stat1T385M/+ mice and CaMKIIa-Stat1+/+ littermates were tested in the (A-B) open field, (C) tail suspension, (D) grooming, and (E) marble burying assays. (A) Open field distance: Two-way ANOVA: ns (B) Open field time in center: Two-way ANOVA: ns; (C) Tail suspension: Two-way ANOVA: ns; (D) Grooming: Two-way ANOVA: ns; (E) Marbles 20 min: Two-way repeated measures ANOVA: ns; Marbles over time: Two-way repeated measures ANOVA: main effect of time ***p<0.0005; n = 9–17 per group.

10

Fig S10 Prolonged STAT1 signaling in myeloid cells did not cause hyperactive behavior. Male and female Csf1R-Stat1T385M/+ mice and Csf1R-Stat1+/+ littermates were tested in the (A-B) open field, (C) tail suspension, (D) grooming, and (E) marble burying assays. (A) Open field distance: Two-way ANOVA: main effect of sex *p<0.05; (B) Open field time in center: Twoway ANOVA: ns; (C) Tail suspension: Two-way ANOVA: ns; (D) Grooming: Two-way ANOVA: NS; (E) Marbles 20 min: Two-way repeated measures ANOVA: main effect of sex **p<0.005; Marbles over time: Two-way repeated measures ANOVA: main effect of group *p<0.05, main effect of time ***p<0.0005. Interaction *p<0.05; n = 11–15 per group.

Highlights.

  1. The Interferon-Stat1 pathway is implicated in many neurodevelopmental disorders.

  2. Modeling prolonged STAT1 signaling in neurons, with a clinical STAT1 gain-of-function mutation, causes hyperactive behaviors in mice.

  3. STAT1 gain-of-function mutation in neurons reduces neuron counts and activation in the caudate putamen.

  4. STAT1 gain-of-function mutation exclusively in dopaminergic neurons, which project to the caudate putamen, causes similar hyperactive behaviors.

Acknowledgements

The authors would like to thank all members of the Filiano lab and the Marcus Center for Cellular Cures for their feedback on data presented in the manuscript. We would also like to thank Drs. Josh D. Milner and John J. O’Shea for providing the Stat1T385M flox mouse and Dr. Abby Polter for providing advice to target dopaminergic neurons.

A.J.F owns intellectual property licensed to Cryo-Cell Internation.

Funding Sources

This work was supported by grants from the National Institutes of Health (NS123084) and the Marcus Foundation. The Stat1T385M flox mouse was generated with funds from the National Institute of Arthritis and Musculoskeletal and Skin Diseases (NIAMS).

Footnotes

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

1

Fig. S1 Stat1T385M schematic. The Stat1T385M flox mouse has a clinical T385M point mutation in exon 14 of the endogenous Stat1 gene. cDNA encoding exons 13–25 of the wildtype Stat1 allele followed by a stop codon and flanked by loxP sites was inserted before the endogenous exon 13. In cell types not expressing Cre, the endogenous Stat1 gene is transcribed through exon 12 and then the wildtype exons 13–25 cDNA are transcribed up until the stop codon, resulting in wildtype Stat1 expression. In cells expressing Cre, the gene is recombined at the loxP sites, resulting in the wildtype cDNA insert and stop codon being excised out, and the entire endogenous Stat1 gene with the T385M mutation being transcribed.

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2

Fig. S2 Timing and location of Cre expression in mouse lines used in behavior experiments. (A) Visualization of brain regions where each Cre is expressed. Allen Brain Atlas was used as a reference to confirm which brain regions expressed Cre in each Cre model. Available at https://connectivity.brain-map.org/transgenic. (B) Age of Cre expression in each line. (C) Representative scatter plots of gating strategies used to FAC sort neurons from Syn1-Stat1T385M/+ mice and genotyping PCR of recombined genomic Stat1 allele from input, isolated neurons (NeuN), or flow through (f.t.). (D) Representative scatter plots of input, MAC sorted microglia (Mg), or flow through (f.t.) from Csf1r-Stat1T385M/+ mice and genotyping PCR. (E) PCR of recombined genomic Stat1 allele of isolated neuronal nuclei from the cortex (CTX) or substantia nigra (SN) of Dat-Stat1T385M/+ mice.

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3

Fig. S3 Prolonged STAT1 signaling in neurons did not cause motor deficits or increased susceptibility to seizure. Male and female Syn1-Stat1T385M/+ mice and Syn1-Stat1+/+ littermates were tested in the (A) pole assay, and (B-C) female mice were subjected to PTZ induced seizures and (B) maximum seizure stage and (C) latency to seizure was measured. (A) Pole descent assay: Two-way ANOVA: NS; n = 14–18. (B) Maximum seizure stage: Unpaired T-test: NS; (C) Latency to seizure: Two-way ANOVA: main effect of score*** p<0.0005; n = 11–12.

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4

Fig S4 Prolonged STAT1 signaling in neurons did not affect neuron counts in motor cortex, thalamus, hippocampus, or substantia nigra. Cellularity of neurons from (A) SynI-Stat1T385M/+ and (B) Dat-Stat1 T385M/+ mice compared to their littermates n = 4–6 mice per group. NeuN positive neurons were counted in the motor cortex and thalamus. The width of the dentate gyrus and CA3 of the hippocampus was measured. The area of TH+ neurons of the substantia nigra was measured.

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5

Fig S5 Prolonged STAT1 signaling in neurons did not affect c-Fos expression in motor cortex, thalamus or hippocampus. Female and male SynI-Stat1T385M/+ and SynI-Stat1+/+ littermates were put through the marble burying assay, then brains were harvested 90 min later and stained for c-Fos. (A) Motor cortex: Two-way ANOVA: NS; n = 6–8. (B) Thalamus: Two-way ANOVA: NS; n = 6–8 (C) Hippocampus: Two-way ANOVA: NS; n = 6–8.

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6

Fig S6 Prolonged STAT1 signaling in inhibitory neurons did not cause hyperactive behavior. Male and female Vgat-Stat1T385M/+ mice and Vgat-Stat1+/+ littermates were tested in the (A-B) open field, (C) tail suspension, (D) grooming, and (E) marble burying assays. (A) Open field distance: Two-way ANOVA: main effect of genotype *p<0.05, main effect of sex ***p<0.0005. (B) Open field time in center: Two-way ANOVA: main effect of genotype *p<0.05, main effect of sex *p<0.05. (C) Tail suspension: Two-way ANOVA: NS (D) Grooming: Two-way ANOVA: main effect of sex *p<0.05; (E) Marble burying after 20 minutes: Two-way repeated measures ANOVA: NS; Marble burying over time: Two-way repeated measures ANOVA: main effect of time ***p<0.0005, interaction *p<0.05; n = 13–18 per group.

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7

Fig S7 Prolonged STAT1 signaling in excitatory neurons did not cause hyperactive behavior. Male and female Vglut1-Stat1T385M/+ mice and Vglut1-Stat1+/+ littermates were tested in (A-B) open field, (C) tail suspension, (D) grooming, and (E) marble burying assays. (A) Open field distance: ns (B) Open field time in center: Two-way ANOVA: interaction of genotype and sex **p<0.005; (C) Tail suspension: Two-way ANOVA: main effect of sex *p<0.05; (D) Grooming: Two-way ANOVA: main effect of genotype p = 0.06, main effect of sex *p<0.05; interaction p=0.05. (E) Marbles 20 min: Two-way ANOVA: main effect of genotype **p<0.005. Marbles time course: Two-way repeated measures ANOVA: main effect of group** p<0.005, main effect of time*** p<0.0005, interaction*** p<0.0005; post-hoc Sidak’s multiple comparisons test: female Cre-vs female GOF** p<0.005; n = 10–13 per group.

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8

Fig S8 Prolonged STAT1 signaling in excitatory neurons did not cause hyperactive behavior. Male and female Vglut2-Stat1T385M/+ mice and Vglut2-Stat1+/+ littermates were tested in the (A-B) open field, (C) tail suspension, (D) grooming, and (E) marble burying assays. (A) Open field distance: ns; (B) Open field time in center: Two-way ANOVA: ns; (C) Tail suspension: Two-way ANOVA: main effect of sex *p<0.05; (D) Grooming: Two-way ANOVA: ns; (D) Marbles 20 min: Two-way repeated measures ANOVA: NS; Marbles over time: Two-way repeated measures ANOVA: main effect of time ***p<0.0005; n = 9–17 per group.

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9

Fig S9 Prolonged STAT1 signaling in excitatory neurons did not cause hyperactive behavior. Male and female CaMKIIa-Stat1T385M/+ mice and CaMKIIa-Stat1+/+ littermates were tested in the (A-B) open field, (C) tail suspension, (D) grooming, and (E) marble burying assays. (A) Open field distance: Two-way ANOVA: ns (B) Open field time in center: Two-way ANOVA: ns; (C) Tail suspension: Two-way ANOVA: ns; (D) Grooming: Two-way ANOVA: ns; (E) Marbles 20 min: Two-way repeated measures ANOVA: ns; Marbles over time: Two-way repeated measures ANOVA: main effect of time ***p<0.0005; n = 9–17 per group.

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10

Fig S10 Prolonged STAT1 signaling in myeloid cells did not cause hyperactive behavior. Male and female Csf1R-Stat1T385M/+ mice and Csf1R-Stat1+/+ littermates were tested in the (A-B) open field, (C) tail suspension, (D) grooming, and (E) marble burying assays. (A) Open field distance: Two-way ANOVA: main effect of sex *p<0.05; (B) Open field time in center: Twoway ANOVA: ns; (C) Tail suspension: Two-way ANOVA: ns; (D) Grooming: Two-way ANOVA: NS; (E) Marbles 20 min: Two-way repeated measures ANOVA: main effect of sex **p<0.005; Marbles over time: Two-way repeated measures ANOVA: main effect of group *p<0.05, main effect of time ***p<0.0005. Interaction *p<0.05; n = 11–15 per group.

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