Selective Neuronal Knockout of STAT3 Function Inhibits Epilepsy Progression, Improves Cognition, and Restores Dysregulated Gene Networks in a Temporal Lobe Epilepsy Model.

OBJECTIVE: Temporal lobe epilepsy (TLE) is a progressive disorder mediated by pathological changes in molecular cascades and hippocampal neural circuit remodeling that results in spontaneous seizures and cognitive dysfunction. Targeting these cascades may provide disease-modifying treatments for TLE patients. Janus Kinase/Signal Transducer and Activator of Transcription (JAK/STAT) inhibitors have emerged as potential disease-modifying therapies; a more detailed understanding of JAK/STAT participation in epileptogenic responses is required, however, to increase the therapeutic efficacy and reduce adverse effects associated with global inhibition. METHODS: We developed a mouse line in which tamoxifen treatment conditionally abolishes STAT3 signaling from forebrain excitatory neurons (nSTAT3KO). Seizure frequency (continuous in vivo electroencephalography) and memory (contextual fear conditioning and motor learning) were analyzed in wild-type and nSTAT3KO mice after intrahippocampal kainate (IHKA) injection as a model of TLE. Hippocampal RNA was obtained 24 h after IHKA and subjected to deep sequencing. RESULTS: Selective STAT3 knock-out in excitatory neurons reduced seizure progression and hippocampal memory deficits without reducing the extent of cell death or mossy fiber sprouting induced by IHKA injection. Gene expression was rescued in major networks associated with response to brain injury, neuronal plasticity, and learning and memory. We also provide the first evidence that neuronal STAT3 may directly influence brain inflammation. INTERPRETATION: Inhibiting neuronal STAT3 signaling improved outcomes in an animal model of TLE, prevented progression of seizures and cognitive co-morbidities while rescuing pathogenic changes in gene expression of major networks associated with epileptogenesis. Specifically targeting neuronal STAT3 may be an effective disease-modifying strategy for TLE. ANN NEUROL 2023;94:106-122.

Binge ethanol exposure in advanced age elevates neuroinflammation and early indicators of neurodegeneration and cognitive impairment in female mice.

Binge drinking is rising among aged adults (>65 years of age), however the contribution of alcohol misuse to neurodegenerative disease development is not well understood. Both advanced age and repeated binge ethanol exposure increase neuroinflammation, which is an important component of neurodegeneration and cognitive dysfunction. Surprisingly, the distinct effects of binge ethanol exposure on neuroinflammation and associated degeneration in the aged brain have not been well characterized. Here, we establish a model of intermittent binge ethanol exposure in young and aged female mice to investigate the effects of advanced age and binge ethanol on these outcomes. Following intermittent binge ethanol exposure, expression of pro-inflammatory mediators (tnf-α, il-1ß, ccl2) was distinctly increased in isolated hippocampal tissue by the combination of advanced age and ethanol. Binge ethanol exposure also increased measures of senescence, the nod like receptor pyrin domain containing 3 (NLRP3) inflammasome, and microglia reactivity in the brains of aged mice compared to young. Binge ethanol exposure also promoted neuropathology in the hippocampus of aged mice, including tau hyperphosphorylation and neuronal death. We further identified advanced age-related deficits in contextual memory that were further negatively impacted by ethanol exposure. These data suggest binge drinking superimposed with advanced age promotes early markers of neurodegenerative disease development and cognitive decline, which may be driven by heightened neuroinflammatory responses to ethanol. Taken together, we propose this novel exposure model of intermittent binge ethanol can be used to identify therapeutic targets to prevent advanced age- and ethanol-related neurodegeneration.

Deep postnatal phenotyping of a new mouse model of nonketotic hyperglycinemia.

Nonketotic hyperglycinemia due to deficient glycine cleavage enzyme activity causes a severe neonatal epileptic encephalopathy. Current therapies based on mitigating glycine excess have only limited impact. An animal model with postnatal phenotyping is needed to explore new therapeutic approaches. We developed a Gldc p.Ala394Val mutant model and bred it to congenic status in two colonies on C57Bl/6J (B6) and J129X1/SvJ (J129) backgrounds. Mutant mice had reduced P-protein and enzyme activity indicating a hypomorphic mutant. Glycine levels were increased in blood and brain regions, exacerbated by dietary glycine, with higher levels in female than male J129 mice. Birth defects were more prevalent in mutant B6 than J129 mice, and hydrocephalus was more frequent in B6 (40%) compared to J129 (none). The hydrocephalus rate was increased by postnatal glycine challenge in B6 mice, more so when delivered from the first neonatal week than from the fourth. Mutant mice had reduced weight gain following weaning until the eighth postnatal week, which was exacerbated by glycine loading. The electrographic spike rate was increased in mutant mice following glycine loading, but no seizures were observed. The alpha/delta band intensity ratio was decreased in the left cortex in female J129 mice, which were less active in an open field test and explored less in a Y-maze, suggesting an encephalopathic effect. Mutant mice showed no evidence of memory dysfunction. This partial recapitulation of human symptoms and biochemistry will facilitate the evaluation of new therapeutic approaches with an early postnatal time window likely most effective.

Effects of a potassium channel opener on brain injury and neurologic outcomes in an animal model of neonatal hypoxic-ischemic injury.

BACKGROUND: Hypoxia-ischemia (HI) is the most common cause of brain injury in newborns and the survivors often develop cognitive and sensorimotor disabilities that undermine the quality of life. In the current study, we examined the effectiveness of flupirtine, a potassium channel opener, shown previously in an animal model to have strong anti-neonatal-seizure efficacy, to provide neuroprotection and alleviate later-life disabilities caused by neonatal hypoxic-ischemic injury. METHODS: The rats were treated with a single dose of flupirtine for 4 days following HI induction in 7-day-old rats. The first dose of flupirtine was given after the induction of HI and during the reperfusion period. The effect of treatment was examined on acute and chronic brain injury, motor functions, and cognitive abilities. RESULTS: Flupirtine treatment significantly reduced HI-induced hippocampal and cortical tissue loss at acute time point. Furthermore, at chronic time point, flupirtine reduced contralateral hippocampal volume loss and partially reversed learning and memory impairments but failed to improve motor deficits. CONCLUSION: The flupirtine treatment regimen used in the current study significantly reduced brain injury at acute time point in an animal model of neonatal hypoxic-ischemic encephalopathy. However, these neuroprotective effects were not persistent and only modest improvement in functional outcomes were observed at chronic time points.

NF-κB/NLRP3 Translational Inhibition by Nanoligomer Therapy Mitigates Ethanol and Advanced Age-Related Neuroinflammation.

Binge alcohol use is increasing among aged adults (>65 years). Alcohol-related toxicity in aged adults is associated with neurodegeneration, yet the molecular underpinnings of age-related sensitivity to alcohol are not well described. Studies utilizing rodent models of neurodegenerative disease reveal heightened activation of Nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) and Nod like receptor 3 (NLRP3) mediate microglia activation and associated neuronal injury. Our group, and others, have implicated hippocampal-resident microglia as key producers of inflammatory mediators, yet the link between inflammation and neurodegeneration has not been established in models of binge ethanol exposure and advanced age. Here, we report binge ethanol increased the proportion of NLRP3+ microglia in the hippocampus of aged (18-20 months) female C57BL/6N mice compared to young (3-4 months). In primary microglia, ethanol-induced expression of reactivity markers and NLRP3 inflammasome activation were more pronounced in microglia from aged mice compared to young. Making use of an NLRP3-specific inhibitor (OLT1177) and a novel brain-penetrant Nanoligomer that inhibits NF-κB and NLRP3 translation (SB_NI_112), we find ethanol-induced microglial reactivity can be attenuated by OLT1177 and SB_NI_112 in microglia from aged mice. In a model of intermittent binge ethanol exposure, SB_NI_112 prevented ethanol-mediated microglia reactivity, IL-1ß production, and tau hyperphosphorylation in the hippocampus of aged mice. These data suggest early indicators of neurodegeneration occurring with advanced age and binge ethanol exposure are NF-κB- and NLRP3-dependent. Further investigation is warranted to explore the use of targeted immunosuppression via Nanoligomers to attenuate neuroinflammation after alcohol consumption in the aged.

Deep postnatal phenotyping of a new mouse model of nonketotic hyperglycinemia.

Nonketotic hyperglycinemia due to deficient glycine cleavage enzyme activity causes a severe neonatal epileptic encephalopathy. Current therapies based on mitigating glycine excess have only limited impact. An animal model with postnatal phenotyping is needed to explore new therapeutic approaches. We developed a Gldc p.Ala394Val mutant model and bred it to congenic status in 2 colonies on C57Bl/6J (B6) and J129X1/SvJ (J129) backgrounds. Mutant mice had reduced P-protein and enzyme activity indicating a hypomorphic mutant. Glycine levels were increased in blood and brain regions, exacerbated by dietary glycine, with higher levels in female than male J129 mice. Birth defects were more prevalent in mutant B6 than J129 mice, and hydrocephalus was more frequent in B6 (40%) compared to J129 (none). The hydrocephalus rate was increased by postnatal glycine challenge in B6 mice, more so when delivered from the first neonatal week than from the fourth. Mutant mice had reduced weight gain following weaning until the eighth postnatal week, which was exacerbated by glycine loading. The electrographic spike rate was increased in mutant mice following glycine loading, but no seizures were observed. The alpha/delta band intensity ratio was decreased in the left cortex in female J129 mice, which were less active in an open field test and explored less in a Y-maze, suggesting an encephalopathic effect. Mutant mice showed no evidence of memory dysfunction. This partial recapitulation of human symptoms and biochemistry will facilitate the evaluation of new therapeutic approaches with an early postnatal time window likely most effective. Take home message: A mouse model of nonketotic hyperglycinemia is described that shows postnatal abnormalities in glycine levels, neural tube defects, body weight, electroencephalographic recordings, and in activity in young mice making it amenable for the evaluation of novel treatment interventions. Author contributions: Study concept and design: JVH, MHM, NB, KNMAnimal study data: MAS, HJ, NB, MHM, JC, CBBiochemical and genetic studies: MAS, RAVH, MWFStatistical analysis: NB, JVHFirst draft writing: JVH, NB, MHMCritical rewriting: MAS, NB, MHM, TAB, JC, MWF, KNM, JVHFinal responsibility, guarantor, and communicating author: JVH. Competing interest statement: The University of Colorado (JVH, MS, KNM, HJ) has the intention to file Intellectual property protection for certain biochemical treatments of NKH. Otherwise, the authors have stated that they had no interests that might be perceived as posing a conflict or bias to this subject matter. Funding support: Financial support is acknowledged form the NKH Crusaders, Brodyn's Friends, Nora Jane Almany Foundation, the Dickens Family Foundation, the Lucas John Foundation, Les Petits Bourdons, Joseph's Fund, the Barnett Family, Maud & Vic Foundation, Lucy's BEElievers fund, Hope for NKH, Madi's Mission NKH fund, and from Dr. and Ms. Shaw, and the University of Colorado Foundation NKH research fund. The study was supported by a grant (CNS-X-19-103) from the University of Colorado School of Medicine and the Colorado Clinical Translational Science Institute, which is supported by NIH/NCATS Colorado CTSA Grant Number UL1 TR002535. Contents are the authors' sole responsibility and do not necessarily represent official NIH views. All funding sources had no role in the design or execution of the study, the interpretation of data, or the writing of the study. Ethics approval on Laboratory Animal Studies: Mouse studies were carried out with approval from the Institutional Animal Care and Use Committee of the University of Colorado Anschutz Medical Campus (IACUC# 00413). Data sharing statement: The data that support the findings of this study are available from the corresponding author upon reasonable request.

CaV3.1 T-type calcium channels are important for spatial memory processing in the dorsal subiculum.

The dorsal subiculum (dSub) is one of the key structures responsible for the formation of hippocampal memory traces but the contribution of individual ionic currents to its cognitive function is not well studied. Although we recently reported that low-voltage-activated T-type calcium channels (T-channels) are crucial for the burst firing pattern regulation in the dSub pyramidal neurons, their potential role in learning and memory remains unclear. Here we used in vivo local field potential recordings and miniscope calcium imaging in freely behaving mice coupled with pharmacological and genetic tools to address this gap in knowledge. We show that the CaV3.1 isoform of T-channels is critically involved in controlling neuronal activity in the dSub in vivo. Altering neuronal excitability by inhibiting T-channel activity markedly affects calcium dynamics, synaptic plasticity, neuronal oscillations and phase-amplitude coupling in the dSub, thereby disrupting spatial learning. These results provide an important causative link between the CaV3.1 channels, burst firing of dSub neurons and memory formation, thus further supporting the notion that changes in neuronal excitability regulate memory processing. We posit that subicular CaV3.1 T-channels could be a promising novel drug target for cognitive disorders.

Triplication of the interferon receptor locus contributes to hallmarks of Down syndrome in a mouse model.

Down syndrome (DS), the genetic condition caused by trisomy 21, is characterized by variable cognitive impairment, immune dysregulation, dysmorphogenesis and increased prevalence of diverse co-occurring conditions. The mechanisms by which trisomy 21 causes these effects remain largely unknown. We demonstrate that triplication of the interferon receptor (IFNR) gene cluster on chromosome 21 is necessary for multiple phenotypes in a mouse model of DS. Whole-blood transcriptome analysis demonstrated that IFNR overexpression associates with chronic interferon hyperactivity and inflammation in people with DS. To define the contribution of this locus to DS phenotypes, we used genome editing to correct its copy number in a mouse model of DS, which normalized antiviral responses, prevented heart malformations, ameliorated developmental delays, improved cognition and attenuated craniofacial anomalies. Triplication of the Ifnr locus modulates hallmarks of DS in mice, suggesting that trisomy 21 elicits an interferonopathy potentially amenable to therapeutic intervention.

Context Fear Conditioning in Down Syndrome Mouse Models: Effects of Trisomic Gene Content, Age, Sex and Genetic Background.

Down syndrome (DS), trisomy of the long arm of human chromosome 21 (Hsa21), is the most common genetic cause of intellectual disability (ID). Currently, there are no effective pharmacotherapies. The success of clinical trials to improve cognition depends in part on the design of preclinical evaluations in mouse models. To broaden understanding of the common limitations of experiments in learning and memory, we report performance in context fear conditioning (CFC) in three mouse models of DS, the Dp(16)1Yey, Dp(17)1Yey and Dp(10)1Yey (abbreviated Dp16, Dp17 and Dp10), separately trisomic for the human Hsa21 orthologs mapping to mouse chromosomes 16, 17 and 10, respectively. We examined female and male mice of the three lines on the standard C57BL/6J background at 3 months of age and Dp17 and Dp10 at 18 months of age. We also examined female and male mice of Dp17 and Dp10 at 3 months of age as F1 hybrids obtained from a cross with the DBA/2J background. Results indicate that genotype, sex, age and genetic background affect CFC performance. These data support the need to use both female and male mice, trisomy of sets of all Hsa21 orthologs, and additional ages and genetic backgrounds to improve the reliability of preclinical evaluations of drugs for ID in DS.

CaV3.1 isoform of T-type calcium channels supports excitability of rat and mouse ventral tegmental area neurons.

Recent data have implicated voltage-gated calcium channels in the regulation of the excitability of neurons within the mesolimbic reward system. While the attention of most research has centered on high voltage L-type calcium channel activity, the presence and role of the low voltage-gated T-type calcium channel (T-channels) has not been well explored. Hence, we investigated T-channel properties in the neurons of the ventral tegmental area (VTA) utilizing wild-type (WT) rats and mice, CaV3.1 knock-out (KO) mice, and TH-eGFP knock-in (KI) rats in acute horizontal brain slices of adolescent animals. In voltage-clamp experiments, we first assessed T-channel activity in WT rats with characteristic properties of voltage-dependent activation and inactivation, as well as characteristic crisscrossing patterns of macroscopic current kinetics. T-current kinetics were similar in WT mice and WT rats but T-currents were abolished in CaV3.1 KO mice. In ensuing current-clamp experiments, we observed the presence of hyperpolarization-induced rebound burst firing in a subset of neurons in WT rats, as well as dopaminergic and non-dopaminergic neurons in TH-eGFP KI rats. Following the application of a pan-selective T-channel blocker TTA-P2, rebound bursting was significantly inhibited in all tested cells. In a behavioral assessment, the acute locomotor increase induced by a MK-801 (Dizocilpine) injection in WT mice was abolished in CaV3.1 KO mice, suggesting a tangible role for 3.1 T-type channels in drug response. We conclude that pharmacological targeting of CaV3.1 isoform of T-channels may be a novel approach for the treatment of disorders of mesolimbic reward system.

Mild myelin disruption elicits early alteration in behavior and proliferation in the subventricular zone.

Myelin, the insulating sheath around axons, supports axon function. An important question is the impact of mild myelin disruption. In the absence of the myelin protein proteolipid protein (PLP1), myelin is generated but with age, axonal function/maintenance is disrupted. Axon disruption occurs in Plp1-null mice as early as 2 months in cortical projection neurons. High-volume cellular quantification techniques revealed a region-specific increase in oligodendrocyte density in the olfactory bulb and rostral corpus callosum that increased during adulthood. A distinct proliferative response of progenitor cells was observed in the subventricular zone (SVZ), while the number and proliferation of parenchymal oligodendrocyte progenitor cells was unchanged. This SVZ proliferative response occurred prior to evidence of axonal disruption. Thus, a novel SVZ response contributes to the region-specific increase in oligodendrocytes in Plp1-null mice. Young adult Plp1-null mice exhibited subtle but substantial behavioral alterations, indicative of an early impact of mild myelin disruption.

Preferences of newborn mice for odours indicating closer genetic relatedness: is experience necessary?

Evidence from studies with adult rodents indicates that individual recognition enables distinctions between familiar individuals irrespective of relatedness (but including close kin) and a separate mechanism enables discriminations based on genetic relatedness without prior familiarity. For example, adult mice could assess the extent of their genetic relatedness to unfamiliar individuals using perceptual similarities between their individual odours. The ontogeny of this genetic relatedness assessment mechanism, however, had not been investigated. Here, in two-choice tests, newborn mice differentially preferred odours of more genetically similar lactating females (paternal aunts to unrelated conspecific and conspecific to heterospecific) even without prior direct exposure to adults with the tested genotypes. The results provide a direct demonstration of genetic relatedness assessment abilities in newborns and show that experience with parental odours is not necessary for genetic relatedness distinctions. Future studies will be necessary to determine whether exposure to odours of other foetuses in the womb or littermates shortly after birth affects this genetic relatedness assessment process.

Galantamine improves olfactory learning in the Ts65Dn mouse model of Down syndrome.

Down syndrome (DS) is the most common form of congenital intellectual disability. Although DS involves multiple disturbances in various tissues, there is little doubt that in terms of quality of life cognitive impairment is the most serious facet and there is no effective treatment for this aspect of the syndrome. The Ts65Dn mouse model of DS recapitulates multiple aspects of DS including cognitive impairment. Here the Ts65Dn mouse model of DS was evaluated in an associative learning paradigm based on olfactory cues. In contrast to disomic controls, trisomic mice exhibited significant deficits in olfactory learning. Treatment of trisomic mice with the acetylcholinesterase inhibitor galantamine resulted in a significant improvement in olfactory learning. Collectively, our study indicates that olfactory learning can be a sensitive tool for evaluating deficits in associative learning in mouse models of DS and that galantamine has therapeutic potential for improving cognitive abilities.