Heterogeneity in the progression of retinal pathologies in mice harboring patient mimicking Impg2 mutations.

Biallelic mutations in interphotoreceptor matrix proteoglycan 2 (IMPG2) in humans cause retinitis pigmentosa (RP) with early macular involvement, albeit the disease progression varies widely due to genetic heterogeneity and IMPG2 mutation type. There are currently no treatments for IMPG2-RP. To aid preclinical studies toward eventual treatments, there is a need to better understand the progression of disease pathology in appropriate animal models. Toward this goal, we developed mouse models with patient mimicking homozygous frameshift (T807Ter) or missense (Y250C) Impg2 mutations, as well as mice with a homozygous frameshift mutation (Q244Ter) designed to completely prevent IMPG2 protein expression, and characterized the trajectory of their retinal pathologies across postnatal development until late adulthood. We found that the Impg2T807Ter/T807Ter and Impg2Q244Ter/Q244Ter mice exhibited early onset gliosis, impaired photoreceptor outer segment maintenance, appearance of subretinal deposits near the optic disc, disruption of the outer retina, and neurosensorial detachment, whereas the Impg2Y250C/Y250C mice exhibited minimal retinal pathology. These results demonstrate the importance of mutation type in disease progression in IMPG2-RP and provide a toolkit and preclinical data for advancing therapeutic approaches.

High Rac1 activity is functionally translated into cytosolic structures with unique nanoscale cytoskeletal architecture.

Rac1 activation is at the core of signaling pathways regulating polarized cell migration. So far, it has not been possible to directly explore the structural changes triggered by Rac1 activation at the molecular level. Here, through a multiscale imaging workflow that combines biosensor imaging of Rac1 dynamics with electron cryotomography, we identified, within the crowded environment of eukaryotic cells, a unique nanoscale architecture of a flexible, signal-dependent actin structure. In cell regions with high Rac1 activity, we found a structural regime that spans from the ventral membrane up to a height of ∼60 nm above that membrane, composed of directionally unaligned, densely packed actin filaments, most shorter than 150 nm. This unique Rac1-induced morphology is markedly different from the dendritic network architecture in which relatively short filaments emanate from existing, longer actin filaments. These Rac1-mediated scaffold assemblies are devoid of large macromolecules such as ribosomes or other filament types, which are abundant at the periphery and within the remainder of the imaged volumes. Cessation of Rac1 activity induces a complete and rapid structural transition, leading to the absence of detectable remnants of such structures within 150 s, providing direct structural evidence for rapid actin filament network turnover induced by GTPase signaling events. It is tempting to speculate that this highly dynamical nanoscaffold system is sensitive to local spatial cues, thus serving to support the formation of more complex actin filament architectures-such as those mandated by epithelial-mesenchymal transition, for example-or resetting the region by completely dissipating.

Enhanced Operant Extinction and Prefrontal Excitability in a Mouse Model of Angelman Syndrome.

Angelman syndrome (AS), a neurodevelopmental disorder associated with intellectual disability, is caused by loss of maternal allele expression of UBE3A in neurons. Mouse models of AS faithfully recapitulate disease phenotypes across multiple domains, including behavior. Yet in AS, there has been only limited study of behaviors encoded by the prefrontal cortex, a region broadly involved in executive function and cognition. Because cognitive impairment is a core feature of AS, it is critical to develop behavioral readouts of prefrontal circuit function in AS mouse models. One such readout is behavioral extinction, which has been well described mechanistically and relies upon prefrontal circuits in rodents. Here we report exaggerated operant extinction in male AS model mice, concomitant with enhanced excitability in medial prefrontal neurons from male and female AS model mice. Abnormal behavior was specific to operant extinction, as two other prefrontally dependent tasks (cued fear extinction and visuospatial discrimination) were largely normal in AS model mice. Inducible deletion of Ube3a during adulthood was not sufficient to drive abnormal extinction, supporting the hypothesis that there is an early critical period for development of cognitive phenotypes in AS. This work represents the first formal experimental analysis of prefrontal circuit function in AS, and identifies operant extinction as a useful experimental paradigm for modeling cognitive aspects of AS in mice.SIGNIFICANCE STATEMENT Prefrontal cortex encodes "high-level" cognitive processes. Thus, understanding prefrontal function is critical in neurodevelopmental disorders where cognitive impairment is highly penetrant. Angelman syndrome is a neurodevelopmental disorder associated with speech and motor impairments, an outwardly happy demeanor, and intellectual disability. We describe a behavioral phenotype in a mouse model of Angelman syndrome and related abnormalities in prefrontal cortex function. We hypothesize that robust and reliable prefrontally encoded behavior may be used to model cognitive impairments in Angelman syndrome.

Environment-sensing merocyanine dyes for live cell imaging applications.

Fluorescent biosensors based on environmentally sensitive dyes enable visualization and quantification of endogenous protein activation within living cells. Merocyanine dyes are especially useful for live cell imaging applications, as they are extraordinarily bright, have long wavelengths of excitation and emission, and can exhibit readily detectable fluorescence changes in response to environment. We sought to systematically examine the effects of structural features on key photophysical properties, including dye brightness, environmental responsiveness, and photostability, through the synthesis of a library of 25 merocyanine dyes, derived from combinatorial reaction of 5 donor and 5 acceptor heterocycles. Four of these dyes showed optimal properties for specific imaging applications and were subsequently prepared with reactive side chains and enhanced aqueous solubility using a one-pot synthetic method. The new dyes were then applied within a biosensor design for Cdc42 activation, where dye mero60 showed a remarkable 1470% increase in fluorescence intensity on binding activated Cdc42 in vitro. The dye-based biosensors were used to report activation of endogenous Cdc42 in living cells.

Visual Sequences Drive Experience-Dependent Plasticity in Mouse Anterior Cingulate Cortex.

Mechanisms of experience-dependent plasticity have been well characterized in mouse primary visual cortex (V1), including a form of potentiation driven by repeated presentations of a familiar visual sequence ("sequence plasticity"). The prefrontal anterior cingulate cortex (ACC) responds to visual stimuli, yet little is known about if and how visual experience modifies ACC circuits. We find that mouse ACC exhibits sequence plasticity, but in contrast to V1, the plasticity expresses as a change in response timing, rather than a change in response magnitude. Sequence plasticity is absent in ACC, but not V1, in a mouse model of a neurodevelopmental disorder associated with intellectual disability and autism-like features. Our results demonstrate that simple sensory stimuli can be used to reveal how experience functionally (or dysfunctionally) modifies higher-order prefrontal circuits and suggest a divergence in how ACC and V1 encode familiarity.

Cannabidiol attenuates seizures and EEG abnormalities in Angelman syndrome model mice.

Angelman syndrome (AS) is a neurodevelopmental disorder characterized by intellectual disability, lack of speech, ataxia, EEG abnormalities, and epilepsy. Seizures in individuals with AS are common, debilitating, and often drug resistant. Thus, there is an unmet need for better treatment options. Cannabidiol (CBD), a major phytocannabinoid constituent of cannabis, has shown antiseizure activity and behavioral benefits in preclinical and clinical studies for some disorders associated with epilepsy, suggesting that the same could be true for AS. Here, we show that acute CBD (100 mg/kg) treatment attenuated hyperthermia- and acoustically induced seizures in a mouse model of AS. However, neither acute CBD nor a 2-week-long course of CBD administered immediately after a kindling protocol could halt the proepileptogenic plasticity observed in AS model mice. CBD had a dose-dependent sedative effect but did not have an impact on motor performance. CBD abrogated the enhanced intracortical local field potential power, including the delta and theta rhythms observed in AS model mice, indicating that CBD administration could also help normalize the EEG deficits observed in individuals with AS. We believe our results provide critical preclinical evidence supporting CBD treatment of seizures and alleviation of EEG abnormalities in AS and will thus help guide the rational development of CBD as a treatment for AS.

Ube3a reinstatement mitigates epileptogenesis in Angelman syndrome model mice.

Angelman syndrome (AS) is a neurodevelopmental disorder in which epilepsy is common (~90%) and often refractory to antiepileptics. AS is caused by mutation of the maternal allele encoding the ubiquitin protein ligase E3A (UBE3A), but it is unclear how this genetic insult confers vulnerability to seizure development and progression (i.e., epileptogenesis). Here, we implemented the flurothyl kindling and retest paradigm in AS model mice to assess epileptogenesis and to gain mechanistic insights owed to loss of maternal Ube3a. AS model mice kindled similarly to wild-type mice, but they displayed a markedly increased sensitivity to flurothyl-, kainic acid-, and hyperthermia-induced seizures measured a month later during retest. Pathological characterization revealed enhanced deposition of perineuronal nets in the dentate gyrus of the hippocampus of AS mice in the absence of overt neuronal loss or mossy fiber sprouting. This pro-epileptogenic phenotype resulted from Ube3a deletion in GABAergic but not glutamatergic neurons, and it was rescued by pancellular reinstatement of Ube3a at postnatal day 21 (P21), but not during adulthood. Our results suggest that epileptogenic susceptibility in AS patients is a consequence of the dysfunctional development of GABAergic circuits, which may be amenable to therapies leveraging juvenile reinstatement of UBE3A.

Loss of UBE3A from TH-expressing neurons suppresses GABA co-release and enhances VTA-NAc optical self-stimulation.

Motivated reward-seeking behaviours are governed by dopaminergic ventral tegmental area projections to the nucleus accumbens. In addition to dopamine, these mesoaccumbal terminals co-release other neurotransmitters including glutamate and GABA, whose roles in regulating motivated behaviours are currently being investigated. Here we demonstrate that loss of the E3-ubiquitin ligase, UBE3A, from tyrosine hydroxylase-expressing neurons impairs mesoaccumbal, non-canonical GABA co-release and enhances reward-seeking behaviour measured by optical self-stimulation.

An orally bioavailable chemical probe of the Lysine Methyltransferases EZH2 and EZH1.

EZH2 or EZH1 is the catalytic subunit of the polycomb repressive complex 2 that catalyzes methylation of histone H3 lysine 27 (H3K27). The trimethylation of H3K27 (H3K27me3) is a transcriptionally repressive post-translational modification. Overexpression of EZH2 and hypertrimethylation of H3K27 have been implicated in a number of cancers. Several selective inhibitors of EZH2 have been reported recently. Herein we disclose UNC1999, the first orally bioavailable inhibitor that has high in vitro potency for wild-type and mutant EZH2 as well as EZH1, a closely related H3K27 methyltransferase that shares 96% sequence identity with EZH2 in their respective catalytic domains. UNC1999 was highly selective for EZH2 and EZH1 over a broad range of epigenetic and non-epigenetic targets, competitive with the cofactor SAM and non-competitive with the peptide substrate. This inhibitor potently reduced H3K27me3 levels in cells and selectively killed diffused large B cell lymphoma cell lines harboring the EZH2(Y641N) mutant. Importantly, UNC1999 was orally bioavailable in mice, making this inhibitor a valuable tool for investigating the role of EZH2 and EZH1 in chronic animal studies. We also designed and synthesized UNC2400, a close analogue of UNC1999 with potency >1,000-fold lower than that of UNC1999 as a negative control for cell-based studies. Finally, we created a biotin-tagged UNC1999 (UNC2399), which enriched EZH2 in pull-down studies, and a UNC1999-dye conjugate (UNC2239) for co-localization studies with EZH2 in live cells. Taken together, these compounds represent a set of useful tools for the biomedical community to investigate the role of EZH2 and EZH1 in health and disease.