Generation of an Alagille Syndrome (ALGS) patient-derived induced pluripotent stem cell line (TRNDi036-A) carrying a heterozygous mutation (p.Cys693*) in the JAG1 gene.

Alagille syndrome (ALGS) is an autosomal dominant, multisystemic disorder due to haploinsufficiency in JAG1 or less frequently, mutations in NOTCH2. The disease has been difficult to diagnose and treat due to variable expression. The generation of this iPSC line (TRNDi036-A) carrying a heterozygous mutation (p.Cys693*) in the JAG1 gene provides a means of studying the disease and developing novel therapeutics towards patient treatment.

Generation of an Alagille syndrome (ALGS) patient-derived induced pluripotent stem cell line (TRNDi032-A) carrying a heterozygous mutation (p.Cys682Leufs*7) in the JAG1 gene.

Alagille syndrome (ALGS) is an autosomal dominant, multisystemic disorder due to haploinsufficiency in either the JAG1 gene (ALGS type 1) or the NOTCH2 gene (ALGS type 2). The disease has been difficult to diagnose and treat due to its muti-system clinical presentation, variable expressivity, and prenatal onset for some of the features. The generation of this iPSC line (TRNDi032-A) carrying a heterozygous mutation, p.Cys682Leufs*7 (c.2044dup), in the JAG1 gene provides a means of studying the disease and developing novel therapeutics towards patient treatment.

Generation and characterization of NGLY1 patient-derived midbrain organoids.

NGLY1 deficiency is an ultra-rare, autosomal recessive genetic disease caused by mutations in the NGLY1 gene encoding N-glycanase one that removes N-linked glycan. Patients with pathogenic mutations in NGLY1 have complex clinical symptoms including global developmental delay, motor disorder and liver dysfunction. To better understand the disease pathogenesis and the neurological symptoms of the NGLY1 deficiency we generated and characterized midbrain organoids using patient-derived iPSCs from two patients with distinct disease-causing mutations-one homozygous for p. Q208X, the other compound heterozygous for p. L318P and p. R390P and CRISPR generated NGLY1 knockout iPSCs. We demonstrate that NGLY1 deficient midbrain organoids show altered neuronal development compared to one wild type (WT) organoid. Both neuronal (TUJ1) and astrocytic glial fibrillary acid protein markers were reduced in NGLY1 patient-derived midbrain organoids along with neurotransmitter GABA. Interestingly, staining for dopaminergic neuronal marker, tyrosine hydroxylase, revealed a significant reduction in patient iPSC derived organoids. These results provide a relevant NGLY1 disease model to investigate disease mechanisms and evaluate therapeutics for treatments of NGLY1 deficiency.

Sex differences and risk factors for bleeding in Alagille syndrome.

Spontaneous bleeds are a leading cause of death in the pediatric JAG1-related liver disease Alagille syndrome (ALGS). We asked whether there are sex differences in bleeding events in patients, whether Jag1Ndr/Ndr mice display bleeds or vascular defects, and whether discovered vascular pathology can be confirmed in patients non-invasively. We performed a systematic review of patients with ALGS and vascular events following PRISMA guidelines, in the context of patient sex, and found significantly more girls than boys reported with spontaneous intracranial hemorrhage. We investigated vascular development, homeostasis, and bleeding in Jag1Ndr/Ndr mice, using retina as a model. Jag1Ndr/Ndr mice displayed sporadic brain bleeds, a thin skull, tortuous blood vessels, sparse arterial smooth muscle cell coverage in multiple organs, which could be aggravated by hypertension, and sex-specific venous defects. Importantly, we demonstrated that retinographs from patients display similar characteristics with significantly increased vascular tortuosity. In conclusion, there are clinically important sex differences in vascular disease in ALGS, and retinography allows non-invasive vascular analysis in patients. Finally, Jag1Ndr/Ndr mice represent a new model for vascular compromise in ALGS.

Generation of two gene corrected human isogenic iPSC lines (NCATS-CL6104 and NCATS-CL6105) from a patient line (NCATS-CL6103) carrying a homozygous p.R401X mutation in the NGLY1 gene using CRISPR/Cas9.

NGLY1 deficiency is a rare recessive genetic disease caused by mutations in the NGLY1 gene which codes for N-glycanase 1 (NGLY1). Here, we report the generation of two gene corrected iPSC lines using a patient-derived iPSC line (NCATS-CL6103) that carried a homozygous p.R401X mutation in the NGLY1 gene. These lines contain either one (NCATS-CL6104) or two (NCATS-CL6105) CRISPR/Cas9 corrected alleles of NGLY1. This pair of NGLY1 mutation corrected iPSC lines can be used as a control for the NCATS-CL6103 which serves as a cell-based NGLY1 disease model for the study of the disease pathophysiology and evaluation of therapeutics under development.

An induced pluripotent stem cell line (NCATS-CL9075) from a patient carrying compound heterozygote mutations, p.R390P and p.L318P, in the NGLY1 gene.

NGLY1 deficiency is a rare disorder caused by mutations in the NGLY1 gene which codes for the highly conserved N-glycanase1 (NGLY1). This enzyme functions in cytosolic deglycosylation of N- linked glycoproteins. An induced pluripotent stem cell (iPSC) line was generated from the dermal fibroblasts of a 2-year-old patient carrying compound heterozygous mutations, p.R390P and p.L318P in the NGLY1 gene. This cell-based iPSC disease model provides a resource to study disease pathophysiology and to develop a cell-based disease model for drug development for NGLY1 patients.

Pharmacological inhibition of phosphodiesterase 7 enhances consolidation processes of spatial memory.

Augmentation of cAMP signaling through inhibition of phosphodiesterases (PDE) is known to enhance plasticity and memory. Inhibition of PDE4 enhances consolidation into memory, but less is known about the role of other cAMP specific PDEs. Here, we tested the effects of oral treatment with a selective inhibitor of PDE7 of nanomolar potency on spatial and contextual memory. In an object location task, doses of 0.3-3 mg/kg administered 3 h after training dose-dependently attenuated time-dependent forgetting in rats. Significant enhancement of memory occurred at a dose of 3 mg/kg with corresponding brain levels consistent with PDE7 inhibition. The same dose given prior to training augmented contextual fear conditioning. In mice, daily dosing before training enhanced spatial memory in two different incremental learning paradigms in the Barnes Maze. Drug treated mice made significantly less errors locating the escape in a probe-test 24 h after the end of training, and they exhibited hippocampal-dependent spatial search strategies more frequently than controls, which tended to show serial sampling of escape locations. Acquisition and short-term memory, in contrast, were unaffected. Our data provide evidence for a role of PDE7 in the consolidation of hippocampal-dependent memory. We suggest that targeting PDE7 for memory enhancement may provide an alternative to PDE4 inhibitors, which tend to have undesirable gastrointestinal side-effects.

Phosphodiesterase 1b (PDE1B) Regulates Spatial and Contextual Memory in Hippocampus.

Augmentation of cyclic nucleotide signaling through inhibition of phosphodiesterase (PDE) activity has long been understood to enhance memory. Efforts in this domain have focused predominantly on PDE4, a cAMP-specific phosphodiesterase implicated in consolidation. But less is known about the function of other PDEs expressed in neuroanatomical regions critical to memory. The PDE1 isoforms are the only PDEs to regulate neuronal cAMP and cGMP levels in a Ca2+/Calmodulin (CaM) dependent manner. Here, we show that knock-down of PDE1B in hippocampus of adult mice enhances contextual and spatial memory without effect on non-cognitive behaviors. Pharmacological augmentation of memory in rats was observed with a selective inhibitor of PDE1 dosed before and immediately after training, but not with drug dosed either 1 h after training or before recall. Our data clearly demonstrate a role for the PDE1B isoforms as negative regulators of memory, and they implicate PDE1 in an early phase of consolidation, but not retrieval. Inhibition of PDE1B is a promising therapeutic mechanism for treating memory impairment.

PDE4D regulates Spine Plasticity and Memory in the Retrosplenial Cortex.

The retrosplenial cortex (RSC) plays a critical role in episodic memory, but the molecular mechanisms governing plasticity in this structure are poorly understood. Diverse studies have demonstrated a role for RSC in acquisition, early consolidation and retrieval similar to the hippocampus (HC), as well as in systems consolidation similar to the anterior cingulate cortex. Here, we asked whether established molecular and structural substrates of memory consolidation in the HC also engage in RSC shortly after learning. We show striking parallels in training induced gene-activation in HC and RSC following contextual conditioning, which is blocked by systemic administration of an NMDA receptor antagonist. Long-term memory is enhanced by retrosplenial and hippocampal knockdown (KD) of the cAMP specific phosphodiesterase Pde4d. However, while training per se induces lasting spine changes in HC, this does not occur in RSC. Instead, increases in the number of mature dendritic spines are found in the RSC only if cAMP signaling is augmented by Pde4d KD, and spine changes are at least partially independent of training. This research highlights parallels and differences in spine plasticity mechanisms between HC and RSC, and provides evidence for a functional dissociation of the two.

Heritable and inducible gene knockdown in astrocytes or neurons in vivo by a combined lentiviral and RNAi approach.

Gene knockout by homologous recombination is a popular method to study gene functions in the mouse in vivo. However, its lack of temporal control has limited the interpretation of knockout studies because the complete elimination of a gene product often alters developmental processes, and can induce severe malformations or lethality. Conditional gene knockdown has emerged as a compelling alternative to gene knockout, an approach well-established in vitro but that remains challenging in vivo, especially in the adult brain. Here, we report a method for conditional and cell-specific gene knockdown in the mouse brain in vivo that combines Cre-mediated RNA interference (RNAi) with classical and lentivirus-mediated transgenesis. The method is based on the inducible expression of a silencing short hairpin RNA (shRNA) introduced in mice by lentivirus-mediated transgenesis, and on its activation by excision of a floxed stop EGFP reporter with an inducible Cre recombinase expressed in astrocytes or in neurons. This dual system should be of broad utility for comparative studies of gene functions in these two cell types in vivo.

Zif268/Egr1 gain of function facilitates hippocampal synaptic plasticity and long-term spatial recognition memory.

It is well established that Zif268/Egr1, a member of the Egr family of transcription factors, is critical for the consolidation of several forms of memory; however, it is as yet uncertain whether increasing expression of Zif268 in neurons can facilitate memory formation. Here, we used an inducible transgenic mouse model to specifically induce Zif268 overexpression in forebrain neurons and examined the effect on recognition memory and hippocampal synaptic transmission and plasticity. We found that Zif268 overexpression during the establishment of memory for objects did not change the ability to form a long-term memory of objects, but enhanced the capacity to form a long-term memory of the spatial location of objects. This enhancement was paralleled by increased long-term potentiation in the dentate gyrus of the hippocampus and by increased activity-dependent expression of Zif268 and selected Zif268 target genes. These results provide novel evidence that transcriptional mechanisms engaging Zif268 contribute to determining the strength of newly encoded memories.

Elimination of dendritic spines with long-term memory is specific to active circuits.

Structural changes in brain circuits active during learning are thought to be important for long-term memory storage. If these changes support long-term information storage, they might be expected to be present at distant time points after learning, as well as to be specific to the circuit activated with learning, and sensitive to the contingencies of the behavioral paradigm. Here, we show such changes in the hippocampus as a result of contextual fear conditioning. There were significantly fewer spines specifically on active neurons of fear-conditioned mice. This spine loss did not occur in homecage mice or in mice exposed to the training context alone. Mice exposed to unpaired shocks showed a generalized reduction in spines. These learning-related changes in spine density could reflect a direct mechanism of encoding or alternately could reflect a compensatory adaptation to previously described enhancement in transmission due to glutamate receptor insertion.

Neural functions of calcineurin in synaptic plasticity and memory.

Major brain functions depend on neuronal processes that favor the plasticity of neuronal circuits while at the same time maintaining their stability. The mechanisms that regulate brain plasticity are complex and engage multiple cascades of molecular components that modulate synaptic efficacy. Protein kinases (PKs) and phosphatases (PPs) are among the most important of these components that act as positive and negative regulators of neuronal signaling and plasticity, respectively. In these cascades, the PP protein phosphatase 2B or calcineurin (CaN) is of particular interest because it is the only Ca(2+)-activated PP in the brain and a major regulator of key proteins essential for synaptic transmission and neuronal excitability. This review describes the primary properties of CaN and illustrates its functions and modes of action by focusing on several representative targets, in particular glutamate receptors, striatal enriched protein phosphatase (STEP), and neuromodulin (GAP43), and their functional significance for synaptic plasticity and memory.

Deletion of the potassium channel Kv12.2 causes hippocampal hyperexcitability and epilepsy.

We found the voltage-gated K+ channel Kv12.2 to be a potent regulator of excitability in hippocampal pyramidal neurons. Genetic deletion and pharmacologic block of Kv12.2 substantially reduced the firing threshold of these neurons. Kv12.2-/- (also known as Kcnh3-/-) mice showed signs of persistent neuronal hyperexcitability including frequent interictal spiking, spontaneous seizures and increased sensitivity to the chemoconvulsant pentylenetetrazol.

Changes in the proteome after neuronal zif268 overexpression.

Long-lasting forms of brain plasticity are a cellular basis for long-term memory, and their disturbance underlies pathological conditions such as dementia and cognitive impairment. Neuronal plasticity is a complex process that utilizes molecular cascades in the cytoplasm and the nucleus and involves numerous transcription factors, in particular, immediate early genes (IEGs). The signaling cascades that control IEGs are fairly well described, but the downstream transcriptional response is poorly understood, especially its late components. Here, we investigated the response induced by the IEG Zif268 in the adult brain in relation to long-term memory. Using a mouse model with increased neuronal expression of Zif268 that leads to improved memory, we identified an ensemble of proteins regulated by Zif268 expression and differentiated between direct and indirect targets based on the presence of a consensus binding motif in their promoter. We show that Zif268 regulates numerous substrates with diverse biological functions including protein modification and degradation (proteasome-core complex), phosphorylation, cell division, sensory perception, metabolism, and metal ion transport. The results provide a comprehensive and quantitative data set characterizing the Zif268-dependent proteome in the adult mouse brain and offers biologically important new insight into activity-dependent pathways downstream of IEGs.

Control of the establishment of aversive memory by calcineurin and Zif268.

Emotional memory is a rapidly acquired and persistent form of memory, and its robustness is in part determined by the initial strength of the memory. Here, we provide new evidence that the protein phosphatase calcineurin (CaN), a potent negative regulator of neuronal signaling that is known to constrain learning and memory, critically regulates the establishment of emotional memory through mechanisms involving the immediate early gene Zif268 (also known as Egr1). We found that CaN is inhibited in the amygdala during the establishment of aversive memory, but Zif268 is activated. Using inducible transgenesis in mice, we further saw that CaN inhibition and Zif268 overexpression during memory establishment strengthen the memory trace and enhance its resistance to extinction. We found that CaN inhibition correlates with increased Zif268 expression and that a common pool of proteins is regulated in the amygdala after CaN inhibition and Zif268 overexpression. Together, these findings reveal a previously unknown mechanism for the control of emotional memory that depends on CaN and Zif268.

Protein phosphatase 1-dependent bidirectional synaptic plasticity controls ischemic recovery in the adult brain.

Protein kinases and phosphatases can alter the impact of excitotoxicity resulting from ischemia by concurrently modulating apoptotic/survival pathways. Here, we show that protein phosphatase 1 (PP1), known to constrain neuronal signaling and synaptic strength (Mansuy et al., 1998; Morishita et al., 2001), critically regulates neuroprotective pathways in the adult brain. When PP1 is inhibited pharmacologically or genetically, recovery from oxygen/glucose deprivation (OGD) in vitro, or ischemia in vivo is impaired. Furthermore, in vitro, inducing LTP shortly before OGD similarly impairs recovery, an effect that correlates with strong PP1 inhibition. Conversely, inducing LTD before OGD elicits full recovery by preserving PP1 activity, an effect that is abolished by PP1 inhibition. The mechanisms of action of PP1 appear to be coupled with several components of apoptotic pathways, in particular ERK1/2 (extracellular signal-regulated kinase 1/2) whose activation is increased by PP1 inhibition both in vitro and in vivo. Together, these results reveal that the mechanisms of recovery in the adult brain critically involve PP1, and highlight a novel physiological function for long-term potentiation and long-term depression in the control of brain damage and repair.

Inducible and neuron-specific gene expression in the adult mouse brain with the rtTA2S-M2 system.

To achieve inducible and reversible gene expression in the adult mouse brain, we exploited an improved version of the tetracycline-controlled transactivator-based system (rtTA2(S)-M2, rtTA2 hereafter) and combined it with the forebrain-specific CaMKIIalpha promoter. Several independent lines of transgenic mice carrying the CaMKIIalpha promoter-rtTA2 gene were generated and examined for anatomical profile, doxycycline (dox)-dependence, time course, and reversibility of gene expression using several lacZ reporter lines. In two independent rtTA2-expressing lines, dox-treatment in the diet induced lacZ reporter expression in neurons of several forebrain structures including cortex, striatum, hippocampus, amygdala, and olfactory bulb. Gene expression was dose-dependent and was fully reversible. Further, a similar pattern of expression was obtained in three independent reporter lines, indicating the consistency of gene expression. Transgene expression could also be activated in the developing brain (P0) by dox-treatment of gestating females. These new rtTA2-expressing mice allowing inducible and reversible gene expression in the adult or developing forebrain represent useful models for future genetic studies of brain functions.

Reversible blockade of experience-dependent plasticity by calcineurin in mouse visual cortex.

Numerous protein kinases have been implicated in visual cortex plasticity, but the role of serine/threonine protein phosphatases has not yet been established. Calcineurin, the only known Ca2+/calmodulin-activated protein phosphatase in the brain, has been identified as a molecular constraint on synaptic plasticity in the hippocampus and on memory. Using transgenic mice overexpressing calcineurin inducibly in forebrain neurons, we now provide evidence that calcineurin is also involved in ocular dominance plasticity. A transient increase in calcineurin activity is found to prevent the shift of responsiveness in the visual cortex following monocular deprivation, and this effect is reversible. These results imply that the balance between protein kinases and phosphatases is critical for visual cortex plasticity.