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1.
Mol Ther Nucleic Acids ; 29: 189-203, 2022 Sep 13.
Article in English | MEDLINE | ID: mdl-35860385

ABSTRACT

Mutations in the TECPR2 gene are the cause of an ultra-rare neurological disorder characterized by intellectual disability, impaired speech, motor delay, and hypotonia evolving to spasticity, central sleep apnea, and premature death (SPG49 or HSAN9; OMIM: 615031). Little is known about the biological function of TECPR2, and there are currently no available disease-modifying therapies for this disease. Here we describe implementation of an antisense oligonucleotide (ASO) exon-skipping strategy targeting TECPR2 c.1319delT (p.Leu440Argfs∗19), a pathogenic variant that results in a premature stop codon within TECPR2 exon 8. We used patient-derived fibroblasts and induced pluripotent stem cell (iPSC)-derived neurons homozygous for the p.Leu440Argfs∗19 mutation to model the disease in vitro. Both patient-derived fibroblasts and neurons showed lack of TECPR2 protein expression. We designed and screened ASOs targeting sequences across the TECPR2 exon 8 region to identify molecules that induce exon 8 skipping and thereby remove the premature stop signal. TECPR2 exon 8 skipping restored in-frame expression of a TECPR2 protein variant (TECPR2ΔEx8) containing 1,300 of 1,411 amino acids. Optimization of ASO sequences generated a lead candidate (ASO-005-02) with ∼27 nM potency in patient-derived fibroblasts. To examine potential functional rescue induced by ASO-005-02, we used iPSC-derived neurons to analyze the neuronal localization of TECPR2ΔEx8 and showed that this form of TECPR2 retains the distinct, punctate neuronal expression pattern of full-length TECPR2. Finally, ASO-005-02 had an acceptable tolerability profile in vivo following a single 20-mg intrathecal dose in cynomolgus monkeys, showing some transient non-adverse behavioral effects with no correlating histopathology. Broad distribution of ASO-005-02 and induction of TECPR2 exon 8 skipping was detected in multiple central nervous system (CNS) tissues, supporting the potential utility of this therapeutic strategy for a subset of patients suffering from this rare disease.

2.
FASEB J ; 35(12): e22053, 2021 12.
Article in English | MEDLINE | ID: mdl-34820911

ABSTRACT

Mutations in KCNC3, the gene that encodes the Kv3.3 voltage dependent potassium channel, cause Spinocerebellar Ataxia type 13 (SCA13), a disease associated with disrupted motor behaviors, progressive cerebellar degeneration, and abnormal auditory processing. The Kv3.3 channel directly binds Hax-1, a cell survival protein. A disease-causing mutation, Kv3.3-G592R, causes overstimulation of Tank Binding Kinase 1 (Tbk1) in the cerebellum, resulting in the degradation of Hax-1 by promoting its trafficking into multivesicular bodies and then to lysosomes. We have now tested the effects of antisense oligonucleotides (ASOs) directed against the Kv3.3 channel on both wild type mice and those bearing the Kv3.3-G592R-encoding mutation. Intracerebroventricular infusion of the Kcnc3-specific ASO suppressed both mRNA and protein levels of the Kv3.3 channel. In wild-type animals, this produced no change in levels of activated Tbk1, Hax-1 or Cd63, a tetraspanin marker for late endosomes/multivesicular bodies. In contrast, in mice homozygous for the Kv3.3-G592R-encoding mutation, the same ASO reduced Tbk1 activation and levels of Cd63, while restoring the expression of Hax-1 in the cerebellum. The motor behavior of the mice was tested using a rotarod assay. Surprisingly, the active ASO had no effects on the motor behavior of wild type mice but restored the behavior of the mutant mice to those of age-matched wild type animals. Our findings indicate that, in mature intact animals, suppression of Kv3.3 expression can reverse the deleterious effects of a SCA13 mutation while having little effect on wild type animals. Thus, targeting Kv3.3 expression may prove a viable therapeutic approach for SCA13.


Subject(s)
Motor Disorders/prevention & control , Mutation , Oligonucleotides, Antisense/administration & dosage , Protein Serine-Threonine Kinases/metabolism , Shaw Potassium Channels/antagonists & inhibitors , Spinocerebellar Ataxias/complications , Animals , Male , Mice , Mice, Inbred C57BL , Mice, Knockout , Motor Disorders/etiology , Motor Disorders/metabolism , Motor Disorders/pathology , Protein Serine-Threonine Kinases/genetics , Shaw Potassium Channels/genetics , Shaw Potassium Channels/metabolism
3.
Drug Discov Today Technol ; 23: 17-25, 2017 Mar.
Article in English | MEDLINE | ID: mdl-28647082

ABSTRACT

There is a pressing need for new and more effective treatments for central nervous system (CNS) disorders. A large body of evidence now suggests that alterations in synaptic transmission and neuronal excitability represent underlying factors for many neurological and psychiatric diseases. However, it has been challenging to target these complex functional domains for therapeutic discovery using traditional neuronal assay methods. Here we review advances in neuronal screening technologies and cellular model systems that enable phenotypic screening of neuronal function as a basis for novel CNS drug discovery approaches.


Subject(s)
Central Nervous System Agents/pharmacology , Central Nervous System Diseases/drug therapy , Drug Discovery , Neurons/drug effects , Cells, Cultured , Central Nervous System Agents/therapeutic use , Central Nervous System Diseases/pathology , Electrodes , High-Throughput Screening Assays , Humans , Induced Pluripotent Stem Cells
4.
J Biol Chem ; 291(4): 1948-1956, 2016 Jan 22.
Article in English | MEDLINE | ID: mdl-26627835

ABSTRACT

Variation in PPP3CC, the gene that encodes the γ isoform of the calcineurin catalytic subunit, has been reported to be associated with schizophrenia. Because of its low expression level in most tissues, there has been little research devoted to the specific function of the calcineurin Aγ (CNAγ) versus the calcineurin Aα (CNAα) and calcineurin Aß (CNAß) catalytic isoforms. Consequently, we have a limited understanding of the role of altered CNAγ function in psychiatric disease. In this study, we demonstrate that CNAγ is present in the rodent and human brain and dephosphorylates a presynaptic substrate of calcineurin. Through a combination of immunocytochemistry and immuno-EM, we further show that CNAγ is localized to presynaptic terminals in hippocampal neurons. Critically, we demonstrate that RNAi-mediated knockdown of CNAγ leads to a disruption of synaptic vesicle cycling in cultured rat hippocampal neurons. These data indicate that CNAγ regulates a critical aspect of synaptic vesicle cycling and suggest that variation in PPP3CC may contribute to psychiatric disease by altering presynaptic function.


Subject(s)
Calcineurin/metabolism , Endocytosis , Synaptic Vesicles/enzymology , Animals , Calcineurin/genetics , Cells, Cultured , Hippocampus/cytology , Hippocampus/enzymology , Humans , Male , Mice , Mice, Inbred BALB C , Neurons/enzymology , Rats , Synaptic Vesicles/genetics
5.
J Physiol ; 592(16): 3463-94, 2014 Aug 15.
Article in English | MEDLINE | ID: mdl-24879872

ABSTRACT

Parvalbumin-containing (PV) neurons, a major class of GABAergic interneurons, are essential circuit elements of learning networks. As levels of acetylcholine rise during active learning tasks, PV neurons become increasingly engaged in network dynamics. Conversely, impairment of either cholinergic or PV interneuron function induces learning deficits. Here, we examined PV interneurons in hippocampus (HC) and prefrontal cortex (PFC) and their modulation by muscarinic acetylcholine receptors (mAChRs). HC PV cells, visualized by crossing PV-CRE mice with Rosa26YFP mice, were anatomically identified as basket cells and PV bistratified cells in the stratum pyramidale; in stratum oriens, HC PV cells were electrophysiologically distinct from somatostatin-containing cells. With glutamatergic transmission pharmacologically blocked, mAChR activation enhanced PV cell excitability in both CA1 HC and PFC; however, CA1 HC PV cells exhibited a stronger postsynaptic depolarization than PFC PV cells. To delete M1 mAChRs genetically from PV interneurons, we created PV-M1 knockout mice by crossing PV-CRE and floxed M1 mice. The elimination of M1 mAChRs from PV cells diminished M1 mAChR immunoreactivity and muscarinic excitation of HC PV cells. Selective cholinergic activation of HC PV interneurons using Designer Receptors Exclusively Activated by Designer Drugs technology enhanced the frequency and amplitude of inhibitory synaptic currents in CA1 pyramidal cells. Finally, relative to wild-type controls, PV-M1 knockout mice exhibited impaired novel object recognition and, to a lesser extent, impaired spatial working memory, but reference memory remained intact. Therefore, the direct activation of M1 mAChRs on PV cells contributes to some forms of learning and memory.


Subject(s)
Action Potentials , Cognition , Inhibitory Postsynaptic Potentials , Interneurons/metabolism , Receptor, Muscarinic M1/metabolism , Animals , CA1 Region, Hippocampal/cytology , CA1 Region, Hippocampal/metabolism , CA1 Region, Hippocampal/physiology , Female , Interneurons/physiology , Male , Mice , Parvalbumins/genetics , Parvalbumins/metabolism , Prefrontal Cortex/cytology , Prefrontal Cortex/metabolism , Prefrontal Cortex/physiology , Receptor, Muscarinic M1/genetics
6.
J Biomol Screen ; 19(2): 205-14, 2014 Feb.
Article in English | MEDLINE | ID: mdl-24088370

ABSTRACT

Alterations in synaptic transmission have been implicated in a number of psychiatric and neurological disorders. The discovery of small-molecule modulators of proteins that regulate neurotransmission represents a novel therapeutic strategy for these diseases. However, high-throughput screening (HTS) approaches in primary neurons have been limited by challenges in preparing and applying primary neuronal cultures under conditions required for generating sufficiently robust and sensitive HTS assays. Synapsin I is an abundant presynaptic protein that plays a critical role in neurotransmission through tethering synaptic vesicles to the actin cytoskeleton. It has several phosphorylation sites that regulate its modulation of synaptic vesicle trafficking and, therefore, the efficacy of synaptic transmission. Here, we describe the development of a rapid, sensitive, and homogeneous assay to detect phospho-synapsin I (pSYN1) in primary cortical neurons in 384-well plates using AlphaScreen technology. From results of a pilot screening campaign, we show that the assay can identify compounds that modulate synapsin I phosphorylation via multiple signaling pathways. The implementation of the AlphaScreen pSYN1 assay and future development of additional primary neuronal HTS assays provides an attractive approach for discovery of novel classes of therapeutic candidates for a variety of CNS disorders.


Subject(s)
Brain/pathology , Neurons/metabolism , Synapsins/isolation & purification , Synaptic Transmission/genetics , Brain/metabolism , Cells, Cultured , High-Throughput Screening Assays , Humans , Nervous System Diseases/drug therapy , Nervous System Diseases/metabolism , Neurons/pathology , Phosphorylation , Signal Transduction , Synapsins/metabolism , Synaptic Vesicles/metabolism
7.
J Neurosci ; 33(27): 10938-49, 2013 Jul 03.
Article in English | MEDLINE | ID: mdl-23825400

ABSTRACT

Working memory is an essential component of higher cognitive function, and its impairment is a core symptom of multiple CNS disorders, including schizophrenia. Neuronal mechanisms supporting working memory under normal conditions have been described and include persistent, high-frequency activity of prefrontal cortical neurons. However, little is known about the molecular and cellular basis of working memory dysfunction in the context of neuropsychiatric disorders. To elucidate synaptic and neuronal mechanisms of working memory dysfunction, we have performed a comprehensive analysis of a mouse model of schizophrenia, the forebrain-specific calcineurin knock-out mouse. Biochemical analyses of cortical tissue from these mice revealed a pronounced hyperphosphorylation of synaptic vesicle cycling proteins known to be necessary for high-frequency synaptic transmission. Examination of the synaptic vesicle cycle in calcineurin-deficient neurons demonstrated an impairment of vesicle release enhancement during periods of intense stimulation. Moreover, brain slice and in vivo electrophysiological analyses showed that loss of calcineurin leads to a gene dose-dependent disruption of high-frequency synaptic transmission and network activity in the PFC, correlating with selective working memory impairment. Finally, we showed that levels of dynamin I, a key presynaptic protein and calcineurin substrate, are significantly reduced in prefrontal cortical samples from schizophrenia patients, extending the disease relevance of our findings. Our data provide support for a model in which impaired synaptic vesicle cycling represents a critical node for disease pathologies underlying the cognitive deficits in schizophrenia.


Subject(s)
Calcineurin/deficiency , Memory Disorders/metabolism , Memory, Short-Term/physiology , Prefrontal Cortex/metabolism , Synaptic Transmission/physiology , Synaptic Vesicles/metabolism , Adult , Animals , Calcineurin/genetics , Female , Humans , Male , Memory Disorders/genetics , Mice , Mice, Knockout , Middle Aged , Nerve Net/metabolism , Organ Culture Techniques , Synaptic Vesicles/genetics
8.
PLoS One ; 6(10): e25999, 2011.
Article in English | MEDLINE | ID: mdl-21998743

ABSTRACT

Unbiased, high-throughput screening has proven invaluable for dissecting complex biological processes. Application of this general approach to synaptic function would have a major impact on neuroscience research and drug discovery. However, existing techniques for studying synaptic physiology are labor intensive and low-throughput. Here, we describe a new high-throughput technology for performing assays of synaptic function in primary neurons cultured in microtiter plates. We show that this system can perform 96 synaptic vesicle cycling assays in parallel with high sensitivity, precision, uniformity, and reproducibility and can detect modulators of presynaptic function. By screening libraries of pharmacologically defined compounds on rat forebrain cultures, we have used this system to identify novel effects of compounds on specific aspects of presynaptic function. As a system for unbiased compound as well as genomic screening, this technology has significant applications for basic neuroscience research and for the discovery of novel, mechanism-based treatments for central nervous system disorders.


Subject(s)
High-Throughput Screening Assays/methods , Synapses/drug effects , Synapses/physiology , Animals , Cells, Cultured , Drug Discovery , Neurons/cytology , Neurons/drug effects , Rats , Synaptic Vesicles/drug effects , Time Factors
9.
Proc Natl Acad Sci U S A ; 104(8): 2815-20, 2007 Feb 20.
Article in English | MEDLINE | ID: mdl-17360599

ABSTRACT

The calcineurin cascade is central to neuronal signal transduction, and genes in this network are intriguing candidate schizophrenia susceptibility genes. To replicate and extend our previously reported association between the PPP3CC gene, encoding the calcineurin catalytic gamma-subunit, and schizophrenia, we examined 84 SNPs from 14 calcineurin-related candidate genes for genetic association by using 124 Japanese schizophrenic pedigrees. Four of these genes (PPP3CC, EGR2, EGR3, and EGR4) showed nominally significant association with schizophrenia. In a postmortem brain study, EGR1, EGR2, and EGR3 transcripts were shown to be down-regulated in the prefrontal cortex of schizophrenic, but not bipolar, patients. These findings raise a potentially important role for EGR genes in schizophrenia pathogenesis. Because EGR3 is an attractive candidate gene based on its chromosomal location close to PPP3CC within 8p21.3 and its functional link to dopamine, glutamate, and neuregulin signaling, we extended our analysis by resequencing the entire EGR3 genomic interval and detected 15 SNPs. One of these, IVS1 + 607A-->G SNP, displayed the strongest evidence for disease association, which was confirmed in 1,140 independent case-control samples. An in vitro promoter assay detected a possible expression-regulatory effect of this SNP. These findings support the previous genetic association of altered calcineurin signaling with schizophrenia pathogenesis and identify EGR3 as a compelling susceptibility gene.


Subject(s)
Calcineurin/genetics , Early Growth Response Protein 3/genetics , Genetic Predisposition to Disease , Multigene Family/genetics , Schizophrenia/genetics , Adult , Brain/metabolism , Calcineurin/metabolism , Case-Control Studies , Early Growth Response Protein 3/metabolism , Female , Gene Expression Regulation , Genetic Testing , Humans , Linkage Disequilibrium/genetics , Male , Middle Aged , Pedigree , Polymorphism, Single Nucleotide/genetics , RNA, Messenger/genetics , RNA, Messenger/metabolism
10.
Trends Pharmacol Sci ; 27(4): 226-33, 2006 Apr.
Article in English | MEDLINE | ID: mdl-16530856

ABSTRACT

Schizophrenia is a devastating psychiatric disorder that affects approximately 1% of the population worldwide. It is characterized by so-called 'positive symptoms'--including delusions and hallucinations--'negative symptoms'--including blunted emotions and social isolation--and cognitive deficits--including impairments in attention and working memory. Studies of the inheritance of schizophrenia have revealed that it is a multifactorial disease that is characterized by multiple genetic susceptibility elements, each contributing a modest degree of risk. Linkage studies have identified several potential schizophrenia susceptibility loci, and in recent years major progress has been made in the identification of positional candidate susceptibility genes from these loci. A central goal of future research will be to use this genetic knowledge to generate specific animal models, characterize genetic interactions, investigate the disease pathophysiology and assist drug-discovery efforts.


Subject(s)
Genetic Predisposition to Disease , Schizophrenia/genetics , Animals , Carrier Proteins/genetics , Catechol O-Methyltransferase/genetics , Chromosome Mapping , Disease Models, Animal , Dysbindin , Dystrophin-Associated Proteins , Genetic Linkage , Humans , Meta-Analysis as Topic , Proline Oxidase/genetics , RGS Proteins/genetics , Schizophrenia/drug therapy , Schizophrenia/metabolism
12.
Proc Natl Acad Sci U S A ; 100(15): 8987-92, 2003 Jul 22.
Article in English | MEDLINE | ID: mdl-12851457

ABSTRACT

Calcineurin (CN), a calcium- and calmodulin-dependent protein phosphatase, plays a significant role in the central nervous system. Previously, we reported that forebrain-specific CN knockout mice (CN mutant mice) have impaired working memory. To further analyze the behavioral effects of CN deficiency, we subjected CN mutant mice to a comprehensive behavioral test battery. Mutant mice showed increased locomotor activity, decreased social interaction, and impairments in prepulse inhibition and latent inhibition. In addition, CN mutant mice displayed an increased response to the locomotor stimulating effects of MK-801. Collectively, the abnormalities of CN mutant mice are strikingly similar to those described for schizophrenia. We propose that alterations affecting CN signaling could comprise a contributing factor in schizophrenia pathogenesis.


Subject(s)
Calcineurin/deficiency , Schizophrenia/etiology , Amphetamine/pharmacology , Animals , Behavior, Animal , Calcineurin/genetics , Calcineurin/physiology , Corpus Striatum/drug effects , Corpus Striatum/physiopathology , Disease Models, Animal , Dizocilpine Maleate/pharmacology , Dopamine/physiology , Glutamic Acid/physiology , Humans , Male , Mice , Mice, Inbred C57BL , Mice, Knockout , Motor Activity/drug effects , Schizophrenia/genetics , Schizophrenia/physiopathology , Signal Transduction , Social Behavior
13.
Proc Natl Acad Sci U S A ; 100(15): 8993-8, 2003 Jul 22.
Article in English | MEDLINE | ID: mdl-12851458

ABSTRACT

Schizophrenia is a severe psychiatric disorder characterized by a complex mode of inheritance. Forebrain-specific CNB knockout mice display a spectrum of behavioral abnormalities related to altered behaviors observed in schizophrenia patients. To examine whether calcineurin dysfunction is involved in schizophrenia etiology, we undertook studies of an initial subset of calcineurin-related genes, prioritizing ones that map to loci previously implicated in schizophrenia by linkage studies. Transmission disequilibrium studies in a large sample of affected families detected association of the PPP3CC gene, which encodes the calcineurin gamma catalytic subunit, with disease. Our results identify PPP3CC, located at 8p21.3, as a potential schizophrenia susceptibility gene and support the proposal that alterations in calcineurin signaling contribute to schizophrenia pathogenesis.


Subject(s)
Calcineurin/genetics , Genetic Variation , Schizophrenia/genetics , Adult , Base Sequence , Calcineurin/chemistry , Calcineurin/physiology , Chromosome Mapping , Chromosomes, Human, Pair 8/genetics , DNA, Complementary/genetics , Haplotypes , Humans , Polymorphism, Genetic , Protein Subunits , Schizophrenia/etiology , Signal Transduction
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