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International Journal of Molecular... Aug 2023The human heart controls blood flow, and therewith enables the adequate supply of oxygen and nutrients to the body. The correct function of the heart is coordinated by...
Knockout of the Cardiac Transcription Factor NKX2-5 Results in Stem Cell-Derived Cardiac Cells with Typical Purkinje Cell-like Signal Transduction and Extracellular Matrix Formation.
The human heart controls blood flow, and therewith enables the adequate supply of oxygen and nutrients to the body. The correct function of the heart is coordinated by the interplay of different cardiac cell types. Thereby, one can distinguish between cells of the working myocardium, the pace-making cells in the sinoatrial node (SAN) and the conduction system cells in the AV-node, the His-bundle or the Purkinje fibres. Tissue-engineering approaches aim to generate hiPSC-derived cardiac tissues for disease modelling and therapeutic usage with a significant improvement in the differentiation quality of myocardium and pace-making cells. The differentiation of cells with cardiac conduction system properties is still challenging, and the produced cell mass and quality is poor. Here, we describe the generation of cardiac cells with properties of the cardiac conduction system, called conduction system-like cells (CSLC). As a primary approach, we introduced a CrispR-Cas9-directed knockout of the NKX2-5 gene in hiPSC. NKX2-5-deficient hiPSC showed altered connexin expression patterns characteristic for the cardiac conduction system with strong connexin 40 and connexin 43 expression and suppressed connexin 45 expression. Application of differentiation protocols for ventricular- or SAN-like cells could not reverse this connexin expression pattern, indicating a stable regulation by NKX2-5 on connexin expression. The contraction behaviour of the hiPSC-derived CSLCs was compared to hiPSC-derived ventricular- and SAN-like cells. We found that the contraction speed of CSLCs resembled the expected contraction rate of human conduction system cells. Overall contraction was reduced in differentiated cells derived from NKX2-5 knockout hiPSC. Comparative transcriptomic data suggest a specification of the cardiac subtype of CSLC that is distinctly different from ventricular or pacemaker-like cells with reduced myocardial gene expression and enhanced extracellular matrix formation for improved electrical insulation. In summary, knockout of NKX2-5 in hiPSC leads to enhanced differentiation of cells with cardiac conduction system features, including connexin expression and contraction behaviour.
Topics: Humans; Cardiac Conduction System Disease; Homeobox Protein Nkx-2.5; Purkinje Cells; Purkinje Fibers; Signal Transduction; Sinoatrial Node; Stem Cells; Transcription Factors; Induced Pluripotent Stem Cells
PubMed: 37686171
DOI: 10.3390/ijms241713366 -
Neuropharmacology Sep 2023Cellular responses to metabotropic glutamate (mGlu) receptor activation are shaped by mechanisms of receptor-receptor interaction. mGlu receptor subtypes form... (Review)
Review
Cellular responses to metabotropic glutamate (mGlu) receptor activation are shaped by mechanisms of receptor-receptor interaction. mGlu receptor subtypes form homodimers, intra- or inter-group heterodimers, and heteromeric complexes with other G protein-coupled receptors (GPCRs). In addition, mGlu receptors may functionally interact with other receptors through the βγ subunits released from G proteins in response to receptor activation or other mechanisms. Here, we discuss the interactions between (i) mGlu and GABA receptors in cerebellar Purkinje cells; (ii) mGlu and 5-HTserotonergic receptors in the prefrontal cortex; (iii) mGlu and A receptors or mGlu and D dopamine receptors in medium spiny projection neurons of the indirect and direct pathways of the basal ganglia motor circuit; (iv) mGlu and A receptors in relation to the pathophysiology of Alzheimer's disease; and (v) mGlu and A adenosine or α- or β adrenergic receptors. In addition, we describe in detail a novel form of non-heterodimeric interaction between mGlu and mGlu receptors, which appears to be critically involved in mechanisms of activity-dependent synaptic plasticity in the prefrontal cortex and hippocampus. Finally, we highlight the potential implication of these interactions in the pathophysiology and treatment of cerebellar disorders, schizophrenia, Alzheimer's disease, Parkinson's disease, l-DOPA-induced dyskinesias, stress-related disorders, and cognitive dysfunctions. This article is part of the Special Issue on "The receptor-receptor interaction as a new target for therapy".
Topics: Humans; Alzheimer Disease; Levodopa; Parkinson Disease; Receptor, Metabotropic Glutamate 5; Basal Ganglia
PubMed: 37142158
DOI: 10.1016/j.neuropharm.2023.109569 -
Neuron May 2024Children diagnosed with autism spectrum disorder (ASD) commonly present with sensory hypersensitivity or abnormally strong reactions to sensory stimuli. Such...
Children diagnosed with autism spectrum disorder (ASD) commonly present with sensory hypersensitivity or abnormally strong reactions to sensory stimuli. Such hypersensitivity can be overwhelming, causing high levels of distress that contribute markedly to the negative aspects of the disorder. Here, we identify a mechanism that underlies hypersensitivity in a sensorimotor reflex found to be altered in humans and in mice with loss of function in the ASD risk-factor gene SCN2A. The cerebellum-dependent vestibulo-ocular reflex (VOR), which helps maintain one's gaze during movement, was hypersensitized due to deficits in cerebellar synaptic plasticity. Heterozygous loss of SCN2A-encoded Na1.2 sodium channels in granule cells impaired high-frequency transmission to Purkinje cells and long-term potentiation, a form of synaptic plasticity important for modulating VOR gain. VOR plasticity could be rescued in mice via a CRISPR-activator approach that increases Scn2a expression, demonstrating that evaluation of a simple reflex can be used to assess and quantify successful therapeutic intervention.
Topics: Animals; NAV1.2 Voltage-Gated Sodium Channel; Mice; Neuronal Plasticity; Cerebellum; Autism Spectrum Disorder; Humans; Reflex, Vestibulo-Ocular; Male; Purkinje Cells; Mice, Inbred C57BL
PubMed: 38412857
DOI: 10.1016/j.neuron.2024.01.029 -
Science (New York, N.Y.) Jul 2023Canonically, each Purkinje cell (PC) in the adult cerebellum receives only one climbing fiber (CF) from the inferior olive. Underlying current theories of cerebellar...
Canonically, each Purkinje cell (PC) in the adult cerebellum receives only one climbing fiber (CF) from the inferior olive. Underlying current theories of cerebellar function is the notion that this highly conserved one-to-one relationship renders Purkinje dendrites into a single computational compartment. However, we discovered that multiple primary dendrites are a near-universal morphological feature in humans. Using tract tracing, immunolabeling, and in vitro electrophysiology, we found that in mice ~25% of mature multibranched cells receive more than one CF input. Two-photon calcium imaging in vivo revealed that separate dendrites can exhibit distinct response properties to sensory stimulation, indicating that some multibranched cells integrate functionally independent CF-receptive fields. These findings indicate that PCs are morphologically and functionally more diverse than previously thought.
Topics: Animals; Humans; Mice; Axons; Dendrites; Purkinje Cells; Synapses
PubMed: 37499000
DOI: 10.1126/science.adi1024 -
BioRxiv : the Preprint Server For... Jul 2023Purkinje cell dysfunction causes movement disorders such as ataxia, however, recent evidence suggests that Purkinje cell dysfunction may also alter sleep regulation....
UNLABELLED
Purkinje cell dysfunction causes movement disorders such as ataxia, however, recent evidence suggests that Purkinje cell dysfunction may also alter sleep regulation. Here, we used an ataxia mouse model generated by silencing Purkinje cell neurotransmission ( ) to better understand how cerebellar dysfunction impacts sleep physiology. We focused our analysis on sleep architecture and electrocorticography (ECoG) patterns based on their relevance to extracting physiological measurements during sleep. We found that circadian activity is unaltered in the mutant mice, although their sleep parameters and ECoG patterns are modified. The mutant mice have decreased wakefulness and rapid eye movement (REM) sleep, while non-rapid eye movement (NREM) sleep is increased. The mutant mice have an extended latency to REM sleep, which is also observed in human ataxia patients. Spectral analysis of ECoG signals revealed alterations in the power distribution across different frequency bands defining sleep. Therefore, Purkinje cell dysfunction may influence wakefulness and equilibrium of distinct sleep stages in ataxia. Our findings posit a connection between cerebellar dysfunction and disrupted sleep and underscore the importance of examining cerebellar circuit function in sleep disorders.
SUMMARY STATEMENT
Utilizing a precise genetic mouse model of ataxia, we provide insights into the cerebellum's role in sleep regulation, highlighting its potential as a therapeutic target for motor disorders-related sleep disruptions.
PubMed: 37461479
DOI: 10.1101/2023.07.03.547586 -
Cell Reports Dec 2023Cerebellar dysfunction has been linked to autism spectrum disorders (ASDs). Although cerebellar pathology has been observed in individuals with fragile X syndrome (FXS)...
Cerebellar dysfunction has been linked to autism spectrum disorders (ASDs). Although cerebellar pathology has been observed in individuals with fragile X syndrome (FXS) and in mouse models of the disorder, a cerebellar functional contribution to ASD-relevant behaviors in FXS has yet to be fully characterized. In this study, we demonstrate a critical cerebellar role for Fmr1 (fragile X messenger ribonucleoprotein 1) in ASD-relevant behaviors. First, we identify reduced social behaviors, sensory hypersensitivity, and cerebellar dysfunction, with loss of cerebellar Fmr1. We then demonstrate that cerebellar-specific expression of Fmr1 is sufficient to impact social, sensory, cerebellar dysfunction, and cerebro-cortical hyperexcitability phenotypes observed in global Fmr1 mutants. Moreover, we demonstrate that targeting the ASD-implicated cerebellar region Crus1 ameliorates behaviors in both cerebellar-specific and global Fmr1 mutants. Together, these results demonstrate a critical role for the cerebellar contribution to FXS-related behaviors, with implications for future therapeutic strategies.
Topics: Animals; Mice; Fragile X Syndrome; Autistic Disorder; Fragile X Mental Retardation Protein; Autism Spectrum Disorder; Disease Models, Animal; Cerebellar Diseases; Mice, Knockout
PubMed: 38048226
DOI: 10.1016/j.celrep.2023.113533 -
Nature Communications Feb 2024In the central nervous system, astrocytes enable appropriate synapse function through glutamate clearance from the synaptic cleft; however, it remains unclear how...
In the central nervous system, astrocytes enable appropriate synapse function through glutamate clearance from the synaptic cleft; however, it remains unclear how astrocytic glutamate transporters function at peri-synaptic contact. Here, we report that Down syndrome cell adhesion molecule (DSCAM) in Purkinje cells controls synapse formation and function in the developing cerebellum. Dscam-mutant mice show defects in CF synapse translocation as is observed in loss of function mutations in the astrocytic glutamate transporter GLAST expressed in Bergmann glia. These mice show impaired glutamate clearance and the delocalization of GLAST away from the cleft of parallel fibre (PF) synapse. GLAST complexes with the extracellular domain of DSCAM. Riluzole, as an activator of GLAST-mediated uptake, rescues the proximal impairment in CF synapse formation in Purkinje cell-selective Dscam-deficient mice. DSCAM is required for motor learning, but not gross motor coordination. In conclusion, the intercellular association of synaptic and astrocyte proteins is important for synapse formation and function in neural transmission.
Topics: Animals; Mice; Amino Acid Transport System X-AG; Cerebellum; Glutamic Acid; Neuroglia; Neurons; Purkinje Cells; Synapses
PubMed: 38302444
DOI: 10.1038/s41467-023-44579-z -
Cureus Aug 2023Lhermitte-Duclos disease (LDD), or dysplastic cerebellar gangliocytoma, is a rare benign tumor characterized by unilateral hemispheric cerebellar expansion. It is linked...
Lhermitte-Duclos disease (LDD), or dysplastic cerebellar gangliocytoma, is a rare benign tumor characterized by unilateral hemispheric cerebellar expansion. It is linked to mutations in the phosphatase and tensin homolog (PTEN) gene, which inhibit the phosphatidylinositol-3'-kinase pathway, leading to increased cell division and defective neuronal migration. This study aims to compare the clinical, radiological, histopathological, surgical resolution, and follow-up characteristics of reported cases of this rare condition. An in-depth search of LDD patients' clinical records at our institute between 2003 and 2023 was conducted, in addition to a systematic literature review on PubMed. Three patients with a diagnosis of LDD were found. Cerebellar abnormalities, varying headaches, and visual impairment were all present clinically. On T2 in the posterior fossa, all three MRI scans displayed the typical hyperintense parallel streak appearance. The histopathological report showed that large ganglion cells had replaced the granular layer, Purkinje cells had degenerated, the molecular layer had become hyper-myelinated, and synaptophysin and chromogranin were positive. Partial tumor resection and avoiding intracranial hypertension were the main goals of treatment. Genetic follow-up was conducted for all three patients. Neurosurgeons must be aware of LDD to provide close genetic monitoring despite the benign nature of the tumor because of its link to Cowden syndrome and elevated risk of cancer in other organs.
PubMed: 37779805
DOI: 10.7759/cureus.44326 -
Acta Neuropathologica Aug 2023Essential Tremor (ET) is a prevalent neurological disease characterized by an 8-10 Hz action tremor. Molecular mechanisms of ET remain poorly understood. Clinical data...
Essential Tremor (ET) is a prevalent neurological disease characterized by an 8-10 Hz action tremor. Molecular mechanisms of ET remain poorly understood. Clinical data suggest the importance of the cerebellum in disease pathophysiology, and pathological studies indicate Purkinje Cells (PCs) incur damage. Our recent cerebellar cortex and PC-specific transcriptome studies identified alterations in calcium (Ca) signaling pathways that included ryanodine receptor type 1 (RyR1) in ET. RyR1 is an intracellular Ca release channel located on the Endoplasmic Reticulum (ER), and in cerebellum is predominantly expressed in PCs. Under stress conditions, RyR1 undergoes several post-translational modifications (protein kinase A [PKA] phosphorylation, oxidation, nitrosylation), coupled with depletion of the channel-stabilizing binding partner calstabin1, which collectively characterize a "leaky channel" biochemical signature. In this study, we found markedly increased PKA phosphorylation at the RyR1-S2844 site, increased RyR1 oxidation and nitrosylation, and calstabin1 depletion from the RyR1 complex in postmortem ET cerebellum. Decreased calstabin1-RyR1-binding affinity correlated with loss of PCs and climbing fiber-PC synapses in ET. This 'leaky' RyR1 signature was not seen in control or Parkinson's disease cerebellum. Microsomes from postmortem cerebellum demonstrated excessive ER Ca leak in ET vs. controls, attenuated by channel stabilization. We further studied the role of RyR1 in tremor using a mouse model harboring a RyR1 point mutation that mimics constitutive site-specific PKA phosphorylation (RyR1-S2844D). RyR1-S2844D homozygous mice develop a 10 Hz action tremor and robust abnormal oscillatory activity in cerebellar physiological recordings. Intra-cerebellar microinfusion of RyR1 agonist or antagonist, respectively, increased or decreased tremor amplitude in RyR1-S2844D mice, supporting a direct role of cerebellar RyR1 leakiness for tremor generation. Treating RyR1-S2844D mice with a novel RyR1 channel-stabilizing compound, Rycal, effectively dampened cerebellar oscillatory activity, suppressed tremor, and normalized cerebellar RyR1-calstabin1 binding. These data collectively support that stress-associated ER Ca leak via RyR1 may contribute to tremor pathophysiology.
Topics: Humans; Ryanodine Receptor Calcium Release Channel; Calcium; Tremor; Cerebellum; Endoplasmic Reticulum; Muscle, Skeletal
PubMed: 37335342
DOI: 10.1007/s00401-023-02602-z