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Cell Reports Jan 2024While ATM loss of function has long been identified as the genetic cause of ataxia-telangiectasia (A-T), how it leads to selective and progressive degeneration of...
While ATM loss of function has long been identified as the genetic cause of ataxia-telangiectasia (A-T), how it leads to selective and progressive degeneration of cerebellar Purkinje and granule neurons remains unclear. ATM expression is enriched in microglia throughout cerebellar development and adulthood. Here, we find evidence of microglial inflammation in the cerebellum of patients with A-T using single-nucleus RNA sequencing. Pseudotime analysis revealed that activation of A-T microglia preceded upregulation of apoptosis-related genes in granule and Purkinje neurons and that microglia exhibited increased neurotoxic cytokine signaling to granule and Purkinje neurons in A-T. To confirm these findings experimentally, we performed transcriptomic profiling of A-T induced pluripotent stem cell (iPSC)-derived microglia, which revealed cell-intrinsic microglial activation of cytokine production and innate immune response pathways compared to controls. Furthermore, A-T microglia co-culture with either control or A-T iPSC-derived neurons was sufficient to induce cytotoxicity. Taken together, these studies reveal that cell-intrinsic microglial activation may promote neurodegeneration in A-T.
Topics: Humans; Ataxia Telangiectasia; Microglia; Ataxia Telangiectasia Mutated Proteins; Neurons; Cytokines
PubMed: 38159274
DOI: 10.1016/j.celrep.2023.113622 -
Nature Communications Aug 2023Synaptic adhesion molecules (SAMs) shape the structural and functional properties of synapses and thereby control the information processing power of neural circuits....
Synaptic adhesion molecules (SAMs) shape the structural and functional properties of synapses and thereby control the information processing power of neural circuits. SAMs are broadly expressed in the brain, suggesting that they may instruct synapse formation and specification via a combinatorial logic. Here, we generate sextuple conditional knockout mice targeting all members of the two major families of presynaptic SAMs, Neurexins and leukocyte common antigen-related-type receptor phospho-tyrosine phosphatases (LAR-PTPRs), which together account for the majority of known trans-synaptic complexes. Using synapses formed by cerebellar Purkinje cells onto deep cerebellar nuclei as a model system, we confirm that Neurexins and LAR-PTPRs themselves are not essential for synapse assembly. The combinatorial deletion of both neurexins and LAR-PTPRs, however, decreases Purkinje-cell synapses on deep cerebellar nuclei, the major output pathway of cerebellar circuits. Consistent with this finding, combined but not separate deletions of neurexins and LAR-PTPRs impair motor behaviors. Thus, Neurexins and LAR-PTPRs are together required for the assembly of a functional cerebellar circuit.
Topics: Animals; Mice; Cerebellum; Purkinje Cells; Brain; Cognition; Mice, Knockout; Phosphotyrosine; Protein Tyrosine Phosphatases; Receptor-Like Protein Tyrosine Phosphatases, Class 2
PubMed: 37591863
DOI: 10.1038/s41467-023-40526-0 -
Brain : a Journal of Neurology Aug 2023Multiple system atrophy (MSA) is a neurodegenerative disease characterized by the accumulation of misfolded α-synuclein (αSyn) and myelin disruption. However, the...
Multiple system atrophy (MSA) is a neurodegenerative disease characterized by the accumulation of misfolded α-synuclein (αSyn) and myelin disruption. However, the mechanism underlying αSyn accumulation in MSA brains remains unclear. Here, we aimed to identify epsin-2 as a potential regulator of αSyn propagation in MSA brains. In the MSA mouse model, PLP-hαSyn mice, and FABP7/αSyn hetero-aggregate-injected mice, we initially discovered that fatty acid-binding protein 7 (FABP7) is related to MSA development and forms hetero-aggregates with αSyn, which exhibit stronger toxicity than αSyn aggregates. Moreover, the injected FABP7/αSyn hetero-aggregates in mice selectively accumulated only in oligodendrocytes and Purkinje neurons, causing cerebellar dysfunction. Furthermore, bioinformatic analyses of whole blood from MSA patients and FABP7 knockdown mice revealed that epsin-2, a protein expressed in both oligodendrocytes and Purkinje cells, could potentially regulate FABP7/αSyn hetero-aggregate propagation via clathrin-dependent endocytosis. Lastly, adeno-associated virus type 5-dependent epsin-2 knockdown mice exhibited decreased levels of αSyn aggregate accumulation in Purkinje neurons and oligodendrocytes, as well as improved myelin levels and Purkinje neuron function in the cerebellum and motor performance. These findings suggest that epsin-2 plays a significant role in αSyn accumulation in MSA, and we propose epsin-2 as a novel therapeutic target for MSA.
Topics: Mice; Animals; Multiple System Atrophy; alpha-Synuclein; Fatty Acid-Binding Protein 7; Mice, Transgenic; Oligodendroglia; Brain
PubMed: 37082980
DOI: 10.1093/brain/awad137 -
Brain : a Journal of Neurology Mar 2024AFG3L2 is a mitochondrial protease exerting protein quality control in the inner mitochondrial membrane. Heterozygous AFG3L2 mutations cause spinocerebellar ataxia type...
AFG3L2 is a mitochondrial protease exerting protein quality control in the inner mitochondrial membrane. Heterozygous AFG3L2 mutations cause spinocerebellar ataxia type 28 (SCA28) or dominant optic atrophy type 12 (DOA12), while biallelic AFG3L2 mutations result in the rare and severe spastic ataxia type 5 (SPAX5). The clinical spectrum of SPAX5 includes childhood-onset cerebellar ataxia, spasticity, dystonia and myoclonic epilepsy. We previously reported that the absence or mutation of AFG3L2 leads to the accumulation of mitochondria-encoded proteins, causing the overactivation of the stress-sensitive protease OMA1, which over-processes OPA1, leading to mitochondrial fragmentation. Recently, OMA1 has been identified as the pivotal player communicating mitochondrial stress to the cytosol via a pathway involving the inner mitochondrial membrane protein DELE1 and the cytosolic kinase HRI, thus eliciting the integrated stress response. In general, the integrated stress response reduces global protein synthesis and drives the expression of cytoprotective genes that allow cells to endure proteotoxic stress. However, the relevance of the OMA1-DELE1-HRI axis in vivo, and especially in a human CNS disease context, has been poorly documented thus far. In this work, we demonstrated that mitochondrial proteotoxicity in the absence/mutation of AFG3L2 activates the OMA1-DELE1-HRI pathway eliciting the integrated stress response. We found enhanced OMA1-dependent processing of DELE1 upon depletion of AFG3L2. Also, in both skin fibroblasts from SPAX5 patients (including a novel case) and in the cerebellum of Afg3l2-/- mice we detected increased phosphorylation of the α-subunit of the eukaryotic translation initiation factor 2 (eIF2α), increased levels of ATF4 and strong upregulation of its downstream targets (Chop, Chac1, Ppp1r15a and Ffg21). Silencing of DELE1 or HRI in SPAX5 fibroblasts (where OMA1 is overactivated at basal state) reduces eIF2α phosphorylation and affects cell growth. In agreement, pharmacological potentiation of integrated stress response via Sephin-1, a drug that selectively inhibits the stress-induced eIF2alpha phosphatase GADD34 (encoded by Ppp1r15a), improved cell growth of SPAX5 fibroblasts and cell survival and dendritic arborization ex vivo in primary Afg3l2-/- Purkinje neurons. Notably, Sephin-1 treatment in vivo extended the lifespan of Afg3l2-/- mice, improved Purkinje neuron morphology, mitochondrial ultrastructure and respiratory capacity. These data indicate that activation of the OMA1-DELE1-HRI pathway is protective in the context of SPAX5. Pharmacological tuning of the integrated stress response may represent a future therapeutic strategy for SPAX5 and other cerebellar ataxias caused by impaired mitochondrial proteostasis.
Topics: Humans; Animals; Mice; Child; Spinocerebellar Ataxias; Muscle Spasticity; Optic Atrophy; Peptide Hydrolases; ATPases Associated with Diverse Cellular Activities; ATP-Dependent Proteases; Mitochondrial Proteins; Metalloproteases; Intellectual Disability
PubMed: 37804316
DOI: 10.1093/brain/awad340 -
Nature Communications Jul 2023The classification of neuronal subpopulations has significantly advanced, yet its relevance for behavior remains unclear. The highly organized flocculus of the...
The classification of neuronal subpopulations has significantly advanced, yet its relevance for behavior remains unclear. The highly organized flocculus of the cerebellum, known to fine-tune multi-axial eye movements, is an ideal substrate for the study of potential functions of neuronal subpopulations. Here, we demonstrate that its recently identified subpopulations of 9+ and 9- Purkinje cells exhibit an intermediate Aldolase C expression and electrophysiological profile, providing evidence for a graded continuum of intrinsic properties among PC subpopulations. By identifying and utilizing two Cre-lines that genetically target these floccular domains, we show with high spatial specificity that these subpopulations of Purkinje cells participate in separate micromodules with topographically organized connections. Finally, optogenetic excitation of the respective subpopulations results in movements around the same axis in space, yet with distinct kinematic profiles. These results indicate that Purkinje cell subpopulations integrate in discrete circuits and mediate particular parameters of single movements.
Topics: Purkinje Cells; Biomechanical Phenomena; Eye Movements; Cerebellum; Movement
PubMed: 37468512
DOI: 10.1038/s41467-023-40111-5 -
Circulation Nov 2023Conotruncal defects due to developmental abnormalities of the outflow tract (OFT) are an important cause of cyanotic congenital heart disease. Dysregulation of...
BACKGROUND
Conotruncal defects due to developmental abnormalities of the outflow tract (OFT) are an important cause of cyanotic congenital heart disease. Dysregulation of transcriptional programs tuned by NKX2-5 (NK2 homeobox 5), GATA6 (GATA binding protein 6), and TBX1 (T-box transcription factor 1) have been implicated in abnormal OFT morphogenesis. However, there remains no consensus on how these transcriptional programs function in a unified gene regulatory network within the OFT.
METHODS
We generated mice harboring a 226-nucleotide deletion of a highly conserved cardiac enhancer containing 2 GATA-binding sites located ≈9.4 kb upstream of the transcription start site of () using CRISPR-Cas9 gene editing and assessed phenotypes. Cardiac defects in mice were structurally characterized using histology and scanning electron microscopy, and physiologically assessed using electrocardiography, echocardiography, and optical mapping. Transcriptome analyses were performed using RNA sequencing and single-cell RNA sequencing data sets. Endogenous GATA6 interaction with and activity on the enhancer was studied using chromatin immunoprecipitation sequencing and transposase-accessible chromatin sequencing in human induced pluripotent stem cell-derived cardiomyocytes.
RESULTS
mice recapitulated cyanotic conotruncal defects seen in patients with , and mutations. mice also exhibited defects in right Purkinje fiber network formation, resulting in right bundle-branch block. Enhancer deletion reduced embryonic expression selectively in the right ventricle and OFT of mutant hearts, indicating that enhancer activity is localized to the anterior second heart field. Transcriptional profiling of the mutant OFT revealed downregulation of important genes involved in OFT rotation and septation, such as , and . Endogenous GATA6 interacted with the highly conserved enhancer in human induced pluripotent stem cell-derived cardiomyocytes and in wild-type mouse hearts. We found critical dose dependency of cardiac enhancer accessibility on gene dosage in human induced pluripotent stem cell-derived cardiomyocytes.
CONCLUSIONS
Our results using human and mouse models reveal an essential gene regulatory network of the OFT that requires an anterior second heart field enhancer to link GATA6 with NKX2-5-dependent rotation and septation gene programs.
Topics: Humans; Mice; Animals; Transcription Factors; Homeodomain Proteins; Gene Regulatory Networks; Homeobox Protein Nkx-2.5; Mice, Transgenic; Induced Pluripotent Stem Cells; Heart; Myocytes, Cardiac; Gene Expression Regulation, Developmental
PubMed: 37772400
DOI: 10.1161/CIRCULATIONAHA.123.065700 -
BioRxiv : the Preprint Server For... Sep 2023The cerebellar cortex contributes to diverse behaviors by transforming mossy fiber inputs into predictions in the form of Purkinje cell (PC) outputs, and then refining...
The cerebellar cortex contributes to diverse behaviors by transforming mossy fiber inputs into predictions in the form of Purkinje cell (PC) outputs, and then refining those predictions. Molecular layer interneurons (MLIs) account for approximately 80% of the inhibitory interneurons in the cerebellar cortex, and are vital to cerebellar processing. MLIs are thought to primarily inhibit PCs and suppress the plasticity of excitatory synapses onto PCs. MLIs also inhibit, and are electrically coupled to, other MLIs, but the functional significance of these connections is not known. Behavioral studies suggest that cerebellar-dependent learning is gated by disinhibition of PCs, but the source of such disinhibition has not been identified. Here we find that two recently recognized MLI subtypes, MLI1 and MLI2, have highly specialized connectivity that allows them to serve very different functional roles. MLI1s primarily inhibit PCs, are electrically coupled to each other, fire synchronously with other MLI1s on the millisecond time scale , and synchronously pause PC firing. MLI2s are not electrically coupled, they primarily inhibit MLI1s and disinhibit PCs, and are well suited to gating cerebellar-dependent learning. These findings require a major reevaluation of processing within the cerebellum in which disinhibition, a powerful circuit motif present in the cerebral cortex and elsewhere, greatly increases the computational power and flexibility of the cerebellum. They also suggest that millisecond time scale synchronous firing of electrically-coupled MLI1s helps regulate the output of the cerebellar cortex by synchronously pausing PC firing, which has been shown to evoke precisely-timed firing in PC targets.
PubMed: 37745401
DOI: 10.1101/2023.09.15.557934 -
Development (Cambridge, England) Nov 2023Reciprocal interactions between non-myocytes and cardiomyocytes regulate cardiac growth and differentiation. Here, we report that the transcription factor Ebf1 is highly...
Reciprocal interactions between non-myocytes and cardiomyocytes regulate cardiac growth and differentiation. Here, we report that the transcription factor Ebf1 is highly expressed in non-myocytes and potently regulates heart development. Ebf1-deficient hearts display myocardial hypercellularity and reduced cardiomyocyte size, ventricular conduction system hypoplasia, and conduction system disease. Growth abnormalities in Ebf1 knockout hearts are observed as early as embryonic day 13.5. Transcriptional profiling of Ebf1-deficient embryonic cardiac non-myocytes demonstrates dysregulation of Polycomb repressive complex 2 targets, and ATAC-Seq reveals altered chromatin accessibility near many of these same genes. Gene set enrichment analysis of differentially expressed genes in cardiomyocytes isolated from E13.5 hearts of wild-type and mutant mice reveals significant enrichment of MYC targets and, consistent with this finding, we observe increased abundance of MYC in mutant hearts. EBF1-deficient non-myocytes, but not wild-type non-myocytes, are sufficient to induce excessive accumulation of MYC in co-cultured wild-type cardiomyocytes. Finally, we demonstrate that BMP signaling induces Ebf1 expression in embryonic heart cultures and controls a gene program enriched in EBF1 targets. These data reveal a previously unreported non-cell-autonomous pathway controlling cardiac growth and differentiation.
Topics: Animals; Mice; Cell Differentiation; Gene Expression Regulation; Myocardium; Myocytes, Cardiac; Transcription Factors
PubMed: 37787076
DOI: 10.1242/dev.202054 -
Life Science Alliance Sep 2023Niemann-Pick disease type C1 (NPC1) is a fatal lysosomal storage disorder characterized by progressive neuronal degeneration. Its key pathogenic events remain largely...
Niemann-Pick disease type C1 (NPC1) is a fatal lysosomal storage disorder characterized by progressive neuronal degeneration. Its key pathogenic events remain largely unknown. We have, herein, found that neonatal BM-derived cell transplantation can ameliorate Purkinje cell degeneration in NPC1 mice. We subsequently addressed the impact of the peripheral immune system on the neuropathogenesis observed in NPC1 mice. The depletion of mature lymphocytes promoted NPC1 phenotypes, thereby suggesting a neuroprotective effect of lymphocytes. Moreover, the peripheral infusion of CD4-positive cells (specifically, of regulatory T cells) from normal healthy donor ameliorated the cerebellar ataxic phenotype and enhanced the survival of Purkinje cells. Conversely, the depletion of regulatory T cells enhanced the onset of the neurological phenotype. On the other hand, circulating inflammatory monocytes were found to be involved in the progression of Purkinje cell degeneration, whereas the depletion of resident microglia had little effect. Our findings reveal a novel role of the adaptive and the innate immune systems in NPC1 neuropathology.
Topics: Mice; Animals; Purkinje Cells; Niemann-Pick Disease, Type C; Cerebellum; Immune System; Microglia
PubMed: 37369603
DOI: 10.26508/lsa.202201881