Modeling antisense oligonucleotide therapy in MECP2 duplication syndrome human iPSC-derived neurons reveals gene expression programs responsive to MeCP2 levels.

Genomic copy-number variations (CNVs) that can cause neurodevelopmental disorders often encompass many genes, which complicates our understanding of how individual genes within a CNV contribute to pathology. MECP2 duplication syndrome (MDS or MRXSL in OMIM; OMIM#300260) is one such CNV disorder caused by duplications spanning methyl CpG-binding protein 2 (MECP2) and other genes on Xq28. Using an antisense oligonucleotide (ASO) to normalize MECP2 dosage is sufficient to rescue abnormal neurological phenotypes in mouse models overexpressing MECP2 alone, implicating the importance of increased MECP2 dosage within CNVs of Xq28. However, because MDS CNVs span MECP2 and additional genes, we generated human neurons from multiple MDS patient-derived induced pluripotent cells (iPSCs) to evaluate the benefit of using an ASO against MECP2 in a MDS human neuronal context. Importantly, we identified a signature of genes that is partially and qualitatively modulated upon ASO treatment, pinpointed genes sensitive to MeCP2 function, and altered in a model of Rett syndrome, a neurological disorder caused by loss of MeCP2 function. Furthermore, the signature contained genes that are aberrantly altered in unaffected control human neurons upon MeCP2 depletion, revealing gene expression programs qualitatively sensitive to MeCP2 levels in human neurons. Lastly, ASO treatment led to a partial rescue of abnormal neuronal morphology in MDS neurons. All together, these data demonstrate that ASOs targeting MECP2 benefit human MDS neurons. Moreover, our study establishes a paradigm by which to evaluate the contribution of individual genes within a CNV to pathogenesis and to assess their potential as a therapeutic target.

A single-cell transcriptomic map of the developing Atoh1 lineage identifies neural fate decisions and neuronal diversity in the hindbrain.

Proneural transcription factors establish molecular cascades to orchestrate neuronal diversity. One such transcription factor, Atonal homolog 1 (Atoh1), gives rise to cerebellar excitatory neurons and over 30 distinct nuclei in the brainstem critical for hearing, breathing, and balance. Although Atoh1 lineage neurons have been qualitatively described, the transcriptional programs that drive their fate decisions and the full extent of their diversity remain unknown. Here, we analyzed single-cell RNA sequencing and ATOH1 DNA binding in Atoh1 lineage neurons of the developing mouse hindbrain. This high-resolution dataset identified markers for specific brainstem nuclei and demonstrated that transcriptionally heterogeneous progenitors require ATOH1 for proper migration. Moreover, we identified a sizable population of proliferating unipolar brush cell progenitors in the mouse Atoh1 lineage, previously described in humans as the origin of one medulloblastoma subtype. Collectively, our data provide insights into the developing mouse hindbrain and markers for functional assessment of understudied neuronal populations.

The big tau splice isoform resists Alzheimer's-related pathological changes.

In Alzheimer's disease (AD), the microtubule-binding protein tau becomes abnormally hyperphosphorylated and aggregated in selective brain regions such as the cortex and hippocampus 1-3 . However, other brain regions like the cerebellum and brain stem remain largely intact despite the universal expression of tau throughout the brain. Here, we found that an understudied splice isoform of tau termed "big tau" is significantly more abundant in the brain regions less vulnerable to tau pathology compared to tau pathology-vulnerable regions. We used various cellular and animal models to demonstrate that big tau possesses multiple properties that can resist AD-related pathological changes. Importantly, human AD patients show a higher expression level of pathology-resisting big tau in the cerebellum, the brain region spared from tau pathology. Our study examines the unique properties of big tau, expanding our current understanding of tau pathophysiology. Altogether, our data suggest that alternative splicing to favor big tau is a viable strategy to modulate tau pathology.

MeCP2 Interacts with the Super Elongation Complex to Regulate Transcription.

Loss-of-function mutations in methyl-CpG binding protein 2 ( MECP2 ) cause Rett syndrome, a postnatal neurodevelopmental disorder that occurs in ∼1/10,000 live female births. MeCP2 binds to methylated cytosines across genomic DNA and recruits various partners to regulate gene expression. MeCP2 has been shown to repress transcription in vitro and interacts with co-repressors such as the Sin3A and NCoR complexes. Based on these observations, MeCP2 has been largely considered as a repressor of transcription. However, a mouse model of RTT displays many down-regulated genes, and those same genes are up-regulated in a MECP2 duplication mouse model. Furthermore, TCF20, which has been associated with transcriptional activation, have recently been identified as a protein interactor of MeCP2. These data broaden the potential functions of MeCP2 as a regulator of gene expression. Yet, the molecular mechanisms underlying MeCP2-dependent gene regulation remain largely unknown. Here, using a human MECP2 gain-of-function Drosophila model, we screened for genetic modifiers of MECP2 -induced phenotypes. Our approach identified several subunits of the Drosophila super elongation complex, a P-TEFb containing RNA polymerase II (RNA pol II) elongation factor required for the release of promoter-proximally paused RNA pol II, as genetic interactors of MECP2 . We discovered that MeCP2 physically interacts with the SEC in human cells and in the mouse brain. Furthermore, we found that MeCP2 directly binds AFF4, the scaffold of the SEC, via the transcriptional repression domain. Finally, loss of MeCP2 in the mouse cortex caused reduced binding of AFF4 specifically on a subset of genes involved in the regulation of synaptic function, which also displayed the strongest decrease in RNA pol II binding in the genebody. Taken together, our study reveals a previously unrecognized mechanism through which MeCP2 regulates transcription, providing a new dimension to its regulatory role in gene expression.

Mapping SCA1 regional vulnerabilities reveals neural and skeletal muscle contributions to disease.

Spinocerebellar ataxia type 1 (SCA1) is a fatal neurodegenerative disease caused by an expanded polyglutamine tract in the widely expressed ataxin-1 (ATXN1) protein. To elucidate anatomical regions and cell types that underlie mutant ATXN1-induced disease phenotypes, we developed a floxed conditional knockin mouse (f-ATXN1146Q/2Q) with mouse Atxn1 coding exons replaced by human ATXN1 exons encoding 146 glutamines. f-ATXN1146Q/2Q mice manifested SCA1-like phenotypes including motor and cognitive deficits, wasting, and decreased survival. Central nervous system (CNS) contributions to disease were revealed using f-ATXN1146Q/2Q;Nestin-Cre mice, which showed improved rotarod, open field, and Barnes maze performance by 6-12 weeks of age. In contrast, striatal contributions to motor deficits using f-ATXN1146Q/2Q;Rgs9-Cre mice revealed that mice lacking ATXN1146Q/2Q in striatal medium-spiny neurons showed a trending improvement in rotarod performance at 30 weeks of age. Surprisingly, a prominent role for muscle contributions to disease was revealed in f-ATXN1146Q/2Q;ACTA1-Cre mice based on their recovery from kyphosis and absence of muscle pathology. Collectively, data from the targeted conditional deletion of the expanded allele demonstrated CNS and peripheral contributions to disease and highlighted the need to consider muscle in addition to the brain for optimal SCA1 therapeutics.

Longitudinal single-cell transcriptional dynamics throughout neurodegeneration in SCA1.

Neurodegeneration is a protracted process involving progressive changes in myriad cell types that ultimately results in the death of vulnerable neuronal populations. To dissect how individual cell types within a heterogeneous tissue contribute to the pathogenesis and progression of a neurodegenerative disorder, we performed longitudinal single-nucleus RNA sequencing of mouse and human spinocerebellar ataxia type 1 (SCA1) cerebellar tissue, establishing continuous dynamic trajectories of each cell population. Importantly, we defined the precise transcriptional changes that precede loss of Purkinje cells and, for the first time, identified robust early transcriptional dysregulation in unipolar brush cells and oligodendroglia. Finally, we applied a deep learning method to predict disease state accurately and identified specific features that enable accurate distinction of wild-type and SCA1 cells. Together, this work reveals new roles for diverse cerebellar cell types in SCA1 and provides a generalizable analysis framework for studying neurodegeneration.

The Atoh1-Cre Knock-In Allele Ectopically Labels a Subpopulation of Amacrine Cells and Bipolar Cells in Mouse Retina.

The retina has diverse neuronal cell types derived from a common pool of retinal progenitors. Many molecular drivers, mostly transcription factors, have been identified to promote different cell fates. In Drosophila, atonal is required for specifying photoreceptors. In mice, there are two closely related atonal homologs, Atoh1 and Atoh7 While Atoh7 is known to promote the genesis of retinal ganglion cells, there is no study on the function of Atoh1 in retinal development. Here, we crossed Atoh1Cre/+ mice to mice carrying a Cre-dependent TdTomato reporter to track potential Atoh1-lineage neurons in retinas. We characterized a heterogeneous group of TdTomato+ retinal neurons that were detected at the postnatal stage, including glutamatergic amacrine cells, AII amacrine cells, and BC3b bipolar cells. Unexpectedly, we did not observe TdTomato+ retinal neurons in the mice with an Atoh1-FlpO knock-in allele and a Flp-dependent TdTomato reporter, suggesting Atoh1 is not expressed in the mouse retina. Consistent with these data, conditional removal of Atoh1 in the retina did not cause any observable phenotypes. Importantly, we did not detect Atoh1 expression in the retina at multiple ages using mice with Atoh1-GFP knock-in allele. Therefore, we conclude that Atoh1Cre/+ mice have ectopic Cre expression in the retina and that Atoh1 is not required for retinal development.

A novel pathogenic mutation of MeCP2 impairs chromatin association independent of protein levels.

Loss-of-function mutations in MECP2 cause Rett syndrome (RTT), a severe neurological disorder that mainly affects girls. Mutations in MECP2 do occur in males occasionally and typically cause severe encephalopathy and premature lethality. Recently, we identified a missense mutation (c.353G>A, p.Gly118Glu [G118E]), which has never been seen before in MECP2, in a young boy who suffered from progressive motor dysfunction and developmental delay. To determine whether this variant caused the clinical symptoms and study its functional consequences, we established two disease models, including human neurons from patient-derived iPSCs and a knock-in mouse line. G118E mutation partially reduces MeCP2 abundance and its DNA binding, and G118E mice manifest RTT-like symptoms seen in the patient, affirming the pathogenicity of this mutation. Using live-cell and single-molecule imaging, we found that G118E mutation alters MeCP2's chromatin interaction properties in live neurons independently of its effect on protein levels. Here we report the generation and characterization of RTT models of a male hypomorphic variant and reveal new insight into the mechanism by which this pathological mutation affects MeCP2's chromatin dynamics. Our ability to quantify protein dynamics in disease models lays the foundation for harnessing high-resolution single-molecule imaging as the next frontier for developing innovative therapies for RTT and other diseases.

Literature-based predictions of Mendelian disease therapies.

In the effort to treat Mendelian disorders, correcting the underlying molecular imbalance may be more effective than symptomatic treatment. Identifying treatments that might accomplish this goal requires extensive and up-to-date knowledge of molecular pathways-including drug-gene and gene-gene relationships. To address this challenge, we present "parsing modifiers via article annotations" (PARMESAN), a computational tool that searches PubMed and PubMed Central for information to assemble these relationships into a central knowledge base. PARMESAN then predicts putatively novel drug-gene relationships, assigning an evidence-based score to each prediction. We compare PARMESAN's drug-gene predictions to all of the drug-gene relationships displayed by the Drug-Gene Interaction Database (DGIdb) and show that higher-scoring relationship predictions are more likely to match the directionality (up- versus down-regulation) indicated by this database. PARMESAN had more than 200,000 drug predictions scoring above 8 (as one example cutoff), for more than 3,700 genes. Among these predicted relationships, 210 were registered in DGIdb and 201 (96%) had matching directionality. This publicly available tool provides an automated way to prioritize drug screens to target the most-promising drugs to test, thereby saving time and resources in the development of therapeutics for genetic disorders.

Atoh1 drives the heterogeneity of the pontine nuclei neurons and promotes their differentiation.

Pontine nuclei (PN) neurons mediate the communication between the cerebral cortex andthe cerebellum to refine skilled motor functions. Prior studies showed that PN neurons fall into two subtypes based on their anatomic location and region-specific connectivity, but the extent of their heterogeneity and its molecular drivers remain unknown. Atoh1 encodes a transcription factor that is expressed in the PN precursors. We previously showed that partial loss of Atoh1 function in mice results in delayed PN development and impaired motor learning. In this study, we performed single-cell RNA sequencing to elucidate the cell state-specific functions of Atoh1 during PN development and found that Atoh1 regulates cell cycle exit, differentiation, migration, and survival of PN neurons. Our data revealed six previously not known PN subtypes that are molecularly and spatially distinct. We found that the PN subtypes exhibit differential vulnerability to partial loss of Atoh1 function, providing insights into the prominence of PN phenotypes in patients with ATOH1 missense mutations.

Delineating regional vulnerability in the neurodegenerative disease SCA1 using a conditional mutant ATXN1 mouse.

Spinocerebellar ataxia type 1 (SCA1) is a fatal neurodegenerative disease caused by an expanded polyglutamine tract in the widely expressed ATXN1 protein. To elucidate anatomical regions and cell types that underlie mutant ATXN1-induced disease phenotypes, we developed a floxed conditional knockout mouse model ( f-ATXN1 146Q/2Q ) having mouse Atxn1 coding exons replaced by human exons encoding 146 glutamines. F-ATXN1 146Q/2Q mice manifest SCA1-like phenotypes including motor and cognitive deficits, wasting, and decreased survival. CNS contributions to disease were revealed using ATXN1 146Q/2Q ; Nestin-Cre mice, that showed improved rotarod, open field and Barnes maze performances. Striatal contributions to motor deficits were examined using f-ATXN1 146Q/2Q ; Rgs9-Cre mice. Mice lacking striatal ATXN1 146Q/2Q had improved rotarod performance late in disease. Muscle contributions to disease were revealed in f-ATXN1 146Q/2Q ; ACTA1-Cre mice which lacked muscle pathology and kyphosis seen in f-ATXN1 146Q/2Q mice. Kyphosis was not improved in f-ATXN1 146Q/2Q ;Nestin - Cre mice. Thus, optimal SCA1 therapeutics will require targeting mutant ATXN1 toxic actions in multiple brain regions and muscle.

MeCP2 regulates Gdf11, a dosage-sensitive gene critical for neurological function.

Loss- and gain-of-function of MeCP2 causes Rett syndrome (RTT) and MECP2 duplication syndrome (MDS), respectively. MeCP2 binds methyl-cytosines to finely tune gene expression in the brain, but identifying genes robustly regulated by MeCP2 has been difficult. By integrating multiple transcriptomics datasets, we revealed that MeCP2 finely regulates growth differentiation factor 11 (Gdf11). Gdf11 is down-regulated in RTT mouse models and, conversely, up-regulated in MDS mouse models. Strikingly, genetically normalizing Gdf11 dosage levels improved several behavioral deficits in a mouse model of MDS. Next, we discovered that losing one copy of Gdf11 alone was sufficient to cause multiple neurobehavioral deficits in mice, most notably hyperactivity and decreased learning and memory. This decrease in learning and memory was not due to changes in proliferation or numbers of progenitor cells in the hippocampus. Lastly, loss of one copy of Gdf11 decreased survival in mice, corroborating its putative role in aging. Our data demonstrate that Gdf11 dosage is important for brain function.

Disruption of the ATXN1-CIC complex reveals the role of additional nuclear ATXN1 interactors in spinocerebellar ataxia type 1.

Spinocerebellar ataxia type 1 (SCA1) is a paradigmatic neurodegenerative disease in that it is caused by a mutation in a broadly expressed protein, ATXN1; however, only select populations of cells degenerate. The interaction of polyglutamine-expanded ATXN1 with the transcriptional repressor CIC drives cerebellar Purkinje cell pathogenesis; however, the importance of this interaction in other vulnerable cells remains unknown. Here, we mutated the 154Q knockin allele of Atxn1154Q/2Q mice to prevent the ATXN1-CIC interaction globally. This normalized genome-wide CIC binding; however, it only partially corrected transcriptional and behavioral phenotypes, suggesting the involvement of additional factors in disease pathogenesis. Using unbiased proteomics, we identified three ATXN1-interacting transcription factors: RFX1, ZBTB5, and ZKSCAN1. We observed altered expression of RFX1 and ZKSCAN1 target genes in SCA1 mice and patient-derived iNeurons, highlighting their potential contributions to disease. Together, these data underscore the complexity of mechanisms driving cellular vulnerability in SCA1.

Decreasing mutant ATXN1 nuclear localization improves a spectrum of SCA1-like phenotypes and brain region transcriptomic profiles.

Spinocerebellar ataxia type 1 (SCA1) is a dominant trinucleotide repeat neurodegenerative disease characterized by motor dysfunction, cognitive impairment, and premature death. Degeneration of cerebellar Purkinje cells is a frequent and prominent pathological feature of SCA1. We previously showed that transport of ATXN1 to Purkinje cell nuclei is required for pathology, where mutant ATXN1 alters transcription. To examine the role of ATXN1 nuclear localization broadly in SCA1-like disease pathogenesis, CRISPR-Cas9 was used to develop a mouse with an amino acid alteration (K772T) in the nuclear localization sequence of the expanded ATXN1 protein. Characterization of these mice indicates that proper nuclear localization of mutant ATXN1 contributes to many disease-like phenotypes including motor dysfunction, cognitive deficits, and premature lethality. RNA sequencing analysis of genes with expression corrected to WT levels in Atxn1175QK772T/2Q mice indicates that transcriptomic aspects of SCA1 pathogenesis differ between the cerebellum, brainstem, cerebral cortex, hippocampus, and striatum.

Gene-based therapeutics for rare genetic neurodevelopmental psychiatric disorders.

We are in an emerging era of gene-based therapeutics with significant promise for rare genetic disorders. The potential is particularly significant for genetic central nervous system disorders that have begun to achieve Food and Drug Administration approval for select patient populations. This review summarizes the discussions and presentations of the National Institute of Mental Health-sponsored workshop "Gene-Based Therapeutics for Rare Genetic Neurodevelopmental Psychiatric Disorders," which was held in January 2021. Here, we distill the points raised regarding various precision medicine approaches related to neurodevelopmental and psychiatric disorders that may be amenable to gene-based therapies.

Cross-species genetic screens identify transglutaminase 5 as a regulator of polyglutamine-expanded ataxin-1.

Many neurodegenerative disorders are caused by abnormal accumulation of misfolded proteins. In spinocerebellar ataxia type 1 (SCA1), accumulation of polyglutamine-expanded (polyQ-expanded) ataxin-1 (ATXN1) causes neuronal toxicity. Lowering total ATXN1, especially the polyQ-expanded form, alleviates disease phenotypes in mice, but the molecular mechanism by which the mutant ATXN1 is specifically modulated is not understood. Here, we identified 22 mutant ATXN1 regulators by performing a cross-species screen of 7787 and 2144 genes in human cells and Drosophila eyes, respectively. Among them, transglutaminase 5 (TG5) preferentially regulated mutant ATXN1 over the WT protein. TG enzymes catalyzed cross-linking of ATXN1 in a polyQ-length-dependent manner, thereby preferentially modulating mutant ATXN1 stability and oligomerization. Perturbing Tg in Drosophila SCA1 models modulated mutant ATXN1 toxicity. Moreover, TG5 was enriched in the nuclei of SCA1-affected neurons and colocalized with nuclear ATXN1 inclusions in brain tissue from patients with SCA1. Our work provides a molecular insight into SCA1 pathogenesis and an opportunity for allele-specific targeting for neurodegenerative disorders.

Reduction of mutant ATXN1 rescues premature death in a conditional SCA1 mouse model.

Spinocerebellar ataxia type 1 (SCA1) is an adult-onset neurodegenerative disorder. As disease progresses, motor neurons are affected, and their dysfunction contributes toward the inability to maintain proper respiratory function, a major driving force for premature death in SCA1. To investigate the isolated role of motor neurons in SCA1, we created a conditional SCA1 (cSCA1) mouse model. This model suppresses expression of the pathogenic SCA1 allele with a floxed stop cassette. cSCA1 mice crossed to a ubiquitous Cre line recapitulate all the major features of the original SCA1 mouse model; however, they took twice as long to develop. We found that the cSCA1 mice produced less than half of the pathogenic protein compared with the unmodified SCA1 mice at 3 weeks of age. In contrast, restricted expression of the pathogenic SCA1 allele in motor neurons only led to a decreased distance traveled of mice in the open field assay and did not affect body weight or survival. We conclude that a 50% or greater reduction of the mutant protein has a dramatic effect on disease onset and progression; furthermore, we conclude that expression of polyglutamine-expanded ATXN1 at this level specifically in motor neurons is not sufficient to cause premature lethality.

A weakened recurrent circuit in the hippocampus of Rett syndrome mice disrupts long-term memory representations.

Successful recall of a contextual memory requires reactivating ensembles of hippocampal cells that were allocated during memory formation. Altering the ratio of excitation-to-inhibition (E/I) during memory retrieval can bias cell participation in an ensemble and hinder memory recall. In the case of Rett syndrome (RTT), a neurological disorder with severe learning and memory deficits, the E/I balance is altered, but the source of this imbalance is unknown. Using in vivo imaging during an associative memory task, we show that during long-term memory retrieval, RTT CA1 cells poorly distinguish mnemonic context and form larger ensembles than wild-type mouse cells. Simultaneous multiple whole-cell recordings revealed that mutant somatostatin expressing interneurons (SOM) are poorly recruited by CA1 pyramidal cells and are less active during long-term memory retrieval in vivo. Chemogenetic manipulation revealed that reduced SOM activity underlies poor long-term memory recall. Our findings reveal a disrupted recurrent CA1 circuit contributing to RTT memory impairment.

Disruption of MeCP2-TCF20 complex underlies distinct neurodevelopmental disorders.

MeCP2 is associated with Rett syndrome (RTT), MECP2 duplication syndrome, and a number of conditions with isolated features of these diseases, including autism, intellectual disability, and motor dysfunction. MeCP2 is known to broadly bind methylated DNA, but the precise molecular mechanism driving disease pathogenesis remains to be determined. Using proximity-dependent biotinylation (BioID), we identified a transcription factor 20 (TCF20) complex that interacts with MeCP2 at the chromatin interface. Importantly, RTT-causing mutations in MECP2 disrupt this interaction. TCF20 and MeCP2 are highly coexpressed in neurons and coregulate the expression of key neuronal genes. Reducing Tcf20 partially rescued the behavioral deficits caused by MECP2 overexpression, demonstrating a functional relationship between MeCP2 and TCF20 in MECP2 duplication syndrome pathogenesis. We identified a patient exhibiting RTT-like neurological features with a missense mutation in the PHF14 subunit of the TCF20 complex that abolishes the MeCP2-PHF14-TCF20 interaction. Our data demonstrate the critical role of the MeCP2-TCF20 complex for brain function.