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.

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.

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.

Evolutionarily conserved regulators of tau identify targets for new therapies.

Tauopathies are neurodegenerative diseases that involve the pathological accumulation of tau proteins; in this family are Alzheimer disease, corticobasal degeneration, and chronic traumatic encephalopathy, among others. Hypothesizing that reducing this accumulation could mitigate pathogenesis, we performed a cross-species genetic screen targeting 6,600 potentially druggable genes in human cells and Drosophila. We found and validated 83 hits in cells and further validated 11 hits in the mouse brain. Three of these hits (USP7, RNF130, and RNF149) converge on the C terminus of Hsc70-interacting protein (CHIP) to regulate tau levels, highlighting the role of CHIP in maintaining tau proteostasis in the brain. Knockdown of each of these three genes in adult tauopathy mice reduced tau levels and rescued the disease phenotypes. This study thus identifies several points of intervention to reduce tau levels and demonstrates that reduction of tau levels via regulation of this pathway is a viable therapeutic strategy for Alzheimer disease and other tauopathies.

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.

The Transcription Factor Pou3f1 Sheds Light on the Development and Molecular Diversity of Glutamatergic Cerebellar Nuclear Neurons in the Mouse.

The cerebellar nuclear (CN) neurons are a molecularly heterogeneous population whose specification into the different cerebellar nuclei is defined by the expression of varying sets of transcription factors. Here, we present a novel molecular marker, Pou3f1, that delineates specific sets of glutamatergic CN neurons. The glutamatergic identity of Pou3f1+ cells was confirmed by: (1) the co-expression of vGluT2, a cell marker of glutamatergic neurons; (2) the lack of co-expression between Pou3f1 and GAD67, a marker of GABAergic neurons; (3) the co-expression of Atoh1, the master regulator required for the production of all cerebellar glutamatergic lineages; and (4) the absence of Pou3f1-expressing cells in the Atoh1-null cerebellum. Furthermore, the lack of Pax6 and Tbr1 expression in Pou3f1+ cells reveals that Pou3f1-expressing CN neurons specifically settle in the interposed and dentate nuclei. In addition, the Pou3f1-labeled glutamatergic CN neurons can be further classified by the expression of Brn2 and Irx3. The results of the present study align with previous findings highlighting that the survival of the interposed and dentate CN neurons is largely independent of Pax6. More importantly, the present study extends the field's collective knowledge of the molecular diversity of cerebellar nuclei.

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.

C. Thomas Caskey (1938-2022).

A visionary architect of genomic medicine.

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.