Loss of Ataxin-1 Potentiates Alzheimer's Pathogenesis by Elevating Cerebral BACE1 Transcription.

Expansion of CAG trinucleotide repeats in ATXN1 causes spinocerebellar ataxia type 1 (SCA1), a neurodegenerative disease that impairs coordination and cognition. While ATXN1 is associated with increased Alzheimer's disease (AD) risk, CAG repeat number in AD patients is not changed. Here, we investigated the consequences of ataxin-1 loss of function and discovered that knockout of Atxn1 reduced CIC-ETV4/5-mediated inhibition of Bace1 transcription, leading to increased BACE1 levels and enhanced amyloidogenic cleavage of APP, selectively in AD-vulnerable brain regions. Elevated BACE1 expression exacerbated Aß deposition and gliosis in AD mouse models and impaired hippocampal neurogenesis and olfactory axonal targeting. In SCA1 mice, polyglutamine-expanded mutant ataxin-1 led to the increase of BACE1 post-transcriptionally, both in cerebrum and cerebellum, and caused axonal-targeting deficit and neurodegeneration in the hippocampal CA2 region. These findings suggest that loss of ataxin-1 elevates BACE1 expression and Aß pathology, rendering it a potential contributor to AD risk and pathogenesis.

A Mild PUM1 Mutation Is Associated with Adult-Onset Ataxia, whereas Haploinsufficiency Causes Developmental Delay and Seizures.

Certain mutations can cause proteins to accumulate in neurons, leading to neurodegeneration. We recently showed, however, that upregulation of a wild-type protein, Ataxin1, caused by haploinsufficiency of its repressor, the RNA-binding protein Pumilio1 (PUM1), also causes neurodegeneration in mice. We therefore searched for human patients with PUM1 mutations. We identified eleven individuals with either PUM1 deletions or de novo missense variants who suffer a developmental syndrome (Pumilio1-associated developmental disability, ataxia, and seizure; PADDAS). We also identified a milder missense mutation in a family with adult-onset ataxia with incomplete penetrance (Pumilio1-related cerebellar ataxia, PRCA). Studies in patient-derived cells revealed that the missense mutations reduced PUM1 protein levels by ∼25% in the adult-onset cases and by ∼50% in the infantile-onset cases; levels of known PUM1 targets increased accordingly. Changes in protein levels thus track with phenotypic severity, and identifying posttranscriptional modulators of protein expression should identify new candidate disease genes.

Rett Syndrome and the Ongoing Legacy of Close Clinical Observation.

This year marks the 50th anniversary of the publication of Andreas Rett's report on 22 girls who developed a peculiar and devastating neurological disorder that later came to bear his name. On this occasion, we reflect on the progress that has occurred in understanding Rett Syndrome, development of potential treatments, and the ramifications that Rett research has had on the fields of neurobiology and genetics.

Post-translational Control of the Temporal Dynamics of Transcription Factor Activity Regulates Neurogenesis.

Neurogenesis is initiated by the transient expression of the highly conserved proneural proteins, bHLH transcriptional regulators. Here, we discover a conserved post-translational switch governing the duration of proneural protein activity that is required for proper neuronal development. Phosphorylation of a single Serine at the same position in Scute and Atonal proneural proteins governs the transition from active to inactive forms by regulating DNA binding. The equivalent Neurogenin2 Threonine also regulates DNA binding and proneural activity in the developing mammalian neocortex. Using genome editing in Drosophila, we show that Atonal outlives its mRNA but is inactivated by phosphorylation. Inhibiting the phosphorylation of the conserved proneural Serine causes quantitative changes in expression dynamics and target gene expression resulting in neuronal number and fate defects. Strikingly, even a subtle change from Serine to Threonine appears to shift the duration of Atonal activity in vivo, resulting in neuronal fate defects.

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.

Pumilio1 haploinsufficiency leads to SCA1-like neurodegeneration by increasing wild-type Ataxin1 levels.

Spinocerebellar ataxia type 1 (SCA1) is a paradigmatic neurodegenerative proteinopathy, in which a mutant protein (in this case, ATAXIN1) accumulates in neurons and exerts toxicity; in SCA1, this process causes progressive deterioration of motor coordination. Seeking to understand how post-translational modification of ATAXIN1 levels influences disease, we discovered that the RNA-binding protein PUMILIO1 (PUM1) not only directly regulates ATAXIN1 but also plays an unexpectedly important role in neuronal function. Loss of Pum1 caused progressive motor dysfunction and SCA1-like neurodegeneration with motor impairment, primarily by increasing Ataxin1 levels. Breeding Pum1(+/-) mice to SCA1 mice (Atxn1(154Q/+)) exacerbated disease progression, whereas breeding them to Atxn1(+/-) mice normalized Ataxin1 levels and largely rescued the Pum1(+/-) phenotype. Thus, both increased wild-type ATAXIN1 levels and PUM1 haploinsufficiency could contribute to human neurodegeneration. These results demonstrate the importance of studying post-transcriptional regulation of disease-driving proteins to reveal factors underlying neurodegenerative disease.

Parkinson's Disease Genetics and Pathophysiology.

Parkinson's disease (PD) is a common neurodegenerative disorder characterized by degeneration of the substantia nigra pars compacta and by accumulation of α-synuclein in Lewy bodies. PD is caused by a combination of environmental factors and genetic variants. These variants range from highly penetrant Mendelian alleles to alleles that only modestly increase disease risk. Here, we review what is known about the genetics of PD. We also describe how PD genetics have solidified the role of endosomal, lysosomal, and mitochondrial dysfunction in PD pathophysiology. Finally, we highlight how all three pathways are affected by α-synuclein and how this knowledge may be harnessed for the development of disease-modifying therapeutics.

From anatomy to electrophysiology: clinical Lasker goes deep.

This year, the Lasker∼DeBakey Clinical Medical Research Award will be shared by Mahlon R. DeLong and Alim-Louis Benabid for elucidating the role of the subthalamic nucleus in mediating the motor dysfunction of Parkinson's disease and for pioneering the use of deep-brain stimulation to alleviate symptoms of the disease.

Identification and characterization of conserved noncoding cis-regulatory elements that impact Mecp2 expression and neurological functions.

While changes in MeCP2 dosage cause Rett syndrome (RTT) and MECP2 duplication syndrome (MDS), its transcriptional regulation is poorly understood. Here, we identified six putative noncoding regulatory elements of Mecp2, two of which are conserved in humans. Upon deletion in mice and human iPSC-derived neurons, these elements altered RNA and protein levels in opposite directions and resulted in a subset of RTT- and MDS-like behavioral deficits in mice. Our discovery provides insight into transcriptional regulation of Mecp2/MECP2 and highlights genomic sites that could serve as diagnostic and therapeutic targets in RTT or MDS.

An AT-hook domain in MeCP2 determines the clinical course of Rett syndrome and related disorders.

Mutations in the X-linked MECP2 cause Rett syndrome, a devastating neurological disorder typified by a period of apparently normal development followed by loss of cognitive and psychomotor skills. Data from rare male patients suggest symptom onset and severity can be influenced by the location of the mutation, with amino acids 270 and 273 marking the difference between neonatal encephalopathy and death, on the one hand, and survival with deficits on the other. We therefore generated two mouse models expressing either MeCP2-R270X or MeCP2-G273X. The mice developed phenotypes at strikingly different rates and showed differential ATRX nuclear localization within the nervous system, over time, coinciding with phenotypic progression. We discovered that MeCP2 contains three AT-hook-like domains over a stretch of 250 amino acids, like HMGA DNA-bending proteins; one conserved AT-hook is disrupted in MeCP2-R270X, lending further support to the notion that one of MeCP2's key functions is to alter chromatin structure.

miR760 regulates ATXN1 levels via interaction with its 5' untranslated region.

Identifying modifiers of dosage-sensitive genes involved in neurodegenerative disorders is imperative to discover novel genetic risk factors and potential therapeutic entry points. In this study, we focus on Ataxin-1 (ATXN1), a dosage-sensitive gene involved in the neurodegenerative disease spinocerebellar ataxia type 1 (SCA1). While the precise maintenance of ATXN1 levels is essential to prevent disease, the mechanisms that regulate ATXN1 expression remain largely unknown. We demonstrate that ATXN1's unusually long 5' untranslated region (5' UTR) negatively regulates its expression via posttranscriptional mechanisms. Based on recent reports that microRNAs (miRNAs) can interact with both 3' and 5' UTRs to regulate their target genes, we identify miR760 as a negative regulator that binds to a conserved site in ATXN1's 5' UTR to induce RNA degradation and translational inhibition. We found that delivery of Adeno-associated virus (AAV)-expressing miR760 in the cerebellum reduces ATXN1 levels in vivo and mitigates motor coordination deficits in a mouse model of SCA1. These findings provide new insights into the regulation of ATXN1 levels, present additional evidence for miRNA-mediated gene regulation via 5' UTR binding, and raise the possibility that noncoding mutations in the ATXN1 locus may act as risk factors for yet to be discovered progressive ataxias.

Presymptomatic training mitigates functional deficits in a mouse model of Rett syndrome.

Mutations in the X-linked gene MECP2 cause Rett syndrome, a progressive neurological disorder in which children develop normally for the first one or two years of life before experiencing profound motor and cognitive decline1-3. At present there are no effective treatments for Rett syndrome, but we hypothesized that using the period of normal development to strengthen motor and memory skills might confer some benefit. Here we find, using a mouse model of Rett syndrome, that intensive training beginning in the presymptomatic period dramatically improves the performance of specific motor and memory tasks, and significantly delays the onset of symptoms. These benefits are not observed when the training begins after symptom onset. Markers of neuronal activity and chemogenetic manipulation reveal that task-specific neurons that are repeatedly activated during training develop more dendritic arbors and have better neurophysiological responses than those in untrained animals, thereby enhancing their functionality and delaying symptom onset. These results provide a rationale for genetic screening of newborns for Rett syndrome, as presymptomatic intervention might mitigate symptoms or delay their onset. Similar strategies should be studied for other childhood neurological disorders.

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.

Dual targeting of brain region-specific kinases potentiates neurological rescue in Spinocerebellar ataxia type 1.

A critical question in neurodegeneration is why the accumulation of disease-driving proteins causes selective neuronal loss despite their brain-wide expression. In Spinocerebellar ataxia type 1 (SCA1), accumulation of polyglutamine-expanded Ataxin-1 (ATXN1) causes selective degeneration of cerebellar and brainstem neurons. Previous studies revealed that inhibiting Msk1 reduces phosphorylation of ATXN1 at S776 as well as its levels leading to improved cerebellar function. However, there are no regulators that modulate ATXN1 in the brainstem-the brain region whose pathology is most closely linked to premature death. To identify new regulators of ATXN1, we performed genetic screens and identified a transcription factor-kinase axis (ZBTB7B-RSK3) that regulates ATXN1 levels. Unlike MSK1, RSK3 is highly expressed in the human and mouse brainstems where it regulates Atxn1 by phosphorylating S776. Reducing Rsk3 rescues brainstem-associated pathologies and deficits, and lowering Rsk3 and Msk1 together improves cerebellar and brainstem function in an SCA1 mouse model. Our results demonstrate that selective vulnerability of brain regions in SCA1 is governed by region-specific regulators of ATXN1, and targeting multiple regulators could rescue multiple degenerating brain areas.

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.

Insufficient Evidence for "Autism-Specific" Genes.

Despite evidence that deleterious variants in the same genes are implicated across multiple neurodevelopmental and neuropsychiatric disorders, there has been considerable interest in identifying genes that, when mutated, confer risk that is largely specific for autism spectrum disorder (ASD). Here, we review the findings and limitations of recent efforts to identify relatively "autism-specific" genes, efforts which focus on rare variants of large effect size that are thought to account for the observed phenotypes. We present a divergent interpretation of published evidence; discuss practical and theoretical issues related to studying the relationships between rare, large-effect deleterious variants and neurodevelopmental phenotypes; and describe potential future directions of this research. We argue that there is currently insufficient evidence to establish meaningful ASD specificity of any genes based on large-effect rare-variant data.

Coexpression enrichment analysis at the single-cell level reveals convergent defects in neural progenitor cells and their cell-type transitions in neurodevelopmental disorders.

A large number of genes have been implicated in neurodevelopmental disorders (NDDs), but their contributions to NDD pathology are difficult to decipher without understanding their diverse roles in different brain cell types. Here, we integrated NDD genetics with single-cell RNA sequencing data to assess coexpression enrichment patterns of various NDD gene sets. We identified midfetal cortical neural progenitor cell development-more specifically, the ventricular radial glia-to-intermediate progenitor cell transition at gestational week 10-as a key point of convergence in autism spectrum disorder (ASD) and epilepsy. Integrated Gene Ontology-based analysis further revealed that ASD genes activate neural differentiation and inhibit cell cycle during the transition, whereas epilepsy genes function as downstream effectors in the same processes, offering one possible explanation for the high comorbidity rate of the two disorders. This approach provides a framework for investigating the cell-type-specific pathophysiology of NDDs.

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.

Recommendations by the ClinGen Rett/Angelman-like expert panel for gene-specific variant interpretation methods.

The genes MECP2, CDKL5, FOXG1, UBE3A, SLC9A6, and TCF4 present unique challenges for current ACMG/AMP variant interpretation guidelines. To address those challenges, the Rett and Angelman-like Disorders Variant Curation Expert Panel (Rett/AS VCEP) drafted gene-specific modifications. A pilot study was conducted to test the clarity and accuracy of using the customized variant interpretation criteria. Multiple curators obtained the same interpretation for 78 out of the 87 variants (~90%), indicating appropriate usage of the modified guidelines the majority of times by all the curators. The classification of 13 variants changed using these criteria specifications compared to when the variants were originally curated and as present in ClinVar. Many of these changes were due to internal data shared from laboratory members however some changes were because of changes in strength of criteria. There were no two-step classification changes and only 1 clinically relevant change (Likely pathogenic to VUS). The Rett/AS VCEP hopes that these gene-specific variant curation rules and the assertions provided help clinicians, clinical laboratories, and others interpret variants in these genes but also other fully penetrant, early-onset genes associated with rare disorders.

Combination of whole exome sequencing and animal modeling identifies TMPRSS9 as a candidate gene for autism spectrum disorder.

Autism spectrum disorders are associated with some degree of developmental regression in up to 30% of all cases. Rarely, however, is the regression so extreme that a developmentally advanced young child would lose almost all ability to communicate and interact with her surroundings. We applied trio whole exome sequencing to a young woman who experienced extreme developmental regression starting at 2.5 years of age and identified compound heterozygous nonsense mutations in TMPRSS9, which encodes for polyserase-1, a transmembrane serine protease of poorly understood physiological function. Using semiquantitative polymerase chain reaction, we showed that Tmprss9 is expressed in various mouse tissues, including the brain. To study the consequences of TMPRSS9 loss of function on the mammalian brain, we generated a knockout mouse model. Through a battery of behavioral assays, we found that Tmprss9-/- mice showed decreased social interest and social recognition. We observed a borderline recognition memory deficit by novel object recognition in aged Tmprss9-/- female mice, but not in aged Tmprss9-/- male mice or younger adult Tmprss9-/- mice in both sexes. This study provides evidence to suggest that loss of function variants in TMPRSS9 are related to an autism spectrum disorder. However, the identification of more individuals with similar phenotypes and TMPRSS9 loss of function variants is required to establish a robust gene-disease relationship.