Circuit-Specific Deep Brain Stimulation Provides Insights into Movement Control.

Deep brain stimulation (DBS), a method in which electrical stimulation is delivered to specific areas of the brain, is an effective treatment for managing symptoms of a number of neurological and neuropsychiatric disorders. Clinical access to neural circuits during DBS provides an opportunity to study the functional link between neural circuits and behavior. This review discusses how the use of DBS in Parkinson's disease and dystonia has provided insights into the brain networks and physiological mechanisms that underlie motor control. In parallel, insights from basic science about how patterns of electrical stimulation impact plasticity and communication within neural circuits are transforming DBS from a therapy for treating symptoms to a therapy for treating circuits, with the goal of training the brain out of its diseased state. Expected final online publication date for the Annual Review of Neuroscience, Volume 47 is July 2024. Please see http://www.annualreviews.org/page/journal/pubdates for revised estimates.

Cerebellar Functions Beyond Movement and Learning.

The cerebellum has a well-established role in controlling motor functions, including coordination, posture, and the learning of skilled movements. The mechanisms for how it carries out motor behavior remain under intense investigation. Interestingly though, in recent years the mechanisms of cerebellar function have faced additional scrutiny since nonmotor behaviors may also be controlled by the cerebellum. With such complexity arising, there is now a pressing need to better understand how cerebellar structure, function, and behavior intersect to influence behaviors that are dynamically called upon as an animal experiences its environment. Here, we discuss recent experimental work that frames possible neural mechanisms for how the cerebellum shapes disparate behaviors and why its dysfunction is catastrophic in hereditary and acquired conditions-both motor and nonmotor. For these reasons, the cerebellum might be the ideal therapeutic target.

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.

MLL4 Is Required to Maintain Broad H3K4me3 Peaks and Super-Enhancers at Tumor Suppressor Genes.

Super-enhancers are large clusters of enhancers that activate gene expression. Broad trimethyl histone H3 lysine 4 (H3K4me3) often defines active tumor suppressor genes. However, how these epigenomic signatures are regulated for tumor suppression is little understood. Here we show that brain-specific knockout of the H3K4 methyltransferase MLL4 (a COMPASS-like enzyme, also known as KMT2D) in mice spontaneously induces medulloblastoma. Mll4 loss upregulates oncogenic Ras and Notch pathways while downregulating neuronal gene expression programs. MLL4 enhances DNMT3A-catalyzed DNA methylation and SIRT1/BCL6-mediated H4K16 deacetylation, which antagonize expression of Ras activators and Notch pathway components, respectively. Notably, Mll4 loss downregulates tumor suppressor genes (e.g., Dnmt3a and Bcl6) by diminishing broad H3K4me3 and super-enhancers and also causes widespread impairment of these epigenomic signatures during medulloblastoma genesis. These findings suggest an anti-tumor role for super-enhancers and provide a unique tumor-suppressive mechanism in which MLL4 is necessary to maintain broad H3K4me3 and super-enhancers at tumor suppressor genes.

Emerging connections between cerebellar development, behaviour and complex brain disorders.

The human cerebellum has a protracted developmental timeline compared with the neocortex, expanding the window of vulnerability to neurological disorders. As the cerebellum is critical for motor behaviour, it is not surprising that most neurodevelopmental disorders share motor deficits as a common sequela. However, evidence gathered since the late 1980s suggests that the cerebellum is involved in motor and non-motor function, including cognition and emotion. More recently, evidence indicates that major neurodevelopmental disorders such as intellectual disability, autism spectrum disorder, attention-deficit hyperactivity disorder and Down syndrome have potential links to abnormal cerebellar development. Out of recent findings from clinical and preclinical studies, the concept of the 'cerebellar connectome' has emerged that can be used as a framework to link the role of cerebellar development to human behaviour, disease states and the design of better therapeutic strategies.

Ankyrin-R Links Kv3.3 to the Spectrin Cytoskeleton and Is Required for Purkinje Neuron Survival.

Ankyrin scaffolding proteins are critical for membrane domain organization and protein stabilization in many different cell types including neurons. In the cerebellum, Ankyrin-R (AnkR) is highly enriched in Purkinje neurons, granule cells, and in the cerebellar nuclei (CN). Using male and female mice with a floxed allele for Ank1 in combination with Nestin-Cre and Pcp2-Cre mice, we found that ablation of AnkR from Purkinje neurons caused ataxia, regional and progressive neurodegeneration, and altered cerebellar output. We show that AnkR interacts with the cytoskeletal protein ß3 spectrin and the potassium channel Kv3.3. Loss of AnkR reduced somatic membrane levels of ß3 spectrin and Kv3.3 in Purkinje neurons. Thus, AnkR links Kv3.3 channels to the ß3 spectrin-based cytoskeleton. Our results may help explain why mutations in ß3 spectrin and Kv3.3 both cause spinocerebellar ataxia.SIGNIFICANCE STATEMENT Ankyrin scaffolding proteins localize and stabilize ion channels in the membrane by linking them to the spectrin-based cytoskeleton. Here, we show that Ankyrin-R (AnkR) links Kv3.3 K+ channels to the ß3 spectrin-based cytoskeleton in Purkinje neurons. Loss of AnkR causes Purkinje neuron degeneration, altered cerebellar physiology, and ataxia, which is consistent with mutations in Kv3.3 and ß3 spectrin causing spinocerebellar ataxia.

Cerebellar Dysfunction as a Source of Dystonic Phenotypes in Mice.

There is now a substantial amount of compelling evidence demonstrating that the cerebellum may be a central locus in dystonia pathogenesis. Studies using spontaneous genetic mutations in rats and mice, engineered genetic alleles in mice, shRNA knockdown in mice, and conditional genetic silencing of fast neurotransmission in mice have all uncovered a common set of behavioral and electrophysiological defects that point to cerebellar cortical and cerebellar nuclei dysfunction as a source of dystonic phenotypes. Here, we revisit the Ptf1aCre/+;Vglut2flox/flox mutant mouse to define fundamental phenotypes and measures that are valuable for testing the cellular, circuit, and behavioral mechanisms that drive dystonia. In this model, excitatory neurotransmission from climbing fibers is genetically eliminated and, as a consequence, Purkinje cell and cerebellar nuclei firing are altered in vivo, with a prominent and lasting irregular burst pattern of spike activity in cerebellar nuclei neurons. The resulting impact on behavior is that the mice have developmental abnormalities, including twisting of the limbs and torso. These behaviors continue into adulthood along with a tremor, which can be measured with a tremor monitor or EMG. Importantly, expression of dystonic behavior is reduced upon cerebellar-targeted deep brain stimulation. The presence of specific combinations of disease-like features and therapeutic responses could reveal the causative mechanisms of different types of dystonia and related conditions. Ultimately, an emerging theme places cerebellar dysfunction at the center of a broader dystonia brain network.

Causal Evidence for a Role of Cerebellar Lobulus Simplex in Prefrontal-Hippocampal Interaction in Spatial Working Memory Decision-Making.

Spatial working memory (SWM) is a cerebrocerebellar cognitive skill supporting survival-relevant behaviors, such as optimizing foraging behavior by remembering recent routes and visited sites. It is known that SWM decision-making in rodents requires the medial prefrontal cortex (mPFC) and dorsal hippocampus. The decision process in SWM tasks carries a specific electrophysiological signature of a brief, decision-related increase in neuronal communication in the form of an increase in the coherence of neuronal theta oscillations (4-12 Hz) between the mPFC and dorsal hippocampus, a finding we replicated here during spontaneous exploration of a plus maze in freely moving mice. We further evaluated SWM decision-related coherence changes within frequency bands above theta. Decision-related coherence increases occurred in seven frequency bands between 4 and 200 Hz and decision-outcome-related differences in coherence modulation occurred within the beta and gamma frequency bands and in higher frequency oscillations up to 130 Hz. With recent evidence that Purkinje cells in the cerebellar lobulus simplex (LS) represent information about the phase and phase differences of gamma oscillations in the mPFC and dorsal hippocampus, we hypothesized that LS might be involved in the modulation of mPFC-hippocampal gamma coherence. We show that optical stimulation of LS significantly impairs SWM performance and decision-related mPFC-dCA1 coherence modulation, providing causal evidence for an involvement of cerebellar LS in SWM decision-making at the behavioral and neuronal level. Our findings suggest that the cerebellum might contribute to SWM decision-making by optimizing the decision-related modulation of mPFC-dCA1 coherence.

Genetically eliminating Purkinje neuron GABAergic neurotransmission increases their response gain to vestibular motion.

Purkinje neurons in the caudal cerebellar vermis combine semicircular canal and otolith signals to segregate linear and gravitational acceleration, evidence for how the cerebellum creates internal models of body motion. However, it is not known which cerebellar circuit connections are necessary to perform this computation. We first showed that this computation is evolutionarily conserved and represented across multiple lobules of the rodent vermis. Then we tested whether Purkinje neuron GABAergic output is required for accurately differentiating linear and gravitational movements through a conditional genetic silencing approach. By using extracellular recordings from lobules VI through X in awake mice, we show that silencing Purkinje neuron output significantly alters their baseline simple spike variability. Moreover, the cerebellum of genetically manipulated mice continues to distinguish linear from gravitational acceleration, suggesting that the underlying computations remain intact. However, response gain is significantly increased in the mutant mice over littermate controls. Altogether, these data argue that Purkinje neuron feedback regulates gain control within the cerebellar circuit.

MeCP2 Levels Regulate the 3D Structure of Heterochromatic Foci in Mouse Neurons.

Methyl-CpG binding protein 2 (MeCP2) is a nuclear protein critical for normal brain function, and both depletion and overexpression of MeCP2 lead to severe neurodevelopmental disease, Rett syndrome (RTT) and MECP2 multiplication disorder, respectively. However, the molecular mechanism by which abnormal MeCP2 dosage causes neuronal dysfunction remains unclear. As MeCP2 expression is nearly equivalent to that of core histones and because it binds DNA throughout the genome, one possible function of MeCP2 is to regulate the 3D structure of chromatin. Here, to examine whether and how MeCP2 levels impact chromatin structure, we used high-resolution confocal and electron microscopy and examined heterochromatic foci of neurons in mice. Using models of RTT and MECP2 triplication syndrome, we found that the heterochromatin structure was significantly affected by the alteration in MeCP2 levels. Analysis of mice expressing either MeCP2-R270X or MeCP2-G273X, which have nonsense mutations in the upstream and downstream regions of the AT-hook 2 domain, respectively, showed that the magnitude of heterochromatin changes was tightly correlated with the phenotypic severity. Postnatal alteration in MeCP2 levels also induced significant changes in the heterochromatin structure, which underscored importance of correct MeCP2 dosage in mature neurons. Finally, functional analysis of MeCP2-overexpressing mice showed that the behavioral and transcriptomic alterations in these mice correlated significantly with the MeCP2 levels and occurred in parallel with the heterochromatin changes. Taken together, our findings demonstrate the essential role of MeCP2 in regulating the 3D structure of neuronal chromatin, which may serve as a potential mechanism that drives pathogenesis of MeCP2-related disorders.SIGNIFICANCE STATEMENT Neuronal function is critically dependent on methyl-CpG binding protein 2 (MeCP2), a nuclear protein abundantly expressed in neurons. The importance of MeCP2 is underscored by the severe childhood neurologic disorders, Rett syndrome (RTT) and MECP2 multiplication disorders, which are caused by depletion and overabundance of MeCP2, respectively. To clarify the molecular function of MeCP2 and to understand the pathogenesis of MECP2-related disorders, we performed detailed structural analyses of neuronal nuclei by using mouse models and high-resolution microscopy. We show that the level of MeCP2 critically regulates 3D structure of heterochromatic foci, and this is mediated in part by the AT-hook 2 domain of MeCP2. Our results demonstrate that one primary function of MeCP2 is to regulate chromatin structure.

Abnormal cerebellar function and tremor in a mouse model for non-manifesting partially penetrant dystonia type 6.

KEY POINTS: Loss-of-function mutations in the Thap1 gene cause partially penetrant dystonia type 6 (DYT6). Some non-manifesting DYT6 mutation carriers have tremor and abnormal cerebello-thalamo-cortical signalling. We show that Thap1 heterozygote mice have action tremor, a reduction in cerebellar neuron number, and abnormal electrophysiological signals in the remaining neurons. These results underscore the importance of Thap1 levels for cerebellar function. These results uncover how cerebellar abnormalities contribute to different dystonia-associated motor symptoms. ABSTRACT: Loss-of-function mutations in the Thanatos-associated domain-containing apoptosis-associated protein 1 (THAP1) gene cause partially penetrant autosomal dominant dystonia type 6 (DYT6). However, the neural abnormalities that promote the resultant motor dysfunctions remain elusive. Studies in humans show that some non-manifesting DYT6 carriers have altered cerebello-thalamo-cortical function with subtle but reproducible tremor. Here, we uncover that Thap1 heterozygote mice have action tremor that rises above normal baseline values even though they do not exhibit overt dystonia-like twisting behaviour. At the neural circuit level, we show using in vivo recordings in awake Thap1+/- mice that Purkinje cells have abnormal firing patterns and that cerebellar nuclei neurons, which connect the cerebellum to the thalamus, fire at a lower frequency. Although the Thap1+/- mice have fewer Purkinje cells and cerebellar nuclei neurons, the number of long-range excitatory outflow projection neurons is unaltered. The preservation of interregional connectivity suggests that abnormal neural function rather than neuron loss instigates the network dysfunction and the tremor in Thap1+/- mice. Accordingly, we report an inverse correlation between the average firing rate of cerebellar nuclei neurons and tremor power. Our data show that cerebellar circuitry is vulnerable to Thap1 mutations and that cerebellar dysfunction may be a primary cause of tremor in non-manifesting DYT6 carriers and a trigger for the abnormal postures in manifesting patients.

Abnormal Cerebellar Development in Autism Spectrum Disorders.

Autism spectrum disorders (ASD) comprise a group of heterogeneous neurodevelopmental conditions characterized by impaired social interactions and repetitive behaviors with symptom onset in early infancy. The genetic risks for ASD have long been appreciated: concordance of ASD diagnosis may be as high as 90% for monozygotic twins and 30% for dizygotic twins, and hundreds of mutations in single genes have been associated with ASD. Nevertheless, only 5-30% of ASD cases can be explained by a known genetic cause, suggesting that genetics is not the only factor at play. More recently, several studies reported that up to 40% of infants with cerebellar hemorrhages and lesions are diagnosed with ASD. These hemorrhages are overrepresented in severely premature infants, who are born during a period of highly dynamic cerebellar development that encompasses an approximately 5-fold size expansion, an increase in structural complexity, and remarkable rearrangements of local neural circuits. The incidence of ASD-causing cerebellar hemorrhages during this window supports the hypothesis that abnormal cerebellar development may be a primary risk factor for ASD. However, the links between developmental deficits in the cerebellum and the neurological dysfunctions underlying ASD are not completely understood. Here, we discuss key processes in cerebellar development, what happens to the cerebellar circuit when development is interrupted, and how impaired cerebellar function leads to social and cognitive impairments. We explore a central question: Is cerebellar development important for the generation of the social and cognitive brain or is the cerebellum part of the social and cognitive brain itself?

Laparoscopic ventral mesh rectopexy for obstructive defecation syndrome: Follow-up in the Indian population.

CONTEXT: Obstructive defecation syndrome (ODS) is a poorly understood cause of constipation. In selected patients not responding to conservative management, surgical options may be offered. Laparoscopic ventral mesh rectopexy (LVMR) is another surgical option which gained popularity in the past decade. AIM: This study aims to identify the efficacy of LVMR in the Indian population. SETTING AND DESIGN: It is a retrospective analysis of prospectively collected data of patients who underwent LVMR from January 2015 to January 2017 at a tertiary centre in India. SUBJECTS AND METHODS: Thirty patients fulfilled the inclusion criteria. Patients were periodically followed for 2 years. Pre- and post-operative modified Longo's ODS scores were recorded and compared. Furthermore, other complications were noted and evaluated. STATISTICAL ANALYSIS USED: Relevant statistical tests were used to analyse the collected data. RESULTS: Thirty patients (28 females, 2 males, mean age: 52.4 years) underwent LVMR for ODS due to anatomical abnormality like rectorectal intussusceptions (RRIs) (36.7%), rectocele (13.3%), or combined RRI with rectocele (50%). The mean pre-operative modified Longo's ODS score was 23.17 ± 4.82 which decreased to 2.37 ± 1.59 at the end of 6 months and 1.23 ± 1.14 and 1.57 ± 1.14 at the end of 12 months and 2 years, respectively. The mean modified Longo's ODS score showed a significant fall of 94.7% at 12-month follow-up and 93.2% fall on 2-year follow-up. The mean operative time was 115 min and the average hospital stay of patients who underwent LVMR was 3.26 days. CONCLUSION: LVMR is a safe surgical procedure with minimal complications and good functional results for ODS patients due to rectal anatomical abnormality. Further larger studies are required to decide the best treatment modality for ODS.

Quantitative assessment of crural closure for laparoscopic anti-reflux surgeries: A novel technique to reduce post-operative dysphagia.

BACKGROUND: Long-term dysphagia is a known complication of laparoscopic anti-reflux surgery (LARS). Of the several factors, inadequate hiatal closure is one of the major reasons for its occurrence. The aim of this study is to develop a technique for the quantitative assessment of crural closure during LARS to reduce dysphagia. MATERIALS AND METHODS: It is an analysis of prospectively collected data of 109 patients who underwent LARS at a tertiary healthcare centre in India. To identify the adequacy of hiatal closure intraoperatively, a 7 French Fogarty catheter was used, and its balloon was inflated with 1 cc air at the repaired hiatus. This inflated balloon in the repaired hiatus following cruroplasty gives an accurate quantitative assessment of the adequate closure and adequate space for food bolus to pass without causing mechanical obstruction after hiatus repair. Pre- and post-operative 12 months' DeMeester scores and lower oesophageal sphincter (LES) pressures were calculated. RESULTS: The patients had a significant reduction in DeMeester scores postoperatively from a mean of 68.5-12.3 (P < 0.0001). None of the patients had long-term dysphagia or the need for long-term proton-pump inhibitors. The mean LES pressures on post-operative manometry showed increase to 15.1 mmHg from a mean of 6.4 mmHg, which was statistically significant (P = 0.0001). None of the patients had a recurrence of hiatus hernia. CONCLUSION: Quantitative assessment of adequacy for crural closure during LARS using a 7 French Fogarty catheter balloon is a novel technique which may decrease the incidence of post-operative dysphagia or intrathoracic wrap migration or recurrence of hiatus hernia.

Redefining the cerebellar cortex as an assembly of non-uniform Purkinje cell microcircuits.

The adult mammalian cerebellar cortex is generally assumed to have a uniform cytoarchitecture. Differences in cerebellar function are thought to arise primarily through distinct patterns of input and output connectivity rather than as a result of variations in cortical microcircuitry. However, evidence from anatomical, physiological and genetic studies is increasingly challenging this orthodoxy, and there are now various lines of evidence indicating that the cerebellar cortex is not uniform. Here, we develop the hypothesis that regional differences in properties of cerebellar cortical microcircuits lead to important differences in information processing.

Current Opinions and Consensus for Studying Tremor in Animal Models.

Tremor is the most common movement disorder; however, we are just beginning to understand the brain circuitry that generates tremor. Various neuroimaging, neuropathological, and physiological studies in human tremor disorders have been performed to further our knowledge of tremor. But, the causal relationship between these observations and tremor is usually difficult to establish and detailed mechanisms are not sufficiently studied. To overcome these obstacles, animal models can provide an important means to look into human tremor disorders. In this manuscript, we will discuss the use of different species of animals (mice, rats, fruit flies, pigs, and monkeys) to model human tremor disorders. Several ways to manipulate the brain circuitry and physiology in these animal models (pharmacology, genetics, and lesioning) will also be discussed. Finally, we will discuss how these animal models can help us to gain knowledge of the pathophysiology of human tremor disorders, which could serve as a platform towards developing novel therapies for tremor.

Consensus Paper: Experimental Neurostimulation of the Cerebellum.

The cerebellum is best known for its role in controlling motor behaviors. However, recent work supports the view that it also influences non-motor behaviors. The contribution of the cerebellum towards different brain functions is underscored by its involvement in a diverse and increasing number of neurological and neuropsychiatric conditions including ataxia, dystonia, essential tremor, Parkinson's disease (PD), epilepsy, stroke, multiple sclerosis, autism spectrum disorders, dyslexia, attention deficit hyperactivity disorder (ADHD), and schizophrenia. Although there are no cures for these conditions, cerebellar stimulation is quickly gaining attention for symptomatic alleviation, as cerebellar circuitry has arisen as a promising target for invasive and non-invasive neuromodulation. This consensus paper brings together experts from the fields of neurophysiology, neurology, and neurosurgery to discuss recent efforts in using the cerebellum as a therapeutic intervention. We report on the most advanced techniques for manipulating cerebellar circuits in humans and animal models and define key hurdles and questions for moving forward.

Real-Time Visualization of Ureters Using Indocyanine Green During Laparoscopic Surgeries: Can We Make Surgery Safer?

Background. Intraoperative ureteral injury is rare, but a grave complication during laparoscopic surgery. Several methods for intraoperative localization of ureters are described with their own pitfalls. Intraoperative localization using near-infrared (NIR) fluorescence with indocyanine green (ICG) is an easier and assured method during laparoscopic pelvic surgeries. Method. From September 2017 to December 2017, patients undergoing laparoscopic pelvic surgeries were administered cystoscopic-guided intraureteral ICG immediately preoperatively with tip of a 6-Fr ureteral catheter. The fluorescence of ureters was visualized in the NIR mode of the camera system, localizing the ureters precisely and in real time. Results. This technique was used to visualize ureters in 30 surgeries. Median age of the patients was 46.7 years with median body mass index of 23.2 kg/m2. Mean duration between administration of dye and insertion of trocar was 10 minutes. Mean duration for insertion of cystoscopically guided intraureteral ICG was 7 minutes. Ureteral fluorescence was visualized in all cases with some variation in intensity of the brightness perceived depending on surrounding fat. Duration of the lengthiest surgery was 240 minutes, and fluorescence was appreciated till the end. There were no intraoperative or postoperative complications attributed to ICG administration. In 10 patients (33%), there was difficulty in identifying the ureters on conventional white light mode, in which ICG localization was extremely helpful. Conclusion. ICG-stained ureteral visualization under NIR light is a safe and feasible method that provides real-time ureteral demarcation. This easily replicable, sensitive, and specific method of ureteral visualization can make complex laparoscopic pelvic surgeries safer.

Extensive cryptic splicing upon loss of RBM17 and TDP43 in neurodegeneration models.

Splicing regulation is an important step of post-transcriptional gene regulation. It is a highly dynamic process orchestrated by RNA-binding proteins (RBPs). RBP dysfunction and global splicing dysregulation have been implicated in many human diseases, but the in vivo functions of most RBPs and the splicing outcome upon their loss remain largely unexplored. Here we report that constitutive deletion of Rbm17, which encodes an RBP with a putative role in splicing, causes early embryonic lethality in mice and that its loss in Purkinje neurons leads to rapid degeneration. Transcriptome profiling of Rbm17-deficient and control neurons and subsequent splicing analyses using CrypSplice, a new computational method that we developed, revealed that more than half of RBM17-dependent splicing changes are cryptic. Importantly, RBM17 represses cryptic splicing of genes that likely contribute to motor coordination and cell survival. This finding prompted us to re-analyze published datasets from a recent report on TDP-43, an RBP implicated in amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD), as it was demonstrated that TDP-43 represses cryptic exon splicing to promote cell survival. We uncovered a large number of TDP-43-dependent splicing defects that were not previously discovered, revealing that TDP-43 extensively regulates cryptic splicing. Moreover, we found a significant overlap in genes that undergo both RBM17- and TDP-43-dependent cryptic splicing repression, many of which are associated with survival. We propose that repression of cryptic splicing by RBPs is critical for neuronal health and survival. CrypSplice is available at www.liuzlab.org/CrypSplice.

Complex taxonomy of the 'brush tail' peregrine earthworm Pontoscolex corethrurus.

Pontoscolex corethrurus is the most widespread earthworm species in tropical and sub-tropical zones and one of the most studied in soil science. Although, ecological interactions of P. corethrurus with its environment are well documented, the taxonomic status of the species remains unclear. In this study, we investigated phylogenetic relationships within the genus Pontoscolex, in particular focusing on morphologically indistinguishable (i.e., cryptic) lineages. A total of 792 specimens collected from 25 different countries and islands all over the world were analyzed using two mitochondrial (COI and 16S rDNA) and two nuclear (internal transcribed spacers 2 and 28S rDNA) markers, and a total of 11 morphological characters both internal and external were investigated in all genetically characterized lineages. A large-scale multilocus sequence data matrix was also obtained for Pontoscolex spp. specimens using the Anchored Hybrid Enrichment (AHE) method. Multilocus phylogenetic and phylogenomic analyses, combined with species delimitation methods; including single locus (mPTP, ABGD) and multilocus (BPP) approaches, revealed congruent results. Four cryptic species were supported within the P. corethrurus species complex, and four potentially new species within the genus Pontoscolex. One widespread lineage (L1), within P. corethrurus complex was observed in the current population of Fritz Müller's garden where P. corethrurus was first described in 1856. Cryptic lineages were observed in sympatry at several localities. This, in combination with observed heteroplasmy in COI gene in one population raises an important question of reproductive isolation between these species.