Timing neurogenesis: a clock or an algorithm?

Emerging evidence supports the existence of dedicated molecular mechanisms under evolutionary selection to control time during neurogenesis. Here, we briefly review these mechanisms and discuss a potentially useful conceptual framework inspired by computer science to think about how these biological mechanisms operate during brain development and evolution.

A New Behavioral Paradigm for Visual Classical Conditioning in Drosophila.

Visual learning in animals is a remarkable cognitive ability that plays a crucial role in their survival and adaptation. Therefore, the ability to learn is highly conserved among animals. Despite lacking a centralized nervous system like vertebrates, invertebrates have demonstrated remarkable learning abilities. Here, we describe a simple behavioral assay that allows the analysis of visual associative learning in individually traceable freely walking adult fruit flies. The setup is based on the simple and widely used behavioral assay to study orientation behavior in flies. A single wing-clipped fly that has been starved for 21 h is placed on a platform where two unreachable opposite visual sets are displayed. This visual learning protocol was initially developed to study the cognitive ability of fruit flies to process numerical information. Through the application of the protocol, flies are able to associate a specific visual set with an appetitive reward. This association is revealed 2 h later during the testing session where we observed a change in their preference upon learning (i.e., change in their spontaneous preference). Moreover, this protocol could potentially be used to associate any other visual object/property to the reward, expanding the opportunities of studying visual learning in freely walking fruit flies at individual level.

Management of Extra-articular Scapular Fractures: A Narrative Review and Proposal of a Treatment Algorithm.

The management of scapular fractures can be either conservative or operative, but it is still unclear how to choose the treatment option. Scapular fractures can be classified anatomically into four types: scapular spine, scapular body, and scapular neck where the treatment is conservative most of the time except for certain indications that are specific to each one, and inferior angle of the scapula where the operative treatment yields the best results but conservative treatment can be contemplated in some cases.

Subscapular sparing approach for total shoulder arthroplasty: a systematic review and meta-analysis of comparative studies.

Background: Total shoulder arthroplasty has been effective in treating primary glenohumeral arthritis surgically. However, the subscapularis (SSC) tendon must be released as part of the surgery's conventional approach, which could lead to postoperative problems and later tendon failure. Using a procedure that spares the tendon, Lafosse et al reported improved postoperative SSC performance. This meta-analysis will compare this approach to the standard one. Methods: PubMed, Cochrane, and Google Scholar (pages 1-20) were searched till October 2022. The clinical outcomes consisted of the postoperative pain, adverse events, and range of motion. Results: Only 3 studies were included in this meta-analysis. The subscapularis sparing approach tended to have better postoperative internal rotation (P = .06) and shoulder elevation (P = .1); however, the results were marginally statistically insignificant. Conclusion: This is the first meta-analysis to compare the subscapularis sparing to the standard approach in total shoulder arthroplasty. Showing better postoperative range of motion (elevation and internal rotation), the SSC approach might be considered as a good substitute to the standard one. However, it is limited by the potentially limited exposure making it harder to use in muscular and obese patients. More randomized clinical trials are needed to reach more significant results and establish clearer indications of this approach.

Numerical discrimination in Drosophila melanogaster.

Sensitivity to numbers is a crucial cognitive ability. The lack of experimental models amenable to systematic genetic and neural manipulation has precluded discovering neural circuits required for numerical cognition. Here, we demonstrate that Drosophila flies spontaneously prefer sets containing larger numbers of objects. This preference is determined by the ratio between the two numerical quantities tested, a characteristic signature of numerical cognition across species. Individual flies maintained their numerical choice over consecutive days. Using a numerical visual conditioning paradigm, we found that flies are capable of associating sucrose with numerical quantities and can be trained to reverse their spontaneous preference for large quantities. Finally, we show that silencing lobula columnar neurons (LC11) reduces the preference for more objects, thus identifying a neuronal substrate for numerical cognition in invertebrates. This discovery paves the way for the systematic analysis of the behavioral and neural mechanisms underlying the evolutionary conserved sensitivity to numerosity.

Developmental transcriptional control of mitochondrial homeostasis is required for activity-dependent synaptic connectivity.

During neuronal circuit formation, local control of axonal organelles ensures proper synaptic connectivity. Whether this process is genetically encoded is unclear and if so, its developmental regulatory mechanisms remain to be identified. We hypothesized that developmental transcription factors regulate critical parameters of organelle homeostasis that contribute to circuit wiring. We combined cell type-specific transcriptomics with a genetic screen to discover such factors. We identified Telomeric Zinc finger-Associated Protein (TZAP) as a temporal developmental regulator of neuronal mitochondrial homeostasis genes, including Pink1 . In Drosophila , loss of dTzap function during visual circuit development leads to loss of activity-dependent synaptic connectivity, that can be rescued by Pink1 expression. At the cellular level, loss of dTzap/TZAP leads to defects in mitochondrial morphology, attenuated calcium uptake and reduced synaptic vesicle release in fly and mammalian neurons. Our findings highlight developmental transcriptional regulation of mitochondrial homeostasis as a key factor in activity-dependent synaptic connectivity.

The temporal balance between self-renewal and differentiation of human neural stem cells requires the amyloid precursor protein.

Neurogenesis in the developing human cerebral cortex occurs at a particularly slow rate owing in part to cortical neural progenitors preserving their progenitor state for a relatively long time, while generating neurons. How this balance between the progenitor and neurogenic state is regulated, and whether it contributes to species-specific brain temporal patterning, is poorly understood. Here, we show that the characteristic potential of human neural progenitor cells (NPCs) to remain in a progenitor state as they generate neurons for a prolonged amount of time requires the amyloid precursor protein (APP). In contrast, APP is dispensable in mouse NPCs, which undergo neurogenesis at a much faster rate. Mechanistically, APP cell-autonomously contributes to protracted neurogenesis through suppression of the proneurogenic activator protein-1 transcription factor and facilitation of canonical WNT signaling. We propose that the fine balance between self-renewal and differentiation is homeostatically regulated by APP, which may contribute to human-specific temporal patterns of neurogenesis.

The brain on time: links between development and neurodegeneration.

Neurodegenerative diseases are characterized by the progressive loss of structure or function of neurons. In this Spotlight, we explore the idea that genetic forms of neurodegenerative disorders might be rooted in neural development. Focusing on Alzheimer's, Parkinson's and Huntington's disease, we first provide a brief overview of the pathology for these diseases. Although neurodegenerative diseases are generally thought of as late-onset diseases, we discuss recent evidence promoting the notion that they might be considered neurodevelopmental disorders. With this view in mind, we consider the suitability of animal models for studying these diseases, highlighting human-specific features of human brain development. We conclude by proposing that one such feature, human-specific regulation of neurogenic time, might be key to understanding the etiology and pathophysiology of human neurodegenerative disease.

Numerosity as a visual property: Evidence from two highly evolutionary distant species.

Most animals, from humans to invertebrates, possess an ability to estimate numbers. This evolutionary advantage facilitates animals' choice of environments with more food sources, more conspecifics to increase mating success, and/or reduced predation risk among others. However, how the brain processes numerical information remains largely unknown. There are currently two lines of research interested in how numerosity of visual objects is perceived and analyzed in the brain. The first argues that numerosity is an advanced cognitive ability processed in high-order brain areas, while the second proposes that "numbers" are attributes of the visual scene and thus numerosity is processed in the visual sensory system. Recent evidence points to a sensory involvement in estimating magnitudes. In this Perspective, we highlight this evidence in two highly evolutionary distant species: humans and flies. We also discuss the advantages of studying numerical processing in fruit flies in order to dissect the neural circuits involved in and required for numerical processing. Based on experimental manipulation and the fly connectome, we propose a plausible neural network for number sense in invertebrates.

Autophagy in synapse formation and brain wiring.

A recent characterization of the role of autophagy in two different neuron types during brain development in Drosophila revealed two different mechanisms to regulate synapse formation. In photoreceptor neurons, autophagosome formation in synaptogenic filopodia destabilizes presumptive synaptic contacts and thereby restricts incorrect synaptic partnerships. In dorsal cluster neurons, autophagy is actively suppressed to keep mature synapses stable during axonal branching. These findings indicate that different neuron types can require activation or suppression of synaptic autophagy during the same developmental period to ensure proper synapse formation and brain connectivity.

EGFR-dependent suppression of synaptic autophagy is required for neuronal circuit development.

The development of neuronal connectivity requires stabilization of dynamic axonal branches at sites of synapse formation. Models that explain how axonal branching is coupled to synaptogenesis postulate molecular regulators acting in a spatiotemporally restricted fashion to ensure branching toward future synaptic partners while also stabilizing the emerging synaptic contacts between such partners. We investigated this question using neuronal circuit development in the Drosophila brain as a model system. We report that epidermal growth factor receptor (EGFR) activity is required in presynaptic axonal branches during two distinct temporal intervals to regulate circuit wiring in the developing Drosophila visual system. EGFR is required early to regulate primary axonal branching. EGFR activity is then independently required at a later stage to prevent degradation of the synaptic active zone protein Bruchpilot (Brp). Inactivation of EGFR results in a local increase of autophagy in presynaptic branches and the translocation of active zone proteins into autophagic vesicles. The protection of synaptic material during this later interval of wiring ensures the stabilization of terminal branches, circuit connectivity, and appropriate visual behavior. Phenotypes of EGFR inactivation can be rescued by increasing Brp levels or downregulating autophagy. In summary, we identify a temporally restricted molecular mechanism required for coupling axonal branching and synaptic stabilization that contributes to the emergence of neuronal wiring specificity.

The ribose methylation enzyme FTSJ1 has a conserved role in neuron morphology and learning performance.

FTSJ1 is a conserved human 2'-O-methyltransferase (Nm-MTase) that modifies several tRNAs at position 32 and the wobble position 34 in the anticodon loop. Its loss of function has been linked to X-linked intellectual disability (XLID), and more recently to cancers. However, the molecular mechanisms underlying these pathologies are currently unclear. Here, we report a novel FTSJ1 pathogenic variant from an X-linked intellectual disability patient. Using blood cells derived from this patient and other affected individuals carrying FTSJ1 mutations, we performed an unbiased and comprehensive RiboMethSeq analysis to map the ribose methylation on all human tRNAs and identify novel targets. In addition, we performed a transcriptome analysis in these cells and found that several genes previously associated with intellectual disability and cancers were deregulated. We also found changes in the miRNA population that suggest potential cross-regulation of some miRNAs with these key mRNA targets. Finally, we show that differentiation of FTSJ1-depleted human neural progenitor cells into neurons displays long and thin spine neurites compared with control cells. These defects are also observed in Drosophila and are associated with long-term memory deficits. Altogether, our study adds insight into FTSJ1 pathologies in humans and flies by the identification of novel FTSJ1 targets and the defect in neuron morphology.

PTPA variants and impaired PP2A activity in early-onset parkinsonism with intellectual disability.

The protein phosphatase 2A complex (PP2A), the major Ser/Thr phosphatase in the brain, is involved in a number of signalling pathways and functions, including the regulation of crucial proteins for neurodegeneration, such as alpha-synuclein, tau and LRRK2. Here, we report the identification of variants in the PTPA/PPP2R4 gene, encoding a major PP2A activator, in two families with early-onset parkinsonism and intellectual disability. We carried out clinical studies and genetic analyses, including genome-wide linkage analysis, whole-exome sequencing, and Sanger sequencing of candidate variants. We next performed functional studies on the disease-associated variants in cultured cells and knock-down of ptpa in Drosophila melanogaster. We first identified a homozygous PTPA variant, c.893T>G (p.Met298Arg), in patients from a South African family with early-onset parkinsonism and intellectual disability. Screening of a large series of additional families yielded a second homozygous variant, c.512C>A (p.Ala171Asp), in a Libyan family with a similar phenotype. Both variants co-segregate with disease in the respective families. The affected subjects display juvenile-onset parkinsonism and intellectual disability. The motor symptoms were responsive to treatment with levodopa and deep brain stimulation of the subthalamic nucleus. In overexpression studies, both the PTPA p.Ala171Asp and p.Met298Arg variants were associated with decreased PTPA RNA stability and decreased PTPA protein levels; the p.Ala171Asp variant additionally displayed decreased PTPA protein stability. Crucially, expression of both variants was associated with decreased PP2A complex levels and impaired PP2A phosphatase activation. PTPA orthologue knock-down in Drosophila neurons induced a significant impairment of locomotion in the climbing test. This defect was age-dependent and fully reversed by L-DOPA treatment. We conclude that bi-allelic missense PTPA variants associated with impaired activation of the PP2A phosphatase cause autosomal recessive early-onset parkinsonism with intellectual disability. Our findings might also provide new insights for understanding the role of the PP2A complex in the pathogenesis of more common forms of neurodegeneration.

Transient regulation of focal adhesion via Tensin3 is required for nascent oligodendrocyte differentiation.

The differentiation of oligodendroglia from oligodendrocyte precursor cells (OPCs) to complex and extensive myelinating oligodendrocytes (OLs) is a multistep process that involves large-scale morphological changes with significant strain on the cytoskeleton. While key chromatin and transcriptional regulators of differentiation have been identified, their target genes responsible for the morphological changes occurring during OL myelination are still largely unknown. Here, we show that the regulator of focal adhesion, Tensin3 (Tns3), is a direct target gene of Olig2, Chd7, and Chd8, transcriptional regulators of OL differentiation. Tns3 is transiently upregulated and localized to cell processes of immature OLs, together with integrin-ß1, a key mediator of survival at this transient stage. Constitutive <i>Tns3</i> loss of function leads to reduced viability in mouse and humans, with surviving knockout mice still expressing Tns3 in oligodendroglia. Acute deletion of <i>Tns3</i> in vivo, either in postnatal neural stem cells (NSCs) or in OPCs, leads to a twofold reduction in OL numbers. We find that the transient upregulation of Tns3 is required to protect differentiating OPCs and immature OLs from cell death by preventing the upregulation of p53, a key regulator of apoptosis. Altogether, our findings reveal a specific time window during which transcriptional upregulation of Tns3 in immature OLs is required for OL differentiation likely by mediating integrin-ß1 survival signaling to the actin cytoskeleton as OL undergo the large morphological changes required for their terminal differentiation.

Arthroscopic release of the pectoralis minor tendon from the coracoid for pectoralis minor syndrome.

PURPOSE: The term "pectoralis minor syndrome" refers to this constellation of symptoms that can occur when the pectoralis minor (Pm) is shortened and contracted. Release of the tendon of the Pm from the coracoid has been reported to provide substantial clinical improvement to patients presenting with pectoralis minor syndrome. The purpose of this study was (1) to describe the technique for endoscopic release of pectoralis minor tendon at the subdeltoid space, (2) to classify the pectoralis minor syndrome according to its severity and (3) and to report the short-term outcomes of this procedure in a consecutive series of patients diagnosed with pectoralis minor syndrome. METHODS: Endoscopic release of the pectoralis minor tendon was performed in a series of 10 patients presenting with pectoralis minor syndrome. There were six females and four males with a median age at the time of surgery of 42 (range from 20 to 58) years. Four shoulders were categorized as grade I (scapular dyskinesis), and six as grade II (intermittent brachial plexopathy). Shoulders were evaluated for pain, motion, satisfaction, subjective shoulder value (SSV), quick-DASH, ASES score, and complications. The mean follow-up time was 19 (range, 6 to 49) months. RESULTS: Arthroscopic release of the tendon of the Pm led to substantial resolution of pectoralis minor syndrome symptoms in all but one shoulder, which was considered a failure. Preoperatively, the median VAS for pain was 8.5 (range, 7-10) and the mean SSV was 20% (range, 10% - 50%). At most recent follow-up the mean VAS for pain was 1 (range, 0-6) and the mean SSV 80% (range, 50% - 90%). Before surgery, mean ASES and quick-DASH scores were 19.1 (range, 10-41.6) and 83.1 (range, 71 and 95.5) points respectively. At most recent follow-up, mean ASES and quick-DASH scores were 80.1 (range, 40-100) and 19.3 (range, 2.3-68) points respectively. No surgical complications occurred in any of the shoulder included in this study. CONCLUSIONS: Endoscopic release of the tendon of the pectoralis minor from the coracoid improves pain, function and patient reported outcomes in the majority of patients presenting with the diagnosis of isolated pectoralis minor syndrome.

Brain connectivity inversely scales with developmental temperature in Drosophila.

Variability of synapse numbers and partners despite identical genes reveals the limits of genetic determinism. Here, we use developmental temperature as a non-genetic perturbation to study variability of brain wiring and behavior in Drosophila. Unexpectedly, slower development at lower temperatures increases axo-dendritic branching, synapse numbers, and non-canonical synaptic partnerships of various neurons, while maintaining robust ratios of canonical synapses. Using R7 photoreceptors as a model, we show that changing the relative availability of synaptic partners using a DIPγ mutant that ablates R7's preferred partner leads to temperature-dependent recruitment of non-canonical partners to reach normal synapse numbers. Hence, R7 synaptic specificity is not absolute but based on the relative availability of postsynaptic partners and presynaptic control of synapse numbers. Behaviorally, movement precision is temperature robust, while movement activity is optimized for the developmentally encountered temperature. These findings suggest genetically encoded relative and scalable synapse formation to develop functional, but not identical, brains and behaviors.

Induction of granule and Purkinje cells from primary cultured mouse cerebellar progenitors.

The architecturally stereotypical structure of cerebellum is ideal for investigating the generation of neuronal diversity, but in vitro models for assessing early cerebellar progenitor differentiation were lacking. Here, we report a detailed protocol for long-term in vitro generation of Pax6+ granule cells and Calbindin+ Purkinje cells from common Sox2+ embryonic cerebellar progenitors. We describe the procedure for dissecting mouse cerebellar anlage, cell seeding, and tamoxifen-induced labeling of progenitor cells, followed by time-lapse video recording of clonal expansion and neuronal differentiation. For complete details on the use and execution of this protocol, please refer to Zhang et al. (2021).

The amyloid precursor protein is a conserved Wnt receptor.

The Amyloid Precursor Protein (APP) and its homologues are transmembrane proteins required for various aspects of neuronal development and activity, whose molecular function is unknown. Specifically, it is unclear whether APP acts as a receptor, and if so what its ligand(s) may be. We show that APP binds the Wnt ligands Wnt3a and Wnt5a and that this binding regulates APP protein levels. Wnt3a binding promotes full-length APP (flAPP) recycling and stability. In contrast, Wnt5a promotes APP targeting to lysosomal compartments and reduces flAPP levels. A conserved Cysteine-Rich Domain (CRD) in the extracellular portion of APP is required for Wnt binding, and deletion of the CRD abrogates the effects of Wnts on flAPP levels and trafficking. Finally, loss of APP results in increased axonal and reduced dendritic growth of mouse embryonic primary cortical neurons. This phenotype can be cell-autonomously rescued by full length, but not CRD-deleted, APP and regulated by Wnt ligands in a CRD-dependent manner.

Notch1 switches progenitor competence in inducing medulloblastoma.

The identity of the cell of origin is a key determinant of cancer subtype, progression, and prognosis. Group 3 medulloblastoma (MB) is a malignant childhood brain cancer with poor prognosis and few candidates as putative cell of origin. We overexpressed the group 3 MB genetic drivers MYC and Gfi1 in different candidate cells of origin in the postnatal mouse cerebellum. We found that S100b+ cells are competent to initiate group 3 MB, and we observed that S100b+ cells have higher levels of Notch1 pathway activity compared to Math1+ cells. We found that additional activation of Notch1 in Math1+ and Sox2+ cells was sufficient to induce group 3 MB upon MYC/Gfi1 expression. Together, our data suggest that the Notch1 pathway plays a critical role in group 3 MB initiation.

Generation of excitatory and inhibitory neurons from common progenitors via Notch signaling in the cerebellum.

Brain neurons arise from relatively few progenitors generating an enormous diversity of neuronal types. Nonetheless, a cardinal feature of mammalian brain neurogenesis is thought to be that excitatory and inhibitory neurons derive from separate, spatially segregated progenitors. Whether bi-potential progenitors with an intrinsic capacity to generate both lineages exist and how such a fate decision may be regulated are unknown. Using cerebellar development as a model, we discover that individual progenitors can give rise to both inhibitory and excitatory lineages. Gradations of Notch activity determine the fates of the progenitors and their daughters. Daughters with the highest levels of Notch activity retain the progenitor fate, while intermediate levels of Notch activity generate inhibitory neurons, and daughters with very low levels of Notch signaling adopt the excitatory fate. Therefore, Notch-mediated binary cell fate choice is a mechanism for regulating the ratio of excitatory to inhibitory neurons from common progenitors.