Lmo4 establishes rostral motor cortex projection neuron subtype diversity.

The mammalian neocortex is parcellated into anatomically and functionally distinct areas. The establishment of area-specific neuronal diversity and circuit connectivity enables distinct neocortical regions to control diverse and specialized functional outputs, yet underlying molecular controls remain largely unknown. Here, we identify a central role for the transcriptional regulator Lim-only 4 (Lmo4) in establishing the diversity of neuronal subtypes within rostral mouse motor cortex, where projection neurons have particularly diverse and multi-projection connectivity compared with caudal motor cortex. In rostral motor cortex, we report that both subcerebral projection neurons (SCPN), which send projections away from the cerebrum, and callosal projection neurons (CPN), which send projections to contralateral cortex, express Lmo4, whereas more caudal SCPN and CPN do not. Lmo4-expressing SCPN and CPN populations are comprised of multiple hodologically distinct subtypes. SCPN in rostral layer Va project largely to brainstem, whereas SCPN in layer Vb project largely to spinal cord, and a subset of both rostral SCPN and CPN sends second ipsilateral caudal (backward) projections in addition to primary projections. Without Lmo4 function, the molecular identity of neurons in rostral motor cortex is disrupted and more homogenous, rostral layer Va SCPN aberrantly project to the spinal cord, and many dual-projection SCPN and CPN fail to send a second backward projection. These molecular and hodological disruptions result in greater overall homogeneity of motor cortex output. Together, these results identify Lmo4 as a central developmental control over the diversity of motor cortex projection neuron subpopulations, establishing their area-specific identity and specialized connectivity.

An inherited TUBB2B mutation alters a kinesin-binding site and causes polymicrogyria, CFEOM and axon dysinnervation.

Microtubules are essential components of axon guidance machinery. Among ß-tubulin mutations, only those in TUBB3 have been shown to cause primary errors in axon guidance. All identified mutations in TUBB2B result in polymicrogyria, but it remains unclear whether TUBB2B mutations can cause axon dysinnervation as a primary phenotype. We have identified a novel inherited heterozygous missense mutation in TUBB2B that results in an E421K amino acid substitution in a family who segregates congenital fibrosis of the extraocular muscles (CFEOM) with polymicrogyria. Diffusion tensor imaging of brains of affected family members reveals aberrations in the trajectories of commissural projection neurons, implying a paucity of homotopic connections. These observations led us to ask whether axon dysinnervation is a primary phenotype, and why the E421K, but not other, TUBB2B substitutions cause CFEOM. Expression of exogenous Tubb2b-E421K in developing callosal projection neurons is sufficient to perturb homotopic connectivity, without affecting neuronal production or migration. Using in vitro biochemical assays and yeast genetics, we find that TUBB2B-E421K αß-heterodimers are incorporated into the microtubule network where they alter microtubule dynamics and can reduce kinesin localization. These data provide evidence that TUBB2B mutations can cause primary axon dysinnervation. Interestingly, by incorporating into microtubules and altering their dynamic properties, the E421K substitution behaves differently than previously identified TUBB2B substitutions, providing mechanistic insight into the divergence between resulting phenotypes. Together with previous studies, these findings highlight that ß-tubulin isotypes function in both conserved and divergent ways to support proper human nervous system development.

ZMYND11 Functions in Bimodal Regulation of Latent Genes and Brain-like Splicing to Safeguard Corticogenesis.

Despite the litany of pathogenic variants linked to neurodevelopmental disorders (NDD) including autism (ASD) and intellectual disability 1,2 , our understanding of the underlying mechanisms caused by risk genes remain unclear. Here, we leveraged a human pluripotent stem cell model to uncover the neurodevelopmental consequences of mutations in ZMYND11 , a newly implicated risk gene 3,4 . ZMYND11, known for its tumor suppressor function, encodes a histone-reader that recognizes sites of transcriptional elongation and acts as a co-repressor 5,6 . Our findings reveal that ZMYND11-deficient cortical neural stem cells showed upregulation of latent developmental pathways, impairing progenitor and neuron production. In addition to its role on histones, ZMYND11 controls a brain-specific isoform switch involving the splicing regulator RBFOX2. Extending our findings to other chromatin-related ASD risk factors revealed similar developmental pathway activation and splicing dysregulation, partially rescuable through ZMYND11's regulatory functions.

Evolving consolidation patterns and outcomes for a large cohort of primary CNS lymphoma patients.

Consolidation strategies for primary central nervous system lymphoma (PCNSL) after induction chemoimmunotherapy include whole-brain radiotherapy (≤24Gy reduced-dose [RD], >24Gy standard-dose [SD] WBRT) and cytarabine, non-myeloablative chemotherapy (NMC), or autologous hematopoietic cell transplantation (AHCT); however, comparative outcomes are lacking. PCNSL outcomes from 1983-2020 were stratified by decade and MSKCC recursive partitioning analysis (RPA) class. Clinicodemographic associations with consolidation were analyzed by multinomial logistic regression. Progression-free (PFS) and overall survival (OS) were analyzed by proportional hazards from consolidation. Of 559 patients, 385 (69%) were consolidated. Median follow-up and OS were 7.4 and 5.7 years, respectively. WBRT use declined (61% in 1990s vs. 12% in 2010s), while AHCT (4% in 1990s vs. 32% in 2010s) and NMC (27% in 1990s vs. 52% in 2010s) rose. Compared to RPA 1 (age<50), RPA 2 (age≥50, KPS≥70) was more likely to receive NMC. Those with partial response to induction were less likely to receive AHCT (OR 0.36, p=0.02). Among 351 with complete response to consolidation, only receipt of R-MPV induction was associated with improved PFS (HR 0.5, p=0.006). Among RPA 1, median PFS and OS were not reached for AHCT or RD-WBRT, vs. 2.5 and 13.0 years, respectively, after NMC. Among RPA class 3 (KPS<70), median PFS and OS post-RD-WBRT were 4.6 and 10 years, respectively, vs. 1.7 and 4.4 years post-NMC. No significant adjusted survival differences were seen across consolidation strategies. NMC is increasingly used in lieu of RD-WBRT despite a trend toward less favorable PFS. RD-WBRT can be considered in patients ineligible for AHCT.

CNS bridging radiotherapy achieves rapid cytoreduction before CAR T-cell therapy for aggressive B-cell lymphomas.

ABSTRACT: Chimeric antigen receptor (CAR) T-cell therapy (CART) for central nervous system lymphoma (CNSL) is a promising strategy, yet responses are frequently not durable. Bridging radiotherapy (BRT) is used for extracranial lymphoma in which it can improve CART outcomes through cytoreduction of high-risk lesions. We hypothesized that BRT would achieve similar, significant cytoreduction before CART for CNSL (CNS-BRT). We identified patients with CNSL with non-Hodgkin B-cell lymphoma who received CNS-BRT before commercial CART. Cytoreduction from CNS-BRT was calculated as change in lesion size before CART. Twelve patients received CNS-BRT, and the median follow-up among survivors is 11.8 months (interquartile range, 8.5-21.9). Ten patients had CNSL (9 secondary, 1 primary) and 2 patients had epidural disease (evaluable for toxicity). All 10 patients with CNSL had progressive disease at the time of CNS-BRT. Of 12 patients, 1 experienced grade ≥3 cytokine release syndrome, and 3 of 12 patients experienced grade ≥3 immune effector cell-associated neurotoxicity syndrome. CNS-BRT achieved a 74.0% (95% confidence interval, 62.0-86.0) mean reduction in lesion size from baseline (P = .014) at a median of 12 days from BRT completion and before CART infusion. Best CNS response included 8 complete responses, 1 partial response, and 1 progressive disease. Three patients experienced CNS relapse outside the BRT field. Preliminary data suggest CNS-BRT achieves rapid cytoreduction and is associated with a favorable CNS response and safety profile. These data support further study of BRT as a bridging modality for CNSL CART.

Activation of HERV-K(HML-2) disrupts cortical patterning and neuronal differentiation by increasing NTRK3.

The biological function and disease association of human endogenous retroviruses (HERVs) are largely elusive. HERV-K(HML-2) has been associated with neurotoxicity, but there is no clear understanding of its role or mechanistic basis. We addressed the physiological functions of HERV-K(HML-2) in neuronal differentiation using CRISPR engineering to activate or repress its expression levels in a human-pluripotent-stem-cell-based system. We found that elevated HERV-K(HML-2) transcription is detrimental for the development and function of cortical neurons. These effects are cell-type-specific, as dopaminergic neurons are unaffected. Moreover, high HERV-K(HML-2) transcription alters cortical layer formation in forebrain organoids. HERV-K(HML-2) transcriptional activation leads to hyperactivation of NTRK3 expression and other neurodegeneration-related genes. Direct activation of NTRK3 phenotypically resembles HERV-K(HML-2) induction, and reducing NTRK3 levels in context of HERV-K(HML-2) induction restores cortical neuron differentiation. Hence, these findings unravel a cell-type-specific role for HERV-K(HML-2) in cortical neuron development.

Lmo4 and Clim1 progressively delineate cortical projection neuron subtypes during development.

Molecular controls over the development of the exceptional neuronal subtype diversity of the cerebral cortex are now beginning to be identified. The initial subtype fate decision early in the life of a neuron, and the malleability of this fate when the balance of key postmitotic signals is modified, reveals not only that a neuron is deterministically set on a general developmental path at its birth, but also that this program must be precisely executed during postmitotic differentiation. Here, we show that callosal projection neurons (CPN) and subcerebral projection neurons (subcerebral PN) in layer V of the neocortex share aspects of molecular identity after their birth that are progressively resolved during differentiation. The LIM-homeodomain-related genes Lmo4 and Clim1 are initially expressed by both CPN and subcerebral PN in layer V, and only during mid to late differentiation does expression of Lmo4 and Clim1 become largely segregated into distinct neuronal subtypes. This progressive postmitotic resolution of molecular identity reveals similarities and possibly shared evolutionary origin between layer V CPN and subcerebral PN, and provides insight into how and when these neuronal subtypes achieve their distinct identities during cortical development.

A Multiplex Human Pluripotent Stem Cell Platform Defines Molecular and Functional Subclasses of Autism-Related Genes.

Autism is a clinically heterogeneous neurodevelopmental disorder characterized by impaired social interactions, restricted interests, and repetitive behaviors. Despite significant advances in the genetics of autism, understanding how genetic changes perturb brain development and affect clinical symptoms remains elusive. Here, we present a multiplex human pluripotent stem cell (hPSC) platform, in which 30 isogenic disease lines are pooled in a single dish and differentiated into prefrontal cortex (PFC) lineages to efficiently test early-developmental hypotheses of autism. We define subgroups of autism mutations that perturb PFC neurogenesis and are correlated to abnormal WNT/ßcatenin responses. Class 1 mutations (8 of 27) inhibit while class 2 mutations (5 of 27) enhance PFC neurogenesis. Remarkably, autism patient data reveal that individuals carrying subclass-specific mutations differ clinically in their corresponding language acquisition profiles. Our study provides a framework to disentangle genetic heterogeneity associated with autism and points toward converging molecular and developmental pathways of diverse autism-associated mutations.

Specification of positional identity in forebrain organoids.

Human brain organoids generated with current technologies recapitulate histological features of the human brain, but they lack a reproducible topographic organization. During development, spatial topography is determined by gradients of signaling molecules released from discrete signaling centers. We hypothesized that introduction of a signaling center into forebrain organoids would specify the positional identity of neural tissue in a distance-dependent manner. Here, we present a system to trigger a Sonic Hedgehog (SHH) protein gradient in developing forebrain organoids that enables ordered self-organization along dorso-ventral and antero-posterior positional axes. SHH-patterned forebrain organoids establish major forebrain subdivisions that are positioned with in vivo-like topography. Consistent with its behavior in vivo, SHH exhibits long-range signaling activity in organoids. Finally, we use SHH-patterned cerebral organoids as a tool to study the role of cholesterol metabolism in SHH signaling. Together, this work identifies inductive signaling as an effective organizing strategy to recapitulate in vivo-like topography in human brain organoids.

Radiation-Associated Secondary Cancer in Patients With Breast Cancer Harboring TP53 Germline Variants.

This cohort study examines the risk of radiation-associated sarcoma in patients with breast cancer harboring germline TP53 variants.

Surgical Decompression of High-Grade Spinal Cord Compression from Hormone Refractory Metastatic Prostate Cancer.

BACKGROUND: Spine and nonspine skeletal metastases occur in more than 80% of patients with prostate cancer. OBJECTIVE: To examine the characteristics of the patient population undergoing surgery for the treatment of prostate cancer metastatic to the spine. METHODS: A retrospective chart review was performed on all patients treated at our institution from June 1993 to August 2014 for surgical management of metastatic spine disease from prostate cancer. RESULTS: During the study period, 139 patients with 157 surgical lesions underwent surgery for metastatic spine disease. Decompression for high-grade epidural spinal cord compression was required for 126 patients with 143 lesions. Preoperatively, 69% had a motor deficit and 21% were nonambulatory, with 32% due to motor weakness. At surgery, 87% of patients had hormone-refractory prostate cancer (HRPC) and 61% failed prior radiation. Median overall survival for HRPC patients was 6.6 mo (95% confidence interval [CI]: 5.6-8.6) while the median overall survival for hormone-sensitive patients was 16.3 mo (95% CI: 4.0-26.6). CONCLUSION: The majority of patients undergoing surgery for prostate cancer metastases to the spine were refractory to hormone therapy, indicating that patients with hormone-sensitive prostate cancer are unlikely to develop symptomatic spinal cord compression or spinal instability. A significant number of HRPC patients presented with neurological deficits attributable to spinal cord compression. Vigilant monitoring for the development of signs and symptoms of epidural spinal cord compression and spinal instability in hormone-refractory patients is recommended. Surgical decision making may be affected by the much shorter postoperative survival for HRPC patients as compared to patients with hormone-sensitive cancer.

The Role of Extent of Resection in IDH1 Wild-Type or Mutant Low-Grade Gliomas.

BACKGROUND: Maximizing extent of resection (EOR) improves outcomes in adults with World Health Organization (WHO) grade II low-grade gliomas (LGG). However, recent studies demonstrate that LGGs bearing a mutation in the isocitrate dehydrogenase 1 (IDH1) gene are a distinct molecular and clinical entity. It remains unclear whether maximizing EOR confers an equivalent clinical benefit in IDH mutated (mtIDH) and IDH wild-type (wtIDH) LGGs. OBJECTIVE: To assess the impact of EOR on malignant progression-free survival (MPFS) and overall survival (OS) in mtIDH and wtIDH LGGs. METHODS: We performed a retrospective review of 74 patients with WHO grade II gliomas and known IDH mutational status undergoing resection at a single institution. EOR was assessed with quantitative 3-dimensional volumetric analysis. The effect of predictor variables on MPFS and OS was analyzed with Cox regression models and the Kaplan-Meier method. RESULTS: Fifty-two (70%) mtIDH patients and 22 (30%) wtIDH patients were included. Median preoperative tumor volume was 37.4 cm3; median EOR of 57.6% was achieved. Univariate Cox regression analysis confirmed EOR as a prognostic factor for the entire cohort. However, stratifying by IDH status demonstrates that greater EOR independently prolonged MPFS and OS for wtIDH patients (hazard ratio [HR] = 0.002 [95% confidence interval {CI} 0.000-0.074] and HR = 0.001 [95% CI 0.00-0.108], respectively), but not for mtIDH patients (HR = 0.84 [95% CI 0.17-4.13] and HR = 2.99 [95% CI 0.15-61.66], respectively). CONCLUSION: Increasing EOR confers oncologic and survival benefits in IDH1 wtLGGs, but the impact on IDH1 mtLGGs requires further study.

Incidence and risk factors for preoperative deep venous thrombosis in 314 consecutive patients undergoing surgery for spinal metastasis.

OBJECTIVE The authors of this study aimed to identify the incidence of and risk factors for preoperative deep venous thrombosis (DVT) in patients undergoing surgical treatment for spinal metastases. METHODS Univariate analysis of patient age, sex, ethnicity, laboratory values, comorbidities, preoperative ambulatory status, histopathological classification, spinal level, and surgical details was performed. Factors significantly associated with DVT univariately were entered into a multivariate logistic regression model. RESULTS The authors identified 314 patients, of whom 232 (73.9%) were screened preoperatively for a DVT. Of those screened, 22 (9.48%) were diagnosed with a DVT. The screened patients were older (median 62 vs 55 years, p = 0.0008), but otherwise similar in baseline characteristics. Nonambulatory status, previous history of DVT, lower partial thromboplastin time, and lower hemoglobin level were statistically significant and independent factors associated with positive results of screening for a DVT. Results of screening were positive in only 6.4% of ambulatory patients in contrast to 24.4% of nonambulatory patients, yielding an odds ratio of 4.73 (95% CI 1.88-11.90). All of the patients who had positive screening results underwent preoperative placement of an inferior vena cava filter. CONCLUSIONS Patients requiring surgery for spinal metastases represent a population with unique risks for venous thromboembolism. This study showed a 9.48% incidence of DVT in patients screened preoperatively. The highest rates of preoperative DVT were identified in nonambulatory patients, who were found to have a 4-fold increase in the likelihood of harboring a DVT. Understanding the preoperative thrombotic status may provide an opportunity for early intervention and risk stratification in this critically ill population.

High-Content Screening in hPSC-Neural Progenitors Identifies Drug Candidates that Inhibit Zika Virus Infection in Fetal-like Organoids and Adult Brain.

Zika virus (ZIKV) infects fetal and adult human brain and is associated with serious neurological complications. To date, no therapeutic treatment is available to treat ZIKV-infected patients. We performed a high-content chemical screen using human pluripotent stem cell-derived cortical neural progenitor cells (hNPCs) and found that hippeastrine hydrobromide (HH) and amodiaquine dihydrochloride dihydrate (AQ) can inhibit ZIKV infection in hNPCs. Further validation showed that HH also rescues ZIKV-induced growth and differentiation defects in hNPCs and human fetal-like forebrain organoids. Finally, HH and AQ inhibit ZIKV infection in adult mouse brain in vivo. Strikingly, HH suppresses viral propagation when administered to adult mice with active ZIKV infection, highlighting its therapeutic potential. Our approach highlights the power of stem cell-based screens and validation in human forebrain organoids and mouse models in identifying drug candidates for treating ZIKV infection and related neurological complications in fetal and adult patients.

Sex, Age, Anatomic Location, and Extent of Resection Influence Outcomes in Children With High-grade Glioma.

BACKGROUND: Survival duration and prognostic factors in adult high-grade glioma have been comprehensively analyzed, but less is known about factors contributing to overall survival (OS) and progression-free survival (PFS) in pediatric patients. OBJECTIVE: To identify these factors in the pediatric population. METHODS: We retrospectively reviewed institutional databases evaluating all patients ≤21 years with high-grade glioma treated between 1988 and 2010. Kaplan-Meier curves and log-rank statistics were used to compare groups univariately. Multivariate analyses were completed using Cox proportional hazards regression models. RESULTS: Ninety-seven patients were identified with a median age of 11 years. Median OS was 1.7 years, and median PFS was 272 days. Location was significant for OS (P < .001). Patients with gross total resection (GTR) had a median OS of 3.4 years vs 1.6 years for subtotal resection and 1.3 years for biopsy patients (P < .001). Female patients had improved OS (P = .01). Female patients with GTR had a mean OS of 8.1 years vs 2.4 years for male patients with GTR and 1.4 years for all other female patients and male patients (P = .001). PFS favored patients ≤3 and ≥13 years and females (P = .003 and .001). CONCLUSION: OS was significantly correlated with the location of the tumor and the extent of resection. GTR significantly improved overall survival for both glioblastoma multiforme and anaplastic astrocytoma patients, and female patients showed a much larger survival benefit from GTR than male patients.

Phenotypic spectrum of the tubulin-related disorders and functional implications of disease-causing mutations.

A spectrum of neurological disorders characterized by abnormal neuronal migration, differentiation, and axon guidance and maintenance have recently been attributed to missense and splice-site mutations in the genes that encode α-tubulin and ß-tubulin isotypes TUBA1A, TUBA8, TUBB2B, and TUBB3, all of which putatively coassemble into neuronal microtubules. The resulting nervous system malformations can include different types of cortical malformations, defects in commissural fiber tracts, and degeneration of motor and sensory axons. Many clinical phenotypes and brain malformations are shared among the various mutations regardless of structural location and/or isotype, while others segregate with distinct amino acids or functional domains within tubulin. Collectively, these disorders provide novel paradigms for understanding the biological functions of microtubules and their core components in normal health and disease.