Spatial Single-cell Analysis Decodes Cortical Layer and Area Specification.

The human cerebral cortex, pivotal for advanced cognitive functions, is composed of six distinct layers and dozens of functionally specialized areas1,2. The layers and areas are distinguished both molecularly, by diverse neuronal and glial cell subtypes, and structurally, through intricate spatial organization3,4. While single-cell transcriptomics studies have advanced molecular characterization of human cortical development, a critical gap exists due to the loss of spatial context during cell dissociation5,6,7,8. Here, we utilized multiplexed error-robust fluorescence in situ hybridization (MERFISH)9, augmented with deep-learning-based cell segmentation, to examine the molecular, cellular, and cytoarchitectural development of human fetal cortex with spatially resolved single-cell resolution. Our extensive spatial atlas, encompassing 16 million single cells, spans eight cortical areas across four time points in the second and third trimesters. We uncovered an early establishment of the six-layer structure, identifiable in the laminar distribution of excitatory neuronal subtypes by mid-gestation, long before the emergence of cytoarchitectural layers. Notably, while anterior-posterior gradients of neuronal subtypes were generally observed in most cortical areas, a striking exception was the sharp molecular border between primary (V1) and secondary visual cortices (V2) at gestational week 20. Here we discovered an abrupt binary shift in neuronal subtype specification at the earliest stages, challenging the notion that continuous morphogen gradients dictate mid-gestation cortical arealization6,10. Moreover, integrating single-nuclei RNA-sequencing and in situ whole transcriptomics revealed an early upregulation of synaptogenesis in V1-specific Layer 4 neurons, suggesting a role of synaptogenesis in this discrete border formation. Collectively, our findings underscore the crucial role of spatial relationships in determining the molecular specification of cortical layers and areas. This work not only provides a valuable resource for the field, but also establishes a spatially resolved single-cell analysis paradigm that paves the way for a comprehensive developmental atlas of the human brain.

Postmortem Assessment of Olfactory Tissue Degeneration and Microvasculopathy in Patients With COVID-19.

Importance: Loss of smell is an early and common presentation of COVID-19 infection. Although it has been speculated that viral infection of olfactory neurons may be the culprit, it is unclear whether viral infection causes injuries in the olfactory bulb region. Objective: To characterize the olfactory pathology associated with COVID-19 infection in a postmortem study. Design, Setting, and Participants: This multicenter postmortem cohort study was conducted from April 7, 2020, to September 11, 2021. Deceased patients with COVID-19 and control individuals were included in the cohort. One infant with congenital anomalies was excluded. Olfactory bulb and tract tissue was collected from deceased patients with COVID-19 and appropriate controls. Histopathology, electron microscopy, droplet digital polymerase chain reaction, and immunofluorescence/immunohistochemistry studies were performed. Data analysis was conducted from February 7 to October 19, 2021. Main Outcomes and Measures: (1) Severity of degeneration, (2) losses of olfactory axons, and (3) severity of microvasculopathy in olfactory tissue. Results: Olfactory tissue from 23 deceased patients with COVID-19 (median [IQR] age, 62 [49-69] years; 14 men [60.9%]) and 14 control individuals (median [IQR] age, 53.5 [33.25-65] years; 7 men [50%]) was included in the analysis. The mean (SD) axon pathology score (range, 1-3) was 1.921 (0.569) in patients with COVID-19 and 1.198 (0.208) in controls (P < .001), whereas axon density was 2.973 (0.963) × 104/mm2 in patients with COVID-19 and 3.867 (0.670) × 104/mm2 in controls (P = .002). Concomitant endothelial injury of the microvasculature was also noted in olfactory tissue. The mean (SD) microvasculopathy score (range, 1-3) was 1.907 (0.490) in patients with COVID-19 and 1.405 (0.233) in control individuals (P < .001). Both the axon and microvascular pathology was worse in patients with COVID-19 with smell alterations than those with intact smell (mean [SD] axon pathology score, 2.260 [0.457] vs 1.63 [0.426]; P = .002; mean [SD] microvasculopathy score, 2.154 [0.528] vs 1.694 [0.329]; P = .02) but was not associated with clinical severity, timing of infection, or presence of virus. Conclusions and Relevance: This study found that COVID-19 infection is associated with axon injuries and microvasculopathy in olfactory tissue. The striking axonal pathology in some cases indicates that olfactory dysfunction in COVID-19 infection may be severe and permanent.

Further Delineation of the Clinical and Pathologic Features of HIKESHI-Related Hypomyelinating Leukodystrophy.

BACKGROUND: A recurrent homozygous missense variant, c.160G>C;p.(Val54Leu) in HIKESHI, was found to cause a hypomyelinating leukodystrophy with high frequency in the Ashkenazi Jewish population. We provide extended phenotypic classification of this disorder based on clinical history of a further seven affected individuals, assess carrier frequency in the Ashkenazi Jewish population, and provide a neuropathological study. METHODS: Clinical information, neuroimaging, and biosamples were collected. Brain autopsy was performed for one case. RESULTS: Individuals with HIKESHI-related disease share common clinical features: early axial hypotonia evolving to dystonia or with progressive spasticity, hyperreflexia and clonus, feeding difficulties with poor growth, and nystagmus. Severe morbidity or death during febrile illness occurred in five of the nine affected individuals. Magnetic resonance images of seven patients were analyzed and demonstrated diffuse hypomyelination and thin corpus callosum. Genotyping data of more than 125,000 Ashkenazi Jewish individuals revealed a carrier frequency of 1 in 216. Gross pathology examination in one case revealed abnormal white matter. Microscopically, there was a near-total absence of myelin with a relative preservation of axons. The cerebral white matter showed several reactive astrocytes and microglia. CONCLUSIONS: We provide pathologic evidence for a primary disorder of the myelin in HIKESHI-related leukodystrophy. These findings are consistent with the hypomyelination seen in brain magnetic resonance imaging and with the clinical features of early-onset spastic/dystonic quadriplegia and nystagmus. The high carrier rate of the recurrent variant seen in the Ashkenazi Jewish population requires increased attention to screening and diagnosis of this condition, particularly in this population.

Streamlined alpha-synuclein RT-QuIC assay for various biospecimens in Parkinson's disease and dementia with Lewy bodies.

Definitive diagnosis of Parkinson's disease (PD) and dementia with Lewy bodies (DLB) relies on postmortem finding of disease-associated alpha-synuclein (αSynD) as misfolded protein aggregates in the central nervous system (CNS). The recent development of the real-time quaking induced conversion (RT-QuIC) assay for ultrasensitive detection of αSynD aggregates has revitalized the diagnostic values of clinically accessible biospecimens, including cerebrospinal fluid (CSF) and peripheral tissues. However, the current αSyn RT-QuIC assay platforms vary widely and are thus challenging to implement and standardize the measurements of αSynD across a wide range of biospecimens and in different laboratories. We have streamlined αSyn RT-QuIC assay based on a second generation assay platform that was assembled entirely with commercial reagents. The streamlined RT-QuIC method consisted of a simplified protocol requiring minimal hands-on time, and allowing for a uniform analysis of αSynD in different types of biospecimens from PD and DLB. Ultrasensitive and specific RT-QuIC detection of αSynD aggregates was achieved in million-fold diluted brain homogenates and in nanoliters of CSF from PD and DLB cases but not from controls. Comparative analysis revealed higher seeding activity of αSynD in DLB than PD in both brain homogenates and CSF. Our assay was further validated with CSF samples of 214 neuropathologically confirmed cases from tissue repositories (88 PD, 58 DLB, and 68 controls), yielding a sensitivity of 98% and a specificity of 100%. Finally, a single RT-QuIC assay protocol was employed uniformly to detect seeding activity of αSynD in PD samples across different types of tissues including the brain, skin, salivary gland, and colon. We anticipate that our streamlined protocol will enable interested laboratories to easily and rapidly implement the αSyn RT-QuIC assay for various clinical specimens from PD and DLB. The utilization of commercial products for all assay components will improve the robustness and standardization of the RT-QuIC assay for diagnostic applications across different sites. Due to ultralow sample consumption, the ultrasensitive RT-QuIC assay will facilitate efficient use and sharing of scarce resources of biospecimens. Our streamlined RT-QuIC assay is suitable to track the distribution of αSynD in CNS and peripheral tissues of affected patients. The ongoing evaluation of RT-QuIC assay of αSynD as a potential biomarker for PD and DLB in clinically accessible biospecimens has broad implications for understanding disease pathogenesis, improving early and differential diagnosis, and monitoring therapeutic efficacies in clinical trials.

Development of a Flow Cytometry-Based Functional Assay to Study Anti-TNF Mechanisms of Action and Capture Donor Heterogeneity.

TNF is a key cytokine in autoimmune diseases like rheumatoid arthritis, and TNF antagonists are commonly prescribed therapeutics. Although anti-TNF drugs have enabled a very significant progress in this field, disease heterogeneity remains and causes diversity in patient response. These challenges increase the need for anti-TNF characterization tools that may open perspectives toward the development of personalized medicine. In this study, we present a novel whole blood-based flow cytometry functional assay that allows, within a given whole blood sample, the characterization of an anti-TNF molecule mechanisms of action. Whole blood from healthy human donors was employed to mimic the physiological state but also to streamline experimental workflows. Samples were incubated with LPS alone or in combination with various anti-TNF molecules such as adalimumab (ADA), etanercept (ETA), and infliximab. A 10-color flow cytometry panel including CD69, transmembrane TNF, CD16, CD62L, CD66b, CD11b, and CD54 as activation markers was used following a centrifugation-free protocol. CD69 expression decreased on NK, NKT, and T cells upon treatment with ADA, ETA, and IFX as a direct indication of forward signaling neutralization. Percentages of transmembrane TNF+ monocytes increased after incubation when using ADA or IFX but not ETA, revealing the potential of the two first molecules to trigger reverse signaling. Ab-dependent cell cytotoxicity was informed by CD16 and CD69 expressions in some donors that showed increasing levels of CD16- CD69+ NK cells when incubated with anti-TNFs. This study proposes a novel approach to assess anti-TNF mechanisms of action and provides a path toward capturing donor heterogeneity.