Para-infectious brain injury in COVID-19 persists at follow-up despite attenuated cytokine and autoantibody responses

To understand neurological complications of COVID-19 better both acutely and for recovery, we measured markers of brain injury, inflammatory mediators, and autoantibodies in 203 hospitalised participants; 111 with acute sera (1–11 days post-admission) and 92 convalescent sera (56 with COVID-19-associated neurological diagnoses). Here we show that compared to 60 uninfected controls, tTau, GFAP, NfL, and UCH-L1 are increased with COVID-19 infection at acute timepoints and NfL and GFAP are significantly higher in participants with neurological complications. Inflammatory mediators (IL-6, IL-12p40, HGF, M-CSF, CCL2, and IL-1RA) are associated with both altered consciousness and markers of brain injury. Autoantibodies are more common in COVID-19 than controls and some (including against MYL7, UCH-L1, and GRIN3B) are more frequent with altered consciousness. Additionally, convalescent participants with neurological complications show elevated GFAP and NfL, unrelated to attenuated systemic inflammatory mediators and to autoantibody responses. Overall, neurological complications of COVID-19 are associated with evidence of neuroglial injury in both acute and late disease and these correlate with dysregulated innate and adaptive immune responses acutely

Genomic epidemiology of SARS-CoV-2 in a UK university identifies dynamics of transmission

AbstractUnderstanding SARS-CoV-2 transmission in higher education settings is important to limit spread between students, and into at-risk populations. In this study, we sequenced 482 SARS-CoV-2 isolates from the University of Cambridge from 5 October to 6 December 2020. We perform a detailed phylogenetic comparison with 972 isolates from the surrounding community, complemented with epidemiological and contact tracing data, to determine transmission dynamics. We observe limited viral introductions into the university; the majority of student cases were linked to a single genetic cluster, likely following social gatherings at a venue outside the university. We identify considerable onward transmission associated with student accommodation and courses; this was effectively contained using local infection control measures and following a national lockdown. Transmission clusters were largely segregated within the university or the community. Our study highlights key determinants of SARS-CoV-2 transmission and effective interventions in a higher education setting that will inform public health policy during pandemics.</jats:p

Super-resolution imaging reveals mechanisms of glutamate transporter localization near neuron-astrocyte contacts

2021 Summer.Includes bibliographical references.Astrocytes contact neurons at several locations, including somatic clusters of Kv2.1 potassium channels and synapses across the brain. A primary function of astrocytes at these locations is to limit the action of extracellular glutamate. Astrocytic glutamate transporters, such as Glt1, ensure the fidelity of glutamic neurotransmission by spatially and temporally limiting glutamate signals. Additionally, they act to limit glutamate induced hyperexcitability by preventing the spread of glutamate to extrasynaptic receptors. The role of Glt1 in limiting neuronal hyperactivity relies heavily on the localization and diffusion of the transporter in the membrane, however, little is known about the mechanisms governing these properties. The work presented in this dissertation examines the mechanisms of Glt1 localization near Kv2.1-mediated neuron-astrocyte contact sites. To that end, in Chapter 2, we used super-resolution imaging to analyze the localization of two splice forms of Glt1, Glt1a and Glt1b. In cultures of primary astrocytes, we find that Glt1a, but not Glt1b, is specifically localized over cortical actin filaments. We go on to discover that this localization is dependent on the Glt1a C-terminus, where Glt1a and Glt1b differ, as exogenous expression of the Glt1a C-terminus was able to prevent localization of Glt1a to cortical actin filaments. In the somatosensory cortex, astrocyte Glt1 forms net-like structures around neuronal Kv2.1 clusters, however the cause of this Glt1 localization pattern is unknown. In Chapter 3, using super-resolution imaging of mixed cultures of astrocytes and neurons, we replicate findings of astrocyte Glt1 in a net-like localization around neuronal Kv2.1 clusters. We discover that both astrocyte actin and ER were excluded from the region across from neuronal Kv2.1 clusters. The actin-Glt1a relationship discussed in Chapter 2 is likely responsible for the net-like appearance of Glt1, as astrocytic Glt1 and actin colocalize in nets around Kv2.1 clusters at points of neuron-astrocyte contact. Neuronal control over the astrocyte cytoskeleton appears central to this Glt1a localization, although the mechanism of this control is still unknown. Together, these data describe a novel interaction between the Glt1a C-terminus and cortical actin filaments, which localizes Glt1 near neuronal structures involved in detecting ischemic insult. Although the mechanism of neuronal control over the astrocyte cytoskeleton remains a mystery, presumably cell-cell contact has a major influence. Contacts between neurons and astrocytes at Kv2.1 clusters could be mediated by the Kv2.1 β-subunit, AMIGO, which acts a cell adhesion molecule. Only one member of the AMIGO family of proteins is known to be an auxiliary β-subunit for Kv2 channels and to modulate Kv2.1 electrical activity. However, the AMIGO family has two additional members of ∼50% similarity that have not yet been characterized as Kv2 β-subunits. In Chapter 4, we show that the surface trafficking and localization of all three AMIGOs are controlled by their interaction with both Kv2.1 and Kv2.2 channels. Additionally, assembly of each AMIGO with either Kv2 alters important electrophysiological properties of these channels. The coregulatory effects of Kv2s and AMIGOs likely fine-tune both electrical and cell adhesion properties of the neurons in which they are expressed. Altogether, the work presented in this dissertation further defines the composition of Kv2.1-induced neuron-astrocyte contact sites, representing the first significant addition to this field in more than a decade

Cellular selectivity of AAV serotypes for gene delivery in neurons and astrocytes by neonatal intracerebroventricular injection

<div><p>The non-pathogenic parvovirus, adeno-associated virus (AAV), is an efficient vector for transgene expression <i>in vivo</i> and shows promise for treatment of brain disorders in clinical trials. Currently, there are more than 100 AAV serotypes identified that differ in the binding capacity of capsid proteins to specific cell surface receptors that can transduce different cell types and brain regions in the CNS. In the current study, multiple AAV serotypes expressing a GFP reporter (AAV1, AAV2/1, AAVDJ, AAV8, AAVDJ8, AAV9, AAVDJ9) were screened for their infectivity in both primary murine astrocyte and neuronal cell cultures. AAV2/1, AAVDJ8 and AAV9 were selected for further investigation of their tropism throughout different brain regions and cell types. Each AAV was administered to P0-neonatal mice via intracerebroventricular injections (ICV). Brains were then systematically analyzed for GFP expression at 3 or 6 weeks post-infection in various regions, including the olfactory bulb, striatum, cortex, hippocampus, substantia nigra (SN) and cerebellum. Cell counting data revealed that AAV2/1 infections were more prevalent in the cortical layers but penetrated to the midbrain less than AAVDJ8 and AAV9. Additionally, there were differences in the persistence of viral transgene expression amongst the three serotypes examined <i>in vivo</i> at 3 and 6 weeks post-infection. Because AAV-mediated transgene expression is of interest in neurodegenerative diseases such as Parkinson’s Disease, we examined the SN with microscopy techniques, such as CLARITY tissue transmutation, to identify AAV serotypes that resulted in optimal transgene expression in either astrocytes or dopaminergic neurons. AAVDJ8 displayed more tropism in astrocytes compared to AAV9 in the SN region. We conclude that ICV injection results in lasting expression of virally encoded transgene when using AAV vectors and that specific AAV serotypes are required to selectively deliver transgenes of interest to different brain regions in both astrocytes and neurons.</p></div

Tropism of AAV9 in multiple brain regions at 3 and 6 weeks post injection.

<p>AAV9 6-week injected tissue of multiple brain regions were immunostained for total neuronal marker MAP2 (red), astrocyte marker S100β (purple) and GFP (green) as depicted in representative 10X montage (top) and 100X high magnification images (bottom) of the olfactory bulb (<b>A</b>), striatum (<b>B</b>), motor cortex (<b>C</b>), hippocampus (<b>D</b>) and cerebellum (<b>E</b>). Notice; red/green co-localize to yellow, green/purple co-localize to cyan. (<b>F</b>) Each region was quantitated for GFP⁺/per 20X objective image field at 3 and 6-weeks post AAV9 injection (<i>n</i> = 3/serotype/time point; *<i>p</i><0.05).</p

Tropism of AAV2/1 in multiple brain regions at 3 and 6 weeks post injection.

<p>AAV2/1 6-week injected tissue of multiple brain regions were immunostained for total neuronal marker MAP2 (red), astrocyte marker S100β (purple) and GFP (green) as depicted in representative 10X montage (top) and 100X high magnification images (bottom) of the olfactory bulb (<b>A</b>), striatum (<b>B</b>), motor cortex (<b>C</b>), hippocampus (<b>D</b>) and cerebellum (<b>E</b>). Notice; red/green co-localize to yellow, green/purple co-localize to cyan. (<b>F</b>) Each region was quantitated for GFP⁺/per 20X objective image field at 3 and 6-weeks post AAV2/1 injection (<i>n</i> = 3-4/serotype/time point).</p

AAVDJ8-GFAP-mCherry targets exclusively astrocytes in the substantia nigra.

<p>(<b>A</b>) Astrocyte-specific promoter, GFAP, was incorporated into suitable serotype AAVDJ8 to drive expression of fluorescent reporter, mCherry for targeting astrocytes, as depicted in plasmid AAV vector map. (<b>B</b>) Primary astrocyte cultures were transduced with AAVDJ8-GFAP-mCherry to confirm astrocyte transduction efficiency <i>in vitro</i> as depicted in representative 100X objective images of immunostaining for mCherry(red) and GFAP(green), 83.2%±6.5 GFAP⁺/mCherry⁺ quantitated. (<b>C</b>) mCherry (red) and S100β(green) immunofluorescence is visualized in 10X objective montage image of sagittal cross section from AAVDJ8-GFAP-mCherry 3-week infected brain. (<b>D</b>) AAVDJ8-GFAP-mCherry expression in 10X objective image of SN region immunostained for mCherry (red), S100β(green), and TH (cyan) from 3 week infected brain, 100X objective inset images represent colocalization of S100β⁺/mCherry⁺ cells. (<b>E</b>) AAVDJ8-GFAP-mCherry expression levels were quantitated for mCherry⁺cells/20X objective image field at 3 and 6 weeks post ICV (<i>n</i> = 3/serotype, groups were not significant). (<b>F</b>) 3D volumetric view in XYZ and XY planes for CLARITY immunofluorescent image stained for TH (cyan), GFAP (green) and mCherry (red) within the SNpc. AAVDJ8-GFAP-mCherry infected astrocytes are exclusiveling co-localizing with GFAP⁺ cells within the vicinity of dopaminergic neurons. Video of 3D projection can be found in <b><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0188830#pone.0188830.s003" target="_blank">S2 Video</a></b>.</p

Tropism of AAVDJ8 in multiple brain regions at 3 and 6 weeks post injection.

<p>AAVDJ8 6-week injected tissue of multiple brain regions were immunostained for total neuronal marker MAP2 (red), astrocyte marker S100β (purple) and GFP (green) as depicted in representative 10X montage (top) and 100X high magnification images (bottom) of the olfactory bulb (<b>A</b>), striatum (<b>B</b>), motor cortex (<b>C</b>), hippocampus (<b>D</b>) and cerebellum (<b>E</b>). Notice; red/green co-localize to yellow, green/purple co-localize to cyan. (<b>F</b>) Each region was quantitated for GFP⁺/per 20X objective image field at 3 and 6-weeks post AAVDJ8 injection (<i>n</i> = 3/serotype/time point).</p

AAV9 and AAVDJ8 transduce DA neurons and astrocytes of the substantia nigra.

<p>10X objective representative images of SN from 6-week tissue immunostained for TH (cyan) to visualize viral GFP expression (native) in the SN pars compacta (white outline) and SN pars reticulata for AAV2/1 (<b>A</b>), AAVDJ8 (<b>B</b>), and AAV9 (<b>C</b>) injected brains. (<b>D</b>) Total GFP⁺ cells/ 20X objective image field were quantitated for both 3 and 6-weeks post injection within the SN region (<i>n</i> = 3/serotype; *<i>p</i><0.05). (<b>E</b>) AAV9 and (<b>F</b>) AAVDJ8 100X objective representative images immunostained for TH and S100β (red) within the SNpc. at 6-weeks post ICV. (<b>G</b>) % S100β-GFP⁺ and TH-GFP⁺ cells in the SNpc were quantitated/per 40X objective image field (<i>n</i> = 3/serotype; <i>p</i><0.05). (<b>H</b>) Representative, three dimensional (3D) 40X objective montage image of clarified SN tissue from a AAVDJ8–GFP infected brain, immunostained for TH (cyan), GFAP (red) and GFP (green) in XYZ and XZ volumetric planes. Video of 3D projection can be found in <b><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0188830#pone.0188830.s002" target="_blank">S1 Video</a></b>.</p