Invited Review: The spectrum of neuropathology in COVID-19.

There is increasing evidence that patients with Coronavirus disease 19 (COVID-19) present with neurological and psychiatric symptoms. Anosmia, hypogeusia, headache, nausea and altered consciousness are commonly described, although there are emerging clinical reports of more serious and specific conditions such as acute cerebrovascular accident, encephalitis and demyelinating disease. Whether these presentations are directly due to viral invasion of the central nervous system (CNS) or caused by indirect mechanisms has yet to be established. Neuropathological examination of brain tissue at autopsy will be essential to establish the neuro-invasive potential of the SARS-CoV-2 virus but, to date, there have been few detailed studies. The pathological changes in the brain probably represent a combination of direct cytopathic effects mediated by SARS-CoV-2 replication or indirect effects due to respiratory failure, injurious cytokine reaction, reduced immune response and cerebrovascular accidents induced by viral infection. Further large-scale molecular and cellular investigations are warranted to clarify the neuropathological correlates of the neurological and psychiatric features seen clinically in COVID-19. In this review, we summarize the current reports of neuropathological examination in COVID-19 patients, in addition to our own experience, and discuss their contribution to the understanding of CNS involvement in this disease.

Neurodegeneration in frontotemporal lobar degeneration and motor neurone disease associated with expansions in C9orf72 is linked to TDP-43 pathology and not associated with aggregated forms of dipeptide repeat proteins.

AIMS: A hexanucleotide expansion in C9orf72 is the major genetic cause of inherited behavioural variant Frontotemporal dementia (bvFTD) and motor neurone disease (MND), although the pathological mechanism(s) underlying disease remains uncertain. METHODS: Using antibodies to poly-GA, poly-GP, poly-GR, poly-AP and poly-PR proteins, we examined sections of cerebral cortex, hippocampus, thalamus, cerebellum and spinal cord, from 20 patients with bvFTD and/or MND bearing an expansion in C9orf72 for aggregated deposits of dipeptide repeat proteins (DPR). RESULTS: Antibodies to poly-GA, poly-GP and poly-GR detected numerous rounded cytoplasmic inclusions (NCI) within granule cells of hippocampal dentate gyrus and those of the cerebellum, as well as 'star-burst' shaped NCI in pyramidal neurones of CA3/4 region of hippocampus. NCI were uncommon in Purkinje cells, and only very rarely seen in anterior horn cells. Poly-PA antibody detected occasional NCI within CA3/4 neurones alone, whereas poly-PR antibody did not identify any NCI but immunostained the nucleus of anterior horn cells, CA3/4 neurones and Purkinje cells, in patients with or without expansion in C9orf72, as well as in normal controls. Poly-GA antibody generally detected more DPR than poly-GP, which in turn was greater than poly-GR. All patients with bvFTD + MND or MND showed plentiful p62/TDP-43 positive inclusions in remaining anterior horn cells. CONCLUSION: Degeneration and loss of anterior horn cells associated with expansions in C9orf72 occurs in the absence of DPR, and implies that changes involving loss of nuclear staining for and a cytoplasmic aggregation of TDP-43 are more likely to be the cause of this.

Proteomic analyses reveal that loss of TDP-43 affects RNA processing and intracellular transport.

Transactive response DNA-binding protein 43 (TDP-43) is a predominantly nuclear, ubiquitously expressed RNA and DNA-binding protein. It recognizes and binds to UG repeats and is involved in pre-mRNA splicing, mRNA stability and microRNA metabolism. TDP-43 is essential in early embryonic development but accumulates in cytoplasmic aggregates in amyotrophic lateral sclerosis (ALS) and tau-negative frontotemporal lobar degeneration (FTLD). It is not known yet whether cytoplasmic aggregates of TDP-43 are toxic or protective but they are often associated with a loss of TDP-43 from the nucleus and neurodegeneration may be caused by a loss of normal TDP-43 function or a gain of toxic function. Here we present a proteomic study to analyze the effect of loss of TDP-43 on the proteome. MS data are available via ProteomeXchange with identifier PXD001668. Our results indicate that TDP-43 is an important regulator of RNA metabolism and intracellular transport. We show that Ran-binding protein 1 (RanBP1), DNA methyltransferase 3 alpha (Dnmt3a) and chromogranin B (CgB) are downregulated upon TDP-43 knockdown. Subsequently, transportin 1 level is increased as a result of RanBP1 depletion. Improper regulation of these proteins and the subsequent disruption of cellular processes may play a role in the pathogenesis of the TDP-43 proteinopathies ALS and FTLD.

Control tissue in brain banking: the importance of thorough neuropathological assessment.

Historically, control brain tissue was classified as such mainly by clinical history, and underwent limited neuropathological analysis. Significant progress has been made in recent years with the collection of more extensive clinical information and more specific classifications of neurodegenerative disease, aided by advances in histological processing and increasingly sensitive detection methods. We hypothesised that this may have resulted in certain pathologies previously going unidentified, due to insufficient block sampling and an inadequate range of stains, resulting in the disease not being recognised. We therefore investigated the significance of changes to our own protocols for examining control brain tissue before and after 2007. Control cases that were originally assessed before 2007 were re-assessed using our current staining protocol and antibodies, and compared with age-matched cases post-2007. We found that almost all cases that were originally described as neuropathologically normal displayed some level of pathology after re-analysis, with four cases displaying what we have termed 'major' pathology that previously went unidentified, emphasising on a small scale the importance of accurate neuropathological analysis of control tissue, and highlighting the inherent difficulty of traditionally classifying tissue simply as 'disease' or 'control'. We hope our findings will stimulate debate within the brain banking community, with the eventual aim being standardisation of protocols for assessing controls across brain banks.

Changes in the expression of genes related to neuroinflammation over the course of sporadic Alzheimer's disease progression: CX3CL1, TREM2, and PPARγ.

The role of neuroinflammation in the pathogenesis of neurodegenerative diseases has become more evident in recent years. Research on the etiology and pathogenesis of sporadic Alzheimer's disease (AD) has focused on the role of chemokines such as CX3CL1, on the triggering receptors expressed by myeloid cells (TREMs), especially TREM2, and on the transcription factor/nuclear hormone receptor peroxisome proliferator-activated receptor gamma (PPARγ). Here we analyzed the expression levels of CX3CL1, TREM2, and PPARγ in tissue homogenates from human brain regions that have different degrees of vulnerability to neuropathological AD-related changes to obtain insights into the pathogenesis and progression of AD. We found that CX3CL1 and TREM2, two genes related to neuroinflammation, are more highly expressed in brain regions with pronounced vulnerability to AD-related changes, such as the hippocampus, and that the expression levels reflect the course of the disease, whereas regions with low vulnerability to AD, seemed generally less affected by neuroinflammation. Furthermore, our results support previous findings of significantly higher CX3CL1 plasma levels in patients with mild to moderate AD than in patients with severe AD. Thus, CX3CL1 should be considered as promising additional marker for the early diagnosis of AD and underlines once more, the involvement of the neuroinflammation in the pathogenesis of this neurodegenerative disease.

Transportin 1 colocalization with Fused in Sarcoma (FUS) inclusions is not characteristic for amyotrophic lateral sclerosis-FUS confirming disrupted nuclear import of mutant FUS and distinguishing it from frontotemporal lobar degeneration with FUS inclusions.

AIMS: Transportin 1 (TNPO 1) is an abundant component of the Fused in Sarcoma (FUS)-immunopositive inclusions seen in a subgroup of frontotemporal lobar degeneration (FTLD-FUS). TNPO 1 has been shown to bind to the C-terminal nuclear localizing signal (NLS) of FUS and mediate its nuclear import. Amyotrophic lateral sclerosis (ALS)-linked C-terminal mutants disrupt TNPO 1 binding to the NLS and impair nuclear import in cell culture. If this held true for human ALS then we predicted that FUS inclusions in patients with C-terminal FUS mutations would not colocalize with TNPO 1. METHODS: Expression of TNPO 1 and colocalization with FUS was studied in the frontal cortex of FTLD-FUS (n = 3) and brain and spinal cord of ALS-FUS (n = 3), ALS-C9orf72 (n = 3), sporadic ALS (n = 7) and controls (n = 7). Expression levels and detergent solubility of TNPO 1 was measured by Western blot. RESULTS: Aggregates of TNPO 1 were abundant and colocalized with FUS inclusions in the cortex of all FTLD-FUS cases. In contrast, no TNPO 1-positive aggregates or FUS colocalization was evident in two-thirds, ALS-FUS cases and was rare in one ALS-FUS case. Nor were they present in C9orf72 or sporadic ALS. No increase in the levels of TNPO 1 was seen in Western blots of spinal cord tissues from all ALS cases compared with controls. CONCLUSIONS: These findings confirm that C-terminal FUS mutations prevent TNPO 1 binding to the NLS, inhibiting nuclear import and promoting cytoplasmic aggregation. The presence of TNPO 1 in wild-type FUS aggregates in FTLD-FUS distinguishes the two pathologies and implicates different disease mechanisms.

Neuropathology of the hippocampus in FTLD-Tau with Pick bodies: a study of the BrainNet Europe Consortium.

AIMS: Frontotemporal lobar degeneration with Pick bodies (Pick's disease) is characterized by the presence of tau immunoreactive spherical structures in the cytoplasm of neurones. In view of confusion about the molecular pathology of Pick's disease, we aimed to evaluate the spectrum of tau pathology and concomitant neurodegeneration-associated protein depositions in the characteristically affected hippocampus. METHODS: We evaluated immunoreactivity (IR) for tau (AT8, 3R, 4R), α-synuclein, TDP43, p62, and ubiquitin in the hippocampus, entorhinal and temporal cortex in 66 archival cases diagnosed neuropathologically as Pick's disease. RESULTS: Mean age at death was 68.2 years (range 49-96). Fifty-two (79%) brains showed 3R immunoreactive spherical inclusions in the granule cells of the dentate gyrus. These typical cases presented mainly with the behavioural variant of frontotemporal dementia, followed by progressive aphasia, mixed syndromes or early memory disturbance. α-Synuclein IR was seen only in occasional spherical tau-positive inclusions, TDP-43 IR was absent, and 4R IR was present only as neurofibrillary tangles in pyramidal neurones. Aß IR was observed in 16 cases; however, the overall level of Alzheimer's disease-related alterations was mainly low or intermediate (n = 3). Furthermore, we identified six cases with unclassifiable tauopathy. CONCLUSIONS: (i) Pick's disease may occur also in elderly patients and is characterized by a relatively uniform pathology with 3R tau inclusions particularly in the granule cells of dentate gyrus; (ii) even minor deviation from these morphological criteria suggests a different disorder; and (iii) immunohistological revision of archival cases expands the spectrum of tauopathies that require further classification.

Anxiety behaviour of the male rat on the elevated plus maze: associated regional increase in c-fos mRNA expression and modulation by early maternal separation.

Stressful stimuli cause region-specific increases in c-fos expression within the rat brain. Early maternal separation (EMS) is a model of early life adversity that results in long lasting changes to stress and anxiety responses. This study examined the regional distribution of c-fos mRNA after exposure to the elevated plus-maze (EPM) and how EMS altered this pattern. On each of post-natal days 5-21 pups were separated from the dam for 6 h -- control rats remained undisturbed. At 70 days old, male offspring were either exposed to the EPM or left undisturbed in the home cage. After exposure to the EPM, c-fos mRNA expression was significantly increased in specific brain areas, including cingulate cortex, medial amygdala and hippocampus. EMS rats displayed greater anxiety behaviour on the EPM vs. controls. Although EMS caused no overall effect on basal c-fos mRNA, a significant interaction between treatment group and exposure to the EPM occurred in the dentate gyrus and piriform cortex, with lower EPM-induced mRNA levels in EMS rats. The region-specific increase in c-fos mRNA reflects activation of neural circuits associated with EPM-induced anxiety. The effect of EMS on this activation in the two regions suggests these areas may contribute to the differential response to the anxiogenic stress of the EPM.

Comparison of the neurokinin-1 antagonist GR205171, alone and in combination with the 5-HT3 antagonist ondansetron, hyoscine and placebo in the prevention of motion-induced nausea in man.

AIMS: In man a neurokinin-1 (NK1) receptor antagonist has previously been shown to be ineffective in the prevention of motion-induced nausea. The antiemetic efficacy of NK1 receptor antagonists against chemotherapy-induced emesis is, however, enhanced when combined with a 5-HT3 receptor antagonist. Hence the efficacy of the NK1 antagonist GR205171 in combination with the 5-HT3 antagonist ondansetron (Zofrantrade mark) was assessed in motion-induced nausea. METHODS: GR205171 25 mg i.v., with and without concomitant administration of ondansetron 8 mg i.v., and hyoscine hydrobromide 0. 6 mg orally (positive control) were compared with placebo in a model of motion-induced nausea. The study was performed to a four-period, randomized, balanced, double-blind, crossover design in 16 healthy subjects. The end-point was the exposure to the motion stimulus required to produce moderate nausea in the subjects. RESULTS: The motion stimulus required to produce moderate nausea was significantly greater for the positive control than placebo (P < 0. 001). There was no significant difference between either GR205171 or GR205171 plus ondansetron and placebo (P = 0.648 and 0.342, respectively). CONCLUSIONS: The enhancement of NK1 receptor antagonist antiemetic activity through combination with a 5-HT3 receptor antagonist is not replicated in motion-induced nausea.