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APOE e4 is the largest genetic risk factor for late-onset Alzheimer’s disease. It has been associated with poorer episodic memory in older age as well as higher default mode network (DMN) connectivity compared to e4 non-carriers during resting state fMRI. Default mode network includes posterior medial, retrosplenial, angular gyrus and medial prefrontal cortex regions. During movie watching, the posterior parietal cortex, retrosplenial cortex and hippocampus have been shown to respond to event boundaries (e.g., changes in scenes or character goals) and have increased connectivity. These event boundaries have been suggested to be critical in helping to organise information in memory. Based on previous findings of differential connectivity at rest in the DMN in e4 carriers, here we will examine whether e4 carriers will show differential response to event boundaries in posterior regions of the DMN network. This study will use data from the Cam-CAN dataset which is a group of around 600 genotyped participants representing the adult lifespan. We will examine whether there are genotype differences in boundary-evoked responses to a short film. Our analysis will be formed of three main components. Firstly, a general linear model will be fitted with event boundaries as a regressor to quantify boundary-evoked activity in each region of interest. This would allow us to test whether univariate event boundary activity differs by genotype and/or by age. Second, we will test the functional connectivity of posterior regions of the default mode network around event boundaries. This analysis will follow previously reported differences in connectivity (Cooper et al., 2021) and examine whether connectivity at event boundaries is different for e4 carriers vs non-carriers. Finally, data will be submitted to a regression model with planned contrasts to compare differences between APOE genotype and age.

Abstract Chapter 2: Circadian and ultradian RNA oscillations from a single cell perspectivePhysiology and behavior of most organisms oscillate in cycles that follow daily environmental changes. These cycles of 24 hours periodicity include general behavior patterns as activity levels, feeding, and sleep-wake cycles; but also include less conspicuous oscillations such as metabolic or immune function. Circadian rhythms are driven by a transcription–translation feedback loop (TTFL) that generates oscillation at the RNA and protein levels of the so-called ́core-clock genes ́. In Drosophila melanogaster, a set of 300 neurons (clock neurons) control the circadian rhythm of locomotor activity and directly or indirectly control rhythms in other behavioral processes as sleep and feeding. In addition, peripheral tissues in Drosophila also display 24-hour rhythms at the gene expression and physiology levels. These peripheral oscillations can be either completely self- sustained by the peripheral tissue, as well as entirely dependent or partially regulated by the central brain through neurohormones like PDF. To this date it is unknow which cells in peripheral tissues harbor a molecular clock. Moreover, it is not known if the cycling genes in these tissues are coming directly from cells with a functional molecular clock or, or from signaling from neighboring cells or the central brain. To answer these questions, we mapped the main core-clock genes, and PDF receptor (Pdfr) in all cells in the fly body using publicly available single-cell data from the fly atlas. We also mapped cycling genes coming from 3 tissues with robust oscillations: fat body, gut and Malpighian tubule. Strikingly, we found that in peripheral tissues, only some cells express core-clock genes. Expression of Pdfr is highly expressed in cell with no core-clock expression particularly high in lamina neurons in the optic lobe. Additionally, we found Lawf2 and Poxn neurons as new putative clock neurons in the optic lobe and the brain respectively. Circadian rhythms are present in most living animals but rhythms with shorter than 24-hours period (ultradian) are also prevalent. In mammals, some examples or ultradian rhythms are body temperature, hormone release and sleep. In mice and flies, ultradian locomotor activity patterns arise with the disruption of the circadian clock. However, there is no knowledge on ultradian gene expression in the fly brain or the molecular mechanism governing these rhythms. To study this, we surveyed RNA expression from fly brains every two hours. We found genes cycling in an ultradian fashion with two and three peaks of expression over the day (period of around 8 and 12 hours). Among the ultradian cyclers, we found various genes implicated in different functions such as Wnt signaling, Serotonin receptor signaling, and response to bacterial infection. Interestingly, a proportion of transcripts cycle in an ultradian fashion in the Drosophila melanogaster brain and are enriched in glial cells. We then explored the functions of both MBL-C and MBL-OP and we found that ubiquitous knockdown (KD) of both isoforms leads to major changes in the brain transcriptomes. Moreover, KD of both mbl isoforms lead to changes in similar sets of genes. We do not see these major effects when we knock down each isoform only in neurons by using the neuronal specific driver elav-gal4 driver. As mbl-C and mbl-OP are expressed in different cell types in the adult head, this indicates that MBL-OP and MBL-C might share some functions over development in elav negative cells. Moreover, we found that in heads MBL-C lead to changes mainly in muscle related genes while in photoreceptors MBL-OP KD leads to changes in alternative splicing of genes related to photoreceptor survival to light. We could validate these results using total RNA sequencing from sorted cells. Moreover, this data allowed us also to map different mbl linear and circular isoforms. Different mbl mRNA isoforms are expressed in a mutually exclusive manner and are co-expressed with different circMbl isoforms: MBL-OP is expressed with circMbl2-4 in photoreceptors, while MBL-C is expressed along with circMbl in muscles and brain cells. By doing knockdown and overexpression experiments we could corroborate that this co-expression pattern is actually co-regulatory: MBL promotes circMbl back-splicing; while circMbl synthesis competes with the formation of the linear mRNA. In photoreceptors MBL-OP, not only promotes circMbl formation but also inhibits splicing of its first two introns. Using this approach, we studied in vivo the role of circMbl, the most abundant circular RNA in the fly brain. We found that knocking it down leads to specific changes in the brain transcriptome. In particular, we found changes in genes enriched in some glial clusters, peptidergic neurons, clock neurons, Lawf2 cells, and photoreceptors. To understand where circMbl is expressed we developed a new bioinformatic approach that allowed us to map circMbl in a single cell dataset from the fly brain. We found circMbl highly enriched in Lawf2 neurons, photoreceptor, peptidergic and clock neurons. We found that circMbl levels correlate well with the signal from the linear mRNA. This can be explained as MBL promotes circMbl synthesis. Abstract Chapter 1: Circular RNA regulation at the single cell and tissue specific levelExonic circular RNAs (circRNAs) are highly abundant RNAs generated mostly from exons of protein-coding genes. Assaying the functions of circRNAs is not straightforward as common approaches for circRNA depletion tend to also alter the levels of mRNAs generated from the hosting gene. In this work we describe an shRNA approach to knock down (at a post- transcriptional level) circular RNAs (circRNAs) without affecting their linear counterparts in vivo. We also describe a computational platform for determining the potential off-target effects as well as for verifying the obtained phenotypes.

Dengue infection is common in 129 countries including Pakistan, with an estimated 390 million dengue virus infections per year. Asia accounts for 70% of the actual burden. Clinical signs and symptoms of dengue include a high fever (40°C/104 °F) accompanied by two of the following symptoms during the febrile phase: severe headache, pain behind the eyes, muscle and joint pains, nausea, vomiting, swollen glands and rash. Severe dengue is a potentially fatal complication, due to plasma leaking, fluid accumulation, respiratory distress, severe bleeding or organ impairment. Dengue virus does not usually cause neurological manifestations, unlike other arboviral infections. Encephalopathy is the most common neurological complication. Encephalitis is rare manifestation. We report a rare case of dengue encephalitis from Pakistan, with extensive brain lesions involving the thalamus, cerebellar vermis and frontoparietal periventricular regions. Therefore, we suggest dengue encephalitis should be considered in the differential diagnosis of fever with altered sensorium, especially in Asian countries such as Pakistan where dengue is rampant as dengue encephalitis can be fatal at times.

A woman in her 30s with medically intractable epilepsy and Lennox-Gastaut Syndrome on multiple antiseizure medications and with a deep brain stimulator presented to the epilepsy monitoring unit with increased seizure frequency. She was noted to have periods of apparent apnoea time linked to bursts of epileptiform activity on continuous video EEG monitoring. Once the clinical seizures were controlled, she was discharged to the sleep laboratory. She was noted to have obstructive and central sleep apnoea, which improved with the use of positive airway pressure. Central sleep apnoeas were time linked to electrographic seizures. Ictal central apnoea can easily be overlooked and is likely more common than currently recognised in patients with epilepsy. Ictal central apnoea may be a biomarker for sudden unexpected death in epilepsy.

Stroke-like migraine attacks after radiation therapy (SMART) syndrome is a rare complication of radiotherapy with complex neurological impairment. Patients present with neurological symptoms and signs such as migraine, hemianopsia, hemiplegia, aphasia and/or seizures—without recurrence of neoplastic disease. In this report, we describe SMART syndrome in two adult patients 4 and 14 years following brain irradiation, respectively.

We describe the case of a woman in her 60s admitted to the intensive care unit after a first generalised tonic-clonic seizure in the context of alcohol withdrawal. She was placed under invasive mechanical ventilation due to persistence of coma despite antiepileptic treatment. Despite continuous sedation with propofol, the frequency and intensity of seizure increased. Seizures were very similar to epileptic tonic-clonic seizures and were recorded with video and electroencephalogram (EEG). A diagnosis of tetanus was considered after a scalp wound was discovered. The patient’s husband revealed that a trismus had appeared a few days before hospital admission after a head trauma. EEG showed a pattern of diffuse spikes, which disappeared after a cisatracurium bolus. The diagnosis of tetanus was later confirmed by cultures from wound samples. Therefore, severe tetanus can mimic both the clinical and EEG features of status epilepticus and could be added to the differential diagnosis of epilepsy.

Investigations of sex differences in the human brain take place on politically sensitive terrain. While some scholars express concern that gendered biases and stereotypes remain embedded in scientific research, others are alarmed about the politicization of science. To help better understand these debates, this review sets out three kinds of conflicts that can arise in the neuroscience of sex differences: academic freedom versus gender equality; frameworks, background assumptions, and dominant methodologies; and inductive risk and social values. The boundaries between fair criticism and politicization are explored for each kind of conflict, pointing to ways in which the academic community can facilitate fair criticism while protecting against politicization.

Laminin-alpha2-related muscular dystrophy (LAMA2-MD) is a genetic condition due to reduced LAMA2, a protein found throughout the nervous system. Late-onset LAMA2-MD may present with proximal muscle weakness, joint contractures, neuropathy, epilepsy and/or cardiorespiratory issues, and is less common than the neonatal form. We describe a novel phenotype of LAMA2-MD with progressive myelopathy and spinal cord abnormalities.A woman was referred for evaluation of multiple sclerosis (MS) with progressive gait difficulty and abnormal neuroimaging showing white matter abnormalities in the brain and spinal cord. Ancillary testing was not consistent with primary neuroinflammatory disorders, systemic autoimmunity or infection. Metabolic workup revealed low cyanocobalamin. Genetic testing identified two LAMA2-MD variants.Genetic disorders can mimic treatable neurological conditions. Chronic progressive course, involvement of the peripheral and central nervous systems, and confluent white matter abnormalities should be investigated with molecular testing that includes LAMA2 sequencing to ensure proper diagnosis and management.