Novel therapeutic strategies in NBIA: A gene therapy approach for PLA2G6-associated neurodegeneration

Infantile neuroaxonal dystrophy (INAD) is a debilitating, intractable and ultimately lethal neurodegenerative disorder. It is caused by mutations in the PLA2G6 gene which encodes for phospholipase A2. INAD patients present neurodegeneration-associated symptoms between six months and three years of age. Severe spasticity, progressive cognitive decline, and visual impairment typically result in death during the first decade (Morgan et al, 2006). There is no disease-modifying treatment available and palliative care focuses on quality of life. Therefore, there is an overwhelming need to develop novel therapies to treat INAD patients. To create a landscape of the behavioural and pathological deficits, we aim to first conduct an in-depth characterization of the PLA2G6 mouse model developed by Wada et al (2009). Additionally, we aim to develop an AAV-mediated gene therapy approach for the treatment of INAD and conduct a pre-clinical study in the pla2g6-inad mouse model. The objective is to be able to prevent or ameliorate both the central and peripheral nervous system phenotype and improve the lifespan and/or quality of life of the animal. Recombinant adeno-associated virus serotype 9 vector (AAV9) will be used to deliver the therapeutic human PLA2G6 gene to the neonatal pla2g6-inad mouse. The strong neuron specific synapsin-I promoter will drive the human PLA2G6 gene. The efficacy of different administration routes including intracerebroventricular (ICV), intravenous (IV) and a combination of intracerebroventricular (ICV)/ intravenous (IV) and intracerbroventricular (ICV)/intraperioteneal (IP) will be investigated in the pla2g6-inad mouse model. AAV9-hSyn1-hPLA2G6 gene therapy treated pla2g6-inad mice showed an increased lifespan with the largest improvements observed in the animal cohort that received a combined administration of AAV9-hPLA2G6. The significant increase in lifespan supplemented with significant improvements in behavioural tests validates the potential beneficial use of gene therapy for infantile neuroaxonal dystrophy (INAD)

Tilstedeværelsen av en akutt fase-reaksjon hos lam med eksperimentell klassisk skrapesjuke indikerer et skifte mot en pro-inflammatorisk tilstand i det kliniske endestadiet

Classical scrapie in sheep is a transmissible and fatal neurodegenerative disease caused by the self-replicating and infectious prion protein, PrPSc, which is a conformational variant of the normal cellular prion protein, PrPC. The prion protein is a highly conserved glycoprotein encoded by the PRNP gene and therefore within the same host both PrPC and PRPSC have the same unique amino acid sequence and they only differ in their three-dimensional folded structure. Specific mutations at codons 136, 154 and 171 of the PRNP gene leads to single amino acid substitutions, and the most common polymorphisms give rise to five possible alleles and 15 PRNP genotypes found in sheep. The different alleles are highly associated with levels of susceptibility to classical scrapie, where A136R154R171 allele provides high genetic resistance and V136R154Q171 allele results in highly susceptible animals. On the basis of this association between PRNP genotype and susceptibility, many EU MSs have implemented national breeding for resistance programme with the aim of increasing distribution of ARR allele and reducing the distribution of VRQ allele. For almost 20 years, the EU TSE regulation has required surveillance within each country to establish prevalence of prion diseases and the different PRNP genotypes. Classical scrapie has a widespread distribution and incidence rate fluctuates due to the complex interaction between prion and host factors, and prevalence can only be estimated by ante mortem testing through active and passive surveillance. Transmission between sheep occurs through direct and indirect contact, and PrPSc can remain infective in the environment for years. The most common route of infection is the oral route, and infected animals can excrete PrPSc through foetal membranes and fluids, saliva, urine, faeces, and milk. Pathogenesis is highly influenced by PRNP genotype, as animals of the most susceptible genotypes have the most effective uptake of PrPSc across small intestine followed by an extensive dissemination and involvement of the SLOs, and an early neuroinvasion with spread of PrPSc within the CNS. The susceptible genotypes will contribute the most to spread of infectivity and environmental contamination. This work describes the results from experimental classical scrapie where homozygous VRQ lambs were inoculated orally at birth with homogenated brain material from either healthy sheep or from natural cases of classical scrapie. This resulted in a worst-case scenario type of classical scrapie with sudden onset of severe clinical signs at 22 wpi followed by a rapid deterioration and euthanasia at 23 wpi. Serum samples were collected at regular intervals and tissue samples from brain and liver were sampled at post mortem examination. Proteomic examinations of serum revealed a downregulation of several protein peaks during the pre-symptomatic incubation period in the scrapie affected group compared to the control group, and a shift to upregulation of protein peaks onwards from 22 wpi. Genomic examinations of serum samples showed a slight downregulation IL1B and TLR4 at 16 wpi, followed by a change at 22 wpi with upregulation of genes encoding TLRs, C3 and APPs. Genomic examination of liver and brain tissues showed an alteration in gene expression of APPs in accordance with an APR. Serum analyses of different APPs showed increased levels of the positive APPs and a reduced concentration of negative APPs. These findings are indicative of a shift from anti-inflammatory to pro-inflammatory systemic innate immune response that coincide with the onset of debilitating clinical disease. In neurodegenerative diseases, the innate immune response in the CNS has a key role in both onset and progression of disease and resolution of inflammation. The accumulation of PrPSc in the CNS has been associated with a chronic activation of the innate immune response, pro-inflammatory activation of microglia, neuroinflammation, and neurodegeneration. The diseases phenotype registered in this work is a result of PRNP genotype, and time and dose of inoculation, which can occur naturally if the right circumstances are in place. New-born homozygous VRQ lambs from an infected dam can get infected at birth. These cases could develop a similar disease progression as described in this work, resulting in an efficient and fast uptake and widespread peripheral and central dissemination of PrPSc, and clinical disease at a young age. These cases would present as a diagnostic challenge and easily missed as classical scrapie. Due to their young age, these cases would not be sampled through active surveillance. If incubation period extends commercial lifespan, these lambs would be slaughtered for human consumption, and due to their PRNP genotype, prions would enter the food chain. Control of classical scrapie can probably not be achieved by absence of infectivity, but absence of clinical disease is possible through breeding for resistance which will provide flock immunity to classical scrapie.Klassisk skrapesyke hos sau er en overførbar og dødelig nevrodegenerativ sykdom forårsaket av det selvrepliserende og smittsomme prionproteinet, PrPSc, som er en variant av det normale cellulære prionproteinet, PrPC. Prionproteinet er et glykoprotein som er kodet for av PRNP-genet. Dette betyr at PrPC og PRPSC hos samme verten, har den samme unike aminosyresekvensen og det er kun den tredimensjonale strukturen som skiller dem. Spesifikke mutasjoner ved kodonene 136, 154 og 171 i PRNP-genet fører til substitusjoner av enkelte aminosyrer, og de vanligste polymorfismer gir opphav til fem mulige alleler, og 15 PRNP-genotyper hos sau. De forskjellige allelene er assosiert med nivå av mottakelighet for klassisk skrapesyke, og A136R154R171-allel fører til genetisk resistens, og V136R154Q171-allel gir høy mottagelighet. På bakgrunn av denne sammenhengen mellom PRNP-genotype og mottakelighet, har mange EU medlemsland innført nasjonale avlsprogram som har mål om å øke utbredelsen av ARR-allel, og samtidig en reduksjon av VRQ-allel. I snart 20 år har EUs TSE-regelverk krevd nasjonale overvåkingsprogram for å bestemme forekomsten av prionsykdommer og kartlegge utbredelsen av de forskjellige PRNP-genotypene. Klassisk skrapesyke er utbredt, men forekomsten vil variere med bakgrunn i det komplekse samspillet mellom prionprotein og vertsfaktorer. Prevalens kan estimeres gjennom ante mortem testing i forbindelse med aktivt og passivt overvåkingsprogram. Smitteoverføring mellom sau skjer ved direkte og indirekte kontakt, og PrPSc er smittsomt i flere år i miljøet. Den vanligste infeksjonsveien er gjennom oralt inntak, og dyr kan skille ut smittsomt PrPSc via fosterhinner og væsker, spytt, urin, feces og melk, og nivå er styrt av PRNP genotype.Research Council of Norwa

Towards a mechanistic understanding of how age increases susceptibility to inflammation induced behavioural and cognitive disturbance

Accumulating evidence implicates inflammation and the innate immune system in the pathophysiology of age-related cognitive decline and neurodegenerative disorders such as Alzheimer’s disease. However, we have a limited understanding of the mechanisms by which age may increase the susceptibility to the behavioural and cognitive impairments associated with inflammation. In this thesis, I developed and validated a new experimental model of mild acute inflammation (IFN-β injection) and integrated it with two different MRI methodologies, behavioural, cognitive, physiological and immunology data to investigate how age modulates the effects of inflammation on the brain. To achieve this, healthy young and old participants underwent two experimental sessions in which they each received IFN-β-1b (EXTAVIA® [100 μg]) and saline in randomised order. Two neuroimaging techniques were used to assess actions on the brain. 1) Resting-state functional magnetic resonance (rsfMRI) which was used to investigate functional connectivity architecture with a particular focus on the efficiency of information transfer. 2) Diffusion-Weighted Magnetic Resonance Spectroscopy (DW-MRS) which was used to explore whether this novel MRI-based method was sensitive to detecting changes in glial cells using a model of mild inflammation. Fatigue, sickness and mood questionnaires, as well as a battery of cognitive tasks (reward/punishment reinforcement learning task, psychomotor retardation and visuospatial memory tasks), were used to assess behavioural and cognitive changes. Physiological monitoring and serial blood draws were used to assess physiological and immunological responses. Key findings include demonstrating that: 1) IFN-β is a safe and robust new experimental model of mild acute inflammation, as shown by the changes observed in the physiological, immune and behavioural responses. 2) That DW-MRS is sufficiently sensitive to detect changes in glial morphometry induced by a model of mild inflammation (IFN-β). 3) That DW-MRS is sensitive to previously reported age-related differences in glial and neuronal densities and glial morphometry. 4) That IFN-β altered global brain functional connectivity architecture (rsfMRI data). Here, IFN-β particularly affected highly connected nodes (as has been reported in Alzheimer’s disease) and, the intensity of the effect varied with respect to age. Together, these data provide support for IFN-β as a model of mild acute inflammation and confirm the effectiveness of employing non-invasive imaging methods, in conjunction with a range of behavioural and cognitive tasks to investigate the impact of experimentally induced neuroinflammation in young and old individuals

Examining the Acute and Chronic Effects of Sepsis on the Circadian Clock in the Mouse

Circadian rhythms are recurring patterns (~24hrs) in behaviour and physiology that are driven primarily by an endogenous biological timekeeping system, with the master pacemaker located in the suprachiasmatic nucleus. Studies have indicated bidirectional relationships between the circadian and the immune systems, however while there is much evidence regarding the regulation of immune function by the circadian system, information regarding the impact of immune processes on the timekeeping system is more limited, including that regarding the long-term modulation of the circadian system following immune challenge. The current set of studies address this gap in the literature by examining the long-term impact of sepsis, a substantial immune challenge, on circadian timekeeping processes, following sepsis induction by peripheral treatment with lipopolysaccharide (5mg/kg). Following recovery, post-septic circadian behaviour, SCN molecular oscillations and SCN responsiveness were assessed. SCN neurochemistry was also assessed both in the acute phase and in the long-term post LPS treatment. LPS induced sepsis did not affect core circadian locomotor rhythmicity parameters, but did result in long-term attenuations in post-septic resetting in response to phase advancing photic stimulation, and alterations in re-entrainment to advances of the photoperiod. Perturbations were observed in SCN neurochemistry in the acute phase following septic LPS treatment, and chronic attenuations were also found in post-septic SCN clock gene protein product expression. LPS induced sepsis caused attenuations in SCN functional activation in response to both photic and immune stimulation, as well as alterations in circadian resetting in response to phase resetting immune stimuli. Overall, these data provide further insight into immune circadian communication, and the long-term impact of immune challenge on timekeeping processes, and describe a previously unknown impact of the chronic effects of experimental sepsis on the circadian timekeeping system

Kainic acid-induced seizures: inflammation and excitotoxic neuronal damage in the developing rat hippocampus

Epileptic seizures are harmful to the developing brain. During epileptic seizures, overactivation of glutamate receptors (GluR) leads to neuronal degeneration, defined as excitotoxicity. The hippocampus is especially vulnerable to excitotoxic neuronal death, but its mechanism has remained incompletely known in the developing brain. Recently, signs of activation of inflammatory processes after epileptic seizures have been detected in the hippocampus. The purpose of this thesis was to study the inflammatory reaction and death mechanisms in excitoxic neurodegeneration induced by the glutamate analogue kainic acid (KA) in the developing hippocampus. Organotypic hippocampal slice cultures (OHCs), prepared from 6-7-day-old rats (P6-7) and treated with KA, served as an in vitro model. KA-induced status epilepticus in P9 and P21 rats was used as an in vivo model. The results showed that the pyramidal cell layers of the hippocampus were the most susceptible to irreversible and age-specific neurodegeneration, which occurred in the juvenile (P21), but not in the immature (P9), rat hippocampus. The primary death mechanism was necrosis as there were no significant changes in the expression of selected apoptosis markers and morphological cellular features of necrosis were found. Inflammatory response was similarly age-dependent after KA treatment as a rapid, fulminant and wide response was detected in the juvenile, but not in the immature, rat brain. An anti-inflammatory drug treatment, given before KA, was not neuroprotective in OHCs, possibly because of the timing of the treatment. In summary, the results suggest that KA induces an age-dependent inflammatory response and necrotic neurodegeneration, which may cause disturbances in hippocampal connectivity and promote epileptogenesis.Siirretty Doriast

The Formyl Peptide Receptor 2 Regulates Microglial Phenotype Through Immunometabolism: Implications for Alzheimer’s Disease

Microglia are key players in the pathology of Alzheimer’s disease (AD), driving chronic inflammation, oxidative stress, and the altered metabolism seen in the brains of patients. With clinical trials continuing to fail, new approaches towards drug development are critical. Strategies to reduce microglial activation may therefore be a viable therapeutic approach to tackling AD. Formyl peptide receptor 2 (Fpr2), which drives peripheral inflammatory resolution, is expressed in microglia. However, its functional role in neuroinflammation is unclear. This thesis provides evidence to support the peripheral findings of Fpr2 stimulation, wherein it may also hold promise for exploitation as a therapeutic for neurodegenerative disorders, including AD. We also highlight novel findings surrounding the modulation of both oxidative stress and microglial metabolism associated with Fpr2 activation. Under inflammatory conditions, we report that selective agonists for Fpr2 modulate the microglial inflammatory response, actively shifting from a pro-inflammatory to a pro-resolving phenotype, emphasised by the reduction of pro-inflammatory cytokines and concomitant increases in both pro-resolving cytokines and phagocytosis. Metabolic shifting away from glycolysis was also observed for pro-resolving microglia. Moreover, we describe for the first time that Fpr2 completely reverses reactive oxygen species (ROS) production from the mitochondria and NADPH oxidase enzymes following an inflammatory stimulus. We also highlight that the toxic oligomeric amyloid (oAβ) facilitates microglial ROS production and subsequent metabolic changes without triggering an inflammatory response. oAβ facilitated NADPH oxidase activation, which in turn resulted in the activation of glucose 6-phosphate dehydrogenase (G6PD), the rate limiting step for the pentose phosphate pathway. This metabolic pathway is responsible for producing NADPH, which in turn NADPH oxidases exploit for further ROS production. These changes resulted in noticeable reductions in both microglial glycolysis and oxidative phosphorylation. We present data underlining that Fpr2/3 stimulation reverses oAβ-induced ROS production, with a resultant reduction in G6PD activity and the return of homeostatic glycolysis. These oAβ-induced microglial changes triggered the apoptosis of SH-SY5Y cells in co-culture with BV-2 microglia. However, supporting our interest in Fpr2/3 for therapeutic approaches to neurodegenerative diseases, post-treatment with a select agonist for the receptor successfully prevented apoptosis of these neuronal like SH-SY5Y cells. This original data unveils novel functions of Fpr2/3 in the central nervous system (CNS), supplementing the well-established pro-resolving functions the receptor facilitates within the periphery. The combination of pro-resolving, anti-oxidative, immunometabolic and anti-apoptotic functions of Fpr2/3 support the exploitation of this receptor for therapeutic research into multiple different CNS disorders, including AD

The microbiota-gut-brain axis: from dysbiosis to neurodegenerative disease

Treballs Finals de Grau de Farmàcia, Facultat de Farmàcia i Ciències de l'Alimentació, Universitat de Barcelona, 2022. Tutor/a: Raquel Martín VenegasThe relation between the gut and the brain has been discussed for years, but the concept of gut microbiota (GM) has not been linked for so long. Having suggested its involvement in neurodegenerative disorders, this literature review aims to summarise the current knowledge on the possible bidirectional pathways that could explain it, while discussing the composition and alterations of the GM and the main pathological features that characterise neurodegeneration. It also presents the existing scientific evidence on the use of GM as a therapeutic target, accompanied by a systematic review-meta-analysis that evaluates the indication of one of the possible interventions: the faecal microbiota transplantation (FMT). GM is made up of the trillions of microorganisms in the gut, mostly bacteria, which interact dynamically with the host, contributing to both health and disease. These, through neural, endocrine and immune pathways, based on mechanisms that require further research, can modify the gut-brain axis promoting neurodegenerative processes such as neuroinflammation, protein misfolding and loss of integrity of the intestinal and blood-brain barriers, thus facilitating the passage of components derived from a deregulated GM that has been characterised in Alzheimer's and Parkinson's patients. Although more studies are needed, the promising results obtained in animal and more limited human trials, using GM modulating interventions such as oral bacteriotherapy or FMT, give hope for the cure and early detection of these increasingly prevalent diseases