Age-dependent hippocampal network dysfuntion in a mouse model of alpha-synucleinopathy

PhD ThesisAggregation of the protein alpha-synuclein (ASYN) is a key pathological feature of the alpha-synucleinopathies, a group of diseases including Lewy body dementia and Parkinson’s disease. A common symptom of alpha-synucleinopathy is cognitive dysfunction, and impairment in hippocampal gamma-frequency oscillations may underlie some of the cognitive deficits associated with ASYN pathology. The Thy-1 A30P mouse model overexpresses human mutant ASYN, with mice developing hippocampal spatial memory impairment by 12 months (Freichel et al. 2007). The aim of this thesis was to explore age-related hippocampal network changes in 2-4 month (A30P2+) mice and 10-13 month (A30P10+) mice to assess the effect of overexpression of mutant ASYN on hippocampal network activity in vitro. Using acute brain slice preparations of isolated hippocampi, A30P2+ mouse slices were found to exhibit excitatory/inhibitory network changes in region CA3 in the form of increased spontaneous sharp wave amplitude, increased frequency and amplitude of inhibitory postsynaptic potentials, and increased power of kainate-induced gamma-frequency oscillations. Immunohistochemistry revealed an increase in the density of parvalbuminpositive interneurons alongside a decrease in calbindin-positive interneurons. This change was accompanied by a more depolarised resting membrane potential in A30P2+ mouse CA3 pyramidal cells, and a sensitivity to interictal discharges in response to either kainate receptor agonism or GABAA receptor antagonism. With ageing, levels of excitability in A30P10+ mice were comparable to WT10+ mice. A30P10+ mice instead exhibited an impairment in cholinergic-induced, but not kainateinduced or spontaneous, gamma-frequency network oscillations. While mitochondrial dysfunction was not detectable with COX/SDH histochemistry until 15+ months in A30P mice, A30P10+ mice did show increased immunoreactivity for Iba1+ microglia. An environment of inflammation and excitotoxicity may be present in older A30P mice as a result of early network hyperexcitability, and this thesis explores early network changes in A30P mice and the wider dysfunction that followsEisai and the Medical Research Council for funding the MRC Industrial CASE Studentship

Protective effect of PDE4B subtype-specific inhibition in an App knock-in mouse model for Alzheimer's disease

Meta-analysis of genome-wide association study data has implicated PDE4B in the pathogenesis of Alzheimer's disease (AD), the leading cause of senile dementia. PDE4B encodes one of four subtypes of cyclic adenosine monophosphate (cAMP)-specific phosphodiesterase-4 (PDE4A-D). To interrogate the involvement of PDE4B in the manifestation of AD-related phenotypes, the effects of a hypomorphic mutation (Pde4bY358C) that decreases PDE4B's cAMP hydrolytic activity were evaluated in the AppNL-G-F knock-in mouse model of AD using the Barnes maze test of spatial memory, 14C-2-deoxyglucose autoradiography, thioflavin-S staining of β-amyloid (Aβ) plaques, and inflammatory marker assay and transcriptomic analysis (RNA sequencing) of cerebral cortical tissue. At 12 months of age, AppNL-G-F mice exhibited spatial memory and brain metabolism deficits, which were prevented by the hypomorphic PDE4B in AppNL-G-F/Pde4bY358C mice, without a decrease in Aβ plaque burden. RNA sequencing revealed that, among the 531 transcripts differentially expressed in AppNL-G-F versus wild-type mice, only 13 transcripts from four genes - Ide, Btaf1, Padi2, and C1qb - were differentially expressed in AppNL-G-F/Pde4bY358C versus AppNL-G-F mice, identifying their potential involvement in the protective effect of hypomorphic PDE4B. Our data demonstrate that spatial memory and cerebral glucose metabolism deficits exhibited by 12-month-old AppNL-G-F mice are prevented by targeted inhibition of PDE4B. To our knowledge, this is the first demonstration of a protective effect of PDE4B subtype-specific inhibition in a preclinical model of AD. It thus identifies PDE4B as a key regulator of disease manifestation in the AppNL-G-F model and a promising therapeutic target for AD

Mitochondrial electron transport chain defects modify Parkinson's disease phenotypes in a Drosophila model

Mitochondrial defects have been implicated in Parkinson's disease (PD) since complex I poisons were found to cause accelerated parkinsonism in young people in the early 1980s. More evidence of mitochondrial involvement arose when many of the genes whose mutations caused inherited PD were discovered to be subcellularly localized to mitochondria or have mitochondrial functions. However, the details of how mitochondrial dysfunction might impact or cause PD remain unclear. The aim of our study was to better understand mitochondrial dysfunction in PD by evaluating mitochondrial respiratory complex mutations in a Drosophila melanogaster (fruit fly) model of PD. We have conducted a targeted heterozygous enhancer/suppressor screen using Drosophila mutations within mitochondrial electron transport chain (ETC) genes against a null PD mutation in parkin. The interactions were assessed by climbing assays at 2-5 days as an indicator of motor function. A strong enhancer mutation in COX5A was examined further for L-dopa rescue, oxygen consumption, mitochondrial content, and reactive oxygen species. A later timepoint of 16-20 days was also investigated for both COX5A and a suppressor mutation in cyclope. Generalized Linear Models and similar statistical tests were used to verify significance of the findings. We have discovered that mutations in individual genes for subunits within the mitochondrial respiratory complexes have interactions with parkin, while others do not, irrespective of complex. One intriguing mutation in a complex IV subunit (cyclope) shows a suppressor rescue effect at early time points, improving the gross motor defects caused by the PD mutation, providing a strong candidate for drug discovery. Most mutations, however, show varying degrees of enhancement or slight suppression of the PD phenotypes. Thus, individual mitochondrial mutations within different oxidative phosphorylation complexes have different interactions with PD with regard to degree and direction. Upon further investigation of the strongest enhancer (COX5A), the mechanism by which these interactions occur initially does not appear to be based on defects in ATP production, but rather may be related to increased levels of reactive oxygen species. Our work highlights some key subunits potentially involved in mechanisms underlying PD pathogenesis, implicating ETC complexes other than complex I in PD. [Abstract copyright: Copyright © 2022. Published by Elsevier Inc.