Comparative genome analysis in rodent models of Parkinson’s disease and spinocerebellar

ataxia type 3

by

Nivretta Thatra

B.S. Neurobiology, The University of Washington, 2014

A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF

THE REQUIREMENTS FOR THE DEGREE OF

MASTER OF SCIENCE

in

THE FACULTY OF GRADUATE AND POSTDOCTORAL STUDIES

(Bioinformatics)

THE UNIVERSITY OF BRITISH COLUMBIA

(Vancouver)

May 2019

© Nivretta Thatra, 2019

The following individuals certify that they have read, and recommend to the Faculty of Graduate and Postdoctoral Studies for acceptance, a thesis/dissertation entitled:

Comparative genome analysis in rodent models of Parkinson’s disease and spinocerebellar ataxia type 3

submitted by Nivretta Thatra in partial fulfillment of the requirements for the degree of Master of Science in Bioinformatics

Examining Committee:

Dr. Jörg Gsponer Co-supervisor

Dr. Paul Pavlidis Co-supervisor

Dr. Sara Mostafavi Supervisory Committee Member

Dr. Weihong Song Additional Examiner

Additional Supervisory Committee Members:

Dr. Martin Hirst Supervisory Committee Member

Supervisory Committee Member

ii

Abstract

The shared hallmarks of neurodegenerative diseases (NDs) – notably, the existence of protein deposits,1 selective vulnerability of cell types,2 chronic neuroimmune response,3 and early dysfunction in brain vasculature4 – support the idea of studying different transgenic models relevant to NDs in concert rather than separately. Indeed, transgenic models of different NDs, namely Parkinson’s disease (PD) and spinocerebellar ataxia type 3 (SCA3), show comparable behavioral abnormalities and some similarities in cell loss. In this project, we hypothesized the reflection of these previously characterized similarities at the transcriptomic level in transgenic models of PD and SCA3, and that prioritizing overlaps in gene expression across transgenic models might allow the identification of genes that are involved in pathological pathways relevant to more than one ND. I show in an unpublished dataset of rodent transcriptomes from two time points and up to three brain regions that most overlaps in gene expression patterns are specific both to the brain region and time point from which samples are obtained. Overlaps in gene expression are found between transgenic models that study the effects of the same gene, synuclein alpha (Snca). In examining the overlaps of the interpretations of gene expression via cell type proportional estimations and functional analysis, I find commonalities across models suggesting changes in endothelial cells at the earlier time point and oligodendrocytes at the later time point.

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Lay Summary

Many neurodegenerative diseases (NDs) share common features, like the death of specific brain cells and protein deposits in brain cells. With therapeutic aims, researchers hope to find genes associated with these protein deposits or dying brain cells that can be targeted by drugs to help those with NDs. Since brain tissue from humans is difficult to obtain, many investigations are conducted with animal models that are relevant to human disease. In this project, I looked for gene overlaps in rodent models that are relevant to two human NDs (Parkinson’s disease and spinocerebellar ataxia type 3). I show that most overlaps in gene expression occur when the rodent models are related to each other. Otherwise, overlaps in gene expression seem to be associated with changes in brain cell type proportion estimations, perhaps due to cell death.

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Preface

The research initiative presented in this paper – to compare different transgenic models of neurodegenerative diseases – was originally proposed in the NeuroGEM project, which both my supervisors Dr. Jörg Gsponer and Dr. Pavlidis are a part of. Our collaborator and NeuroGEM member Dr. Olaf Riess of The University of Tübingen was responsible for overseeing collection of the data by members of his group before it was sent our way. Following data transfer, I was responsible for the analysis of the data presented here. I wrote this thesis with suggestions from

Drs. Gsponer and Pavlidis. All chapters and results are as yet unpublished, though manuscripts are planned.

v

Table of Contents

Abstract ...... iii

Lay Summary ...... iv

Preface ...... v

Table of Contents ...... vi

List of Tables ...... xii

List of Figures ...... xiii

List of Abbreviations ...... xiv

Acknowledgements ...... xv

Dedication ...... xvi

Chapter 1: Introduction ...... 1

1.1 Parkinson’s disease ...... 2

1.1.1 SNCA ...... 3

1.1.2 Aggregation and toxicity of α-synuclein ...... 4

1.2 Spinocerebellar ataxia type 3 ...... 5

1.2.1 ATXN3 ...... 5

1.2.2 Aggregation and toxicity of ataxin-3 ...... 5

1.3 Overlapping pathways in neurodegenerative diseases ...... 6

1.3.1 Aggregation...... 7

1.3.2 Specific cell types ...... 8

1.3.2.1 Cell types in PD ...... 9

1.3.2.2 Cell types in SCA3...... 10 vi

1.3.2.3 Cell type proportion estimation ...... 10

1.3.3 Microglia and inflammation ...... 11

1.3.4 Parkinsonism and levodopa-responsive PD in SCA3 ...... 12

1.4 Current state of rodent models of NDs ...... 13

1.4.1 Current state of rodent models of PD ...... 14

1.4.2 SNCA overexpression rat model relevant to PD ...... 15

1.4.3 Snca KO mouse model relevant to PD ...... 16

1.4.4 Snca KO and SNCA overexpression mouse model relevant to PD ...... 16

1.4.5 Current state of rodent models of SCA3 ...... 17

1.4.6 Mutant ATXN3 model of SCA3 ...... 18

1.5 Transcriptional analyses...... 18

1.5.1 Transcriptional analyses in rodent models of PD ...... 19

1.5.2 Transcriptional analyses in rodent models of SCA3...... 19

1.6 Methods of assessing overlaps of gene lists ...... 20

1.7 Aims ...... 20

Chapter 2: Materials and methods ...... 22

2.1 Data ...... 22

2.2 Data quality control and pre-processing ...... 24

2.3 Fitting linear models ...... 27

2.3.1 Sub-setting the data by age ...... 27

2.3.2 Sub-setting the data by age and tissue ...... 28

2.4 Functional enrichment ...... 29

2.5 Overlaps ...... 30 vii

2.6 Estimating cell type proportions ...... 30

Chapter 3: Results...... 32

3.1 Results overview ...... 32

3.2 Differential expression overview ...... 33

3.2.1 Differential expression in SCA3-84q...... 35

3.2.2 Differential expression in PD-ovx ...... 35

3.2.3 Differential expression in PD-ko ...... 36

3.2.4 Differential expression in PD-ko+ovx ...... 36

3.3 Gene-to-gene overlaps ...... 37

3.4 Differential expression in PD-ko+ovx in comparison to PD-ko ...... 40

3.5 Cell types analysis...... 40

3.6 Overlaps in cell type proportional changes ...... 41

3.7 Functional enrichment overview...... 43

3.7.1 Functional enrichment in SCA3-84q ...... 43

3.7.2 Functional enrichment in PD-ovx ...... 44

3.7.3 Functional enrichment in PD-ko ...... 45

3.7.4 Functional enrichment in PD-ko+ovx ...... 45

3.8 Functional enrichment overlaps ...... 45

Chapter 4: Discussion and conclusions ...... 47

4.1 SCA3-84q differential expression results in context of existing literature on transgenic

lines …………………………………………………………………………………………47

4.2 Overlap between PD-ko and PD-ko+ovx transgenic models ...... 49

viii

4.3 MGPs of endothelial cells and oligodendrocytes show proportional changes in three

models...... 50

4.4 Overlaps in functional enrichment ...... 51

4.5 Limitations and future work...... 52

4.6 Conclusions ...... 53

Bibliography ...... 55

Appendices ...... 65

Appendix A ...... 65

A.1 Upregulated genes (FDR = 0.05) in SCA3-84q at 2 months in cerebellum ...... 65

A.2 Downregulated genes (FDR = 0.05) in SCA3-84q at 2 months in cerebellum ...... 67

A.3 Upregulated genes (FDR = 0.05) in SCA3-84q at 12 months, common to all

tissues……………………………………………………………………………………….68

A.4 Upregulated genes (FDR = 0.05) in SCA3-84q at 12 months in striatum ...... 69

A.5 Downregulated genes (FDR = 0.05) in SCA3-84q at 12 months in striatum ...... 71

A.6 Upregulated genes (FDR = 0.05) in PD-ovx at 5 months, common to all tissues .... 72

A.7 Downregulated genes (FDR = 0.05) in PD-ovx at 5 months, common to all tissues 74

A.8 Upregulated genes (FDR = 0.05) in PD-ovx at 5 months in frontal cortex ...... 76

A.9 Downregulated genes (FDR = 0.05) in PD-ovx at 5 months in frontal cortex ...... 78

A.10 Upregulated genes (FDR = 0.05) in PD-ovx at 5 months in striatum ...... 80

A.11 Downregulated genes (FDR = 0.05) in PD-ovx at 5 months in striatum ...... 81

A.12 Upregulated genes (FDR = 0.05) in PD-ovx at 5 months in cerebellum ...... 83

A.13 Downregulated genes (FDR = 0.05) in PD-ovx at 5 months in cerebellum ...... 85

A.14 Upregulated genes (FDR = 0.05) in PD-ovx at 12 months, common to all tissues .. 87 ix

A.15 Downregulated genes (FDR = 0.05) in PD-ovx at 12 months, common to all tissues

………………………………………………………………………………………89

A.16 Upregulated genes (FDR = 0.05) in PD-ovx at 12 months in frontal cortex ...... 90

A.17 Downregulated genes (FDR = 0.05) in PD-ovx at 12 months in frontal cortex ...... 99

A.18 Upregulated genes (FDR = 0.05) in PD-ovx at 12 months in striatum ...... 105

A.19 Downregulated genes (FDR = 0.05) in PD-ovx at 12 months in striatum ...... 107

A.20 Downregulated genes (FDR = 0.05) in PD-ovx at 12 months in frontal cortex ..... 109

A.21 Upregulated genes (FDR = 0.05) in PD-ovx at 12 months in cerebellum ...... 110

A.22 Downregulated genes (FDR = 0.05) in PD-ovx at 12 months in cerebellum ...... 112

A.23 Upregulated genes (FDR = 0.05) in PD-ko at 2 months in frontal cortex ...... 114

A.24 Downregulated genes (FDR = 0.05) in PD-ko at 2 months in frontal cortex ...... 114

A.25 Upregulated genes (FDR = 0.05) in PD-ko at 2 months in striatum ...... 115

A.26 Downregulated genes (FDR = 0.05) in PD-ko at 2 months in striatum ...... 115

A.27 Upregulated genes (FDR = 0.05) in PD-ko at 12 months in frontal cortex ...... 115

A.28 Downregulated genes (FDR = 0.05) in PD-ko at 12 months in frontal cortex ...... 117

A.29 Upregulated genes (FDR = 0.05) in PD-ko+ovx at 2 months, common to all tissues

………………………………………………………………………………….…119

A.30 Downregulated genes (FDR = 0.05) in PD-ko+ovx at 2 months, common to all tissues ……………………………………………………………………………………..119

A.31 Upregulated genes (FDR = 0.05) in PD-ko+ovx at 2 months in frontal cortex ...... 120

A.32 Downregulated genes (FDR = 0.05) in PD-ko+ovx at 2 months in frontal cortex . 120

A.33 Downregulated genes (FDR = 0.05) in PD-ko+ovx at 2 months in cerebellum ..... 121

A.34 Upregulated genes (FDR = 0.05) in PD-ko+ovx at 12 months in frontal cortex .... 121 x

A.35 Downregulated genes (FDR = 0.05) in PD-ko+ovx at 12 months in frontal cortex

……………………………………………………………………………………..123

xi

List of Tables

Table 1 Summary of samples obtained from transgenic models and changes to data after pre- processing ...... 26

Table 2 Differential expression summary ...... 34

xii

List of Figures

Figure 1 Workflow summary ...... 22

Figure 2 Expression of transgenes in all four transgenic models is as expected ...... 34

Figure 3 Top 50 upregulated and downregulated genes in individual tissues ...... 35

Figure 4 Differential expression results in PD-ko are remarkably similar to PD-ko+ovx ...... 38

Figure 5 Highest correlation is between PD-ko and PD-ko+ovx (a) transgenic models ...... 38

Figure 6 Number of overlapping genes common across brain regions and within tissues show similar trends ...... 39

Figure 7 Summary of inferred cell type proportional changes between wildtype and transgenic models ...... 42

Figure 8 Marker gene profiles (MGPs) for oligodendrocytes in SCA3-84q mice ...... 42

Figure 9 Overlaps in functionally enriched GO terms at the 12-month time point ...... 46

Figure 10 Summary of differential expression in the context of model behavioural and physiological characteristics ...... 49

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List of Abbreviations

NDs – neurodegenerative diseases AD – amyotrophic lateral sclerosis HD – Huntington’s disease PD – Parkinson’s disease SCA – spinocerebellar ataxias SCA3 – spinocerebellar ataxia type 3 RNAseq – ribonucleic acid-sequencing SNCA – synuclein alpha LRRK2 – leucine rich repeat kinase 2 ATXN2 – ataxin 2 ATXN3 – ataxin 3 MAPT – microtubule associated protein tau GCH1 – guanosine triphosphaste cyclohydrolase 1 DCTN1 – dynactin subunit 1 VPS35 – vacuolar protein sorting-associated protein 35 PARK2 – Parkin PINK1 or PARK6 – PTEN induced kinase 1 CAG – cytosine-adenine-guanine DNA – deoxyribonucleic acid MGPs – marker gene profiles BAC – bacterial artificial chromosome PD-ovx – rat strain overexpressing human SNCA PD-ko – mouse strain with knock-out of endogenous Snca PD-ko+ovx – mouse strain overexpressing human SNCA in a background of endogenous Snca knocked out YAC – yeast artificial chromosome SCA3-84q/MJD84.2 – mouse strain expressing mutant human ATXN3 DE – differential expression or differentially expressed FASTQ – proper name for file format FASTQC – proper name for software BAM – proper name for file format STAR – Spliced Transcripts Alignment to a Reference RSEM – RNA-seq by expectation-maximization ANOVA – analysis of variance RIN – RNA integrity number GO – Gene Ontology ORA – over-representation analysis FDR – false discovery rate NADH – nicotinamide adenine dinucleotide MSA – multiple system atrophy PFFs – preformed fibrils

xiv

Acknowledgements

Unlike the trait of scientific brilliance, which is expected from tenured academics, defaulting to a critical skepticism regarding one’s own field is often a thankless self-initiated endeavor. For sharing with me their brilliance and realistic skepticism, I extend my gratitude to my supervisors Dr. Jörg Gsponer and Dr. Paul Pavlidis. Much of my growth in the field of bioinformatics is due to their mentorship. I also thank my colleagues in their labs, especially

Manuel Belmadani, who brought my wet-lab skills up to dry-lab speed. Additionally, I acknowledge the essential financial support I received from NSERC’s Collaborative Research and Training Experience Program and UBC’s Cordula and Gunter Paetzold Fellowship.

There is no hyperbole in saying that this work would not have been possible without the support of my family and friends. My family – Murlidharan Thatra, Usha Murlidharan, and

Vishruth Murlidharan – are consistently the wisest, talented, and ruthlessly kind people I know.

Thank you to two of my dearest friends, Addyson Frattura and Kara Jackson, for building me homes where we learned, together, how to perceive the kinds of words and sounds that “cleanse the doors of perception.” Finally, for the type of companionship that pithy remarks cannot capture, I thank A.K., A.C., A.C.B., C.P., F.B., G.U.P., J.K., M.C., M.M., N.M., S.R., W.B.,

W.V., and The Ubyssey 2017-2018 editorial board.

xv

Dedication

For my friend and teacher Dr. Tracy Larson

xvi

Chapter 1: Introduction

Many neurodegenerative diseases (NDs) – including Alzheimer’s disease (AD), amyotrophic lateral sclerosis, Huntington’s disease (HD), multiple system atrophy, Parkinson’s disease (PD) and the spinocerebellar ataxias (SCA) – share the common feature of intra- and extracellular protein deposits.1,5 These toxic deposits are caused by aggregation of misfolded proteins, and by the breakdown in the mechanisms that usually allow the cell to sequester and neutralize aberrantly folded proteins.6 Though these hallmarks of disease have been known for decades, generally, there are no cures or even dramatically beneficial therapeutics for many NDs.

As such, there is continued motivation to compare NDs and propose therapeutic strategies – for example, promoting autophagy with existing compounds like rapamycin to help the clearing of aggregated proteins7 – for a number of these diseases. Understandably, with therapeutic aims, the field has devoted substantial resources to the study of NDs through the use of next generation sequencing with the hopes of finding actionable proteins to target.

Most NDs are idiopathic in nature, illustrated by the fact that only five to ten per cent of

PD8 and less than one per cent of AD9 cases are thought to be inherited in a monogenic

Mendelian way. Still, animal models based on familial mutations are used in these studies of idiopathic forms of NDs, as a shared mechanism of disease in idiopathic and familial NDs is supported by almost identical clinical and pathological findings in the two forms.10 One method of prioritizing genes after sequencing in these analyses of transgenic models is by examining a single model alone. In this project, we hypothesized that the shared characteristic of aggregated proteins supports the prioritization of overlaps in gene expression across transgenic models. This might allow the identification of genes that are involved in pathological pathways relevant to more than one ND, namely PD and spinocerebellar ataxia type 3 (SCA3). To introduce these 1

topics and my aims, I first review the relevant background on PD and SCA3 pathology. Second,

I expand on the overlapping mechanisms in NDs generally as well as PD and SCA3 specifically, to further justify studying these diseases in concert. Third, I provide context for the transgenic models of NDs which I employ, and finally, review methods of transcriptional analysis with the use of RNA-sequencing (RNAseq) data.

1.1 Parkinson’s disease

Parkinson’s disease (PD) and the resulting motor symptoms of rigidity, bradykinesia, postural instability, and tremor are the subject of much research. These symptoms begin with the loss of dopaminergic neurons and the resulting depletion of dopamine in the substantia nigra pars compacta.11 In healthy brains these dopaminergic neurons contribute to fine motor control via excitation of the striatum.12 In PD brains the remaining neurons of the substantia nigra show positive immunostaining for Lewy bodies, which are cytoplastic inclusions that contain α- synuclein,11 ubiquitin, and over 300 other aggregated proteins.13 The majority of PD patients have idiopathic forms of the disease but inherited forms of this ND cannot be ignored as they are similarly debilitating.11 Some forms of idiopathic PD presentation are responsive to the drug levodopa14 – a precursor to neurotransmitters that are lacking in PD, such as dopamine – and these cases are termed levodopa-responsive PD. This introduction focuses on the protein α- synuclein and its coding gene synuclein alpha (SNCA) as they are key components in all PD cases, but there are, notably, other genes with clinical implications for PD: leucine rich repeat kinase 2 (LRRK2), ataxin 2 (ATXN2), ataxin 3 (ATXN3), microtubule associated protein tau (MAPT), guanosine triphosphaste cyclohydrolase 1 (GCH1), dynactin subunit 1

(DCTN1) and vacuolar protein sorting-associated protein 35 (VPS35).15

2

1.1.1 SNCA

Although pathological inclusions in PD were described outside the substantia nigra in

1912 – around the same time as the discovery of the degeneration of neurons in the substantia nigra – α-synuclein’s role as the main component of Lewy pathology was only discovered in the

1990s. A missense mutation (Ala53Thr or A53T) of the SNCA gene, which encodes α-synuclein and is found on chromosome four, was shown to cause autosomal dominant PD in 1997. Two other missense mutations in SNCA (Ala30Pro or A30P and Glu46Lys or E46K) were later discovered.16 Mutations in SNCA can, among other effects, lead to disruption of the axonal transport of α-synuclein,17 in turn causing its accumulation. Idiopathic cases further showed that there are six stages of α-synuclein progression. Pathological fibrils reach the substantia nigra by stage 3. Taken all together this work ingeniously linked familial and idiopathic PD: mutations in

SNCA can cause familial PD, and α-synuclein-containing Lewy bodies are found in all PD cases.

Indeed, recent genome-wide association studies of idiopathic PD show that sequence variants of the regulatory region of SNCA are the predominant genetic risk factor for idiopathic PD.16

Heterozygous duplications or triplications of SNCA, resulting in the overexpression of α- synuclein, were also identified in familial cases of PD; increased multiplication of the gene increases disease penetrance.16 Carriers of triplication tend to present with disease a decade sooner than those with duplication errors,15 suggesting that there could be a simple dose relationship between disease severity and levels of α-synuclein. Other well-studied genes associated with PD are PARK2 and LRRK2, respectively coding for the proteins parkin and leucine-rich repeat kinsase-2. Parkin is well studied, and is known to ubiquitinate a form of α- synuclein as well as interact with LRRK2.18 Parkin also interacts with the protein product of

3

PTEN induced kinase 1 (PINK1 or PARK6) in the removal of damaged mitochondria by mitophagy.19

1.1.2 Aggregation and toxicity of α-synuclein

What exactly makes Lewy bodies pathological, or whether they are pathological at all, is still an outstanding question. Especially intriguing is that the normal function of α-synuclein is unclear, though there are indications for its involvement in synaptic plasticity, the supply of synaptic vesicles, and the regulation of dopamine synthesis.17 The evidence for the deleterious effects of α-synuclein is also varied. Aggregates of α-synuclein are cytotoxic to neurons in vitro and are thereby assumed to be cytotoxic in the human brain, in cultured human cells α-synuclein induces the degeneration of dopaminergic neurons when dopamine is present,20 and there is evidence that α-synuclein protofibrils may themselves be cytotoxic.21 The protein was also found to sequester neurotransmitter synthetic enzymes in neurons and thus decrease the level of these key enzymes below basal levels.13

The gaps in knowledge of the exact mechanisms of α-synuclein toxicity leave some room to argue for the neuroprotective20 function of these inclusions. An increase in α-synuclein could simply be an adaptive response to toxic stimuli, as evidenced by an increase in α-synuclein expression in neurons exposed to oxidative stress. These neurons, compared to neurons lacking

α-synuclein, were relatively resistant to apoptotic changes. Moreover, in transgenic mice the overexpression of α-synuclein does not consistently result in neurodegeneration.20 Indeed, even in human cases, there is a poor correlation between Lewy bodies and neuronal loss in both early and late stages of the disease.22

4

1.2 Spinocerebellar ataxia type 3

Machado-Joseph disease or spinocerebellar ataxia type 3 (SCA3), the most common form of the spinocerebellar ataxias, is a disorder of motor coordination, affecting balance, gait, speech, and gaze. However, there are a range of other clinical presentations for SCA3, with some patients showing levodopa-responsive parkinsonism, along with non-motor symptoms including cognitive disturbances, dysfunctions in attention, and depressed mood. Alterations of the central nervous system are present in SCA3, consisting of neuronal degeneration especially in the dentate nucleus of the cerebellum, the substantia nigra, the nerve motor nuclei, and the spinal cord.23

1.2.1 ATXN3

SCA3 is caused by a mutation in a single gene – ATXN3 on chromosome 1424 – which results in the aberrant expansion of its protein product, ataxin-3. The mutation in ATXN3 qualifies SCA3 as a polyglutamine disease, a group of disorders that includes nine inherited NDs such as HD, and spinocerebellar ataxias 1, 2, 6, 7 and 17. These polyglutamine disease are all caused by expansions of cytosine‐adenine‐guanine (CAG) trinucleotides in the coding regions of single, and otherwise not linked, genes. The simple monogenic cause of SCA3 has been defined for decades, but the mechanisms resulting in neurodegeneration are as yet unidentified.

Polyglutamine repeat expansions are also found in the healthy population, with their CAG repeats being limited to 10 to 44, while SCA3 patients have 61 to 87 repeats. There is an inverse correlation between the number of CAG repeats and the age of onset of SCA3.23

1.2.2 Aggregation and toxicity of ataxin-3

The protein ataxin-3 in its most common form has 361 amino acids23 and is widely expressed in non-neural and neural human tissues.24 Its functions are claimed to be numerous; 5

various studies support its involvement in quality control via proteasome (protein degradation) pathways, regulation of the cytoskeleton, the regulation of transcription, and deoxyribonucleic acid (DNA) repair. Interestingly, ataxin-3 is likely related to the ubiquitin‐proteasome pathway.23

This is supported by evidence that ataxin-3 deubiquitinates parkin and other proteins directly.25

The aggregates of ataxin-3 – containing mutant and normal forms of the gene along with ubiquitin and other proteins – are intraneuronal inclusions that are mostly localized to the nucleus, but similar aggregates have been shown in the cytoplasm and axons of neurons in regions known to undergo neurodegeneration in SCA3. These aggregates can lead to toxic species that obstruct important cellular pathways: autophagy, transcription, and mitochondrial function. Still, the exact role of the inclusions remains nebulous. In sequestering functional proteins, the aggregates could be deleterious, or by retaining the pathogenic proteins, they could be neuroprotective.23

The occurrence of nuclear inclusions containing ataxin-3 are not associated with the distribution of neurodegeneration, nor are they associated with other variables of SCA3 patients such as length of CAG repeats and age at disease onset.26 Furthermore, in a recent neuropathological report of 12 genetically confirmed cases of SCA3, neither the presence nor the abundance of these intraneuronal inclusions of ataxin-3 correlated with the degree of tissue destruction.27 This suggests that the inclusions are secondary effects of disease; the aggregates themselves are perhaps not as toxic as the expanded form of ataxin-3, which itself could be causing damage to organelles in the cytoplasm of neurons.27

1.3 Overlapping pathways in neurodegenerative diseases

As much as the varying etiologies of NDs paint each disease as separate clinical entities that affect different brain regions resulting in distinct symptoms, there are patterns common to 6

many NDs. The aggregation of misfolded proteins,1 selective vulnerability of cell types,2 chronic neuroimmune response,3 and early dysfunction in brain vasculature4 are a few salient commonalities.

The commonalities between NDs support the idea of studying different transgenic models of diseases in concert rather than separately. In genomics approaches such as the ones I have employed in this thesis, genes can be prioritized by comparing across transgenic models, rather than within one specific strain. With increasing tissue and cell-type-specific expression studies of

NDs, the genetic loci identified within one ND have been used to identify other genes that show similar expression profiles.2 Since these genes map to particular biological pathways, and since these biological pathways are similar in more than one ND, there could also be as-yet unidentified genes that overlap between NDs. The common pathways could point to common pathological causes at pre-clinical stages, or common compensations in late stages of disease.

Below I will expand on these common pathways.

1.3.1 Aggregation

There are distinct proteins that aggregate in specific NDs: as described above, these are ataxin-3 in SCA3 and α-synuclein in PD. However, the processes of misfolding and aggregation are similar across illnesses such as PD, the spinocerebellar ataxias, AD, HD, frontotemporal dementia, and dementia with Lewy bodies. Disease-associated proteins all undergo abnormal folding to form intermolecular β-sheet-rich structures, which then cluster together in highly ordered aggregates known as amyloid. Amyloid is large and insoluble.1 One pathogenic result of these aggregates is their sequestration of molecular chaperones, which may disrupt normal protein biosynthesis and degradation. There is also evidence that aggregates provide a scaffold for procaspase oligomerization, causing a cascade of caspase-dependent events that can lead to 7

apoptosis.28 Moreover, a 2016 microarray analysis of 500 healthy brain samples was able to predict the tissue-specific spread of AD based on the expression pattern of genes that correspond to proteins that co-aggregate in amyloid plaques.29 The transcriptional analysis revealed the expression of these genes, even at ages before the onset of disease, in tissues that are known to be vulnerable in AD based on post-mortem detection of insoluble fibrils.29

Yet, given the histological evidence that aggregation does not correlate with neural toxicity in cases of SCA326,27 and PD,22 there is support for the idea that the formation of these aggregates is not itself pathogenic. Recent studies suggest that smaller soluble precursors to these large protein aggregates are the actual causes of neurodegeneration. The precursors are intermediary oligomers in the formation of amyloid, but can also be the final products of protein aggregation.1 The larger aggregates can themselves be toxic if they serve as reservoirs of the smaller oligomers.30 Thus far these misfolded precursors to amyloid have proven hard to capture for structural and mechanistic characterizations,31 and therefore there is a relative lack of knowledge of their relationship to disease.1 Overall, protein aggregation seems to symbolize the end of a molecular cascade of events, with earlier components of the cascade being more pathogenic than the aggregates themselves.

1.3.2 Specific cell types

Specific neuronal vulnerability is a hallmark of NDs. Somehow, in many of these diseases, degeneration is apparent in a set of cells in one area of the brain, while nearby cells of similar types are unaffected.32 Further indications of the cell type-specific phenotypes of NDs are the cell type-specific aggregation of proteins that normally show widespread expression.2

Though protein aggregation itself is not strictly correlated with neurodegeneration, the aggregates imply the existence of smaller toxic precursors. Thus the mechanisms causing 8

inclusions in specific cell types are still of relevance. A recent review of these mechanisms across NDs suggests that vulnerable neurons have intrinsic anatomy and biochemistry that allows their susceptibility to protein aggregation.2

1.3.2.1 Cell types in PD

In PD, about 90 per cent of the dopaminergic neurons of the ventral area of the substantia nigra degenerate, yet dopaminergic neurons of the nearby dorsal area only show a loss of about

25 per cent.33 In the ventral tegmental area, there is an even less loss of dopaminergic neurons.34

Other (non-dopaminergic) nuclei also show neuronal degeneration or pathological Lewy bodies.

For example, neuronal loss is exhibited by cholinergic neurons in the basal forebrain, and glutamatergic neurons in the intralaminar nuclei of the basolateral amygdala and the thalamus.2

Within the dorsal motor nucleus of the vagus, only cholinergic and catecholaminergic neurons are positive for Lewy bodies. Particularly interesting is that GABAergic neurons in all regions seem to be resistant to Lewy bodies. A possible explanation can be found within the view that the spread of Lewy bodies in PD is prion-like;5,35,36 in this hypothesis, environmental pathogens or infectious agents cause Lewy bodies, and these agents are transferred from synapse to synapse to other neurons.22 The widespread manifestation of prion diseases is unlike the confinement of

Lewy bodies to particular neurons, but there may be selective factors that presuppose some neurons to take-up Lewy bodies following their prion-like spread. There are numerous hypotheses37–39 as to why dopaminergic neurons and other brain nuclei in PD show heightened vulnerability, but the explanation with the largest amount of convincing experimental evidence is tied to the bioenergetic needs of dopaminergic neurons.22 There is now evidence that the highly branched axonal structure of vulnerable substantia nigra dopaminergic neurons demands higher levels of bioenergetic support in order to maintain electrical excitability, in comparison to less 9

vulnerable neurons of the ventral tegmental area. This, in turn, leads to mitochondrial oxidative stress. In fact, it has been shown that reducing the size of the axonal arbor decreases measured levels of this stress.40

1.3.2.2 Cell types in SCA3

Relatively less is known about selective neuronal vulnerability in SCA3, with most pathological reports focusing on nuclei-level rather than cell type-specific characterizations,23,24,26 but translatable knowledge can be gleaned from other polyglutamine diseases such as HD. In HD, the medium spiny GABAergic neurons of the striatum are selectively affected, while other striatal cells are in comparison spared from degeneration.32 As the disease advances, other striatal cells such as parvalbuminergic neurons also degenerate,41 though region-specific vulnerabilities continue well into disease progression.42 The specific biochemistry of HD neurons in the striatum that presupposes them to vulnerability is thought to be associated with glutamate excitotoxicity. Medium spiny neurons are known to highly express glutamate receptors, and it is believed that changes to the normal expression patterns of genes coding these receptors may underly the vulnerability of these neurons. Furthermore, bioenergetic requirements are a factor in HD vulnerability as with PD. In HD, the high energy demands of medium spiny neurons, which they require in order to remain hyperpolarized, is thought to be associated with their susceptibility to mitochondrial dysfunction.32

1.3.2.3 Cell type proportion estimation

Assessing the effect of NDs on specific cell types, and overlaps in these effects, is of relevance to this thesis as I hoped to investigate the cell type-specific transcriptomic effects in the available transgenic models; a traditional analysis of differential expression does not account for changes in cell type proportions, and thus shows overall average gene expression for all cell 10

types present in their respective tissues. Furthermore, the datasets used are from specific brain regions without any available histological characterization of specific cell types present.

However, methods of estimating cell types in transcriptomic data have been developed with the aid of the discovery of genes which are expressed in a specific set of cells.43 The expression pattern of these known marker genes can be used by computational methods to estimate cell type-specific proportion changes in RNAseq data.44,45 In this thesis, I used the analysis approach developed in Mancarci et al. (2017),46 which uses marker gene profiles (MGPs) as proxies for relative cell type abundance in each sample. MGPs are collections of genes pooled from existing literature, which results in about 15 to 100 markers for each cell type. By summarizing the expression of marker genes as the first principal component of their expression level, an MGP is obtained for each cell type. MGPs can then serve as estimations of cell type proportional increases and decreases across samples.

1.3.3 Microglia and inflammation

The role of microglia in neurodegeneration is a recurring pattern in NDs; as part of the neuroimmune system, they are responsible for damaging and killing neurons when triggered by specific stimuli or by inflammation. In PD, AD, HD, and prion diseases, neuron death is caused by inflammation as well as disruptions to the housekeeping functions of microglia. There are several checkpoints to prevent microglial overreaction, including the Trem2 and Cx3cr1– fractalkine pathways. The gene Trem2, for example, encodes an immune receptor, and mutations in this gene have been identified as a risk factor for AD in genome-wide association studies.

Furthermore, Trem2 variants are associated with tauopathies and PD.3

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1.3.4 Parkinsonism and levodopa-responsive PD in SCA3

Parkinsonism is a clinical syndrome sharing symptoms with PD, but unlike PD, can be caused by a variety of agents other than degeneration of the substantia nigra. Parkinsonism is one of the reported diverse phenotypes of the spinocerebellar ataxias. In 1995, SCA3 became the first genetically confirmed subtype in which there was evidence of levodopa-responsive PD, as opposed to only parkinsonism. Other subtypes of spinocerebellar ataxia have since been associated with levodopa-responsive PD and parkinsonism. SCA3, especially in African ethnicities, is now regarded as one of the genetic causes of familial parkinsonism. There have been numerous studies on the genotype and phenotype correlations in SCA3 from which information about parkinsonism in SCA3 could be gained, but there have been no findings other than those about the inverse correlation between the number of CAG repeats and the age of disease onset.14

The affected brain regions in PD and SCA3 also show overlaps; the lesions present in the dentate nucleus and the substantia nigra in SCA3 are responsible for the ataxic and Parkinsonian phenotypes, respectively. Unlike cases of idiopathic PD, the lesions to the substantia nigra in

SCA3 are relatively more focal and do not diffuse across the compact and lateral areas of the nucleus. Notably, following studies of human cases of SCA3, much less is known about the destruction to the substantia nigra in SCA3, while much attention has been devoted to the dentate nucleus.27 Moreover, a 2011 study showed that both wild-type and expanded ataxin-3 interact directly with parkin by deubiquitination. The same study also remarkably revealed that in a transgenic mouse model of SCA3, mutant ataxin-3 but not wild-type ataxin-3 plays a role in the clearance of parkin via the autophagy pathway. Taken together, the findings imply that decreased parkin levels may contribute to and account for parkinsonism in SCA3.25 12

1.4 Current state of rodent models of NDs

With the identification of many overlapping pathways in NDs, a relevant question for further research arises in distinguishing correlative associations between pathways and disease from actual causative mechanisms of disease. Much of the difficulty in teasing apart correlations from causations is due to the limitation that most human ND tissue can only be obtained post- mortem.24 Though post-mortem tissues tell us much about the compensations for cell loss, many details are lost in late-stages tissues. These missing details include early disease progression, the mechanisms that engender nuclear inclusions/aggregates, and expression pattern changes at pre- clinical stages of the disease. Insights can be gained from human cellular models of NDs, which are becoming increasingly available, but their simplistic nature does not capture the workings of a complete nervous system.47 Instead, precise answers to these questions, especially the effect of disease-relevant mutations on early functional changes to susceptible neurons and circuits, can be evaluated with the use of experimental rodent models.

The problems inherent in the use of animal models of NDs cannot be denied. No animal model can perfectly phenocopy human disease.48 Many models only mimic specific phases of disease, such as initial proteinopathy or later neurodegeneration. Even when models do develop a neurodegenerative cascade of events, there is no guarantee that the sequence follows human disease. Finally, mouse models have historically had poor predictive power for the efficacy of therapeutics in humans.49,50 The need for new, more relevant animal models is an area of ongoing research.

However, there have been many positive outcomes of the numerous transgenic rodent models of NDs that have been created with the use of genetic “guideposts” after the discovery of clearly deterministic genes that drive these disorders.49 The phenotypes of idiopathic disease 13

often do not exactly mimic genetic forms, but in many cases the genetic forms exhibit very similar pathology and are good surrogates for studying idiopathic disease. Accordingly, animal models based on genetic forms of NDs have produced novel insights regarding the mechanisms of human disease, as well as changes in disease progression over time.51 These insights have led to molecular targets for disease-alleviating therapies. When the appropriate conservative measures are in place with the use of these models, they have led to beneficial results.

With these measures in mind, I investigated transgenic rodent models in this body of work. These models have been behaviorally and histologically characterized in past literature,52–

55 but their temporal genetic expression profiles are less established. To ensure than the models are relevant to early and late stages of disease, I studied an early and late time point from each transgenic model. To ensure that these time points can be compared across models, I aimed to keep the time points similar across models. In this section I will further outline the state of rodent models in PD and SCA3, and the specific models I investigated.

1.4.1 Current state of rodent models of PD

There are three broad approaches to the modeling of PD.49 The historically oldest one is the use of drugs which can, for example, bluntly mimic the immobility caused by PD. One such model was used in the discovery of levodopa’s beneficial effects.56 Their further use has been limited. A second category of models focusses on genetic forms of PD not associated with α- synuclein pathology; these models overexpress mutations of the LRRK or PARK2 genes. These models show varying degrees of relevance to disease; knockouts of both LRRK1 and LRRK2 or only PARK2 display age-dependent dopaminergic neuron loss. Finally, numerous models attempt to examine α-synuclein pathology.49 These leverage the missense mutations – A30P, A53T, or

E46K – or the multiplications of α-synuclein that can cause PD to create transgenic models. The 14

models can show a range of phenotypes – decreased striatal levels of dopamine, behavioral impairment, accumulation of α-synuclein, and nigrostriatal neurodegeneration – depending on which promoter was used to drive expression of the transgene, which form of the protein is expressed, and the level of expression of α-synuclein.57 All PD models in this thesis examine α- synuclein pathology.

1.4.2 SNCA overexpression rat model relevant to PD

This model attempts to recapitulate α-synuclein pathology related to the overexpression of the protein caused by multiplications of the SNCA gene in some forms of PD.52 Some overexpression models of α-synuclein drive overexpression by the use of various promotors, which drive high expression broadly in the central nervous system.51 However, α-synuclein transgenic mice using this technique often do not show nigrostriatal pathology of dopaminergic neurons. This may be due to effects of the promoters used or could be due to differences in mouse strains. To address these concerns, our collaborators created a novel model of α-synuclein overexpression in rats (model termed PD-ovx in this thesis), based on suggestions in the literature that rats may be more sensitive to stressors on the dopamine pathway than mice.52 They used bacterial artificial chromosome (BAC) transgenics,52 which allowed a more native-like expression pattern of α-synuclein.51 The PD-ovx model carries the full-length human wild-type

SNCA locus as well as the human SNCA promoter.

In this rat model, by 16 months, there is a two-fold increase in the total measured levels of human α-synuclein when compared to the almost unchanged level of endogenous rat α- synuclein. At three months, the rats display subtle locomotor deficits and a disruption in olfactory discrimination. At 12 months, there is a marked decrease in striatal dopamine. Between

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the ages of 12 and 16 months, these rats develop an increased morbidity due to weight loss. By

18 months, there is a decrease of 39 per cent of dopaminergic neurons in the substantia nigra.52

1.4.3 Snca KO mouse model relevant to PD

In this model, a targeting vector was used to disrupt endogenous mouse Snca exons in embryonic stem cells, essentially preventing the expression of α-synuclein.53 It is noteworthy that the value of α-synuclein knockout models (PD-ko) are highly debated; earlier reports in the field mention the validity of studying the neuroprotective effects of the protein in transgenic knockout models,58 but a recent review reports that due to the apparent lack of phenotypes in knockout mice, many have concluded that α-synuclein does not have a substantial role in dopaminergic survival and that its role can be compensated.51 However, the study of these PD-ko models continues because the mechanisms of α-synuclein function are still to be elucidated.

From the ages of one to two months, these PD-ko animals experience a reduction of total striatal dopamine. The deficiency of α-synuclein does not alter locomotor activity nor does it cause any gross deficits of brain morphology. By 14 months, the mice fail to learn in a passive avoidance test. By 26 months, there is a substantial decline in dopamine level without the reduction of the number of dopaminergic neurons in the substantia nigra.53

1.4.4 Snca KO and SNCA overexpression mouse model relevant to PD

This mouse model (termed PD-ko+ovx) was created with the use of two transgenic lines.

One line used a BAC construct to overexpress human SCNA using the endogenous mouse promoter to mimic pathogenic levels while ensuring correct spatiotemporal expression. This line was then bred to a mouse model in which endogenous α-synuclein was knocked out, allowing for the creation of a model that overexpressed SNCA and lacked the endogenous gene. In this

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manner, the model allows the study of SCNA without the confounding interactions between the human and endogenous gene.

In these PD-ko+ovx mice, the levels of human α-synuclein are 1.9-fold higher in the striatum in comparison to endogenous mouse α-synuclein in age-matched control animals.

Soluble α-synuclein species – similar to those found in substantia nigra tissue from a PD patient

– and increased clustering of vesicles in dopamine terminals in the dorsal striatum are observed by three months of age. Insoluble aggregation of α-synuclein, however, was not observed even at

18 months. Even without overt protein aggregation, at 18 months, there is a 30 per cent reduction in dopamine neurons in the substantia nigra. Furthermore, the mice display Parkinsonian phenotypes at 18 months, such as motor abnormalities in forepaw stride length tests.54

1.4.5 Current state of rodent models of SCA3

As with PD, there are several animal models of SCA3 that have provided insights about disease mechanisms and allow for the study of intranuclear aggregations that post-mortem human tissues do not allow.24,27 Based on the specific polyglutamine origin of SCA3, some models employ the expression of full-length human mutant ataxin-3. They vary in which isoform of ATXN3 and which gene promoter they employ, as well as the number of CAG repeats in the human gene. These models have been compared in the literature to mice expressing a portion of human mutant ataxin-3; the studies surprisingly show that neurological deterioration is more evident in models expressing a portion of the mutant protein when compared to those expressing full-length mutant ataxin-3. In the context of such results, a hypothesis for the toxicity of fragmented ataxin-3 has been proposed which states that while all neurons express full-length ataxin-3, affected neurons express a protease that cleaves the full-length protein to release the toxic fragment.59 17

1.4.6 Mutant ATXN3 model of SCA3

Our collaborators sequenced the transcriptome of MJD84.2 transgenic mouse models of

SCA3 (model termed SCA3-84q), which carries the full ATXN3 gene with 84 CAG repeats and the human promoter using yeast artificial chromosome (YAC) transgenics. All ataxin-3 isoforms were detected in the model.55 In comparison to other transgenic models expressing the full human ATXN3 gene, the phenotypes of this model are intermediate in their intensity based on severity of behaviors, the total number of abnormalities, and age of occurrence. The slower progression of phenotypes in this model makes it an appropriate one to study the mechanisms that affect the rate of progression of symptoms.59

MJD84.2 has been reported to show abnormal gait by one month of age, and reduced balance and coordination is seen by 7.5 months. After about six months, the SCA3-84q mice progressively lose weight. At 12 months, there was a loss of 40% of the neurons of the dentate nucleus of the cerebellum. The remaining neurons in the dentate contained intranuclear inclusions.55 However, another more recent study focusing on the transcriptome of these mice reports that these mice do not develop any motor symptoms even at 17.5 months, without referencing the earlier study.60

1.5 Transcriptional analyses

In the study of the transcriptome and prioritization of disease-relevant gene lists from humans and transgenic models, analysis of RNAseq datasets is replacing the use of microarrays, given the former technique’s ability to investigate all RNAs at a high resolution.61 In keeping with this development, RNAseq data was collected from transgenic models by our collaborators.

However, these results can still be understood in the context of transcriptional results obtained from microarray studies. 18

1.5.1 Transcriptional analyses in rodent models of PD

Transcriptional characterization of transgenic rodent models of PD are available, but to my knowledge, the gene expressions profiles of these models are rarely compared to each other within the same study. In a model that overexpressed mutant human A53T-α-synuclein, there was upregulation of Foxp1 in the brainstem, which is interesting given the context of downregulation of Foxp1 in α-synuclein knockout mice.62 A study of a different mouse model overexpressing human SNCA using the mouse Thy1 promoter found that genes associated with phosphoproteins were downregulated in the striatum, while those associated with intracellular signaling cascade and the MAPK signaling pathway were upregulated.63

1.5.2 Transcriptional analyses in rodent models of SCA3

Genomic studies in rodent models of SCA3 are sparse, but a microarray analysis of a transgenic line expressing mutant ATXN3 with 79 CAG repeats shows differential expression of genes involved in synaptic transmission, signal transduction, and intracellular calcium mobilization.64 A recent characterization of the MJD84.2 line at 17.5 months showed an enrichment of genes associated with cellular signaling pathways across all four brain tissues which were sampled. Other groups of significantly altered genes include those associated with transmission across chemical synapses, cholesterol biosynthesis, and myelination. They also examined differential gene expression in blood samples from the same animals at nine and 17.5 months of age. There was only one gene found to be differentially expressed at nine months, while there was a total of 142 genes differentially expressed at 17.5 months. Enriched functional categories at 17.5 months include those associated with respiratory electron transport and mitochondria.60

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1.6 Methods of assessing overlaps of gene lists

I analyzed the transcriptomes of each transgenic model in this thesis as an independent experiment, which then allowed the comparison of gene expression patterns across models. The increasing amounts of available RNAseq data have resulted in the development of different analysis pipelines61 to illuminate gene expression patterns. As with microarray data, differential expression (DE) in RNAseq can be assessed with general linear models (GLMs).65,66 GLMs can account for the effects of many parameters on the response variable, as well as the interactions between the parameters. In my case, the response variable of gene expression can be modelled as a linear response to the parameters of genotype and tissue.

After obtaining lists of genes that show DE, these lists can be directly tested for overlaps, correlations, and enrichment of sets of genes. The guiding hypothesis is that by identifying genes that appear as differentially expressed in two transgenic disease models, these common genes could be involved in similar pathways or mechanisms is each disease model, which researchers have proposed in the past,67 but not specifically with these transgenic models. I also ascertained overlaps after functional enrichment of the gene lists and after cell type proportion estimation. A hypergeometric distribution – equivalent to the one-tailed Fisher’s exact test – is usually used to measure the significance of these overlaps. These tests for overlaps have been used widely in with microarrays, and, by extension, can be applied to RNAseq data.67,68

1.7 Aims

Transgenic models of NDs, specifically PD and SCA3, have been created with the intention of further understanding the mechanisms that underly the pathological nature of the mutant proteins found in each disease. Transcriptional analyses of PD and SCA3 do exist52–55 but how their temporal genetic expression profiles compare to one and another is less established. 20

Also, the overlapping pathways in PD and SCA3 suggest that similar genes may be commonly implicated in these diseases. In this thesis I aim to study DE in four of these transgenic models –

PD-ovx, PD-ko, PD-ko+ovx, and SCA3-84q – at novel early and late stage time points.

Furthermore, by looking at estimations of cell type proportional changes in these models, I aim to understand whether these changes in DE can be explained by selective neuronal vulnerability.

While it is already known that protein aggregation and selective neuronal vulnerability are hallmarks of both PD and SCA3, looking at overlaps in these models may allow the pinpointing of genes that are associated with overlapping mechanisms at early stages in the transgenic models or compensations for the transgenes’ effects at later stages.

In the following chapter, I will further specify the details of the RNAseq data I analyzed, describe the processes involved in filtering and normalizing the data before fitting linear models, applying the specific linear models used, using marker gene profiles to obtain cell type proportion estimations, implementing functional enrichment, and testing for overlaps. Chapter 3 contains the results of these analyses. Finally, Chapter 4 will detail a discussion of observed overlaps, as well as the limitations and the conclusions suggested by these data.

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Chapter 2: Materials and methods

With the motivation of finding overlapping genes and pathways in four transgenic models relevant to Parkinson’s disease (PD) and spinocerebellar ataxia type 3 (SCA3), specifically the

PD-ovx, PD-ko, PD-ko+ovx, and SCA3-84q models which have been described in detail in sections 1.4.1-6, I analyzed the gene expression patterns of two time points from up to three tissues from each of four transgenic models. Figure 1 is an overview of the workflow I implemented. Analyses were conducted in R (version 3.5.0),69 except when other software are noted.

Figure 1 Workflow summary All steps after RNA sequencing were conducted by the author of this thesis. QC: quality control. Raw data files were received as FASTQ (proper name, not an acronym) files, which is a text-based file format that stores a biological sequence and quality scores. Alignment files are in the BAM (proper name) file format, which is a binary file format for storing sequence data. Analyses were conducted in R, except when noted in below specific steps. Local installations of FASTQC (proper name), Spliced Transcripts Alignment to a Reference (STAR), and RNA-seq by expectation-maximization (RSEM) were implemented using existing pipeline scripts.

2.1 Data

The lab of Dr. Olaf Riess, of The University of Tübingen, performed RNA sequencing

(RNAseq) on four transgenic models at two time points each. After the appropriate breeding and housing of the rodent transgenic models, animals were euthanized and brains were immediately dissected for regions relevant to this study (frontal cortex, striatum, and cerebellum). Library

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preparation was performed using the Qiagen AllPrep DNA/RNA/miRNA Universal Kit (80224) resulting in read lengths of 68 base pairs for all transgenic models except the PD-ovx samples at both time points and the PD-ko+ovx and PD-ko samples at the later 12-month time point, which had read lengths of 125 base pairs. Before RNAseq library preparation, the Reiss lab confirmed the expression of the transgene in each sample by quantitative polymerase chain reaction

(qPCR). RNAseq was performed on up to three different brain regions from each model and time point. The brain regions were chosen based on relevance to the transgenic model as well as with the consideration that the models would eventually be compared to each other, therefore requiring similar tissue from each model. From the SCA3-84q model, tissue was collected at two and 12 months of age from transgenic and age-matched wild-type mice, from the frontal cortex, striatum, and cerebellum for both time points, with five replicates for each age and tissue group.

This resulted in 60 samples in total, including wild-type controls (see Table 1). Similar tissue and locations were obtained from the PD-ovx model at five months and 12 months, resulting in 60 total samples. For the PD-ko and PD-ko+ovx models, RNAseq was performed on tissue from five replicates from frontal cortex, striatal, and cerebellar tissues at two months. For the 12- month samples, RNAseq was performed only on frontal cortex. This resulted in 20 samples each for the two transgenic PD models (see Table 1), with 20 control samples shared across these two models. Thus, in total we started with 180 samples for the entire study. For paired-end sequencing after library preparation, Illumina’s high-throughput sequencing platform HiSeq2500 was used with the software bcl2fastq70 version 1.8.4 for base calling. At this point, the raw data from the Reiss lab were sent to us.

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2.2 Data quality control and pre-processing

I obtained the raw RNAseq files in standard FASTQ (proper name) format and used existing pipeline scripts71 to implement software necessary to obtain read count matrices. First, for quality control of the raw files, I used a local installation of the widely used72 software

FASTQC73 (proper name) which implements standard quality-control metrics on Illumina high throughput sequence data. All samples were rated to be of good quality. The next step was aligning the files to the reference using Spliced Transcripts Alignment to a Reference (STAR)74 version 2.4.0h; the assembly version used for mouse samples was Ensembl GRCm38,75 packaged by Illumina for the iGenomes collection, and Rnor_6.076 for rat samples. I modified the assemblies to include the relevant human gene, either SNCA or ATXN3. This allowed a confirmation of the expression of the human transgene, or lack thereof, at appropriate levels in all samples. Next, the “rsem-prepare-reference” script provided by the RNA-seq by expectation- maximization (RSEM) RNAseq transcript quantifier77 v1.2.31 was used to quantify the number of reads per gene.

Using the resulting four read count matrices (one each for model), I checked for the correlations of each sample’s read counts with values of other samples in the same transgenic model. Two samples displayed low correlation within their respective relevant biological replicates and were removed (noted in Table 1). The outliers from the SCA3-84q data and PD-ko data showed a correlation values of 0.88 and 0.74 to other relevant samples, respectively, while the rest of the samples had correlation values around 0.92. I further filtered out genes that showed expression below counts of 5-10 in less than 70 per cent of samples. This removed genes which are near background levels and thus unlikely to be reliably measured, and also allowed for a better estimation of the expression level mean-variance relationship for each data set, a step 24

important to downstream differential expression analysis.78 Finally, the data for each model were quantile normalized.

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Table 1 Summary of samples obtained from transgenic models and changes to data after pre-processing Transgenic Time point, Tissue Transgenic Control Total samples Genes Genes Mean Min - max model months samples samples before after library size, lib size, filtering filtering millions millions Frontal cortex 5 5 4 (1 outlier Striatum 5 2 removed) SCA3-84q Cerebellum 5 5 59 45706 15826 29.6 21.8 - 39.2 Frontal cortex 5 5 12 Striatum 5 5 Cerebellum 5 5 Frontal cortex 5 5 5 Striatum 5 5 Cerebellum 5 5 PD-ovx 60 32285 13861 18.2 13.1 - 21.9 Frontal cortex 5 5 12 Striatum 5 5 Cerebellum 5 5 Frontal cortex 5 5 2 Striatum 5 5 Cerebellum 5 5 PD-ko+ovx 40 45706 16401 36.4 23.6 - 71.3 Frontal cortex 5 5 12 Striatum - - Cerebellum - - 4 (1 outlier Frontal cortex 5 removed) 2 Striatum 5 5 PD-ko Cerebellum 5 5 39 45706 16393 36.8 24.3 - 66.9 Frontal cortex 5 5 12 Striatum - - Cerebellum - -

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2.3 Fitting linear models

I analyzed each transgenic model as its own independent experiment. Accordingly, I implemented two linear models for each transgenic group to find differential expression (DE) between wild-type controls and the transgenic models using limma,79 a widely used R package for linear modelling of transcriptomic data.78

Other popular methods for performing DE include edgeR, which performs DE analysis based on an assumption of a negative binomial distribution for count data.72 The limma package considers that modeling the underlying data distribution is not as important as modeling the mean-variance (heteroscedasticity) in the data.78 In fact, limma has been shown to perform well in comparison to edgeR’s discrete distribution approach, especially because data can lose this supposed distribution after batch correction and normalization procedures.72 Significantly differentially expressed genes were selected so as to control the false discovery rate (FDR) at

0.05, which was estimated by the Benjamini-Hochberg procedure.80 The p-value distributions of all analyses were plotted and checked to ensure that there was a right-skewed distribution where there was DE – as opposed to an uniform distribution consistent with a transcriptome-wide lack of DE – unless otherwise noted. In sections 2.3.1-2, I describe the two linear models which I implemented.

2.3.1 Sub-setting the data by age

For one linear model, each of the two time points from each transgenic model was analyzed separately, treating genotype and brain region as covariates. This allowed for a two- way factorial design. The mathematical model underlying this analysis is, with expression level for one gene (푌) varying over samples (푘):

푌푘 = 휇 + 훽1푔푘 + 훽2푡푘 + 훽3(푔푘푡푘) + 휀푘 27

2 with 휀푘 ~ 푁(0, 휎 )

1, 푓푟표푛푡푎푙 푐표푟푡푒푥 1, 푡푟푎푛푠푔푒푛푖푐 푔 = { 푡 = {2, 푠푡푟푖푎푡푢푚 푘 0, 푤푖푙푑푡푦푝푒 푘 3, 푐푒푟푒푏푒푙푙푢푚

휇 is the mean, 훽1 is the coefficient associated with genotype, 훽2 is the coefficient associated with tissue, and 훽3 is the coefficient for the interaction effect of genotype and tissue. 휀푘 represents random residual noise, assumed to be normally distributed with mean zero and variance 휎2.

2.3.2 Sub-setting the data by age and tissue

I chose to also subset the data by tissue and examine differential expression trends within tissues, allowing for a one-way analysis of variance (ANOVA). In this model, genotype was the only covariate. This was because the differences underlying each tissue can preclude the use of one of these tissue as the ‘baseline’ tissue for comparison. Moreover, since tissue was only available from one brain region (frontal cortex) for two of the transgenic models, an analysis including the tissue as a covariate was unavailable in these instances. The mathematical model underlying this second analysis is, with expression level for one gene (푌) varying over samples

(푘):

푌푘 = 휇 + 훽1푔푘 + 휀푘

2 with 휀푘 ~ 푁(0, 휎 )

1, 푡푟푎푛푠푔푒푛푖푐 푔 = { 0, 푤푖푙푑푡푦푝푒

휇 is the mean, 훽1 is the effect size coefficient of the effect of genotype. 휀푘 is the contribution of error, assumed to be normally distributed with variance 휎2.

I also implemented a third linear model, which did not improve model fits, including two other covariates that might affect differential expression; these covariates are a quality control

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RNA integrity number (RIN) for each sample and the library preparation method (resulting in two different read lengths, 68 and 125 basepairs) used before sequencing. Differential expression between wildtype and transgenic models was not explained by these covariates, and in fact they did not contribute to variance in the data – the RIN scores for these samples, for example, did not vary by more than a few decimal points, and this is known to not affect DE to a great degree.81

Therefore I did not include them in further analysis.

2.4 Functional enrichment

The results of DE analysis (section 2.3) were rankings of all genes, which were divided into two sets of ranked gene lists after thresholding to control the FDR, one of upregulated genes and the other of downregulated genes, for each test. I used an R wrapper82 for ErmineJ version

3.1.283 to find enriched Gene Ontology (GO) terms in these ranked gene lists, with the requirement that each list contain a minimum of 20 genes. Each gene list was enriched for GO terms that fall into three categories: “biological process,” “molecular function,” and “cellular component.”84 Functionally enriched terms such as “glial cell differentiation” and

“oligodendrocyte development” can serve as a proxy for cell type proportions, while terms like and “fatty acid catabolic process” suggest molecular functions. ErmineJ offers various methods for enrichment analysis, one of which is the over-representation analysis (ORA) method;85 this specific method asks which terms are “over-represented” in the “hit list” of genes that fall into the FDR = 0.05 gene score threshold which I used in my DE analyses. A key element of ErmineJ is its ability to assess the impact of gene multifunctionality, which, in brief, accounts for the fact that highly annotated genes can have outsized and undesirable effects on GO enrichment results.86 With this in mind, GO terms were prioritized by their multifunctionality score such that terms with low multifunctionality scores were ranked higher in significance. 29

2.5 Overlaps

There are multiple ways to assess the significance of overlaps between lists of genes, including the chi-squared test and the hypergeometric distribution or the equivalent one-tailed version of Fisher’s exact test assumes.67,68 The chi-squared test is often used for larger samples sizes than what is presented in this thesis. A more conservative test uses the hypergeometric distribution, which assumes: if there are n genes total in two investigations, if x genes are in a list of differential expression in either investigation, and if m genes are in the lists of both investigations, that m has a hypergeometric distribution. A traditional p-value is the result from this test for significance. However, since those methods are based on a strict arbitrary threshold to define differentially expressed genes, some similarities in expression patterns could be lost.68

To help ensure that this was not the case, I also calculated rank correlations between all the p- values obtained from each transgenic model compared to its relevant control.

2.6 Estimating cell type proportions

For each tissue available for each transgenic model, I applied the cell type proportions estimation technique described in Mancarci et al. (2017),46 allowing for the use of marker gene profiles to characterize the cell types present in each sample. This cell type estimation technique was developed with the intuition that a change in gene expression could be due to different causes: global transcriptional change, cell-type specific transcriptional changes, or changes in cellular proportions. Pooling a group of literature-verified markers associated with cell types that are correlated well with each other, followed by testing whether these markers show a significant trend between experimental groups, allows for the ability to distinguish whether DE is due to changes in cellular proportions as opposed to the other two listed possibilities. In order to construct a group of markers – or marker gene profile (MGP) – for each cell type, Mancarci et al. 30

pooled data from 36 brain cell types from existing literature, resulting in 50-100 markers for each cell type. MGPs are then calculated by applying principal component analysis to the marker genes; an MGP is the first principal component of the marker genes. These can then be interpreted as a relative measure of cell type proportions, and an estimation of cell type proportional increases and decreases across samples can be obtained.

I implemented the appropriate quality control measures for the markers available in the dataset. This involves two criteria. First, the first principal component should explain a large proportion of variance – ideally greater than 40% – in the expression of marker genes. Secondly, a significant portion of the marker genes – again, ideally greater than 40% – should correlate well with each other. If the marker genes do not correlate well with each other, it might indicate that the variance explained by the first principal component cannot be explained by changes in cellular abundance; for example, this can happen if some of the genes are highly regulated in a subset of the samples or if a substantial proportion of the genes are not sufficiently expressed or not specific to the cell type. If these two criteria did not hold for markers of a cell type in a particular tissue, those markers were dropped, as was the proportion estimation for that cell type.

Finally, to find significant differences in cell type proportion estimations between wild type rodents and transgenic models, I used the Wilcoxon rank sum test and estimated the FDR using the Benjamini-Hochberg procedure.80

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Chapter 3: Results

With data from four transgenic rodent models, I performed differential expression analyses to obtain gene-lists that could then be compared for overlaps. The transgenic models are relevant to Parkinson’s disease (PD) and spinocerebellar ataxia-3 (SCA3). The four rodent models analyzed are: a mouse model expressing mutant human ATXN3 containing 84 CAG repeats (SCA3-84q), a rat model (PD-ovx) which overexpresses the human SNCA gene, a mouse knockout model of the endogenous Snca gene (PD-ko), and finally, a mouse model which is both a knockout of the endogenous Snca gene and overexpresses the human SNCA gene (PD-ko+ovx)

(see section 1.4 for further details of the transgenic models). The RNAseq data – three tissues and two time points from each transgenic model (see Table 1) – was taken through a pre- processing pipeline that ensured quality control and the removal of outliers. I used two linear models to find differentially expressed genes between wild-type controls and the transgenic models, and these gene lists were functionally enriched. The two linear models allowed for the discernment of differential expression common to all brain tissues as well as differential expression specific to one brain tissue, therefore differentially expressed (DE) genes and enriched functions were obtained across tissues as well as within each tissue specifically.

Estimations of cell type proportions were based on sets of correlated marker genes for each cell type (see Chapter 2: Materials and methods for details and Figure 1 for workflow). Finally, in assessing the significance of the overlaps of these analyses across transgenic models, a hypergeometric distribution was used.

3.1 Results overview

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Overall, I found that two transgenic models, the SCA3-84q and PD-ovx models, showed substantial differential expression at the earlier and later time points, while the transgenic models

PD-ko and PD-ko+ovx only showed differential expression at the later 12-month time point. An assessment of the direct gene-to-gene overlaps of differential expression analyses showed a highly significant overlap between the PD-ko and PD-ko+ovx transgenic models. Estimations of cell type proportions were consistent with the trends in DE analysis; where there were dramatic

DE, I observed changes in estimated cell type proportions. Two cell types, endothelial cells and oligodendrocytes, showed evidence of proportional changes in three transgenic models.

Functional enrichment revealed enrichment of various GO terms including those associated with protein degradation, synapse plasticity, and oligodendrocyte differentiation. An assessment of the overlaps in functional enrichment, however, highlighted terms associated with cell types, rather than protein degradation or plasticity pathways. These results are consistent with an interpretation that where similarities in differential expression profiles are observed in the transgenic models, these similarities are predominantly due to cell type proportion changes, rather than overlaps in transgene-dependent regulation of transcription within extant cells. A detailed explication of the results is contained in the coming sections, specifically 3.1-5.

3.2 Differential expression overview

Aligning the data to custom references including the transgene allowed a confirmation of the expression of each transgene and ensured that the transgenic samples were in fact annotated correctly. Figure 2 shows these results. A summary of differential expression findings is presented in Table 2, Appendix A contains the top 50 differentially expressed genes in each comparison, and the results are expanded on in the sections below.

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Figure 2 Expression of transgenes in all four transgenic models is as expected Background expression is shown by the grey dotted line. A) In the SCA3-84q dataset, expression of the endogenous Atxn3 gene is similar in both wildtype and transgenic models. Expression of human ATXN3 mimics levels of endogenous expression, and only in the transgenic model, as expected. B) In the PD-ovx dataset, expression of the endogenous rat Snca gene is similar in wildtype and transgenic models. Human SNCA is overexpressed in only the transgenic models. C) In the PD-ko+ovx dataset, expression of the endogenous mouse Snca gene is at biological levels in wildtype mice, and is not expressed in PD-ko+ovx mice, as they should overexpress human SNCA in a knockout background. Human SNCA is overexpressed in only the transgenic models. D) The PD-ko mice do not show expression of the mouse Snca gene nor human SNCA, as expected.

Table 2 Differential expression summary The number of significantly differentially expressed (DE) genes (FDR = 0.05) in transgenic models compared to wildtype are presented. Uniform p-value distributions, and therefore a lack of DE, are marked as *. Transgenic Time point, DE genes common DE genes in DE genes in DE genes in model months to all tissues frontal cortex striatum cerebellum 2 0 0 1* 768 SCA3-84q 12 3 2* 1535 1

5 114 2986 113 130 PD-ovx 12 595 412 279 539

2 7* 9 6 4* PD-ko 12 - 3061 - -

PD- 2 10 15 6* 4 ko+ovx 12 - 3053 - -

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3.2.1 Differential expression in SCA3-84q

I analyzed 30 samples from SCA3-84q transgenic mice in comparison to 29 samples of wild-type mice. The analysis at the early, two-month time point showed significant differential expression only in the cerebellum, which had 768 DE genes at an FDR of 0.05. The expression patterns of the top 50 DE genes are shown in Figure 3a. At the later 12-month time point, common to all brain regions, there were 3 DE genes (FDR = 0.05), with two of these being protein coding genes Psat1 and Prob1. In the individual tissue analysis, only the striatum showed

DE with 1,535 DE genes (FDR = 0.05) (Figure 3b).

Figure 3 Top 50 upregulated and downregulated genes in individual tissues A, B) Striatal and cerebellar data is presented for the SCA3-84q model. C, D, E, F) Frontal cortex data is presented for the PD-ovx, PD-ko, and PD-ko+ovx models. Each column is one brain sample, with each row as the z-score transformed expression of the log transformed counts per million expression for one gene. In the bars at the top of the figure, wildtypes (wt) samples are indicated in light grey and transgenic samples are labeled in dark grey. Genes above the white horizontal deviation are upregulated in the transgenic models, and genes below the deviation are downregulated.

3.2.2 Differential expression in PD-ovx

The analysis of 30 samples from the rat transgenic model PD-ovx showed substantial differential expression at both five months and 12 months. There were 114 DE genes

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(FDR = 0.05) common to all brain regions at five months compared to wild-type mice. When examining each brain region individually, the results were most dramatic in the frontal cortex which had 2,986 DE genes (Figure 3c), while the striatum had 113 DE genes, and the cerebellum had 130 DE genes (FDR = 0.05).

At 12 months, there were 595 DE genes common to all brain regions and the effect observed in the frontal cortex at five months was dampened; the individual tissue analysis showed 412 DE genes (FDR = 0.05) (Figure 3d). There were 279 and 539 DE genes

(FDR = 0.05) in the striatum and cerebellum respectively.

3.2.3 Differential expression in PD-ko

The analysis of 19 samples from a mouse transgenic model, PD-ko, with a knockout of endogenous Snca, showed very subtle effects at the early two-month time point, with more differential expression present at 12 months. In the analysis across brain regions, at two months, a uniform p-value distribution was observed, suggestive of a lack of true DE. The analysis of the individual tissues showed 9 DE genes in the frontal cortex, 6 genes in the striatum, and a lack of differential expression in the cerebellum (FDR = 0.05). At 12 months, only the frontal cortex was available for analysis. There were 3,061 DE genes in this tissue (FDR = 0.05) (Figure 3e).

3.2.4 Differential expression in PD-ko+ovx

From 20 samples of a mouse transgenic model, PD-ko+ovx, which overexpressed human

SNCA in the background of a knockout of endogenous Snca, there were slight effects observed at the early two-month time point with more dramatic effects at 12 months. At two months, across brain regions, there were 10 DE genes (FDR = 0.05). When examining individual regions, there were 15 DE genes in frontal cortex, a lack of DE genes in the striatum, and 4 DE genes in the

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cerebellum (FDR = 0.05). At 12 months, only the frontal cortex was available for analysis, and there were 3,053 DE genes (FDR = 0.05) (Figure 3f).

3.3 Gene-to-gene overlaps

Examination of genes that overlap across brain regions – i.e., genes that were DE at specific time points in more than one transgenic model – showed the highly significant overlap of differential expression in the PD-ko and PD-ko+ovx mice, calling into question the effect of overexpression of human SNCA. In these two transgenic models, at the two-month time point, one gene was commonly upregulated out of four genes in the PD-ko+ovx model and one gene in the PD-ko models. This overlap was significant (p value = 0.00024). Out of six downregulated genes in the PD-ko+ovx model and six downregulated genes in the PD-ko model, four genes were commonly downregulated (see Figure 4). This overlap was also significant (p value =

7.45e-14). At the 12-month time point, 843 genes were commonly upregulated (p value = 2.22e-

308) out of 1,457 in the PD-ko+ovx model and 1,444 in the PD-ko model. Out of 1,596 downregulated genes in the PD-ko+ovx model and 1,617 downregulated genes in the PD-ko model, 1,144 were commonly downregulated (p value = 2.22e-308) (see Figure 4). Looking for overlaps using a threshold to create a “hit list” of genes did not result in the omission of overlaps in gene ranks, as shown by the correlation plots in Figure 5.

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Figure 4 Differential expression results in PD-ko are remarkably similar to PD-ko+ovx A) At the two-month time point, one gene, Hk2, was commonly upregulated (p value = 0.00024) out of 4 total upregulated genes in PD-ko+ovx and 1 upregulated gene in PD-ko; 4 genes (Ggct, Inmt, Pde1c, and Pyurf) were commonly downregulated (p value = 7.45e-14) out of 6 genes downregulated in PD-ko+ovx and 6 gene downregulated in PD-ko. B) At the 12-month time point, 843 genes were commonly upregulated (p value = 2.22e- 308) and 1,144 were commonly downregulated (p value = 2.22e-308).

Figure 5 Highest correlation is between PD-ko and PD-ko+ovx (a) transgenic models A) Correlation between PD-ko+ovx and PD-ko: two months r = 0.012, 12 months r = 0.470 B) Correlation between PD-ovx and PD-ko: two months r = -0.011, 12 months r = -0.015 C) Correlation between PD-ovx and PD-ko+ovx: two/five months r = 0.053, 12 months r = -0.020 D) Correlation between PD-ovx and SCA3-84q: two/five months r = -0.020, 12 months, r = 0.048 E) Correlation between PD-ko+ovx and SCA3-84q: two months r = 0.070, 12 months r = -0.068 F) Correlation between PD-ko and SCA3-84q: two months r = 0.001, 12 months r = -0.060

Overlaps were also observed from the tissue-specific linear models (see Figure 6b).

Notably, the PD-ovx transgenic model, a rat strain, showed some overlaps with its orthologous

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genes in the mouse PD-ko+ovx and PD-ko models at the 12-month time point, however, these overlaps were not statistically significant when examined with the tissue-specific linear model

(see Figure 3.3). Otherwise, the trends were unchanged from those observed common to all three brain regions, with the PD-ko and PD-ko+ovx models showing overlaps across tissues at the earlier time point. There were overlaps between the PD-ovx and PD-ko models in the frontal cortex, the PD-ovx and PD-ko+ovx models in the frontal cortex, and the PD-ovx and SCA3-84q models in the striatum at the 12-month time point, but these were not significant.

All three-way overlaps – that is, overlaps between three transgenic models – were also ascertained; the only observed overlap was of nine upregulated and 11 downregulated genes in the frontal cortex at the 12-month time point of the PD-ovx (rat strain), PD-ko (mouse), and PD- ko+ovx (mouse) transgenic models. No four-way overlaps were observed.

Figure 6 Number of overlapping genes common across brain regions and within tissues show similar trends Each cell shows the number of overlapping DE genes in the comparison listed on the left, out of the number of total possible genes. A) Overlapping DE genes common to all three brain regions. At 12 months, since only the frontal cortex tissue was available for the PD-ko+ovx & PD-ko mice, this analysis was not applied thus this cell is marked NA. B) Overlapping DE genes within individual tissues; frontal cortex (FC), striatum (ST), and cerebellum (CB). A hypergeometric distribution – equivalent to the one-tailed Fisher’s exact test – is usually used to measure the significance of these overlaps.

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3.4 Differential expression in PD-ko+ovx in comparison to PD-ko

To extend the examination of overlaps, I tested for differential expression in the PD- ko+ovx model directly against the PD-ko model. (To reiterate, the previous analyses of these two transgenic models compared each model to wildtype controls.) The analysis showed very little differential expression at both time points; at two months there were no DE genes common to all tissues (FDR = 0.05), no DE within the frontal cortex (FDR = 0.05), one DE gene in the striatum

(FDR = 0.05), four DE genes in the cerebellum. At 12 months there was one DE gene in the frontal cortex (FDR = 0.05), even though there is substantial differential expression at 12 months in both the PD-ko and PD-ko+ovx in comparison to wildtype.

3.5 Cell types analysis

The selective vulnerability and the eventual death of specific neurons is a trait of many

NDs, with certain cell types showing bioenergetic properties that entail a vulnerability to pathological processes like protein aggregation.2,32 PD patients show loss of a substantial portion of the dopaminergic neurons of the ventral area of the substantia nigra, yet dopaminergic neurons of the dorsal area of the substantia nigra and the ventral tegmental area do not exhibit such dramatic loss.33,34 In SCA3, neuronal degeneration is present in the dentate nucleus of the cerebellum, the substantia nigra, nerve motor nuclei, and the spinal cord.23 Since the transcriptomic profiles described in the sections above represent overall average gene expression for all cell types present in their respective tissues, a cell-type proportion estimation is needed. I further characterized the effect of transgenic perturbations on specific cell type proportions within specific tissues using marker gene profiles (MGPs),44,45 which can be interpreted as computational proxies for relative cell type proportions.46 In my analysis, MGP estimations were

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consistent with the results of differential expression analysis; where there was substantial differential expression, there tended to be changes in cell type proportions (see Figure 7).

3.6 Overlaps in cell type proportional changes

Though more changes in cell type proportions were observed at the 12-month time point in keeping with the differential expression analysis, it is notable that endothelial cells showed changes in their corresponding MGPs at the early time point in three transgenic models. The

MGPs corresponding to endothelial cells decreased in the PD-ovx model, a rat strain, at two months compared to wild-type rats (FDR < 0.05) and increased in PD-ko mice and PD-ko+ovx mice (FDR < 0.1).

Across three transgenic models at the 12-month time point, the MGPs of oligodendrocytes suggest a change in proportion of this cell type when compared to wild type animals (Figure 7). Specifically, the SCA3-84q (Figure 8) striatum and PD-ovx frontal cortex tissues show an increase of MPGs consistent with a relative increase in oligodendrocytes when compared to controls, while the PD-ko (FDR = 0.05) show a decrease. Although it was not significant, it should be noted that the PD-ko+ovx frontal cortex also shows a trend of decreasing oligodendrocyte MGPs.

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Figure 7 Summary of inferred cell type proportional changes between wildtype and transgenic models Data is split into the two time points; early (two or five months), and late (12 months). Column names show the transgenic models, which are further divided into tissues: frontal cortex (FC), ST (striatum), and CB (cerebellum). Each row is a single cell type. Blue cells indicate the analyses where there was a significant difference between wild type and the transgenic model. Red cells indicate analyses where there was no significant difference. Grey cells are those that do not exist in that specific tissue, i.e. Purkinje cells are only found in the cerebellum. White cells indicate the cell type was dropped due to quality control failure in the MGP analysis.

Figure 8 Marker gene profiles (MGPs) for oligodendrocytes in SCA3-84q mice MGPs, normalized to 0 and 1, are plotted for all samples of SCA-84q mice in comparison to wildtype. A) two- month time point B) Significant differences are seen at 12 months in the striatum. FDR ** < 0.1. Each data point represents one sample and are shown along with the box plot for the same data.

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3.7 Functional enrichment overview

The lists of upregulated and downregulated DE genes were analyzed for the detection of enriched Gene Ontology (GO) terms. I found enrichment of terms that, as will be expanded below, are consistent with the effects observed in the MGP analysis. This was especially evident at the later 12-month time point. The following sections elaborate on these results.

3.7.1 Functional enrichment in SCA3-84q

At two months in the cerebellum, upregulated genes in the SCA3-84q mice were significantly enriched for the GO terms “ubiquitin conjugating enzyme activity”, “ubiquitin-like protein conjugating enzyme activity,” and “regulation of long-term neuronal synaptic plasticity.”

Downregulated genes were enriched for GO terms “regulation of axonogenesis” and “cerebellar cortex development.” The ubiquitin terms suggest the early activation of protein degradation pathways and the synaptic plasticity terms implicate early pathological effects on synapses in the cerebellum, but the association of these terms with cell types cannot be ruled out. Functional enrichment was only conducted on analyses which identified more than 20 DE genes, therefore this analysis could not be performed on analyses other than the cerebellum.

Similar analyses were conducted at the later 12-month time point in the striatum; the top

10 enriched GO terms in 862 upregulated genes included six terms associated with ribosomal subunits (Figure 3b). In the 673 downregulated genes, significantly enriched GO terms were

“regulation of synaptic transmission, glutamatergic” and “regulation of synapse structure or activity.” However, since we see MGP effects at this time point in the striatum, these terms could be associated with cell type proportional changes rather than the degradation of synaptic function.

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3.7.2 Functional enrichment in PD-ovx

Significantly DE genes and therefore functionally enriched GO terms were observed at both time points in PD-ovx rats. Upregulated genes in the frontal cortex were enriched in terms such as “gamma-aminobutyric acid signaling pathway,” “GABA receptor complex,” “protein polyubiquitination,” and “ubiquitin-like protein-specific protease activity;” these terms could be related to MGP changes in this tissue at two months. Downregulated genes in the frontal cortex were associated with nicotinamide adenine dinucleotide (NADH). At five months in the cerebellum, upregulated terms included “alcohol binding,” “response to alkaloid,” and

“neurotransmitter transport.” Downregulated terms were associated with cholesterol and sterol regulation. In the striatum, terms including “semaphorin receptor binding,” “response to water,” and “organic cation transport” were upregulated. Enzyme activity-terms were downregulated, as indicated with terms “metalloexopeptidase activity,” “carboxypeptidase activity,” and

“exopeptidase activity.”

At 12 months, frontal cortex genes that were upregulated were enriched for GO terms associated with myelination as well as “oligodendrocyte differentiation” and “glial cell development,” consistent with changes in oligodendrocyte MGPs at this time point and in this tissue. The top upregulated terms in the cerebellum were “regulation of DNA biosynthetic process” and “phospholipid translocation.” Downregulated terms in the cerebellum included

“nucleotide catabolic process” and “nucleoside phosphate catabolic process.” At this later 12- month time point, in the striatum, terms associated with neural cell types such as “perikaryon” and “glial cell projection” were upregulated. Downregulated terms included “purinergic receptor activity,” “purinergic receptor signaling pathway,” and “fatty acid catabolic process.”

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3.7.3 Functional enrichment in PD-ko

Significant differential expression was observed only at the later time point in the PD-ko mice. Only the frontal cortex was available for analysis at 12 months. In this tissue, there was an enrichment of Ras and Rho signaling G protein associated GO terms in significantly up- regulated genes, and an enrichment of ribosomal subunit GO terms in down-regulated genes.

3.7.4 Functional enrichment in PD-ko+ovx

Similar to the PD-ko mice, functional enrichment in the PD-ko+ovx mice was conducted only on the 12-month frontal cortex tissue. In the 1,457 upregulated genes, the top 10 significantly enriched GO terms included four terms for protein ubiquitination, pointing to protein degradation processes in the frontal cortex that could be associated with changes in cell type proportions. Downregulated genes were mostly enriched for GO terms associated with ribosomal cellular components.

3.8 Functional enrichment overlaps

The only overlaps in enriched GO terms across models were at the 12-month time point.

Of these overlaps (see Figure 9), three were considered significant (hypergeometric test, p value

< 0.05). As observed in the analysis of gene-to-gene overlaps, the PD-ko and PD-ko+ovx models showed a high degree of overlap (Figure 9a). The enriched GO terms between the PD-ovx (a rat strain) and PD-ko+ovx (mouse) and PD-ko transgenic models (mouse) (Figure 9b, 9d) are associated with changes in cell type proportions, specifically glia and oligodendrocytes.

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Figure 9 Overlaps in functionally enriched GO terms at the 12-month time point B) The seven overlapping GO terms are, listed in order of significance: ensheathment of neurons, axon ensheathment, glial cell differentiation, oligodendrocyte development, glial cell development, myelination, and oligodendrocyte differentiation. C) The three overlapping GO terms are: glial cell differentiation, glial cell development, and basal part of cell.

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Chapter 4: Discussion and conclusions

My analysis of RNAseq data from four transgenic rodent models – SCA3-84q, PD-ovx,

PD-ko+ovx, and PD-ko – was conducted at two different time points and in three different tissues for each model. The results of differential expression analysis generally showed more dramatic changes at the later time points in two models (PD-ko and PD-ko+ovx), while the other two models (SCA3-84q and PD-ovx) had changes at both early and late time points. Overlaps of genes yielded a significantly similar result; the PD-ko+ovx and PD-ko mice have extremely similar expression profiles, to such an extent that the effect of the overexpressed transgene unexpectedly appears negligible. Direct gene-to-gene overlaps were not significant between models other than the two noted previously, but there were significant overlaps in the functionally enriched terms in the PD-ovx and PD-ko+ovx and the PD-ovx and SCA3-84q comparisons. Estimations of cell type proportional changes in all four transgenic models are consistent with the overlap in functionally enrichment terms – i.e. functionally enriched terms can also serve as proxies for cell type proportional changes. The analysis revealed commonalities across models in the marker gene profiles (MGPs) associated with endothelial cells and oligodendrocytes. Here I will discuss these findings along with limitations and implications for future work.

4.1 SCA3-84q differential expression results in context of existing literature on transgenic lines

I identified differentially expressed (DE) genes in the four available transgenic models across and within the three available brain regions. DE genes in the SCA3-84q model were evident in the cerebellum at two months (Figure 10a), but this effect was not maintained at 12 months, which does not exactly match the original characterization of this transgenic model. The 47

original characterization found evidence of Purkinje cell loss in the cerebellum,55 which should be evident in transcriptomic changes in this tissue, and which I did not observe. However, in support of my findings, another very recent study of this transgenic line in which RNAseq was performed at 17.5 months in the cerebellum also found very little differential expression in this tissue.60 The same study also reported no dramatic phenotypes in these mice even at 17.5 months, which is contrary to the original characterization of this transgenic line.55 This apparent contradiction is unresolved but suggests some genetic drift from the initial studies of the models.

Genetic drift is known to occur in transgenic models, and it is for this reason that the Jackson

Laboratory suggests refreshing breeders on a regular basis.

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Figure 10 Summary of differential expression in the context of model behavioural and physiological characteristics The known characteristics of each transgenic model are shown on the top of each timeline, with the appropriate citations. Differential expression is shown below each timeline; Uniform p-value distributions, and therefore a lack of DE are marked with a *, and a lack of tissue samples are marked as -.

4.2 Overlap between PD-ko and PD-ko+ovx transgenic models

My differential expression analysis revealed an overlap of DE genes in two models; the

PD-ko model, which is a knockout of endogenous mouse Snca, and the PD-ko+ovx model, which overexpresses human SNCA in a Scna-KO background. The overlaps were consistently observed in the analysis of differential expression common to all available regions, within tissue differential expression analysis, functional enrichment, and cell type proportional estimation.

Differential expression analysis directly comparing the PD-ko+ovx model to the PD-ko model

(rather than to wildtype controls) revealed very little to no differential expression, even at 12 months. An intriguing area of future work, then, is that of the effect of human SNCA on the mouse transcriptome. This could be assisted with the genomic examination of and comparison to

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a related existing transgenic mouse line,87 which overexpresses human SNCA while maintaining expression of the endogenous mouse Snca gene.

While I found some direct gene-to-gene overlaps between the rat strain overexpressing the human SNCA gene (PD-ovx) and the mouse strains PD-ko and PD-ko+ovx, these results were not significant. The three models did share commonalities in their functionally enriched terms at the 12-month time point, with the similarities between the two mouse lines being much greater than that of the similarities between the rat and mouse strains. This suggests either that the effect of human SNCA is different on the transcriptomes of different species, or that the existence of the endogenous rat Snca in the PD-ovx model compensates for whatever effects the human gene may have on the transcriptome.

4.3 MGPs of endothelial cells and oligodendrocytes show proportional changes in three models

At the early time point, endothelial cells showed decreases in their corresponding MGPs in the PD-ovx model and increases in PD-ko mice and PD-ko+ovx mice. Endothelial cells are part of the vascular network that supports the brain, which is key in the regulation of cerebral blood flow and the maintenance of the blood-brain barrier. Early changes to both these areas are found in PD, AD, and HD. Thus far abnormalities with regard to vascularization have been suggested as biomarkers of preclinical stages of disease, rather than as causative agents.4

Therefore, shifts in endothelial cell proportions may be early markers of disease-like phenotypes in the transgenic models.

Shifts in oligodendrocyte MGPs, suggestive of changes in the proportion of this cell type at the 12-month time point (Figure 7, 8) showed that the SCA3-84q striatum (FDR < 0.1) and

PD-ovx frontal cortex tissues share an increase of oligodendrocyte MGPs when compared to 50

controls, while the PD-ko and PD-ko+ovx models show a decrease (FDR < 0.05). Interestingly, mature oligodendrocytes do not normally express α-synuclein88 (it is thought to be expressed exclusively in neurons89) which would imply that knocking out Snca should only have indirect effects on oligodendrocytes. The degree to which Snca affects oligodendrocytes has been investigated previously due to evidence that multiple system atrophy (MSA, another ND) causes cytoplasmic α-synuclein inclusions in oligodendrocytes,88 suggesting that aberrant expression of

α-synuclein is possible in this cell type. A study in which transgenic α-synuclein was expressed in oligodendrocytes without expression in any other brain cell types, insoluble fibrils were seen in this cell type, which is also a phenotype of MSA patients.88 The latest work in this field reports that α-synuclein does show expression in oligodendrocyte precursor cells, and that incubating these precursor cells with pre-formed α-synuclein fibrils results in the differentiation of the precursors into oligodendrocytes with α-synuclein aggregates.89 While the source of α- synuclein in mature oligodendrocytes remains nebulous, my results suggest that oligodendrocytes may be sensitive to changes in α-synuclein expression.

4.4 Overlaps in functional enrichment

After conducting functional enrichment on the DE genes from each transgenic model, I assessed the overlap in functionally enriched Gene Ontology (GO) terms. Overlaps were only observed at the later 12-month time point (Figure 9). These overlapping terms, when significant, were associated with genes which are expressed specifically in glia. My interpretation is that the

GO enrichment results are a less direct reflection of the effects we observe more directly with the

MGP analysis, and therefore are consistent with changes in oligodendrocyte proportions.

At the earlier time points, functionally enriched terms associated with ubiquitination, and therefore protein degradation, were shown in the SCA3-84q and PD-ovx models, but were not 51

found in the overlap analysis. One explanation can be found in the noisiness of the various GO terms associated with protein degradation and ubiquitin – “ubiquitin conjugating enzyme activity”, “ubiquitin-like protein conjugating enzyme activity”, “protein polyubiquitination,” and

“ubiquitin-like protein-specific protease activity” are a few examples. Another possibility is that these processes are also associated with cell types – likely microglia – but because I did not have accurate MGPs for all possible rodent brain cell types, this is hard to evaluate.

It should be noted that a 2011 study showed that both wild-type and expanded ataxin-3 interact directly with parkin by deubiquitination.25 The same study also intriguingly revealed that in a transgenic mouse model of SCA3, mutant ataxin-3 but not wild-type ataxin-3 plays a role in the clearance of parkin via the autophagy pathway.25 Taken together, the findings imply that decreased parkin levels may contribute to the pathology in SCA3 and could account for parkinsonism in SCA3. My findings add evidence to the already existing knowledge of pathological protein aggregation in both SCA3 and PD.

4.5 Limitations and future work

While this study revealed some overlaps in DE in transgenic models of PD and SCA3, some experimental characteristics limited their interpretability. As I have discussed, observed overlaps corroborated evidence of commonalities in pathways that are altered by NDs, mainly in pathways related to protein aggregation, and the selective vulnerability of cell types,2 but did not allow the exploration of common early mechanisms that may cause aggregation and other similarities that characterize NDs. A lack of differential expression at the two-month time point in two models, PD-ko and PD-ko+ovx, suggests that this time point was too early for ascertaining overlaps in mechanisms via differential expression analysis, or that the phenotypes of these transgenic models are too subtle to observe transcriptomic overlaps at this level of 52

analysis. Another possibility is that these four rodent models simply do not share many overlaps in mechanisms at early time points. A further limitation was that at the later time point, data availability was limited to the frontal cortex in two of the rodent models, which further limited interpretation of overlaps to this specific tissue.

The use of transgenic models to study NDs has drawbacks, as the phenotypes of human diseases are quite difficult to recapitulate exactly.48–50 With regard to protein aggregation in the transgenic lines used in this thesis, for example, only the SCA3-84q model was shown to contain intranuclear inclusions.55 In this vein, the results of my cell type proportion estimations can be tested directly with histochemical techniques, and could add more data to the ability of these transgenic models to mimic neuronal vulnerability in human NDs. In addition to further biological validation of my results, the use of different transgenic models within the same analysis pipeline may better allow the examination of overlaps between NDs. Specific mechanisms or downstream compensations for protein aggregation could better be ascertained with newer animal models in which α-synuclein preformed fibrils (PFFs) are injected intrastriatally and trigger normal endogenous levels of α-synuclein to misfold and accumulate into Lewy body-like inclusions.90 This PFF model mimics many features of human idiopathic

PD, including a protracted course of aggregation and degeneration events that provide investigators the ability to focus on particular phases of disease. The model also shows degeneration of specific neuronal subpopulations.90

4.6 Conclusions

Genomics approaches using transgenic models of disease will continue to remain one of the avenues of investigation in the search for understanding the mechanisms that underly NDs. In this thesis I have implemented a comparative analysis of four transgenic models relevant to 53

SCA3 and PD. My analysis highlighted the unreported similarity in the expression pattern of two transgenic models, calling to attention the need to treat these models as related perturbations rather than two separate transgenic examinations. I have also shown that with an examination of changes in cell type proportions, overlaps in differential expression were revealed to be associated with proportional shifts in specific cell types, as opposed to global transcriptional changes or cell-type specific transcriptional changes. An application of a similar approach with more relevant transgenic models and controlling for the effects of cell type proportional changes may revel mechanisms common to transgenic models of different NDs.

54

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64

Appendices

Appendix A

The top 50 differentially expressed genes, from Table 2, with their log fold changes, full gene name, and FDRs are shown here.

A.1 Upregulated genes (FDR = 0.05) in SCA3-84q at 2 months in cerebellum

ENSEMBL SYMBOL GENENAME logFC adj.P.Val ENSMUSG00000003657 Calb2 calbindin 2 0.325415 0.03429 ENSMUSG00000004771 Rab11a RAB11A, member RAS oncogene family 0.389603 0.03429 ENSMUSG00000018589 Glra2 glycine receptor, alpha 2 subunit 0.627586 0.036862 ENSMUSG00000018846 Pank3 pantothenate kinase 3 0.326121 0.036862 ENSMUSG00000001687 Ubl3 ubiquitin-like 3 0.220402 0.037198 ENSMUSG00000017412 Cacnb4 calcium channel, voltage-dependent, beta 4 subunit 0.37779 0.037198 ENSMUSG00000017418 Arl5b ADP-ribosylation factor-like 5B 0.373267 0.037198 ENSMUSG00000020362 Cnot6 CCR4-NOT transcription complex, subunit 6 0.343822 0.037198 ENSMUSG00000020572 Nampt nicotinamide phosphoribosyltransferase 0.329646 0.037198 ENSMUSG00000021072 Tmx1 thioredoxin-related transmembrane protein 1 0.374952 0.037198 ENSMUSG00000021796 Bmpr1a bone morphogenetic protein receptor, type 1A 0.248682 0.037198 ENSMUSG00000010290 AI597479 expressed sequence AI597479 0.28476 0.037376 ENSMUSG00000012422 Tmem167 transmembrane protein 167 0.400865 0.037376 ENSMUSG00000010803 Gabra1 gamma-aminobutyric acid (GABA) A receptor, subunit alpha 1 0.269744 0.03738 ENSMUSG00000014956 Ppp1cb protein phosphatase 1 catalytic subunit beta 0.46781 0.03738 ENSMUSG00000020189 Osbpl8 oxysterol binding protein-like 8 0.322683 0.03738 ENSMUSG00000019874 Fabp7 fatty acid binding protein 7, brain 0.616214 0.0386 ENSMUSG00000000058 Cav2 caveolin 2 0.516155 0.039144 dual specificity phosphatase 3 (vaccinia virus phosphatase VH1- ENSMUSG00000003518 Dusp3 related) 0.2328 0.039144 ENSMUSG00000006699 Cdc42 cell division cycle 42 0.30618 0.039144 ENSMUSG00000019775 Rgs17 regulator of G-protein signaling 17 0.360357 0.039144 65

ENSEMBL SYMBOL GENENAME logFC adj.P.Val ENSMUSG00000021712 Trim23 tripartite motif-containing 23 0.358992 0.039144 ENSMUSG00000019966 Kitl kit ligand 0.305779 0.039364 ENSMUSG00000011427 Zfp790 zinc finger protein 790 0.246536 0.04039 ENSMUSG00000020577 Tspan13 tetraspanin 13 0.260718 0.040523 ENSMUSG00000007812 Zfp655 zinc finger protein 655 0.296655 0.041062 ENSMUSG00000000948 NA NA 0.623442 0.041136 ENSMUSG00000007653 Gabrb2 gamma-aminobutyric acid (GABA) A receptor, subunit beta 2 0.261221 0.041136 ENSMUSG00000009207 Lnpk lunapark, ER junction formation factor 0.295975 0.041136 ENSMUSG00000015932 Dstn destrin 0.221164 0.041136 ENSMUSG00000019877 Serinc1 serine incorporator 1 0.266497 0.042149 ENSMUSG00000015733 Capza2 capping protein (actin filament) muscle Z-line, alpha 2 0.327251 0.042166 ENSMUSG00000019779 Frk fyn-related kinase 0.818042 0.043688 ENSMUSG00000020385 Clk4 CDC like kinase 4 0.231622 0.043723 ENSMUSG00000017776 Crk v-crk avian sarcoma virus CT10 oncogene homolog 0.291605 0.043978 ENSMUSG00000019906 Lin7a lin-7 homolog A (C. elegans) 0.22696 0.045097 ENSMUSG00000020130 Tbc1d15 TBC1 domain family, member 15 0.247395 0.045097 ENSMUSG00000020925 Ccdc43 coiled-coil domain containing 43 0.247421 0.045564 ENSMUSG00000020859 Spag9 sperm associated antigen 9 0.286628 0.045778 ENSMUSG00000001260 Gabrg1 gamma-aminobutyric acid (GABA) A receptor, subunit gamma 1 0.356564 0.046144 ENSMUSG00000020794 Ube2g1 ubiquitin-conjugating enzyme E2G 1 0.274944 0.046144 ENSMUSG00000009030 Pdcl phosducin-like 0.207537 0.046731 ENSMUSG00000020390 Ube2b ubiquitin-conjugating enzyme E2B 0.244671 0.046848 ENSMUSG00000020590 Snx13 sorting nexin 13 0.235735 0.047034 ENSMUSG00000005674 Tomm40l translocase of outer mitochondrial membrane 40-like 0.257432 0.047843 ENSMUSG00000017057 Il13ra1 interleukin 13 receptor, alpha 1 0.365658 0.047857 ENSMUSG00000000581 C1d C1D nuclear receptor co-repressor 0.42113 0.048796 ENSMUSG00000019818 Cd164 CD164 antigen 0.213247 0.049719 ENSMUSG00000015341 Golga7 golgi autoantigen, golgin subfamily a, 7 0.262368 0.049895 ENSMUSG00000017831 Rab5a RAB5A, member RAS oncogene family 0.292616 0.049895

66

A.2 Downregulated genes (FDR = 0.05) in SCA3-84q at 2 months in cerebellum

ENSEMBL SYMBOL GENENAME logFC adj.P.Val ENSMUSG00000001151 Pcnt pericentrin (kendrin) -0.48031 0.03429 ENSMUSG00000002771 Grin2d glutamate receptor, ionotropic, NMDA2D (epsilon 4) -0.53849 0.03429 ENSMUSG00000004562 Arhgef40 Rho guanine nucleotide exchange factor (GEF) 40 -0.36969 0.036853 ENSMUSG00000000567 Sox9 SRY (sex determining region Y)-box 9 -0.43406 0.037198 ENSMUSG00000001270 Ckb creatine kinase, brain -0.33838 0.037198 ENSMUSG00000001855 Nup214 nucleoporin 214 -0.43507 0.037198 ENSMUSG00000001985 Grik3 glutamate receptor, ionotropic, kainate 3 -0.35314 0.037198 ENSMUSG00000004054 Map3k11 mitogen-activated protein kinase kinase kinase 11 -0.34194 0.037198 ENSMUSG00000005397 Nid1 nidogen 1 -0.279 0.037198 ENSMUSG00000007817 Zmiz1 zinc finger, MIZ-type containing 1 -0.54574 0.037198 ENSMUSG00000011751 Sptbn4 spectrin beta, non-erythrocytic 4 -0.45186 0.037198 ENSMUSG00000008496 Pou2f2 POU domain, class 2, transcription factor 2 -0.43236 0.037376 ENSMUSG00000004929 Thop1 thimet oligopeptidase 1 -0.29275 0.03738 ENSMUSG00000001435 Col18a1 collagen, type XVIII, alpha 1 -0.34277 0.038267 ENSMUSG00000003660 Snrnp200 small nuclear ribonucleoprotein 200 (U5) -0.41587 0.038267 ENSMUSG00000005045 Chd5 chromodomain helicase DNA binding protein 5 -0.42572 0.038267 ENSMUSG00000010825 Grid2ip glutamate receptor, ionotropic, delta 2 (Grid2) interacting protein 1 -0.33028 0.038267 ENSMUSG00000000631 Myo18a myosin XVIIIA -0.3828 0.0386 ENSMUSG00000001507 Itga3 integrin alpha 3 -0.35548 0.0386 ENSMUSG00000006276 Eps15l1 epidermal growth factor receptor pathway substrate 15-like 1 -0.25981 0.0386 ENSMUSG00000001034 Mapk7 mitogen-activated protein kinase 7 -0.29334 0.039144 ENSMUSG00000001424 Snd1 staphylococcal nuclease and tudor domain containing 1 -0.29204 0.039144 ENSMUSG00000001632 Brpf1 bromodomain and PHD finger containing, 1 -0.31079 0.039144 ENSMUSG00000002486 Tchp trichoplein, keratin filament binding -0.31711 0.039144 ENSMUSG00000006930 Hap1 huntingtin-associated protein 1 -0.31508 0.039144 ENSMUSG00000010066 Cacna2d2 calcium channel, voltage-dependent, alpha 2/delta subunit 2 -0.37344 0.039144 ENSMUSG00000007880 Arid1a AT rich interactive domain 1A (SWI-like) -0.44701 0.039926 ENSMUSG00000000538 Tom1l2 target of myb1-like 2 (chicken) -0.21489 0.040523 67

ENSEMBL SYMBOL GENENAME logFC adj.P.Val ENSMUSG00000003352 Cacnb3 calcium channel, voltage-dependent, beta 3 subunit -0.26747 0.040523 ENSMUSG00000002968 Med25 mediator complex subunit 25 -0.35847 0.040839 ENSMUSG00000001930 Vwf Von Willebrand factor -0.56445 0.041136 ENSMUSG00000002496 Tsc2 tuberous sclerosis 2 -0.33867 0.041136 ENSMUSG00000003068 Stk11 serine/threonine kinase 11 -0.22217 0.041136 ENSMUSG00000003378 Grik5 glutamate receptor, ionotropic, kainate 5 (gamma 2) -0.29113 0.041136 ENSMUSG00000004263 Atn1 atrophin 1 -0.5412 0.041136 ENSMUSG00000007030 Vwa7 von Willebrand factor A domain containing 7 -0.30915 0.041136 ENSMUSG00000007216 Zfp775 zinc finger protein 775 -0.29152 0.041136 ENSMUSG00000007594 Hapln4 hyaluronan and proteoglycan link protein 4 -0.3001 0.041136 ENSMUSG00000002489 Tiam1 T cell lymphoma invasion and metastasis 1 -0.29691 0.041272 ENSMUSG00000006307 Kmt2b lysine (K)-specific methyltransferase 2B -0.35934 0.043586 sema domain, transmembrane domain (TM), and cytoplasmic domain, ENSMUSG00000001227 Sema6b (semaphorin) 6B -0.19958 0.043688 ENSMUSG00000005514 Por P450 (cytochrome) oxidoreductase -0.26839 0.043688 ENSMUSG00000003363 Pld3 phospholipase D family, member 3 -0.34398 0.043723 ENSMUSG00000007029 Vars valyl-tRNA synthetase -0.33299 0.043763 ENSMUSG00000003575 Crtc1 CREB regulated transcription coactivator 1 -0.33341 0.045778 ENSMUSG00000005198 Polr2a polymerase (RNA) II (DNA directed) polypeptide A -0.45062 0.046144 ENSMUSG00000007833 Aldh16a1 aldehyde dehydrogenase 16 family, member A1 -0.2997 0.046731 ENSMUSG00000002052 Supt6 suppressor of Ty 6 -0.29849 0.046738 ENSMUSG00000001911 Nfix nuclear factor I/X -0.3401 0.046979 ENSMUSG00000009292 Trpm2 transient receptor potential cation channel, subfamily M, member 2 -0.2439 0.04748

A.3 Upregulated genes (FDR = 0.05) in SCA3-84q at 12 months, common to all tissues

ENSEMBL SYMBOL GENENAME logFC adj.P.Val ENSMUSG00000106793 NA NA 3.799103 6.16E-07

68

ENSEMBL SYMBOL GENENAME logFC adj.P.Val ENSMUSG00000024640 Psat1 phosphoserine aminotransferase 1 0.309502 0.011951 ENSMUSG00000073600 Prob1 proline rich basic protein 1 0.526761 0.011951

A.4 Upregulated genes (FDR = 0.05) in SCA3-84q at 12 months in striatum

ENSEMBL SYMBOL GENENAME logFC adj.P.Val ENSMUSG00000002910 Arrdc2 arrestin domain containing 2 0.677928 0.017813 ENSMUSG00000001999 Blvra biliverdin reductase A 0.452284 0.023874 ENSMUSG00000003380 Rabac1 Rab acceptor 1 (prenylated) 0.6717 0.023874 ENSMUSG00000005069 Pex5 peroxisomal biogenesis factor 5 0.459946 0.023874 ENSMUSG00000000399 Ndufa9 NADH:ubiquinone oxidoreductase subunit A9 0.520459 0.024274 ENSMUSG00000000743 Chmp1a charged multivesicular body protein 1A 0.561365 0.024274 ENSMUSG00000000869 Il4 interleukin 4 1.02803 0.024274 ENSMUSG00000001741 Il16 interleukin 16 0.550168 0.024274 ENSMUSG00000003166 Dgcr2 DiGeorge syndrome critical region gene 2 0.27595 0.024274 ENSMUSG00000004610 Etfb electron transferring flavoprotein, beta polypeptide 0.629681 0.024274 ENSMUSG00000005779 Psmb4 proteasome (prosome, macropain) subunit, beta type 4 0.719015 0.024274 ENSMUSG00000002064 Sdf2 stromal cell derived factor 2 0.564125 0.026175 ENSMUSG00000005161 Prdx2 peroxiredoxin 2 0.485149 0.027736 ENSMUSG00000002379 Ndufa11 NADH:ubiquinone oxidoreductase subunit A11 0.396001 0.028216 ENSMUSG00000004945 Tmem242 transmembrane protein 242 0.54002 0.03019 ENSMUSG00000004268 Emg1 EMG1 N1-specific pseudouridine methyltransferase 0.417183 0.030207 ENSMUSG00000001416 Cct3 chaperonin containing Tcp1, subunit 3 (gamma) 0.508593 0.030467 ENSMUSG00000000194 Gpr107 G protein-coupled receptor 107 0.252962 0.030973 ENSMUSG00000002416 Ndufb2 NADH:ubiquinone oxidoreductase subunit B2 0.579867 0.030973 ENSMUSG00000003849 Nqo1 NAD(P)H dehydrogenase, quinone 1 0.461005 0.030973 ENSMUSG00000004207 Psap prosaposin 0.368706 0.03155 ENSMUSG00000000088 Cox5a cytochrome c oxidase subunit 5A 0.689049 0.031722 69

ENSEMBL SYMBOL GENENAME logFC adj.P.Val ENSMUSG00000001098 Kctd10 potassium channel tetramerisation domain containing 10 0.290899 0.032022 ENSMUSG00000004849 Ap1s1 adaptor protein complex AP-1, sigma 1 0.361509 0.033034 ENSMUSG00000001366 Fbxo9 f-box protein 9 0.264955 0.033431 ENSMUSG00000002345 Borcs8 BLOC-1 related complex subunit 8 0.315342 0.033518 ENSMUSG00000004071 Cdip1 cell death inducing Trp53 target 1 0.223741 0.033795 ENSMUSG00000003923 Tfam transcription factor A, mitochondrial 0.353262 0.034029 ENSMUSG00000000441 Raf1 v-raf-leukemia viral oncogene 1 0.269771 0.034444 ENSMUSG00000004393 Ddx56 DEAD (Asp-Glu-Ala-Asp) box polypeptide 56 0.419999 0.034502 ENSMUSG00000003062 Stard3nl STARD3 N-terminal like 0.67799 0.034522 ENSMUSG00000005674 Tomm40l translocase of outer mitochondrial membrane 40-like 0.359047 0.035579 ENSMUSG00000003355 Fkbp11 FK506 binding protein 11 0.815575 0.035955 ENSMUSG00000000738 Spg7 SPG7, paraplegin matrix AAA peptidase subunit 0.407718 0.037361 ENSMUSG00000002455 Prpf6 pre-mRNA splicing factor 6 0.262504 0.03766 succinate dehydrogenase complex, subunit D, integral membrane ENSMUSG00000000171 Sdhd protein 0.344405 0.037851 ENSMUSG00000003199 Mpnd MPN domain containing 0.386644 0.037868 dual specificity phosphatase 3 (vaccinia virus phosphatase VH1- ENSMUSG00000003518 Dusp3 related) 0.238499 0.037868 ENSMUSG00000001482 Def8 differentially expressed in FDCP 8 0.205403 0.038334 ENSMUSG00000004394 Tmed4 transmembrane p24 trafficking protein 4 0.216237 0.040779 ENSMUSG00000003429 Rps11 ribosomal protein S11 0.455175 0.041349 ENSMUSG00000002983 Relb avian reticuloendotheliosis viral (v-rel) oncogene related B 0.545266 0.041926 ENSMUSG00000001100 Poldip2 polymerase (DNA-directed), delta interacting protein 2 0.210376 0.042727 ENSMUSG00000001946 Esam endothelial cell-specific adhesion molecule 0.363215 0.04281 ENSMUSG00000005699 Pard6a par-6 family cell polarity regulator alpha 0.377779 0.045312 sema domain, immunoglobulin domain (Ig), TM domain, and short ENSMUSG00000000627 Sema4f cytoplasmic domain 0.37388 0.045463 ENSMUSG00000001642 Akr1b3 aldo-keto reductase family 1, member B3 (aldose reductase) 0.358088 0.045679 ENSMUSG00000000149 Gna12 guanine nucleotide binding protein, alpha 12 0.243866 0.046726 ENSMUSG00000004936 Map2k1 mitogen-activated protein kinase kinase 1 0.219248 0.048064 70

ENSEMBL SYMBOL GENENAME logFC adj.P.Val ENSMUSG00000003721 Insig2 insulin induced gene 2 0.283592 0.048711

A.5 Downregulated genes (FDR = 0.05) in SCA3-84q at 12 months in striatum

ENSEMBL SYMBOL GENENAME logFC adj.P.Val ENSMUSG00000009216 Fam163b family with sequence similarity 163, member B -0.56457 0.002809 ENSMUSG00000000567 Sox9 SRY (sex determining region Y)-box 9 -0.66314 0.008594 ENSMUSG00000008496 Pou2f2 POU domain, class 2, transcription factor 2 -0.58486 0.017316 ENSMUSG00000001833 Sep7 septin 7 -0.50556 0.020996 ENSMUSG00000007682 Dio2 deiodinase, iodothyronine, type II -0.5284 0.020996 ENSMUSG00000010608 Rbm25 RNA binding motif protein 25 -0.39349 0.023874 ENSMUSG00000002578 Ikzf4 IKAROS family zinc finger 4 -0.43195 0.024274 ENSMUSG00000006586 Runx1t1 RUNX1 translocation partner 1 -0.6452 0.024274 ENSMUSG00000008200 Fnbp4 formin binding protein 4 -0.2851 0.024274 ENSMUSG00000003929 Zfp81 zinc finger protein 81 -0.3934 0.025214 ENSMUSG00000000861 Bcl11a B cell CLL/lymphoma 11A (zinc finger protein) -0.35676 0.02634 ENSMUSG00000006262 Mob1b MOB kinase activator 1B -0.55363 0.027201 ENSMUSG00000007817 Zmiz1 zinc finger, MIZ-type containing 1 -0.55273 0.027511 ENSMUSG00000003279 Dlgap1 DLG associated protein 1 -0.4432 0.028216 ENSMUSG00000009687 Fxyd5 FXYD domain-containing ion transport regulator 5 -0.50992 0.028311 ENSMUSG00000003411 Rab3b RAB3B, member RAS oncogene family -0.3175 0.030973 ENSMUSG00000004187 Kifc2 kinesin family member C2 -0.23031 0.030973 ENSMUSG00000005442 Cic capicua transcriptional repressor -0.38982 0.030973 ENSMUSG00000014592 Camta1 calmodulin binding transcription activator 1 -0.51594 0.030973 ENSMUSG00000002617 Zfp40 zinc finger protein 40 -0.45366 0.031172 ENSMUSG00000008859 Rala v-ral simian leukemia viral oncogene A (ras related) -0.33168 0.031291 ENSMUSG00000014767 Tbp TATA box binding protein -0.33648 0.031291 ENSMUSG00000008658 Rbfox1 RNA binding protein, fox-1 homolog (C. elegans) 1 -0.4356 0.03154 ENSMUSG00000003974 Grm3 glutamate receptor, metabotropic 3 -0.31303 0.032022 ENSMUSG00000005732 Ranbp1 RAN binding protein 1 -0.29784 0.032974 ENSMUSG00000004110 Cacna1e calcium channel, voltage-dependent, R type, alpha 1E subunit -0.36538 0.033034 71

ENSEMBL SYMBOL GENENAME logFC adj.P.Val ENSMUSG00000000632 Sez6 seizure related gene 6 -0.30187 0.033431 ENSMUSG00000007880 Arid1a AT rich interactive domain 1A (SWI-like) -0.40104 0.033431 ENSMUSG00000013921 Clip3 CAP-GLY domain containing linker protein 3 -0.27003 0.033795 ENSMUSG00000012114 Med15 mediator complex subunit 15 -0.24305 0.033837 ENSMUSG00000000600 Krit1 KRIT1, ankyrin repeat containing -0.29511 0.034444 ENSMUSG00000007030 Vwa7 von Willebrand factor A domain containing 7 -0.6556 0.035043 ENSMUSG00000003949 Hlf hepatic leukemia factor -0.41019 0.035905 ENSMUSG00000007670 Khsrp KH-type splicing regulatory protein -0.28128 0.036309 ENSMUSG00000005583 Mef2c myocyte enhancer factor 2C -0.43438 0.037225 ENSMUSG00000005371 Fbxo11 F-box protein 11 -0.33044 0.03766 ENSMUSG00000014813 Stc1 stanniocalcin 1 -0.51709 0.037851 ENSMUSG00000007877 Tcap titin-cap -0.96828 0.039551 ENSMUSG00000005686 Ampd3 adenosine monophosphate deaminase 3 -0.35249 0.043698 ENSMUSG00000001985 Grik3 glutamate receptor, ionotropic, kainate 3 -0.23308 0.043862 ENSMUSG00000002107 Celf2 CUGBP, Elav-like family member 2 -0.32485 0.043862 ENSMUSG00000004364 Cul3 cullin 3 -0.3049 0.045172 ENSMUSG00000000560 Gabra2 gamma-aminobutyric acid (GABA) A receptor, subunit alpha 2 -0.54001 0.045463 ENSMUSG00000002341 Ncan neurocan -0.20331 0.045846 ENSMUSG00000001911 Nfix nuclear factor I/X -0.3981 0.047031 ENSMUSG00000005312 Ubqln1 ubiquilin 1 -0.25589 0.048667 ENSMUSG00000008307 1700109H08Rik RIKEN cDNA 1700109H08 gene -0.81672 0.049844 ENSMUSG00000007815 Rhoa ras homolog family member A -0.19109 0.049852 ENSMUSG00000004263 Atn1 atrophin 1 -0.47024 0.049895 ENSMUSG00000000402 Egfl6 EGF-like-domain, multiple 6 -0.50581 0.049918

A.6 Upregulated genes (FDR = 0.05) in PD-ovx at 5 months, common to all tissues

ENSEMBL SYMBOL GENENAME logFC adj.P.Val ENSRNOG00000017286 Ephx2 epoxide hydrolase 2 3.648385 6.32E-15 ENSRNOG00000012067 Fam111a family with sequence similarity 111, member A 5.464553 3.71E-14 ENSRNOG00000055020 NA NA 3.784522 2.20E-12 72

ENSEMBL SYMBOL GENENAME logFC adj.P.Val ENSRNOG00000058006 Sncg synuclein, gamma 3.563522 1.07E-11 ENSRNOG00000006639 Scn9a sodium voltage-gated channel alpha subunit 9 3.131844 1.63E-08 ENSRNOG00000007657 Col27a1 collagen type XXVII alpha 1 chain 1.64911 4.68E-07 ENSRNOG00000038905 NA NA 1.078492 2.38E-06 ENSRNOG00000007033 Sorcs2 sortilin-related VPS10 domain containing receptor 2 0.767323 1.04E-05 ENSRNOG00000020424 Plpp4 phospholipid phosphatase 4 1.588305 3.49E-05 ENSRNOG00000001427 Orai2 ORAI calcium release-activated calcium modulator 2 0.849777 4.11E-05 ENSRNOG00000001368 Rph3a rabphilin 3A 0.740247 6.21E-05 ENSRNOG00000005016 Tmed4 transmembrane p24 trafficking protein 4 0.465794 6.21E-05 ENSRNOG00000009372 Tacr3 tachykinin receptor 3 4.503797 8.66E-05 ENSRNOG00000005093 Lgr6 leucine-rich repeat-containing G protein-coupled receptor 6 2.07384 0.000133 ENSRNOG00000014166 Smoc2 SPARC related modular calcium binding 2 2.59236 0.000157 ENSRNOG00000025890 Opa3 OPA3, outer mitochondrial membrane lipid metabolism regulator 0.431418 0.000164 ENSRNOG00000000407 Dcbld1 discoidin, CUB and LCCL domain containing 1 1.239161 0.000264 ENSRNOG00000028041 Tnnt1 troponin T1, slow skeletal type 1.616227 0.000512 ENSRNOG00000005960 RGD1311744 similar to RIKEN cDNA 5830475I06 1.502798 0.005807 ENSRNOG00000016326 Cx3cl1 C-X3-C motif chemokine ligand 1 0.632082 0.006558 ENSRNOG00000014090 Retsat retinol saturase 0.761791 0.008034 ENSRNOG00000061102 Taf9b TATA-box binding protein associated factor 9b 0.488628 0.008243 ENSRNOG00000010011 Osbpl3 oxysterol binding protein-like 3 0.756536 0.008474 ENSRNOG00000007748 Tex15 testis expressed 15, meiosis and synapsis associated 2.318155 0.012811 ENSRNOG00000022983 Trim17 tripartite motif-containing 17 1.2371 0.012904 ENSRNOG00000002866 Rassf6 Ras association domain family member 6 1.433201 0.013385 ENSRNOG00000000963 Tmem132c transmembrane protein 132C 1.370769 0.013567 ENSRNOG00000008554 Slc9a9 solute carrier family 9 member A9 0.519996 0.014431 ENSRNOG00000016434 Prkd2 protein kinase D2 0.733241 0.01568 ENSRNOG00000058611 NA NA 1.200001 0.01568 ENSRNOG00000037595 Gpbp1l1 GC-rich promoter binding protein 1-like 1 0.278478 0.016145 ENSRNOG00000058938 Camkv CaM kinase-like vesicle-associated 0.507431 0.016581 ENSRNOG00000004281 Cobl cordon-bleu WH2 repeat protein 0.608148 0.016703 73

ENSEMBL SYMBOL GENENAME logFC adj.P.Val ENSRNOG00000061779 Man2b2 mannosidase, alpha, class 2B, member 2 0.4741 0.016703 ENSRNOG00000028910 NA NA 0.880611 0.020058 ENSRNOG00000010107 NA NA 0.752058 0.022244 ENSRNOG00000042201 Efcab2 EF-hand calcium binding domain 2 0.973395 0.022244 ENSRNOG00000019825 Zdhhc24 zinc finger, DHHC-type containing 24 0.484717 0.026718 ENSRNOG00000009571 Wipf3 WAS/WASL interacting protein family, member 3 0.645549 0.030188 ENSRNOG00000052519 Ttc30a1 tetratricopeptide repeat domain 30A1 0.961346 0.030379 ENSRNOG00000001707 Vwa5b2 von Willebrand factor A domain containing 5B2 0.488027 0.033685 ENSRNOG00000043866 NA NA 0.37061 0.033815 ENSRNOG00000030983 B3galt5 Beta-1,3-galactosyltransferase 5 0.430571 0.041151 ENSRNOG00000021249 Ap5s1 adaptor related protein complex 5 subunit sigma 1 0.720937 0.041671 ENSRNOG00000033251 Slc25a52 solute carrier family 25, member 52 1.298573 0.04189 ENSRNOG00000017468 Trappc6a trafficking protein particle complex 6A 0.768223 0.045478 ENSRNOG00000019376 Zfp329 zinc finger protein 329 0.493177 0.045478

A.7 Downregulated genes (FDR = 0.05) in PD-ovx at 5 months, common to all tissues

ENSEMBL SYMBOL GENENAME logFC adj.P.Val ENSRNOG00000001300 P2rx4 purinergic receptor P2X 4 -1.37547 2.26E-10 ENSRNOG00000007117 Cluap1 clusterin associated protein 1 -1.06257 9.84E-06 ENSRNOG00000021521 Chst5 carbohydrate sulfotransferase 5 -1.77279 2.21E-05 ENSRNOG00000000279 Rtn4ip1 reticulon 4 interacting protein 1 -0.93249 0.000333 ENSRNOG00000005141 Hus1 HUS1 checkpoint clamp component -0.45863 0.000408 ENSRNOG00000015441 Il4r interleukin 4 receptor -1.01987 0.000853 ENSRNOG00000010834 Mpped1 metallophosphoesterase domain containing 1 -0.55832 0.000864 ENSRNOG00000001739 Meltf melanotransferrin -1.32294 0.000999 ENSRNOG00000007041 Abcg2 ATP binding cassette subfamily G member 2 -1.36939 0.00104 ENSRNOG00000010802 Ube3d ubiquitin protein ligase E3D -1.24416 0.00233 ENSRNOG00000008214 Fbxo9 f-box protein 9 -0.39837 0.002339 ENSRNOG00000007564 Evc EvC ciliary complex subunit 1 -0.87863 0.007077 74

ENSEMBL SYMBOL GENENAME logFC adj.P.Val ENSRNOG00000010940 Acad11 acyl-CoA dehydrogenase family, member 11 -0.59789 0.007077 ENSRNOG00000018822 Slc5a5 solute carrier family 5 member 5 -1.4417 0.008034 ENSRNOG00000004763 Sirpa signal-regulatory protein alpha -0.22436 0.008243 ENSRNOG00000017895 Eno1 enolase 1 -0.40506 0.009334 ENSRNOG00000021270 Trmt6 tRNA methyltransferase 6 -0.46893 0.009334 ENSRNOG00000005350 Pwp1 PWP1 homolog, endonuclein -0.56117 0.012107 ENSRNOG00000007025 Evc2 EvC ciliary complex subunit 2 -0.90958 0.012107 ENSRNOG00000014143 Col24a1 collagen type XXIV alpha 1 chain -1.58725 0.012107 ENSRNOG00000005809 Arhgdib Rho GDP dissociation inhibitor beta -0.64342 0.012811 ENSRNOG00000006591 Fam163b family with sequence similarity 163, member B -0.5045 0.013348 ENSRNOG00000021086 Dtx4 deltex E3 ubiquitin ligase 4 -0.69615 0.013567 ENSRNOG00000016696 Angpt2 angiopoietin 2 -1.04314 0.016703 ENSRNOG00000003114 B4galt4 beta-1,4-galactosyltransferase 4 -0.83252 0.016954 ENSRNOG00000013981 Ptpn5 protein tyrosine phosphatase, non-receptor type 5 -0.48391 0.018024 ENSRNOG00000013720 Aebp1 AE binding protein 1 -1.19541 0.018881 ENSRNOG00000001254 Col6a2 collagen type VI alpha 2 chain -0.83312 0.019616 ENSRNOG00000017693 Slc2a5 solute carrier family 2 member 5 -0.86013 0.019616 ENSRNOG00000001960 Sult1d1 sulfotransferase family 1D, member 1 -1.19687 0.021397 ENSRNOG00000020389 Capn12 calpain 12 -1.0723 0.021397 ENSRNOG00000008316 Vps39 VPS39 HOPS complex subunit -0.3044 0.022244 ENSRNOG00000012811 Spint1 serine peptidase inhibitor, Kunitz type 1 -0.83762 0.022244 ENSRNOG00000002343 Uchl1 ubiquitin C-terminal hydrolase L1 -0.58591 0.022557 ENSRNOG00000006595 Htr3a 5-hydroxytryptamine receptor 3A -0.82765 0.026743 ENSRNOG00000011831 Nudt18 nudix hydrolase 18 -0.45951 0.030188 ENSRNOG00000014265 Tnfrsf19 TNF receptor superfamily member 19 -0.57614 0.031613 ENSRNOG00000005457 Lamp5 lysosomal-associated membrane protein family, member 5 -0.52644 0.033815 ENSRNOG00000007862 Acat1 acetyl-CoA acetyltransferase 1 -0.42232 0.033927 ENSRNOG00000011936 Abhd14a abhydrolase domain containing 14A -0.76098 0.034584 ENSRNOG00000006033 Spon2 spondin 2 -0.76828 0.039036 ENSRNOG00000006033 LOC100910790 spondin-2-like -0.76828 0.039036 ENSRNOG00000019118 Slc13a3 solute carrier family 13 member 3 -0.81805 0.039384 ENSRNOG00000000704 Cmklr1 chemerin chemokine-like receptor 1 -0.81365 0.04189 75

ENSEMBL SYMBOL GENENAME logFC adj.P.Val ENSRNOG00000001383 Slc8b1 solute carrier family 8 member B1 -0.63982 0.04189 ENSRNOG00000012290 Gchfr GTP cyclohydrolase I feedback regulator -0.90004 0.04189 ENSRNOG00000018524 Ezr ezrin -0.53588 0.041898 ENSRNOG00000011379 Ccndbp1 cyclin D1 binding protein 1 -0.37089 0.045478 ENSRNOG00000018689 RGD1305464 similar to human chromosome 15 open reading frame 39 -0.71471 0.049589 ENSRNOG00000001158 Abcg1 ATP binding cassette subfamily G member 1 -0.39721 0.04997

A.8 Upregulated genes (FDR = 0.05) in PD-ovx at 5 months in frontal cortex

ENSEMBL SYMBOL GENENAME logFC adj.P.Val ENSRNOG00000001427 Orai2 ORAI calcium release-activated calcium modulator 2 1.229458 4.72E-06 ENSRNOG00000000811 Pkib cAMP-dependent protein kinase inhibitor beta 1.535721 0.003501 ENSRNOG00000000879 Slc9a6 solute carrier family 9 member A6 0.394611 0.009415 ENSRNOG00000000718 Cggbp1 CGG triplet repeat binding protein 1 0.392266 0.012291 ENSRNOG00000001074 Mphosph9 M-phase phosphoprotein 9 0.516135 0.013673 ENSRNOG00000000304 Cd164 CD164 molecule 0.364427 0.014097 ENSRNOG00000000161 Chm CHM, Rab escort protein 1 0.905184 0.01432 synaptotagmin binding, cytoplasmic RNA interacting ENSRNOG00000000204 Syncrip protein 0.659666 0.014501 ENSRNOG00000001311 Rab36 RAB36, member RAS oncogene family 0.685262 0.015237 ENSRNOG00000000916 Katnal1 katanin catalytic subunit A1 like 1 3.267375 0.017387 ENSRNOG00000000916 LOC100910196 katanin p60 ATPase-containing subunit A-like 1-like 3.267375 0.017387 ENSRNOG00000000916 LOC103690050 katanin p60 ATPase-containing subunit A-like 1 3.267375 0.017387 ENSRNOG00000001329 Gjc3 gap junction protein, gamma 3 1.616471 0.017803 ENSRNOG00000001130 Nos1 nitric oxide synthase 1 0.818823 0.019199 ENSRNOG00000000412 Slc35f1 solute carrier family 35, member F1 0.432454 0.019205 HECT domain and ankyrin repeat containing, E3 ubiquitin ENSRNOG00000000327 Hace1 protein ligase 1 0.406361 0.020046 ENSRNOG00000000823 Gcc2 GRIP and coiled-coil domain containing 2 0.306059 0.020771 ENSRNOG00000000082 Hltf helicase-like transcription factor 0.483365 0.021219 76

ENSEMBL SYMBOL GENENAME logFC adj.P.Val ENSRNOG00000000881 Mmgt1 membrane magnesium transporter 1 0.262566 0.021753 ENSRNOG00000001098 Pds5b PDS5 cohesin associated factor B 0.312597 0.024295 ENSRNOG00000000796 Ranbp2 RAN binding protein 2 0.477364 0.024546 ENSRNOG00000000397 Ccar1 cell division cycle and apoptosis regulator 1 0.265022 0.02853 ENSRNOG00000001064 Sbno1 strawberry notch homolog 1 0.405981 0.029504 ENSRNOG00000001317 Zfp68 zinc finger protein 68 0.364099 0.029631 ENSRNOG00000000657 Nek7 NIMA-related kinase 7 0.838181 0.031454 ENSRNOG00000001573 Usp25 ubiquitin specific peptidase 25 0.434153 0.032553 ENSRNOG00000000127 Kpna6 karyopherin subunit alpha 6 0.351386 0.032837 ENSRNOG00000001062 Kmt5a lysine methyltransferase 5A 0.305977 0.033816 ENSRNOG00000001335 Zkscan1 zinc finger with KRAB and SCAN domains 1 0.661674 0.034699 ENSRNOG00000000236 Zfp207 zinc finger protein 207 0.236881 0.034706 ENSRNOG00000000170 Slc30a4 solute carrier family 30 member 4 0.372796 0.034831 ENSRNOG00000001242 Gstt3 glutathione S-transferase, theta 3 0.421125 0.034831 ENSRNOG00000000237 RGD1310429 similar to Protein Njmu-R1 0.589583 0.035547 ENSRNOG00000000886 Caln1 calneuron 1 0.783702 0.035639 ENSRNOG00000000277 Tet1 tet methylcytosine dioxygenase 1 0.944598 0.036716 ENSRNOG00000000636 Rtkn2 rhotekin 2 0.87912 0.038206 ENSRNOG00000000010 Cbln1 cerebellin 1 precursor 0.69377 0.03884 ENSRNOG00000000075 Mtf2 metal response element binding transcription factor 2 0.474307 0.04054 ENSRNOG00000001368 Rph3a rabphilin 3A 0.287075 0.041018 MAM domain containing glycosylphosphatidylinositol ENSRNOG00000000618 Mdga2 anchor 2 0.491234 0.041735 similar to DNA segment, Chr 16, ERATO Doi 472, ENSRNOG00000001554 RGD1563888 expressed 1.014126 0.042782 ENSRNOG00000000942 Pan3 poly(A) specific ribonuclease subunit PAN3 0.702983 0.0436 ENSRNOG00000001484 Castor2 cytosolic arginine sensor for mTORC1 subunit 2 0.763224 0.043612 ENSRNOG00000001557 Cxadr CXADR, Ig-like cell adhesion molecule 0.913191 0.0444 ENSRNOG00000000073 Tmed5 transmembrane p24 trafficking protein 5 0.583498 0.04649 ENSRNOG00000000411 Nus1 NUS1 dehydrodolichyl diphosphate synthase subunit 0.178233 0.047318 77

ENSEMBL SYMBOL GENENAME logFC adj.P.Val ENSRNOG00000000593 Rev3l REV3 like, DNA directed polymerase zeta catalytic subunit 0.385559 0.047787 ENSRNOG00000001456 Nup153 nucleoporin 153 0.502145 0.049387 ENSRNOG00000000387 Slc25a16 solute carrier family 25 member 16 0.368463 0.049827 ENSRNOG00000001331 Rnf34 ring finger protein 34 0.207524 0.049827

A.9 Downregulated genes (FDR = 0.05) in PD-ovx at 5 months in frontal cortex

ENSEMBL SYMBOL GENENAME logFC adj.P.Val ENSRNOG00000000279 Rtn4ip1 reticulon 4 interacting protein 1 -0.89859 0.003501 ENSRNOG00000000288 Scarf2 scavenger receptor class F, member 2 -0.78003 0.003739 ENSRNOG00000000281 Prodh1 proline dehydrogenase 1 -0.51252 0.008205 ENSRNOG00000000487 Grm4 glutamate metabotropic receptor 4 -1.02488 0.008205 ENSRNOG00000000692 Ung uracil-DNA glycosylase -0.95249 0.008205 ENSRNOG00000000054 Abhd8 abhydrolase domain containing 8 -0.44091 0.009297 ENSRNOG00000000522 Cpne5 copine 5 -0.40185 0.010163 ENSRNOG00000000442 Notch4 notch 4 -0.7326 0.011298 ENSRNOG00000000306 Smpd2 sphingomyelin phosphodiesterase 2 -0.71177 0.013737 ENSRNOG00000000704 Cmklr1 chemerin chemokine-like receptor 1 -1.05411 0.013737 ENSRNOG00000000467 Ring1 ring finger protein 1 -0.51324 0.015247 ENSRNOG00000000490 Rps10 ribosomal protein S10 -0.50255 0.017936 ENSRNOG00000000465 Slc39a7 solute carrier family 39 member 7 -0.33079 0.019242 ENSRNOG00000000175 Mier2 MIER family member 2 -0.44315 0.019868 ENSRNOG00000000172 Sqor sulfide quinone oxidoreductase -0.70956 0.020697 ENSRNOG00000000569 Vsir V-set immunoregulatory receptor -0.45874 0.022462 ENSRNOG00000000421 Skiv2l Ski2 like RNA helicase -0.32997 0.023004 ENSRNOG00000000436 Egfl8 EGF-like-domain, multiple 8 -0.69082 0.02315 ENSRNOG00000000634 Cabcoco1 ciliary associated calcium binding coiled-coil 1 -0.42991 0.023613 ENSRNOG00000000622 Hint1 histidine triad nucleotide binding protein 1 -0.26364 0.024668 ENSRNOG00000000786 Rpp21 ribonuclease P/MRP subunit p21 -0.61307 0.024675 ENSRNOG00000000481 Cuta cutA divalent cation tolerance homolog -0.41441 0.024788

78

ENSEMBL SYMBOL GENENAME logFC adj.P.Val ENSRNOG00000000456 Psmb8 proteasome subunit beta 8 -0.75612 0.024916 ENSRNOG00000000504 Fance FA complementation group E -0.52003 0.026508 ENSRNOG00000000441 Gpsm3 G-protein signaling modulator 3 -0.60917 0.027074 ENSRNOG00000000420 Nelfe negative elongation factor complex member E -0.38063 0.0273 ENSRNOG00000000186 Tst thiosulfate sulfurtransferase -0.4338 0.028103 ENSRNOG00000000571 Psap prosaposin -0.14929 0.028483 ENSRNOG00000000505 Rpl10a ribosomal protein L10A -0.30512 0.029504 ENSRNOG00000000505 RGD1566137 similar to ribosomal protein L10a -0.30512 0.029504 ENSRNOG00000000506 Tead3 TEA domain transcription factor 3 -0.52754 0.030397 ENSRNOG00000000464 Rxrb retinoid X receptor beta -0.26014 0.030795 ENSRNOG00000000697 Coro1c coronin 1C -0.18431 0.031061 ENSRNOG00000000435 Ppt2 palmitoyl-protein thioesterase 2 -0.36643 0.033246 ENSRNOG00000000728 Clic2 chloride intracellular channel 2 -1.25699 0.033418 ENSRNOG00000000431 Atf6b activating transcription factor 6 beta -0.24282 0.03383 ENSRNOG00000000488 Hmga1 high mobility group AT-hook 1 -0.54649 0.034227 ENSRNOG00000000561 Pald1 phosphatase domain containing, paladin 1 -0.46873 0.036061 ENSRNOG00000000501 Zfp523 zinc finger protein 523 -0.2148 0.036896 ENSRNOG00000000455 Tap2 transporter 2, ATP binding cassette subfamily B member -0.56019 0.038075 ENSRNOG00000000185 Mpst mercaptopyruvate sulfurtransferase -0.45599 0.038691 ENSRNOG00000000433 Prrt1 proline-rich transmembrane protein 1 -0.3137 0.039509 microtubule associated monooxygenase, calponin and ENSRNOG00000000307 Mical1 LIM domain containing 1 -0.50013 0.040064 ENSRNOG00000000474 Rgl2 ral guanine nucleotide dissociation stimulator-like 2 -0.26415 0.040974 ENSRNOG00000000156 Megf6 multiple EGF-like-domains 6 -0.87161 0.046266 multiple epidermal growth factor-like domains protein 6- ENSRNOG00000000156 LOC100911486 like -0.87161 0.046266 ENSRNOG00000000024 Hebp1 heme binding protein 1 -0.45759 0.047034 ENSRNOG00000000513 Mapk14 mitogen activated protein kinase 14 -0.20748 0.047244 ENSRNOG00000000443 LOC103689965 complement C4-like -0.58609 0.047835 ENSRNOG00000000231 Kctd17 potassium channel tetramerization domain containing 17 -0.2567 0.048938

79

A.10 Upregulated genes (FDR = 0.05) in PD-ovx at 5 months in striatum

ENSEMBL SYMBOL GENENAME logFC adj.P.Val ENSRNOG00000012067 Fam111a family with sequence similarity 111, member A 5.208463 7.85E-08 ENSRNOG00000017286 Ephx2 epoxide hydrolase 2 3.922294 6.56E-07 ENSRNOG00000055020 NA NA 3.880347 3.94E-06 ENSRNOG00000038905 NA NA 1.457788 0.002892 ENSRNOG00000001427 Orai2 ORAI calcium release-activated calcium modulator 2 1.128923 0.004135 ENSRNOG00000058006 Sncg synuclein, gamma 1.778875 0.015663 ENSRNOG00000017767 Mrvi1 murine retrovirus integration site 1 homolog 1.215672 0.019455 ENSRNOG00000001229 Col18a1 collagen type XVIII alpha 1 chain 1.443343 0.020456 ENSRNOG00000005960 RGD1311744 similar to RIKEN cDNA 5830475I06 1.75911 0.028589 ENSRNOG00000006226 Cacng2 calcium voltage-gated channel auxiliary subunit gamma 2 0.689748 0.028589 ENSRNOG00000014089 Map3k2 mitogen activated protein kinase kinase kinase 2 1.941725 0.028589 ENSRNOG00000015440 Wrn Werner syndrome RecQ like helicase 0.948699 0.028589 ENSRNOG00000021474 Siglec5 sialic acid binding Ig-like lectin 5 1.702936 0.028589 ENSRNOG00000033251 Slc25a52 solute carrier family 25, member 52 1.658964 0.028589 ENSRNOG00000038948 Stab2 stabilin 2 1.903098 0.028589 ENSRNOG00000058750 Vkorc1l1 vitamin K epoxide reductase complex, subunit 1-like 1 0.902284 0.028589 ENSRNOG00000007271 Map3k9 mitogen-activated protein kinase kinase kinase 9 0.784448 0.028653 ENSRNOG00000017108 Syngr1 synaptogyrin 1 0.696168 0.030964 ENSRNOG00000058611 NA NA 1.290339 0.030964 ENSRNOG00000023509 Irs2 insulin receptor substrate 2 0.784091 0.030985 ENSRNOG00000028622 Pnpla1 patatin-like phospholipase domain containing 1 2.440873 0.030985 phosphatidylinositol-specific phospholipase C, X domain ENSRNOG00000042289 Plcxd2 containing 2 1.131964 0.031571 ENSRNOG00000006305 Slc38a2 solute carrier family 38, member 2 0.603063 0.032472 ENSRNOG00000010597 Slc5a7 solute carrier family 5 member 7 1.256935 0.033147 ENSRNOG00000049259 LOC102549842 zinc finger protein 91-like 0.968025 0.033785 ENSRNOG00000009265 Kcnk9 potassium two pore domain channel subfamily K member 9 1.689034 0.03423 ENSRNOG00000042702 Nipa1 NIPA magnesium transporter 1 0.966971 0.035026 80

ENSEMBL SYMBOL GENENAME logFC adj.P.Val ENSRNOG00000000708 Sgsm1 small G protein signaling modulator 1 0.448221 0.037597 ENSRNOG00000017428 Map1b microtubule-associated protein 1B 0.890967 0.037597 ENSRNOG00000004812 Sema6d semaphorin 6D 0.885883 0.043467 ENSRNOG00000001329 Gjc3 gap junction protein, gamma 3 2.003456 0.044611 ENSRNOG00000013851 Spry4 sprouty RTK signaling antagonist 4 0.704532 0.044611 ENSRNOG00000001602 Ltn1 listerin E3 ubiquitin protein ligase 1 0.581568 0.044895 ENSRNOG00000015269 Atf7 activating transcription factor 7 1.14612 0.044895 ENSRNOG00000053122 Scn1a sodium voltage-gated channel alpha subunit 1 1.002014 0.045114 ENSRNOG00000007687 Sema7a semaphorin 7A (John Milton Hagen blood group) 0.406671 0.0454 ENSRNOG00000021525 Nbeal1 neurobeachin-like 1 1.141028 0.046101 ENSRNOG00000008053 Atp8a2 ATPase phospholipid transporting 8A2 1.270379 0.047373 ENSRNOG00000001484 Castor2 cytosolic arginine sensor for mTORC1 subunit 2 1.570398 0.047832 ENSRNOG00000015763 Nat8f3 N-acetyltransferase 8 (GCN5-related) family member 3 1.080754 0.047832 ENSRNOG00000003680 Gabrb2 gamma-aminobutyric acid type A receptor beta 2 subunit 2.138104 0.048589

A.11 Downregulated genes (FDR = 0.05) in PD-ovx at 5 months in striatum

ENSEMBL SYMBOL GENENAME logFC adj.P.Val ENSRNOG00000001300 P2rx4 purinergic receptor P2X 4 -1.3422 0.000278 ENSRNOG00000016336 NA NA -3.31365 0.00268 ENSRNOG00000007117 Cluap1 clusterin associated protein 1 -1.08883 0.006254 ENSRNOG00000011831 Nudt18 nudix hydrolase 18 -0.9377 0.011667 ENSRNOG00000000704 Cmklr1 chemerin chemokine-like receptor 1 -1.41757 0.013691 ENSRNOG00000001289 LOC498154 hypothetical protein LOC498154 -0.86128 0.013691 ENSRNOG00000006591 Fam163b family with sequence similarity 163, member B -0.55756 0.013691 ENSRNOG00000007041 Abcg2 ATP binding cassette subfamily G member 2 -1.64877 0.013691 ENSRNOG00000011936 Abhd14a abhydrolase domain containing 14A -1.26571 0.013691 ENSRNOG00000013720 Aebp1 AE binding protein 1 -1.55311 0.013691 ENSRNOG00000021243 Siglec1 sialic acid binding Ig like lectin 1 -1.4316 0.013691 81

ENSEMBL SYMBOL GENENAME logFC adj.P.Val ENSRNOG00000017621 Spns1 sphingolipid transporter 1 -0.68054 0.01379 ENSRNOG00000016967 Hfe homeostatic iron regulator -1.33379 0.015663 ENSRNOG00000009694 Bmp4 bone morphogenetic protein 4 -3.1486 0.019455 ENSRNOG00000018680 Rpl17 ribosomal protein L17 -1.07719 0.019455 ENSRNOG00000000279 Rtn4ip1 reticulon 4 interacting protein 1 -1.03681 0.020724 ENSRNOG00000000288 Scarf2 scavenger receptor class F, member 2 -1.0165 0.028589 ENSRNOG00000017895 Eno1 enolase 1 -0.52974 0.028653 ENSRNOG00000021230 Ubox5 U-box domain containing 5 -0.61739 0.028653 ENSRNOG00000000893 Tmem248 transmembrane protein 248 -0.59322 0.030118 ENSRNOG00000017820 Nqo2 N-ribosyldihydronicotinamide:quinone reductase 2 -1.68841 0.030964 ENSRNOG00000005690 Lmcd1 LIM and cysteine-rich domains 1 -0.87813 0.032472 ENSRNOG00000009761 Tmod1 tropomodulin 1 -0.49664 0.032472 ENSRNOG00000017328 Pter phosphotriesterase related -1.05299 0.032472 ENSRNOG00000001383 Slc8b1 solute carrier family 8 member B1 -0.91921 0.033147 ENSRNOG00000007564 Evc EvC ciliary complex subunit 1 -1.13695 0.033147 ENSRNOG00000007862 Acat1 acetyl-CoA acetyltransferase 1 -0.6029 0.033147 ENSRNOG00000021104 Emp3 epithelial membrane protein 3 -1.04766 0.033147 ENSRNOG00000021521 Chst5 carbohydrate sulfotransferase 5 -1.41917 0.033147 ENSRNOG00000010412 Ccdc180 coiled-coil domain containing 180 -1.34104 0.03423 ENSRNOG00000010616 Ndor1 NADPH dependent diflavin oxidoreductase 1 -0.6769 0.03423 ENSRNOG00000011861 Aadat aminoadipate aminotransferase -0.96254 0.034966 ENSRNOG00000020864 Kirrel2 kirre like nephrin family adhesion molecule 2 -1.20736 0.034966 ENSRNOG00000007600 Igsf1 immunoglobulin superfamily, member 1 -0.72262 0.035437 ENSRNOG00000005809 Arhgdib Rho GDP dissociation inhibitor beta -0.69506 0.037391 ENSRNOG00000019772 Dnpep aspartyl aminopeptidase -0.53903 0.03757 ENSRNOG00000000487 Grm4 glutamate metabotropic receptor 4 -0.7853 0.037597 ENSRNOG00000018412 Sfi1 SFI1 centrin binding protein -0.63373 0.037597 ENSRNOG00000021087 Lgi4 leucine-rich repeat LGI family, member 4 -0.63013 0.037686 ENSRNOG00000015155 Tnnc2 troponin C2, fast skeletal type -0.98328 0.037914 ENSRNOG00000001404 Agfg2 ArfGAP with FG repeats 2 -0.63328 0.038172

82

ENSEMBL SYMBOL GENENAME logFC adj.P.Val ENSRNOG00000001344 Aldh2 aldehyde dehydrogenase 2 family (mitochondrial) -0.67829 0.043467 ENSRNOG00000003809 Sat1 spermidine/spermine N1-acetyl transferase 1 -0.58358 0.043467 ENSRNOG00000005931 Cpq carboxypeptidase Q -0.4489 0.043467 ENSRNOG00000017693 Slc2a5 solute carrier family 2 member 5 -1.0549 0.043467 ENSRNOG00000008312 Stra6 stimulated by retinoic acid 6 -0.96487 0.043627 ENSRNOG00000017901 Acy3 aminoacylase 3 -0.90509 0.044895 ENSRNOG00000005275 Shmt1 serine hydroxymethyltransferase 1 -1.06094 0.0454 ENSRNOG00000000172 Sqor sulfide quinone oxidoreductase -0.91878 0.046101 ENSRNOG00000021117 Rps6ka4 ribosomal protein S6 kinase A4 -0.42156 0.047832

A.12 Upregulated genes (FDR = 0.05) in PD-ovx at 5 months in cerebellum

ENSEMBL SYMBOL GENENAME logFC adj.P.Val ENSRNOG00000017286 Ephx2 epoxide hydrolase 2 3.648385 5.76E-08 ENSRNOG00000012067 Fam111a family with sequence similarity 111, member A 5.464553 1.37E-06 ENSRNOG00000020424 Plpp4 phospholipid phosphatase 4 1.588305 5.96E-05 ENSRNOG00000000963 Tmem132c transmembrane protein 132C 1.370769 0.00026 ENSRNOG00000001368 Rph3a rabphilin 3A 0.740247 0.00026 ENSRNOG00000006639 Scn9a sodium voltage-gated channel alpha subunit 9 3.131844 0.000269 ENSRNOG00000038905 NA NA 1.078492 0.000285 ENSRNOG00000007657 Col27a1 collagen type XXVII alpha 1 chain 1.64911 0.00039 ENSRNOG00000016326 Cx3cl1 C-X3-C motif chemokine ligand 1 0.632082 0.00075 ENSRNOG00000005016 Tmed4 transmembrane p24 trafficking protein 4 0.465794 0.000775 ENSRNOG00000005093 Lgr6 leucine-rich repeat-containing G protein-coupled receptor 6 2.07384 0.000853 ENSRNOG00000000811 Pkib cAMP-dependent protein kinase inhibitor beta 0.674262 0.001732 ENSRNOG00000028041 Tnnt1 troponin T1, slow skeletal type 1.616227 0.002468 ENSRNOG00000001427 Orai2 ORAI calcium release-activated calcium modulator 2 0.849777 0.002505 ENSRNOG00000014166 Smoc2 SPARC related modular calcium binding 2 2.59236 0.003127 ENSRNOG00000010107 NA NA 0.752058 0.003342

83

ENSEMBL SYMBOL GENENAME logFC adj.P.Val ENSRNOG00000031662 Slc6a5 solute carrier family 6 member 5 0.77742 0.005285 ENSRNOG00000007033 Sorcs2 sortilin-related VPS10 domain containing receptor 2 0.767323 0.005598 ENSRNOG00000000407 Dcbld1 discoidin, CUB and LCCL domain containing 1 1.239161 0.006314 ENSRNOG00000010011 Osbpl3 oxysterol binding protein-like 3 0.756536 0.006314 OPA3, outer mitochondrial membrane lipid metabolism ENSRNOG00000025890 Opa3 regulator 0.431418 0.006314 ENSRNOG00000006235 Nell2 neural EGFL like 2 0.324038 0.010883 ENSRNOG00000046834 C3 complement C3 0.566726 0.011029 ENSRNOG00000050534 Gcnt1 glucosaminyl (N-acetyl) transferase 1, core 2 0.719401 0.01327 ENSRNOG00000014090 Retsat retinol saturase 0.761791 0.016089 ENSRNOG00000028910 NA NA 0.880611 0.016441 ENSRNOG00000011992 Slc18a1 solute carrier family 18 member A1 1.110578 0.025971 ENSRNOG00000002866 Rassf6 Ras association domain family member 6 1.433201 0.026061 ENSRNOG00000022983 Trim17 tripartite motif-containing 17 1.2371 0.027671 ENSRNOG00000051563 Giot1 gonadotropin inducible ovarian transcription factor 1 0.707529 0.030245 ENSRNOG00000030983 B3galt5 Beta-1,3-galactosyltransferase 5 0.430571 0.031676 ENSRNOG00000013766 Acaa2 acetyl-CoA acyltransferase 2 0.651338 0.032056 ENSRNOG00000018700 Mobp myelin-associated oligodendrocyte basic protein 0.3225 0.033158 ENSRNOG00000011994 Perp PERP, TP53 apoptosis effector 0.589407 0.03331 ENSRNOG00000017468 Trappc6a trafficking protein particle complex 6A 0.768223 0.03331 ENSRNOG00000033517 LOC100360791 tumor protein, translationally-controlled 1 1.756224 0.037301 ENSRNOG00000020468 Stard4 StAR-related lipid transfer domain containing 4 0.585475 0.037717 ENSRNOG00000001707 Vwa5b2 von Willebrand factor A domain containing 5B2 0.488027 0.041024 ENSRNOG00000005195 Cst3 cystatin C 0.422552 0.043021 ENSRNOG00000005960 RGD1311744 similar to RIKEN cDNA 5830475I06 1.502798 0.043363 ENSRNOG00000008554 Slc9a9 solute carrier family 9 member A9 0.519996 0.043363 ENSRNOG00000009372 Tacr3 tachykinin receptor 3 4.503797 0.043363 ENSRNOG00000019825 Zdhhc24 zinc finger, DHHC-type containing 24 0.484717 0.044518 HECT domain and ankyrin repeat containing, E3 ubiquitin ENSRNOG00000000327 Hace1 protein ligase 1 0.390845 0.0466

84

ENSEMBL SYMBOL GENENAME logFC adj.P.Val ENSRNOG00000021249 Ap5s1 adaptor related protein complex 5 subunit sigma 1 0.720937 0.0466 ENSRNOG00000004327 Ddc dopa decarboxylase 0.555527 0.048398 ENSRNOG00000052113 Ppp1r9b protein phosphatase 1, regulatory subunit 9B 0.208483 0.049191 ENSRNOG00000025463 LOC100125362 hypothetical protein LOC100125362 0.531515 0.049752 ENSRNOG00000031033 ND2 NADH dehydrogenase subunit 2 0.231182 0.049752 ENSRNOG00000024039 Serinc5 serine incorporator 5 0.353811 0.049819

A.13 Downregulated genes (FDR = 0.05) in PD-ovx at 5 months in cerebellum

ENSEMBL SYMBOL GENENAME logFC adj.P.Val ENSRNOG00000001300 P2rx4 purinergic receptor P2X 4 -1.37547 1.82E-05 ENSRNOG00000007117 Cluap1 clusterin associated protein 1 -1.06257 0.000137 ENSRNOG00000015441 Il4r interleukin 4 receptor -1.01987 0.000413 ENSRNOG00000005457 Lamp5 lysosomal-associated membrane protein family, member 5 -0.52644 0.001907 ENSRNOG00000000279 Rtn4ip1 reticulon 4 interacting protein 1 -0.93249 0.003127 ENSRNOG00000005141 Hus1 HUS1 checkpoint clamp component -0.45863 0.003127 ENSRNOG00000007564 Evc EvC ciliary complex subunit 1 -0.87863 0.003188 ENSRNOG00000007041 Abcg2 ATP binding cassette subfamily G member 2 -1.36939 0.003984 ENSRNOG00000005492 Hpcal1 hippocalcin-like 1 -0.30375 0.006314 ENSRNOG00000016177 Scara3 scavenger receptor class A, member 3 -0.41573 0.006314 ENSRNOG00000010802 Ube3d ubiquitin protein ligase E3D -1.24416 0.007178 ENSRNOG00000007025 Evc2 EvC ciliary complex subunit 2 -0.90958 0.00886 ENSRNOG00000021086 Dtx4 deltex E3 ubiquitin ligase 4 -0.69615 0.00886 ENSRNOG00000002343 Uchl1 ubiquitin C-terminal hydrolase L1 -0.58591 0.011167 ENSRNOG00000008214 Fbxo9 f-box protein 9 -0.39837 0.012306 ENSRNOG00000011936 Abhd14a abhydrolase domain containing 14A -0.76098 0.012306 ENSRNOG00000018126 Abca1 ATP binding cassette subfamily A member 1 -0.62222 0.01327 ENSRNOG00000018822 Slc5a5 solute carrier family 5 member 5 -1.4417 0.01651 ENSRNOG00000005809 Arhgdib Rho GDP dissociation inhibitor beta -0.64342 0.01777 85

ENSEMBL SYMBOL GENENAME logFC adj.P.Val ENSRNOG00000014265 Tnfrsf19 TNF receptor superfamily member 19 -0.57614 0.01777 ENSRNOG00000020922 Hspb6 heat shock protein family B (small) member 6 -0.39 0.019907 ENSRNOG00000019118 Slc13a3 solute carrier family 13 member 3 -0.81805 0.02277 ENSRNOG00000005257 Prkaca protein kinase cAMP-activated catalytic subunit alpha -0.28252 0.028248 ENSRNOG00000010940 Acad11 acyl-CoA dehydrogenase family, member 11 -0.59789 0.031042 ENSRNOG00000007219 LOC103692716 heat shock protein HSP 90-alpha -0.60722 0.031676 ENSRNOG00000008316 Vps39 VPS39 HOPS complex subunit -0.3044 0.031676 ENSRNOG00000004794 Rtn1 reticulon 1 -0.26596 0.032056 ENSRNOG00000001254 Col6a2 collagen type VI alpha 2 chain -0.83312 0.03331 ENSRNOG00000016460 Clu clusterin -0.34497 0.03331 ENSRNOG00000017895 Eno1 enolase 1 -0.40506 0.033497 ENSRNOG00000011831 Nudt18 nudix hydrolase 18 -0.45951 0.035077 ENSRNOG00000001739 Meltf melanotransferrin -1.32294 0.035613 ENSRNOG00000005350 Pwp1 PWP1 homolog, endonuclein -0.56117 0.035613 ENSRNOG00000013720 Aebp1 AE binding protein 1 -1.19541 0.035613 ENSRNOG00000003114 B4galt4 beta-1,4-galactosyltransferase 4 -0.83252 0.037301 ENSRNOG00000011379 Ccndbp1 cyclin D1 binding protein 1 -0.37089 0.037717 ENSRNOG00000021084 Mpeg1 macrophage expressed 1 -1.84635 0.038371 ENSRNOG00000010480 Vstm5 V-set and transmembrane domain containing 5 -0.45883 0.041811 ENSRNOG00000000704 Cmklr1 chemerin chemokine-like receptor 1 -0.81365 0.043009 ENSRNOG00000017693 Slc2a5 solute carrier family 2 member 5 -0.86013 0.043021 ENSRNOG00000001158 Abcg1 ATP binding cassette subfamily G member 1 -0.39721 0.043363 ENSRNOG00000017819 Cd14 CD14 molecule -0.9402 0.045196 ENSRNOG00000014143 Col24a1 collagen type XXIV alpha 1 chain -1.58725 0.046478 ENSRNOG00000016696 Angpt2 angiopoietin 2 -1.04314 0.046478 ENSRNOG00000020736 Nadsyn1 NAD synthetase 1 -0.57713 0.046581 ENSRNOG00000017820 Nqo2 N-ribosyldihydronicotinamide:quinone reductase 2 -1.01114 0.047339 ENSRNOG00000018680 Rpl17 ribosomal protein L17 -0.62981 0.047537 ENSRNOG00000001752 Nrros negative regulator of reactive oxygen species -0.52427 0.049191 ENSRNOG00000010370 Tnip1 TNFAIP3 interacting protein 1 -0.36979 0.049752 86

ENSEMBL SYMBOL GENENAME logFC adj.P.Val ENSRNOG00000013172 Nr1h3 nuclear receptor subfamily 1, group H, member 3 -0.49108 0.049752

A.14 Upregulated genes (FDR = 0.05) in PD-ovx at 12 months, common to all tissues

ENSEMBL SYMBOL GENENAME logFC adj.P.Val ENSRNOG00000001427 Orai2 ORAI calcium release-activated calcium modulator 2 1.030112 3.76E-12 ENSRNOG00000001368 Rph3a rabphilin 3A 0.806918 7.89E-11 ENSRNOG00000000700 Tmem119 transmembrane protein 119 0.77617 6.54E-05 ENSRNOG00000005519 Grm3 glutamate metabotropic receptor 3 0.455733 0.000316 ENSRNOG00000001711 Hrasls HRAS-like suppressor 0.71222 0.000337 ENSRNOG00000001329 Gjc3 gap junction protein, gamma 3 0.930176 0.00065 ENSRNOG00000004327 Ddc dopa decarboxylase 0.727862 0.000888 HECT domain and ankyrin repeat containing, E3 ubiquitin protein ENSRNOG00000000327 Hace1 ligase 1 0.315867 0.00104 ENSRNOG00000004011 Nedd1 neural precursor cell expressed, developmentally down-regulated 1 0.614207 0.001128 ENSRNOG00000005195 Cst3 cystatin C 0.482509 0.001476 ENSRNOG00000000699 Selplg selectin P ligand 0.725004 0.001709 ENSRNOG00000002345 Rasgef1b RasGEF domain family, member 1B 0.650729 0.003197 ENSRNOG00000001602 Ltn1 listerin E3 ubiquitin protein ligase 1 0.30744 0.003258 ENSRNOG00000000811 Pkib cAMP-dependent protein kinase inhibitor beta 0.769092 0.003785 ENSRNOG00000002863 Cacna1e calcium voltage-gated channel subunit alpha1 E 0.522209 0.003951 ENSRNOG00000000204 Syncrip synaptotagmin binding, cytoplasmic RNA interacting protein 0.365849 0.004109 ENSRNOG00000002705 Vps4b vacuolar protein sorting 4 homolog B 0.301744 0.00498 ENSRNOG00000000815 Smpdl3a sphingomyelin phosphodiesterase, acid-like 3A 0.343727 0.00525 ENSRNOG00000002592 Rps6ka6 ribosomal protein S6 kinase A6 0.613967 0.005927 ENSRNOG00000002028 Tmem50b transmembrane protein 50B 0.181763 0.006516 ENSRNOG00000004458 Ston2 stonin 2 0.639318 0.006851 ENSRNOG00000000775 Mog myelin oligodendrocyte glycoprotein 0.474422 0.006905 ENSRNOG00000004693 Pbx1 PBX homeobox 1 0.326988 0.007378

87

ENSRNOG00000002349 Gabra2 gamma-aminobutyric acid type A receptor alpha2 subunit 0.472553 0.007562 ENSRNOG00000004322 Sh3kbp1 SH3 domain-containing kinase-binding protein 1 0.355111 0.008533 ENSRNOG00000003746 Gjb1 gap junction protein, beta 1 0.595803 0.008659 ENSRNOG00000004067 Nrcam neuronal cell adhesion molecule 0.346759 0.009698 ENSRNOG00000005561 Brinp1 BMP/retinoic acid inducible neural specific 1 0.338382 0.009698 ENSRNOG00000003213 Helz helicase with zinc finger 0.330063 0.015999 ENSRNOG00000001609 Cep97 centrosomal protein 97 0.379963 0.019157 ENSRNOG00000000142 Plxdc2 plexin domain containing 2 0.257374 0.02126 ENSRNOG00000004713 Kcnj16 potassium voltage-gated channel subfamily J member 16 0.688593 0.02126 ENSRNOG00000002210 Hsd17b11 hydroxysteroid (17-beta) dehydrogenase 11 0.287371 0.021671 ENSRNOG00000002866 Rassf6 Ras association domain family member 6 1.25384 0.022812 ENSRNOG00000003873 Cpd carboxypeptidase D 0.460389 0.023562 ENSRNOG00000004226 Irak3 interleukin-1 receptor-associated kinase 3 0.567361 0.026067 ENSRNOG00000000805 Gja1 gap junction protein, alpha 1 0.281389 0.026173 ENSRNOG00000003253 Qdpr quinoid dihydropteridine reductase 0.37788 0.029417 ENSRNOG00000005726 Pclo piccolo (presynaptic cytomatrix protein) 0.323804 0.031554 ENSRNOG00000001657 Cldnd1 claudin domain containing 1 0.194409 0.032251 ENSRNOG00000001555 Btg3 BTG anti-proliferation factor 3 0.393091 0.032333 ENSRNOG00000002579 Parm1 prostate androgen-regulated mucin-like protein 1 0.332352 0.032716 ENSRNOG00000004160 Prps2 phosphoribosyl pyrophosphate synthetase 2 0.357979 0.036023 ENSRNOG00000003694 Prox1 prospero homeobox 1 0.354647 0.0387 ENSRNOG00000004998 Jakmip1 janus kinase and microtubule interacting protein 1 0.259343 0.039865 ENSRNOG00000000407 Dcbld1 discoidin, CUB and LCCL domain containing 1 0.469047 0.040575 ENSRNOG00000002280 Sh3bgrl SH3 domain binding glutamate-rich protein like 0.417398 0.040803 ENSRNOG00000003890 Nap1l1 nucleosome assembly protein 1-like 1 0.187302 0.040842 ENSRNOG00000001817 Tm7sf3 transmembrane 7 superfamily member 3 0.209598 0.042595 ENSRNOG00000000657 Nek7 NIMA-related kinase 7 0.405536 0.047465

88

A.15 Downregulated genes (FDR = 0.05) in PD-ovx at 12 months, common to all tissues

ENSEMBL SYMBOL GENENAME logFC adj.P.Val ENSRNOG00000001300 P2rx4 purinergic receptor P2X 4 -1.33141 3.89E-14 ENSRNOG00000000279 Rtn4ip1 reticulon 4 interacting protein 1 -0.90581 3.87E-10 ENSRNOG00000002343 Uchl1 ubiquitin C-terminal hydrolase L1 -0.76148 0.000107 ENSRNOG00000005809 Arhgdib Rho GDP dissociation inhibitor beta -0.66992 0.000121 ENSRNOG00000001374 Rasal1 RAS protein activator like 1 -0.99009 0.00036 ENSRNOG00000005257 Prkaca protein kinase cAMP-activated catalytic subunit alpha -0.32645 0.000643 ENSRNOG00000005003 Ptprn2 protein tyrosine phosphatase, receptor type N2 -0.26798 0.000991 ENSRNOG00000001837 Sst somatostatin -0.98255 0.00111 ENSRNOG00000004794 Rtn1 reticulon 1 -0.24985 0.001111 ENSRNOG00000005007 Scn3a sodium voltage-gated channel alpha subunit 3 -0.76384 0.001163 ENSRNOG00000005330 Crebbp CREB binding protein -0.30446 0.001564 ENSRNOG00000004687 Thbd thrombomodulin -0.71655 0.001901 ENSRNOG00000006033 Spon2 spondin 2 -0.90125 0.00214 ENSRNOG00000005141 Hus1 HUS1 checkpoint clamp component -0.30833 0.002435 ENSRNOG00000001351 Trafd1 TRAF type zinc finger domain containing 1 -0.42875 0.002981 ENSRNOG00000005623 Ankmy2 ankyrin repeat and MYND domain containing 2 -0.24897 0.003183 ENSRNOG00000002630 Cnot8 CCR4-NOT transcription complex, subunit 8 -0.30405 0.003533 ENSRNOG00000005550 Lrfn5 leucine rich repeat and fibronectin type III domain containing 5 -0.4068 0.004336 ENSRNOG00000001254 Col6a2 collagen type VI alpha 2 chain -1.00799 0.005702 ENSRNOG00000001752 Nrros negative regulator of reactive oxygen species -0.50446 0.006851 ENSRNOG00000003497 Thoc6 THO complex 6 -0.43158 0.007182 ENSRNOG00000001469 Eln elastin -0.65642 0.007398 ENSRNOG00000001738 Eif4g1 eukaryotic translation initiation factor 4 gamma, 1 -0.22205 0.007427 ENSRNOG00000001888 Arvcf ARVCF, delta catenin family member -0.50812 0.008138 ENSRNOG00000004276 Itga3 integrin subunit alpha 3 -0.28261 0.009321 ENSRNOG00000003114 B4galt4 beta-1,4-galactosyltransferase 4 -0.45471 0.009698 ENSRNOG00000004229 Tac3 tachykinin 3 -1.02052 0.009698 ENSRNOG00000000172 Sqor sulfide quinone oxidoreductase -0.48118 0.011036 ENSRNOG00000001316 Anapc5 anaphase-promoting complex subunit 5 -0.2142 0.01397 ENSRNOG00000001124 Rnft2 ring finger protein, transmembrane 2 -0.19327 0.017378 89

ENSEMBL SYMBOL GENENAME logFC adj.P.Val ENSRNOG00000000840 Atp6v1g2 ATPase H+ transporting V1 subunit G2 -0.16086 0.018227 ENSRNOG00000003504 Rnaseh2a ribonuclease H2, subunit A -0.30404 0.022368 ENSRNOG00000000780 Ppp1r11 protein phosphatase 1, regulatory (inhibitor) subunit 11 -0.17707 0.022446 tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation ENSRNOG00000001436 Ywhag protein, gamma -0.16523 0.023743 ENSRNOG00000002229 Adcy5 adenylate cyclase 5 -0.2804 0.024094 ENSRNOG00000001792 Slc12a8 solute carrier family 12, member 8 -1.05552 0.025061 ENSRNOG00000001710 Abcf3 ATP binding cassette subfamily F member 3 -0.21058 0.025676 ENSRNOG00000001289 LOC498154 hypothetical protein LOC498154 -0.36366 0.031166 ENSRNOG00000001431 Rasa4 RAS p21 protein activator 4 -0.60992 0.033005 ENSRNOG00000001960 Sult1d1 sulfotransferase family 1D, member 1 -0.98141 0.034165 ENSRNOG00000003835 Slc43a2 solute carrier family 43 member 2 -0.23812 0.035182 ENSRNOG00000004014 Chmp6 charged multivesicular body protein 6 -0.24332 0.040471 alpha-1,3-mannosyl-glycoprotein 4-beta-N- ENSRNOG00000003235 Mgat4b acetylglucosaminyltransferase B -0.21994 0.041282 ENSRNOG00000003125 Rogdi rogdi homolog -0.22113 0.041911 ENSRNOG00000001408 Actl6b actin-like 6B -0.21485 0.043092 ENSRNOG00000005482 Sap30bp SAP30 binding protein -0.19587 0.043187 ENSRNOG00000001708 Dvl3 dishevelled segment polarity protein 3 -0.19397 0.044605 ENSRNOG00000004091 Cwc25 CWC25 spliceosome-associated protein homolog -0.31473 0.045848 ENSRNOG00000003815 Slc25a11 solute carrier family 25 member 11 -0.21187 0.047663 ENSRNOG00000000704 Cmklr1 chemerin chemokine-like receptor 1 -0.62833 0.047785

A.16 Upregulated genes (FDR = 0.05) in PD-ovx at 12 months in frontal cortex

ENSEMBL SYMBOL GENENAME logFC adj.P.Val ENSRNOG00000058006 Sncg synuclein, gamma 2.497551 4.43E-08 ENSRNOG00000001427 Orai2 ORAI calcium release-activated calcium modulator 2 1.286495 7.01E-08 ENSRNOG00000012067 Fam111a family with sequence similarity 111, member A 6.792384 7.01E-08 ENSRNOG00000005960 RGD1311744 similar to RIKEN cDNA 5830475I06 2.709647 1.03E-07

90

ENSEMBL SYMBOL GENENAME logFC adj.P.Val ENSRNOG00000055020 NA NA 3.82388 1.03E-07 ENSRNOG00000016516 Mbp myelin basic protein 0.528985 3.48E-06 ENSRNOG00000018029 Doc2g double C2-like domains, gamma 1.398921 3.48E-06 ENSRNOG00000059510 NA NA 0.579789 3.48E-06 ENSRNOG00000003253 Qdpr quinoid dihydropteridine reductase 0.739439 4.37E-06 ENSRNOG00000061779 Man2b2 mannosidase, alpha, class 2B, member 2 0.842968 6.06E-06 ENSRNOG00000038905 NA NA 1.394624 1.14E-05 ENSRNOG00000000775 Mog myelin oligodendrocyte glycoprotein 0.750658 1.66E-05 ENSRNOG00000031033 ND2 NADH dehydrogenase subunit 2 0.305408 0.000107 ENSRNOG00000012490 Amph amphiphysin 0.887349 0.000125 ENSRNOG00000052204 Tbc1d24 TBC1 domain family, member 24 0.497875 0.000166 ENSRNOG00000024039 Serinc5 serine incorporator 5 0.598488 0.000171 ENSRNOG00000021474 Siglec5 sialic acid binding Ig-like lectin 5 1.796088 0.000222 ENSRNOG00000000567 Unc5b unc-5 netrin receptor B 0.513338 0.000235 ENSRNOG00000001329 Gjc3 gap junction protein, gamma 3 1.340972 0.000238 ENSRNOG00000016558 Pllp plasmolipin 0.486949 0.00024 ENSRNOG00000022764 Evi2a ecotropic viral integration site 2A 0.606823 0.000284 ENSRNOG00000025110 Vwa3a von Willebrand factor A domain containing 3A 1.970142 0.000355 ENSRNOG00000030449 Gsta4 glutathione S-transferase alpha 4 0.793081 0.000396 ENSRNOG00000014090 Retsat retinol saturase 0.628221 0.000437 ENSRNOG00000022386 Opalin oligodendrocytic myelin paranodal and inner loop protein 0.840999 0.000446 ENSRNOG00000048273 Apod apolipoprotein D 0.462282 0.000446 ENSRNOG00000006639 Scn9a sodium voltage-gated channel alpha subunit 9 1.780229 0.000468 ENSRNOG00000009629 Car2 carbonic anhydrase 2 0.403341 0.000564 ENSRNOG00000004089 Enpp2 ectonucleotide pyrophosphatase/phosphodiesterase 2 0.414482 0.000634 ENSRNOG00000033251 Slc25a52 solute carrier family 25, member 52 2.171275 0.000641 ENSRNOG00000002524 Gpr37 G protein-coupled receptor 37 0.494967 0.000662 ENSRNOG00000017496 Cnp 2',3'-cyclic nucleotide 3' phosphodiesterase 0.49434 0.000692 ENSRNOG00000010263 Cldn11 claudin 11 0.573531 0.00075 ENSRNOG00000008533 Ago2 argonaute 2, RISC catalytic component 0.786116 0.000846 91

ENSEMBL SYMBOL GENENAME logFC adj.P.Val ENSRNOG00000057125 Ddr1 discoidin domain receptor tyrosine kinase 1 0.426604 0.000846 ENSRNOG00000021023 Mag myelin-associated glycoprotein 0.650317 0.000966 ENSRNOG00000009734 Akr1b8 aldo-keto reductase family 1, member B8 1.158476 0.001027 ENSRNOG00000009734 Akr1b10 aldo-keto reductase family 1 member B10 1.158476 0.001027 ENSRNOG00000015150 Spg7 SPG7, paraplegin matrix AAA peptidase subunit 0.403176 0.001035 OPA3, outer mitochondrial membrane lipid metabolism ENSRNOG00000025890 Opa3 regulator 0.375191 0.001057 ENSRNOG00000017767 Mrvi1 murine retrovirus integration site 1 homolog 1.009249 0.001068 ENSRNOG00000018491 Chchd5 coiled-coil-helix-coiled-coil-helix domain containing 5 0.523675 0.001157 ENSRNOG00000030704 NA NA 1.121723 0.001157 ENSRNOG00000017577 Bphl biphenyl hydrolase like 0.955051 0.001175 ENSRNOG00000009345 Ugt8 UDP glycosyltransferase 8 0.565882 0.001208 ENSRNOG00000003296 Dck deoxycytidine kinase 0.655748 0.001431 ENSRNOG00000000700 Tmem119 transmembrane protein 119 0.787864 0.001482 ENSRNOG00000003276 Myo1d myosin ID 0.527877 0.001482 ADAM metallopeptidase with thrombospondin type 1 motif, ENSRNOG00000003538 Adamts4 4 0.683362 0.001532 ENSRNOG00000033059 Galnt6 polypeptide N-acetylgalactosaminyltransferase 6 0.636118 0.001532 ENSRNOG00000002419 Plp1 proteolipid protein 1 0.718819 0.0017 ENSRNOG00000018627 Plekhb1 pleckstrin homology domain containing B1 0.303817 0.0017 ENSRNOG00000017468 Trappc6a trafficking protein particle complex 6A 0.823659 0.00173 ENSRNOG00000028274 Myrf myelin regulatory factor 0.548274 0.001824 ENSRNOG00000017510 Mfge8 milk fat globule-EGF factor 8 protein 0.407291 0.001886 ENSRNOG00000047300 Bdkrb2 bradykinin receptor B2 2.549871 0.001886 ENSRNOG00000024082 Gldn gliomedin 1.125433 0.00194 ENSRNOG00000007393 Ndrg1 N-myc downstream regulated 1 0.393138 0.002103 ENSRNOG00000026306 Clec5a C-type lectin domain containing 5A 1.287667 0.002205 SPARC/osteonectin, cwcv and kazal like domains ENSRNOG00000029903 Spock3 proteoglycan 3 0.439029 0.002205 ENSRNOG00000042702 Nipa1 NIPA magnesium transporter 1 0.432805 0.002471 92

ENSEMBL SYMBOL GENENAME logFC adj.P.Val ENSRNOG00000007726 Mcam melanoma cell adhesion molecule 0.654482 0.002555 LSM6 homolog, U6 small nuclear RNA and mRNA ENSRNOG00000049370 Lsm6 degradation associated 1.066306 0.002815 ENSRNOG00000049370 LOC100360750 Sm protein F-like 1.066306 0.002815 ENSRNOG00000007367 4-Sep septin 4 0.312347 0.002925 ENSRNOG00000058909 Rps9 ribosomal protein S9 0.437835 0.003037 ENSRNOG00000058909 Rps9l1 ribosomal protein S9-like 1 0.437835 0.003037 ENSRNOG00000017512 Aldh3b1 aldehyde dehydrogenase 3 family, member B1 0.446202 0.003246 ENSRNOG00000056894 St6galnac3 ST6 N-acetylgalactosaminide alpha-2,6-sialyltransferase 3 0.639428 0.003246 ENSRNOG00000021472 Ermn ermin 0.644753 0.003446 ENSRNOG00000030614 LOC689899 similar to 60S ribosomal protein L23a 1.096719 0.003495 ENSRNOG00000000699 Selplg selectin P ligand 0.742463 0.003601 ENSRNOG00000022196 Bmpr2 bone morphogenetic protein receptor type 2 0.340604 0.003725 ENSRNOG00000024728 Arhgap22 Rho GTPase activating protein 22 0.535329 0.003767 ENSRNOG00000018603 Carns1 carnosine synthase 1 0.778874 0.003855 ENSRNOG00000061102 Taf9b TATA-box binding protein associated factor 9b 0.289826 0.003855 pleckstrin homology, MyTH4 and FERM domain containing ENSRNOG00000010650 Plekhh1 H1 0.568219 0.004273 ENSRNOG00000036869 Tmem88b transmembrane protein 88B 0.634931 0.004483 ENSRNOG00000047321 Hba-a2 hemoglobin alpha, adult chain 2 0.803241 0.004784 ENSRNOG00000019825 Zdhhc24 zinc finger, DHHC-type containing 24 0.589003 0.005021 ENSRNOG00000019484 Slc6a9 solute carrier family 6 member 9 0.331418 0.005506 ENSRNOG00000004964 Erbb3 erb-b2 receptor tyrosine kinase 3 0.461676 0.005947 ENSRNOG00000001311 Rab36 RAB36, member RAS oncogene family 0.728354 0.00602 ENSRNOG00000004147 Abca8a ATP-binding cassette, subfamily A (ABC1), member 8a 0.575455 0.006054 ring finger and FYVE-like domain containing E3 ubiquitin ENSRNOG00000007596 Rffl protein ligase 0.360424 0.006225 ENSRNOG00000009945 Pls1 plastin 1 0.526252 0.006225 ENSRNOG00000013679 Sema4d semaphorin 4D 0.329497 0.006225 ENSRNOG00000024578 Ttyh2 tweety family member 2 0.391471 0.006225 93

ENSEMBL SYMBOL GENENAME logFC adj.P.Val ENSRNOG00000052687 Megf8 multiple EGF-like-domains 8 0.303669 0.006225 ENSRNOG00000002735 Dhx9 DExH-box helicase 9 0.231104 0.006355 ENSRNOG00000002928 Guk1 guanylate kinase 1 0.273644 0.006424 ENSRNOG00000029886 Hba1 hemoglobin, alpha 1 0.747087 0.006569 ENSRNOG00000009636 Scrn1 secernin 1 0.207351 0.006631 ENSRNOG00000018991 Gsn gelsolin 0.429363 0.006631 ENSRNOG00000014684 Npr1 natriuretic peptide receptor 1 0.571789 0.006683 ENSRNOG00000022784 Fzd10 frizzled class receptor 10 0.617534 0.006697 ENSRNOG00000013279 LOC681458 similar to stearoyl-coenzyme A desaturase 3 1.171628 0.006768 ENSRNOG00000030124 Ptpn11 protein tyrosine phosphatase, non-receptor type 11 0.176081 0.006768 ENSRNOG00000043212 Dip2a disco-interacting protein 2 homolog A 0.3011 0.006842 ENSRNOG00000017154 Atp11a ATPase phospholipid transporting 11A 0.345205 0.00691 ENSRNOG00000019276 Dele1 DAP3 binding cell death enhancer 1 0.39801 0.006981 ENSRNOG00000022286 NA NA 0.510085 0.007103 ENSRNOG00000009582 Rpe65 RPE65, retinoid isomerohydrolase 1.480393 0.007114 ENSRNOG00000010598 Hs3st1 heparan sulfate-glucosamine 3-sulfotransferase 1 0.402912 0.007114 ENSRNOG00000030625 Tf transferrin 0.361469 0.007221 ENSRNOG00000006200 St18 ST18, C2H2C-type zinc finger 0.817389 0.007293 ENSRNOG00000019556 Cd9 CD9 molecule 0.273764 0.007308 ENSRNOG00000001262 LOC108348157 speriolin-like protein 1.500864 0.007751 ENSRNOG00000003746 Gjb1 gap junction protein, beta 1 0.659987 0.007813 ENSRNOG00000000811 Pkib cAMP-dependent protein kinase inhibitor beta 1.022774 0.008132 ENSRNOG00000020813 Ltbp3 latent transforming growth factor beta binding protein 3 0.282209 0.008132 ENSRNOG00000004327 Ddc dopa decarboxylase 0.800013 0.008535 ENSRNOG00000019659 Aspa aspartoacylase 0.451644 0.008535 ENSRNOG00000023657 Gprin3 GPRIN family member 3 1.167763 0.008535 ENSRNOG00000012148 Trio trio Rho guanine nucleotide exchange factor 0.274585 0.008779 ENSRNOG00000020084 Pcdhb5 protocadherin beta 5 0.864093 0.008779 ENSRNOG00000018870 Hapln2 hyaluronan and proteoglycan link protein 2 0.887082 0.008937 ENSRNOG00000004322 Sh3kbp1 SH3 domain-containing kinase-binding protein 1 0.290822 0.009113 94

ENSEMBL SYMBOL GENENAME logFC adj.P.Val ENSRNOG00000061299 LOC100134871 beta globin minor gene 0.58521 0.009113 ENSRNOG00000018700 Mobp myelin-associated oligodendrocyte basic protein 0.351071 0.009151 ENSRNOG00000010184 Brk1 BRICK1, SCAR/WAVE actin-nucleating complex subunit 0.285409 0.009254 ENSRNOG00000011305 Sox10 SRY box 10 0.420325 0.009901 ENSRNOG00000007206 LOC361016 similar to RIKEN cDNA 4933406L09 0.481429 0.009998 ENSRNOG00000017841 Fam192a family with sequence similarity 192, member A 0.268499 0.010199 ENSRNOG00000026985 Phldb1 pleckstrin homology-like domain, family B, member 1 0.342174 0.010199 ENSRNOG00000010765 Vcl vinculin 0.28432 0.010249 ENSRNOG00000015625 Kiaa0895l hypothetical protein LOC688736 0.409539 0.010249 ENSRNOG00000016975 Pxmp4 peroxisomal membrane protein 4 0.839374 0.010717 sparc/osteonectin, cwcv and kazal like domains proteoglycan ENSRNOG00000012747 Spock1 1 0.228737 0.010844 ENSRNOG00000002773 Rgs4 regulator of G-protein signaling 4 0.263132 0.011124 ENSRNOG00000026336 Tmem255b transmembrane protein 255B 0.734955 0.01151 ENSRNOG00000028261 Tppp tubulin polymerization promoting protein 0.266951 0.01151 ENSRNOG00000004672 Sec14l2 SEC14-like lipid binding 2 0.193433 0.011856 ENSRNOG00000014744 Pacs2 phosphofurin acidic cluster sorting protein 2 0.191717 0.012472 ENSRNOG00000021249 Ap5s1 adaptor related protein complex 5 subunit sigma 1 0.601391 0.012472 ENSRNOG00000002610 Carhsp1 calcium regulated heat stable protein 1 0.42064 0.012994 ENSRNOG00000016434 Prkd2 protein kinase D2 0.545832 0.012994 ENSRNOG00000022730 Zfp57 zinc finger protein 57 0.443331 0.012994 ENSRNOG00000020393 Rhog ras homolog family member G 0.37691 0.014634 ENSRNOG00000002682 Zfp692 zinc finger protein 692 0.334792 0.014774 ENSRNOG00000038328 Gjc2 gap junction protein, gamma 2 0.543109 0.015049 ENSRNOG00000001911 Map6d1 MAP6 domain containing 1 0.315489 0.015232 ENSRNOG00000006570 Plekhg3 pleckstrin homology and RhoGEF domain containing G3 0.434554 0.01524 ENSRNOG00000002941 Uhmk1 U2AF homology motif kinase 1 0.250375 0.015455 ENSRNOG00000058249 Pgk1 phosphoglycerate kinase 1 0.195107 0.016109 ENSRNOG00000002028 Tmem50b transmembrane protein 50B 0.175265 0.01624

95

ENSEMBL SYMBOL GENENAME logFC adj.P.Val microtubule associated serine/threonine kinase family ENSRNOG00000027002 Mast4 member 4 0.321603 0.01624 microtubule associated serine/threonine kinase family ENSRNOG00000027002 NEWGENE_1310139 member 4 0.321603 0.01624 ENSRNOG00000006841 Ano4 anoctamin 4 0.43091 0.016285 ENSRNOG00000007606 Rnf43 ring finger protein 43 0.52791 0.01651 ENSRNOG00000000151 Ldlrap1 low density lipoprotein receptor adaptor protein 1 0.672 0.018814 ENSRNOG00000052519 Ttc30a1 tetratricopeptide repeat domain 30A1 0.955406 0.01885 ENSRNOG00000023338 Tspan2 tetraspanin 2 0.421485 0.018931 ENSRNOG00000007925 Pak6 p21 (RAC1) activated kinase 6 0.333855 0.019819 ENSRNOG00000001417 Plod3 procollagen-lysine, 2-oxoglutarate 5-dioxygenase 3 0.343666 0.020092 ENSRNOG00000010977 Igfbp6 insulin-like growth factor binding protein 6 1.470782 0.020237 ENSRNOG00000025528 Efr3a EFR3 homolog A 0.183095 0.02047 ENSRNOG00000028448 Elovl1 ELOVL fatty acid elongase 1 0.303865 0.02047 ENSRNOG00000004841 Akap6 A-kinase anchoring protein 6 0.215721 0.02058 ENSRNOG00000020676 Ppp1r14a protein phosphatase 1, regulatory (inhibitor) subunit 14A 0.81275 0.02058 ENSRNOG00000017879 Gab1 GRB2-associated binding protein 1 0.372523 0.020604 ENSRNOG00000001834 Mzt2b mitotic spindle organizing protein 2B 0.504528 0.020702 ENSRNOG00000015445 Mal mal, T-cell differentiation protein 0.399117 0.020894 ENSRNOG00000007281 Flnc filamin C 0.438436 0.021018 ENSRNOG00000015179 Tradd TNFRSF1A-associated via death domain 0.958727 0.021018 ENSRNOG00000001764 Vps8 VPS8 CORVET complex subunit 0.207641 0.022386 ENSRNOG00000004680 Kif5c kinesin family member 5C 0.29598 0.022386 ENSRNOG00000010896 Tprn taperin 0.413952 0.023032 ENSRNOG00000001192 Gltp glycolipid transfer protein 0.286289 0.023301 ENSRNOG00000002866 Rassf6 Ras association domain family member 6 1.527359 0.023301 ENSRNOG00000008915 Prima1 proline rich membrane anchor 1 0.65691 0.023301 ENSRNOG00000059116 NA NA 0.416126 0.023301 ENSRNOG00000031211 Acsm5 acyl-CoA synthetase medium-chain family member 5 1.624733 0.023448 ENSRNOG00000007441 Klhl32 kelch-like family member 32 0.409731 0.023553 96

ENSEMBL SYMBOL GENENAME logFC adj.P.Val ENSRNOG00000021220 Cpxm1 carboxypeptidase X (M14 family), member 1 0.586738 0.023553 ENSRNOG00000033316 Foxo4 forkhead box O4 0.312823 0.023553 ENSRNOG00000004805 Stac2 SH3 and cysteine rich domain 2 0.225046 0.023553 ENSRNOG00000051483 Selenow selenoprotein W 0.206796 0.023553 ENSRNOG00000051483 LOC103689961 selenoprotein W-like 0.206796 0.023553 ENSRNOG00000008364 Cat catalase 0.274911 0.024106 ENSRNOG00000007748 Tex15 testis expressed 15, meiosis and synapsis associated 2.087546 0.024502 ENSRNOG00000012561 Arhgef10 Rho guanine nucleotide exchange factor 10 0.265398 0.025117 ENSRNOG00000008622 Creb5 cAMP responsive element binding protein 5 0.679555 0.025517 ENSRNOG00000007869 Wscd1 WSC domain containing 1 0.261174 0.025987 ENSRNOG00000052498 Grb14 growth factor receptor bound protein 14 0.3503 0.026012 ENSRNOG00000019921 Rhbdl1 rhomboid like 1 0.383186 0.026178 ENSRNOG00000028814 Oasl2 2'-5' oligoadenylate synthetase-like 2 0.556948 0.026811 ENSRNOG00000001482 Gtf2ird2 GTF2I repeat domain containing 2 0.356168 0.026908 ENSRNOG00000061379 C7 complement C7 1.366121 0.027923 ENSRNOG00000061768 Slc43a3 solute carrier family 43, member 3 0.652378 0.02805 ENSRNOG00000055089 Slc44a1 solute carrier family 44 member 1 0.222516 0.028073 ENSRNOG00000011572 Klc1 kinesin light chain 1 0.149796 0.028363 ENSRNOG00000013656 Lpar1 lysophosphatidic acid receptor 1 0.413223 0.028363 ENSRNOG00000024411 Cbr4 carbonyl reductase 4 0.39305 0.028469 ENSRNOG00000016828 Cmtm5 CKLF-like MARVEL transmembrane domain containing 5 0.232392 0.028496 ENSRNOG00000021062 Fxyd5 FXYD domain-containing ion transport regulator 5 0.485134 0.028563 ENSRNOG00000005342 Rassf5 Ras association domain family member 5 0.24018 0.029609 ENSRNOG00000010673 Eral1 Era-like 12S mitochondrial rRNA chaperone 1 0.29937 0.02965 ENSRNOG00000010718 Gpr153 G protein-coupled receptor 153 0.449804 0.029691 ENSRNOG00000002126 Ncam2 neural cell adhesion molecule 2 0.30696 0.030921 ENSRNOG00000001493 NA NA 0.295027 0.030944 ENSRNOG00000020495 Eif3k eukaryotic translation initiation factor 3, subunit K 0.216841 0.033098 ENSRNOG00000027124 Tdg thymine-DNA glycosylase 0.238136 0.033098 ENSRNOG00000013934 St5 suppression of tumorigenicity 5 0.266778 0.033288 97

ENSEMBL SYMBOL GENENAME logFC adj.P.Val ENSRNOG00000007457 Serping1 serpin family G member 1 0.877566 0.033561 ENSRNOG00000012906 Bcas1 breast carcinoma amplified sequence 1 0.338158 0.034108 ENSRNOG00000018841 Sox8 SRY box 8 0.337861 0.034523 ENSRNOG00000058105 Hbb hemoglobin subunit beta 0.476549 0.0347 ENSRNOG00000027405 Ccp110 centriolar coiled-coil protein 110 0.272151 0.034949 ENSRNOG00000013874 Rplp1 ribosomal protein lateral stalk subunit P1 0.246842 0.034967 ENSRNOG00000013874 LOC100360522 ribosomal protein P1-like 0.246842 0.034967 ENSRNOG00000013229 Mthfs methenyltetrahydrofolate synthetase 0.406915 0.036021 ENSRNOG00000015616 Rgs14 regulator of G-protein signaling 14 0.232504 0.036564 ENSRNOG00000048152 Myo1b myosin Ib 0.260596 0.036564 ENSRNOG00000017601 Srd5a1 steroid 5 alpha-reductase 1 0.286207 0.037131 ENSRNOG00000006255 Nipal4 NIPA-like domain containing 4 0.532457 0.037432 ENSRNOG00000014293 Nkd1 naked cuticle homolog 1 0.402309 0.039565 ENSRNOG00000001827 Masp1 mannan-binding lectin serine peptidase 1 0.363211 0.039617 ENSRNOG00000006747 Cc2d1a coiled-coil and C2 domain containing 1A 0.215777 0.039617 glycerophosphodiester phosphodiesterase domain ENSRNOG00000002800 Gdpd2 containing 2 0.880651 0.039771 ENSRNOG00000047505 Tubb4a tubulin, beta 4A class IVa 0.207699 0.039771 ENSRNOG00000004400 Avpr1a arginine vasopressin receptor 1A 1.010152 0.040799 ENSRNOG00000054978 Hist1h1c histone cluster 1 H1 family member c 0.43991 0.040939 ENSRNOG00000001757 Tm4sf19 transmembrane 4 L six family member 19 0.840174 0.041198 ENSRNOG00000009411 Chn2 chimerin 2 0.342096 0.041544 ENSRNOG00000004581 Zdhhc9 zinc finger, DHHC-type containing 9 0.215334 0.04235 ENSRNOG00000001833 Mcm4 minichromosome maintenance complex component 4 0.327897 0.042984 ENSRNOG00000017917 Cdhr2 cadherin-related family member 2 0.358528 0.044142 ENSRNOG00000000316 Sobp sine oculis binding protein homolog 0.228563 0.044359 ENSRNOG00000022636 Alpk1 alpha-kinase 1 0.270049 0.044489 ENSRNOG00000032328 Diras2 DIRAS family GTPase 2 0.361533 0.044928 ENSRNOG00000053560 NA NA 0.462451 0.045221 ENSRNOG00000013223 Fah fumarylacetoacetate hydrolase 0.422943 0.045775 98

ENSEMBL SYMBOL GENENAME logFC adj.P.Val ENSRNOG00000003863 Trir telomerase RNA component interacting RNase 0.502479 0.045816 ENSRNOG00000004314 Ppm1h protein phosphatase, Mg2+/Mn2+ dependent, 1H 0.202712 0.048384 ENSRNOG00000029841 Cdh19 cadherin 19 0.512904 0.048406 ENSRNOG00000052386 Nlrx1 NLR family member X1 0.477633 0.048416 ENSRNOG00000014268 Abca2 ATP binding cassette subfamily A member 2 0.238964 0.048474 ENSRNOG00000020014 Myh14 myosin heavy chain 14 0.285515 0.04959 ENSRNOG00000024712 Insc INSC, spindle orientation adaptor protein 0.695218 0.04959 ENSRNOG00000031849 Armh4 armadillo-like helical domain containing 4 0.309967 0.04959 ENSRNOG00000054360 Tspan11 tetraspanin 11 0.542224 0.04959 ENSRNOG00000012701 Map7 microtubule-associated protein 7 0.251646 0.049691 ENSRNOG00000014840 Gna14 G protein subunit alpha 14 0.621371 0.049691 ENSRNOG00000020935 Capn1 calpain 1 0.396841 0.049691 ENSRNOG00000030877 Htr2c 5-hydroxytryptamine receptor 2C 0.314506 0.049691

A.17 Downregulated genes (FDR = 0.05) in PD-ovx at 12 months in frontal cortex

ENSEMBL SYMBOL GENENAME logFC adj.P.Val ENSRNOG00000007117 Cluap1 clusterin associated protein 1 -0.80063 1.21E-07 ENSRNOG00000001300 P2rx4 purinergic receptor P2X 4 -1.05302 4.99E-07 ENSRNOG00000017820 Nqo2 N-ribosyldihydronicotinamide:quinone reductase 2 -1.62416 3.12E-06 ENSRNOG00000001404 Agfg2 ArfGAP with FG repeats 2 -0.48946 6.53E-05 ENSRNOG00000017962 Serpinb6a serpin family B member 6A -0.58456 0.000165 ENSRNOG00000032585 NA NA -0.5764 0.000344 ENSRNOG00000020411 Sec23ip SEC23 interacting protein -0.36735 0.000446 ENSRNOG00000001383 Slc8b1 solute carrier family 8 member B1 -0.76215 0.000521 ENSRNOG00000014761 Rasd2 RASD family, member 2 -0.30822 0.000692 ENSRNOG00000017895 Eno1 enolase 1 -0.26171 0.000762 ENSRNOG00000012811 Spint1 serine peptidase inhibitor, Kunitz type 1 -1.11322 0.00078 ENSRNOG00000003063 Phka1 phosphorylase kinase regulatory subunit alpha 1 -0.38178 0.000966 99

ENSEMBL SYMBOL GENENAME logFC adj.P.Val ENSRNOG00000042971 LOC363337 similar to RIKEN cDNA 1700081O22 -1.02864 0.001035 ENSRNOG00000000279 Rtn4ip1 reticulon 4 interacting protein 1 -0.55674 0.001057 ENSRNOG00000005007 Scn3a sodium voltage-gated channel alpha subunit 3 -0.41423 0.001368 ENSRNOG00000011936 Abhd14a abhydrolase domain containing 14A -0.63175 0.001482 ENSRNOG00000037815 Acad10 acyl-CoA dehydrogenase family, member 10 -0.54623 0.001482 ENSRNOG00000002630 Cnot8 CCR4-NOT transcription complex, subunit 8 -0.34294 0.001886 ENSRNOG00000006956 NA NA -0.94262 0.002024 ENSRNOG00000018126 Abca1 ATP binding cassette subfamily A member 1 -0.78507 0.002024 ENSRNOG00000056150 Purb purine rich element binding protein B -0.48967 0.002192 ENSRNOG00000011348 Snx14 sorting nexin 14 -0.38456 0.002346 ENSRNOG00000017876 Dnajc21 DnaJ heat shock protein family (Hsp40) member C21 -0.70485 0.002619 ENSRNOG00000015003 Pex11a peroxisomal biogenesis factor 11 alpha -0.89682 0.002815 ENSRNOG00000020206 Ctsd cathepsin D -0.22617 0.002925 ENSRNOG00000001323 Zfp157 zinc finger protein 157 -0.37347 0.003329 ENSRNOG00000031889 NA NA -0.62152 0.003559 ENSRNOG00000025936 Rpl6 ribosomal protein L6 -0.2474 0.003855 ENSRNOG00000028523 Tctn1 tectonic family member 1 -0.44829 0.003855 ENSRNOG00000012782 Cemip2 cell migration inducing hyaluronidase 2 -0.40451 0.003885 ENSRNOG00000038025 Rpp38 ribonuclease P/MRP subunit p38 -0.83921 0.005153 ENSRNOG00000024127 Atp13a5 ATPase 13A5 -0.49568 0.005463 ENSRNOG00000016346 Prkcd protein kinase C, delta -0.51108 0.006225 ENSRNOG00000029939 Gypc glycophorin C (Gerbich blood group) -0.74289 0.006225 ENSRNOG00000050134 LOC108348293 uncharacterized LOC108348293 -1.6031 0.006225 ENSRNOG00000051135 Tug1 taurine up-regulated 1 -0.27119 0.006424 human immunodeficiency virus type I enhancer binding protein ENSRNOG00000014460 Hivep1 1 -0.36577 0.006726 ENSRNOG00000013902 P2ry12 purinergic receptor P2Y12 -0.37178 0.006977 ENSRNOG00000021084 Mpeg1 macrophage expressed 1 -2.02023 0.007113 ENSRNOG00000018122 Tspan17 tetraspanin 17 -0.32077 0.007114 ENSRNOG00000033906 Zfp667 zinc finger protein 667 -0.49111 0.007114 100

ENSEMBL SYMBOL GENENAME logFC adj.P.Val ENSRNOG00000039559 Fam219a family with sequence similarity 219, member A -0.19321 0.007114 ENSRNOG00000045747 Capns1 calpain, small subunit 1 -0.29198 0.007114 ENSRNOG00000048194 LOC100912380 calpain small subunit 1-like -0.29198 0.007114 ENSRNOG00000005457 Lamp5 lysosomal-associated membrane protein family, member 5 -0.29168 0.008132 ENSRNOG00000008312 Stra6 stimulated by retinoic acid 6 -0.78384 0.008132 ENSRNOG00000058790 Fam120b family with sequence similarity 120B -0.25888 0.008447 ENSRNOG00000002369 Rgs8 regulator of G-protein signaling 8 -0.35809 0.008535 ENSRNOG00000001351 Trafd1 TRAF type zinc finger domain containing 1 -0.35515 0.008779 ENSRNOG00000005350 Pwp1 PWP1 homolog, endonuclein -0.5368 0.008779 ENSRNOG00000029165 Stx1a syntaxin 1A -0.20354 0.008937 ENSRNOG00000003929 Pcdh19 protocadherin 19 -0.35052 0.009045 ENSRNOG00000017693 Slc2a5 solute carrier family 2 member 5 -0.75202 0.009755 ENSRNOG00000009563 Krt2 keratin 2 -1.26361 0.009998 ENSRNOG00000020480 Fads1 fatty acid desaturase 1 -0.30709 0.010075 ENSRNOG00000003461 Zfp330 zinc finger protein 330 -0.24329 0.010199 ENSRNOG00000059857 Rnd1 Rho family GTPase 1 -0.29131 0.010199 ENSRNOG00000060438 NA NA -1.71539 0.010199 ENSRNOG00000018242 Camkk1 calcium/calmodulin-dependent protein kinase kinase 1 -0.2147 0.010613 ENSRNOG00000001418 Znhit1 zinc finger, HIT-type containing 1 -0.37936 0.01151 ENSRNOG00000005330 Crebbp CREB binding protein -0.24559 0.01151 ENSRNOG00000016776 Extl1 exostosin-like glycosyltransferase 1 -0.24006 0.011909 ENSRNOG00000010412 Ccdc180 coiled-coil domain containing 180 -0.94367 0.012235 ENSRNOG00000021243 Siglec1 sialic acid binding Ig like lectin 1 -1.4801 0.012426 ENSRNOG00000011947 Tifab TIFA inhibitor -0.77856 0.012472 ENSRNOG00000024479 Klhl34 kelch-like family member 34 -0.89078 0.012472 ENSRNOG00000005141 Hus1 HUS1 checkpoint clamp component -0.28245 0.012524 ENSRNOG00000000007 Gad1 glutamate decarboxylase 1 -0.26073 0.014472 ENSRNOG00000010753 Aig1 androgen-induced 1 -0.2592 0.014634 ENSRNOG00000054246 LOC679711 similar to RIKEN cDNA 5031410I06 -1.45059 0.014634 ENSRNOG00000033744 Rasgrp4 RAS guanyl releasing protein 4 -0.74353 0.014675 101

ENSEMBL SYMBOL GENENAME logFC adj.P.Val ENSRNOG00000009263 Ifi27 interferon, alpha-inducible protein 27 -0.56868 0.015049 ENSRNOG00000016810 Stmn1 stathmin 1 -0.17648 0.015168 ENSRNOG00000020770 Arl4d ADP-ribosylation factor like GTPase 4D -0.58471 0.015168 ENSRNOG00000033940 Adgrl4 adhesion G protein-coupled receptor L4 -0.39636 0.015168 ENSRNOG00000042878 Wdr74 WD repeat domain 74 -0.34675 0.015187 ENSRNOG00000046309 Gpr68 G protein-coupled receptor 68 -0.57989 0.015187 ENSRNOG00000017621 Spns1 sphingolipid transporter 1 -0.34457 0.015203 ENSRNOG00000005257 Prkaca protein kinase cAMP-activated catalytic subunit alpha -0.25743 0.01529 ENSRNOG00000060356 Kif15 kinesin family member 15 -1.06841 0.015445 ENSRNOG00000059702 Tex9 testis expressed 9 -0.58817 0.015543 ENSRNOG00000029366 Prrt2 proline-rich transmembrane protein 2 -0.24211 0.01624 ENSRNOG00000036641 LOC689065 hypothetical protein LOC689065 -0.93519 0.016744 ENSRNOG00000023467 Fam168b family with sequence similarity 168, member B -0.15213 0.016793 ENSRNOG00000022523 Fkbp5 FK506 binding protein 5 -0.29684 0.017133 ENSRNOG00000023077 Cpne9 copine family member 9 -0.27866 0.017168 ENSRNOG00000012664 Polr1e RNA polymerase I subunit E -0.46936 0.017879 ENSRNOG00000008474 Acox3 acyl-CoA oxidase 3, pristanoyl -0.36412 0.01815 ENSRNOG00000012442 Cemip cell migration inducing hyaluronidase -0.29821 0.01815 ENSRNOG00000010555 Phyhip phytanoyl-CoA 2-hydroxylase interacting protein -0.15172 0.01829 ENSRNOG00000010555 LOC108348161 phytanoyl-CoA hydroxylase-interacting protein -0.15172 0.01829 ENSRNOG00000029707 ND4 NADH dehydrogenase subunit 4 -0.1655 0.018777 UDP-GlcNAc:betaGal beta-1,3-N-acetylglucosaminyltransferase ENSRNOG00000009267 B3gnt2 2 -0.34207 0.018814 ENSRNOG00000020557 Ryr1 ryanodine receptor 1 -0.50597 0.018814 ENSRNOG00000030216 Abcg3l1 ATP-binding cassette, subfamily G (WHITE), member 3-like 1 -0.79469 0.018906 ENSRNOG00000018336 Eps8l2 EPS8-like 2 -0.32596 0.019873 ENSRNOG00000018797 Myrip myosin VIIA and Rab interacting protein -0.18532 0.019994 ENSRNOG00000057214 NA NA -1.22254 0.020079 ENSRNOG00000007600 Igsf1 immunoglobulin superfamily, member 1 -0.45153 0.020237 ENSRNOG00000013949 Idh2 isocitrate dehydrogenase (NADP(+)) 2, mitochondrial -0.23404 0.02058 102

ENSEMBL SYMBOL GENENAME logFC adj.P.Val ENSRNOG00000026514 Wdr93 WD repeat domain 93 -1.52055 0.02058 ENSRNOG00000021234 Slc4a11 solute carrier family 4 member 11 -0.53961 0.020657 ENSRNOG00000018445 Agt angiotensinogen -0.33505 0.020702 ENSRNOG00000005809 Arhgdib Rho GDP dissociation inhibitor beta -0.40743 0.02074 ENSRNOG00000030568 Rgs12 regulator of G-protein signaling 12 -0.2487 0.02074 ENSRNOG00000018487 Slc3a2 solute carrier family 3 member 2 -0.24369 0.021067 ENSRNOG00000015713 Parva parvin, alpha -0.25453 0.022386 transient receptor potential cation channel, subfamily C, ENSRNOG00000006324 Trpc6 member 6 -0.42938 0.023301 ENSRNOG00000016748 Poll DNA polymerase lambda -0.28494 0.023301 ENSRNOG00000002730 Rgs5 regulator of G-protein signaling 5 -0.30923 0.023553 ENSRNOG00000007862 Acat1 acetyl-CoA acetyltransferase 1 -0.19841 0.023553 ENSRNOG00000021663 Vxn vexin -0.19523 0.023553 ENSRNOG00000000800 Man1a1 mannosidase, alpha, class 1A, member 1 -0.38957 0.024057 ENSRNOG00000012110 Col17a1 collagen type XVII alpha 1 chain -1.22321 0.024302 ENSRNOG00000052968 Hnrnpa3 heterogeneous nuclear ribonucleoprotein A3 -0.26059 0.024317 ENSRNOG00000021911 Camlg calcium modulating ligand -0.30557 0.024338 ENSRNOG00000024904 Pla2g4e phospholipase A2, group IVE -0.28809 0.024398 ENSRNOG00000055344 Trnau1ap tRNA selenocysteine 1 associated protein 1 -0.31805 0.024458 ENSRNOG00000027592 Rerg RAS-like, estrogen-regulated, growth-inhibitor -0.40951 0.024502 ENSRNOG00000046947 LOC365985 similar to adenylate kinase 5 isoform 1 -0.21797 0.024666 ENSRNOG00000014801 Exog exo/endonuclease G -0.37732 0.025122 ENSRNOG00000031041 Rps4y2 ribosomal protein S4, Y-linked 2 -1.11127 0.02656 ENSRNOG00000007014 Cnksr2 connector enhancer of kinase suppressor of Ras 2 -0.39175 0.026687 ENSRNOG00000021824 Dnajb1 DnaJ heat shock protein family (Hsp40) member B1 -0.33246 0.027868 ENSRNOG00000015093 Sparcl1 SPARC like 1 -0.32141 0.027923 ENSRNOG00000048230 LOC300308 similar to hypothetical protein 4930509O22 -0.55545 0.027923 ENSRNOG00000060047 Aldh18a1 aldehyde dehydrogenase 18 family, member A1 -4.16539 0.028563 ENSRNOG00000022288 Pafah2 platelet-activating factor acetylhydrolase 2 -0.5038 0.029859 ENSRNOG00000004516 Itgbl1 integrin subunit beta like 1 -0.64634 0.030365 103

ENSEMBL SYMBOL GENENAME logFC adj.P.Val ENSRNOG00000021013 Stx3 syntaxin 3 -0.39165 0.030693 ENSRNOG00000015084 Necab2 N-terminal EF-hand calcium binding protein 2 -0.23798 0.031132 ENSRNOG00000017766 Car12 carbonic anhydrase 12 -0.26745 0.031372 ENSRNOG00000057099 NA NA -0.75151 0.032156 ENSRNOG00000037162 Slbp stem-loop binding protein -0.31237 0.03231 ENSRNOG00000014890 Mrpl43 mitochondrial ribosomal protein L43 -0.5299 0.033561 ENSRNOG00000018704 Nolc1 nucleolar and coiled-body phosphoprotein 1 -0.16736 0.034108 ENSRNOG00000012723 Trim55 tripartite motif-containing 55 -0.68971 0.034967 ENSRNOG00000024230 Tnfaip8l3 TNF alpha induced protein 8 like 3 -0.48084 0.034967 ENSRNOG00000027175 Cnpy4 canopy FGF signaling regulator 4 -0.40277 0.034967 ENSRNOG00000061345 LOC690414 hypothetical protein LOC690414 -1.48129 0.034967 ENSRNOG00000055319 Mrfap1 Morf4 family associated protein 1 -0.16785 0.036276 ENSRNOG00000020717 Bod1 biorientation of chromosomes in cell division 1 -0.23645 0.036564 ENSRNOG00000016867 Zfp346 zinc finger protein 346 -0.22817 0.036848 ENSRNOG00000032070 Dync2h1 dynein cytoplasmic 2 heavy chain 1 -0.25571 0.037351 ENSRNOG00000016336 NA NA -1.86495 0.037432 ENSRNOG00000013215 Dctd dCMP deaminase -0.51398 0.037549 ENSRNOG00000008786 Ap1b1 adaptor related protein complex 1 subunit beta 1 -0.17894 0.038166 ENSRNOG00000012106 Dnaja4 DnaJ heat shock protein family (Hsp40) member A4 -0.17917 0.038166 ENSRNOG00000006375 Vdac1 voltage-dependent anion channel 1 -0.16195 0.039153 ENSRNOG00000028047 Mecr mitochondrial trans-2-enoyl-CoA reductase -0.39994 0.039153 ENSRNOG00000051772 NA NA -0.4539 0.039287 ENSRNOG00000000805 Gja1 gap junction protein, alpha 1 -0.23274 0.039617 ENSRNOG00000008316 Vps39 VPS39 HOPS complex subunit -0.18525 0.039617 ENSRNOG00000014762 Ppie peptidylprolyl isomerase E -0.31209 0.039617 ENSRNOG00000013011 Dnajb4 DnaJ heat shock protein family (Hsp40) member B4 -0.20897 0.040367 ENSRNOG00000000778 Znrd1as1 ZNRD1 antisense RNA 1 -0.58824 0.04228 ENSRNOG00000011214 Polr2f RNA polymerase II subunit F -0.26164 0.04334 ENSRNOG00000018680 Rpl17 ribosomal protein L17 -0.40392 0.043357 ENSRNOG00000015218 Nsmce1 NSE1 homolog, SMC5-SMC6 complex component -0.2943 0.043397 104

ENSEMBL SYMBOL GENENAME logFC adj.P.Val ENSRNOG00000001289 LOC498154 hypothetical protein LOC498154 -0.34829 0.044275 ENSRNOG00000033693 NA NA -0.97028 0.044275 ENSRNOG00000031342 NA NA -1.33614 0.044571 ENSRNOG00000006749 Tmtc3 transmembrane and tetratricopeptide repeat containing 3 -0.29227 0.044609 ENSRNOG00000045837 NA NA -1.15523 0.044609 ENSRNOG00000046536 NA NA -0.29287 0.044928 ENSRNOG00000053410 Adcy10 adenylate cyclase 10 -0.80123 0.045493 ENSRNOG00000043007 Rcn3 reticulocalbin 3 -0.36948 0.045775 ENSRNOG00000009683 Sdcbp syndecan binding protein -0.14037 0.048474 ENSRNOG00000049150 NA NA -0.5973 0.048474 ENSRNOG00000012357 Unc45a unc-45 myosin chaperone A -0.21293 0.049078 ENSRNOG00000013039 Add1 adducin 1 -0.11955 0.049078 ENSRNOG00000005984 Etv6 ets variant 6 -0.56208 0.04959 ENSRNOG00000011826 Lzts1 leucine zipper tumor suppressor 1 -0.14045 0.04959

A.18 Upregulated genes (FDR = 0.05) in PD-ovx at 12 months in striatum

ENSEMBL SYMBOL GENENAME logFC adj.P.Val ENSRNOG00000000190 NA NA 0.466278 0.045434 ENSRNOG00000000700 Tmem119 transmembrane protein 119 0.597062 0.00818 ENSRNOG00000000775 Mog myelin oligodendrocyte glycoprotein 0.340936 0.033186 ENSRNOG00000001130 Nos1 nitric oxide synthase 1 0.517419 0.020958 ENSRNOG00000001229 Col18a1 collagen type XVIII alpha 1 chain 2.056593 3.54E-05 ENSRNOG00000001329 Gjc3 gap junction protein, gamma 3 1.177328 0.00117 ENSRNOG00000001335 Zkscan1 zinc finger with KRAB and SCAN domains 1 0.554651 0.028042 ENSRNOG00000001368 Rph3a rabphilin 3A 0.522816 0.000515 ENSRNOG00000001427 Orai2 ORAI calcium release-activated calcium modulator 2 1.230787 1.04E-07 ENSRNOG00000001484 Castor2 cytosolic arginine sensor for mTORC1 subunit 2 0.606531 0.04855 ENSRNOG00000001602 Ltn1 listerin E3 ubiquitin protein ligase 1 0.274906 0.045165 105

ENSEMBL SYMBOL GENENAME logFC adj.P.Val ENSRNOG00000001607 Adamts1 ADAM metallopeptidase with thrombospondin type 1 motif, 1 0.687339 0.003345 ENSRNOG00000001854 Tmtc1 transmembrane and tetratricopeptide repeat containing 1 0.634335 0.033186 ENSRNOG00000002163 Klf3 Kruppel like factor 3 0.368836 0.012763 ENSRNOG00000002215 Mylk myosin light chain kinase 0.439235 0.022329 ENSRNOG00000002248 Fryl FRY like transcription coactivator 0.38021 0.030347 ENSRNOG00000002318 Limch1 LIM and calponin homology domains 1 0.543466 0.014837 ENSRNOG00000002863 Cacna1e calcium voltage-gated channel subunit alpha1 E 0.710472 0.011813 ENSRNOG00000003253 Qdpr quinoid dihydropteridine reductase 0.313566 0.045587 ENSRNOG00000003491 Prkca protein kinase C, alpha 0.546161 0.033006 ENSRNOG00000003882 Cep350 centrosomal protein 350 0.604752 0.015786 ENSRNOG00000004147 Abca8a ATP-binding cassette, subfamily A (ABC1), member 8a 0.342164 0.031733 ENSRNOG00000004226 Irak3 interleukin-1 receptor-associated kinase 3 0.788009 0.028707 ENSRNOG00000004232 G2e3 G2/M-phase specific E3 ubiquitin protein ligase 0.479513 0.043184 ENSRNOG00000004327 Ddc dopa decarboxylase 0.655303 0.000799 ENSRNOG00000004680 Kif5c kinesin family member 5C 0.296973 0.022376 ENSRNOG00000004693 Pbx1 PBX homeobox 1 0.48614 0.02678 ENSRNOG00000004812 Sema6d semaphorin 6D 0.611798 3.54E-05 ENSRNOG00000004947 NA NA 1.113759 0.034936 ENSRNOG00000004964 Erbb3 erb-b2 receptor tyrosine kinase 3 0.400054 0.005572 ENSRNOG00000005159 Fam135b family with sequence similarity 135, member B 0.749281 0.039555 ENSRNOG00000005299 Kif5a kinesin family member 5A 0.328587 0.026148 ENSRNOG00000005519 Grm3 glutamate metabotropic receptor 3 0.325475 0.022329 ENSRNOG00000005711 Ptprd protein tyrosine phosphatase, receptor type, D 0.594551 0.031334 ENSRNOG00000005726 Pclo piccolo (presynaptic cytomatrix protein) 0.382288 0.045712 ENSRNOG00000005865 Itprid2 ITPR interacting domain containing 2 0.265378 0.040332 ENSRNOG00000005960 RGD1311744 similar to RIKEN cDNA 5830475I06 3.220081 3.20E-05 ENSRNOG00000006255 Nipal4 NIPA-like domain containing 4 0.310397 0.047572 ENSRNOG00000006718 Rbm33 RNA binding motif protein 33 0.510021 0.014823 ENSRNOG00000006841 Ano4 anoctamin 4 0.375935 0.033343 ENSRNOG00000007202 Sema3d semaphorin 3D 1.080701 0.004745 106

ENSEMBL SYMBOL GENENAME logFC adj.P.Val ENSRNOG00000007246 Atxn7 ataxin 7 0.771783 0.018789 ENSRNOG00000007705 Kcnj10 potassium voltage-gated channel subfamily J member 10 0.606665 0.030347 ENSRNOG00000007726 Mcam melanoma cell adhesion molecule 0.66048 0.000126 ENSRNOG00000008036 Dennd4c DENN domain containing 4C 0.485354 0.045165 ENSRNOG00000008039 Cul5 cullin 5 0.622368 0.035495 ENSRNOG00000008157 Syn2 synapsin II 0.235483 0.030893 ENSRNOG00000008173 Sesn3 sestrin 3 0.550191 0.045434 ENSRNOG00000008244 LOC690035 similar to Protein KIAA0586 0.6576 0.012079 ENSRNOG00000008533 Ago2 argonaute 2, RISC catalytic component 0.798679 0.001619

A.19 Downregulated genes (FDR = 0.05) in PD-ovx at 12 months in striatum

ENSEMBL SYMBOL GENENAME logFC adj.P.Val ENSRNOG00000000279 Rtn4ip1 reticulon 4 interacting protein 1 -0.88864 3.52E-05 ENSRNOG00000000288 Scarf2 scavenger receptor class F, member 2 -0.99964 0.004951 ENSRNOG00000000525 Pi16 peptidase inhibitor 16 -0.49693 0.045486 ENSRNOG00000000704 Cmklr1 chemerin chemokine-like receptor 1 -0.95702 0.009601 ENSRNOG00000000893 Tmem248 transmembrane protein 248 -0.39373 0.021312 ENSRNOG00000001289 LOC498154 hypothetical protein LOC498154 -0.47677 0.03475 ENSRNOG00000001300 P2rx4 purinergic receptor P2X 4 -1.16222 1.16E-06 ENSRNOG00000001302 Adora2a adenosine A2a receptor -0.36191 0.020292 ENSRNOG00000001404 Agfg2 ArfGAP with FG repeats 2 -0.4893 0.00128 ENSRNOG00000001418 Znhit1 zinc finger, HIT-type containing 1 -0.62195 0.004988 ENSRNOG00000001726 Tmem44 transmembrane protein 44 -0.41772 0.043723 ENSRNOG00000001770 Ehhadh enoyl-CoA hydratase and 3-hydroxyacyl CoA dehydrogenase -0.49548 0.042117 ENSRNOG00000002630 Cnot8 CCR4-NOT transcription complex, subunit 8 -0.44651 0.006573 ENSRNOG00000003114 B4galt4 beta-1,4-galactosyltransferase 4 -0.51784 0.033402 ENSRNOG00000004091 Cwc25 CWC25 spliceosome-associated protein homolog -0.39768 0.026726 ENSRNOG00000004359 Wars tryptophanyl-tRNA synthetase -0.22866 0.045165 ENSRNOG00000004687 Thbd thrombomodulin -0.59602 0.026188 107

ENSEMBL SYMBOL GENENAME logFC adj.P.Val ENSRNOG00000004794 Rtn1 reticulon 1 -0.23761 0.04628 ENSRNOG00000005350 Pwp1 PWP1 homolog, endonuclein -0.40821 0.046211 ENSRNOG00000005809 Arhgdib Rho GDP dissociation inhibitor beta -0.48972 0.005572 ENSRNOG00000006235 Nell2 neural EGFL like 2 -0.20722 0.03765 ENSRNOG00000006591 Fam163b family with sequence similarity 163, member B -0.43801 0.00318 ENSRNOG00000006595 Htr3a 5-hydroxytryptamine receptor 3A -0.63797 0.00128 ENSRNOG00000006860 Itk IL2-inducible T-cell kinase -1.1648 0.000227 ENSRNOG00000007046 Cfap70 cilia and flagella associated protein 70 -0.40032 0.045165 ENSRNOG00000007117 Cluap1 clusterin associated protein 1 -0.81216 4.90E-05 ENSRNOG00000007564 Evc EvC ciliary complex subunit 1 -0.80991 0.000841 ENSRNOG00000007862 Acat1 acetyl-CoA acetyltransferase 1 -0.33236 0.018789 ENSRNOG00000007882 Ablim2 actin binding LIM protein family, member 2 -0.36811 0.02678 ENSRNOG00000008312 Stra6 stimulated by retinoic acid 6 -0.81819 0.004944 ENSRNOG00000008994 Arpc4 actin related protein 2/3 complex, subunit 4 -0.28612 0.029307 ENSRNOG00000009563 Krt2 keratin 2 -1.36859 0.030893 ENSRNOG00000009968 Ercc8 ERCC excision repair 8, CSA ubiquitin ligase complex subunit -0.33023 0.038347 ENSRNOG00000010141 Ttll1 tubulin tyrosine ligase like 1 -0.37269 0.030559 ENSRNOG00000010412 Ccdc180 coiled-coil domain containing 180 -1.15115 0.000952 ENSRNOG00000010753 Aig1 androgen-induced 1 -0.2861 0.030991 ENSRNOG00000011348 Snx14 sorting nexin 14 -0.27461 0.043387 ENSRNOG00000011379 Ccndbp1 cyclin D1 binding protein 1 -0.30311 0.029307 ENSRNOG00000011663 Rnf7 ring finger protein 7 -0.34183 0.044147 ENSRNOG00000011758 Fkbp7 FK506 binding protein 7 -0.55498 0.045165 ENSRNOG00000011936 Abhd14a abhydrolase domain containing 14A -0.8563 0.042898 ENSRNOG00000011947 Tifab TIFA inhibitor -0.70752 0.030559 ENSRNOG00000012106 Dnaja4 DnaJ heat shock protein family (Hsp40) member A4 -0.30049 0.00851 ENSRNOG00000012811 Spint1 serine peptidase inhibitor, Kunitz type 1 -1.20688 0.001619 ENSRNOG00000013794 Rbp1 retinol binding protein 1 -0.53333 0.032366 ENSRNOG00000014117 Hmox1 heme oxygenase 1 -0.54584 0.030893 ENSRNOG00000014371 Cdh13 cadherin 13 -0.22524 0.043387 ENSRNOG00000015003 Pex11a peroxisomal biogenesis factor 11 alpha -1.21925 0.001619 ENSRNOG00000015157 Smtnl2 smoothelin-like 2 -0.95693 0.022848 108

ENSEMBL SYMBOL GENENAME logFC adj.P.Val ENSRNOG00000015869 Pccb propionyl-CoA carboxylase subunit beta -0.28406 0.04855

A.20 Downregulated genes (FDR = 0.05) in PD-ovx at 12 months in frontal cortex

ENSEMBL SYMBOL GENENAME logFC adj.P.Val ENSRNOG00000000007 Gad1 glutamate decarboxylase 1 -0.26073 0.014472 ENSRNOG00000000279 Rtn4ip1 reticulon 4 interacting protein 1 -0.55674 0.001057 ENSRNOG00000000778 Znrd1as1 ZNRD1 antisense RNA 1 -0.58824 0.04228 ENSRNOG00000000800 Man1a1 mannosidase, alpha, class 1A, member 1 -0.38957 0.024057 ENSRNOG00000000805 Gja1 gap junction protein, alpha 1 -0.23274 0.039617 ENSRNOG00000001289 LOC498154 hypothetical protein LOC498154 -0.34829 0.044275 ENSRNOG00000001300 P2rx4 purinergic receptor P2X 4 -1.05302 4.99E-07 ENSRNOG00000001323 Zfp157 zinc finger protein 157 -0.37347 0.003329 ENSRNOG00000001351 Trafd1 TRAF type zinc finger domain containing 1 -0.35515 0.008779 ENSRNOG00000001383 Slc8b1 solute carrier family 8 member B1 -0.76215 0.000521 ENSRNOG00000001404 Agfg2 ArfGAP with FG repeats 2 -0.48946 6.53E-05 ENSRNOG00000001418 Znhit1 zinc finger, HIT-type containing 1 -0.37936 0.01151 ENSRNOG00000002369 Rgs8 regulator of G-protein signaling 8 -0.35809 0.008535 ENSRNOG00000002630 Cnot8 CCR4-NOT transcription complex, subunit 8 -0.34294 0.001886 ENSRNOG00000002730 Rgs5 regulator of G-protein signaling 5 -0.30923 0.023553 ENSRNOG00000003063 Phka1 phosphorylase kinase regulatory subunit alpha 1 -0.38178 0.000966 ENSRNOG00000003461 Zfp330 zinc finger protein 330 -0.24329 0.010199 ENSRNOG00000003929 Pcdh19 protocadherin 19 -0.35052 0.009045 ENSRNOG00000004516 Itgbl1 integrin subunit beta like 1 -0.64634 0.030365 ENSRNOG00000005007 Scn3a sodium voltage-gated channel alpha subunit 3 -0.41423 0.001368 ENSRNOG00000005141 Hus1 HUS1 checkpoint clamp component -0.28245 0.012524 ENSRNOG00000005257 Prkaca protein kinase cAMP-activated catalytic subunit alpha -0.25743 0.01529 ENSRNOG00000005330 Crebbp CREB binding protein -0.24559 0.01151 ENSRNOG00000005350 Pwp1 PWP1 homolog, endonuclein -0.5368 0.008779 ENSRNOG00000005457 Lamp5 lysosomal-associated membrane protein family, member 5 -0.29168 0.008132 ENSRNOG00000005809 Arhgdib Rho GDP dissociation inhibitor beta -0.40743 0.02074 109

ENSEMBL SYMBOL GENENAME logFC adj.P.Val ENSRNOG00000005984 Etv6 ets variant 6 -0.56208 0.04959 transient receptor potential cation channel, subfamily C, ENSRNOG00000006324 Trpc6 member 6 -0.42938 0.023301 ENSRNOG00000006375 Vdac1 voltage-dependent anion channel 1 -0.16195 0.039153 ENSRNOG00000006749 Tmtc3 transmembrane and tetratricopeptide repeat containing 3 -0.29227 0.044609 ENSRNOG00000006956 NA NA -0.94262 0.002024 ENSRNOG00000007014 Cnksr2 connector enhancer of kinase suppressor of Ras 2 -0.39175 0.026687 ENSRNOG00000007117 Cluap1 clusterin associated protein 1 -0.80063 1.21E-07 ENSRNOG00000007600 Igsf1 immunoglobulin superfamily, member 1 -0.45153 0.020237 ENSRNOG00000007862 Acat1 acetyl-CoA acetyltransferase 1 -0.19841 0.023553 ENSRNOG00000008312 Stra6 stimulated by retinoic acid 6 -0.78384 0.008132 ENSRNOG00000008316 Vps39 VPS39 HOPS complex subunit -0.18525 0.039617 ENSRNOG00000008474 Acox3 acyl-CoA oxidase 3, pristanoyl -0.36412 0.01815 ENSRNOG00000008786 Ap1b1 adaptor related protein complex 1 subunit beta 1 -0.17894 0.038166 ENSRNOG00000009263 Ifi27 interferon, alpha-inducible protein 27 -0.56868 0.015049 UDP-GlcNAc:betaGal beta-1,3-N-acetylglucosaminyltransferase ENSRNOG00000009267 B3gnt2 2 -0.34207 0.018814 ENSRNOG00000009563 Krt2 keratin 2 -1.26361 0.009998 ENSRNOG00000009683 Sdcbp syndecan binding protein -0.14037 0.048474 ENSRNOG00000010412 Ccdc180 coiled-coil domain containing 180 -0.94367 0.012235 ENSRNOG00000010555 Phyhip phytanoyl-CoA 2-hydroxylase interacting protein -0.15172 0.01829 ENSRNOG00000010555 LOC108348161 phytanoyl-CoA hydroxylase-interacting protein -0.15172 0.01829 ENSRNOG00000010753 Aig1 androgen-induced 1 -0.2592 0.014634 ENSRNOG00000011214 Polr2f RNA polymerase II subunit F -0.26164 0.04334 ENSRNOG00000011348 Snx14 sorting nexin 14 -0.38456 0.002346 ENSRNOG00000011826 Lzts1 leucine zipper tumor suppressor 1 -0.14045 0.04959

A.21 Upregulated genes (FDR = 0.05) in PD-ovx at 12 months in cerebellum

ENSEMBL SYMBOL GENENAME logFC adj.P.Val ENSRNOG00000001427 Orai2 ORAI calcium release-activated calcium modulator 2 1.030112 1.96E-06 110

ENSEMBL SYMBOL GENENAME logFC adj.P.Val ENSRNOG00000001368 Rph3a rabphilin 3A 0.806918 1.69E-05 ENSRNOG00000000811 Pkib cAMP-dependent protein kinase inhibitor beta 0.769092 0.001561 ENSRNOG00000000700 Tmem119 transmembrane protein 119 0.77617 0.002929 ENSRNOG00000001329 Gjc3 gap junction protein, gamma 3 0.930176 0.004717 HECT domain and ankyrin repeat containing, E3 ubiquitin protein ENSRNOG00000000327 Hace1 ligase 1 0.315867 0.007183 ENSRNOG00000000699 Selplg selectin P ligand 0.725004 0.007669 ENSRNOG00000001602 Ltn1 listerin E3 ubiquitin protein ligase 1 0.30744 0.007696 ENSRNOG00000001609 Cep97 centrosomal protein 97 0.379963 0.007696 ENSRNOG00000002705 Vps4b vacuolar protein sorting 4 homolog B 0.301744 0.008328 ENSRNOG00000004327 Ddc dopa decarboxylase 0.727862 0.008328 ENSRNOG00000002254 Tmem33 transmembrane protein 33 0.238901 0.010032 ENSRNOG00000002863 Cacna1e calcium voltage-gated channel subunit alpha1 E 0.522209 0.011306 ENSRNOG00000004011 Nedd1 neural precursor cell expressed, developmentally down-regulated 1 0.614207 0.013758 ENSRNOG00000002750 Rc3h1 ring finger and CCCH-type domains 1 0.46339 0.014708 ENSRNOG00000001730 Acap2 ArfGAP with coiled-coil, ankyrin repeat and PH domains 2 0.280042 0.014976 ENSRNOG00000001711 Hrasls HRAS-like suppressor 0.71222 0.018621 ENSRNOG00000000204 Syncrip synaptotagmin binding, cytoplasmic RNA interacting protein 0.365849 0.02046 ENSRNOG00000002232 Aff1 AF4/FMR2 family, member 1 0.464414 0.02167 ENSRNOG00000003213 Helz helicase with zinc finger 0.330063 0.021966 ENSRNOG00000004218 Klhl28 kelch-like family member 28 0.392006 0.02275 ENSRNOG00000003781 Atp10b ATPase phospholipid transporting 10B (putative) 0.596675 0.02509 ENSRNOG00000001774 Lrch3 leucine rich repeats and calponin homology domain containing 3 0.300984 0.025391 ENSRNOG00000002866 Rassf6 Ras association domain family member 6 1.25384 0.026408 ENSRNOG00000003694 Prox1 prospero homeobox 1 0.354647 0.027243 ENSRNOG00000003434 Trove2 TROVE domain family, member 2 0.428121 0.029645 ENSRNOG00000002941 Uhmk1 U2AF homology motif kinase 1 0.428755 0.030028 ENSRNOG00000002592 Rps6ka6 ribosomal protein S6 kinase A6 0.613967 0.030132 ENSRNOG00000003873 Cpd carboxypeptidase D 0.460389 0.033543 ENSRNOG00000004186 Snx13 sorting nexin 13 0.228149 0.033888 111

ENSEMBL SYMBOL GENENAME logFC adj.P.Val ENSRNOG00000001657 Cldnd1 claudin domain containing 1 0.194409 0.034354 ENSRNOG00000000815 Smpdl3a sphingomyelin phosphodiesterase, acid-like 3A 0.343727 0.036114 ENSRNOG00000004402 Lpgat1 lysophosphatidylglycerol acyltransferase 1 0.305606 0.036119 ENSRNOG00000002318 Limch1 LIM and calponin homology domains 1 0.210768 0.03615 ENSRNOG00000002028 Tmem50b transmembrane protein 50B 0.181763 0.036309 ENSRNOG00000003890 Nap1l1 nucleosome assembly protein 1-like 1 0.187302 0.036378 ENSRNOG00000002538 Epb41l5 erythrocyte membrane protein band 4.1 like 5 0.413097 0.036494 ENSRNOG00000002349 Gabra2 gamma-aminobutyric acid type A receptor alpha2 subunit 0.472553 0.03804 ENSRNOG00000000657 Nek7 NIMA-related kinase 7 0.405536 0.038546 ENSRNOG00000004226 Irak3 interleukin-1 receptor-associated kinase 3 0.567361 0.041742 ENSRNOG00000002529 Rap2c RAP2C, member of RAS oncogene family 0.282447 0.044324 ENSRNOG00000003241 Gabrg2 gamma-aminobutyric acid type A receptor gamma 2 subunit 0.216492 0.044792 ENSRNOG00000002833 Gsk3b glycogen synthase kinase 3 beta 0.256189 0.04689 ENSRNOG00000000142 Plxdc2 plexin domain containing 2 0.257374 0.046934 ENSRNOG00000001108 N4bp2l2 NEDD4 binding protein 2-like 2 0.215268 0.047587 ENSRNOG00000001834 Mzt2b mitotic spindle organizing protein 2B 0.326082 0.047587 ENSRNOG00000004057 Ccdc88a coiled coil domain containing 88A 0.300843 0.047587 ENSRNOG00000001335 Zkscan1 zinc finger with KRAB and SCAN domains 1 0.232018 0.048334 ENSRNOG00000001956 Dzip3 DAZ interacting zinc finger protein 3 0.350328 0.048742 ENSRNOG00000002225 Scarb2 scavenger receptor class B, member 2 0.275122 0.049863

A.22 Downregulated genes (FDR = 0.05) in PD-ovx at 12 months in cerebellum

ENSEMBL SYMBOL GENENAME logFC adj.P.Val ENSRNOG00000001300 P2rx4 purinergic receptor P2X 4 -1.33141 7.84E-07 ENSRNOG00000000279 Rtn4ip1 reticulon 4 interacting protein 1 -0.90581 1.86E-05 ENSRNOG00000001351 Trafd1 TRAF type zinc finger domain containing 1 -0.42875 0.002587 ENSRNOG00000001374 Rasal1 RAS protein activator like 1 -0.99009 0.004717 ENSRNOG00000004794 Rtn1 reticulon 1 -0.24985 0.006251 112

ENSEMBL SYMBOL GENENAME logFC adj.P.Val ENSRNOG00000005257 Prkaca protein kinase cAMP-activated catalytic subunit alpha -0.32645 0.00849 ENSRNOG00000006033 Spon2 spondin 2 -0.90125 0.009163 ENSRNOG00000006033 LOC100910790 spondin-2-like -0.90125 0.009163 ENSRNOG00000003497 Thoc6 THO complex 6 -0.43158 0.010104 ENSRNOG00000002630 Cnot8 CCR4-NOT transcription complex, subunit 8 -0.30405 0.011232 ENSRNOG00000001888 Arvcf ARVCF, delta catenin family member -0.50812 0.012244 ENSRNOG00000005623 Ankmy2 ankyrin repeat and MYND domain containing 2 -0.24897 0.0123 ENSRNOG00000006375 Vdac1 voltage-dependent anion channel 1 -0.2025 0.012913 ENSRNOG00000002343 Uchl1 ubiquitin C-terminal hydrolase L1 -0.76148 0.013532 ENSRNOG00000004276 Itga3 integrin subunit alpha 3 -0.28261 0.013585 ENSRNOG00000001792 Slc12a8 solute carrier family 12, member 8 -1.05552 0.014411 ENSRNOG00000001254 Col6a2 collagen type VI alpha 2 chain -1.00799 0.015285 ENSRNOG00000005809 Arhgdib Rho GDP dissociation inhibitor beta -0.66992 0.01644 ENSRNOG00000001710 Abcf3 ATP binding cassette subfamily F member 3 -0.21058 0.01654 ENSRNOG00000001738 Eif4g1 eukaryotic translation initiation factor 4 gamma, 1 -0.22205 0.016677 ENSRNOG00000003887 Lgi2 leucine-rich repeat LGI family, member 2 -0.22372 0.020619 ENSRNOG00000001752 Nrros negative regulator of reactive oxygen species -0.50446 0.02069 ENSRNOG00000004687 Thbd thrombomodulin -0.71655 0.021392 ENSRNOG00000005330 Crebbp CREB binding protein -0.30446 0.021966 ENSRNOG00000001469 Eln elastin -0.65642 0.024109 ENSRNOG00000001837 Sst somatostatin -0.98255 0.02509 ENSRNOG00000000172 Sqor sulfide quinone oxidoreductase -0.48118 0.025884 ENSRNOG00000005141 Hus1 HUS1 checkpoint clamp component -0.30833 0.027339 ENSRNOG00000003504 Rnaseh2a ribonuclease H2, subunit A -0.30404 0.027779 ENSRNOG00000004091 Cwc25 CWC25 spliceosome-associated protein homolog -0.31473 0.027779 ENSRNOG00000005003 Ptprn2 protein tyrosine phosphatase, receptor type N2 -0.26798 0.027779 ENSRNOG00000001316 Anapc5 anaphase-promoting complex subunit 5 -0.2142 0.028749 ENSRNOG00000003835 Slc43a2 solute carrier family 43 member 2 -0.23812 0.030351 ENSRNOG00000000487 Grm4 glutamate metabotropic receptor 4 -0.51098 0.031928 tyrosine 3-monooxygenase/tryptophan 5-monooxygenase ENSRNOG00000001436 Ywhag activation protein, gamma -0.16523 0.03369 ENSRNOG00000003114 B4galt4 beta-1,4-galactosyltransferase 4 -0.45471 0.03369 113

ENSEMBL SYMBOL GENENAME logFC adj.P.Val ENSRNOG00000005007 Scn3a sodium voltage-gated channel alpha subunit 3 -0.76384 0.033888 ENSRNOG00000001232 Slc19a1 solute carrier family 19 member 1 -0.39489 0.034546 ENSRNOG00000001715 Ece2 endothelin-converting enzyme 2 -0.3219 0.034546 ENSRNOG00000003815 Slc25a11 solute carrier family 25 member 11 -0.21187 0.034546 ENSRNOG00000004763 Sirpa signal-regulatory protein alpha -0.16337 0.035826 ENSRNOG00000001408 Actl6b actin-like 6B -0.21485 0.036243 ENSRNOG00000001431 Rasa4 RAS p21 protein activator 4 -0.60992 0.036309 leucine rich repeat and fibronectin type III domain containing ENSRNOG00000005550 Lrfn5 5 -0.4068 0.03706 ENSRNOG00000006930 Casq1 calsequestrin 1 -0.65324 0.038018 ENSRNOG00000000262 Zfp821 zinc finger protein 821 -0.23068 0.038272 ENSRNOG00000007046 Cfap70 cilia and flagella associated protein 70 -0.75848 0.042995 ENSRNOG00000001124 Rnft2 ring finger protein, transmembrane 2 -0.19327 0.043095 ENSRNOG00000004900 Crhr1 corticotropin releasing hormone receptor 1 -0.40281 0.044792 ENSRNOG00000004014 Chmp6 charged multivesicular body protein 6 -0.24332 0.045316

A.23 Upregulated genes (FDR = 0.05) in PD-ko at 2 months in frontal cortex

ENSEMBL SYMBOL GENENAME logFC adj.P.Val ENSMUSG00000052861 Dnah6 dynein, axonemal, heavy chain 6 3.38956 1.16E-07

A.24 Downregulated genes (FDR = 0.05) in PD-ko at 2 months in frontal cortex

ENSEMBL SYMBOL GENENAME logFC adj.P.Val ENSMUSG00000029798 Herc6 hect domain and RLD 6 -2.05777 1.33E-06 ENSMUSG00000062190 Lancl2 LanC (bacterial lantibiotic synthetase component C)-like 2 -0.70838 0.00019 ENSMUSG00000003477 Inmt indolethylamine N-methyltransferase -2.04032 0.001657 ENSMUSG00000029780 Nt5c3 5'-nucleotidase, cytosolic III -0.45334 0.001657 ENSMUSG00000043162 Pyurf Pigy upstream reading frame -0.53642 0.001657 ENSMUSG00000037826 Ppm1k protein phosphatase 1K (PP2C domain containing) -0.49987 0.004591 114

ENSMUSG00000037788 Vopp1 vesicular, overexpressed in cancer, prosurvival protein 1 -0.31149 0.012776 ENSMUSG00000083287 NA NA -1.1745 0.024139

A.25 Upregulated genes (FDR = 0.05) in PD-ko at 2 months in striatum

ENSEMBL SYMBOL GENENAME logFC adj.P.Val ENSMUSG00000052861 Dnah6 dynein, axonemal, heavy chain 6 4.329593 1.60E-07

A.26 Downregulated genes (FDR = 0.05) in PD-ko at 2 months in striatum

ENSEMBL SYMBOL GENENAME logFC adj.P.Val ENSMUSG00000003477 Inmt indolethylamine N-methyltransferase -3.59736 1.25E-06 ENSMUSG00000029781 Fkbp9 FK506 binding protein 9 -0.56764 0.001282 ENSMUSG00000043162 Pyurf Pigy upstream reading frame -0.61869 0.013946 ENSMUSG00000062190 Lancl2 LanC (bacterial lantibiotic synthetase component C)-like 2 -0.40474 0.025885 ENSMUSG00000014554 Dguok deoxyguanosine kinase -0.41745 0.034371

A.27 Upregulated genes (FDR = 0.05) in PD-ko at 12 months in frontal cortex

ENSEMBL SYMBOL GENENAME logFC adj.P.Val ENSMUSG00000003974 Grm3 glutamate receptor, metabotropic 3 0.292633 0.006163 ENSMUSG00000000804 Usp32 ubiquitin specific peptidase 32 0.280827 0.006376 ENSMUSG00000000416 Cttnbp2 cortactin binding protein 2 0.279405 0.006797 ENSMUSG00000003847 Nfat5 nuclear factor of activated T cells 5 0.322441 0.007341 ENSMUSG00000003228 Grk5 G protein-coupled receptor kinase 5 0.357463 0.010006 ENSMUSG00000002475 Abhd3 abhydrolase domain containing 3 0.229229 0.01017 ENSMUSG00000001260 Gabrg1 gamma-aminobutyric acid (GABA) A receptor, subunit gamma 1 0.297163 0.010381 ENSMUSG00000003623 Crot carnitine O-octanoyltransferase 0.230802 0.011647 ENSMUSG00000002413 Braf Braf transforming gene 0.264001 0.012892 115

ENSEMBL SYMBOL GENENAME logFC adj.P.Val ENSMUSG00000002265 Peg3 paternally expressed 3 0.277481 0.013198 ENSMUSG00000005124 Wisp1 WNT1 inducible signaling pathway protein 1 0.643546 0.013547 ENSMUSG00000000184 Ccnd2 cyclin D2 0.350615 0.015777 ENSMUSG00000003178 NA NA 0.379208 0.01667 ENSMUSG00000002028 Kmt2a lysine (K)-specific methyltransferase 2A 0.255727 0.017512 ENSMUSG00000004591 Pkn2 protein kinase N2 0.261866 0.018114 ENSMUSG00000001986 Gria3 glutamate receptor, ionotropic, AMPA3 (alpha 3) 0.201751 0.018563 ENSMUSG00000000560 Gabra2 gamma-aminobutyric acid (GABA) A receptor, subunit alpha 2 0.298092 0.021898 ENSMUSG00000003452 Bicd1 BICD cargo adaptor 1 0.323559 0.02194 ENSMUSG00000000001 Gnai3 guanine nucleotide binding protein (G protein), alpha inhibiting 3 0.254025 0.022116 ENSMUSG00000004609 Cd33 CD33 antigen 0.453424 0.022288 ENSMUSG00000003226 Ranbp2 RAN binding protein 2 0.307101 0.024888 ENSMUSG00000000711 Rab5b RAB5B, member RAS oncogene family 0.149913 0.025243 ENSMUSG00000004698 Hdac9 histone deacetylase 9 0.288674 0.026665 ENSMUSG00000000838 Fmr1 fragile X mental retardation 1 0.276115 0.027746 ENSMUSG00000004798 Ulk2 unc-51 like kinase 2 0.184979 0.027856 ENSMUSG00000004110 Cacna1e calcium channel, voltage-dependent, R type, alpha 1E subunit 0.195017 0.027941 ENSMUSG00000004360 9330159F19Rik RIKEN cDNA 9330159F19 gene 0.192573 0.028143 ENSMUSG00000004319 Clcn3 chloride channel, voltage-sensitive 3 0.219603 0.028168 ENSMUSG00000003418 St8sia6 ST8 alpha-N-acetyl-neuraminide alpha-2,8-sialyltransferase 6 0.543186 0.028541 solute carrier family 1 (glial high affinity glutamate transporter), ENSMUSG00000005089 Slc1a2 member 2 0.226462 0.032433 ENSMUSG00000000600 Krit1 KRIT1, ankyrin repeat containing 0.209285 0.03414 ENSMUSG00000001089 Luzp1 leucine zipper protein 1 0.217873 0.035341 ENSMUSG00000001280 Sp1 trans-acting transcription factor 1 0.260623 0.035934 cysteine and histidine-rich domain (CHORD)-containing, zinc- ENSMUSG00000001774 Chordc1 binding protein 1 0.249093 0.037156 ENSMUSG00000002107 Celf2 CUGBP, Elav-like family member 2 0.159389 0.037488 ENSMUSG00000001741 Il16 interleukin 16 0.461483 0.038444 ENSMUSG00000003119 Cdk12 cyclin-dependent kinase 12 0.266702 0.039814 116

ENSEMBL SYMBOL GENENAME logFC adj.P.Val ENSMUSG00000000266 Mid2 midline 2 0.297353 0.040048 ENSMUSG00000004364 Cul3 cullin 3 0.219364 0.04063 ENSMUSG00000000420 Galnt1 polypeptide N-acetylgalactosaminyltransferase 1 0.192795 0.041257 ENSMUSG00000005267 Zfp287 zinc finger protein 287 0.286069 0.041686 ENSMUSG00000001173 Ocrl OCRL, inositol polyphosphate-5-phosphatase 0.176316 0.041753 ENSMUSG00000004221 Ikbkg inhibitor of kappaB kinase gamma 0.170045 0.041965 ENSMUSG00000001998 Ap4e1 adaptor-related protein complex AP-4, epsilon 1 0.352386 0.042419 potassium intermediate/small conductance calcium-activated ENSMUSG00000000794 Kcnn3 channel, subfamily N, member 3 0.312345 0.043555 ENSMUSG00000001323 Srr serine racemase 0.166365 0.044148 ENSMUSG00000003282 Plag1 pleiomorphic adenoma gene 1 0.536182 0.044681 ENSMUSG00000001870 Ltbp1 latent transforming growth factor beta binding protein 1 0.245346 0.046515 ENSMUSG00000003746 Man1a mannosidase 1, alpha 0.261996 0.047517 ENSMUSG00000000305 Cdh4 cadherin 4 0.211813 0.048347

A.28 Downregulated genes (FDR = 0.05) in PD-ko at 12 months in frontal cortex

ENSEMBL SYMBOL GENENAME logFC adj.P.Val ENSMUSG00000001506 Col1a1 collagen, type I, alpha 1 -1.2126 0.002664 ENSMUSG00000000326 Comt catechol-O-methyltransferase -0.30297 0.003942 ENSMUSG00000000552 Zfp385a zinc finger protein 385A -0.42515 0.004763 ENSMUSG00000001270 Ckb creatine kinase, brain -0.6471 0.005455 ENSMUSG00000002343 Armc6 armadillo repeat containing 6 -0.46703 0.005976 sema domain, transmembrane domain (TM), and cytoplasmic ENSMUSG00000001227 Sema6b domain, (semaphorin) 6B -0.42853 0.007828 ENSMUSG00000002210 Smg9 smg-9 homolog, nonsense mediated mRNA decay factor (C. elegans) -0.4707 0.007929 ENSMUSG00000000325 Arvcf armadillo repeat gene deleted in velocardiofacial syndrome -0.46048 0.008781 ENSMUSG00000001424 Snd1 staphylococcal nuclease and tudor domain containing 1 -0.2207 0.00889

117

ENSEMBL SYMBOL GENENAME logFC adj.P.Val solute carrier family 9 (sodium/hydrogen exchanger), member 3 ENSMUSG00000002504 Slc9a3r2 regulator 2 -0.42359 0.00952 ENSMUSG00000002379 Ndufa11 NADH:ubiquinone oxidoreductase subunit A11 -0.46578 0.009584 ENSMUSG00000001552 Jup junction plakoglobin -0.44806 0.009951 ENSMUSG00000002102 Psmc3 proteasome (prosome, macropain) 26S subunit, ATPase 3 -0.1843 0.01072 ENSMUSG00000000740 Rpl13 ribosomal protein L13 -0.35989 0.011044 ENSMUSG00000002372 Ranbp3 RAN binding protein 3 -0.3048 0.011844 ENSMUSG00000001666 Ddt D-dopachrome tautomerase -0.45203 0.012459 ENSMUSG00000001054 Rmnd5b required for meiotic nuclear division 5 homolog B -0.25641 0.015142 ENSMUSG00000001988 Npas1 neuronal PAS domain protein 1 -0.67902 0.01667 ENSMUSG00000001802 Lrp3 low density lipoprotein receptor-related protein 3 -0.35172 0.01755 ENSMUSG00000002524 Puf60 poly-U binding splicing factor 60 -0.21179 0.017751 ENSMUSG00000002658 Gtf2f1 general transcription factor IIF, polypeptide 1 -0.27043 0.018466 ENSMUSG00000000915 Hip1r huntingtin interacting protein 1 related -0.3488 0.018982 ENSMUSG00000001525 Tubb5 tubulin, beta 5 class I -0.21077 0.019025 ENSMUSG00000000149 Gna12 guanine nucleotide binding protein, alpha 12 -0.29904 0.020001 ENSMUSG00000002105 Slc39a13 solute carrier family 39 (metal ion transporter), member 13 -0.32761 0.020087 ENSMUSG00000002280 Narfl nuclear prelamin A recognition factor-like -0.22225 0.020375 ENSMUSG00000002043 Trappc6a trafficking protein particle complex 6A -0.59425 0.020938 ENSMUSG00000002393 Nr2f6 nuclear receptor subfamily 2, group F, member 6 -0.32371 0.020938 ENSMUSG00000001794 Capns1 calpain, small subunit 1 -0.16168 0.021417 ENSMUSG00000002274 Metrn meteorin, glial cell differentiation regulator -0.48867 0.021442 ENSMUSG00000000253 Gmpr guanosine monophosphate reductase -0.24334 0.023373 ENSMUSG00000002608 Ccdc97 coiled-coil domain containing 97 -0.26156 0.025329 ENSMUSG00000001751 Naglu alpha-N-acetylglucosaminidase (Sanfilippo disease IIIB) -0.31708 0.026371 ENSMUSG00000002395 Use1 unconventional SNARE in the ER 1 homolog (S. cerevisiae) -0.3371 0.026746 ENSMUSG00000001082 Mfsd10 major facilitator superfamily domain containing 10 -0.53577 0.027856 ENSMUSG00000002319 Ipo4 importin 4 -0.20269 0.028485 ENSMUSG00000000632 Sez6 seizure related gene 6 -0.26838 0.028648 ENSMUSG00000002228 Ppm1j protein phosphatase 1J -1.05597 0.031107 118

ENSEMBL SYMBOL GENENAME logFC adj.P.Val ENSMUSG00000001034 Mapk7 mitogen-activated protein kinase 7 -0.41654 0.031958 ENSMUSG00000001288 Rarg retinoic acid receptor, gamma -0.38876 0.033952 ENSMUSG00000000861 Bcl11a B cell CLL/lymphoma 11A (zinc finger protein) -0.17718 0.036317 ENSMUSG00000001844 Zdhhc4 zinc finger, DHHC domain containing 4 -0.24268 0.037019 ENSMUSG00000001436 Slc19a1 solute carrier family 19 (folate transporter), member 1 -0.32129 0.038485 ENSMUSG00000002486 Tchp trichoplein, keratin filament binding -0.28334 0.038511 ENSMUSG00000001062 Vps9d1 VPS9 domain containing 1 -0.2517 0.040615 ENSMUSG00000002233 Rhoc ras homolog family member C -0.3407 0.041736 ENSMUSG00000000384 Tbrg4 transforming growth factor beta regulated gene 4 -0.22364 0.042257 nucleus accumbens associated 1, BEN and BTB (POZ) domain ENSMUSG00000001910 Nacc1 containing -0.16113 0.044681 ENSMUSG00000001380 Hars histidyl-tRNA synthetase -0.13768 0.046948 ENSMUSG00000001755 Coasy Coenzyme A synthase -0.22864 0.049865

A.29 Upregulated genes (FDR = 0.05) in PD-ko+ovx at 2 months, common to all tissues

ENSEMBL SYMBOL GENENAME logFC adj.P.Val ENSMUSG00000000247 Lhx2 LIM homeobox protein 2 0.64351 0.000189 ENSMUSG00000000628 Hk2 hexokinase 2 0.479842 0.010793 ENSMUSG00000019124 Scrn1 secernin 1 0.179539 0.045142 ENSMUSG00000049281 Scn3b sodium channel, voltage-gated, type III, beta 0.247222 0.045142

A.30 Downregulated genes (FDR = 0.05) in PD-ko+ovx at 2 months, common to all tissues

ENSEMBL SYMBOL GENENAME logFC adj.P.Val ENSMUSG00000043162 Pyurf Pigy upstream reading frame -0.74318 2.02E-08 ENSMUSG00000004347 Pde1c phosphodiesterase 1C -1.2911 9.79E-07 ENSMUSG00000002797 Ggct gamma-glutamyl cyclotransferase -0.52256 0.000117 ENSMUSG00000003477 Inmt indolethylamine N-methyltransferase -2.04926 0.000117 119

ENSEMBL SYMBOL GENENAME logFC adj.P.Val ENSMUSG00000003273 Car11 carbonic anhydrase 11 -0.32914 0.045142 ENSMUSG00000040613 Apobec1 apolipoprotein B mRNA editing enzyme, catalytic polypeptide 1 -0.66464 0.045142

A.31 Upregulated genes (FDR = 0.05) in PD-ko+ovx at 2 months in frontal cortex

ENSEMBL SYMBOL GENENAME logFC adj.P.Val ENSMUSG00000052861 Dnah6 dynein, axonemal, heavy chain 6 2.969871 3.25E-05 ENSMUSG00000040249 Lrp1 low density lipoprotein receptor-related protein 1 0.32927 0.016685 ENSMUSG00000031502 Col4a1 collagen, type IV, alpha 1 0.337192 0.032332 ENSMUSG00000031785 Adgrg1 adhesion G protein-coupled receptor G1 0.339037 0.043504

A.32 Downregulated genes (FDR = 0.05) in PD-ko+ovx at 2 months in frontal cortex

ENSEMBL SYMBOL GENENAME logFC adj.P.Val ENSMUSG00000029798 Herc6 hect domain and RLD 6 -2.16712 1.87E-05 ENSMUSG00000003477 Inmt indolethylamine N-methyltransferase -2.29067 0.000144 ENSMUSG00000029780 Nt5c3 5'-nucleotidase, cytosolic III -0.37984 0.000687 ENSMUSG00000062190 Lancl2 LanC (bacterial lantibiotic synthetase component C)-like 2 -0.71925 0.000687 ENSMUSG00000037826 Ppm1k protein phosphatase 1K (PP2C domain containing) -0.44953 0.010615 ENSMUSG00000043162 Pyurf Pigy upstream reading frame -0.52881 0.013537 ENSMUSG00000051671 Coa6 cytochrome c oxidase assembly factor 6 -0.51341 0.013537 ENSMUSG00000028719 Cmpk1 cytidine monophosphate (UMP-CMP) kinase 1 -0.28286 0.020895 ENSMUSG00000029781 Fkbp9 FK506 binding protein 9 -0.36259 0.029913 ENSMUSG00000072704 Smim10l1 small integral membrane protein 10 like 1 -0.24657 0.032452 ENSMUSG00000044934 Zfp367 zinc finger protein 367 -0.3519 0.040946

120

A.33 Downregulated genes (FDR = 0.05) in PD-ko+ovx at 2 months in cerebellum

ENSEMBL SYMBOL GENENAME logFC adj.P.Val ENSMUSG00000043162 Pyurf Pigy upstream reading frame -0.74318 0.000289 ENSMUSG00000004347 Pde1c phosphodiesterase 1C -1.2911 0.000454 ENSMUSG00000003477 Inmt indolethylamine N-methyltransferase -2.04926 0.029389 ENSMUSG00000002797 Ggct gamma-glutamyl cyclotransferase -0.52256 0.043429

A.34 Upregulated genes (FDR = 0.05) in PD-ko+ovx at 12 months in frontal cortex

ENSEMBL SYMBOL GENENAME logFC adj.P.Val ENSMUSG00000005871 Apc adenomatosis polyposis coli 0.349291 0.011298 ENSMUSG00000003226 Ranbp2 RAN binding protein 2 0.377424 0.0135 ENSMUSG00000005371 Fbxo11 F-box protein 11 0.279636 0.014245 ENSMUSG00000000804 Usp32 ubiquitin specific peptidase 32 0.284281 0.015114 ENSMUSG00000003847 Nfat5 nuclear factor of activated T cells 5 0.298653 0.015665 ENSMUSG00000005893 Nr2c2 nuclear receptor subfamily 2, group C, member 2 0.327367 0.015676 ENSMUSG00000004364 Cul3 cullin 3 0.309435 0.016586 ENSMUSG00000004319 Clcn3 chloride channel, voltage-sensitive 3 0.272693 0.016619 ENSMUSG00000005583 Mef2c myocyte enhancer factor 2C 0.353217 0.016754 ENSMUSG00000003452 Bicd1 BICD cargo adaptor 1 0.410493 0.017108 ENSMUSG00000002428 Hltf helicase-like transcription factor 0.287883 0.019773 ENSMUSG00000001089 Luzp1 leucine zipper protein 1 0.226785 0.021625 ENSMUSG00000004609 Cd33 CD33 antigen 0.391442 0.022171 ENSMUSG00000000560 Gabra2 gamma-aminobutyric acid (GABA) A receptor, subunit alpha 2 0.2876 0.022998 ENSMUSG00000001376 Vps50 VPS50 EARP/GARPII complex subunit 0.237454 0.022998 cysteine and histidine-rich domain (CHORD)-containing, zinc-binding ENSMUSG00000001774 Chordc1 protein 1 0.315574 0.022998 ENSMUSG00000005124 Wisp1 WNT1 inducible signaling pathway protein 1 0.613506 0.023923 pleckstrin homology domain containing, family A (phosphoinositide ENSMUSG00000005225 Plekha8 binding specific) member 8 0.242687 0.023928 121

ENSEMBL SYMBOL GENENAME logFC adj.P.Val ENSMUSG00000002413 Braf Braf transforming gene 0.253771 0.024697 ENSMUSG00000003746 Man1a mannosidase 1, alpha 0.450221 0.025746 ENSMUSG00000004317 Clcn5 chloride channel, voltage-sensitive 5 0.297699 0.026654 ENSMUSG00000000838 Fmr1 fragile X mental retardation 1 0.294039 0.026855 ENSMUSG00000000001 Gnai3 guanine nucleotide binding protein (G protein), alpha inhibiting 3 0.261392 0.028218 ENSMUSG00000005534 Insr insulin receptor 0.244539 0.028789 ENSMUSG00000004221 Ikbkg inhibitor of kappaB kinase gamma 0.202344 0.028938 ENSMUSG00000003228 Grk5 G protein-coupled receptor kinase 5 0.40425 0.029089 ENSMUSG00000001173 Ocrl OCRL, inositol polyphosphate-5-phosphatase 0.222172 0.029389 ENSMUSG00000003178 NA NA 0.382732 0.030108 ENSMUSG00000000197 Nalcn sodium leak channel, non-selective 0.270203 0.030713 ENSMUSG00000004508 Gab2 growth factor receptor bound protein 2-associated protein 2 0.192555 0.031302 ENSMUSG00000004127 Trmt10a tRNA methyltransferase 10A 0.388056 0.031437 ENSMUSG00000000711 Rab5b RAB5B, member RAS oncogene family 0.166676 0.033121 ENSMUSG00000005102 Eif2ak4 eukaryotic translation initiation factor 2 alpha kinase 4 0.220139 0.033854 ENSMUSG00000004591 Pkn2 protein kinase N2 0.263526 0.035958 ENSMUSG00000000266 Mid2 midline 2 0.318805 0.036764 ENSMUSG00000004360 9330159F19Rik RIKEN cDNA 9330159F19 gene 0.21278 0.037098 ENSMUSG00000000263 Glra1 glycine receptor, alpha 1 subunit 1.319294 0.037224 ENSMUSG00000004798 Ulk2 unc-51 like kinase 2 0.175222 0.037577 ENSMUSG00000004233 Wars2 tryptophanyl tRNA synthetase 2 (mitochondrial) 0.446826 0.03865 ENSMUSG00000000184 Ccnd2 cyclin D2 0.248596 0.038988 ENSMUSG00000005802 Slc30a4 solute carrier family 30 (zinc transporter), member 4 0.262776 0.039056 ENSMUSG00000000948 NA NA 0.663058 0.042239 ENSMUSG00000001986 Gria3 glutamate receptor, ionotropic, AMPA3 (alpha 3) 0.190515 0.042531 pleckstrin homology domain-containing, family A (phosphoinositide ENSMUSG00000002733 Plekha3 binding specific) member 3 0.18594 0.043146 ENSMUSG00000000568 Hnrnpd heterogeneous nuclear ribonucleoprotein D 0.172522 0.043476 ENSMUSG00000005886 Ncoa2 nuclear receptor coactivator 2 0.207682 0.044244 ENSMUSG00000003418 St8sia6 ST8 alpha-N-acetyl-neuraminide alpha-2,8-sialyltransferase 6 0.455431 0.044415

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ENSEMBL SYMBOL GENENAME logFC adj.P.Val ENSMUSG00000003721 Insig2 insulin induced gene 2 0.194642 0.048426 ENSMUSG00000004110 Cacna1e calcium channel, voltage-dependent, R type, alpha 1E subunit 0.202429 0.049507 ENSMUSG00000000787 Ddx3x DEAD/H (Asp-Glu-Ala-Asp/His) box polypeptide 3, X-linked 0.226318 0.049714

A.35 Downregulated genes (FDR = 0.05) in PD-ko+ovx at 12 months in frontal cortex

ENSEMBL SYMBOL GENENAME logFC adj.P.Val ENSMUSG00000000915 Hip1r huntingtin interacting protein 1 related -0.4106 0.011008 ENSMUSG00000001270 Ckb creatine kinase, brain -0.71435 0.011612 ENSMUSG00000001288 Rarg retinoic acid receptor, gamma -0.48641 0.011612 ENSMUSG00000002210 Smg9 smg-9 homolog, nonsense mediated mRNA decay factor (C. elegans) -0.5149 0.011612 ENSMUSG00000000552 Zfp385a zinc finger protein 385A -0.46874 0.011674 ENSMUSG00000001082 Mfsd10 major facilitator superfamily domain containing 10 -0.67382 0.011674 ENSMUSG00000000142 Axin2 axin 2 -0.32589 0.013121 ENSMUSG00000001666 Ddt D-dopachrome tautomerase -0.45724 0.013453 ENSMUSG00000001034 Mapk7 mitogen-activated protein kinase 7 -0.54694 0.0138 ENSMUSG00000001802 Lrp3 low density lipoprotein receptor-related protein 3 -0.42708 0.014021 ENSMUSG00000000740 Rpl13 ribosomal protein L13 -0.41042 0.014843 ENSMUSG00000000384 Tbrg4 transforming growth factor beta regulated gene 4 -0.29148 0.01493 ENSMUSG00000001911 Nfix nuclear factor I/X -0.31014 0.016305 ENSMUSG00000000247 Lhx2 LIM homeobox protein 2 -0.30233 0.01644 ENSMUSG00000000326 Comt catechol-O-methyltransferase -0.25146 0.016619 ENSMUSG00000001062 Vps9d1 VPS9 domain containing 1 -0.34166 0.016894 ENSMUSG00000000693 Loxl3 lysyl oxidase-like 3 -0.57892 0.017723 sema domain, transmembrane domain (TM), and cytoplasmic domain, ENSMUSG00000001227 Sema6b (semaphorin) 6B -0.41986 0.017933 ENSMUSG00000001313 Rnd2 Rho family GTPase 2 -0.32693 0.017933 ENSMUSG00000001751 Naglu alpha-N-acetylglucosaminidase (Sanfilippo disease IIIB) -0.35516 0.019342 ENSMUSG00000002274 Metrn meteorin, glial cell differentiation regulator -0.6564 0.019342 ENSMUSG00000001552 Jup junction plakoglobin -0.3978 0.020414

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ENSEMBL SYMBOL GENENAME logFC adj.P.Val ENSMUSG00000002343 Armc6 armadillo repeat containing 6 -0.38292 0.020427 ENSMUSG00000002083 Bbc3 BCL2 binding component 3 -1.0605 0.020609 ENSMUSG00000000149 Gna12 guanine nucleotide binding protein, alpha 12 -0.35068 0.022634 ENSMUSG00000002105 Slc39a13 solute carrier family 39 (metal ion transporter), member 13 -0.40972 0.022687 ENSMUSG00000000530 Acvrl1 activin A receptor, type II-like 1 -0.30194 0.023093 ENSMUSG00000002280 Narfl nuclear prelamin A recognition factor-like -0.30568 0.024697 ENSMUSG00000000743 Chmp1a charged multivesicular body protein 1A -0.22587 0.024888 ENSMUSG00000001436 Slc19a1 solute carrier family 19 (folate transporter), member 1 -0.33334 0.025278 ENSMUSG00000002342 Tmem161a transmembrane protein 161A -0.28362 0.026368 ENSMUSG00000001229 Dpp9 dipeptidylpeptidase 9 -0.25552 0.02754 ENSMUSG00000001506 Col1a1 collagen, type I, alpha 1 -0.90956 0.032303 ENSMUSG00000000325 Arvcf armadillo repeat gene deleted in velocardiofacial syndrome -0.41542 0.034401 ENSMUSG00000001053 N4bp3 NEDD4 binding protein 3 -0.53379 0.035958 ENSMUSG00000000959 Oxa1l oxidase assembly 1-like -0.17331 0.037913 ENSMUSG00000001418 Glmp glycosylated lysosomal membrane protein -0.20609 0.038643 ENSMUSG00000002307 Daxx Fas death domain-associated protein -0.22449 0.039449 ENSMUSG00000002250 Ppard peroxisome proliferator activator receptor delta -0.3657 0.040542 ENSMUSG00000002250 1810013A23Rik RIKEN cDNA 1810013A23 gene -0.3657 0.040542 ENSMUSG00000001128 Cfp complement factor properdin -0.5034 0.040943 ENSMUSG00000001054 Rmnd5b required for meiotic nuclear division 5 homolog B -0.18354 0.041864 ENSMUSG00000002227 Mov10 Moloney leukemia virus 10 -0.33518 0.043841 ENSMUSG00000001910 Nacc1 nucleus accumbens associated 1, BEN and BTB (POZ) domain containing -0.19047 0.043904 ENSMUSG00000000282 Mnt max binding protein -0.22319 0.044656 ENSMUSG00000001794 Capns1 calpain, small subunit 1 -0.14458 0.044686 ENSMUSG00000000148 Brat1 BRCA1-associated ATM activator 1 -0.3413 0.047401 ENSMUSG00000001729 Akt1 thymoma viral proto-oncogene 1 -0.26976 0.047621 ENSMUSG00000000738 Spg7 SPG7, paraplegin matrix AAA peptidase subunit -0.21836 0.048078 T cell, immune regulator 1, ATPase, H+ transporting, lysosomal V0 protein ENSMUSG00000001750 Tcirg1 A3 -0.38477 0.04812

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