Elucidating the Lysosomal Function of Granulins Open Access

Abstract

Loss of function mutations in the gene granulin (GRN) decrease levels of the protein progranulin (PGRN) and cause the neurodegenerative diseases frontotemporal dementia (FTD) and neuronal ceroid lipofuscinosis (CLN11). Although, lysosomal dysfunction is a common feature of PGRN deficiency disorders, the underlying function of PGRN and its constituent granulin subunits in the lysosome remains unclear. The studies detailed below demonstrate that granulins have the ability to rescue disease-like phenotypes in vivo, and that these phenotypes of interest are conserved across models of PGRN deficiency. This work contributes to the field’s understanding of the molecular mechanisms underlying lysosomal dysfunction in models of PGRN deficiency, and provide insight into novel therapeutic avenues for PGRN based therapies.

Table of Contents

1     Introduction:

1.1      A Brief History of Frontotemporal Dementia (FTD)

1.2      FTD Clinical Subtypes

1.3      Pathology of Frontotemporal Lobar Degeneration

1.3.1    FTLD-Tau

1.3.2    FTLD-TDP

1.3.3    FTLD-FET

1.4      Genetics of FTD

1.4.1    C9orf72

1.4.2    MAPT

1.4.3    GRN

1.4.4    GRN in other Neurodegenerative Diseases

1.5      Functions of PGRN

1.6      PGRN and Neuronal Ceroid Lipofuscinosis

1.6.1    Overlap Between CLN11 and FTD-GRN

1.7      Lysosomal Functions of PGRN

1.8      Gene Therapy PGRN Treatments

1.9      Granulins

1.10    Production of Granulins

1.11    Functions of GRNs

1.11.1      Granulins in the Lysosome

1.11.2      Contrasting Functions of GRN and PGRNs

1.12    GRNs in FTD-GRN

1.13    Dissertation Aims and Hypothesis

2     Granulins rescue inflammation, lysosome dysfunction, and neuropathology in a mouse model of progranulin deficiency

2.1      Abstract

2.2      Introduction

2.3      RESULTS: ICV injection of rAAV at birth leads to widespread expression of granulins, PGRN, and GFP throughout the mouse brain.

2.4      Proteome-wide dysregulation in the thalamus of Grn^-/- mice is ameliorated by expression of granulins.

2.5      Individual granulins rescue dysregulated proteins in the thalamus of Grn^-/- mice.

2.6      Markers of Lysosomal Dysfunction are rescued by granulin expression across brain regions.

2.7      Microglial activation and inflammatory markers are reduced by hGRNs

2.8      Lysosomal lipid dysregulation is rescued by a single granulin.

2.9      Lipofuscin accumulation in Grn^-/- brains is alleviated by expression of human granulins.

2.10    Behavioral Phenotypes

2.11    Discussion

2.12    Acknowledgements

2.13    Supplemental Figures

2.14    Experimental models and subject details

2.15    Methods

Sample preparation for lipidomics and metabolomics analyses.

Lipidomics analysis.

Metabolomics analysis.

Analysis of glucosyl- and galactosyl-sphingolipids.

3     Chapter 3: HeLa cells Recapitulate Phenotypes of Lipid Dysregulation after loss of PGRN

3.1      Introduction

3.2      Confirming that HeLa GRN^-/- cell line is PGRN deficient

3.3      Characterizing Levels of lysosomal Proteins in GRN^-/- HeLas

3.4      Activity of Cysteine Cathepsins are dysregulated in GRN^-/- HeLa cells

3.5      Levels of LMP proteins and Lysosomal Membrane Integrity

3.6      Lipid Dysregulation is a characteristic of GRN^-/- HeLa cells

3.7      Discussion

3.8      Methods

Sample preparation for lipidomics and metabolomics analyses.

3.9      Lipidomics analysis.

4     Discussion and Future Directions

4.1      Summary of Findings:

4.2      Shared phenotypes between models:

4.3      GRNs are beneficial proteins in the lysosome

4.4      A New Model of GRN Function

4.5      Role of GRNs in Lipid Metabolism:

4.5.1    BMP

4.5.2    Glycosylsphingosines

4.6      The roles of individual GRNs, interchangeable, or divergent?

4.7      Reconciling findings that GRNs are beneficial with previous findings

4.8      Autonomous vs. Non-cell Autonomous Benefits of GRN expression

4.9      Therapeutic Role of GRNs, Outstanding Questions and Future pre-clinical studies.

4.9.1    Time of administration

4.9.2    Peripheral administration

4.9.3    Limitations of PGRN Deficient Mouse Models

4.9.4    Patient Derived iPSC lines as a platform for assessing therapeutic potential of GRNs.

4.10    Investigating Proposed mechanisms for GRNs

4.11    Isolation of Lysosomes for PGRN deficient iPSCs

4.12    Exploring Cell Type Specific Effects

4.13    Understanding Inflammatory Roles of GRNs

4.14    Closing Remarks

5     References

About this Dissertation

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School	Laney Graduate School
Department	Biological and Biomedical Sciences
Subfield / Discipline	Neuroscience
Degree	Ph.D.
Submission	Dissertation
Language	English
Research Field	Health Sciences, Pharmacology Biology, Molecular Biology, Neuroscience
Keyword	lysosome frontotemporal dementia progranulin granulins
Committee Chair / Thesis Advisor	Thomas Kukar, Emory University
Committee Members	Chadwick Hales, Emory University Eric Ortlund, Emory University Victor Faundez, Emory University Nicholas Seyfried, Emory University

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