Loss of tyrosine hydroxylase, motor deficits and elevated iron in a mouse model of phospholipase A2G6-associated neurodegeneration (PLAN)

Highlights

• Mouse model of phospholipase A2G6-associated neurodegeneration (PLAN).

• PLAN mice displayed body tremors and diminished wire hang performance.

• PLAN mice had reduced tyrosine hydroxylase but no neuronal loss in substantia nigra.

• PLAN mice had spheroid neuropathology and iron accumulation in the substantia nigra.

• Reduced tyrosine hydroxylase and spheroid neuropathology correlated with motor deficits.

Abstract

Phospholipase A2G6-associated neurodegeneration (PLAN) is a rare early-onset monogenic neurodegenerative movement disorder which targets the basal ganglia and other regions in the central and peripheral nervous system; presenting as a series of heterogenous subtypes in patients. We describe here a B6.C3-Pla2g6^m1J/CxRwb mouse model of PLAN which presents with early-onset neurodegeneration at 90 days which is analogous of the disease progression that is observed in PLAN patients. Homozygous mice had a progressively worsening motor deficit, which presented as tremors starting at 65 days and progressed to severe motor dysfunction and increased falls on the wire hang test at 90 days. This motor deficit positively correlated with a reduction in tyrosine hydroxylase (TH) protein expression in dopaminergic neurons of the substantia nigra (SN) without any neuronal loss. Fluorescence imaging of Thy1-YFP revealed spheroid formation in the SN. The spheroids in homozygous mice strongly mirrors those observed in patients and were demonstrated to correlate strongly with the motor deficits as measured by the wire hang test. The appearance of spheroids preceded TH loss and increased spheroid numbers negatively correlated with TH expression. Perls/DAB staining revealed the presence of iron accumulation within the SN of mice. This mouse model captures many of the major hallmarks of PLAN including severe-early onset neurodegeneration, a motor deficit that correlates directly to TH levels, spheroid formation and iron accumulation within the basal ganglia. Thus, this mouse line is a useful tool for further research efforts to improve understanding of how these disease mechanisms give rise to the disease presentations seen in PLAN patients as well as to test novel therapies.

Introduction

Neurodegeneration with brain iron accumulation (NBIA) is a group of rare monogenic disorders (Gregory et al., 2009, Hogarth, 2015, Levi and Finazzi, 2014, Schneider et al., 2012). These disorders share the common hallmarks of early-onset neurodegeneration, extrapyramidal motor symptoms and the accumulation of iron inside of the basal ganglia (BG) (Gregory et al., 2009, Hogarth, 2015, Levi and Finazzi, 2014, Schneider et al., 2012). To date, at least 10 different NBIA disorders have been genetically characterized (Gregory et al., 2009, Hogarth, 2015, Levi and Finazzi, 2014, Schneider et al., 2012). Following their genetic characterization, work in a series of animal models of NBIA has begun to provide insights into the processes underlying the development, progression and presentation of NBIA due to these genetic mutations (Beck et al., 2011, Malik et al., 2008, Shinzawa et al., 2008, Strokin et al., 2012, Strokin and Reiser, 2017, Strokin and Reiser, 2016, Wada et al., 2009, Zhao et al., 2011, Zhou et al., 2016). Despite this characterization many aspects of the pathophysiology of these disorders still remain to be determined.

Phospholipase A2G6-associated neurodegeneration (PLAN) is a form of NBIA that is caused by mutations in the PLA2G6 gene encoding the calcium independent phospholipase A2G6 (iPLA2β) (Morgan et al., 2006). PLA2G6 has proposed roles in membrane dynamics and homeostasis through phospholipid remodeling, as well as leukotriene and prostaglandin synthesis, apoptosis and inflammation (Morgan et al., 2006, Schneider et al., 2009, Sun et al., 2010). Pathogenic mutations found in PLAN generate enzymatically inactive isoforms which inhibit the actions of the phospholipase and render it nonfunctional (Morgan et al., 2006).

The presentation and symptomology of PLAN can be broadly separated into 3 distinct disorders, infantile neuroaxonal dystrophy (INAD), atypical neuroaxonal dystrophy (aNAD) and a later onset progressive dystonia-parkinsonism syndrome which has also been classified as a form of Parkinson’s disease (PD), PARK14 (Gregory et al., 2009, Hogarth, 2015, Paisan-Ruiz et al., 2009, Yoshino et al., 2010, Zhou et al., 2016). INAD presents early in life, between 6 months and 3 years of age, and has a rapid and devastating progression (Gregory et al., 2009, Hogarth, 2015). aNAD represents a small subset of INAD cases with a later emergence and delayed progression. PARK14 is a progressive dystonia-parkinsonism syndrome which manifests in late adolescence or early adulthood (Paisan-Ruiz et al., 2009, Yoshino et al., 2010), but can also be causative factor in adult-onset PD (Zhou et al., 2016). The shared classification of this presentation of PLAN as both NBIA and PD provides evidence for the strong parallels between NBIA and PD. Thus, research efforts in PLAN potentially provide novel insights applicable not only to the small group of patients with NBIA, but also to the larger group of individuals with PD.

Iron accumulation inside the BG is often, but not universally seen in INAD and aNAD. This accumulation is predominantly in the globus pallidus (GP), but it is also frequently seen in the substantia nigra (SN) on MRI (Gregory et al., 2009, Hogarth, 2015). Iron accumulation is seen with variable severity in the GP and SN of many of the patients with PLAN/PARK14 (Agarwal et al., 2012, Paisan-Ruiz et al., 2009, Yoshino et al., 2010). Furthermore, dystrophic neuroaxonal spheroids characteristic of PLAN are found in the brainstem, peripheral nerves, BG and spinal cord of patients (Gregory et al., 2009, Hogarth, 2015).

A study revealed that the nature and location of mutations in the PLA2G6 gene may be responsible for the large degree of heterogeneity between the various forms of PLAN (Engel et al., 2010). The severity of impairment of iPLA2β function may be directly related to the phenotypic presentation of PLAN, with a more severe mutation resulting in the more rapid onset and quicker progression of INAD, which also sees a more widespread pathology and a later onset dystonia-parkinsonism phenotype seen in mutations with less impact on iPLA2β catalytic activity. This relationship could explain much of the heterogeneity seen in the presentation of PLA2G6 mutations in various mouse models of PLAN (Beck et al., 2016, Beck et al., 2015, Beck et al., 2011, Strokin and Reiser, 2016, Malik et al., 2008, Shinzawa et al., 2008, Strokin et al., 2012, Strokin and Reiser, 2017, Wada et al., 2009, Zhao et al., 2011, Zhou et al., 2016).

Recently a novel mouse model of Phospholipase A2G6-Associated Neurodegeneration (PLAN) has been highlighted by the work of Strokin et al. (2012), which notably features prominent early-onset neurodegeneration as young as 50 days of age which is visible by an accompanying grip strength motor deficit. The presence of neurodegenerative symptoms which closely match those seen in NBIA patients makes this model a promising candidate for research into NBIA. The work done by Strokin et al., 2012, Strokin and Reiser, 2017, Strokin and Reiser, 2016 in this model provide novel insights into the impact of iPLA2β mutations on cellular regulation and handling of Ca²⁺. However, these investigations did not provide insight into two key features of PLAN and other NBIA disorders: the degeneration and iron accumulation inside the BG. Furthermore, there was no examination of tyrosine hydroxylase (TH) positive dopaminergic neuronal loss in this mouse model unlike other mouse models of PLAN (Beck et al., 2015, Zhou et al., 2016). Characterization of these two hallmarks of NBIA and TH expression will support future research efforts using this model and provide novel insights into the disease processes in PLAN. Also further characterization of this mouse model will help us determine where it fits in the spectrum of PLAN in so far as the severity of disorder and its phenotypic categorization as either INAD, aNAD or dystonia-parkinsonism syndrome. It is clear from the work in other models, that neurodegenerative symptoms are present in the later stages of the disease. However, questions remain as to the pathological changes which occur early in the disease course. Exploring the pathophysiology of early-disease changes is crucial to understanding how disease progression occurs in PLAN. Ultimately, better understanding of these pathological changes is needed to understand how they can lead to the phenotypic presentation of PLAN as either INAD, aNAD or dystonia-parkinsonism syndrome.

In this study a mouse line, which was a cross of the Strokin and Reiser iPLA2β mutant mouse line (Strokin and Reiser, 2016) and a Thy1-YFP mouse line, was used to facilitate assessment of subcellular neuronal morphology and pathophysiology of the iPLA2β mutation up to 90 days of age. The Thy1-YFP mouse line has been a valuable tool to study neuronal morphology and neuropathology in other nervous system disorders (Bannerman et al., 2005, Bannerman and Hahn, 2007). We selected to study up to 90 days since this appeared to be the early time course of the disease in the Strokin and Reiser iPLA2β mutant mouse line as weight loss began shortly after this age. This investigation was conducted based on the hypothesis that during the early-stages of the disorder significant degeneration of the BG occurs in B6.C3-Pla2g6^m1J/CxRwb mice including a loss of dopaminergic function within the SN, which may contribute to the observed motor symptoms previously reported by Strokin et al. (2012). Additionally, we hypothesized that, like PLAN patients, these mice would also show abnormal iron metabolism resulting in iron accumulation within the BG. To address these hypotheses this investigation assessed motor performance and weight change in the B6.C3-Pla2g6^m1J/CxRwb mice, measured the potential capacity for dopaminergic function based on TH protein expression in these mice, assessed the presence of neurodegeneration within the substantia nigra pars compacta (SNc) and substantia nigra pars reticulata (SNr) and evaluated the SN and other midbrain areas for the presence of overt iron accumulation in order to assess whether this mouse model can recapitulate some of the phenotypes found in PLAN human patients.