Atxn2-CAG100-KnockIn mouse spinal cord shows progressive TDP43 pathology associated with cholesterol biosynthesis suppression

Abstract

Large polyglutamine expansions in Ataxin-2 (ATXN2) cause multi-system nervous atrophy in Spinocerebellar Ataxia type 2 (SCA2). Intermediate size expansions carry a risk for selective motor neuron degeneration, known as Amyotrophic Lateral Sclerosis (ALS). Conversely, the depletion of ATXN2 prevents disease progression in ALS. Although ATXN2 interacts directly with RNA, and in ALS pathogenesis there is a crucial role of RNA toxicity, the affected functional pathways remain ill defined. Here, we examined an authentic SCA2 mouse model with Atxn2-CAG100-KnockIn for a first definition of molecular mechanisms in spinal cord pathology.Neurophysiology of lower limbs detected sensory neuropathy rather than motor denervation. Triple immunofluorescence demonstrated cytosolic ATXN2 aggregates sequestrating TDP43 and TIA1 from the nucleus. In immunoblots, this was accompanied by elevated CASP3, RIPK1 and PQBP1 abundance. RT-qPCR showed increase of Grn, Tlr7 and Rnaset2 mRNA versus Eif5a2, Dcp2, Uhmk1 and Kif5a decrease. These SCA2 findings overlap well with known ALS features. Similar to other ataxias and dystonias, decreased mRNA levels for Unc80, Tacr1, Gnal, Ano3, Kcna2, Elovl5 and Cdr1 contrasted with Gpnmb increase. Preterminal stage tissue showed strongly activated microglia containing ATXN2 aggregates, with parallel astrogliosis. Global transcriptome profiles from stages of incipient motor deficit versus preterminal age identified molecules with progressive downregulation, where a cluster of cholesterol biosynthesis enzymes including Dhcr24, Msmo1, Idi1and Hmgcs1 was prominent. Gas chromatography demonstrated a massive loss of crucial cholesterol precursor metabolites. Overall, the ATXN2 protein aggregation process affects diverse subcellular compartments, in particular stress granules, endoplasmic reticulum and receptor tyrosine kinase signaling. These findings identify new targets and potential biomarkers for neuroprotective therapies. Introduction:Within the dynamic research field into neurodegenerative disorders, the rare monogenic variant SCA2 (Spinocerebellar Ataxia type 2) gained much attention over the past decade, since its pathogenesis is intertwined with the motor neuron diseases ALS/FTLD (Amyotrophic Lateral Sclerosis / Fronto-Temporal Lobar Dementia) and with extrapyramidal syndromes such as Parkinson/PSP (Progressive Supranuclear Palsy). Particularly in the past year, it received massive interest since antisense-oligonucleotides were shown to effectively prevent the disease in mouse models, with clinical trials now being imminent. Still, there is an urgent unmet need to define the molecular events of pathogenesis and to identify biomarkers of progression in SCA2 patients, which reflect therapeutic benefits much more rapidly than clinical rating scales, brain imaging volumetry or neurophysiology measures.SCA2 was first described as separate entity in Indian patients, based on the characteristic early slowing of eye saccadic movements [222]. A molecularly homogeneous founder population of ~1,000 patients in...

In Human and Mouse Spino-Cerebellar Tissue, Ataxin-2 Expansion Affects Ceramide-Sphingomyelin Metabolism

“…Sphingomyelin of C24 length interacts with cholesterol in lipid bilayers as important stabilizing elements for the plasma membrane, particularly in myelinating glia cells [134,135]. Although our study is limited to the analysis of one patient cerebellum and six mutant versus six WT mice, the results gain credibility in light of our previous report on sphingolipid anomalies also in the Atxn2 -KO brain [50] and in view of our findings submitted in parallel on the suppression of cholesterol biosynthesis in the nervous tissue of our new Atxn2 -CAG100-KIN mouse [136]. Contrary to the scenario in SCA2, C24 sphingomyelin accumulates with cholesterol in adrenoleukodystrophy [137] throughout the white matter, also leading to a demyelinating process.…”

Mouse Ataxin-2 Expansion Downregulates CamKII and Other Calcium Signaling Factors, Impairing Granule—Purkinje Neuron Synaptic Strength

“…This observation is consistent with the normally nuclear localization of Sam68, its potential redistribution into RNP granules and potential interaction with ATXN2. We therefore questioned whether Sam68 could be trapped in cytosolic ATXN2 aggregates like many other nuclear RNA processing proteins such as TDP-43, FUS, TIA-1 [ 52 , 69 ], which would explain its increased abundance at the terminal stage. Immunohistochemical staining of Sam68 and ATXN2 confirmed the nuclear localization of Sam68 in Purkinje and granule neurons of the WT cerebellum at 14 mo of age, while ATXN2 showed a diffuse cytosolic localization ( Supplementary Figure S2 ).…”

“…Increased amounts and activation of ATXN2 under stress by phosphorylation cascades were shown to suppress mTORC1 activity, due to sequestration of its components into SGs for energetic sustainability [ 34 , 49 , 50 ]. Global transcriptome and metabolome profiling of spinal cord tissue from two SCA2 mouse models highlighted a pronounced effect of ATXN2 and its sequence homolog ATXN2L on cholesterol and membrane lipid homeostasis [ 46 , 51 , 52 , 53 ]. Indeed, the loss of ATXN2 function in mouse leads to a metabolic excess syndrome manifested as diabetes mellitus with insulin resistance, lipid droplet accumulation in the liver, and hypercholesterolemia [ 48 ], whereas the loss of ATXN2L leads to mid-gestation embryonic lethality [ 53 ].…”

The RNA‐binding protein and stress granule component ATAXIN‐2 is expressed in mouse and human tissues associated with glaucoma pathogenesis

“…RGC autophagy can be affected by acute and chronic IOP elevations (Deng et al, 2013;Park et al, 2012;Rodríguez-Muela et al, 2012;Su et al, 2014) with αRGCs responding with autophagic arrest, loss of synapses, and apoptosis (Della Santina et al, 2013;Guttenplan et al, 2020). While it remains to be seen whether manipulation of ocular ATXN2 reduces autophagic and apoptotic stress in hypertensive RGCs, it is worth noting that ATXN2 depletion is neuroprotective in SCA2 and ALS mouse models as well as ataxic flies (Auburger et al, 2017;Bakthavachalu et al, 2018;Becker et al, 2017;Lessing & Bonini, 2008;Scoles et al, 2017;Watanabe et al, 2020), possibly due to the loss of TDP-43 recruitment (Becker et al, 2017;Canet-Pons et al, 2021;Elden et al, 2010). It may also be of interest to determine potential ATXN2 colocalization with mTORC1 (mTOR, raptor, and S6 ribosomal proteins), which function as auxiliary nutrient sensors and autophagy suppressors in the inner retina, given their physical interaction described in flies and modulatory activity described in mouse fibroblasts and human neuroblastoma cells (Lastres-Becker et al, 2016;Takahara & Maeda, 2012).…”

“…Scale bars: 25 μm (a,d,g,j,m,p) and 10 μm (b,c,e,f,h,i,k,l,n,o,q,r) [Color figure can be viewed at wileyonlinelibrary.com] expression patterns in Purkinje cells and RGCs thus points at potential significance of non-ER mRNA signaling and stress granule assembly in projection neurons. Loss of TDP-43, a binding nuclear partner that is recruited by ATXN2 into stress granules(Becker et al, 2017;Canet-Pons et al, 2021;Elden et al, 2010;Kim et al, 2014), has been linked to retinal neurodegeneration, RGC axonal loss, and frontotemporal dementia(Atkinson et al, 2021;Ward et al, 2014;Watanabe et al, 2020). Depletion of ATXN2 has been shown to reduce TDP-43-mediated cytotoxicity(Becker et al, 2017) in Purkinje neurons, the predominant ATXN2 + cell type in the cerebellum, which may suggest a similar treatment strategy for POAG may be effective.F I G U R E 7 (a-c) Primary human trabecular meshwork cells show ATXN2-ir (FITC) in the cytoplasm and the perinuclear region.…”