Pathological tau drives ectopic nuclear speckle scaffold protein SRRM2 accumulation in neuron cytoplasm in Alzheimer's disease

Abstract

Several conserved nuclear RNA binding proteins (sut-1, sut-2, and parn-2) control tau aggregation and toxicity in C. elegans, mice, and human cells. MSUT2 protein normally resides in nuclear speckles, membraneless organelles composed of phase-separated RNAs and RNA-binding proteins that mediate critical steps in mRNA processing including mRNA splicing. We used human pathological tissue and transgenic mice to identify Alzheimer's disease-specific cellular changes related to nuclear speckles. We observed that nuclear speckle constituent scaffold protein SRRM2 is mislocalized and accumulates in cytoplasmic lesions in AD brain tissue. Furthermore, progression of tauopathy in transgenic mice is accompanied by increasing mislocalization of SRRM2 from the neuronal nucleus to the soma. In AD brain tissue, SRRM2 mislocalization associates with increased severity of pathological tau deposition. These findings suggest potential mechanisms by which pathological tau impacts nuclear speckle function in diverse organisms ranging from C. elegans to mice to humans. Future translational studies aimed at restoring nuclear speckle homeostasis may provide novel candidate therapeutic targets for pharmacological intervention.

Keywords: Alzheimer’s disease; MSUT2; MapT tauopathy PS19; Nuclear speckles; PABPN1; SC-35; SRRM2; Tau; ZC3H14.

Fig. 1

Derangement of nuclear speckle marker pSRRM2 (ab SC-35) in AD. Immunohistochemistry using a pSRRM2 recognizing antibody raised against purified human spliceosomes (clone SC-35, see Table 2). Human pSRRM2 stains nuclear speckles within neurons in the frontal cortex of the normal adult human brain (a). A subset of AD donors (30%) also exhibit normal nuclear speckle staining in cortical neurons (b), however, the majority of AD donors (72%) exhibit striking mis-localization of pSRRM2 to the cytoplasm in neurons in the frontal cortex (c). Additionally, all AD donors exhibited mis-localization of pSRRM2 in the hippocampus (d) and amygdala (e), brain regions where tauopathy occurs earlier in disease progression. AD donors with aberrant cytoplasmic pSRRM2 in tauopathy rich regions showed normal nuclear pSRRM2 distribution in cerebellar neurons (f), which do not typically exhibit any tau pathology. Scale bars, 50 µm

Fig. 2

Abnormal pSRRM2 mis-localization occurs in a transgenic mouse model of tauopathy. Immunostaining with SC-35 antibody (a, c, e, g) demonstrates that pSRRM2 is mis-localized to the neuronal cytoplasm in brain regions of PS19 mice where Gallyas positive NFTs (b, d, f) are prevalent. At three months, pSRRM2 appears as diffuse mild immunoreactivity in the cytoplasm (a) and there are no tangles (b). At seven months, a small number of Gallyas positive NFTs are present and neurons with robust cytoplasmic distribution are detectable in these same regions (c, d). By nine months, PS19 mice with robust tangle burden display abnormal pSRRM2 cytoplasmic localization in a large number of neurons (e, f and quantified in h). In contrast, pSRRM2 is not detectable in a nine-month-old non-Tg mouse brain (g). (*p = 0.04 by two-tailed Student’s t test) Scale bars, 100 µm

Fig. 3

pSRRM2 co-localizes with pathological tau in AD. a pSRRM2 (Red) is localized to the nucleus and pTau (pS422-Green) is cytoplasmic in cognitively normal elderly controls; visible overlapping signal is visualized as yellow (Pearson Coefficient of Colocalization = 0.017), (Panel image scale bar = 100um; inset scale bar = 25um). b pSRRM2 (Red) relocalizes to the cytoplasm and colocalizes with pTau (Green), as visualized in yellow in AD patients (Pearson Coefficient of Colocalization = 0.99). (Panel image scale bar = 100um; inset scale bar = 10um). c Proximity Ligation Assay imaging between pSRRM2 and pTau. Top: Panel image and inset imaging of PLA colocalization in a cognitively normal elderly control donor shows minimal cytoplasmic colocalization (Panel image scale bar = 100um; inset scale bar = 10um). Bottom: Panel image and inset imaging of PLA colocalization in an AD donor show significant colocalization in the cytoplasm (Panel image scale bar = 100um; inset scale bar = 10um)

Fig. 4

pSRRM2 mis-localization in AD associates with disease severity. Cytoplasmic pSRRM2 accumulation in AD brain is associated with a more aggressive disease progression. a AD donors with predominately cytoplasmic pSRRM2 in postmortem frontal cortex (n = 20) had an earlier age of disease onset compared to those with primarily nuclear pSRRM2 distribution (n = 9) (**p = 0.0042 by two-tailed Student’s t test). b Representative brain sections from frontal cortex of an AD donor with normal nuclear pSRRM2 distribution (top) compared to an AD donor with abnormal cytoplasmic accumulation of pSRRM2 (bottom) stained with the anti-phosphorylated tau antibody AT180. Brain tissue from cases with cytoplasmic pSRRM2 exhibited more pathological tau. Densitometry analysis of AT180-positive reactivity in AD cases with nuclear (n = 8) or cytoplasmic (n = 19) pSRRM2 (***p = 0.0002 by two-tailed Student’s t test). c Representative brain sections from postmortem brain frontal cortex of an AD donor with normal nuclear pSRRM2 distribution (top) compared to an AD donor with abnormal cytoplasmic accumulation of pSRRM2 (bottom) stained with the neuronal marker NeuN. Brain tissue from cases with cytoplasmic pSRRM2 exhibited decreased NeuN immunoreactivity (indicative of more neuronal loss). Densitometry analysis of NeuN-positive reactivity in AD cases with nuclear (n = 9) or cytoplasmic (n = 18) pSRRM2 (**p = 0.0025 by two-tailed Student’s t test). Scale bars, 250 µm

Fig. 5

Nuclear Speckle disruption occurs as a result of pathological tau in AD. a Changes associated with aging, including decreased proteostasis, increased neuroinflammation, and metabolic challenge, contribute to the initiation of amyloid plaque deposition. Plaques have been shown to trigger a tauopathy cascade. Here we demonstrate that pathological tau deposition coincides with depletion of nuclear speckle scaffold protein SRRM2 from the nucleus. Nuclear SRRM2 function within nuclear speckles is required for appropriate mRNA processing. Inefficient mRNA processing causes neurodegeneration. b Phosphorylation of SRRM2 at pS1068 drives SRRM2 transit from the nucleus to cytoplasm and we propose that pathological tau provokes phosphorylation and mislocalization of SRRM2 through an ER stress and ERK1/2 mediated pathway which leads to neurodegeneration. c Modulation of nuclear speckle content via loss of regulatory RNA binding proteins resident in nuclear speckles such as SUT-1 or SUT-2/MSUT2 can fully rescue tauopathy phenotypes. We propose that the sut RNA binding protein pathway modulates tauopathy by restoring nuclear speckle function in the face of pathological tau