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Published in final edited form as: Brain Res. 2011 Mar 2;1386:191–199. doi: 10.1016/j.brainres.2011.02.052

β-amyloid triggers ALS-associated TDP-43 pathology in AD models

Alexander M Herman b, Preeti J Khandelwal a, Brenna B Stanczyk a, G William Rebeck a, Charbel E-H Moussa a,b
PMCID: PMC3073036  NIHMSID: NIHMS278701  PMID: 21376022

Abstract

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease associated with loss of motor neurons in the brain and spinal cord. ALS is occasionally diagnosed with frontotemporal lobar dementia with ubiquitin positive inclusions (FTLD-U). Alzheimer's disease (AD) is the most common type of age-associated dementia. Abnormal levels of aggregated Tar-DNA binding protein-43 (TDP-43) are detected in the majority of patients with ALS, FTLD and AD. We observed a significant increase (200%) in the levels of TDP-43 in cortical autopsies of late stage AD patients. Lentiviral expression of Aβ1-42 in the rat motor cortex led to an increase in TDP-43 pathology, including up-regulation of the mature ~44kDa protein, identical to the pathological changes seen in AD. Furthermore, expression of Aβ1-42 was associated with TDP-43 phosphorylation and accumulation in the cytosol. Clearance of Aβ with parkin prevented TDP-43 pathology. TDP-43 modifications were also observed in 3xTransgenic AD (3xTg-AD) compared to wild type mice, but these changes were attenuated in parkin-injected hippocampi, even in the presence of Tau pathology, suggesting that TDP-43 pathology is triggered by Aβ, independent of Tau. Increased levels of casein kinase (CK1 and CK2), which are associated with TDP-43 phosphorylation, were also observed in Aβ1-42 expressing brains. These data indicate an overlap in TDP-43 pathology between AD and ALS-FTLD and suggest that Aβ triggers modifications of TDP-43.

1-Introduction

The number of neurodegenerative diseases associated with pathological aggregates of transactivation response element (TAR)-DNA-binding protein 43 (TDP-43) has increased in the last decade. TDP-43 is a 414-amino acid protein with a theoretical molecular mass of 44.74 kDa. The expressed protein contains two highly conserved RNA recognition motifs (RRM1 and RRM2), as well as a glycine-rich C-terminal, which mediates protein-protein binding (Ardley et al., 2001; Morett and Bork, 1999). Full-length TDP-43 is localized predominantly to the nucleus, with small amounts of cytosolic presence under normal conditions (Buratti et al., 2005; Buratti and Baralle, 2008; Wang et al., 2004; Winton et al., 2008). TDP-43 pathology both in the brain and spinal cord is characterized by decreased solubility, ubiquitination, hyper-phosphorylation and cleavage of TDP-43 into 25 and 35kDa fragments, as well as cellular translocation from nuclear to cytosolic compartments (Amador-Ortiz et al., 2007; Geser et al., 2008; Hasegawa et al., 2007; Mackenzie et al., 2007; Neumann et al., 2006; Neumann et al., 2007a; Neumann et al., 2007b; Tan et al., 2007; Zhang et al., 2007). TDP-43 was identified in the inclusions of frontotemporal lobar degeneration with ubiquitin-positive inclusions (FTLD-U) and Amyotrophic Lateral Sclerosis (ALS) (Neumann et al., 2006). FTLD is one of the major causes of dementia in young adults (Ratnavalli et al., 2002; Snowden et al., 2002) and comprises a group of heterogeneous neurodegenerative disorders that are occasionally associated with motor neuron disease (MND) (Bennett et al., 1999; Neary et al., 1990; Neary et al., 2000; Tofaris et al., 2001).

TDP-43 is a major constituent of inclusions in motor and non-motor neurons in ALS and FTLDMND (Arai et al., 2006; Neumann et al., 2007a; Tan et al., 2007). ALS is a neurodegenerative disorder that affects both upper and lower motor neurons, leading to progressive paralysis and death (Pasinelli and Brown, 2006). Only ~20% of ALS cases are familial associated with missense mutation in Cu/Zn superoxide dismutase gene (SOD1) (Gros-Louis et al., 2006; Rosen, 1993). Most ALS cases are sporadic with 50% of patients display coincident deterioration of both motor and cognitive function (Morita et al., 2006; Talbot and Ansorge, 2006) and 20% develop clinical features suggestive of FTLD (Lomen-Hoerth et al., 2002; Lomen-Hoerth et al., 2003). Pathologically, ALS patients have TDP-43 accumulation in motor neurons (Ayala et al., 2005; Neumann et al., 2006) and Tau-negative ubiquitin inclusions identical to those of FTLD-U patients (Forman et al., 2006). Although no TDP-43 mutations have been associated with FTLD-U, several mutations (Q331K, M337V, G294A, A90V) have been identified in MND/ALS (Gitcho et al., 2008; Sreedharan et al., 2008). TDP-43 is also altered in AD, the most common cause of dementia in the elderly. A large number (75%) of AD cases show TDP-43 pathology (Amador-Ortiz et al., 2007). Lewy body disorders also demonstrate TDP-43 pathology in AD with LBD, PD and PD with dementia (Nakashima-Yasuda et al., 2007). Co-localization between TDP-43 and NFTs and TDP-43 and α-Synuclein in dystrophic neurites were also identified, despite studies showing lack of co-existence between TDP-43 and Tau pathologies (Arai et al., 2006; Nakashima-Yasuda et al., 2007; Neumann et al., 2007b).

The pathology of AD is characterized by intraneuronal deposition of hyper-phosphorylated Tau as well as extracellular β-amyloid (Aβ) plaques (Hardy and Selkoe, 2002). Aβ is produced intracellularly via the endosomal system and secretory pathways that mediate the processing of amyloid precursor protein (Haass et al., 1992; Koo and Squazzo, 1994). Aβ1-40 and Aβ1-42 are produced intracellularly (Cook et al., 1997; Greenfield et al., 1999; Lee et al., 1998; Skovronsky et al., 1998; Xu et al., 1997), and accumulate in the brain of individuals with AD [Wilson et al., 1999; Gouras et al., 2000]. Both intracellular and extracellular oligomeric Aβ have been implicated in AD pathology, but intracellular oligomeric species may be formed first to act in the earlier stages of disease (Gouras et al., 2000; Wilson et al., 1999). Immunocytochemical studies on AD, Down's syndrome (DS) and APP transgenic mouse brains reveal abundant intraneuronal Aβ (Hanihara et al., 1995; Hof et al., 1992; Hyun et al., 2002; Ito et al., 2008; Lee et al., 2001; Morales et al., 2002; Murray et al., 2005; Ros et al., 2008; Sanchez et al., 2002; van de Warrenburg et al., 2001).

Since neurodegenerative diseases often involve the accumulation of several misfolded proteins, we sought to ask whether specific pathogenic proteins cause the accumulation of TDP-43. We generated animal models using lentiviral gene delivery of Aβ1-42 (Burns et al., 2009) and α-Synuclein (Khandelwal et al., 2010) to mimic the pathology of AD and PD, respectively. We also used 3xTg-AD mice, which display Aβ and hyper-phosphorylation of Tau. We compared TDP-43 pathology in animal models to autopsies of human patients diagnosed with late stage AD. Finally, we analyzed the effects of the E3-ubiquitin ligase parkin to reduce Aβ and α-Synuclein levels in gene transfer models and 3xTg-AD mice. These studies allow us to better understand the overlapping biochemical pathways underlying neurodegeneration at the interface between different neurological diseases, including FTLD, ALS, PD and AD.

2-Results

TPD-43 pathology is increased in AD brains

To examine TDP-43 pathology in the human brain autopsies, we analyzed motor cortex from late stage AD (N=12) patients and age-matched (N=7) control subjects (John Hopkins University Medical Institution) using Western blots. Analysis of lysates from AD patients (Fig. 1A, top blot) showed an increase in full-length APP and C-terminal fragments (CTFs) compared to age-matched controls. Probing these blots with anti-TDP-43 (2E2-D3) antibody (Fig. 1A, upper middle blot) revealed an increase in the level of full length ~44 kDa TDP-43. A smaller TDP-43 band (~15kDa) was also detected, perhaps due to TDP-43 cleavage. A protein smear was also observed between 62-98Kd, suggesting aggregation of TDP-43 in AD patients compared to control subjects. An increase in phosphorylated TDP-43 (pTDP-43) was also observed in AD (Fig. 1A lower middle blot) compared to control brains, which included both full length and cleaved TDP-43. Densitometric analysis (Fig.1B) of blots revealed a significant (~200%) increase in the level of the ~44kDa band of TDP-43 (P<0.05, Neuman Keuls with multiple comparison) in AD patients compared to control subejcts, suggesting that TDP-43 pathology co-exists with Aβ accumulation in AD.

Fig. 1. TPD-43 pathology in induced in brain autopsies from AD patients.

Fig. 1

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A). WB of lysates from human motor cortex were probed with anti-APP (C1.61) antibody (upper blot). Anti-TDP-43 (Abnova) antibody (upper middle blot) showed an increase in TDP-43 level, the appearance of a small ~15kDa band and a protein smear on the upper gel in AD brains not control. Anti phospho-TDP-43 (pTDP-43) antibody (lower middle blot) shows an increase in pTDP-43 levels and cleavage and aggregation in AD not control. Bottom blot is an actin loading control, molecular weight markers are indicated. B). Densitometric analysis of blots, Asterisk is significantly different to control, P<0.05, Neuman Keuls with multiple comparison, N=7 control; 12 AD patients.

Intracellular Aβ1-42 induces TPD-43 up-regulation, cleavage, and aggregation in gene transfer animal models

To test whether TDP-43 pathology is associated with either Aβ or α-Synuclein expression, we used lentiviral gene transfer animal models to express intracellular Aβ1-42 (N=8) or α-Synuclein (N=8) and examined for changes in endogenous TDP-43 processing. All lentiviral clones, including parkin, Aβ1-42 and α-Synuclein, were injected with lentiviral LacZ to control for viral m.o.i. (2×1010) used. Lentiviral Aβ1-42 was injected into the motor cortex in the presence or absence of lentiviral parkin and lentiviral α-Synuclein was injected into the striatum and animals were sacrificed 4 weeks post-injection. The brains were dissected out and analyzed by Western blots. As expected, animals injected with lentiviral Aβ1-42 had a significant (200%, N=8, P<0.05) increase in the ~44kDa band of TDP-43 (Fig. 2A) in the rat primary motor cortex and the appearance of bands at ~28kDa and 62-98kDa compared to lentiviral LacZ, lentiviral parkin or lentiviral α-Synuclein expressing rats. We previously showed that co-expression of lentiviral Aβ1-42 and lentiviral parkin leads to clearance of intracellular Aβ1-42 (Burns et al., 2009). Injection with lentiviral parkin led to a significant increase (50%) in parkin levels in injected hemispheres compared to endogenous levels. Lentiviral parkin expression mediated the clearance of Aβ1-42 (lower blot) and prevented the changes in TDP-43 in the rat brain (Fig. 2A). Expression of lentiviral Aβ1-42 also resulted in phosphorylation (asterisks) of TDP-43 (Fig. 2B) and the appearance of a cleavage band ~28Kda and another unknown smaller ~15kDa band compared to lentiviral LacZ or lentiviral α-Synuclein injected rats. To examine the levels of kinases that are associated with TDP-43 phosphorylation, we examined the expression levels of casein kinase 1 (CK1) and 2 (CK2). The levels of CK1 and CK2 (Fig. 2B) were increased in Aβ1-42 compared to lentiviral LacZ, parkin and α-Synuclein injected brains, and parkin clearance of Aβ1-42 prevented the increase in CK1 and CK2 levels, suggesting that changes in kinase activity may alter TDP-43 post-translational modification. These data suggest that intracellular Aβ1-42 increases TDP-43 levels, processing and aggregation.

Fig. 2. Intracellular Aβ1-42 induces TPD-43 protein up-regulation, cleavage, and aggregation in gene transfer animal models.

Fig. 2

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A). WB analysis of brain lysates on 4-12% NuPAGE gel probed with anti-parkin antibody (PRK8) and anti-TDP-43 antibody (Abnova) showing an increase in the level of TDP-43 expression, cleavage and aggregation in lentiviral-Aβ1-42 compared to lentiviral LacZ, parkin or α-Synuclein injected brains. Middle blot shows Aβ1-42 and bottom blot shows α-Synuclein expression. B). WB analysis with anti p-TDP-43 (Millipore) antibody (upper blot) shows an increase in the level of pTDP-43 expression (asterisks), and cleavage of TDP-43 in lentiviral-Aβ1-42 compared to lentiviral LacZ or α-Synuclein injected brains. Middle blots show casein kinase 1 and 2 (CK1 and CK2) levels, and bottom blot shows actin loading control. C). Blots show parkin expression and TDP-43 immunoreactivity in brain lysates of 3xTg-AD mice. D). Blots show TDP-43 immunoreactivity in brain lysates of 3xTg-AD mice compared to non-transgenic wild type controls. E). Shows blots of GSK-3β and different Tau epitopes in 3xTg-AD mice. Lentiviral-α-Synuclein, Lv-Syn, Lentiviral LacZ: Lv-LacZ, Lentiviral Aβ1-42: Lv-Aβ1-42, N=4 animals per treatment.

To test the effects of Aβ on TDP-43 pathology over longer periods of time, we tested TDP-43 modification in 3xTg-AD mice (that express the APPsw, PS1 and TauP301L) 3 months post-injection with lentiviral parkin into the right hippocampus (ipsilateral) and lentiviral LacZ into the left (contralateral) hippocampus. Lentiviral parkin led to a significant (50%) increase in parkin expression levels in the ispsilateral compared to the contralateral hemispheres of 3xTg-AD mice. Accumulation and cleavage of TDP-43 (Fig. 2C) were decreased in parkin-injected hemispheres compared to contralateral LacZ-injected hemispheres in 3xTg-AD mice. To further test the effects of Aβ expression in 3xTg-AD mice on TDP-43 pathology, we compared these transgenic mice to wild type controls (Fig. 2D). Western blot analysis showed 50% (P<0.05) increase in mature (44kDa) TDP-43 and the appearance of 35kDa bands, suggesting cleavage of TDP-43 in 3xTg-AD mice compared to wild type control. Lentiviral parkin expression did not cause any changes to GSK-3β activity (Fig. 2E) nor to the hyper-phosphorylation levels of Tau, suggesting that β-amyloid triggers TDP-43 modification independent of Tau.

Intracellular Aβ1-42 increases cytosolic accumulation of TDP-43

We performed an independent method to investigate changes in TDP-43 processing using immunohistochemical (IHC) staining of the motor cortex. Immunoreactivity of TDP-43 was predominantly visible within the DAPI stained nuclei (Fig. 3A) in the motor cortex of control (LacZ injected) rats. Injection of lentiviral LacZ+Lentiviral Aβ1-42 resulted in cytosolic accumulation of TDP-43 (Fig. 3B), and higher magnification images (Fig. 3C&D) revealed TDP-43 accumulation in the cytosol. These data suggest that Aβ1-42 can affect the modification of TDP-43 and accumulation in the cytosol. To further examine the effects of Aβ1-42 on TDP-43, we co-stained sections of motor cortex from lentiviral LacZ+lentiviral-Aβ1-42 injected rat brains. As expected we observed Aβ1-42 expression (Fig. 3E) and co-localization with TDP-43 (Fig. 3F&G), suggesting that cells that express Aβ1-42 also up-regulate the endogenous levels and cytosolic accumulation of TDP-43.

Fig. 3. Intracellular Aβ1-42 increases cytosolic accumulation of TDP-43.

Fig. 3

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A). Staining of 20 micron thick cortical rat brain sections injected with 2×1010 m.o.i LacZ lentivirus and B). 1×1010 each of lentiviral LacZ and lentiviral Aβ1-42. C) High magnification (60×) of 20 micron-thick sections of rat motor cortex stained with DAPI and TPD-43 antibody (ProteinTech) in lentiviral LacZ and D). Aβ1-42 injected rat brains. E). Co-staining against human Aβ1-42 (Zymed) and F). TDP-43 (Abnova) shows G). co-localization of these proteins in Aβ1-42 expressing cells and up-regulation and cytosolic accumulation of TDP-43. Lentiviral lacZ: Lv-LacZ, Lentiviral Aβ1-42: Lv-Aβ1-42 . N=4 animals per treatment.

3-Discussion

This work shows the effects of amyloid proteins involved in the pathogenesis of a number of neurodegenerative diseases on the modification of TDP-43. An increase in TDP-43 protein level was observed in autopsies of AD patients and animal model expressing intraneuronal Aβ. Expression of intraneuronal Aβ1-42 was also associated with cytosolic TDP-43 accumulation, in agreement with previously reported data that show TDP-43 accumulation in the cytosol under abnormal conditions (Buratti et al., 2005; Buratti and Baralle, 2008; Caccamo et al., 2009; Wang et al., 2004; Wegorzewska et al., 2009; Winton et al., 2008; Zhang et al., 2009). TDP-43 phosphorylation, which we detected in AD brains and Aβ expressing animals, is also a prominent change in ALS and FTLD-U (Hasegawa et al., 2008). These data suggest that intracellular Aβ triggers TDP-43 pathology, which was identified in the inclusions of FTLD-U and ALS (Amador-Ortiz et al., 2007; Geser et al., 2008; Hasegawa et al., 2007; Mackenzie et al., 2007; Neumann et al., 2006; Neumann et al., 2007a; Neumann et al., 2007b; Tan et al., 2007; Zhang et al., 2007)). FTLD-U is one of the major causes of dementia in young adults (Ratnavalli et al., 2002; Snowden et al., 2002) and is associated with ALS (Bennett et al., 1999; Neary et al., 1990; Neary et al., 2000; Tofaris et al., 2001). This type of dementia may be symptomatically related to AD-dementia and the coincident cognitive decline reported in ALS patients (Morita et al., 2006; Talbot and Ansorge, 2006). Most ALS cases are sporadic with 50% of patients display coincident deterioration of both motor and cognitive function (Morita et al., 2006; Talbot and Ansorge, 2006) and 20% develop clinical symptoms similar to FTLD (Lomen-Hoerth et al., 2002; Lomen-Hoerth et al., 2003). Furthermore, our data are consistent with recent findings, which showed that an increase in TDP-43 pathology is linked to accumulation of Aβ oligomers in 3xTg-AD mice and Aβ clearance with immunization reverses TDP-43 pathology without affecting Tau levels (Caccamo et al., 2010). Although animal models that express Aβ are different than AD brains, the effects observed in transgenic and gene transfer animals expressing Aβ are comparable to results obtained from AD brains, suggesting that Aβ may be a driving force of TDP-43 pathology.

The TDP-43 pathology associated with FTLD-ALS is a non-Tauopathy in which neuronal and glial inclusions are negative for Tau and α-Synuclein (Forman et al., 2006; Neumann et al., 2007a). Our animal models expressing intraneuronal Aβ1-42 or α-Synuclein show an increase in Tau phosphorylartion, but lentiviral parkin expression reverses Tau modification in these animals (Khandelwal et al., 2010; Rebeck et al., 2010). However, Tau changes did not correlate with changes to TDP-43 in these animal models. Furthermore, lentiviral parkin did not change the levels of P301L Tau in the 3xTg-AD mice but reduced Aβ accumulation and TDP-43 levels, further suggesting that TDP-43 pathology is Tau-independent, consistent with other studies (Caccamo et al., 2010; Neumann et al., 2007a; Neumann et al., 2007b). Expression of lentiviral Aβ1-42 increased the levels of casein kinases associated with TDP-43 phosphorylation (Hasegawa et al., 2008), suggesting that intraneuronal β-amlyoid accumulation activates kinases known to increase protein phosphorylation (Khandelwal et al., 2010; Rebeck et al., 2010). TDP-43 pathology has not been identified in primary Tauopathies (Davidson et al., 2007), but Tau pathology associated with AD is observed with TDP-43 pathology (Amador-Ortiz et al., 2007). Co-localization between TDP-43 and neurofibrillary tangles (NFTs) and TDP-43 in dystrophic neurites were identified, although other studies show lack of co-existence between TDP-43 and Tau pathologies (Arai et al., 2006; Nakashima-Yasuda et al., 2007; Neumann et al., 2007b). Taken together, these data are consistent with previous findings that show TDP-43 pathology in most (75%) AD cases (Amador-Ortiz et al., 2007). Lewy body disorders also display TDP-43 pathology in 30% AD with LBD, and 19% PD with dementia (Nakashima-Yasuda et al., 2007). However, the absence of α-Synuclein aggregation in these studies is notable and may be sufficient to explain the lack of α-Synuclein effects on TDP-43 pathology. β-amyloid induced TDP-43 pathology, particularly in brain regions involved in cognitive function, may be associated with dementia in neurodegenerative diseases.

These studies showed the effects of pathogenic accumulation of β-amyloid on FTLD/ALS-associated TDP-43 pathology, independent of Tau modification. TDP-43 pathology in animal models was similar to autopsies of human patients diagnosed with late stage AD, suggesting an overlap of biochemical pathways underlying neurodegeneration at the interface between different neurological diseases, including ALS, FTLD-U and AD. Our data suggest that Aβ triggers modifications of TDP-43 leading to its accumulation. More work is required to delineate the mechanisms of how Aβ1-42 increases TDP-43 pathology, particularly in cortical motor neurons in the brain and spinal cord, thus mimicking TDP-43-associated ALS pathology. We need to better understand the susceptibility of neurons in different brain regions to the accumulation of Aβ or TDP-43 and how the accumulation of one pathogenic protein affects the pathology of the other. Future studies that determine the mutual effects of TDP-43 and Aβ and the susceptibility of different brain regions to the pathogenic changes of these proteins may help understand the onset of dementia and cognitive deterioration in several neurodegenerative diseases.

4- Methods and Materials

Human brain autopsies

Autopsies of human motor cortex were collected 2-4 hours postmortem from late stage AD (N=12) patients and age-matched (N=7) control subjects at John Hopkins University Medical Institution. The average ages of these groups are 75 years old. The ages of AD patients were 65 to 83 years. Control subjects were 64 to 81 years old.

Stereotaxic injection

Lentiviral constructs were used to generate the animal models. Stereotaxic surgery was performed to inject the lentiviral constructs encoding LacZ, parkin, or α-Synuclein into the rat striatum (Khandelwal et al., 2010) and LacZ, Aβ1-42, parkin, parkin+Aβ1-42 into the primary motor cortex (Burns et al., 2009) of male Sprague-Dawley rats weighing between 170-220g. Lentiviral parkin was also injected into the hippocampus of 3xTg-AD mice. All animals were anesthetized (50mg/kg body weight) with a cocktail of Ketamine and Xylazine (50:8). The stereotaxic coordinates were according to Paxinos and Watson brain atlas. Lentiviral stocks were injected through a Microsyringe pump controller (Micro4) using total pump (World Precision Instruments, Inc) delivery of 6 μl at a rate of 0.2 μl/min. Animals were injected into one side of the brain with a lentiviral-LacZ vector at 2×1010 m.o.i; or with 1×1010 m.o.i lentiviral-Aβ1-42 + 1×1010 m.o.i lentiviral-LacZ; or 1×1010 m.o.i lentiviral-Aβ1-42 + 1×1010 m.o.i lentiviral-parkin; and 1×1010 m.o.i lentiviral-Synuclein + 1×1010 m.o.i lentiviral-LacZ, and 1×1010 m.o.i lentiviral-parkin + 1×1010 m.o.i lentiviral-LacZ. Lentiviral gene transfer rats were sacrificed 4 weeks post-injection and 3xTg-AD mice were sacrificed 3 months post-injection with lentiviral parkin.

Western blot analysis

Brain tissues were homogenized in 1× STEN buffer (50 mM Tris (pH 7.6), 150 mM NaCl, 2 mM EDTA, 0.2 % NP-40, 0.2 % BSA, 20 mM PMSF and protease cocktail inhibitor), centrifuged at 10,000 × g for 20 min at 4°C and the supernatant containing the soluble fraction of proteins were collected. The supernatant were analyzed on SDS NuPAGE 4-12% Bis-Tris gel (Invitrogen, Inc). Protein estimation was performed using BioRad protein assay (BioRad Laboratories Inc, Hercules, CA). Human anti-Aβ1-42 was immunoprobed with 1:1000 mouse monoclonal (Zymed) antibody and parkin (1:1000) with rabbit polyclonal (PRK8) antibody (Upstate). Total TDP-43 was probed either with (1:1000) rabbit polyclonal antibody generated against N-terminal 260 amino acids of the full length protein (ProteinTech) or (1:1000) mouse monoclonal (2E2-D3) TDP-43 (Abnova). Phosphorylated TDP-43 was probed (1:1000) with mouse monoclonal (1D3) phospho-TDP-43 (Millipore). Mouse monoclonal (1:1000) CK1 (Abnova) and rabbit polyclonal CK2 (1:1000) antibodies (Abnova) were used to probe for casein kinases. Anti-α-Synuclein (1:1000) antibody (Chemicon International) was used to probe against α-Synuclein. Anti-Tau (1:1000) antibodies (Chemicon International) were used to probe for different Tau epitopes. Western blots were quantified by densitometry using Quantity One 4.6.3 software (Bio Rad).

Immunocytochemical and histological analysis of brain sections

Immunohistochemistry was performed on 20 micron-thick brain sections. TDP-43 was probed (1:200) with Rabbit polyclonal (ProteinTech) or (1:200) mouse monoclonal (Abnova) antibodies. Staining against human Aβ1-42 was performed using (1:200) rabbit polyclonal antibody (Zymed). TDP-43 staining was coupled with nuclear DAPI staining to determine nuclear and cytosolic immunoreactivity of TDP-43.

Acknowledgments

This work was supported by NIH AG30378 award to Dr. Charbel E-H Moussa.

Abbreviations

PD

Parkinson's disease

AD

Alzheimer's disease

FTLD-U

Fronto-temporal lobar dementia with ubiquitin positive inclusions

ALS

Amyotrophic Lateral Sclerosis

MND

Motor Neuron Disease

TDP-43

Tar-DNA binding protein-43

Aβ

β-amyloid

CK1

Casein kinase 1

Footnotes

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