Declining phosphatases underlie aging-related hyperphosphorylation of neurofilaments

Abstract

Cytoskeletal protein phosphorylation is frequently altered in neuropathologic states but little is known about changes during normal aging. Here we report that declining protein phosphatase activity, rather than activation of kinases, underlies aging-related neurofilament hyperphosphorylation. Purified PP2A or PP2B dephosphorylated the heavy neurofilament (NFH) subunit or its extensively phorphorylated carboxyl-terminal domain in vitro. In cultured primary hippocampal neurons, inhibiting either phosphatase induced NFH phosphorylation without activating known neurofilament kinases. Neurofilament phosphorylation in the mouse CNS, as reflected by levels of the RT-97 phosphoepitope associated with late axon maturation, more than doubled during the 12-month period after NFH expression plateaued at p21. This was accompanied by declines in levels and activity of PP2A but not PP2B, and no rise in activities of neurofilament kinases (Erk1,2, cdk5 and JNK1,2). Inhibiting PP2A in mice in vivo restored brain RT-97 to levels seen in young mice. Declining PP2A activity, therefore, can account for rising neurofilament phosphorylation in maturing brain, potentially compounding similar changes associated with adult-onset neurodegenerative diseases.

Figure 1

A cartoon of NF-H shows its polypeptide domains (A) and sequences of the KSPXXXK and KSPXK type of recombinant proteins derived from the rat and human NF-H tail domains respectively (B).

Figure 2. Identification of NF protein phosphatases

(A) Isolated neurofilaments contain tightly associated PP2A and PP2B: Neurofilaments isolated from mouse spinal cord (10 µg protein) were subjected to SDS PAGE on a 4–20% gradient gel. One lane of the gel was stained with Coomassie Brilliant Blue. (lane 1) The remaining gel was subjected to immunoblot analysis with antibodies to PP2A (Lane 3) or PP2B (Lane 2). (B–D) Dephosphorylation of NF proteins by purified PP2A and PP2B in vitro. Recombinant NFH was phosphorylated by purified cdk5 (B) or Erk2 (C) and a recombinant KSPXXXK 24 repeat fusion protein derived from NFH tail sequence was phosphorylated by Erk2 (D). In each case, the autoradiograph (upper panel), and the Western blot analysis (lower panel) immunolabeled with RT-97 monoclonal antibody are presented. Shown are (E) western blot analyses of PP2B mediated dephosphorylation of native NFH in the NF pellet from spinal cord probed with antibodies to RT-97 (left panel) or SMI-33 (right panel)( representative data are presented)

Figure 3. Okadaic acid inhibition of PP2A and PP2B elevate RT-97 IR levels in hippocampal neurons

Hippocampal neurons in culture (7DIV) untreated (A) or treated (B) with 10 nM Okadaic acid overnight were fixed in 4% paraformaldehyde and immune stained with RT-97 monoclonal antibody. that specifically stains neurofilaments phosphorylated at KSP repeats. Western blot analysis of lysates of hippocampal neurons treated with and without 10 nM okadaic acid overnight and lysed in MPER (Thermo Scientific Rockford, IL) in the presence of protease and phosphatase inhibitors (panel C). A bar graph in panel D indicates the relative band intensities of pJNK1,2, pErk 1,2 and RT-97 on gels (n=3,*p<0.05)

Figure 4. Okadaic acid injection to mouse brain enhances NF phosphorylation

Vibratome sections of mouse brain, injected with PBS (A) or okadaic acid (B) in the striatun, were immunostained with monoclonal antibody RT-97 that specifically stains neurofilaments phosphorylated at KSP repeats. Images were taken from the striatum showing increased RT-97 immunostaining of nerve fiber bundles in okadaic acid-injected mouse brain. (Scale bar = 100 µM) (C) RT-97 IR rises in Okadaic acid-injected mice striatum: Erk1,2, pErk1,2, JNK1,2, pJNK1,2, and RT-97 immunoreactivities were assessed by Western blot analyses of whole homogenates from three sets of striata dissected from three months old mice injected with 100 nM Okadaic acid. Blots were developed using a HRP-based chemiluminescence probe (D) Graphic representation of pJNK1,2, pErk1,2 and RT-97 immunoreactivities. The band intensities were quantified using NIH image software (n=3 *p<0.05).

Figure 5. Cyclosporine A blocks veratridine-induced dephosphorylation of the RT-97 epitope mediated through PP2B in hippocampal neurons

(A) Western blot analysis of whole homogenates of hippocampal neurons treated with vehicle (Cont;1) veratridine ( Ver ; 2,3), Cyclosporine A (Cyclo; 4,5) and both together (6,7) at 1 and 5 µM. Blots were developed using a HRP based chemiluminescence method (B) Graphic representation of RT-97 immunoreactivities in figure 5A. The band intensities were quantified using NIH image software (n=3*p<0.05). (C–F) Hippocampal neurons in culture (7 DIV) following treatment with veratridine, (E) cyclosporine A, (D) and both together (F) at 1 and 5 µM concentration for 24 hours were fixed in 4% paraformaldehyde along with untreated control neurons (C) and then subjected to double immunostaining using monoclonal antibody RT-97 together with polyclonal antibody raised against NFL. A mixture of anti-mouse and anti-rabbit secondary antibodies tagged to fluorophores Alexa 568 red and Alexa 488 green, respectively were used to develop and visualize the neurons.

Figure 6. RT-97 IR increases during brain maturation and aging

Vibratome sections of brain from mice aged 5 days, 30 days and 4 months were immunostained with monoclonal antibody RT-97 that specifically stains neurofilaments phosphorylated at KSP repeats. Images taken from the striatum of mice at 5 days, 30 days and 4 months of age show changes of RT-97 immunostaining during brain maturation. Scale bar = 20 µm. (B) Western blot analysis were performed on whole homogenates of of spinal cords from mice at postnatal ages of 3, 7, 12, 21, 30, 180, 365 and 660 days using antibodies for neurofilaments, RT-97, SMI-33, neurofilament kinases and phosphatases, loaded in respective lanes 1–8. Blots were developed using HRP-based chemiluminescence or a DAB kit from Vector laboratories. Quantification of band intensities was done using Multi gauge V2.3 software. (C–G) The graphic representations of RT-97, and SMI-33 along with NF kinases or phosphatases are shown. Band densities were quantified using Multi gauge V2.3 software and graphed using Microsoft Excel. (H) The graphic representations of RT-97 and SMI-33 along with PP2A and PP1 activities in spinal cords are shown. Phosphatase activities were monitored by radiometric assays as described in the text and are expressed as % dephosphorylation. (The data represent mean ± SEM, n=3)

Figure 7. The expression of phosphatases declines in sciatic nerve during maturity and aging

(A) Western blot analysis performed on whole homogenates of sciatic nerves from mice at postnatal ages of 7, 12, 21, 30, 180, 365 and 660 days using antibodies for neurofilaments, RT-97, SMI-33, neurofilament kinases and phosphatases, loaded in respective lanes 1–7. Blots were developed using HRP-based chemiluminescence or a DAB kit from Vector laboratories. Quantification of band intensities was done using Multi gauge V2.3 software. (B–D) The graphic representations of RT-97, and SMI-33 along with NF phosphatases are shown. Band densities were quantified using Multi gauge V2.3 software and graphed using Microsoft Excel (The data represent mean +/− SEM, n=3)

Figure 8. Both kinases and phosphatases regulate NF phosphorylation

Phosphorylation at the RT-97 epitope in NFH based on this study (italicised) and Veeranna et al., 2008, is affected by kinases and phosphatases shown in the diagram‥ Also note that various upstream factors and cross talk among kinases influence NF phosphorylation.