Dysfunction of microtubule-associated proteins of MAP2/tau family in Prion disease

Abstract

The aggregation of PrP^Sc is thought to be crucial for the neuropathology of prion diseases. A growing body of evidence demonstrates that the perturbation of the microtubule network contributes to PrP^Sc-mediated neurodegeneration. Microtubules are a component of the cytoskeleton and play a central role in organelle transport, axonal elongation and cellular architecture in neurons. The polymerization, stabilization, arrangement of microtubules can be modulated by interactions with a series of microtubule-associated proteins (MAPs). Recent studies have proposed the abnormal alterations of two major microtubule-associated proteins, tau and MAP2, in the brain tissues of naturally occurred and experimental human and animal prion diseases. Increased total tau protein and hyperphosphorylation of tau at multiple residues are observed at the terminal stage of prion disease. The abnormal aggregation of tau protein disturbs its binding ability to microtubules and affects the microtubule dynamic. Significantly downregulated MAP2 is detected in the brain tissues of scrapie-infected hamsters and PrP106–126 treated cells, which corresponds well with the remarkably low levels of tubulin. In conclusion, dysfunction of MAP2/tau family leads to disruption of microtubule structure and impairment of axonal transport, and eventually triggers apoptosis in neurons, which becomes an essential pathway for prion to induce the neuropathology.

Introduction

Prion diseases are infectious and fatal neurodegenerative disorders of human and animals which are characterized by the aggregation of partially-protease resistant isoform, PrP^Sc and spongiform degeneration in the central nervous system.¹ Severe loss of neurons is a key characteristic for all prion diseases. Although the molecular mechanisms associated with neuronal death induced by PrP^Sc are not well-understood, ER stress, mitochondrial dysfunction and autophagy have been demonstrated to response for killing neurons in prion diseases.^2,3 Moreover, more and more evidences support that destabilization of microtubule network may play a primary actor of neurotoxicity induced by PrP^Sc.^4–7

Microtubules mediate many functions in neurons, including organelle transport, cell shape establishment and maintenance, as well as axonal elongation and growth cone steering.⁸ Neurons cannot synthesize proteins along the axon and proper neuronal function is dependent on transport of cargo across long distance. Interference with microtubule dynamics is likely to contribute to impairment of axonal transport of various vesicles and organelles, leads to energy depletion in the neuron and provides a source for oxidative damage in the cell and initiation of apoptosis. Microtubules are polymers of α- and β-tubulin dimmers. The polymerization, stabilization, and dynamic properties of microtubules are influenced by interactions with microtubule-associated proteins (MAPs).⁹ Members of MAPs have been largely divided into two categories: Type I (MAP1a/1b family) and Type II (MAP2/tau family). MAP2/tau family consists of the neuronal proteins MAP2 and tau, and non-neuronal protein MAP3/MAP4 (MAP3 and MAP4 are identical protein). Both of MAP2 and tau appear to stabilize microtubules in cells and facilitate the polymerization of tubulin in vitro. MAP2, especially high molecular MAP2 isoforms (HMWMAP2) are located mainly in dendrites of mature neurons, while tau is only associated to axonal microtubules. Many previous studies have repeatedly identified that the levels of tubulin decreased in central nervous system (CNS) tissues of either naturally occurred or experimental human and animal TSEs, which accompanied by the change of those two microtubule-associated proteins.^4,6 Dysfunction of MAP2/tau family leads to disruption of microtubule structure and impairment of axonal transport, eventually triggers apoptosis of neurons.

Tau

Structure

The microtubule-associated protein tau is a group of molecular mass of 45–66 kDa proteins encoded by a single gene localized at human chromosome 17. Tau proteins are found primarily in the CNS tissues, which are abundant in neurons and are also expressed at very low levels in astrocytes and oligodendrocytes.¹⁰ Six tau isoforms are identified in adult human brain, produced by alternative mRNA splicing. Tau protein contains several repeated 18-residue microtubule-binding domains (TBD) in the C-terminal region, which are linked through a proline-rich domain (PRD) to an acidic domain (AD) in N-terminal (Fig. 1).

Figure 1. Schematic diagram showing structures of tau and MAP2. Tau, MAP2a/2b and MAP2c/2d are indicated on the right. AD, acidic domain; PRD, proline-rich domain; TBD, tubulin-binding domain; CD, central domain.

Regulatory mechanism of tau function in neuron

Tau plays a key role in regulating microtubule dynamics, axonal transport and neurite outgrowth in neuron, especially to modulate the stability of axonal microtubules. There are more than 80 Ser/Thr phosphorylation sites and 5 potential tyrosine phosphorylation sites in human tau. The biological activity of tau is regulated by the degree of its phosphorylation. Low phosphorylation level of tau in normal brain is required for its interaction with tubulin, in order to promote its assembly into microtubules and help stabilize their structure. Some kinases and phosphatases have been implicated in the abnormal hyperphosphorylation of tau. The main aberrantly hyperphosphorylated sites on tau include the phospho-sites Ser202/Thr205, Thr-231/Ser-235, Ser-262, Ser-396/404 and Ser422.^11-14 Phosphorylation of Ser202/Thr205, Thr-231/Ser-235 and Ser-262 significantly decreases the interaction of tau with microtubules.^12,13 Phosphorylation of Ser-396/404 increases the propensity of tau to oligomerize and eventually form filamentous aggregates.^11,14 Cyclin-dependent kinase 5 (CDK5) and glycogen synthase kinase 3β (GSK3β) have been identified as major candidates mediating tau phosphorylation at disease-associated sites. Ser-396/404 is a major target of GSK3β, while Ser202/Thr205, Thr-231/Ser-235 and Ser-262 are major targets for CDK5. CDK5 is an atypical cyclin-dependent kinase and its activity is dependent upon interaction with p35/p39. The calcium-dependent protease calpain can cleave CDK5/p35 into CDK5/p25 which is more stable and thus causes a more prolonged activation of CDK5. GSK3β can be inhibited by phosphorylation of a Ser residue (Ser9) and be activated by phosphorylation of a Try residue(Try216).

Dysfunction of tau in prion disease

Abnormal hyperphosphorylation of tau leads to its dissociation from microtubules and in turn to microtubule destabilization. To date, several studies have reported the deposits of hyperphosphorylated tau in the brains from the patients with prion disease and in experimental animal TSEs.^15,16 In Gerstmann-Sträussler-Scheinker syndrome (GSS) with the special PRNP mutation, neurofibrillary tangles (NFTs) of abundant hyperphosphorylated tau protein have even been found around PrP^Sc plaques. Indeed, the concept has been well-accepted that GSS is a kind of tauopathy. However, hyperphosphorylated tau in NFT form is not frequently observed in sporadic CJD (sCJD) that is the most predominant type of human prion diseases. Additionally, the change of tau level in cerebrospinal fluid (CSF) in human TSEs seems to be disease-related. The diagnostic value of CSF tau for CJD has been widely evaluated, showing total tau (t-tau) protein and the ratio of p-tau protein/t-tau protein can be used as a diagnostic biochemical parameter to discriminate CJD from other neurodegenerative diseases.¹⁷ Recently, a sCJD-associated tau profile in CSF has been proposed by western blots with tau Exon-2 and -10 specific antibodies.¹⁸

Increased CDK5 expression has been demonstrated in the brains of scrapie-infected animals.⁶ In addition, an increased activity of calpain may be involved in prion disease and result in cleavage of p35/p39 to p25/p29.⁴ An increase of p-tau (Ser202/Thr205) corresponds well with stimulated CDK5 activity in scrapie-infected animals.⁶ In spite of the ability of cytotoxic peptide PrP106–126 to induce activity of GSK3β in cultured neuron,¹⁹ the expression of GSK3β in the brains of scrapie-infected animals is downregulated and its target p-tau (Ser396/Ser404) is depression as well.⁶ In contrast, the activities of both CDK5 and GSK3β are demonstrated to be increased, which are involved to regulate tau hyperphosphorylation in Alzheimer disease.²⁰ The different pattern of two kinases between AD and prion disease may lead to different pathological effects of tau. In prion disease, the predominant phosphorylation of tau are those that decrease the ability of tau to bind microtubules by CDK5 rather than those increase the ability of tau to self-associate and aggregation by GSK3β. That might explain why it is hard to observe NFTs in most types of prion diseases, except in some GSS cases.

Direct molecular interaction between tau and PrP, regardless of PrP^C or PrP^Sc, have been addressed.^21,22 The tandem repeats region (aa186–283) of tau protein and the N-terminus (aa23–91) of PrP are responsible for the interaction. Interestingly, the familial CJD-related PrPs (PrP-PG7 and PrP-PG12 with more numbers of octarepeats) and GSS-related mutant PrP-P102L show remarkably higher binding activities with tau than wild-type PrP. Stronger interacting activities of those CJD-associated PrP mutants with tau will lead to greater disturbance on the tau-associated stabilization of microtubule.

MAP2

Structure

MAP2 is a group of heat-stable proteins that are very abundant in the CNS tissues of mammalian. The multiple human MAP2 isoforms expressed in neurons result from the alternative splicing of an mRNA transcribed from a single gene located in chromosome 2. The main forms of MAP2 are divided into high-molecular weight MAP2 (HMWMAP2) and low-molecular weight MAP2 (LMWMAP2). HMWMAP2 consists of MAP2a and MAP2b that are specially expressed in neurons, while LMWMAP2 includes MAP2c and MAP2d that are also present in glial cells.²³ LMWMAP2 contains the N- and C-terminal regions of HMWMAP2 linked together but lacks the central domain (CD) (Fig. 1). At the C-terminal region, all MAP2 isoforms have a proline-rich domain (PRD) and a tubulin-binding domain (TBD), which are highly conserved with tau protein.

Regulatory mechanism of MAP2 function in neuron

MAP2 can interact with microtubule through its tubulin-binding domain and regulate neurite outgrowth, microtubule dynamics and organelle transport in axons and dendrites. Similar to tau protein, phosphorylation state of MAP2 alters its binding affinity to microtubules. MAP2 contains a great number of serines and threonines. Thirty percent of ser/thr residues locate in the regions of PRD (having Ser/Thr-Pro motifs) and TBD (having KXGS motifs). Several kinases have been identified to phosphorylate MAP2 at special sites, including CDKs, GSK3 and ERKs which mainly phosphorylate Ser/Thr-Pro motifs in PRD, and PKA, PKC and MARKs which mainly phosphorylated KXGS motifs in TBD.²⁴ A high level of phosphorylation decreases the association of MAP2 with tubulin. Moreover, MAP2 is rich in PEST sequences, which are putative signals for rapid proteolytic degradation. Studies show calpain can inhibit microtubule assembly through MAP2 degradation.^25,26 Calpain is a ubiquitous calcium-dependent protease which is essential for physiological neuronal function. Activation of calpain can be triggered by calcium influx and oxidative stress. This could be another important mechanism to alter MAP2-tubulin association.

Dysfunction of MAP2 in prion disease

It has been reported that the levels of MAP2 are usually decreased in ischemia-induced neurodegeneration and Alzheimer diseases in several animal models.²⁷ Our recent study shows the loss of MAP2 has also been observed in prion disease.⁴ Dynamic analysis of scrapie-infected rodents identify that the disappearance of MAP2 seems to occur at the early stage of infection. It suggests the expression of MAP2 in neurons is more vulnerable during the progression of prion diseases. Calpain activity may be increased in prion disease because brain-derived infectious PrP^Sc and synthetic PrP peptides may affect calcium homeostasis.²⁸ During scrapie infection, a time-dependent increase in calpain level has been found along with a gradually decrease in tubulin and MAP2 levels. Inhibition of calpain activity not only reverses the reduction of cellular MAP2 level, but also partially rescues PrP106–126 induced cytotoxicity. It is reasonable to speculate that the abrupt reduction of MAP2 in brain tissues is associated with the rapid increase of calpain in prion diseases. Until now, it is short of directly evidence that abnormal phosphorylation state of MAP2 plays a role in prion disease. But several relevant kinases, such as GSK3β, CDK5 and MARK have been modified in prion disease.⁶ Meanwhile, there is few research to demonstrate a directly interaction between MAP2 and PrP. It will be interesting to see a possible interaction between those two proteins, as it may disclose a different molecular mechanism for MAP2 to mediate disruption of microtubule in prion disease from tau.

Conclusion and Future Study

Based on the present literatures, we hypothesize a representative pathway that prion caused cell death mediated by alteration of MAP2/tau family in Figure 2. Abnormal aggregation of PrP^Sc in cytoplasm stimulates ER stress and affects calcium homeostasis. Calcium influx can trigger activation of calpain, and subsequently, result in cleavage of p35/p39 to p25/p29 and cause a more prolonged activation of CDK5. Increased calpain directly degrades MAP2 that inhibits microtubule assembly. Meanwhile, PrP^Sc deposit leads to a reduction of GSK3β. The changes of CDK5 and GSK3β induce abnormal alterations for the phosphorylation status of tau and MAP2 and cause their dissociation from microtubules. Dysfunction of MAP2/tau family leads to disruption of microtubule structure and impairment of axonal transport, and eventually triggers apoptosis in neurons, although some key steps, such as the mechanism of transcriptional regulations for GSK3β, CDK5 and calpain and the role of abnormal phosphorylation state of MAP2 in prion disease are still not clear. Further characterizations of modification of tau and MAP2 in the pathogenesis of prion diseases are specially needed for understanding prion neurotoxicity and developing potential therapy for prion disease.

Figure 2. A representation of possible mechanisms of PrP^Sc neurotoxicity mediated by dysfunction of MAP2/tau family. Aggregation of PrP^Sc stimulates ER stress and affects calcium homeostasis. Calcium influx activates calpain and results in cleavage of p35/p39 to p25/p29, which cause a more prolonged activation of CDK5. Increased calpain directly degrades MAP2 that inhibits microtubule assembly. Increased CDK5 and decreased GSK3β induce abnormal alterations of phosphorylating statuses of tau and MAP2, which lead to the dissociation of microtubules, and eventually trigger apoptosis in neurons.