Philip Grant

Differential expression of multiple transglutaminases in human brain. Increased expression and cross-linking by transglutaminases 1 and 2 in Alzheimer's disease.

The transglutaminase (TGase) family of enzymes, of which seven different members are known in the human genome, participate in many biological processes involving cross-linking proteins into large macromolecular assemblies. The TGase 2 enzyme is known to be present in neuronal tissues and may play a role in neuronal degenerative diseases such as Alzheimers disease (AD) by aberrantly cross-linking proteins. In this paper, we demonstrate by reverse transcriptase-polymerase chain reaction and immunological methods with specific antibodies that in fact three members, the TGase 1, TGase 2, and TGase 3 enzymes, and are differentially expressed in various regions of normal human brain tissues. Interestingly, the TGase 1 and 3 enzymes and their proteolytically processed forms are involved in terminal differentiation programs of epithelial cell development and barrier function. In addition, we found that the levels of expression and activity of the TGase 1 and 2 enzymes were both increased in the cortex and cerebellum of AD patients. Furthermore, whereas normal brain tissues contain ≈1 residue of cross-link/10,000 residues, AD patient cortex and cerebellum tissues contain 30–50 residues of cross-link/10,000 residues. Together, these findings suggest that multiple TGase enzymes are involved in normal neuronal structure and function, but their elevated expression and cross-linking activity may also contribute to neuronal degenerative disease.

Neurofilament protein synthesis and phosphorylation.

Neurofilament proteins, a major intermediate filament component of the neuronal cytoskeleton, are organized as 10 nm thick filaments in axons and dendrites. They are large, abundantly phosphorylated proteins with numerous phosphate acceptor sites, up to 100 in some cases, organized as numerous repeat motifs. Together with other cytoskeletal components such as microtubules, MAPs, actin and plectin-like linking molecules, they make up a dynamic lattice that sustains neuronal function from neuronal “birthday” to apoptotic cell death. The activity of the neuronal cytoskeleton is regulated by phosphorylation, dephosphorylation reactions mediated by numerous associated kinases, phosphatases and their regulators. Factors regulating multisite phosphorylation of NFs are topographically localized, with maximum phosphorylation of NF proteins consigned to axons. Phosphorylation defines the nature of NF interactions with one another and with other cytoskeletal components such as microtubules, MAPs and actin. To understand how these functional interactions are regulated by phosphorylation we attempt to identify the relevant kinases and phosphatases, their specific targets and the factors modulating their activity. As an initial working model we propose that NF phosphorylation is regulated topographically in neurons by compartment-specific macromolecular complexes of substrates, kinases and phosphatases. This implies that axonal complexes differ structurally and functionally from those in cell bodies and dendrites. Such protein assemblies, by virtue of conformational changes within proteins, facilitate ordered, sequential multisite phosphorylations that modulate dynamic cytoskeletal interactions.

A Cdk5 inhibitory peptide reduces tau hyperphosphorylation and apoptosis in neurons

The extracellular aggregation of amyloid β (Aβ) peptides and the intracellular hyperphosphorylation of tau at specific epitopes are pathological hallmarks of neurodegenerative diseases such as Alzheimers disease (AD). Cdk5 phosphorylates tau at AD‐specific phospho‐epitopes when it associates with p25. p25 is a truncated activator, which is produced from the physiological Cdk5 activator p35 upon exposure to Aβ peptides. We show that neuronal infections with Cdk5 inhibitory peptide (CIP) selectively inhibit p25/Cdk5 activity and suppress the aberrant tau phosphorylation in cortical neurons. Furthermore, Aβ1−42‐induced apoptosis of these cortical neurons was also reduced by coinfection with CIP. Of particular importance is our finding that CIP did not inhibit endogenous or transfected p35/Cdk5 activity, nor did it inhibit the other cyclin‐dependent kinases such as Cdc2, Cdk2, Cdk4 and Cdk6. These results, therefore, provide a strategy to address, and possibly ameliorate, the pathology of neurodegenerative diseases that may be a consequence of aberrant p25 activation of Cdk5, without affecting ‘normal’ Cdk5 activity.

Cdk5 and MAPK are associated with complexes of cytoskeletal proteins in rat brain.

Neurofilament proteins, the major cytoskeletal components of large myelinated axons, are highly phosphorylated by second messenger-dependent and -independent kinases. These kinases, together with tubulins and other cytoskeletal proteins, have been shown to bind to neurofilament preparations. Cdk5 and Erk2, proline-directed kinases in neuronal tissues, phosphorylate the Lys-Ser-Pro (KSP) repeats in tail domains of NF-H, NF-M, and other axonal proteins such as tau and synapsin. In neurofilament and microtubule preparations from rat brain, we demonstrated by Western blot analysis that cdk5, a neuronal cyclin dependent kinase and Erk1/2 were associated with complexes of NF proteins, tubulins and tau. Using P13(suc1) affinity chromatography, a procedure known to bind cdc2-like kinases in proliferating cells with high affinity, we obtained a P13 complex from a rat brain extract exhibiting the same profiles of cdk5 and Erk2 bound to cytoskeletal proteins. The phosphorylation activities of these preparations and the effect of the cdk5 inhibitor, butyrolactone, were consistent with the presence of active kinases. Finally, during a column fractionation and purification of Erk kinases from rat brain extracts, fractions enriched in Erk kinase activity also exhibited co-elution of phosphorylated NF-H, tubulin, tau and cdk5. We suggest that in mammalian brain, different kinases, their regulators and phosphatases form multimeric complexes with cytoskeletal proteins and regulate multisite phosphorylation from synthesis in the cell body to transport and assembly in the axon.

A truncated peptide from p35, a Cdk5 activator, prevents Alzheimer's disease phenotypes in model mice

Alzheimers disease (AD), one of the leading neurodegenerative disorders of older adults, which causes major socioeconomic burdens globally, lacks effective therapeutics without significant side effects. Besides the hallmark pathology of amyloid plaques and neurofibrillary tangles (NFTs), it has been reported that cyclin‐dependent kinase 5 (Cdk5), a critical neuronal kinase, is hyperactivated in AD brains and is, in part, responsible for the above pathology. Here we show that a modified truncated 24‐aa peptide (TFP5), derived from the Cdk5 activator p35, penetrates the blood‐brain barrier after intraperitoneal injections, inhibits abnormal Cdk5 hyperactivity, and significantly rescues AD pathology (up to 70–80%) in 5XFAD AD model mice. The mutant mice, injected with TFP5 exhibit behavioral rescue, whereas no rescue was observed in mutant mice injected with either saline or scrambled peptide. However, TFP5 does not inhibit cell cycle Cdks or normal Cdk5/p35 activity, and thereby has no toxic side effects (even at 200 mg/kg), a common problem in most current therapeutics for AD. In addition, treated mice displayed decreased inflammation, amyloid plaques, NFTs, cell death, and an extended life by 2 mo. These results suggest TFP5 as a potential therapeutic, toxicity‐free candidate for AD.—Shukla, V., Zheng, Y.‐L., Mishra, S. K., Amin, N. D., Steiner, J., Grant, P., Kesavapany, S., Pant, H. C. A truncated peptide from p35, a Cdk5 activator, prevents Alzheimers disease phenotypes in model mice. FASEB J. 27, 174–186 (2013). www.fasebj.org

Cyclin-Dependent Kinase 5 Phosphorylation of Human Septin SEPT5 (hCDCrel-1) Modulates Exocytosis

Cyclin-dependent kinase 5 (Cdk5) is predominantly expressed in the nervous system, where it is involved in neuronal migration, synaptic transmission, and survival. The role of Cdk5 in synaptic transmission is mediated by regulating the cellular functions of presynaptic proteins such as synapsin, Munc18, and dynamin 1. Its multifunctional role at the synapse is complex and probably involves other novel substrates. To explore this possibility, we used a yeast two-hybrid screen of a human cDNA library with p35 as bait and isolated human septin 5 (SEPT5), known also as hCDCrel-1, as an interacting clone. Here we report that p35 associates with SEPT5 in GST (glutathione S-transferase)-pull-down and coimmunoprecipitation assays. We confirmed that Cdk5/p35 phosphorylates SEPT5 in vitro and in vivo and identified S327 of SEPT5 as a major phosphorylation site. A serine (S)-to-alanine (A) 327 mutant of SEPT5 bound syntaxin more efficiently than SEPT5 wild type. Additionally, coimmunoprecipitation from synaptic vesicle fractions and Cdk5 wild-type and knock-out lysates showed that phosphorylation of septin 5 by Cdk5/p35 decreases its binding to syntaxin-1. Moreover, mutant nonphosphorylated SEPT5 potentiated regulated exocytosis more than the wild type when each was expressed in PC12 cells. These data suggest that Cdk5 phosphorylation of human septin SEPT5 at S327 plays a role in modulating exocytotic secretion.

p35/Cyclin-Dependent Kinase 5 Phosphorylation of Ras Guanine Nucleotide Releasing Factor 2 (RasGRF2) Mediates Rac-Dependent Extracellular Signal-Regulated Kinase 1/2 Activity, Altering RasGRF2 and Microtubule-Associated Protein 1b Distribution in Neurons

Cyclin-dependent kinase 5 (Cdk5) is a proline-directed kinase the activity of which is dependent on association with its neuron-specific activators, p35 and p39. Cdk5 activity is critical for the proper formation of cortical structures and lamination during development. In the adult nervous system, Cdk5 function is implicated in cellular adhesion, dopamine signaling, neurotransmitter release, and synaptic activity. In addition, Cdk5 is also involved in “cross-talk” with other signal transduction pathways. To further examine its involvement in cross-talk with other pathways, we identified proteins that interacted with p35 using the yeast two-hybrid system. We report here that p35 associates with Ras guanine nucleotide releasing factor 2 (RasGRF2) in coimmunoprecipitation and colocalization studies using transfected cell lines as well as primary cortical neurons. Additionally, Cdk5 phosphorylates RasGRF2 both in vitro and in vivo, leading to a decrease in Rac–guanidine exchange factor activity and a subsequent reduction in extracellular signal-regulated kinase 1/2 activity. We show that p35/Cdk5 phosphorylates RasGRF2 on serine737, which leads to an accumulation of RasGRF2 in the neuronal cell bodies coinciding with an accumulation of microtubule-associated protein 1b. The membrane association of p35 and subsequent localization of Cdk5 activity toward RasGRF2 and Rac provide insights into important cellular signaling processes that occur at the membrane, resulting in downstream effects on signal transduction cascades.

A 24-Residue Peptide (p5), Derived from p35, the Cdk5 Neuronal Activator, Specifically Inhibits Cdk5-p25 Hyperactivity and Tau Hyperphosphorylation

The activity of Cdk5-p35 is tightly regulated in the developing and mature nervous system. Stress-induced cleavage of the activator p35 to p25 and a p10 N-terminal domain induces deregulated Cdk5 hyperactivity and perikaryal aggregations of hyperphosphorylated Tau and neurofilaments, pathogenic hallmarks in neurodegenerative diseases, such as Alzheimer disease and amyotrophic lateral sclerosis, respectively. Previously, we identified a 125-residue truncated fragment of p35 called CIP that effectively and specifically inhibited Cdk5-p25 activity and Tau hyperphosphorylation induced by Aβ peptides in vitro, in HEK293 cells, and in neuronal cells. Although these results offer a possible therapeutic approach to those neurodegenerative diseases assumed to derive from Cdk5-p25 hyperactivity and/or Aβ induced pathology, CIP is too large for successful therapeutic regimens. To identify a smaller, more effective peptide, in this study we prepared a 24-residue peptide, p5, spanning CIP residues Lys245–Ala277. p5 more effectively inhibited Cdk5-p25 activity than did CIP in vitro. In neuron cells, p5 inhibited deregulated Cdk5-p25 activity but had no effect on the activity of endogenous Cdk5-p35 or on any related endogenous cyclin-dependent kinases in HEK293 cells. Specificity of p5 inhibition in cortical neurons may depend on the p10 domain in p35, which is absent in p25. Furthermore, we have demonstrated that p5 reduced Aβ(1–42)-induced Tau hyperphosphorylation and apoptosis in cortical neurons. These results suggest that p5 peptide may be a unique and useful candidate for therapeutic studies of certain neurodegenerative diseases.

Quantitative phosphoproteomic analysis of neuronal intermediate filament proteins (NF-M/H) in Alzheimer's disease by iTRAQ

Aberrant hyperphosphorylation of neuronal cytoskeletal proteins is one of the major pathological hallmarks of neurodegenerative disorders such as Alzheimer disease (AD), amyotrophic lateral sclerosis (ALS), and Parkinsons disease (PD). Human NF‐M/H display a large number of multiple KSP repeats in the carboxy‐terminal tail domain, which are phosphorylation sites of proline‐directed serine/threonine (pSer/Thr‐Pro, KS/T‐P) kinases. The phosphorylation sites of NF‐M/H have not been characterized in AD brain. Here, we use quantitative phosphoproteomic methodology, isobaric tag for relative and absolute quantitation (iTRAQ), for the characterization of NF‐M/H phosphorylation sites in AD brain. We identified 13 hyperphosphorylated sites of NF‐M;9 Lys‐Ser‐Pro (KSP) sites;2 variant motifs, Glu‐Ser‐Pro (ESP) Ser‐736 and Leu‐Ser‐Pro (LSP) Ser‐837;and 2 non‐S/T‐P motifs, Ser‐783 and Ser‐788. All the Ser/Thr residues are phosphorylated at significantly greater abundance in AD brain compared with control brain. Ten hyperphosphorylated KSP sites have been identified on the C‐terminal tail domain of NF‐H, with greater abundance of phosphorylation in AD brain compared with control brain. Our data provide the direct evidence that NF‐M/H are hyperphosphorylated in AD compared with control brain and suggest the role of both proline‐directed and non‐proline‐directed protein kinases in AD. This study represents the first comprehensive iTRAQ analyses and quantification of phosphorylation sites of human NF‐M and NF‐H from AD brain and suggests that aberrant hyperphosphorylation of neuronal intermediate filament proteins is involved in AD.—Rudrabhatla, P., Grant, P., Jaffe, H., Strong, M. J., Pant, H. C. Quantitative phosphoproteomic analysis of neuronal intermediate filament proteins (NF‐M/H) in Alzheimers disease by iTRAQ. FASEB J. 24, 4396–4407 (2010). www.fasebj.org

P13suc1 associates with a cdc2-like kinase in a multimeric cytoskeletal complex in squid axoplasm

P13suc1 sepharose-conjugated beads were used to extract the kinases that phosphorylate neurofilaments in the squid giant axon. Using Western blots and in vitro kinase assays, we demonstrated the presence of an active cdc2-like kinase and its putative regulators such as cyclin E, p13, and p67 in axoplasm and a P13-axoplasm complex (P13-Ax). Protein kinase A (PKA) and casein kinase (CK) I and II were also found in the P13-Ax. Western blot analysis of the P13-Ax also demonstrated several axonal cytoskeletal components; e.g., neurofilaments (NFs; NF 60, 70, and 220), tubulin, actin, and microtubule-associated proteins. NF 220 and tubulin were phosphorylated by the kinases in the P13-Ax. To determine whether NFs bound directly to the P13 beads, or bound indirectly by association with cdc2 kinase, a washed, axon-derived neurofilament preparation that contained NFs, PKA, CKl, and tubulin, but no cdc2-like kinase, yielded no bound proteins after incubation with P13suc1. The wash supernatant from the neurofilament preparation, however, containing the cdc2-like kinase, did yield cytoskeletal components that bound to P13suc1. Moreover, a bacterial-expressed cdk5 associated with P13 beads was able to complex with selected cytoskeletal components in the washed neurofilament preparation. These data indicate that direct binding of P13 beads with a cdc2-like kinase could extract active multimeric complexes composed of axonal cytoskeletal proteins and kinases. Application of P13 chromatography may be useful in characterizing the network of functional interactions among cytoskeletal elements and protein kinases in neurons.