Investigation of the Effect of Peptide p5 Targeting CDK5-p25 Hyperactivity on Munc18-1 (P67) Regulating Neuronal Exocytosis Using Molecular Simulations.

Munc18-1 is an SM (sec1/munc-like) family protein involved in vesicle fusion and neuronal exocytosis. Munc18-1 is known to regulate the exocytosis process by binding with closed- and open-state conformations of Syntaxin1, a protein belonging to the SNARE family established to be central to the exocytosis process. Our previous work studied peptide p5 as a promising drug candidate for CDK5-p25 complex, an Alzheimer's disease (AD) pathological target. Experimental in vivo and in vitro studies suggest that Munc18-1 promotes p5 to selectively inhibit the CDK5-p25 complex without affecting the endogenous CDK5 activity, a characteristic of remarkable therapeutic implications. In this paper, we identify several binding modes of p5 with Munc18-1 that could potentially affect the Munc18-1 binding with SNARE proteins and lead to off-target effects on neuronal communication using molecular dynamics simulations. Recent studies indicate that disruption of Munc18-1 function not only disrupts neurotransmitter release but also results in neurodegeneration, exhibiting clinical resemblance to other neurodegenerative conditions such as AD, causing diagnostic and treatment challenges. We characterize such interactions between p5 and Munc18-1, define the corresponding pharmacophores, and provide guidance for the in vitro validation of our findings to improve therapeutic efficacy and safety of p5.

Cyclin-Dependent Kinase 5 Regulates cPLA2 Activity and Neuroinflammation in Parkinson's Disease.

Hyperactivation of cyclin-dependent kinase 5 (Cdk5) by p25, contributes to neuroinflammation causing neurodegeneration in Parkinson's disease (PD) and Alzheimer's disease. However, the mechanism by which Cdk5 induces neuroinflammation in the PD brain is largely unexplored. Here, we show that Cdk5 phosphorylates cytosolic phospholipase A2 (cPLA2) at Thr-268 and Ser-505 sites lead to its activation and generation of eicosanoid products. Mutational studies using site-directed mutagenesis and molecular simulations show that the architecture of the protein changes on each single-point mutation. Interestingly, double mutations also led to a severe decline in the activity of cPLA2 and to the disruption of its translocation to the plasma membrane. Further, the brain lysates of transgenic PD mouse models show hyperactivation of Cdk5, resulting in enhanced phosphorylation of Thr-268 and Ser-505 of cPLA2 and its heightened activity, confirming the findings observed in the cell culture model of PD. These phosphorylation sites of cPLA2 and Cdk5 could be explored as the future therapeutic targets against neuroinflammation in PD. Further, conjoint transcriptomic analysis of the publicly available human PD datasets strengthens the hypothesis that genes of the arachidonic acid, prostaglandin synthesis, and inflammatory pathways are significantly upregulated in the case of PD patients compared with that of healthy control subjects.

Computational Study of the Allosteric Effects of p5 on CDK5-p25 Hyperactivity as Alternative Inhibitory Mechanisms in Neurodegeneration.

The cyclin-dependent kinase (CDK5) forms a stable complex with its activator p25, leading to the hyperphosphorylation of tau proteins and to the formation of plaques and tangles that are considered to be one of the typical causes of Alzheimer's disease (AD). Hence, the pathological CDK5-p25 complex is a promising therapeutic target for AD. Small peptides, obtained from the truncation of CDK5 physiological activator p35, have shown promise in inhibiting the pathological complex effectively while also crossing the blood-brain barrier. One such small 24-residue peptide, p5, has shown selective inhibition toward the pathological complex in vivo. Our previous research focused on the characterization of a computationally predicted CDK5-p5 binding mode and of its pharmacophore, which was consistent with competitive inhibition. In continuation of our previous work, herein, we investigate four additional binding modes to explore other possible mechanisms of interaction between CDK5 and p5. The quantitative description of the pharmacophore is consistent with both competitive and allosteric p5-induced inhibition mechanisms of CDK5-p25 pathology. The gained insights can direct further in vivo/in vitro tests and help design small peptides, linear or cyclic, or peptidomimetic compounds as adjuvants of orthosteric inhibitors or as part of a cocktail of drugs with enhanced effectiveness and lower side effects.

Rapid, quantitative therapeutic screening for Alzheimer's enzymes enabled by optimal signal transduction with transistors.

We show that commercially sourced n-channel silicon field-effect transistors (nFETs) operating above their threshold voltage with closed loop feedback to maintain a constant channel current allow a pH readout resolution of (7.2 ± 0.3) × 10-3 at a bandwidth of 10 Hz, or ≈3-fold better than the open loop operation commonly employed by integrated ion-sensitive field-effect transistors (ISFETs). We leveraged the improved nFET performance to measure the change in solution pH arising from the activity of a pathological form of the kinase Cdk5, an enzyme implicated in Alzheimer's disease, and showed quantitative agreement with previous measurements. The improved pH resolution was realized while the devices were operated in a remote sensing configuration with the pH sensing element off-chip and connected electrically to the FET gate terminal. We compared these results with those measured by using a custom-built dual-gate 2D field-effect transistor (dg2DFET) fabricated with 2D semi-conducting MoS2 channels and a signal amplification of 8. Under identical solution conditions the nFET performance approached the dg2DFETs pH resolution of (3.9 ± 0.7) × 10-3. Finally, using the nFETs, we demonstrated the effectiveness of a custom polypeptide, p5, as a therapeutic agent in restoring the function of Cdk5. We expect that the straight-forward modifications to commercially sourced nFETs demonstrated here will lower the barrier to widespread adoption of these remote-gate devices and enable sensitive bioanalytical measurements for high throughput screening in drug discovery and precision medicine applications.

Quantum capacitance-limited MoS2 biosensors enable remote label-free enzyme measurements.

We have demonstrated atomically thin, quantum capacitance-limited, field-effect transistors (FETs) that enable the detection of pH changes with 75-fold higher sensitivity (≈4.4 V per pH) over the Nernst value of 59 mV per pH at room temperature when used as a biosensor. The transistors, which are fabricated from monolayer films of MoS2, use a room temperature ionic liquid (RTIL) in place of a conventional oxide gate dielectric and exhibit very low intrinsic noise resulting in a pH resolution of 92 × 10-6 at 10 Hz. This high device performance, which is a function of the structure of our device, is achieved by remotely connecting the gate to a pH sensing element allowing the FETs to be reused. Because pH measurements are fundamentally important in biotechnology, the increased resolution demonstrated here will benefit numerous applications ranging from pharmaceutical manufacturing to clinical diagnostics. As an example, we experimentally quantified the function of the kinase Cdk5, an enzyme implicated in Alzheimer's disease, at concentrations that are 5-fold lower than physiological values, and with sufficient time-resolution to allow the estimation of both steady-state and kinetic parameters in a single experiment. The high sensitivity, increased resolution, and fast turnaround time of the measurements will allow the development of early diagnostic tools and novel therapeutics to detect and treat neurological conditions years before currently possible.

Overexpression of the Cdk5 inhibitory peptide in motor neurons rescue of amyotrophic lateral sclerosis phenotype in a mouse model.

Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disease that affects motor nerve cells in the brain and the spinal cord. Etiological mechanisms underlying the disease remain poorly understood; recent studies suggest that deregulation of p25/Cyclin-dependent kinase 5 (Cdk5) activity leads to the hyperphosphorylation of Tau and neurofilament (NF) proteins in ALS transgenic mouse model (SOD1G37R). A Cdk5 involvement in motor neuron degeneration is supported by analysis of three SOD1G37R mouse lines exhibiting perikaryal inclusions of NF proteins and hyperphosphorylation of Tau. Here, we tested the hypothesis that inhibition of Cdk5/p25 hyperactivation in vivo is a neuroprotective factor during ALS pathogenesis by crossing the new transgenic mouse line that overexpresses Cdk5 inhibitory peptide (CIP) in motor neurons with the SOD1G37R, ALS mouse model (TriTg mouse line). The overexpression of CIP in the motor neurons significantly improves motor deficits, extends survival and delays pathology in brain and spinal cord of TriTg mice. In addition, overexpression of CIP in motor neurons significantly delays neuroinflammatory responses in TriTg mouse. Taken together, these data suggest that CIP may serve as a novel therapeutic agent for the treatment of neurodegenerative diseases.

Phosphorylation, Dephosphorylation, and Multiprotein Assemblies Regulate Dynamic Behavior of Neuronal Cytoskeleton: A Mini-Review.

Cellular localization, assembly and abnormal aggregation of neurofilaments depend on phosphorylation. Pathological processes associated with neurodegeneration exhibit aberrant accumulation of microtubule associated aggregated forms of hyperphosphorylated neuronal protein tau in cell bodies. These processes are critical for the disease progression in patients suffering from Alzheimer's disease, Parkinson's disease, and Amyotrophic Lateral Sclerosis. In healthy cells, tau is localized in axons. Topographic regulation suggests that whereas the sites of synthesis of kinases and neurofilaments are the cell bodies, and sites of their functional assemblies are axons, phosphorylation/dephosphorylation are the key processes that arrange the molecules at their precise locations. Phosphorylation sites in the dynamic developmental and degenerative processes differ. Not all these processes are well understood. New advancements identify epigenetic factors involved in AD which account for the influence of age-related environment/genome interactions leading to the disease. Progress in proteomics highlights previously found major proteins and adds more to the list of those involved in AD. New key elements of specificity provide determinants of molecular recognition important for the assembly of macromolecular complexes. In this review, we discuss aberrant spatial distribution of neuronal polypeptides observed in neuropathies: aggregation, association with proteins of the neuronal cytoskeleton, and phosphorylation dependent dynamics. Particularly, we emphasize recent advancements in understanding the function and determinants of specific association of molecules involved in Alzheimer's disease with respect to the topographic regulation of phosphorylation in neuronal cytoskeleton and implications for the design of new therapies. Further, we address the role of various filament systems in maintenance of the shape, rigidity and dynamics of the cytoskeleton.

Phosphorylation of the Transient Receptor Potential Ankyrin 1 by Cyclin-dependent Kinase 5 affects Chemo-nociception.

Cyclin-dependent kinase 5 (Cdk5) is a key neuronal kinase that is upregulated during inflammation, and can subsequently modulate sensitivity to nociceptive stimuli. We conducted an in silico screen for Cdk5 phosphorylation sites within proteins whose expression was enriched in nociceptors and identified the chemo-responsive ion channel Transient Receptor Potential Ankyrin 1 (TRPA1) as a possible Cdk5 substrate. Immunoprecipitated full length TRPA1 was shown to be phosphorylated by Cdk5 and this interaction was blocked by TFP5, an inhibitor that prevents activation of Cdk5. In vitro peptide-based kinase assay revealed that four of six TRPA1 Cdk5 consensus sites acted as substrates for Cdk5, and modeling of the ankyrin repeats disclosed that phosphorylation would occur at characteristic pockets within the (T/S)PLH motifs. Calcium imaging of trigeminal ganglion neurons from genetically engineered mice overexpressing or lacking the Cdk5 activator p35 displayed increased or decreased responsiveness, respectively, to stimulation with the TRPA1 agonist allylisothiocyanate (AITC). AITC-induced chemo-nociceptive behavior was also heightened in vivo in mice overexpressing p35 while being reduced in p35 knockout mice. Our findings demonstrate that TRPA1 is a substrate of Cdk5 and that Cdk5 activity is also able to modulate TRPA1 agonist-induced calcium influx and chemo-nociceptive behavioral responses.

Peripheral and orofacial pain sensation is unaffected by the loss of p39.

Abstract: Cdk5 is a key neuronal kinase necessary for proper brain development, which has recently been implicated in modulating nociception. Conditional deletion of Cdk5 in pain-sensing neurons attenuates pain responses to heat in both the periphery and orofacial regions. Cdk5 activity is regulated by binding to the activators p35 and p39, both of which possess a cyclin box. Our previous examination of the nociceptive role of the well-characterized Cdk5 activator p35 using mice that either lack or overexpress this regulatory subunit demonstrated that Cdk5/p35 activity affects mechanical, chemical, and thermal nociception. In contrast, the nociceptive role of Cdk5's other less-studied activator p39 is unknown. Here, we report that the knockout of p39 in mice did not affect orofacial and peripheral nociception. The lack of any algesic response to nociceptive stimuli in the p39 knockout mice contrasts with the hypoalgesic effects that result from the deletion of p35. Our data demonstrate different and nonoverlapping roles of Cdk5 activators in the regulation of orofacial as well as peripheral nociception with a crucial role for Cdk5/p35 in pain signaling.

Curcumin Ameliorates Neuroinflammation, Neurodegeneration, and Memory Deficits in p25 Transgenic Mouse Model that Bears Hallmarks of Alzheimer's Disease.

Several studies have indicated that neuroinflammation is indeed associated with neurodegenerative disease pathology. However, failures of recent clinical trials of anti-inflammatory agents in neurodegenerative disorders have emphasized the need to better understand the complexity of the neuroinflammatory process in order to unravel its link with neurodegeneration. Deregulation of Cyclin-dependent kinase 5 (Cdk5) activity by production of its hyperactivator p25 is involved in the formation of tau and amyloid pathology reminiscent of Alzheimer's disease (AD). Recent studies show an association between p25/Cdk5 hyperactivation and robust neuroinflammation. In addition, we recently reported the novel link between the p25/Cdk5 hyperactivation-induced inflammatory responses and neurodegenerative changes using a transgenic mouse that overexpresses p25 (p25Tg). In this study, we aimed to understand the effects of early intervention with a potent natural anti-inflammatory agent, curcumin, on p25-mediated neuroinflammation and the progression of neurodegeneration in p25Tg mice. The results from this study showed that curcumin effectively counteracted the p25-mediated glial activation and pro-inflammatory chemokines/cytokines production in p25Tg mice. Moreover, this curcumin-mediated suppression of neuroinflammation reduced the progression of p25-induced tau/amyloid pathology and in turn ameliorated the p25-induced cognitive impairments. It is widely acknowledged that to treat AD, one must target the early-stage of pathological changes to protect neurons from irreversible damage. In line with this, our results demonstrated that early intervention of inflammation could reduce the progression of AD-like pathological outcomes. Moreover, our data provide a rationale for the potential use of curcuminoids in the treatment of inflammation associated neurodegenerative diseases.

Influence of a GSK3ß phosphorylation site within the proximal C-terminus of Neurofilament-H on neurofilament dynamics.

Phosphorylation of the C-terminal tail of the heavy neurofilament subunit (NF-H) impacts neurofilament (NF) axonal transport and residence within axons by fostering NF-NF associations that compete with transport. We tested the role of phosphorylation of a GSK-3ß consensus site (S493) located in the proximal portion of the NF-H tail in NF dynamics by transfection of NB2a/d1 cells with NF-H, where S493 was mutated to aspartic acid (S493D) or to alanine (S493A) to mimic constitutive phosphorylation and non-phosphorylation. S493D underwent increased transport into axonal neurites, while S493A displayed increased perikaryal NF aggregates that were decorated by anti-kinesin. Increased levels of S493A co-precipitated with anti-kinesin indicating that reduced transport of S493A was not due to reduced kinesin association but due to premature NF-NF interactions within perikarya. S493D displayed increased phospho-immunoreactivity within axonal neurites at downstream C-terminal sites attributable to mitogen-activated protein kinase and cyclin-dependent kinase 5. However, S493D was more prone to proteolysis following kinase inhibition, suggesting that S493 phosphorylation is an early event that alters sidearm configuration in a manner that promotes appropriate NF distribution. We propose a novel model for sidearm configuration.

TFP5, a Peptide Inhibitor of Aberrant and Hyperactive Cdk5/p25, Attenuates Pathological Phenotypes and Restores Synaptic Function in CK-p25Tg Mice.

It has been reported that cyclin-dependent kinase 5 (cdk5), a critical neuronal kinase, is hyperactivated in Alzheimer's disease (AD) and may be, in part, responsible for the hallmark pathology of amyloid plaques and neurofibrillary tangles (NFTs). It has been proposed by several laboratories that hyperactive cdk5 results from the overexpression of p25 (a truncated fragment of p35, the normal cdk5 regulator), which, when complexed to cdk5, induces hyperactivity, hyperphosphorylated tau/NFTs, amyloid-ß plaques, and neuronal death. It has previously been shown that intraperitoneal (i.p.) injections of a modified truncated 24-aa peptide (TFP5), derived from the cdk5 activator p35, penetrated the blood-brain barrier and significantly rescued AD-like pathology in 5XFAD model mice. The principal pathology in the 5XFAD mutant, however, is extensive amyloid plaques; hence, as a proof of concept, we believe it is essential to demonstrate the peptide's efficacy in a mouse model expressing high levels of p25, such as the inducible CK-p25Tg model mouse that overexpresses p25 in CamKII positive neurons. Using a modified TFP5 treatment, here we show that peptide i.p. injections in these mice decrease cdk5 hyperactivity, tau, neurofilament-M/H hyperphosphorylation, and restore synaptic function and behavior (i.e., spatial working memory, motor deficit using Rota-rod). It is noteworthy that TFP5 does not inhibit endogenous cdk5/p35 activity, nor other cdks in vivo suggesting it might have no toxic side effects, and may serve as an excellent therapeutic candidate for neurodegenerative disorders expressing abnormally high brain levels of p25 and hyperactive cdk5.

Squid Giant Axon Contains Neurofilament Protein mRNA but does not Synthesize Neurofilament Proteins.

When isolated squid giant axons are incubated in radioactive amino acids, abundant newly synthesized proteins are found in the axoplasm. These proteins are translated in the adaxonal Schwann cells and subsequently transferred into the giant axon. The question as to whether any de novo protein synthesis occurs in the giant axon itself is difficult to resolve because the small contribution of the proteins possibly synthesized intra-axonally is not easily distinguished from the large amounts of the proteins being supplied from the Schwann cells. In this paper, we reexamine this issue by studying the synthesis of endogenous neurofilament (NF) proteins in the axon. Our laboratory previously showed that NF mRNA and protein are present in the squid giant axon, but not in the surrounding adaxonal glia. Therefore, if the isolated squid axon could be shown to contain newly synthesized NF protein de novo, it could not arise from the adaxonal glia. The results of experiments in this paper show that abundant 3H-labeled NF protein is synthesized in the squid giant fiber lobe containing the giant axon's neuronal cell bodies, but despite the presence of NF mRNA in the giant axon no labeled NF protein is detected in the giant axon. This lends support to the glia-axon protein transfer hypothesis which posits that the squid giant axon obtains newly synthesized protein by Schwann cell transfer and not through intra-axonal protein synthesis, and further suggests that the NF mRNA in the axon is in a translationally repressed state.

The interaction of Munc 18 (p67) with the p10 domain of p35 protects in vivo Cdk5/p35 activity from inhibition by TFP5, a peptide derived from p35.

In a series of studies, we have identified TFP5, a truncated fragment of p35, the Cdk5 kinase regulatory protein, which inhibits Cdk5/p35 and the hyperactive Cdk5/p25 activities in test tube experiments. In cortical neurons, however, and in vivo in Alzheimer's disease (AD) model mice, the peptide specifically inhibits the Cdk5/p25 complex and not the endogenous Cdk5/p35. To account for the selective inhibition of Cdk5/p25 activity, we propose that the "p10" N-terminal domain of p35, absent in p25, spares Cdk5/p35 because p10 binds to macromolecules (e.g., tubulin and actin) as a membrane-bound multimeric complex that favors p35 binding to Cdk5 and catalysis. To test this hypothesis, we focused on Munc 18, a key synapse-associated neuronal protein, one of many proteins copurifying with Cdk5/p35 in membrane-bound multimeric complexes. Here we show that, in vitro, the addition of p67 protects Cdk5/p35 and has no effect on Cdk5/p25 activity in the presence of TFP5. In cortical neurons transfected with p67siRNA, we also show that TFP5 inhibits Cdk5/p35 activity, whereas in the presence of p67 the activity is protected. It does so without affecting any other kinases of the Cdk family of cyclin kinases. This difference may be of significant therapeutic value because the accumulation of the deregulated, hyperactive Cdk5/p25 complex in human brains has been implicated in pathology of AD and other neurodegenerative disorders.

Profiling of p5, a 24 Amino Acid Inhibitory Peptide Derived from the CDK5 Activator, p35 CDKR1 Against 70 Protein Kinases.

Cyclin-dependent kinase 5 (CDK5) is a multifunctional serine/threonine kinase that regulates a large number of neuronal processes essential for nervous system development and function with its activator p35 CDK5R1. Upon neuronal insults, p35 is proteolyzed and cleaved to p25 producing deregulation and hyperactivation of CDK5 (CDK5/p25), implicated in tau hyperphosphorylation, a pathology in some neurodegenerative diseases. A truncated, 24 amino acid peptide, p5, derived from p35 inhibits the deregulated CDK5 phosphotransferase activity and ameliorates Alzheimer's disease (AD) phenotypes in AD model mice. In the present study, we have screened a diverse panel of 70 human protein kinases for their sensitivities to p5, and a subset of these to p35. At least 16 of the tested protein kinases exhibited IC50 values that were 250 µM or less, with CAMK4, ZAP70, SGK1, and PIM1 showing greater sensitivity to inhibition by p5 than CDK5/p35 and CDK5/p25. In contrast, the p5 peptide modestly activated LKB1 and GSK3ß. A sub set of kinases screened against p35 showed that activity of CAMK4 in the absence of calcium and calmodulin was also markedly inhibited by p35. The Cyclin Y-dependent kinases PFTK1 (CDK14) and PCTK1 (CDK16) were activated by p35 at least 10-fold in the absence of Cyclin Y and by approximately 50% in its presence. These findings provide additional insights into the mechanisms of action for p5 and p35 in the regulation of protein phosphorylation in the nervous system.

TFP5/TP5 peptide provides neuroprotection in the MPTP model of Parkinson's disease.

Cyclin-dependent kinase 5 (Cdk5) is a member of the serine-threonine kinase family of cyclin-dependent kinases. Cdk5 is critical to normal mammalian nervous system development and plays important regulatory roles in multiple cellular functions. Recent evidence indicates that Cdk5 is inappropriately activated in several neurodegenerative conditions, including Parkinson's disease (PD). PD is a chronic neurodegenerative disorder characterized by the loss of dopamine neurons in the substantia nigra, decreased striatal dopamine levels, and consequent extrapyramidal motor dysfunction. During neurotoxicity, p35 is cleaved to form p25. Binding of p25 with Cdk5 leads deregulation of Cdk5 resulting in number of neurodegenerative pathologies. To date, strategies to specifically inhibit Cdk5 hyperactivity have not been successful without affecting normal Cdk5 activity. Here we show that inhibition of p25/Cdk5 hyperactivation through TFP5/TP5, truncated 24-aa peptide derived from the Cdk5 activator p35 rescues nigrostriatal dopaminergic neurodegeneration induced by 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP/MPP(+)) in a mouse model of PD. TP5 peptide treatment also blocked dopamine depletion in the striatum and improved gait dysfunction after MPTP administration. The neuroprotective effect of TFP5/TP5 peptide is also associated with marked reduction in neuroinflammation and apoptosis. Here we show inhibition of Cdk5/p25-hyperactivation by TFP5/TP5 peptide, which identifies Cdk5/p25 as a potential therapeutic target to reduce neurodegeneration in PD.

Compartment-Specific Phosphorylation of Squid Neurofilaments.

Studies of the giant axon and synapse of third-order neurons in the squid stellate ganglion have provided a vast literature on neuronal physiology and axon transport. Large neuronal size also lends itself to comparative biochemical studies of cell body versus axon. These have focused on the regulation of synthesis, assembly, posttranslational modification and function of neuronal cytoskeletal proteins (microtubules (MTs) and neurofilaments (NFs)), the predominant proteins in axoplasm. These contribute to axonal organization, stability, transport, and impulse transmission responsible for rapid contractions of mantle muscles underlying jet propulsion. Studies of vertebrate NFs have established an extensive literature on NF structure, organization, and function; studies of squid NFs, however, have made it possible to compare compartment-specific regulation of NF synthesis, assembly, and function in soma versus axoplasm. Since NFs contain over 100 eligible sites for phosphorylation by protein kinases, the compartment-specific patterns of phosphorylation have been a primary focus of biochemical studies. We have learned that NF phosphorylation is tightly compartmentalized; extensive phosphorylation occurs only in the axonal compartment in squid and in vertebrate neurons. This extensive phosphorylation plays a key role in organizing NFs, in association with microtubules (MTs), into a stable, dynamic functional lattice that supports axon growth, diameter, impulse transmission, and synaptic activity. To understand how cytoskeletal phosphorylation is topographically regulated, the kinases and phosphatases, bound to NFs isolated from cell bodies and axoplasm, have also been studied.

Analysis of the Inhibitory Elements in the p5 Peptide Fragment of the CDK5 Activator, p35, CDKR1 Protein.

Besides the hallmark pathology of amyloid plaques and neurofibrillary tangles, it is well documented that cyclin-dependent kinase 5 (CDK5), a critical neuronal protein kinase in nervous system development, function, and survival, when deregulated and hyperactivated induces Alzheimer's disease (AD) and amyotrophic lateral sclerosis and Parkinson's disease-like phenotypes in mice. In a recent study, we demonstrated that p5, a small, truncated fragment of 24 amino acid residues derived from the CDK5 activator protein 35 (NCK5A, p35), selectively inhibited deregulated CDK5 hyperactivity and ameliorated AD phenotypes in model mice. In this study, we identified the most inhibitory elements in the p5 peptide fragment. Each amino acid residue in p5 was systematically replaced with its homologous residues that may still be able to functionally substitute. The effects of these p5 peptide analogs were studied on the phosphotransferase activities of CDK5/p35, CDK5/p25, ERK1, and GSK3ß. The mimetic p5 peptide (A/V substitution at the C-terminus of the peptide) in the sequence, KNAFYERALSIINLMTSKMVQINV (p5-MT) was the most effective inhibitor of CDK5 kinase activity of 79 tested mimetic peptides including the original p5 peptide, KEAFWDRCLSVINLMSSKMLQINA (p5-WT). Replacement of the residues in C-terminus end of the peptide affected CDK5 phosphotransferase activity most significantly. These peptides were strong inhibitors of CDK5, but not the related proline-directed kinases, ERK1 and GSK3ß.

Peptide TFP5/TP5 derived from Cdk5 activator P35 provides neuroprotection in the MPTP model of Parkinson's disease.

Parkinson's disease (PD) is a chronic neurodegenerative disorder characterized by the loss of dopamine neurons in the substantia nigra, decreased striatal dopamine levels, and consequent extrapyramidal motor dysfunction. Recent evidence indicates that cyclin-dependent kinase 5 (Cdk5) is inappropriately activated in several neurodegenerative conditions, including PD. To date, strategies to specifically inhibit Cdk5 hyperactivity have not been successful without affecting normal Cdk5 activity. Previously we reported that TFP5 peptide has neuroprotective effects in animal models of Alzheimer's disease. Here we show that TFP5/TP5 selective inhibition of Cdk5/p25 hyperactivation in vivo and in vitro rescues nigrostriatal dopaminergic neurodegeneration induced by 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP/MPP+) in a mouse model of PD. TP5 peptide treatment also blocked dopamine depletion in the striatum and improved gait dysfunction after MPTP administration. The neuroprotective effect of TFP5/TP5 peptide is also associated with marked reduction in neuroinflammation and apoptosis. Here we show selective inhibition of Cdk5/p25 -hyperactivation by TFP5/TP5 peptide, which identifies the kinase as a potential therapeutic target to reduce neurodegeneration in Parkinson's disease.

The homeodomain transcription factor Phox2 in the stellate ganglion of the squid Loligo pealei.

Homeodomain transcription factors regulate development of embryos and cellular physiology in adult systems. Paired-type homeodomain genes constitute a subclass that has been particularly implicated in establishment of neuronal identity in the mammalian nervous system. We isolated fragments of eight homeodomain genes of this subclass expressed in the stellate ganglion of the North Atlantic long finned squid Loligo pealei (lp) [Note: Loligo pealei has been officially renamed Doryteuthis pealei. For reasons of uniformity and clarity Loligo pealei (lp) is used here]. Of the most abundant ones, we cloned a full length cDNA which encoded the squid ortholog of the paired-type homeodomain proteins Phox2a/b. The homology of lpPhox2 to invertebrate and mammalian Phox2 was limited to the homeodomain. In contrast to mouse Phox2b, lpPhox2 was unable to transactivate the dopamine beta-hydroxylase (DBH) promoter in a heterologous mammalian transfection system. In vivo, lpPhox2 was expressed in the developing stellate ganglion of stage 27 squid embryos and continued to be expressed in the adult stellate neurons where expression was confined to the giant fiber lobe containing the neurons that form the giant axons. The expression of lpPhox was similarly timed and distributed as the Fmrf gene. Furthermore, the Fmrf upstream region contained putative Phox2a/b binding sites. These results suggest a role of lpPhox2 in the developmental specification of neuronal identity and regulation of neurons of the squid giant axon.