John Ryder

The Parkinson’s disease kinase LRRK2 autophosphorylates its GTPase domain at multiple sites

Mutations in leucine-rich repeat kinase 2 (LRRK2) are a common cause of inherited Parkinsons disease (PD). The protein is large and complex, but pathogenic mutations cluster in a region containing GTPase and kinase domains. LRRK2 can autophosphorylate in vitro within a dimer pair, although the significance of this reaction is unclear. Here, we mapped the sites of autophosphorylation within LRRK2 and found several potential phosphorylation sites within the GTPase domain. Using mass spectrometry, we found that Thr1343 is phosphorylated and, using kinase dead versions of LRRK2, show that this is an autophosphorylation site. However, we also find evidence for additional sites in the GTPase domain and in other regions of the protein suggesting that there may be multiple autophosphorylation sites within LRRK2. These data suggest that the kinase and GTPase activities of LRRK2 may exhibit complex autoregulatory interdependence.

Regulation of amyloid precursor protein (APP) phosphorylation and processing by p35/Cdk5 and p25/Cdk5

The phosphorylation status of amyloid precursor protein (APP) at Thr668 is suggested to play a critical role in the proteolytic cleavage of APP, which generates either soluble APPβ (sAPPβ) and β‐amyloid peptide (Aβ), the major component of senile plaques in patient brains inflicted with Alzheimers disease (AD), or soluble APPα (sAPPα) and a peptide smaller than Aβ. One of the protein kinases known to phosphorylate APPThr668 is cyclin‐dependent kinase 5 (Cdk5). Cdk5 is activated by the association with its regulatory partner p35 or its truncated form, p25, which is elevated in AD brains. The comparative effects of p35 and p25 on APPThr668 phosphorylation and APP processing, however, have not been reported. In this study, we investigated APPThr668 phosphorylation and APP processing mediated by p35/Cdk5 and p25/Cdk5 in the human neuroblastoma cell line SH‐SY5Y. Transient overexpression of p35 and p25 elicited distinct patterns of APPThr668 phosphorylation, specifically, p35 increasing the phosphorylation of both mature and immature APP, whereas p25 primarily elevated the phosphorylation of immature APP. Despite these differential effects on APP phosphorylation, both p35 and p25 overexpression enhanced the secretion of Aβ, sAPPβ, as well as sAPPα. These results confirm the involvement of Cdk5 in APP processing, and suggest that p35‐ and p25‐mediated Cdk5 activities lead to discrete APP phosphorylation.

Neurochemical and behavioral profiling of the selective GlyT1 inhibitors ALX5407 and LY2365109 indicate a preferential action in caudal vs. cortical brain areas

Selective inhibitors of the glycine transporter 1 (GlyT1) have been implicated in central nervous system disorders related to hypoglutamatergic function such as schizophrenia. The selective GlyT1 inhibitors ALX5407 (NFPS) and LY2365109 {[2-(4-benzo[1,3]dioxol-5-yl-2-tert-butylphenoxy)ethyl]-methylamino}-acetic acid increased cerebrospinal fluid levels of glycine and potentiated NMDA-induced increases in dialysate levels of neurotransmitters in the prefrontal cortex (PFC) and the striatum. However, higher doses produced both stimulatory and inhibitory effects on motor performance and impaired respiration, suggesting significant involvement of cerebellar and brain stem areas. A dual probe microdialysis study showed that ALX5407 transiently elevated extracellular levels of glycine in the PFC with more sustained increases in the cerebellum. In support of these findings, immuno-staining with pan-GlyT1 and GlyT1a antibodies showed a higher abundance of immunoreactivity in the brain stem/cerebellum as compared to the frontal cortical/hippocampal brain areas in four different species studied, including the mouse, rat, monkey and human. In addition, the inhibitory effects of ALX5407 on cerebellar levels of cGMP in the mouse could be reversed by the glycine A receptor antagonist strychnine but not the glycine B receptor antagonist L-701324. We propose that the adverse events seen with higher doses of ALX5407 and LY2365109 are the result of high GlyT1 inhibitory activity in caudal areas of the brain with sustained elevations of extracellular glycine. High levels of glycine in these brain areas may result in activation of strychnine-sensitive glycine A receptors that are inhibitory on both motor activity and critical brain stem functions such as respiration.

Quantitative Measurement of Intact Alpha-Synuclein Proteoforms from Post-Mortem Control and Parkinson's Disease Brain Tissue by Intact Protein Mass Spectrometry

A robust top down proteomics method is presented for profiling alpha-synuclein species from autopsied human frontal cortex brain tissue from Parkinsons cases and controls. The method was used to test the hypothesis that pathology associated brain tissue will have a different profile of post-translationally modified alpha-synuclein than the control samples. Validation of the sample processing steps, mass spectrometry based measurements, and data processing steps were performed. The intact protein quantitation method features extraction and integration of m/z data from each charge state of a detected alpha-synuclein species and fitting of the data to a simple linear model which accounts for concentration and charge state variability. The quantitation method was validated with serial dilutions of intact protein standards. Using the method on the human brain samples, several previously unreported modifications in alpha-synuclein were identified. Low levels of phosphorylated alpha synuclein were detected in brain tissue fractions enriched for Lewy body pathology and were marginally significant between PD cases and controls (p = 0.03).

Inhibition of Aβ production and APP maturation by a specific PKA inhibitor

Alzheimers disease is characterized pathologically by extracellular amyloid β protein (Aβ) deposition in the brain. The Aβ peptide, a 39–42 amino acid fragment, is derived from defined proteolysis of the amyloid precursor protein (APP) [Glenner et al., Appl. Pathol. 2 (1984) 357–369; Selkoe, Neuron 6 (1991) 487–498] and is the primary component of senile plaques. Although it is known that intracellular APP is subjected to posttranslational modification, the molecular mechanism that regulates the APP processing is not completely clear. In the present study, we demonstrates that H89, a specific inhibitor for cAMP dependent protein kinase A (PKA), inhibits Aβ production and APP secretion in a dose dependent manner in cells stably transfected with human APP bearing a ‘Swedish mutation’. Concurrent with the effect, H89 inhibits C‐terminal fragment of the APP. We also found that the PKA inhibitor abolishes the mature form of intracellular APP and accumulates the immature form. Finally, direct administration of H89 into brains of transgenic mice overexpressing human APP shows that the compound inhibits Aβ production in the hippocampal region. Our data suggests that PKA plays an important role in the maturation of APP associated with APP processing.

Overexpression of GSK3βS9A Resulted in Tau Hyperphosphorylation and Morphology Reminiscent of Pretangle-Like Neurons in the Brain of PDGSK3β Transgenic Mice

It has been demonstrated that GSK3β is involved in Alzheimer Disease (AD) pathogenesis. In order to understand the underlying mechanism, we have generated and characterized transgenic mice in which the constitutively active human GSK3β(with S9A mutation) was overexpressed in the brain under the control of the platelet-derived growth factor (PDGF) B-chain promoter. Varying levels of human GSK3βS9A transgene protein expression was observed in six of the seven founders generated. Line 3083, 3107, 3112 and 3125 displayed higher GSK3βS9A protein expression levels. Immunostaining analysis demonstrated that transgene expression was observed mainly in cortex and hippocampus of transgenic brain. Expression of human GSK3β transgene did not significantly change the brain total GSK3β protein levels in any of the generated mouse lines, as comparing to age matched wild type mice. Although significant kinase activity was detected in human GSK3βS9A transgene protein extracted from brains of all six expressing lines, significant increase in total GSK3βS9A kinase activity was observed only in the offspring of line 3083 and 3107. By analyzing the offspring from several transgenic mouse lines, including lines other than 3083 and 3107, it was found that overexpressed constitutively active human GSK3βS9A resulted in hyperphosphorylation of tau and morphology reminiscent of pretangle-like neurons in cortex and hippocampus.

FRAT1 peptide decreases Aβ production in swAPP751 cells

Recently, LiCl has been shown to inhibit amyloid β peptide secretion in association with diminished glycogen synthase kinase β (GSK3β) activity. However, it remains unclear if direct inhibition of GSK3β activity will result in decreased Aβ production. requently earranged in dvanced ‐cell lymphomas (FRAT1) protein is a negative regulator of GSK3α/β kinase activity. To examine whether direct inhibition of GSK3α/β kinase activity can lower Aβ production, a FRAT1 peptide was expressed in swAPP751 cells that produce high levels of Aβ. Our data demonstrate that cellular expression of FRAT1 peptide in swAPP751 cells increases both GSK3α and β phosphorylation on Ser21 and Ser9, respectively, while inhibiting kinase activity of both isoforms. Moreover, as a result of FRAT1 expression, the production of both total Aβ and Aβ1‐42 was significantly decreased. Thus, we provide evidence that direct regulation of GSK3α/β by FRAT1 peptide significantly decreases Aβ production in swAPP751 cells.

Pharmacological Characterization of cGMP Regulation by the Biarylpropylsulfonamide Class of Positive, Allosteric Modulators of α-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic Acid Receptors

The biarylpropylsulfonamide class of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) potentiators represented by N-2-(4-(4-cyanophenol)phenol)propyl-2-propanesulfonamide (LY404187) and (R)-4′-[1-fluoro-1-methyl-2-(propane-2-sulfonylamino)-ethyl]-biphenyl-4-carboxylic acid methylamide (LY503430) are positive, allosteric AMPA receptor activators, which enhance AMPA receptor-mediated neurotransmission by reducing desensitization of the ion channel. Although these compounds have efficacy in in vivo rodent models of cognition, depression, and Parkinsons disease, little is known about biochemical pathways activated by these agents. Given the well established regulation of the nitric oxide/cGMP pathway by excitatory neurotransmission, the current study characterized AMPA receptor potentiator-mediated cGMP response in mouse cerebellum. Acute treatment by both LY404187 and LY503430 [2.0, 5.0, or 10 mg/kg subcutaneously (s.c.)] elevated basal cerebellar cGMP levels in a dose-dependent manner. Pretreatment with the noncompetitive, allosteric AMPA receptor-selective antagonist 7H-1,3-dioxolo[4,5-h][2,3]benzodiazepine-7-carboxamide, 5-(4-aminophenyl)-8,9-dihydro-N,8-dimethyl-monohydrochloride-(9CI) (GYKI 53655) [3.0 mg/kg intraperitoneally (i.p.)], completely blocked the effect of LY404187, demonstrating that activation of AMPA receptors induces cGMP levels. Interestingly, pretreatment with the N-methyl-d-aspartate (NMDA) open channel blocker dizocilpine (0.3 and 1.0 mg/kg i.p.) also abolished the AMPA receptor potentiator-mediated cGMP accumulation, indicating that activation of AMPA receptors leads to NMDA receptor-mediated transmission involved in cGMP regulation. Pharmacological augmentation of the endogenous glutamate tone via the alkaloid harmaline (20-60 mg/kg i.p.) synergized with AMPA potentiator activity and provided further direct evidence of in vivo allosteric activation of AMPA receptors by LY404187. The synergism between harmaline and LY404187 was specific, since cGMP accumulation induced by foot-shock stress was not augmented by the AMPA receptor potentiator. Taken together, these data indicate that the cGMP system may play an important role in pharmacological efficacy of the biarylpropylsulfonamide class of AMPA receptor potentiators.

RIFLE: A novel ring zinc finger‐leucine‐rich repeat containing protein, regulates select cell adhesion molecules in PC12 cells

Cell adhesion molecules play a critical role in cell contacts, whether cell–cell or cell–matrix, and are regulated by multiple signaling pathways. In this report, we identify a novel ring zinc finger‐leucine‐rich repeat containing protein (RIFLE) and show that RIFLE, expressed in PC12 cells, enhances the Serine (Ser)21/9 phosphorylation of glycogen synthase kinase‐3α/β (GSK‐3α/β) resulting in the inhibition of GSK‐3 kinase activity and increase of β‐catenin levels. RIFLE expression also is associated with elevated E‐cadherin protein levels but not N‐cadherin. The regulation of these cell adhesion‐associated molecules by RIFLE is accompanied by a significant increase in cell–cell and cell–matrix adhesion. Moreover, increase in cell–cell adhesion but not cell–matrix adhesion by RIFLE can be mimicked by selective inhibition of GSK‐3. Our results suggest that RIFLE represents a novel signaling protein that mediates components of the Wnt/wingless signaling pathway and cell adhesion in PC12 cells.

Preclinical profile of a dopamine D1 potentiator suggests therapeutic utility in neurological and psychiatric disorders

ABSTRACT DETQ, an allosteric potentiator of the dopamine D1 receptor, was tested in therapeutic models that were known to respond to D1 agonists. Because of a species difference in affinity for DETQ, all rodent experiments used transgenic mice expressing the human D1 receptor (hD1 mice). When given alone, DETQ reversed the locomotor depression caused by a low dose of reserpine. DETQ also acted synergistically with L‐DOPA to reverse the strong hypokinesia seen with a higher dose of reserpine. These results indicate potential as both monotherapy and adjunct treatment in Parkinsons disease. DETQ markedly increased release of both acetylcholine and histamine in the prefrontal cortex, and increased levels of histamine metabolites in the striatum. In the hippocampus, the combination of DETQ and the cholinesterase inhibitor rivastigmine increased ACh to a greater degree than either agent alone. DETQ also increased phosphorylation of the AMPA receptor (GluR1) and the transcription factor CREB in the striatum, consistent with enhanced synaptic plasticity. In the Y‐maze, DETQ increased arm entries but (unlike a D1 agonist) did not reduce spontaneous alternation between arms at high doses. DETQ enhanced wakefulness in EEG studies in hD1 mice and decreased immobility in the forced‐swim test, a model for antidepressant‐like activity. In rhesus monkeys, DETQ increased spontaneous eye‐blink rate, a measure that is known to be depressed in Parkinsons disease. Together, these results provide support for potential utility of D1 potentiators in the treatment of several neuropsychiatric disorders, including Parkinsons disease, Alzheimers disease, cognitive impairment in schizophrenia, and major depressive disorder. HIGHLIGHTSThe dopamine D1 potentiator DETQ was tested in humanized D1 mice and rhesus monkeys.Actions of DETQ were dependent on endogenous dopaminergic tone.DETQ displayed a behavioral profile consistent with central D1 receptor activation.Neurochemical actions of DETQ support potential pro‐cognitive effects.D1 potentiators show promise for Parkinsons disease and other CNS disorders.