Crosstalk between Cdk5 and GSK3β: Implications for Alzheimer's Disease

GSK3β and Cdk5 are the two kinases in the center of research on Alzheimer's disease (AD), involved in the pathological symptoms of AD, Aβ plaque formation, tau hyperphosphorylation and neurodegeneration. So far, both kinases have mostly been examined in isolation, leading to a schism of the research field into defenders of the GSK3β-versus the Cdk5 hypotheses of AD. However, in this debate the fact that activities of GSK3β and Cdk5 can influence each other deserves more attention. Recent evidence from p25 transgenic mice suggests that there is a dynamic crosstalk: during aging or prolonged overactivation of Cdk5, GSK3β activity may alter in favor of AD pathogenesis. In this review we summarize the connections between GSK3β and Cdk5 and discuss implications for AD hypotheses

PKMζ Differentially Utilized between Sexes for Remote Long-Term Spatial Memory

It is well established that male rats have an advantage in acquiring place-learning strategies, allowing them to learn spatial tasks more readily than female rats. However many of these differences have been examined solely during acquisition or in 24h memory retention. Here, we investigated whether sex differences exist in remote long-term memory, lasting 30d after training, and whether there are differences in the expression pattern of molecular markers associated with long-term memory maintenance. Specifically, we analyzed the expression of protein kinase M zeta (PKMζ) and the α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor subunit GluA2. To adequately evaluate memory retention, we used a robust training protocol to attenuate sex differences in acquisition and found differential effects in memory retention 1d and 30d after training. Female cohorts tested for memory retention 1d after 60 training trials outperformed males by making significantly fewer reference memory errors at test. In contrast, male cohorts tested 30d after 60 training trials outperformed females of the same condition, making fewer reference memory errors and achieving significantly higher retention test scores. Furthermore, given 60 training trials, females tested 30d later showed significantly worse memory compared to females tested 1d later, while males tested 30d later did not differ from males tested 1d later. Together these data suggest that with robust training males do no retain spatial information as well as females do 24h post-training but maintain this spatial information for longer. Males also showed a significant increase in synaptic PKMζ expression and a positive correlation with retention test scores, while females did not. Interestingly, both sexes showed a positive correlation between retention test scores and synaptic GluA2 expression. Furthermore, the increased expression of synaptic PKMζ, associated with male memory but not with female memory, identifies another potential sex-mediated difference in memory processing

Transcriptional analysis of sex differences in hippocampal plasticity in the mouse.

The neuronal representation of experience as stable memories requires a process termed consolidation, which engages the hippocampus. Sexual dimorphisms in the performance of a number of tasks requiring hippocampus-dependent memory formation have previously been described. These sex differences are generally attributed to gonadal hormone-mediated mechanisms which impact on neuroanatomy and modulate memory formation. At the molecular level, memory consolidation requires de novo transcription, activating the transcription factor CREB. This activation can be accomplished by a variety of signalling pathways including the CaM kinase cascade. Male mutant mice bearing a genetic deletion of CaMKK/, an element of this cascade, are impaired in spatial memory formation in the Morris water maze (MWM), and fail to activate CREB after spatial training. Remarkably, female mutants performed equally to their WT counterparts, indicating a sex-specific requirement for this kinase in spatial memory consolidation. This mutant line was used as a tool to investigate dimorphisms in the molecular mechanisms underlying memory formation. First, comparison of hippocampal transcriptional profiles between WT and CaMKK/ mutants by Affymetrix Microarray analysis identified four CaMKK/ regulated genes in males. Second, quantitative real-time PCR was used to compare hippocampal transcriptional profiles of these genes in naive males and females, and after training in two hippocampus-dependent tasks: the MWM and contextual fear conditioning (CFC). This study identified three genes with altered transcription thirty minutes after spatial training in the MWM and CFC in male mice: PSF, Gaa1 and SRp20. Naive females expressed lower levels of all three genes than naive males, and two of them (Gaa1 and SRp20) were not regulated specifically by training in these tasks at the same time point in females. The work described in this thesis has identified two male-specific molecular markers for hippocampal activity, and provided insights into sexual dimorphisms in the molecular mechanisms underlying memory consolidation

Biochemical analysis of genetically-modified mice with learning and memory phenotypes.

Recent advances in molecular genetics have enabled generation of sophisticated genetically-modified mouse models to study specific molecules and their biological function in vivo. Here, I investigated biochemical changes in two different genetically-modified mouse lines with previously described learning and memory phenotypes. Firstly, I analysed biochemical changes in a mouse line carrying a threonine to alanine point mutation at Thr286 of alpha Ca2+/calmoduline-dependent kinase II (aCaMKII), which disenables this phosphorylation site. Autophosphorylation at Thr286 switches aCaMKII into an autonomous activity mode. The T286A mutant mice displayed changes in basal phosphorylation levels. In order to study biochemical changes after activity-dependent synaptic potentiation, an in vivo long-term potentiation (LTP) approach was established and validated by assessing activity-dependent changes in phosphorylation levels of well-characterised marker molecules including synapsin I and NR2B. Both aCaMKII and pCaMKII exhibited elevated levels of autophosphorylation after LTP stimulation in hippocampal area CA1 and dentate gyrus (DG). This finding indicates that pCaMKII may compensate for the loss of autonomous aCaMKII activity in T286A mutants. Furthermore, induction of LTP triggered phosphorylation of glycogen synthase kinase 3 (GSK3) at its inhibitory site suggesting a role for GSK3 in synaptic plasticity. Secondly, I investigated a transgenic (TG) mouse line expressing the cyclin-dependent kinase (Cdk5) activator protein, p25, a protein previously linked to some aspects of Alzheimer's disease (AD). The forebrain restricted expression of p25 started postnatally and stayed constant throughout the life-span of the TG mice. The expression of p25 triggered constitutive over-activation of Cdk5 in the TG mice. The p25 TG mice displayed age-dependent hyperphosphorylation of the microtubule-associated protein tail and age-dependent alterations in the processing of amyloid precursor protein (APP). Furthermore, p25-induced over-activation of Cdk5 led to inhibition of GSK3. This negative regulation of GSK3 was lost in aged p25 TG mice and correlated with the increased tau hyperphosphorylation. The levels of tau phosphorylation in aged p25 mice were reduced after treatment with lithium, an inhibitor of GSK3. These results indicate that GSK3 directly mediates tau hyperphosphorylation, whereas Cdk5 acts indirectly via inhibitory control of GSK3

The role of mediators of neuronal plasticity in the circadian regulation of suprachiasmatic nucleus by light

Circadian rhythms are highly conserved physiological functions that are present in almost all living organisms. In mammals, circadian rhythms are synchronized to the environmental light:dark cycle by daily adjustments in the hypothalamic suprachiasmatic nucleus (SCN), the location of the master circadian pacemaker. We hypothesize that light entrainment of the circadian clock involves neural plastic adaptations in the SCN. The mechanism of neural plasticity has been intensively studied in the hippocampus and the dentate gyrus. However, the cell and molecular mechanism underlying circadian clock resetting in the SCN remains poorly understood. Thus, we sought to investigate whether modulators that are known to regulate neural plasticity in the hippocampus play a role in the signal transduction of circadian clock resetting. Light induced expression of tissue-type plasminogen activator (tPA) in the SCN, maximal induction was seen one hour following a light pulse at circadian time (CT) 16. A corresponding increase in the tPA proteolytic activity was also observed. tPA-STOPTM (an inhibitor of tPA)-infused animals exhibited attenuated light-induced phase delay of circadian wheel running activity. The levels of cyclin-dependent kinase 5 activators, p35 and p25 were decreased at Zeitgeber Time (ZT) 16, at ZT22 the levels were increased whereas, no change was observed at ZT6. The bi-transgenic animal, CK-p25 demonstrated increased phase delay at CT16 and attenuated phase advance at CT22 following transient overexpression of p25. SCN neurons expressing p25 co-localized with phosphorylated-extracellular signal-regulated kinase and Gastrin Releasing Peptide. This is one of the first studies to report the involvement of these neuromodulators in circadian light entrainment. Mounting evidence shows that circadian rhythm disturbances may be associated with increased health risks, such as jet-lag, cancer development, cardiovascular and metabolic disorders. The findings of this study have improved our understanding of the complex and intricate pathways involved in light entrainment and may lead to development of novel therapeutic avenues in treating circadian rhythm disturbances.  Ph.D

Microglia Activity in the Mouse Brain Lacking Prostaglandin E2-Connection to Autism

Prostaglandin E2 (PGE2) is a signaling molecule produced by cyclooxygenase2 (COX-2) that is important for brain development and microglial activation. Abnormal COX2/PGE2 signaling has been linked to Autism Spectrum Disorders (ASD). We aim to determine the impact of abnormal signaling of the COX-2/PGE2 pathway on microglial density and morphology in the developing brain of COX-2-KI mice. Using immunohistochemistry, we show at embryonic day 19 (E19) and postnatal day 25 (P25) COX-2-KI mice had sex and regional specific differences in microglial density, activation state, branch length, and branching networks. We show for the first time that these trajectories were influenced not only by the COX-2 deficiency, but also by sex, brain region, and developmental stage. These novel findings provide additional evidence that abnormal COX-2/PGE2 signaling can result in a disruption of microglial density and morphology during development and contribute to brain pathologies that result in ASDs

Dual role of CDK5 on cognitive deficits and striatal vulnerability in Huntington’s disease

[eng] Huntington’s disease (HD) is a neurodegenerative disorder caused by an autosomic mutation on the Huntingtin (HTT) coding gene. HD is mainly characterized by the appearance of motor symptoms or choreas, which are associated to the selective degeneration of striatal neurons, and by the presence of cognitive disturbances, which are attributed to alterations in corticostriatal connectivity and to hippocampal dysfunction. For this reason, finding targets involved both on striatal vulnerability and cognitive disturbances, might result in therapeutic strategies able to act simultaneously on HD’s motor and cognitive symptoms. In this Thesis we have focused on Cyclin-dependent kinase 5 (Cdk5) as one of these putative targets. Cdk5 acts mainly in the central nervous system, where its activator p35 is expressed, and it plays a major role on synaptic plasticity regulation. In addition, altered Cdk5 activity has been described in several neurodegenerative disorders, including HD, where Cdk5 deregulation has been associated to increased striatal vulnerability to excitotoxicity. Moreover, alteration of Cdk5 activity and/or subcellular distribution has also been linked to neuronal cell cycle re-entry, which has been proposed as a possible mechanism leading to neuronal dysfunction and eventual death in several neurodegenerative conditions. Therefore, on one hand, we aimed to study Cdk5 involvement in cognitive deficits and synaptic plasticity alterations in HD. To this end, we generated a new double mutant mice model which expresses one copy of mutant HTT (mHTT) (knock-in or KI), and is conditionally heterozygous for Cdk5 (Cdk5+/). We described that double mutant mice (KI:Cdk5+/-) presented restored corticostriatal and hippocampal cognitive function when compared to their KI littermates. We also observed that preserved corticostriatal function correlated with recovery of corticostriatal NR2B surface levels, which were reduced in KI mice. Moreover, recovery of NR2B surface levels was associated to normalization of NR2B total levels and of the pSrc/pNR2B pathway in the cortex of KI:Cdk5+/- mice. On the other hand, preserved hippocampal cognitive function correlated with recovery of CA1 dendritic spine density, as well as, with increased Rac1 activity in KI:Cdk5+/- mice. Restoration of dendritic spine density was also observed in layer V cortical neurons, in a Rac1-independent manner. Finally, we described that KI mice showed reduced physiological p35 plasma membrane levels in the cortex, which was recovered in KI:Cdk5+/- mice, correlating with preferential alteration of Cdk5 substrates phosphorylation levels in this brain region. In sum, our results demonstrate Cdk5 complex and brain region-specific involvement in cognitive deficits appearance and in synaptic alterations in HD. On the other hand, we also assessed whether Cdk5 deregulation might cause cell cycle re-entry of striatal neurons in HD. Cdk5 forms a nuclear complex with p27 and E2F1 in differentiated neurons, thus preventing E2F1 from binding to its coactivator DP1 and from activating transcription of cell cycle progression genes. For this reason, we analysed nuclear levels of Cdk5 and p27, and we observed that KI mice showed reduced Cdk5 and p27 nuclear levels, which could induce neuronal cell cycle re-entry. In agreement, we also observed increased levels of CyclinD1 in the striatum of KI mice since early symptomatic stages, and increased Cdk4 levels at late disease stages. Finally, we observed that NMDA treatment of striatal primary cultures caused a general reduction of cell cycle proteins neuronal expression, and importantly, it altered their subcellular distribution, reducing nuclear localization of the cell cycle inhibitor p27 and inducing nuclear presence of cell cycle progression proteins, E2F1 and Cdk4. Our results also suggested that presence of mHTT might further potentiate NMDA-induced subcellular distribution alteration of cell cycle proteins. Therefore, we suggest that reduction of Cdk5 nuclear levels might induce cell cycle re-entry of striatal neurons, a process which could be favoured by alterations in NMDA receptors activation, present in HD.[cat] La malaltia de Huntington (MH) és un desordre neurodegeneratiu causat per una mutació al gen que codifica per la proteïna Huntingtina (HTT), i que consisteix principalment en l’aparició de dèficits motors, associats a la degeneració selectiva de l’estriat; i en l’aparició de dèficits cognitius, associats a una alteració en la connectivitat corticoestriatal i a una disfunció hipocampal. En aquesta Tesi, hem analitzat la implicació de la cinasa Cdk5, per una banda, en l’aparició dels dèficits cognitius; i per l’altre banda, en la reentrada neuronal al cicle cel·lular com a un possible mecanisme de susceptibilitat a la vulnerabilitat estriatal en la MH. Els nostres resultats han mostrat que la reducció genètica de Cdk5 en un model murí de la MH (KI), prevé l’aparició dels dèficits cognitius corticoestriatal i hipocampals. Aquesta millora cognitiva està associada a la recuperació dels nivells de membrana de NR2B a nivell corticoestriatal, i a la restauració de la densitat d’espines dendrítiques a l’hipocamp i a l’escorça, indicant una implicació de Cdk5, complexa i específica de regió cerebral, en les alteracions sinàptiques i l’aparició dels dèficits cognitius en la MH. D’altre banda, hem observat que els nivells nuclears de Cdk5 estan disminuïts a l’estriat dels ratolins KI, cosa que podria alterar la seva funció com a inhibidor de la progressió del cicle cel·lular en neurones diferenciades. En concordança amb aquesta hipòtesi, diferents proteïnes del cicle cel·lular presenten una alteració en els seus nivells proteics, tant en ratolins KI, com en mostres de pacients humans. A més, l’activació dels receptors NMDA en neurones estriatals porta a una alteració de la distribució subcel·lular de les proteïnes del cicle cel·lular prèviament analitzades, un efecte que podria ser potenciat per la presència de la HTT mutada. En conclusió, els resultats d’aquesta Tesi, mostren la complexa implicació de Cdk5 en l’aparició dels dèficits cognitius en la MH, i suggereixen que l’alteració de la localització nuclear de Cdk5 podria portar a la desregulació de diferents proteïnes del cicle cel·lular, un mecanisme que es podria veure afavorit per alteracions en l’activació dels receptors NMDA, presents en la MH

Decreased locomotor activity in mice expressing tTA under control of the CaMKIIΑ promoter

Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/72144/1/j.1601-183X.2007.00339.x.pdfhttp://deepblue.lib.umich.edu/bitstream/2027.42/72144/2/GBB339Figs_S1-3.pdfhttp://deepblue.lib.umich.edu/bitstream/2027.42/72144/3/GBB_339_sm_FigureS1-3.pd

Characterisation of transgenic mouse models of Alzheimer's disease

A number of transgenic mouse models have been developed to study the molecular and pathological alterations associated with Alzheimer’s disease (AD). The 3xTg mouse is widely used as a research tool and carries mutations in the amyloid precursor protein (Swedish APP K670N/M671L), presenilin 1 (PS1M146V) and tau (TauP301L) which results in the development of pathological features simlar to the plaques and tangles observed in human AD. The TASTPM mouse carries both the APP K670N/M671L and PS1M146V mutations but does not possess a tau transgene, so develops only plaque-like structures in the brain. This thesis aims to systematically characterise biochemical, electrophysiological and behavioural changes present in the 3xTg and TASTPM mouse models of AD. The widely studied amyloid cascade hypothesis proposes that the generation of Aß through abnormal APP processing is a key initiating process in AD, and so molecular or electrophysiological changes which are observed in both models could represent a common pathway of disease development. In addition, comparison between the two models could help to elucidate the role of the tau transgene in early phenotypic changes. The studies of hippocampal electrophysiology presented in this thesis show that the marked deficits in long-term potentiation (LTP) originally reported at the age of 6 months (Oddo et al., 2003) are not present in our colony of 3xTg mice. In support of this, although these mice do overexpress APP and tau the expected hyperphosphorylation of tau is not observed even at the advanced ages of 12-17 months. This suggests that some of the processes associated with the development of pathological features are occurring more slowly in our colony. In addition, studies using a T-maze paradigm in 6 month 3xTg mice suggest that major cognitive deficits are not present at this age. This suggests that drift of the phenotype has occured in the 3xTg mouse and has implications for further studies using this model. The 3xTg mouse does, however, present a deficit in basal synaptic transmission which is progressive with increasing age from 6-17 months. Similarly, hippocampal synaptic function is normal in TASTPM mice studied at 2 months, when no biochemical changes are present, but is markedly reduced at the age of 6 months when it proved difficult to make any electrophysiological recordings. The data in this thesis shows that treatment with 1mM kynurenic acid during the slicing process markedly improved baseline synaptic transmission to the level observed in control mice. This shows that kynurenic acid can improve the viability of the slices, and as the compound is a glutamate receptor antagonist, suggests that reduction of glutamate-induced excitotoxicity during the slicing process results in its neuroprotective effects. This data suggests that alterations common to the 3xTg and TASTPM models, and therefore due to the presence of the APP or PS1 transgenes, may result in an increased susceptibility of hippocampal neurons to cellular stressors such as excitotoxicity. To summarise, this thesis presents data which characterises in detail aspects of the electrophysiological, biochemical and behavioural phenotype of the 3xTg and TASTPM mouse models of AD, with the aim of observing early changes which may be associated with the mechanisms of AD development.EThOS - Electronic Theses Online ServiceGBUnited Kingdo

Characterisation of transgenic mouse models of Alzheimer's disease

A number of transgenic mouse models have been developed to study the molecular and pathological alterations associated with Alzheimer’s disease (AD). The 3xTg mouse is widely used as a research tool and carries mutations in the amyloid precursor protein (Swedish APP K670N/M671L), presenilin 1 (PS1M146V) and tau (TauP301L) which results in the development of pathological features simlar to the plaques and tangles observed in human AD. The TASTPM mouse carries both the APP K670N/M671L and PS1M146V mutations but does not possess a tau transgene, so develops only plaque-like structures in the brain. This thesis aims to systematically characterise biochemical, electrophysiological and behavioural changes present in the 3xTg and TASTPM mouse models of AD. The widely studied amyloid cascade hypothesis proposes that the generation of Aß through abnormal APP processing is a key initiating process in AD, and so molecular or electrophysiological changes which are observed in both models could represent a common pathway of disease development. In addition, comparison between the two models could help to elucidate the role of the tau transgene in early phenotypic changes. The studies of hippocampal electrophysiology presented in this thesis show that the marked deficits in long-term potentiation (LTP) originally reported at the age of 6 months (Oddo et al., 2003) are not present in our colony of 3xTg mice. In support of this, although these mice do overexpress APP and tau the expected hyperphosphorylation of tau is not observed even at the advanced ages of 12-17 months. This suggests that some of the processes associated with the development of pathological features are occurring more slowly in our colony. In addition, studies using a T-maze paradigm in 6 month 3xTg mice suggest that major cognitive deficits are not present at this age. This suggests that drift of the phenotype has occured in the 3xTg mouse and has implications for further studies using this model. The 3xTg mouse does, however, present a deficit in basal synaptic transmission which is progressive with increasing age from 6-17 months. Similarly, hippocampal synaptic function is normal in TASTPM mice studied at 2 months, when no biochemical changes are present, but is markedly reduced at the age of 6 months when it proved difficult to make any electrophysiological recordings. The data in this thesis shows that treatment with 1mM kynurenic acid during the slicing process markedly improved baseline synaptic transmission to the level observed in control mice. This shows that kynurenic acid can improve the viability of the slices, and as the compound is a glutamate receptor antagonist, suggests that reduction of glutamate-induced excitotoxicity during the slicing process results in its neuroprotective effects. This data suggests that alterations common to the 3xTg and TASTPM models, and therefore due to the presence of the APP or PS1 transgenes, may result in an increased susceptibility of hippocampal neurons to cellular stressors such as excitotoxicity. To summarise, this thesis presents data which characterises in detail aspects of the electrophysiological, biochemical and behavioural phenotype of the 3xTg and TASTPM mouse models of AD, with the aim of observing early changes which may be associated with the mechanisms of AD development.EThOS - Electronic Theses Online ServiceGBUnited Kingdo