Daniel TS Pak - US grants

[unreadable] DESCRIPTION (provided by applicant): The proper development of neuronal connections depends on precisely controlling the balance between formation and elimination of synapses. These opposing processes also play important roles in synaptic plasticity, learning and memory. Compared to the current wealth of data regarding synaptogenesis, however, relatively little is known about the molecular events underlying the loss of central synapses. We have recently identified a novel mechanism of synapse loss centered on the serum-inducible kinase (SNK), a serine-threonine protein kinase of the polo family. In cortex and hippocampus, the short-lived SNK is induced by synaptic activity and subsequently promotes loss of excitatory synapses and dendritic spines (the primary loci of excitatory synapses in the CMS). SNK exerts its effect on synapses via the phosphorylation- and ubiquitin-dependent degradation of specific postsynaptic substrates such as SPAR, an important morphogen for dendritic spines. Because SNK regulates the number, structure and composition of synapses and spines, this kinase is likely to be important for shaping the long-term functional properties of neurons. We have identified an additional potential substrate of SNK, the abundant postsynaptic Ras regulator SynGAP. Ras signaling is of critical importance for a wide variety of neurobiological processes including synaptic plasticity, development, and protection from excitotoxic insults. Biochemical and molecular approaches will be employed in Aim 1 to determine whether SynGAP and SNK physically interact and whether SynGAP is a direct phosphorylation substrate of SNK. In Aim 2 we will analyze the functional relationship between SNK and SynGAP in vitro and determine whether SNK regulates SynGAP and Ras in neurons. Finally, the role of a putative SNK/SynGAP/Ras regulatory network in regulating dendritic spine morphology will be addressed in Aim 3. The experiments described in this proposal are essential for understanding the mechanisms of SNK action and may have broad impact in the fields of synaptic plasticity, synapse development, and pathological synapse loss. Elucidating these pathways is likely to be of clinical significance and highly relevant to public health in view of the fact that neocortical synapse loss is a hallmark of human neurodegeneration and is the major correlate of cognitive decline in many forms of dementia. [unreadable] [unreadable]

[unreadable] DESCRIPTION (provided by applicant): The proper development of neuronal connections depends on precisely controlling the balance between formation and elimination of synapses. These opposing processes also play important roles in synaptic plasticity, learning and memory. Compared to the current wealth of data regarding synaptogenesis, however, relatively little is known about the molecular events underlying the loss of central synapses. We have recently identified a novel mechanism of synapse loss centered on the serum-inducible kinase (SNK), a serine-threonine protein kinase of the polo family. In cortex and hippocampus, the short-lived SNK is induced by synaptic activity and subsequently promotes loss of excitatory synapses and dendritic spines (the primary loci of excitatory synapses in the CMS). SNK exerts its effect on synapses via the phosphorylation- and ubiquitin-dependent degradation of specific postsynaptic substrates such as SPAR, an important morphogen for dendritic spines. Because SNK regulates the number, structure and composition of synapses and spines, this kinase is likely to be important for shaping the long-term functional properties of neurons. We have identified an additional potential substrate of SNK, the abundant postsynaptic Ras regulator SynGAP. Ras signaling is of critical importance for a wide variety of neurobiological processes including synaptic plasticity, development, and protection from excitotoxic insults. Biochemical and molecular approaches will be employed in Aim 1 to determine whether SynGAP and SNK physically interact and whether SynGAP is a direct phosphorylation substrate of SNK. In Aim 2 we will analyze the functional relationship between SNK and SynGAP in vitro and determine whether SNK regulates SynGAP and Ras in neurons. Finally, the role of a putative SNK/SynGAP/Ras regulatory network in regulating dendritic spine morphology will be addressed in Aim 3. The experiments described in this proposal are essential for understanding the mechanisms of SNK action and may have broad impact in the fields of synaptic plasticity, synapse development, and pathological synapse loss. Elucidating these pathways is likely to be of clinical significance and highly relevant to public health in view of the fact that neocortical synapse loss is a hallmark of human neurodegeneration and is the major correlate of cognitive decline in many forms of dementia. [unreadable] [unreadable]

Regulating synaptic activity within a suitable working range is important for the stability of neuronal function. Homeostatic synaptic plasticity (HSP) utilizes compensatory feedback mechanisms to combat excessively low or high firing rates despite fluctuations in neuronal input, but underlying molecular pathways are not well understood. We discovered that chronic inactivity induces the formation of giant excitatory synapses specifically in proximal dendrites, with no morphological changes detected at distal synapses. These enlarged proximal dendritic structures were composed of complex clusters of synapses arranged on unusually large and elaborate dendritic spine-like protrusions, and were enriched for AMPARs and N-type calcium channels, but not NMDA receptors. Taken together, these properties were reminiscent of [unreadable]thorny excrescences,[unreadable] large branched dendritic spines of unclear function on proximal dendrites of hippocampal CA3 pyramidal neurons and mossy cells in vivo. Thus, our overarching hypotheses are that complex synapses represent the in vitro correlates of thorny excrescences, and that thorny excrescences therefore act as homeostatic control devices in independently tunable proximal dendritic zones. In this proposal, we will pursue the following Specific Aims: 1) Directly test homeostatic regulation of thorny excrescence formation in cultured neurons, hippocampal slices, and in vivo, and examine the ultrastructure of in vitro excrescences;2) Investigate the molecular mechanisms that regulate thorny excrescence formation, focusing in particular on the role of Cav2.2 [unreadable] Stargazin interaction as an anchor for AMPA receptor delivery and the role of CaMKIIb in promoting assembly of this ternary complex;and 3) Analyzing the functional properties of proximal dendritic cluster synapses using electrophysiological techniques. The results obtained will be important for understanding neuronal responses to extreme changes in synaptic activity levels, and will have clinical significance for various neurological disorders involving aberrant brain activity.

? DESCRIPTION (provided by applicant): Alzheimer's disease (AD) is the most common form of dementia. Currently, there are no treatments that can reverse the underlying disease progression, and thus new strategies and treatments are essential. Accumulation of amyloid beta (Abeta), a proteolytic cleavage product of amyloid precursor protein (APP), is considered the initiating step leading to cognitive impairment and neuronal cell death, but the signaling pathways that control proteolytic processing of APP are not well understood. Accumulating evidence implicates synaptic activity as a key driver of APP amyloidogenic processing, and hyperexcitation is a characteristic feature of the AD brain during early stages. However, the molecular links between neuronal activity and Abeta production are unclear. Plk2 is a synaptic activity-inducible member of the polo-like kinase (Plk) family that plays an important role in homeostatic weakening of excitatory synapses in response to prolonged overexcitation. Our preliminary data demonstrate that Plk2 is upregulated in AD brain, directly phosphorylates APP and is required for neuronal activity-dependent APP amyloidogenic processing in vitro. Additionally, genetic inhibition of Plk2 reduced soluble Abeta levels and plaque deposition in AD model mice under basal conditions and eliminated the increase in Abeta formation in response to heightened synaptic activity. Finally, administration of a Plk inhibitor, BI-6727 (volasertib), o AD model mice also led to a marked reduction in soluble Abeta levels. In this proposal we will test the hypotheses that (1) BI-6727 (volasertib) reduces plaque deposition and ameliorates cognitive impairment and other pathological features in the 5xFAD mouse model of AD; and (2) Plk2 plays a selective role among Plk family members in APP processing, and functions in response to moderate and well as strong neuronal hyperactivity as evoked by graded optogenetic stimulation. This proposal is highly significant as it will help elucidate a novel mechanistic link between excitatory synaptic activity and APP processing. The identification of this regulatory pathway for APP processing presents Plk2 as an innovative and potentially attractive target for therapeutic interventions in AD.

Alzheimer's disease (AD) is the most common neurodegenerative disorder. There are no existing therapies that can reverse disease progression, and thus new approaches are urgently needed. Accumulation of amyloid beta (A?), a proteolytic cleavage product of amyloid precursor protein (APP), is considered the initiating step in pathogenesis, but the signaling pathways that control this process are not well defined. Synaptic activity is thought to be a key driver of APP amyloidogenic processing, but the molecular links between neuronal activity and A? production are unclear. An attractive candidate is Plk2, a synaptic activity-inducible member of the polo-like kinase (Plk) family that functions in homeostatic synaptic plasticity to weaken synapses in response to prolonged overexcitation. Plk2 is upregulated in AD brain, directly phosphorylates APP and is required for APP amyloidogenic processing in response to chronic hyperexcitation in vitro. Importantly, inhibition of Plk2 (using transgenic dominant negative interference or a small molecule Plk inhibitor) shows encouraging evidence of slowing pathogenesis in AD model mice. The physiological function of APP has remained elusive, but as a Plk2 substrate, APP may play a role in synaptic depression. We identified Plk2 phosphorylation sites within APP C-terminus that was critical for downregulation of AMPA receptors during homeostatic synaptic plasticity. Interestingly, distinct APP phosphorylation sites are required for AMPA receptor internalization during a different form of plasticity, NMDA-receptor dependent long term depression (LTD). In this proposal we will test the hypotheses that (1) pharmacological inhibition of Plk2 using 2 different small molecule inhibitors slows disease progression in mouse models of AD; (2) Plk2 plays a selective role among Plk family members in activity-regulated APP processing, as determined by analysis of conventional and conditional knockout mice for Plk1-3; and 3) APP is centrally involved in synaptic depression of AMPA receptors at excitatory synapses under diverse plasticity paradigms, triggered by different kinases (Plk2 or GSK3) signaling through a combinatorial ?phosphorylation code? in the APP C-terminus. This proposal is highly significant as it will help elucidate novel physiological mechanisms that link different forms of synaptic activity to APP amyloidogenic processing. These studies will advance basic understanding of APP function and synaptic plasticity, while uncovering and validating novel targets for therapeutic interventions in AD.

The profound loss of basal forebrain cholinergic neurons (BFCNs) is an early hallmark in Alzheimer?s disease (AD). As cholinergic innervation is essential for cognition, degeneration of BFCNs may be linked to mental decline in AD patients. Current AD therapies involving cholinergic drugs provide modest benefits but are not based on disease mechanisms and do not halt BFCN degeneration. The reasons for the vulnerability of BFCNs to cell death in AD are largely unknown, but BFCN loss predicts degeneration in cortex, and cholinesterase inhibitors reduce atrophy in basal forebrain as well as cortex and hippocampus. These observations support the premise that protection of BFCNs could slow pathogenesis in AD. Thus, there is an urgent need to identify molecular mechanisms of cell death in BFCNs. In this proposal, we will investigate molecular events associated with BFCN dysfunction. We focus on neuronal hyperexcitability, which is a prominent, early feature in AD patients linked to cognitive deficits. Hyperactivity induces homeostatic synaptic plasticity (HSP), a compensatory mechanism that tunes synaptic strength in response to perturbations in neuronal activity, thereby maintaining excitation within an optimal range and preserving network stability. However, little is known regarding HSP in mammalian CNS cholinergic synapses, in normal conditions or in AD models. We will test the hypothesis that hyperexcitation and HSP mechanisms exacerbate AD pathogenesis. Furthermore, we propose that BFCNs, which are highly vulnerable and affected early in AD, provide a sensitive readout for detecting such dysfunctions. We propose the following aims: 1) Using an optimized septal-hippocampal co-culture system, we will examine the course of normal BFCN and cholinergic synapse development; determine morphological and functional changes that occur in cholinergic neurons and synapses during overexcitation conditions; and utilize similar co-cultures prepared from an AD mouse model to examine the perturbations to BFCNs in their normal development, response to hyperexcitation, and susceptibility to distinct forms of cell death. 2) We will analyze BFCNs and target hippocampal neurons in vivo with multidisciplinary approaches to examine the homeostatic responses to hyperexcitation, and use ChAT-Cre mice crossed to an AD mouse model to identify impairments in BFCN structure or synaptic function, under both basal and hyperexcitation conditions. These significant studies use innovative technology to investigate questions of basic and translational importance. If successful, the findings may lead to improved therapies against BFCN neurodegeneration in AD.

Alzheimer?s disease (AD) is a neurodegenerative disorder characterized by plaques comprised of A?, and neurofibrillary tangles (NFTs) containing the microtubule associated protein tau. Tau pathology closely correlates with neuronal degeneration and cognitive deficits. As the loss of tau protects against A?-induced neurotoxicity, tau is thought to act as a pathogenic downstream effector of A? to induce neuronal injury. Furthermore, aggregated tau can propagate in a prion-like manner to initiate a self-perpetuating toxic cascade. For these reasons, tau-directed approaches and novel mechanisms of treatment are essential. Hyperphosphorylation of tau is a consistent feature of all tauopathies, suggesting it may be obligatory step in pathogenesis and hence a rational target of modulation. Phosphorylation dissociates tau from microtubules and promotes tau aggregation into paired helical filaments that further accumulate to form NFTs. Tau is natively unfolded, and under normal conditions has little tendency to aggregate; therefore hyperphosphorylated tau in NFTs is a prominent sign of neurodegeneration, and understanding the etiology and pattern of phosphorylated tau is essential. A complication in this analysis is that there are over 80 potential tau phosphorylation sites. Due to the complexity of this problem, tau ?hyperphosphorylation? is not precisely defined. Additionally, tau plays important roles during both associative and homeostatic forms of synaptic plasticity, but little is known regarding the phosphorylation events involved in these pathways. Homeostatic responses to hyperexcitation are of particular interest as this type of aberrant overactivity is observed in early stage AD and may be relevant for the initiation of pathogenesis. Here, we will use unbiased mass spectrometry (MS) to perform comprehensive mapping of tau phosphorylation patterns during specific physiological conditions as well as in disease models. Compiling phosphomaps into an ?atlas? of tau modifications will begin to decipher the phosphorylation code that governs tau function in physiology and pathology. In Aim 1, we will use qualitative and quantitative MS approaches to define tau phosphorylation patterns during different forms of synaptic plasticity in cultured hippocampal neurons, examining a time course after each paradigm to observe time-dependent changes. We will also use specific kinase inhibitors with the above stimulation protocols to facilitate identification of endogenous tau kinases and signaling pathways. In Aim 2, we will use MS to identify tau phosphorylation patterns in a humanized double knock-in model of familial AD. As a primary tauopathy model of frontotemporal dementia, we will use P301L-tau in the background of humanized tau knock-in mice. We will examine different ages to understand the earliest tau modifications and profile of disease progression, as well as male vs. female mice to elucidate sex differences in disease severity. Defining the physiological regulation and phosphorylation code of tau is highly significant for understanding basic mechanisms involved in synaptic plasticity. Furthermore, this work will help guide new therapeutic approaches for targeting pathogenic tau in AD and other tauopathies.