Todd C. Sacktor - US grants

protein transport; protein kinase C; synapses; neurotransmitters; isozymes; hippocampus; phorbols; membrane proteins; diacylglycerols; brain electronic stimulator; dentate gyrus; brain electrical activity; brain mapping; calcium; genes; neurons; immunoprecipitation; immunofluorescence technique; Gastropoda; laboratory rat; western blottings; immunocytochemistry;

DESCRIPTION (provided by applicant): Long-term changes in synaptic strength are thought to contribute to the cellular basis of memory. These changes can be divided into two phases: a brief induction, triggering the modification, and a persistent maintenance, sustaining it over time. We have found a new form of atypical PKC, called PKMzeta, increases during the maintenance of long-term potentiation (LTP), a widely studied, putative cellular model of memory. PKC is usually held in an inactive state because its regulatory domain inhibits its catalytic domain; second messengers activate the enzyme by transiently releasing this autoinhibition. In contrast, PKMzeta is an independent PKC catalytic domain and, lacking the inhibition from a regulatory domain, is autonomously active. We found that increased function of PKMzeta is sufficient to enhance synaptic transmission when the kinase is introduced into CA1 pyramidal cells. The persistently increased activity of PKMzeta is also necessary for maintenance, because specific inhibitors of PKMzeta reverse established LTP. Our overall goal then is to elucidate the physiological mechanisms that increase PKMzeta function in LTP maintenance. The 1st aim is to examine posttranslational mechanisms for increasing PKMzeta function. Preliminary data suggest that PKMzeta is phosphorylated through the PI3-kinase/PDKl pathway. Using phosphorylation state-specific antisera to PKMzeta, we will test the hypotheses that this modification increases PKMzeta's function in LTP and dephosphorylation of this site decreases its activity in LTD. The 2nd aim is to determine the translational mechanisms for increasing PKMzeta function. PKM, the independent catalytic domain of PKC, is usually thought of as a proteolytic fragment of PKC. Our preliminary data, however, show that brain PKMzeta, is formed physiologically, not by proteolysis, but by translation of a brain-specific PKMzeta mRNA. This PKMzeta, mRNA is targeted to dendrites, and we will examine the regulation of increased local translation by the MAP-kinase and rapamycin-sensitive pathways. The 3rd aim examines the transcriptional mechanisms for increasing PKMzeta function. New data indicate that increased PKMzeta function is critical for late LTP. We find that an internal promoter within the PKCS, gene transcribes the PKMzeta mRNA. A long 5'untranslated region (5'UTR) on some PKMzeta mRNAs inhibits its translation. Regulation of the PKMzeta transcription start site during LTP may produce PKMC, mRNAs with shorter 5'UTRs, thus increasing the message's translational capacity and providing a long-term mechanism for maintaining increased PKMzeta function. These 3 goals aim to understand in mechanistic detail the physiological increase of PKMzeta function in LTP maintenance, which might help provide a unifying framework for the complex signaling pathways of memory.

DESCRIPTION (Adapted from applicant's abstract): PKC has been implicated in both short-term and long-term forms of synaptic plasticity which are believed to contribute to the physiological mechanisms of memory, and thus to behavioral and mental states. One widely held hypothesis for the persistence of changes in synaptic strength is that enzymes, such as PKC, which can enhance synaptic transmission in the short-term, become persistently active in the long-term. We have examined the regulation of PKC following a high-frequency tetanus of afferents in the CA1 region of the hippocampus that produces a long-term synaptic enhancement, LTP. We have observed that during LTP, PKC becomes persistently active through the proteolytic removal of the enzyme's autoinhibitory regulatory domain, increasing the level of the free, independently active catalytic fragment, termed PKM. This proteolytic activation, however, is specifically directed towards the ayptical zeta isoform of the enzyme, only one of the 10 isozymes of PKC. In addition, we have observed that low-frequency stimulation of afferents that produces LTD decreases the level of PKMzeta by proteolytic downregulation. The overall goal of this grant is to investigate how the unique properties of a typical PKCzeta, working in combination with other signal transduction pathways, regulate proteolytic processing to produce increases or decreases in PKMzeta during LTP and LTD, respectively. We will use a combination of biochemical and physiological techniques, both in vitro and in the hippocampal slice preparation, to address the following 3 Specific Aims: (1) to develop an assay to study the proteolytic processing of zeta. Preliminary results in vitro indicate that the calcium-dependent protease, calpain, can either form or degrade PKMzeta, depending upon the site of proteolytic cleavage on zeta. (2) To characterize the mechanisms that modulate this proteolytic processing. We have observed that the atypical isoform zeta, but not the conventional isoforms of PKC, has 2 potential CaM-kinase II phosphorylation sites in its catalytic domain. We will examine whether these, or other forms of post-translational modification of zeta, regulate the accessibility of zeta's cleavage sites to determine the direction of the change in PKMzeta. (3) To determine the functional consequences of the proteolytic activation of zeta on the long-term modulation of synaptic transmission. We have found that zeta phosphorylation can be isolated by the application of specific combinations of pharmacological agents. We will use these combinations in hippocampal slices to isolate the role of zeta phosphorylation during the maintenance of long-term synaptic plasticity. These specific aims will help to achieve a detailed molecular and functional analysis of the proteolytic regulation of PKMzeta that causes both increases and decreases of constitutively active PKC during bidirectional synaptic plasticity.

Long-term memories are believed to be due to persistent changes in synaptic strength. Although the molecular mechanisms initiating these changes have been extensively studied, the mechanisms maintaining these changes, which may contribute to storing long-term memory, have been unknown. Recently, however, a candidate molecular mechanism has emerged for maintaining a persistent form of synaptic enhancement triggered by strong afferent stimulation of synapses, known as long-term potentiation (LTP). The key molecule in this maintenance mechanism is a brain-specific, protein kinase C isoform, PKM?. Unlike other PKC isoforms that require second messengers for activation, PKM? consists of an independent PKC catalytic domain that is constitutively active. PKM? is produced from a PKM? mRNA, and the amount of the kinase increases with LTP induction. The persistent activity of the kinase is then both necessary and sufficient for maintaining the synaptic enhancement. Postsynaptic perfusion of PKM? enhances synaptic transmission, and inhibition of PKM? activity reverses previously established LTP. Recently, PKM? inhibition has been found to disrupt the storage of previously established long-term memories. These data indicate that PKM? is a candidate molecule uniquely important for information storage at synapses and during behavior. Thus the overall goal of this application is to elucidate in mechanistic detail the function of PKM? in persistent synaptic enhancement and memory storage. Our 3 Specific Aims are: 1) To characterize the mechanisms by which PKM? enhances synaptic strength. We found that PKM? potentiates synaptic strength by increasing the number of postsynaptic AMPA receptors (AMPARs) through interactions between the AMPAR GluR2 subunit and the trafficking protein NSF. We will examine whether this potentiation is through increased exocytosis and/or decreased endocytosis of postsynaptic AMPARs and the function of this altered trafficking in memory maintained by PKM?. 2) To determine whether preexisting and newly translated PKM? mediate distinct phases of potentiation during LTP. Preliminary evidence indicates that antisense oligodeoxynucleotides blocking new PKM? synthesis prevents the persistence of a phase of LTP. We will determine whether this new synthesis occurs at dendritic sites. 3) To determine the role of preexisting, newly translated, and new gene transcription of PKM? in distinct phase of memory. PKM? maintains memory up to several months after training. We will employ both antisense to block translation of PKM? mRNA and conditional genetic deletion of PKM? to examine the function of distinct mechanisms of expression of PKM? in different phases of memory. These 3 aims will provide fundamental new information on a potential molecular mechanism for maintaining synaptic and behavioral information storage, which may be relevant to both normal memory and its disorders.

DESCRIPTION (provided by applicant): Long-term changes of synaptic strength are thought to contribute to the underlying physiological substrate of memory. The molecular mechanisms mediating this plasticity can be divided into 2 phases: induction, triggering the change, and maintenance, sustaining it over time. One model to study these mechanisms is a long-term increase in synaptic strength induced by afferent tetanic stimulation, called long-term potentiation (LTP). Whereas the signaling pathways of LTP induction are exceedingly complex, much less is known about LTP maintenance. These maintenance mechanisms, however, may underlie memory storage. Our laboratory has focused on protein kinase C (PKC) in LTP maintenance. By examining the complete PKC isoform family, we identified a new PKC isoform, called PKMzeta, which persistently increases in LTP maintenance. Most PKCs consist of an N-terminal autoinhibitory regulatory domain and a C-terminal catalytic domain;second messengers activate PKC by releasing this intramolecular autoinhibition. PKMzeta, in contrast, is the independent catalytic domain of the atypical PKCzeta isoform, and, lacking a regulatory domain, is constitutively active. In Work Accomplished, we found whole-cell perfusion of PKMzeta into CA1 pyramidal cells potentiates AMPA receptor-mediated synaptic transmission. Furthermore, PKMzeta inhibitors reverse established LTP. Thus our overall goal now is to elucidate the mechanisms of PKMzeta function. Our 1st Aim is to determine the receptors mediating PKMzeta enhancement of excitatory synaptic transmission. AMPARs are composed of subunits, GluR1-4;we will use knock-out mice for each subunit to determine the subunit targets of AMPAR potentiation by PKMzeta. Our 2nd aim is to examine the molecular mechanisms of PKMzeta-mediated synaptic enhancement. Preliminary data indicate that PKMzeta increases the number of postsynaptic AMPARs through interaction between the GluR2 subunit and a critical AMPAR- trafficking protein called NSF (N-ethylmaleimide sensitive fusion protein). Our 3rd aim is to define the phase of LTP maintained by synaptically activated PKMzeta and to begin to determine the kinase's role in hippocampus-dependent spatial memory. These 3 aims will elucidate the function of PKMzeta and might provide core molecular mechanisms for maintaining long-term synaptic plasticity. These mechanisms may be important for both normal memory storage and memory dysfunction in amnestic disorders.

How molecular mechanisms modify neuronal networks to maintain long-term memory is a fundamental question in neuroscience, with relevance for disorders of persistent, memory-like dysfunction of brain circuits. Atypical PKCs (aPKC), the persistently active isoform PKM? and PKC?/?, are core molecules maintaining late- phase synaptic long-term potentiation (LTP) and several forms of long-term memory. Unlike most PKCs that are active only briefly after stimulation, aPKCs have persistent actions. After strong synaptic stimulation, PKM? increases by new synthesis, and the persistent increase in the autonomously active isoform enhances synaptic transmission during LTP maintenance and lasts for days to weeks during long-term memory storage. The other aPKC, PKC?/?, can also maintain LTP and long-term memory, as revealed by PKM?-knockout (KO) mice. Inhibitors of aPKC disrupt memory even weeks after it is formed and ameliorate persistent symptoms of PTSD, addiction, and chronic neuropathic pain in specific brain circuits in animal models. Conversely, overexpressing PKM? enhances long-term memory and alleviates persistent deficits in disorders in which decreased PKM? is implicated. Thus, understanding how aPKCs contribute to maintaining memory by sustaining representations of memory in brain circuits will provide fundamental information to assess their roles in pathological memory. Therefore, our Specific Aims are: Aim 1: Is there a hierarchy of PKCs in memory maintenance that store representations differently in networks of neurons? Spatial memory representations depend on the discharge of hippocampus place cell ensembles. We will examine if the properties of hippocampus place cell ensemble representations of spatial memories differ when maintained by PKM? or PKC?/?, and if other PKCs can also maintain spatial memory. Aim 2: How are spatial memory-related place cell ensemble representations modified when memory is erased by inhibiting individual PKCs in wild-type and PKM?- KO mice? Using novel isoform-selective antagonists and conditional KO (cKO) mice, we will test the necessity of aPKC-mediated enhanced synaptic connectivity for representing spatial memory by examining whether reversing this connectivity concurrently erases memory and destabilizes memory-related place cell ensemble representations. Aim 3: Does persistently increased synthesis of PKM? maintain very long-term memory? Strong conditioning produces increases in PKM? in the hippocampus that last a month. We will use PKM?-antisense and PKM?-cKOs to determine if these persistent increases are due to persistent increased synthesis and/or decreased degradation. To test sufficiency of PKM? for maintaining memory and memory- related representations of space, we will use overexpression of PKM? that prolongs long-term memory to examine if increased PKM? synthesis also perpetuates memory-related place cell ensemble representations. Our aims will elucidate the persistent molecular mechanisms maintaining long-term memories, causally test their role in memory persistence, and so establish a basis for understanding disorders of pathological memory.

Therapeutic time window is a key element of any drug to treat TBI. Patients with moderate to severe TBI can be  treated  hours after  injury;?  those  with  mild  TBI  may  delay  treatment  for  days  until  their  symptoms  do  not abate.  Few drugs have been developed with therapeutic time windows long enough to treat TBI, in part, because little  is  known  about  which  cellular  functions  can  be  targeted  by  drugs  dosed  hours  to  days  post-­injury  (PI).  The  combination  of  minocycline  (MINO)  plus  N-­acetylcysteine  (NAC)  retains  high  potency  when  first  dosed  12h  PI  (MN12). Published and preliminary data suggest that MN12 prevents neuronal loss and protects dendrites in the  hippocampal  ipsilateral  to  the  impact  site,  allows  learning  of  an  active  place  avoidance  task  that  requires both  hippocampi;? and restores late long-­term potentiation (LTP) to both hippocampi. Preliminary data suggests that  a  first  dose  of  MINO  plus  NAC  at  72H  PI  (MN72)  is  less  potent  than  MN12  yet  restores  acquisition  of  Barnes  maze, a task that requires only one hippocampus, and late LTP in the hippocampus contralateral to the impact  site. MN72 also increases protein synthesis in the contralateral hippocampus. This proposal examines if MN12  and  MN72 target dendrites,  synapses,  spines,  and  synaptic  protein  synthesis after  closed head  injury  (CHI)  in  mice.  Proposed  studies  will  examine  whether  MN12  and  MN72  target protein  kinase  M  zeta  (PKMz?),  which  is  essential for late LTP and retention of hippocampal-­dependent tasks. Studies will also examine whether MINO  plus  NAC  remains  potent  when  dosed  later  than  72H  PI.  These  data  support  3  specific  aims  (SA)  that  test  a  central hypothesis that: Dosing of MINO plus NAC at clinically relevant therapeutic time windows limits  gray matter injury and improves cognition and memory. SA1: Where does a first dose of MINO plus NAC  at 12 or 72h after CHI repair dendrites, spines and synapses? The working hypothesis of SA1 is that MN12  acts bilaterally to prevent injury and induce repair while MN72 acts only on the contralateral hippocampus. SA1  will  also  assay  neuroinflammation,  oxidative  stress  and  mitochondrial  morphology  after  MN12  or  MN72  treatment.  SA2:  Does  MN12  and  MN72  target  PKMz?  expression  to  restore  synaptic  plasticity  and  acquisition  of  hippocampal-­dependent  tasks?  SA2  will  examine  a  role  for  PKMz?  expression  by  MN12  or  MN72 using NTSA, a novel and specific inhibitor of PKMz?, or in conditional PKMz? mutant mice. SA2 is predicted  to show that MN12 or MN72 target PKMz? to restore late LTP and long-­term memory. SA 3: Does MINO plus  NAC limits brain injury and restore function in the subacute (14D PI) or chronic (45D PI) stages of TBI?  The  utility  of  MINO  plus  NAC  would  be  greatly  increased  if  the  drugs  retained  potency  when  dosed  in  later  phases  of  TBI.  These  studies  have  high  potential  significance  since  they  show  that  a  combination  of  FDA-­ approved  drugs  with  clinically  useful  windows  can  restore  cognition  and  memory,  which  are  central  deficits  produced by TBI. These studies have potentially high impact since the absence of effective drugs make people  with TBI less likely to seek treatment. MN12 and MN72 are attractive candidates for clinical trials to treat TBI.