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A Critical Role for Protein Tyrosine Phosphatase Nonreceptor Type 5 in Determining Individual Susceptibility to Develop Stress-Related Cognitive and Morphological Changes.

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A Critical Role for Protein Tyrosine Phosphatase Nonreceptor Type 5 in Determining Individual Susceptibility to Develop Stress-Related Cognitive and Morphological Changes.

… or how I learned to quit caring and love the Dendritic Spine Density of the Dorsal Hippocampus CA1Pyramidal Neuron and OLM voodoo.


Where we will be going today
Where we will be going today

Low PTPN5 expression  Increased ERK1/2 activation  Increase in CaV1.2 expression and (phosphorylation of ) KV4.2 channel recovery delay from inactivation  Glutamate (and Bay K8644) excitotoxicity  Influx of Ca2+into cell  cellular degradation  “BAM!” susceptibility to stress/physical degradation of nervous system!


Where we are looking

Bonus: This exact picture showed up in another advanced seminar presentation. First to tell me which one gets a shiny Zachary Taylor

Where we are looking

  • Dorsal Hippocampus

    • Memory consolidation

    • Spatial navigation

  • What we are interested in

    • CA1 area

      • Stuff this area does?

Do you know? Because Andy doesn’t


Protein tyrosine phosphatase nonreceptor type 5 ptpn5
Protein tyrosine seminar presentation. First to tell me which one gets a shiny Zachary Taylorphosphatasenonreceptor type 5 PTPN5

  • “…functions in regulating the duration of extracellular signal-regulated kinase (ERK) activation and downstream transcriptional responses after NMDA receptor stimulation, NMDA receptor endocytosis by amyloid, and AMPA receptor endocytosis after metabotropic glutamate receptor stimulation, suggesting that PTPN5 signaling may function as a tonic suppressor of synaptic strengthening.”


What is erk
What is ERK? seminar presentation. First to tell me which one gets a shiny Zachary Taylor

Erk!

Erk!

Erk!

Erk!

In this case not a silly sound

Erk!


Erk 1 2
ERK seminar presentation. First to tell me which one gets a shiny Zachary Taylor1/2

  • Extracellular signal-related kinase.

    • Controls

      • Ion channels

      • Neurtotransmitter receptors

      • Protein kinases

      • Transcription factors

      • And more. Like lions, tigers, and bear.

Erk?


What you need to know about erk1 2
What you need to know about ERK1/2 seminar presentation. First to tell me which one gets a shiny Zachary Taylor

  • Responsible for a cascade of events, that eventually results in an increased expression of Cav1.2 channels and recovery delay of KV4.2 channels from inactivation.


Glutamate
Glutamate seminar presentation. First to tell me which one gets a shiny Zachary Taylor

  • Massively important (excitatory) neurotransmitter

    • Its just about in every nerve cell, and is incorporated into the proteins of just about every cell in the body

    • Two main receptors.

      • NMDA

      • AMPA


Nmda receptor action
NMDA receptor action seminar presentation. First to tell me which one gets a shiny Zachary Taylor

Shamelessly stole…er borrowed from Cliffs webpage.


Action potential
Action Potential seminar presentation. First to tell me which one gets a shiny Zachary Taylor



Excitotoxicity
Excitotoxicity LDP?)

  • Cell death by Ca2+ overload

    • In our case causedby over-active NMDAr


Methods
Methods LDP?)

  • Rats

    • Male adult Sprague Dawleyrats.

    • Rats were testedduring light phase(b/n 10a – 3p)

  • Stress Protocol

    • Tail shock while held immobile in a plexiglass tube.

      • 1 mA for 1 s, 30-90 sec apart

Why?!


Object location memory test olm
Object location memory test (OLM) LDP?)

  • Open-field box (40x40x40 cm)

    • 10min exploration/acclimation

  • Test

    • 5min exploration

      • Score time spent investigatingobjects

    • Discrimination Index (Cognitive Performance)

      • [time on novel / (time on + time on )] x 100%

    • 3hrs, and 1wk post stress


Methods1
Methods LDP?)

  • Other Stress measurements:

    • Sucrose preference

      • Seek less sweet,  SAD

    • Open Field tests

      • Like more hiding,  SAD

    • Resident-intruder test

      • Less aggressive to intruder, not  SAD?

    • Forced-swimming test

      • Less swim,  SAD

    • Plasma corticosterone recording

      • High corticosterone,  SAD (stressed really)


Corticosterone stress
Corticosterone = Stress LDP?)

Do you expect me to talk?

No Mr. Bond. I expect you to have a marked increase in corticosterone levels!


Methods2
Methods LDP?)

  • Lentivirus injection

    • Knockouts and overexpression!

    • Recombinant Lentivirus

      • Shut stuff down

        • shRNA(small hairpin RNA)

          • Leads to RNA interference. This results in perfect cleaving of mRNA or simply prevents mRNA translation.

      • Overexpression

        • Overexpression of PTPN5 ( specifically STEP46 which is six-fold more active than STEP61)



Results things done
Results/Things Done LDP?)

  • Individual differences in susceptibility to stress-related cognitive and morphological changes

  • PTPN5 determines individual susceptibility to develop stress-related cognitive and morphological changes

  • PTPN5 loss of function increases susceptibility to develop stress-related cognitive and morphological changes, and vice-versa.

  • PTPN5 gain of function decreases susceptibility to develop stress-related cognitive and morphological changes.

  • PTPN5 loss of function increases excitatory overload.


Individual differences in susceptibility to stress related cognitive and morphological changes
Individual differences in susceptibility to stress-related cognitive and morphological changes

  • Rats respond differently to stress! Although initial response, at least as quantified here, was similar. Long term responses differed.

    • Based on these responses rats could be grouped into susceptible and unsusceptible categories.

      • OLM scores (AKA Voodoo)

      • Dendritic spine density in pyramidal CA1 neurons

      • Other measurements of stress


Graphs figure 1
Graphs: Figure 1 cognitive and morphological changes

A: shows how the rats are distributed that weren’t stressed,

before and after(3hr after and 1wk) stress event. B: dividng rats into susceptible and non-susceptible groups, based off of OLM score.


C: Break down of Susceptible vs. Unsusceptible individuals post shockD: Chart showing dendritic spine density in hippocampal CA1 pyramidal cells


E: spine density in response to acute stress. 1wk after exposure.

F: Pretty picture of Dendritic Spines



Graphs figure 2
Graphs: Figure 2 post stress

A: % Susceptible. NS 0% Post-S 1 wk 20% Post-S 2 wk 24%

B: % Susceptible. NS 0% Post-S 1 wk 24% Post-S 2 wk 22%



D: Forced Swim results – all rats, post shock (+control) and 2 weeks post stress.

E: Corticosteroid levels – all rats, post swim test (+control)


PTPN5 determines individual susceptibility to develop stress-related cognitive and morphological changes

  • Susceptible rats expressed lower PTPN5 levels compared to nonstressed and unsusceptible rats.

  • Susceptible rats had lower PTPN5 mRNA levels in the DH

  • PTPN5 levels were significantly correlated with OLM performance.

  • Likewise dendritic spine density was significantly correlated with OLM performance.


Figure 3
Figure 3 stress-related cognitive and morphological changes

Fig A: PTPN5 protein expression in hippocampal CA1 regionFig B: PTPN5 activity in CA1 tissue


Fig: C 11 brain enriched protein tyrosine-phosphatases. Note difference in PTPN5 in susceptible and unsusceptible rats.


Fig. D: Correlation analysis of discrimination index and PTPN5 expression.

Fig. E: Correlation analysis of dendritic spine density and PTPN5 expression.


PTPN5 loss of function increases susceptibility to develop stress-related cognitive and morphological changes.

  • Knockout PTPN5 expression.

    • Lentivirus coexpressing EGFP (electric green florescent protein) and anti-PTPN5 shRNA or control sh-DsRed.

    • Injected into CA1 region

    • 1wk recover

    • Test

      • OLM

      • Dendritic Spine Density


Figure 4
Figure 4 stress-related cognitive and morphological changes.

Fig. C: PTPN5 Silencing confirmed (sh-DsRed)

Fig. D: charts showing susceptibility and performance in OLM. 1wk post stress


1 week or immediately post stress
1 week or immediately post stress stress-related cognitive and morphological changes.

Fig. E: Charts and graphs showing susceptibility based on spine density.

Fig. F: Control injection vs PTPN5 silenced individuals LTP induction (Pre and immediate post shock) Help? Still a little fuzzy on what this tells us exactly.

Fig. G: Control Injection vs PTPN5 silenced individuals in LTD induction (Pre and immediate post shock)


24h post stress
24h post stress stress-related cognitive and morphological changes.

Fig. H: ERK 1/2 phosphorylation in PTPN5 silenced and control rats

Fig. I: 24hr post shock LTP induction in naïve, control injection, and PTPN5 silenced individuals.

Fig. J: 24hr post shock LDP induction in naïve, control injection, and PTPN5 silenced individuals.


PTPN5 gain of function decreases susceptibility to develop stress-related cognitive and morphological changes

  • Overexpression of a very active PTPN5 variant.

    • Normally STEP61, researchers injected STEP46. Sixfold more active. Think SUPER PTPN5 (unless Summers says not to)

    • Due to possibility that PTPN5 overexpression would lead to a decrease in surface expression of NMDA receptors, viral titers were manipulated to yield an approximate 2-3 fold increase in PTPn5 activity.


1hr post stress
1hr post stress stress-related cognitive and morphological changes

Fig. C: Confirmation of PTPN5 overexpression

Fig. D: control, control injection, and PTPN5 overexpression LTP induction response

Fig. E: control, control injection, and PTPN5 overexpression LTD induction.


Fig. F: ERK 1/2 phosphorylation in control injection and PTPN5 over expression rats. No Stress, immediate post stress, 1hr post stress, and 24hr post stress.

Fig. G: graphs and charts of OLM performance of naïve, control injection, and PTPN5 over expression. 1wk post stress.

Fig. H: graphs and charts of dendritic spine density in naïve, control injection, and PTPN5 over expression rats. 1wk post stress.


Ptpn5 loss of function increases excitatory overload
PTPN5 loss of function increases excitatory overload. PTPN5 over expression rats. No Stress, immediate post stress, 1hr post stress, and 24hr post stress.

  • Take a look at ERK1/2 dependent signaling

  • PTPN5 silenced rats showed significantly prolonged ERK1/2 activation.

    • Shown by ERK1/2 and KV4.2 channel phosphorylation at Thr607 compared to controls

      • Phosphorylation = activity suppression

    • Delayed increase in CaV1.2 channel expression after stress, which was significantly promoted in PTPN5 silenced rats.

Shut er’ down!


Loss of function cont
Loss of function cont. PTPN5 over expression rats. No Stress, immediate post stress, 1hr post stress, and 24hr post stress.

  • A closer look at the role of ERK in CaV1.2 and KV4.2 phosphorylation.

    • Administered ERKI (ERK1/2 docking site inhibitor), into DH 24hr post silencing.

      • Resulted in significantly reduced CaV1.2 channel expression which prevented stress-related cognitive and morphological changes in PTPN5 silenced rats 1wk post injection.

      • Decreased dendritic spine loss and OLM performance impairment.

    • Suggests that sustained ERK1/2 activation in low PTPN5 expression individuals leads to stress


Loss of function cont1
Loss of function cont. PTPN5 over expression rats. No Stress, immediate post stress, 1hr post stress, and 24hr post stress.

  • Susceptible rats significant increase in CaV1.2 channel expression compared to nonstressed and unsusceptible rats 1wk post procedure.

  • Glutamate (fig. 7 d-g)

    • Curious if increased CaV1.2 channel expression and KV4.2 phosphorylation would increase cell vulnerability to Glu.

      • Administered CaV1.2 and KV4.2 agonists (Bay K8644)

        • Brain slices from control injection and silenced individuals. 5d post procedure.

          • PTPN5 individuals showed increased susceptibility to Glu and Bay K8644 toxicity

        • Blocking ERK and CaV1.2 in PTPN5 silenced rats significantly reduced Glu. Vulnerability


  • Loss of function final
    Loss of function (final) PTPN5 over expression rats. No Stress, immediate post stress, 1hr post stress, and 24hr post stress.

    • Block L type CaV1.2 channels after stress to reduce occurrence of stress phenotypes

      • Used nifedipine 48hrs post stress or 5d post stress

        • Resulted in significant reduction in stress phenotype

      • Final experiment, knockout CaV1.2 in PTPN5 silenced rats. 24hr post procedure.

        • OLM and Dendritic spine density measured

      • Unsurprisingly, rats expressed extremely low CaV1.2 levels. And as a result less stress phenotype.


    Figure 6
    Figure 6 PTPN5 over expression rats. No Stress, immediate post stress, 1hr post stress, and 24hr post stress.

    Fig. A: Various expressions based off of PTPN5 knockout vs control infected individuals.

    Fig. B: various concentrations for control infected rats vs PTPN5 knockout rats.


    Fig. D: Ca PTPN5 over expression rats. No Stress, immediate post stress, 1hr post stress, and 24hr post stress.V1.2 protein expression in PTPN5 silenced individual with control vehicle or ERKI (ERK1/2 docking site inhibitor)

    Fig. E: Graphs and Charts of OLM criteria in PTPN5 silenced individuals with vehicle or ERKI

    Fig. F: Graphs and Charts of OLM criteria in PTPN5 silenced indivuals with vehicle or ERKI


    Figure 7
    Figure 7 PTPN5 over expression rats. No Stress, immediate post stress, 1hr post stress, and 24hr post stress.

    Susceptible

    Fig. A: CaV1.2 protein expression levels in control, unsusceptible and susceptible rats. 1 week post stress.

    Fig. B: Correlation analysis of CaV1.2 expression and OLM discrimination index 1wk post stress.

    Fig. C: Correlation analysis of CaV1.2 expression and dendritic spine density.


    Fig. D: comparison of cell death by [glutamate] in PTPN5 silenced and control injection.

    Fig. E: comparison of [LDH] in PTPN5 silenced and control injection rats.

    Fig. F: comparison of cell death by [Bay K8644] ] in PTPN5 silenced and control injection rats.

    Fig. G: comparison of [LDH] ] in PTPN5 silenced and control injection rats.

    LDH = Lactate Dehydrogenase. Another metric used to measure cell death. Results from tissue breakdown.

    Bay K8644 = Calcium channel agonist.


    Fig. I: Charts and graphs of OLM performance 1wk post stress for control, early and late Nifedipine injections

    Fig. J: Charts and graphs of dendritic spine density 1wk post stress in control and early and late Nifedipine injections.


    Figure 8
    Figure 8 for control, early and late Nifedipine injections

    Fig. B: CaV1.2 protein expression in DH of PTPN5 silenced individuals with either control injection vs CaV1.2 knockout, plus control 1wk post stress.

    Fig. C: Charts and graphs of OLM index in PTPN5 silenced rats either control, vehicle control, or CaV1.2 silenced 1 wk post stressed.

    Fig. D: Charts and graphs of dendritic spine density in PTPN5 silenced rats either control, vehicle control, or CaV1.2 silenced 1 wk post stressed.


    What happened
    What happened for control, early and late Nifedipine injections

    • Low PTPN5 expression  Increased ERK1/2 activation  increase in CaV1.2 expression and KV4.2 (phosphorylation) channel recovery delay from inactivation  Glutamate (and Bay K8644) excitotoxicity  cell degradation  BAM! susceptibility to stress!


    Fin for control, early and late Nifedipine injections


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