1. Investigating a Fragment of the Leptin C-D loop: Neuroprotective and Behavioural Effects
Alison Holiday

2. Alzheimer’s Disease (AD)
Toxic amyloid beta (Aβ)
Aberrant synaptic function
Neuronal degeneration
Soluble oligomers influence activity-dependent hippocampal synaptic plasticity
Inhibits LTP and exacerbates LTD
Alters AMPA receptor trafficking – synaptic dysfunction
Profound cognitive deficits – memory
Accumulation of toxic amyloid plaques and tau tangles
Toxic amyloid beta (Aβ) – aberrant synaptic function, neuronal degeneration
Soluble Aβ oligomers – influence activity-dependent hippocampal synaptic plasticity -> learning and memory deficits
Inhibits LTP and exacerbates LTD
Alters AMPA receptor trafficking to/from synapses – contributes to hippocampal synaptic dysfunction
- What is AMPA

3. Leptin
Leptin = satiety and energy homeostatsis
Increase
Increased clearance of AB and Tau
Linked with breast cancer and chronic pain
Solution: neuroactive fragment
Decrease
Occurs with age
Impaired LTP, synaptic plasticity and spatial learning
Receptors in hippocampus
Insensitivity
Obesity
Increased cognitive decline in AD
Increases with age
Zhang, Y., Proenca, R., Maffei, M., Barone, M., Leopold, L., & Friedman, J. M. (1995). Positional cloning of the mouse obese gene and its human homologue. Nature, 372(6505), 479.
Diet and lifestyle are considered to be risk factors for AD. Specifically disruption of metabolic systems has been linked to AD.
Leptin is a hormone produced by adipose tissue and acts in the hypothalamus to inhibit hunger and maintain energy balance.
In obesity a decreased sensitivity to leptin occurs so that the person can no longer detect when they are “full” despite the body having plentiful energy stores.
Obesity significantly increases cognitive decline in AD
In humans higher levels of leptin + decreased dementia and AD reported
In rodents leptin modulates the production and clearance of AB
Mice with leptin receptor disruption show impairments in LTP, synaptic plasticity and spatial learning, when treated with leptin – increased AB and tau clearance
(Pedrós, I., Petrov, D., Artiach, G., Abad, S., Ramon-Duaso, C., Sureda, F., … Camins, A. (2015). Adipokine pathways are altered in hippocampus of an experimental mouse model of Alzheimer’s disease. The Journal of Nutrition, Health & Aging, 19(4), 403–12. doi: /s )
Leptin receptors in hippocampus (Harvey J. Leptin regulation of neuronal excitability and cognitive function. Curr Opin Pharmacol 2007;7:643–7.) which is involved in learning and memory suggesting leptin plays a role
Although leptin sounds like it could bean excellent drug for AD there are problems with using the whole molecule. Because leptin receptors are found in many areas of the brain and throughout the body a leptin drug would have widespread effects beyond the potentially beneficial effects seen in animal studies, Leptin is also a large molecule so it would be expensive to manufacture as a drug in its whole form, finally increased leptin has been linked to problems such as promoting proliferation in breast cancer (García-Robles, M. J., Segura-Ortega, J. E., & Fafutis-Morris, M. (2013). The Biology of Leptin and Its Implications in Breast Cancer: A General View. Journal of Interferon & Cytokine Research, 33(12), 717–727. doi: /jir ) and endometriosis (chronic pain) (Ectopic endometrium-derived leptin produces estrogen-dependent chronic pain in a rat model of endometriosisAlvarez, P.;  Bogen, O.;  Chen, X.;  Giudice, L.C.;  Levine, J.D. (2014)Neuroscience vol. 258 p )
In order to make a leptin a feasible contender for alzheimer’s treatment it is possible that a fragment of the leptin protein could be used that has the positive neuroprotective effects of leptin without the negative widespread actions of the whole protein. A bioactive fragment has already been discovered and so one of my first tasks is to see if the fragment is as effective as leptin.
What it is
How it is linked to AD etc
Fragment??
LEPTIN LEVELS DECLINE WITH AGE, LEPTIN INSENSITIVITY INCREASES WITH AGE
FLOW CHART?
Leptin usually acts to induce satiety and maintain energy homeostasis
Obese patients demonstrate an insensitivity to leptin which is linked to an increased cognitive decline in Alzheimer’s disease (AD)
In rodents treatment with Leptin has lead to an increased clearance of Amyloid B and Tau
Leptin receptors are also located in the hippocampus suggesting it plays a role in learning and memory
Mice without leptin are impaired in LTP, synaptic plasticity and spatial learning.
Leptin signalling is widespread, costly to make, linked to cancer and chronic pain
Solution: a bioactive fragment of leptin

4. Leptin116-130 prevents copper induced cell death
CuCl2-treated
cultures
Crystal violet assay on leptin and leptin treated cells with
copper
CuCl2-treated
cultures
SH-SY5Y human neuroblastoma cells were differentiated into neuronal-like cells and treated with a range of concentrations from nM of full length human leptin and leptin and 5uM copper chloride for 5 days. Here I demonstrate the results of this with two assays an LDH assay and a crystal violet assay.
LDH assay detects the level of lactate dehydrogenase in the cell culture medium. LDH is an enzyme normally found in cells, higher level of detection of ldh in the medium indicates cell membranes have burst – this is commonly associated with necrosis a form of cell death. In this assay lower levels of LDH were detected than in the kill condition for all concentrations of leptin.
Crystal violet assay stains DNA and proteins in cells. This is a useful assay for determining levels of cell survival within a culture. Cells are fixed and washed prior to staining so that only adherent cells will be stained which indicates they are still alive… Results showed all concentrations of leptin/fragment had significantly more surviving cells than the kill condition
FRAGMENT 16AMINO ACIDS
CD LOOP – SHOWN TO BE BIOACTIVE
FRAGMENTS – DIFFERENT EFFECTS – THIS IS NEUROACTIVE (NS RATHER THAN PERIPHERY)

5. Leptin116-130 prevents amyloid beta induced cell death
Ab1-42-treated
cultures
*** ** *
LDH assay on leptin and leptin treated cells with amyloid beta
Ab1-42-treated
cultures
*** ** *
Crystal violet assay on leptin and leptin treated cells with
amyloid beta
These experiments were repeated again using 10uM amyloid beta 1-42 as this is the toxic species of amyloid found in alzheimers disease. Once again leptin/fragment were able to protect against increased LDH and showed more living cells than the kill condition
Together these results indicate the leptin was able to protect against induced cell death.

6. Leptin116-130 acts via STAT3 and Akt pathways
Ratio of phosphorylated-STAT3 to pan-STAT3 in protein extracts from untreated and leptin pre-treated cells
Ratio of phosphorylated-Akt to pan-Akt in protein extracts from untreated and leptin pre-treated cells **
We next wanted to assess what downstream signaling was involved in the protective mechanisms of leptin hence we used ELISA to look at cell survival pathways specifically STAT3 and PIS-kinase-dependant pathways as these have previously been highlighted to be important in leptin signalling. Cells were exposed to 1nM leptin for 3 hours or left untreated prior to protein extraction. The levels of phosphorylated or pan STAT3 and Akt were measured. An increased in the ratio of phosophorylated STAT3 to pan STAT3 and phosphorylated Akt to pan Akt were seen. Indicating leptin reduced cell death via activation of these pathways.
In addition we have pharmacological data which shows these pathways have to be active in order to give leptin fragments neuroprotective effects

7. Leptin116-130 improves episodic memory
Object-place-context recognition test
Healthy C57 mice injected with saline, leptin or leptin fragment
Discrimination
index =
Novel - Familiar
Novel + Familiar

8. Leptin116-130 improves episodic memory
Total exploration times for mice in each treatment group
Discrimination index for mice in each treatment group

9. Leptin116-130 effects hippocampal synaptic function
Promotes conversion of short-term potentiation into persistent increase in synaptic transmission in juvenile rat hippocampus
LTP
NMDA receptor dependent
Enhanced synaptic transmission in adult rat hippocampal slices
Promotes trafficking of AMPA receptors
Increased surface expression of GluA1 in cultured hippocampal neurons
Facilitates hippocampal synaptic plasticity
Prevents aberrant effects of Aβ on hippocampal synaptic function
Promotes conversion of short-term potentiation into persistent increase in synaptic transmission in juvenile rat hippocampus – LTP
NMDA receptor dependent
Enhanced synaptic transmission in adult rat hippocampal slices
Promotes trafficking of AMPA receptors – increased surface expression of GluA1 in cultured hippocampal neurons
Facilitates hippocampal synaptic plasticity
Prevents aberrant effects of Aβ on hippocampal synaptic function

10. Leptin
Leptin enhances the survival of human neural cells from Aβ1-42 and Cu2+ insults
Signalling via PI3-kinase and STAT3 is essential for leptin mediated neuroprotection
Leptin is a cognitive enhancer
Mice performing an OPC test for episodic-like memory improved with leptin and leptin treatment
Leptin promotes trafficking of AMPA receptor subunit GluA1 to synapses and facilitates hippocampal synaptic plasticity
Leptin may be useful in the development of leptin-mimetic agents for therapeutic use
Conclusion

11. Acknowledgements Collaborators from Dundee: Supervisors:
Dr Jenni Harvey
Yasaman Malekizadeh
Supervisors:
Dr Gayle Doherty ( andrews.ac.uk/ )
Dr James Ainge
Devon Redfearn
Publication: Malekizadeh, Y., Holiday, A., Redfearn, D., Ainge, J. A., Doherty, G. and Harvey, J. (2016) A Leptin Fragment Mirrors the Cognitive Enhancing and Neuroprotective Actions of Leptin. Cerebral Cortex, In press.

12. Questions