5. Metabolic Diseases 3: Hypothalamus

Question 1

Why are chronic diseases complex to understand?

Answer 1

1 disease can disrupt homeostasis in a way that leads to the development of other diseases - multi-morbidity

Question 2

What do we need to understand for adult stem cells to be used in future therapies?

Answer 2

• Need to understand the behaviour and control of adult stem cells and how differentiated cells arise from them and are maintained

Question 3

What are the 3 distinct subunits made by cleaving Pomc involved in?

Answer 3

• 2 involved in energy expenditure
• 1 is adrenocorticotropic hormone (ACTH) which triggers release of cortisol in stress pathway
• Energy expenditure and stress pathway are inherently linked

Question 4

Where is the arcuate nucleus located?

What does this mean for NPY/Pomc neurons?

Answer 4

• Arcuate nuclei sit bilaterally either side of the 3rd ventricle
• Meaning NPY/Pomc neurons sit very close to 3rd ventricle

Question 5

What is located beneath the 3rd ventricle?

What is the significance of this area?

Answer 5

• Median eminence

- There is no blood-brain barrier allowing direct 2 way communication between the brain and the body

Question 6

What does the lack of blood-brain barrier in the median eminence allow?

Answer 6

• Allows neurohormones made by hypothalamic neurons to enter the bloodstream and access body
• Allows small molecules in bloodstream (e.g. leptin) to access the brain and hypothalamic neurons

Question 7

What are hypothalamic stem cells called?

What do these cells appear like?

Answer 7

• Tanycytes
• They have a radial glia-like appearance with their cell body at the 3rd ventricle and a basal process that projects to the arcuate nucleus

Question 8

How can tanycytes divide?

Answer 8

They can divide asymmetrically to give:

• 1 daughter that is retained at 3rd ventricle as a radial glia -like cell
• 1 daughter that differentiates into a neuron and uses scaffold of radial glia to migrate into arcuate nucleus

Question 9

How do glucose requirements change during life?

What is the current idea on how this affects NPY/Pomc neurons?

Answer 9

• During puberty and pregnancy, glucose requirements increase
• NPY and Pomc neurons can be generated from hypothalamic stem cells to anticipate and/or respond to changing glucose requirements (e.g. during puberty generate more NPY neurons to increase food intake to cope with increased glucose requirements)

Question 10

What studies provide the evidence that NPY/Pomc neurons may be generated through life from hypothalamic stem cells?

Answer 10

Genetic lineage tracing studies have shown adult tanycytes can give rise to NPY neurons in the adult mice

Question 11

What are the experiments that have shown NPY/Pomc neurons can be generated from tanycytes throughout life?

Answer 11

1. Identify populations of cells that develop over time using markers (transcriptional profile of each cells type)
2. Lineage trace the stem cell - use cell-specific promoter to create construct that traces stem cell and all of its descendants
3. Identify key genes required to maintain each cell type - - e.g. if TF identified that is key for maintaining Arcuate progenitors, if KO this TF would expect to see loss of Arcuate progenitors and all its descendants
4. Use promoters of these genes to perform genetic ablation studies to eliminate particular populations and observe effect (different to TF KO as losing whole cell)

Question 12

What did Nasif et al (2015) discover that defines identity of arcuate progenitors that sit upstream of Pomc neurons?

Answer 12

Expression of the TF Islet 1

Question 13

What did Nasif et al (2015) report Islet 1 promotes?

Answer 13

Terminal differentiation of Pomc neurons

Question 14

What were the results from Nasif et al (2015) study on Islet 1?
What did they conclude about the requirement for Islet 1 expression

Answer 14

• Islet 1 was detected in the prospective hypothalamus just before the expression of Pomc
• Islet 1 binds in vitro and in vivo to homeodomain binding DNA motifs in the enhancers of Pomc gene (Islet 1 unregulates Pomc expression)
• Mutation of these binding sites disrupted the ability of Islet 1 to drive reporter gene expression to Pomc neurons
• Conditional KO of Islet 1 from Pomc neurons impairs Pomc expression leading to hyperphagia and obesity
• Islet 1 is required for Pomc-induced satiety throughout entire life

Question 15

What have studies on the extrinsic signals regulating hypothalamic stem cell behaviour shown?

Answer 15

• Fgf10 promotes self-renewal

- Shh promotes differentiation

Question 16

Describe the method to generate a lineage tracing model.

Answer 16

1. Identify a cell-specific promoter that is only expressed in the cell to be traced
2. Clone Cre-ER(T2) downstream of this cell-specific promoter and make 1st transgenic mouse
   - Encodes a cre recombinase that is fused to a mutant oestrogen ligand-binding domain that requires tamoxifen for activity (Cre recombinase only active when tamoxifen is injected
3. Make 2nd transgenic mouse where a stable reporter (GFP) is downstream of a constitutive promoter, but separated from the promoter by a floxed STOP sequence
4. Cross 2 transgenic mice
5. Add tamoxifen at any stage in life to activate Cre recombinase which will recombine out the STOP sequence in cell to be traced. Reporter will now be expressed in that cell and all its descendants

Question 17

Describe an example of a genetic lineage tracing experiment.

Answer 17

• In first cohort, tamoxifen is added and mice are immediately sacrificed to show that reporter is only expressed in original cell
• In second cohort, tamoxifen added and mice are left for 6-9months
• If original cell is a stem cell, expect to see expression of reporter in same place as original cell (self-renewal) and in the arcuate nucleus (differentiation)
• Then place marker on differentiated daughters (e.g. NPY) to show stem cell gives rise to NPY neurons

Question 18

What technique was used by Robins et al (2013) in the study of α tanycytes?

Answer 18

• Long term lineage tracing using a GLAST::CreER(T2) conditional driver (GLAST = α tanycyte-specific promoter)
• GLAST::CreER(T2) mice were crossed with 1 of 2 reporter lines (GFP or lacZ)
• Tamoxifen was then added to activate Cre recombinase

Question 19

What were the results of Robins et al (2013) study of α tanycytes?

Answer 19

• Immediate sacrifice showed only a subset of tanycytes are labelled
• Longer lineage tracing showed α tanycytes can self-renew, or give rise to new tanycyte subsets, astrocytes and neurons of the arcuate nucleus

Question 20

What did the subsequent study on tanycytes show?

Answer 20

Used lineage tracing of tanycytes to show that tanycytes give rise to neurons that populate the arcuate nucleus and a subset of these express NPY

Question 21

What did Robins et al (2013) show with caged mice?

Answer 21

• In a caged mouse there is no need for additional NPY neurons therefore tanycytes show little proliferation
• Intracerebroventricular infusion of FGF2 into these mice stimulated tanycyte proliferation
• In control, BrdU showed little tanycyte proliferation
• In FGF2 infusion, BrdU showed increased tanycyte proliferation and migration of daughters along tanycyte processes

Question 22

Why is it hard to investigate if the balance of NPY and Pomc neurons is disrupted in obesity/T2D?

Answer 22

• Required to compare Pomc neurons before and after onset of T2D (required to kill individual to analyse Pomc neuron numbers)
• Can only compare with tissue from same person which is impossible as can’t kill twice

Question 23

What did Scarlett et al (2016) show in there animal model study to see if stimulating tanycytes can promote diabetes remission?

Answer 23

• An intracerebroventricular injection of FGF1 induced sustained diabetes remission in mouse and rat T2D models, at a dose 1/10th of that required for anti-diabetic efficacy following peripheral injection
• Anti-diabteic effects were not secondary to weight loss, did not increase risk of hypoglycaemia and involves a novel, incompletely understood mechanism for increasing blood glucose clearance

Question 24

What were the problems with the Scarlett et al (2016) study (intracerebroventicular injection of FGF1)?

Answer 24

• Normally, an experiment should repeat what has previously been shown (e.g. FGF infusion stimulates increased tanycyte proliferation) however they did not do this
• Instead they looked at the expression of HSP25 gene and stated that tanycytes become more active after FGF injection as they upregulate HSP25 expression (function of HSP25 is unknown)

Question 25

Why were tanycytes lining the 3rd ventricle said to have interesting potential as mediators of FGF1-induced diabetes remission?

Answer 25

They respond to glucose and FGF signalling

Question 26

How did Scarlett et al (2016) show tanycytes are activated following FGF1 injection?

Answer 26

• Used immunohistochemistry to detect c-FOS expression (marker for cellular activation) in response to FGF1 injection (induces diabetes remission) and FGF19 (does not induce diabetes remission)
• FGF1 injection induced robust expression of c-FOS expression in tanycytes
• FGF19 did not
• This suggests a functional link between activation of tanycyctes and diabetes remission induced by FGF1 injection