5. Metabolic Diseases 3: Hypothalamus Flashcards Preview

BMS336 - Modelling Human Disease and Dysfunction > 5. Metabolic Diseases 3: Hypothalamus > Flashcards

Flashcards in 5. Metabolic Diseases 3: Hypothalamus Deck (26)
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1
Q

Why are chronic diseases complex to understand?

A

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

2
Q

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

A
  • Need to understand the behaviour and control of adult stem cells and how differentiated cells arise from them and are maintained
3
Q

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

A
  • 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
4
Q

Where is the arcuate nucleus located?

What does this mean for NPY/Pomc neurons?

A
  • Arcuate nuclei sit bilaterally either side of the 3rd ventricle
  • Meaning NPY/Pomc neurons sit very close to 3rd ventricle
5
Q

What is located beneath the 3rd ventricle?

What is the significance of this area?

A
  • Median eminence

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

6
Q

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

A
  • 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
7
Q

What are hypothalamic stem cells called?

What do these cells appear like?

A
  • 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
8
Q

How can tanycytes divide?

A

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
9
Q

How do glucose requirements change during life?

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

A
  • 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)
10
Q

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

A

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

11
Q

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

A
  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)
12
Q

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

A

Expression of the TF Islet 1

13
Q

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

A

Terminal differentiation of Pomc neurons

14
Q

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

A
  • 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
15
Q

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

A
  • Fgf10 promotes self-renewal

- Shh promotes differentiation

16
Q

Describe the method to generate a lineage tracing model.

A
  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
17
Q

Describe an example of a genetic lineage tracing experiment.

A
  • 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
18
Q

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

A
  • 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
19
Q

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

A
  • 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
20
Q

What did the subsequent study on tanycytes show?

A

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

21
Q

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

A
  • 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
22
Q

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

A
  • 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
23
Q

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

A
  • 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
24
Q

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

A
  • 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)
25
Q

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

A

They respond to glucose and FGF signalling

26
Q

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

A
  • 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