BRAIN SEX DIAMORPHISMS AND DISEASE

Question 1

What diseases do males generally have more of?

Answer 1

ADHD, autism, dyslexia, taurettes, parkinson’s, substance abuse, schizophrenia (early onset and more severe) and stroke until menopause

Question 2

What do females generally have more of?

Answer 2

depression, anxiety disorders, eating disorders, senile dementia – alzheimers type, and MS

Question 3

What does gender epidemiological data suggest?

Answer 3

THESE ALL SUGGEST A ROLE FOR OESTROGENS IN THE PATHOPHYSIOLOGY

Question 4

What has been one of the key studies in looking at the effects of oestrogen? What did it find

Answer 4

Main clinical focus: ovarian steroids in menopause and hormone replacement therapy – The Women’s Health Initiative (WHI) study. -2002- prospective, double blind crossover clinical trial intended to run for 5 years to test whether HRT was beneficial for CVS system. However, stopped after 2 years because incidence of stroke increased
-However, the group of women they chose to do this were in their 60s- had been without E2 for 10 yrs. If without E2 for long time, system adapts to be without it so when you add it back, it becomes harmful

Question 5

What has the main pre-clinical focus been? Problems

Answer 5

Main pre-clinical experimental focus has been to investigate estrogen therapy in ovariectomised females (mainly rodents, occasionally monkeys)
-Ovariecromise, so take away oestrogen. Then give E2 back, to see if protective. But when people give back E2 in experimental situations, they give a continuous level, rather than a cyclical manner.
Few studies have considered the role of androgens or the effects of hormone treatments in male

Question 6

What role does oestrogen generally have?

Answer 6

Pleiotropic: many targets in the brain. 
Development: potent neurotropic factor (influencing brain development and organization). 
–	Migration
–	Neurite outgrowth
–	Synaptogenesis
–	Growth factor production
–	Apoptosis
In the adult brain they are thought to play a role in memory and cognition as well as postural stabililty, movement and fine motor skills and Brain plasticity, e.g. neurogenesis, dendritic spine density

Question 7

What are oestrogen indirect and direct actions?

Answer 7

Direct: Brain
Indirect: cholesterol and alters immune function that impacts brain.
Brain - promote survival by acting on cell death cascades
Promote synaptic plasticity - neurite sprouting and regeneration and modulates neuronal function and synaptic transmission

Question 8

What is generally the difference between ERs?

Answer 8

ERα- mRNA widely distributed in many brain regions in males and females
ERβ- mRNA more restricted distribution
ERα and Erβ may co-localise; Proteins can form homo and hetero-dimers

Question 9

Which area is ER alpha mainly found in? Evidence

Answer 9

Hypothalamus
 ER mRNA expression in adjacent brain sections (coronal) of 26 yr old male - looking at radioactive ligands binding to receptor. Particularly PVN, ARC, VMH

Amygdala
Regulation of fear, emotional & stress responses, social behaviour, cognition and has Major connections with areas strongly associated with memory
Key structure implicated in affective disorders (depression, anxiety)
Therefore this provides anatomical evidence that these regions could be affected by hormones

Question 10

Which area is ER beta mainly found in

Answer 10

Hippocampus
Subiculum - ER β dominated where information leaves the hippocampus to influence amygdala, cortical and subcortical structures

Cortex- ERb dominant regions

Question 11

Are oestrogen actions in the brain ER mediated?

Answer 11

• rapid actions in culture (Many studies in cultured cells-but brain neurons only grow in culture at a very young age, how relevant to actual adult cells?)
• ERKO studies - conflicting reports
• some suggestions ER-dependent effects occur at physiological concentrations whilst ER-independent effects occur at pharmacological doses (is the relevance of using 10/100 thousand fold concs of estrogen- higher levels may not be working via the estrogen receptor)

Question 12

What are experimental ways to damage the brain cells? Eg for different disease? And what tissue is used

Answer 12

Generally use primary neuronal cultures e.g. neuroblastoma and pheochromocytoma.
Post stroke – glutamate released. Glutamate is an excitotoxin, but too much is toxic.

For parkinsons disease, selectively use dopaminergic killing such as 6-ohda and mptp

Mitochondrial toxins
Hydrogen peroxide

Serum- needed to grow, has nutrients and growth factors to allow healing to occur. Serum deprivation causes cell to die.

Add amyloid B peptide to cell lines and cultures- Alzheimers.

Question 13

In the absence of any disease model, one experiment to show the effects of oestrogen?

Answer 13

Gave peroxide to challenge cells and then gave oestrogen treatment to see if cells survived. Was a signficant increase in cell survival numbers.

Protective effects not blocked by ICI182 780 (ER antagonist), which has some protective effects of its own.

Question 14

What are the two hallmark features of Alzheimer’s disease? What correlates with memory and cognitive decline>

Answer 14

– Diffuse neuritic plaques predominantly composed of Amyloid β peptide (underlies synaptic dysfunction)
– Neurofibrillary tangles composed of filamentous aggregates of hyperphosphorylated tau protein
• Loss of neuronal synaptic density correlates with memory and cognitive decline better than the extracellular plaque or tangle formation and precedes neuronal loss.

Question 15

Which animal model is used to model Alzheimer’s?

Answer 15

The mouse triple transgenic model:
• PS1(Presenilin-1) Mutations in gene causally linked to many cases of early onset AD. Up-regulation linked to increased vulnerability to excitotoxic injury
• APP (amyloid precursor protein)
• Tau protein
The increase in Aβ accumulation and tau hyperphosphorylation in the hippocampus, subiculum and frontal cortex. decline in working memory performance with aging occur to a far greater extent in 3x-tg AD mice compared with WT.

Question 16

What 3 bits of evidence is there for oestrogen’s actions in Alzheimer’s disease?

Answer 16

1: in TTG mice, oestrogen lead to decreased BETA AMYLOID deposition in the prefrontal cortex, the subiculum and Ca1. This was measured by ovx sham (low), ovx (high) and ovx and E2 (low)
2: at 6 months of age, oestrogen lead to decreased TAU PHOSPHORYLATION. Same results as above. But there were low n numbers and large error bars.
3: E2 regulates spontaneous alternation behaviour (Y-maze, memory test) in 3xTg-AD but not WT mice

In 3xTg-AD, decreased spontaneous alternation behaviour in ovariectomised animals. Treating with E2 in 3xTg-AD transgenic animals can restore memory.
Ovx wild mice had no effect

Question 17

What is the effect of progesterone on alzheimers? 2 evidence - think 2 different ways that alzheimers acts

Answer 17

Progesterone attenuates the effects of E2 on AB accumulation in 3xTg-AD mice
OVX (When you ovariectomise, also removing progesterone), OVX+P4, OVX +E2+P4
So OVX+P4- does not have an effect
OVX+P4+E2- also prevents E2 from having its effect on decreasing AB load
But in this study, did not give OVX+E2 on its own. Should have had this as another control

P4 reduces tau hyperphosphorylation in ovx 3xTg-AD mice
In this case, progesterone doesn’t interfere with effect of estrogen
Progesterone- beneficial in effects on tau, but not AB
Only used one dose of these hormones- limitation

Question 18

What do oestrogen and progesterone results for alzheimer’s suggest?

Answer 18

That there could be an optimum hormonal therapy to treat alzheimers. Due to the different presence of ERa and ERb across different brain regions, oestrogen has different effects in different areas in beta-amyloid accumulation. These results support continued investigation of SERMs as an alternative to oestrogen-based HT in reducing the risk of AD in postmenopausal women.

Question 19

How might oestrogen be acting in alzheimer’s disease?

Answer 19

Genomic signalling- Estrogen may be signalling through CRE and ERE on BCL-2 family- genes involved in anti-apoptotic pathway
Non-genomic signalling- can activate PI3K, PKC cascades- these reduce apoptosis
Affects non-amyloidogenic processing of AB precursor protein
Increased expression of Neprilysin, enzyme that breaks down AB, and IDE enzymes

Question 20

How do male and female transgender mice naturally progress?

Answer 20

With aging, mice exhibit higher levels of Aβ-IR & number of plaques than males CA1, subiculum and FCX.
Spontaneous alternation behavior females deteriorated to a greater extent t- so sex has effect on behavior

Hormones and hormone mimics offer novel potential as targets for neuroprotective strategies in AD- further investigation needed, looking at dose levels.

Question 21

What epidemiological evidence is there for stroke?

Answer 21

• Risk of stroke is lower in premenopausal women than in age-matched men but increases post-menopausally to equal men by 65 years.

Conflicting studies on the use of HRT
• Some studies report that continued use of estrogen may reduce risk of stroke post-menopausally by up to 50%
• Other studies find that ERT can increase mortality and morbidity after stroke

Question 22

What is an animal model for stroke? What mechanism? What is used as a control?

Answer 22

Take mice. Ovx and then E2/ vehicle. WT and ERKO mouse> occlude middle cerebral artery>24hours collect brain slices. Oestrogen replacement reduces the size of these infarcts in mice.

Estradiol prevented the injury-induced downregulation of
bcl-2 mRNA in the injured cortex
• On lesioned side of the brain, E increased the antiapoptotic factor bcl-2 compared to control
• Non-lesioned side was also used as a control- treatment did not have any effect

• Perikaryal damage- damage to the neurone itself- dose dependent decrease after E2 treatment
• Axonal pathology (look at amyloid precursor and neurofilament)- no effect of E2 treatment

Question 23

What are the problems with using oestrogen in modelling stroke?

Answer 23

Dose (physiological vs pharmacological), formulation/ route of administration (oral, injection, transdermal), duration

• high doses: production of thrombogenic factors (transdermal route is safer and less side effects, no 1st pass metabolism)
• Should consider effects in intact animals (males and females) Would be ideal to give E2 after insult, to show heres lesion and E2 can prevent effects. Not Ovx
• Clinical studies vary in timing of HRT in relation to onset of disease - prior to onset may be protective; not effective, even deleterious, after onset
• clinical studies have used a variety of estrogenic substances (conjugated equine estrogens) alone or in combination with progestins (eg. WHI study) vs principal use of oestradiol in animal studies
• animals models may poorly reflect the processes underlying human disease.

Question 24

What happens in Schizophrenia?

Answer 24

Severe mental illness, typically manifesting as severe psychotic illness with onset in early adulthood; characterised by bizarre delusions, auditory hallucinations, thought disorder, odd behaviour and progressive deterioration in personal, domestic, social and occupational competence, all occurring in clear consciousness.

Question 25

What is the difference between male and females in schizophrenia

Answer 25

There is overall equal prevalence, however men after affected at a younger age (women more bimodal) and men have it more severe. Males are more likely to have negative symptoms, women positive symptoms.

Question 26

What is the aetiology of schizophrenia?

Answer 26

• Decreased brain weight and volume (especially reduced volume of the hippocampus, amygdala and the parahippocampal gyrus) (MRI studies)
• Enlarged ventricles. Enlarged lateral and third ventricle
• Decreased activation of frontal lobe and increased activation of temporal lobe (functional neuroimaging studies) Smaller medial temporal lobes
• Pre-pulse inhibition disruption
• Reduced cortical volume
• Reduced cortical grey matter

Question 27

What is the hypothesis alzheimers pathophysiology? 4

Answer 27

Dopamine hypothesis:
• Due to excess dopaminergic activity
• Amphetamines and levodopa increase dopamine and cause positive symptoms in non-schizophrenics
• All effective antipsychotic medications are dopamine D2 receptor antagonists
Serotonin hypothesis:
• SZ due to excess serotonergic activity
• LSD and psilocybin (5HT receptor agonists) cause positive symptoms in non-schizophrenics
• Newer antipsychotics are potent 5HT receptor antagonists
EAAs:
• Insufficient EAAs or receptors (eg NMDA receptors) are implicated in SZ
• Low CSF glutamate; decreased glutamate receptors in temporal lobes
• Single dose of PCP (non-competitive NMDA receptor antagonist) causes positive and negative symptoms in non-schizophrenics
Phospholipid membrane hypothesis:
• Abnormalities of phospholipid metabolism (neuronal membranes) implicated in SZ
• Use of omega-3 fatty acids in SZ

Question 28

What evidence is there concerning oestrogen in schizophrenia?

Answer 28

• Estrogens protective against psychosis
• Correlation of fluctuating estrogen levels with severity of symptoms
• Antenatal vs. postpartum psychosis. Psychotic episodes increase greatly in schizophrenics post pregnancy
-Variants in oestrogen receptor alpha are associated with varying degrees of schizophrenia

Question 29

What are pre-pulse inhibition studies?

Answer 29

Pre-pulse inhibtion studies – a particular physiological phenomenon. If we are exposed to a particular stimulus eg auditory. If we before we are exposed to a mini stimulus, our response to secondary stimulus is less. sensorimotor gating deficiencies reproduced using a 5-HT1A agonist in rodent models. In schizophremcis there is inhibition of the pre pulse stimulus.
In support of this theory, male mice which lack the aromatase enzyme, and hence estrogen synthesis, develop a disruption in baseline PPI characteristics around 12 months of age, whereas females do not15. A similar effect is induced by ovariectomy of female rats.
Treatment with estrogen conversely inhibits the disruption of PPI. In male rats, castration significantly reduces the effect of 8-OH-DPAT, while testosterone treatment reversed this effect15. Thus, estrogen appears to be protective against disruption of PPI in rats, at least by 5-HT1A receptor stimulation, while testosterone was facilitatory. In women, disruption of PPI by treatment with the 5-HT1A receptor partial agonist buspirone, could be prevented by concurrent treatment with estrogen, evidence that supports that the above may happen in humans too

Question 30

For schizophrenia, what evidence is there for oestrogen receptors?

Answer 30

variants in the estrogen receptor alpha gene and its mRNA may also contribute to risk for schizophrenia. Thus, the variation in the ESR1 gene is associated with schizophrenia >may involve alternative gene regulation and transcript processing. This means that is not the circulating hormones, but the brain response to them that may be dysrupted in schizophrenia due to inheritance of mutated forms of the gene. Similarly, androgen receptor gene mutations, may confer vulnerability in men

Question 31

Epidemiology and symptoms of parkinson’s?

What is the biggest risk factor?

Answer 31

• Mask like face, pill rolling tremor, flexion of trunk, shuffling movement (Primary symptoms)
• Depression, anxiety, dementia and problems with sleep, speech and swallowing (2o symptoms)
• Second most common neurological disorder, 5% linked to genetic causes such as PINK
• After age, biggest risk factor is being male (1.5:1). However, the ratio of males to females does not change after the menopause – may not just be hormonal.
  Prominent is environmental toxins that dopaminergic neurons are particularly sensitive to. Eg agricultural workers.

Question 32

What is the main pathological finding in Parkinson’s?

Answer 32

loss of DA neurones in the SNc that project to the striatum, forming the NSDA pathway. This controls fine movements

Question 33

What physical evidence can be seen?

Answer 33

• Can see that lose pigmentation (neuromelanin) in brain in parkinsons. Lewy bodies – alpha sy-nuclean found (genetic links). Bodies that store protein, not clear if they are causing damage or are there to prevent the damage. They are characteristic of parkinsons disease

Question 34

What is current treatment for Parkinson’s? Problems

Answer 34

Current treatment relies on the replacement of dopamine, not the loss of neurons. As neurons are lost have to replace more and more dopamine. Give L dopa, precursor, so can cross the blood brain barrier but this relies on having a few surviving neurons.
Bromocriptine is an agonist that can be used but is not selective for the striatum meaning that can affect other regions of the brain such as the pre frontal cortex leading to schizophrenia like symptoms.
Need compound to modify the disease and doesn’t just treat the symptoms.

Question 35

What factors contribute to sex differences? What sex differences can be seen?

Answer 35

• genetic factors, including factors linked to the sex chromosomes. SRY expressed in dopaminergic neurons in SNC. Would have thought that SRY only expressed in testes.
• life-style, especially agri-toxins
• sex hormones
Sex differences in the clinical profile
- females present with a milder phenotype in early clinical stages (but tend to get worse depression); less frequent rigidity, but greater incidence of PD-associated depression;
later onset than men.

Sex differences in treatment responses: eg men and women have difference liver metabolism

Question 36

What evidence is there the pathway is sexually dimorphic?

Answer 36

1) After age, biggest risk factor is being male and this difference is maintained after menopause
2) Menstrual cycle variatiors:
- Sensorimotor function in normal women (menstrual cycle, studies have found they have better fine motor control pre-oestrous when oestrogen levels are high)
- Premenopausal women with parkinsons, symptoms improve during pre-oestrous stage
3) Retrospective epidemiological data of women on HRT
- studies suggest that, in postmenopausal women, PD symptoms worsen after withdrawal of HRT
- HRT lowered UPDRS score (united Parkinson’s disease rating scale)
- Oestradiol replacement therapy was associated with a reduced incidence of PD, a delayed onset of PD, and a better cognitive performance in women with PD, but not all studies are in agreement.
- Prospective, double-blind studies have been contradictory, but used relatively small patient samples.
- Properly controlled, large scale prospective studies are needed. Clinical studies do not permit investigations into the nature of oestrogen’s actions
- These do not tell us information about men
4) Rodent studies-evidence for sex differences and hormonal influence on NSDA pathway.
Cocaine=blocks DA uptake in VTA
Females have greater locomotor response to cocaine.
5)Oestrogen receptors are expressed in the nigrostriatal pathway

Question 37

What experimental models are there for PD? 2 major overall types

Answer 37

1. Lesion models
   • Rodents
   – MPTP. 6-OHDA: Anaesthatise animal and surgery>Only need to lesion one SNc and has no effect on contralateral side, other side as its own control. It mimics key features of PD such as oxidative stress, infiltration of microglia, excitotoxicity and is progressive in nature.
   – Methamphetamine (MA)
   • Can only model dopaminergic> MPTP, 6OHDA (toxins that get taken up by dopaminergic neuron reuptake transporter and then destroy neuron. MTPT injected peripherally however and can affect other pathways, seem to cause some cardiac causes of death
   • DON’T GET LEWY BODIES IN THE RAT MODEL, BUT RATS THEN DO NOT ACTUALLY DEVELOP PARKINSONS

•	Non-human primates (MPTP)
•	Mimic aspects of processes that may initiate or propagate neurodegenerative processes
–	Mitochondrial dysfunction
–	Excitotoxicity
–	Inflammation
–	Glial activation
–	Trophic factor deficiency
–	apoptosis
–	Oxidative stress

2. Transgenic models (may mimic hereditory forms of disease) with disruptions to genes for
   – Alpha synuclein
   – Parkin
   – Ubiquitin C-terminal hydrolase (UCHL-1)
   – Superoxide dismutase

HOWEVER ARE LACKING STUDIES FOR HORMONAL INFLUENCES

Question 38

Which PD studies have shown oestrogen being protecting.

Answer 38

OVX Mice in MPTP studies looking at dopamine in striatum. Intact (more), MTPT (less), MPTP +17b E2 (more), MTPT +17aE2 (less - biologically inactive isomer)
OVX rats and 6OHDA in striatum. No difference when no lesion but when lesioned, E2 increased number of dopaminergic pathways.

Looking at dopamine and dopamine metabolites in females di oestrous and pre-oestrous, at di oestrous less oestrogen and less dopamine.

Question 39

What is the role of ER alpha/beta?

Answer 39

DO NOT CLEARLY KNOW WHAT THE RELEVANCE OF ER ALPHA AND BETA IN THE SNC AND STRIATUM IS. (Only Snc seems to be lacking er alpha).

Question 40

What did gender studies on PD show?

Answer 40

In striatum, giving lesion 6OHDA. Much bigger reduction in males compared to females (lesioned versus non lesioned side). Both in striatal dopamine content and substantia pars compact

Question 41

What experiment was there to show what happens in males? What did it show about males? Conclusion?

Answer 41

Give lesion at baseline, 20% loss in females compared to 50% loss in males. Remove oxv greater lesion but then improves with oestrogen treatment. Oestrogen appears to be the main factor missing, protective in females.

Castrate the male – lesion gets smaller. Testicular factor that could be making the male animals more susceptible. If you treat animals with DHT, did not reverse the effects of castration. Must not be testosterone, perhaps testoserone converted to e2. This was seen because e2 then reversed these effects, lesion bigger.
Suggest that aromatization of testosterone to oestradiol in brain seems to worsen dopamine

Males: gonadal factors are not protective and may even exacerbate striatal DA loss. Females: they are

Question 42

However, why is the striatum not the whole story with PD

Answer 42

•Hormonal manipulations that affect DA levels in the injured striatum do NOT affect DA cell loss in the SNc

Question 43

Describe experiment to look at substantia nigra? What was the conclusion?

Answer 43

Results
1.Measure TH (tyrosine hydroxylase-rate limiting step in DA synthesis) in SNc Control =MORE CELL numbers in M than F

LESION WITH 60HDA Dose dependant loss of TH in both sexes
LARGER LOSS MALES-replicated in several other studies.
Despite there being more neurones in the M SNc, lesioning seems to have a larger reduction in neurones in males than in F.

In animals not treated with 6-OHDA, when Gdx+placebo, Gdx+DHT or GDX+E2, no effect on TH-IR cell number
Something else must be accountable for differences in DA cells in SNc between males and females (M>F).
Hormonal influences on 6-OHDA-induced striatal dopamine depletion are independent of effects on cell survival

Question 44

What might be happening?

Answer 44

In humans suffering from PD, surviving neurones and their associated circuitry have a remarkable capacity to compensate for cell loss:
• Increased TH expression & DA synthesis
• Increased DA release per impulse
• Reduced DAT
Therefore high capacity to adapt keeps system going for long time.

Circulating oestradiol positively influences process involved in adaptive mechanisms in female, not male, rats

Question 45

How do you test the male versus female adaptations?

Answer 45

OVX both sexes and then replace oestrogen/ testosterone.
Oestrogen increases only in females: TH expression and DA synthesis, improved effects of psychotropic drugs and decreases only females (striatal dat levels and uptake, slight decrease in males) and gabaminergic inhibitory tone

Question 46

Which other system is involved in plasticity? And gender roles?

Answer 46

Nor-adrenergic transmission to the NSDA system from
the Locus Coerulus
influences NSDA activity and is known to affect its adaptive responses to injury
•In female mice TH expression in the LC is up-regulated by circulating E2
•In male mice TH expression in the LC is down-regulated by circulating T after conversion to E2
Female rats have improved recovery from 6-OHDA lesions compared with males,
as observed from locomotor deficits discernable in specific tests. Compensation
mechanisms may be better in females

Question 47

Looking at the substantia nigra not the striatum, what conclusions can be drawn

Answer 47

• Prevailing endogenous sex hormone levels are not responsible for sex differences in DA cell loss in the SNc in experimental PD
• Instead, hormonal influences on adaptive compensatory mechanisms within the surviving neurones appear to be the targets for circulating hormonal influences in models of early PD.
• However, the findings that E2 in the systemic circulation (E2 or T metabolized to E2) protects against striatal DA loss in females but not males suggests that the plastic compensatory responses to injury are sexually dimorphic.
Together these findings could account for why physiological levels of oestrogens appear neuroprotective at the level of the striatal DA terminals, but not at the cell bodies in the SNc.

Question 48

What might be a next point to discuss with relation to gender differences?

Answer 48

Hypothesis
Sex differences in organization of the NSDA system and its regulatory input underlie sex-specific effects of oestrogens.
Although the NSDA system performs essentially the same functions in males and females, several lines of evidence suggest that the similarities are achieved by different means
Imaging studies- NMR, PET- tests of cognition- males and females perform same but different parts of the brain lights up
Sex differences are apparent in the shape of the rat and mouse SNc and the numbers and topographical organization of SNc dopaminergic neurons
-VTA already shown to be sexually dimorphic in structure
-SNC- female volume is much bigger, but males have increased cell number

Sexually differentiated inputs to the NSDA system that are oestrogen-sensitive
(at least in females) include the mesocortical DA pathway and the serotonergic
system of the dorsal raphe
Suggests sex differences in input systems

Question 49

A final general reason why males may develop PD?

Answer 49

In the normal brain, genes involved in signal transduction and neuronal maturation were up-regulated in women, whereas genes implicated in PD pathogenesis, when harbouring specific mutations (e.g. α-synuclein and PINK-1), were up-regulated in men. These studies demonstrate that gene expression profiles in normal SNc DAergic neurons are sex-specific and suggest a bias in males which may underlie the predisposition to develop PD