Neurodevelopment & Neurogenetics

Process of neural induction (early neural tube formation)

BLASTULA STAGE: zygote is single-layered, hollow, spherical structure
↓
GASTRULATION: becomes multilayered, tri-laminar structure

• Ectoderm (skin, nervous system)

• Mesoderm (muscle, connective tissue, blood vessels)

• Endoderm (viscera i.e., gut, lungs, liver)

↓
INVOLUTION: dorsal endoderm rises up via lip of blastula, dragging dorsal mesoderm to form a parallel strip of tissue
↓
NEURAL INDUCTION: notochord ‘organiser’ (part of dorsal mesoderm)

• Antagonists to inhibit BMPs and induce default neural pathway over epidermal

Produces forebrain neural tissue
↓
CAUDALISATION & NEURAL PLATE:

• Signal gradient begins to create posterior neural structures (midbrain, hindbrain, spinal cord)

Column of epithelial cells thickens to form neural plate
↓
NEURULATION: neural plate folds in and down on itself at neural folds

• Neural folds meet at midline and fuse separating the neural tube (becomes CNS)

• Neural crest cells migrate inside that form PNS

• Epidermis cells form a continuous superficial layer

Main subdivisions of CNS at 3 vesicle stage + derivatives

Anterior portion of neural tube swells to form 3 primary vesicles
*from rostral to caudal

1. Prosencephalon → forebrain

2. Mesencephalon → midbrain

3. Rhombencephalon → hindbrain

Posterior portion aka caudal neural tube → spinal cord

Main subdivisions of CNS at 5 vesicle stage + derivatives

Begins to show hints of structure

*from rostral to caudal

Prosencephalon forms:

1. Telencephalon → ‘c’ shape of forebrain, cortex, lateral ventricle

2. Diencephalon → thalamic (epi, sub, dorsal, hypo), 3rd ventricle
   Optic cups → forms the eye and optic nerve (actually a CNS tract)
   

3. Mesencephalon → midbrain (tectum, tegmentum, cerebral peduncles), cerebral aqueduct
   

Rhombencephalon forms:

4. Metencephalon → pons and cerebellum, 4th ventricle
   <separated by pontine flexure>

5. Myelencephalon → medulla, 4th ventricle
   

Caudal neural tube → spinal cord, central canal

Derivates of neural crest

Neural crest cells migrate out from dorsal neural tube:

• Sensory neurons

• Autonomic ganglia

• Enteric neurons

• Schwann cells

• Melanocytes

• Chromaffin cells

• Cranial sensory ganglia

Axes of organisation during development

3 axes of organisation during development:

1. Rostro-caudal axis (aka ant.-post., NEURAXIS)

2. Ventro-dorsal axis (mediolateral prior to neural tube closure)

3. Ventricular-pial (aka deep-superficial, radial dimension)
   → from generation in inner VZ to migration to outer pial surface

Determination of rostro-caudal axis in nervous system regions

<pre-neural plate formation>

1. Neuraxis establishment

• Posteriorizing agents are released from somites, most concentrated at caudal neural plate

• Causes regionalisation via diffusible molecule gradient i.e., brain enlargement (crude axial orientation)
  

2. Neuromere formation: neuraxis is divided into smaller subunits
   

3. Rhombomere segmentation: 8 bulges in developing hindbrain

• Encoded by HOX gene expression (with receptors for RA)

  • Modulated by an A-P gradient of the morphogen, retinoic acid (RA)

• Produce specific cranial nerves, neurons, hindbrain structures

  • Autonomic and motor functions (CN V, VII, IX)

  • 2 segment pattern (exits even #, innervates to odd below)

Determination of dorso-ventral axis in nervous system regions

AT VENTRAL:

• Sonic hedgehog (Shh) molecule released by notochord

  • Induces floor plate cell (FPC) formation at midline
    → like a 2nd notochord, releases more Shh

  • Shh induces neural tube cells at midline to form motor neuron precursors (no notochord = no motor neurons)

• Ventral-to-dorsal gradient of Shh (high to low)

  • High Shh concentration = V3 int., motor neurons [ventral]

  • Low Shh concentrations = V0, V1/V2 int. [dorsal-ventral]
    

AT DORSAL:

• BMP induces differentiation at dorsal end of spinal cord

  • Higher dose at dorsal midline, lower at lateral neural plate

• BMP signalling induces crest cell differentiation

  • Low BMP = DRG (sensory neurons), ANS neurons

  • Medium BMP = Sensory interneuron precursors,

  • High BMP = roof plate cells (specialised glia), epidermis

Mechanisms shaping nervous system region differentiation and their interactions

1. Morphogens
   i.e., Shh, BMPs, FGFs, RA
   → diffusible, activate or repress transcription factors

2. Transcription factor-encoding genes
   i.e., RA receptors, Hox genes
   → regulatory, control expression of other genes

3. Cell-surface or secreted signalling molecule-encoding genes
   i.e., Eph receptors, ephrins, neurotrophins
   → direct, regulate cell interactions

Pattern of neurogenesis and migration for brainstem, spinal cord and thalamus

1. Neurogenesis: occurs in ventricular zone

2. Proliferation: neuroepithelial stem cells undergo mitosis to increase progenitor number

3. Differentiation: radial glia first form a scaffold

4. Migration: postmitotic neurons climb scaffold to migrate superficially, accumulating at the deep border to form the mantle zone

Oldest neurons = superficial
Youngest neurons = deep

Pattern of cortical neurogenesis and cortical neuron migration

1. Neurogenesis: of neuroepithelial stem cells occurs in ventricular zone and subventricular zone

2. Proliferation: radial glial cells (RGSs) undergo mitosis symmetrically to increase progenitor number (split later on)

3. Differentiation & migration:
   1) First postmitotic neurons migrate a short distance to form disorganised preplate (primordial plexiform layer, PPL)
   2) Further neurogenesis forms cell-sparse intermediate zone (IZ) below preplate (becomes white matter)
   3) Waves of superficially-migrating neurons split PPL into 2 layers, divided by the cortical plate (future II-VI)
   → superficial marginal zone (MZ)
   → deeper subplate (SP)
   4) Neurons migrate radially through IZ and past older neurons to take stack above cortical plate
   5) After reaching point of migration, neurons undergo terminal soma translocation and move into position beneath the marginal zone

Inverse pattern to most of the nervous system, where the deeper layers (VI, V) form first, and the superficial layers (I, II) are younger*
*applies only to excitatory neurons (~80%), inhibitory interneurons migrate parallel to cortical layers, less discrete pattern

Correlation between:
> Tangential (parallel) organisation of cortex
AND
> Expression of early signalling molecules

Concentration gradients of morphogens (signalling molecules) in the VZ spatially dictate progenitor cell identity and guide parallel migration of inhibitory interneurons

• Borders of early cerebral cortex release different signalling molecules (i.e., FGFs, EGFs, Wnts, BMPs)

• Creates transcription factor gradients that activate/repress different genes

  • Correlation between molecular signals, structural, and functional cortical organisation

• GABAergic interneurons generated in basal ganglia forerunner (ganglionic eminences) migrate tangentially

  • Directed via molecular signals like NRG1 and Shh

= creates a ‘protomap’ that guides further axonal pathfinding and neuronal connectivity

General mechanisms that establish neural connectivity

• Early identity acquisition and programming post-neurogenesis

  • based on time and location of birth

  • determines exposure to transcriptional factors and therefore differentiation

  • i.e., connectivity, NT, electrophysiological properties

• Axon pathfinding and target selection

  • guided to target via growth cones

  • uses attractive/repulsive chemical cues in environment + fasciculation and guidepost axons

• Synapse formation

  • selecting the correct partner cell, recognised using cell adhesion molecules

  • high specificity, targeting certain laminar or regions of target neuron

How do molecular cues guide topographic map formation?

Via growth cones, the expanded growing tip of an axon with expanding and retracting: lamellipodia (flat processes) & filopodia (thin processes)

• Processes are covered in receptors programmed to what the growing axon is likely to find in its own, ideal environment

  • Unique complement of receptors driving attractive, repulsive, or neutral responses to different things

  • Enables steering through complex environments

• Movement patterns include: extension, adhesion, translocation, and de-adhesion

  • Molecular guidance cues
    1) Soluble (diffusible, chemoattraction/chemorepulsion): netrins, secreted semaphorins, neurotrophins (NGF, BDNF)
    2) Cell-surface (contact-mediated): ephrins, CAMs, membrane-bound semaphorins

  • Extracellular matrix (ECM) adhesion molecules: laminin, fibronectin, CAMs

  • Electrical stimuli: depolarisation/APs can cause retraction/inhibition and reverse the effects of other chemical signals

  • Mechanical & physical stimuli: tension/force to trigger growth or directional change, also fasciculation (attachment to other axons)

How does topographic map formation correlate to brain function?

allows neurons in one region to project to another while preserving spatial relationships

1. Neurons have programmed intrinsic positional identities (anterior vs posterior retina)

2. Growth cones extend axons, course projection guided by molecular gradients i.e., RCGs with cell gradients of Eph receptors, target tissues with inverse gradient of ephrin ligands
   → high Eph retinal neurons are repelled more strongly by high ephrin regions = CHEMOAFFINITY

3. Local cues (contact-mediated, soluble, ECM, electrical, physical) refine direction

4. Activity-dependent refinement of mapping
   → correlated firing strengthens synapses, uncorrelated connections are pruned

Process of neural projection refinement

1. Early development → neurons form excess connections due to axon and dendrite overgrowth, often imprecise

2. Sensory-independent refinement (pre-sensory experience) → spontaneous, correlated firing begins initial circuit structuring

3. Experience-dependent refinement (post-birth/sensory onset) → environmental input and experience promote fine-scale refinement

4. Maturation and myelination → transmission speeds improve, and circuits are stabilized

Involves: synaptic pruning, axon retraction/degeneration (axosome shedding or local degeneration), glial-mediated clearance, and molecular cues

Adult brain regions that correlate with embryonic structures

Telencephalon → forebrain

• cortex, amygdala, hippocampus, striatum, olfactory bulb

• lateral ventricle

Diencephalon → thalamic structures

• thalamus, hypothalamus, epithalamus, subthalamus, retina

• 3rd ventricle

Mesencephalon → midbrain

• superior and inferior colliculi, tegmentum

• cerebral aqueduct

Metencephalon → hindbrain

• pons, cerebellum

• 4th ventricle

Myelencephalon → hindbrain

• medulla

• 4th ventricle

Caudal neural tube → spinal cord

• central canal

Mechanisms of axon guidance and corticogenesis

Relationship between concurrent neural development and formation of functional neural circuits

Decision points along retinocollicular pathway

1. Exiting of neurons at retina to enter optic nerve head

   • Nasal and temporal neurons adhere to laminin (substrate) following to exit point at optic disc

   • Performs 90^o turn when exposed to netrin (diffusible & chemoattractive) to move through optic nerve
     

2. Partial decussation at optic chiasm (EphB1 receptors only present in ventrotemporal RGCs → interact with EphB2 ligands at midline)

   • NASAL crosses at chiasm (contralateral) = absence of EphB1 receptors
     → no response to Eph-B2 signal and therefore decussate

   • TEMPORAL stays on same side (ipsilateral) = presence of EphB1 receptors
     → repelled away from midline and doesn’t decussate
     

3. Target selection & topographic mapping

   • Growth cones identify their topographically appreciate location along A-P and M-L axes
     → A-P driven by EphA (temporal are repelled by high levels in posterior SC, move anteriorly)
     → M-L driven by EphB

   • Rate is slowed and changes from rapid axon extension to complex branching (more filopodia)

Evidence for molecular cues guiding topographic map formation

Membrane stripe assay

Transplanting of different cell membranes to observe chosen growth paths of different axons

Nasal axons demonstrate no bias (ant/post)
→ low expression of EphA receptor → not repelled by ligand and projects caudally

Temporal axons show clear bias for anterior/rostral
→ high expression of EphA receptor → repelled by ligand and projects rostrally

Wnt signalling pathway

Key in rostral-caudal patterning of the CNS (also dorsal-ventral)

• Caudal brain formation: its ligands have dose-dependent action on the caudal neuroepithelium

  • Inhibited by Dkk1 expression in neuroectoderm (anterior)

• Neural tube gradient: progressive caudalisation from forebrain to hindbrain

Key signalling pathways in neurodevelopment [!]*

Wnt: induction, neural plate and crest cell development, early patterning

BMP: inhibition of neural induction, dorsal-ventral patterning

SHH: stem cell proliferation and differentiation, ventral neural tube structures (forebrain)

Notch: balances stem cell maintenance and differentiation (lateral inhibition)

FGF: inhibits BMP, early neural tube induction

BMP signalling pathway

An anti-neural factor, determining whether tissues become neural or non-neural

• Highest concentration at ventral regions (superficial/epidermis)

  • Bind to receptors and phosphorylate to activate Smad proteins
    → R-Smads regulate promoter activity via transcriptional coactivators OR co-repressors

  • Blocked by noggin, chordin

• Controls CNS and PNS development

  • Represses neurogenesis to maintain pluripotency
    → defines from which tissues NCCs are generated (PNS)

  • Cell proliferation and patterning

  • Germ layer formation, gastrulation, organogenesis
    

High conc. = non-neural ectoderm develops (epidermis)

Mid conc. = neural crest cells

Low conc. = neural plate specification from antagonists

FGF signalling

Creates a pre-patterning of neural induction

• Released form organiser precursors prior to gastrulation

  • Activates Sox3

• High concentration in posterior regions

  • with Wnt and Ra specialise posterior neural structures

SHH signalling pathway

Concentrated in floor plate cells of notochord

• Development of the neural tube

  • Acts in opposition to WNT/BMP

• Ventral structure patterning and ventral forebrain

• Neuronal differentiation and proliferation

Notch signalling pathway

Determines left-right asymmetry and segmentation in the mesoderm, for rostro-caudal axis

• Maintains neural progenitors/stem cells, delaying maturity

  • via activation of transcriptional repressor genes

  • suppresses differentiation and proliferation

• Influences timing and location of neurogenesis (when and where in the brain)

  • modulates morphology (neurons, astrocytes, or oligodendrocytes)

  • also migration, plasticity, radial glia maintenance, dendrite development

Transcription factors (and how do they regulate gene expression?)

Regulatory proteins that activate (or inhibit) DNA transcription
→ by binding to specific DNA sequences to activate specific genes

• Specific binding domain (with very high affinity) + effector domain

  • Classified either by binding domain or 3D protein structure

• Key decider in cellular differentiation
  = regulation of gene expression in a tissue-specific manner

• Homeodomain proteins and basic helix-loop-helix (bHLH) are key classes involved in regional identity and axes i.e.,

  • Pax5-6

  • Otx2

  • Hox genes

  • Emx2

Pax6 gene

A transcription factor: promotes development of rostral-lateral cortical regions (upper & outer)

• Opposing gradient to Emx2 on anteroposterior axis

  • Presence downregulates Emx2

• Higher expression in front of cortex

  • Motor (M1) and somatosensory (S1) specialization

  • Promotes glutamatergic

  • Also maintains cortical progenitor cells

Emx2 gene

A transcription factor: promotes development of caudal-medial cortical regions (lower & inner)

• Opposing gradient to Pax6 on anteroposterior axis

  • Presence downregulates Pax6

• Higher concentration in back of cortex

  • Visual specialization (V1) hippocampus and dentate gyrus

  • Establishes ‘protomap’, early regionalisation

Otx2 gene

A transcription factor: creates the midbrain/hindbrain boundary (MHB) via a sharp border with GBX2

• The ‘isthmic organiser’, promotes midbrain & ventral forebrain expression

  • Neurogenesis and oligodendrogenesis, also cortical interneurons

Hox gene

A transcription factor: establishes rostral-caudal (or AP) axis patterning

• Key in early neurodevelopment

  • Progenitor cell specification, neuronal migration, cell survival, axon guidance, dendrite morphogenesis

• Hindbrain and spinal cord formation

  • Hox1-5 in hindbrain VS Hox4-11 in spinal cord*

  • 4 clusters in mammals with spatial and temporal colinearity

*NOTE: HoxA-D refer to cluster location, Hox1-13 refer to paralog group

Transcriptomic studies in neurodevelopment

The complete sequencing of RNA in a cell population = coding + non-coding

• Actively changing, allows identification of expression, what’s switched on/off

  • Measured by number of RNA transcripts to infer activity

  • Can map expression across time stages and spatial regions in vitro

• RNA sequencing: cell suspension → extract RNA → transcribe mRNA fragments → convert to cDNA → sequence and put against genome

• Types: BULK (average expression in a tissue) and SINGLE-CELL: (patient variability via clustering and cell type)

• Uses:

  • Gene responses to environmental stressors (sample exposure)

  • Targeted treatments for specific disorders i.e., comparing cell sequencing before and after treatment

  • Identifying which drugs target which pathways in disease states
    → via changes in up/downregulation

Central dogma of molecular biology

The ‘one-way flow’ of genetic information:
DNA
↓ transcription (specialised enzymes read base pairs)
RNA
↓ translation (instructions for how protein is to be assembled)
Protein

---

• We thought complete DNA sequencing would enable disease and risk understanding at a population level

  • Human Genome Project → genome-wide association & twin studies

  • BUT only a small proportion of risk was explained by DNA

• Hence, majority of human diseases are not explained by single-gene mutations
  = epigenetics

Epigenetic marker + 2 examples of epigenetic mechanisms

Biochemical markers placed on top of DNA that control which genes are switched on/off

• Enables different expression at different times = specialised functions

• Heavily influenced by lifestyle and environment, reversible & dynamic

  • Usually post-translational = performed after removal from ribosome

  • i.e., chromatin remodelling, non-coding RNAs, microRNAs
    

1. DNA methylation = gene silencing
   → addition of a methyl group to a cytosine residue in a CpG sequence

2. Histone modification = transcription activation or repression
   → direct addition of groups (acetyl, methyl, phosphate etc.) to histone tails

DNA methylation

Addition of a methyl group onto cytosine sites via DNA methyltransferases

• Usually on CpG sites or islands (cytosine sites next to guanine nucleotide)
  → located near promoters, have a key role in gene expression

• Causes closing of chromatin structures = less accessible for transcription

  • Net effect of downstream gene silencing

Histone modifications

Post-translational addition of groups to histone tails that can either activate (open) or deactivate (close) chromatin

• Acetyl, methyl, phosphate groups usually control how chromatin is wrapped

  • Inserting proteins into a structure that take up space, forcing it to open

  • Generally open chromatin and increase gene transcription

• Can have ‘writer’ enzymes (i.e., HAT) and ‘eraser’ enzymes (HDAC)

Role of epigenetic changes in normal neurodevelopment

• Gestation and early life are sensitive periods of brain development

  • Parental DNA is demethylated and re-initiated de novo establishing new patterns

  • DNA methylation has an important role in foetal development

• 10% of neurodevelopmental disorders are single-gene mutations
  → 90% are vulnerability genes + environmental risks + epigenetic modifications

  • Most patients exhibit immune dysregulation

  • Elevated pro-inflammatory cytokines, dysregulated peripheral signalling and microglia activity, increased comorbid immune conditions

Maternal immune activation hypothesis

Exposure to maternal inflammation during pregnancy increases risk of neurodevelopmental and neuropsychiatric disorders in offspring

---

Mother with own vulnerability genes has immune activation during pregnancy
(includes infection, autoimmune, asthma, allergy, obesity, poor diet, smoking, pollution, mental illness, psychosocial conditions)
↓
Change in environment of growing foetus altering brain and immune cell development
↓
Foetus with unique set of vulnerability genes is born with an increased risk of x neurodevelopmental disorder
↓
Greater susceptibility to environmental factors later in life i.e., trauma, illness
↓
Higher likelihood of expressing neurodevelopmental or neuropsychiatric disorders

Examples of mutations in genes encoding pathogenic epigenetic modifiers

Single-gene mutations can cause chromatin condensation, alternative splicing, transcriptional activation

Rett syndrome: random single-gene mutation on MECP₂ of X chromosome

• ‘Methyl-CpG-binding protein 2’, affects only females

• Loss of motor skills, language/breathing irregular, seizures, slow growth, ASD-like symptoms

Kabuki syndrome: random single-gene mutation on KMT₂D

• Affects histone methyltransferase (opening chromatin) = too much closure

• Growth delays, musculoskeletal and cardiac abnormalities, intellectual disability

Mendelian/monogenic disorders vs complex/polygenic disorders

Mendelian disorders: from mutations in a single gene following predictable inheritance patterns (rare)

Complex disorders: the combined effect of many small genes (‘unlucky coupling’), influenced by environmental risk factors

Types of Mendelian inheritance patterns

• AUTOSOMAL

  • Dominant: need 1 copy for expression
    → i.e., Huntington’s disease, 50% chance offspring is affected

  • Recessive: need 2 copies for expression
    → i.e., Cystic fibrosis, 25% chance offspring is affected

• CHROMOSOMAL

  • X-linked dominant
    → i.e., Fragile X, Rett syndrome

  • X-linked recessive
    → i.e., red-green colorblindness

  • Y-linked
    

• DE NOVO: arises for the first time in gametes or immediately post-fertilization

  • Can appear ‘recessive’, cannot be determined without sequencing

  • Most produce no effect, located in regions that don’t affect gene function

  • Most people have ~70 de novo variants/gene
    

Types of point mutations
→ including their effects on gene coding sequence and protein function

May or may not affect gene function depending on positioning
(i.e., intergenic, up/downstream, 3’/5’ UTR, introns, exons, splice sites)

• Substitution

• Insertion

• Deletion
  ^ last 2 can cause frameshift if not in multiples of 3^

How do repeat expansions arise (+ their effects)?

Short tandem repeats (STRs) are unstable during DNA replication

• Can expand or contract in length from mother to daughter DNA

  • Usually 3-6 base pairs long, can arise in any position in a genome

• If this repetition occurs in a gene + expands beyond a certain size = pathogenic

  • Size of expansion/no. of copies affects severity and/or phenotype

  • i.e., toxic protein/peptide, silenced expression, RNA can’t be translated

Types of variants (functional consequence of gene mutations)

Can only occur if mutation is in coding sequence (intron)

---

Synonymous: ‘silent’, often base change won’t alter the amino acid produced

• 64 possible triplet combinations, only 20 amino acids
  

Nonsense: aka stopgain, introduces premature stop codon

• Causes truncated or non-existent protein (if nonsense-mediated decay occurs)

• Termination codon is reached and protein is folded = drastic effect on function
  

Missense: aka nonsynonymous, swapping one amino acid for another
→ more subtle effects than truncation, effect depends on:

• How conserved across species the mutated region is (location):

  • CONSERVED: high similarity across mammals = important

  • NON-CONSERVED: less detrimental

• Nature of swapped amino acid:

  • CONSERVATIVE: similar chemical properties

  • NON-CONSERVATIVE: dissimilar chemical properties (worse)
    

Splice-site: in the special conserved sequences at intron-exon boundary needed for normal splicing

• Can cause whole or partial exon skipping and intron retention, insertion/deletion of AAs, frameshift, alternative splice site activation

• Downstream can cause premature stop codons = truncation or total protein loss

Morphogen

A long-range signalling molecule that patterns developing tissues (pluripotent, unspecialized stem cells) in a concentration-dependent manner
↓
Triggers expression of specific transcription factors
↓
Targeted gene expression creates differentiated cell types

Types of genetic mutations

from largest to smallest

---

Gross chromosomal abnormalities: i.e., 3 copies of chr 21 (down syndrome)

Structural variation: whole genes/exons

Repeat expansions: of codons

Point variants: 1 or a few nucleotides

Typical gene structure

Transcribed from 5’ end (promoter) → 3’ end

• Amino assembly starts from initiator codon

• Codes only exons (coding sequences), situated between introns

• Stops at termination codon downstream

Developmental and Epileptic Encephalopathy (DEE)

Clinical features: slowing or regression in development/cognition, epileptiform EEG activity (abnormal spikes), frequent seizures, motor impairment, behavioural issues

Common inheritance patterns: typically de novo and Mendelian (autosomal recessive, AR)

Affected structures & pathways: VGICs & LGICs, synapse proteins, cell signalling (mTOR, GPCRs)
→ SCN1A: non-conservative change in highly conserved protein (α subunit, VGIC)
→ PPP3A: truncation, also highly conserved, affected synaptic vesicle recycling

Epigenetic regulators: mutation affects transcription factors, chromatin remodelers, histone modifiers

Fragile X Syndrome

Clinical features: (evolving over time) severe behavioural changes, social anxiety, language delays, seizures (monogenic form of ASD)

Genetic cause:

• X-linked intellectual disability affecting FMR1

• Repeat expansion disorder in 5’ UTR region (not coding region)

• Normally 6-50x CGG sequences, excess causes late onset toxicity

• Full mutation = histone methylation of promoter, hence epigenetic silencing

FMR1 function & phenotype: RNA binding protein regulating mRNA translation in post-synaptic neuron

• #of FMR1 repeats is unstable during DNA replication and repair (can increase as well in subsequent generations)

• Intermediate → premutation → full mutation

Attention Deficit Hyperactivity Disorder (ADHD)

• ~80% heritability

• Multifactorial/polygenic disease: subtle variations in several susceptibility genes + environment-lifestyle interactions

• Susceptibility genes identified via association analysis
  ^(compare allele frequencies between unrelated unaffected vs affected people for a common point variant (SNP))^

  • Most ADHD variants don’t affect coding sequence, but gene expression levels (amount of protein produced)

  • Risk alleles are variants of small effect (increases risk by 5-10%) → insight into biological pathways for treatment

Autism Spectrum Disorder (ASD)

• ~80% heritability

• Typically combination of de novo + rare/common variants + environmental factors

  • Causative mutations = Mendelian genes (<10%

  • Rare point mutations = larger effect (also copy-number variants), (~10-20%) in synaptic proteins

  • Common variants = smaller effect (shared with scz, ADHD, depression)

• Environmental risk factors: older parents, birth trauma, gestational diabetes, valproate

Genome-wide linkage analysis and next-generation sequencing for mendelian vs complex disorders

Previous gene identification was slow, sequencing individual candidate genes from other epilepsy syndromes, requiring large families

↓

1. GWLA: identify regions with variants shared by affected relatives (uses familial data, SNP markers)
   → how older epilepsy genes were identified i.e., SCN1A on Chr 2
   → more effective for Mendelian disorders

2. NGS (WGS): directly sequences DNA to identify specific, rare variants on unrelated individuals
   → base-pair resolution, shows structural changes
   → more effective for complex or polygenic disorders

Causative genes VS susceptibility genes

Causative genes: mutations directly cause the disease (often Mendelian), in rare, high-penetrance mutations
→ typically rare, familial, early-onset

Susceptibility genes: increase the risk or predispose to a disease (often complex/polygenic), usually common, low-penetrance variants
→ typically multi-factorial, sporadic, late-onset

Next generation sequencing vs genome-wide linkage analysis (GWLA)

• NGS:

  • Whole-exome: enriching protein-coding regions where most mutations have identified to date

  • Whole-genome: high-through put comparison of patient’s whole genome to human reference genome

• GWLA: maps disease genes by tracing large chromosomal segments in families (good for rare, high-effect Mendelian disorders)

Disease heritability VS inheritance

Heritability: how much a trait varies in a population due to genetic factors

• h² = V_g/V_p (proportion of phenotypic variation attributable to genetic variation)

• H² ranges from 0 to 1

Inheritance: the patterns and mechanisms by which genetic traits pass from parents to offspring (i.e., Mendelian or polygenic)

Evidence for heritability in brain disorders (i.e., tinnitus phenotype)

1. Twin studies: more common in monozygotic vs dizygotic twins

2. Adoptive studies: higher concordance between biological parents and adoptees

3. GWAS on self-reported tinnitus

4. Familial aggregation: multiple familial cases with higher prevalence than general population

Common vs rare genetic variation in disease

Common variants (>1-5% frequency) = SMALL effects on complex, polygenic diseases
→ higher allelic frequency, low phenotype penetrance

Rare variants (<1% frequency) = LARGE effects, can also drive Mendelian diseases
→ lower allelic frequency, high phenotype penetrance

Research strategies for new gene discovery in neurodevelopmental disorders (i.e., ME)

• Genotyping (GWAS) (coding OR non-coding): microarray scanning for pre-selected common variants (SNPs) across large populations to find disease associations + ancestry and association test is helpful
  → 'reading some letters of a sentence’, harder to infer meaning

• Exome-sequencing
  → ‘reading keywords of a sentence’

• Genome sequencing (coding OR non-coding): reading the entire DNA sequence to identify an individual’s common and rare variants
  → ‘the complete sentence’, good because non-coding regions have a lot of regulatory information (common variants)

Allele frequency

How common a specific variant of a gene is in a population
→ alternative forms of a gene that arise by mutation found at the same place on a chromosome

What is the rationale for using animal models in neuroscience & neurodevelopmental disorder research?

Genetics are a central factor in neurodevelopmental disorders

• Ethical neccessity

• High control over variables (mice are all also very similar to one another)

• ‘Bridge’ between cells and humans, allowing study of functional consequences i.e., movement

• High degree of conservation in critical pathways (98% homology in causative gene)

Validity and translational relevance of different animal models

Overall, depends on what you’re studying and for how long

i.e., an model for Parkinson’s disease that uses an injection to kill DA neurons
→ GOOD for evaluating neurological changes i.e., electrophysiology
→ POOR for evaluating development and progression of symptoms

i.e., Tourette syndrome models (stronger symptomology in males, good response to human drug treatments)

1. Selective depletion of striatal cholinergic interneurons

2. KO of a high-risk TS gene
   

Example methodologies

• Genetic induction (transgenic, KO, KI, CRISPR-Cas9)

• Pharmacological (neurotoxin or vectors)

• Surgical lesion (i.e., compression injury of spinal cord, arterial occlusion for stroke)

• Environmental or behavioural manipulation (stress, diet, hypoxia/ischemia, sleep deprivation, social isolation)

• Autoimmune or autoinflammation (MS models)

• Humanized (xenografted, organoids)

• Spontaneous/naturally occurring

Ethical frameworks in the context of animal models

REDUCE the number of animals being used

REFINE tests to expose animals to minimal stress, use multiple models

REPLACE animal studies with alternative methods when possible, but must maintain statistical power

consider:

• severity of procedures, pain & distress, long-term housing and wellbeing (avoid single cells when possible), end-of-life decisions

• when is an animal model vs cell model needed (contextual)

• avoid assigning human complexity to rodents, over-interpreting behaviour

How can animal models inform mechanisms and treatments for neurodevelopmental disorders?

Example: The shaker rat is a spontaneous model for cerebellar degeneration

• Animals experience complete Purkinje cell death over time resulting in loss of coordination

• Lack of Slc9a6 due to frameshift mutation
  → encoding protein comprises endosome, performs cell trafficking and recycling

• Similar mutation associated with Christianson syndrome in humans, extreme intellectual disability, epileptic seizures, cerebellar degeneration

  • Affected protein also heavily expressed in hippocampus and frontal cortex

  • Causes cognitive deficits, epilepsy, autism-like symptoms, ataxia

  • Non-motor symptoms = absence seizures (spike-wave, loss of consciousness), cognitive & behavioural deficits

• Loss of incoordination was preventable using AAV

Disease modelling challenges for brain-related research

• New medicines used to require animal testing before human trials
  → limitations as many rodent models lacked similar symptomology to humans for rare gene variant diseases

• Humans have a very high encephalization quotient (actual brain mass / expected brain mass based on body size) and a lot of neurons

• Challenges in recapitulating human physiology and developmental milestones

• Lack of physiological complexity and genetic similarity

2D vs 3D cellular models

2D cultures are a single layer

• Lose cellular interactions

• BUT high throughput and high reproducibility

• Limited phenotype and circuitry for disease modelling

3D brain organoid are spheres, the size of a pea

• Spontaneously grow astrocytes but lack glia and blood vessels

• Better for modelling only certain brain regions (cortex, inhibitory neurons) as forms similar patterned layering to cortex

• Good for neurodegenerative and neurodevelopmental disorders

• Harder to reproduce, higher cost, more complex imaging/analysis

Induced pluripotent stem cells (iPSCs)

Takahashi and Yamanaka transcription factors
→ Oct4, Soc2, Klf4, c-Myc

• Could transform any somatic cell into a stem cell via epigenetic wipeout

  • Reprogrammed via ectopic co-expression of defined pluripotency factors
    ^then also tested for Yamanaka factor expression via immunofluorescence^

  • Using either peripheral blood mononuclear cells (PMBCs) or skin fibroblasts

• Can self-renewal indefinitely in culture and differentiate into all specialised cell types (including neurons, which can’t divide)

• Pluripotent, meaning they can differentiate into all 3 embryonic germ layers

• Can be generated from any healthy person or patient

  • Individual-specific

  • No concern of species differences

• Replicate key features of the human brain like cortical folding and electrical activity

  • In the form of assembloids (spine + skeletal organoids) can create more functional and holistic body systems

Pluripotent stem cells in a dish → form embryoid bodies (ecto, meso, endo) → proliferate → differentiate → organoid balls

USES: cellular mechanisms, connectivity in development, organoids, excitatory-inhibitory imbalance (MEA), synaptic plasticity, drug development, environmental contributions, disease-specific modelling

Utility of modelling Rett syndrome using 3D brain organoids

• iPSC samples are taken from children with the specific gene mutations

  • Human relevance, specific genetic provide

  • Offers insights mouse models can’t provide

• Accurately represents complex diseases (conditions specific to humans)

• Personalized medicine and drug treatments

• Ethical advantage

  • Reduces reliance on animal testing

• Direct insight into disease mechanism

  • How genetic mutations manifest in cells

  • And the effects of genetic manipulation

• Can transplant organoids onto mice for whole-organism physiological testing

• Can be modelled using 2D (neurons, microglia, astrocytes, oligodendrocytes) OR 3D

1 MONTH: neuronal morphology (immunofluorescence)

3 MONTHS: synaptic function (MEA, Ca²⁺ signalling)

6 MONTHS: synaptic phenotype (electron microscopy)

EXAMPLE: can observe synaptic dysfunction on calcium signalling recordings from Rett organoids
→ neurons from Rett syndrome patients have a very low scale bar, low/poor excitability

Rett syndrome

Neurodevelopmental disorder caused by loss of function variants in MECP2 gene

• X-linked, mostly affects females

• Symptoms in 6-18 months of life

• Cannot speak, require constant care, apnea, scoliosis, seizures

• Autism-like symptoms i.e., loss of social interaction

MECP2 has:

1. a MBD = binds to methylated DNA on chromatic

2. a TRD = can stop or start transcription