Stimulation of Endogenous Adult Neural Stem Cell Populations

1
Stimulation of Endogenous Adult Neural
StemCell Populations
2
What is Parkinsons Disease?

• Affects 2 of individuals over 65 worldwide
• Caused by the degeneration of dopaminergic
  neurons in the SNc
• Origin unknown, although some genetic links
• Chronic and progressive
• Symptoms include
• Problems initiating movement
• Impairments in gait and posture
• Speech and swallowing disturbances
• Tremors at rest
• Micrographia

3
Circuitry

• Pyramidal system (motor cortex) voluntary
  movements
• Generates motor commands to muscles
• Extrapyramidal system (basal ganglia)
  modulation
• Also receives motor command to determine its
  context and nature ? sends info back to MC via
  thalamus to make movements smooth and precise
• SNc provides input to
• striatum (part of BG)
• Progressive loss of input
• affects extrapyramidal
• system, causing deficits
• in modulation

4
Treatment

• There is no cure for PD
• Current treatments can help alleviate the
  symptoms, but cannot stop PD progression
• Treatment plans typically include both drugs and
  supportive physical therapy
• Use drugs to increase DA levels (e.g. Levo-dopa)
  and decrease ACh levels (e.g. anticholinergics)
• Require careful monitoring ? aversive side
  effects
• Stem cells as a potential cure?

5
Use of Endogenous Neural Stem Cells

• Main idea activate the brains ability to repair
  itself
• Plan of Action
• Stimulate on-going adult neurogenesis
• Direct migration to area of damage
• Differentiation and replacement of degenerated
  and damaged neurons

6
Transplant vs Endogenous

• Embryonic stem cell transplant requires surgery
• Concerns about cell rejection, longevity of
  cells, effects of long-term culturing,
  complications involved with surgery and
  post-surgery care
• Use of endogenous neural stem cells as
  non-invasive cell replacement therapy
• Concerns about errors in migration, long-term
  effects of increased proliferation
• Mechanisms and regulation of adult neurogenesis
  not yet fully understood
• Both concerned with cells ability to form
  correctly functioning afferent and efferent
  projections, tumors, overgrowth

7
Transplant vs Endogenous

• Bjorklund et al (Feb 2002) reported successful
  ESC transplant in Parkinson rat model
• Mouse ESCs transplanted to striatum developed
  into fully differentiated, functional
  dopaminergic neurons
• Evidenced behavioral recovery
• Redmond et al (Jan 2008) reported successful
  transplant of fetal ventral mesencephalic tissue
  into primates rendered Parkinsonian
• Animals showed significant behavioral
  improvements
• Fetal VM tissue grafted to either putamen or
  caudate
• Reported no dyskinesias, tumors, deformations,
  inappropriate stem cell migration, or overgrowth
• Has been proposed to use both transplantation and
  stimulation of endogenous neurogenic response

8
Fallon et al (Dec 2000)

• Unilateral lesion of SN DA neurons using 6-OHDA
  injection at rostral border of SN and VTA
• Transforming growth factor (TGF?) infusions
  delivered using an osmotic minipump (placed SC)
  attached to a cannula (implanted into ipsilateral
  striatum)
• 200 ?l solution with 10, 50, or 100 ?g TGF? in
  aCSF
• Plain aCSF for controls
• Simultaneous infusions with lesion
• Infusions two weeks after lesion
• Controls (infusions without lesion)
• Intraventricular infusion with lesion
• Lesion without infusion

9
Fallon et al (Dec 2000)

• First determined conditions sufficient to induce
  massive proliferation of multipotent stem cells
  originating in SVZ and their subsequent directed
  migration to striatum
• Has been shown that TGF?-responsive stem and
  neuroprogenitor cells in SVZ express EGFr mRNA
• Utilized in situ nucleic acid hybridization

10
Proliferation and Migration

• Lesion with aCSF control infusion no change (a)
• No lesion with TGF? infusion significant
  increase in EGFr mRNA expression in SVZ (b)
• Lesion with TGF? infusion significant increase
  as well
• gt9 days of infusion showed another ridge of EGFr
  mRNA positive cells encroaching into striatum
  from SVZ (c)
• Most pronounced in concurrent lesion and infusion
  but still seen in infusion after 2 wks
• 50 and 100?g appear
• equally effective 10?g
• minimally less effective
• Lack of EGFr in situ signal
• confirms 6-OHDA injection
• lesioned majority of EGFr
• expressing DA neurons in
• SN-VTA (d f)

11
Proliferation and Migration

• Serial time course analysis
• 4-6 days continuous infusion in lesioned show
  thickening of SVZ (a)
• By 9 days, ridge separates en masse from SVZ
  and seen progressively lateral toward
  infusion site at striatum (b)
• Progresses to midstriatum by day 14 (c)
• Seen in concurrent lesion/infusion and in
  infusion after 2 wks

12
Proliferation and Migration

• Directed migration limited to striatum?
• Septal infusions produced ridge
• medially in septum toward infusion site
• originating from mSVZ (d a)
• Infusion into LV of lesioned showed
• temporary proliferation of SVZ cells
• but no migratory ridge

13
Ridge Characterization

• 50mg/kg BrdU IP hourly for 3 days from day
  of surgery
• Increased incorporation in first 3 days
• BrdU cells seen in ridge, striatum,
  external capsule, and cortex adjacent to
  infusion cannula
• Stained for nestin, a cytoskeletal marker for
  early lineage neuronal and glial progenitors
• SVZ and ridge cells nestin from 4th infusion
  day

14
Ridge Characterization

• Stained for s100?, a marker for astrocytic
  lineage, and for ?-III tubulin, a marker for
  neuronal lineage
• 1, 4, 7, and 9 days no significant positive
  staining
• 14 days ?-III tubulin cells (e) as well as
  s100? cells were seen

15
Ridge Characterization

• Doublecortin, a marker for migrating young
  neurons, also present in ridge cells
• Carried out TH and DAT immunoreactivity
• 2nd-3rd week of infusion occasional TH
  and DAT cells seen
• 3rd-4th week of infusion increasing numbers
  of TH (f, g) and DAT cells in striatum
• Some BrdU neurons also DAT or TH

16
Functional Recovery

• Apomorphine-induced rotation technique used to
  test behavioral changes in both lesioned and
  nonlesioned animals
• Apomorphine (DA agonist) causes
  contralateral rotation
• Animal set on rotometer bowl and behavior
  observed for 5 mins
• SC injection of 2.5mg/kg apomorphine
• After 10 mins, apomorphine- induced rotations
  observed at 10-min intervals, each
  session lasting 5 mins for a total of
  15 mins

17
Functional Recovery

• Two sets of behavioral tests
• Unilateral lesion 2 wks later, received 14-day
  continuous infusion of either aCSF or TGF? into
  ipsilateral striatum
• Behavior tested before lesion and weekly for 4
  weeks after, both before and after infusions
• Statistically significant (Plt0.05) 31.5
  improvement in TGF? treated animals vs aCSF
  control group
• Unilateral lesion TGF?, aCSF, or no infusion
  received contemporaneously
• Groups receiving no or aCSF infusion exhibited
  contralateral rotational behavior in response to
  apomorphine injection
• 10 of the 11 TGF? treated did not exhibit
  rotational behavior asymmetry following
  apomorphine injections during entire
  4-week period
• Other behaviors (exploratory, consummatory) did
  not appear adversely affected

18
Adult Neurogenesis in the SN?

• Lie et al (Aug 2002) found adult rat SN contains
  population of progenitors, but only gave rise to
  glial cells in situ
• Zhao et al (Jun 2003) reported evidence for
  neurogenesis in the adult mammalian SN
• Suggests DA neurons die and are replaced
  constantly, but at very low rate (20 new
  cells/day)
• Significant portion of new neurons seem to
  reestablish connection to striatum
• Rate of turnover doubled when mature DA neurons
  were killed off with toxin
• Frielingsdorf et al (Jun 2004) found no evidence
  for new DA neurons in the adult mammalian SN
• Existence controversial ? if it does exist, could
  bypass migration

19
Conclusion

• Results show brain may have ability to repair
  itself with sufficient initiation of repair
  process
• Massive proliferation, directed migration, and
  phenotypic differentiation into TH and DAT
  neurons
• Showed behavioral recovery
• Brain contains stem cells capable of PMD in
  response to exogenously administered growth
  factor
• Uncertain as to whether TGF? injections directly
  or indirectly cause increased proliferation and
  migration
• TGF? may have several mechanisms of action
• Injury signal may promote migration
• Adult neurogenesis in the SN may exist
• Future therapeutic potential for wide range of
  degenerative diseases and acute neural trauma