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Unveiling Pathogenic mechanisms in Niemann-Pick type C disease

Presentation for Parseghian Foundation Conference 2013

Michael Castello

on 9 November 2013

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Transcript of Unveiling Pathogenic mechanisms in Niemann-Pick type C disease

Unveiling pathogenic mechanisms in Niemann-Pick type C disease
Michael A. Castello
Relevant Background
Molecular basis of NPC
Several views of pathogenesis
Our Expansive Approach
Preliminary Results
Widespread gene expression analysis of pre-symptomatic NPC mice
Surprising effect of APP, typically associated with Alzheimer's disease
Early results from large-scale metabolic simulations
Future Possibilities
Model system
Generate gene expression database
Relevant Background
Molecular Basis of NPC
NPC is caused by mutations in the genes encoding either NPC1 or NPC2 protein
These mutations have multiple effects:
Accumulation of cholesterol and other lipids in endosomes
Production of
amyloid beta (Aβ)
phosphorylated tau protein
Neurodegeneration and death
Two Hypotheses
NPC is primarily a disorder of cholesterol transport
NPC is primarily a disorder of
sphingosine transport;
cholesterol accumulation is a
Classical Hypothesis
Observations of cholesterol buildup in endosomes prompts the assumption that NPC1 and NPC2 are responsible for cholesterol transport
Genetic mutation
Functionally impaired protein
Cholesterol accumulation
Alternative Hypothesis
Sphingosine transport
Calcium ion depletion
Platt Laboratory [1]
Lipid storage
What is the actual mechanism of neurodegeneration in NPC?
Where do Aβ and tau fit in?
What practical use is this information?
We can observe and attempt to alleviate the effects of the mutation; however, it remains unclear why they occur
Knowledge concerning the function of NPC proteins creates a foundation for the design of treatments that target the earliest stages of the disease, where intervention would be most effective
Currently we can treat symptoms
Ideally, we would target the source
Learn more about the mechanisms of NPC in order to design more effective treatments
Our Expansive Approach
Model System
Gene Expression Database
Mice with mutation in NPC1 gene, faithfully recreating the symptoms observed in the human disease [2]
Analysis performed on pre-symptomatic mice, before widespread dysregulation has occurred
Should allow for separation of ultimate causes from subsequent consequences
Collected cerebella and cortex from mice
Extract RNA from brain tissue and analyze using Agilent mouse v2 microarray platform
Probes for changes in approximately 40,000 genes compared to control mice
Preliminary Results
Significant Changes in Gene Expression
Initial data from microarray
Examples of Relevant Genes
Group into functional relationships
Very low-density lipoprotein receptor
Cholesterol metabolism, membrane trafficking, myelination
decreased 2-3 fold
in NPC mice,
in cortex and in cerebellum
Acid ceramidase
Expression is
in cerebellum of NPC mice
Tetraspanin 2
Expression is
cortex and cerebellum of NPC mice
Catalyzes metabolism of
Mutations cause Farber disease, a
lysosomal storage disorder
Present in
Involved in
neuronal migration
during development
Cerebellar changes are
with changes previously observed by Liao et al [3]
uncharacterized in humans
Based on similarities to other proteins, may help
form and stabilize
myelin sheath
Amyloid Precursor Protein: Unexpected Importance
APP is typically associated with
Alzheimer’s disease
Parent protein responsible for
amyloid beta (Aβ)
also shows
pathology involving
Aβ and tau
Cholesterol accumulation
Tau aggregation
Neurological Degeneration
NPC Mice with APP Knockout
In work first shown here, Dr. Ana Nunes
removed APP
NPC mice
Aβ aggregation
Possible Sequence
When APP is removed, the NPC phenotype is
severely exacerbated
newfound significanc
e of APP underscores the need to understand the mechanism of NPC
Collaboration with Dr. Clyde Phelix at the University of Texas San Antonio
Transcriptome-to-Metabolome™ Biosimulation
Novel use of our microarray data to supplement a large-scale model of cellular metabolism
Beginning with gene expression data, the TTM simulation calculates reaction kinetics including...
Enzyme activity
...ultimately using these values to determine metabolic output
Simulation model validated in human brains [6]
Using this model, a non-functional NPC1 protein predicts
early changes in sphingosine
This simulation model can be
to predict
changes in localization
Future Possibilities
We are uncovering
valuable pieces of information
on the
sequence of events
in NPC disease progression
Identifying which genes are most
directly affected
by NPC helps us select the
most valuable genetic targets
for further study
Metabolic simulation can create a
virtual time course
that suggests
targets for early intervention
that may be able avert the widespread downstream consequences
Overall, our data is creating a foundation from which we can more intelligently select and design potential therapeutic interventions
Dr. Salvador Soriano
Kristy D. Howard
Dr. Ana Nunes
Dr. Clyde F. Phelix
Longo Lab

Oberg Lab

Kirsch Lab
Loma Linda University Department of Anatomy
Presentation CC-BY-SA 2013 Michael A. Castello. Literature references follow and are also available upon request. Branding by Caitlyn Mayers Design. This has been a modulate-free presentation.
Great Scott!
Biosimulation Results
This map traces the
cholesterol synthesis pathway
from beginning to end
Differences between
control and NPC mice
are highlighted
Neurobiol Dis. 2011 Jun;42(3):349-59. doi: 10.1016/j.nbd.2011.01.028. Epub 2011 Feb 17.
APP-Related Genes
NAD(P) dependent steroid dehydrogenase-like protein
Mutations cause
buildup of cholesterol


resulting in
CHILD syndrome
Active in
cholesterol biosynthesis
decreased in cortex
of APPko and APPko/NPC mice
Hydroxysteroid (17-beta) dehydrogenase 7
decreased in cerebellum
of APPko and APPko/NPC mice
Active in
cholesterol biosynthesis
Important for fetal neuronal development [5]
RAB3 GTPase activating protein subunit 1
increased in both cortex and cerebellum
of APPko and APPko/NPC mice
Active in
membrane trafficking
Mutations affect
neurological development
(Warburg Micro syndrome)
Exploring Combined Effects
Microarray analysis was performed to search for
combined effects
of the two conditions
Alzheimer's Research Trust, UK
Medical Research Council, UK
[1]E. Lloyd-Evans, A. J. Morgan, X. He, D. A. Smith, E. Elliot-Smith, D. J. Sillence, G. C. Churchill, E. H. Schuchman, A. Galione, and F. M. Platt, “Niemann-Pick disease type C1 is a sphingosine storage disease that causes deregulation of lysosomal calcium,” Nat Med, vol. 14, no. 11, pp. 1247–1255, Oct. 2008.

[2]S. K. Loftus, J. A. Morris, E. D. Carstea, J. Z. Gu, C. Cummings, A. Brown, J. Ellison, K. Ohno, M. A. Rosenfeld, D. A. Tagle, P. G. Pentchev, and W. J. Pavan, “Murine Model of Niemann-Pick C Disease: Mutation in a Cholesterol Homeostasis Gene,” Science, vol. 277, no. 5323, pp. 232 –235, Jul. 1997.

[3]G. Liao, Z. Wen, K. Irizarry, Y. Huang, K. Mitsouras, M. Darmani, T. Leon, L. Shi, and X. Bi, “Abnormal gene expression in cerebellum of Npc1-/- mice during postnatal development,” Brain Res, vol. 1325, pp. 128–140, Apr. 2010.

[4]A. Nunes, S. N. R. Pressey, J. D. Cooper, and S. Soriano, “Loss of amyloid precursor protein in a mouse model of Niemann-Pick type C disease exacerbates its phenotype and disrupts tau homeostasis,” Neurobiology of Disease, vol. 42, no. 3, pp. 349–359, Jun. 2011.

[5]H. Jokela, P. Rantakari, T. Lamminen, L. Strauss, R. Ola, A.-L. Mutka, H. Gylling, T. Miettinen, P. Pakarinen, K. Sainio, and M. Poutanen, “Hydroxysteroid (17beta) dehydrogenase 7 activity is essential for fetal de novo cholesterol synthesis and for neuroectodermal survival and cardiovascular differentiation in early mouse embryos,” Endocrinology, vol. 151, no. 4, pp. 1884–1892, Apr. 2010.

[6]C. F. Phelix, R. G. LeBaron, D. J. Roberson, R. E. Villanueva, G. Villareal, O. B. Rahimi, S. Siedlak, X. Zhu, and G. Perry, “Transcriptome-To-Metabolome Biosimulation Reveals Human Hippocampal Hypometabolism with Age and Alzheimer’s Disease,” IJKDB, vol. 2, no. 2, 2011.
Michael A. Castello
June 13, 2013
Full transcript