This document summarizes research on lysosomal membrane permeability (LMP) and its implications for neurodegenerative diseases like neuronal ceroid lipofuscinoses (NCLs). The researchers found evidence that LMP occurs in mouse models of two forms of NCL, CLN2 and CLN3, resulting in the formation of protein aggregates. Normally, LMP triggers a response to sequester damaged lysosomes, but this "lysophagy" response is impaired in NCL models. As a result, aggregates containing lysosomal contents accumulate and may contribute to neurodegeneration. The researchers are testing the drug arimoclomol, which could prevent LMP, in hopes of reducing neurodegeneration in CL

1. Lysosomal Membrane Permeability: Implications for Disease and Therapeutic Potential
Matthew C. Micsenyi, Ph.D. & Steven U. Walkley, D.V.M., Ph.D.
Sidney Weisner Laboratory of Genetic Neurological Disease, Albert Einstein College of Medicine, Bronx, NY
matthew.micsenyi@phd.einstein.yu.edu
Rose F. Kennedy Intellectual and
Developmental Disabilities Research Center
WHAT THIS
MEANS FOR
THERAPY
LMP might be a target for
therapeutic intervention in
NCL disease.
The drug arimoclomol
upregulates the heat shock
response and expression of
the protein HSP70. HSP70 is
known to stabilize lysosomal
membranes and prevent LMP.
Arimoclomol is currently in
phase II/III clinical trials in the
U.S. for the treatment of ALS.
We are evaluating the use of
arimoclomol in treating CLN2
and CLN3 mice, with the goal
of preventing LMP and
neurodegeneration.
Lysosomal Membrane Permeability, Protein Aggregates & Neurodegeneration
 Lysosomes are the major recycling centers of cells, and are impaired in NCL disease.
 Our studies have shown that lysosomes rupture in brain cells in CLN2 and CLN3 mouse models.
 Lysosome rupture is known as lysosomal membrane permeability (LMP), and this causes cell death.
 We have found that LMP results in a response that stimulates the formation of protein aggregates.
 Importantly, we have found a deficiency in CLN2 & CLN3 disease to further deal with LMP.
 We are currently testing drugs to prevent LMP in mouse models of CLN2 & CLN3 disease.
Research Support
Evidence of LMP in CLN2 and CLN3 Mice
ML
PCL
GCL
CLN3 Cerebral Cortex
CLN3 Cerebellum
p62 Lysosomes SCMAS merge
N N
5μm
Neuron Microglia
N N
Large p62
aggregates
(green). Note the
shell-like
morphology of
p62. Lysosomes
are labeled in
blue and SCMAS
storage is labeled
in red.
ML: molecular
layer;
PCL: Purkinje cell
layer;
GCL: granule cell
layer.
35μm
Cortical neuron and
activated microglia
(based on morphology)
in the CLN3 mouse
brain exhibiting p62
aggregate
accumulation. p62 is
localized in a shell-like
pattern around
lysosomes and SCMAS
storage indicating LMP
is occurring in both cell
types. The area of p62
accumulation is shown
at a higher magnification
below each channel.
“N” represents the
nucleus of the neuron.
Cover Image from Micsenyi et al. (2013).
CLN2 mouse cerebellum showing p62 aggregates
(green). p62 is a marker for LMP.
CLN3: p62 Aggregate in Cerebellum
p62 Lysosomes SCMAS merge
2μm
Large p62 aggregate in CLN3 mouse cerebellum localized with
lysosomes and SCMAS storage. p62 is a marker for LMP.
What is Lysosomal Membrane
Permeability (LMP)?
Lysosomal membrane permeability (LMP) is a disease related event whereby the membrane
of the lysosome becomes damaged and permeable. Normally, LMP results in a response that
stimulates the clearance of the damaged lysosome by a mechanism called lysophagy. A
failure in lysophagy to appropriately respond to LMP causes cell death. Our studies suggest
that LMP occurs in NCL disease, and that there is a failure to respond to this event.
Lysosomal Membrane Permeability in NCL disease
Acute Chronic
Lysophagy
Forming autophagosome Impairment
p62
NBR1
ROS
Ubiquitin
SCMAS Protein Mitochondria Enzyme
Lysosome: TPP1 or CLN3 Deficient
1.Potential Causes
Acute: LMP may be caused by reactive oxygen species (ROS) generated by
mitochondria that damage lysosomal membranes. Increased ROS combined
with lysosomal storage of the SCMAS protein may cause LMP.
Chronic: A failure in constitutively active lysophagy may lead to
accumulation of old lysosomes, resulting in LMP.
2.Response
We are the first to show that LMP occurs in the brain of CLN2 and CLN3
mouse models. This is important because LMP directly causes cell death and
is likely involved in neurodegeneration in the NCLs. Our studies have
identified a novel mechanism in which LMP stimulates an initial response
within cells by which the proteins p62, NBR1 and ubiquitin localize to
damaged lysosomal membrane. These proteins surround the lysosome in
order to sequester released lysosomal contents. The resulting structure is a
protein aggregate.
3.Consequences & Failures in NCL
Normally when LMP is stimulated (A), the damaged
lysosomes and their contents are sequestered by p62, NBR1
and ubiquitin as described above (B). These proteins then
facilitate the engulfment of the damaged lysosome by an
autophagosome (C). The autophagosome is then
transported to fuse with an intact lysosome, thereby
redelivering the contents back to the lysosomal system (D).
This process has been called lysophagy.
Our studies have shown that LMP occurs in CLN2 and
CLN3 resulting in protein aggregate formation.
However, lysophagy is impaired (E). As a result, cells
accumulate p62, NBR1 and ubiquitin-positive protein
aggregates containing sequestered lysosomal contents (F).
These aggregates also contain the primary storage protein
SCMAS.
Importantly, LMP can result in the release of lysosomal
enzymes called cathepsins (G). The release of lysosomal
cathepsins in the cell following LMP is known to directly
stimulate cell death pathways (G and H). Furthermore, the
failure to clear aggregates is associated with cell death (F
and H). Our studies have identified that LMP occurs in
CLN2 and CLN3 disease. In addition to evaluating how LMP
contributes NCL pathogenesis, we are currently conducting
a preclinical trial in CLN2 and CLN3 mice by administering a
drug purported to stabilize lysosomal membranes and
prevent LMP. We propose that this therapeutic strategy may
prevent neurodegeneration.
Normal
LMP
p62
NBR1
Ubiquitin
A
B
Forming
C
Autophagosome
Autophagosome
Intact
D
Lysosome
E
F
NCL
p62
NBR1
Ubiquitin
LMP
Lysophagy Initiation
Failure
Aggregate
Released
Cathepsins
Cell
Death
G
H