Lysosomal Membrane Permeability: Implications for Disease and Therapeutic PotentialMatthew 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

WHAT THIS MEANS FOR

THERAPYLMP 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

Rose F. Kennedy Intellectual and Developmental Disabilities Research Center

p62 Lysosomes mergeSCMAS

NN

5μm

Neuron Microglia

NN

CLN3 Cerebral Cortex

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.

Evidence of LMP in CLN2 and CLN3 Mice

CLN3: p62 Aggregate in Cerebellum

p62 Lysosomes mergeSCMAS

2μm

Large p62 aggregate in CLN3 mouse cerebellum localized with lysosomes and SCMAS storage. p62 is a marker for LMP.

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.

ML

PCL

GCL

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CLN3 Cerebellum

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.

Acute Chronic

XLysophagyImpairmentForming autophagosome

Lysosomal Membrane Permeability in NCL disease

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NBR1

ROS

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SCMAS Protein Mitochondria Enzyme

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Lysosome: TPP1 or CLN3 Deficient1.Potential CausesAcute: 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.ResponseWe 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 NCLNormally 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.

NormalLMP

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Forming Autophagosome

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NCL

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