PSEN1 and mitophagy

This post is background information on a post describing the role of the role of PSEN1 in assembly of CIC7 in the lysosome and the mitophagy of dysfunctional mitochondria in Alzheimer’s Disease. This concept has relevance to all neurodegernative diseases in which mitochondria become damaged and need to be removed. The Martín-Maestro study is nice in that it takes the reader though each stage of mitophagy and relates it back to PSEN1 and lsysome function.

Martín-Maestro P, Gargini R, A Sproul A, García E, Antón LC, Noggle S, Arancio O, Avila J, García-Escudero V. ( 2017) Mitophagy Failure in Fibroblasts and iPSC-Derived Neurons of Alzheimer’s Disease-Associated Presenilin 1 Mutation. Front Mol Neurosci. 2017 Sep 14;10:291. PMC free article

The life of a fibroblast might not be as prone to stress the mitochondria to the limit, but compounds like CCCP can destroy the membrane potential. This image was created from a variety of Internet sources

• Pink1 is a mitochondrial protein kinase that attaches phosphate groups to serine and threonines of PARK2 and ubiquitin.  PINK1 can localize to the outer membrane of the mitochondria where it senses depolarization, i.e. a decrease in membrane potential.
• PARK2 codes for Parkin, a ubiquitin ligase that attaches ubiquitin tags on damaged proteins
• Ubiquitin is a protein of which polymers may be attached to larger proteins by ubiquitin ligases.
• P62/ SQSTM1 is generally thought of as an adaptor protein, in the mitophagy process, that binds poly ubiquitination and light chain 3.
• LC3, microtubule associated protein light chain 3 is associated with the fusion and expansion of the autophagosome.
• LAMP2, lysosome associated membrane glycoprotein 2 is a protein on lysosome organelle membranes that mediates fusion with the autophagosome in chaperone mediated.

The following table summarizes the Western blot results after CCP treatment.  

parameter	Wild type control	FAD1	Comment
Mito surface, a.u.	~1000	~1200*	The difference seems small but significant. Mitochondria do swell when dysfunctional.
PINK2 level		80% less	proteolytic activated, comparison is to wild type
PARK2 level		Almost 2x control	Not seen w/out CCCP
PARK2 mito overlap	~40%	~60%	More overlap in FAD1 suggests that (1) defective mitochondria are not being cleared or (2) mitochondra are more poisoned by CCCP.
Mito membrane pot %	~75→62	~62→60	CCCP reversable→CCCP total
p62 levels		~200% more	Is there more p62 because the autophagosomes are not fusing with lysosomes and getting degraded?
p62 accumulation	~1.4	~1.7	Degradation blocked w/NH4Cl The difference between WT and FAD1 is not significant
P62 degradation	~1.5	~0.8*	The degradation of p62 is slower in FAD1 fibroblasts with damaged mitochondria
LC3 ii level		2x control	LC3ii is the proteolytic version in response to CCCP
LC3ii/LC3i ratio	~1	~1.8 *	Truncated/ full length
LCIii synthesis	2	1.4	Degradation blocked w/NH4Cl
LCii degradation	1.4	0.8 *	Lyso dysfunction
LAMP2 size, a.u.	0.4	0.4 ***	Estimate of lyso size
Mito overlap LC3ii, au	~40→30	~40→50*	cont→CCCP
Procathepsin B a.u.	~0.04	~0.07 *	Pro-cathepsin is full length inactive protease
Active / pro	~10	~4 *	FAD1 lysosomes are acidification defective. Cathepsins are acid activated proteaseases.

Up regulated gene transcripts in FAD1

We need to remember that PSEN1 regulates insertion of CIC7 and the ATPase into lysosome membranes, and ultimately the ability to acidify. This is covered in a sister post. All of these transcripts are are higher in the FAD1 fibroblasts compared to fibroblasts from donors without PSEN1 mutations.

• ATP6VOE1 is one of several sub units in the lysosome H⁺ ATPase that uses the energy in ATP to pump H⁺ against an electrochemical gradient into lysosomes.
• LAMP1 together with the LAMP2 gene product, this protein comprises over 50% of lysosome surface proteins.
• LAMP2, pretty much the same story as LAMP1 on this Wikipedia page. It may be noteworthy that both isoforms are transcribed more in FAD1
• EEA1, the early endosome antigen. This transcript is only significant at p-0.06, not really at the traditional thresh hold of p<0.05
• TFEB, is the transcription factor that controls transcription of genes that code for lysosome proteins. This transcription factor transcript is upregulated in many neurodegenerative diseases and lysosomal storage disorders.
• CTSL1 codes for cathepsin L1, one of the proteases in lysosomes.
• ARSB is the gene for arylsulfatase B, an enzyme that degrades condroitin sulfate, among other things.
• GNS codes for N-acetylglucosamine-6-sulfatase, another lysosomal enzyme that degrades sulfated polysaccharides. Mutations in this gene cause San Filippo Syndrome, a lysosomal storage disorder.

The transcription factor TFEB should be turned off by properly functioning lysosomes, yet many of the mRNAs that get transcribed when TFEB enters the nucleus are increased suggesting that TFEB is turned on. We have to remember that the FAD1 fibroblasts have mutations in PSEN1, a gene that codes for a protein that helps load lysosomes with the v-ATPase H⁺ pump and CIC7. We also have to remember that the Parkinson’s defective protein PARK2 is what starts the process of clearing cells of defective mitochondria. It is highly significant that a patient with a CLCN7 mutation can be treated with a Parkinson’s medication.