SerThr-PhosphoProteome of Brain from Aged PINK1-KO+A53T-SNCA Mice Reveals pT1928-MAP1B and pS3781-ANK2 Deficits, as Hub between Autophagy and Synapse Changes

Abstract

Hereditary Parkinson’s disease (PD) can be triggered by an autosomal dominant overdose of alpha-Synuclein (SNCA) as stressor or the autosomal recessive deficiency of PINK1 Serine/Threonine-phosphorylation activity as stress-response. We demonstrated the combination of PINK1-knockout with overexpression of SNCAA53T in double mutant (DM) mice to exacerbate locomotor deficits and to reduce lifespan. To survey posttranslational modifications of proteins underlying the pathology, brain hemispheres of old DM mice underwent quantitative label-free global proteomic mass spectrometry, focused on Ser/Thr-phosphorylations. As exceptionally strong effect, we detected >300-fold reductions of phosphoThr1928 in MAP1B, a microtubule-associated protein, and a similar reduction of phosphoSer3781 in ANK2, an interactor of microtubules. MAP1B depletion is known to trigger perturbations of microtubular mitochondria trafficking, neurite extension and synaptic function, so it was noteworthy that relevantly decreased phosphorylation was detected also for other microtubule and microfilament factors, namely MAP2S1801, MARK1S394, MAP1AT1794, KIF1AS1537, 4.1NS541, 4.1GS86 and ADD2S528. While the MAP1B heavy chain supports regeneration and growth cones, its light-chain assists DAPK1-mediated autophagy. Interestingly, relevant phosphorylation decreases of DAPK2S299, VPS13DS2429 and VPS13CS2480 in the DM brain affected regulators of autophagy, which are implicated in PD. Overall, significant downregulations were enriched for PFAM C2 domains, other kinases, and synaptic transmission factors upon automated bioinformatics, while upregulations were not enriched for selective motifs or pathways. Validation experiments confirmed the change of LC3 processing as reflection of excessive autophagy in DM brain, and dependence of ANK2/MAP1B expression on PINK1 levels. Our new data provide independent confirmation in a mouse model with combined PARK1/PARK4/PARK6 pathology that MAP1B/ANK2 phosphorylation events are implicated in Parkinsonian neurodegeneration. These findings expand on previous observations in D. melanogaster that the MAP1B ortholog futsch in the presynapse is a primary target of the PARK8 protein LRRK2, and on a report that MAP1B is a component of the pathological Lewy body aggregates in PD patient brains. Similarly, ANK2 gene locus variants are associated with the risk of PD, ANK2 interacts with PINK1/Parkin-target proteins such as MIRO1 or ATP1A2, and ANK2-derived peptides are potent inhibitors of autophagy.

Systematic Surveys of Iron Homeostasis Mechanisms reveal Ferritin Superfamily and Nucleotide Surveillance Regulation to be Modified by PINK1 Absence

“…Mitophagy is relevant for only a few among hundreds or thousands of mitochondria per cell at any given time, and the accumulation of iron is an insidious process over decades in PD patient brain, so the compensatory efforts needed are minimal. Therefore, global expression profiles of Pink1 -/-mouse brain showed only subtle evidence of deficient mitophagy and altered mitochondrial biogenesis [16,[29][30][31], the dysregulated expression of heme-related transcripts Hmox1 and Hebp1 was noted only upon culture of mouse Pink1 -/-primary cortical neurons [31], and limited survival of the Pink1 -/-mouse was observed only after additional overexpression of toxic alpha-synuclein [16,32]. In general, PINK1-and PARKIN-deficient mice show signs of altered mitophagy and neurodegeneration only in the presence of further stressors such as mitochondrial mutations, exhaustive exercise, or bacterial infections [33][34][35][36].…”

4.1N-Mediated Interactions and Functions in Nerve System and Cancer

“…The 4.1N is correlated with tumor progression, aggressive behaviors in NSCLC and EOC, and chemotherapy resistance in EOC. 4.1N is also related to liver disease ( Stepanova et al, 2010 ), cardiac disease ( JBirks et al, 2003 ; Taylor-Harris et al, 2005 ; Pinder et al, 2012 ), nonsyndromic intellectual disability ( Hamdan et al, 2011 ), hereditary Parkinson’s disease ( Auburger et al, 2019 ), and reproductive system disease ( Wang et al, 2020b ; Wang et al, 2021 ). The FERM and CTD represent two adaptors where a number of regulations converge on the association of protein 4.1N with its partners, through which 4.1N locates, supports, and coordinates partners in signaling transduction.…”

“…However, the Trk receptor that directly phosphorylates the tyrosine residue of 4.1N and mediates 4.1N insertion into the nucleus under nerve growth factor (NGF) stimulation has been referred to in PC12 cells ( Ye et al, 1999 ). A study focused on protein Ser/Thr-phosphorylation modifications in mice brain hemispheres underlying hereditary Parkinson’s disease has documented 2.2-fold increases of pS541, pS544, and pS546 in 4.1N protein ( Auburger et al, 2019 ).…”

Systematic Surveys of Iron Homeostasis Mechanisms Reveal Ferritin Superfamily and Nucleotide Surveillance Regulation to be Modified by PINK1 Absence

“…Mitophagy is relevant for only a few among hundreds or thousands of mitochondria per cell at any given time, and the accumulation of iron is an insidious process over decades in PD patient brain, so the compensatory efforts needed are minimal. Therefore, global expression profiles of Pink1 −/− mouse brain showed only subtle evidence of deficient mitophagy and altered mitochondrial biogenesis [ 20 , 33 , 34 , 35 ], the dysregulated expression of heme-related transcripts Hmox1 (Heme oxygenase 1) and Hebp1 (Heme binding protein 1) was noted only upon culture of mouse Pink1 −/− primary cortical neurons [ 35 ], and limited survival of the Pink1 −/− mouse was observed only after additional overexpression of toxic alpha-synuclein (SNCA) [ 20 , 36 ]. In general, PINK1- and PARKIN-deficient mice show signs of altered mitophagy and neurodegeneration only in the presence of further stressors such as mitochondrial mutations, exhaustive exercise, or bacterial infections [ 37 , 38 , 39 , 40 ].…”