Synj1 haploinsufficiency causes dopamine neuron vulnerability and alpha-synuclein accumulation in mice

Abstract

Synaptojanin1 (synj1) is a phosphoinositide phosphatase with dual SAC1 and 5′-phosphatase enzymatic activities in regulating phospholipid signaling. The brain-enriched isoform has been shown to participate in synaptic vesicle (SV) recycling. More recently, recessive human mutations were identified in the two phosphatase domains of SYNJ1, including R258Q, R459P and R839C, which are linked to rare forms of early-onset Parkinsonism. We now demonstrate that Synj1 heterozygous deletion (Synj1+/−), which is associated with an impaired 5′-phosphatase activity, also leads to Parkinson’s disease (PD)-like pathologies in mice. We report that male Synj1+/− mice display age-dependent motor function abnormalities as well as alpha-synuclein accumulation, impaired autophagy and dopaminergic terminal degeneration. Synj1+/− mice contain elevated 5′-phosphatase substrate, PI(4,5)P2, particularly in the midbrain neurons. Moreover, pharmacological elevation of membrane PI(4,5)P2 in cultured neurons impairs SV endocytosis, specifically in midbrain neurons, and further exacerbates SV trafficking defects in Synj1+/− midbrain neurons. We demonstrate down-regulation of SYNJ1 transcript in a subset of sporadic PD brains, implicating a potential role of Synj1 deficiency in the decline of dopaminergic function during aging.

Synaptojanin1 deficiency upregulates basal level autophagosome formation in astrocytes

“…In the Synj1+/-mouse, our previous study didn't reveal any changes in LC3 immunofluorescence in the midbrain dopamine neurons, but found a reduction of p62 at 3 months of age with a robust increase at 18 months [25]. These results suggest an agedependent reversal of the autolysosomal function in the dopamine neurons.…”

“…Constructs -pEGFPC1-FLAG-WT hSYNJ1-145 kDa, pEGFPC1-FLAG-R258Q hSYNJ1-145 kDa and pEGFPC1-FLAG-R839C hSYNJ1-145 kDa [25] Immunofluorescence analysis -The following antibodies were used for immunofluorescence: mouse anti-PI(4,5)P2 (Echelon Biosciences, z-P045, 1:100) [25], chicken anti-GFP (Thermo Fisher, A-10262, 1:1000), guinea pig anti-p62 (Progene, GPP62-C, 1:1000), rabbit anti-EEA1 (Abcam, ab109110, 1:1000), mouse anti-Rab7 (Abcam, ab50533, 1:200). Immunocytochemistry was performed following previously published procedures [16,25,62]. Immuofluorescence was analyzed using a Nikon Ti-2 wide-field microscope with Spectra-X (Lumencor) as the light source and an Andor Ultra 897 EMCCD camera.…”

“…We thank Dr. Wei-xing Zong for discussion of the work and suggestions for the manuscript preparation. [25]. b-e) WT hSynj1 (WTSJ1), R258Q hSynj1 (RQSJ1) or R839C hSynj1 (RCSJ1) were expressed in the Synj1 KO astrocytes and compared to the KO and its littermate WT astrocyte culture.…”

“…The Parkinsonism linked R258Q mutation abolishes the SAC1 activity by ~80% [22, 25] and leads to dystrophic changes in both GABAergic and dopaminergic synapses in mice [25, 26]. The R839C mutation, which results in similar clinical phenotypes, has a more profound impact on both phosphatases as it reduces the 5’-phosphatase activity by ~60% and PI4P hydrolysis by 80% [25]. The functional relevance of the R839C mutation, however, hasn’t been explored.…”

“…On the other hand, missense mutations in both the SAC1 and 5'phosphophatase domains of SYNJ1 have been found to associate with families of early onset atypical Parkinsonism with seizure [22][23][24]. The Parkinsonism linked R258Q mutation abolishes the SAC1 activity by ~80% [22,25] and leads to dystrophic changes in both GABAergic and dopaminergic synapses in mice [25,26]. The R839C mutation, which results in similar clinical phenotypes, has a more profound impact on both phosphatases as it reduces the 5'phosphatase activity by ~60% and PI4P hydrolysis by 80% [25].…”