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.

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<abstract> <p>It has been established that the CA3 region of the hippocampus is involved in consolidating short-term memory to long-term memory and aids in spatial navigation retention. Seizures and many neurologic diseases induce damage to that region of the hippocampus, resulting in deficits in memory consolidation and spatial navigation. Drug treatments have been proven to have limited effectiveness, but cell replacement therapy has demonstrated to be more promising. Celavie Biosciences have developed a multipotent, nontumorigenic human neural progenitor cell (hNPC) line shown to have the ability to migrate <italic>in situ</italic>, reducing structural and functional deficits in neurodegenerative animal models. Here, we examined whether transplanted hNPCs would reestablish the memories of Han-Wistar rats subjected to hippocampal excitotoxic lesioning. The rats were lesioned in the CA3c regions at 50 days bilaterally with the neurotoxin NMDA (1 µl containing 7.5 mg/ml; −3.5 mm AP; ±2.0 L and −2.5 V). At 54 days of age, live hNPCs (500000 cells in 5 µl cell suspension media), frozen-killed hNPCs (500000 cells/5 µl), HEK293T cells (500000 cells/5 µl) or vehicle (cell suspension media; 5 µl) were bilaterally implanted directly into the NMDA damaged area. The rats were tested two weeks later with three different memory tests: novel and place-object assays and the water-maze task. Results showed that rats receiving live hNPC implantation performed significantly better in the water maze task than control groups; yet, novel and place-object test results showed no significant differences among treatments. Histology confirmed the survival of implanted hNPCs after 28 days post-implantation as well as showing neuroprotective effects. This study showed that Celavie's hNPCs were able to survive and improve some but not all hippocampal functionality, emphasizing the promise for cell replacement therapeutics for neurodegenerative disorders.</p> </abstract>

The dopamine transporter (DAT) mediated DA reuptake is a major molecular mechanism for termination of dopaminergic signaling in the brain. Psychoactive substances such as cocaine act by inhibition of plasma membrane DAT function as well as by altering its expression. The precise manner and mechanism by which cocaine regulates DAT trafficking, especially at neuronal processes, are poorly understood. We have now engineered a novel pH-sensitive reporter for DAT by conjugating pHluorin to the second exofacial loop of human DAT. We show that DAT-pHluorin can be used to study DAT localization and its dynamic trafficking at neuronal processes. Using DAT-pHluorin we show that unlike neuronal soma and dendrites, which contain majority of the DATs in weakly acidic intracellular compartments, axonal DATs at both shafts and boutons are primarily (75%) localized to the plasma membrane, while varicosities contain abundant intracellular DAT within acidic intracellular structures. Using this novel reporter, we show, for the first time, that cocaine exposure leads to a brief DAT internalization followed by membrane reinsertion that lasts for days. We further show that the cocaine-induced DAT trafficking is sensitive to the activities of Synaptojanin1 phosphatase. Thus, our study using the newly engineered DAT optical reporter reveals the previously unknown dynamics and molecular regulation for cocaine-regulated DAT trafficking in neuronal processes.

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