Summary Mutations in α-synuclein and Leucine-rich repeat kinase 2 (LRRK2) are linked to autosomal dominant forms of Parkinson’s disease (PD). However, little is known about any potential pathophysiological interplay between these two PD-related genes. Here we show in transgenic mice that although over-expression of LRRK2 alone did not cause neurodegeneration, the presence of excess LRRK2 greatly accelerated the progression of neuropathological abnormalities developed in PD-related A53T α-synuclein transgenic mice. Moreover, we found that LRRK2 promoted the abnormal aggregation and somatic accumulation of α-synuclein in A53T mice, likely resulted from the impairment of microtubule dynamics, Golgi organization, and ubiquitin-proteasome pathway. Conversely, genetic ablation of LRRK2 preserved the Golgi structure, suppressed the aggregation and somatic accumulation of α-synuclein, and thereby delayed the progression of neuropathology in A53T mice. These findings demonstrate that over-expression of LRRK2 enhances α-synuclein-mediated cytotoxicity and suggest inhibition of LRRK2 expression as a potential therapeutic option for ameliorating α-synuclein-induced neurodegeneration.

We recently showed by electron microscopy that the postsynaptic density (PSD) from hippocampal cultures undergoes rapid structural changes after ischemia-like conditions. Here we report that similar structural changes occur after delay in transcardial perfusion fixation of the mouse brain. Delay in perfusion fixation, a condition that mimics ischemic stress, resulted in 70%, 90%, and 23% increases in the thickness of PSDs from the hippocampus (CA1), cerebral cortex (layer III), and cerebellar cortex (Purkinje spines), respectively. In step with PSD thickening, the amount of PSD-associated alpha-calcium calmodulin-dependent protein kinase II (alpha- CaMKII) label increased more in cerebral cortical spines than in Purkinje spines. Although the Purkinje PSDs thickened only slightly after delayed fixation, they became highly curved, and many formed sub-PSD spheres approximately 80 nm in diameter that labeled for CaMKII. Delayed perfusion fixation also produced more cytoplamic CaMKII clusters ( approximately 110 nm in diameter) in the somas of pyramidal cells (from hippocampus and cerebral cortex) than in Purkinje cells. Thus a short delay in perfusion fixation produces cell-specific structural changes at PSDs and neuronal somas. Purkinje cells respond somewhat differently to delayed perfusion fixation, perhaps owing to their lower levels of CaMKII, and CaMKII binding proteins at PSDs. We present here a catalogue of structural changes that signal a perfusion fixation delay, thereby providing criteria by which to assess perfusion fixation quality in experimental structural studies of brain and to shed light on the subtle changes that occur in intact brain following metabolic stress.

A major issue in the slow transport of cytoskeletal proteins is the form in which they are transported. We have investigated the possibility that unpolymerized as well as polymerized cytoskeletal proteins can be actively transported in axons. We report the active transport of highly diffusible tubulin oligomers, as well as transport of the less diffusible neurofilament polymers. After injection into the squid giant axon, tubulin was transported in an anterograde direction at an average rate of 2.3 mm͞day, whereas neurofilament was moved at 1.1 mm͞day. Addition of the metabolic poisons cyanide or dinitrophenol reduced the active transport of both proteins to less than 10% of control values, whereas disruption of microtubules by treatment of the axon with cold in the presence of nocodazole reduced transport of both proteins to Ϸ20% of control levels. Passive diffusion of these proteins occurred in parallel with transport. The diffusion coefficient of the moving tubulin in axoplasm was 8.6 m 2 ͞s compared with only 0.43 m 2 ͞s for neurofilament. These results suggest that the tubulin was transported in the unpolymerized state and that the neurofilament was transported in the polymerized state by an energy-dependent nocodazole͞cold-sensitive transport mechanism.