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Background: Dynactin p150 Glued , the largest subunit of the dynactin macromolecular complex, binds to both microtubules and tubulin dimers through the N-terminal cytoskeleton-associated protein and glycine-rich (CAP-Gly) and basic domains, and serves as an anti-catastrophe factor in stabilizing microtubules in neurons. P150 Glued also initiates dynein-mediated axonal retrograde transport. Multiple missense mutations at the CAP-Gly domain of p150 Glued are associated with motor neuron diseases and other neurodegenerative disorders, further supporting the importance of microtubule domains (MTBDs) in p150 Glued functions. However, most functional studies were performed in vitro. Whether p150 Glued is required for neuronal function and survival in vivo is unknown., Methods: Using Cre-loxP genetic manipulation, we first generated a line of p150 Glued knock-in mice by inserting two LoxP sites flanking the MTBD-coding exons 2 to 4 of p150 Glued -encoding Dctn1 gene (Dctn1 LoxP/ ), and then crossbred the resulting Dctn1 LoxP/ mice with Thy1-Cre mice to generate the bigenic p150 Glued (Dctn1 LoxP/LoxP ; Thy1-Cre) conditional knockout (cKO) mice for the downstream motor behavioral and neuropathological studies., Results: P150 Glued expression was completely abolished in Cre-expressing postnatal neurons, including corticospinal motor neurons (CSMNs) and spinal motor neurons (SMNs), while the MTBD-truncated forms remained. P150 Glued ablation did not affect the formation of dynein/dynactin complex in neurons. The p150 Glued cKO mice did not show any obvious developmental phenotypes, but exhibited impairments in motor coordination and rearing after 12 months of age. Around 20% loss of SMNs was found in the lumbar spinal cord of 18-month-old cKO mice, in company with increased gliosis, neuromuscular junction (NMJ) disintegration and muscle atrophy. By contrast, no obvious degeneration of CSMNs, striatal neurons, midbrain dopaminergic neurons, cerebellar granule cells or Purkinje cells was observed. Abnormal accumulation of acetylated α-tubulin, and autophagosome/lysosome proteins was found in the SMNs of aged cKO mice. Additionally, the total and cell surface levels of glutamate receptors were also substantially elevated in the p150 Glued -depleted spinal neurons, in correlation with increased vulnerability to excitotoxicity., Conclusion: Overall, our findings demonstrate that p150 Glued is particularly required to maintain the function and survival of SMNs during aging. P150 Glued may exert its protective function through regulating the transportation of autophagosomes, lysosomes, and postsynaptic glutamate receptors in neurons.

Background: Parkinson's disease (PD) is the most common movement disorder. While neuronal deposition of alpha-synuclein serves as a pathological hallmark of PD and Dementia with Lewy Bodies, alpha-synuclein-positive protein aggregates are also present in astrocytes. The pathological consequence of astrocytic accumulation of alpha-synuclein, however, is unclear., Results: Here we show that PD-related A53T mutant alpha-synuclein, when selectively expressed in astrocytes, induced rapidly progressed paralysis in mice. Increasing accumulation of alpha-synuclein aggregates was found in presymptomatic and symptomatic mouse brains and correlated with the expansion of reactive astrogliosis. The normal function of astrocytes was compromised as evidenced by cerebral microhemorrhage and down-regulation of astrocytic glutamate transporters, which also led to increased inflammatory responses and microglial activation. Interestingly, the activation of microglia was mainly detected in the midbrain, brainstem and spinal cord, where a significant loss of dopaminergic and motor neurons was observed. Consistent with the activation of microglia, the expression level of cyclooxygenase 1 (COX-1) was significantly up-regulated in the brain of symptomatic mice and in cultured microglia treated with conditioned medium derived from astrocytes over-expressing A53T alpha-synuclein. Consequently, the suppression of COX-1 activities extended the survival of mutant mice, suggesting that excess inflammatory responses elicited by reactive astrocytes may contribute to the degeneration of neurons., Conclusions: Our findings demonstrate a critical involvement of astrocytic alpha-synuclein in initiating the non-cell autonomous killing of neurons, suggesting the viability of reactive astrocytes and microglia as potential therapeutic targets for PD and other neurodegenerative diseases.

Mutations in alpha-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 overexpression of LRRK2 alone did not cause neurodegeneration, the presence of excess LRRK2 greatly accelerated the progression of neuropathological abnormalities developed in PD-related A53T alpha-synuclein transgenic mice. Moreover, we found that LRRK2 promoted the abnormal aggregation and somatic accumulation of alpha-synuclein in A53T mice, which likely resulted from the impairment of microtubule dynamics, Golgi organization, and the ubiquitin-proteasome pathway. Conversely, genetic ablation of LRRK2 preserved the Golgi structure and suppressed the aggregation and somatic accumulation of alpha-synuclein, and thereby delayed the progression of neuropathology in A53T mice. These findings demonstrate that overexpression of LRRK2 enhances alpha-synuclein-mediated cytotoxicity and suggest inhibition of LRRK2 expression as a potential therapeutic option for ameliorating alpha-synuclein-induced neurodegeneration., (2009 Elsevier Inc. All rights reserved.)

Amyotrophic lateral sclerosis (ALS), the most common adult-onset motor neuron disease is caused by a selective loss of motor neurons. One form of juvenile onset autosomal recessive ALS (ALS2) has been linked to the loss of function of the ALS2 gene. The pathogenic mechanism of ALS2-deficiency, however, remains unclear. To further understand the function of alsin that is encoded by the full-length ALS2 gene, we screened proteins interacting with alsin. Here, we report that alsin interacted with glutamate receptor interacting protein 1 (GRIP1) both in vitro and in vivo, and colocalized with GRIP1 in neurons. In support of the physiological interaction between alsin and GRIP1, the subcellular distribution of GRIP1 was altered in ALS2(-/-) spinal motor neurons, which correlates with a significant reduction of AMPA-type glutamate receptor subunit 2 (GluR2) at the synaptic/cell surface of ALS2(-/-) neurons. The decrease of calcium-impermeable GluR2-containing AMPA receptors at the cell/synaptic surface rendered ALS2(-/-) neurons more susceptible to glutamate receptor-mediated neurotoxicity. Our findings reveal a novel function of alsin in AMPA receptor trafficking and provide a novel pathogenic link between ALS2-deficiency and motor neuron degeneration, suggesting a protective role of alsin in maintaining the survival of motor neurons.

Amyotrophic lateral sclerosis (ALS), the most common motor neuron disease, is caused by a selective loss of motor neurons in the CNS. Mutations in the ALS2 gene have been linked to one form of autosomal recessive juvenile onset ALS (ALS2). To investigate the pathogenic mechanisms of ALS2, we generated ALS2 knock-out (ALS2(-/-)) mice. Although ALS2(-/-) mice lacked obvious developmental abnormalities, they exhibited age-dependent deficits in motor coordination and motor learning. Moreover, ALS2(-/-) mice showed a higher anxiety response in the open-field and elevated plus-maze tasks. Although they failed to recapitulate clinical or neuropathological phenotypes consistent with motor neuron disease by 20 months of age, ALS2(-/-) mice or primary cultured neurons derived from these mice were more susceptible to oxidative stress compared with wild-type controls. These observations suggest that loss of ALS2 function is insufficient to cause major motor deficits or motor neuron degeneration in a mouse model but predisposes neurons to oxidative stress.

Wilms' tumor suppressor (WT1), a 52- to 54-kda transcription factor, is the gene product of Wilms' tumor 1 (wt1), one of at least three genes involved in the development of a pediatric kidney cancer. Expression patterns of WT1 indicate that it is not restricted to the kidney but may play a role in the development and homeostasis of other tissues as well. WT1 has been implicated in various cellular processes including proliferation, differentiation, and apoptosis. High levels of WT1 induce apoptosis independent of p53, whereas low levels of WT1 inhibit apoptosis. Because apoptosis has been suggested to play a role in neurodegeneration in Alzheimer's disease (AD), immunohistochemistry of WT1 and paired helical filament (PHF) in serial sections was carried out. Immunohistochemical localization of WT1 and PHF showed the presence of WT1 in approximately 42% of PHF-positive neurofibrillary tangle containing-neurons. Laser confocal microscopy of hippocampal neuron cultures undergoing apoptosis induced by amyloid beta peptide (Abeta) or staurosporine demonstrated significant time-dependent elevations of WT1 correlating with increased levels of apoptosis. Blockade of WT1 transcription by antisense oligonucleotide reduced WT1 expression and prevented neuronal apoptosis in both Abeta- and staurosporine-treated cultures. Together, these data suggest a role for WT1 in the neurodegeneration observed in AD brain.