Xian Lin

Leucine-Rich Repeat Kinase 2 Regulates the Progression of Neuropathology Induced by Parkinson’s Disease-related Mutant α-synuclein

Mutations in alpha-synuclein and Leucine-rich repeat kinase 2 (LRRK2) are linked to autosomal dominant forms of Parkinsons 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.

Phosphorylation of Ezrin/Radixin/Moesin Proteins by LRRK2 Promotes the Rearrangement of Actin Cytoskeleton in Neuronal Morphogenesis

Leucine-rich repeat kinase 2 (LRRK2) functions as a putative protein kinase of ezrin, radixin, and moesin (ERM) family proteins. A Parkinsons disease-related G2019S substitution in the kinase domain of LRRK2 further enhances the phosphorylation of ERM proteins. The phosphorylated ERM (pERM) proteins are restricted to the filopodia of growing neurites in which they tether filamentous actin (F-actin) to the cytoplasmic membrane and regulate the dynamics of filopodia protrusion. Here, we show that, in cultured neurons derived from LRRK2 G2019S transgenic mice, the number of pERM-positive and F-actin-enriched filopodia was significantly increased, and this correlates with the retardation of neurite outgrowth. Conversely, deletion of LRRK2, which lowered the pERM and F-actin contents in filopodia, promoted neurite outgrowth. Furthermore, inhibition of ERM phosphorylation or actin polymerization rescued the G2019S-dependent neuronal growth defects. These data support a model in which the G2019S mutation of LRRK2 causes a gain-of-function effect that perturbs the homeostasis of pERM and F-actin in sprouting neurites critical for neuronal morphogenesis.

Deletion at ITPR1 underlies ataxia in mice and spinocerebellar ataxia 15 in humans.

We observed a severe autosomal recessive movement disorder in mice used within our laboratory. We pursued a series of experiments to define the genetic lesion underlying this disorder and to identify a cognate disease in humans with mutation at the same locus. Through linkage and sequence analysis we show here that this disorder is caused by a homozygous in-frame 18-bp deletion in Itpr1 (Itpr1Δ18/Δ18), encoding inositol 1,4,5-triphosphate receptor 1. A previously reported spontaneous Itpr1 mutation in mice causes a phenotype identical to that observed here. In both models in-frame deletion within Itpr1 leads to a decrease in the normally high level of Itpr1 expression in cerebellar Purkinje cells. Spinocerebellar ataxia 15 (SCA15), a human autosomal dominant disorder, maps to the genomic region containing ITPR1; however, to date no causal mutations had been identified. Because ataxia is a prominent feature in Itpr1 mutant mice, we performed a series of experiments to test the hypothesis that mutation at ITPR1 may be the cause of SCA15. We show here that heterozygous deletion of the 5′ part of the ITPR1 gene, encompassing exons 1–10, 1–40, and 1–44 in three studied families, underlies SCA15 in humans.

The Chaperone Activity of Heat Shock Protein 90 Is Critical for Maintaining the Stability of Leucine-Rich Repeat Kinase 2

Parkinsons disease (PD), a progressive neurodegenerative disease characterized by bradykinesia, rigidity, and resting tremor, is the most common neurodegenerative movement disorder. Although the majority of PD cases are sporadic, some are inherited, including those caused by leucine-rich repeat kinase 2 (LRRK2) mutations. The substitution of serine for glycine at position 2019 (G2019S) in the kinase domain of LRRK2 represents the most prevalent genetic mutation in both familial and apparently sporadic cases of PD. Because mutations in LRRK2 are likely associated with a toxic gain of function, destabilization of LRRK2 may be a novel way to limit its detrimental effects. Here we show that LRRK2 forms a complex with heat shock protein 90 (Hsp90) in vivo and that inhibition of Hsp90 disrupts the association of Hsp90 with LRRK2 and leads to proteasomal degradation of LRRK2. Hsp90 inhibitors may therefore limit the mutant LRRK2-elicited toxicity to neurons. As a proof of principle, we show that Hsp90 inhibitors rescue the axon growth retardation caused by overexpression of the LRRK2 G2019S mutation in neurons. Therefore, inhibition of LRRK2 kinase activity can be achieved by blocking Hsp90-mediated chaperone activity and Hsp90 inhibitors may serve as potential anti-PD drugs.

Astrocytic expression of Parkinson's disease-related A53T α-synuclein causes neurodegeneration in mice

BackgroundParkinsons disease (PD) is the most common movement disorder. While neuronal deposition of α-synuclein serves as a pathological hallmark of PD and Dementia with Lewy Bodies, α-synuclein-positive protein aggregates are also present in astrocytes. The pathological consequence of astrocytic accumulation of α-synuclein, however, is unclear.ResultsHere we show that PD-related A53T mutant α-synuclein, when selectively expressed in astrocytes, induced rapidly progressed paralysis in mice. Increasing accumulation of α-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 α-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.ConclusionsOur findings demonstrate a critical involvement of astrocytic α-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.

Conditional expression of Parkinson's disease-related mutant α-synuclein in the midbrain dopaminergic neurons causes progressive neurodegeneration and degradation of transcription factor nuclear receptor related 1.

α-Synuclein (α-syn) plays a prominent role in the degeneration of midbrain dopaminergic (mDA) neurons in Parkinsons disease (PD). However, only a few studies on α-syn have been performed in the mDA neurons in vivo, which may be attributed to a lack of α-syn transgenic mice that develop PD-like severe degeneration of mDA neurons. To gain mechanistic insights into the α-syn-induced mDA neurodegeneration, we generated a new line of tetracycline-regulated inducible transgenic mice that overexpressed the PD-related α-syn A53T missense mutation in the mDA neurons. Here we show that the mutant mice developed profound motor disabilities and robust mDA neurodegeneration, resembling some key motor and pathological phenotypes of PD. We also systematically examined the subcellular abnormalities that appeared in the mDA neurons of mutant mice and observed a profound decrease of dopamine release, the fragmentation of Golgi apparatus, and the impairments of autophagy/lysosome degradation pathways in these neurons. To further understand the specific molecular events leading to the α-syn-dependent degeneration of mDA neurons, we found that overexpression of α-syn promoted a proteasome-dependent degradation of nuclear receptor-related 1 protein (Nurr1), whereas inhibition of Nurr1 degradation ameliorated the α-syn-induced loss of mDA neurons. Given that Nurr1 plays an essential role in maintaining the normal function and survival of mDA neurons, our studies suggest that the α-syn-mediated suppression of Nurr1 protein expression may contribute to the preferential vulnerability of mDA neurons in the pathogenesis of PD.

Loss of ALS2 Function Is Insufficient to Trigger Motor Neuron Degeneration in Knock-Out Mice But Predisposes Neurons to Oxidative Stress

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.

The G59S Mutation in p150glued Causes Dysfunction of Dynactin in Mice

The G59S missense mutation at the conserved microtubule-binding domain of p150glued, a major component of dynein/dynactin complex, has been linked to an autosomal dominant form of motor neuron disease (MND). To study how this mutation affects the function of the dynein/dynactin complex and contributes to motor neuron degeneration, we generated p150glued G59S knock-in mice. We found that the G59S mutation destabilizes p150glued and disrupts the function of dynein/dynactin complex, resulting in early embryonic lethality of homozygous knock-in mice. Heterozygous knock-in mice, which developed normally, displayed MND-like phenotypes after 10 months of age, including excessive accumulation of cytoskeletal and synaptic vesicle proteins at neuromuscular junctions, loss of spinal motor neurons, increase of reactive astrogliosis, and shortening of gait compared with wild-type littermates and age-matched p150glued heterozygous knock-out mice. Our findings indicate that the G59S mutation in p150glued abrogates the normal function of p150glued and accelerates motor neuron degeneration.

LRRK2 regulates synaptogenesis and dopamine receptor activation through modulation of PKA activity

Leucine-rich repeat kinase 2 (LRRK2) is enriched in the striatal projection neurons (SPNs). We found that LRRK2 negatively regulates protein kinase A (PKA) activity in the SPNs during synaptogenesis and in response to dopamine receptor Drd1 activation. LRRK2 interacted with PKA regulatory subunit IIβ (PKARIIβ). A lack of LRRK2 promoted the synaptic translocation of PKA and increased PKA-mediated phosphorylation of actin-disassembling enzyme cofilin and glutamate receptor GluR1, resulting in abnormal synaptogenesis and transmission in the developing SPNs. Furthermore, PKA-dependent phosphorylation of GluR1 was also aberrantly enhanced in the striatum of young and aged Lrrk2(-/-) mice after treatment with a Drd1 agonist. Notably, a Parkinsons disease-related Lrrk2 R1441C missense mutation that impaired the interaction of LRRK2 with PKARIIβ also induced excessive PKA activity in the SPNs. Our findings reveal a previously unknown regulatory role for LRRK2 in PKA signaling and suggest a pathogenic mechanism of SPN dysfunction in Parkinsons disease.

Amyotrophic Lateral Sclerosis 2-Deficiency Leads to Neuronal Degeneration in Amyotrophic Lateral Sclerosis through Altered AMPA Receptor Trafficking

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.