A mutation in dynein rescues axonal transport defects and extends the life span of ALS mice

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative condition characterized by motoneuron degeneration and muscle paralysis. Although the precise pathogenesis of ALS remains unclear, mutations in Cu/Zn superoxide dismutase (SOD1) account for approximately 20-25% of familial ALS cases, and transgenic mice overexpressing human mutant SOD1 develop an ALS-like phenotype. Evidence suggests that defects in axonal transport play an important role in neurodegeneration. In Legs at odd angles (Loa) mice, mutations in the motor protein dynein are associated with axonal transport defects and motoneuron degeneration. Here, we show that retrograde axonal transport defects are already present in motoneurons of SOD1(G93A) mice during embryonic development. Surprisingly, crossing SOD1(G93A) mice with Loa/+ mice delays disease progression and significantly increases life span in Loa/SOD1(G93A) mice. Moreover, there is a complete recovery in axonal transport deficits in motoneurons of these mice, which may be responsible for the amelioration of disease. We propose that impaired axonal transport is a prime cause of neuronal death in neurodegenerative disorders such as ALS.     

Cocaine decreases expression of neurogranin via alterations in thyroid receptor/retinoid X receptor signaling

The molecule responsible for the enzyme activity plasma membrane (PM) aminophospholipid translocase (APLT), which catalyzes phosphatidylserine (PS) translocation from the outer to the inner leaflet of the plasma membrane, is unknown in mammals. A Caenorhabditis elegans study has shown that ablation of transbilayer amphipath transporter-1 (TAT-1), which is an ortholog of a mammalian P-type ATPase, Atp8a1, causes PS externalization in the germ cells. We demonstrate here that the hippocampal cells of the dentate gyrus, and Cornu Ammonis (CA1, CA3) in mice lacking Atp8a1 exhibit a dramatic increase in PS externalization. Although their hippocampi showed no abnormal morphology or heightened apoptosis, these mice displayed increased activity and a marked deficiency in hippocampus-dependent learning, but no hyper-anxiety. Such observations indicate that Atp8a1 plays a crucial role in PM-APLT activity in the neuronal cells. In corroboration, ectopic expression of Atp8a1 but not its close homolog, Atp8a2, caused an increase in the population (V(max) ) of PM-APLT without any change in its signature parameter K(m) in the neuronal N18 cells. Conversely, expression of a P-type phosphorylation-site mutant of Atp8a1 (Atp8a1*) caused a decrease in V(max) of PM-APLT without significantly altering its K(m) . The Atp8a1*-expressing N18 cells also exhibited PS externalization without apoptosis. Together, our data strongly indicate that Atp8a1 plays a central role in the PM-APLT activity of some mammalian cells, such as the neuronal N18 and hippocampal cells.     

Combining comparative proteomics and molecular genetics uncovers regulators of synaptic and axonal stability and degeneration in vivo

Degeneration of synaptic and axonal compartments of neurons is an early event contributing to the pathogenesis of many neurodegenerative diseases, but the underlying molecular mechanisms remain unclear. Here, we demonstrate the effectiveness of a novel "top-down" approach for identifying proteins and functional pathways regulating neurodegeneration in distal compartments of neurons. A series of comparative quantitative proteomic screens on synapse-enriched fractions isolated from the mouse brain following injury identified dynamic perturbations occurring within the proteome during both initiation and onset phases of degeneration. In silico analyses highlighted significant clustering of proteins contributing to functional pathways regulating synaptic transmission and neurite development. Molecular markers of degeneration were conserved in injury and disease, with comparable responses observed in synapse-enriched fractions isolated from mouse models of Huntington's disease (HD) and spinocerebellar ataxia type 5. An initial screen targeting thirteen degeneration-associated proteins using mutant Drosophila lines revealed six potential regulators of synaptic and axonal degeneration in vivo. Mutations in CALB2, ROCK2, DNAJC5/CSP, and HIBCH partially delayed injury-induced neurodegeneration. Conversely, mutations in DNAJC6 and ALDHA1 led to spontaneous degeneration of distal axons and synapses. A more detailed genetic analysis of DNAJC5/CSP mutants confirmed that loss of DNAJC5/CSP was neuroprotective, robustly delaying degeneration in axonal and synaptic compartments. Our study has identified conserved molecular responses occurring within synapse-enriched fractions of the mouse brain during the early stages of neurodegeneration, focused on functional networks modulating synaptic transmission and incorporating molecular chaperones, cytoskeletal modifiers, and calcium-binding proteins. We propose that the proteins and functional pathways identified in the current study represent attractive targets for developing therapeutics aimed at modulating synaptic and axonal stability and neurodegeneration in vivo.     

Neuronal autophagy: going the distance to the axon

Autophagy, a regulated cellular degradation process responsible for the turnover of long-lived proteins and organelles, has been increasingly implicated in neurological disorders. Although autophagy is mostly viewed as a stress-induced process, recent studies have indicated that it is constitutively active in central nervous system (CNS) neurons and is protective against neurodegeneration. Neurons are highly specialized, post-mitotic cells that are typically composed of a soma (cell body), a dendritic tree and an axon. The detailed process of autophagy in such a highly differentiated cell type remains to be characterized. To elucidate the physiological role of neuronal autophagy, we generated mutant mice containing a neural cell type-specific deletion of Atg7, an essential gene for autophagy. Establishment of these mutant mice allowed us to examine cell-autonomous events in cerebellar Purkinje cells deficient in autophagy. Our data reveal the indispensability of autophagy in the maintenance of axonal homeostasis and the prevention of axonal dystrophy and degeneration. Furthermore, our study implicates dysfunction of axonal autophagy as a potential mechanism underlying axonopathy, which is linked to neurodegeneration associated with numerous human neurological disorders. Finally, our study has raised a possibility that "constitutive autophagy" in neurons involves processes that are not typical of autophagy in other cell types, but rather is highly adapted to local physiological function in the axon, which is projected in a distance from one neuron to another for transducing neural signals.     

Neuronal autophagy: Going the distance to the axon

Autophagy, a regulated cellular degradation process responsible for the turnover of long-lived proteins and organelles, has been increasingly implicated in neurological disorders. Although autophagy is mostly viewed as a stress-induced process, recent studies have indicated that it is constitutively active in central nervous system (CNS) neurons and is protective against neurodegeneration. Neurons are highly specialized, post-mitotic cells that are typically composed of a soma (cell body), a dendritic tree and an axon. The detailed process of autophagy in such a highly differentiated cell type remains to be characterized. To elucidate the physiological role of neuronal autophagy, we generated mutant mice containing a neural cell type-specific deletion of Atg7, an essential gene for autophagy. Establishment of these mutant mice allowed us to examine cell-autonomous events in cerebellar Purkinje cells deficient in autophagy. Our data reveal the indispensability of autophagy in the maintenance of axonal homeostasis and the prevention of axonal dystrophy and degeneration. Furthermore, our study implicates dysfunction of axonal autophagy as a potential mechanism underlying axonopathy, which is linked to neurodegeneration associated with numerous human neurological disorders. Finally, our study has raised a possibility that “constitutive autophagy” in neurons involves processes that are not typical of autophagy in other cell types, but rather is highly adapted to local physiological function in the axon, which is projected in a distance from one neuron to another for transducing neural signals.

Addendum to: Komatsu M, Wang QJ, Holstein GR, Friedrich Jr. VL, Iwata J, Kominami E, Chait BT, Tanaka K, Yue Z. Essential role for autophagy protein Atg7 in the maintenance of axonal homeostasis and the prevention of axonal degeneration. Proc Natl Acad Sci USA 2007; 104:14489-94.     

P4 ATPases - Lipid flippases and their role in disease

P4 ATPases (type 4 P-type ATPases) are multispan transmembrane proteins that have been implicated in phospholipid translocation from the exoplasmic to the cytoplasmic leaflet of biological membranes. Studies in Saccharomyces cerevisiae have indicated that P4 ATPases are important in vesicle biogenesis and are required for vesicular trafficking along several intracellular vesicular transport routes. Although little is known about mammalian P4 ATPases, some members of this subfamily appear to be associated with human disease or mouse pathophysiology. ATP8B1, a phosphatidylserine translocase, is the most extensively studied mammalian P4 ATPase. This protein is important for maintaining the detergent resistant properties of the apical membrane of the hepatocyte. Mutations in ATP8B1 give rise to severe liver disease. Furthermore, a role for Atp8b3 in mouse sperm cell capacitation has been suggested, whereas deficiency of Atp10a and Atp10d leads to insulin resistance and obesity in mice. Here we review the present status on the pathophysiological consequences of P4 ATPase deficiency.     

Transport through recycling endosomes requires EHD1 recruitment by a phosphatidylserine translocase

P₄-ATPases translocate aminophospholipids, such as phosphatidylserine (PS), to the cytosolic leaflet of membranes. PS is highly enriched in recycling endosomes (REs) and is essential for endosomal membrane traffic. Here, we show that PS flipping by an RE-localized P₄-ATPase is required for the recruitment of the membrane fission protein EHD1. Depletion of ATP8A1 impaired the asymmetric transbilayer distribution of PS in REs, dissociated EHD1 from REs, and generated aberrant endosomal tubules that appear resistant to fission. EHD1 did not show membrane localization in cells defective in PS synthesis. ATP8A2, a tissue-specific ATP8A1 paralogue, is associated with a neurodegenerative disease (CAMRQ). ATP8A2, but not the disease-causative ATP8A2 mutant, rescued the endosomal defects in ATP8A1-depleted cells. Primary neurons from Atp8a2^−/− mice showed a reduced level of transferrin receptors at the cell surface compared to Atp8a2^+/+ mice. These findings demonstrate the role of P₄-ATPase in membrane fission and give insight into the molecular basis of CAMRQ.     

A search for radiosensitive mouse mutants by use of the micronucleus technique

In order to identify radiosensitive mutations in mice, 26 genetically well defined mutations in 26 different combinations of homozygous, hemizygous or heterozygous conditions, together with normal mice and mutagen-sensitive MS/Ae mice were analysed for the induction of micronuclei by X-rays in bone-marrow cells. For each mutant two doses of 0.5 and 1.0 Gy, two sampling times of 18 and 27 h after irradiation and unirradiated controls were studied. Using our criteria, homozygous contrasted allele of steel (Slcon), scabby (scb), viable dominant spotting (Wv), quaking (qk), fidget (fi) and postaxial hemimelia (px), heterozygous lurcher (Lc), hemizygous gyro (Gy), the compounds Slcon/grizzle-belly (SlgbH) and Wv/rump-white (Rw) and MS/Ae mice, were regarded as radiosensitive, with Slcon/Slcon the highest in rank order. Homozygous wabbler-lethal (wl) and wasted (wst) showed hyposensitivity which for the latter may be connected with enhanced cell killing.     

Regulation of neuronal autophagy in axon: implication of autophagy in axonal function and dysfunction/degeneration

Autophagy has recently emerged as potential drug target for prevention of neurodegeneration. However, the details of autophagy process and regulation in the central nervous system (CNS) are unclear. By using a neuronal excitotoxicity model mice, we engineered expression of a fluorescent autophagic marker and systematically investigated autophagic activity under neurodegenerative condition. The study reveals an early response of Purkinje cells to excitotoxic insult by induction of autophagy in axon terminals, and that axonal autophagy is particularly robust in comparison to the cell body and dendrites. The accessibility of axons to rapid autophagy induction suggests local biogenesis of autophagosomes in axons. Characterization of functional interaction between autophagosome protein LC3 and microtubule-associated protein 1B (MAP1B), which is involved in axonal growth, injury and transport provides evidence for neuron or axon-specific regulation of autophagosomes. Furthermore, we propose that p62/SQSTM1, a putative autophagic substrate can serve as a marker for evaluating impairment of autophagic degradation, which helps resolve the controversy over autophagy levels under various pathological conditions. Future study of the relationship between autophagy and axonal function (e.g., transport) will provide insight into the mechanism underlying axonopathy which is directly linked to neurodegeneration.     

Progranulin is neurotrophic in vivo and protects against a mutant TDP-43 induced axonopathy

Mislocalization, aberrant processing and aggregation of TAR DNA-binding protein 43 (TDP-43) is found in the neurons affected by two related diseases, amyotrophic lateral sclerosis (ALS) and frontotemporal lobe dementia (FTLD). These TDP-43 abnormalities are seen when TDP-43 is mutated, such as in familial ALS, but also in FTLD, caused by null mutations in the progranulin gene. They are also found in many patients with sporadic ALS and FTLD, conditions in which only wild type TDP-43 is present. The common pathological hallmarks and symptomatic cross over between the two diseases suggest that TDP-43 and progranulin may be mechanistically linked. In this study we aimed to address this link by establishing whether overexpression of mutant TDP-43 or knock-down of progranulin in zebrafish embryos results in motor neuron phenotypes and whether human progranulin is neuroprotective against such phenotypes. Mutant TDP-43 (A315T mutation) induced a motor axonopathy characterized by short axonal outgrowth and aberrant branching, similar, but more severe, than that induced by mutant SOD1. Knockdown of the two zebrafish progranulin genes, grna and grnb, produced a substantial decrease in axonal length, with knockdown of grna alone producing a greater decrease in axonal length than grnb. Progranulin overexpression rescued the axonopathy induced by progranulin knockdown. Interestingly, progranulin also rescued the mutant TDP-43 induced axonopathy, whilst it failed to affect the mutant SOD1-induced phenotype. TDP-43 was found to be nuclear in all conditions described. The findings described here demonstrate that progranulin is neuroprotective in vivo and may have therapeutic potential for at least some forms of motor neuron degeneration.     

Changed conformation of mutant Tau-P301L underlies the moribund tauopathy, absent in progressive, nonlethal axonopathy of Tau-4R/2N transgenic mice

Protein tau-3R/4R isoform ratio and phosphorylation regulates binding to microtubules and, when disturbed by aging or mutations, results in diverse tauopathies and in neurodegeneration. The underlying mechanisms were studied here in three transgenic mouse strains with identical genetic background, all expressing the tau-4R/2N isoform driven specifically in neurons by the thy1 gene promoter. Two strains, expressing human tau-4R/2N or mutant tau-4R/2N-P301L at similar, moderate levels, developed very different phenotypes. Tau-4R/2N mice became motor-impaired already around age 6-8 weeks, accompanied by axonopathy (dilatations, spheroids), but no tau aggregates, and surviving normally. In contrast, tau-P301L mice developed neurofibrillary tangles from age 6 months, without axonal dilatations and, despite only minor motor problems, all succumbing before the age of 13 months. The third strain, obtained by tau knock-out/knock-in (tau-KOKI), expressed normal levels of wild-type human tau-4R/2N replacing all mouse tau isoforms. Tau-KOKI mice survived normally with minor motor problems late in life and without any obvious pathology. Biochemically, a fraction of neuronal tau in aging tau-P301L mice was hyperphosphorylated concomitant with conformational changes and aggregation, but overall, tau-4R/2N was actually more phosphorylated than tau-P301L. Significantly, tau with changed conformation and with hyperphosphorylation colocalized in the same neurons in aging tau-P301L mice. Taken together, we conclude that excessive binding of tau-4R/2N as opposed to reduced binding of tau-P301L to microtubules is responsible for the development of axonopathy and tauopathy, respectively, in tau-4R/2N and tau-P301L mice and that the conformational change of tau-P301L is a major determinant in triggering the tauopathy.     

Linking axonal degeneration to microtubule remodeling by Spastin-mediated microtubule severing

Mutations in the AAA adenosine triphosphatase (ATPase) Spastin (SPG4) cause an autosomal dominant form of hereditary spastic paraplegia, which is a retrograde axonopathy primarily characterized pathologically by the degeneration of long spinal neurons in the corticospinal tracts and the dorsal columns. Using recombinant Spastin, we find that six mutant forms of Spastin, including three disease-associated forms, are severely impaired in ATPase activity. In contrast to a mutation designed to prevent adenosine triphosphate (ATP) binding, an ATP hydrolysis-deficient Spastin mutant predicted to remain kinetically trapped on target proteins decorates microtubules in transfected cells. Analysis of disease-associated missense mutations shows that some more closely resemble the canonical hydrolysis mutant, whereas others resemble the ATP-binding mutant. Using real-time imaging, we show that Spastin severs microtubules when added to permeabilized, cytosol-depleted cells stably expressing GFP-tubulin. Using purified components, we also show that Spastin interacts directly with microtubules and is sufficient for severing. These studies suggest that defects in microtubule severing are a cause of axonal degeneration in human disease.     

Peripheral neuropathy in the Twitcher mouse involves the activation of axonal caspase 3

Infantile Krabbe disease results in the accumulation of lipid-raft-associated galactosylsphingosine (psychosine), demyelination, neurodegeneration and premature death. Recently, axonopathy has been depicted as a contributing factor in the progression of neurodegeneration in the Twitcher mouse, a bona fide mouse model of Krabbe disease. Analysis of the temporal-expression profile of MBP (myelin basic protein) isoforms showed unexpected increases of the 14, 17 and 18.5 kDa isoforms in the sciatic nerve of 1-week-old Twitcher mice, suggesting an abnormal regulation of the myelination process during early postnatal life in this mutant. Our studies showed an elevated activation of the pro-apoptotic protease caspase 3 in sciatic nerves of 15- and 30-day-old Twitcher mice, in parallel with increasing demyelination. Interestingly, while active caspase 3 was clearly contained in peripheral axons at all ages, we found no evidence of caspase accumulation in the soma of corresponding mutant spinal cord motor neurons. Furthermore, active caspase 3 was found not only in unmyelinated axons, but also in myelinated axons of the mutant sciatic nerve. These results suggest that axonal caspase activation occurs before demyelination and following a dying-back pattern. Finally, we showed that psychosine was sufficient to activate caspase 3 in motor neuronal cells in vitro in the absence of myelinating glia. Taken together, these findings indicate that degenerating mechanisms actively and specifically mediate axonal dysfunction in Krabbe disease and support the idea that psychosine is a pathogenic sphingolipid sufficient to cause axonal defects independently of demyelination.     

A functional misexpression screen uncovers a role for enabled in progressive neurodegeneration

Drosophila is a well-established model to study the molecular basis of neurodegenerative diseases. We carried out a misexpression screen to identify genes involved in neurodegeneration examining locomotor behavior in young and aged flies. We hypothesized that a progressive loss of rhythmic activity could reveal novel genes involved in neurodegenerative mechanisms. One of the interesting candidates showing progressive arrhythmicity has reduced enabled (ena) levels. ena down-regulation gave rise to progressive vacuolization in specific regions of the adult brain. Abnormal staining of pre-synaptic markers such as cystein string protein (CSP) suggest that axonal transport could underlie the neurodegeneration observed in the mutant. Reduced ena levels correlated with increased apoptosis, which could be rescued in the presence of p35, a general Caspase inhibitor. Thus, this mutant recapitulates two important features of human neurodegenerative diseases, i.e., vulnerability of certain neuronal populations and progressive degeneration, offering a unique scenario in which to unravel the specific mechanisms in an easily tractable organism.     

A cytoplasmic dynein tail mutation impairs motor processivity

Mutations in the tail of the cytoplasmic dynein molecule have been reported to cause neurodegenerative disease in mice. The mutant mouse strain Legs at odd angles (Loa) has impaired retrograde axonal transport, but the molecular deficiencies in the mutant dynein molecule, and how they contribute to neurodegeneration, are unknown. To address these questions, we purified dynein from wild-type mice and the Legs at odd angles mutant mice. Using biochemical, single-molecule, and live-cell-imaging techniques, we find a marked inhibition of motor run-length in vitro and in vivo, and significantly altered motor domain coordination in the dynein from mutant mice. These results suggest a potential role for the dynein tail in motor function, and provide direct evidence for a link between single-motor processivity and disease.     

Transgenic mouse model of tauopathies with glial pathology and nervous system degeneration

Frontotemporal dementias (FTDs), including corticobasal degeneration (CBD) and progressive supranuclear palsy (PSP), are neurodegenerative tauopathies characterized by widespread CNS neuronal and glial tau pathologies, but there are no tau transgenic (Tg) mice that model neurodegeneration with glia tau lesions. Thus, we generated Tg mice overexpressing human tau in neurons and glia. No neuronal tau aggregates were detected, but old mice developed Thioflavin S- and Gallyas-positive glial tau pathology resembling CBD astrocytic plaques. Tau-immunoreactive and Gallyas-positive oligodendroglial coiled bodies (similar to CBD and PSP), glial degeneration, and motor deficits were associated with age-dependent accumulations of insoluble hyperphosphorylated human tau and tau immunopositive filaments in degenerating glial cells. Thus, tau-positive glial lesions similar to human FTDs occur in these Tg mice, and these pathologies are linked to glial and axonal degeneration.     

Depression-like behavior in the forced swimming test in PACAP-deficient mice: amelioration by the atypical antipsychotic risperidone

Axonal degeneration is a key component of many neurodegenerative diseases. Injured axons undergo a program of self-destruction termed Wallerian degeneration that is an active, well-regulated process. The pathways leading to axon fragmentation are uncharacterized, but experiments with wld ^s mutant mice led to the discovery that over-expression of NMN adenylyltransferase 1 or treatment with NAD⁺ can inhibit axonal degeneration. In this study, we show that the purine nucleosides adenosine and guanosine, but not inosine, inhibit injury-induced axonal degeneration in cultured dorsal root ganglia neurons. Axons can be preserved by adding adenosine within 6 h of the axonal injury. The presence of adenosine was required continuously after the injury to maintain axonal protection. Together these results suggest that adenosine does not alter the neuronal response to injury, but instead inhibits a local axonal pathway necessary for the commitment and/or execution of the axon destructive program.     

Proteomic and histochemical analysis of proteins involved in the dying-back-type of axonal degeneration in the gracile axonal dystrophy (gad) mouse

Local axonal degeneration is a common pathological feature of peripheral neuropathies and neurodegenerative disorders of the central nervous system, including Alzheimer's disease, Parkinson's disease, and stroke; however, the underlying molecular mechanism is not known. Here, we analyzed the gracile axonal dystrophy (gad) mouse, which displays the dying-back-type of axonal degeneration in sensory neurons, to find the molecules involved in the mechanism of axonal degeneration. The gad mouse is analogous to a null mutant of ubiquitin carboxyl-terminal hydrolase L1 (UCH-L1). UCH-L1 is a deubiquitinating enzyme expressed at high levels in neurons, as well as testis and ovary. In addition, we recently discovered a new function of UCH-L1—namely to bind to and stabilize mono-ubiquitin in neurons, and found that the level of mono-ubiquitin was decreased in neurons, especially in axons of the sciatic nerve, in gad mice. The low level of ubiquitin suggests that the target proteins of the ubiquitin proteasome system are not sufficiently ubiquitinated and thus degraded in the gad mouse; therefore, these proteins may be the key molecules involved in axonal degeneration. To identify molecules involved in axonal degeneration in gad mice, we compared protein expression in sciatic nerves between gad and wild-type mice at 2 and 12 weeks old, using two-dimensional difference gel electrophoresis. As a result, we found age-dependent accumulation of several proteins, including glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and 14-3-3, in gad mice compared with wild-type mice. Histochemical analyses demonstrated that GAPDH and 14-3-3 were localized throughout axons in both gad and wild-type mice, but GAPDH accumulated in the axons of gad mice. Recently, it has been suggested that a wide range of neurodegenerative diseases are characterized by the accumulation of intracellular and extracellular protein aggregates, and it has been reported that oxidative stress causes the aggregation of GAPDH. Furthermore, histochemical analysis demonstrated that sulfonated GAPDH, a sensor of oxidative stress that elicits cellular dysfunction, was expressed in the axons of gad mice, and 4-hydroxy-2-nonenal, a major marker of oxidative stress, was also only detected in gad mice. Our findings suggest that GAPDH may participate in a process of the dying-back-type of axonal degeneration in gad mice and may provide valuable insight into the mechanisms of axonal degeneration.     

Dopaminergic neuronal loss, reduced neurite complexity and autophagic abnormalities in transgenic mice expressing G2019S mutant LRRK2

Mutations in the leucine-rich repeat kinase 2 (LRRK2) gene cause late-onset, autosomal dominant familial Parkinson's disease (PD) and also contribute to idiopathic PD. LRRK2 mutations represent the most common cause of PD with clinical and neurochemical features that are largely indistinguishable from idiopathic disease. Currently, transgenic mice expressing wild-type or disease-causing mutants of LRRK2 have failed to produce overt neurodegeneration, although abnormalities in nigrostriatal dopaminergic neurotransmission have been observed. Here, we describe the development and characterization of transgenic mice expressing human LRRK2 bearing the familial PD mutations, R1441C and G2019S. Our study demonstrates that expression of G2019S mutant LRRK2 induces the degeneration of nigrostriatal pathway dopaminergic neurons in an age-dependent manner. In addition, we observe autophagic and mitochondrial abnormalities in the brains of aged G2019S LRRK2 mice and markedly reduced neurite complexity of cultured dopaminergic neurons. These new LRRK2 transgenic mice will provide important tools for understanding the mechanism(s) through which familial mutations precipitate neuronal degeneration and PD.