The structure of the brain as a product of morphogenesis is difficult to reconcile with the observed complexity of cerebral connectivity. We therefore analyzed relationships of adjacency and crossing between cerebral fiber pathways in four nonhuman primate species and in humans by using diffusion magnetic resonance imaging. The cerebral fiber pathways formed a rectilinear three-dimensional grid continuous with the three principal axes of development. Cortico-cortical pathways formed parallel sheets of interwoven paths in the longitudinal and medio-lateral axes, in which major pathways were local condensations. Cross-species homology was strong and showed emergence of complex gyral connectivity by continuous elaboration of this grid structure. This architecture naturally supports functional spatio-temporal coherence, developmental path-finding, and incremental rewiring with correlated adaptation of structure and function in cerebral plasticity and evolution.

Recessive mutations in parkin are the most common cause of familial early-onset Parkinson's disease (PD). Recent studies suggest that certain parkin mutants may exert dominant toxic effects to cultured cells and such dominant toxicity can lead to progressive dopaminergic (DA) neuron degeneration in Drosophila. To explore whether mutant parkin could exert similar pathogenic effects to mammalian DA neurons in vivo, we developed a BAC (bacterial artificial chromosome) transgenic mouse model expressing a C-terminal truncated human mutant parkin (Parkin-Q311X) in DA neurons driven by a dopamine transporter promoter. Parkin-Q311X mice exhibit multiple late-onset and progressive hypokinetic motor deficits. Stereological analyses reveal that the mutant mice develop age-dependent DA neuron degeneration in substantia nigra accompanied by a significant loss of DA neuron terminals in the striatum. Neurochemical analyses reveal a significant reduction of the striatal dopamine level in mutant mice, which is significantly correlated with their hypokinetic motor deficits. Finally, mutant Parkin-Q311X mice, but not wild-type controls, exhibit age-dependent accumulation of proteinase K-resistant endogenous ␣-synuclein in substantia nigra and colocalized with 3-nitrotyrosine, a marker for oxidative protein damage. Hence, our study provides the first mammalian genetic evidence that dominant toxicity of a parkin mutant is sufficient to elicit agedependent hypokinetic motor deficits and DA neuron loss in vivo, and uncovers a causal relationship between dominant parkin toxicity and progressive ␣-synuclein accumulation in DA neurons. Our study underscores the need to further explore the putative link between parkin dominant toxicity and PD.

The etiology of Parkinson disease (PD) is unclear but may involve environmental toxins such as pesticides leading to dysfunction of the ubiquitin proteasome system (UPS). Here, we measured the relative toxicity of ziram (a UPS inhibitor) and analogs to dopaminergic neurons and examined the mechanism of cell death. UPS (26 S) activity was measured in cell lines after exposure to ziram and related compounds. Dimethyl-and diethyldithiocarbamates including ziram were potent UPS inhibitors. Primary ventral mesencephalic cultures were exposed to ziram, and cell toxicity was assessed by staining for tyrosine hydroxylase (TH) and NeuN antigen. Ziram caused a preferential damage to TH؉ neurons and elevated ␣-synuclein levels but did not increase aggregate formation. Mechanistically, ziram altered UPS function through interfering with the targeting of substrates by inhibiting ubiquitin E1 ligase. Sodium dimethyldithiocarbamate administered to mice for 2 weeks resulted in persistent motor deficits and a mild reduction in striatal TH staining but no nigral cell loss. These results demonstrate that ziram causes selective dopaminergic cell damage in vitro by inhibiting an important degradative pathway implicated in the etiology of PD. Chronic exposure to widely used dithiocarbamate fungicides may contribute to the development of PD, and elucidation of its mechanism would identify a new potential therapeutic target. Parkinson disease (PD)2 is a common neurodegenerative disease characterized by relatively selective degeneration of dopaminergic (DA) neurons in the substantia nigra (nigrostriatal neurons). The etiology probably involves both environmental and genetic factors including pesticide exposure (1-3). Hundreds of pesticides are used alone or in combinations making it difficult to separate individual effects. Because no individual pesticide has been established by epidemiologic studies, we chose to perform an unbiased screen of potential toxicants for their ability to interfere with the ubiquitin-proteasome system (UPS), a biological pathway implicated in the etiology of PD. Impaired UPS activity has been reported in the brains of patients with PD, and mutations in two UPS genes, Parkin and UCHL-1, cause rare genetic forms of PD (4). Although these results are not universally reproduced (5-7), in some studies administration of UPS inhibitors to rodents recapitulates many of the clinical and pathological aspects of PD (8 -10). We hypothesized that chronic pesticide exposure may increase the risk of developing PD by inhibiting the UPS. We screened several pesticides for their ability to inhibit the UPS and found a number of toxicants that can lower activity at relevant concentrations (11). We then focused on dithiocarbamate fungicides because they were found to be one of the most potent UPS inhibitors and are widely used in crop protection.In the present study, zinc dimethyldithiocarbamate (ziram) was one of several dimethyl-and diethyldithiocarbamates found to inhibit the UPS at 0.15-1 M. Furthermore, ziram increased ␣-syn...

Mutation of the lysosomal hydrolase acid-β-glucosidase (GCase), which leads to reduced GCase activity, is one of the most frequent genetic risk factors for Parkinson's disease (PD) and promotes α-synuclein accumulation in the brain, a hallmark of PD and other synucleinopathies. Whether targeting GCase pharmacologically is a valid therapeutic strategy for sporadic PD in the absence of GCase mutation is unknown. We have investigated whether increasing the stability, trafficking, and activity of wild-type GCase could be beneficial in synucleinopathies by administering the pharmacological chaperone AT2101 (afegostat-tartrate, isofagomine) to mice that overexpress human wild-type α-synuclein (Thy1-aSyn mice). AT2101 administered orally for 4 months to Thy1-aSyn mice improved motor and nonmotor function, abolished microglial inflammatory response in the substantia nigra, reduced α-synuclein immunoreactivity in nigral dopaminergic neurons, and reduced the number of small α-synuclein aggregates, while increasing the number of large α-synuclein aggregates. These data support the further investigation of pharmacological chaperones that target GCase as a therapeutic approach for sporadic PD and other synucleinopathies, even in the absence of glucocerebrosidase mutations.

Individual variation in the addiction liability of amphetamines has a heritable genetic component. We previously identified Hnrnph1 (heterogeneous nuclear ribonucleoprotein H1) as a quantitative trait gene underlying decreased methamphetamine-induced locomotor activity in mice. Here, we showed that mice (both females and males) with a heterozygous mutation in the first coding exon of Hnrnph1 (H1 ϩ/Ϫ) showed reduced methamphetamine reinforcement and intake and dose-dependent changes in methamphetamine reward as measured via conditioned place preference. Furthermore, H1 ϩ/Ϫ mice showed a robust decrease in methamphetamine-induced dopamine release in the NAc with no change in baseline extracellular dopamine, striatal whole-tissue dopamine, dopamine transporter protein, dopamine uptake, or striatal methamphetamine and amphetamine metabolite levels. Immunohistochemical and immunoblot staining of midbrain dopaminergic neurons and their forebrain projections for TH did not reveal any major changes in staining intensity, cell number, or forebrain puncta counts. Surprisingly, there was a twofold increase in hnRNP H protein in the striatal synaptosome of H1 ϩ/Ϫ mice with no change in whole-tissue levels. To gain insight into the mechanisms linking increased synaptic hnRNP H with decreased methamphetamine-induced dopamine release and behaviors, synaptosomal proteomic analysis identified an increased baseline abundance of several mitochondrial complex I and V proteins that rapidly decreased at 30 min after methamphetamine administration in H1 ϩ/Ϫ mice. In contrast, the much lower level of basal synaptosomal mitochondrial proteins in WT mice showed a rapid increase. We conclude that H1 ϩ/Ϫ decreases methamphetamine-induced dopamine release, reward, and reinforcement and induces dynamic changes in basal and methamphetamine-induced synaptic mitochondrial function.

Parkinson's disease (PD) is a neurodegenerative disorder characterized by the loss of nigrostriatal dopaminergic neurons and the accumulation of alpha-synuclein. Both traumatic brain injury (TBI) and pesticides are risk factors for PD, but whether TBI causes nigrostriatal dopaminergic cell loss in experimental models and whether it acts synergistically with pesticides is unknown. We have examined the acute and long-term effects of TBI and exposure to low doses of the pesticide paraquat, separately and in combination, on nigrostriatal dopaminergic neurons in adult male rats. In an acute study, rats received moderate TBI by lateral fluid percussion (LFP) injury, were injected with saline or paraquat (10 mg/kg IP) 3 and 6 days after LFP, were sacrificed 5 days later, and their brains processed for immunohistochemistry. TBI alone increased microglial activation in the substantia nigra, and caused a 15% loss of dopaminergic neurons ipsilaterally. Paraquat increased the TBI effect, causing a 30% bilateral loss of dopaminergic neurons, reduced striatal tyrosine hydroxylase (TH) immunoreactivity more than TBI alone, and induced alpha-synuclein accumulation in the substantia nigra pars compacta. In a long-term study, rats received moderate LFP, were injected with saline or paraquat at 21 and 22 weeks post-injury, and were sacrificed 4 weeks later. At 26 weeks post injury, TBI alone induced a 30% bilateral loss of dopaminergic neurons that was not exacerbated by paraquat. These data suggest that TBI is sufficient to induce a progressive degeneration of nigrostriatal dopaminergic neurons. Furthermore, TBI and pesticide exposure, when occurring within a defined time frame, could combine to increase the PD risk.

While cognitive decline is observed in the normal aging monkey, neurons are not lost with age. Instead, frontal white matter is lost as myelin degenerates and both correlate with age-related cognitive decline. As age-related myelin damage increases, there should be an increase in clearance of damaged myelin by microglial phagocytosis. In this study, brains of behaviorally tested rhesus monkeys were assessed using unbiased stereology to quantify the density of activated microglia (LN3 antibody positive) and phagocytic microglia (galectin-3 (Gal-3) antibody positive) in three white matter regions: the corpus callosum, cingulum bundle (CGB), and frontal white matter (FWM). LN3 cell density was significantly increased in the CGB, whereas Gal-3 cell density was significantly increased in all regions. Increases in Gal-3 cell density in the FWM were associated with cognitive impairment. In the FWM of old animals, Gal-3-positive microglia were classified by morphological subtype as ramified, hypertrophic, or amoeboid. The densities of hypertrophic and amoeboid microglia significantly correlated with cognitive impairment. Finally, microglia were double-labeled with LN3 and Gal-3 showing that 91% of Gal-3 cells were also LN3 positive, thus expressing an Bactivated^phenotype. Furthermore, 15% of all double-labeled cells formed phagocytic cups. Overall, these results suggest that microglia become activated in white matter with age where the majority express a phagocytic phenotype. We hypothesize that age-related phagocytic activation of microglia is a response to accumulating myelin pathology. The association of Gal-3 in the FWM with cognitive impairment may reflect regional differences in damage or dysfunction of normal clearance mechanisms.

Abnormal accumulation of α-synuclein is associated with several neurodegenerative disorders (synucleinopathies), including sporadic Parkinson's disease (PD). Genetic mutations and multiplication of α-synuclein cause familial forms of PD and polymorphisms in the α-synuclein gene are associated with PD risk. Overexpression of α-synuclein can impair essential functions within the cell such as microtubule-dependent transport, suggesting that compounds that act on the microtubule system may have therapeutic benefit for synucleinopathies. In this study, mice overexpressing human wildtype α-synuclein under the Thy1 promoter (Thy1-aSyn) and littermate wildtype control mice were administered daily the microtubule-interacting peptide NAPVSIPQ (NAP; also known as davunetide or AL-108) intranasally for two months starting at one month of age, in a regimen known to produce effective concentrations of the peptide in mouse brain. Motor performance, coordination, and activity were assessed at the end of treatment. Olfactory function, which is altered in PD, was measured one month later. Mice were sacrificed at 4.5 months of age, and their brains examined for proteinase K-resistant α-synuclein inclusions in the substantia nigra and olfactory bulb. NAP-treated Thy1-aSyn mice showed a 38% decrease in the number of errors per step in the challenging beam traversal test and a reduction in proteinase K-resistant α-synuclein inclusions in the substantia nigra compared to vehicle treated transgenics. The data indicate a significant behavioral benefit and a long lasting improvement of α-synuclein pathology following administration of a short term (2 month) NAP administration in a mouse model of synucleinopathy.