Chunni Zhu

Behavioral and immunohistochemical effects of chronic intravenous and subcutaneous infusions of varying doses of rotenone

Mitochondrial toxins such as the complex 1 inhibitor rotenone are widely used as pesticides and may be present in military environments. Administration of rotenone can induce biochemical and histological alterations similar to those of Parkinsons disease in rats. However, only a subset of animals show these effects and it is unclear whether more subtle alterations are caused by chronic administration of rotenone in those animals that appear resistant to its toxic effects on dopaminergic nerve terminals. To address this question, vehicle or rotenone (2.0, 2.5, or 3.5 mg/kg/day) was administered intravenously or subcutaneously for 21 days to adult rats, and rotenone effects on survival, motor behavior, and striatal tyrosine hydroxylase immunoreactivity (TH-IR) were examined. Both intravenous and subcutaneous rotenone induced a dose-dependent decrease in survival rates. Surviving animals showed a decrease in spontaneous rearing. Locomotor activity and movement initiation time were also altered in some of the experimental groups. Confirming previous results, TH-IR in the striatum was markedly decreased in rats that fell ill early in the study and in a few of the surviving rats with high rotenone doses. However, none of the surviving rats receiving 2.0 mg/kg/day showed TH-IR loss reminiscent of Parkinsons disease, and loss of striatal TH-IR across doses was not correlated with motor behavior in individual rats. Thus, chronic administration of low doses of rotenone induces motor anomalies even in animals that do not develop histological signs of Parkinsons disease, indicating a pervasive neurological effect of moderate mitochondrial dysfunction in vivo.

Neurons express hemoglobin α‐ and β‐chains in rat and human brains

Hemoglobin is the oxygen carrier in vertebrate blood erythrocytes. Here we report that hemoglobin chains are expressed in mammalian brain neurons and are regulated by a mitochondrial toxin. Transcriptome analyses of laser‐capture microdissected nigral dopaminergic neurons in rats and striatal neurons in mice revealed the presence of hemoglobin α, adult chain 2 (Hba‐a2) and hemoglobin β (Hbb) transcripts, whereas other erythroid markers were not detected. Quantitative reverse transcriptase‐polymerase chain reaction (RT‐PCR) analysis confirmed the expression of Hba‐a2 and Hbb in nigral dopaminergic neurons, striatal γ‐aminobutyric acid (GABA)ergic neurons, and cortical pyramidal neurons in rats. Combined in situ hybridization histochemistry and immunohistochemistry with the neuronal marker neuronal nuclear antigen (NeuN) in rat brain further confirmed the presence of hemoglobin mRNAs in neurons. Immunohistochemistry identified hemoglobin α‐ and β‐chains in both rat and human brains, and hemoglobin proteins were detected by Western blotting in whole rat brain tissue as well as in cultures of mesencephalic neurons, further excluding the possibility of blood contamination. Systemic administration of the mitochondrial inhibitor rotenone (2 mg/kg/d, 7d, s.c.) induced a marked decrease in Hba‐a2 and Hbb but not neuroglobin or cytoglobin mRNA in transcriptome analyses of nigral dopaminergic neurons. Quantitative RT‐PCR confirmed the transcriptional downregulation of Hba‐a2 and Hbb in nigral, striatal, and cortical neurons. Thus, hemoglobin chains are expressed in neurons and are regulated by treatments that affect mitochondria, opening up the possibility that they may play a novel role in neuronal function and response to injury. J. Comp. Neurol. 515:538–547, 2009.

A Progressive Mouse Model of Parkinson’s Disease: The Thy1-aSyn (“Line 61”) Mice

Identification of mutations that cause rare familial forms of Parkinson’s disease (PD) and subsequent studies of genetic risk factors for sporadic PD have led to an improved understanding of the pathological mechanisms that may cause nonfamilial PD. In particular, genetic and pathological studies strongly suggest that alpha-synuclein, albeit very rarely mutated in PD patients, plays a critical role in the vast majority of individuals with the sporadic form of the disease. We have extensively characterized a mouse model over-expressing full-length, human, wild-type alpha-synuclein under the Thy-1 promoter. We have also shown that this model reproduces many features of sporadic PD, including progressive changes in dopamine release and striatal content, alpha-synuclein pathology, deficits in motor and nonmotor functions that are affected in pre-manifest and manifest phases of PD, inflammation, and biochemical and molecular changes similar to those observed in PD. Preclinical studies have already demonstrated improvement with promising new drugs in this model, which provides an opportunity to test novel neuroprotective strategies during different phases of the disorder using endpoint measures with high power to detect drug effects.

Variable effects of chronic subcutaneous administration of rotenone on striatal histology.

When infused in rats, rotenone, a mitochondrial complex I inhibitor, induces alterations that resemble the histological changes of Parkinsons disease, particularly degeneration of the nigrostriatal dopaminergic system. However, the specificity of rotenone effects has been challenged recently. We have re‐examined the alterations caused by rotenone in the substantia nigra and the striatum of rats after infusion of rotenone (2 mg/kg per day s.c.) for 21 days. Three patterns of striatal tyrosine‐hydroxylase immunoreactivity (TH‐IR) were observed: 46% of animals showed no reduction, and 46% of animals showed diffuse reduction in TH‐IR, whereas one animal presented a focal loss of TH‐IR in the striatum. Confocal microscopy analysis showed that the vesicular monoamine transporter (VMAT2) was decreased in parallel with TH‐IR, strongly suggesting a loss of striatal DA nerve terminals in animals with diffuse or central TH‐IR loss. However, no significant loss of TH‐IR neurons was observed in the substantia nigra. Analysis of NeuN and DARPP‐32 immunoreactivity, and Nissl staining, in the striatum showed no striatal neuronal loss in animals with either preserved TH‐IR or diffuse TH‐IR reduction. However, in the animal with focal TH‐IR loss, severe neuronal loss was evident in the center and the periphery of the striatum, together with microglial activation detected by OX‐6 and OX‐42 staining. Thus, in most cases, chronic subcutaneous infusion of low doses of rotenone does not induce significant striatal neuronal loss, despite TH‐IR and VMAT‐IR reduction in a subset of animals, supporting the use of rotenone as a model of Parkinsons disease under carefully controlled experimental conditions. J. Comp. Neurol. 478:418–426, 2004.

Improvement of neuropathology and transcriptional deficits in CAG 140 knock-in mice supports a beneficial effect of dietary curcumin in Huntington's disease

BackgoundNo disease modifying treatment currently exists for Huntingtons disease (HD), a fatal neurodegenerative disorder characterized by the formation of amyloid-like aggregates of the mutated huntingtin protein. Curcumin is a naturally occurring polyphenolic compound with Congo red-like amyloid binding properties and the ability to cross the blood brain barrier. CAG140 mice, a knock-in (KI) mouse model of HD, display abnormal aggregates of mutant huntingtin and striatal transcriptional deficits, as well as early motor, cognitive and affective abnormalities, many months prior to exhibiting spontaneous gait deficits, decreased striatal volume, and neuronal loss. We have examined the ability of life-long dietary curcumin to improve the early pathological phenotype of CAG140 mice.ResultsKI mice fed a curcumin-containing diet since conception showed decreased huntingtin aggregates and increased striatal DARPP-32 and D1 receptor mRNAs, as well as an amelioration of rearing deficits. However, similar to other antioxidants, curcumin impaired rotarod behavior in both WT and KI mice and climbing in WT mice. These behavioral effects were also noted in WT C57Bl/6 J mice exposed to the same curcumin regime as adults. However, neither locomotor function, behavioral despair, muscle strength or food utilization were affected by curcumin in this latter study. The clinical significance of curcumins impairment of motor performance in mice remains unclear because curcumin has an excellent blood chemistry and adverse event safety profile, even in the elderly and in patients with Alzheimers disease.ConclusionTogether with this clinical experience, the improvement in several transgene-dependent parameters by curcumin in our study supports a net beneficial effect of dietary curcumin in HD.

Cognitive deficits in a mouse model of pre-manifest Parkinson’s disease

Early cognitive deficits are increasingly recognized in patients with Parkinson’s disease (PD), and represent an unmet need for the treatment of PD. These early deficits have been difficult to model in mice, and their mechanisms are poorly understood. α‐Synuclein is linked to both familial and sporadic forms of PD, and is believed to accumulate in brains of patients with PD before cell loss. Mice expressing human wild‐type α‐synuclein under the Thy1 promoter (Thy1‐aSyn mice) exhibit broad overexpression of α‐synuclein throughout the brain and dynamic alterations in dopamine release several months before striatal dopamine loss. We now show that these mice exhibit deficits in cholinergic systems involved in cognition, and cognitive deficits in domains affected in early PD. Together with an increase in extracellular dopamine and a decrease in cortical acetylcholine at 4–6 months of age, Thy1‐aSyn mice made fewer spontaneous alternations in the Y‐maze and showed deficits in tests of novel object recognition (NOR), object–place recognition, and operant reversal learning, as compared with age‐matched wild‐type littermates. These data indicate that cognitive impairments that resemble early PD manifestations are reproduced by α‐synuclein overexpression in a murine genetic model of PD. With high power to detect drug effects, these anomalies provide a novel platform for testing improved treatments for these pervasive cognitive deficits.

A pilot trial of the microtubule-interacting peptide (NAP) in mice overexpressing alpha-synuclein shows improvement in motor function and reduction of alpha-synuclein inclusions.

Abnormal accumulation of α-synuclein is associated with several neurodegenerative disorders (synucleinopathies), including sporadic Parkinsons 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 2 months starting at 1 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 1 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 months) NAP administration in a mouse model of synucleinopathy.

Striatal atrophy and dendritic alterations in a knock-in mouse model of Huntington's disease

Huntingtons disease (HD) is a progressive neurodegenerative disease characterized by progressive atrophy of the striatum, cerebral cortex, and white matter tracks. Major pathological hallmarks of HD include neuronal loss, primarily in the striatum, and dendritic anomalies in surviving striatal neurons. Although many mouse models of HD have been generated, their success at reproducing all pathological features of the disease is not fully known. Previously, we demonstrated extensive striatal neuronal loss and striatal atrophy at 20-26 months of age in a knock-in (KI) mouse model of HD. To further investigate this model, which carries a human exon 1 with ∼119 CAG repeats inserted into the mouse gene (initially 140 repeats), we have examined whether these mice exhibit the atrophy and neuronal anomalies characteristic of HD. Stereological analyses revealed no changes in the striatal volume of male and female homozygote mice at 4 months, however striatal atrophy was already present at 12 months in both sexes. Analysis of cortical and corpus callosum volume in male homozygotes revealed a loss in corpus callosum volume by 20-26 months. At this later age, the surviving striatal neurons displayed extensive loss of spines in distal branch orders that affected both immature and mature spines. Mirroring late stage HD striatal neuronal morphology, the striatal neurons at this late age also showed reduced dendritic complexity, as revealed by Sholl analysis. Tyrosine hydroxylase immunoreactivity was also decreased in the striatum of 20-26 month old KI mice, suggesting an alteration in striatal inputs. These data further indicate that CAG140 homozygote KI mice exhibit HD-like pathological features and are a useful model to test the effects of early and/or sustained administration of novel neuroprotective treatments.

Context Dependent Neuroprotective Properties of Prion Protein (Prp)

Although it has been known for more than twenty years that an aberrant conformation of the prion protein (PrP) is the causative agent in prion diseases, the role of PrP in normal biology is undetermined. Numerous studies have suggested a protective function for PrP, including protection from ischemic and excitotoxic lesions and several apoptotic insults. On the other hand, many observations have suggested the contrary, linking changes in PrP localization or domain structure—independent of infectious prion conformation—to severe neuronal damage. Surprisingly, a recent report suggests that PrP is a receptor for toxic oligomeric species of a-β, a pathogenic fragment of the amyloid precursor protein, and likely contributes to disease pathogenesis of Alzheimer’s disease. We sought to access the role of PrP in diverse neurological disorders. First, we confirmed that PrP confers protection against ischemic damage using an acute stroke model, a well characterized association. After ischemic insult, PrP knockouts had dramatically increased infarct volumes and decreased behavioral performance compared to controls. To examine the potential of PrP’s neuroprotective or neurotoxic properties in the context of other pathologies, we deleted PrP from several transgenic models of neurodegenerative disease. Deletion of PrP did not substantially alter the disease phenotypes of mouse models of Parkinson’s disease or tauopathy. Deletion of PrP in one of two Huntington’s disease models tested, R6/2, modestly slowed motor deterioration as measured on an accelerating rotarod but otherwise did not alter other major features of the disease. Finally, transgenic overexpression of PrP did not exacerbate the Huntington’s motor phenotype. These results suggest that PrP has a context-dependent neuroprotective function and does not broadly contribute to the disease models tested herein.

Evidence for behavioral benefits of early dietary supplementation with CoEnzymeQ10 in a slowly progressing mouse model of Huntington's disease.

Controversies surround the usefulness of Coenzyme Q10 (CoQ10) in Huntingtons disease (HD), an autosomal dominant, fatal, neurodegenerative disease with no cure or disease modifying treatment. CoQ10, an endogenous substrate for electron transport and an anti-oxidant, has been shown in some but not all studies to improve symptoms and survival in mouse models of HD. Previous studies have been conducted in fast-progressing models that better mimic the juvenile forms of HD than the much more common middle-age onset form, possibly accounting for mixed results. Establishing the usefulness of CoQ10 to alter HD disease course in a model that better recapitulates the progressive features of the human disorder is important because clinical trials of CoQ10, which is safe and well tolerated, are being planned in patients. The CAG140 knock-in (KI) mouse model of HD in which an expanded (approximately 120) CAG repeat is inserted in the mouse gene provides a model of the mutation in the proper genomic and protein context. These mice display progressive motor, cognitive and emotional anomalies, transcriptional disturbances and late striatal degeneration. Homozygote mutant CAG140 KI mice and wild-type littermates were fed CoQ10 (0.2%, 0.6%) in chow, and behavioral and pathological markers of disease were examined. CoQ10 improved early behavioral deficits and normalized some transcriptional deficits without altering huntingtin aggregates in striatum. The lower dose (0.2%) was more beneficial than 0.6%. Similar to previous studies, this low dose also induced deleterious effects in open field and rotarod in WT mice, however these effects are of unclear clinical significance in view of the excellent safety profile of CoQ10 in humans. These data confirm that CoQ10 may be beneficial in HD but suggest that maximum benefit may be observed when treatment is begun at early stages of the disease and that dosage may be critical.