Journal of Neuroscience Neurobiology of Disease

T-Cell Infiltration and Signaling in the Adult Dorsal Spinal Cord Is a Major Contributor to Neuropathic Pain-Like Hypersensitivity

Wed, 18 Nov 2009 10:02:12 PST

Partial peripheral nerve injury in adult rats results in neuropathic pain-like hypersensitivity, while that in neonatal rats does not, a phenomenon also observed in humans. We therefore compared gene expression profiles in the dorsal horn of adult and neonatal rats in response to the spared nerve injury (SNI) model of peripheral neuropathic pain. The 148 differentially regulated genes in adult, but not young, rat spinal cords indicate a greater microglial and T-cell response in adult than in young animals. T-cells show a large infiltration in the adult dorsal horn but not in the neonate after SNI. T-cell-deficient Rag1-null adult mice develop less neuropathic mechanical allodynia than controls, and central expression of cytokines involved in T-cell signaling exhibits large relative differences between young and adult animals after SNI. One such cytokine, interferon- (IFN), is upregulated in the dorsal horn after nerve injury in the adult but not neonate, and we show that IFN signaling is required for full expression of adult neuropathic hypersensitivity. These data reveal that T-cell infiltration and activation in the dorsal horn of the spinal cord following peripheral nerve injury contribute to the evolution of neuropathic pain-like hypersensitivity. The neuroimmune interaction following peripheral nerve injury has therefore a substantial adaptive immune component, which is absent or suppressed in the young CNS.

Divergent Pathways Mediate Spine Alterations and Cell Death Induced by Amyloid-{beta}, Wild-Type Tau, and R406W Tau

Tackenberg, C., Brandt, R. — Wed, 18 Nov 2009 10:02:12 PST

Alzheimer's disease is characterized by synaptic alterations and neurodegeneration. Histopathological hallmarks represent amyloid plaques composed of amyloid-β (Aβ) and neurofibrillary tangles containing hyperphosphorylated tau. To determine whether synaptic changes and neurodegeneration share common pathways, we established an ex vivo model using organotypic hippocampal slice cultures from amyloid precursor protein transgenic mice combined with virus-mediated expression of EGFP-tagged tau constructs. Confocal high-resolution imaging, algorithm-based evaluation of spines, and live imaging were used to determine spine changes and neurodegeneration. We report that Aβ but not tau induces spine loss and shifts spine shape from mushroom to stubby through a mechanism involving NMDA receptor (NMDAR), calcineurin, and GSK-3β activation. In contrast, Aβ alone does not cause neurodegeneration but induces toxicity through phosphorylation of wild-type (wt) tau in an NMDAR-dependent pathway. We show that GSK-3β levels are elevated in APP transgenic cultures and that inhibiting GSK-3β activity or use of phosphorylation-blocking tau mutations prevented Aβ-induced toxicity of tau. FTDP-17 tau mutants are differentially affected by Aβ. While R406W tau shows increased toxicity in the presence of Aβ, no change is observed with P301L tau. While blocking NMDAR activity abolishes toxicity of both wt and R406W tau, the inhibition of GSK-3β only protects against toxicity of wt tau but not of R406W tau induced by Aβ. Tau aggregation does not correlate with toxicity. We propose that Aβ-induced spine pathology and tau-dependent neurodegeneration are mediated by divergent pathways downstream of NMDAR activation and suggest that Aβ affects wt and R406W tau toxicity by different pathways downstream of NMDAR activity.

Amyloid Precursor Protein Mediates a Tyrosine Kinase-Dependent Activation Response in Endothelial Cells

Austin, S. A., Sens, M. A., Combs, C. K. — Wed, 18 Nov 2009 10:02:12 PST

Amyloid precursor protein (APP) is a ubiquitously expressed type 1 integral membrane protein. It has the ability to bind numerous extracellular matrix components and propagate signaling responses via its cytoplasmic phospho-tyrosine, ₆₈₂YENPTY₆₈₇, binding motif. We recently demonstrated increased protein levels of APP, phosphorylated APP (Tyr682), and β-amyloid (Aβ) in brain vasculature of atherosclerotic and Alzheimer's disease (AD) tissue colocalizing primarily within the endothelial layer. This study demonstrates similar APP changes in peripheral vasculature from human and mouse apoE^–/– aorta, suggesting that APP-related changes are not restricted to brain vasculature. Therefore, primary mouse aortic endothelial cells and human umbilical vein endothelial cells were used as a model system to examine the function of APP in endothelial cells. APP multimerization with an anti-N-terminal APP antibody, 22C11, to simulate ligand binding stimulated an Src kinase family-dependent increase in protein phospho-tyrosine levels, APP phosphorylation, and Aβ secretion. Furthermore, APP multimerization stimulated increased protein levels of the proinflammatory proteins, cyclooxygenase-2 and vascular cell adhesion molecule-1 also in an Src kinase family-dependent manner. Endothelial APP was also involved in mediating monocytic cell adhesion. Collectively, these data demonstrate that endothelial APP regulates immune cell adhesion and stimulates a tyrosine kinase-dependent response driving acquisition of a reactive endothelial phenotype. These APP-mediated events may serve as therapeutic targets for intervention in progressive vascular changes common to cerebrovascular disease and AD.

APP Anterograde Transport Requires Rab3A GTPase Activity for Assembly of the Transport Vesicle

Wed, 18 Nov 2009 10:02:12 PST

The amyloid precursor protein (APP) is anterogradely transported by conventional kinesin in a distinct transport vesicle, but both the biochemical composition of such a vesicle and the specific kinesin-1 motor responsible for transport are poorly defined. APP may be sequentially cleaved by β- and -secretases leading to accumulation of β-amyloid (Aβ) peptides in brains of Alzheimer's disease patients, whereas cleavage of APP by -secretases prevents Aβ generation. Here, we demonstrate by time-lapse analysis and immunoisolations that APP is a cargo of a vesicle containing the kinesin heavy chain isoform kinesin-1C, the small GTPase Rab3A, and a specific subset of presynaptic protein components. Moreover, we report that assembly of kinesin-1C and APP in this vesicle type requires Rab3A GTPase activity. Finally, we show cleavage of APP in transport vesicles by -secretase activity, likely mediated by ADAM10. Together, these data indicate that maturation of APP transport vesicles, including recruitment of conventional kinesin, requires Rab3 GTPase activity.

Ghrelin Promotes and Protects Nigrostriatal Dopamine Function via a UCP2-Dependent Mitochondrial Mechanism

Wed, 11 Nov 2009 10:02:14 PST

Ghrelin targets the hypothalamus to regulate food intake and adiposity. Endogenous ghrelin receptors [growth hormone secretagogue receptor (GHSR)] are also present in extrahypothalamic sites where they promote circuit activity associated with learning and memory, and reward seeking behavior. Here, we show that the substantia nigra pars compacta (SNpc), a brain region where dopamine (DA) cell degeneration leads to Parkinson's disease (PD), expresses GHSR. Ghrelin binds to SNpc cells, electrically activates SNpc DA neurons, increases tyrosine hydroxylase mRNA and increases DA concentration in the dorsal striatum. Exogenous ghrelin administration decreased SNpc DA cell loss and restricted striatal dopamine loss after 1-methyl-4-phenyl-1,2,5,6 tetrahydropyridine (MPTP) treatment. Genetic ablation of ghrelin or the ghrelin receptor (GHSR) increased SNpc DA cell loss and lowered striatal dopamine levels after MPTP treatment, an effect that was reversed by selective reactivation of GHSR in catecholaminergic neurons. Ghrelin-induced neuroprotection was dependent on the mitochondrial redox state via uncoupling protein 2 (UCP2)-dependent alterations in mitochondrial respiration, reactive oxygen species production, and biogenesis. Together, our data reveal that peripheral ghrelin plays an important role in the maintenance and protection of normal nigrostriatal dopamine function by activating UCP2-dependent mitochondrial mechanisms. These studies support ghrelin as a novel therapeutic strategy to combat neurodegeneration, loss of appetite and body weight associated with PD. Finally, we discuss the potential implications of these studies on the link between obesity and neurodegeneration.

A{beta} Immunotherapy Protects Morphology and Survival of Adult-Born Neurons in Doubly Transgenic APP/PS1 Mice

Biscaro, B., Lindvall, O., Hock, C., Ekdahl, C. T., Nitsch, R. M. — Wed, 11 Nov 2009 10:02:14 PST

The hippocampus is heavily affected by progressive neurodegeneration and β-amyloid pathology in Alzheimer's disease (AD). The hippocampus is also one of the few brain regions that generate new neurons throughout adulthood. Because hippocampal neurogenesis is regulated by both endogenous and environmental factors, we determined whether it benefits from therapeutic reduction of β-amyloid peptide (Aβ)-related toxicity induced by passive Aβ immunotherapy. Aβ immunotherapy of 8–9-month-old mice expressing familial AD-causing mutations in the amyloid precursor protein and presenilin-1 genes with an antibody against Aβ decreased compact β-amyloid plaque burden and promoted survival of newly born neurons in the hippocampal dentate gyrus. As these neurons matured, they exhibited longer dendrites with more complex arborization compared with newly born neurons in control-treated transgenic littermates. The newly born neurons showed signs of functional integration indicated by expression of the immediate-early gene Zif268 in response to exposure to a novel object. Aβ immunotherapy was associated with higher numbers of synaptophysin-positive synaptic boutons. Labeling dividing progenitor cells with a retroviral vector encoding green fluorescent protein (GFP) showed that Aβ immunotherapy restored the impaired dendritic branching, as well as the density of dendritic spines in new mature neurons. The presence of cellular prion protein (PrP^c) on the dendrites of the GFP⁺ newly born neurons is compatible with a putative role of PrP^c in mediating Aβ-related toxicity in these cells. In addition, passive Aβ immunotherapy was accompanied by increased angiogenesis. Our data establish that passive Aβ immunotherapy can restore the morphological maturation of the newly formed neurons in the adult hippocampus and promote angiogenesis. These findings provide evidence for a role of Aβ immunotherapy in stimulating neurogenesis and angiogenesis in transgenic mouse models of AD, and they suggest the possibility that Aβ immunotherapy can recover neuronal and vascular functions in brains with β-amyloidosis.

NOS2 Gene Deficiency Protects from Sepsis-Induced Long-Term Cognitive Deficits

Wed, 11 Nov 2009 10:02:14 PST

To date, long-term consequences of septic encephalopathy on cerebral metabolism, cognition, learning, and memory capabilities and factors involved are poorly understood. In this study, we used a murine sepsis model to demonstrate that bacterial lipopolysaccharide (LPS) causes long-term cognitive deficits in mice. Two months after LPS treatment, wild-type mice committed more working and reference memory errors than controls. The behavioral impairment was independent of the cerebral glucose uptake as evidenced by ¹⁸F-Fluordeoxyglucose small animal positron emission tomography. In contrast, mice deficient for the inducible nitric oxide synthase gene (NOS2–/–) did not show any cognitive changes when challenged with LPS. Immunohistochemical analysis demonstrated that LPS did not lead to neuronal cell death but caused sustained microglial activation in wild-type as compared to NOS2–/– mice. Expression analysis showed that LPS-treated NOS2–/– mice had lower brain mRNA levels for proinflammatory factors compared with wild-type mice. Expression analysis demonstrated distinct changes in the content of synaptic proteins in wild-type mice, which were not observed in the NOS2–/– mice. Together, this data set outlines the importance of the NOS2 activation for long-term cerebral changes after severe sepsis.

A Selective Allosteric Potentiator of the M1 Muscarinic Acetylcholine Receptor Increases Activity of Medial Prefrontal Cortical Neurons and Restores Impairments in Reversal Learning

Wed, 11 Nov 2009 10:02:14 PST

M₁ muscarinic acetylcholine receptors (mAChRs) may represent a viable target for treatment of disorders involving impaired cognitive function. However, a major limitation to testing this hypothesis has been a lack of highly selective ligands for individual mAChR subtypes. We now report the rigorous molecular characterization of a novel compound, benzylquinolone carboxylic acid (BQCA), which acts as a potent, highly selective positive allosteric modulator (PAM) of the rat M₁ receptor. This compound does not directly activate the receptor, but acts at an allosteric site to increase functional responses to orthosteric agonists. Radioligand binding studies revealed that BQCA increases M₁ receptor affinity for acetylcholine. We found that activation of the M₁ receptor by BQCA induces a robust inward current and increases spontaneous EPSCs in medial prefrontal cortex (mPFC) pyramidal cells, effects which are absent in acute slices from M₁ receptor knock-out mice. Furthermore, to determine the effect of BQCA on intact and functioning brain circuits, multiple single-unit recordings were obtained from the mPFC of rats that showed BQCA increases firing of mPFC pyramidal cells in vivo. BQCA also restored discrimination reversal learning in a transgenic mouse model of Alzheimer's disease and was found to regulate non-amyloidogenic APP processing in vitro, suggesting that M₁ receptor PAMs have the potential to provide both symptomatic and disease modifying effects in Alzheimer's disease patients. Together, these studies provide compelling evidence that M₁ receptor activation induces a dramatic excitation of PFC neurons and suggest that selectively activating the M₁ mAChR subtype may ameliorate impairments in cognitive function.

Trafficking of Membrane Proteins to Cone But Not Rod Outer Segments Is Dependent on Heterotrimeric Kinesin-II

Wed, 11 Nov 2009 10:02:15 PST

Heterotrimeric kinesin-II is a molecular motor localized to the inner segment, connecting cilium and axoneme of mammalian photoreceptors. Our purpose was to identify the role of kinesin-II in anterograde intraflagellar transport by photoreceptor-specific deletions of kinesin family member 3A (KIF3A), its obligatory motor subunit. In cones lacking KIF3A, membrane proteins involved in phototransduction did not traffic to the outer segments resulting in complete absence of a photopic electroretinogram and progressive cone degeneration. Rod photoreceptors lacking KIF3A degenerated rapidly between 2 and 4 weeks postnatally, but the phototransduction components including rhodopsin trafficked to the outer segments during the course of degeneration. Furthermore, KIF3A deletion did not affect synaptic anterograde trafficking. The results indicate that trafficking of membrane proteins to the outer segment is dependent on kinesin-II in cone, but not rod photoreceptors, even though rods and cones share similar structures, and closely related phototransduction polypeptides.

A Nanomedicine Transports a Peptide Caspase-3 Inhibitor across the Blood-Brain Barrier and Provides Neuroprotection

Wed, 04 Nov 2009 10:02:51 PST

Caspases play an important role as mediators of cell death in acute and chronic neurological disorders. Although peptide inhibitors of caspases provide neuroprotection, they have to be administered intracerebroventricularly because they cannot cross the blood–brain barrier (BBB). Herein, we present a nanocarrier system that can transfer chitosan nanospheres loaded with N-benzyloxycarbonyl-Asp(OMe)-Glu(OMe)-Val-Asp(OMe)-fluoromethyl ketone (Z-DEVD-FMK), a relatively specific caspase-3 inhibitor, across BBB. Caspase-3 was chosen as a pharmacological target because of its central role in cell death. Polyethylene glycol-coated nanospheres were conjugated to an anti-mouse transferrin receptor monoclonal antibody (TfRMAb) that selectively recognizes the TfR type 1 on the cerebral vasculature. We demonstrate with intravital microscopy that this nanomedicine is rapidly transported across the BBB without being measurably taken up by liver and spleen. Pre- or post-treatment (2 h) with intravenously injected Z-DEVD-FMK-loaded nanospheres dose dependently decreased the infarct volume, neurological deficit, and ischemia-induced caspase-3 activity in mice subjected to 2 h of MCA occlusion and 24 h of reperfusion, suggesting that they released an amount of peptide sufficient to inhibit caspase activity. Similarly, nanospheres inhibited physiological caspase-3 activity during development in the neonatal mouse cerebellum on postnatal day 17 after closure of the BBB. Neither nanospheres functionalized with TfRMAb but not loaded with Z-DEVD-FMK nor nanospheres lacking TfRMAb but loaded with Z-DEVD-FMK had any effect on either paradigm, suggesting that inhibition of caspase activity and subsequent neuroprotection were due to efficient penetration of the peptide into brain. Thus, chitosan nanospheres open new and exciting opportunities for brain delivery of biologically active peptides that are useful for the treatment of CNS disorders.

Estrogen Attenuates Ischemic Oxidative Damage via an Estrogen Receptor {alpha}-Mediated Inhibition of NADPH Oxidase Activation

Wed, 04 Nov 2009 10:02:51 PST

The goal of this study was to elucidate the mechanisms of 17β-estradiol (E₂) antioxidant and neuroprotective actions in stroke. The results reveal a novel extranuclear receptor-mediated antioxidant mechanism for E₂ during stroke, as well as a hypersensitivity of the CA3/CA4 region to ischemic injury after prolonged hypoestrogenicity. E₂ neuroprotection was shown to involve a profound attenuation of NADPH oxidase activation and superoxide production in hippocampal CA1 pyramidal neurons after stroke, an effect mediated by extranuclear estrogen receptor (ER)-mediated nongenomic signaling, involving Akt activation and subsequent phosphorylation/inactivation of Rac1, a factor critical for activation of NOX2 NADPH oxidase. Intriguingly, E₂ nongenomic signaling, antioxidant action, and neuroprotection in the CA1 region were lost after long-term E₂ deprivation, and this loss was tissue specific because the uterus remained responsive to E₂. Correspondingly, a remarkable loss of ER, but not ERβ, was observed in the CA1 after long-term E₂ deprivation, with no change observed in the uterus. As a whole, the study reveals a novel, membrane-mediated antioxidant mechanism in neurons by E₂ provides support and mechanistic insights for a "critical period" of E₂ replacement in the hippocampus and demonstrates a heretofore unknown hypersensitivity of the CA3/CA4 to ischemic injury after prolonged hypoestrogenicity.

Reticulon-4A (Nogo-A) Redistributes Protein Disulfide Isomerase to Protect Mice from SOD1-Dependent Amyotrophic Lateral Sclerosis

Yang, Y. S., Harel, N. Y., Strittmatter, S. M. — Wed, 04 Nov 2009 10:02:51 PST

Amyotrophic lateral sclerosis (ALS) is a fatal motor neuron disease inherited in a small subset of patients. The SOD1(G93A) transgenic mouse models this subset of patients, and studies of this strain have suggested that endoplasmic reticulum (ER) stress and deficits in ER chaperone function are contributors to ALS pathophysiology. Here, we demonstrate that the reticulon family of proteins is a novel regulator of the ER chaperone protein disulfide isomerase (PDI), and that through PDI, reticulon-4A (Nogo-A) can protect mice against the neurodegeneration that characterizes ALS. We show that overexpressing reticulon protein induces a punctate redistribution of PDI intracellularly, both in vitro and in vivo. Conversely, reduction of endogenous NogoA expression causes a more homogeneous expression pattern in vivo. These effects occur without induction of the unfolded protein response. To examine the effect of PDI redistribution on ALS disease progression, we conducted survival and behavior studies of SOD1(G93A) mice. Deletion of a single copy of the NogoA,B gene accelerates disease onset and progression, while deletion of both copies further worsens disease. We conclude that NogoA contributes to the proper function of the ER resident chaperone PDI, and is protective against ALS-like neurodegeneration. Our results provide a novel intracellular role for reticulon proteins and support the hypothesis that modulation of PDI function is a potential therapeutic approach to ALS.

Main Immunogenic Region Structure Promotes Binding of Conformation-Dependent Myasthenia Gravis Autoantibodies, Nicotinic Acetylcholine Receptor Conformation Maturation, and Agonist Sensitivity

Luo, J., Taylor, P., Losen, M., de Baets, M. H., Shelton, G. D., Lindstrom, J. — Wed, 04 Nov 2009 10:02:51 PST

The main immunogenic region (MIR) is a conformation-dependent region at the extracellular apex of 1 subunits of muscle nicotinic acetylcholine receptor (AChR) that is the target of half or more of the autoantibodies to muscle AChRs in human myasthenia gravis and rat experimental autoimmune myasthenia gravis. By making chimeras of human 1 subunits with 7 subunits, both MIR epitopes recognized by rat mAbs and by the patient-derived human mAb 637 to the MIR were determined to consist of two discontiguous sequences, which are adjacent only in the native conformation. The MIR, including loop 1 67–76 in combination with the N-terminal helix 1 1–14, conferred high-affinity binding for most rat mAbs to the MIR. However, an additional sequence corresponding to 1 15–32 was required for high-affinity binding of human mAb 637. A water soluble chimera of Aplysia acetylcholine binding protein with the same 1 MIR sequences substituted was recognized by a majority of human, feline, and canine myasthenia gravis sera. The presence of the 1 MIR sequences in 1/7 chimeras greatly promoted AChR expression and significantly altered the sensitivity to activation. This reveals a structural and functional, as well as antigenic, significance of the MIR.

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

Wed, 04 Nov 2009 10:02:51 PST

Leucine-rich repeat kinase 2 (LRRK2) functions as a putative protein kinase of ezrin, radixin, and moesin (ERM) family proteins. A Parkinson's 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.

Identification of Two Distinct Macrophage Subsets with Divergent Effects Causing either Neurotoxicity or Regeneration in the Injured Mouse Spinal Cord

Wed, 28 Oct 2009 10:32:16 PDT

Macrophages dominate sites of CNS injury in which they promote both injury and repair. These divergent effects may be caused by distinct macrophage subsets, i.e., "classically activated" proinflammatory (M1) or "alternatively activated" anti-inflammatory (M2) cells. Here, we show that an M1 macrophage response is rapidly induced and then maintained at sites of traumatic spinal cord injury and that this response overwhelms a comparatively smaller and transient M2 macrophage response. The high M1/M2 macrophage ratio has significant implications for CNS repair. Indeed, we present novel data showing that only M1 macrophages are neurotoxic and M2 macrophages promote a regenerative growth response in adult sensory axons, even in the context of inhibitory substrates that dominate sites of CNS injury (e.g., proteoglycans and myelin). Together, these data suggest that polarizing the differentiation of resident microglia and infiltrating blood monocytes toward an M2 or "alternatively" activated macrophage phenotype could promote CNS repair while limiting secondary inflammatory-mediated injury.

Simvastatin Inhibits the Activation of p21ras and Prevents the Loss of Dopaminergic Neurons in a Mouse Model of Parkinson's Disease

Ghosh, A., Roy, A., Matras, J., Brahmachari, S., Gendelman, H. E., Pahan, K. — Wed, 28 Oct 2009 10:32:16 PDT

Parkinson's disease (PD) is second only to Alzheimer's disease as the most common devastating human neurodegenerative disorder. Despite intense investigation, no interdictive therapy is available for PD. We investigated whether simvastatin, a Food and Drug Administration-approved cholesterol-lowering drug, could protect against nigrostriatal degeneration after 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) intoxication to model PD in mice. First, MPP⁺ induced the activation of p21^ras and nuclear factor-B (NF-B) in mouse microglial cells. Inhibition of MPP⁺-induced activation of NF-B by p21^ras, a dominant-negative mutant of p21^ras, supported the involvement of p21^ras in MPP⁺-induced microglial activation of NF-B. Interestingly, simvastatin attenuated activation of both p21^ras and NF-B in MPP⁺-stimulated microglial cells. Consistently, we found a very rapid activation of p21^ras in vivo in the substantia nigra pars compacta of MPTP-intoxicated mice. However, after oral administration, simvastatin entered into the nigra, reduced nigral activation of p21^ras, attenuated nigral activation of NF-B, inhibited nigral expression of proinflammatory molecules, and suppressed nigral activation of glial cells. These findings paralleled dopaminergic neuronal protection, normalized striatal neurotransmitters, and improved motor functions in MPTP-intoxicated mice. Similarly, pravastatin, another cholesterol-lowering drug, suppressed microglial inflammatory responses and protected dopaminergic neurons in MPTP-intoxicated mice, but at levels less than simvastatin. Furthermore, both the statins administered 2 d after initiation of the disease were still capable of inhibiting the demise of dopaminergic neurons and concomitant loss of neurotransmitters, suggesting that statins are capable of slowing down the progression of neuronal loss in the MPTP mouse model. Therefore, we conclude that statins may be of therapeutic benefit for PD patients.

Beclin 1 Gene Transfer Activates Autophagy and Ameliorates the Neurodegenerative Pathology in {alpha}-Synuclein Models of Parkinson's and Lewy Body Diseases

Wed, 28 Oct 2009 10:32:16 PDT

Accumulation of the synaptic protein -synuclein (-syn) is a hallmark of Parkinson's disease (PD) and Lewy body disease (LBD), a heterogeneous group of disorders with dementia and parkinsonism, where Alzheimer's disease and PD interact. Accumulation of -syn in these patients might be associated with alterations in the autophagy pathway. Therefore, we postulate that delivery of beclin 1, a regulator of the autophagy pathway, might constitute a strategy toward developing a therapy for LBD/PD. Overexpression of -syn from lentivirus transduction in a neuronal cell line resulted in lysosomal accumulation and alterations in autophagy. Coexpression of beclin 1 activated autophagy, reduced accumulation of -syn, and ameliorated associated neuritic alterations. The effects of beclin 1 overexpression on LC3 and -syn accumulation were partially blocked by 3-MA and completely blocked by bafilomycin A1. In contrast, rapamycin enhanced the effects of beclin 1. To evaluate the potential effects of activating autophagy in vivo, a lentivirus expressing beclin 1 was delivered to the brain of a -syn transgenic mouse. Neuropathological analysis demonstrated that beclin 1 injections ameliorated the synaptic and dendritic pathology in the tg mice and reduced the accumulation of -syn in the limbic system without any significant deleterious effects. This was accompanied by enhanced lysosomal activation and reduced alterations in the autophagy pathway. Thus, beclin 1 plays an important role in the intracellular degradation of -syn either directly or indirectly through the autophagy pathway and may present a novel therapeutic target for LBD/PD.

Intrabody Gene Therapy Ameliorates Motor, Cognitive, and Neuropathological Symptoms in Multiple Mouse Models of Huntington's Disease

Southwell, A. L., Ko, J., Patterson, P. H. — Wed, 28 Oct 2009 10:32:16 PDT

Huntington's disease (HD) is an autosomal dominant neurodegenerative disease resulting from the expansion of a glutamine repeat in the huntingtin (Htt) protein. Current therapies are directed at managing symptoms such as chorea and psychiatric disturbances. In an effort to develop a therapy directed at disease prevention we investigated the utility of highly specific, anti-Htt intracellular antibodies (intrabodies). We previously showed that V_L12.3, an intrabody recognizing the N terminus of Htt, and Happ1, an intrabody recognizing the proline-rich domain of Htt, both reduce mHtt-induced toxicity and aggregation in cell culture and brain slice models of HD. Due to the different mechanisms of action of these two intrabodies, we then tested both in the brains of five mouse models of HD using a chimeric adeno-associated virus 2/1 (AAV2/1) vector with a modified CMV enhancer/chicken β-actin promoter. V_L12.3 treatment, while beneficial in a lentiviral model of HD, has no effect on the YAC128 HD model and actually increases severity of phenotype and mortality in the R6/2 HD model. In contrast, Happ1 treatment confers significant beneficial effects in a variety of assays of motor and cognitive deficits. Happ1 also strongly ameliorates the neuropathology found in the lentiviral, R6/2, N171-82Q, YAC128, and BACHD models of HD. Moreover, Happ1 significantly prolongs the life span of N171-82Q mice. These results indicate that increasing the turnover of mHtt using AAV-Happ1 gene therapy represents a highly specific and effective treatment in diverse mouse models of HD.