Genetic ablation of acid ceramidase in Krabbe disease confirms the psychosine hypothesis and identifies a new therapeutic target.

Infantile globoid cell leukodystrophy (GLD, Krabbe disease) is a fatal demyelinating disorder caused by a deficiency in the lysosomal enzyme galactosylceramidase (GALC). GALC deficiency leads to the accumulation of the cytotoxic glycolipid, galactosylsphingosine (psychosine). Complementary evidence suggested that psychosine is synthesized via an anabolic pathway. Here, we show instead that psychosine is generated catabolically through the deacylation of galactosylceramide by acid ceramidase (ACDase). This reaction uncouples GALC deficiency from psychosine accumulation, allowing us to test the long-standing "psychosine hypothesis." We demonstrate that genetic loss of ACDase activity (Farber disease) in the GALC-deficient mouse model of human GLD (twitcher) eliminates psychosine accumulation and cures GLD. These data suggest that ACDase could be a target for substrate reduction therapy (SRT) in Krabbe patients. We show that pharmacological inhibition of ACDase activity with carmofur significantly decreases psychosine accumulation in cells from a Krabbe patient and prolongs the life span of the twitcher (Twi) mouse. Previous SRT experiments in the Twi mouse utilized l-cycloserine, which inhibits an enzyme several steps upstream of psychosine synthesis, thus altering the balance of other important lipids. Drugs that directly inhibit ACDase may have a more acceptable safety profile due to their mechanistic proximity to psychosine biogenesis. In total, these data clarify our understanding of psychosine synthesis, confirm the long-held psychosine hypothesis, and provide the impetus to discover safe and effective inhibitors of ACDase to treat Krabbe disease.

Targeting Heat Shock Protein 70 to Ameliorate c-Jun Expression and Improve Demyelinating Neuropathy.

Increased expression of the c-jun transcription factor occurs in a variety of human neuropathies and is critical in promoting Schwann cell (SC) dedifferentiation and loss of the myelinated phenotype. Using cell culture models, we previously identified KU-32 as a novobiocin-based C-terminal heat shock protein 90 (Hsp90) inhibitor that decreased c-jun expression and the extent of demyelination. Additional chemical optimization has yielded KU-596 as a neuroprotective novologue whose mechanistic efficacy to improve a metabolic neuropathy requires the expression of Hsp70. The current study examined whether KU-596 therapy could decrease c-jun expression and improve motor function in an inducible transgenic model of a SC-specific demyelinating neuropathy (MPZ-Raf mice). Treating MPZ-Raf mice with tamoxifen activates the MAPK kinase pathway, increases c-jun expression and produces a profound demyelinating neuropathy characterized by a loss of motor function and paraparesis. KU-596 therapy did not interfere with MAPK activation but reduced c-jun expression, significantly improved motor performance, and ameliorated the extent of peripheral nerve demyelination in both prevention and intervention studies. Hsp70 was necessary for the drug's neuroprotective efficacy since MPZ-Raf × Hsp70 knockout mice did not respond to KU-596 therapy. Collectively, our data indicate that modulating Hsp70 may provide a novel therapeutic approach to attenuate SC c-jun expression and ameliorate the onset of certain demyelinating neuropathies in humans.

Allopregnanolone mediates the exacerbation of Tourette-like responses by acute stress in mouse models.

Tourette syndrome (TS) is a neuropsychiatric disorder characterized by multiple tics and sensorimotor abnormalities, the severity of which is typically increased by stress. The neurobiological underpinnings of this exacerbation, however, remain elusive. We recently reported that spatial confinement (SC), a moderate environmental stressor, increases tic-like responses and elicits TS-like sensorimotor gating deficits in the D1CT-7 mouse, one of the best-validated models of TS. Here, we hypothesized that these adverse effects may be mediated by neurosteroids, given their well-documented role in stress-response orchestration. Indeed, SC increased the levels of progesterone, as well as its derivatives 5α-dihydroprogesterone and allopregnanolone, in the prefrontal cortex (PFC) of D1CT-7 mice. Among these steroids, however, only allopregnanolone (5-15 mg/kg, IP) dose-dependently exacerbated TS-like manifestations in D1CT-7, but not wild-type littermates; these effects were countered by the benchmark anti-tic therapy haloperidol (0.3 mg/kg, IP). Furthermore, the phenotypic effects of spatial confinement in D1CT-7 mice were suppressed by finasteride (25-50 mg/kg, IP), an inhibitor of the main rate-limiting enzyme in allopregnanolone synthesis. These findings collectively suggest that stress may exacerbate TS symptoms by promoting allopregnanolone synthesis in the PFC, and corroborate previous clinical results pointing to finasteride as a novel therapeutic avenue to curb symptom fluctuations in TS.

An anti-neuroinflammatory that targets dysregulated glia enhances the efficacy of CNS-directed gene therapy in murine infantile neuronal ceroid lipofuscinosis.

Infantile neuronal ceroid lipofuscinosis (INCL) is an inherited neurodegenerative lysosomal storage disease (LSD) caused by a deficiency in palmitoyl protein thioesterase-1 (PPT1). Studies in Ppt1(-/-) mice demonstrate that glial activation is central to the pathogenesis of INCL. Astrocyte activation precedes neuronal loss, while cytokine upregulation associated with microglial reactivity occurs before and concurrent with neurodegeneration. Therefore, we hypothesized that cytokine cascades associated with neuroinflammation are important therapeutic targets for the treatment of INCL. MW01-2-151SRM (MW151) is a blood-brain barrier penetrant, small-molecule anti-neuroinflammatory that attenuates glial cytokine upregulation in models of neuroinflammation such as traumatic brain injury, Alzheimer's disease, and kainic acid toxicity. Thus, we used MW151, alone and in combination with CNS-directed, AAV-mediated gene therapy, as a possible treatment for INCL. MW151 alone decreased seizure susceptibility. When combined with AAV-mediated gene therapy, treated INCL mice had increased life spans, improved motor performance, and eradication of seizures. Combination-treated INCL mice also had decreased brain atrophy, astrocytosis, and microglial activation, as well as intermediary effects on cytokine upregulation. These data suggest that MW151 can attenuate seizure susceptibility but is most effective when used in conjunction with a therapy that targets the primary genetic defect.

Regulation of motor function and behavior by atypical chemokine receptor 1.

Atypical Chemokine Receptor 1 (ACKR1), previously known as Duffy Antigen Receptor for Chemokines, stands out among chemokine receptors for high selective expression on cerebellar Purkinje neurons. Although ACKR1 ligands activate Purkinje cells in vitro, evidence for ACKR1 regulation of brain function in vivo is lacking. Here we demonstrate that Ackr1 (-/-) mice have markedly impaired balance and ataxia on a rotating rod and increased tremor when injected with harmaline, which induces whole-body tremor by activating Purkinje cells. Ackr1 (-/-) mice also exhibited impaired exploratory behavior, increased anxiety-like behavior and frequent episodes of marked hypoactivity under low-stress conditions. Surprisingly, Ackr1 (+/-) had similar behavioral abnormalities, indicating pronounced haploinsufficiency. The behavioral phenotype of Ackr1 (-/-) mice was the opposite of mouse models of cerebellar degeneration, and the defects persisted when Ackr1 was deficient only on non-hematopoietic cells. Together, the results suggest that normal motor function and behavior may partly depend on negative regulation of Purkinje cell activity by Ackr1.

Differential sensitivity of cranial and limb motor function to nigrostriatal dopamine depletion.

The present study determined the differential effects of unilateral striatal dopamine depletion on cranial motor versus limb motor function. Forty male Long Evans rats were first trained on a comprehensive motor testing battery that dissociated cranial versus limb motor function and included: cylinder forepaw placement, single pellet reaching, vermicelli pasta handling; sunflower seed opening, pasta biting acoustics, and a licking task. Following baseline testing, animals were randomized to either a 6-hydroxydopamine (6-OHDA) (n=20) or control (n=20) group. Animals in the 6-OHDA group received unilateral intrastriatal 6-OHDA infusions to induce striatal dopamine depletion. Six-weeks following infusion, all animals were re-tested on the same battery of motor tests. Near infrared densitometry was performed on sections taken through the striatum that were immunohistochemically stained for tyrosine hydroxylase (TH). Animals in the 6-OHDA condition showed a mean reduction in TH staining of 88.27%. Although 6-OHDA animals were significantly impaired on all motor tasks, limb motor deficits were more severe than cranial motor impairments. Further, performance on limb motor tasks was correlated with degree of TH depletion while performance on cranial motor impairments showed no significant correlation. These results suggest that limb motor function may be more sensitive to striatal dopaminergic depletion than cranial motor function and is consistent with the clinical observation that therapies targeting the nigrostriatal dopaminergic system in Parkinson's disease are more effective for limb motor symptoms than cranial motor impairments.

Bone Marrow Transplantation Alters the Tremor Phenotype in the Murine Model of Globoid-Cell Leukodystrophy.

Tremor is a prominent phenotype of the twitcher mouse, an authentic genetic model of Globoid-Cell Leukodystrophy (GLD, Krabbe's disease). In the current study, the tremor was quantified using a force-plate actometer designed to accommodate low-weight mice. The actometer records the force oscillations caused by a mouse's movements, and the rhythmic structure of the force variations can be revealed. Results showed that twitcher mice had significantly increased power across a broad band of higher frequencies compared to wildtype mice. Bone marrow transplantation (BMT), the only available therapy for GLD, worsened the tremor in the twitcher mice and induced a measureable alteration of movement phenotype in the wildtype mice. These data highlight the damaging effects of conditioning radiation and BMT in the neonatal period. The behavioral methodology used herein provides a quantitative approach for assessing the efficacy of potential therapeutic interventions for Krabbe's disease.

Bone marrow transplantation augments the effect of brain- and spinal cord-directed adeno-associated virus 2/5 gene therapy by altering inflammation in the murine model of globoid-cell leukodystrophy.

Globoid-cell leukodystrophy (GLD) is an inherited demyelinating disease caused by the deficiency of the lysosomal enzyme galactosylceramidase (GALC). A previous study in the murine model of GLD (twitcher) demonstrated a dramatic synergy between CNS-directed adeno-associated virus 2/5 (AAV2/5) gene therapy and myeloreductive bone marrow transplantation (BMT). However, the mechanism by which these two disparate therapeutic approaches synergize is not clear. In addition, the therapeutic efficacy may have been limited since the CNS-directed gene therapy was restricted to the forebrain and thalamus. In the current study, intrathecal and intracerebellar injections were added to the therapeutic regimen and the mechanism of synergy between BMT and gene therapy was determined. Although AAV2/5 alone provided supraphysiological levels of GALC activity and reduced psychosine levels in both the brain and spinal cord, it significantly increased CNS inflammation. Bone marrow transplantation alone provided essentially no GALC activity to the CNS and did not reduce psychosine levels. When AAV2/5 is combined with BMT, there are sustained improvements in motor function and the median life span is increased to 123 d (range, 92-282 d) compared with 41 d in the untreated twitcher mice. Interestingly, addition of BMT virtually eliminates both the disease and AAV2/5-associated inflammatory response. These data suggest that the efficacy of AAV2/5-mediated gene therapy is limited by the associated inflammatory response and BMT synergizes with AAV2/5 by modulating inflammation.

Developmental effects of dietary n-3 fatty acids on activity and response to novelty.

Insufficient availability of n-3 polyunsaturated fatty acids (PUFA) during pre- and neonatal development decreases accretion of docosahexaenoic acid (DHA, 22:6n-3) in the developing brain. Low tissue levels of DHA are associated with neurodevelopmental disorders including attention deficit hyperactivity disorder (ADHD). In this study, 1st- and 2nd-litter male Long-Evans rats were raised from conception on a Control diet containing alpha-linolenic acid (4.20 g/kg diet), the dietarily essential fatty acid precursor of DHA, or a diet Deficient in alpha-linolenic acid (0.38 g/kg diet). The Deficient diet resulted in a decrease in brain phospholipid DHA of 48% in 1st-litter pups and 65% in 2nd-litter pups. Activity, habituation, and response to spatial change in a familiar environment were assessed in a single-session behavioral paradigm at postnatal days 28 and 70, inclusive. Activity and habituation varied by age with younger rats exhibiting higher activity, less habituation, and less stimulation of activity induced by spatial novelty. During the first and second exposures to the test chamber, 2nd-litter Deficient pups exhibited higher levels of activity than Control rats or 1st-litter Deficient pups, and less habituation during the first exposure, but were not more active after introduction of a novel spatial stimulus. The higher level of activity in a familiar environment, but not after introduction of a novel stimulus is consistent with clinical observations in ADHD. The observation of this effect only in 2nd-litter rats fed the Deficient diet suggests that brain DHA content, rather than dietary n-3 PUFA content, likely underlies these effects.

Caloric restriction alleviates abnormal locomotor activity and dopamine levels in the brain of the methionine sulfoxide reductase A knockout mouse.

Oxidative stress is associated with the aging process, a risk factor for neurodegenerative diseases, and decreased by reduced energy intake. Oxidative modifications can affect protein function; the sulfur-containing amino acids, including methionine, are particularly susceptible to oxidation. A methionine sulfoxide can be enzymatically reduced by the methionine sulfoxide reductase (Msr) system. Previously, we have shown that MsrA(-/-) mice exhibit altered locomotor activity and brain dopamine levels as function of age. Previous studies have demonstrated that a caloric restriction enhances antioxidant defense and reduces the action of reactive oxygen species. Here we examine locomotor behavior and dopamine levels of MsrA(-/-) mice after caloric restriction starting at eight months of age and ending at 17 months. The MsrA(-/-) mice did not have any significant difference in spontaneous distance traveled when compared to controls at 17 months of age. In contrast, our previous report showed decreased locomotor activity in the MsrA(-/-) mice at 12 months of age and older when fed ad-libitum. After completion of the caloric restriction diet, dopamine levels were comparable to control mice. This differs from the abnormal dopamine levels previously observed in MsrA(-/-) mice fed ad-libitum. Thus, caloric restriction had a neutralization effect on MsrA ablation. In summary, it is suggested that caloric restriction alleviates abnormal locomotor activity and dopamine levels in the brain of the methionine sulfoxide reductase A knockout mouse.

Striatal expression of a calmodulin fragment improved motor function, weight loss, and neuropathology in the R6/2 mouse model of Huntington's disease.

Huntington's disease (HD) is an autosomal dominant neurodegenerative disorder, caused by a polyglutamine expansion in the huntingtin protein (htt). Increasing evidence suggests that transglutaminase (TGase) plays a critical role in the pathophysiology of HD possibly by stabilizing monomeric, polymeric and aggregated htt. We previously reported that in HEK293 and SH-SY5Y cells expression of a calmodulin (CaM)-fragment, consisting of amino acids 76-121 of CaM, decreased binding of CaM to mutant htt, TGase-modified htt and cytotoxicity associated with mutant htt and normalized intracellular calcium release. In this study, an adeno-associated virus (AAV) that expresses the CaM-fragment was injected into the striatum of HD transgenic R6/2 mice. The CaM-fragment significantly reduced body weight loss and improved motor function as indicated by improved rotarod performance, longer stride length, lower stride frequency, fewer low mobility bouts and longer travel distance than HD controls. A small but insignificant increase in survival was observed in R6/2 mice with CaM-fragment expression. Immunoprecipitation studies show that expression of the CaM-fragment reduced TGase-modified htt in the striatum of R6/2 mice. The percentage of htt-positive nuclei and the size of intranuclear htt aggregates were reduced by the CaM-fragment without striatal volume changes. The effects of CaM-fragment appear to be selective, as activity of another CaM-dependent enzyme, CaM-dependent kinase II, was not altered. Moreover, inhibition of TGase-modified htt was substrate-specific since overall TGase activity in the striatum was not altered by treatment with the CaM-fragment. Together, these results suggest that disrupting CaM-htt interaction may provide a new therapeutic strategy for HD.

Demonstration of functional coupling between dopamine synthesis and its packaging into synaptic vesicles.

We have previously shown that the membrane-associated form of the GABA-synthesizing enzyme, glutamate decarboxylase 65 (GAD(65)), is activated by synaptic vesicle proton gradient-mediated protein phosphorylation. We now report that the rate-limiting enzyme in dopamine (DA) biosynthesis, tyrosine hydroxylase (TH), is regulated similarly to GAD(65). The membrane-associated form of TH (MTH) was activated by conditions favoring protein phosphorylation (e.g. ATP) and was inhibited by phosphatase (e.g. calf intestine phosphatase). Furthermore, the ATP-mediated activation of MTH was abolished by conditions that disrupted the proton gradient of synaptic vesicles, e.g. the presence of carbonyl cyanide M-chorophenylhydrazone, gramicidin, or the V-type ATPase inhibitor (bafilomycin), but not the P-type ATPase inhibitor (vanadate). Moreover, DA newly synthesized from tyrosine by MTH and membrane-associated aromatic amino acid decarboxylase was taken up preferentially rather than pre-existing DA. Therefore, the previously proposed model showing close coupling between GABA synthesis and GABA packaging into synaptic vesicles by vesicular GABA transporters is also applicable to the DA system. Hence, it is concluded that there is a general coupling mechanism between neurotransmitter synthesis and packaging of transmitter into synaptic vesicles.