Jordi Alberch

Brain-Derived Neurotrophic Factor Regulates the Onset and Severity of Motor Dysfunction Associated with Enkephalinergic Neuronal Degeneration in Huntington's Disease

The mechanism that controls the selective vulnerability of striatal neurons in Huntingtons disease is unclear. Brain-derived neurotrophic factor (BDNF) protects striatal neurons and is regulated by Huntingtin through the interaction with the neuron-restrictive silencer factor. Here, we demonstrate that the downregulation of BDNF by mutant Huntingtin depends on the length and levels of expression of the CAG repeats in cell cultures. To analyze the functional effects of these changes in BDNF in Huntingtons disease, we disrupted the expression of bdnf in a transgenic mouse model by cross-mating bdnf+/ - mice with R6/1 mice. Thus, we compared transgenic mice for mutant Huntingtin with different levels of BDNF. Using this double mutant mouse line, we show that the deficit of endogenous BDNF modulates the pathology of Huntingtons disease. The decreased levels of this neurotrophin advance the onset of motor dysfunctions and produce more severe uncoordinated movements. This behavioral pathology correlates with the loss of striatal dopamine and cAMP-regulated phosphoprotein-32-positive projection neurons. In particular, the insufficient levels of BDNF cause specific degeneration of the enkephalinergic striatal projection neurons, which are the most affected cells in Huntingtons disease. This neuronal dysfunction can specifically be restored by administration of exogenous BDNF. Therefore, the decrease in BDNF levels plays a key role in the specific pathology observed in Huntingtons disease by inducing dysfunction of striatal enkephalinergic neurons that produce severe motor dysfunctions. Hence, administration of exogenous BDNF may delay or stop illness progression.

Cystamine and cysteamine increase brain levels of BDNF in Huntington disease via HSJ1b and transglutaminase

There is no treatment for the neurodegenerative disorder Huntington disease (HD). Cystamine is a candidate drug; however, the mechanisms by which it operates remain unclear. We show here that cystamine increases levels of the heat shock DnaJ-containing protein 1b (HSJ1b) that are low in HD patients. HSJ1b inhibits polyQ-huntingtin-induced death of striatal neurons and neuronal dysfunction in Caenorhabditis elegans. This neuroprotective effect involves stimulation of the secretory pathway through formation of clathrin-coated vesicles containing brain-derived neurotrophic factor (BDNF). Cystamine increases BDNF secretion from the Golgi region that is blocked by reducing HSJ1b levels or by overexpressing transglutaminase. We demonstrate that cysteamine, the FDA-approved reduced form of cystamine, is neuroprotective in HD mice by increasing BDNF levels in brain. Finally, cysteamine increases serum levels of BDNF in mouse and primate models of HD. Therefore, cysteamine is a potential treatment for HD, and serum BDNF levels can be used as a biomarker for drug efficacy.

Differential effects of glial cell line-derived neurotrophic factor and neurturin on developing and adult substantia nigra dopaminergic neurons.

Abstract: Neurturin (NTN) and glial cell line‐derived neurotrophic factor (GDNF), two members of the GDNF family of growth factors, exert very similar biological activities in different systems, including the substantia nigra. Our goal in the present work was to compare their function and define whether nonoverlapping biological activities on midbrain dopaminergic neurons exist. We first found that NTN and GDNF are differentially regulated during postnatal development. NTN mRNA progressively decreased in the ventral mesencephalon and progressively increased in the striatum, coincident with a decrease in GDNF mRNA expression. This finding suggested distinct physiological roles for each factor in the nigrostriatal system. We therefore examined their function in ventral mesencephalon cultures and found that NTN promoted survival comparable with GDNF, but only GDNF induced sprouting and hypertrophy of developing dopaminergic neurons. We subsequently examined the ability of NTN to prevent the 6‐hydroxydopamine‐induced degeneration of adult dopaminergic neurons in vivo. Fibroblasts genetically engineered to deliver high levels of GDNF or NTN were grafted supranigrally. NTN was found to be as potent as GDNF at preventing the death of nigral dopaminergic neurons, but only GDNF induced tyrosine hydroxylase staining, sprouting, or hypertrophy of dopaminergic neurons. In conclusion, our results show selective survival‐promoting effects of NTN over wider survival, neuritogenic, and hypertrophic effects of GDNF on dopaminergic neurons in vitro and in vivo. Such differences are likely to underlie unique roles for each factor in postnatal development and may ultimately be exploited in the treatment of Parkinson’s disease.

Brain-derived neurotrophic factor, neurotrophin-3, and neurotrophin-4/5 prevent the death of striatal projection neurons in a rodent model of Huntington's disease

Abstract: Intrastriatal injection of quinolinate has been proven to be a very useful animal model to study the pathogenesis and treatment of Huntingtons disease. To determine whether growth factors of the neurotrophin family are able to prevent the degeneration of striatal projection neurons, cell lines expressing brain‐derived neurotrophic factor (BDNF), neurotrophin‐3 (NT‐3), or neurotrophin‐4/5 (NT‐4/5) were grafted in the adult rat striatum before quinolinate injection. Three days after lesioning, ongoing cell death was assessed by in situ detection of DNA fragmentation. In animals grafted with the control cell line, quinolinate injection induced a gradual cell loss that was differentially prevented by intrastriatal grafting of BDNF‐, NT‐3‐, or NT‐4/5‐secreting cells. Seven days after lesioning, we characterized striatal projection neurons that were protected by neurotrophins. Quinolinate injection, alone or in combination with the control cell line, induced a selective loss of striatal projection neurons. Grafting of a BDNF‐secreting cell line prevented the loss of all types of striatal projection neurons analyzed. Glutamic acid decarboxylase 67‐, preproenkephalin‐, and preprotachykinin A‐ but not prodynorphin‐expressing neurons were protected by grafting of NT‐3‐ or NT‐4/5‐secreting cells but with less efficiency than the BDNF‐secreting cells. Our findings show that neurotrophins are able to promote the survival of striatal projection neurons in vivo and suggest that BDNF might be beneficial for the treatment of striatonigral degenerative disorders, including Huntingtons disease.

Altered P2X7-receptor level and function in mouse models of Huntington’s disease and therapeutic efficacy of antagonist administration

The precise mechanism by which mutant huntingtin elicits its toxicity remains unknown. However synaptic alterations and increased susceptibility to neuronal death are known contributors to Huntingtons disease (HD) symptomatology. While decreased metabolism has long been associated with HD, recent findings have surprisingly demonstrated reduced neuronal apoptosis in Caenorhabditis elegans and Drosophila models of HD by drugs that diminish ATP production. Interestingly, extracellular ATP has been recently reported to elicit neuronal death through stimulation of P2X7 receptors. These are ATP‐gated cation channels known to modulate neurotransmitter release from neuronal presynaptic terminals and to regulate cytokine production and release from microglia. We hypothesized that alteration in P2X7‐mediated calcium permeability may contribute to HD synaptic dysfunction and increased neuronal apoptosis. Using mouse and cellular models of HD, we demonstrate increased P2X7‐receptor level and altered P2X7‐mediated calcium permeability in somata and terminals of HD neurons. Furthermore, cultured neurons expressing mutant huntingtin showed increased susceptibility to apoptosis triggered by P2X7‐receptor stimulation. Finally, in vivo administration of the P2X7‐antagonist Brilliant Blue‐G (BBG) to HD mice prevented neuronal apoptosis and attenuated body weight loss and motor‐coordination deficits. These in vivo data strongly suggest that altered P2X7‐receptor level and function contribute to HD pathogenesis and highlight the therapeutic potential of P2X7 receptor antagonists.—Diaz‐Hernandez, M., Diez‐Zaera, M., Sanchez‐Nogueiro, J., Gomez‐Villafuertes, R., Canals, J.M., Alberch, J., Miras‐Portugal, M.T., Lucas, J.J. Altered P2X7‐receptor level and function in mouse models of Huntingtons disease and therapeutic efficacy of antagonist administration. FASEB J. 23, 1893–1906 (2009)

Loss of striatal type 1 cannabinoid receptors is a key pathogenic factor in Huntington’s disease

Endocannabinoids act as neuromodulatory and neuroprotective cues by engaging type 1 cannabinoid receptors. These receptors are highly abundant in the basal ganglia and play a pivotal role in the control of motor behaviour. An early downregulation of type 1 cannabinoid receptors has been documented in the basal ganglia of patients with Huntingtons disease and animal models. However, the pathophysiological impact of this loss of receptors in Huntingtons disease is as yet unknown. Here, we generated a double-mutant mouse model that expresses human mutant huntingtin exon 1 in a type 1 cannabinoid receptor-null background, and found that receptor deletion aggravates the symptoms, neuropathology and molecular pathology of the disease. Moreover, pharmacological administration of the cannabinoid Δ(9)-tetrahydrocannabinol to mice expressing human mutant huntingtin exon 1 exerted a therapeutic effect and ameliorated those parameters. Experiments conducted in striatal cells show that the mutant huntingtin-dependent downregulation of the receptors involves the control of the type 1 cannabinoid receptor gene promoter by repressor element 1 silencing transcription factor and sensitizes cells to excitotoxic damage. We also provide in vitro and in vivo evidence that supports type 1 cannabinoid receptor control of striatal brain-derived neurotrophic factor expression and the decrease in brain-derived neurotrophic factor levels concomitant with type 1 cannabinoid receptor loss, which may contribute significantly to striatal damage in Huntingtons disease. Altogether, these results support the notion that downregulation of type 1 cannabinoid receptors is a key pathogenic event in Huntingtons disease, and suggest that activation of these receptors in patients with Huntingtons disease may attenuate disease progression.

Mutant huntingtin impairs post-Golgi trafficking to lysosomes by delocalizing optineurin/Rab8 complex from the Golgi apparatus.

Huntingtin regulates post-Golgi trafficking of secreted proteins. Here, we studied the mechanism by which mutant huntingtin impairs this process. Colocalization studies and Western blot analysis of isolated Golgi membranes showed a reduction of huntingtin in the Golgi apparatus of cells expressing mutant huntingtin. These findings correlated with a decrease in the levels of optineurin and Rab8 in the Golgi apparatus that can be reverted by overexpression of full-length wild-type huntingtin. In addition, immunoprecipitation studies showed reduced interaction between mutant huntingtin and optineurin/Rab8. Cells expressing mutant huntingtin produced both an accumulation of clathrin adaptor complex 1 at the Golgi and an increase of clathrin-coated vesicles in the vicinity of Golgi cisternae as revealed by electron microscopy. Furthermore, inverse fluorescence recovery after photobleaching analysis for lysosomal-associated membrane protein-1 and mannose-6-phosphate receptor showed that the optineurin/Rab8-dependent post-Golgi trafficking to lysosomes was impaired in cells expressing mutant huntingtin or reducing huntingtin levels by small interfering RNA. Accordingly, these cells showed a lower content of cathepsin D in lysosomes, which led to an overall reduction of lysosomal activity. Together, our results indicate that mutant huntingtin perturbs post-Golgi trafficking to lysosomal compartments by delocalizing the optineurin/Rab8 complex, which, in turn, affects the lysosomal function.

THE EXPANDING CLINICAL PROFILE OF ANTI-AMPA RECEPTOR ENCEPHALITIS

Antibodies to the glutamate receptor 1 (GluR1) and GluR2 subunits of the α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor (AMPAR-ab) were recently described in 10 patients from a series of 109 with limbic encephalitis (LE).1 Besides the hippocampus, GluR1/2 subunits of AMPAR are also widely expressed in the cerebral cortex, basal ganglia, and cerebellum,2 suggesting that the clinical presentation could also extend beyond the clinical profile of LE. In this study, we analyzed the presence of AMPAR-ab in a consecutive series of patients whose serum or CSF was sent to our laboratory for analysis of antineuronal antibodies in the last 2 years. ### Methods. We review all patients with final diagnosis of limbic encephalitis3 or with an immunohistochemical pattern of NMDAR-ab or AMPAR-ab in the serum or CSF (figure e-1 on the Neurology ® Web site at www.neurology.org). AMPAR-ab immunoreactivity on brain sections was confirmed on HEK293 cells transfected with plasmids containing rodent GluR1/2 subunits as described (figure).1 The study was approved by the Ethical Committee of the Hospital Clinic. Figure Immunoreactivity of antibodies against AMPA receptors (A) Section of paraformaldehyde-perfused rat brain incubated with the CSF of a patient with AMPAR-ab. There is a robust reactivity with the neuropil of hippocampus. Bar = 25 μm. (B) HEK293 cells transfected with GluR1/2 AMPAR subunits show intense reactivity with the positive CSF (green) …

Neuroprotection by neurotrophins and GDNF family members in the excitotoxic model of Huntington's disease.

Huntingtons disease is a neurodegenerative disorder characterized by a selective degeneration of striatal projection neurons, which deal with choreic movements. Neuroprotective therapy could be achieved with the knowledge of the specific trophic requirements of these neuronal populations. Thus, the induction of endogenous trophic response or the exogenous administration of neurotrophic factors may help to prevent or stop the progression of the illness. Excitotoxicity has been implicated in the etiology of Huntingtons disease, because intrastriatal injection of glutamate receptor agonists reproduces some of the neuropathological features of this disorder. Activation of glutamate receptors in the striatum differentially regulates the expression of neurotrophins, glial cell line-derived neurotrophic factor (GDNF), neurturin, and their receptors in the striatum and in its connections, cortex, and substantia nigra, showing a selective trophic response against excitotoxic insults. Transplantation of cells genetically engineered to release neurotrophic factors in the striatum has been used to study the neuroprotective effects of neurotrophin and GDNF family members in the excitotoxic model of Huntingtons disease. Neurotrophins (brain-derived neurotrophic factor [BDNF], neurotrophin-3, and neurotrophin-4) protected striatal projection neurons against quinolinic or kainic acid treatment. However, GDNF family members showed a more specific action. Neurturin only protected gamma-aminobutyric acid (GABA)/enkephalinergic neurons that project to the external segment of the globus pallidus, whereas GDNF exerts its effects on GABA/substance P positive neurons, which project to the substantia nigra pars compacta and the internal segment of the globus pallidus. In conclusion, the trophic requirements of each population of striatal projection neurons are due to a complex interaction between several neurotrophic factors, such as neurotrophins and GDNF family members, which can be modified, in different pathological conditions. Moreover, these neurotrophic factors may be able to provide selective protection for basal ganglia circuits, which are affected in striatonigral degenerative disorders, such as Huntingtons disease or multisystem atrophy.

Reduced expression of the TrkB receptor in Huntington's disease mouse models and in human brain

Deficits of neurotrophic support caused by reduced levels of brain‐derived neurotrophic factor (BDNF) have been implicated in the selective vulnerability of striatal neurones in Huntingtons disease (HD). Therapeutic strategies based on BDNF administration have been proposed to slow or prevent the disease progression. However, the effectiveness of BDNF may depend on the proper expression of its receptor TrkB. In this study, we analysed the expression of TrkB in several HD models and in postmortem HD brains. We found a specific reduction of TrkB receptors in transgenic exon‐1 and full‐length knock‐in HD mouse models and also in the motor cortex and caudate nucleus of HD brains. Our findings also demonstrated that continuous expression of mutant huntingtin is required to down‐regulate TrkB levels. This was shown by findings in an inducible HD mouse model showing rescue of TrkB by turning off mutant huntingtin expression. Interestingly, the length of the polyglutamine tract in huntingtin appears to modulate the reduction of TrkB. Finally, to analyse the effect of BDNF in TrkB we compared TrkB expression in mutant huntingtin R6/1 and double mutant (R6/1 : BDNF+/–) mice. Similar TrkB expression was found in both transgenic mice suggesting that reduced TrkB is not a direct consequence of decreased BDNF. Therefore, taken together our findings identify TrkB as an additional component that potentially might contribute to the altered neurotrophic support in HD.