Jesús F. Torres-Peraza

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.

Full Motor Recovery Despite Striatal Neuron Loss and Formation of Irreversible Amyloid-Like Inclusions in a Conditional Mouse Model of Huntington's Disease

The primary mechanism responsible for Huntingtons disease remains unknown. Postulated early pathogenic events include the following: impaired protein folding, altered protein degradation, mitochondrial dysfunction, and transcriptional dysregulation. Although related therapies can delay disease progression in mouse models, they target downstream and probably indirect effects of mutant-huntingtin expression. Accordingly, in case they prove beneficial in humans, they might only palliate some aspects of disease. Our previous studies in the Tet/HD94 conditional model and the recently reported efficacy of RNA interference against mutant huntingtin in another mouse model support silencing mutant-huntingtin expression as a valid therapeutic approach that has the advantage of targeting toxicity at its root. Here, we address whether gene silencing can still be beneficial in the late stages of disease with detectable striatal neuron loss. Stereological analysis was applied to determine an age at which Tet/HD94 mice show a decrease in the number of striatal neurons. Then, progression of neuropathology and motor phenotype were analyzed in mice that were allowed to continue expressing mutant huntingtin and in mice that no longer expressed it. Neuronal loss did not revert in gene-off mice, but the additional loss that takes place in gene-on mice was prevented. The total number of huntingtin-containing inclusions dramatically reverted, but a small fraction of inclusions positive for the amyloid dye thioflavin-S remained. Interestingly, despite a 20% decrease in striatal neurons and the presence of amyloid-like irreversible inclusions, gene-off mice fully recover from their motor deficit, thus ruling out amyloid-like huntingtin inclusions as the main toxic species and suggesting that gene-silencing therapies might work in late stages of disease.

Suppressing aberrant GluN3A expression rescues synaptic and behavioral impairments in Huntington's disease models

Huntingtons disease is caused by an expanded polyglutamine repeat in the huntingtin protein (HTT), but the pathophysiological sequence of events that trigger synaptic failure and neuronal loss are not fully understood. Alterations in N-methyl-D-aspartate (NMDA)-type glutamate receptors (NMDARs) have been implicated. Yet, it remains unclear how the HTT mutation affects NMDAR function, and direct evidence for a causative role is missing. Here we show that mutant HTT redirects an intracellular store of juvenile NMDARs containing GluN3A subunits to the surface of striatal neurons by sequestering and disrupting the subcellular localization of the endocytic adaptor PACSIN1, which is specific for GluN3A. Overexpressing GluN3A in wild-type mouse striatum mimicked the synapse loss observed in Huntingtons disease mouse models, whereas genetic deletion of GluN3A prevented synapse degeneration, ameliorated motor and cognitive decline and reduced striatal atrophy and neuronal loss in the YAC128 Huntingtons disease mouse model. Furthermore, GluN3A deletion corrected the abnormally enhanced NMDAR currents, which have been linked to cell death in Huntingtons disease and other neurodegenerative conditions. Our findings reveal an early pathogenic role of GluN3A dysregulation in Huntingtons disease and suggest that therapies targeting GluN3A or pathogenic HTT-PACSIN1 interactions might prevent or delay disease progression.

Effect of glatiramer acetate (Copaxone) on the immunophenotypic and cytokine profile and BDNF production in multiple sclerosis: a longitudinal study.

We assessed the effect of glatiramer acetate (GA) on the immunophenotypic and cytokine profile and the BDNF production by peripheral blood mononuclear cells, and their association with the clinical response in 19 naïve-treated MS patients prospectively followed-up after GA therapy. Two patients withdrew the therapy. After a median follow-up of 21 months, twelve were considered responders and five as non-responders. Non-responder patients had significant longer disease duration and a higher EDSS score at baseline. In the responder group, a significant decrease in the percentage of INF-gamma producing total lymphocytes, CD4+ and CD8+ T cells, and reduced percentage of IL-2 producing CD4+ and CD8+ T cells were observed at 12, 18 and 24 months. These changes were associated with a significant increase in the percentage of CD3+, CD4+ and CD4(+) CD45RA(+) T cells, and BDNF production from month 6 that remained significant throughout the study. We did not observe significant changes in the nonresponder group for any of the parameters studied. Our data suggest that GA treatment induces a downmodulation of proinflammatory cytokines associated with the regulation of the peripheral T cell compartment and with increased production of BDNF that might be related to the clinical response.

Transgenic mice overexpressing the full-length neurotrophin receptor TrkC exhibit increased catecholaminergic neuron density in specific brain areas and increased anxiety-like behavior and panic reaction

Accumulating evidence has suggested that neurotrophins participate in the pathophysiology of mood disorders. We have developed transgenic mice overexpressing the full-length neurotrophin-3 receptor TrkC (TgNTRK3) in the central nervous system. TgNTRK3 mice show increased anxiety-like behavior and enhancement of panic reaction in the mouse defense test battery, along with an increase in the number and density of catecholaminergic (tyrosine hydroxylase positive) neurons in locus coeruleus and substantia nigra. Furthermore, treatment of TgNTRK3 mice with diazepam significantly attenuated the anxiety-like behaviors in the plus maze. These results provide evidence for the involvement of TrkC in the development of noradrenergic neurons in the central nervous system with consequences on anxiety-like behavior and panic reaction. Thus, changes in TrkC expression levels could contribute to the phenotypic expression of panic disorder through a trophic effect on noradrenergic neurons in the locus coeruleus. Our results demonstrate that the elevated NT3-TrkC tone via overexpression of TrkC in the brain may constitute a molecular mechanism for the expression of anxiety and anxiety.

PH domain leucine-rich repeat protein phosphatase 1 contributes to maintain the activation of the PI3K/Akt pro-survival pathway in Huntington's disease striatum

Dysregulation of gene expression is one of the mechanisms involved in the pathophysiology of Huntingtons disease (HD). Here, we examined whether mutant huntingtin regulates the levels of PH domain leucine-rich repeat protein phosphatase 1 (PHLPP1), a phosphatase that specifically dephosphorylates Akt at Ser473. Our results show decreased PHLPP1 protein levels in knock-in models (HdhQ111/Q111 mouse striatum and STHdhQ111/Q111 cells), in the striatum of N-terminal exon-1 mutant huntingtin transgenic mouse models (R6/1; R6/1 : BDNF +/−, R6/2 and Tet/HD94) and in the putamen of HD patients. Quantitative PCR analysis revealed a reduction in PHLPP1 mRNA levels in the striatum of R6/1 compared with wild-type mice. Coincident with reduced PHLPP1 protein levels, we observed increased phosphorylated Akt (Ser473) levels specifically in the striatum. The analysis of the conditional mouse model Tet/HD94 disclosed that after mutant huntingtin shutdown PHLPP1 levels returned to wild-type levels whereas phospho-Akt levels were partially reduced. In conclusion, our results show that mutant huntingtin downregulates PHLPP1 expression. In the striatum, these reduced levels of PHLPP1 can contribute to maintain high levels of activated Akt that may delay cell death and allow the recovery of neuronal viability after mutant huntingtin silencing.

Protective neuronal induction of ATF5 in endoplasmic reticulum stress induced by status epilepticus

Activating transcription factor 5 (ATF5) is a basic-leucine-zipper transcription factor of the ATF/CREB family. The Atf5 gene generates two transcripts, Atf5α and Atf5β, of which Atf5α is known to be selectively translated upon endoplasmic reticulum stress response in non-neuronal cells. ATF5 is highly expressed in the developing brain where it modulates proliferation of neural progenitor cells. These cells show a high level of ATF5 that has to decrease to allow them to differentiate into mature neurons or glial cells. This has led to the extended notion that differentiated neural cells do not express ATF5 unless they undergo tumourigenic transformation. However, no systematic analysis of the distribution of ATF5 in adult brain or of its potential role in neuronal endoplasmic reticulum stress response has been reported. By immunostaining here we confirm highest ATF5 levels in neuroprogenitor cells of the embryonic and adult subventricular zone but also found ATF5 in a large variety of neurons in adult mouse brain. By combining Atf5 in situ hybridization and immunohistochemistry for the neuronal marker NeuN we further confirmed Atf5 messenger RNA in adult mouse neurons. Quantitative reverse transcriptase polymerase chain reaction demonstrated that Atf5α is the most abundant transcript in adult mouse encephalon and injection of the endoplasmic reticulum stress inducer tunicamycin into adult mouse brain increased neuronal ATF5 levels. Accordingly, ATF5 levels increased in hippocampal neurons of a mouse model of status epilepticus triggered by intra-amygdala injection of kainic acid, which leads to abnormal hippocampal neuronal activity and endoplasmic reticulum stress. Interestingly, ATF5 upregulation occurred mainly in hippocampal neuronal fields that do not undergo apoptosis in this status epilepticus model such as CA1 and dentate gyrus, thus suggesting a neuroprotective role. This was confirmed in a primary neuronal culture model in which ATF5 overexpression resulted in decreased endoplasmic reticulum stress-induced apoptosis and the opposite result was achieved by Atf5 RNA interference. Furthermore, in vivo administration of the eIF2α phosphatase inhibitor salubrinal resulted in increased ATF5 hippocampal levels and attenuated status epilepticus-induced neuronal death in the vulnerable CA3 subfield. In good agreement with the neuroprotective effect of increased ATF5, we found that apoptosis-resistant epileptogenic foci from patients with temporal lobe epilepsy also showed increased levels of ATF5. Thus, our results demonstrate that adult neurons express ATF5 and that they increase its levels upon endoplasmic reticulum stress as a pro-survival mechanism, thus opening a new field for neuroprotective strategies focused on ATF5 modulation.

Age-dependent decline of motor neocortex but not hippocampal performance in heterozygous BDNF mice correlates with a decrease of cortical PSD-95 but an increase of hippocampal TrkB levels.

Brain-derived neurotrophic factor (BDNF) is a key player in learning and memory processes. However, little is known about brain area-specific functions of this neurotrophin. Here we investigated whether BDNF could differently affect motor neocortical and hippocampal-related cognitive and plastic morphologic changes in young (12-week-old) and middle-aged (30-week-old) BDNF heterozygous (BDNF⁺/⁻) and wild type (wt) mice. We found that at 30 weeks of age, BDNF⁺/⁻ mice showed impaired performance in accelerating rotarod and grasping tests while preserved spatial learning in a T-maze and recognition memory in an object recognition task compared with wt mice suggesting a specific neocortical dysfunction. Accordingly, a significant reduction of synaptic markers (PSD-95 and GluR1) and corresponding puncta was observed in motor neocortex but not in hippocampus of BDNF⁺/⁻ mice. Interestingly, 30-week-old BDNF⁺/⁻ mice displayed increased TrkB levels in the hippocampus but not in the motor neocortex, which suggests specific hippocampal compensatory mechanisms as a consequence of BDNF decrease. In conclusion, our data indicates that BDNF could differentially regulate the neuronal micro-structures and cognition in a region-specific and in an age-dependent manner.

Evolution of brain-derived neurotrophic factor levels after autologous hematopietic stem cell transplantation in multiple sclerosis

A neuroprotective role of inflammation has been suggested based on that immune cells are the main source of brain-derived neurotrophic factor (BDNF). We investigated the 3-year evolution of BDNF levels in serum, CSF and culture supernatant of peripheral blood mononuclear cells (PBMC), unstimulated and stimulated with anti-CD3 and soluble anti-CD28 antibodies, in 14 multiple sclerosis patients who underwent an autologous hematopoietic stem cell transplantation (AHSCT). BDNF levels were correlated with previously reported MRI measures that showed a reduction of T2 lesion load and increased brain atrophy, mainly at first year post-transplant. A significant decrease of serum BDNF levels was seen at 12 months post-transplant. BDNF values were found significantly lower in stimulated but not in unstimulated PBMC supernatants during the follow-up, supporting that AHSCT may induce a down-regulation of BDNF production. The only significant correlation was found between CSF BDNF levels and T2 lesion load before and 1 year after AHSCT, suggesting that BDNF reflects the past and ongoing inflammatory activity and demyelination of these highly active patients. Our study suggests that AHSCT can reduce BDNF levels to values associated with lower activity. This decrease does not seem to correlate with the brain atrophy measures observed in the MRI.

Mice heterozygous for neurotrophin-3 display enhanced vulnerability to excitotoxicity in the striatum through increased expression of N-methyl-d-aspartate receptors

The striatum is one of the brain areas most vulnerable to excitotoxicity, a lesion that can be prevented by neurotrophins. In the present study, intrastriatal injection of the N-methyl-d-aspartate receptor (NMDAR) agonist quinolinate (QUIN) was performed in mice heterozygous for neurotrophin-3 (NT3 +/-) or brain-derived neurotrophic factor (BDNF +/-) to analyze the role of endogenous neurotrophins on the regulation of striatal neurons susceptibility to excitotoxic injury. QUIN injection induced a decrease in dopamine- and cyclic AMP-regulated phosphoprotein of 32 kDa (DARPP-32) protein levels that was higher in NT-3 +/- than in BDNF+/- or wild type animals. This enhanced susceptibility was specific for enkephalin- and tachykinin-positive projection neurons, and also for parvalbumin-positive interneurons. However the excitotoxic damage in large interneurons was not modified in NT-3 +/- mice compared with wild type animals. This effect can be related to the regulation of NMDARs by endogenous NT-3. Thus, our results show that there is an age-dependent regulation of NMDAR subunits NR1 and NR2A, but not NR2B, in NT-3 +/- mice. The deficit of endogenous NT-3 induced a decrease in NR1 and NR2A subunits at postnatal day (P) 0 and P3 mice respectively, whereas an upregulation was observed in 12 week old NT-3 +/- mice. This differential effect was also observed after administration of exogenous NT-3. In primary striatal cultures, NT-3 treatment induced an enhancement in NR2A, but not NR2B, protein levels. However, intrastriatal grafting of NT-3 secreting-cells in adult wild type mice produced a down-regulation of NR2A subunit. In conclusion, NT-3 regulates the expression of NMDAR subunits modifying striatal neuronal properties that confers the differential vulnerability to excitotoxicity in projection neurons and interneurons in the striatum.