Improved health by combining dietary restriction and promoting muscle growth in DNA repair-deficient progeroid mice.

BACKGROUND: Ageing is a complex multifactorial process, impacting all organs and tissues, with DNA damage accumulation serving as a common underlying cause. To decelerate ageing, various strategies have been applied to model organisms and evaluated for health and lifespan benefits. Dietary restriction (DR, also known as caloric restriction) is a well-established long-term intervention recognized for its universal anti-ageing effects. DR temporarily suppresses growth, and when applied to progeroid DNA repair-deficient mice doubles lifespan with systemic health benefits. Counterintuitively, attenuation of myostatin/activin signalling by soluble activin receptor (sActRIIB), boosts the growth of muscle and, in these animals, prevents muscle wasting, improves kidney functioning, and compresses morbidity. METHODS: Here, we investigated a combined approach, applying an anabolic regime (sActRIIB) at the same time as DR to Ercc1Δ/- progeroid mice. Following both single treatments and combined, we monitored global effects on body weight, lifespan and behaviour, and local effects on muscle and tissue weight, muscle morphology and function, and ultrastructural and transcriptomic changes in muscle and kidney. RESULTS: Lifespan was mostly influenced by DR (extended from approximately 20 to 40 weeks; P < 0.001), with sActRIIB clearly increasing muscle mass (35-65%) and tetanic force (P < 0.001). The combined regime yielded a stable uniform body weight, but increased compared with DR alone, synergistically improved motor coordination and further delayed the onset and development of balance problems. sActRIIB significantly increased muscle fibre size (P < 0.05) in mice subjected to DR and lowered all signs of muscle damage. Ercc1Δ/- mice showed abnormal neuromuscular junctions. Single interventions by sActRIIB treatment or DR only partially rescued this phenotype, while in the double intervention group, the regularly shaped junctional foldings were maintained. In kidney of Ercc1Δ/- mice, we observed a mild but significant foot process effacement, which was restored by either intervention. Transcriptome analysis also pointed towards reduced levels of DNA damage in muscle and kidney by DR, but not sActRIIB, while these levels retained lower in the double intervention. CONCLUSIONS: In muscle, we found synergistic effects of combining sActRIIB with DR, but not in kidney, with an overall better health in the double intervention group. Crucially, the benefits of each single intervention are not lost when administered in combination, but rather strengthened, even when sActRIIB was applied late in life, opening opportunities for translation to human.

Metabolomic analysis of dietary-restriction-induced attenuation of sarcopenia in prematurely aging DNA repair-deficient mice.

BACKGROUND: Sarcopenia is characterized by loss of skeletal muscle mass and function, and is a major risk factor for disability and independence in the elderly. Effective medication is not available. Dietary restriction (DR) has been found to attenuate aging and aging-related diseases, including sarcopenia, but the mechanism of both DR and sarcopenia are incompletely understood. METHODS: In this study, mice body weight, fore and all limb grip strength, and motor learning and coordination performance were first analysed to evaluate the DR effects on muscle functioning. Liquid chromatography-mass spectrometry (LC-MS) was utilized for the metabolomics study of the DR effects on sarcopenia in progeroid DNA repair-deficient Ercc1∆/- and Xpg-/- mice, to identify potential biomarkers for attenuation of sarcopenia. RESULTS: Muscle mass was significantly (P < 0.05) decreased (13-20%) by DR; however, the muscle quality was improved with retained fore limbs and all limbs grip strength in Ercc1∆/- and Xpg-/- mice. The LC-MS results revealed that metabolites and pathways related to oxidative-stress, that is, GSSG/GSH (P < 0.01); inflammation, that is, 9-HODE, 11-HETE (P < 0.05), PGE2, PGD2, and TXB2 (P < 0.01); and muscle growth (PGF2α) (P < 0.01) and regeneration stimulation (PGE2) (P < 0.05) are significantly downregulated by DR. On the other hand, anti-inflammatory indicator and several related metabolites, that is, ß-hydroxybutyrate (P < 0.01), 14,15-DiHETE (P < 0.0001), 8,9-EET, 12,13-DiHODE, and PGF1 (P < 0.05); consumption of sources of energy (i.e., muscle and liver glycogen); and energy production pathways, that is, glycolysis (glucose, glucose-6-P, fructose-6-P) (P < 0.01), tricarboxylic acid cycle (succinyl-CoA, malate) (P < 0.001), and gluconeogenesis-related metabolite, alanine (P < 0.01), are significantly upregulated by DR. The notably (P < 0.01) down-modulated muscle growth (PGF2α) and regeneration (PGE2) stimulation metabolite and the increased consumption of glycogen in muscle and liver may be related to the significantly (P < 0.01) lower body weight and muscle mass by DR. The downregulated oxidative stress, pro-inflammatory mediators, and upregulated anti-inflammatory metabolites resulted in a lower energy expenditure, which contributed to enhanced muscle quality together with upregulated energy production pathways by DR. The improved muscle quality may explain why grip strength is maintained and motor coordination and learning performance are improved by DR in Ercc1∆/- and Xpg-/- mice. CONCLUSIONS: This study provides fundamental supporting information on biomarkers and pathways related to the attenuation of sarcopenia, which might facilitate its diagnosis, prevention, and clinical therapy.

Pathomechanisms of ALS8: altered autophagy and defective RNA binding protein (RBP) homeostasis due to the VAPB P56S mutation.

Mutations in RNA binding proteins (RBPs) and in genes regulating autophagy are frequent causes of familial amyotrophic lateral sclerosis (fALS). The P56S mutation in vesicle-associated membrane protein-associated protein B (VAPB) leads to fALS (ALS8) and spinal muscular atrophy (SMA). While VAPB is primarily involved in the unfolded protein response (UPR), vesicular trafficking and in initial steps of the autophagy pathway, the effect of mutant P56S-VAPB on autophagy regulation in connection with RBP homeostasis has not been explored yet. Examining the muscle biopsy of our index ALS8 patient of European origin revealed globular accumulations of VAPB aggregates co-localised with autophagy markers LC3 and p62 in partially atrophic and atrophic muscle fibres. In line with this skin fibroblasts obtained from the same patient showed accumulation of P56S-VAPB aggregates together with LC3 and p62. Detailed investigations of autophagic flux in cell culture models revealed that P56S-VAPB alters both initial and late steps of the autophagy pathway. Accordingly, electron microscopy complemented with live cell imaging highlighted the impaired fusion of accumulated autophagosomes with lysosomes in cells expressing P56S-VAPB. Consistent with these observations, neuropathological studies of brain and spinal cord of P56S-VAPB transgenic mice revealed signs of neurodegeneration associated with altered protein quality control and defective autophagy. Autophagy and RBP homeostasis are interdependent, as demonstrated by the cytoplasmic mis-localisation of several RBPs including pTDP-43, FUS, Matrin 3 which often sequestered with P56S-VAPB aggregates both in cell culture and in the muscle biopsy of the ALS8 patient. Further confirming the notion that aggregation of the RBPs proceeds through the stress granule (SG) pathway, we found persistent G3BP- and TIAR1-positive SGs in P56S-VAPB expressing cells as well as in the ALS8 patient muscle biopsy. We conclude that P56S-VAPB-ALS8 involves a cohesive pathomechanism of aberrant RBP homeostasis together with dysfunctional autophagy.

Region-specific preservation of Purkinje cell morphology and motor behavior in the ATXN1[82Q] mouse model of spinocerebellar ataxia 1.

Purkinje cells are the primary processing units of the cerebellar cortex and display molecular heterogeneity that aligns with differences in physiological properties, projection patterns, and susceptibility to disease. In particular, multiple mouse models that feature Purkinje cell degeneration are characterized by incomplete and patterned Purkinje cell degeneration, suggestive of relative sparing of Purkinje cell subpopulations, such as those expressing Aldolase C/zebrinII (AldoC) or residing in the vestibulo-cerebellum. Here, we investigated a well-characterized Purkinje cell-specific mouse model for spinocerebellar ataxia type 1 (SCA1) that expresses human ATXN1 with a polyQ expansion (82Q). Our pathological analysis confirms previous findings that Purkinje cells of the vestibulo-cerebellum, i.e., the flocculonodular lobes, and crus I are relatively spared from key pathological hallmarks: somatodendritic atrophy, and the appearance of p62/SQSTM1-positive inclusions. However, immunohistological analysis of transgene expression revealed that spared Purkinje cells do not express mutant ATXN1 protein, indicating the sparing of Purkinje cells can be explained by an absence of transgene expression. Additionally, we found that Purkinje cells in other cerebellar lobules that typically express AldoC, not only display severe pathology but also show loss of AldoC expression. The relatively preserved flocculonodular lobes and crus I showed a substantial fraction of Purkinje cells that expressed the mutant protein and displayed pathology as well as loss of AldoC expression. Despite considerable pathology in these lobules, behavioral analyses demonstrated a relative sparing of related functions, suggestive of sufficient functional cerebellar reserve. Together, the data indicate that mutant ATXN1 affects both AldoC-positive and AldoC-negative Purkinje cells and disrupts normal parasagittal AldoC expression in Purkinje cells. Our results show that, in a mouse model otherwise characterized by widespread Purkinje cell degeneration, sparing of specific subpopulations is sufficient to maintain normal performance of specific behaviors within the context of the functional, modular map of the cerebellum.

Mechanisms underlying cognitive deficits in a mouse model for Costello Syndrome are distinct from other RASopathy mouse models.

RASopathies, characterized by germline mutations in genes encoding proteins of the RAS-ERK signaling pathway, show overlapping phenotypes, which manifest themselves with a varying severity of intellectual disability. However, it is unclear to what extent they share the same downstream pathophysiology that underlies the cognitive deficits. Costello syndrome (CS) is a rare RASopathy caused by activating mutations in the HRAS gene. Here we investigated the mechanisms underlying the cognitive deficits of HRas G12V/G12V mice. HRas G12V/G12V mice showed robust upregulation of ERK signaling, neuronal hypertrophy, increased brain volume, spatial learning deficits, and impaired mGluR-dependent long-term depression (LTD). In contrast, long-term potentiation (LTP), which is affected in other RASopathy mouse models was unaffected. Treatment with lovastatin, a HMG-CoA-Reductase inhibitor which has been shown to rescue the behavioral phenotypes of mouse models of NF1 and Noonan syndrome, was unable to restore ERK signaling and the cognitive deficits of HRas G12V/G12V mice. Administration of a potent mitogen-activated protein kinase (MEK) inhibitor rescued the ERK upregulation and the mGluR-LTD deficit of HRas G12V/G12V mice, but failed to rescue the cognitive deficits. Taken together, this study indicates that the fundamental molecular and cellular mechanisms underlying the cognitive aspects of different RASopathies are remarkably distinct, and may require disease specific treatments.

Dynein Regulator NDEL1 Controls Polarized Cargo Transport at the Axon Initial Segment.

The development and homeostasis of neurons relies heavily on the selective targeting of vesicles into axon and dendrites. Microtubule-based motor proteins play an important role in polarized transport; however, the sorting mechanism to exclude dendritic cargo from the axon is unclear. We show that the dynein regulator NDEL1 controls somatodendritic cargo transport at the axon initial segment (AIS). NDEL1 localizes to the AIS via an interaction with the scaffold protein Ankyrin-G. Depletion of NDEL1 or its binding partner LIS1 results in both cell-wide and local defects, including the non-polarized trafficking of dendritic cargo through the AIS. We propose a model in which LIS1 is a critical mediator of local NDEL1-based dynein activation at the AIS. By localizing to the AIS, NDEL1 facilitates the reversal of somatodendritic cargos in the proximal axon.

Cell-autonomous progeroid changes in conditional mouse models for repair endonuclease XPG deficiency.

As part of the Nucleotide Excision Repair (NER) process, the endonuclease XPG is involved in repair of helix-distorting DNA lesions, but the protein has also been implicated in several other DNA repair systems, complicating genotype-phenotype relationship in XPG patients. Defects in XPG can cause either the cancer-prone condition xeroderma pigmentosum (XP) alone, or XP combined with the severe neurodevelopmental disorder Cockayne Syndrome (CS), or the infantile lethal cerebro-oculo-facio-skeletal (COFS) syndrome, characterized by dramatic growth failure, progressive neurodevelopmental abnormalities and greatly reduced life expectancy. Here, we present a novel (conditional) Xpg-/- mouse model which -in a C57BL6/FVB F1 hybrid genetic background- displays many progeroid features, including cessation of growth, loss of subcutaneous fat, kyphosis, osteoporosis, retinal photoreceptor loss, liver aging, extensive neurodegeneration, and a short lifespan of 4-5 months. We show that deletion of XPG specifically in the liver reproduces the progeroid features in the liver, yet abolishes the effect on growth or lifespan. In addition, specific XPG deletion in neurons and glia of the forebrain creates a progressive neurodegenerative phenotype that shows many characteristics of human XPG deficiency. Our findings therefore exclude that both the liver as well as the neurological phenotype are a secondary consequence of derailment in other cell types, organs or tissues (e.g. vascular abnormalities) and support a cell-autonomous origin caused by the DNA repair defect itself. In addition they allow the dissection of the complex aging process in tissue- and cell-type-specific components. Moreover, our data highlight the critical importance of genetic background in mouse aging studies, establish the Xpg-/- mouse as a valid model for the severe form of human XPG patients and segmental accelerated aging, and strengthen the link between DNA damage and aging.

The Copper Metabolism MURR1 domain protein 1 (COMMD1) modulates the aggregation of misfolded protein species in a client-specific manner.

The Copper Metabolism MURR1 domain protein 1 (COMMD1) is a protein involved in multiple cellular pathways, including copper homeostasis, NF-κB and hypoxia signalling. Acting as a scaffold protein, COMMD1 mediates the levels, stability and proteolysis of its substrates (e.g. the copper-transporters ATP7B and ATP7A, RELA and HIF-1α). Recently, we established an interaction between the Cu/Zn superoxide dismutase 1 (SOD1) and COMMD1, resulting in a decreased maturation and activation of SOD1. Mutations in SOD1, associated with the progressive neurodegenerative disorder Amyotrophic Lateral Sclerosis (ALS), cause misfolding and aggregation of the mutant SOD1 (mSOD1) protein. Here, we identify COMMD1 as a novel regulator of misfolded protein aggregation as it enhances the formation of mSOD1 aggregates upon binding. Interestingly, COMMD1 co-localizes to the sites of mSOD1 inclusions and forms high molecular weight complexes in the presence of mSOD1. The effect of COMMD1 on protein aggregation is client-specific as, in contrast to mSOD1, COMMD1 decreases the abundance of mutant Parkin inclusions, associated with Parkinson's disease. Aggregation of a polyglutamine-expanded Huntingtin, causative of Huntington's disease, appears unaltered by COMMD1. Altogether, this study offers new research directions to expand our current knowledge on the mechanisms underlying aggregation disease pathologies.

Characterization of the mouse neuroinvasiveness of selected European strains of West Nile virus.

West Nile virus (WNV) has caused outbreaks and sporadic infections in Central, Eastern and Mediterranean Europe for over 45 years. Most strains responsible for the European and Mediterranean basin outbreaks are classified as lineage 1. In recent years, WNV strains belonging to lineage 1 and 2 have been causing outbreaks of neuroinvasive disease in humans in countries such as Italy, Hungary and Greece, while mass mortality among birds was not reported. This study characterizes three European strains of WNV isolated in Italy (FIN and Ita09) and Hungary (578/10) in terms of in vitro replication kinetics on neuroblastoma cells, LD50 values in C57BL/6 mice, median day mortality, cumulative mortality, concentration of virus in the brain and spinal cord, and the response to infection in the brain. Overall, the results indicate that strains circulating in Europe belonging to both lineage 1 and 2 are highly virulent and that Ita09 and 578/10 are more neurovirulent compared to the FIN strain.

Age-related neuronal degeneration: complementary roles of nucleotide excision repair and transcription-coupled repair in preventing neuropathology.

Neuronal degeneration is a hallmark of many DNA repair syndromes. Yet, how DNA damage causes neuronal degeneration and whether defects in different repair systems affect the brain differently is largely unknown. Here, we performed a systematic detailed analysis of neurodegenerative changes in mouse models deficient in nucleotide excision repair (NER) and transcription-coupled repair (TCR), two partially overlapping DNA repair systems that remove helix-distorting and transcription-blocking lesions, respectively, and that are associated with the UV-sensitive syndromes xeroderma pigmentosum (XP) and Cockayne syndrome (CS). TCR-deficient Csa(-/-) and Csb(-/-) CS mice showed activated microglia cells surrounding oligodendrocytes in regions with myelinated axons throughout the nervous system. This white matter microglia activation was not observed in NER-deficient Xpa(-/-) and Xpc(-/-) XP mice, but also occurred in Xpd(XPCS) mice carrying a point mutation (G602D) in the Xpd gene that is associated with a combined XPCS disorder and causes a partial NER and TCR defect. The white matter abnormalities in TCR-deficient mice are compatible with focal dysmyelination in CS patients. Both TCR-deficient and NER-deficient mice showed no evidence for neuronal degeneration apart from p53 activation in sporadic (Csa(-/-), Csb(-/-)) or highly sporadic (Xpa(-/-), Xpc(-/-)) neurons and astrocytes. To examine to what extent overlap occurs between both repair systems, we generated TCR-deficient mice with selective inactivation of NER in postnatal neurons. These mice develop dramatic age-related cumulative neuronal loss indicating DNA damage substrate overlap and synergism between TCR and NER pathways in neurons, and they uncover the occurrence of spontaneous DNA injury that may trigger neuronal degeneration. We propose that, while Csa(-/-) and Csb(-/-) TCR-deficient mice represent powerful animal models to study the mechanisms underlying myelin abnormalities in CS, neuron-specific inactivation of NER in TCR-deficient mice represents a valuable model for the role of NER in neuronal maintenance and survival.

Accelerated age-related cognitive decline and neurodegeneration, caused by deficient DNA repair.

Age-related cognitive decline and neurodegenerative diseases are a growing challenge for our societies with their aging populations. Accumulation of DNA damage has been proposed to contribute to these impairments, but direct proof that DNA damage results in impaired neuronal plasticity and memory is lacking. Here we take advantage of Ercc1(Δ/-) mutant mice, which are impaired in DNA nucleotide excision repair, interstrand crosslink repair, and double-strand break repair. We show that these mice exhibit an age-dependent decrease in neuronal plasticity and progressive neuronal pathology, suggestive of neurodegenerative processes. A similar phenotype is observed in mice where the mutation is restricted to excitatory forebrain neurons. Moreover, these neuron-specific mutants develop a learning impairment. Together, these results suggest a causal relationship between unrepaired, accumulating DNA damage, and age-dependent cognitive decline and neurodegeneration. Hence, accumulated DNA damage could therefore be an important factor in the onset and progression of age-related cognitive decline and neurodegenerative diseases.

Spinal inhibitory interneuron pathology follows motor neuron degeneration independent of glial mutant superoxide dismutase 1 expression in SOD1-ALS mice.

Motor neuron degeneration and skeletal muscle denervation are hallmarks of amyotrophic lateral sclerosis (ALS), but other neuron populations and glial cells are also involved in ALS pathogenesis. We examined changes in inhibitory interneurons in spinal cords of the ALS model low-copy Gurney G93A-SOD1 (G1del) mice and found reduced expression of markers of glycinergic and GABAergic neurons, that is, glycine transporter 2 (GlyT2) and glutamic acid decarboxylase (GAD65/67), specifically in the ventral horns of clinically affected mice. There was also loss of GlyT2 and GAD67 messenger RNA-labeled neurons in the intermediate zone. Ubiquitinated inclusions appeared in interneurons before 20 weeks of age, that is, after their development in motor neurons but before the onset of clinical signs and major motor neuron degeneration, which starts from 25 weeks of age. Because mutant superoxide dismutase 1 (SOD1) in glia might contribute to the pathogenesis, we also examined neuron-specific G93A-SOD1 mice; they also had loss of inhibitory interneuron markers in ventral horns and ubiquitinated interneuron inclusions. These data suggest that, in mutant SOD1-associated ALS, pathological changes may spread from motor neurons to interneuronsin a relatively early phase of the disease, independent of the presence of mutant SOD1 in glia. The degeneration of spinal inhibitory interneurons may in turn facilitate degeneration of motor neurons and contribute to disease progression.

Bicaudal D2, dynein, and kinesin-1 associate with nuclear pore complexes and regulate centrosome and nuclear positioning during mitotic entry.

BICD2 is one of the two mammalian homologues of the Drosophila Bicaudal D, an evolutionarily conserved adaptor between microtubule motors and their cargo that was previously shown to link vesicles and mRNP complexes to the dynein motor. Here, we identified a G2-specific role for BICD2 in the relative positioning of the nucleus and centrosomes in dividing cells. By combining mass spectrometry, biochemical and cell biological approaches, we show that the nuclear pore complex (NPC) component RanBP2 directly binds to BICD2 and recruits it to NPCs specifically in G2 phase of the cell cycle. BICD2, in turn, recruits dynein-dynactin to NPCs and as such is needed to keep centrosomes closely tethered to the nucleus prior to mitotic entry. When dynein function is suppressed by RNA interference-mediated depletion or antibody microinjection, centrosomes and nuclei are actively pushed apart in late G2 and we show that this is due to the action of kinesin-1. Surprisingly, depletion of BICD2 inhibits both dynein and kinesin-1-dependent movements of the nucleus and cytoplasmic NPCs, demonstrating that BICD2 is needed not only for the dynein function at the nuclear pores but also for the antagonistic activity of kinesin-1. Our study demonstrates that the nucleus is subject to opposing activities of dynein and kinesin-1 motors and that BICD2 contributes to nuclear and centrosomal positioning prior to mitotic entry through regulation of both dynein and kinesin-1.

A new paraneoplastic encephalomyelitis autoantibody reactive with the axon initial segment.

Serum from a patient with paraneoplastic encephalomyelitis (PEM) and small cell lung cancer (SCLC) showed high titer immunohistochemical staining of the axon initial segment (AIS) on rat and human brain sections. EM studies showed that the antigen was localized in close proximity of the microtubules in the AIS. Double labeling experiments and absence of staining at the nodes of Ranvier excluded the previously identified betaIV spectrin as autoantigen. Screening a rat hippocampal cDNA library resulted in the isolation of ubiquitin-conjugating enzyme E2E1 (UBE2E1). However, blocking and elution experiments excluded UBE2E1 as the AIS autoantigen.

A novel mouse model with impaired dynein/dynactin function develops amyotrophic lateral sclerosis (ALS)-like features in motor neurons and improves lifespan in SOD1-ALS mice.

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative condition characterized by progressive motor neuron degeneration and muscle paralysis. Genetic evidence from man and mouse has indicated that mutations in the dynein/dynactin motor complex are correlated with motor neuron degeneration. In this study, we have generated transgenic mice with neuron-specific expression of Bicaudal D2 N-terminus (BICD2-N) to chronically impair dynein/dynactin function. Motor neurons expressing BICD2-N showed accumulation of dynein and dynactin in the cell body, Golgi fragmentation and several signs of impaired retrograde trafficking: the appearance of giant neurofilament swellings in the proximal axon, reduced retrograde labelling by tracer injected in the muscle and delayed expression of the injury transcription factor ATF3 after axon transection. Despite these abnormalities, BICD2-N mice did not develop signs of motor neuron degeneration and motor abnormalities. Interestingly, the BICD2-N transgene increased lifespan in 'low copy' SOD1-G93A ALS transgenic mice. Our findings indicate that impaired dynein/dynactin function can explain several pathological features observed in ALS patients, but may be beneficial in some forms of ALS.

Distribution of RET immunoreactivity in the rodent spinal cord and changes after nerve injury.

RET (for "rearranged during transfection") is a transmembrane tyrosine kinase signaling receptor for members of the glial cell line-derived neurotrophic factor (GDNF) family of ligands. We used RET immunohistochemistry (IHC), double-labeling immunofluorescence (IF), and in situ hybridization (ISH) in adult naïve and nerve-injured rats to study the distribution of RET in the spinal cord. In the dorsal horn, strong RET-immunoreactive (-ir) fibers were abundant in lamina II-inner (II(i)), although this labeling was preferentially observed after an antigen-unmasking procedure. After dorsal rhizotomy, RET-ir fibers in lamina II(i) completely disappeared from the dorsal horn, indicating that they were all primary afferents. After peripheral axotomy, RET-ir in primary afferents decreased in lamina II(i) and appeared to increase slightly in laminae III and IV. RET-ir was also observed in neurons and dendrites throughout the dorsal horn. Some RET-ir neurons in lamina I had the morphological appearance of nociceptive projection neurons, which was confirmed by the finding that 53% of RET-ir neurons in lamina I colocalized with neurokinin-1. GDNF-ir terminals were in close proximity to RET-ir neurons in the superficial dorsal horn. In the ventral horn, RET-ir was strongly expressed by motoneurons, with the strongest staining in small, presumably gamma-motoneurons. Increased RET expression following peripheral axotomy was most pronounced in alpha-motoneurons. The expression and regulation pattern of RET in the spinal cord are in line with its involvement in regenerative processes following nerve injury. The presence of RET in dorsal horn neurons, including nociceptive projection neurons, suggests that RET also has a role in signal transduction at the spinal level. This role may include mediating the effects of GDNF released from nociceptive afferent fibers.

ATF3 expression precedes death of spinal motoneurons in amyotrophic lateral sclerosis-SOD1 transgenic mice and correlates with c-Jun phosphorylation, CHOP expression, somato-dendritic ubiquitination and Golgi fragmentation.

To obtain insight into the morphological and molecular correlates of motoneuron degeneration in amyotrophic lateral sclerosis (ALS) mice that express G93A mutant superoxide dismutase (SOD)1 (G93A mice), we have mapped and characterized 'sick' motoneurons labelled by the 'stress transcription factors' ATF3 and phospho-c-Jun. Immunocytochemistry and in situ hybridization showed that a subset of motoneurons express ATF3 from a relatively early phase of disease before the onset of active caspase 3 expression and motoneuron loss. The highest number of ATF3-expressing motoneurons occurred at symptom onset. The onset of ATF3 expression correlated with the appearance of ubiquitinated neurites. Confocal double-labelling immunofluorescence showed that all ATF3-positive motoneurons were immunoreactive for phosphorylated c-Jun. Furthermore, the majority of ATF3 and phospho-c-Jun-positive motoneurons were also immunoreactive for CHOP (GADD153) and showed Golgi fragmentation. A subset of ATF3 and phosphorylated c-Jun-immunoreactive motoneurons showed an abnormal appearance characterized by a number of distinctive features, including an eccentric flattened nucleus, perikaryal accumulation of ubiquitin immunoreactivity, juxta-nuclear accumulation of the Golgi apparatus and the endoplasmic reticulum, and intense Hsp70 immunoreactivity. These abnormal cells were not immunoreactive for active caspase 3. We conclude that motoneurons in ALS-SOD1 mice prior to their death and disappearance experience a prolonged sick phase, characterized by the gradual accumulation of ubiquitinated material first in the neurites and subsequently the cell body.

Modulation of presynaptic plasticity and learning by the H-ras/extracellular signal-regulated kinase/synapsin I signaling pathway.

Molecular and cellular studies of the mechanisms underlying mammalian learning and memory have focused almost exclusively on postsynaptic function. We now reveal an experience-dependent presynaptic mechanism that modulates learning and synaptic plasticity in mice. Consistent with a presynaptic function for endogenous H-ras/extracellular signal-regulated kinase (ERK) signaling, we observed that, under normal physiologic conditions in wild-type mice, hippocampus-dependent learning stimulated the ERK-dependent phosphorylation of synapsin I, and MEK (MAP kinase kinase)/ERK inhibition selectively decreased the frequency of miniature EPSCs. By generating transgenic mice expressing a constitutively active form of H-ras (H-rasG12V), which is abundantly localized in axon terminals, we were able to increase the ERK-dependent phosphorylation of synapsin I. This resulted in several presynaptic changes, including a higher density of docked neurotransmitter vesicles in glutamatergic terminals, an increased frequency of miniature EPSCs, and increased paired-pulse facilitation. In addition, we observed facilitated neurotransmitter release selectively during high-frequency activity with consequent increases in long-term potentiation. Moreover, these mice showed dramatic enhancements in hippocampus-dependent learning. Importantly, deletion of synapsin I, an exclusively presynaptic protein, blocked the enhancements of learning, presynaptic plasticity, and long-term potentiation. Together with previous invertebrate studies, these results demonstrate that presynaptic plasticity represents an important evolutionarily conserved mechanism for modulating learning and memory.

Decrease of Hsp25 protein expression precedes degeneration of motoneurons in ALS-SOD1 mice.

We have investigated the expression of Hsp25, a heat shock protein constitutively expressed in motoneurons, in amyotrophic lateral sclerosis (ALS) mice that express G93A mutant SOD1 (G93A mice). Immunocytochemistry and Western blotting showed that a decrease of Hsp25 protein expression occurred in motoneurons of G93A mice prior to the onset of motoneuron death and muscle weakness. This decrease in Hsp25 expression also preceded the appearance of SOD1 aggregates as identified by cellulose acetate filtration and Western blot analysis. In contrast to Hsp25 protein levels, Hsp25 mRNA as determined by in situ hybridization and RT-PCR, remained unchanged. This suggests that the decrease in Hsp25 protein levels occurs post-transcriptionally. In view of the cytoprotective properties of Hsp25 and the temporal relationship between decreased Hsp25 expression and the onset of motoneuron death, it is feasible that reduced Hsp25 concentration contributes to the degeneration of motoneurons in G93A mice. These data are consistent with the idea that mutant SOD1 may reduce the availability of the protein quality control machinery in motoneurons.

Long term proteasome inhibition does not preferentially afflict motor neurons in organotypical spinal cord cultures.

Ubiquitinated inclusions are a constant feature of amyotrophic lateral sclerosis (ALS). It has been hypothesised that these inclusions reflect overload or failure of the ubiquitin-proteasome system, and that this failure contributes to the degeneration of motor neurons. In the present study we have examined the effect of low concentrations of proteasome inhibitors on protein aggregation and viability of neurons in organotypical spinal cord cultures. We found a dose-dependent degeneration of neurons after a one-week exposure to the proteasome inhibitors lactacystin and epoxomicin. Neuronal degeneration was associated with an increase in poly-ubiquitination, consistent with failure of the ubiquitin-proteasome system. Proteasome inhibition caused degeneration of both motor neurons and interneurons, and no difference in survival between motor neurons and interneurons was observed. Since protein aggregation may particularly play a role in ALS patients with superoxide dismutase 1 (SOD1) mutations, we have compared the effect of proteasome inhibition between spinal cord cultures from non-transgenic and SOD1(G93A) transgenic mice. There was no difference between the viability of motor neurons from transgenic and non-transgenic mice.