Motoneurons In Lumbar Spinal Cord Research Articles

Selective Loss of MATR3 in Spinal Interneurons, Upper Motor Neurons and Hippocampal CA1 Neurons in a MATR3 S85C Knock-In Mouse Model of Amyotrophic Lateral Sclerosis.

Simple SummaryAmyotrophic lateral sclerosis (ALS) is a neurodegenerative disease affecting the motor neurons in the brain and spinal cord. Mutations in the gene Matr3 have been linked to ALS, including the autosomal dominant missense mutation S85C. We previously created a mouse model containing the S85C mutation in the Matr3 gene to understand how it causes ALS. The S85C mice exhibited MATR3 staining loss in selective populations of degenerating neurons, such as Purkinje cells in the cerebellum and α-motor neurons in the lumbar spinal cord. However, studies have shown that neurons other than motor neurons may be involved in contributing to ALS; therefore, we investigated additional neuronal cell types in the spinal cord and brain. Here, we found that MATR3 staining is selectively reduced in interneurons and α-motor neurons of the cervical and thoracic regions of the spinal cord, as well as in subsets of upper motor neurons and hippocampal neurons. These neurons did not exhibit cell body loss; however, how the MATR3 loss affects neuronal function remains to be determined. Overall, these findings demonstrate that the MATR3 S85C mutation affects other neuronal types of the brain and spinal cord in addition to motor neurons, suggesting that these additional neuronal types are involved in ALS pathogenesis.The neuropathological hallmark of amyotrophic lateral sclerosis (ALS) is motor neuron degeneration in the spinal cord and cortex. Accumulating studies report that other neurons in the central nervous system (CNS) are also affected in ALS. Mutations in Matr3, which encodes a nuclear matrix protein involved in RNA splicing, have been linked to ALS. Previously, we generated a MATR3 S85C knock-in (KI) mouse model that recapitulates early-stage features of ALS. We reported that MATR3 S85C KI mice exhibit defects in lumbar spinal cord motor neurons and in cerebellar Purkinje cells, which are associated with reduced MATR3 immunoreactivity. Here, we show that neurons in various other regions of the CNS are affected in MATR3 S85C KI mice. Using histological analyses, we found selective loss of MATR3 staining in α-motor neurons, but not γ-motor neurons in the cervical and thoracic spinal cord. Loss of MATR3 was also found in parvalbumin-positive interneurons in the cervical, thoracic and lumbar spinal cord. In addition, we found the loss of MATR3 in subsets of upper motor neurons and hippocampal CA1 neurons. Collectively, our findings suggest that these additional neuronal types may contribute to the disease process in MATR3 S85C KI mice.

Plasticity in Motoneurons Following Spinal Cord Injury in Fructose-induced Diabetic Rats.

Spinal cord injury (SCI) causes motor impairment and the proper excitation/inhibition balance in motoneurons is important for recovery. Diabetes mellitus impairs regenerative capacity following SCI. The purpose of this study was to assess the short-term plasticity (STP) of lumbar spinal cord motoneurons in conditions of (1) lateral hemisection (SCI), (2) fructose-induced diabetes (D), and (3) diabetes associated with hemisection (D + SCI). We show that in the cases of SCI, D, and D + SCI, the ratio of percentage share of excitatory and inhibitory combinations of motoneurons responses to high-frequence stimulation of sciatic nerve is multidirectional. In the SCI and D + SCI groups, the cumulative changes in generalized baseline frequencies decreased significantly. When we compared the cumulative changes in the intensity of excitatory and inhibitory responses relative to baseline during high-frequency stimulation (tetanization epoch), we found that there was a significant intensification in tetanic depression in the D + SCI groups versus SCI, as well as an intensification in tetanic potentiation in the D + SCI vs. D and D + SCI vs. SCI groups. Thus, in conditions of traumatic and/or metabolic pathology, the distinct synaptic inputs exhibit opposing plasticity for homeostatic control of neurotransmission and these integral changes most likely shape postsynaptic STP in the spinal motor network.

The Spinal Cord Damage in a Rat Asphyxial Cardiac Arrest/Resuscitation Model.

After cardiac arrest/resuscitation (CA/R), animals often had massive functional restrictions including spastic paralysis of hind legs, disturbed balance and reflex abnormalities. Patients who have survived CA also develop movement restrictions/disorders. A successful therapy requires detailed knowledge of the intrinsic damage pattern and the respective mechanisms. Beside neurodegenerations in the cerebellum and cortex, neuronal loss in the spinal cord could be a further origin of such movement artifacts. Thus, we aimed to evaluate the CA/R-induced degeneration pattern of the lumbar medulla spinalis by immunocytochemical expression of SMI 311 (marker of neuronal perikarya and dendrites), IBA1 (microglia marker), GFAP (marker of astroglia), calbindin D28k (marker of the cellular neuroprotective calcium-buffering system), MnSOD (neuroprotective antioxidant), the transcription factor PPARγ and the mitochondrial marker protein PDH after survival times of 7 and 21days. The CA/R specimens were compared with those from sham-operated and completely naïve rats. RESULTS& CONCLUSION: The main ACA/R-mediated results were: (1) degeneration of lumbar spinal cord motor neurons, characterized by neuronal pyknotization and peri-neuronal tissue artifacts; (2) attendant activation of microglia in the short-term group; (3) attendant activation of astroglia in the long-term group; (4) degenerative pattern in the intermediate gray matter; (5) activation of the endogenous anti-oxidative defense systems calbindin D28k and MnSOD; (6) activation of the transcription factor PPARγ, especially in glial cells of the gray matter penumbra; and (7) activation of mitochondria. Moreover, marginal signs of anesthesia-induced cell stress were already evident in sham animals when compared with completely naïve spinal cords. A correlation between the NDS and the motor neuronal loss could not be verified. Thus, the NDS appears to be unsuitable as prognostic tool.

CD34 Identifies a Subset of Proliferating Microglial Cells Associated with Degenerating Motor Neurons in ALS.

Amyotrophic lateral sclerosis (ALS) is characterized by degeneration of upper and lower motor neurons accompanied by proliferation of reactive microglia in affected regions. However, it is unknown whether the hematopoietic marker CD34 can identify a subpopulation of proliferating microglial cells in the ALS degenerating spinal cord. Immunohistochemistry for CD34 and microglia markers was performed in lumbar spinal cords of ALS rats bearing the SOD1G93A mutation and autopsied ALS and control human subjects. Characterization of CD34-positive cells was also performed in primary cell cultures of the rat spinal cords. CD34 was expressed in a large number of cells that closely interacted with degenerating lumbar spinal cord motor neurons in symptomatic SOD1G93A rats, but not in controls. Most CD34+ cells co-expressed the myeloid marker CD11b, while only a subpopulation was stained for Iba1 or CD68. Notably, CD34+ cells actively proliferated and formed clusters adjacent to damaged motor neurons bearing misfolded SOD1. CD34+ cells were identified in the proximity of motor neurons in autopsied spinal cord from sporadic ALS subjects but not in controls. Cell culture of symptomatic SOD1G93A rat spinal cords yielded a large number of CD34+ cells exclusively in the non-adherent phase, which generated microglia after successive passaging. A yet unrecognized CD34+ cells, expressing or not the microglial marker Iba1, proliferate and accumulate adjacent to degenerating spinal motor neurons, representing an intriguing cell target for approaching ALS pathogenesis and therapeutics.

Characterisation of the pathogenic effects of the in vivo expression of an ALS-linked mutation in D-amino acid oxidase: Phenotype and loss of spinal cord motor neurons.

Amyotrophic lateral sclerosis (ALS) is the most common adult-onset neuromuscular disorder characterised by selective loss of motor neurons leading to fatal paralysis. Current therapeutic approaches are limited in their effectiveness. Substantial advances in understanding ALS disease mechanisms has come from the identification of pathogenic mutations in dominantly inherited familial ALS (FALS). We previously reported a coding mutation in D-amino acid oxidase (DAOR199W) associated with FALS. DAO metabolises D-serine, an essential co-agonist at the N-Methyl-D-aspartic acid glutamate receptor subtype (NMDAR). Using primary motor neuron cultures or motor neuron cell lines we demonstrated that expression of DAOR199W, promoted the formation of ubiquitinated protein aggregates, activated autophagy and increased apoptosis. The aim of this study was to characterise the effects of DAOR199W in vivo, using transgenic mice overexpressing DAOR199W. Marked abnormal motor features, e.g. kyphosis, were evident in mice expressing DAOR199W, which were associated with a significant loss (19%) of lumbar spinal cord motor neurons, analysed at 14 months. When separated by gender, this effect was greater in females (26%; p< 0.0132). In addition, we crossed the DAOR199W transgenic mouse line with the SOD1G93A mouse model of ALS to determine whether the effects of SOD1G93A were potentiated in the double transgenic line (DAOR199W/SOD1G93A). Although overall survival was not affected, onset of neurological signs was significantly earlier in female double transgenic animals than their female SOD1G93A littermates (125 days vs 131 days, P = 0.0239). In summary, some significant in vivo effects of DAOR199W on motor neuron function (i.e. kyphosis and loss of motor neurons) were detected which were most marked in females and could contribute to the earlier onset of neurological signs in double transgenic females compared to SOD1G93A littermates, highlighting the importance of recognizing gender effects present in animal models of ALS.

Expression of aromatase and estrogen receptors in lumbar motoneurons of mice

Estrogen exerts protective roles in amyotrophic lateral sclerosis (ALS). However, the expression of aromatase (ARO) and estrogen receptors (ERs) in the motoneurons of spinal cord, has not yet been elucidated. By immunohistochemistry, we found that ARO and ERs were present in the ventral horn of adult mice lumbar spinal cord, and colocalized with SMI-32, a motoneuron specific marker. Within motoneurons, we observed that ARO is detected primarily in the cytoplasm, with fewer ARO in the nucleus; ERα and ERβ mainly localized in the nucleus with less in the cytoplasm; while GPR30 is located in soma and processes. In conclusion, we found that ERs and ARO are expressed in the motoneurons of lumbar spinal cord in adult mice. These findings suggest that estrogen may be useful as a promising therapeutic agent for prevention of damage and improvement of locomotor function in ALS.

Immunohistochemical research of reaction of motoneurons of lumbar spinal cord of the mice that were in 30-day flight on the BION-M1 biosatellite on a week readaptation to conditions of Earth gravitation

Immunohistochemical research of reaction of motoneurons of lumbar spinal cord of the mice that were in 30-day flight on the BION-M1 biosatellite on a week readaptation to conditions of Earth gravitation

Enforced DNA repair enzymes rescue neurons from apoptosis induced by target deprivation and axotomy in mouse models of neurodegeneration

It is unknown whether DNA damage accumulation is an upstream instigator or secondary effect of the cell death process in different populations of adult postmitotic neurons in the central nervous system. In two different mouse models of injury-induced neurodegeneration characterized by relatively synchronous accumulation of mitochondria, oxidative stress, and DNA damage prior to neuronal apoptosis, we enforced the expression of human 8-oxoguanine DNA glycosylase (hOGG1) and human apurinic-apyrimidinic endonuclease-1/Ref1 (hAPE) using recombinant adenoviruses (Ad). Thalamic lateral geniculate neurons and lumbar spinal cord motor neurons were transduced by Ad-hOGG1 and Ad-hAPE injections into the occipital cortex and skeletal muscle, respectively, prior to their target deprivation- and axotomy-induced retrograde apoptosis. Enforced expression of hOGG1 and hAPE in thalamus and spinal cord was confirmed by western blotting and immunohistochemistry. In injured populations of neurons in thalamus and spinal cord, a DNA damage response (DDR) was registered, as shown by localization of phospho-activated p53, Rad17, and replication protein A-32 immunoreactivities, and this DDR was attenuated more effectively by enforced hAPE expression than by hOGG1 expression. Enforced expression of hOGG1 and hAPE significantly protected thalamic neurons and motor neurons from retrograde apoptosis induced by target deprivation and axotomy. We conclude that a DDR response is engaged pre-apoptotically in different types of injured mature CNS neurons and that DNA repair enzymes can regulate the survival of retrogradely dying neurons, suggesting that DNA damage and activation of DDR are upstream mechanisms for this form of adult neurodegeneration in vivo, thus identifying DNA repair as a therapeutic target for neuroprotection.

Notch Signaling Pathway Is Activated in Motoneurons of Spinal Muscular Atrophy

Spinal muscular atrophy (SMA) is a neurodegenerative disease produced by low levels of Survival Motor Neuron (SMN) protein that affects alpha motoneurons in the spinal cord. Notch signaling is a cell-cell communication system well known as a master regulator of neural development, but also with important roles in the adult central nervous system. Aberrant Notch function is associated with several developmental neurological disorders; however, the potential implication of the Notch pathway in SMA pathogenesis has not been studied yet. We report here that SMN deficiency, induced in the astroglioma cell line U87MG after lentiviral transduction with a shSMN construct, was associated with an increase in the expression of the main components of Notch signaling pathway, namely its ligands, Jagged1 and Delta1, the Notch receptor and its active intracellular form (NICD). In the SMNΔ7 mouse model of SMA we also found increased astrocyte processes positive for Jagged1 and Delta1 in intimate contact with lumbar spinal cord motoneurons. In these motoneurons an increased Notch signaling was found, as denoted by increased NICD levels and reduced expression of the proneural gene neurogenin 3, whose transcription is negatively regulated by Notch. Together, these findings may be relevant to understand some pathologic attributes of SMA motoneurons.

Expression pattern of the endoplasmic reticulum stress protein gp96 in monophasic and chronic relapsing form of experimental autoimmune encephalomyelitis in rats.

Gp96 is the endoplasmic reticulum (ER)-resident molecular chaperone, which is involved in the correction of unfolded proteins, in the activation of proteasome-dependent ER-associated degradation of the misfolded proteins, and in activation of the protein translation that modulates polypeptide traffic into the ER. Furthermore, owing to its peptide chaperone capacity and ability to interact with professional antigen-presenting cells, as well as with growth factors, integrins and Toll-like receptors, it is also endowed with crucial immunological functions acting as a "danger signal" to the innate and adaptive immunity. Considering these properties, in the present study the tissue expression of gp96 was examined during the monophasic and chronic relapsing form of experimental autoimmune encephalomyelitis (CR-EAE), induced in genetically susceptible DA rats by subcutaneous injection of myelin basic protein (MBP) or bovine brain homogenate in complete Freund's adjuvant (CFA). Immunohistochemical analyses were done in periods of attacks and remissions of EAE, and the results were compared with findings in intact rats and those treated only with CFA. The data revealed that the constitutive gp96 expression, found in several neurons and glial cells in the brain and spinal cord of intact animals, significantly diminished during the attacks of CR-EAE. On the contrary, the remission of disease was followed by high upregulation of gp96, mainly in the oligodendrocytes within the white matter, in the neurons of the hippocampal area, as well as in the motoneurons of lumbar spinal cord, suggesting that gp96 might be involved in proteostasis and immune-related pathways linked with the reparative processes in the CNS.

Treadmill training stimulates brain-derived neurotrophic factor mRNA expression in motor neurons of the lumbar spinal cord in spinally transected rats

Brain-derived neurotrophic factor (BDNF) induces plasticity within the lumbar spinal circuits thereby improving locomotor recovery in spinal cord-injured animals. We examined whether lumbar spinal cord motor neurons and other ventral horn cells of spinally transected (ST) rats were stimulated to produce BDNF mRNA in response to treadmill training. Rats received complete spinal cord transections as neonates (n=20) and one month later, received four weeks of either a low (100 steps/training session; n=10) or high (1000 steps/training session; n=10) amount of robotic-assisted treadmill training. Using combined non-radioactive in situ hybridization and immunohistochemical techniques, we found BDNF mRNA expression in heat shock protein 27-labeled motor neurons and in non-motor neuron cells was greater after 1000 steps/training session compared to the 100 steps/training session and was similar to BDNF mRNA labeling in untrained Intact rats. In addition, there were significantly more motor neurons that contained BDNF mRNA labeling within processes in the ST rats that received the higher amount of treadmill training. These findings suggested that motor neurons and other ventral horn cells in ST rats synthesized BDNF in response to treadmill training. The findings support a mechanism by which postsynaptic release of BDNF from motor neurons contributed to synaptic plasticity.

Glucose is an adequate energy substrate for the depolarizing action of GABA and glycine in the neonatal rat spinal cord in vitro

In vitro studies have repeatedly demonstrated that the neurotransmitters γ-aminobutyric acid (GABA) and glycine depolarize immature neurons in many areas of the CNS, including the spinal cord. This widely accepted phenomenon was recently challenged by experiments showing that the depolarizing action of GABA on neonatal hippocampus and neocortex in vitro was prevented by adding energy substrates (ES), such as the ketone body metabolite dl-β-hydroxybutyric acid (DL-BHB), lactate, or pyruvate to the artificial cerebrospinal fluid (ACSF). It was suggested that GABA-induced depolarizations in vitro might be an artifact due to inadequate energy supply when glucose is the sole energy source, consistent with the energy metabolism of neonatal rat brain being largely dependent on ESs other than glucose. Here we examined the effects of these ESs (DL-BHB, lactate, pyruvate) on inhibitory postsynaptic potentials (IPSPs) recorded from neonatal rat lumbar spinal cord motoneurons (MNs), in vitro. We report that supplementing the ACSF with physiologic concentrations of DL-BHB, lactate, or pyruvate does not alter the reversal potential of IPSPs (E(IPSP)). Only high concentrations of pyruvate hyperpolarized E(IPSP). In addition, the depolarizing action of GABA on primary afferent terminals was not affected by supplementing the ACSF with ES at physiologic concentrations. We conclude that depolarizing IPSPs in immature MNs and the primary afferent depolarizations are not caused by inadequate energy supply. Glucose at its standard concentration appears to be an adequate ES for the neonatal spinal cord in vitro.

Excitatory effect of histamine on rat spinal motoneurons by activation of both H1 and H2 receptors in vitro

The central histaminergic nervous system, originating from the tuberomammillary nucleus of the hypothalamus, widely innervates almost the whole brain as well as the spinal cord. However, the effect of histamine on spinal motoneurons, the final common path for motor control, is still unknown. By using 8-14-day-old rat spinal slice preparations and intracellular recordings, the effect of histamine on motoneurons in lumbar spinal cord and the underlying mechanisms were studied. Bath application of histamine (30-300 μM) induced a membrane depolarization in the majority of recorded spinal motoneurons (78/90, 86%). Perfusing slices with tetrodotoxin or low-Ca(2+) /high-Mg(2+) medium did not block the histamine-induced excitation, indicating a direct postsynaptic action of histamine on motoneurons. Separate application of the selective histamine H(1) receptor antagonist mepyramine or the selective histamine H(2) receptor antagonist ranitidine partially suppressed the histamine-induced excitation, whereas a combination of ranitidine and mepyramine totally blocked the excitatory effect of histamine on motoneurons. On the other hand, both the selective histamine H(1) receptor agonist 2-pyridylethylamine and the selective histamine H(2) receptor agonist dimaprit mimicked the excitation of histamine on spinal motoneurons. These agonist-induced excitations were also blocked by mepyramine or ranitidine. Furthermore, histamine affected membrane input resistance and potentiated repetitive firing behavior of spinal motoneurons. These results demonstrate that histamine excites rat spinal motoneurons via the histamine H(1) and H(2) receptors and increases their excitability, suggesting that the hypothalamospinal histaminergic fibers may directly modulate final motor outputs and actively regulate ongoing motor execution andspinal motor reflexes.

Downregulation of the Potassium Chloride Cotransporter KCC2 in Vulnerable Motoneurons in the SOD1-G93A Mouse Model of Amyotrophic Lateral Sclerosis

The balance between excitatory and inhibitory synaptic inputs is critical for the physiological control of motoneurons. The maintenance of a low-intracellular chloride concentration by the potassium chloride cotransporter 2 (KCC2) is essential for the efficacy of fast synaptic inhibition of mature motoneurons in response to the activation of ionotropic γ-aminobutyric acid A and glycine receptors. Altered synaptic balance and excitotoxicity have been proposed as candidate pathophysiological processes in amyotrophic lateral sclerosis (ALS). Therefore, we investigated the expression patterns of KCC2 and its functional opponent, the chloride influx-mediating sodium-potassium chloride cotransporter 1 (NKCC1), in the superoxide dismutase 1 (SOD1-G93A) mouse model of ALS. We detected reduced KCC2 messenger RNA levels and less membrane-bound KCC2 immunoreactivity in ALS-vulnerable motoneurons in lumbar spinal cord and hypoglossal nuclei of SOD1-G93A mice but not in degeneration-resistant oculomotor nuclei. Downregulation of KCC2 started during late presymptomatic stages and accelerated in parallel to hind limb and tongue motor function deficits. In contrast, NKCC1 messenger RNA levels were unaltered in postnatal lumbar spinal cord motoneurons. Our data indicate that reductions in KCC2 gene expression may contribute to selective motor deficits and disease progression in vulnerable motoneurons in a mouse model of ALS.

Proteomic Profiling of Cervical and Lumbar Spinal Cord Reveals Potential Protective Mechanisms in the Wobbler Mouse, a Model of Motor Neuron Degeneration

The wobbler mouse is a model of selective motor neuron degeneration in the cervical spinal cord. Comparing cervical and lumbar tracts of control and diseased mice at the early stage of pathology by proteomic analysis, we identified 31 proteins by peptide mass fingerprint after tryptic digestion and MALDI-TOF analysis, that were differently represented among the four experimental groups. In healthy mice, patterns of protein expression differed between cervical and lumbar tract: proteins of cellular energetic metabolism pathway showed lower expression in the cervical tract, while cellular trafficking proteins were overrepresented. In wobbler mice, these differences disappeared and the expression pattern was similar between cervical and lumbar spinal cord. We found that most of the proteins differentially regulated in wobbler with respect to control cervical tract were related to astrogliosis or involved in glutamate-glutamine cycle, energy transduction and redox functions. Proteins overrepresented in the wobbler lumbar spinal cord were cytoskeleton proteins and cellular transport proteins, in particular the vesicle fusing ATPase and the isoform 2 of syntaxin-binding protein 1, involved in vesicle trafficking. We suggest that overexpression of proteins involved in vesicle trafficking, together with proteins counteracting mitochondrial dysfunction can have neuroprotective effects, preserving lumbar spinal cord motor neurons in wobbler mice.

Immune activation is required for NT-3-induced axonal plasticity in chronic spinal cord injury

After an unilateral lesion of the corticospinal tract (CST) at the level of the medulla over-expression of Neurotrophin-3 (NT-3) in lumbar spinal cord motoneurons induced axonal sprouting of the intact CST in the acutely injured but not uninjured or chronically injured spinal cord in rats. This suggested that processes associated with immune-mediated wound healing may act with NT-3 to induce neuroplasticity. To test whether immune processes were involved we measured NT-3-induced axonal sprouting in immunosuppressed compared to immunocompetent rats. Rats were immunosuppressed with anti-leukocyte antibodies 1 day before receiving a CST lesion and then 2 weeks later NT-3 was over-expressed in the lumbar spinal motoneurons with an adenoviral vector carrying the NT-3 gene targeted to the motoneurons by retrograde transport. At 35 days post-lesion no axonal sprouting was measured in immunosuppressed rats whereas axonal sprouting was measured in the immunocompetent rats. We then tested whether re-evoking an immune response in chronically lesioned rats would induce neuroplasticity. Rats received CST lesions and then 4 months later were treated with systemic injections of lipopolysaccharide (LPS) 7 days before NT-3 was over-expressed in the lumbar spinal motoneurons. Axonal sprouting was observed in the LPS treated rats but not in control animals that were not treated with LPS. Further studies showed that lesioning the CST activated and LPS reactivated microglia and CD4 + T-cells in the acutely lesioned and chronically lesioned rats, respectively. However, immunosuppression only decreased the number of activated CD4 + T-cells suggesting they were responsible for the support of axonal growth. These observations demonstrate that processes associated with immune-mediated wound healing play a role in NT-3-induced neuroplasticity after injury.

Intraparenchymal spinal cord delivery of adeno-associated virus IGF-1 is protective in the SOD1 G93A model of ALS

The potent neuroprotective activities of neurotrophic factors, including insulin-like growth factor 1 (IGF-1), make them promising candidates for treatment of amyotrophic lateral sclerosis (ALS). In an effort to maximize rate of motor neuron transduction, achieve high levels of spinal IGF-1 and thus enhance therapeutic benefit, we injected an adeno-associated virus 2 (AAV2)-based vector encoding human IGF-1 (CERE-130) into lumbar spinal cord parenchyma of SOD1 G93A mice. We observed robust and long-term intraspinal IGF-1 expression and partial rescue of lumbar spinal cord motor neurons, as well as sex-specific delayed disease onset, weight loss, decline in hindlimb grip strength and increased animal survival.

Exogenous testosterone reverses age-related atrophy in a spinal neuromuscular system

Aging is associated with a variety of pathologies, including motor dysfunctions and reductions in sexual behavior. In male rats, declines in sexual behavior during the aging process may be caused in part by the loss of the lumbar spinal cord motoneurons that innervate the penile musculature. Alternatively, declining sexual behavior may be caused by the precipitous reductions in circulating testosterone that occur during aging. In this paper, we report two experiments examining these issues. In Experiment 1, we counted motoneurons in the lumbar motor nuclei and measured several androgen-sensitive morphological properties of the penile muscles and their innervating motoneurons at several time points during the aging process. Motoneuron number in the lumbar nuclei did not change over time, even with very advanced age. In contrast, the penile muscles and their innervating motoneurons underwent profound atrophy, with muscle weight and motoneuron dendritic length declining to less than 50% of young adult levels. In Experiment 2, we treated aged animals with exogenous testosterone, and then examined their penile neuromuscular systems for morphological changes. Testosterone treatment, both acute and chronic, completely reversed age-related declines in the weight of the penile muscles and in the soma size and dendritic length of their innervating motoneurons. Together, these data suggest that reductions in male sexual behavior during the aging process are caused primarily by declines in testosterone levels rather than motoneuron loss. Furthermore, they raise the possibility that testosterone treatment could play an important role in maintaining neuronal connectivity in the aging body.

Motor neuron pathology in experimental autoimmune encephalomyelitis: studies in THY1-YFP transgenic mice

Using adult male C57BL/6 mice that express a yellow fluorescent protein transgene in their motor neurons, we induced experimental autoimmune encephalomyelitis (EAE) by immunization with myelin oligodendrocyte glycoprotein peptide 35-55 (MOG peptide) in complete Freund's adjuvant (CFA). Control mice of the same transgenic strain received CFA without MOG peptide. Early in the course of their illness, the EAE mice showed lumbosacral spinal cord inflammation, demyelination and axonal fragmentation. By 14 weeks post-MOG peptide, these abnormalities were much less prominent, but the mice remained weak and, as in patients with progressive multiple sclerosis, spinal cord atrophy had developed. There was no significant loss of lumbar spinal cord motor neurons in the MOG peptide-EAE mice. However, early in the course of the illness, motor neuron dendrites were disrupted and motor neuron expression of hypophosphorylated neurofilament-H (hypoP-NF-H) immunoreactivity was diminished. By 14 weeks post-MOG peptide, hypoP-NF-H expression had returned to normal, but motor neuron dendritic abnormalities persisted and motor neuron perikaryal atrophy had appeared. We hypothesize that these motor neuron abnormalities contribute to weakness in this form of EAE and speculate that similar motor neuron abnormalities are present in patients with progressive multiple sclerosis.

β-amyloid 42 accumulation in the lumbar spinal cord motor neurons of amyotrophic lateral sclerosis patients

Amyotrophic lateral sclerosis (ALS) is characterized by a progressive loss of large motor neurons in the brain and spinal cord. Amyloid precursor protein (APP), the transmembrane precursor of beta-amyloid (A beta), accumulates in the anterior horn motor neurons of ALS patients with mild lesions. APP undergoes an alternative proteolysis mediated by caspase-3, which is activated in motor neurons in a mouse model of ALS. The ALS spinal cord motor neurons also show evidence of increased oxidative damage, which is thought to alter APP processing. We sought to determine whether A beta42, the more pathogenic A beta species, accumulates in the postmortem lumbar spinal cord of ALS patients. While there was little or no A beta42 labeling in control spinal cord tissues, elevated A beta42 immunoreactivity occurred in ALS motor neuronal perikarya and axonal swellings in the anterior horn. A few A beta42-positive neurons exhibited thioflavine S staining. No extracellular A beta42 deposits were found. A beta42 coexisted with the oxidative damage markers malondialdehyde, 8-hydroxydeoxyguanosine, heme oxygenase-1, and nitrotyrosine in abnormal neurons. The neurons with intracellular A beta42 accumulation also displayed robust cleaved caspase-3 immunoreactivity. Very little A beta40 immunoreactivity occurred in motor neurons of both control and ALS. These results suggest that aberrant accumulation of A beta42 in ALS spinal cord motor neurons is associated with oxidative stress, and may play a role in the pathogenesis of neurodegeneration in ALS.