Loss of Mfn2 results in progressive, retrograde degeneration of dopaminergic neurons in the nigrostriatal circuit.

Mitochondria continually undergo fusion and fission, and these dynamic processes play a major role in regulating mitochondrial function. Studies of several genes associated with familial Parkinson's disease (PD) have implicated aberrant mitochondrial dynamics in the disease pathology, but the importance of these processes in dopaminergic neurons remains poorly understood. Because the mitofusins Mfn1 and Mfn2 are essential for mitochondrial fusion, we deleted these genes from a subset of dopaminergic neurons in mice. Loss of Mfn2 results in a movement defect characterized by reduced activity and rearing. In open field tests, Mfn2 mutants show severe, age-dependent motor deficits that can be rescued with L-3,4 dihydroxyphenylalanine. These motor deficits are preceded by the loss of dopaminergic terminals in the striatum. However, the loss of dopaminergic neurons in the midbrain occurs weeks after the onset of these motor and striatal deficits, suggesting a retrograde mode of neurodegeneration. In our conditional knockout strategy, we incorporated a mitochondrially targeted fluorescent reporter to facilitate tracking of mitochondria in the affected neurons. Using an organotypic slice culture system, we detected fragmented mitochondria in the soma and proximal processes of these neurons. In addition, we found markedly reduced mitochondrial mass and transport, which may contribute to the neuronal loss. These effects are specific for Mfn2, as the loss of Mfn1 yielded no corresponding defects in the nigrostriatal circuit. Our findings indicate that perturbations of mitochondrial dynamics can cause nigrostriatal defects and may be a risk factor for the neurodegeneration in PD.

Hind limb muscle atrophy precedes cerebral neuronal degeneration in G93A-SOD1 mouse model of amyotrophic lateral sclerosis: a longitudinal MRI study.

Amyotrophic lateral sclerosis (ALS) is a progressive, fatal, neurodegenerative disorder caused by the degeneration of motor neurons in the CNS, which results in complete paralysis of skeletal muscles. Recent experimental studies have suggested that the disease could initiate in skeletal muscle, rather than in the motor neurons. To establish the timeframe of motor neuron degeneration in relation to muscle atrophy in motor neuron disease, we have used MRI to monitor changes throughout disease in brain and skeletal muscle of G93A-SOD1 mice, a purported model of ALS. Longitudinal MRI examination of the same animals indicated that muscle volume in the G93A-SOD1 mice was significantly reduced from as early as week 8 of life, 4 weeks prior to clinical onset. Progressive muscle atrophy from week 8 onwards was confirmed by histological analysis. In contrast, brain MRI indicated that neurodegeneration occurs later in G93A-SOD1 mice, with hyperintensity MRI signals detected only at weeks 10-18. Neurodegenerative changes were observed only in the motor nuclei areas of the brainstem; MRI changes indicative of neurodegeneration were not detected in the motor cortex where first motor neurons originate, even at the late disease stage. This longitudinal MRI study establishes unequivocally that, in the experimental murine model of ALS, muscle degeneration occurs before any evidence of neurodegeneration and clinical signs, supporting the postulate that motor neuron disease can initiate from muscle damage and result from retrograde dying-back of the motor neurons.

Neuronal FLT1 receptor and its selective ligand VEGF-B protect against retrograde degeneration of sensory neurons.

Even though VEGF-B is a homologue of the potent angiogenic factor VEGF, its angiogenic activities have been controversial. Intrigued by findings that VEGF-B may also affect neuronal cells, we assessed the neuroprotective and vasculoprotective effects of VEGF-B in the skin, in which vessels and nerves are functionally intertwined. Although VEGF-B and its FLT1 receptor were prominently expressed in dorsal root ganglion (DRG) neurons innervating the hindlimb skin, they were not essential for nerve function or vascularization of the skin. However, primary DRG cultures lacking VEGF-B or FLT1 exhibited increased neuronal stress and were more susceptible to paclitaxel-induced cell death. Concomitantly, mice lacking VEGF-B or a functional FLT1 developed more retrograde degeneration of sensory neurons in a model of distal neuropathy. On the other hand, the addition of the VEGF-B isoform, VEGF-B(186), to DRG cultures antagonized neuronal stress, maintained the mitochondrial membrane potential and stimulated neuronal survival. Mice overexpressing VEGF-B(186) or FLT1 selectively in neurons were protected against the distal neuropathy, whereas exogenous VEGF-B(186), either delivered by gene transfer or as a recombinant factor, was protective by directly affecting sensory neurons and not the surrounding vasculature. Overall, this indicates that VEGF-B, instead of acting as an angiogenic factor, exerts direct neuroprotective effects through FLT1. These findings also suggest a clinically relevant role for VEGF-B in preventing distal neuropathies.

JNK2 and JNK3 combined are essential for apoptosis in dopamine neurons of the substantia nigra, but are not required for axon degeneration.

Activation of c-jun N-terminal kinase (JNK) by the mitogen-activated protein kinase cascade has been shown to play an important role in the death of dopamine neurons of the substantia nigra, one of the principal neuronal populations affected in Parkinson's disease. However, it has remained unknown whether the JNK2 and JNK3 isoforms, either singly or in combination, are essential for apoptotic death, and, if so, the mechanisms involved. In addition, it has been unclear whether they play a role in axonal degeneration of these neurons in disease models. To address these issues we have examined the effect of single and double jnk2 and jnk3 null mutations on apoptosis in a highly destructive neurotoxin model, that induced by intrastriatal 6-hydroxydopamine. We find that homozygous jnk2/3 double null mutations result in a complete abrogation of apoptosis and a prolonged survival of the entire population of dopamine neurons. In spite of this complete protection at the cell soma level, there was no protection of axons. These studies provide a striking demonstration of the distinctiveness of the mechanisms that mediate cell soma and axon degeneration, and they illustrate the need to identify and target pathways of axon degeneration in the development of neuroprotective therapeutics.

[Alzheimer's disease. Molecular pathology, animal models, and current treatment]. / Alzheimer-Demenz. Molekulare Pathologie, Tiermodelle und Therapiestrategien.

The currently approved but only mildly efficient drugs against Alzheimer's disease treat merely the symptoms. Genetic, neuropathological, and biochemical data support the importance of the amyloid hypothesis of Alzheimer's disease, at the moment the most influential hypothesis. Many treatment strategies have been performed based on this hypothesis and were markedly successful in preclinical animal models. Unfortunately the treatment is still unsuccessful in humans. This could be due to the animal models showing marginal behavioural deficits but no Alzheimer-like nerve cell loss, although they all developed a more or less pronounced plaque load. Today we know however that Alzheimer plaques are not mainly responsible for the cell loss. Therefore novel animal models have been developed that show age-dependent axonal degeneration, massive neuronal loss, and robust behavioural deficits. Successful treatment of an animal model with such robust deficits would be very likely better suited to transferral into the clinic. The final validation or disproof of individual Alzheimer hypotheses and their resulting treatment strategies can however be obtained only after clinical proof.

Altered expression of Smad family members in injured motor neurons of rat.

We examined changes in the expression of Smad family members, which transduce signals from TGF-beta superfamily ligands, following hypoglossal nerve injury. RT-PCR and in situ hybridization revealed that Smad1, 2, 3 and 4 mRNAs were significantly up-regulated in injured side, whereas Smad8 mRNA was down-regulated. Immunohistochemistry and Western blotting analysis confirmed the alterations of Smad1, 2 and 4 in injured neurons. These results suggest that the Smad signaling may be important for nerve regeneration.

Major myelin protein gene (P0) mutation causes a novel form of axonal degeneration.

Mutations in the major peripheral nervous system (PNS) myelin protein, myelin protein zero (MPZ), cause Charcot-Marie-Tooth Disease type 1B (CMT1B), typically thought of as a demyelinating peripheral neuropathy. Certain MPZ mutations, however, cause adult onset neuropathy with minimal demyelination but pronounced axonal degeneration. Mechanism(s) for this phenotype are unknown. We performed an autopsy of a 73-year-old woman with a late-onset neuropathy caused by an H10P MPZ mutation whose nerve conduction studies suggested severe axonal loss but no demyelination. The autopsy demonstrated axonal loss and reorganization of the molecular architecture of the axolemma. Segmental demyelination was negligible. In addition, we identified focal nerve enlargements containing MPZ and ubiquitin either in the inner myelin intralaminar and/or periaxonal space that separates axons from myelinating Schwann cells. Taken together, these data confirmed that a mutation in MPZ can cause axonal neuropathy, in the absence of segmental demyelination, thus uncoupling the two pathological processes. More important, it also provided potential molecular mechanisms as to how the axonal degeneration occurred: either by disruption of glial-axon interaction by protein aggregates or by alterations in the molecular architecture of internodes and paranodes. This report represents the first study in which the molecular basis of axonal degeneration in the late-onset CMT1B has been explored in human tissue.

Retinoic acid receptor-dependent survival of olfactory sensory neurons in postnatal and adult mice.

To address the hypothesis that retinoids produced by synthesizing enzymes present in the primary olfactory system influence the mouse olfactory sensory map, we expressed a dominant-negative retinoic acid receptor selectively in olfactory sensory neurons. We show that neurons deficient in nuclear retinoid signaling are responsive to odors and form correct odorant receptor-specific axonal projections to target neurons in the olfactory bulb of the brain. Subsequent to the formation of the map, the neurons die prematurely by retrograde-driven caspase-3 activation, which resembles the previously described mechanism of neural death after olfactory bulb ablation. This neurodegenerative event is initiated the second postnatal week and occurs in the adult animal without a compensatory increase of progenitor cell proliferation. In addition, we find that nuclear retinoid signaling is required for the expression of a retinoic acid-degrading enzyme, Cyp26B1, in a small fraction of mature neurons. Collectively, the results provide evidence for a role of locally regulated retinoid metabolism in neuroprotection and in determining population size of neurons at a late stage of neural circuit formation.

Effects of axotomy on telomere length, telomerase activity, and protein in activated microglia.

The adult central nervous system (CNS) is generally thought of as a postmitotic organ. However, DNA labeling studies have shown that one major population of nonneuronal cells, called microglia, retain significant mitotic potential. Microglial cell division is prominent during acute CNS injury involving neuronal damage or death. Prior work from this laboratory has shown that purified microglia maintained in vitro with continual mitogenic stimulation exhibit telomere shortening before entering senescence. In the current study, we sought to investigate whether telomere shortening occurs in dividing microglia in vivo. For this purpose, we used a nerve injury model that is known to trigger localized microglial proliferation in a well-defined CNS region, the facial motor nucleus. Adult Sprague-Dawley rats underwent facial nerve axotomy, and facial motor nuclei were microdissected after 1, 4, 7, and 10 days. Whole tissue samples were subjected to measurements of telomere length, telomerase activity, and telomerase protein. Results revealed a tendency for all of these parameters to be increased in lesioned samples. In addition, microglial cells isolated directly from axotomized facial nuclei with fluorescence-activated cell sorting (FACS) showed increased telomerase activity relative to unoperated controls, suggesting that microglia are the primary cell type responsible for the increases observed in whole tissue samples. Overall, the results show that microglia activated by injury are capable of maintaining telomere length via telomerase during periods of high proliferation in vivo. We conclude that molecular mechanisms pertaining to telomere maintenance are active in the injured CNS.

Hereditary neuropathy with liability to pressure palsy: fulminant development with axonal loss during military training.

Hereditary neuropathy with liability to pressure palsy (HNPP) is characterised by recurrent mononeuropathies following minor trauma. We describe a case of fulminant HNPP beginning on the first day of military physical training. Protracted weakness, muscle atrophy, hand contractures, and multifocal sensory loss developed during a further three weeks of basic training. Nerve conduction changes were typical of HNPP, but without segmental slowing. Electromyographically, there was prominent acute denervation in muscles of the hands and right shoulder. Sural nerve biopsy demonstrated tomaculae and remyelination. Genetic testing revealed PMP-22 gene deletion. This case report demonstrates that HNPP can present with rapidly progressive peripheral nerve dysfunction and electrophysiological evidence of focal axonal loss.

Activation of the c-Jun transcription factor following neurodegeneration in vivo.

Neurodegenerative diseases often result in neuronal cell death, but the molecular mechanisms responsible are not fully understood. The expression and activation by phosphorylation of the c-Jun transcription factor plays an important role for the fate of affected neurons in response to injury. c-Jun is phosphorylated at serines 63 and 73 by the c-Jun N-terminal kinases and c-Jun N-terminal phosphorylation augments the transcriptional activity of c-Jun. Two approaches in neurodegeneration were investigated: The transection of the medial forebrain bundle to study neuronal cell body response in the derived neuronal populations of the substantia nigra pars compacta (SNC). This model of central axotomy leads as a long-term reaction to degeneration of the affected SNC neurons. A central component of the axotomy-induced alterations leading to neuronal degeneration is the rapid induction, lasting expression and activation of the c-Jun transcription factor. The focal cerebral ischemia, induced by occlusion of the arteria cerebri media and the subsequent reperfusion, serves as a suitable in vivo model for stroke. Also, ischemia leads to upregulation and activation of c-Jun and its target genes. Here the key role of c-Jun for the fate of neurons following degeneration is discussed with data received from experiments performed in Manfred Zimmermann's department investigating the effects of c-Jun on its target genes and on factors influencing c-Jun expression and activation.

Axoplasmic importins enable retrograde injury signaling in lesioned nerve.

Axoplasmic proteins containing nuclear localization signals (NLS) signal retrogradely by an unknown mechanism in injured nerve. Here we demonstrate that the importin/karyopherin alpha and beta families underlie this process. We show that importins are found in axons at significant distances from the cell body and that importin beta protein is increased after nerve lesion by local translation of axonal mRNA. This leads to formation of a high-affinity NLS binding complex that traffics retrogradely with the motor protein dynein. Trituration of synthetic NLS peptide at the injury site of axotomized dorsal root ganglion (DRG) neurons delays their regenerative outgrowth, and NLS introduction to sciatic nerve concomitantly with a crush injury suppresses the conditioning lesion induced transition from arborizing to elongating growth in L4/L5 DRG neurons. These data suggest a model whereby lesion-induced upregulation of axonal importin beta may enable retrograde transport of signals that modulate the regeneration of injured neurons.

Lymphocyte regulation of neuropeptide gene expression after neuronal injury.

The neuropeptides vasoactive intestinal peptide (VIP) and pituitary adenylyl cyclase-activating peptide (PACAP) are induced strongly in neurons after several types of injury, and exhibit neuroprotective actions in vitro and in vivo. It is thought that changes in expression of neuropeptides and other molecules in injured neurons are mediated by new factors produced in Schwann and immune cells at the injury site, a loss of target-derived factors, or a combination of mediators. To begin to determine the role of the inflammatory mediators, we investigated axotomy-induced changes in VIP and PACAP gene expression in the facial motor nucleus in severe combined immunodeficient (SCID) mice, and in mice with targeted mutations in specific cytokine genes. In normal mice, VIP and PACAP mRNA was induced strongly in facial motor neurons 4 days after axotomy. The increase in PACAP mRNA was blocked selectively in SCID mice, indicating that mechanisms responsible for VIP and PACAP gene induction are not identical. The loss of PACAP gene expression in SCID mice after axotomy was fully reversed by an infusion of normal splenocytes, suggesting that PACAP mRNA induction requires inflammatory mediators. PACAP and VIP mRNA inductions, however, were maintained in mice lacking leukemia inhibitory factor (LIF) and interleukin-6 (IL-6), and in mice lacking both receptors for tumor necrosis factor alpha (TNFalpha). The data suggest that an inflammatory response, most likely involving T lymphocytes, is necessary for the axotomy-induced increase in PACAP but not in VIP. LIF, IL-6, and TNFalpha, however, are not required for this response to injury.

IL-2 gene knockout affects T lymphocyte trafficking and the microglial response to regenerating facial motor neurons.

Following facial nerve axotomy in mice, T cells cross the intact blood-brain barrier (BBB), home to nerve cell bodies in the facial motor nucleus (FMN), and augment neuroregenerative processes. The pivotal T cell immunoregulatory cytokine, IL-2, appears to have bidirectional effects on neuronal and microglial cell function, suggesting rival hypotheses that IL-2 could either enhance or disrupt processes associated with regeneration of axotomized facial motor neurons. We tested these competing hypotheses by comparing the effect of facial nerve axotomy on C57BL/6-IL-2(-/-) knockout and C57BL/6-IL-2(+/+) wild-type littermates. Since IL-2 may also be produced endogenously in the brain, we also sought to determine whether differences between the knockout and wild-type mice were attributable to loss of IL-2 gene expression in the CNS, loss of peripheral sources of IL-2 and the associated effects on T cell function, or a combination of these factors. To address this question, we bred novel congenic mice with the SCID mutation (mice lacking T cell derived IL-2) that were homozygous for either the IL-2 knockout or wild-type gene alleles (C57BL/6scid-IL-2(-/-) and C57BL/6scid-IL-2(+/+) littermates, respectively). Groups were assessed for differences in (1) T lymphocytes entering the axotomized FMN; (2) perineuronal CD11b(+) microglial phagocytic clusters, a measure of motor neuron death; and (3) activated microglial cells as measured by MHC-II positivity. C57BL/6-IL-2(-/-) knockout mice had significantly higher numbers of T cells and lower numbers of activated MHC-II-positive microglial cells in the regenerating FMN than wild-type littermates, although the number of CD11b(+) phagocytic microglia clusters did not differ. Thus, despite the significant impairment of T cell function known to be associated with loss of peripheral IL-2, the increased number of T cells entering the axotomized FMN appears to have sufficient activity to support neuroregenerative processes. Congenic C57BL/6scid-IL-2(-/-) knockout mice had lower numbers of CD11b(+) microglial phagocytic clusters than congenic C57BL/6scid-IL-2(+/+) wild-type littermates, suggesting that loss of the IL-2 gene in the CNS (and possibly the loss of other unknown sources of the gene) enhanced neuronal regeneration. Further study of IL-2's complex actions in neuronal injury may provide greater understanding of key variables that determine whether or not immunological processes in the brain are proregenerative.

Thalamic retrograde degeneration in the congenitally hydrocephalic rat is attributable to apoptotic cell death.

Congenitally hydrocephalic HTX rats develop ventricular dilatation with extensive damage of the cerebral white matter. Recently, we have reported that neuronal cell death also occurs in the thalamus of HTX rats. To investigate the mechanism underlying this thalamic degeneration in these animals, we carried out a histopathological study of the brain at different phases of postnatal development. Eosinophilic neurons with condensed chromatin or fragmented nuclei were observed in the thalamus from postnatal day 17 onward. The incidence of cell death in the thalamus increased with the progression of hydrocephalus. Ultrastructurally, thalamic neurons occasionally had apoptotic features including nuclear chromatin condensation and marginalization. Immunohistochemically, single-stranded DNA-positive neuronal nuclei were found in the thalamus. They were also positively stained with the TUNEL method. Marked loss of myelin and axons with many TUNEL-positive oligodendrocytes were found in the cerebral white matter. These findings suggest that the neuronal cell death observed in the thalamus in hydrocephalic HTX rats is retrograde degeneration due to extensive damage of axons in the cerebral white matter and that the thalamic retrograde degeneration is attributable to apoptotic cell death.

Inherited neuropathies.

Inherited neuropathies are common and are usually caused by mutations in genes that are expressed by myelinating Schwann cells or neurons, which is the biological basis for long-standing distinction between primary demyelinating and axonal neuropathies. Neuropathies can be isolated, the primary manifestation of a more complex syndrome, or overshadowed by other aspects of the inherited disease. Increasing knowledge of the molecular-genetic causes of inherited neuropathies facilitates faster, more accurate diagnosis, and sets the stage for development of specific therapeutic interventions.

Hereditary spastic paraplegia.

The hereditary spastic paraplegias are a large group of clinically similar disorders. Seventeen different HSP loci have been discovered thus far. Different genetic forms of uncomplicated HSP are clinically very similar. Except for the average age at which symptoms appear, different genetic types of uncomplicated HSP cannot be distinguished reliably by clinical parameters alone. For most subjects, HSP is a diagnosis of exclusion. The differential diagnosis includes treatable disorders as well as those for which the prognosis is quite different from HSP. Even with the emerging availability of laboratory testing for HSP gene mutations, it is still essential that alternative disorders be excluded by careful history, examination, laboratory studies, neuroimaging, and neurophysiologic evaluation. Uncomplicated HSP is due to axonal degeneration at the ends of the longest motor (corticospinal tract) and sensory (dorsal column fibers) in the spinal cord. The observation that some forms begin in childhood and are essentially nonprogressive while other forms begin in adulthood and are slowly progressive raises the possibility that some forms of HSP (e.g.; those associated with LICAM gene mutations and possibly those due to SPG3A mutations) are neurodevelopmental disorders; and other forms are truly neurodegenerative disorders. The mechanisms by which spastin, atlastin, and paraplegin mutations cause axonal degeneration that results in clinically similar forms of HSP are not known. Nonetheless, the identification of these genes and the ability to generate animal models of these forms of HSP will permit direct exploration of the molecular basis of HSP.

The effects of sciatic nerve axotomy on spinal motoneurons in neonatal Bax-deficient mice.

During development, the survival of spinal motoneurons depends on the integrity of the connection to their peripheral targets. Peripheral nerve axotomy induces apoptosis in neonatal neurons supplying axons to the nerve. Bax is known to promote apoptosis among developing neurons. To examine the effect of axotomy on spinal motoneurons in Bax-deficient (Bax-/-) and wild-type neonatal mice (Bax+/+), the sciatic nerve was axotomized on postnatal day (P) 0, and motoneurons in the fourth lumbar (L4) segment were visualized at P7 by acetylcholinesterase (AChE) histochemical staining. Presumably due to the reduction in naturally occurring cell death resulting from the deficiency of Bax, there were about 50% more AChE-positive cells in Bax-/- than in Bax+/+. Motoneurons in the dorsolateral motor pool of L4 project through the sciatic nerve. In Bax+/+, axotomy of the sciatic nerve induced significant cell loss in the pool. Most motoneurons survived such axotomy in Bax-/-, although they appeared atrophic and their AChE expression was decreased. Motoneurons may receive vital support retrogradely from their targets, and loss of such support may lead to hypofunction of spinal motoneurons, as indicated by the reduced production of AChE by axotomized motoneurons and their small size in Bax-/-.

Microarray analysis suggests the involvement of proteasomes, lysosomes, and matrix metalloproteinases in the response of motor neurons to root avulsion.

We used microarray analysis of RNA expression from punch samples from ventral horn of spinal cord to identify alterations in gene expression in motor neurons 3 days after proximal spinal root avulsion, a traumatic injury that results in the death of 80% of the motor neurons. This analysis identified the anticipated increases in expression of genes coding for proteins involved in the apoptosis cascades and abortive cell cycle re-entry, as well as decreases in expression of genes coding for proteins related to neuronal functional activity, including groups of genes related to energy metabolism, transporter proteins, ion channels, and receptors. It was also found that cathepsins, metalloproteinases, and proteasome-related protein products were highly up-regulated in motor neurons following axotomy. Each of these products represent pathways that have been implicated in other models of neuronal damage, but which have not previously been described as a response to axotomy.