Multimodal actions of neural stem cells in a mouse model of ALS: a meta-analysis.

Amyotrophic lateral sclerosis (ALS) is a lethal disease characterized by the unremitting degeneration of motor neurons. Multiple processes involving motor neurons and other cell types have been implicated in its pathogenesis. Neural stem cells (NSCs) perform multiple actions within the nervous system to fulfill their functions of organogenesis and homeostasis. We test the hypothesis that transplanted, undifferentiated multipotent migratory NSCs may help to ameliorate an array of pathological mechanisms in the SOD1(G93A) transgenic mouse model of ALS. On the basis of a meta-analysis of 11 independent studies performed by a consortium of ALS investigators, we propose that transplanted NSCs (both mouse and human) can slow both the onset and the progression of clinical signs and prolong survival in ALS mice, particularly if regions sustaining vital functions such as respiration are rendered chimeric. The beneficial effects of transplanted NSCs seem to be mediated by a number of actions including their ability to produce trophic factors, preserve neuromuscular function, and reduce astrogliosis and inflammation. We conclude that the widespread, pleiotropic, modulatory actions exerted by transplanted NSCs may represent an accessible therapeutic application of stem cells for treating ALS and other untreatable degenerative diseases.

Corticospinal motor neurons and related subcerebral projection neurons undergo early and specific neurodegeneration in hSOD1G9³A transgenic ALS mice.

Amyotrophic lateral sclerosis (ALS) is characterized by predominant vulnerability and central degeneration of both corticospinal/corticobulbar motor neurons (CSMN; "upper motor neurons") in cerebral cortex, and spinal/bulbar motor neurons (SMN; "lower motor neurons") in spinal cord and brainstem. Increasing evidence indicates broader cerebral cortex pathology in cognitive, sensory, and association systems in select cases. It remains unclear whether widely accepted transgenic ALS models, in particular hSOD1(G93A) mice, undergo degeneration of CSMN and molecularly/developmentally closely related populations of nonmotor projection neurons [e.g., other subcerebral projection neurons (SCPN)], and whether potential CSMN/SCPN degeneration is specific and early. This relative lack of knowledge regarding upper motor neuron pathology in these ALS model mice has hindered both molecular-pathophysiologic understanding of ALS and their use toward potential CSMN therapeutic approaches. Here, using a combination of anatomic, cellular, transgenic labeling, and newly available neuronal subtype-specific molecular analyses, we identify that CSMN and related nonmotor SCPN specifically and progressively degenerate in hSOD1(G93A) mice. Degeneration starts quite early and presymptomatically, by postnatal day 30. Other neocortical layers, cortical interneurons, and other projection neuron populations, even within layer V, are not similarly affected. Nonneuronal pathology in neocortex (activated astroglia and microglia) is consistent with findings in human ALS cortex and in affected mouse and human spinal cord. These results indicate previously unknown neuron type-specific vulnerability of CSMN/sensory and association SCPN, and identify that characteristic dual CSMN and SMN degeneration is conserved in hSOD1(G93A) mice. These results provide a foundation for detailed investigation of CSMN/SCPN vulnerability and toward potential CSMN therapeutics in ALS.

Thrombopoietin is ineffective in a mouse model of motor neuron disease.

This study assessed the therapeutic efficacy of thrombopoietin (TPO) in the mouse model of ALS using two treatment paradigms. TPO was administered either daily or in 13-day treatment cycles to SOD1-G93A mice. Quantitative analysis of platelet levels, VEGF and TGF-beta1 trophic factors were assessed. The effect of TPO on disease progression was analyzed by behavioral analysis and clinical examination. TPO treatment increased levels of platelets and TGF-beta1 but not VEGF. This treatment did not affect onset or survival in these mice. Although biologically active, demonstrated by increased platelet and TGF-beta1 levels, rmTPO did not attenuate disease progression in ALS mice.

Ro5-4864 promotes neonatal motor neuron survival and nerve regeneration in adult rats.

The translocator protein (18 kDa; TSPO), formerly known as the peripheral benzodiazepine receptor, is an outer mitochondrial membrane protein that associates with the mitochondrial permeability transition pore to regulate both steroidogenesis and apoptosis. TSPO expression is induced in adult dorsal root ganglion (DRG) sensory neurons after peripheral nerve injury and a TSPO receptor ligand, Ro5-4864, enhances DRG neurite growth in vitro and axonal regeneration in vivo. We have now found that TSPO is induced in neonatal motor neurons after peripheral nerve injury and have evaluated its involvement in neonatal and adult sensory and motor neuron survival, and in adult motor neuron regeneration. The TSPO ligand Ro5-4864 rescued cultured neonatal DRG neurons from nerve growth factor withdrawal-induced apoptosis and protected neonatal spinal cord motor neurons from death due to sciatic nerve axotomy. However, Ro5-4864 had only a small neuroprotective effect on adult facial motor neurons after axotomy, did not delay onset or prolong survival in SOD1 mutant mice, and failed to protect adult DRG neurons from sciatic nerve injury-induced death. In contrast, Ro5-4864 substantially enhanced adult facial motor neuron nerve regeneration and restoration of function after facial nerve axotomy. These data indicate a selective sensitivity of neonatal sensory and motor neurons to survival in response to Ro5-4864, which highlights that survival in injured immature neurons cannot necessarily predict success in adults. Furthermore, although Ro5-4864 is only a very weak promoter of survival in adult neurons, it significantly enhances regeneration and functional recovery in adults.

A glial cell line-derived neurotrophic factor (GDNF):tetanus toxin fragment C protein conjugate improves delivery of GDNF to spinal cord motor neurons in mice.

Glial cell line-derived neurotrophic factor (GDNF) has shown robust neuroprotective and neuroreparative activities in various animal models of Parkinson's Disease or amyotrophic lateral sclerosis (ALS). The successful use of GDNF as a therapeutic in humans, however, appears to have been hindered by its poor bioavailability to target neurons in the central nervous system (CNS). To improve delivery of exogenous GDNF protein to CNS motor neurons, we employed chemical conjugation techniques to link recombinant human GDNF to the neuronal binding fragment of tetanus toxin (tetanus toxin fragment C, or TTC). The predominant species present in the purified conjugate sample, GDNF:TTC, had a molecular weight of approximately 80 kDa as determined by non-reducing SDS-PAGE. Like GDNF, addition of GDNF:TTC to culture media of neuroblastoma cells expressing GFRalpha-1/c-RET produced a dose-dependent increase in cellular phospho-c-RET levels. Treatment of cultured midbrain dopaminergic neurons with either GDNF or the conjugate similarly promoted both DA neuron survival and neurite outgrowth. However, in contrast to mice treated with GDNF by intramuscular injection, mice receiving GDNF:TTC revealed intense GDNF immunostaining associated with spinal cord motor neurons in fixed tissue sections. That GDNF:TTC provided neuroprotection of axotomized motor neurons in neonatal rats further revealed that the conjugate retained its GDNF activity in vivo. These results indicate that TTC can serve as a non-viral vehicle to substantially improve the delivery of functionally active growth factors to motor neurons in the mammalian CNS.

Mice deficient in the Rab5 guanine nucleotide exchange factor ALS2/alsin exhibit age-dependent neurological deficits and altered endosome trafficking.

ALS2/alsin is a member of guanine nucleotide exchange factors for the small GTPase Rab5 (Rab5GEFs), which act as modulators in endocytic pathway. Loss-of-function mutations in human ALS2 account for a number of juvenile recessive motor neuron diseases (MNDs). However, the normal physiological role of ALS2 in vivo and the molecular mechanisms underlying motor dysfunction are still unknown. To address these issues, we have generated mice homozygous for disruption of the Als2 gene. The Als2-null mice observed through 21 months of age demonstrated no obvious developmental, reproductive or motor abnormalities. However, immunohistochemical and electrophysiological analyses identified an age-dependent, slowly progressive loss of cerebellar Purkinje cells and disturbance of spinal motor neurons associated with astrocytosis and microglial cell activation, indicating a subclinical dysfunction of motor system in Als2-null mice. Further, quantitative epidermal growth factor (EGF)-uptake analysis identified significantly smaller-sized EGF-positive endosomes in Als2-null fibroblasts, suggesting an alteration of endosome/vesicle trafficking in the cells. Collectively, while loss of ALS2 does not produce a severe disease phenotype in mice, these Als2-null animals should provide a useful model with which to understand the interplay between endosomal dynamics and the long-term viability of large neurons such as Purkinje cells and spinal motor neurons.

Tetanus toxin fragment C fusion facilitates protein delivery to CNS neurons from cerebrospinal fluid in mice.

To improve protein delivery to the CNS following intracerebroventricular administration, we compared the distribution of a human Cu/Zn superoxide dismutase:tetanus toxin fragment C fusion protein (SOD1:TTC) in mouse brain and spinal cord with that of tetanus toxin fragment C (TTC) or human SOD1 (hSOD1) alone, following continuous infusion into the lateral ventricle. Mice infused with TTC or SOD1:TTC showed intense anti-TTC or anti-hSOD1 labeling, respectively, throughout the CNS. In contrast, animals treated with hSOD1 revealed moderate staining in periventricular tissues. In spinal cord sections from animals infused with SOD1:TTC, the fusion protein was found in neuron nuclear antigen-positive (NeuN+) neurons and not glial fibrillary acidic protein-positive (GFAP+) astrocytes. The percentage of NeuN+ ventral horn cells that were co-labeled with hSOD1 antibody was greater in mice treated with SOD1:TTC (cervical cord = 73 +/- 8.5%; lumbar cord = 62 +/- 7.7%) than in mice treated with hSOD1 alone (cervical cord = 15 +/- 3.9%; lumbar cord = 27 +/-4.7%). Enzyme-linked immunosorbent assay for hSOD1 further demonstrated that SOD1:TTC-infused mice had higher levels of immunoreactive hSOD1 in CNS tissue extracts than hSOD1-infused mice. Following 24 h of drug washout, tissue extracts from SOD1:TTC-treated mice still contained substantial amounts of hSOD1, while extracts from hSOD1-treated mice lacked detectable hSOD1. Immunoprecipitation of SOD1:TTC from these extracts using anti-TTC antibody revealed that the recovered fusion protein was structurally intact and enzymatically active. These results indicate that TTC may serve as a useful prototype for development as a non-viral vehicle for improving delivery of therapeutic proteins to the CNS.

How do adult neurons survive?

Extract: Apoptotic cell death, also known as cell suicide or programmed cell death, is a series of intracellular biochemical steps that lead to a cell's controlled but inevitable death. Apoptosis plays a crucial role in the normal development of the embryonic nervous system. Many developing neurons are destined to die by apoptosis unless they are "rescued" by their exposure to growth factors that shut off the cell suicide program, enabling their survival. Naturally too many neurons are generated, forcing them to compete for a limited supply of critical growth factors. Only the "fittest" survive -- those that make the right connections at the right time. This enables the survival of only those neurons needed for the appropriate formation and function of the nervous system; surplus neurons are discarded, creating order by cleaning out what is not needed. This is a seemingly wasteful, but effective strategy for setting up the complex and intricate circuits of the nervous system. In stark contrast, the neurons in the adult do not divide and are irreplaceably lost once they are dead, so they need to survive for the entire lifetime of the organism. Their premature death can lead to irreversible functional deficits that underlie many neurodegenerative diseases. Mature neurons possess, through multiple inherent or intrinsic molecular mechanisms, the ability to control or repress inadvertent activation of the cell suicide program that lies dormant within every cell.

Adult neuron survival strategies--slamming on the brakes.

Developing neurons are programmed to die by an apoptotic pathway unless they are rescued by extrinsic growth factors that generate an anti-apoptotic response. By contrast, adult neurons need to survive for the lifetime of the organism, and their premature death can cause irreversible functional deficits. The default apoptotic pathway is shut down when development is complete, and consequently growth factors are no longer required to prevent death. To protect against accidental apoptotic cell death, anti-apoptotic mechanisms are activated in mature neurons in response to stress. Loss or reduced activity of these intrinsic anti-apoptotic 'brakes' might contribute to or accelerate neurodegeneration, whereas their activation might rescue neurons from injury or genetic abnormalities.

Hsp27 upregulation and phosphorylation is required for injured sensory and motor neuron survival.

Peripheral nerve transection results in the rapid death by apoptosis of neonatal but not adult sensory and motor neurons. We show that this is due to induction and phosphorylation in all adult axotomized neurons of the small heat shock protein Hsp27 and the failure of such induction in most neonatal neurons. In vivo delivery of human Hsp27 but not a nonphosphorylatable mutant prevents neonatal rat motor neurons from nerve injury-induced death, while knockdown in vitro and in vivo of Hsp27 in adult injured sensory neurons results in apoptosis. Hsp27's neuroprotective action is downstream of cytochrome c release from mitochondria and upstream of caspase-3 activation. Transcriptional and posttranslational regulation of Hsp27 is necessary for sensory and motor neuron survival following peripheral nerve injury.