Heterogeneity of Baló's concentric sclerosis: a study of eight cases with different therapeutic concepts.

BACKGROUND: Baló's Concentric Sclerosis (BCS) is a rare heterogeneous demyelinating disease with a variety of phenotypes on Magnetic Resonance Imaging (MRI). Existing literature lacks data especially on the therapeutic approach of the disease which we intended to elucidate by means of suggesting a new possible BCS classification and introducing different therapeutic concepts based on each BCS-subgroup characteristics. METHODS: We present a retrospective study of eight treated patients with BCS-type lesions, emphasizing on MRI characteristics and differences on therapeutic maneuvers. RESULTS: Data analysis showed: at disease onset the BCS-type lesion was tumefactive (size ≥2 cm) in 6 patients, with a mean size of 2.7 cm (± 0.80 SD); a coexistence of MS-like plaques on brain MRI was identified in 7 patients of our cohort. The mean age was 26.3 years (±7.3 SD) at disease onset and the mean follow-up period was 56.8 months (range 9-132 months). According to radiological characteristics and response to therapies, we further categorized them into 3 subgroups: a) Group-1; BCS with or without coexisting nonspecific white matter lesions; poor response to intravenous methylprednisolone (IVMP); treated with high doses of immunosuppressive agents (4 patients), b) Group-2; BCS with typical MS lesions; good response to IVMP; treated with MS-disease modifying therapies (2 patients), c) Group-3; BCS with typical MS lesions; poor response to IVMP; treated with rituximab (2 patients). CONCLUSIONS: Our study introduces a new insight regarding the categorization of BCS into three subgroups depending on radiological features at onset and during the course of the disease, in combination with the response to different immunotherapies. Immunosuppressive agents such as cyclophosphamide are usually effective in BCS. However, therapeutic alternatives like anti-CD20 monoclonal antibodies or more classical disease-modifying MS therapies can be considered when BCS has also mixed lesions similar to MS. Future studies with a larger sample size are necessary to further establish these findings, thus leading to better treatment algorithms and improved clinical outcomes.

A case of Alemtuzumab-induced neutropenia in multiple sclerosis in association with the expansion of large granular lymphocytes.

BACKGROUND: Alemtuzumab has been demonstrated to reduce the risks of relapse and accumulation of sustained disability in Multiple Sclerosis (MS) patients compared to ß-interferon. It acts against CD52, leading primarily to lymphopenia. Recent data have shown that mild neutropenia is observed in 16% of treated MS-patients whereas severe neutropenia occurred in 0.6%. CASE PRESENTATION: Herein, we present the case of a 34-year-old woman with relapsing-remitting MS, with a history of treatment with glatiramer acetate and natalizumab, who subsequently received Alemtuzumab (12 mg / 24 h × 5 days). 70-days after the last Alemtuzumab administration, the patient displayed neutropenia (500 neutrophils/µL) with virtual absence of B-cells (0.6% of total lymphocytes), low values of CD4-T-cells (6.6%) and predominance of CD8-T-cells (48%) and NK-cells (47%); while large granular lymphocytes (LGL) predominated in the blood-smear examination. Due to prolonged neutropenia (5-days) the patient was placed on low-dose corticosteroids leading to sustained remission. CONCLUSION: This is the first case of a patient with relapsing-remitting MS with neutropenia two months post-Alemtuzumab, with simultaneous presence of LGL cells in the blood and a robust therapeutic response to prednisolone. We recommend testing with a complete blood count every 15 days in the first 3 months after the 1st Alemtuzumab administration and searching for large granular lymphocytes cell expansion on microscopic examination of the peripheral blood if neutropenia develops.

The frontal assessment battery is not useful to discriminate progressive supranuclear palsy from frontotemporal dementias.

BACKGROUND: The frontal assessment battery (FAB) has been suggested as a useful tool in the differential diagnosis of progressive supranuclear palsy (PSP) from Parkinson's disease (PD) and multiple system atrophy with parkinsonism (MSA-P). However, the utility of the FAB in the differential diagnosis of PSP from frontotemporal dementia (FTD) phenotypes is still under research. METHODS: We performed the FAB, in a multi-centre cohort of 70 PSP, 103 FTD (N = 84 behavioral variant FTD, N = 10 semantic dementia, N = 9 progressive non-fluent aphasia), 26 PD and 11 MSA-P patients, diagnosed according to established criteria. Patients were also rated with the mini mental state examination and motor scales. RESULTS: The FAB total score showed a poor discriminatory power between PSP and FTD as a group [area under the curve (AUC) = 0.523]. Moreover, the FAB score showed no correlation with disease duration in PSP (r = 0.05) or FTD group (r = 0.04). In contrast, we confirmed that the FAB is clinically useful to differentiate PSP from PD and MSA-P (AUC = 0.927). In fact, the sum of two FAB subscores together (verbal fluency and Luria motor series) were as good as the total score in differentiating PSP from PD and MSA-P (AUC = 0.957). CONCLUSIONS: The FAB may not be a useful tool to differentiate PSP from FTDs, and shows no correlation with disease duration in these disorders. On the other hand, the essential information to differentiate PSP from PD and MSA-P is contained in the sum of only two FAB subscores. This should be taken into consideration in both clinical practice and the planning of clinical trials.

Rare Late-Onset Presentation of Glutaric Aciduria Type I in a 16-Year-Old Woman with a Novel GCDH Mutation.

Glutaric acidemia type I (GA-I) is a treatable autosomal recessive disorder of lysine, hydroxylysine, and tryptophan metabolism caused by glutaryl-CoA dehydrogenase (GCDH) deficiency. Presentation and progression of disease are variable ranging from asymptomatic carrier state to catastrophic encephalopathy. GA-I usually presents before age 18 months, usually triggered by childhood infection, with mild or severe acute encephalopathy, striatal degeneration, and movement disorder, most often acute dystonia. At a presymptomatic stage diagnosis is suggested clinically by macrocephaly, radiologically by widened Sylvian fissures and biochemically by the presence of excess 3-hydroxyglutaric acid and glutaric acid in urine. Treatment consists of lysine-restricted diet and carnitine supplementation, specific diet restrictions, as well as symptomatic and anticatabolic treatment of intercurrent illness. Presymptomatic diagnosis and treatment are essential to prognosis. We report the case of 16-year-old macrocephalic female with late-onset GA-I and unusual paucisymptomatic presentation with fainting after exercise and widespread white matter signal changes at MRI. She was compound heterozygote for a novel mutation (IVS10-2A>G) affecting splicing at GCDH and a common missense mutation (c. 1240C>T; p.Arg402Trp, R402W). Interestingly, the site of the novel mutation is the nucleotide position of a common mutation found almost exclusively in patients of Chinese/Taiwanese origin (IVS10-2A>C).

Protective role of olesoxime against wild-type α-synuclein-induced toxicity in human neuronally differentiated SHSY-5Y cells.

BACKGROUND AND PURPOSE: Parkinson's disease (PD) is usually diagnosed clinically from classical motor symptoms, while definitive diagnosis is made postmortem, based on the presence of Lewy bodies and nigral neuron cell loss. α-Synuclein (ASYN), the main protein component of Lewy bodies, clearly plays a role in the neurodegeneration that characterizes PD. Additionally, mutation in the SNCA gene or copy number variations are associated with some forms of familial PD. Here, the objective of the study was to evaluate whether olesoxime, a promising neuroprotective drug can prevent ASYN-mediated neurotoxicity. EXPERIMENTAL APPROACH: We used here a novel, mechanistically approachable and attractive cellular model based on the inducible overexpression of human wild-type ASYN in neuronally differentiated human neuroblastoma (SHSY-5Y) cells. This model demonstrates gradual cellular degeneration, coinciding temporally with the appearance of soluble and membrane-bound ASYN oligomers and cell death combining both apoptotic and non-apoptotic pathways. KEY RESULTS: Olesoxime fully protected differentiated SHSY-5Y cells from cell death, neurite retraction and cytoplasmic shrinkage induced by moderate ASYN overexpression. This protection was associated with a reduction in cytochrome c release from mitochondria and caspase-9 activation suggesting that olesoxime prevented ASYN toxicity by preserving mitochondrial integrity and function. In addition, olesoxime displayed neurotrophic effects on neuronally differentiated SHSY-5Y cells, independent of ASYN expression, by promoting their differentiation. CONCLUSIONS AND IMPLICATIONS: Because ASYN is a common underlying factor in many cases of PD, olesoxime could be a promising therapy to slow neurodegeneration in PD.

Genetic assessment of familial and early-onset Parkinson's disease in a Greek population.

BACKGROUND AND PURPOSE: Although the first mutation associated with Parkinson's disease (PD) was identified several years ago in the alpha-synuclein (SNCA) gene in families of Greek and Italian ancestry, a more systematic study of this and other known PD mutations has not been performed in the Greek population. METHODS: A genetic analysis in 111 familial or sporadic with early-onset (≤50 years, EO) PD patients was performed for the presence of the A53T SNCA mutation. In separate subgroups of these patients, further mutations in the SNCA, LRRK2, Parkin, PINK1 and DJ-1 genes were searched for. Additionally, a subgroup of familial cases was analysed for mutations in the glucocerebrosidase (GBA) gene. RESULTS: In total, five patients (4.5% of our whole population) were identified with the A53T SNCA mutation, two with a heterozygote dosage mutation and one with a heterozygote point mutation in the Parkin gene, and seven patients (10.3% of our familial cohort) with GBA gene mutations. CONCLUSIONS: The A53T mutation in the SNCA gene, although uncommon, does represent a cause of PD in the Greek population, especially of familial EOPD with autosomal dominant inheritance. GBA mutations in the familial cohort tested here were as common as in a cohort of sporadic cases previously examined from the same centres. For the remainder of the genes, genetic defects that could definitively account for the disease were not identified. These results suggest that further Mendelian traits that lead to PD in the Greek population remain to be identified.

Multicenter external validation of the ABCD2 score in triaging TIA patients.

OBJECTIVES: A simple clinical score (ABCD(2) score) has been introduced to triage TIA patients with a high early risk of stroke. External validation studies have yielded inconsistent results regarding the predictive ability of the ABCD(2) score. We aimed to prospectively validate the former score in a multicenter case series study. METHODS: We prospectively calculated the ABCD(2) score (age [> or = 60 years: 1 point]; blood pressure [systolic >140 mm Hg or diastolic >90 mm Hg: 1[; clinical features [unilateral weakness: 2, speech disturbance without weakness: 1, other symptom: 0]; duration of symptoms [ <10 minutes: 0, 10-59 minutes: 1, > or = 60 minutes: 2]; diabetes mellitus [yes: 1]) in consecutive TIA patients hospitalized in 3 tertiary care neurology departments across 2 different racial populations (white and Asian). RESULTS: The 7-day and 90-day risks of stroke in the present case series (n = 148) were 8% (95% CI 4%-12%) and 16% (95% CI 10%-22%). The ABCD(2) score accurately discriminated between TIA patients with high 7-day (c statistic 0.72, 95% CI 0.57-0.88) and 90-day (c statistic 0.75, 95% CI 0.65-0.86) risks of stroke. The 90-day risk of stroke was 7-fold higher in patients with an ABCD(2) score >3 points (28%, 95% CI 18%-38%) than in patients with an ABCD(2) score < or = 3 points (4%, 95% CI 0%-9%). After adjustment for stroke risk factors, race, history of previous TIA, medication use before the index TIA and secondary prevention treatment strategies, an ABCD(2) score of >2 was associated with a nearly 5-fold greater 90-day risk of stroke (hazard ratio 4.65, 95% CI 1.04-20.84, p = 0.045). CONCLUSION: Our findings externally validate the usefulness of the ABCD(2) score in triaging TIA patients with a high risk of early stroke in a multiethnic sample of hospitalized patients. The present data support current guidelines endorsing the immediate hospitalization of patients with an ABCD(2) score >2.

RAIDD is required for apoptosis of PC12 cells and sympathetic neurons induced by trophic factor withdrawal.

Caspase 2 has been implicated in trophic deprivation-induced neuronal death. We have shown that overexpression of the caspase 2-binding protein RAIDD induces neuronal apoptosis, acting synergistically with trophic deprivation. Currently, we examine the role of endogenous RAIDD in apoptosis of PC12 cells and sympathetic neurons. Expression of a truncated caspase recruitment domain-only form of caspase 2, which presumably disrupts the RAIDD interaction with endogenous caspase 2, attenuated trophic deprivation-induced apoptosis. Furthermore, downregulation of RAIDD by small interfering RNA led to inhibition of trophic deprivation-induced death, whereas death induced by DNA damage, which is not caspase 2-mediated, was not inhibited. Therefore, RAIDD, likely through interaction with caspase 2, is involved in trophic deprivation-induced neuronal apoptosis. This is the first demonstration of the involvement of RAIDD in apoptosis, and provides further support for the idea that apoptotic pathways in the same system may differ depending on the initiating stimulus.

Apoptosis and the conformational change of Bax induced by proteasomal inhibition of PC12 cells are inhibited by bcl-xL and bcl-2.

The function of the proteasome has been linked to various pathologies, including cancer and neurodegeneration. Proteasomal inhibition can lead to death in a variety of cell types, however the manner in which this occurs is unclear, and may depend on the particular cell type. In this work we have extended previous findings pertaining to the effects of pharmacological proteasomal inhibitors on PC12 cells, by examining in more detail the induced death pathway. We find that cell death is apoptotic by ultrastructural criteria. Caspase 9 and 3 are processed, cytochrome c is released from the mitochondria and a dominant negative form of caspase 9 prevents death. Furthermore, Bax undergoes a conformational change and is translocated to the mitochondria in a caspase-independent fashion. Total cell levels of Bax however do not change, whereas levels of the BH3-only protein Bim increase with proteasomal inhibition. Transient overexpression of bcl-xL or, to a lesser extent, of bcl-2, significantly decreased apoptotic death and prevented Bax conformational change. We conclude that death elicited by proteasomal inhibition of PC12 cells follows a classical "intrinsic" pathway. Significantly, antiapoptotic bcl-2 family members prevent apoptosis by inhibiting Bax conformational change. Increased levels of Bim may contribute to cell death in this model.

Caspase-dependent and -independent neuronal death: two distinct pathways to neuronal injury.

Caspases are cysteine proteases that mediate apoptotic death in a variety of cellular systems, including neurons. Caspases are activated through extrinsic or intrinsic pathways. The latter is used by most neurons in most situations. In this pathway, release of mitochondrial cytochrome c into the cytoplasm induces formation of the apoptosome, which leads to the activation of caspase 9 and subsequently other caspases. Recent data demonstrate that when caspase activation is inhibited at or downstream of the apoptosome, neurons undergo a delayed, caspase-independent death. Furthermore, there are instances, most notably following excitotoxic injury and calcium overload, in which the direct cell death pathway elicited differs from classical apoptosis. The molecular and biochemical features of such caspase-independent, nonapoptotic forms of neuronal death are just beginning to be elucidated, but alterations at the level of the mitochondria and noncaspase proteases play significant roles. Mitochondrial alterations in caspase-independent death may include energy depletion, generation of free radicals, opening of the permeability transition pore, and release of cytotoxic proteins, such as apoptosis-inducing factor. The particular mechanisms employed can be context dependent. In disease states, in which a combination of apoptotic and nonapoptotic death occurs, therapeutic strategies need to take into account both caspase-dependent and -independent pathways.

RAIDD aggregation facilitates apoptotic death of PC12 cells and sympathetic neurons.

In human cell lines, the caspase 2 adaptor RAIDD interacts selectively with caspase 2 through its caspase recruitment domain (CARD) and leads to caspase 2-dependent death. Whether RAIDD induces such effects in neuronal cells is unknown. We have previously shown that caspase 2 is essential for apoptosis of trophic factor-deprived PC12 cells and rat sympathetic neurons. We report here that rat RAIDD, cloned from PC12 cells, interacts with rat caspase 2 CARD. RAIDD overexpression induced caspase 2 CARD- and caspase 9-dependent apoptosis of PC12 cells and sympathetic neurons. Apoptosis correlated with the formation of discrete perinuclear aggregates. Both death and aggregates required the expression of full-length RAIDD. Such aggregates may enable more effective activation of caspase 2 through close proximity. Following trophic deprivation, RAIDD overexpression increased death and aggregate formation. Therefore, RAIDD aggregation is important for its death-promoting effects and may play a role in trophic factor withdrawal-induced neuronal apoptosis.

Ubiquitinated inclusions and neuronal cell death.

Ubiquitinated inclusions and selective neuronal cell death are considered the pathological hallmarks of Parkinson's disease and other neurodegenerative diseases. Recent genetic, pathological and biochemical evidence suggests that dysfunction of ubiquitin-dependent protein degradation by the proteasome might be a contributing, if not initiating factor in the pathogenesis of these diseases. In neuronal cell culture models inhibition of the proteasome leads to cell death and formation of fibrillar ubiquitin and alpha-synuclein-positive inclusions, thus modeling some aspects of Lewy body diseases. The processes of inclusion formation and neuronal cell death share some common mechanisms, but can also be dissociated at a certain level.

Inhibitors of trypsin-like serine proteases prevent DNA damage-induced neuronal death by acting upstream of the mitochondrial checkpoint and of p53 induction.

We have previously shown that the pharmacological agents 4-(2-aminoethyl)=benzenesulfonylfluoride hydrochloride (AEBSF) and Na-p-tosyl-L-lysine chloromethylketone (TLCK), inhibitors of trypsin-like serine proteases, prevent the death of trophic factor-deprived PC12 cells and sympathetic neurons. Both AEBSF and TLCK inhibit caspase activation in this model, but it is unclear whether they do so indirectly or through a direct effect at the level of the caspases. In the current study, we have used these agents in another model of neuronal death that is induced by DNA damage. We find that both agents delay the death of DNA-damaged PC12 cells, neonatal rat sympathetic neurons and embryonic rat cortical neurons. As in the trophic deprivation model, they act upstream of the caspases. In addition, they prevent mitochondrial alterations, such as cytochrome c release or loss of transmembrane potential. In contrast, the general caspase inhibitor bok-asp-fmk does not prevent cytochrome c release and has only a partial and transient effect on loss of transmembrane potential. Interestingly, both AEBSF and TLCK prevent the induction and nuclear accumulation of p53 that is induced by DNA damage in cortical neurons. Therefore, these serine protease inhibitors act at a point upstream in the apoptotic pathway, prior to p53 induction and the mitochondrial checkpoint, to delay neuronal death in this model, and do not act at the level of the caspases. We conclude that therapeutic strategies based on serine protease inhibition may be useful in preventing neuronal cell death.

Expression of A53T mutant but not wild-type alpha-synuclein in PC12 cells induces alterations of the ubiquitin-dependent degradation system, loss of dopamine release, and autophagic cell death.

Alpha-synuclein mutations have been identified in certain families with Parkinson's disease (PD), and alpha-synuclein is a major component of Lewy bodies. Other genetic data indicate that the ubiquitin-dependent proteolytic system is involved in PD pathogenesis. We have generated stable PC12 cell lines expressing wild-type or A53T mutant human alpha-synuclein. Lines expressing mutant but not wild-type alpha-synuclein show: (1) disruption of the ubiquitin-dependent proteolytic system, manifested by small cytoplasmic ubiquitinated aggregates and by an increase in polyubiquitinated proteins; (2) enhanced baseline nonapoptotic death; (3) marked accumulation of autophagic-vesicular structures; (4) impairment of lysosomal hydrolysis and proteasomal function; and (5) loss of catecholamine-secreting dense core granules and an absence of depolarization-induced dopamine release. Such findings raise the possibility that the primary abnormality in these cells may involve one or more deficits in the lysosomal and/or proteasomal degradation pathways, which in turn lead to loss of dopaminergic capacity and, ultimately, to death. These cells may serve as a model to study the effects of aberrant alpha-synuclein on dopaminergic cell function and survival.

Caspase inhibition: a potential therapeutic strategy in neurological diseases.

Caspases are intracellular proteases that participate in apoptotic pathways in mammalian cells, including neurons. Here we review evidence that caspase inhibition, through pharmacological or molecular means, may inhibit neuronal cell death in a number of in vitro and in vivo models of neurological disease. It has recently become clear that, at least in most cell culture models, caspase inhibition offers only transient protection, and that a caspase-independent death eventually occurs. This may be due to irreversible caspase-independent alterations at the level of the mitochondria. Despite concerns that targeting caspases alone may prove insufficient to truly reverse the effects of various death stimuli, in vivo studies indicate that caspase inhibition promotes survival and functional outcome in a variety of neurological disease models. In addition, studies of human post-mortem material suggest that caspases are activated in certain human neurological diseases. Caspase inhibition may therefore provide a novel strategy for the treatment of such disorders. Caspases, through the generation of toxic fragments of critical protein substrates, may also be involved in earlier steps of neuronal dysfunction, such as protein aggregation in Huntington's and Alzheimer's disease, and therefore caspase inhibition may be of additional value in the treatment of these particular disorders.

Proteasomal inhibition leads to formation of ubiquitin/alpha-synuclein-immunoreactive inclusions in PC12 cells.

Proteasomal dysfunction has been recently implicated in the pathogenesis of several neurodegenerative diseases, including Parkinson's disease and diffuse Lewy body disease. We have developed an in vitro model of proteasomal dysfunction by applying pharmacological inhibitors of the proteasome, lactacystin or ZIE[O-tBu]-A-leucinal (PSI), to dopaminergic PC12 cells. Proteasomal inhibition caused a dose-dependent increase in death of both naive and neuronally differentiated PC12 cells, which could be prevented by caspase inhibition or CPT-cAMP. A percentage of the surviving cells contained discrete cytoplasmic ubiquitinated inclusions, some of which also contained synuclein-1, the rat homologue of human alpha-synuclein. However the total level of synuclein-1 was not altered by proteasomal inhibition. The ubiquitinated inclusions were present only within surviving cells, and their number was increased if cell death was prevented. We have thus replicated, in this model system, the two cardinal pathological features of Lewy body diseases, neuronal death and the formation of cytoplasmic ubiquitinated inclusions. Our findings suggest that inclusion body formation and cell death may be dissociated from one another.

Cyclin-dependent kinases and P53 pathways are activated independently and mediate Bax activation in neurons after DNA damage.

DNA damage has been implicated as one important initiator of cell death in neuropathological conditions such as stroke. Accordingly, it is important to understand the signaling processes that control neuronal death induced by this stimulus. Previous evidence has shown that the death of embryonic cortical neurons treated with the DNA-damaging agent camptothecin is dependent on the tumor suppressor p53 and cyclin-dependent kinase (CDK) activity and that the inhibition of either pathway alone leads to enhanced and prolonged survival. We presently show that p53 and CDKs are activated independently on parallel pathways. An increase in p53 protein levels, nuclear localization, and DNA binding that result from DNA damage are not affected by the inhibition of CDK activity. Conversely, no decrease in retinoblastoma protein (pRb) phosphorylation was observed in p53-deficient neurons that were treated with camptothecin. However, either p53 deficiency or the inhibition of CDK activity alone inhibited Bax translocation, cytochrome c release, and caspase-3-like activation. Taken together, our results indicate that p53 and CDK are activated independently and then act in concert to control Bax-mediated apoptosis.

Synuclein-1 is selectively up-regulated in response to nerve growth factor treatment in PC12 cells.

Mutations in the alpha-synuclein gene have recently been identified in families with inherited Parkinson's disease and the protein product of this gene is a component of Lewy bodies, indicating that alpha-synuclein is involved in Parkinson's disease pathogenesis. A role for normal alpha-synuclein in synaptic function, apoptosis or plasticity responses has been suggested. We show here that in rat pheochromocytoma PC12 cells synuclein-1, the rat homolog of human alpha-synuclein, is highly and selectively up-regulated at the mRNA and protein levels after 7 days of nerve growth factor treatment. Synuclein-1 expression appears neither sufficient nor necessary for the neuritic sprouting that occurs within 1-2 days of nerve growth factor treatment. Rather, it likely represents a component of a late neuronal maturational response. Synuclein-1 redistributes diffusely within the cell soma and the neuritic processes in nerve growth factor-treated PC12 cells. Cultured neonatal rat sympathetic neurones express high levels of synuclein-1, with a diffuse intracellular distribution, similar to neuronal PC12 cells. These results suggest that levels of synuclein-1 may be regulated by neurotrophic factors in the nervous system and reinforce a role for alpha-synuclein in plasticity-maturational responses. In contrast, there is no correlation between synuclein expression and apoptotic death following trophic deprivation.

Involvement of caspase 3 in apoptotic death of cortical neurons evoked by DNA damage.

Previous reports have shown that DNA-damage-evoked death of embryonic cortical neurons is delayed by general caspase inhibitors and is accompanied by an increase in DEVD-AFC cleavage activity. We show here that this cleavage activity is lacking in camptothecin-treated caspase 3-deficient neurons. Moreover, we report that death of camptothecin-treated caspase 3-deficient neurons cultured from E16 embryos is delayed and that no significant increase in survival is observed with cotreatment with the general caspase inhibitor BAF. These results indicate that caspase-dependent death of camptothecin-treated cortical neurons requires caspase 3 activity. The delay in death is accompanied by impairment of DNA fragmentation. However, Bax-dependent cytochrome c release still occurs in camptothecin-treated caspase 3-deficient cortical neurons. Accordingly, we hypothesize that the delayed death which occurs in the absence of caspase 3 activity may be due to mitochondrial dysfunction. Finally, we show that the delay in death observed with E16 caspase 3-deficient neurons does not occur in neurons cultured from E19 embryos. This suggests that the requirement for caspase 3 in death of neurons evoked by DNA damage may differ depending upon the developmental state of the cell.

Delaying caspase activation by Bcl-2: A clue to disease retardation in a transgenic mouse model of amyotrophic lateral sclerosis.

Molecular mechanisms of apoptosis may participate in motor neuron degeneration produced by mutant copper/zinc superoxide dismutase (mSOD1), the only proven cause of amyotrophic lateral sclerosis (ALS). Consistent with this, herein we show that the spinal cord of transgenic mSOD1 mice is the site of the sequential activation of caspase-1 and caspase-3. Activated caspase-3 and its produced beta-actin cleavage fragments are found in apoptotic neurons in the anterior horn of the spinal cord of affected transgenic mSOD1 mice; although such neurons are few, their scarcity should not undermine the potential importance of apoptosis in the overall mSOD1-related neurodegeneration. Overexpression of the anti-apoptotic protein Bcl-2 attenuates neurodegeneration and delays activation of the caspases and fragmentation of beta-actin. These data demonstrate that caspase activation occurs in this mouse model of ALS during neurodegeneration. Our study also suggests that modulation of caspase activity may provide protective benefit in the treatment of ALS, a view that is consistent with our recent demonstration of caspase inhibition extending the survival of transgenic mSOD1 mice.