Apoptotic Factors and Mitochondrial Complexes Assist Determination of Strain-Specific Susceptibility of Mice to Parkinsonian Neurotoxin MPTP.

Identification of genetic mutations in Parkinson's disease (PD) promulgates the genetic nature of disease susceptibility. Resilience-associated genes being unknown till date, the normal genetic makeup of an individual may be determinative too. Our earlier studies comparing the substantia nigra (SN) and striatum of C57BL/6J, CD-1 mice, and their F1-crossbreds demonstrated the neuroprotective role of admixing against the neurotoxin MPTP. Furthermore, the differences in levels of mitochondrial fission/fusion proteins in the SN of parent strains imply effects on mitochondrial biogenesis. Our present investigations suggest that the baseline levels of apoptotic factors Bcl-2, Bax, and AIF differ across the three strains and are differentially altered in SN following MPTP administration. The reduction in complex-I levels exclusively in MPTP-injected C57BL/6J reiterates mitochondrial involvement in PD pathogenesis. The MPTP-induced increase in complex-IV, in the nigra of both parent strains, may be compensatory in nature. The ultrastructural evaluation showed fairly preserved mitochondria in the dopaminergic neurons of CD-1 and F1-crossbreds. However, in CD-1, the endoplasmic reticulum demonstrated distinct luminal enlargement, bordering onto ballooning, suggesting proteinopathy as a possible initial trigger.The increase in α-synuclein in the pars reticulata of crossbreds suggests a supportive role for this output nucleus in compensating for the lost function of pars compacta. Alternatively, since α-synuclein over-expression occurs in different brain regions in PD, the α-synuclein increase here may suggest a similar pathogenic outcome. Further understanding is required to resolve this biological contraption. Nevertheless, admixing reduces the risk to MPTP by favoring anti-apoptotic consequences. Similar neuroprotection may be envisaged in the admixed populace of Anglo-Indians.

Fibrinogen and Complement Factor H Are Promising CSF Protein Biomarkers for Parkinson's Disease with Cognitive Impairment─A Proteomics-ELISA-Based Study.

Parkinson's disease (PD) with cognitive impairment (PDCI) is essentially diagnosed through clinical and neuropsychological examinations. There is a need to identify biomarkers to foresee cognitive decline in them. We performed label-free unbiased nontargeted proteomics (Q-TOF LC/MS-MS) on the CSF of non-neurological control; PDCI; PD; and normal pressure hydrocephalus (NPH) patients, followed by targeted ELISA for validation. Of the 281 proteins identified, 42 were differentially altered in PD, PDCI, and NPH. With a certain overlap, 28 proteins were altered in PDCI and 25 proteins were altered in NPH. Five significantly upregulated proteins in PDCI were fibrinogen, gelsolin, complement factor-H, and apolipoproteins A-I and A-IV, whereas carnosine dipeptidase-1, carboxypeptidase-E, dickkopf-3, and secretogranin-3 precursor proteins were downregulated. Those uniquely altered in NPH were the insulin-like growth factor-binding protein, ceruloplasmin, α-1 antitrypsin, VGF nerve growth factor, and neural cell adhesion molecule L1-like protein. The ELISA-derived protein concentrations correlated with neuropsychological scores of certain cognitive domains. In PDCI, the Wisconsin card sorting percentile correlated negatively with fibrinogen. Intraperitoneal injection of native fibrinogen caused motor deficits in C57BL/6J mice as assessed by the pole test. Thus, a battery of proteins such as fibrinogen-α-chain, CFAH, and APOA-I/APOA-IV alongside neuropsychological assessment could be reliable biomarkers to distinguish PDCI and NPH.

Differences in Neuronal Numbers, Morphology, and Developmental Apoptosis in Mice Nigra Provide Experimental Evidence of Ontogenic Origin of Vulnerability to Parkinson's Disease.

Parkinson disease (PD) prevalence varies by ethnicity. In an earlier study, we replicated the reduced vulnerability to PD in an admixed population, using 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-susceptible C57BL/6 J, MPTP-resistant CD-1 and their F1 crossbreds. In the present study, we investigated if the differences have a developmental origin. Substantia nigra was evaluated at postnatal days 2 (P2), P6, P10, P14, P18, and P22. C57BL/6 J mice had smaller nigra and fewer dopaminergic neurons than the CD-1 and crossbreds at P2, which persisted through development. A significant increase in numbers and nigral volume was observed across strains until P14. A drastic decline thereafter was specific to C57BL/6 J. CD-1 and crossbreds retained their numbers from P14 to stabilize with supernumerary neurons at adulthood. The neuronal size increased gradually to attain adult morphology at P10 in the resistant strains, vis-à-vis at P22 in C57BL/6 J. Accordingly, in comparison to C57BL/6 J, the nigra of CD-1 and reciprocal crossbreds possessed cytomorphological features of resilience, since birth. The considerably lesser dopaminergic neuronal loss in the CD-1 and crossbreds was seen at P2 and P14 and thereafter was complemented by attenuated developmental cell death. The differences in programmed cell death were confirmed by reduced TUNEL labelling, AIF, and caspase-3 expression. GDNF expression aligned with the cell death pattern at P2 and P14 in both nigra and striatum. Earlier maturity of nigra and its neurons appears to be better features that reflect as MPTP resistance at adulthood. Thus, variable MPTP vulnerability in mice and also differential susceptibility to PD in humans may arise early during nigral development.

Chitotriosidase, a biomarker of amyotrophic lateral sclerosis, accentuates neurodegeneration in spinal motor neurons through neuroinflammation.

BACKGROUND: Cerebrospinal fluid from amyotrophic lateral sclerosis patients (ALS-CSF) induces neurodegenerative changes in motor neurons and gliosis in sporadic ALS models. Search for identification of toxic factor(s) in CSF revealed an enhancement in the level and enzyme activity of chitotriosidase (CHIT-1). Here, we have investigated its upregulation in a large cohort of samples and more importantly its role in ALS pathogenesis in a rat model. METHODS: CHIT-1 level in CSF samples from ALS (n = 158), non-ALS (n = 12) and normal (n = 48) subjects were measured using ELISA. Enzyme activity was also assessed (ALS, n = 56; non-ALS, n = 10 and normal-CSF, n = 45). Recombinant CHIT-1 was intrathecally injected into Wistar rat neonates. Lumbar spinal cord sections were stained for Iba1, glial fibrillary acidic protein and choline acetyl transferase to identify microglia, astrocytes and motor neurons respectively after 48 h of injection. Levels of tumour necrosis factor-α and interleukin-6 were measured by ELISA. FINDINGS: CHIT-1 level in ALS-CSF samples was increased by 20-fold and it can distinguish ALS patients with a sensitivity of 87% and specificity of 83.3% at a cut off level of 1405.43 pg/ml. Enzyme activity of CHIT-1 was also 15-fold higher in ALS-CSF and has a sensitivity of 80.4% and specificity of 80% at cut off value of 0.1077989 µmol/µl/min. Combining CHIT-1 level and activity together gave a positive predictive value of 97.78% and negative predictive value of 100%. Administration of CHIT-1 increased microglial numbers and astrogliosis in the ventral horn with a concomitant increase in the levels of pro-inflammatory cytokines. Amoeboid-shaped microglial and astroglial cells were also present around the central canal. CHIT-1 administration also resulted in the reduction of motor neurons. CONCLUSIONS: CHIT-1, an early diagnostic biomarker of sporadic ALS, activates glia priming them to attain a toxic phenotype resulting in neuroinflammation leading to motor neuronal death.

Small molecule modulator of aggrephagy regulates neuroinflammation to curb pathogenesis of neurodegeneration.

BACKGROUND: Plethora of efforts fails to yield a single drug to reverse the pathogenesis of Parkinson's disease (PD) and related α-synucleopathies. METHODS: Using chemical biology, we identified a small molecule inhibitor of c-abl kinase, PD180970 that could potentially clear the toxic protein aggregates. Genetic, molecular, cell biological and immunological assays were performed to understand the mechanism of action. In vivo preclinical disease model of PD was used to assess its neuroprotection efficacy. FINDINGS: In this report, we show the ability of a small molecule inhibitor of tyrosine kinases, PD180970, to induce autophagy (cell lines and mice midbrain) in an mTOR-independent manner and ameliorate the α-synuclein mediated toxicity. PD180970 also exerts anti-neuroinflammatory potential by inhibiting the release of proinflammatory cytokines such as IL-6 (interleukin-6) and MCP-1 (monocyte chemoattractant protein-1) through reduction of TLR-4 (toll like receptor-4) mediated NF-κB (nuclear factor kappa-light-chain-enhancer of activated B cells) activation. In vivo studies show that PD180970 is neuroprotective by degrading the toxic protein oligomers through induction of autophagy and subsiding the microglial activation. INTERPRETATION: These protective mechanisms ensure the negation of Parkinson's disease related motor impairments. FUND: This work was supported by Wellcome Trust/DBT India Alliance Intermediate Fellowship (500159-Z-09-Z), DST-SERB grant (EMR/2015/001946), DBT (BT/INF/22/SP27679/2018) and JNCASR intramural funds to RM, and SERB, DST (SR/SO/HS/0121/2012) to PAA, and DST-SERB (SB/YS/LS-215/2013) to JPC and BIRAC funding to ETA C-CAMP.

Divergent Expression Patterns of Drp1 and HSD10 in the Nigro-Striatum of Two Mice Strains Based on their MPTP Susceptibility.

Alterations in the basal ganglia circuitry are critical events in the pathophysiology of Parkinson's disease (PD). We earlier compared MPTP-susceptible C57BL/6J and MPTP-resistant CD-1 mice to understand the differential prevalence of PD in different ethnic populations like Caucasians and Asian-Indians. The MPTP-resistant CD-1 mice had 33% more nigral neurons and lost only 15-17% of them following MPTP administration. In addition to other cytomorphological features, their basal ganglia neurons had higher calcium-buffering protein levels. During disease pathogenesis as well as in MPTP-induced parkinsonian models, the loss of nigral neurons is associated with reduction in mitochondrial complex-1. Under these conditions, mitochondria respond by undergoing fusion or fission. 17ß-hydroxysteroid type 10, i.e., hydroxysteroid dehydrogenase10 (HSD10) and dynamin-related peptide1 (Drp1) are proteins involved in mitochondrial hyperfusion and fission, respectively. Each plays an important role in mitochondrial structure and homeostasis. Their role in determining susceptibility to the neurotoxin MPTP in basal ganglia is however unclear. We studied their expression using immunohistochemistry and Western blotting in the dorsolateral striatum, ventral tegmental area, and substantia nigra pars compacta (SNpc) of C57BL/6J and CD-1 mice. In the SNpc, which exhibits more neuron loss following MPTP, C57BL/6J had higher baseline Drp1 levels; suggesting persistence of fission under normal conditions. Whereas, HSD10 levels increased in CD-1 following MPTP administration. This suggests mitochondrial hyperfusion, as an attempt towards neuroprotection. Thus, the baseline differences in HSD10 and DRP1 levels as well as their contrasting MPTP-responses may be critical determinants of the magnitude of neuronal loss/survival. Similar differences may determine the variable susceptibility to PD in humans.

Aging mildly affects dendritic arborisation and synaptic protein expression in human substantia nigra pars compacta.

The protein α-synuclein, a major component of Lewy bodies in nigral neurons of aged and Parkinson's disease (PD) patients, normally co-localizes with synaptophysin and regulates the pool of synaptic vesicles. Our earlier study on substantia nigra pars compacta (SNpc) in an Asian-Indian population, demonstrated an age-associated linear but non-logarithmic increase in soluble α-synuclein without any loss of nigral neurons. Another distinctive finding was the presence of activated microglia in the ventrolateral region of the aged nigra, suggesting sub-threshold neurodegeneration. Since microglia prune dendrites, we evaluated the alterations in dendritic arborisation in the SNpc from autopsied midbrains of Asian-Indians through aging, using Golgi-Kopsch protocol. Further, we evaluated the expression of synaptic proteins, synaptophysin and synaptotagmin-11 as parallel markers of synaptic transmission anomalies. The dendritic arborization pattern was typical of large multipolar neurons. A subtle but non-significant decline in parameters like dendritic length and number of intersections was noted. Thus, the alterations were milder than those reported in PD. In the neurons of the young (till 10 years), faint cytoplasmic immunoreactivity of synaptic proteins was noted. In the adults and elderly, it was membrane-bound or appeared as punctae within neuropil. Both proteins showed a slight age-related decline, suggesting a mild decrease in the synaptic vesicular traffic, affecting the dopamine transmission with age that may manifest as minor motor disabilities in the elderly. Mapping the differences in synaptic profiles in differentially susceptible ethnic populations, could reveal interesting insights. Thus, nigra of aged individuals and PD patients share pathogenic features that differ in magnitude.

Admixing MPTP-resistant and MPTP-vulnerable mice enhances striatal field potentials and calbindin-D28K expression to avert motor behaviour deficits.

Asian-Indians are less vulnerable to Parkinson's disease (PD) than the Caucasians. Their admixed populace has even lesser risk. Studying this phenomenon using 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-susceptible C57BL/6J, MPTP-resistant CD-1 and their resistant crossbred mice revealed differences in the nigrostriatal cyto-molecular features. Here, we investigated the electrophysiological and behavioural correlates for differential MPTP-susceptibility and their outcome upon admixing. We recorded local field potentials (LFPs) from dorsal striatum and assessed motor co-ordination using rotarod and grip strength measures. Nigral calbindin-D28K expression, a regulator of striatal activity through nigrostriatal projections was evaluated using immunohistochemistry. The crossbreds had significantly higher baseline striatal LFPs. MPTP significantly increased the neuronal activity in delta (0.5-4 Hz) and low beta (12-16 Hz) ranges in C57BL/6J; significant increase across frequency bands till high beta (0.5-30 Hz) in CD-1, and caused no alterations in crossbreds. MPTP further depleted the already low nigral calbindin-D28K expression in C57BL/6J. While in crossbreds, it was further up-regulated. MPTP affected the rotarod and grip strength performance of the C57BL/6J, while the injected CD-1 and crossbreds performed well. The increased striatal ß-oscillations are comparable to that in PD patients. Higher power in CD-1 may be compensatory in nature, which were also reported in pre-symptomatic monkeys. Concurrent up-regulation of nigral calbindin-D28K may assist maintenance of striatal activity by buffering calcium overload in nigra. Thus, preserved motor behaviour in PD reminiscent conditions in CD-1 and crossbreds complement compensated/unaffected striatal LFPs. Similar electrophysiological correlates and cytomorphological features are envisaged in human phenomenon of differential PD prevalence, which are modulated upon admixing.

Sporadic amyotrophic lateral sclerosis (SALS) - skeletal muscle response to cerebrospinal fluid from SALS patients in a rat model.

Skeletal muscle atrophy is the most prominent feature of amyotrophic lateral sclerosis (ALS), an adult-onset neurodegenerative disease of motor neurons. However, the contribution of skeletal muscle to disease progression remains elusive. Our previous studies have shown that intrathecal injection of cerebrospinal fluid from sporadic ALS patients (ALS-CSF) induces several degenerative changes in motor neurons and glia of neonatal rats. Here, we describe various pathologic events in the rat extensor digitorum longus muscle following intrathecal injection of ALS-CSF. Adenosine triphosphatase staining and electron microscopic (EM) analysis revealed significant atrophy and grouping of type 2 fibres in ALS-CSF-injected rats. Profound neuromuscular junction (NMJ) damage, such as fragmentation accompanied by denervation, were revealed by α-bungarotoxin immunostaining. Altered expression of key NMJ proteins, rapsyn and calpain, was also observed by immunoblotting. In addition, EM analysis showed sarcolemmal folding, Z-line streaming, structural alterations of mitochondria and dilated sarcoplasmic reticulum. The expression of trophic factors was affected, with significant downregulation of vascular endothelial growth factor (VEGF), marginal reduction in insulin-like growth factor-1 (IGF-1), and upregulation of brain-derived neurotrophic factor (BDNF) and glial-derived neurotrophic factor (GDNF). However, motor neurons might be unable to harness the enhanced levels of BDNF and GDNF, owing to impaired NMJs. We propose that ALS-CSF triggers motor neuronal degeneration, resulting in pathological changes in the skeletal muscle. Muscle damage further aggravates the motor neuronal pathology, because of the interdependency between them. This sets in a vicious cycle, leading to rapid and progressive loss of motor neurons, which could explain the relentless course of ALS.This article has an associated First Person interview with the first author of the paper.

Baseline striatal and nigral interneuronal protein levels in two distinct mice strains differ in accordance with their MPTP susceptibility.

Epidemiological studies reveal an ethnicity-based bias in prevalence of Parkinson's disease (PD), deriving from the differences that exist between Caucasians and African or Asian populations. Experimental mice models provide a scope to analyse the cellular mechanisms of differential susceptibility to PD. C57BL/6J mice, for instance, are more susceptible to MPTP-induced Parkinsonism whereas CD-1 mice are resistant. In PD-pathogenesis, interneuronal contribution is also likely, although they comprise only 5-10% of the striatal cells. The interneurons harbour calcium binding proteins, like calretinin (Cal-R) and parvalbumin (PV), which are crucial in Ca2+ homeostasis for preventing calcium-induced excitotoxicity. GAD-67-immunoreactive interneurons are the other prominent set of GABAergic interneurons. In PD, dopamine loss up-regulates GAD-67 expression in striatal projection neurons and other basal ganglia circuit. We studied the possible contribution of interneurons in determining variable susceptibility by assessing the expression of calretinin, PV and GAD-67 in both striatum and substantia nigra pars compacta (SNpc) in two distinct mice strains, i.e. C57BL/6J and CD-1 under normal conditions, using unbiased stereology for quantification of immunoreactive cells and immunoblotting. The vulnerable C57BL/6J had lesser basal parvalbumin expression in both nigra and striatum whereas the calretinin levels were low only in the striatum. GAD-67 expression showed no perceptible differences in the striatum or SNpc of either of the strains. Differential expression of calcium buffering/binding proteins under normal physiological condition proffers a role for interneurons in the differential susceptibility to PD. Thus, even the baseline susceptibility indices i.e. without using the neurotoxin; can provide vital mechanistic insights into PD pathogenesis.

Etiogenic factors present in the cerebrospinal fluid from amyotrophic lateral sclerosis patients induce predominantly pro-inflammatory responses in microglia.

BACKGROUND: Microglial cell-associated neuroinflammation is considered as a potential contributor to the pathophysiology of sporadic amyotrophic lateral sclerosis. However, the specific role of microglia in the disease pathogenesis remains to be elucidated. METHODS: We studied the activation profiles of the microglial cultures exposed to the cerebrospinal fluid from these patients which recapitulates the neurodegeneration seen in sporadic amyotrophic lateral sclerosis. This was done by investigating the morphological and functional changes including the expression levels of prostaglandin E2 (PGE2), cyclooxygenase-2 (COX-2), TNF-α, IL-6, IFN-γ, IL-10, inducible nitric oxide synthase (iNOS), arginase, and trophic factors. We also studied the effect of chitotriosidase, the inflammatory protein found upregulated in the cerebrospinal fluid from amyotrophic lateral sclerosis patients, on these cultures. RESULTS: We report that the cerebrospinal fluid from amyotrophic lateral sclerosis patients could induce an early and potent response in the form of microglial activation, skewed primarily towards a pro-inflammatory profile. It was seen in the form of upregulation of the pro-inflammatory cytokines and factors including IL-6, TNF-α, iNOS, COX-2, and PGE2. Concomitantly, a downregulation of beneficial trophic factors and anti-inflammatory markers including VEGF, glial cell line-derived neurotrophic factor, and IFN-γ was seen. In addition, chitotriosidase-1 appeared to act specifically via the microglial cells. CONCLUSION: Our findings demonstrate that the cerebrospinal fluid from amyotrophic lateral sclerosis patients holds enough cues to induce microglial inflammatory processes as an early event, which may contribute to the neurodegeneration seen in the sporadic amyotrophic lateral sclerosis. These findings highlight the dynamic role of microglial cells in the pathogenesis of the disease, thus suggesting the need for a multidimensional and temporally guarded therapeutic approach targeting the inflammatory pathways for its treatment.

Manganese- and 1-methyl-4-phenylpyridinium-induced neurotoxicity display differences in morphological, electrophysiological and genome-wide alterations: implications for idiopathic Parkinson's disease.

Idiopathic Parkinson's disease and manganese-induced atypical parkinsonism are characterized by movement disorder and nigrostriatal pathology. Although clinical features, brain region involved and responsiveness to levodopa distinguish both, differences at the neuronal level are largely unknown. We studied the morphological, neurophysiological and molecular differences in dopaminergic neurons exposed to the Parkinson's disease toxin 1-methyl-4-phenylpyridinium ion (MPP+ ) and manganese (Mn), followed by validation in the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine and Mn mouse models. Morphological analysis highlighted loss of neuronal processes in the MPP+ and not the Mn model. Cellular network dynamics of dopaminergic neurons characterized by spike frequency and inter-spike intervals indicated major neuronal population (~ 93%) with slow discharge rates (0-5 Hz). While MPP+ exposure suppressed the firing of these neurons, Mn neither suppressed nor elevated the neuronal activity. High-throughput transcriptomic analysis revealed up-regulation of 694 and 603 genes and down-regulation of 428 and 255 genes in the MPP+ and Mn models respectively. Many differentially expressed genes were unique to either models and contributed to neuroinflammation, metabolic/mitochondrial function, apoptosis and nuclear function, synaptic plasticity, neurotransmission and cytoskeleton. Analysis of the Janus kinase-signal transducer and activator of transcription pathway with implications for neuritogenesis and neuronal proliferation revealed contrasting profile in both models. Genome-wide DNA methylomics revealed differences between both models and substantiated the epigenetic basis of the difference in the Janus kinase-signal transducer and activator of transcription pathway. We conclude that idiopathic Parkinson's disease and atypical parkinsonism have divergent neurotoxicological manifestation at the dopaminergic neuronal level with implications for pathobiology and evolution of novel therapeutics. Cover Image for this issue: doi. 10.1111/jnc.13821.

A novel autophagy modulator 6-Bio ameliorates SNCA/α-synuclein toxicity.

Parkinson disease (PD) is a life-threatening neurodegenerative movement disorder with unmet therapeutic intervention. We have identified a small molecule autophagy modulator, 6-Bio that shows clearance of toxic SNCA/α-synuclein (a protein implicated in synucleopathies) aggregates in yeast and mammalian cell lines. 6-Bio induces autophagy and dramatically enhances autolysosome formation resulting in SNCA degradation. Importantly, neuroprotective function of 6-Bio as envisaged by immunohistology and behavior analyses in a preclinical model of PD where it induces autophagy in dopaminergic (DAergic) neurons of mice midbrain to clear toxic protein aggregates suggesting that it could be a potential therapeutic candidate for protein conformational disorders.

VEGF alleviates ALS-CSF induced cytoplasmic accumulations of TDP-43 and FUS/TLS in NSC-34 cells.

Cytoplasmic mislocalisation and aggregation of TDP-43 and FUS/TLS proteins in spinal motor neurons contribute to the pathogenesis of the highly fatal disorder amyotrophic lateral sclerosis (ALS). We investigated the neuroprotective effect of VEGF on expression of these proteins in the motor neuronal cell line NSC-34 modelled to reminisce sporadic form of ALS. We studied the expression of TDP-43 and FUS/TLS proteins after exposure to ALS-CSF and following VEGF supplementation by quantitative confocal microscopy and electron microscopy. ALS-CSF caused cytoplasmic overexpression of both the proteins and stress-granule formation in the cells. These alterations were alleviated by VEGF supplementation. The related ultrastructural changes like nuclear membrane dysmorphism and p-bodies associated changes were also reversed. However the protein expression did not completely translocate to the nucleus, as some cells continued to show to cytoplasmic mislocalisation. Thus, the present findings indicate that VEGF alleviates TDP43 and FUS pathology by complimenting its role in controlling apoptosis and reversing choline acetyl transferase expression. Hence, VEGF appears to target multiple pathogenic processes in the neurodegenerative cascade of ALS.

Admixing of MPTP-Resistant and Susceptible Mice Strains Augments Nigrostriatal Neuronal Correlates to Resist MPTP-Induced Neurodegeneration.

Disease genetics in admixed populations like Hispanic-Americans, African-Americans, etc. are gaining importance due to high disease burden in them. Furthermore, epidemiological studies conclusively prove ethnicity-based differential prevalence of Parkinson's disease (PD), since the American-Caucasians are more susceptible than Asian-Indians and Africans. Contradictorily, Anglo-Indians, an admixture of Europeans and Asian-Indians are five-times less susceptible than Indians. We evaluated the neural basis of this phenomenon using the cytomorphological features of susceptibility to nigrostriatal neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). The nigral dopaminergic neuronal numbers, their size and tyrosine hydroxylase (TH), PitX3 and Nurr1 expression were compared in MPTP-susceptible C57BL/6J mice, MPTP-resistant CD-1 mice and their crossbreds using stereology, morphometry and densitometry. Apoptotic index was evaluated by TUNEL-assay and caspase-3 expression. Striatal volume, TH and glial derived neurotrophic factor (GDNF) expression were studied. The normal CD-1 and crossbreds had significantly more, although smaller, nigral dopaminergic neurons than C57BL/6J, and a larger striatum. The crossbreds had higher TH, Nurr1 and PitX3 levels. MPTP administration caused loss of ~50-60 % nigral dopaminergic neurons in C57BL/6J and ~15 % in CD-1, but none in crossbreds. MPTP-induced cellular shrinkage in C57BL/6J was contrasted by nuclear enlargement without somal alterations in resistant strains. MPTP lowered the striatal TH and GDNF in C57BL/6J. Elevated striatal GDNF in CD-1 and crossbreds could be of compensatory nature and complemented the reduced nigral caspase-3 expression to attenuate and/or block apoptosis. Similar neural correlates of resilience are envisaged in the Anglo-Indian population. Thus, we present the core neuroanatomical features of resilience against PD and evidence for ethnicity-based differential prevalence.

Brain-Derived Neurotrophic Factor Facilitates Functional Recovery from ALS-Cerebral Spinal Fluid-Induced Neurodegenerative Changes in the NSC-34 Motor Neuron Cell Line.

BACKGROUND: The survival of motor neurons is dependent upon neurotrophic factors both during childhood and adolescence and during adult life. In disease conditions, such as in patients with amyotrophic lateral sclerosis (ALS), the mRNA levels of trophic factors like brain-derived neurotrophic factor (BDNF), insulin-like growth factor-1 (IGF-1), fibroblast growth factor-2 (FGF-2), and vascular endothelial growth factor are downregulated. This was replicated in our in vivo experimental system following the injection of cerebral spinal fluid (CSF) of sporadic ALS (ALS-CSF) patients. OBJECTIVE: To evaluate the protective role of BDNF in a model of sporadic ALS patients. METHODS: The expressions of endogenous BDNF, its receptor TrkB, the enzyme choline acetyl transferase (ChAT), and phosphorylated neurofilaments were studied in NSC-34 cells. The calcium-buffering and proapoptotic effects were assessed by calbindin-D28K and caspase-3 expression, respectively. RESULTS: ALS-CSF considerably depleted the endogenous BDNF protein, while its effect on IGF-1 and FGF-2 was inconsequential; this indirectly indicates a key role for BDNF in supporting motor neuronal survival. The exogenous supplementation of BDNF reversed autocrine expression; however, it may not be completely receptor mediated, as the TrkB levels were not restored. BDNF completely revived ChAT expression. It may inhibit apoptosis by restoring Ca2+ homeostasis, since caspase-3 and calbindin-D28K expression was back to normal. The organellar ultrastructural changes were only partially reversed. CONCLUSION: Our study provides evidence that BDNF supplementation ameliorates most but not all degenerative changes. The incomplete revival at the ultrastructural level signifies the requirement of factors other than BDNF for near-total protection of motor neurons, and, to an extent, it explains why only a partial success is achieved in clinical trials with BDNF in ALS patients.

Astroglia acquires a toxic neuroinflammatory role in response to the cerebrospinal fluid from amyotrophic lateral sclerosis patients.

BACKGROUND: Non-cell autonomous toxicity is one of the potential mechanisms implicated in the etiopathogenesis of amyotrophic lateral sclerosis (ALS). However, the exact role of glial cells in ALS pathology is yet to be fully understood. In a cellular model recapitulating the pathology of sporadic ALS, we have studied the inflammatory response of astroglia following exposure to the cerebrospinal fluid from ALS patients (ALS-CSF). METHODS: Various inflammatory markers including pro-inflammatory and anti-inflammatory cytokines, COX-2, PGE-2, trophic factors, glutamate, nitric oxide (NO), and reactive oxygen species (ROS) were analyzed in the rat astroglial cultures exposed to ALS-CSF and compared with the disease control or normal controls. We used immunofluorescence, ELISA, and immunoblotting techniques to investigate the protein expression and real-time PCR to study the messenger RNA (mRNA) expression. Glutamate, NO, and ROS were estimated using appropriate biochemical assays. Further, the effect of conditioned medium from the astroglial cultures exposed to ALS-CSF on NSC-34 motor neuronal cell line was detected using the MTT assay. Statistical analysis was carried out using one-way ANOVA followed by Tukey's post hoc test, or Student's t test, as applicable. RESULTS: Here, we report that the ALS-CSF enhanced the production and release of inflammatory cytokines IL-6 and TNF-α, as well as COX-2 and PGE-2. Concomitantly, anti-inflammatory cytokine IL-10 and the beneficial trophic factors, namely VEGF and GDNF, were down-regulated. We also found impaired regulation of glutamate, NO, and ROS in the astroglial cultures treated with ALS-CSF. The conditioned medium from the ALS-CSF exposed astroglial cultures induced degeneration in NSC-34 cells. CONCLUSIONS: Our study demonstrates that the astroglial cells contribute to the neuroinflammation-mediated neurodegeneration in the in vitro model of sporadic ALS.

Differential expression of calbindin in nigral dopaminergic neurons in two mice strains with differential susceptibility to 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine.

Parkinson's disease (PD) affects the A9 dopaminergic (DA) neurons of substantia nigra pars compacta (SNpc) whereas other DA neuronal subtypes are spared. The role of calbindin in this differential vulnerability has been long elicited, and is seen in the MPTP induced mice models of PD. A peculiar feature of mice models is the strain specific differences in the susceptibility to MPTP. Here, calbindin-D28K expression in DA neurons of SNpc of MPTP susceptible C57BL/6 mice and MPTP resistant CD-1 mice was studied as a susceptibility marker of degeneration. Unbiased stereological estimation of immunoperoxidase stained midbrain sections revealed significantly higher number of calbindin immunoreactive cells in SNpc of CD-1 mice compared to that of C57BL/6 strain. Western blotting showed minimal differences in the levels. Calbindin-tyrosine hydroxylase immunofluorescence co-labeling was performed to map the calbindin immunoreactive DA neurons in SNpc and ventral tegmental area (VTA) and to quantify the calbindin expression at cellular level. While the levels were comparable in VTA of both mice strains, the SNpc of CD-1 mice showed significantly higher calbindin expression. Within the SNpc, the medial and dorsal subdivisions showed higher calbindin expression in CD-1. The expression in the ventrolateral SNpc of both strains remained comparable. Our observations clearly point at overall higher levels and sizeable percentage of cells expressing more calbindin in SNpc of CD-1 mice, which might confer neuroprotection against MPTP, while its lower expression makes C57BL/6 mice more susceptible. Similar mechanism may be attributed to the phenomenon of differential prevalence of PD in different ethnic populations.

Cerebrospinal Fluid from Sporadic Amyotrophic Lateral Sclerosis Patients Induces Mitochondrial and Lysosomal Dysfunction.

In our laboratory, we have developed (1) an in vitro model of sporadic Amyotrophic Lateral Sclerosis (sALS) involving exposure of motor neurons to cerebrospinal fluid (CSF) from sALS patients and (2) an in vivo model involving intrathecal injection of sALS-CSF into rat pups. In the current study, we observed that spinal cord extract from the in vivo sALS model displayed elevated reactive oxygen species (ROS) and mitochondrial dysfunction. Quantitative proteomic analysis of sub-cellular fractions from spinal cord of the in vivo sALS model revealed down-regulation of 35 mitochondrial proteins and 4 lysosomal proteins. Many of the down-regulated mitochondrial proteins contribute to alterations in respiratory chain complexes and organellar morphology. Down-regulated lysosomal proteins Hexosaminidase, Sialidase and Aryl sulfatase also displayed lowered enzyme activity, thus validating the mass spectrometry data. Proteomic analysis and validation by western blot indicated that sALS-CSF induced the over-expression of the pro-apoptotic mitochondrial protein BNIP3L. In the in vitro model, sALS-CSF induced neurotoxicity and elevated ROS, while it lowered the mitochondrial membrane potential in rat spinal cord mitochondria in the in vivo model. Ultra structural alterations were evident in mitochondria of cultured motor neurons exposed to ALS-CSF. These observations indicate the first line evidence that sALS-CSF mediated mitochondrial and lysosomal defects collectively contribute to the pathogenesis underlying sALS.