OBJECTIVE Posthemorrhagic hydrocephalus (PHH) following preterm intraventricular hemorrhage (IVH) is among the most severe sequelae of extreme prematurity and a significant contributor to preterm morbidity and mortality. The authors have previously shown hemoglobin and ferritin to be elevated in the lumbar puncture cerebrospinal fluid (CSF) of neonates with PHH. Herein, they evaluated CSF from serial ventricular taps to determine whether neonates with PHH following severe initial ventriculomegaly had higher initial levels and prolonged clearance of CSF hemoglobin and hemoglobin degradation products compared to those in neonates with PHH following moderate initial ventriculomegaly. METHODS In this observational cohort study, CSF samples were obtained from serial ventricular taps in premature neonates with severe IVH and subsequent PHH. CSF hemoglobin, ferritin, total iron, total bilirubin, and total protein were quantified using ELISA. Ventriculomegaly on cranial imaging was assessed using the frontal occipital horn ratio (FOHR) and was categorized as severe (FOHR > 0.6) or moderate (FOHR ≤ 0.6). RESULTS Ventricular tap CSF hemoglobin (mean) and ferritin (initial and mean) were higher in neonates with severe versus moderate initial ventriculomegaly. CSF hemoglobin, ferritin, total iron, total bilirubin, and total protein decreased in a nonlinear fashion over the weeks following severe IVH. Significantly higher levels of CSF ferritin and total iron were observed in the early weeks following IVH in neonates with severe initial ventriculomegaly than in those with initial moderate ventriculomegaly. CONCLUSIONS Among preterm neonates with PHH following severe IVH, elevated CSF hemoglobin, ferritin, and iron were associated with more severe early ventricular enlargement (FOHR > 0.6 vs ≤ 0.6 at first ventricular tap).

Abstract Objective Parkinson disease (PD) is defined by the accumulation of misfolded α‐synuclein (α‐syn) in Lewy bodies and Lewy neurites. It affects multiple cortical and subcortical neuronal populations. The majority of people with PD develop dementia, which is associated with Lewy bodies in neocortex and referred to as Lewy body dementia (LBD). Other neuropathologic changes, including amyloid β (Aβ) and tau accumulation, occur in some LBD cases. We sought to quantify α‐syn, Aβ, and tau accumulation in neocortical, limbic, and basal ganglia regions. Methods We isolated insoluble protein from fresh frozen postmortem brain tissue samples for eight brains regions from 15 LBD, seven Alzheimer disease (AD), and six control cases. We measured insoluble α‐syn, Aβ, and tau with recently developed sandwich ELISAs. Results We detected a wide range of insoluble α‐syn accumulation in LBD cases. The majority had substantial α‐syn accumulation in most regions, and dementia severity correlated with neocortical α‐syn. However, three cases had low neocortical levels that were indistinguishable from controls. Eight LBD cases had substantial Aβ accumulation, although the mean Aβ level in LBD was lower than in AD. The presence of Aβ was associated with greater α‐syn accumulation. Tau accumulation accompanied Aβ in only one LBD case. Interpretation LBD is associated with insoluble α‐syn accumulation in neocortical regions, but the relatively low neocortical levels in some cases suggest that other changes contribute to impaired function, such as loss of neocortical innervation from subcortical regions. The correlation between Aβ and α‐syn accumulation suggests a pathophysiologic relationship between these two processes.

ObjectiveIron has been implicated in the pathogenesis of brain injury and hydrocephalus after preterm germinal matrix hemorrhage‐intraventricular hemorrhage, however, it is unknown how external or endogenous intraventricular clearance of iron pathway proteins affect the outcome in this group.MethodsThis prospective multicenter cohort included patients with posthemorrhagic hydrocephalus (PHH) who underwent (1) temporary and permanent cerebrospinal fluid (CSF) diversion and (2) Bayley Scales of Infant Development‐III testing around 2 years of age. CSF proteins in the iron handling pathway were analyzed longitudinally and compared to ventricle size and neurodevelopmental outcomes.ResultsThirty‐seven patients met inclusion criteria with a median estimated gestational age at birth of 25 weeks; 65% were boys. Ventricular CSF levels of hemoglobin, iron, total bilirubin, and ferritin decreased between temporary and permanent CSF diversion with no change in CSF levels of ceruloplasmin, transferrin, haptoglobin, and hepcidin. There was an increase in CSF hemopexin during this interval. Larger ventricle size at permanent CSF diversion was associated with elevated CSF ferritin (p = 0.015) and decreased CSF hemopexin (p = 0.007). CSF levels of proteins at temporary CSF diversion were not associated with outcome, however, higher CSF transferrin at permanent CSF diversion was associated with improved cognitive outcome (p = 0.015). Importantly, longitudinal change in CSF iron pathway proteins, ferritin (decrease), and transferrin (increase) were associated with improved cognitive (p = 0.04) and motor (p = 0.03) scores and improved cognitive (p = 0.04), language (p = 0.035), and motor (p = 0.008) scores, respectively.InterpretationLongitudinal changes in CSF transferrin (increase) and ferritin (decrease) are associated with improved neurodevelopmental outcomes in neonatal PHH, with implications for understanding the pathogenesis of poor outcomes in PHH. ANN NEUROL 2021;90:217–226

Background and Purpose- Preterm neonates with intraventricular hemorrhage (IVH) are at risk for posthemorrhagic hydrocephalus and poor neurological outcomes. Iron has been implicated in ventriculomegaly, hippocampal injury, and poor outcomes following IVH. We hypothesized that levels of cerebrospinal fluid blood breakdown products and endogenous iron clearance proteins in neonates with IVH differ from those of neonates with IVH who subsequently develop posthemorrhagic hydrocephalus. Methods- Premature neonates with an estimated gestational age at birth <30 weeks who underwent lumbar puncture for clinical evaluation an average of 2 weeks after birth were evaluated. Groups consisted of controls (n=16), low-grade IVH (grades I-II; n=4), high-grade IVH (grades III-IV; n=6), and posthemorrhagic hydrocephalus (n=9). Control subjects were preterm neonates born at <30 weeks' gestation without brain abnormality or hemorrhage on cranial ultrasound, who underwent lumbar puncture for clinical purposes. Cerebrospinal fluid hemoglobin, total bilirubin, total iron, ferritin, ceruloplasmin, transferrin, haptoglobin, and hemopexin were quantified. Results- Cerebrospinal fluid hemoglobin levels were increased in posthemorrhagic hydrocephalus compared with high-grade IVH (9.45 versus 6.06 µg/mL, P<0.05) and cerebrospinal fluid ferritin levels were increased in posthemorrhagic hydrocephalus compared with controls (511.33 versus 67.08, P<0.01). No significant group differences existed for the other cerebrospinal fluid blood breakdown and iron-handling proteins tested. We observed positive correlations between ventricular enlargement (frontal occipital horn ratio) and ferritin (Pearson r=0.67), hemoglobin (Pearson r=0.68), and total bilirubin (Pearson r=0.69). Conclusions- Neonates with posthemorrhagic hydrocephalus had significantly higher levels of hemoglobin than those with high-grade IVH. Levels of blood breakdown products, hemoglobin, ferritin, and bilirubin correlated with ventricular size. There was no elevation of several iron-scavenging proteins in cerebrospinal fluid in neonates with posthemorrhagic hydrocpehalus, indicative of posthemorrhagic hydrocephalus as a disease state occurring when endogenous iron clearance mechanisms are overwhelmed.

Buddhala, Chandana PhD; Paturu, Mounica; Morales, Diego BA; Smyser, Christopher MD; Limbrick, David D MD, PhD; Mahaney, Kelly B MD, MS; Strahle, Jennifer MD Author Information

Objective: The purpose of this study was to investigate the relationship between disruption of MRI-measured resting-state functional connectivity (rs-fcMRI) brain networks and CSF levels of potentially pathogenic proteins that reflect brain pathology in Parkinson disease (PD). Methods: PD participants without dementia (n = 43) and age-matched controls (n = 22) had lumbar punctures to measure CSF protein levels, Pittsburgh compound B (PiB)–PET imaging, and rs-fcMRI while off medication. Imaging analyses focused on 5 major resting-state networks as well as the striatum. Results: Participants with PD had significantly reduced sensorimotor functional connectivity, which correlated with reduced CSF levels of α-synuclein. The PD group also had significantly stronger default mode network functional connectivity that did not correlate with CSF β-amyloid (Aβ)42 or PiB uptake. In contrast, default mode network functional connectivity in the control group did correlate with CSF Aβ42 levels. Functional connectivity was similar between groups in the dorsal attention, control, and salience networks. Conclusion: These results suggest that abnormal α-synuclein accumulation, but not Aβ, contributes to the disruption of motor-related functional connectivity in PD. Furthermore, correlating CSF protein measures with the strength of resting-state networks provides a direct link between abnormal α-synuclein metabolism and disrupted brain function in PD.

Accumulation of misfolded alpha-synuclein (alpha-syn) protein in Lewy bodies and neurites is the cardinal pathologic feature of Parkinson disease (PD), but abnormal deposition of other proteins may also play a role. Cerebrospinal fluid (CSF) levels of proteins known to accumulate in PD may provide insight into disease-associated changes in protein metabolism and their relationship to disease progression. We measured CSF beta-syn, amyloid beta(1-42) (A beta(1-42)), and tau from 77 nondemented PD and 30 control participants. CSF a-syn and A beta(1-42) were significantly lower in PD compared with controls. In contrast with increased CSF tau in Alzheimer disease, CSF tau did not significantly differ between PD and controls. CSF protein levels did not significantly correlate with ratings of motor function or performance on neuropsychological testing. As expected, CSF A beta(1-42) inversely correlated with [ C-11]-Pittsburgh compound B (PiB) mean cortical binding potential, with PiB(+) PD participants having lower CSF A beta(1-42) compared with PiB-PD participants. Furthermore, CSF beta-syn positively correlated with A beta(1-42) in PD participants but not in controls, suggesting a pathophysiologic connection between the metabolisms of these proteins in PD. (C) 2015 Elsevier Inc. All rights reserved.

OBJECTIVE:People with Parkinson disease (PD) frequently develop dementia, which is associated with neocortical deposition of alpha-synuclein (α-syn) in Lewy bodies and Lewy neurites. In addition, neuronal loss and deposition of aggregated α-syn also occur in multiple subcortical nuclei that project to neocortical, limbic, and basal ganglia regions. Therefore, we quantified regional deficits in innervation from these PD-affected subcortical nuclei, by measuring the neurotransmitters and neurotransmitter transporter proteins originating from projections of dopaminergic neurons in substantia nigra pars compacta, serotonergic neurons in dorsal raphé nuclei, noradrenergic neurons in locus coeruleus, and cholinergic neurons in nucleus basalis of Meynert.METHODS:High-performance liquid chromatography and novel enzyme-linked immunosorbent assays were performed to quantify dopaminergic, serotonergic, noradrenergic, and cholinergic innervation in postmortem brain tissue. Eight brain regions from 15 PD participants (with dementia and Braak stage 6 α-syn deposition) and six age-matched controls were tested.RESULTS:PD participants compared to controls had widespread reductions of dopamine transporter in caudate, amygdala, hippocampus, inferior parietal lobule (IPL), precuneus, and visual association cortex (VAC) that exceeded loss of dopamine, which was only significantly reduced in caudate and amygdala. In contrast, PD participants had comparable deficits of both serotonin and serotonin transporter in caudate, middle frontal gyrus, IPL, and VAC. PD participants also had significantly reduced norepinephrine levels for all eight brain regions tested. Vesicular acetylcholine transporter levels were only quantifiable in caudate and hippocampus and did not differ between PD and control groups.INTERPRETATION:These results demonstrate widespread deficits in dopaminergic, serotonergic, and noradrenergic innervation of neocortical, limbic, and basal ganglia regions in advanced PD with dementia.

Accumulation of misfolded α-synuclein (α-syn) protein in Lewy bodies and neurites is the cardinal pathologic feature of Parkinson disease (PD), but abnormal deposition of other proteins may also play a role. Cerebrospinal fluid (CSF) levels of proteins known to accumulate in PD may provide insight into disease-associated changes in protein metabolism and their relationship to disease progression. We measured CSF α-syn, amyloid β1–42 (Aβ1–42), and tau from 77 nondemented PD and 30 control participants. CSF α-syn and Aβ1–42 were significantly lower in PD compared with controls. In contrast with increased CSF tau in Alzheimer disease, CSF tau did not significantly differ between PD and controls. CSF protein levels did not significantly correlate with ratings of motor function or performance on neuropsychological testing. As expected, CSF Aβ1–42 inversely correlated with [11C]-Pittsburgh compound B (PiB) mean cortical binding potential, with PiB+ PD participants having lower CSF Aβ1–42 compared with PiB− PD participants. Furthermore, CSF α-syn positively correlated with Aβ1–42 in PD participants but not in controls, suggesting a pathophysiologic connection between the metabolisms of these proteins in PD.

Accumulation of misfolded α-synuclein (α-syn) protein in Lewy bodies and neurites is the cardinal pathologic feature of Parkinson disease (PD), but abnormal deposition of other proteins may also play a role. Cerebrospinal fluid (CSF) levels of proteins known to accumulate in PD may provide insight into disease-associated changes in protein metabolism and their relationship to disease progression. We measured CSF α-syn, amyloid β₁₋₄₂ (Aβ₁₋₄₂), and tau from 77 nondemented PD and 30 control participants. CSF α-syn and Aβ₁₋₄₂ were significantly lower in PD compared with controls. In contrast with increased CSF tau in Alzheimer disease, CSF tau did not significantly differ between PD and controls. CSF protein levels did not significantly correlate with ratings of motor function or performance on neuropsychological testing. As expected, CSF Aβ₁₋₄₂ inversely correlated with [(11)C]-Pittsburgh compound B (PiB) mean cortical binding potential, with PiB(+) PD participants having lower CSF Aβ₁₋₄₂ compared with PiB(-) PD participants. Furthermore, CSF α-syn positively correlated with Aβ₁₋₄₂ in PD participants but not in controls, suggesting a pathophysiologic connection between the metabolisms of these proteins in PD.

New therapeutic targets are becoming increasingly popular for the treatment of a wide array of neurodegenerative diseases, the preferred targets being those that prevent neuronal apoptosis at multiple levels or those that can cross the blood-brain barrier in order to replace degenerated cells and promote neuronal regeneration. One such rapidly emerging neuroprotective agents is taurine. Taurine is a ubiquitous amino acid that satisfies most criteria to be classified as a neurotransmitter. Because of a wide spectrum of effects that taurine can induce on intrinsic apoptosis pathways, such as modulating mitochondrial pore permeability, attenuating endoplasmic reticulum stress, maintaining calcium homeostasis, and downregulating the activities of a range of pro-apoptotic proteins, including calpain and caspases, while upregulating a variety of anti-apoptotic proteins involved in glutamate and hypoxia-induced toxicity, taurine is being extensively studied and successfully applied for the treatment of neurodegenerative diseases. Another potential molecule being researched for combating neurodegenerative diseases is granulocyte colony-stimulating factor (G-CSF), which originates from the cytokine family of growth factors. G-CSF has gained widespread attention because of its ability to cross the blood-brain barrier, the presence of its receptors in the central nervous system, anti-apoptotic functions, and its proliferative role in the restoration of tissue survival via neurogenesis. In this review from the available current literature, the modes of action of taurine and G-CSF are discussed. Further mechanistic studies are warranted in order to fully realize the potential of these two molecules.

Previously, we have shown that the GABA synthesizing enzyme, L-glutamic acid decarboxylase 65 (GAD65) is cleaved to form its truncated form (tGAD65) which is 2-3 times more active than the full length form (fGAD65). The enzyme responsible for cleavage was later identified as calpain. Calpain is known to cleave its substrates either under a transient physiological stimulus or upon a sustained pathological insult. However, the precise role of calpain cleavage of fGAD65 is poorly understood. In this communication, we examined the cleavage of fGAD65 under diverse pathological conditions including rats under ischemia/reperfusion insult as well as rat brain synaptosomes and primary neuronal cultures subjected to excessive stimulation with high concentration of KCl. We have shown that the formation of tGAD65 progressively increases with increasing stimulus concentration both in rat brain synaptosomes and primary rat embryo cultures. More importantly, direct cleavage of synaptic vesicle - associated fGAD65 by calpain was demonstrated and the resulting tGAD65 bearing the active site of the enzyme was detached from the synaptic vesicles. Vesicular GABA transport of the newly synthesized GABA was found to be reduced in calpain treated SVs. Furthermore, we also observed that the levels of tGAD65 in the focal cerebral ischemic rat brain tissue increased corresponding to the elevation of local glutamate as indicated by microdialysis. Moreover, the levels of tGAD65 was also proportional to the degree of cell death when the primary neuronal cultures were exposed to high KCl. Based on these observations, we conclude that calpain-mediated cleavage of fGAD65 is pathological, presumably due to decrease in the activity of synaptic vesicle - associated fGAD65 resulting in a decrease in the GABA synthesis - packaging coupling process leading to reduced GABA neurotransmission.

Previously, we showed that taurine protects neurons against glutamate‐induced excitotoxicity by inhibiting the glutamate‐induced increase of [Ca2+]i. In this study, we report that taurine prevents glutamate‐induced chromosomal condensation, indicating that taurine inhibits glutamate‐induced apoptosis. We found that Bcl‐2 was down‐regulated while Bax was up‐regulated by glutamate treatment, and these changes were prevented in the presence of taurine. We have also shown that taurine inhibits glutamate‐induced activation of calpain. Furthermore, calpastatin, a specific calpain inhibitor, also prevented glutamate‐induced cell death. Here we propose the mechanisms underlying glutamate‐induced apoptosis and taurine's inhibition of glutamate‐induced apoptosis to be as follows: glutamate stimulation induces [Ca2+]i elevation, which in turn activates calpain; activation of calpain leads to a reduction of Bcl‐2:Bax ratios; with decreased Bcl‐2:Bax ratios Bax homodimers form, Bax homodimerization, and translocation to the mitochondria result in the release of cytochrome c; released cytochrome c in turn activates a downstream caspase cascade leading to apoptosis. The antiapoptotic function of taurine is due to its inhibition of glutamate‐induced membrane depolarization. © 2008 Wiley‐Liss, Inc.

This review focuses on the recent advances that were made in understanding the fundamental mechanisms of the regulation of l-glutamic acid decarboxylase (GAD; E.C. 4.1.1.15), the enzyme responsible for the synthesis of the major inhibitory neurotransmitter γ-amino butyric acid (GABA). In the brain, there are two isoforms of GAD- GAD67 and GAD65, where 67 and 65 refer to their respective molecular weights in kDa. A number of neurodegenerative diseases are known to occur as a result of insufficient inhibition due to failure of GABA neurotransmission. Since the rate-limiting step in GABA biosynthesis is the decarboxylation of glutamate by GAD, it is important to understand how GAD is regulated. So far, we know that GAD is regulated at the transcriptional level by alternate splicing and at the post-translational level by protein phosphorylation, palmitoylation and activity-dependent cleavage. Here, we present new evidence of the presence of GAD65 associated with mitochondria in the axon terminal and project a model in which ATP generated by mitochondrial GAD65 may serve an important function in providing energy for GAD65 mediated GABA biosynthesis and packaging into synaptic vesicles by vesicular GABA transporter (VGAT).

Brain L‐glutamate decarboxylase (GAD) is responsible for the synthesis of neurotransmitter GABA. There are two GAD isoforms, namely GAD65 and GAD67. The two are regulated differentially by protein phosphorylation. GAD65 is activated by phosphorylation mediated by PKC‐ε whereas GAD67 is inhibited by phosphorylation mediated by PKA. The phosphorylation site of GAD67 was identified as Threonine 91 based on MALDI‐TOF and site‐directed mutation analysis. (Biochemistry 43:6182–6189, 2004). Here we further report several lines of evidence on the regulation of GAD activity during neuronal activity with special focus on GAD65. Under stimulation condition, GAD65 activity is increased due to increase in phosphorylation through translocation of PKC‐ε to become membrane‐anchored. Several potential phosphorylation sites have been implicated in the regulation of GAD65 activity. In addition, cleavage of GAD65 by calpain also increased GAD65 activity. A model depicting activity‐dependent regulation of GAD65 by phosphorylation and proteolytic cleavage is proposed.

Glutamic acid decarboxylase (GAD) is the rate-limiting enzyme for gamma-aminobutyric acid (GABA) biosynthesis. Previously, we reported the presence of truncated forms of GAD in vivo and in vitro. In addition, an unidentified endogenous protease responsible for proteolytic cleavage of full-length GAD (fGAD) to its truncated form (tGAD) was also observed. In this communication, we report that mu-calpain is a good candidate for conversion of fGAD(67) to tGAD(67). This conclusion is based on the following observations: 1. purified recombinant GAD(67) is cleaved by mu-calpain at specific sites; 2. in brain synaptosomal preparation, GAD(67) is cleaved to its truncated form by an endogenous protease which is inhibited by specific calpain inhibitors; 3. in mu-calpain knockout mice, the level of tGAD in the brain is greatly reduced compared with the wild type; 4. when mu-calpain gene is silenced by siRNA, the level of tGAD is also markedly reduced compared to the control group; and 5. mu-calpain is activated by neuronal stimulation and Ca(2+)-influx. The physiological significance of calpain in regulation of GABA synthesis and GABAergic neurotransmission is also discussed.

In GAD65-knockout mice, lack of GAD65 expression was confirmed. The expression level of vesicular GABA transporter (VGAT) was upregulated, and no change in the synaptic vesicles (SV)-associated GAD67 was found. GAD65(-/-) SV transported cytosolic GABA much more efficiently than that of the wild type, further supporting our model that there is a structural and functional coupling between GABA synthesis and packaging into SV. Both full-length and truncated forms of GAD65 could bind to GABAergic SV, indicating the N-terminus is not required for the anchoring of GAD65 to SV. Although both GAD65(-/-) SV reconstituted with either GAD65 or GAD67 could synthesize GABA from [3H] glutamate and transport this newly synthesized GABA into SV, the combined evidence suggests that GAD65 plays a major role in GABA transmission in normal physiological condition. However, GAD67 could serve this role under some pathological conditions.